CD4 mimics targeting the HIV entry mechanism and their hybrid molecules with a CXCR4 antagonist

Article abstract of DOI:10.1016/j.bmcl.2010.07.106

Small molecules behaving as CD4 mimics were previously reported as HIV-1 entry inhibitors that block the gp120-CD4 interaction and induce a conformational change in gp120, exposing its co-receptor-binding site. A structure-activity relationship (SAR) study of a series of CD4 mimic analogs was conducted to investigate the contribution from the piperidine moiety of CD4 mimic 1 to anti-HIV activity, cytotoxicity, and CD4 mimicry effects on conformational changes of gp120. In addition, several hybrid molecules based on conjugation of a CD4 mimic analog with a selective CXCR4 antagonist were also synthesized and their utility evaluated.

Full text of DOI:10.1016/j.bmcl.2010.07.106

Bioorganic & Medicinal Chemistry Letters 20 (2010) 5853–5858
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
CD4 mimics targeting the HIV entry mechanism and their hybrid molecules
with a CXCR4 antagonist
Tetsuo Narumi a, Chihiro Ochiai a, Kazuhisa Yoshimura b, Shigeyoshi Harada b, Tomohiro Tanaka a,
Wataru Nomura a, Hiroshi Arai a, Taro Ozaki a, Nami Ohashi 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:
Small molecules behaving as CD4 mimics were previously reported as HIV-1 entry inhibitors that block
the gp120–CD4 interaction and induce a conformational change in gp120, exposing its co-receptor-bind-
ing site. A structure–activity relationship (SAR) study of a series of CD4 mimic analogs was conducted to
investigate the contribution from the piperidine moiety of CD4 mimic 1 to anti-HIV activity, cytotoxicity,
and CD4 mimicry effects on conformational changes of gp120. In addition, several hybrid molecules
based on conjugation of a CD4 mimic analog with a selective CXCR4 antagonist were also synthesized
and their utility evaluated.
Received 14 June 2010
Revised 22 July 2010
Accepted 26 July 2010
Available online 3 August 2010
Keywords:
CD4
HIV entry
Hybrid molecule
gp120
Ó 2010 Elsevier Ltd. All rights reserved.
The infection of host cells by HIV-1 takes place in multiple steps
via a dynamic supramolecular mechanism mediated by two viral
envelope glycoproteins (gp41, gp120) and several cell surface pro-
teins (CD4, CCR5/CXCR4).1 Cell penetration begins with the inter-
action of gp120 with the primary receptor CD4. This induces
conformational changes in gp120, leading to the exposure of its
V3 loop allowing the subsequent binding of gp120 to a co-receptor,
CCR52 or CXCR4.3
residues, Val255, Asp368, Glu370, Ser375, Ile424, Trp427, Val430, and
Val475 are located within a 4.4 Å-radius of 1 and that a large cavity
exists around the p-position of the aromatic ring of 1.4e Based on
these observations, we conducted a structure–activity relationship
(SAR) study of a series of analogs of CD4 mimics with substituents
at the p-position of the aromatic ring. This study revealed that a
certain size and electron-withdrawing ability of the substituents
are indispensable for potent anti-HIV activity.4e
N-(4-Chlorophenyl)-N0-(2,2,6,6-tetramethyl-piperidin-4-yl)ox-
alamide (NBD-556: 1) and the related compounds NBD-557 (2)
and YYA-021 (3) have been identified as a novel class of HIV-1 en-
try inhibitors, which exert potent cell fusion and virus cell fusion
inhibitory activity at low micromolar levels (Fig. 1).4 Furthermore,
compound 1 can also induce thermodynamically favored confor-
mational changes in gp120 similar to those caused by CD4 bind-
ing. The X-ray crystal structure of gp120 complexed with CD4
revealed the presence of a hydrophobic cavity, the Phe43 cavity,
which is penetrated by the aromatic ring of Phe43 of CD4.5 Molec-
ular modeling revealed that compound 1 is also inserted into the
Phe43 cavity, the para-chlorophenyl group of 1 entering more dee-
ply than the phenyl ring of Phe43 of CD4 and interacting with the
Although several reported SAR studies of 1 have revealed the
contributions of the phenyl ring and the oxalamide linker of 1 to
the binding affinity with gp120, the anti-HIV activity and the
CD4 mimicry on conformational changes of gp120,4 there has been,
to the best of our knowledge, no prior report describing SAR stud-
ies of the piperidine ring of 1. In this paper, the contributions of the
piperidine ring of 1 to the anti-HIV activity, CD4 mimicry and cyto-
toxicity were investigated through the SAR studies focused on the
piperidine ring of 1. Furthermore, to apply the utility of CD4 mim-
ics to the development of potent anti-HIV agents, a series of the
O
NH
conserved gp120 residues such as Trp427, Phe382, and Trp112 4c
The
.
