New C15-substituted active vitamin D3

Article abstract of DOI:10.1021/ol200828s

C15-Substituted 1α,25-dihydroxyvitamin D₃ analogs were synthesized for the first time to investigate the effects of the modified CD-ring on biological activity concerning the agonistic positioning of helix-3 and helix-12 of the vitamin D recept

Full text of DOI:10.1021/ol200828s

ORGANIC
LETTERS
2011
Vol. 13, No. 11
2852–2855
New C15-Substituted Active Vitamin D3
Kanako Shindo,† Go Kumagai,† Masashi Takano,† Daisuke Sawada,† Nozomi Saito,†,§
Hiroshi Saito,‡ Shinji Kakuda,‡ Ken-ichiro Takagi,‡ Eiji Ochiai,‡ Kyohei Horie,‡
Midori Takimoto-Kamimura,‡ Seiichi Ishizuka,‡, Kazuya Takenouchi,‡ and
Atsushi Kittaka*,†
Faculty of Pharmaceutical Sciences, Teikyo University, Sagamihara, Kanagawa
252-5195, Japan, and Teijin Institute for Bio-medical Research, Teijin Pharma Ltd.,
Hino, Tokyo 191-8512, Japan
akittaka@pharm.teikyo-u.ac.jp
Received March 30, 2011
ABSTRACT
C15-Substituted 1R,25-dihydroxyvitamin D3 analogs were synthesized for the first time to investigate the effects of the modified CD-ring on
biological activity concerning the agonistic positioning of helix-3 and helix-12 of the vitamin D receptor (VDR). X-ray cocrystallographic analysis
˚
proved that 0.6 A shifts of the CD-ring and shrinking of the side chain were necessary to maintain the position of the 25-hydroxy group for proper
interaction with helix-12. The 15-hydroxy-16-ene derivative showed higher binding affinity for hVDR than the natural hormone.
The physiologically active metabolite of vitamin D3,
1R,25-dihydroxyvitamin D3 [1R,25(OH)2D3, 1], regulates
cellular growth, differentiation or apoptosis, and immu-
nomodulation, in addition to its classical role in serum
calcium/phosphorus homeostasis as well as bone metabo-
lism in the human body.1 Hormonal vitamin D mediates
most of its actions by binding to the specific nuclear vitamin
D receptor (VDR) in the target tissues. VDR belongs to the
nuclear receptor superfamily acting as a ligand-dependent
transcription factor. When 1R,25(OH)2D3 binds to the
VDR, the protein undergoes a conformational change that
allows it to interact with coactivators to form an active
transcriptional complex with its heterodimeric partner, the
retinoid X receptor (RXR); that is, the liganded VDR-RXR
heterodimer binds to vitamin D response elements (VDRE) in
the promoter regions of target genes with high affinity and
modulates gene expression after recruiting coactivators in a
wide variety of cells.2 During the 1R,25(OH)2D3 signaling
process, the stabilization of the ligand binding domain (LBD)
of the VDR by moving helix 12 (H12) to the agonistic position
is the most important step. In the presence of 1R,25(OH)2D3,
H12 of the human VDR occupies the optimum position for
charge clamp formation between the Glu420 residue as the
minus charge on H12 and the Lys246 residue as the plus
charge on helix 3 (H3). The correct positioning of H12 and the
† Teikyo University.
(2) Carlberg, C.; Polly, P. Crit. Rev. Eukaryot. Gene Expr. 1998, 8,
19–42.
‡ Teijin Pharma Ltd.
§ Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-
0812, Japan.
Division of Hematology/Oncology, University of Pittsburgh School of
Medicine, Pittsburgh, PA 15240, USA.
€ €
€
€
€
€
(3) (a) Vaisanen, S.; Perakyla, M.; Karkkainen, J. I.; Uskokovic,
€ €
M. R.; Carlberg, C. Mol. Pharmacol. 2003, 63, 1230–1237. (b) Vaisanen,
€
€
€
€
S.; Perakyla, M.; Karkkainen, J. I.; Steinmeyer, A.; Carlberg, C. J. Mol.
