JPS61501447A - Method for promoting bone mineralization of poultry using 1α-hydroxyvitamin D2 analog - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] □ 1α-hydroxyvitamin D2 analog and method for producing the same The present invention was developed by The Department of Health and Human Services. Shizu (the Department of Health and) Iu+w NIH Grant No. iAM14881 granted by (NIH Grant No. AM14881) It is what was done. The Government has certain rights in this invention.

Tao Agricultural Engineering The present invention relates to biologically active vitamin D. More specifically, the present invention , concerning 1α-hydroxyvitamin D2 analogues that exhibit unexpected biological properties. do.

background 1α-Hydroxyvitamin D compounds are useful for calcium and phosphatide in animals or humans. It has a well-known and clearly demonstrated activity in regulating homeostasis of Preparation of natural metabolites and analogs with useful biological properties There has been a lot of interest in the discovery of the body. In this way, various compounds that exhibit biological activity Things have been made (for example, Topics in Current Chemistry). Topics in Current Chew, Volume 83, Page 1 (197 DeLuca et al.'s paper published in 1999), Annual Review of Biochemistry (Ann, Rev, Biochem,) No. 52@, p. 411 (1983), Russian Chemical Reviews Yakuhi published in Ches, Rev.) Volume 49, Page 371 (1980) (see paper by Yakhimovich), in this type of compounds , certain vitamin D9 conductors, especially lα,25-dihydroxyvitamin D3 and and 1α-hydroxyvitamin D3 as regulators of calcium homeostasis. In addition to the typical mechanisms of cellular differentiation, tion) process and suppress the growth and proliferation of leukemia cells. Currently, it is attracting particular attention because it can be controlled (such as Suda). No. 4,391,802, Proceedings of the Nation. Academy of Sciences of the United States Proc, Natl, Acad, USA, Volume 80, No. 2 Suda et al.'s paper published on page 01 (1983), Nature re) Rights published in volume t5306, pages 492 to 494 (1983) Paper by Reits et al.).

Most of the known vitamins 0 and 11 and their analogues are derivatives of the vitamin D3 family. ie, they have saturated steroid side chains. However, the side Vitamin D compounds with unsaturated chains, i.e. 2S-hydroxyvitamin D2 (U.S. No. 3,585,221), 1α.

25-dihydroxyvitamin D2 (U.S. Pat. No. 3,880.894), 24 -Hydroxy and 24.25-dihydroxyvitamin D2 ITh quality (archive Buzz, Talent, Biochemistry and Biophysics (Arch, Biochew+, Biophys,) 2028. Page 450 (1980) Jones published in? paper), lα-hydroxyvitamin D2 (U.S. Patent No. 3,907,843) U.S. Pat. No. 3,786°062 and Bogoslovski ( Bogos 1ovski i) paper (Journal of General Chemistry) Story Talent (J, Gen, Chew, tl ssR) Volume 48 (4), Page 828 (1978), Chemical Abstracts Chew, Abst’), Volume 89, Page 163848j, No. 89, page 209016s], the 24-methyl 4 substituent Certain related 22-trans-dehydro compounds are also absent & Structural formula A novel vitamin D analogue is obtained which is characterized by It is a droxy group. Compounds of the present invention in which R is hydrogen, compounds in which R is hydrogen, It can be obtained as an intermediate in the production of compounds.

Structurally, the compounds of the invention are hydroxyvitamins lacking the 24-methyl substituent. are analogs of the MinD2 compounds, i.e., the compounds are each lα-hydroxy -28-norvitamin D2 and lα, 25-dihydroxy-28-norvitamin It is D2.

Both the product and the intermediate exhibit high biological activity and, as further described below, Characterized by unexpected and novel patterns of activity.

An example of a chemical method for obtaining the compounds described above is shown in Process Scheme I. child In the following description of the method, as well as in the examples and tables, Arabic numerals ( For example, according to (1), (罎, (and)...(1dan), (yoyu), (12)) Compound designation refers to the structure so numbered in the process mechanism. Is Umono.

The starting material is a diene-protected aldehyde of structure (1). protected aldehyde) (see process mechanism 1), and this The method of Rarton et al. [Journal of Chemical Society] Tee (J.

