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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
ChemMedChem. Author manuscript; available in PMC 2013 July 1.
Published in final edited form as:
PMCID: PMC3517065
NIHMSID: NIHMS416242

Synthesis and Structure-Activity Relationship Studies of HIV-1 Virion Infectivity Factor (Vif) Inhibitors that Block Viral Replication

Abstract

The HIV-1 Vif protein, essential for in vivo viral replication, protects the virus from innate antiviral cellular factor APOBEC3G (A3G), and is an attractive target for developing antiviral therapeutics. Here we have evaluated the structure-activity relationships of RN18, a small molecule recently identified as an inhibitor of Vif function that blocks viral replication only in non-permissive cells expressing A3G, by inhibiting Vif-A3G interactions. Microwave-assisted cross-coupling reactions were developed to prepare a series of RN18 analogues with diverse linkages and substitutions on the phenyl rings. A dual cell-based assay system was used to assess antiviral activity against wild-type HIV-1 in both non-permissive (H9) and permissive (MT-4) cells that also allowed evaluation of specificity. In general, variations of phenyl substitutions were detrimental for antiviral potency and specificity, but isosteric replacements of amide and ether linkages were relatively well tolerated. These SAR data define structural requirements for Vif-specific activity, identify new compounds with improved antiviral potency and specificity, and provide leads for further exploration to develop new antiviral therapeutics.

Keywords: APOBEC3G, HIV-1 Vif, inhibitors, structure-activity relationships, drug discovery, antiviral agents

Introduction

Since the start of the AIDS epidemic in 1981, this disease has led to the death of nearly 30 million people globally. Although the overall growth of the epidemic appears to be slowing, nearly three million new infections and an estimated 1.8 million AIDS-related deaths are still very high.[1] Over the past two decades, more than 25 anti-HIV drugs have been developed targeting several different stages of the virus life cycle.[2] Among them inhibitors of HIV-1 reverse transcriptase and protease, when used in combinations in the highly active antiretroviral therapy (HAART),[3] have proven to be highly effective in reducing AIDS-related mortality throughout the world.[1] However, the development of drug resistance and toxic side effects associated with HAART have created a need for more potent and less toxic therapies against other viral targets and host-virus interactions.[4]

The HIV-1 virion infectivity factor (Vif) is one of only 6 regulatory proteins encoded by the virus, and is required for in vivo viral replication.[57] Vif targets innate antiviral cellular factor APOBEC3G (A3G),[8] a human DNA-editing enzyme, which, along with other APOBEC proteins, inhibits replication of retroviruses and retrotransposons.[912] In the absence of Vif, A3G incorporates into virions and causes extensive mutations during reverse transcription by catalyzing Zn-dependent hydrolytic deamination of deoxycytidine (dC) to deoxyuridine (dU) in the newly synthesized minus strand of viral DNA, rendering the virus noninfectious.[13] In addition to this deaminase-dependent mechanism, A3G can act in a deaminase-independent mechanism by directly inhibiting reverse transcription.[14] Vif overcomes the innate antiviral activity of A3G in several different ways, including promoting its degradation in the E3-ubiquitin-proteosome pathway,[1517] modulating its expression by inhibiting translation,[18] and directly interfering with packaging,[19] thus protecting viral progeny from this innate antiviral defense mechanism.

Since HIV-1 Vif has no known cellular homologs, this protein represents an extremely attractive, yet unrealized, target for antiviral intervention. Although zinc-chelating agent N,N,N',N'-tetrakis-(2-pyridylmethyl) ethylenediamine (PTEN) is reported to inhibit Vif activity and restore A3G function,[20] this effect is due the requirement of zinc for Vif function and is unrelated to its interactions with A3G. Small molecules that specifically inhibit Vif function will restore cellular levels of A3G along with its antiviral activity, leading to the inhibition of viral replication.

We recently described the development of a florescence-based high throughput screening (HTS) assay to identify small molecules that specifically inhibit Vif-A3G interactions.[21] Screening of a diverse 30K small molecule library identified two compounds, RN18 and RN19 (Figure 1), that specifically antagonize HIV-1 Vif function and inhibit viral replication only in the presence of A3G.[21] RN18 and RN19 inhibit HIV-1 replication in a dose-dependent manner (IC50 = 6 µM and 25 µM, respectively) only in non-permissive cells (H9, CEM) expressing A3G, and not in permissive cells (MT-4), which do not express A3G. In addition, RN18 increases cellular levels of A3G in a Vif-dependent manner and increases A3G incorporation into the virions, leading to the production of less infectious viruses. These results strongly suggest that RN18 is a Vif-specific inhibitor of HIV-1 replication, and that targeting the Vif-A3G axis is a valid strategy for developing antiviral therapeutics for HIV-1 infection and for enhancing innate immunity against viruses.

Figure 1
Chemical Structures of HIV-1 Vif Inhibitors RN18 and RN19.

Recently, Cen et al. reported the identification of two small molecules, IMB26 and IMB35, that inhibit HIV-1 replication by specifically stabilizing A3G.[22] Unlike RN18, these molecules increase cellular A3G levels in a Vif-independent manner, suggesting a different mechanism of action unrelated to Vif. RN18 and RN19 remain the only Vif antagonists that inhibit HIV-1 replication by specifically targeting Vif-A3G interactions. To identify structural features required for the Vif-specific activity of RN18 and to improve antiviral potency and pharmacological properties, we prepared a series of closely related analogues with diverse ring linkages and substitutions. These analogues were tested for antiviral activity against wild-type HIV-1 in both non-permissive (H9) and permissive (MT-4) cells to determine their specificity. In addition, cytotoxicity was assessed to rule out non-specific antiviral activity. We report here the design, synthesis and structure-activity relationship studies of RN18 analogues, leading to the identification of several new compounds with improved antiviral potency, specificity and toxicity profiles.

Design and Synthesis

We envisioned preparing RN18 analogues with diverse ring linkages and substitutions using the two synthetic routes outlined in Figure 2. Both methods involve cross-coupling of substituted aryl halides with either thiols or phenols using Cu-based catalysts. The direct coupling of pre-assembled aryl iodides with substituted thiophenols can provide easy access to RN18 and A-ring analogues. This convergent method is particularly attractive as it allows access to analogues with diverse linkages between phenyl rings B and C, such as reverse amide, sulfonamide, and reverse sulfonamide. The second route involving initial coupling of aryl iodides and methyl 2-mercaptobenzoate is suitable for quickly assembling diverse C-ring analogues after ester hydrolysis followed by coupling with aryl or alkyl amines.

Figure 2
a) A convergent route for the synthesis of RN18 and analogues; b) alternate route for the synthesis of RN18 and C-ring analogues.

In recent years, a number of metal-catalyzed cross-coupling reactions have been developed for the coupling of aryl iodides and thiophenols.[2325] Among them, Ulmann-type Cu-catalyzed coupling methods are highly attractive because of their efficiency, mild reaction conditions, and broad substrate scope. Due to its simplicity of operation, we chose to use the cross-coupling method developed by Kwong and Buchwald using ethylene glycol as a ligand and potassium carbonate as a base in 2-propanol.[26] Thus the coupling of 2-iodo-N-(2-methoxyphenyl)benzamide (1a) and thiophenol 2 under these conditions provided the aryl sulfide product 4a in 60% yield after purification (Scheme 1). However, attempts to couple 1a with 4-nitrothiophenol 3 under the same conditions were unsuccessful, presumably due to low reactivity of 3 in the cross-coupling reaction. Reaction conditions of Bates et al. involving neocuproine as a ligand and NaOt-Bu as a base also failed to provide the desired aryl sulfide 5a.[27] Microwave irradiation has been shown to enhance the efficiency of cross-coupling reactions, including copper(I) iodide-catalyzed coupling of aryl halides and thiophenols as observed by Wu et al.[28] After initial optimization of microwave conditions, reaction of 1a and 3 with copper(I) iodide, ethylene glycol, and potassium carbonate in 2-propanol followed by irradiating the resulting mixture in a microwave reactor (CEM Explorer) at 80 °C for 30 min twice provided the desired product 5a (RN18) in 63% yield after purification by flash chromatography.

Scheme 1
Reagents and conditions: a) Cu(I)I, K2CO3, iPrOH, HOCH2CH2OH, MW (150 WT), 80 °C, 30 min (2 ×); b) SnCl2[bullet]2H2O, EtOAc, 80 °C, 3 h.

As illustrated in Scheme 1, the optimized cross-coupling conditions proved to be efficient, allowing the coupling of a variety of 2- and 3-substituted aryl iodide substrates 1a–g with 4-nitrothiophenol 3. The intermediate aryl iodides 1a–g, were prepared by the reaction of appropriate anilines and acid chlorides or sulfonyl chlorides (see Supporting Information for details). Parallel reactions of aryl iodides 1a–g and 4-nitrothiophenol under the optimized cross-coupling conditions provided the corresponding aryl sulfides 5a–g in 25–62% isolated yield. The RN18 analogues 5a–g contain a variety of linkages between rings B and C, including reverse amide (5c, 5f–g), sulfonamide (5d), and reverse sulfonamides (5e). Reduction of the 4-nitro group in 5a–e using SnCl2.2H2O in ethyl acetate provided the corresponding 4-amino analogues 6a–e.

To prepare RN18 and series of C-ring analogues, we developed an alternate synthetic route as outlined in Scheme 2. This method involved initial cross-coupling of aryl iodides and methyl 2-mercaptobenzoate (7) to provide the corresponding esters 10–11. In this case, re-optimization of microwave-assisted cross-coupling conditions was necessary due to the presence of a methyl ester in the thiophenol. Thus, reacting 7 and 8 with copper(I) iodide and potassium carbonate in 1,2-dimethoxyethane (DME) instead of 2-propanol and irradiating the resulting mixture in a microwave reactor at 80 °C for 30 min twice provided the ester 10 in 86% isolated yield; the reaction was scaled up to 30 mmol using open vessel microwave irradiation. The resulting esters 10–11 were hydrolyzed to obtain the corresponding acids 12–13 in excellent yield. Acid activation using oxalyl chloride followed by reaction of the resulting benzoyl chloride with a variety of aromatic and aliphatic amines 14a–h afforded C-ring analogues 15–16. Similarly, activation of commercially available acid 17 with oxalyl chloride followed by reaction of the resulting benzoyl chloride with amines provided the ether-linked benzamide derivatives 18. Reduction of the 4-nitro group in 18a provided the corresponding 4-amino analogue 19a (Scheme 2).

