Side-Chain tfasaturated l~Hydroxyvitaπιin D Compounds

Ωiis invention was made with Government support under OTH Grant No. AM 14881 awarded by the Department of Health and Human Services. The Government has certain rights in this invention. Technical Pield

Ωie invention relates to biologically-active vitamin D cαπpounds. More specifically, this invention relates to l-hyd_xayvitamin T> ccπpounds containing a 22,23-cis-αbαble bend in the side chain. Background

Because of the well-known and clearly established activity of l(__-hyd__Oxyvitamin D compounds in regulating calcium and phosphate hαπeostasis in the animal or human, there has been interest in the preparation of the natural metabolites and in the discovery of analogs with useful medicinal properties _* This has led to the preparation of a variety of compounds {for examples, see DeLuca et al., Topics in Current Chemistry, vol. j|3, p. 1 (1979); i-nn. Rev. Biochan. 52, 411 (1983); Yakhirrovich, Russian Chem. Rev. 49, 371 (1980) some of which, e.g. lo hydroxyvitamin D ₃ (U.S. Patent 3,741,996) or lα,25-d_ihydrc*yvita_r-in D ₃ (U.S. Patent 3,697,559) already find use in medical practice. Interest in such cαπpounds is continuing especially new that it has been recognized that in addition to their classical function as regulators of calcium hαreostasis, lα25-dihyά^xyvitamin D, an its analog, l<__-hydrαxyvi amin D ₃ , also affect cellular differentiation processes and are capable of ÷inhibiting the growth and proliferation of certain malignant cells [Suda et al., U.S. Patent 4,391,802; Suda et al., Proc. Natl. Acad. Sci. USA 80, 201 (1983); Reits a et al., Nature, Vol. 306, p.

492-494 (1983)3 _* There is increasing-evidence to show, that expression of biological activity by vitamin D metabolites and analogs involves binding to an intracellular receptor protein at some stage of the overall process (see DeLuca et al. , supra) . High affinity for this receptor protein is thus a prerequisite for high potency, and desirable vitamin D analogs are those which cαπpete effectively with the natural hormone, foe "the receptor binding site.

Shewn side chain unsaturated vitamin D compounds include the hydroxy derivatives of vitamin D _2# namely vitaπ n D_ (U.S. Patent 3,585,221) , lα,25-di_φd_ra_^vitamin D ₂

(U.S. Patent 3,880,894), 24-hydrcocy- and 24,25-d__hydro_y- vitamin D- (Jones et al., Arch. Biocham. Bicpiiys. 202, 450

(1980)), D- (U.S. Patent 3,907,843) and certain 24-de_rethylvitamin D ₂ compounds (U.S. Patent 3,786,062; Bogoslovskϋ et al., J. Gen. Chan. USSR 48(4) , 828

(1978); Chan Abstr. JJ9, 163848J and JJ9, 209016s) . One example of a compound with a cis-double bond in the side chain is also known (Bogoslovskϋ et al., supra) . Disclosure of Invention

The novel compounds of the present invention are characterized by the structures A and B shown belσwt

where the hydroxy group (or protected hydroxy group) at carbon 1 nay have the α- or β-stereochemical orientation, and where X. and . represent hydrogen or a hydroxy-^rotecting group, e.g. acyl, alkylsilyl, irethoxyiretbyl or tetral_ryd__opyranyl.

These compounds are thus characterized by a 22,23-cis- double bond (22Z-double bond) in the side chain.

Preferred hydros^-protecting groups are acyl (alkanoyl) groups of 1 to 6 carbons (e.g. foπryl, acetyl, propionyl, butyryl, etc.) or aroyl groups, such as benzqyl, halo- or nitrobenzoyl, or carboxyalkanoyl groups of 2 to 6 carbons, such as oxalyl, malonyl, succinyl, glutaryl or adipyl.

