2-METHYL AND 2-METHYLENE ANALOGS OF 1αα-HYDROXY-19-NORVITAMIN D3: SYNTHESIS,BIOLOGICAL ACTIVITIES AND DOCKING TO THE LIGAND BINDING DOMAIN OF RAT VITAMIN D RECEPTOR

Pawel Grzywacz, Lori A. Plum, Wanda Sicinska, Rafal R. Sicinski, and Hector F. DeLucaDepartment of Biochemistry, College of Agriculture and Life Sciences, University of Wisconsin-Madison, Madison, WI 53706, USA

T h e d i s c o v e r y o f t h e m o s t a c t i v e m e t a b o l i t e o f v i t a m i n D 3 , 1α,25-dihydroxyvitamin D3 (1α,25-(OH)2D3, calcitriol, 1; Figure 1), has greatly stimulated research into its physiology and chemistry.1,2 It has been established that 1 supports mineralization in animals and humans,3 a f f ec t s t he immune sy s t em and exe r t s po t en t e f f ec t s upon ce l l proliferation and cellular differentiation.4 We synthesized interesting analogs of the natural hormone,5 in which the exocyclic methylene group is transposed, in comparison with 1, from C-10 to C-2. Compound 2 (2MD), with an unnatural configuration at C-20, exhibits the same affinity to the VDR as calcitriol. It also shows a preferential activity on bone relative to intestine. Recent studies confirmed its high ability to induce bone formation both in vitro and in vivo .6 In view of the very high calcemic potency of 2MD, we synthesized analog 3 considering it to be a possible biological precursor of 2MD. It might be expected that in a living organism, this compound should undergo 25-hydroxylation in the liver and s lowly form the more act ive metabol i te 2 . In our cont inuing investigation of the structure-activity relationship in this series of 19-norvitamin D compounds, we also prepared dihydro analogs of 3, namely, 2α- and 2β-methyl-19-norvitamins 4 and 5 , as well as its 25-methyl derivative 6, that cannot be “activated” by 25-hydroxylation process.

The strategy of our synthesis of 2-substituted 19-norvitamins was based on the Lythgoe type Wittig-Horner coupling.7 The A-ring fragment, phosphine oxide 12 ,5 was prepared from commercially available (-)-quinic acid (Scheme 1). The corresponding CD-fragments 17 (Scheme 2) and 21 (Scheme 3) required for the synthesis of vitamins 3 and 6, respect ively, were obtained from the Inhoffen-Lythgoe diol 13 . Epimerization at C-20 was accomplished at the stage of intermediate 22-aldehydes.8 The alkyl side-chain fragments were attached in the cuprate-catalyzed reaction of the corresponding 22-tosylates with Grignard reagents 16 and 21.9 The coupling of 8-ketones, 17 and 21, with the anion generated from 12, followed by hydroxyls deprotection gave the corresponding 2-methylene-19-norvi tamins 3 and 6 . Selec t ive hydrogenation of 3 provided isomeric 2-methyl-substituted vitamins 4 and 5. Their stereochemistry at C-2 was tentatively assigned on the basis of 1H NMR spectroscopy.

Docking simulations (100 000 iterations each) were performed by FlexiDock software from TRIPOS. Internal rotations around the single bonds of the ligand were allowed during the simulations. For each of the six theoretical vitamin D conformers (two 6-s-trans and four 6-s-cis), at least four docking experiments were done, starting with different pre-positioning of the ligand.10 For final consideration, the lowest energy complex, possessing parallel orientation of tryptophan ring to the steroid 5,7-diene moiety was selected. The 3.5 ≈ contacts between 3 and rVDR were detected by the program Biodesigner.