H
N
N
H
modeling also suggested that an oxalamide linker forms hydrogen
bonds with carbonyl groups of the gp120 backbone peptide bonds.
Our model of 1 docked into gp120 revealed that eight other gp120
O
R
1 (R = Cl)
2 (R = Br)
3
(R = Me)
* Corresponding author.
E-mail address: tamamura.mr@tmd.ac.jp (H. Tamamura).
Figure 1. NBD-556 (1) and related compounds.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.07.106

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T. Narumi et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5853–5858
hybrid molecules that combined CD4 mimic analogs with a selec-
tive CXCR4 antagonist were also synthesized and bioevaluated.
For the design of novel CD4 mimic analogs, we initially tried to
directly derivatize the nitrogen atom of piperidine group. However,
direct alkylation and acylation of 1 failed probably as a result of
steric hindrance from the methyl groups on the piperidine ring
so we synthesized several derivatives lacking the methyl groups
and evaluated their anti-HIV activity, cytotoxicity, and ability to
mimic CD4. According to the previous SAR study,4e the p-Cl (4),
p-Br (5) and p-methyl derivatives (6) lacking the methyl groups
on the piperidine ring were prepared. Compounds 4–6 were syn-
thesized by published methods as shown in Scheme 1. Briefly, cou-
pling of aniline derivatives with ethyl chloroglyoxalate in the
presence of Et3N and subsequent saponification gave the corre-
sponding acids (10–12). Condensation of these acids with 4-ami-
no-N-benzylpiperidine in the presence of EDC–HOBt system,
followed by debenzylation under von Braun conditions with 1-
chloroethyl choroformate6 produced the desired compounds 4–6.7
The anti-HIV activity of each of the synthetic compounds was
evaluated against MNA (R5) strain, with the results shown in Table
1. IC50 values were determined by the 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide (MTT) method8 as the con-
centrations of the compounds which conferred 50% protection
against HIV-1-induced cytopathogenicity in PM1/CCR5 cells. Cyto-
toxicity of the compounds based on the viability of mock-infected
PM1/CCR5 cells was also evaluated using the MTT method. CC50
values, the concentrations achieving 50% reduction of the viability
of mock-infected cells, were also determined. Compounds 1 and 3
showed potent anti-HIV activity. The anti-HIV IC50 of compound 2
was previously reported to be comparable to that of compound 1,
and thus was not determined in this study. Novel derivatives 4 and
6 without the methyl groups on the piperidine ring, showed signif-
icant anti-HIV activity comparable to that of the parent compounds
1 and 3, respectively. The p-methyl derivative 6 has slightly lower
activity than the p-Cl derivative 4 and the p-Br derivative 5. These
results are consistent with our previous SAR studies on the parent
compounds 1–3. Compound 5 was found to exhibit relatively
strong cytotoxicity (CC50 = 50 lM) and compounds 4 and 6 have
cytotoxicities comparable to that of compounds 1 and 3, respec-
tively. This observation indicates that the methyl groups on the
piperidine ring do not contribute significantly to the anti-HIV
activity or the cytotoxicity.
Compound 1 and the newly synthesized derivatives 4–6 were
also evaluated for their effects on conformational changes of
gp120 by a fluorescence activated cell sorting (FACS) analysis.
