KR940003360B1 - Side chain unsaturated 1 alpha-hydroxyvitamin d homologs - Google Patents

Abstract

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Description

[Name of invention]

1α-hydroxyvitamin D Homolog with Unsaturated Side Chains

Detailed description of the invention

The present invention relates to novel vitamin D compounds having specific activity in causing differentiation of tumor cells into normal cells. More specifically, the present invention relates to analogs of 1α, 25-dihydroxyvitamin D ₃ [1,25- (OH) ₂ D ₃ ] with unsaturated side chains and extended side chains, which increase in differentiating tumor cells. The selective action as an anti-tumor agent by exhibiting a much reduced action on calcium metabolism.

[Background of invention]

The activity of the D-vitamin group (vitamin (D ₃ or D ₂ )) in regulating calcium metabolism and normal bone growth and development is known to require metabolism of the parent vitamin in the characteristic hydroxylated form. Specifically, 1α, 25-dihydroxyvitamin D ₃ [1,25- (OH) ₂ D ₃ ], a dihydroxylated metabolite commonly formed from vitamin D ₃ in animals or humans, transports calcium in the intestine And is involved in stimulating calcium uptake (bone migration) from bone and thus has been demonstrated to be an active species that regulates the total blood calcium concentration in living organisms (see below for the calcium-related action of vitamin D metabolites or analogs). Collectively referred to herein as the “calcemic activity” or “calcemic action” of a compound). Also, for example, 1α- hydroxy-vitamin D _3, 1α- hydroxy-vitamin D _2, 1α, 25- dihydroxy vitamin D ₂ or 1,25- (OH) ₂ D ₃ of the fluoro substituted derivatives, such as Certain structural analogues of 1,25- (OH) ₂ D _{3 have} also been known to be highly active calcemic agents, resulting in 1,25- (OH) ₂ D ₃ and its active analogs. Iii), has been used and recommended as a medicament in the prevention and treatment of various calcium metabolism and bone diseases such as vitamin D-resistant rickets or osteoporosis and related diseases.

More recently, it has been found that 1,25- (OH) ₂ D ₃ exhibits biological functions other than the well-known “calcemic action” above. For example, 1,25- (OH) ₂ D ₃ and closely related analogues (1α-OH-D ₃ , 1,25 (OH) ₂ D ₂ , fluoro-substituted analogs, etc.) may cause cell differentiation. [Abe et al., Proc. Natl. Acad. Sci. USA, Vol. 78, p. 4990 (1981); Honma et al., Proc. Natl. Acad. Sci. USA Vol. 80, p. 201 (1983). Specifically 1,25- (OH) ₂ D ₃ and its analogs have been found to inhibit the proliferation of tumor cells (ie human leukemia cells) in culture and convert them into normal macrophages. (Hereinafter, this form of action is collectively referred to as the “differentiating action” of the vitamin D compound). Because of their excellent efficacy as differentiation inducing agents, these vitamin D derivatives are potentially useful as anticancer agents, and in fact their use for the treatment of human leukemia has been proposed. (Suda et al., US Pat. No. 4,391,802). However, while these compounds show a high potency in differentiating tumor cells in culture, their equally high calcemic action in vivo limits or prevents their use as substantial anticancer agents. Thus 1,25- (OH) ₂ D ₃ or its fluorinated derivatives are very potent compounds. These similar compounds can dangerously increase blood calcium levels by their intrinsic calcemic action at the concentrations necessary in vivo to be effective for use as anticancer (leukemia inhibitor) agents. Other known vitamin D derivatives present similar limitations between differentiation and calcemic action and their practical use as potential anticancer agents and therefore have the same limitations and risks. These observations have clearly recognized the need for compounds with higher specificity and selectivity as anticancer agents, i.e. compounds with improved differentiation / calcemic effect ratios, which encouraged these studies to promote differentiation by recent studies. Several vitamin D derivatives have been produced. For example, certain 1,25- (OH) ₂ D ₃ homologs whose side chains are extended by one carbon atom (in or at the end of the chain) may be described as 1,25- (OH) ₂ for leukemia cells in culture. It was found that D ₃ exhibits a significantly higher (about 10-fold) differentiation activity than itself. DuLuca et al., US Pat. No. 4,717,721; Ostrem and DeLuca, Steroid, Vol. 49, pp. 73-102 (1988); Ostrem et al., J. Biol. Chem, Vol. 262, p. 14864 (1987)]. However, these homologues are still very powerful calcemic agents which exhibit calcemic action which is almost equivalent to the calcemic action of 1,25- (OH) ₂ D ₃ . Thus, these compounds have the property of an enhanced differentiation / calcemic activity ratio but they do not solve the problem of the undesirable powerful calcemic action discussed above. Other vitamin D-related compounds reported to have selective differentiation have been prepared [Ostrem et al., Supra; Kubodera et al., Chem. Pharm. Bull., Vol. 34, pp. 2286-2289 (1986); Ikekawa et al., Chem. Pharm. Bull, Vol. 35, page 4362 (1987), which are structurally separate and different from the compounds of the present invention.

[Summary of invention]

We have found that vitamin D-related compounds exhibit a preferred and very advantageous type of action in terms of their differentiation versus calcemic response. These novel vitamin analogs have very good activity [similar to or greater than that of 1,25- (OH) ₂ D ₃ ] in inhibiting the proliferation of tumor cells and inducing their differentiation into normal monocyte-like cells. It has much less activity than 1,25- (OH) ₂ D _{3 as} far as calcemic action is concerned. Thus, these novel compounds exhibit dramatically increased differentiation / calcemic activity ratios and, by this feature, these compounds represent preferred reagents for the treatment of tumor diseases. When the activity is high in the induction of differentiation and much lower in the activity of the calcemic agent, administration of these compounds does not excessively increase the blood calcium concentration, thereby overcoming the main practical problems associated with the high calcemic action.

These novel compounds have structural features as homologues of 1,25- (OH) ₂ D ₃ with unsaturated side chains, in which side chains are stretched by inserting two or three methylene units into the carbon chain. Therefore, they are represented by the following general structure.

Wherein X, Y and Z may be the same as or different from each other, are selected from the group consisting of a hydrogen atom and a hydroxy protecting group, and n is 3 or 4.

Specific and preferred examples of these compounds include 24-dihomo-1,25-dihydroxy- ₂₂ -dehydrovitamin D ₃ (the above compounds wherein X, Y and Z are hydrogen atoms and n is 3) and 24-trihomo -1,25-dihydroxy- ₂₂ -dehydrovitamin D ₃ (the above compounds wherein X, Y and Z are hydrogen atoms and n is 4).

It is evident that these novel compounds are associated with 24-homo-vitamin D compounds with unsaturated side chains as shown in US Pat. No. 4,717,721. However, new compounds have distinct structural and biological properties. Structurally, the distinguishing feature is the unsaturated side chains homologized by the insertion of two or three methylene units, and biologically the compound is a very potent cell differentiator with no or significantly reduced calcemic action.

