Design, synthesis, and biological evaluation of N-carboxyphenylpyrrole derivatives as potent HIV fusion inhibitors targeting gp41

Article abstract of DOI:10.1021/jm800869t

On the basis of the structures of small-molecule hits targeting the HIV-1 gp41, N-(4-carboxy-3-hydroxy)phenyl-2,5-dimethylpyrrole (2, NB-2), and N-(3-carboxy-4-chloro)phenylpyrrole (A₁, NB-64), 42 N-carboxyphenylpyrrole derivatives in two categories (A and B series) were designed and synthesized. We found that 11 compounds exhibited promising anti-HIV-1 activity at micromolar level and their antiviral activity was correlated with their inhibitory activity on gp41 six-helix bundle formation, suggesting that these compounds block HIV fusion and entry by disrupting gp41 core formation. The structure-activity relationship and molecular docking analysis revealed that the carboxyl group could interact with either Arg579 or Lys574 to form salt bridges and two methyl groups on the pyrrole ring were favorable for interaction with the residues in gp41 pocket. The most active compound, N-(3-carboxy-4-hydroxy)phenyl-2,5-dimethylpyrrole (A₁₂), partially occupied the deep hydrophobic pocket, suggesting that enlarging the molecular size of A₁₂ could improve its binding affinity and anti-HIV-1 activity for further development as a small-molecule HIV fusion and entry inhibitor.

Full text of DOI:10.1021/jm800869t

J. Med. Chem. 2008, 51, 7843–7854
7843
Design, Synthesis, and Biological Evaluation of N-Carboxyphenylpyrrole Derivatives as Potent
HIV Fusion Inhibitors Targeting gp41
Kun Liu,† Hong Lu,‡ Ling Hou,† Zhi Qi,‡ Ca´tia Teixeira,§ Florent Barbault,§ Bo-Tao Fan,§ Shuwen Liu,| Shibo Jiang,*,‡,| and
Lan Xie*,†
Beijing Institute of Pharmacology & Toxicology, 27 Tai-Ping Road, Beijing, 100850, China, Lindsley F. Kimball Research Institute, New York
Blood Center, New York, New York 10065, ITODYS, UniVersity Paris 7sCNRS UMR 7086, 1 rue Guy de la Brosse, 75005 Paris, France,
School of Pharmaceutical Sciences, Southern Medical UniVersity, Guangzhou, 510515, China
ReceiVed July 14, 2008
On the basis of the structures of small-molecule hits targeting the HIV-1 gp41, N-(4-carboxy-3-
hydroxy)phenyl-2,5-dimethylpyrrole (2, NB-2), and N-(3-carboxy-4-chloro)phenylpyrrole (A1, NB-64), 42
N-carboxyphenylpyrrole derivatives in two categories (A and B series) were designed and synthesized. We
found that 11 compounds exhibited promising anti-HIV-1 activity at micromolar level and their antiviral
activity was correlated with their inhibitory activity on gp41 six-helix bundle formation, suggesting that
these compounds block HIV fusion and entry by disrupting gp41 core formation. The structure-activity
relationship and molecular docking analysis revealed that the carboxyl group could interact with either
Arg579 or Lys574 to form salt bridges and two methyl groups on the pyrrole ring were favorable for
interaction with the residues in gp41 pocket. The most active compound, N-(3-carboxy-4-hydroxy)phenyl-
2,5-dimethylpyrrole (A12), partially occupied the deep hydrophobic pocket, suggesting that enlarging the
molecular size of A12 could improve its binding affinity and anti-HIV-1 activity for further development as
a small-molecule HIV fusion and entry inhibitor.
Introduction
According to the estimate of UNAIDS, about 33.2 million
people worldwide are living with HIV and more than 25 million
patients have died of AIDS (www.unaids.org/en/Knowledge-
Centre/HIVData/EpiUpdate/EpiUpdArchive/2007/). Thus far, 28
anti-HIV drugs have been licensed by the United States Food
and Drug Administration (FDA) (http://www.hivandhepatitis-
.com/hiv_and_aids/hiv_treat.html). Most of these drugs belong
to two categories: reverse transcriptase inhibitors (RTI) and
Figure 1. Structures of hits 2 and A1.
protease inhibitors (PI). Combined application of these antiret-
HB) core formation, thereby inhibiting fusion between the viral
roviral drugs has shown significant synergistic effects.1 How-
and target cell membranes.10,13,14 Although 1 is very potent in
ever, an increasing number of patients with HIV infection/AIDS
inhibiting HIV infection, it has two critical limitations as a drug:
can no longer use such drugs as a result of drug resistance and
lack of oral availability and high production cost.15 Therefore,
serious adverse effects.2-4 Therefore, it is essential to develop
it is necessary to develop orally available nonpeptide small-
novel anti-HIV drugs targeting HIV entry.
molecule fusion inhibitors but with a mechanism of action
HIV-1 envelope glycoprotein (Env) transmembrane subunit
similar to C-peptides. By screening a drug-like chemical library
gp41 plays an important role in virus fusion and entry5 and can
using a high-throughput screening technique,16 we previously
serve as a target for the development of HIV-1 fusion
identified two small molecules, N-(4-carboxy-3-hydroxy)phenyl-
inhibitors.6,7 The gp41 ectodomain contains a fusion peptide
2,5-dimethylpyrrole (2, NB-2) and N-(3-carboxy-4-chloro)phe-
(FP), the N- and C-terminal heptad repeat (NHR and CHR,
nylpyrrole (A1, NB-64)17 (Figure 1), which inhibit HIV-1 fusion
respectively). The peptides derived from the gp41 CHR region
and entry by interfering with the gp41 6-HB formation. These
(designated C-peptides) are potent HIV fusion inhibitors.8-10
promising hits prompted us to focus on the modification of 2
One of the C-peptides, T-20 (1, Enfuvirtide, a 36-amino acid
and A1 to discover and develop new lead compounds with novel
synthetic peptide)11,12 was approved by the US FDA in 2003
scaffold and higher potency. Here we report the results of our
as the first member of a new class of anti-HIV drugssHIV
hit-to-lead process, including design, synthesis, and biological
fusion inhibitors for treating HIV/AIDS patients who have failed
evaluation of 42 N-carboxyphenyl pyrroles and related deriva-
to respond to RTI and PI. Drug 1 was believed to interact with
tives. Their primary structure-activity relationships are discussed.
the HIV-1 gp41 NHR and block the gp41 six-helix bundle (6-
Design. Previous studies have identified an attractive target
in gp41 for small-molecule HIV fusion inhibitors, i.e., the deep
* To whom correspondence should be addressed. L. Xie and S. Jiang,
For L.X.: phone, 86-10-6818-1014; Fax, 86-10-6821-1656, E-mail:
lanxieshi@yahoo.com. For S. J.: phone, 212-570-3058; fax, 212-570-3099;
hydrophobic pocket (∼16 Å long, ∼7 Å wide, and 5-6 Å deep)
on the surface of the internal N-helix trimer that is filled by
E-mail: SJiang@NYBloodCenter.org.
three conserved hydrophobic residues with large side chains
†
Beijing Institute of Pharmacology & Toxicology.
Lindsley F. Kimball Research Institute, New York Blood Center.
ITODYS, University Paris 7sCNRS UMR 7086.
School of Pharmaceutical Sciences, Southern Medical University.
(Ile635, Trp631, and Trp628) in the gp41 CHR region.13,18 The
‡
§
combined molecular mass of these residues inside the pocket
is ∼600 Da, which is within the size range for binding of an
|
10.1021/jm800869t CCC: $40.75
2008 American Chemical Society
Published on Web 11/18/2008

7844 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Liu et al.
Figure 2. Molecular modeling: the docking conformation of 2 inside the hydrophobic pocket of gp41.
orally bioavailable, small-molecule drug.19 On the basis of this
information, we docked a known hit 2 into the gp41 hydrophobic
pocket (Figure 2). This step allowed us to image the gp41
binding “pocket” shape, the orientation and effective binding
conformation of inhibitor 2, and more surface amino acids such
as Trp571, Gln575, Arg579, Leu581, Gln577, Lys574, and
Ile573. The docking results indicated that 2 partially occupies
the pocket. Our previous studies indicated that the carboxyl
group of A1 orients to the positively charged Lys574, which is
a key surface amino acid in forming a salt “bridge” with an
inhibitor.20-22 On the other hand, the carboxyl group of 2
interacts with Arg579 rather than Lys574 (Figure 2). Thus, it
can be hypothesized that the acid group in the compounds can
interact with either of these two positively charged residues
around the pocket to form a salt bridge, which stabilizes the
interaction between the compound and the HIV-1 gp41.
Consequently, our hit-to-lead optimization and rational design
aimed at improving the shape complementation with the binding
pocket in order to provide more binding points and increase
affinity with the biologic target. It is anticipated that these results
will, in turn, provide the basis for discovering more potent leads
with a new scaffold.