Ergo according to Chew, Soc, ) (C) 1968 (1971) Obtained from sterol acetate. In the presence of a strong organic base in an organic solvent, a (CH”)CHC)I2CH2-So□phco2 of Rudehyde (1) By reaction with 3-methyl-1-methylphenylsulfone, which has the structure As shown in the process engineering, a hydroxy-sulfone intermediate of structure (2) can be obtained. Ru. Treatment of intermediate (S) in a buffered alcohol solution yields 22.2 of structure (3). A 3-)-lance olefin (22E-olefin) is obtained.

The 22-olefin (and) is treated with a strong hydrogen reducing reagent (e.g., Li) in an ether solvent. A or H4) gives a 5,7-diene intermediate (hill). When the intermediate is irradiated with ultraviolet light in an organic solvent, the corresponding previtamin D derivative is obtained. This is separated and heated in an organic solvent at temperatures ranging from room temperature to reflux. to isomerize the previtamin D chromophore to vitamin D chromophore, thus forming the structure A (mutual) 22-dehydrovitamin DJ compound is obtained. Compound (dan) is known is a vitamin D analogue of method).

The intermediate (dan) was then prepared using the general procedure of the DeLuca book (U.S. Patent No. 4,195,0 27 and 4,260°549). These anti The reaction step comprises reacting the compound (and) with toluenesulfonyl chloride in a conventional manner, The structure (first ) to produce the 3,5-cyclovitamin D intermediate shown by the corresponding 3 3,5-cyclovitamin D intermediate including the step of obtaining a β-tosylate derivative. treated with selenium oxide and tert-butyl hydroperoxide and purified by chromatography. When produced, a pair consisting mainly of 1α-hydroxycyclovitamin D of structure (7) is obtained. The corresponding 1α-hydroxylated S product is obtained. A small amount of the corresponding lβ-hydro Xyepimer too. Isolation of epi7 may be present in the product 0-order, which is possible by chromatography but not necessary at this stage. Then, the l-hydroxycyclovitamin D intermediate was dissolved in wood acetic acid at 40 to 60°C. Heating at C gives a mixture of 3-acetylated solvolysis products, which By chromatography, 5.6-cis and 5.6 of structures (dan) and (2) were obtained. -)-lance compounds are isolated respectively. 1-Hydrox subjected to solvolysis If the cyclovitamin D product contains any 1β-hydroxy epimer, Also solvolysis mixtures. lβ-hydro corresponding to compound Th (8) or (9) roxy epimers, which can also be chromatographically analyzed if desired. It can also be isolated.

When the acetate compound of compound (Dan) is subjected to conventional base hydrolysis, the structure (10) is obtained. A diol product is obtained.

This diol (10) was then added (as described in U.S. Pat. No. 4,307.025) Using liver homogenates obtained from vitamin D-deficient mice (as shown in By the way, in vitro enzymatic hydroxylation and chromatography of the product mixture Separation is performed to obtain the desired 1α,25-dihydroxy compound represented by structure (12). The zylated product is obtained in pure form.

In the following detailed description of the above synthetic method, the physicochemical data are as follows: Obtained using the methods and equipment referenced below. high pressure liquid chromatography (HPLC) is Zorbax-Si1 (DuPont (Dupont)) Column (6.2mmX 2Sc+a column, flow rate 4m Waters using approx. 105 kg/crn’ (1500 psi) for 11 minutes. ・Associates Model・AL C/G P C204 (Waters As 5ociates Model ALC/GPC204). column black Matography is silica gel (Silica Ge1) 60.770-23 0 preparative thin layer chromatography performed on 0AST mesh (Merck) (TLC) is Silica 60PF-254 (Silica60PF-254 ) (20 x 20 cm 7' rate, 1 ■ silica gel), irradiation is done with Vico Panovia 608A 36 with Vycor filter (Hanov ia 508A36) A mercury arc lamp was used. Both reactions Preferably, it is carried out in an active atmosphere (eg argon).

Ph　S　Oz　Na　(1.97g　, 12mmol) in 3-methyl-1 -D M F of bromobutane (1.5°C g, 1.2 kb, 10 mmol) (20-1) Wl;-) was added to the 75°C solution. Heat the mixture at 75°C for 5 hours. Heat, then cool and infuse into water.

Extracted with benzene. Wash the organic phase with 5%-HC solution, 5% NaHCO] and water. Cleaned, dried and evaporated. Oily sulfonate The produced e+ (1,69 g, 80%) is almost pure and can be used without purification. Ta.

