Biological evaluation of epoxy analogs of 1α,25-dihydroxyvitamin D3

r~l.J TT E RW0 R T H [~E I N E M A N N

Biological evaluation of epoxy analogs of la,25-dihydroxyvitamin D 3 Katrien Allewaert,* Xu-yang Zhao,t Jie Zhao,* Franqoise Glibert,* Dimitri Branisteanu,* Pierre De Clercq,t Maurits Vandewalle,t and Roger Bouillon* *Laboratory o f Experimental Medicine and Endocrinology, Gasthuisberg, Katholieke Universiteit Leuven, Belgium; and "i-Laboratory f o r Organic Chemistry, Universiteit Gent, Belgium

The biological activity of 16-epoxy side-chain analogs of le~,25-dihydroxyvitamin D 3 (let,25(OH)2D3) was evaluated in vitro and in vivo. Compared to 1a,25(OH)eD ~, all analogs had lower affinities for the pig duodenal vitamin D receptor and also for the human serum vitamin D binding protein. The in vitro effects on cell proliferation or differentiation of human promyeloid leukemia (induction of superoxide production in HL-60 cells), human osteosarcoma MG-63 cells (osteocalcin secretion), or human breast cancer cells (incorporation of thymidine in MCF-7 cells), was markedly inhibited by several epoxy analogs, compared to Ic~,25(OH)2D 3, but the rank order of their activi~' widely varied among different cancer cells. The most potent analogs (24S,25S24-hydroxy-25,26-epo~'-22-ene-let-OHD> 25,26-epoxv-23-yne-le~-OHD 3 and 25,26-epoxy-23-yne-20-epi-le~OHD 3 or compounds 16, 5, and 7, respectively) were equipotent (16 and 5) or 30-fold (compound 7 on MG-63 cells) to 40-fold (compound 7 on MCF-7 cells) more active than 1e~,25-(OH)eD~. These analogs were nevertheless poorly antirachitic (<3%) when tested in vitamin D-deficient chicks (using serum and bone calcium, serum osteocalcin and duodenal calbindin D-28K, as end points). Compound 7 was also lO0-fold more active than la,25-(OH)2D J in inhibition of proliferation of human foreskin keratinocytes. Some epoxy analogs of la,25-(OH)2D 3 thus display interesting dissociations between their receptor affinity and their potency to induce cell differentiation, whereas their effect on cell proli['eration/differentiution exceed their calcemic effects more than 100- to lO00-fold. (Steroids 61):324-332. 1995)

Keywords:vitamin D; epoxy analogs; antirachitic; differentiation: receptor: binding protein

Introduction The parent vitamin D structure is a very flexible molecule, partly due to its long side-chain and partly due to its seco B-ring structure. To become biologically active, vitamin D3 must be hydroxylated at C-25 and C-1. The natural metabolism of 25-hydroxyvitamin D 3 (25-OH-D 3) involves additional hydroxylation of the side-chain, sometimes resulting in active (24,25-(OH)2D 3 and l a , 2 5 ( O H ) z D 3 - 2 6 , 2 3 lactone) but mostly in inactive degradation products. 1-2 Up to now, more than 400 vitamin D analogs have been synthesized. Most of the structural modifications of la,25(OH)zD 3 occur in the side-chain, followed by the C/D ring, the A-ring, and the seco-B-ring alterations. 4"5 Several of these side-chain analogs have an oxygen instead of a carbon 1 20, 22, 23, or 24. 6-12 Only the 22-oxa-analog, at pos'tion Address reprint requests to R. Bouillon, Legendo, Onderwijs en Navorsing, Gasthuisberg, B-3000 Leuven Belgi& Received August 16, 1994; accepted November 11, 1994. Steroids 60:324-332, 1995 © 1995 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010

alone or in combination with other side-chain modifications, created interestin~ analogs with increased celldifferentiating properties. 13-17 A keto modification at C-24 decreases the biological activity. 3"18 The oxygen function can, however, also be introduced as an epoxide, and such modification could provide additional information regarding the structure-function relationship of vitamin D. Indeed, the vitamin D hormone is not only a key calciotropic hormone but also shows remarkable effects on cell differentiation (e.g., psoriasis and cancer cells) and on the immune function. W'2° We therefore synthesized 2~ and analyzed 16 analogs with epoxy structures in the side-chain.

