L

Vitamin D Derivatives and Methods for Preparing Same

Technical Field

This invention relates to novel vi ^* t_amin D derivatives.

More specifically, this invention relates to 26-homo- vitamins.

Still more specifically this invention relates to hydroxylated 26-hcmovitamins.

Vitamin D is known to regulate calcium and phosphorous metabolism in animals and humans and it has now been firmly established that the biological efficacy of vitamin D depends upon its metabolic conversion, in vivo, to hydroxylated derivatives. Thus vitamin D_ is hydroxylated in vivo to 25-hydroxyvitamin D_ in the liver which in turn is converted into l ,25-dihyσroxyvitamin D-. in the kidneys. It is the latter compound which is now recognized as being the circulating hormonal form of vitamin D.

Because of their biological activity in prαroting calcium and phosphorous transport in the intestine and the mobilization and mineralization of bone these forms of vitamin D are important pharmaceutical products which are eminently suitable for use in the treatment of various bone disorders. Background Art

Vi-tamin D derivatives and their preparation and application are discussed in many references in the patent and other literature. For example, U.S. Patent No. 3,565,924 is directed to 25-hydroxycholecalciferol; U.S. Patent No. 3,697,559 is directed to 1,25-dihydroxycholecalciferol; U.S. Patent No. 3,741,996 is directed to lα-hydroxycholecalciferol; U.S. Patent No. 3,786,062 is directed to 22-dehydro-25-hy- droxycholecalciferol; U.S. Patent No. 3,880,894 is directed to 1,25-dihydroxyergocalciferol; U.S. Patent No. 4,201,881 is

directed to 24,24-diflιιoι^-lα,25-dihydroxycholecalciferol; U.S. Patent No. 4,196,133 is directed to 24,24-difluoro-lα,25- dϋydroxycholecalciferol. Disclosure of Invention

New derivatives of vitamin D ₃ have now been fcwnd which express excellent vitamin D-like activity and which, for that reason, could readily serve as a substitute for viiamin D ₃ , as well as various of its derivative.;, in kncwn applications, such as, for example the treatment of various disease states manifesting calcium and phosphorous imbalance as hypcparathyroidism, osteodystrophy, osteomalacia and osteoporosis.

These derivatives are 26-_xιπcvita_r ns and particularly lα,25--dihydro_^-22E-dehydro-26-homi-Λ7i ^* taιn__n D_ and lα,25- d__hydroxy-26-hαrevitamin D-.

Cαrpounds of the present invention can be conveniently represented by the formula:

where R, , R~ and R, are each selected from the group consisting of hydrogen, an acyl group having frcm 1 to about 4 carbon atoms, and benzoyl and R. and R _j . each represent hydrogen atoms or taken together form a carbon to carbon double bond. Best Mode for Carrying Out the Invention

The compounds of this invention can be prepared in accordance with the process shown in the following schematic and process description. In the schematic and detailed description of the process like numbers identify like ccmpounds.

Schematic

lt

In accordance with the process of the present invention: lα,3β-d__χjet_ιoxyrι_ei_ho_Q*-23,24-dinorchol-5-en-22-a l was reacted with vinyl magnesium brαnide to provide the allyl alcohol (1) . This alcohol was subjected to Claisen rearrangement by reacting it with triinethyl ortho-n-butylate and a catalytic amount of propionic acid to afford the ester (2.) in good yield (97%) . (.αrpound (2) was converted to its enolate form by treatment with n-butyllithium and THF, which was then treated with oxygen and subsequently reduced with triethylphosphite to introduce an hydroxyl group at the C-25 position in the molecule. Then, the 27-ester (3) was converted to the alcohol (4) by successive treatment with lithium aluminum hydride to obtain the corresponding diol, followed by treatment with methanesulfonyl chloride and pyridine to provide the mesylate which, in turn was treated with lithium aluminum hydride.

