Development of 14-epi-19-nortachysterol and its unprecedented binding configuration for the human vitamin D receptor

Article abstract of DOI:10.1021/ja201481j

In the study of the synthesis of 14-epi-19-norprevitamin D3, we found 14-epi-19-nortachysterol derivatives through C6,7-cis/trans isomerization. We also succeeded in their chemical synthesis and revealed their marked stability and potent VDR binding affin

Full text of DOI:10.1021/ja201481j

ARTICLE
pubs.acs.org/JACS
Development of 14-epi-19-Nortachysterol and Its
Unprecedented Binding Configuration for the Human
Vitamin D Receptor
Daisuke Sawada,† Yuya Tsukuda,† Hiroshi Saito,‡ Shinji Kakuda,‡ Midori Takimoto-Kamimura,‡
Eiji Ochiai,‡ Kazuya Takenouchi,‡ and Atsushi Kittaka*,†
†Faculty of Pharmaceutical Sciences, Teikyo University, 1091-1, Suwarashi, Midori-ku, Sagamihara, Kanagawa 252-5195, Japan
‡Teijin Institute for Bio-medical Research, Teijin Pharma Ltd., Hino, Tokyo 191-8512, Japan
S Supporting Information
b
ABSTRACT:
In the study of the synthesis of 14-epi-19-norprevitamin D3, we found 14-epi-19-nortachysterol derivatives through C6,7-cis/trans
isomerization. We also succeeded in their chemical synthesis and revealed their marked stability and potent VDR binding affinity. To
the best of our knowledge, this is the first isolation of stable tachysterol analogues. Surprisingly, 14-epi-19-nortachysterol derivatives
exhibited an unprecedented binding configurations for the ligand binding pocket in hVDR, C5,6-s-trans and C7,8-s-trans triene
configurations, which were opposite the natural C7,8-ene-configuration of 1R,25(OH)2D3.
’ INTRODUCTION
Among these compounds, the 2R-methyl-substituted analogue
(2R-methyl-14-epi-P1) indicated moderate VDR binding ac-
tivity (8.4% of the natural hormone, Table 1) and transactiva-
tion activity of osteocalcin promoter in HOS cells. These
results provided important information; compounds in pre-
form could have potent genomic activity and encouraged us in
the further synthesis of new derivatives. Throughout these
studies, however, we were concerned about the minor isomer
in equilibrium, 14-epi-1, which must be transformed from
isolated 14-epi-P1 during biological evaluation and might
show some biological activity. To clarify the precise activity
of the previtamin D form, we designed new target compounds,
14-epi-1R,25(OH)2-19-norprevitamin D3 (14-epi-19-norP1),
which led neither to the [1,7]-hydride shift nor to thermal
equilibrium as natural vitamin D3. By comparison of the
activities of 14-epi-19-norP1 and 14-epi-P1, we could under-
stand the real activity of previtamin D form; therefore, we
started the synthesis of 14-epi-19-norP1 and its 2-methyl
substituted analogues.
