Flexibility of small molecular CD4 mimics as HIV entry inhibitors

Article abstract of DOI:10.1016/j.bmc.2018.10.011

CD4 mimics such as YIR-821 and its derivatives are small molecules which inhibit the interaction between the Phe43 cavity of HIV-1 gp120 with host CD4, an interaction that is involved in the entry of HIV to cells. Known CD4 mimics generally possess three

Full text of DOI:10.1016/j.bmc.2018.10.011

Accepted Manuscript
Flexibility of small molecular CD4 mimics as HIV entry inhibitors
Takuya Kobayakawa, Nami Ohashi, Yuki Hirota, Kohei Takahashi, Yuko
Yamada, Tetsuo Narumi, Kazuhisa Yoshimura, Shuzo Matsushita, Shigeyoshi
Harada, Hirokazu Tamamura
PII:
S0968-0896(18)31677-8
https://doi.org/10.1016/j.bmc.2018.10.011
BMC 14570
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Accepted Date:
26 September 2018
16 October 2018
Please cite this article as: Kobayakawa, T., Ohashi, N., Hirota, Y., Takahashi, K., Yamada, Y., Narumi, T.,
Yoshimura, K., Matsushita, S., Harada, S., Tamamura, H., Flexibility of small molecular CD4 mimics as HIV entry
inhibitors, Bioorganic & Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.2018.10.011
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Flexibility of small molecular CD4 mimics as HIV entry inhibitors
#
#
Takuya Kobayakawaa, , Nami Ohashi a, , Yuki Hirotaa, Kohei Takahashia, Yuko Yamadaa, Tetsuo
Narumia,$, Kazuhisa Yoshimura b,c, Shuzo Matsushitac, Shigeyoshi Haradab,*, Hirokazu Tamamuraa,*
aInstitute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10
Kandasurugadai, Chiyoda-ku, Tokyo 101-0062, Japan
bAIDS Research Center, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo
162-8640, Japan
cCenter for AIDS Research, Kumamoto University, Kumamoto 860-0811, Japan
#equally contributed
$present address: Graduate School of Integrated Science and Technology, Shizuoka University, 3-5-1 Johoku,
Hamamatsu, Shizuoka 432-8561, Japan
RECEIVED DATE (to be automatically inserted after your manuscript is accepted if required
according to the journal that you are submitting your paper to)
*To whom correspondence should be addressed; shigeh@nih.go.jp, phone; +81-3-4582-2813, fax; +81-3-
5285-5037, or tamamura.mr@tmd.ac.jp, phone; +81-3-5280-8036, fax; +81-3-5280-8039
1

Abstract
CD4 mimics such as YIR-821 and its derivatives are small molecules which inhibit the interaction
between the Phe43 cavity of HIV-1 gp120 with host CD4, an interaction that is involved in the entry of
HIV to cells. Known CD4 mimics generally possess three structural features, an aromatic ring, an
oxalamide linker and a piperidine moiety. We have shown previously that introduction of a cyclohexyl
group and a guanidine group into the piperidine moiety and a fluorine atom at the meta-position of the
aromatic ring leads to a significant increase in the anti-HIV activity. In the current study, the effects of
conformational flexibility were investigated by introduction of an indole-type group in the junction
between the oxalamide linker and the aromatic moiety or by replacement of the oxalamide linker with a
glycine linker. This led to the development of compounds with high anti-HIV activity, showing the
importance of the junction region for the expression of high anti-HIV activity. The present data are
expected to be useful in the future design of novel CD4 mimic molecules.
Keywords
CD4 mimic
oxalamide linker
indole
entry inhibitor
anti-HIV
2

1. Introduction
In the treatment of patients with AIDS and human immunodeficiency virus type 1 (HIV-1), anti-retroviral
therapy (ART), which uses anti-HIV drugs such as protease inhibitors, integrase inhibitors and reverse
transferase inhibitors, has made a major contribution.1 There are still some serious problems however
regarding side effects, emergence of resistant viruses and high costs. To overcome these problems, the
development of novel drugs and an increase of the options concerning anti-HIV drugs, are required. An
important target of highly desirable HIV-1 drugs is entry of the virus into host cells.2 The first step of this
viral entry is the interaction between a viral envelope glycoprotein, gp120 and the host cell surface
protein CD4. This interaction triggers conformational changes in gp120 exposing variable loops and
enabling the subsequent binding of gp120 to a co-receptor, either CCR5 or CXCR4.3,4 Small molecule
CD4 mimics such as N1-(4-chlorophenyl)-N2-(2,2,6,6-tetramethylpiperidin-4-yl)oxalamide (NBD-556),
which have attracted attention as HIV-1 entry inhibitors (Fig. 1).5 Furthermore, the interaction of CD4
mimics with the conserved “Phe43 cavity” on gp120 induces a conformational change in gp120 and
causes a favorable enthalpy change similar to that caused by interaction of soluble CD4 with gp120.
These properties make CD4 mimics valuable not only as entry inhibitors but also as “envelop protein
openers”. CD4 mimics based on NBD-556 are composed essentially of three regions: an aromatic ring,
an oxalamide linker and a piperidine moiety. Our previous studies on the structure-activity relationships
(SARs) of such compounds have provided insight into the modification of the piperidine moiety,
suggesting that bulky hydrophobic groups such as a cyclohexyl ring and cationic groups such as a
guanidine group on its piperidine ring might contribute to the anti-HIV activity and lower cytotoxicity as
was shown in the CD4 mimic YIR-821.6,7 Our SAR studies on the aromatic ring have shown that
introduction of an electron-withdrawing group at the p-position of the phenyl ring might be associated
with potent anti-HIV activity, and that a CD4 mimic, containing a m-fluoro-p-chlorophenyl aromatic
group and a cyclohexyl group on the piperidine ring, has highly potent anti-HIV activity, but its
cytotoxicity is also relatively high.8 We have also discovered the relatively low cytotoxic CD4 mimic
compounds YYA-021 and MTA-003, which have a p-methylphenyl group or a 1,3-benzodioxolyl group,
3

