Synthesis of 14-epi-2ti-hydroxypropoxy-1ti,25-dihydroxy-19-nortachysterol and its hVDR binding
Daisuke Sawada a , Yuya Tsukuda a , Hiroshi Saito b , Kenichiro Takagi b , Shinji Kakuda b , Midori Takimoto-Kamimura b , Eiji Ochiai b , Kazuya Takenouchi b , Atsushi Kittaka a,∗
aFaculty of Pharmaceutical Sciences, Teikyo University, 2-11-1, Kaga, Itabashi, Tokyo 173-8605, Japan
bTeijin Institute for Bio-medical Research, Teijin Pharma Ltd., 4-3-2, Hino, Tokyo 191-8512, Japan

a r t i c l e i n f o

Article history:
Received 27 June 2012
Received in revised form 3 October 2012 Accepted 28 November 2012

Keywords:
Vitamin D3
14-epi-19-Nortahcysterol Vitamin D receptor Vitamin D analogs Binding affinity
a b s t r a c t

Recently, we evaluated a novel skeleton in the vitamin D family, 14-epi-1ti,25(OH)2 -19-nortachysterol, and discovered its unique binding configuration in the human vitamin D receptor (VDR) with the C5,6- and C7,8-s-trans triene configuration. Because of its unprecedented form, this skeleton has a promising characteristic profile for clinical use, and also the synthesis of its derivatives should be versatile. Therefore, we synthesized the novel analog, 2ti-hydroxypropoxy substituted 14-epi-1ti,25(OH)2 -19-nortachysterol, and evaluated its human VDR binding affinity. Although this substitution is one of the promising modi- fication of vitamin D3 such as eldecalcitol (ED-71), it had negative effects on the binding affinity, and the compound showed lower affinity than 1ti,25(OH)2 D3 and its parent compound, 14-epi-1ti,25(OH)2 -19- nortachysterol. It was thought that the unprecedented binding configuration of this skeleton should not allow the terminal hydroxyl group of the 2ti-substituent to construct effective hydrogen bond networks around the amino acid residues in the binding pocket.
This article is part of a Special Issue entitled ‘Vitamin D Workshop’.
© 2012 Elsevier Ltd. All rights reserved.

Contents

1.Introduction 27
2.Results and discussion 29
3.Conclusion 29
Acknowledgments 29
References 29

1.Introduction

1ti,25(OH)2 D3 (1), an active metabolite of vitamin D3 , is a hormone that controls calcium and phosphorus homeostasis in humans and animals, and has also been used as a medicine for many years [1]. Moreover, its novel functions and multiple roles related to immunomodulation, differentiation, proliferation, apo- ptosis, and so on, have been disclosed by recent research [2], and the potential of new treatments for various diseases such as autoimmu- nity and cancer have been brought to light [3]. Therefore, vitamin D and its synthetic analogs are attractive candidates for clinical use,

∗ Corresponding author. Tel.: +81 3 3964 8109; fax: +81 3 3964 8117.
E-mail address: [email protected] (A. Kittaka).

0960-0760/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jsbmb.2012.11.014
and are one of the most interesting topics in the chemotherapy regimen.
In our group, many vitamin D analogs have been synthesized so far, and we especially evaluated 2ti-substitution effects on the biological properties [4]. Among them, 2ti -hydroxypropoxy substitution has potent positive effects [5], such as the essential modification of eldecalcitol (ED-71) at 2ti -position [6], which will be a strong tool for new derivatizations. Recently, we evaluated a novel skeleton in the vitamin D family, 14-epi-1ti ,25(OH)2 -19- nortachysterol (2), and discovered its unique binding configuration in the human vitamin D receptor (VDR) with the C5,6- and C7,8- s-trans triene configuration [7]. Because of its unprecedented form, 14-epi-19-nortachysterol has a promising characteristic pro- file for clinical use, and also the synthesis of its derivatives should be versatile. So, the 2ti-hydroxypropoxy-substituted ana- log of 14-epi-19-nortachysterol is a promising target compound,

28
D. Sawada et al. / Journal of Steroid Biochemistry & Molecular Biology 136 (2013) 27–29

Scheme 1. Natural vitamin D3 and tachysterol analogs.

