1α,25-Difluorovitamin D3: An inert vitamin D analog

ARCHIVES

Vol.

OF BIOCHEMISTRY

209, No. 2, July,

AND

BIOPHYSICS

pp. 579-583,

1981

la,25Difluorovitamin HERBERT

E. PAAREN,

D,: An Inert Vitamin D Analog’ MARY A. FIVIZZANI, HEINRICH HECTOR F. DE LUCA2

K. SCHNOES,

AND

Department University

of Biochemistry, College of Agricultural and Life Sciences, of Wisconsin-Madison, Mad&m, Wisconsin 53706 Received

November

19, 1980

la@-Difluorovitamin D3 has been synthesized by reacting 1,25-dihydroxyvitamin Ds3-acetate with diethylaminosulfurtrifluoride followed by hydrolysis. Retention of configuration of the fluoro group in this reaction was demonstrated by physical studies using la-fluoro and l@-fluorovitamin D3 models. The 1,25-difluorovitamin D3 compound possessed no vitamin D-like activity demonstrating the importance of la- and 25-hydroxylations of vitamin D for activity. However, 1,25-difluorovitamin D3 had no anti-25-hydroxylation activity and no antivitamin D activity. Since 25-fluorovitamin D3 has anti25-hydroxylase activity, it appears the introduction of a fluoro group on the 1 position diminishes interaction of the vitamin D molecule with the 25-hydroxylase system.

Many areas of biochemical research have benefited from the use of fluorinated derivates as metabolic probes (1, 2). Fluorine’s utility in this area arises from its unique physical, chemical, and electronic properties. Because of its small atomic radius, approximtely equal to that of a hydrogen atom, it readily substitutes for hydrogen (3). The strength of the carbonfluorine bond, reflected by its stability with respect to biochemical transformations, makes it a useful metabolic block and its highly electronegative nature can perturb binding affinities. Recently, a number of fluorinated analogs have been used to explore different aspects of the vitamin D endocrine system (4-6). The initial metabolic conversion of vitamin D3 to 25-hydroxyvitamin D3 (25OH-D3)3 is a unique control point in the 1 Supported by program-project Grant AM-14881 from the National Institutes of Health and the Harry Steenbock Research Fund of the Wisconsin Alumni Research Foundation. ’ To whom correspondence should be addressed. 3 Abbreviations used: 25-OH-4, 25-hydroxyvitamin D3; 25-F-4,25-fluorovitamin Da, l-F-4, l-fluorovitamin Da; 25-OH[sH] D,,, 25-hydroxy[SHlvitamin Da; la,25-FzD3, l&Z-difluorovitamin D3; la,25-

expression of vitamin D-like activity because all known active metabolites of vitamin D are 25-hydroxylated (7). A potential inhibitor of this enzymatic conversion, 25-fluorovitamin D3 (25-F-D3) (8) has been synthesized and bioassayed. It was found to be an effective inhibitor of the in viwo conversion of [3H]vitamin D3 to 25hydroxy[3H]vitamin D3 (25-OH-[3H]D3). However, at high concentrations 25-F-D3 itself possesses biological activity probably because it is metabolized to a biologically active form, presumably a la-hydroxylated form. 1-Fluoro-vitamin D3 (lF-DQ) has also been synthesized and shown to possess biological activity presumably because of 25-hydroxylation (9). It therefore appeared that blocking both the laand 25-positions with fluoro groups should provide a form of vitamin D that would be devoid of vitamin D activity and might be a potent vitamin D antagonist. MATERIALS

AND

METHODS

Vitamin D3 Compounds. Vitamin from Phillips-Duphar (Amsterdam,

D8 was purchased Holland). la,25-

(OH)zD3, la,25-dihydroxyvitamin Ds; DAST, diethylaminosulfurtrifluoride; tic, thin-layer chromatography; hplc, high-pressure liquid chromatography.

579

0003-9861/81/080579-05$02.00/0 Copyright

0 1981 by Academic

Press. Inc.

