Note: Descriptions are shown in the official language in which they were submitted.
9~35~
-- 1 --
Description
24,24-difluoro-25-hydroxycholecalciferol
Technical Field
This invention relates to a compound which is chara~terized
S by vitamin D-like activity.
More specifically this invention relates to a derivative of
vitamin D3.
Vitamin D3 is a well-known agent for the control of calcium
and phosphorous homeostasis. In ~he normal animal or human
this compound is known to stimulate intestinal calciurn transport
and boné-calcium mobilization ànd is effective in preventing rickets.
It is also now well known that to be effective vitamin D3 must
be converted in vivo to its hydroxylated forms. Por exam~le, the
vitamin is first hydroxylated in the liver to form 25-hydroxy
vitamin D3 and is further hydroxylated in the kidney to produce
1~,25-dihydroxy vitamin D3 or 24,25-dihydroxy vitamin D3. The
lcy-hydroxylated form of the vitamin is generally considered to be
the physiologically active or hormonal form of the vitamin and to
be responsible for what are termed the vitamin D-like activities, L
such as increasing intestinal absorption o~ calcium and phosphate,
mobilizing bone mineral, and retaining calcium in the kidneys.
Background Art
P~eferences to various of vitamin D derivatives are extant in
the patent and other literature. See, for example, U. S~ Patent
Numbers: 3,565,924 directed to 25-hydroxycholecalciferol;
3,697,559 directed to 1,25-dihydroxy cholecalci~erol; 3J 741,996
directed to 1~-hydroxycholecalciferol; 3,907,843 directed to la!~
hydroxyergocalciferol; 3,715,374 directed to 24,25-dihydroxy-
cholecalciferol; 3,739,001 directed to 25,26-dihydroxycholecal-
3B5~
ciferol; 3, 786, 062 directed to 22-dehydro-25-hydroxycholecalci-
ferol; 3~ 847, 955 directed to 1, 24, 25-trihydroxycholecalciferol;
3, 906, 014 directed to 3-deoxy- l~-hydroxycholecalciferol; 4~ 069, 321
directed to the preparation of various side chain fluorinated vitamin
5 D3 derivatives and side chain fluorinated dihydrotachysterol3
analogs.
Disclosure of Invention
.
A new derivative of vitamin D3 has now been found which
expresses excellent vitamin D-like activity and which, therefore,
10 could sexve as a substitute for vitamin D3 in its variou~ known
applications and would be useful in the treatment of various diseases
such as osteomalacia, osteodystrophy and hypoparathyroidism.
This derivative has been identified as 24,24-difluoro-25-
hydroxycholecalciferol (24,24-difluoro-25-hydroxy vitamin D3 or
24 F2,25-OH D3).
Best Mode for Carrying Out the Invention
The compound of this invention was synthesized in accordance
with the following description and abbreviated schematic:
o ~flc
Cot~ H ~ ~
l~o~co ~0~ 1
t . Z~ 3
. .
Aco ACO Aco
6 5 Y
~o H ~1/
5(
- 4 -
Cholenic acid 1 was treated with dihydropyran in a suitable
organic solvent (CH2C12) at 0~ in the presence of P-toluene sulfonic
acid and then with IN Na OH in ethanol at ~0 to form the cholenic
acid tetrahydropyranyl ether (protection of the hydroxyl group in
the A ring). ~his compound W2S then treated with an excess of
CH3Li in tetrahydrofuran (THF ) - ethyl ether at 0 C for four hours
after which the protective tetrahydropyranyl group was removed by
treatment with ~ -TsOH in CH2C12 - methanol for 24 hours at
20C, Subsequent acetylation (Ac20-pyridine-CH2Cl2, 200, 24
hours)gave the methylketone 2 (mp 148-151, ~2. 12 (3H,s,C-25),~
m/e 354 (M-60)) (Yield = 6% overall ~rom _).
The methylketone 2 was refluxed for seven hours in acetic
anhydride in the presence of ~ -~sOH (enolacetylation) to give the
diacetate3(mplO9-llOo, &'5.02(1H, m, C-23), 1.90(3H,s,C-25)
m/e 396 (M-60)). The diacetate was then converted tO the difluoro-
cyclopropane 4 by heating with sodium chlorodifluoro-acetate in
diglyme at 170~ for 0. 5 hours, Yield, !34~; mp 112-llS, 5. 38
(lH,m, C-6), 4. 60 (lH, m, C-3), 2. 05 (3H, s,24-OAc), 2. 02 (3H, s,
3-OAc), 1. 60 (3H, m, C-26), m/e 446 (M-60)).