respectively.9,10 As such, we speculate that our earlier SAR studies leave the modification of the main
structure of the aromatic ring and the oxalamide linker undecided, and we have investigated the
modification of the oxalamide linker and the aromatic ring. Initially, molecules which have a constrained
structure in the region containing a part of oxalamide linker and the aromatic ring of NBD-556 were
designed. The X-ray crystal structure of gp120 complexed with NBD-556 (PDB: 3TGS) shows that the
oxalamide linker forms a hydrogen bond with the backbone carbonyl oxygen atoms of Asn425 of the
protein and that the aromatic ring forms a structure that is coplanar with a segment of the oxalamide.11
Accordingly, we used an indole structure to serve as a constrained substituent of the oxalamide linker that
would be coplanar with an aromatic ring. In a different strategy, molecules with a glycine linker in place
of the oxalamide linker were designed to investigate the effect of the introduction of a flexible linker (Fig.
1).
4

Figure 1. New design of CD4 mimic molecules. Indole-type structures were designed for conformational
rigidity and Gly-linker type structures for conformational flexibility.
2. Results and Discussion
2.1. Chemistry
Indole-type compounds (9a,b-21a,b) and (24-26) were synthesized as described in Schemes 1 and 2.
Compound 1a is commercially available. Compounds 2a-5a were synthesized by previously reported
methods.12 Treatment of 1a-5a with methyl iodide followed by saponification gave N-methylated indole
derivatives 1b-5b. Piperidine moieties, described as R2-H, were purchased and synthesized by previously
reported procedures.13 The condensation of a primary or secondly amine of the piperidine moieties (6-8)
with the carboxylic acids of the indole derivatives (1a,b-5a,b) using EDCI/HOBt provided the desired
compounds (9a,b-21a,b). Compound 23 were prepared from 5-amino-6-iodo-1,3-benzodioxole (22) by
palladium-catalyzed annulation,14 and the subsequent condensation of 23 with piperidine moieties (6-8)
using EDCI/HOBt gave 5,6-methylenedioxyindole derivatives 24-26 (Scheme 2).
The synthesis of CD4 mimic molecules, which include a Gly-linker in place of an oxalamide
linker, is described in Schemes 3 and 4. Protection of the amine (27) with a 4-nitrobenzenesulfonyl (Ns)
group gave the amine (28). The amine (6) was reacted with bromoacetyl bromide to give compound 29,
and this was followed by an SN2 reaction with amine 28 to yield compound 30. Removal of the Ns group
of 30 gave compound 31, which possesses a glycine linker. Coupling of 27 with ethyl bromoacetate gave
compound 32. Hydrolysis of the ester (32) with LiOH15 gave the acid (33), which was coupled with the amine
(7) gave the desired product (34). Boc-protection of 32 and subsequent hydrolysis15 gave the acid (35),
which was coupled with the amine (8). Subsequent deprotection of the Boc group with TFA gave
compound 37. CD4 mimics (41-43), including a 1,3-benzodioxole moiety and a Gly-linker were
synthesized as shown in Scheme 4. Starting from a commercially available compound (38), the procedure
that was used for the synthesis of compound 34 gave compounds 41-43.
5

Scheme 1. Synthesis of indole-type derivatives. Reagents and conditions: (a) MeI, K2CO3, DMF, rt; (b) 1
M LiOH aq., THF/H2O, reflux; (c) R2-H (6, 7, 8), HOBt·H2O, EDCI·HCl, Et3N, DMF.
6

Scheme 2. Synthesis of 5,6-methylenedioxyindole-type derivatives. Reagents and conditions: (a)
1,4-diazabicyclo[2.2.2]octane (DABCO), pyruvic acid, Pd(OAc)2, DMF, 27%; (b) R2-H (6, 7, 8),
HOBt·H2O, EDCI·HCl, Et3N, DMF, 24: 29%, 25: 36%, 26: 28%, respectively.
Scheme 3. Synthesis of Gly-linker type CD4 mimic molecules. Reagents and conditions for 31: (a) NsCl,
NaOAc, MeOH/H2O, 60 °C, 71%; (b) bromoacetyl bromide, K2CO3, CH2Cl2, 89%; (c) 28, K2CO3, DMF,
6%.; (d) thiophenol, K2CO3, DMF, 67%. For 34: (e) ethyl bromoacetate, NaOAc, EtOH, reflux, 53%; (f)
1 M LiOH aq., THF/H2O, reflux, 88%; (g) 7, HOBt·H2O, EDCI·HCl, Et3N, DMF, 35%. For 37: (h)
Boc2O, n-heptane, 90 °C (i) 1 M LiOH aq., THF/H2O, reflux, 99% in 2 steps; (j) 8, HOBt·H2O,
EDCI·HCl, Et3N, DMF, 32%; (k) TFA, 0 °C, 89%.
Scheme 4. Synthesis of 1,3-benzodioxole-type compounds including a Gly-linker. Reagents and
7

