AU770292B2 - Compounds active at a novel site on receptor-operated calcium channels useful for treatment of neurological disorders and diseases - Google Patents

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S&F Ref: 444260D1

AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT

ORIGINAL

Name and Address of Applicant: Actual Inventor(s): Address for Service: NPS Pharmaceuticals, Inc.

Suite 240 420 Chipeta Way Salt Lake City Utah 84108 United States of America Alan L Mueller Scott T Moe Manuel F Balandrin Bradford C Vanwagenen Eric G Delmar Linda D Artman Robert M Barmore Daryl L Smith Spruson Ferguson St Martins Tower 31 Market Street Sydney NSW 2000 Invention Title: Compounds Active at a Novel Site on Receptor-operated Calcium Channels Useful for Treatment of Neurological Disorders and Diseases The following statement is a full description of this invention, including the best method of performing it known to me/us:- 5845c [I:\DAYLIB\LIBF]08337.doc:vsg Description Compounds Active at a Novel Site on Receptor-Operated Calcium Channels Useful for Treatment of Neurological Disorders and Diseases Reference is made to co-pending International Patent Application No. PCT/US96/10201, filed June 7, 1996, which is a continuation-in-part of co-pending application U.S. Serial No.

08/485,038, filed June 7, 1995, which is a continuation-in-part of co-pending International Patent Application No. PCT/US94/12293, filed October 26, 1994, designating the United States, which is a continuation-in-part of co-pending application U.S. Serial No. 08/288,688, filed August 9, 1994, now abandoned, which was a continuation-in-part of co-pending International Patent Application No.

PCT/US94/01462, filed February 8, 1994, which is a continuation-in-part of U.S. Serial No.

08/014,813, filed February 8, 1993, now abandoned, all of which are hereby incorporated by reference herein in their entirety.

Field of the Invention This invention relates to compounds useful as neuroprotectants, anticonvulsants, anxiolytics, analgesics, muscle relaxants or adjuvants to general anesthetics. The invention relates as well to methods useful for the treatment of neurological disorders and diseases, including, but not limited to, global and focal ischemic and hemorrhagic stroke, head trauma, spinal cord injury, hypoxiainduced nerve cell damage such as in cardiac arrest or neonatal distress, [R:\LIBZZ]444260DI speci.doc:gym epilepsy, anxiety, and neurodegenerative diseases such as Alzheimer's Disease, Huntington's Disease, Parkinson's Disease, and amyotrophic lateral sclerosis (ALS). The invention relates as well to methods of screening for compounds active at a novel site on receptor-operated calcium channels, and thereby possessing therapeutic utility as neuroprotectants, anticonvulsants, anxiolytics, analgesics, muscle relaxants or adjuvants to general anesthetics, and/or possessing potential therapeutic utility for the treatment of neurological disorders and diseases as described above.

Background of the Invention The following is a description of relevant art, none of which is admitted to be prior art to the claims.

Glutamate is the major excitatory neurotransmitter in the mammalian brain. Glutamate binds or interacts with one or more glutamate receptors which can be differentiated pharmacologically into several subtypes. In the mammalian central nervous system (CNS) there are three main subtypes of ionotropic glutamate receptors, defined pharmacologically by the selective agonists N-methyl-D-aspartate (NMDA), kainate and a-amino-3-hydroxy-5-methylisoxazdle-4-propionic acid (AMPA). The NMDA receptor has been implicated in a variety of neurological pathologies including stroke, head trauma, spinal cord injury, epilepsy, anxiety, and neurodegenerative diseases such as Alzheimer's Disease (Watkins and Collingridge, The NMDA Receptor, Oxford: IRL Press, 1989). A role for NMDA receptors in nociception and analgesia has been postulated as well (Dickenson, A cure for wind-up: NMDA receptor antagonists as potential analgesics. Trends Pharmacol.

Sci. 11: 307, 1990). More recently, AMPA receptors have been widely studied for their possible contributions to such neurological pathologies (Fisher and Bogousslavsky, Evolving toward effective therapy for acute ischemic stroke. J. Amer. Med. Assoc. 270: 360, 1993; Yamaguchi et al., Anticonvulsant activity of AMPA/kainate antagonists: Comparison of GYKI 52466 and NBQX in S. maximal electroshock and chemoconvulsant seizure models.

Epilepsy Res. 15: 179, 1993).

When activated by glutamate, the endogenous neurotransmitter, the NMDA receptor permits the influx of extracellular calcium (Ca 2 and sodium through an associated ion channel. The NMDA receptor allows considerably more influx of Ca 2 than do kainate or AMPA receptors (but see below), and is an example of a receptor-operated Ca 2 channel. Normally, the channel is opened only briefly, allowing a localized and transient increase in the concentration of intracellular Ca 2 ([Ca 2 which, in turn, alters the functional activity of the cell. However, prolonged increases in [Ca 2 li, resulting from chronic stimulation of the NMDA receptor, are toxic to the cell and lead to cell death. The chronic elevation in [Ca 2 resulting from stimulation of NMDA receptors, is said to be a primary cause of neuronal degeneration following a stroke (Choi, Glutamate neurotoxicity and diseases of the nervous system. Neuron 1: 623, 1988). Overstimulation of NMDA 4 receptors is also said to be involved in the pathogenesis of some forms of epilepsy (Dingledine et al., Excitatory amino acid receptors in epilepsy.

Trends Pharmacol. Sci. 11: 334, 1990), anxiety (Wiley and Balster, Preclinical evaluation of N-methyl-D-aspartate antagonists for antianxiety effects: A review. In: Multiple Sigma and PCP Receptor Ligands: Mechanisms for Neuromodulation and Neuroprotection? NPP Books, Ann Arbor, Michigan, pp.

801-815, 1992), neurodegenerative diseases (Meldrum.and Garthwaite, Excitatory amino acid neurotoxicity and S.neurodegenerative disease. Trends Pharmacol. Sci. 11: 379, 1990), and hyperalgesic states (Dickenson, A cure for wind-up: NMDA receptor antagonists as potential analgesics. Trends Pharmacol. Sci. 11: 307, 1990).

The activity of the NMDA receptor-ionophore complex is regulated by a variety of modulatory sites that can be targeted by selective antagonists.

Competitive antagonists, such as the phosphonate act at the glutamate binding site, whereas noncompetitive antagonists, such as phencyclidine (PCP), MK-801 or magnesium (Mg 2 act within the associated ion channel (ionophore). There is also a glycine binding site that can be blocked selectively with compounds such as 7-chlorokynurenic acid. There is evidence suggesting that glycine acts as a co-agonist, so that both glutamate and glycine are necessary to fully elicit NMDA receptor-mediated responses. Other potential sites for modulation of NMDA receptor function include a zinc (Zn 2 binding site and a sigma ligand binding site.

Additionally, endogenous polyamines such as spermine are believed to bind to a specific site and so potentiate NMDA receptor function (Ransom and Stec, Cooperative modulation of 3 H]MK-801 binding to the NMDA receptor-ion channel complex by glutamate, glycine and polyamines.

J. Neurochem. 51: 830, 1988). The potentiating effect of polyamines on NMDA receptor function may be mediated via a specific receptor site for polyamines; polyamines demonstrating agonist, antagonist, and inverse agonist activity have been described (Reynolds, Arcaine is a 10 competitive antagonist of the polyamine site on the NMDA receptor. Europ. J. Pharmacol. 177: 215, 1990; Williams et al., Characterization of polyamines having agonist, antagonist, and inverse agonist effects at the polyamine recognition site of the NMDA receptor. Neuron 5: 199, 1990). Radioligand binding studies have demonstrated additionally that higher concentrations of polyamines inhibit NMDA receptor function (Reynolds and Miller, Ifenprodil is a novel type of NMDA receptor antagonist: Interaction with polyamines. Molec. Pharmacol. 36: 758, 1989; Williams et al., Effects of polyamines on the binding of 3 H]MK-801 to the NMDA receptor: Pharmacological evidence for the existence of a polyamine recognition site. Molec. Pharmacol. 36: 575, 1989; Sacaan and Johnson, Characterization of the stimulatory and inhibitory effects of polyamines on [H]TCP binding to the NMDA receptor-ionophore complex.

Molec. Pharmacol. 37: 572, 1990). This inhibitory effect .of polyamines on NMDA receptors is probably a nonspecific effect not mediated via the polyamine receptor) because patch clamp electro-physiological studies have demonstrated that this inhibition is produced by compounds previously shown to act at the polyamine receptor as either agonists or antagonists (Donevan et al., Arcaine Blocks N-Methyl-D-Aspartate Receptor Responses by an Open Channel Mechanism: Whole-Cell and Single-Channel Recording Studies in Cultured Hippocampal Neurons. Molec. Pharmacol. 41: 727, 1992; Rock and Macdonald, Spermine and Related Polyamines Produce a Voltage-Dependent Reduction of NMDA Receptor Single-Channel Conductance. Molec. Pharmacol.

42: 157, 1992).

Recent studies have demonstrated the molecular diversity of glutamate receptors (reviewed by Nakanishi, Molecular Diversity of Glutamate Receptors and Implications for Brain Function. Science 258: 597, 1992). At least five distinct NMDA receptor subunits (NMDAR1 and NMDAR2A through NMDAR2D), each encoded by a distinct gene, have been identified to date. Also, in NMDAR1, alternative splicing gives rise to at least six additional isoforms. It appears that NMDAR1 is a necessary subunit, and that combination of NMDAR1 with different members of NMDAR2 forms the fully functional NMDA receptor-ionophore complex. The NMDA receptor-ionophore complex, thus, can be defined as a hetero-oligomeric structure composed of at least NMDAR1 and NMDAR2 subunits; the existence of additional, as yet undiscovered, subunits is not excluded by this definition. NMDAR1 has been shown to possess binding sites for glutamate, glycine, Mg 2 MK-801, and Zn 2 The binding sites for sigma ligands and polyamines have not yet been localized on NMDA receptor subunits, although ifenprodil recently has been reported to be more potent at the NMDAR2B subunit than at the NMDAR2A subunit (Williams, Ifenprodil discriminates subtypes of the N-methyl-D-aspartate .receptor: selectivity and mechanisms at recombinant heteromeric receptors. Mol.

Pharmacol. 44: 851, 1993).

Several distinct subtypes of AMPA and kainate receptors have been cloned as well (reviewed by Nakanishi, Molecular diversity of glutamate receptors and implications for brain function. Science 258: 597, 1992). Of particular relevance are the AMPA receptors designated GluR1, GluR2, GluR3, and GluR4 (also termed GluRA through GluRD), each of which exists in one of two forms, termed flip and flop, which arise by RNA alternative splicing. GluR1, GluR3 and GluR4, when expressed as homomeric or heteromeric receptors, are permeable to Ca 2 and are therefore examples of receptor-operated Ca 2 channels. Expression of GluR2 alone or in combination with the other subunits gives rise to a receptor which is largely impermeable to Ca 2 As most native AMPA receptors studied in situ are not SCa 2 -permeable (discussed above), it is believed that such receptors in situ possess at least one GluR2 subunit.

Furthermore, it is hypothesized that the GluR2 subunit is functionally distinct by virtue of the fact that it contains an arginine residue within the putative pore-forming transmembrane region II; GluR1, GluR3 and GluR4 all contain a glutamine residue in this critical region (termed the Q/R site, where Q and R are the single letter designations for glutamine and arginine, respectively). The activity of the AMPA receptor is regulated by a number of modulatory sites that can be targeted by selective antagonists (Honore et al., Quinoxalinediones: potent competitive non-NMDA glutamate receptor antagonists. Science 241: 701, 1988; Donevan and Rogawski, GYKI 52466, a 2,3-benzodiazepine, is a highly selective, noncompetitive antagonist of AMPA/kainate receptor responses. Neuron 10: 51, 1993).

Competitive antagonists such as NBQX act at the glutamate binding site, whereas compounds such as GYKI 52466 appear to act noncompetitively at an associated allosteric site.

Compounds that act as competitive or noncompetitive antagonists at the NMDA receptor are said to be effective in preventing neuronal cell death in various in vitro neurotoxicity assays (Meldrum and Garthwaite, Excitatory amino acid neurotoxicity and neurodegenerative disease. Trends Pharmacol. Sci. 11: 379, 1990) and in in vivo models of stroke (Scatton, F Therapeutic potential of NMDA receptor antagonists in ischemic cerebrovascular disease in Drug Strategies in S. the Prevention and Treatment of Stroke, IBC Technical Services Ltd., 1990). Such compounds are also effective anticonvulsants (Meldrum, Excitatory amino acid neurotransmission in epilepsy and anticonvulsant therapy in Excitatory Amino Acids. Meldrum, Moroni, Simon, and Woods New York: Raven Press, p. 655, 1991), anxiolytics (Wiley and Balster, Preclinical evaluation of N-methyl-D-aspartate antagonists for antianxiety effects: A review. In: Multiple Sigma and PCP Receptor Ligands: Mechanisms for Neuromodulation and Neuroprotection? NPP Books, Ann Arbor, Michigan, pp.

801-815, 1992), and analgesics (Dickenson, A cure for wind-up: NMDA receptor antagonists as potential analgesics. Trends Pharmacol. Sci. 11: 307, 1990), and certain NMDA receptor antagonists.may lessen dementia associated with Alzheimer's Disease (Hughes, Merz' novel approach to the treatment of dementia. Script No. 1666: 24, 1991).

Similarly, AMPA receptor antagonists have come under intense scrutiny as potential therapeutic agents for the treatment of such neurological disorders and diseases. AMPA receptor antagonists have been shown to possess neuroprotectant (Fisher and Bogousslavsky, Evolving toward effective therapy for acute ischemic stroke. J. Amer. Med. Assoc. 270: 360, 1993) and anticonvulsant (Yamaguchi et al., Anticonvulsant activity of AMPA/kainate antagonists: comparison of GYKI 52466 and NBQX in maximal electroshock and chemoconvulsant seizure models. Epilepsy Res. 15: 179, 1993) activity in animal models of ischemic stroke and epilepsy, respectively.

The nicotinic cholinergic receptor present in the mammalian CNS is another example of a receptor-operated Ca2+ channel (Deneris et al., Pharmacological and functional diversity of neuronal nicotinic acetylcholine receptors. Trends Pharmacol.

Sci. 12: 34, 1991). Several distinct receptor subunits have been cloned, and these subunits can be expressed, in Xenopus oocytes for example, to form functional receptors with their associated cation channels. It is hypothesized that such receptor-ionophore complexes are heteropentameric structures. The possible role of nicotinic receptor-operated Ca 2 channels in the pathology of neurological disorders and diseases such as ischemic stroke, epilepsy and neurodegenerative diseases has been largely unexplored.

It has been demonstrated previously that certain spider and wasp venoms contain arylalkylamine.

toxins (also called polyamine toxins, arylamine toxins, acylpolyamine toxins or polyamine amide toxins) with activity against glutamate receptors in the mammalian CNS (for reviews see Jackson and Usherwood, Spider toxins as tools for dissecting elements of excitatory -amino acid transmission. Trends Neurosci. 11: 278, 1988; Jackson and Parks, Spider Toxins: Recent Applications In Neurobiology. Annu. Rev. Neurosci. 12: 405, 1989; Saccomano et al., Polyamine spider toxins: SUnique pharmacological tools. Annu. Rep. Med. Chem. 24: 287, 1989; Usherwood and Blagbrough, Spider Toxins S....Affecting Glutamate Receptors: Polyamines in Therapeutic Neurochemistry. Pharmacol. Therap. 52: 245, 1991; Kawai, Neuroactive Toxins of Spider Venoms. J. Toxicol.

S. Toxin Rev. 10: 131, 1991). Arylalkylamine toxins were initially reported to be selective antagonists of the AMPA/kainate subtypes of glutamate receptors in the mammalian CNS (Kawai et al., Effect of a spider toxin on glutaminergic synapses in the mammalian brain. Biomed.

Res. 3: 353, 1982; Saito et al., Spider Toxin (JSTX) blocks glutamate synapse in hippocampal pyramidal neurons. Brain Res. 346: 397, 1985; Saito et al., Effects of a spider toxin (JSTX) on hippocampal CA1 neurons in vitro. Brain Res. 481: 16, 1989; Akaike et al., Spider toxin blocks excitatory amino acid responses in isolated hippocampal pyramidal neurons. Neurosci.

Lett. 79: 326, 1987; Ashe et al., Argiotoxin-636 blocks excitatory synaptic transmission in rat hippocampal CA1 pyramidal neurons. Brain Res. 480: 234, 1989; Jones et al., Philanthotoxin blocks quisqualate-induced, AMPA-induced and kainate-induced, but not NMDA-induced excitation of rat brainstem neurones in vivo. Br. J.

Pharmacol. 101: 968, 1990). Subsequent studies have demonstrated that while certain arylalkylamine toxins are both nonpotent and nonselective at various glutamate receptors, other arylalkylamines are both very potent and selective at antagonizing responses mediated by NMDA 'o receptor activation in the mammalian CNS (Mueller et al., Effects of polyamine spider toxins on NMDA receptor-mediated transmission in rat hippocampus in vitro. Soc. Neurosci. Abst. 15: 945, 1989; Mueller et Arylamine spider toxins antagonize NMDA receptor-mediated synaptic transmission in rat hippocampal slices. Synapse 9: 244, 1991; Parks et al., Polyamine spider toxins block NMDA receptor-mediated increases in cytosolic calcium in cerebellar granule neurons. Soc. Neurosci. Abst. 15: 1169, 1989; Parks et al., Arylamine toxins from funnel-web spider (Agelenopsis aperta) venom antagonize N-methyl-Daspartate receptor function in mammalian brain. J.

Biol. Chem. 266: 21523, 1991; Priestley et al., Antagonism of responses to excitatory amino acids on rat cortical neurones by the spider toxin, argiotoxin-636.

Br. J. Pharmacol. 97: 1315, 1989; Draguhn et al., Argiotoxin-636 inhibits NMDA-activated ion channels expressed in Xenopus oocytes. Neurosci. Lett. 132: 187, V 1991; Kiskin et al., A highly potent and selective N-methyl-D-aspartate receptor antagonist from the venom of the Agelenopsis aperta spider. Neuroscience 51: 11, 1992; Brackley et al., Selective antagonism of native and cloned kainate and NMDA receptors by polyamine-containing toxins. J. Pharmacol. Exptl.

Therap. 266: 1573, 1993; Williams, Effects of Agelenopsis aperta toxins on the N-methyl-D-aspartate receptor: Polyamine-like and high-affinity antagonist actions. J. Pharmacol. Exptl. Therap. 266: 231, 1993) Inhibition of nicotinic cholinergic receptors by the arylalkylamine toxin philanthotoxin has also been reported (Rozental et al., Allosteric inhibition of nicotinic acetylcholine receptors of vertebrates and insects by philanthotoxin. J. Pharmacol. Exptl. Therap.

249: 123, 1989).

Parks et al. (Arylamine toxins from funnel-web spider (Agelenopsis aperta) venom antagonize N-methyl-D-aspartate receptor function in mammalian brain. J. Biol. Chem. 266: 21523, 1991), describe arylalkylamine spider toxins a-agatoxins) which antagonize NMDA receptor function in mammalian brain.

The authors discuss the mechanism of action of arylalkylamine toxins, and indicate that an NMDA receptor-operated ion channel is the probable site of action of the a-agatoxins, and most probably other spider venom arylalkylamines. They state: The discovery that endogenous polyamines in the vertebrate brain modulate the function of NMDA receptors suggests that the arylamine toxins may produce their Santagonism via a polyamine-binding site on glutamate receptors.

Brackley et al. studied the effects of spermine and philanthotoxin 433 on the responses evoked by application of excitatory amino acids in Xenopus oocytes injected with mRNA from rat or chick brain.

These authors reported that, at concentrations below those that antagonize glutamate receptor function, both spermine and philanthotoxin potentiate the effects of excitatory amino acids and some other neurotransmitters.

On the basis of these and other data, Brackley et al. concluded that the arylamine toxins may, by binding nonspecifically to the membranes of excitable cells, reduce membrane fluidity and alter receptor function. The validity of this intriguing idea for NMDA receptor function is not well supported by 25 two recent binding studies.

Reynolds reported that argiotoxin 636 inhibits the binding of 3 H]MK-801 to rat brain membranes in a manner that is insensitive to glutamate, glycine, or spermidine.

This author concluded that argiotoxin 636 exerts a novel inhibitory effect on the NMDA receptor complex by binding to one of the Mg 2 sites located within the NMDA-gated ion channel. Binding data reported by Williams et al.

also support the conclusion that argiotoxin 636 does not act primarily at the polyamine modulatory site on the NMDA receptor, but rather acts directly to produce an activity-dependent block of the ion channel. It is already known that compounds such as phencyclidine and ketamine can block the ion channels associated with both arthropod muscle glutamate receptors and mammalian NMDA receptors. Thus, it seems possible that vertebrate and invertebrate glutamate receptors share additional binding sites for allosteric modulators of receptor function, perhaps related to divalent cation-binding sites. Clearly, considerable additional work will be needed to determine if the arylamines define such a novel regulatory site.

15 Usherwood and Blagbrough (Spider Toxins 0**o*o Affecting Glutamate Receptors: Polyamines in Therapeutic Neurochemistry. Pharmacol. Therap. 52: 245, 1991) describe a proposed intracellular binding site for arylalkylamine toxins (polyamine amide toxins) located within the membrane potential field referred to as the QUIS-R channel selectivity filter. The authors postulate that the binding site for polyamine amide toxins may occur close to the internal entrance of the channel gated by the QUIS-R of locust muscle. The 25 authors also note that one such toxin, argiotoxin-636, selectively antagonizes the NMDA receptor in cultured rat cortical neurons.

Gullak et al. (CNS binding sites of the novel NMDA antagonist Arg-636. Soc. Neurosci. Abst. 15: 1168, 1989), describe argiotoxin-636 (Arg-636) as a polyamine (arylalkylamine) toxin component of a spider venom.

This toxin is said to block NMDA-induced elevation of cGMP in a noncompetitive fashion. The authors state that: [1 25 ]Arg-636 bound to rat forebrain membranes with Kd and values of 11.25 fM and 28.95 pmol/mg protein specific). The ability of other known polyamines and recently discovered polyamines from Agelenopsis aperta to inhibit binding paralleled neuroactivity as functional NMDA antagonists. No other compounds tested were able to block specific binding.

The authors then stated that polyamines (arylalkylamines) may antagonize responses to NMDA by interacting with membrane ion channels.

Seymour and Mena (In vivo NMDA antagonist 15 activity of the polyamine spider venom component, argiotoxin-636. Soc. Neurosci. Abst. 15: 1168, 1989) describe studies that are said to show that argiotoxin-636 does not significantly affect locomotor activity at doses that are effective against audiogenic seizures in DBA/2 mice, and that it significantly antagonizes NMDA-induced seizures with a minimal effective dose of 32 mg/kg given subcutaneously r Herold and Yaksh (Anesthesia and muscle relaxation with intrathecal injections of AR636 and AG489, two acylpolyamine spider toxins, in rats.

Anesthesiology 77: 507, 1992) describe studies that are said to show that the arylalkylamine argiotoxin-636 (AR636), but not agatoxin-489 (AG489), produces muscle relaxation and anesthesia following intrathecal administration in rats.

Williams (Effects of Agelenopsis aperta toxins on the N-methyl-D-aspartate receptor: Polyamine-like and high-affinity antagonist actions, J. Pharmacol. Exptl.

I

WI Therap. 266: 231, 1993) reports that the a-agatoxins (arylalkylamines) Agel-489 and Agel-505 enhance the binding of 3 H]MK-801 to NMDA receptors on membranes prepared from rat brain by an action at the stimulatory polyamine receptor; polyamine receptor agonists occluded the stimulatory effects of Agel-489 and Agel-505 and.

polyamine receptor antagonists inhibited the stimulatory effect of Agel-505. Higher concentrations of Agel-489 and Agel-505, and argiotoxin-636 at all concentrations tested, had inhibitory effects on the binding of 3 H]MK-801. In Xenopus oocytes voltage-clamped at mV, Agel-505 inhibited responses to NMDA with an IC 5 s of 13 nM; this effect of Agel-505 occurred at concentrations approximately 10,000-fold lower than those that affected P[H]MK-801 binding. Responses to kainate were inhibited only 11% by 30 nM Agel-505. The antagonism of NMDA-induced currents by Agel-505 was strongly voltage-dependent, consistent with an open-channel blocking effect of the toxin. Williams states: Although a-agatoxins can interact S. at the positive allosteric polyamine site on the NMDA receptor, stimulatory effects produced by this interaction may be masked in functional assays due to a separate action of the toxins as high-affinity, noncompetitive antagonists of the receptor.

Brackley et al. (Selective antagonism of native and cloned kainate and NMDA receptors by polyamine-containing toxins, J. Pharmacol. Exp. Therap.

266: 1573, 1993) report that the polyamine-containing toxins (arylalkylamines) philanthotoxin-343 (PhTX-343) and argiotoxin-636 (Arg-636) produce reversible, noncompetitive, partly voltage-dependent antagonism of kainate- and NMDA-induced currents in Xenopus oocytes injected with rat brain RNA. Arg-636 was demonstrated to be selective for NMDA-induced responses (ICs 5 0.04 4M) compared to kainate-induced responses (ICs 0.07 AM), while PhTX-343 was selective for kainate-induced responses (IC 50 0.12 uM) compared to NMDA-induced responses (ICs, 2.5 sM). Arg-636 more potently antagonized responses to NMDA in Xenopus 99** oocytes expressing cloned NMDAR1 subunits (IC 5 0.09 gM) than responses to kainate in oocytes expressing either cloned GluRi (ICs 5 3.4 uM) or GluRl+GluR2 subunits (ICs, 300 uM). PhTX-343, on the other hand, was equipotent at antagonizing NMDAR1 (ICsO 2.19 kM) and GluRl (IC, 5 2.8 MM), but much less potent against GluRl+GluR2 subunits (ICso 270 pM).

9 Raditsch et al. (Subunit-specific block of cloned NMDA receptors by argiotoxin-636. FEBS Lett.

324: 63, 1993) report that Arg-636 more potently antagonizes responses in Xenopus oocytes expressing NMDAR1+NMDAR2A subunits (ICs 5 9 nM) or NMDAR1+NMDAR2B subunits (IC, 0 2.5 nM) than NMDAR1+NMDAR2C subunits (ICs 5 460 nM), even though all of the receptor subunits contain an asparagine residue in the putative pore-forming transmembrane region II (the Q/R site, as discussed above). The authors state that the large difference in Arg-636 sensitivity between NMDAR1+NMDAR2A and NMDAR1+NMDAR2C channels "must be conferred by other structural determinants." W Herlitz et al. (Argiotoxin detects molecular differences in AMPA receptor channels. Neuron 10: 1131, 1993) report that Arg-636 antagonizes subtypes of AMPA receptors in a voltage- and use-dependent manner consistent with open-channel blockade. Arg-636 potently antagonizes Ca2*-permeable AMPA receptors comprised of GluRAi (Ki 0.35 gM), GluRCi (K i 0.23 AM), or GluRDi subunits (K i 0.43 pM), while being essentially ineffective against Ca 2 *-impermeable GluRBi subunits at concentrations up to 10 1M.

Other data reported by these investigators strongly suggest that the Q/R site in the putative pore-forming transmembrane region II is of primary importance in determining Arg-636 potency and Ca 2 permeability.

Blaschke et al. (A single amino acid *see determines the subunit-specific spider toxin block of a-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainate receptor channels. Proc. Natl. Acad. Sci. USA 90: 6528, 1993) report that the arylalkylamine JSTX-3 potently antagonizes responses to kainate in Xenopus oocytes expressing GluRl (ICs 5 0.04 gM) or GluR3 (IC 50 0.03 1M) subunits, but that expressed receptors in which a GluR2 subunit is present are essentially unaffected by the toxin. Site-directed mutagenesis studies strongly implicate the Q/R site as the primary site influencing toxin potency.

Nakanishi et al. (Bioorganic studies of transmitter receptors with philanthotoxin analogs. Pure Appl. Chem., in press) have synthesized a number of highly potent photoaffinity labeled philanthotoxin (PhTX) analogs. Such analogs have been studied on expressed nicotinic cholinergic receptors as a model system for receptor-operated calcium channels. These investigators suggest that these PhTX analogs block the ion channel with the hydrophobic headpiece of the toxin binding to a site near the cytoplasmic surface while the polyamine tail extends into the ion channel from the cytoplasmic side.

Summary of the Invention 10 Applicant has examined the structural diversity and biological activity of arylalkylamines (sometimes referred to as arylamine toxins, polyamine toxins, acylpolyamine toxins or polyamine amide toxins) in spider and wasp.venoms, and determined that some of 15 the arylalkylamines present in these venoms act as potent noncompetitive antagonists of glutamate receptor-operated Ca 2 channels in the mammalian CNS.

Although these arylalkylamine compounds contain within their structure a polyamine moiety, they are unlike 20 other known simple polyamines in possessing extremely

O

O potent and specific effects on certain types of receptor-operated Ca 2 channels.

Using native arylalkylamines as lead structures, a number of analogs were synthesized and tested. Initial findings on arylalkylamines isolated and purified from venom were confirmed utilizing synthetic arylalkylamines. These compounds are small molecules (mol. wt. <800) with demonstrated efficacy in in vivo models of stroke and epilepsy. The NMDA receptor-ionophore complex was used as a model of receptor-operated Ca 2 channels. Selected arylalkylamines were shown to block NMDA receptor-mediated responses by a novel mechanism.

Moreover, the unique behavioral pharmacological profile of these compounds suggests that they are unlikely to cause the PCP-like psychotomimetic activity and cognitive deficits that characterize other inhibitors of the NMDA receptor. Finally, the arylalkylamines are unique amongst NMDA receptor antagonists in that they are able to antagonize certain subtypes of cloned and expressed AMPA receptors, namely, those permeable to Ca*. The arylalkylamines, therefore, are the only known **class of compounds able to antagonize glutamate Sreceptor-mediated increases in cytosolic Ca 2 regardless of the pharmacological definition of receptor subtype.

Additionally, the arylalkylamines inhibit an ther receptor-operated Ca 2 channel, the nicotinic cholinergic receptor. Given that excessive and prolonged increases in cytosolic Ca have been implicated in the etiology of several.neurological disorders and diseases, such arylalkylamines are valuable small molecule leads for the development of novel therapeutics for various neurological disorders and diseases.

Applicant has determined that the selected arylalkylamines bind with high affinity at a novel site on the NMDA receptor-ionophore complex which has heretofore been unidentified, and that said arylalkylamines do not bind with high affinity at any of the known sites (glutamate binding site, glycine binding site, MK-801 binding site, Mg 2 binding site, Zn 2 binding site, polyamine binding site, sigma binding site) on said NMDA receptor-ionophore complex. This determination has allowed applicant to develop methods and protocols by which useful compounds can be identified which provide both therapeutically useful compounds and lead compounds for the development of other therapeutically useful compounds. These compounds can be identified by screening for compounds that bind at this novel arylalkylamine binding site, and by determining whether such a compound has the required biological, pharmacological and physiological properties.

The method includes the step of identifying a compound which binds to the receptor-operated Ca 2 channel at that site bound by the arylalkylamine compounds referred to herein as Compound 1, Compound 2 or Compound 3, and having the structures shown below.

1H N H H NLN M N N NH, 0 H 0 NH Compound 1

*N*

H

Compound 2 0 NH N N NN N H H H H Fe Compound 3 By "therapeutically useful compound" is meant a compound that is potentially useful in the treatment of a disorder or disease state. A compound uncovered by the screening method is characterized as having potential therapeutic utility in treatment because clinical tests have not yet been conducted to determine actual therapeutic utility.

By "neurological disorder or disease" is meant 10 a disorder or disease of the nervous system including, but not limited to, global and focal ischemic and hemorrhagic stroke, head trauma, spinal cord injury, spinal cord ischemia, ischemia- or hypoxia-induced nerve cell damage, hypoxia-induced nerve cell damage as in 15 cardiac arrest or neonatal distress, epilepsy, anxiety, neuropsychiatric or cognitive deficits due to ischemia or hypoxia such as those that frequently occur as a consequence of cardiac surgery under cardiopulmonary bypass, and neurodegenerative disease. Also meant by "neurological disorder or disease" are those disease states and conditions in which a neuroprotectant, anticonvulsant, anxiolytic, analgesic, muscle relaxant and/or adjunct in general anesthesia may be indicated, useful, recommended or prescribed.

By "neurodegenerative disease" is meant diseases including, but not limited to, Alzheimer's Disease, Huntington's Disease, Parkinson's Disease, and amyotrophic lateral sclerosis (ALS).

By "neuroprotectant" is meant a compound capable of preventing the neuronal damage or death associated with a neurological disorder or disease.

By "anticonvulsant" is meant a compound capable of reducing convulsions produced by conditions such as simple partial seizures, complex partial seizures, status epilepticus, and trauma-induced seizures such as occur following head injury, including head surgery.

By "anxiolytic" is meant a compound capable of relieving the feelings of apprehension, uncertainty and fear that are characteristic of anxiety.

By "analgesic" is meant a compound capable of 15 relieving pain by altering perception of nociceptive stimuli without producing anesthesia or loss of consciousness.

By "muscle relaxant" is meant a compound that reduces muscular tension.

20 By "adjunct in general anesthesia" is meant a compound useful in conjunction with anesthetic agents in producing the loss of ability to perceive pain associated with the loss of consciousness.

By "potent" or "active" is meant that the compound has activity at receptor-operated calcium channels, including NMDA receptors, Ca 2 *-permeable AMPA receptors, and nicotinic cholinergic receptors, with an ICs 5 value less than 10 M, more preferably less than 100 nM, and even more preferably less than 1 nM.

By "selective", is meant that the compound is potent at receptor-operated calcium channels as defined above, but is less potent by greater than 10-fold, more preferably 50-fold, and even more preferably 100-fold, at other neurotransmitter receptors, neurotransmitter receptor-operated ion channels, or voltage-dependent ion channels.

By "biochemical and electrophysiological assays of receptor-operated calcium channel function" is meant assays designed to detect by biochemical or electrophysiological means the functional activity of receptor-operated calcium channels. Examples of such assays include, but are not limited to, the fura-2 *fluorimetric assay for cytosolic calcium in cultured rat cerebellar granule cells (see Example 1 and Example 2), patch clamp electrophysiolocial assays (see Example 3 and Example 27), rat hippocampal slice synaptic transmission assays (see Example radioligand binding assays (see Example 4, Example 24, Example 25, and Example 26), and in vitro neuroprotectant assays (see Example 6).

By "efficacy" is meant that a statistically significant level of the desired activity is detectable with a chosen compound; by "significant" is meant a statistical significance at the p 0.05 level.

By "neuroprotectant activity" is meant efficacy in treatment of neurological disorders or diseases including, but not limited to, global and focal ischemic and hemorrhagic stroke, head trauma, spinal cord injury, spinal cord ischemia, ischemia- or hypoxiainduced nerve cell damage, hypoxia-induced nerve cell damage as in cardiac arrest or neonatal distress, neuropsychiatric or cognitive deficits due to ischemia or hypoxia such as those that frequently occur as a consequence of cardiac surgery under cardiopulmonary bypass, and neurodegenerative diseases such as Alzheimer's Disease, Huntington's Disease, Parkinson's Disease, and amyotrophic lateral sclerosis (ALS) (see Examples 7 and 8, below).

By "anticonvulsant activity" is meant efficacy in reducing convulsions produced by conditions such as simple partial seizures, complex partial seizures, status epilepticus, and trauma-induced seizures such as occur following head injury, including head surgery (see Examples 9 and 10, below).

By "anxiolytic activity" is meant that a compound reduces the feelings of apprehension, uncertainty and fear that are characteristic of anxiety.

By "analgesic activity" is meant that a compound produces the absence of pain in response to a stimulus that would normally be painful. Such activity would be useful in clinical conditions of acute and 'chronic pain including, but not limited to the following: preemptive preoperative analgesia; peripheral neuropathies such as occur with diabetes mellitus and multiple sclerosis; phantom limb pain; causalgia; neuralgias such as occur with herpes zoster; central pain such as that seen with spinal cord lesions; hyperalgesia; and allodynia.

By "causalgia" is meant a painful disorder associated with injury of peripheral nerves.

By "neuralgia" is meant pain in the distribution of a nerve or nerves.

By "central pain" is meant pain associated with a lesion of the central nervous system.

By "hyperalgesia" is meant an increased response to a stimulus that is normally painful.

By "allodynia" is meant pain due to a stimulus that does not normally provoke pain (see Examples 11 through 14, below).

By "induction of long-term potentiation in rat hippocampal slices" is meant the ability of tetanic electrical stimulation of afferent Schaffer collateral fibers to elicit long-term increases in the strength of i synaptic transmission at the Schaffer collateral-CAl pyramidal cell pathway in rat hippocampal slices maintained in vitro (see Example 19).

By "therapeutic dose" is meant an amount of a compound that relieves to some extent one or more symptoms of the disease or condition of the patient.

Additionally, by "therapeutic dose" is meant an amount S" that returns to normal, either partially or completely, physiological or biochemical parameters associated with or causative of the disease or condition. Generally, it is an amount between about 1 nmole and 1 jgmole of the compound, dependent on its EC 50 (ICsO in the case of an antagonist) and on the age, size, and disease associated with the patient.

By "impair cognition" is meant the ability to impair the acquisition of memory or the performance of a learned task (see Example 20). Also by "impair congnition" is meant the ability to interfere with normal rational thought processes and reasoning.

By "disrupt motor function" is meant the ability to significantly alter locomotor activity (see Example 15) or elicit significant ataxia, loss of the righting reflex, sedation or muscle relaxation (see Example 16).

By "locomotor activity" is meant the ability to perform normal ambulatory movements.

By "loss of the righting reflex" is meant the ability of an animal, typically a rodent, to right itself after being placed in a supine position.

By "neuronal vacuolization" is meant the production of vacuoles in neurons of the cingulate cortex or retrosplenial cortex (see Example 18).

By "cardiovascular activity is meant the ability to elicit significant changes in parameters including, but not limited to, mean arterial blood pressure and heart rate (see Examples 21 and 22).

By "hyperexcitability" is meant an enhanced susceptibility to an excitatory stimulus.

Hyperexcitability is often manifested as a significant increase in locomotor activity in rodents administered a drug (see Example By "sedation" is meant a calmative effect, or the allaying of activity and excitement. Sedation is often manifested as a significant decrease in locomotor activity in rodents administered a drug (see Example By "PCP-like abuse potential" is meant the potential of a drug to be wrongfully used, as in the recreational use of PCP "angel dust") by man. It is believed that PCP-like abuse potential can be predicted by the ability of a drug to generalize to PCP W in rodents trained to discriminate PCP from saline (see Example 17.) By "generalization to PCP" is meant that a compound is perceived as being PCP in rodents trained to discriminate PCP from saline (see Example 17).

By "PCP-like psychotomimetic activity" is meant the ability of a drug to elicit in man a behavioral syndrome resembling acute psychosis, including visual hallucinations, paranoia, agitation, and confusion. It is believed that PCP-like psychotomimetic activity can be predicted in rodents by the ability of a drug to produce PCP-like stereotypic behaviors including ataxia, head weaving, hyperexcitability, and generalization to PCP in rodents trained to discriminate PCP from saline (see Example Example 16, and Example 17).

By "ataxia" is meant a deficit in muscular coordination.

S By "head weaving" is meant the stereotypic behavior elicited in rodents by PCP in which the head is repeatedly moved slowly and broadly from side to side.

By "pharmaceutical composition" is meant a therapeutically effective amount of a compound of the present invention in a pharmaceutically acceptable carrier, a formulation to which the compound can be added to dissolve or otherwise facilitate administration of the compound. Examples of pharmaceutically acceptable carriers include water, saline, and physiologically buffered saline. Such a pharmaceutical composition is provided in a suitable dose. Such compositions are generally those which are approved for use in treatment of a specified disorder by the FDA or its equivalent in non-U.S. countries.

In a related aspect, the invention features a method for treating a neurological disease or disorder, comprising the step of administering a pharmaceutical composition comprising a compound which binds to a receptor-operated calcium channel at the site bound by one of the arylalkylamines Compound 1, Compound 2 and Compound 3, said compound being a potent and selective noncompetitive antagonist at such a receptor-operated calcium channel, and having one or more of the following pharmacological and physiological properties: efficacy in in vitro biochemical and electrophysiological assays of receptor-operated calcium channel function, in vivo anticonvulsant activity, in vivo neuroprotectant activity, in vivo anxiolytic activity, and in vivo analgesic activity; said compound also possessing one or more of the following pharmacological effects: the •compound does not interfere with the induction of long-term potentiation in rat hippocampal slices, and, at a therapeutic dose, does not impair cognition, does not disrupt motor performance, does not produce neuronal vacuolization, has minimal cardiovascular activity, does not produce sedation or hyperexcitability, has minimal PCP-like abuse potential, and has minimal PCP-like psychotomimetic activity. By "minimal" is meant that any side effect of the drug is tolerated by an average individual, and thus that the drug can be used for therapy of the target disease. Such side effects are well known in the art and are routinely regarded by the FDA as minimal when it approves a drug for a target disease.

Treatment involves the steps of first identifying a patient that suffers from a neurological disease or disorder by standard clinical methodology and then treating such a patient with a composition of the present invention.

