Effects of fluorines on nonsecosteroidal vitamin D receptor agonists

Article abstract of DOI:10.1016/j.bmc.2012.11.029

From our research of nonsecosteroidal vitamin D₃ derivatives with gamma hydroxy carboxylic acid, we identified compound 6, with two CF ₃ groups in the side chain, as a most potent vitamin D receptor (VDR) agonist that shows superagon

Full text of DOI:10.1016/j.bmc.2012.11.029

Bioorganic & Medicinal Chemistry 21 (2013) 712–721
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Effects of fluorines on nonsecosteroidal vitamin D receptor agonists
⇑
Hirotaka Kashiwagi , Masateru Ohta, Yoshiyuki Ono, Kenji Morikami, Susumu Itoh, Hideki Sato,
Tadakatsu Takahashi
Research Division, Chugai Pharmaceutical Co. Ltd, 1-135 Komakado, Gotemba, Shizuoka 412-8513, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
From our research of nonsecosteroidal vitamin D3 derivatives with gamma hydroxy carboxylic acid, we
identified compound 6, with two CF3 groups in the side chain, as a most potent vitamin D receptor
(VDR) agonist that shows superagonistic activity in VDRE reporter gene assay, MG-63 osteocalcin produc-
tion assay and HL-60 cell differentiation assay. Compound 6 demonstrated that fluorination is as effective
in the case of our nonsecosteroidal scaffold as in the case of secosteroidal VD3 analogs. X-ray analysis of
the VDR with compound 6 revealed all of the six fluorine atoms of the hexafluoropropanol (HFP) moiety
in the side chain effectively interacting with the VDR by both steric (van der Waals) and electrostatic
(hydrogen bond, NH–F and CH–F) interactions. The HFP moiety of 6 effectively interacts with helix 12
(H12) of the VDR and stabilizes the position and the orientation of H12, which could result in stabilizing
the coactivator and enhancing the VDR agonistic activity.
Received 29 October 2012
Revised 20 November 2012
Accepted 21 November 2012
Available online 2 December 2012
Keywords:
Vitamin D3
VDR agonist
Nonsecosteroidal
Bisphenylmethane
Fluorine interaction
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
its derivatives.24–30 We have synthesized the bisphenyl core com-
pounds with gamma hydroxy carboxylic acid moiety in the A-part,
In the field of medicinal chemistry, introducing a fluorine atom
is a very useful way of increasing activity and selectivity, blocking
metabolic sites, and modifying physicochemical properties such as
solubility and permeability.1 Recently, structural information
gained from database surveys and numerous published works pro-
vides information on various types of fluorine-related interactions,
such as orbital interaction, closed-shell interaction, anti-parallel
dipolar interaction, orthogonal multipolar interaction, and hydro-
gen bonding, to help rationalize the contribution of fluorine to pro-
tein binding.2–15
In research on secosteroidal vitamin D3 analogs, fluorine substi-
tutions were attempted with a replacement of the hydrogen atoms
on carbon atoms at 24, 26 and 27 positions in the side chain moi-
ety,16–20 and a replacement of the hydroxy group at 1, 3 and 25
positions.21–23 From these many efforts, hexafluoro-substituted
such as 4, and found this class of compounds showed vitamin D3
agonist activity. Through systematic modification of the side chain
part, compound 4 with an (E)-olefin linker and terminal diethyl
group in the side chain showed a potency equal to that of
1,25(OH)2D3 in vitro (Fig. 1).31,32
After the discovery of 4, we attempted to introduce a hexafluo-
ropropanol (HFP) moiety into the side chain to increase vitamin D3
receptor (VDR) agonistic activity further. Additionally, in order to
examine in detail the interactions between VDR and a molecule
with an HFP moiety, we determined the 3D structures of VDR in
complex with compounds 5 and 6 by X-ray crystallography.
2. Chemistry
The synthesis of compounds 5–7 is shown in Scheme 1. Com-
pound 8 was prepared by the method reported previously.32 Lith-
iation of 8 with n-butyllithium and consequent treatment of
hexafluoroacetone gas gave acetylenealcohol 9 in 68% yield. This
acetylene 9 was selectively reduced to (E)-olefin 11 by Red-AlÒ
in 80% yield. Before O-alkylation of the phenol group, the acidic hy-
droxy groups in the side chain of 9 and 11 were protected by a
methoxymethyl (MOM) group to avoid di-O-alkylation. The alkyl-
ation of phenol groups in 10 and 12 with (S)-(+)-dihydro-5-(p-tol-
ylsulfonyloxymethyl)-2(3H)-furanone (13) in the presence of
potassium carbonate at 100 °C afforded lactones 14 and 15 in good
yields. The MOM groups of 14 and 15 were removed by concen-
trated hydrogen chloride with excellent yields (95% and 96%).
derivative of 1a,25-dihydroxyvitamin D3 (1,25(OH)2D3, 1), falecal-
citriol (2) was discovered (Fig. 1). The structural feature of 2 was
two trifluoromethyl (CF3) groups at 26 and 27 positions in the side
chain part of the secosteroidal scaffold. It exhibited very potent
activity in vitro and also in vivo18,19 and was launched as an
anti-hyperparathyroidism drug in 2001. Thus, fluorination of the
side chain is an attractive modification in vitamin D3 research.
In 1999, Boehm et al. reported the first nonsecosteroidal vita-
min D3, LG190178 (3) and since then various groups have reported
⇑
Corresponding author.
E-mail address: kashiwagihrt@chugai-pharm.co.jp (H. Kashiwagi).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.11.029

H. Kashiwagi et al. / Bioorg. Med. Chem. 21 (2013) 712–721
713
Methylene analog 18 was prepared with hydrogenation of acety-
lenealcohol 16 in the presence of a catalytic amount of palladium
hydroxide in 95% yield. The lactones 16, 17 and 18 were hydrolized
to gamma hydroxy carboxylic acid free form by being treated with
1 M potassium hydroxide solution and then acidified with satu-
rated ammonium chloride solution. These carboxylic acids were
converted to sodium salts 5, 6, and 7 using 1.0 equiv amount of
1 M sodium hydroxide solution to avoid retro lactone formation
during long time storage.
Side Chain
25
H
CF3
OH
H
OH
F3C
H
H
3
HO
A ring
OH
HO
OH
1
1: 1α,25-dihydroxyvitamin D3
2: falecalcitriol
3. Results and discussion
Side Chain
In order to evaluate the VDR agonistic activity, the compounds
synthesized were evaluated by three different assays. The first one
is a reporter gene assay (RGA) in MG-63 cells which contains the
VDRE sequence derived from mouse osteopontin promoter. Sec-
ondly, the osteocalcin production activity in MG-63 cells was eval-
uated to measure bone formation activity of functional vitamin D
agonistic effect.33–35 Thirdly, cell differentiation activity in HL-60
cells was evaluated to measure the standard VDR agonistic effect.
All types of activity are represented by an EC50 value relative to
the EC50 value of 1,25(OH)2D3, which is assigned as 100% (a higher
figure means stronger activity).
As expected, the HFP analogs with various linkers (5, 6 and 7)
exhibited very potent levels of activity (Table 1). In VDRE RGA,
the acetylene linker analog 5 showed moderate activity (30%),
and the methylene linker analog 7 showed activity comparable
to 1,25(OH)2D3 but the (E)-olefin linker analog 6 showed sixfold
stronger activity than that of 1,25(OH)2D3. In the HL-60 cell
OH
O
R
OH
R
X
Y
O
(S)
O
A part
HO
NaOOC
OH
OH
3: LG190178
4: X-Y; (E)-CH=CH, R = Et
5: X-Y; C≡C, R = CF3
6: X-Y; (E)-CH=CH, R = CF3
7: X-Y; CH2CH2, R=CF3
Figure 1. Structures of secosteroids 1a,25-dihydroxyvitamin D3 (1), falecalcitriol
(2), nonsecosteroidal analog LG190178 (3) and a series of nonsecosteroidal analogs
with carboxylic acid (4–7). Part labeled A of 4–7 shows the structural correspon-
dence to the A ring of secosteroids (upper left).