conditions for 41-43: (a) ethyl bromoacetate, NaOAc, EtOH, reflux, 77%; (b) 1 M LiOH aq., THF/H2O,
reflux, 95%; (c) R2-H (6, 7, 8), HOBt·H2O, EDCI·HCl, Et3N, DMF, 41: 7%, 42: 32%, 43: 39%,
respectively.
2.2. Biological evaluation
The synthesized compounds were evaluated for anti-HIV activity by the WST-8 assay using the cYTA48P
strain and PM1/CCR5 cells. Cytotoxicity was determined by examining the viability of PM1/CCR5 cells.
The ability of the synthesized compounds to induce the conformational change of gp120 was evaluated by
fluorescence activated cell sorting (FACS) analysis using cYTA48P-infected PM1/CCR5 cells and a
primary antibody 4C11, which is a monoclonal antibody (MAb) against a CD4-induced epitope (CD4i).
The conformational change in gp120 was determined by mean fluorescence intensity (MFI) obtained
from a fluorescent dye, which was labeled in the secondary antibody.
The assay results for the conformationally rigid compounds with an indole moiety (9a,b-21a,b
and 24-26) are shown in Table 1. Among these indole derivatives, compounds 9a and 18a showed
relatively high anti-HIV activity with IC50 values of 3.65 M and 3.60 M, respectively. The cytotoxicity
of compound 9a is lower than that of compound 18a. The CC50 of 9a = 48.6 M and of 18a = 11. 4 M,
but the anti-HIV activity of compound 9a or 18a is less than that of NBD-556 (IC50 = 0.63 M), and the
cytotoxicities of 9a and 18a are higher than that of NBD-556. The compounds with an N-methylated
indole moiety did not show significant anti-HIV activity, particularly compounds 9b and 18b, which are
N-methylated derivatives of 9a and 18a, respectively, strongly suggesting that N-methylation of the indole
group might cause a decrease in anti-HIV activity. Fig. 2 shows the most stable conformations of the
5-chloroindole compound (9a), the 6-chloroindole compound (12a) and NBD-556. These were calculated
using Spartan 10 software (Wavefunction Inc., California, USA).16 Superposition of compounds 9a and
NBD-556 showed that the chlorine atom of compound 9a is distant from that of NBD-556. Our previous
SAR study on the aromatic ring showed that an electron withdrawing group contributes positively to
anti-HIV activity,9 and we speculated that this mismatch of the chlorine atom locations might be the
reason for the low anti-HIV activity of compound 9a. Consequently, compounds such as 12a, with a
8

6-chloroindole moiety were adopted. When compound 12a and NBD-556 were superimposed, the
chlorine atoms were found in the almost same location (Fig. 2), although 12a did not show significant
anti-HIV activity. In the rigid design, the replacement of a part of the oxalamide linker and the aromatic
ring with indole caused a remarkable decrease of anti-HIV activity, although the position of the chlorine
atom in, for example compound 12a was appropriate. A possible reason for this is that these CD4 mimics
is relatively inaccessible to the inside of the Phe43 cavity due to fixation by cyclization of a part of the
oxalamide linker. Among compounds with a 6-chloroindole moiety, compound 13a with
6-chloro-5-fluoro-indole showed moderate anti-HIV activity. Substitution of fluorine, chlorine and a
methyl group on the indole ring showed minimal influence on anti-HIV activity.
5,6-Methylenedioxyindole-type compounds showed moderate to high anti-HIV activity; the IC50 values
of 24, 25 and 26 are 23.0 M, 63.0 M, and 1.00 M, respectively. Compound 26, which has a
dicyclohexylpiperidine moistyI, shows high anti-HIV activity but no significant cytotoxicity. This
suggests that a methylenedioxy group might be suitable as a substituent on the aromatic ring as in
MTA-103, whose structure is shown in Fig. 4.10 The CD4 mimicry of these compounds on the
conformational change of gp120 was evaluated by fluorescence activated cell sorting (FACS) analysis.
The binding of an anti-envelope CD4-induced (CD4i) monoclonal antibody (4C11) to the cell surface
pretreated with the synthetic compounds is related to the mean fluorescence intensity (MFI). Since our
previous study on the FACS analysis of the binding of 4C11 to the cell surface indicated that the
treatment of NBD-556 shows behavior similar to that of soluble CD4,9,13b,c,17 the present study calculated
the MFI relative to that of NBD-556 for each compound. In the FACS analysis, pretreatment with
compounds 9a, 10a and 14b which contain a 5-chloro-4-fluoro- or 5-chloroindole moiety caused
remarkable enhancements of binding affinity for 4C11 as shown in the pretreatment with NBD-556. The
relative MFI of 9a, 10a and 14b are 8.70 (0.82), 8.55 (0.68) and 8.63 (0.76), respectively. These results
suggest that the introduction of chlorine and fluorine atoms at the positions 5 and 4, respectively, on the
indole ring contribute to an increase of the binding of 4C11 to the cell surface. In addition, the
substitution of the piperidine moieties in 5-chloro-4-fluoro-indole-type compounds might contribute to
9

