Design, synthesis, and biological evaluation of highly potent small molecule-peptide conjugates as new HIV-1 fusion inhibitors

Article abstract of DOI:10.1021/jm3018964

The small molecule fusion inhibitors N-(4-carboxy-3-hydroxyphenyl)-2,5- dimethylpyrrole (NB-2) and N-(3-carboxy-4-hydroxyphenyl)-2,5-dimethylpyrrole (A₁₂) target a hydrophobic pocket of HIV-1 gp41 and have moderate anti-HIV-1 activity. In this

Full text of DOI:10.1021/jm3018964

Article
pubs.acs.org/jmc
Design, Synthesis, and Biological Evaluation of Highly Potent Small
Molecule−Peptide Conjugates as New HIV‑1 Fusion Inhibitors
Chao Wang,† Weiguo Shi,† Lifeng Cai,† Lu Lu,‡ Qian Wang,‡ Tianhong Zhang,† Jinglai Li,†
Zhenqing Zhang,† Kun Wang,† Liang Xu,† Xifeng Jiang,† Shibo Jiang,*,‡ and Keliang Liu*,†
†Beijing Institute of Pharmacology & Toxicology, 27 Tai-Ping Road, Beijing 100850, China
‡Key Laboratory of Medical Molecular Virology of the Ministries of Education and Health, Shanghai Medical College and Institute of
Medical Microbiology, Fudan University, Shanghai 200032, China
S
* Supporting Information
ABSTRACT: The small molecule fusion inhibitors N-(4-
carboxy-3-hydroxyphenyl)-2,5-dimethylpyrrole (NB-2) and N-
(3-carboxy-4-hydroxyphenyl)-2,5-dimethylpyrrole (A12) target
a hydrophobic pocket of HIV-1 gp41 and have moderate anti-
HIV-1 activity. In this paper, we report the design, synthesis,
and structure−activity relationship of a group of hybrid
molecules in which the pocket-binding domain segment of
the C34 peptide was replaced with NB-2 and A12 derivatives.
In addition, the synergistic effect between the small molecule
and peptide moieties was analyzed, and lead compounds with a
novel scaffold were discovered. We found that either the nonpeptide or peptide part alone showed weak activity against HIV-1-
mediated cell−cell fusion, but the conjugates properly generated a strong synergistic effect. Among them, conjugates Aoc−βAla−
P26 and Noc−βAla−P26 exhibited a low nanomolar IC50 in the cell−cell fusion assay and effectively inhibited T20-sensitive and
-resistant HIV-1 strains. Furthermore, the new molecules exhibited better stability against proteinase K digestion than T20 and
C34.
hydrophobic deep pocket (∼16 Å long, ∼7 Å wide, and 5−6 Å
deep) in the N-trimer. C34 contains a pocket-binding domain
(PBD) through which it can interact with the primary cavity to
form a stable 6HB with an N-helical coiled-coil motif.15
Although C-peptide fusion inhibitors are very potent at
inhibiting HIV-1 infection, appearance of T20-resisitant strains,
high production cost, and lack of oral bioavailability hinder its
further development; thus, these drawbacks have led to a
blooming discovery of small molecule fusion inhibitors
targeting the gp41 fusion intermediate.16,17
Because the primary cavity with its highly conserved residues
plays a crucial role in gp41-mediated membrane fusion, it
becomes an attractive target for discovering nonpeptide fusion
inhibitors that target gp41.18 Using high-throughput screening
assays,19 Jiang and co-workers have identified two N-
substituted pyrroles, NB-2 and NB-64, which inhibit HIV-1
replication at a micromolar level by interfering with the
formation of the gp41 6HB to block HIV-1 entry.20,21 On the
basis of the structures of NB-2 and NB-64, Xie’s group has
designed and synthesized a series of N-(carboxyphenyl)pyrrole
derivatives and found that one-quarter of them exhibit
enhanced antiviral potency. The most active compound, A12,
has potent inhibitory activity against HIV-1 replication with an
EC50 value of 0.69 μM; however, its inhibitory potency on cell−
INTRODUCTION
■
The envelope glycoproteins (Env) of human immunodeficiency
virus type 1 (HIV-1) play critical roles in the formation of a
coiled-coil six-helical bundle (6HB), thus providing the energy
to promote fusion of the viral and cellular membranes.1 For the
HIV-1 Env complex, the initially produced gp160 is cleaved
into the surface subunit (gp120) and transmembrane subunit
(gp41), which are noncovalently associated.2 After gp120 binds
to its CD4 T cell receptors and chemokine coreceptor CCR5 or
CXCR4,3 a series of conformational changes of gp120 occur,
resulting in dissociation of the gp120−gp41 complex.4 The
ectodomain of gp41 contains three important functional motifs
(Figure 1A): a fusion peptide (FP), an N-terminal heptad
repeat (NHR), and a C-terminal heptad repeat (CHR).5,6 The
6HB formation between the intermediate NHR trimer and the
three folded-back CHRs7,8 provides the energy to drive the viral
and target cell membranes into close apposition, resulting in
membrane fusion.9
Peptides derived from the HIV-1 gp41 CHR, such as T20
and C34, are potent HIV-1 fusion inhibitors.10,11 They interact
with their counterpart in gp41 to form a heterogeneous 6HB
and prevent fusogenic gp41core formation, thus interrupting
the fusion process.12 T20 (brand name, Fuzeon; generic name,
enfuvirtide) was approved by the U.S. FDA for clinical use in
2003,13,14 whereas C34 has been widely used for studying the
structure and function of HIV-1 gp41. The X-ray crystal
structure of the N36/C34 complex shows a highly conserved
Received: December 24, 2012
© XXXX American Chemical Society
A
dx.doi.org/10.1021/jm3018964 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 1. Schematic representation of HIV-1 gp41 and the designed small molecule−peptide conjugates. (A) HIV-1 gp41 contains a fusion peptide
(FP), an N-terminal heptad repeat (NHR), a C-terminal heptad repeat (CHR), a transmembrane domain (TM), and a cytoplasm domain (CP). The
NHR contains a putative T20 interaction domain, a helix zone, and a pocket domain; CHR contains a pocket-binding domain (PBD), a helix-zone-
binding domain, and a lipid-binding domain. The amino acid sequences of the representative peptide fusion inhibitors T20 and C34, along with their
respective targets, N46 and N36, are also shown and are highlighted in different colors. (B) Interaction between the NHR and CHR peptides. The
green lines between the NHR and CHR indicate the interaction between the residues located at the a and d positions in the CHR and the e and g
positions in the NHR to form the 6HB. The interaction between the PBD of the CHR and the deep pocket of the NHR is critical for stabilization of
the 6HB. (C) P26 is a peptide that was engineered by deleting the PBD of C34. Small molecule−peptide conjugates were designed by covalently
linking N-(carboxyphenyl)pyrrole derivatives and P26 with a flexible linker.
cell fusion was moderate .22,23 An increasing number of small
molecule HIV-1 fusion inhibitors have been reported with IC50
values in the micromolar range by cell−cell fusion assay.
However, this does not represent a significant step forward in
improving potency.24,25 Moreover, no conclusive evidence has
shown that the primary cavity is indeed the target of these small
molecule inhibitors.26,27
e.g., the N36/C34 6HB, which has been widely used as a tool to
study the mechanism of HIV fusion inhibitors.1 Residues
Trp628, Trp631, and Ile635 (WWI) of C34 peptide insert into
the pocket formed by a cluster of residues in the NHR coiled
coil (Figure 1B). This “WWI motif” is critical for the NHR and
CHR interhelical interactions and is essential for the peptide’s
anti-HIV potency.29,30 C34 peptide was used as a lead
compound, and to design fusion inhibitors with novel scaffolds,
we replaced the WWI motif with small molecule fusion
inhibitors targeting the deep pocket of the HIV-1 gp41 NHR.
To serve as a control, P26 peptide, which does not contain the
PBD of C34, was synthesized (Figure 1C). Next, small
molecules were covalently attached to the N-terminus of P26
through a flexible linker (β-alanine, βAla) to maintain the
proper binding orientation and conformation.
For small molecule fusion inhibitors, the carboxyl group in
NB-2 is critical31 to its inhibitory activity, since NB-177, which
has the same parent structure as NB-2 but without the
carboxylic acid group, is inactive. The hydrophobic pyrrole ring
of NB-2 is also favorable, since B15, which has a maleimide ring
replacing the pyrrole ring, shows weak inhibitory activity on
HIV-1 replication and 6HB formation. A12, an isomer of NB-2,
exhibits promising anti-HIV-1 potency. Structure−activity
relationship (SAR) analysis22,23 has shown that the carboxylic
acid group of A12 and the hydrophobic pyrrole ring are also key
contact motifs, whereas the phenolic group does not contribute
significantly to the overall binding affinity and has plenty of
room for modifications. On the basis of this reasoning, we
designed two parallel series (the Xoc series and the Xpc series)
Previous reports have indicated that conjugation of an active
peptide with an active nonpeptide moiety elicits a strong
synergistic effect,28 suggesting a more intuitive design for new
drugs with improved activity. In this study, we aimed to
discover new lead compounds with a novel scaffold and higher
potency. Accordingly, we designed and synthesized a set of
conjugates in which the P26 peptide, containing the partial C34
peptide sequence without the PBD, and two series of N-
(carboxyphenyl)pyrrole derivatives were covalently linked
together. The activities of the conjugates were tested in HIV-
1 Env-mediated cell−cell fusion and HIV-1 infection assays
using the laboratory-adapted HIV-1 IIIB strain. In addition,
they were tested against both T20-sensitive and T20-resistant
HIV-1 strains. Furthermore, to better understand and interpret
the bioassay results and the nature of the interaction between
the conjugated peptides and gp41, computational modeling
studies were performed. The results of this study will provide
useful information for the rational design of novel antiviral
peptides against HIV and other viruses with class I fusion
proteins.
Design. Using the protein dissection strategy, Kim’s group
resolved the crystal structure of the HIV-1 gp41 fusion core,
B
dx.doi.org/10.1021/jm3018964 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
of N-substituted pyrrole compounds based on the structures of
were obtained after removal of all protection groups by
trifluoroacetic acid (TFA), including the tert-butyl group on 6
(Figure 3A). As shown in Scheme 2, the Paal−Knorr reaction
was used to synthesize 14 by the condensation of anilines with
chemical intermediate 9, and a tert-butyl ester was introduced at
the 3-position on the pyrrole ring, which could be removed by
6 M HCl/EtOAc to afford 13. On the other hand, the benzyl
group could be deprotected by 1 M KOH to obtain 14.
