Systematic SAR study of the side chain of nonsecosteroidal vitamin D 3 analogs

Article abstract of DOI:10.1016/j.bmc.2012.05.023

A series of nonsecosteroidal vitamin D₃ analogs with carboxylic acid were explored. Through our systematic SAR studies on the side chain moiety, compound 6b was identified as the optimal compound showing excellent vitamin D receptor (VDR) agoni

Full text of DOI:10.1016/j.bmc.2012.05.023

Bioorganic & Medicinal Chemistry 20 (2012) 4495–4506
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Systematic SAR study of the side chain of nonsecosteroidal vitamin D3 analogs
⇑
Hirotaka Kashiwagi , Yoshiyuki Ono, Masateru Ohta, Kenji Morikami, 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:
A series of nonsecosteroidal vitamin D3 analogs with carboxylic acid were explored. Through our system-
atic SAR studies on the side chain moiety, compound 6b was identified as the optimal compound showing
excellent vitamin D receptor (VDR) agonistic activity. Compound 6b had the diethyl group in the terminal
which was bound by (E)-olefin linker to the bisphenyl core. Calculating the volume of the side chain
showed that the diethyl group in 6b filled the hydrophobic region of VDR with the ideal packing coeffi-
cient based on the 55% rule, and that this resulted in the most potent in vitro activity.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 16 April 2012
Revised 11 May 2012
Accepted 11 May 2012
Available online 18 May 2012
Keywords:
Vitamin D3
VDR agonist
Nonsecosteroidal
Packing coefficient
55% Rule
1. Introduction
hydrophobic volume.9 The 25-hydroxy group in secosteroidal
vitamin D3 has a very important role in the vitamin D activation
Since the first cloning of steroid receptors in the mid-1980s, 48
nuclear receptors have been reported in human and classified into
seven subfamilies.1–5 Nuclear receptors are ligand-dependent tran-
scription factors which are located in cytosol. They need specific li-
gands, such as retinoids, thyroid hormone, or vitamin D3, to move
into the nucleus and bind to DNA, and then regulate the expression
of genes. Despite ligand variation, the structural components of
nuclear receptors are similar to each other with few exceptions.
They consist of two major parts which bind to a hinge region;
one is the N-terminus region which contains a DNA-binding
domain and an activation function 1 (AF-1) domain, the other
one is the C-terminus region which contains a ligand-binding do-
main (LBD) including an AF-2 domain. The DNA-binding domain
is the site at which hormone responsive element (HRE) binds.
The AF-1 domain is capable of ligand-independent transcription
activity but, in contrast, transcription in the AF-2 domain is acti-
vated ligand-dependently.1,6
Because many genes associated to various diseases are regu-
lated by nuclear receptors, the ligands binding to nuclear receptors
are very attractive drug targets.7 Extended research is now ongoing
on various nuclear receptors, such as androgen receptor, estrogen
receptor, glucocorticoid receptor, PPARs, RXR, and VDR, using clas-
sic steroidal compounds and unique nonsteroidal compounds.8 In
VDR, many groups reported new ligands and their various types
of activity, as well as the SAR around 25-hydroxyl moiety of the
secosteroidal skeleton, such as side chain linker length and
pathway and also in binding to VDR. Furthermore, modifying the
hydrophobic volume around 26 and 27 methyl groups in
1,25(OH)2D3 (1) is an effective way to modulate VDR agonistic
activity.9–11
In 1999, Boehm et al. reported the first nonsecosteroidal vitamin
D3, LG190178 (2).12 Since then, some groups reported derivatives of
LG190178; however, the information on the side chain part is very
limited and a detailed SAR is still not available.13–18
We previously reported compound 3 as a novel nonsecosteroi-
dal vitamin D3 agonist which has a VDR agonistic effect both
in vitro and in vivo.19 Compound 3 has very unique carboxylic acid
and hydroxy groups in the A-part (Fig. 1). From X-ray crystallo-
graphic analysis of 4/VDR complex, these functional groups made
a salt bridge interaction and formed a tight hydrogen bonding
network in VDR.
To the bisphenyl skeleton with gamma hydroxyl carboxylic acid
moiety in the A-part, in this study we attempted to introduce mul-
tiple-bond linkers ((E) and (Z)-olefins, and acetylene) to minimize
the flexibility and to fix the direction of the side chain, and then
modified the terminal hydrophobic volume of the side chain
systematically. As a result, we found potent VDR agonists and ob-
tained detailed SAR information of the side chain.
In VDR, the LBD, especially the region occupied by the side
chain of the ligand is composed of a lot of hydrophobic amino
acids, such as valine, leucine, isoleucine, and phenylalanine. The
other interesting feature of the VDR/ligand complex is that the
ligand is completely surrounded by VDR. In the area of host–guest
chemistry, Mecozzi and Rebek proposed that when a guest mole-
cule is completely surrounded by the host, the recognition of the
⇑
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.2012.05.023

4496
H. Kashiwagi et al. / Bioorg. Med. Chem. 20 (2012) 4495–4506
Side Chain
solution after adding an equivalent amount of 1 N NaOH to avoid
retro lactone formation.
OH
25
O
3. Results and discussion
H
OH
All synthesized compounds were evaluated by reporter gene as-
say (RGA) in MG-63 cells which contains vitamin D responsive ele-
ment (VDRE) sequence derived from mouse osteopontine
promoter as a vitamin D agonistic activity.19 VDRE RGA activity
is represented by a relative EC50 value based on 1,25(OH)2D3 being
assigned as 100% (a higher figure means stronger activity).
At first, we examined the impact of the linker between the
bisphenyl core and the terminal hydroxy group of the side chain
moiety, X–Y in Table 1. We considered that an ether linker, as in
LG190178, is flexible and the position of the hydroxy group, which
has to make hydrogen bond with VDR, on the side chain with ether
linker is not fixed and could be distributed to the widespread areas
of conformational space. To reduce the flexibility of the side chain
and to find a more potent VDR agonist by restricting the positions
occupied by the terminal hydroxy group on the side chain, we re-
placed the flexible ether linker with acetylene (5b) and olefins (6b
and 7b). These multiple-bond linkers were expected to rigidify the
conformation of the side chain and define the position of the termi-
nal hydrophobic moiety and hydroxy group in a restricted space.
Methylene linker (8b) was used as a standard of the flexible carbon
linker (Table 1). Compared to 3, which has an identical side chain
to LG190178, 5b with acetylene linker and 8b with methylene lin-
ker exhibited the same level of activity as 3 in VDRE RGA. In olefin
analogs, (E)-olefin 6b showed the activity five time higher than
that of 3 and being equal to 1,25(OH)2D3 in VDRE RGA, but the
activity of (Z)-olefin 7b was reduced dramatically when compared
to 3. It was speculated that the difference in activity of (E) and (Z)-
olefins is because the terminal diethyl group of (Z)-olefin could
collide with VDR and cause significant steric repulsion, while the
hydroxy group of the side chain of (E)-olefin could make the
hydrogen bond interactions with VDR properly so that the diethyl
group of (E)-olefin could occupy the hydrophobic pocket of VDR
appropriately.
H
A ring
O
3
HO
OH
1
HO
OH
LG190178
1 1α, 25-dihydroxyvitamin D3
2
Side Chain
OH
X
R2
OH
R1
X
Y
5: X-Y; acetylene
6: X-Y; (E)-olefin
7: X-Y; (Z)-olefin
8: X-Y; methylene
O
O
A part
(S)
(S)
a: R1 and R2 = Me
b: R1 and R2 = Et
c: R1 and R2 = n-Pr
d: R1 , R2 = c-Bu
e: R1 , R2 = c-Pen
f : R1 , R2 = c-Hex
HOOC
HOOC
OH
OH
3: X = O
4: X = CH2
Figure 1. Structures of secosteroids 1
oidal analog LG190178 (2) and
derivatives (3, 4, 5a–f, 6a–f, 7b, and 8b). Part labeled A of 3 and 4 (lower left)
shows the structural correspondence to the A ring of secosteroids (upper left).
a
, 25-dihydroxyvitamin D3 (1), nonsecoster-
series of nonsecosteroidal carboxylic acid
a
guest by the host is firstly governed by the volume of the guest,
and they then showed that the ideal volume occupancy of an inclu-
sion guest compound in hydrophobic and confined spaces of a host
receptor was 55 9%.20 In order to examine whether the concept of
host–guest chemistry could be applied to the VDR/ligand system or
not, we performed volume calculations similar to those done by
Mecozzi et al. to our compounds and VDR. After calculating the
packing coefficient (PC) between the side chain and the corre-
sponding hydrophobic pocket of VDR, we were interested to find
that the optimal PC for the hydrophobic side chain moiety of our
compounds and the SAR of the side chains of VDR ligand follow
the rule for host–guest recognition.
In order to estimate the difference in activity between 5b with
acetylene linker and 6b with (E)-olefin linker, the docking models
of 5b and 6b were constructed based on crystallographic structure
of compound 4 in complex with VDR (PDB code: 3AZ3) (Fig. 2).19
The compounds 5b and 6b were manually docked into the crystal
21
structure of VDR using software MOLOC
.
Compounds 5b and 6b
were optimized using the MAB force field with fixed VDR structure.
In the models, the carboxylic acid of A-part is formed a salt bridge
with Arg274 and hydrogen-bonded to the crystallographic water.
The hydroxy group of A-part makes hydrogen bond to the crystal
water and makes a hydrogen bonding network with Ser278,
Tyr143 and Arg274 via crystal water, in the same manner as com-
pound 4. Hydrophobic diethyl moieties of the side chain of 5b and
6b are superposed well to each other as shown in Figure 2; thus,
we could not explain the difference in activity between 5b and
6b from the position and shape of the hydrophobic moiety.
The difference in the activity of (E)-olefin and acetylene analogs
could be explained by the loss of rotational entropy in the bonds of
the side chain. Database searches of crystallographic structures of
small molecules by Cambridge structural database (CSD) indicated
that the bond rotation of CC triple bond is free (Fig. 3a).22 This re-
sult accords with the description that the barrier of the rotation of
CC triple bond is low.23 In contrast, the bond rotation along (E)-ole-
fin is very restricted (Fig. 3b), although the number of hits in a CSD
search for the query was three. Thus, no significant loss of bond
rotational entropy along (E)-olefin bonds occurs when (E)-olefin
linker analogs bind to VDR. But, in the case of acetylene linker
2. Chemistry
The synthesis of compounds 5a–f, 6a–f, 7b, and 8b is shown in
Scheme 1. Compound 9 was prepared by a previously reported
method.19 Sonogashira coupling with trimethylsilylacetylene in
the presence of palladium catalyst afforded acetylene 10 and the
consequent removal of trimethylsilyl group using tetrabutylam-
monium fluoride (TBAF) gave acetylene 11 in very good yield.
Alkylation of 11 with n-butyl lithium and various ketones gave cor-
responding alcohols 12a–f in moderate yields. These acetylene
alcohols were selectively reduced to (E)-olefins 13a–f by Red-AlÒ.
Acetylene 12b was converted to (Z)-olefin 14b and saturated
methylene 15b in the hydrogenation condition of Lindlar catalyst
and palladium hydroxide on carbon, respectively. All phenol inter-
mediate was alkylated by tosylate 16 in the presence of potassium
carbonate to afford lactones 17a–f, 18a–f, 19b, and 20b in good
yield. These lactones were hydrolyzed to gamma hydroxyl carbox-
ylic acids 5a–f, 6a–f, 7b, and 8b in good yield. All compounds
(5a–f, 6a–f, 7b, and 8b) were stored in a freezer as an ethanol

