U.S. patent application number 11/374782 was filed with the patent office on 2006-07-06 for (20s)-1alpha-hydroxy-2alpha-methyl and 2beta-methyl-19-nor-vitamin d3 and their uses.
Invention is credited to Hector F. DeLuca, Pawel K. Grzywacz, Rafal R. Sicinski.
Application Number | 20060148769 11/374782 |
Document ID | / |
Family ID | 29248431 |
Filed Date | 2006-07-06 |
United States Patent
Application |
20060148769 |
Kind Code |
A1 |
DeLuca; Hector F. ; et
al. |
July 6, 2006 |
(20S)-1alpha-hydroxy-2alpha-methyl and 2beta-methyl-19-nor-vitamin
D3 and their uses
Abstract
This invention discloses
(20S)-1.alpha.-hydroxy-2.alpha.-methyl-19-nor-vitamin D.sub.3 and
(20S)-1.alpha.-hydroxy-2.beta.-methyl-19-nor-vitamin D.sub.3 and
pharmaceutical uses therefor. These compounds exhibit pronounced
activity in arresting the proliferation of undifferentiated cells
and inducing their differentiation to the monocyte thus evidencing
use as an anti-cancer agent and for the treatment of skin diseases
such as psoriasis as well as skin conditions such as wrinkles,
slack skin, dry skin and insufficient sebum secretion. These
compounds also have very significant calcemic activity and
therefore may be used to treat immune disorders in humans as well
as metabolic bone diseases such as osteoporosis.
Inventors: |
DeLuca; Hector F.;
(Deerfield, WI) ; Sicinski; Rafal R.; (Warsaw,
PL) ; Grzywacz; Pawel K.; (Madison, WI) |
Correspondence
Address: |
ANDRUS, SCEALES, STARKE & SAWALL, LLP
St. 110
100 East Wisconsin Avenue
Milwaukee
WI
53202
US
|
Family ID: |
29248431 |
Appl. No.: |
11/374782 |
Filed: |
March 14, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11037876 |
Jan 18, 2005 |
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11374782 |
Mar 14, 2006 |
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10127180 |
Apr 22, 2002 |
6846811 |
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11037876 |
Jan 18, 2005 |
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Current U.S.
Class: |
514/167 |
Current CPC
Class: |
A61P 17/16 20180101;
A61P 37/06 20180101; A61P 3/10 20180101; A61P 19/10 20180101; A61P
1/04 20180101; A61P 29/00 20180101; A61P 17/06 20180101; A61P 19/08
20180101; A61P 35/02 20180101; A61P 25/00 20180101; A61P 19/02
20180101; A61P 35/00 20180101; A61P 11/06 20180101; C07C 49/443
20130101; A61K 31/593 20130101; C07C 49/433 20130101 |
Class at
Publication: |
514/167 |
International
Class: |
A61K 31/59 20060101
A61K031/59 |
Claims
1-76. (canceled)
77. A compound having the formula: ##STR5##
Description
BACKGROUND OF THE INVENTION
[0001] This invention relates to vitamin D compounds, and more
particularly to the pro-drugs
(20S)-1.alpha.-hydroxy-2.alpha.-methyl and
2.beta.-methyl-19-nor-vitamin D.sub.3 and their pharmaceutical
uses.
[0002] The natural hormone, 1.alpha.,25-dihydroxyvitamin D.sub.3
and its analog in ergosterol series, i.e.
1.alpha.,25-dihydroxyvitamin D.sub.2 are known to be highly potent
regulators of calcium homeostasis in animals and humans, and their
activity in cellular differentiation has also been established,
Ostrem et al., Proc. Natl. Acad. Sci. USA, 84, 2610 (1987). Many
structural analogs of these metabolites have been prepared and
tested, including 1.alpha.-hydroxyvitamin D.sub.3,
1.alpha.-hydroxyvitamin D.sub.2, various side chain homologated
vitamins and fluorinated analogs. Some of these compounds exhibit
an interesting separation of activities in cell differentiation and
calcium regulation. This difference in activity may be useful in
the treatment of a variety of diseases as renal osteodystrophy,
vitamin D-resistant rickets, osteoporosis, psoriasis, and certain
malignancies.
[0003] Recently, a new class of vitamin D analogs has been
discovered, i.e. the so called 19-nor-vitamin D compounds, which
are characterized by the replacement of the A-ring exocyclic
methylene group (carbon 19), typical of the vitamin D system, by
two hydrogen atoms. Biological testing of such 19-nor-analogs
(e.g., 1.alpha.,25-dihydroxy-19-nor-vitamin D.sub.3) revealed a
selective activity profile with high potency in inducing cellular
differentiation, and very low calcium mobilizing activity. Thus,
these compounds are potentially useful as therapeutic agents for
the treatment of malignancies, or the treatment of various skin
disorders. Two different methods of synthesis of such
19-nor-vitamin D analogs have been described (Perlman et al.,
Tetrahedron Lett. 31, 1823 (1990); Perlman et al., Tetrahedron
Lett. 32, 7663 (1991), and DeLuca et al., U.S. Pat. No.
5,086,191).
[0004] In U.S. Pat. No. 4,666,634, 2-hydroxy and alkoxy (e.g.,
ED-71) analogs of 1.alpha.,25-dihydroxyvitamin D.sub.3 have been
described and examined by Chugai group as potential drugs for
osteoporosis and as antitumor agents. See also Okano et al.,
Biochem. Biophys. Res. Commun. 163, 1444 (1989). Other
2-substituted (with hydroxyalkyl, e.g., ED-120, and fluoroalkyl
groups) A-ring analogs of 1.alpha.,25-dihydroxyvitamin D.sub.3 have
also been prepared and tested (Miyamoto et al., Chem. Pharm. Bull.
41, 1111 (1993); Nishii et al., Osteoporosis Int. Suppl. 1, 190
(1993); Posner et al., J. Org. Chem. 59, 7855 (1994), and J. Org.
Chem. 60, 4617 (1995)).
[0005] Recently, 2-substituted analogs of
1.alpha.,25-dihydroxy-19-nor-vitamin D.sub.3 have also been
synthesized, i.e. compounds substituted at 2-position with hydroxy
or alkoxy groups (DeLuca et al., U.S. Pat. No. 5,536,713), with
2-alkyl groups (DeLuca et al U.S. Pat. No. 5,945,410), and with
2-alkylidene groups (DeLuca et al U.S. Pat. No. 5,843,928), which
exhibit interesting and selective activity profiles. All these
studies indicate that binding sites in vitamin D receptors can
accommodate different substituents at C-2 in the synthesized
vitamin D analogs.
[0006] In a continuing effort to explore the 19-nor class of
pharmacologically important vitamin D compounds, two analogs which
are characterized by the presence of a methyl substituent at the
carbon 2 (C-2) and the absence of a hydroxyl group at carbon 25
(C-25) in the side chain have been synthesized and tested. These
two analogs are characterized by a hydroxyl group at carbon 1 and a
vitamin D.sub.3 side chain with the methyl group attached to carbon
20 in the unnatural or epi orientation, i.e.
(20S)-1.alpha.-hydroxy-2.alpha.-methyl and
2.beta.-methyl-19-nor-vitamin D.sub.3. These vitamin D analogs
seemed interesting targets because the relatively small methyl
group at C-2 should not interfere with the vitamin D receptor.