Experimental Vitamin D3, 25-OH-D3, and loL,25-(OH)2D3 were obtained from B. Borsje (Solvay-Duphar, Weesp, The Netherlands). [3H]la,25(OH)2D 3 (180 Ci/mmol and [methyl-3H]thymidine (2 Ci/mmol) were obtained from Amersham (Buckinghamshire, UK). Culture media were obtained from Gibco (Roskilde, Denmark). 4-Nitro blue tetrazolium (NBT), phorbol 12-myristate-13-acetate, and the

0039-128X/95/$10.00 SSDI 0039-128X(94)00072-K

Epoxy analogs of vitamin D: Allewaert et aL Sigma kit 90 for measurement of nonspecific acid esterase were obtained from Sigma (St. Louis, MO, USA). Human serum vitamin D binding protein (hDBP) was prepared by affinity chromatography as described previously.22 Human promyeloid leukemia (HL-60), osteosarcoma (MG-63), or breast carcinoma (MCF-7) cells were obtained from the American Type Culture Collection (Rockville, MD, USA).

Synthesis Analogs 2-9 were obtained via epoxidation of the corresponding olefinic precursors constructed by 22-23 bond formation starting with the Inhoffen-Lythgoe diol (unpublished personal results). They were obtained as diastereomeric mixtures at the epoxy oxygen. Epoxy analogs 10-17 of loL,25-(OH)2D 3 were synthesized on the basis of the Sharpless kinetic resolution of secondary alcohols and further included the Julia procedure for side-chain construction and the Lythgoe A-ring coupling procedure. 2~

Binding studies The affinity of Ic~,25-(OH)2D 3 and its epoxy analogs for the vitamin D receptor from normal pig duodenal mucosa was measured essentially as described23'24 by incubation at 4°C, pH 7.5 (0.05 M Tris-HCl, 0.5 M KC1, 5 mM dithiothreitol, 10 mM Na2MoO4, 1.5 mM EDTA) for 20 h. The relative affinity for hDBP was measured by incubating [3H]Ict,25-(OH)2D3 and increasing concentrations of Iot,25-(OH)2D3 or its epoxy analogs with purified hDBP (0.2 p.M) in 1 mL (0.01 M Tris-HC1, 0.154 M NaC1, pH 7.4) for 3 h at 4°C, followed by phase separation by addition of cold dextrancoated charcoal. 23"24

Cell cultures HL-60 cells were seeded at 4 × 10 4 cells/mL and let,25-(OH)2D 3 or its analogs were added in ethanol (final concentration < 0.2%) in RPMI 1640 medium supplemented with 20% heat-inactivated fetal calf serum (FCS) for 4 days at 37°C. Cells were then assayed for maturation by NBT reduction assay as described24 using a hemacytometer, or for proliferation by cell counting and [3H]thymidine incorporation. MG-63 cells, seeded at 5 × 103 cells/mL in 96-well flat-bottomed culture plates (Falcon, Becton Dickinson, NJ, USA) in a volume of 0.2 mL of DMEM and 2% FCS were incubated with 1et,25-(OH)2D 3 or its analogs for 72 h. Osteocalcin was then measured in the culture medium using a homologous human osteocalcin RIA. 25 Breast carcinoma cells (MCF-7) were grown in DMEM/nutrient mix F-12 (HAM) medium supplemented with 10% FCS. Cells (5000/well) were incubated over 24 h in 96 well-tissue culture plates (Falcon 3072) followed by a 72 h incubation with or without Ict,25-(OH)2D3 or analogs. The cells were then incubated with [3H]thymidine (1 IxCi/well) for 4 h and harvested thereafter in NaOH (0.1 M) and the radioactivity counted. The protein content of the cells was measured by the Pierce BCA protein assay (Rockford, IL, USA).

