JPH0455425B2 - - Google Patents

Description

TECHNICAL FIELD This invention relates to biologically active vitamin D compounds. In particular, the present invention relates to a novel 1α,25-dihydroxylated vitamin D compound having a 22,23-cis double bond in its side chain and a method for producing the same.

BACKGROUND Calcium and phosphate homeostasis in animals and humans is regulated by vitamin D metabolites, and 1α,25-dihydroxyvitamin _D3 is generally the most active and most active member of the normal calcium and phosphate balance. It is considered to be an important vitamin D induction regulator. This natural metabolite and its structurally related compounds are therefore of great pharmaceutical interest as effective agents for the prevention and treatment of bone diseases and related disorders of calcium metabolism. Besides the natural D ₃ metabolites, a number of compounds have recently been created that have medicinal properties and have considerable promise as therapeutic agents, including 1α-hydroxyvitamin D ₃ , 1α-hydroxyvitamin D ₂ , 1α, 25
- dihydroxyvitamin D ₂ and certain fluorinated analogs (U.S. Pat. No. 3,741,996;
No. 3907843, No. 3880894, No. 4226788, No.
No. 4358406). Most of the known active analogs are characterized by the type of sterol side chain (ie, saturated side chain), as sterol side chains occur in vitamin _D3 . Known analogues with 22,23-unsaturated side chains include the vitamin D ₂ series of compounds (i.e. C-
In addition to the above-mentioned compounds, 25-hydroxyvitamin D ₂ (U.S. Pat.
3585221), 24- and 24,25-dihydroxy derivatives [Jones et al., Archives.
Of Biochemistry and Biophysics (Arch.Biochem.Biophys.) Volume 202,
No. 450 (1980)] and three compounds lacking the 24-methyl substituent [US Pat. No. 3,786,062; Bogoslovskii et al., Journal of General Chemistry USSR (J.Gen.Chem. USSR) Volume 48(4), 828
(1978); Chemical Abstracts (Chem.Abstr.) Vol. 89j, 163848j, 209016s).

Disclosure of invention A new vitamin D analogue has been discovered which can be represented by the structure:

This new compound is characterized by a 22,23 double bond in the side chain with cis (ie Z) geometric isomerism.
A normal saturated side chain (such as in 1α,25-dihydroxyvitamin _D3 ) or a
25-dihydroxyvitamin D (as in ₂ )
The presence of this 22Z double bond, resulting in a side chain geometric isomerism that is markedly different from that typical of compounds with 22,23-trans (22E) unsaturated side chains, makes this cis-unsaturated product a low biomolecule. It was thought to be active. Surprisingly, this substance, despite its unusual side chain structure, shows high activity and exhibits similar activity to 1α,25-dihydroxyvitamin D with respect to its ability to increase serum calcium levels in test animals. It is.

Preparation of Compounds The above-mentioned compounds, which are novel products of the present invention, are prepared using liver homogenate obtained from vitamin D-deficient mice and carbon 25
by in vitro enzymatic hydroxylation in It was made from a 22Z-dehydro-vitamin D precursor having the following structure.