Moreover, molecular mechanics studies seem to indicate that such
molecular modification substantially alters the conformation of the
cyclohexanediol ring A, shifting its conformational equilibrium
toward the chair form with equatorially oriented methyl substituent
at C-2.
SUMMARY OF THE INVENTION
[0007] The present invention is directed toward the pro-drugs
(20S)-1.alpha.-hydroxy-2.alpha.-methyl-19-nor-vitamin D.sub.3
(formula Ia below) and
(20S)-1.alpha.-hydroxy-2.beta.-methyl-19-nor-vitamin D.sub.3
(formula Ib below), their biological activity, and various
pharmaceutical uses for these compounds.
[0008] Structurally these 2.alpha.-methyl and 2.beta.-methyl 19-nor
analogs are characterized by formula Ia and Ib, respectively shown
below: ##STR1##
[0009] The above two compounds exhibit a desired, and highly
advantageous, pattern of biological activity. These compounds do
not bind or bind poorly to the vitamin D receptor. However, the
2.alpha.-methyl compound has greater intestinal calcium transport
activity, as compared to that of 1.alpha.,25-dihydroxyvitamin
D.sub.3, and has greater ability to mobilize calcium from bone, as
compared to 1.alpha.,25-dihydroxyvitamin D.sub.3. The
2.beta.-methyl compound has intestinal calcium transport activity
and bone calcium mobilization activity about the same as
1.alpha.,25-dihydroxyvitamin D.sub.3. Hence, these compounds can be
characterized as having very potent calcemic activity, and are
highly specific in their calcemic activity. Their activity on
mobilizing calcium from bone and either high or normal intestinal
calcium transport activity allows the in vivo administration of
these compounds for the treatment of metabolic bone diseases where
bone loss is a major concern. Because of their activity on bone,
these compounds would be preferred therapeutic agents for the
treatment of diseases where bone formation is desired, such as
osteoporosis, especially low bone turnover osteoporosis, steroid
induced osteoporosis, senile osteoporosis or postmenopausal
osteoporosis, as well as osteomalacia.
[0010] The compounds of the invention have also been discovered to
be especially suited for treatment and prophylaxis of human
disorders which are characterized by an imbalance in the immune
system, e.g. in autoimmune diseases, including multiple sclerosis,
lupis, diabetes mellitus, host versus graft reaction, and rejection
of organ transplants; and additionally for the treatment of
inflammatory diseases, such as rheumatoid arthritis, asthma, and
inflammatory bowel diseases such as celiac disease and Crohns
disease. Acne, alopecia and hypertension are other conditions which
may be treated with the compounds of the invention.
[0011] The above compounds are also characterized by high or
significant cell differentiation activity. Thus, these compounds
also provide a therapeutic agent for the treatment of psoriasis, or
as an anti-cancer agent, especially against leukemia, colon cancer,
breast cancer and prostate cancer. In addition, due to their
relatively high cell differentiation activity, these compounds
provide a therapeutic agent for the treatment of various skin
conditions including wrinkles, lack of adequate dermal hydration,
i.e. dry skin, lack of adequate skin firmness, i.e. slack skin, and
insufficient sebum secretion. Use of these compounds thus not only
results in moisturizing of skin but also improves the barrier
function of skin.
[0012] The compounds may be present in a composition to treat the
above-noted diseases and disorders in an amount from about 0.01
.mu.g/gm to about 100 .mu.g/gm of the composition, and may be
administered topically, transdermally, orally or parenterally in
dosages of from about 0.01 .mu.g/day to about 100 .mu.g/day.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1 is a graph illustrating the relative activity of
(20S)-1.alpha.-hydroxy-2.alpha.-methyl-19-nor-vitamin D.sub.3,
(20S)-1.alpha.-hydroxy-2.beta.-methyl-19-nor-vitamin D.sub.3, and
1.alpha.,25-dihydroxyvitamin D.sub.3 to compete for binding of
[.sup.3H]-1,25-(OH).sub.2-D.sub.3 to the vitamin D pig intestinal
nuclear receptor; and
[0014] FIG. 2 is a graph illustrating the percent HL-60 cell
differentiation as a function of the concentration of
(20S)-1.alpha.-hydroxy-2.alpha.-methyl-19-nor-vitamin D.sub.3,
(20S)-1.alpha.-hydroxy-2.beta.-methyl-19-nor-vitamin D.sub.3, and
of 1.alpha.,25-dihydroxyvitamin D.sub.3.
DETAILED DESCRIPTION OF THE INVENTION
[0015] (20S)-1.alpha.-Hydroxy-2.alpha.-methyl-19-nor-vitamin
D.sub.3 and (20S)-1.alpha.-hydroxy-2.beta.-methyl-19-nor-vitamin
D.sub.3 were synthesized and tested. Structurally, these 19-nor
analogs are characterized by the formula Ia and Ib, respectively,
previously illustrated herein.
[0016] The preparation of (20S)-1.alpha.-hydroxy-2.beta.-methyl-
and (20S)-1.alpha.-hydroxy-2.beta.-methyl-19-nor-vitamin D.sub.3
having structures Ia and Ib can be accomplished by a common general
method, i.e. the condensation of a bicyclic Windaus-Grundmann type
ketone II with the allylic phosphine oxide III to the corresponding
2-methylene-19-nor-vitamin D analog IV followed by deprotection of
hydroxyls at C-1 and C-3 in the latter compound; and then followed
by a selective reduction of the exomethylene group at C-2 in
compound V to provide the 2.alpha.-methyl isomer (Ia) and
2.beta.-methyl isomer (Ib): ##STR2## In the structures III and IV
groups Y.sub.1 and Y.sub.2 are hydroxy-protecting groups,
preferably tBuMe.sub.2Si groups, it being also understood that any
functionalities that might be sensitive, or that interfere with the
condensation reaction, be suitably protected as is well-known in
the art. The process shown above represents an application of the
convergent synthesis concept, which has been applied effectively
for the preparation of vitamin D compounds [e.g. Lythgoe et al., J.
Chem. Soc. Perkin Trans. I, 590 (1978); Lythgoe, Chem. Soc. Rev. 9,
449 (1983); Toh et al., J. Org. Chem. 48, 1414 (1983); Baggiolini
et al., J. Org. Chem. 51, 3098 (1986); Sardina et al., J. Org.
Chem. 51, 1264 (1986); J. Org. Chem. 51, 1269 (1986); DeLuca et
al., U.S. Pat. No. 5,086,191; DeLuca et al., U.S. Pat. No.
5,536,713].
[0017] A hydrindanone of the structure II is a new compound that
can be prepared from commercial vitamin D.sub.2 by modification of
known methods. Thus, the starting alcohol 1 was prepared from
commercial vitamin D.sub.2 in 3 steps (Scheme 1). The resulting
C-22 alcohol 1 was oxidized to the aldehyde 2, which then was
equilibrated at C-20. The mixture of (20R)- and (20S)-aldehydes was
reduced and (20R)-alcohol 3 was isolated by chromatography. This,
in turn, was tosylated and the tosylate 4 coupled with the Grignard
reagent 5 in the presence of dilithium tetrachlorocuprate. The
obtained hydroindanol 6 was oxidized to the new (20S)-Grundmann
ketone analog II.
[0018] For the preparation of the required phosphine oxides of
general structure III, a new synthetic route has been developed
starting from a methyl quinicate derivative which is easily
obtained from commercial (1R,3R,4S,5R)-(-)-quinic acid as described
by Perlman et al., Tetrahedron Lett. 32, 7663 (1991) and DeLuca et
al., U.S. Pat. No. 5,086,191.
