key: cord-0792878-7a6nkppa
authors: Liu, Yonghua; Li, Jianrui; Gu, Yuxi; Ma, Ling; Cen, Shan; Peng, Zonggen; Hu, Laixing
title: Synthesis and structure-activity relationship study of new biaryl amide derivatives and their inhibitory effects against hepatitis C virus
date: 2022-01-15
journal: Eur J Med Chem
DOI: 10.1016/j.ejmech.2021.114033
sha: a53f0eb894da95ce11cb8f15f8f598b7cd64185f
doc_id: 792878
cord_uid: 7a6nkppa

A series of novel biaryl amide derivatives were synthesized and evaluated for anti-HCV virus activity. Some significant SARs were uncovered. The intensive structural modifications led to fifteen novel compounds with more potent inhibitory activity compared to the hit compounds IMB 26 and IMB1f. Among them, compound 80 was the most active, with EC(50) values almost equivalent to the clinical drug telaprevir (EC(50) = 15 nM). Furthermore, it also had a good safety and in vitro and oral pharmacokinetic (oral bioavailability in rats: 34%) profile, suggesting a highly drug-like nature. Compound 80represents a more promising scaffold for anti-HCV virus activity for further study.

Hepatitis C virus (HCV) infection seriously threatens global health, with approximately 170 million individuals infected and causing up to 350,000 related deaths per year worldwide [1, 2] . HCV infection is an insidious disease, and the early stages of infection are largely asymptomatic such that most are unaware of their infection. HCV-infected people are at high risk for developing chronic liver disease, cirrhosis, and hepatocellular carcinoma [3] . As many as 50e80% of patients newly infected with HCV develop chronic infection; of those chronically infected individuals, approximately 30% progress to liver cirrhosis, and up to 4% will go on to develop life-threatening hepatocellular carcinoma and end-stage liver disease [2, 4] . The combination of pegylatedinterferon (PEG-IFN) with ribavirin is the conventional treatment for HCV infection, which requires 24e48 weeks and causes frequent, sometimes severe, side effects. Moreover, the regimen is only effective in the range of one half to two-thirds of persons treated, depending on clinical stage and genotype [5] . The recent approval of several small-molecule direct-acting antiviral (DAA) therapies for HCV infection has dramatically improved the standard of care for HCV. These drugs target viral proteins (NS3/4 A protease, NS5B polymerase, and NS5A) involved in the replication stage of HCV infection [6] . For example, Epclusa: a combination of sofosbuvir (a nucleotide analog inhibitor of HCV NS5B polymerase) with velpatasvir (NS5A inhibitor), is the backbone of the first oral, pangenotype, single-tablet regimen for the treatment of adults with genotype 1e6 chronic HCV infection [7] . Two newly approved combination hepatitis C drugs (Zepatier: elbasvir þ grazoprevir and Viekira and PaK: ombitasvir þ paritaprevir þ ritonavir þ dasabuvir) have demonstrated improved safety and efficacy for treating genotype 1 or 4 HCV-infected persons [8] . Although these treatments offer renewed hope toward curing HCV infection, the issues of drug resistance, narrow genotype specificity, lack of vaccines, and high cost remain [9e12] . It is still imperative to develop new anti-HCV agents, especially those with novel mechanisms of action (MOAs) and new molecular structures.

Many compounds with biaryl amide moieties have been studied continuously because of their diverse roles in biological functions and diseases, such as viral infection [13] , bacterial infection [14] , diabetes [15] , spinal muscular atrophy [16] , human African trypanosomiasis [17] and cancers [18e22] . As shown in Fig. 1 , ML336 was found to inhibit potently several Venezuelan equine encephalitis viruses in the low nanomolar range without cytotoxicity [13] . Compound 2 was found to have antibiofilm activity as an adjuvant that enhances the susceptibility of drug-resistant strains of bacteria, such as Acinetobacter baumannii and Pseudomonas aeruginosa, to meropenem [14] . Compound 3 showed potent activity in ex vivo diabetic retinopathy models as a new class of selective Rho kinase inhibitors [15] . SCYX-7158 was used to treat human African trypanosomiasis(HAT) and has begun human clinical trials [17] . Compounds 5e9 show effective anticancer activities. Compound 5inhibited autotoxin-dependent invasion of A2058 human melanoma cells in vitro and reduced B16 melanoma metastasis in vivo [18] . Compound 8 was found to be an efficacious RAF protein inhibitor targeting RAS mutant cancer [21] . Compound 9 (Ponatinib) is an orally active multitargeted kinase inhibitor and has been approved to treat chronic myeloid leukemia by the FDA [22] .

Human APOBEC3G (apolipoprotein B messenger RNA [mRNA]editing enzyme catalytic polypeptide-like 3G, hA3G) is a cytidine deaminase and belongs to the APOBEC superfamily. Accumulated evidence shows that hA3G in human T lymphocytes represents an innate immunity factor that displays broad-spectrum antiviral activity, including inhibiting human immunodeficiency virus type 1(HIV-1) [23e26], hepatitis B virus (HBV) [27] , HCV [28, 29] , paramyxovirus [30] , enterovirus 71(EV71) [31, 32] , and T-cell leukemia virus type 1(HTLV-1) [33] . In continuation of our research on antiviral drugs, some biaryl amid ederivatives were found to display significant anti-HIV-1, anti-HCV, and anti-EV17 activities (Fig. 2 ). An antiviral mechanism study demonstrated that IMB-26, as an hA3G stabilizer, directly binds to the hA3G protein and infectively protects hA3G from Vif-mediated degradation and inhibits HIV-1 viral replication [23] . IMB-1f, as an analog of IMB-26, inhibited hepatitis C virus replication [29, 31] . IMB-Z was found to increase hA3G encapsidation into EV17 progeny virion particles and to inhibit EV17 replication [32] . In addition, Young et al. reported that biaryl amide derivative 10, as a small molecule inhibitor of microRNA miR-122, can reduce HCV RNA levels [34] . Since these biaryl amide derivatives target host innate components (hA3G is an innate immunity factor, and microRNA miR-122 is a human liver-specific miRNA), the virus will most likely not be able to develop resistance to these molecules. Therefore, biaryl amide derivatives could be a new class of broad spectrum antiviral agents that merit exploration.