H. Kashiwagi et al. / Bioorg. Med. Chem. 20 (2012) 4495–4506
4497
R2
OH
R3
R1
R2
OH
R3
OTf
a
c
d
a: R2 and R3 = Me
b: R2 and R3 = Et
c: R2 and R3 = n-Pr
d: R2 , R3 = c-Bu
e: R2 , R3 = c-Pen
f : R2 , R3 = c-Hex
OH
OH
OH
OH
9
10: R1 = TMS
11: R1 = H
12a-f
13a-f
b
OH
OH
e
f
OH
OH
OH
OH
14b
12b
15b
TsO
R2
R2
OH
R3
OH
R3
X
X
16
Y
Y
R2
O
OH
R3
X
Y
O
g
h
O
O
OH
HOOC
OH
O
12a-f: X-Y; acetylene
13a-f: X-Y; (E)-olefin
14b: X-Y; (Z)-olefin
15b: X-Y; methylene
5a-f: X-Y; acetylene
6a-f: X-Y; (E)-olefin
7b: X-Y; (Z)-olefin
8b: X-Y; methylene
O
17a-f: X-Y; acetylene
18a-f: X-Y; (E)-olefin
19b: X-Y; (Z)-olefin
20b: X-Y; methylene
Scheme 1. Reagents and conditions: (a) TMS acetylene, CuI, Pd(PPh3)4, 110 °C, acetonitrile, 74%; (b) TBAF, room temp, THF, 97%; (c) n-BuLi, ketones, ꢀ78 °C, THF, 54–90%; (d)
Red-AlÒ, 0 °C, THF, 74–94%; (e) Lindlar catalyst, room temp, MeOH, 62%; (f) Pd(OH)2/C, room temp, MeOH, 67%; (g) K2CO3, 16, 100 °C, DMF, 54ꢀ90%; (h) 1 N KOH, room temp,
MeOH, 71ꢀ98%.
Table 1
VDRE reporter gene activity of various side chain analogs
Compound
XꢀY
Ratio of VDRE
reporter
activitya (%)
5b
6b
7b
C„C
(E)-CH@CH 105
(Z)-CH@CH 0.62
CH2CH2
21
OH
X
Y
8b
26
3
20
LG190178
1,25(OH)2D3
6.7
100
O
HOOC
OH
a
1,25(OH)2D3 ratio of EC50. 1,25(OH)2D3 was assigned as 100% (higher figure
means stronger activity).
analogs, the loss of rotational entropy along CC triple bond was
plain to see because the hydroxy group at the terminal hydropho-
bic moiety has to interact with two His residues of VDR making the
bond rotation of CC triple bond fixed when the acetylene linker
analog binds to VDR.
Figure 2. Superposition of 5b and 6b. Compound 6b, which has a (E)-olefin linker, is
depicted in cyan. Compound 5b, which has an acetylene linker, is depicted in
yellow. The side chains of the residue of VDR, which make hydrogen bond with 6b,
are depicted in white. Crystallographic waters, which make hydrogen bonds with
6b, are depicted as red spheres. Hydrogen bonds are depicted by red dotted line and
their distances are annotated in red characters.
The other possible explanation for the difference in activity be-
tween 5b and 6b is the hydrophobicity of the side chain. The CLogP

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H. Kashiwagi et al. / Bioorg. Med. Chem. 20 (2012) 4495–4506
Table 2
(a)
A
VDRE RGA activity of acetylene and (E)-olefin side chain analogs
1
3
35
Compound
R1
R2
Ratio of VDRE
reporter
2
30
25
20
15
10
5
activitya (%)
4
5a
5b
5c
5d
5e
5f
Me
Et
Me
Et
5.2
21
R2
OH
R1
n-Pr n-Pr 0.25
c-Bu
c-Pen
c-Hex
3.0
20
7.8
O
HOOC
OH
0
6a
6b
6c
6d
6e
6f
Me
Et
Me
Et
10
105
R2
OH
R1
n-Pr n-Pr 1.6
c-Bu
c-Pen
c-Hex
9.0
31
55
Torsion range (degree)
(b)
1
3
O
2
HOOC
OH
LG190178
1,25(OH)2D3
6.7
100
4
a
1,25(OH)2D3 ratio of EC50. 1,25(OH)2D3 was assigned as 100% (higher figure
means stronger activity).
compared to the activity of 5b, the cyclic alkyl analogs of the ter-
minal hydrophobic group (5d–f) exhibited the same activity in
5e with a cyclopentyl group, seven times reduction in activity in
5d with a cyclobutyl group, and 2.7 times reduction in 5f with a
cyclohexyl group. In the case of (E)-olefin analogs, diethyl group
(6b) showed the most potent activity (105%) and the smaller
dimethyl group (6a) exhibited ten times reduction in activity and
the larger di-n-propyl group (6c) exhibited more than 65 times
reduction, respectively when compared to 6b. In the case of cyclic
alkyl analogs of (E)-olefin, VDRE RGA activity rose as ring size
became larger, 9% for cyclobutyl (6d), 31% for cyclopentyl (6e)
and 55% for cyclohexyl (6f), respectively.
Torsion range (degree)
Figure 3. Histograms of the torsion distribution along acetylene and (E)-olefin.
Substructure queries for CSD search are depicted on the histogram. Torsion is
defined for atoms 1-2-3-4. (a) Histogram for torsion along acetylene. (b) Histogram
for torsion along (E)-olefin.
From the results of the optimization of hydrophobic moiety
using both acetylene and (E)-olefin linkers, (E)-olefin analogs
exhibited more potent in vitro activity than the corresponding
acetylene analogs. The reason for the stronger activity of (E)-olefin
analogs is thought to be a small loss of entropy of bond rotation in
the side chain and/or higher hydrophobicity as described above for
5b and 6b.
value for acetylene 5b is 6.65 and that for (E)-olefin 6b is 7.30.24
Hansch–Fujita hydrophobic
p constant of the acetylene group is
0.40 and of the olefin group is 0.82. These values indicated that
(E)-olefin analogs are more hydrophobic than acetylenes, and this
difference in hydrophobicity could affect the VDR activity.
Regarding the in vitro rat metabolic stability, acetylene (5b) and
(E)-olefin (6b) showed good stability. On the other hand, methy-
lene analog (8b) showed significantly poor stability, possibly due
to the benzylic oxidation. From both the activity and the metabolic
stability viewpoints, we selected acetylene (5b) and (E)-olefin (6b)
as linkers for further modification to investigate the SAR for the
side chain, especially for the terminal hydrophobic moiety.
We systematically modified the terminal hydrophobic moiety
of the side chain using acetylene and (E)-olefin linkers (Table 2).
The length of the terminal alkyl chain was changed from one to
three for branched alkyl analogs 5a–c and 6a–c. The ring size
was changed from four to six for cyclic alkyl analogs 5d–f and
6d–f. With an acetylene linker, the substitution of dimethyl (5a)
for diethyl group (5b) showed four times reduction in VDRE RGA
activity. The replacement of diethyl group (5b) with di-n-propyl
(5c) exhibited more than 80 times reduction in activity. When
The trend of the SARs for acetylene and (E)-olefin analogs was
the same for the acyclic alkyl series; diethyl group was the optimal
for both linkers. But, the SARs were slightly different for the cyclic
alkyl series; cyclopentyl was most active for acetylene linker and
cyclohexyl was most potent for (E)-olefin linker.
The docking model of 6b suggested, as shown in Figure 4, that
two ethyl groups of 6b are located in the hydrophobic pocket,
which is formed by Leu230, Val234, Ile268, Tyr401, Leu414,
Val418, and Phe422 of VDR. The SAR of the side chain indicated
that the size and shape of the hydrophobic moiety in the side chain
could have a strong influence on the VDR agonistic activity. To
evaluate the effect of the hydrophobic moiety in the side chain of
the ligand on VDR agonistic activity, we modeled the docking pose
for each compounds and calculated the volume of the side chain
and its packing coefficient (PC) as a ratio of the volume of VDR
pocket accommodating the side chain of the ligand.

H. Kashiwagi et al. / Bioorg. Med. Chem. 20 (2012) 4495–4506
4499
Figure 4. Hydrophobic pocket around the side chain of compound 6b. Amino acid residues of VDR within 6 Å from compound 6b are depicted by white sticks. Key hydrogen
bonding interactions are shown by red dotted lines and each distance is indicated in angstrom (Å).
Table 3 shows the calculated volume Vside chain and PC for com-
pounds 5a–f and 6a–f. (E)-Olefin analogs showed slightly larger
volume (1–2 Å3) than that of corresponding acetylene analogs.
There was a difference of 51 Å3 between Vside chain of the smallest
dimethyl group analogs (5a and 6a) and that of the largest di-n-
propyl group analogs (5c and 6c). The increasing volume order
was diMe < c-Bu < c-Pen < diEt < c-Hex < di-n-Pr, which was the
same tendency in both acetylenes and (E)-olefins.
Figure 6 shows the correlations between PC and VDRE RGA
activity. In acetylene analogs, cyclopentyl group (5e, PC = 52%)
and diethyl group (5b, PC = 55%) showed higher RGA activity than
the other four analogs. Meanwhile, in (E)-olefin analogs, diethyl
group in 6b showed optimal PC (56%) and either decreasing the
volume to have dimethyl group (6a, PC = 44%) or increasing the
volume to have di-n-propyl group (6c, PC = 68%) reduced the activ-
ity. Cyclopentyl group (6e) and cyclohexyl group (6f) both had
moderate RGA activity of 31% and 55%, respectively, and the PC val-
ues of these analogs were in the range of 56 3%. Almost the same
tendency was seen in acetylene analogs in spite of the maximum
VDRE reporter activity being less potent than in (E)-olefins.
Figure 5. Side chain volume (Vside chain, 118 Å3, cyan), Vvoid
(90 Å3, magenta)
space
and the corresponding LBD volume (VLBD, 208 Å3, magenta plus cyan) within the
defined rectangular box (blue) in the co-crystal structure of 4 and VDR (PDB 3AZ3).
PC of 4 was calculated as 57% by Vside chain/VLBD ꢁ 100 (%).
These results suggest that the lipophilic VDR region occupied by
the side chain of a ligand has an optimal PC value for exhibiting
high activity. It was thought that, when the PC value is lower than
optimal, the number of van der Waals interactions between the
side chain atoms and VDR becomes lower and the enthalpic gain
from various non-bonded interactions is reduced. On the other
hand, when the PC value is higher than the optimal, two reasons
could be considered. One of the reasons might be that a large num-
ber of van der Waals interactions are formed resulting in a large
enthalpic gain. However, at the same time, the dynamic mobility
of both protein and ligand becomes reduced, which leads to unfa-
vorable complexation entropy that compensates for the enthalpic
gain.25 So the optimal PC value is achieved by the most moderate
balance between enthalpic gain from hydrophobic interaction
and entropy loss from limited mobility. The other reason might
be a ligand with a large volume makes van der Waals collisions
The compounds 5a–f and 6a–f were manually docked into the
crystallographic structure of VDR and the structure of the ligand
was optimized in the same manner as the docking of 6b to VDR.
The volume occupied by the side chain of the compound
(Vside chain), the volume not occupied by either the ligand or VDR
(Vvoid space) in the VDR pocket, and the volume of the VDR pocket
(VLBD) were calculated by in-house programs (see Fig. 5 for the
graphical image of the values Vside chain, Vvoid space and VLBD and refer
to Section 5.3 for details of the calculations). VLBD was calculated as
208 Å3.
The packing coefficient (PC) values were calculated as follows:
PCð%Þ ¼ ðVside chain=VLBDÞ ꢁ 100
where VLBD = Vside chain + Vvoid space
.

