1.alpha.,25-dihydroxy-22Z-dehydroxyvitamin D compound

The invention provides a novel vitamin D derivative, 1.alpha.,25-dihydroxy-22Z-dehydrovitamin D.sub.2. The compound is characterized by unexpectedly high ability to raise serum calcium levels. The compound could therefore, find ready application as a substitute for vitamin D or its metabolites in the treatment of metabolic bone diseases or in other of their known applications.

TECHNICAL FIELD 
This invention relates to a biologically active vitamin D compound. 
Specifically, the invention relates to a novel 1.alpha.,25-dihydroxylated 
vitamin D compound with a 22,23-cis-double bond in the side chain, and to 
a method for its preparation. 
BACKGROUND 
Calcium and phosphate homeostasis in animals and humans is regulated by 
vitamin D metabolites, and the compound 1.alpha.,25-dihydroxyvitamin 
D.sub.3 is generally considered as the most active and most important 
vitamin D-derived regulator of normal calcium and phosphate balance. This 
natural metabolite and compounds structurally related to it are therefore 
of great pharmaceutical interest as effective agents for the prevention 
and treatment of bone diseases and related calcium metabolism disorders. 
In addition to the natural D.sub.3 metabolites, a number of compounds have 
been prepared in recent years which, because of their high potency, find 
use or show very considerable promise as therapeutic agents, among them 
1.alpha.-hydroxyvitamin D.sub.3, 1.alpha.-hydroxyvitamin D.sub.2, 
1.alpha.,25-dihydroxyvitamin D.sub.2 and certain fluorinated analogs (U.S. 
Pat. Nos. 3,741,996; 3,907,843; 3,880,894; 4,226,788; 4,358,406). Most of 
the known active analogs are characterized by the type of sterol side 
chain as it occurs in vitamin D.sub.3 (i.e. saturated side chain). Known 
analogs with 22,23-unsaturated side chain are represented by compounds of 
the vitamin D.sub.2 series (i.e. 22,23-trans-unsaturated with a 
C-24-methyl substituent), and include, in addition to the compounds named 
above, 25-hydroxyvitamin D.sub.2 (U.S. Pat. No. 3,585,221) and the 24- and 
24,25-dihydroxy derivatives (Jones et al. Arch. Biochem. Biophys. 202, 450 
(1980)) and three compounds lacking the 24-methyl substituent (U.S. Pat. 
No. 3,786,062; Bogoslovskii et al. J. Gen. Chem. USSR, 48(4) 828 (1978); 
Chem. Abstr. 89, 163848j, 209016s).

DISCLOSURE OF INVENTION 
A novel vitamin D analog has now been found which may be represented by 
structure I shown below: 
##STR1## 
This novel compound is characterized by a 22,23-double bond in the side 
chain having the cis (or Z) geometry. Because of the presence of this 
22Z-double bond, which results in a quite different side chain geometry 
from that pertaining to compounds having the normal saturated side chain 
(e.g. as in 1.alpha.,25-dihydroxyvitamin D.sub.3) or a 22,23-trans 
(22E)-unsaturated side chain (e.g. as in 1.alpha.,25-dihydroxyvitamin 
D.sub.2), it was assumed that this cis-unsaturated product would exhibit 
low biological activity, if any. Surprisingly, this material, in spite of 
its altered side chain structure, shows high activity, being as active as 
1.alpha.,25-dihydroxyvitamin D.sub.3 in its ability to raise serum calcium 
levels in test animals. 
PREATION OF THE COMPOUND 
The novel product of this invention, compound I, above, was prepared from a 
22Z-dehydrovitamin D precursor having the structure II shown below, by in 
vitro enzymatic hydroxylation at carbon 25 using a liver homogenate 
preparation from vitamin D-deficient rats. 
