Accepted Manuscript

Small molecule fusion inhibitors: design, synthesis and biological evaluation of

(Z)-3-(5- (3-benzyl-4-oxo-2-thioxothiazolidinylidene)methyl)-N-(3-car‐

boxy-4-hydroxy)phenyl-2,5- dimethylpyrroles and related derivatives targeting

HIV-1 gp41

Xiao-Yang He, Lu Lu, Jiayin Qiu, Peng Zou, Fei Yu, Xing-Kai Jiang, Lin Li,

Shibo Jiang, Shuwen Liu, Lan Xie

PII: S0968-0896(13)00372-6

DOI: http://dx.doi.org/10.1016/j.bmc.2013.04.046

Reference: BMC 10786

To appear in: Bioorganic & Medicinal Chemistry

Received Date: 2 February 2013

Revised Date: 16 April 2013

Accepted Date: 17 April 2013

Please cite this article as: He, X-Y., Lu, L., Qiu, J., Zou, P., Yu, F., Jiang, X-K., Li, L., Jiang, S., Liu, S., Xie, L.,

Small molecule fusion inhibitors: design, synthesis and biological evaluation of (Z)-3-(5- (3-benzyl-4-oxo-2-

thioxothiazolidinylidene)methyl)-N-(3-carboxy-4-hydroxy)phenyl-2,5- dimethylpyrroles and related derivatives

targeting HIV-1 gp41, Bioorganic & Medicinal Chemistry (2013), doi: http://dx.doi.org/10.1016/j.bmc.2013.04.046

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ACCEPTED MANUSCRIPT

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Small molecule fusion inhibitors: design, synthesis and biological evaluation of (Z)-3-(5-

(3-benzyl-4-oxo-2-thioxothiazolidinylidene)methyl)-N-(3-carboxy-4-hydroxy)phenyl-2,5-

dimethylpyrroles and related derivatives targeting HIV-1 gp41

Xiao-Yang Hea, #, Lu Lub, Jiayin Qiuc, Peng Zoud, Fei Yub,c,d, Xing-Kai Jianga, Lin Lic, Shibo Jiangb,

d, Shuwen Liuc, Lan Xiea*

a Beijing Institute of Pharmacology and Toxicology, Beijing 100850, China

b Key Laboratory of Medical Molecular Virology of Ministries of Education and Health, Shanghai

Medical College and Institute of Medical Microbiology, Fudan University, Shanghai 200032, China

c School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515, China

d Lindsley F. Kimball Research Institute, New York Blood Center, NY 10065, USA

*Corresponding author, e-mail: lanxieshi@yahoo.com, (L. Xie); Tel/Fax: 86-10-6931690 # Current address: Beijing Institute of Radiation Medicine, Beijing, 100850, China

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Abstract:

By a scaffold elongation strategy, a series of (Z)-3-(5-(3-benzyl-4-oxo-2-thioxothiazolidinyl

idene)methyl)-N-(3-carboxy-4-hydroxy)phenyl-2,5-dimethylpyrroles and related derivatives with a

linear multi- aromatic ring skeleton were designed, synthesized, and evaluated in HIV-1 gp41 and

cellular assays. Among them, the most active compounds, 12e, 12g, and 12k with a one-carbon

linker (n = 1) between the rhodanine (C) and phenyl (D) rings, exhibited very promising inhibitory

potency with IC50 values of 1.8-2.6 µM and EC50 values of 0.3-1.5 µM against gp41 6-HB formation

and HIV-1 replication in MT-2 cells, respectively. Additionally, they were almost equally effective

against both T20-sensitive and resistant strains. The related SAR studies and molecular modeling

results provided potential for further developing a new class of non-peptide small molecular fusion

inhibitors targeting the HIV-1 gp41.

Key words: 3-Substituted N-(3-carboxy-4-hydroxy)phenyl-2,5-dimethylpyrrole derivatives,

Small-molecule fusion inhibitors, HIV-1 gp-41, Anti-HIV agents

Graphic Abstract

COOHHO

N

S N

O

S

A

B

CD

COOHHO

N

IC50 37.4 µM (gp41- 6HB)EC50 0.7 µM (cells)

IC50 1.8-2.6 µM (gp41- 6HB)EC50 0.3-1.5 µM (cells)

2 12e, 12g, 12k

R

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1. Introduction

The HIV-1 envelope glycoprotein subunit gp41 is considered one of the most attractive targets

for anti-HIV drugs because it plays a crucial role in the fusion process between virus and host cell

membranes. Gp41 is originally buried within glycoprotein gp120 by non-covalent bond, but it

becomes exposed when gp120 binds to receptor CD4 on the T cells. This causes the formation of a

stable six-helix bundle (6-HB) core structure by an N-terminal heptad repeat (NHR) trimer with three

C-terminal heptad repeats (CHR) in an anti-parallel manner, thus bringing the viral and host cell

membrane into proximity and fusion.1-4 By mimicking the CHR peptide sequence of gp41, the

synthetic peptide drug T-20,5, 6 as the first fusion inhibitor targeting gp41, has been successfully used

to treat HIV infection and AIDS patients. However, the clinical use of this peptide has been limited

by high cost, lack of oral availability and metabolic instability. Therefore, to overcome the

limitations of current peptide HIV-fusion inhibitors,7 new strategies for discovering nonpeptide

small-molecule fusion inhibitors with high potency are needed. Indeed, we have already identified

the binding site on HIV-1 gp41 for small molecule inhibitors as a highly conserved hydrophobic

pocket on the surface of inner NHR trimer of gp41.8, 9 It is a linear cavity (ca. 16 Å long, 7 Å wide

and 5 – 6 Å) and can be filled by three conserved hydrophobic CHR residues, W628, W631 and I635,

to form stable 6-HB of gp41. In particular, amino acid K574 on the binding site is demonstrated to

play a critical role in 6-HB stability10-12 by forming a “salt bridge” with D632 of CHR. Therefore,

some small molecules that can bind to this pocket with high affinity should be able to block the 6-HB

formation of gp41 and thereby inhibit HIV-1 entry and replication. So far, several series of

small-molecule compounds with inhibitory activities against 6-HB of HIV-1 gp41 and/or cell-cell

fusion (CCF) have been reported, such as N-phenyl pyrroles NB-2, NB-64,13 phenyl furan

derivatives,14, 15 indole derivatives,16, 17 benzamide,18 biphenyl ethylene ethers,19 �-helical

peptidomimetic compounds,20, 21 and plant natural products.22-24 However, their potency is mostly at

�M level, much less than those of T20 and other peptide derivatives at nM level. Thus, more research

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is required to discover highly potent small molecules and design them as HIV-1 fusion inhibitors

targeting gp41.

In our hit-to-lead optimization process for small-molecule inhibitors targeting gp41 (Figure

1), the initial structural modification based on hit NB-64 discovered two lead compounds, 1 and 2,

both N-phenyl-2,5-dimethylpyrroles, which showed promising inhibitory potency against gp41 6-HB

formation (IC50 25.6 and 37.4 �M, respectively) and against HIV-1 replication (EC50 7.7 and 0.7 �M,

respectively) in cellular assay.25 Since it occupied only a small space in the gp41 binding pocket,

lead 1 was modified by elongating the molecular skeleton to increase molecular volume and shape

complement. Subsequently, a new lead 3 with a linear multi-aromatic-ring skeleton was discovered.

It exhibited improved inhibitory activity against gp41 6-HB formation (IC50 4.4 �M) and maintained

high potency against HIV-1 replication (EC50 3.2 �M) in cellular assay.26 Molecular modeling of 3

demonstrated that the new linear multi-aromatic-ring scaffold fit very well in the linear binding

cavity of gp41, suggesting that it would be beneficial in increasing the affinity between the small-

molecule inhibitor and its biologic target. Prior structure-activity relationship (SAR) analysis also

indicated that an introduced N-substituted rhodanine fragment was a preferable moiety for

maintaining and/or enhancing molecular inhibitory potency against gp41 6-HB formation and HIV-1

replication, thus offering a new linear scaffold that improves lead potency. In this continuing study,

the optimization of lead 2 was conducted to find even more potent small-molecule inhibitors

targeting gp41 and more SARs. As shown in Figure 1, a series of new 3-substituted

N-(3-carboxy)phenyl-2,5-dimethylpyrrole compounds with a linear multi-aromatic-ring scaffold

were designed, synthesized, and evaluated against gp41 6-HB formation and HIV-1 replication in

cellular assay. An N-aryl rhodanine moiety was first introduced at the 3-position of the pyrrole ring

in lead 2 to build up the required linear molecular scaffold, while the structure of 2 was maintained.

Subsequent modifications were performed on (1) the linker length (n = 0, 1, 2) between the

rhodanine ring (C-ring) and a phenyl ring (D-ring), (2) various substitutents (R1) and their positions

(p-, m-, o-) on the D ring, (3) heteroatom replacement X/Y on the rhodanine ring (C ring), and (4) the

ortho-substitutent of the carboxy group (R = OH, Cl, H ) on the A-ring, successively. Meanwhile,

molecular modeling was also used to assist us in designing the new compounds in the context of

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their interaction with the hydrophobic pocket of HIV-1 gp41, especially for the key amino acid

residue K574. Related new results are reported herein.

