DE3030683A1 - 25-HYDROXYCHOLECALCIFEROL-26,23-LACTONE - Google Patents

Description

Wisconsin Alumni Fiesearch Foundation
614 North Walnut Street Madison
Wisconsin 53705 / V.St.A.

Honor exercise
25-Hydroxycholecalciferol-26,23-lactone

Technical ^ Gehiet

The invention relates to a compound produced by a vitamin D-like Effectiveness is characterized.

In particular, the invention relates to a derivative of vitamin D.

The invention particularly relates to 25-hydroxycholecalciferol-26 »23-1actone.

The ability of the D vitamins »the serum calcium concentrations to increase and increase bone growth »is known and the use of these vitamins as food additives has become established.

It is also known that "in order to be effective" these vitamins must be metabolized in vivo to obtain the physiological ones To express functions »with which they are connected». The vitamin is first hydroxylated in the liver with formation of 25-hydroxyvitamin-D, which is believed to that it is the main circulating in the bloodstream

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Represents metabolites. This compound is then further hydroxylated in the kidney to form 1oc _f 25-dihydroxyvitamin-D or 24,25-dihydroxyvitamin-D. The la-hydroxylated form of vitamin D is generally regarded as the physiologically active or hormonal form of the vitamin and responsible for the so-called vitamin D-like effects, such as the increased intestinal absorption of calcium and phosphate, the mobilization of bone minerals and the Retention of calcium in the kidneys. However, it remains di ^ possibility that a further metabolism of 1oc, 25-dihydroxyvitamin D is required to any or all of ^these: elicit reactions. , .. ■ '.

References can be found in the patent literature or other literature on various vitamin D derivatives. See, for example, U.S. Patent Nos .: 3,565,924, directed to 25-hydroxycholecalciferol; 3,697,559 to 1,25-dihydroxycholecalciferol; 3,741,996 to loc-hydroxycholecalciferol; 3,907,843 to Icc-Hydroxyergocalciferol; 3,715,374, directed to 24,25-dihydroxycholecalciferol; 3,739,001, directed to 25,26-dihydroxycholecalciferol; 3,786,062, directed to 22-dehydro-25-hydroxycholecalciferol; 3,847,955 directed to 1,24,25-trihydroxycholecalciferol; 3 9Ο6 014 directed to 3-deoxy-ia-hydroxycholecalciferol.

Description of the invention

A new derivative of vitamin D has now been found that develops a vitamin D-like activity and is believed to that it can be a metabolically active form of the vitamin, responsible for some of the biological reactions mentioned above. This derivative was identified as 25-Hydroxycholecalciferol-26,23-lactone (or 23,25-dihydroxy-26-carboxy-vitamin-D, -7'-lactone). The formation of this compound in high yields from vitamin D is consistent with the assumption that that it is possibly a metabolically active form of the vitamin responsible for some of the biological ones

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Reactions caused by vitamin D.

Isolation and characterization of the vitamin D derivative (metabolite) of the invention was carried out using in one case plasma from chicks which had received normal vitamin D levels in their diet and in the second case plasma from chicks which large doses of vitamin D had been administered. The respective isolation and identification procedures performed "~ were listed in the following examples.

example 1

Isolation and characterization of the metabolite from plasma from chicks raised on maintenance levels of vitamin D.

became. - ———

Twenty-four liters of chick blood was obtained from 8-week-old "roast poultry" (A-G Coop, Arcadia, Wisconsin) who received maintenance levels of vitamin D in their diet ((SO 1000 IU of vitamin D, per pound of food). During production To prevent coagulation, 10% by volume of 0.1 m sodium oxalate, pH 7 »0, added. The blood was separated in a De Laval blood separator to produce 16 liters of plasma.

The plasma was prepared in 1 liter batches using the following procedure extracted:

Plasma was heated with stirring at 70 ° C. for 1 hour and then centrifuged at 13,000 rpm for 1 hour in a Beckman J-21C centrifuge equipped with a JA-14 rotor (Beckman Instruments, Inc.). The pellet obtained was suspended in 400 ml of distilled water and ^{extracted for 1 hour at 4 °} C. with 800 ml of methanol and 400 ml of chloroform. Four hundred milliliters of chloroform were then added, the chloroform layer was removed and the aqueous phase washed with a further four hundred milliliters of chloroform. From the

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the combined chloroform phases, the solvent was removed by evaporation.