Keratinocyte culture Human skin keratinocytes were isolated and cultured using a modification of the method of Kitano and Okada. 26 Briefly, the skin from biopsies of patients with breast tumors, or tissue from foreskin, was cut into pieces measuring 3-5 mm and soaked overnight at 4°C in a solution of dispase (20 Boehringer units/mL). The epidermis was peeled from the dermis, washed with calcium- and magnesium-free phosphate buffer saline, and shaken in a 0.25% trypsin solution for 10 min at room temperature. The reaction was then stopped by addition of PBS containing 10% FCS. The cells were collected after centrifugation at 4°C for 10 min at 800 rpm.

After an additional washing with PBS, the pellet was suspended in culture medium into 25 cm: primaria flasks from Becton Dickinson. The keratinocytes were cultivated at 37°C in an atmosphere of 5% CO 2 in air. A few hours later, the medium was replaced by new one. The medium (Keratinocyte Medium from Gibco conmining Epidermal Growth Factor [5 ng/mL], Bovine Pituitary Extract [35-50 Ixg/mL] and antibiotics) was renewed every other day until confluency. Keratinocytes were finally cultured in 96-well plates and, after 24 h, were treated with various concentrations of vitamin D analogs, followed by pulse-labeling with 1 p,Ci of [3H]thymidine for 3 h. Cultures were washed 3 times with PBS and twice with 10% (v/v) ice-cold trichloroacetic acid. Cells were solubilized with 1 M NaOH and radioactivity was counted in a scintillation counter.

Calcemic effects in vivo The antirachitic activity of the epoxy analog was tested in 3-weekold vitamin D-deficient chicks injected for 10 consecutive days with lct,25-(OH)2D 3 or its analogs. 23"24 Serum calcium (by atomic absorptiometry) and osteocalcin (by specific RIA), duodenal calbindin D-28K (by RIA), and bone calcium content were measured. The hypercalcemic effect of the most interesting analogs was also tested in vitamin D-replete normal Naval Medicinal Research Institute (NMRI) mice by daily subcutaneous injection of la,25-(OH)2D 3, its analogs, or the solvent for 7 consecutive days, using serum, bone, and urinary calcium concentration and serum osteocalcin (by specific mouse RIA) as parameters. 27

Statistical analysis Mean -+ SD or SEM (as indicated) were calculated and differences were calculated using a Student's t-test. Affinity constants were calculated using a computer program to correct for non-specific binding according to Scatchard. 28

Results

Binding studies The affinity of the Ia,25-(OH)2D3 for hDBP was 3.3 - 0.8 × 107 M - ~ (Mean - SD, n = 10), whereas its affinity for the pig duodenal mucosa vitamin D receptor was 1.06 --0.38 × 10 l° M - 1 (n = 10), respectively. All side-chain epoxy analogs showed decreased binding to both the receptor and hDBP (Table 1 and Table 2). Epoxy analogs with two oxygen atoms in the side-chain bound less well to the vitamin D receptor than side-chain epoxides with a single oxygen atom, whereas their affinity for DBP was widely variable. The affinity for the receptor and hDBP o f three analogs with the most interesting biological activity is demonstrated in Figure 1.

Cell differentiation in vitro Three different human cell lines were used for evaluation o f cell differentiating or antiproliferative effects o f the epoxy analogs: leukemia (HL-60), osteosarcoma (MG-63), and breast cancer (MCF-7) cells; [3H]thymidine incorporation (MCF-7), enzyme (HL-60), or specific protein (MG-63) induction were used as end points. A single oxygen in the side-chain present as 24,25- or 25,26-epoxide (both as diastereomeric mixtures at C-24 or C-25, respectively) resulted in similar receptor-and cell-differentiating properties, but the 25,26-epoxy analog bound better to hDBP than the 24,25-epoxy analog (Table 1 and Table 2). Replacement of C-26 and C-27 by a dimethyl group on C-23 virtually abol-

Steroids, 1995, vol. 60, April

325

Papers Table 1

Biological activity profile of epoxy analogs of 1(~,25-(OH)2D3 with a single oxygen atom in the side-chain Inhibition of cell proliferation or differentiation induction

Affinity for Number

1

Vitamin receptor hDBP

Chemical structure

/,%,..f,,.~,,,,~,~AA~"

HL60

MG63

Antirachitic activity (chick)