Figure 1 Lead optimization strategy and new target compounds

2. Chemistry

All target compounds were synthesized as described in Schemes 1-2. For elongation of

molecular skeleton, lead 2 was first introduced an aldehyde group at the 3-position of the pyrrole

ring by using Vilsmeier-Hacck reaction with DMF/POCl3 to obtain N-(3-carboxy-4-hydroxy)

phenyl-2,5-dimethyl-3-formylpyrrole (4). Different N-substituted rhodanine building blocks,

including 8 (n = 0), 9 (n = 1), or 10 (n = 2), were synthesized from commercially available reagents

5-7, respectively, by using methods in our previous studies.25 The Knoevenagel condensation

reactions between 4 and 8, or 9 and 10, were then carried out in the presence of ammonium acetate in

toluene and methanol refluxed for 3-5 h to afford a series of new target compounds 11, 12 (a-e, g-k,

m-n), and 13. Compounds 12e and 12k with an ester group on the phenyl (D-ring) were hydrolyzed

in acidic condition to provide corresponding carboxylic compounds 12f and 12l, respectively.

Similar to the preparation of 4, an aldehyde group was introduced in reagents

N-(4-chloro-3-carboxy)phenyl-2,5-dimethylpyrrole and N-(3-carboxy)phenyl-2,5-dimethylpyrrole to

produce intermediates 14 (R’ = Cl) and 15 (R’ = H), respectively. Subsequently, a coupling reaction

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with 9a yielded compounds 16 and 17, respectively. The condensation of 4 with 18, which was

prepared from thiazolidine-2,4-dione and benzyl bromide in the presence of K2CO3 in DMF, yielded

compound 19, containing an isostere of rhodanine as the C-ring. Building block 21 with a

pyrrolin-2,4-dione ring was prepared by treatment of maleic anhydride with phenyl ethylamine,

followed by a condensation in acetic anhydride.27 After an esterification of 4, the produced

compound 20 was coupled with 21 in the presence of PPh3 in ethanol to afford product 22. Finally,

compound 22 was hydrolyzed under acidic condition to afford carboxylic compound 23.

All new target compounds were identified by 1H NMR and MS spectroscopic data.

Consistent with our prior studies, these new 5-arylidene rhodanine derivatives were also identified as

thermodynamically stable cis-isomers (Z-isomer) because their olefin proton signals of the exocyclic

C=C bond showed a range of 7.66-7.83 ppm (literature � 7.70-7.82 ppm) by the deshielding effect of

the cis-carbonyl (C=O). However, thiazolin-2,4-dione compound 23 was assigned as trans-isomer

(E-isomer) based on the group priority on the exocyclic C=C bond.

9,12 R a H b p-CF3 c p-Br d p-OCH3 e p-COOCH3 f p-COOH g p-t-Bu h m-CF3 i m-Br j m-OCH3 k m-COOCH3 l m-COOH

m o-CF3 n o-Br

�

Scheme 1. Reagents and conditions: (i) POCl3/DMF, <10 ºC; (ii) for 5, bis(carboxylmethyl)

trithiocarbonate, H2O, 100 ºC, 19 h; (iii) for 6 and 7: (1) CS2 in Et2O, 30 min and (2)

BrCH2CO2H/EtOH, reflux, 1 h; (iv) NH4OAc, toluene/CH3OH, reflux, 3-5 h; (v) conc. HCl/HOAc,

under N2, reflux, 24 h.

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R' COOH

N

CHO

N

O

O

OO ROOC

HON

N

O

O

HO COOCH3

N

CHO

i

ii

4 R' = OH14 R' = Cl15 R' = H

2022 R = Me23 R = H

S N

O

OHOOC

HO N SN

O

O

4

18

19

COOH

N

R'

SN

O

S16 R' = Cl17 R' = H

9a

i

21

iii

iv

Scheme 2 Reagents and conditions: (i) NH4OAc, toluene/CH3OH, reflux, 5 h; (ii) CH3I, Cs2CO3,

DMF, rt, 24 h; (iii) Ph3P, EtOH, under N2, reflux, 12 h; (iv) conc. HCl/HOAc, under N2, reflux, 24 h.

3. Results and Discussion

Twenty newly synthesized target compounds (11-13, 16, 17, 19, and 23) with a linear

multi-aromatic-ring scaffold were evaluated against both HIV-1 gp41 6-HB formation (IC50) and

HIV replication in MT-2 cell line (EC50), respectively, by enzyme-linked immunosorbent assays

(ELISA). The colorimetric XTT assay was also performed to test the cytotoxicity of these

compounds on MT-2 cells (CC50). All assay data were summarized in Table 1 and NB-64 was

included as a reference in the same assays.

Based on the known binding site of gp41 for small-molecule inhibitors, we inferred that a

suitably elongated molecule would be the best fit to accommodate the space and shape

complementation requirements for binding the gp41 pocket in order to afford more interactions with

amino acids on the surface of the binding site. As the first attempt, we synthesized compounds 11 (n

= 0), 12a, and 13 with a one-atom (n = 1) or two-atom (n = 2) linker between the rhodanine (C-ring)

and its N-phenyl (D-ring) ring. In comparison to 11, compounds 12a and 13 exhibited significantly

improved potency against gp41 6-HB formation with IC50 values of 11.9 and 12.5 µM, respectively,

suggesting that a linker with at least one atom between C-ring and D-ring might favor ligand binding

to the gp41 binding cavity. Both 12a and 13 also showed high potency against HIV-1 replication

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with EC50 values of 3.2 and 6.2 µM, respectively, in cellular assays. These results prompted us to

investigate a series of compounds 12b-n (n = 1) with diverse substituents at different positions on the

phenyl (D-ring). As shown in Table 1, most of the series 12 compounds, except 12f and 12n,

displayed improved potency in inhibiting gp41 6-HB formation with an IC50 value range of 0.9-22.6

µM. The most potent compound, 12k with an m-CO2CH3 on the D-ring, exhibited an IC50 value of

1.8µM and an EC50 value of 0.3 µM against gp41 6-HB formation and HIV-1 replication,

respectively. Compounds 12e and 12g are also very promising with IC50 values of 1.8 and 2.6 µM

and EC50 values of 1.5 and 0.9 µM in both molecular and cellular assays, respectively. The biologic

data of series 12 compounds revealed some relationships between the substituents on the D-ring and

anti-HIV activity. Compared with 12a, the presence of a bromo (12c), methoxy (12d), methyl ester

(12e), or tert-butyl group (12g) at the para-position on the D-ring obviously improved the inhibitory

activity (IC50 0.9-10.3 µM) for gp41 6-HB formation, but trifluoromethyl (12b) and very polar

carboxy (12f) groups decreased inhibitory potency (IC50 18.8 and 68.0 µM, respectively) in the same

assay. A similar potency pattern was also observed in compounds 12h-12l with an m-substituent on

the D-ring. These compounds were as potent as (IC50 1.8-20.8 µM), or a little better than, the

corresponding p-substituted compounds 12b-12g. Therefore, the presence of a suitable substituent on

the D-ring could increase the affinity of small-molecule inhibitors with gp41 6-HB binding site

regardless of p- or m-substituted position. On the other hand, compounds 12m (R = o-CF3) and 12n

(R = o-Br) with an o-substituted phenyl moiety (D-ring) exhibited obviously reduced or abolished

potency in the gp41 6-HB formation assay, suggesting that the o-position on the D-ring was not

modifiable. Consistent with the results of gp41-6HB formation assay, most of the above active 12

compounds also exhibited high anti-HIV potency in the cellular assay. In addition, when the

hydroxyl group on the A-ring was replaced with a chloro- or hydrogen, the resultant compounds 16

(R’ = Cl) and 17 (R’ = H) showed less potency in both molecular and cellular assays than 12a,

suggesting that the hydroxyl group on the A-ring might be better than others for anti-HIV activity.

Finally, the isostere replacement of the rhodanine ring (C ring) with pyrrolin-2,4-dione or

thiazolin-2,4-dione moiety resulted in compounds 19 and 23 with much less anti-HIV activities in

both assays (data not shown) than 12a and 13. These results supported our previous results25

suggesting that the rhodanine ring was preferred to another five-membered ring as the C-ring moiety.

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Notably, the cytotoxicity, based on the CC50 value, of 12k is about 4-fold higher than that of

12a. However, the anti-HIV-1 potency, based on the IC50 value, of 12k is about 11-fold higher than

that of 12a. Therefore, the selectivity index of 12k (SI = 128) is much higher than that of 12a (SI =

50)(Table 1). We will select 12k as a lead for further structure optimization, in order to identify a

drug candidate with reduced cytotoxicity and increased anti-HIV-1 potency for future pre-clinical

study.