In the hit-to-lead optimization process, both the structural
similarities and differences between 2 and A1 led us to explore
the structural impact of the phenylpyrrole compounds. Two
parallel series of N-phenylpyrrole and N-phenyl-2,5-dimeth-
ylpyrrole derivatives (A1-A10 and A11-A20) with a p- or
m-COOH on the benzene ring were first designed and synthe-
sized to determine which structural moieties are necessary and
which position of the carboxyl group is most favorable for anti-
HIV potency. Specifically, it was possible that the carboxyl
group on the benzene ring could significantly affect the
molecular geometry and binding orientation because it is
expected to form a salt bridge with Lys574 or Arg579 on the
surface of binding site.20-22 Next, more boundary substituents
were introduced on the N-phenylpyrrole skeleton to produce
more potential target-inhibitor interaction points for enhancing
binding affinity. This resulted in an increase of anti-HIV
potency. Meanwhile, an isosteric replacement of the carboxyl
group was also performed by a tetrazole moiety. Next, the
strategy of isosteric replacement was then applied by using
several five-membered heterocycles instead of the pyrrole ring
to investigate if the pyrrole ring necessary for anti-HIV potency
and, hopefully, to identify new potential structural moieties.
Therefore, a series of carboxyphenyl compounds with an 1,2,4-
oxadiazole (B1-B11), thiadiazole (B12), maleimide (B13-B15),
or rhodanine (B16-B22) ring, respectively, were designed and
synthesized. In the B series of compounds, a polar functional
group, or side chain on the five-membered ring could enlarge
the size and change the shape of inhibitors. Both the design
and modification of all new compounds were driven by
bioassays for anti-HIV-1 activity against HIV-1 replication and
gp41 six-helix bundle formation. Meanwhile, molecular model-
ing studies were performed to elucidate the structure and activity
relationship for these small-molecule fusion inhibitors.
Chemistry. As shown in Scheme 1, the Paal-Knorr reaction
was used to synthesize A1-A9 and A11-A20 by the condensation
of anilines or benzylamines with 2,5-dimethoxytetrahydrofuran
or acetonylacetone (hexane-2,5-dione),23 respectively. The
Paal-Knorr reaction was performed with or without glacial
acetic acid as solvent under microwave irradiation in a sealed
tube at 100-150 °C for 10 min.24 Microwave irradiation greatly
reduced reaction time and byproducts, resulting in yields ranging
from 50 to 88%, which was higher than the 28-56% in the
traditional condition. N-Benzylpyrrole A10 was prepared by
treatment of 4-(bromomethyl)phenylacetic acid and pyrrole in
the presence of potassium tert-butoxide (t-BuOK) under mi-
crowave irradiation with a yield of 84%. However, the several
syntheses to obtain pure 2 as a positive control in our bioassays
were unsuccessful even though N-(3-carboxy-4-hydroxy)phenyl-
2,5-dimethylpyrrole (A12), an isomer of 2, was successfully
synthesized with a 79% yield. This appeared to be caused by
the instability of 2 during workup and purification when color
changed rapidly from white to deep-red.
The 1,2,4-oxadiazole derivatives B1-B11 were synthesized
by the acylation of amidoxime,24 as shown in Scheme 2.
Cyanobenzoic acids or ester were treated with hydroxylamine
hydrochloride in the presence of 8-hydroxyquinoline25 to
produce corresponding benzamidoximes 4-6, respectively.
Compound 6 reacted with either bromoacetyl bromide or

N-Carboxyphenylpyrrole DeriVatiVes
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 7845
a
Scheme 1. Synthesis of A1-A20
a (i) and (ii) AcOH, microwave, 150 °C, 10 min; (iii) t-BuOK, DMSO, microwave, 193 °C, 10 min.
a
Scheme 2. Synthesis of B1-B11
a (i) Na2CO3, EtOH, reflux, 4 h; (ii) THF, reflux, 8 h; (iii) DMF, 90 °C, 3 h; (iv) 1N NaOH, EtOH, rt, 4 h; (v) HCl or HBr/AcOH (1:1), 100 °C, 8-12
h; (vi) pyridine, reflux, 2 h.
chloroacetyl chloride in THF at reflux temperature to afford
5-substituted 3-aryl-1,2,4-oxadiazoles B1 or B2, respectively.
5-Chloromethyl group on the oxadiazole ring of B2 was then
converted into 5-thiocyanomethyl by treatment with ammonium
thiocyanate in DMF at 90 °C to produce B3. Following a basic
hydrolysis of B1 in EtOH, an ester exchange product 3-(4′-
ethoxycarbonyl)phenyl-5-hydroxymethyl oxadiazole (B4) was
produced. However, hydrolysis of B2 in the basic condition
yielded 3-(4′-carboxyl)phenyl-5-hydroxymethyl oxadiazole (B5).
Otherwise, esters B2 and B3 were hydrolyzed in the presence
of HCl/AcOH (1:1) to yield corresponding 3-(4′-carboxyl)phe-
nyl-1,2,4-oxadiazoles B6 and B7, respectively. The preparation
of 5-bromomethyl oxadiazole B8 have to hydrolyze B1 in HBr/
AcOH (1:1), whereas the bromide in B1 could be replaced by
chloride if the hydrolysis was carried out in HCl/AcOH. By
treating with trifluoroacetic anhydride in pyridine, 4 and 5
afforded corresponding 5-trifluoromethyl-1,2,4-oxadiazoles B9
and B10, respectively. Compound B11 was synthesized from 4
in one step with a yield of 37%.
The syntheses of B12-B22 are summarized in Scheme 3.
These compounds possess a five-membered heterocyclic ring
moiety instead of the pyrrole ring in the A series of compounds.
According to methods found in the literature,26 thiosemicarba-
zone (7) was prepared from p-carboxylbenzaldehyde by treating
with thiosemicarbazide in ethanol followed by a cyclization in
aq ammonium ferric sulfate solution to produce 4-carboxyphe-
nyl-1,3,4-thiadiazole (B12). Anilines reacted with maleic anhy-
dride afforded intermediate N-aryl maleic monoamides 8 and
9, followed by dehydration and cyclization in acetic anhydride
and triethylamine,26 which produced corresponding N-aryl
maleimide derivatives B13 and B14, respectively. The acidic
hydrolysis of B14 produced an expected compound B15, but a
basic hydrolysis destroyed the maleimide ring. Subsequently,
N-phenyl-substituted rhodanine derivatives B16-B22 were pre-
pared from various anilines by treatment of bis(carboxymethyl)-
trithio carbonate in water with a 28-88% yield, respectively.27
A direct substitution between NH of rhodanine and benzene
bromide failed in spite of using various bases, such as potassium
tert-butoxide, sodium hydride, potassium (or sodium) hydroxide,
and potassium carbonate, even though this reaction seemed
simple and convenient.
Results and Discussion
A total of 42 newly synthesized N-arylpyrrole derivatives (20
in the A series and 22 in the B series) were tested in parallel
with A1 for their inhibitory activity on HIV-1 replication (as
determined by p24 production), HIV-1-mediated cell-cell
fusion, and gp41 6-HB formation. The compounds A2-A10 and
A11-A20 contain the same backbone structures of A1 and 2,
respectively. A majority of these compounds exhibited signifi-
cant inhibitory activity on HIV-1 replication and gp41 6-HB
formation as determined by ELISA and native-PAGE (Table 1
and Figure 3). The anti-HIV-1 activities of these compounds
were correlated with their inhibitory activities on 6-HB forma-
tion (Figure 4), suggesting that these compounds, like 2 and
A1, may interact with the gp41 NHR region to disrupt the fusion-
active core formation, resulting in blockage of gp41-mediated
membrane fusion and inhibition of HIV-1 replication. Indeed,
most of the active compounds displayed inhibitory activity on
HIV-1-mediated cell-cell fusion (data not shown). But unlike
the majority of the active compounds, a few compounds did
not have good correlation between their anti-HIV-1 activity and

7846 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Liu et al.
a
Scheme 3. Synthesis of B12-B22
a (i) EtOH, reflux, 1.5 h; (ii) H2O, reflux, 10 h; (iii) EtOAc, rt, 1 h; (iv) NEt3, 60 °C, 1 h; (v) HCl/AcOH (1:1), 100 °C, 8 h; (vi) H2O, 100 °C, 19 h.