In a further aspect, the invention features compounds useful for treating a patient having a neurological disease or disorder wherein said compound is a polyamine-type compound or an analog thereof a polyheteroatomic molecule) having the formula 'e

R

1 R R 1

R

1 an aromatic carbocyclic aryl groups such as phenyl and bicyclic carbocyclic aryl ring systems such as naphthyl, 1,2,3,4-tetrahydronaphthyl, indanyl, and indenyl), heteroaromatic indolyl, dihydroindolyl, quinolinyl and isoquinolinyl,.and their respective 1,2,3,4-tetrahydro- and 2-oxo- derivatives), alicyclic (cycloaliphatic), or heteroalicyclic ring or ring system (mono-, bi-, or tricyclic), having 5- to 7-membered ring(s) optionally substituted with 1 to 5 substituents independently selected from lower alkyl of 1 to 5 carbon atoms, lower haloalkyl of 1 to S carbon atoms substituted with 1 to 7 halogen atoms, lower alkoxy of 1 to 5 carbon atoms, halogen, nitro, amino, lower alkylamino of 1 to 5 carbon atoms, amido, lower alkylamido of 1 to 5 carbon atoms, cyano, hydroxyl, sulfhydryl, lower acyl of 2 to 4 carbon atoms, sulfonamido, lower alkylsulfonamido of 1 to 5 carbon atoms, lower alkylsulfoxide of 1 to 5 carbon atoms, lower hydroxyalkyl of 1 to 5 carbon atoms, lower alkylketo of 1 to 5 carbon atoms, or lower thioalkyl of 1 to 5 carbon atoms, each m is an integer from 0 to 3, inclusive, eack k is an integer from 1 to 10, inclusive, each j is the same or different and is an integer from 1 to 12, inclusive, S• each R' and R 2 independently is selected from the group consisting of hydrogen, lower alkyl of 1 to carbon atoms, lower alkylamino of 1 to 5 carbon atoms, lower alkylamido of 1 to 5 carbon atoms, lower mono-, di-, or trifluoroalkyl of 1 to 5 carbon atoms, hydroxy, *i amidino, guanidino, or typical common amino acid side S 20 chain or with an associated carbon atom R I and R 2 taken together form a carbonyl, and each Z is selected from the group consisting of nitrogen, oxygen, sulfur, amido, sulfonamido, and carbon.

Preferred aromatic headgroups include, but are not limited to, the following:

OCH

3

Y

Headaroup A

OH

Headgroup C Headgroup B 1~ 0 *00.

Headgroup D ed ru I-{eadgroup F

OR

Headcgroup where Y= R1 R1 R1 R Ar- (C)j Ik-N

R

2

R

2

R

2 c R 2 Preferably the claims- claiming a compound exclude known compounds whose chemical structures are enabled.

In further preferred embodiments, the compound is selected from the group of Compounds 4 through 18, where such compounds have the formulae:

NH

2 Compound 4 00H 3

YO

o N N N_ NH 2 H

NH

2 Compound

OCH

3 IN Y 0 NH H NNH2

NH

2 Compound 6 00H 3 H0 0 NH N0- CH NH 2

CH

3 Compound 7

OCH

3 0 I NH2

NH

2 Compound 8 F W %~SNH Compound 9 SNH2 NH

NH

2 Compound F s

CH

3 Compound 11 FN 0 0 0

HNH

2 Compound 12

OH

Ilk 0 NH 2 Compound 13 0 0

NH

2 Compound 14

SOH

Br Compound Br O O

NH

2 Compound 16 5 O O 0NH2 ee Compound 17 OCH3 o 3 M 0 0 NH2 Compound 18 Applicant has also determined (see Example 23 10 below) that simplified arylalkylamines (see below) are potent, noncompetitive antagonists of the NMDA receptor-ionophore complex. The simplified arylalkylamines are distinct from the arylalkylamines exemplified by Compounds 4-18 as described above. For example, such compounds bind to the site labeled by [PH]MK-801 at concentrations ranging approximately 1 to 400-fold higher than those which antagonize NMDA receptor-mediated function. Such simplified arylalkylamines possess one or more of the following additional biological properties: significant neuroprotectant activity, significant anticonvulsant activity, significant analgesic activity, no PCP-like stereotypic behavior in rodents (hyperexcitability and head weaving) at effective neuroprotectant, anticonvulsant and analgesic doses, no generalization to PCP in a PCP discrimination assay at effective neuroprotectant, anticonvulsant and analgesic doses, no neuronal vacuolization at effective neuroprotectant, anticonvulsant and analgesic does, significantly less potent activity against voltage-sensitive calcium channels, and minimal hypotensive activity at effective neuroprotectant, anticonvulsant and analgesic doses.

Such compounds may, however, inhibit the induction of LTP in rat hippocampal slices and may produce motor impairment at neuroprotectant, anticonvulsant and analgesic doses.

One aspect of the invention includes a compound of Formula VIII: 2 3 S\4

RI

Z

NHR

9 6 R 2 2 7

(X

2 8 FORMULA

VIII

wherein: Z is selected from the group consisting of -CH 2

CH

2

-CH

2

CH(CH

3 and -CH=CH-,-O-CH2-, -S-CH2-;

X

1 and X 2 are independently selected from the group consisting of -CI, -CH 3 -OH and lower O-alkyl in the or 9-substituent positions; i m is independently an integer from 0 to 2; provided that at least one of m is not 0; -NHR is selected from the group consisting of -NH 2

-NHCH

3 and -NHC 2

H

5

R

1 is selected from the group consisting of alkyl, hydroxyalkyl, -OH, -O-alkyl, and- 0acyl, and

R

2 is selected from the group consisting of alkyl, hydroxyalkyl, and pharmaceutically acceptable salts and complexes thereof, wherein the compound is active at an NMDA receptor.

*.9.99 [R:\LIBZZ]444260D speci.doc:gym By "patient" is meant any animal that has a cell with an NMDA receptor. Preferably the animal is a mammal. Most preferably, the animal is a human.

By "alkyl" is meant a branched or unbranched hydrocarbon chain containing between 1 and 6, preferably between 1 and 4, carbon atoms, such as, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, 2-methylpentyl, [R:\LIBZZ]444260D1speci.doc:gym cyclopropylmethyl, allyl, and cyclobutylmethyl.

By "lower alkyl" is meant a branched or unbranched hydrocarbon chain containing between 1 and 4 carbon atoms, of which examples are listed herein.

By "hydroxyalkyl" is meant an alkyl group as defined above, substituted with a hydroxyl group.

By "alkylphenyl" is meant an alkyl group as defined above, substituted with a phenyl group.

By "acyl" is meant where R is H or alkyl as defined above, such as, formyl, acetyl, propionyl, or butyryl; or, R is -O-alkyl such as in alkyl carbonates or R is N-alkyl such as in alkyl carbamates, By "cycloalkyl" is meant a branched or 15 unbranched cyclic hydrocarbon chain containing between 3 and 12 carbon atoms.

In preferred aspects of the invention, Y is selected from the group consisting of -NH 2 and -NH-methyl; 20 R 4 is thiofuran:, pyridyl, phenyl, benzyl, phenoxy, or phenylthio, each of which is optionally substituted with -Cl, -Br, -CF 3 alkyl, -OH, -OCF 3 -O-alkyl, or -0-acyl; X is independently selected from the group consisting of meta-fluoro, meta-chloro, ortho-O-lower alkyl, ortho-methyl, ortho-fluoro, ortho-chloro, meta-Olower alkyl, meta-methyl, ortho-OH, and meta-OH; and either

R

1

R

2 RS, and R 6 are -H; or R 2 is methyl, and R 1

R

5 and R 6 are -H; or R 1 is methyl, and R 2

R

5 and R 6 are -H.

In other preferred aspects of the present invention, RI and R 5 are independently selected from the group consisting of lower alkyl, hydroxyalkyl, -OH, -O-alkyl, and -0-acyl;

R

2 and R 6 are independently selected from the group consisting of lower alkyl, and hydroxyalkyl; or R 1 and R 2 together are -(CH 2 or

-(CH

2 )n-N(R 3 and Y is H;

R

3 is independently selected from the group consisting of -H and lower alkyl;

R

4 is selected from the group consisting of thiofuran, pyridyl, phenyl, benzyl, phenoxy, and phenylthio (each of which-is optionally substituted with S 15 lower alkyl, -Cl, -Br, -CF 3 -OH, -OCF 3 -O-alkyl, or -0-acyl), lower alkyl, and cycloalkyl; X is independently selected from the group consisting of -Cl, -Br, -CF 3 lower alkyl, -OH, and -OCF 3 S 20 m is independently an integer from 0 to Y is -NHR 3 or hydrogen when R 1 and R 2 together are

-(CH

2 )n-N(R 3 and pharmaceutically acceptable salts and complexes thereof, with the provisos that when R' and R 2 together are -(CH 2 3 )-,then

R

5

R

6 and Y are hydrogens; and when R' and R 2 together are not(CH 2 3 then Y is -NHR.

In one preferred aspect, the invention features a method for treating a patient having a neurological disease or disorder comprising administering a compound of Formula II:

R'

R2'NR 3

R

3 R2 Formula II wherein: X is independently selected from the group consisting of -Br, -Cl, -CF3, alkyl, -OH,

-OCF

3 -O-alkyl, and -O-acyl; R is independently selected from the group consisting of alkyl, hydroxyalkyl, -OH, -0-alkyl, and -O-acyl; "oo 10 R 2 is independently selected from the group consisting of alkyl, and hydroxyalkyl, or both R 2 s together are imino; R 3 is independently selected from the group consisting of alkyl, 2-hydroxyethyl, and alkylphenyl; and m is independently an integer from 0 to S 15 Thus, in this preferred aspect, the compounds include the compound of Formula I, wherein: X is independently selected from the group consisting of -Cl, -Br, -CF3, alkyl, -OH, -OCF 3 -O-alkyl, and -O-acyl;

R

1 is selected from the group consisting of -H, alkyl, hydroxyalkyl, -OH, -O-alkyl, and -0-acyl;

R

2 and R 6 are independently selected from the group consisting of alkyl, and hydroxyalkyl, or R 2 and R 6 together are imino;

R

5 is selected from the group consisting of -H, alkyl, hydroxyalkyl, -OH, -0-alkyl, and -O-acyl; Y is NR 3

R

3 and

R

4 is phenyl, optionally substituted with alkyl, -F, -Cl, -Br, -CF 3 -OH, -OCF 3 -O-alkyl, or -O-acyl.

In another preferred aspect, the administered compound has the structure of Formula III:

.NR

3

R

3

S

Formula III wherein: X is independently selected from the group consisting of -Br, -Cl, -CF3, alkyl, -OH,

-OCF

3 -O-alkyl, and -0-acyl.

RI is independently selected from the group consisting of alkyl, hydroxyalkyl, -OH, -O-alkyl, and -O-acyl;

R

2 is independently selected from the group consisting of alkyl, and hydroxyalkyl, or both R 2 s together are imino;

R

3 is independently selected from the group consisting of alkyl, 2-hydroxyethyl, and alkylphenyl;

R

4 is selected from the group consisting of thiofuran, pyridyl, phenyl, benzyl, phenoxy, and phenylthio, (each of which is optionally substituted with alkyl, and cycloalkyl; and, m is independently an integer from 0 to Thus, in the preferred aspect, the compounds include the compound of Formula I, wherein: X is independently selected from the group consisting of -Cl, -Br, -CF 3 alkyl, -OH, -OCF 3 -O-alkyl, and -0-acyl;

R

1 is selected from the group consisting of -H, alkyl, hydroxyalkyl, -OH, -O-alkyl, and -O-acyl;

R

2 and R 6 are selected from the group consisting of alkyl, and hydroxyalkyl, or R 2 and R 6 together are imino; Rs is independently selected from the group consisting of alkyl, hydroxyalkyl, -OH, -O-alkyl, 15 and -O-acyl; and Y is NR 3

R

3 In another preferred aspect, the administered compound has the structure of Formulas IV and V.

N (CH2n (CH 2) n

NR

NR

3 3

NR

3 (X)M (X)m S NR R Formula V Formula

IV

wherein: n is an integer from 1 to 6; X is independently selected from the group consisting of -Br, -Cl, -I -CF 3 alkyl, -OH, *43

-OCF

3 -O-alkyl, and -O-acyl;

R

3 is independently selected from the group consisting of alkyl, 2-hydroxyethyl, and alkylphenyl; and m is independently an integer from 0 to Thus, in this preferred aspect, the administered compounds include the compound of Formula I, wherein:

R

3 is independently selected from the group consisting of and alkyl;

R

4 is phenyl, optionally substituted with alkyl, -F, -Cl, -Br, -CF3, -OH, -OCF 3 -O-alkyl, or -O-acyl; and

R

1 and R 2 together are -(CH 2 or -(CH 2

-N(R

3 In another preferred aspect, the administered 15 compound has the structure of Formulas VI and VII: (CH )n (CH2)n Rr R4 N R 3 *i NR 3

R

3 (X)m I Formula VII Formula VI wherein: n is an integer from 1 to 6; X is independently selected from the group consisting of -Br, -Cl, -CF 3 alkyl, -OH,

-OCF

3 -O-alkyl, and -0-acyl;

R

3 is independently selected from the group consisting of alkyl, 2-hydroxyethyl, and alkylphenyl;

R

4 is selected from the group consisting of thiofuran, pyridyl, phenyl, benzyl, phenoxy, and phenylthio (each of which is optionally substituted with alkyl, and cycloalkyl; and m is independently an integer from 0 to Thus, in this preferred aspect, the administered compounds include the compound of Formula I, wherein: X is independently selected from the group consisting of -Cl, -Br, CF3, alkyl, -OH, -OCF 3 -0-alkyl, and -O-acyl-; and

R

I and R 2 together are -(CH 2 or -(CH 2 n-N(R 3 15 More preferred aspects are those embodiments in which: X is independently selected from the group consisting of meta-fluoro, meta-chloro, ortho-O-lower alkyl, ortho-methyl, ortho-fluoro, ortho-chloro, meta-O-lower alkyl, meta-methyl, ortho-OH, and meta-OH;

NR

3 is selected from the group consisting of NH, N-methyl, and N-ethyl; S3 NR 3

R

3 is selected from the group consisting of NH, NH-methyl, and NH-ethyl;

R

1 is selected from the group consisting of -H and methyl;

R

2 is selected from the group consisting of -H and methyl; and

R

4 is selected from the group consisting of phenyl, benzyl, and phenoxy, each of which is optionally substituted with alkyl, -Cl, -Br, -CF3, -OH,

-OCF

3 -O-alkyl, or -O-acyl.

Especially preferred aspects are those embodiments in which: X is meta-fluoro; each R 1 and R 2 is -H;

NR

3 is selected from the group consisting of NH and N-methyl;

NR

3

R

3 is selected from the group consisting of NH 2 and NH-methyl; and

R

4 is selected from the group consisting of phenyl, benzyl, and phenoxy, each of which is optionally substituted with alkyl, -Cl, -Br, -OH,

-OCF

3 -O-alkyl, or -0-acyl.

S15 In a further aspect, the invention features a method for treating a patient having a neurological disease or disorder comprising administering the compounds of Formula VIII: ooo (X )m 2 R1

NHR

Z

9 6 R2 (X2)m 8 FORMULA VIII wherein: 17 DEC. 2003 15:22 SPRUSON AND FERGUSON 61292615486 NO, 3950 P. 6/7 Z is selected from the group consisting of -CH2CH2-, -CH2CH(CH 3 -CH=CH-, -O-CH 2 -S-CH2-;

X

1 and X 2 are independently selected from the group consisting of -Cl, -CH 3 -OH, and lower O-alkyl in the or 9-substituent positions; s m is independently an integer from 0 to 2; provided that at least one of m is not 0; -NHR is selected from the group consisting of -NH2, -NHCH3, and -NHC 2 Hs;

R

1 is selected from the group consisting of alkyl, hydroxyalkyl, -OH, -0-alkyl, and -0-acyl, and

R

2 is selected from the group consisting of alkyl, hydroxyalkyl, and pharmaceutically to acceptable salts and complexes thereof, wherein the compound is active at an NMDA receptor, Especially preferred aspects are those embodiments in which: Z is -CH2CH2-;

X

1 or X z is-F, or both X1 and X 2 are -F; either R' or R 2 is methyl or both R 1 and R 2 are -H; 15 and -NHR is selected from the group consisting of-NHzor-NHCH 3 A further peferred aspect of the invention is a method for treating a patient having a neurological disease or disorder, the method comprising administering a compound selected from the group consisting of S 0

S.

0 a *0@ a a

S

a.

S

5@* a Compound 156 Compound 184

F

NH

2 Compound 195F Compound 195 Compound 193 Compound 194 R:\LIBZZ]444260Dlspi4idoc:gym COMS ID No: SMBI-00540003 Received by IP Australia: Time 15:23 Date 2003-12-17

NH

2

NH

2 F F Compound 196 Compound 197 and pharmaceutically acceptable salts and complexes thereof.

In other preferred embodiments, the invention features a method for treating a patient having a neurological disease or disorder comprising administering the compounds of Formula IX:

S

S

.J 9

S

9

S

S

[R:\LIBZZ]444260D1 speci.doc:gym

()NHR

W

R

1

(X

2 ml FORMULA IX wherein: W is selected from the group consisting of -CH2-, and 5 X 1 and X 2 are independently selected from the group consisting of -Cl, -CH 3 -OH, and lower O-alkyl; m is independently an integer from 0 to 2; -NHR is selected from the group consisting of -NH,,

-NHCH

3 and -NHC 2

H

s S 10 R 1 is selected from the group consisting of -H, alkyl, hydroxyalkyl, -OH, -O-alkyl, and -O-acyl; and

R

2 is selected from the group consisting of -H,

S*

alkyl, hydroxyalkyl, and pharmaceutically acceptable salts and complexes thereof, wherein the compound is 15 active at an NMDA receptor.

In preferred aspects, the administered compound is selected from the group consisting of Compound 128, 129, 130, 131, 132, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, and 215.

In preferred embodiments, the methods of treatment include administration of a compound selected from Compounds 19 through 215, or pharmaceutically acceptable salts and complexes thereof. Preferably, the compound has an IC, 5 10 AM at an NMDA receptor, more preferably 2.5 AM, and most preferably s 0.5 LM at an NMDA receptor.

In further preferred embodiments, the methods of treatment include administration of a compound selected from the group consisting of Compound 19, 21, 22, 23, 24, 25, 27, 28, 29, 30, 31, 32, 33, 34, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 10 66, 67, 68, 69, 70, 71, 72, 73, 75, 76, 77, 78, 79, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 96, 97, 98, 100, 101, 102, 103, 105, 106, 107, 108, 109, 111, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, .138 (potential prodrug), 139, 141, 142, 143, 144, 145,- 146, 147, 148, 149, and 150, and pharmaceutically acceptable salts and complexes thereof.

These compounds have an IC 50 s 10im at an NMDA receptor.

In more preferred embodiments, the methods of treatment include administration of a compound selected from the group consisting of Compound 19, 21, 22, 23, 24, 25, 27, 28, 29, 30, 31, 32, 33, 34, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,.63, 64, 66, 69, 70, 75, 76, 81, 82, 83, 85, 86, 87, 88, 89, 91, 92, 93, 94, 95, 96, 97, 100, 101, 102, 103, 105, 106, 108, 109, 111, 115, 118, 119, 120, 121, 122, 125, 126, 127, 128, 129, 130, 131, 132, 133, 135, 136, 137, 138 (potential prodrug), 139, 142, 144, 145, 146, 147, 148, 149, and 150, and pharmaceutically acceptable salts and complexes thereof. These compounds have an ICso pM at an NMDA receptor In other embodiments, the compound is selected from the group consisting of Compound 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 69, 76, 82, 83, 88, 89, 90, 92, 93, 94, 95, 96, 101, 102, 103, 105, 109, 111, 115, 118, 119, 120, 121, 122, 125, 126, 127, 129, 130, 131, 135, 136, 137, 138, 139, 142, 144, 145, 148, 149, and 150, and pharmaceutically acceptable salts and 10 complexes thereof.

In particularly preferred embodiments, the methods of treatment include administration of a compound selected from the group consisting of Compound 19, 20, 21, 22, 23, 24, 25, 27, 28, 30, 31, 32, 33, 38, 15 39, 43, 44, 46, 47, 49, 50, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 69, 82, 83, 89, 90, 91, 93, 94, 95, 96, 97, 103, 111, 118, 119, 120, 122, 126, 135, 136, 137, 138 (potential prodrug), 142, 144, 145, 147, 148, 149, and 150, and pharmaceutically acceptable S 20 salts and complexes thereof. These compounds have an ICs 0 0.5 pM at an NMDA receptor.

In more preferred embodiments, the methods of treatment include administration of a compound selected from the group consisting of Compound 20, 24, 25, 33, 50, 60, 66, 69, 103, 111, 118, 119, 120, 122, 136, 137, 138 (potential prodrug), 142, 144, 145, 148, 149, and 150, and pharmaceutically acceptable salts and complexes thereof.

In particularly preferred embodiments, the methods of treatment include administration of a compound selected from the group consisting of Compound _r 33, 50, 60, 119, and 144, and pharmaceutically acceptable salts and complexes thereof.

In other particularly preferred embodiments, the methods of treatment include administration of a compound selected from the group consisting of Compound 33, 50, 60, 119, and 144, and pharmaceutically acceptable salts and complexes thereof.

In preferred aspects, the invention provides a method for treating a patient having a neurological disease or disorder, comprising administering a compound which is selected from the group consisting of Compound 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 181, 182,.183, 184, 185, 186, 187, and pharmaceutically acceptable salts and complexes thereof. These compounds have an IC5 0 5 10gM at an NMDA receptor.

In further preferred aspects, the invention provides a method for treating a patient having a neurological disease or disorder, comprising administering a compound which is selected from the group consisting of Compound 157, 158, 159, 163, 164, 166, 167, 168, 169, 170, 171, 181, 185, 186, and pharmaceutically acceptable salts and complexes thereof.

These compounds have an IC 50 10gM at an NMDA receptor.

In other more preferred aspects, the invention provides a method for treating a patient having a neurological disease or disorder, comprising administering a compound which is selected from the group consisting of Compound 156, 157, 158, 159, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 181, 183, 184, 185, 186, 187, and pharmaceutically acceptable salts and complexes thereof. These compounds have an

IC

50 2.5 AsM at an NMDA receptor.

In further preferred aspects, the invention provides a method for treating a patient having a neurological disease or disorder, comprising administering a compound which is selected from the group consisting of Compound 157, 158, 159, 163, 164, 166, 167, 168, 169, 170, 171, 181, 185, 186, and pharmaceutically acceptable salts and complexes thereof.

10 These compounds have an IC 50 2.5 AM at an NMDA receptor.

In other particularly preferred aspects, the invention provides a method for treating a patient having a neurological disease or disorder, comprising administering a compound which is selected from the group consisting of Compound 156, 157, 158, 159, 161, 163, 164, 165, 167, 168, 169, 170, 171, 181, 186 and pharmaceutically acceptable salts and complexes thereof.

These compounds have an ICs 0 i 0.5 AM at an NMDA receptor.

In further preferred aspects, the invention provides a method for treating a patient having a neurological disease or disorder, comprising administering a compound which is selected from the group consisting of Compound 157, 158, 159, 163, 164, 167, 168, 169, 170, 171, 181, 186 and pharmaceutically acceptable salts and complexes thereof. These compounds have an IC 50 0.5 AM at an NMDA receptor.

In other preferred aspects, the invention provides a method for treating a patient having a neurological disease or disorder comprising administering a compound selected from the group consisting of Compounds 151 215, and pharmaceutically acceptable salts and complexes thereof.

In more preferred aspects, the invention provides a method for treating a patient having a neurological disease or disorder comprising administering a compound selected from the group consisting of Compound 151, 152, 153, 154, 155, 157, 158, 159, 163, 164, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 181, 185, 186, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215 and pharmaceutically acceptable salts and *e complexes thereof.

The present invention provides simplified arylalkylamines comprising the compounds of Formulas I-IX and all preferred aspects of Formulas I-IX as set out above.

Examples of such simplified arylalkylamines S 20 include, but are not limited to, Compounds 19 215, whose structures are provided below. Preferably, the compound has an IC, 0 10 tM at an NMDA receptor. More preferably, the compound has an IC 0 s 5 AM, more preferably 2.5 AM, .and most preferably s 0.5 fM at an NMDA receptor.

In preferred embodiments, the compound is selected from the group consisting of Compound 21, 22, 23, 24, 25, 26, 27, 28, 29, 33, 34, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 69, 76, 78, 79, 82, 83, 84, 88, 89, 90, 92, 93, 94, 95, 96, 98, 101, 53 102, 103, 105, 107, 108, 109, 111, 115, 116, 118, 119, 120, 121, 122, 124, 125, 126, 127, 129, 130, 131, 134, 135, 136, 137, 138 (potential prodrug), 139, 141, 142, 143, 144, 145, 148, 149, and 150. These compounds have an IC 5 so 10 AM at an NMDA receptor.

In other embodiments, the compound is selected from the group consisting of Compound 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 69, 76, 82, 83, 88, 89, 90, 92, 93, 94, 95, 96, 101, 102, 103, 105, 109, 111, 115, 118, 119, 120, 121, 122, 125, 126, 127, 129, 130, 131, 135, 136, 137, 138, 139, 142, 144, 145, 148, 149, and 150.

In more preferred embodiments, the compound is selected from the group consisting of Compound 21, 22, 23, 24, 25, 27, 28, 29, 33, 34, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 69, 76, 82, 83, 88, 89, 90, 92, 93, 94, 95, 96, 101, 102, 103, 105, 108, 109, 111, 115, 118, 119, 120, 121, 122, 125, 126, 127, S 20 129, 130, 131, 135, 136, 137, 138 (potential prodrug), 139, 142, 144, 145, 148, 149, and 150. These compounds have an IC 5 so 2.5 AM at an NMDA receptor.

In particularly preferred embodiments, the compound is selected from the group consisting of Compound 21, 22, 23, 24, 25, 27, 28, 33, 38, 39, 43, 44, 46, 47, 49, 50, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 69, 82, 83, 89, 90, 93, 94, 95, 96, 103, 111, 118, 119, 120, 122, 126, 135, 136, 137, 138 (potential prodrug), 142, 144, 145, 148, 149, and 150.

These compounds have an ICso 0.5 ~M at an NMDA receptor.

In preferred embodiments, the compound is selected from the group consisting of Compound 24, 33, 50, 60, 66, 69, 103, 111, 118, 119, 120, 122, 136, 137, 138, 142, 144, 145, 148, 149, and 150.

In particularly preferred embodiments, the compound is selected from the group consisting of Compound 20, 33, 50, 60, 119, and 144.

In more particularly preferred embodiments, the compound is selected from the group consisting of 10 Compound 33, 50, 60, 119, and 144.

In other preferred aspects, the compound is selected from the group consisting of Compound 151, 152, 153, 154, 155, 157, 158, 159, 163, 164, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 181, 185, 186, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215 and pharmaceutically acceptable salts and complexes thereof.

In other preferred aspects, the compound is 20 selected from the group consisting of Compound 157, 158, 159, 163, 164, 166, 167, 168, 169, 170, 171, 181, 185, 186, and pharmaceutically acceptable salts and complexes thereof. These compounds have an ICs 5 s 10 AM at an NMDA receptor.

In more preferred aspects, the compound is selected from the group consisting of Compound 157, 158, 159, 163, 164, 167, 168, 169, 170, 171, 181, 185, 186, and pharmaceutically acceptable salts and complexes thereof. These compounds have an IC 50 2.5 LM at an NMDA receptor.

In most preferred aspects, the compound is selected from the group consisting of Compound 157, 158, 159, 163, 164, 167, 168, 169, 170, 171, 181, 186, and pharmaceutically acceptable salts and complexes thereof.

These compounds have an IC 50 0.5 .M at an NMDA receptor.

Excluded from the composition of matter aspect of the present invention are known compounds whose chemical structures are covered by the generic 10 formulae presented above.

Also provided in an aspect of the invention are pharmaceutical compositions useful for treating a patient having a neurological disease or disorder. The pharmaceutical compositions are provided in a 15 pharmaceutically acceptable carrier and appropriate dose. The pharmaceutical compositions may be in the form of pharmaceutically acceptable salts and complexes, *as is known to those skilled in the art.

The pharmaceutical compositions comprise the 20 compounds of Formulas I-IX and all preferred aspects of Formulas I-IX as set out above.

Preferred pharmaceutical compositions comprise Compounds 19 215. Preferably, the compound has an ICs 0 s 10 AM at an NMDA receptor. More preferably the compound has an IC 50 5 more preferably g 2.5 AtM, and most preferably 0.5 iM at an NMDA receptor.

In further preferred embodiments, the pharmaceutical composition comprises a compound selected from the group consisting of Compound 20, 21, 22, 23, 24, 25, 27, 28, 29, 30, 31, 32, 33, 34, 37, 38, 39, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 75, 76, 77, 78, 79, 81, 82, 83, 84, 86, 87, 88, 89, 9.0, 91, 92, 93, 94, 95, 96, 97, 98, 100, 101, 102, 103, 105, 106, 107, 108, 109, 111, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, *128, 129, 130., 131, 132, 133, 134, 135, 136, 137, 138 (potential prodrug), 139, 141, 142, 143, 144, 145, 146, 147, 148, 149, and 150. These compounds have an IC 50 10l~gM at an NMvDA receptor.

.Preferably, the-compound is selected from the group consisting of 21, 22, 23, 24, 25, 26, 27, 28, 29, 33, 34, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 69, 76, 78, 79, 82, 83, 84, 88, 89, 92, 93, 94, 95, 96, 98, 101, 102, 103, 105, 107, 108, 109, 111, 115, 116, 118, 119, 120, 121, 122, 124, 125, 126, .127, 129, 130, 131, 134, 135, 136, 137, 138 (potential prodrug) 139, 141, 142, 143, 144, 145, 148, 149, and 150.

In other embodiments, the compound is selected from the group consisting of 54, 55, 56, 57, 58, 59, 61, 62, 63, 64, 65, 66, 69, 76, 82,- 83, 88, 89, 90, 92, :93, 94, 95, 96, 101, 102, 103, 105, 109, 111, 115, 118, 119, 120, 121, 122, 125, 126, 127, 129, 130, 131, 135, 136, 137, 138, 139, 142,, 144, 145, 148, 149, and .150.

In more preferred embodiments, the pharmaceutical composition comprises a compound selected from the group consisting of Compound 20, 21, 22, 23, 24, 25, 27, 28, 29, 30, 31, 32, 33, 34, 37, 38, 39, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51# 52, 53, 54, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 69, 76, 81, 82, 83, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 100, 101, 102, 103, 105, 106, 108, 109, 111, 115, 118, 119, 120, 121, 122, 125, 126, 127, 128, 129, 130, 131, 132, 133, 135, 136, 137, 138 (potential prodrug), 139, 142, 144, 145, 146, 148, 149, and 150.

These compounds have an IC 50 s 2.5 yM at an NMDA receptor.

Preferably, the compound is selected from the group consisting of 21, 22, 23, 24, 25, 27, 28, 29, 33, 34, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 69, 76, 82, 83, 88, 89, 90, 92, 93, 94, 96, 101, 102, 103, 105, 108, 109, 111, 115, 118, 119, 120, 121, 122, 125, 126, 127, 129, 130, 131, 135, 136, 137, 138 (potential prodrug), 139, 142, 144, 145, 148, 149, and 150.

In particularly preferred embodiments, the pharmaceutical composition comprises a compound is selected from the group consisting of Compound 20, 21, S 20 22, 23, 24, 25, 27, 28, 30, 31, 32, 33, 38, 39, 43, 44, 46, 47, 49, 50, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 69, 82, 83, 89, 90, 91, 93, 94, 96, 97, 103, 111, 118, 119, 120, 122, 126, 135, 136, 137, 138 (potential prodrug), 142, 144, 145, 148, 149, and 150. .These compounds have an IC, 0 0.5 jiM at an NMDA receptor.

Preferably, the compound is selected from the group consisting of 21,.22, 23, 24, 25, 27, 28, 33, 38, 39, 43, 44, 46, 47, 49, 50, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 69, 82, 83, 89, 90, 93, 94, 95, 96, 103, 111, 118, 119, 120, 122, 126, 135, 136, 137, 138 (potential prodrug), 142, 144, 145, 148, 149, and 150.

In more preferred embodiments, the pharmaceutical composition comprises a compound selected from the group consisting of Compound 20, 24, 25, 33, 60, 66, 69, 103, 111, 118, 119, 120, 122, 136, 137, 138, 142, 144, 145, 148, 149, and 150.

Preferably, the compound is selected from the group consisting of Compound 24, 25, 33, 50, 60, 66, 69, 103, 111, 118, 119, 120, 122, 136, 137, 138, 142, 144, 145, 148, 149, and 150.

In most particularly preferred embodiments, the pharmaceutical composition comprises a compound selected from the group consisting of Compound 20, 33, 50, 60, 119, and 144.

Preferably, the compound is selected from the group consisting of 33, 50, 60, 119, and 144.

In other preferred aspects, the pharmaceutical composition comprises a compound selected from the group 20 consisting of compound 151, 152, 153, 154, 155, 157, 158, 159, 163, 164, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176,.181, 185, 186, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215 and pharmaceutically acceptable salts and complexes thereof, and a pharmaceutically acceptable carrier.

In other preferred aspects the pharmaceutical composition comprises a compound which is selected from the group consisting of Compound 157, 158, 159, 163, 164, 166, 167, 168, 169, 170, 171, 181, 185, 186, and pharmaceutically acceptable salts and complexes thereof, and a pharmaceutically acceptable carrier. These compounds have an ICs 5 10 pM at an NMDA receptor.

In more preferred aspects, the pharmaceutical composition comprises a compound which is selected from the group consisting of Compound 157, 158, 159, 163, 164, 166, 167, 168, 169, 170, 171, 181, 185, 186, and pharmaceutically acceptable salts and complexes thereof, and a pharmaceutically acceptable carrier. These compounds have an IC 50 2.5 AM at an NMDA receptor.

In most preferred aspects, the pharmaceutical composition comprises a compound which is selected from the group consisting of Compound 157, 158, 159, 163, 164, 167, 168, 169, 170, 171, 181, 186, and pharmaceutically acceptable salts and complexes thereof, and a pharmaceutically acceptable carrier. These compounds have an IC 0 0.5 iM at an NMDA receptor.

Structural modifications can be made to compounds such as 20 or 60 which do not add materially to the structure-activity relationships (SAR) illustrated here. For example, successful bioisosteric replacement or substitution of optionally substituted phenyl groups, such as those occurring in Compounds or 60, can be accomplished with other lipophilic or semi-polar aromatic naphthyl, naphthoxy, benzyl, phenoxy, phenylthio), aliphatic (alkyl, e.g., isopropyl), cycloaliphatic (cycloalkyl, e.g., cyclohexyl), heterocyclic pyridyl, furanyl, thiofuranyl (thiophenyl)], or other functional groups or systems, as is well known in the art, will afford clinically useful compounds (structural homologs, Vanalogs, and/or congeners) with similar biopharmaceutical properties and activity at the NMDA receptor cf. Compounds 37, 75, 79, 83, 89, 119- 122, 125, 126, 128, 130, 132, 137, 144, and 145). For example, such replacements or substitutions have been used to great effect in the development of SAR among other groups of highly clinically and commercially successful synthetic pharmaceutical agents such as the classical H,-antihistamines, anticholinergics (antimuscarinics; anti-Parkinsonians), antidepressants (including tricyclic compounds), and opioid analgesics [See, Foye et al. Principles of Medicinal Chemistry, 4th ed., Lea and Febiger/Williams and.Wilkins, Philadelphia, PA, 1995, 15 pp. 233, 265, 281-282, 340-341, 418-427, and 430; Prous, The Year's Drug News, Therapeutic Targets 1995 Edition, Prous Science Publishers, Barcelona, Spain, 1995, pp. 13, 55-56, 58-59, 74, 89, 144-145, 152, 296- 297, and 317]. Similarly, bioisosteric replacement or 20 substitution of the methylene or methine groups in the propyl backbone of compounds such as 20 or 60 with, oxygen, sulfur, or nitrogen, will afford clinically useful NMDA receptor-active compounds with similarly useful biopharmaceutical properties, such as Compound 88 (a modified "classical Hi-antihistamine-type" structure), which.can be further optimized for activity at the NMDA receptor by preparing, the corresponding compound(s) containing, (bis)(3fluorophenyl) group(s), as taught by the present invention. The propyl backbone of compounds such as and 60 may also be modified successfully by the incorporation of ring systems (as in Compounds 102 and 111ii) and/or unsaturation a double bond, as in Compounds 81, 106, 109, and 139) to afford further clinically useful NMDA receptor-active compounds of the present invention (cf. compounds cited above).

In a related aspect, the invention features a method for making a therapeutic agent comprising the steps of screening for said agent by determining whether said agent is active on a receptor-operated calcium channel, and synthesizing said therapeutic agent in an amount sufficient to provide said agent in a therapeutically effective amount to a patient. Said screening may be performed by methods known to those of S"ordinary skill in the art, and may, for example be performed by the methods set out herein. Those skilled in the art are also familiar with methods used to synthesize therapeutic agents in amounts sufficient to be provided in a therapeutically effective amount.

In a preferred aspect, said receptor-operated S 20 calcium channel is an NMDA receptor. In a more preferred aspect, said method further comprises the step of adding a pharmaceutically acceptable carrier to said agent. In a further preferred aspect said therapeutic agent comprises a compound of Formula I, as set out herein. In a further preferred aspect said therapeutic agent comprises a compound of Formula II, III, IV, V, VI, VII, VIII, or IX, as set out herein. In particularly preferred aspects, said therapeutic agent comprises a compound having a structure selected from the group consisting of Formulas I-IX, and all preferred aspects of said formulas as set out herein. In further 62 preferred aspects, said therapeutic agent is selected from the group consisting of Compounds 19-215. In a particularly preferred aspect, said therapeutic agent is provided to a patient having a neurological disease or disorder. In a related aspect, said screening comprises the step of identifying a compound which binds to said receptor-operated calcium channel at a site bound by one of the arylalkylamines Compound 1, Compound 2, and Compound 3.

i

NH

2

NH

2 F HNH2 F

F

F

F

Compound 19 Comound 20 ComDound 21 '4 I OCH 3 F ,NH 2 F /NH 2

NH

2 I CH 3 I CH 3 F I I F F NFN rnmnound 22 Comoound 23 Crnmnnnd 24 Comnound 22 Compound 23 Comoound 2

H

3 CQ

NH

2

CH

3 Comnound 39 Compound 37 Comoound 38 FNNoCH3

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2 FCH3HICH

CHHCH

3 H

FF~

ComoDound 40 lCompound 41. Compound 42 A NH 2

F

Compound 53 F A"

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2

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Compound 56 N CH 3 F

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Compound 55 F A H NH 2

ACH

3

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Compound 58 ICompond, 57

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2 c K z ,0 *9* Compound 163 Compound 164 Compound 165,

OCH

3 1

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:1 167 JCompound 168 Compound 169 Con

NIP

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3 Compound 172 Con Compound 175 Compound 176 Compound 177 Compound 175 Compound 176 Compound 177

II-

F

Compound 208 Compound 209 Compound 210

NH

2

F

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2

SNNH

2

F

Compound 211 Compound 212 Compound 213

F"

N

NHz22 F NH2 Compound 214 Compound 215 Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.

Description of the Preferred Embodiments The following is a detailed description of the methods and tests by which therapeutically useful compounds can be identified and utilized for the treatment of neurological disorders and diseases. The tests are exemplified by use of Compound 1, Compound 2 or Compound 3, but other compounds which have similar biological activity in these assays can also be used (as discovered) to improve on the tests. Lead compounds such as Compound 1, Compound 2 or Compound 3 can be used for molecular modeling using standard procedures, or existing or novel compounds in natural product libraries can be screened by the methods described below.

One key method is the means by which compounds can be quickly screened with standard radioligand binding techniques (a radiolabeled arylalkylamine binding assay) to identify those which bind at the.same site on receptor-operated Ca 2 channels as Compound 1, Compound 2 or Compound 3. Data from such radioligand binding studies will also confirm that said compounds do not inhibit [3H]arylalkylamine binding via an action at the known sites on receptor-operated Ca 2 channels (such as the glutamate binding site, glycine binding site, MK-801 binding site, Zn 2 binding site, Mg 2 binding site, sigma binding site, or polyamine binding site on the NMDA receptor-ionophore complex). This screening test allows vast numbers of potentially useful compounds to be identified and screened for activity in the other assays. Those skilled in the art will recognize that other rapid assays for detection of binding to the arylalkylamine site on receptor-operated Ca 2 channels can be devised and used in this invention.

Additional testing utilizes electrophysiological (patch clamp) methodology to extend the results obtained with the above-mentioned radioligand binding assay. Such results will confirm that compounds binding to the arylalkylamine site are functional, noncompetitive antagonists of receptor-operated Ca 2 channels with the following properties in common with the arylalkylamines themselves: open-channel block manifested as use-dependent block, and voltage-dependent onset and reversal from block. Such results will also confirm that said compounds do not have their primary activity at the previously described sites on receptor-operated Ca 2 channels (such as the glutamate binding site, o glycine binding site, MK-801 binding site, Zn 2 binding site, Mg 2 binding site, sigma binding site, or polyamine binding site on the NMDA receptor-ionophore complex).

In addition, recombinant DNA technology can be used to make such testing even more rapid. For example, using standard procedures, the gene(s) encoding the novel arylalkylamine binding site receptor) can be identified and cloned. This can be accomplished in one of several ways. For example, an arylalkylamine affinity column can be prepared, and solubilized membranes from cells or tissues containing the arylalkylamine receptor passed over the column. The receptor molecules bind to the column and are thus isolated. Partial amino acid sequence information is then obtained which allows for the isolation of the gene encoding the receptor. Alternatively, cDNA expression libraries are prepared and subfractions of the library are tested for their ability to impart arylalkylamine receptors on cells which do not normally express such receptors CHO cells, mouse L cells, HEK 293 cells, or Xenopus oocytes). In this way, the library fraction containing the clone encoding the receptor is identified. Sequential subfractionation of active library fractions and assay eventually results in a single clone.encoding the arylalkylamine receptor.

