Synthesis of 2α-substituted-14-epi-previtamin D3 and its genomic activity

Article abstract of DOI:10.1016/j.bmcl.2009.07.112

We synthesized and isolated 2α-substituted analogs of 14-epi-previtamin D₃ after thermal isomerization at 80 °C for the first time. The VDR binding affinity and transactivation activity of osteocalcin promoter in HOS cells were evaluated, and t

Full text of DOI:10.1016/j.bmcl.2009.07.112

Bioorganic & Medicinal Chemistry Letters 19 (2009) 5397–5400
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of 2
a
-substituted-14-epi-previtamin D3 and its genomic activity
Daisuke Sawada a, Tomoyuki Katayama a, , Yuya Tsukuda a, Nozomi Saito a, , Masashi Takano a,
Hiroshi Saito b, Ken-ichiro Takagi b, Eiji Ochiai b, Seiichi Ishizuka b, Kazuya Takenouchi b, Atsushi Kittaka a,
*
a Faculty of Pharmaceutical Sciences, Teikyo University, Sagamiko, Kanagawa 229-0195, Japan
b Teijin Institute for Bio-medical Research, Teijin Pharma Ltd., Tokyo 191-8512, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
We synthesized and isolated 2
at 80 °C for the first time. The VDR binding affinity and transactivation activity of osteocalcin promoter in
HOS cells were evaluated, and the 2
activity than 14-epi-previtamin D3.
a
-substituted analogs of 14-epi-previtamin D3 after thermal isomerization
Received 1 July 2009
Revised 23 July 2009
Accepted 24 July 2009
Available online 28 July 2009
a
-methyl-substituted analog was found to have greater genomic
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Vitamin D3
14-Epi-previtamin D3
Osteocalcin
Vitamin D receptor
Vitamin D3 is present in thermal equilibrium with previtamin
D3 via [1,7]-sigmatropic rearrangement. In this equilibrium, the
6-s-trans isomer, that is, the vitamin D form (A), is more stable
and major than the 6-cis isomer of the previtamin D form (B)
previtamin D form, because previtamin D3 is easily transformed
to vitamin D3 through thermal equilibrium and is almost impossi-
ble to isolate in the pure form.1 While 1 is a ligand of the nucleic
receptor (vitamin D receptor, VDR), regulates gene transcription,
and exhibits various biological responses as a hormone,2 pre-1 is
believed to be a weak ligand of VDR and a poor activator of the
above genomic actions;3 however, pre-1 has been studied as a li-
gand of a putative membrane vitamin D receptor for a long time,4
and it is well-known that pre-1 causes various biological rapid
(Scheme 1). Active vitamin D3, 1a,25(OH)2D3 (1), which is the bio-
logically most active metabolite of vitamin D3, contains 5–10% of
its previtamin D form, 1a,25(OH)2preD3 (pre-1) at 37 °C in similar
equilibrium.1 Most scientists have focused on the analogs of the
major vitamin D form for therapeutic evaluation rather than the
25
R
H
14α−H
25
H
14
R
R
H
14
1
H
HO
14β−H
1
HO
R
(A)
(B)
previtamin D3: R = H (14α−H)
pre-1: R = OH, 1α,25(OH)2preD3 (14α−H)
14-epi-pre-1: R = OH, 14-epi-1α,25(OH)2preD3 (14β−H)
vitamin D3: R = H (14α−H)
1: R = OH, 1α,25(OH)2D3 (14α−H, 1α,25-dihydroxyvitamin D3)
14-epi-1: R = OH, 14-epi-1α,25(OH)2D3 (14β−H)
Scheme 1. Equilibrium between vitamin D3 and previtamin D3.
* Corresponding author. Tel./fax: +81 42 685 3713.
E-mail address: akittaka@pharm.teikyo-u.ac.jp (A. Kittaka).
Present address: Faculty of Pharmaceutical Sciences, Hokkaido University,
Sapporo 060-0812, Japan.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.07.112

5398
D. Sawada et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5397–5400
OTES
H
OH
H
Vitamin D3
H
OH
H
OH
O
2
R
H
H
HO
P(O)Ph2
OTBS
14-epi-pre-1a : R = Me
HO
OH
14-epi-pre-1b : R = (CH2)3OH
14-epi-pre-1c : R = O(CH2)3OH
14-epi-pre-1d : R = Bu
14-epi-pre-1e : R = Ph
14-epi-pre-1f : R = Bn
R
Methyl α-D-glucopyranoside
1a-f
TBSO
R
3a-f
Scheme 2. Retrosynthetic analysis of 2
a-substituted 14-epi-1a,25(OH)2preD3.
responses, for example, stimulation of intestinal Ca2+ transport
(transcaltachia),5 activation of PKC6 and MAP7 kinases, and so on,
which are called non-genomic actions.
