J. Nutr. Sci. Vitaminol., 22, 299-306, 1976

SYNTHESIS AND BIOLOGICAL ACTIVITY OF 5, 6-TRAINS- D3 IN ANEPHRIC RATS1

Tadashi KOBAYASHI,2 Sachiko MORIUCHI,3 Fumio SHIMURA,3 and Goichiro KATSUI4

2 Department of Hygienic Chemistry, Kobe Women's College of Pharmacy, Higashinada-ku, Kobe 658, Japan 3 Nutritional Laboratory, School of Health Sciences, Faculty of Medicine, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan 4Eisai Research Laboratories , Bunkyo-ku, Tokyo 112, Japan (Received February 17, 1976)

Summary A modified procedure for the purification of 5, 6-trans-D3 from a reaction mixture of vitamin D3 with iodine and its biological activity in sham-operated and anephric rats are described. When a solution of , in n-hexane was reacted with small quantities of iodine under influence of visible light, the reaction mixture gave four or five spots, including vitamin D3 and 5, 6-trans-D3, on a thin-layer chro matogram. After purifying the mixture by alumina column chromatog raphy, a colorless oil from the separated 5, 6-trans-D3 fractions was crystallized from n-hexane and snow white crystalline 5, 6-trans-D3 was ob tained. The purification method was thought to be better than the previ ously reported methods (14-16) because it gave good yield without the

procedure of esterification using expensive p-phenylazobenzoyl chloride. A dose of 25ƒÊg of 5, 6-trans-D3 obtained thus gave significant elevation on intestinal transport in both sham-operated and anephric rats, whereas the dose did not give positive effects on serum calcium levels in anephric rats. Serum phosphorous levels were extremely elevated by neph rectomy, but in both sham-operated and anephric rats they were unaf fected by the administration of 25ƒÊg of 5, 6-trans-D3.

It has been documented that vitamin D, is hydroxylated on the 25-position in the liver and then on the lƒ¿-position in the kidney to give lƒ¿, 25-(OH)2-D3, the

1 Following abbreviations are used: 5 , 6-trans-D,-D, and -D3, 5, 6-traps-calciferol, -ergo calciferol and -cholecalciferol; lƒ¿, 25-(OH)2-D3, 1ƒ¿, 25-dihydroxycholecalciferol; lƒ¿-OH-D3, lƒ¿-hydroxycholecalciferol; pyro-D3, pyrocholecalciferol; isopyro-D3, isopyrocholecalciferol; TLC, thin-layer chromatography; GLC, gas-liquid chromatography; UV, ; IR, in frared; NMR, nuclear magnetic resonance; TMS, trimethylsilyl for GLC and tetramethylsilane for NMR; I. U., . 2 小 林 正 , 3森 内 幸 子,志 村 二 三 夫, 4勝 井五 一 郎

299 300 T. KOBAYASHI, S. MORIUCHI, F. SHIMURA , and G. KATSUI

active form of the vitamin (1-3). This metabolite has the biological activity in stimulating intestinal calcium transport and bone calcium mobilization (4 , 5). Since lƒ¿-OH-D3, a synthetic analog, also showed similar activity in both normal and anephric rats, the lƒ¿-OH group in the molecule of 1ƒ¿ , 25-(OH)2-D3 was thought to be the most important function for the initiation of the activity (6-8) . Although 1ƒ¿-OH-D3 obtained by many attractive synthetic courses (7 , 9-12) is a valuable substitute for 1ƒ¿, 25-(OH)2-D3, the syntheses are still complicated and

expensive. Therefore, investigations to find more convenient substitutes are being continued. HOLICK et al. (13) reported that 5, 6-traps-D3, an isomer of

vitamin D3 (5, 6-cis form), showed similar biological activity as 1ƒ¿, 25-(OH)-D3 in both normal and anephric rats though that of the former was weaker than that of

the latter. It was assumed that the 3ƒÀ-OH group in 5, 6-trnas-D3 might act as a

pseudo lƒ¿-OH group because the configuration between 3ƒÀ-OH and 5, 6-double bond resembles that between 1ƒ¿-OH and the double bond in 1ƒ¿, 25(OH)2-D3 or

1ƒ¿-OH-D3 as shown in Fig . 1(13).