Here, we synthesized a series of new N-aryl benzamide analogs by changing R 1 , R 2 , and R 3 (Fig. 2) and evaluated their ability to inhibit hepatitis C virus replication in acutely infected Huh7.5 cells. The medicinal chemistry effort led to the discovery of more potent new lead compounds of anti-HCV 68, 78, and 80, which exhibited strong anti-HCV activity comparable to the clinical drug VX950 (EC 50 ¼ 0.015e0.083 mM). More importantly, a novel pharmacophore of N-aryl-(3-nitro-4-alkoxy)benzamide against HCV infection was revealed by structure-activity relationship (SAR) analysis. The physicochemical and ADME properties of compound 80 were evaluated. The primary study of some compounds inhibiting Vifmediated hA3G degradation progressed.

The synthesis of compounds 13 (IMB-1f) and 14 was performed according to a previously reported method (Scheme 1) [31] . Hydrogenation of the nitro group followed by amide coupling reaction with propanoyl chloride furnished compounds13 and 14, respectively. Compounds 16e20 were obtained by amide coupling reaction between various substituted anilines and benzoic acid derivative 15, which was obtained by a selective amide coupling reaction between 4-methoxy-3-aminobenzoic acid and propanoyl chloride.

Compounds 22e35 were obtained as depicted in Scheme 2. 4hydroxy-3-nitrobenzoic acid acted as a starting compound through an amide coupling reaction with 4-methoxy aniline to afford intermediate 21, which was reacted with various desired alkyl bromides by nucleophilic substitution to afford corresponding nitro intermediates 22e25. Hydrogenation of the nitro group offered corresponding amino derivatives 29e32, which were then reacted with propanoyl chloride to give final products 36e39. Compound 25 was reacted with various secondary amines in the presence of potassium carbonate to afford corresponding nitro compounds 26e28. Hydrogenation of the nitro group offered corresponding amino intermediates 33e35, which were reacted with propanoyl chloride to give final products 40e42.

Compound 45 was synthesized through a 4-step reaction, including two amide coupling reactions, an intramolecular nucleophilic substitution, and a hydrolysis reaction. Using methyl 3,4dihydro-2H-benzo [1, 4] oxazine-6-carboxylate as the starting compound, compound 48 was synthesized through hydrolysis reactions and an amide coupling reaction (Scheme 3).

Compounds 50e57 were obtained as depicted in Scheme 4. Starting from methyl 4-hydroxy-3-nitrobenzoate, a nucleophilic substitution reaction with 2-bromo propane and subsequent hydrolysis reaction under basic conditions afforded 49, which was coupled with substituted anilines to afford corresponding nitro compounds 50 and 51. Reduction of the nitro group with palladium-catalyzed hydrogenation offered corresponding amino derivatives 52 and 53. Compounds 52 and 53 were reacted with propanoyl chloride or 2-bromo propanoyl chloride in the presence of Et 3 N to give products 54, 56, 55, and 57, respectively.

Compounds 60e70 were obtained according to the synthetic route depicted in Scheme 5. Starting from various R 2 -substituted methyl 4-hydroxy benzoate analogs 58aee, a nucleophilic substituted reaction with 1-bromo-3-chloropropane and subsequent hydrolysis reaction yielded the corresponding benzoic acid derivatives 59aee. Compounds 59aee were coupled with 3trifluoromethyl-4-(4-methylpiperazin-1-yl)-aniline in the presence of EDCI and DMAP to afford the corresponding amide derivatives 60e64. Reduction of the nitro compound 60 via palladium-catalyzed hydrogenation offered the corresponding amino product 65, which was reacted with propanoyl chloride to give the desired product 66. Compound 58a was reacted with different brominated alkanes and subsequently hydrolyzed to give intermediates 67aec, which were coupled with 3-trifluoromethyl-4-(4-methylpiperazin-1-yl)-aniline to give compounds 68e70. Compounds 71e80 were prepared as described above through an amide coupling reaction between compound 59a or 67a and various substituted anilines (Scheme 6). The substituted anilines 83aef, 86, and 88 that were not commercially available were readily synthesized by a short three-step sequence (Scheme 7). Briefly, the bromination of 2-trifluoromethyl-4-nitrotoluene with N-bromosuccinimide followed by a nucleophilic substituted reaction with various appropriate second amines gave nitro derivatives 82aef, which were hydrogenated by palladium-catalyzed hydrogenation reduction to afford corresponding amino derivatives 83aef. Compound 88 was synthesized by a nucleophilic substituted reaction followed by a reduction of the nitro group.