~reatment of 4 with LiOH in THF-methanol-water at 20 C
for two hours followed by:acetylation (AC20-pyridine-CH2C12, ~0,
24 hr. ) gave, after chromatography on silica gelJ the difluoro-
ketone 5 (9.3%yield, mp 135-136-5, S2.26 (3H,t,~F = 1 Hz,C-26),
m/e 404 (M-60)). The difluoroketone was obtained in a mixture
with the 23(E)- and the 23(Z)- conjugated ketone, with the difluoro-
ketone being separated by chromatography on silica gel).
'rhe difluoroketone 5 was reacted with an excess o~ CH3MgI
in ethyl ether at 0 C for 15 minutes and was then acetaylated
(AC20-pyridine-CH2C12, 200, 20 hr. ) to furnish the 25-ca~binol,
38~;~
-- 5 -
6, in 85%yield (mp 163-164-5, Sl.~28 (6H,s,C-26,27), m/e 420
(M-60)). The carbinol, 6, was allylically brominated by reacting
it with N-bromo-succinimide in refluxing CC14 for 25 minutes,
The brominated compound was then dehydrobrominated by treatment
S with s-collidine in refluxing xylene for 15 minutes to give a mixture
of the 4, 6-diene and the S, 7-diene, 7. The 5, 7-diene (il max 263,
272, 282 and 292 nm, m/e 4 L9 (M-59)) was isolated in 28~C yield,
by treatment with p TsOH in acetone at 200 for lS hours followed
by preparative thin-layer chromatography (benzene-ethyl acetate
~15:1), 3 times); The recovered 5,7:diene was saponified b~
treatment with S~ KOH-methanol at 200 C for 15 hours and then
irradiated (Hanovia high pressure quartz mercury vapor lamp,
model 654A36; 200 W) in a mixture of ethanol and benzene for
2, 5 minutes at 0 C to give the previtamin 8 in solution. The
lS irradiated solution was refluxed for one hour and then fractionated
with thin-layer chromatography (silicagel, benzene-ethyl acetate,
(5:1), 3 times) and high pressure liquid chromatography (Zorbax
SLI" 25 cm x 2. 1 mm i. d., available through the DuPont Co.,
Willmin~ton, Delaware) CH2C12) to yield 24, 24-difluoro-25-hydroxy
20 vitaminD3, 9, ( A max264nm, ,~min228nm, m/e436(M+),
421, 418, ~03, 377, 271, 253, 136, 118).
9~
-6-
Statement
If it is desired for certain purposes the acetylated 5,
7-diene compound 7, after recovery as described above, can be
szponified by well known means (5% KOH in MeOH, 20, 15 hours)
to convert the acetoxy group at the 3-position to hydroxyl.
The compound thus obtained, 24,24-difluoro-cholest-5,7-dien-
3,25-diol, has the following characteristics:
W : ~ (EtOH), 262(sh), 271, 282, 293 nm
max
Mass: m/e 436 (M~)
NMR (CDC13): ~ 0.82(3H, s, 18-Me), 0.95 (3H, s,
l9-Me), 1.28 (6H, s, 26,27-Me), 3.5 (lH, m,
3-H), 5.5 (2H, m, 6-H, 7-H~
Also, if desired, the previtamin 8 can be recovered by
evaporation or solvent at 5 and the subseguent chromatograph
on silica gel, and subsequently converted to the vitamin.
Biological Activity
Male weanling rats were housed in hanging wire cages and
~ed ad libitum the low calcium, vitamin D deficient diet des-
cribed by Suda et al ~J. Nutr. 100, 1049 (1970)) for three weeks
prior to their use in the following assays.
I Intestinal Calcium Transport
Groups of five or six rats fed as above were given respect-
ively a single dose (650~ mole) of either 24,24-difluoro-25-
hydroxy vitamin D3 (24 F2,25-OH2) or 25-hydroxy vitamin D3
~25-OHD3) dissolved in 0~05 ml of 95% ethanol intrajugularly 8,
23 or 30 hours prior to sacrifice. The rats in the control
sroup were given the ethanol vehicle only. They were then killed
by decapitation after the respective times prescribed and their
1 30 duodena were use~ to measure the intestinal calcium transport
.
s~
6a-
activity in accordance with the techniques of Martin and DeLuca
(Am. J. Physiology 216, 1351 (1969)). Results are shown in
the table below.