Especially preferred are the ∞ pounds of type A above, having a los-hydroxy group, because these compounds show unexpectedly high affinity for the receptor protein. These compounds, as l-hydro_^vitamin D analogs, are related to the known l-]_ydrαxylated vitamin D cαipcunds, but because of the presence of a 22ϊ-double bond, were expected to exhibit low, if any, affinity for the receptor since this 22,23-cis-double bond forces the side-chain into a quite different geometry than that assumed by the fully saturated side-chain as it occurs in lΛ-byd__Oxyvii_amin D- or in the natural hormone, l,25-(OH) _> ₃ , both compounds of known high affinity for the receptor protein (DeLuca et al., supra) . Surprisingly and unexpectedly, it was found, however, that the lα-hydroxy- 22 -dehydro compounds actually exhibit higher affinity for the receptor than does lα-hydroxyvitamin D_.

Hie synthesis of the novel compounds of this invention is surrmarized by Process Scheme 1. "In the fo_ σwing description of this process and in the examples, compound designations by numeral (e.g. (1) , (2), (3) . . . . etc.) refer to structures so numbered in Process Schane I, or in the specification.

The starting material for the synthetic process is the diene-protected aldehyde of structure (1) where R is a methosymethyl group. This starting material is prepared from ergosterol according to the method of Morris et al. (J. Org. Chem. 4_6, 3422 (1981)) . Reaction of compound (1) with a ^" ittig reagent, 'having-the structure shown below,

(CH ₃ ) ₂ CHC _H2 CH ₂ -Ph ₃ Br in an organic solvent and in the presence of a strong base, provides product (_2) featuring the desired 22Z-olefinic side ch in.

By removal of the hydros-protecting group under acidic conditions, product (2) is converted to compound (3_) , which is subjected to reduction with a strong hydride reducing agent in an organic solvent to obtain the 5,7-diene sterol, compound

Irradiation of this material, dissolved in an organic solvent, with ultraviolet light converts the 5,7-diene to the corresponding previta in intermediate, which after isolation and purification is isomerized to the 22j2-d£=__ιyd__o-vi1a-nin D- analog of structure (5) '(R=H) by gentle heating in an organic solvent at a temperature ranging from room temperature to reflux.

The intermediate of structure (5) is a known vitamin D analog, having been prepared previously by Bogoslovskϋ et al. (J. Gen. Chan. USSR, 4jH4), 828 (1978)) fcy a less convenient p__ocedure.

-intermediate (5) is then converted to the desired final products by 1-hydroxylation using the general method of DeLuca et al. (U.S. Patents 4,195,027, 4,260,549). Coπpound (5) is first tosylated to give the 3β-tosylate of structure (6) , which is then solvolyzed inbuffered methanol to obtain the novel 3,5-cyclcvita_t_in D interirediate of structure (7) (R=H). This product is then treated with selenium dioxide, and tert.-butyl_rψdrσperoxide in an organic solvent, to obtain as the major product the lα-hydroxycyclovitamin D analog of structure (8) , where R is a hydroxy group. It is notable that this allylic hydroxylation at carbon 1 proceeds without complications for a compound like intermediate (7) having the unusual cis- ouble bond in the side chain with two allylic positions.

Th intermediate l-hyd__oxycyclσvitamin D product is then solvolyzed in glacial acetic acid to obtain in admixture the 5,6-cis and 5,6-trans vitamin D corrpounds of structures (9) and (10) respectively, having a 3β-acetoxy function. These acetate derivatives (9) and (10) are then separated, and

Process Scheme X

(±) R = CH ₂ 0CH, 12) R = CH _jj OCH, H

(5) R*H

individually hydrolyzed in nάld base to.produce the desired free diol products, characterized by structures 11 and 12 where X. represents hydrogen.

It has been found, that the 1-hydroxylated cyclcvitamin D product described above, of which the lα-h d__oxy-3,5-cyclσ- vitamin D coπpound of structure (8) is the major component, also contains a small amount of the corresponding lβ-hydroxy- 3,5-cyclσvitam__n D epimer, i.e. the product of structure (13) below, upon solvolysis in glacial acetic acid, this Iβ-hydrαxy-epxraer givesrise to the corresponding 56-cis and 5,6-tra^-lβ-hy^__O_^-3β-aceto^-vitamin D analogs represented by st_n_jci_ures (14) and (15) , respectively, below, which, if desired, may also be isolated from the solvolysis mixture by cih__omatography,. and then can be separately hydrolyzed in mild base, as described above, to the lβ,3β-diol epi ers characterized by structures (16) and (17) , respectively.