All analogs were screened for inhibition of HCV RNA replication Scheme 5. Synthesis of compounds 60e70. Reagents and conditions: (i) a. 1-bromo-3-chloropropane (1-bromo-2-chloroethane for 67a;1-bromo-4-chlorobutane for 67b;1-bromo-5-chloropentane for 67c), K 2 CO 3 , NaI, DMF, 65 C, 5 h; b. LiOH, MeOH/THF (1:1), 0 C, 1 h. (ii) 3-trifluoromethyl-4-(4-methylpiperazin-1-yl)-aniline, EDCI, DMAP, CH 2 Cl 2 , rt, 6 h (iii) H 2 ( displayed suitable values of physicochemical parameters, such as the calculated LogP (cLogP < 5) and topological polar surface area (tPSA between 50 and 100 Å), and could have good bioavailability and drug-like features [35, 36] .

As shown in Fig. 2 , the structural elements of R 1 , R 2 , and R 3 were first investigated during the SAR study. In a previous report, 4-OCH 3 in the A ring was considered to be important for antiviral activity, and replacement of the a-bromocarbonyl group with a propionyl group led to lower cytotoxicity [29, 31] . Therefore, we first fixed R 2 as a propionamino group and R 3 as a methyl group and varied the R 1 moiety. Compounds 14 and 16e20 were synthesized to probe the effect of R 1 moieties on anti-HCV activity. As shown in Table 1 , compound 18 with a 4-(4-methylpiperazin-1-yl)-methyl-3trifluoromethyl-phenyl group, which is an important part of ponatinib, a clinical antitumor drug with multitargeted tyrosinekinase inhibition, exhibited definitive anti-HCV activity similar to the reported compounds, IMB-26 and 13 (IMB-1f), with selectively index (SI) values higher than 20 and EC 50 values lower than 1.5 mM. Compounds 14, 19 , and 20 showed rather modest anti-HCV activity, but IS values were higher than those of IMB-26. Compounds 16 and 17 completely lost their antiviral activity. The SAR data pointed to the choice of the R 1 moieties being crucial for high potency, which was not limited to lipophilic or hydrophilic groups but would need suitable volume and polarity distributions.

As compound 13 had a high SI value, we fixed R 1 as a 4-methoxy phenyl group and R 2 as a propionamino group and varied the R 3 moiety to synthesize compounds 36e42 ( Table 2) . Replacing the methyl group in the R 3 moiety with ethyl or propanyl groups, as shown in compounds 36 and 37, slightly decreased anti-HCV activities. Compound 38 with isopropyl substitution displayed higher anti-HCV activity and SI value than IMB-26 and compound 13. Introduction of the 3-chloro group in compound 37, as shown in compound 39, retained partial antiviral activities. Installation of bimethylamino, 4-methyl piperizin-1-yl, or morpholinyl hydrophilic moieties on the head of R 3 (40, 41, and 42) resulted in significant activity loss. Cyclizing R 2 and R 3 moieties gave compounds 45 and 48. Compound 45 retained partial antiviral activities, while 48 lost activity. The above results showed that the R 3 position may be lipophilic required for anti-HCV activity, and the isopropyl moiety is the most advantageous group, as shown in Table 2 .

Next, we investigated the importance of the R 2 moiety for anti-HCV activity. As shown in Table 3 , because intermediates 12 and 24e26 with nitro groups in the R 2 position showed modest inhibitory activity against HCV with EC 50 values in the range of 2.39e8.96 mM, we synthesized two series of compounds (50e53 and 54e57) with four different R 2 moieties and intensively investigated the relationship between R 2 moieties and anti-HCV activity. In the two series of compounds, compounds 50 and 54 with nitro groups displayed the most dominant anti-HCV activity (EC 50 ¼ 0.095 and 3.10 mM, respectively) in the four kinds of substituent derivatives, and the corresponding reduction products 51 and 55 showed the weakest activity. Compounds 52, 53, 56, and 57 with amide groups in the R 2 moiety showed moderate inhibitory activity. Compounds 53 and 57 with a-bromo propionyl groups displayed higher cytotoxicity than compounds 52 and 56 with propionyl groups, which was consistent with previous results [29] . Notably, compounds 54e57 exhibited higher potent activity than IMB 26 and compound 13 with EC 50 values in the range of 0.09e1.32 mM, which means that the 4-(4-methylpiperazin-1-yl)methyl-3-trifluoromethyl-phenyl group may be more dominant than the 4-methoxy phenyl group in the anti-HCV activity. Compound 25 with a 3-chloropropyl group at the R 3 position exhibited similar antiviral activity to compound 24 with an isopropyl group but possessed lower cytotoxicity. To obtain analogs with various structures and good solubility, compound 60 was synthesized. Compound 60 not only unexpectedly displayed the highest inhibitory activity (EC 50 ¼ 0.044 mM) but also exhibited the highest SI value (SI ¼ 154) among the above target compounds. Replacing the nitro group (60) with a fluoro (61), chloro (62) trifluoromethyl (63) or sulfamoyl group (64) deteriorated the anti-HCV activity. Replacing the nitro group (60) with an amino or amido group (65 and 66) decreased the activity, which is consistent with the above two series of compounds 50e53 and 54e57. These data indicate that the nitro group in the R 2 fragment would be an advantage in the anti-HCV activity of these compounds. Decreasing the tether length by one eCH 3 group in the R 3 fragment (68) retained similar activity as compound 60. Increasing the tether length by one or two eCH 3 groups in the R 3 fragment (69 and 70, respectively) did not improve the potency but rather the cell toxicity.