Table 1. Anti-HIV Activity Data of Pyrrole Derivatives A1-A20 as HIV-1 gp-41 Inhibitors
R on benzene ring
inhibiting p24 production
cytotoxicity
CC50 (µM)
inhibition of 6-HB
formation IC50 (µM)
code no.
p-
m-
o-
n-
EC50 (µM)
SIa
A1 (NB-64)
A2
A3
A4
A5
A6
A7
A8
A9
A10
A11
A12
A13
A14
A15
A16
A17
A18
A19
A20
Cl
OH
H
CO2H
CO2H
CO2H
tetrazolyl
OH
H
H
OH
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
0
0
0
0
0
0
0
0
1
1
0
0
0
0
0
0
0
0
1
0
2.39
9.66
335.72
492.61
301.93
436.11
379.12
534.76
497.51
442.56
497.51
465.12
210.76
133.46
88.42
140.47
50.99
6.74
10.63
10.70
7.72
1.34
5.42
7.78
58.74
48.72
210.7
44.81
41.04
35.44
69.25
371.24
81.67
63.93
465.12
1.52
0.69
11.81
7.7
59.14
173.72
2.1
H
226.11
460.83
129.47
497.51
70.84
156.72
465.12
78.68
37.36
42.23
25.61
286.7
188.74
225.15
60.34
160.22
182.44
CO2CH3
CO2H
CH2CO2H
CO2H
CO2H
CH2CO2H
Cl
OH
H
H
CO2CH3
CO2H
CH2CO2H
Cl
CO2H
SO2NH2
H
CO2H
CO2H
CO2H
tetrazolyl
OH
H
H
H
H
138.66
193.42
7.49
32.40
1.50
249.46
88.9
136.28
203.84
400.00
298.56
116.8
0.78
97.07
46.73
19.20
1.18
CO2H
H
H
8.56
15.55
99.08
H
a SI ) selectivity index, CC50/EC50.
their inhibitory activity on 6-helix bundle formation. One of
the explanations is that these active compounds may target more
than one step of the HIV life cycle. For example, betulinic acid,
a bifunctional anti-HIV-1 compound, inhibits HIV infection by
targeting both HIV-1 entry and maturation steps.28 Therefore,
it is worthwhile to further investigate the mechanism(s) of action
of those compounds that do not have good correlation between
their activities against HIV-1 infection and 6-helix bundle
formation.
Notably, the 2,5-dimethylpyrrole compounds A11-A14, A17,
and A19 were significantly more potent than the corresponding
pyrrole compounds A1-A4, A7, and A9, respectively. This fact,
along with previous theoretical work,29 indicated that the two
methyl groups on the pyrrole ring and a near perpendicular
conformation between benzene and pyrrole rings by steric
crowding effect may be favorable for targeting the gp41 binding
site, as shown in Figure 2. Further comparisons between A13
(EC50 11.81 µM) and A16(EC50 173.72 µM), A2 (EC50 9.66 µM)
and A8(EC50 81.67 µM), and A3 (EC50 44.81 µM) and A6(EC50
69.25 µM) indicated that a carboxyl group at the m-position of
the benzene ring is more suitable than the p-position for
enhancing anti-HIV potency. After an isosteric replacement of
carboxyl, A14 with a tetrazolyl moiety at the m-position of the
benzene ring was more potent (EC50 7.70 µM) than A13. More
interestingly, compound A14 exhibited more potent inhibitory
activity against six-helix bundle formation (IC50 25.61 µM) than
A1 (IC50 58.74 µM), suggesting that tetrazolyl moiety could be
a potential structural fragment for designing novel small-

N-Carboxyphenylpyrrole DeriVatiVes
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 7847
tively. The following main interactions between A12 and gp41
were expected: (1) interaction of two H-bonds from the hydroxyl
group of A12 with the main-chain carbonyl oxygen of Gln575
and that of the m-carboxyl group of A12 with the main-chain
amine of Gln577, respectively, (2) an electrostatic interaction
between the carboxyl group of A12 and Arg579, and (3)
hydrophobic interactions of the pyrrole ring with Lys574 and
Ile573 and that of the phenyl ring with the Trp571. The
electrostatic force oriented A12 in the direction of Arg579, and
the steric force of the methyl groups of A12 resulted in a
T-shaped effective binding conformation between the benzene
and pyrrole rings. However, A14 with a three-aromatic-ring
skeleton occupied more binding site space, thus providing more
hydrophobic interactions between A14 and surface amino acid
residues of the gp41 binding site, i.e., the benzene ring with
Ile573 and the pyrrole ring with the hydrocarbon chain of
Lys574. The tetrazolyl ring with more negative charges orients
A14 closer to Arg579 and serves as an H donor to form an
H-bond with main-chain carbonyl oxygen of Gln572. However,
the molecular modeling results indicated that, while both A12
and A14 have similar binding orientation and perpendicular
conformation between aromatic rings, the three-ring angle
geometric conformation of A14 might match the binding site
better, thus enhancing binding affinity.
Figure 3. Inhibition of the HIV-1 gp41 6-HB formation by small-
molecule compounds (N-PAGE). N36 alone (lane 1) exhibited no band
because it carries net positive charges and may migrate off the gel.35
C34 alone (lane 2) displayed a band at a lower position in the gel. The
mixture of N36 and C34 (lane 3) showed a band at the upper position
in gel, corresponding to that of 6-HB.35 In the presence of A3 (lane 4)
and A4 (lane 5), N36 and C34 could still form the 6-HB, while addition
of A1 (lane 6), A2 (lane 8), A12 (lane 9), and A14 (lane 7) resulted in
disappearance of the 6-HB band and reappearance of the C34 band,
suggesting that these compounds can block the 6-HB formation between
N36 and C34.
Among compounds B1-B11 with an oxadiazole ring, only a
few (e.g., B6 and B10) displayed moderate inhibitory activity
against HIV-1 replication and 6-HB formation with selectivity
index (SI) values >16. However, the B series of compounds
with a thiadiazole B12, maleimide B13-B15, or rhodanine ring
B16-B22, respectively, showed no significant inhibitory activity
on HIV-1 replication and 6-HB formation (Table 2). The results
suggest that these heterocyclic structures may be less effective
than pyrrole in interacting with the gp41 pocket and, therefore,
less effective in inhibiting HIV-1 fusion and entry.
In conclusion, we have designed and synthesized 42 N-
carboxyphenylpyrrole derivatives in both the A and B categories.
These novel compounds were based on the structures of small-
molecule hits targeting the HIV-1 gp41, 2 and A1. We found
that a majority of the compounds in the A series exhibited
significant inhibitory activity on HIV-1 entry and replication
as well as 6-HB formation. Their anti-HIV-1 activity is also
correlated with their ability to disrupt the gp41 fusion-active
core formation, suggesting that these compounds inhibit HIV-1
fusion and entry in agreement with the mechanism of action of
2 and A1 and the anti-HIV peptide C34.15,30 Furthermore, 2,5-
dimethylpyrrole compounds were generally more potent than
the corresponding pyrrole compounds, suggesting that the
presence of dimethyl groups resulted in a favorable T-shape
conformation between the benzene and pyrrole rings. Compound
Figure 4. Correlation between the inhibitory activities of the A series
compounds on HIV-1 replication and gp41 6-HB formation.
molecule HIV fusion inhibitors targeting gp41. By introducing
an additional hydroxyl or chloride on the phenyl ring, com-
pounds A1, A2, A11, A12, and A18 all exhibited potent anti-HIV
activity with an EC50 value range of 0.69-9.66 µM. This
suggests that more boundary substituents may greatly enhance
molecular affinity with the binding site. Meanwhile, compound
A17, with more linear molecular size exhibited higher potency
(EC50 2.10 µM) than its counterpart A16 (EC50 173.72 µM),
thus guiding us to expand molecular size in order to enhance
the affinity of inhibitor with the binding site.
A12 was the most active compound; therefore, further develop-
Among the phenylpyrrole derivatives, A12 was the most
effective in inhibiting p24 production (EC50 0.69 µM) and gp41
6-HB formation (IC50 37.36 µM). It also effectively inhibited
HIV-1-mediated cytopathic effect (CPE) and cell-cell fusion
(data not shown). The other two N-carboxylphenyl-2,5-dimet-
hypyrroles, A11 and A17, also showed promising potency.
To understand and interpret the bioassay results, molecular
modeling studies (2D-, 3D-quantitative structure-activity re-
lationship and molecular docking) were performed for the
compounds from series A.29 The docking analysis for the two
active compounds A12 and A14 with the hydrophobic pocket of
the gp41 N-trimer (Figures 5,6) indicates lower calculated
binding free energies of ∆G, -6.9 and -5.6 kcal/mol, respec-
ment is justified. In contrast, the majority of the compounds in
the B series showed no significant anti-HIV-1 activity, indicating
that these heterocyclic structures might be unfavorable for
binding to the gp41 pocket. The docking analysis also suggests
that the positively charged residues Arg579 and Lys574 are
important for interaction with the compounds by forming salt
bridges for stabilizing the binding of the compounds to the
hydrophobic pocket. On the other hand, all compounds in the
A and B series may not be large enough (<300 Da) to fully
occupy the deep hydrophobic pocket, which can accommodate
a compound with a mass of ∼600 Da. Therefore, current active
compounds should be increased in molecular size in order to

7848 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Liu et al.
Figure 5. Surface representation of the hydrophobic pocket of HIV-1 gp41 with the docked compound A12 (left) or A14 (right).
