Small molecular CD4 mimics as HIV entry inhibitors

Article abstract of DOI:10.1016/j.bmc.2011.09.045

Derivatives of CD4 mimics were designed and synthesized to interact with the conserved residues of the Phe43 cavity in gp120 to investigate their anti-HIV activity, cytotoxicity, and CD4 mimicry effects on conformational changes of gp120. Significant potency gains were made by installation of bulky hydrophobic groups into the piperidine moiety, resulting in discovery of a potent compound with a higher selective index and CD4 mimicry. The current study identified a novel lead compound 11 with significant anti-HIV activity and lower cytotoxicity than those of known CD4 mimics.

Full text of DOI:10.1016/j.bmc.2011.09.045

Bioorganic & Medicinal Chemistry 19 (2011) 6735–6742
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Small molecular CD4 mimics as HIV entry inhibitors
Tetsuo Narumi a, Hiroshi Arai a, Kazuhisa Yoshimura b, Shigeyoshi Harada b, Wataru Nomura a,
Shuzo Matsushita b, Hirokazu Tamamura a,
⇑
a Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Chiyoda-ku, Tokyo 101-0062, Japan
b Center for AIDS Research, Kumamoto University, Kumamoto 860-0811, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Derivatives of CD4 mimics were designed and synthesized to interact with the conserved residues of the
Phe43 cavity in gp120 to investigate their anti-HIV activity, cytotoxicity, and CD4 mimicry effects on con-
formational changes of gp120. Significant potency gains were made by installation of bulky hydrophobic
groups into the piperidine moiety, resulting in discovery of a potent compound with a higher selective index
and CD4 mimicry. The current study identified a novel lead compound 11 with significant anti-HIV activity
and lower cytotoxicity than those of known CD4 mimics.
Received 5 August 2011
Revised 23 September 2011
Accepted 24 September 2011
Available online 29 September 2011
Keywords:
CD4 mimic
Ó 2011 Elsevier Ltd. All rights reserved.
HIV entry
gp120-CD4 interaction
Phe43 cavity
1. Introduction
more sensitive to neutralizing antibodies.9 While such properties
are promising for the development of HIV entry inhibitors and
The dynamic supramolecular mechanism of HIV cellular inva-
sion has emerged as a key target for blocking HIV entry into host
cells.1 HIV entry begins with the interaction of a viral envelope gly-
coprotein gp120 and a cell surface protein CD4.2 This triggers
extensive conformational changes in gp120 exposing co-receptor
binding domains and allowing the subsequent binding of gp120
to a co-receptor, CCR53/CXCR4.4 Following the viral attachment
and co-receptor binding, gp41, another viral envelope glycoprotein
mediates the fusion of the viral and cell membranes, thus complet-
ing the infection. Molecules interacting with each of these steps are
potential candidates for anti-HIV-1 drugs. In particular, discovery
and development of novel drugs that inhibit the viral attachment
are required for blocking the HIV infection at an early stage.5
In 2005, small molecular CD4 mimics targeting the viral attach-
ment were identified by an HIV syncytium formation assay and
shown to bind within the Phe43 cavity, a highly conserved pocket
on gp120,6 which is a hydrophobic cavity occupied by the aromatic
ring of Phe43 of CD4.7 These molecules are comprised of three
essential moieties: an aromatic ring, an oxalamide linker, and a
piperidine ring (Fig. 1) and show micromolar order potency against
diverse HIV-1 strains including laboratory and primary isolates.
Furthermore, they possess the unique ability to induce the confor-
mational changes in gp120 required for binding with soluble CD4.8
Such CD4 mimicry can be an advantage for rendering the envelope
the use combinatorially with neutralizing antibodies, cytotoxicity
is one of the drawbacks of CD4 mimics.
To date, we and others have performed structure–activity rela-
tionship (SAR) studies of CD4 mimics based on modifications of the
aromatic ring, the oxalamide linker, and the piperidine moiety of
CD4 mimics. In an initial survey of SAR studies of NBD-556 and
NBD-557, Madani et al. revealed that potency (i.e., CD4 binding
and mimicry) was highly sensitive to modifications of the aromatic
ring, which is thought to bind in the Phe43 cavity of gp120 (Fig. 1).
The CD4 mimic analogs (JRC-II-191) with a para-chloro-meta-
fluorophenyl ring had significantly increased affinity for gp120.10
Our SAR studies also revealed that a certain size and electron-
withdrawing ability of the para-substituents are indispensable
for potent anti-HIV activity.11 Furthermore, the replacement of
the chlorine group at the para position with a methyl group which
is almost as bulky as a bromine atom leads to improvement of sol-
ubility of the compounds in buffer to provide the reproducibility in
the biological studies with comparable biological activities.