There are several isomers of vitamin D in its biosynthesis
pathway; provitamin D, vitamin D, lumisterol, tachysterol, and
so on.1 It is well-known that the most biologically active
compound for mammals is the metabolite of vitamin D3,
1R,25(OH)2D3 (1), which is a ligand of the specific nuclear
receptor (vitamin D receptor, VDR), regulates gene transcrip-
tion, and exhibits various biological responses as a hormone.2,3
Among them the minor isomers and unstable isomers including
their metabolites are not well-established, and details of their
biological properties remain to be uncovered. For therapeutic
evaluation, most scientists have administered the analogues of
the major isomer of vitamin D3, although it contains 6% of its
previtamin D form, 1R,25(OH)2preD3 (P1),4,5 which is gener-
ated from 1 in thermal equilibrium through a [1,7]-hydride shift
(Scheme 1).6
Regarding this equilibrium, 14-epi-vitamin D3 shows a unique
characteristic: the pre-form, 14-epi-previtamin D3 is major and
dominant over 14-epi-vitamin D3 in equilibrium, and 14-epi-
previtamin D3 should be isolated as the stable compound.5
Recently, we have focused on the biological activity of the
previtamin D form by synthesis of 14-epi-1R,25(OH)2previtamin
D3 (14-epi-P1) and its analogues using this inverse equilibrium.7
Received: February 17, 2011
Published: April 18, 2011
r
2011 American Chemical Society
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Scheme 1. Equilibrium of Vitamin D3, 14-epi-Vitamin D3, and Their 19-Nor Analogues
Scheme 2. Synthesis of 14-epi-19-Norprevitamin D3 Analoguesa
a Reagents and conditions: (a) LDA, PhN(Tf)2, THF, yield 82%; (b) POCl3, pyridine, yield 85%; (c) DIBAL-H, toluene, yield 87%; (d) TPAP, NMO,
MS4A, CH2Cl2; (e) TMSCHN2, nBuLi, THF, yield 61% (2 steps); (f) Pd(PPh3)2(OAc)2, CuI, Et2NH, DMF; (g) TBAF, THF, yield 80% (7, 2 steps),
yield 99% (8, 2 steps); (h) (Ph3P)3RhCl, H2, benzene, CH2Cl2, yield 43% (9a), yield 47% (9b); (i) Lindlar cat. quinoline, MeOH, H2, yield 39%,
conversion yield 73% (14-epi-19-norP1), yield 89% (10a), 57% (10b). b Reference 9e.
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Figure 1. Representative NOE values of compounds 9a, 9b, 10a, and 10b.
Table 1. Relative Binding Affinity for VDR
homologation with TMSCHN2.8c,12 Thus, we had both A- and
CD-ring fragments, and we attempted the Sonogashira coupling
reaction.8a These reactions proceeded smoothly and gave the
coupled compounds which were successively reacted with TBAF
to afford the fully deprotected compounds 7 and 8 in excellent
yield. By selective reduction of the 2-methylene moiety of 8 with
Wilkinson’s catalyst,12 2-methyl substitution was effectively
performed without any influence on the other conjugated
unsaturated bonds, and the resultant diastereomers 9a and
9b were separated using HPLC. Finally, the partial reduction
of C6,7-alkyne moiety of 7, 9a, and 9b with Lindlar catalyst
afforded the three target compounds 14-epi-19-norP1, 10a,
and 10b.8b
compound
VDRa
The stereochemistry of the C2-position in 9a, 9b and 10a, 10b
was determined by NOE experiments. The representative NOE
values are described in Figure 1, which reasonably explained
the stereochemistry, and we determined that 9a and 10a have
2R-methyl substitution and 9b and 10b have the 2β-methyl
substitution.
Using the new compounds obtained above, we tested their
human VDR (hVDR) binding affinity,13,14 and the results are
summarized in Table 1. Ene-yne-enes 8, 9a, and 9b showed
moderate affinity, and compound 8 with the 2-methylene
substitution was better than the 2-methyl-substituted analo-
gues of diastereomers 9a and 9b. Compounds in the pre-
form, 14-epi-19-norP1, 10a, and 10b showed low affinity,
lower than 2R-methyl-14-epi-1R,25(OH)2previtamin D3
(2R-methyl-14-epi-P1), but slightly better than 14-epi-P1.7
Judging from these results, for the hVDR binding affinity
of 2R-methyl-14-epi-P1, there should be some contribu-
tion of its isomer (2R-methyl-14-epi-1) in the equilibrium
(Table 1).
Next, we examined the stability of the new compounds in pre-
form with the conjugated triene system (14-epi-19-norP1, 10a,
and 10b), which were thought to be labile. We found that
transformation occurred in the presence of acidic protons, at
least in 1 mM HCl solution. Using HPLC analysis, we were able
to isolate the transformed compounds, and their spectral
data strongly suggested the cis/trans isomerization of C6,7-
double bonds to give 19-nortachysterol skeleton. To confirm
1R,25(OH)2D3 (1)
100
53
8
9a
21
9b
9.3
1.2
1.0
2.9
0.5
8.4
14-epi-19-norP1
10a
10b
14-epi-P1b
2r-methyl-14-epi-P1b
a The potency of 1 is normalized to 100. b Reference 7.