Similarly, hybrid-arrest or hybrid-depletion cloning can be used. Xenopus oocytes are injected with mRNA from an appropriate tissue or cell source human brain tissue). Expression of the arylalkylamine receptor is detected as, for example, an NMDA- or glutamate-stimulated influx of calcium which can be blocked by Compound 1, Compound 2 or Compound 3. cDNA clones are tested for their ability to block expression of this receptor when cDNA or cRNA are hybridized to the mRNA of choice, prior to injection into Xenopus oocytes.

The clone responsible for this effect is then isolated by the process described above. Once the receptor gene is isolated, standard techniques are used to identify the polypeptide or portion(s) thereof which is (are) sufficient for binding arylalkylamines (the arylalkylamine binding domain[s]). Further, using standard procedures, the entire receptor or arylalkylamine binding domain(s) can be expressed by recombinant technology. Said receptor or binding domain(s) can be isolated and used as a biochemical reagent such that, rather than using a competitive assay exemplified below, a simple direct binding assay can be used. That is, a screen is set up for compounds which bind at the novel arylalkylamine receptor. In this way large numbers of compounds can be simultaneously screened, by passage through a column containing the novel arylalkylamine receptor or arylalkylamine binding domain, and analysis performed on compounds which bind to the column.

Additional testing utilizes the combination of molecular biological techniques (expression of cloned NMDA, AMPA or nicotinic cholinergic receptors) and patch clamp electrophysiological techniques. Specifically, arylalkyl-amine analogs can be rapidly screened for potency at cloned and expressed subunits of the above-mentioned receptor-ionophore complexes.

Site-directed mutagenesis can be utilized in an effort to identify which amino acid residues may be important in determining arylalkylamine potency.

Assays for Potent and Selective Antagonists of Receptor-Operated Calcium Channels in the Mammalian CNS Desired properties of a drug include: high affinity and selectivity for receptor-operated Ca 2 channels, such as those present in. NMDA, AMPA and nicotinic cholinergic receptor-ionophore complexes (compared to responses mediated via other neurotransmitter receptors, neurotransmitter receptor-operated ion channels, or voltage-dependent ion channels) and a noncompetitive antagonism of said receptor-operated Ca 2 channels.

The NMDA receptor-ionophore complex is utilized as an example of a receptor-operated Ca 2 channel. Activation of the NMDA receptor opens a cation-selective channel that allows the influx of extracellular Ca 2 and Na', resulting in increases in [Ca 2 ]i and depolarization of the cell membrane.

Measurements of [Ca 2 were used as primary assays for detecting the activity of arylalkylamine compounds on NMDA receptors. Purified arylalkylamines, synthetic aryl-alkylamines, and synthetic analogs of arylalkylamines were examined for activity in in vitro assays capable of measuring glutamate receptor activity.

Selected for detailed study were the arylalkylamines present in the venom of various spider species. The arylalkylamines present in these venoms are structurally distinct but have the basic structure of the class represented by Compounds 1 through 3. Other more simplified synthetic analogs generally consist of suitably substituted aromatic chromophoric groups attached to an alkyl(poly)amine moiety (see Compounds 19 S: through 215 below).

A primary assay that provides a functional index of glutamate receptor activity and that allows high-throughput screening was developed. Primary cultures of rat cerebellar granule.cells loaded with the fluorimetric indicator fura-2 were used to measure changes in [Ca 2 ielicited by NMDA and its coagonist glycine. This assay provides an extremely sensitive and precise index of NMDA receptor activity. Increases in [Ca 2 ]i evoked by NMDA are dependent on the presence of glycine, and are blocked by extracellular Mg 2 or antagonists acting at the glutamate, glycine, or MK-801 binding sites. Increases in [Ca 2 ielicited by NMDA/glycine are readily distinguished from those resulting from depolarization by their refractoriness to inhibition by blockers of voltage-sensitive Ca 2 channels. The fidelity with which measurements of [Ca 2 i corroborate results obtained by electrophysiological and ligand-binding studies suggests that such measurements mirror closely activation of the NMDA receptor-ionophore complex.

Example 1: Potent noncompetitive inhibition of NMDA receptor function Preferential inhibitory effects of arylalkylamines on NMDA receptor-mediated increases in (a 15 [Ca 2 i in cultured rat cerebellar granule cells were measured. Increases in [Ca2] ,were elicited by the addition of NMDA/glycine (50 uM/1 MM) in the presence or absence of different concentrations of each test compound. The IC 50 values were derived for each test 20 compound using from 2 to 8 separate experiments per test compound, and the standard error level was less than of the mean value for each compound.

All of the arylalkylamines tested blocked increases in [Ca 2 ]i in cerebellar granule cells elicited by NMDA/glycine. Certain arylalkylamines similar in structure to Compound 1 or Compound 2 were nearly as potent as MK-801 (IC 50 34 nM) which is the most potent compound in the literature known to preferentially block NMDA receptors. Compound 3 had an IC 0 2 nM, that is, 17-fold more potent than MK-801. Many of the arylalkylamines tested were more potent than competitive antagonists such as AP5 (ICs 15 gM). The inhibitory effects of the arylalkylamines were not overcome by increasing the concentrations of NMDA or glycine. That is, no change was observed in the EC 50 for either NMDA or glycine. The arylalkylamines are thus noncompetitive antagonists at the NMDA receptor-ionophore complex, and act neither at the glutamate nor the glycine binding sites.

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S

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0 o

S

S

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S

00 0 00 06 10 Example 2: Activity against Kainate and AMPA receptor function Measurements of [Ca 2 ]i in cerebellar granule cells can also be used to monitor activation of the native kainate or AMPA receptors present in this tissue.

15 Although the increases in [Ca2]i evoked by these agonists are of a lesser magnitude than those evoked by NMDA/glycine, such responses are robust and can be used to precisely assess the specificity of action of arylalkylamines on pharmacologically defined glutamate 20 receptor subtypes. Comparative measurements of [Ca 2 +]i revealed a clear distinction in the receptor selectivity of the arylalkylamines. Some, like JSTX-3 (Joro Spider toxin from the spider Nephila clavata), were more potent antagonists of responses elicited by kainate (100 pM) or AMPA (30 On the other hand, arylalkylamines within the two structural classes defined by Compound 1 and by Compound 2 were found to inhibit preferentially responses evoked by NMDA (showing about a 100-fold difference in potency). Thus, arylalkylamines such as Compound 1 and Compound 2 are potent and selective 82 inhibitors of NMDA receptor-mediated responses in cerebellar granule cells.

Example 3: Patch clamp electrophysiology studies Patch clamp electrophysiological studies on isolated cortical or hippocampal neurons from adult rat brain have provided additional insight into the mechanism of action of Compound 1, Compound 2 and Compound 3. These studies revealed potent and selective inhibitory effects of arylalkylamines on responses mediated by NMDA receptors. Thus, compounds such as Coipound 1 blocked responses to NMDA at nanomolar Sconcentrations without affecting the responses to kainate. These results, which show selective inhibitory effects of the arylalkylamines in cortical and hippocampal neurons, indicate that the arylalkylamines target NMDA receptors in different regions within the mammalian CNS. Moreover, it was found that the inhibitory effects of these compounds were use- and *i voltage-dependent. This strongly suggests that these compounds are blocking the open channel and, by this action, behave as noncompetitive NMDA receptor antagonists. Importantly, however, the arylalkylamines could be distinguished from both Mg 2 and MK-801, especially with respect to the voltage-dependence of their onset of action and reversibility of effect.

Example 4: Radioligand binding assays Radioligand binding studies have demonstrated that arylalkylamines such as Compound 1 and Compound 2 have a unique site of action. Although they act like 83 MK-801 in some respects (noncompetitive open-channel blockade, discussed above), they fail to displace 3 H]MK-801 binding at concentrations that completely block NMDA receptor-mediated responses. Assays such as these also demonstrate that the arylalkylamines do not bind with high affinity to the known MK-801, Mg 2 or polyamine binding sites on the NMDA receptor-ionophore complex. Neither do the arylalkylamines bind directly to either the glutamate, glycine or sigma binding sites at concentrations that block NMDA receptor-mediated responses. 3 H]Compound 2 was synthesized as a radioligand for use in binding studies to further explore the mechanism of action of Compound 2 and particularly for use in a high-throughput screen to 15 assess the activity of other analogs and to detect new lead structures. A similar approach was taken for 3 H]Compound 5. It is clear that compounds like Compound e 1 and Compound 2 target a site on the NMDA receptor-ionophore complex for which no other known 20 compounds presently exist. The novel site of action of the arylalkylamines at the molecular level translates S, into pronounced therapeutic advantages at the behavioral level. As described below, the arylalkylamines possess a quite different behavioral profile from other noncompetitive antagonists of the NMDA receptor.

Example 5: Synaptic transmission studies The above findings demonstrate that certain arylalkylamines, specifically those related in structure to Compound 1 and Compound 2, act through a novel mechanism and site of action to potently and selectively oooo *o ooooo ooooo ooo inhibit NMDA receptor-mediated responses on neurons from several different brain areas. To further assess the selective inhibitory actions of the arylalkylamines, their effects on synaptic transmission mediated by NMDA or AMPA receptors were assessed.

Glutamate-mediated transmission at synapses of Schaffer collateral fibers and CA1 pyramidal cells was measured in slices of rat brain containing the hippocampus. This assay measures electrophysiologically the postsynaptic depolarization caused by the presynaptic release of glutamate, and can readily distinguish synaptic transmission mediated by NMDA or AMPA receptors. Arylalkylamines like Compound 1, Compound 2 and Compound 3 were again found to exert 15 preferential inhibitory effects on NMDA receptor-mediated responses, and depressed responses mediated by AMPA receptors only at much higher concentrations. For example, Compound 1 had an ICs 0 for the NMDA receptor-mediated response of 20 gM, but an ICs 0 for the AMPA receptor-mediated response of 647 gM. These results show that arylalkylamines can selectively inhibit synaptic transmission mediated by NMDA receptors. Other naturally occurring arylalkylamines present in the venom of Agelenopsis aperta likewise exert potent and selective inhibitory effects on NMDA receptor-mediated responses in the rat hippocampus.

In the aggregate, then, the results of these various studies are complementary and together identify a structurally novel class of compounds with potent and selective inhibitory activity on NMDA receptors in the mammalian CNS. Additionally, these compounds target a unique site on the NMDA receptor-ionophore complex.

Compound 1, Compound 2 and Compound 3 were selected for additional study in a variety of in vitro and in vivo assays that model therapeutically important endpoints.

Neuroprotectant activity Desired properties of a neuroprotectant drug include the following. The drug can be administered by oral or injectable routes it is not significantly broken down in the stomach, intestine or vascular system and thus reaches the tissues to be treated in a therapeutically effective amount). Such drugs are easily tested in rodents to determine their bioavailability. The drug exhibits neuroprotectant activity efficacy) when given after an ischemic insult (stroke, asphyxia) or traumatic injury (head trauma, spinal cord injury). The drug is devoid of or has minimal side effects such as impairment of cognition, disruption of motor performance, sedation or hyperexcitability, neuronal vacuolization, cardiovascular activity, PCP-like abuse potential, or PCP-like psychotomimetic activity.

Although glutamate is the physiological synaptic transmitter, chronic exposure to glutamate leads to neuronal cell death. Much of the neurodegeneration caused by glutamate appears to be mediated by NMDA receptors and results directly from chronically elevated levels of cytosolic Ca 2 There is now extensive experimental support for the view that NMDA and AMPA receptors play a major role in mediating the neuronal degeneration following a stroke and other ischemic/hypoxic events (Choi, Glutamate neurotoxicity and diseases of the nervous system. Neuron 1: 623, 1988). Most of this evidence is based on the ability of competitive or noncompetitive antagonists of the NMDA or AMPA receptor to effectively block neuronal cell death in both in vitro and in vivo models of stroke. Compound 1, Compound 2 and Compound 4 were therefore examined for neuroprotectant effects in standard assays designed to detect such activity.

Example 6: Cortical neuron protection To assess the in vitro neuroprotectant effect of arylalkylamines, mouse cortical neurons grown in S..culture were exposed for 5 minutes to NMDA, and cell death after 24 hours was monitored by measuring the release of lactate dehydrogenase (LDH), a cytoplasmic enzyme that is released from dying.cells (Choi et al., Glutamate neurotoxicity in cortical cell culture. J.

Neurosci. 7: 357, 1987). Exposure to NMDA killed about 80% of the cortical neurons. Compound 1 or Compound 2, included along with NMDA, prevented cell death with ICso values of 70 uM and 30 uM, respectively. The effective concentrations of the arylalkylamines are higher than those of other noncompetitive NMDA receptor antagonists, but similar to those of competitive antagonists. The effective concentrations of NMDA receptor antagonists vary depending on the particular experimental conditions and the type of cell studied (cortical, hippocampal, striatal). This neuroprotectant effect likely results from the ability of these compounds to block the influx of extracellular Ca 2 triggered by the NMDA receptor.

87 More rigorous testing to determine.potential therapeutic efficacy involved in vivo stroke models. In these models, the blood supply is temporarily blocked by clamping the main arteries to the brain. Two in vivo models of this sort were used to determine the ability of Compound 1, Compound 2 and Compound 4 to prevent neuronal cell loss.

Example 7: Bilateral carotid artery occlusion The first assay was the bilateral common carotid artery occlusion model of forebrain ischemia performed in the gerbil (Karpiak et al., Animal models.

for the study of drugs in ischemic stroke. Ann. Rev.

Pharmacol. Toxicol. 29: 403, 1989; Ginsberg and Busto; Rodent models of cerebral ischemia. Stroke 20: 1627, 1989). Blood flow to the brain was interrupted for 7 minutes by clamping the carotid arteries. The test compounds were administered as a single dose given intraperitoneally 30 minutes after reinstating blood flow. During the course of these experiments, the core body temperature of the animals was maintained at 37 0 C to prevent any hypothermic reaction. It has been shown that many NMDA receptor antagonists cause hypothermia and this effect can account for much of the protective effect of these compounds. The brains were examined for neuronal cell death 4 days later by silver staining sections of the brain and quantifying death by morphometric analysis. Compound 2 (20 mg/kg) significantly (p 0.05) protected against neuronal cell death in all areas of the brain examined (region CA1 of hippocampus, striatum and neocortex). Doses as low as 1 mg/kg afforded complete protection of the striatum. The degree of protection is comparable to that achieved with similar doses of the noncompetitive NMDA antagonist, MK-801.

In subsequent experiments, Compound 1 mg/kg) produced a 23% reduction in the amount of neuronal death in region CA1 of the gerbil hippocampus measured at 7 days post-ischemia, while Compound 4 mg/kg) provided 90% protection.

Example 8: Middle cerebral artery occlusion The middle cerebral artery model of stroke performed in the rat (Karpiak et al., Animal models for the study of drugs in ischemic stroke. Ann. Rev.

Pharmacol. Toxicol. 29: 403, 1989; Ginsberg and Busto, Rodent models of cerebral ischemia. Stroke 20: 1627, 1989) is different from the gerbil model because it results in a more restricted brain infarct, and thereby approximates a different kind of stroke (focal thrombotic stroke). In the first study using this -stroke model, one cerebral artery was permanently occluded by surgical ligation. The test compounds were administered 30 minutes after the occlusion by a single intraperitoneal injection. During the course of these experiments, the core body temperature of the animals was maintained at 37 0 C to prevent any hypothermic reaction. Brains were assessed histologically for neuronal cell loss 24 hours later.

Infarct volumes were calculated using the area of histological pallor from 10 slides and integrating the distance between each successive section. A single dose S. S mg/kg) of Compound 1 was found to significantly (p 0.05) protect against neuronal cell loss equally as well as a maximally effective dose (10 mg/kg) of MK-801 (approximately 15% protection). Preliminary studies with Compound 2 (20 mg/kg) indicated a similar trend.

In the second study of focal cerebral ischemia in the rat, the middle cerebral artery was permanently occluded by passing a small piece of suture thread through the carotid artery to the region of the middle cerebral artery. Core body temperature was maintained at 37 0 C. Compound 4, 10 mg/kg i.p. administered immediately after the onset of the ischemic event, produced a statistically significant reduction in the volume of the brain infarct recorded 24 hr later.

In a third model of focal cerebral ischemia in the rat, an ischemic infarct was produced by a photothrombotic method using the dye Rose Bengal.

Compound 4, 10 mg/kg i.p. administered 30 min after the ischemic event, produced a 20% reduction in the volume of the infarct, similar to that seen with the -noncompetitive NMDA receptor antagonist, MK-801.

In a fourth model of focal cerebral ischemia in the rat, the middle cerebral artery was temporarily occluded by passing a small piece of suture thread through the carotid artery to the region of the middle cerebral artery. The suture thread was withdrawn after an ischemic period of 2 hr. Core body temperature was maintained at 37 0 C. Compound 4 administered at 10 mg/kg i.p. immediately after the onset of the ischemic event, produced a statistically significant reduction in the volume of the brain infarct recorded 72 hr later.

Several important features of the lead compounds emerge from these in.vivo results. First, and most importantly, Compound 1, Compound 2 and Compound 4 demonstrate neuroprotectant effects in several different in vivo models of stroke. The gerbil assay is a model for transient global cerebral ischemia and hypoxia such as cardiac arrest or perinatal hypoxia. The rat assays are models of permanent and.temporary focal cerebral ischemia. The finding that Compound 1 and Compound 4 are neuroprotective in the permanent focal stroke models is surprising because the accessibility of the drug to the site of infarction is limited to the penumbral region which generally is not large: Nonetheless, Compound 1 and Compound 4 significantly (p 0.05) limited the extent of damage. Second, the compounds are effective when administered after the ischemic event.

This is important because there is believed to be a "window of opportunity" following an infarct during which drugs may effectively halt necrotic damage. How 20 long this time is in humans has not been defined precisely, and will likely vary depending upon the type of infarct. The essential observation, however, is that these compounds can prevent the spread of neuronal cell death once the degenerative process has commenced.

Finally, Compounds 1, 2, and 4 are effective when administered parenterally, demonstrating that they penetrate the blood-brain barrier.

Anticonvulsant activity Desired properties of an anticonvulsant drug include: the drug can be administered by oral or C C a a injectable routes, the drug exhibits effective anticonvulsant activity against several seizure types, including, but not limited to, simple partial seizures, complex partial seizures, status epilepticus, and trauma-induced seizures such as occur following head injury, including head surgery; and the drug is devoid of or has minimal side effects such as impairment of cognition, disruption of motor performance, sedation or hyperexcitability, neuronal vacuolization, cardiovascular activity, PCP-like abuse potential, or PCP-like psychotomimetic activity.

Glutamate is the major excitatory transmitter in the brain, and thus may play a major role in seizure activity, and contribute to the pathogenesis of epilepsy. Much of the evidence favoring a major role for glutamate receptors in epilepsy derives from pharmacological studies demonstrating that glutamate receptor agonists elicit seizures, and that NMDA and AMPA receptor antagonists are effective anticonvulsants 20 when administered in vivo. There are numerous in vivo models involving different kinds of seizures and behavioral effects that are relevant for clinically distinct forms of epilepsy. It is thus prudent to test for effects in several models, because it may be an oversimplification to suppose that the same mechanism underlies all forms of seizure activity.

Example 9: Convulsant blocking activity In initial studies, the ability of arylalkylamines to block seizures induced by kainate, picrotoxin or bicuculline were examined. Each of these a; 0 0 0 0 0 0 0 0 0 0 0 0 convulsants acts through a different mechanism and seizures elicited by kainate are qualitatively different from those elicited by.picrotoxin or bicuculline. In these experiments, a fraction of Agelenopsis aperta venom containing several arylalkylamine toxins was administered intravenously (iv) 5 min before picrotoxin or bicuculline, and 5 min after kainate administration.

The arylalkylamines diminished the seizures induced by all three of these agents. The effects of picrotoxin or bicuculline were so severe that all 19 control animals died within 25 minutes. In contrast, there were no deaths in the 9 animals pretreated with the arylalkylamines. In fact, only about half the animals treated with the arylalkylamines showed any convulsions at all and those symptoms abated within an hour. These results demonstrate clear anticonvulsant effects of arylalkylamines and prompted further studies using purified arylalkylamines and their analogs.

Example 1.0: Seizure stimuli Three different seizure-inducing test paradigms were used initially in this second group of studies and arylalkylamines such as Compound 1 proved to be effective anticonvulsants in two such paradigms. The first two models used DBA/2 mice which are prone to audiogenic seizures. Seizures were elicited by sound (bell tone at 109 dBs) or the intraperitoneal (ip) administration of NMDA (56 mg/kg). The test substances were administered 15-30 min before the convulsant stimulus. The number of clonic seizures was recorded for 1 min following the audiogenic stimulus or for "-sy a a a, min following the administration of NMDA. Compound 1, Compound 2, and several other arylalkylamines such as Compound 3 and Compound 4 depressed seizures evoked by either stimulus. For example, Compound 2 had an ED, 5 of 0.13 mg/kg s.c. for audiogenic stimulus and 0.083 mg/kg s.c. for NMDA stimulus. Similarly, the ECs, for Compound 4 in the audiogenic seizure model (0.08 mg/kg) approached that for MK-801 (0.02 mg/kg). In contrast, neither Compound 1 nor Compound 2 was effective at doses up to 50 mg/kg s.c. in reducing seizures in CF-1 mice elicited by i.p. NMDA.

In a second independent series of experiments, Compound 1 and Compound 4 were found to prevent seizures induced by sound in another genetically susceptible 15 mouse model of reflex epilepsy (Frings mice) following intraperitoneal injection with IC 50 values of 14.3 mg/kg and -15 mg/kg, respectively. These compounds were considerably more potent against audiogenic seizures in Frings mice following intracerebroventricular injection, with IC 50 values of 0.63 ug (Compound 1) and 4.77 ug (Compound Compound 1 was also found to be effective against seizures elicited by maximal electroshock in CF1 mice at a dose of 4 gg i.c.v.

In further studies using the genetically susceptible mouse model of reflex epilepsy (Frings mice), Compound 9, Compound 12 and Compound 14, administered by i.c.v. injection, prevented sound-induced seizures with ICs values of 4.77 12.2 cg and 13.9 Mg, respectively.

These collective findings demonstrate that arylalkylamines such as Compound 1, Compound 2 and 94 Compound 4 are effective in preventing epileptic (audiogenic) and nonepileptic (chemoconvulsant) seizures. This generalized pattern of activity suggests that arylalkylamines are clinically useful in controlling seizure activity. In addition, the potency of Compound 1, Compound 2 and especially Compound 4 in in vivo models of seizure activity shows that these compounds can have the therapeutically relevant effects when administrated parenterally in low doses, and are especially potent when administered directly into the cerebral ventricles.

Analgesic activity Desired properties of an analgesic drug include: the drug can be administered by oral or injectable routes, the drug exhibits analgesic activity, the drug is devoid of or has minimal side effects such as impairment of cognition, disruption of motor performance, sedation or hyperexcitability, neuronal vacuolization, cardiovascular activity, PCP-like abuse potential, or PCP-like psychotomimetic activity.

Glutamate and NMDA receptor-mediated responses may play a role in certain kinds of pain perception (Dickenson, A cure for wind up: NMDA receptor antagonists as potential analgesics. Trends Pharmacol.

Sci. 11: 302, 1990). The possible analgesic effects of Compound 1, Compound 2, Compound 3 and Compound 4 were therefore examined.

Example 11: Writhing response test In the first series of experiments, the animals were administered an unpleasant stimulus (2-phenyl.-l,4-benzoquinone, PBQ) which elicits a writhing response (abdominal stretching). Typically, the number of writhes occurring in a 5 min observation period is recorded. Classic analgesic drugs, such as morphine, are effective at decreasing the number of PBQ-elicited writhes (100% block of the writhing response at 4 mg/kg Nonsteroidal antiinflammatory agents are likewise effective in this model. Compound 1 (2 mg/kg), Compound 2 (2 mg/kg) and Compound 3 (1 mg/kg) depressed the writhing response by greater than 95% when administered s.c. or i.p. 15 minutes before PBQ. These results demonstrate that Compound 1, Compound 2 and Compound 3 alleviate visceral pain.

t In a similar series of studies, Compound 1 and Compound 4 were found to inhibit acetic acid-induced 20 writhing in mice following i.p. injection with ICs, values of 10 mg/kg and 1 mg/kg, respectively.

Example 12: Hot plate test Compound 1 was tested for analgesic activity in an additional assay. In this model of analgesic activity, mice were administered test.substances s.c.

min before being placed on a hot plate (50oC). The time taken to lick the feet or jump off the plate is an index of analgesic activity, and effective analgesics increase the latency to licking or jumping. Morphine (5.6 mg/kg) increased the latency to jump by 765%. (5.6 mg/kg) increased the latency to jump by 765%.

&see

S

5030 0 0e a 0

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96 Compound 1.was likewise effective in this assay and, at doses of 4 and 32 mg/kg, increased the latency to foot licking by 136% and the latency to jumping by 360%, respectively.

It is noteworthy that the analgesic effects of Compound 1 in the hot plate assay were not accompanied by a decreased performance in the inverted grid assay (see below). This shows that the increase in the latency to jump off the hot plate does not simply reflect impaired motor capabilities. Together, these data suggest that Compound 1 possesses significant analgesic activity.

In a later series of experiments, Compound 1 and Compound 4 were demonstrated to possess significant 15 analgesic activity in rats when administered by the intrathecal route. In these experiments, a 52 0

C

hot plate was used as the nociceptive stimulus.

Compound 1 (0.3 3 nmol) and Compound 4 (0.3 3 nmol) produced dose- and time-dependent antinociceptive 20 effects; these arylalkylamines were similar to morphine (0.3 3 nmol) in terms of potency and efficacy. The NMDA receptor antagonist, MK-801, on the other hand, was ineffective in this assay (3-30 nmol).

Example 13. Tail flick test In this standard assay, the thermal nociceptive stimulus was 520C warm water with the latency to tail flick or withdrawal taken as the endpoint. Compound 1 (0.3 3 nmol) and Compound 4 (0.3 3 nmol) produced a dose- and time-dependent analgesic effect following i.th. administration. These 5 00

S

0@ 97 arylalkylamines were similar to morphine (0.3 3 nmol) in terms of potency and efficacy. The NMDA receptor antagonist, MK-801, on the other hand, was ineffective in this assay (3-30 nmol).

Example 14. Formalin test Male Sprague-Dawley rats were habituated to an observation chamber for at least 1 hr before receiving an injection of dilute formalin in a volume of Al into the left rear paw. Behavioral responses were S 10 monitored immediately after s.c. injection of formalin S•into the dorsal surface of the paw by counting the number of flinches exhibited by the animal. Behaviors were monitored for at least 50 min after formalin injection and were recorded as early phase responses 15 (0 10 min post-formalin) and late phase responses 50 min post-formalin). Compounds were injected intrathecally 10 min prior to formalin (pre-treatment) or 10 min after formalin (post-treatment) in a volume of 5 .l.

0 20 Intraplantal administration of formalin produced a typical biphasic response of flinching behavior, commonly described as the early and late phase responses. Intrathecal administration of Compound 1 (0.3 10 nmol) or Compound 4 (0.3 10 nmol) given as a pretreatment to formalin effectively inhibited both early- and late-phase flinching behaviors. This effect of pretreatment with the arylalkylamines was similar to that seen with pretreatment with morphine (1 10 nmol) or MK-801 (1 30 nmol).

98 Compound 1 (0.3 10 nmol i.th.) administered after the formalin produced some inhibition of late-phase flinching, though significance was achieved only at the 10 nmol dose. Compound 4 (0.3 10 nmol i.th.) administered after the formalin produced significant inhibition of late-phase flinching, with significance achieved at the 3 and 10 nmol doses. This analgesic profile of activity of the arylalkylamines is similar to that seen with post-formalin administration of morphine (1 10 nmol); post-formalin administration oo of MK-801 (1 30 nmol), however, failed to affect late-phase flinching.

Taken together, the results obtained with the hot plate, tail flick and formalin assays demonstrate that arylalkylamines such as Compound 1 and Compound 4 have significant analgesic activity in several rodent models of acute pain. The formalin assay additionally demonstrates that arylalkylamines are effective in an "i animal model of chronic pain. Importantly, the arylalkylamines possess significant analgesic activity :when administered after the formalin stimulus. This profile of activity clearly distinguishes the arylalkylamines from standard NMDA receptor antagonists such as MK-801.

Side effects of arylalkylamines Given the important role NMDA receptors play in diverse brain functions, it is not surprising to find that antagonists of this receptor are typically associated with certain unwelcome side effects. In fact, it is this property that provides the major 99 obstacle to developing therapies that target NMDA receptors. The principal side effects, which characterize both competitive and noncompetitive antagonists, are a PCP-like psychotomimetic activity, impairment of motor performance, sedation or hyperexcitability, impairment of cognitive abilities, neuronal vacuolization, or cardiovascular effects (Willetts et al., The behavioral pharmacology of NMDA receptor antagonists. Trends Pharmacol. Sci. 11: 423, 1990; Olney et al., Pathological changes induced in cerebrocortical neurons by phencyclidine and related drugs. Science 244: 1360, 1989). The psychotomimetic effect associated with inhibition of NMDA receptor-mediated responses is epitomized in the response to phencyclidine (PCP) or "angel dust" which acts at the MK-801 binding site. Impairment of cognitive ability is associated with the important role that NMDA receptors normally play in learning and memory.

Relatively less is known concerning the side effect profile of AMPA receptor antagonists. However, it is becoming clear that such compounds also elicit motor impairment, ataxia and profound sedation.

The activity of arylalkylamines was examined in animal models that index motor impairment, sedation and psychotomimetic activity as well as both in vitro and in vivo models of learning and memory.

PCP-like Psychotomimetic Activity In rodents, both competitive and noncompetitive antagonists of the NMDA receptor produce 100 a PCP-like stereotypic behavior characterized by hyperactivity, head-weaving, and ataxia (Willetts et al., The behavioral pharmacology of NMDA receptor antagonists. Trends Pharmacol. Sci. 11: 423, 1990; Snell and Johnson, In: Excitatory Amino Acids in Health and Disease, John Wiley Sons, p. 261, 1988). We investigated whether the arylalkylamines would elicit such behaviors. In addition, we investigated whether the arylalkylamines would substitute for PCP in rats trained to discriminate PCP from saline (Willetts et The behavioral pharmacology of NMDA receptor antagonists. Trends Pharmacol. Sci. 11: 423, 1990), and Swhether the arylalkylamines would elicit a PCP-like neuronal vacuolization (Olney et al., Pathological changes induced in cerebrocortical neurons by phencyclidine and related drugs. Science 244: 1360, 1989).

Example 15: Locomotor activity The first assay simply monitors locomotor activity during the first hour following peripheral or administration of test substance. Mice received a dose of Compound 1 15 min before being placed into activity chambers. Activity was quantified by counting the number of breaks in a phototube grid in a 60 min period. In this assay, MK-801 (0.25 mg/kg p.o.) causes a 2- to 3-fold increase in locomotor activity.

However, Compound 1, even when tested at 32 mg/kg s.c., did not elicit hyperactivity and, in fact, tended to depress it. This result, using a purifiedarylalkylamine in mice, complements earlier results 101 obtained in rats where the entire arylalkylamine-containing fraction from Agelenopsis aperta, when injected intravenously, did not elicit a PCP-like behavioral syndrome but seemed to produce a mild sedative effect.

Example 16: Motor impairment In the first assay for generalized motor impairment, Compound 1 was examined in the inverted grid assay. In this assay, animals are placed on a wire-holed grid suspended from a rotating metal bar which can be inverted. The animals are then scored for their ability to climb to the top or hang on to the grid. Animals with severe motor impairment fall off the grid. This assay provides an index of "behavioral 15 disruption" that may result from ataxia, loss of the righting reflex, sedation, or muscle relaxation. In these tests, Compound 1, administered at 32 mg/kg s.c., did not lessen the ability of DBA/2 mice to right themselves when the grid was inverted (p 0.05) Compound 2 was likewise without effect (p 0.05) on motor performance in DBA/2 mice when administered at a dose of 20 mg/kg s.c. These doses are considerably higher than those required to prevent sound-induced seizures in DBA/2 mice (see Example 10 above).

The second assay of acute motor impairment was the rotorod assay. In this assay, Frings and CF1 mice were injected with test compound and placed on a knurled rod which rotated at a speed of 6 rpm. The ability of the mice to maintain equilibrium for long periods of time was determined; those mice that were unable to 102 maintain equilibrium on the rotorod for 1 min in each of 3 trials were considered impaired. Compound 1 produced acute motor impairment in Frings mice with a TD, 5 (that dose which produced motor toxicity in 50% of the test animals) of 16.8 mg/kg i.p. This dose is similar to that which prevents sound-induced seizures in Frings mice (see Example 10 above). There is a much clearer separation between effective and toxic doses of Compound 1 in Frings mice, however, when the Compound is administered i.c.v. In this case, no apparent motor toxicity was evident until the dose of Compound 1 Sexceeded 1.56 4g i.c.v. times the EDs 5 of 0.63 ig).

Finally, motor impairment in CF1 mice was noted with Compound 1 following i.c.v. administration of 4 /g.

Compound 4, Compound 9, Compound 12 and Compound 14 were administered to Frings mice by i.c.v.

injection, and acute motor impairment was measured. The

TD,

0 values for Compounds 9, 12 and 14 were 8-16 gg, 14.8 g, 30.2 4g and 30.8 zg, respectively. These TD, values were 2-3 times higher than the effective ICsO values for anticonvulsant potency (see Example above); a clear separation between effective and toxic doses was noted.

Example 17. PCP discrimination In this assay, rats who have been trained to lever press for food reinforcement must select which of two levers in their cages is correct. The only stimulus they have for selecting the correct lever is their ability to detect whether they received a PCP or vehicle injection. After about two months of training, rats 103 become very good at discriminating PCP from vehicle injections and can then be tested with other drugs to determine if they are discriminated as PCP. When tested in this procedure, other drugs which are known to produce a PCP-like intoxication substitute for PCP.

These drugs include various PCP analogs such as ketamine and the noncompetitive NMDA receptor antagonist, MK-801.

Compound 1 (1 30 mg/kg did not substitute for PCP, and thus was completely devoid of 10 PCP-like discriminative stimulus effects. At 30 mg/kg only 1 of the 7 animals tested responded at all on either lever. It is thus clear that a behaviorally effective dosage range of Compound 1 was evaluated. As the ability of test compounds to produce PCP-like effects in rats is believed to be predictive of their ability to produce PCP-like psychotomimetic activity and abuse liability in humans, these results strongly suggest that the arylalkylamines such as Compound 1 will lack such deleterious side effects in man.

20 Example 18 The administration of compounds such as PCP and MK-801 to rats produces a neurotoxic effect termed neuronal vacuolization. Following a single dose of such compounds, vacuoles are found in particular central neurons, especially those in the cingulate cortex and retrosplenial cortex. No such vacuolization was present in rats treated with Compound 1 at the single high dose of 100 mg/kg i.p.

Taken together, the results on locomotor activity, motor impairment, PCP discrimination and 104 neuronal vacuolization strongly suggest that arylalkylamines will be devoid of PCP-like side effects in man.

Cognitive impairment One of the major reasons for postulating a role of NMDA receptors in memory and learning derives from cellular studies on long-term potentiation (LTP) in the rat hippocampus. LTP is a long-lasting increase in the magnitude of synaptic responses produced by brief yet intense synaptic stimulation. Since the discovery of this phenomenon, it has become the preeminent cellular model of learning in the vertebrate brain (Teyler and Discenna, Long-term potentiation. Annu.

Rev. Neurosci. 10: 131, 1987). Transmission at synapses formed by Schaffer collaterals onto CA1 pyramidal cells is mediated by NMDA and AMPA receptors. Following a brief tetanizing stimulus, the magnitude of the population spike (a measure of synaptic transmission) is greatly increased and remains so for hours. It has been 20 shown that all known competitive and noncompetitive antagonists of NMDA receptors block LTP in the rat hippocampus, whereas antagonists of non-NMDA receptors are without effect (Collingridge and Davis, In: The NMDA Receptor, IRL Press. p. 123, 1989). This supports a role of NMDA receptors in memory and learning.

105 Example 19: LTP assay The effects of selected arylalkylamines and literature standards were examined for effects on LTP in slices of rat hippocampus. As anticipated, all the conventional competitive (AP5 and AP7) and noncompetitive (MK-801 and ifenprodil) NMDA receptor antagonists inhibited the induction of LTP in the hippocampus. Slices of rat hippocampus were superfused for 30-60 min with a test compound before delivering a tetanizing stimulus consisting of 3 trains, separated by 500 msec, of 100 Hz for 1 sec each. The response amplitude was monitored for an additional 15 minutes post-tetanus. The tetanizing stimulus caused a mean increase in the amplitude of the synaptic response. The induction of LTP was significantly blocked (p 0.05) by competitive (AP5, AP7) or noncompetitive (MK-801, ifenprodil) NMDA receptor antagonists. Quite surprisingly, none of the arylalkylamines tested (Compound 1, Compound 2, Compound 3 and others) blocked S 20 the induction of LTP (p 0.05), even when used at high concentrations (100-300 /2M) that caused some inhibition of the control response.

These results highlight yet another unique and important feature of arylalkylamines. Arylalkylaminesare the first, and at present the only, class of compounds shown to be selective and potent antagonists of the NMDA receptor that do not block the induction of LTP. This likely reflects the novel mechanism and site of action of arylalkylamines and suggests that drugs which target the novel site on the NMDA receptor will similarly lack effects on LTP. As LTP is the primary 106 cellular model for learning and memory in the mammalian CNS, it additionally suggests that such drugs will lack deleterious effects on cognitive performance.

Example 20: Learning tests Preliminary experiments using one of the more potent synthetic arylalkylamine analogs, Compound 3, in an in vivo learning paradigm demonstrate that these drugs lack effects on cognitive performance. In this test, rats were trained to alternate turning in aT maze for a food reward. MK-801 was included for comparison.

Test compounds were administered i.p. 15 min before testing. Control animals made the correct choice about of the time. Increasing doses of MK-801 progressively decreased the number of correct choices 15 and this decrement in behavior was accompanied by hyperactivity. In contrast, Compound 3 did not impair the ability of the animals to make the correct choices ooeoi (p 0.05) At the highest doses tested, Compound 3 caused some decrease in locomotor activity, exactly the 20 opposite effect observed with MK-801.

Although MK-801 decreased learning performance in parallel with increases in locomotor activity, other studies using different paradigms inrodents and primates have shown a clear dissociation between the effects on learning and locomotion. Thus, both competitive and noncompetitive NMDA receptor antagonists impair learning at doses which do not cause any overt change in motor behavior. This demonstrates that conventional NMDA receptor antagonists impair learning independently of other side effects. The results of the 107 T-maze assay demonstrate that Compound 3, and other arylalkylamines, do not impair learning even at doses that cause some decrease in locomotor activity.

One additional observation emerged from these learning tests. The animals' first response on the second day of testing was random and was therefore not dependent on the last response of the previous day's testing. Control animals thus correctly made the first choice about 50% of the time. MK-801 has no effect on this first choice. However, animals administered Compound 3 on the previous day made the first choice 0 correctly considerably more often. Unlike control r animals then, the animals treated with Compound 3 behaved as if they remembered the last choice of the previous day.

In a second series of experiments, the effect of Compound 4 on learning in the Morris water maze task was evaluated. In this test, a hidden platform was •placed in a fixed location in a circular steel tank, and 20 submerged 2 cm below the surface of the water. Each rat *see S: was given 3 trials per day with a 10 min intertrial interval for 5 days. A trial was initiated by placing the rat in the water, nose facing the wall of the tank, at one of three predetermined starting locations. The order of the start location was varied daily. Learning was measured as a decrease in time required to swim to the platform. If an animal failed to locate the platform within 60 sec after the start of the trial, the rat was hand-guided to it. The animals remained on the platform for 10 sec before being removed from the tank.

Ten min after the last training trial on day 5, the 108 animals received a probe test. The platform was removed for this 1 trial task and the animals were allowed to swim for 60 sec to assess the spatial bias for the platform location. Two measures were recorded from this task: latency to first crossing the area where the platform had been, and total number of crossings. A total of 5 injections of Compound 4 were given to each rat. In the first series of experiments, Compound 4 was administered at 10 mg/kg i.p. daily for 5 days. This treatment regimen impaired learning; however, these *e 906e animals experienced significant weight loss and unusual behavioral signs ("shivering," motor impairment, difficulty in swimming) with repeated dosing of Compound 4. In a subsequent study, six animals received 1 mg/kg i.p. for the first 4 days of training, while two animals received 5 mg/kg i.p. during this period. On the last day of training, both groups received 10 mg/kg. Neither the 1 mg/kg nor the 5 mg/kg animals showed any

F

impairment in learning the location of the hidden 20 platform, nor did the final 10 mg/kg dose produce any impairment in the ability of the animal to perform the already learned task.

The results of these learning tasks are encouraging. They suggest that arylalkylamines lack the learning and memory deficits that typify other NMDA receptor antagonists. In fact, there is a suggestion that the arylalkylamines may even be nootropic (memory enhancers).

109 Cardiovascular effects In vivo studies with certain arylalkylamines revealed a hypotensive effect of these compounds, especially at high doses. On the basis of these results, a systematic study of the effects of arylalkylamines on cardiovascular function was performed.

Example 21: Ca 2 channel inhibition We have discovered that some of the arylalkylamines are quite potent inhibitors of voltage-sensitive Ca 2 channels, specifically those sensitive to inhibition by dihydropyridines (L-type channels). Such effects on vascular smooth muscle would be expected to dilate blood vessels and cause a drop in 15 blood pressure, thus producing hypotension.

