Synthesis and preliminary biological evaluation of 20-epi-eldecalcitol [20-epi-1α,25-dihydroxy-2β-(3-hydroxypropoxy)vitamin D3: 20-epi-ED-71]

Article abstract of DOI:10.1016/j.jsbmb.2010.03.041

Eldecalcitol [1α,25-dihydroxy-2β-(3-hydroxypropoxy)vitamin D₃, developing code: ED-71] is an analog of active vitamin D₃, 1α,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] that possesses a hydroxypropoxy substituent at the 2β-position of 1,25(OH)₂D₃. Eldecalcitol has potent biological effects on bone and is now in preparation for approval as a promising medicine for the treatment of osteoporosis in Japan. To explore chemical structure-biological activity relationships between eldecalcitol and related analogs, we have already synthesized 1-epi-eldecalcitol, 3-epi-eldecalcitol, and 1,3-diepi-eldecalcitol with inherent biological interests of each targeted analog and evaluated their biological responses. It has been reported that 20-epi-1,25(OH)₂D₃, a diastereomer of 1,25(OH)₂D₃ that possesses an inverted methyl substituent at the 20-position of the side chain, shows remarkably enhanced biological activities compared to parental compound, 1,25(OH)₂D₃. As a continuation of our modification studies on eldecalcitol, we took great interest in 20-epi-eldecalcitol and its biological responses. In this paper, the synthesis of 20-epi-eldecalcitol by the Trost coupling reaction between the A-ring fragment and the C/D-ring fragment as well as in vitro preliminary biological evaluation of 20-epi-eldecalcitol are described. In the induction of human myeloid leukemia cell (HL-60) differentiation, inhibition of the human histiocytic lymphoma cell (U937) proliferation, and increase in osteocalcin concentration in the human osteosarcoma cell (MG-63), 20-epi-eldecalcitol showed significantly enhanced activity compared to eldecalcitol.

Full text of DOI:10.1016/j.jsbmb.2010.03.041

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Journal of Steroid Biochemistry & Molecular Biology 121 (2010) 25–28
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Journal of Steroid Biochemistry and Molecular Biology
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Synthesis and preliminary biological evaluation of 20-epi-eldecalcitol
[20-epi-1␣,25-dihydroxy-2␤-(3-hydroxypropoxy)vitamin D3: 20-epi-ED-71]ଝ
Susumi Hatakeyamaa, Madoka Yoshinoa, Kohei Etoa, Keisuke Takahashia, Jun Ishiharaa,
Yoshiyuki Onob, Hitoshi Saitob, Noboru Kuboderab,∗
a Graduate School of Biomedical Sciences, Nagasaki University, 1-14, Bunkyomachi, Nagasaki 852-8521, Japan
b Chugai Pharmaceutical Co., Ltd., 2-1-1, Nihonbashi-Muromachi, Chuo-ku, Tokyo 103-8324, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Eldecalcitol [1␣,25-dihydroxy-2␤-(3-hydroxypropoxy)vitamin D3, developing code: ED-71] is an ana-
log of active vitamin D3, 1␣,25-dihydroxyvitamin D3 [1,25(OH)2D3] that possesses a hydroxypropoxy
substituent at the 2␤-position of 1,25(OH)2D3. Eldecalcitol has potent biological effects on bone and is
now in preparation for approval as a promising medicine for the treatment of osteoporosis in Japan. To
explore chemical structure–biological activity relationships between eldecalcitol and related analogs,
we have already synthesized 1-epi-eldecalcitol, 3-epi-eldecalcitol, and 1,3-diepi-eldecalcitol with inher-
ent biological interests of each targeted analog and evaluated their biological responses. It has been
reported that 20-epi-1,25(OH)2D3, a diastereomer of 1,25(OH)2D3 that possesses an inverted methyl
substituent at the 20-position of the side chain, shows remarkably enhanced biological activities com-
pared to parental compound, 1,25(OH)2D3. As a continuation of our modification studies on eldecalcitol,
we took great interest in 20-epi-eldecalcitol and its biological responses. In this paper, the synthesis of 20-
epi-eldecalcitol by the Trost coupling reaction between the A-ring fragment and the C/D-ring fragment as
well as in vitro preliminary biological evaluation of 20-epi-eldecalcitol are described. In the induction of
human myeloid leukemia cell (HL-60) differentiation, inhibition of the human histiocytic lymphoma cell
(U937) proliferation, and increase in osteocalcin concentration in the human osteosarcoma cell (MG-63),
20-epi-eldecalcitol showed significantly enhanced activity compared to eldecalcitol.