##STR2## 
The following procedure was used: Male weanling rats were fed a low calcium 
and vitamin D-deficient diet as described by Suda et al. [J. Nutr. 100, 
1049 (1970)] for 2 weeks. They were killed by decapitation and their 
livers were removed. A 20% (w/v) homogenate was prepared in ice-cold 0.25 
M sucrose. Incubation was carried out in 10 ml incubation medium in a 125 
ml Erlenmayer flask containing an aliquot of liver homogenate representing 
1 g of tissue, 0.125M sucrose, 50 mM phosphate buffer (pH 7.4), 22.4 mM 
glucose-6-phosphate, 20 m ATP, 160 mM nicotinamide, 25 mM succinate, 0.4 
mM NADP, 5 mM MgCl.sub.2, 0.1M KCl and 0.5 units 
glucose-6-phosphatedehydrogenase. The reaction was initiated by addition 
of 400 .mu.g of the substrate, compound II above, dissolved in 100 .mu.l 
95% ethanol. The incubation mixture was incubated at 37.degree. C. with 
shaking at 80 oscillations/min for 3 h. The reaction was stopped by 
addition of 20 ml methanol and 10 ml dichloromethane. After further 
addition of 10 ml dichloromethane, the organic phase was collected while 
the aqueous phase was re-extracted with 10 ml dichloromethane. The organic 
phases from total of three extractions were combined and evaporated with a 
rotary evaporator. The residue containing the desired product was 
dissolved in 1 ml of CHCl.sub.3 :hexane (65:35) mixture and applied to a 
Sephadex LH-20 column (0.7 cm.times.14 cm) packed, equilibrated and eluted 
with the same solvent. The first 10 ml was discarded while next 40 ml was 
collected and evaporated. The residue was then dissolved in 8% 2-propanol 
in hexane and subjected to high performance liquid chromatography (Model 
LC/GPC 204 HPLC, Waters Associates, Medford, MA) using a Zorbax-SIL column 
(4.6 mm.times.25 cm, Dupont, Wilmington, Del.) operating under pressure of 
1000 psi with a flow rate of 2 ml/min. The desired 25-hydroxylated product 
was eluted at 44 ml. This product was further purified by high performance 
liquid chromatography using a reversed phase column (Richrosorb Rp-18, 4.6 
mm.times.25 cm, E. Merck, Darmstadt, West Germany) operated under pressure 
of 1200 psi and a flow rate of 2 ml/min. The column was eluted with 22% 
H.sub.2 O in methanol, and the compound was eluted at 50 ml. The product 
was further purified by HPLC using the Zorbax-SIL column and conditions as 
described above. The resulting product was then subjected to physical 
characterization. 
CHARACTERIZATION OF THE PRODUCT 
The UV absorption of the product in 95% ethanol exhibited a 
.lambda..sub.max =265 nm and a .lambda..sub.min =228 nm indicating the 
presence of the 5,6-cis-triene chromophore. 
The mass spectrum of the substance contains a molecular ion at m/e 414 as 
required for a 25-hydroxylated product. Elimination of one and two 
molecules of H.sub.2 O gives fragment ions at m/e 396 and 378. Loss of the 
entire steroid side chain (cleavage of C.sub.17 /C.sub.20 bond) results in 
the fragment of m/e 287, which by elimination of one and two molecules of 
H.sub.2 O, gives rise to the peaks at m/e 269 and 251. The spectrum shows 
prominent peaks at m/e 152 and 234 (152-H.sub.2 O) which represent ring A 
fragments and are diagnostic for 1.alpha.,3.beta.-dihydroxyvitamin D 
compounds. In addition, the spectrum shows a very prominent fragment peak 
at m/e 59 which results from cleavage of the C.sub.24 /C.sub.25 bond. The 
presence of this ion confirmed the presence of 25-hydroxy group in the 
product. Thus, these data established the structure of the product 
obtained as the 1.alpha.,25-dihydroxylated compound, as represented by 
structure I, above. 
BIOLOGICAL ACTIVITY 
The biological activities of the novel analog was demonstrated by in vivo 
assay in the rat. Male weanling rats were fed the low calcium vitamin 
D-deficient diet of Suda et al. (supra) for 3 weeks. They were then 
divided into groups of 5 rats each. Rats in a control group received 0.05 
ml 95% ethanol intrajugularly while rats in the other groups were given 
325 pmole of either compound I or 1.alpha.,25-dihydroxyvitamin D.sub.3 
dissolved in 0.05 ml 95% ethanol. Eighteen hours later, they were killed 
by decapitation and blood was collected. Serum obtained by centrifugation 
of the blood was diluted with 0.1% lanthanum chloride solution (1:20) and 
serum calcium concentration was determined with an atomic absorption 
spectrophotometer. Results are shown in the following Table: 
______________________________________ 
Increase in serum calcium concentration in response to a 
single dose of 325 pmole of either compound I or 1.alpha.,25- 
dihydroxyvitamin D.sub.3 given 18 h prior to sacrifice 
Serum Calcium Concentration 
Compound Given (mg/100 ml) .+-. standard deviation 
______________________________________ 
ethanol 4.2 .+-. 0.1.sup.a 
compound I 5.2 .+-. 0.2.sup.b 
1.alpha.,25-dihydroxyvitamin D.sub.3 
5.4 .+-. 0.4.sup.b 
______________________________________ 
.sup.b is significantly different from 
.sup.a p &lt; 0.001 
The above results show the new analog to be highly potent and to exhibit 
biological activity essentially equivalent to that of 
1.alpha.,25-dihydroxyvitamin D.sub.3. 