4500
H. Kashiwagi et al. / Bioorg. Med. Chem. 20 (2012) 4495–4506
Table 3
with VDR and the collisions induce the conformational change of
VDR resulting in high energy state VDR.
Calculated volume results of the side chain moieties of 4, 5a–f, and 6a–f and
corresponding PCs
Mecozzi and Rebek showed that the ideal volume occupancy of
guest compounds included in hydrophobic confined spaces of host
capsular synthetic receptors was 55 9%.20 Diederich et al. applied
this 55% rule in the enzyme pocket and showed optimal packing
coefficiency of hydrophobic moiety of inhibitors in the apolar
pocket to be around 55%.25,26 These ratios are consistent with our
results, thus, our case is a second example that the 55% rule is val-
idated for protein–ligand recognition.
From these results, compound 6b was the ligand with an opti-
mum size of side chain moiety in VDR. When one is trying to fill
the vacant hydrophobic region of a protein by modifying the li-
gand, it might be difficult to design a molecule that will exhibit
optimal activity just by checking its shape and its fit to the receptor
via modeling. In such a case, optimizing the ligand size by consid-
ering PC with the protein could be a useful tool for analog design
and optimization.
Com- R1
pound
R2
Volume
PCb
a
Vside chain (%)
(Å3)
4
118
57
5a
5b
Me
Et
Me
Et
n-Pr
89
115
141
98
109
121
43
55
68
47
52
58
R2
OH
R1
5c
5d
5e
5f
n-Pr
c-Bu
c-Pen
c-Hex
O
HOOC
OH
6a
6b
Me
Et
Me
Et
n-Pr
c-Bu
c-Pen
c-Hex
91
117
142
99
110
122
44
56
68
48
53
59
R2
OH
R1
6c
6d
6e
6f
n-Pr
4. Conclusion
A series of nonsecosteroidal vitamin D3 analogs with carboxylic
acid were explored. Through our systematic SAR studies on the
side chain moiety, compound 6b was identified as the optimal
compound, showing excellent in vitro potency and metabolic sta-
bility. Compound 6b had the diethyl group connected by (E)-olefin
linker to the bisphenyl core. (E)-Olefin linker of 6b has a conforma-
tional rigidity and thus has the advantage that the entropy loss of
bond rotation is less than that of acetylene analog 5b when binding
to VDR. The diethyl group at the terminal of the side chain of 6b
has an ideal PC (56%) for the hydrophobic pocket of VDR. When fill-
ing a hydrophobic region of a protein by a ligand, the 55% rule
could be a useful tool for analog design and optimization.
O
HOOC
OH
a
Calculated volume of extracted side chain moiety from whole molecule (high-
lighted in blue in the structure).
PC was calculated as follows; (Vside chain/208 Å3) ꢁ 100 (%).
b
(a): acetylene
25
5b
5e
5. Experimental
20
5.1. Chemistry: general
15
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 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).
5f
10
5a
5d
5
5c
0
40.0
45.0
50.0
55.0
60.0
65.0
70.0
Packing coefficient (%)
(b): (E)-olefin
120
6b
100
80
60
40
20
0
5.1.1. 4-[1-Ethyl-1-(3-methyl-4-trimethylsilanylethynylphenyl)
propyl]-2-methylphenol (10)
To a solution of 9 (25 g, 60.0 mmol) in MeCN (300 mL) were
added triethylamine (25.1 mL, 180.1 mmol), (trimethylsilyl)acety-
lene (25.5 mL, 180.1 mmol), CuI (1.14 g, 6.00 mmol), and Pd(PPh3)4
(6.93 g, 6.00 mmol). The mixture was stirred at 110 °C for 18 h un-
der nitrogen atmosphere and concentrated. The mixture was
poured into H2O and products were extracted with AcOEt. The
extracts were dried over anhydrous MgSO4 and concentrated.
The obtained residue was chromatographed on silica gel (n-hex-
ane/AcOEt = 15:1) to afford 10 (16 g, 74%) as colorless oil. 1H
NMR (CDCl3) d: 0.24 (9H, s), 0.58 (6H, t, J = 7.3 Hz), 2.02 (4H, q,
J = 7.3 Hz), 2.17 (3H, s), 2.38 (3H, s), 4.58 (1H, br s), 6.64 (1H, d,
J = 9.0 Hz), 6.81–6.86 (2H, m), 6.92 (1H, dd, J = 8.1, 1.7 Hz), 6.99
6f
6e
6a
6d
6c
40.0
45.0
50.0
55.0
60.0
65.0
70.0
Packing coefficient (%)
Figure 6. The correlation between activity and PC; (a) 5a–f (acetylene linker series)
and (b) 6a–f ((E)-olefin linker series).

H. Kashiwagi et al. / Bioorg. Med. Chem. 20 (2012) 4495–4506
4501
(1H, s), 7.30 (1H, d, J = 8.0 Hz). 13C NMR (CDCl3) d: 151.5, 149.9,
5.1.7. 4-{1-Ethyl-1-[4-(1-hydroxycyclobutylethynyl)-3-methyl
140.4, 139.7, 131.3, 130.6, 129.0, 126.5, 125.4, 122.8, 119.6,
114.0, 104.4, 97.4, 49.0, 28.9, 20.9, 16.0, 8.3, 0.0. MS (ESI negative):
363 (MꢀH)ꢀ.
phenyl]-propyl}-2-methylphenol (12d)
The yield was 69%. Colorless oil. 1H NMR (CDCl3) d: 0.59 (6H, t,
J = 7.3 Hz), 1.82–1.90 (2H, m), 2.03 (4H, q, J = 7.3 Hz), 2.18 (3H, s),
2.33–2.37 (2H, m), 2.38 (3H, s), 2.49–2.55 (2H, m), 4.69 (1H, br
s), 6.65 (1H, d, J = 8.0 Hz), 6.82–6.86 (2H, m), 6.94 (1H, dd, J = 8.1,
1.9 Hz), 7.01 (1H, s), 7.28 (1H, d, J = 8.2 Hz). 13C NMR (CDCl3) d:
151.7, 149.7, 140.0, 139.2, 131.0, 130.5, 129.0, 126.3, 125.4,
123.0, 119.1, 114.0, 95.8, 82.5, 68.4, 48.9, 38.8, 28.9, 20.8, 16.1,
12.9, 8.3. MS (ESI negative): 361 (MꢀH)ꢀ.
5.1.2. 4-[1-Ethyl-1-(4-ethynyl-3-methylphenyl)-propyl]-2-
methyl phenol (11)
To a solution of 10 (18.02 g, 49.4 mmol) in THF (247 mL) was
added tetrabutylammonium fluoride (1 M in THF, 74.1 mL,
74.1 mmol). The mixture was stirred for 30 min and concentrated.
The residue was diluted with AcOEt and the mixture was washed
with H2O and brine, dried over anhydrous MgSO4, and concen-
trated. The obtained residue was chromatographed on silica gel
(n-hexane/AcOEt = 10:1) to afford 11 (14.08 g, 97%) as pale yellow
oil. 1H NMR (CDCl3) d: 0.59 (6H, t, J = 7.3 Hz), 2.03 (4H, q,
J = 7.4 Hz), 2.19 (3H, s), 2.40 (3H, s), 3.22 (1H, s), 4.53 (1H, br s),
6.65 (1H, d, J = 8.0 Hz), 6.83–6.87 (2H, m), 6.94 (1H, dd, J = 8.0,
1.8 Hz), 7.01 (1H, s), 7.33 (1H, d, J = 8.0 Hz). 13C NMR (CDCl3) d:
151.5, 150.1, 140.4, 139.8, 131.8, 130.6, 129.1, 126.6, 125.5,
122.8, 118.7, 114.1, 82.9, 80.3, 49.1, 29.0, 20.9, 16.1, 8.3. MS (ESI
negative): 291 (MꢀH)ꢀ.
5.1.8. 4-{1-Ethyl-1-[4-(1-hydroxycyclopentylethynyl)-3-methyl
phenyl]-propyl}-2-methylphenol (12e)
The yield was 70%. Colorless oil. 1H NMR (CDCl3) d: 0.59 (6H, t,
J = 7.3 Hz), 1.70–1.88 (4H, m), 1.99–2.06 (8H, m), 2.18 (3H, s), 2.36
(3H, s), 4.69 (1H, br s), 6.65 (1H, d, J = 8.8 Hz), 6.82–6.86 (2H, m),
6.93 (1H, dd, J = 8.0, 1.6 Hz), 7.00 (1H, d, J = 1.4 Hz), 7.26 (1H, d,
J = 8.0 Hz). 13C NMR (CDCl3) d: 152.0, 149.4, 139.3, 138.8, 130.7,
130.2, 128.8, 126.0, 125.2, 123.0, 119.2, 113.8, 96.4, 82.5, 74.6,
48.7, 42.3, 28.7, 23.6, 20.6, 16.1, 8.1. MS (ESI negative): 375
(MꢀH)ꢀ.
5.1.3. General procedure for alkylation of acetylene group of 11
To a solution of 11 (4.28 g, 14.69 mmol) in THF (92 mL) was
added n-butyllithium (2.5 M in hexane, 14.7 mL, 36.73 mmol) at
ꢀ78 °C and the mixture was stirred for 30 min. To the mixture
was added ketone (44.07 mmol) at ꢀ78 °C and the mixture was
stirred for 3 h. Then the mixture was poured into satd NH4Cl aq
solution and products were extracted with AcOEt. The extracts
were dried over anhydrous Na2SO4 and concentrated. The obtained
residue was chromatographed on silica gel (n-hexane/AcOEt = 6:1)
to afford alcohol.
5.1.9. 4-{1-Ethyl-1-[4-(1-hydroxycyclohexylethynyl)-3-methyl
phenyl]-propyl}-2-methylphenol (12f)
The yield was 90%. Colorless oil. 1H NMR (CDCl3) d: 0.59 (6H, t,
J = 7.3 Hz), 1.57–1.78 (8H, m), 1.99–2.06 (6H, m), 2.18 (3H, s), 2.38
(3H, s), 4.71 (1H, br s), 6.65 (1H, d, J = 8.0 Hz), 6.82–6.86 (2H, m),
6.94 (1H, dd, J = 8.1, 1.9 Hz), 7.00 (1H, d, J = 1.8 Hz), 7.28 (1H, d,
J = 8.2 Hz). 13C NMR (CDCl3) d: 151.8, 149.6, 139.8, 139.0, 131.0,
130.4, 129.0, 126.3, 125.3, 123.0, 119.2, 114.0, 95.8, 83.5, 69.5,
48.9, 40.0, 28.9, 25.1, 23.5, 21.0, 16.2, 8.3. MS (ESI negative): 389
(MꢀH)ꢀ.
5.1.4. 4-{1-Ethyl-1-[4-(3-hydroxy-3-methyI-1-butynyl)-3-
methyl phenyl]-propyl}-2-methylphenol (12a)
5.1.10. General procedure for reduction of acetylene group of
12a–f
The yield was 56%. Colorless oil. 1H NMR (CDCl3) d: 0.58 (6H, t,
J = 7.2 Hz), 1.62 (6H, s), 2.02 (4H, q, J = 7.3 Hz), 2.17 (3H, s), 2.35
(3H, s), 5.79 (1H, br s), 6.65 (1H, d, J = 8.2 Hz), 6.82–6.85 (2H, m),
6.93 (1H, dd, J = 8.0, 1.6 Hz), 7.00 (1H, s), 7.25 (1H, d, J = 8.0 Hz).
13C NMR (CDCl3) d: 151.7, 149.7, 140.1, 139.2, 131.0, 130.6,
129.1, 126.4, 125.5, 122.9, 119.1, 114.1, 97.1, 81.3, 65.9, 49.0,
31.6, 29.0, 20.9, 16.2, 8.3. MS (ESI negative): 349 (MꢀH)ꢀ.
To a solution of acetylene (0.327 mmol) in THF (3.0 mL) was
added Red-AlÒ (65% in toluene, 0.294 mL, 0.980 mmol) at 0 °C
and the mixture was stirred for 2 h. The mixture was quenched
with satd NaHCO3 aq solution, diluted with AcOEt, and Celite
was added. After stirring overnight, the mixture was filtered
through Celite pad and concentrated. The obtained residue was
purified by preparative TLC (n-hexane/AcOEt = 1:1) to afford olefin.
5.1.5. 4-{1-Ethyl-1-[4-(3-ethyl-3-hydroxy-1-pentynyl)-3-methyl
phenyl]-propyl}-2-methylphenol (12b)
5.1.11. 4-{1-Ethyl-1-[4-((E)-3-hydroxy-3-methyl-1-butenyl)-3-
methylphenyl]-propyl}-2-methylphenol (13a)
The yield was 62%. Colorless oil. 1H NMR (CDCl3) d: 0.59 (6H, t,
J = 7.2 Hz), 1.11 (6H, t, J = 7.3 Hz), 1.72–1.78 (4H, m), 2.02 (4H, q,
J = 7.2 Hz), 2.18 (3H, s), 2.37 (3H, s), 4.83 (1H, br s), 6.65 (1H, d,
J = 8.2 Hz), 6.82–6.86 (2H, m), 6.93 (1H, dd, J = 8.1, 1.9 Hz), 7.00
(1H, s), 7.27 (1H, d, J = 8.2 Hz). 13C NMR (CDCl3) d: 151.4, 149.6,
140.4, 139.1, 131.3, 130.6, 129.1, 126.6, 125.4, 122.6, 119.4,
114.0, 94.9, 83.5, 72.8, 49.0, 34.6, 29.0, 21.0, 16.0, 8.7, 8.3. MS
(ESI negative): 377 (MꢀH)ꢀ.
The yield was 82%. Colorless oil. 1H NMR (CDCl3) d: 0.60 (6H, t,
J = 7.2 Hz), 1.42 (6H, s), 2.03 (4H, q, J = 7.3 Hz), 2.18 (3H, s), 2.29
(3H, s), 5.25 (1H, br s), 6.22 (1H, d, J = 15.8 Hz), 6.65 (1H, d,
J = 8.4 Hz), 6.77 (1H, d, J = 16.0 Hz), 6.84 (1H, dd, J = 8.1, 2.2 Hz),
6.89 (1H, d, J = 2.0 Hz), 6.93–6.97 (2H, m), 7.31 (1H, d, J = 8.4 Hz).
13C NMR (CDCl3) d: 151.5, 148.3, 140.6, 137.9, 134.5, 132.6,
130.6, 129.9, 126.6, 126.0, 124.6, 123.9, 122.7, 114.0, 71.4, 48.8,
29.9, 29.1, 20.1, 16.1, 8.4. MS (ESI negative): 351 (MꢀH)ꢀ.
5.1.6. 4-{1-Ethyl-1-[4-(3-hydroxy-3-propyl-1-hexynyl)-3-
methyl phenyl]-propyl}-2-methylphenol (12c)
5.1.12. 4-{1-Ethyl-1-[4-((E)-3-ethyl-3-hydroxy-1-pentenyl)-3-
methylphenyl]-propyl}-2-methylphenol (13b)
The yield was 54%. Colorless oil. 1H NMR (CDCl3) d: 0.59 (6H, t,
J = 7.3 Hz), 0.98 (6H, t, J = 7.2 Hz), 1.59–1.65 (4H, m), 1.68–1.73
(4H, m), 2.02 (4H, q, J = 7.3 Hz), 2.18 (3H, s), 2.36 (3H, s), 4.57
(1H, br s), 6.65 (1H, d, J = 8.6 Hz), 6.82–6.86 (2H, m), 6.93 (1H,
dd, J = 8.1, 1.9 Hz), 7.00 (1H, d, J = 1.8 Hz), 7.26 (1H, d, J = 8.0 Hz).
13C NMR (CDCl3) d: 151.8, 149.6, 140.0, 139.1, 131.2, 130.6,
129.1, 126.4, 125.4, 123.0, 119.3, 114.0, 95.4, 83.4, 72.0, 49.0,
44.5, 29.0, 21.0, 17.7, 16.1, 14.3, 8.3. MS (ESI negative): 405
(MꢀH)ꢀ.
The yield was 85%. Colorless oil. 1H NMR (CDCl3) d: 0.61 (6H, t,
J = 7.3 Hz), 0.92 (6H, t, J = 7.4 Hz), 1.64 (4H, q, J = 7.5 Hz), 2.04 (4H,
q, J = 7.4 Hz), 2.19 (3H, s), 2.30 (3H, s), 4.73 (1H, br s), 6.01 (1H, d,
J = 16.0 Hz), 6.65 (1H, d, J = 8.2 Hz), 6.75 (1H, d, J = 16.0 Hz), 6.87
(1H, dd, J = 8.4, 2.3 Hz), 6.91 (1H, d, J = 2.0 Hz), 6.93–6.97 (2H, m),
7.30 (1H, d, J = 8.8 Hz). 13C NMR (CDCl3) d: 151.4, 148.0, 140.7,
135.8, 134.4, 133.2, 130.6, 129.9, 126.7, 125.9, 124.7, 122.5,
114.0, 76.1, 48.8, 33.3, 29.1, 20.2, 16.1, 8.4, 7.9. MS (ESI negative):
379 (MꢀH)ꢀ.