Received 20 October 2009
Received in revised form 9 February 2010
Accepted 12 March 2010
This paper is dedicated to the memory of
the late Dr. Etsuro Ogata, Professor
Emeritus, Tokyo University/Director
Emeritus, Cancer Institute Hospital, Japan,
who passed away on November 1, 2009.
Keywords:
Active vitamin D3
1␣,25-Dihydroxyvitamin D3
1␣,25-Dihydroxy-2␤-(3-hydroxypropoxy)
vitamin D3
ED-71
Eldecalcitol
© 2010 Elsevier Ltd. All rights reserved.
20-Epi-eldecalcitol
1. Introduction
als of 2 for bone fracture prevention produced excellent results
[3,4].
There is intense interest in obtaining analogs more potent
than active vitamin D3, 1␣,25-dihydroxyvitamin D3 [1,25(OH)2D3,
1] in terms of regulatory effects on calcium and phospho-
rus metabolism with the objective of treating bone diseases
such as osteoporosis [1]. Eldecalcitol [1␣,25-dihydroxy-2␤-(3-
hydroxypropoxy)vitamin D3, developing code: ED-71, 2], an analog
of 1,25(OH)2D3 (1) from which a hydroxypropoxy substituent at
the 2␤-position is appended, is such an analog that shows potent
effects on bone therapy [2]. Eldecalcitol (2) is now in prepara-
tion for approval as a promising medicine for the treatment of
osteoporosis in Japan. Recent completion of phase III clinical tri-
To explore chemical structure–biological activity relationships
between eldecalcitol (2) and related analogs, we have already
synthesized 1-epi-eldecalcitol [5], 3-epi-eldecalcitol [6], and 1,3-
diepi-eldecalcitol [7] with inherent biological interests of each
targeted analogs and evaluated their biological responses [8]. It
has been reported that 20-epi-1,25(OH)2D3 (3), a diastereomer of
1,25(OH)2D3 (1) which possesses an inverted C-21 methyl sub-
stituent at the 20-position of the side chain, shows remarkably
enhanced biological activities compared to parental compound,
1. For example, 20-epi-1,25(OH)2D3 (3) exhibits 18 times the
potency of induction of human myeloid leukemia cell (HL-60)
differentiation [9], 50 times the inhibition of the human histio-
cytic lymphoma cell (U937) proliferation [10], and 4.5 times the
increase in osteocalcin concentrations in the human osteosar-
coma cell (MG-63) [11] compared to 1. These findings prompted
our interest in analog of eldecalcitol (2) epimerized at the 20-
position and its biological responses [12]. In this paper, we describe
the synthesis of 20-epi-eldecalcitol (4) as a continuation of our
modification studies on 2 and its preliminary biological evalua-
ଝ
Special issue selected article from the 14th Vitamin D Workshop held at Brugge,
Belgium on October 4–8, 2009.
∗
Corresponding author at: Disease Area Strategy Dept. 2, Chugai Pharmaceutical
Co., Ltd., 2-1-1, Nihonbashi-Muromachi, Chuo-ku, Tokyo 103-8324, Japan.
Tel.: +81 3 3273 1010; fax: +81 3 3281 0191.
E-mail address: kuboderanbr@chugai-pharm.co.jp (N. Kubodera).
0960-0760/$ – see front matter © 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jsbmb.2010.03.041

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S. Hatakeyama et al. / Journal of Steroid Biochemistry & Molecular Biology 121 (2010) 25–28
Fig. 1. Chemical structures of active vitamin D3 analogs.