Removal of the MOM group by treatment with acid gave (22E, 25 )-lα,3β,25--t_rihydro_^-26-horro-cholesta-5,22-diene (5) which was acetylated to provide the diacetate (6) . Allylie brcmination of 6) with N-broirosuccinimide and then with tetra- n-butylarπmonium fluoride gave the 5,7,22-triene (7) which was irradiated and iso erized by heating to provide the (22E)- dehydroxy-26-hc_rovitamin D_ (8) .

(25 )-lα,25-^ihydro_ψ-26-hc-r__vitamin D_ (11) was obtained by selectively hydrogenating the 5,22-diene (6) to provide the 5-ene {9} , which was converted to the 5,7-diene (10) and then to the 26-hαro-vitamin (11) as described above. Detailed Description of Process

In the following detailed description of the process of this invention melting points were determined with a hot-stage microscope and were uncorrected. ^" TI-NMR spectra were taken with a Hitachi R-24A (60 MHz) in CDCl., with Me.Si as an internal standard, unless otherwise noted. Mass spectra were obtained with a Shimadzu QP-1000 mass spectrometer at 70 eV. UV spectra were obtained in ethanol solution with a Shimadzu

UV-200 double beam spectrcphotαneter. Column chromatography was effected using silica gel (E. Merck, Kieselgel 60, 70-230 mesh) . Preparative thin layer chrαπatography was carried out on precoated plates of silica gel (E. Merck, Kieselgel 60 1.254' 0.25 mm thickness) . The usual work-up refers to dilution with water, extraction with an organic solvent, indicated in parenthesis, washing the extract to neutrality, drying over anhydrous magnesium sulphate, filtration, and removal of the solvent under reduced pressure. The following abbreviations were used; THP - tetrahydropyranyl; THF - tetrahydrofuran; ether - diethyl ether, MeOH - methanol, MCM - rrei±oxyrrethyl, IDA - lithium diisopropyl amide. Ternperatures cure in ° centigrade.

(22E,25 )-lα,3β-D ^* ure'i oxyτrethyloxy-26-hcmx:holesta-5,22-dien— 27-oic acid methyl ester (2)

A solution of the allylie alcohol (1) (390 g, 0.844 irrrol) , trimethyl ortho-n-butylate (0.7 ml) and propionic acid (3 drops) in toluene (6 ml) was refluxed under argon for 2 hr. Removal of the solvent under reduced pressure gave a crude product, which was applied to a column of silica gel (20 g) . Elution with hexane-ethyl acetate (5 : 1) gave the ester (2) (446 mg, 97%) as an oil. ^- MR 5:0.68 (3H, s, 18-H ₃ ) , 0.88 (3H, t, J = 7 Hz, -CH ₂ CH ₃ ) , 0.98 (3H, d, J = 6 Hz, 21-H.) β.03 (3H, s, 19-H ₃ ) , 3.38 (3H, s, -0CH ₃ ) , 3.43 (3H, s, -0C__ ₃ ) , 3.68 (3H, s, -C0 ₂ CH ₃ ) , 3.76 (15H, m, lβ-H) , 4.68 (2H, s, 3β-O-CH ₂ -0-) , 4.69 (2H, ABq, J = 7 Hz, ΔAB = 11 Hz, lα-0-CH ₂ -0), 5.27 (2H, m, 22-H and 23-H) , and 5.56 (1H, m, 6-H).

(22E,251)-lα,3β-D:Lrre-t±ιoxymethyloxy^ 5,22-dien-27-oic acid methyl ester (3_)