4502
H. Kashiwagi et al. / Bioorg. Med. Chem. 20 (2012) 4495–4506
5.1.13. 4-{1-Ethyl-1-[4-((E)-3-hydroxy-3-propyl-1-hexenyl)-3-
methylphenyl]-propyl}-2-methylphenol (13c)
5.1.18. 4-{1-Ethyl-1-[4-(3-ethyl-3-hydroxypentyl)-3-methyl
phenyl]-propyl}-2-methylphenol (15b)
The yield was 89%. Colorless oil. 1H NMR (CDCl3) d: 0.61 (6H,
t, J = 7.3 Hz), 0.92 (6H, t, J = 7.3 Hz), 1.33–1.43 (4H, m), 1.55–1.62
(4H, m), 2.04 (4H, q, J = 7.2 Hz), 2.19 (3H, s), 2.30 (3H, s), 5.04
(1H, br s), 6.05 (1H, d, J = 15.8 Hz), 6.65 (1H, d, J = 8.2 Hz), 6.73
(1H, d, J = 15.8 Hz), 6.86 (1H, dd, J = 8.4, 2.2 Hz), 6.91 (1H, d,
J = 2.0 Hz), 6.94–6.97 (2H, m), 7.29 (1H, d, J = 8.6 Hz). 13C
NMR (CDCl3) d: 151.4, 148.0, 140.6, 136.3, 134.4, 133.1, 130.6,
129.9, 126.6, 125.9, 125.2, 124.7, 122.6, 114.0, 75.9, 48.8, 43.7,
29.1, 20.2, 16.9, 16.1, 14.5, 8.4. MS (ESI negative): 407
(MꢀH)ꢀ.
To a solution of 12b (33.0 mg, 0.087 mmol) in MeOH (2 mL) was
added 10% palladium hydroxide (20 mg), the mixture was stirred
at room temperature under hydrogen overnight. The mixture was
filtered through Celite pad and concentrated. The obtained residue
was purified by preparative TLC (n-hexane/AcOEt = 2:1) to afford
15b (22.4 mg, 67%) as colorless oil. 1H NMR (CDCl3) d: 0.59 (6H,
t, J = 7.3 Hz), 0.91 (6H, t, J = 7.5 Hz), 1.56 (4H, q, J = 7.5 Hz), 1.64–
1.69 (2H, m), 2.03 (4H, q, J = 7.4 Hz), 2.19 (3H, s), 2.25 (3H, s),
2.54–2.59 (2H, m), 4.96 (1H, br s), 6.64 (1H, d, J = 8.4 Hz), 6.85
(1H, dd, J = 8.4, 2.2 Hz), 6.90–6.93 (3H, m), 7.00 (1H, d, J = 8.6 Hz).
13C NMR (CDCl3) d: 151.4, 146.3, 140.8, 137.4, 134.6, 130.6,
129.8, 127.7, 126.7, 125.7, 122.5, 114.0, 74.9, 48.7, 38.8, 30.9,
29.2, 26.7, 19.6, 16.1, 8.4, 7.8. MS (ESI positive): 405 (M+Na)+.
5.1.14. 4-(1-Ethyl-1-{4-[(E)-2-(1-hydroxycyclobutyl)-vinyl]-3-
methylphenyl}-propyl)-2-methylphenol (13d)
The yield was 91%. Colorless oil. 1H NMR (CDCl3) d: 0.60 (6H, t,
J = 7.2 Hz), 1.61–1.70 (1H, m), 1.79–1.89 (1H, m), 1.96–2.07
(2H, m), 2.04 (4H, q, J = 7.3 Hz), 2.18 (3H, s), 2.23–2.30 (2H, m),
2.30 (3H, s), 5.63 (1H, br s), 6.34 (1H, d, J = 15.8 Hz), 6.65 (1H,
d, J = 8.4 Hz), 6.82 (1H, d, J = 15.8 Hz), 6.84 (1H, dd, J = 8.1,
2.1 Hz), 6.89 (1H, d, J = 1.8 Hz), 6.94–6.98 (2H, m), 7.35 (1H, d,
J = 8.0 Hz). 13C NMR (CDCl3) d: 151.6, 148.4, 140.3, 134.5, 134.2,
132.5, 130.5, 129.9, 126.5, 125.9, 124.7, 124.4, 122.8, 114.0,
75.4, 48.8, 36.3, 29.1, 20.1, 16.1, 12.2, 8.4. MS (ESI negative):
363 (MꢀH)ꢀ.
5.1.19. General procedure for alkylation of phenol group of12a–
f, 13a–f, 14b, and 15b
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, toluene-4-sulfonic acid (S)-5-oxo-tetrahy-
dro-furan-2-ylmethyl ester (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 extracts were washed with H2O and brine, dried over
anhydrous Na2SO4, and concentrated. The obtained residue was
chromatographed on silica gel (n-hexane/AcOEt = 5:1–3:1) to af-
ford lactone.
5.1.15. 4-(1-Ethyl-1-{4-[(E)-2-(1-hydroxycyclopentyl)-vinyl]-3-
methylphenyl}-propyl)-2-methylphenol (13e)
The yield was 74%. Colorless oil. 1H NMR (CDCl3) d: 0.60 (6H, t,
J = 7.1 Hz), 1.70–1.83 (6H, m), 1.87–1.96 (2H, m), 2.03 (4H, q,
J = 7.2 Hz), 2.17 (3H, s), 2.28 (3H, s), 6.12 (1H, br s), 6.25 (1H, d,
J = 15.8 Hz), 6.65 (1H, d, J = 8.2 Hz), 6.80–6.88 (3H, m), 6.93–6.97
(2H, m), 7.31 (1H, d, J = 8.0 Hz). 13C NMR (CDCl3) d: 151.7, 148.2,
140.2, 136.1, 134.3, 132.7, 130.5, 129.9, 126.4, 125.9, 124.5,
124.2, 122.9, 113.9, 75.9, 48.7, 40.6, 29.1, 23.6, 20.1, 16.1, 8.4. MS
(ESI negative): 377 (MꢀH)ꢀ.
5.1.20. (S)-5-(4-{1-Ethyl-1-[4-(3-hydroxy-3-methyl-1-butynyl)-
3-methylphenyl]-propyl}-2-methylphenoxymethyl)-dihydro-
furan-2-one (17a)
The yield was 75%. Colorless oil. ½a D25
ꢂ
+17.7 (c 1.17, CHCl3). 1H
NMR (CDCl3) d: 0.58 (6H, t, J = 7.3 Hz), 1.62 (6H, s), 2.03 (4H, q,
J = 7.3 Hz), 2.14 (3H, s), 2.29–2.31 (1H, m), 2.36 (3H, s), 2.42–2.45
(1H, m), 2.52–2.61 (1H, m), 2.72–2.81 (1H, m), 4.06 (1H, dd,
J = 10.4, 3.3 Hz), 4.16 (1H, dd, J = 10.3, 3.2 Hz), 4.87–4.89 (1H, m),
6.66 (1H, d, J = 8.6 Hz), 6.86 (1H, d, J = 2.0 Hz), 6.91–6.94 (2H, m),
6.98 (1H, s), 7.26 (1H, d, J = 7.2 Hz). 13C NMR (CDCl3) d: 177.2,
154.0, 149.4, 140.9, 139.2, 131.0, 130.7, 129.0, 126.1, 125.6,
125.4, 119.2, 109.8, 97.3, 81.1, 77.8, 69.3, 65.7, 49.0, 31.6, 28.9,
28.3, 24.0, 20.9, 16.6, 8.3. MS (ESI positive): 471 (M+Na)+.
5.1.16. 4-(1-Ethyl-1-{4-[(E)-2-(1-hydroxycyclohexyl)-vinyl]-3-
methylphenyl}-propyl)-2-methylphenol (13f)
The yield was 94%. Colorless oil. 1H NMR (CDCl3) d: 0.60 (6H, t,
J = 7.2 Hz), 1.50–1.75 (10H, m), 2.03 (4H, q, J = 7.0 Hz), 2.17 (3H, s),
2.28 (3H, s), 5.65 (1H, br s), 6.20 (1H, d, J = 16.0 Hz), 6.65 (1H, d,
J = 8.2 Hz), 6.80–6.84 (2H, m), 6.88 (1H, d, J = 2.0 Hz), 6.93–6.97
(2H, m), 7.31 (1H, d, J = 7.8 Hz). 13C NMR (CDCl3) d: 151.6, 148.3,
140.4, 137.5, 134.5, 132.8, 130.5, 129.9, 126.5, 125.9, 124.8,
124.6, 122.8, 114.0, 72.3, 48.8, 38.1, 29.1, 25.4, 22.1, 20.1, 16.1,
8.4. MS (ESI negative): 391 (MꢀH)ꢀ.
5.1.21. (S)-5-(4-{1-Ethyl-1-[4-(3-ethyl-3-hydroxy-1-pentynyl)-3-
methylphenyl]-propyl}-2-methylphenoxymethyl)-
dihydrofuran-2-one (17b)
The yield was 73%. Colorless oil. ½a D25
ꢂ
+13.6 (c 1.10, CHCl3). 1H
NMR (CDCl3) d: 0.59 (6H, t, J = 7.3 Hz), 1.11 (6H, t, J = 7.4 Hz),
1.70–1.83 (4H, m), 2.03 (4H, q, J = 7.2 Hz), 2.14 (3H, s), 2.27–2.35
(1H, m), 2.37 (3H, s), 2.40–2.48 (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), 4.86–4.92 (1H, m), 6.66 (1H, d, J = 8.6 Hz), 6.87 (1H, d,
J = 1.8 Hz), 6.92–6.94 (2H, m), 6.99 (1H, s), 7.27 (1H, d, J = 8.2 Hz).
13C NMR (CDCl3) d: 177.2, 154.0, 149.4, 140.9, 139.2, 131.3,
130.7, 129.1, 126.1, 125.7, 125.4, 119.5, 109.9, 95.0, 83.4, 77.8,
72.7, 69.3, 49.0, 34.6, 28.9, 28.3, 24.0, 21.0, 16.6, 8.7, 8.3. MS (ESI
positive): 499 (M+Na)+.
5.1.17. 4-{1-Ethyl-1-[4-((Z)-3-ethyl-3-hydroxy-1-pentenyl)-3-
methylphenyl]-propyl}-2-methylphenol (14b)
To a solution of 12b (100 mg, 0.264 mmol) in MeOH (4 mL) was
added Lindlar catalyst (5% palladium on calcium carbonate; poi-
soned with lead, 5 mg), the mixture was stirred at room tempera-
ture under hydrogen overnight. Then the mixture was filtered
through Celite pad and concentrated. The obtained residue was
purified by preparative TLC (n-hexane/AcOEt = 2:1) to afford 14b
(62 mg, 62%) as a white powder. 1H NMR (CDCl3) d: 0.58 (6H, t,
J = 7.2 Hz), 0.92 (6H, t, J = 7.4 Hz), 1.54 (4H, q, J = 7.4 Hz), 2.02
(4H, q, J = 7.2 Hz), 2.19 (3H, s), 2.22 (3H, s), 4.70 (1H, br s), 5.55
(1H, d, J = 12.7 Hz), 6.49 (1H, d, J = 12.5 Hz), 6.63 (1H, d,
J = 8.4 Hz), 6.84 (1H, dd, J = 8.2, 2.2 Hz), 6.90 (1H, d, J = 1.8 Hz),
6.94–6.97 (2H, m), 7.11 (1H, d, J = 8.2 Hz). 13C NMR (CDCl3) d:
151.4, 148.4, 140.7, 136.7, 135.5, 133.5, 130.5, 129.6, 127.8,
127.6, 126.7, 125.3, 122.5, 114.1, 78.0, 48.8, 34.2, 29.1, 20.6, 16.1,
8.4, 8.3. MS (ESI positive): 403 (M+Na)+.
5.1.22. (S)-5-(4-{1-Ethyl-1-[4-(3-hydroxy-3-propyl-1-hexynyl)-
3-methylphenyl]-propyl}-2-methylphenoxymethyl)-dihydro-
furan-2-one (17c)
The yield was 78%. Colorless oil. ½a D25
ꢂ
+12.1 (c 0.73, CHCl3). 1H
NMR (CDCl3) d: 0.58 (6H, t, J = 7.3 Hz), 0.98 (6H, t, J = 7.1 Hz),
1.55–1.60 (4H, m), 1.68–1.73 (4H, m), 2.03 (4H, q, J = 7.3 Hz),
2.14 (3H, s), 2.27–2.35 (1H, m), 2.36 (3H, s), 2.39–2.49 (1H, m),
2.53–2.61 (1H, m), 2.73–2.82 (1H, m), 4.06 (1H, dd, J = 10.3,