In practice, it has been found that the 5,6-trans-lβ- hydroxy derivative of structure (15) above, often represents such a minor component of the solvolysis mixture that its direct isolation may be unduly laborious. It is generally more convenient in such cases to prepare 5,6-trans-lβ-hydroxy analogs, by the known iodine-catalyzed i≤cmerizatiαn process of Verloσp et l. (Sec. Trav. Ch±m. Pays-Bas 78, 1004 (1969)) from the corresponding 5,6-cis compounds. Thus treatment of product (14) with a catalytic amount of iodine in a hydrocarbon or ether solvent gives 5,6-trans product (15) , and

the analogous iscmerization of (16) provides the corresponding trans compound of structure (17) .

Acylated derivatives of the products .of this invention are readily prepared by conventional methods. Thus, mono-acylates of structures (9) , (10) or (14) and (15) result directly from solvolysis; such monoacylates may be further acylated to the corresponding 1,3-diacylates, or desired acylates may be prepared by conventional acylatiσn of the free diols of structures (11) , (12) or (16) and (17) . It is to be noted also.that the D intermediates of structure (8) or (13) , can be acylated to the corresponding 1-0-acyl derivatives. Subsequent solvolysis of such acyl derivatives in glacial acetic acid, or in an acidic aqueous medium (e.g. according to the method of DeLuca et al., U.S. Patent 4,195,027) yields the 5,6-cis and 5,6-trans D analogs as their l,3-di ⁱ -0-acyl or 1-0-acyl derivatives, respectively.

A noteworthy property of the novel compounds of this invention is their high potency as expressed by high binding affinity for the protein receptor. It was assumed that the change in side chain geometry dictated by the presence of a 22,23-cis-double bond (22Z^double bond) would aboϋsh binding affinity, .or at.least.result in a marked decrease in binding affinity, since it is known (e.g. see DeLuca et al., Topics in Curr. Chem., supra) that even subtle changes in stereochem¬ istry (e.g. the change from 24R-hydroxy to 24 -hyάroxy) can result in pronounced differences in binding properties, and the compounds were indeed prepared for the purpose of confirming that assumption. Surprisingly, it was found by cαtpetitive binding assays .(performed according to the protocol of Shepard et al., Biochan. J. 182, 55 (1979)) that the lα-hydrαxy~22 -dehydro analog of structure (11) exhibits 3-5-fold higher affinity for the receptor protein than does lα-hycroxyvitamin D ₃ a known and highly potent vitamin D_ derivative. The other products of this invention exhibit

lower but still substantial binding affinitywhich is .in each case higher than that of the corresponding compound featuring a saturated side chain as it occurs in natural metabolites or other known analogs.

Because of this high binding affinity the compounds of this invention can be highly useful substitutes for the known metaboϋtes in the therapy or prophylaxis of calcium disorders such as rickets, hypσparathyrσidism, ostecx-^strophy, osteαralacia or osteoporosis in the human, or related calcium deficiency diseases (e.g. milk fever) in animals. likewise these compounds may be used for the treatment of certain malignancies, such as human leukania. Particularly preferred for the above appϋcatiαns is the analog depicted by structure (11) in Process Scheme I, or the corresponding 5,6-trans cciiipσund of structure (12) or their acylated derivatives. Suitable mixtures of the above products may also be used in medical or veterinary appϋcatiαns, e.g. the cxsπbination of the products represented by structures (ϋ) and (12) .

For therapeutic purposes, the compounds may be administered by any conventional route of administration and in any form suitable far the method of administration selected. The compounds may be formulated with any acceptable and innocuous phaπnacsutical carrier, in the form of pills, tablets, gelatin capsules, or suppositories, or as solutions, aπulsions, dispersions or suspensions in innocuous solvents or oils, and such formulation may contain also other therapeutically active and beneficial ingredients as may be appropriate for the specific applications. For human applications, the compounds are advantageously administered in amounts from about 0.5 to about 10 ug per day, the specific dosage being adjusted in accordance with the specific compound administered, the disease to be treated and the medical history, condition and response of the subject, as is well understood by those skilled in the art.