Table 1. Inhibitory activities against HIV-1 gp41 6-helix bundle (6-HB) formation and HIV-1

replication a

R'

HOOC

NS

N

S

O

nR

gp41 6-HB MT-2 cell line

# R’ n R IC50b (µM) EC50

c (µM) CC50d (µM) SIe

11 OH 0 H NA 35.6 ± 4.7 >100 >2.8

12a OH 1 H 11.9 ± 0.5 3.2 ± 0.1 158.7 ± 0.0 50.2

13 OH 2 H 12.5 ± 0.0 6.2 ± 0.6 26.0 ± 2.9 4.2

12b OH 1 p-CF3 18.8 ± 0.4 17.2 ± 1.8 45.9 ± 2.2 2.7

12c OH 1 p-Br 10.3 ± 0.8 42.7 ± 7.0 94.4 ± 6.4 2.2

12d OH 1 p-OCH3 0.9 ± 0.0 19.8 ± 3.5 75.3 ± 6.9 3.8

12e OH 1 p-CO2CH3 1.8 ± 1.1 1.5 ± 0.3 36.9 ± 3.4 24.6

12f OH 1 p-CO2H 68.0 ± 4.4 7.1 ± 2.4 105.1 ± 10.3 14.7

12g OH 1 p-t-Bu 2.6 ± 0.0 0.9 ± 0.1 55.2 ± 6.3 61.3

12h OH 1 m-CF3 9.2 ± 0.6 3.9 ± 0.4 26.3 ± 4.1 6.7

12i OH 1 m-Br 7.8 ± 0.3 32.4 ± 1.3 48.2 ± 5.1 1.5

12j OH 1 m-OCH3 2.1 ± 0.2 23.5 ± 3.9 102.3 ± 4.4 4.4

12k OH 1 m-CO2CH3 1.8 ± 0.8 0.3 ± 0.2 38.5 ± 30.2 128

12l OH 1 m-CO2H 20.8 ± 0.2 12.1 ± 1.6 >100 >8.3

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12m OH 1 o-CF3 22.6 ± 2.2 29.1 ± 3.3 28.8 ± 4.3 1

12n OH 1 o-Br ND 41.1 ± 7.7 25.7 ± 1.2 0.6

16 Cl 1 H 34.3 ± 4.7 19.1 ± 2.3 68.3 ± 2.7 3.6

17 H 1 H 45.7 ± 8.6 15.3 ± 2.8 83.4 ± 11.5 5.5

NB-2 37.4 0.7 133.4 191

NB-64 58.7 2.4 335.7 140

a Compounds were tested in triplicate, and the data were presented as the mean ± SD; b Inhibiting

gp41 6-helix bundle formation ; c Inhibiting p24 production in the MT-2 cells; d Cytotoxicity; e SI: selectivity index (CC50/EC50); f

Not determined.

Figure 2 Docking small-molecule inhibitors 2 and 12k with gp41 (PDB: 1aik). (a): surface

representation of the gp41 core with lead 2; (b): surface representation of the gp41 binding site with

active compound 12k. (c): stereoview of 12k docked to gp41 with amino acids on the surface of

binding pocket. The hydrogen bond and cation-� interaction was shown in black dashed line and

yellow line, respectively. Compound 12k was shown by stick model with green color, and all

residues were shown by stick model with gray color, respectively.

To further assess the potential of these active compounds as leads for further development,

we tested the inhibitory activity of the most active compounds 12e, 12g, and 12k against HIV-1

strains resistant to T20 (enfuvirtide), the first HIV-1 fusion inhibitor drug for clinical use, and T20

was included as a control. As shown in Table 2, T20 was effective against the T20-senstive strain

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NL4-3 (36G), but was more than 20-fold less effective against the T20-resistant strains with

mutations in the GIV motif (residues 36 - 44) at the N-terminal region of the gp41 NHR domain32.

Interestingly, the three most active compounds 12k, 12e, and 12g are almost equally effective against

both T20-sensitive and resistant strains. These results suggest that these small molecule HIV-1 fusion

inhibitors mainly target the pocket in the C-terminal region of gp41 NHR domain, rather than the

GIV motif in the N-terminal region of gp41 NHR domain that is targeted by T2032. Therefore, these

compounds can be used as leads for development of small molecule HIV-1 fusion inhibitors for

treatment of HIV-1-infected patients refractory to current T20 therapy.

Table 2. Inhibitory activity (IC50, µM) of compounds on infection by T20-sensitive and resistant

HIV-1 strains

HIV-1 strains 12k 12e 12g T20

aNL4-3 (36G) N42S 2.222 ± 1.276 2.007 ± 0.034 0.473 ± 0.011 0.077 ± 0.014

bNL4-3 (36G) V38A/N42D 0.725 ± 0.237 2.204 ± 0.390 2.024 ± 0.075 1.542 ± 0.101

bNL4-3 (36G) V38A 1.743 ± 1.164 4.852 ± 5.702 4.252 ± 0.285 >2

bNL4-3 (36G) N42T/N43K 2.017 ± 1.427 2.300 ± 0.042 1.963 ± 0.037 >2

bNL4-3 (36G) V38E/N42S 3.367 ± 0.651 2.746 ± 1.429 2.670 ± 0.117 >2

bNL4-3 (36G) V38A/N42T 2.824 ± 2.009 1.674 ± 0.013 1.776 ± 0.197 1.659 ± 0.088

aT20-sensitive strain. bT20-resistant strains. The experiments were performed in triplicate and the

data were presented in mean ± SD.

To gain insight into small-molecule inhibitor interaction with gp41, the most active new

compound 12k was docked into the gp41 hydrophobic pocket (PDB: 1aik) by using Accelrys

Discovery Studio 3.0 software. As shown in Figure 2, compound 12k, which was elongated from

lead 2, occupied more space in the hydrophobic pocket of gp41 and fit well with complementary

shape. Similar to 2, the salicylic acid moiety (A-ring) is deeply anchored into the groove between

Q577 and W571 side chains, and the carboxy group on the A-ring forms an H-bond with amino acid

G572 on the backbone of NHR. The characteristic T-shaped conformation between the salicylic

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phenyl and pyrrole rings is maintained; thus, the 2,5-dimethylpyrrole is inserted into the hydrophobic

cavity, which was supposed to be occupied by residue W631 of CHR upon formation of 6-HB. The

newly introduced rhodanine moiety (C-ring) is located near the key amino acid K574 with a positive

charge. In addition, the presence of a one-carbon linker between the C-ring and D-ring increased

molecular flexibility, allowing the C and D rings to adjust binding conformation around key residue

K574 and produce a salt bridge between the thioxo and the side chain of K574 and the cation-�

interaction between the phenyl ring and K574, suggesting that a flexible linker between C and D

rings might be important to enhance biological activities. Meanwhile, the D-ring part filled a

hydrophobic hollow formed by residues Q567, V570, and W571 from the NHR, and the m-methoxy

carbonyl (m-CO2CH3) group was deeply inserted into this hollow to provide more interactions with

the target cavity. Therefore, this docking model of compound 12k illustrated that the

multi-aromatic-ring scaffold, alone with molecular flexibility, is favorable for increasing binding

affinity by more binding interaction points, especially the key residue K574. These results also

supported the improved inhibitory potency of 12k in both assays.

4. Conclusion

As a continuing structural optimization, a series of (Z)-3-(5-(3-benzyl-4-oxo-2-thioxothi

azolidinylidene)methyl)-N-(3-carboxy-4-hydroxy)phenyl-2,5-dimethylpyrroles and related

derivatives were designed, synthesized, and evaluated in HIV-1 gp41 and cellular assays. Among

them, the most active compounds, 12k, 12e, and 12g with a one-carbon linker between the C and D

rings, exhibited very promising inhibitory potency with IC50 values of 1.8-2.6 µM and EC50 values of

0.3-1.5 µM against gp41 6-HB formation and HIV-1 replication, respectively, as well as an SI range

of 24-128. As such, they are more potent than our prior leads 1-3. The SAR studies revealed that (1)

the 3-carboxy-4-hydroxyphenyl (A-ring) is a favorable moiety, which might play an anchoring role

to maintain the binding orientation of small-molecule inhibitors; (2) a one-atom linker (n = 1)

between C and D rings provided the molecular flexibility to fit the gp41 binding pocket; (3)

substituents on the D-ring greatly affect anti-HIV potency, and an ester group at the p- or m-position

is preferable to others; (4) the p- and m-position on the D-ring were modifiable, whereas the

ortho-position was not; (5) the rhodanine ring was a preferable moiety compared with other hetero

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five-membered rings. The molecular docking model of active compound 12k illustrated the ability of

this multi-aromatic-ring scaffold to fill the linear binding cavity with shape complementation and

interact with the key residue K574. These results show promise for the further development of

nonpeptide small-molecule fusion inhibitors targeting the gp41subunit.

5. Experimental section

5.1. Chemistry

The proton nuclear magnetic resonance (1H NMR) spectra were measured on a JNM-ECA-400

(400MHz) spectrometer using tetramethylsilane (TMS) as internal standard. The solvent used was

DMSO-d6, unless otherwise indicated. Mass spectra (MS) were measured on an ABI Perkin-Elmer

Sciex API-150 mass spectrometer with electrospray ionization, and the relative intensity of each ion

peak is presented as a percent (%). Melting points were measured with an SGW X-4 Micro-Melting

apparatus without correction. The microwave reactions were performed on a microwave reactor from

Biotage, Inc. Thin-layer chromatography (TLC) on silica gel GF254 plates was used to monitor

reactions. Medium-pressure column chromatography was performed using a CombiFlash Companion

purification system from ISCO, Inc. All chemicals and solvents were obtained from Beijing

Chemical Works or Sigma-Aldrich, Inc. Purities of all target compounds were determined by an

Agilent 1200 HPLC system with an Eclipse XDB-C18 column, UV detector at 254 nm, mobile

MeOH/H2O/HCO2H (95/5/0.001) and flow rate of 1 mL/min.