Figure 6. Docking conformations of active compounds A12 (left) and A14 (right) inside the hydrophobic pocket of HIV-1 gp41and residues of the
pocket surrounding ligands.
irradiated under microwave at 150 °C for 10 min as monitored by
TLC (petroleum ether/EtOAc 3:1). The mixture was poured into
ice-water. The solid was filtered out and washed with water and
then crude product was purified by flash column (gradual eluant:
EtOAc/petroleum ether with AcOH (v/v 1: 0.005) to obtain pure
expected product.
improve their efficacy in blocking the 6-HB formation and
inhibiting HIV fusion and entry.
Experimental Section
Chemistry. Melting points were measured with a RY-1 melting
apparatus without correction. The proton nuclear magnetic reso-
nance (1H NMR) spectra were measured on a JNM-ECA-400 (400
MHz) spectrometer using tetramethylsilane (TMS) as internal
standard. The solvent used was DMSO-d6 unless indicated. Mass
spectra (MS) were measured on either a Micromass ZabSpec or an
ABI Perkin-Elmer Sciex API-3000 mass spectrometer with elec-
trospray ionization. Elementary analyses were performed with a
model-1106 analyzer. Active target compounds were analyzed for
C, H, and N and gave values within (0.4% of the theoretical values.
HPLC analyses were performed using an Agilent 1100 series and
an Eclipse XDB-C18 column eluting with a mixture of solvents A
and B (condition 1: A/B ) water/acetonitrile 30:70, flow rate 0.8
mL/min; condition 2: A/B ) water/methanol 20:80, flow rate 0.6
mL/min; UV 254 nm). The microwave reactions were performed
on a microwave reactor from Biotage, Inc. Thin-layer chromatog-
raphy (TLC) was performed on silica gel GF254 plates. Silica gel
GF254 (200-300 mesh) from Qingdao Haiyang Chemical Company
was used for TLC, preparative TLC, and column chromatography.
Medium-pressure column chromatography was performed using a
CombiFlash Companion purification system. All chemicals were
obtained from Beijing Chemical Works or Sigma-Aldrich, Inc.
General Procedure for the Preparation of N-Phenylpyrrole
Derivatives A1-A7, A9. To a solution of aminobenzoic acid
analogues (1 equiv) in 3 mL of glacial acetic acid was added 2,5-
dimethoxy-tetrahydrofuran (1.1 equiv). The mixture was stirred and
N-(3-Carboxy-4-chloro)phenylpyrrole (A1, NB-64). Yield:
82%, starting with 331 mg (1.93 mmol) of 5-amino-2-chlorobenzoic
1
acid to afford A1, white solid, mp 125-128 °C. H NMR δ ppm
7.90 (1H, d, J ) 2.0 Hz, ArH-2), 7.75 (1H, dd, J ) 8.4 and 2.0
Hz, ArH-6), 7.60 (1H, d, J ) 8.4 Hz, ArH-5), 7.43 (2H, m, PyH-
2,5), 6.29 (2H, t, J ) 2.2 Hz, PyH-3,4). MS m/z (%) 221 (M+,
100), 223 (M + 2, 36). Anal. (C11H8ClNO2) C, H, N.
N-(3-Carboxy-4-hydroxy)phenylpyrrole (A2). Yield: 51%,
starting with 306 mg (2 mmol) of 5-aminosalicylic acid to afford
1
A2, white solid, mp 178-180 °C. H NMR δ ppm 11.34 (1H, br,
COOH), 7.82 (1H, d, J ) 2.8 Hz, ArH-2), 7.73 (1H, dd, J ) 9.2
and 2.8 Hz, ArH-6), 7.26 (2H, m, PyH-2,5), 7.07 (1H, d, J ) 9.2
Hz, ArH-5), 6.24 (2H, t, J ) 2.2 Hz, PyH-3,4). MS m/z (%) 203
(M+, 100). Anal. (C11H9NO3) C, H.
N-(3-Carboxy)phenylpyrrole (A3). Yield: 57%, starting with
274 mg (2 mmol) of 3-aminobenzoic acid to afford A3, white solid,
1
mp 175-178 °C. H NMR δ ppm 13.18 (1H, br, COOH), 7.98
(1H, d, J ) 2.0 Hz, ArH-2), 7.80 (2H, dd, J ) 8.4 and 2.0 Hz,
ArH-4,6), 7.57 (1H, t, J ) 8.4 Hz, ArH-5), 7.40 (2H, m, PyH-2,5),
6.26 (2H, t, J ) 2.2 Hz, PyH-3,4). MS m/z (%) 187 (M+, 100).
Anal. (C11H9NO2) C, H, N.
N-(3-(1H-tetrazol-5-yl)phenylpyrrole (A4). Yield: 88%, starting
with 161 mg (1 mmol) of 3-tetrazolylaniline to afford A4, white
solid, mp 210-212 °C. 1H NMR δ ppm 8.19 (1H, d, J ) 2.0 Hz,

N-Carboxyphenylpyrrole DeriVatiVes
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 7849
Table 2. Anti-HIV Activity Data of Five-Membered Heterocyclic Compounds B1-B22 as HIV-1 gp41 Inhibitorsa
a SI* ) selectivity index, CC50/EC50.

7850 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Liu et al.
ArH-2), 7.92 (1H, d, J ) 8.4 Hz, ArH-4), 7.83 (1H, dd, J ) 8.4
and 2.0 Hz, ArH-6), 7.71 (1H, t, J ) 8.4 Hz, ArH-5), 7.47 (2H, m,
PyH-2,5), 6.35 (2H, t, J ) 2.2 Hz, PyH-3,4). MS m/z (%) 211
(M+, 100). Anal. (C11H9N5) C, H, N.
N-(3-Hydroxy-4-methoxycarbonyl)phenylpyrrole (A5). Yield:
71%, starting with 167 mg (1 mmol) of methyl 4-aminosalicylate
to afford A5, white solid, mp 121-124 °C. 1H NMR δ ppm 10.75
(1H, s, OH), 7.85 (1H, d, J ) 8.4 Hz, ArH-5), 7.52 (2H, m, PyH-
2,5), 7.25 (1H, d, J ) 8.4 Hz, ArH-6), 7.23 (1H, s, ArH-2), 6.31
(2H, t, J ) 2.2 Hz, PyH-3,4), 3.91 (3H, s, OCH3). MS m/z (%)
217 (M+, 100). Anal. (C12H11NO3 ·1/8H2O) C, H, N.
N-(3-Carboxy-4-chloro)phenyl-2,5-dimethylpyrrole (A11).
Yield: 66%, starting with 343 mg (2 mmol) of 5-amino-2-
chlorobenzoic acid to afford A11, white solid, mp 140-142 °C. 1H
NMR δ ppm 13.63 (1H, br, COOH), 7.69 (1H, d, J ) 8.4 Hz,
ArH-5), 7.61 (1H, d, J ) 2.0 Hz, ArH-2), 7.48 (1H, dd, J ) 8.4
and 2.0 Hz, ArH-6), 5.83 (2H, s, PyH), 1.98 (6H, s, Py-CH3 × 2).
MS m/z (%) 249 (M+, 100), 251 (M + 2, 42). Anal. (C13H12ClNO2)
C, H, N.
N-(3-Carboxy-4-hydroxy)phenyl-2,5-dimethylpyrrole (A12).
Yield: 79%, starting with 153 mg (1 mmol) of 5-aminosalicylic
1
acid to afford A12, white solid, mp 169-171 °C. H NMR δ ppm
11.50 (1H, br, COOH), 7.54 (1H, d, J ) 2.0 Hz, ArH-2), 7.43
(1H, dd, J ) 8.4 and 2.0 Hz, ArH-6), 7.09 (1H, d, J ) 8.4 Hz,
ArH-5), 5.78 (2H, s, PyH), 1.94 (6H, s, Py-CH3 × 2). MS m/z (%)
231 (M+, 100). Anal. (C13H13NO3 ·1/8H2O) C, H, N.
N-(4-Carboxy)phenylpyrrole (A6). Yield: 65%, starting with
274 mg (2 mmol) of 4-aminobenzoic acid to afford A6, white solid,
1
mp 199-201 °C. H NMR δ ppm 12.92 (1H, br, COOH), 8.01
(2H, d, J ) 8.4 Hz, ArH-3,5), 7.73 (2H, d, J ) 8.4 Hz, ArH-2,6),
7.50 (2H, m, PyH-2,5), 6.32 (2H, t, J ) 2.2 Hz, PyH-3,4). MS m/z
(%)187 (M+, 86). Anal. (C11H9NO2) C, H, N.
N-(3-Carboxy)phenyl-2,5-dimethylpyrrole (A13). Yield: 50%,
starting with 274 mg (2 mmol) of 3-aminobenzoic acid to afford
A13, white solid, mp 145-148 °C. 1H NMR δ ppm 13.18 (1H, br,
COOH), 7.97 (1H, d, J ) 8.4 Hz, ArH-4), 7.65 (1H, s, ArH-2),
7.62 (1H, t, J ) 8.4 Hz, ArH-5), 7.52 (1H, d, J ) 8.4 Hz, ArH-6),
5.78 (2H, s, PyH), 1.92 (6H, s, Py-CH3 × 2). MS m/z (%) 214
(M-H, 100). Anal. (C13H13NO2) C, H, N.