[0019] The final step of the process is the selective homogeneous
catalytic hydrogenation of the exomethylene unit at carbon 2 in the
vitamin V performed efficiently in the presence of
tris(triphenylphosphine)rhodium(I) chloride [Wilkinson's catalyst,
(Ph.sub.3P).sub.3RhCl]. Such reduction conditions allowed to reduce
only C(2)=CH.sub.2 unit leaving C(5)-C(8) butadiene moiety
unaffected. The isolated material is an epimeric mixture (ca. 1:1)
of 2-methyl-19-nor-vitamins Ia and Ib differing in configuration at
C-2. The mixture can be used without separation or, if desired, the
individual 2.alpha.- and 2.beta.-isomers can be separated by an
efficient HPLC system.
[0020] The overall process of the synthesis of compounds Ia and Ib
is illustrated and described more completely in U.S. Pat. No.
5,945,410 entitled "2-Alkyl-19-Nor-Vitamin D Compounds" the
specification of which is specifically incorporated herein by
reference.
[0021] Specifically, the preparation of hydrindanone II is
described hereinafter and illustrated in Scheme I. The final steps
of the convergent synthesis, i.e. the coupling of this compound
with phosphine oxide 7 followed by hydroxyl deprotection in the
vitamin D compound 8 and reduction/hydrogenation of the
exomethylene unit in 2-methylene-19-nor-vitamin D compound V is
also hereinafter described and illustrated in Scheme 2.
Preparation of
(20S)-de-A,B-8.beta.-benzoyloxy-20-(hydroxymethyl)pregnane (1).
[0022] The starting alcohol 1 was prepared from commercial vitamin
D.sub.2 in 70% yield, according to the procedure published by J. C.
Hanekamp, R. B. Rookhuizen, H. J. T. Bos, L. Brandsma Tetrahedron,
1992, 48, 9283-9294.
[0023] Ozone was passed through a solution of vitamin D.sub.2 (3 g,
7.6 mmol) in methanol (250 mL) and pyridine (2.44 g, 2.5 mL, 31
mmol) for 50 min at -78.degree. C. The reaction mixture was then
flushed with an oxygen for 15 min to remove the residual ozone and
the solution was treated with NaBH.sub.4 (0.75 g, 20 mmol). After
20 min the second portion of NaBH.sub.4 (0.75 g, 20 mmol) was added
and the mixture was allowed to warm to room temperature. The third
portion of NaBH.sub.4 (0.75 g, 20 mmol) was then added and the
reaction mixture was stirred for 18 h. The reaction was quenched
with water (40 mL) and the solution was concentrated under reduced
pressure. The residue was extracted with ethyl acetate (3.times.80
mL) and the combined organic phase was washed with 1M aq. HCl,
saturated aq. NaHCO.sub.3, dried (Na.sub.2SO.sub.4) and
concentrated under reduced pressure. The residue was
chromatographed on silica gel with hexane/ethyl acetate (75:25) to
give (20S)-de-A,B-20-(hydroxymethyl)pregnan-8.beta.-ol (1.21 g, 75%
yield) as white crystals.
[0024] Benzoyl chloride (2.4 g, 2 mL, 17 mmol) was added to a
solution of the 8.beta.,20-diol (1.2 g, 5.7 mmol) and DMAP (30 mg,
0.2 mmol) in anhydrous pyridine (20 mL) at 0.degree. C. The
reaction mixture was stirred at 4.degree. C. for 24 h, diluted with
methylene chloride (100 mL), washed with 5% aq. HCl, water,
saturated aq. NaHCO.sub.3, dried (Na.sub.2SO.sub.4) and
concentrated under reduced pressure. The residue (3.39 g) was
treated with solution of KOH (1 g, 15.5 mmol) in anhydrous ethanol
(30 mL) at room temperature. After stirring of the reaction mixture
for 3 h, ice and 5% aq. HCl were added until pH=6. The solution was
extracted with ethyl acetate (3.times.50 mL) and the combined
organic phase was washed with saturated aq. NaHCO.sub.3, dried
(Na.sub.2SO.sub.4) and concentrated under reduced pressure. The
residue was chromatographed on silica gel with hexane/ethyl acetate
(75:25) to give the alcohol 1 (1.67 g, 93% yield) as a colorless
oil: [.alpha.].sub.D+56.0 (c 0.48, CHCl.sub.3); .sup.1H NMR (400
MHz, CDCl.sub.3+TMS) .delta. 8.08-8.02 (2H, m, o-H.sub.Bz),
7.59-7.53 (1H, m, p-H.sub.Bz), 7.50-7.40 (2H, m, m-H.sub.Bz), 5.42
(1H, d, J=2.4 Hz, 8.alpha.-H), 3.65 (1H, dd, J=10.5, 3.2 Hz, 22-H),
3.39 (1H, dd, J=10.5, 6.8 Hz, 22-H), 1.08 (3H, d, J=5.3 Hz,
21-H.sub.3), 1.07 (3H, s, 18-H.sub.3); .sup.13C NMR (125 MHz)
.delta. 166.70 (s, C.dbd.O), 132.93 (d, p-C.sub.Bz), 131.04 (s,
i-C.sub.Bz), 129.75 (d, o-C.sub.Bz), 128.57 (d, m-C.sub.Bz), 72.27
(d, C-8), 67.95 (t, C-22), 52.96 (d), 51.60 (d), 42.15 (s, C-13),
39.98 (t), 38.61 (d), 30.73 (t), 26.81 (t), 22.91 (t), 18.20 (t),
16.87 (q, C-21), 13.81 (q, C-18); MS (EI) m/z 316 (5, M.sup.+), 301
(3, M.sup.+-Me), 299 (1, M.sup.+-OH), 298 (2, M.sup.+-H.sub.2O),
285 (10, M.sup.+-CH.sub.2OH), 257 (6), 230 (9), 194 (80), 135 (84),
105 (100); exact mass calculated for C.sub.20H.sub.28O.sub.3
316.2038, found 316.2019.
Preparation of (20S)-de-A,B-8.beta.-benzoyloxy-20-formylpregnane
(2).
[0025] A mixture of alcohol 1 (1.63 g, 5.2 mmol), pyridinium
dichromate (6.05 g, 16.1 mmol) and pyridinium p-toluenesulfonate
(100 mg, 0.4 mmol) in anhydrous methylene chloride (30 mL) was
stirred at room temperature for 12 h. The resulting suspension was
filtered through a short layer of Celite. The adsorbent was washed
with ether, solvents were removed under reduced pressure and a
residue was purified by column chromatography on silica gel with
hexane/ethyl acetate (90:10) to give the aldehyde 2 (1.36 g, 83%
yield) as an oil: .sup.1H NMR (400 MHz, CDCl.sub.3+TMS) .delta.