However, the synthesis of Npc using the same route was
unsuccessful as a result of the instability of 4-(3-(tert-
butoxycarbonyl)-2,5-dimethyl-1H-pyrrol-1-yl)-2-hydroxyben-
zoic acid, an isomer of 11, during workup and purification when
the color changed rapidly from white to deep red. Therefore,
Npc was prepared as shown in Scheme 3. The amino group of
commercially available 4-aminosalicylic acid was protected by
Boc2O before coupling with benzyl alcohol and deprotection of
the amino group with HCl in ethyl acetate (6 M). In this series,
compounds 13 and 20 were covalently linked to the N-
terminus of the peptide, and the corresponding conjugates
Apc−βAla−P26 and Npc−βAla−P26 were obtained after
removal of all peptide protection groups by hydrofluoric acid
(HF), including the benzyl group on the nonpeptide moiety
(Figure 3B).
A12 and NB-2. In our modifications, a carboxylic acid group was
introduced into the parent structure of these two small
molecules as the intermediate to couple to P26 via an amide
bond. First, a carboxymethyl group was introduced into the
phenolic groups of A12 and NB-2 to obtain Aoc and Noc,
respectively. We assumed that this modification would have
little impact on the overall structures of these two compounds.
Second, a new carboxylic acid group was introduced to the
pyrrole groups of A12 and NB-2 to obtain Apc and Npc,
respectively. We suspected that this modification would change
the electron density of the pyrrole ring and further impact the
interaction with its target (Figure 2).
RESULTS AND DISCUSSION
■
Inhibitory Activities of the Small Molecule−Peptide
Conjugates on HIV-1 Env-Mediated Cell−Cell Fusion. We
used a cell−cell fusion assay to measure the inhibitory activities
of the small molecule−P26 conjugates. The activities of the
compounds are shown in Table 1. In our assay, the positive
control peptides C34 and T20 had IC50 values of 1.4 0.17
Figure 2. Chemical structures of Aoc, Noc, Apc, and Npc.
Chemistry. As shown in Scheme 1, derivatives of the Xoc
series were obtained from either 5-nitrosalicylic acid or 4-
nitrosalicylic acid by esterification with tert-butyl alcohol, and
the phenolic group was further alkylated with ethyl
bromoacetate to provide 3a and 3b. Then the nitro group
was converted into an amine by catalytic reduction. Without
purification, the Paal−Knorr reaction was used to synthesize 5
by the condensation of anilines with acetonylacetone (hexane-
2,5-dione). By saponification, 5 was converted into the acids 6a
and 6b, to which the N-terminus of the peptide was attached,
and the corresponding Noc−βAla−P26 (P26 conjugated with
an NB-2 derivative through a β-alanine) and Aoc−βAla−P26
(P26 conjugated with an A12 derivative through a β-alanine)
and 10
terminal PBD, P26 (Asn636−Leu661), had an IC50 value of
2540 630 nM. Thus, removal of the PBD reduced the
1.4 nM, respectively. The peptide without the N-
inhibitory activity by ∼1800-fold, from which we can infer that
the PBD region plays a critical role in inhibiting HIV-1-
mediated cell−cell fusion by interacting with the primary
pocket in the gp41 NHR. In the Xpc series, compounds 14 and
21 had no inhibitory activity in the cell−cell fusion assay; thus,
an introduced polar group on the five-membered ring could
significantly affect its electron density and further disrupt the
hydrophobic interactions with its binding sites. On the
a
Scheme 1. Synthesis of Compounds 7a and 7b
a
Reagents and conditions: (i) tert-butyl alcohol, DCC, DMAP, 60 °C; (ii) BrCH2CO2Et, K2CO3, 2-butanone, reflux, 5 h; (iii) 10% Pd/C, CH3OH,
50 psi; (iv) 2,5-hexanedione, p-TsOH, NMM, toluene, reflux; (v) 1 M NaOH, CH3OH; (vi) TFA, DCM, rt.
C
dx.doi.org/10.1021/jm3018964 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 3. Strategy for preparation of small molecule−peptide conjugates.
a
Scheme 2. Synthesis of Compound 14
a
Reagents and conditions: (i) EtONa, BrCH2COCH3, 0 °C to rt; (ii) 9, p-TsOH, NMM, toluene, reflux; (iii) PhCH2OH, DCC, DMF/DCM, reflux;
(iv) 6 M HCl/EtOAc, rt; (v) 1 M KOH, dioxane.
contrary, in the Xoc series, compounds 7a and 7b both
displayed moderate inhibitory activity, indicating that this
modification maintained the interactions of the pyrrole and
carboxylic acid group on the benzene ring with their targets
very well. Attaching 21 or 14 to the N-terminus of P26
dramatically increased the activity of the P26 peptide with IC50
values of 130 41.2 and 300 33.9 nM, respectively, about an
8−20-fold increase compared with that of P26 alone. Notably,
the inhibitory activity was further increased when 7a and 7b
were conjugated to P26, with IC50 values of 22 3.2 and 14
3.0 nM, respectively, resulting in about 170-fold more potency
than that of P26 alone, reaching the potency of T20. These
results suggest that the nonpeptide moiety might be favorable
for binding to the cavity, resulting in a strong synergistic effect
that significantly enhances the antiviral potency of the small
molecule−peptide conjugates. To test whether the synergistic
effect resulted from the linker (βAla) or a simple hydrophobic
interaction, we covalently linked βAla to P26 or Phe to the N-
terminus of βAla−P26. The results suggested that the non-
pocket-specific binding portion in the small molecule−peptide
conjugates makes limited contact with the hydrophobic cavity,
and these control peptides could not reach the goals described
above.
D
dx.doi.org/10.1021/jm3018964 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
a
Scheme 3. Synthesis of Compound 21
a
Reagents and conditions: (i) Boc2O, NaOH, dioxane/H2O, rt; (ii) PhCH2OH, DCC, DMF/DCM; (iii) 2 M HCl/EtOAc, rt; (iv) 9, p-TsOH,
NMM, toluene, reflux; (v) 6 M HCl/EtOAc, rt; (vi) HF.
Table 1. Inhibitory Activities of Small Molecules and Small Molecule−P26 Conjugates on HIV-1 Env-Mediated Cell−Cell
a
Fusion
compd
structure
IC50 (nM)
b
C34
WMEWDREINNYTSLIHSLIEESQNQQEKNEQELL
NNYTSLIHSLIEESQNQQEKNEQELL
1.40 0.17
2540 630
4553 262
3430 630
130 41.20
300 33.90
22.5 3.18
14.9 2.99
b
P26
b
βAla−P26
Phe−βAla−P26
Npc−βAla−P26
Apc−βAla−P26
Noc−βAla−P26
βAla−NNYTSLIHSLIEESQNQQEKNEQELL
b
c
Phe−βAla−NNYTSLIHSLIEESQNQQEKNEQELL
Npc−βAla−NNYTSLIHSLIEESQNQQEKNEQELL
Apc−βAla−NNYTSLIHSLIEESQNQQEKNEQELL
Noc−βAla−NNYTSLIHSLIEESQNQQEKNEQELL
Aoc−βAla−NNYTSLIHSLIEESQNQQEKNEQELL
1-(4-carboxy-3-hydroxyphenyl)-2,5-dimethyl-1H-pyrrole-3-carboxylic acid
1-(3-carboxy-4-hydroxyphenyl)-2,5-dimethyl-1H-pyrrole-3-carboxylic acid
2-(carboxymethoxy)-4-(2,5-dimethyl-1H-pyrrol-1-yl)benzoic acid
2-(carboxymethoxy)-5-(2,5-dimethyl-1H-pyrrol-1-yl)benzoic acid
c
c
c
Aoc−βAla−P26
21 (Npc)
d
NA
d
14 (Apc)
NA
7a (Noc)
>100000
7b (Aoc)
>100000
b
T20
YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF
10.1 1.40
a
b
The compounds were tested in triplicate, and the data are presented as the mean standard deviation. These peptides have an acetyl group at the
c
d
N-terminus and carboxyamide at the C-terminus. These peptides have carboxyamide at the C-terminus. NA = not active.
Inhibitory Activities of the Conjugated Peptides on
Viral Replication and Their Cytotoxicities. Very good
agreement between inhibition of HIV-1-mediated cell−cell
fusion and inhibition of viral replication was observed for the
small molecule−peptide conjugates. The inhibitory activities of
the small molecule−peptide conjugates on HIV-1IIIB replication
in MT-2 cells were assessed using a p24 enzyme-linked
immunosorbent assay (ELISA) as previously described.20 Two
of the compounds, Aoc−βAla−P26 and Noc−βAla−P26,
Table 2. Antiviral Activities of Conjugates against
Laboratory-Adapted HIV-1 Strains and Their Cytotoxicities
a
inhibition of
p24
production,
EC50 (nM)
inhibition of
6HB
formation, IC50
CC50
(μM)
b
compd
SI
(μM)
Npc−βAla−P26
Apc−βAla−P26
Noc−βAla−P26
Aoc−βAla−P26
T20
175 35.6
458 32.7
14.5 0.5
14.1 0.2
20.0 3.0
>500
>500
>500
>500
>2000
>1000
167 88.2
123 98.4
24.9 1.7
7.8 0.4
>30000
>30000
inhibited HIV-1IIIB infection with IC50 values of 14.1
0.2
and 14.5 0.5 nM, respectively, similar to the antiviral potency
of T20. The other two compounds, Apc−βAla−P26 and
Npc−βAla−P26, had IC50 values of 458 32.7 and 175 35.6
nM, respectively. We also tested the cytotoxicities of these
compounds on TZM-b1 cells using an MTS cytotoxicity assay
[MTS = 3-(4,5-dimethylthiazol-2-yl)-5-((3-carboxymethoxy)-
phenyl)-2-(4-sulfophenyl)-2H-tetrazolium]. The CC50 (con-
centration causing 50% cytotoxicity) values were all greater
than 500 μM, with selectivity indices (SIs) from 1000 to 30000
(Table 2).
a
The compounds were tested in triplicate, and the data are presented
b
as the mean standard deviation. SI =selectivity index, CC50/EC50
disrupt the interaction between the NHR and CHR of HIV-1
gp41 by using a fluorescence resonance energy transfer
assay.12,32 The ligand bpy (2,2′-bipyridine-5′-carboxylate) was
linked to the N-terminus of the N-peptide (GQAVEAQQH-
LLQLTVWGIKQLQARILAVEKK) containing the hydropho-
bic pocket of gp41 with a suitable linker. The probe peptide
CP2-LY (MTWBEWDREIBNYTSLIC-LY), which specifically
binds to the N-peptide, was modified with Lucifer yellow dye at
its C-terminus. The bpy-N-peptide formed a trimeric coiled
Small Molecule−Peptide Conjugates Interact with
HIV-1 gp41 NHR and Inhibit 6HB Formation of the gp41
NHR and CHR. Next we tested the conjugates’ ability to
E
dx.doi.org/10.1021/jm3018964 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
coil, namely, env2.0, by chelation with Fe(II) and acted as a
fluorescence quencher of the probe peptide. Competitive
inhibition of the env2.0−probe interaction by these small
molecule−P26 conjugates, with a concomitant increase in
fluorescence according to their potency, could be detected in
this assay. As shown in Table 2 and Figure 4, P26 exhibited no
peptide eluted at the earlier retention time. However, no new
peak corresponding to Xpc−βAla−P26 or P26 peptide (data
not shown) with N36 at the corresponding retention time was
observed. These results indicated that Noc−βAla−P26 and
Aoc−βAla−P26 are able to interact with N36 to form
complexes in PBS.