Further SAR studies were focused on the piperidine moiety of
CD4 mimics to investigate its contribution to biological activities,
and we found that the piperidine ring is critical for the CD4 mim-
icry on the conformational changes in gp120 and that substituents
on the nitrogen of the piperidine moiety can contribute signifi-
cantly to both anti-HIV activity and cytotoxicity.12 Based on these
SARs and our modeling study, we speculate that interactions of the
piperidine moiety with several amino acids in the vicinity of the
Phe43 cavity in gp120, specifically an electrostatic interaction with
⇑
Corresponding author. Tel.: +81 3 5280 8036; fax: +81 3 5280 8039.
E-mail address: tamamura.mr@tmd.ac.jp (H. Tamamura).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.09.045

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T. Narumi et al. / Bioorg. Med. Chem. 19 (2011) 6735–6742
biological activity, including anti-HIV activity and CD4 mimicry,
similar to that of the parent compound NBD-556.12 Furthermore,
to interact with Val430 by hydrophobic interaction, the methyl
groups on the piperidine ring were replaced with cyclohexyl
groups to prepare a novel CD4 mimic analog with enhanced
hydrophobicity.
NH
O
H
N
N
H
O
X
Y
X = Cl, Br, Me
Y = H, F
2.1. Chemistry
Figure 1. CD4 mimics.
The syntheses of CD4 mimics are outlined in Scheme 1. CD4
mimics with guanidine, thiourea, and urea groups on the piperidine
moiety were prepared using our previously reported method.12
Coupling of p-chloroaniline with ethyl chloroglyoxylate followed
by aminolysis of the ethyl ester with 4-amino-N-benzylpiperidine
under microwave conditions (150 °C, 3 h) gave the corresponding
amide. Removal of the benzyl group with 1-chloroethyl chlorofor-
mate14 gave the free piperidine moiety, which was modified to pro-
duce the desired compounds 4–8 (Scheme 1).
For synthesis of a CD4 mimic derivative with two cyclohexyl
groups, treatment of 2,2,6,6-tetramethylpiperidin-4-one 9 with
cyclohexanone in the presence of ammonium chloride furnished
a 2,6-substituted piperidin-4-one derivative,15 and reductive ami-
nation with benzylamine and subsequent removal of benzyl group
provided a primary amine 10. Microwave-assisted aminolysis of
ester 2 with amine 10 yielded the desired dicyclohexyl-substituted
analog 11 (Scheme 2). The synthesis of the other compounds is
described in Supplementary data.
Asp368 and a hydrophobic interaction with Val430, are critical for
biological activity. LaLonde et al. focused on modifications of the
piperidine moiety using computational approaches, adducing evi-
dence for the importance of these interactions to the binding affin-
ity against gp120.13 Based on these results, we envisioned that an
enhancement of the interaction of CD4 mimics with residues asso-
ciated with the Phe43 cavity in gp120 would lead to the increase of
their potency and CD4 mimicry inducing the conformational
changes of gp120, and the decrease of their cytotoxicity. Thus, in
this study a series of CD4 mimics, which were designed to interact
with the conserved residues in the Phe43 cavity, were synthesized
to increase binding affinity for gp120, and the appropriate SAR
studies were performed.
2. Results and discussion
Two types of CD4 mimic analogs were designed: (1) CD4 mim-
ics with the ability to interact electrostatically with Asp368, and
(2) CD4 mimics with the ability to interact hydrophobically with
Val430 ( Fig. 2). The X-ray structure of gp120 bound to soluble
CD4 (PDB: 1RZJ) revealed that the guanidino group of Arg59 of
CD4 is involved in a hydrogen bond with Asp368 of gp120. In order
to mimic this interaction, a guanidino and related groups such as
thiourea and urea were introduced to the piperidine moiety of
the CD4 mimic derivative COC-021, which was developed in order
to modify the nitrogen of the piperidine moiety and which showed
2.2. Biological studies
The anti-HIV activity of synthetic CD4 mimics was evaluated
in a single-round viral infective assay. Inhibition of HIV-1 infec-
tion was measured as reduction in b-galactosidase gene expres-
sion after a single-round of virus infection of TZM-bl cells as
described previously.9 IC50 was defined as the concentration that
caused a 50% reduction in the b-galactosidase activity (relative
light units [RLU]) compared to virus control wells. Cytotoxicity
Val430
Asp368
NH
O
H
N
N
H
O
X
Y
CD4 mimics
NH
O
H
N
N
H
O
Cl
COC-021
Z
O
N
NH2
O
NH
H
N
H
N
N
H
N
H
electrostatic
interaction
hydrophobic
interaction
O
O
Cl
Cl
Z = N, O S
Figure 2. Design strategy for novel CD4 mimics with enhanced electrostatic/hydrophobic interaction.