Okamura et al. reported that the thermal equilibrium ratio be-
tween vitamin D form (A) and previtamin D form (B) at 80 °C
was reversed by epimerizing the CD-ring bridgehead hydrogen of
porary first targets. This strategy would help us to understand the
equilibrium between vitamin D3 and previtamin D3. The 14-epi-1
analogs were divided into two fragments, which were CD-ring
and A-ring fragments (Scheme 2). The CD-ring fragment could be
obtained by epimerization at H14 in Grundmann’s ketone deriva-
tive, which was derived from vitamin D3.9 The A-ring fragment
C14, that is, 14-epi-1
and dominant to 14-epi-1
a
,25(OH)2preD3 (14-epi-pre-1) was major
could be synthesized from methyl
introduce various alkyl groups at the 2
previously.10 We found that 2
-alkyl and 2a x
a
-
D
-glucoside, and we could
-position as we reported
-( -hydroxyalkyl)
a
,25(OH)2D3 (14-epi-1).8 Since it re-
a
quires a high temperature (80 °C) to reach thermal equilibrium,
14-epi-pre-1 is expected to be isolated stable at room tempera-
ture. Using this reverse equilibrium, we focused on the synthesis
of the 14-epi-pre-1 analogs with A-ring modification, and aimed
to identify the more detailed biological properties and potential
as therapeutic agents of the previtamin D3 skeleton.
a
substitution afforded great improvements VDR binding affinity
and the subsequent genomic actions.11 We therefore decided to
prepare analogs with 2
Letter.
a-substitutions (14-epi-pre-1aꢀ1f) in this
The CD-ring fragment (2) was synthesized from the known ke-
tone of TES-protected 25-hydroxy Grundmann’s ketone (4).9
According to the literature, epimerization of H14 was successfully
conducted by NaOMe with recovery of the starting material
(Scheme 3).8
14-epi-pre-1 could be prepared from 14-epi-1 by thermal
isomerization, so we planed to synthesize 14-epi-1 analogs as tem-
A-ring fragments (8a–f) were prepared from the known
enynes 3a–f (Scheme 4),10a–d which reacted with n-BuLi and then
(CH2O)n to give alcohols 5a–f in good to excellent yields. Then,
hydroalumination and subsequent iodination of the alkyne gave
the vinyl iodides 6a–f. Next, cyclization by Heck reaction pro-
OTES
H
OTES
H
NaOMe
MeOH
H
H
O
2
O
4
conv. y. 93%
(4 : 2 = 1.2 : 1)
ceeded smoothly to afford
a
six-membered A-ring,12 whose
hydroxyls were easily transformed into phosphine oxides in three
Scheme 3. Synthesis of the CD-ring fragment.
OH
OH
°
n-BuLi, −78
C
1) Red-Al
I
TBSO
OTBS
°
(CH2O)n
2) I2, −78
C
TBSO
OTBS
R
TBSO
OTBS
R
3a : R = Me
R
3b : R = (CH2)3OTBS
3c : R = O(CH2)3OTBS
3d : R = Bu
3e : R = Ph
3f : R = Bn
5a : R = Me y. 91%
6a : R = Me y. 73%
5b : R = (CH2)3OTBS y. 92%
5c : R = O(CH2)3OTBS y. 89%
5d : R = Bu y. 91%
5e : R = Ph y. 81%
5f : R = Bn y. 94%
6b : R = (CH2)3OTBS y. 70%
6c : R = O(CH2)3OTBS y. 75%
6d : R = Bu y. 75%
6e : R = Ph y. 67%
6f : R = Bn y. 76%
P(O)Ph2
OH
1) NCS, S(CH3)2
2) PHPh2, n-BuLi, −78
°
C
Pd(PPh3)4
3) H2O2
Et3N, reflux
TBSO
OTBS
R
TBSO
R
OTBS
8a : R = Me y. 77%
7a : R = Me y. 93%
8b : R = (CH2)3OTBS y. 74%
8c : R = O(CH2)3OTBS y. 76%
8d : R = Bu y. 76%
8e : R = Ph y. 39%
8f : R = Bn y. 83%
7b : R = (CH2)3OTBS y. 89%
7c : R = O(CH2)3OTBS y. 82%
7d : R = Bu y. 93%
7e : R = Ph y. 99%
7f : R = Bn y. 92%
Scheme 4. Synthesis of the A-ring fragments.