the anti-HIV activity and the binding affinity for 4C11, but this is clearly different from the SAR studies
with NBD-556 derivatives having a 4-chloro-3-fluoro phenyl group, in which a dicyclohexyl group was
most suitable.13c The interaction mode of the piperidine moieties with the surface of the Phe43 cavity
might be affected by cyclization of a part of the oxalamide linker, producing a more rigid structure.
Table 1
Anti-HIV-activity, cytotoxicity and gp120 conformational change inducibility of conformationally rigid
indole-type compounds.
anti-HIV activity
cytotoxicity
(CC50 [M])b
70.4
FACS
(MFI)c (Rel. MFI)d
FACS
Compd.
R1
X
Y
R2
(IC50 [M])a
0.63
8.91
8.70
8.55
8.24
8.05
8.08
8.14
5.27
6.65
6.74
8.63
6.45
7.61
8.11
7.96
ND
1
NBD-556
9a
A
A
B
C
A
B
C
A
A
B
C
A
B
C
A
B
C
A
H
5-Cl
5-Cl
5-Cl
5-Cl
H
H
H
H
H
H
H
3.65
48.6
0.82
4-F
4-F
4-F
19.4
0.68
10a
14a
18a
11a
>10 (10 M, 32%)
>100
>200
11.4
0.42
3.60
0.25
4-Me 6-Me
4-Me 6-Me
4-Me 6-Me
>100
60.3
0.28
>100
>200
9.6
0.33
15a
19a
9b
>100
-
H
5-Cl Me
5-Cl Me
5-Cl Me
5-Cl Me
>100
37.5
-
4-F
4-F
4-F
13.9
-
10b
14b
18b
11b
15b
19b
12a
16a
20a
13a
>100 (10 M, 32%)
>100
>63.5
12.3
0.76
>100
-
4-Me 6-Me Me
4-Me 6-Me Me
4-Me 6-Me Me
>100
>37.5
>50.2
>13.5
37.4
-
>100
0.30
>100
>100
0.17
5-H
5-H
5-H
5-F
6-Cl
6-Cl
6-Cl
6-Cl
H
H
H
H
-
-
-
-
>100
52.6
ND
10.7
ND
>100 (10 M, 11%)
88.7
103
ND
10

B
C
A
B
C
A
B
C
A
B
C
5-F
5-F
5-H
5-H
5-H
5-F
5-F
5-F
-
6-Cl
6-Cl
H
H
>100
121
11.3
33.2
78.4
33.9
18.7
41.1
29.4
NDe
ND
ND
ND
ND
ND
ND
ND
ND
ND
30.7
26.2
36.4
-
17a
21a
12b
16b
20b
13b
17b
21b
24
>100
-
6-Cl Me
6-Cl Me
6-Cl Me
6-Cl Me
6-Cl Me
6-Cl Me
-
>100 (10 M, 27%)
>100
>100
>100
>100
>100
23.0
-
-
-
-
-
-
-
-
H
H
H
0.23
0.13
0.36
-
63.0
25
-
1.00
>100
26
a IC50 values were determined using the cYTA48P strain and PM1/CCR5 cells.
b CC50 values were determined using PM1/CCR5 cells.
c Mean fluorescence intensity. FACS analysis was performed using cYTA48P-infected PM1/CCR5 cells, a
test compound (100 M) and an anti-envelope CD4-induced antibody 4C11 (3.5 mg/mL).
d Relative MFI (MFI normalized to NBD-556 (control)) = ([MFI of compound]-[MFI of 4C11])/([MFI of
NBD-556]-[MFI of 4C11]). MFI of 4C11: 7.76.
e ND: not done
Figure 2. Comparison of modeled structures of 5-substituted indole-type (9a) and 6-substituted
indole-type compounds (12a) based on superposition on the NBD-556 structure. Spartan’ 10 was used for
calculation.16 The chlorine group in the NBD-556 model structure (gray) is colored green.
The conformationally flexible compounds 31, 34, 37 and 41-43 with a Gly-linker were evaluated
11

similarly (Table 2). Among the compounds containing a 4-chlorophenyl group, a tetramethylpiperidine
compound (31) has an IC50 = 36.9 M and a dicyclohexylpiperidine compound (37) has IC50 = 2.20 M
The anti-HIV activity of compound 37 is approximately one-third of that of NBD-556, but the
cytotoxicity of compound 37 is lower than that of NBD-556. These results suggest that the carbonyl
oxygen in the oxalamide linker of NBD-556, which is located near the aromatic ring and is absent from
compounds 31 and 37, might contribute positively to the interaction with the Phe43 cavity. Among the
compounds containing a 1,3-benzodioxole group, a dicyclohexylpiperidine-type compound (43) showed
moderate anti-HIV activity with IC50 = 10.8 M. All conformationally flexible compounds showed
relatively low MFI values compared with NBD-556 and some conformationally rigid compounds,
indicating that an increase of flexibility by the substitution of the Gly-linker caused a significant decrease
of the ability to change the conformation of gp120. Conformational restriction of the linker might be
critical for the induction of the conformational change of gp120.
Table 2
Anti-HIV activity, cytotoxicity and gp120 conformational change inducibility of conformationally
flexible compounds with the Gly-linker.
anti-HIV activity
(IC50 [M])a
0.63
cytotoxicity
(CC50 [M])b
70.4
FACS
(MFI)c (Rel. MFI)d
FACS
R1
Compd.
65.3
30.2
23.8
19.6
23.4
18.5
28.7
1
0.22
0.081
-
NBD-556
A
B
C
A
B
C
36.9
ND
31
34
37
41
42
43
>100
ND
2.20
>100
>30.0
ND
0.073
-
>100
ND
10.8
ND
0.19
a IC50 values were determined using the cYTA48P strain and PM1/CCR5 cells.
12

b CC50 values were determined using PM1/CCR5 cells.
c FACS analysis was performed using cYTA48P-infected PM1/CCR5 cells, a test compound (100 M)
and 4C11 (3.5 mg/mL).
d Relative MFI (MFI normalized to NBD-556 (control)) = ([MFI of compound]-[MFI of 4C11])/([MFI of
NBD-556]-[MFI of 4C11]). MFI of 4C11: 20.1.
e ND: not done
2.3. Docking studies
To obtain a plausible model of the binding of CD4 mimic molecules in the Phe43 cavity of gp120,
docking studies were performed based on the X-ray crystal structure of the complex of gp120 and
NBD-556 (PDB: 3TGS).11 Docking simulations were performed with Molecular Operating Environment
(MOE) (MOLSIS Inc., Tokyo).18 The docking structures of compounds 26, 37 and 43, which have high or
moderate anti-HIV activity, are shown in Fig. 3. I Docking studies revealed that when the nitrogen atoms
of the piperidine ring and the linker are closer to Val430 and Asn425, respectively, these compounds have
higher anti-HIV activity. This suggests that the interaction of CD4 mimic molecules with Val430 and
Asn425 is important.
Val430
Asn425
Gly473
13