Because of the dominance of the 4-(4-methylpiperazin-1-yl)methyl-3-trifluoromethyl-phenyl group in the anti-HCV activity, novel analogs 71e79 were synthesized to explore further the structure-activity relationship (Table 4 ). Replacing the trifluoromethyl group in the B ring with a cyano group afforded less potent activity, as shown in compounds 60 and 71. Replacing 4methylpiperazin-1-yl with 4-(dimethylamino)piperidinyl afforded a less potent analog, as shown in compounds 60 and 72. However, the introduction of smaller substitutions, such as 4-(dimethylamino)cyclopentylamino and 3-(dimethylamino)azetidin-1-yl groups, as shown in compounds 73 and 74, restored and even increased anti-HCV activity. Replacing the N,N-dimethyl group in compound 74 with a carboxy group resulted in a distinct loss of activity (75). Introduction of morpholinyl and dimethyl-substituted morpholinyl groups, as in 76 and 77, compared to compound 60, led to 3e6-fold decreased activity. Introduction of 4-(4methylpiperazin-1-yl)-methyl-pyridin-3-yl group, as shown in compound 78, led to dropped potency of only 2-fold (compared 78 to 60, Table 4 ), showing that the pyridine-3-yl moiety was tolerated in the place of 3-trifluoromethyl-phenyl. Replacing the (4methylpiperazin-1-yl)-methyl group (78) with 4methylpiperidinyl (79) led to 8-fold decreased potency. Compound 80 with a 3-(dimethylamino)azetidin-1-yl group in the R 1 fragment and a 2-chloroethyl group in the R 3 fragment displayed the highest activity (EC 50 ¼ 0.015 mM) and SI value (SI ¼ 431) among all synthesized novel target compounds.

Because compounds 60 and 80 showed strong anti-HCV activities (EC 50 : 0.044 and 0.015 mM) and high selectivity indices (SI: 154 and 431), we chose compounds 60 and 80 to investigate their safety profiles. Acute toxicity tests of compounds 60 and 80 were performed in KunMing mice. Each compound was given intraperitoneally in a single-dosing experiment at 50, 100, 150, or 200 mg/kg (n ¼ 6 per group). The mice were closely monitored for 7 days. Compound 60 displayed low safety profiles with median lethal dose (LD 50 ) values lower than 100 mg/kg. Compound 80 demonstrated modest safety profiles with LD 50 values higher than 150 mg/kg. The results suggested that compound 80 was relatively safe in vivo.

Compound 80 showed the highest activity among all synthesized novel compounds and low toxicity. While compound 80 has poor solubility in water (<5 mg/mL), the aqueous solubility of the corresponding hydrochloride salt was improved to 7.8 mg/mL (at pH 7.0). Thus, compound 80 was further profiled in four assays to assess in vitro drug-like properties: logD, microsomal stability, cell permeability and plasma stability (Table 5) . Compound 80 showed decent plasma stability (t 1/2, rat ¼ 16.9 h and t 1/2, human ¼ 19.9 h), which could ensure that a high concentration of the compound reached the bloodstream. Compound 80 showed moderate permeability (0.5 < P app < 2.5 ( Â 10 À6 cm/s)) and was likely an efflux transporter substrate based on Caco-2 assays. In data from HLM/RLM, it appeared that compound 80 had low to medium metabolic stability based on liver microsome assays.

Given its favorable in vitro ADME profile, the in vivo pharmacokinetics of compound 80 were evaluated in a rat (Sprague-Dawley) model after a single dose of 2 mg/kg through the intravenous (i.v.) route and 10 mg/kg via the oral route of administration (Fig. 3) . The plasma profiles obtained from the pharmacokinetic experiments are shown in Table 6 and Fig. 3 . The results indicated that compound 80 has satisfying PK properties with an oral total exposure (AUC) of 1502 ng h/mL, medium in vivo clearance (38.3 mL/min/kg), C max of 452 ng/mL, and moderate bioavailability of 34%. Considering that sustained exposure to PK in vivo should exceed at least several times the in vitro EC 50 expected to be useful in human efficacy studies, we used 100 ng/mL, equating to 10-fold above the EC 50 for HCV, as a minimum requirement efficacy concentration. At the 10 mg/kg dose, plasma concentrations remained above 100 ng/mL for over 4 h, indicating a modest stability to metabolism in vivo of this kind of compound.

Twenty-two compounds were subjected to a preliminary screening test to identify their inhibition of Vif-mediated hA3G degradation using our previously reported assay [23] . Briefly, 293T cells were cotransfected with the expression vectors for hA3G and Vif and then treated with 20 mM test compounds and MG132, a well-known proteasome inhibitor, as a positive control. The results in Fig. 4 show that compared with that in the cells treated with DMSO, seven compounds (12, 13, 18, 19, 20, 40 , and 41) were effective in inhibiting Vif-mediated hA3G degradation in this assay (>50%). Four compounds (17, 36, 37 , and 45) displayed modest activity (25%e50%). Eleven compounds showed weak activity (<25%). According to the structure-action relationship, the amido group in the R 2 moiety was superior in inhibiting Vif-mediated hA3G degradation, while nitro and amino groups were adverse (except compound 12). R 1 and R 3 moieties would be versatile and tolerant to hydrophobic or hydrophilic groups. Compounds 13 and 18 with propionyl moieties in the R 2 moiety had good anti-HCV activity and simultaneously displayed potent inhibition of Vifmediated hA3G degradation. Compound 54 with a nitro group displayed excellent anti-HCV activity but poor inhibition of Vifmediated hA3G degradation. Since most nitro compounds displayed poor inhibition of Vif-mediated hA3G degradation, subsequent synthesized compounds were not evaluated for activity. The antiviral mechanism of these nitro compounds is still in process.