Because of this high potency, the compound of this invention will find 
application as a therapeutic agent in the therapy or prophylaxis of 
disorders such as the various types of rickets, hypoparathyroidism, 
osteodystrophy, osteomalacia or osteoporosis in the human, or for the 
treatment of related calcium deficiency diseases (e.g. milk fever, leg 
weakness, egg shell thinness) in animals. Likewise the compound may be 
used for the treatment of certain malignancies, such as human leukemia. 
For therapeutic purposes, the compound may be administered by any 
conventional route of administration and in any form suitable for the 
method of administration selected. The compound may be formulated with any 
acceptable and innocuous pharmaceutical carrier, in the form of pills, 
tablets, gelatin capsules, or suppositories, or as solutions, emulsions, 
dispersions or suspensions in innocuous solvents or oils, and such 
formulations may contain also other therapeutically active and beneficial 
ingredients as may be appropriate for the specific applications. For human 
applications, the compound is advantageously administered in amounts from 
0.25 to 10 .mu.g per day, the specific dosage being adjusted in accordance 
with 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 22Z-dehydro precursor substrate, compound II above, required for the 
preparation of the novel product of this invention is itself prepared by 
the process depicted in Process Scheme I, appended, and described below. 
In the description, compound designation by Arabic numerals (e.g. (b 1), 
(2), (3), etc.) refer to the structures so numbered in the Process Scheme. 
The desired substrate (compound II) for the above described 
25-hydroxylation reaction is identified by Arabic numeral (11) in Process 
Scheme I and in the following description. 
(22Z)-3.beta.-(Methoxymethoxy)-5.alpha.,8.alpha.-(4-phenyl-1,2-urazolo)chol 
esta-6,22-dien (2). 
Isopentyl phosphonium bromide [(CH.sub.3).sub.2 CHCH.sub.2 CH.sub.2 
PPh.sub.3 Br] (1.67 g, 4.04 mmol) in dry tetrahydrofuran (73 ml) was 
treated with n-butyllithium (1.7M solution in hexane, 2.42 ml, 4.11 mmol) 
at 3.degree.-5.degree. C. with stirring. After stirring for 1 h at room 
temperature, the orange-red solution was cooled to 3.degree. C. and 
aldehyde (1) (1.84 g, 3.36 mmol) 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% HCl, saturated sodium bicarbonate and water, dried 
(Na.sub.2 SO.sub.4) 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 .delta. 0.83 (3H, 
s, 18-H.sub.3), 0.89 and 0.91 (6H, each d, J=6.8 Hz, 26-H.sub.3 and 
27-H.sub.3), 0.97 (3H, d, J=6.8 Hz, 21-H.sub.3), 0.98 (3H, s, 19-H.sub.3), 
3.30 (1H, dd, J.sub.1 =4.4 Hz, J.sub.2 =14 Hz, 9-H), 3.38 (3H, s, 
OCH.sub.3), 4.33 (1H, m, 3-H), 4.70 and 4.81 (2H, ABq, J=6.8 Hz, 
OCH.sub.2 O), 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 (5H, br m, Ar-H); IR: 1756, 1703, 1601, 1397, 
1046 cm.sup.-1 ; mass spectrum, m/z 601 (M.sup.+, 1%), 426 (4), 364 (61), 
349 (16), 253 (18), 251 (18), 119 (PhNCO, 100). 
(22Z)-5.alpha.,8.alpha.-(4-phenyl-1,2-urazolo)cholesta-6,22-dien-3.beta.-ol 
(3). 