MCF- Serum 7 calcium

Serum osteoBone caicin calcium

Duodenal calbindin

100~

100a

100a

100a

100a

100b

100b

100b

100b

25,26-Epoxylct-OHD 3

27

76

37

18

22

14

25

9

17

24,25-Epoxylc=-OHD3

16

27

6

40

18

1

11

2

2

1

0

0

5

0

<0.1

<0.1

<0.1

<0.1

25,26-Epoxy23-yne-1eOHD3

30

6

240

150

127

1

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25,26-Epoxy23-yne-19nor-lc~-OHD3

20

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12

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All values reported in the table are relative to the values obtained with 1~x,25-(OH)2D3 ( = 100%). aThe affinity of 1~,25-(OH)2D3 for the pig receptor was 1.06 -+ 0.38 × 10l° M -1 and for hDBP 3.3 +- 0.8 × 107 M -1. The concentration of l~,25-(OH)2D3 needed for 50% inhibition of [3H]thymidine incorporation in MCF-7 cells was 3.0 +- 2.5 x 10 -8 M whereas the concentration needed for half-maximal differentiation of HL-60 and MG-63 cells was 1.4 +- 1.2 and 0.12 +- 0.14 × 10 -8 M, respectively. bThe in vivo biological activity (serum calcium, osteocalcin, bone calcium, and calbindin-D 28K) was measured in rachitic chicks and compared with the dose-response curve obtained after intramuscular injections with 1~,25-(OH)2D3 for 10 consecutive days. Compounds 2, 5, 6, end 7 are mixtures of diastereomers at C-25. Compounds 3, 4, and 8 are mixtures of diastereomers at C-24.

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S t e r o i d s , 1995, v o l . 60, A p r i l

Epoxy analogs of vitamin D: Allewaert et al. Table 2

Biological activity profile of epoxy analogs of 1cx,25-(OH)2D3with two oxygen atoms in the side-chain Inhibition of cell proliferation or differentiation induction

Affinity for Numbet 9

Chemical structure A

'..r~ I

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R

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R

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3

0

4

1

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5

3

7

15

20

2

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20

73

12

160

45

1

3

1

<1

10

7

44

7

20

3

2

1

2

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9

30

33

4

34

1

2

4

1

(24R,25S)-24Hydroxy-25, 26-epoxy-27nor-l(~-OH D3

3

21

<1

<1

ND

<1

<1

<1

<1

2

5

<1

<1

ND

<1

<1

<1

<1

(24S,25S)-24Hydroxy-25, 26-epoxy-22ene-l(x-OH Dz

3

15

100

65

1

1

1

1

(24S,25R)-24o Hydroxy-25, 26-epoxy-22ene-l(~-OH D3

2

30

140

22

1

3

2

1

(24S,25S)-24Hydroxy-25, 26-epoxy- 1eOH D3 (24R,25Rl-24Hydroxy-25, 26-epoxy-l(~OH

OH .~

12

24-Dihomo-24', 24"-epoxy23-yne-1~-OH D3

HL- MG- MCF- Serum 7 calcium hDBP 60 63

I -oH

..~ ~ 1 / O

R 11

Vitamin receptor

Antirachitic activity (chick)

~" ~'O

O3

(24S,25R)-24-

Hydroxy-25, 26-epoxy-1 (~OH D3

R 13

OH [ ~ ~ / O //,,,..

14

OH

~ ~ ~ / O /s,,,..

(24R,25S)-24-

R OH

15

/S,ao,"~ ~ , , , ~ . ~ , 1 0

R

OH _

16

OH 17

R

(24~25R)-24Hydroxy-25, 26-epoxy-27nor-le-OH D3

aND = not determined.