Biol. 2002, 315, 229–238.
(1) Vitamin D, 2nd ed.; Feldman, D., Pike, J. W., Glorieux, F. H., Eds.;
Elsevier Academic Press: Burlington, MA, 2005.
(4) Rochel, N.; Wurtz, J. M.; Mitscher, A.; Klaholz, B.; Moras, D.
Mol. Cell 2000, 5, 173–179.
r
10.1021/ol200828s
Published on Web 05/03/2011
2011 American Chemical Society

subsequent precise charge clamp formation are critical for
effective VDRꢀcoactivator interaction.3
16β-epoxy-17-keto sterols afforded only an E-ethylidene
epoxide by the corresponding Wittig reaction.11
Moras’X-raycrystallographicanalysisoftheengineered
human VDR LBD-1R,25(OH)2D3 complex showed that
H3 covered the CD-ring of the ligand molecule.4 H3
contacts with H12 in the agonistic position through both
hydrophobic and polarinteractions. We thought that if H3
was pushed upward by a newly introduced functional group
at the C15 position of the ligand CD-ring, the position of H3
would move and affect the agonistic location of H12, and
the new ligand with C15-substitution would exhibit a unique
biological profile. First, 1R,15R,25-trihydroxyvitamin D3
(2) and 15R-methoxy-1R,25(OH)2D3 (3) were designed,
synthesized, and biologically evaluated.
Scheme 2. (Z)-Ethylidene Synthesis and NOE Experiments of
14
The CD-ring parts were synthesized from an Inhoffen-
Lythgoe diol (6), which wasconvertedtothe knownketone
(7).5 Vinyl acetate 8 from ketone 7 via transacetylation
with isopropenyl acetate was treated with allyl methyl
carbonate in the presence of catalysts Pd(OAc)2 and
tributyltin methoxide to give R,β-unsaturated ketone 9 in
good yield with recovery of 8.6 Reduction of enone 9 with
DIBAL-H gave allyl alcohol 10 stereoselectively and sub-
sequent mCPBA epoxidation afforded 15R,16R-epoxyhy-
drindan derivative 11 (steroidal numbering) exclusively,
whose stereochemistry was determined by X-ray crystal-
lographic analysis of its acetate 12 (Scheme 1).7,8
1,4-Addition of the magnesium cyanocuprate derivative
of 5-bromo-2-methyl-2-pentanol MOM ether12 to ethyli-
dene epoxide 14 afforded the 15R-hydroxy-16-ene CD ring
15 with 20R (natural) and 20S side chains in a ratio of 11:1
(Scheme 3). The stereochemistry at C20 of the major
isomer 15a results from attack of the alkyl cuprate on the
β-face of the Z-alkene 14 in an SN20 manner.11c The Δ16
double bond of the major isomer 15a was hydrogenated using
Pd/C to give 16 with 17R(natural) and 17Sconfiguration in a
ratio of 14:1. To confirm the stereochemistry at C17 and C20
of the major isomer 16a, the 15R-hydroxy group of 16a was
reduced by BartonꢀMcCombie radical deoxygenation13
followed by oxidation at the C8-hydroxy group to give
ketone 19, whose spectral properties were identical to the
known ketone14 derived from vitamin D3 (Scheme 3).