[Production of New Compound]

Synthesis examples of the novel compounds of the present invention are shown schematically in Schemes 1,2 and 3. Scheme 1 represents the desired 1α-hydroxyvitamin D-22-aldehyde intermediate, which is the desired vitamin D homolog when combined with the appropriate alkylphenylsulfone side chain unit as shown in Scheme 2 [eg, each compound (25) And (26). Scheme 3 illustrates the preparation of alkylphenylsulfone units required for side chain coupling. The experimental detailed procedure for the chemical reaction step shown in the scheme is shown in the following specific examples. As used in these examples, compounds represented by Arabic numerals (eg, 1, 2, 3, etc.) refer to structures with the same numbers in this scheme.

[Common way]

3β-acetoxy-22,23-bisnor 5-cholic acid (1) was purchased from Stealoids (Walken, NH). All other chemicals were of the highest quality purchased from commercially available sources. The solvent was purified by standard methods.

Thin layer chromatography (TLC) was performed using a precoated silica gel sheet with UV indicator from EM Sciences, Gibsontown, NJ. The solvent system used was A [chloroform-ethanol 18:15 (v / v)], B [hexane-ethyl acetate 1: 1] and C [hexane-ethyl acetate 3: 1].

High performance liquid chromatography (HPLC) was performed using Waters Associates liquid chromatography with a Model 6000A solvent delivery system, a Model 6 UK Universal Injector, and a detector capable of detecting various wavelengths of the Model 450. . A Zorbax-Sil (Zorbax-Sil, Phenomenex) column (6.2 mm x 25 cm and 10 mm x 25 cm) was used. The solvent system is A (3% 2-propanol in hexane), B (2% 2-propanol in hexane), C (6% 2-propanol in hexane), (D: 10% 2-propanol in hexane) And E (20% 2-propanol in hexane). Silica gel Sep-Pak (Water Associates) cartridges were used for prefiltration of HPLC samples.

Electron impact mass spectra (MS) were recorded at 70 eV with a Kratos MS-50 TC mass spectrometer equipped with a Kratos S-55 data system.

Ultraviolet (IV) absorption spectra were recorded with a Hitachi Model 60-100 UV-Vis spectrophotometer.

Infrared spectra were recorded on a Nicolet MX-1 FT-IR spectrometer using a film of oily material or carbon tetrachloride solution.

Proton magnetic resonance spectra ( ¹ H-NMR) were recorded on a Bruker 270,400 or 500 MHz spectrometer in a CDCl ₃ solution containing tetramethylsilane (TMS) as an internal reference.

Example 1

Synthesis of Protective C-22-aldehyde (Compound 18; Scheme 1)

This aldehyde is described by Kutner et al, Tet. Letters, Vol. 28, pp. 6129-6132 (1987).

10 g of compound (1) was dissolved in 420 ml of 5% KOH in methanol and the solution was stirred at ambient temperature for 15 minutes until no starting material was detected by TLC (solvent system A).

To this solution was added dropwise 160 ml of 10% sulfuric acid in methanol with stirring and the resulting suspension was diluted with 400 ml of 1% sulfuric acid in methanol. The mixture was heated under reflux for 48 hours to complete esterification (TLC, solvent system A).

Compound (2) (the ester) was extracted with ethyl acetate. The organic layer was washed with 5% NaHCO ₃ and saturated NaCl and dried over magnesium sulfate. 9.0 g (88%) of the resulting compound (2) was used in the next step without further purification.

To a solution of 4.4 g (12 mmol) of compound (2) in 135 ml of anhydrous dimethylformamide (DMF) was added 3.6 g (52.8 mmol) of imidazole followed by 4.0 g (26.4 mmol) of tert-butyldimethylsilyl chloride. The solution was stirred for 5 minutes at room temperature until a mass precipitate formed and then stirring was continued for an additional 15 minutes. The reaction mixture was extracted with 400 ml of hexane, washed with water and saturated NaCl solution and dried over magnesium sulfate. The solvent was evaporated to give the TLC pure product (solvent system B) and 5.3 g (91%) of this compound (3) was used in the next step without further purification. Analytical samples were obtained by flash chromatography using 2% ethyl acetate in hexanes.

Mixture of compound (3) (1.0 g, 2.1 mmol), dibroanthine (0.42 g, 1.5 mmol) and anhydrous sodium bicarbonate (0.91 g, 10 mmol) in 20 ml of hexane under reflux until no starting material was detected Heated for 30 minutes (TTC, system C). The precipitate was filtered off, and the solution was dried under reduced pressure. The residue was redissolved in 5 ml of dry THF and 0.06 g (0.19 mmol) of tetrabutylammonium bromide were added and the mixture was stirred at room temperature under nitrogen atmosphere for 30 minutes. Tetrabutylammonium fluoride (10 ml, 1M solution in THF) was then added, followed by 0.7 ml of S-collidine, and the mixture was stirred for 1 hour at room temperature under a nitrogen atmosphere. Again 5 ml of tetrabutylammonium fluoride solution was added and stirring continued for 3 hours. 50 ml of ether was added and the organic layer was washed with water, cold 1N HCl, 10% NaHCO ₃ and dried over anhydrous magnesium sulfate. Compound (4) as a product was dissolved in benzene and chromatographed with silica gel 70-230 mesh (30 g). 0.44 (58%) of compound (4) was eluted with ethyl acetate in hexane. Analytical samples were obtained by HPLC. (System A, Rv 77ml):

IR (film): 1737, 1604, 1495, 1082, 1030 cm ^-1 ; UV (3% 2-propanol in hexane) λ _max 262 nm (ε 7,000), λ _max 272 nm (ε 9,800), λ _max 282 nm (ε 10,500), λ _max 293 nm (ε 6,000); ¹ H NMR (CDCl ₃ ) δ0.54 (3H, s, 18-CH ₃ ), 0.94 (3H, s, 19-CH ₃ ), 1.22 (3H, d, J = 6Hz, 2-CH ₃ ), 3.6 (1H, m, 3-H), 3.68 (3H, s, CO ₂ CH ₃ ), 5.42 (1H, m, 6-H), 5.58 (1H, m, 7-H); MS m / z (relative strength) 358 (61), 340 (12), 325 (100), 299 (68), 271 (7), 253 (17), 237 (26), 211 (27), 143 ( 72), 119 (35).

A solution of 830 mg (2.3 mmol) of compound (4) in 350 ml of a benzene-ethyl ether 1: 4 (v / v) mixture was transferred to Hannovia in a water-cooled quartz impregnation well equipped with a nitrogen bubbler and Vycor filter. Irradiation was conducted under a nitrogen atmosphere for 40 minutes (4 × 10 minutes) using a (Hanovia) 608A36 medium pressure UV lamp. The reaction was examined at 265 nm by HPLC using 2% 2-propane in hexanes. The solution was dried under reduced pressure and redissolved in 100 ml of pure ethanol and then heated under reflux for 3 hours under nitrogen atmosphere. The solution was then concentrated, dissolved in 1 ml of 10% ethyl acetate in hexanes and chromatographed with silica gel 70-230 mesh (30 g). 298 mg (36%) of vitamin ester (5) were eluted using a 15% ethyl acetate mixture in hexanes. Analytical samples were obtained by HPLC (System B, Rv 94 ml).