H. Kashiwagi et al. / Bioorg. Med. Chem. 20 (2012) 4495–4506
4503
3.4 Hz), 4.17 (1H, dd, J = 10.2, 3.3 Hz), 4.86–4.92 (1H, m), 6.66 (1H,
d, J = 8.4 Hz), 6.87 (1H, d, J = 2.0 Hz), 6.90–6.94 (2H, m), 6.98 (1H, s),
7.26 (1H, d, J = 8.0 Hz). 13C NMR (CDCl3) d: 177.2, 154.0, 149.4,
141.0, 139.2, 131.3, 130.7, 129.1, 126.1, 125.7, 125.4, 119.5,
109.8, 95.6, 83.3, 77.9, 71.8, 69.3, 49.1, 44.5, 29.0, 28.3, 24.1,
21.1, 17.7, 16.6, 14.3, 8.3. MS (ESI positive): 527 (M+Na)+.
5.1.27. (S)-5-(4-{1-Ethyl-1-[4-((E)-3-ethyl-3-hydroxy-1-
pentenyl)-3-methylphenyl]-propyl}-2-methylphenoxymethyl)-
dihydrofuran-2-one (18b)
The yield was 90%. Colorless oil. ½a D25
ꢂ
+11.9 (c 0.24, CHCl3). 1H
NMR (CDCl3) d: 0.60 (6H, t, J = 7.3 Hz), 0.92 (6H, t, J = 7.4 Hz),
1.60–1.67 (4H, m), 2.05 (4H, q, J = 7.3 Hz), 2.15 (3H, s), 2.28–2.38
(1H, m), 2.31 (3H, s), 2.39–2.49 (1H, m), 2.52–2.61 (1H, m), 2.73–
2.82 (1H, m), 4.06 (1H, dd, J = 10.3, 3.4 Hz), 4.17 (1H, dd, J = 10.3,
3.4 Hz), 4.86–4.91 (1H, m), 6.01 (1H, d, J = 16.0 Hz), 6.66 (1H, d,
J = 8.4 Hz), 6.74 (1H, d, J = 16.0 Hz), 6.93–6.97 (4H, m), 7.30 (1H,
d, J = 8.8 Hz). 13C NMR (CDCl3) d: 177.2, 153.9, 147.9, 141.3,
135.9, 134.4, 133.3, 130.7, 129.9, 126.2, 125.8, 125.6, 124.8,
109.9, 77.9, 76.0, 69.3, 48.8, 33.3, 29.1, 28.3, 24.1, 20.2, 16.6, 8.4,
7.9. MS (ESI positive): 496 (M+NH4)+.
5.1.23. (S)-5-(4-{1-Ethyl-1-[4-(1-hydroxycyclobutylethynyl)-3-
methylphenyl]-propyl}-2-methylphenoxymethyl)-dihydro-
furan-2-one (17d)
The yield was 83%. Colorless oil. ½a D25
ꢂ
+18.4 (c 1.43, CHCl3). 1H
NMR (CDCl3) d: 0.59 (6H, t, J = 7.3 Hz), 1.82–1.88 (2H, m), 2.04
(4H, q, J = 7.3 Hz), 2.14 (3H, s), 2.30–2.35 (2H, m), 2.38 (3H, s),
2.41–2.61 (5H, m), 2.73–2.81 (1H, m), 4.06 (1H, dd, J = 10.4,
3.5 Hz), 4.17 (1H, dd, J = 10.7, 3.0 Hz), 4.86–4.91 (1H, m), 6.66
(1H, d, J = 8.4 Hz), 6.87 (1H, d, J = 1.8 Hz), 6.93 (2H, dd, J = 8.2,
1.8 Hz), 7.00 (1H, s), 7.28 (1H, d, J = 8.2 Hz). 13C NMR (CDCl3) d:
177.2, 154.0, 149.5, 140.9, 139.3, 131.1, 130.7, 129.0, 126.1,
125.7, 125.4, 119.2, 109.8, 96.0, 82.4, 77.9, 69.3, 68.4, 49.0, 38.8,
28.9, 28.3, 24.0, 20.9, 16.6, 12.9, 8.3. MS (ESI positive): 483
(M+Na)+.
5.1.28. (S)-5-(4-{1-Ethyl-1-[4-((E)-3-hydroxy-3-propyl-1-
hexenyl)-3-methylphenyl]-propyl}-2-methylphenoxymethyl)-
dihydrofuran-2-one (18c)
The yield was 72%. Colorless oil. ½a D25
ꢂ
+12.5 (c 1.19, CHCl3). 1H
NMR (CDCl3) d: 0.60 (6H, t, J = 7.3 Hz), 0.92 (6H, t, J = 7.3 Hz),
1.33–1.43 (4H, m), 1.55–1.61 (4H, m), 2.04 (4H, q, J = 7.2 Hz),
2.15 (3H, s), 2.27–2.37 (1H, m), 2.30 (3H, s), 2.39–2.48 (1H, m),
2.52–2.60 (1H, m), 2.73–2.82 (1H, m), 4.06 (1H, dd, J = 10.4,
3.3 Hz), 4.16 (1H, dd, J = 10.4, 3.3 Hz), 4.85–4.91 (1H, m), 6.05
(1H, d, J = 15.8 Hz), 6.66 (1H, d, J = 8.4 Hz), 6.73 (1H, d,
J = 16.0 Hz), 6.91–6.98 (4H, m), 7.29 (1H, d, J = 8.8 Hz). 13C NMR
(CDCl3) d: 177.2, 153.9, 147.8, 141.2, 136.5, 134.4, 133.2, 130.7,
129.8, 126.2, 125.8, 125.6, 125.1, 124.7, 109.8, 77.9, 75.7, 69.3,
48.8, 43.7, 29.1, 28.3, 24.1, 20.2, 16.9, 16.6, 14.5, 8.4. MS (ESI posi-
tive): 529 (M+Na)+.
5.1.24. (S)-5-(4-{1-Ethyl-1-[4-(1-hydroxycyclopentylethynyl)-3-
methylphenyl]-propyl}-2-methylphenoxymethyl)-
dihydrofuran-2-one (17e)
The yield was 58%. Colorless oil. ½a D25
ꢂ
+13.7 (c 0.83, CHCl3). 1H
NMR (CDCl3) d: 0.58 (6H, t, J = 7.3 Hz), 1.75–1.90 (4H, m), 1.99–
2.07 (8H, m), 2.14 (3H, s), 2.27–2.34 (1H, m), 2.36 (3H, s), 2.41–
2.48 (1H, m), 2.52–2.61 (1H, m), 2.72–2.81 (1H, m), 4.06 (1H, dd,
J = 10.4, 3.5 Hz), 4.16 (1H, dd, J = 10.4, 3.3 Hz), 4.85–4.91 (1H, m),
6.66 (1H, d, J = 8.6 Hz), 6.86 (1H, d, J = 2.0 Hz), 6.90–6.94 (2H, m),
6.98 (1H, s), 7.26 (1H, d, J = 8.0 Hz). 13C NMR (CDCl3) d: 177.2,
154.0, 149.4, 141.0, 139.2, 131.0, 130.7, 129.0, 126.1, 125.7,
125.4, 119.4, 109.8, 96.3, 82.1, 77.9, 75.1, 69.3, 49.0, 42.6, 28.9,
28.3, 24.1, 23.4, 20.9, 16.6, 8.3. MS (ESI positive): 497 (M+Na)+.
5.1.29. (S)-5-[4-(1-Ethyl-1-{4-[(E)-2-(1-hydroxycyclobutyl)-
vinyl]-3-methylphenyl}-propyl)-2-methylphenoxymethyl]-
dihydrofuran-2-one (18d)
The yield was 90%. Colorless oil. ½a D25
ꢂ
+21.4 (c 0.95, CHCl3). 1H
NMR (CDCl3) d: 0.60 (6H, t, J = 7.1 Hz), 1.61–1.71 (1H, m), 1.79–
1.89 (1H, m), 2.05 (4H, q, J = 7.2 Hz), 2.15 (3H, s), 2.20–2.35 (5H,
m), 2.31 (3H, s), 2.37–2.48 (1H, m), 2.49–2.59 (1H, m), 2.71–2.80
(1H, m), 4.05 (1H, dd, J = 10.5, 3.4 Hz), 4.16 (1H, dd, J = 9.9,
3.0 Hz), 4.84–4.90 (1H, m), 6.35 (1H, d, J = 15.8 Hz), 6.66 (1H, d,
J = 8.4 Hz), 6.82 (1H, d, J = 15.8 Hz), 6.91–6.98 (4H, m), 7.36 (1H,
d, J = 8.4 Hz). 13C NMR (CDCl3) d: 177.1, 153.9, 148.1, 141.2,
134.5, 130.7, 130.0, 129.9, 127.9, 126.2, 125.9, 125.6, 124.7,
124.3, 110.0, 77.9, 75.2, 69.3, 48.8, 36.4, 29.1, 28.2, 24.0, 20.1,
16.5, 12.2, 8.3. MS (ESI positive): 485 (M+Na)+.
5.1.25. (S)-5-(4-{1-Ethyl-1-[4-(1-hydroxycyclohexylethynyl)-3-
methylphenyl]-propyl}-2-methylphenoxymethyl)-
dihydrofuran-2-one (17f)
The yield was 72%. Colorless oil. ½a D25
ꢂ
+13.4 (c 1.17, CHCl3). 1H
NMR (CDCl3) d: 0.59 (6H, t, J = 7.2 Hz), 1.56–1.77 (8H, m), 1.99–
2.06 (6H, m), 2.14 (3H, s), 2.26–2.35 (1H, m), 2.38 (3H, s), 2.40–
2.48 (1H, m), 2.52–2.60 (1H, m), 2.72–2.81 (1H, m), 4.06 (1H, dd,
J = 10.3, 3.4 Hz), 4.16 (1H, dd, J = 10.3, 3.2 Hz), 4.85–4.90 (1H, m),
6.66 (1H, d, J = 8.6 Hz), 6.87 (1H, d, J = 2.0 Hz), 6.91–6.95 (2H, m),
6.99 (1H, s), 7.28 (1H, d, J = 8.2 Hz). 13C NMR (CDCl3) d: 177.2,
154.0, 149.4, 140.9, 139.2, 131.1, 130.7, 129.0, 126.1, 125.6,
125.4, 119.4, 109.8, 96.2, 83.3, 77.8, 77.2, 69.3, 49.0, 40.1, 28.9,
28.3, 25.2, 24.0, 23.5, 21.1, 16.6, 8.3. MS (ESI positive): 506
(M+NH4)+.
5.1.30. (S)-5-[4-(1-Ethyl-1-{4-[(E)-2-(1-hydroxycyclopentyl)-
vinyl] -3-methylphenyl}-propyl)-2-methylphenoxymethyl]-
dihydrofuran-2-one (18e)
The yield was 54%. Colorless oil. ½a D25
ꢂ
+13.2 (c 1.20, CHCl3). 1H
NMR (CDCl3) d: 0.60 (6H, t, J = 7.3 Hz), 1.72–1.95 (8H, m), 2.04
(4H, q, J = 7.2 Hz), 2.14 (3H, s), 2.28–2.36 (1H, m), 2.31 (3H, s),
2.38–2.48 (1H, m), 2.52–2.60 (1H, m), 2.72–2.81 (1H, m), 4.06
(1H, dd, J = 10.3, 3.4 Hz), 4.16 (1H, dd, J = 10.1, 3.2 Hz), 4.85–4.90
(1H, m), 6.26 (1H, d, J = 16.0 Hz), 6.66 (1H, d, J = 8.6 Hz), 6.83 (1H,
d, J = 16.0 Hz), 6.91–6.97 (4H, m), 7.33 (1H, d, J = 8.6 Hz). 13C
NMR (CDCl3) d: 177.2, 153.9, 147.9, 141.3, 136.4, 134.4, 132.8,
130.7, 129.9, 126.2, 125.9, 125.5, 124.6, 124.1, 109.8, 82.3, 77.9,
69.3, 48.8, 40.7, 29.0, 28.3, 24.0, 23.7, 20.2, 16.6, 8.4. MS (ESI posi-
tive): 499 (M+Na)+.
5.1.26. (S)-5-(4-{1-Ethyl-1-[4-((E)-3-hydroxy-3-methyl-1-
butenyl)-3-methylphenyl]-propyl}-2-methylphenoxymethyl)-
dihydrofuran-2-one (18a)
The yield was 84%. Colorless oil. ½a D25
ꢂ
+20.8 (c 1.25, CHCl3). 1H
NMR (CDCl3) d: 0.60 (6H, t, J = 7.2 Hz), 1.42 (6H, s), 2.04 (4H, q,
J = 7.2 Hz), 2.14 (3H, s), 2.28–2.36 (1H, m), 2.30 (3H, s), 2.40–2.48
(1H, m), 2.50–2.62 (1H, m), 2.72–2.81 (1H, m), 4.06 (1H, dd,
J = 10.4, 3.3 Hz), 4.16 (1H, dd, J = 10.4, 3.1 Hz), 4.86–4.91 (1H, m),
6.23 (1H, d, J = 15.8 Hz), 6.66 (1H, d, J = 8.6 Hz), 6.77 (1H, d,
J = 16.0 Hz), 6.90–6.97 (4H, m), 7.32 (1H, d, J = 8.8 Hz). 13C NMR
(CDCl3) d: 177.2, 153.9, 148.0, 141.2, 138.0, 134.5, 130.7, 130.0,
129.9, 127.9, 126.2, 125.9, 124.6, 123.7, 109.8, 77.9, 71.2, 69.3,
48.8, 29.9, 29.0, 28.3, 24.0, 20.1, 16.6, 8.4. MS (ESI positive): 473
(M+Na)+.
5.1.31. (S)-5-[4-(1-Ethyl-1-{4-[(E)-2-(1-hydroxycyclohexyl)-
vinyl]-3-methylphenyl}-propyl)-2-methylphenoxymethyl]-
dihydrofuran-2-one (18f)
The yield was 71%. Colorless oil. ½a D25
ꢂ
+18.1 (c 1.29, CHCl3). 1H
NMR (CDCl3) d: 0.60 (6H, t, J = 7.2 Hz), 1.52–1.74 (10H, m), 2.04