5.1.1. N-(3-Carboxy-4-hydroxy)phenyl-2,5-dimethyl-3-formylpyrrole (4). Anhydrous DMF (20

mL) was placed in a dried 3-neck flask (250-mL) equipped with a thermometer. Then 7 mL of POCl3

(74.1 mmol) was added dropwise into the above flask with stirring and controlled below 10 °C in an ice

bath. After another 30 min stirring to ensure complete conversion to the Vilsmeier reagent, a solution

of N-(3-carboxy-4-hydroxy)phenyl-2,5-dimethylpyrrole (2) (10.75 g, 50 mmol) in 40 mL of DMF was

added slowly for 30 min, keeping the temperature below 10 °C and then stirring for another 2 h at room

temperature. After quenching with ice, the solution was adjusted with 5% NaOH (aq) to pH 9-10 and

rapidly heated to reflux for 30 min. The mixture was slowly poured into ice water with stirring and left

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to set overnight. The precipitated solid was filtered out, washed with water to neutral, and dried to

afford 10.7 g of 4, gray solid, 88% yield. mp 190 °C (dec.); 1H NMR (CDCl3) � ppm 11.18 (1H, br s,

COOH), 9.81 (1H, s, CHO), 7.92 (1H, d, J = 2.4 Hz, ArH-6), 7.30 (1H, dd, J = 2.4 & 8.8 Hz, ArH-4),

7.14 (1H, d, J = 8.8 Hz, ArH-3), 6.42 (1H, s, PyH), 2.31 & 2.00 (each 3H, s, CH3 × 2); MS m/z (%) 258

(M – 1, 100).

5.1.2. N-Phenyl-2-thioxothiazolidin-4-one (8). A suspension of aniline (465 mg, 5 mmol) in water

(10 mL) was heated to 95 °C until the aniline was fully dissolved. Then

bis(carboxylmethyl)trithiocarbonate (1.25 g, 5.5 mmol) was added and heated at 100 °C for 19 h.

After cooling to room temperature, the precipitated solid was filtered out and washed with water.

The dried solid was purified by a flash silica column (gradual eluent: EtOAc/petroleum ether, 0 ~

50%) to afford 730 mg of 8, pale-yellow solid, 70% yield. mp 184-186 °C; 1H NMR (CDCl3) � ppm

7.57~7.19 (5H, m, ArH × 5), 4.20 (2H, s, SCH2); MS m/z (%) 208 (M 1, 100).

5.1.3. General procedure for preparation of 3-substituted 2-thioxothiazolidin-4-one

intermediates (9 and 10). To a solution of substituted benzylamine (6) or phenylethylamine (7) (1.0

eq.) in ether was added CS2 (1.0 eq. in ether) dropwise in an ice bath with stirring for an additional 1 h.

The precipitated solid was filtered, washed with ether, and dried at room temperature. The collected

ammonium dithiocarbamate was then added into the solution of bromoacetic acid (0.5 eq.) in ethanol,

and the mixture was heated to reflux for 3-5 h. After cooling to room temperature, the mixture was

poured into ice water. The 3-substituted rhodanine product (9 or 10) was either filtered out or

extracted with EtOAc and purified by flash silica column (eluent: EtOAc/petroleum ether, 0 ~ 40%).

5.1.3.1. 3-Benzyl-2-thioxothiazolidin-4-one (9a). Starting from benzylamine 6a (5 mL, 45.8 mmol)

to afford 4.18 g of 9a, light yellow solid, 41% yield. mp 72-74 °C; 1H NMR (CDCl3) � ppm 7.22 ~

7.39 (5H, m, ArH), 4.59 (2H, s, NCH2Ar), 4.00 (2H, s, SCH2); MS m/z (%) 224 (M + 1, 50), 91

(PhCH2, 100).

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5.1.3.2. 3-(4-(Trifluoromethyl)benzyl)-2-thioxothiazolidin-4-one (9b). Starting from

4-trifluoromethyl benzylamine 6b (1.75 g, 10 mmol) to afford 1.02 g of 9b, white solid, 35% yield.

mp 102-104 °C; 1H NMR (CDCl3) � ppm 7.53 ~ 7.59 (4H, m, ArH), 5.23 (2H, s, ArCH2N), 4.03 (2H,

s, SCH2); MS m/z (%) 290 (M – 1, 100).

5.1.3.3. 3-(4-Bromobenzyl)-2-thioxothiazolidin-4-one (9c). Starting from 4-bromobenzylamine 6c

(1.86 g, 10 mmol) to afford 745 mg of 9c, light yellow solid, 25% yield. mp 84-86 °C; 1H NMR

(CDCl3) � ppm 7.45 (2H, d, J = 8.4 Hz, ArH-3, 5), 7.34 (2H, d, J = 8.4 Hz, ArH-2, 6), 5.13 (2H, s,

ArCH2N), 4.00 (2H, s, SCH2); MS m/z (%) 169 (M – 132, p-BrBn, 100), 171 (169 + 2, 95).

5.1.3.4. 3-(4-Methoxybenzyl)-2-thioxothiazolidin-4-one (9d). Starting from

4-methoxybenzylamine 6d (1.37 g, 10 mmol) to afford 895 mg of 9d, white solid, 35% yield. mp

90-92 °C; 1H NMR (CDCl3) � ppm 7.43 (2H, d, J = 8.4 Hz, ArH-2, 6), 6.84 (2H, d, J = 8.4 Hz, ArH-3,

5), 5.12 (2H, s, ArCH2N), 3.96 (2H, s, SCH2), 3.78 (3H, s, OCH3); MS m/z (%) 254 (M + 1, 100).

5.1.3.5. 3-(4-(Methoxycarbonyl)benzyl)-2-thioxothiazolidin-4-one (9e). Starting from

4-methoxycarbonyl benzylamine 6e (3.0 g, 18 mmol) to afford 1.34 g of 9e, light yellow solid, 26%

yield. mp 154-156 °C; 1H NMR (CDCl3) � ppm 7.99 (2H, d, J = 8.4 Hz, ArH-3, 5), 7.48 (2H, d, J = 8.4

Hz, ArH-2, 6), 5.23 (2H, s, ArCH2N), 4.03 (2H, s, SCH2), 3.91 (3H, s, OCH3); MS m/z (%) 282 (M +

1, 100).

5.1.3.6. 3-(4-tert-Butylbenzyl)-2-thioxothiazolidin-4-one (9g). Starting from 4-chlorobenzylamine

6g (1.63 g, 10 mmol) to afford 1.07 g of 9g, light yellow oil, 41% yield. mp 100-102 °C; 1H NMR

(CDCl3) � ppm 7.40 (2H, d, J = 8.4 Hz, ArH-3, 5), 7.34 (2H, d, J = 8.4 Hz, ArH-2, 6), 5.15 (2H, s,

NCH2Ar), 3.97 (2H, s, SCH2), 1.30 (9H, s, CH3 × 3); MS m/z (%) 280 (M + 1, 40), 297 (M + 18, 100).

5.1.3.7. 3-(3-(Trifluoromethyl)benzyl)-2-thioxothiazolidin-4-one (9h). Starting from

3-trifluoromethyl benzylamine 6h (1.75 g, 10 mmol) to afford 1.02 g of 9h, light yellow solid, 35%

yield. mp 68-70 °C; 1H NMR (CDCl3) � ppm 7.70 (1H, s, ArH-2), 7.64 (1H, d, J = 8.0 Hz, ArH-4),

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7.57 (1H, d, J = 8.0 Hz, ArH-6), 7.44 (1H, t, J = 8.0 Hz, ArH-5), 5.22 (2H, s, NCH2Ar), 4.03 (s, 2H,

SCH2); MS m/z (%) 290 (M 1, 100).

5.1.3.8. 3-(3-Bromobenzyl)-2-thioxothiazolidin-4-one (9i). Starting from 3-bromobenzylamine 6i

(1.86 g, 10 mmol) to afford 890 mg of 9i, white solid, 30% yield. mp 87-89 °C; 1H NMR (CDCl3) �

ppm 7.57 (1H, s, ArH-2), 7.44 (1H, d, J = 7.6 Hz, ArH-4), 7.38 (1H, d, J = 7.6 Hz, ArH-6), 7.19 (1H, t,

J = 7.6 Hz, ArH-5), 5.14 (2H, s, ArCH2N), 4.02 (2H, s, SCH2); MS m/z (%) 169 (M 132, BrBn,

100), 171 (169 + 2, 85).

5.1.3.9. 3-(3-Methoxybenzyl)-2-thioxothiazolidin-4-one (9j). Starting from 3-methoxybenzylamine

6j (1.37 g, 10 mmol) to afford 1 g of 9j, white solid, 39% yield. mp 78-80 °C; 1H NMR (CDCl3) �

ppm 7.23 (1H, t, J = 8.0 Hz, ArH-5), 6.99 ~7.02 (2H, m, ArH), 6.84 (1H, dd, J = 2.4 & 8.0 Hz, ArH-4),

5.16 (2H, s, ArCH2N), 3.99 (2H, s, SCH2), 3.79 (3H, s, OCH3); MS m/z (%) 254 (M + 1, 60), 276 (M

+ 23, 100).