To a solution of IDA (prepared with diiscpropy-amine (0.13 ml, 0.929 mmol) , 1.56 M n-butyllithium (0.59 ml) and THF (2 ml) , the ester (2) (437 mg, 0.800 mol) in THF (5 ml) was added and the ir xture was stirred under argon at -78°C for 30 min. Oxygen was bubbled into this solution and then triethylphosphite (0.14 ml, 0.817 mmol) was added. The usual work-up (ether for extraction) gave a crude product, which was applied to a column of silica gel (25 g) . Elution with hexane-ethyl acetate (5 : 1) provided the hydroxy ester (3_) (303 mg, 67%) as an oil. ϊ-WSR 6:0.68 (3H, s, 18-Hg) , 0.85 (3H, t, J = 7 Hz, -CH-CE-) , 0.98 (3H, d, J = 6 Hz, 21-H..) , 1.02 (3H, s, 19-H ₃ ), 3.08 (1H, bs, W _1/2 = 3 Hz, -OH), 3.38 (3H, s, -CCH ₃ ), 3.42 (3H, s, -0O_ ₃ ) , 3.76 (3H, s, -C0 ₂ CH ₃ ) , 4.68 (2H, s, 3β-C-CH ₂ -0-) , 4.68 (2H, ABq, J = 7Hz, ΔAB = 11 Hz, lα-0-CH ₂ -0-) , 5.32 (2H, m, 22-H and 23-H) , 5.55 (1H, m, 6-H).

(22E,25 )-l ,3β-D-jret_αoxyme1_hyloxy-25-hydroxy-26-hατ_ocholesta- -5,22-diene (4)

To a solution of the hydroxyester (3) (294 mg, 0.539 mmol) in THF (5 ml) , lithium aluirdnum hydride (20 mg, 0.526 mmol) was added and this mixture was stirred at room temperature for 30 min. The usual work- p (ether for extraction) gave a crude diol. This was treated with methane- sulfonyl chloride (0.04 ml, 0.517 mmol) and pyridine (1.5 ml) at room temperature for 30 min. The usual work-up (ether for extraction) gave a crude mesylate. To a solution of the crude mesylate in THF (5 ml) , lithium aluminum hydride (20 g, 0.526 mmol) was added and the irdxture was refluxed for 30 min. The usual work-up (ether for extraction) gave a crude product, which was applied to a column of silica gel (20 g) . Elution with hexane-ethyl acetate (5 : 1) provided the alcohol (4) (190 rrg, 70%) as an oil. ^-NMR 6:0.71 (3H, s, 18-H ₃ ) , 0.90 (3H, t, J = 7 Hz, →_H ₂ CH ₃ ) , 1.03 (3H, d, J = 6 Hz, 1-H ₃ ) ,

1.03 (3H, s , 19-H-.) , 1.12 (3H, s, 27-H ₃ ) , 3.36 (3H, s, -0CH ₃ )

3.40 (3H, s , -0CH ₃ ) , 3.74 (1H, m, lβ-H) , 4.66 (2H, s,

3β-0-CH ₂ -0-) , 4.67 (2H, ABq, J = 7 Hz , ΔftB = 11 Hz, lα-0- H ₂ -0-) , 5.35 (2H, , 22-H and 23-H) and 5.54 (1H, m,

6-H) .

(22E,25»-lα,3 ,25-Tr_Jιydroxy-26-h rocholesta-5,22-diene (5)

A solution of the djj-rethoxyirethyl ester (4) 181 mg, 0.349 mmol) in THF (5 ml) was treated with 6N HCl (1 ml) at 50°C for 1.5 hr. The usual work-up (ethyl acetate for extraction) gave a crude product, which was applied to a column of silica gel (15 g) . Elution with hexane-ethyl acetate (1 : 2) provided the triol (5) (147 rrg, 98%) , m.p. 85-87°C (hexane-dichloro- irethane) . ^" ^Ϊ-NMR 6:0.69 (3H, s, 18-H ₃ ) , 0.89 (3H, t, J = 7 Hz, -CH ₂ CH ₃ ) , 1.02 (3H, s, 19-H ₃ ) , 1.13 (3H, s, 27-H ₃ ) , 3.85 (]5H, m, Iβ-H) , 3.98 (1H, m, 3α-H) , 5.40 (2H, m, 22-H and 23-H), and 5.60 (1H, m, 6-H).