Steroids, 1995, vol. 60, April

327

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Figure 1 Affinity of 1c~,25-(OH)2D3 and three most interesting epoxy analogs for the pig intestinal vitamin D receptor (left panel) and human vitamin D-binding protein (right panel), means ± SEM. • 1a,25-(OH)2D3; ~ 25,26-epoxy-23-yne-1c~-OHD3, compound 5; (2) 25,26-epoxy-23-yne-20-epi-1~-OHDa, compound 7; • (24S, 25S)-24-hydroxy-25,26-epoxy-22-ene-1a-OHD3, compound 16.

ished all biological activity. The introduction of a 23-yne group (compounds 5-7), however, with a normal or 19-nor A-ring, decreased the binding to DBP without a similar reduction in receptor binding. The 23-yne configuration. however, increased the cell differentiation capability, but only in the presence of a normal (and not the 19-nor) A-ring. The 20-epi-23-yne configuration (compound 7) further decreased the DBP binding without affecting the receptor affinity and markedly enhanced its cell differentiating effect (Table 1) in several malignant cells. The introduction of a 22-yne bond in the 24-25-epoxy analog (compound 8) did not enhance its cell differentiation effect compared with the simple 24,25-epoxy homolog (compound 3). On the other hand, the four possible diastereomers at C-24 and C-25 of 25,26-epoxy-lo~,24-(OH)zD 3 (compounds 10-13) had widely variable receptor and hDBP affinities (more than 10-fold differences) but their effect on cell differentiation was unpredictable. Indeed, no uniform pattern was obtained as some of these stereoisomers were more active on one cell type (24R,25R or compound 11 being most active on MG-63 cells) and a totally different rank order of effects was observed in other cell types (24S,24R or compound 12 being most active on HL-60 cells). Loss of C-27 destroyed the biological properties, whereas the introduction of a double bond between C-22 and C-23 slightly increased its cell differentiation activity. Several epoxy analogs displayed interesting cell-differentiating properties with low calcemic effects. Indeed, 5, 11, 16, 17, and especially 7 were at least equipment to or more potent than let,25-(OH)zD 3 in inhibiting MCF-7 cell proliferation or in inducing cell differentiation in MG-63 or HL-60 cells. The 20-epi analog (compound 7) indeed exceeded the activity of let,25-(OH)zD 3 more than 20- to 40-fold, depending on the cancer cell line used (Figure 2).

Effects on keratinocyte proliferation Two analogs, 5 and 7, were studied for their antiproliferarive properties in human keratinocytes. Analog 5 was equi-

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Steroids, 1995, vol. 60, April

potent to Ic~,25-(OH)2D 3 but analog 7 had a 100-fold greater potency (Figure 3). Among the other analogs none was substantially more active than la,25-(OH)2D 3 (data not shown).

Calcemic effects in vivo The in vivo calcemic effect was tested in vitamin D-deftcient chicks treated for 10 consecutive days with the analogs. All epoxy analogs were markedly less antirachitic than Ia,25-(OH)2D 3. Indeed, only 25,26-epoxy-lo~-OHD 3 (compound 2) retained about 20% of the calcemic effect of the natural hormone, whereas most other analogs were substantially less active (~<2% of the activity of lc~,25(OH)zD3; Figure 3). The two most interesting analogs, 25,26-epoxy-23-yne-la-OHD3 (compound 5) and 25,26epoxy-23-yne-20-epi-la-OHD3 (compound 7) were also tested in vitamin D-repleted normal mice by daily administration of the compounds for 7 days. Compound 5 retained only ~1% of the calcemic effect, whereas 7 was slightly more potent depending on the parameter measured (20% of the effect of la,25-(OH)2D 3 on serum calcium; Figure 4). Discussion

The introduction of an epoxy group in the vitamin D 3 structure has been reported previously as an intermediate step in the synthesis of other analogs or for study of its oxidative degradation 29-35 without reporting their biological activity. 25-Hydroxyvitamin D 3 endoperoxide (6,19-dihydro-6,19epidioxy-25-OH-D3) was 2-I0 times more active than 25OH-D 3 in the induction of HL-60 cell differentiation, but its l c~,25-(OH)2D 3 homolog had only 1% of the potency of the natural hormone. 34'35 Moreover the dioxy structure was only active after transformation into 6,19-epoxy-25-OH-D 3 or 6,19-epoxy- hx,25-(OH)2D3. 35 The affinity for the vitamin D receptor and hDBP was decreased by the introduction of an epoxy group in the side-chain, and the calcemic activity of nearly all analogs was reduced to very low values. On the contrary, a few