The profile of binding of an anti-envelope CD4-induced monoclo-
nal antibody (4C11) to the Env-expressing cell surface (an R5-
HIV-1 strain, JR-FL, -infected PM1 cells) pretreated with the above
derivatives was examined. Comparison of the binding of 4C11 to
the cell surface was measured in terms of the mean fluorescence
intensity (MFI), as shown in Figure 2. Pretreatment of the Env-
expressing cell surface with compound 1 (MFI = 53.66) produced
a significant increase in binding affinity for 4C11, consistent with
that reported previously.4e This indicates that compound 1 en-
hances the binding affinity of gp120 with the 17b monoclonal anti-
body which recognizes CD4-induced epitopes on gp120. The Env-
expressing cells without CD4 mimic-pretreatment failed to show
significant binding affinity to 4C11. On the other hand, the profiles
of the binding of 4C11 to the Env-expressing cell surface pretreated
with compound 4 (Cl derivative) and 5 (Br derivative) (MFI = 49.88
and 52.34) were similar to that of compound 1. Pretreatment of the
cell surface with compound 6 (Me derivative) (MFI = 45.99) pro-
duced slightly lower enhancement but significant levels of binding
affinity for 4C11, compared to that of compound 1 as pretreat-
ments. These results suggested that the removal of the methyl
groups on the piperidine moiety might not affect the CD4 mimicry
effects on conformational changes of gp120 and it was conjectured
that the phenyl ring of CD4 mimic might be a key moiety for the
interaction with gp120 to induce the conformational changes of
gp120. This is consistent with the results in the previous paper
where it was reported that CD4 mimics having suitable substitu-
ent(s) on the phenyl ring cause a conformational change, resulting
in external exposure of the co-receptor-binding site of gp120.4e
Based on these results, a series of N-alkylated and N-acylated
piperidine derivatives 13–18 with no methyl groups were prepared.
Several compounds with 6-membered rings were also prepared to
determine whether or not the piperidine ring is mandatory. The
synthesis of these derivatives is shown in Scheme 2. Since the p-Cl
derivative 4 showed potent anti-HIV activity and relatively low
cytotoxicity, compared to the p-Br derivative 5, chlorine was se-
lected as the substituent at the p-position of the phenyl ring. The
N-methyl derivative 13 was synthesized by coupling of 10 with
4-amine-1-methylpiperidine. Alkylation of 4 with tert-butyl bromo-
acetate, followed by deprotection of tert-butyl ester provided com-
pound 14. The N-isopropyl derivative 15 was prepared by
reductive amination of 4 with isopropyl aldehyde. The N-acyl deriv-
atives 16–18 were prepared by simple acylation or condensation
with the corresponding substrate. The synthesis of other derivatives
19–23 with different 6-membered rings is depicted in Scheme 3. The
6-membered ring derivatives with the exception of 21 were pre-
pared by coupling of acid 10 with the corresponding amines. Com-
pound 21 was prepared by reaction of 10 with thionyl chloride to
give the corresponding acid chloride, which was subsequently cou-
pled with 4-aminopyridine.
O
H
N
NH2
a,b
OH
R
O
R
7 (R = Cl)
10 (R = Cl)
11 (R = Br)
12 (R = Me)
8 (R = Br)
9 (R = Me)
O
NH
H
N
c,d
N
H
O
R
4 (R = Cl)
5 (R = Br)
6 (R = Me)
Scheme 1. Synthesis of compounds 4–6. Reagents and conditions: (a) ethyl
chloroglyoxylate, Et3N, THF; (b) 1 M aq NaOH, THF, 67%–quant.; (c) 1-benzyl-4-
aminopiperidine, EDCꢀHCl, HOBtꢀH2O, Et3N, THF; (d) (i) 1-chloroethyl chlorofor-
mate, CH2Cl2; (ii) MeOH, 8–47%.
Table 1
Effects of the methyl groups on anti-HIV activity and cytotoxicity of CD4 mimic
analogsa
Compd
R
IC50
(lM)
CC50 (lM)
MNA (R5)
1
2
3
4
5
6
Cl
Br
Me
Cl
Br
12
ND
15
8
6
20
110
93
210
100
50
Me
190
a
All data with standard deviation are the mean values for at least three inde-
pendent experiments (ND = not determined).
Compounds 1, 3, and 13–18 were evaluated for their CD4 mim-
icry effects on conformational changes of gp120 by the FACS anal-

T. Narumi et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5853–5858
5855
Figure 2. FACS analysis of compounds 1 and 4–6. JR-FL (R5, Sub B) chronically infected PM1 cells were preincubated with 100
l
M of a CD4 mimic 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 goat anti-human IgG antibody
was used for antibody-staining. Flow cytometry data for the binding of 4C11 (green lines) 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). Data are representative of the results from a minimum of two independent experiments. The
number at the bottom of each graph shows the mean fluorescence intensity (MFI) of the antibody 4C11.