F3C
OR1
CF3
F3C
OR1
CF3
a
b
8
9: R1 = OH
10: R1=OMOM
OH
11: R1 = OH
12: R1 = OMOM
OH
OH
c
d
OTs
13
F3C
Y
F3C
Y
OMOM
CF3
OH
CF3
F3C
X
X
OMOM
CF3
O
X
Y
O
e
f
O
10; X-Y; C≡C
O
OH
12; X-Y; (E)-CH=CH
O
O
O
O
14; X-Y; C≡C
16; X-Y; C≡C
15; X-Y; (E)-CH=CH
17; X-Y; (E)-CH=CH
18; X-Y; CH2CH2
g
F3C
OH
CF3
X
Y
h
5; X-Y; C≡C
O
6; X-Y; (E)-CH=CH
7: X-Y; CH2CH2
NaOOC
OH
Scheme 1. Reagents and conditions: (a) n-BuLi, hexafluoroacetone gas, 0 °C, THF, 68%; (b) Red-AlÒ, 0 °C, THF, 80%; (c) K2CO3, MOMCl, room temp, DMF, 89%; (d) NaH, MOMCl,
50 °C, DMF, 41%; (e) K2CO3, 13, 100 °C, DMF, 85–90%; (f) concd HCl, room temp, 1,4-dioxane, 95–96%; (g) Pd(OH)2/C, H2, room temp, MeOH, 95%; (h) (i) 1 M KOH, room temp,
MeOH, (ii) satd NH4Cl aq, (iii) 1 M NaOH, EtOH, 49–83%.

714
H. Kashiwagi et al. / Bioorg. Med. Chem. 21 (2013) 712–721
Table 1
In vitro activities and calculated side chain volumes and PCs
R
OH
R
X
Y
O
NaOOC
OH
b
Compound
–X–Y–
R
Ratio of VDRE reporter genea (%)
Ratio of HL-60 differentiationa (%)
Ratio of Osteocalcina (%) Vside
(Å3)
PCc (%)
chain
4
5
6
7
–(E)–CH@CH– Et
105
30
592
91
6.7
100
126
227
252
106
71
447
546
3746
429
185
100
117
131
132
134
56
63
64
65
–C„C–
CF3
–(E)–CH@CH– CF3
–CH2CH2–
CF3
LG190178
1,25(OH)2D3
100
a
b
c
1,25(OH)2D3 ratio of EC50. 1,25(OH)2D3 was assigned to 100% (higher figure means stronger activity).
Calculated volume of extracted side chain moiety from whole molecule (highlighted in blue on structure).
PC was calculated as follows: (Vside chain/208 Å3) ꢀ 100 (%).
differentiation assay, all of the HFP analogs were equally potent to
or more potent than 1,25(OH)2D3: respectively, compounds 5 and 6
showed 2.2-fold and 2.5-fold the activity of 1,25(OH)2D3 and 1.8-
fold and 2.0-fold the activity of compound 4, which is the most ac-
tive bisphenyl core compound with an alkyl side chain. In the oste-
ocalcin assay, all of the HFP analogs showed stronger activity than
1,25(OH)2D3: respectively, compounds 5 and 7 showed 5.5-fold
and 4.3-fold more potency than 1,25(OH)2D3, and the activity of
6 was a remarkable 37-fold. In most cases in the three assays,
the HFP analogs showed activity equal to or more potent than
1,25(OH)2D3. The (E)-olefin linker analog 6 showed the most po-
tent activity among the three HFP analogs and exhibited a super
agonistic character in all of the three assays.
(A)
H8
H10
H4
H12
H2
H6
To confirm the detailed interactions between the HFP moiety
and the VDR, we cocrystallized the ligand-binding domain of hu-
man VDR with compounds 5 and 6 in accordance with a method
previously reported.31 The 3D structure of the VDRs in both com-
plexes showed a typical nuclear hormone receptor fold consisting
of 12 helices (Fig. 2A and B). The positions and the binding modes
of 5 and 6 in the VDR were identical to those of non-fluorinated
bisphenyl compounds.31 In spite of the linker difference, the posi-
tions of each atom (except the linker atoms) of 5 and 6 in the VDR
were nearly identical and finely superimposed (Fig. 3A and B). In
detail, the carboxyl group in the A-part of 5 and 6 had three key
interactions: the salt bridge with Arg274 from helix 5 (H5) of the
VDR, the hydrogen bond with the crystal water, and the intramo-
lecular hydrogen bond with the gamma hydroxy group of the A
part of 5 and 6 (Fig. 3A). The gamma hydroxy group of the A part
made a hydrogen-bonding interaction also with the crystal water
which made a hydrogen bond with the phenol oxygen of Tyr143
from helix 1 (H1). The phenyl group containing the A part was
(B)
H1
H9
H5
H3
H11
sandwiched between CH–
(H5) and with the C of Leu233 from helix 3 (H3) (Fig. 3B). The
bis-ethyl moiety between the phenyl rings made good CH– inter-
p interactions with the Cb of Ser275
e
p
actions with the indole ring of Trp286 from the loop between H5
and helix 6 (H6). The phenyl group containing the side chains of
Figure 2. (A) X-ray crystal structure of VDR/compound 6 (slate) complex. (PDB ID:
3W0C) (B) X-ray crystal structure of VDR/compound 5 (pink) complex (PDB ID:
3W0A). Compounds 5 and 6 are represented by the spheres. VDRs are represented
by their secondary structures. Different colors are assigned for each helix from 1 to
12.
5 and 6 was also sandwiched between CH–p interactions with
the C of Val234 (H3) and with the C of Val300 (H6).
c
c
Figure 4A shows the hydroxy group of the HFP moiety of 5 and 6
located at nearly identical positions, in spite of their linker differ-
ences. The hydroxy group of the HFP moiety had interactions with
the NHs of the imidazole of His305 from the loop between H6 and
helix 7 (H7) and His397 from helix 11 (H11) via a hydrogen bond,
just as the 25-OH group of secosteroidal VD3 analogs did (Fig. 4B).

H. Kashiwagi et al. / Bioorg. Med. Chem. 21 (2013) 712–721
715
(A)
(A)
Tyr143
Arg274
(B)
F6
(B)
Trp286
Val300
F1
Ser275
His305
His397
Leu233
Val234
Figure 4. (A) The superposition of VDR bound conformation of compound 5 (pink)
and 6 (slate) in each crystal. (B) The interactions between the HFP moiety of
compound
6 (slate) and the VDR (pale cyan). Hydrogen bonds and NH–F
interactions are depicted by orange dashed lines.
Figure 3. The superposition of the 3D structure of compound 5 (pink) and 6 (slate)
in the VDR. The VDR complexed with 6 is colored in pale cyan and the VDR
complexed with 5 is colored in pink. The crystallographic waters are depicted as red
spheres. (A) The interactions between the A part of the ligand and the VDR.
Hydrogen-bonding interactions are depicted by orange dashed lines. (B) The
Two CF3 groups of the HFP moiety of 5 and 6 were buried in the
hydrophobic pocket formed by Leu227 (H3), Val234 (H3), Ile268
(H5), Tyr401 (H11), Leu414 from the loop between H11 and helix
12 (H12), and Val418 (H12), and Phe422 (H12) of the VDR
(Fig. 5A). Furthermore, the two CF3 groups were filling this hydro-
phobic pocket tightly. The atoms of the VDR located within 4 Å dis-
tance from the fluorine atoms of the HFP moiety are listed in
Table 2. The number of contacts between fluorine atoms and the
VDR atoms within 4 Å distance were 24 for 6 and 25 for 5. These
VDR atoms made good van der Waals (vdW) interactions with fluo-
rine atoms and the tight vdW interactions increased the affinity of
5 and 6 with the VDR further.
Table 2 shows the detailed fluorine interactions around the side
chain moiety of 5 and 6. This detailed analysis revealed the exis-
tence of a large number of CH–F interactions around the HFP moi-
ety (Fig. 5B). The hydrogen atom attached to one of the Cd atoms of
Leu404 interacted with F1 and F2 and the Cd of Leu414 interacted
with F1. With respect to Ala231, F3 interacted with the hydrogens
interactions between the bisphenyl core of the ligand and the VDR. CH–
interactions are depicted by orange dashed lines.
p
The distance of the hydrogen bond from the oxygen of the hydroxy
group in the side chain of 5 to the imidazole nitrogen N of His397
was 2.7 Å, and that to the N of His305 was 2.9 Å. The distance
from the hydroxy group of 6 to the N of His397 was 2.7 Å and that
to the N of His305 was 2.7 Å. These short hydrogen bond dis-
e
e
e
e
tances indicated the strong hydrogen bond interaction of the hy-
droxy group of the HFP moiety. In the case of compounds 5–7,
the pKa value of the hydroxy group of the HFP moiety was remark-
ably reduced because of the strong electron-withdrawing effect of
two CF3 groups. The calculated pKa values of the hydroxy group of
the HFP moiety by MoKa36 are 6.7 for 5, 8.5 for 6, and 9.4 for 7.