The procedure was as follows. Regarding male child mice, Suda et al. [Journal of Nutrition (J. Nutr.), Vol. 100, No.
1049 (1970)] and fed a diet low in calcium and deficient in vitamin D for two weeks. The mouse was killed by decapitation and the liver was removed. 20%
(Weight/volume) homogeneity was made in ice-cold 0.25M sucrose. tissue, 0.125M sucrose, 50mM phosphate buffer (PEPH7.4),
22.4mM glucose-6-phosphate,
20mM ATP, 160mM nicotinamide,
25mM succinate, 0.4mM NADP, 5mM
Cultures were grown in 10 ml of culture medium in a 125 ml Erlenmeyer flask containing an aliquot of liver homogenate equivalent to 1 g of MgCl ₂ , 0.1 M KCl and 0.5 units of glucose-6-phosphate-dehydrogenase. I did it. The reaction was started by adding 400 μg of the above compound substrate dissolved in 100 μl of 95% ethanol. culture medium mixture for 1 min.
The culture was incubated at 37° C. for 3 hours with shaking 80 times. The reaction was stopped by adding 20 ml methanol and 10 ml dichloromethane. Add another 10 ml of dichloromethane, then collect the organic phase and dissolve the aqueous phase in dichloromethane.
Re-extracted with 10ml. The organic phases obtained from all three extractions were combined and evaporated on a rotary evaporator. The residue containing the desired product was purified with _CHCl3 :hexane (65:35)
Dissolve in 1 ml of the mixture containing the same solvent,
Sephadex, which is subjected to equilibrium elution with this solvent,
It was applied to a Sephadex LH-20 column (0.7 cm x 14 cm). The first 10ml was discarded and the next 40ml was collected and evaporated. Next, the residue was dissolved in 8% 2-propanol in hexane to give a solution of about 70.3
Zorbax-SIL operating at a flow rate of 2 ml/min under pressure of Kg/cm ² (1000 psi)
High performance liquid chromatography using a column (4.6 mm x 25 cm, manufactured by Dupont, Wilmington, Delaware) [Model LC/GP.C 204 HPC (Model LC/GP.C)] 204 HPLC),
Medford, Massachusetts
Waters Associates
Associates). 44ml desired 25−
The hydroxylated product eluted. This product was transferred to a reverse phase column operated at a flow rate of 2 ml/min under approximately 84 Kg/cm ² (1200 psi) [E.
Refrosolv Elpe 18 manufactured by Merck
(Richro-sorbRP-18), 4.6 cm x 25 cm] was further purified by high performance liquid chromatography. The column was eluted with 22% H ₂ O in ethanol to elute 50 ml of compound. The product was purified using a Zolbax-sil column under the conditions described above.
Further purification was done by HPLC. The resulting product was then subjected to processing for physical property measurements.

Determination of Product Characteristics The ultraviolet absorption of the product in 95% ethanol is
λ _nax =265 nm and λ _nio 228 nm, indicating the presence of a 5,6-cis-triene chromophore.

The mass spectrum of this material contains a molecular ion of m/e 414 as required for the 25-hydroxylated product. Removal of one and two molecules of H ₂ O yields fragment ions with m/e of 396 and 378. loses the entire steroid side chain ( _C17 /
C ₂₀ bond cleavage) yields a fragment of m/e287, from which one and two molecules of H ₂ O are removed,
Peaks with m/e of 269 and 251 are obtained. The spectrum shows prominent peaks at m/e 152 and 134 (152-H ₂ O), which represent the ring A fragment and are characteristic of 1α,3β-dihydroxyvitamin D compounds. Furthermore, the spectrum is
It showed a very prominent fragment peak at m/e59 caused by cleavage of the _C24 / _C25 bond. The presence of this ion confirmed the presence of 25-hydroxy groups in the product. These data thus confirm that the structure of the product obtained is a 1α,25-hydroxylated compound as represented by the structure described above.

Biological Activity The biological activity of the novel congener was confirmed by in vivo analysis in mice. To a male baby mouse,
A low calcium, vitamin D deficient diet such as Suda (described above) was fed for 3 weeks. These were then divided into groups of 5 mice each. Mice in the control group received 0.05 ml of 95% ethanol intrajugularly, while mice in other groups received 0.05 ml of 95% ethanol dissolved in 0.05 ml of 95% ethanol.
325 pmol of compound 1 or 1α,25-dihydroxyvitamin D ₃ was given. After 18 hours, mice were killed by decapitation and blood was collected. Serum obtained by centrifuging blood was diluted (1:20) with 0.1% lanthanum chloride solution, and serum calcium concentration was measured using an atomic absorption spectrometer. The results are shown in the table below.

[Table] From the above results, it can be seen that the compound of the present invention has remarkable medicinal efficacy and exhibits biological activity almost equivalent to that of 1α,25-dihydroxyvitamin _D3 .

Such high medicinal efficacy makes the compounds of the invention useful in the treatment or prevention of diseases such as various types of rickets, hypoparathyroidism, osteodystrophy, osteomalacia or osteoporosis in humans. It finds use as a therapeutic prophylactic agent or for the treatment of associated calcium deficiencies in animals (eg lactation fever, leg weakness, eggshell thinning). This compound can also be used in the treatment of certain cancers, such as leukemia in humans.