4C11, similar to that observed in the pretreatment with compound
1. The profile of the binding of 4C11 to the cell surface pretreated
with compounds 14 and 17 was similar to that of controls, suggest-
ing that these derivatives offer no significant enhancement of bind-
ing affinity for 4C11 and that the carboxylic moiety in the terminal
of piperidine ring is not suited to CD4 mimicry. It is hypothesized
that the carboxylic moieties of compounds 14 and 17 might pre-
vent the interaction of CD4 mimic with gp120 by their multiple
contacts with side chain(s) of amino acid(s) around the Phe43 cav-
ity, such as Asp368 and Glu370. Replacement of the piperidine moi-
ety with the different 6-membered rings resulted in a significant
loss of binding affinity for 4C11 in the FACS analysis of compound
19–23 (MFI(19) = 11.44, MFI(20) = 12.84, MFI(21) = 12.47, in MFI
(blank) = 11.34; MFI(22) = 26.67, MFI(23) = 20.21, in MFI(blank) =
26.79, data not shown), indicating a significant contribution from
the piperidine ring which interacts with gp120 inducing conforma-
tional changes.
Me
N
O
H
N
a
N
H
10
O
Cl
13
R
O
N
b for 14
c for 15
d for 16
H
N
N
H
4
O
Cl
e for 17
f for 18
14 (R =
)
CO2H
15
16
(R = iBu)
(R = Ac)
O
17 (R =
)
CO2H
In view of their ability to induce conformational changes of
gp120, the anti-HIV activity and cytotoxicity of the piperidine
derivatives 13–18 were further evaluated, with the results shown
in Table 2. The anti-HIV activity of the synthetic compounds was
evaluated against various viral strains including both laboratory
and primary isolates and IC50 and CC50 values were determined
as those of compounds 4–6. The N-methylpiperidine compound
13, was not found to possess significant anti-HIV activity against
a primary isolate, but was found to possess moderate anti-HIV
O
18
(R =
)
Scheme 2. Synthesis of N-alkylated and N-acylated piperidine derivatives 13–18.
Reagents and conditions: (a) 4-amine-1-methylpiperidine, EDCꢀHCl, HOBtꢀH2O,
Et3N, THF, 16%; (b) (i) tert-butyl bromoacetate, NaH, DMF; (ii) TFA, 6%; (c)
isobutylaldehyde, NaBH(OAc)3, AcOH, DCE, quant.; (d) acetyl chloride, Et3N, DMF,
quant.; (e) succinic anhydride, Et3N, THF, 37%; (f) isobutyric acid, EDCꢀHCl,
HOBtꢀH2O, Et3N, THF, 95%.
activity against a laboratory isolate, a IIIB strain (IC50 = 67
Anti-HIV activity was not observed however, even at concentra-
tions of 100 M of 13 against an 89.6 strain. The potency was
approximately eight-fold lower than that of the parent compound
1 (IC50 = 8 M), indicating a partial contribution of the hydrogen
atom of the amino group of the piperidine ring to the bioactivity
of CD4 mimic. Although compound 15, with an N-isobutylpiperi-
dine moiety, failed to show significant anti-HIV activity against
laboratory isolates, relativity potent activity was observed against
lM).
O
H
l
19 20 22 23
a for
,
,
,
N
10
R
b for 21
l
O
Cl
N
O
N
R =
N
N
H
N
H
H
a primary isolate, an MTA strain (IC50 = 28 lM). Compounds 16 and
18, which are N-acylpiperidines, were tested against laboratory
isolates and significant anti-HIV activity was not observed even
20
23
19
21
Me
Me
N
at 100
failed to show significant anti-HIV activity against laboratory iso-
lates even at 100 M, which are compatible with the FACS analysis.
lM. Compounds 14 and 17, with the carboxylic moieties,
N
N
N
H
l
22
These results suggest that the N-substituent on the piperidine ring
of CD4 mimic analogs may contribute to a critical interaction re-
quired for binding to gp120. Compounds 19–23 showed no signif-
Scheme 3. Synthesis of 6-membered ring derivatives 19–23. Reagents and condi-
tions: (a) the corresponding amine, EDCꢀHCl, HOBtꢀH2O, Et3N, THF, 22%–quant.; (b)
4-aminopyridine, SOCl2, MeOH, 38%.
icant anti-HIV activity against a IIIB strain even at 100
are compatible with the FACS analysis (data not shown).