The ability of arylalkylamines to inhibit dihydropyridine-sensitive Ca 2 channels was examined in cerebellar granule cells and a rat aortic smooth muscle cell line, Ar5 cells. In cerebellar granule cells, 20 Compound 2 inhibited depolarization-induced increases in [Ca 2 i at concentrations 100-fold higher than those required to block responses to NMDA (ICo 0 values of 24 PM and 161 nM, respectively). Overall, we have observed a wide range of potencies against voltage-sensitive Ca 2 channels that does not correlate with potency against NMDA receptors. This strongly suggests that further structure-activity work based on chemical modification of the arylalkylamine molecule will lead to the development of compounds that are very potent NMDA antagonists with low potency against voltage-sensitive Ca 2 channels. Indeed, Compound 1 (with an ICs 0 of 102 nM 110 against NMDA receptor-mediated responses in cerebellar granule cells) is a relatively poor inhibitor of voltage-sensitive Ca 2 channels in cerebellar granule cells (IC 50 257 M) and is virtually without effect on voltage-sensitive Ca 2 influx in Ar5 cells (IC, 5 808

MM).

Arylalkylamines are not, however, indiscriminate blockers of voltage-sensitive Ca 2 channels. They do not, for example, affect voltage-sensitive Ca 2 channels in cerebellar Purkinje cells (P-type channels) or those channels thought to be involved in neurotransmitter release (N-channels). The "arylalkylamines that do block voltage-sensitive Ca 2 channels appear to target specifically L-type Ca 2 15 channels. Moreover, as mentioned above, there is a high degree of structural specificity in this effect. For example, one arylalkylamine is 57.times more potent than another arylalkylamine in blocking Ca 2 influx through L-type channels, where the only structural difference 20 between the compounds is the presence or absence of a hydroxyl group.

Example 22: In vivo cardiovascular studies The arylalkylamines Compound 1 and Compound 2 produce moderate drops (20-40 mm Hg) in mean arterial blood pressure (MABP) in anesthetized rats at doses which are effective in the in vivo stroke models (10-30 mg/kg The hypotensive effect of Compound 4 has been evaluated in greater detail. Compound 4 elicited a marked drop (40 mm Hg) in mean arterial pressure which persisted for approximately 90-120 min when administered 111 at the dose of 10 mg/kg it was in this same group of rats that Compound 4 afforded significant neuroprotection in the suture model of middle cerebral artery occlusion (see Example 8 above). Similar results were obtained in the rat study in which Compound 4 demonstrated neuroprotectant activity in the Rose Bengal photothrombotic model of focal ischemic stroke (see Example 8 above). Further studies using the pithed rat preparation strongly suggest that the hypotensive activity of Compound 4 is a peripherally mediated effect. The hypotension and bradycardia produced by Compound 4 was maintained in rats pretreated with atropine, suggesting that these effects are not mediated by a cholinergic mechanism. Similarly, Compound 4 15 elicited hypotension and bradycardia in chemically sympathectomized rats (pretreated with a ganglionic blocker), suggesting that these effects are not mediated via the sympathetic nervous system.

On the basis of these findings, it is 20 anticipated that chemical efforts will minimize the cardiovascular side effects by enhancing the uptake of arylalkylamine into the brain such that lower doses are required for neuroprotection, and increasing the selectivity (potency ratio) of arylalkylamines for receptor-operated Ca 2 channels over voltage-sensitive Ca 2 channels.

Example 23: Biological activity of Compound 19 and analogs Compounds 19 215 had high potencies against NMDA-induced increases in [Ca 2 ]i in rat cerebellar 112 granule cells grown in culture (Table The inhibitory effect of Compound 19 on responses to NMDA was noncompetitive. Compounds 19 215 inhibited 3 H]MK-801 binding in membranes prepared from rat hippocampal and cortical tissue (Table 1).

Compound 19 possessed the following additional biological activities: significant (p 0.05 compared to control) anticonvulsant activity against maximal electroshock-induced seizures in mice following i.p.

administration (ED 0 s 26.4 mg/kg and TDso (rotorod) 43.8 mg/kg); significant anticonvulsant activity against maximal electroshock-induced seizures in mice following oral administration (EDso 35 mg/kg), but with motor impairment at 30 mg/kg; significant analgesic 15 activity in the hot-plate and PBQ-induced writhing assays at 16 mg/kg no PCP-like stereotypic behavior (hyperexcitability and head weaving) at mg/kg i.p. in rats; no generalization to PCP in the PCP discrimination assay in rats at doses up to the 20 behaviorally active dose of 30 mg/kg i.p. Compound 19 was significantly less potent in antagonizing increases in [Ca2+]i elicited by depolarizing concentrations of KC1 in rat cerebellar granule cells (ICso 10.2 A4M), and was without effect on blood pressure when administered s.c.

in rats at doses up to 100 mg/kg. Compound 19, however, did block the induction of LTP in rat hippocampal slices when tested at 100 gM.

Compound 20 possessed the following additional biological activities: significant anticonvulsant activity against maximal electroshock-induced seizures in mice following i.p. administration (EDso 20.1 mg/kg 113 and TDs 5 (rotorod) 20.6 mg/kg); no significant anticonvulsant activity against maximal electroshock-induced seizures in mice following oral administration at doses up to 30 mg/kg, but with motor impairment at 30 mg/kg; significant anticonvulsant activity against sound-induced seizures in a genetically susceptible mouse model of reflex epilepsy (Frings mice) following i.p. (ED 5 o 2.1 mg/kg and TDo 0 19.9 mg/kg) and oral (ED 5 o 9.7 mg/kg and TD 50 21.8 mg/kg) administration; significant anticonvulsant activity against maximal electroshock-induced seizures in rats following oral administration with an ED 5 s value of 33.64 mg/kg and an TDso value of 55.87 mg/kg; an increase in seizure threshold as indexed by the i.v. Metrazol 15 test in mice at the dose of 10 mg/kg significant neuroprotectant activity in a rat model of temporary focal ischemia (a 51% reduction in the infarct volume following the administration of two doses of 1 mg/kg the first given immediately after middle cerebral 20 artery occlusion and the second given 6 hr later; a 43% reduction in the infarct volume following the administration of two doses of 1 mg/kg the first given 2 hr after middle cerebral artery occlusion at the time of reperfusion) and the second given 6 hr later); significant neuroprotectant activity (a 24% reduction in the infarct volume) in a rat model of permanent focal ischemia following the administration of 1 mg/kg i.p. at 30 min and again 4 hr post-occlusion; significant neuroprotectant activity (a 50% reduction in the infarct volume) in a rat photothrombotic model of focal ischemia following the administration of 10 mg/kg i.p. at 15 min, 3 hr, and again 6 hr post-occlusion; no significant analgesic activity at the dose of 25 mg/kg i.p. in the rat 52 0 C hot plate test or the rat 48 0 C tail flick test; significant analgesic activity, not blocked by the opiate receptor antagonist naloxone, in the rat formalin test at the dose of 10 mg/kg significant analgesic activity, not blocked by naloxone, against acetic acid-induced abdominal writhing in mice at the dose of 10 mg/kg no generalization to PCP in the PCP discrimination assay in rats at doses up to the behaviorally active dose of 10 mg/kg no neuronal vacuolization in rats when administered at doses of .and 30 mg/kg no significant cardiovascular activity in anesthetized rats at doses up to 15 15 Amoles/kg i.v. or 10 mg/kg no significant cardiovascular activity in conscious beagle dogs at doses of 0.3 or 1 mg/kg i.v. (60 sec bolus injection); transient increases in mean arterial pressure and heart rate in conscious beagle dogs at the dose of 3 mg/kg 20 with larger magnitude and longer duration effects seen at the dose of 10 mg/kg i.v. (60 sec bolus injection); increased motor activity, agitation and anxiousness, slight tremors, licking of the mouth, whining,and urination in conscious beagle dogs at the dose of 3 mg/kg i.v. (60 sec bolus injection); dilated pupils, whole body tremors, incoordination, licking of the mouth, salivation, panting, rapid blinking of the eyes, whining, anxiousness, seizures, and death in conscious beagle dogs at the dose of 10 mg/kg i.v. sec bolus injection); no behavioral effects in conscious male NMRI mice at the doses of 2 and 4 mg/kg i.p.; 115 excitation and increased reactivity to touch in conscious male NMRI mice at the dose of 8 mg/kg i.p.; excitation, Straub tail, tremor, stereotypies, hypothermia, and mydriasis in conscious male NMRI mice at the doses of 16 and 32 mg/kg convulsions and death in conscious male NMRI mice at the dose of 64 mg/kg convulsions and death in conscious male NMRI mice at the doses of 128 and 256 mg/kg i.p; no behavioral effects in conscious male Wistar rats at the dose of 2 mg/kg excitation, stereotypies, increased reactivity to touch, increased muscle tone, and tremor in conscious male Wistar rats at doses ranging from 4 to 16 mg/kg Straub tail, !convulsions, and death in conscious male Wistar rats at 15 the dose of 32 mg/kg i.v.

Compound 21 possessed the following additional biological activities: significant anticonvulsant activity against sound-induced seizures in a genetically susceptible mouse model of reflex epilepsy (Frings mice) 20 following i.p. administration (EDs, 3.41 mg/kg and TDs (motor impairment)= 15.3 mg/kg).

Compound 33 (an enantiomer of Compound 21) possessed the following additional biological activities: significant anticonvulsant activity against sound-induced seizures in a genetically susceptible mouse model of reflex epilepsy (Frings mice) following i.p. administration (ED, 5 4.6 mg/kg and TD 50 (motor impairment) 27.8 mg/kg); significant anticonvulsant activity against maximal electroshock-induced seizures in rats following oral administration at the dose of mg/kg, with no motor toxicity apparent at this dose; 116 significant neuroprotectant activity in a rat model of focal ischemic stroke following i.p. administration of 2 mg/kg 30 min prior -to vessel occlusion and 2 mg/kg 3 hr post-occlusion; no significant analgesic activity at the dose of 25 mg/kg i.p. in the rat 520C hot plate test or the rat 48 0 C tail flick test; significant analgesic activity in a rat model of chronic neuropathic pain following i.th. administration of doses ranging from 15 to 80 ug; significant analgesic activity in a rat model of chronic neuropathic pain following i.p.

administration of doses of 3-10 mg/kg; no neuronal vacuolization when administered to rats at the dose of mg/kg no significant cardiovascular activity in anesthetized rats at doses up to 3 mg/kg no 15 significant cardiovascular activity in conscious beagle dogs at the dose of 0.3 mg/kg i.v. (60 sec bolus injection); transient increases in mean arterial pressure in conscious beagle dogs at the dose of 1 mg/kg with larger magnitude and longer duration effects S 20 seen at the doses of 3 and 10 mg/kg i.v. (60 sec bolus injection); a transient increase in heart rate in conscious beagle dogs at the dose of 10 mg/kg i.v.

sec bolus injection); licking of the mouth in conscious beagle dogs at the dose of 3 mg/kg i.v.

(60 sec bolus injection); dilated pupils, whole body tremors, incoordination, licking of the mouth, salivation, and panting in conscious beagle dogs at the dose of 10 mg/kg.i.v. (60 sec bolus injection); no significant drug-induced changes in the ECG in conscious beagle dogs at doses up to 10 mg/kg i.v. (60 sec bolus injection); no behavioral effects in conscious male NMRI 117 mice at the doses of 2 and 4 mg/kg excitation, increased reactivity to touch, and hypothermia in conscious male NMRI mice at the dose of 8 mg/kg i.p.; excitation, Straub tail, tremor, jumping, stereotypies, hypothermia, and mydriasis in conscious male NMRI mice at the doses of 16 and 32 mg/kg convulsions in conscious male NMRI mice at the dose of 64 mg/kg i.p.; convulsions and death in conscious male NMRI mice at the doses of 128 and 256 mg/kg i.p.

Compound 34 (an enantiomer of Compound 21) possessed the following additional biological activities: significant anticonvulsant activity against sound-induced seizures in a genetically susceptible S: mouse model of reflex epilepsy (Frings mice) following i.p. administration (EDso 22 mg/kg and TD,, (motor impairment) between 10 and 15 mg/kg); hyperthermia in conscious male NMRI mice at the dose of 2 mg/kg no behavioral effects in conscious male NMRI mice at the dose of 4 mg/kg excitation, increased reactivity to touch, and hypothermia in conscious male NMRI mice at the dose of 8 mg/kg excitation, Straub tail, tremor, jumping, stereotypies, hypothermia, and mydriasis in conscious male NMRI mice at the doses of 16 and 32 mg/kg convulsions in conscious male NMRI mice at the dose of 64 mg/kg convulsions and death in conscious male NMRI mice at the doses of 128 and 256 mg/kg i.p.

Compound 22 possessed the following additional biological activities: significant anticonvulsant activity against sound-induced seizures in a genetically susceptible mouse model of reflex epilepsy (Frings mice) 118 following i.p. (EDso 4.9 mg/kg and TDs 5 (inverted grid) 26.8 mg/kg) and oral (EDso 5.1 mg/kg and LD, 5 18.3 mg/kg) administration; and no significant cardiovascular activity in anesthetized rats at doses up to 15 gmoles/kg (4.47 mg/kg) i.v.

Compound 50 (an enantiomer of Compound 22) possessed the following additional biological activities: significant anticonvulsant activity against sound-induced seizures in a genetically susceptible mouse model of reflex epilepsy (Frings mice) following i.p. administration (EDso 2.7 mg/kg and TDsO (motor impairment) 17.4 mg/kg); significant anticonvulsant o* activity against sound-induced seizures in a genetically susceptible mouse model of reflex epilepsy (Frings mice) 15 following p.o. administration (EDso 9.0 mg/kg and TDs (motor impairment) 18.9 mg/kg); significant anticonvulsant activity against maximal electroshock-induced seizures in rats following oral administration with EDo 0 28 mg/kg and TDso 20 mg/kg; significant neuroprotectant activity in a rat model of focal ischemic stroke following i.p. administration of :2 mg/kg 30 min prior to vessel occlusion and 2 mg/kg 3 hr post-occlusion; no significant analgesic activity at the dose of 25 mg/kg i.p. in the rat 520C hot plate test or the rat 480C tail flick test; and no significant cardiovascular activity in anesthetized rats at doses up to 5 mg/kg i.v.

Compound 51 (an enantiomer of Compound 22) possessed the following additional biological activities: significant anticonvulsant activity against sound-induced seizures in a genetically susceptible 119 mouse model of reflex epilepsy (Frings mice) following i.p. administration (ED 5 9.1 mg/kg and TD 50 (motor impairment) 13.6 mg/kg).

Compound 24 possessed the following additional biological activities: significant anticonvulsant activity against sound-induced seizures in a genetically susceptible mouse model of reflex epilepsy (Frings mice) following i.p. administration (EDso 5 mg/kg and TDs, (motor impairment) 16 mg/kg); significant anticonvulsant activity against maximal electroshock-induced seizures in rats following oral administration with EDsO 46 mg/kg and TD 50 51 mg/kg; no significant neuroprotectant activity in a rat model of focal ischemic stroke following i.p. administration of 2 mg/kg 30 min prior to vessel occlusion and 2 mg/kg 3 hr post-occlusion; and no significant cardiovascular activity in anesthetized rats at doses up to 10 mg/kg i.v.

0Compound 25 possessed the following additional biological activities: significant anticonvulsant activity against maximal electroshock-induced seizures in mice following i.p. administration with an ED, 12.47 mg/kg and a TDs 5 32.18 mg/kg; significant anticonvulsant activity against maximal electroshock-induced seizures in rats following oral administration with an ED 5 46.43 mg/kg and a TDs between 163 and 326 mg/kg.

Compound 31 possessed the following additional biological activities: significant anticonvulsant activity against sound-induced seizures in a genetically susceptible mouse model of reflex epilepsy (Frings mice) 120 following i.p. administration (EDso 6 mg/kg and TDsO (motor impairment) between 10 and 20 mg/kg).

Compound 46 possessed the following additional biological activities: significant anticonvulsant activity against sound-induced seizures in a genetically susceptible mouse model of reflex epilepsy (Frings mice) following i.p. administration (ED50 25 mg/kg and TDs, (motor impairment) between 18 and 21 mg/kg); and no significant neuroprotectant activity in a rat model of focal ischemic stroke following i.p. administration of 2 mg/kg 30 min prior to vessel occlusion and 2 mg/kg 3 hr post-occlusion.

Compound 57 possessed the following additional biological activities: significant anticonvulsant 15 activity against sound-induced -seizures in a genetically susceptible mouse model of reflex epilepsy (Frings mice) following i.p. administration (ED50 1 mg/kg and TD 50 (motor impairment) between-6 and 8 mg/kg).

Compound 58 possessed the following additional biological activities: significant anticonvulsant activity against sound-induced seizures in a genetically susceptible mouse model of reflex epilepsy (Frings mice) following i.p. administration (EDs 0.9 mg/kg and TD, 0 (motor impairment) 14.5 mg/kg); no significant neuroprotectant activity in a rat model of focal ischemic stroke following i.p. administration of 2 mg/kg min prior to vessel occlusion and 2 mg/kg 3 hr post-occlusion; and no significant cardiovascular activity in anesthetized rats at doses up to 2 mg/kg i.v.

121 Compound 59 possessed the following additional biological activities: significant anticonvulsant activity against sound-induced seizures in a genetically susceptible mouse model of reflex epilepsy (Frings mice) following i.p. administration (EDsO 2.7 mg/kg and TD, 0 (motor impairment) 7.8 mg/kg); a reduction in seizure threshold as indexed by the i.v. Metrazol test in mice at the dose of 11.7 mg/kg no significant neuroprotectant activity in a rat model of focal ischemic stroke following i.p. administration of 2 mg/kg min prior to vessel occlusion and 2 mg/kg 3 hr post-occlusion; and no significant cardiovascular activity in anesthetized rats at doses up to 10 mg/kg 'i.v.

i 15 Compound 60 possessed-the following additional biological activities: significant anticonvulsant activity against sound-induced seizures in a genetically susceptible mouse model of reflex epilepsy (Frings mice) following i.p. administration (EDs 5 4.4 mg/kg and TDs, 20 (motor impairment) 9.2 mg/kg); significant anticonvulsant activity against sound-induced seizures in a genetically susceptible mouse model of reflex epilepsy (Frings mice) following oral administration (ED,0 10 mg/kg and TD, 0 (motor impairment) 25.6 mg/kg); significant anticonvulsant activity against maximal electroshock-induced seizures in mice following i.p. administration (EDs, 8.17 mg/kg and TDs 5 (rotorod) 17.30 mg/kg); no effect on seizure threshold as indexed by the i.v. Metrazol test in mice at the doses of 1 and 4 mg/kg a reduction in seizure threshold as indexed by the i.v. Metrazol test in mice at the 122 doses of 8 and 17 mg/kg significant neuroprotectant activity in a rat model of temporary focal ischemic stroke following i.p. administration of 2 mg/kg 30 min prior to vessel occlusion and 2 mg/kg 3 hr post-occlusion; significant neuroprotectant activity in a rat model of temporary focal ischemic stroke following i.p. or i.v. administration of 1 mg/kg 2 hr and again 8 hr post-occlusion; significant neuroprotectant activity in a rat model of temporary focal ischemic stroke following i.v. administration of 1 mg/kg 2 hr post-occlusion; no significant neuroprotectant activity in a rat photothrombotic model of focal ischemia following the administration of 10 mg/kg i.p. at 15 min, 3 hr, and again 6 hr 15 post-occlusion; no neuronal vacuolization when administered at doses of 20 mg/kg i.p. or 10 mg/kg i.v.; no significant cardiovascular activity in conscious beagle dogs at the dose of 0.3 mg/kg i.v. (60 sec bolus injection); transient increases in mean arterial i 20 pressure in conscious beagle dogs at the doses of 1 and 3 mg/kg with larger magnitude and longer duration effects seen at the dose of 10 mg/kg i.v. (60 sec bolus injection); transient increases in heart rate in conscious beagle dogs at the doses of 3 and 10 mg/kg i.v. (60 sec bolus injection); no significant changes in the ECG in conscious beagle dogs at doses ranging from 0.3 to 10 mg/kg i.v. (60 sec bolus injection); no significant behavioral effects in conscious beagle dogs at the doses of 0.3 and 1 mg/kg i.v. (60 sec bolus injection); a slight increase in respiratory rate in conscious beagle dogs at the dose of 3 mg/kg i.v.

123 sec bolus injection); dilated pupils, whole body tremors, salivation, and urination in conscious beagle dogs at the dose of 10 mg/kg i.v. (60 sec bolus injection); no significant behavioral effects in conscious male Wistar rats at doses up to 4 mg/kg i.v.; excitation, stereotypies, increased reactivity to touch, increased muscle tone, and tremor in conscious male Wistar rats at the dose of 8 mg/kg Straub tail, convulsions, and death in conscious male Wistar rats at the dose of 16 mg/kg i.v.

Compound 119 possessed the following additional biological activities: significant S.anticonvulsant activity against sound-induced seizures in a genetically susceptible mouse model of reflex 15 epilepsy (Frings mice) following i.p. administration with an EDso 7.0 mg/kg and TDso (motor impairment) 26.3 mg/kg.

Compound 120 possessed the following additional biological activities: significant 20 anticonvulsant activity against sound-induced seizures in a genetically susceptible'mouse model of reflex epilepsy (Frings mice) following i.p. administration with an ED 50 4.77 mg/kg and TD 50 (motor impairment) between 20 and 30 mg/kg.

Compound 122 possessed the following additional biological activities: significant anticonvulsant activity against sound-induced seizures in a genetically susceptible mouse model of reflect epilepsy (Frings mice) following i.p. administration with an ED 50 4.7 mg/kg and TD 50 (motor impairment) 15.3 mg/kg.

124 S S

S

Compound 138 possessed the following additional biological activities: significant anticonvulsant activity against maximal electroshockinduced seizures in mice following i.p. administration with an ED 50 51.9 mg/kg and TD 50 (motor impairment) 100.7 mg/kg.

Compound 151 possessed the following additional biological activities: significant anticonvulsant activity against maximal electroshockinduced seizures in mice following i.p. administration with an ED 50 36.5 mg/kg and TD 50 (motor impairment) 108.4 mg/kg; a significant increase in seizure threshold as indexed by the i.v. Metrazol test in mice at the doses of 36.5 and 108 mg/kg i.p.

15 Compound 156 possessed the following additional biological activites: significant anticonvulsant activity against sound-induced seizures in a genetically susceptible mouse model of reflect epilepsy (Frings mice) following i.p. administration with an ED 50 5.0 mg/kg and TD 50 (motor impairment) 17.4 mg/kg.

Taken together, the results obtained with these simplified synthetic arylalkylamines suggest that such simplified molecules do not interact specifically with the arylalkylamine binding site on receptor-operated Ca 2 channels as do Compounds 1, 2 and 3. Specifically, Compounds 19 215 bind to the site labeled by 3 H]MK-801 at concentrations ranging approximately 1 to 400-fold higher than those which antagonize the function of the NMDA receptor-ionophore complex. The fact that Compounds 19 215 at S. 1 ae 5* 125 therapeutic doses do not generally produce PCP-like stereotypic behavior, substitute for PCP in drug discrimination assays, or elicit neuronal vacuolization suggests; however, that such compounds might be useful either as lead compounds or drug candidates for neurological disorders and diseases. It has been reported that compounds which bind with low affinity (relative to MK-801) to the site labeled by 1[H]MK-801 might possess therapeutic utility and possess a more favorable side effect profile than that possessed by a high affinity antagonist such as MK-801 itself (Rogawski, Therapeutic potential of excitatory amino see*: acid antagonists: channel blockers and 2,3-benzodiazepines. Trends Pharmacol. Sci. 14: 325, 15 1993). The low affinity of certain compounds within the group of Compounds 19 215 (relative to MK-801) for the site labeled by 3 H]MK-801 may place these compounds into *e this general class of low affinity noncompetitive antagonists.

C

0CC

CCCC

C.

C C 20 Identification of a novel modulatory site on receptor-operated calcium channels Having identified arylalkylamines which have therapeutically useful properties as defined above, compounds can now be identified which act at the critical arylalkylamine binding site on receptor-operated Ca 2 channels, such as those present within NMDA, AMPA and nicotinic cholinergic receptor-ionophore complexes.

Examples of suitable tests now follow: 126 0000 Example 24: Radioligand binding in rat cortex or cerebellum.

The following assay can be utilized as a high throughput assay to screen product libraries natural product libraries and compound files at major pharmaceutical companies) to identify new classes of compounds with activity at this unique arylalkylamine site. These new classes of compounds are then utilized as chemical lead structures for a drug development program targeting the arylalkylamine binding site on receptor-operated Ca 2 channels. The compounds identified by this assay offer a novel therapeutic approach to treatment of neurological disorders or diseases. Examples of such compounds include those 15 provided in the generic chemical formulae above.

Routine experiments can be performed to identify those compounds having the desired activities.

Rat brain membranes are prepared according to the method of Williams et al. (Effects of polyamines on the binding of 3 H]MK-801 to the NMDA receptor: Pharmacological evidence for the existence of a polyamine recognition site. Molec. Pharmacol. 36: 575, 1989) with the following alterations: Male Sprague-Dawley rats (Harlan Laboratories) weighing 100-200 g are sacrificed by decapitation. The cortex or cerebellum from 20 rats are cleaned and dissected. The resulting brain tissue is homogenized at 40C with a polytron homogenizer at the lowest setting in 300 ml 0.32 M sucrose containing 5 mM K-EDTA (pH The homogenate is centrifuged for 10 min at 1,000 x g and the supernatant removed and centrifuged at 30,000 x g 127 for 30 minutes. The resulting pellet is resuspended in 250 ml 5 mM K-EDTA (pH 7.0) stirred on ice for 15 min, and then centrifuged at 30,000 x g for 30 minutes. The pellet is resuspended in 300 ml 5 mM K-EDTA (pH 7.0) and incubated at 320C for 30 min. The suspension is then centrifuged at 100,000 x g for 30 min. Membranes are washed by resuspension in 500 ml 5 mM K-EDTA (pH incubated at 32 0 C for 30 min, and centrifuged at 100,000 x g for 30 minutes. The wash procedure, including the 30 min incubation, is repeated. The final pellet is resuspended in 60 ml 5 mM K-EDTA (pH 7.0) and stored in aliquots at -800C. The extensive washing procedure utilized in this assay was designed in an effort to minimize the concentrations of glutamate and glycine (co-agonists at the NMDA-receptor-ionophore complex) present in the membrane preparation.

To perform a binding assay with 3 H]arylalkylamine, aliquots of SPMs (Synaptic Plasma Membranes) are thawed, resuspended in 30 mls of 30 mM EPPS/lmM K-EDTA, pH 7.0, and centrifuged at 100,000 x g for 30 minutes. SPMs are resuspended in buffer A (30 mM EPPS/1 mM K-EDTA, pH The 3 H]arylalkylamine is added to this reaction mixture. Binding assays are carried out in polypropylene test tubes. The final incubation volume is 500 il. Nonspecific binding is determined in the presence of 100 MM nonradioactive arylalkylamine. Duplicate samples are incubated at 0 C for 1 hour. Assays are terminated by the addition of 3 ml of ice-cold buffer A, followed by filtration over glass-fiber filters (Schleicher Schuell No. 30) that are presoaked in 0.33% polyethyleneimine (PEI). The 128 filters are washed with another 3 x 3 ml of buffer A, and radioactivity is determined by scintillation counting at an efficiency of 35-40% for 3

H.

In order to validate the above assay, the following experiments are also performed: The amount of nonspecific binding of the 3 H]arylalkylamine to the filters is determined by passing 500 1l of buffer A containing various concentrations of [H]arylalkylamine through the presoaked glass-fiber filters. The filters are washed with another 4 x 3 ml of buffer A, and radioactivity bound to the filters is determined by scintillation counting at an efficiency of 35-40% for 3H. In filters that are not pretreated with 0.33% PEI, it was found that 87% of the 3 H-ligand was bound to the filter.

Presoaking with 0.33% PEI reduces the nonspecific binding to 0.5 1.0% of the total.ligand added.

A saturation curve is constructed by resuspending SPMs in buffer A. The assay buffer (500 41) contains 60 Ag of protein. Concentrations of 3 H]arylalkylamine are used, ranging from 1.0 nM to 400 4M in half-log units. A saturation curve is constructed from the data, and an apparent KD value and Bax value determined by Scatchard analysis (Scatchard, The attractions of proteins for small molecules and ions. Ann. N.Y. Acad. Sci. 51: 660, 1949). The cooperativity of binding of the [3H]arylalkylamine is determined by the construction of a Hill plot (Hill, A new mathematical treatment of changes of ionic concentrations in muscle and nerve under the action of 129 electric currents, with a theory to their mode of excitation. J. Physiol. 40: 190, 1910).

The dependence of binding on protein (receptor) concentration is determined by resuspending SPMs in buffer A. The assay buffer (500 pl) contains a concentration of 3 H]arylalkylamine equal to its KD value and increasing concentrations of protein. The specific binding of 3 H]arylalkylamine should be linearly related to the amount of protein (receptor) present.

The time course of ligand-receptor binding is determined by resuspending SPMs in buffer A.

The assay buffer (500 l) contains a concentration of 3 H]arylalkylamine equal to its Ko value and 100 jg of protein. Duplicate samples are incubated at 0°C for varying lengths of time; the time at which equilibrium is reached is determined, and this time point is routinely used in all subsequent assays.

•o The pharmacology of the binding site can be analyzed by competition experiments. In such 20 experiments, the concentration of 3 H]arylalkylamine and the amount of protein are kept constant, while the concentration of test (competing) drug is varied. This assay allows for the determination of an ICsO and an apparent K, for the competing drug (Cheng and Prusoff, Relationship between the inhibition constant (K i and the concentration of inhibitor which causes 50 percent inhibition (IC 0 of an enzymatic reaction. J. Biochem.

Pharmacol. 22: 3099, 1973). The cooperativity of binding of the competing drug is determined by Hill plot analysis.

130 Specific binding of the 3 H]arylalkylamine represents binding to a novel site on receptor-operated Ca 2 channels such as those present'within NMDA-, AMPAand nicotinic cholinergic receptor-ionophore complexes.

As such, other arylalkylamines should compete with the binding of [3H]arylalkylamine in a competitive fashion, and their potencies in this assay should correlate with their inhibitory potencies in a functional assay of receptor-operated Ca 2 channel antagonism inhibition of NMDA receptor-induced increases in [Ca 2 ']i in cultures of rat cerebellar granule cells).

Conversely, compounds which have activity at the other known sites on receptor-operated Ca 2 channels MK-801, Mg 2 polyamines) should not displace 15 [3H]arylalkylamine binding in a competitive manner.

Rather, complex allosteric modulation of 3 PH]arylalkylamine binding, indicative of noncompetitive ooo" interactions, might be expected to occur. In preliminary experiments, MK-801 did not displace 20 3 H]arylalykylamine binding at concentrations up to 100 AM.

Studies to estimate the dissociation kinetics are performed by measuring the binding of ['H]arylalkylamine after it is allowed to come to equilibrium (see above), and a large excess of nonradioactive competing drug is added to the reaction mixture. Binding of the 3 H]arylalkylamine is then assayed at various time intervals. With this assay, the association and dissociation rates of binding of the PH]arylalkylamine are determined (Titeler, Multiple Dopamine Receptors: Receptor Binding Studies in Dopamine Pharmacology. Marcel Dekker, Inc., New York, 1983). Additional experiments involve varying the reaction temperature (0°C to 37 0 C) in order to understand the temperature dependence of this parameter.

Example 25: Radioligand binding in cerebellar granule cells Primary cultures of cerebellar granule neurons are obtained from 8-day-old rats and plated onto squares of Aclar plastic coated with poly-L-lysine. The plastic squares are placed in 24-well culture plates, and approximately 7.5 X 10 s granule cells are added to each well. Cultures are maintained in Eagles' medium (HyClone Laboratories) containing 25 mM KC1, 10% fetal calf serum (HyClone Laboratories), 2 mM glutamine, 100 /g/ml gentamicin, 50 U/ml penicillin, and 50 kg/ml streptomycin at 37 0 C in a humid atmosphere of 5% CO 2 in air for 24 hr before the addition of cytosine arabinoside (10 AM, final). No changes of culture medium are made until the cells are used for receptor binding studies 6-8 days after plating.

To perform a binding assay with S" [PH]arylalkylamine, the reaction mixture consists of 200 4l of buffer A (20 mM K-HEPES, 1 mM K-EDTA, pH in each well of the 24-well plate. The P[H]arylalkylamine is added to this reaction mixture.

Nonspecific binding is determined in the presence of 100 AM nonradioactive arylalkylamine. Triplicate samples are incubated at 0 C for 1 hour. Assays are terminated by manually scraping the cells off the Aclar squares and placing them into polypropylene test tubes.

The membranes prepared from whole cells in this manner are suspended in 10 ml of ice-cold buffer A, and filtered over glass-fiber filters (Schleicher Schuell No. 30) that are presoaked in 0.33% PEI. The filters are washed with another 3 x 3 ml of buffer A, and radioactivity on the filters is determined by scintillation counting at an efficiency of 35-40% for 3

H.

The assay may be terminated by centrifugation rather than filtration in order to minimize nonspecific binding.

Specific experiments to characterize and validate the assay are performed essentially as above, except that cells are used in place of membranes for the initial binding. The binding assay allows for the 15 determination of an IC 5 s value and an apparent KD for the competing drug as described by Scatchard analysis (The attractions of proteins for small molecules and ions.

Ann. N.Y. Acad. Sci. 51: 660, 1949). Cooperativity of binding of the competing drug is determined by Hill plot 20 analysis (A new mathematical treatment of changes of ionic concentrations in muscle and nerve under the action of electric currents, with a theory to their mode of excitation. J. Physiol. 40: 190, 1910). The specific binding of the [1H]arylalkylamine represents binding to a novel site on receptor-operated calcium channels.

Example 26: Recombinant receptor binding assay The following is one example of a rapid screening assay for useful compounds of this invention.

In this assay, a cDNA or gene clone encoding the arylalkylamine binding site (receptor) from a suitable organism such as a human is obtained using standard procedures. Distinct fragments of the clone are expressed in an appropriate expression vector to produce the smallest polypeptide(s) obtainable from the receptor which retain the ability to bind Compound 1, Compound 2 or Compound 3. In this way, the polypeptide(s) which includes the novel arylalkylamine receptor for these compounds can be identified. Such experiments can be facilitated by utilizing a stably transfected mammalian cell line HEK 293 cells) expressing the i: arylalkylamine receptor.

Alternatively, the arylalkylamine receptor can be chemically reacted with chemically modified .*o Compound 1, Compound 2 or Compound 3 in such a way that amino acid residues of the arylalkylamine receptor which contact (or are adjacent to) the selected compound are modified and thereby identifiable. The fragment(s) of the arylalkylamine receptor containing those amino acids which are determined to interact with Compound 1, Compound 2 or Compound 3 and are sufficient for binding to said molecules, can then be recombinantly expressed, as described above, using a standard expression vector(s).

The recombinant polypeptide(s) having the desired binding properties can be bound to a solid phase support using standard chemical procedures. This solid phase, or affinity matrix, may then be contacted with Compound 1, Compound 2 or Compound 3 to demonstrate that those compounds can bind to the column, and to identify conditions by which the compounds may be removed from 134 the solid phase. This procedure may then be repeated using a large library of compounds to determine those compounds which are able to bind to the affinity matrix, and then can be released in a manner similar to Compound 1, Compound 2 or Compound 3. However, alternative binding and release conditions may be utilized in order to obtain compounds capable of binding under conditions distinct from those used for arylalkylamine binding conditions which better mimic physiological conditions encountered especially in pathological states). Those compounds which do bind can thus be selected from a very large collection of compounds present in a liquid medium or extract.

Once compounds able to bind to the arylalkylamine binding polypeptide(s) described above are identified, those compounds can then be readily tested in the various assays described above to determine whether they, or-simple derivatives thereof, are useful compounds for therapeutic treatment of neurological disorders and diseases described above.

In an alternate method, native arylalkylamine receptor can be bound to a column or other solid phase support. Those compounds which are not competed off by reagents which bind other sites on the receptor can then be identified. Such compounds define novel binding sites on the receptor. Compounds which are competed off by other known compounds thus bind to known sites, or bind to novel siteswhich overlap known binding sites.

Regardless, such compounds may be structurally distinct from known compounds and thus may define novel chemical classes of agonists or antagonist which may be useful as 135 therapeutics. In summary, a competition assay can be used to identify useful compounds of this invention.

Example 27: Patch-clamp electrophysiology assay The following assay is performed for selected compounds identified in the above-mentioned radioligand binding assays as interacting in a highly potent and competitive fashion at the novel arylalkylamine binding site on receptor-operated Ca 2 channels, such as those present in NMDA-, AMPA- or nicotinic cholinergic 10 receptor-ionophore complexes. This patch-clamp assay provides additional relevant data about the site and mechanism of action of said previously selected compounds. Specifically, the following pharmacological and physiological properties of the compounds 15 interacting at the arylalkylamine binding site are determined, utilizing the NMDA receptor-ionophore complex as an example of receptor-operated Ca 2 channels: potency and efficacy at blocking NMDA receptor-mediated ionic currents, the noncompetitive nature of block with 20 respect to glutamate and glycine, use-dependence of action, voltage-dependence of action, both with respect to onset and reversal of blocking, the kinetics of blocking and unblocking (reversal), and open-channel mechanism of blocking. Such data confirm that the compounds interacting at the arylalkylamine binding site retain the unique biological profile of the arylalkylamines, and do not have their primary activity at the known sites on the NMDA receptor-ionophore complex (glutamate binding site, glycine binding site; 136 MK-801 binding site, Mg 2 binding site, Zn 2 binding site, sigma binding site, polyamine binding site).

Patch-clamp recordings of mammalian neurons (hippocampal, cortical, cerebellar granule cells) are carried out utilizing standard procedures (Donevan et al., Arcaine blocks N-methyl-D-aspartate receptor responses by an open channel mechanism: whole-cell and single-channel recording studies in cultured hippocampal neurons. Molec. Pharmacol. 41: 727, 1992; Rock and Macdonald, Spermine and related polyamines produce a voltage-dependent reduction of NMDA receptor *l single-channel conductance. Molec. Pharmacol. 42: 157, 1992).

Alternatively, patch-clamp experiments can be performed on Xenopus oocytes or on a stably transfected mammalian cell line HEK 293 cells) expressing specific subunits of receptor-operated Ca 2 channels. In this manner, for example, potency and efficacy at various glutamate receptor subtypes NMDAR1, NMDAR2A through NMDAR2D, GluRI through GluR4) can be determined. Further information regarding the site of action of the arylalkylamines on these glutamate receptor subtypes can be obtained by using site-directed mutagenesis.

Example 28: Synthesis of arylalkylamines Arylalkylamines such as Compound 1, Compound 2 and Compound 3 are synthesized by standard procedures (Jasys et al., The total synthesis of argiotoxins 636, 659 and 673. Tetrahedron Lett. 29: 6223, 1988; Nason et al., Synthesis of neurotoxic Nephila spider venoms: 137 NSTX-3 and JSTX-3. Tetrahedron Lett. 30: 2337, 1989).

Specific examples of syntheses of arylalkylamine analogs 4-18 are provided in co-pending application U.S. Serial No. 08/485,038, filed June 7, 1995, and co-pending International Patent Application No. PCT/US94/12293, published as W095/21612,filed October 26, 1994, hereby incorporated by reference herein in their entirety.

Example 29: Synthesis of simplified arylalkylamines Synthesis of Compound 20 was accomplished as follows.

A solution of sodium hydride (1.21 g, 50 mmol) in dimethoxyethane was treated with diethyl cyanomethylphosphonate (8.86 g, 50 mmol) and the reaction stirred 4 hr at room temperature. To this was added 3,3'-difluorobenzophenone (10 g, 46 mmol) in DME. The reaction was stirred 24 hr at room temperature, quenched with H0, and partitioned between diethyl ether and water. The ether fraction was dried over Na 2

SO

4 and concentrated. GC/MS of this material showed 90% of the product A and 10% starting benzophenone.

A solution of this material in ethanol with a catalytic amount of Pd(OH) 2 was hydrogenated at 55 psi hydrogen for 4 hr at room temperature. The reaction was filtered and the catalyst washed with ethanol The filtrate and ethanol washes were combined and concentrated. GC/MS of this material showed 90% of the product B and 10% of the starting benzophenone.

A solution of this material in THF was treated with 70 ml 1 M B 2 H6 (70 mmol) in THF and refluxed 1 hr.

After cooling the reaction was treated with 6 N HC1 138 ml) and refluxed an additional hour. After cooling the reaction was basified to pH 14 with 10 N NaOH and equilibrated with ether. The ether layer was removed and washed with 10% HC1 The acidic washes were combined, basified to pH 14 with 10 N NaOH and extracted with dichloromethane The organic washes were combined, dried over Na 2

SO

4 and concentrated to yield an oil. GC/MS of this material showed 100% Compound GC/EI-MS (Re =7.11 min) m/z (relative intensity) 247 (M, 31), 230 (100) 215 (30) 201 (52) 183 134 (23), 121 101 95 77 This material in diethyl ether was filtered and treated with 35 ml 1 M HC1 in ether. The precipitate was collected, dried, and recrystallized from water-ethanol to afford 1.045 g of Compound 20, as the hydrochloride salt. !H-NMR (CDC13) d 8.28 (3H, br 7.28-7.17 (2 H, 7.02-6.86 (6 H, m), 4.11 (1H, t, J=8 Hz), 2.89 (2H, br t, J=8 Hz), 2.48 (2H, br t, J=7 Hz); 1 3 C-NMR (CDC13) d 164.6, 161.3, 144.8, 144.7, 130.4, 130.3, 123.3, 123.2, 114.7, 114.5, 114.1, 113.8, 47.4, 38.4, 32.7.