All of the chloroform extract was partitioned into 500 ml of hexane and 500 ml of 10 % water in methanol with shaking for 1 hour. Five hundred milliliters of chloroform were then added to the water-methanol phase. The chloroform phase was collected and the aqueous phase was washed with 500 ml of chloroform twice. The combined chloroform phases were concentrated and used for subsequent chromatography.

The concentrated chloroform extract was chromatographed in four batches on a 3 χ 30 cm Sephadex LH-20 (a hydroxypropyl ether derivative of a polydextran, commercial product of Pharmacia Fine Chemicals, Inc., Piscataway, NJ) column, eluted with νιΛιΛ hexane: chloroform: methanol. The column fractions were determined using the competitive protein binding Eadioassay method of Haddad et al. (Arch. Biochem. Biophys. 182 , 390, 1977) and the binding peak that eluted in the 618 to 807 ml Begion was pooled and concentrated.

The combined fractions from this peak were further chromatographed in three batches on a 2 × 55 cm Sephadex LH-20 column, eluted with 70:30 chloroform: hexane. The fractions were scored as above and the binding fractions which eluted in the 24,25-dihydroxyvitamin-D, (24,25- (0H) ₂ D,) area (2 ^ 2 to 318 ml) were pooled and concentrated.

This pooled fraction was further chromatographed in three batches on a 1 × 58 cm Sephadex LH-20 column, eluted with 9: 1: 1 hexane: chloroform: methanol. The fractions were scored as above, and those which eluted in the 25i26-dihydroxTvitamin-D, (25.26- (0H) ₂ D,) area (133-162 ml) D were pooled and concentrated.

A high pressure liquid chromatography (HPLC) was performed on this Component performed using a

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Father's model ALP / 6PC 204-Instruments (Waters Associates, MiIford, Mass.) Equipped with a Model 440 absorbance detector that "monitored at 254 mm and a 0.46 χ 25 cm Partisil ODS- (octadecylsilane bound to silicon dioxide, commercial product from Whatman, Inc., Clifton, New Jersey) column ₄ eluted with 25% water in methanol, fractions were scored and the binding peak corresponding to the UV (254 nm) absorption peak eluting from 25.5 to 28.5 ml, was pooled. This component was subjected to another HPIX3 on a 0.46 χ 25 cm microparticle silica gel column (Waters Associates), eluted with 8 % isopropanol in Hexar *. Fractions were scored as above and the only binding component (and Major UV (254 nm) absorption peak eluting from 14.5 to 16.5 ml »was pooled, this fraction was chromatographed again using the same system and the single UV absorption peak was collected.

The UV spectra of the collected compound in ethanol gave ^{264 1} ^ ¹ '^ min * ²² ^ ¹ ^ ^10D 264' ^{/ 0D} 229 ^{β 1} I ⁴⁰ * ^a

of 8 µg was assumed to have an extinction coefficient of 18,600 and a molecular weight of 428.

The mass spectrometry of the compound gave the following ions and intensities: m / e 428, 27.6 #, M ⁺ ; m / e 410, 4.1%, M ⁺ -H ₂ O; m / e 395, 12%, M ⁺ -H ₂ O-CH ₅ ; m / e 2? 1; 4.5%, M ⁺ side chain; m / e 253, 9.3%, M ⁺ side chain H ₂ O; m / e 136, 100%, A-Hing + C-6, C-7 and C-19; m / e 118, 94%, Α-ring and C-6, C-?, C-19-H ₂ O.

High-resolution mass spectrometry of the molecular ion of the compound gave a weight of 428.2901 for a formula of ^C 27 ^H 40 ° 4 ( ^calculated weight «= 428.2926).