These pKa values are smaller than 17.1, which is the calculated
pKa value of the corresponding hydroxy group in the non-fluori-
nated compound 4. This acidic pKa is thought to contribute to the
strong hydrogen bonding with two histidines of the VDR.
attached to the C
Val234, the hydrogens attached to all of the side chain carbons, Cb
and two C s, interacted with F4 and the hydrogen attached to one
of the C atoms interacted also with F6. With regard to Val418, the
hydrogen attached to one of the C atoms interacted with F5 and
that on the other C interacted with F4. The hydrogens attached
to one of the Cd and one of the C of Tyr401 interacted with F5
a and one of the Cb atoms of Ala231. In the case of
In addition to the hydrogen-bonding interactions of the hydro-
xy group of the HFP moiety, one of the fluorine atoms, F1, of the
HFP moiety of 5 and 6 made an NH–F interaction with the imidaz-
c
c
ole nitrogen N
3.3 Å for 5 and 3.1 Å for 6) and the other fluorine atom, F6, also
made an NH–F interaction with His305 (the distance between N
e of His397 (the distance between Ne and F1 was
c
c
e
e
and F6 was 3.2 Å for 5 and 3.4 Å for 6) (Fig. 4B). In the case of an
NH–F interaction, a positively-charged hydrogen (d+) attached to
a nitrogen electrostatically interacts with a negatively charged
fluorine (dꢁ) favorably. These NH–F interactions further strengthen
the attractive interaction between the HFP moiety and the VDR.
and the hydrogen attached to the Cd of Ile268 interacted with F6.
In all of the CH–F interactions, a negatively-charged fluorine (dꢁ)
atom attractively interacts with a positively-charged (d+) hydrogen
atom attached to carbon, although the d+ of the hydrogen is small.
Thus, the large number of small and attractive electrostatic

716
H. Kashiwagi et al. / Bioorg. Med. Chem. 21 (2013) 712–721
interactions enhances further the favorable interaction between
the HFP moiety and the VDR.
(A)
Leu227
As described above, four factors are thought to be the cause of
the favorable interactions between the HFP moiety and the VDR
in view of the 3D structure. Firstly, the hydroxy group of the HFP
moiety acidified by two electron-withdrawing CF3 groups makes
strong hydrogen bonds with two histidines of the VDR. Secondly,
two NH–F interactions between two fluorine atoms and two histi-
dines of the VDR increase favorable electrostatic interactions.
Thirdly, two CF3 groups fill the hydrophobic pocket of the VDR
tightly and this results in a gain of vdW interaction energy be-
tween the HFP moiety and the VDR. Fourthly, a large number of
weak but attractive CH–F interactions strengthen the favorable
interaction between the HFP and the VDR.
Leu404
Tyr401
Phe422
We previously reported that there exists an optimal packing
coefficient (PC) of around 55% in the hydrophobic pocket of the
VDR occupied by the side chain of the ligand, and the PC of the side
chain of compound 4 was very close to the ideal PC.32 In order to
check whether the 55% rule can also be applied to HFP analogs,
the PC values of 5, 6 and 7 were calculated in the manner we re-
ported previously.32 The calculated PC values were 63% for 5, 64%
for 6, and 65% for 7. It is apparent that the PC values of these fluo-
rinated analogs were larger than the optimal value (55%) and the
SAR of the fluorinated analogs could not be explained by the PC ef-
fect. The 55% rule is originally a rule that was derived from the case
where a hydrophobic guest molecule binds to a hydrophobic pock-
et of the host molecule. Thus, the interactions involved in the 55%
rule are mainly hydrophobic interactions. But, in the case of the
interaction between a molecule with a HFP moiety and the VDR,
electrostatic interactions such as hydrogen bonds, NH–F, and
CH–F are included, as described above. These electrostatic interac-
tions induced by the HFP moiety seem to be a reason for the dis-
crepancy between the 55% rule and the SAR of compounds with
a HFP moiety.
Regarding the activation of nuclear hormone receptors (NHR), it
is known that H12 of the NHR interacts with their coactivator and
plays a key role in exhibiting agonistic activity. 3D structures of
NHR/coactivator/agonist complex revealed that the side chain of
Glu from H12 of NHR makes hydrogen bonds with the main chain
NHs of the coactivator peptides and these hydrogen bonds contrib-
ute to recruit the coactivator.37 Three secosteroidal VD3 analogs
(Fig. 6) with the HFP moiety in the side chain were reported and
all of them showed super agonistic activity.38,39 The 3D structures
of zebra fish (z)VDR in complex with the coactivator SRC-1 peptide
with a LXXLL-motif, and these three secosteroidal VD3 analogs,
CD578 (PDB ID: 3DR1),38 Gemini 0072 (PDB ID: 3O1E),39 and Gem-
ini 0097 (PDB ID: 3O1D),39 were also reported. Figure 7A and B
show the superposition of the three secosteroidal VD3 analogs
and two nonsecosteroidal analogs, 5 and 6. The positions and the
interactions of the HFP moiety of these five molecules are almost
the same (Fig. 8A and B). The HFP moiety of both 5 and 6 interacts
with Val418 and Phe422 from H12 and Leu414 from the loop be-
tween H11 and H12. These interactions stabilize the position of
H12. In the case of the three secosteroidal analogs, the HFP moiety
also interacts with Val444 and Phe448 from H12 of zVDR and
Leu440 from the loop between H11 and H12, which are, respec-
tively, the residues corresponding to Val418, Phe422 and Leu414
of human VDR, and these interactions stabilize the H12 and also
stabilize the coactivator peptide via the interaction between
Glu446 from H12 of zVDR and the coactivator peptide. Figure 8A
and B show the superposition of the side chains of Val, Phe, Leu
and Glu and the main chain traces of the 3D structures of these five
VDR complexes. The positions and the orientation of the side
chains of Val, Phe, Leu and Glu and the interactions between the
Val, Phe and Leu residues and the HFP moiety are almost identical.
These commonalities of the position of H12 and of the interactions
Ile268
Leu227
(B)
Leu414
Leu404
Ala231
Val234
F2
F3
F4
Tyr401
Val418
F5
F6
Phe422
Ile268
Figure 5. (A) Hydrophobic pocket of the VDR around the HFP moiety of 6.
Compound 6 is colored slate. VDR is colored in pale cyan. (B) CH–F interactions
between the HFP moiety of 6 and the VDR. CH–F interactions are depicted by orange
dashed lines.
Table 2
The list of fluorine atoms of compounds 6 and 5 and the residue atoms of the VDR
within 4 Å distance of each other
6
5
F
Residue Atom Distance (Å)
F
Residue Atom Distance (Å)
F1 Leu227
His305
CD1
CD2
NE2
CD2
CD1
CD2
CD2
CD1
CA
CB
N
CB
CG1
CG2
CG2
CD1
CE1
CG1
CD1
3.1
3.5
3.2
3.8
3.4
3.5
3.5
3.1
3.3
3.5
3.4
3.3
3.4
3.7
3.8
3.5
3.7
3.4
3.7
F1 Leu227
His305
His305
Leu404
F2 Leu227
Leu404
Leu414
F3 Leu227
Ala231
Ala231
Ala231
F4 Val234
Val234
CD1
CD2
NE2
CD2
CD1
CD2
CD2
CD1
CA
3.1
3.7
3.4
3.8
3.4
3.4
3.4
3.3
3.6
3.8
3.7
3.5
3.4
3.7
His305
Leu404
F2 Leu227
Leu404
Leu414
F3 Leu227
Ala231
Ala231
Ala231
CB
N
F4 Val234
Val234
CB
CG1
CG2
Val234
Val418
Val234
F5 Tyr401
Tyr401
F5 Tyr401
Tyr401
CD1
CE1
CG1
CD1
CE1
CG1
CD1
CE1
NE2
CE1
CZ
3.5
3.6
3.4
3.6
3.9
3.6
3.9
3.5
3.1
3.4
3.8
Val418
Phe422
Val418
Phe422
Phe422
F6 Val234
Ile268
F6 Val234
Ile268
CG1
CD1
CE1
NE2
CE1
3.9
3.9
3.6
3.3
3.5
His397
His397
Phe422
His397
His397
Phe422
Phe422

H. Kashiwagi et al. / Bioorg. Med. Chem. 21 (2013) 712–721
717
D3C
F3C
HO
HO
D3C
F3C
H
H
CF3
OH
CF3
OH
CD3
OH
F3C
F3C
D3C
H
H
HO
HO
OH
OH
HO
OH
Gemini 0097
Gemini 0072
CD578
Figure 6. Fluorinated secosteroidal VD3 analogs.