A solution of adduct (2) (601 mg, 1 mmol) 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 (Na.sub.2 SO.sub.4) and evaporated under reduced 
pressure. Purification of the crude product by column chromatography 
(benzene ether 70:30 as eluant) gave the hydroxy adduct (3) (550 mg, 99%) 
as a foam: NMR .delta. 0.83 (3H, s, 18-H.sub.3), 0.89 and 0.91 (6H, each 
d, J=6.8 Hz, 26-H.sub.3 and 27-H.sub.3), 0.95 (3H, s, 19-H.sub.3), 0.98 
(3H, d, J=6.8 Hz, 21-H.sub.3), 3.16 (1H, dd, J.sub.1 =4.4 Hz, J.sub.2 =14 
Hz, 9-H), 4.44 (1H, m, 3-H), 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 m, Ar-H); IR: 3447, 1754, 
1700, 1600, 1397 cm.sup.-1 ; mass spectrum, m/z (557 (M.sup.+, 1%) 382 
(35), 349 (33), 253 (20), 251 (33), 119 (100), 55 (82). 
(22Z)-Cholesta-5,7,22-trien-3.beta.-ol (4) 
The adduct (3) (530 mg, 0.95 mmol) was converted to the diene (4) by 
reduction with lithium aluminum hydride (1 g), in tetrahydrofuran (60 ml) 
at reflux for 18 h. After conventional work-up, the product was purified 
by chromatography over silica (benzene-ether 94:6 as eluant) to afford 
pure diene (4) (290 mg, 76%) after crystallization from ethanol:mp 
148.degree.-151.degree. C.; [.alpha.].sub.D.sup.24 =-132.degree. (c=0.9, 
CHCl.sub.3); NMR .delta. 0.66 (3H, s, 18-H.sub.3), 0.90 and 0.91 (6H, each 
d, J=6.8 Hz, 26-H.sub.3 and 27-H.sub.3), 0.96 (3H, s, 19-H.sub.3), 0.98 
(3H, d, J=6.9 Hz, 21-H.sub.3), 3.64 (1H, m, 3-H), 5.20 (2H, br m, 22-H and 
23-H), 5.39 and 5.57 (2H, ABq, J=6 Hz, 7-H and 6-H); UV .lambda..sub.max 
281 nm; IR: 3346, 1463, 1375, 1364, 1067, 1040, 831 cm.sup.-1 ; mass 
spectrum, m/z 382 (M.sup.+, 100), 349 (65); 323 (32), 271 (15), 253 (30). 
(5Z,7E,22Z)-9,10-Secocholesta-5,7,10(19),22-tetraen-3.beta.-ol (5). 
Irradiation of 5,7-diene (4) (150 mg, 0.39 mmol) dissolved in ether (120 
ml) and benzene (30 ml) (degassed with argon for 40 min) was performed at 
0.degree. C. for 13 min using a UV-lamp and Vycor filter. HPLC (1% of 
2-propanol in hexane) of the resulting mixture afforded the previtamin 
(56.9 mg, 38%) as a colorless oil: NMR .delta.0.75 (3H, s, 18-CH.sub.3), 
0.90 and 0.91 (6H, each d, J=6.7 Hz, 26-H.sub.3 and 27-H.sub.3), 0.99 (3H, 
d, J=6.8 Hz, 21-H.sub.3), 1.64 (3H, s, 19-H.sub.3), 3.90 (1H, m, 3-H), 
5.20 (2H, br m, 22-H and 23-H), 5.69 and 5.95 (2H, ABq, J=12 Hz, 7-H and 
6-H); UV .lambda..sub.max 261 nm, .lambda..sub.min 234 nm. 
Thermal isomerization of this previtamin intermediate (56 mg, 0.15 mmol) in 
refluxing ethanol (3 h) gave the oily vitamin analog (5) (43 mg, 77%) 
after separation by HPLC. NMR .delta. 0.60 (3H, s, 18-H.sub.3), 0.89 and 
0.90 (6H, each d, J=6.7 Hz, 26-H.sub.3 and 27-H.sub.3), 0.97 (3H, d, J=6.6 
Hz, 21-H.sub.3), 3.96 (1H, s, 3-H), 4.82 and 5.05 (2H, each narr. m, 
19-H.sub.2), 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 .lambda..sub.max 265.5 nm, .lambda..sub.min 
228 nm; IR: 3427, 1458, 1379, 1048, 966, 943, 892 cm.sup.-1 ; mass 
spectrum, m/z 382 (M.sup.+, 21), 349 (5), 271 (8), 253 (14), 136 (100), 
118 (82). Vitamin analog (5) is a known compound (Bogoslovskii et al., 
supra). 