(22E,25.|)-lα,3β-Diacetoxy-25-hydro_^-26-homocholesta-5 ,22- diene (6)

A solution of the triol {5) (100 mg, 0.233 rrirol) in pyridine (1 ml) was treated with acetic anhydride (1 ml) at room temperature for 15 hr. The usual work-up (ethyl acetate for extraction) gave a crude product, which was applied to a column of silica gel (10 g) . Elution with hexane-ethyl acetate (5 : 1) provided the diacetate (6) (101 mg, 85%) as an amorphous solid. TΪ-NMR 6:0.68 (3H, s, 18-H , 0.88 (3H, t, J = 7 Hz, -CH ₂ CH ₃ ) , 0.98 (3H, d, J = 6Hz, 21-H , 1.08 (3H, s, 19-H ₃ ) , 1.12 (3H, s, 27-H ₃ ) , 2.03 (3H, s, acetyl) , 2.06 (3H, s, acetyl), 4.98 (1H, m, 3α-H) , 5.06 (1H, m, 16-H) , 5.37 (2H, m, 22-H and 23-H) , and 5.53 (1H- m, 6-H) . (22E,25 )-lα,3β,25-Tr_5hydroxy-26-hc)_rocholesta-5,7,22-triene (7)

A solution of the 5,22-diene (6) (38 mg, 0.0739 rrπol) and N-brc_rosucc__niπιide (19 mg, 0.107 itirol) in carbontetrachloride (3 ml) was refluxed under argon for 20 min. After cooling to 0°C, the resulting precipitate was filtered off. The filtrate

was concentrated below 40°C to leave the residue. The THF (5 ml) solution of this residue was treated with a catalytic amount of tetra-n-butyl _ατ_τx.nium bromide at room temperature for 50 min. Then, the mixture was treated with a solution of tetra-n-butylairrronium fluoride in THF (0.3 ml, 0.3 mmol) at room temperature for 30 min. The usual work-up (ethyl acetate for extraction) gave a crude triene. This triene in THF (5 ml) was treated with 5% KOH-MsOH (4 ml) at room temperature for 14 hr. The usual work-up (ethyl acetate for extraction) gave a crude product, which was submitted to preparative thin layer chrcmatography (benzene-ethyl acetate, 1 : 1, developed six times) . The band of Rf value 0.45 was scraped off and eluted with ethyl acetate. Removal of the solvent provided the 5,7,22-triene (7) (8.7 mg, 40%). UV λ ^EtCE : 293, 282,

— ³ max

271 m.

(22E,25J)-lα,25-DJhydroxy-22-dehydro-26-hc_rcryita_rιin P., (8)

A solution of the triene (7) (4.4 mg, 0.0103 mmol) in benzene (90 ml) and ethanol (40 ml) was irradiated with a medium pressure mercury lamp through a Vycor filter at 0°C under argon for 2.5 min. The reaction mixture was refluxed under argon for 1 hr. I-temσval of the solvent under reduced pressure gave a crude product, which was submitted to preparative thin layer chrcmatography (benzene-ethyl acetate,

1 : 1, developed six times) . The band of Rf value 0.49 was scraped off and eluted with ethyl acetate. Removal of the solvent provided the vitamin D ₃ analogue (8) (0.91 g, 21%) .

(400 MHz) 6 : 0.56 (3H, s, 18-H..) , 0.91 (3H, t, J = 7.6 Hz, -CH ₉ CH ₃ ) , 1.04 (3H, d, J = 6.8 Hz, 21-Hg) , 1.13 (3H, s, 27-H ₃ ) , 4.23 (1H, m, _1/2 = 18.4 Hz, 3α-H) , 4.43 (1H, m, W _1/2 = 16.9 Hz, lβ-H) , 5.00 (1H, bs, - _/2 = 3.2 Hz, 19-H) , 5.32 (1H, bs, _1/2 = 3.2 Hz, 19-H), 5.37 (2H, m, 22-H and 23-H), 6.02 (IE, d, J - 11.5 Hz, 7-H) , and 6.38 (1H, d, J = 11.5 Hz, 6-H).