9.60 (1H, d, J=3.1 Hz, CHO), 8.05 (2H, m, o-H.sub.Bz), 7.57 (1H, m,
p-H.sub.Bz), 7.45 (2H, m, m-H.sub.Bz), 5.44 (1H, s, 8.alpha.-H),
2.39 (1H, m, 20-H), 2.03 (2H, dm, J=11.5 Hz), 1.15 (3H, d, J=6.9
Hz, 21-H.sub.3), 1.10 (3H, s, 18-H.sub.3); MS (EI) m/z 314 (1,
M.sup.+), 299 (0.5, M.sup.+-Me), 286 (1, M.sup.+-CO), 285 (5,
M.sup.+-CHO), 257 (1, M.sup.+-C.sub.3H.sub.5O), 209 (10,
M.sup.+-PhCO), 192 (38), 134 (60), 105 (100), 77 (50); exact mass
calculated for C.sub.20H.sub.26O.sub.3 314.1882, found
314.1887.
Preparation of
(20R)-de-A,B-8.beta.-benzoyloxy-20-(hydroxymethyl)pregnane (3).
[0026] The aldehyde 2 (1.36 g, 4.3 mmol) was dissolved in
CH.sub.2Cl.sub.2 (15 mL) and a 40% aq. n-Bu.sub.4NOH solution (5.6
mL, 5.57 g, 8.6 mmol) was added. The resulting mixture was stirred
at room temperature for 16 h, diluted with methylene chloride (30
mL), washed with water, dried (Na.sub.2SO.sub.4) and concentrated
under reduced pressure. A residue was chromatographed on silica gel
with hexane/ethyl acetate (95:5) to afford a mixture of aldehyde 2
and its 20-epimer (730 mg, 53% yield) in ca. 1:1.7 ratio (by
.sup.1H NMR).
[0027] This mixture of aldehydes (730 mg, 2.3 mmol) was dissolved
in THF (5 mL) and NaBH.sub.4 (175 mg, 4.6 mmol) was added, followed
by a dropwise addition of ethanol (5 mL). The reaction mixture was
stirred at room temperature for 30 min and it was quenched with a
saturated aq. NH.sub.4Cl solution. The mixture was extracted with
ether (3.times.30 mL) and the combined organic phase was washed
with with water, dried (Na.sub.2SO.sub.4) and concentrated under
reduced pressure. The residue was chromatographed on silica gel
with hexane/ethyl acetate (95:5.fwdarw.80:20) to give the desired,
pure (20R)-alcohol 3 (366 mg, 52% yield) as an oil and a mixture of
3 and its 20-epimer 1 (325 mg, 45% yield) in ca. 1:4 ratio (by
.sup.1H NMR).
[0028] 3: [.alpha.].sub.D+43.0 (c 0.54, CHCl.sub.3); .sup.1H NMR
(500 MHz CDCl.sub.3+TMS) .delta. 8.10-8.00 (2H, m, o-H.sub.Bz),
7.60-7.53 (1H, m, p-H.sub.Bz), 7.48-7.41 (2H, m, m-H.sub.Bz), 5.42
(1H, br s, 8.alpha.-H), 3.75 (1H, dd, J=10.6, 3.5 Hz, 22-H), 3.48
(1H, dd, J=10.6, 7.0 Hz, 22-H), 1.069 (3H, s, 18-H.sub.3), 0.973
(3H, d, J=6.7 Hz, 21-H.sub.3); .sup.13C NMR (125 MHz) .delta.
166.70 (s, C.dbd.O), 132.94 (d, p-C.sub.Bz), 131.05 (s,
i-C.sub.Bz), 129.76 (d, o-C.sub.Bz), 128.59 (d, m-C.sub.Bz), 72.28
(d, C-8), 66.95 (t, C-22), 52.94 (d), 51.77 (d), 41.96 (s, C-13),
39.56 (t), 37.78 (d), 30.75 (t), 26.67 (t), 22.71 (t), 18.25 (t),
16.76 (q, C-21), 14.14 (q, C-18); MS (EI) m/z 316 (16, M.sup.+),
301 (5, M.sup.+-Me), 299 (2, M.sup.+-OH), 298 (3,
M.sup.+-H.sub.2O), 285 (9, M.sup.+-CH.sub.2OH), 257 (5), 242 (11),
230 (8), 194 (60), 147 (71), 105 (100); exact mass calculated for
C.sub.20H.sub.28O.sub.3 316.2038, found 316.2050.
Preparation of
(20R)-de-A,B-8-benzoyloxy-20-[(p-toluenesulfonyl)oxymethyl]pregnane
(4).
[0029] To a stirred solution of the alcohol 3 (393 mg, 1.24 mmol),
DMAP (10 mg, 0.08 mmol) and Et.sub.3N (0.7 mL, 0.51 g, 5.04 mmol)
in anhydrous methylene chloride (10 mL) was added p-toluenesulfonyl
chloride (320 mg, 1.68 mmol) at 0.degree. C. The reaction mixture
was allowed to warm to room temperature (4 h) and stirring was
continued for additional 22 h. Methylene chloride (60 mL) was added
and the mixture was washed with a saturated aq. NaHCO.sub.3
solution, dried (Na.sub.2SO.sub.4) and concentrated under reduced
pressure. A residue was chromatographed on silica gel with
hexane/ethyl acetate (95:5) to afford a tosylate 4 (533 mg, 91%
yield) as a colorless oil: [.alpha.].sub.D=+15.0 (c 0.54,
CHCl.sub.3); .sup.1H NMR (500 MHz, CDCl.sub.3+TMS) .delta. 8.02
(2H, m, o-H.sub.Bz), 7.80 (2H, d, J=8.2 Hz, o-H.sub.Ts), 7.55 (1H,
m, p-H.sub.Bz), 7.44 (2H, m, m-H.sub.Bz), 7.35 (2H, d, J=8.2 Hz,
m-H.sub.Ts), 5.39 (1H, br s, 8.alpha.-H), 4.15 (1H, dd, J=9.4, 3.4
Hz, 22-H), 3.83 (1H, dd, J=9.4, 7.1 Hz, 22-H), 2.457 (3H, s,
Me.sub.Ts), 1.98 (1H, m), 0.978 (3H, s, 18-H.sub.3), 0.898 (3H, d,
J=6.6 Hz, 21-H.sub.3); .sup.13C NMR (125 MHz) .delta. 166.60 (s,
C.dbd.O), 144.87 (s, p-C.sub.Ts), 133.35 (s, i-C.sub.Ts), 132.98
(d, p-C.sub.Bz), 130.94 (s, i-C.sub.Bz), 129.97 (d, m-C.sub.Ts),
129.72 (d, o-C.sub.Bz), 128.58 (d, m-C.sub.Bz), 128.13 (d,
o-C.sub.Ts), 74.21 (t, C-22), 72.03 (d, C-8), 52.44 (d), 51.52 (d),
41.82 (s, C-13), 39.30 (t), 35.00 (d), 30.57 (t), 26.56 (t), 22.54
(t), 21.85 (q, Me.sub.Ts), 18.12 (t), 16.85 (q, C-21), 14.09 (q,
C-18); MS (EI) m/z 470 (1, M.sup.+), 365 (33, M.sup.+-PhCO), 348
(64, M.sup.+-PhCOOH), 193 (52), 176 (71), 134 (72), 105 (100);
exact mass calculated for C.sub.27H.sub.34O.sub.5S 470.2127, found
470.2091.
Preparation of (20S)-de-A,B-cholestan-8.beta.-ol (6).
[0030] Magnesium turnings (1.32 g, 55 mmol),
1-chloro-3-methylbutane (3.3 mL, 2.9 g, 27.2 mmol) and iodine (2
crystals) were refluxed in anhydrous THF (18 mL) for 10 h. The
solution of the formed Grignard reagent 5 was cooled to -78.degree.