Scheme 2
Reagents and conditions: a) Cu(I)I, K2CO3, 1,2-DME, MW (150 WT), 80 °C, 30 min (2 ×); (b) Ba(OH)2[bullet]8H2O, MeOH, 80 °C, 2 h; (c) (i) (COCl)2, CH2Cl2, DMF, RT, 8 h, (ii) R′R″NH (14a–h), Et3N, CH2Cl2 ...

Next, we prepared a series of analogues with aryl ether and benzyl ether linkages between rings A and B using synthetic methods outlined in Scheme 3. A set of ether-linked analogues with variations on the A-ring was prepared using a modified Ullmann cross-coupling method developed by Buck et al., involving copper(I) chloride and 2,2,6,6-tetramethylheptane-3,5-dione (TMHD) as the catalyst system.[29] Reactions of aryl bromides 1j–l, prepared by the reaction of appropriate anilines with acid chloride or sulfonyl chloride (see Supporting Information for details), and substituted phenols 20–21 with copper(I) chloride, TMHD, and cesium carbonate in N-methylpyrrolidinone followed by irradiating the reaction mixture in a microwave reactor provided the desired ether-linked analogues 22–23. Another set of ether-linked analogues was prepared using aryl boronic acids as donors in cross-coupling reactions with phenol (Scheme 3). Reaction of 2-substituted phenol 1k and a variety of 4-substitued aryl boronic acids 24–28 using copper acetate as the catalyst and Et3N as a base provided the corresponding ether-linked reverse amide derivatives 29–33.

Scheme 5
Reagents and conditions: (a) Cu(I)Cl, Cs2CO3, CH2(Cot-Bu)2, NMP, MW (150 WT), 120 °C, 30 min (2 ×); b) Cu(OAc)2, CH2Cl2, PhOH, Et3N, 4A MS, RT, overnight; c) K2CO3, DMF, MW (150 WT), 90 °C, 30 min (2 ×).

The benzyl ether-linked analogues 40–45 were prepared from 2-substituted phenols and a variety of substituted benzyl bromides (Scheme 3). The SN2 displacement reactions, involving phenols 1j–k and benzyl bromides 34–39, were carried out in dimethylformamide (DMF) using K2CO3 as a base, followed by irradiating the reaction mixture in a microwave reactor at 90 °C for 30 min twice, providing the desired benzyl ether-linked derivatives 40–45 in good yield.

Results and Discussion

We determined the antiviral activities of RN18 analogues against wild-type HIV-1 in non-permissive H9 cells expressing A3G, as well as in permissive MT-4 cells, which do not express A3G, and examined cytotoxicity using MTT-based cell viability assays (Table 1). For antiviral assays, cells were infected with an X4-tropic HIV-1 variant (HIV-1LAI) in the presence and absence of inhibitors and viral replication was assessed by measuring the reverse transcriptase (RT) activity. RN18 (5a), used as a control, inhibited RT activity and viral replication in a dose-dependent manner in non-permissive H9 cells with an IC50 of 6 µM, but did not affect RT activity in permissive MT4 cells up to 100 µM.

Table 1
Antiviral potency in permissive and non-premissive cells and cytotoxicity of HIV-1 Vif inhibitors.

The isosteric replacement of amide linkage between rings B and C did not significantly affect the antiviral potency, but had a major effect on specificity, as some analogues showed activity in both H9 and MT4 cells. The reverse amide analogue 5c inhibited viral replication in H9 cells (H9 IC50 = 5.5 µM) with similar potency as RN18, but also showed weak activity in MT4 cells (MT4 IC50 = 52 µM). The sulfonamide analogue 5d, which is also equipotent to RN18 in H9 cells (H9 IC50 = 5.9 µM), did not show activity in MT4 cells up to 100 µM. Similar to the reverse amide analogue 5c, the reverse sulfonamide compound 5e also showed activity in both H9 (H9 IC50 = 21.5 µM) and MT4 cells (MT4 IC50 = 39 µM). The reverse amide derivative 5f has an activity profile similar to 5c, exhibiting potent activity in H9 cells (H9 IC50 = 5.2 µM) and relatively weak activity in MT4 cells (MT4 IC50 = 53.6 µM). These results suggest that amide or sulfonamide linkage between phenyl rings B and C is important for Vif-specific antiviral activity, as reversing the linkage may be detrimental for the specificity.

Compared to the A-ring 4-nitro compounds 5a–e, the corresponding 4-amino analogues 6a–e were generally less potent. Similar to the corresponding nitro compounds, the amide and sulfonamide analogues 6a and 6d maintained specificity and inhibited viral replication only in H9 cells, but the reverse amide and reverse sulfonamide compounds 6c and 6e inhibited replication in both H9 and MT-4 cells. Only the sulfonamide analogue 6d (H9 IC50 = 9.3 µM) exhibited antiviral potency and specificity profile similar to the parent nitro compound 5d, suggesting that the phenyl ring A may be amenable to various substitutions.

The C-ring analogues 15b–f with a variety of substitutions on the aromatic ring were all significantly less potent than RN18. Interestingly, the C-ring analogue 15h with a non-aromatic group showed potent antiviral activity, but no specificity; it inhibited replication in both H9 (IC50 = 3.9 µM) and MT-4 cells (IC50 = 3.7 µM). However, unlike other non-specific analogues, 15h was significantly less toxic, suggesting that its mechanism of action may be different from RN18. Similarly, analogues 16a and 16d with the 2-chloro-4-nitrophenyl A-ring exhibited potent antiviral activity, but lacked specificity, even though both contain amide linkage between rings B and C. In general, compounds with C-ring modifications showed reduced antiviral activity and specificity.

Next, we examined the role of thio-ether linkage between phenyl rings A and B by replacing it with either ether or benzyl ether. Compared to RN18, the corresponding ether analogue 18a inhibited viral replication in both H9 (IC50 = 4.7 µM) and MT4 cells (IC50 = 15.2 µM), showing significant loss of specificity. Since 18a also exhibited significant toxicity (CC50 = 15 µM), its antiviral effects are probably unrelated to Vif inhibition. The ether-linked analogues 19–33 with variations on the A ring provided interesting structure-activity relationships in this series. The A ring 1,3-benzodioxolane derivative 23h selectively inhibited viral replication in H9 cells (IC50 = 12.6 µM) with no toxicity up to 100 µM. The reverse amide derivative 30k with the dimethylaminophenyl group potently inhibited replication in H9 cells (IC50 = 2.5 µM), weak activity in MT-4 cells (IC50 = 68 µM), and no toxicity up to 100 µM, indicating significant specificity. The 4-methoxyphenyl derivative 31k (H9 IC50 = 9.7 µM), though less potent than 30k, also showed some specificity. These results suggest that, unlike the reverse amide thio-ether analogues, the ether-linked reverse amide analogues possess significant antiviral activity and their specificity can be influenced by variations at the phenyl ring A.

The benzyl ether-linked analogue 40j potently inhibited HIV-1 replication in H9 cells (H9 IC50 = 2.8 µM), showed no activity in MT-4 cells (MT-4 IC50 >100 µM) and no toxicity up to 100 µM, exhibiting a specificity profile similar to RN18. The reverse amide compound 40k (H9 IC50 = 34 µM) has reduced antiviral potency but exhibited no specificity (MT-4 IC50 = 26.6 µM). Compared to 40j, the 3-nitrophenyl analogue 41j showed slightly reduced potency in H9 cells (IC50 = 7.1 µM) with a similar specificity profile (MT-4 IC50 >100 µM). In contrast, the 4-cyanophenyl analogue 42j exhibited potent activity in H9 cells (IC50 = 3.1 µM), but has reduced specificity as it also inhibited replication in MT-4 cells (MT-4 IC50 = 22.8 µM). Surprisingly, the 4-methoxyphenyl (43j) and 4-methylbenzoate (45k) derivatives did not show any activity up to 100 µM, further supporting the earlier observation that variations at the A ring significantly influence the activity and specificity profiles. Interestingly, the activity profiles of the benzyl ether-linked analogues are similar to the thio-ether compounds.

Overall, our structure-activity relationship studies indicate that variations of phenyl substitutions at rings A and C significantly affect the potency and specificity profiles of RN18 analogues, and that the isosteric replacement of ring linkages are fairly well tolerated. Although not entirely conclusive, rather stringent requirements for an amide or sulfonamide linkage between rings B and C, a thio or benzyl ether linkage between rings A and B, and a 2-methoxyphenyl group as the C-ring fragment, are evident for the Vif-specific antiviral activity and specificity. Nonetheless, we have identified several analogues with antiviral potency, specificity, and toxicity profiles similar to or better than RN18. Specifically, the sulfonamide derivatives 5d and 6d and the benzyl ether-linked analogues 40j and 41j are interesting lead compounds warranting further SAR exploration.

One explanation for the observed SAR trends could be that we directly tested the compounds for cellular antiviral potency, which can be affected not only by in vivo specificity, but also by factors such as cell membrane permeability, solubility, protein binding, and stability under physiological conditions. Another reason could be the mechanism of action of RN18 and analogues, which likely involves inhibition of Vif-A3G protein-protein interactions. We have previously demonstrated that RN18 inhibits HIV-1 replication by specifically antagonizing Vif function, only in the presence of A3G, by inhibiting Vif-A3G interactions. It can be argued that RN18 analogues with similar activity and specificity profiles also exert their antiviral activity by specifically antagonizing Vif function, likely by inhibiting Vif-A3G interactions.

Conclusions

We have evaluated the structure-activity relationships of HIV-1 Vif inhibitor RN18, a small molecule recently discovered as a specific inhibitor of Vif function that blocks viral replication by targeting the Vif-A3G interactions. Two synthetic methods were developed, both involving microwave-assisted cross-coupling of aryl iodides and thiophenols, to provide excess to RN18 and series of analogues with diverse linkages and substitutions at the phenyl rings. Another series of analogues was prepared by replacing the thio-ether linkage with ether and benzyl ether. Compounds were tested for cytotoxicity and antiviral activity against wild-type HIV-1 in non-permissive H9 cells expressing A3G and permissive MT-4 cells, which do not express A3G.

Although not exhaustive, the SAR data indicate fairly stringent requirements for the Vif-A3G-specific antiviral activity of RN18 and analogues. An amide or sulfonamide linkage between rings B and C appears to be important for Vif-specific antiviral activity, as reversing the linkage may be detrimental for the specificity. A thio or benzyl ether linkage between rings A and B is generally preferred over the ether linkage, although some ether-linked reverse amide analogues show interesting activity and specificity profile that can be influenced by variations at the A ring. A limited exploration of the C-ring phenyl substitutions suggests a strong preference for a 2-methoxyphenyl group. However, variations at the phenyl ring A, involving both electron-withdrawing and donating groups, though influence the activity and specificity profile, appear to be relatively well tolerated. These SAR studies resulted in the identification of new analogues, such as 5d and 40j, which potently and selectively inhibit HIV-1 replication only in the presence of A3G, likely by inhibiting Vif-A3G interactions. These compounds are potential lead structures for further SAR exploration to enhance antiviral potency and pharmacological properties, and may serve as chemical probes of Vif function to understand HIV-1 biology and the APOBEC-mediated innate immune response.