’ RESULTS AND DISCUSSION
19-Norprevitamin D3 analogues were previously developed
by Okamura,4,8 Mouri~no,9 Gotor,10 and De Clercq’s groups,11
and we followed their methodology using the coupling reaction
between the A-ring fragment and the CD-ring fragment
(Scheme 2). The CD-ring fragment 3 was obtained through
the known compound 27 from vitamin D3 using LDA and then
PhNTf2 in one step.9a The known compound 6 was used for the
A-ring precursor,9e and compound 5, used for the 2-methyl
substituted A-ring precursor, was derived from (À)-quinic acid
through the known compound 4 by dehydration with POCl3,
reduction of methyl ester, oxidation to aldehyde, and then
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Scheme 3. Synthesis of 14-epi-19-Nortachysterol Analoguesa
a Reagents and conditions: (a) Bu3SnH, AIBN, toluene; (b) Pd2(dba)3 CHCl3, Ph3As, LiCl, DMF, THF; (c) TBAF, THF, yield 46% (13, 3 steps), yield
3
62% (14, 3 steps); (d) (Ph3P)3RhCl, H2, benzene, CH2Cl2, yield 45% (15a), yield 47% (15b).
Scheme 4. Synthesis of 1R,25(OH)2-19-Nortachysterol
the tachysterol structures, we embarked upon the chemical
synthesis of the most probable compounds, 14-epi-19-nortachys-
terol analogues (13, 15a, and 15b, Scheme 3). As in the above
synthesis, we utilized the intermediates 5 and 6 as A-ring
precursors, and the reaction with tributyltin hydride in the
presence of AIBN transformed them into vinylstannanes 11
and 12 after basic column chromatography, which were imme-
diately coupled with CD-ring fragment 3 under Stille coupling
conditions. The coupled compounds were deprotected using
TBAF, and we were able to synthesize 14-epi-1R,25(OH)2-19-
nortachysterol (13) and its 2-methylene-substituted compound
(14). Regioselective reduction of compound 14 was successfully
accomplished with Wilkinson’s catalyst to give 2-methyl-substi-
tuted diastereomers 15a and 15b.12 The spectral data of the
compounds 13, 15a, and 15b were identical to the corresponding
data of isomerized products derived from 14-epi-19-norP1, 10a,
and 10b under acidic conditions.
Table 2. Relative Binding Affinity for VDR13,14
compound
VDRa
1R,25(OH)2D3 (1)
100
15
13
14
83
15a
71
15b
48
a The potency of 1 is normalized to 100.
slowly decomposed even under neutral conditions at room tem-
perature, and it was impossible to determine its biological properties
(Scheme 4). Above all, since the 14-epi-CD-ring system, cis-
hydrindane, prefers the C8,9-endo double bond rather than
C7,8-exo double bond,17 it is thought that 14-epimerization is
essential for stabilizing the tachysterol skeleton.
Here again, we investigated the hVDR binding affinity of
tachysterol analogues and, as we expected, the affinity was greatly
improved and C2-substitution had a marked effect (Table 2).18
As in the case of ene-yne-ene compounds in Table 1, 2-methy-
lene substitution (compound 14) had the highest binding affinity
for the hVDR.12
The new compounds of the 14-epi-19-nortachysterol
skeleton indicated particular stability in comparison with natural
tachysterol, which is easily converted to vitamin D3 by UV
irradiation15 and oxidized by O2.16 To the best of our knowledge,
these compounds are the first example of stable tachysterol
analogues.
At this point, we were interested in the interaction be-
tween our new synthetic molecules and hVDR, and X-ray
Additionally, 1R,25(OH)2-19-nortachysterol (17), synthe-
sized with 6 and 1611 in the same manner, was unstable and
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Figure 2. Binding configurations and crystallographic data of 15a and 15b in hVDR. The electron density omit maps contoured at 1.0σ are shown above
the molecules, respectively (BÀD, FÀH). These pictures show Fo À Fc maps in purple and 2Fo ÀFc maps in green.
