NO166185B - ANALOGY PROCEDURE FOR THE PREPARATION OF THERAPEUTIC ACTIVE 2-AMINO-7- (2-THIENTYLMETHYL) -4-PYRROLO (2,3-D) PYRIMIDONE OR A PHARMACEUTICAL ACCEPTABLE BASE OR ACID ADDICTION. - Google Patents

Abstract

New 7-deazaguanins with the formula. wherein R 9 is OH or SH, R 5 is hydrogen or NH, R 7 is independently hydrogen or NH. provided that both cannot simultaneously be NH, n is an integer from one to four, Ar is (i) unsubstituted phenyl or phenyl substituted by halogen, trifluoromethyl, alkyl of one to four carbon atoms ,. hydroxy or alkoxy having from one to four carbon atoms, (ii) 2- or 3-thienyl or (iii) - or 3-furanyl or a pharmaceutically acceptable base or acid addition salt thereof. The compounds are active as immunomodulators. Their preparation is described.

Description

The pyrrolo[2,3-d]pyrimidin-4-ones of the following formulas 2, 3 and 4

2, R is n-C3H7

3, R is CH 2 C 6 H 5

4, R is cyclopentyl

are known. R. K. Robbins et al., synthesized the compounds of formula 2 and 3 as described in J. Het. Chem. 1964, 34, but indicated no biological activity for any of the compounds. M. Legraverend et al., described the synthesis of the compounds of formulas 3 and 4 in Tetrahedron Letters, 1985, 2001, but again no biological activity was reported for either compound.

The following references form a background of minor interest, where a 7-(substituted phenyl)pyrrolo[2,3-d]pyrimidin-4-one with a methyl group in each of the five (5) and six (6) positions of treatment of CNS diseases or inflammations, is generically discussed in US patent 4,229,453. Similarly, 4-mercapto-7-(substituted or unsubstituted phenyl)pyrrolo[2,3-d]pyrimidine derivatives, which require an alkyl group or phenyl group in the five (5) or six (6) positions, are discussed in German 3145287 (Derwent Abstract No. 49344 K/21). Other pyrrolo[2,3-d]pyrimidin-4-ones or thiones, distinguished by having different substituents in the five (5) position, are found in US Patents 4,435,570 and 4,140,851, European Publications 160,910 ( Derwent Abstract No. 85-284574/46), 89,055 (corresponding to US Patent 4,571,423), 119,591 (Derwent Abstract No. 84-238735/39), 79,447 (corresponding to US Patent 4,435,569), German 3,306-390 (Derwent Abstract No. 39438 E/20), German 3145287 (Derwent Abstract No. 49344 K/21), British Patent 981,458 (Derwent Abstract No. 15,454), Japanese J6 0204,788 (Derwent Abstract No. 85/298810/48) and Japanese J5 9036625 (Derwent Abstract No. 84-086061/84).

Finally, hydroxy- and mercapto-analogues of the antibiotic sparsomycin A, pyrrolo[2,3-d]pyrimidin-4-one or -thione with a 3 iicker moiety in the seven (7) position, are described by Upjohn in Dutch 6,407,785 ( Derwent Abstract no. 15,466) and correspondingly by Warner-Lambert in European publication 57,548 (Derwent Abstract no. 68572 E/33).

The widely available Norwegian patent applications 85.4050 and 87.3584 deal with similar activity to that which has now been found for the present invention for other ring systems.

The present invention relates to an analogous method for the preparation of a compound of formula I

wherein Ar is 2-thienyl, or a pharmaceutically acceptable base or acid addition salt thereof.

The hitherto unknown intermediate used in the process according to the present invention is a compound of formula X

where Ar is as above.

The method according to the present invention for producing the hitherto unknown compound of formula I is characterized by treating a compound of formula X

wherein Ar is as defined above, with HCl in ethanol under reflux to obtain the compound of formula I. The compound of formula X can be prepared by treating a compound of formula IX

where Ar has the meaning given above, with HC1 in ethanol at

room temperature, and then with a base such as ammonium hydroxide to obtain the compound of formula X.

The above preparations employ standard synthetic techniques or techniques as shown or equivalent to those shown in the following examples. The starting materials in jr production are easily available, known or can be produced using known methods.

The production method according to the present invention is summarized in the following form I.