This study provides further evidence that the Vif-A3G axis is a valid target for developing small molecule-based antiviral therapies for HIV-1 infection, and that more potent and selective inhibitors of Vif-A3G interactions may serve as effective anti-HIV-1 therapeutics.

Experimental Section

Chemistry

General

1H and 13C NMR spectra were recorded on a Varian Mercury 400 MHz spectrometer. Chemical shifts are reported in ppm (δ scale) relative to the solvent signal and coupling constant (J) values are reported in hertz (Hz). Data are presented as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br = broad), coupling constant in Hz, and integration. High-resolution mass spectra (HRMS) were recorded on Waters Q-TOF Premier mass spectrometer by direct infusion of solutions of each compound using electrospray ionization (ESI) in positive mode. Low-resolution mass spectra were obtained using Waters Alliance HT/Micromass ZQ system (ESI). Flash and column chromatography was performed using silica gel (230–400 mesh, Merck KGA). Analytical thin-layer chromatography (TLC) was performed using silica gel (60 F-254) coated aluminum plates (Merck KGA) and spots were visualized by exposure to ultraviolet light (UV) and/or exposure to an acidic solution of p-anisaldehyde followed by brief heating. Solvents and chemicals were purchased from Acros and Aldrich and were used as received.

Typical Procedure for the Synthesis of RN18 (5a) and Analogues

N-(2-Methoxyphenyl)-2-((4-nitrophenyl)thio)benzamide (5a) (RN-18)

Compound 1a (0.355 g, 1.0 mmol), Cu(I)I (20 mg, 0.1 mmol), K2CO3 (0.277 g, 2.0 mmol), and 4-nitrothiophenol 3 (0.155g, 1 mmol) were added to a 10 mL microwave reaction vessel with Teflon-lined septum. The tube was evacuated and backfilled with dry N2 (3 cycles). Ethylene glycol (0.11 mL, 2.0 mmol) and 2-propanol (1.5 mL) were added by syringe at room temperature. The reaction vessel was irradiated in a microwave reactor (CEM, Explorer) at 80 °C and 150 Watt power for 30 min (2 ×). The reaction mixture was allowed to cool to room temperature, diluted with ethyl acetate (10 mL), filtered and concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel eluting with EtOAc-hexanes (1:5) mixture to afford the product 5a (RN18) as a pale yellow crystalline solid (0.24 g, 63%). A similar procedure was used to prepare multi-grams of RN18; details are provided in the Supporting Information. 1H NMR (400 MHz, CDCl3) δ 8.40 (d, J = 8.0 Hz, 1H), 8.38 (s, 1H, overlapping), 8.08–8.04 (m, 2H), 7.78 (dd, J = 7.6, 2.0 Hz, 1H), 7.57–7.48 (m, 3H), 7.30–7.26 (m, 2H), 7.06 (ddd, J = 8.0, 8.0, 1.6 Hz, 1H), 6.97 (ddd, J = 7.6, 7.6, 1.2 Hz, 1H), 6.85 (dd, J = 8.0, 1.2 Hz, 1H), 3.79 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 165.44, 148.22, 146.87, 146.15, 140.63, 135.76, 131.66, 130.09, 129.87, 129.49, 128.79 (2C), 127.54, 124.54, 124.35 (2C), 121.37, 120.05, 110.19, 55.88; HRMS (ESI) m/z: calcd for C20H17N2O4S [M + H]+ 381.0909; found 381.0916.

N-(2-Methoxyphenyl)-2-(phenylthio)benzamide (4a)

Reaction of 1a and thiophenol gave the product 4a as a white solid in 98% isolated yield. 1H NMR (400 MHz, CDCl3) δ 8.65 (s, 1H) 8.51 (d, J = 7.2 Hz, 1H), 7.72 (m, 1H), 7.42–7.38 (m, 2H), 7.34–7.28 (m, 5H), 7.21–7.19 (m, 1H), 7.07 (ddd, J = 8.0, 8.0, 2.0 Hz, 1H), 7.0 (ddd, J = 7.6, 7.6, 1.2 Hz, 1H), 6.89 (dd, J = 8.0, 1.6 Hz, 1H), 3.83 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 165.90, 148.37, 136.87, 136.12, 134.63, 132.81 (2C), 131.80, 131.17, 129.66 (2C), 128.97, 128.12, 127.95, 126.96, 124.21, 121.38, 120.20, 110.17, 55.56; HRMS (ESI) m/z: calcd for C20H17NO2S [M + H]+ 335.0980; found 335.0984.

N-(2-Methoxyphenyl)-3-(4-nitrophenylthio)benzamide (5b)

Reaction of 1b and 4-nitrothiophenol gave the product 5b as a pale yellow solid in 62% isolated yield. 1H NMR (400 MHz, CDCl3) δ 8.51 (s, 1H), 8.48 (dd, J = 8.0, 1.6 Hz, 1H), 8.13–8.09 (m, 2H), 8.06 (t, J = 1.6 Hz, 1H), 7.93 (ddd, J = 7.6, 1.6, 1.2 Hz, 1H), 7.69 (ddd, J = 7.6, 1.6, 1.2 Hz, 1H), 7.58 (t, J = 8.0 Hz, 1H), 7.28–7.25 (m, 2H, overlapping), 7.11 (ddd, J = 8.0, 8.0, 1.6 Hz, 1H), 7.03 (ddd, J = 8.0, 8.0, 1.2 Hz, 1H), 6.93 (dd, J = 8.0, 1.2 Hz, 1H), 3.92 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 164.16, 148.35, 147.18, 146.05, 137.46, 137.29, 133.05, 132.63, 130.58, 127.99, 127.89 (2C), 127.58, 124.55, 124.48, 121.49 (2C), 120.16, 110.20, 56.08; HRMS (ESI) m/z: calcd for C20H17N2O4S [M + H]+ 381.0909; found 381.0907.

N-(2-(4-Nitrophenylthio)phenyl)-2-methoxybenzamide (5c)

Reaction of 1c and 4-nitrothiophenol gave the product 5c as a pale yellow solid in 53% isolated yield. 1H NMR (400 MHz, CDCl3) δ 10.86 (s, 1H), 8.86 (dd, J = 8.4, 1.2 Hz, 1H), 8.24 (dd, J = 8.2, 1.8 Hz, 1H), 8.07–8.03 (m, 2H), 7.63 (dd, J = 7.6, 1.2 Hz, 1H), 7.60 (ddd, J = 8.0, 8.0, 1.6 Hz, 1H), 7.46 (ddd, J = 8.0, 8.0, 2.0 Hz, 1H), 7.20 (ddd, J = 7.6, 7.6, 1.2 Hz, 1H), 7.12–7.07 (m, 3H), 6.93 (d, J = 8.4 Hz, 1H), 3.83 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 163.58, 157.29, 146.86, 145.49, 142.18, 137.45, 133.75, 132.64, 132.43, 125.46 (2C), 124.89, 124.37 (2C), 122.15, 121.56, 121.27, 117.05, 111.52, 55.98; HRMS (ESI) m/z: calcd for C20H17N2O4S [M + H]+ 381.0909; found 381.0906.

N-(2-Methoxyphenyl)-2-((4-nitrophenyl)thio)benzenesulfonamide (5d)

Reaction of 1d and 4-nitrothiophenol gave the product 5d as an orange-brown crystalline solid in 47% isolated yield. 1H NMR (400 MHz, CDCl3) δ 8.21–8.18 (m, 1H), 8.05–8.02 (m, 2H), 7.81 (s, 1H), 7.51–7.45 (m, 4H), 7.15–7.11 (m, 2H), 6.99 (ddd, J = 8.0, 8.0, 2.0 Hz, 1H), 6.85 (ddd, J = 7.6, 7.6, 1.6 Hz, 1H), 6.65 (dd, J = 8.4, 1.2 Hz, 1H), 3.49 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 148.77, 146.25, 141.51, 136.95, 134.02, 131.98, 131.50, 129.43, 129.02 (2C), 125.86, 125.04, 124.27 (2C), 121.37, 119.40, 110.54, 105.0, 55.51; HRMS (ESI) m/z: calcd for C19H17N2O5S2 [M + H]+ 417.0579; found 417.0587.

2-Methoxy-N-(2-(4-nitrophenylthio)phenyl)benzenesulfonamide (5e)

Reaction of 1e and 4-nitrothiophenol gave the product 5e as a pale yellow crystalline solid in 25% isolated yield. 1H NMR (400 MHz, CDCl3) δ 8.10 (s, 1H), 7.99 (m, 2H), 7.91 (dd, J = 8.0, 1.2 Hz, 1H), 7.81 (d, J = 8.4 Hz, 1H), 7.47–7.39 (m, 3H), 7.09 (ddd, J = 7.6, 7.6, 1.2 Hz, 1H), 7.01 (ddd, J = 8.0, 8.0, 0.8 Hz, 1H), 6.91 (m, 2H), 6.76 (d, J = 8.4 Hz, 1H), 3.72 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 156.45, 146.68, 145.73, 140.66, 137.95, 135.53, 132.64, 131.03, 126.39, 125.84 (2C), 125.26, 124.40 (2C), 120.66, 119.46, 117.24, 112.18, 56.22; HRMS (ESI) m/z: calcd for C19H17N2O5S2 [M + H]+ 417.0579; found 417.0596.

2-(2-Methoxyphenyl)-N-(2-((4-nitrophenyl)thio)phenyl)acetamide (5f)

Reaction of 1f and 4-nitrothiophenol gave the product 5f in 45% isolated yield. 1H NMR (400 MHz, CDCl3) δ 8.59 (dd, J = 8.0, 1.2 Hz, 1H), 8.46 (s, 1H), 7.98–7.94 (m, 2H), 7.54–7.49 (m, 2H), 7.18–7.11 (m, 2H), 7.07 (dd, J = 7.6, 1.6 Hz, 1H), 6.83–6.79 (m, 3H), 6.66 (d, J = 8.4 Hz, 1H), 3.67 (s, 3H), 3.65 (s, 2H); 13C NMR (100 MHz, CDCl3) δ 169.91, 157.10, 146.10, 145.63, 141.23, 137.35, 132.64, 131.42, 129.43, 125.55 (2C), 124.96, 124.26 (2C), 122.50, 121.31, 121.29, 116.54, 110.74, 55.59, 40.30; HRMS (ESI) m/z: calcd for C21H19N2O4S [M + H]+ 395.1066; found 395.1058.