580

FIG. 1. Synthesis of la-25-difluorovitamin

PAAREN

Dz.

Dihydroxyvitamin Dz (la-25-(OH)zD,) was a gift from Hoffmann-LaRoche, Inc. (Nutley, N. J.). [3a3H]Vitamin Dz was synthesized according to the procedure of Yamada, Schnoes, and DeLuca (unpublished results). la,25-Difluorovitamin Dz (la,25-FzD,) was synthesized from either la,25-(0H)zDz acetate (Scheme a) or la,25-(OH)*-cyclovitamin Dz (Scheme b) (see Fig. 1). Diethylaminosulfurtrifluoride (DAST) was synthesized by the method of Middleton (10). Scheme a. To 1.6 mg of la,25-(0H)zDz acetate (11) in 400 ~1 of dry CHzClz at -70°C was added 10 ~1 of DAST. The reaction was removed from the cooling bath for 3 min then quenched with 10% NaHCO,. The aqueous reaction mixture was diluted with Hz0 and extracted with CHzClz. The organic extract was washed with water, dried over MgS04, and concentrated in oacuo. The crude oil was then taken up in 0.5 ml of ethanol and treated with 100 ~1 of 0.1 M KOH/HzO for 2.0 h. After addition of HzO, the reaction was extracted twice with lo-ml portions of EtzO, and the organic phase was washed with HzO, dried over MgS04 and concentrated to an oil which was chromatographed on a 10 X 20-cm, 750~pm silica gel plate in 35% ethyl acetate in hexane to yield 560 pg of la,25-Fz-Da: uv: X,,, 265 nm; NMR, 6 0.53 (3H, s, l%CHz), 0.93 (3H, d, J = 6.0 Hz, 21-CHz), 1.34 (6H, d, J = 22 Hz, 26- and 27-CHz). 3.98 (1H. m, 3-H), 5.02 (lH-d-m, J = 52 Hz, I-H), 5.12 (lH, m(sharp), 19(Z)H), 5.41 (lH, m(sharp), 19(E)-H), 6.03 (lH, d, J= 11.0 Hz, 7-H), 6.44 (lH, d, J = 11.0 Hz, 6-H); mass spectrum, m/e, 420 (M+, 20), 400 (70), 382 (35), 135 (100). Scheme b. To 5.0 mg of l&5-(OH),-3,5-cyclovitamin D3 (11) in 1.0 ml of CHzClz at -70°C was added 25 ~1 of DAST. The reaction was taken from the cooling bath and 3.0 min later quenched with 10% NaHCOz. After diluting with HzO. the crude mixture was extracted with CHzClz, the organic phase washed with HzO, dried over MgSO,, and concentrated in vacua to an oil. The oily product was purified on preparative thin-layer chromatography (tic) (750 pm, 10 X 20-cm silica gel plate developed in 40% ethyl acetate/hexane) to yield 3.8 mg of la,25-difluoro-3,5cyclovitamin Da: NMR, 60.53 (3H, s, 18-CHz), 0.93 (3H, d, J = 6.0 Hz, 21-CHe). 1.34 (6H, d, J = 22 Hz, 26- and 27-CHz), 3.25 (3H, s, 6-OCH3) ,4.18 (lH, d, J= 9.6 Hz, 6-H), 4.97 (lH, d, J= 9.6 Hz, 7-H), 5.18 (lH, dd, J= 58 Hz, l-H), 5.45 (lH, d, J= 4.0 Hz, 19(2&H), and 5.51 (lH, d, J = 4.0 Hz, 19(E)-H); mass spec-