The trimethylsilyl derivative of the compound was prepared from 1 µg using 30 λ pyridine and 25 Ti N, O-bis (trimethylsilyl) trifluoroacetamide at 55 ° C for 40 minutes. The reaction mixture was directly mass spectrometry

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- / Λ

subjected to and found: m / e 572, 20%, M ⁺ ; m / e 482, 15 %, M ⁺ -HOTMS; m / e 467 »IO%, M ⁺ -HOTMS-CH ,; m / e 208, 34 #, A-in fragment; m / e 118, 100 #, A-fiing-Fragment-HOTMS.

Example 2

Isolation of the metabolite from plasma of chicks given large doses of vitamin D ^ .

Seventy-one, 10 week old male chicks were intramuscularly administered 10 IU vitamin D ₅ in 50 A) (U-0.001 cc) ethanol x daily for three days. They were given

followed by 10 'IU of vitamin D intramuscularly in four dosages administered in 50 TV ethanol, five days after the last Dosing, the chicks were bled by cardiac puncture using a small amount of heparin for avoidance the coagulation. The blood was immediately centrifuged to produce 1100 ml of plasma.

The plasma was extracted in approximately 400 ml batches with 800 ml methanol and 400 ml chloroform. After standing for 1 hour at 4 ^° C., a further 400 ml of chloroform were added and the chloroform phase was collected. The aqueous phase was washed with 400 ml of chloroform and the combined chloroform phases were concentrated by evaporation of the solvent for chromatography.

The entire concentrated extract was at a 3 * 30 cm Sephadex LH-20 column chromatographed, eluted at 9: 1: 1 Hexane: Chloroform: Methanol «The fractions were evaluated as above and the binding component used in the field from 608 to 931 ml eluted, pooled and concentrated. This peak was then chromatographed on a 2 × 55 cm Sephadex LH-20 column, eluted with 70:30 chloroform: Hexane. The fractions were scored and the binding component that eluted in the 240-258 ml area was pooled and focused. This fraction was an HPLC on a

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0.46 × 25 cm Partisil-ODS column, eluted with 25 % water in methanol. The major UV (254 nm) absorption peak was collected (18-22.5 ml) and chromatographed again using the same system. The UV (254 nm) absorbing substance was further purified by HFLC on a 0.46 × 25 cm microparticle silica gel column, eluted with 8 % isopropanol in hexane. The UV (254 nm) absorbing compound eluting from 15-17 ml was collected and purified again on the same system.

The UV spectra of these samples and those produced in the same way gave / l _max »264 nm, 1 _min ^{= 228 nm} ' ^OD 264 ^{/ OD} 228 ^{= 1} ' ⁵¹ · A total mass of 56 jig was obtained, calculated assuming £« 18,600 .

The high-resolution mass spectrometry on this compound gave: m / e 428, 27.6%, M ⁺ (27 ² ^ oV ^{; m / e} m / e 395 »12%, M ⁺ -H ₂ O-CH ₃ ; m / e 271, 4.5%, M ⁺ side chain; m / e 253, 9.3% »M ⁺ side chain-H ₂ O; m / e 136, 100%, A-in ⁺ ; m / e 118, 94 %, A- ⁺

The mass spectra of the TMS derivative of the compound (prepared as described above) found: m / e 572 x 20%, M; m / e 482, 15%, M ⁺ -HOTMS-ZmZe 467, 10%, M ⁺ -HOTMS-CH ₃ ; m / e 208.34%, A-ring ⁺ ; m / e 118, 100%, A-Ring ⁺ -HOTMS.

The UV spectra and mass spectra of the isolated compound, as well as the mass spectra of the TMS (trimethylsilyl) derivative, were identical to those of the compound obtained from the plasma of chicks _{raised with maintenance levels of vitamin D 3} , indicating that the identical compound was obtained for each isolation.

The compound was again purified by HPLC on silica gel, purified as described above and proton NMR was performed in CDCli solution recorded on a 270 megahertz instrument. The spectrum obtained was identical to that of

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25-Hydroxyvitamin-D, (25-OH-D,) with the exception of the following resonances: £ 4.46, m, 1H, C-23-H; 6 1.56, s, 3H ₁ C-27-H ,; • 5 1.09, d, J »6.3 Hz, sH, C-21-H ₃ ; £ 0.63, s, 3H, C-18-H ,.