Scheme 1. Epoxyhydrindan (11) Synthesis and ORTEP Draw-
ing of X-ray Molecular Structure of Its Acetate 12
The secondary hydroxy group at C15 of 16a was meth-
oxymethylated(20) or methylated (21) followedby C8-OH
oxidation using TPAP/NMO after deprotection of its TBS
group to give C8-ketone. The resulting ketones 22 and 23
were coupled with A-ring phosphine oxide 2415 by a
WittigꢀHorner reaction to afford the coupling products
25 (13%) and 26 (9%) with the eliminated Δ14-CD-ring
(27, 4ꢀ17%) and recovered 22 (62%) and 23 (78%),
respectively.16 The subsequent deprotection and HPLC
TPAP oxidation of 11 afforded R-epoxy ketone 13,9 and
subsequent Wittig reaction gave ethylidene 1410 as a single
isomer with Z-configration determined by NOE experi-
ments (Scheme 2), although it was reported that 15β,
(11) (a) Takahashi, T.; Ootake, A.; Tsuji, J.; Tachibana, K. Tetra-
hedron 1985, 41, 5747–5754. (b) Marino, J. P.; Abe, H. J. Am. Chem. Soc.
1981, 103, 2907–2909. (c) Moon, S.; Stuhmiller, L. M.; Chadha, R. K.;
McMorris, T. C. Tetrahedron 1990, 46, 2287–2306.
(12) Larsen, S. D.; Spilman, C. H.; Yagi, Y.; Dinh, D. M.; Hart,
K. L.; Hess, G. F. J. Med. Chem. 1994, 37, 2343–2351.
(13) Barton, D. H. R.; McCombie, S. W. J. Chem. Soc., Perkin Trans.
1 1975, 1574–1585.
ꢀ
ꢀ
(5) Fernandez, B.; Martınez Perez, J. A.; Granja, J. R.; Castedo, L.;
´
~
Mourino, A. J. Org. Chem. 1992, 57, 3173–3178.
(6) Tsuji, J.; Minami, I.; Shimizu, I. Tetrahedron Lett. 1983, 24, 5639–
5640.
(7) Crystallographic data: empirical formula = C18H32O4Si; formula
weight = 340.53; crystal system = orthorhombic; space group =
P212121 (#19); a = 8.03971(19) A; b = 11.3007(3) A; c = 22.2692(6)
A; V = 2023.26(9) A3; Z = 4; Dcalc = 1.118 g/cm3; crystal dimensions =
0.20 ꢁ 0.15ꢁ 1.00 mm3; datacollection temp = 296.0( 1 K; radiation=
Cu KR (λ = 1.54187 A); μ (Cu KR) = 11.53 cmꢀ1; number of reflections
measured = 20 529; number of unique reflections = 3674 (Rint = 0.079);
Residuals: R1(I > 2.00σ(I)) = 0.0801; Residuals: wR2 = 0.0926.
(8) The direct epoxidation of enone 9 with m-CPBA was unsucessful,
since the BaeyerꢀVilliger reaction gave a tetrahydroqoumarin derivative.
(9) Griffith, W. P.; Ley, S. V.; Whitcombe, G. P.; White, A. D.
J. Chem. Soc., Chem. Commun. 1987, 1625–1627.
~
(14) (a) Torneiro, M.; Fall, Y.; Castedo, L.; Mourino, A. Tetrahedron
1997, 53, 10851–10870. (b) Ono, K.; Yoshida, A.; Saito, N.; Fujishima,
T.; Honzawa, S.; Suhara, Y.; Kishimoto, S.; Sugiura, T.; Waku, K.;
Takayama, T.; Kittaka, A. J. Org. Chem. 2003, 68, 7407–7415.
(15) (a) Hatakeyama, S.; Irie, H.; Shintani, T.; Noguchi, Y.; Yamada,
H.; Nishizawa, M. Tetrahedron 1994, 50, 13369–13376. (b) Baggiolini,
E. G.; Iacobelli, J. A.; Hennessy, B. M.; Batcho, A. D.; Sereno, J. F.;
Uskokovic, M. R. J. Org. Chem. 1986, 51, 3098–3108. (c) Miles, W. H.;
Connell, K. B.; Ulas, G.; Tuson, H. H.; Dethoff, E. A.; Mehta, V.;
Thrall, A. J. J. Org. Chem. 2010, 75, 6820–6829.