C. and added dropwise via cannula to a solution of the tosylate 4
(348 mg, 0.74 mmol) in anhydrous TBF (5 mL) at -78.degree. C. Then
6 mL of the solution of Li.sub.2CuCl.sub.4 [prepared by dissolving
of a dry LiCl (232 mg, 5.46 mmol) and dry CuCl.sub.2 (368 mg, 2.75
mmol) in anhydrous THF (27 mL)] was added dropwise via cannula to
the reaction mixture at -78.degree. C. The cooling bath was removed
and the mixture was stirred at room temperature for 20 h and then
poured into 1M aq. H.sub.2SO.sub.4 solution (25 mL) containing ice
(ca. 100 g). The mixture was extracted with methylene chloride
(3.times.50 mL) and the combined organic layers were washed with
saturated aq. NH.sub.4Cl, saturated aq. NaHCO.sub.3, dried
(Na.sub.2SO.sub.4) and concentrated under reduced pressure. The
residue was chromatographed on silica gel with chloroform to give
alcohol 6 (149 mg, 76% yield) as a colorless oil: .sup.1H NMR (400
MHz, CDCl.sub.3+TMS) .delta. 4.07 (1H, d, J=2.2 Hz, 8.alpha.-H),
1.98 (1H, dm, J=13.1 Hz), 0.93 (3H, s, 18-H.sub.3), 0.86 (6H, d,
J=6.6 Hz, 26- and 27-H.sub.3), 0.81 (3H, d, J=6.6 Hz, 21-H.sub.3);
.sup.13C NMR (125 MHz) .delta. 69.41 (d, C-8), 56.27 (d), 52.62
(d), 41.84 (s, C-13), 40.28 (t), 39.38 (t), 35.40 (t), 34.83 (d),
33.51 (t), 28.03 (d), 27.10 (t), 23.93 (t), 22.72 (q, C-26/27),
22.63 (q, C-26/27), 22.40 (t), 18.53 (q, C-21), 17.47 (t), 13.73
(q, C-18); MS (EI) m/z 266 (7, M.sup.+), 251 (6, M.sup.+-Me), 248
(2, M.sup.+-H.sub.2O), 233 (4, M.sup.+-Me-H.sub.2O), 163 (6), 152
(11), 135 (38), 111 (100); exact mass calculated for
C.sub.18H.sub.34O 266.2610, found 266.2601.
Preparation of (20S)-de-A,B-cholestan-8-one (II).
[0031] Pyridinium dichromate (90 mg, 239 .mu.mol) was added to a
solution of the alcohol 6 (15 mg, 56 .mu.mol) and pyridinium
p-toluenesulfonate (2 mg, 8 .mu.mol) in anhydrous methylene
chloride (6 mL). The resulting suspension was stirred at room
temperature for 3.5 h. The reaction mixture was filtered through a
Waters silica Sep-Pak cartridge (2 g) that was further washed with
CHCl.sub.3. After removal of solvents ketone II (13 mg, 88% yield)
was obtained as a colorless oil: .sup.1H NMR (400 MHz,
CDCl.sub.3+TMS) .delta. 2.46 (1H, dd, J=11.5, 7.6 Hz), 0.89 (6H, d,
J=6.6 Hz, 26- and 27-H.sub.3), 0.87 (3H, d, J=6.1 Hz, 21-H.sub.3),
0.65 (3H, s, 18-H.sub.3); MS (EI) m/z 264 (41, M.sup.+), 249 (37,
M.sup.+-Me), 246 (3, M.sup.+-H.sub.2O), 231 (3,
M.sup.+-Me-H.sub.2O), 221 (50, M.sup.+-C.sub.3H.sub.7), 152 (34),
125 (100), 111 (69); exact mass calculated for C.sub.18H.sub.32O
264.2453, found 264.2454.
Preparation of (20S)-1.alpha.-hydroxy-2-methylene-19-norvitamin
D.sub.3 (V).
[0032] To a solution of phosphine oxide 7 (34 mg, 58 .mu.mol) in
anhydrous THF (450 .mu.L) at -20.degree. C. was slowly added PhLi
(1.7 M in cyclohexane-ether, 75 .mu.L, 128 .mu.mol) under argon
with stirring. The solution turned deep orange. After 30 min the
mixture was cooled to -78.degree. C. and a precooled (-78.degree.
C.) solution of ketone II (12 mg, 45 .mu.mol) in anhydrous THF
(200+100 .mu.L) was slowly added. The mixture was stirred under
argon at -78.degree. C. for 3 h and at 0.degree. C. for 18 h. Ethyl
acetate was added, and the organic phase was washed with brine,
dried (Na.sub.2SO.sub.4) and evaporated. The residue was dissolved
in hexane and applied on a Waters silica Sep-Pak cartridge (2 g).
The cartridge was washed with hexane and hexane/ethyl acetate
(99.5:0.5) to give 19-norvitamin derivative 8 (12 mg). The Sep-Pak
was then washed with hexane/ethyl acetate (96:4) to recover the
unchanged C,D-ring ketone II (7 mg), and with ethyl acetate to
recover diphenylphosphine oxide 7 (19 mg). The protected vitamin 8
was further purified by HPLC (10.times.250 mm Zorbax-Silica column,
4 mL/min) using hexane/2-propanol (99.9:0.1) solvent system. Pure
compound 8 (10 mg, 36% yield) was eluted at R.sub.V=15 mL as a
colorless oil: UV (in hexane) .lamda..sub.max 262.5, 252.5, 243.5
nm; .sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 6.21 and 5.82 (1H and
1H, each d, J=11.1 Hz, 6- and 7-H), 4.95 and 4.90 (1H and 1H, each
s, .dbd.CH.sub.2), 4.41 (2H, m, 1.beta.- and 3.alpha.-H), 2.80 (1H,
dd, J=11.9, 3.5 Hz, 9.beta.-H), 2.49 (1H, dd, J=13.2, 6.0Hz,
10.alpha.-H), 2.44 (1H, dd, J=12.7, 4.6 Hz, 4.alpha.-H), 2.32 (1H,
dd, J=13.2, 3.1 Hz, 10.beta.-H), 2.16 (1H, dd, J=12.7, 8.2 Hz,
4.beta.-H), 1.98 (2H, m), 1.84 (1H, m), 0.876 (9H, s, Si-t-Bu),
0.851 (6H, d, J=6.0 Hz, 26- and 27-H.sub.3), 0.845 (9H, s,
Si-t-Bu), 0.820 (3H, d, J=6.5 Hz, 21-H.sub.3), 0.521 (3H, s,
18-H.sub.3), 0.060, 0.046, 0.029 and 0.006 (each 3H, each s,
4.times.Si--CH.sub.3); MS (EI) m/z 628 (3, M.sup.+), 613 (1,
M.sup.+-Me), 571 (3, M.sup.+-t-Bu), 496 (63,
M.sup.+-t-BuMe.sub.2SiOH), 383 (4,
M.sup.+-t-BuMe.sub.2SiOH--C.sub.8H.sub.17), 366 (21), 234 (20), 129
(41), 75 (100); exact mass calculated for
C.sub.39H.sub.72O.sub.2Si.sub.2 628.5071, found 628.5068.