(A)
(A)
H12
Leu414
SRC-1
Glu420
Val418
Phe422
(B)
(B)
H12
Leu414
Glu420
Val418
Figure 7. (A) The superposition of nonsecosteroidal VD3 analogs, CD578 (pale
yellow, PDB ID: 3DR1), Gemini 0097(pale green, PDB ID: 3O1D), Gemini 0072 (light
orange, PDB ID: 3O1E), and nonsecosteroidal analogs, 6 (slate) and 5 (pink) in the
VDR. (B) The superposition of CD578 (pale yellow) and compound 6 (slate).
Phe422
Figure 8. (A) The interactions between the HFP moiety of CD578 (pale yellow),
Gemini 0097(pale green), Gemini 0072 (light orange), compound (pink),
5
compound 6 (slate), the H12 of VDR, and SRC-1 coactivator peptide (purpleblue).
The VDRs, other than the one in complex with 6, are colored the same as their
ligand. The VDR complexed with 6 is colored in pale cyan. The species of the VDRs
in complex with CD578, Gemini 0097 and Gemini 0072 are zebra fish. SRC-1
coactivator peptide binds to zebra fish VDR (zVDR). The residue numbers depicted
are those of human VDR (hVDR). The hydrogen bonds between Glu446 (Glu420 in
hVDR number) and SRC-1 peptide are depicted by orange dashed lines. (B) The
interactions between the H12 of hVDR and compound 6. Compound 6 is colored in
slate. The H12 of hVDR is colored in pale cyan.
between H12 and the HFP moiety are assumed to be the reason for
the superagonistic character exhibited by 5 and 6.
Regarding the difference between the activity between acety-
lene linker 5 and (E)-olefin linker 6, we could not explain the dif-
ference in the activity from their 3D information alone because
the binding conformations of 5 and 6 were superposed well when
bound to the VDR. Two factors to explain this difference are possi-
ble, just as in the case of the increase in activity of compound 4
(with ethyl groups at the terminal of the side chain) compared to
that of the corresponding compound with an acetylene linker.32
The first possible reason is the hydrophobicity of the molecule.
The ClogP40 value of acetylene 5 is 6.13 and that of (E)-olefin 6
is 6.78. Thus, 6 is more hydrophobic than 5 and this could be the
reason for the stronger activity of 6. The second reason could be
a loss of entropy from the bond rotation of the side chain. The rota-
tion around a triple bond was reported to be almost free,41 but the
rotation of the double bond in the olefin linker of 6 is restricted.
When 5, which has a triple bond, binds to the VDR and the bond
rotation becomes fixed, there is a larger loss of entropy than when
6, with a double bond, binds to the VDR. Therefore the larger loss of
entropy of 5 compared to 6 could decrease the activity of 5. It
should be noted that, as described above, the pKa value of 5 is more
acidic than that of 6. Thus, the favorable electrostatic interaction
energy of the hydroxy group of 5 with two histidines is thought
to be larger than that of 6. It is thought that the free energy gain
of 6 by the two factors, hydrophobicity and entropy loss of the
rotatable bond, is larger than the loss of electrostatic interaction
energy caused by having a less acidic hydroxy group.
4. Conclusion
From our research of nonsecosteroidal vitamin D3 derivatives
with gamma hydroxy carboxylic acid, we identified compound 6,
which has two CF3 groups in the side chain, as a most potent
VDR agonist showing superagonistic activity in VDRE RGA,
MG-63 osteocalcin production assay and HL-60 cell differentiation

718
H. Kashiwagi et al. / Bioorg. Med. Chem. 21 (2013) 712–721
assay. Compound 6 demonstrated that fluorination is effective in
the case of our nonsecosteridal scaffold, as it is in the case of secos-
teroidal VD3 analogs. X-ray analysis of the VDR with compound 6
revealed that all of the six fluorine atoms of the HFP moiety in
the side chain effectively interact with the VDR by both steric
(vdW) and electrostatic (hydrogen bond, NH–F and CH–F) interac-
tions. The HFP moiety of 6 effectively interacts especially with H12
of the VDR and stabilizes the position and the orientation of H12,
which could result in stabilizing the coactivator and enhancing
the VDR agonistic activity.
5.1.3. 4-{1-Ethyl-1-[3-methyl-4-((E)-4,4,4-trifluoro-3-
hydroxy-3-trifluoromethyl-1-butenyl)phenyl]propyl}-
2-methylphenol (11)
To a solution of 9 (8.67 g, 18.91 mmol) in THF (100 mL) was
added Red-AlÒ (65 wt% in toluene, 17 mL, 56.74 mmol) at 0 °C
and the mixture was stirred for 1 h. The mixture was poured into
1 N HCl and products were extracted with CH2Cl2. The extracts
were dried over anhydrous MgSO4 and concentrated. The obtained
residue was purified with silicagel chromatography (n-hexane/
AcOEt = 10:1 to 4:1) to afford 11 (6.11 g, 70%) as a colorless oil.
1H NMR (CDCl3) d: 0.61 (6H, t, J = 7.3 Hz), 2.05 (4H, q, J = 7.4 Hz),
2.19 (3H, s), 2.34 (3H, s), 4.59 (1H, br s), 6.08 (1H, d, J = 15.8 Hz),
6.66 (1H, d, J = 8.2 Hz), 6.85 (1H, dd, J = 8.4, 2.3 Hz), 6.88 (1H, d,
J = 2.2 Hz), 7.00–7.02 (2H, m), 7.34 (1H, d, J = 7.6 Hz), 7.37 (1H, d,
J = 15.7 Hz). 13C NMR (CDCl3) d: 151.5, 150.4, 140.4, 135.5, 135.1,
130.7, 130.5, 130.2, 126.5, 126.1, 125.3, 123.9, 122.8, 121.0,
116.4, 114.1, 49.0, 29.0, 19.9, 16.1, 8.3. MS (ESI negative): 459
(MꢁH)ꢁ.
5. Experimental
5.1. Chemistry: general
Purchased reagents and solvents were used without further
purification unless otherwise noted. 1H and 13C NMR spectra were
carried out on VARIAN 400-MR spectrophotometers; chemical
shifts are reported in parts per million (ppm) downfield from that
of internal tetramethylsilane (TMS). Mass spectrophotometry was
measured with a Waters ACQUITY SQD electrospray ionization
(ESI) system. High-resolution mass spectra (HRMS) were recorded
on Thermo Fisher Scientific LTQ Orbitrap XL (ESI) instruments.
Optical rotations were measured on a HORIBA SEPA-200 polarim-
eter (sodium D line at 25 °C). Chromatographic purification was
carried out using Merck silica gel 60 (column) or Merck silica gel
60 PF254 (preparative TLC).
5.1.4. 4-{1-Ethyl-1-[3-methyl-4-((E)-4,4,4-trifluoro-3-
methoxymethoxy-3-trifluoromethyl-1-butenyl)phenyl]propyl}-
2-methylphenol (12)
To a solution of 11 (400 mg, 0.869 mmol) in DMF (5 mL) was
added NaH (60% in mineral oil, 34.7 mg, 0.869 mmol) and the mix-
ture was stirred for 30 min. To the mixture was added methoxy-
methyl chloride (0.132 mL, 1.74 mmol) and the mixture was
stirred at 50 °C overnight. Then the mixture was poured into satd
NH4CI aq solution and products were extracted with Et2O. The ex-
tracts were washed with H2O and brine, dried over anhydrous
Na2SO4, and concentrated. The obtained residue was chromato-
graphed on silica gel (n-hexane/AcOEt/CH2Cl2 = 10:1:1) to afford
12 (180 mg, 41%) as a colorless oil. 1H NMR (CDCl3) d: 0.61 (6H,
t, J = 7.3 Hz), 2.05 (4H, q, J = 7.4 Hz), 2.20 (3H, s), 2.32 (3H, s),
3.50 (3H, s), 4.60 (1H, br s), 4.97 (2H, s), 6.07 (1H, d, J = 16.6 Hz),
6.66 (1H, d, J = 8.2 Hz), 6.85 (1H, dd, J = 8.3, 2.2 Hz), 6.88 (1H, d,
J = 2.2 Hz), 7.00–7.03 (2H, m), 7.34 (1H, d, J = 17.4 Hz), 7.35 (1H,
d, J = 7.8 Hz). 13C NMR (CDCl3) d: 151.5, 150.6, 140.4, 137.7,
135.6, 131.1, 130.6, 130.2, 126.6, 126.3, 125.2, 123.8, 122.6,
121.0, 114.7, 114.1, 93.3, 56.4, 49.0, 29.0, 19.9, 16.1, 8.4. MS (ESI
negative): 503 (MꢁH)ꢁ.