The present invention is further described in the foϋowing detailed description which is intended to be iϋustrative only and not limiting of the appended claims.. In this description the physico-chemical data ?as obtaned using the referenced methods and apparatus. High pressure liquid iroπatography (HPLC) was performed on a Waters Associates Model ALC/GPC 204 using a Zorbax-S (DuPont) (6.2 max 25 cm column, flow rate 4 ml/min, 1500 psi). Column chrcπatography was performed on Silica Gel 60, 70-230 mesh ASM (Merck). I ^» reparative thin-layer chromatography (TLC) was carried out on Siϋca 60 PF-254 (20 x 20 cm plates, 1 irm siϋca gel). Irradiations were carried out nsing a H*_nσvia 608A36 mercury arc lamp fitted with a Vycor filter. All reactions are preferably performed under an inert atmosphere (e.g. argon) .

(22Z)-3β-(Methoxy ethoxy)-5 ,8α-(4-phenyl-l,2-urazolo)cholest- a-6,22-dien (2_) . Isopentyl phosphonium bromide [ (CH ) ₂ CHCH ₂ CH ₂ PPh Br] (1.67 g, 4.04 m ol) in dry tetrahydrofuran (73 ml) was treated with n-butylϋthium (1.7 M solution in hexane, 2.42 ml, 4.11 mol) at 3-5°C with stirring. After stirring for 1 h at room temperature, the orange-red solution was cooled to 3°C and aldehyde (1) (1.84 g, 3.36 irrrol) in dry THF (24 ml) was added. The colorless reaction mixture was stirred overnight at room temperature and then poured into water and extracted with benzene. The organic extract was washed with 5% HC1, saturated sodium bicarbonate and water, dried (Na^SO.) and concentrated in a vacuo to an oil, which was purified on a column of silica gel. Elution with benzene-ether (94:6) mixture afforded adduct (2) (1.38 g, 68%) .as a foam; NMR 6 O.83 (3H, s, I8-H3) , O.S9 and 0.91 (6H, each d, J=6.8 Hz, 26-H ₃ and 27-iy , 0.97 (3H, d, J=6.8 Hz, 21-H ₃ ), 0.98 (3H, s, 19-H..) , 3.30 (IB, dd, J.^4.4

Hz, J ₂ =14Hz, 9-H), 3.38 (3H, s, CCH^ , 4.33 (IH, m, 3-H) , 4.70 and 4.81 (.2H, ABq, J=6.8 Hz, CCH_ ₂ 0) , 5.21 (2H, br m, 22-H and 23-H), 6.23 and 6.39 (2H, ABq, J=8.5 Hz, 6-H and 7-H) , 7.41

(Si, brm, Ar-H); IR: 1756,1703,1601,1397,1046 cπT ¹ ; mass spectrum, m/z.601 (M ⁺ ,<1%), 426 (4) , 364 (61), 349 (16), 253 (18), 251 (18) , 119 (EhNCO, 100).

(22Z)-5α,Set-(4-pheπyl-l,2-urazolo)cholesta-6,22-dien^3 -ol (3). A solution of adduct (2) (601 g, 1 irmol) and p-toluenesulfonic acid (523 mg, 2.75 mmol) in methanol (20 ml)-THF (12 ml) mixture was stirred for 2 days at room temperature. The reaction mixture was poured into saturated sodium bicarbonate and extracted several times with benzene. Extracts were washed with water, dried (I^SO.) and evaporated under reduced pressure. Purification of the crude product by coluπn ch_r___atography (benzene ether 70:30 as eluant) gave the hydroxy adduct (3) (550 mg, 99%) as a foam: NMB.60.83 (3H, s, 18-H.), 0.89 and 0.91 (6H, each d, J=6.8 Hz, 26-H ₃ and 27-H ₃ ) , 0.95 (3H, s, 19-H ₃ ), 0.98 (3H, d, J=6.8 Hz, 21-H ₃ ) , 3.16 (IH, dd, J _χ =4.4 Hz, J ₂ =14 Hz, 9-H) , 4.44 (IH, , 3→i), 5.22 (2H, br m, 22-H and 23-H) , 6.22 and 6.39 (2H, ABq, J=8.5 Hz, 6-H and 7-H), 7.40 (5H, br , Ar-H); IR: 3447,1754,1700,1600,1397 cm ^" ; mass spectrum, m/:z (557 (M ⁺ , <1%) , 382 (35) , 349 (33) , 253 (20), 251 (33), 119 (100), 55 (82).