[0033] Protected vitamin 8 (10 mg, 16 .mu.mol) was dissolved in
anhydrous THF (3 mL) and a solution of tetrabutylammonium fluoride
(1 M in THF, 160 .mu.L, 160 .mu.mol) was added, followed by freshly
activated molecular sieves 4A (300 mg). The mixture was stirred
under argon at room temperature for 2 h, then diluted with 2 mL of
hexane/ethyl acetate (6:4) and applied on a Waters silica Sep-Pak
cartridge (2 g). Elution with the same solvent system gave the
crude product V that was further purified by HPLC (10.times.250 mm
Zorbax-Silica column, 4 mL/min) using hexane/2-propanol (9:1)
solvent system. Analytically pure 2-methylene-19-norvitamin V (3.3
mg, 52% yield) was collected at R.sub.V=32 mL as a colorless oil:
UV (in EtOH) .lamda..sub.max 261.5, 251.5, 243.5 nm; .sup.1H NMR
(500 MHz, CDCl.sub.3+TMS) .delta. 6.36 and 5.88 (1H and 1H, each d,
J=11.3 Hz, 6- and 7-H), 5.11 and 5.09 (each 1H, each s,
.dbd.CH.sub.2), 4.47 (2H, m, 1.beta.- and 3.alpha.-H), 2.85 (1H,
dd, J=13.4, 4.6 Hz, 10.beta.-H), 2.81 (1H, br d, J=13.9 Hz,
9.beta.-H), 2.58 (1H, dd, J=13.2, 3.7 Hz, 4.alpha.-H), 2.33 (1H,
dd, J=13.2, 6.1 Hz, 4.beta.-H), 2.29 (1H, dd, J=13.4, 8.4 Hz,
10.alpha.-H), 1.99 (2H, m), 1.86 (1H, m), 0.867 (6H, d, J=6.6 Hz,
26- and 27-H.sub.3), 0.839 (3H, d, J=6.5 Hz, 21-H.sub.3), 0.547
(3H, s, 18-H.sub.3); MS (EI) m/z 400 (100, M.sup.+), 385 (5,
M.sup.+-Me), 382 (16, M.sup.+-H.sub.2O), 367 (6,
M.sup.+-Me-H.sub.2O), 349 (3, M.sup.+-Me-2H.sub.2O), 315 (46), 287
(56, M.sup.+-C.sub.8H.sub.17), 269 (52), 247 (42); exact mass
calculated for C.sub.27H.sub.44O.sub.2 400.3341, found
400.3346.
Preparation of (20S)-1.alpha.-hydroxy-2.alpha.-methyl-19-norvitamin
D.sub.3 (Ia) and
(20S)-1.alpha.-hydroxy-2.beta.-methyl-19-norvitamin D.sub.3
(Ib).
[0034] Tris(triphenylphosphine)rhodium (I) chloride (3.5 mg, 3.8
.mu.mol) was added to dry benzene (2.5 mL) presaturated with
hydrogen. The mixture was stirred at room temperature until a
homogeneous solution was formed (ca. 45 min). A solution of vitamin
V (1.8 mg, 4.5 .mu.mol) in dry benzene (400+400 .mu.L) was then
added and the reaction was allowed to proceed under a continuous
stream of hydrogen for 3 h. Benzene was removed under vacuum, the
residue was redissolved in hexane/ethyl acetate (1:1) and applied
on a Waters silica Sep-Pak cartridge (2 g). A mixture of 2-methyl
vitamins was eluted with the same solvent system. The compounds
were further purified by HPLC (10.times.250 mm Zorbax-Silica
column, 4 mL/min) using hexane/2-propanol (9:1) solvent system. The
mixture of 2-methyl-19-norvitamins Ia and Ib gave a single peak at
R.sub.V=34 mL. Separation of both epimers was achieved by
reversed-phase HPLC (10.times.250 mm Chromegabond C18 column, 3
mL/min) using methanol/water (9:1) solvent system. 2.beta.-Methyl
vitamin Ib (280 .mu.g, 15% yield) was collected at R.sub.V=47 mL
and its 2.alpha.-epimer Ia (382 .mu.g, 21% yield) at R.sub.V51
mL.
[0035] Ia: UV (in EtOH) .lamda..sub.max 260.5, 250.5, 242.5 nm;
.sup.1H NMR (500 MHz, CDCl.sub.3+TMS) .delta. 6.37 and 5.82 (1H and
1H, each d, J=11.1 Hz, 6- and 7-H), 3.96 (1H, m, w/2=14 Hz,
1.beta.-H), 3.61 (1H, m, w/2=20 Hz, 3.alpha.-H), 2.80 (2H, br m,
9.beta.- and 10.alpha.-H), 2.60 (1H, dd, J=13.0, 4.5 Hz,
4.alpha.-H), 2.22 (1H, br d, J12.8 Hz, 10.beta.-H), 2.13 (1H,
.about.t, J=13.0 Hz, 4.beta.-H), 1.133 (3H, d, J=6.8 Hz,
2.alpha.-CH.sub.3), 0.866 (6H, d, J=6.6 Hz, 26- and 27-H.sub.3),
0.833 (3H, d, J=6.4 Hz, 21-H.sub.3), 0.530 (3H, s, 18-H.sub.3); MS
(EI) m/z 402 (100, M.sup.+), 387 (4, M.sup.+-Me), 384 (7,
M.sup.+-H.sub.2O), 369 (3, M.sup.+-Me-H.sub.2O), 317 (24), 289 (60,
M.sup.+-C.sub.8H.sub.17), 271(33), 259 (40), 247 (63); exact mass
calculated for C.sub.27H.sub.46O.sub.2 402.3498, found
402.3496.
[0036] Ib: UV (in EtOH) .lamda..sub.max 260.5, 250.0, 242.0 nm;
.sup.1H NMR (500 MHz, CDCl.sub.3+TMS) .delta. 6.26 and 5.87 (1H and
1H, each d, J=11.3 Hz, 6-H and 7-H), 3.90 (1H, m, w/2=14 Hz,
3.alpha.-H), 3.50 (1H, m, w/2=26 Hz, 1.beta.-H), 3.08 (1H, dd,
J=12.6, 4.3 Hz, 10.beta.-H), 2.80 (1H, dd, J=12.5, 3.8 Hz,
9.beta.-H), 2.43 (1H, br d, J=ca. 14 Hz, 4.alpha.-H), 2.34 (1H, dd,
J=13.9, 3.0 Hz, 4.beta.-H), 1.143 (3H, d, J=6.8 Hz,
2.beta.-CH.sub.3) 0.867 (6H, d, J=6.6 Hz, 26- and 27-H.sub.3),
0.839 (3H, d, J=6.5 Hz, 21-H.sub.3), 0.543 (3H, s, 18-H.sub.3); MS
(EI) m/z 402 (100, M.sup.+), 387 (8, M.sup.+-Me), 384 (8,
M.sup.+-H.sub.2O), 369 (5, M.sup.+-Me-H.sub.2O), 317 (42), 289 (88,
M.sup.+-C.sub.8H.sub.17), 271 (52), 259 (55), 247 (66); exact mass
calculated for C.sub.27H.sub.46O.sub.2 402.3498, found 402.3486.
##STR3## ##STR4## Biological Activity of
(20S)-1.alpha.-Hydroxy-2.alpha.-Methyl and
2.beta.-Methyl-19-nor-vitamin D.sub.3
[0037] The 2.beta.-methyl-(20S)-1.alpha.-hydroxyvitamin D.sub.3
does not bind to the vitamin D receptor, while the
2.alpha.-methyl-(20S)-1.alpha.-hydroxyvitamin D.sub.3 binds the
receptor but at a 100-fold less affinity than
1.alpha.,25-dihydroxyvitamin D.sub.3 (1,25-(OH).sub.2D.sub.3) (FIG.