N-(4-Carboxymethene)phenylpyrrole (A7). Yield: 77%, starting
with 151 mg (1 mmol) of 4-aminophenylacetic acid to afford A7,
white solid, mp 161-164 °C. 1H NMR δ ppm 12.38 (1H, br,
COOH), 7.52 (2H, d, J ) 8.4 Hz, ArH-2,6), 7.34 (2H, m, PyH-
2,5), 7.32 (2H, d, J ) 8.4 Hz, ArH-3,5), 6.26 (2H, t, J ) 2.2 Hz,
PyH-3,4), 3.59 (2H, s, CH2); MS m/z (%) 201 (M+, 64). Anal.
(C12H11NO2 ·1/8H2O) C, H, N.
N-(3-(1H-Tetrazol-5-yl)phenyl-2,5-dimethylpyrrole (A14). The
mixture of 3-tetrazolylaniline (161 mg, 1 mmol) and hexane-2,5-
dione (0.3 mL, 2.5 mmol), without glacial acetic acid, was irradiated
under microwave at 100 °C for 10 min to afford 137 mg of A14,
N-(4-Carboxy-3-hydroxy)phenylpyrrole (A8). To a solution of
A5 (538 mg, 2.48 mmol) in 10 mL methanol was added 15 mL of
40% aq NaOH, and the mixture was stirred at room temperature
for 6 h monitored by TLC (petroleum ether/EtOAc 3:1). The
mixture was then poured into water and acidified with 5% aq HCl
to pH 3. The solid was collected, washed with water, and purified
by flash chromatography [eluant: EtOAc/petroleum ether with
AcOH (4: 0.02) 0-20%] to afford 157 mg of A8, 31% yield, white
1
57% yield, white solid, mp 147-148 °C. H NMR δ ppm 8.14
(1H, d, J ) 8.4 Hz, ArH-4), 7.87 (1H, s, ArH-2), 7.76 (1H, t, J )
8.4 Hz, ArH-5), 7.53 (1H, d, J ) 8.4 Hz, ArH-6), 5.85 (2H, s,
PyH), 2.02 (6H, s, Py-CH3 × 2). MS m/z (%) 239 (M+, 76). Anal.
(C13H13N5) C, H, N.
N-(3-Hydroxy-4-methoxycarbonyl)phenyl-2,5-dimethylpyr-
role (A15). Yield: 62%, starting with 378 mg (2.26 mmol) of methyl
1
1
solid, mp 208-210 °C. H NMR δ ppm 13.94 (1H, br, COOH),
4-aminosalicylate to afford A15, yellow solid, mp 58-61 °C. H
10.83 (1H, s, OH), 7.52 (1H, d, J ) 8.4 Hz, ArH-5), 7.51 (2H, m,
PyH-2,5), 7.25 (1H, d, J ) 8.4 Hz, ArH-6), 7.22 (1H, s, ArH-2),
6.30 (2H, t, J ) 2.2 Hz, PyH-3,4). MS m/z (%): 203 (M+, 100).
Anal. (C11H9NO3) calcd C, H, N.
NMR (CDCl3) δ ppm 10.92 (1H, s, OH), 7.94 (1H, d, J ) 8.4 Hz,
ArH-5), 6.86 (1H, d, J ) 2.0 Hz, ArH-2), 6.76 (1H, dd, J ) 8.4
and 2.0 Hz, ArH-6), 5.91 (2H, s, PyH), 3.99 (3H, s, OCH3), 2.08
(6H, s, Py-CH3 × 2). MS m/z (%) 245 (M+, 100); HPLC purity
98.6%.
N-(4-Carboxy)benzylpyrrole (A9). Yield: 70%, starting with
151 mg (1 mmol) of 4-aminomethylbenzoic acid to afford A9, white
N-(4-Carboxy)phenyl-2,5-dimethylpyrrole (A16). Yield: 83%,
starting with 274 mg (2 mmol) of 4-aminobenzoic acid to afford
A16, white solid, mp 177-179 °C. 1H NMR δ ppm 13.07 (1H, br,
COOH), 8.06 (2H, d, J ) 8.4 Hz, ArH-3,5), 7.40 (2H, d, J ) 8.4
Hz, ArH-2,6), 5.83 (2H, s, PyH), 1.99 (6H, s, Py-CH3 × 2). MS
m/z (%): 215 (M+, 100). Anal. (C13H13NO2) C, H, N.
1
solid, mp 168-170 °C. H NMR δ ppm 12.92 (1H, br, COOH),
7.90 (2H, d, J ) 8.4 Hz, ArH-3,5), 7.24 (2H, d, J ) 8.4 Hz, ArH-
2,6), 6.83 (2H, m, PyH-2,5), 6.64 (2H, t, J ) 2.2 Hz, PyH-3,4),
5.19 (2H, s, CH2). MS m/z (%) 201 (M+, 93). Anal. (C12H11NO2)
C, H, N.
N-(4-Carboxymethene)phenyl-2,5-dimethylpyrrole (A17).
Yield 42%, starting with 521 mg (3.45 mmol) of 4-aminopheny-
lacetic acid to afford A17, white solid, mp 110-112 °C. H NMR
δ ppm 7.40 (2H, d, J ) 8.4 Hz, ArH-2,6), 7.20 (2H, d, J ) 8.4 Hz,
ArH-3,5), 5.78 (2H, s, PyH), 3.65 (2H, s, -CH2), 1.93 (6H, s, Py-
CH3 × 2). MS m/z (%): 229 (M+, 100); Anal. (C14H15NO2) C, H,
N.
N-(4-Carboxymethene)benzylpyrrole (A10). To a solution of
(4-bromomethyl)-phenylacetic acid (57 mg, 0.25 mmol) and pyrrole
(0.02 mL, 0.29 mmol) in 2 mL of DMSO was added t-BuOK (70
mg, 0.63 mmol). The mixture was stirred and irradiated under
microwave at 193 °C for 20 min monitored by TLC (chloroform/
methanol/ammonia 6:1:0.1). The mixture was poured into ice-water
and acidified with 2N HCl to pH 3. The solid was collected, washed
with water, and purified with flash column [eluant: EtOAc/
petroleum ether with AcOH (4:0.02), 0-20%] to afford 45 mg of
1
N-(4-Chloro-2-carboxy)phenyl-2,5-dimethylpyrrole (A18). The
mixture of 172 mg (1 mmol) of 2-amino-5-chlorobenzoic acid and
0.3 mL of hexane-2,5-dione (2.5 mmol), without acetic acid, was
irradiated under microwave at 100 °C for 10 min to produce 109
1
A10, 84% yield, pale-yellow solid, mp 106-109 °C. H NMR δ
ppm 12.28 (1H, br, COOH), 7.21 (2H, d, J ) 8.4 Hz, ArH-3,5),
7.12 (2H, d, J ) 8.4 Hz, ArH-2,6), 6.80 (2H, m, PyH-2,5), 6.01
(2H, t, J ) 2.2 Hz, PyH-3,4), 5.06 (2H, s, N CH2), 3.53 (2H, s,
-CH2CO); MS m/z (%) 215 (M+, 98). Anal. (C13H13NO2 ·1/8H2O)
C, H, N.
1
mg of A18, 44% yield, pink solid, mp 124-127 °C. H NMR δ
ppm 13.16 (1H, br, COOH), 7.86 (1H, d, J ) 2.0 Hz, ArH-3),
7.75 (1H, dd, J ) 8.4 and 2.0 Hz, ArH-5), 7.33 (1H, d, J ) 8.4
Hz, ArH-6), 5.74 (2H, s, PyH), 1.86 (6H, s, Py-CH3 × 2). MS m/z
(%) 248 (M - H, 100), 251 (M + 2, 47). Anal. (C13H12ClNO2 ·1/
8H2O) C, H, N.
General Procedure for the Preparation of N-Aryl-2,5-dim-
ethylpyrroles A11-A20. To a solution of amino benzoic acid
analogues (1 equiv) in 3 mL of glacial acetic acid was added
hexane-2,5-dione (1.1 equiv). The reaction mixture was irradiated
under microwave at 150 °C for 10 min and monitored by TLC
(petroleum ether/EtOAc 3:1). The mixture was poured into ice-water
and left overnight. The precipitated solid was filtered, washed with
water, and purified by a flash column [gradual eluant: EtOAc/
petroleum ether with AcOH (4:0.02), 0-20%] to afford pure
expected products.