(22Z)-Cholesta-5,7,22-trien-3β-ol (4). The adduct (3) (530 mg, 0.95 mmol) was converted to the diene (4) by reduction with ϋthiu aluminum hydride (1 g) , in tetrahydrofuran (60 mL) at reflux for 18 h. After conventional work-up, the product was purified by ch__omatography over silica (benzene-ether 94:6 as eluant) to afford pure diene (4) (290 mg, 76%) after crystaϋization from ethanol:mp 148-151°C;

24 [α] = -132° (c=0.9, σiCl ₃ ); NMR δ 0.66.(3H, s, 18-H ₃ ), 0.90 and 0.91 (6H, each d, J=6.8 Hz, 26-H and 27-HJ , 0.96 (3H, s,

•19-E _j ), 0.98 (3H, d, J=6.9 Hz, 1-^)., 3.54 (IH, m _t 3-H) , 5.20

(2H, brm, 22-H and 23-H) , 5.39 and 5.57 (2H, ABq, J=6 Hz, 7-H and 6-H); Wλ 281 πm IR: 3346,1463,1375,1364,1067,1040, . max '

831 cm ; irass spectrum, m/z 382 (M , 100) , 349 (65); 323 (32), 271 (15), 253 (30).

(5Z,7E,22Z)-9,10-Secocholesta-5,7,10(19),22-tetraen-3β-ol (5) .

Irradiation of 5,7-diene (4) (150 mg, 0.39 mmol) dissolved in ether (120 ml) and benzene (30ml) (degassed with argon for 40 min) was performed at 0°C for 13 min using a UV-lamp and ^" Vycor filter. HPLC (1% of 2-propanol in hexane) of the resulting mixture afforded the provitamin (56.9 mg, 38%) as a colorless oil: NMR 60.75 (3H, s, 18-CH ₃ ) , -0.90 and 0.91 (6H, each d,

J=6.7 Hz, 26-H ₃ and 27-H ₃ ) , 0.99 (3H, d, J=6.8 Hz, 21-H ₃ ) ,

1.64 (3H, s, 19-H ₃ ), 3.90 (IH, m, 3-H) , 5.20 (2H, brm, 22-H and 23-H), 5.69 and 5.95 (2H, ABq, J=12 Hz, 7-H and 6-H); UV λ 261 nm, λ . 234 nm. max ' min

Thermal isαπerization of this provitamin intermediate (56 g, 0.15 mmol) in refluxing ethanol (3 h) .gave the oily vitamin analog (5) (43 mg, 77%) after separation by HPLC. NMR 60.60 (3H, s, 18-H ₃ ), 0.89 and 0.90 (6H, each d, J=6.7 Hz, 26-1^ and 27-H ₃ ) , 0.97 (3H, d, J=6.6 Hz, 21-H ₃ ) , 3.96 (IH, s, 3-H), 4.82 and 5.05 (2H, each narr. m, 19-H ), 5.20 (2H, br m.

22-H and 23-H) , 6.04 and 6.24 (2H, ABq, J=11.4 Hz, 7-H and

6-H); UV λ 265.5 nm, λ . 228 nm; IR: 3427,1458,1379, max , min ₊

1048,966,943,892 c ; mass spectrum, m/z 382 (M , 21), 349 (5), 271 (8), 253 (14)., 036 (100), ϋ8 (82).

1-Hydroxylation of compound (5). Ereshly recrystalϋzed p-toluenesulfcnyl chloride (50 mg,.0.26 irmol) was added to a solution of vitamin (5) (50 mg, 0.13 i iol) in dry pyridine (300 μl) . After 30 h at 4°C, the reaction mixture was poured into ice/saturated NaHC0_ with stirring, ϊhe mixture was stirred for 15 min and extracted with benzene. The organic extract was washed with saturated NaHC0 ₃ , saturated copper sulfate and water, dried (Na ₂ S0 ₄ ) and concentrated in vacuo to obtain the oϋy tosylate (6).. The crude tosylate (6) was treated with TTaHC0 ₃ (150 τπg) in anhydrous methanol (10 ml) and the mixture was stirred for 8 _^ 5 h at 55°C. After cooling and concentration tov2 ml the mixture was diluted with benzene (80 ml) , washed with water, dried (Na ₂ S0.) and evaporated under reduced pressure. The oily 3,5-cyclovitamin D compound