1). The absence of a 25-hydroxyl group in these compounds is
largely responsible (see Eisman, J. A. and H. F. DeLuca, Steroids
30, 245-257, 1977) for this diminished activity. Importantly, the
2.alpha.-methyl derivative is superior to the 2.beta.-methyl analog
in binding to the receptor.
[0038] Surprisingly, FIG. 2 illustrates
(20S)-1.alpha.-hydroxy-2.alpha.-methyl-19-nor-vitamin D.sub.3 is
almost as potent as 1,25-(OH).sub.2D.sub.3 on HL-60
differentiation, making it an excellent candidate for the treatment
of psoriasis and cancer, especially against leukemia, colon cancer,
breast cancer and prostate cancer. In addition, due to its
relatively high cell differentiation activity, this compound
provides a therapeutic agent for the treatment of various skin
conditions including wrinkles, lack of adequate dermal hydration,
i.e. dry skin, lack of adequate skin firmness, i.e. slack skin, and
insufficient sebum secretion. Use of this compound thus not only
results in moisturizing of skin but also improves the barrier
function of skin. The 2.beta. derivative is 100 times less active
than 1,25(OH).sub.2D.sub.3 making it less effective in these
areas.
[0039] The data in Table 1 show that
(20S)-1.alpha.-hydroxy-2.beta.-methyl-19-nor-vitamin D.sub.3 has
high activity relative to that of 1,25-(OH).sub.2D.sub.3, the
natural hormone, in stimulating intestinal calcium transport. Also,
(20S)-1.alpha.-hydroxy-2.beta.-methyl-19-nor-vitamin D.sub.3 has
significant activity in stimulating intestinal calcium transport,
and its activity is about the same as 1,25-(OH).sub.2D.sub.3.
[0040] The data in Table 1 also demonstrate that
(20S)-1.alpha.-hydroxy-2.alpha.-methyl-19-nor-vitamin D.sub.3 has
higher bone calcium mobilization activity, as compared to
1,25-(OH).sub.2D.sub.3. Also,
(20S)-1.alpha.-hydroxy-2.beta.-methyl-19-nor-vitamin D.sub.3 has
significant bone calcium mobilization activity, and its activity is
about the same as 1,25-(OH).sub.2D.sub.3.
[0041] A very important feature of these analogs is that they bind
poorly or not at all to the vitamin D receptor, while having
biological activity either higher than or equal to
1,25-(OH).sub.2D.sub.3. This suggests that these analogs are pro
drugs. That is, they are probably activated in vivo by being
25-hydroxylated. Once 25-hydroxylated, they are then able to bind
the vitamin D receptor and provide activity. These results suggest
that these compounds might be preferable to the final drug in that
they are slowly activated within the body providing a more
controlled and prolonged activity.
[0042] The data in Table 1 thus illustrate that
(20S)-1.alpha.-hydroxy-2.alpha.-methyl-19-nor-vitamin D.sub.3 may
be characterized as having significant and very potent calcemic
activity which is greater than 1,25-(OH).sub.2D.sub.3, and that
(20S)-1.alpha.-hydroxy-2.beta.-methyl-19-nor-vitamin D.sub.3 also
has significant and very potent calcemic activity that is about the
same as 1,25-(OH).sub.2D.sub.3.
[0043] Competitive binding of the analogs to the porcine intestinal
receptor was carried out by the method described by Dame et al.
(Biochemistry 25, 4523-4534, 1986).
[0044] The differentation of HL-60 promyelocytic into monoctyes was
determined as described by Ostrem et al. (J. Boil. Chem. 262,
14164-14171, 1987).
[0045] Intestinal calcium transport was determined as described by
Perlman et al. (Biochemistry 29, 190-196, 1990).
Interpretation of Data
[0046] The in vivo tests to determine serum calcium of rats on a
low calcium diet provides an insight to osteoblastic or bone
activity of (20S)-1.alpha.-hydroxy-2.alpha.-methyl-19-nor-vitamin
D.sub.3 and (20S)-1.alpha.-hydroxy-2.beta.-methyl-19-nor-vitamin
D.sub.3. The data in Table 1 show that
(20S)-1.alpha.-hydroxy-2.alpha.-methyl-19-nor-vitamin D.sub.3 is
significantly more potent than 1,25(OH).sub.2D.sub.3 in raising
calcium in the plasma via the stimulation of the osteoblasts. At
the same time, the activity of
(20S)-1.alpha.-hydroxy-2.alpha.-methyl-19-nor-vitamin D.sub.3 on
intestinal calcium transport is also significantly greater than
that of 1,25-(OH).sub.2D.sub.3 (Table 1). Therefore, these data
show (20S)-1.alpha.-hydroxy-2.alpha.-methyl-19-nor-vitamin D.sub.3
to have significant and very potent activity on bone which is
higher than 1,25(OH).sub.2D.sub.3.
[0047] The data in Table 1 also show that
(20S)-1.alpha.-hydroxy-2.beta.-methyl-19-nor-vitamin D.sub.3 is
only slightly less potent than 1,25(OH).sub.2D.sub.3 in raising
calcium in the plasma calcium via the stimulation of the
osteoblasts. At the same time, the activity of
(20S)-1.alpha.-hydroxy-2.beta.-methyl-19-nor-vitamin D.sub.3 on
intestinal calcium transport is about the same as that of
1,25-(OH).sub.2D.sub.3 (Table 1). Therefore, these data show
(20S)-1.alpha.-hydroxy-2.beta.-methyl-19-nor-vitamin D.sub.3 to
have significant and very potent activity on bone about equal to
1,25(OH).sub.2D.sub.3.
[0048] The compounds Ia and Ib exhibit a desired, and highly
advantageous, pattern of biological activity. These compounds are
characterized by relatively high intestinal calcium transport
activity, as compared to that of 1.alpha.,25-dihydroxyvitamin
D.sub.3, while also exhibiting relatively high activity, as
compared to 1.alpha.,25-dihydroxyvitamin D.sub.3, in their ability
to mobilize calcium from bone. Hence, these compounds are highly
specific in their calcemic activity. Their activity on mobilizing
calcium from bone and either high or normal intestinal calcium
transport activity allows the in vivo administration of these
compounds for the treatment of metabolic bone diseases where bone
loss is a major concern. Because of their calcemic activity on
bone, these compounds would be preferred therapeutic agents for the
treatment of diseases where bone formation is desired, such as
osteoporosis, especially low bone turnover osteoporosis, steroid
induced osteoporosis, senile osteoporosis or postmenopausal
osteoporosis, as well as osteomalacia.
[0049] (20S)-1.alpha.-Hydroxy-2.alpha.-methyl-19-nor-vitamin
D.sub.3 and (20S)-1.alpha.-hydroxy-2.beta.-methyl-19-nor-vitamin
D.sub.3 are much less active than 1,25(OH).sub.2D.sub.3 in binding
to the vitamin D receptor (FIG. 1), and they are both also only
slightly less active than 1,25-(OH).sub.2D.sub.3 in causing
differentiation of the promyelocyte, HL-60, into the monocyte (FIG.