Figure 3. Docking models of compounds 26, 37 and 43 with the Phe43 cavity of gp120 (PDB: 3TGS).11
Numerical values mean distances (Ǻ).
The superimposed structures of compounds MTA-103, 24 and 41, which have 1,3-benzodioxole
and tetramethylpiperidine moieties, are shown in Fig. 4. The IC50 value of MTA-103 is 6.20 M,10 that of
24 is 23 M and of 41 is >30.0 M. The aromatic moieties of these docking structures are similar, but the
piperidine moieties are clearly different. When the piperidine nitrogen atoms are closer to Val430, these
compounds have higher anti-HIV activity. This suggests that the tetramethylpiperidine moiety in
compound 24 is not close to Val430 because the conformationally rigid moiety, 5,6-methylenedioxyindole,
binds to the Phe43 cavity, ant that the tetramethylpiperidine moiety in compound 41 is distant from
Val430 because of the flexible Gly-linker. The oxalamide might be the most suitable linker. Flexibility
and rigidity in the junction region between the aromatic ring and the amide might have a significant effect
on the interaction of the piperidine moieties with Val430.
Val430
Figure 4. Superposition of docking structures of MTA-10310 (blue), compounds 24 (green) and 41
(orange).
3. Conclusion
In this study, the introduction of an indole moiety as a scaffold in a CD4 mimic molecule was successful,
14

producing new compounds with significant anti-HIV activity and low cytotoxicity. Furthermore, judging
from molecular modeling, flexibility and rigidity in the junction between the oxalamide linker and the
aromatic moiety of CD4 mimic molecules could have a significant effect on the interaction of the
piperidine moiety with Val430, which contributes to high anti-HIV activity. These results will be useful in
the future design of HIV entry inhibitors based on CD4 mimic.
4. Experimental
4.1. Chemistry
All of the compounds were synthesized as shown in Schemes 1-4. Detailed procedures and
characterization data are described in Supplementary data.
4.2. Anti-HIV assay and cytotoxicity assay
The anti-HIV activities of the synthetic compounds were evaluated against an R5 primary isolate
cYTA48P strain17b in PM1/CCR5 cells and the IC50 values were determined by a single round assay as the
concentrations of the compounds that led to 50% reduction in relative light units (RLU) relative to virus
control wells after subtraction of background RLU in cell control wells. Cytotoxicity of the compounds
based on the viability of mock-infected PM1/CCR5 cells was evaluated using the WST-8 method.
4.3. FACS analysis
cYTA48P chronically infected PM1 cells were pre-incubated with 100 M of a test compound for 15 min,
and then incubated with an anti-HIV-1 mAb, 4C11, at 4 °C for 15 min. The cells were washed with PBS,
and fluorescein isothiocyanate (FITC)-conjugated mouse anti-human IgG antibody was used for
antibody-staining. Flow cytometry data for the binding of 4C11 to the Env-expressing cell surface in the
presence of a test compound were obtained. Data are values of the mean fluorescence intensity (MFI)
from a minimum of two independent experiments.
15

4.4. Molecular modeling
Molecular modeling calculations were performed using compounds docked into gp120 (PDB ID: 3TGS)
with MOE (version 2012.10).11,18 In the molecular modeling, global energy minimization of compounds
docked into gp120 was searched.
Supplementary data
Supplementary data (synthetic procedures and characterization data of compounds) associated
with this article can be found, in the online version, at doi: .bmc.
.
Acknowledgments
This work was supported in part by the grant for Research Program on HIV/AIDS from Japan Agency for
Medical Research and Development, (AMED Grant Numbers 18fk0410002h0003 and
18fk0410006h0303), and Grant-in-Aid for Scientific Research from the Ministry of Education, Culture,
Sports, Science, and Technology of Japan (JSPS KAKENHI Grant Number JP15H04652). It was also
supported in part by the Platform for Drug Discovery, Informatics, and Structural Life Science of MEXT,
Japan, and the Cooperative Research Project of Research Center for Biomedical Engineering. We are
grateful to Prof. Yoshio Hayashi and Dr. Akihiro Taguchi, Tokyo University of Pharmacy and Life
Sciences for their assistance in the molecular modeling.
References and notes
1. (a) Ohashi N, Tamamura H. Peptide-derived mid-sized anti-HIV agents. In: Ryadnov M, Hudecz F,
eds. Amino Acids, Peptides and Proteins. Cambridge, UK: The Royal Society of Chemistry;
2017;41:1−29; (b) Tamamura H, Kobayakawa T, Ohashi N. In: Mid-size drugs based on peptides and
peptidomimetics: A new drug category. Singapore: Springer Briefs in Pharmaceutical Science &
Drug Development; 2018:1-100.
16