1-Hydroxylation of compound (5). 
Freshly recrystallized p-toluenesulfonyl chloride (50 mg, 0.26 mmol) was 
added to a solution of vitamin (5) (50 mg, 0.13 mmol) in dry pyridine (300 
.mu.l). After 30 h at 4.degree. C., the reaction mixture was poured into 
ice/saturated NaHCO.sub.3 with stirring. The mixture was stirred for 15 
min and extracted with benzene. The organic extract was washed with 
saturated NaHCO.sub.3, saturated copper sulfate and water, dried (Na.sub.2 
SO.sub.4) and concentrated in vacuo to obtain the oily tosylate (6). The 
crude tosylate (6) was treated with NaHCO.sub.3 (150 mg) in anhydrous 
methanol (10 ml) and the mixture was stirred for 8.5 h at 55.degree. C. 
After cooling and concentration to 2 ml the mixture was diluted with 
benzene (80 ml), washed with water, dried (Na.sub.2 SO.sub.4) 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 SeO.sub.2 (5.1 mg, 0.046 mmol) in dry CH.sub.2 Cl.sub.2 (5 
ml), tert-butylhydroperoxide (16.5 .mu.l, 0.118 mmol) was added. After 30 
min dry pyridine (50 .mu.l) was added and the mixture was stirred for 
additional 25 min at room temperature, diluted with CH.sub.2 Cl.sub.2 (3 
ml) and cooled to 0.degree. C. The crude 3,5-cyclovitamin product (7) in 
CH.sub.2 Cl.sub.2 (4.5 ml) was then added. The reaction proceeded at 
0.degree. C. for 15 min and then it was allowed to warm slowly (30 min) to 
room temperature. The mixture was transferred to a separatory funnel and 
shaken with 30 ml of 10% NaOH. Ether (150 ml) was added and the separate 
organic phase was washed with 10% NaOH, water and dried over Na.sub.2 
SO.sub.4. Concentration to dryness in vacuo gave a yellow oily residue 
which was purified on silica gel TLC plate developed in 7:3 hexane-ethyl 
acetate giving 1-hydroxycyclovitamin product (20 mg, 37%): NMR .delta. 
0.59 (3H, s, 18-H.sub.3), 0.63 (1H, m, 3-H), 0.89 and 0.90 (6H, each d, 
J=6.9 Hz, 26-H.sub.3 and 27-H.sub.3), 0.96 (3H, d, J=6.9 Hz, 21-H.sub.3), 
3.25 (3H, s, --OCH.sub.3), 4.17 (2H, m, 1-H and 6-H), 4.96 (1H, d, J=9.3 
Hz, 7-H), 5.1-5.4 (4H, br m, 19-H.sub.2, 22-H nd 23-H); mass spectrum, m/z 
412 (M.sup.+, 26), 380 (48), 339 (22), 269 (28), 245 (20), 135 (100). This 
product is composed chiefly of the 1.alpha.-hydroxycyclovitamin D compound 
of structure (8), as well as small amount of the corresponding 
1.beta.-hydroxy-epimer. These components may be separated at this stage, 
if desired, but such separation is not required. 