Small Molecule−Peptide Conjugates Are Highly
Potent against T20-Sensitive and T20-Resistant HIV-1
Strains. Although T20 is effective against a broad spectrum of
HIV-1 strains, including the wild type and those resistant to
multiple reverse transcriptase inhibitors (RTIs) or protease
inhibitors (PIs), drug-resistant HIV-1 variants were still easily
induced in patients after T20 therapy.33,34 Therefore, we
determined whether our hybrid molecule was effective against
HIV-1 strains resistant to T20. A panel of HIV-1NL4−3 mutants,
including one T20-sensitive strain and five T20-resistant strains,
was used in our experiments. As shown in Table 3, T20 was
much less effective against T20-resistant strains. We found that
double mutations in the gp41 NHR also conferred cross-
resistance to C34. Strikingly, Aoc−βAla−P26 and Noc−βAla−
P26 were highly potent against both T20-sensitive and T20-
resistant strains with IC50 values ranging from 130 to 250 nM,
whereas T20 was inactive against these resistant HIV-1 strains
at concentrations up to 2000 nM. Early in vitro studies
indicated that HIV-1 acquired T20 resistance by mutating the
amino acid residues in the “GIV” motif (amino acids
GIVQQQNNLL located in the NHR region of gp41).35,36
Our previous studies indicated that CHR peptides containing
PBD, such as C34, are much more effective against T20-
resistant strains with mutations in the GIV motif than T20,
which lacks PBD.37 The small molecule moiety in our
conjugated peptides mimics the PBD and can specifically
interact with the NHR pocket, thus providing additional
binding sites other than T20 and rendering the conjugated
peptides effective against T20-resistant HIV-1 isolates. These
results indicated that small molecule−peptide conjugates could
be developed into a new generation of fusion inhibitors to
overcome T20-resistant variants.
Figure 4. Inhibitory activities of the five C-peptides on 6HB formation
between env2.0 and CP2-LY as determined by the fluorescence
resonance energy transfer experiment.
significant competitive inhibition with env2.0, whereas Npc−
and Apc−P26 conjugates showed moderate inhibitory activities
with IC50 values of 167
88.2 and 123
98.4 μM,
respectively. Both Noc− and Aoc−P26 conjugates exhibited
high inhibitory potencies against env2.0−probe formation with
IC50 values of 24.9 1.70 and 7.79 0.366 μM, respectively.
Their inhibitory activities on the env2.0−probe complex
strongly correlated with the inhibitory activities of these
compounds on cell−cell fusion and their antiviral activity
against the HIV-1IIIB strain. The specific interactions between
small molecule−peptide conjugates and HIV-1 gp41 NHR were
further confirmed by size-exclusion HPLC (SE-HPLC) (Figure
5). The solid line chromatograms of Figure 5 show the
conjugated peptides alone. Next the N36 peptide was mixed
with each conjugated peptide. After a 30 min incubation period,
the mixture was applied onto an SE-HPLC column, and the
chromatograms are shown as dotted lines. The complex formed
between Noc−βAla−P26 and Aoc−βAla−P26 with the N36
Aoc−P26 Conjugates Are Resistant to Proteinase K
Digestion. Some of the major disadvantages of T20 are its
poor pharmacokinetics in vivo and its high sensitivity to
proteolytic enzymes in the blood. We speculated that our
designed conjugated peptides would be more resistant than
T20 to proteolytic enzymes. Therefore, we determined the
stability of Aoc−βAla−P26 with proteinase K (a broad-
spectrum serine proteinase) digestion.38 When incubated with
20 ng/mL proteinase K in PBS for 1 h, the T20 concentration
Figure 5. Determination of complex formation between small molecule−P26 conjugates and N36 using SE-HPLC analysis.
F
dx.doi.org/10.1021/jm3018964 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
a
Table 3. Activities of Small Molecule−Peptide Conjugates against T20-Sensitive and T20-Resistant Strains
IC50 (nM)
NL4-3 mutant
T20
C34
Aoc−βAla−P26
Noc−βAla−P26
b
D36G
(36G)V38A
(36G)V38A/N42D
50 10
5.12 0.51
130 12
170 10
c
>2000 (>40)
>2000 (>40)
>2000 (>40)
>2000 (>40)
>2000 (>40)
3.26 1.1 (0.6)
37.2 4.1 (7.3)
192 9 (38)
136 12 (27)
85.5 10 (17)
210 10 (1.6)
220 10 (1.7)
230 1 (1.8)
190 80 (1.5)
240 1 (1.8)
240 30 (1.4)
250 80 (1.5)
240 30 (1.4)
190 10 (1.1)
220 30 (1.3)
c
c
(36G)N42T/N43K
c
(36G)V38E/N42S
c
(36G)V38A/N42T
a
The compounds were tested in triplicate, and the data are presented as the mean standard deviation; the values in parentheses indicate relative
b
c
changes (n-fold) in the IC50 compared with the IC50 in the presence of the D36G substitution. T20-sensitive strain. T20-resistant strain.
Figure 6. Digestion of C-peptide inhibitors with proteinase K treatment, as determined by HPLC. (A) Time course of residual T20, C34, and
Aoc−βAla−P26 with proteinase K digestion. The chromatograms of T20 (B), C34 (C), and Aoc−βAla−P26 (D) at 0 h (purple), 0.5 h (black), and
1 h (red) are also shown, respectively.
decreased quickly, and only 40% of the original amount was
detected by HPLC. In contrast, C34 retained about 70% of the
original amount. Interestingly, Aoc−βAla−P26 maintained
about 80% of the original amount, as detected by HPLC,
suggesting that the conjugated peptide was much more
resistant to proteinase K than T20 and even better than C34
(Figure 6). Subsequently, we investigated the pharmacokinetic
(PK) profile of Aoc−βAla−P26 in rats after a single-dose
intravenous administration and determined the in vivo and in
vitro half-lives of Aoc−βAla−P26 and T20, respectively.
Aoc−βAla−P26 exhibited a shorter in vivo half-life than T20
(0.223 h vs 0.997 h). However, its in vitro half-life in the liver
and kidney homogenates was longer than that of T20,
consistent with the stability in proteinase K digestion assay
(detailed PK data can be found in the Supporting Information).
Therefore, we speculate that the shorter in vivo half-life of
Aoc−βAla−P26 may be due to its higher tissue-binding rate
compared to that of T20.
N36−C34 complex (PDB 1AIK).1 Molecular dynamics
simulations were conducted on the peptide complex using
AMBER 8.039 on an SGI Altix 350 workstation (Fremont, CA).
From the calculated result of the negative total binding free
energy, we learned that the binding free energies of Aoc−βAla−
P26 (−306.61
(−283.71 25.40 kcal/mol) were notably less than the
binding free energy of P26 peptide (−236.21 12.32 kcal/
mol), indicating that the complexes formed between
Aoc−βAla−P26 or Noc−βAla−P26 and NHR were more
stable than P26. Figure 7A shows the computational model of
the complex formed between Aoc−βAla−P26 (yellow and red)
and the N36 peptide (blue) of gp41. Aoc−βAla−P26 and N36
interacted with each other in an antiparallel manner to form a
stable coiled coil; however, the conjugated peptide did not fully
adopt an α-helical structure. We hypothesized the following to
explain this result. In 6HB composed of N36 and C34, residues
Trp628 and Trp631 of C34 were at the a and d positions,
respectively, in the same helix. Ile635 was at the a position in
the next helix. Removing the PBD destroyed the two helices.
Consequently, the small molecule was unable to attach the
residues at the a or d position, preventing it from inducing high
α-helicity. Figure 7B shows the local enlargement of the
interaction between Aoc in the conjugates with the
corresponding residues in the primary cavity. The hydrophilic
15.65 kcal/mol) and Noc−βAla−P26
Computational Modeling. To better understand and
interpret the bioassay results and the nature of the interaction
between the conjugated peptides and gp41, computational
modeling studies were performed to estimate the binding free
energy for the association between the predominant con-
formations of the designed small molecule−peptide conjugates
and the NHR on the basis of the X-ray crystal structure of the
G
dx.doi.org/10.1021/jm3018964 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
CONCLUSION
■
In conclusion, novel highly potent small molecule−peptide
conjugates with low nanomolar levels of anti-HIV fusion
activity were designed and synthesized. We found that all four
small molecule−peptide conjugates exhibited significant
inhibitory activity on HIV-1-mediated cell−cell fusion and
viral replication, as well as 6HB formation. A small molecule
moiety can act as a substitute for the PBD of the C34 peptide
and maintain highly potent anti-HIV-1 fusion activity. Unlike
T20, the hybrid molecule was less sensitive to proteinase K
digestion and highly effective against T20-sensitive and T20-
resistant HIV-1 strains. Therefore, the rational combination of
small molecule and peptide anti-HIV-1 fusion inhibitors into
one molecule has shed new light on the design of novel HIV-1
fusion inhibitors targeting gp41. Our results provide important
information for understanding the interaction between the
NHR deep pocket and small molecule fusion inhibitors.
Figure 7. Aoc−βAla−P26 and gp41 N-trimer interaction. Computer
modeling is based on the crystal structure of the HIV-1 gp41 6HB
(PDB code 1AIK). (A) Representation of the N36 coiled coil as a
molecular surface (light blue), with Aoc−βAla−P26 (yellow and red)
depicted as rods. Three C-peptides were expected to interact with the
N-trimer, but for clarity, only one C-peptide is shown. In the
conjugated peptide, Aoc and P26 are shown by a stick model (yellow)
and a ribbon diagram (red), respectively. The left panel shows a front
view of the conjugated peptide−N36 complex, while the right panel
shows a view from the side. The small molecule−P26 conjugated
peptide packs against a conserved groove on the surface of the coiled
coil. (B) The top of the coiled coil has a large cavity that provides a
binding pocket for Aoc (gray). The hydrogen bond between the
carboxylic acid and Gln575 is shown with a light green dashed line.