For therapeutic purposes, the compounds can be administered by conventional routes of administration and in a form appropriate to the chosen method of administration. The compound can be formulated with an acceptable and non-toxic pharmaceutical carrier to
It may be provided in the form of a pill, tablet, gelatin capsule or suppository, or as a solution, emulsion, dispersion or suspension in a non-toxic solvent or oil, and such formulations may be Suitable therapeutically active and beneficial ingredients may also be included. For human use, the compound is advantageously administered in amounts of 0.25 to 10 μg per day, with the particular dosage being dependent on the disease to be treated, the medical history, and Adjusted according to patient condition and response.

The 22Z-dehydro precursor, the above-mentioned compound, required for the preparation of the novel products of the invention itself is obtained by the method shown in the reaction steps below. In the reaction process, Arabic numerals (e.g. 1, 2,
3, etc.) refers to the structure numbered in the reaction process.
The desired substance (compound) for the 25-hydroxylation reaction described above is designated by the Arabic numeral 11 in the reaction process and in the description below.

(22Z)-3β-(methoxymethoxy)-5α,8α-
(4-phenyl-1,2-urazolo)cholester 6,22-diene (2). isopentyl phosphonium in dry tetrahydrofuran (73 ml).
Pramid [(CH ₃ ) ₂ CHCH ₂ CH ₂ PPh ₃ Br] (1.67g,
4.04 mmol) was prepared using n-butyllithium (1.7M solution in hexane, 2.42 ml, 4.11 mmol) at 3.
The treatment was carried out at 5°C to 5°C with stirring. After stirring for 1 hour at room temperature, the orange red solution was cooled to 3°C and dissolved in aldehyde (1) in dry THF (24 ml).
(1.84 g, 3.36 mmol) was added. The colorless reaction mixture was stirred at room temperature overnight and then poured into water.
Extracted with benzene. The organic extracts were washed with 5% HCl, saturated sodium bicarbonate and water, dried (Na ₂ SO ₄ ) and concentrated in vacuo to an oil that was purified on a column of silica gel. Benzene-
Elution with an ether (94:6) mixture gave adduct (2) (1.38 g, 68%) as a foam. Nuclear magnetic resonance or NMR is 0.83 (3H, s, 18−H ₃ ),
0.89 and 0.91 (6H, each d, J = 6.8Hz, 26-H ₃ and
27−H ₃ ), 0.97 (3H, d, J= _6.8Hz , 21−H ₃ ),
0.98 (3H, s, 19−H ₃ ), 3.30 (1H, dd, J ₁ =
4.4H _z , J ₂ = 14H _z , 9-H), 3.38 (3H, s,
OCH ₃ ), 4.33 (1H, m, 3-H), 4.70 and 4.81
(2H, ABq, J=6.8Hz, OCH ₂ O), 5.21 (2H,
brm, 22-H and 23-H), 6.23 and 6.39 (2H,
ABq, J=8.5Hz, 6-H and 7-H), 7.41
(5H, brm, Ar-H), IR is 1756, 1703, 1601,
1397, 1046cm ^-1 , mass spectrum is m/z601
(M ⁺ , 1%), 426(4), 364(61), 1349(16), 253(1
8), 251(18), 119 (PhNCO, 100).

(22Z)-5α,8α-(4-phenyl-1,2-urazolo)cholester 6,22-dien-3β-ol (3). Adduct (2) (601 mg 1 mmol) and p-
A solution of toluenesulfonic acid (523 mg, 2.75 mmol) was stirred at room temperature for 2 days. The reaction mixture was poured into saturated sodium bicarbonate and extracted several times with benzene. The extract was washed with water, dried (Na ₂ SO ₄ ),
Evaporated under reduced pressure. Column chromatography of the crude product (eluent, benzene:ether 70:30)
When purified, the hydroxy adduct (3) (550 mg,
99%) was obtained as a foam. NMR is 0.83 (3H,
s, 18−H ₃ ), 0.89 and 0.91 (6H, each d, J = 6.8
Hz, 26−H ₃ and 27−H ₃ ), 0.95 (3H, s, 19−
_H3 ), 0.98 (3H, d, J=6.8Hz, 21- _H3 ), 3.16
(1H, dd, J ₁ = 4.4Hz, J ₂ = 14Hz, 9-H), 4.44
(1H, m, 3-H), 5.22 (2H, brm, 22-H and
23-H), 6.22 and 6.39 (2H, ABq, J=8.5Hz,
6-H and 7-H), 7.40 (5H, brm, Ar-H);
IR is 3447, 1754, 1700, 1600, 1397 cm ^-1 ; Mass spectrum is m/z (557 (M ⁺ , 1%), 382 (35),
They were 349(33), 253(20), 251(33), 119(110), and 55(82).