A series of novel biaryl amide derivatives were synthesized and assayed for anti-HCV activity in vitro. Intensive structural modifications led to fifteen novel compounds with higher potent inhibitory activity than IMB 26, especially compound 80, with EC 50 values almost equivalent to those of the clinical drug telaprevir. Additionally, some significant SARs were uncovered. Among the structures of the anti-HCV compounds, R 1 moieties are apt to be hydrophobic moieties (for example, an aromatic nucleus) through a methylene linked to hydrophilic moieties (for example, cyclic amine), R 2 moieties should be a hydrogen bond acceptor (for example, a nitro group) and R 3 moieties prefer to be hydrophobic moieties as requirements for anti-HCV activity. Such SARs provided valuable implications for further lead optimizations. Compound 18 showed comparable inhibitory activity against HCV to IMB-26 and moreover displayed effective inhibitory activity against Vifmediated hA3G degradation, although it possessed obviously different structures at the R 1 position. Most compounds with nitro groups, however, displayed poor inhibition of Vif-mediated hA3G degradation. Compound 80 displayed the highest anti-HCV activity and SI value and possessed good physicochemical properties, making it a more promising scaffold for further study.

All reagents and solvents were purchased from commercial sources and used as obtained. All reactions were carried out in flamedried glassware and monitored by thin layer chromatography using aluminum TLC plate 60F254D (Merck Millipore) and visualized under UV light. 1 H NMR and 13 C NMR spectra were recorded with a Bruker 400 or a Varian Inova 500 or 600 NMR spectrometer. Chemical shifts are reported in parts per million (ppm) and are referenced to the residual solvent peak. The following notations are used: singlet (s); doublet (d); triplet (t); quartet (q); multiplet (m); broad (br). Data are reported in the following manner: chemical shift (multiplicity, coupling constant if appropriate, integration). Signals are quoted as d values in ppm and coupling constants (J) are reported in Hertz. Using General Procedure A: Coupling of 4-substituted-3-nitrobenzoic acid and various substituted anilines fragment. To a mixture of 4substituted-3-nitrobenzoic acid, substituted anilines (1.0e1.2 equiv), DMAP (0.1 equiv) in CH 2 Cl 2 (1.5e4 mL, ca. 0.05 M) was added N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (2.0 equiv). The reaction was stirred at room temperature for 16 h. The reaction mixture was extracted with CH 2 Cl 2 , washed with water and dried with anhydrous Na 2 SO 4 . The solvent was removed under vacuum, and the residue was purified by silica gel flash chromatography (eluents: 10e50% THF in petroleum ether) to offer the coupled nitro product in 78e93% yield.

General Procedure B: Hydrogenation of nitro compounds. The nitro compound (0.5 mmoL) was dissolved in ethyl acetate (5 mL), and methanol (5 mL) and Pa/C (0.05 g, 10%) was added. The resulting mixture was hydrogenated at hydrogen gas pressure of 14e35 psi for 2.5e24 h. The catalyst was removed by filtration and the filtrate was concentrated under vacuo to give amino derivatives, which was used in the next step without further purification.

General procedure C: Coupling of various substituted anilines and acyl chloride. Propionyl chloride (1.0 eq) was added dropwise to a solution of substituted aniline (1.0 mmoL) and TEA (1. 5 mmoL) in dichloromethane at 0 C. The mixture was then stirred at room temperature until the starting material was completely disappeared. The reaction was quenched with water and extracted with dichloromethane. The organic layer was washed with brine, dried over Na 2 SO 4 , concentrated in vacuo, and further purified by flash chromatography on silica gel (eluents: 1e10% MeOH in dichloromethane with 0.2% NH 4 OH (aq) ) or C-18 functionalized silica chromatography (eluents: 5e90% MeOH in deionized water with 0.5% NH 4 OH (aq) ) to give the product. 

Compound 15 (0.223 g, 1.0 mmoL) and 4-(1H-indol-2-yl)aniline (0.208 g, 1.0 mmoL) were reacted according to general procedure A to afford compound 19 (0.338 g, yield 82%). 1 

4-hydroxy-3-nitrobenzoic acid (5.00 g, 27.3mmoL) and 4-methoxylaniline (4.03 g, 32.8 mmoL) were reacted according to general procedure A to afford 21 (4.08 g, yield 52%). 1 

To a solution of compound 21 (0.289 g, 1.0 mmoL) in tetrahydrofuran (8.0 mL) was added ethanol (67 mL, 1.2 mmoL) and triphenylphosphine (0.520 g, 2.0 mmoL). The mixture was cooled at 0 C and Diethyl azodicarboxylate in toluene (40%, 0.77 mL, 1.7 mmoL) was added dropwise. The mixture was then stirred at room temperature until the starting material was completely disappeared. The reaction was quenched with water and extracted with dichloromethane. The organic layer was washed with brine, dried over Na 2 SO 4 , concentrated in vacuo, and further purified by flash chromatography on silica gel to give 22 (0.240 g, 76%). 1 

Compound 21 was reacted with isopropanol (93 mL, 1. 2 mmoL) according to a method similar to that of compound 22 to afford 24 (0.264 g, 80%). 1 

Compound 21 was reacted with 3-chloropropanol (0.10 mL, 1. 