The synthesized vitamin D compound 3 is a 25-deoxy analog of the biologically active 2MD. Its binding potency for VDR is 5.9 times lower in comparison with 2MD. This experimental result is in agreement with the energy of modeled complexes of both these vitamins with VDR. Calculated energy of the most stable complex of VDR and 2MD was – 65.9 kcal /mol , whereas a -51.6 kcal /mol value was found for i t s counterpart with 25-deoxy compound 3. The latter analog is settled in the binding pocket (Figure 2) similar to the parent hormone 1, with 6-s-trans conformation of the 5,7-diene moiety, and equatorial orintation of 1α-OH group. Also the vitamin orientation in the binding pocket, with an A ring directed towards Y143 and a side chain between His 301 and His 393, resembles position of 1α,25-(OH)2D3 in hVDR deletion mutant.11 The same set of amino acids, found in the hormone-hVDR crystall ine complex, creates hydrogen bonds with 1α- and 3β-hydroxyls (Figure 3). Hydrophilic amino acids: R270 and S233 contact 1α-OH, while S274 and Y143 contact 3β-OH group. The lengths of these hydrogen bonds are: 2.94, 3.72, 3.03, and 2.46 Å, respectively.

The side chain, with the 25-OH group removed, is stabilized in the VDR pocket by efficient hydrophobic interactions (Figure 4). The short (<3.5 ≈) specific contacts were found between methyl groups of amino acids L223, L226, A227, V230, A299, L410 and 26- and 27-methyls from the ligand. Also hydrophobic interactions between 21-methyl group and I264, L305, L309 help to anchor side chain in a manner similar to that existing in the hormone complex.

1. Measurement of Binding to the Porcine Intestinal Vitamin D Receptor. The presented results (Figure 5) indicate that a binding ability of synthesized 2-methylene analogs, 3 and 6, is ca. 8 times lower than that of the hormone 1. Two other tested compounds with 2-methyl substituents (4 and 5) are much less effective in this assay. 2. Measurement of HL-60 Cellular Differentiation. The established cellular activity of the 2-methylene compounds, 3 and 6, is slightly weaker in comparison with 1 (Figure 6). The 2-methyl substituted analogs, 4 and 5, possess much weaker differentiation activity. 3 . I n t e s t i n a l c a l c i u m t r a n s p o r t . 2 - M e t h y l e n e - a n d 2α-methyl-19-norvitamins 3 and 4 are more potent than hormone 1 in the intestine (Figure 7 and 8), whereas the 2β-methyl analog 5 is slightly less potent (Figure 8). Surprisingly, the 25-methyl compound 6 shows similar intestinal activity as 1α,25-(OH)2D3 (Figure 9). 4 . B o n e c a l c i u m m o b i l i z a t i o n . 2 - M e t h y l e n e - a n d 2α-methyl-19-norvitamins 3 and 4 exhibit an extremely high ability to mobilize calcium from bone (Figure 7 and 8). The 2β-methyl analog 5 has a similar calcium mobilization response as 1 (Figure 8). Unexpectedly, 25-methyl substituted vitamin 6 retains bone calcium mobilizing activity

(Figure 9). Highly elevated calcemic activities of 2-methylene and 2α-methyl substituted 1α-hydroxy-19-norvitamin D3 analogs 3 and 4 , approaching these of the corresponding 25-hydroxy counterparts,5 suggest in vivo 25-hydroxylation of the tested analogs. However, replacing the 25-hydroxyl group with a methyl substituent does not abolish the in vivo activity as shown by the results of

biological tests of 25-methyl vitamin 6.(1) H.F. DeLuca, J. Burmester, H. Darwish, J. Krisinger, Molecular mechanism of the action of

1,25-dihydroxyvitamin D3, in: Comprehensive Medicinal Chemistry; C. Hansch, P.G. Sammes, J.B. Taylor (Ed.), Pergamon Press, Oxford, 1991; vol. 3, pp. 1129-1143.

(2) (a) A.W. Norman, R. Bouillon, M. Thomasset, Vitamin D, a pluripotent steroid hormone: Structural studies, molecular endocrinology and clinical applications, W. de Gruyter (Ed.), Berlin, 1994. (b) A.W. Norman (Ed.), Vitamin D, the calcium homeostatic steroid hormone, Academic Press, New York, 1979.