Fig. 2. Retrosynthesis of 20-epi-eldecalcitol (4).
tion using HL-60, U937, and MG-63 compared to eldecalcitol (2)
(Fig. 1).
J = 11.0 Hz), 5.42 (t, 1H, J = 2.0 Hz), 4.98 (t, 1H, J = 2.0 Hz), 4.23 (d,
1H, J = 8.0 Hz), 4.19 (m, 1H), 3.75–3.71 (m, 2H), 3.71–3.60 (m, 6H),
3.10 (dd, 1H, J = 14.8, 4.5 Hz), 2.86 (dd, 1H, J = 10.0, 4.0 Hz), 2.47
(dd, 1H, J = 14.0, 6.0 Hz), 2.35 (d, 1H, J = 13.5 Hz), 2.04–1.98 (m, 2H),
1.88–1.79 (m, 4H), 1.72–1.65 (m, 2H), 1.56–1.24 (m, 12H), 1.17 (m,
6H), 0.87 (d, 3H, J = 7.0 Hz), 0.57 (s, 3H); 13C NMR (75 MHz, CDCl3) ı
144.2, 142.9, 132.1, 124.4, 117.2, 111.8, 85.4, 71.5, 71.1, 68.2, 66.5,
61.1, 60.4, 56.2, 45.9, 44.3, 40.3, 36.6, 35.4, 31.8, 29.7, 29.2, 27.3,
23.7, 22.2, 20.9, 18.5, 14.2, 12.2, 1.0; EIMS m/z 490 (M+), 472 (100%);
HRMS (EI) m/z calcd for C30H50O5 (M+) 490.3676, found 490.3641.
2. Materials and methods
2.1. (1R,2R,3R,Z)-5-((E)-2-((1R,3aS,7aR)-1-((S)-6-Hydroxy-6-
methylheptan-2-yl)-7a-methyldihydro-1H-inden-4-
(2H,5H,6H,7H,7aH)-ylidene)ethylidene)-2-
(3-hydroxypropoxy)-4-methylenecyclohexane-1,3-diol (4)
20-Epi-eldecalcitol (4) was prepared from the coupling reaction
of A-ring fragment 5 with C/D-ring fragment 7 and the subse-
2.2. Induction of HL-60 differentiation
quent desilylation reaction as a colorless oil; [␣]D −74.6◦ (c, 0.54,
20
The induction of HL-60 differentiation activity by eldecalcitol
(2), 20-epi-eldecalcitol (4), and 1,25(OH)2D3 (1) was measured
according to Kittaka et al. [9].
CH3OH); FTIR (neat) 3407, 1463, 1375, 1260, 1215, 1105 cm−1
;
1H NMR (500 MHz, CD3OD) ı 6.30 (d, 1H, J = 11.0 Hz), 6.06 (d, 1H,
Fig. 3. Synthesis of A-ring fragment 5. (a) HO(CH2)3OH, t-BuOK, 120 ◦C; (b) t-BuCOCl, pyridine, CH2Cl2, rt; (c) (1) H2, Pd(OH)2, MeOH, rt; (2) Me2C(OMe)2, TsOH, rt; (d) (1)
DMSO, (COCl)2, CH2Cl2, −60 ◦C; (2) CH2 CHMgBr, THF, −60 ◦C; (3) t-BuCOCl. Et3N, DMAP, CH2Cl2, rt; (e) 1 M HCl, MeOH, rt; (f) Ph3P, DEAD, benzene, reflux; (g) (1) LiC CTMS,
BF3-OEt2, THF, −78 ◦C; (2) 10N NaOH, MeOH, rt; (3) TBSOTf, Et3N, CH2Cl2, 0 ◦C.