All but one of the compounds 13–18 have no significant cyto-
toxicity to PM1/CCR5 cells (CC50 P260 M); the exception is
compound 18 (CC50 = 45 M). Compounds 13 and 15 show rela-
tively potent anti-HIV activity without significant cytotoxicity.
lM, which
l
ysis, and the results are shown in Figure 3. Pretreatment of the
Env-expressing cells with the N-substituted compounds 13, 15,
16, and 18 produced a notable increase in binding affinity to
l

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T. Narumi et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5853–5858
Figure 3. FACS analysis of compounds 1, 3, and 13–18. The experimental procedures are described in Figure 2. The lanes of (a), (b) and (c) show independent experiments.
Table 2
Anti-HIV activity and cytotoxicity of compounds 13–18a
Compd
R
IC50
(lM)
CC50 (lM)
Laboratory isolates
Primary isolates
MTA (R5)
IIIB (X4)
89.6 (dual)
1
4
8
ND
67
>100
>100
>100
>100
>100
10
ND
>100
ND
ND
>100
>100
ND
ND
ND
ND
ND
28
ND
ND
ND
150
100
>300
260
>300
>300
>300
45
H
Me
CH2CO2H
iBu
13
14
15
16
17
18
Ac
C(O)CH2CH2CO2H
C(O)iPr
a
All data with standard deviation are the mean values for at least three independent experiments.
The results for 15 showed it to have 3–6 times less cytotoxicity
than 4 and 18. This observation indicates that the alkylation of
the piperidine nitrogen may be favorable because it lowers the
cytotoxicity of CD4 mimic analogs.
lengths were designed. Based on the fact that alkylation of the
piperidine nitrogen, having no deleterious effects on bioactivity,
is an acceptable modification of CD4 mimic analogs, the alkyla-
mine moiety was incorporated into the nitrogen atom of the piper-
idine moiety to conjugate CD4 mimic analogs with 4F-benzoyl-
In the course of the SAR studies on CD4 mimic analogs, we have
already found that a CD4 mimic or sCD4 exhibited a remarkable
synergistic effect4e with a 14-mer peptide CXCR4 antagonist
T140.9 This result indicates that the interaction of CD4 mimic with
gp120 could facilitate the approach of CXCR4 to gp120 by exposing
the co-receptor binding site of gp120. It was thought that the CD4
mimic analogs conjugated with a selective CXCR4 antagonist might
as a consequence show a higher synergistic effect for the improve-
ment of anti-HIV activity. In this context, efforts were made to syn-
thesize and bioevaluate hybrid molecules that combined a CD4
mimic analog with 4F-benzoyl-TZ14011, which is a derivative of
T140 optimized for CXCR4 binding and stability in vivo.10
a
TZ14011. Reductive alkylation of 4 with N -Boc-pyrrolidin-2-ol
24, which exists in equilibrium with the corresponding aldehyde,
and successive treatment with TFA and HCl/dioxane provided the
amine hydrochloride 25. Treatment of 25 with succinic anhydride
under basic condition gave the corresponding acid 26, which was
subjected to condensation with the side chain of D
-Lys8 of 4F-ben-
zoyl-TZ14011 in an EDC–HOBt system to give the desired hybrid
molecule 27 with a tetramethylene linker.11 Other hybrid mole-
cules 28 and 29 bearing hexa- and octamethylene linkers, respec-
tively, were prepared using the corresponding aldehydes 30 and
31.
The synthesis of hybrid molecules 27–29 is outlined in Scheme
4. To examine the influence of the linker length on anti-HIV activity
and cytotoxicity, three hybrid molecules with linkers of different
The assay results for these hybrid molecules 27–29 are shown
in Table 3. To investigate the effect of conjugation of two molecules
on binding activity against CXCR4, the inhibitory potency against

T. Narumi et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5853–5858
5857
R
O
N
NH3
Cl
H
a, b
25
26
R =
R =
OH
N
+
4
O
N
c
N
H
H
Boc
N
O
OH
O
Cl
24
O
d
Cl
O
H
N
N
H
n
O
O
O
N
N
H
NH
a-d
NHBoc
27, n = 1
28, n = 3
29, n = 5
+
4
Arg1
O
O
Nal3
Tyr 5
O
Arg7
O
n
H
H
H
H
N
N
N
N
30, n= 3
31, n= 5
N
N
H
N
H
N
H
H
O
Arg2
O
O
O
O
Cit6
Arg11
S
F
S
O
O
O
N
H
N
H
N
H
N
H2N
N
H
N
Pro9
H
Arg14
Cit12
Tyr 10
O
O
O
Scheme 4. Synthesis of hybrid molecules 27–29. Reagents and conditions: (a) NaBH(OAc)3, AcOH, DCE; (b) TFA, then 4 M HCl/1,4-dioxane; (c) succinic anhydride, pyridine,
DMF, then 4 M HCl/1,4-dioxane; (d) 4F-benzoyl-TZ14011, EDCꢀHCl, HOBtꢀH2O, Et3N, DMF. Nal =
L-3-(2-naphthyl)alanine, Cit = L-citrulline.