2. (a) Tamamura H, Xu Y, Hattori T, Zhang X, Arakaki R, Kanbara K, Omagari A, Otaka A, Ibuka T,
Yamamoto N, Nakashima H, Fujii N. A low-molecular-weight inhibitor against the chemokine
receptor CXCR4: a strong anti-HIV peptide T140. Biochem Biophys Res Commun.
1998;253:877−882;
(b) Tamamura H, Omagari A, Oishi S, Kanamoto T, Yamamoto N, Peiper SC, Nakashima H, Otaka A,
Fujii, N. Pharmacophore identification of a specific CXCR4 inhibitor, T140, leads to development of
effective anti-HIV agents with very high selectivity indexes. Bioorg Med Chem Lett.
2000;10:2633−2637;
(c) Fujii N, Oishi S, Hiramatsu K, Araki T, Ueda S, Tamamura H, Otaka A, Kusano S, Terakubo S,
Nakashima H, Broach JA, Trent JO, Wang Z, Peiper SC. Molecular-size reduction of a potent
CXCR4-chemokine antagonist using orthogonal combination of conformation- and sequence-based
libraries. Angew Chem, Int Ed. 2003;42:3251−3253;
(d) Tamamura H, Hiramatsu K, Ueda S, Wang Z, Kusano S, Terakubo S, Trent JO, Peiper SC,
Yamamoto N, Nakashima H, Otaka A, Fujii N. Stereoselective synthesis of
[L-Arg-L/D-3-(2-naphthyl)alanine]-type (E)-alkene dipeptide isosteres and its application to the
synthesis and biological evaluation of pseudopeptide analogues of the CXCR4 antagonist FC131. J
Med Chem. 2005;48:380−391;
(e) Tamamura H, Araki T, Ueda S, Wang Z, Oishi S, Esaka A, Trent JO, Nakashima H, Yamamoto N,
Peiper SC, Otaka A, Fujii N. Identification of novel low molecular weight CXCR4 antagonists by
structural tuning of cyclic tetrapeptide scaffolds. J Med Chem. 2005;48:3280−3289.
3. Holz-Smith SL, Sun IC, Jin L, Matthews TJ, Lee KH, Chen CH. Role of human immunodeficiency
virus (HIV) type 1 envelope in the anti-HIV activity of the betulinic acid derivative IC9564.
Antimicrob Agents Chemother. 2001;45:60−66.
4. Lin P-f, Blair WS, Wang T, Spicer TP, Guo Q, Zhou N, Gong Y-F, Wang H.-GH, Rose R, Yamanaka
GA, Robinson BS, Li C.-B, Demers GS, Deminie C, Fridell RA, Yang Z, Zadjura LM, Meanwell NA,
Colonno RJ. A small molecule HIV-1 inhibitor that targets the HIV-1 envelope and inhibits CD4
17

receptor binding. Proc Natl Acad Sci U S A. 2003;100:11013−11018.
5. (a) Zhao Q, Ma L, Jiang S, Lu H, Liu S, He Y, Strick N, Neamati N, Debnath AK. Identification of
N-phenyl-N’-(2,2,6,6-tetramethyl-piperidin-4-yl)-oxalamides as a new class of HIV-1 entry inhibitors
that prevent gp120 binding to CD4. Virology. 2005;339:213−225.
(b) Madani N, Sch n A, Princiotto AM, Lalonde JM, Courter JR, Soeta T, Ng D, Wang L, Brower ET,
Xiang SH, Kwon YD, Huang CC, Wyatt R, Kwong PD, Freire E, Smith AB 3rd, Sodroski J.
Small-molecule CD4 mimics interact with a highly conserved pocket on HIV-1 gp120. Structure.
2008;16:1689−1701.
(c) Lalonde JM, Elban MA, Courter JR, Sugawara A, Soeta T, Madani N, Princiotto AM, Kwon YD,
Kwong PD, Schon A, Freire E, Sodroski J, Smith AB 3rd. Design, synthesis and biological evaluation
of small molecule inhibitors of CD4-gp120 binding based on virtual screening. Bioorg. Med. Chem.
2011;19:91−101.
(d) Lalonde JM, Kwon YD, Jones DM, Sun AW, Courter JR, Soeta T, Kobayashi T, Princiotto AM,
Wu X, Sch n A, Freire E, Kwong PD, Mascola JR, Sodroski J, Madani N, Smith AB 3rd.
Structure-based design, synthesis, and characterization of dual hotspot small-molecule HIV-1 entry
inhibitors. J. Med. Chem. 2012;55:4382−4396.
(e) Courter JR, Madani N, Sodroski J, Schon A, Freire E, Kwong PD, Hendrickson WA, Chaiken IM,
Lalonde JM, Smith AB 3rd. Structure-based design, synthesis and validation of CD4-mimetic small
molecule inhibitors of HIV‑ 1 entry: conversion of a viral entry agonist to an antagonist. Acc. Chem.
Res. 2014;47:1228−1237.
f adani Princiotto ch n , LaLonde J, Feng Y, Freire E, Park J, Courter JR, Jones DM,
Robinson J, Liao HX, Moody MA, Permar S, Haynes B, Smith AB, Wyatt R, Sodroski, J.
CD4-mimetic small molecules sensitize human immunodeficiency virus to vaccine-elicited
antibodies. J. Virol. 2014;88:6542−6555.
(g) Curreli F, Choudhury S, Pyatkin I, Zagorodnikov VP, Bulay AK, Altieri A, Kwon YD, Kwong PD,
Debnath AK. Design, synthesis, and antiviral activity of entry inhibitors that target the CD4-binding
18

