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Design, Synthesis, and Biological Evaluation of Highly Potent Small Molecule−Peptide Conjugates as New HIV‑1 Fusion Inhibitors Chao Wang,† Weiguo Shi,† Lifeng Cai,† Lu Lu,‡ Qian Wang,‡ Tianhong Zhang,† Jinglai Li,† Zhenqing Zhang,† Kun Wang,† Liang Xu,† Xifeng Jiang,† Shibo Jiang,*,‡ and Keliang Liu*,† †

Beijing Institute of Pharmacology & Toxicology, 27 Tai-Ping Road, Beijing 100850, China Key Laboratory of Medical Molecular Virology of the Ministries of Education and Health, Shanghai Medical College and Institute of Medical Microbiology, Fudan University, Shanghai 200032, China

‡

S Supporting Information *

ABSTRACT: The small molecule fusion inhibitors N-(4carboxy-3-hydroxyphenyl)-2,5-dimethylpyrrole (NB-2) and N(3-carboxy-4-hydroxyphenyl)-2,5-dimethylpyrrole (A12) target a hydrophobic pocket of HIV-1 gp41 and have moderate antiHIV-1 activity. In this paper, we report the design, synthesis, and structure−activity relationship of a group of hybrid molecules in which the pocket-binding domain segment of the C34 peptide was replaced with NB-2 and A12 derivatives. In addition, the synergistic effect between the small molecule and peptide moieties was analyzed, and lead compounds with a novel scaffold were discovered. We found that either the nonpeptide or peptide part alone showed weak activity against HIV-1mediated cell−cell fusion, but the conjugates properly generated a strong synergistic effect. Among them, conjugates Aoc−βAla− P26 and Noc−βAla−P26 exhibited a low nanomolar IC50 in the cell−cell fusion assay and effectively inhibited T20-sensitive and -resistant HIV-1 strains. Furthermore, the new molecules exhibited better stability against proteinase K digestion than T20 and C34.

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hydrophobic deep pocket (∼16 Å long, ∼7 Å wide, and 5−6 Å deep) in the N-trimer. C34 contains a pocket-binding domain (PBD) through which it can interact with the primary cavity to form a stable 6HB with an N-helical coiled-coil motif.15 Although C-peptide fusion inhibitors are very potent at inhibiting HIV-1 infection, appearance of T20-resisitant strains, high production cost, and lack of oral bioavailability hinder its further development; thus, these drawbacks have led to a blooming discovery of small molecule fusion inhibitors targeting the gp41 fusion intermediate.16,17 Because the primary cavity with its highly conserved residues plays a crucial role in gp41-mediated membrane fusion, it becomes an attractive target for discovering nonpeptide fusion inhibitors that target gp41.18 Using high-throughput screening assays,19 Jiang and co-workers have identified two Nsubstituted pyrroles, NB-2 and NB-64, which inhibit HIV-1 replication at a micromolar level by interfering with the formation of the gp41 6HB to block HIV-1 entry.20,21 On the basis of the structures of NB-2 and NB-64, Xie’s group has designed and synthesized a series of N-(carboxyphenyl)pyrrole derivatives and found that one-quarter of them exhibit enhanced antiviral potency. The most active compound, A12, has potent inhibitory activity against HIV-1 replication with an EC50 value of 0.69 μM; however, its inhibitory potency on cell−

INTRODUCTION The envelope glycoproteins (Env) of human immunodeficiency virus type 1 (HIV-1) play critical roles in the formation of a coiled-coil six-helical bundle (6HB), thus providing the energy to promote fusion of the viral and cellular membranes.1 For the HIV-1 Env complex, the initially produced gp160 is cleaved into the surface subunit (gp120) and transmembrane subunit (gp41), which are noncovalently associated.2 After gp120 binds to its CD4 T cell receptors and chemokine coreceptor CCR5 or CXCR4,3 a series of conformational changes of gp120 occur, resulting in dissociation of the gp120−gp41 complex.4 The ectodomain of gp41 contains three important functional motifs (Figure 1A): a fusion peptide (FP), an N-terminal heptad repeat (NHR), and a C-terminal heptad repeat (CHR).5,6 The 6HB formation between the intermediate NHR trimer and the three folded-back CHRs7,8 provides the energy to drive the viral and target cell membranes into close apposition, resulting in membrane fusion.9 Peptides derived from the HIV-1 gp41 CHR, such as T20 and C34, are potent HIV-1 fusion inhibitors.10,11 They interact with their counterpart in gp41 to form a heterogeneous 6HB and prevent fusogenic gp41core formation, thus interrupting the fusion process.12 T20 (brand name, Fuzeon; generic name, enfuvirtide) was approved by the U.S. FDA for clinical use in 2003,13,14 whereas C34 has been widely used for studying the structure and function of HIV-1 gp41. The X-ray crystal structure of the N36/C34 complex shows a highly conserved © XXXX American Chemical Society

Received: December 24, 2012

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Figure 1. Schematic representation of HIV-1 gp41 and the designed small molecule−peptide conjugates. (A) HIV-1 gp41 contains a fusion peptide (FP), an N-terminal heptad repeat (NHR), a C-terminal heptad repeat (CHR), a transmembrane domain (TM), and a cytoplasm domain (CP). The NHR contains a putative T20 interaction domain, a helix zone, and a pocket domain; CHR contains a pocket-binding domain (PBD), a helix-zonebinding domain, and a lipid-binding domain. The amino acid sequences of the representative peptide fusion inhibitors T20 and C34, along with their respective targets, N46 and N36, are also shown and are highlighted in different colors. (B) Interaction between the NHR and CHR peptides. The green lines between the NHR and CHR indicate the interaction between the residues located at the a and d positions in the CHR and the e and g positions in the NHR to form the 6HB. The interaction between the PBD of the CHR and the deep pocket of the NHR is critical for stabilization of the 6HB. (C) P26 is a peptide that was engineered by deleting the PBD of C34. Small molecule−peptide conjugates were designed by covalently linking N-(carboxyphenyl)pyrrole derivatives and P26 with a flexible linker.

cell fusion was moderate .22,23 An increasing number of small molecule HIV-1 fusion inhibitors have been reported with IC50 values in the micromolar range by cell−cell fusion assay. However, this does not represent a significant step forward in improving potency.24,25 Moreover, no conclusive evidence has shown that the primary cavity is indeed the target of these small molecule inhibitors.26,27 Previous reports have indicated that conjugation of an active peptide with an active nonpeptide moiety elicits a strong synergistic effect,28 suggesting a more intuitive design for new drugs with improved activity. In this study, we aimed to discover new lead compounds with a novel scaffold and higher potency. Accordingly, we designed and synthesized a set of conjugates in which the P26 peptide, containing the partial C34 peptide sequence without the PBD, and two series of N(carboxyphenyl)pyrrole derivatives were covalently linked together. The activities of the conjugates were tested in HIV1 Env-mediated cell−cell fusion and HIV-1 infection assays using the laboratory-adapted HIV-1 IIIB strain. In addition, they were tested against both T20-sensitive and T20-resistant HIV-1 strains. Furthermore, to better understand and interpret the bioassay results and the nature of the interaction between the conjugated peptides and gp41, computational modeling studies were performed. The results of this study will provide useful information for the rational design of novel antiviral peptides against HIV and other viruses with class I fusion proteins. Design. Using the protein dissection strategy, Kim’s group resolved the crystal structure of the HIV-1 gp41 fusion core,

e.g., the N36/C34 6HB, which has been widely used as a tool to study the mechanism of HIV fusion inhibitors.1 Residues Trp628, Trp631, and Ile635 (WWI) of C34 peptide insert into the pocket formed by a cluster of residues in the NHR coiled coil (Figure 1B). This “WWI motif” is critical for the NHR and CHR interhelical interactions and is essential for the peptide’s anti-HIV potency.29,30 C34 peptide was used as a lead compound, and to design fusion inhibitors with novel scaffolds, we replaced the WWI motif with small molecule fusion inhibitors targeting the deep pocket of the HIV-1 gp41 NHR. To serve as a control, P26 peptide, which does not contain the PBD of C34, was synthesized (Figure 1C). Next, small molecules were covalently attached to the N-terminus of P26 through a flexible linker (β-alanine, βAla) to maintain the proper binding orientation and conformation. For small molecule fusion inhibitors, the carboxyl group in NB-2 is critical31 to its inhibitory activity, since NB-177, which has the same parent structure as NB-2 but without the carboxylic acid group, is inactive. The hydrophobic pyrrole ring of NB-2 is also favorable, since B15, which has a maleimide ring replacing the pyrrole ring, shows weak inhibitory activity on HIV-1 replication and 6HB formation. A12, an isomer of NB-2, exhibits promising anti-HIV-1 potency. Structure−activity relationship (SAR) analysis22,23 has shown that the carboxylic acid group of A12 and the hydrophobic pyrrole ring are also key contact motifs, whereas the phenolic group does not contribute significantly to the overall binding affinity and has plenty of room for modifications. On the basis of this reasoning, we designed two parallel series (the Xoc series and the Xpc series) B

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were obtained after removal of all protection groups by trifluoroacetic acid (TFA), including the tert-butyl group on 6 (Figure 3A). As shown in Scheme 2, the Paal−Knorr reaction was used to synthesize 14 by the condensation of anilines with chemical intermediate 9, and a tert-butyl ester was introduced at the 3-position on the pyrrole ring, which could be removed by 6 M HCl/EtOAc to afford 13. On the other hand, the benzyl group could be deprotected by 1 M KOH to obtain 14. However, the synthesis of Npc using the same route was unsuccessful as a result of the instability of 4-(3-(tertbutoxycarbonyl)-2,5-dimethyl-1H-pyrrol-1-yl)-2-hydroxybenzoic acid, an isomer of 11, during workup and purification when the color changed rapidly from white to deep red. Therefore, Npc was prepared as shown in Scheme 3. The amino group of commercially available 4-aminosalicylic acid was protected by Boc2O before coupling with benzyl alcohol and deprotection of the amino group with HCl in ethyl acetate (6 M). In this series, compounds 13 and 20 were covalently linked to the Nterminus of the peptide, and the corresponding conjugates Apc−βAla−P26 and Npc−βAla−P26 were obtained after removal of all peptide protection groups by hydrofluoric acid (HF), including the benzyl group on the nonpeptide moiety (Figure 3B).

of N-substituted pyrrole compounds based on the structures of A12 and NB-2. In our modifications, a carboxylic acid group was introduced into the parent structure of these two small molecules as the intermediate to couple to P26 via an amide bond. First, a carboxymethyl group was introduced into the phenolic groups of A12 and NB-2 to obtain Aoc and Noc, respectively. We assumed that this modification would have little impact on the overall structures of these two compounds. Second, a new carboxylic acid group was introduced to the pyrrole groups of A12 and NB-2 to obtain Apc and Npc, respectively. We suspected that this modification would change the electron density of the pyrrole ring and further impact the interaction with its target (Figure 2).