Table 3
synergistic effect, the lower potency of hybrid molecules may be
attributed to the inadequacy in the structure and/or the characters
of the linkers. All the hybrid molecules 27–29 have relatively strong
CXCR4-binding activity, anti-HIV activity and cytotoxicity of hybrid molecules 27–29a
b
c
d
Compd
EC50
(lM) IC50
(
lM) CC50
(l
M) SI (CC50/IC50
)
cytotoxicity [CC50(27) = 8.6
lM; CC50(28) = 8.0 lM; CC50(29) =
4F-benzoyl-
TZ14011
1 (NBD-556)
27 (C4)
28 (C6)
29 (C8)
0.0059
0.0131
ND
ND
8.6 M]. However, selectivity indexes (SI = CC50/IC50) were 169 for
l
ND
0 2.10
0.0509
0.0365
0.0353
0.0493
ND
19.2e
169
219
244
ND
27, 219 for 28, and 244 for 29, all 9–13 times higher than that of 1
(SI = 9.2). This result indicates that conjugation of a CD4 mimic ana-
log with a selective CXCR4 antagonist can improve the SI of CD4
mimic.
0.0044
0.0187
0.0071
ND
8.60
8.00
8.60
ND
AZT
The SAR study of a series of CD4 mimic analogs was conducted
to investigate the contribution of the piperidine moiety of 1 to
anti-HIV activity, cytotoxicity, and CD4 mimicry on conformational
changes of gp120. The results indicate that (i) the methyl groups
on the piperidine ring of 1 have no great influence on the activities
of CD4 mimic; (ii) the presence of piperidine moiety is important
for the CD4 mimicry; and (iii) N-substituents of the piperidine
moiety contribute significantly to anti-HIV activity and cytotoxic-
ity, as observed with N-alkyl groups such as methyl and isobutyl
groups which show moderate anti-HIV activity and lower
cytotoxicity.
Several hybrid molecules based on conjugation of a CD4 mimic
with a selective CXCR4 antagonist were also synthesized and bio-
evaluated. All the hybrid molecules showed significant binding
activity against CXCR4 comparable to the parent antagonist and
exhibited potent anti-HIV activity. Although no significant syner-
gistic effect was observed, conjugation of a CD4 mimic with a
selective CXCR4 antagonist might lead to the development of novel
type of CD4 mimic-based HIV-1 entry inhibitors, which possess
higher selective indexes than a simple CD4 mimic. These results
will be useful for the rational design and synthesis of a new type
of HIV-1 entry inhibitors. Further structural modification studies
of CD4 mimic are the subject of an ongoing project.
a
All data with standard deviation are the mean values for at least three inde-
pendent experiments.
b
EC50 values are based on the inhibition of [125I]-SDF-1
a binding to CXCR4
transfectants of CHO cells.
c
IC50 values are based on the inhibition of HIV-1-induced cytopathogenicity in
MT-2 cells.
d
CC50 values are based on the reduction of the viability of mock-infected MT-2
cells.
e
This value is based on the CC50 and IC50 values from Table 1.
binding of [125I]-SDF-1
molecules 27–29 significantly inhibited the SDF-1
CXCR4. The corresponding EC50 values are: EC50(27) = 0.0044
EC50(28) = 0.0187 M; EC50(29) = 0.0071 M. These potencies are
comparable to that of 4F-benzoyl-TZ14011 (EC50 = 0.0059 M),
indicating that introduction of the CD4 mimic analog into the
-Lys8 residue of 4F-benzoyl-TZ14011 does not affect binding
activity against CXCR4. Comparison of the binding activities of
27–29 showed that all hybrid molecules were essentially equipo-
tent in inhibition of the binding of SDF-1a to CXCR4. This observa-
tion indicates that the linker length between two molecules has no
a
to CXCR4 was measured. All the hybrid
binding to
M;
a
l
l
l
l
D
effect on the binding inhibition.
Anti-HIV activity based on the inhibition of HIV-1 entry into the
target cells was examined by the MTT assay using a IIIB(X4) strain.