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S. Hatakeyama et al. / Journal of Steroid Biochemistry & Molecular Biology 121 (2010) 25–28
27
Fig. 4. Synthesis of C/D-ring fragment 7 and coupling with A-ring fragment 5 to obtain 20-epi-eldecalcitol (4). (a) TsCl, DMAP, pyridine, rt; (b) TBSOTf, 2,6-lutidine, CH2Cl2,
−40 ◦C; (c) DMSO, s-collidine, 150 ◦C; (d) n-Bu4NOH, CH2Cl2, rt; (e) NaBH4, EtOH, THF, 0 ◦C; (f) TsCl, DMPA, pyridine, rt; (g) NaI, DMF, 85 ◦C; (h) MVK, Zn, CuI, EtOH, H2O,
20–30 ◦C; (i) MeMgI, THF, 0 ◦C; (j) 47% HF, MeCN, THF, 0 ◦C; (k) TPAP, NMO, CH2Cl2, rt; (l) Ph3P+CH2Br/Br−, NaHMDS, rt; (m) A-ring fragment 5, Pd(PPh3)4, Et3N, toluene,
reflux; (n) 47% HF, MeCN, rt.
2.3. Inhibition of U937 proliferation
Inhoffen-Lythgoe diol (8) obtained by ozonolysis of vitamin D2
as shown in Fig. 4 [18–20]. With A-ring fragment 5 and C/D-ring
fragment 7 in hand, we finally investigated the Trost coupling
reaction. Upon treatment of an excess 5 with 7 in the presence
of tetrakis(triphenylphosphine)palladium (0) [Pd(PPh3)4] and tri-
ethylamine (Et3N) in toluene, the coupled product 28 was obtained
in 42% yield, which was desilylated using 47% HF in acetonitrile
(MeCN) to afford 20-epi-eldecalcitol (4) in 73% yield (Fig. 4). The
1H NMR chemical shift of the C-21 methyl resonance in 20-epi-
eldecalcitol (4) (0.87 ppm) shifted upfield by 0.04 ppm relative to
natural configuration, eldecalcitol (2) (0.91 ppm) [2] and is consis-
tent with this general pattern [21].
The inhibitory activity of U937 proliferation by eldecalcitol (2),
20-epi-eldecalcitol (4), and 1,25(OH)2D3 (1) was measured accord-
ing to Binderup et al. [10].
2.4. Osteocalcin concentration in MG-63
The osteocalcin concentrations in MG-63 with eldecalcitol
(2), 20-epi-eldecalcitol (4), and 1,25(OH)2D3 (1) were measured
according to Ryhanen et al. [11].
3. Results
3.1. Synthesis of 20-epi-eldecalcitol (4)
3.2. Preliminary biological evaluation of 20-epi-eldecalcitol (4)
Our synthesis of 20-epi-eldecalcitol (4) was envisioned using
the coupling reaction of A-ring fragment 5, derived from C2-
symmetrical epoxide 6, with C/D-ring fragment 7, which can
be obtained from Inhoffen-Lythgoe diol (8) [13,14] (Fig. 2).
The required A-ring fragment 5 for the synthesis of 20-epi-
eldecalcitol (4) was synthesized based on the methodology that
has been previously established by us [15–17] (Fig. 3). Next,
we performed the synthesis of C/D-ring fragment 7 from the
The results of preliminary in vitro biological evaluation of syn-
thetic 20-epi-eldecalcitol (4) in comparison with eldecalcitol (2)
and 1,25(OH)2D3 (1) are summarized in Table 1. As anticipated,
20-epi-eldecalcitol (4) showed greatly enhanced activity toward
the induction of HL-60 differentiation (6085.99/49.6 = 122.7 times),
inhibition of U937 proliferation (738.74/4.15 = 178.0 times), and
increase in osteocalcine concentrations in MG-63 (2980/15 = 198.7
times), compared to eldecalcitol (2).
Table 1
Preliminary biological evaluation of 20-epi-eldecalcitol (4) in comparison with
1,25(OH)2D3 (1) and eldecalcitol (2).
4. Conclusion
HL-60
U937
100
4.15
738.74
MG-63
Based on the Trost coupling methodology involving A-ring
fragment 5 and C/D-ring fragment 7, the synthesis of 20-epi-
eldecalcitol (4) has been successfully achieved. In preliminary
biological evaluations, 20-epi-eldecalcitol (4) showed greatly
enhanced potencies toward HL-60, U937, and MG-63 cell lines.