Fig. 1. Chemical structures of vitamin D3 and related compounds .

5,6-Trans-D2 was first synthesized by VERLLOOPet al. (14, 15) through the reaction of vitamin D2 with small quantities of iodine under influence of visible light, while INHOFFENet al. (16) prepared 5,6-traps-D3 from vitamin D3 by a similar method. We reinvestigated the methods and modified the purification procedure in order to get a good yield without using an expensive process. The biological activity in sham-operated and anephric rats was investigated by using the compound thus obtained. In this paper, the modified procedure for puri fi cation of synthetic 5,6-traps-D3 and its biological activity are described.

EXPERIMENTAL UV absorption spectra were obtained on a Hitachi 323 spectrophotometer with ethanol as a solvent; IR spectra were obtained on a Shimadzu IR-27G infracord spectrophotometer with CCl4 as a solvent; NMR spectra were obtained on a Varian A-60D spectrometer (60 MHz) with CDCl3 as a solvent and tetra methylsilane (TMS) was used as an internal standard; mass spectra were re SYNTHESIS AND BIOLOGICAL ACTIVITY OF 5, 6-TRANS-D3 301 corded on a double focus high-resolution spectrometer (Japan Electron Optics JMS-01SG) equipped with a direct inlet system. Melting points were estimated in open capillary tubes and are uncorrected.

Gas-liquid chromatography (GLC)

GLC was carried out with a Shimadzu GC-3BF gas chromatograph equipped with a hydrogen flame ionization detector. A glass column (0.3•~260cm) packed

with 1.5% OV-17 on Shimalite W (80-100 mesh) was run at 225•Ž with a nitrogen flow of 120 ml/min. Trimethylsilylation was performed by mixing a sample

solution in pyridine containing 5-10mg/ml of the solute concentration (1ml) with hexamethyldisilazane (0.5ml) and trimethylchlorosilane (0.1ml). After

allowing the mixture to stand for 10 min at room temperature, the precipitation formed by addition of reagents was removed by centrifugation for 15 min, and

then on 5ƒÊl of the supernatant taken with a microsyringe GLC was performed.

Thin-layer chromatography (TLC) A sample solution in a suitable organic solvent was spotted on a Kieselgel

GF254 plate (250ƒÊ-thick, 20•~20cm) activated at 110•Ž for 1 hr and then devel oped with a mixed solvent of benzene-acetone (95:5). Spots were detected under

a 254nm UV light or visualized by spraying with 20% p-toluene sulfonic acid in ethanol and heating.

5, 6-Trans-vitamin D3

To a solution of vitamin D3 (10g) in n-hexane (900ml), a solution of iodine

(20 mg) in n-hexane (100ml) was added, and the mixture was stirred at room tem

perature for 30 min under the visible light of an ordinary 20 W light bulb. The isomerization reaction was stopped by adding 1% sodium thiosulfate solution

(500 ml). The mixed solution was transferred to a separatory funnel, separated, and the water layer was discarded. The organic layer was washed with water, dried over anhydrous sodium sulfate and then filtered. The filtrate was evaporated

to dryness under reduced pressure and 10g of slightly yellow oil was obtained. The oil was dissolved in 50ml of 50% ethyl ether in n-hexane and applied to 350g

of Merck Brockmann neutral alumina column (3.2•~47cm) packed with the same solvent mixture. The column was developed by 50% ethyl ether in n-hexane and

each 200ml fraction was collected. The 5, 6-trans-D3 fractions (fractions 3-6) detected by TLC were collected and evaporated to dryness under reduced pressure.