To a solution of compound 25 (0.300 g, 0.82 mmoL) in DMF (5.0 mL) was added sodiumiodide (0.246 g, 1.64 mmoL) and dimethylamine (0.82 mL, 2.0 M in THF). The mixture was stirred at 70 C for 4 h. After cooling to rt, the solvent was removed, and the residue was purified by C-18 functionalized silica chromatography (eluents 5e90% MeOH in deionized water with 0.5% NH 4 OH (aq) to afford compound 26 (0.178 g, 58%). 1 2-bromoacetyl bromide (0.48 mL, 5.0 mmol) was added dropwise to a solution of methyl 3-amino-4-hydroxybenzoate (0.84 g, 5.0 mmol) and NaHCO 3 (0.69 g, 8.25 mmol) in EtOAc/H 2 O (40 mL, 1:1) at 0 C. The mixture was then stirred at room temperature until the starting material was completely disappeared. The reaction was extracted with EtOAc (50 mL). The organic layer was orderly washed with 10% HCl, water, and brine, dried over Na 2 SO 4 , and concentrated in vacuo to give methyl 3-(2-bromoacetamido)-4hydroxybenzoate (43, 1.07 g). The compound was dissolved in DMF (5.0 mL) and K 2 CO 3 (0.56 g, 5.25 mmol) was added the above mixture. The mixture was heated to 80 C and stirred for 3 h until the starting material was completely disappeared. After cooling to room temperature, the solvent was removed, and the residue was extracted with ethyl acetate, washed with water and brine in turn, and dried over anhydrous Na 2 SO 4 . After filtration and concentration, compound 44 was obtained. 1 

To a solution of compound 44 (0.72 g, 3.48 mmoL) in MeOH/H 2 O (12 mL, 2:1) was added sodium hydroxide (0.28 g, 7.0 mmoL). The mixture was stirred at 70 C for 3 h to give substituted benzoic acid, which was reacted 4-methoxy aniline according to general procedure A to give the target compound 45 (0.593 g, 82%). 1 To the solution of methyl 3,4-dihydro-2H-benzo[b] [1, 4] oxazine-6-carboxylate (0.20 g, 1.03 mmol) in acetonitrile (2.5 mL) was added DMAP (0.025 g, 0.2 mmol) and Ditertbutyl dicarbonate (0.23 g, 1.14 mmol). The mixture was stirred at room temperature for 3 h until the starting material was completely disappeared. The solvent was removed, and the residue was extracted with ethyl acetate, washed with water and brine in turn, and dried over anhydrous Na 2 SO 4 . After filtration and concentration, the residual material was purified by flash column chromatography (silica gel) eluted with petroleum ether and ethyl acetate (20:1) to yield N-Boc substituted product. The compound was dissolved in MeOH/H 2 O (5 mL, 2:1) and added sodium hydroxide (0.054 g, 1. 36 mmoL) . The mixture was stirred at 70 C for 3 h to give substituted benzoic acid (46). 1 

To a solution of methyl 4-hydroxy-3-nitrobenzoate (1.50 g, 7.6 mmoL) in DMF (15.0 mL) was added 2-bromopropane (1.10 mL, 11.4 mmoL) and potassium carbonate (1.57 g, 11.4 mmoL). The mixture was stirred at 60 C for 12 h. After cooling to rt, the solvent was removed, and the residue was extracted with ethyl acetate, washed with water and brine in turn, and dried over anhydrous Na 2 SO 4 . After filtration and concentration, the obtained residue was dissolved in methanol and THF (10 mL, 1:1) and 1 N NaOH (10 mL) was added. The mixture was stirred at 60 C for 1.5 h. After cooling to rt, the solvent was removed, and the residue was extracted with ethyl acetate, washed with 1 N HCl, water, and brine in turns, and dried over anhydrous Na 2 SO 4 . The mixture was filtered, and the solvent was removed to give compound 49 (1.10 g, 64%). LC/MS (ESI, m/z):

Compound 49 (0.450 g, 2.0 mmoL) and 4-dimethylamino-aniline (0.272 g, 2.0mmoL) were reacted according to general procedure A to afford 50 (0.597 g, yield 87%). 1 To a solution of methyl 4-hydroxy-3-nitrobenzoate (58a, 1.50 g, 7.6 mmoL) in DMF (15.0 mL) was added 1-bromo-3-chloropropane (1.12 mL, 11.4 mmoL) and potassium carbonate (1.56 g, 11.4 mmoL). The mixture was stirred at 60 C for 12 h. After cooling to rt, the solvent was removed, and the residue was extracted with ethyl acetate, washed with water and brine in turn, and dried over anhydrous Na 2 SO 4 . After filtration and concentration, the obtained residue was purified by flash column chromatography (silica gel) eluted with petroleum ether and ethyl acetate (V ¼ 5:1) to yield methyl 3-chloropropoxy-3-nitrobenzoate (1.95 g, 94%). 1 25 mmoL) was dissolved in tetrahydrofunan (10 mL) and methanol (10 mL) and lithium hydroxide (0.117 g, 5.10 mmoL) was added. The mixture was stirred at room temperature for about 3.0 h. The solvent was removed, and the residue was extracted with ethyl acetate, washed with 1 N HCl, water, and brine in turns, and dried over anhydrous Na 2 SO 4 . After 2 h, the mixture was filtered, and the solvent was removed to give 4-(3-chloropropoxyl)-3-nitrobenzoic acid (59a, 1.10 g, 98%). 1 

Compound 64 was synthesized using a method similar to that of 60. Methyl 4-hydroxy-3-(sulfamoyl)benzoate (58e, 0.231 g, 1.00 mmoL) and 1-bromo-3-chloropropane (0.13 mL, 1.36 mmoL) was using as the starting materials to yield the intermediate methyl 4-(3-chloropropoxyl)-3-(trifluoromethyl)benzoate. The intermediate was hydrolyzed to yield 4-(3-chloropropoxyl)-3-(trifluoromethyl) benzoic acid (59e, 0.223 g, 76%). 1 

500 MHz, CDCl 3 ) d 8.83 (s, 1H), 8.53 (s, 1H), 7.91 (s, 1H), 7.85 (m, 2H), 7.74 (d, J ¼ 8.5 Hz, 1H), 7.68 (d, J ¼ 8.5 Hz, 1H)