EXPERIMENTAL SECTION
■
Chemistry. Melting points were measured with an RY-1 melting
point apparatus without correction. 1H (400 MHz) and 13C (100
MHz) NMR spectra were measured on a JNM-ECA-400 spectrometer
using TMS as the internal standard. The solvent used was DMSO-d6
or CD3OD. Mass spectra were measured on an API-150 mass
spectrometer with the electrospray ionization source from ABI Inc.
TLC was performed on silica gel GF254 plates. Silica gel (200−300
mesh) from Qingdao Haiyang Chemical Co. was used for column
chromatography. All chemicals were obtained from Beijing Chemical
Works or Sigma-Aldrich, Inc. and were distilled and dried before use
according to literature procedures. For small molecule compounds,
purity was determined by analytical RP-HPLC using an Agilent 1200
series and an RP-C18 column (Agilent Eclipse XDB-C18, 5 μm, 4.6 ×
250 mm) using two different solvent systems (conditions 1 and 2 in
the Supporting Information) and a flow rate of 1 mL/min with the
detection wavelength at 254 nm. For peptides, purity was determined
by analytical RP-HPLC on an RP-C8 column (Zorbax Eclipse XDB-
C8, 5 μm, 4.6 × 150 mm) using two different solvent systems
(methods A and B in the Supporting Information) and a flow rate of 1
mL/min with the detection wavelength at 210 nm. All target
compounds were purified, which resulted in ≥95% purity for assay
testing (details of HPLC characterization of the compounds are given
in the Supporting Information).
residue Gln575 could form hydrogen bonds with the carboxylic
acid group of Aoc, and the hydrophobic cavity nicely
complements the shape of the pyrrole ring of Aoc to form
hydrophobic interactions.
Currently, more and more small molecule HIV-1 fusion
inhibitors targeting the primary hydrophobic cavity on the
groove of the gp41 N-trimer have been identified. However,
until now, no significant improvement in the antiviral potency
of the small molecule inhibitors has been reported. The crystal
structures of these compounds binding to the target have also
not been reported. Therefore, it is reasonable to question
whether the pocket is the target of these nonpeptide fusion
inhibitors. NB-2 and A12 are N-substituted pyrroles that were
designed to target this cavity. In this study, their derivatives
were covalently attached to the N-terminus of the peptide P26
through a linker, replacing the PBD of C34. We found that the
nonpeptide small molecule moiety could largely restore the
function of the PBD in HIV-1-mediated cell−cell fusion and
caused a significant effect on the whole antiviral potency of the
conjugates. Furthermore, the fusion inhibitory activity of the
conjugates had a strong correlation with these N-
(carboxyphenyl)pyrroles alone. A fluorescence resonance
energy transfer experiment further confirmed that N-
substituted pyrrole derivatives did indeed interact with the
hydrophobic pocket, and the binding affinity was significantly
improved with the increased number of critical functional
groups on these small molecules. The interactions between the
hybrid peptides and N36 were also characterized by SE-HPLC.
The combined results indicated that these N-(carboxyphenyl)-
pyrrole derivatives could largely act as substitutes for the PBD
of C34 by targeting the hydrophobic cavity on the surface of
the N-trimer coiled coil. Furthermore, the rationally designed
N-(carboxyphenyl)pyrrole−peptide conjugates could be used
as molecular probes to provide obvious evidence that the cavity
is the target of these nonpeptide fusion inhibitors.
Synthesis of tert-Butyl 2-Hydroxy-4-nitrobenzoate (2a). DCC
(1.2 equiv, 0.62 g, 3 mmol) dissolved in dry THF (6 mL) was added
dropwise over 5 min to a stirred solution of 4-nitrosalicylic acid (1a;
0.5 g, 2.73 mmol) and DMAP (1 equiv, 0.33 g, 2.73 mmol) in dry tert-
butyl alcohol (12.5 mL). The mixture was stirred and heated to reflux
overnight. The reaction was monitored by TLC. After the reaction was
complete, the solvents were removed under reduced pressure. Then 15
mL of ethyl acetate was added, and the solution was filtered to remove
the 1,3-dicyclohexylurea (DCU) formed. After removal of the solvent
under reduced pressure, the crude product was purified by column
chromatography using petroleum ether/ethyl acetate (30:1) as the
eluent to afford 0.48 g of 2a as pale yellow crystals, 73% yield, mp 84−
86 °C. MS (m/z): calcd for C11H13NO5, 239. LC−MS (m/z (rel
intens)): 239.5 (M+, 60). 1H NMR (DMSO-d6): δ 11.00 (s, 1H), 7.88
(d, 1H), 7.71 (m, 2H), 1.60 (s, 9H). 13C NMR (DMSO-d6): δ 166.10,
159.16, 150.56, 131.60, 121.84, 113.45, 111.85, 83.26, 27.68.
Data for tert-Butyl 2-Hydroxy-5-nitrobenzoate (2b). Mp: 82−84
°C. MS (m/z): calcd for C11H13NO5, 239. LC−MS (m/z (rel intens)):
1
239.5 (M+, 60). H NMR (DMSO-d6): δ 11.55 (s, 1H), 8.47 (d, J =
4.0 Hz, 1H), 8.33 (dd, J = 8.0, 4.0 Hz, 1H), 7.16 (d, J = 8.0 Hz, 1H),
1.59 (s, 9H). 13C NMR (DMSO-d6): δ 165.96, 164.58, 139.20, 129.69,
126.35, 118.57, 115.63, 83.58, 27.67.
Synthesis of tert-Butyl 2-(2-Ethoxy-2-oxoethoxy)-4-nitroben-
zoate (3a). To a solution of 2a (0.42 g, 1.75 mmol) in 2-butanone
(10 mL) was added anhydrous potassium carbonate (1.1 equiv, 0.26 g,
H
dx.doi.org/10.1021/jm3018964 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
1.88 mmol), and the mixture was heated to reflux for 30 min. Then
ethyl bromoacetate (1.2 equiv, 0.36 g, 2.15 mmol) was added. The
mixture was continuously stirred, was heated to reflux for 3 h, and was
monitored by TLC. After the reaction was finished, water (15 mL) was
added. The product was extracted with chloroform three times,
successively, and dried over Na2SO4. After removal of the solvent
under reduced pressure, the crude product was purified by column
chromatography using petroleum ether/ethyl acetate (20:1) as the
eluent to afford 0.45 g of 3a as a yellow oil, 80% yield, mp 50−52 °C.
MS (m/z): calcd for C15H19NO7, 325. LC−MS (m/z (rel intens)):
326.2 (M + H, 27). 1H NMR (DMSO-d6): δ 7.89 (dd, J = 8.4, 1.9 Hz,
1H), 7.84 (d, J = 1.9 Hz, 1H), 7.77 (d, J = 8.4 Hz, 1H), 5.04 (s, 2H),
4.19 (q, J = 7.0 Hz, 2H), 1.54 (s, 9H), 1.22 (t, J = 7.0 Hz, 3H). 13C
NMR (DMSO-d6): δ 167.83, 164.06, 155.90, 149.45, 130.59, 129.05,
116.02, 108.66, 82.35, 65.81, 60.87, 27.69, 14.04.
Data for 2-(2-(tert-Butoxycarbonyl)-4-(2,5-dimethyl-1H-pyrrol-1-
yl)phenoxy)acetic Acid (6b). Mp: 150−152 °C. MS (m/z): calcd for
C19H23NO5, 345. LC−MS (m/z (rel intens)): 346.3 (M + H, 100). 1H
NMR (CD3OD): δ 7.44 (d, J = 2.8 Hz, 1H), 7.30 (dd, J = 8.7, 2.8 Hz,
1H), 7.13 (d, J = 8,7 Hz, 1H), 5.78 (s, 2H), 4.79 (s, 2H), 1.96 (s, 6H),
1.58 (s, 9H). 13C NMR (DMSO-d6): δ 169.80, 164.23, 155.47, 131.99,
130.88, 129.34, 127.70, 122.93, 114.26, 105.88, 81.21, 65.40, 27.75,
12.87.
Synthesis of 2-(Carboxymethoxy)-4-(2,5-dimethyl-1H-pyrrol-1-
yl)benzoic Acid (7a). The protected 6a (0.34 g, 1 mmol) was
acidolysed by stirring in TFA (1.47 g, 13 mmol) and dichloromethane
(DCM; 0.26 g, 32 mmol) in the presence of triethylsilane (0.29 g, 2.5
mmol) at room temperature. When the reaction was completed, the
solvent was removed, the residue was triturated with H2O, and the
precipitated solid was isolated by filtration. The precipitated solid was
washed with water and dried to afford 0.10 g of 7a as a red solid, 36%
yield, mp 190−192 °C. MS (m/z): calcd for C15H15NO5, 289. LC−
MS (m/z (rel intens)): 290.1 (M + H, 100). 1H NMR (DMSO-d6): δ
12.99 (s, 2H), 7.76 (d, J = 8.1 Hz, 1H), 6.17 (dd, J = 8.1, 1.6 Hz, 1H),
6.87 (d, J = 1.6 Hz, 1H), 5.81 (s, 2H), 4.86 (s, 2H), 1.98 (s, 6H). 13C
NMR (DMSO-d6): δ 169.96, 166.60, 156.99, 141.94, 131.34, 127.57,
120.55, 120.01, 113.40, 106.36, 65.17, 12.79.
Data for tert-Butyl 2-(2-Ethoxy-2-oxoethoxy)-5-nitrobenzoate
(3b). Mp: 56−58 °C. MS (m/z): calcd for C15H19NO7, 325. LC−
1
MS (m/z (rel intens)): 326.2 (M + H, 27). H NMR (DMSO-d6): δ
8.36 (dd, J = 9.2, 2.8 Hz, 1H), 8.33 (d, J = 2.8 Hz, 1H), 7.29 (d, J = 9.2
Hz, 1H), 5.06 (s, 2H), 4.19 (q, J = 7.0 Hz, 2H), 1.55 (s, 9H), 1.23 (t, J
= 7.0 Hz, 3H). 13C NMR (DMSO-d6): δ 167.58, 163.21, 160.81,
140.52, 128.01, 125.66, 122.88, 114.35, 82.16, 65.73, 60.98, 27.70,
14.03.
General Procedure for the Preparation of 4a,b. A 180 mg amount
of 3a,b was dissolved in methanol (20 mL), followed by addition of
10% Pd on charcoal catalyst (18 mg), and the mixture was
hydrogenated at 50 psi and room temperature. After the reaction
was finished, the catalyst was removed by filtration, and the solvent
was evaporated. The product was directly used for the next step
without further purification.