(16) The coupling yield of the 15R-substituted CD-ring (22,23) was
low; however, the 15β-OMOM counterpart gave the corresponding
triene product in 49% yield under the same reaction conditions.
(10) Ethylidene epoxide 14 was unstable, and it was used within 24 h
for the next step.
Org. Lett., Vol. 13, No. 11, 2011
2853

25-dihydroxyvitamin D3 (1);17 therefore, synthesis of 16-
ene analogs was suggested as a candidate drug in the
1990s.17e We therefore revisited 16-ene-vitamin D3 analog
synthesis. The WittigꢀHorner coupling yield of the 15R-
substituted CD-ring was low, and here, we utilizedthe Trost
Pd-mediated coupling reaction between CD-ring bromoo-
lefin 29 and enyne 30 as shown in Scheme 5.18 The free
secondary hydroxy group of 15a was protected by MOM
followed by deprotection of the TBS group and TPAP/
NMO oxidation to give C8-ketone, which was converted to
bromoolefin 29 in good yield. The coupling reaction pro-
ceeded in moderate yield to give 16-ene analog 32, which
was deprotected under mild acidic conditions to afford the
desired molecule 4. Since we were interested in the syner-
getic effects of the 2R-methyl group in the A-ring and 16-ene
modification in the CD-ring on biological activity, com-
pound 5 was also synthesized using enyne 31.19,20
Scheme 3. Introduction of Side Chain to Ethylidene Epoxide 14
via SN20 Type 1,4-Addition
We tested the preliminary biological activity of the new
compounds 2ꢀ5: (1) binding affinity for VDR was 13%
(for 2), 1.5% (for 3), 65% (for 4), and 278% (for 5) of that
of the natural hormone, 1R,25(OH)2D3. (2) EC50 values of
transactivation activity of an osteocalcin promoter in HOS
cells were 0.12, 0.12, 0.08, and 0.12 nM, respectively, while
purification gave the desired C15-modified analogs 2 and 3
(Scheme 4).
Scheme 4. WittigꢀHorner Coupling Reaction
Scheme 5. Trost Coupling Reaction
a value of 0.09nM was observed in the case of 1R,25(OH)2-
D3. 16-Ene analogs 4 and 5 showed comparable and even
strongeraffinityfor VDR and transactivation activitythan
1R,25(OH)2D3 (Table 1).
Next, we studied the crystal structure of the complex
between truncated hVDR LBD and the new ligand 3 to see
new interactions of the 15R-OCH3 group and amino acid
residues (Figure 1).21 The hVDR LBD-3 complex was
In our synthetic route, the new C15-substituted 16-ene-
active vitamin D3 analogs were also available from 15a. It
is known that 1R,25-dihydroxy-16-ene-vitamin D3 shows
higher binding affinity for VDR, lower affinity for the
vitamin D binding protein in serum, and greater resistance
in an active form to catabolism as compared with 1R,
(18) Trost, B. M.; Dumas, J.; Villa, M. J. Am. Chem. Soc. 1992, 114,
9836–9845.
(19) Konno, K.; Fujishima, T.; Maki, S.; Liu, Z.; Miura, D.; Chokki,
M.; Ishizuka, S.; Yamaguchi, K.; Kan, Y.; Kurihara, M.; Miyata, N.;
Smith, C.; DeLuca, H. F.; Takayama, H. J. Med. Chem. 2000, 43, 4247–
4265.
(20) For the effects of 2R-functionalization on VDR binding and
biological activities, see: (a) Saito, N.; Honzawa, S.; Kittaka, A. Curr.
Top. Med. Chem. 2006, 6, 1273–1288. (b) Hourai, S.; Fujishima, T.;
Kittaka, A.; Suhara, Y.; Takayama, H.; Rochel, N.; Moras, D. J. Med.
Chem. 2006, 49, 5199–5205.