Scheme 2. Retrosynthetic analysis of 14-epi-2ti-hydroxypropoxy-1ti,25(OH)2 -19-nortachysterol (3).

Scheme 3. Synthesis of 14-epi-2ti-hydroxypropoxy-1ti,25(OH)2 -19-nortachysterol (3).

D. Sawada et al. / Journal of Steroid Biochemistry & Molecular Biology 136 (2013) 27–29 29

Table 1
Relative binding affinity for human VDR [10]. Compound
1ti ,25(OH)2 D3 (1)
2
3
a The potency of 1 is normalized to 100.

Affinity for VDRa
100
14
3.7
the amino acid residues in the binding pocket. Further studies on the interaction of this new skeleton with the VDR including X-ray co-crystallographic studies and various molecular modifications are now in due course.

Acknowledgments

This work was supported in part by Grants-in-Aid from the Japan Society for the Promotion of Science (No. 23590015 to D.S. and No.

and here we report the synthesis and binding affinity of 14-epi- 2ti -(3-hydroxypropoxy)-1ti,25(OH)2 -19-nortachysterol (3) for the human VDR (Scheme 1).

2.Results and discussion

According to the previous method we developed for the synthesis of 14-epi-1ti,25(OH)2 -19-nortachysterol (2), the target compound (3) was synthesized from the two fragments, the known CD-ring fragment (4) derived from vitamin D3 [7] and the A-ring fragment (5) derived from shikimic acid, followed by the Stille cou- pling reaction through the hydrostannation of the alkyne moiety of compound 5 (Scheme 2).
The A-ring fragment (5) was obtained from the known com- pound 6 [8] via etherification with alkyl bromide 7 [9], but had some problems: compound 5 showed very poor reactivity, and the neighboring tert-butyldimethylsilyl (TBS) group easily migrated to the reactant alkoxide ion in situ. In spite of the low yield, however, this reaction finally afforded the desired compound 5 with 1 day reaction time at -10 ◦ C in a dimethylformamide (DMF) and hexam- ethylphosphoramide (HMPA) mixed solvent system. Next, for the coupling reaction, the A-ring fragment 5 was reacted with Bu3 SnH in the presence of 2,2′ -azobisisobutyronitrile (AIBN) to give the pre- cursor, vinylstannane 8, and then, without further purification, it was coupled with the CD-ring fragment 4 under Stille coupling con- ditions. As we expected, this reaction afforded the triene molecule (9) whose four silyl groups were immediately removed by tetra- butylammonium fluoride (TBAF) to give the target compound 3 (Scheme 3).
Using the new compound obtained above (3), we tested its bind- ing affinity for human VDR [10]. The results are summarized in Table 1 in comparison with the natural hormone 1 and its parent compound 2. Compound 3 showed lower affinity than the natural hormone 1 and, contrary to our expectation, was also lower than compound 2. In the ligand binding pocket of the VDR, the terminal hydroxy group of the substituent, the 2ti-hydroxypropoxy group of the active vitamin D3 analog, forms hydrogen bond networks both with Arg-274 and one of the water molecules, and consequently, affords a benefit for binding affinity [11]. In this case, however, the 2ti-substitution should not be advantageous to construct the hydrogen bond networks in the unique binding configuration of this skeleton, C5,6- and C7,8-s-trans triene configuration [7]. In addition, this long substituent would become a steric hindrance to fit the binding pocket without forming any hydrogen bond networks.

3.Conclusion

We synthesized 14-epi-2ti-hydroxypropoxy-1ti,25(OH)2 -19- nortachysterol (3), and evaluated its human VDR binding affinity. This substitution at 2ti-position had negative effects on the bind- ing affinity, and compound 3 showed lower affinity than 1 and 2. It was thought that the unprecedented binding configuration of this skeleton should not allow the terminal hydroxy group of the 2ti- substituent to construct effective hydrogen bond networks around
24590021 to A.K.). References
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