Data fro 2-(Carboxymethoxy)-5-(2,5-dimethyl-1H-pyrrol-1-yl)-
benzoic Acid (7b). Mp: 144−146 °C. MS (m/z): calcd for
C15H15NO5, 289. LC−MS (m/z (rel intens)): 290.1 (M + H, 100).
1H NMR (DMSO-d6): δ 11.79 (s, 1H), 7.53 (d, J = 2.8 Hz, 1H), 7.43
(dd, J = 8.6, 2.8 Hz, 1H), 7.12 (d, J = 8.6 Hz, 1H), 5.78 (s, 2H), 4.80
(s, 2H), 1.94 (s, 6H). 13C NMR (DMSO-d6): δ 168.98, 166.59,
158.72, 135.09, 129.58, 129.29, 127.71, 118.58, 114.12, 105.78, 61.77,
12.72.
Synthesis of tert-Butyl 2-Acetyl-4-oxopentanoate (9). To a
solution of tert-butyl acetoacetate (8; 5 mL, 30.6 mmol) in ethanol
was added sodium ethoxide (12.3 mL, 30.6 mmol) dropwise in an ice
bath for 15 min with stirring. Then the mixture was added dropwise to
a solution of bromopropanone (2.2 mL, 25.4 mmol) in ethanol/
toluene (30 mL, 2:1) at 0 °C with continued stirring at room
temperature for 4 h and then adjusted to pH 7.0 using 2 M HCl. After
removal of the solvent under reduced pressure, 200 mL of ethyl
acetate was added, the solid precipitate was filtered, and the filtrate was
washed with water to neutral pH and dried over Na2SO4. The crude
product was purified by silica gel column chromatography to afford
3.79 g of 9 as a yellow oil, 70.1% yield. MS (m/z): calcd for
C11H118NO4, 214. LC−MS (m/z (rel intens)): 165.0 (M-C4H9O+Na,
100). H NMR (DMSO-d6): δ 3.85 (t, J = 8.0 Hz, 1H), 2.92 (m, J =
8.0 Hz, 2H), 2.23 (s, 3H), 2.11 (s, 3H), 1.41 (s, 9H). 13C NMR
(DMSO-d6): δ 205.75, 202.81, 167.82, 81.28, 54.63, 40.97, 29.45,
27.45.
Synthesis of 5-(3-(tert-Butoxycarbonyl)-2,5-dimethyl-1H-pyrrol-
1-yl)-2-hydroxybenzoic Acid (11). To a solution of 5-amino-2-
hydroxybenzoic acid (10; 1.1 equiv, 0.39 g, 2.57 mmol) in 10 mL of
toluene was added 9 (1 equiv, 0.50 g, 2.33 mmol). The mixture was
heated to reflux, 20 mg of p-toluenesulfonic acid was added, and the
mixture continued to reflux for 3 h with monitoring by TLC. After the
reaction was complete, the solvents were removed under reduced
pressure. Then 20 mL of ethyl acetate was added, and the organic
phase was washed with brine and dried over Na2SO4. After the mixture
was filtered and the filtrate was evaporated under reduced pressure, the
residue was purified by silica gel column chromatography to yield 0.54
g of intermediate 11 as a white solid, yield 70%, mp 146−152 °C. MS
(m/z): calcd for C18H21NO5, 331. LC−MS (m/z (rel intens)): 332.2
(M + H, 40). 1H NMR (DMSO-d6): δ 11.43 (s, 1H), 7.58 (d, J = 2.5
Hz, 1H), 7.44 (dd, J = 8.7, 2.8 Hz, 1H), 7.11 (d, J = 8.7 Hz, 1H), 6.18
(s, 1H), 2.17 (s, 3H), 1.90 (s, 3H), 1.48 (s, 9H). 13C NMR (DMSO-
d6): δ 171.53, 164.68, 161.27, 135.77, 135.41, 129.94, 128.72, 118.90,
114.23, 112.70, 107.99, 78.89, 28.68, 12.85, 12.66.
Synthesis of tert-Butyl 4-(2,5-Dimethyl-1H-pyrrol-1-yl)-2-(2-
ethoxy-2-oxoethoxy)benzoate (5a). To a solution of 4a (1.0 g,
3.38 mmol) in 20 mL of toluene were added N-methylmorpholine (1
equiv, 0.34 g, 3.38 mmol) and 2,5-hexanedione (2 equiv, 0.74 g, 6.49
mmol). The mixture was heated to reflux, followed by the addition of
30 mg of p-toluenesulfonic acid (PTSA), and the mixture continued to
reflux for 3 h with monitoring by TLC. After the reaction was
complete, the solvents were removed under reduced pressure. Then 20
mL of ethyl acetate was added, and the organic phase was washed with
brine, successively, and dried over Na2SO4. After filtration and
evaporation under reduced pressure, the residue was purified by silica
gel column chromatography to yield 0.94 g of intermediate 5a as a
colorless oil, yield 75%. MS (m/z): calcd for C21H27NO5, 373. LC−
1
MS (m/z (rel intens)): 374.2 (M + H, 20). H NMR (DMSO-d6): δ
7.67 (d, J = 8.1, 1H), 6.94 (d, J = 1.9 Hz, 1H), 6.92 (dd, J = 8.1, 1.9
Hz, 1H), 5.81 (s, 2H), 4.92 (s, 2H), 4.14 (q, J = 7.0 Hz, 2H), 1.98 (s,
6H), 1.54 (s, 9H), 1.19 (t, J = 7.0 Hz, 3H). 13C NMR (DMSO-d6): δ
168.29, 164.61, 156.67, 141.81, 130.86, 127.60, 121.71, 120.31, 113.58,
106.36, 81.01, 65.40, 60.70, 27.79, 14.02, 12.82.
Data for tert-Butyl 5-(2,5-Dimethyl-1H-pyrrol-1-yl)-2-(2-ethoxy-
2-oxoethoxy)benzoate (5b). MS (m/z): calcd for C21H27NO5, 373.
1
LC−MS (m/z (rel intens)): 374.4 (M + H, 40). H NMR (DMSO-
d6): δ 7.37 (m, 2H), 7.13 (m, 1H), 5.79 (s, 2H), 4.91 (s, 2H), 4.20 (q,
J = 7.0 Hz, 2H), 1.95 (s, 6H), 1.52 (s, 9H), 1.22 (t, J = 7.0 Hz, 3H).
13C NMR (DMSO-d6): δ 168.27, 164.23, 155.21, 131.95, 131.09,
129.29, 127.69, 123.15, 114.42, 105.88, 81.26, 65.55, 60.74, 27.73,
14.04, 12.82.
Synthesis of 2-(2-(tert-Butoxycarbonyl)-5-(2,5-dimethyl-1H-pyr-
rol-1-yl)phenoxy)acetic Acid (6a). A 20 mg amount of 5a was
dissolved in 4 mL of methanol, and 1 mL of 25% NaOH in H2O was
added. The mixture was stirred for 30 min at room temperature and
then adjusted to pH 3.0 using 1 M HCl. The precipitated solid was
filtered, washed with water until neutral pH, and dried to afford 18 mg
of 6a as a white solid, 98% yield, mp 170−172 °C. MS (m/z): calcd for
C19H23NO5, 345. LC−MS (m/z (rel intens)): 346.3 (M + H, 98). 1H
NMR (CD3OD): δ 7.78 (d, J = 8.1 Hz, 1H), 6.88 (dd, J = 8.1, 1.9 Hz,
1H), 6.82 (d, 1.9 Hz, 1H), 5.80 (s, 2H), 4.76 (s, 2H), 1.98 (s, 6H),
1.57 (s, 9H). 13C NMR (DMSO-d6): δ 169.83, 164.66, 157.79, 141.73,
130.85, 127.58, 120.90, 119.17, 113.60, 106.32, 80.74, 67.08, 27.84,
12.88.
Synthesis of tert-Butyl 1-(3-((Benzyloxy)carbonyl)-4-hydroxy-
phenyl)-2,5-dimethyl-1H-pyrrole-3-carboxylate (12). DCC (1.1
equiv, 0.27 g, 1.32 mmol) dissolved in dry THF (6 mL) was added
dropwise over 5 min to a stirred solution of 11 (0.4 g, 1.2 mmol),
benzyl alcohol (3 equiv, 0.39 g, 3.6 mmol), and DMAP (0.1 equiv,
I
dx.doi.org/10.1021/jm3018964 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
0.03 g, 0.27 mmol) in dry DCM/N,N-dimethylformamide (DMF) (12
mL, 5:1). The mixture was stirred and heated to reflux overnight. The
reaction was monitored by TLC. After the reaction was complete, the
solvents were removed under reduced pressure. Then 30 mL of ethyl
acetate was added, the mixture was filtered to remove the DCU
formed, and the organic phase was washed with brine and dried over
Na2SO4. After filtration and removal of the solvent under reduced
pressure, the crude product was purified by silica gel column
chromatography using petroleum ether/ethyl acetate (50:1) as the
eluent to afford 0.35 g of 12 as a colorless oil, 70% yield. MS (m/z):
calcd for C25H27NO5, 421. LC−MS (m/z (rel intens)): 422.3 (M + H,
10). 1H NMR (DMSO-d6): δ 10.71 (s, 1H), 7.59 (d, J = 2.5 Hz, 1H),
7.48 (m, 5H), 7.44 (dd, J = 8.7, 2.8 Hz, 1H), 7.14 (d, J = 8.7 Hz, 1H),
6.17 (s, 1H), 5.38 (s, 2H), 2.16 (s, 3H), 1.89 (s, 3H), 1.48 (s, 9H). 13C
NMR (DMSO-d6): δ 167.69, 164.66, 159.95, 136.09, 135.56, 135.39,
129.93, 129.06, 128.85, 128.79, 128.71, 128.66, 119.22, 114.95, 112.75,
108.02, 78.89. 67.29, 28.68, 12.85, 12.66.