T. Narumi et al. / Bioorg. Med. Chem. 19 (2011) 6735–6742
6737
Table 1
O
H
NH2
Effects of the nitrogen-substituents on anti-HIV activity and cytotoxicity of CD4
a
N
mimic analogsa
OEt
Cl
O
O
H
Cl
1
2
b
c
N
IC50
M)
CC50
M)
SI (CC50/IC50
)
R
(
l
(
l
O
X
O
O
NH
H
b,c,d
N
Compd
X
R
YTA (R5)
51
N
H
H
N
O
3
3d
4e
Cl
Cl
7.0
7.3
12
NH
N
Cl
NH
H
N
e for 4
f for 5
g for 6
6.1
5.5
8.3
72
42
R
N
NH2
S
H
N
N
H
H
N
N
N
h for 7
i for 8
5
6
Cl
Cl
7.6
37
O
NH2
O
Cl
H
N
O
O
NH
NH2
310
7
Ph
)
4 (R =
)
)
(R =
NH2
O
N
H
H
N
S
N
N
7
8
Cl
Cl
11
6.2
0.56
—
5
6
(R =
(R =
8 (R =
)
HN Ph
O
NH2
Ph
H
N
5.1
ND
35
O
Ph
)
NH2
H
N
12 (NBD-556) Cl
NH
NH
0.61
8.4
57
31
Scheme 1. Synthesis of N-modified piperidine derivatives 4–8. Reagents and
conditions: (a) Ethyl chloroglyoxylate, Et3N, THF, quant.; (b) 1-benzyl-4-aminopi-
peridine, Et3N, EtOH, 150 °C, microwave, 78%; (c) 1-chloroethyl chloroformate,
CH2Cl2; (d) MeOH, reflux, 64% in two steps; (e) 1H-pyrazole-1-carboxamidine
hydrochloride, Et3N, DMF, 61%; (f) (trimethylsilyl)isothiocyanate, CHCl3, 36%;
(g) (trimethylsilyl)isocyanate, CHCl3, 30%; (h) phenyl isocyanate, CHCl3, 32%,
(i) benzoyl chloride, Et3N, CH2Cl2, 68%.
H
N
13
Me
260
a
All data with standard deviation are the mean values for at least three inde-
pendent experiments (ND = not determined)
b
IC50 values are based on the reduction in the b-galactosidase activity in TZM-bl
cells.
a,b,c
NH
c
NH
CC50 values are based on the reduction of the viability of mock-infected PM1/
CCR5 cells.
O
d
H2N
O
Desalted by satd NaHCO3 aq.
TFA salts.
e
9
10
d
NH
H
N
the different assay system. All of the synthesized novel deriva-
tives of compound 12 showed moderate to potent anti-HIV activ-
ity. A guanidine derivative 4 and thiourea derivative 5 showed
potent anti-HIV activities (IC50 of 4 = 6.1
5 = 5.5 M) but their potency was approximately 10-fold lower
than that of the parent compound 12. A urea derivative 6 also
showed potent anti-HIV activity (IC50 = 8.3 M) and exhibited
lower cytotoxicity (CC50 = 310 M). On the other hand, introduc-
N
H
O
11
Cl
lM and IC50 of
l
Scheme 2. Synthesis of dicyclohexyl derivative 11. Reagents and conditions: (a)
Cyclohexanone, NH4Cl, DMSO, 60 °C; (b) benzylamine, NaBH4, MeOH; (c) 10% Pd/C,
H2, MeOH, 7% from 9; (d) 2, Et3N, EtOH, 150 °C, microwave, 17%.
l
l
tion of a phenyl group in the urea derivative 6, led to an N-phe-
nylurea derivative 7, with an increase of cytotoxicity
of the compounds based on the viability of mock-infected PM1/
CCR5 cells was evaluated using WST-8 method. The assay results
for the CD4 mimics 3–8 are shown in Table 1. Compound 12
(NBD-556) showed potent anti-HIV activity; its IC50 value was
(CC50 = 6.2
anti-HIV activity, an N-benzoyl derivative 8 was also tested. The
IC50 value of 8 was 5.1 M, which is equipotent with the thioam-
lM). To examine the influence of the N–H group on
l
ide derivative 5. The N-benzoyl derivative 8 was essentially equi-
potent with 3 and this result suggests the presence of the
hydrogen atom of the N–H group does not contribute to an in-
crease in anti-HIV activity. The thiourea derivative 5 and the
N-phenylurea derivative 7, which have more acidic protons (pKa
of thiourea and N-phenylurea; 21.0 and 19.5,16 respectively) than
the urea derivative 6 (pKa of urea; 26.916), were found to exhibit
relatively strong cytotoxicity. This observation indicates that
0.61 lM, and it is thus 13–20-fold more potent than the reported
values.11,12 Although previous studies found that compound 13,
with a methyl group at the p-position of the phenyl ring, and
compound 3, with no dimethyl groups on the piperidine ring,
showed potent anti-HIV activity, only moderate activities were
observed in the current study; this is about 12–14-fold less
potency than reported for compound 12 and is probably due to

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T. Narumi et al. / Bioorg. Med. Chem. 19 (2011) 6735–6742
Table 2
Table 3
Anti-HIV activity and cytotoxicity of CD4 mimic analogs 11, 12, and 14–17a
Anti-HIV activity of CD4 mimic 12 and dicyclohexyl derivative 11a
b
c
Compd
R
IC50
(l
M)
IC50 (lM)
O
H
Single-round assay Multi-round assay
N
b
c
R
IC50
(lM) CC50 (lM)
SI (CC50/IC50
)
O
H
N
Cl
12 (NBD-556)
NH
NH
0.61
0.68
0.90
0.56
Compd
R
YTA (R5)
H
N
H
N
11
NH
0.68
120
176
11
H
N
a
14
15
16
F
3.1
>500
>500
>500
>160
—
All data with standard deviation are the mean values for at least three inde-
pendent experiments.