2-(2-Fluorophenyl)-N-(2-((4-nitrophenyl)thio)phenyl)acetamide (5g)

Reaction of 1g and 4-nitrothiophenol gave the product 5g in 37% isolated yield. 1H NMR (400 MHz, CDCl3) δ 8.57 (dd, J = 8.0, 1.2 Hz, 1H), 8.19 (s, 1H), 8.02–7.98 (m, 2H), 7.57–7.52 (m, 2H), 7.22–7.11 (m, 3H), 7.0 (ddd, J = 8.0, 8.0, 1.2 Hz, 1H), 6.87–6.84 (m, 3H), 3.67 (d, J = 1.2 Hz, 2H); 13C NMR (100 MHz, CDCl3) δ 168.26, 160.92 (d, J = 245.4 Hz), 145.80, 145.74, 140.62, 137.43, 132.76, 131.76 (d, J = 3.7 Hz), 129.96 (d, J = 8.1 Hz), 125.52 (2C), 125.32, 124.94 (d, J = 3.7 Hz), 124.36 (2C), 121.21, 121.07, 116.68, 115.88 (d, J = 22.0 Hz), 38.56 (J = 2.9 Hz); HRMS (ESI) m/z: calcd for C20H16FN2O3S [M + H]+ 383.0866; found 383.0862.

Typical Procedure for the Synthesis of Analogues 6a–e

2-((4-Aminophenyl)thio)-N-(2-methoxyphenyl)benzamide (6a)

A solution of the nitro compound 5a (0.5 mmol) and SnCl2.2H2O (0.56 g, 2.5 mmol) in EtOAc (10 mL) was heated at 80 °C for 3 hours. The reaction mixture was allowed to reach room temperature and treated with saturated aqueous NaHCO3 solution till basic. The aqueous layer was extracted with ethyl acetate (3 × 25 mL); the combined organic portion was washed with saturated aqueous NaCl solution (25 mL), dried (Na2SO4), filtered, and evaporated. The residue was purified by flash chromatography on silica gel eluting with EtOAc-hexanes (2:3) mixture to afford the product 6a as an off-white solid in 90% isolated yield. 1H NMR (400 MHz, CDCl3) δ 8.58 (d, J = 3.2 Hz, 1H), 8.56 (s, 1H), 7.62 (dd, J = 7.6, 1.6 Hz, 1H), 7.33–7.29 (m, 2H), 7.24 (ddd, J = 7.6, 7.6, 1.6 Hz, 1H), 7.17 (ddd, J = 7.6, 7.6, 1.6 Hz, 1H), 7.08 (ddd, J = 7.6, 7.6, 1.2 Hz, 1H), 7.02 (ddd, J = 8.0, 8.0, 1.2 Hz, 1H), 6.98 (dd, J = 7.6, 1.2 Hz, 1H), 6.91 (dd, J = 8.0, 1.6 Hz, 1H), 6.69–6.65 (m, 2H), 3.89 (s, 3H), 3.84 (br s, 2H); 13C NMR (100 MHz, CDCl3) δ 166.15, 148.38, 146.77, 139.92, 136.82 (2C), 134.64, 130.98, 128.85, 128.82, 128.39, 128.04, 125.36, 124.16, 121.43, 120.83, 120.23, 116.60 (2C), 110.22, 56.03; HRMS (ESI) m/z: calcd for C20H19N2O2S [M + H]+ 351.1167; found 351.1169.

3-(4-Aminophenylthio)-N-(2-methoxyphenyl)benzamide (6b)

Reduction of the nitro group in 5b gave the product 6b as an off-white solid in 65% isolated yield. 1H NMR (400 MHz, CDCl3) δ 8.48 (dd, J = 8.0, 1.6 Hz, 1H), 8.44 (s, 1H), 7.59 (m, 2H), 7.37–7.30 (m, 3H), 7.25 (m, 1H), 7.07 (ddd, J = 7.6, 7.6, 1.6 Hz, 1H), 7.00 (ddd, J = 7.6, 7.6, 1.2 Hz, 1H), 6.90 (dd, J = 8.0, 1.2 Hz, 1H), 6.72–6.69 (m, 2H), 3.90 (s, 3H), 3.87 (s, 2H); 13C NMR (100 MHz, CDCl3) δ 165.09, 148.34, 147.80, 141.68, 136.86 (2C), 136.19, 130.00, 129.34, 127.90, 125.29, 124.16, 123.95, 121.40, 120.02, 119.15, 116.23 (2C), 110.14, 56.02; HRMS (ESI) m/z: calcd for C20H19N2O2S [M + H]+ 351.1167; found 351.1170.

N-(2-(4-Aminophenylthio)phenyl)-2-methoxybenzamide (6c)

Reduction of the nitro group in 5c gave the product 6c in 78% isolated yield. 1H NMR (400 MHz, CDCl3) δ 10.96 (s, 1H), 8.67 (dd, J = 8.4, 1.2 Hz, 1H), 8.29 (dd, J = 8.0, 1.6 Hz, 1H), 7.49 (ddd, J = 8.0, 8.0, 2.0 Hz, 2H), 7.38 (dd, J = 8.0, 1.6 Hz, 1H), 7.33 (ddd, J = 8.0, 8.0, 1.6 Hz, 1H), 7.13–7.09 (m, 3H), 7.02 (ddd, J = 7.6, 7.6, 1.2 Hz, 1H), 7.01 (d, J = 8.4 Hz, 1H, overlapping), 6.59–6.54 (m, 2H), 3.99 (s, 3H), 3.71 (s, 2H); 13C NMR (100 MHz, CDCl3) δ 163.73, 157.66, 146.34, 139.70, 134.11, 133.53, 132.80, 132.41 (2C), 129.30, 125.06, 124.46, 122.71, 122.10, 121.67, 121.54, 116.19 (2C), 111.63, 56.27; HRMS (ESI) m/z: calcd for C20H19N2O2S [M + H]+ 351.1167; found 351.1173.

2-((4-Aminophenyl)thio)-N-(2-methoxyphenyl)benzenesulfonamide (6d)

Reduction of the nitro group in 5d gave the product 6d as an off-white solid in 83% isolated yield. 1H NMR (400 MHz, CDCl3) δ 8.11 (s, 1H), 7.93 (dd, J = 8.0, 1.6 Hz, 1H), 7.54 (dd, J = 8.4, 1.6 Hz, 1H), 7.24–7.18 (m, 3H), 7.08 (ddd, J = 7.6, 7.6, 1.2 Hz, 1H), 6.99 (ddd, J = 7.6, 7.6, 1.6 Hz, 1H), 6.91 (dd, J = 8.4, 1.2 Hz, 1H), 6.85 (ddd, J = 8.0, 8.0, 1.2 Hz, 1H), 6.78 (dd, J = 8.0, 1.2 Hz, 1H), 6.69–6.65 (m, 2H), 3.89 (br s, 2H), 3.71 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 149.10, 148.06, 140.76, 137.09 (2C), 135.40, 132.87, 130.63, 129.29, 126.52, 124.75, 124.63, 121.31, 119.56, 118.93, 116.19 (2C), 110.74, 55.92; HRMS (ESI) m/z: calcd for C19H19N2O3S2 [M + H]+ 387.0837; found 387.0843.

N-(2-(4-aminophenylthio)phenyl)-2-methoxybenzenesulfonamide (6e)

Reduction of the nitro group in 5e gave the product 6e in 75% isolated yield. 1H NMR (400 MHz, CDCl3) δ 8.21 (s, 1H), 7.91 (dd, J = 8.4, 2.0 Hz, 1H), 7.61 (dd, J = 8.4, 1.2 Hz, 1H), 7.48 (ddd, J = 8.0, 8.0, 1.6 Hz, 1H), 7.25 (dd, J = 8.0, 1.2 Hz, 1H, overlapping), 7.15 (ddd, J = 8.0, 8.0, 1.6 Hz, 1H), 7.0–6.87 (m, 5H), 6.56–6.52 (m, 2H), 3.85 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 156.71, 146.44, 138.16, 135.26, 134.85, 132.33 (2C), 131.18, 129.51 (2C), 126.60, 125.15, 124.66, 122.47, 120.37, 119.21, 116.13, 112.12, 56.32; HRMS (ESI) m/z: calcd for C19H19N2O3S2 [M + H]+ 387.0837; found 387.0842.

Typical Procedure for the Synthesis of Intermediate Esters 10–11

Methyl 2-(4-nitrophenylthio)benzoate (10)

4-Iodonitrobenzene 8 (0.5 g, 2.0 mmol), Cu(I)I (50 mg, 0.26 mmol), and K2CO3 (0.552 g, 4.0 mmol) were added to a 10 mL microwave reaction vessel with Teflon-lined septum. The tube was evacuated and backfilled with dry N2 (3 cycles). 1,2-Dimethoxyethane (2 mL) was added by syringe at room temperature followed by methyl thiosalicylate 7 (0.34 g, 2 mmol). The reaction vessel was irradiated in a microwave reactor (CEM, Explorer) at 80 °C and 150 Watt power for 30 min (2 ×). The reaction mixture was allowed to reach room temperature, diluted with EtOAc (10 mL) and filtered; solids were repeatedly washed with EtOAc. The combined filtrate was concentrated to provide an orange solid. The crude product was purified by flash column chromatography on silica gel eluting with EtOAc-hexanes (1:6) mixture to afford the product 10 as a pale yellow crystalline solid (0.5 g, 86%). 1H NMR (400 MHz, CDCl3) δ 8.15 (m, 2H), 7.95 (dd, J = 7.6, 1.6 Hz, 1H), 7.47 (m, 2H), 7.39 (ddd, J = 8.0, 8.0, 1.6 Hz, 1H), 7.32 (ddd, J = 8.0, 8.0, 1.6 Hz, 1H), 7.17 (dd, J = 8.0, 1.6 Hz, 1H), 3.89 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 166.82, 146.93, 144.95, 136.71, 132.71, 131.88 (2 C), 131.72, 131.25, 131.07, 127.30, 124.42 (2 C), 52.56; MS (ESI): m/z 312.50 (M + Na)+.