N-(4-Carboxy)benzyl-2,5-dimethylpyrrole (A19). Yield: 77%,
starting with 302 mg (2 mmol) of 4-aminomethylbenzoic acid to
afford A19, white solid, mp 171-175 °C. 1H NMR (CDCl3) δ ppm
8.04 (2H, d, J ) 8.4 Hz, ArH-3,5), 6.98 (2H, d, J ) 8.4 Hz, ArH-
2,6), 5.88 (2H, s, PyH), 5.07 (2H, s, CH2), 2.13 (6H, s, Py-CH3 ×
2). MS m/z (%) 229 (M+, 100). Anal. (C14H15NO2) C, H, N.
N-(4-Aminosulfonyl)phenyl-2,5-dimethylpyrrole (A20). The
mixture of sulfanilamide (172 mg, 1 mmol) and hexane-2,5-dione
(0.3 mL, 2.5 mmol), without glacial acetic acid, was irradiated under

N-Carboxyphenylpyrrole DeriVatiVes
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 7851
microwave at 180 °C for 15 min to afford 188 mg of A20, purified
with flash chromatography [eluant: EtOAc/petroleum ether with
Et3N (4: 0.02), 0-20%], 75% yield, white solid, mp 158-159 °C.
1H NMR (CDCl3) δ ppm 8.05 (2H, d, J ) 8.4 Hz, ArH-2,6), 7.39
(2H, d, J ) 8.4 Hz, ArH-3,5), 5.94 (2H, s, PyH), 4.95 (2H, s,
-SO2NH2), 2.06 (6H, s, Py-CH3 × 2). MS m/z (%) 250 (M+, 100).
Anal. (C12H14N2O2S) C, H, N.
ArH-3,5), 7.85 (2H, d, J ) 8.4 Hz, ArH-2,6), 4.49 (2H, s,
-CH2OH), 4.42 (2H, q, -OCH2CH3), 1.44 (3H, t, -CH2CH3). ESI-
MS m/z (%) (C12H12N2O4) 248 (M+, 100); HPLC purity 97.1%.
3-(4-Carboxy)phenyl-5-hydroxymethyl-1,2,4-oxadiazole (B5).
A mixture of B2 (30 mg, 0.12 mmol) in ethanol (3 mL) and 1 mL
of aq NaOH (1N) was stirred at room temperature for 4 h. After
removal of ethanol in vacuo, more water (ca. 8 mL) was added,
washed with EtOAc (2 × 5 mL), and acidified with 5% HCl to pH
2. Solid was filtered out, washed with water to neutral pH, and
dried to afford B5: 18 mg, 68% yield, white solid, mp 240-242
(Z)-(3-Carboxyl)benzamidoxime (4). To a solution of 3-cy-
anobenzoic acid (588 mg, 4 mmol) in 30 mL ethanol was added
hydroxylamine hydrochloric acid (595 mg, 8.56 mmol) in water
(3 mL) and sodium carbonate (687 mg, 6.48 mmol) in water (6
mL), successively, in the presence of 8-hydroxyquinoline (2 mg,
0.013 mmol). The mixture was heated to reflux for 4 h monitored
by TLC (petroleum ether/EtOAc/AcOH 1:1:0.03). After removal
of ethanol solvent under reduced pressure, the residue was diluted
with 30 mL water, and the water solution was slowly acidified with
10% HCl to pH 3.2. The white precipitate was filtrated, washed
with water to neutral pH, and dried to afford 668 mg of 4, 93%
1
°C. H NMR δ ppm 13.27 (1H, br, COOH), 11.53 (1H, s, OH),
8.03 (2H, d, J ) 8.4 Hz, ArH-3,5), 7.88 (2H, d, J ) 8.4 Hz, ArH-
2,6), 4.41 (2H, s, CH2). MS m/z (%) 219 (M-H, 100); HPLC purity
99.6%.
3-(4-Carboxy)phenyl-5-chloromethyl-1,2,4-oxadiazole (B6). To
a solution of B2 (30 mg, 0.12 mmol) in glacial acetic acid (1 mL)
was dropped 1 mL of hydrochloric acid (36-38%). The mixture
was heated at 100 °C for 8 h. After cooling to room temperature,
precipitated solid was filtered out, washed with water to neutral
pH, and dried to afford B6: 16 mg, 55% yield, white solid, mp
1
yield, white solid, mp 197-199 °C. H NMR δ ppm 13.03 (1H,
br, COOH), 9.77 (1H, s, OH), 8.27 (1H, s, ArH-2), 7.95 (2H, dd,
J ) 8.4 Hz, ArH-4,6), 7.53 (1H, t, J ) 8.4 Hz, ArH-5), 5.95 (2H,
br, NH2).
1
210-212 °C. H NMR δ ppm 13.35 (1H, br, COOH), 8.17 (4H,
dd, J ) 8.4 Hz, ArH), 5.22 (2H, s, CH2). MS m/z (%): 237 (M -
H, 94), 239 (M - H + 2, 28); HPLC purity 99.2%.
(Z)-(4-Carboxyl)benzamidoxime (5). The preparation was the
same as the synthesis of 4. Starting with 735 mg (5 mmol) of
4-cyanobenzoic acid to afford 611 mg of 5, 68% yield, white solid,
mp 228 °C dec. 1H NMR δ ppm 13.02 (1H, br, COOH), 9.89 (1H,
s, OH), 7.94 (2H, d, J ) 8.4 Hz, ArH-3,5), 7.80 (2H, d, J ) 8.4
Hz, ArH-2,6), 5.94 (2H, s, NH2). MS m/z (%) 179 (M - H, 100).
(Z)-(4-Methoxycarbonyl)benzamidoxime (6). The preparation
was the same as that of 4. Starting with methyl 4-cyanobenzoate
(3) (806 mg, 5 mmol) to afford 901 mg of 6, white solid, 93%
3-(4-Carboxy)phenyl-5-thiocyanatomethyl-1,2,4-oxadiazole
(B7). Preparation was the same as that described for B6. Starting
with 27 mg of B3 to afford B7, 38% yield, white solid, mp 240-244
°C. 1H NMR δ ppm 13.26 (1H, br, COOH), 8.08 (4H, dd, J ) 8.4
Hz, ArH), 4.53 (2H, s, CH2). MS m/z (%) 274 (M - CN + K,
100); HPLC purity 96.2%.
5-Bromomethyl-3-(4-carboxy)phenyl-1,2,4-oxadiazole (B8). A
mixture of B1 (40 mg, 0.13 mmol) in 1 mL of glacial acetic acid
and 1 mL hydrobromic acid (40%) was heated at 100 °C for 12 h
to afford 25 mg of B8, white solid, 65% yield, mp 209-211 °C.1H
NMR δ ppm 13.31 (1H, br, COOH), 8.16 (4H, dd, J ) 8.4 Hz,
ArH), 5.02 (2H, s, CH2). MS m/z (%) 283 (M+, 69), 281 (M - 2,
100); HPLC purity 99.8%.
1
yield, mp 164-166 °C. H NMR δ ppm 9.91 (1H, s, OH), 7.96
(2H, d, J ) 8.4 Hz, ArH-3,5), 7.83 (2H, d, J ) 8.4 Hz, ArH-2,6),
5.95 (2H, br, NH2), 3.86 (3H, s, -OCH3). MS m/z (%) 195 (M +
H, 100).
5-Bromomethyl-3-(4-methoxycarbonyl)phenyl-1,2,4-oxadia-
zole (B1). The mixture of 6 (388 mg, 2 mmol) and bromoacetyl
bromide (0.19 mL, 2.2 mmol) in THF (10 mL) was heated to reflux
for 8 h and monitored by TLC (petroleum ether/EtOAc 4:1). After
solvent removal, residue was purified by a silica gel column
(petroleum ether/EtOAc 4:1) to afford 378 mg of B1, yield 64%,
3-(3-Carboxyphenyl)-5-(trifluoromethyl)-1,2,4-oxadiazole (B9).
A mixture of 4 (180 mg, 1 mmol) and trifluoroacetic anhydride
(0.42 mL, 3 mmol) in anhydrous pyridine (3 mL) was heated to
reflux for 3 h and monitored by TLC (petroleum ether/EtOAc/AcOH
3:1:0.03). The mixture was poured into ice-water and adjusted
with HCl (10%) to pH 4, then extracted with EtOAc three times.
After solvent was removed, the residue was purified with flash silica
column [eluant: EtOAc/petroleum ether with AcOH (4: 0.03)
3-20%] to afford B9: 107 mg, 42% yield, white solid, mp 114-117
1
white solid, 100-102 °C. H NMR δ ppm 8.19 (4H, dd, J ) 8.4
Hz, ArH), 5.03 (2H, s, CH2), 3.90 (3H, s, -OCH3). MS m/z (%):
296 (M+, 48), 298 (M + 2, 44). Anal. (C11H9BrN2O3) C, H, N.
5-Chloromethyl-3-(4-methoxycarbonyl)phenyl-1,2,4-oxadia-
zole (B2). Preparation was the same as that described for B1.