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RESULTS AND DISCUSSION Inhibitory Activities of the Small Molecule−Peptide Conjugates on HIV-1 Env-Mediated Cell−Cell Fusion. We used a cell−cell fusion assay to measure the inhibitory activities of the small molecule−P26 conjugates. The activities of the compounds are shown in Table 1. In our assay, the positive control peptides C34 and T20 had IC50 values of 1.4 ± 0.17 and 10 ± 1.4 nM, respectively. The peptide without the Nterminal PBD, P26 (Asn636−Leu661), had an IC50 value of 2540 ± 630 nM. Thus, removal of the PBD reduced the inhibitory activity by ∼1800-fold, from which we can infer that the PBD region plays a critical role in inhibiting HIV-1mediated cell−cell fusion by interacting with the primary pocket in the gp41 NHR. In the Xpc series, compounds 14 and 21 had no inhibitory activity in the cell−cell fusion assay; thus, an introduced polar group on the five-membered ring could significantly affect its electron density and further disrupt the hydrophobic interactions with its binding sites. On the

Figure 2. Chemical structures of Aoc, Noc, Apc, and Npc.

Chemistry. As shown in Scheme 1, derivatives of the Xoc series were obtained from either 5-nitrosalicylic acid or 4nitrosalicylic acid by esterification with tert-butyl alcohol, and the phenolic group was further alkylated with ethyl bromoacetate to provide 3a and 3b. Then the nitro group was converted into an amine by catalytic reduction. Without purification, the Paal−Knorr reaction was used to synthesize 5 by the condensation of anilines with acetonylacetone (hexane2,5-dione). By saponification, 5 was converted into the acids 6a and 6b, to which the N-terminus of the peptide was attached, and the corresponding Noc−βAla−P26 (P26 conjugated with an NB-2 derivative through a β-alanine) and Aoc−βAla−P26 (P26 conjugated with an A12 derivative through a β-alanine) Scheme 1. Synthesis of Compounds 7a and 7ba

Reagents and conditions: (i) tert-butyl alcohol, DCC, DMAP, 60 °C; (ii) BrCH2CO2Et, K2CO3, 2-butanone, reflux, 5 h; (iii) 10% Pd/C, CH3OH, 50 psi; (iv) 2,5-hexanedione, p-TsOH, NMM, toluene, reflux; (v) 1 M NaOH, CH3OH; (vi) TFA, DCM, rt. a

C

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Figure 3. Strategy for preparation of small molecule−peptide conjugates.

Scheme 2. Synthesis of Compound 14a

Reagents and conditions: (i) EtONa, BrCH2COCH3, 0 °C to rt; (ii) 9, p-TsOH, NMM, toluene, reflux; (iii) PhCH2OH, DCC, DMF/DCM, reflux; (iv) 6 M HCl/EtOAc, rt; (v) 1 M KOH, dioxane. a

contrary, in the Xoc series, compounds 7a and 7b both displayed moderate inhibitory activity, indicating that this modification maintained the interactions of the pyrrole and carboxylic acid group on the benzene ring with their targets very well. Attaching 21 or 14 to the N-terminus of P26 dramatically increased the activity of the P26 peptide with IC50 values of 130 ± 41.2 and 300 ± 33.9 nM, respectively, about an 8−20-fold increase compared with that of P26 alone. Notably, the inhibitory activity was further increased when 7a and 7b were conjugated to P26, with IC50 values of 22 ± 3.2 and 14 ± 3.0 nM, respectively, resulting in about 170-fold more potency than that of P26 alone, reaching the potency of T20. These

results suggest that the nonpeptide moiety might be favorable for binding to the cavity, resulting in a strong synergistic effect that significantly enhances the antiviral potency of the small molecule−peptide conjugates. To test whether the synergistic effect resulted from the linker (βAla) or a simple hydrophobic interaction, we covalently linked βAla to P26 or Phe to the Nterminus of βAla−P26. The results suggested that the nonpocket-specific binding portion in the small molecule−peptide conjugates makes limited contact with the hydrophobic cavity, and these control peptides could not reach the goals described above. D

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Scheme 3. Synthesis of Compound 21a

a

Reagents and conditions: (i) Boc2O, NaOH, dioxane/H2O, rt; (ii) PhCH2OH, DCC, DMF/DCM; (iii) 2 M HCl/EtOAc, rt; (iv) 9, p-TsOH, NMM, toluene, reflux; (v) 6 M HCl/EtOAc, rt; (vi) HF.

Table 1. Inhibitory Activities of Small Molecules and Small Molecule−P26 Conjugates on HIV-1 Env-Mediated Cell−Cell Fusiona compd

structure

IC50 (nM)

C34b P26b βAla−P26b Phe−βAla−P26b Npc−βAla−P26c Apc−βAla−P26c Noc−βAla−P26c Aoc−βAla−P26c 21 (Npc) 14 (Apc) 7a (Noc) 7b (Aoc) T20b

WMEWDREINNYTSLIHSLIEESQNQQEKNEQELL NNYTSLIHSLIEESQNQQEKNEQELL βAla−NNYTSLIHSLIEESQNQQEKNEQELL Phe−βAla−NNYTSLIHSLIEESQNQQEKNEQELL Npc−βAla−NNYTSLIHSLIEESQNQQEKNEQELL Apc−βAla−NNYTSLIHSLIEESQNQQEKNEQELL Noc−βAla−NNYTSLIHSLIEESQNQQEKNEQELL Aoc−βAla−NNYTSLIHSLIEESQNQQEKNEQELL 1-(4-carboxy-3-hydroxyphenyl)-2,5-dimethyl-1H-pyrrole-3-carboxylic acid 1-(3-carboxy-4-hydroxyphenyl)-2,5-dimethyl-1H-pyrrole-3-carboxylic acid 2-(carboxymethoxy)-4-(2,5-dimethyl-1H-pyrrol-1-yl)benzoic acid 2-(carboxymethoxy)-5-(2,5-dimethyl-1H-pyrrol-1-yl)benzoic acid YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF

1.40 ± 0.17 2540 ± 630 4553 ± 262 3430 ± 630 130 ± 41.20 300 ± 33.90 22.5 ± 3.18 14.9 ± 2.99 NAd NAd >100000 >100000 10.1 ± 1.40

The compounds were tested in triplicate, and the data are presented as the mean ± standard deviation. bThese peptides have an acetyl group at the N-terminus and carboxyamide at the C-terminus. cThese peptides have carboxyamide at the C-terminus. dNA = not active.

a

Inhibitory Activities of the Conjugated Peptides on Viral Replication and Their Cytotoxicities. Very good agreement between inhibition of HIV-1-mediated cell−cell fusion and inhibition of viral replication was observed for the small molecule−peptide conjugates. The inhibitory activities of the small molecule−peptide conjugates on HIV-1IIIB replication in MT-2 cells were assessed using a p24 enzyme-linked immunosorbent assay (ELISA) as previously described.20 Two of the compounds, Aoc−βAla−P26 and Noc−βAla−P26, inhibited HIV-1IIIB infection with IC50 values of 14.1 ± 0.2 and 14.5 ± 0.5 nM, respectively, similar to the antiviral potency of T20. The other two compounds, Apc−βAla−P26 and Npc−βAla−P26, had IC50 values of 458 ± 32.7 and 175 ± 35.6 nM, respectively. We also tested the cytotoxicities of these compounds on TZM-b1 cells using an MTS cytotoxicity assay [MTS = 3-(4,5-dimethylthiazol-2-yl)-5-((3-carboxymethoxy)phenyl)-2-(4-sulfophenyl)-2H-tetrazolium]. The CC50 (concentration causing 50% cytotoxicity) values were all greater than 500 μM, with selectivity indices (SIs) from 1000 to 30000 (Table 2). Small Molecule−Peptide Conjugates Interact with HIV-1 gp41 NHR and Inhibit 6HB Formation of the gp41 NHR and CHR. Next we tested the conjugates’ ability to

Table 2. Antiviral Activities of Conjugates against Laboratory-Adapted HIV-1 Strains and Their Cytotoxicitiesa

compd

inhibition of p24 production, EC50 (nM)

Npc−βAla−P26 Apc−βAla−P26 Noc−βAla−P26 Aoc−βAla−P26 T20

175 ± 35.6 458 ± 32.7 14.5 ± 0.5 14.1 ± 0.2 20.0 ± 3.0

CC50 (μM)

SIb

inhibition of 6HB formation, IC50 (μM)

>500 >500 >500 >500

>2000 >1000 >30000 >30000

167 ± 88.2 123 ± 98.4 24.9 ± 1.7 7.8 ± 0.4

a

The compounds were tested in triplicate, and the data are presented as the mean ± standard deviation. bSI =selectivity index, CC50/EC50

disrupt the interaction between the NHR and CHR of HIV-1 gp41 by using a fluorescence resonance energy transfer assay.12,32 The ligand bpy (2,2′-bipyridine-5′-carboxylate) was linked to the N-terminus of the N-peptide (GQAVEAQQHLLQLTVWGIKQLQARILAVEKK) containing the hydrophobic pocket of gp41 with a suitable linker. The probe peptide CP2-LY (MTWBEWDREIBNYTSLIC-LY), which specifically binds to the N-peptide, was modified with Lucifer yellow dye at its C-terminus. The bpy-N-peptide formed a trimeric coiled E