In certain circumstances it may be necessary to protect either N or O in the intermediates of the above process, with suitable protecting groups, which are known. Introduction and removal of such suitable oxygen and nitrogen protecting groups are well known within organic chemistry, see e.g. (1) "Protective Groups in Organic Chemistry", J.F.W. McOmie, ed., (New York, 1987), pp. 43, 95, (2) J.F.W. VicOmie, "Advances in Organic Chemistry", vol. 3, 191-281

(.1963); (3) R.A. Borssonas, "Advances in Organic Chemistry", vol. 3, 159-190 (1963); and (4) J.F.W. McOmie, Chem & Ind., 603 (.1979) .

Examples of suitable oxygen-protecting groups are benzyl, t-butyl, dimethylsilyl, methyl, isopropyl, ethyl, tertiary butyl, ethoxyethyl and the like. Protection of an N-H-containing part is necessary for some of the methods described here for preparing compounds according to the invention. Suitable nitrogen protecting groups are benzyl, triphenylmethyl, trialkylsilyl, trichloroethylcarbamate, trichloroethoxycarbonyl, vinyloxycarbamate and the like.

In certain circumstances it is necessary to protect two different oxygen atoms with different protecting groups so that one can be selectively removed while the other is retained. The benzyl and t-butyldimethylsilyl groups are used in this way, as one can be removed in the presence of the other, whereby benzyl can be removed by catalytic hydrogenolysis and t-butyldimethylsilyl can be removed by reaction with e.g. tetra-n-butylammonium fluoride.

In the method described here for the preparation of compounds according to the invention, the requirements for protective groups are well known to those skilled in organic chemistry, and the use of suitable protective groups is therefore necessarily implied in the method according to the scheme shown here, even if it is not directly shown .

The products from the method described here are isolated using conventional techniques, such as extraction, distillation, chromatography and the like.

The salts of the above-described compound of formula I are prepared by reacting the appropriate base with a stoichiometrically equivalent amount of the acid compounds of formula I, to obtain pharmacologically acceptable salts thereof.

The compound produced according to the invention can also exist in hydrated or solvated forms.

The compound of formula I prepared according to the present invention exists in tautomeric forms as shown below. Both forms are covered by formula I.

The compound of formula I is useful both in the free base form and in the form of base salts, where possible, and in the form of acid addition salts. Production of the three forms falls within the scope of the invention. In practice, use of the salt form corresponds to use of the base form. Suitable pharmaceutically acceptable salts are those derived from mineral acids such as hydrochloric acid and sulfuric acid as well as organic acids such as methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonate and the like, or those derived from bases such as suitable organic and inorganic bases. Examples of suitable inorganic bases for forming salts of the compound prepared according to the invention include the hydroxides of ammonia, sodium, lithium, potassium, calcium, magnesium, aluminium, zinc and the like.

Salts can also be formed with suitable organic bases. Bases suitable for forming pharmaceutically acceptable base addition salts with the compound of the present invention include organic bases which are non-toxic and attractively strong to form such salts. These organic bases form a class whose limits are readily understood by the person skilled in the art. By way of illustration, the class may be said to include mono-, di- and trialkylamines such as methylamine, dimethylamine and triethylamine, mono-, di- or trihydroxyalkylamines such as mono-, di- and triethanolamine, amino acids such as arginine and lysine, guanidine, N-methyl-glucamine, L-glutamine, N-methylpiperazine, morpholine, ethylenediamine, N-benzylphenethylamine, tri(hydroxymethyl)aminomethane and the like. (See, e.g., "Pharmaceutical Salts", J. Pharm. Sei.,

(1977), 66(1):1-19).

The acid addition salts of the basic compound are prepared either by dissolving the free base of compound I in aqueous or aqueous alcohol solution or suitable solvents containing the appropriate acid or base, and isolating the salt by evaporation of the solution or by reacting the free base of compound I with a acid, as well as by reacting compound I with an acidic group and then with a base, so that the reactions take place in an organic solvent, in which case the salt is separated directly or can be obtained by concentrating the solution.

The compound of formula I produced according to the present invention has been shown to have significant enzyme inhibitory activity and cytotoxic activity. In the purine nucleoside phosphorylase (PNP-4) enzyme assay, an IC5Q value is obtained at a dose of 1.0 micromoles of the compound of the present invention. PNP-4 activity for the compound of formula I is measured radiochemically by measuring the formation of [<14->C]hypoxanthine from [14-C]inosine (Biomedicine, 33, 39 (1980)) using human erythrocytes as enzyme source.