ET AL. trum, m/e: 434 (M+, 40), 402 (50),. 271 (50), 245 (40), 135 (100). A 3.0-mg portion of the above product was taken up in 300 ~1 of glacial acetic acid and heated to 55’C for 15 min. The reaction was cooled, quenched with ice/saturated NaHCOe, and extracted with EtzO. The organic extract was washed with water, dried over MgS04, and concentrated to an oil which was taken up in 1.0 ml of EtOH and treated with 200 ~1 of 0.1 M KOH/HzO for 2.0 h at room temperature. After workup and preparative tic (see Scheme a) 1.3 mg of l&5-Fz-Dz was obtained which was identical in all respects to the product obtained from Scheme a. Animals. Male albino weanling rats were obtained from Holtzman Company (Madison, Wis.), housed in overhanging wire cages and fed ad lititum a vitamin D-deficient, low-calcium diet (12). The animals were maintained on this diet for 3.0 to 3.5 weeks prior to their use in bioassays. Weight-matched (140 + 10 g) male albino rats were raised on a vitamin D-deficient diet containing adequate calcium levels for 5.0 weeks prior to use in the metabolism studies. Biological assays and metabolism studies. The biological activity of la,25-FzDz was tested by administering graded doses to vitamin D-deficient rats in 50 pl of ethanol intrajugularly under light ether anesthesia. The inhibitory properties of la,25-FzDz were tested by administering the fluoro analog as above followed 2 h later by a second dose of vitamin De by the same route of administration. The animals were sacrificed and serum calcium was determined in 0.1% LaClz by atomic absorption spectroscopy 24 h after the second dose. Duodenal calcium transport was determined using the everted gut sac technique of Martin and DeLuca (13). For the metabolic studies vitamin D-deficient rats received either 54 c(g of la,25-FzDz (1000 fold excess) in 50 pl of ethanol or 50 ~1 of ethanol intrajugularly under light ether anesthesia followed 2 h later by 50 ng of [3cY-3H]-vitamin Dz (1.0 &i/dose) in 50 pl of ethanol intrajugularly. The animals were killed 24 h later and the serum was extracted according to Bligh and Dyer (14). The serum extracts were chromatographed on a column (1 X 37 cm) of Lipidex-5000 eluted with hexanes:CHCle (9O:lO). Then 3.0-ml fractions were collected and evaporated, and total radioactivity was determined in toluene-counting solution (2 g of 2,5-diphenyloxazole and 0.1 g of 1,4-bis-2-(4methyl-5-phenyloxazolyl)benzene per 1 liter of toluene). Metabolite levels are expressed as nanograms of metabolite per milliliter of serum. RESULTS

The synthesis of la,25-FzD3 was accomplished via two related pathways. The first involved direct fluorination of la,25(OH)2D, 3-acetate with DAST in CH&%

l&5-DIFLUOROVITAMIN

at -78°C. This produced the 1,25-FzD3 acetate in -80% yield which was subjected to mild basic hydrolysis (0.1 N KOH/EtOH, 25”C, 2.0 h) to remove the 3/3-acetoxy function. Although sulfur fluorides are known to replace hydroxyl groups by fluorine with retention of configuration (15) it was impossible to establish the stereochemistry of the C-l fluorine by conventional NMR techniques. A rigorous proof of stereochemistry was accomplished by comparative fluorination reactions conducted on lcu-hydroxycyclovitamin D3 and its I@hydroxy epimer. Since NMR analysis easily distinguished between these isomers, the corresponding 1-fluoro derivatives should also be easily characterized. This was found to be the case. DAST fluorination of la-hydroxycyclovitamin D3 at -78°C produced one monofluoro analog which retained NMR chemical shifts analogous to the la-hydroxy starting material. Fluorination of Ifi-hydroxycyclovitamin D3 under the same conditions produced two monofluoro analogs in a 3:2 ratio. The predominant product was shown to be identical to that obtained from the fluorination of la-hydroxycyclovitamin Da. While the minor product possessed all the spectral characteristics expected of the l/3fluoro analog. It is evident from these results that steric factors can influence the stereochemical outcome of DAST fluorination reactions. When the la- and I@fluorocyclovitamin D mixture was cycloreverted under standard conditions (glacial HOAc, 55”C, 15 min) followed by mild basic hydrolysis (0.1 N KOH/EtOH, 25”C, 2.0 h) and subjected to hplc, 5,6-cis-lcu-fluoro- and I@fluorovitamin D3 were isolated in addition to their 5,6-bans isomers. The la-fluorovitamin formed from the cyclovitamin scheme was found to be identical in all respects to that obtained by direct fluorination and subsequent hydrolysis of la-OH-D3 acetate while the lp-fluoro analog exhibited markedly different spectral characteristics (e.g., uv spectrum: lb-fluor0 Lax 274 (lS,OOO), 248 (16,600) vs lafluoro X,,, 264 (18,066); NMR, 2’70 MHz, 6: Ifi-fluoro, 5.14, C(l)-H, doublet J = 49 Hz vs la-fluoro 5.03, C(l)-H, doublet J = 52 Hz. DAST fluorination followed by cyclo-