D. Sawada et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5397–5400
5399
OTES
H
OTES
H
H
H
2
O
TBAF or HF/Py
THF
n-BuLi
THF
+
TBSO
OTBS
P(O)Ph2
R
9a: R = Me y. 44%
9b: R = (CH2)3OTBS y. 52%
9c: R = O(CH2)3OTBS y. 55%
9d: R = Bu y. 24%
9e: R = Ph y. 9%
9f: R = Bn y. 59%
TBSO
OTBS
R
8a-f
OH
H
OH
H
OH
R
H
H
HO
benzene
°
80
C
14-epi-pre-1a: R = Me y. 39%*
14-epi-pre-1b: R = (CH2)3OH y. 41%*
14-epi-pre-1c: R = O(CH2)3OH y. 42%*
14-epi-pre-1d: R = Bu y. 96%
HO
OH
R
14-epi-pre-1e: R = Ph y. 81%
14-epi-pre-1f: R = Bn y. 92%
1a-f
*Deprotection was conducted by HF/Py.
Scheme 5. Coupling reaction and synthesis of 2a-substituted 14-epi-1a,25(OH)2preD3.
steps to give 8a–f, respectively.8 As above, we were able to prepare
A-ring fragments in good overall yield.
Table 1
Relative binding affinity for chick intestinal VDR and osteocalcin promoter transac-
tivation activity in HOS cells of 2a-substituted 14-epi-1a,25(OH)2preD3
14
Since both fragments were available, we tried the coupling
reaction under basic conditions using n-BuLi (Scheme 5).8 Small
excess amounts of the A-ring fragment worked well and we ob-
tained the coupled products 9a–f, although some were obtained
in low yield. The silyl protected 14-epi-1 analogs (9a–f) tend not
to lead to isomerization to their previtamin D form, probably be-
cause TBS groups at the A-ring should show steric hindrance to
reaching the transition state of the [1,7]-sigmatropic hydrogen
shift existing between the vitamin D form and previtamin D form.
Then, all silyl groups in 9a–f were removed in one step with excess
TBAF or HF/pyridine, and most of the deprotected compounds re-
mained in the vitamin D form (1a–f), and only small amounts of
the previtamin D form (pre-1aꢀ1f) were produced under these
reaction conditions. Isomerization was therefore examined at
80 °C in benzene, and fortunately the portions of the desired 14-
epi-pre-1aꢀ1f increased, and most vitamin D forms were con-
verted into the previtamin D form easily in less than two hours.
After thermal equilibrium had been established, the ratio of the
compounds was about 5/95 (vitamin D/previtamin D) based on
1H NMR studies. Using HPLC, the mixture of both forms was sepa-
rated, and pure 14-epi-pre-1aꢀ1f were used for further biological
studies.13
Compound
VDRa
Osteocalcin transactivation
activity (ED50 (nM))
1
100
0.5
8.4
1.4
0.03
0.46
0.12
0.69
0.95
5.77
0.88
30.2
14-epi-pre-1
14-epi-pre-1a
14-epi-pre-1b
14-epi-pre-1c
14-epi-pre-1d
14-epi-pre-1e
14-epi-pre-1f
0.17
0.27
<0.03
0.03
and 2
the biological actions of the previtamin D form.
In conclusion, we synthesized the 2 -substituted analogs of 14-
a-substitution on the A-ring seems to have great effects on
a
epi-pre-1 for the first time and were able to isolate these new ana-
logs (14-epi-pre1aꢀ1f) after thermal isomerization at 80 °C. Using
them, we evaluated the VDR binding affinity and transactivation
activity of osteocalcin promoter in HOS cells, among which, the
2a-methyl-substituted analog (14-epi-pre-1a) was found to have
greater genomic activity than 14-epi-pre-1. Further studies of
the action mechanisms of 14-epi-pre-1 analogs as well as studies
on non-genomic activity are currently in progress in our
laboratory.
The VDR binding affinity and the osteocalcin promoter transac-
tivation activity of the new compounds were evaluated using the
chick intestinal VDR and HOS cells, respectively.14 The results are
summarized in Table 1 in comparison with the natural hormone
1 and 14-epi-pre-1, which was synthesized in a similar manner
in our laboratory. The new compounds showed lower activity than
the natural hormone 1; however, some showed higher activity
Acknowledgments
We are grateful to Ms. Junko Shimode and Ms. Ayako Kawaji
(Teikyo University) for the spectroscopic measurements. This work
was supported in part by Grants-in-Aid from the Ministry of Edu-
cation, Culture, Sports, Science and Technology, Japan (to D.S and
N.S.) and by a Grant-in-Aid from the Japan Society for the Promo-
tion of Science (to A.K.).
than 14-epi-1a,25(OH)2preD3 (14-epi-pre-1). In particular, 14-
epi-pre-1a, the 2a-methyl substituted analog indicated a remark-
able increase in VDR binding affinity and transactivation activity. It
is worth noting that 14-epi-pre-1 analogs gain genomic activity,

5400
D. Sawada et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5397–5400
6. (a) Shumitz, J. P.; Schwartz, Z.; Sylvia, V. L.; Dean, D. D.; Calderon, F.; Boyan, B.