Methyl 2-(2-chloro-4-nitrophenylthio)benzoate (11)

Reaction of methyl thiosalicylate 7 and 2-chloro-1-iodo-4-nitrobenzene 9 gave the product 11 as a yellow crystalline solid in 70% isolated yield. 1H NMR (400 MHz, CDCl3) δ 8.26 (d, J = 2.4 Hz, 1H), 7.99–7.95 (m, 2H), 7.50–7.43 (m, 2H), 7.27 (dd, J = 7.6, 1.6 Hz, 1H), 7.16 (d, J = 8.4 Hz, 1H), 3.87 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 166.81, 146.72, 145.46, 134.60, 133.83, 133.36, 133.17, 133.02, 131.59, 131.40, 128.80, 125.04, 121.22, 52.76; MS (ESI): m/z 346.50 (M + Na)+.

Typical Procedure for Synthesis of Analogues 15–16

2-(4-Nitrophenylthio)-N-(2-methoxyphenyl)benzamide (15a) (RN-18)

A solution of ester 10 (0.72 g, 2.5 mmol) in MeOH (25 ml) was treated with Ba(OH)2[bullet]8H2O (1.18 g, 3.75 mmol) and the resulting mixture was heated at 80 °C for 3 hours. Reaction mixture was cooled to room temperature and solvents were removed under reduced pressure. The residue was treated with 1M HCl solution in Et2O (25 mL) and filtered; the solids were washed with Et2O (40 mL) and filtered. The combined filtrate was dried (MgSO4), filtered, and concentrated to provide the acid 12 as a light brown solid (0.66 g, 96%), which was used as such in the next reaction.

To a solution of the above acid 12 (0.66 g, 2.4 mmol) in dry CH2Cl2 (8 mL) was added oxalyl chloride (1 mL) followed by a drop of dimethylformamide (DMF). The mixture was stirred at room temperature for 4 hours. Solvents were removed under reduced pressure and the residue was dried using high vacuum to give the corresponding acid chloride as a yellow solid. A solution of 2-methoxyaniline (0.3 g, 2.4 mmol) in dry CH2Cl2 (10 mL) under nitrogen atmosphere was cooled to 0 °C and Et3N (0.75 mL, 5.4 mmol) was added followed by the slow addition of the above acid chloride solution in dry CH2Cl2 (10 mL). The resulting mixture was warmed to room temperature and stirred overnight. Reaction mixture was diluted with CH2Cl2 (20 mL) and water (10 mL) and layers were separated; the organic portion was washed with saturated aqueous NaCl solution, dried (Na2SO4), filtered, and evaporated to afford a yellow solid. The product was purified by flash chromatography on silica gel eluting with EtOAc-hexanes (1:5) mixture to give the product 15a (RN18) as a yellow crystalline solid (0.83 g, 90%). Analytical data is identical to the product 5a prepared by direct cross-coupling method.

N-(2-Hydroxyphenyl)-2-((4-nitrophenyl)thio)benzamide 15b

Reaction of acid 12 and 2-hydroxyaniline 14b gave the product 15b in 66% isolated yield. 1H NMR (400 MHz, CDCl3) δ 8.41 (br s, 1H), 8.11–8.08 (m, 2H), 8.04 (s, 1H), 7.92 (m, 1H), 7.63–7.56 (m, 3H), 7.30–7.26 (m, 2H, overlapping), 7.14 (ddd, J = 8.0, 8.0, 1.6 Hz, 1H), 7.09 (dd, J = 7.6, 1.2 Hz, 1H), 7.0 (dd, J = 8.0, 1.2 Hz, 1H), 6.88 (ddd, J = 7.6, 7.6, 1.2 Hz, 1H); 13C NMR (100 MHz, CD3OD) δ 168.77, 149.67, 148.20, 147.28, 142.24, 136.97, 132.54, 130.91, 130.67, 130.15, 129.48 (2C), 126.99, 126.79, 125.09 (2C), 123.50, 120.54, 116.80; HRMS (ESI) m/z: calcd for C19H15N2O4S [M + H]+ 367.0753; found 367.0758.

N-(2-Fluorophenyl)-2-((4-nitrophenyl)thio)benzamide 15c

Reaction of acid 12 and 2-fluoroaniline 14c gave the product 15c in 79% isolated yield. 1H NMR (400 MHz, CDCl3) δ 8.34 (dd, J = 8.0, 1.6 Hz, 1H), 8.25 (s, 1H), 8.07 (m, 2H), 7.86 (dd, J = 7.6, 2.0 Hz, 1H), 7.60–7.53 (m, 3H), 7.28 (m, 2H), 7.13 (m, 1H), 7.08 (m, 2H); 13C NMR (100 MHz, CDCl3) δ 165.37, 152.78 (J = 241.8 Hz), 146.33, 146.19, 139.49, 136.06, 132.17, 130.13, 130.06, 129.78, 128.61 (2C), 126.21 (J = 10.3 Hz), 125.14 (J = 8.1 Hz), 124.94 (J = 3.7 Hz), 124.50 (2C), 121.90, 115.09 (J = 19.0 Hz); HRMS (ESI) m/z: calcd for C19H14FN2O3S [M + H]+ 369.0709; found 369.0711.

N-(Benzo[d][1,3]dioxol-5-yl)-2-((4-nitrophenyl)thio)benzamide 15d

Reaction of acid 12 and benzo[d][1,3]dioxol-5-amine 14d gave the product 15d in 87% isolated yield. 1H NMR (400 MHz, CDCl3) δ 8.09–8.06 (m, 2H), 7.81 (m, 2H), 7.58–7.50 (m, 3H), 7.28–7.25 (m, 2H, overlapping), 7.20 (s, 1H), 6.74 (d, J = 1.2 Hz, 2H), 5.95 (s, 2H); 13C NMR (100 MHz, CDCl3) δ 165.50, 148.10, 146.24, 144.90, 140.03, 135.94, 131.91, 131.80, 130.14, 129.93, 128.50 (2C), 124.52 (2C), 113.40, 108.36, 105.0, 102.98, 101.64; HRMS (ESI) m/z: calcd for C20H15N2O5S [M + H]+ 395.0702; found 395.0692.

N-(2-(Hydroxymethyl)phenyl)-2-((4-nitrophenyl)thio)benzamide 15e

Reaction of acid 12 and (2-aminophenyl)methanol 14e gave the product 15e in 84% isolated yield. 1H NMR (400 MHz, CDCl3) δ 9.13 (s, 1H), 8.12–8.06 (m, 3H), 7.76 (m, 1H), 7.54–7.48 (m, 3H), 7.36–7.30 (m, 3H), 7.19 (dd, J = 8.0, 1.2 Hz, 1H), 7.11 (ddd, J = 8.0, 8.0, 1.2 Hz, 1H), 4.69 (s, 2H), 2.06 (br s, 1H); 13C NMR (100 MHz, CDCl3) δ 166.17, 146.77, 146.25, 140.27, 137.40, 135.34, 131.74, 130.66, 129.82, 129.69, 129.53, 129.08, 129.05, 129.03 (2C), 125.04, 124.43 (2C), 122.74, 64.75; HRMS (ESI) m/z: calcd for C20H17N2O4S [M + H]+ 381.0909; found 389.0909.

N-(3-(Hydroxymethyl)phenyl)-2-((4-nitrophenyl)thio)benzamide 15f

Reaction of acid 12 and (3-aminophenyl)methanol 14f gave the product 15f in 90% isolated yield. 1H NMR (400 MHz, CDCl3) δ 8.07–8.03 (m, 2H), 7.96 (br s, 1H), 7.78 (m, 1H), 7.58–7.49 (m, 4H), 7.37 (d, J = 7.6 Hz, 1H), 7.31–7.25 (m, 3H, overlapping), 7.12 (d, J = 7.6 Hz, 1H), 4.65 (s, 2H), 1.93 (br s, 1H); 13C NMR (100 MHz, CDCl3) δ 165.53, 146.08, 145.96, 142.06, 139.78, 137.56, 135.51, 131.57, 129.71, 129.47, 129.38, 129.19, 128.39 (2C), 124.18 (2C), 123.24, 119.07, 118.35, 67.73; HRMS (ESI) m/z: calcd for C20H17N2O4S [M + H]+ 381.0909; found 389.0911.

Morpholino(2-((4-nitrophenyl)thio)phenyl)methanone 15g

Reaction of acid 12 and morpholine 14g gave the product 15g in 99% isolated yield. 1H NMR (400 MHz, CDCl3) δ 8.10–8.06 (m, 2H), 7.57–7.44 (m, 3H), 7.39 (dd, J = 7.6, 1.6 Hz, 1H), 7.26–7.21 (m, 2H), 3.75 (br s, 4H), 3.55 (t, J = 4.8 Hz, 2H), 3.21 (t, J = 5.2 Hz, 2H); 13C NMR (100 MHz, CDCl3) δ 167.61, 146.60, 145.69, 141.27, 136.17, 130.41, 130.29, 127.56, 127.48 (2C), 124.03 (2C), 66.63, 66.58, 47.28, 41.94; HRMS (ESI) m/z: calcd for C17H17N2O4S [M + H]+ 345.0909; found 345.0900.

tert-Butyl 4-(2-((4-nitrophenyl)thio)benzoyl)piperazine-1-carboxylate 15h

Reaction of acid 12 and 1-Boc-piperazine 14h gave the product 15h in 99% isolated yield. 1H NMR (400 MHz, CDCl3) δ 8.10–8.06 (m, 2H), 7.57–7.44 (m, 3H), 7.38 (dd, J = 7.2, 1.2 Hz, 1H), 7.25–7.21 (m, 2H), 3.71 (br s, 2H), 3.49 (t, J = 5.2 Hz, 2H), 3.35 (t, J = 4.8 Hz, 2H), 3.18 (t, J = 5.6 Hz, 2H), 1.45 (s, 9H); 13C NMR (100 MHz, CDCl3) δ 168.01, 154.67, 146.77, 146.03, 141.62, 136.39, 130.73, 130.57, 127.86, 127.84 (2C), 127.77, 124.36 (2C), 80.72, 47.03 (2C), 41.76 (2C), 28.57 (3C); HRMS (ESI) m/z: calcd for C22H25N3O5SNa [M + Na]+ 466.1413; found 466.1401.

2-(2-Chloro-4-nitrophenylthio)-N-(2-methoxyphenyl)benzamide 16a

Reaction of acid 13 and 2-methoxyaniline 14a gave the product 16a as an orange solid in 92% isolated yield. 1H NMR (400 MHz, CDCl3) δ 8.42 (br s, 1H), 8.36 (dd, J = 8.0, 1.6 Hz, 1H), 8.15 (d, J = 2.4 Hz, 1H), 7.90 (dd, J = 8.8, 2.4 Hz, 1H), 7.84 (dd, J = 7.6, 1.2 Hz, 1H), 7.62–7.52 (m, 3H), 7.05 (ddd, J = 8.0, 8.0, 1.6 Hz, 1H), 6.94 (m, 2H), 6.85 (dd, J = 8.0, 0.8 Hz, 1H), 3.79 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 165.04, 148.21, 146.62, 145.85, 141.54, 136.70, 132.02, 131.88, 130.69, 129.79, 128.50, 127.87, 127.37, 124.65, 124.51, 122.14, 121.20, 120.03, 110.12, 55.80; MS (ESI): m/z 437.61 (M + Na)+; HRMS (ESI) m/z: calcd for C20H16ClN2O4S [M + H]+ 415.0519; found 415.0511.