4504
H. Kashiwagi et al. / Bioorg. Med. Chem. 20 (2012) 4495–4506
(4H, q, J = 7.2 Hz), 2.15 (3H, s), 2.27–2.36 (1H, m), 2.30 (3H, s),
2.38–2.48 (1H, m), 2.51–2.61 (1H, m), 2.73–2.81 (1H, m), 4.06
(1H, dd, J = 10.3, 3.4 Hz), 4.16 (1H, dd, J = 10.2, 3.3 Hz), 4.85–4.90
(1H, m), 6.21 (1H, d, J = 16.0 Hz), 6.66 (1H, d, J = 8.4 Hz), 6.81 (1H,
d, J = 16.0 Hz), 6.91–6.97 (4H, m), 7.32 (1H, d, J = 8.8 Hz). 13C
NMR (CDCl3) d: 177.2, 153.9, 148.0, 141.2, 138.0, 134.5, 132.9,
130.7, 129.9, 126.2, 125.9, 125.5, 124.6, 124.5, 109.8, 77.9, 71.9,
69.3, 48.8, 38.1, 29.0, 28.3, 25.5, 24.0, 22.1, 20.1, 16.6, 8.4. MS
(ESI positive): 513 (M+Na)+.
5.1.36. (S)-5-(4-{1-Ethyl-1-[4-(3-ethyl-3-hydroxy-1-pentynyl)-3-
methylphenyl]-propyl}-2-methylphenoxy)-4-hydroxypentanoic
acid (5b)
The yield was 76%. Colorless amorphous. ½a D25
ꢂ
+1.68 (c 0.60,
CHCl3). 1H NMR (CD3OD) d: 0.59 (6H, t, J = 7.2 Hz), 1.09 (6H, t,
J = 7.4 Hz), 1.66–1.85 (5H, m), 1.95–2.03 (1H, m), 2.07 (4H, q,
J = 7.3 Hz), 2.15 (3H, s), 2.34 (3H, s), 2.41–2.57 (2H, m), 3.90 (2H,
d, J = 5.1 Hz), 3.93–3.99 (1H, m), 6.76 (1H, d, J = 8.6 Hz), 6.85 (1H,
d, J = 2.0 Hz), 6.94 (2H, dd, J = 8.2, 1.8 Hz), 7.00 (1H, s), 7.23 (1H,
d, J = 8.0 Hz). 13C NMR (CD3OD) d: 178.4, 157.1, 151.8, 142.3,
141.1, 133.1, 132.4, 131.2, 128.1, 127.9, 127.4, 122.0, 112.3, 97.3,
84.9, 74.2, 74.0, 71.2, 50.0, 36.5, 32.0, 31.0, 30.8, 22.1, 17.6, 10.1,
9.6. HRMS (ESI negative): Calcd for C31H41O5 493.2959. Found:
493.2958 (MꢀH)ꢀ.
5.1.32. (S)-5-(4-{1-Ethyl-1-[4-((Z)-3-ethyl-3-hydroxy-1-
pentenyl)-3-methylphenyl]-propyl}-2-methylphenoxymethyl)-
dihydrofuran-2-one (19b)
The yield was 85%. Colorless oil. ½a D25
ꢂ
+31.7 (c 0.36, CHCl3). 1H
NMR (CDCl3) d: 0.58 (6H, t, J = 7.2 Hz), 0.92 (6H, t, J = 7.4 Hz),
1.54 (4H, q, J = 7.5 Hz), 2.03 (4H, q, J = 7.4 Hz), 2.16 (3H, s), 2.22
(3H, s), 2.27–2.37 (1H, m), 2.40–2.49 (1H, m), 2.53–2.61 (1H, m),
2.74–2.82 (1H, m), 4.06 (1H, dd, J = 10.3, 3.4 Hz), 4.17 (1H, dd,
J = 10.4, 3.3 Hz), 4.86–4.91 (1H, m), 5.55 (1H, d, J = 12.5 Hz), 6.49
(1H, d, J = 12.7 Hz), 6.66 (1H, d, J = 9.2 Hz), 6.91–6.96 (4H, m),
7.11 (1H, d, J = 8.2 Hz). 13C NMR (CDCl3) d: 177.2, 153.9, 148.2,
141.2, 136.7, 135.5, 133.6, 130.6, 129.6, 127.7, 127.6, 126.3,
125.6, 125.2, 109.9, 78.0, 77.9, 69.3, 48.8, 34.2, 29.1, 28.3, 24.1,
20.6, 16.6, 8.4, 8.3. MS (ESI positive): 501 (M+Na)+.
5.1.37. (S)-5-(4-{1-Ethyl-1-[4-(3-hydroxy-3-propyl-1-hexynyl)-
3-methylphenyl]-propyl}-2-methylphenoxy)-4-
hydroxypentanoic acid (5c)
The yield was 95%. Colorless amorphous. ½a D25
ꢂ
+2.00 (c 0.50,
CHCl3). 1H NMR (CD3OD) d: 0.59 (6H, t, J = 7.3 Hz), 0.98 (6H, t,
J = 7.1 Hz), 1.57–1.72 (8H, m), 1.78–1.88 (1H, m), 1.93–2.02 (1H,
m), 2.06 (4H, q, J = 7.3 Hz), 2.15 (3H, s), 2.33 (3H, s), 2.37–2.42
(2H, m), 3.90 (2H, d, J = 5.3 Hz), 3.93–3.99 (1H, m), 6.77 (1H, d,
J = 8.6 Hz), 6.83 (1H, d, J = 2.0 Hz), 6.92–6.96 (2H, m), 7.00 (1H, s),
7.21 (1H, d, J = 8.0 Hz). 13C NMR (CD3OD) d: 182.5, 157.2, 151.8,
142.2, 141.0, 133.0, 132.3, 131.2, 128.1, 127.9, 127.4, 122.0,
112.2, 97.8, 84.7, 74.1, 73.2, 72.1, 49.8, 46.7, 35.5, 32.1, 30.8,
22.1, 19.8, 17.6, 15.7, 9.6. HRMS (ESI negative): Calcd for
5.1.33. (S)-5-(4-{1-Ethyl-1-[4-(3-ethyl-3-hydroxypentyl)-3-
methyl phenyl]-propyl}-2-methylphenoxymethyl)-
dihydrofuran-2-one (20b)
The yield was 78%. Colorless oil. ½a D25
ꢂ
+12.9 (c 1.09, CHCl3). 1H
C
33H45O5 521.3272. Found: 521.3279 (MꢀH)ꢀ.
NMR (CDCl3) d: 0.59 (6H, t, J = 7.2 Hz), 0.90 (6H, t, J = 7.4 Hz),
1.55 (4H, q, J = 7.6 Hz), 1.63–1.68 (2H, m), 2.03 (4H, q,
J = 14.9 Hz), 2.15 (3H, s), 2.25 (3H, s), 2.28–2.36 (1H, m), 2.38–
2.49 (1H, m), 2.52–2.61 (3H, m), 2.73–2.81 (1H, m), 4.06 (1H, dd,
J = 10.4, 3.5 Hz), 4.16 (1H, dd, J = 10.3, 3.2 Hz), 4.85–4.91 (1H, m),
6.66 (1H, d, J = 8.4 Hz), 6.89–6.97 (4H, m), 7.00 (1H, d, J = 8.6 Hz).
13C NMR (CDCl3) d: 177.2, 153.8, 146.2, 141.5, 137.5, 134.7,
130.7, 129.8, 127.8, 126.2, 125.7, 125.5, 109.8, 77.9, 74.6, 69.3,
48.7, 38.8, 30.9, 29.1, 28.3, 26.7, 24.1, 19.6, 16.6, 8.4, 7.8. MS (ESI
positive): 503 (M+Na)+.
5.1.38. (S)-5-(4-{1-Ethyl-1-[4-(1-hydroxycyclobutylethynyl)-3-
methylphenyl]-propyl}-2-methylphenoxy)-4-hydroxypentanoic
acid (5d)
The yield was 95%. Colorless amorphous. ½a D25
ꢂ
+2.52 (c 0.95,
CHCl3). 1H NMR (CD3OD) d: 0.59 (6H, t, J = 7.2 Hz), 1.80–1.90 (3H,
m), 1.92–2.01 (1H, m), 2.07 (4H, q, J = 7.3 Hz), 2.15 (3H, s), 2.27–
2.39 (4H, m), 2.36 (3H, s), 2.43–2.49 (2H, m), 3.90 (2H, d,
J = 4.9 Hz), 3.93–3.99 (1H, m), 6.78 (1H, d, J = 8.6 Hz), 6.84 (1H, d,
J = 1.8 Hz), 6.93–6.97 (2H, m), 7.01 (1H, d, J = 1.4 Hz), 7.24 (1H, d,
J = 8.0 Hz). 13C NMR (CD3OD) d: 183.5, 157.2, 151.9, 142.1, 141.1,
132.9, 132.3, 131.2, 128.1, 127.9, 127.4, 121.9, 112.2, 98.4, 83.5,
74.1, 72.3, 69.8, 49.8, 40.8, 36.4, 32.4, 30.8, 21.9, 17.6, 14.7, 9.6.
5.1.34. General procedure for hydrolysis of lactone group of
17a–f, 18a–f, 19b, and 20b
To a solution of lactone (0.355 mmol) in methanol (7.5 ml), 1 N
KOH solution (0.7 ml, 0.700 mmol) was added. The mixture was stir-
red at room temperature for 2 h. The mixture was poured into satd
NH4Cl aq solution and products were extracted with AcOEt. The ex-
tracts were washed with brine, dried over MgSO4, and concentrated.
The obtained residue was chromatographed on silica gel (5% MeOH/
CH2Cl2) to afford gamma hydroxyl carboxylic acid. All compounds
were stocked at ꢀ30 °C as a 1 mg/mL EtOH solution with 1.0 equiv
of 1 N NaOH solution to avoid retro lactone formation.
HRMS (ESI negative): Calcd for C30H37O5 477.2646. Found:
477.2639 (MꢀH)ꢀ.
5.1.39. (S)-5-(4-{1-Ethyl-1-[4-(1-hydroxycyclopentylethynyl)-3-
methylphenyl]-propyl}-2-methylphenoxy)-4-hydroxypentanoic
acid (5e)
The yield was 83%. Colorless amorphous. ½a D25
ꢂ
+2.22 (c 0.63,
CHCl3). 1H NMR (CD3OD) d: 0.59 (6H, t, J = 7.3 Hz), 1.75–1.88 (5H,
m), 1.93–2.03 (5H, m), 2.06 (4H, q, J = 7.4 Hz), 2.15 (3H, s), 2.33
(3H, s), 2.35–2.40 (2H, m), 3.90 (2H, d, J = 5.1 Hz), 3.93–3.99 (1H,
m), 6.77 (1H, d, J = 8.6 Hz), 6.83 (1H, d, J = 1.8 Hz), 6.94 (2H, dd,
J = 8.2, 2.0 Hz), 6.99 (1H, s), 7.21 (1H, d, J = 8.0 Hz). 13C NMR
(CD3OD) d: 183.2, 157.2, 151.8, 142.2, 141.1, 132.8, 132.3, 131.1,
128.1, 127.9, 127.4, 122.1, 112.2, 98.7, 83.4, 76.4, 74.1, 72.3, 50.0,
44.2, 36.1, 32.3, 30.8, 25.2, 21.9, 17.6, 9.6. HRMS (ESI negative):
Calcd for C31H39O5 491.2803. Found: 491.2813 (MꢀH)ꢀ.
5.1.35. (S)-5-(4-{1-Ethyl-1-[4-(3-hydroxy-3-methyl-1-butynyl)-
3-methylphenyl]-propyl}-2-methylphenoxy)-4-
hydroxypentanoic acid (5a)
The yield was 71%. Colorless amorphous. ½a D25
ꢂ
+2.04 (c 0.49,
CHCl3). 1H NMR (CD3OD) d: 0.59 (6H, t, J = 7.2 Hz), 1.56 (6H, s),
1.78–1.88 (1H, m), 1.93–2.01 (1H, m), 2.06 (4H, q, J = 7.3 Hz),
2.15 (3H, s), 2.33 (3H, s), 2.34–2.39 (2H, m), 3.90 (2H, d,
J = 5.1 Hz), 3.93–3.99 (1H, m), 6.77 (1H, d, J = 8.6 Hz), 6.83 (1H, d,
J = 1.8 Hz), 6.94 (2H, dd, J = 8.3, 1.9 Hz), 7.00 (1H, s), 7.21 (1H, d,
J = 8.2 Hz). 13C NMR (CD3OD) d: 183.5, 157.2, 151.8, 142.1, 141.1,
132.8, 132.3, 131.1, 128.1, 127.9, 127.4, 121.9, 112.2, 99.6, 82.5,
74.1, 72.3, 66.9, 49.8, 36.4, 32.8, 32.4, 30.8, 21.9, 17.6, 9.6. HRMS
(ESI negative): Calcd for C29H37O5 465.2646. Found: 465.2642
(MꢀH)ꢀ.
5.1.40. (S)-5-(4-{1-Ethyl-1-[4-(1-hydroxycyclohexylethynyl)-3-
methylphenyl]-propyl}-2-methylphenoxy)-4-hydroxypentanoic
acid (5f)
The yield was 92%. Colorless amorphous. ½a D25
ꢂ
+2.36 (c 0.76,
CHCl3). 1H NMR (CD3OD) d: 0.59 (6H, t, J = 7.2 Hz), 1.57–1.77 (8H,
m), 1.80–1.88 (1H, m), 1.93–2.02 (3H, m), 2.07 (4H, q, J = 7.3 Hz),
2.15 (3H, s), 2.34–2.39 (2H, m), 2.35 (3H, s), 3.90 (2H, d,