co-crystallographic analyses were performed using the complex
of compound 15a and 15b with the ligand binding pocket (LBP)
of the hVDR. Surprisingly, as shown in Figure 2, they revealed
that the compounds fit the LBP with the C7,8-s-trans diene
configuration as not (A) but (B), and not (E) but (F), which
were opposite the natural C7,8-ene-configuration of 1R,25-
(OH)2D3 (1). Also, there were three possible configurations
of the CD-ring, and each electron density map correspond-
ing to each configuration is shown in Figure 2. These show
the mixture of the three configurations of the CD-ring (B, C,
and D for 15a and F, G, and H for 15b) in Figure 2. The
occupancy of each configuration of the CD-ring cannot be
refined by the limit of the resolution.19
Concerning the orientation of the 2-methyl substitution,
which was thought to make a hydrophobic interaction with
Phe150, Leu233, and Ser237 on the R-side,18b compound 15b
made it on the β-side (F, G, H in Figure 2). Therefore, C5,6-s-
trans configuration was preferred regardless of the stereo-
chemistry of 2-methyl substitution. As far as we know, this
binding configuration is unprecedented among the ligand
molecules for VDR, and it is worth noting that this unique
binding mode exhibited comparable VDR binding affinity to
the natural hormone. Furthermore, we focused on the linker
configuration between the A-ring and the CD-ring (from C5
to C8 positions) of the above molecules. By X-ray co-crystal-
lographic analyses of 9a and 9b, their ene-yne-ene linkers also
fit the LBP of the hVDR, and the 2-methyl substitutions were
located on the R-side regardless of their C2-stereochemistries
(Figure 3).
The superposed binding configuration among 1R,25(OH)2D3
(1), compounds 9a, and 15a(B) in the LBP of the hVDR was
described, and the C5ÀC8 linkers successfully placed themselves
in the specific space sandwiched between Ser275 and Trp288 on
one side and Leu233 on the other side (Figure 4).2 Also, the three
hydroxy groups (C1, C3, and C25 positions) of the compounds
well overlapped, respectively, and the CD-ring moiety indicated
various conformational changes including the orientation of
C20-methyl groups. These results showed the suitability of the
linker and flexibility of the CD-ring structure; the appropriate
distribution of both the A-ring and the side chain in the LBP was
critical for high affinity.20
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unique binding configuration for hVDR. We believe that this
new skeleton could have potential as a new drug candidate.
Further studies of other new tachysterol analogues and their
thermodynamic and biological properties are now in progress.
’ ASSOCIATED CONTENT
S
Supporting Information. Experimental procedures,
b
spectral data for all new compounds (1H NMR, 13C NMR, IR,
HRMS), and crystal data (Protein Data Bank accession numbers
3AUQ for 9a and 3AUR for 9b). This material is available free of
charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
akittaka@pharm.teikyo-u.ac.jp
’ ACKNOWLEDGMENT
This work was supported by Yokohama Academic Foundation
(to D.S.), in part by Grant-in-Aid from the Ministry of Education,
Culture, Sports, Science and Technology, Japan (No. 20790019
to D.S), and in part by Grants-in-Aid for Scientific Research from
Japan Society for the Promotion of Science (No. 19590016 and
21590022 to A.K.).
Figure 3. Superimposed three-dimensional structures of 9a and 9b
based on X-ray crystallographic analysis in the hVDR ligand binding
pocket. The 9a complex is shown in blue and the 9b complex in red.
Protein Data Bank accession numbers are 3AUQ for 9a and 3AUR
for 9b.
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’ CONCLUSIONS
In conclusion, we disclosed 14-epi-19-nortachysterol deriva-
tives from 14-epi-19-norprevitamin D3 by proton-mediated
C6,7-cis/trans isomerization and succeeded in their chemical
synthesis. They showed marked stability and potent hVDR binding
affinity with the unprecedented binding configuration for hVDR.
To the best of our knowledge, this is the first example of the
isolation of stable tachysterol analogues, and revealed their
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(19) In Figure 2, all structures were determined on the basis of the
electron density map. The X-ray crystallographic data in Figure 2 are not
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(20) EC50 values of osteocalcin promoter transactivation activity in
human osteosarcoma (HOS) cells under serum free conditions for
compounds 9a, 13, and 15a were 0.04, 1.06, and 0.27 nM, respectively,
when 1R,25(OH)2D3 (1) showed 0.03 nM as the positive control.
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