NMR is δ0.88 (6H, d, J=6.5Hz, 2xCH3), 1.6 1 (3) 1. m.

-C84-C! i), 3.08 (211, m, 302-C) I2-), 7.50 -7.95 (5H, m.

Ar4); IR is 1300 (br), 1147. 1088. 745.　 692. 564゜539cm"; ff1.fit Spect J Shiha Ale 21 2 (M”, Ko), 143 (92).

77 (57), 71 (7), 70 (7), 55 (42), 43 (10G), 41 (47) n-butyl lithium (1,7M hexane solution, 4.121 see, 7 mmol), diisopropylamine (707-gt 11 sentences, 7 mmol) of dry THF (14X) and the mixture was incubated at room temperature for 15 min. did. Sulfone prepared in Example 1 (1, SOg, 7.07 mmol) Dry TI (F (11 ml) solution) was added dropwise over 10 minutes.The solution was further heated at room temperature. After pressing for 15 minutes and cooling to 0℃, add aldehyde (1) (545 g, 1 ml). A dry THF (7 ml) solution of RIMOLE was added. Continue stirring at 0°C for 2 hours to remove the solution. was slowly warmed to room temperature (30 minutes), containing the hydroxysulfone product(s). ) Add the mixture to a saturated solution of N az HP O4 in methanol (200 sEL). Sodium amalgam (5,65X, Illg) was added and the reaction mixture was The mixture was stirred and weighed at 4°C for 17 hours. The precipitated mercury was filtered out and the reaction mixture was reduced to approx. After concentrating to 5 m2, it was diluted with water and extracted with methylene chloride. tree organic extracts Wash with water, dry in vacuo, and remove the oily residue with silica. Chromatography was performed on a Kel column. Remove excess sulfone reagent with benzene. The mixture was eluted with ether (7:3)W. It was eluted with benzene ether (64). The pure adduct (3) (275 g, 671 g) was then produced as a foam.

NMR is δ0.8] (3H, s, +8-83), 0.86 (5H, d, J -6,71(z, 25-H, and 27-H, 0.97(3H,s,!9 -Hj), 1.03 (3) 1. d, J-5, 8Hz, 21)1. ), 3.16 (IH, dd, J, -4,4Hz.

J = 14Hz, 9-H), 4.44 (IH, m, 3-H), 5.25 (2H, br　m.

22-H and 23-H), 6.22 and 6J9 (2N, ABq, J=8. 5Hz, 6-H and 7-H), 7.40 (5H, br　Ar-H); IR is 3444.　1754゜1701, 1599.1402.969.757 cm", mass spectrum is */e 577 (M", 1%), 38Z (7G) , 349(51), 253(211), 251(45), 119(PhNC0, 8), 55 (1 [10)].

(22E)-Cholesta-5,7,22-trien-3β-ol (4) Adduct (3) (130 mg, 0.5 mmol) and lithium aluminum hydride (700 mg) of dry THF (401 sentences) liquid was heated in a heated oven for 18 hours. did. Excess reagents were destroyed with defeated water. Add anhydrous Na25o4 and remove the organic phase. A crystalline residue was obtained which was decanted, evaporated and purified on a column of silica gel. Be When eluted with a mixture of alcohol and ether (94:6), it crystallized from methanol. Pure diene (4) (180 g, 80-g) was obtained. Melting point is 119.5 or 122.5°C5(α)24=-118° (C-1,2, CHC intersection 3), NM R is δ0.63 (koH, s, 18-H3), 0.87(6)1. d, J-6 ,7Hz, 26-H3 and 27-H), 0.95(:tH,s, +9-83 ), 1.01(lt(,d, J=6.8Hz.

Ko 2]-) 13), '1.64 (l) I, Sono, 3-1+), 5.25 (211, br @, 22-H and 2.1-H), 5. :18 and 5.57 (2H, A Bq, J-5Hz, 7-H and 6-H), UV input is 2fLnm, IR is ko4koS, 146], +382°ax 1:156. +062.1036.968c shoulder -1, mass spectrum is ■/e 3 (M", IIJO), 149 (M"-1120-Me, 71), Ko23 ( 34), 271 (M”-side chain, 16), 253 (M”-side chain-820, 32 ) was there.