H. Kashiwagi et al. / Bioorg. Med. Chem. 20 (2012) 4495–4506
4505
J = 4.9 Hz), 3.93–3.99 (1H, m), 6.77 (1H, d, J = 8.6 Hz), 6.83 (1H, d,
J = 2.0 Hz), 6.93–6.97 (2H, m), 7.00 (1H, s), 7.23 (1H, d, J = 8.0 Hz).
13C NMR (CD3OD) d: 183.5, 157.2, 151.8, 142.2, 141.1, 132.9,
132.3, 131.2, 128.1, 127.9, 127.4, 122.0, 112.2, 98.4, 84.9, 74.1,
72.3, 70.6, 50.0, 42.0, 36.4, 32.4, 30.8, 27.4, 25.5, 22.1, 17.6, 9.6.
(3H, m), 7.35 (1H, d, J = 8.0 Hz). 13C NMR (CD3OD) d: 183.5,
157.1, 150.4, 142.6, 136.8, 136.5, 135.4, 132.4, 131.9, 128.1,
127.9, 127.8, 126.7, 126.1, 112.2, 76.8, 74.1, 72.3, 49.4, 38.1, 36.4,
32.4, 30.9, 21.1, 17.6, 14.0, 9.6. HRMS (ESI negative): Calcd for
C
30H39O5 479.2803. Found: 479.2795 (MꢀH)ꢀ.
HRMS (ESI negative): Calcd for
C32H41O5 505.2959. Found:
505.2953 (MꢀH)ꢀ.
5.1.45. (S)-5-[4-(1-Ethyl-1-{4-[(E)-2-(1-hydroxycyclopentyl)-
vinyl] -3-methylphenyl}-propyl)-2-methylphenoxy]-4-
hydroxypentanoic acid (6e)
5.1.41. (S)-5-(4-{1-Ethyl-1-[4-((E)-3-hydroxy-3-methyl-1-
butenyl)-3-methylphenyl]-propyl}-2-methylphenoxy)-4-
hydroxypentanoic acid (6a)
The yield was 97%. Colorless amorphous. ½a D25
ꢂ
+2.77 (c 0.87,
CHCl3). 1H NMR (CDCl3) d: 0.60 (6H, t, J = 7.2 Hz), 1.71ꢀ1.78 (8H,
m), 1.79ꢀ1.88 (1H, m), 1.93ꢀ2.01 (1H, m), 2.07 (4H, q,
J = 7.3 Hz), 2.15 (3H, s), 2.28 (3H, s), 2.36ꢀ2.41 (2H, m), 3.91 (2H,
d, J = 5.1 Hz), 3.94ꢀ3.99 (1H, m), 6.22 (1H, d, J = 15.8 Hz), 6.77
(1H, d, J = 8.6 Hz), 6.84 (1H, d, J = 16.0 Hz), 6.85 (1H, d, J = 2.0 Hz),
6.91ꢀ6.98 (3H, m), 7.32 (1H, d, J = 8.0 Hz). 13C NMR (CD3OD) d:
183.2, 157.1, 150.2, 142.6, 138.7, 136.3, 135.6, 132.4, 131.8,
128.1, 127.8, 127.8, 126.7, 126.2, 112.2, 83.9, 74.1, 72.3, 50.0,
42.2, 36.1, 32.3, 30.9, 25.6, 21.1, 17.6, 9.6. HRMS (ESI negative):
Calcd for C31H41O5 493.2959. Found: 493.2955 (MꢀH)ꢀ.
The yield was 90%. Colorless amorphous. ½a D25
ꢂ
+2.73 (c 0.73,
CHCl3). 1H NMR (CD3OD) d: 0.59 (6H, t, J = 7.2 Hz), 1.37 (6H, s),
1.78–1.88 (1H, m), 1.93–2.01 (1H, m), 2.06 (4H, q, J = 7.3 Hz),
2.14 (3H, s), 2.27 (3H, s), 2.34–2.40 (2H, m), 3.90 (2H, d,
J = 5.3 Hz), 3.93–3.98 (1H, m), 6.19 (1H, d, J = 15.8 Hz), 6.76 (1H,
d, J = 8.6 Hz), 6.77 (1H, d, J = 16.0 Hz), 6.84 (1H, d, J = 2.0 Hz),
6.91–6.97 (3H, m), 7.30 (1H, d, J = 8.0 Hz). 13C NMR (CD3OD) d:
183.5, 157.1, 150.3, 142.6, 140.3, 136.5, 135.5, 132.4, 131.8,
128.1, 127.9, 127.8, 126.7, 125.8, 112.2, 74.1, 72.6, 72.3, 50.0,
36.4, 32.4, 31.0, 30.9, 21.1, 17.6, 9.6. HRMS (ESI negative): Calcd
for C29H39O5 467.2803. Found: 467.2795 (MꢀH)ꢀ.
5.1.46. (S)-5-[4-(1-Ethyl-1-{4-[(E)-2-(1-hydroxycyclohexyl)-
vinyl]-3-methylphenyl}-propyl)-2-methylphenoxy]-4-
hydroxypentanoic acid (6f)
The yield was 98%. Colorless amorphous. ½a D25
ꢂ
+2.26 (c 1.06,
5.1.42. (S)-5-(4-{1-Ethyl-1-[4-((E)-3-ethyl-3-hydroxy-1-
penteny1)-3-methylphenyl]-propyl}-2-methylphenoxy)-4-
hydroxypentanoic acid (6b)
CHCl3). 1H NMR (CD3OD) d: 0.59 (6H, t, J = 7.3 Hz), 1.30ꢀ1.41 (2H,
m), 1.51ꢀ1.77 (8H, m), 1.79ꢀ1.89 (1H, m), 1.93ꢀ2.02 (1H, m), 2.06
(4H, q, J = 7.3 Hz), 2.14 (3H, s), 2.27 (3H, s), 2.34ꢀ2.40 (2H, m),
3.90 (2H, d, J = 5.1 Hz), 3.93ꢀ3.99 (1H, m), 6.16 (1H, d, J = 15.8 Hz),
6.76 (1H, d, J = 8.6 Hz), 6.81 (1H, d, J = 16.0 Hz), 6.84 (1H, d,
J = 2.0 Hz), 6.91ꢀ6.97 (3H, m), 7.30 (1H, d, J = 8.2 Hz). 13C NMR
(CD3OD) d: 183.5, 157.1, 150.3, 142.6, 140.4, 136.4, 135.7, 132.4,
131.8, 128.1, 127.8, 127.8, 126.6, 126.5, 112.2, 74.1, 73.5, 72.3,
49.8, 39.8, 36.4, 32.4, 30.9, 27.7, 24.1, 21.1, 17.6, 9.6. HRMS (ESI neg-
ative): Calcd for C32H43O5 507.3116. Found: 507.3108 (MꢀH)ꢀ.
The yield was 85%. Colorless amorphous. ½a D25
ꢂ
+4.10 (c 0.20,
CHCl3). 1H NMR (CD3OD) d: 0.60 (6H, t, J = 7.2 Hz), 0.92 (6H, t,
J = 7.4 Hz), 1.63 (4H, q, J = 7.4 Hz), 1.78–1.88 (1H, m), 1.93–2.00
(1H, m), 2.06 (4H, q, J = 7.3 Hz), 2.15 (3H, s), 2.27 (3H, s), 2.34–
2.40 (2H, m), 3.90 (2H, d, J = 5.1 Hz), 3.93–3.99 (1H, m), 5.99 (1H,
d, J = 16.0 Hz), 6.75 (1H, d, J = 16.2 Hz), 6.76 (1H, d, J = 8.4 Hz),
6.85 (1H, d, J = 2.0 Hz), 6.92 (1H, s), 6.96 (2H, dd, J = 8.5, 2.2 Hz),
7.28 (1H, d, J = 8.0 Hz). 13C NMR (CD3OD) d: 183.5, 157.1, 150.2,
142.6, 137.8, 136.3, 135.8, 132.4, 131.8, 128.1, 127.9, 127.8,
127.8, 126.7, 112.1, 77.7, 74.1, 72.3, 49.4, 36.4, 35.0, 32.4, 30.9,
21.2, 17.6, 9.7, 9.3. HRMS (ESI negative): Calcd for C31H43O5
495.3116. Found: 495.3124 (MꢀH)ꢀ.
5.1.47. (S)-5-(4-{1-Ethyl-1-[4-((Z)-3-ethyl-3-hydroxy-1-
pentenyl)-3-methylphenyl]-propyl}-2-methylphenoxy)-4-
hydroxypentanoic acid (7b)
The yield was 71%. Colorless amorphous. ½a D25
ꢂ
+4.84 (c 0.33,
CHCl3). 1H NMR (CD3OD) d: 0.59 (6H, t, J = 7.3 Hz), 0.86 (6H, t,
J = 7.4 Hz), 1.43ꢀ1.55 (4H, m), 1.77ꢀ1.87 (1H, m), 1.94ꢀ2.01 (1H,
m), 2.07 (4H, q, J = 7.3 Hz), 2.14 (3H, s), 2.19 (3H, s), 2.38ꢀ2.46
(2H, m), 3.90 (2H, d, J = 5.1 Hz), 3.95ꢀ3.98 (1H, m), 5.60 (1H, d,
J = 12.9 Hz), 6.47 (1H, d, J = 12.7 Hz), 6.76 (1H, d, J = 8.6 Hz), 6.84
(1H, d, J = 2.0 Hz), 6.92ꢀ6.98 (3H, m), 7.11 (1H, d, J = 8.6 Hz). 13C
NMR (CD3OD) d: 181.5, 157.1, 150.3, 142.6, 139.2, 137.1, 136.4,
132.5, 131.3, 130.3, 130.1, 128.1, 127.8, 126.8, 112.2, 79.0, 74.1,
71.9, 50.0, 35.4, 34.6, 31.8, 30.9, 21.5, 17.6, 9.6, 9.4. HRMS (ESI neg-
ative): Calcd. for C31H43O5 495.3116. Found: 495.3126 (MꢀH)ꢀ.
5.1.43. (S)-5-(4-{1-Ethyl-1-[4-((E)-3-hydroxy-3-propyl-1-
hexenyl)-3-methylphenyl]-propyl}-2-methylphenoxy)-4-
hydroxypentanoic acid (6c)
The yield was 93%. Colorless amorphous. ½a D25
ꢂ
+2.58 (c 1.01,
CHCl3). 1H NMR (CD3OD) d: 0.59 (6H, t, J = 7.2 Hz), 0.92 (6H, t,
J = 7.3 Hz), 1.32–1.46 (4H, m), 1.54–1.59 (4H, m), 1.78–1.88 (1H,
m), 1.92–2.01 (1H, m), 2.05 (4H, q, J = 7.3 Hz), 2.14 (3H, s), 2.26
(3H, s), 2.33–2.39 (2H, m), 3.89 (2H, d, J = 5.3 Hz), 3.92–3.98 (1H,
m), 6.01 (1H, d, J = 16.0 Hz), 6.72 (1H, d, J = 16.2 Hz), 6.76 (1H, d,
J = 8.6 Hz), 6.84 (1H, d, J = 2.0 Hz), 6.91 (1H, s), 6.95 (2H, dd,
J = 8.4, 2.2 Hz), 7.26 (1H, d, J = 8.0 Hz). 13C NMR (CD3OD) d: 183.5,
157.1, 150.2, 142.6, 138.5, 136.3, 135.8, 132.4, 131.8, 128.1,
127.8, 127.8, 127.3, 126.6, 112.2, 77.3, 74.1, 72.3, 49.5, 45.6, 36.4,
32.4, 30.9, 21.2, 18.9, 17.6, 15.9, 9.7. HRMS (ESI negative): Calcd
for C33H47O5 523.3429. Found: 523.3432 (MꢀH)ꢀ.
5.1.48. (S)-5-(4-{1-Ethyl-1-[4-(3-ethyl-3-hydroxypentyl)-3-
methyl phenyl]-propyl}-2-methylphenoxy)-4-
hydroxypentanoic acid (8b)
The yield was 91%. Colorless amorphous. ½a D25
ꢂ
+1.88 (c 0.64,
CHCl3). 1H NMR (CD3OD) d: 0.59 (6H, t, J = 7.2 Hz), 0.92 (6H, t,
J = 7.5 Hz), 1.55 (4H, q, J = 7.5 Hz), 1.60ꢀ1.64 (2H, m), 1.79ꢀ1.89
(1H, m), 1.94ꢀ2.01 (1H, m), 2.05 (4H, q, J = 7.3 Hz), 2.15 (3H, s),
2.25 (3H, s), 2.35ꢀ2.41 (2H, m), 2.55ꢀ2.59 (2H, m), 3.91 (2H, d,
J = 5.1 Hz), 3.94ꢀ4.00 (1H, m), 6.76 (1H, d, J = 8.4 Hz), 6.85 (1H, d,
J = 2.0 Hz), 6.88ꢀ6.93 (2H, m), 6.95 (1H, dd, J = 8.5, 2.2 Hz), 6.99
(1H, d, J = 8.0 Hz). 13C NMR (CD3OD) d: 183.5, 157.1, 148.6, 142.8,
140.0, 136.6, 132.4, 131.9, 129.9, 128.1, 127.7, 112.2, 76.4, 74.1,
72.3, 50.0, 41.0, 36.4, 32.4, 32.4, 31.0, 28.7, 20.6, 17.6, 9.7, 9.1.
5.1.44. (S)-5-[4-(1-Ethyl-1-{4-[(E)-2-(1-hydroxycyclobutyl)-
vinyl]-3-methylphenyl}-propyl)-2-methylphenoxy]-4-
hydroxypentanoic acid (6d)
The yield was 98%. Colorless amorphous. ½a D25
ꢂ
+2.11 (c 0.87,
CHCl3). 1H NMR (CD3OD) d: 0.60 (6H, t, J = 7.2 Hz), 1.64–1.76 (1H,
m), 1.79–1.89 (2H, m), 1.93–2.01 (1H, m), 2.07 (4H, q, J = 7.3 Hz),
2.15 (3H, s), 2.18ꢀ2.30 (4H, m), 2.28 (3H, s), 2.34ꢀ2.40 (2H, m),
3.90 (2H, d, J = 5.1 Hz), 3.93ꢀ3.98 (1H, m), 6.33 (1H, d,
J = 15.8 Hz), 6.77 (1H, d, J = 8.6 Hz), 6.81ꢀ6.85 (2H, m), 6.92ꢀ6.98
HRMS (ESI negative): Calcd. for
C31H45O5 497.3272. Found:
497.3270 (MꢀH)ꢀ.