H
N
b
IC50 values of the single-round assay are based on the reduction in the b-
>100
Cl
galactosidase activity in TZM-bl cells.
c
IC50 values of the multi-round assay are based on the inhibition of HIV-1-
H
N
Br >100
—
induced cytopathogenicity in PM1/CCR5 cells.
H
N
17
19.8
480
35
24
57
N
the cytotoxicity but also the anti-HIV activity. This is possibly due
to the stability in the assay condition derived from the hydrophobic-
ity of cyclohexyl group(s). These results are consistent with a previ-
ous study of the analog with one hydrophobic gem-dimethyl group
on the piperidine moiety, a compound with potent anti-HIV activity
and efficient binding affinity for gp120.13
H
N
12 (NBD-556)
NH
0.61
a
All data with standard deviation are the mean values for at least three inde-
To gain insight into the interactions involved in the binding,
molecular modeling of compound 11 docked into gp120 (1RZJ)
was carried with Sybyl 7.1 (Fig. 3). The binding mode of compound
11 in the Phe43 cavity suggested that the orientation of the piper-
idine moiety of 11 is different from that in compound 12, and that
the cyclohexyl group can be positioned near Val430 with whose
isopropyl group it can interact hydrophobically.
pendent experiments
b
IC50 values are based on the reduction in the b-galactosidase activity in TZM-bl
cells.
c
CC50 values are based on the reduction of the viability of mock-infected PM1/
CCR5 cells.
Fluorescence activated cell sorting (FACS) analysis was per-
formed as previously reported,11,12 to evaluate the CD4 mimicry
effects on conformational changes of gp120 and the results are
shown in Figure 4. Comparison of the binding of an anti-envelope
CD4-induced monoclonal antibody (4C11) to the cell surface pre-
treated with the above CD4 mimics was measured in terms of
the mean fluorescence intensity (MFI). Our previous studies
revealed that the profile of the binding of 4C11 to the Env-express-
ing cell surface pretreated with compound 12 was entirely similar
to that of pretreatment of soluble CD4. In this FACS analysis, the
MFI of pretreatment with compound 12 is 23.13. The profiles of
the binding of 4C11 to the cell surface pretreated with compounds
3, 4 and 5 were comparable to that of compound 12 [MFI
(3) = 20.54, MFI (4) = 20.85, MFI (5) = 20.24, respectively], suggest-
ing that these derivatives offer a significant enhancement of bind-
ing affinity for 4C11. On the other hand, pretreatment with 6 and 8
did not cause significant enhancement of the binding affinity for
4C11, indicating that introduction of a carbonyl group on the
piperidine nitrogen is not conducive to CD4 mimicry. The profile
of the binding of 4C11 to the Env-expressing cell surface pretreated
with compound 11, which had significant anti-HIV activity and
lower cytotoxicity than compound 12, (MFI (11) = 22.17) was sim-
ilar to that of compound 12, suggesting that compound 11 offers
significant enhancement of binding affinity for 4C11. This result
indicates that compound 11 retains the CD4 mimicry on the
conformational changes of gp120. Although compound 14 and
compound 17 showed potent anti-HIV activity and no significant
cytotoxicity, the profiles pretreated with (MFI (14 and
17) = 15.20 and 15.38) were similar to that of the control
(MFI = 14.94), suggesting that these compounds 14 and 17 failed
to produce a significant increase in binding affinity for 4C11. These
substitution on the piperidine moiety of acidic functional groups
was unfavorable.
The assay results for CD4 mimics that target hydrophobic inter-
actions are shown in Table 2. Compound 11 showed significant
anti-HIV activity (IC50 = 0.68 lM) comparable to that of the lead
compound 12, but exhibited lower cytotoxicity. Compound 11
showed approximately four-fold less cytotoxicity than 12. The SI
of 11 is 176, 3 times higher than that of 12 (SI = 57). This result
suggests that substitution of bulky hydrophobic groups into the
piperidine moiety may be consistent with lower cytotoxicity of
CD4 mimics. It is noteworthy that compound 14, which has a p-
fluoroanilino group in place of the piperidine ring, exhibits potent
anti-HIV activity (IC50 = 3.1
lM) without significant cytotoxicity
(CC50 >500 M). The SI of compound 14 is >160, which is compara-
l
ble to that of 11. However, replacement of the piperidine moiety
with a p-bromo- or p-chloroanilino group resulted in the loss of
anti-HIV activity. These results suggest that the introduction of a
fluorine atom to the piperidine moiety might be consistent with
improvement of the anti-HIV activity. Extension of the alkyl chain
by two carbons, as in 17 resulted in a 30-fold loss of anti-HIV activ-
ity, indicating that relatively rigid structures are preferable for
anti-HIV activity.