~L , .
... . . . . . ... ..
-- 7 --
l~able 1
Compound !; _4~Ca sexosal/~5Ca rnucosal
Given 8 h 23 h 30 h
Control 2, 7~0, 2*a~ 2. 5+0. 4a) 2. 6 tO. 2a~
24~2,25~0HD3 6. 6_1, 2b) 5; 9~ 6b) 8 2-- b)
25-OHD 5, 0+0. 7C) 5. 5+0. 8~) 5. 7+1, 4c~ _
3 ~
.
Signlficance b) & c) from b) & c) from b) & c) from
of difference a) a) - a)
~ ~ O. 001 ~ ~ O. 001 - 1~ C 0. 001
b) from c) b) from c) b) from c)
~C 0. 025 - N.S. ~ 0, 05
*Standard deviation of the mean
To show the effect of small doses of 24F2, 25-OHD3 on
intestinal calcium transport rats fed the low calcium diet as
15 indicated above, in groups of 5 or 6 were given a single dose
of 29:F2~ 2S-OHD3 or 2~-OHD3 dissolved in 0. 05 ml of 95%
ethanol intrajugularly. Rats in the control group received the
vehicle alone, Either 20 hours or 168 hours after receiving
the dose the rats were killed and their duodena were used to
20 measure the intestinal calcium transport activity in accordance
with the Martin and DeLuca procedure referenced above. Results
~re ~hown in T--~le Z below.
.
- 8
Table 2
Compound Dosage 45Ca serosal/45Ca mucosal
Given ( ~ mole/rat) 20 h 168 h
____ .. __ ~
Control 1. 5+0, 5*a)2, 0+0. 4aJ,
24F2, 25-OHD3 6. 5 1. 9+0. 6b) 2. 1 tO. lb)
32. 5 1. 9~ o. 3b)3. 7 ~0, gc~
25-OHD '6. 5 1. 8-~0. 4b) , 2. 1~, 2b)
- ......... .... : - -32. 5.. : .. - - 2, 2:~0, 6b) 3. 8 ~0. 7
.
Significance . b) from a) b) from a)
of difference . N. S. ' N. S.
. . . c) from a) -
~CO.OS
. d) from a)
.. . ........ . . ........ ~ C 0. 001
15 *Standard derivative of the mean
.
Serum Calc'iu'm''C'o'n'cë-ntr'atio'n'
.
~ ats fed as indicated above were divided into groups of six
rats each. The rat~ in one group were given a single dose of 650
mole of 24F2, 25-OHD3, in the second gr ,oup a dose of 650 ~ mole
20 of 25-OHD3 (in each case the vitamin D3 derivative was dissolved
- in 0. 05 ml of 95% ethanol) while the third group (control) was
given the vehicle alone. The materials were administered intra-
jugul~rly either 8 or 29 hours prior to sacrifice.
The :rats were killed by decapitation after the indicated times,
25 the blood collected and centrifuged to obtain the serum. The
serum (O. 1 ml) was mixed with 1. 9 ml of 0. 1% NaCC solution and
the calcium concentration was measured with an atomic absorption
spectraphotometer (Perkin-Elmer Model HO-214). Results are
shown in the table below.
o
Table 3
Serum calcium (mg/100 ml)
Compound Given 8 h 24 h
_ ~ . .. .
Control 7. 7~0. 2*a)3. 9+0. 1 ~
24F , 25-OHD ~ b~5. 2t 0. 2b)
2 3 ~ b)5. 3+0.2b)
Significance b)frorn a)b)from a)
of difference - - --t3<0. 001~0~ 001
*Standard derivative of the mean
10 Antirachitic Activity
Male weanling rats (Holtzman Co., Madison,- Wisconsin)
maintained in hanging wire cages were fed, in groups of six the
low phosphorous diet described in Am. J. Physiol 204, 833
(1963) (Guroff, DeLuca and Steenbock) and were simultaneously
given e^ither 24F2,25-OHD3 dissolved in 0.1 ml ethanol/propylene
glycol ( /95, v/v) subcutaneously every day for two weeks. Rats
in the control group were fed in like manner but received only
the vehicle subcutaneously.