2). This result suggests that both
(20S)-1.alpha.-hydroxy-2.alpha.-methyl-19-nor-vitamin D.sub.3 and
(20S)-1.alpha.-hydroxy-2.beta.-methyl-19-nor-vitamin D.sub.3 will
be very effective in psoriasis because they have direct cellular
activity in causing cell differentiation and in suppressing cell
growth. It also indicates that they both will have significant
activity as an anti-cancer agent, especially against leukemia,
colon cancer, breast cancer and prostate cancer, as well as against
skin conditions such as dry skin (lack of dermal hydration), undue
skin slackness (insufficient skin firmness), insufficient sebum
secretion and wrinkles. These results also illustrate that
(20S)-1.alpha.-hydroxy-2.alpha.-methyl-19-nor-vitamin D.sub.3 and
(20S)-1.alpha.-hydroxy-2.beta.-methyl-19-nor-vitamin D.sub.3 are
both excellent candidates for numerous human therapies and that
they may be useful in a number of circumstances in addition to
cancer and psoriasis such as autoimmune diseases.
[0050] Male, weanling Sprague-Dawley rats were placed on Diet 11
(0.47% Ca) diet+AEK for 11 days, followed by Diet 11 (0.02% Ca)
+AEK for 31 days. Dosing (i.p.) began 7 days prior to sacrifice.
Doses were given on a daily basis, 24 hours apart. The first 10 cm
of the intestine was collected for gut transport studies and serum
was collected for bone Ca mobilization analysis. The results are
reported in Table 1. TABLE-US-00001 TABLE 1 Response of Intestinal
Calcium Transport and Serum Calcium (Bone Calcium Mobilization)
Activity to Chronic Doses of 1,25-(OH).sub.2D.sub.3 and
(20S)-1.alpha.-Hydroxy- 2.alpha.-methyl-19-nor-vitamin D.sub.3 and
(20S)-1.alpha.-Hydroxy-2.beta.-methyl-19-nor-vitamin D.sub.3 Amount
Ca transport S/M Serum Ca Compound (pmol/day) (mean .+-. SEM) (mean
.+-. SEM) none (control) 0 4.5 .+-. 0.40 4.4 .+-. 0.07
1.alpha.,25-(OH).sub.2D.sub.3 130 5.3 .+-. 0.42 5.0 .+-. 0.05 260
6.5 .+-. 0.84 5.5 .+-. 0.16
(20S)-1.alpha.-(OH)-2.alpha.-methyl-19-nor-D.sub.3 130 8.6 .+-.
0.90 10.0 .+-. 0.20 260 6.7 .+-. 0.68 12.7 .+-. 0.15
(20S)-1.alpha.-(OH)-2.beta.-methyl-19-nor-D.sub.3 130 6.8 .+-. 0.73
4.8 .+-. 0.04 260 5.7 .+-. 0.45 5.1 .+-. 0.04 *The above data are
the average and standard error (SE) from 5 animals.
[0051] For treatment purposes, the compounds of this invention
defined by formula Ia and Ib may be formulated for pharmaceutical
applications as a solution in innocuous solvents, or as an
emulsion, suspension or dispersion in suitable solvents or
carriers, or as pills, tablets or capsules, together with solid
carriers, according to conventional methods known in the art. Any
such formulations may also contain other
pharmaceutically-acceptable and non-toxic excipients such as
stabilizers, anti-oxidants, binders, coloring agents or emulsifying
or taste-modifying agents.
[0052] The compounds may be administered orally, topically,
parenterally or transdermally. The compounds are advantageously
administered by injection or by intravenous infusion or suitable
sterile solutions, or in the form of liquid or solid doses via the
alimentary canal, or in the form of creams, ointments, patches, or
similar vehicles suitable for transdermal applications. Doses of
from 0.01 .mu.g to 100 .mu.g per day of the compounds are
appropriate for treatment purposes, such doses being adjusted
according to the disease to be treated, its severity and the
response of the subject as is well understood in the art. Since the
compounds exhibit specificity of action, each may be suitably
administered alone, or together with graded doses of another active
vitamin D compound--e.g. 1.alpha.-hydroxyvitamin D.sub.2 or
D.sub.3, or 1.alpha.,25-dihydroxyvitamin D.sub.3--in situations
where different degrees of bone mineral mobilization and calcium
transport stimulation is found to be advantageous.
[0053] Compositions for use in the above-mentioned treatments
comprise an effective amount of the
(20S)-1.alpha.-hydroxy-2.alpha.-methyl-19-nor-vitamin D.sub.3 or
(20S)-1.alpha.-hydroxy-2.beta.-methyl-19-nor-vitamin D.sub.3 as
defined by the above formula Ia and Ib as the active ingredient,
and a suitable carrier. An effective amount of such compound for
use in accordance with this invention is from about 0.01 .mu.g to
about 100 .mu.g per gm of composition, and may be administered
topically, transdermally, orally or parenterally in dosages of from
about 0.01 .mu.g/day to about 100 .mu.g/day.
[0054] The compounds may be formulated as creams, lotions,
ointments, topical patches, pills, capsules or tablets, or in
liquid form as solutions, emulsions, dispersions, or suspensions in
pharmaceutically innocuous and acceptable solvent or oils, and such
preparations may contain in addition other pharmaceutically
innocuous or beneficial components, such as stabilizers,
antioxidants, emulsifiers, coloring agents, binders or
taste-modifying agents.
[0055] The compounds are advantageously administered in amounts
sufficient to effect the differentiation of promyelocytes to normal
macrophages. Dosages as described above are suitable, it being
understood that the amounts given are to be adjusted in accordance
with the severity of the disease, and the condition and response of
the subject as is well understood in the art.
[0056] The formulations of the present invention comprise an active
ingredient in association with a pharmaceutically acceptable
carrier therefore and optionally other therapeutic ingredients. The
carrier must be "acceptable" in the sense of being compatible with
the other ingredients of the formulations and not deleterious to
the recipient thereof.
[0057] Formulations of the present invention suitable for oral
administration may be in the form of discrete units as capsules,
sachets, tablets or lozenges, each containing a predetermined
amount of the active ingredient; in the form of a powder or
granules; in the form of a solution or a suspension in an aqueous
liquid or non-aqueous liquid; or in the form of an oil-in-water
emulsion or a water-in-oil emulsion.
[0058] Formulations for rectal administration may be in the form of
a suppository incorporating the active ingredient and carrier such
as cocoa butter, or in the form of an enema.
[0059] Formulations suitable for parenteral administration
conveniently comprise a sterile oily or aqueous preparation of the
active ingredient which is preferably isotonic with the blood of
the recipient.
[0060] Formulations suitable for topical administration include
liquid or semi-liquid preparations such as liniments, lotions,
applicants, oil-in-water or water-in-oil emulsions such as creams,
ointments or pastes; or solutions or suspensions such as drops; or
as sprays.
[0061] For asthma treatment, inhalation of powder, self-propelling
or spray formulations, dispensed with a spray can, a nebulizer or
an atomizer can be used. The formulations, when dispensed,
preferably have a particle size in the range of 10 to 100.mu..
[0062] The formulations may conveniently be presented in dosage
unit form and may be prepared by any of the methods well known in
the art of pharmacy. By the term "dosage unit" is meant a unitary,
i.e. a single dose which is capable of being administered to a
patient as a physically and chemically stable unit dose comprising
either the active ingredient as such or a mixture of it with solid
or liquid pharmaceutical diluents or carriers.
* * * * *