(25j)-lα,3β-Diacetoxy-25-hydroxy-26-hαrocholest-5-ene (9)

A mixture of the 5,22-diene (6) (35 mg, 0.0681 mmol) and 10% Pd-C (4 mg) in ethyl acetate (4 ml) was stirred at room temperature under hydrogen for 3 hr. The Pd catalyst was filtered off and the filtrate was concentrated to leave the residue, which was submitted to preparative thin layer chrαnatography (hexane-ethyl acetate, 2 : 1, developed once) . The band of Rf value 0.46 was scraped off. Elution with ethyl acetate provided the 5-ene (9) (30 mg, 85%) as an amorphous solid. ^- MR 6 : 0.66 (3H, s, 18-Hj) , 0.88 (3H, t, J = 7 Hz, -CH ₂ CH ₃ ) , 1.08 (3H, s, 19-H ) , 1.12 (3H, s, 27-H ₃ ) , 2.02 (3H, s, acetyl), 2.04 (3H, s, acetyl), 4.97 (1H, , 3α-H) , 5.04 (1H, m, lβ-H) , and 5.51 (1H, m, 6-H) . (25g)-lα,3β,25-Tr_-hydroxy-26-hαrocholesta-5,7-diene (10)

The 5-ene (22 mg, 0.0426 rrmol) was converted, as described for ( ) , to the 5,7-diene 10 (6.7 mg, 37%) . UV λ

^Et0H : 293, 282, 271 nm. max ' '

(25 )-la,25-Dihydroxy-26-homcvita_nin D- (11)

The diene (10) (4.8 mg, 0.0112 rrmol) was converted, as described for {8) , to the vitamin D_ analogue (11) (1.3 mg,

27%) . UV λ ^Et0H : 265 nm, λ ^E * ^CH : 228 nm. MS m/z: 430 (M ⁺ ) , max min

412, 394, 379, 376, 287, 269, 251, 152, 134, 116, 73, 55.

If desired, the c tpounds of this invention can be readily obtained in crystalline form by crystallization fran suitable solvents, e.g. hexane, ethers, alcohols, or mixture thereof as will be apparent to those skilled in the art.

Biological Activity

Bone calcium mobilization activity of lα,25-(OH)g-26-hαno-D, compounds

Male weanling rats were purchased frcm Holtzman Co. ,

Madison, Wis. and fed ad libitum a low calcium, vitamin D deficient diet as described by Suda et al (J. Nutrition 100:

1049, 1970) and water for 3 weeks. The rats were then divided into 4 groups of 6 each and were intrajugularly given respectively 650 prole of either lα,25-(OH) ₉ -26-horro-D ₃ ,

lα,25-(OH) ₂ -(22E)Δ ²² -26-homo-D ₃ or lα,25-(0H) ^ dissolved in 0.05 ml of 95% ethanol 7 hrs. prior to sacrifice. The rats in the control group were given 0.05 ml of 95% ethanol 7 hrs. prior to sacrifice. The rats in the control group were given 0.05 ml of ethanol vehicle in the same manner. They were killed by decapitation, the blood was collected and centrifuged to obtain serum. Serum calcium concentration was determined with an atomic absorption _"pecτ_rσphoτ_αreter (Perkin-El er Model 214) in presence of 0.1% lanthanum chloride. Results are shown in the table below:

Table 1

Serum Calcium Concentration

C rpound Administered (ιrg/100 ml)

ethanol 3.4 + 0.3 lα, 25- (OH) --26-hc_ro-D- 4.6 + 0.2 b) lα, 25- (OH) ₂ - ( 22E) Δ ,22 - ^J 26-homo-D ₃ 4.6 + 0.3 b) lα,25- (OH) ₂ D ₃ 4.5 + 0.2 b)

*standard deviation from the mean b) is significantly different frαu a) P 0.001

It can be concluded from the foregoing data that in the vitamin D responsive systems of vitamin D-deficient animals the cc pounds of this invention exhibited the same activity as lα,25-hyάroxyvitamin D_, the circulating hormonal form of the vitamin.

The compounds of this invention may be readily adrriinistered for purposes of their calcemic activity in sterile parenteral solutions by injection or intravenously or by alimentary canal in the form of oral dosages, or by sυppository or even transcutaneously. Doses of from about 0.1 μg to about 2.5 μg per day are effective in obteining the physiological calcium balance responses characteristic of

vitamin D-like activity with maintenance dosage of from about 0.1 μg to about 0.5 μg being suitable.