site of HIV-1. J. Med. Chem. 2012;55:4764−4775.
h ch n , Madani N, Klein JC, Hubicki A, Ng D, Yang X, Smith AB 3rd, Sodroski J, Freire E.
Thermodynamics of binding of a low-molecular-weight CD4 mimetic to HIV-1 gp120. Biochemistry.
2006;45:10973−10980.
(i) ch n , Lam SY, Freire E. Thermodynamics-based drug design: strategies for inhibiting
protein–protein interactions. Future Med. Chem. 2011;3:1129−1137.
(j) Kwon YD, Finzi A, Wu X, Dogo-Isonagie C, Lee LK, Moore LR, Schmidt SD, Stuckey J, Yang Y,
Zhou T, Zhu J, Vicic DA, Debnath AK, Shapiro L, Bewley CA, Mascola JR, Sodroski JG, Kwong PD.
Unliganded HIV-1 gp120 core structures assume the CD4-bound conformation with regulation by
quaternary interactions and variable loops. Proc Natl Acad Sci U S A. 2012;109:5663−5668;
(k) Shrivastava I, Lalonde JM. Enhanced dynamics of HIV gp120 glycoprotein by small molecule
binding. Biochemistry. 2011;50:4173−4183.
(l) Tintori C, Selvaraj M, Badia R, Clotet B, Esté JA, Botta M. Computational studies identifying
entry inhibitor scaffolds targeting the Pheꢀ43 cavity of HIV ‐ 1 gp120. ChemMedChem.
2013;8:475−483.
(m) Lalonde JM, Le-Khac M, Jones DM, Courter JR, Park J, Sch n A, Princiotto AM, Wu X,
Mascola JR, Freire E, Sodroski J, Madani N, Hendrickson WA, Smith AB 3rd. Structure-based design
and synthesis of an HIV-1 entry inhibitor exploiting X-ray and thermodynamic characterization. ACS
Med. Chem. Lett. 2013;4:338−343.
(n) Curreli F, Haque K, Xie L, Qiu Q, Xu J, Yong W, Tong X, Debnath K. Synthesis, antiviral activity
and resistance of a novel small molecule HIV-1 entry inhibitor. Bioorg. Med. Chem.
2015;23:7618−7628.
(o) Curreli F, Kwon Y, Zhang H, Scacalossi D, Belov DS, Tikhonov AA, Andreev IA, Altieri A,
Kurkin AV, Kwong PD, Debnath AK. Structure-based design of a small molecule CD4-antagonist
with broad spectrum anti-HIV‑ 1 activity. J. Med. Chem. 2015;58:6909−6927.
(p) Curreli F, Belov DS, Ramesh RR, Patel N, Altieri A, Kurkin AV, Debnath AK. Design, synthesis
19

and evaluation of small molecule CD4-mimics as entry inhibitors possessing broad spectrum
anti-HIV-1 activity. Bioorg. Med. Chem. 2016;24:5988−6003.
(q) Melillo B, Liang S, Park J, Schon A, Courter JR, LaLonde JM, Wendler DJ, Princiotto AM,
Seaman MS, Freire E, Sodroski J, Madani N, Hendrickson WA, Smith AB 3rd. Small-molecule
CD4-mimics: structure-based optimization of HIV‑ 1 entry inhibition. ACS Med. Chem. Lett. 2016;7:
330−334.
(r) Curreli F, Young YD, Belov DS, Ramesh RR, Kurkin AV, Altieri A, Kwong PD, Debnath AK.
Synthesis, antiviral potency, in vitro ADMET, and X‑ ray structure of potent CD4 mimics as entry
inhibitors that target the Phe43 cavity of HIV‑ 1 gp120. J. Med. Chem. 2017;60:3124−3153.
(s) Madani N, Princiotto AM, Mach L, Ding SL, Prevost J, Richard J, Hora B, Sutherland L, Zhao
CA, Conn, BP, Bradley T, Moody MA, Melillo B, Finzi A, Haynes BF, Smith AB 3rd, Santra S,
Sodroski J. A CD4-mimetic compound enhances vaccine efficacy against stringent
immunodeficiency
virus
challenge.
Nat.
Commun.
2018.
https://doi.org/10.1038/s41467-018-04758-9.
6. Narumi T, Arai H, Yoshimura K, Harada S, Nomura W, Matsushita S, Tamamura H. Small molecular
CD4 mimics as HIV entry inhibitors. Bioorg Med Chem. 2011;19:6735−6742.
7. Ohashi N, Harada S, Mizuguchi T, Irahara Y, Yamada Y, Kotani M, Nomura W, Matsushita S,
Yoshimura K, Tamamura H. Small-molecule CD4 mimics containing mono-cyclohexyl moieties as
HIV entry inhibitors. ChemMedChem. 2016;11:940−946.
8. Narumi T, Arai H, Yoshimura K, Harada S, Hirota Y, Ohashi N, Hashimoto C, Nomura W, Matsushita
S, Tamamura H. CD4 mimics as HIV entry inhibitors: Lead optimization studies of the aromatic
substituents. Bioorg Med Chem. 2013;21:2518−2526.
9. Yamada Y, Ochiai C, Yoshimura K, Tanaka T, Ohashi N, Narumi T, Nomura W, Harada S, Matsushita
S, Tamamura H. CD4 mimics targeting the mechanism of HIV entry. Bioorg Med Chem Lett.
2010;20:354−358.
10. Mizuguchi T, Harada S, Miura T, Ohashi N, Narumi T, Mori H, Irahara Y, Yamada Y, Nomura W,
20