(5Z, 7E, 22E)-9,10-Secokoresta-5,7,10(19),2 2-tetraene-3β-(1201)-benzene (]0tfL) mixture solution Degassed with argon for 40 minutes. In a quartz immersion cell with UV lamp and filter The solution was then irradiated for 13 minutes at 0°C. Remove the solvent under reduced pressure and combine with the eluent. and separated by HPLC using 1% hexane solution of 2-propanol. A pure previtamin D derivative was obtained and collected in 24ml. NMR is δQ, 72 (3t l, s, 18-H3), G-87 (61 (, d.

J-6, 71 (z, 26-H, and 27-1 (J), t, Q4 (3) 1. d, J-6,8Hz.

21%), 1.65 (314, s, +5-HJ), Ko, 91(1) 1. @ ,]-IH.

5.28(2)1. br m, 22-II and 23-1 (), 5.50 ( LH, m, II-H).

5.69 and 5.96 (2H, AB q, J-12, 5Hz, 7-H and 6 -H), IV input was 260.5Nm, λ was 234n-, Brevi ma X l1In A solution of Tamine (401 g, 0.1 mmol) in ethanol (100 μl) was refluxed. It was heated for 3 hours. After removing the solvent, the produced 'j@i compound was separated by HPLC. (eluted with 1% hexane solution of 2-propertool), yield of vitamin (5) (34 ml) 30.81 g ($77) (collected at C, NMR is δ0.55(coH,s, 18-t(3), G-88(6tl.

d, J-6-7Hz, 25-Hl and 27-H3), 1.02 (3H, d , +4. SHz.

21%), 196 (11 (, L FH), 4.82 and 5.05 (2B 8n arr.

m, 19-) 12), 5.27 (2H, br m, 22-H and 23-H ), 6.03 and 6-24 (2H, ABq, J=11-4) 1z, 7-H and 6H), UV-included wax is 265Nm, to. is 228 nm, (R is 3420 ．． 14511.1441°137B, +365.1050. ! 170.94 3.891.862 cm-1, mass spectrum is snow/e(M”, 22), 3 49CII! "-H20-Me, 4), 271(M"-side chain, 8), 2 53 (M”-side chain-H20,13), 1:15 (10G), 118 new Recrystallized p-)-luenesulfonyl chloride (50 mg, 0.26 mmol) dried pidgin (30 mg, 0.08 mmol) and vitamin (tan) (30 mg, 0.08 mmol) 0h sentence) was added to the solution. After 30 hours at 4°C, the reaction mixture was placed on ice/NaH. Pour into CO:l with H4. Extract the mixture with benzene for 15 minutes. did. The organic extract was washed with saturated N a HCO3, saturated copper sulfate and water and dried ( Naz5o4)L/, concentrated in vacuo to obtain oily 3β-tosyl derivative . The crude tosylate was dissolved in anhydrous methanol (10mM>N a HCO, (15 Qs+g) and the mixture was stirred at 55°C for 8.5 hours. Cool.

After concentrating to about 2 mi, the mixture was diluted with benzene (80%) and washed with water. It was then dried (N a 2 S O 4 ) and evaporated under reduced pressure. the resulting oil The 3,5-cyclovitamin D analogue (6) is sufficiently pure and does not require any purification. It was used in the next oxidation step without further treatment. SeO□ (4 mg.

Vigorous stirring of a dry CH2C (SaM) suspension of 0.0:16 mmol) To that, tert-butylhydroberoxide (13,2 minato, 0.094 mmol) was added. ) was added. After 30 minutes, dry pyridine (40 minutes) was added and the mixture was allowed to stand at room temperature. Incubate for another 25 minutes, dilute with CH20 (3×), and cool to 30℃. Ta. CH2C intersection. Next, the crude 3゜5-cyclovitamin production Th (6) added to The mixture was then allowed to stand and slowly (3 0 minutes) Warm to room temperature. Transfer the mixture to a separatory funnel and add 10$N a It was shaken off with OH.

Add ether (150 x 9.) and wash the separated organic phase with 10 ml of NaOH. Cleaned and dried over Na2SO4.

When Cm was dried in a vacuum, an oily residue with a value color of 7.3 was obtained. Silica gel TLC developed with hexane ethyl acetate (R, 0, 35) Plate and purify 1-hydroxyvitamin production% J (14.4 servings, 45z) Obtained. NMR is 80.55 (IH, s, L8-113), (1,64 (I Lm.