IR (film): 1738 cm ^-1 ; UV (EtOH) λ _max 264 nm, λ _min 288 nm; ¹ H NMR (CDCl ₃ ) δ: 0.56 (3H, s, 18-CH ₃ ), 1.20 (3H, d, J = 7 Hz, 21-CH ₃ ), 3.66 (3H, s, CO ₂ CH ₃ ), 3.95 (1H, m, 3-H), 4.80 (1H, d, J = 1.2 Hz, 19Z-H), 5.05 (1H, d, J = 1.2 Hz, 19E-H), 6.03 (1H, d, J = 11 Hz, 7-H), 6.23 (1H, d, J = 11 Hz, 6-H); MS m / z (relative strength), M ⁺ 358 (45), 340 (9), 325 (45), 299 (22), 253 (19), 237 (18), 136 (60), 118 (100) .

A solution of 10 mg (0.028 mmol) of dry toluene 5 ml Compound (5) was cooled to −70 ° C. under a nitrogen atmosphere in a dry ice-acetone bath. Diisobutylaluminum hydride (DIBAL-H, 50 µl, 25% solution in toluene, 0.088 mmol) was added dropwise to this solution with stirring. The reaction mixture was stirred at −70 ° C. for 10 minutes, then 2 ml of methanol was added slowly. The mixture was warmed to room temperature and diluted with ethyl ether, then washed with 5% HCl, 5% NaHCO ₃ , water and saturated NaCl and dried over anhydrous magnesium sulfate. Silica gel chromatography (15% ethyl acetate in hexane) was carried out to obtain 4.9 mg (54%) of compound (6) showing the following spectral statistics.

MS: 328 (M ⁺ , 29), 310 (5), 295 (31), 269 (11), 253 (6), 136 (47), 118 (86), 29 (100); ¹ H NMR (CDCl ₃ ) δ: 0.59 (3H, s, 180-CH ₃ ), 1.14 (3H, d, J = 7Hz, 21-CH ₃ ), 4.0 (1H, m, 3-H), 4.81 ( 1H, d, J = 1.2 Hz, 19E-H), 5.05 (1H, d, J = 1.2 Hz, 19Z-H), 6.05 (1H, d, J = 11 Hz, 7-H), 6.23 (1H, d , J = 11 Hz, 6-H), 9.58 (1H, d, J = 3.8 Hz, 22-H).

Further elution on silica gel column with 5% 2-propanol in hexane gave 2.7 mg (29%) of compound (7) which is C-22-alcohol.

Compound (5) was converted to compound (8) using p-toluenesulfonyl chloride in pyrimidine at 4 ° C. for 20 hours. 102 mg (0.2 mmol) of the compound (8) dissolved in 2 ml of anhydrous dichloromethane in methanol solution (15 ml) in 250 mg of anhydrous potassium bicarbonate were added at 55 degreeC, stirring. The mixture was stirred at 55 ° C. for 24 hours under a nitrogen atmosphere. The solvent was then removed under reduced pressure and the residue was extracted with ether. The organic layer was washed with water and dried over anhydrous magnesium sulfate. The product compound (9) was purified by silica gel chromatography using 20% ethyl acetate in hexanes (50 mg, 68%).

tert-butyl hydroperoxide (112 μl, 3.0 M solution in toluene, 0.34 mmol) was added to a suspension of 9 mg (0.8 mmol) of selenium dioxide in 2 ml of dry methylene chloride. The mixture was stirred at room temperature under a nitrogen atmosphere until a clear solution formed. Then, anhydrous pyridine (12 [mu] l1, 0.15 mmol) was added followed by 50 mg of compound (9) dissolved in 2 ml of anhydrous dichloromethane. The mixture was stirred for 30 minutes under a nitrogen atmosphere. 2 ml of cold 10% sodium bicarbonate was added and the mixture was extracted with ether. The organic layer was washed with cold 10% sodium bicarbonate, ice water, and then dried over anhydrous magnesium sulfate. Silicakel chromatography (10-20% ethyl acetate in hexane) was carried out to give 12.5 mg of compound (10). The product was immediately dissolved in 0.5 ml of glacial acetic acid and the solution was heated at 55 ° C. for 15 minutes with stirring under a nitrogen atmosphere. The reaction mixture was poured into ice, extracted with ether and washed with ice-cold saturated sodium bicarbonate. The combined ether extracts were washed with water and dried over dry magnesium sulfate. Analytical samples of (11) and (12), which are the (5Z, 7E) and (5E, 7E) isomers, respectively, were obtained 2.5: 1 by preparative HPLC.

=2.07 ;¹H NMR(CDCl₃)δ, 0.56(3H, s, 18-CH₃), 1.20(3H, d, J=6.5Hz, 21-CH₃), 2.04(3H, s, 3β-아세틸), 3.66(3H, s, 22-CO₂CH₃), 4.4(1H, m, 1-H), 5.2(1H, m, 3-H), 5.01(1H, br s, 19E-H), 5.34(1H, br s, 19Z-H), 6.01(1H, d, J=10Hz, 7-H), 6.33(1H, d, J=10Hz, 6-H) ; MS m/z(상대강도), 416(M⁺, 4), 356(100), 338(21), 251(13), 134(95).Compound 11: HPLC, R _v 68 mL; UV (EtOH) λ _max 264 nm, λ _min 277 nm,

= 2.07; ¹ H NMR (CDCl ₃ ) δ, 0.56 (3H, s, 18-CH ₃ ), 1.20 (3H, d, J = 6.5 Hz, 21-CH ₃ ), 2.04 (3H, s, 3β-acetyl), 3.66 (3H, s, 22-CO ₂ CH ₃ ), 4.4 (1H, m, 1-H), 5.2 (1H, m, 3-H), 5.01 (1H, br s, 19E-H), 5.34 (1H , br s, 19Z-H), 6.01 (1H, d, J = 10 Hz, 7-H), 6.33 (1H, d, J = 10 Hz, 6-H); MS m / z (relative intensity), 416 (M ⁺ , 4), 356 (100), 338 (21), 251 (13), 134 (95).

=3.51 ;¹H NMR(CDCl₃)δ, 0.56(3H, s, 18-CH₃), 1.20(3H, d, J=6. 5Hz, 21-CH₃), 2.04(3H, s, 3β-OAc), 3.66(3H, s, 22-CO₂CH₃), 4.5(1H, m, 1-H), 5.3(1H, m, 3-H), 4.99(1H, br s, 19E-H), 5.13(1H, br s, 19Z-H), 5.81(1H, d, J=10Hz, 7-H), 6.56(1H, d, J=10Hz, 6-H).Compound 12: HPLC, R _v 78 ml; UV (EtOH) λ _max 267 nm, λ _min 277 nm,

= 3.51; ¹ H NMR (CDCl ₃ ) δ, 0.56 (3H, s, 18-CH ₃ ), 1.20 (3H, d, J = 6.5 Hz, 21-CH ₃ ), 2.04 (3H, s, 3β-OAc), 3.66 (3H, s, 22-CO ₂ CH ₃ ), 4.5 (1H, m, 1-H), 5.3 (1H, m, 3-H), 4.99 (1H, br s, 19E-H), 5.13 ( 1 H, br s, 19 Z-H), 5.81 (1 H, d, J = 10 Hz, 7-H), 6.56 (1 H, d, J = 10 Hz, 6-H).

For large scale production, isomers 11 and 12 can also be advantageously separated effectively by the maleic anhydride method described in US Pat. No. 4,554,106.