5.1.1. 4-{1-Ethyl-1-[3-methyl-4-(4,4,4-trifluoro-3-hydroxy-3-
trifluoromethyl-1-butynyl)phenyl]propyl}-2-methylphenol (9)
To a solution of 8 (16.44 g, 56.22 mmol) in THF (250 mL) was
added n-butyllithium (2.71 M in n-hexane, 51.9 mL, 140.55 mmol)
at 0 °C and the mixture was stirred for 30 min. To the mixture was
added hexafluoroacetone by bubbling and the mixture was stirred
at 0 °C for 30 min. Then the mixture was poured into satd NH4Cl aq
solution and products were extracted with AcOEt. The extracts
were dried over anhydrous MgSO4 and concentrated. The obtained
residue was chromatographed on silica gel (n-hexane/AcOEt = 10:1
to 4:1) to afford 9 (17.62 g, 68%) as a colorless oil. 1H NMR (CDCl3)
d: 0.59 (6H, t, J = 7.3 Hz), 2.04 (4H, q, J = 7.3 Hz), 2.19 (3H, s), 2.38
(3H, s), 4.71 (1H, br s), 6.66 (1H, d, J = 8.0 Hz), 6.82–6.86 (2H, m),
6.99 (1H, d, J = 8.0 Hz), 7.05 (1H, s), 7.34 (1H, d, J = 8.2 Hz). 13C
NMR (CDCl3) d: 152.2, 151.5, 140.8, 140.1, 131.8, 130.6, 129.4,
126.6, 125.7, 122.8, 122.6, 119.7, 116.2, 114.2, 89.5, 80.0, 49.3,
28.9, 20.6, 16.0, 8.3. MS (ESI negative): 457 (MꢁH)ꢁ.
5.1.5. General procedure for alkylation of the phenol group of
10 and 12
To a solution of phenol (0.394 mmol) in DMF (4 mL) was added
K2CO3 (0.985 mmol) and the mixture was stirred at room temper-
ature for 30 min. Then, (S)-(+)-dihydro-5-(p-tolylsulfonyloxymeth-
yl)-2(3H)-furanone (13) (0.591 mmol) was added and the mixture
was stirred at 100 °C for 15 h. The mixture was poured into satd
NH4Cl aq solution and products were extracted with AcOEt. The ex-
tracts were washed with H2O and brine, dried over anhydrous
Na2SO4, and concentrated. The obtained residue was chromato-
graphed on silica gel (n-hexane/AcOEt = 5:1 to 3:1) to afford
lactone.
5.1.2. 4-{1-Ethyl-1-[3-methyl-4-(4,4,4-trifluoro-3-
methoxymethoxy-3-trifluoromethyl-1-butynyl)phenyl]propyl}-
2-methylphenol (10)
To a solution of 9 (356 mg, 0.777 mmol) in DMF (5 mL) was
added K2CO3 (268 mg, 1.94 mmol) and the mixture was stirred
for 20 min. To the mixture was added methoxymethyl chloride
(0.071 mL, 0.935 mmol) and the mixture was stirred for 1 h. Then
the mixture was poured into satd NH4CI aq solution and products
were extracted with AcOEt. The extracts were washed with H2O
and brine, dried over anhydrous MgSO4, and concentrated. The ob-
tained residue was chromatographed on silica gel (n-hexane/
AcOEt = 6:1) to afford 10 (349 mg, 89%) as a colorless oil. 1H
NMR (CDCl3) d: 0.59 (6H, t, J = 7.3 Hz), 2.04 (4H, q, J = 7.3 Hz),
2.19 (3H, s), 2.39 (3H, s), 3.48 (3H, s), 4.57 (1H, s), 5.15 (2H, s),
6.66 (1H, d, J = 8.8 Hz), 6.82–6.85 (2H, m), 6.99 (1H, dd, J = 8.1,
1.9 Hz), 7.05 (1H, d, J = 1.6 Hz), 7.37 (1H, d, J = 8.2 Hz). 13C NMR
(CDCl3) d: 152.3, 151.9, 140.8, 139.7, 132.0, 130.5, 129.4, 126.4,
125.7, 123.1, 122.6, 119.7, 116.3, 114.1, 95.0, 93.3, 76.4, 56.4,
49.2, 28.9, 20.5, 16.1, 8.2. MS (ESI negative): 501 (MꢁH)ꢁ.
5.1.6. (S)-5-(4-{1-Ethyl-1-[3-methyl-4-(4,4,4-trifluoro-3-
methoxymethoxy-3-trifluoromethyl-1-butynyl)phenyl]propyl}-
2-methylphenoxymethyl)dihydrofuran-2-one (14)
The yield was 85%. Colorless oil. ½a2D5ꢂ+9.58 (c 1.36, CHCl3). 1H
NMR (CDCl3) d: 0.59 (6H, t, J = 7.2 Hz), 2.05 (4H, q, J = 7.3 Hz),
2.15 (3H, s), 2.27–2.36 (1H, m), 2.39 (3H, s), 2.41–2.49 (1H, m),
2.53–2.61 (1H, m), 2.72–2.81 (1H, m), 3.47 (3H, s), 4.07 (1H, dd,
J = 10.3, 3.4 Hz), 4.17 (1H, dd, J = 10.4, 3.3 Hz), 4.86–4.91 (1H, m),
5.15 (2H, s), 6.67 (1H, d, J = 8.4 Hz), 6.86 (1H, d, J = 2.0 Hz), 6.92
(1H, dd, J = 8.5, 2.4 Hz), 6.98 (1H, dd, J = 8.0, 1.4 Hz), 7.04 (1H, s),
7.37 (1H, d, J = 8.0 Hz). 13C NMR (CDCl3) d: 177.1, 154.2, 152.0,
140.9, 140.5, 132.0, 130.6, 129.4, 126.1, 125.9, 125.6, 122.6,

H. Kashiwagi et al. / Bioorg. Med. Chem. 21 (2013) 712–721
719
119.7, 116.4, 110.0, 95.0, 93.2, 77.8, 77.2, 76.5, 69.4, 56.5, 49.3,
28.9, 28.3, 24.1, 20.6, 16.6, 8.3. MS (ESI positive): 618 (M+NH4)+.
2.30–2.35 (1H, m), 2.38–2.48 (1H, m), 2.52–2.61 (1H, m), 2.73–
2.83 (3H, m), 4.05 (1H, dd, J = 10.4, 3.5 Hz), 4.16 (1H, dd, J = 10.4,
3.3 Hz), 4.86–4.92 (1H, m), 6.66 (1H, d, J = 8.4 Hz), 6.91–6.94 (4H,
m), 7.00 (1H, d, J = 8.6 Hz). 13C NMR (CDCl3) d: 177.8, 153.9,
147.1, 141.3, 135.2, 134.8, 130.7, 130.1, 127.9, 126.2, 126.0,
125.6, 124.7, 121.8, 109.9, 78.2, 76.1, 69.3, 48.7, 31.0, 29.1, 28.4,
25.2, 24.0, 19.2, 16.6, 8.4. MS (ESI positive): 561 (M+H)+.
5.1.7. (S)-5-(4-{1-Ethyl-1-[3-methyl-4-((E)-4,4,4-trifluoro-3-
methoxymethoxy-3-trifluoromethyl-1-butenyl)phenyl]propyl}-
2-methylphenoxymethyl)dihydrofuran-2-one (15)
The yield was 90%. Colorless oil. 1H NMR (CDCl3) d: 0.61 (6H, t,
J = 7.2 Hz), 2.06 (4H, q, J = 7.3 Hz), 2.16 (3H, s), 2.25–2.39 (1H, m),
2.32 (3H, s), 2.39–2.50 (1H, m), 2.53–2.61 (1H, m), 2.72–2.82
(1H, m), 3.50 (3H, s), 4.07 (1H, dd, J = 10.4, 3.5 Hz), 4.17 (1H, dd,
J = 10.2, 3.3 Hz), 4.86–4.91 (1H, m), 4.96 (2H, s), 6.07 (1H, d,
J = 16.6 Hz), 6.67 (1H, d, J = 8.4 Hz), 6.90 (1H, d, J = 2.0 Hz), 6.94
(1H, dd, J = 8.4, 2.2 Hz), 6.99 (1H, s), 7.00 (1H, d, J = 8.4 Hz), 7.34
(1H, d, J = 17.2 Hz), 7.35 (1H, d, J = 7.8 Hz). 13C NMR (CDCl3) d:
177.1, 154.1, 150.5, 140.9, 137.7, 135.6, 131.1, 130.6, 130.2,
126.2, 126.2, 125.8, 125.2, 123.8, 121.0, 114.7, 110.0, 93.3, 77.9,
77.2, 69.3, 56.4, 49.0, 29.0, 28.3, 24.1, 19.9, 16.6, 8.4. MS (ESI posi-
tive): 620 (M+NH4)+.