]-+1), 0-88 (6H, d, J-5, 9Hz, 26-l and 27-HJ ), 1.01(koH,d,J-6,9Hz,21-H),126(3t(,S, -0CH3), 4.2 (2H, m, l-H and 6-H), 4.95 (1N, d , J-9-3Hz, 7-H).

5.1-5.4 (4tl, br 1.19-H2, 22-H and 2koH), The Qii spectrum is ale 412 (M”, 27), 380 (M”-@eOH , 46), 339 (22), 269 (M”-side%3-MeOtl, 29) , 245 (18), 1 co 5 (100), the product described in 1 is mainly composed of The lα-hydroxycyclovitamin D analogues and small amounts of the corresponding lβ-e It consisted of pimmer. This hydroxycyclovitamin D product (12 A solution of glacial acetic acid (0.5°C) of 10g) was heated at 55°C for 15 minutes. ice the mixture / Saturated N a carefully poured into HCO3 and pumped out with benzene and ether 1 The combined extracts were washed with water, dried (NazSO4) and evaporated. obtained The resulting product mixture was subjected to HPLC (eluent: t, SZ-\K of 2-propanol). 4.91g of compound (8) (dissolved in 42ml) and compound (9) 3.1 mg (5 (1 @ molten a) was obtained.

The compound (dan) has an NMR of 60.51i (3H,s, 18-Hl).

0.88 (68, d, J = 7.011z, 26-HJ and 27-1 (3), 1.02(ko)I, d.

J=5.8Hz, 2l-11x), 2.04(:IH,s,-0COC)+ 3), 4-41 (凰■.

1-11), 5.02(Ill, narr, @, +9-t(), 5. 1-5.4 (4H, br　m.

3-,! 9-. Z2 and 2coH), 5. [)3 and 6, Ko5 (2H ,AHq.

J=11.4Hz, 7-H and 6-H), UV included or 254na, λwin+S aX or 227. Sn■,'fJ quantity spectrum or ale 440 (M", Is), 38 0 (M”-NoAC, 84), 352 (kl”-IIOAc-H2O, 9 ), 269 (M”-side).

Compound (9 is NMR: 60.57 (3H, 18-Hl).

0.89 (6H, d, J-7, 0Hz, 25-H and 27-tl,), 1 ．． (1301(, d.

Ko J=5.8Hz, 21-H), 2.04(:l)I,s,-0(:OCH,), 4.49 (18, Sono.

1-H), 5.00 and S-14 (2H, each narr, m, 19-H,) , s, zs (ko H1br Maro, 3-, 22- and 23-H), 5.81 and 6.511 (2H.

ARq, J=+2.0Hz, 7-H and 6-1(), UV included or 2S9. S rv, ax Input: 228 nm, mass spectrum: 440 (M'', 5).

1ml1l+ :180(47), 26(1(is), 135(IOQ), 134(62) )Met.

Furthermore, from the solvolysis product mixture, a small amount (0,3 7) 1β-hydroxy-shrimp 7- was obtained, which is characterized by the following data. there were.

NMR is δ0.55(3)1. s, 18-) 13), 0.87 (6H, d, J=6.9’Hz.

25-HJ and 27-HJ), 1.01 (3H, d, J-6, 9H7,, 2 1-HJ).

2.06 (3H, s, -0COCH3), 4.17 (IH, m, 1-H) , 4.99 (2H.

(3-H and 19-) 1), 5.1-5.4 (31(, br　m, 19- , 22- and 2l-1 (), 6.00 and 5.38 (2) 1. 8Bq, J- 111Hz, 7-H and 6-H), UV input is 262.5βm, λwin is 227nm, ats 1a× The vectors are ale 440 (1+1”, 27), 380 (78), 362 ( 12), 269 (28), 251 (20), lko5 (100), 134 ( 78).

The effect of the 3β-acetoxy group in compounds (8) and (9)! 3β-acetoxyvitamin derivative (dan) (0,7-6-g) in ethanol (0 , 1tQ) solution is treated with KOH 10% methanol C0,811) solution, The mixture was treated at 50°C for 1 hour during normal work-up and at NHP. When LC purification (eluent: 8% hexane solution of 2-propatool) was performed, the structure was The synthesized 1α,3β-diol was obtained in a yield of 84%. NMR is δ0. 56 (ko H, s, 18-H), 0.87 (68, d, J-7,0Hz, 26-1 (and 27-) 1), 1.02 (3H, d, J-6, 8Hz, 2 l-HJ), 4.22 (IH, @, :1-H), 4.42 (Ill, m , I-H).