Compound 58a (0.20 g, 1.0 mmoL)was reacted with 1-bromo-2-chloroethane (0.125 mL, 1.5 mmoL) and potassium carbonate (0.28 g, 2.0 mmoL) to give methyl 2-chloroethoxy-3-nitrobenzoate (0.181 g, 70%). The compound was hydrolyzed to give 4-(2-chloroethoxy)-3-nitrobenzoic acid (67a, 0.168 g, 98%). 1 H

64 (s, 2H), 3.58 (t, J ¼ 6.0 Hz, 2H), 2.53 (m, 8H), 2.32 (s, 3H), 1.88 (m, 6H); 13 C NMR (100 MHz

-methylpiperazin-1-yl)methyl)phenyl

-(dimethylamino)piperidin-1-yl)methyl)aniline (83a, 0.302 g, 1.00 mmoL) were reacted according to general procedure A to afford 72 (0.411 g, yield 76%). 1 H NMR (400 MHz, CDCl 3 ) d 8.42 (s, 1H), 8.36 (s, 1H), 8.16 (d, J ¼ 8

-dimethylaminopyrrolidin-1-yl) methyl)phenyl)-3-nitro-4-(3-chloropropoxy)-benzamide (73) Compound 59a (0.259 g, 1.00 mmoL) and 3-(trifluoromethyl)-4-(3-dimethylamino)-pyrrolidin-1-yl)methylaniline (83b, 0.288 g, 1.00 mmoL) were reacted according to general procedure A to afford 73 (0.205 g, yield 39%). 1 H NMR (500 MHz, CDCl 3 ) d 9.12 (br s, 1H), 8.39 (s, 1H), 8.12 (d, J ¼ 9

yield 41%). 1 H NMR (500 MHz, CDCl 3 ) d 8.39 (d, J ¼ 1.5 Hz, 1H), 8.17 (s, 1H), 8.14 (dd, J ¼ 7.5, 1.5 Hz, 1H), 7.89 (s, 1H), 7.84 (d, J ¼ 7.0 Hz, 1H), 7.65 (d, J ¼ 7.0 Hz, 1H)

3-chloropropoxy)-3-nitrobenzamido)-2-(trifluoromethyl)benzyl)azetidine-3-carboxylic acid (75) Compound 59a (0.259 g, 1.00 mmoL) and methyl 1-(4-amino-2-(trifluoromethyl)benzyl)azetidine-3-carboxylate

38 mmoL) in MeOH/THF (4 mL, 1:1) was added lithium hydroxide (18 mg, 0.76 mmoL). The mixture was stirred for 4 h, the reaction was neutralized by diluted hydrochloric acid to give white solid, then filtering and drying

4-(morpholinmethyl)phenyl)-3-nitro-4-(3-chloropropoxy)-benzamide (76) Compound 59a (0.164 g, 0.63 mmoL) and 3-(trifluoromethyl)-4-(morpholinmethyl)aniline (83e, 0.151 g, 0.58 mmoL) were reacted according to general procedure A

Hz, 1H), 8.02 (s, 1H), 7.87 (s, 1H), 7.85 (d, J ¼ 7.5 Hz, 1H), 7.80 (d, J ¼ 6.0 Hz, 1H), 7.22 (d, J ¼ 7.5 Hz, 1H)

5-dimethylmorpholin)methyl)aniline (83f, 0.185 g, 0.58 mmoL) were reacted according to general procedure A to afford 77 (0.241 g, yield 79%). 1 H NMR (600 MHz

g, 1.00 mmoL) were reacted according to general procedure A to afford 78 (0.205 g, yield 46%). 1 H NMR (600 MHz, CDCl 3 ) d 8.65 (d, J ¼ 2.4 Hz, 1H)

Compound 59a (0.380 g, 1.48 mmoL) and 5-amine-2-(4-methylpiperidin-1-yl)pyridine (88, 0.260 g, 1.35 mmoL) were reacted according to general procedure A to afford 79 (0.250 g, yield 43%). 1 H NMR (600 MHz

Hz, 1H), 7.95 (s, 1H), 7.84 (d, J ¼ 7.8 Hz, 1H), 7.15 (d, J ¼ 8.0 Hz, 1H), 6.66 (d, J ¼ 8.0 Hz, 1H), 4.33 (q, J ¼ 5.4 Hz, 2H)

-dimethylaminoazetidin-1-yl) methyl)phenyl)-3-nitro-4-(2-chloroethoxy)-benzamide (80) 4-(2-chloroethoxyl)-3-nitrobenzoic acid (67a, 0.245 g

DMSO-d 6 ) d 10.57 (s, 1H), 8.54 (d, J ¼ 1.8 Hz, 1H), 8.28 (dd, J ¼ 9.0, 1.8 Hz, 1H), 8.16 (s, 1H), 8.03 (d

Synthesis of compound 83a-f. Taking 83a as an example: To a solution of 1-(bromomethyl)-4-nitro-2-(trifluoromethyl)benzene (81, 0.65 g, 2.3 mmoL) in anhy

Synthesis of 83c was similar to that of compound 83a: compound 81 (1.55 g, 5.47 mmoL) and 3-(dimethylamino)azetidine dihydrochloride (0.95 g, 5.47 mmoL) were reacted to afford 3-(trifluoromethyl)-4-(3-dimethylamino)azetidin-1-yl)methyl-nitrobenzene (82c, 1.00 g, 60%). 1 H NMR (500 MHz