Hydrogenated diisobutylaluminum (15 μl, 1.5 M solution in toluene) was added to the solution of 2 mg of compound (11) in 0.5 ml of anhydrous toluene at −70 ° C. under nitrogen atmosphere. The mixture was stirred at −70 ° C. for 10 minutes and 0.2 ml of methanol was slowly added to decompose the organometallic complex. The mixture was warmed to room temperature and extracted with ethyl ether. The organic layer was washed with water and dried over anhydrous magnesium sulfate. Compound (13) and compound (14) were obtained by preparative HPLC using solvent system E. Compound (13) showed the following spectral data.

334 (M ⁺ , 22), 326 (13), 311 (2), 285 (4), 269 (4), 152 (29), 134 (100); ¹ H NMR (CDCl ₃ ) δ, 0.59 (3H, s, 18-CH ₃ ), 1.15 (3H, d, J = 7 Hz, 21-CH ₃ ), 4.2 (1H, m, 3-H), 4.4 ( 1H, m, 1-H), 4.99 (1H, d, J = 1.2 Hz, 19Z-H), 5.31 (1H, d, J = 1.2 Hz, 19E-H), 6.02 (1H, d, J = 11 Hz , 7-H), 6.36 (1H, d, J = 11 Hz, 6-H), 9.56 (1H, d, J = 4 Hz, 22-H).

A 0.1 N KOH solution in 10 ml of methanol was added to a stirred solution of 100 mg (0.24 mmol) of compound (11) in 10 ml of ethyl ether. The resulting solution was stirred for 90 minutes at room temperature until no starting material was detected by TLC (solvent system B). Compound (15) was isolated by standard extraction methods (ethyl acetate, saturated NaCl, anhydrous sodium sulfate) to give 86.2 mg (96%) of a colorless oil.

A mixture of 250 mg (3.6 mmol) of imidazole and 250 mg (1.6 mmol) of tert-butyldimethyl chloride in 2 ml of DMF was added to a stirred solution of 86.2 mg (0.23 mmol) of compound (15) in 4 ml of dimethyl formamide. The resulting homogeneous mixture was stirred at 55 ° C. for 15 minutes until no starting material was detected by TLC (solvent system B). This product was isolated by hexane extraction of the reaction mixture. The organic extract was washed with brine and dried over anhydrous magnesium sulfate. The hexane solution of the crude product was filtered through a silica gel Sep-Pak cartridge to give 136 ml (98%) of the mixture (16).

=1.91 ;¹H NMR(CDCl₃)δ, 0.07[12H, s, Si(CH₃)₂], 0.55(3H, s, 18-CH₃),[18H, s, C(CH₃)₃], 1.20(3H, d, J=6.8Hz, 21-CH₃), 3.65(3H, s, O-CH₃), 4.18(1H, m, 3-H), 4.36(1H, m, 1-H), 4.84(1H, d, J=1.2Hz, 19Z-H), 5.16(1H, d, J=1.2Hz, 19E-H), 5.96(1H, d, J=11.2Hz, 7-H), 6.19(1H, d, J=11.2Hz, 6-H) ; MS m/z(M/e 248까지 표준화시킨 강도)IR (film): 2974, 2930, 1736, 1447, 1286, 1258, 1150, 1085 cm ^-1 ; UV (hexane) λ _max 264nm, λ _min 227nm

= 1.91; ¹ H NMR (CDCl ₃ ) δ, 0.07 [12H, s, Si (CH ₃ ) ₂ ], 0.55 (3H, s, 18-CH ₃ ), [18H, s, C (CH ₃ ) ₃ ], 1.20 ( 3H, d, J = 6.8 Hz, 21-CH ₃ ), 3.65 (3H, s, O-CH ₃ ), 4.18 (1H, m, 3-H), 4.36 (1H, m, 1-H), 4.84 (1H, d, J = 1.2 Hz, 19Z-H), 5.16 (1H, d, J = 1.2 Hz, 19E-H), 5.96 (1H, d, J = 11.2 Hz, 7-H), 6.19 (1H , d, J = 11.2 Hz, 6-H); MS m / z (strength normalized to M / e 248)

602 (M ⁺ , 10), 470 (59), 413 (7), 338 (10), 248 (100).

25 mg (0.65 mmol) of lithium aluminum hydride were added to a stirred solution of 136.2 mg (0.23 mmol) of compound (16) in 5 ml of dry THF at 0 ° C. under argon. The suspension was stirred at 0 ° C. for 15 minutes and excess lithium aluminum hydride was decomposed by dropwise addition of 10% water in THF. This suspension was diluted with 10 ml THF and stirring continued for another 15 minutes at room temperature. The product was isolated by standard extraction using ethyl acetate. 118.4 mg of compound (17) was obtained as a colorless oil in 91% yield.

=1.57 ;¹H NMR(CDCl₃)δ, 0.00[12H, s, Si-CH₃), 0.53(3H, s, 18-CH₃), 0.85[18H, s, Si-C(CH₃)₃], 1.04(3H, d, J=6.4Hz, 21-CH₃), 3.37 and 3.63(1H 및 1H, 각각 m,22-CH₂), 4.17(1H, m, 3-H), 4.35(1H, m, 1-H), 4.84(1H, br s, 19Z-H), 5.16(1H, br, s, 19E-H), 6.00(1H, d, J=12.2Hz, 7=H), 6.21(1H, d, J=12.2Hz, 6-H) ; MS m/z(m/z 248까지 표준화시킨 강도), 574(M⁺, 17), 442(67), 383(11), 308(17), 248(100).IR (film): 3450, 2952, 2886, 1447, 1258, 1105, 1085, 834 cm ^-1 ; UV (EtOH) λ _max 264 nm, λ _min 277 nm,

= 1.57; ¹ H NMR (CDCl ₃ ) δ, 0.00 [12H, s, Si-CH ₃ ), 0.53 (3H, s, 18-CH ₃ ), 0.85 [18H, s, Si-C (CH ₃ ) ₃ ], 1.04 (3H, d, J = 6.4 Hz, 21-CH ₃ ), 3.37 and 3.63 (1H and 1H, m, 22-CH ₂ ), 4.17 (1H, m, 3-H), 4.35 (1H, m, 1-H), 4.84 (1 H, br s, 19 Z-H), 5.16 (1 H, br, s, 19 E-H), 6.00 (1 H, d, J = 12.2 Hz, 7 = H), 6.21 (1 H, d, J = 12.2 Hz, 6-H); MS m / z (strength normalized to m / z 248), 574 (M ⁺ , 17), 442 (67), 383 (11), 308 (17), 248 (100).

A solution of 30 μl (0.34 mmol) of oxalyl chloride in 0.5 ml of dichloromethane was added to a stirred solution of DMSO (50 μl (0.7 mmol)) in 3 ml of dichloromethane at −60 ° C. under a nitrogen atmosphere. The resulting solution was stirred at −60 ° C. for 10 minutes and 27 mg (0.05 mmol) of compound (17) in 1 ml of dichloromethane were slowly added. The mixture was stirred at -60 ° C for 30 minutes. Then 0.2 ml of triethylamine was added and the solution was further stirred for 5 minutes. The product, compound (18), was extracted with ethyl ether and the organic layer was washed with saturated NaCl solution and dried over anhydrous magnesium sulfate. Silicagel Sep-Pak filtration gave 17 mg (62%) of TLC pure product.