139 OH Oil F 0 Li* (H 2 CCN) F CN H 2 Ni-Al F A B HC A F- NH 3 CI H2/caIalyst F NHICI C Compound (hydrochloride salt) Synthesis of Compound 21, Compound 33 and Compound 34 was accomplished as follows.

A 100 ml round-bottomed flask equipped with 5 stir bar, septa, and argon source was charged with Compound 1 (2.43 g, 10 mmol) in 30 ml THF. The solution was cooled to -78 0 C and treated dropwise with 11 ml lithium bis(trimethylsilyl)amide (1M in THF) (11 mmol). The reaction was stirred at -78 0 C for 30 min 10 and treated dropwise with excess iodomethane (3.1 ml, mmol). The reaction was stirred 30 min at -58 0

C.

GC/EI-MS analysis of an aliquot from the reaction showed consumption of the starting nitrile 1. The reaction was quenched with water, diluted with diethyl ether and transferred to a separatory funnel. The ether layer was washed with 10% HC1 brine dried with anhydrous MgSO 4 and concentrated to a brown oil. This material was distilled (Kugelrohr, 100 0 C) at reduced pressure to afford 1.5 g of a clear oil. GC/EI-MS of this material showed it to contain the desired product 2, (R,=7.35 min). m/z (rel. int.) 257 3), 203 (100), 183 170 133 109 'H-NMR 140 (CDC13) d 7.4-6.9 (8H, 4.01 (1H, d, J=10 Hz), 3.38 (1H, dq, J=7, 10 Hz), 1.32 (3H, d, J=7 Hz); 13

C-NMR

(CDC1 3 d 19.4, 30.5, 54.2, 114.5, 114.6, 114.7, 114.9, 115.0, 115.3, 123.3, 123.4, 123.6, 123.7, 130.5, 130.6, 131.7.

Product 3 was synthesized by the catalytic reduction of 2 using Raney nickel in 95:5 EtOH:aqueous sodium hydroxide (2 Eq.) under 60 psi hydrogen.

GC/EI-MS (R,=7.25 min) m/z (rel. int.) 261 20), 244 S 10 229 215 201 183 (100), 133 (42), 115 109 95 'H-NMR (CDC1 3 d 7.3-6.8 I* (8H, 3.62 (1H, d, J=10 Hz), 2.70 (1H, 2.40 (2H, 1.73 (2H, 0.91 (3H, d, J=7 Hz). Note that product 3 in this reaction sequence corresponds to 15 Compound 21.

Product 2 in 10% IPA-hexane (100 mg/ml) was chromatographed, in 500 /l aliquots, through Chiral Cel OD (2.0 x 25 cm) using 10% IPA-hexane at 10 ml/min measuring optical density at 254 nm. This afforded the 20 two optically pure enantiomers 4 and 5 (as determined by analytical chiral HPLC; Note, the stereochemistry of these two compounds has not been assigned at this time).

These two compounds were identical in their GC/EI-MS and 'H-NMR spectra as product 2 (data above).

Each of the enantiomers 4 and 5 was reduced separately using dimethyl sulfideborane complex in the following manner. A solution of compound (4 or 5 in THF was heated to reflux and treated with excess (2 Eq.) 1M (in THF) dimethyl sulfideborane complex and the 141 reaction refluxed 30 min. After this time the reaction was cooled to 0°C and treated with 6 N HCI. The reaction was set to reflux for 30 min. After this time the reaction was transferred to a separatory funnel, basified to pH 12 with 10ON NaOH, and the product (6 or 7) extracted into ether. The ether layer was washed with brine, dried over anhydrous MgSO, and concentrated to an oil. The product was purified by prep-TLC using methanol-chlorform. Each of the individual D 10 enantiomers (6 and 7) were found to be identical in their GC/EI-MS and H-NMR spectra as product 3 (data above). Note that products 6 and 7 in this scheme correspond to Compounds 33 and 34. Compound 33-HC1: mp 260-270°C (dec), [-]36526 +6.6 (c 1.0 in EtOH), []D26 +0.4 (c 1.0 in EtOH). Compound 34-HC1: c] 36523 -6.1 (c 1.0 in EtOH), [o]D23 +0.1 (c 1.0 in EtOH).

Compound 33-HI: The free base of Compound 33 was dissolved in EtOH and 47% hydriodic acid (1.1 equivt.) was added. The solvent was evaporated under vacuum and 20 the resulting solid hydroiodide was recrystallized twice from heptane/EtOAc by slow evaporation: mp 195-197 0

C.

The absolute configuration of Compound 33-HI was determined to be R by single-crystal (monoclinic colorless needle, 0.50 x 0.05 x 0.03 mm) X-ray diffraction analysis using a Siemens R3m/V diffractometer (3887 observed reflections).

142 I) (Mc,Si) 2 NL, d(W'H) Raney nckcel I NH, CN 2) Mel CN H' F NH Compound 21 IPA I Hela F CN F CN SiF 1) Mc S-BH) 2) HCI Ft NHI F l! F.6 **(Compound 33) Compound 34) Synthesis of Compound 22 was accomplished as described below. Compound 23 was synthesized in a similar manner.

o. Triethyl phosphonoacetate (17.2 g, 76.8 mmol) was slowly added to a suspension of sodium hydride (3.07 g, 76.8 mmol) in N,N-dimethylformamide (350 ml). After 15 minutes 3,3'-difluorobenzophenone (15.2 g, 69.8 mmol) was added to the solution and stirred an additional 18 hr. The reaction mixture was quenched with water and partitioned between water and ether. The combined organic layers were washed with brine and dried over anhydrous magnesium sulfate. The solvent was evaporated in vacuo to give 19.7 g of ethyl 3,3-bis(3-fluorophenyl)acrylate as a yellow oil.

To a solution of ethyl 3,3-bis(3-fluorophenyl) -acrylate (19.7 g, 68.4 mmol) in.200 ml of ethanol was added palladium hydroxide on carbon (3.5 The mixture was shaken under 60 psi of hydrogen for 3 hours, 143 then filtered and evaporated in vacuo to give 19.5 g of product A as a colorless oil.

The ethyl ester A (19.2 g) was hydrolyzed by stirring for 6 days with 50 ml of 10 N sodium hydroxide.

The reaction mixture was then diluted with 50 ml of water and acidified to pH 0 with concentrated HC1. The aqueous mixture was extracted 3 times with ether and the ether extracts dried over magnesium sulfate and evaporated to give 3,3-bis(3-fluorophenyl)propionic acid 10 as a white powder.

~3,3-bis(3-fluorophenyl)propionic acid (13 g, 49.6 mmol) was dissolved in 50 ml (685 mmol) of thionyl chloride and stirred overnight at room temperature. The excess thionyl chloride was removed in vacuo on a rotary 15 evaporator to give 13.7 g of product B as a yellow oil.

o* To acid chloride B (13.7 g, 49 mmol) dissolved in 100 ml of dry THF was added iron(III) acetylacetonate (0.52 g, 1.47 mmol). Methylmagnesium chloride (16.3 ml, 49 mmol) was then added over a period of 1 hr by syringe 20 pump. The reaction was stirred for an additional hour, then quenched by pouring into ether/5% HC1. The ether layer was separated, washed with 5% HC1 and saturated NaC1, and dried over sodium sulfate. The solvent was evaporated in vacuo to give 4,4-bis(3-fluorophenyl)-2-butanone as a yellow oil. The crude oil was purified on silica gel using heptane/ethyl acetate as the elutant.

To 4,4-bis(3-fluorophenyl)-2-butanone (5.7 g, 21.9 mmol) in 25 ml of ethanol was added pyridine 144 (1.91 g, 24.1 mmol) and methoxylamine hydrochloride (2.01 g, 24.1 mmol). The reaction was stirred overnight at room temperature, then poured into ether/5% HC1. The ether layer was separated, washed with 5% HC1 and saturated NaC1, and dried over sodium sulfate. The solvent was evaporated in vacuo to give 6.26 g of the O-methyl oxime of 4,4-bis(3-fluorophenyl)-2-butanone.

To sodium borohydride (4.1 g, 108.3 mmol) in 15 ml of THF was slowly added zirconium tetrachloride (6.31 g, 10 27.1 mmol). This mixture was stirred for 15 min, then o: the oxime (6.26 g, 21.7 mmol) in 6 ml of THF was added over 5 min. After 3 hours of stirring at room temperature, the reaction was worked up by slowly adding mM sodium hydroxide followed by ether. The aqueous 15 layer was extracted 4 times with ether, and the combined ether extracts were dried over sodium sulfate. The solvent was evaporated in vacuo to give 5.3 g of Compound 22.

145 EtO )P COOEt 1O EtO F 0 L F NaH, DMF F COOEt 2. H 2 Pd(OH) 2 Ethanol F F

A

N' Cl 1. NaOH/MeOH/H 2 0 F 2. SOCl2

F

1. Fe(Acac) 3 MeMgBr N NH 2 2. H 2 N-OMe, pyridine

CH

3 *9 3. NaBH4/ rC4 TH Compound 22 Synthesis of Compound 24 was accomplished as described below. Compounds 25-29, 52-53, 65, 76-78, 83, 96-97, 115, and 135-136 were prepared in a similar manner.

A suspension of magnesium turnings (0.95 g, 39.2 mmol) in 150 ml anhydrous diethyl ether was treated with l-bromo-3-fluorobenzene (6.83 g, 39.2 mmol) dropwise via syringe. After 1.5 hr the solution was transfered via cannula to a flask containing o-anisaldehyde (5.0 g, 36.7 mmol) in 100 ml anhydrous diethyl ether at 0 0 C and stirred 2hr. The reaction mixture was quenched with water and partitioned between water and ether. The combined organic layers were 146 washed with brine and dried over anhydrous magnesium sulfate to afford 7.90g (93% yield) of product A.

Pyridinium dichromate (16.0 g, 42.5 mmol) was added to a solution of the. alcohol A (7.90 g, 34.0 mmol) in dichloromethane (100 ml), and the reaction was stirred 12 hr. Diethyl ether (300 ml) was added to the reaction mixture and the black solution was filtered through a silica gel plug (30 cm) and washed with an additional 500 ml ether. After evaporation of the solvent in vacuo, the solid was recrystallized from acetone to give 7.45 g (95% yield) of product B.

Diethyl cyanomethylphosphonate (7.0 g, 39.5 mmol) was slowly added to a suspension of sodium hydride (1.58 g, 39.5 mmol) in 100 ml N,N-dimethylformamide. After 30 minutes the ketone B was added to the solution and stirred an additional 2 hr. The reaction mixture was quenched with water, and partitioned between water and ether. The combined organic layers were washed with brine and dried over S: 20 anhydrous magnesium sulfate. The solvent was evaporated in vacuo to give a pale yellow oil.

In a glass bomb, the oil was dissolved in 100 ml ethanol and 20 ml 10N NaOH. A catalytic amount of Raney Nickel suspened in water (ca. 15 mol percent) was added to the solution. The reaction mixture was shaken under 60 psi H 2 for 12 hr on a Parr Hydrogenator.

After filtering off excess Raney Nickel, the solution was extracted with chloroform. The combined organic layers were washed with brine and dried over anhydrous 147 magnesium sulfate. After filtration, the oil was run through a silica gel column in chloroform and methanol.

The solvent was evaporated in vacuo to give a pale yellow oil. GC/EI-MS (R,=8.10 min) m/z (rel. intensity) 259 (100), 242 213 183 136 109 91 77 The oil was then acidified with hydrogen chloride in diethyl ether. Evaporation of the ether afforded a pale yellow solid that was recrystallized in hot acetonitrile to afford 3.45 g (42.1% yield) white needles of Compound 24, as the hydrochloride salt.

oo *°oo r 148 1) Mg, ether

OCH~

B r PCC or PDC CH,Ck

N

EL'P,CN, NaH-DMF 2) Raney Ni, EIOH. NaOH-

H

2 60 p.s.i.

3) HCI-ether 0OCH 3

I

.0

S

0.0 **too:

NH

3

CI

Compound 24 (HC1 salt) Compounds 101 and 103 were synthesized from Compounds 25 and 24, respectively, by cleavage of their 0-methyl ethers with borane tribromide in the normal manner.

Synthesis of Compound 30 was accomplished as described below. Compound 31 was prepared in a similar manner.

A suspension containing-magnesium turnings- (0.95 g, 39.1 mmol) in 150 ml anhydro us diethyl ether was treated with 1-bromo-3-fluorobenzene (6.85 g, 149 39.1 mmol) dropwise via syringe. After 1.5 hr the solution was transfered via cannula to a flask containing 3-chlorobenzaldehyde (5.0 g, 35.6 mmol) in 100 ml anhydrous diethyl ether at 0°C and stirred 2 hr.

The reaction mixture was quenched with water and partitioned between water and ether. The combined organic layers were washed with brine and dried over anyhydrous magnesium sulfate to afford 8.40 g (>99% yield) of product A.

Pyridinium chlorochromate (15.0 g, 39.8 mmol)

S

was added to a solution of the alcohol A (8.40 g, 35.5 mmol) in 100 ml dichloromethane and stirred 18 hr.

000 Diethyl ether (300 ml) was added to the reaction mixture and the black solution was filtered through a silica gel 15 plug (30 cm), and washed with an additional 500 ml fee* ether. After evaporation of the solvent the solid was recrystallized from acetone to give 6.31 g (76% yield) of product B.

Diethyl cyanomethylphosphonate (5.2 g, 20 29.6 mmol) was slowly added to a suspension of sodium hydride (1.2 g, 29.6 mmol) in N,N-dimethylformamide (100 ml). After 30 minutes the ketone B was added to the solution and stirred an additional 6 hr. The reaction mixture was quenched with water and partitioned between water and ether. The combined organic layers were washed with brine and dried over anhydrous magnesium sulfate. The solvent was evaporated in vacuo to give a yellow oil.

150 In a glass bomb, the oil was dissolved in ethanol (100 ml) and 10N NaOH (20 ml). A catalytic amount of rhodium suspended on alumina (ca. 35 mol percent) was added to the solution. The reaction mixture was shaken under 60 psi H 2 for 24 hr on a Parr Hydrogenator. After filtering off excess catalyst, the solution was extracted with chloroform. The combined organic layers were washed with brine and dried over anhydrous magnesium sulfate. After filtration and 10 evaporation of the solvent in vacuo, the oil was taken up in tetrahydrofuran (100 ml). Diborane (23.4 ml, 1.0 M) was added and the solution was refluxed for hr. The solvent was evaporated in vacuo and 6N HC1 (50 ml) was added carefully. The solution was refluxed for 1 hr. After cooling, the mixture was basified with 10N NaOH to pH 14 and partitioned between dichloromethane and water. The combined organic layers were dried over anhydrous magnesium sulfate and filtered. After evaporation of the solvent, the yellow oil was run through a silica gel column in chloroform and methanol. The solvent was evaporated in vacuo to give a yellow oil.. GC/EI-MS (Rt=8.15 min) m/z (rel.

intensity) 263 246 211 196 183 (100), 165 133 The oil was then acidified with hydrogen chloride in diethyl ether. Evaporation of the ether afforded 0.96 g of a white solid, Compound as the hydrochloride salt.

151 1) Mg, ether 2) H CI 0 Fi Br Pcc-cH 2 CI2 0 .0.

CN, NaH-DMF 2) RhlAlumrina, EtOH

H

2 60 p.s.i.

3) B 2

H

6

-THF

4) 6N HCI HCI-cther

NH

3

CI

Compound 30 (HCl salt) Synthesis of Compound 35 was accomplished as described below.. Compounds 36-37 were prepared in a similar manner.

A solution of 3-fluorobenzaldehyde (3.0 g, 24.2 mmol) at O 0 C in 150 ml diethyl ether was treated with 3.0 M ethyl magnesium chloride (12.7 ml, 25.4 mmol) in tetrahydofuran (TI{F) via syringe. After 4. hr, the reaction mixture was quenched with water and partitioned between water and ether.' The combined organic layers 152 were washed with brine and dried over anyhydrous magnesium sulfate to afford 4.25 g of product A.

Pyridinium chlorochromate (6.53 g, 30.3 mmol) was added to a solution of A in dichloromethane (100 ml) and stirred 18 hr. Diethyl ether (300 ml) was added to the reaction mixture and the black solution was filtered through a silica gel plug (30 cm) and washed with an additional 500 ml ether. After evaporation of the solvent the solid was recrystallized from acetone to give 3.05 g of product B. The solvent was evaporated in vacuo to give a pale yellow oil.

Diethyl cyanomethylphosphonate (4.7 g, 26.4 mmol) was slowly added to a suspension of sodium hydride (1.1 g, 26.4 mmol) in 100 ml N,N-dimethylformamide. After 30 minutes the ketone B was added to the solution and stirred an additional 6 hr. The reaction mixture was quenched with water and partitioned between water and ether. The combined organic layers were washed with brine and dried over anhydrous magnesium sulfate. The solvent was evaporated in vacuo to give a yellow oil.

In a glass bomb, the oil was dissolved in 100 ml ethanol and 20 ml 10N NaOH. A catalytic amount of Raney Nickel suspended in water (ca. 15 mol percent) was added to the solution. The reaction mixture was shaken under 60 psi H 2 for 24 hr on a Parr Hydrogenator.

After filtering off excess catalyst, the solution was extracted with chloroform. The combined organic layers were washed with brine and dried over anhydrous 153 magnesium sulfate. After filtration, the oil was run through a silica gel column in chloroform and methanol.

The solvent was evaporated in vacuo to give a pale yellow oil. GC/EI-MS (Rt=3.45 min) m/z (rel. intensity) 167 150 135 109 (100), 96 (53), The oil was then acidified with hydrogen chloride in diethyl ether. Evaporation of the ether left a pale yellow solid that was recrystallized in hot acetonitrile to afford 2.2 g of Compound 35, as the S 10 hydrochloride salt.

OH -i r

CH

3

CH

2 MgIr, ether PCC.CH 2

CI

2 1) o 'P CN.,NaH-DMF ElO 2) Raney Ni, EtOH, NaOH

H

2 60 p.s.i.

3) HCl-ether

NH

3

CI

Compound 35 (HC1 salt) 154 Synthesis of Compound 38 was accomplished as described below.

To a solution of 3,3-bis(3-fluorophenyl) -propionitrile (1.5 g, 6.17 mmol) in 250 ml of THF at -70 0 C was added butyl lithium (4.25 ml in hexanes, 6.8 mmol) by syringe over 5 minutes. The solution was stirred for 5 min then methyl iodide (1.75 g, 12.3 mmol) was added over 1 min. The reaction mixture was then allowed to warm up to room temperature and worked up by diluting with ether and washing with 5% HC1 and water.

The ether layer was dried over sodium sulfate and evaporated to give 1.5 g of the methylated nitrile as a yellow oil.

To the 3,3-bis(3-fluorophenyl)- 2-methyl-propionitrile (1.46 g, 5.7 mmol) in 50 ml of dichloromethane at 0°C was added diisobutylaluminum hydride (1.02 ml, 5.7 mmol) by syringe over a 10 min period. The reaction was stirred for 30 min at 0°C followed by 2 additional hours at room temperature. The reaction was worked up by adding 200 ml of 10% HC1 and stirring at 40 0 C for 30 min followed by extraction of the product with dichloromethane. The organic layer was dried over sodium sulfate and evaporated to give 1.36 g of the product A.

To a solution of the aldehyde A (1.36 g, 5.23 mmol) in 40 ml of ether at 0°C was added methylmagnesium bromide (5.23 ml in ether, 5.23 mmol).

The reaction was stirred for 3 hr at room temperature, and then quenched with dilute HC1. The ether layer was 155 separated, dried over sodium sulfate and evaporated to give 1.48 g of 4,4-bis(3-fluorophenyl) -3-methylbutan-2-ol.

To a solution of the alcohol (1.4 g, 5.07 mmol) in 300 ml of dichloromethane was added pyridinium chlorochromate (1.2 g, 5.58 mmol), and the mixture was stirred overnight. The reaction was then diluted with 100 ml of ether and filtered through a silica plug. The solvent was evaporated to give 1.39 g 10 of product B.

The ketone B (1.3 g, 4.9 mmol) was added to a solution of methoxylamine hydrochloride (0.45 g, 5.38 mmol) and pyridine (0.44 ml, 5.38 mmol) in 30 ml of ethanol, and stirred overnight. The ethanol was then 15 evaporated, and the residue taken up in ether and HCl. The ether layer was separated, washed once with HC1, dried over sodium sulfate and evaporated to give 1.4 g of the O-methyl oxime.

To a suspension of sodium borohydride (0.87 g, 20 23.1 mmol) in 5 ml of THF was added zirconium tetrachloride (1.35 g, 5.8 mmol), and the solution was stirred for 15 min followed by the addition of another ml of THF. The O-methyl oxime (1.4 g, 4.6 mmol) in ml of THF was then added, and the mixture stirred overnight. The THF was removed by evaporation in vacuo, and the residue treated with 10% sodium hydroxide.

After the bubbling ceased ether was added and the layers separated. The aqueous layer was extracted four times with ether, and the combined ether extracts were dried 156 *0* 0 00 over sodium sulfate. The ether was evaporated to give 1.25 g of Compound 38.

F N 1BuLj, Mel

CH

CNF

CHO

F 2. DIBAH, CH 2

CI

2

N

FF

A

CH

3 I. MeMgBr F CH 3 2. PCC ICH 2 Cl 2 0

F

B

ZYCH

3 I- H 2 N e.(2epyridine F NNH 2 2.teaB 4 ZrCI7 CH 3 Compound 38 157 Compound 32 and Compounds 39 53 were synthesized according to standard procedures as described above.

Compounds 107, 116, 139, and 143 were prepared as synthetic intermediates used in the preparation of Compounds 32, 115, 20, and 25, respectively.

Compound 50 was also prepared using the chiral synthesis described below.

To an ice-cold solution of 10 N-benzyl-(S)-a-methylbenzylamine (18.0 g, 85.2 mmol) in THF (75 ml) was added butyl lithium (2.5 M in hexane; 37.5 ml, 93.8 mmol) via a syringe over a period of 10 min at such a rate as to keep the reaction temperature below 10 0 C during the addition. The 15 reaction was then stirred at 0°C for 15 min. The *reaction was cooled to -78 0 C in a dry ice/isopropanol bath and then a solution of benzyl crotonate (15.0 g, S" 85.2 mmol) in THF (100 ml) was added dropwise over a period of 45 min. The reaction was stirred at -78 0 C for 20 15 min, and then saturated NH 4 C1 (50 ml) was added. The reaction mixture was then quickly transferred to a separatory funnel containing saturated NaC1 (500 ml) and ether (200 ml). The layers were separated and the aqueous layer extracted with ether (200 ml). The combined organic layers were dried, evaporated, and chromatographed on silica gel (50 mm x 30 cm) (hexaneethyl acetate to yield 21.0 g, 63.7% of product A. 1 H-NMR showed that the diastereoselectivity of the reaction is 158 A mixture of magnesium (2.58 g, 106 mmol), THF (200 ml), and l-bromo-3-fluorobenzene (18.60 g, 106.3 mmol) was refluxed for 45 min. While still under reflux, product A (16.45 g, 42.45 mmol) was added via syringe with THF (25 ml) over a 2 min period. The reaction was refluxed for 1 hr, and then allowed to cool to room temperature. Saturated NH 4 Cl(aq., 200 ml) was added. The reaction mixture was then transferred to a separatory funnel containing saturated NaCl(aq) (500 ml) and diethyl ether (200 ml). The layers were separated and the aqueous layer extracted with ether (200 ml).

The combined organic layers were dried over sodium sulfate and evaporated to give 21.41 g of product B as a yellow liquid.

Product B (20.02 g, 42.45 mmol, theoretical) was dissolved in acetic acid (120 ml) and sulfuric acid ml). The reaction was stirred at 90 0 C for 1 hr.

The acetic acid was rotary evaporated giving a brown sludge. This material was placed in an ice bath and cold water (400 ml) was added. The product immediately precipitated. To the reaction was slowly added 10 N NaOH (150 ml) to neutral pH. Diethyl ether (200 ml) was added to this mixture. The mixture was shaken until there was no undissolved material. The ether layer was separated, washed with water (2 x 100 ml), dried over sodium sulfate, and rotary evaporated yielding 13.14 g (68.2% based on ester) of a thick brown oil. This oil was taken up in ether and converted to the hydrochloride O 159 salt with hydrogen chloride in diethyl ether to give product C as a yellow-white solid.

Product C (7.17 g, 14.6 mmol) was taken up in absolute ethanol (200 ml). Pearlman's catalyst (Pd(OH) 2 2.00 g) was added. The reaction was shaken under 70 psi hydrogen gas at 70 0 C for 20 hr, and the reaction mixture was filtered through Celite. The filtrate was rotary evaporated to give 3.54 g of a light yellow glass. This material was taken up in diethyl ether (100 ml) and was basified with 1 N NaOH (25 ml).

The ether layer was washed with water (1 x 25 ml), dried over sodium sulfate, and rotary evaporated to give 2.45 g of a light yellow oil. This material was Kugelrohr distilled (90-100 0 C, 1 mm Hg) to give 1.17 g S 15 of a colorless liquid. This material was taken up in diethyl ether and converted to the hydrochloride salt with ethereal hydrogen chloride. After rotary evaporation, the salt was recrystallized from 0.12 N HCl (200 mg/ml). The crystals were filtered off and were 20 washed with cold 0.12 N HC1 yielding 0.77 g of Compound 50 as silvery white crystals (as the hydrochloride salt).

Compound 51 was synthesized in a similar manner to Compound 50 utilizing N-benzyl-(R)-amethylbenzylamine as a chiral starting material.

160 0' -Br

H

2

NG

EH3 KF/Celitt

DMI'U

N.

EH3 MgI~r THF. reflux 1) BuLi. THF. -78C 0 o o.

AcOH. H 2 S0 4

H

2 0 ZCH3

HCI

1) Pd(O4).

2 EtOH- 2) HCI-Et 2

O

Compound 50 (HC1 salt) Synthesis of Compound 54 was accomplished as described below.

To a solution of 3,3'-difluorobenzophenone g, 22.9 mmol) and methyl cyanoacetate (3.4 g, 34.4 mmol) in 15 ml of ether was added titanium isopropoxide (16.9 ml, 57.25 mmol). This solution was stirred for 6 days at room temperature then quenched with 0.5 mol of HC1 in 300 ml of water. The mixture was diluted with 100 ml of ether, and the layers separated.

10 The ether layer was washed with 5% HCl and saturated brine, then dried over sodium sulfate. The solvents were evaporated in vacuo to give 8 g of product A.

Compound A was dissolved in 50 ml of isopropanol, followed by the addition of a small amount 15 of bromocresol green. Sodium cyanoborohydride (1.52 g, 24.2 mmol) was added all at once followed immediately with the dropwise addition of concentrated HC1, added at such a rate as to keep the solution yellow. After 2 hours the reaction was worked up by partitioning 20 between ether and water. The ether layer was washed with water and saturated brine, dried over sodium sulfate, and concentrated to give the product B.

To a solution of lithium aluminum hydride (30.4 ml, 30.4 mmol) in THF was added product B (1 g, 3.04 mmol) in 2 ml of THF over a period of 30 seconds.

This solution was stirred overnight at room temperature, then quenched with the addition of 20 ml of ethyl acetate. The solvents were then removed in vacuo, and the resulting oil was dissolved in aqueous HC1 and 162 acetonitrile. The product was then purified on a C-18 column with a gradient of 0.1% HCl to acetonitrile to give .82 mg of Compound 54, as the hydrochloride salt.

EI-MS m/z (relative intensity) 277 100), 260 242 229 215 204 183 133 124 109 30 (22).

-CN

F0 NC,,COOMe 0 Cl- 3 Ti(i-OPrh Ether0

CH

F

F

S.

A

OH

N .NH 3

CI

NaCNBH 3 F 0 Y C3 I. LAH

F

Isopopaol.HCI0

CH

3 2. Chromatography Ispoao. C in HCI/AcCN F

F

B

Compound 54 (1i salt) Compound 55 was synthesized analogously to Compound 21 except that ethyl iodide was used in the' alkylation step. GC/EI-MS 7.43 min) m/z (relative intensity) 275 100) ,28(66) 229 204 (57) 201 183 134 124 109 72 (72).

The synthesis of Compound 56 was accomplished as follows.

The alcohol A was synthesized from 3-f luorobromobenzene and 3-fluoro-2-methylbenzaldehyde 0 163 as described for product A in the synthesis of Compound 24.

The alcohol A (8.4 g, 36.2 mmol) was stirred with manganese dioxide (12.6 g, 144.8 mmol) in 100 ml of dichloromethane for 4 days. The reaction mixture was then diluted with ether and filtered through a 0.2 micron teflon membrane filter. The filtrate was concentrated to give 7.6 g of the ketone B.

The substituted acrylonitrile C was synthesized as described for product A in the Compound 20 synthesis.

To the nitrile C (4 g, 15.7 mmol) in 240 ml of ethanol was added 2 g of 10% palladium dihydroxide on carbon. This mixture was hydrogenated at 60-40 psi for 3 days. The reaction mixture was then filtered and concentrated. The resulting oil was dissolved in chloroform and chromatographed on silica gel isopropylamine in chloroform) to give the amine. This amine was dissolved in aqueous 20 HCl/acetonitrile and purified via HPLC on C-18 acetonitrile/0.1% HC1 to 50% acetonitrile/0.1% HC1 over min) then lyophilized to give 800 mg of Compound 56, as the hydrochloride salt. GC/EI-MS (Re 7.39 min) m/z (relative intensity) 261 64), 244 229 (57), 215 (100), 203 183 133 122 109 (32).

164 1) Mg, ether 2 H F OH F N O F O H MnO2 H3C F Br I CH 2 Cl 2

NZ

F

F

A B 0 I NH 3

CI

Eo _CN F CN 1)Pd(OH)2, H 2 Ethanol F EtO P C N

H

3

OH

3 1A 2) Chromatography in NaH-DMFF p HCI/AcCN p

F

Compound 56 (HCi salt) The synthesis of Compound 57 was accomplished as follows.

To a solution of 5-fluoro-2-methylbenzonitrile g, 37 mmol) in 50 ml of THF was added S 5 3-fluorophenylmagnesium bromide (46 ml, 40 mmol) and copper cyanide (0.072 g, 0.8 mmol). This solution was refluxed for 4 hours, then poured into ether/20% HC1 and stirred for a further 2 hours. The layers were separated, and the ether layer washed with water and 10 saturated brine. The solution was dried over sodium sulfate and concentrated. The crude oil was purified on silica (h-exane to 50% dichloromethane in hexane over min) to give 6.7 g of the ketone A.

The ketone A was converted to Compound 57 as described for Compound 56. GC/EI-MS (Re 7.35 min) m/z (relative intensity) 261 52), 244 229 215 (100), 203 201 183 133 122 109 (26).

0 165 A O i) Mg. cthcr F

CH

3 F Br 2

F

2 CN CuCN F

A

Et

NH

3 Cl EtO CN F CN 1) Pd(OH) 2

H

2 Ethanol EtO C N CH3 NaH-DMF CH3 2) Chromatography in NaHDF HClAcCN

F

F

Compound 57 (HC1 salt) The synthesis of Compound 58 was accomplished as follows.

To a solution of 5-fluoro-2-methylbenzoyl chloride (2.24 g, 13 mmol) in 10 ml of dry THF was added 5 iron III acetylacetonate (0.16 g, 0.44 mmol). The solution was cooled to OoC, and a THF solution of 5-fluoro-2-methylphenylmagnesium bromide (20 ml, 15.5 mmol) was added by syringe over a period of 30 min.

The reaction was stirred for another 30 min, then poured 10 slowly into ether/5% HC1. The ether layer was separated, washed with saturated brine, dried over sodium sulfate, and concentrated to give 3.2 g of ketone A.

Dry THF (30 was cooled to -780C followed by the addition of butyl lithium (5.85 ml, 14.6 mmol, M solution in hexanes). Acetonitrile (0.76 ml, 14.62 mmol) was then added over a period of 2 min, then allowed to stir at -780C for 15 min. To this solution 166 was added ketone A (3 g, 12.2 mmol) in 5 ml of THF. The solution was stirred for 30 min at -78 0 C then allowed to warm to room temperature and stirred overnight. The reaction mixture was partitioned between ether and HC1. The ether layer was separated, washed with saturated brine, dried over sodium sulfate, and concentrated to give 2.2 g of the nitrile B.

The nitrile B (1 g, 3.48 mmol) was dissolved in 30 ml of ethanol and 3 ml of 10 N sodium hydroxide.

To this solution was added 1 g of a 50% aqueous slurry of Raney nickel, and the mixture was hydrogenated at 60 psi for 20 hours. The reaction was filtered and concentrated to a white solid. This residue was taken up in ether/water and the ether layer separated. The 15 ether solution was dried over sodium sulfate and concentrated to give 0.96 g of the hydroxyamine

C.

The hydroxyamine C (0.96 g, 3.3 mmol) was o taken up in concentrated HC1 and heated to 70 0 C which caused brief solution, and then precipitation of the 20 alkene D. The alkene was collected by filtration and 0* a dissolved in 30 ml of ethanol and 1 ml of conc. HC1.

Palladium dihydroxide on carbon (0.4 g) was added to the solution;:and the mixture hydrogenated at 60 psi for 24 hours. The product was isolated by filtering off the catalyst and evaporating the solvent. The residue was dissolved in 0.1% HC1 and acetonitrile, and purified on C-18 (15% acetonitrile/0.1% HCl to acetonitrile) to give 0.6 g of Compound 58, as the hydrochloride salt.

GC/EI-MS 7.82 min) m/z (relative intensity) 275 167 100) 258 (20) 243 (74) 229 (38) 214 (65) 201 (31) 196 (32) 183 (20) 148 (35) 138 (42) 133 (48) 122 (69) 109 (41).

CH

3 F MgBr

CH

3 C Fc(Acac) 3 r ;I THIF CH 3 0

F

BuLi AcCN

THIF

'CN Raney Ni H 2 NaOH

,CH

3 Ethanol F CH NH 3

CI

CH

3

NH

3 Cl conc. HCI heat PdCQH)/C. H 2

HCI

Ethanol D Compound 58 (HC1 salt) 168 Synthesis of Compound 59 was accomplished as follows.

Compound 20 (2.0 g, 7.05 mmol) was dissolved in abs. EtOH (200 ml) and cooled to 5-10 0 C in an ice bath. Acetaldehyde (0.395 ml, 7.05 mmol, cooled to -4oC) was added followed by nickel-aluminum alloy (200 mg, Fluka Chemika), and the reaction was hydrogenated on a Parr apparatus at 50 psi for 2 hr.

GC/MS showed 75% yield of the product and 2% of the N,N-diethyl side-reaction product. The reaction mixture was filtered through diatomaceous earth and the filtrate was evaporated under reduced pressure. The crude product was dissolved in isopropanol (5 ml)/ether ml)/ethereal HC1 (1 and then hexane (5 ml) was added to the cloud point. The cloudy mixture was filtered through paper, then hexane (10 ml) was added to the cloud point, and the solution was filtered again. The filtrate was stoppered and the product was allowed to crystallize at room temperature. The crystals were 20 collected and dried to provide 0.325 g (14.8% yield) of Compound 59, as the hydrochloride salt (colorless needles).

:The synthesis of Compound 60 was accomplished as follows. Compounds 66, 69, 108, 123, 142, and 145 can be synthesized in a similar manner starting from Compounds 33, 50, 32, 60, 25 and 119, respectively.

Compound 20 (as the free base) (1.0 g, mmol) was refluxed in ethyl formate (150 ml) for 2 hr. The solvent was then removed under reduced 169 pressure to provide 1.1 g, 99% yield of formamide A as a colorless oil. GC/MS showed the product to be 100.0% pure and was used in the following step without further purification.

The formamide A (1.1 g, 4.0 mmol) was dissolved in dry THF (100 ml) and heated to reflux (no condenser). Borane-methyl sulfide complex (1.2 ml, 12 mmol, 10.5 M) was added dropwise over a period of 3 min to the refluxing solution. Reflux was maintained for approximately 15 min, open to the air, until the reaction volume was reduced to approximately 30 ml. The reaction was then cooled in an ice bath, and ice (5 g, small pieces) was carefully added followed by H 2 0 (25 ml) and conc. HC1 (25 ml). The acidic solution was refluxed for 30 min. The reaction mixture was then cooled in an ice bath, basified with NaOH (10N), extracted with ether (3 X 100 ml), dried (Na 2 SO,, anhydrous), and evaporated under reduced pressure. The crude product was dissolved in ether (10 ml)/hexane (50 ml) and ethereal HCL (1 M) was added dropwise to precipitate the hydrochloride Ssalt. The salt was collected and recrystallized from isopropanol (3 ml)/ether (40 ml) to provide 0.5 g of as the hydrochloride salt.

170 N i Ethyl formate

H

NH2 N H F reflux F 0 F F Compound 20 (as free base) A 1) Borane-methyl sulfide, THF CH3

\NH

2

CI

2) HCI F

F

Compound 60 (HC1 salt) .:...Alternatively, Compound 60 was synthesized from commercially available starting materials in the following four step reaction sequence. The first intermediate in this synthetic route, ethyl-N-benzyl-N-methyl-3-aminopropionate, was prepared by conjugate addition of N-benzylmethylamine to ethyl acrylate. The ester functionality of the first intermediate was then reacted with two equivalents of Grignard reagent (prepared from 1-bromo-3-fluorobenzene) to provide N-benzyl-N-methyl-3-hydroxy-3- (bis-3-fluorophenyl) propylamine. The Grignard reaction product was then dehydrated in a mixture of 6N HCl/acetic acid to yield N-benzyl-N-methyl-3-(bis-3-fluorophenyl)-2-propenamine.

Catalytic hydrogenation of this material as its hydrochloride salt in ethanol over Pearlman's catalyst provided, after recrystallization from ethyl acetate, colorless, needles of Compound 60 as the hydrochloride salt.

In a 500-mL, 3-necked flask equipped with thermometer, reflux condenser, and a 125-mL addition funnel [charged with ethyl acrylate (88.3 mL, 81.5 g, 0.815 mol)] was placed N-benzylmethylamine (100 mL, 94.0 g, 0.776 mol). The ethyl acrylate was added dropwise to the stirring reaction mixture over a period of 80 min. After stirring for 18 h at room temperature, the product was vacuum distilled and the fraction containing product was collected at 78-95 0

C

(0.12-0.25 mm Hg), (138 g, 80% yield): Bp 78-95 0

C

(0.12-0.25 mm Hg); TLC, Rf 0.23 [hexane-EtOAc

S

R 0.57 [MeOH-CHC13 GC, t. 6.06 min; MS, 221 206 (M-CH 3 192 (M-C 2

H

5 176 (M-OC 2 H) 144 (M-C 6

H

5 134 [CH 2 N (CH) CH 2 Ph] 120 [N(CH 3 )CHPh] es 91 (CH 7 77 (CHs) 42 (CH 2

CH

2 -H NMR (free base, 6CDCl 3 d 1.25 ppm J 7.1, 3H, CH 2

CH

3 2.20 3H, NCH,), 2.51 J 7.3, 2H, COCH,), 2.74 J 7.2, 2H, CH2N), 3.51 2H, NCH2Ph), 4.13 J 7.1, 2H, OCCH) 7.18-7.35 5H, ArH); "C NMR (free base, CDCl 3 d 15.2 (CH 2 34.0 (COCH 2 42.9 (NH, 3 53.8 (NCH 2 61.4 (OCH 2

CH

3 63.1 (CH 2 Ph), 128.0 (CH), 129.2 130.0 139.9 173.7 In a 5-L, four-necked, round-bottom flask, under nitrogen, were placed Mg [51.5 g, 2.12 mol, turnings, washed with THF (2 x 300 mL)] and THF (2 L).

An addition funnel was charged with l-bromo-3-fluorobenzene (neat, 392.8 g, 2.24 mol).

172 One-twentieth of the bromide was added to the magnesium suspension followed by one crystal of iodine. After initiation of the Grignard reaction the remaining 1-bromo-3-fluorobenzene was then added to the refluxing mixture over a period of 50 min. The reaction was refluxed for an additional 45 min. To the refluxing solution of Grignard reagent was added a solution of ethyl N-benzyl-N-methyl-3-aminopropionate (187.5 g, 0.847 mol) in THF (100 mL) over a period of 20 min.

10 After the ester addition was complete, the reaction was refluxed for 1h. The reaction was then cooled in an ice bath. Saturated NHCl 400 mL) and H 2 0 (400 mL) were added and the mixture was transferred to a separatory funnel. The organic layer was separated and 15 the aqueous layer was extracted once with THF (400 mL).