The anti-HIV activities of 12 and compound 11, which has a high-
er SI than the parent compound 12 were evaluated in a multi-round
viral infective assay and the results are shown in Table 3. In this as-
say, the IC50 value of 12 was 0.90
than measured in a single-round assay (IC50 = 0.61
11 showed higher anti-HIV activity (IC50 = 0.56 M) than compound
lM, which was slightly larger value
lM). Compound
l
12, indicating that the introduction of hydrophobic cyclohexyl
groups into the piperdine moiety has a positive effect on not only

T. Narumi et al. / Bioorg. Med. Chem. 19 (2011) 6735–6742
6739
Figure 3. Docking structures of (a) compound 11 and (b) compound 12 bound in the Phe43 cavity of gp120 (1RZJ); (c) merge image of compounds 11 and 12. Compounds 11
and 12 are represented in yellow and green sticks, respectively. Key residues in the cavity forming interactions with compounds are represented in gray sticks.
Figure 4. FACS analysis of compounds 12, 3–6, 8 (Table 1), 11, 14, and 17 (Table 2).
results are consistent with our previous finding that the piperidine
ring is critical to the CD4 mimicry of the conformational changes in
gp120.
to the substitution on the piperidine moiety have been ongoing
studies.
4. Experimentals
1H NMR and 13C NMR spectra were recorded using a Bruker
Avance III spectrometer. Chemical shifts are reported in d (ppm)
relative to Me4Si (in CDCl3) as internal standard. Low- and high-
resolution mass spectra were recorded on a Bruker Daltonics
microTOF focus in the positive and negative detection mode. For
flash chromatography, Wakogel C-200 (Wako Pure Chemical
Industries, Ltd) and silica gel 60 N (Kanto Chemical Co., Inc.) were
employed. For analytical HPLC, a Cosmosil 5C18-ARII column
(4.6 Â 250 mm, Nacalai Tesque, Inc., Kyoto, Japan) was employed
with a linear gradient of CH3CN containing 0.1% (v/v) TFA at a flow
rate of 1 cm3 minÀ1 on a JASCO PU-2089 plus (JASCO Corporation,
Ltd., Tokyo, Japan), and eluting products were detected by UV at
3. Conclusion
A series of CD4 mimics were designed and synthesized to inter-
act with the conserved residues in the Phe43 cavity of gp120 to
investigate their anti-HIV activity, cytotoxicity, and CD4 mimicry
as a function of conformational change of gp120. The biological
activities of the synthetic compounds indicate that (1) the hydro-
gen atom of the piperidine moieties contributes significantly to
cytotoxicity, and (2) installation of bulky hydrophobic groups into
the piperidine moiety can increase anti-HIV activity and decrease
cytotoxicity thus providing a novel compound with higher selec-
tive index than those of the original CD4 mimics. Furthermore, this
modification has no great influence on the CD4 mimicry on the
conformational change of gp120. Thus, compound 11 is promising
for further studies. More detailed SAR investigations with respect
220 nm. Preparative HPLC was performed using a Cosmosil 5C18
-
ARII column (20 Â 250 mm, Nacalai Tesque, Inc.) on a JASCO
PU-2087 plus (JASCO Corporation, Ltd, Tokyo, Japan) in a suitable

6740
T. Narumi et al. / Bioorg. Med. Chem. 19 (2011) 6735–6742
gradient mode of CH3CN solution containing 0.1% (v/v) TFA at a
flow rate of 7 cm3 minÀ1. Microwave reactions were performed in
Biotage Microwave Reaction Kit (sealed vials) in an Initiator™
(Biotage). The wattage was automatically adjusted to maintain
the desired temperature for the desired period of time.
1.00 mmol) and stirred at room temperature for 1 h. The precipi-
tate was collected and washed with cold CHCl3, and dried under
reduced pressure to provide the title compound 5 as white powder.
(62.0 mg, 36% yield).
1H NMR (400 MHz, DMSO) d 1.45–1.69 (m, 2H), 1.69–1.81 (m,
2H), 2.67–2.81 (m, 2H), 3.02–3.16 (m, 2H), 3.75–3.89 (m, 1H),
7.41 (d, J = 9.00 Hz, 2H), 7.85 (d, J = 9.00 Hz, 2H), 9.00 (d,
J = 8.50 Hz, 1H), 10.80 (s, 1H); 13C NMR (125 MHz, DMSO) d 27.8
(2C), 42.3 (2C), 44.4, 122.0 (2C), 128.2, 128.6 (2C), 129.5, 136.6,
158.6, 159.4; Anal. calcd for C14H18ClN4O2S: C, 49.34; H, 5.03; N,
16.44. Found: C, 49.32; H, 4.76; N, 16.11.
4.1. Chemistry
4.1.1. N1-(4-Chlorophenyl)-N2-(piperidin-4-yl)oxalamide (3)
To a stirred solution of p-choroaniline (1) (14.0 g, 110 mmol) in
THF (146 mL) were added ethyl chloroglyoxylate (8.13 mL,
73.2 mmol) and triethylamine (Et3N) (15.2 mL, 110 mmol) at 0 °C.