The 1-hydroxycyclovitamin product (18 mg) as obtained above was heated 
(55.degree. C./15 min) in glacial acetic acid (0.8 ml), the mixture was 
neutralized (ice/saturated NaHCO.sub.3) and extracted with benzene and 
ether, to yield after HPLC (1.5% of 2-propanol in hexane as eluent) 
separation pure 1.alpha.-hydroxy-3.beta.-acetoxyvitamins (9) (6.60 mg, 
34%, eluting at 42 ml) and (10) (4.20 mg, 22%, eluting at 50 ml). Compound 
(9): NMR .delta. 0.60 (3H, s, 18-H.sub.3), 0.90 and 0.92 (6H, each d, 
J=7.0 Hz, 26-H.sub.3 and 27-H.sub.3), 0.97 (3H, d, J=6.8 Hz, 21-H.sub.3), 
2.04 (3H, s, --OCOCH.sub.3), 4.41 (1H, m, 1-H), 5.02 (1H, narr. m, 19-H), 
5.1-5.4 (4H, br m, 3-, 19-, 22- and 23-H), 6.03 and 6.35 (2H, ABq, J=11.4 
Hz, 7-H and 6-H); UV .lambda..sub.max 264.5 nm, .lambda..sub.min 227.5 nm; 
mass spectrum, m/z 440 (M.sup.+, 10), 380 (72), 362 (7), 269 (31), 251 
(12), 135 (100), 134 (99). Compound (10): NMR .delta. 0.60 (3H, s, 
18-H.sub.3), 0.90 and 0.91 (6H, each d, J=7.0 Hz, 26-H.sub.3 and 
27-H.sub.3), 0.97 (3H, d, J=6.9 Hz, 21-H.sub.3), 2.05 (3H, s, 
--OCOCH.sub.3), 4.49 (1H, m, 1-H), 5.00 and 5.14 (2H, each narr. m, 
19-H.sub.2), 5.20 (3H, br m, 3-, 22- and 23-H), 5.82 and 6.59 (2H, ABq, 
J=12.0 Hz, 7-H and 6-H); UV .lambda..sub.max 270 nm; .lambda..sub.min 228 
nm; mass spectrum, m/z 440 (M.sup.+, 4), 380 (30), 269 (10), 135 (100), 
134 (52). 
Hydrolysis of 3.beta.-acetoxy group in compounds (9) and (10). 
Each of the 3.beta.-acetoxy-derivatives (9) or (10) was separately 
hydrolyzed, using the same procedure. A solution of 3.beta.-acetoxyvitamin 
(0.7-6 mg) in ethanol (0.1 ml) was treated with 10% KOH in methanol (0.8 
ml) and the mixture was heated for 1 h at 50.degree. 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 67 0.59 (3H, s, 18-H.sub.3), 0.89 and 0.90 (6H, each d, J=7.0 Hz, 
26-H.sub.3 and 27-H.sub.3), 0.96 (3H, d, J=6.8 Hz, 21-H.sub.3), 4.23 (1H, 
m, 3-H), 4.43 (1H, m, 1-H), 5.00 (1H, narr. m, 19-H), 5.1-5.4 (3H, br m, 
19-, 22-, and 23-H), 6.02 and 6.39 (2H, ABq, J=11.4 Hz, 7-H and 6-H); UV 
.lambda..sub.max 264.5 nm, .lambda..sub.min 227.5 nm; mass spectrum, m/z 
398 (M.sup.+, 21), 380 (8), 287 (6), 269 (7), 251 (5), 152 (36), 134 (100 
). (Elution volume 39 ml). Compound (12): NMR .delta. 0.61 (3H, s, 
18-H.sub.3), 0.89 and 0.91 (6H, each d, J=7.0 Hz, 26-H.sub.3 and 
27-H.sub.3), 0.97 (3H, d, J=6.9 Hz, 21-H.sub.3), 4.25 (1H, m, 3-H), 4.51 
(1H, m, 1-H), 4.98 and 5.13 (2H, each narr. m., 19-H.sub.2), 5.21 (2H, br 
m, 22-H and 23-H), 5.89 and 6.59 (2H, ABq, J=11.5 Hz, 7-H and 6-H); UV 
.lambda..sub.max 273 nm, .lambda..sub.min 229.5 nm; mass spectrum, m/z 398 
(M.sup.+, 17), 380 (4), 287 (5), 269 (5), 251 (4), 152 (29), 134 (100). 
(Elution volume 38 ml). 
In the above described process, high pressure liquid chromatography (HPLC) 
was performed on a Waters Associates Model ALC/GPC 204 using a Zorbax-Sil 
(DuPont) (6.2 mm.times.25 cm column, flow rate 4 ml/min, 1500 psi). Column 
chromatography was performed on Silica Gel 60, 70-230 mesh ASTM (Merck). 
Preparative thin-layer chromatography (TLC) was carried out on Silica 60 
PF-254 (20.times.20 cm plates, 1 mm silica gel). Irradiations were carried 
out using a Hanovia 608A36 mercury arc lamp fitted with a Vycor filter. 
All reactions are preferably performed under an inert atmosphere (e.g. 
argon). 
The compound of this invention can, if desired, be readily obtained in 
crystalline form by crystallization from suitable solvents such as hexane, 
ethers and alcohols (absolute or aqueous), and mixtures thereof as will be 
evident and well known to those skilled in the art. 
##STR3##