Synthesis of 1-(3-((Benzyloxy)carbonyl)-4-hydroxyphenyl)-2,5-
dimethyl-1H-pyrrole-3-carboxylic Acid (13). A solution of 12 (0.2
g, 0.48 mmol) in 5 mL of HCl in ethyl acetate (6 M) was stirred at
room temperature for 10 min. The reaction mixture was evaporated to
remove the solvent. The residue was dissolved in 20 mL of ethyl
acetate, and the solution was evaporated to dryness. The resulting
product was purified by silica gel column chromatography to yield 0.17
g of 13 as a pink solid, yield 98%, mp 82−84 °C. MS (m/z): calcd for
C21H19NO5, 365. LC−MS (m/z (rel intens)): 366.2 (M + H, 37). 1H
NMR (DMSO-d6): δ 11.69 (s, 1H), 10.72 (s, 1H), 7.61 (d, J = 2.5 Hz,
1H), 7.47 (dd, J = 8.6, 2.5 Hz, 1H), 7.33−7.42 (m, 5H), 7.15 (d, J =
8.6 Hz, 1H), 6.21 (s, 1H), 5.38 (s, 2H), 2.18 (s, 3H), 1.90 (s, 3H). 13C
NMR (DMSO-d6): δ 167.17, 166.22, 159.40, 135.59, 135.30, 135.03,
129.39, 128.55, 128.36, 128.28, 128.20, 128.14, 118.68, 114.44, 111.35,
107.66, 66.77, 12.40, 12.10.
Synthesis of 1-(3-Carboxy-4-hydroxyphenyl)-2,5-dimethyl-1H-
pyrrole-3-carboxylic Acid (14). A 20 mg amount of 13 was dissolved
in 4 mL of dioxane, and 1 mL of 1 M KOH was added. The mixture
was stirred for 3 h at room temperature and then adjusted to pH 2−3
using 1 M HCl. The solution was extracted with ethyl acetate three
times. After removal of the solvent under reduced pressure, the residue
was purified by silica gel column chromatography to afford 3.2 mg of
14 as a white solid, 21% yield, mp 164−166 °C. MS (m/z): calcd for
C14H13NO5, 275. LC−MS (m/z (rel intens)): 276.1 (M + H, 100). 1H
NMR (DMSO-d6): δ11.54 (s, 2H), 7.60 (d, J = 2.5 Hz, 1H), 7.46 (dd,
J = 8.7, 2.5 Hz, 1H), 7.11 (d, J = 8.7 Hz, 1H), 6.21 (s, 1H), 2.19 (s,
3H), 1.92 (s, 3H). 13C NMR (DMSO-d6): δ 171.00, 166.21, 160.70,
135.32, 135.26, 129.39, 128.21, 118.35, 113.66, 111.31, 107.61, 12.36,
12.06.
was monitored by TLC. After the reaction was complete, the solvents
were removed under reduced pressure. Then 200 mL of ethyl acetate
was added, the mixture was filtered to remove the DCU formed, and
the organic phase was washed with brine and dried over Na2SO4. After
filtration and removal of the solvent under reduced pressure, the crude
product was purified by silica gel column chromatography using
petroleum ether/ethyl acetate (30:1) as the eluent to afford 6.2 g of 17
as a white solid, 46% yield, mp 112−114 °C. MS (m/z): calcd for
C19H21NO5, 343. LC−MS (m/z (rel intens)): 344.2 (M + H, 67). 1H
NMR (CD3OD): δ 7.71 (d, J = 8.9 Hz, 1H), 7.32−7.45 (m, 5H), 7.12
(d, J = 2.0 Hz, 1H), 6.89 (dd, J = 8.9, 2.0 Hz, 1H), 5.32 (s, 2H), 1.50
(s, 9H). 13C NMR (CD3OD): δ 170.37, 163.52, 153.93, 147.41,
136.75, 131.20, 129.16, 128.91, 128.82, 110.13, 107.06, 105.65, 80.93,
67.15, 28.07.
Synthesis of Benzyl 4-Amino-2-hydroxybenzoate (18). The
solution of 17 (10.76 g, 31.3 mmol) in 50 mL of HCl in ethyl
acetate (6 M) was stirred at room temperature for 2 h. The
precipitated solid was filtered and washed with petroleum ether to
afford 7.0 g of 18 as a white solid, 95% yield. MS (m/z): calcd for
C14H13NO3, 243. LC−MS (m/z (rel intens)): 244.1 (M + H, 100). 1H
NMR (DMSO-d6): δ 10.76 (s, 1H), 7.50 (dd, J = 8.7, 2.2 Hz, 1H),
7.33−7.47 (m, 5H), 6.48−6.70 (m, 4H), 6.16 (dd, J = 8.7, 2.2 Hz,
1H), 6.05 (d, J = 2.0 Hz, 1H), 5.32 (s, 2H). 13C NMR (DMSO-d6): δ
169.11, 162.80, 136.18, 131.18, 128.53, 128.11, 127.96, 107.10, 99.20,
65.52.
Synthesis of tert-Butyl 1-(4-((benzyloxy)carbonyl)-3-hydroxy-
phenyl)-2,5-dimethyl-1H-pyrrole-3-carboxylate (19). To a solution
of 18 (6.8 g, 24 mmol) in 100 mL of toluene were added N-
methylmorpholine (1 equiv, 2.69 mL, 24 mmol) and 9 (1.1 equiv, 5.72
g, 26.4 mmol). The mixture was heated to reflux, 0.34 g of p-
toluenesulfonic acid was added, and the mixture continued to reflux for
3 h with monitoring by TLC. After the reaction was complete, the
solvents were removed under reduced pressure. Then 80 mL of ethyl
acetate was added, and the organic phase was washed with brine and
dried over Na2SO4. After filtration and evaporation under reduced
pressure, the residue was purified by silica gel column chromatography
to yield 6.04 g of 19 as a colorless oil, yield 60%. MS (m/z): calcd for
C25H27NO5, 421. LC−MS (m/z (rel intens)): 422.2 (M + H, 10). 1H
NMR (DMSO-d6): δ 10.74 (s, 1H), 7.93 (d, J = 8.4 Hz, 1H), 7.37−
7.52 (m, 5H), 6.94 (d, J = 2.0 Hz, 1H), 6.86 (dd, J = 8.4, 2.0 Hz, 1H),
6.20 (s, 1H), 5.41 (s, 2H), 2.21 (s, 3H), 1.95 (s, 3H), 1.48 (s, 9H). 13C
NMR (DMSO-d6): δ 167.45, 164.07, 160.24, 142.79, 135.64, 134.33,
131.43, 128.59, 128.32, 128.15, 127.77, 119.32, 117.03, 114.02, 112.76,
108.11, 78.53, 66.71, 28.15, 12.34, 12.13.
Synthesis of 1-(4-((Benzyloxy)carbonyl)-3-hydroxyphenyl)-2,5-
dimethyl-1H-pyrrole-3-carboxylic Acid (20). The solution of 19
(6.04 g, 14.3 mmol) in 50 mL of HCl in ethyl acetate (6 M) was
stirred at room temperature for 1 h. The reaction mixture was
evaporated to remove the solvent. The precipitated solid was filtered
and washed with petroleum ether to afford 3.58 g of 20 as a brown
solid, 69% yield, mp 190−192 °C. MS (m/z): calcd for C21H19NO5,
365. LC−MS (m/z (rel intens)): 366.1 (M + H, 43). 1H NMR
(DMSO-d6): δ 11.73 (s, 1H), 10.72 (s, 1H), 7.93 (d, J = 8.4 Hz, 1H),
7.37−7.52 (m, 5H), 6.96 (d, J = 2.0 Hz, 1H), 6.88 (dd, J = 8.4, 2.0 Hz,
1H), 6.24 (s, 1H), 5.41 (s, 2H), 2.23 (s, 3H), 1.96 (s, 3H). 13C NMR
(DMSO-d6): δ 167.46, 166.12, 160.26, 142.82, 135.62, 134.75, 131.37,
128.55, 128.28, 128.13, 127.76, 119.29, 116.98, 113.92, 111.90, 108.25,
66.69, 12.37, 12.05.
Synthesis of 4-(tert-Butoxycarbonyl)-2-hydroxybenzoic Acid (16).
A solution of 4-amino-2-hydroxybenzoic acid (15; 1 equiv, 3.06 g, 20
mmol) in 1 M NaOH (20 mL) was stirred and cooled in an ice−water
bath. Di-tert-butyl pyrocarbonate (1.2 equiv, 5.24 g, 24 mmol) in 30
mL of dioxane was added, and stirring was continued at room
temperature for 2 h. The solution was concentrated in vacuo to
approximately 10−15 mL, cooled in an ice−water bath, covered with a
layer of ethyl acetate (30 mL), and acidified with a dilute solution of
KHSO4 to pH 2−3. The aqueous phase was extracted with ethyl
acetate (15 mL), and the extraction was repeated. The ethyl acetate
extracts were pooled, washed with water, dried over anhydrous
Na2SO4, and evaporated in vacuo. The residue was recrystallized with
DCM to afford 3.79 g of 16 as a white solid, 75% yield, mp 160−162
°C. MS (m/z): calcd for C12H15NO5, 253. LC−MS (m/z (rel intens)):
Synthesis of 1-(4-Carboxy-3-hydroxyphenyl)-2,5-dimethyl-1H-
pyrrole-3-carboxylic Acid (21). At 0 °C, a solution of 20 (0.5 g,
1.37 mmol) in 10 mL of HF was stirred for 1 h. After evaporation, the
residue was dissolved in ether and evaporated under reduced pressure
to remove the solvent. The residue was recrystallized with ethyl acetate
to provide 0.10 g of 21 as a gray solid, 27% yield, mp 204−206 °C. MS
(m/z): calcd for C14H13NO5, 275. LC−MS (m/z (rel intens)): 276.1
(M + H, 100). 1HNMR: δ 11.83 (s, 2H), 7.93 (d, J = 8.4 Hz, 1H), 6.95
(d, J = 1.7 Hz, 1H), 6.86 (dd, J = 8.4, 1.7 Hz. 1H), 6.24 (s, 1H), 2.24
(s, 3H), 1.97 (s, 3H). 13C NMR (DMSO-d6): δ 171.23, 166.16,
161.54, 143.01, 134.80, 131.38, 127.78, 119.11, 116.73, 113.29, 111.86,
108.23, 12.40, 12.08.
1
254.3 (M + H, 63). H NMR (CD3OD): δ 7.71 (d, J = 8.9 Hz, 1H),
7.11 (d, J = 2.0 Hz, 1H), 6.85 (dd, J = 8.7, 2.0 Hz, 1H), 1.52 (s, 9H).
13C NMR (CD3OD): δ 172.75, 163.79, 154.03, 147.11, 131.68, 109.85,
107.42, 105.59, 80.86, 28.10.
Synthesis of Benzyl 4-(tert-Butoxycarbonyl)-2-hydroxybenzoate
(17). DCC (1.1 equiv, 9.0 g, 43.45 mmol) dissolved in DCM (5 mL)
was added dropwise over 30 min to a stirred solution of 16 (10.0 g,
39.5 mmol), benzyl alcohol (3 equiv, 12.85 g, 118.5 mmol), and
DMAP (0.1 equiv, 0.4 g, 0.39 mmol) in dry DCM/DMF (35 mL, 6:1).