Chloroacetyl chloride (0.18 mL, 2.2 mmol) was reacted with 6 (388
mg, 2 mmol) to afford B2: 367 mg, 73% yield, white solid, mp
57-58 °C. 1H NMR δ ppm 8.19 (4H, dd, J ) 8.4 Hz, ArH), 5.22
(2H, s, CH2), 3.90 (3H, s, OCH3). MS m/z (%) 252 (M+, 37), 254
(M + 2, 13). Anal. (C11H9ClN2O3 ·1/2H2O) C, H, N.
1
°C. H NMR δ ppm 13.48 (1H, s, COOH), 8.58 (1H, s, ArH-2),
8.32 (2H, dd, J ) 8.4 Hz, ArH-4,6), 7.80 (1H, t, J ) 8.4 Hz, ArH-
5). MS m/z (%) 257 (M-H, 65); HPLC purity 97.1%.
3-(4-Carboxyphenyl)-5-(trifluoromethyl)-1,2,4-oxadiazole (B10).
The preparation was the same as that of B9. Starting with 5 (180
mg, 1 mmol) to afford 136 mg of B10, white solid, 53% yield, mp
1
3-(4-Methoxycarbonyl)phenyl-5-(thiocyanatomethyl)-1,2,4-
oxadiazole (B3). A mixture of B2 (184 mg, 0.73 mmol) and
ammonium thiocyanate (228 mg, 3 mmol) in 5 mL of DMF was
heated at 90 °C for 3 h and monitored by TLC (petroleum ether/
EtOAc 4: 1). The solution was poured into ice-water and a
precipitated yellow solid was filtered, washed with water, and
purified by a silica gel column (petroleum ether/EtOAc 4:1) to
obtain B3: 89 mg, 44% yield, pale-yellow solid, mp 124-126 °C.
1H NMR δ ppm 8.20 (4H, dd, J ) 8.4 Hz, ArH), 4.88 (2H, s,
CH2), 3.91 (3H, s, OCH3). MS m/z (%) 275 (M+, 46); HPLC purity
98.7%.
3-(4-Ethoxycarbonyl)phenyl-5-hydroxymethyl-1,2,4-oxadia-
zole (B4). A solution of B1 (90 mg, 0.3 mmol) in ethanol (3 mL)
in the presence of 1 mL of aq NaOH (1N) was stirred at room
temperature for 4 h. The mixture was poured into water, acidified
with 5% HCl to pH 5, and then extracted with EtOAc three times.
Then organic solvent was removed in vacuo, and residue was
purified by a silica gel column (petroleum ether/EtOAc 4:1) to
afford B4: 52 mg, 70% yield, white solid, mp 145-148 °C. 1H
NMR (CDCl3) δ ppm 9.05 (1H, s, OH), 8.17 (2H, d, J ) 8.4 Hz,
169-171 °C. H NMR δ ppm 13.44 (1H, br, COOH), 8.21 (2H,
d, J ) 8.4 Hz, ArH-3,5), 8.15 (2H, d, J ) 8.4 Hz, ArH-2,6). MS
m/z (%) 258 (M+, 100); HPLC purity 97.1%.
3-(3-Carboxy)phenyl-5-chloromethyl-1,2,4-oxadiazole (B11).
As described for the preparation of B2, a mixture of 4 (270 mg, 1.5
mmol) and chloroacetyl chloride (0.16 mL, 1.9 mmol) in 8 mL
anhydrous THF was refluxed for 4 h. The mixture was poured into
ice-water, and precipitated solid was collected, washed with water
to neutral pH, and dried in vacuo to afford B11: 132 mg, 37% yield,
white solid, mp 128-132 °C. 1H NMR δ ppm 13.39 (1H, s,
COOH), 8.56 (1H, s, ArH-2), 8.28 (1H, d, J ) 8.4 Hz, ArH-4),
8.18 (1H, d, J ) 8.4 Hz, ArH-6), 7.76 (1H, t, J ) 8.4 Hz, ArH-5).
MS m/z (%) 237 (M - H, 62), 239 (M - H + 2, 36); HPLC purity
96.9%.
4-Carboxylbenzaldehyde thiosemicarbazone (7). A solution
of 4-carboxybenzaldehyde (450 mg, 3 mmol) and thiosemicarbazide
(300 mg, 3.3 mmol) in ethanol (6 mL) was heated to reflux for
1.5 h and monitored by TLC (CHCl3/CH3OH/AcOH 3:1:0.05). After
reaction mixture was cooled, the solid was filtered out, washed with
ethanol, and dried to give 606 mg of 7, 91% yield, yellow solid,

7852 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Liu et al.
1
mp 250 °C dec. H NMR δ ppm 13.04 (1H, br, COOH), 11.56
(1H, d, J ) 8.4 Hz, ArH-4), 7.70 (1H, t, J ) 8.4 Hz, ArH-5), 7.51
(1H, s, ArH-2), 7.43 (1H, d, J ) 8.4 Hz, ArH-6), 4.23 (2H, s, CH2).
MS m/z (%) 278 (M + H, 17); Anal. (C10H6F3NOS2) C, H, N.
N-(4-Methoxyphenyl)rhodanine (B17). Yield: 87%, starting with
492 mg (4 mmol) of 4-methoxyaniline to afford B17, pale-yellow
solid, mp 124-127 °C. 1H NMR (CDCl3) δ ppm 7.13 (2H, d, J )
8.4 Hz, ArH-2,6), 7.05 (2H, d, J ) 8.4 Hz, ArH-3,5), 4.18 (2H, s,
CH2), 3.85 (3H, s, OCH3). MS m/z (%) 240 (M + H, 14). Anal.
(C10H9NO2S2) C, H, N.
(1H, s, -CHd), 8.29 (1H, br, NH), 8.11 (2H, br, NH2), 7.93 (4H,
m, ArH). MS m/z (%) 222 (M - H, 100).
5-Amino-3-(4-carboxy)phenyl-1,3,4-thiadiazole (B12). Thi-
osemicarbazone 7 (370 mg, 1.66 mmol) and ammonium ferric
sulfate (3.2 g, 6.64 mmol) were soluble in H2O (15 mL), and the
mixture was heated to reflux for 10 h. The mixture was then poured
into ice-water, basified with 10% NaOH aq to pH 5, and extracted
with EtOAc three times. Next, organic solvent was removed in
vacuo, and residue was purified by a silica gel column (CHCl3/
CH3OH/AcOH 9:1:0.02) to obtain 171 mg of B12, 47% yield, yellow
N-(4-Hydroxyphenyl)rhodanine (B18). Yield: 88%, starting with
436 mg (4 mmol) of 4-aminophenol to afford B18, pale-yellow solid,
mp 180-182 °C. 1H NMR (CDCl3) δ ppm 9.12 (1H, s, OH), 7.00
(4H, dd, J ) 8.4 Hz, ArH-2,3,5,6), 4.17 (2H, s, CH2). MS m/z (%)
224 (M - H, 52); HPLC purity 99.6%.
1
solid, mp 240 °C dec. H NMR δ ppm 8.00 (2H, d, J ) 8.4 Hz,
ArH-3,5), 7.84 (2H, d, J ) 8.4 Hz, ArH-2,6), 7.57 (2H, br, NH2).
MS m/z (%): 222 (M + H, 100); HPLC purity 98.9%.
(Z)-4-[(4-Carboxy)phenylamino]-4-oxobut-2-enoic acid (8). A
solution of maleic anhydride (108 mg, 1.1 mmol) and 4-aminoben-
zoic acid (137 mg, 1 mmol) in EtOAc (7 mL) was stirred for 1 h
at rt, resulting in production of abundant crystal solid. The solid
was filtered and washed with EtOAc to afford 223 mg of 8, 95%
N-(4-Carboxyphenyl)rhodanine (B19). Yield: 77%, starting with
822 mg (6 mmol) of 4-aminobenzoic acid to afford B19, pale-white
1
solid, mp 188-190 °C. H NMR δ ppm 13.23 (1H, br, COOH),
8.09 (2H, d, J ) 8.4 Hz, ArH-3,5), 7.43 (2H, d, J ) 8.4 Hz, ArH-
2,6), 4.40 (2H, s, CH2). MS m/z (%) 252 (M - H, 84); HPLC
purity 99.9%.
1
yield, yellow solid, mp 218-220 °C. H NMR δ ppm 12.82 (1H,
br, COOH), 10.60 (1H, s, NH), 7.92 (2H, d, J ) 8.4 Hz, ArH-3,5),
7.74 (2H, d, J ) 8.4 Hz, ArH-2,6), 6.51 (1H, d, J ) 12.0 Hz, dCH-
2), 6.34 (1H, d, J ) 12.0 Hz, dCH-3). MS m/z (%) 234 (M - H,
100).