The resulting colorless oil (4.2g) was dissolved in n-hexane (40ml) and left over night to crystallize in a freezer. The resulting snow white crystals were recrystal

lized from the same solvent. Yield 3.0 g (30%); mp 89-90•Ž; UV (nm), ƒÉAmax 273

(ƒÃmax 23,600); IR (cm-1), 3630 (3-OH), 890 (19-=CH2); NMR (ƒÂ), 0.56 (s, 18-CH3), 0.89 (d, J=5.5 Hz, 26 and 27-CH3), 0.95 (d, J=4.5 Hz, 21-CH3), 1.58 (1 H, s, 302 T. KOBAYASHI, S. MORIUCHI, F. SHIMURA, and G. KATSUI

3-OH), 3.92 (1H, m, 3-CH), 4.53 and 5.00 (2H, m, 19-=CH2), 5.87 and 6.57 (2H, d,d of AB type, J=12 Hz, 6, 7-CH=CH-); mass spectrum (m/e, rel. intensity), 384 (M+, 31), 271 (11), 253 (13), 136 (100), 118 (82); Anal Calcd. for C27H44O, C 84.31, H 11.53, Found, C 84.02, H 11.25.

Biological activity of 5, 6-trans-vitamin D3

1) Animals. Weanling female rats of the Wistar strain (40-60g) were fed ad libitum a vitamin D-deficient diet (Ca, 0.47%; P, 0.3%) (17) for 6 weeks and then a vitamin D-deficient low calcium diet (Ca, 0.02%; P, 0.3%) (17) for one more week. The animals, weighing about 130g, showed low serum calcium concentrations (5.0-5.5mg/dl). They were starved 24hr before sacrifice. The animals were divided into four groups of four rats each. Two groups were bilaterally nephrectomized, while the others were sham-operated. One of the nephrectomized groups and one of the sham-operated groups received intra jugularly a solution of 5, 6-trans-D3 (25ƒÊg) in 95% ethanol (50ƒÊl), while the other group of each (controls) similarly received 95% ethanol (50ƒÊl) only. At 24hr after dosing, the animals were decapitated and the duodena and blood were collected for the following experiments. 2) Intestinal calcium transport assay. Intestinal calcium transport assay using everted sac and 45Ca was performed according to the procedure of MARTIN and DELUCA (18).

3) Serum calcium assay. Serum obtained by centrifuging the blood was diluted with 0.5% La2O3 solution in 25% hydrogen chloride to make a suitable concentration (19). Serum calcium concentration was estimated with a Shimadzu

AA-610S atomic absorption spectrometer. 4) Serum phosphorous assay. Serum phosphorous concentration was esti mated according to the procedure of CHEN et al. (20).

RESULTS AND DISCUSSION 1.Synthesis of 5,6-traps-vitamin D3 When vitamin D3 was reacted with iodine according to EXPERIMENTAL, the reaction mixture showed an absorption maximum at 272 nm in the UV spectrum. Four or five spots, including those of vitamin D3 and 5, 6-trans-D3, were observed on a thin-layer chromatogram as shown in Fig. 2, while five large peaks, including those of pyro and isopyro-D3 TMS ethers derived from thermal cyclization of vitamin D3, were observed on the gas chromatogram as shown in Fig. 3. After purifying the mixture by alumina column chromatography, 5,6 trans-D3 was separated from other compounds as shown in the thin-layer chromato grams of each fraction (Fig. 2). The colorless oil obtained from the 5,6-trans-D3 fractions was very easily crystallized from n-hexane. The NMR and mass spectra of 5,6-trans-D3 are shown in Figs. 4 and 5. The gas chromatogram of purified SYNTHESIS AND BIOLOGICAL ACTIVITY OF 5, 6-TRANS-D3 303

Fig. 2. Thin-layer chromatogram of vitamin D3, 5, 6-trans-D3, reaction mixture of vitamin D3 with iodine and the fractions obtained by alumina column chromatography. D3, vitamin D3; t. D3, 5, 6-trans-D3; C, crude reaction mixture.

Fig. 3. Gas chromatograms of vitamin D3, 5, 6-trans-D3 and reaction mixture of vitamin D3 with iodine. The numbers described in the gas chromatograms indicate retention times.