4506
H. Kashiwagi et al. / Bioorg. Med. Chem. 20 (2012) 4495–4506
5.2. VDRE reporter gene assay
MG-63 cells were plated at 2 ꢁ 103 cells/200
Vvoid space, which is the volume in the VDR pocket not occupied
by either ligand atoms or VDR atoms, (see the grid points colored
by magenta in Fig. 5) was calculated for each compound as follows;
all the grid points within the rectangular box were examined to see
whether they lay outside the vdW sphere of each atom of VDR and
outside the vdW sphere of each atom of the compound. The num-
ber of grid point m was defined as the number of grid points that
are not within the vdW radius for each atom of either the
compound or VDR. Vvoid space was calculated as m ꢁ (0.1 Å)3.
VLBD, which represents a volume of the pocket of VDR, is defined
as a summation of Vside chain and Vviod space. The value of VLBD was
208 Å3.
l
L/well in a 96-
well white cell-culture plate and were incubated at 37 °C in 5%
CO2 incubator for 24 h. Then, MG-63 cells were cotransfected with
0.05
three repeats of the VDRE sequence from mouse osteopontin
promoter and 0.001 g of pRL-SV40 vector (Promega Corporation,
lg of the pGV2-basic/VDRE-luciferase vector which contained
l
WI, USA) using Lipofectamine (Invitrogen). The cells were added
to minimum essential medium (MEM) containing 5% fetal bovine
serum 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 10ꢀ7–10ꢀ11 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
Pharmaceuticals, Weesp, The Netherlands) which was used as a
positive control.
The packing coefficient (PC) values were calculated as follows;
PCð%Þ ¼ Vsidechain=VLBD ꢁ 100
where Vside chain = n ꢁ (0.1 Å)3, Vvoid space = m ꢁ (0.1 Å)3, VLBD
=
Vside chain + Vvoid space
.
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.
5.3. Modeling and volume calculations
References and notes
The compounds 5a–f and 6a–f were manually docked in the
crystal structure of VDR (PDB code 3AZ3). The conformations of
compounds were optimized using the MAB force field as imple-
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21
mented in a program MOLOC by fixing the VDR structure.
In order to calculate the PC values, the volume occupied by the
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To calculate Vside chain, the space around the volumes occupied
by all of the side chains of 5a–f and 6a–f should be defined. The
rectangular box (see the rectangular box depicted by blue lines
in Fig. 5) was defined so as to include all of the side chain volume
of the compounds 5a–f and 6a–f. To determine the size of the rect-
angular box, the minimum and maximum limits for x, y and z
directions of all side chain atoms for each compound were calcu-
lated by comparing x coordinate of the atom van der Waals
(vdW) radius, y vdW and z vdW for each atom of the side chain.
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C-C„C-C(OH)-R1R2 and C–CH@CH-C(OH)R1R2, of the compound.
3D coordinates of the side chain moiety were extracted from the
modeled structure. The radii for atomic spheres were 1.70 Å for
carbon atoms, 1.55 Å for nitrogen atoms, 1.52 Å for oxygen atoms,
and 1.80 Å for sulfur atoms as defined by Bondi.27 Hydrogen atoms
were ignored in the calculation. Minimum and maximum limits for
x, y, z directions for all of the compounds were determined by com-
paring the minimum and maximum limits of 5a–f and 6a–f. The
rectangular box for the side chain was defined as minimum and
maximum limits for each x, y and z direction with 0.75 Å margins.
The calculations of minimum and maximum limits for x, y, z direc-
tions plus 0.75 Å margins resulted in a rectangular box of
8.3 Å ꢁ 11.3 Å ꢁ 7.4 Å. The grid points were generated with 0.1 Å
grid spacing within the rectangular box.
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Vside
for each compound (see the grid points colored by
chain
cyan in Fig. 5) was calculated as follows; all the grid points within
the rectangular box were examined to see whether they were
within the vdW sphere of each atom of the side chain of the com-
pound. The number n is defined as the number of the grid points
within the vdW radius for each atom of the side chain. Vside chain
was calculated as n ꢁ (0.1 Å)3.
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