It is known that an in vivo inhibition of purine nucleoside phosphorylase (HPLC-1) enzyme test can also be used substantially as described in "Annals of New York Academy of Sciences", vol. 451, p. 313 (1985) to further demonstrate the activity of the compound of formula I prepared according to the present invention. The described compositions have also been shown by a standard test (HTBA-1) (Science, 214, 1137, (1981)) to be selectively cytotoxic for T cells in the presence of 2'-deoxyguanosine at a corresponding concentration range, and not -toxic to B cells in the presence of the same amount of 2'-deoxyguanosine as of the compound according to Example 2, demonstrating the utility of the compound of formula

In the pharmaceutical compositions described here. Die. PNP inhibition and removal of T cells or modulation of T cells are known to be properties of compounds useful in the treatment of psoriasis, transplant rejection and autoimmune diseases, the disclosed compositions of the compound, which are selectively cytotoxic to T -cells and which are PNP inhibitors, therefore also be valuable for such treatment. For example, 8-aminoguanosine, a known PNP inhibitor, has been shown to be effective in inhibiting rejection of skin grafts in dogs. (J.B. Benear et al., Transplantation, 1986, 41:274). Clinically, it has been shown that modulation and/or removal of T cells by means of breast duct drainage, lymphophoresis or total lymphoid irradiation provided partial to complete relief of rheumatoid arthritis in patients who were totally refractory to other forms of therapy (A. Tanay et al., Arthritis and Rheumatism, Vol. 30, No. 1. p.

1 (1987). S. Strober et al., Annual of Internal Medicine, vol. 102, No. 4, 441-449 (1985), H.G. Nusslein et al., Arthritis and Rheumatism, vol. 28, No. 11, 1205-1210 (1985), E. Brahn et al., ibid, Vol. 27, No. 5, 481-487 (1984) and J. Karsh et al., ibid, Vol. 24, No. 7. 867-873 (1981)). Cyclosporin A, a T-cell modulator, showed beneficial effects in the treatment of juvenile diabetes. (A. Assan et al., The Lancet, January 12, p. 67

(1985). Furthermore, cyclosporin A is today the drug of choice for alleviating transplant rejection (R.M. Merion et al., New England. J. Med., (1984) 148). Later, cyclosporin A has been shown to be useful in the treatment of psoriasis. Furthermore, it is understood that cyclosporine therapy has been shown to markedly reduce activated T cells in psoriatic lesions. Therefore, it is reasonable to assume that the basis for successful treatment of psoriasis is modulation of T-cell activity as demonstrated by compounds of the present invention. (See C.N. Ellis et al., JAMA, vol. 256, no. 22, December 12, 1986, 3110-3116). Finally, cyclosporin A has been shown to be effective in rheumatoid arthritis. (M.E. Weinblatt et al., Arthritis and Rheumatism, vol. 30, no. 1, 11-17 (January 1987). 0. Forre et al., Arthritis and Rheumatism, vol. 30, no. 1, 88-92 ( January 1987), M. Dougados et al., Arthritis and Rheumatism, vol., 30, no. 1, 83-87 (January 1987).

Results obtained with the compound according to the present invention are shown in the following activity table.

In vivo studies based on the above can be used to determine activity in the particularly stated disease states.

As T cells play a central role in the immune system, the mentioned compound is believed to be able to be used for immuno-regulation to prevent rejection during transplantation, or for the treatment of psoriasis and for the treatment of autoimmune diseases, such as rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease, multiple sclerosis, myasthenia gravis, gout or gouty arthritis, juvenile diabetes, cancer and viral diseases. The mentioned properties of the compound can be utilized by administering to a warm-blooded animal an effective amount of a pharmaceutical composition containing at least 0.1% by weight of active ingredient, based on the total weight of the composition of the compound prepared according to the invention.

Pharmaceutical compositions may be formulated in any suitable manner, preferably with an inert carrier, for administration orally, parenterally, ophthalmically, topically or as suppositories.

For example, the mentioned compound is formulated in dosage forms, such as tablets or syrups, by mixing with an inert pharmaceutical carrier, such as lactose or ordinary syrup, using methods known per se. For injectable dosage forms, it is formulated with carriers such as water, propylene glycol, peanut oil, sesame oil and the like. In these dosage forms, the active ingredient is from approx. 0.05 to 0.5 g per unit dose.

The invention is further illustrated by means of the following examples.