The combined organic layers were washed with satd. NaC1 (2 x 200 mL, dried (anh. Na 2 filtered .through paper, and rotary evaporated vacuum to yield 281.6 g of crude product as an orange, viscous oil. This 20 material (281.6 g, 0.766 mol) was dissolved in acetonitrile (1.4 Concentrated hydrochloric acid (65.0 mL, 0.786 mol, 12N) was added to the stirring filtrate.. The crystallizing mixture was then cooled to oC for 17 h. The product was collected, washed with cold acetonitrile (800 mL), and dried to provide a white solid, 235.6 g (69% yield from the ester). For analytical purposes, the hydrochloride salt was further purified by recrystallization from acetonitrile: mp 194-197 0 C (uncorr.); TLC, Rf 0.23 [hexane-EtOAc 173 R, 0.85 [MeOH-CHC 3 1 R, 0.72 [MeOH-CHC1 3 GC, tR 10.93 min; MS, 367 272 (M-C 6

H

4 F) 258 (M-CH 2 Ph-H 2 0) 219 [(CH4F) 2 CH] 148 [CH2CH 2

N(CH

3

)CH

2 Ph] 134 [CHN (CH) CH 2 Ph] 91 42 (CH 2

CH

2 'H NMR (free base, CDC13) d 2.18 3H,

NCH

3 2.41 2H, CHCH,), 2.58 2H, CH 2 3.42 (s, 2H, CH2Ph) 6.86 (dt, J 8.5, J2 1.8, 2H, Ar-H), 7.18-7.30 10H, Ar-H), 8.33 (bs, 1H, OH); "C NMR (free base, CDC1 3 d 35.6 (CHCH 2 41.5 (CH 3

NCH

3 54.3 (CH 2 CH2N), 62.6 (CH 2 CH2Ph), 113.1 J 23, CH, Ar-Cs,,) 113.5 J 23, CH) 121.2 J 3, CH), oo* 127.5 128.5 129.2 129.5 (CH), 129.6 137.0 150.2 162.8 J 243, q, Ar-C 3 3 In a 5-L, 3-necked reaction vessel, equipped with an overhead mechanical stirrer, reflux condenser, and thermometer, was placed N-benzyl-N-methyl-3-hydroxy-3-bis (3-fluorophenyl)propylamine hydrochloride (225.4 g, 0.559 mol), 6N HC1 (1392 mL) and glacial HOAc (464 mL).

The suspension was heated in a water bath (80-85 oC) and stirred for 18 h. After 18 h of heating, the reaction mixture was cooled in an ice/MeOH bath. Ethyl acetate (500 mL) was added to the cooled reaction mixture. NaOH (10N, 1.7 L) was then added to the cooled mixture over a period of 25 min at such a rate as to keep the temperature below 40 oC. The mixture was transferred to a 6-L separatory funnel. The organic layer was separated and the aqueous layer was extracted with ethyl 174 acetate (2 x 500 mL). The combined organic layers were washed with satd. NaC1 (2 x 100 mL, dried Na 2

SO,

(250 rotary evaporated, and then dried under vacuum to provide 185.6 g (95% yield) of the free base as a fluid, brownish-colored oil.

The material above was stirred with hexane The resulting solution was filtered through paper. 4M HC1 in dioxane (146 mL) was added dropwise with stirring to the filtrate over a period of 5 min.

The semi-translucent solvent was then decanted away from the light-yellow colored, semisolid precipitate. The crude hydrochloride salt was dissolved in refluxing ethyl acetate (600 mL) and was filtered. The filtrate was then thoroughly cooled in an ice bath, and hexane 15 (110 mL) was slowly added, with vigorous stirring.

SAfter cooling in an ice bath for 2 h, the entire flask e filled with a white crystalline solid. This material was collected on a filter funnel, washed with ice-cold hexane/ethyl acetate 400 mL], and dried to yield 128.7 g, 59.7% of a white solid. On standing the mother liquor precipitated another 14.8 g of an off-white solid. Total yield 128.7 g 14.8 g 143.5 Mp 141-142 °C (uncorr.); TLC, Rf 0.20 [hexane-EtOAc Rf 0.75 [MeOH-CHC1 3 Rf 0.49 [MeOH-CHC1 3 GC tR 10.40 min; MS, 349 330, 301, 281, 258 (M-CH 2 Ph), 240, 229 [M-N(CH 3

)CH

2 Ph, 201, 183, 146, 133, 109, 91 (CHC6Hs) 65, 42 (CHNHCH 3 IH NMR (free base, CDC1 3 d 2.20 ppm 3H, NCH 3 3.08 J 6.8, 2H, CH2N) 3.47 J 1, 2H, CH 2 Ph), 175 6.29 J 6.8, 1H, CH), 6.85-7.04 6H, ArH), 7.19-7.35 7H, ArH).

N-Benzyl-N-methyl-3-bis(3-fluorophenyl)allylamine hydrochloride (120.0 g, 0.311 mol) was dissolved in abs.

EtOH (1250 mL). Pd(OH) 2 /charcoal (10.0 g, -20% Pd, Fluka Chemical) was added. The reaction mixture was stirred under a steady flow of hydrogen gas for 18 h at 25 °C (atmospheric pressure). The mixture was then filtered through Celite'/fritted glass, the catalyst was washed with EtOH (2 x 50 mL), and the solvent was removed under reduced pressure to yield 95.4 g, 103% of crude product.

This material was dissolved in refluxing ethyl acetate (300 mL) with vigorous stirring and filtered. The flask was allowed to stand for 2 h at 25 OC, during which time the hydrochloride salt began to crystallize as needles.

The flask was then cooled, the product was collected, washed with ice-cold ethyl acetate (20 mL), and dried to yield 73.7 g, 80%, of Compound 60 as a white, crystalline solid. Mp 129-130 0 C; UV/Vis, e 2.1 x 103 LmolL-cm- 1 (264 nm, EtOH, 25 oC, linear range: 0.05-0.20 mg/mL); TLC, Rf 0.00 [hexane-EtOAc Rf 0.07 [MeOH-CHCl 3 Rf 0.19 [MeOH-CHC1 3

-NH

4 0H GC, t, 7.45 min; MS, 261 229, 215, 201, 183, 164, 150, 138, 122, 101, 83, 75, 57, 42

[CH

2

NHCH

3 'H NMR (HC1 salt, CDC1 3 1 gtt MeOD) 5 2.56 2H, NCH 2 2.60 3H, NCH,), 2.85 J 2H, CHCH 2 4.11 J 8.0, 1H, CH), 6.87-6.98 4H, ArH), 7.06 J 7.7, 2H, Ar 2 2 7.25 (dd, J, 6, J 2 176 8, ArH); 1 3 C NMR (HC1 salt, CDCl 3 1 gt MeOD) 5 30.9

(CH

2 CHCH)., 32.7 (CH 3 ,I NCH 3 47.6 (CH, CHCH 2 47.8

(CH

2

CH

2 113.9 (J 21, ArC 22 or ArC4 4 114.5

J

22, ArC 2 2 or ArC 4 4 123.2 J 3, Ar-C 6 130.3 J 9, Ar-C 5 144.7 J 7, Ar-C, 1 .62.9 (d, J 245, Ar-C 3 3 IR: KBr pellet 3436.9, 2963.4, 2778.5, 2453.7, 1610.6, 1589.3, 1487.0, 1-445.3, 1246.0, 764.5; solubility: 2 g/mL (H120), 1 g/mL (EtOH) anal. calcd. for C 16 H,NF,.HC1 (Karl Fischer: 0.26% 1H20) C, 64.37; H, 6.11; N, 4.69; found: C, 64.14; H, 6.13; N, 4.69.

C2H 3- F-H 4 MgBr 0 CHl 4 CHU (2 equvi.) 0

OH

NH.i F

H

2

I

NC(C~na Pd(ON), /C Convound so (MCI waIt 177 Compound 105 was prepared by selective reduction of its corresponding alkene by catalytic hydrogenation over Pd/C.

SCompound 61 was prepared from 2-bromo-4-fluoroanisole and 3-fluorobenzaldehyde as described for Compound 24. GC/EI-MS (Re 9.22 min) m/z (relative intensity) 277 74), 260 245 231 229 217 203 201 183 154 133 109 (100).

Compound 62 was prepared from 2-bromoanisole and 2-methoxybenzaldehyde as described for Compound 24.

GC/EI-MS (Re 9.30 min) m/z (relative intensity) 271 100), 254 (17) 240 225 (40) 223 (45) 207 181 165 136 121 91 (83).

15 The synthesis of Compound 63 was accomplished as follows.

Alcohol A was obtained from 3-fluorobenzaldehyde as described for product A of the Compound 24 synthesis.

20 To alcohol A (10.275 g, 47 mmol) in 200 ml of ethanol was added 1.6 g of 10% Pd/C and 1 ml of concentrated HC1. This mixture was hydrogenated for 3 h?-a-60 psi, then filtered and concentrated to give the diphenylmethane B.

Product B (2.01 g, 9.86 mmol) was dissolved in ml of THF and cooled to -78 0 C. Butyl lithium (4.4 ml, 10.8 mmol, 2.5 M in hexanes) was added slowly by syringe, and then the reaction stirred for another 30 min at -78 0 C. To this orange solution was added cyclopentene oxide (0.9 ml, 10.3 mmol). The reaction was allowed to stir 3 hours while warming slowly to room temperature.

178 The reaction was quenched with 150 ml of 10% HC1 and extracted 3 times with ether. The ether layer was dried over sodium sulfate and concentrated to give 2.5 g of the alcohol C.

To the alcohol C (1 g, 3.5 mmol) in 10 ml of dry THF was added triphenylphosphine (1.37 g, 5.2 mmol) in 5 ml of THF and p-nitrobenzoic acid (0.87 g, 5.2 mmol) in 5 ml of THF. This solution was cooled to 0°C followed by the addition of DEAD (0.82 ml, 5.2 mmol), and allowed to stir overnight. The reaction was partitioned between water and ether. The ether was rembved in vacuo and the resulting oil was chromatographed on silica gel in hexane/ethyl acetate to yield 365 mg of the cis-ester. This ester was hydrolyzed in methanol with potassium carbonate by stirring overnight. After removal of the methanol, the residue was taken up in ether, washed with water, dried over sodium sulfate and concentrated to give 250 mg of the cis alcohol D.

20 To the alcohol D (.25 g, 0.9 mmol) in 5 ml of dry THF was added triphenylphosphine (342 mg, 1.3 mmol) in 5 ml of THF and phthalimide (191.3 mg, 1.3 mmol) in 5 ml of THF. This solution was cooled to 0°C followed by the'addition of DEAD (0.205 ml, 1.3 mmol), and allowed to stir overnight. The reaction was partitioned between water and ether. The ether was removed in vacuo and the resulting oil was chromatographed on silica gel in hexane/ethyl acetate to yield 100 mg of the phthalimide E.

To a solution of the phthalimide E (100 mg) in ml of ethanol was added 8.8 mg of hydrazine hydrate.

179 The solution was refluxed for 5 hours then stirred at room temperature overnight. The reaction was worked up by adding 1 ml of conc. HC1 and filtering off the white solid. The resulting solution was concentrated to dryness and the solid taken up in ether and aqueous sodium hydroxide. The ether layer was dried over sodium sulfate and concentrated to a white solid. This was taken up in a small amount of ether and treated with drops of 1M HCl in ether. After stirring overnight, the white solid was collected by filtration and dried to give 50 mg of Compound 63, as the hydrochloride salt.

GC/EI-MS 9.22 min) m/z (relative intensity) 287 45), 270 201 183 (81) 133 (38), 109 83 56 (100), 43 (37) *o* 180 Mg, ether 2F F

O.

F Br Pd/C H 2 EtOH, HCI 1) BuLi THF 2) Cyclopentene Oxide 1) p-Nitrobenzoic Acid, PhP DEAD THF 2) Na 2 CO3, MeOH r Phthalimide Ph 3

P,

DEAD, THF 1) Hydrazine, EtOH 2) HCI, Ether

NH

3

CI

E Compound 63 (HC1 salt) The synthesis of Compound 64 was done as described for Compound 63 except that the inversion step (product C to D) was omitted in order to obtain the cis amine as the final product. GC/EI-MS (Re 8.28 min) m/z (relative intensity) 287 (M 15), 270 201 183 133 109 84 56 (100), 43 (32).

The synthesis of Compound 65 was accomplished as follows.

The ketone A was synthesized similarly to ketone B in the Compound 24 synthesis using 2-methylphenylmagnesium bromide and 2-methylbenzaldehyde as starting materials. This ketone was converted to the final product using the procedure outlined for Compound 58.. GC/EI-MS (Re 7.84 min) m/z (relative intensity) 181 239 88) 222 (14) 207 (100) 193 (46) 178 (71) 165 (60) 130 (39) 120 (40) 115 (51) 104(4) 91 (38) 77 (21).

1) Mg, ether (IBr

CH

3

CH

3

OH

H

3

A

Pcc

CH

2

CI

2 A CM 3 0

H

3

A

NH

2 BuLi ACCN

THF

Raney Ni H 2 NaOH Ethanol A CH 3 N A

NH

3

C

H

3

A

conc. HC1 heat Pd(OH) 2 /C H 2

HCI

Ethanol A CH 3

NH

3

CI

H

3

A

Compound .H1- sal t) 182 MCompound 119 was synthesized in a seven-step reaction sequence starting from commercially-available trans-3fluorocinnamic acid. This synthetic route is conceptually similar to that reported in the literature Patent 4,313,896 (1982)] for related analogs.

However, the three final steps were performed using a significantly different reaction sequence than that reported. The cinnamic acid was reduced and chlorinated in three steps to the corresponding 3-(3-fluorophenyl)propylchloride. This compound was brominated with NBS (N-bromosuccinimide) and the Soo. resulting trihalide was then reacted with S3-fluorophenol. The resulting ether was converted to the final product using a Gabriel synthesis.

Trans-3-fluorocinnamic acid (25.0 g, 150.4 mmol) was dissolved in abs. EtOH (250 mL) and hydrogenated over 10% Pd/C (2.5 g) in a Parr apparatus at 60 psig, 50 0 C, for 1 h (hydrogen uptake: calcd. 245 psig; found 260 psig). The reaction mixture was filtered and evaporated to yield a 20 crystalline product (23.0 g, GC, tR 4.43 min; MS, 168 Under a stream of dry nitrogen, at .0-10 0 C, a solution of 3-fluorohydrocinnamic acid (22.0 g, 131 mmol) in THF (100 mL) was added dropwise, over a period of 15 min, to a suspension of LiAlH 4 (4.23 g, 111 mmol) in THF (200 mL) The reaction was heated to reflux for a period of 1 h and then worked-up according to Fieser Fieser's Reagents for Organic Synthesis (Vol. 1, 1967) to provide a white solid (20.1 g, GC, tR 3.74 min; MS, 154. A solution of 3-(3-fluorophenyl)-l-propanol (15.0 g, 97.4 mmol) and triphenylphosphine (36.0 g, 183 137.3 mmol) in CC14 (150 mL) was refluxed for 19 h.

Additional P(C6Hs) 3 (3 x 3.0 g, 3 x 11.4 mmol) was added periodically over a period of 24 h. The resulting precipitate was removed by filtration and the solids were washed with hexane. The filtrate was evaporated under vacuum and the residue was suspended in hexane (200 mL) and then filtered. Evaporation of the filtrate provided 16.0 g of crude product which was purification by silica gel flash chromatography, elution with hexane, to provide 14.7 g of a colorless liquid. GC, t. 3.63 min; MS, 172/174 A solution of the above chloride (12.0 g, ,o 69.5 mmol), N-bromosuccinimide (17.3 g, 97.2 mmol), and dibenzoyl peroxide (0.06 g) in CC14 (75 mL) was refluxed for 15 1 h. The reaction mixture was then cooled in an ice bath, filtered, and the solids were washed with hexane. The filtrate was evaporated to provide 17.9 g (100%) of product.

GC, t, 5.21 min; MS, 251/253 A mixture of 3-bromo-3-(3-fluorophenyl) 20 -1-propylchloride (4.0 g, 15.9 mmol), 3-fluorophenol (1.98 g, 17.7 mmol), and K2CO 3 (2.65 g, 19.2 mmol) suspended in acetone (80 mL) was refluxed for 15 h. The volatiles were the-removed under vacuum and the resulting residue was suspended.in a mixture of hexane (200 mL) and NaOH (0.1N, 100 mL). The layers were separated and the organic layer washed, 0.1N NaOH (100 mL) and H 2 0 (100 mL), dried (anh. Na 2 and evaporated in vacuuo. The resulting residue was chromatographed on silica gel, elution with hexane followed by hexane/EtOAc [100:1] then [40:1] to provide 1.64 g of product as a colorless oil. -GC, tR 7.28 min; MS, 282/283 TLC r, 0.3, hexane/EtOAc 184 [40:1]) A solution of 3-(3-fluorophenyl)-3- (3-fluorophenoxy)-1-propylchloride (1.52 g, 5.38 mmol) and potassium phthalate (1.20 g, 6.48 mmol) was heated to 90 0

C

in DMF (30 mL) for a period of 2 h in a nitrogen atmosphere.

The reaction mixture was then cooled and poured into H 2 0 (100 mL). The resulting solution was extracted with EtzO (2 x 100 mL). The organic extract was washed, sat.

NaCl (100 mL) and H20 (2 x 100 mL), dried (anh. NaSO 4 and evaporated under vacuum to provide 2.17 g of crude product.

The material was chromatographed on. silica gel, elution wiph hexane/EtOAc [40:1] and then [20:1) to provide after evaporation 1.81 g of product as a glass.

S 5 A solution of N-phthaloyl-3-(3-fluorophenyl)-3 15 -(fluorophenoxy)-1-propylamine (1.74 g, 4.42 mmol) and anh. hydrazine (1.43 g, 44.6 mmol) in abs. EtOH '30 mL) was refluxed for 1 h. The reaction was cooled and evaporated under vacuum. The resulting material was suspended in EtO (75 mL) and washed with 0.2N NaOH (2 x 25 mL). The organic 20 layer was dried (anh. Na 2

SO

4 and evaporated under vacuum to -provide 1.04 g which was purified by reverse-phase chromatography [Vydac Prep. C18; 264 nm; 50 mL/min; gradient elution ACN/0.1% HC1 aq., 10%-50% over 20 min; re 17.4 min], to yield 0.89 g of Compound 119 as a hygroscopic hydrochloride salt.

185 0 0

NI

F-

CI

q0

F

N nc

-NM

3

CI

F

CflWmMrd 1g In( 8") Compounds 118, 120-122 and 137 were prepared in a manner similar to the procedures used for the preparation of Compound 119.

Compound 113 was synthesized from commercially available 4,4-diphenylcyclohexenone in three steps. First, the alkene in the starting material was reduced by means of catalytic hydrogenation. Methoxylamine formation followed by reduction using standard procedures.

The synthesis of Compounds 188 and 189 was accomplished as follows.

186

SNH

2 CIHPLC

NH

2

NH

2

N

k HPLC C Cr HCO HCO

HCOO

Compound 136 Compound iII Compound 1 9 Compounds 188 and 189 The enantiomers of Compound 136 were separated by analytical chiral HPLC. Aliquots (20 kg) were injected onto a Chiralcel-OD-R (Chiral Technologies, Inc., Exton, PA) 5 reversed-phase HPLC column (0.46 x 250 mm) using the following conditions: gradient elution, 40%-70% ACN 0.5N KTFA) over 30 min; flow rate, 1 mL/min; detector, 264 nm. Two identically-sized peaks were collected at 21.0 and 24.4 min. GC/MS analysis of the two samples 10 indicate that both materials have identical GC retention times as well as identical mass spectra.

The synthesis of Compound 151 was accomplished as follows.

foe.

NH

3 F

NH

Compound 151 187 3, 3-Bis(3-fluorophenyl)propanamide (Compound 151) A solution of liquid anh. ammonia (10 mL) in

CH

2 C1 2 (50 mL) at -78 0 C.was treated with a solution of 3,3-bis(3-fluorophenyl)propionyl chloride (2.19 g, 7.81 mmol) in CH2C12 (25 mL). The reaction was then stirred at ambient temperature for 15 min and was then diluted with diethyl ether (500 mL), washed three times with 10% HC1, three times with IN NaOH, and finally once with HO. The organic layer was dried (anh. Na 2

SO

4 and evaporated to give the primary amide as a white solid (2.01 g, 98%).

The synthesis of Compound 156 was accomplished as follows.

*st *Compound 156 mL) was added NaH (60% dispersion, 2.20 g, 55.0 mmol) over a period of 2 min. The reaction was stirred for 10 min and then a solution of dibenzosuberone (10.3 g, 49.6 mmol) (9.66 g, 54.5 mmol) in dry N,N-dimethylformamide

(DMF,

mL) was added NaH (60% dispersion, 2.20 g, 55.0 mmol) over a period of 2 min. The reaction was stirred for 10 min and then a solution of dibenzosuberone (10.3 g, 49.6 mmol) 188 Win dry DMF (10 mL) was added over a period of 2 min. The reaction was stirred at 800C for 4 h under Water (200 mL) was added and the reaction mixture was extracted with Et 2 O (2 x 100 mL). The combined organic layers were rotary evaporated to less than 50 mL. The resulting crystals were collected and washed with cold Et,O (2 x 50 mL) to yield 7.48 g 5-(2-Aminoethyl)-5H-10,11-dihydrodibenzo[a,d]cycloheptene hydrochloride (Compound 156) 5-Cyanomethylidino-10,11-dihydrodibenzo(a,d]cycloheptene was dissolved in EtOH (100 mL). 1N NaOH (10 mL) and Raney® nickel (aq. suspension, 0.50 g) were added. The reaction *iS mixture was shaken under 60 psig H 2 at 50°C for 22 h, and was *0 0 ace then filtered through Celite®. The filtrate was rotary 15 evaporated and the residue was dissolved in Et 2 0 (100 mL), washed with satd. aq. NaCl (50 mL) and H0O (50 mL) The EtO layer was dried (anh. Na 2

SO

4 and rotary evaporated to give the crude product (850 mg) as a colorless oil. This oil was dissolved in EtOAc (5 mL) and filtered. 1.OM HC1 (5 mL) in 20 Et,O was added to the filtrate and a white, crystalline solid precipitated. This material was recrystallized from EtOH 0@ (5 mL)-EtO (12 mL) to yield 600 mg of product as a white powder.

The synthesis of Compound 167 was accomplished as follows.

CH

3 LI CO3 O O 1) T i (i-PrO CH3 OH

OCH

3 2) NaBICN Compound 167 189 2-Methoxypropiophenone A mixture of 2-hydroxypropiophenone (3.00 g, 20.0 mmol), iodomethane (3.40 g, 24.0 mmol), and K 2

CO

3 (granular, anh.; 13.8 g, 99.9.mmol)-was refluxed in acetone (75.mL) for 18 h. The reaction mixture was cooled to room temperature and the inorganic salts were removed by filtration. The filtrate was evaporated under vacuum to give an oil which was subsequently dissolved in diethyl ether (200 mL) and then washed with 0.1N NaOH (3 x 50 mL) followed by H 2 0 (50 mL). The organic layer was dried (anh.

Na 2

SO

4 filtered, and evaporated to an orange oil (3.17 g, This material was used in the following step without further purification. TLC, Rf 0.55 MeOH:l% IPA:CHCl 3 GC, tr 4.58 min; MS, m/z 164 S) (2-methoxyphenylpropyl) -3,3 -diphenylpropylamine (Compound 167) A solution of 2-methoxypropiophenone (0.848 g, 5.17 mmol), 3,3-diphenylpropylamine (1.00 g, 4.70 mmol), and titanium(IV) isopropoxide [Ti(OCH(CH 3 2 4 (1.76 mL, 5.88 mmol, 1.25 equiv)] was stirred at room temperature for 6 h. EtOH- (2 mL) was then added, followed by sodium cyanoborohydride (0.295 g, 4.70 mmol) in portions over a period of 10 min, and the reaction was then stirred for 18 h. The reaction mixture was then poured into diethyl ether (200 mL) and the resulting suspension was centrifuged to remove the titanium precipitate. The supernatant was collected and the pellet was rinsed with diethyl ether (200 mL). The combined organic washings were evaporated under vacuum to give a crude oil which was chromatographed on silica gel (elution with 4% MeOH-CH 2 Cl 2 to provide 647 mg of product. The material was then dissolved in 190 diethyl ether (50 mL), filtered, and excess ethereal Hc1 was added to precipi tate the hydrochloride salt (125 mg, 7.416) as a white. solid; TLC, Rf 0. 25 (411 MeOH-C1 2 C1 2

GC,

tr =11.2 min; MS, rn/z 359 The synthesis of Compounds 172 -176 was accomplished as follows.

CHO

OHOH

N m-FCfiH 4 MgBr F PCC F%

CH

3

CN,

F4F

OCH

3

FF

OcR.

OCH

3 b OC*4 3 .a 1) BH3 S(Cli) 2 2) HLI

NH

2

-HCI

F

Pd/C

KC

B0 5

CE

Compound 173 Compound 174 Compound 176 Compound 175 R, -Diffluoro-4 -me thoxybenzhydrol A mixture of Mg 0 turnings (2.45 g, 101 mrnol), 1-bromo-3-fluorobeflzene (17.6 g, 100 mmol), and dry THE (200 mL) was carefully heated to reflux for 30 min. While still refluxing, 3-fluoro-p-anisaldehyde (15.3 g, 99.3 mmol) in THF (100 mL) was added over a period of 5 min. The reaction temperature was maintained for 30 min, cooled to room temperature, and then the reaction was quenched with .satd. aq. NH 4 Cl (200 mL). The organic layer was separated, washed with satd. aq. NaC1 (2 x 200 mL), dried (anh. Na 2

SO

4 and rotary evaporated to yield 23.5 g of product as an orange-brown oil.

10 3,3'-Difluoro-4-methoxybenzophenone Pyridinium chlorochromate (22.3 g, 103 mmol) was \added to a solution of 3,3'-difluoro-4-methoxybenzhydrol- (23.5 g, 93.8 mmol) in CH 2 C12 (300 mL) and the reaction mixture was stirred for 16 h. Diethyl ether (500 mL) was 15 added and the reaction mixture was filtered through Celite'.

The filtrate was rotary evaporated and the resulting oil was flash chromatographed (gradient elution of hexanes to 1:1 hex-EtOAc). The TLC-pure fractions were rotary evaporated to yield 1.58 g of a white solid. The rest of S 20 the impure fractions containing product were combined and rotary evaporated to the point where crystals began to form.

Additional hexane (300 mL) was added and the crystallizing solution was allowed to stand. The resulting crystals were collected and washed with hexanes (2 x 50 mL) to yield 6.81 g of product. The two batches were combined to afford a total yield of 8.39 g (R,S)-a-Cyanomethyl-3,3'-difluoro-4-methoxybenzhydrol To dry THF (100 mL) was added butyllithium (2.6M in heptane; 16.0 mL, 41.6 mmol) at -78 0 C. Acetonitrile (2.20 mL, 42.1 mmol) was added over a period of 1 min and 192 the reaction was stirred at -78 0 C under N, for 30 min. A solution of 3,3'-difluoro-4-methoxybenzophenone (8.38 g, 33.8 mmol) in anh. THF (50 mL) was added to the reaction over a period of 5 min and the solution was stirred at -78 0

C

for 30 min. The cold bath was removed and the reaction was allowed to warm for 30 min. Satd. aq. NH 4 Cl (100 mL) was added to quench the reaction. The THF layer was separated, washed with satd. aq. NaCl (2 x 25 mL), dried (anh. Na 2

SO

4 rotary evaporated, and dried under vacuum to yield 10.1 g 10 (103%) of product as a yellow oil.

and (3-Fluoro-4-methoxy)-3- (3-fluorophenyl)allylamine hydrochloride (Compound 172) (R,S)-a-Cyanomethyl-3,3'-difluoro-4-methoxybenzhydrol .15 (9.77 g, 33.8 mmol) was dissolved in dry THF (200 mL) and heated to boiling (no condenser). Under a stream of nitrogen, borane-dimethyl sulfide complex (BH 3

,S(CH

3 10.1M; 16.8 mL, 170 mmol) was added carefully over a period of 2 min to the boiling solution. Boiling was then maintained for 15 min until most of the THF was.gone. The reaction mixture was then cooled in an ice bath. Ice (10 g) was carefully added, followed by H 2 0 (50 mL). The reaction was then.-heated to near boiling and 12.1N HC1 (100 mL) was added. The reaction was boiled (no condenser) for 30 min and was then cooled in an ice bath, basified with 10N NaOH (100 mL), and extracted twice with Et20 (200 mL, 100 mL) The combined ether layers were washed with lN NaOH (50 mL) and H 2 0 (50 mL), dried (anh. Na 2

SO

4 and rotary evaporated.

The resulting oil was flash chromatographed (CHC13; 1:100 MeOH-CHCl 3 1:10 MeOH-CHCl 3 through flash silica gel to afford 6.83 g of a yellow oil. This oil was dissolved in 193 EtOH (2 mL) and Et 2 O (10 mL). 1.OM HC1 in Et20 (27 mL) was added and the solution was rotary evaporated to yield 7.10 g of product as a solid, yellow foam.

(3-Fluoro-4-methoxy) (3 -fluorophenyl)propylamine maleate (Compound 173) The mixture of and (Z)-3-(3-fluoro-4methoxy)-3-(3-fluorophenyl)-allylamine hydrochlorides (7.10 g, 22.8 mmol) was dissolved in EtOH (200 mL) and a suspension of palladium on charcoal (10% Pd; 0.71 g) in H 2 0 10 (3.5 mL) was added. The reaction mixture was then shaken *o under 60 psig H 2 for 18 h and subsequently filtered through Celite'. The filtrate was rotary evaporated, the residue was dissolved in EtOAc (25 mL) and Et 2 O (100 mL), and was basified with sat. aq. NaHCO 3 (25 mL). The organic layer was separated, dried (anh. Na 2

SO

4 and rotary evaporated to yield 6.28 g of an oil. This oil and maleic acid (2.59 g) were dissolved into hot EtOAc (100 Diethyl ether (70 mL) was added and crystals soon began to form. The crystals were collected and dried to yield 2.45 g of a white powder. The combined filtrate and washings afforded more crystalline product out upon standing. The second crop was filtered, washed with 1:1 EtOAc-Et 2 O (2 x 25 mL) and Et20--(1-x 25 mL), and dried to provide 3.69 g of a white powder. The total yield was thus 6.14 g (3-Fluoro-4-methoxy) (3fluorophenyl)propylformamide (R,S)-3-(3-Fluoro-4-methoxy)-3-(3fluorophenyl)propyl amine maleate (3.12 g, 7.93 mmol) was free-based in a mixture of EtOAc (25 mL), EtO2 (100 mL), and satd. aq. NaHCO 3 (25 mL). The organic layer was separated, dried (anh. Na 2

SO

4 and rotary evaporated. A solution of 194 W the amine in ethyl formate (75 mL, 930 mmol) was refluxed for 17 h. The reaction solution was rotary evaporated to yield 2.38 g of formamide as a light-orange, viscous oil.

(R,S)-N-Methyl-3-(3-fluoro-4-methoxy)-3-(3fluorophenyl)propyl amine maleate (Compound 174) (R,S)-3-(3-Fluoro-4-methoxy)- 3 3 fluorophenyl)propyl formamide (2.27, 7.43 mmol) in THF.

(100 mL) was heated to boiling (no condenser). Boranedimethyl sulfide complex (10.1M; 2.30 mL, 23.2 mmol) was Sadded carefully over a period of 2 min to the boiling solution. Boiling was then maintained for 15 min. The reaction was then cooled in an ice bath. Ice (10 g) was carefully added, followed by H 2 0 (30 mL), followed by 12.1N HC1 (50 mL). The reaction was then boiled (no condenser) for 30 min. The reaction was subsequently cooled in an ice bath, basified with 10N NaOH (50 mL), and extracted with EtzO (200 mL). The ether layer was washed with satd. aq. NaCl (100 mL), dried (anh. Na 2

SO

4 and rotary evaporated to yield 2.03 g of a yellow oil. This material was purified by RP- HPLC (20-60% acetonitrile-0.1% aq. HC1 over 20 min). The collected fractions were frozen and lyophilized to yield 1.28 g of a white solid. The free-base of the purified amine was dissolved in EtOAc. Maleic acid (305 mg) was added and the mixture was heated until everything had dissolved. The product was crystallized by adding mL). The crystals were filtered and washed with 195 1:1 EtOAc-Et 2 O (10 mL) followed by Et2O (10 mL) to yield 967 mg of product as a white, finely crystalline solid.

S) 3 (3-Fluoro 4 -hydroxy) -3-(3-fluorophenyl)propylamine maleate (Compound 175) (R,S)-3-(3-Fluoro-4-methoxy)-3-(3fluorophenyl)propyl-amine maleate (2.45 g, 6.23 mmol) was free-based in the normal manner and dissolved in CH 2 Cl 2 mL). The resulting solution was cooled to -78 0 C. Under

N

2 flow, boron tribromide (1.OM in CH 2 Cl,; 15 mL, 15 mmol) o 10 was added over a period of 5 min. The cold bath was removed and the reaction mixture was allowed to warm to room temperature. After 30 min at 25 0 C, the reaction was hydrolyzed with 12.1N HC1 (10 mL). The aqueous layer was neutralized (pH 7) by the careful addition of 10N NaOH 15 (14 mL). Satd. aq. NaHC03 (50 mL) was added along with EtzO (100 mL) and EtOAc (20 mL). This mixture was shaken vigorously and the organic layer was separated. The aqueous layer was extracted with EtOAc (20 mL). The combined organic layers were dried (anh. Na 2 SO,) and rotary evaporated. The resulting oil was dissolved in EtOH, .1.OM HC1 in Et 2 O (7 mL) was added, and the solution was rotary evaporated. This material was then purified by RP-HP-LC- [20-60% acetonitrile-0.1% HC1 over 20 min] The collected fractions were frozen and lyophilized, affording 716 mg of a white solid. The free-base of the purified amine (315 mg) was dissolved in EtOAc. Maleic acid (138 mg) was added and the mixture was heated until everything dissolved. The EtOAc was rotary evaporated to give a hard glass which was dissolved in MeOH (5 mL). Water (100 mL) was then added and the solution was subsequently frozen and lyophilized. The above procedure yielded 445 mg 196 of product as a white solid.

-N-Me thyl 3 (3 fluoro 4 -hydroxy) 3 (3fluorophenyl)propyl amine hydrochloride (Compound 176) A solution of (R,S)-N-methyl-3-(3-fluoro-4methoxy)-3-(3-fluorophenyl)propylamin e (703 mg, 2.41 mmol) in CH 2 C1 2 (10 mL) was cooled to -780C. Under nitrogen, boron tribromide (1.OM in CH 2 C1 2 6.0 mL, 6.0 mmol) was added over a period of 5 min. The cooling bath was then removed and the reaction was allowed to warm to room temperature. After S 10 1 h, the reaction was quenched with 12.1N HC1 (5 mL). The.

aqueous layer was then neutralized (pH 7) by the careful addition of 10N NaOH mL). Satd. aq. NaHCO 3 (25 mL) was added along with Et 2 O (50 mL), EtOAc (15 mL), and CHC13 mL). This mixture was shaken vigorously, and the organic 15 layer was separated, dried (anh. Na 2

SO

4 and filtered e through paper. The crude product was then purified by RP-HPLC (20-60% acetonitrile-0.1% aq. HC1 gradient over.

20 min). The fractions were frozen and lyophilized to afford 602 mg of product as a white solid.

20 The synthesis of Compound 185 was accomplished as -follows.

I a m.FCLtOH 0J£ a 0 F O

F

OH6H

DEAD

Compound 185 (3-Fl uorophenoxy) -3 -phenylpropyl chloride Following a similar procedure for the chiral synthesis of fluoxetine [Srebnik, M. et al., J. Org. Chem.

53(13), 2916-20 (1988), hereby incorporated by reference herein], a solution of (S)-(-)3-chloro-l-phenyl-l-propanol 197 (4.00 g, 23.4 mmol), 3-fluorophenol (2.63 g, 23.4 mmol), and Sdiethyl azodicarboxylate (4.00 g, 23.4 mmol) were dissolved in THF (200 mL). The mixture was cooled to 0°C and triphenylphosphine (6.77 g, 25.8 mmol, 1.1 equiv) was added slowly over 10 min. The reaction mixture was then stirred at room temperature for 18 h. The THF was subsequently evaporated under vacuum to afford a gel which was washed with pentane (3 x 50 mL). The pentane washings were filtered and the filtrate was evaporated under vacuum to 10 give a clear oil. This oil was dissolved in diethyl ether (150 mL) and washed with 1% HCl-satd. NaCI (25 mL), 0.1N NaOH-satd. NaCi (2 x 25 mL), and finally H 2 0 (2 x 25 mL).

The organic layer was then dried (anh. Na 2

SO

4 filtered, and evaporated to dryness under vacuum to give an orange oil.

15 The crude product was chromatographed on silica gel 0 x 180 mm, gravity column), elution with 40:1 hexane-EtOAc, to provide 971 mg of product as a colorless oil.

(3-Fluorophenoxy)-3-phenylpropylamine 20 (Compound 185) *A solution of (R)-3-(3-fluorophenoxy)-3phenylpropyl chloride (0.971 g, 3.96 mmol), conc. NH 4

OH

and EtOH (20 mL) were shaken at 90 0 C on a Parr' apparatus.(50-90 psig) for 18 h. The mixture was then evaporated under vacuum and the residue was dissolved in EtzO (100 mL) and washed with H0O (2 x 25 mL). The organic layer was dried (anh. Na 2

SO

4 filtered, and evaporated under vacuum to provide a yellow oil. This material was then dissolved in EtOAc (50 mL) and filtered. A solution of maleic acid (0.272 g, 2.6 mmol, 0.93 equiv) dissolved in hot EtOAc (5 mL) was added to precipitate the maleate salt 198 (519 mg, 53.5%) as a white solid: TLC Rf 0.25 (1% MeOH-CHC1 3 GC, t, 7.37 min; MS, m/z 245 The synthesis of Compound 187 was accomplished as follows.

ScN

N

NCCHPO(OEtj C Al(Hg) C LAH Compound 187 11-Cyanomethylene- 1G,11 -dihydrodibenzo coxepine To a solution of diethyl cyanomethylphosphonate (5.06 g, 28.6 mmol) in dry DMF (15 mL) was added NaH mineral oil dispersion; 1.14 g, 28.5 mmol) over a period-of 2 min. The reaction was stirred for 10 min and then a solution of 6,11-dihydrodibenzo b,c]oxepin-11-one [Kurokawa 10 M. et al., Chem. Pharm. Bull. 39(10), 2564-2573 (1991), hereby incorporated by reference herein] (4.00 g, 19.0 mmol) in dry DMF (5 mL) was added. The reaction mixture was stirred under argon for 21 h. Water (100 mL) was then added and the product was extracted with EtOAc (2 x 50 mL). The S 15 combined organic layers were washed with satd. aq. NaCl (2 x 50 mU dried (anh. Na 2

SO

4 and rotary evaporated. The resulting solid was recrystallized from hot EtOAc (10 mL)hexanes (40 mL) to provide 2.43 g of product.

11-Cyanomethyl -11H-1O, 1 -dihydrodibenzo[b,c]oxepine Following a procedure described in Great Britain Patent 1,129,029 (1968) (Chem. Abstr. 70:37664), hereby incorporated by reference herein], for the preparation of aluminum amalgam, A10 granules (2.00 g, 74.1 mmol) were first etched with 0.5N NaOH (100 mL) and then washed with (100 mL) followed by EtOH (100 mL). A solution of HgCl 2 (2.00 g, 7.37 mmol) in Et20 (100 mL) was added. The reaction 199 mixture was stirred for 5 min and the supernatant was decanted. The solid Al(Hg) amalgam was washed with H 2 0 (100 mL), EtOH (100 and then Et20 (100 mL). The amalgam was covered with Et 2 O (100 mL) and a solution of ll-cyanomethylene-10,11-dihydrodibenzo[b,c]oxepine (2.00 g, 8.57 mmol) in EtOAc (30 mL) and EtOH (20 mL) was added.

Water (2 mL) was added and the reaction mixture was stirred for 18 h and then filtered. The filtrate was rotary evaporated to yield 1.65 g of product as a white, crystalline solid.

ll-(2-Aminoethyl)-11H-10,ll-dihydrodibenzo[b,cloxepine hydrochloride (Compound 187) To a stirring suspension of lithium aluminum hydride (0.67 g, 18 mmol) in anh. Et,O (30 mL) was added a solution of 11-cyanomethyl-11H-10, 11-dihydrodibenzo[b,c] oxepine (1.65 g, 7.01 mmol) in dry THF (5 mL)/anh. mL) over a period of 2 min. The reaction was stirred for 30 min. In the following order, H 2 0 (0.7 mL), 5N NaOH (0.7 mL), and H 2 0 (2.1 mL) were added to the reaction mixture. Diethyl ether (30 mL) was added and the mixture was filtered. The filtrate was rotary evaporated and the resulting oil was dissolved in EtOH (10 mL)-Et20 (65 mL).

1.0M- -H1 in Et20 (10 mL) was added and the solution was allowed to crystallize, giving 1.42 g (73.4 of the title compound.

Compounds 67-68, 70-75, 79-82, 84-89, 91-95,.98- 100, 102, 104-106, 109-114, 117, 124-134, 138, and 140-150 were synthesized by standard procedures known to those skilled in the art, as described above.

Gas Chromatography of Simplified Arylalkylamines Gas chromatographic and mass spectral data were 200 obtained on a Hewlett-Packard 5890 Series II Gas Chromatograph equipped with a 5972. Series Mass Selective Detector [Ultra-2 Ultra Performance Capillary Column (crosslinked 5% phenyl methyl silicone); column length, 25 m, column 0.20 mm; The flow rate, 60 mL/min; injector temp., 2500C; gradient temperature program, 200C/min from 125 to 3250C for 10 min, then held constant at 3250C for 6 min).