(22Z)-cholester 5,7,22-triene-
3β-ol (4). Adduct (3) (530 mg, 0.95 mmol)
was converted to diene (4) by reduction in tetrahydrofuran (60 ml) with lithium aluminum hydride (1 g) at reflux for 18 hours.
After conventional work-up, the product was purified by chromatography on silica (eluent, benzene-ether 94:6) and crystallized from ethanol to give the pure diene (4) (290 mg, 76%). Obtained. Melting point is 148~
151℃, [α] ^24/D = -132° (C = 0.9, CHCl ₃ ),
NMR is 0.66 (3H, s, 18-H ₃ ), 0.90 and 0.91
(6H, each d, J=6.8Hz, 26−H ₃ and 27−H ₃ ),
0.96 (3H, s, 19−H ₃ ), 0.98 (3H, d, J=6.9
Hz, 21- _H3 ), 3.64 (1H, m, 3-H), 5.20
(2H, brm, 22-H and 23-H), 5.39 and 5.57
(2H, ABq, J=6Hz, 7-H and 6-H),
UV is λ _nax 281nm, IR is 3346, 1463, 1375,
1364, 1067, 1040, 831cm ^-1 , mass spectrum is m/z382 (M ⁺ , 100), 349(65), 323(32), 271(15),
It was 253(30).

(5Z, 7E, 22Z) −9, 10 − Secoco Star 5,
7,10(19),22-tetraen-3β-ol(5). 5,7 diene (4) (150 mg, 0.39 mmol) dissolved in ether (120 ml) and benzene (30 ml) (degassed with argon for 40 minutes) was heated with an ultraviolet (UV) lamp and a Vycor filter. Irradiation was carried out for 13 minutes at 0°C. HPLC (1% hexane solution of 2-propanol) of the resulting mixture revealed that the previtamin (56.9
mg, 38%) was obtained as a colorless oil.
NMR is 0.75 (3H, s, 18-CH ₃ ), 0.90 and 0.91
(6H, each d, J=6.7Hz, 26−H ₃ and 27−H ₃ ),
0.99 (3H, d, J=6.8Hz, 21−H ₃ ), 1.64 (3H,
s, 19-H ₃ ), 3.90 (1H, m, 3-H), 5.20
(2H, brm, 22-H and 23-H), 5.69 and 5.95
(2H, ABq, J=12Hz, 7-H and 6-H), and the UV was λ _nax 261 nm and λ _nio 234 nm.

Thermal isomerization (3 hours) of this previtamin intermediate (56 mg, 0.15 mmol) in refluxing ethanol yielded the oily vitamin congener (5) (43 mg, 77%) after separation by HPLC. It was done.
NMR is 0.60 (3H, s, 18-H ₃ ), 0.89 and 0.90
(6H, each d, J = _6.7Hz , 26- _H3 and 27- _H3 ),
0.97 (3H, d, J=6.6H _z , 21−H ₃ ), 3.96 (1H,
s, 3-H), 4.82 and 5.05 (2H, each narr.m, 19
-H ₂ ), 5.20 (2H, brm, 22-H and 23-H),
6.04 and 6.24 (2H, ABq, J=11.4Hz, 7-H and 6-H), UV is λ _nax 265.5nm, λ _nio 228nm, IR
is 3427, 1458, 1379, 1048, 966, 943, 892 cm ^-1 ,
The mass spectrum is m/z382 (M ⁺ , 21), 349(5),
They were 271(8), 253(14), 136(100), and 118(32). Vitamin homolog (5) is a known compound (see Bogoslovsky, cited above).