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peptide eluted at the earlier retention time. However, no new peak corresponding to Xpc−βAla−P26 or P26 peptide (data not shown) with N36 at the corresponding retention time was observed. These results indicated that Noc−βAla−P26 and Aoc−βAla−P26 are able to interact with N36 to form complexes in PBS. Small Molecule−Peptide Conjugates Are Highly Potent against T20-Sensitive and T20-Resistant HIV-1 Strains. Although T20 is effective against a broad spectrum of HIV-1 strains, including the wild type and those resistant to multiple reverse transcriptase inhibitors (RTIs) or protease inhibitors (PIs), drug-resistant HIV-1 variants were still easily induced in patients after T20 therapy.33,34 Therefore, we determined whether our hybrid molecule was effective against HIV-1 strains resistant to T20. A panel of HIV-1NL4−3 mutants, including one T20-sensitive strain and five T20-resistant strains, was used in our experiments. As shown in Table 3, T20 was much less effective against T20-resistant strains. We found that double mutations in the gp41 NHR also conferred crossresistance to C34. Strikingly, Aoc−βAla−P26 and Noc−βAla− P26 were highly potent against both T20-sensitive and T20resistant strains with IC50 values ranging from 130 to 250 nM, whereas T20 was inactive against these resistant HIV-1 strains at concentrations up to 2000 nM. Early in vitro studies indicated that HIV-1 acquired T20 resistance by mutating the amino acid residues in the “GIV” motif (amino acids GIVQQQNNLL located in the NHR region of gp41).35,36 Our previous studies indicated that CHR peptides containing PBD, such as C34, are much more effective against T20resistant strains with mutations in the GIV motif than T20, which lacks PBD.37 The small molecule moiety in our conjugated peptides mimics the PBD and can specifically interact with the NHR pocket, thus providing additional binding sites other than T20 and rendering the conjugated peptides effective against T20-resistant HIV-1 isolates. These results indicated that small molecule−peptide conjugates could be developed into a new generation of fusion inhibitors to overcome T20-resistant variants. Aoc−P26 Conjugates Are Resistant to Proteinase K Digestion. Some of the major disadvantages of T20 are its poor pharmacokinetics in vivo and its high sensitivity to proteolytic enzymes in the blood. We speculated that our designed conjugated peptides would be more resistant than T20 to proteolytic enzymes. Therefore, we determined the stability of Aoc−βAla−P26 with proteinase K (a broadspectrum serine proteinase) digestion.38 When incubated with 20 ng/mL proteinase K in PBS for 1 h, the T20 concentration

coil, namely, env2.0, by chelation with Fe(II) and acted as a fluorescence quencher of the probe peptide. Competitive inhibition of the env2.0−probe interaction by these small molecule−P26 conjugates, with a concomitant increase in fluorescence according to their potency, could be detected in this assay. As shown in Table 2 and Figure 4, P26 exhibited no

Figure 4. Inhibitory activities of the five C-peptides on 6HB formation between env2.0 and CP2-LY as determined by the fluorescence resonance energy transfer experiment.

significant competitive inhibition with env2.0, whereas Npc− and Apc−P26 conjugates showed moderate inhibitory activities with IC50 values of 167 ± 88.2 and 123 ± 98.4 μM, respectively. Both Noc− and Aoc−P26 conjugates exhibited high inhibitory potencies against env2.0−probe formation with IC50 values of 24.9 ± 1.70 and 7.79 ± 0.366 μM, respectively. Their inhibitory activities on the env2.0−probe complex strongly correlated with the inhibitory activities of these compounds on cell−cell fusion and their antiviral activity against the HIV-1IIIB strain. The specific interactions between small molecule−peptide conjugates and HIV-1 gp41 NHR were further confirmed by size-exclusion HPLC (SE-HPLC) (Figure 5). The solid line chromatograms of Figure 5 show the conjugated peptides alone. Next the N36 peptide was mixed with each conjugated peptide. After a 30 min incubation period, the mixture was applied onto an SE-HPLC column, and the chromatograms are shown as dotted lines. The complex formed between Noc−βAla−P26 and Aoc−βAla−P26 with the N36

Figure 5. Determination of complex formation between small molecule−P26 conjugates and N36 using SE-HPLC analysis. F

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Table 3. Activities of Small Molecule−Peptide Conjugates against T20-Sensitive and T20-Resistant Strainsa IC50 (nM) NL4-3 mutant b

D36G (36G)V38Ac (36G)V38A/N42Dc (36G)N42T/N43Kc (36G)V38E/N42Sc (36G)V38A/N42Tc

T20

C34

50 ± 10 >2000 (>40) >2000 (>40) >2000 (>40) >2000 (>40) >2000 (>40)

5.12 ± 0.51 3.26 ± 1.1 (0.6) 37.2 ± 4.1 (7.3) 192 ± 9 (38) 136 ± 12 (27) 85.5 ± 10 (17)

Aoc−βAla−P26 130 210 220 230 190 240

± ± ± ± ± ±

12 10 (1.6) 10 (1.7) 1 (1.8) 80 (1.5) 1 (1.8)

Noc−βAla−P26 170 240 250 240 190 220

± ± ± ± ± ±

10 30 80 30 10 30

(1.4) (1.5) (1.4) (1.1) (1.3)

The compounds were tested in triplicate, and the data are presented as the mean ± standard deviation; the values in parentheses indicate relative changes (n-fold) in the IC50 compared with the IC50 in the presence of the D36G substitution. bT20-sensitive strain. cT20-resistant strain.

a

Figure 6. Digestion of C-peptide inhibitors with proteinase K treatment, as determined by HPLC. (A) Time course of residual T20, C34, and Aoc−βAla−P26 with proteinase K digestion. The chromatograms of T20 (B), C34 (C), and Aoc−βAla−P26 (D) at 0 h (purple), 0.5 h (black), and 1 h (red) are also shown, respectively.

N36−C34 complex (PDB 1AIK).1 Molecular dynamics simulations were conducted on the peptide complex using AMBER 8.039 on an SGI Altix 350 workstation (Fremont, CA). From the calculated result of the negative total binding free energy, we learned that the binding free energies of Aoc−βAla− P26 (−306.61 ± 15.65 kcal/mol) and Noc−βAla−P26 (−283.71 ± 25.40 kcal/mol) were notably less than the binding free energy of P26 peptide (−236.21 ± 12.32 kcal/ mol), indicating that the complexes formed between Aoc−βAla−P26 or Noc−βAla−P26 and NHR were more stable than P26. Figure 7A shows the computational model of the complex formed between Aoc−βAla−P26 (yellow and red) and the N36 peptide (blue) of gp41. Aoc−βAla−P26 and N36 interacted with each other in an antiparallel manner to form a stable coiled coil; however, the conjugated peptide did not fully adopt an α-helical structure. We hypothesized the following to explain this result. In 6HB composed of N36 and C34, residues Trp628 and Trp631 of C34 were at the a and d positions, respectively, in the same helix. Ile635 was at the a position in the next helix. Removing the PBD destroyed the two helices. Consequently, the small molecule was unable to attach the residues at the a or d position, preventing it from inducing high α-helicity. Figure 7B shows the local enlargement of the interaction between Aoc in the conjugates with the corresponding residues in the primary cavity. The hydrophilic

decreased quickly, and only 40% of the original amount was detected by HPLC. In contrast, C34 retained about 70% of the original amount. Interestingly, Aoc−βAla−P26 maintained about 80% of the original amount, as detected by HPLC, suggesting that the conjugated peptide was much more resistant to proteinase K than T20 and even better than C34 (Figure 6). Subsequently, we investigated the pharmacokinetic (PK) profile of Aoc−βAla−P26 in rats after a single-dose intravenous administration and determined the in vivo and in vitro half-lives of Aoc−βAla−P26 and T20, respectively. Aoc−βAla−P26 exhibited a shorter in vivo half-life than T20 (0.223 h vs 0.997 h). However, its in vitro half-life in the liver and kidney homogenates was longer than that of T20, consistent with the stability in proteinase K digestion assay (detailed PK data can be found in the Supporting Information). Therefore, we speculate that the shorter in vivo half-life of Aoc−βAla−P26 may be due to its higher tissue-binding rate compared to that of T20. Computational Modeling. To better understand and interpret the bioassay results and the nature of the interaction between the conjugated peptides and gp41, computational modeling studies were performed to estimate the binding free energy for the association between the predominant conformations of the designed small molecule−peptide conjugates and the NHR on the basis of the X-ray crystal structure of the G

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CONCLUSION

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EXPERIMENTAL SECTION

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In conclusion, novel highly potent small molecule−peptide conjugates with low nanomolar levels of anti-HIV fusion activity were designed and synthesized. We found that all four small molecule−peptide conjugates exhibited significant inhibitory activity on HIV-1-mediated cell−cell fusion and viral replication, as well as 6HB formation. A small molecule moiety can act as a substitute for the PBD of the C34 peptide and maintain highly potent anti-HIV-1 fusion activity. Unlike T20, the hybrid molecule was less sensitive to proteinase K digestion and highly effective against T20-sensitive and T20resistant HIV-1 strains. Therefore, the rational combination of small molecule and peptide anti-HIV-1 fusion inhibitors into one molecule has shed new light on the design of novel HIV-1 fusion inhibitors targeting gp41. Our results provide important information for understanding the interaction between the NHR deep pocket and small molecule fusion inhibitors.

Figure 7. Aoc−βAla−P26 and gp41 N-trimer interaction. Computer modeling is based on the crystal structure of the HIV-1 gp41 6HB (PDB code 1AIK). (A) Representation of the N36 coiled coil as a molecular surface (light blue), with Aoc−βAla−P26 (yellow and red) depicted as rods. Three C-peptides were expected to interact with the N-trimer, but for clarity, only one C-peptide is shown. In the conjugated peptide, Aoc and P26 are shown by a stick model (yellow) and a ribbon diagram (red), respectively. The left panel shows a front view of the conjugated peptide−N36 complex, while the right panel shows a view from the side. The small molecule−P26 conjugated peptide packs against a conserved groove on the surface of the coiled coil. (B) The top of the coiled coil has a large cavity that provides a binding pocket for Aoc (gray). The hydrogen bond between the carboxylic acid and Gln575 is shown with a light green dashed line.