N-(Benzo[d][1,3]dioxol-5-yl)-2-((2-chloro-4-nitrophenyl)thio)benzamide 16d

Reaction of acid 13 and benzo[d][1,3]dioxol-5-amine 14d gave the product 16d as an orange solid in 79% isolated yield. 1H NMR (400 MHz, CDCl3) δ 8.20 (d, J = 2.4 Hz, 1H), 7.92 (dd, J = 8.8, 2.0 Hz, 1H), 7.91–7.86 (m, 2H), 7.65–7.56 (m, 3H), 7.19 (d, J = 1.6 Hz, 1H), 6.92 (d, J = 8.8 Hz, 1H), 6.78–6.72 (m, 2H), 5.95 (s, 2H); 13C NMR (100 MHz, CDCl3) δ 165.10, 148.14, 146.12, 145.07, 140.96, 136.94, 132.20, 131.62, 131.06, 130.51, 130.39, 128.34, 127.40, 124.88, 122.41, 113.61, 108.37, 103.11, 101.64; HRMS (ESI) m/z: calcd for C20H14ClN2O5S [M + H]+ 429.0312; found 429.0310.

Typical Procedure for Synthesis of Analogues 18a,e,g

N-(2-Methoxyphenyl)-2-(4-nitrophenoxy)benzamide 18a

To a solution of acid 17 (0.5 g, 1.93 mmol) in dry CH2Cl2 (8 mL) was added oxalyl chloride (1 mL) followed by a drop of dimethylformamide (DMF). The reaction mixture was stirred at room temperature for 4 hours. Solvents were removed under reduced pressure and the residue was dried under high vacuum to give the corresponding acid chloride as a pale yellow solid. A solution of 2-methoxyaniline (0.26 g, 2.11 mmol) in dry CH2Cl2 (10 mL) under nitrogen atmosphere was cooled to 0 °C and Et3N (0.47 mL, 3.37 mmol) was added followed by the slow addition of the above acid chloride solution in dry CH2Cl2 (10 mL). The resulting mixture was warmed to room temperature and stirred overnight. Reaction mixture was diluted with CH2Cl2 (20 mL) and water (10 mL) and layers were separated; the organic portion was washed with saturated aqueous NaCl solution, dried (Na2SO4), filtered, and evaporated to afford a yellow solid. The product was purified by flash chromatography on silica gel eluting with EtOAc-hexanes (1:4) mixture to give the product 18a as a pale yellow crystalline solid (0.63 g, 90%). 1H NMR (400 MHz, CDCl3) δ 9.77 (s, 1H), 8.54 (dd, J = 8.0, 1.2 Hz, 1H), 8.32 (dd, J = 8.0, 1.6 Hz, 1H), 8.29–8.25 (m, 2H), 7.54 (ddd, J = 8.0, 8.0, 1.6 Hz, 1H), 7.41 (ddd, J = 8.0, 8.0, 1.2 Hz, 1H), 7.18–7.14 (m, 2H), 7.07–7.03 (m, 2H), 6.98 (ddd, J = 8.0, 8.0, 1.2 Hz, 1H), 6.86 (dd, J = 7.6, 1.2 Hz, 1H), 3.75 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 162.04, 161.86, 152.84, 148.30, 143.91, 133.51, 132.91, 128.01, 126.73, 126.33 (2C), 126.12, 124.25, 121.50, 120.66, 120.39, 118.42 (2C), 110.16, 55.88; HRMS (ESI) m/z: calcd for C20H17N2O5 [M + H]+ 365.1137; found 365.1129.

N-(2-(hydroxymethyl)phenyl)-2-(4-nitrophenoxy)benzamide 18e

Reaction of acid 17 and 2-aminobenzylalcohol 14e gave the product 18e as a light orange crystalline solid in 86% isolated yield. 1H NMR (400 MHz, CDCl3) δ 9.82 (s, 1H), 8.26–8.21 (m, 3H), 8.13 (d, J = 8.0 Hz, 1H), 7.53 (ddd, J = 8.0, 8.0, 1.6 Hz, 1H), 7.40–7.33 (m, 2H), 7.22–7.16 (m, 3H), 7.10 (ddd, J = 7.6, 7.6, 1.2 Hz, 1H), 7.01 (dd, J = 8.0, 0.8 Hz, 1H), 4.61 (d, J = 5.2 Hz, 2H), 2.01 (t, J = 6.0 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 163.07, 161.52, 152.86, 143.58, 137.04, 133.22, 132.32, 130.44, 129.02, 128.99, 126.46, 125.96 (2C), 125.53, 124.71, 123.09, 119.99, 118.53 (2C), 63.88; HRMS (ESI) m/z: calcd for C20H17N2O5 [M + H]+ 365.1137; found 365.1145.

Morpholino(2-(4-nitrophenoxy)phenyl)methanone 18g

Reaction of acid 17 and morpholine gave the product 18g as a colorless crystalline solid in 93% isolated yield. 1H NMR (400 MHz, CDCl3) δ 8.23–8.18 (m, 2H), 7.50–7.43 (m, 2H), 7.34 (ddd, J = 7.6, 7.6, 1.2 Hz, 1H), 7.06 (dd, J = 8.0, 0.8 Hz, 1H), 7.03–6.99 (m, 2H), 3.70–3.62 (m, 6H), 3.39–3.32 (m, 2H); 13C NMR (100 MHz, CDCl3) δ 166.46, 162.77, 150.60, 143.32, 131.58, 129.48, 129.35, 126.44, 126.26 (2C), 121.24, 117.33 (2C), 67.12, 67.01, 47.70, 42.41; HRMS (ESI) m/z: calcd for C17H17N2O5 [M + H]+ 329.1137; found 329.1132.

2-(4-Aminophenoxy)-N-(2-methoxyphenyl)benzamide 19a

Reduction of the nitro group in 18a gave the product 19a in 78% isolated yield. 1H NMR (400 MHz, CDCl3) δ 10.70 (s, 1H), 8.63 (dd, J = 7.6, 2.0 Hz, 1H), 8.33 (dd, J = 8.0, 2.0 Hz, 1H), 7.36 (ddd, J = 7.6, 7.6, 2.0 Hz, 1H), 7.17 (ddd, J = 8.0, 8.0, 1.2 Hz, 1H), 7.05–6.96 (m, 4H), 6.86–6.83 (m, 2H), 6.76–6.72 (m, 2H), 3.65 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 162.58, 156.85, 148.36, 146.76, 143.69, 132.72, 132.15, 128.53, 123.41, 123.23, 122.62, 121.39 (2C), 121.08, 120.05, 116.49, 116.18 (2C), 109.94, 55.61; HRMS (ESI) m/z: calcd for C20H19N2O3 [M + H]+ 335.1396; found 335.1393.

Typical Procedure for the Synthesis of Analogues 22–23

2-(4-Methoxyphenoxy)-N-(2-methoxyphenyl)benzamide 22h

2-Bromo-N-(2-methoxyphenyl)benzamide 1h (0.20 g, 0.65 mmol), 4-methoxyphenol 20 (0.19 g, 1.25 mmol), Cu(I) chloride (32.0 mg, 0.33 mmol), and Cs2CO3 (0.43 g, 1.31 mmol) were added to a 10 mL microwave reaction vessel with Teflon-lined septum. The tube was evacuated and backfilled with dry N2 (3 cycles). 2,2,6,6-tetramethylheptane-3,5-dione (TMHD) (0.014 mL, 0.065 mmol) and N-methylpyrrolidone (2 mL) were added by syringe at room temperature and the reaction vessel was irradiated in a microwave reactor (CEM, Explorer) at 120 °C and 150 Watt power for 30 min (2 ×). The reaction mixture was cooled to reach room temperature, diluted with ethyl acetate (10 mL) and filtered; the filtrate was concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel eluting with EtOAc-hexanes (1:8) mixture to give the compound 22h (0.08 g, 36%). 1H NMR (400 MHz, CDCl3) δ 10.63 (s, 1H), 8.63 (dd, J = 7.2, 2.0 Hz, 1H), 8.35 (dd, J = 7.6, 1.6 Hz, 1H), 7.38 (ddd, J = 7.6, 7.6, 2.0 Hz, 1H), 7.20 (ddd, J = 7.6, 7.6, 1.2 Hz, 1H), 7.13–7.09 (m, 2H), 7.06–6.98 (m, 2H), 6.97–6.93 (m, 2H), 6.84 (m, 2H), 3.84 (s, 3H), 3.65 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 162.74, 156.96, 156.73, 148.72, 148.59, 133.06, 132.56, 128.74, 123.87, 123.76, 123.29, 121.54 (2C), 121.43, 120.37, 117.13, 115.32 (2C), 110.18, 55.92, 55.89; HRMS (ESI) m/z: calcd for C21H20NO4 [M + H]+ 350.1392; found 350.1381.

2-(4-Methoxyphenoxy)-N-(2-methoxyphenyl)benzenesulfonamide 22i

Reaction of 2-bromo-N-(2-methoxyphenyl)benzenesulfonamide 1i and 4-methoxyphenol 20 gave compound 22i in 32% isolated yield. 1H NMR (400 MHz, CDCl3) δ 8.00 (dd, J = 8.0, 2.0 Hz, 1H), 7.70 (s, 1H), 7.52 (dd, J = 7.6, 1.6 Hz, 1H), 7.34 (ddd, J = 8.0, 8.0, 2.0 Hz, 1H), 7.05 (ddd, J = 7.6, 7.6, 1.2 Hz, 1H), 6.97 (ddd, J = 7.6, 7.6, 1.6 Hz, 1H), 6.92–6.86 (m, 4H), 6.83 (ddd, J = 7.6, 7.6, 1.2 Hz, 1H), 6.76 (dd, J = 8.0, 1.2 Hz, 1H), 6.70 (dd, J = 8.0, 0.8 Hz, 1H), 3.82 (s, 3H), 3.62 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 157.10, 156.30, 148.52, 148.42, 134.72, 131.43, 128.00, 126.74, 124.26, 121.98 (2C), 121.93, 121.29, 118.52, 116.77, 115.16 (2C), 110.55, 55.92, 55.66; HRMS (ESI) m/z: calcd for C20H20NO5S [M + H]+ 386.1062; found 386.1058.