(7) thus obtained was sufficiently pure to be used for the following oxidation step without any purification. To a vigorously stirred suspension of Se0_ (5,1 mg, O.046 ππol) in dry CH Cϋ (5 ml) , tert-burylhydroperαxide {16.5 μl, 0.U8 πrnol) was added. After 30 min dry pyridine (50 μl) was added and the mixture was stirred for additional 25 min at room teπperature, diluted with CH ₂ C1 ₂ (3 ml) and cooled to 0 ^β C. The crude 3,5-cyclσvitamin product (7) in CH Cϋ (4.5 ml) was then added. Ωie reaction proceeded at 0°C for 15 min and then it was allowed to warm slowly (30 min) to room tarperature. The mixture wasvtransferred to a separatαry funnel and shaken with 30 ml of 10% NaCH. Ether (150 ml) was addedand the separate organic phase was washedwith 10% NaOH, water and dried over Na-SO.. Concentration to dryness in vacuo gave a yeϋσw oϋy residue which was purified on sϋica gel TLC plate developed in 7:3 hexane-etbyl acetate giving 1-hydroxy- cyclσvitamin product (20 mg, 37%) : NMR 60.59 (3H, s, 18- ^" H_), 0.63 (IH, m, 3-H), 0.89 and 0.90 (6H, each d, J=6.9 Hz, 26-H ₃ and 27-H ₃ ), 0.96 (3H, d, J=6.9 Hz, 21-H ₃ ) , 3.25 (3H, s, -CCH ₃ ), 4.17 (2H, , 1-H and 6-H) , 4.96 (IH, d, J=9.3 Hz, 7-H) , 5.1-5.4 (4H, br m, 29-^, 22-H and 23-H); mass spectrum, m/ 412 (M ⁺ , 26) , 380 (48) , 339 (22) , 269 (28) , 245 (20) , 135 (100). This product is composed chiefly of the lα-hydroxy- cyclovitamin D compound of structure (8) , as well as small amount of the corresponding lβ-hydroxy-epimer (13) . These components may be separated at this stage, if desired, but such separation is. not required.

The 1-hydroxyyclσvitamin product (18 irg) as .obtained above was heated (55°C/15 min) in glacial acetic acid (0.8 ml), .the mixture was neutralized (ice/sat ra±ed NaHCO.) and extracted with benzene and ether, to yield after HPLC (1.5% of 2-prσpanol in hexane as eluent) separation pure 3β-acetα__y- vita ins (9_) (6.60 g, 34%, eluting at 42 ml) , (10) (4.20 mg, 22%, eluting at 50 ml), and (14) (1.44 mg, 7%, eluting at 36 ml).

Compound (9): NMR 60.60 (3H, s, 18-H ₃ ), 0-.90 and 0.92 (6H, each d, J=7.0 Hz, 26-H ₃ and 27-H ₃ ) , 0.97 (3H, d, J=6.8 Hz,

21-^) , 2.04 (3H, s, -OC0CH ₃ ), 4.41 (IH, , 1-H) , 5.02 (IH, narrowm, 19-H) , 5.1-5.4 (4H, br m, 3-„19-,22- and 23-H) , 6.03 and 6.35 (2H, ABq, J=ϋ.4 Hz, 7-H and 6-H); UV λ 264.5 nm, 440 (M , 10) , 380 (72) , 362

(7), 269 (31), 251 (12), 135 (100), 134 (99). Compound (10): NMR 60.60 (3H, s, 18-H ₃ ) , 0.90 and 0.91 (6H, each d, J=7.0 Hz, 26-H. and 27-H , 0.97 (3H, d, J=6.9 Hz, 21-Kj , 2.05 (3H, s, -0C0CH ₃ ), 4.49 (H, m, 1-H), 5.CO and 5.14 (2H, each narr. m, l^IL), 5.20 (3H, br m, 3-,22- and 23HH) . 5 2 and 6.59 (2H _T ABq, J=12.0 Hz, 7-H and 6-H); UV λ 270 nm; λ . 228 nm; mass spectrum, m/z 440 (M , 4) , 380 max min

(30), 269 (10), 135 (100), 134 (52).