Chemistry. Melting points were measured with an RY-1 melting point apparatus without correction. 1H (400 MHz) and 13C (100 MHz) NMR spectra were measured on a JNM-ECA-400 spectrometer using TMS as the internal standard. The solvent used was DMSO-d6 or CD3OD. Mass spectra were measured on an API-150 mass spectrometer with the electrospray ionization source from ABI Inc. TLC was performed on silica gel GF254 plates. Silica gel (200−300 mesh) from Qingdao Haiyang Chemical Co. was used for column chromatography. All chemicals were obtained from Beijing Chemical Works or Sigma-Aldrich, Inc. and were distilled and dried before use according to literature procedures. For small molecule compounds, purity was determined by analytical RP-HPLC using an Agilent 1200 series and an RP-C18 column (Agilent Eclipse XDB-C18, 5 μm, 4.6 × 250 mm) using two different solvent systems (conditions 1 and 2 in the Supporting Information) and a flow rate of 1 mL/min with the detection wavelength at 254 nm. For peptides, purity was determined by analytical RP-HPLC on an RP-C8 column (Zorbax Eclipse XDBC8, 5 μm, 4.6 × 150 mm) using two different solvent systems (methods A and B in the Supporting Information) and a flow rate of 1 mL/min with the detection wavelength at 210 nm. All target compounds were purified, which resulted in ≥95% purity for assay testing (details of HPLC characterization of the compounds are given in the Supporting Information). Synthesis of tert-Butyl 2-Hydroxy-4-nitrobenzoate (2a). DCC (1.2 equiv, 0.62 g, 3 mmol) dissolved in dry THF (6 mL) was added dropwise over 5 min to a stirred solution of 4-nitrosalicylic acid (1a; 0.5 g, 2.73 mmol) and DMAP (1 equiv, 0.33 g, 2.73 mmol) in dry tertbutyl alcohol (12.5 mL). The mixture was stirred and heated to reflux overnight. The reaction was monitored by TLC. After the reaction was complete, the solvents were removed under reduced pressure. Then 15 mL of ethyl acetate was added, and the solution was filtered to remove the 1,3-dicyclohexylurea (DCU) formed. After removal of the solvent under reduced pressure, the crude product was purified by column chromatography using petroleum ether/ethyl acetate (30:1) as the eluent to afford 0.48 g of 2a as pale yellow crystals, 73% yield, mp 84− 86 °C. MS (m/z): calcd for C11H13NO5, 239. LC−MS (m/z (rel intens)): 239.5 (M+, 60). 1H NMR (DMSO-d6): δ 11.00 (s, 1H), 7.88 (d, 1H), 7.71 (m, 2H), 1.60 (s, 9H). 13C NMR (DMSO-d6): δ 166.10, 159.16, 150.56, 131.60, 121.84, 113.45, 111.85, 83.26, 27.68. Data for tert-Butyl 2-Hydroxy-5-nitrobenzoate (2b). Mp: 82−84 °C. MS (m/z): calcd for C11H13NO5, 239. LC−MS (m/z (rel intens)): 239.5 (M+, 60). 1H NMR (DMSO-d6): δ 11.55 (s, 1H), 8.47 (d, J = 4.0 Hz, 1H), 8.33 (dd, J = 8.0, 4.0 Hz, 1H), 7.16 (d, J = 8.0 Hz, 1H), 1.59 (s, 9H). 13C NMR (DMSO-d6): δ 165.96, 164.58, 139.20, 129.69, 126.35, 118.57, 115.63, 83.58, 27.67. Synthesis of tert-Butyl 2-(2-Ethoxy-2-oxoethoxy)-4-nitrobenzoate (3a). To a solution of 2a (0.42 g, 1.75 mmol) in 2-butanone (10 mL) was added anhydrous potassium carbonate (1.1 equiv, 0.26 g,

residue Gln575 could form hydrogen bonds with the carboxylic acid group of Aoc, and the hydrophobic cavity nicely complements the shape of the pyrrole ring of Aoc to form hydrophobic interactions. Currently, more and more small molecule HIV-1 fusion inhibitors targeting the primary hydrophobic cavity on the groove of the gp41 N-trimer have been identified. However, until now, no significant improvement in the antiviral potency of the small molecule inhibitors has been reported. The crystal structures of these compounds binding to the target have also not been reported. Therefore, it is reasonable to question whether the pocket is the target of these nonpeptide fusion inhibitors. NB-2 and A12 are N-substituted pyrroles that were designed to target this cavity. In this study, their derivatives were covalently attached to the N-terminus of the peptide P26 through a linker, replacing the PBD of C34. We found that the nonpeptide small molecule moiety could largely restore the function of the PBD in HIV-1-mediated cell−cell fusion and caused a significant effect on the whole antiviral potency of the conjugates. Furthermore, the fusion inhibitory activity of the conjugates had a strong correlation with these N(carboxyphenyl)pyrroles alone. A fluorescence resonance energy transfer experiment further confirmed that Nsubstituted pyrrole derivatives did indeed interact with the hydrophobic pocket, and the binding affinity was significantly improved with the increased number of critical functional groups on these small molecules. The interactions between the hybrid peptides and N36 were also characterized by SE-HPLC. The combined results indicated that these N-(carboxyphenyl)pyrrole derivatives could largely act as substitutes for the PBD of C34 by targeting the hydrophobic cavity on the surface of the N-trimer coiled coil. Furthermore, the rationally designed N-(carboxyphenyl)pyrrole−peptide conjugates could be used as molecular probes to provide obvious evidence that the cavity is the target of these nonpeptide fusion inhibitors. H

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Data for 2-(2-(tert-Butoxycarbonyl)-4-(2,5-dimethyl-1H-pyrrol-1yl)phenoxy)acetic Acid (6b). Mp: 150−152 °C. MS (m/z): calcd for C19H23NO5, 345. LC−MS (m/z (rel intens)): 346.3 (M + H, 100). 1H NMR (CD3OD): δ 7.44 (d, J = 2.8 Hz, 1H), 7.30 (dd, J = 8.7, 2.8 Hz, 1H), 7.13 (d, J = 8,7 Hz, 1H), 5.78 (s, 2H), 4.79 (s, 2H), 1.96 (s, 6H), 1.58 (s, 9H). 13C NMR (DMSO-d6): δ 169.80, 164.23, 155.47, 131.99, 130.88, 129.34, 127.70, 122.93, 114.26, 105.88, 81.21, 65.40, 27.75, 12.87. Synthesis of 2-(Carboxymethoxy)-4-(2,5-dimethyl-1H-pyrrol-1yl)benzoic Acid (7a). The protected 6a (0.34 g, 1 mmol) was acidolysed by stirring in TFA (1.47 g, 13 mmol) and dichloromethane (DCM; 0.26 g, 32 mmol) in the presence of triethylsilane (0.29 g, 2.5 mmol) at room temperature. When the reaction was completed, the solvent was removed, the residue was triturated with H2O, and the precipitated solid was isolated by filtration. The precipitated solid was washed with water and dried to afford 0.10 g of 7a as a red solid, 36% yield, mp 190−192 °C. MS (m/z): calcd for C15H15NO5, 289. LC− MS (m/z (rel intens)): 290.1 (M + H, 100). 1H NMR (DMSO-d6): δ 12.99 (s, 2H), 7.76 (d, J = 8.1 Hz, 1H), 6.17 (dd, J = 8.1, 1.6 Hz, 1H), 6.87 (d, J = 1.6 Hz, 1H), 5.81 (s, 2H), 4.86 (s, 2H), 1.98 (s, 6H). 13C NMR (DMSO-d6): δ 169.96, 166.60, 156.99, 141.94, 131.34, 127.57, 120.55, 120.01, 113.40, 106.36, 65.17, 12.79. Data fro 2-(Carboxymethoxy)-5-(2,5-dimethyl-1H-pyrrol-1-yl)benzoic Acid (7b). Mp: 144−146 °C. MS (m/z): calcd for C15H15NO5, 289. LC−MS (m/z (rel intens)): 290.1 (M + H, 100). 1 H NMR (DMSO-d6): δ 11.79 (s, 1H), 7.53 (d, J = 2.8 Hz, 1H), 7.43 (dd, J = 8.6, 2.8 Hz, 1H), 7.12 (d, J = 8.6 Hz, 1H), 5.78 (s, 2H), 4.80 (s, 2H), 1.94 (s, 6H). 13C NMR (DMSO-d6): δ 168.98, 166.59, 158.72, 135.09, 129.58, 129.29, 127.71, 118.58, 114.12, 105.78, 61.77, 12.72. Synthesis of tert-Butyl 2-Acetyl-4-oxopentanoate (9). To a solution of tert-butyl acetoacetate (8; 5 mL, 30.6 mmol) in ethanol was added sodium ethoxide (12.3 mL, 30.6 mmol) dropwise in an ice bath for 15 min with stirring. Then the mixture was added dropwise to a solution of bromopropanone (2.2 mL, 25.4 mmol) in ethanol/ toluene (30 mL, 2:1) at 0 °C with continued stirring at room temperature for 4 h and then adjusted to pH 7.0 using 2 M HCl. After removal of the solvent under reduced pressure, 200 mL of ethyl acetate was added, the solid precipitate was filtered, and the filtrate was washed with water to neutral pH and dried over Na2SO4. The crude product was purified by silica gel column chromatography to afford 3.79 g of 9 as a yellow oil, 70.1% yield. MS (m/z): calcd for C11H18NO4, 214. LC−MS (m/z (rel intens)): 165.0 (M-C4H9O+Na, 100). 1H NMR (DMSO-d6): δ 3.85 (t, J = 8.0 Hz, 1H), 2.92 (m, J = 8.0 Hz, 2H), 2.23 (s, 3H), 2.11 (s, 3H), 1.41 (s, 9H). 13C NMR (DMSO-d6): δ 205.75, 202.81, 167.82, 81.28, 54.63, 40.97, 29.45, 27.45. Synthesis of 5-(3-(tert-Butoxycarbonyl)-2,5-dimethyl-1H-pyrrol1-yl)-2-hydroxybenzoic Acid (11). To a solution of 5-amino-2hydroxybenzoic acid (10; 1.1 equiv, 0.39 g, 2.57 mmol) in 10 mL of toluene was added 9 (1 equiv, 0.50 g, 2.33 mmol). The mixture was heated to reflux, 20 mg of p-toluenesulfonic acid was added, and the mixture continued to reflux for 3 h with monitoring by TLC. After the reaction was complete, the solvents were removed under reduced pressure. Then 20 mL of ethyl acetate was added, and the organic phase was washed with brine and dried over Na2SO4. After the mixture was filtered and the filtrate was evaporated under reduced pressure, the residue was purified by silica gel column chromatography to yield 0.54 g of intermediate 11 as a white solid, yield 70%, mp 146−152 °C. MS (m/z): calcd for C18H21NO5, 331. LC−MS (m/z (rel intens)): 332.2 (M + H, 40). 1H NMR (DMSO-d6): δ 11.43 (s, 1H), 7.58 (d, J = 2.5 Hz, 1H), 7.44 (dd, J = 8.7, 2.8 Hz, 1H), 7.11 (d, J = 8.7 Hz, 1H), 6.18 (s, 1H), 2.17 (s, 3H), 1.90 (s, 3H), 1.48 (s, 9H). 13C NMR (DMSOd6): δ 171.53, 164.68, 161.27, 135.77, 135.41, 129.94, 128.72, 118.90, 114.23, 112.70, 107.99, 78.89, 28.68, 12.85, 12.66. Synthesis of tert-Butyl 1-(3-((Benzyloxy)carbonyl)-4-hydroxyphenyl)-2,5-dimethyl-1H-pyrrole-3-carboxylate (12). DCC (1.1 equiv, 0.27 g, 1.32 mmol) dissolved in dry THF (6 mL) was added dropwise over 5 min to a stirred solution of 11 (0.4 g, 1.2 mmol), benzyl alcohol (3 equiv, 0.39 g, 3.6 mmol), and DMAP (0.1 equiv,