We are very much interested in in vivo biological activity of 20-
epi-eldecalcitol (4) on bone. Detailed experimental conditions and
further biological studies will be reported elsewhere.
1,25(OH)2D3 (1)
Eldecalcitol (2)
20-Epi-eldecalcitol (4)
100
49.6
6085.99
100
15
2980
HL-60: relative potency of induction of human myeloid leukemia cell (HL-60) dif-
ferentiation [9].
U937: relative potency of inhibition of the human histiocytic lymphoma (U937) pro-
liferation [10].
MG-63: relative potency of transcriptional activity of osteocalcin of human osteosar-
coma cell (MG-63) [11].

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S. Hatakeyama et al. / Journal of Steroid Biochemistry & Molecular Biology 121 (2010) 25–28
Acknowledgments
D3 (E-71) using C2-symmetrical epoxide as a common starting material, Anti-
cancer Res. 29 (2009) 3571–3578.
[9] N. Saito, Y. Suhara, M. Kurihara, T. Fujishima, S. Honzawa, H. Takayanagi, T.
Kozono, M. Matsumoto, M. Ohmori, N. Miyata, H. Takayama, A. Kittaka, Design
and efficient synthesis of 2␣-(␻-hydroxyalkoxy)-1␣,25-dihydroxyvitamin D3
analogues, including 2-epi-ED-71 and their 20-epimers with HL-60 cell differ-
entiation activity, J. Org. Chem. 69 (2004) 7463–7471.
We are grateful to Professor David Horne of Department of
Molecular Medicine, City of Hope, for helpful suggestions and read-
ing of the manuscript.
[10] L. Binderup, S. Latini, E. Binderup, C. Bretting, M. Calverley, K. Hansen, 20-Epi-
vitamin D3 analogues: a novel class of potent regulators of cell growth and
immune responses, Biochem. Pharmacol. 42 (1991) 1569–1575.
[11] S. Pyhanen, A. Mahonen, T. Jaaskelainen, P.H. Maenpaa, Synthetic 20-epi
analogs of calcitriol are potent inducers of target-gene activation in osteoblastic
cells, Eur. J. Biochem. 238 (1996) 97–103.
[12] L. Binderup, E. Binderup, W.O. Godtfredsen, A.-M. Kissmeyer, Development of
new vitamin D analogs, in: D. Feldman, J.W. Pike, F.H. Glorieux (Eds.), Vitamin
D, 2nd ed., Elsevier Academic Press, Burlington, 2005, pp. 1489–1510.
[13] B.M. Trost, J. Dumas, New strategy for the total synthesis of 1␣-hydroxyvitamin
D derivatives, J. Am. Chem. Soc. 114 (1992) 1924–1925.
[14] B.M. Trost, J. Dumas, M. Villa, New strategies for the synthesis of vitamin D
metabolites, J. Am. Chem. Soc. 114 (1992) 9836–9845.
[15] J. Maeyama, H. Hiyamizu, K. Takahashi, J. Ishihara, S. Hatakeyama, N. Kubodera,
Two convergent approaches to the synthesis of 1␣,25-dihydroxy-2␤-(3-
hydroxypropoxy)vitamin D3 (ED-71) by the Lythgoe and the Trost coupling
reactions, Heterocycles 70 (2006) 295–307.
[16] K.C. Nicolaou, D.P. Papahatjis, D.A. Claremon, R.L. Magolda, R.E. Dolle, Total
synthesis of ionophore antibiotic X-14547A, J. Org. Chem. 50 (1985) 1440–1456.
[17] O. Mitsunobu, The use of diethyl azocarboxylate and triphenylphosphine in
synthesis and transformation of natural products, Synthesis (1981) 1–28.