Cαrpound (14): NMR δ 0.58 (3H, s, 18-H ₃ ) , 0.89 and 0.90 (6H, each d, J=6.9 Hz, 26-H ₃ and 27-H ₃ ), 0.96 (3H, d, J=6.9 Hz, 21-H , 2.06 (3H, s, -OCOOy , 4.16 (IH, m, 1-H) , 4.98 (2H, m, 3-H and 19-H) , 5.1-5.4 (3H, br m, 19-,22- and 23-H); UV λ _m 263 nm, λ .^ 227 nm; mass spectrum, m/z 440 (M , 32) , 380

(78), 362 (21), 269 (28), 251 (19), 135 (100), 134 (82).

Hydrolysis of 3β-acetoxy group in compounds (9) , (10) and

(14). Each of the 3β-acetoxy-derivatives {9) , (10) and (14) was separately hydrolyzed, using the same procedure. A solution of 3β-acetoxyvitamin (0.7-6 mg) in ethanol (0.1 ml) was treated with 10% KOH in methanol (0.8 ml) and the π xture was heated for 1 h at 50°C. After usual work-up and final

HPLC purification (8% of 2-propanol in hexane as eluent) the corresponding 1-hydroxyvitamins were obtained, namely:

Compound (11): NMR δ 0.59 (3H, s, 18-H ₃ ) , 0.89 and 0.90 (6H, each d, J=7.0 Hz, 26-H. and 27-H-), 0.96 (3H, d, J=6.8 Hz,

21-H ₃ ), 4.23 (IH, m, 3-H) , 4.43 (IH, m, 1-H) , 5.00 (IH, narr. m, 19-H), 5.1-5.4 (3H, brm, 19-, 22-, and 23-H) , 6.02 and

6.39 (2H, AEq, J=11.4 Hz, 7-H and 6-H)j UV λ 264.5 ran, λ . ¹ max mxn

227.5 nm; mass spectrum, m/z 398 (M , 21), 380 (8), 287 (6), 269 (7) , 251 (5) , 152 (36) , 134 (100) . (Elution volume 39 ml) .

Ccπpound (12): WSR δ 0.61 (3H, s, 18-^), 0.89 and 0.91 (6H, each d, J=7.0 Hz, 26-H ₃ and 27-^), 0.97 (3H, d, J=6.9 Hz, 21-iy , 4.25 (IH, , 3-H) , 4.51 (IH, , 1-H) , 4.98 and 5.13 (2H, each narr. m„ 19-H ₂ ), 5.21 (2H _t brm, 22-H and 23-H), 5.89 and 6.59 (2H, ABq, J=ϋ.5 Hz, 7-H and €-H); UV λ _m 273 n , λ . 229.5 nm; mass spectrum, m/z. ³⁹⁸ < ^M ' ¹⁷ ^ * ³⁸ ° t4 , 287 (5), 269 (5), 251 (4), 152 (29), 134 (100). (Elution volume 38 ml) .

Compound (16): NMR δ 0.60 (3H, s, 18-H ) , 0.89 and 0.91 (6H, each d, J=7.O Hz, 26-H ₃ and 27-H ₃ ) , 0.97 (3H, d, J=6.9 Hz, 21-H ₃ ) , 4.10 (IH, , 3-H) , 4.36 (IH, m, 1-H) , 5.01 (IH, d, J=2 Hz, 19-H), 5.1-5.4 (3H, brm, 19-, 22- and 23-H), 6.06 and

6.45 (2Ξ, ABq, J=ϋ.3 Hz, 7-H and 6-H); UV λ 262.5 nm, λ . ^' max mxn

226.5 nm; mass spectrum, m/z 398 (M , 20), 380 (19), 269 (ϋ), 251 (10) , 152 (100) , 134 (60) . (Elution volurre 32 ml) .

If desired, the ccis σurids of this invention can be , readily obtained by crysta11ization from suitable solvents such as ethers, hexane, alcohols, and mixtures thereof as will be evident and well known to those skilled in the art.