1.88 mmol), and the mixture was heated to reflux for 30 min. Then ethyl bromoacetate (1.2 equiv, 0.36 g, 2.15 mmol) was added. The mixture was continuously stirred, was heated to reflux for 3 h, and was monitored by TLC. After the reaction was finished, water (15 mL) was added. The product was extracted with chloroform three times, successively, and dried over Na2SO4. After removal of the solvent under reduced pressure, the crude product was purified by column chromatography using petroleum ether/ethyl acetate (20:1) as the eluent to afford 0.45 g of 3a as a yellow oil, 80% yield, mp 50−52 °C. MS (m/z): calcd for C15H19NO7, 325. LC−MS (m/z (rel intens)): 326.2 (M + H, 27). 1H NMR (DMSO-d6): δ 7.89 (dd, J = 8.4, 1.9 Hz, 1H), 7.84 (d, J = 1.9 Hz, 1H), 7.77 (d, J = 8.4 Hz, 1H), 5.04 (s, 2H), 4.19 (q, J = 7.0 Hz, 2H), 1.54 (s, 9H), 1.22 (t, J = 7.0 Hz, 3H). 13C NMR (DMSO-d6): δ 167.83, 164.06, 155.90, 149.45, 130.59, 129.05, 116.02, 108.66, 82.35, 65.81, 60.87, 27.69, 14.04. Data for tert-Butyl 2-(2-Ethoxy-2-oxoethoxy)-5-nitrobenzoate (3b). Mp: 56−58 °C. MS (m/z): calcd for C15H19NO7, 325. LC− MS (m/z (rel intens)): 326.2 (M + H, 27). 1H NMR (DMSO-d6): δ 8.36 (dd, J = 9.2, 2.8 Hz, 1H), 8.33 (d, J = 2.8 Hz, 1H), 7.29 (d, J = 9.2 Hz, 1H), 5.06 (s, 2H), 4.19 (q, J = 7.0 Hz, 2H), 1.55 (s, 9H), 1.23 (t, J = 7.0 Hz, 3H). 13C NMR (DMSO-d6): δ 167.58, 163.21, 160.81, 140.52, 128.01, 125.66, 122.88, 114.35, 82.16, 65.73, 60.98, 27.70, 14.03. General Procedure for the Preparation of 4a,b. A 180 mg amount of 3a,b was dissolved in methanol (20 mL), followed by addition of 10% Pd on charcoal catalyst (18 mg), and the mixture was hydrogenated at 50 psi and room temperature. After the reaction was finished, the catalyst was removed by filtration, and the solvent was evaporated. The product was directly used for the next step without further purification. Synthesis of tert-Butyl 4-(2,5-Dimethyl-1H-pyrrol-1-yl)-2-(2ethoxy-2-oxoethoxy)benzoate (5a). To a solution of 4a (1.0 g, 3.38 mmol) in 20 mL of toluene were added N-methylmorpholine (1 equiv, 0.34 g, 3.38 mmol) and 2,5-hexanedione (2 equiv, 0.74 g, 6.49 mmol). The mixture was heated to reflux, followed by the addition of 30 mg of p-toluenesulfonic acid (PTSA), and the mixture continued to reflux for 3 h with monitoring by TLC. After the reaction was complete, the solvents were removed under reduced pressure. Then 20 mL of ethyl acetate was added, and the organic phase was washed with brine, successively, and dried over Na2SO4. After filtration and evaporation under reduced pressure, the residue was purified by silica gel column chromatography to yield 0.94 g of intermediate 5a as a colorless oil, yield 75%. MS (m/z): calcd for C21H27NO5, 373. LC− MS (m/z (rel intens)): 374.2 (M + H, 20). 1H NMR (DMSO-d6): δ 7.67 (d, J = 8.1, 1H), 6.94 (d, J = 1.9 Hz, 1H), 6.92 (dd, J = 8.1, 1.9 Hz, 1H), 5.81 (s, 2H), 4.92 (s, 2H), 4.14 (q, J = 7.0 Hz, 2H), 1.98 (s, 6H), 1.54 (s, 9H), 1.19 (t, J = 7.0 Hz, 3H). 13C NMR (DMSO-d6): δ 168.29, 164.61, 156.67, 141.81, 130.86, 127.60, 121.71, 120.31, 113.58, 106.36, 81.01, 65.40, 60.70, 27.79, 14.02, 12.82. Data for tert-Butyl 5-(2,5-Dimethyl-1H-pyrrol-1-yl)-2-(2-ethoxy2-oxoethoxy)benzoate (5b). MS (m/z): calcd for C21H27NO5, 373. LC−MS (m/z (rel intens)): 374.4 (M + H, 40). 1H NMR (DMSOd6): δ 7.37 (m, 2H), 7.13 (m, 1H), 5.79 (s, 2H), 4.91 (s, 2H), 4.20 (q, J = 7.0 Hz, 2H), 1.95 (s, 6H), 1.52 (s, 9H), 1.22 (t, J = 7.0 Hz, 3H). 13 C NMR (DMSO-d6): δ 168.27, 164.23, 155.21, 131.95, 131.09, 129.29, 127.69, 123.15, 114.42, 105.88, 81.26, 65.55, 60.74, 27.73, 14.04, 12.82. Synthesis of 2-(2-(tert-Butoxycarbonyl)-5-(2,5-dimethyl-1H-pyrrol-1-yl)phenoxy)acetic Acid (6a). A 20 mg amount of 5a was dissolved in 4 mL of methanol, and 1 mL of 25% NaOH in H2O was added. The mixture was stirred for 30 min at room temperature and then adjusted to pH 3.0 using 1 M HCl. The precipitated solid was filtered, washed with water until neutral pH, and dried to afford 18 mg of 6a as a white solid, 98% yield, mp 170−172 °C. MS (m/z): calcd for C19H23NO5, 345. LC−MS (m/z (rel intens)): 346.3 (M + H, 98). 1H NMR (CD3OD): δ 7.78 (d, J = 8.1 Hz, 1H), 6.88 (dd, J = 8.1, 1.9 Hz, 1H), 6.82 (d, 1.9 Hz, 1H), 5.80 (s, 2H), 4.76 (s, 2H), 1.98 (s, 6H), 1.57 (s, 9H). 13C NMR (DMSO-d6): δ 169.83, 164.66, 157.79, 141.73, 130.85, 127.58, 120.90, 119.17, 113.60, 106.32, 80.74, 67.08, 27.84, 12.88. I