N-(3-Carboxyphenyl)rhodanine (B20). Yield: 86%, starting with
418 mg (3 mmol) of 3-aminobenzoic acid to afford B20, white solid,
1
mp 185-187 °C. H NMR δ ppm 13.27 (1H, br, COOH), 8.05
(1H, d, J ) 8.4 Hz, ArH-6), 7.87 (1H, s, ArH-2), 7.69 (1H, t, J )
8.4 Hz, ArH-5), 7.55 (1H, d, J ) 8.4 Hz, ArH-4), 4.37 (2H, s,
CH2). MS m/z (%) 252 (M - H, 100); HPLC purity 99.8%.
N-(3-Carboxy-4-chlorophenyl)rhodanine (B21). Yield: 81%,
starting with 343 mg (2 mmol) of 5-amino-2-chlorobenzoic acid
to afford B21, white solid, mp 184-186 °C. 1H NMR δ ppm 13.71
(1H, br, COOH), 7.78 (1H, d, J ) 2.4 Hz, ArH-6), 7.75 (1H, d, J
) 8.4 Hz, ArH-3), 7.50 (1H, dd, J ) 8.4 and 2.4 Hz, ArH-4), 4.35
(2H, s, CH2). MS m/z (%) 286 (M - H, 100), 288 (M - H + 2,
44); HPLC purity 99.5%.
(Z)-4-[(3-Hydroxy-4-methoxycarbonyl)phenylamino]-4-oxobut-
2-enoic acid (9). The method of synthesis was the same as that of
8. Starting with 334 mg (2 mmol) of methyl 4-aminosalicylate,
1
234 mg, 88% yield, yellow solid, mp 204-207 °C. H NMR δ
ppm 12.87 (1H, br, COOH), 10.61 (1H, s, OH), 10.56 (1H, s, NH),
7.76 (1H, d, J ) 8.4 Hz, ArH-5), 7.41 (1H, s, ArH-2), 7.13 (1H, d,
J ) 8.4 Hz, ArH-6), 6.49 (1H, d, J ) 12.0 Hz, dCH-3), 6.34 (1H,
d, J ) 12.0 Hz, dCH-2), 3.87 (3H, s, OCH3). MS m/z (%) 264 (M
- H, 100).
N-(3-Carboxy-4-hydroxyphenyl)rhodanine (B22). Yield: 28%,
N-(4-Carboxy)phenylmaleimide (B13). A mixture of 8 (223 mg,
0.95 mmol) and triethylamine (0.06 mL, 0.475 mmol) in 3 mL of
acetic anhydride was stirred and heated at 60 °C for 1 h. The
mixture was poured into ice-water, and the solid isolated by
filtration was washed with water to neutral pH and dried in vacuo
to afford B13: 98 mg, 47% yield, white solid, mp 160-163 °C. 1H
NMR δ ppm 12.91 (1H, br, COOH), 8.05 (2H, d, J ) 8.4 Hz,
ArH-3,5), 7.51 (2H, d, J ) 8.4 Hz, ArH-2,6), 7.23 (2H, s,
maleimide-H). MS m/z (%) 217 (M+, 100); HPLC purity 98.5%.
N-(3-Hydroxy-4-methoxycarbonyl)phenylmaleimide(B14).Prepa-
ration was the same as that of B13. Solid 9 (389 mg, 1.47 mmol)
was reacted with triethylamine (0.1 mL, 0.73 mmol) in 5 mL of
acetic anhydride to afford 132 mg of B14, 27% yield, white solid,
mp 121-123 °C. 1H NMR δ ppm 10.62 (1H, s, OH), 7.88 (1H, d,
J ) 8.4 Hz, ArH-5), 7.22 (2H, s, maleimide-H), 7.04 (1H, d, J )
2.0 Hz, ArH-2), 7.00 (1H, dd, J ) 8.4 and 2.0 Hz, ArH-6), 3.90
(3H, s, OCH3). MS m/z (%) 247 (M+, 54). Anal. (C12H9NO5) C,
H, N.
N-(4-Carboxy-3-hydroxy)phenylmaleimide (B15). Compound
B14 (40 mg, 0.16 mmol) was hydrolyzed in the same condition as
B6 to afford B15, yield 79%, white solid, mp 141-144 °C. 1H NMR
δ ppm 12.81 (1H, br, COOH), 11.42 (1H, s, OH), 7.43 (1H, d, J
) 8.4 Hz, ArH-5), 6.09 (1H, dd, J ) 8.4 and 2.0 Hz, ArH-6), 6.06
(2H, s, maleimide-H), 5.96 (1H, d, J ) 2.0 Hz, ArH-2). MS m/z
(%) 232 (M - H, 11); HPLC purity 97.5%.
General Procedure for the Preparation of N-Phenylrhoda-
nine Derivatives B16-B22. A suspension of aniline analogues (1
equiv) in 4 mL of water was heated to 95 °C until the aniline was
fully dissolved. Then bis(carboxymethyl) trithiocarbonate (1.1
equiv) was added. The resulting solution was heated at 100 °C for
19 h and was cooled to room temperature. The precipitated solid
was filtrated out and washed with water. The dried solid was
purified by silica gel column (petroleum ether/acetone 3:1) or flash
silica column [eluant: EtOAc/petroleum ether with HOAc (3:0.03),
20-30%] to obtain pure product.
starting with 306 mg (2 mmol) of 5-aminosalicylic acid to afford
1
B22, yellow solid, mp 174-176 °C. H NMR δ ppm: 13.09 (1H,
br, COOH), 11.52 (1H, s, OH), 7.72 (1H, d, J ) 2.4 Hz, ArH-6),
7.41 (1H, dd, J ) 8.4 and 2.4 Hz, ArH-4), 7.10 (1H, d, J ) 8.4
Hz, ArH-3), 4.33 (2H, s, CH2).MS m/z (%) 269 (M+, 20), 268 (M
- H, 100); HPLC purity 99.7%.
Determination of the Inhibitory Activity of the Compounds
on HIV-1 Replication. The inhibitory activity of compounds on
HIV-1 IIIB replication in MT-2 cells was determined as previously
described. In brief, 1 × 104 MT-2 cells were infected with an HIV-1
strain (100 TCID50) in 200 µL of RPMI 1640 medium containing
10% FBS in the presence or absence of a test compound at graded
concentrations overnight. Then the culture supernatants were
removed and fresh media containing no test compounds were added.
On the fourth day postinfection, 100 µL of culture supernatants
were collected from each well, mixed with equal volumes of 5%
Triton X-100, and assayed for p24 antigen, which was quantitated
by ELISA. Briefly, wells of polystyrene plates (Immulon 1B, Dynex
Technology, Chantilly, VA) were coated with HIV immunoglobulin
(HIVIG), which was prepared from plasma of HIV-seropositive
donors with high neutralizing titers against HIV-1IIIB, in 0.085 M
carbonate-bicarbonate buffer (pH 9.6) at 4 °C overnight, followed
by washes with washing buffer (0.01 M PBS containing 0.05%
Tween-20) and blocking with PBS containing 1% dry fat-free milk
(Bio-Rad, Inc., Hercules, CA). Virus lysates were added to the wells
and incubated at 37 °C for 1 h. After extensive washes, anti-p24
mAb (183-12H-5C), biotin-labeled antimouse IgG1 (Santa Cruz
Biotech, Santa Cruz, CA), streptavidin-labeled horseradish peroxi-
dase (Zymed, San Francisco, CA), and the substrate 3,3′,5,5′-
tetramethylbenzidine (Sigma Chemical Co., St. Louis, MO) were
added sequentially. Reactions were terminated by addition of 1N
H2SO4. Absorbance at 450 nm was recorded in an ELISA reader
(Ultra 386, TECAN, Research Triangle Park, NC). Recombinant
protein p24 purchased from US Biological (Swampscott, MA) was
included for establishing standard dose-response curves. Each
sample was tested in triplicate. The percentage of inhibition of p24
production was calculated as previously described.31 The effective
N-(3-(Trifluoromethyl)phenyl)rhodanine (B16). Yield: 54%,
starting with 2 mL (16 mmol) of 3-(trifluoromethyl)aniline to afford
B16, yellow solid, mp 150-152 °C. 1H NMR (CDCl3) δ ppm 7.77

N-Carboxyphenylpyrrole DeriVatiVes
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 7853
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absence of the compounds tested. After incubation at 37 °C for
30 min, the mixture was added to wells of a 96-well polystyrene
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rabbit antisera directed against the gp41 six-helix bundle. Then,
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TMB were added sequentially. Absorbance at 450 nm was read,
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compounds on the gp41 6-HB formation. In brief, the N-peptide
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Acknowledgment. This investigation was supported by
grants 2006AA02Z319 and 2006DFA33560 from the Ministry
of Science and Technology in China and 2007G06 from Beijing
Municipal Science & Technology Commission to L. Xie, grants
2005Z2-E4041 from Guangzhou Science and Technology Key
Project and RO1 AI46221 from the U.S. National Institutes of
Health to S. Jiang, and the Ph.D. fellowship SFRH/BD/22190/
2005 from Fundac¸a˜o para a Cieˆncia e a Tecnologia (Portugal)
to Ca´tia Teixeira.
Supporting Information Available: Elemental analysis and
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