5.1.3.10. 3-(3-(Methoxycarbonyl)benzyl)-2-thioxothiazolidin-4-one (9k). Starting from

3-methoxycarbonyl benzylamine 6k (1.0 g, 6.05 mmol) to afford 360 mg of 9k, light yellow solid,

21% yield. mp 126-128 °C; 1H NMR (CDCl3) � ppm 8.08 (1H, s, ArH-2), 7.98 (1H, d, J = 8.0 Hz,

ArH-4), 7.63 (1H, d, J = 8.0 Hz, ArH-6), 7.40 (1H, t, J = 8.0 Hz, ArH-5), 5.23 (2H, s, ArCH2N), 4.04

(2H, s, SCH2), 3.92 (3H, s, OCH3); MS m/z (%) 282 (M + 1, 80).

5.1.3.11. 3-(2-(Trifluoromethyl)benzyl)-2-thioxothiazolidin-4-one (9m). Starting from

2-trifluoromethyl benzylamine 6m (1.75 g 10 mmol) to afford 970 mg of 9m, white solid, 33%

yield. mp 119-122 °C; 1H NMR (CDCl3) � ppm 7.91 (1H, d, J = 7.6 Hz, ArH-3), 7.46 (1H, t, J = 7.6

Hz, ArH-5), 7.38 (1H, t, J = 7.6 Hz, ArH-4), 6.91 (1H, d, J = 7.6 Hz, ArH-6), 5.41 (2H, s, ArCH2N),

4.14 (2H, s, SCH2); MS m/z (%) 292 (M + 1, 60).

5.1.3.12. 3-(2-Bromobenzyl)-2-thioxothiazolidin-4-one (9n). Starting from 2-bromobenzylamine

6n (930 mg, 5 mmol) to afford 590 mg of 9n, white solid, 39% yield. mp 86-88 °C; 1H NMR (CDCl3)

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� ppm 7.59 (1H, d, J = 8.0 Hz, ArH-3), 7.24 (1H, t, J = 8.0 Hz, ArH-5), 7.14 (1H, t, J = 8.0 Hz, ArH-4),

6.86 (1H, d, J = 8.0 Hz, ArH-6), 5.26 (2H, s, ArCH2N), 4.11 (2H, s, SCH2); MS m/z (%) 169 (M

132, BrBn, 100), 171 (169 + 2, 100).

5.1.3.13. 3-Phenethyl-2-thioxothiazolidin-4-one (10). Starting from 2-phenyl ethylamine 7 (1 mL,

8 mmol) to afford 700 mg of 10, light yellow solid, 37% yield. mp 86-88 °C; 1H NMR (CDCl3) �

ppm 7.22 ~ 7.35 (5H, m, ArH), 4.22 (2H, t, J = 8.0 Hz, NCH2), 3.93 (2H, s, SCH2), 2.93 (2H, t, J =

8.0 Hz, CH2Ar); MS m/z (%) 238 (M + 1, 100).

5.1.4. General procedure for Knoevenagel condensation reaction to prepare target compounds

11-13, 16, and 17. A mixture of an aldehyde compound (4, 15, or 16) and a rhodanine derivative (8, 9

or 10, mole ratio 1:1) in the presence of NH4OAc (100 mg) in toluene (20 mL/mmol) and methanol (10

mL/mmol) was heated to reflux for 3-5 h. After the reaction was finished, ethyl acetate was added to

the solution, and organic phase was washed with water and brine, successively, and then dried over

Na2SO4. After removal of solvent under reduced pressure, the residue was purified by flash silica

column (gradual eluent: EtOAc/petroleum ether with AcOH (3%), 0 ~ 60%) to give the pure product.

5.1.4.1. (Z)-N-(3-Carboxy-4-hydroxy)phenyl-2,5-dimethyl-3-(5-(4-oxo-3-phenyl-2-thioxothiazol

idinylidene)methyl)pyrrole (11). Starting from 4 (50 mg, 0.19 mmol) and 8 (41 mg, 0.19 mmol) to

afford 60 mg of 11, yellow solid, 69% yield. mp 200 °C (dec.); 1H NMR � ppm 7.71 (1H, s, =CH),

7.67 (1H, d, J = 2.8 Hz, ArH-6), 7.49 ~ 7.59 (4H, m, ArH), 7.34 ~ 7.39 (2H, m, ArH), 7.13 (1H, d, J =

8.8 Hz, ArH-3), 6.31 (1H, s, PyH), 2.17 & 2.02 (each 3H, s, CH3 × 2); MS m/z 449 (M – 1, 30), 405 (M

– 44, 100); HPLC purity 98.62%.

5.1.4.2. (Z)-3-(5-(3-Benzyl-4-oxo-2-thioxothiazolidinylidene)methyl)-N-(3-carboxy-4-hydroxy)

phenyl-2,5-dimethylpyrrole (12a). Starting from 4 (130 mg, 0.5 mmol) and 9a (112 mg, 0.5 mmol)

to afford 162 mg of 12a, yellow solid, 69% yield. mp 226-228 °C (dec.); 1H NMR � ppm 7.74 (1H, s,

=CH), 7.66 (1H, d, J = 2.4 Hz, ArH-6), 7.32 ~ 7.52 (6H, m, ArH-4, ArH on D-ring), 7.14 (1H, d, J =

8.8 Hz, ArH-3), 6.22 (1H, s, PyH), 4.69 (2H, s, ArCH2N), 2.16 & 1.98 (each 3H, s, CH3); MS m/z (%)

463 (M – 1, 100); HPLC purity 96.49%.

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5.1.4.3. (Z)-N-(3-Carboxy-4-hydroxy)phenyl-2,5-dimethyl-3-(5-(3-(4-trifluoromethylbenzyl)-4-

oxo-2-thioxothiazolidinylidene)methyl)pyrrole (12b). Starting from 4 (130 mg, 0.5 mmol) and 9b

(145 mg, 0.5 mmol) to afford 188 mg of 12b, yellow solid, 71% yield. mp 144-146 °C; 1H NMR �

ppm 7.71 ~ 7.75 (3H, m, =CH, ArH-3’, 5’), 7.66 (1H, d, J = 2.8 Hz, ArH-6), 7.50 ~ 7.53 (3H, m, ArH),

7.14 (1H, d, J = 8.8 Hz, ArH-3), 6.27 (1H, s, PyH), 5.32 (2H, s, ArCH2N), 2.17 & 2.00 (each 3H, s,

CH3); MS m/z (%) 531 (M – 1, 100); HPLC purity 95.62%.

5.1.4.4. (Z)-3-(5-(3-(4-Bromobenzyl)-4-oxo-2-thioxothiazolidinylidene)methyl)-N-(3-carboxy-4-

hydroxy)phenyl-2,5-dimethylpyrrole (12c). Starting from 4 (100 mg, 0.39 mmol) and 9c (117 mg,

0.39 mmol) to afford 130 mg of 12c, yellow solid, 55% yield. mp 252-254 °C; 1H NMR � ppm 7.73

(1H, s, =CH), 7.67 (1H, d, J = 2.8 Hz, ArH-6), 7.55 (2H, d, J = 8.4 Hz, ArH-3’, 5’), 7.52 (1H, dd, J =

2.8 & 8.8 Hz, ArH-4), 7.29 (2H, d, J = 8.4 Hz, ArH-2’, 6’), 7.14 (1H, d, J = 8.8 Hz, ArH-3), 6.26 (1H,

s, PyH), 5.20 (2H, s, NCH2Ar), 2.16 & 2.00 (each 3H, s, CH3); MS m/z (%) 541 (M – 1, 80), 543 (M +

1, 100); HPLC purity 98.14%.

5.1.4.5. (Z)-N-(3-Carboxy-4-hydroxy)phenyl-2,5-dimethyl-(3-(5-(3-(4-methoxybenzyl)-4-oxo-2-

thioxothiazolidinylidene)methyl)pyrrole (12d). Starting from 4 (130 mg, 0.5 mmol) and 9d (127

mg, 0.5 mmol) to afford186 mg of 12d, yellow solid, 75% yield. mp 248-250 °C (dec.); 1H NMR �

ppm 7.72 (1H, s, =CH), 7.66 (1H, d, J = 2.8 Hz, ArH-6), 7.50 (1H, dd, J = 2.8 & 8.8 Hz, ArH-4), 7.31

(2H, d, J = 8.4 Hz, ArH-2’, 6’), 7.13 (1H, s, J = 8.8 Hz, ArH-3), 6.90 (2H, d, J = 8.4 Hz, ArH-3’, 5’),

6.24 (1H, s, PyH-4), 5.16 (2H, s, ArCH2N), 3.72 (3H, s, OCH3), 2.16 & 1.99 (each 3H, s, CH3 × 2); MS

m/z (%) 493.2 (M – 1, 100); HPLC purity 96.58%.

5.1.4.6. (Z)-N-(3-Carboxy-4-hydroxy)phenyl-2,5-dimethyl-(3-(5-

(3-(4-(methoxycarbonyl)benzyl) -4-oxo-2-thioxothiazolidinylidene)methyl)pyrrole (12e).