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0.03 g, 0.27 mmol) in dry DCM/N,N-dimethylformamide (DMF) (12 mL, 5:1). The mixture was stirred and heated to reflux overnight. The reaction was monitored by TLC. After the reaction was complete, the solvents were removed under reduced pressure. Then 30 mL of ethyl acetate was added, the mixture was filtered to remove the DCU formed, and the organic phase was washed with brine and dried over Na2SO4. After filtration and removal of the solvent under reduced pressure, the crude product was purified by silica gel column chromatography using petroleum ether/ethyl acetate (50:1) as the eluent to afford 0.35 g of 12 as a colorless oil, 70% yield. MS (m/z): calcd for C25H27NO5, 421. LC−MS (m/z (rel intens)): 422.3 (M + H, 10). 1H NMR (DMSO-d6): δ 10.71 (s, 1H), 7.59 (d, J = 2.5 Hz, 1H), 7.48 (m, 5H), 7.44 (dd, J = 8.7, 2.8 Hz, 1H), 7.14 (d, J = 8.7 Hz, 1H), 6.17 (s, 1H), 5.38 (s, 2H), 2.16 (s, 3H), 1.89 (s, 3H), 1.48 (s, 9H). 13C NMR (DMSO-d6): δ 167.69, 164.66, 159.95, 136.09, 135.56, 135.39, 129.93, 129.06, 128.85, 128.79, 128.71, 128.66, 119.22, 114.95, 112.75, 108.02, 78.89. 67.29, 28.68, 12.85, 12.66. Synthesis of 1-(3-((Benzyloxy)carbonyl)-4-hydroxyphenyl)-2,5dimethyl-1H-pyrrole-3-carboxylic Acid (13). A solution of 12 (0.2 g, 0.48 mmol) in 5 mL of HCl in ethyl acetate (6 M) was stirred at room temperature for 10 min. The reaction mixture was evaporated to remove the solvent. The residue was dissolved in 20 mL of ethyl acetate, and the solution was evaporated to dryness. The resulting product was purified by silica gel column chromatography to yield 0.17 g of 13 as a pink solid, yield 98%, mp 82−84 °C. MS (m/z): calcd for C21H19NO5, 365. LC−MS (m/z (rel intens)): 366.2 (M + H, 37). 1H NMR (DMSO-d6): δ 11.69 (s, 1H), 10.72 (s, 1H), 7.61 (d, J = 2.5 Hz, 1H), 7.47 (dd, J = 8.6, 2.5 Hz, 1H), 7.33−7.42 (m, 5H), 7.15 (d, J = 8.6 Hz, 1H), 6.21 (s, 1H), 5.38 (s, 2H), 2.18 (s, 3H), 1.90 (s, 3H). 13C NMR (DMSO-d6): δ 167.17, 166.22, 159.40, 135.59, 135.30, 135.03, 129.39, 128.55, 128.36, 128.28, 128.20, 128.14, 118.68, 114.44, 111.35, 107.66, 66.77, 12.40, 12.10. Synthesis of 1-(3-Carboxy-4-hydroxyphenyl)-2,5-dimethyl-1Hpyrrole-3-carboxylic Acid (14). A 20 mg amount of 13 was dissolved in 4 mL of dioxane, and 1 mL of 1 M KOH was added. The mixture was stirred for 3 h at room temperature and then adjusted to pH 2−3 using 1 M HCl. The solution was extracted with ethyl acetate three times. After removal of the solvent under reduced pressure, the residue was purified by silica gel column chromatography to afford 3.2 mg of 14 as a white solid, 21% yield, mp 164−166 °C. MS (m/z): calcd for C14H13NO5, 275. LC−MS (m/z (rel intens)): 276.1 (M + H, 100). 1H NMR (DMSO-d6): δ11.54 (s, 2H), 7.60 (d, J = 2.5 Hz, 1H), 7.46 (dd, J = 8.7, 2.5 Hz, 1H), 7.11 (d, J = 8.7 Hz, 1H), 6.21 (s, 1H), 2.19 (s, 3H), 1.92 (s, 3H). 13C NMR (DMSO-d6): δ 171.00, 166.21, 160.70, 135.32, 135.26, 129.39, 128.21, 118.35, 113.66, 111.31, 107.61, 12.36, 12.06. Synthesis of 4-(tert-Butoxycarbonyl)-2-hydroxybenzoic Acid (16). A solution of 4-amino-2-hydroxybenzoic acid (15; 1 equiv, 3.06 g, 20 mmol) in 1 M NaOH (20 mL) was stirred and cooled in an ice−water bath. Di-tert-butyl pyrocarbonate (1.2 equiv, 5.24 g, 24 mmol) in 30 mL of dioxane was added, and stirring was continued at room temperature for 2 h. The solution was concentrated in vacuo to approximately 10−15 mL, cooled in an ice−water bath, covered with a layer of ethyl acetate (30 mL), and acidified with a dilute solution of KHSO4 to pH 2−3. The aqueous phase was extracted with ethyl acetate (15 mL), and the extraction was repeated. The ethyl acetate extracts were pooled, washed with water, dried over anhydrous Na2SO4, and evaporated in vacuo. The residue was recrystallized with DCM to afford 3.79 g of 16 as a white solid, 75% yield, mp 160−162 °C. MS (m/z): calcd for C12H15NO5, 253. LC−MS (m/z (rel intens)): 254.3 (M + H, 63). 1H NMR (CD3OD): δ 7.71 (d, J = 8.9 Hz, 1H), 7.11 (d, J = 2.0 Hz, 1H), 6.85 (dd, J = 8.7, 2.0 Hz, 1H), 1.52 (s, 9H). 13 C NMR (CD3OD): δ 172.75, 163.79, 154.03, 147.11, 131.68, 109.85, 107.42, 105.59, 80.86, 28.10. Synthesis of Benzyl 4-(tert-Butoxycarbonyl)-2-hydroxybenzoate (17). DCC (1.1 equiv, 9.0 g, 43.45 mmol) dissolved in DCM (5 mL) was added dropwise over 30 min to a stirred solution of 16 (10.0 g, 39.5 mmol), benzyl alcohol (3 equiv, 12.85 g, 118.5 mmol), and DMAP (0.1 equiv, 0.4 g, 0.39 mmol) in dry DCM/DMF (35 mL, 6:1). The mixture was stirred and heated to reflux overnight. The reaction

was monitored by TLC. After the reaction was complete, the solvents were removed under reduced pressure. Then 200 mL of ethyl acetate was added, the mixture was filtered to remove the DCU formed, and the organic phase was washed with brine and dried over Na2SO4. After filtration and removal of the solvent under reduced pressure, the crude product was purified by silica gel column chromatography using petroleum ether/ethyl acetate (30:1) as the eluent to afford 6.2 g of 17 as a white solid, 46% yield, mp 112−114 °C. MS (m/z): calcd for C19H21NO5, 343. LC−MS (m/z (rel intens)): 344.2 (M + H, 67). 1H NMR (CD3OD): δ 7.71 (d, J = 8.9 Hz, 1H), 7.32−7.45 (m, 5H), 7.12 (d, J = 2.0 Hz, 1H), 6.89 (dd, J = 8.9, 2.0 Hz, 1H), 5.32 (s, 2H), 1.50 (s, 9H). 13C NMR (CD3OD): δ 170.37, 163.52, 153.93, 147.41, 136.75, 131.20, 129.16, 128.91, 128.82, 110.13, 107.06, 105.65, 80.93, 67.15, 28.07. Synthesis of Benzyl 4-Amino-2-hydroxybenzoate (18). The solution of 17 (10.76 g, 31.3 mmol) in 50 mL of HCl in ethyl acetate (6 M) was stirred at room temperature for 2 h. The precipitated solid was filtered and washed with petroleum ether to afford 7.0 g of 18 as a white solid, 95% yield. MS (m/z): calcd for C14H13NO3, 243. LC−MS (m/z (rel intens)): 244.1 (M + H, 100). 1H NMR (DMSO-d6): δ 10.76 (s, 1H), 7.50 (dd, J = 8.7, 2.2 Hz, 1H), 7.33−7.47 (m, 5H), 6.48−6.70 (m, 4H), 6.16 (dd, J = 8.7, 2.2 Hz, 1H), 6.05 (d, J = 2.0 Hz, 1H), 5.32 (s, 2H). 13C NMR (DMSO-d6): δ 169.11, 162.80, 136.18, 131.18, 128.53, 128.11, 127.96, 107.10, 99.20, 65.52. Synthesis of tert-Butyl 1-(4-((benzyloxy)carbonyl)-3-hydroxyphenyl)-2,5-dimethyl-1H-pyrrole-3-carboxylate (19). To a solution of 18 (6.8 g, 24 mmol) in 100 mL of toluene were added Nmethylmorpholine (1 equiv, 2.69 mL, 24 mmol) and 9 (1.1 equiv, 5.72 g, 26.4 mmol). The mixture was heated to reflux, 0.34 g of ptoluenesulfonic acid was added, and the mixture continued to reflux for 3 h with monitoring by TLC. After the reaction was complete, the solvents were removed under reduced pressure. Then 80 mL of ethyl acetate was added, and the organic phase was washed with brine and dried over Na2SO4. After filtration and evaporation under reduced pressure, the residue was purified by silica gel column chromatography to yield 6.04 g of 19 as a colorless oil, yield 60%. MS (m/z): calcd for C25H27NO5, 421. LC−MS (m/z (rel intens)): 422.2 (M + H, 10). 1H NMR (DMSO-d6): δ 10.74 (s, 1H), 7.93 (d, J = 8.4 Hz, 1H), 7.37− 7.52 (m, 5H), 6.94 (d, J = 2.0 Hz, 1H), 6.86 (dd, J = 8.4, 2.0 Hz, 1H), 6.20 (s, 1H), 5.41 (s, 2H), 2.21 (s, 3H), 1.95 (s, 3H), 1.48 (s, 9H). 13C NMR (DMSO-d6): δ 167.45, 164.07, 160.24, 142.79, 135.64, 134.33, 131.43, 128.59, 128.32, 128.15, 127.77, 119.32, 117.03, 114.02, 112.76, 108.11, 78.53, 66.71, 28.15, 12.34, 12.13. Synthesis of 1-(4-((Benzyloxy)carbonyl)-3-hydroxyphenyl)-2,5dimethyl-1H-pyrrole-3-carboxylic Acid (20). The solution of 19 (6.04 g, 14.3 mmol) in 50 mL of HCl in ethyl acetate (6 M) was stirred at room temperature for 1 h. The reaction mixture was evaporated to remove the solvent. The precipitated solid was filtered and washed with petroleum ether to afford 3.58 g of 20 as a brown solid, 69% yield, mp 190−192 °C. MS (m/z): calcd for C21H19NO5, 365. LC−MS (m/z (rel intens)): 366.1 (M + H, 43). 1H NMR (DMSO-d6): δ 11.73 (s, 1H), 10.72 (s, 1H), 7.93 (d, J = 8.4 Hz, 1H), 7.37−7.52 (m, 5H), 6.96 (d, J = 2.0 Hz, 1H), 6.88 (dd, J = 8.4, 2.0 Hz, 1H), 6.24 (s, 1H), 5.41 (s, 2H), 2.23 (s, 3H), 1.96 (s, 3H). 13C NMR (DMSO-d6): δ 167.46, 166.12, 160.26, 142.82, 135.62, 134.75, 131.37, 128.55, 128.28, 128.13, 127.76, 119.29, 116.98, 113.92, 111.90, 108.25, 66.69, 12.37, 12.05. Synthesis of 1-(4-Carboxy-3-hydroxyphenyl)-2,5-dimethyl-1Hpyrrole-3-carboxylic Acid (21). At 0 °C, a solution of 20 (0.5 g, 1.37 mmol) in 10 mL of HF was stirred for 1 h. After evaporation, the residue was dissolved in ether and evaporated under reduced pressure to remove the solvent. The residue was recrystallized with ethyl acetate to provide 0.10 g of 21 as a gray solid, 27% yield, mp 204−206 °C. MS (m/z): calcd for C14H13NO5, 275. LC−MS (m/z (rel intens)): 276.1 (M + H, 100). 1HNMR: δ 11.83 (s, 2H), 7.93 (d, J = 8.4 Hz, 1H), 6.95 (d, J = 1.7 Hz, 1H), 6.86 (dd, J = 8.4, 1.7 Hz. 1H), 6.24 (s, 1H), 2.24 (s, 3H), 1.97 (s, 3H). 13C NMR (DMSO-d6): δ 171.23, 166.16, 161.54, 143.01, 134.80, 131.38, 127.78, 119.11, 116.73, 113.29, 111.86, 108.23, 12.40, 12.08. J