[18] H.H. Inhoffen, G. Quinkert, S. Schutz, G. Friedrich, E. Tober, Studien in der
vitamin D-reihe, XXV. Abbau der vitamin D2 und D3 zum 8-methyl-trans-
hydrindanol-(4)-on-(1), Chem. Ber. 91 (1958) 781–791.
[19] G.H. Posner, Z. Li, M.C. White, V. Victoria, K. Takeuchi, S.E. Guggio, P. Dolan,
T.W. Kensler, 1␣,25-Dihydroxyvitamin D3 analogs featuring aromatic and het-
eroaromatic rings: Design, synthesis, and preliminary biological testing, J. Med.
Chem. 38 (1995) 4529–4537.
[20] T. Fujishima, K. Konno, K. Nakagawa, M. Kurobe, T. Okano, H. Takayama,
Efficient synthesis and biological evaluation of all A-ring diastereomers of
1␣,25-dihydroxyvitamin D3 and its 20-epimer, Bioorg. Med. Chem. 8 (2000)
123–134.
[21] C.H. Robinson, P. Hofer, N.M.R. spectra of C(20)-substituted pregnans, Chem.
Ind. 26 (1966) 377–378.
References
[1] G.H. Posner, M. Kahraman, Overview: rational design of 1␣,25-
dihydroxyvitamin D3 analogs (deltanoids), in: D. Feldman, J.W. Pike,
F.H. Glorieux (Eds.), Vitamin D, 2nd ed., Elsevier Academic Press, Burlington,
2005, pp. 1405–1422.
[2] K. Miyamoto, E. Murayama, K. Ochi, H. Watanabe, N. Kubodera, Synthetic stud-
ies of vitamin D analogs. XIV. Synthesis and calcium regulating activity of
vitamin D3 analogs bearing a hydroxyalkoxy group at the 2␤-position, Chem.
Pharm. Bull. 41 (1993) 1111–1113.
[3] N. Kubodera, N. Tsuji, Y. Uchiyama, K. Endo, A new active vitamin D analog,
ED-71, causes increase in bone mass with preferential effects on bone in osteo-
porotic patients, J. Cell. Biochem. 88 (2003) 286–289, and references cited
therein.
[4] T. Matsumoto, T. Miki, H. Hagino, T. Sugimoto, S. Okamoto, T. Hirota, Y. Tani-
gawara, Y. Hayashi, M. Fukunaga, M. Shiraki, T. Nakamura, A new active vitamin
D, ED-71, increases bone mass in osteoporotic patients under vitamin D sup-
plementation: a randomized, double-blind, placebo-controlled clinical trial, J.
Clin. Endocrinol. Metab. 90 (2005) 5031–5036.
[5] K. Eto, A. Fujiyama, M. Kaneko, K. Takahashi, J. Ishihara, S. Hatakeyama, Y. Ono,
N. Kubodera, An improved synthesis of 1-epi-ED-71, a biologically interesting
diastereomer of 1␣,25-dihydroxy-2␤-(3-hydroxypropoxy)vitamin D3 (ED-71),
Heterocycles 77 (2009) 323–331.
[6] S. Hatakeyama, S. Nagashima, N. Imai, K. Takahashi, J. Ishihara, A. Sugita, T.
Nihei, H. Saito, F. Takahashi, N. Kubodera, Synthesis and evaluation of a 3-
position diastereomer of 1␣,25-dihydroxy-2␤-(3-hydroxypropoxy)vitamin D3
(ED-71), Bioorg. Med. Chem. 14 (2006) 8050–8056.
[7] A. Fujiyama, M. Kaneko, K. Takahashi, J. Ishihara, S. Hatakeyama, N. Kubodera,
Synthesis of 1,3-diepi-ED-71,
1␣,25-dihydroxy-2␤-(3-hydroxypropoxy)vitamin D3 (ED-71), Heterocycles 71
(2007) 2263–2271.
a biologically important diastereomer of
[8] N. Kubodera, S. Hatakeyama, Synthesis of all possible A-ring diastereomers
at the 1- and 3-positions of 1␣,25-dihydroxy-2␤-(3-hydroxypropoxy)vitamin