The mixture was stirred for 6 h at room temperature. After the pre-
cipitate was filtrated off, the filtrate solution was concentrated un-
der reduced pressure. The residue was dissolve in EtOAc, and
washed with 1 M HCl, saturated NaHCO3 and brine, then dried over
MgSO4. Concentration under reduced pressure gave the crude ethyl
oxalamate, which was used without further purification. To a solu-
tion of the above ethyl oxalamate (1.27 g, 5.25 mmol) in EtOH
(13.0 mL) were added Et3N (1.46 mL, 10.5 mmol) and 4-amino-
1-benzylpiperidine (2.97 mL, 15.8 mmol). The reaction mixture
was stirred for 3 h at 150 °C under microwave irradiation. After
being cooled to room temperature, the crystal was collected and
washed with cold EtOH and n-hexane, and dried under reduced
pressure to provide the corresponding amide (1.58 g, 81% yield)
as colorless crystals. To a stirred solution of S1 (1.46 g, 3.90 mmol)
in CH2Cl2 (39.0 mL) was added dropwise 1-chloroethyl chlorofor-
mate (0.860 mL, 7.80 mmol) at 0 °C. After being stirred at room
temperature for 30 min, the mixture was refluxed for 1 h. After con-
centration under reduced pressure, the residue was dissolved in
MeOH and then refluxed for 1 h. After concentration under reduced
pressure, the residue was diluted with EtOAc and washed with sat-
urated NaHCO3 and brine, then dried over MgSO4. After concentra-
tion under reduced pressure, the residue was washed with cold
EtOAc, and dried under reduced pressure to provide the title com-
pound 3 (778 mg, 71% yield) as white powder.
4.1.4. N1-(1-Carbamoylpiperidin-4-yl)-N2-(4-chlorophenyl)
oxalamide (6)
To a stirred solution of 3 (60.0 mg, 0.213 mmol) in CHCl3
(1.10 mL) was added trimethylsilyl isocyanate (56.0 lL,
0.421 mmol), and the mixture was stirred at room temperature
for 1 h. The precipitate was collected and washed with cold CHCl3,
and dried under reduced pressure to provide the title compound 6
(20.1 mg, 30% yield) as white powder.
1H NMR (500 MHz, DMSO) d 1.44–1.55 (m, 2H), 1.58–1.71 (m,
2H), 2.65–2.78 (m, 2H), 3.76–3.87 (m, 1H), 3.87–4.01 (m, 2H),
5.94 (s, 1H), 7.42 (d, J = 9.00 Hz, 2H), 7.86 (d, J = 9.00 Hz, 2H),
8.95 (d, J = 9.00 Hz, 1H), 10.80 (s, 1H); 13C NMR (125 MHz, DMSO)
d 30.8 (2C), 42.6 (2C), 47.1, 122.0 (2C), 128.1, 128.6 (2C), 136.7,
157.8, 158.8, 159.0; HRMS (ESI), m/z calcd for C14H18ClN4O3
(MH+) 325.1062, found 325.1060.
4.1.5. N1-(4-Chlorophenyl)-N2-(1-(phenylcarbamoyl)piperidin-
4-yl)oxalamide (7)
To a stirred solution of 3 (140 mg, 0.498 mmol) in CHCl3
(5.00 mL) was added phenyl isocyanate (54.0 lL, 0.500 mmol)
and stirred at room temperature for 1 h. The precipitate was
collected and washed with cold CHCl3, and dried under reduced
pressure to provide the title compound 7 as white powder.
(64.1 mg, 32% yield).
1H NMR (500 MHz, DMSO) d 1.52–1.66 (m, 2H), 1.68–1.80 (m,
2H), 2.81–2.95 (m, 2H), 3.84–3.96 (m, 1H), 4.08–4.20 (m, 2H),
6.91–6.94 (m, 2H), 7.21–7.24 (m, 2H), 7.36–7.52 (m, 4H), 7.86 (d,
J = 9.00 Hz, 2H), 8.53 (s, 1H), 8.99 (d, J = 8.50 Hz, 2H), 10.81 (s,
1H); 13C NMR (125 MHz, DMSO) d 31.3 (2C), 43.4 (2C), 47.5,
120.0 (2C), 122.0, 122.4 (2C), 128.6, 128.7 (2C), 129.1 (2C), 137.1,
141.1, 155.2, 159.2, 159.5; HRMS (ESI), m/z calcd for C20H22ClN4O3
(MH+) 401.1375, found 401.1372.