Supplementary data
D. J. Cell Physiol. 1996, 168, 570; (b) Slater, S. J.; Kelly, M. B.; Taddeo, F. J.; Larkin,
J. D.; Yeager, M. D.; McLane, J. A.; Ho, C.; Stubbs, C. D. J. Biol. Chem. 1995, 270,
6639; (c) Bissonnette, M.; Tien, X.-Y.; Niedziela, S. M.; Hartmann, S. C.; Frawley,
B. P., Jr.; Roy, H. K.; Sitrin, M. D.; Perlman, R. L.; Brasitus, T. A. Am. J. Physiol.
1994, 267, G465.
Supplementary data (spectroscopic data of 14-epi-pre-1bꢀ1f
and experimental details of testing binding affinity for the chick
intestinal VDR and osteocalcin promoter transactivation activity)
associated with this article can be found, in the online version, at
doi:10.1016/j.bmcl.2009.07.112.
7. (a) Song, X.; Bishop, J. E.; Norman, A. W. Endocrinology 1998, 139, 457; (b) De
Boland, A. R.; Norman, A. W. J. Cell. Biochem. 1998, 69, 470; (c) Beno, D. W. A.;
Brady, L. M.; Bissonnette, M.; Davis, B. H. J. Biol. Chem. 1995, 270, 3642.
8. Maynard, D. F.; Trankle, W. G.; Norman, A. W.; Okamura, W. H. J. Med. Chem.
1994, 37, 2387.
9. (a) Inhoffen, H. H.; Quinkert, G.; Siegismund, S.; Kampe, D.; Domagk, G. F. Chem.
Ber. 1957, 90, 664; (b) Kiegel, J.; Wovkulich, P. M.; Uskokovic, M. R. Tetrahedron
Lett. 1991, 32, 6057.
10. (a) Kittaka, A.; Suhara, Y.; Takayanagi, H.; Fujishima, T.; Kurihara, M.;
Takayama, H. Org. Lett. 2000, 2, 2619; (b) Saito, N.; Suhara, Y.; Kurihara, M.;
Fujishima, T.; Honzawa, S.; Takayanagi, H.; Kozono, T.; Matsumoto, M.;
Ohmori, M.; Miyata, N.; Takayama, H.; Kittaka, A. J. Org. Chem. 2004, 69,
7463; (c) Suhara, Y.; Kittaka, A.; Kishimoto, S.; Calverley, M. J.; Fujishima, T.;
Saito, N.; Sugiura, T.; Waku, K.; Takayama, H. Bioorg. Med. Chem. Lett. 2002, 12,
3255; (d) Saito, N.; Masuda, M.; Matsunaga, T.; Saito, H.; Anzai, M.; Takenouchi,
K.; Miura, D.; Ishizuka, S.; Takimoto-Kamimura, M.; Kittaka, A. Tetrahedron
2004, 60, 7951; (e) Hourai, S.; Fujishima, T.; Kittaka, A.; Suhara, Y.; Takayama,
H.; Rochel, N.; Moras, D. J. Med. Chem. 2006, 49, 5199.
References and notes
1. Curtin, M. L.; Okamura, W. H. J. Am. Chem. Soc. 1991, 113, 6958.
2. (a)Vitamin D; Feldman, D., Pike, J. W., Glorieux, F. H., Eds., 2nd ed.; Elsevier
Academic Press: New York, 2005. For mechanism of action, see: Section II; for
target organs and tissues, see: Section IV; (b) DeLuca, H. F. Nutri. Rev. 2008, 66,
S73; (c) Brown, A. J.; Slatopolsky, E. Mol. Aspects Med. 2008, 29, 433; (d)
Bouillon, R.; Okamura, W. H.; Norman, A. W. Endocr. Rev. 1995, 16, 200; (e) Zhu,
G. D.; Okamura, W. H. Chem. Rev. 1995, 95, 1877; (f) Ettinger, R. A.; DeLuca, H. F.
Adv. Drug Res. 1996, 28, 269.