It has recently been discovered that lα,25-d_5hydroxy- vitamin D ₃ (lα,25-(OH) -£.,) and its structural analog lα-hydroxyvitamin D _g (lα-0H-D ₃ ) , in addition to their well-established calcemic action referred to above, also express potent anti-cancer activity. Specifically, it was shown that the above-named compounds were effective in causing differentiation of malignant human cells, such as leukemia cells in culture, to non-malignant macrophages, and the anti-cancer activity on cells in vitro could be correlated with beneficial effects in vivo by showing that the adrrdnistration of these cαrpounds extended the life span of leukemic mice (compared to controls) and markedly improved the condition of human leukemia patients. Based on these observations, lα-hydroxylated vitamin D cαπpounds have been proposed as therapeutic agents for the treatment of leukemoid diseases (Suda et al., U.S. Patent No. 4,391,802).

Although these known lα-hydroxyvitamin D compounds tested by Suda et al_. (supra) , namely lα-hydroxyvitamin D_ (lα-OH-D.,) and lα,25-dihydroxyviiamin D, (lα,25-(OH) ~D_) , are indeed highly effective in causing differentiation of leukemic cells, a serious disadvantage to their use as antileukemic agents is the inherent, and hence unavoidable high calcemic activity of these substances. Thus, lα,25-(OH) „D_, the most potent vitemin-derived antileukemic agent known thus far, is also the most potent calcemic agent, and the antileukemic potency of lα-0H-D ₃ is likewise correlated with high calcemic activity. The administration of these cαrpounds, at the dosage level where they are effective as antileukemic drugs (e.g. 1 ug/day as specified in the examples of the Suda et al. patent) , would necessarily produce elevated, potentially excessive, calcium levels with attendant serious medical cαrplications, particularly in patients already suffering from debilitating disease. Because of the high intrinsic potency of the known

lα-hydroxyvitamin D compounds in raising calcium levels, their use as antileukemic agents may be precluded.

A preferred method of -treatment of malignant disease states clearly would be the administration of compounds characterized by a high antileukemic to calcemic activity ratio, that is, of cαrpσunds ex_ι__biting an enhanced potency in causing differentiation of leukemic cells as compared to their potency in raising serum calcium levels.

The ααrpounds of this invention are also preferentially active in inducing the differentiation of malignant cells to non-malignant cells, i.e. in antineoplastic activity as measured by leukemia cell differentiation, while being no more active than lα,25-dihydroxyvitamin D ₃ in their effect on calcium etabolian. Because of this unique and unexpected ccmbination of properties, the novel side-chain hαrcvitamin D compounds of this invention represent superior and preferred agents for the treatment of leukemias and other neoplastic diseases. Jhen administered to human prσmyelocytic leukemia cells (HL-60 cells) grown in culture, the side-chain hcrrøvitairάn D compounds of this invention induce the differentiation of these cells to macrophages (monocytes) . In several standard assays for measuring differentiation activity, these cαπpounds were sham to be more effective than lα,25-(OH) _,D_, the most active vitamin D derivative kncwn thus far. These assays were performed as follows: Assay of hαrovitamin D compounds for differentiation activity.

The human prαrryelocytic leukemia cell line (HL-60) was maintained in suspension culture in PPM11640 medium (Gibco, Grand Island, NY) supplemented with 10% (v/v) heat inactivated fetal calf serum, 100 μg/ml penicillin, 100 μg/ml streptααycin and 0.25 μg/ml fungizone. Cells were cultured in a humidified atmosphere with 5% CO.,. Cell viability was assessed by standard assays, e.g. trypan blue exclusion. Moηphological evaluations were done on Wright stained slide preparations.