The mixture was stirred and heated to reflux overnight. The reaction
J
dx.doi.org/10.1021/jm3018964 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Peptide Synthesis. Peptides were synthesized using a Liberty
automated microwave peptide synthesizer (CEM Co., Matthews, NC)
with a standard solid-phase Fmoc chemistry protocol. All protected
amino acids used were purchased from GL Biochem Ltd. (Shanghai,
China). Rink amide resin and MBHA resin (0.38−0.45 mmol/g,
Nankai Hecheng S&T Co. Ltd., Tianjin, China) were used. Coupling
of the amino acids was achieved using O-benzotriazol-1-yl-N,N,N′,N′-
tetramethyluronium hexafluorophosphate (HBTU; GL Biochem,
Shanghai, China) and diisopropylethylamine (DIEA; Acrose) as an
activator and an active base, respectively, in DMF solution. The Fmoc
protective group was removed using 20% piperidine/DMF. Between
every coupling or Fmoc removal, the resin was washed five times with
DMF and three times with DCM. The carboxyl termini were amidated
upon cleavage from the resin, and the amino termini were capped with
acetic acid anhydride, except conjugated peptides. The peptides were
cleaved from the resin and deprotected with reagent K, which
contained 82.5% trifluoroacetic acid, 5% thioanisole, 5% m-cresol, 5%
water, and 2.5% ethanedithiol. The crude products were precipitated
with cold diethyl ether and lyophilized. The crude peptide products
were purified by preparative reversed-phase HPLC using a Waters
preparative HPLC system (PrepLC 4000): gradient elution of 30−
50% solvent B in solvent A (0.1% trifluoroacetic acid in H2O, solvent
A; 0.1% trifluoroacetic acid in 70% CH3CN/H2O, solvent B) over 60
min at 16 mL/min on a Waters X-bridge C8, 10 μm, 19.5 mm × 250
mm column. Analytical RP-HPLC was performed on an RP-C8
column (Zorbax Eclipse XDB-C8, 5 μm, 4.6 × 150 mm) with gradient
elution of 5−100% solvent B in solvent A over 25 min at a flow rate of
1 mL/min. The compounds were detected by UV absorption at 220
nm with SHIMADZU SPD-10A. All peptides were purified to >98%
purity. The molecular weight of the peptides was confirmed by
MALDI-TOF-MS (Autoflex III, Bruker Daltonics).
Cell−Cell Fusion Assay. Cell−cell fusion assays were performed
as previously described.40 HL2/3 cells (Drs. Barbara Felber and
George Pavlakis), which stably express HIV Gag, Env, Tat, Rev, and
Nef proteins, and TZM-bl cells (Drs. John C. Kappes, Xiaoyun Wu),
which stably express large amounts of CD4 and CCR5, were obtained
from the NIH AIDS Reference and Reagent Program. TZM-bl cells
(2.5 × 104 cells/well) and HL2/3 cells (7.5 × 104 cells/well) were
coincubated in 96-well plates (Corning Costar) at 37 °C in 5% CO2 in
the presence of different concentrations of inhibitors. After a 6−8-h
incubation, the medium was aspirated, the cells were washed and lysed,
and luciferase activity was measured using the Luciferase Assay System
(Promega, Madison, WI) on a SpectraMax M5 plate reader (Molecular
Devices, Sunnyvale, CA).
HIV-1 Infection Assay. For measuring the inhibitory activity of
the peptides on infection of HIV-1 IIIB and T20-resistant HIV-1
isolate, 1 × 104 MT-2 cells were infected with 100 TCID50 HIV-1IIIB
in the presence or absence of the peptides at graded concentrations.
On the fourth day postinfection, the culture supernatants were
collected for detection of HIV-1 Gag antigen p24 using an ELISA.
Cytotoxicity Assay. For determining the cytotoxicity of the
peptides, TZM-b1 cells were seeded in 96-well cell culture plates
(Corning, Inc., Corning, NY) at a density of approximately 1 × 104
cells/well in DMEM supplemented with 10% (v/v) FBS and incubated
for 24 h. The cells were washed twice with PBS and incubated with
100 μL of peptide at the indicated concentration at 37 °C for 24 h.
The cells were washed with PBS to remove the peptide. Thereafter, 20
μL of Cell Titer 96 AQueous One Solution Reagent (Promega Corp,
Madison, WI) was added to each well, followed by an incubation
period of 1 h. The solution absorbance was measured at 490 nm using
a SpectraMax M5 (Molecular Devices). PBS was used as a negative
control. Cell viability data were obtained by calculating the ratio of
viable cells in the treated cultures to the untreated control cells. The
experiment was repeated five times for each peptide concentration.
Binding Affinity Assay. The ability of the conjugated peptides to
bind to the hydrophobic pocket on the gp41 N-peptide coiled coil of
HIV-1 was determined using a fluorescence intensity assay as
previously described.32,41 Briefly, a metallopeptide receptor,
FeII(env2.0)3, was used to mimic the hydrophobic pocket in the
gp41 coiled coil. env2.0 contains 17 hydrophobic pocket residues
flanked by five residues on either side. Mixing FeII(env2.0)3 with a
fluorescein-labeled pocket-binding C-peptide, C18-FL, caused quench-
ing of fluorescence, which could be reversed in the presence of a
competitive inhibitor. Typically, 7 μM binding sites (three per receptor
trimer) and 1 μM C18-FL were used in the assay.
Binding Assays by Size-Exclusion Chromatography. N36 was
mixed with conjugated peptides (final concentration 0.20 mM) at a
molar ratio of 1:1 in 50 mM sodium phosphate/150 mM NaCl (pH
7.2) and incubated at 37 °C for 30 min. The mixture or peptide (20
μL) was applied to the Phenomenex BioSep-SEC-s2000, 300 × 7.80
mm HPLC column equilibrated with H2O and eluted at 0.8 mL/min,
and fractions were monitored at 210 nm.
Proteinase K Digestion Experiments. To test the proteinase K
sensitivity of C-peptides, each peptide (1 mg/mL, 500 μL) was mixed
with proteinase K (working concentration of 20 ng/mL), and then the
mixture was incubated at 37 °C. At different times points (0, 5, 15, 30,
60, 120, and 240 min), 10 μL samples were removed and mixed with
30 μL of CH3CN to stop the reaction. The samples were centrifuged,
and the supernatant was analyzed by HPLC using the same conditions
as described above.
Computational Modeling. The conformation of the coiled-coil
chimera protein TLT was studied by molecular dynamics simulation
using AMBER 8.0 on an SGI Altix 350 workstation (Fremont, CA).39
Molecular models were constructed using SWISS-MODEL Web
servers on the basis of the structure of the 6HB (PDB 1AIK). The
peptide was immersed in a rectangular box with TIP3P water before
energy minimization. Two stages of minimization and two stages of
equilibration were conducted to obtain an initial state for a further 5
nm simulation, which was conducted at a constant temperature of 300
K and a constant pressure of 1 atm, without restraints on the system.
The coordinates of the entire system at each time point (10 ps) of the
output trajectories were saved. PBTOT/GBTOT was calculated using
the mm_pbsa.pl program.
ASSOCIATED CONTENT
* Supporting Information
■
S
HPLC purity and characterization data of the compounds and
pharmacokinetic experiments and results. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
■
*Phone: 86-10-6816-9363 (K.L.); 86-21-54237673 (S.J.). Fax:
86-10-6821-1656 (K.L.); 86-21-54237465 (S. J.). E-mail:
keliangliu55@126.com (K.L.); shibojiang@fudan.edu.cn (S.J.).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This research was supported, in part, by grants from the
National Science Foundation of China (81072581 and
81273560) and a National Science and Technology Major
Project of China grant (2012ZX09301003).
ABBREVIATIONS USED
■
CHR, C-terminal heptad repeat; NHR, N-terminal heptad
repeat; 6HB, six-helix bundle; FDA, Food and Drug
Administration; Aoc-βAla-P26, P26 conjugated with an A12
derivative through a β-alanine; Noc-βAla-P26, P26 conjugated
with an NB-2 derivative through a β-alanine; DCC, N,N′-
dicyclohexylcarbodiimide; DMAP, 4-(dimethylamino)pyridine
REFERENCES
■
(1) Chan, D. C.; Fass, D.; Berger, J. M.; Kim, P. S. Core structure of
gp41 from the HIV envelope glycoprotein. Cell 1997, 89, 263−273.
K
dx.doi.org/10.1021/jm3018964 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(2) Chan, D. C.; Kim, P. S. HIV entry and its inhibition. Cell 1998,
93, 681−684.
activity evaluation of potent fusion inhibitors targeting HIV-1 gp41.
Med. Chem. 2011, 7, 309−316.
(3) Ashkenazi, A.; Shai, Y. Insights into the mechanism of HIV-1
envelope induced membrane fusion as revealed by its inhibitory
peptides. Eur. Biophys. J. 2011, 40 (4), 349−357.
(22) Liu, K.; Lu, H.; Hou, L.; Qi, Z.; Teixeira, C.; Barbault, F.; Fan,
B.; Liu, S.; Jiang, S.; Xie, L. Design, synthesis, and biological evaluation
of N-carboxyphenylpyrrole derivatives as potent HIV fusion inhibitors
targeting gp41. J. Med. Chem. 2008, 51 (24), 7843−7854.
(23) Wang, Y.; Lu, H.; Zhu, Q.; Jiang, S.; Liao, Y. Structure-based
design, synthesis and biological evaluation of new N-carboxyphenyl-
pyrrole derivatives as HIV fusion inhibitors targeting gp41. Bioorg.
Med. Chem. Lett. 2010, 20 (1), 189−192.
(4) Cai, L.; Jiang, S. Development of peptide and small-molecule
HIV-1 fusion inhibitors that target gp41. ChemMedChem 2010, 5 (11),
1813−1824.
(5) Naider, F.; Anglister, J. Peptides in the treatment of AIDS. Curr.
Opin. Struct. Biol. 2009, 19, 473−482.
(6) Gallo, S. A.; Puri, A.; Blumenthal, R. HIV-1 gp41 six-helix bundle
formation occurs rapidly after the engagement of gp120 by CXCR4 in
the HIV-1 Env-mediated fusion process. Biochemistry (Moscow) 2001,
40, 12231−12236.
(7) Liu, S.; Wu, S.; Jiang, S. HIV entry inhibitors targeting gp41: from
polypeptides to small-molecule compounds. Curr. Pharm. Des. 2007,
13 (2), 143−162.
(8) Pan, C.; Liu, S.; Jiang, S. HIV-1 gp41 fusion intermediate: a target
for HIV therapeutics. J. Formosan Med. Assoc. 2010, 109 (2), 94−105.