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Figure 2

Effects of selected side-chain epoxy analogs of 1cx,25-(OH)2Dz on cell proliferation or differentiation of malignant human cells. Left panel: Differentiation of HL-60 cells: the effect of compounds 5, 7, and 16 on cell differentiation of HL-60 was evaluated by their ability to induce superoxide production, measured by NBT reduction. All points are means -+ SEM (n 1> 4). Middle panel: Production of osteocalcin by MG-63 cells: the effect of compounds 5, 7, and 16 on cell differentiation was evaluated by osteocalcin secretion in the medium of human osteosarcoma cells, MG-63 (n /> 6). Right panel: Proliferation of MCF-7 cells: the effect of compounds 5, 7, and 16 on cell proliferation of MCF-7 cells was evaluated by [3H]thymidine incorporation (n = 6). • 1a,25-(OH)2D3; A 25,26_epoxy-23-yne-1a-OHD3, compound 5; O 25,26-epoxy-23-yne-20-epi-1a-OHD3, compound 7; • (24S, 25S)-24-hydroxy-25,26epoxy-22-ene-1~x-OHD3, compound 16.

analogs showed remarkably good potency for inhibition of cell proliferation or induction of cell differentiation in malignant cells. The reason for the discrepancy between receptor/DBP binding, the biological activity in vitro, and the calcemic effects in vivo is not obvious. A decreased binding to DBP can enhance the biological activity in vitro with low calcemic activity in vivo, due to differences in pharmacokinetics23'36"37 but can probably not explain the good biological activity in vitro of 5 and 7 as their effect is equally

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75

[..

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50

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lo-tO

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Figure 3

Effects of selected side chain epoxy analogs of 1¢x,25(OH)2D3 on cell proliferation of human keratinocytes in culture as evaluated by [ZH]thymidine incorporation. • 1~,25-(OH)2D3; A 25,26-epoxy-23-yne-1a-OHDz, compound 5; © 25,26-epoxy23-yne-20-epi-lcx-OHDz, compound 7.

present when tested in cell cultures grown in DBP-free fetal calf serum. Moreover the biological potency of 5 and 7 also largely exceeded their receptor affinity when tested in human keratinocytes grown in a serum-free medium (Figure 3). Epoxides are known to be sensitive to hydrolysis into dihydroxy metabolites whereby 25,26-epoxy and 24,25epoxy groups would result in the intracellular generation of lo~,25,26-(OH)3D 3 and lot,24,25-(OH)3D 3, respectively. However, the biological profile of the epoxy analogs did not correspond with the previously reported biological activity of the natural trihydroxylated vitamin D 3 metabolites. Firstly, the affinity of several trihydroxylated lot,25(OH)2D 3 metabolites for the vitamin D receptor is lower than that of the corresponding epoxy analogs. Indeed, lot,25R,26-(OH)3D3, let,25S,26-(OH)aD 3, and lct,24R,25(OH)3D 3 all have an affinity between 5% and 10% of that of lot,25-(OH)~TD3, whereas that of lct,24S,25-(OH)aD 3 is even lower. J By contrast, the 24,25- and 25,26-epoxy analogs have a relative affinity of 16% and 27%, respectively (Table 1). Secondly, the stimulation of the intestinal calcium absorption by lot,25,26-(OH)3D 3 was between 1% and 10% of that of Ict,25-(OH)2D 338 whereas the corresponding 25,26-epoxy analog (compound 2) was more hypercalcemic (Table 1). The 24,25-epoxy analog (compound 3) displayed only minimal calcemic activity (<3%) whereas the corresponding let,24,25-(OH)aD 3 metabolite had a relative activity of nearly 50% for stimulation of intestinal absorption. 39 Finally lot,24,25-(OH)3D 3 was more potent in inhibiting cell proliferation in vitro (75% and 30% growth inhibition of fibroblasts and HL-60 cells, respectively)4°'41 than 24,25-epoxy-letOHD 3 (Table 1). These comparisons make it unlikely that the biological activity of the epoxy analogs would be simply due to their metabolism into dihydroxylated side-chain analogs. However, final proof will depend on the availability of pure isomers and the study of their metabolism in different cell types. The biological effect of the stereoisomers of 25,26-ep-

Steroids, 1995, vol. 60, April

329

Papers 1oi

A2

A1

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eoi

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f

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tO*

10 "

, 10"

10

.