2-(Benzo[d][1,3]dioxol-5-yloxy)-N-(2-methoxyphenyl)benzamide 23h

Reaction of 2-bromo-N-(2-methoxyphenyl)benzamide 1h and benzo[d][1,3]dioxol-5-ol 21 gave the product 23h in 30% isolated yield. 1H NMR (400 MHz, CDCl3) δ 10.50 (s, 1H), 8.63 (dd, J = 8.0, 2.0 Hz, 1H), 8.34 (dd, J = 8.0, 2.0 Hz, 1H), 7.40 (ddd, J = 7.6, 7.6, 2.0 Hz, 1H), 7.21 (ddd, J = 8.0, 8.0, 0.8 Hz, 1H), 7.06–6.98 (m, 2H), 6.90–6.82 (m, 3H), 6.69 (d, J = 2.4 Hz, 1H), 6.63 (dd, J = 8.4, 2.4 Hz, 1H), 6.02 (s, 2H), 3.70 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 162.64, 156.36, 149.90, 148.88, 148.57, 144.96, 133.11, 132.58, 128.67, 124.06, 123.81, 123.59, 121.43, 120.38, 117.47, 112.80, 110.20, 108.71, 102.68, 102.07, 55.94; HRMS (ESI) m/z: calcd for C21H18NO5 [M + H]+ 364.1185; found 364.1184.

Typical Procedure for the Synthesis of Analogues 29–33

2-Methoxy-N-(2-(4-nitrophenoxy)phenyl)benzamide 29k

Et3N (0.57 mL, 4.10 mmol) was added to a mixture of N-(2-hydroxyphenyl)-2-methoxybenzamide 1k (0.20 g, 0.82 mmol), 4-nitrophenylboronic acid 24 (0.15 g, 0.82 mmol), Cu(OAc)2 (0.15 g, 0.82 mmol) and powdered 4A molecular sieves in CH2C12. The resulting heterogeneous mixture was stirred at room temperature overnight. The resulting slurry was filtered and the filtrate was evaporated to dryness. The residue was purified by flash chromatography eluting with EtOAc-hexanes (1:4) mixture to provide the product 29k (0.04 g, 12.7%). 1H NMR (400 MHz, CDCl3) δ 10.34 (s, 1H), 8.76 (dd, J = 8.0, 1.2 Hz, 1H), 8.27 (dd, J = 7.6, 2.0 Hz, 1H), 8.24 (m, 2H), 7.47 (ddd, J = 7.6, 7.6, 2.0 Hz, 1H), 7.32 (ddd, J = 8.0, 8.0, 1.2 Hz, 1H), 7.15–7.08 (m, 4H), 7.03 (dd, J = 8.4, 1.2 Hz, 1H), 6.94 (d, J = 8.0 Hz, 1H), 3.75 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 163.51, 162.99, 157.40, 143.70, 143.32, 133.78, 132.77, 131.97, 126.77, 126.42 (2C), 124.56, 122.41, 121.92, 121.59, 120.30, 117.07 (2C), 111.75, 56.14; HRMS (ESI) m/z: calcd for C20H17N2O5 [M + H]+ 365.1137; found 365.1140.

N-(2-(4-(Dimethylamino)phenoxy)phenyl)-2-methoxybenzamide 30k

Reaction of N-(2-hydroxyphenyl)-2-methoxybenzamide 1k and (4-(dimethylamino)phenyl)boronic acid 25 gave the product 30k in 24% isolated yield. 1H NMR (400 MHz, CDCl3) δ 10.84 (s, 1H), 8.72 (dd, J = 8.0, 1.6 Hz, 1H), 8.32 (dd, J = 8.0, 1.6 Hz, 1H), 7.47 (ddd, J = 8.0, 8.0, 2.0 Hz, 1H), 7.14–7.07 (m, 2H), 7.02–6.94 (m, 4H), 6.80 (dd, J = 8.0, 1.6 Hz, 1H), 6.77–6.73 (m, 2H), 3.84 (s, 3H), 2.94 (s, 6H); 13C NMR (100 MHz, CDCl3) δ 163.36, 157.65, 147.94, 147.69, 133.33, 132.63, 130.44, 123.67, 123.35, 122.25, 121.64, 120.90, 120.36 (2C), 116.33, 114.17 (2C), 111.70, 56.16, 41.41 (2C); HRMS (ESI) m/z: calcd for C22H23N2O3 [M + H]+ 363.1709; found 363.1704.

2-Methoxy-N-(2-(4-methoxyphenoxy)phenyl)benzamide 31k

Reaction of N-(2-hydroxyphenyl)-2-methoxybenzamide 1k and 4-methoxyphenylboronic acid 26 gave the product 31k in 18% isolated yield. 1H NMR (400 MHz, CDCl3) δ 10.75 (s, 1H), 8.72 (dd, J = 7.6, 1.6 Hz, 1H), 8.31 (dd, J = 7.6, 1.6 Hz, 1H), 7.47 (ddd, J = 8.0, 8.0, 2.0 Hz, 1H), 7.15–7.10 (m, 2H), 7.04–6.95 (m, 4H), 6.92–6.88 (m, 2H), 6.82 (dd, J = 8.4, 1.6 Hz, 1H), 3.81 (s, 6H); 13C NMR (100 MHz, CDCl3) δ 163.39, 157.61, 156.18, 150.56, 147.02, 133.40, 132.66, 130.81, 123.98, 123.81, 122.15, 121.70, 121.14, 120.10 (2C), 117.07, 115.22 (2C), 111.70, 56.10, 55.91; HRMS (ESI) m/z: calcd for C21H20NO4 [M + H]+ 350.1392; found 350.1372.

4-(2-(2-Methoxybenzamido)phenoxy)phenyl acetate 32k

Reaction of N-(2-hydroxyphenyl)-2-methoxybenzamide 1k and (4-acetoxyphenyl)boronic acid 27 gave the product 32k in 12% isolated yield. 1H NMR (400 MHz, CDCl3) δ 10.58 (s, 1H), 8.75 (dd, J = 8.4, 1.6 Hz, 1H), 8.29 (dd, J = 8.0, 2.0 Hz, 1H), 7.47 (ddd, J = 8.0, 8.0, 2.0 Hz, 1H), 7.19 (ddd, J = 8.0, 8.0, 1.6 Hz, 1H), 7.11 (ddd, J = 8.0, 8.0, 1.2 Hz, 1H), 7.08–7.02 (m, 5H), 6.96–6.92 (m, 2H), 3.75 (s, 3H), 2.30 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 169.87, 163.51, 157.58, 154.96, 146.47, 145.70, 133.52, 132.66, 131.43, 124.99, 124.06, 123.23 (2C), 121.98, 121.70, 121.50, 118.91 (2C), 118.64, 111.71, 56.11, 21.32; HRMS (ESI) m/z: calcd for C22H20NO5 [M + H]+ 378.1341; found 378.1339.

Methyl 4-(2-(2-methoxybenzamido)phenoxy)benzoate 33k

Reaction of N-(2-hydroxyphenyl)-2-methoxybenzamide 1k and (4-(methoxycarbonyl)phenyl)boronic acid 28 gave the product 33k in 22% isolated yield. 1H NMR (400 MHz, CDCl3) δ 10.45 (s, 1H), 8.76 (dd, J = 8.0, 1.6 Hz, 1H), 8.28 (dd, J = 7.6, 2.0 Hz, 1H), 8.06–8.02 (m, 2H), 7.46 (ddd, J = 7.6, 7.6, 2.0 Hz, 1H), 7.27 (ddd, J = 8.0, 8.0, 1.6 Hz, 1H, overlapping), 7.13–7.04 (m, 4H), 7.0 (dd, J = 8.4, 1.6 Hz, 1H), 6.92 (d, J = 8.4 Hz, 1H), 3.90 (s, 3H), 3.69 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 166.66, 163.50, 161.69, 157.49, 144.28, 133.60, 132.72, 132.15 (2C), 131.98, 126.01, 125.21, 124.28, 121.90, 121.81, 121.77, 119.98, 116.93 (2C), 111.68, 56.03, 52.35; HRMS (ESI) m/z: calcd for C22H20NO5 [M + H]+ 378.1341; found 378.1336.

Typical Procedure for the Synthesis of Analogues 40–45

N-(2-Methoxyphenyl)-2-((4-nitrobenzyl)oxy)benzamide 40j

2-Hydroxy-N-(2-methoxyphenyl)benzamide 1j (0.245 g, 1.0 mmol), 1-(bromomethyl)-4-nitrobenzene 34 (0.22 g, 1.0 mmol), and K2CO3 (0.29 g, 2.1 mmol) were added to a 10 mL microwave reaction vessel with Teflon-lined septum. The tube was evacuated and backfilled with dry N2 (3 cycles). Dry DMF (4 mL) was added by syringe at room temperature and the reaction mixture was irradiated in a microwave reactor (CEM, Explorer) at 90 °C and 150 Watt power for 30 min (2 ×). The reaction mixture was allowed to reach room temperature, diluted with EtOAc (10 mL) and filtered through a Celite pad; the filter cake was repeatedly washed with EtOAc. The combined filtrate was concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel eluting with EtOAc-hexanes (1:4) mixture to provide the product 40j as a yellow crystalline solid (0.2 g, 53%). 1H NMR (400 MHz, DMSO-d6) δ 10.38 (s, 1H), 8.40 (dd, J = 8.0, 1.6 Hz, 1H), 8.25 (d, J = 8.8 Hz, 2H), 8.05 (dd, J = 7.6, 1.6 Hz, 1H), 7.78 (d, J = 8.8 Hz, 2H), 7.52 (m, 1H), 7.29 (d, J = 8.4 Hz, 1H), 7.14 (t, J = 7.2 Hz, 1H), 7.09–6.99 (m, 2H), 6.94 (ddd, J = 8.0, 8.0, 1.2 Hz, 1H), 5.61 (s, 2H), 3.55 (s, 3H); 13C NMR (100 MHz, DMSO-d6) δ 162.96, 156.26, 148.91, 147.98, 144.59, 134.09, 132.27, 129.37 (2C), 128.30, 124.62, 124.44 (2C), 122.79, 122.39, 121.23, 120.46, 114.54, 111.34, 69.91, 56.26; HRMS (ESI) m/z: calcd for C21H19N2O5 [M + H]+ 379.1294; found 379.1284.