5, G (1 (IH, narr, m, 19-) 1), 5.1-5.4 (ko) l, b r m, 19-, 22- and 23-11), 6.01 and 6.311 (2H , AB Q, J-11, 4Hz, 7-H and tl), UV on, 2 is 21i 4. Sns, entered, originally 227. Snm, quality (it suhe') toru lf al e 39801+, 21), 380 (M+-) 1,0.9) 2.87 (M"- Side j’fl, 6), 289 (M”-side M-H, 0,8), 251 (5), +(38),! 34 (+00) It is, the compound (1 side) is added to the above HPC system. and elutes at 40 mu.

Similar treatment was performed on acetoxy derivative (2).

HPLC purification revealed that 5.6-trans-1α, 3β of 4I4 structure (11) -diol was obtained with a yield of 72%. NMR is 60.58 (]Il, s, +8-f(i, (1,87(6H,d.

J = 7-1)) lz, 26-Ha and 27-HJ), 1.03 (3 H, d, J = 5-8 tl z - and 5.13 (ZH, each narr, *, 19-H), 5.25(2t(,br*.

22-H and 23-) 1), 5.88 and 6.58 (2H, AHq, J-1 1.5Hz.

7-H and 6-H), UV input-, is 273 nm, input, (t22' 1. Snm, mass spectrum is ale 39g (M” 21), 380 (5) , 287 (6), 269 (S), 251 (4), 152 (33), 1 piece 4 (110) I was looking. Compound (11) elutes at 38 m.

25-hydroxylation of compound Th(10) The structure obtained as above (once ), the 5.6-cis-1α,3β-diol was then converted to 25-hydroxy Sillized. Journal of Nutricicon (J, Suda (Nut Otsu) published in Volume 100, Page 1049 (1970) ), etc., a low-calcium vitamin D or vitamin D-deficient diet was given for 2 weeks. I got it. I killed the mouse with 4 slashes and took out its liver. 20% (W/V) homogenate in water Made in cooled 0.25M sucrose. Liver equivalent to 1 g of tissue, 2 yen Aliquot of material, 0.125M sucrose, 50L mM phosphate # buffer (pH 7,4), 22.4mM crucose-6-phosphate, 20mATP, 160mM Nicotinamide, 25mM Succinate, 0.4mM NADP, 5m MMgCl-2,0,2MKCl and 0.5 units of glucose-6-phosphate One of the 125mM Erlenmeyer flasks containing Funito dehydrosinase. Tests were carried out in 0 m2 culture medium. Dissolved in 95% ethanol of 100 pots λ The reaction was started by adding 400 hg of compound (IO).

The mixture was incubated at 37° C. with shaking at a rate of 80 vibrations/min for 3 hours. 20m Stop the reaction by adding m methanol and m m dichloromethane. I let it happen. An additional 1.0 ml of dichloromethane was added, then the organic phase was collected while , the aqueous phase was re-extracted with 10 m of dichloromethane. Obtained from a total of three extractions The organic phases were combined and evaporated on a rotary evaporator.

The remaining residue containing the desired product was mixed with CHC mixture and hexane (65:35). Dissolve in 1 m solution and apply Sephadex L H-20 column. (0.7 cm x 14 cm), loaded, equilibrated and eluted with the same solvent.

Discard the first 10m sentence, collect the next 40m sentence and evaporate it. Zorbax-3il (Zorbax-3il) dissolved in 8% hexane Column (4.5mm x 2Sc++, Wilenton, Praue (DE)) High temperature using DuPont Inc. Performance Liquid Chromatography (Massachusetts (MA)).

Wate of Medford Model L C/GPC20 manufactured by rs As5ociates 9) (PLC) to a pressure of approximately 70-3 kg/cm' (1 (IQG psi) It was activated at a rapid speed of 2m11 minutes.

The desired 25-hydroxylated product was eluted in a 42 m column. 7 (6) The product was transferred to a reversed-phase column [Darmstad, West Germany]. E. Merck's Richrosord Using Rp-18, 4.6m@X25cm, approximately 8+k g/c rr+ High-performance liquid chromatograph operated at a flow rate of 1200 psm l 7 min. It was further refined by fee. The column was eluted with 22% methanol in H2O and the raw The product was eluted in 50ml. Prepare the product as described above using Solpak Susil. The resulting product, which was further purified by HPLC under the same conditions, had the following data characteristics: had.