=1.9 ; MS m/z(m/z에 대한 상대 강도 248) 572(M⁺, 13), 440(53), 383(11), 308(14), 248(100) ;IR (film): 2954, 2929, 2884, 2857, 1727, 1472, 1375, 1256, 1085, 909, 880, 835 cm ⁻¹ ; NMR (CDCl ₃ ) δ, 0.00 (12H, s, Si-CH ₃ ), 0.60 (3H, s, 18-CH ₃ ), 0.88 [18H, d, Si-C (CH ₃ ) ₃ ], 1.11 (3H , d, J = 6.9 Hz, 21-CH ₃ ), 4.23 (1H, m, 3-H), 4.43 (1H, m, 1-H), 4.93 (1H, br s, 19Z-H), 5.19 ( 1H, br s, 19E-H), 6.07 (1H, d, J = 10.0 Hz, 7-H), 6.26 (1H, d, J = 10.0 Hz, 6-H), 9.54 (1H, d, J = 3 Hz, 22-H); UV (hexane) λ _max 264 nm, λ _min 277 nm,

= 1.9; MS m / z (relative intensity 248 against m / z) 572 (M ⁺ , 13), 440 (53), 383 (11), 308 (14), 248 (100);

Mass spectrometry for C ₃₄ H ₆₀ O ₃ Si ₂

Theoretic: 572.4081

Found: 572.4117

The aldehyde 18 was obtained in increased yield when the oxidation step was carried out under the following conditions; A solution of 15 μl (0.17 mmol) of oxalyl chloride in 0.75 mL of anhydrous dichloromethane was added dropwise a stirred solution of 25 μL (0.36 mmol) of dimethyl sulfoxide in 0.25 mL of anhydrous dichloromethane at -60 ° C. under argon atmosphere. After the mixture was stirred at −60 ° C. for 10 minutes, a solution of 20.3 mg (0.035 mmol) of alcohol (17) in 0.5 ml of anhydrous dichloromethane was slowly added and the flash was washed with additional 0.2 ml of anhydrous dichloromethane. . The mixture was stirred at −60 ° C. for 30 minutes before 0.3 ml (2.15 mmol) of triethylamine were added at −60 ° C. The mixture was stirred for 5 minutes, then warmed to 0 ° C. and extracted with ether. The ether layer was washed with brine and dried (MgSO ₄ ). Colorless oil (18) was obtained by silica gel Sep-Pak filtration and purified by HPLC (Zorbax-Sil 9.4 × 25 cm, 10% EtOAc in hexane) to obtain 19 mg (96%) of pure aldehyde (18); Only a small amount of alcohol was recovered (0.12 mg).

Example 2

Side chain attachment: Synthesis of 24-dihomo-1α, 25-dihydroxy- ₂₂ -dehydrovitamin D ₃ (Compound 25, Scheme 2)

(a) Preparation of hydroxysulfones (19)

2-methyl-6-phenylsulfonyl) -2- (triethylsilyloxy) -hexane in 300 μl of anhydrous tetrahydrofuran (containing 1,10-phenanthroline as indicator) at −78 ° C. under argon atmosphere. (Compound 31, Scheme 3) To 31 mg (84 μmol) of a stirred solution was added 13 μl (90 μmol) of diisopropylamine followed by n-BuLi (91 μmol) (1.30 M solution in hexane). The solution was stirred for 30 minutes at -78 ° C under argon atmosphere, and then 6 mg (10 mmol) of C-22-aldehyde (Compound 18) in 300 µl of anhydrous tetrahydrofuran were added and stirred at -78 ° C for 1 hour. . The mixture was decomposed by adding 1 ml of saturated NH ₄ Cl solution, heated to 0 ° C., and extracted with ethyl acetate. Ethyl acetate was washed with water and brine, dried over anhydrous MgSO ₄ , filtered and evaporated. Preparative HPLC (Zorbax-Sil column 9.6 × 25 cm, solvent system: 10% ethyl acetate in hexane) gave 0.6 mg of unreacted aldehyde and 6.6 mg of hydroxysulfone (19) as epimer mixture (yield 77%) .

(b) 24-dihomo-1α, 25-dihydroxy- ₂₂ -dehydro-vitamin D ₃ (25)

A saturated solution of Na ₂ HPO ₄ in 1.0 ml of methanol was added to a stirred solution of 3.3 mg of hydroxysulfone (19) in 1.0 ml of anhydrous tetrahydrofuran followed by 160 mg of dry powder Na ₂ HPO ₄ . The mixture was stirred for 30 minutes under argon and then cooled to 0 ° C. Then about 400 mg of fresh 5% amalgam sodium was added and the mixture was stirred at 5 ° C. for 16 h. The mixture was diluted with 5 ml of hexane and stirring continued for 15 minutes. The solvent was decanted and the solid was washed with hexane (5 ml x 3 times). Ice and saturated NaCl solution were added to the combined organic solution. The organic layer was separated and passed through a Sep-pak cartridge in hexane. HPLC purification (Zorbax-Sil 9.4 × 25 columns, 10% EtOAc in hexanes) of ^di-homo-22 -24- -1,25- (OH), protected by a _{2 D 3 (21) 2.0mg (} 71%) and 22- A small amount of the hydroxylated product (22) was obtained. 2 mg of protected triol (21) was dissolved in 1.0 ml of dry THF and to this solution was added a 1M solution of tetrabutylammonium fluoride in 50 μl THF. The mixture was stirred at 50 ° C. under argon for 1 hour. Then 8 ml of ether was added and the organic layer was washed with saturated NaCl. After removal of the solvent the residue was dissolved in 10% 2-propanol in hexanes and filtered through silica Sep-Pak. HPLC (0.6% 20% 2-propanol in hexane, Zorbax-Sil 9.4 × 25 cm) gave 0.6 mg of the dihomo compound (25) as the desired product.

=1.87 ;¹H NMR(CDCl₃), 0.55(3H, s, 18-CH₃), 1.00(3H, d, J=6.6Hz, 21-CH₃), 1.23(6H, s, 26, 27-CH₃), 4.23(1H, m, 3-H), 4.43(1H, m, 1-H), 5.00(1H, brs, 19Z-H), 5.32(1H, brs, 19E-H), 5.29(2H, m, 22H 및 23H), 6.01(1H, d, J=11.3Hz, 7-H) ; MS m/z(상대강도) 442(M⁺, 15), 424(23), 406(33), 391(7), 287(11), 285(10), 269(27), 251(23), 152(33), 134(100), 116(6), 59(20) ;UV (EtOH) λ _max 264 nm, λ _min 228 nm,

= 1.87; ¹ H NMR (CDCl ₃ ), 0.55 (3H, s, 18-CH ₃ ), 1.00 (3H, d, J = 6.6 Hz, 21-CH ₃ ), 1.23 (6H, s, 26, 27-CH ₃ ) , 4.23 (1H, m, 3-H), 4.43 (1H, m, 1-H), 5.00 (1H, brs, 19Z-H), 5.32 (1H, brs, 19E-H), 5.29 (2H, m , 22H and 23H), 6.01 (1H, d, J = 11.3 Hz, 7-H); MS m / z (relative strength) 442 (M ⁺ , 15), 424 (23), 406 (33), 391 (7), 287 (11), 285 (10), 269 (27), 251 (23) 152 (33), 134 (100), 116 (6), 59 (20);