Hydroxylation of compound (5). Freshly recrystallized p-toluenesulfonyl chloride (50 mg, 0.26
mmol) to vitamin (5) (50 mg, 0.13 mmol)
of dry pyridine (300 μl). at 4℃
After 30 hours, the reaction mixture was poured onto ice/saturated _Na HCO ₃ with stirring. The mixture was stirred for 15 minutes and extracted with benzene. The organic extract was purified with saturated _{NaHCO 3} and saturated copper sulfate. and washed with water and dried (Na ₂ SO ₄ ),
Concentration in vacuo gave tosylate 6 as an oil. The crude tosylate 6 was dissolved in anhydrous methanol (10
ml) with N _a HCO ₃ (150 mg) and the mixture was stirred at 55° C. for 8.5 h. After cooling and concentrating to 2ml, the mixture was diluted with benzene (80ml), washed with water, dried (Na ₂ SO ₄ ) and evaporated under reduced pressure. The oily 3,5-cyclovitamin D compound thus obtained was sufficiently pure to be used in the next oxidation step without purification. in dry CH ₂ Cl ₂ (5 ml)
To a vigorously stirred suspension of S _e O ₂ (5.1 mg, 0.046 mmol) was added tert-butyl hydroperoxide (16.5 μl, 0.118 mmol). After 30 minutes, dry pyridine (50 μl) was added and the mixture was further incubated at room temperature.
Stirred for 25 minutes, diluted with CH ₂ Cl ₂ (3 ml) and cooled to 0°C. The crude 3,5-cyclovitamin product (7) in CH ₂ Cl ₂ was then added. Reaction at 0℃
This was allowed to proceed for 15 minutes and then left to warm up to room temperature (30 minutes). Transfer the mixture to a separatory funnel and add 30
Shake with ml of 10% NaOH. Ether (150 ml) was added and the separated organic phase was diluted with 10% NaOH,
Washed _with water and dried over _Na2SO4 . Concentration to dryness in vacuo gave a yellow oily residue, which was purified on a silica gel TLC plate developed in 7:3 hexane-ethyl acetate to yield the 1-hydroxycyclovitamin product (20
mg, 37%) was obtained. NMR is 0.59 (3H, s,
18- _H3 ), 0.63 (1H, m, 3-H), 0.89 and 0.90
(6H, each d, J=6.9Hz, 26−H ₃ and 27−H ₃ ),
0.96 (3H, d, J=6.9Hz, 21−H ₃ ), 3.25 (3H,
s, -OCH ₃ ), 4.17 (2H, m, 1-H and 6-
H), 4.96 (1H, d, J=9.3Hz, 7-H), 5.1-
5.4 (4H, brm, 19-H ₂ , 22-H and 23-H), mass spectra are m/z 412 (M ⁺ , 26), 380 (48),
They were 339(22), 269(28), 245(20), and 135(100).
This product is mainly composed of a 1α-hydroxycyclovitamin D compound of structure (8) and also contains small amounts of the corresponding 1β-hydroxy-epimer. These components can be separated at this stage if desired, but such separation is not necessary.

The 1-hydroxycyclovitamin product (18 mg) obtained above was heated (55°C, 15 min) in glacial acetic acid (0.8 ml) to neutralize the mixture (ice/
saturated _NaHCO3 ), extracted with benzene and ether,
After HPLC separation (eluent: 2-propanol in 1.5% hexane), pure 1α-hydroxy-3β-acetoxyvitamin (9) (6.60 mg, 34%,
42 ml of elution) and (10) (4.20 mg, 22%, 50 ml of elution) were obtained.

Compound (9) has an NMR of 0.60 (3H, s, 18-H ₃ ),
0.90 and 0.92 (6H, each d, J = _7.0Hz , 26- _H3 ,
and 27−H ₃ ), 0.97 (3H, d, J=6.8H _z , 21−
H ₃ ), 2.04 (3H, s, −OCOCH ₃ ), 4.41 (1H, m,
1-H), 5.02 (1H, narr.m, 19-H), 5.1-5.4
(4H, brm, 3-, 19-, 22- and 23-H),
6.03 and 6.35 (2H, ABq, J= _11.4Hz , 7-H and 6-H), UV is λ _nax 264.5 nm, λ _nio 227.5 nm,
The mass spectrum is m/z440 (M ⁺ , 10), 380(72),
They were 362(7), 269(31), 251(12), 135(100), and 134(99).