3. (a) Chen, Y.-J.; Gao, L.-J.; Murad, I.; Verstuyf, A.; Verlinden, L.; Verboven, C.;
Bouillon, R.; Viterbo, D.; Milanesio, M.; Haver, D. V.; Vandewalle, M.; De Clercq,
P. J. Org. Biomol. Chem. 2003, 1, 257; (b) Verlinden, L.; Verstuyf, A.; Verboven, C.;
Eelen, G.; De Ranter, C.; Gao, L.-J.; Chen, Y.-J.; Murad, I.; Choi, M.; Yamamoto, K.;
Yamada, S.; Van Haver, D.; Vandewalle, M.; De Clercq, P. J.; Bouillon, R. J. Biol.
Chem. 2003, 278, 35476.
11. For our review articles, see: (a) Saito, N.; Honzawa, S.; Kittaka, A. Curr. Top. Med.
Chem. 2006, 6, 1273; (b) Saito, N.; Kittaka, A. ChemBioChem 2006, 7, 1478; (c)
Saito, N.; Kittaka, A. J. Synth. Org. Chem. Jpn. 2007, 65, 947.
4. (a) Mizwicki, M. T.; Keidel, D.; Bula, C. M.; Bishop, J. E.; Zanello, L. P.; Wurtz, J.-
M.; Moras, D.; Norman, A. W. PNAS 2004, 101, 12876; (b) Nemere, I.;
Dormanen, M. C.; Hammond, M. W.; Okamura, W. H.; Norman, A. W. J. Biol.
Chem. 1994, 269, 23750; (c) Norman, A. W.; Okamura, W. H.; Hammond, M. W.;
Bishop, J. E.; Dormanen, M. C.; Bouillon, R.; Van Baelen, H.; Ridall, A. L.; Daane,
E.; Khoury, R.; Farach-Carson, M. C. Mol. Endocrinol. 1997, 11, 1518; (d) Norman,
A. W. Endocrinology 2006, 147, 5542; (e) Huhtakangas, J. A.; Olivera, C. J.;
Bishop, J. E.; Zanello, L. P.; Norman, A. W. Mol. Endocrinol. 2004, 18, 2660; (f)
Nguyen, T. M.; Lieberherr, M.; Fritsch, J.; Guillozo, H.; Alvarez, M. L.; Fitouri, Z.;
Jehan, F.; Carabedian, M. J. Biol. Chem. 2004, 279, 7591.
12. Hatakeyama, S.; Irie, H.; Shintani, T.; Noguchi, Y.; Yamada, H.; Nishizawa, M.
Tetrahedron 1994, 50, 13369.
13. Data for 14-epi-pre-1a: ½a D22
ꢁ
ꢂ11.64 (c 1.61, CHCl3); UV (EtOH) kmax 252.5 nm,
kmin 229.5 nm; IR (neat) 3374, 2961, 1456, 1375 cmꢂ1
;
1H NMR (400 MHz,
CDCl3) d 0.84 (s, 3H), 0.87 (d, J = 6.4 Hz, 3H), 0.99–1.98 (m, 34H), 2.50 (dd,
J = 16.8, 5.1 Hz, 1H), 3.65 (dt, J = 10.0, 5.5 Hz, 1H), 3.87 (d, J = 2.0 Hz, 1H), 5.58
(d, J = 3.4 Hz, 1H), 5.71 (d, J = 13.1 Hz, 1H), 5.75 (d, J = 13.1 Hz, 1H); 13C NMR
(100 MHz, CDCl3) d 12.8, 17.6, 19.6, 20.9, 21.8, 22.9, 28.6, 29.1, 29.5, 29.6, 33.9,
34.1, 35.7, 38.4, 41.1, 41.7, 44.3, 51.0, 51.8, 68.2, 71.1, 74.5, 125.6, 127.4, 130.4,
130.8, 132.7, 138.7; EI-LRMS m/z 430 (M+), 412, 396, 374, 350, 169; EI-HRMS
calcd for C28
H46O3 (M+) 430.3446, found 430.3430. For spectroscopic data of
5. (a) Zhou, L.-X.; Nemere, I.; Norman, A. W. J. Bone Miner. Res. 1992, 7, 457; (b) De
Boland, A. R.; Norman, A. W. Endocrinology 1990, 127, 2475; (c) Nemere, I.;
Yoshimoto, Y.; Norman, A. W. Endocrinology 1984, 115, 1476; (d) Norman, A.
W.; Mizwicki, M. T.; Norman, D. P. G. Nat. Rev. Drug Disc. 2004, 3, 27.
14-epi-pre-1bꢀ1f, see: Supplementary data.
14. For experimental details of testing binding affinity for the chick intestinal VDR
and osteocalcin promoter transactivation activity, see: Supplementary data.