D3 SYNTHESIS

581

reversion and simple hydrolysis of la,25(OH)pcyclovitamin D3 provided, as expected, material identical to that obtained from the direct fluorination of the lcr,25(OH)zD3 3-acetate. The lcQ5-FzD3 had no demonstrable biological activity. Using 60 pmol (25 ng) of la,25-(OH)2D3 as a positive control dose, we found that l-, lo-, and lOO-fold doses of lcu,25-FzD3 showed no vitamin D-related responses with respect to the elevation of serum calcium and on the intestinal transport of calcium (Table I). The inability of 1a,25-F2D3 to elicit any response at the 2.7pg or 6000-pmol dose when compared to the full biological activity of 5.0 rcg of 25F-D3 (8) would indicate that the presence of the la- and 25-fluoro substituents are effectively preventing the bioactivation of the analog. The anti-vitamin D properties of l&,25F2D3 were explored using the standard protocol of inhibitor injection, followed by a physiologic dose (50 ng) of vitamin D3 2 h later. Serum calcium and intestinal transport responses were determined 20 h after the second dose and are shown in Table II. It is quite evident from these results that la,25-FzD3, even at a lOOO-fold excess (55 pg), failed to block the expression of vitamin D activity. The surprising lack of anti-vitamin D activity prompted an investigation of whether this analog, like 25-F-D3, could block the 25-hydroxylase system in vivo. This study was conducted by administering to weight-matched, vitamin D-deficient rats a lOOO-fold (55 pg) excess of la,25-FzD3 2 h before a radiolabeled 50-ng dose (1.0 &i) of 3a-[3H]vitamin Da. The normal control received an ethanol vehicle followed by [3H]vitamin D3 2 h later. After 22 h the blood was collected and serum levels of [3H]vitamin D3 and 25-OH-[3H]D3 were determined by Lipidex-5000 chromatography with the results given in Table III. Circulating serum levels of 25(OH)D$H] are unchanged by the presence of the difluoro analog while [3H]vitamin D3 levels are twice as high in the animal predosed with la,25-FzD3. Even though uptake of vitamin D3 would seem to be slightly impaired, 25-hydroxylation ob-

PAAREN TABLE

ET AL. I

INTESTINAL AND SERUM CALCIUM (BONE) RESPONSE TO lcr,25-(OH)2D3 AND la,25-F1D3 Serum calcium (mg/lOO ml)

45Ca serosal/ 45Ca mucosal

Group

Dose

1 2 3 4 5

Ethanol 60 pmol lc~,25-(OH)~D~ 60 pmol la,25-FzD3 600 pmol la,25-F2D3 6000 pmol la,25-F~Ds

1.9 2.9 1.9 1.9 2.0

+ 0.2 + 0.1 * 0.1” _+ 0.3” It 0.2”

4.0 5.2 3.8 4.1 4.1

k + k + +

0.1 0.1 0.1” 0.2” 0.1”

Note. Data given as the mean + SEM of six rats per group. ’ Significantly different from la,25-(OH)rD3 control, P < 0.001.

viously is not. This metabolic profile can be contrasted to that obtained for a similar dose (50 Mg) of 25-F-D3 which effectively prevents 25hydroxylation of the vitamin (16). DISCUSSION