A Fourier Transform Infrared Spectrum (PT-IR) was obtained from this connection on a Nicolet-7199-ϊΤ-IR instrument (Nicolet Instrument Corp., Madison, Vis.). Bas spectrum obtained was very similar to that of a vitamin D spectrum with the exception of an intense absorbance at 1787 cm, which is an indication of a 7 * "- La et on.

The methyl lactol product of the TMS derivative of the compound was prepared by mixing 1 ug of (TMS ^ compound with 25 % 0.01 M MeLi in diethyl ether at room temperature for 1 hour. The reaction mixture was quenched with 50 of water and twice with 200 λ. The reaction mixture was purified by HPLC on a 0.4 x 25 cm silica gel column, eluted with 5 % ethyl acetate in hexane. The UV (254 nm) absorbing compound (eluted from 30 to 42 ml) was collected and subjected to mass spectrometry The following diagnostic ions of methyl lactol were obtained: m / e 588, 4.4%, M ⁺ ; m / e 570, 7.3%, M ⁺ -H ₂ O; m / e 528, 1.5%, M ⁺ -HOAc; m / e 498, 2%, M ⁺ -HOTMS; m / e 208, 33 %, A-ring ⁺ ; m / e 118, 100%, A-ring ⁺ - HOTMS.

The methyl lactol was silylated as above and the mass spectra gave the following diagnostic ions: m / e 660, 12.7%, M ⁺ ; m / e 645, 1.8%, M ⁺ -CH ₅ ; m / e ^{570.4%, M +} -HOTMS; m / e ^{555.2%, M +} -HOTMS-CH ,; m / e 528, 10%, M ⁺ -AcOTMS; m / e 208, 54%, A-ring ⁺ ; m / e 118, 100%, A-Ring ⁺ -HOTMS.

The only structure consistent with all of the above spectral data, i.e. H. UV, IR, CMR and mass spectrometry is consistent is the 25-hydroxycholecalciferol-26,23-lactone or 3β, 25-dihydroxy-9,10-seco-5,7,110 (19) -cholestÄ-trieno-26,23-lactone.

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The compound according to the invention can be characterized by the following formula:

Male batten (Holtzman Co., Madison, Vis.) Were individually placed in hanging wire cages and given food and water ad libitum. They were on the purified low calcium diet described by Suda et al. For three weeks. (J. Nutr. 100, 1049-1050 (197O)). The rats were administered lactone 25-hydroxy-cholecalciferol-26,23-in groups of seven intrajugularly 50 ^ ethanol (control) or 50 \ ethanol containing 300 ng.

Twelve hours after the administration, the sats were beheaded and the blood was collected. It was centrifuged immediately and 0.1 ml of serum was diluted with 1.9 × al 0.1% lanthanum chloride solution. Serum calcium concentrations were determined using a Model 403 Atomic Absorption Spectrometer (Perkin-Elmer, Horwalk, Conn.). The results obtained are shown in the following table:

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Control Serum calcium (mg / 100 ml) rat rat Lactone No. 4.52 Mr. 1 4.26 1 4.33 2 3.94 2 4.48 3 4.20 3 4.67 4th 4.39 4th 4.84 5 4.70 5 4.72 6th 4.50 6th 5, cn 7th "-" "'7, - 4.63

Average - S.D. 4.36 ± 0.25 avg ± S.D. 4.67-0.22

The calcium levels of the lactone-dosed rats differed differs considerably from that of the controls with P <0.05, indicating that the 25-hydroxycholecalciferol-26,23-lactone has a vitamin D-like effectiveness in mobilizing calcium from bones.

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Claims (1)

.3- Wisconsin Alumni Besearch Foundation 614 North Walnut Street Madison Wisconsin 53705 / V.St.A claim 1. 25-Hydroxycholcalciferol-26,23-lactone. 030606/0050