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flanked by five residues on either side. Mixing FeII(env2.0)3 with a fluorescein-labeled pocket-binding C-peptide, C18-FL, caused quenching of fluorescence, which could be reversed in the presence of a competitive inhibitor. Typically, 7 μM binding sites (three per receptor trimer) and 1 μM C18-FL were used in the assay. Binding Assays by Size-Exclusion Chromatography. N36 was mixed with conjugated peptides (final concentration 0.20 mM) at a molar ratio of 1:1 in 50 mM sodium phosphate/150 mM NaCl (pH 7.2) and incubated at 37 °C for 30 min. The mixture or peptide (20 μL) was applied to the Phenomenex BioSep-SEC-s2000, 300 × 7.80 mm HPLC column equilibrated with H2O and eluted at 0.8 mL/min, and fractions were monitored at 210 nm. Proteinase K Digestion Experiments. To test the proteinase K sensitivity of C-peptides, each peptide (1 mg/mL, 500 μL) was mixed with proteinase K (working concentration of 20 ng/mL), and then the mixture was incubated at 37 °C. At different times points (0, 5, 15, 30, 60, 120, and 240 min), 10 μL samples were removed and mixed with 30 μL of CH3CN to stop the reaction. The samples were centrifuged, and the supernatant was analyzed by HPLC using the same conditions as described above. Computational Modeling. The conformation of the coiled-coil chimera protein TLT was studied by molecular dynamics simulation using AMBER 8.0 on an SGI Altix 350 workstation (Fremont, CA).39 Molecular models were constructed using SWISS-MODEL Web servers on the basis of the structure of the 6HB (PDB 1AIK). The peptide was immersed in a rectangular box with TIP3P water before energy minimization. Two stages of minimization and two stages of equilibration were conducted to obtain an initial state for a further 5 nm simulation, which was conducted at a constant temperature of 300 K and a constant pressure of 1 atm, without restraints on the system. The coordinates of the entire system at each time point (10 ps) of the output trajectories were saved. PBTOT/GBTOT was calculated using the mm_pbsa.pl program.

Peptide Synthesis. Peptides were synthesized using a Liberty automated microwave peptide synthesizer (CEM Co., Matthews, NC) with a standard solid-phase Fmoc chemistry protocol. All protected amino acids used were purchased from GL Biochem Ltd. (Shanghai, China). Rink amide resin and MBHA resin (0.38−0.45 mmol/g, Nankai Hecheng S&T Co. Ltd., Tianjin, China) were used. Coupling of the amino acids was achieved using O-benzotriazol-1-yl-N,N,N′,N′tetramethyluronium hexafluorophosphate (HBTU; GL Biochem, Shanghai, China) and diisopropylethylamine (DIEA; Acrose) as an activator and an active base, respectively, in DMF solution. The Fmoc protective group was removed using 20% piperidine/DMF. Between every coupling or Fmoc removal, the resin was washed five times with DMF and three times with DCM. The carboxyl termini were amidated upon cleavage from the resin, and the amino termini were capped with acetic acid anhydride, except conjugated peptides. The peptides were cleaved from the resin and deprotected with reagent K, which contained 82.5% trifluoroacetic acid, 5% thioanisole, 5% m-cresol, 5% water, and 2.5% ethanedithiol. The crude products were precipitated with cold diethyl ether and lyophilized. The crude peptide products were purified by preparative reversed-phase HPLC using a Waters preparative HPLC system (PrepLC 4000): gradient elution of 30− 50% solvent B in solvent A (0.1% trifluoroacetic acid in H2O, solvent A; 0.1% trifluoroacetic acid in 70% CH3CN/H2O, solvent B) over 60 min at 16 mL/min on a Waters X-bridge C8, 10 μm, 19.5 mm × 250 mm column. Analytical RP-HPLC was performed on an RP-C8 column (Zorbax Eclipse XDB-C8, 5 μm, 4.6 × 150 mm) with gradient elution of 5−100% solvent B in solvent A over 25 min at a flow rate of 1 mL/min. The compounds were detected by UV absorption at 220 nm with SHIMADZU SPD-10A. All peptides were purified to >98% purity. The molecular weight of the peptides was confirmed by MALDI-TOF-MS (Autoflex III, Bruker Daltonics). Cell−Cell Fusion Assay. Cell−cell fusion assays were performed as previously described.40 HL2/3 cells (Drs. Barbara Felber and George Pavlakis), which stably express HIV Gag, Env, Tat, Rev, and Nef proteins, and TZM-bl cells (Drs. John C. Kappes, Xiaoyun Wu), which stably express large amounts of CD4 and CCR5, were obtained from the NIH AIDS Reference and Reagent Program. TZM-bl cells (2.5 × 104 cells/well) and HL2/3 cells (7.5 × 104 cells/well) were coincubated in 96-well plates (Corning Costar) at 37 °C in 5% CO2 in the presence of different concentrations of inhibitors. After a 6−8-h incubation, the medium was aspirated, the cells were washed and lysed, and luciferase activity was measured using the Luciferase Assay System (Promega, Madison, WI) on a SpectraMax M5 plate reader (Molecular Devices, Sunnyvale, CA). HIV-1 Infection Assay. For measuring the inhibitory activity of the peptides on infection of HIV-1 IIIB and T20-resistant HIV-1 isolate, 1 × 104 MT-2 cells were infected with 100 TCID50 HIV-1IIIB in the presence or absence of the peptides at graded concentrations. On the fourth day postinfection, the culture supernatants were collected for detection of HIV-1 Gag antigen p24 using an ELISA. Cytotoxicity Assay. For determining the cytotoxicity of the peptides, TZM-b1 cells were seeded in 96-well cell culture plates (Corning, Inc., Corning, NY) at a density of approximately 1 × 104 cells/well in DMEM supplemented with 10% (v/v) FBS and incubated for 24 h. The cells were washed twice with PBS and incubated with 100 μL of peptide at the indicated concentration at 37 °C for 24 h. The cells were washed with PBS to remove the peptide. Thereafter, 20 μL of Cell Titer 96 AQueous One Solution Reagent (Promega Corp, Madison, WI) was added to each well, followed by an incubation period of 1 h. The solution absorbance was measured at 490 nm using a SpectraMax M5 (Molecular Devices). PBS was used as a negative control. Cell viability data were obtained by calculating the ratio of viable cells in the treated cultures to the untreated control cells. The experiment was repeated five times for each peptide concentration. Binding Affinity Assay. The ability of the conjugated peptides to bind to the hydrophobic pocket on the gp41 N-peptide coiled coil of HIV-1 was determined using a fluorescence intensity assay as previously described. 32,41 Briefly, a metallopeptide receptor, FeII(env2.0)3, was used to mimic the hydrophobic pocket in the gp41 coiled coil. env2.0 contains 17 hydrophobic pocket residues

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ASSOCIATED CONTENT

S Supporting Information *

HPLC purity and characterization data of the compounds and pharmacokinetic experiments and results. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: 86-10-6816-9363 (K.L.); 86-21-54237673 (S.J.). Fax: 86-10-6821-1656 (K.L.); 86-21-54237465 (S. J.). E-mail: [email protected] (K.L.); [email protected] (S.J.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported, in part, by grants from the National Science Foundation of China (81072581 and 81273560) and a National Science and Technology Major Project of China grant (2012ZX09301003).

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ABBREVIATIONS USED CHR, C-terminal heptad repeat; NHR, N-terminal heptad repeat; 6HB, six-helix bundle; FDA, Food and Drug Administration; Aoc-βAla-P26, P26 conjugated with an A12 derivative through a β-alanine; Noc-βAla-P26, P26 conjugated with an NB-2 derivative through a β-alanine; DCC, N,N′dicyclohexylcarbodiimide; DMAP, 4-(dimethylamino)pyridine

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REFERENCES

(1) Chan, D. C.; Fass, D.; Berger, J. M.; Kim, P. S. Core structure of gp41 from the HIV envelope glycoprotein. Cell 1997, 89, 263−273.