EXAMPLE 1

2-amino-6-f(2-thienylmethyl)amino1-4-pyrimidinol

2-Amino-6-chloro-4-pyrimidinol, monohydrate (85%, 100 g, 0.5197 mol) was suspended in methoxyethanol (700 mL), and 2-thienyl-methylamine (96%, 61.3 g, 0 .5197 mol) was added to the suspension. The mixture was heated under reflux for 2 hours and then 73 mL (d = 0.726, 0.52 mol) of triethylamine was added and refluxing was continued for another 18 hours. The reaction mixture was poured into ice water (1000 ml), acidified with acetic acid (pH 4.0) and the precipitated solid filtered off, washed and dried. Yield: 110 g (72.6%). This was used in the next step without further purification.

EXAMPLE 2

2- amino- 7-( 2- thienylmethyl)- 4- pvrrolo r 2. 3- d] pyrimidones

Chloroacetaldehyde-dimethyl acetal (14 mL) was added to water (50 mL) and concentrated HCl (2.0 mL). The mixture was heated to reflux for 30 minutes and neutralized with sodium acetate (10 g). The resulting solution was added in one portion to a mixture of 2-amino-4-(2-thienylmethyl)-6-pyrimidone (10 g, 45 mmol), sodium acetate (5.0 g) and hot water (50 mL). The mixture was allowed to stand under stirring on a steam bath (80°C) for 30 minutes, the precipitated solid was filtered off, washed with water and dried in vacuum. The crude product was dissolved in methanol and concentrated HCl and treated with activated charcoal to remove dye. The product thus obtained (3.2 g) was recrystallized from methanol and 1N HCl (100 mL) to give 1.68 g (13.5%) of the desired product as a light brown solid, m.p. 243-245°C (decomp.).

2- amino- 4- chloro- 6- r( 2- thienylmethyl) amino1- 5-( 2. 2- diethoxyethyl)-<p>yrimidine

A solution of 2-amino-4,6-dichloro-5-(2,2-diethoxyethyl)-pyrimidine (M. Legraverend et al., J. Med. Chem., 1985, 28:1477)

(906 mg, 3.20 mmol) in 40 mL of n-butanol containing Et3N (1 mL)

and 2-thienylamine (425 mg, 3.75 mmol), was heated to 100°C for 48 h. The reaction mixture was cooled to 25°C and concentrated. The residue was purified by column chromatography on silica gel eluting with chloroform to give the desired product (1.045 g) (91.5%) as a yellow oil.

2- amino- 4Tchloro- 7-( 2- thienylmethyl) pyrrolo 2. 3- dlpyrimidine

A suspension of 2-amino-4-chloro-6-[(2-thienylmethyl)amino]-5-(2,2-diethoxyethyl)pyrimidine, (1.0 g, 2.80 mmol) in 65 mL of 0.3N HCl and ethanol (2.25:1) were stirred at 25°C for 24 hours. The reaction mixture was neutralized with ammonium hydroxide solution, and the product was collected by filtration. TLC analysis showed that the reaction was not complete and the product was therefore resuspended in 50 ml of 0.2N HCl and stirred for 48 hours. The reaction mixture was neutralized with NH 4 OH solution and concentrated. The residue was taken up in water and then evaporated to dryness to give a yellow solid (573 mg) (77.3%). This was used in the next step without further purification.

2- amino- 7-( 2- thienylmethyl)- 4- pyrrolo f 2. 3- d1pyrimidone

2-amino-4-chloro-7-(2-thienylmethyl)pyrrolo[2,3-d]pyrimidine

(563 mg, 2.10 mmol) was suspended in 30 mL of 1N HCl and ethanol (1:1) and the mixture heated under reflux for 8 h. Removal of solvent gave a residue which was chromatographed over silica gel eluting with a mixture of hexane-ethyl acetate (10:1) to give 139 mg of a mixture of 4-chloro and 4-ethoxy derivatives. This was then dissolved in 30 ml of 3N HCl, and the solution was heated under reflux for 2 hours and then allowed to cool. The precipitated solid was filtered off and then crystallized from a methanol-1N HCl (1:1) mixture to give 55 mg of the desired product as the hydrochloride salt, m.p. 235-237°C (decomp.).

Claims (1)

Analogous process for the preparation of a therapeutically active compound of formula I wherein Ar is 2-thienyl, or a pharmaceutically acceptable base or acid addition salt thereof, characterized in that a compound of formula X is treated wherein Ar is as defined above, with HCl in ethanol under reflux to obtain a compound of formula I, which is optionally converted to a pharmaceutically acceptable salt.