5 Cells were seeded at 1.5 - 2 x 10 cells/ml in 10 ml of medium in tissue culture dishes. After 20 hr, duplicate dishes were then treated with each of the test compounds at various concentrations as indicated in the tables below. The test compounds were added as solutions in 100% ethanol so that the total ethanol concentration in each culture dish did not exceed 0.2%. Control cultures were treated with the same concentration of ethanol. After four days (96 hr) of incubation with test cαπpounds, the cells were harvested from these culture dishes and cell number and viability were determined. The extent of differentiation induced by the tested vitamin D derivatives was expressed as the percentage of cells that exhibit funchonal and enzymatic markers characteristic of monocytes. The two markers assayed were a) the ability of the cells to phagocytize dead yeast, and b) the ability of the cells to produce superoxide (reduce nitro- tetrazalium blue) when stimulated with pharbol esters. a) Phagocytosis Assay for Differentiation Activity: The harvested cells were resuspended in RPMl medium containing 20%

AB serum and 20% fetal calf serum, to give a preparation containing 2 x 10 cells/ml. To 0.5 ml (10 cells) of the above cell suspension was then added 0.5 ml of a suspension

(in phosphate-buffered saline) of heat-killed saccharαπyces p cerevisiae cells (1 x 10 cells) which had been stained with trypan blue. After incubation of this mixture for 1 hr at 37°C, the number of phagocytic cells was counted (as deteiTuined by the trypan blue stained yeast appearing intracellularly) and expressed as a percent of the total viable cells present. This "% phagcicytic cells" indicates the percent of differentiation induced by the test compounds. Results are summarized in Table 2 below.

Table 2

Percent phagocytic (differentiated) cells produced in HL-60 cell cultures treated with vitamin D compounds at various concentrations

Cαrpound Concentration (moles/liter) Administered 0 (a,b) -,^-10 _ _π _-10 _ ,,.-9(b) _ -,,.-8(b) -7 (b) -7 3x10 5x10 1x10 1x10 1x10 3x10

1 , 25- (0H) ₂ D ₃ 10+1.5 17 23 28+4 47+1 67+6 69 hαro-cpd I* 10+1.5 28 38 44+5 72+2 76+3 77 hαno-cpd II** 10+1.5 22 42 48+6 70+0 78+4 83 J_.

^Control level; cell cultures were treated with solvent ethanol only. Results tabulated in these columns represent the mean +_ SEM of three different experiments, each done in duplicate.

*lα,25-^ilydroxy-26-hc ^* movitamin D_ **lα,25-dihydroxy-22E-^ehydro-26-hc_ι_ovitamin D_

The results in Table 2 show that the hαno compounds are significantly more potent than l,25-(OH) ₂ D ₃ . At all concentrations, the hcmo cαrpounds achieve a greater degree of differentiation of the leukemia cells than lα,25-(OH) _> ₃ , the most active cαrpound known thus far. For example, at a

—8 concentration of 10 molar the hcmo cαrpσunds achieve a differentiation of 70%, whereas l,25-(OH) _> ₃ at the same concentration gives only about 47% differentiated cells. To achieve 50% differentiation requires a concentration of 1 x

—9 —8

10 M of the hαmo compounds, but about 1 x 10 M of lα,25-(OH) ₂ D ₃ , i.e. a difference in potency of about 10-fold. b) NBT-Reduction Assay for Differentiation: This assay depends on the ability of rronocyte-like leukemia cells to reduce the nitroblue tetrazoliu (NBT) reagent to a black-blue precipitate (formazan) when stimulated by phorbol esters. The assay was performed according to the general procedure given by Yen et al (J. Cellular Physiol. 118, 277 (1984)). The cells were harvested as above and then suspended in- PMl medium; to 0.2 ml of this suspension (containing about 1.4 x

10 cells/ml) was added 0.2 ml of the nitroblue tetrazolium

(NBT) reagent. (The NBT reagent was prepared by mixing a solution contaiiiing 50 mg of nitroblue tetrazolium in 50 ml of phosphate-buffered saline with 10 microliters of an acetone/water (1:1) solution containing 0.5 mg/rαl of

4β-phorbol- 12-i_ryristate-13-acetate) . After standing in a water bath for 30 min, the differentiated cells (i.e. the cells showing formazan blue deposits indicative of NBT reduction) were counted with a herr_ocytometer and expressed as the percent of total viable cells present. The results of this assay are shown in Table 3 below.