Starting from 4 (130 mg, 0.5 mmol) and 9e (140 mg, 0.5 mmol) to afford 204 mg of 12e, yellow solid,

78% yield. mp 158-160 °C; 1H NMR � ppm 7.95 (2H, d, J = 8.4 Hz, ArH-3’, 5’), 7.75 (1H, s, =CH),

7.66 (1H, d, J = 2.8 Hz, ArH-6), 7.48 (1H, dd, J = 2.8 & 8.4 Hz, ArH-4), 7.44 (2H, d, J = 8.4 Hz,

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ArH-2’, 6’), 7.12 (1H, d, J = 8.4 Hz, ArH-3), 6.27 (1H, s, PyH), 5.31 (2H, s, ArCH2N), 3.84 (2H, s,

OCH3), 2.17 & 2.00 (each 3H, s, CH3 × 2); MS m/z (%) 522 (M – 1, 100); HPLC purity 95.59%.

5.1.4.7. (Z)-3-(5-(3-(4-Carboxybenzyl)-4-oxo-2-thioxothiazolidinylidene)methyl)-N-(3-carboxy-

4-hydroxy)phenyl-2,5-dimethylpyrrole (12f). To a mixture of the methyl ester of 12e (100 mg, 0.19

mmol) in 10 mL glacial acetic acid was added 2 mL 36% HCl under nitrogen atmosphere, and the

mixture was heated to reflux for 24 h. After cooling to room temperature, the solution was poured into

40 mL ice water, and the precipitate was filtered out and purified by flash silica column [gradual

eluent: EtOAc/petroleum ether with AcOH (3%), 0 ~ 50%] to afford 67 mg of 12f, yellow solid, 65%

yield. mp 298-300 °C (dec.); 1H NMR � ppm 7.92 (2H, d, J = 7.6 Hz, ArH-2’, 6’), 7.75 (1H, s, =CH),

7.66 (1H, d, J = 2.8 Hz, ArH-6), 7.51 (1H, dd, J = 2.8 & 8.8 Hz, ArH-4), 7.41 (2H, d, J = 7.6 Hz,

ArH-3’, 5’), 7.14 (d, 1H, J = 8.8 Hz, ArH-3), 6.27 (1H, s, PyH), 5.30 (2H, s, ArCH2N), 2.17 & 2.00

(each 3H, s, CH3 × 2); MS m/z (%) 507 (M – 1, 100); HPLC purity 96.04%.

5.1.4.8. (Z)-3-(5-(3-(4-tert-Butylbenzyl)-4-oxo-2-thioxothiazolidinylidene)methyl)-N-(3-carboxy

-4-hydroxy)phenyl-2,5-dimethylpyrrole (12g). Starting from 4 (50 mg, 0.19 mmol) and 9g (54 mg,

0.19 mmol) to afford 67 mg of 12g, yellow solid, 67% yield. mp 140-142 °C; 1H NMR � ppm 7.72

(1H, s, =CH), 7.67 (1H, d, J = 2.8 Hz, ArH-6), 7.50 (1H, dd, J = 2.8 & 8.8 Hz, ArH-4), 7.37 (2H, d, J =

8.0 Hz, ArH-2’, 6’), 7.27 (2H, d, J = 8.0 Hz, ArH-3’, 5’), 7.15 (1H, d, J = 8.8 Hz, ArH-3), 6.26 (1H, s,

PyH-4), 5.20 (2H, s, ArCH2N), 2.16 & 1.99 (each 3H, s, CH3 × 2), 1.25 (9H, s, t-Bu); MS m/z (%) 519

(M – 1, 100); HPLC purity 95.37%.

5.1.4.9. (Z)-N-(3-Carboxy-4-hydroxy)phenyl-2,5-dimethyl-3-(5-(4-oxo-2-thioxo-3-(3-trifluoro

methylbenzyl)thiazolidinylidene)methyl)pyrrole (12h). Starting from 4 (130 mg, 0.5 mmol) and 9h

(145 mg, 0.5 mmol) to afford 100 mg of 12h, yellow solid, 38% yield. mp 124-126 °C; 1H NMR �

ppm 7.75 (1H, s, =CH), 7.59 ~ 7.71 (5H, m, ArH-6, ArH on D-ring), 7.50 (1H, dd, J = 2.8 & 8.8 Hz,

ArH-4), 7.13 (1H, d, J = 8.8 Hz, ArH-3), 6.26 (1H, s, PyH), 5.32 (2H, s, ArCH2N), 2.17 & 2.00 (each

3H, s, CH3 × 2); MS m/z (%) 531 (M – 1, 100), 487 (M – 44, 50); HPLC purity 96.02%.

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5.1.4.10. (Z)-3-(5-(3-(3-Bromobenzyl)-4-oxo-2-thioxothiazolidinylidene)methyl)-N-(3-carboxy

-4-hydroxy)phenyl-2,5-dimethylpyrrole (12i). Starting from 4 (100 mg, 0.39 mmol) and 9i (117 mg,

0.39 mmol) to afford 130 mg of 12i, red solid, 62% yield. mp 208-210 °C; 1H NMR � ppm 7.74 (1H,

s, =CH), 7.67 (1H, d, J = 2.8 Hz, ArH-6), 7.48 ~ 7.53 (3H, m, ArH), 7.30 ~ 7.32 (2H, m, ArH), 7.14

(1H, d, J = 8.8 Hz, ArH-3), 6.26 (1H, s, PyH), 5.22 (2H, s, ArCH2N), 2.17 & 2.00 (each 3H, s, CH3 ×

2); MS m/z (%) 543 (M + 1, 100), 541 (M – 1, 80); HPLC purity 99.13%.

5.1.4.11. (Z)-N-(3-Carboxy-4-hydroxy)phenyl-2,5-dimethyl-3-(5-(3-(3-methoxybenzyl)-4-oxo-2-

thioxothiazolidinylidene)methyl)pyrrole (12j). Starting from 4 (130 mg, 0.5 mmol) and 9j (127 mg,

0.5 mmol) to afford 163 mg of 12j, 66% yield. mp 244-246 °C; 1H NMR � ppm 7.73 (1H, s, =CH),

7.62 (1H, d, J = 2.4 Hz, ArH-6), 7.52 (1H, dd, J = 2.4 & 8.4 Hz, ArH-4), 7.25 (1H, t, J = 8.0 Hz,

ArH-5’), 7.14 (1H, d, J = 8.4 Hz, ArH-3), 6.84 ~ 6.88 (3H, m, ArH on D-ring), 6.26 (1H, s, PyH), 5.20

(2H, s, ArCH2N), 3.72 (3H, s, OCH3), 2.16 & 2.00 (each 3H, s, CH3 × 2); MS m/z (%) 493 (M – 1,

100); HPLC purity 96.28%.

5.1.4.12. (Z)-N-(3-Carboxy-4-hydroxy)phenyl-2,5-dimethyl-3-(5-(3-(3-(methoxycarbonyl)

benzyl)-4-oxo-2-thioxothiazolidinylidene)methyl)pyrrole (12k). Starting from 4 (100 mg, 0.39

mmol) and 9k (110 mg, 0.39 mmol) to afford 189 mg of 12k, yellow solid, 93% yield. mp 214-216

°C; 1H NMR � ppm 7.92 (1H, s, ArH-2’), 7.89 (1H, d, J = 7.6 Hz, ArH-4’), 7.75 (1H, s, =CH), 7.67

(1H, d, J = 2.4 Hz, ArH-6), 7.63 (1H, d, J = 7.6 Hz, ArH-6’), 7.50 ~ 7.54 (2H, m, ArH-4, ArH-5’), 7.15

(1H, d, J = 8.4 Hz, ArH-3), 6.27 (1H, s, PyH-4), 5.30 (2H, s, ArCH2N), 3.85 (3H, s, OCH3), 2.17 &

2.00 (each 3H, s, CH3 × 2); MS m/z (%) 521 (M – 1, 100); HPLC purity 96.08%.

5.1.4.13. (Z)-3-((3-(3-Carboxybenzyl)-4-oxo-2-thioxothiazolidinylidene)methyl)-N-(3-carboxy-4

-hydroxy)phenyl-2,5-dimethylpyrrole (12l). The preparation was the same as 12f. 12k (100 mg,

0.19 mmol) was hydrolyzed to afford 70 mg of 12l, yellow solid, 72% yield. mp 292-294 °C (dec.);

1H NMR � ppm 7.89 (1H, s, ArH-2’), 7.87 (1H, d, J = 7.6 Hz, ArH-4’), 7.76 (1H, s, =CH), 7.68 (1H, d,

J = 2.8 Hz, ArH-6), 7.61 (1H, d, J = 7.6 Hz, ArH-6’), 7.47 ~ 7.53 (2H, m, ArH-4, ArH-5’), 7.15 (1H, d,

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J = 8.4 Hz, ArH-3), 6.27 (1H, s, PyH), 5.30 (2H, s, ArCH2N), 2.17 & 2.00 (each 3H, s, CH3 × 2); MS

m/z (%) 507 (M – 1, 80), 463 (M- – 44, 100); HPLC purity 98.95%.

5.1.4.14.