Acknowledgements
In this assay, the IC50 value of 4F-benzoyl-TZ14011 was 0.0131
All hybrid molecules 27–29 showed significant anti-HIV activity
[IC50(27) = 0.0509 M; IC50(28) = 0.0365 M; IC50(29) = 0.0353
M]; however, the potency was 2- to 4-fold lower than that of
the parent compound 4F-benzoyl-TZ14011, indicating that the
conjugation of CD4 mimic with a CXCR4 antagonist did not provide
a significant synergistic effect. In view of the fact that the combina-
tional uses of CD4 mimic with T140 produced a highly remarkable
lM.
This work was supported by Grant-in-Aid for Scientific
Research from the Ministry of Education, Culture, Sports, Science,
and Technology of Japan, Japan Human Science Foundation, and
Health and Labour Sciences Research Grants from Japanese Minis-
try of Health, Labor, and Welfare. T.T. and N.O. are grateful for the
JSPS Reseach Fellowships for Young Scientist.
l
l
l

5858
T. Narumi et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5853–5858
The extract was washed with aq saturated NaHCO3 and brine, and dried over
References and notes
MgSO4. Concentration under reduced pressure followed by flash chroma-
tography over silica gel with CHCl3–MeOH (20:1) including 1% Et3N gave the
crude benzyl amine as a white powder. To the solution of the above crude
benzyl amine (95 mg, 0.26 mmol) in dry CH2Cl2 (10 mL), 1-chloroethyl
1. Chan, D. C.; Kim, P. S. Cell 1998, 93, 681.
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chloroformate (110 lL, 0.68 mmol) was added dropwise with stirring at 0 °C.
The mixture was then refluxed for 3 h under N2 atmosphere. After
concentration under reduced pressure, the residue was resolved in MeOH
(10 mL) and then refluxed for 1 h. Concentration under reduced pressure gave a
crude product. Reprecipitation with MeOH–Et2O afforded a white powder of
the title compound 4 (33 mg, 46% yield). dH(400 MHz; CD3OD) 1.83–1.92 (2H,
m, CH2), 2.10–2.17 (2H, m, CH2), 3.13 (2H, t, J 12.5, CH2), 3.34 (1H, m, NH),
3.42–3.49 (1H, m, CH2), 4.04 (1H, m, CH), 7.34 (2H, m, ArH), 7.51 (1H, m, NH),
7.73 (2H, m, ArH), 8.84 (1H, m, NH); LRMS (ESI), m/z calcd for C13H17ClN3O2
(MH)+ 282.10, found 282.14.
3. Feng, Y.; Broder, C. C.; Kennedy, P. E.; Berger, E. A. Science 1996, 272, 872.
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11. The synthesis of a hybrid molecule 27: To the solution of compound 26
(2.6 mg, 4.6
23
mol) and EDCIꢀHCl (4.5 mg, 23
stirred for 1 h at room temperature. To the mixture 4F-benzoyl-TZ14011
(15 mg, 4.1 mol) was then added and the mixture was stirred for 24 h at room
l
mol) in DMF (1.0 mL), Et3N (26
lL, 92 lmol), HOBtꢀH2O (3.5 mg,
l
lmol) were added with stirring at 0 °C, and
5. Protein Data Bank (PDB) (entry 1RZJ).
6. Olofson, R. A.; Abbott, D. E. J. Org. Chem. 1984, 49, 2795.
7. The synthesis of compound 4: To the solution of compound 10 (104 mg,
l
temperature under N2 atmosphere. After concentration under reduced
pressure, the residue was purified by reversed phase HPLC (tR = 23 min,
elution: a linear gradient of 27–31% acetonitrile containing 0.1% TFA over
30 min) to afford a fluffy white powder of the desired compound 27 (1.3 mg,
9.8%). LRMS (ESI), m/z 2621.20 [M+H]+, calcd 2620.25.
0.52 mmol) in dry THF (4.0 mL), Et3N (159
0.57 mmol), EDCIꢀHCl (109 mg, 0.57 mmol) and 4-amino-1-benzylpiperidine
(109 L, 0.57 mmol) were added with stirring at 0 °C, and continuously stirred
lL, 1.15 mmol), HOBtꢀH2O (87 mg,
l
for 6 h with warming to room temperature under N2 atmosphere. After
concentration under reduced pressure, the residue was extracted with EtOAc.