Matsushita S, Yoshimura K, Tamamura H. A minimally cytotoxic CD4 mimic as an HIV entry
inhibitor. Bioorg Med Chem Lett. 2016;26:397−400.
11. Huang CC, Venturi M, Majeed S, Moore MJ, Phogat S, Zhang MY, Dimitrov DS, Hendrickson WA,
Robinson J, Sodroski J, Wyatt R, Choe H, Farzan M, Kwong PD. Structural basis of tyrosine
sulfation and VH-gene usage in antibodies that recognize the HIV type 1 coreceptor-binding site on
gp120. Proc Natl Acad Sci U S A. 2004;101:2706−2711.
12. (a) Rydon HN, Tweddle JC. Experiments on the synthesis of Bz-substituted indoles and tryptophans.
Part III. The synthesis of the four Bz-chloro-indoles and -tryptophans. J Chem Soc. 1955;3499−3503;
(b) Rosauer KG, Ogawa AK, Willoughby CA, Ellsworth KP, Geissler WM, Myers WR, Chapman KT,
Harris G, Moller DE. Novel 3,4-dihydroquinolin-2(1H)-one inhibitors of human glycogen
phosphorylase a. Bioorg Med Chem. 2003;13:4385−4388;
c Lüth Lӧwe W. yntheses of 4-(indole-3-yl)quinazolines – A new class of epidermal growth
factor receptor tyrosine kinase inhibitors. Eur J Med Chem. 2008;43:1478−1488;
(d) Yoshikawa, K.; Yokomizo, A.; Naito, H.; Haginoya, N.; Kobayashi, S.; Yoshino, T.; Nagata, T.;
Mochizuki, A.; Osanai, K.; Watanabe, K.; Kanno, H.; Ohta, T. Design, synthesis, and SAR of
cis-1,2-diaminocyclohexane derivatives as potent factor Xa inhibitors. Part I: Exploration of 5–6
fused rings as alternative S1 moieties. Bioorg Med Chem. 2009;17:8206−8220;
(e) Mayes BA, Chaudhuri NC, Hencken CP, Jeannot F, Latham GM, Mathieu S, McGarry FP, Stewart
AJ, Wang J, Moussa A. Robust synthesis of methyl 5-chloro-4-fluoro-1H-indole-2-carboxylate: a key
intermediate in the preparation of an HIV NNRTI candidate. Org Process Res Dev.
2010;14:1248−1253;
(f) Onajole OK, Pieroni M, Tipparaju SK, Lun S, Stec J, Chen G, Gunosewoyo H, Guo H,
Ammerman NC, Bishai WR, Kozikowski AP. Preliminary structure−activity relationships and
biological evaluation of novel antitubercular indolecarboxamide derivatives against drug-susceptible
and drug-resistant Mycobacterium tuberculosis strains. J Med Chem. 2013;56:4093−4103;
(g) Hayashi S, Ueno N, Murase A, Takada J. Design, synthesis and structure–activity relationship
21

studies of novel and diverse cyclooxygenase-2 inhibitors as anti-inflammatory drugs. J Enzyme Inhib
Med Chem. 2014;29:846−867;
(h) Franz ND, Belardinelli JM, Kaminski MA, Dunn LC, de Moura VCN, Blaha, MA, Truong DD, Li
W, Jackson M, North EJ. Design, synthesis and evaluation of indole-2-carboxamides with pan
anti-mycobacterial activity. Bioorg Med Chem. 2017;25:3746–3755.
13. (a) Nguyen W, Howard BL, Jenkins DP, Wulff H, Thompson P E, Manallack DT. Structure–activity
relationship exploration of Kv1.3 blockers based on diphenoxylate. Bioorg Med Chem Lett.
2012;22:7106−7109;
(b) Narumi T, Arai H, Yoshimura K, Harada S, Nomura W, Matsushita S, Tamamura H. Small
molecular CD4 mimics as HIV entry inhibitors. Bioorg Med Chem. 2011;19:6735−6742;
(c) Narumi T, Arai H, Yoshimura K, Harada S, Hirota Y, Ohashi N, Hashimoto C, Nomura W,
Matsushita S, Tamamura H. CD4 mimics as HIV entry inhibitors: Lead optimization studies of the
aromatic substituents. Bioorg Med Chem. 2013;21:2518−2526.
14. Chen C, Liebermann DR, Larsen RD, Verhoeven, TR, Reider, PJ. Syntheses of indoles via a
palladium-catalyzed annulation between iodoanilines and ketones. J Org Chem. 1997;62:2676−2677.
15. Asundaria ST, Patel NS, Patel KC. Novel 3-[4-(diethylamino)phenyl]-4-substituted-1-ylsulfonyl)
sydnones: Synthesis, characterization and antimicrobial studies. Org Commun. 2010;3:30−38.
16. The optimized geometries calculated by P 3 Density Functional with the partan’10 version 1.1.0:
Wavefunction Inc., Irvine, California) were used as starting geometries for DFT calculations. DFT
calculations were carried out with the same program. The geometries were fully optimized in vacuo
by using the B3LYP/6-31G* level of theory.
17. (a) Narumi T, Ochiai C, Yoshimura K, Harada S, Tanaka T, Nomura W, Arai H, Ozaki T, Ohashi N,
Matsushita S, Tamamura H. CD4 mimics targeting the HIV entry mechanism and their hybrid
molecules with a CXCR4 antagonist. Bioorg Med Chem Lett. 2010;20:5853−5858;
(b) Yoshimura K, Harada S, Shibata J, Hatada M, Yamada Y, Ochiai C, Tamamura H, Matsushita S.
Enhanced exposure of human immunodeficiency virus type 1 primary isolate neutralization epitopes
22

through binding of CD4 mimetic compounds. J Virol. 2010;84:7558−7568.
18. Molecular Operating Environment (MOE), 2012.10; Chemical Computing Group Inc., Montreal, QC,
Canada, 2012.
23