5.1.11. General procedure for hydrolysis of the lactone group of
16, 17 and 18
To a solution of lactone (0.355 mmol) in methanol (7.5 mL), 1 M
KOH solution (0.7 mL, 0.700 mmol) was added. The mixture was
stirred at room temperature for 2 h. The mixture was poured into
satd NH4Cl aq solution and products were extracted with AcOEt.
The extracts were washed with brine, dried over MgSO4, and con-
centrated. The obtained residue was chromatographed on silica gel
(5% MeOH/CH2Cl2) to afford gamma hydroxycarboxylic acid. Then,
the free form of carboxylic acid was diluted in EtOH, 1.0 equiv of
1 M NaOH solution was added and then concentrated. The ob-
tained salt was stored at ꢁ30 °C as 1 mg/mL EtOH solution.
5.1.8. (S)-5-(4-{1-Ethyl-1-[3-methyl-4-(4,4,4-trifluoro-3-
hydroxy-3-trifluoromethyl-1-butynyl)phenyl]propyl}-2-
methylphenoxymethyl)dihydrofuran-2-one (16)
5.1.12. (S)-5-(4-{1-Ethyl-1-[3-methyl-4-(4,4,4-trifluoro-3-
hydroxy-3-trifluoromethyl-1-butynyl)phenyl]propyl}-2-
To a solution of 14 (19.5 mg, 0.032 mmol) in 1,4-dioxane
(3.0 mL) was added concd HCl and the mixture was stirred at room
temperature overnight. The mixture was poured into H2O and
products were extracted with CH2Cl2. The extracts were dried over
anhydrous MgSO4 and concentrated. The obtained residue was
purified by preparative TLC (n-hexane/AcOEt = 2:1) to afford 16
(17.1 mg, 96%) as a colorless oil. ½a2D5ꢂ +11.1 (c 0.78, CHCl3). 1H
NMR (CDCl3) d: 0.59 (6H, t, J = 7.3 Hz), 2.04 (4H, q, J = 7.2 Hz),
2.13 (3H, s), 2.25–2.35 (1H, m), 2.37 (3H, s), 2.40–2.48 (1H, m),
2.53–2.61 (1H, m), 2.73–2.82 (1H, m), 4.02–4.07 (1H, m), 4.13–
4.17 (1H, m), 4.85–4.90 (1H, m), 6.65 (1H, d, J = 8.4 Hz), 6.85 (1H,
d, J = 2.2 Hz), 6.91 (1H, dd, J = 8.5, 2.1 Hz), 6.97 (1H, d, J = 8.0 Hz),
7.03 (1H, s), 7.34 (1H, d, J = 8.2 Hz). 13C NMR (CDCl3) d: 177.6,
154.1, 151.9, 140.8, 140.6, 131.8, 130.6, 129.4, 126.1, 125.9,
125.7, 122.6, 119.8, 116.4, 110.0, 89.3, 80.1, 78.0, 77.2, 69.3, 49.3,
28.9, 28.4, 24.0, 20.5, 16.6, 8.3. MS (ESI positive): 579 (M+Na)+.
methylphenoxy)-4-hydroxypentanoic acid sodium salt (5)
The yield was 52%. Colorless amorphous. ½a2D5ꢂ+2.53 (c 0.40,
CHCl3). 1H NMR (CD3OD) d: 0.60 (6H, t, J = 7.3 Hz), 1.80–1.87 (1H,
m), 1.95–2.03 (1H, m), 2.09 (4H, q, J = 7.3 Hz), 2.15 (3H, s), 2.36
(3H, s), 2.39–2.47 (2H, m), 3.90 (2H, d, J = 5.1 Hz), 3.95–3.98 (1H,
m), 6.77 (1H, d, J = 8.6 Hz), 6.85 (1H, d, J = 1.8 Hz), 6.94 (1H, dd,
J = 8.6, 2.5 Hz), 7.03 (1H, dd, J = 8.0, 1.6 Hz), 7.09 (1H, s), 7.34 (1H,
d, J = 8.0 Hz). 13C NMR (CD3OD) d: 183.2, 157.3, 154.3, 142.6,
141.9, 133.5, 132.3, 131.5, 128.1, 128.0, 127.8, 125.5, 122.7,
118.9, 112.3, 90.1, 83.0, 74.1, 71.7, 51.3, 49.4, 34.5, 31.7, 30.8,
21.5, 17.6, 9.6. HRMS (ESI negative): Calcd for
C29H31F6O5
573.2081. Found: 573.2077 (MꢁH)ꢁ.
5.1.13. (S)-5-(4-{1-Ethyl-1-[3-methyl-4-((E)-4,4,4-trifluoro-3-
hydroxy-3-trifluoromethyl-1-butenyl)phenyl]propyl}-2-
methylphenoxy)-4-hydroxypentanoic acid sodium salt (6)
The yield was 49%. Colorless amorphous. ½a2D5ꢂ+2.25 (c 0.62,
CHCl3). 1H NMR (CD3OD) d: 0.59 (6H, t, J = 7.2 Hz), 1.78–1.88 (1H,
m), 1.92–2.00 (1H, m), 2.07 (4H, q, J = 7.4 Hz), 2.14 (3H, s), 2.31
(3H, s), 2.33–2.39 (2H, m), 3.89 (2H, d, J = 5.1 Hz), 3.92–3.98 (1H,
m), 6.16 (1H, d, J = 15.8 Hz), 6.77 (1H, d, J = 8.6 Hz), 6.84 (1H, d,
J = 2.0 Hz), 6.93–7.01 (3H, m), 7.33 (1H, d, J = 8.0 Hz), 7.40 (1H, d,
J = 15.8 Hz). 13C NMR (CD3OD) d: 183.5, 157.2, 152.2, 142.3,
137.4, 136.5, 133.7, 132.4, 132.2, 128.1, 128.1, 127.9, 127.1,
124.3, 121.3, 112.2, 74.1, 72.3, 59.2, 49.8, 36.4, 32.4, 30.9, 21.0,
17.6, 9.6. HRMS (ESI negative): Calcd for C29H33F6O5 575.2238.
Found: 575.2224 (MꢁH)ꢁ.
5.1.9. (S)-5-(4-{1-Ethyl-1-[3-methyl-4-((E)-4,4,4-trifluoro-3-
hydroxy-3-trifluoromethyl-1-butenyl)phenyl]propyl}-2-
methylphenoxymethyl)dihydrofuran-2-one (17)
The yield was 95%. Colorless oil. ½a2D5ꢂ+11.1 (c 0.71, CHCl3). 1H
NMR (CDCl3) d: 0.60 (6H, t, J = 7.2 Hz), 2.05 (4H, q, J = 7.2 Hz),
2.15 (3H, s), 2.27–2.35 (1H, m), 2.33 (3H, s), 2.39–2.48 (1H, m),
2.52–2.61 (1H, m), 2.73–2.81 (1H, m), 4.06 (1H, dd, J = 10.3,
3.4 Hz), 4.16 (1H, dd, J = 10.4, 3.3 Hz), 4.86–4.91 (1H, m), 6.08
(1H, d, J = 15.8 Hz), 6.66 (1H, d, J = 8.4 Hz), 6.90 (1H, d, J = 1.8 Hz),
6.94 (1H, dd, J = 8.5, 2.2 Hz), 6.97–7.01 (2H, m), 7.34 (1H, d,
J = 8.4 Hz), 7.37 (1H, d, J = 16.4 Hz). 13C NMR (CDCl3) d: 177.4,
154.0, 150.2, 140.9, 135.6, 135.1, 130.7, 130.7, 130.2, 126.2,
126.1, 125.7, 125.3, 123.8, 121.0, 116.3, 110.0, 78.0, 69.3, 67.0,
49.0, 29.0, 28.3, 24.0, 20.0, 16.6, 8.3. MS (ESI positive): 581
(M+Na)+.