The recent advances in our understanding of the functional metabolism of vitamin D and the mechanism of action of the active forms have kindled renewed interest in the synthesis of antivitamin D compounds. Of the attempts made, only agents that block vitamin D 25-hydroxylation have proved to have significant antivitamin D activity. Thus the 19-hydroxy10(19)-dihydrovitamin D3 (17) and the 25TABLE

II

FAILURE OF l&45-FzD8 TO BLOCK INTESTINAL AND SERUM CALCIUM RESPONSESTO VITAMIN DS

Group 1” 2b f 56

Dose of la,25-FrDs (wnol) None None 1.3 6.5 13

‘%a serosal/ 45Ca mucosal 2.1 5.0 5.4 5.4 5.1

+ * + + +

0.2 0.T 0.4” 0.4” 0.7”

azavitamin D3 (18) have proven to have significant antivitamin D activity by virture of blocking vitamin D 25-hydroxylation. 1-Fluorovitamin D3 which was synthesized with the intent of blocking 1-hydroxylation failed as an antagonist because it possessed significant biological activity in its own right, presumably by virture of 25-hydroxylation with perhaps other additional modifications (9). Inasmuch as 25-OH-D has significant biological activity in nephrectomized animals, it is apparent that it can act directly at high concentrations with the intestinal, bone, and other receptors (19). To obtain a true blockage of vitamin D activity it therefore appears that the most successful antivitamin should block the entry of vitamin D into the activation sequence, namely, at the 25-hydroxylation stage. This led to the chemical synthesis of 24,25-dehydrovitamin D3 and 25,26-dehydrovitamin D3 as well as 25-F-D3 (8). All of these compounds blocked 25-hydroxylation of radioactive vitamin D in viva but failed as antagonists

Serum calcium (mg/lOO ml) 4.0 4.9 5.2 4.8 5.0

-t f 2 f 2

0.1 0.1’ 0.2” 0.1” 0.1’

Note. Data given as the mean Itr SEM of six rats per group. ’ This group received only ethanol vehicle. b These groups received 50 ng vitamin Da 2 h after the dose of la,25-FzDs or ethanol vehicle. ‘Significantly different from control, P c 0.001.

TABLE

III

EFFECT OF la,25-FzDs ON SERUM 25-OH-D3 LEVELS Dose 0.13 (50 ng) nmol [‘HIvitamin Ds 0.13 pmol la,25-FZDs + [‘HIvitamin Da

Vitamin Da h3hnl)

25-OH-Da (w/ml)

0.15

0.70

0.34

0.63

Note. lcu,25-FzDs was dosed 2.0 h prior to a 50-ng dose of [‘HIvitamin Ds.

la,25-DIFLUOROVITAMIN

of vitamin D action by virture of their conversion to active metabolites (8). It therefore appeared that the 25-F-D3 likely became l- and 24-hydroxylated providing an active form. To prevent alternative hydroxylations it was conceived that blocking both the 1 and the 25 position should bring about a vitamin D compound with significant anti-vitamin D activity. This paper reports the chemical synthesis of 1,25-FzD3 that should satisfy these criteria. As expected, this compound elicited no vitamin D activity in its own right providing strong evidence that l- and 25hydroxylation are essential aspects of vitamin D function. This compound, therefore, would be expected to interact with the 25-hydroxylase and prevent 25-hydroxylation of vitamin D thus providing significant antivitamin D activity. Surprisingly, however, 1,25-FzD3 elicited no antivitamin D activity whatsoever. This suggested, therefore, that perhaps the la,25-F2D3 might be blocking 25-hydroxylation but for some unknown reason vitamin D could be activated. A test of whether this compound could in fact block 25-hydroxylation in viva was made. The results show that 1,25-FzD3 does not block in viva 25-hydroxylation of vitamin D in contrast to previous experience in which 25-F-D was fully ably to block 25-hydroxylation in viva under identical circumstances. Several possible explanations can be visualized for these phenomena. First, it is possible that the 1,25-FzD3 compound is not transported or transferred into the hepatic tissues in an identical fashion with vitamin D3. Another possibility is that 1,25-FzD3 may be rapidly metabolized and excreted rendering it ineffective as an antagonist. A final and perhaps most plausible explanation is that the introduction of a 1-fluoro group imparts electronic characteristics in the A-ring that diminishes interaction of the vitamin molecule with the 25-hydroxylase enzyme. It is not possible at this time to provide evidence for or against any of these possibilities. Nevertheless, the 1,25-FzD3 compound provides additional evidence for the importance of l- and 25-hydroxylation for the function of vitamin D and it demonstrates that the introduction of a 1-fluoro group