(Z)-N-(3-Carboxy-4-hydroxy)phenyl-2,5-dimethyl-(3-(5-(3-(2-(Trifluoromethyl)benzyl)

-4-oxo-2-thioxothiazolidinylidene)methyl)pyrrole (12m). Starting from 4 (100 mg, 0.39 mmol) and

9m (112 mg, 0.39 mmol) to afford 138 mg of 12m, yellow solid, 67% yield. mp 250-252 °C; 1H NMR

� ppm 7.81 (1H, d, J = 8.0 Hz, ArH-4’), 7.76 (1H, s, =CH), 7.68 (1H, d, J = 2.4 Hz, ArH-6), 7.62 (1H,

t, J = 8.0 Hz, ArH-5’), 7.49 ~ 7.53 (2H, m, ArH-4, ArH-5’), 7.15 (1H, d, J = 8.8 Hz, ArH-3), 6.99 (1H,

d, J = 8.0 Hz, ArH-6’), 6.31 (1H, s, PyH), 5.39 (2H, s, ArCH2N), 2.17 & 2.02 (each 3H, s, CH3 × 2);

MS (%) m/z 531 (M – 1, 100); HPLC purity 98.95%

5.1.4.15. (Z)-3-(5-(3-(2-Bromobenzyl)-4-oxo-2-thioxothiazolidinylidene)methyl)-N-(3-carboxy-

4-hydroxy)phenyl-2,5-dimethylpyrrole (12n). Starting from 4 (50 mg, 0.19 mmol) and 9n (60 mg,

0.19 mmol) to afford 130 mg of 12n, yellow solid, 62% yield. mp 286-288 °C (dec.); 1H NMR � ppm

7.76 (1H, s, =CH), 7.67 ~ 7.70 (2H, m, ArH-6, ArH-2’), 7.53 (1H, dd, J = 2.4 & 8.8 Hz, ArH-4), 7.33

(1H, t, J = 7.6 Hz, ArH-4’), 7.25 (1H, t, J = 7.6 Hz, ArH-5’), 7.15 (1H, d, J = 8.4 Hz, ArH-3), 6.86 (1H,

d, J = 7.6 Hz, ArH-6’), 6.31 (1H, s, PyH-4), 5.22 (2H, s, ArCH2N), 2.18 & 2.01 (each 3H, s, CH3 × 2);

MS m/z (%) 541 (M – 1, 40), 543 (M + 1, 50); 497 (M – 44, 100), 499 (497 + 2, 80); HPLC purity

98.92%.

5.1.4.17. (Z)-N-(3-Carboxy-4-hydroxy)phenyl-2,5-dimethyl-3-(5-(4-oxo-3-phenethyl-2-thioxo

thiazolidinylidene)methyl)pyrrole (13). Starting from 4 (130 mg, 0.5 mmol) and 10 (120 mg, 0.5

mmol) to afford 140 mg of 13, yellow solid, 58% yield. mp 228-230 °C; 1H NMR � ppm 7.66 ~ 7.67

(2H, m, ArH-6, =CH), 7.51 (1H, dd, J = 2.8 & 8.8 Hz, ArH-4), 7.20 ~ 7.31 (5H, m, ArH × 5 on

D-ring), 7.14 (1H, d, J = 8.8 Hz, ArH-3), 6.23 (1H, s, PyH), 4.23 (2H, t, J = 8.0 Hz, CH2N), 2.95(t, J

= 8.0 Hz, ArCH2), 2.15 & 1.99 (each 3H, s, CH3 × 2); MS m/z (%) 477 (M – 1, 100); HPLC purity

96.71%.

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5.1.5. N-(3-Carboxy-4-chloro)phenyl-2,5-dimethyl-3-formylpyrrole (14). The preparation was the

same as 4. Starting with POCl3 (6 mL, 63.5 mmol), DMF (20 mL) and

2-chloro-3-(2,5-dimethyl-1H-pyrrol-1-yl)benzoic acid (10 g, 40 mmol) to afford 8.17 g of 14, gray

solid, 73% yield. mp 186-188 °C (dec.); 1H NMR (CDCl3) � ppm 9.87 (1H, s, CHO), 7.88 (1H, d, J =

2.4 Hz, ArH-6), 7.68 (1H, d, J = 8.4 Hz, ArH-3), 7.35 (1H, dd, J = 2.4 & 8.4 Hz, ArH-4), 6.43 (1H, s,

PyH), 2.32 & 2.02 (each 3H, s, CH3 × 2); MS m/z (%) 276 (M – 1, 100), 278 (M 1, 30).

5.1.6. N-(3-Carboxy)phenyl-2,5-dimethyl-3-formylpyrrole (15). The preparation was the same as

4. Starting with POCl3 (6 mL, 63.5 mmol), DMF (20 mL) and

3-(2,5-dimethyl-1H-pyrrol-1-yl)benzoic acid (9.25 g, 40 mmol) to afford 8.2 g of 15, gray solid, 79%

yield. mp 198-200 °C (dec.); 1H NMR (CDCl3) � ppm 9.89 (1H, s, CHO), 8.27 (1H, d, J = 8.0 Hz,

ArH-6), 7.98 (1H, s, ArH-2), 7.67 (1H, t, J = 8.0 Hz, ArH-5), 7.49 (1H, d, J = 8.0 Hz, ArH-4), 6.43

(1H, s, PyH), 2.31 & 2.01 (each 3H, s, CH3 × 2); MS m/z (%) 242 (M – 1, 100).

5.1.7. (Z)-3-(5-(3-Benzyl-4-oxo-2-thioxothiazolidin-5-ylidene)methyl)-N-(3-carboxy-4-chloro)

phenyl-2,5-dimethylpyrrole (16). Starting from 14 (140 mg, 0.5 mmol) and 9a (112 mg, 0.5 mmol)

to afford 180 mg of 16, red solid, 75% yield. mp 214-216 °C; 1H NMR � ppm 7.74 ~ 7.75 (3H, m,

ArH-3, 6, =CH), 7.58 (1H, dd, J = 2.4 & 8.4 Hz, ArH-4), 7.32 ~ 7.51 (5H, m, ArH × 5 on D-ring),

6.25 (1H, s, PyH), 4.70 (2H, s, ArCH2N), 2.18 & 2.01(each 3H, s, CH3 × 2); MS m/z (%) 481 (M – 1,

30), 483 (M + 1, 10), 437 (M- – 45, 100), 439 (437 + 2, 40); HPLC purity 98.15%.

5.1.7. (Z)-3-(5-(3-Benzyl-4-oxo-2-thioxothiazolidinylidene)methyl)-N-(3-carboxy)phenyl-2,5-

dimethylpyrrole (17). Starting from 15 (97 mg, 0.4 mmol) and 9a (90 mg, 0.4 mmol) to afford 118

mg of 17, 66% yield. mp 220-222 °C (dec.); 1H NMR � ppm 8.10 (1H, d, J = 8.0 Hz, ArH-6), 7.80

(1H, s, ArH-2), 7.76 (1H, s, =CH), 7.72 (1H, t, J = 8.0 Hz, ArH-5), 7.67 (1H, d, J = 8.0 Hz, ArH-4),

7.32 ~ 7.51 (m, 5H, ArH × 5 on D-ring), 6.26 (1H, s, PyH), 4.70 (2H, s, ArCH2N), 2.17 & 2.00 (each

3H, s, CH3 × 2); MS m/z (%) 447 (M – 1, 100); HPLC purity 97.12%.

5.1.9. 3-Benzylthiazolin-2,4-dione (18). To a solution of thiazolin-2,4-dione (500 mg, 4.27 mmol)

and benzyl bromide (800 mg, 4.7 mmol) in 15 mL DMF was added K2CO3 (1.18 g, 8.55 mmol). The

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reaction was stirred for 3 h under room temperature, the solid filtered off, and the filter extracted by

EtOAc. After the removal of the solvent, the residue was purified by flash silica column [gradual

eluent: EtOAc/petroleum ether, 0 ~ 30%] to afford 793 mg of 18, colorless oil, 90% yield. 1H NMR

(CDCl3) � ppm 7.31 ~ 7.39 (5H, m, ArH × 5), 4.77 (2H, s, ArCH2N), 3.94(2H, s, SCH2).

5.1.10. (Z)- 3-(5-(3-Benzyl-2,4-dioxothiazolidinylidene)methyl)-N-(3-carboxy-4-hydroxy)phenyl

-2,5-dimethylpyrrole (19). The preparation was the same as 12. Starting with 9 (50 mg, 0.19 mmol)

and 18 (40 mg, 0.19 mmol) to afford 53 mg of 19, light yellow solid, 61% yield. Amorphous; 1H

NMR � ppm 7.83 (1H, s, =CH), 7.63 (1H, d, J = 2.4 Hz, ArH-6), 7.46 (1H, dd, J = 2.4 & 8.8 Hz,

ArH-4), 7.38 ~ 7.29 (5H, m, ArH × 5 on D-ring), 7.10 (1H, d, J = 8.8 Hz, ArH-3), 6.20 (1H, s, PyH),

4.82 (2H, s, ArCH2N), 2.13 & 1.99 (each 3H, s, CH3 × 2); MS m/z (%) 447 (M – 1, 40), 404 (M – 44,

100); HPLC purity 96.19%.

5.1.11. N-(3-Carbmethoxy-4-hydroxy)phenyl-2,5-dimethyl-3-formyl-pyrrole (20). To a solution

of 4 (260 mg, 1 mmol) and Cs2CO3 (163 mg, 0.5 mmol) in 10 mL DMF was added CH3I (142 mg, 1

mmol). The solution was stirred at room temperature for 24 h and then poured into ice water. The

precipitate was filtered out and dried to afford 206 mg of 20, white needle, 75% yield. mp 136-138 °C;

1H NMR (CDCl3) � ppm 10.96 (1H, s, COOH), 9.87 (1H, s, CHO), 7.71 (1H, d, J = 2.4 Hz, ArH-6),

7.29 (1H, dd, J = 2.4 & 8.8 Hz, ArH-4), 7.13 (1H, d, J = 8.8 Hz, ArH-3), 6.37 (1H, s, PyH), 3.98 (3H,

s, OCH3), 2.18 & 1.99 (each 3H, s, CH3 × 2); MS m/z (%) 274 (M + 1, 40).