5.1.14. (S)-5-(4-{1-Ethyl-1-[3-methyl-4-(4,4,4-trifluoro-3-
hydroxy-3-trifluoromethylbutyl)phenyl]propyl}-2-
methylphenoxy)-4-hydroxypentanoic acid sodium salt (7)
The yield was 83%. Colorless amorphous. ½a2D5ꢂ+1.38 (c 0.86,
CHCl3). 1H NMR (CD3OD) d: 0.58 (6H, t, J = 7.3 Hz), 1.77–1.87 (2H,
m), 1.95–2.02 (2H, m), 2.06 (4H, q, J = 7.1 Hz), 2.15 (3H, s), 2.23
(3H, s), 2.41–2.55 (2H, m), 2.76–2.81 (2H, m), 3.89 (2H, dd,
J = 5.1, 1.2 Hz), 3.94–4.00 (1H, m), 6.73 (1H, d, J = 8.3 Hz), 6.87
(1H, d, J = 2.0 Hz), 6.92–6.96 (3H, m), 6.99 (1H, d, J = 8.8 Hz). 13C
NMR (CD3OD) d: 178.0, 155.9, 148.3, 141.6, 136.8, 135.6, 131.3,
131.0, 128.8, 127.1, 126.9, 126.6, 126.3, 123.5, 111.2, 73.0, 70.2,
68.6, 49.6, 33.0, 30.5, 30.0, 29.9, 26.2, 19.1, 16.4, 8.5. HRMS (ESI
5.1.10. (S)-5-(4-{1-Ethyl-1-[3-methyl-4-(4,4,4-trifluoro-3-
hydroxy-3-trifluoromethylbutyl)phenyl]propyl}-2-
methylphenoxymethyl)dihydrofuran-2-one (18)
To a solution of 16 (55 mg, 0.099 mmol) in AcOEt (5 mL) was
added Pd(OH)2/C (20 wt%, 14 mg, 0.020 mmol) and the mixture
was stirred at room temperature under H2 atmosphere overnight.
The mixture was filtered through a celite pad and concentrated.
The obtained residue was purified by preparative TLC (n-hexane/
AcOEt = 2:1) to afford 18 (52.5 mg, 95%) as colorless oil.
½
a2D5ꢂ+12.0 (c 1.05, CHCl3). 1H NMR (CDCl3) d: 0.59 (6H, t,
negative): Calcd for
C29H35F6O5 577.2394. Found: 575.2389
(MꢁH)ꢁ.
J = 7.3 Hz), 2.04 (4H, q, J = 7.3 Hz), 2.11–2.17 (5H, m), 2.24 (3H, s),

720
H. Kashiwagi et al. / Bioorg. Med. Chem. 21 (2013) 712–721
Table 3
5.2. VDRE reporter gene assay
Crystal and diffraction data of VDR with compounds 6 and 5
MG-63 cells were plated at 2 ꢀ 103 cells/200
lL/well in a 96-
well white cell-culture plate and were incubated at 37 °C in 5%
6
5
Wavelength (Å)
1.0
1.0
CO2 incubator for 24 h. Then, MG-63 cells were cotransfected with
Cell (Å)
a
b
c
44.4
51.9
132.1
1.9
44.6
51.8
132.5
1.8
0.05
three repeats of the VDRE sequence from mouse osteopontin pro-
moter and 0.001 g of pRL-SV40 vector (Promega Corporation, WI,
lg of the pGV2-basic/VDRE-luciferase vector which contained
l
Resolution (Å)
USA) using Lipofectamine (Invitrogen). The cells were added to
minimum essential medium (MEM) containing 5% fetal bovine ser-
um treated with dextran-coated charcoal (DCC-FBS) and were
incubated for 8 h. The cells were treated with the serial diluted
compounds (final concentrations were 10ꢁ7 to 10ꢁ11 mol/L with
0.1% DMSO) and were incubated for an additional 3 days. After
removing the supernatants, the cells were lysed in cell-lysis buffer
and luciferase activity was measured by DLR™ Assay System (Pro-
mega Corporation, WI, USA) and the luminescence was detected by
Wallac ALVO SX 1420 multi-label counter (PerkinElmer, Inc., MA,
USA). The half maximal effective concentrations (EC50) were deter-
mined and the inductive activity was calculated as the ratio of the
EC50 value of the compounds to that of 1,25(OH)2D3 (Solvay Phar-
maceuticals, Weesp, The Netherlands), which was used as a posi-
tive control.
Completeness (last shell) (%)
Rsym (last shell) (%)
I/Sigma (last shell)
Rcryst
Rmsd bond length (Å)
Rmsd bond angles (deg)
No. of non-hydrogen protein atoms
No. of water molecules
93.5 (89.3)
12.2 (35.8)
4.0 (2.0)
20.1
0.010
0.98
91.3 (72.1)
6.2 (15.7)
6.4 (4.3)
19.4
0.010
0.94
1993
195
1993
266
and data processing were carried out by generally accepted meth-
ods. After brief soaking in the buffer containing 30% glycerol, 0.6 M
ammonium sulfate and 0.1 M MES (pH 6.0), crystals were trapped
in the fiber loops and flash-cooled with liquid nitrogen. Crystals
were stored and transported in a dry shipper. Data collections were
carried out at the synchrotron beamline BL32B2 of the Spring-8
facility in Hyogo, Japan, operated by the Pharmaceutical Consor-
tium for Protein Structure Analysis. Crystals were kept frozen dur-
ing data collection with a vapor stream of liquid nitrogen. X-ray
diffraction data was collected with the R-AXIS V imaging plate area
detector. The crystals were isomorphous with the space group
P212121 reported by Moras et al.42,43 for the 1,25(OH)2D3-VDR
complex. Data were processed with CCP4 and MOSFLM. The initial
structure model for the refinement was constructed by removing
all the water and the ligand atoms in the 1,25(OH)2D3–VDR com-
plex structure in the Protein Databank (PDB ID: 1db1). A rigid body
refinement was followed by simulated annealing using the CNX.
Electron density in the ligand-binding pocket clearly and unambig-
uously showed the ligand conformation. After fitting the ligand
model into the electron density, structure refinement was contin-
ued with the software autoBUSTER and the water molecules were
placed automatically. The data collection and the refinement sta-
tistics are summarized in Table 3.
5.3. Osteocalcin induction assay
MG-63 cells were plated at 2 ꢀ 103 cells/well in 200
lL of ser-
um-free MEM in a 96-well plate and were incubated at 37 °C in
5% CO2 for 24 h. After washing cells with 5% DCC-FBS/MEM (cul-
ture medium), the cells were added to culture medium and treated
with the serial diluted compounds (final concentrations were 10ꢁ7
to 10ꢁ11 mol/L with 0.1% DMSO), and incubated for 8 h. After
changing the culture medium to a fresh one, the cells were incu-
bated for an additional 4 days, and then supernatant was collected
and stored at ꢁ80 °C.
The frozen supernatant was thawed slowly at room tempera-
ture and Gla-type osteocalcin EIA kit (Takara Bio. Inc, Tokyo, Japan)
was used to measure osteocalcin. Absorbance at 450 nm was mea-
sured on a plate reader (Model 3550, Bio-Rad Laboratories, CA,
USA) and each concentration was calculated by comparison with
standards using
lplate Manager I software (Bio-Rad Laborato-
ries). The EC50 value was determined and the inductive activity
was calculated as the ratio of the EC50 value of the compounds to
that of 1,25(OH)2D3.
5.6. Modeling and volume calculations
The compounds 5, 6, and 7 were manually docked in the crystal
structure of the VDR (PDB ID: 3AZ3). The conformations of com-
pounds were optimized using the MAB force field as implemented
in the program MOLOC44 with the VDR structure fixed. Based on
the docking structures of the VDR with 5, 6, and 7, the PC values
(the volume occupied by the side chain of the compound) of com-
pounds 5, 6, and 7 were calculated as described in Ref. 32.
5.4. HL-60 cell differentiation assay
Human promyelocytic leukemic HL-60 cells (2 ꢀ 105 cells/mL)
in a 96-well micro-titer plate were seeded in 100 lL of PRMI-
1640 medium with antibiotics and 10% heat-inactivated fetal bo-
vine serum at 37 °C in a humidified atmosphere of 5% CO2 in air.
To examine the effects of the analogs on cell differentiation, HL-
60 cells were cultured for 4 days with or without different concen-
trations (10ꢁ10 to 10ꢁ7 M) of analogs. On the last day of incubation,
Acknowledgements
The authors thank Ms. Hitomi Suda of Chugai Pharmaceutical
Co., Ltd for HRMS measurements of the compounds and Editing
Services of Chugai Pharmaceutical Co., Ltd for proofreading the
manuscript.
20 lL of detection mixture reagent consisting of PMA (final 100 ng/
mL) and WST-8 reagent (final 20%), was added and incubated for
an additional 90 minutes in the same conditions. After incubation,
the change of absorbance at 450 nm was measured with the plate
reader. The EC50 value was determined and the cell differentiation
activity was calculated as the ratio of the EC50 value of the com-
pounds to that of 1,25(OH)2D3.