Mass Spectrometry for C ₂₉ H ₄₆ O ₃

Theoretic: 442.3446

Found: 442.3441

Example 3

Side chain attachment: Synthesis of 24-trihomo-1α, 25-dihydroxy-22-dehydrovitamin D ₃ (Compound 26, Scheme 2)

(a) Preparation of hydroxysulfones (20)

2-methyl-7- (phenylsulfonyl) -2- (triethylsilyloxy) -heptane in 500 μl of anhydrous tetrahydrofuran (containing 1,10-phenanthroline as indicator) at −78 ° C. under argon atmosphere. (Compound 35, Scheme 3) To 58 mg (151 μmol) of a stirred solution was added 23 μl of diisopropylamine (160 μmol) followed by 160 μM of n-BuLi- (1.5 M solution in hexane). This solution was stirred for 30 min at -78 ° C under argon atmosphere, then 7 mg (12 μmol) of C-22-aldehyde (Compound 18) in 300 μl of anhydrous tetrahydrofuran were added and stirred for 1 hour. The mixture was decomposed by adding 1 ml of saturated NH ₄ Cl solution, warmed to 0 ° C., and extracted with ethyl acetate. Ethyl acetate was washed with water and brine, dried over anhydrous MgSO ₄ , filtered and evaporated. Preparative HPLC [Zorbax-Sil column 9.4 × 25 cm, solvent system: 10% ethyl acetate in hexanes] was carried out to obtain 7.5 mg of hydroxysulfone (20) as a mixture of 0.4 mg of unreacted aldehyde and epimer (yield 78%) ).

(b) 24-trihomo-1α, 25-dihydroxy-22-dihydro vitamin D ₃ (26)

A saturated solution of Na ₂ HPO ₄ in 1.0 ml of methanol was added to a stirred solution of 7.5 mg of hydroxysulfone (20) in 1.0 ml of anhydrous tetrahydrofuran followed by 160 mg of anhydrous powdered Na ₂ HPO ₄ . The mixture was stirred for 30 minutes under argon and then cooled to 0 ° C. Fresh 5% sodium amalgam (about 400 mg) was then added and the mixture was stirred at 5 ° C. for 16 hours. The mixture was diluted with 5 ml of hexane and stirring continued for 15 minutes. The solvent was decanted and the solid was washed with hexane (5 ml x 3 times). The combined organic layers were washed with brine, separated, dried and evaporated. The residue was passed through a Sep-Pak cartridge in 10% ethyl acetate in hexanes. 2.12 mg of 22-24-trihomo-1,25- (OH) ₂ D ₃ (23) and 1.33 mg of 22-hydroxylation product (24) protected by HPLC purification were obtained. (Zorbax-Sil 9.4 × 25 column, 10% EtOAc in hexanes). 2.1 mg of compound (23) was dissolved in 1.0 ml of anhydrous THF, and 50 µl of tetrabutylammonium fluoride (1M solution) in THF was added to this solution. The mixture was stirred at 5 ° C. under argon for 1 hour. Ether was then added and the organic layer was washed with brine. The ether layer was dried over anhydrous MgSO ₄ , filtered and evaporated. The residue was dissolved in 30% 2-propanol in hexanes and passed through silica Scp-Pak. Purification by HPLC (20% 2-propanol in hexane, Zorbax-Sil 9.4 × 25 cm column) gave 0.8 mg of trihomo compound (26) as the desired product.

=1.81 ;¹H NMR : (CDCl₃) 0.56(3H, s18-CH₃), 1.00(3H, d, J=6.6Hz, 21-CH₃), 1.23(6H, s, 26, 27-CH₃), 4.23(1H, m, 3-H), 4.43(1H, m, 1-H), 5.00(1H, brs, 19Z-H), 5.32(1H, brs, 19E-H), 5.29(2H, m, 22H 및 23H), 6.01(1H, d, J=11.3Hz, 7-H) ; MS m/z (상대강도) 456(M⁺)(11)438(50), 420(30), 402(8), 287(10), 269(23), 251(23), 152(35), 134(100).UV (EtOH) λ _max 264 nm, λ _min 228 nm,

= 1.81; ¹ H NMR: (CDCl ₃ ) 0.56 (3H, s18-CH ₃ ), 1.00 (3H, d, J = 6.6 Hz, 21-CH ₃ ), 1.23 (6H, s, 26, 27-CH ₃ ), 4.23 (1H, m, 3-H), 4.43 (1H, m, 1-H), 5.00 (1H, brs, 19Z-H), 5.32 (1H, brs, 19E-H), 5.29 (2H, m, 22H And 23H), 6.01 (1H, d, J = 11.3 Hz, 7-H); MS m / z (relative strength) 456 (M ⁺ ) (11) 438 (50), 420 (30), 402 (8), 287 (10), 269 (23), 251 (23), 152 (35) , 134 (100).

Example 4

Synthesis of sulfone side chain units (Scheme 3)

(a) Preparation of sulfone side chain residues (32)

A solution of 3 g of 4-chlorovalerylchloride 27 (from Aldrich; 19.2 mmol) in 25 ml of anhydrous THF over 30 minutes under argon with vigorous stirring at -10 ° C. methyl bromide (12.9 ml, in ether, 25 ml of anhydrous THT) 3M solution) was added dropwise to the solution. The reaction mixture was then warmed up to room temperature in 2 hours, then quenched with water and neutralized with dilute hydrochloric acid. The mixture was extracted with ether and the combined organic layers were washed with water and then dried over sodium sulfate. After removal of the solvent the residue was distilled in vacuo to yield 2.1 kg (70%) of chloro-alcohol (28) as a colorless liquid. Then 1.5 g (10 mmol) of chloro-alcohol 28 in 5 ml of anhydrous dimethylformamide were added to a stirred solution of 1.32 g (12 mmol) of thiophenyl and 1.32 g (11.3 mmol) of t-butoxylated in 25 ml of anhydrous dimethylformamide. The reaction mixture was stirred overnight at room temperature before the solution was partitioned between dichloromethane and water. The organic layer was washed with aqueous sodium bicarbonate solution, water and dried over anhydrous magnesium sulfate.

The solvent was evaporated in vacuo and the crude oil was purified by silica gel flash chromatography using hexane-ethyl acetate. 2.2 g (98%) of sulfide 29 was obtained as a colorless liquid. This sulfide 29 (1.01 g, 4.5 mmol) was then dissolved in 40 ml of dry dichloromethane and partially added with 3-cloperbenzoic acid (2.5 g (11.6 mmol); 80-85% from Aldrich) with stirring and occasional cooling. It was. The reaction mixture was stirred for 2 hours and then quenched with 10% sodium bicarbonate. The combined extracts were washed with sodium sulfite solution and brine and dried over magnesium sulfate. The solvent was removed in vacuo and the crude oil was purified by silica gel flash chromatography using hexane-ethyl acetate mixture to give 1.1 g (97%) of sulfone 30 as a colorless liquid. To a stirred solution of 1.3 g (5.1 mmol) of sulfone 30 and 1.5 g (22.7 mmol) of imidazole in 50 ml of dry dimethylformamide was added 1.15 g (77 mmol) of triethylsilyl chloride. The reaction mixture was kept at room temperature for 2 hours and then diluted with dichloromethane. The mixture was washed with aqueous ammonium chloride solution and water. The organic layer was dried over sodium sulfate and the solvent was removed in vacuo. The residue was purified by silica gel flash chromatography. First hexaethyldisiloxane was eluted with hexane. Triethylsilyl-protected sulfone 31.1.8 (97%) was eluted with a hexane-ethyl acetate 9: 1 mixture as a colorless liquid.

