Novel nonsecosteroidal vitamin D3 carboxylic acid analogs for osteoporosis, and SAR analysis

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Abstract

Novel vitamin D3 analogs with carboxylic acid were explored, focusing on a nonsecosteroidal analog, LG190178, with a bisphenyl skeleton. From X-ray analysis of these analogs with vitamin D receptor (VDR), the carboxyl groups had very unique hydrogen bonding interactions in VDR and mimicked 1α-hydroxy group and/or 3β-hydroxy group of 1α,25-dihydroxyvitamin D3. A highly potent analog, 6a, with good in vitro activity and pharmacokinetic profiles was identified from an SAR study. Compound 6a showed significant prevention of bone loss in a rat osteoporosis model by oral administration.

Introduction

1α,25-Dihydroxyvitamin D3, [1,25(OH)2D3], 1, is a known classic ligand of vitamin D receptor (VDR) which is synthesized in skin, liver, and kidney and plays an essential role in calcium homeostasis.1, 2, 3 It stimulates calcium absorption in intestine, regulates bone formation and resorption, enhances calcium reabsorption in kidney, and inhibits the synthesis and secretion of parathyroid hormone (PTH) in the parathyroid gland.4 Physiological actions of 1 are exhibited by binding to VDR and the consequent formation of a complex with several co-factors which binds to vitamin D responsive element (VDRE) in the promoter moiety of target genes controlling the transcription.5, 6, 7, 8, 9

Over three decades, derivatizations of vitamin D3 have focused on the secosteroidal skeleton. Chemical modifications have led to a wide range of derivatives, especially around the side chain moiety and the A ring moiety of secosteroidal vitamin D3 structure. Accordingly, more potent VDR agonists than 1,25(OH)2D3 (1) have been reported.10

Meanwhile, there has been a growing interest in nonsecosteroidal VDR agonists, since a bisphenyl compound, LG190178 (2), showed characteristics of a vitamin D3 analog in vitro.11, 12, 13, 14, 15, 16

Recently the crystal structure of YR301 (3), which is a stereoisomer of 2, with VDR was reported and it has revealed that the secondary hydroxyl group in the A-part and the secondary hydroxyl group in the side chain of 3 worked like the 1-OH and 25-OH groups of 1, respectively.17, 18, 19

Hydrogen bonding of the 2-OH group in the A-part of 3 with Ser237 and Arg274 would be of especial interest, because Arg274 in VDR is an essential amino acid which binds the 1-OH group of 1, and indeed the lack of this interaction, such as in 25-OH vitamin D3, decreased the binding affinity to VDR. However, the structure–activity relationships (SAR) of the A-part of 2 is still unclear.

Here we describe the SAR of nonsecosteroidal carboxylic acid derivatives focusing on an interaction of the A-part of the ligand with Arg274. We attempted to introduce a carboxyl group with methylene linker into the A-part of the bisphenyl skeleton (Fig. 1). The SAR study revealed the carboxyl group and methylene linker length significantly influence the VDR agonistic activity. As a result, we successfully confirmed that our carboxyl group had a salt bridge interaction to Arg274 in VDR from X-ray analysis of these derivatives.

We evaluated these analogs in the reporter gene activity of VDRE and osteocalcin production activity as a VDR agonist in vitro. Also, in vivo effects on bone and serum calcium levels were evaluated using a rat osteoporosis model, which is commonly used in the evaluation of the activity of vitamin D3 analogs.20, 21

Section snippets

Chemistry

The synthesis of compounds 4ad and 6a is shown in Scheme 1. Compound 7 was prepared from bisphenol by the method reported by Boehm.11 Alkylation of 7 with K2CO3 and various alkylbromides gave corresponding ester (8a, 8c, and 8d) in moderate yields. Hydrolysis of ester and the following reduction of ketone using NaBH4 gave carboxylic acid analogs in good yields (4a, 4c, and 4d).

Compound 4b was prepared from alcohol 9, which was obtained from the alkylation of phenol 7 using 3-bromo-1-propanol.

Results and discussion

All synthesized compounds were evaluated by reporter gene assay in MG-63 cells, which contained VDRE sequence derived from mouse osteopontin promoter for vitamin D agonistic activity. The osteocalcin production activity in MG-63 cells was evaluated to measure bone formation activity of functional vitamin D agonistic effect.22, 23, 24 VDRE reporter gene activity and osteocalcin activity are represented by a relative EC50 value which is the EC50 value of 1,25(OH)2D3 assigned as 100% (a larger

Conclusion

Novel nonsecosteroidal vitamin D3 agonists were explored. They have carboxylic acid instead of 1,3-diol of 1,25(OH)2D3 and compounds 4d and 5a were identified as the optimized linker length analogs. Additional hydroxyl group in gamma position of the carboxyl group in 4d and 5a significantly improved metabolic stability. X-ray structure of 6b exhibited the carboxyl group tightly interacting with Ser237 and Arg274 and the gamma hydroxyl group interacting with Ser278 and Tyr134 via crystal water.

Chemistry: General

Purchased reagents and solvents were used without further purification unless otherwise noted. 1H and 13C NMR spectra were carried out on VARIAN 400-MR spectrophotometers; chemical shifts are reported in parts per million (ppm) downfield from that of internal tetramethylsilane (TMS). Mass spectrophotometry was measured with a Waters ZQ2000 electrospray ionization (ESI) system. High-resolution mass spectra (HRMS) were recorded on Thermo Fisher Scientific LTQ Orbitrap XL (ESI) instruments.

Acknowledgments

The authors thank Ms. Hitomi Suda of Chugai Pharmaceutical Co., Ltd for HRMS measurements of the compounds, and Dr. Hiromasa Hamada and Editing Services of Chugai Pharmaceutical Co., Ltd for proofreading the manuscript.

References and notes (27)

  • M.F. Boehm et al.

    Chem. Biol.

    (1999)
  • S. Hosoda et al.

    Bioorg. Med. Chem. Lett.

    (2005)
  • S. Hosoda et al.

    Bioorg. Med. Chem.

    (2006)
  • W. Hakamata et al.

    Bioorg. Med. Chem.

    (2008)
  • S. Harada et al.

    J. Steroid Biochem. Mol. Biol.

    (2010)
  • J.P. Brown et al.

    Lancet

    (1984)
  • J.B. Lian et al.

    Steroids

    (2001)
  • R. Paredes et al.

    J. Steroid Biochem. Mol. Biol.

    (2004)
  • N. Rochel et al.

    Mol. Cell

    (2000)
  • R. Bouillon et al.

    Endocr. Rev.

    (1995)
  • J.L. Omdahl et al.

    Annu. Rev. Nutr.

    (2002)
  • H. Reichel et al.

    N. Engl. J. Med.

    (1989)
  • R. Brommage et al.

    Endocr. Rev.

    (1985)
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