UV absorption (95% ethanol) is included 265. Enter sinaX zza, the mass spectrum is m/z 414 (mol, ion M+), 396 ( M''-H, 0), 3713 (M'''-2zH, O), 287 (M''-side chain) , 269 (M”-(Mjl-H2O), 2!:1 (MP-IJI49Q -2J: + (, O), 152 (Ring A, C7/8 bond cleavage), 134 (1 52-H,0) and 59 above data, especially characteristic ultraviolet absorption and mass spectra From the prominent and characteristic peak at m/z 152.134 and 5g in The desired 25-hydro product represented by structure (12) (process scheme I) It can be seen that it is a xylated compound.

If desired, the compounds of the invention may be combined with any suitable compound, as will be clear to those skilled in the art. Solvent 1 Crystallization from e.g. hexane, ether, alcohol or mixtures thereof can be easily obtained in crystalline form.

Physical activity of side chain dehydrovitamin D compounds Physical efficacy was determined by in vivo efficacy assay in mice and by la-hydro This was confirmed by comparison with a xyvitamin D compound.

Tests are carried out on both the product (1st) and the compound (1st), which is an important intermediate. It was found that there was significant activity.

Physical activity of compound (10) The efficacy test was conducted as follows. A male baby mouse was taken from Wisconsin (W T), Holtzman Corporation of Madison Zman Co., Ltd.), and published the paper by Suda et al. ・Nutrition (J, Nutr) Volume 100, 1LO page 49 (1970) )], it contains enough phosphorus with any amount of water and low calcium, and contains vitamins. After feeding the mice a diet deficient in D for 3 weeks, the mice were divided into groups of 6 mice each. Compound (Yodan) or 1α-hydro dissolved in 0.05 mJl of 95% ethanol Kill 50 pmol of X-vitamin D3 (LA-0H-D) at 18:00 It was given immediately intrajugularly. control group Mice were given ethanol vehicle in the same manner and killed by decapitation. , blood was collected. Serum obtained by centrifugation of blood was added to 0.1% lanthanum chloride solution. (l; 20), and serum calcium concentration was measured by atomic absorption spectrometry. Ta. The results are shown in Table 1.

Serum calcium concentration for one administration of la-OH-D3650pmo1 increase Compound given Serum calcium concentration (mg/roomJ1) Soil standard deviation Ethanol 3.2±O, la) Compound (Yodan) 4.5±0.4b) 1α-0H-D3 4.7±0.5b) Efficacy assay was conducted as follows. male Child mice were fed a diet low in calcium and deficient in vitamin D as described above. The mice were then divided into groups of 5 mice each for 3 weeks. control group 0.05m sentence for a mouse? 5% ethanol was given into the jugular foramen, while the test group For mice in the loop, add the compound to the solution (prepared to 100%) or 0.05 m solution in 95% ethanol. decomposed la, 25-dihydroxyvitamin D3 (1α, 25-(OH) 2 D3) was used for 325 Pmol. After 6:00 p.m., he was killed by decapitation and his blood was drained. Collected. Serum calcium C level was measured by atomic absorption spectrometry as described above. Established. The results are shown in Table H.

Serum calci for a single dose of Lcx, 25-(OH)2D, 325pmol Compounds that gave an increase in serum calcium concentration once (mg/roomλ) standard deviation Ethanol 4.2±0.1a) Compound (12) 5.0±0.4b) 1α, 25-(OH) D 5.4 ± 0.4 b) b) is significantly different from a) . p<0.001 From the above results, the compound of the present invention (1dan) and the final product (dan), Both have been shown to promote an increase in serum calcium levels in vitamin D-deficient mice. It has remarkable activity. In fact, these substances are highly effective and useful as medicines. In the general and patent literature (see, e.g., U.S. Pat. No. 4,225,596) This has been widely demonstrated in numerous reports. Corresponding known side chain saturated compounds 1α-OH-D, and 1α, 25-(O)()U DJ, and biological activity. gender or equivalent.

Particularly remarkable and unsustainable is compound (1).

Animal (chick) bones make a huge difference to the physiological availability of vitamin D2 compounds. It has excellent activity in imparting mineralization to minerals. This is the data below This is shown in more detail.