Compound 19. (Rt 7.40 min), m/z (rel. int.) 211 195 (16) 194 (100) 193 (73) 180 179 (33) 178 (19), 168 (24) 167 166 (23) 165 (72) 164 153 152 117 116 115 106 104 (14), 103 102 91 78 77 63 51- (17) Compound 20. (Rt =7.34 min), m/z (rel. int.)' 247 231 (16) 230 (100) 229 (45) 215 (29) 214 (14) 1204 (43), 203 (37),'202 201 184 183 181 151 135 (13) 134 133 124 122 (16), o 121 (19) 2.09 (15) 101 (29) 96 (18) 95 (11) 83 (11) 57 42 (9) Compound 21-; (Rt =-7.53 min), m/z (rel. int.) 261 (M+i,69), 262 (13) 245 (17) 244 (100) 230 (11) 229 (42) 216 (11), 215 214 (14) 204 (45) 203 (35) 202 (16) 201 (63), 184 4(124 183 (61) 148 (11) 136 135 (27) 133 (36) 124 (21) ,,115 (16) 109 (43) 83 (12) 74 58 (14) 57 (11) Compound 22. (Rt =7.37 min), m/z (rel. int.) 261 244 229 204 203 201 183 (16), 138 133 109 101 75 58 57 44 (100) 42 (7) Compound 24. (Rt 8.21 min), m/z (rel. int.) 259 (M+,122), 260 242 241 228 227 216 201 213 212 211 199 196 185 (34), 184 183 171 170 165 151 150 146 136 134 133 123 121 (22) .120 109 (100), 91 77 51 Compound 25. (Rt 8.49 min), m/z (rel. int.) 259 243 242 241 227 217 216 (100), 215 212 211 201 200 199 196 185 184 183 171 170 (28), 165 146 136 134 133 121 (21), 77 (9) Compound 26.. (Rt 8.69 min), m/z (rel. int.) 259 243 242 (100), 241 227 215 212 211 184 183 172 171 170 (23), 165 (13),+147 146 134- (19) 133 121 (13), 91 (11, 77 Compound 27. (Rt 8.80 min), m/z (rel. int.) 243 226 212 21 1 200 199 198 197 (100), 196 185 184 183 179 (13), 178 165 134 133 120 117 (16), 20 115 (27) 104 (13) 101 (11) 91 (23) 77 (13) Compound 2S8. (Rt 8.77 min), m/z (rel. int.) 243 227 226 225 212 211 (100), 200 (22), .:199 (1 79, 197 196 185 (46) 184 (35) 183 (64), 179 165 134 133 121 120 (18), 117 115 101 91 77 65 51 (9) Compound 29. (Rt 7.89 min), m/z (rel. int.) 243 227 226 225 212 211 (100), 199 (13)f 197 196 185 184 183 .179 134 (11) 133 120 117 115 91.(14) Compound 30. (Rt =8.36 min), mlz (rel. int.) 263 202 -qw 246 220 (13) 212 (17) 211 (10.0) 197 (10) 196 185 (43) 184 (30) 183 (69) 181 165 133 (18), 115 101 (15) 75 Compound -31. (Rt 9.31 min), m/z (rel. int.) 279 281.(11) 262 (10) 236 (10) 229 (33) 228 (17) 227 (100), 203 201 (33) 199 (15) 192 (15) 178 (19) 166 (18), 165 164 163 140 115 103 (9) Compound 32. (Rt 7.30 min), m/z (rel. int.) 229 213 212 (100), 211 197 196 194 (14), 186 (26) 185 (30) 184 (19) 183 (69) 170 (17) 166 (16), 165 134 133 116 115 103 (18), (11) 78 77 75 51 43 (13) 42 (13) Compound 33. (Rt =7.56 min), m/z (rel. int.) 261 245 (18) 244 (100) 229 (43) 215 (16) ,214 (15) ,204 (57) *203 202 201 184 183 148 (16), *136 1-35 133 (60) 124 (51) 115 (27) 111 (14) 109 107 96 (14) 83 (27) 75 (20) 58 (96),:57 56 (23) 41 Compound 34.. 7.39 min), m/z (rel. int.) 261 262 (14) 245 (18) 244 (100) 229 (42) 216 215 214 (14) 204 (52) 203 202 (14) 201 (54) 184 (12), :183 181 (10) 148 (13) 136 135 (31) 133 124 115 109. 107 83' 58 57 (11) Compound 35. (Rt 4.45 min), m/z (rel. int.) 181 165 164 138 136 135 133 (12), 123 122 121 110 109 (100), 101 (13), 96 83' (14),.75 56 45 (21) 44 (40) 42 41 Compound 37. (Rt 4.87 min) m/z (rel. int.) 196 203 195 178 163 152 150 137 (12), 136 135 133 124 123 122 (49), 121 110 109 (100), 101 96 83 (17), 56 55 45 44 43 41 Compound 38. (Rt 7.68 min), m/z (rel. int.) 275 203 201 183 135 133 109 71 45 44 (100), 42 (4) Compound.39. (Rt 7.67min), m/z (rel. int.) 289 203 201 183 135 133 109 85 59 58 (100) Compound 40. (Rt 7.63min), m/z (rel. int.) 289 203 201 183 152 135 133 109 85 70 58 (100) Compound 41. (Rt 7.93 min), m/z (rel. int.) 275 258 203 202 201 184 183 (59), 181 150 149 147 135 134 (14), 133 124 123 109 107 103 83 75 72 (100), 71 57 56 (41) o Compound 43. (Rt 9.18 min), m/z (rel. int.) 293 276 243 (i1) 241 236 235 201 (18), 199-(m22 179 (11) 178 176 166 165 164 163 103 102 75 44 (100), 43 42 Compound 46. (Rt 9.34 min), m/z (rel. int.) 293 295 276 243 242 241 237 (12), 236 201 199 178 176 166 (31), 165 (100), 164 163 152 151 149 (12), 140 139 129 127 125 117 (26), 116 115 91 89 77 75 (22),+63 204 (14) 58 (51) 57 (15) 56 (19) 41 (19) compound 50. (RL 7.37 min), m/z (rel. int.) 261 244 229 204 203 201 183 101 58 44 (100) 42 (7) Compound 51. (Rt 7.30 min), m/z (rel. int.) 261 244 229 204 203 202 201 183 133 121 101 75 58 44 (100), 43 42 (11) Compound 52. (Rt 7.24 min), m/z (rel. int.) 247 231 230 229 216 215 214 (16), 204 (29) 203 (31) 202 (16) 201 (63) 196 (21) 184 (20)4 183 (100), 182 181 170 151 150 (11 135 134 133 124 122 121 (21), 109 (13) 101 (27) 96 (21) 75 (23) 43 (14) 42 Compound 53. (Rt 7.21 min), m/z (rel. int.) 247 248 231 230 229 215 214 (16), 203 (33)1- 202 (16) 201 (68) 196 (26) 184 (16) 183 (100) 181 (15) 151.(21) 150 135 (14) 134 (35) 133 (24), 124 (19) 122 (23) 121 (25) Ill (13) 101 (31) 96 (19), 75 (19) Compound 55. (Rt 7.86 min) m/z (rel. int.) 275 276 258 229 216 215 214 (19), 204 203 (41) 202 (21) 201 (82) 184 (18) 183 (100) 181 150 (21)'l 135 133 124 115 (13), 109 (90) 101 (15) 83 (20) 75 (16) 72 (23) 57 (13) 56 (24) Compound 56. (Rt 7.79 min) m/z (rel. int.) 261 262 244 229 218 217 216 (19), 215 (100) 214 (45) 203 202 (32) 201 (51) 197 (16), 196 183 13 8 135 134 133 (39), 122 121 109 101 96 83 205 (13) Compound 57. (Rt 7.65min), m/z (rel. int.) 261 244 229 218 217 216 215 (100), 214 203 202 201 197 196 (19), 183 138 135 134 133 122 (29), 109 (25) 101 (13) Compound 58. (Rt 8.15 min), m/z (rel. int.) 275 (M+,134), 276 258 244 243 (100), 232 229.(53), 217 216 (23) 215 214 (97) 201 (44) 197 (21), 196 (43) 183 (23) 148 (38) 147 (21) 138 (46) 135 (46.) 134 (18) 133 (64) 125 (25) 123 (28) 122 (81) 115 (27) 109 107 83 44 43 (19).

Compound 59. (Rt =7.61 min), m/z (rel. int.) 275 27) 204 203 (10) 201 (19) 183 (25) 109 101 58 (100), 57 56 44 (9) Compound 60. (Rt 7.34 min), m/z (rel. int.) 261 262 204 203 201 183 133 (11), 122 (11) 121 (10) 109 101, (16) ,*96 (11) 75 (10) 57 44 (100), 42 (11) Compound 61.. (Rt 8. 07mn) in/z (rel. jut.) 277 68), 278 260 246 245 234 231 (32), 229 (26) 217 (20) 203 (23) 201 (24) 188 (12) 183 (22), 154 151 150 133 124 (10) 109 (100) 44 (14) Compound 62. (Rt 8.93 min), m/z (rel. int. 271 115) 272 (22) 254.(16) 239 (22) 225 (36) 223 (40) 181 (33), 165 153 152 136 132 (13) 131 (16) 123 122 121 (89) 1.19 (13) 115 (23) 105 (17) 91 (100) 77 (22.) Compound 63. (Rt 8.47mmn), m/z (rel. jut.) 287 241 204 (27) 203 (20) 202 201 (30) 183 (38), 206 150 (13) 133 (20) 109 (27) 84 (45) 83 (43) 82 (11) 57 (18) 56 (100) 43 Compound 64. (Rt =8.57 min), 288 270 242 (16), 204 (35) 203 (27) 202 (18), 147 (16) 146 (17) 135 (16), 83 82 75 57 Compound 65. (Rt 8.18 min), 240 222 208 (18), 192 181 180 (32), 165 152 130 (36), 116 115 107 (20), 91 (37) 77 (20) 65 (17) Compound 66. (Rt 7.46 min), M/z 241 201 133 (21) m/z 207 179 129 105 (rel. int.) 287 215 214 (18), 183 150 (45) 109 (45) 84 (31), 156 (100), 43 (44) (rel1. 2 39 88) (100) 195 (24) 193 (48), 178 166 (16) 120 117 (34), 104 103 (11),

S

m/z (rel. int.) 275 201 183 133 109 71 45 44 (100)Y, 42 (3) Compound 67. (Rt 7.56 min), m/z (rel. int.) 225 194 193 (12) 179 168 (10) 167 (12) 166 165 152 120 116 115 103 77 51 44 .(100) Compound 68. (Rt 7.85 min), m/z (rel. int.). 239 194 193 168 167 (12),.166 165 (19), 152 134 116 115 91 77 59 58 (100), 44 (8) Compound 69. (Rt 7.35mmn), m/z (rel. int.) 275 ,11), 203 202 201 183 122 121 101 58 (100) 57 56 Compound 72. (Rt 7.90 min), m/z (rel. int.) 253 238 193 168 167 (14) 165 (17) 152 .115 91 73 72 (100), 58 56 44 43 42 (8) 207 Compound 73. (Rt 7.29 min), m/z (rel. int.) 239 240 167 165 152 115 77 59 58 (100), 44 42 Compound 74. (R~t 8.01l min), m/z (rel. int.) 267 7) 167 165 152 91 87 86 (100) 72 58 (10) 56 42 (4) Compound 79. (Rt =.7.89 min), m/z (rel. int..) 230 214 (15) 213 (100) 212 (62) 201 (26) 200 (72) 198 (21), 195 188 187 .186 185 184 157 135 133 109 107 106 (62), 79 78 51 Compound 81. (Rt 7.40 min), m/z (rel. int.) 209 (M+,891, 210 208 (100), 193 192 191 189 (12), 178 166 (11) 165 (4 5) 152 132 131 15 130 117 (22) 115 (48) 106 105 104 (12), 103 (16) 91 (16) 77 (22) 51 Compound 82. (Rt 7. 93mn) m/z (rel. int.) 275 124), *276 232 215 214 204 (14) 203 (100), 291 196 183 150 138 136 (14), 135 133 125 124 123 121 (14), .115 111l (72) 110 109 101 (14) 83 (8) *Compound 83. (Rt 7.22 min), m/z (rel. int.) 235 219 (17),-218,(100) 217 203 (20) 192 (10) 191 (38) 190 189 185 (17) 183 171 165 147 146 (11) 134 (12) 133 (17) 121 109 97 45 (7) Compound 85. (Rt 7.73 min), m/z (rel. int.) 239 222. (15) 179 178 168 (16) 167 166 (12) 165 (43) 161 152 (20) 146 (17) 129 120 (15) 118 117 (19) 115 (25) 91 (25) 77 72 44 208 (100), 42 (6) Compound 86. (Rt 7.66 min), m/z (rel. int.) 239 222 168 167 166 165 152 120 117 115 91 72 44 (100), 42 (3) Compound 87. (Rt 7.33 min), m/z (rel. int.) 239 222 179 178 168 167 166 (13), 165 161 152 146 128 120 (11), 118 117 115 91 77 72 51 44 (100), 42 (9) Compound 88. (Rt 7.4 min), m/z (rel. int.) 227 183 168 167 (100), 166 165 164 163 153 152 139 115 105 77 51 45 (23) Compound 89. (Rt 8.74 min), m/z (rel. int.) 260 (M+,220), 261 259 242 203 202 201 (61), 183 165 (100), 150 148 138 137 (61), 122 121 111 101 96 75 44 43 (29) SCompound 90. (Rt 7.32min), m/z (rel. int.) 235 219 218 (100), 217 206 205 204 203 202 193 192 191 190 189 185 171 159 147 146 134 133 121 109 101 97 Compound 91. (Rt 10.67 min), m/z (rel. int.) 329 301 300 167 166 165 152 132 120 119 118 117 115 (11), 106 105 104 103 92 91 77 41 (6) Compound 92. (Rt 10.37min), m/z (rel. int.) 337 209 338 204 203 201 183 (10)f' 133 121 120 106 92 91 (100) Compound 93. (Rt 10.25 min), m/z (rel. int.) 351 352 337 336 203 201 183 (17), 135 134 133 132 120 118 109 106 105 (100) 104 (13j, 103 91 79 77 (12) Compound 94. (Rt 10.48 min), m/z (rel. int.) 365 337 (25) 336 (100) 203 201 183 (14) 133 132 120 119 118 115 109 106 104 (10) 91 (52) Compound 95. (Rt 6.68min), m/z (rel. it.) 283 .0:284 267 266 265 251 250 241 240 (100), 239 237 232 220 (17), 219 199 152 151 142 140 (13), *139 127 (22) 119 (24) 114 (12) 101 (10) 63 @00 44 (9) Compound 96. (Rt =6.93 min), m/z (rel. init.) 265 249 (16) 248 (100) 247 (34) 233 (27) 232 (11) 223 20 222 221 (3 220 (10) 219 202 201. (54),

P

152 .151 133 124 119 109 101 90*0(14), 75 (9) :Compound 97. (Rt 8.10 min), m/z (rel. int.) 241 (M+,101), 242 224 223 210 209 197 (12), 196 195 194 193. 181 178 167 (38) 166 (16) 165 (52) 153 (12) 152 (36) 136 (27), 133 132 116 115 (25) 103 91 (100), 77 (18) Compound 98. (Rt 6.69 min) m/z (rel. int.) 232 204 203 202 201 (100), 188 184 (14), 183 (84) 182 (10) 181 170 109 107 210 83 75 57 (7) Compound 99. (Rt 6.75 min), m/z (rel. int.) 233 204 (12) 203 (68) 202 (26) 20. (100) 200 188 184 183 182 181 170 133 109 107 83 81 75 57 (9) Compound 100. (RL 7.66 min), m/z (rel. int.) 261 (M+,150), 262 (29) 217 (11) 216 (70) ,.215 (28) 214 (11) 203 202 (31) 201 (100) 196 (10) 184 (15) 183 (90) 181 (11), 133 (20) 124 (12) 122 (20) 109 (39) 101 (14) 83 75 (10) 45 (43) Compound 101. (Rt 7.72 min), m/z (rel. int.) 245 229 (16) 228 (100) 227 (36) 213 (21) 211 (22) 202 (57), 201 (30) 199 (21) 183 (50) 181 (14)t, 171 (15) 170 (26), 165 (12) 152 (21) 134 (19) 133 (35) 122 (28) 120 15 120 119 109 107 106 101 94 (15) 91 (20) 77 (18) 74 (15) 65 (20) 63 (14) 51 (15) 44 (27),t 43 (17) 42 (14) Compound 102. (Rt 8.33 min), m/z (rel. int.) 273 204 203 201 183 177 133 109 70 69 (100), 68 43 42 41 Compound 103. (Rt =8.59 min), m/z (rel. int.) 245 (M+,118), *246 -(-2D4 229 (15) ,228 (100) 227 (85) 213 (27) 211 (23), 209 (15) ,-207 (12) 202 (19) 201 (32) 200 (17) 199 (84), 196 (10) 183 (38) 181 (15) 171 170 (23) 152 (19), 151 (15) 150 (10) 134 133 (32) 131 (12) 122 (36), 119 (15) 109 (24) 107 106 (12) 91 (19) 77 (12) Compound 104. (Rt 7.72 min), m/z (rel. int.) 261 262 (17) 217 (15) 216 (92) 215 (18) 204 (12) 203 (86), 202 (25) 201 (100) 184 (10) 183 (69) 148 (12) 133 (13), 122 109 (26) 101 83 45 (33) 211 Comp~ound 105. (Rt 10. 24 min), m/z (rel. it.) 351 7), 201 183 135 134 133 109 92 91 (100) 65 42 (7) Compound 106. (Rt 7.52 min), m/z (rel. int.) 259 (M+i,77), 260 258 244 228 227 214 (14), 201 -165 164 (100), 162.(29), 133 109 (44), (13) 44 (80) 42 (56) Compound 107. (Rt 7.45 min), m/z .(rel. int. 227 101) 228 (16) 226 (100) 211 (22) 210 (68) 209 (49) 207 (13), 196 (22) 184 (15) 183 150 (50) 148 (31) ,'133 (44), 132 (53) 130 117 (15) 115 (29) 106 77 (18), 52. (14) Comp~ound 108. (Rt =7.46 min) m/z (rel. int.) 243 244 212 211 197 186 185 184 183 (19) 165 (15) 133 120 1.03 77 44 (100) 2(6) Compound 109. (Rt 8.68 min), m/z (rel. int.) 285 (M+,110), 286 284 256 228 227 225 220 207 201 191 190 (100), 163 (11), 162 (85) 161 (10) 147 (11) ,146 133 (32) ,109 83 (12) 8.2 (36) Compound 110. (R~t 8.66 min), m/z (rel. int.) 285 286 284 (100), 243. (16),1 227 (26) 225 (11) 221 (10) 220 214 207 201 147 146 (16), 133 (17) '109 (20) 42 Compound (Rt =8.81 min)-, m/z (rel. int.) 287 (M+4,29) 214 204 203 202 201 183 (42), 135 3 133 (28) 109 84 (47) 83 (100), 82 (19) 70 (16) 68 (13) 57 56 (28) 44 (16) 43 42 (14) Compound 112. (Rt 8.85 min), m/z (rel. int.) 287 (M+i,141), 212 288 (29) 286 202 201 183 133 (23), 109 84 (100), 83 82 57 56 43 42 Compound 113. (Rt 9.08 min), m/z (rel. int.) 251 180 (38) 179 (36) 178 (39) 174 (15) 173 (100) 166 (11), 165 158 152 132 115 91 82 77 56 51 43 (23) Compound 114. (Rt 8.71 min), m/z (rel. int.) 237 (M+i,197), 238 (37) 236 (67) 193 (15) 179 (30) 178 165 (41), 159 158 132 130 116 .115 (37), 106 103 91 77 57 56 (100), 51 (32) 43 (50) 42 (34) Compound 115. (Rt 9.45 min), rn/z (rel. int.) 271 255 254 253 239 229 228 (100), 227 224 223 213 212 211 197 (34) 196 (17) 195 (11) 181 (18) 169 (10) 165 (22), 153 152 146 145 141 139 136 (22) 134 (11) 133 (41) 122 (16) 121 (31) 115 91 (18) 77 (15) 65 (11) 63 (10) 44 Compound 116. (Rt 9.50 min), m/z (rel. int.) 269 268 (32) 254 253 (21) 252 (100) 251 (14) 238 (23), 237 (18) 221 (10) 209 178 165 (19) ,+162 (22), 5:160 (19) 152 (18) ,147 (11) 146 ,145 (18) 139 (9) 130 (11) 115 Compound 117. (Rt =7.64 min), m/z (rel. int.) 212 183 (16) 182 (100) ,-180 ,,167 152 104:(27) 91 78 77 (41) 51 (13) Compound 118. (Rt 7.46 min), ni/z (rel. int.) 245 153 152 150 135 133 124 123 122 121 109 101 96 (16) 94 (100) 93 83 77 (21) 75 (11) 66 213 (30) 63 (10) 51 (14) 50 (6) Compound 119. (Rt 7.39 min), m/z (rel. it.) 263 171 170 152 151 150 141 135 133 (23) 123 122 (100), 121 120 (11), 113 112 111 109 107 103 (13), 102 101 97 96 95 94 84 83 82 81 77 75 74 69 64 63 57 56 51.

50 42 (8) Compound 120. (Rt 8.48 min), m/z (rel. mnt.) 279 159 157 153 152 (100), 150 133 (114, 130 128 123 122 121 111 109 101 99 96 95 83 1P 0. 73 65 64 63 51 50 (8) 15 Compound 121. (Rt =8.30 min), m/z (rel. int.) 275 152 125 124 (100), 122 121 109 *96 95 (10) ,'81 (14) 77 65 52 (11) Compound 122. (Rt 7.39 min), m/z (rel. int.) 263 170 152 151 150 141 135 133 123 122 121 112 (100), 11l (18), *109 -107 103 102 101 96 92 83 81 77 75 64 ,*63 57 (61) 56 (14) 51 (14) 50 (11) Compound_123. (Rt 5.88 min), m/z (rel. int.) 275 276 202 201 183 133 109 101' 71 59 (12) 58 .(100) 44 42 (26) Compound 124. (Rt 7.05 min), m/z (rel. int.) 229*(M+,15), 213 (15)1 212 211 198 197 (100), 196 (24), 186 185 184 183 179 178 177 176 171 170 169 166 165 152 133 75 57 56 (4) 214 Cormpound 125.

226 209 178 (20) 165 104 (94) r 103 (13) 51 (20), Compound 126.

244 (31) 152 133 (21) 122 83 (27), Compound 127.

208 (20) 207 178 (36) 167 130 (26) 129 65 (7) 15 Compound 128.

212 15) 194 179 178 152 (24) 120 7.54 min), m/z (tel. int.) 225 208 193 180.(14)., 179 (21), 130 117 115 105 (18), (45) 91 (100) 78 (30) 77 (38) 65 (36) 63 45 (17) (Rt 7.81 min), m/z (rel. int.) 261 151 150 136 135 (100), 115 110 109 107 96 56 (7) (Rt 7.93 min), m/z (rel. int.) 225 193 (13) 181 180 179 (100), 166 165 152 134 117 (18) 115 (22) 104 91 (38) 77 060 *set 00 *too *00 40 .00 (Rt (36) (60) (39) 7.42 min), m/z (rel. int.) 211.(M+,83), 193 (18) 182 181 (20) 180 (17), 176 (11) 167 (57) 166 (44) 165 *(100), 116 (12) 115 (28) 104 (22) 103 91 (46) 89 (16) 78 (10) 77 (20) 65 (15) 63 (12) 51 (12) Compound 1-29. (Rt =7.39 min), m/z (rel. int.) 229 230 212 211 201 200 199 (22), 198 (4:,197 196 185 184 183 (100), 179 178 177 176 170 165 (33), 152 (12) 133 (22) 120 (57) 115 (17) 109 (44) 104 (23), 103 91 89 83 78 77 63 (16) 51 (13) Compound 130. (Rt =7.38 min), m/z (rel. int.) 229 (M+,133), 230 212 211 (1 200 199 198 (16), 197 (53) 196 (64) 185 (49) 184 (43) 183 (100) 179 (28), 178 177 170 165 133 120 215 115 109 104 103 91 89 83 77 63 (16) Compound 131. (Rt 7.40 min), m/z (rel. int.) 229 (M+,146), 230 212 211 200 199 198 (16), 197 196 185 184 183 (100), 179 (28), 178 170 165 133 120 115 109 104 103 91 89 83 77 (22) Compound 132. (Rt 7.03 min), m/z (rel. int.) 0 185 184 (100), 183 181 165 155 (12), 153 152 120 119 115 106 (16) 91 89 78 77 51 (16) Compound 133.

195 194 166 165 115 104 77 (42) Compound 134.

183 182 166 165 89 Compound 135.

257 (-14 256 213 211 183 181 134 133 106 91 Compound 136.

277 260 234 233 224 223 (Rt 7.09 min), m/z (rel. int.) 211 (100) 181 (27) 180 179 (31) 178 (28), 152 120 119 118 (12), 103 102 .91 89 78 65 51 (13) (Rt 7.45 min), m/z (rel. int.) 211 (100), 181 179 178 167 (27), 152 115 104 103 91 78 77 65 (7) (Rt 8.60 min), m/z (rel. int.) 273 231 230 (100), 228 227 (57), 202 201 199 184 (13), 171 170 152 150 (19), 122 121 109 107 (13), 65 (12) (Rt 9.26 min), m/z (rel. int.) 275 259 258 257 243 232 (100), 231 229 (15) 227 (42), 208 197 196 195 (13), 216 182 (14) 181 (33) ,179 (11) ,178 (18) ,166 (22) ,165 164 (12) ,163 (10) ,153 (32) ,152 (55) ,151 (18) ,149 139 (11) ,137 (17) ,136 (19) ,121 (13) ,115 (25) ,102 (11) 91 (16) 77 (17) Compound 137. (Rt =7.42 min), rn/z (rel. int.) 245 153 152 141 135 134 (100), 132 (11), 117 115 112 105 104 103 (32), 91 84 83 78 77 75 63 57 51 (9) Compound 138. 9.24 min), m/z (rel. int.) 289 (M+,77)t 290 (16) 230 (20) 229 (21) 215 (15) 203 (22) 201 (32) 183 134 133 124 121 109 (10) 73 (100) ,43 (23) Compound 139. (Rt =7.25 min), m/z (rel. int.) 245 246 (15) 244 (67) 229 (16) 228 (63) 227 (46) 225 224 214 201 183 151 150 (100), 149 (14) 148 (58) 135 (22) 133 (54) 124 (14) 122-(12), 109 (18) 101 (15) ,75 (13) *~oe Compound 140a. (Rt =8.64 min), m/z (rel. int.) 271 20 272 (14) 270 (3 7) 255 254 (100), 242 227 (14), *.226 (63),-225 199 197 196 183 (32), 176 170 150 148 146 133. (32), 131 121 (11) Compound-4ob. (Rt =8.68 min), m/z (rel. int.) 271 (M+157), 272 270 255 254 (100), 242 227 (12), 226 225 209 199 197 196 (19), 183 (25) 176 (21) 170 (16) 150 (33) 148 (22) 146 133 (20) 131 Compound 141. 8.44 min), m/z (rel. int. 257 48), 258 256 (36) 241 (21) 240 (100) 239 (19) 226 (22), 225 (20) 209 (11) 197 (14) 196 (18) 183 (25) 170 (16), 217 162 160 150 (28) 148 (26) 147 146 145 (13) ,133 (20) ,130 121 Compound 142. B. 847 min), m/z (rel. int.) 273 14), 217 216. 215 183 170 150 121 58 45 44 (100) Compound 143. 9. 39 min), m/z (rel. it.) 273 275 (16) 274 (19) 272 (36) 258 (39) 257 (26) 256 (100), 255 (17) 242 (25) 241 (15) 221 (23) 178 (25) 177 (11), 176 (14) 168 (14) 167 (11) 166 (54) 165 (34) 164 (34), 163 (16) 162 (45) 160 (19) 152 (28) 151-(22) 149 (19), 147 (18) 145 (24) 139 (11) 136 131 (15) 130 121 (15) 115 1:11 (11) 103 102 (19) 89 77 75 63 51 (12) Compound 14.5. (Rt 7.35 min), m/z (rel. int.) 277 122 109 96 95 83 75 63 57 44 (100) 42 Compound 148. (Rt 8.43 min), m/z (rel. int.) 261 3), 170 169 168 153. 151 140 139 138 132 125 123 (4 115 103 20 102 101 95 94 (100), 89 77 (22), 66- 65 63 51 50 Compound 149. (Rt =9.28 min), m/z (rel. int.) 295 170 169 (12) 168 (100) 166 159 (22) 157 (66), 152.(11), 140 139 138 132 130 (32), 129 128 127 125 115 111 103 (55) 102 (18) 101 (15) 99 89 (10)i, 77. (26) 76 75 (27) 73 (11)1 65 (11) 64 (10) 63 (22) 51 (11).

Compound 150. (Rt 8.32 min), m/z (rel.-int.) 279 171 170 169 168 (100), 166 142 141 140 139 138 132 130 125 (16) 115 (12) 113 (10) 112 (89) 111 (11) 104 218 103 102 (19) 101 (12) 95 (14) 89 (11) 84 (11) 83 (24) 77 (29) 76 75 (24) 63 (13) 57 (17) 51 (11) Compound 151. (Rt 7. 68 min), m/z (rel. int. 261 62), 244 ,216 (79) 203 (65) 201 183 (100) 121 101 (40) 75 (35) 44 (52).

Compound 152. (Rt B. 8097+min), m/z (rel. int. 42 (34) 43 44 (42) 56 (13) 57 (10) 58 (72) 71. 72 (74), 73 (14) 74 (14) 75 (14) 86 (15) 95 96 (10) 100 (42) 101 (31) 114 (90) 115 120 (10) 121 122 123 138 (10) 149 164 170 181 183 (100) 184 (15) 188 194 195 196 (10) 201 202 203 204 (10) 214 (12) 215 (12), *216 (12) 317 (92) 318 Compound 153. (Rt 7. 88 min), m/z (rel. int. 42 43 44 (32) 46 (16) 72 (24) 75 (11) 86 (21) 95 96 101 (20) 109 (14) 121 (30) 122 (14) 123 139 149 (10) 170 181 183 (59) 184 188 194 (11) 195 196 (10) 201 (51) 202 (17) 203 (56), 204 214 215 (18) 216 217 (13) 289 (100) 290 Compound 1-54. (Rt 7. 74 min), m/z (rel. int. 42 (16) 44 45 46 (15) 58 (20) 72 (59) 73 (17) 75 (12), 8 6 101 (18) 121 (22) 183 (52) 194 201 (44) 202 (15) 203 (41) 214 215 (11) 216 (20) 217 (10) ,+289 (100) 2 90 Compound 155. (Rt 7. 67 min), m/z (rel. int.) 58 (44) 95 96 (11) 101 (22) 109 (16) 121 (33) 122 125 (12) 149 183 (62) 184 (10) 196 (12) 201 202 (19) 203 (53) 214 (11) 215 (19) 217 (13) 275 (100) 276 (18).

Compound 156. (Rt 8. 93 mi, m/z (rel. int.) 237 11) 219 220 (41) 219 (30) 219 (30) 206 205 (39) 204 194 (28) 193 (100) 192 (21) 191 (31) 190 189 (17), 179 (15) 178 (50) 177 176 165 (20) 152 128 116 ,115 (39) 91 (11) Compound 157. (Rt 10.88 min), m/z (rel. int.) 343 300 (100), 167 166 166 165 152 133 120 118 117 115 104 92 91 (62) 77 Compound 158. (Rt 10.74 min), m/z (rel. int.) 342 300 (100) 167 (13) 166 165 (13) 152 120 (22), 118 -115 106 104 91 (31).

Compound 159. (Rt =11.41 min), m/z (rel.. mt.) 363 33 6 (33) 3 35 (24) ,334 (95) 182 (10) 181 168 (14), *167 (40) ,166 (18) ,165 (36) ,156 (27) 155 (15) 154 153 (27) 152 (29) 140 139 138 (14) .127 (32), 126 125 (100) 117 116 115 (24) 103 *91 (43) 77 72 41 (12).

Compound 160. (Rt =11.48 min),.m/z (rel. int.) 0 :336 (35) 335 (25) ,334 182.(5) 181 (11) 168 167 (29) 166 (13) 165 (29) 156 (11) 155 (11) 154 (37), 153 (26) ,-152 (23) ,140 139 138 (12) 127 (26).

o .:126 125 (81) 125 (81) 117 (13) 115 (17) 103 91 Compound-161. (Rt =11.83 min), m/z (rel. int.) 408 407 (12) 381 (24) 380 (98) 379 (25) 378 (100) 200 (77), 199 (31) 198 (87) 197 (24) 184 (17) 182 (15) 181 (16), 171 (75) 169 (77) 168 (18) 167 (60) 166 (22) 165 (58), 152 (32) 118 (27) 117 (47) 116 (13) 11S (37) 104 (13), 103 (19) 91 (64) 90 (17) 77 Compound 162. (Rt 12.02 min), m/z (rel. int.) 408*(M+,3), 380 (100) 379 (25) 378 (99) 200 (40) 199 (32) 198 (48), 220 197 184 182 (16) 181 (23) 171 (83) 169 (85) 168 167 166 (18) 165 (50) 152 (28) 119 (11) 118 (32) ,117 (46) ,116 (12) 115 (34) .104 (11) 103 (17) 91 (63) 90 (16) 89 (10) 77 (23).

Compound 163. (Rt =10.58 min), m/z (rel. int.) 347 (M+,14)1 318 (100) 181 168 167 (24) 166 (17) 165 (26), 152 150 139 138 137 137 (23), 136 124 122 117 115 110 109 (100) 103 91 (22) 77 Compound 164. (Rt 10.59 min), m/z (rel. int.) 347 318 (100) 181 178 168 167 (27) 166 (17), 165 152 150 139 138 137 (18); 136 122 117 115 110 109 (79), 103 91 (20) 91 (20) 77 Compound 165. (Rt 10.61 min), m/z (rel. int.) 347 318 181 167 166 165 152 (13), 138 (34) 137 (27) 136 (11) 136 (11) 122 (14) 117 115 (11) 110 109 (100) 91 (22) 77 Compound 166. (Rt 11.62 min), m/z (rel'. int.) 359 330 (100) 167 165 (14) 152 150 149 (38), 148 135 134 122 122 121 117 115 91 77 Compou~nd 167. (Rt 11.18 min), m/z (rel. int.) 359 330 (100OL, 136 121 (6) Compound 168. (Rt =10.86 min), m/z (rel. int.) 343 314 (100) 167 (16) 166 165 (16) 152 134 (17), 133 (13) 132 118 (14) 117 115 (10) 106 106 105 (59) 91 (20) 77 Compound 169. (Rt 10.94 min), m/z (rel. int.) 343 314 (100) 167 (14) 166 165 (15) 152.(8) 134 133 (16) 132 132 118 (14) 117 115 106 105 (62) 91 (18) 77 Compound 170. (Rt 12.52 min), m/z (rel. int.) 374 345 315 207 194 193 (16) 179 168 167 166 (16) 165 (100) 164 164 152 136 (45) 117 (13) 115 (11) 104 103 91 90 77 Compound 171. (Rt 11.16 min), m/z (rel. int.) 341 182 181 168 167 167 166 165 (21) 152 (11) 144 132 (18) 131 (100) 129 (10) 128 120 (12) 118 117 116 (10) 115 (15) 106 104 103 91 77 Compound 172. (Rt =8.53 min), m/z (rel. int.) 275 (M+,165), 274 (95) 260 (18) ,259 (24) 258 (87) 257 (28) 254 (17), 244 (41) 243 (46) ,242 (18) 233 (30) 214 (26) 201 (46), 189 (18) 188 (36) ,183 (27) 181 (22) 180 (100) 178 (24), 165 (38) 163 (28) ,154 (17) 150 (39) 149 (25) 148 (82), 139 (58) 135 (20) ,133 (35) 109 Compound 173. (Rt =8.44 min), m/z (rel. int.) 277 (M+i,11), 260 (80) 259 (34) ,245 241 234 233 (23) 230 (19) 229 (100), 214 203 202 201 190 189 (25) 188 (18) 183 (18) 171 170 (15) 165 (14) 164 154 152 151, 134 133 (23) 121 109 (13) 101 Compound 174. (Rt 8.49 min) m/z (rel. int.) 291 260 (27) 259 (17) ,234 (18) 233 (14) 230 (10) ,229 203 202 (10) 201 189 (20) 188 183 (15) 170 (12) 169 168 (13) 165 (14) 164 152 151 138 137 134 133 (13) 10.9 (12) 101 57 44- (100) 42 Compound 175. (Rt 9.64 min), m/z (rel. int.) 303 123 (47) 109 96 95 (40) 85 (26) 84 (100) 82 222 75 68 56 55 43 42 (16).

Compound 176. (Rt 8.35 min), m/z (rel. int.) 277 245 220 219 183 171 170 151 138 109 57 44 (100), 42 Compound 177. (Rt 7.83 min), m/z (rel. int.) 241 134 109 108 (100), 107 104 103 91 90 79 78 77 65 51 Compound 178. (Rt 8.29 min), m/z (rel. int.) 257 134 125 124 (100), 109 104 103 95 91 81 78 77 65 52 51 eoVe 0 0Compound 179. (Rt 7.88 min), m/z (rel. int.) 255 148 115 108 107 104 (12) 103 91 79 78 77 65 51 44 (100), 42 15 oee.

Compound 180. (Rt 7.28 min), m/z (rel. int.) 295 183 162 145 143 135 134 (100), •133 132 117 115 114 113 112 105 104 103 102 95 91 (18), 89 83 79 78 77 75 65 63 (11) 51 Compound 181. (Rt 7.7 min), m/z (rel. int.) 259 137 135 122 121 109 108 (100), 107 96 91 79 78 77 65 51 Compound 182. (Rt 8.00 min), m/z (rel. int.) 225 208 207 182 181 (100), 165 152 (24), 151 74 Compound 183. (Rt 8.98 min), m/z (rel. int.) 241 224 223 199 198 197 (100), 178 165 152 (13) 150 223 Compound 184. (Rt 8.90 min), m/z (rel. int.) 235 218 217 203 202 193 192 (67), 191 (100), 190 189 178 165 152 Compound 185. (Rt 7.37 min), m/z (rel. int.) 245 152 141 135 134 (100), 132 115 (14), 112 105 104 103 102 95 91 89 84 83 79 (10) 78 77 (44), 65 64 63 57 56 52 51 50 Compound 186. (Rt 7.31 min), m/z (rel. int.) 245 152 141 135 134 (100) 132 117 115 112 (38) 77 75 65 64 63 57 52 51 50 Compound 187. (Rt 8.64 min), m/z (rel. int.) 239 221 220 207 196 195 194 (14), 193 192 191 181 179 178 (100), 168 167 166 165 164 153 152 139 128 115 91 89 77 63 51 44 Example 30: Biological properties of synthesized arylalkylamines Compounds synthesized as described in Example 28 and Example 29 were tested for various biological properties detailed in the examples.

224 Table 1 Compound vs. NMDA a

IC

5 0 (12M) vs. 3 HIMK-801C Compound 1 0.102 126 (4) Compound 2 0.192 not tested Compound 3 0.003 not tested Compoun d 4 0.184 89 (1) Compound 5 0.102 15.2 (2) 0.070 3 )b Compound 6 0.129 >100 (1) at 100 'UM)d Compound 7 0.163 129 (1) Compound 8 0.099 219 (1) Compound 9 1.2 100 (2) at 100 zd Compound 10 0.082 80 (1) (57% at 80 g~ Compound 11 4.0 not tested Compound 12 6.0 (11) 98 (1) Compound 13 not tested not. tested Compoound 14 8.8 100 aum Compound 15 4.9 100 kiM -Compound 16 5.1 28.8 (1) Compgund 17 9.6 36.3 (1) Compound 18 5.1 34 (1) Compound 19 0:435 (11) 2.1 Compound 20 0.070 (15) 0.252 (9) Compound 21 0.038 0.457 (2) Compound 22 0.145 3.45 (2) Compound 23 0.267 5.4 (1) 225 a *8*aa* a Compound 24 0.206 0.591 (6) Compound 25 0.279 0.871 (2) Compound 26 27 34 (2) Compound 27 0.071 0.180 (2) Compound 28 0.380 2.3 (3) Compound 29 1.9 5.8 (3) Compound 30 0.035 0.407 (2) Compound 31 0.052 1.3 (2) Compound 32 0.284 0.799 (3) Compound 33 0.060 0.181 (6) Compound 34 0.426 2.7 (3) Compound 35 6.2 25.1 (1) Compound 36 not tested not tested Compound 37 0.944 11.1 (2) Compound 38 0.407 2.3 (2) Compound 39 0.251 2.9 (3) Compound 40 0.933.(1) 18.1 (3) Compound 41 0.724 14.0 (3) Compound 42 not tested not tested Compound 43 0.232 7.5 (2) Compound 44 not tested not tested Compound 45 not tested not tested Compound 46 0.013 5.2 (2) Compound 47 not tested not tested Compound 48 not tested not tested not tested not tested Compound 49 Compound 50 0.089 0.762 (4) 226 i.

a a.

o.