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i

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|,,

!

B2

B1

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t O '11

,

t O 1"

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oL//

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,

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Figure 4 Antirachitic and hypercalcemic activity of some side-chain epoxy analogs of la,25-(OH)2D3. A. Vitamin D-deficient chicks were treated daily with vitamin D analogs for 10 days and serum (A1) and bone (A2) calcium, duodenal calbindin D-28K (A3), and serum osteocalcin (A4) were measured. B. Vitamin D-replete mice were treated daily for 7 days and serum calcium (B1) and osteocalcin (B2) were measured as end points. All data are means -+ SEM (n /> 5). • 1a,25-(OH)2Ds; A 25,26-epoxy-23-yne-1tx-OHD3, compound 5; (3 25,26-epoxy-23-yne-20-epi-1c~-OHD3,compound 7; • (24S, 25S)-24-hydroxy-25,26-epoxy-22-ene-1c=-OHD3,compound 16.

oxy-lot,24-(OH)2D3 was widely variably depending on the configuration at C-24 and C-25 (Table 2). This applied for receptor and DBP binding as well as for their inhibition of cell proliferation in different cancer lines. No obvious explanation can be offered, but the observations of very potent cell-differentiating effects of some analogs further argue for intrinsic biological activity as their further hydrolysis products lot,24,25,26-(OH)4D3 would have a poor biological activity. One of the remarkable aspects of the epoxy analogs is the widely different results on cell differentiation or proliferation that can be obtained with the same analog when tested in different cell lines (Table 1 and Table 2). This cannot be due to differences in end points in several test systems used (HL-60, MG-63, and MCF-7) because markers of cell differentiation (e.g., NBT reduction in HL-60 and osteocalcin secretion by MG-63 cells) and cell proliferation ([3H]thymidine incorporation) are usually mirror images. 23'24 Indeed, the inhibition of thymidine uptake by several epoxy analogs was obtained by concentrations similar to those

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needed for stimulation of osteocalcin secretion by MG-63 cells (data not shown). This suggests that testing of vitamin D analogs for anticancer therapy cannot be reliably based on a single cell line. The biochemical or molecular background of these differences is not known, but differences in degradation or transactivation by the steroid-receptor complex are possible mechanisms. The most potent epoxy analogs (compounds 5, 7, and 16) resemble other vitamin analogs with simple hydroxylation in the side-chain. Analog 5 resembles the potent 16-ene-23-yne-hx,25-(OH)zD3, which has a low calcemic activity combined with high cellular differentiation properties 23'4z'43 but this is partly due to the 16-ene configuration. Compound 7 resembles the recently published 20-epi analogs, 44 •45 which showed an increased activity for cell differentiation and immune effects, 19'44'45 but, unlike the 20-epi analogs with hydroxylated side-chains, the 20-epiepoxy analogs only displayed reduced antirachitic and hypercalcemic effects. The difference in calcemic effects in vitamin D-deficient chicks and vitamin D--replete rats is

Epoxy analogs of vitamin D: Allewaert et aL unusual and has not been observed for other vitamin D 3 analogs, except for other 20-epi analogs (KH 1060) which also have decreased antirachitic properties (35% of let,25(OH)2D 3 in rachitic chicks [unpublished personal results]) in contrast to equipotent effects in ratsJ 4 In conclusion, we observed that the introduction of an epoxy group in the side-chain created some interesting vitamin D analogs whose cell differentiating activity exceeded their calcemic effects more than 100-fold and make these and possible related compounds interesting structures for further preclinical trials. Acknowledgments The studies were supported by a grant from the Belgian National Foundation for Medical Research, grant no. 3.0044.89. A.W. Norman (Riverside) kindly provided the chick Calbindin D-28K protein for the development of this RIA. The technical assistance of R. Convents, S. Marcelis, and B.K. Tan, and the secretarial help of B. Minten is kindly appreciated. References 1. 2. 3. 4. 5. 6.

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