(3) (a) H. Reichel, H.P. Koeffler, A.W. Norman, The role of the vitamin D endocrine system in health and disease, N. Engl. J. Med. 320 (1989) 980-991. (b) H. F. DeLuca, The vitamin D story: A collaborative effort of basic science and clinical medicine, FASEB J. 2 (1988) 224-236. (c) N. Ikekawa, Structures and biological activities of vitamin D metabolites and their analogs, Med. Res. Rev. 7 (1987) 333-366.

(4) (a) G. Jones, S.A. Strugnell, H.F. DeLuca, Current understanding of the molecular action of vitamin D, Physiol. Rev. 78 (1998) 1193-1231. (b) T. Suda, T. Shinki, N. Takahashi, The role of vitamin D in bone and intestinal cell differentiation, Annu. Rev. Nutr. 10 (1990) 195-211. (c) T. Suda, The role of 1α,25-dihydroxyvitamin D3 in the myeloid cell differentiation, Proc. Soc. Exp. Biol. Med. 191 (1989) 214-220. (d) V.K. Ostrem, H.F. DeLuca, The vitamin D-induced differentiation of HL-60 cells: Structural requirements, Steroids 49 (1987) 73-102. (e) V.K. Ostrem, W.F. Lau, S.H. Lee, K. Perlman, J. Prahl, H.K. Schnoes, H.F. DeLuca, N. Ikekawa, Induction of monocytic differentiation of HL-60 cells by 1,25-dihydroxyvitamin D analogs, J. Biol. Chem. 262 (1987) 14164-14171.

(5) R.R. Sicinski, J.M. Prahl, C.M. Smith, H.F. DeLuca, New 1α,25-dihydroxy-19-norvitamin D3 compounds o f h igh b io log ica l ac t iv i ty : Syn thes i s and b io log ica l eva lua t ion o f 2-hydroxymethyl, 2-methyl and 2-methylene analogues, J. Med. Chem. 41 (1998) 4662-4674.

(6) N.K. Shevde, L.A. Plum, M. Clagett-Dame, H. Yamamoto, J.W. Pike, H.F. DeLuca, A potent analog of 1α,25-dihydroxyvitamin D3 selectively induces bone formation, Proc. Natl. Acad. Sci. USA, 99 (2002) 13487-13491.

(7) (a) B. Lythgoe, Synthetic approaches to vitamin D and its relatives, Chem. Soc. Rev. (1981) 449-475. (b) B. Lythgoe, T.A. Moran, M.E.N. Nambudiry, S. Ruston, Allylic phosphine oxides as precursors of conjugated dienes of defined geometry, J. Chem. Soc., Perkin Trans. 1 (1976) 2386-2390.

(8) G.H. Posner, Z. Li, M.C. White, V. Vinader, K. Takeuchi, S.E. Guggino, P. Dolan, T.W. Kensler, 1α,25-dihydroxyvitamin D3 analogs featuring aromatic and heteroaromatic rings: Design, synthesis and preliminary biological testing, J. Med. Chem. 38 (1995) 4529-4537.

(9) S.A. Barrack, R.A. Gibbs, W.H. Okamura, Potential inhibitors of vitamin D metabolism: An oxa analog of vitamin D, J. Org. Chem. 53 (1988) 1790-1796.

(10) P. Rotkiewicz, W. Sicinska, A. Kolinski, H.F. DeLuca, Model of three-dimensional structure of vitamin D receptor and its binding mechanism with 1α,25-dihydroxyvitamin D3, Proteins, 44 (2001) 188-199.

(11) N. Rochel, J. M. Wurtz, A. Mitschler, B. Klaholz, D. Moras, The crystal structure of the nuclear receptor for vitamin D bound to its natural ligand, Mol. Cell, 5 (2000) 173-179.