Table 3

Percent of cells in HL-60 cell cultures exhibiting nitroblue tetrazolium (NBT) reduction activity af er treatment with Vitamin D Cαnpσunds at various concentrations

Compound Concentration (moles/liter)

₀ (a,b) -10 _r , __.-

Administered 3x10 ^" 5x10 ^■ 10 lxlθ ^"9(b) I lO ^"8 ]χKf ⁷ < ^b > 3xlO~ ⁷ vO 1,25-(0H) ₂ D ₃ 10+1.5 15 27 31+4 45+4 69+7 65 homo-cpd I* 10+1.5 27 41 47+7 72+5 79+2 78 homo-cpd II** 10+1.5 22 44 49+4 70+2 79+5 80

Control level; cell cultures treated with solvent ethanol only. Data represent the mean + SEM of three separate experiments, each assayed in duplicate.

*lα,25-^ihydro_^-26-hσmcvitemin D_ **lα,25-^_Jhydroxy-22E-^ehydro-26-hαnovita_rάn D_.

r*. f.

5o _.

00 o

The results shown in Table 3 again establish that the homo cαπpσunds tested are more active than lα,25-(OH) ₂ D_ in inducing the differentiation of human myeloid leukemia cells to normal cells, in vitro. To achieve 60% differentiation of the leukemic cells as measured by this NBT reduction assay,

-9 requires a concentration of 2 x 10 M of the hcmo compounds; to achieve the same degree of differentiation with

—8 lα,25-(OH) _> ₃ requires a concentration of 3.5 x 10 M—a

17-fold difference in potency.

Thus, both of the above assays confirm the high potency of the hcrrovitamin D compounds in inducing the differentiation of leukemic cells. In addition, the above results show that in this differentiation activity these _x _ovitamin D α__tpounds are significantly more potent than lα,25-(OH) ₂ D ₃ .

Since this differentiating activity is expressed in the case of human leukemia cells (HL-60) , it is clear that these novel horrovitamin D cαtpounds can be used effectively against leukemias in human subjects. At the same time, these compounds do not exhibit enhanced calcemic activity, but are about as active as lα,25-(OH) ₂ D_. Thus, these hαrovitamin D compounds are characterized by a high antineoplastic to calcemic activity ratio. By virtue of this novel and desirable biological property, these side-chain homo compounds would function as superior therapeutic agents for the treatment of malignant diseases.

For the treatment of human leukemia, the hαrcvitamin D cαrpounds of this invention are administered to subjects in dosages sufficient to induce the differentiation of leukemic cells to macrophages. Suitable dosage amounts are from 0.2 μg to 5 μg per day, it being understood that dosages can be adjusted according to the severity of the disease or the reponse or the condition of subject as is well-understood in the art.

For treatment purposes, dosage forms of the cαrpounds can be prepared by cαribining them with a non-toxic pharmaceuti-

cally acceptable carrier as is well known in the art. Such carriers may be either solid or liquid such as, for example, corn starch, lactose, sucrose, peanut oil, olive oil, sesame oil and water. If a solid carrier is used the dosage forms of the compounds of the invention may be tablets, capsules, powders, troches or lozenges. If a liquid carrier is used, soft gelatin capsules, or syrup or liquid suspensions, emulsions or solutions may be the dosage form. The dosage forms may also contain adjuvants, such as preserving, stabilizing, wetting or emulsi__ying agents, solution prαtioters, etc. They may also contain other therapeutically valuable substances.

It should be understood that although dosage ranges are given the particular dose to be adrr nistered to a host will depend upon the specific disease state being treated, the end results being sought in a particular case, the physical size of the host, as well as other factors known to those skilled in the art in the therapeutic use of such medicinal agents.

The cαrpounds can be advantageously adrninistered by injection, or by intravenous infusion of suitable sterile solutions, or in the form of oral doses via the alimentary canal.