583

D3 SYNTHESIS

in the vitamin D molecule brings about some change that diminishes interaction with either the liver and/or the vitamin D3 25-hydroxylase system. REFERENCES 1. HEIDELBERGER, B. J., MOOREN,

C., GRIESBACH, L., MONTAG, D., AND CRUZ, 0. (1958) Cancer

Res. l&305-317. 2. FRIED, J., AND BORMAN, A. (1958) Vitam. Harm. 16,303-374. 3. PAULING, L. (1960) The Nature of the Chemical Bend, 3rd ed., p. 88, Cornell Univ. Press, Ithaca, N. Y. 4. TANAKA, Y., DELUCA, H. F., KOBAYASHI,Y.,TAGUCHI, T., IKEKAWA, N., AND MORISAKI, M. (1979) J. Biol. Chem. 254,7X%3-7167. 5. NAPOLI, J. L., FIVIZZANI, M. A., SCHNOES, H. K., AND DELUCA, H. F. (1978) Biochemistry 17, 2387-2392. 6. NAPOLI, J. L., MELLON, W. S., FIVIZZANI, M. A., SCHNOES, H. K., AND DELUCA, H. F. (1979) J.

Biol. Chem. 254,2017-2022. 7. DELUCA, H. F., PAAREN, H. E., AND SCHNOES, H. K. (1979) in Topics in Current Chemistry (Dewar, M. J. S., Hafner, K., Heilbronner, E., Ito, S., Lehn, J.-M., Niedenzu, K., Rees, C. W., Shgfer, K., Wittig, G., and Boschke, F. L., eds.), Vol. 83, pp. 7-63, Springer-Verlag, New York. 8. ONISKO, B. L., SCHNOES, H. K., DELUCA, H. F., AND GLOVER, R. S. (1979) Biochem. J. 182. l9. 9. NAPOLI, J. L., FIVIZZANI, M. A., SCHNOES, H. K., AND DELUCA, H. F. (1979) Biochemistry 18, 1641-1646. 10. MIDDLETON, W. J. (1975) J. Org. Chem. 40, 574-

578. 11. PAAREN, H. E., DELUCA, H. F., AND SCHNOES, H. K. (1980) J. Org. Chem. 45.3253-3258. 12. SUDA, T., DELUCA, H. F. AND TANAKA, Y. (1970) J. Nutr. 100,1049-1052. 13. MARTIN, D. L., AND DELUCA, H. F. (1969) Amer. .I Physiol. 216.1351-1359. 14. BLIGH, E. G., AND DYER, W. J. (1959) Canad J. B&hem. Physiol. 37,911-917. 15. SHEPPARD, W. A., AND SHARTS, C. M. (1969) Organic Fluorine Chemistry, pp. 164-166, Benjamin, New York. 16. ONISKO, B. L., SCHNOES, H. K., AND DELUCA, H. F. (1980) Bioovg. Chem. 9. 187-198. 17. PAAREN, H. E., MORIARTY, R. M., SCHNOES, H. K., AND DELUCA, H. F. (1980) Biochemistry 19.5335-5339. 18. ONISKO, B. L., SCHNOES, H. K., AND DELUCA, H. F. (1978) J. Biol. Chem. 254,3493-3496. 19. RAISZ, L. G., TRUMMEL, C. L., HOLICK, M. F., AND DELUCA, H. F. (1972) Science 175, 768-769.