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Article

(2) Chan, D. C.; Kim, P. S. HIV entry and its inhibition. Cell 1998, 93, 681−684. (3) Ashkenazi, A.; Shai, Y. Insights into the mechanism of HIV-1 envelope induced membrane fusion as revealed by its inhibitory peptides. Eur. Biophys. J. 2011, 40 (4), 349−357. (4) Cai, L.; Jiang, S. Development of peptide and small-molecule HIV-1 fusion inhibitors that target gp41. ChemMedChem 2010, 5 (11), 1813−1824. (5) Naider, F.; Anglister, J. Peptides in the treatment of AIDS. Curr. Opin. Struct. Biol. 2009, 19, 473−482. (6) Gallo, S. A.; Puri, A.; Blumenthal, R. HIV-1 gp41 six-helix bundle formation occurs rapidly after the engagement of gp120 by CXCR4 in the HIV-1 Env-mediated fusion process. Biochemistry (Moscow) 2001, 40, 12231−12236. (7) Liu, S.; Wu, S.; Jiang, S. HIV entry inhibitors targeting gp41: from polypeptides to small-molecule compounds. Curr. Pharm. Des. 2007, 13 (2), 143−162. (8) Pan, C.; Liu, S.; Jiang, S. HIV-1 gp41 fusion intermediate: a target for HIV therapeutics. J. Formosan Med. Assoc. 2010, 109 (2), 94−105. (9) Liu, S.; Zhao, Q.; Jiang, S. Determination of the HIV-1 gp41 fusogenic core conformation modeled by synthetic peptides: applicable for identification of HIV-1 fusion inhibitors. Peptides 2003, 24 (9), 1303−1313. (10) Jiang, S.; Zhao, Q.; Debnath, A. K. Peptide and non-peptide HIV fusion inhibitors. Curr. Pharm. Des. 2002, 8, 563−580. (11) Wild, C. T.; Shugars, D. C.; Greenwell, T. K.; McDanal, C. B.; Matthews, T. J. Peptides corresponding to a predictive α-helical domain of human immunodeficiency virus type 1 gp41 are potent inhibitors of virus infection. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 9770−9774. (12) Cai, L.; Balogh, E.; Gochin, M. Stable extended human immunodeficiency virus type 1 gp41 coiled coil as an effective target in an assay for high-affinity fusion inhibitors. Antimicrob. Agents Chemother. 2009, 53 (6), 2444−2449. (13) He, Y.; Cheng, J.; Lu, H.; Li, J.; Hu, J.; Qi, Z.; Liu, Z.; Jiang, S.; Dai, Q. Potent HIV fusion inhibitors against enfuvirtide-resistant HIV1 strains. Proc. Natl. Acad. Sci. U.S.A. 2008, 105 (42), 16332−16337. (14) Matthews, T.; Salgo, M.; Greenberg, M.; Chung, J.; DeMasi, R.; Bolognesi, D. Enfuvirtide: the first therapy to inhibit the entry of HIV1 into host CD4 lymphocytes. Nat. Rev. Drug Discovery 2004, 3, 215− 225. (15) Ferrer, M.; Kapoor, T. M.; Strassmaier, T.; Weissenhorn, W.; Skehel, J. J.; Oprian, D.; Schreiber, S. L.; Wiley, D. C.; Harrison, S. C. Selection of gp41-mediated HIV-1 cell entry inhibitors from biased combinatorial libraries of non-natural binding elements. Nat. Struct. Biol. 1999, 6 (10), 953−960. (16) Gochin, M.; Zhou, G. Amphipathic properties of HIV-1 gp41 fusion inhibitors. Curr. Top. Med. Chem. 2011, 11, 3022−3032. (17) Jiang, S.; Tala, S. R.; Lu, H.; Abo-Dya, N. E.; Avan, I.; Gyanda, K.; Lu, L.; Katritzky, A. R.; Debnath, A. K. Design, synthesis, and biological activity of novel 5-((arylfuran/1H-pyrrol-2-yl)methylene)-2thioxo-3-(3-(trifluoromethyl)phenyl)thiazolidin-4-ones as HIV-1 fusion inhibitors targeting gp41. J. Med. Chem. 2011, 54, 572−579. (18) Zhou, G.; Ferrer, M.; Chopra, R.; Kapoor, T. M.; Strassmaier, T.; Weissenhorn, W.; Skehel, J. J.; Oprian, D.; Schreiber, S. L.; Harrison, S. C.; Wiley, D. C. The structure of an HIV-1 specific cell entry inhibitor in complex with the HIV-1 gp41 trimeric core. Bioorg. Med. Chem. 2000, 8, 2219−2238. (19) Liu, S.; Boyer-Chatenet, L.; Lu, H.; Jiang, S. Rapid and automated fluorescence-linked immunosorbent assay for high throughput screening of HIV-1 fusion inhibitors targeting gp41. J. Biomol. Screening 2003, 8, 685−693. (20) Jiang, S.; Lu, H.; Liu, S.; Zhao, Q.; He, Y.; Debnath, A. K. Nsubstituted pyrrole derivatives as novel human immunodeficiency virus type 1 entry inhibitors that interfere with the gp41 six-helix bundle formation and block virus fusion. Antimicrob. Agents Chemother. 2004, 48 (11), 4349−4359. (21) Tan, J.; Zhang, B.; Cong, X.; Yang, L.; Liu, B.; Kong, R.; Kui, Z.; Wang, C.; Hu, L. Computer-aided design, synthesis, and biological

activity evaluation of potent fusion inhibitors targeting HIV-1 gp41. Med. Chem. 2011, 7, 309−316. (22) Liu, K.; Lu, H.; Hou, L.; Qi, Z.; Teixeira, C.; Barbault, F.; Fan, B.; Liu, S.; Jiang, S.; Xie, L. Design, synthesis, and biological evaluation of N-carboxyphenylpyrrole derivatives as potent HIV fusion inhibitors targeting gp41. J. Med. Chem. 2008, 51 (24), 7843−7854. (23) Wang, Y.; Lu, H.; Zhu, Q.; Jiang, S.; Liao, Y. Structure-based design, synthesis and biological evaluation of new N-carboxyphenylpyrrole derivatives as HIV fusion inhibitors targeting gp41. Bioorg. Med. Chem. Lett. 2010, 20 (1), 189−192. (24) Frey, G.; Rits-Volloch, S.; Zhang, X. Q.; Schooley, R. T.; Chen, B.; Harrison, S. C. Small molecules that bind the inner core of gp41 and inhibit HIV envelope-mediated fusion. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 13938−13943. (25) Xu, Y.; Lu, H.; Kennedy, J. P.; Yan, X.; McAllister, L. A.; Yamamoto, N.; Moss, J. A.; Boldt, G. E.; Jiang, S.; Janda, K. D. Evaluation of “credit card” libraries for inhibition of HIV-1 gp41 fusogenic core formation. J. Comb. Chem. 2006, 8, 531−539. (26) Balogh, E.; Wu, D.; Zhou, G.; Gochin, M. NMR second site screening for structure determination of ligands bound in the hydrophobic pocket of HIV-1 gp41. J. Am. Chem. Soc. 2009, 131, 2821−2823. (27) Gochin, M.; Zhou, G.; Phillips, A. H. Paramagnetic relaxation assisted docking of a small indole compound in the HIV-1 gp41 hydrophobic pocket. ACS Chem. Biol. 2010, 6, 267−274. (28) Wang, C.; Zhao, M.; Qiu, X.; Peng, S. The synthesis and immunosuppressive activities of steroid-urotoxin linkers. Bioorg. Med. Chem. 2004, 12, 4403−4421. (29) Chan, D. C.; Chutkowski, C. T.; Kim, P. S. Evidence that a prominent cavity in the coiled coil of HIV type 1 gp41 is an attractive drug target. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 15613−15617. (30) Mo, H.; Konstantinidis, A. K.; Stewart, K. D.; Dekhtyar, T.; Ng, T.; Swift, K.; Matayoshi, E. D.; Kati, W.; Kohlbrenner, W.; Molla, A. Conserved residues in the coiled-coil pocket of human immunodeficiency virus type 1 gp41 are essential for viral replication and interhelical interaction. Virology 2004, 329, 319−327. (31) Teixeira, C.; Barbault, F.; Rebehmed, J.; Liu, K.; Xie, L.; Lu, H.; Jiang, S.; Fan, B.; Maurel, F. Molecular modeling studies of Nsubstituted pyrrole derivatives-Potential HIV-1 gp41 inhibitors. Bioorg. Med. Chem. 2008, 16 (6), 3039−3048. (32) Cai, L.; Gochin, M. A novel fluorescence intensity screening assay identifies new low-molecular-weight Inhibitors of the gp41 coiled-coil domain of human immunodeficiency virus type 1. Antimicrob. Agents Chemother. 2007, 51 (7), 2388−2395. (33) Lu, L.; Tong, P.; Yu, X.; Pan, C.; Zou, P.; Chen, Y.; Jiang, S. HIV-1 variants with a single-point mutation in the gp41 pocket region exhibiting different susceptibility to HIV fusion inhibitors with pocketor membrane-binding domain. Biochim. Biophys. Acta 2012, 1818, 2950−2957. (34) Naito, T.; Izumi, K.; Kodama, E.; Sakagami, Y.; Kajiwara, K.; Nishikawa, H.; Watanabe, K.; Sarafianos, S. G.; Oishi, S.; Fujii, N.; Matsuoka, M. SC29EK, a peptide fusion inhibitor with enhanced αhelicity, inhibits replication of human immunodeficiency virus type 1 mutants resistant to enfuvirtide. Antimicrob. Agents Chemother. 2009, 53 (3), 1013−1018. (35) Mink, M.; Mosier, S. M.; Janumpalli, S.; Davison, D.; Jin, L.; Melby, T.; Sista, P.; Erickson, J.; Lambert, D.; Stanfield-Oakley, S. A.; Salgo, M.; Cammack, N.; Matthews, T.; Greenberg, M. L. Impact of human immunodeficiency virus type 1 gp41 amino acid substitutions selected during enfuvirtide treatment on gp41 binding and antiviral potency of enfuvirtide in vitro. J. Virol. 2005, 79, 12447−12454. (36) Wei, X.; Decker, J. M.; Liu, H.; Zhang, Z.; Arani, R. B.; Kilby, J. M.; Saag, M. S.; Wu, X.; Shaw, G. M.; Kappes, J. C. Emergence of resistant human immunodeficiency virus type 1 in patients receiving fusion inhibitor (T-20) monotherapy. Antimicrob. Agents Chemother. 2002, 46, 1896−1905. (37) Liu, S.; Jing, W.; Cheung, B.; Lu, H.; Sun, J.; Yan, X.; Niu, J.; Farmar, J.; Wu, S.; Jiang, S. HIV gp41 C-terminal heptad repeat L

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Journal of Medicinal Chemistry

Article

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

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