References and notes
1. Muller, K.; Faeh, C.; Diederich, F. Science 1881, 2007, 317.
2. Paulini, R.; Muller, K.; Diederich, F. Angew. Chem., Int. Ed. 2005, 44, 1788.
3. Zhou, P.; Zou, J.; Tian, F.; Shang, Z. J. Chem. Inf. Model. 2009, 49, 2344.
4. Iwaoka, M.; Komatsu, H.; Katsuda, T.; Tomoda, S. J. Am. Chem. Soc. 1902, 2002,
124.
5.5. Crystallographic structure analysis
Protein sample preparation and crystallization were based on
the methods by Moras et al.42,43 X-ray diffraction data collection
5. Matta, C. F.; Castillo, N.; Boyd, R. J. Phys. Chem. A 2005, 109, 3669.
6. Lee, S.; Mallik, A. B.; Fredrickson, D. C. Cryst. Growth Des. 2004, 4, 279.

H. Kashiwagi et al. / Bioorg. Med. Chem. 21 (2013) 712–721
721
7. Olsen, J. A.; Banner, D. W.; Seiler, P.; Sander, U. O.; D’Arcy, A.; Stihle, M.;
Mueller, K.; Diederich, F. Angew. Chem., Int. Ed. 2003, 42, 2507.
8. Plenio, H. ChemBioChem 2004, 5, 650.
27. Hosoda, S.; Tanatani, A.; Wakabayashi, K.; Nakano, Y.; Miyachi, H.; Nagasawa,
K.; Hashimoto, Y. Bioorg. Med. Chem. Lett. 2005, 15, 4327–4331.
28. Ma, Y.; Khalifa, B.; Yee, Y. K.; Lu, J.; Memezawa, A.; Savkur, R. S.; Yamamoto, Y.;
Chintalacharuvu, S. R.; Yamaoka, K.; Stayrook, K. R.; Bramlett, K. S.; Zeng, Q. Q.;
Chandrasekhar, S.; Yu, X. P.; Linebarger, J. H.; Iturria, S. J.; Burris, T. P.; Kato, S.;
Chin, W. W.; Nagpal, S. J. Clin. Invest. 2006, 116, 892–904.
29. Hosoda, S.; Tanatani, A.; Wakabayashi, K.; Makishima, M.; Imai, K.; Miyachi, H.;
Nagasawa, K.; Hashimoto, Y. Bioorg. Med. Chem. 2006, 14, 5489–5502.
30. Demizu, Y.; Takahashi, T.; Kaneko, F.; Sato, Y.; Okuda, Y.; Ochiai, E.; Horie, K.;
Takagi, K.; Kakuda, S.; Takimoto-Kamimura, M.; Kurihara, M. Bioorg. Med.
Chem. Lett. 2011, 21, 6104–6107.
9. Bettinger, H. F. ChemPhysChem 2005, 6, 1169.
10. Lu, Y.; Zou, J.; Yu, Q.; Jiang, Y.; Zhao, W. Chem. Phys. Lett. 2007, 449, 6.
11. Awwadi, F. F.; Willett, R. D.; Peterson, K. A.; Twamley, B. J. Phys. Chem. A 2007,
111, 2319.
12. Awwadi, F. F.; Willett, R. D.; Peterson, K. A.; Twamley, B. Chem. -Eur. J. 2006, 12,
8952.
13. Kawahara, S.; Tsuzuki, S.; Uchimaru, T. J. Phys. Chem. A 2004, 108, 6744.
14. Riley, K. E.; Merz, K. M., Jr. J. Phys. Chem. B 2005, 109, 17752.
15. Chopra, D.; Nagarajan, K.; Row, T. N. G. J. Mol. Struct. 2008, 888, 70.
16. Tanaka, Y.; DeLuca, H. F.; Schnoes, H. K.; Ikekawa, N.; Kobayashi, Y. Arch.
Biochem. Biophys. 1980, 199, 473.
31. Kashiwagi, H.; Ono, Y.; Shimizu, K.; Haneisihi, T.; Ito, S.; Iijima, S.; Kobayashi,
T.; Ichikawa, F.; Harada, S.; Sato, H.; Sekiguchi, N.; Ishigai, M.; Takahashi, T.
Bioorg. Med. Chem. 2011, 19, 4721.
17. Kobayashi, Y.; Taguchi, T.; Mitsuhashi, S.; Eguchi, T.; Ohshima, E.; Ikekawa, N.
Chem. Pharm. Bull. 1982, 30, 4297.
32. Kashiwagi, H.; Ono, Y.; Ohta, M.; Morikami, K.; Takahashi, T. Bioorg. Med. Chem.
2012, 20, 4495.
18. Tanaka, Y.; DeLuca, H. F.; Kobayashi, Y.; Ikekawa, N. Arch. Biochem. Biophys.
1984, 229, 348.
33. Brown, J. P.; Malaval, L.; Chapuy, M. C.; Delmas, P. D.; Edouard, C.; Meunierp, J.
Lancet 1984, 323, 1091.
19. Inaba, M.; Okuno, S.; Nishizawa, Y.; Yukioka, K.; Otani, S.; Matsui-Yuasa, I.;
Morisawa, S.; DeLuca, H. F.; Morii, H. Arch. Biochem. Biophys. 1987, 258, 421.
20. Inaba, M.; Okuno, S.; Nishizawa, Y.; Imanishi, Y.; Katsumata, T.; Sugata, I.;
Morii, H. Biochem. Pharmacol. 1993, 45, 2331.
21. Sheves, M.; Sialom, B.; Mazur, Y. J. C. S. Chem. Commun. 1978, 13, 554.
22. Napoli, J. L.; Fivizzani, M. A.; Schnoes, H. K.; DeLuca, H. F. Biochemistry 1978, 17,
2387.
23. Ohshima, E.; Sai, H.; Takatsuto, S.; Ikekawa, N.; Kobayashi, Y.; Tanaka, Y.;
DeLuca, H. F. Chem. Pharm. Bull. 1984, 32, 3525.
24. Boehm, M. F.; Fitzgerald, P.; Zou, A.; Elgort, M. G.; Bischoff, E. D.; Mere, L.; Mais,
D. E.; Bissonnette, R. P.; Heyman, R. A.; Nadzan, A. M.; Reichman, M.; Allegretto,
E. A. Chem. Biol. 1999, 6, 265.
34. Lian, J. B.; Stein, J. L.; Stein, G. S.; Montecino, M.; van Wijnen, A. J.; Javed, A.;
Gutierrez, S. Steroids 2001, 66, 159.
35. Paredes, R.; Arriagada, G.; Cruzat, F.; Olate, J.; van Wijnen, A.; Lian, J.; Stein, G.;
Stein, J.; Montecino, M. J. Steroid Biochem. Mol. Biol. 2004, 89–90, 269.
36. Milletti, F.; Storchi, L.; Sforna, G.; Cruciani, G. J. Chem. Inf. Model. 2007, 47, 2172.
37. Svensson, S.; Östberg, T.; Jacobsson, M.; Norström, C.; Stefansson, K.; Hallén, D.;
Johansson, I. C.; Zachrisson, K.; Ogg, D.; Jendeberg, L. EMBO J. 2003, 22, 4625.
38. Eelen, G.; Valle, N.; Sato, Y.; Rochel, N.; Verlinden, L.; De Clercq, P.; Moras, D.;
Bouillon, R.; Muñoz, A.; Verstuyf, A. Chem. Biol. 2008, 15, 1029.
39. Huet, T.; Maehr, H.; Lee, H. J.; Uskokovic, M. R.; Suh, N.; Moras, D.; Rochel, N.
Med. Chem. Commun. 2011, 2, 424.
40. Daylight Chemical Information Systems, Inc.: Laguna Niguel, CA 92677, USA.
41. Toyota, S. Chem. Rev. 2010, 110, 5398.
42. Juntunen, K.; Rochel, N.; Moras, D.; Vihko, P. Biochem. J. 1999, 344, 297.
43. Rochel, N.; Wurtz, J. M.; Mitschler, A.; Klaholz, B.; Moras, D. Mol. Cell 2000, 5, 173.
44. Gerber, P. R.; Müller, K. J. Comput. Aided Mol. Des. 1995, 9, 251.
25. Swann, S. L.; Bergh, J.; Farach-Carson, M. C.; Ocasio, C. A.; Koh, J. T. J. Am. Chem.
Soc. 2002, 124, 13795–13805.
26. Peräkylä, M.; Malinen, M.; Herzig, K. H.; Carlberg, C. Mol. Endocrinol. 2005, 19,
2060–2073.