10-610-8 10-8

10-8 10-710-10 10-9 10-8 10-710-9

10-9 10-710-1010-120

5000

10000

15000

20000163

DPM

's B

ound

DPM

's B

ound

% D

iffer

entia

tion

% D

iffer

entia

tion

0

20

0

25

50

75

100

40

60

80

100

Molar Concentration Molar Concentration

0

5000

10000

15000

Molar Concentration Molar Concentration

145

OH

OHOH

H

HOH

OHOH

H

H

OHOH

H

H

OHOH

H

H

OHOH

H

H

OHOH

H

H

1

2 3

4 5 6

OHH

H OH

BzOH

H OH

BzOH

H OH

OH

H

CH2POPh2

OTBSTBSO

OHOH

H

H

OHOH

H

H

OHOH

H

H

ClMg

1. BzCl, Py

2. KOH, EtOH

1. PDC

2. n-Bu4NOH3. NaBH4

1. TsCl, Et3N2. Li2CuCl4

3. PDC

1. PhLi,

2. TBAF

+

H2, (Ph3P)3RhCl

13 14 (93% yield) 15 (27% yield)

17 (60% yield)

3 (45% yield)

4 (21% yield) 5 (15% yield)

16

12

OH

OH

OH

OH

HOOC

OTBS

OH

OH

TBSO

MeOOC

OTBS

OH

TBSO

O

OTBS

O

TBSO

COOMe

OTBSTBSO

CH2OH

OTBSTBSO

CH2POPh2

1. MeOH, TsOH

2. TBSCl, Et3N

1. DIBALH

2. NaIO4

1. RuCl3, NaIO42. LDA, TMSCH2COOMe

1. n-BuLi, MePh3P Br

2. DIBALH

1. n-BuLi, TsCl

2. n-BuLi, Ph2PH3. H2O2

(-)-Quinic acid 7

8 (48% yield) 9 (51% yield)

10 (44% yield)11 (81% yield)12 (87% yield)

+ -

OHH

H OH

TBSOH

H OH

TBSOH

H OH

OH

H

CH2POPh2

OTBSTBSO

OHOH

H

H

ClMg

1. TBSOTf

2. TBAF

1. SO3.Py

2. n-Bu4NOH3. NaBH4

1. TsCl, Et3N2. Li2CuCl4

3. HF.Py4. PDC

1. PhLi,

2. TBAF

13 18 (93% yield) 19 (36% yield)

21 (59% yield)

6 (36% yield)

20

12

Figure 1

Scheme 1

Scheme 2

Scheme 3

Docking

Figure 2

Biology

Figure 5 VDR Competitive Binding

Figure 6 HL-60 Cell Differentiation

Conclusion

References

Figure 9

Figure 7

Figure 8

Figure 3

Figure 4

Introduction

Bone Calcium Mobilization

0

2

4

6

8

10

12

14

Vehicle 50 ug/kg 1 450 ug/kg 10.5 ug/kg 6 1.5 ug/kg 6 4.5 ug/kg 6 13.5 ug/kg 6

Seru

m C

a (m

g/dL

)

Intestinal Calcium

0

2

4

6

8

10

12

40.5 ug/kg 1 Vehicle 40.5 ug/kg 6

Sero

sal C

a/M

ucos

al C

a

Intestinal Calcium Transport

02468

1012

Vehicle 260 pmol 1 130 pmol 3 260 pmol 3Sero

sal C

a/M

ucos

al C

a

Bone Calicum Mobilization

02468

101214

Vehicle 260 pmol 1 130 pmol 3 260 pmol 3

Seru

m C

a (m

g/dL

)

Intestinal Calcium Transport

02468

1012

Vehicle 130pmol 1

260pmol 1

130pmol 4

260pmol 4

130pmol 5

260pmol 5

Sero

sal C

a/M

ucos

al C

a

Bone Calcium Mobilization

02468

101214

Vehicle 130pmol 1

260pmol 1

130pmol 4

260pmol 4

130pmol 5

260pmol 5

Seru

m C

a (m

g/dL

)

163

145