(17) (a) Chen, T. C.; Persons, K.; Uskokovic, M. R.; Horst, R. L.;
Holick, M. F. J. Nutr. Biochem. 1993, 4, 49–57. (b) Bishop, J. E.; Collins,
E. D.; Okamura, W. H.; Norman, A. W. J. Bone Mineral Res. 1994, 9,
1277–1288. (c) Siu-Caldera, M. L.; Clark, J. W.; Santos-Moore, A.;
ꢀ
Peleg, S.; Liu, Y. Y.; Uskokovic, M. R.; Sharma, S.; Reddy, G. S. J.
Steroid Biochem. Mol. Biol. 1996, 59, 405–412. (d) Jung, S. J.; Lee, Y. Y.;
Pakkala, S.; de Vos, S.; Elstner, E.; Norman, A. W.; Green, J.;
Uskokovic, M. R.; Koeffler, H. P. Leuk. Res. 1994, 18, 453–463.
ꢀ
(e) Uskokovic, M. R.; Studzinski, G. P.; Reddy, D. G. In Vitamin D;
(21) The Protein Data Bank accession number for the coordinates of
the structures of the vitamin D receptor complex with 3 reported in this
article is 3AX8.
Feldman, D., Glorieux, F. H., Pike, J. W., Eds.; Academic Press: New York,
1997; Chapter 62, pp 1045ꢀ1070.
2854
Org. Lett., Vol. 13, No. 11, 2011

Table 1. VDR Binding Affinity and Osteocalcin Promoter
Transactivation Activity in HOS Cells
relative VDR
binding affinity
(%)
osteocalcin transactivation
activity
compound
in HOS/SF cells (EC50, nM)
1R,25(OH)2D3 (1)
100a,b
13a
0.09
0.12
0.12
0.08
0.12
2
3
4
5
1.5a
65b
278b
a Chick intestinal VDR. b Human VDR.
superposed to hVDR LBD-1 with an rmsd of 0.308 A on
all CR atoms. In this study, the methyl group of 15R-OCH3
made a new CH O interaction at a 3.1 A distance with a
3 3 3
Ser275Oγ atom, which had never been found in the
reported hVDR LBD-ligand complexes so far. The 25-
OH group of 1 formed hydrogen bonds to His305 and
His397 in the active hVDR-ligand complex, and the 25-
OH group of 3 was located at the same position as in 1 in
the LBD of the hVDR for the stable complex formation,
and it caused the CD-ring of 3 to push upward in 0.6 A and
the C17 side chain was shrunk to keep the original position
of the 25-OH group that formed hydrogen bonds with
His305 and His397. The H3 and H12 of this study took
similar positions with those in the hVDR LBD-natural
ligand complex.4 However, the interaction between the
C15-substituted group and Ser275Oγ could be used posi-
tively to design new profile ligands, which would affect the
dynamic process of a ligandꢀreceptor complex formation
including cofactor recruiting steps to show unique biolo-
gical profiles.22
Figure 1. X-ray studies on hVDR-3 complex.
greater activity than the natural hormone. We are studying
furthermodifications atthe C15 positiontounderstandthe
structural function on both the ligands and the receptor.
Acknowledgment. We are grateful to Professor Tai C.
Chen (Boston University School of Medicine) for discus-
sions about the biological strong points of 16-ene active
vitamin D3 analogs. This work was supported in part by
Grants-in-Aid for Scientific Research from the Japan
Society for the Promotion of Science (No. 19590016 and
21590022).
In summary, we synthesized C15-modified active vita-
min D3 derivatives for the first time and tested their
preliminary biological activities. C16-Ene analogs showed
Supporting Information Available. Detailed experimen-
tal procedures and spectral data for all new compounds
and X-ray structural data of 12. This material is available
free of charge via the Internet at http://pubs.acs.org.
(22) Arai, M. A.; Takeyama, K.; Ito, S.; Kato, S.; Chen, T. C.;
Kittaka, A. Bioconjugate Chem. 2007, 18, 614–620.
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