N-(2-(4-Nitrobenzyloxy)phenyl)-2-methoxybenzamide 40k

Reaction of N-(2-hydroxyphenyl)-2-methoxybenzamide 1k and 1-(bromomethyl)-4-nitrobenzene 34 gave the product 40k in 51% isolated yield. 1H NMR (400 MHz, CDCl3) δ 10.47 (s, 1H), 8.68 (m, 1H), 8.32–8.26 (m, 3H), 7.67 (d, J = 8.8 Hz, 2H), 7.48 (ddd, J = 7.6, 7.6, 1.8 Hz, 1H), 7.13 (t, J = 7.6 Hz, 1H), 7.09–7.02 (m, 2H), 6.94 (m, 2H), 5.26 (s, 2H), 3.56 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 163.33, 157.46, 148.09, 147.35, 144.11, 133.48, 132.77, 128.96, 128.65 (2C), 124.19, 123.84, 122.46, 122.17, 121.86, 121.37, 111.78, 111.56, 69.84, 55.94; HRMS (ESI) m/z: calcd for C21H19N2O5 [M + H]+ 379.1294; found 379.1289.

N-(2-Methoxyphenyl)-2-((3-nitrobenzyl)oxy)benzamide 41j

Reaction of 2-hydroxy-N-(2-methoxyphenyl)benzamide 1j and 1-(bromomethyl)-3-nitrobenzene 35 gave the product 41j as a yellow solid in 86% isolated yield. 1H NMR (400 MHz, CDCl3) δ 10.20 (s, 1H), 8.62 (dd, J = 8.0, 2.0 Hz, 1H), 8.35 (t, J = 2.0 Hz, 1H), 8.27 (dd, J = 7.6, 2.0 Hz, 1H), 8.20 (dd, J = 8.4, 2.0, 1H), 7.84 (dd, J = 8.0, 1.2 Hz, 1H), 7.56 (t, J = 8.0 Hz, 1H), 7.47–7.43 (m, 1H), 7.17 (ddd, J = 7.6, 7.6, 0.8 Hz, 1H), 7.07–6.98 (m, 3H), 6.82 (dd, J = 8.0, 1.6 Hz, 1H), 5.42 (s, 2H), 3.55 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 163.13, 155.94, 148.68, 148.37, 138.32, 133.54, 133.22, 132.91, 130.19, 128.35, 123.96, 123.75, 123.62, 122.72, 122.46, 121.47, 120.66, 113.51, 110.09, 70.49, 55.63; HRMS (ESI) m/z: calcd for C21H19N2O5 [M + H]+ 379.1294; found 379.1291.

2-(4-nitrilebenzyloxy)-N-(2-methoxyphenyl)benzamide 42j

Reaction of 2-hydroxy-N-(2-methoxyphenyl)benzamide 1j and 4-(bromomethyl)benzonitrile 36 gave the product 42j in 40% isolated yield. 1H NMR (400 MHz, CDCl3) δ 10.23 (s, 1H), 8.63 (dd, J = 7.6, 2.0 Hz, 1H), 8.28 (dd, J = 8.0, 1.6 Hz, 1H), 7.68 (m, 2H), 7.59 (d, J = 8.0 Hz, 2H), 7.44 (ddd, J = 8.0, 8.0, 2.0 Hz, 1H), 7.16 (ddd, J = 8.0, 8.0, 0.8 Hz, 1H), 7.08–6.99 (m, 2H), 6.97 (d, J = 8.0 Hz, 1H), 6.84 (dd, J = 8.0, 1.6 Hz, 1H), 5.39 (s, 2H), 3.52 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 163.13, 155.95, 148.31, 141.51, 133.22, 132.91, 132.80 (2C), 128.38, 127.97 (2C), 123.98, 123.52, 122.58, 121.48, 120.63, 118.63, 113.25, 112.48, 110.03, 70.47, 55.54; HRMS (ESI) m/z: calcd for C22H19N2O3 [M + H]+ 359.1396; found 359.1387.

2-(4-methoxybenzyloxy)-N-(2-methoxyphenyl)benzamide 43j

Reaction of 2-hydroxy-N-(2-methoxyphenyl)benzamide 1j and 1-(bromomethyl)-4-methoxybenzene 37 gave the product 43j in 50% isolated yield. 1H NMR (400 MHz, CDCl3) δ 10.51 (s, 1H), 8.64 (dd, J = 8.0, 2.0 Hz, 1H), 8.31 (dd, J = 7.6, 1.6 Hz, 1H), 7.45–7.38 (m, 3H), 7.11 (ddd, J = 7.6, 7.6, 1.2 Hz, 1H), 7.08–6.97 (m, 3H), 6.91–6.88 (m, 2H), 6.82 (dd, J = 7.6, 1.6 Hz, 1H), 5.26 (s, 2H), 3.80 (s, 3H), 3.51 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 163.40, 159.96, 156.74, 148.66, 133.15, 132.77, 129.51 (2C), 128.61, 128.10, 123.74, 123.04, 121.88, 121.21, 120.80, 114.34 (2C), 113.54, 109.91, 71.35, 55.57, 55.52; HRMS (ESI) m/z: calcd for C22H22NO4 [M + H]+ 364.1549; found 364.1547.

2-((benzo[d][1,3]dioxol-6-yl)methoxy)-N-(2-methoxyphenyl)benzamide 44j

Reaction of 2-hydroxy-N-(2-methoxyphenyl)benzamide 1j and 5-(bromomethyl)benzo[d][1,3]dioxole 38 gave the product 44j in 47% isolated yield. 1H NMR (400 MHz, CDCl3) δ 10.44 (s, 1H), 8.63 (dd, J = 8.0, 2.0 Hz, 1H), 8.30 (dd, J = 7.6, 2.0 Hz, 1H), 7.42 (ddd, J = 8.4, 7.6, 2.0 Hz, 1H), 7.12 (ddd, J = 7.6, 7.6, 1.2 Hz, 1H), 7.07–6.97 (m, 4H), 6.94 (dd, J = 8.0, 1.6 Hz, 1H), 6.85 (dd, J = 8.0, 2.0 Hz, 1H), 6.79 (d, J = 8.4 Hz, 1H), 5.95 (s, 2H), 5.23 (s, 2H), 3.63 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 163.35, 156.54, 148.66, 148.32, 147.98, 133.13, 132.79, 129.86, 128.56, 123.80, 123.13, 121.97, 121.59, 121.27, 120.84, 113.51, 109.97, 108.58, 108.49, 101.46, 71.47, 55.62; HRMS (ESI) m/z: calcd for C22H20NO5 [M + H]+ 378.1341; found 378.1340.

Methyl 4-((2-(2-methoxybenzamido)phenoxy)methyl)benzoate 45k

Reaction of N-(2-hydroxyphenyl)-2-methoxybenzamide 1k and methyl 4-(bromomethyl)benzoate 39 gave the product 45k in 76% isolated yield. 1H NMR (400 MHz, CDCl3) δ 10.52 (s, 1H), 8.71 (m, 1H), 8.30 (dd, J = 7.6, 1.6 Hz, 1H), 8.09 (m, 2H), 7.58 (m, 2H), 7.45 (ddd, J = 7.6, 7.6, 1.6 Hz, 1H), 7.11 (ddd, J = 7.6, 7.6, 1.2 Hz, 1H), 7.06 (m, 2H), 6.98 (m, 1H), 6.90 (dd, J = 8.4, 0.8 Hz, 1H), 5.18 (s, 2H), 3.94 (s, 3H), 3.40 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 166.90, 163.33, 157.54, 147.68, 141.77, 133.33, 132.73, 130.46, 130.25 (2C), 128.96, 128.40 (2C), 123.70, 122.16, 122.07, 121.63, 121.04, 111.62, 111.33, 70.50, 55.67, 52.53; HRMS (ESI) m/z: calcd for C23H22NO5 [M + H]+ 392.1498; found 392.1484.

Biology

Antiviral assays

Antiviral assays were carried out as previously described.[21] Briefly, H9 or MT4 cells were maintained in RPMI Medium 1640 (Invitrogen) containing 25 mM HEPES and L-glutamine with 10% FBS, 100 units/ml penicillin, and 100 mg/ml streptomycin (Invitrogen) at 37 °C in a humidified incubator (5% CO2). Cells were seeded at 2 × 105 per well in a 24-well plate, treated overnight with various concentrations of RN18 or analogues (all at 0.1% DMSO) and infected with HIV-1 (X4-tropic HIV-1 variant [HIV-1LAI]) at 2 × 105 c.p.m. reverse transcriptase per well. All cells were maintained in the presence of DMSO, RN18 or analogues for 14 days, and viral replication was monitored every 2 days by measuring reverse transcriptase activity in culture supernatants. The average % relative infectivity at day 7 was determined from 3 separate reverse transcriptase assays. Grafit software was used to fit curves and to determine IC50 values. CAUTION! Use extreme caution when handling the X4-tropic HIV-1 variant strain, limiting all laboratory procedures to the safety of a BL2+ laboratory facility.

Cell viability assays

The cytotoxicity of RN18 analogues was assessed in HeLa cells using an MTT-based cell viability assay.[30] Briefly, HeLa cells were harvested in the log phase of growth and seeded at 1×103 cells per well (180 µL) in a 96-well plate by using DMEM (Invitrogen) supplemented with fetal bovine serum (10%; Invitrogen). Cells were incubated under a humidified atmosphere at 37 °C with CO2 (5%) for the duration of the assay. At 24 h post-seeding, 10 × stock solutions (20 µL; RN18 or analogues in DMEM) were added to the appropriate wells. Each test plate included two negative controls: DMEM (20 µL), and 2) media (20 µL) with DMSO (10 %; the final concentration of DMSO was 1%, the highest added to the cells). At 72 h post drug addition, the MTT solution (50 µL of 2 mg mL−1 solution) prepared in PBS (Invitrogen) was carefully dispensed into each well. After 4 h incubation at 37 °C, CO2 (5 %), the medium was aspirated from all wells and DMSO (100 µL) was added and mixed by using a multichannel pipette. Absorbance was read at 550 nm by using a Safire plate reader (TECAN) and the percentage viability was calculated for each specific concentration. The results were expressed as RA (drug concentrations inhibiting relative MTT absorbance, RA, by 50%), by using the equation: RA (%) = (Atreatedgroup/Acontrolgroup) × 100%. The CC50 for each compound was calculated by using Grafit software.

Supplementary Material

Supporting Information

ACKNOWLEDGMENTS

We thank Dr. Maloy K. Parai for assistance with NMR data acquisition, the University of Massachusetts Medical School (UMMS) Center for AIDS Research (CFAR) for virology support, and members of the Rana laboratory for helpful discussions. This work was supported in part by NIH grants (AI043198, AI041404, U19MH081836) to T.M.R. and M.S.

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