1H NMR (400 MHz, CDCl3) d 1.39–1.52 (m, 2H), 1.92–2.01 (m,
2H), 2.67–2.79 (m, 2H), 3.06–3.19 (m, 2H), 3.83–3.95 (m, 1H),
7.34 (d, J = 8.80 Hz, 2H), 7.44 (d, J = 7.64 Hz, 1H), 7.59 (d,
J = 8.80 Hz, 2H), 9.28 (s, 1H); 13C NMR (125 MHz, CDCl3) d 33.0
(2C), 45.2 (2C), 47.9, 21.0 (2C), 129.3 (2C), 130.5, 135.0, 157.6,
158.8; HRMS (ESI), m/z calcd for C13H17ClN3O2 (MH+) 282.1004,
found 282.1002.
4.1.2. N1-(1-Carbamimidoylpiperidin-4-yl)-N2-(4-chlorophenyl)
oxalamide (4)
To a stirred solution of 3 (50.0 mg, 0.178 mmol) in DMF
(20.0 mL) was added 1-aminopyrazole hydrochlride (312 mg,
2.13 mmol) and Et3N (0.390 mL, 28.1 mmol). The reaction mixture
was stirred at room temperature for 24 h. After concentration un-
der reduced pressure, purification by preparative HPLC gave the
4.1.6. N1-(1-Benzoylpiperidin-4-yl)-N2-(4-chlorophenyl)
oxalamide (8)
To a stirred solution of 3 (500 mg, 1.78 mmol) in CHCl3
(17.8 mL) was added benzoyl chloride (307 lL, 2.67 mmol) and
the mixture was stirred at room temperature for 1 h. The precipi-
tate was collected and washed with cold EtOAc, and dried under
reduced pressure to provide the title compound 8 (232 mg, 34%
yield).
trifluoroacetate of the title compound
(36.0 mg, 61% yield).
4 as white powder
1H NMR (500 MHz, CDCl3) d 1.21–1.68 (br, 4H), 1.96–2.08 (br,
2H), 3.02–3.16 (br, 2H), 4.04–4.07 (m, 1H), 7.35 (d, J = 9.00 Hz,
2H), 7.41–7.43 (m, 5H), 7.52 (d, J = 8.00 Hz, 1H), 7.59 (d,
J = 9.00 Hz, 2H), 9.25 (s, 1H); 13C NMR (125 MHz, CDCl3) d 31.4
(2C), 41.0 (2C), 47.6, 121.0 (2C), 126.9 (2C), 128.6 (2C), 129.3
(2C), 129.9, 130.6, 134.8, 135.6, 157.2, 159.0, 170.5; HRMS (ESI),
m/z calcd for C20H21ClN3O3 (MH+) 386.1266, found 386.1276.
1H NMR (500 MHz, DMSO) d 1.41–1.55 (m, 2H), 1.59–1.71 (m,
2H), 2.70–2.74 (m, 2H), 3.74–3.87 (m, 1H), 3.88–4.03 (m, 2H),
5.93 (s, 2H), 7.42 (d, J = 9.00 Hz, 2H), 7.85 (d, J = 9.00 Hz, 2H),
8.95 (d, J = 9.00 Hz, 1H), 10.80 (s, 1H); 13C NMR (125 MHz, DMSO)
d 31.3 (2C), 43.0 (2C), 47.6, 122.4 (2C), 128.6, 129.1 (2C), 137.1,
158.2, 159.3, 159.5; HRMS (ESI), m/z calcd for C14H19ClN5O2
(MH+) 324.1222, found 324.1213.
4.1.7. Amine (10)
4.1.3. N1-(1-Carbamothioylpiperidin-4-yl)-N2-(4-chlorophenyl)
oxalamide (5)
To a stirred solution of 3 (140 mg, 0.498 mmol) in CHCl3
(5.00 mL) was added trimethylsilyl isothiocyanate (141 mL,
To
a stirred solution of 2,2,6,6-tetramethylpiperidin-4-one
(7.75 g, 50.0 mmol) and cyclohexanone (15.5 mL, 150 mmol) in
DMSO (71.0 mL) was added NH4Cl (16.1 g, 300 mmol) and stirred
at 60 °C for 5 h. The reaction mixture was diluted with H2O

T. Narumi et al. / Bioorg. Med. Chem. 19 (2011) 6735–6742
6741
(150 mL), acidified with 7% aq HCl, and extracted with Et2O
(200 mL Â 3). The water layer was adjusted to pH 9 using 10% aq
K2CO3 and then back-extracted with EtOAc. The extract was
washed with brine and dried over Na2SO4. After concentration un-
der reduced pressure, the residue was dissolve in MeOH (60.0 mL)
and benzylamine (10.9 mL, 100 mmol) was added. After being stir-
red at room temperature for 1 h, sodium cyanoborohydride was
added and stirred at room temperature for 6 h. The reaction mix-
ture was poured into saturated NaHCO3 and extracted with EtOAc,
then dried over MgSO4. After concentration under reduced pres-
sure, the residue was dissolve in MeOH (150 mL) and 10% Pd/C
(5.32 g, 5.00 mmol) was added and stirred at room temperature
for 24 h under hydrogen atmosphere. After the reaction mixture
was filtered through celite, the filtrate solution was concentrated
under reduced pressure followed by flash chromatography over
silica gel with EtOAc–EtOH (4:1) to gave the title compound 10
(820 mg, 7 % yield) as a colorless oil.