Compound (10) has an NMR of 0.60 (3H, s, 18-H ₃ ),
0.90 and 0.91 (6H, each d, J = _7.0Hz , 26- _H3 and 27- _H3 ), 0.97 (3H, d, J = 6.9Hz _, 21-
H ₃ ), 2.05 (3H, s, -OCOCH ₃ ), 4.49 (1H, m,
1-H), 5.00 and 5.14 (2H, each narr.m, 19-
H ₂ ), 5.20 (3H, brm, 3-, 22- and 23-H),
5.82 and 6.59 (2H, ABq, J = 12.0Hz _, 7-H and 6-H), UV is λ _nax 270 nm, λ _nio 228 nm, mass spectrum is m/z 440 (M ⁺ , 4), 380(30) ,269
(10), 135(100), and 134(52).

Hydrolysis of the 3β-acetoxy group of compounds (9) and (10) Each of the 3β-acetoxy derivatives 9 and 10 was 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 mixture was heated at 50° C. for 1 hour. After normal treatment and finally HPLC purification (eluent: 8% hexane solution of 2-propanol), the corresponding 1
- Hydroxyvitamin was obtained. Compound (11)
has NMR of 0.59 (3H, s, 18−H ₃ ), 0.89 and
0.90 (6H, each d, J = _7.0Hz , 26−H ₃ and 27−
H ₃ ), 0.96 (3H, d, J = _6.8Hz , 21-H 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,
brm, 19-, 22- and 23-H), 6.02 and 6.39
(2H, ABq, _J =11.4Hz, 7-H and 6-H),
UV is λ _nax 264.5nm, λ _nio 227.5nm, mass spectrum is m/z 398 (M ⁺ , 21), 380(8), 287(6), 269(7),
They were 251(5), 152(36), and 134(100). (The elution amount is
39ml).

Compound (12) has an NMR of 0.61 (3H, s, 18-
H ₃ ), 0.89 and 0.91 (6H, each d, J = 7.0Hz, 26−
_H3 and 27− _H3 ), 0.97 (3H, d, J= _6.9Hz , 21
-H ₃ ), 4.25 (1H, m, 3-H), 4.51 (1H, m,
1-H), 4.98 and 5.13 (2H, each narr.m, 19-
H ₂ ), 5.21 (2H, brm, 22-H and 23-H), 5.89
and 6.59 (2H, ABq, J=11.5Hz, 7-H and 6
-H), UV is λ _nax 273nm, λ _nio 229.5nm, mass spectrum is m/z 398 (M ⁺ .17), 380(4), 287(5),
They were 269(5), 251(4), 152(29), and 134(100). (The elution volume is 38 ml.) In the above method, high pressure liquid chromatography (HPLC) is
[6.2mm x 25cm column, flow rate 4ml/min, approx. 105Kg/
cm ² (1500psi)
I did it on 204. Column chromatography was performed using Silica Gel 60, 70-230 ASTM mesh (Merck & Co.). Preparative thin layer chromatography (TLC) uses silica 60 PF-254
(Silica60PF-254) (20cm x 20cm plate, 1
mm silica gel). Irradiation is done using Hanovia (Hanovia608A36) equipped with a Vicol filter.
This was done using a mercury arc lamp. Preferably, all reactions are conducted under an inert atmosphere (eg, argon).

Compounds of the invention can be used, if desired, in hexane, ethers and alcohols (anhydrous or aqueous), as will be clear and well known to those skilled in the art.
in crystalline form by crystallization from a suitable solvent such as
and can be easily obtained as a mixture.

Claims (1)

[Claims] 1 A compound with the structure 2. The compound according to claim 1, which is in a crystalline form. The invention is directed to the Department of Health and Human Services.
Health and Human Services) N.I.
NIH Grant No. 14881 (NIH Grant No. 14881)
No. 14881) with the support of the government.