Ten thousand words A one-day-old male White Refhorn chick was taken to Northern Batchleys (N other Hatcheries) (Wisconsin, WI) Beaver.

Obtained from Dam). Chick, Omtal (0+dahl) etc. paper (bye) Biochemistry Volume 10.

No. 2935-2940, 1971] Vitamin D-deficient soybean A protein diet was fed for 3 weeks, at which point the chicks became vitamin D deficient. These were then divided into groups of 6 chicks each. One group has Uesso One group received the oil vehicle by mouth, and the other group received the same amount of urinary veneer. The indicated compounds (see Table 1) dissolved in Esson oil were given daily for 7 days.

All chicks were sacrificed by cervical dislocation 24 hours after the last dose. remove the tibia Remove the sticky soft connective tissue, and apply Soxhlet ) using an extractor for 24 hours with ethanol followed by 24 hours with diethyl ether. After extraction, these bones were heated to approximately 38°C (100°F) to a constant volume. After drying and weighing, these were heated in a Matsufuru furnace at 650"C for 24 hours. It was subjected to intermediate iodization treatment. The ash was weighed and the percentage of ash in each tibia was calculated. The results are shown in table m. show.

rotation Bone mineralization in rickets chicks Compound Amount Number of Animals Ash % (pmol/α/7 days) 1.25-(OH)2 C3130641, O±3.4 pieces l a-OHC313 0642,0±3　,2　*1　a-OHD2　1300　6　38.0　±4 -0 *Compound (10) 130 6 38. Of3.9* From these results, 1.25 (OH)2 C3 or 1α-0H-D or the novel compound of the present invention Item (10) increases the mineral content of the tibia in patients with rickets. In particular, it can be seen that it is as effective as C1. To obtain efficacy, 1300 p m o 10') lα-0H-D2 is required. It was. This means that to obtain the same mineralization of the rickets chicken, lα--OH - Requires 10 times more lα-〇H-D2 than D. It was found that 9 is consistent with the previous result. From these results, the compound Th(10 ) is an analogue of 1α-0H-D2, but it is not known for its physiological use as vitamin D2. In mineralizing the bones of animals known to differentiate against compounds, It can be seen that it exhibits unexpectedly excellent activity. This predicts that this novel analogue It shows unexpected features and usefulness. This unexpected compound (Kodan) The in vivo activity of the corresponding 1α,25-dihydroxy compound (compound ( It appears after hydroxylation to 12)), so the known 1α,25-dihydroxy Compound (12), a 24-desmethyl analog of civitamin D3, is administered to chicks. mineralization of bone equivalent to that of 1α,25-dihydroxyvitamin D3. It is clear that this novel vitamin D2 analogue It is found to be sufficiently active in chicks. For vitamin D type 2 derivatives Such high efficacy of compounds (1dan) and (1u) in discriminating animals , which is new and a distinct feature of these materials.

This remarkable biological activity makes the compounds of the invention useful for treating human rickets and parathyroid mechanisms. hypovolemia, osteodystrophy, osteomalacia or osteoporosis or associated calcium in animals. In the treatment and prevention of calcium metabolism disorders such as sium deficiency (eg lactation fever) It is easy to find use as a substitute for various known vitamin D metabolites. can. These compounds may also cause certain types of malignant disease, such as human leukemia. ! 4 Can be used to treat The present invention has three advantages in promoting mineralization of bird bones. Considering that the compound W4 exhibits remarkable and unexpected activity, all known Calcium imbalance in poultry showing significantly low activity of vitamin D2 derivatives specific for the prevention and treatment of conditions caused by cancer (e.g. thinning of eggshells, weakened legs in poultry). It is recognized that it is useful for

For therapeutic purposes, the compounds may be administered by conventional methods of administration.

Claims (7)

[Claims] 1. formula ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ A compound having the above formula, where R is hydrogen or hydroxy. 2. 2. A compound according to claim 1, wherein R is hydrogen. 3. The compound according to claim 2, which has a crystalline form. 4. 3. A compound according to claim 2, wherein R is hydroxy. 5. 5. A compound according to claim 4, which has a crystalline form. 6. A compound comprising the compound of claim 2 and a pharmaceutically acceptable excipient. Pharmaceutical composition. 7. A compound comprising the compound of claim 4 and a pharmaceutically acceptable excipient. Pharmaceutical composition.