(9) Liu, S.; Zhao, Q.; Jiang, S. Determination of the HIV-1 gp41
fusogenic core conformation modeled by synthetic peptides:
applicable for identification of HIV-1 fusion inhibitors. Peptides
2003, 24 (9), 1303−1313.
(10) Jiang, S.; Zhao, Q.; Debnath, A. K. Peptide and non-peptide
HIV fusion inhibitors. Curr. Pharm. Des. 2002, 8, 563−580.
(11) Wild, C. T.; Shugars, D. C.; Greenwell, T. K.; McDanal, C. B.;
Matthews, T. J. Peptides corresponding to a predictive α-helical
domain of human immunodeficiency virus type 1 gp41 are potent
inhibitors of virus infection. Proc. Natl. Acad. Sci. U.S.A. 1994, 91,
9770−9774.
(12) Cai, L.; Balogh, E.; Gochin, M. Stable extended human
immunodeficiency virus type 1 gp41 coiled coil as an effective target in
an assay for high-affinity fusion inhibitors. Antimicrob. Agents
Chemother. 2009, 53 (6), 2444−2449.
(13) He, Y.; Cheng, J.; Lu, H.; Li, J.; Hu, J.; Qi, Z.; Liu, Z.; Jiang, S.;
Dai, Q. Potent HIV fusion inhibitors against enfuvirtide-resistant HIV-
1 strains. Proc. Natl. Acad. Sci. U.S.A. 2008, 105 (42), 16332−16337.
(14) Matthews, T.; Salgo, M.; Greenberg, M.; Chung, J.; DeMasi, R.;
Bolognesi, D. Enfuvirtide: the first therapy to inhibit the entry of HIV-
1 into host CD4 lymphocytes. Nat. Rev. Drug Discovery 2004, 3, 215−
225.
(24) Frey, G.; Rits-Volloch, S.; Zhang, X. Q.; Schooley, R. T.; Chen,
B.; Harrison, S. C. Small molecules that bind the inner core of gp41
and inhibit HIV envelope-mediated fusion. Proc. Natl. Acad. Sci. U.S.A.
2006, 103, 13938−13943.
(25) Xu, Y.; Lu, H.; Kennedy, J. P.; Yan, X.; McAllister, L. A.;
Yamamoto, N.; Moss, J. A.; Boldt, G. E.; Jiang, S.; Janda, K. D.
Evaluation of “credit card” libraries for inhibition of HIV-1 gp41
fusogenic core formation. J. Comb. Chem. 2006, 8, 531−539.
(26) Balogh, E.; Wu, D.; Zhou, G.; Gochin, M. NMR second site
screening for structure determination of ligands bound in the
hydrophobic pocket of HIV-1 gp41. J. Am. Chem. Soc. 2009, 131,
2821−2823.
(27) Gochin, M.; Zhou, G.; Phillips, A. H. Paramagnetic relaxation
assisted docking of a small indole compound in the HIV-1 gp41
hydrophobic pocket. ACS Chem. Biol. 2010, 6, 267−274.
(28) Wang, C.; Zhao, M.; Qiu, X.; Peng, S. The synthesis and
immunosuppressive activities of steroid-urotoxin linkers. Bioorg. Med.
Chem. 2004, 12, 4403−4421.
(29) Chan, D. C.; Chutkowski, C. T.; Kim, P. S. Evidence that a
prominent cavity in the coiled coil of HIV type 1 gp41 is an attractive
drug target. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 15613−15617.
(30) Mo, H.; Konstantinidis, A. K.; Stewart, K. D.; Dekhtyar, T.; Ng,
T.; Swift, K.; Matayoshi, E. D.; Kati, W.; Kohlbrenner, W.; Molla, A.
Conserved residues in the coiled-coil pocket of human immunodefi-
ciency virus type 1 gp41 are essential for viral replication and
interhelical interaction. Virology 2004, 329, 319−327.
(31) Teixeira, C.; Barbault, F.; Rebehmed, J.; Liu, K.; Xie, L.; Lu, H.;
Jiang, S.; Fan, B.; Maurel, F. Molecular modeling studies of N-
substituted pyrrole derivatives-Potential HIV-1 gp41 inhibitors. Bioorg.
Med. Chem. 2008, 16 (6), 3039−3048.
(32) Cai, L.; Gochin, M. A novel fluorescence intensity screening
assay identifies new low-molecular-weight Inhibitors of the gp41
coiled-coil domain of human immunodeficiency virus type 1.
Antimicrob. Agents Chemother. 2007, 51 (7), 2388−2395.
(33) Lu, L.; Tong, P.; Yu, X.; Pan, C.; Zou, P.; Chen, Y.; Jiang, S.
HIV-1 variants with a single-point mutation in the gp41 pocket region
exhibiting different susceptibility to HIV fusion inhibitors with pocket-
or membrane-binding domain. Biochim. Biophys. Acta 2012, 1818,
2950−2957.
(34) Naito, T.; Izumi, K.; Kodama, E.; Sakagami, Y.; Kajiwara, K.;
Nishikawa, H.; Watanabe, K.; Sarafianos, S. G.; Oishi, S.; Fujii, N.;
Matsuoka, M. SC29EK, a peptide fusion inhibitor with enhanced α-
helicity, inhibits replication of human immunodeficiency virus type 1
mutants resistant to enfuvirtide. Antimicrob. Agents Chemother. 2009,
53 (3), 1013−1018.
(15) Ferrer, M.; Kapoor, T. M.; Strassmaier, T.; Weissenhorn, W.;
Skehel, J. J.; Oprian, D.; Schreiber, S. L.; Wiley, D. C.; Harrison, S. C.
Selection of gp41-mediated HIV-1 cell entry inhibitors from biased
combinatorial libraries of non-natural binding elements. Nat. Struct.
Biol. 1999, 6 (10), 953−960.
(16) Gochin, M.; Zhou, G. Amphipathic properties of HIV-1 gp41
fusion inhibitors. Curr. Top. Med. Chem. 2011, 11, 3022−3032.
(17) Jiang, S.; Tala, S. R.; Lu, H.; Abo-Dya, N. E.; Avan, I.; Gyanda,
K.; Lu, L.; Katritzky, A. R.; Debnath, A. K. Design, synthesis, and
biological activity of novel 5-((arylfuran/1H-pyrrol-2-yl)methylene)-2-
thioxo-3-(3-(trifluoromethyl)phenyl)thiazolidin-4-ones as HIV-1 fu-
sion inhibitors targeting gp41. J. Med. Chem. 2011, 54, 572−579.
(18) Zhou, G.; Ferrer, M.; Chopra, R.; Kapoor, T. M.; Strassmaier,
T.; Weissenhorn, W.; Skehel, J. J.; Oprian, D.; Schreiber, S. L.;
Harrison, S. C.; Wiley, D. C. The structure of an HIV-1 specific cell
entry inhibitor in complex with the HIV-1 gp41 trimeric core. Bioorg.
Med. Chem. 2000, 8, 2219−2238.
(35) Mink, M.; Mosier, S. M.; Janumpalli, S.; Davison, D.; Jin, L.;
Melby, T.; Sista, P.; Erickson, J.; Lambert, D.; Stanfield-Oakley, S. A.;
Salgo, M.; Cammack, N.; Matthews, T.; Greenberg, M. L. Impact of
human immunodeficiency virus type 1 gp41 amino acid substitutions
selected during enfuvirtide treatment on gp41 binding and antiviral
potency of enfuvirtide in vitro. J. Virol. 2005, 79, 12447−12454.
(36) Wei, X.; Decker, J. M.; Liu, H.; Zhang, Z.; Arani, R. B.; Kilby, J.
M.; Saag, M. S.; Wu, X.; Shaw, G. M.; Kappes, J. C. Emergence of
resistant human immunodeficiency virus type 1 in patients receiving
fusion inhibitor (T-20) monotherapy. Antimicrob. Agents Chemother.
2002, 46, 1896−1905.
(19) Liu, S.; Boyer-Chatenet, L.; Lu, H.; Jiang, S. Rapid and
automated fluorescence-linked immunosorbent assay for high
throughput screening of HIV-1 fusion inhibitors targeting gp41. J.
Biomol. Screening 2003, 8, 685−693.
(20) Jiang, S.; Lu, H.; Liu, S.; Zhao, Q.; He, Y.; Debnath, A. K. N-
substituted pyrrole derivatives as novel human immunodeficiency virus
type 1 entry inhibitors that interfere with the gp41 six-helix bundle
formation and block virus fusion. Antimicrob. Agents Chemother. 2004,
48 (11), 4349−4359.
(37) Liu, S.; Jing, W.; Cheung, B.; Lu, H.; Sun, J.; Yan, X.; Niu, J.;
Farmar, J.; Wu, S.; Jiang, S. HIV gp41 C-terminal heptad repeat
(21) Tan, J.; Zhang, B.; Cong, X.; Yang, L.; Liu, B.; Kong, R.; Kui, Z.;
Wang, C.; Hu, L. Computer-aided design, synthesis, and biological
L
dx.doi.org/10.1021/jm3018964 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
contains multifunctional domains. Relation to mechanisims of action
of anti-HIV peptides. J. Biol. Chem. 2007, 282 (13), 9612−9620.
(38) Pang, W.; Wang, R.; Yang, L.; Liu, C.; Tien, P.; Zheng, Y.
Recombinant protein of heptad-repeat HR212, a stable fusion inhibitor
with potent anti-HIV action in vitro. Virology 2008, 377, 80−87.
(39) Case, D. A.; Darden, T. A.; Cheatham, T.; Simmerling, C.;
Wang, J.; Duke, R. E.; Luo, R.; Merz, K. M.; Wang, B.; Pearlman, D.
A.; Crowley, M.; Brozell, S.; Tsui, V.; Gohlke, H.; Mongan, J.; Hornak,
V.; Cui, G.; Beroza, P.; Schafmeister, C.; Caldwell, J. W.; Ross, W. S.;
Kollman, P. A. AMBER 8; University of California: San Francisco,
2004.
(40) Wexler-Cohen, Y.; Shai, Y. Demonstrating the C-terminal
boundary of the HIV-1 fusion conformation in a dynamic ongoing
fusion process and implication for fusion inhibition. FASEB J. 2007, 21
(13), 3677−3684.
(41) Zhou, G.; Wu, D.; Hermel, E.; Balogh, E.; Gochin, M. Design,
synthesis, and evaluation of indole compounds as novel inhibitors
targeting Gp41. Bioorg. Med. Chem. Lett. 2010, 20, 1500−1503.
M
dx.doi.org/10.1021/jm3018964 | J. Med. Chem. XXXX, XXX, XXX−XXX