4 *ooo a a Compound 51 1.1 4.5 (2) Compound 52 0.102 0.380.(2) Compound 53 0.217 4.2 (2) Compound 54 0.036 0.046 (3) Compound 55 0.035 0.153 (2) Compound 56 0.218 0.955 (2) Compound 57 0.028 0.063 (2) Compound 58 0.028 0.203 (3) Compound 59 0.272 0.453 (3) Compound 60 0.416 (11) 0.641 (9) Compound 61 0.134 0.324 (2) Compound 62 0.177 0.617 (1) Compound 63 0.093 0.245 (3) Compound 64 0.309 0.851 (2) Compound 65 0.167 2.0 (1) Compound 66 0.236 1.2 (2) Compound 67 10.95 2.9 (1) Compound 68 2.9 not tested Compound 69 0.224 0.366 (1) Compound 70 1.7 not tested Compound 71 6.35 not tested Compound 72 7.4 not tested Compound 73 12.6 not tested Compound 74 27.5 not tested Compound 75 0.94 not tested Compound 76 0.73 not tested Compound 77 5.5 not tested Compound 78 10.2 not tested 227

'.OS

4 0e 5 er '2 *C

S

10 Compound 79 12.6 10.2 (2) Compound 80 28 182 (1) Compound 81 1.4 6.1 (2) Compound 82 0.106 0.794 (1) Compound 83 0.342 0.794 (1) Compound 84 7.9 23.4 (1) Compound 85 1.2 3.5 (1) Compound 86 1.2 6.0 (1) Compound 87 0.657 3.0 (1) Compound 88 2.5 10.6 (2) Compound 89 0.240 1.2 (2) Compound 90 0.270 1.4 (2) Compound 91 0.162 14.1 (2) Compound 92 1.3 20.2 (2) Compound 93 0.486 26.9 (2) Compound 94 0.248 22.6 (2) Compound 95 0.311 3.0 (2) Compound 96 0.187 1.1 (2) Compound 97 0.410 2.6 (1) Compound 98 7.9 52.5 (2) Compound 99 100 105 Compound 100 0.602 3.2 (1) Compound 101 0.912 2.0 (1) Compound 102 1.01 3.3 (1) Compound 103 0.380 0.661 (2) Compound 104 7.983 10 (1) Compound 105 1.03 3 (1) Compound 106 0.767 1.31 (1) a B004 0S40 4O 0 *4 228 Compound 107 2.67 3.83 (1) Compound 108 1.06 0.942 (1) Compound 109 1.95 1.08 (3) Compound 110 42.7 13.3 (1) Compound 111 0.645 0.167 (2) Compound 112 28.0 21.0 (1) Compound 113 13.5 not tested Compound 114 3.4 not tested Compound 115 1.4 1.0 (1) Compound 116 3.6 not tested Compound 117 19.6 6.0 (2) Compound 118 0.409 0.240 (3) Compound 119 0.115 0.087 (3) Compound 120 0.101 0.074 (3) Compound 121 0.656 0.670 (3) Compound 122 0.209 0.342 (2) Compound 123 9.6 3 (2) Compound 124 3.5 14.3 (3) Compound 125 1.7 6.7 (2) Compound 126 0.398 6.0 (1) Compound 127 1.2 17.5 (2) Compound 128 0.646 5.5 1 Compound 129 1.26 not tested Compound 130 0..851 not tested Compound 131 1.23 not tested Compound 132 1.3 6.4 (1) Compound 133 0.760 3.0 (1) Compound 134 2.5 10 (1) 229 Compound 135 0.244 1.185 (2) Compound 136 0.139 0.706 (1) Compound 137 0.232 0.074 (2) Compound 138 107 100 (1) Compound 139 1.97 5.6 (2) Compound 140 20.8 not tested Compound 141 4.26 8.97 (1) Compound 142 1.013 1.54 (2) Compound 143 2.82 not tested Compound 144 not tested not tested Compound 145 0.098(1) 0.626.(1) Compound 146 0.829 0.372 (1) Compound 147 0.894 not tested Compound 148 0.549 0.373 (2) Compound 149 0.085 0.150 (3) Compound 150 0.195 0.351 (2) Compound 151 54.9 100 (1) Compound 152 not tested not tested Compound 153 not tested not tested Compound 154 not tested not tested Compound 155 not tested not tested Compound 156 0.069 0.090 (2) Compound 157 0.142 23.16 (2) Compound 158 0.351 39.64 (1) Compound 159 0.185 10.41 (1) Compound 160 7.35 48.94 (1) Compound 161 0.247 5.62 (1) Compound 162 1.138 76.41 (1) 230 Compound 163 Compound 164 0.326 (2) 10.34 (1)

I

0.475 (2) 18.30 (1) Compound 165 0.337 171 (1) Compound 166 0.619 36.7 (1) Compound 167 0.080 14.5 (1) Compound 168 0.092 17.4 (1) Compound 169 0.298 26.7 (1) Compound 170 0.238 57.0 (1) Compound 171 0.310 39.6 (1) Compound 172 38.0 37.3 (1) Compound 173 22.9 24.1 (1) Compound 174 not tested 57.0 (1) Compound 175 not tested 5.1 (1) Compound 176 not tested 10.0 (1) Compound 177 not tested 0.754 (1) Compound 178 not tested 1.25 (1) Compound 179 not tested 1.67 (1) Compound 180 <100 <10(1) Compound 181 0.081 0.632(1) Compound 182 2.6 7.05(1) Compound 183 0.676 5.01(1) Compound 184 1.5 1.51(1) Compound 185 0.646 0.639(1) Compound 186 0..155 0.123(1) Compound 187 1.78 2.01(1) Compound 188 not tested not tested Compound 189 not tested not tested Compound 190 not tested not tested 231 Compound 191 not tested not tested Compound 192 not tested not tested Compound 193 not tested not tested Compound 194 not tested not tested Compound 195 not tested not tested Compound 196 not tested not tested Compound 197 not tested not tested Compound 198 not tested not tested Compound 199 not tested not tested Compound 200 not tested not tested Compound 201 not tested not tested Compound 202 not tested not tested Compound 203 not tested not tested Compound 204 not tested not tested Compound 205 not tested not tested Compound 206 not tested not tested Compound 207 not tested not tested Compound 208 not tested not tested Compound 209 not tested not tested Compound 210 not tested not tested Compound 211 not tested not tested Compound 212 not tested not tested Compound 213 not tested not tested Compound 214 not tested not tested Compound 215 not tested not tested a:Inhibition of NMDA/glycine-induced increases in intracellular calcium in cultured rat cerebellar granule cells (RCGC's) (see Example in parentheses 20 232 indicates the number of experiments).

b:TFA salt.

c:Inhibition of 3 H]MK-801 binding in rat cortical/ hippocampal washed membrane preparations (see Example 4).

d:ICs, study incomplete. inhibition at the stated concentration.

A comparison of the IC 5 0 values in the RCGC assay with the IC 50 values in the 3 H]MK-801 binding assay (Table 1) illustrates that the arylalkylamines of the invention inhibit NMDA receptor activity by a mechanism different than that of binding to the MK-801 S.binding site; the concentration of the compound that i" inhibits NMDA receptor function is several orders of magnitude less than the concentration that competes at the site labeled by 3 H]MK-801. This is not the case, however, with the simplified arylalkylamines exemplified by Compounds 19 215. Such compounds bind to the site labeled by [(H]MK-801 at concentrations ranging approximately 1 to 400-fold higher than those which antagonize-NMDA receptor-mediated function in the rat cerebellar granule cell assay.

Some of the simplified arylalkylamines disclosed.have structural features similar to portions of other compounds which are utilized as, for example, anticholinergics, antiparkinsonians, antihistamines, antidepressants, calcium channel blockers, coronary vasodilators, opiate analgesics, and antiarrhythmics.

However, when certain of these compounds were evaluated for NMDA receptor antagonist potency (Example as can be seen in Table 2, none of the compounds tested, with 233 the exception of (BJ- and CS) -fendiline, nisoxetine, and the Eli Lilly compound, had IC 50 values less than 1 suM.

These data are summarized in Table 2.

Table 2 Compound Structure

ICS

0

(IIM)

and vs. NMDAa Therapeutic utility (R)-fendiline -H 0.719 (calcium channelN blocker; coronary

C

vasodi lator) 0.686 (calcium channel blocker; coronary

CH

prenylamine -H (calcium channel QCi 3 blocker; coronary v asodilator) pheniramine 0.j 3 22 (antihistamine) N H

NI

234 Table 2 chlorpheniramine C) a- 3 >100 (antihistamine) N H brompheniramine 0-13 138 (antihistamine)

H

diphenhydramine 26 (antihistamine) 011 H

OH

3 doxyl amine H 3 62 (antihistamine; H hypnotic) OH 3 chiorcyclizine 'N CH3 (antihistamine) N C1 cycl izine C 3 28 nor-cyclizine N 23 (pharmaceutical N7 intermediate)

T

lidoflazine (calcium channel blocker; coronary vasodilator) pimozide F (antipsychocic) dispyamdeH 3 C CH 3 >100 disopramide 0 H 3 CH 87 (anticrhl-iric) 3

SH

3 C OH 3 8 pridinol I OH107 (anticholi:.ergic; antiparkisonian)

NN

(antihistam-ine)

CH

3 trihexyph7dnidyl OH .1 antiparkinsoiian) 236

SOW.

*00*0 Novo-{',ordisk c ompoundd (calcium channel blocker; neuronrotectant) 2 37 9 9 9*99 9 9*99** *99999 9 9* nisoxetifle IH 0.894 (monoamine uptake N, CH 3 inhibitor;0 antidepressant)

OCH

3 terodiline H CH 3 not tested (calcium channel H blocker;

OH

3

OH

3 anticholinergic; vasodilator) tomoxetine Hnot tested- (monoamine uptake N H inhibitor;o antidepres sant)

H

amitriptyline C3not tested (serotonin uptake inhibitor; antidepressant) imipramine not tested

-CH

(serotonin uptakeNo inhibitor; antidepressant) cldnffpramine C3not tested (serotonin uptake

OH

3 inhibitor;

/C

antidepressant) C1 238 it.

doxepine- C3not tested :serotonin uptake /OH inhibitor; antidepressant) -h--orprornazine not tested dopamine neuroleptic)

C

des ipramine 2.3 (antidepressant)

H

protriptyline 5 (antidepressant)//

H

Li rlly Compound NNDA receptor antagonist 6

KNH

2 0.609 aa: Inhibition of NMDA/glycine-induced increases in intracellular calcium in cultured rat cerebellar granule cells (RCGC's) (see Example 1).

239 b: Disclosed as compound 2 in Table 4 in Marcusson et al., Inhibition of [3H]paroxetine binding by various serotonin uptake inhibitors: structure-activity relationships. Europ. J. Pharmacol. 215: 191-198, 1992.

c: Disclosed as compound 17 in Jakobsen et al., Aryloxy-phenylpropylamines and their calcium overload blocking compositions and methods of use. U.S. Patent No. 5,310,756, May 10, 1994.

d: Disclosed as compound 25 in Jakobsen et al., Aryloxy-phenylpropylamines and their calcium overload blocking compositions and methods of use. U.S. Patent No. 5,310,756, May 10, 1994.

e: Disclosed as Compound 1 in McQuaid et Inhibition of 3 H]-MK801 binding and protection against NMDA-induced lethality in mice by a series of-imipramine analogs Res.

Comm. in Pathol. and Pharm. 77:171-178, 1992.

Structure-activity relationship studies were initiated using Compound 19 as the lead structure. An examination of the side chain demonstrated that the propyl side.chain was optimal for NMDA receptor antagonist potency (Table This finding was verified "using Compound 20 as the lead structure (Table 3).

Table 3 Compound Structure Tx-Cs 0 =fAa 2,2-diphenylethylanine 4.

N24.

3,3-diphenylpropylamine 0.435 (Compound 19) I-NH 2 se* 4,4-dp1nylutylarune N1.7 (Compound

C

so..

-diphenylpentylamine N6.4 (Compound 71) INF 2 IleI so:2,2-bis(3-fluoropbhenyl)-l- N7.9 (Compound 98) 3,3-bis(3-fluorophenyl)-l- N0.070 propylamine NH2 (Compound

N.

4,4-bis(3-wfluoroplefyl)-l- N Q602 butylamine F-NH 2 (Compound 100) 241 a:Inhibition of NMDA/glycine-induced increases in intracellular calcium in cultured rat cerebellar granule cells (RCGC's) (see Example 1).

Further SAR studies examined the optimal pattern of phenyl ring substitution. Initial studies demonstrated that substitution of a halogen group (fluoro or chloro) at the meta position was optimal for NMDA receptor antagonist potency (Table Increasing the number of fluoro substituents led to an apparent decrease in potency (Table 4).

*fee too* $0800 *9 0* O *m S0 2 42 Table compound Structure 1C 50 (H)vs.

NMA a 3,3-dipheny--propy1Xlmfle N0.43S.

(Compound 19) I NH 2 3-(2-fJ~uoropDhefly1)-3-(4-0.3 fJlucrophenyl)-l-

WH

2 propylamine

J

(Compound 76) IN.

F

3,3-bis(4-fluorophenyl)-l- FN propylamine

NH

2 ::::*(Compound 77)

F

3,3-bis(3-fluorophenyl)-- 0.070 *propylamine

NHI

(Compound 3-(2-fJluorophenyl)-3-(3- NF 0.102 fluoroplienyl) -1-I propylamine .(Compound 52) 243 3,3-bis(2-fluorophenyl)-l-1- 0.217 propyl amine

H

(Compound 53)

F

N.

3 3-bis(3-chloroohenyl)-l- 0.052 propylamine NH (Coz~ound 31)

C

3-(3-fluorophenvl)-3-(3- 0 0.035 chiorophenyl) cta 2 propylamine (Compound 30) N 3-(3-f2.uorophenyl)-3- N.0.284 phenyl-1-propylarmine

INH

2 (Compound 32) 0000.

F

0003- 5-difluorophenyl) F0.187 (3-f luoropheny.) -1-I 0 NH2 propylamiine (Compound 96).

FE

-f 4'10 bis (3D 5, difluoxophenyl)

N

propylamine F (Compound 97) _F F 3,3-bis(3- N.10.2 (trifluoromethyl)phenyl] cF 3 2 1-propyamnine (Compound 78) .I 244 a:Inhibition of NMDA/glycine-induced increases in intracellular calcium in cultured rat cerebellar granule cells (RCGC's) (see Example 1).

Replacement of one of the fluoro groups on one phenyl ring with a methyl, methoxy or.hydroxy group led to no change or a decrease in the in vitro NMDA receptor antagonist potency. The ortho position was optimal for this methyl, methoxy or hydroxy group, and the rank order of potency for this substitution was methyl 10 methoxy hydroxy (Table Also illustrated in Table 5 are those compounds possessing the 3,3-bis(3fluorophenyl) moiety with additional methyl or methoxy substitutions on the phenyl rings, often leading to an increase in NMDA receptor antagonist potency. Table also illustrates those compounds possessing the 3,3bis(2-methylphenyl) or 3,3-bis(2-methoxyphenyl) moiety in place of the 3,3-bis(3-fluorophenyl) moiety; these substitutions are acceptable, although a decrease in potency is noted.

245 Table Compo uzd Structure

IC

50 ORi) VS.

NMDA&

3,3-bis(3-fluorophenyl)-l- propylamine I (Compound 3-(3-fluorophenyl)-3-(2- C8 3 0.7 methyiphenyl) W2-I propylamine (Compound 27) 3-(3-fluorophenyl)-3-(3- N.0.80 methyiphenyl)

H

3 C N4 propylaznine (Compound 28) 3 -(3--fluorophenyl)-3-(4-

H

3 1.9 methylphenyl)-1-I N8 propyJlamine (Compound 29) 3 fluorophenyl) 3-(2

N.OCH

3 0.206 methoxyphenyl)

NFI

,proppylamine (Compound 24)

N

246 3-.(3-fluorophenyl)-3-(3- 0.279 methoxyphenyl) H3CO H propylamine (Compound 3-(3-fluorophenyl)-3-(4- H 3 C N27 methoxyphenyl)

NH

propylamie (Compound 26) 3 -methoxyphenyl) -3 N CH phenyl-l-propylamine (Compound 97) 3-(2-hydroxyphenyl)-3-(3- N0.380 f fluorolphenyl) 1 F propylamineIl (Compound 103) z:- 3-(3-hydroxyphenyl)-3-3- N0.912 fluorophenyl) F N1 propylamine (Compound 101)I *3-(3-fluorophenyl)-3-(2- N0.218 methyl-3-fluorophenyl) FNH 2 _propylamine H 3 I.0 (Compound 56)N 3-(3-fluorophenyl)-3-(3- 0.02 fJluoro-6-methylphenyl)

H

propylainine CF 3 (Compound 57) 247 0 0 0.

3,3-bis(3-fluoro-6- C30.028 methylphenyl)

N

propylamine CH3 (Compound 58) 3-(3-fluorophenyl)-3-(3- 020.134 fJluoro-6-methoxyphenyl) -1propylanine (Compound 61) 3, 3-bis (2-methylphenyl) CH 3 lt16 propylandne (Compound 65) H 3, 3-bis (2-methoxyphenyl)- -C3 .7 1-propylamine -NK2 (Compound 62) H 2

C

3,3-bis(3-methoxyphenyl)-1.

1-propylamine

H

3 CO

F

(Compound 115) H3M 248 a:Inhibition of NMDA/glycine-induced increases in intracellular calcium in cultured rat cerebellar granule cells (RCGC's) (see Example 1).

The next series of SAR experiments investigated the effect of alkyl chain substitutions (branching patterns) on NMDA receptor antagonist potency in vitro. The addition of a methyl group on either the a or 6 carbon on the propyl side chain led to a decrease or no change in potency, respectively (Table 6).

1 249 Table 6 Compound -Structure

IC

50 !flDA a 3,3-bis(3-fluorophenyi)-1- 0.070 propylamine NH2 (Compound

NI

3,3-bis(3-fluorophefly1)-2- CH310m0-38 methyl-i-propylamine

H

(Compound 21) ;1 3,3-bi.s(3-fluorophenyl)-2- N CH 3 0.060methyi-l-propylamine F- H (Compound 33) .3,3-bis(3-fluorophenyl)-2- N CIH 3 0.426 methyl-i -propylamine

F

(Compound 34) 3,3-bis(3-fiuorophenyl)-1- N0.145 methyi-l-propylamine

H

(Compound 22) CH3

N

3,3-bis(3-fiuorophenyi)-i- N*1 0.089 methyl-i -propylamine

INH

2 (Compound 50) CH 3 250 3, 3-bis (3-fluorophel))-1methyl -i-propylamifle (Compound 51) :1.1 0 0 0 0000 0.09.

CH

3 0.3 3, 3 bis (3 -f luorophel) 2 0.035H ethyl-l-propylalUife F eNH (Compound

SSF)

3,3-bis(3-fluoropheflyl)-l- '026 ethyl -1-propylamine (Compound 23) H 3,3-bis (3-fluorophelyl) OH 003 hydroxyethyl -1-propylainfe F N2 (Compound 54)

F

3, 3-bis (3-f luorophenyl)

H

2 .0 ethyl-1-propylamile F

H

(Compound 82) 3,3-bis(3-fluorophelyl)'--

CH

3 0.407 1,2 -dimethy.-1 -propylamifle

F-NHI

(Compound 38)

CH

3 3,3-bis(3-fluorophelyl)-

CH

3 0.724 2, 2-dimethyl--propyalUife CH2 (Compound 41) C3 3, 3-bis (3-fluorophefyl) 2, 2-diethyl-1-propylamifle (Compound F

NH

2

CH

3 251 a:Inhibition of NMDA/glycifle-iflduced increases in intracellular calcium in cultured rat cerebellar granule cells (RCGC's) (see Example 1).

The next series of SAR experiments investigated the effect of incorporation of a double bond within the propyl chain on NMDA receptor antagonist potency in vitro (Table 7) As can be seen in Table 7, the incorporation of a double bond decreased potency in a consistent manner.

Table Compound Structure

ICS()

(M)Vs.

NMDAa 3,3-bis(3-fluorophenyl)-1- 0.070 propylamine

NH

2 (Compound 3,3-bis(3-fluorophelyl)-

IN

2 1.4 prop-2 -ene-1-anane

I

(Compound 139) 3,3 -diphenyl--propyamile 0.435 (Compound 19)

NH

2 Ile 252 *too 0 S 0 000 3 ,3-diphenyl-prop-2-e .ne-l- 1.4 amine

NH

2 (Compound 81) 3-(3-fluoromhenyl)-3- 0.284 phenyl-l-propylamine

N.H

2 (Compound 32) 3-(3-fluorophenyl)-3- 2.67 phenyl-prop-2-ene-1-amine ;e, (Compound 107) ;Ol SNH2 of 2 comnounds) 3,3-bis(3-methoxyphenyl)- 1-propylamine H3,CO H (Compound 115) 3 C0 3,3-bis(3-met-hoxypienyl)- 47 prop-2-ene-1-amine H 3 CO NH (Compound 116)

HSC

253 a:Inhibition of NMDA/glycine-induced increases in intracellular calcium in cultured rat cerebellar granule cells (RCGC's) (see Example 1).

"The next series of SAR experiments investigated the effect of incorporation of the propylamine chain into a ring structure on NMDA receptor antagonist potency in vitro (Table 8).

***ee 254 255 a:Inhibition of NMDA/glycine-induced increases in intracellular calcium in cultured rat cerebellar granule cells (RCGC's) (see Example 1).

The next series of SAR experiments investigated the effect of simple alkyl substitution on the nitrogen on NMDA receptor antagonist potency in vitro (Table 9).

o o 256 T abl1e 9 compound IStructure Ic 5 0 3,3-bis(3-fluoropoheflyl)-1- 0.070 Oronylamine (Compound N-methyl-3, 3-bis(3- H 0.416 f luorotphenyl) -1

NH

2 aropyIAmni ne (Compound N-ethyl-3, 3-bis H 0.272 fluoropheny.) F

CH

3 propyJlamine (Compound 59)

F.

N,N-d.methyl-3,3-bis(3- 9.6 f luorophenyl) 1- ~CHpropylamine (Compound 123) 3-(3-fluorophenyl)-3- 0.284 phenyl-l-propylam e 0NH 2 (Compound 32)

F.

N-methyl-3-(3- H 1.06 fluorophenyl) -3-phenyl-1- CH3 propylamine (Compound 108)

N.

3, 3-diphenylpropylamn~fe (Compound 19.) 0.435 257 N-met-hyl-3,3- H -10.95 d.iphenylpr opylamine N H (Compound 67) N-ethyl-3,3- NH'2.9 diphenylpropylam.ine N%-"H (Compound. 68) N, N-dimethyl-3, 3- NH 12.6 diphenylpropylamine

CH

3 (Compound 73) N-isopropyl-3, 3- diphenylpropylamine y H (Compound 72) CH3 o ,N-diethyl-3,3- NH 27.5 diphenylpropylantjne K.1--.C8 3 oro..

258 a:Inhibition of NMDA/glycine-induced increases in intracellular calcium in cultured rat cerebellar granule cells (RCGC's) (see Example 1).

Certain simplified arylalkylamine compounds were selected for evaluation of activity in a battery of neurotransmitter receptor binding assays, and for activity against the L-type calcium channel and delayed rectifier potassium channel. The compounds were inactive (less than 50% inhibition at concentrations up to 10 4M) in the following assays: nonselective a2 adrenergic receptor ([PH]RX 821002 binding in rat cortex), HI histamine receptor 3 H]pyrilamine binding in bovine cerebellum), nonselective sigma receptor H]DTG binding in guinea pig brain), nonselective opiate 15 receptor ([PH]naloxone binding in rat forebrain), monoamine oxidase (MAO) activity, both MAO-A ([C])serotonin metabolism in rat liver mitochondria) and MAO-B ([4"C]phenylethylamine metabolism in rat liver mitochondria) 20 As can be seen in Table 10, activity was noted for several compounds at concentrations below 10 uM in Sthe following assays: L-type calcium channel, delayed rectifier potassium channel, central muscarinic cholinergic receptor binding, and monoamine (dopamine, norepinephrine, and serotonin) uptake binding assays.

This profile of activity in the central muscarinic cholinergic receptor and monoamine uptake binding assays is not unexpected,- given the chemical structures of our simplified arylalkylamines (refer to Table 2 above).

With the exceptions, however, of the activity of Compound 19 in the serotonin uptake binding assay, the 259 555*** activity of Compound 34 in the dopamine uptake binding assay, the activity of Compound 50 in the seroconin uptake binding assay, the activity of Compounds G3 and G4 in the dopamine uptake binding assay, and the activity of Compound G0 in the dopamine and serotonin uptake binding assays, the simplified arylalkylamine compounds were most potent at the NIVDA receptor.

Table Compound IcS 0 (AsM) L-type Delayed Central Monoacmine vs. NMDAI calcium rectifier muscarinic uptake charineib potassium cholinergic bindd~ng channelc receptord assays- 0 Compound 0.435 10.2 1-10 4%I at 701 at 19 0.174t at 0.1175-% at 101311 at 0.19530-1 at 10918%; at 0 .1h 89* at Compound 0.070 2.2 1-10 8%6 at 6%i at 20 0.1901 at 0.11.81t at at 0 .19581 at 10928*1 at 0 .1h 9411 at 1 Qk Compound 0.060 1.6 10 4211 at 23%* at 33 0.199t at 0.1186-1 at 101201 at 0 .1g54 -0 at 1091426 at 0.1h89-* at lOh 260 0 .0 0 0.

0 0 :0 Compound 0.426 not -10 25%1 at 609, at 34 tested 0.199k at 0.1199% at 10110*6 at 0.1964-0 at lQgl~k at 0.1-h79*- at loh Compound 0.089 not 10 11%0 at. 1796 at tested 0.184k at 0.11 9 3 at lot 101c at 0.1978*6 at 10975k at 0 119 7 at Compound 0.013 0.676 -3 33k at 40%6 at 46 0.189k at 0. 1197*6 at 10 1017*6 at 0 -19646 at 10910k at Q h 7556 at 1 Qh Compound 0.093 1.9 not 11% at 64%* at 63 tested 0.181% at 0. 119801 at 10179k at 0. 1976*- at lQgl3%- at 0.1h850% at loh Compound 0.309 not not 11%0 at 50%; at 64 tested tested 0.183%- at 0. 1 1 9 9 *6 at l0, at 0Q. l65%- at l0g290i at 0 .1h 6809, at lOb Compound 0.416 2. not I j at U.91 60 tested 0.193% at at 0 10 at 0.

a:Inhibition of NMDA/glycine-induced increases in intracellular calcium in cultured rat cerebellar granule cells (RCGC's) (see Example 1).

:Inhibition of KC1 depolarization-induced increases in intracellular calcium in cultured rat cerebellar granule cells (RCGCs); estimated ICs, value in M.

c:Inhibition of delayed rectifier potassium channel in S. cultured N1E-115 neuroblastoma cells; estimated IC,, value in M.

d:Inhibition of the binding of. quinuclidinylbenzilate (QNB) to rat cortical membranes; percent block at indicated concentration in AM.

e:Inhibition of the binding of [PH]WIN-35,428 to guinea pig striatal membranes (dopamine uptake binding assay), P[H]desipramine to rat cortical membranes (norepinephrine uptake binding assay), or [H]citalopram to rat forebrain membranes (serotonin uptake binding assay); percent 262 block at indicated concentration in M, or IC 0 when available.

f:dopamine uptake binding assay 9:norepinephrine uptake binding assay h:serotonin uptake binding assay Advantageous properties of the arylalkylamine compounds of the present invention are illustrated by the fact that concentrations which suppress NMDA receptor-mediated synaptic transmission fail to inhibit LTP. Furthermore, while compounds such as Compound 9, and 11 do produce a hypotensive response following systemic administration in rats, the hypotensive effect produced by these compounds is of a relatively short duration (approximately 30 min). Additionally, 15 Compounds 12 and 14 have no cardiovascular activity at doses up to 37.3 Amoles/kg i.v. and 15.Mmoles/kg i.v., respectively.

263 Table 11 Compound suppression of NMA LTP Assayb Drop in Mean Receptor-Mediated Arterial Blood Synaptic Pressurec Transmnission' Compound 1 10 30.juM no block at 65 mm Hg at 300 AiM 1.5 4moles/kg 60 min duration 3 0 uM no block at 40 mm Hg at Compound 2 100 AM 1.5 /zmoles/kg 120 min duration Compound 3 10 30 MM not tested 20 mm Hg att mg/kg 60 min duration Compound 4 10 100 no block at 40 mm Hg at 0 1.5 Mmoles/kg 120 min duration Compound 9 10 100 M no block at 75 mm Hg at 300 MiM 4.5 umoles/kg 90 min duration Compound 11 riot tested not tested 20 mm Hg ati mg/kg min duration not tested not tested no effect At Compound 12 doses up to 37.3 gmoles/kgi .v.

Compound 14 not tested not tested no effect at doses up to gmoles/kg i.v.

Compound 19 100 300 pM block at 100 not tested

M

264 Compound 20 30 300 jM block at 100 no effect at AM doses up to kmoles/kg i.v.

Compound 22 not tested not tested no effect at doses up to Amoles/kg i.v.

a:Concentration which suppresses NMDA receptor-mediated synaptic transmission (see Example b:Concentration that does not block the induction of LTP (see Example 19).

C:Drop in systemic blood pressure produced by administration of compound in rats (see Example 22).

a a o 0e o,* 265

C.

S

S.

CCC

C

Formulation and Administration As demonstrated herein, useful compounds of this invention and their pharmaceutically acceptable salts may be used to treat neurological disorders or diseases. While these compounds will typically be used in therapy for human patients, they may also be used to treat similar or identical diseases in other vertebrates such as other primates, farm animals such as swine, cattle and poultry, and sports animals and pets such as 10 horses, dogs and cats.

In therapeutic and/or diagnostic applications, the compounds of the invention can be formulated for a variety of modes of administration, including systemic and topical or localized administration. Techniques and formulations generally may be found in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton PA (18th ed. 1990).

Pharmaceutically acceptable salts are generally well known to those of ordinary skill in the 20 art, and may include, by way of example but not limitation, acetate, benzenesulfonate, besylate, benzoate, bicarbonate, bitartrate, calcium edetate, camsylate, carbonate, citrate, edetate, edisylate, estolate, esylate, fumarate, gluceptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate,- iodide, isethionate, lactate, lactobionate, malate, maleate, mandelate, mesylate, mucate, napsylate, nitrate, pamoate (embonate), pantothenate, phosphate/disphosphate, polygalacturonate, so.: of C

CCC

C..

266 salicylate, stearate, subacetate, succinate, sulfate, tannate, tartrate, or teoclate. Other pharmaceutically acceptable salts may be found in, for example, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA (18th ed. 1990).

Preferred pharmaceutically acceptable salts include, for example, acetate, benzoate, bromide, carbonate, citrate, gluconate, hydrobromide, hydrochloride, maleate, mesylate, napsylate pamoate (embonate), phosphate, salicylate, succinate, sulfate, e* or tartrate.

The useful compounds of this invention may also be in the form of pharmaceutically acceptable :0 complexes. Pharmaceutically acceptable complexes are s 15 known to those of ordinary skill in the art and include, g*oe ,0604. by way of example but not limitation, 0000 8-chlorotheophyllinate (teoclate).

The exact formulation, route of administration 0 and dosage can be chosen by the individual physician in 0 20 view of the patient's condition. (See Fingl et al., in The Pharmacological Basis of Therapeutics, 1975, Ch. 1 p. 1).

It should be noted that the attending physician would know how and when to terminate, interrupt, or adjust administration due to toxicity or organ dysfunction. Conversely, the attending physician would also know to adjust treatment to higher levels if the clinical responses were not adequate (precluding toxicity). The magnitude of an administered dose in the management of the disorder of interest will vary with 267 the severity of the condition to be treated and to the route of administration. The severity of the condition may, for example, be evaluated in part, by standard prognostic evaluation methods. Further, the dose and perhaps dose frequency, will also vary according to the age, body weight, and response of the individual patient. A program comparable to that discussed above may be used in veterinary medicine.

Depending on the specific conditions being treated, such agents may be formulated into liquid or solid dosage forms and administered systemically or locally. The agents may be delivered, for example, in a timed or sustained-release form as is known to those skilled in the art. Techniques for formulation and administration may be found in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA (18th ed. 1990). Suitable routes may include oral, buccal, sublingual, rectal, transdermal, vaginal, transmucosal, nasal or intestinal administration; parenteral. delivery, including intramuscular, subcutaneous, intramedullary injections, as well as .e intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections, just to name a few.

For injection, the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological saline buffer. For such transmucosal administration, penetrants appropriate to the barrier to be permeated 268 are used in the formulation. Such penetrants are generally known in the art.

Use of pharmaceutically acceptable carriers to formulate the compounds herein disclosed for the practice of the invention into dosages suitable for systemic administration is within the scope of the invention. With proper choice of carrier and suitable manufacturing practice, the compositions of the present invention, in particular, those formulated as solutions, may be administered parenterally, such as by intravenous injection. The compounds can be formulated readily using pharmaceutically acceptable carriers well known in the art into dosages suitable for oral administration.

Such carriers enable the compounds of the invention to be formulated as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions and the like, for

O.

oral ingestion by a patient to be treated.

Agents intended to be administered intracellularly may be administered using techniques well known to those of ordinary skill in the art. For example, such agents may be encapsulated into liposomes, 00 then. administered as described above. Liposomes are spherical lipid bilayers with aqueous interiors. All molecules present in an aqueous solution at the time of liposome formation are incorporated into the aqueous interior. The liposomal contents are both protected o from the external-microenvironment and, because liposomes fuse with cell membranes, are efficiently delivered into the cell cytoplasm. Additionally, due to their hydrophobicity, small organic molecules may be 269 directly administered intracellularly.

Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve its intended purpose. Determination of the effective amounts is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

In addition to the active ingredients, these pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. The preparations formulated for oraladministration may be in the form of tablets, dragees, capsules, or solutions.

eeleo2 The pharmaceutical compositions of the present invention may be manufactured in a manner that is itself known, by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspension. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid ester, such as ethyl oleate or triglycerides, or liposomes. Aqueous 270 injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.

Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.

Pharmaceutical preparations for oral usecan be obtained by combining the active compounds with solid excipients, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose (CMC), and/or polyvinylpyrrolidone (PVP: povidone). If desired, disintegrating agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dye-stuffs or pigments may be added 271 to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin, and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols (PEGs). In addition, stabilizers may be added.

Other embodiments are within the following claims claims.

I. 0*e

Claims (27)

1. A compound of Formula VIII: 2 3 IHR FORMULA VIII (2 8 wherein: Z is selected from the group consisting of-CH 2 CH 2 -CH 2 CH(CH3)-, and -CH=CH-,-O-CH2-, -S-CH 2 X 1 and X 2 are independently selected from the group consisting of -CI, -CH 3 -OH and lower O-alkyl in the or 9-substituent positions; m is independently an integer from 0 to 2; provided that at least one of m is not 0; -NHR is selected from the group consisting of-NH2, -NHCH 3 and -NHC 2 H 5 R 1 is selected from the group consisting of alkyl, hydroxyalkyl, -OH, -0-alkyl, and- 0- acyl, and R 2 is selected from the group consisting of alkyl, hydroxyalkyl, and pharmaceutically acceptable salts and complexes thereof, wherein the compound is active at an NMDA receptor.

2. A compound selected from the group consisting of F NH 2 FCompound 195 Compound 195 Compound 193 Compound 194 C NH 2 FCompound 197 Compound 197 Compound 196 [R:\LIBZZ]444260D1speci.doc: gym 273 and pharmaceutically acceptable salts and complexes thereof. 3 The compound of claim 2, wherein said compound is NH 2 F F Compound 193 and a pharmaceutically acceptable salt and complex thereof.

4. The compound of claim 2, wherein said compound is F NH 2 F Compound 194 and a pharmaceutically acceptable salt and complex thereof. 1o 5. The compound of claim 2, wherein said compound is F x NH 2 F Compound 195 and a pharmaceutically acceptable salt and complex thereof.

6. The compound of claim 2, wherein said compound is x- NH 2 F Compound 196 and a pharmaceutically acceptable salt and complex thereof. [R:\LIBZZ]444260D1 speci.doc:gym

17. DEC. 2003 15:22 SPRUSON AND FERGUSON. 61292615486 NO. 3950 7/7 7. The compound of claim 2, wherein said compound is F Compound 197 and a pharmaceutically acceptable salt and complex thereof, 8. A method for treating a patient having a neurological disease or disorder, the method comprising administering a compound selected from the group consisting of esse S 'SW' S. I lb. S S C. S Compound 156 NH 2 Compound 184 F p d NH 2 F Compound 195 0 C S C S 09 S 5O S nest. S S. .9 S .C 055 S Compound 193 Compound 194 NH 2 NH 2 F F Compound 196 Compound 197 and pharmaceutically acceptable sails and complexes thereof. [RALlEZIl444260Dlss ci.doc:;m COMS ID No: SMBI-00540003 Received by IP Australia: Time 15:23 Date 2003-12-17 275 9. The method of claim 8, wherein said compound is selected from the group consisting F Compound 193 NH 2 F Compound 195 ,NH 2 Compound 194 r Compound 196 and pharmaceutically acceptable salts and complexes thereof. The method of claim 8, wherein said compound is NH 2 F F Compound 193 and a pharmaceutically acceptable salt and complex thereof. 11. The method of claim 8, wherein said compound is F x NH 2 F Compound 194 and a pharmaceutically acceptable salt and complex thereof. Compound 197 [R:\LIBZZ]444260D1 speci.doc:gym 276 12. The method of claim 8, wherein said compound is F NH 2 F Compound 195 and a pharmaceutically acceptable salt and complex thereof. 13. The method of claim 8, wherein said compound is NH 2 F Compound 196 and a pharmaceutically acceptable salt and complex thereof. 14. The method of claim 8, wherein said compound is /NH 2 F l Compound 197 and a pharmaceutically acceptable salt and complex thereof. A pharmaceutical composition, comprising a compound selected from the group consisting of F F NH 2 x NH 2 NH 2 F 15 F F Compound 193 Compound 194 Compound 195 [R:\LIBZZ]444260D1speci.doc:gym r 277 x NH 2 NH 2 F F F Compound 196 Compound 197 and pharmaceutically acceptable salts and complexes thereof, and a pharmaceutically acceptable carrier. 16. The pharmaceutical composition of claim 15, wherein said compound is NH 2 F F Compound 193 and a pharmaceutically acceptable salt and complex thereof. 17. The pharmaceutical composition of claim 15, wherein said compound F NH 2 10 F Compound 194 and a pharmaceutically acceptable salt and complex thereof. 18 The pharmaceutical composition of claim 15, wherein said compound is F NH 2 F 15 Compound 195 and a pharmaceutically acceptable salt and complex thereof. [R:\LIBZZ]444260D1 speci.doc:gym Iu 278

19. The pharmaceutical composition of claim 15, wherein said compound is NH 2 F Compound 196 and a pharmaceutically acceptable salt and complex thereof.

20. The pharmaceutical composition of claim 15, wherein said compound is NH 2 F Compound 197 and a pharmaceutically acceptable salt and complex thereof.

21. The method of any one of claims 8 to 14, wherein said neurological disease or disorder is selected from the group consisting of stroke, head trauma, spinal cord injury, epilepsy, anxiety, Alzheimer's disease, Huntington's disease, Parkinsons's disease, and amyotrophic lateral sclerosis.

22. The method of claim 21, wherein said neurological disease or disorder is stroke.

23. The method of claim 18, wherein said neurological disease or disorder is head trauma. i 524. The method of claim 21, wherein said neurological disease or disorder is spinal cord injury.

25. The method claim 21, wherein said neurological disease or disorder is epilepsy. Soooo

26. The method of claim 21, wherein said neurological disease or disease is anxiety.

27. The method of claim 21, wherein said neurological disease or disorder is Alzheimer's disease.

28. The method of claim 21, wherein said neurological disease or disorder is Huntington's 0; disease. 0°00 29. The method of claim 21, wherein said neurological disease or disorder is Parkinson's .oooo 0 "disease. 0 [R:\LIBZZ]444260DI speci.doc:gym Ir 279 The method of claim 21, wherein said neurological disease or disorder is amyotrophic lateral sclerosis.

31. The method of claim 22, wherein said stroke is global ischemic.

32. The method of claim 22, wherein said stroke is hemorrhagic.

33. The method of claim 22, wherein said stroke is focal ischemic.

34. A method for providing neuroprotection to a patient, comprising administering a compound of any one of claims 1, 2 or A pharmaceutical composition, comprising a compound of any one of claims 1, 2 or and a pharmaceutically acceptable carrier.

36. The pharmaceutical composition of claim 35, wherein said pharmaceutical composition is adapted for the treatment of a neurological disease or disorder.

37. The pharmaceutical composition of claim 35, wherein said pharmaceutical composition is adapted to provide neuroprotection to a patient.

38. The pharmaceutical composition of claim 35, wherein said compound is a Is hydrochloride salt.

39. Compound 156 or 184 or a pharmaceutically acceptable salt and complex thereof when used in treating a patient having a neurological disease or disorder. A compound as defined in claims 1 to 7 or a pharmaceutically acceptable salt and complex thereof when used in treating a patient having a neurological disease or disorder.

41. A compound of claim 39 or 40 wherein the neurological disease or disorder is defined in the method of any one of claims 21 to 33.

42. Use of compound 156 or 184 or a pharmaceutically acceptable salt and complex thereof in the manufacture of a medicament for treating a neurological disease or disorder. i 43. Use of a compound or a pharmaceutically acceptable salt and complex thereof as defined in any one of claims 1 to 7 in the manufacture of a medicament for treating a neurological disease or disorder.

44. Use of a composition of any one of claims 35 to 38 in the manufacture of a medicament for treating a neurological disease or disorder. Use as defined in claim 42, 43 or 44 wherein the neurological disease or disorder is 0 o defined in the method of any one of claims 21 to 33.

46. A compound of Formula VIII and a pharmaceutically acceptable salt and complex •thereof substantially as hereinbefore described with reference to any one of the examples. [R:\LIBZZ]444260D1 speci.doc:gym A, I k I' I 280

47. A method of treating a patient having a neurological disease or disorder, comprising administering a compound selected from the group consisting of Compound 156, 184, 193, 194, 195, 196 and 197 substantially as hereinbefore described with reference to any one of the examples. Dated 11 November, 2003 NPS Pharmaceuticals, Inc. Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON sees *se s: love *ses [R:\LIBZZ]444260D I speci.doc:gym