5, 6-traps-D3 gave a main peak with a few minor peaks as shown in Fig. 3. These minor peaks might be due to thermal decomposition products formed from 5,6 trans-D3 during GLC, because VERLOOPet al. (15) mentioned that 5, 6-trans-D3 304 T. KOBAYASHI, S. MORIUCHI, F. SHIMURA, and G. KATSUI

Fig. 4. NMR spectrum of 5, 6-traps-D3. TMS, tetramethylsilane.

Fig. 5. Mass spectrum of 5, 6-trans-D3.

was decomposed by heating at 180•Ž even in nitrogen gas and the temperature of the GLC was higher than 180•Ž.

VERLOOP et al. (14, 15) and INHOFFEN et al. (16) purified 5, 6-trans-D2 and-D3 as the crystalline p-phenylazobenzoates from the reaction mixtures, respectively.

Crystalline 5, 6-trans-D2 and-D3 were individually obtained from the unsaponi fi able matters of the corresponding esters. Both 5, 6-trans-D and their esters were

crystallized from acetone, but the methods were very difficult and gave poor

yield when we reinvestigated them. On the other hand, the presently proposed crystallization method using n-hexane is very easy and gives good yield without

the procedure through esterification, using expensive p-phenylazobenzoyl chloride.

2. Biological activity of 5, 6-trans-vitamin D3 The effects of the synthesized 5, 6-trans-D3 on intestinal calcium transport and

serum calcium and phosphorous concentrations were determined and the results

are shown in Table 1. A dose of 25ƒÊg of 5, 6-trans-D3 gave significantly higher elevation on intestinal calcium transport in both the sham-operated and nephrecto

mized groups when these were compared with those of the corresponding control

groups. Therefore, 5, 6-trans-D3 was confirmed to have a similar activity as SYNTHESIS AND BIOLOGICAL ACTIVITY OF 5, 6-TRANS-D3 305

Table 1. Effects of 5, 6-trans-D3 on intestinal calcium transport, serum calcium and phosphorous concentrations in sham-operated and nephrectomized rats.

Each value is shown as M•}SE. Sham, sham-operated; Nx, nephrectomized. The comparison was individually made between corresponding sham-operated and

nephrectomized rats. The values of a are significantly different from those of b (P<0 .001).

1ƒ¿, 25-(OH)2-D3 on the intestinal calcium transport, though a comparatively large dose was necessary.

The serum calcium levels of the sham-operated rats were clearly elevated by the administration of 5, 6-trans-D3 like as 1ƒ¿, 25-(OH)2-D3 or 1ƒ¿-OH-D3, whereas

those of nephrectomized rats were unaffected by dose. The results were slightly different from those of HOLICK et al. (13). In the previous investigations on 1

ƒ¿ OH-D3 using similarly fed rats (8), the serum calcium levels of nephrectomized rats were elevated by a dose of 6,250 pmole but not by that of 312 pmole (prob

ably physiological dose) of lƒ¿-OH-D3. The results were also slightly different from those of HOLICK et al. (6) and KANEKO et al. (7), and we assumed that the

difference might be derived from the difference of feeding conditions (8). From

these considerations, higher doses than 25ƒÊg of 5, 6-traps-D3 might have positive effects on the serum calcium levels of the nephrectomized rats in this experiment,

too. However, since 25ƒÊg of 5, 6-traps-D3, corresponding to 1,000 I. U. of vitamin D3, was thought already to be overdoses, doses higher than 25ƒÊg were not in

vestigated at this time. Such experiments will be done in future. Serum phosphorous levels showed extreme elevation by nephrectomy, but

these in both sham-operated and nephrectomized rats were unaffected by the administrations of 5, 6-trans-D3 as shown in Table 1. The results were not corre lated with those of serum calcium levels. Therefore, the simple assumption that

elevation of serum phosphorous levels may give inhibitory effects on elevation of

serum calcium levels must be denied. From these results and considerations, we assumed that some unidentified metabolite active to bone might exist besides

1ƒ¿, 25-(OH)2-D3.

The authors wish to thank Miss H. Endo and Miss K. Saiki of Kobe Women's College of Pharmacy for making elemental analysis and mass spectra measurements. 306 T. KOBAYASHI, S. MORIUCHI, F. SHIMURA, and G. KATSUI

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