5.1.12. 2,5-Dioxo-1-phenethylpyrrolidin-3-yl acetate (21). To a solution of maleic anhydride (0.98

g, 10 mmol) in 15 mL ether, phenylethylamine (1.21 g, 10 mmol) was added dropwise in 15 mL ether,

and the reaction was stirred for 1.5 h at room temperature. Next, the white precipitate was filtered out,

washed with ether, and dried to afford 2.08 g of N-phenylethylmaleamic acid. Then the mixture of

maleamic acid and 100 mg NaOAc in 20 mL acetic anhydride was heated to reflux for 12 h. The

reaction was cooled to room temperature and poured into 100 mL ice water. The precipitate was

collected, washed with water, and dried to afford 1.44 g of 21, white solid, 55% yield. mp 108-110 °C;

1H NMR (CDCl3) � ppm 7.20 ~ 7.31 (5H, m, ArH × 5), 5.36 (1H, dd, J = 4.8 & 8.8 Hz, 3-H), 3.79 (2H,

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t, J = 8.0 Hz, CH2N), 3.12 (1H, dd, J = 8.8 & 14.4 Hz, 4-H), 2.91 (2H, t, J = 8.0 Hz, ArCH2), 2.63 (1H,

dd, J = 4.8 & 8.8 Hz, 4-H), 2.16 (3H, s, COCH3); MS m/z (%) 262 (M + 1, 30).

5.1.13. (E)-N-(3-Carbmethoxy-4-hydroxy)phenyl-2,5-dimethyl-3-(3-(2,5-dioxo-1-phenethyl

pyrrolidinylidene)methyl)pyrrole (22). The mixture of 20 (136 mg, 0.5 mmol) and 21 (260 mg, 1

mmol) in 10 mL anhydrous ethanol in the presence of PPh3 (262 mg, 1 mmol) was heated to reflux

under nitrogen atmosphere for 12 h. After cooling to room temperature, the precipitate was filtered out,

washed with cold ethanol, and dried to afford 195 mg of 22, gray solid, 85% yield. mp 184-186 °C

(dec.); 1H NMR (CDCl3) � ppm 10.94 (1H, s, OH), 7.70 (1H, d, J = 2.8 Hz, ArH-6), 7.59 (1H, t, J = 2.0

Hz, =CH), 7.22 ~ 7.30 (6H, m, ArH-4, ArH × 5), 7.12 (1H, d, J = 8.8 Hz, ArH-3), 6.10 (1H, s, PyH),

3.97 (3H, s, OCH3), 3.85 (2H, t, J = 8.0 Hz, CH2N), 3.45 (2H, d, J = 2.0 Hz, COCH2), 2.95 (2H, t, J =

8.0 Hz, ArCH2), 2.15 & 2.02 (each 3H, s, CH3 × 2); MS m/z 459 (M + 1, 100).

5.1.14. (E)-N-(3-Carboxy-4-hydroxy)phenyl-2,5-dimethyl-3-(3-(2,5-dioxo-1-phenethylpyrroli

dinylidene)methyl)pyrrole (23). Compound 22 (200 mg, 0.44 mmol) in 10 mL glacial acetic acid and

2 mL 36% HCl under nitrogen atmosphere was heated to reflux for 24 h. The solution was then poured

into ice water. The precipitate was filtered out and purified by flash silica column [gradual eluent:

EtOAc/petroleum ether with AcOH (3%), 0 ~ 50%] to afford 60 mg of 23, gray solid, 33% yield.

Amorphous; 1H NMR � ppm 7.60 (1H, d, J = 2.8 Hz, ArH-6), 7.47 (1H, dd, J = 2.8 & 8.8 Hz,

ArH-4), 7.36 (1H, s, =CH), 7.19 ~ 7.31 (5H, m, ArH × 5), 7.13 (1H, d, J = 8.8 Hz, ArH-3), 6.25 (1H,

s, PyH), 3.69 (2H, t, J = 8.0 Hz, CH2N), 3.48 (2H, d, J = 2.0 Hz, COCH2), 2.85 (2H, t, J = 8.0 Hz,

ArCH2), 2.09 & 1.97 (each 3H, s, CH3 × 2); MS m/z (%) 443 (M – 1, 70), 399 (M – 44, 100); HPLC

purity 98.13%.

5.2.1. Sandwich ELISA to detect gp41 6-HB formation

A sandwich ELISA, as previously described,28 was used to test the inhibitory activity of the

compounds on gp41 six-helix bundle formation. Briefly, peptide N36 (2 �M) was preincubated with

the compound at graded concentrations at 37 °C for 30 min, followed by addition of C34 (2 �M). In

the control experiments, N36 was preincubated with C34 or PBS at 37 °C for 30 min in the absence

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of the compounds tested. After incubation at 37 °C for 30 min, the mixture was added to wells of a

96-well polystyrene plate which were precoated with IgG (10 �g/mL) purified from rabbit antisera

directed against the gp41 six-helix bundle. Then, the mAb NC-1 recognizing gp41-6HB specifically,

biotin-labeled goat-anti-mouse IgG (Santa Cruz Biotechnolgy, Santa Cruz, CA), streptavidin-labeled

horseradish peroxidase (SA-HRP, Zymed, San Francisco, CA), and the substrate

3,3’,5,5’-tetramethylbenzidine (TMB, Sigma Chemical Co., St. Louis, MO), were added

sequentially. Absorbance at 450 nm was read in an ELISA reader (Ultra 386, TECAN Research Triangle

Park, NC), and the percentage of inhibition by the compounds was calculated as previously

described28. IC50 values were calculated using the computer program CalcuSyn, kindly provided by

Dr. T. C. Chou (Sloan-Kettering Cancer Center, New York). All the samples were tested in triplicate.

5.2.2. Determination of the inhibitory activity of the compounds on HIV-1 replication

The inhibitory activity of compounds on HIV-1IIIB replication in MT-2 cells was determined as

previously described29, 30. Briefly, 1 × 104 MT-2 cells were infected with an HIV-1IIIB strain or

T20-sensitive and resistant HIV-1 strains (100 TCID50) in 200 �L of RPMI 1640 medium containing

10% PBS in the presence or absence of a test compound at graded concentrations overnight. Next,

the culture supernatants were removed, and fresh media containing no test compounds were added.

On the fourth day post-infection, 100 �L 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 washing with 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

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washes, anti-p24 mAb (183-12H-5C), biotin-labeled anti-mouse IgG1, SA-HRP, and TMB were

added sequentially. Reactions were terminated by addition of 1 N H2SO4. Absorbance at 450 nm was

recorded in an ELISA reader (Ultra 386, TECAN Research, Triangle Park, NC). Recombinant

protein p24 purchased from United States Biological (Swampscott, MA) was included to establish

standard dose-response curves. Each sample was tested in triplicate. The percentage of inhibition of

p24 production was calculated as previously described31. EC50 values were calculated using the

computer program CalcuSyn program.

5.2.3. Assessment of in Vitro Cytotoxicity

The in vitro cytotoxicity of compounds on MT-2 cells was measured by XTT assay.30 Briefly, 100

µL of the test compound at graded concentrations were added to equal volumes of cells (5

105/mL) in wells of 96-well plates. After incubation at 37 for 4 days, 50 µL of XTT solution (1

mg/mL) containing 0.02 µM of phenazine methosulphate (PMS, Sigma) was added. After 4 h, the

absorbance at 450 nm was measured with an ELISA reader. The CC50 (concentration for 50%

cytotoxicity) values were calculated using the CalcuSyn program.

5.3. Molecular docking of 14m onto the hydrophobic pocket

Molecular docking was performed by using Discovery Studio 3.0 (Accelrys Inc.). The default atom

types and parameters supplied with Discovery Studio 3.0 were used throughout the docking. The

ligand was prepared using the ligand preparation tool available in the software and then minimized

using the minimization tool with CHARMM Force Field. The X-ray crystal structure of the gp41

core (1aik) was available from the Protein Data Bank (PDB) at the Research Collaboratory for

Structural Bioinformatics (RCSB). The protein was prepared using the protein preparation tool; then

one of the three CHRs was deleted to expose the hydrophobic pocket. The binding sphere was

defined from the selection of the residues K574, Q577, L568, and W571, as shown in Figure 2, with

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the radius value of 11 Å. The docking was performed using the CDOCKER tool in Discovery Studio,

and the pose was selected on the basis of the score and hydrophobic and hydrogen bond interactions.

Acknowledgement

This investigation was supported by grants 2006AA02Z319, 2006DFA33560, and 2009ZX09103-011

from the Ministry of Science and Technology in China, and grants U0832001/L02 and 81173098 from

the National Science Foundation of China (NSFC).

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Graphic Abstract

COOHHO

N

S N

O

S

A

B

CD

COOHHO

N

IC50 37.4 µM (gp41- 6HB)EC50 0.7 µM (cells)

IC50 1.8-2.6 µM (gp41- 6HB)EC50 0.3-1.5 µM (cells)

2 12e, 12g, 12k

R