1H NMR (500 MHz, CDCl3) d 3.08 (t, J = 6.50 Hz, 2H), 3.82 (q,
J = 6.50 Hz, 2H), 7.12–7.21 (m, 2H), 7.30–7.37 (m, 2H),7.54–7.66
(m 3H), 8.40 (s, 1H), 8.60 (d, J = 5.00 Hz, 1H), 9.26 (s, 1H); 13C
NMR (125 MHz, CDCl3) d 36.5, 39.0, 121.0 (2C), 121.8, 123.4,
129.2 (2C), 130.3, 135.1, 136.7, 149.5, 157.5, 158.6, 159.6; HRMS
(ESI), m/z calcd for C15H15ClN3O2 (MH+) 304.0847, found 304.0850.
4.2. Molecular modeling
The structures of compounds 11 and 12 were built in Sybyl and
minimized with the MMFF94 force field and partial charges.17
Dockings were then performed using FlexSIS through its SYBYL
module, into the crystal structure of gp120 (PDB: 1RZJ).
4.3. FACS analysis
JR-FL (R5, Sub B) chronically infected PM1 cells were pre-incu-
bated with 100 lM of a CD4 mimic for 15 min, and then incubated
1H NMR (500 MHz, CDCl3) d 0.730 (t, J = 12.0 Hz, 2H), 1.15–1.85
(m, 23H), 2.01–3.7 (m, 2H), 2.95–3.05 (m, 1H); 13C NMR (125 MHz,
CDCl3) d 22.2 (2C), 22.8 (2C), 26.2 (2C), 37.3 (2C), 42.3 (2C), 43.6
(2C), 47.0, 53.2 (2C); HRMS (ESI), m/z calcd for C15H29N2 (MH+)
237.2325, found 237.2321.
with an anti-HIV-1 mAb, 4C11, at 4 °C for 15 min. The cells were
washed with PBS, and fluorescein isothiocyanate (FITC)-conjugated
goat anti-human IgG antibody was used for antibody-staining.
Flow cytometry data for the binding of 4C11 (green lines, Fig. 4)
to the Env-expressing cell surface in the presence of a CD4 mimic
are shown among gated PM1 cells along with a control antibody
(anti-human CD19: black lines, Fig. 4). Data are representative of
the results from a minimum of two independent experiments.
The number at the bottom of each graph in Figure 4 shows the
mean fluorescence intensity (MFI) of the antibody 4C11.
4.1.8. N1-(4-Chlorophenyl)-N2-(2,6-dicyclohexylpiperidin-4-yl)
oxalamide (11)
To a solution of 10 (722 mg, 3.05 mmol) in EtOH (15.0 mL) was
added ethyl 2-((4-chlorophenyl)amino)-2-oxoacetate (363 mg,
1.50 mmol) and triethylamine (0.415 mL, 3.00 mmol) and stirred
for 3 h at 150 °C under microwave irradiation. The mixture was fil-
tered and the precipitate was collected and washed with cold
EtOH, and dried under reduced pressure to provide the compound
11 (108 mg, 17% yield) as white powder.
Acknowledgments
This work was supported in part by Grant-in-Aid for Scientific
Research from the Ministry of Education, Culture, Sports, Science,
and Technology of Japan, and Health and Labour Sciences Research
Grants from Japanese Ministry of Health, Labor, and Welfare.
1H NMR (500 MHz, DMSO) d 1.12–1.91 (br, 24H), 4.02–4.07 (m,
1H), 7.42 (d, J = 9.00 Hz, 2H), 7.84 (d, J = 9.00 Hz, 2H), 8.76 (br, 1H),
9.25 (s, 1H); 13C NMR (125 MHz, CDCl3) d 22.1 (2C), 22.7 (2C), 26.0
(2C), 37.2 (2C), 42.5 (2C), 42.9 (2C), 43.6, 52.7 (2C), 120.9 (2C),
129.3 (2C), 130.4, 135.0, 157.6, 158.8; HRMS (ESI), m/z calcd for
Supplementary data
C
23H33ClN3O2 (MH+) 418.2256, found 418.2261.
Supplementary data (NMR charts of compounds) associated with
this article can be found, in the online version, at doi:10.1016/
j.bmc.2011.09.045.
4.1.9. N1-(4-Chlorophenyl)-N2-(4-fluorophenyl)oxalamide (14)
To a solution of the ethyl 2-((4-chlorophenyl)amino)-2-oxoace-
tate (1.21 g, 5.00 mmol) in EtOH (25.0 mL) were added Et3N
(1.38 mL, 10.0 mmol) and 4-fluoroaniline 12 (1.44 mL, 15.0 mmol).
The reaction mixture was stirred for 3 h at 150 °C under micro-
wave irradiation. After being cooled to room temperature, the crys-
tal was collected and washed with cold EtOH and n-hexane, and
dried under reduced pressure to provide the compound 14
(601 mg, 41% yield) as colorless crystals. Compounds 15 and 16
were similarly synthesized.
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