IR (knit): 3.45, 2940, 1440, 1360, 1130, 1020 cm ⁻¹ ; ¹ H NMR (400 MHz, CDCl ₃ ) δ 0.518 (6H, q, J = 6.2 Hz, Si-CH ₂ ), 0.899 (9H, t, J = 6.2 Hz, Si-C-CH ₃ ) δ 0.518 (6H , q, J = 6.2 Hz, Si-CH ₂ ), 0.899 (9H, t, J = 6.2 Hz, Si-C-CH ₃ ), 1.142 (6H, s, CH ₃ ), 1.307-1.462 (4H, m ), 1.655-1.738 (2H, m, H-4), 3.080-3.122 (2H, m, H-2), 7.567 (2H, t, J = 6.8HZ, H-aryl meta), 7.648 (1H, t , J = 6.8 Hz, H-aryl para), 7.916 (2H, d, J = 6.83 Hz, H-aryl ortho); MS (EI, 70 eV): m / z (relative strength) 372 (M ⁺ , 2), 341 (100), 229 (2), 227 (18), 173 (24), 103 (22), 75 (45 ), 55 (33).

(b) Preparation of sulfone side chain unit 35

Bromination of a solution of 3.8 g (2.8 ml, 18 mmol) of 6-bromohexyl chloride (32) in 10 ml of anhydrous tetrahydrofuran in 15 ml of anhydrous tetrahydrofuran at -10 ° C. with vigorous stirring under argon atmosphere for 15-20 minutes. To a solution of 14 ml of methylmagnesium (3M solution in ether) was added dropwise. The mixture was stirred at room temperature for 2 hours, then cooled to 0 ° C. and carefully digested with 1: 1 diluted hydrochloric acid. The mixture was extracted with ether, and the combined organic layers were washed with water, dried over anhydrous magnesium sulfate and evaporated to give 3.6 g (94%) of bromo alcohol (33) as a colorless oil. 3.4 g (16 mmol) of this bromo-alcohol were treated with 3.3 g (20 mol) of sodium benzene sulfinate in anhydrous dimethylformamide for 4-1 / 2 hours at 70 ° C. The mixture was poured onto ice, extracted with dichloromethane, dried over anhydrous MgSO ₄ washed with 1N HCl, water, 10% NaHCO ₃ solution, filtered and evaporated to give sulfone (34) which was purified by flash chromatography on silica gel. Then eluted with 40-50% ethyl acetate in hexanes to give a sulfone containing 4.18 g (98%) of some corresponding sulfinic acid ester.

Ms, m / z 270 (M ⁺ ), 255 (M ⁺ -15), 77, 59.

To a stirred solution of 4 g (14 mmol) of sulfone (34) and 3.8 g (55 mol) of imidazole in 13 ml of anhydrous dimethylformamide was added 4.6 g (5.1 ml, 30 mmol) of triethylsilyl chloride. The reaction mixture was stirred at rt for 2 h, then poured into ice water, extracted with ether, dried over anhydrous MgSO ₄ , filtered and evaporated. The residue was purified by flash chromatography. First hexaethyldisiloxane was eluted with hexane. 3% ethyl acetate in hexane eluted the sulfinic acid ester with some sulfone and 10% ethyl acetate in hexane eluted 3.4 g (60%) of protected pure sulfone (35).

Elemental Analysis for C ₂₀ H ₃₆ O ₃ SSi

Theoretic value: C; 62.45%, H; 9.43%, S; 38.34%

Found: C; 61.97%, H; 9.45%, S; 8.33%

Ms, m / z (relative strength)

355 (100) (M ⁺ -29), 277 (15), 173 (35), 103 (43), 75 (95), 55 (23), NMR (400 MHz, CDCl ₃ ), 0.54 (6H, q, J = 7 Hz, Si-CH ₂ ), 0.94 (9H, t, J = 8 Hz, Si-C-CH ₃ ), 1.15 (6H, s, CH ₃ ), 1.31-1.36 (4H, m), 3.08-3.12 (2H, m, H = 2), 7.57 (2H, t, J = 6.8 Hz, H-aryl meta), 7.66 (1H, t,), H-aryl para), 7.92 (2H, d, J = 6.8 Hz, H-aryl ortho).

Biological activity

Established assays known in the art were used to test for both differentiation and calcemic action of the novel homolog 25. The analytical method and the results obtained are described in more detail in the following examples.

Example 5

Measurement of the Differentiation Activity of Di Homo Compound (25) in HL-60 Cells (Table 1)

The degree of differentiation of HL-60 cells (human leukemia cells) for the test compounds was confirmed by three different assays (NBT-reduction, phagocytosis and esterase activity). The first two assays were performed by the general method provided by DeLuca et al. US Pat. No. 4,717,721. A third assay, measuring nonspecific acid esterase activity as a marker for differentiation, was performed according to the method given in Sigma Kit No. 90 which is readily available from Sigma Chemical Corp., St. Louis, Montana. Osc et al., Proc. Natl. Acad. Sci. USA 84, 2610-2614 (1987); Osterm et al. J. Biol. Chem. Vol. 262, pp. 1164-14171 (1987)]. The results are shown in Table 1 below. The data is presented as the percentage of differentiated cells as a result of treatment of various concentrations of 1,25- (OH) ₂ D ₃ (used as a comparative reference) or vitamin D test compound.

TABLE 1

Comparison of Differentiation Activity of 1,25 (OH) ₂ D ₃ and Side Chain Di-Homo Compounds in Cultured HL-60 Cells

Example 6

Calcemic Action of Dihomo Compound (25)

(a) Intestinal calcium transport activity (Table 2)

Weaning male rats were obtained from Harlan-Sprague Dawley Company of Madison, Wisconsin, et al. [Suda et al, J. Nutr. Volume 100, pages 1049-1052, (1970)] were fed low calcium rickets feed (0.02% calcium, 0.3% phosphorus). They were fed any amount of this feed for a total of 4 weeks. At the end of the third week, animals were divided into groups of six animals. One group received daily intraperitoneal injection of basic agent (0.1% 95% propylene glycol, 5% ethanol) for 7 days. The remaining groups were injected with the same amount of base over the same period but containing the following compounds in the following dosages: 2.5 ng or 25 ng of 1,25- (OH) ₂ D ₃ , or 24-dihi-1 la, 125 ng of 25-dihydroxy-22-dehydrovitamin D ₃ (compound 25). Animals were killed 24 hours after the last dose, intestinal debridement followed by duodenal sections [Halloran and Deluca, Arch, Biochem. Biophys. Vol. 208, pp. 477-486, (1981)] was used to measure the intestinal calcium transport. The results are shown in Table 2 below.