CDCl 3 ) d 145.2, 130.9, 128.8 (q, J ¼ 24.7 Hz), 126.3, 124.3 (q, J ¼ 227.1 Hz), 117.7, 112.2 (q, J ¼ 4.9 Hz), 59.6, 58.9, 56.9, 42.0. ESI-HRMS calcd for C 13 H 19 N 3 F 3 [M þ H] þ 274.1526; found 274.1525. Synthesis of compound 83d was similar to that of 83a: compound 81 (0.50 g, 1.76 mmoL) and methyl azetidine-3-carboxylate hydrochloride (0.27 g, 1.76 mmoL) were reacted to afford methyl 1-(4-nitro-2-(trifluoromethyl)benzyl)azetidine-3-carboxylate (82e, 0.274 g, 49%). 1 H NMR (600 MHz

CDCl 3 ) d 8.51 (s, 1H), 8.37 (d, J ¼ 8.4 Hz, 1H), 8.11 (d, J ¼ 8.4 Hz, 1H), 3.74 (s, 6H), 2.51 (s, 4H); 13 C NMR (150 MHz

5 (q, J ¼ 272.4 Hz), 121.6 (q, J ¼ 6.0 Hz), 67.2, 58.4, 53.7. ESI-HRMS calcd for C 12 H 16 N 2 F 3

,5-dimethylmorpholin)-nitrobenzene (82f, 0.270 g, 73%). 1 H NMR (600 MHz

ESI-HRMS calcd for C 14 H 20 ON 2 F 3 [M þ H] þ 289.1522; found 289.1522. Synthesis of compound 86 was similar to that of 83a: 2-(bromomethyl)-5-nitropyridine (84, 0.43 g, 2.0 mmoL) and 4-methylpiperazine (0.22 mL, 2.0 mmoL) were reacted to afford 5-nitro-2-((4-methylpiperazin-1-yl)methylpyridine (85, 0.315 g, 67%). 1 H NMR (600 MHz, CDCl 3 ) d 8.48 (d, J ¼ 2.4 Hz, 1H), 8.37 (dd, J ¼ 8.4, 1.8 Hz, 1H), 7.80 (d, J ¼ 8.4 Hz, 1H), 3.77 (s, 2H), 2.55 (m, 4H), 2.45 (m, 4H), 2.28 (s, 3H); 13 C NMR (150 MHz

0 Hz, 1H), 6.57 (d, J ¼ 9

) Huh7.5 cells were cultured in Dulbecco's modified eagle medium (DMEM, Invitrogen, CA) supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen) and 1% penicillinestreptomycin (Invitrogen)

Agents Telaprevir (HY-10235, VX-950) was purchased from the Med-ChemExpress

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Small molecular compounds that inhibit hepatitis C virus replication through destabilizing heat shock cognate 70 messenger RNA

This work was supported by the CAMS Innovation Fund for Medical Sciences (2017-I2M-3-012).

phosphate dehydrogenase (GAPDH) (10494-1-AP) were from Protein Tech Inc. All test compounds were dissolved supplied in DMSO at 10 mM and then diluted in Dulbecco's modified Eagle's medium culture medium. For EC 50 and CC 50 determinations, test compounds were serially diluted in eight steps of 1:5 dilutions in 96well plates.

Anti-HCV activity assay in vitro [37] Huh7.5 cells were seeded into 96-well or 6-well plates (Costar) at a density of 3 Â 10 4 cells/cm 2 . After 24 h of incubation, the cells were infected with HCV viral stock (recombination virus strain J6/ JFH/JC, 45 IU/cell) and simultaneously treated with different concentration of compounds or solvent control. The culture medium was removed after 72 h of incubation, and the intracellular total RNA (in 96-well plates) was extracted with RNeasy Mini Kit (Qiagen) and quantified with qRT-PCR. It was performed on a 7500 Fast Real-Time PCR system (Applied Biosystems, Singapore) using an AgPath-ID One-Step RT-PCR Kit (Applied Biosystems, Foster, CA, USA) according to the manufacturer's instructions. All quantifications were normalized to the level of the internal control gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), the levels of HCV RNA were analyzed with the 2 -△△CT method, and a value of half maximal effective concentration (EC 50 ) was calculated with the Reed-Muench Method.

Huh7.5 cells were seeded into 96-well plates (Costar) at a density of 3.0 Â 10 4 cells/cm 2 . After 24 h of incubation, fresh culture medium containing test compounds at various concentrations were added. Cytotoxicity was evaluated with the tetrazolium-based MTT assay at 96 h.

Hydrochloride salts of compound 80 were added to distilled water (1.0 mL). After shaking for 1.0 h at 25 C and then centrifuging at 3000 rpm for 10 min, the saturated supernatants were measured the volume and then lyophilized to determine the concentration dissolved in water. For compound 80, the saturated supernatants were transferred to other vials for analysis by HPLC-UV. Each sample was performed in triplicate. For quantification, a model 1200 HPLC-UV (Agilent) system was used with an Agilent TC-C18 column (250 Â 4.6 mm, 5 mm) and elution of 2 mM HCO 2 NH 4 /methanolwater (95:5). The flow rate was 1.0 mL/min and injection volume was 10 mL with the detection wavelength at 254 nm. Aqueous concentration was determined by comparison of the peak area of the saturated solution with a standard curve plotted peak area versus known concentrations, which were prepared by solutions of test compound in methanol at 135.0, 45.0, 15.0, 5.0, and 2.5 mg/mL.In vivo toxicity, In vitro and In vivo pharmacokinetic, and Compounds protecting hA3G from Vif-mediated degradation assessment methods. See supporting information. All in vivo studies were in accordance with the Animal Care and Use Committee of People's Republic of China.

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.