Twenty-four hours after receiving the last subcutaneous
20 dose the rats were killed by decapitation and their duodena were
used for measurement of intestinal calcium transport as
described above. Their radii and ulnae were removed for
measurment of widened epiphyseal plates, and femurs for
determination of ash content (femurs were dried to constant
25 weight and then ashed in a muffle furnace at 650~ C for 8 hours. )
Results obtained are shown in the table below
- 10-
u c~ ~ o o O O â~ O
~ r~ o oo ~i oo ~ ~ ~ V ~ ~ V
¢-- -- ~--~--~
~o ~ ~o~o ~o
1 ~o ~ o
I~ ~ ~ ~ ~ ~
~l ~ o~ ~ ~ --
. c~ u) ~
~ . -- ~
P. . ~
E V ^V
o+~ olo+l ~ '
CY~ --i o _, o A 'O O
,_ _ ~ _l
, ~ A o'~ ~:1 0 o:~l
., ~ ~ 0 ~^'Y
._ ~ C~ ~ ~
. E~
Cd ~ ~ U~ ln ~' ~o
~ . . o
~ . . ~
~ ~ 3 . ~ ~ ~
Male weanling rats were fed the low phosphorous diet
referenced above and then divided into groups of five or six
rats each, The rats in each group were given respectively a
single dose (as shown in the table below) of either 24F2, 25-OHD3
or 25-OHD3 dissolved in 0. 05 ml of 95~ ethanol intrajugularly,
Rats in the control group received the ethanol vehicle above,
168 hours' after receiving the indicated dosage the rats were
killed by decapitation, the blood of each group was collected --
and the radii and ulnae were removed to determine antirachitic
activity in accordance with the rat line test (U, S. Pharmacopoeia,
15th Pcev., Mack Publishing Co,, Easton, Pa,, 1955, p, 889),
~he blood was centrifuged immediately after collection to yield
the serum. 'rhe inorganic phosphorous in the serum was
determined by the method of Chen et al (Anal, Chem., 28,
1756, (1956)).
Results obtained are shown in the table below,
- 12 -
Table 5
Compound Dosage Serum lnorganicLine Test Score
Given ( g mole) Phvsphorous(Unit)
(mg/l_0 ml) __
Control 1. 6~0. 2*a~ 0
24F2, 25-OHD3 130 3. 0+0. 2b)d~, 4~1, 4
325 3.5t0.4) 5
25-OHD3 130 3, 3+0. lb) 2. 6+0, 6 )
325 ~ 3. 6+0. 4b) 3, 5+0, 6
Significance b)from a)b)from a)
of difference - - - - p ~0. 001~ cQ 025
*Standard deviation of the mean
To determine the antLrachitic activity in response to a daily
dose of 24F2,25-OHD3 rats were fed the low phosphorous diet
15 referenced above for three weeks. They were then given either
24~2,25-OHD3 or 25-OHD3 (in each case, 65 ~ mole dissolved
in 0.1 ml ethanol/propylene glycol ( /95, V/v)) subcutaneously
every day for 8 days while being maintained on the same diet
(9 rats were in each group). The rats in the control group (4 rats)
20 were given only the ethanol/propylene glycol vehicle in ~he same
manner.
Twenty-four hours after receiving the last dose they were
killed and their radii and ulnae were removed and used for
measuring antirachitic activity (rat line test, supra) while the
25 femurs were removed and ashed as described above.
Results obtained are shown in the table below.
- 13 -
Table 6
CompoundLine Test ~otal Femur Ash P~rcent Ash
GivenScore (Unit) (mg) (%)
. _ _
Control 0 23, 80~3, 98*a) 19. 5+3. 4a)
24F2J 25-OHD3~5 37, 03+4, 94b)26, ~ 8b)
25 -OHD3 - >~ 5 -38. 56+5, ~gb)~ b)
Significance b) from a)b) from a)
of difference - - - - - - - eC 0. 001~c 0, 005
*Standard deviation of the mean
It is evident from the foregoing data that 24, 24-difluoro-25-
hydroxy vita.min D3 exhibits pronounced vitamin D-like activity
and appears to be wholly as effective in this regard as 25-hydroxy
vitamin D3 (see U. S. Lettexs Patent No. 3, 565, 924).