TABLE 2

Intestinal Calcium Transport Activity of 1,25- (OH) ₂ D ₃ and Side Chain Homologs in Mice

(b) Measurement of calcium mobilization of bone (Table 3)

Weaning male rats were obtained from Harlan Sprague Dawley Campani and talked [J. Nutr. Volume 100, pages 1049-1052, (1970) were fed low calcium (0.02% calcium, 0.3% phosphorus) vitamin D-deficient feed for 4 weeks. At the end of week 3, the animals were divided into groups of 6 animals and injected with the doses indicated in Table 3 dissolved in 0.1 ml of 95% propylene glycol and 5% ethanol. Only the solvent base was injected into the control. The other groups were injected with indicated doses of 1,25- (OH) ₂ D ₃ or dihomo compound 25 for 7 days, respectively. Serum calcium was measured at the end of 7 days of administration by automatic absorption. The results of the two tests are shown in Table 3 below.

TABLE 3

Bone calcium migration (serum calcium concentration) of 1,25- (OH) ₂ D ₃ and side chain homologues in rats

The results shown in Table 1 clearly show that Dihomo analog 25 is much more potent than 1,25- (OH) ₂ D ₃ in differentiating leukemia cells into normal monocytes. For example, at 1 × 10 ⁻⁸ molar concentration of 1,25- (OH) ₂ D ₃ , 55-61% of differentiated cells are produced, while the same concentration of compound (25) causes 78% of differentiation. . It should be noted that 1,25- (OH) ₂ D ₃ at a concentration of 1 × 10 ^-7 M is required to induce the same degree of differentiation (90%) as produced by the 5 × 10 ^-8 M dihomo analogs (about 92%). In consideration, it can be concluded that this analog 25 is about five times more potent than 1,25- (OH) ₂ D ₃ as a differentiating agent.

In contrast, dihomo compounds show very low calcemic action compared to 1,25- (OH) ₂ D ₃ . This conclusion is supported by the results in Tables 2 and 3. For example, the intestinal calcium transport assay shown in Table 2 is predicted when known active metabolite 1,25- (OH) ₂ D ₃ is administered for 7 days at a dose of 12.5 or 25 ng / day, Compared to the control group). However, for the new dihomo compound (25), a dose of 125 mg / day was required for 7 days to induce a response, and even at such high concentrations, a moderate response was obtained and 1,25- (OH) ₂ D Slightly more than half of the responses caused by the 10-fold lower dose of ₃ . Thus, the new dihomo analogs in this assay have calcemic activity that is at least 10-fold weaker than 1,25- (OH) ₂ D ₃ .

The same conclusion can be deduced from the results of the calcium mobilization analysis of bone shown in Table 3. To obtain the same degree of response as caused by 1,25- (OH) ₂ D ₃ 12.5 and 25 ng respectively, 125 doses of dihomo analog (25) and 250 ng / day (administered for 7 days) are required. Do. It is also noteworthy that increasing the dose of the dihomo compound (about 500 ng / day) does not increase the calcium mobilization response of the bone any more if other conditions are inhibited (see Table 3). In the second experiment shown in Table 2, 1,25- (OH) ₂ D ₃ elicited a very important response at doses of 12.5 and 25 ng / day (relative to the control), respectively, and the dihomo analogs were administered at 125 ng / day. No action was shown in the dose. In a third test where dihomo analog 25 was tested over a dosage range of up to 1000 ng / day, the compound did not cause a calcium mobilization response at any dose concentration, which essentially consumed bone. No action to produce serum calcium. Thus, the analysis of the migration of these bones is in full agreement with the calcium transport data in Table 2, indicating that the new dihomo analog 25 is several times less potent than 1,25- (OH) ₂ D ₃ in its calcemic action.

The same type of activity is observed for trihomo compound 26 of the present invention. This substance also shows the predicted activity in inducing differentiation of HL-60 cells, while it does not produce any significant response to rat serum calcium concentrations (relative to the control) and is also very good and dramatically increases the differentiation / calcemic ratio. Indicates.

This form of action is, of course, exactly what is planned for differentiation in the treatment of tumor diseases. The cell differentiation of tumor cells, which is the desired effect, is very good, while the calcemic action, which is an undesirable action, is dramatically reduced, resulting in a significantly increased differentiation / calcemic action ratio. Known 1α-hydroxy vitamin D compounds have been known as effective therapeutic agents for the treatment of leukemia (see Suda et al., US Pat. No. 4,391,802). Based on the bioanalytical data cited herein, there is no undesirable calcemic action of conventional known compounds when the novel side chain homo compounds of the present invention are administered at the same concentration as the compounds according to the prior art. By appearing below 10, it can be concluded that most of the problems causing increased calcium levels in the treated subject can be eliminated. In addition, based on the results shown in Table 1, it can be foreseen that the novel homo compounds exhibit very high differentiation activity against tumor cells, in particular leukemia cells, thereby increasing their therapeutic benefits. Thus, the novel compounds of the present invention present effective and practical embodiments of the concept of the differentiation treatment of tumor diseases so that their mode of action clearly suggests that these compounds are the preferred therapeutic agents for such treatment.

For the purpose of treatment, these compounds are employed as solutions in harmless solvents, as emulsions, suspensions or dispersants in suitable and harmless solvents or carriers, or as pills, tablets, capsules by conventional methods known in the art. It can be formulated. Such formulations may also contain other pharmaceutically acceptable and nontoxic excipients such as stabilizers, antioxidants, binders, colorants or emulsifiers or taste enhancers.

The compounds are advantageously administered in the form of oral administration by injection or by intravenous infusion of a suitable sterile solution or via the digestive tract. For the treatment of human leukemia, the homovitamin D compounds of the invention are administered to a subject in a sufficient dosage to induce differentiation of leukemia cells into macrophages. Suitable dosages are from 0.5 μg to 50 μg per day, and it is to be understood that the dose may be adjusted (eg, further increased) depending on the extent or reactivity or condition of the patient's disease as is acceptable in the art. do.

Claims (10)

A compound having the structure

In the above formula, X, Y and Z may be the same or different from each other, a hydrogen atom or a triethylsilyl group, and n is 3 or 4. The compound of claim 1 wherein X, Y and Z are each hydrogen atoms. A compound having the structure

In the above formula, X, Y and Z may be the same or different from each other, a hydrogen atom or a triethylsilyl group, and n is 3 or 4. A pharmaceutical composition for treating leukemia, comprising at least one compound according to claim 1 and a pharmaceutically acceptable excipient. The pharmaceutical composition of claim 4, wherein the compound is 24-dihomo-1α, 25-dihydroxy-22-dehydrovitamin D ₃ . The pharmaceutical composition of claim 4, wherein the compound is 24-trihomo-1α, 25-dihydroxy-22-dehydrovitamin D ₃ . 6. A pharmaceutical composition according to claim 5 which contains about 0.5 to about 50 μg of said vitamin homologue. The pharmaceutical composition of claim 6, which contains about 0.5 to about 50 μg of the batamine homologue. The compound of claim 1, wherein the compound is 24-dihomo-1α, 25-dihydroxy-22-dehydrovitamin D ₃ . The compound of claim 1, wherein the compound is 24-trihomo-1α, 25-dihydroxy-22-dehydrovitamin D ₃ .

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