Structural Optimizations of Thieno[3,2-b]pyrrole

Article pubs.acs.org/jmc

Structural Optimizations of Thieno[3,2‑b]pyrrole Derivatives for the Development of Metabolically Stable Inhibitors of Chikungunya Virus Kuan-Chieh Ching,†,‡ Thi Ngoc Quy Tran,‡ Siti Naqiah Amrun,§ Yiu-Wing Kam,§ Lisa F. P. Ng,§,∥ and Christina L. L. Chai*,†,‡,⊥ †

NUS Graduate School for Integrative Sciences and Engineering, Centre for Life Sciences, No. 05-01, 28 Medical Drive, 117456, Singapore ‡ Department of Pharmacy, Faculty of Science, National University of Singapore, Block S4A, Level 3, 18 Science Drive 4, 117543, Singapore § Singapore Immunology Network, A*STAR, 8A Biomedical Grove, Immunos Building, No. 04-06, 138648, Singapore ∥ Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Block MD6, Centre for Translational Medicine, 14 Medical Drive, No. 14-01T, 117599, Singapore ⊥ Institute of Chemical and Engineering Sciences, A*STAR, 8 Biomedical Grove, Neuros Building, No. 07-01/02/03, 138665, Singapore S Supporting Information *

ABSTRACT: Chikungunya virus (CHIKV) is a re-emerging vector-borne alphavirus, and there is no approved effective antiviral treatment currently available for CHIKV. We previously reported the discovery of thieno[3,2-b]pyrrole 1b that displayed good antiviral activity against CHIKV infection in vitro. However, it has a short half-life in the presence of human liver microsomes (HLMs) (T1/2 = 2.91 min). Herein, we report further optimization studies in which potential metabolically labile sites on compound 1b were removed or modified, resulting in the identification of thieno[3,2-b]pyrrole 20 and pyrrolo[2,3-d]thiazole 23c possessing up to 17-fold increase in metabolic half-lives in HLMs and good in vivo pharmacokinetic properties. Compound 20 not only attenuated viral RNA production and displayed broad-spectrum antiviral activity against other alphaviruses and CHIKV isolates but also exhibited limited cytotoxic liability (CC50 > 100 μM). These studies have identified two compounds that have the potential for further development as antiviral drugs against CHIKV infection.

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tion.9−11 There are currently no specific drugs to prevent or cure CHIKF.12 Treatment is primarily directed at relieving the symptoms.12 Although a number of known antimicrobial agents have demonstrated potent in vitro activity against CHIKV, their utilization for the treatment of CHIKV infection is limited.13−18 Up to now, examples of inhibitors of CHIKV replication such as natural products and synthetic molecules have been reported, but none of these compounds have progressed

INTRODUCTION

Chikungunya fever (CHIKF) is a febrile illness caused by an arthropod-borne alphavirus, the chikungunya virus (CHIKV).1,2 The disease is commonly characterized by acute onset of high-grade fever and arthralgia that may persist for months, resulting in high morbidity.3,4 Outbreaks have occurred in countries throughout the world such as Africa, Europe, in the Indian Ocean and Pacific Islands, Southeast Asia, and more recently in the Americas.5−8 The recent resurgence of CHIKF has drawn global attention due to its explosive onset, rapid spread, high morbidity, and myriad clinical presenta© 2017 American Chemical Society

Received: February 7, 2017 Published: March 28, 2017 3165

DOI: 10.1021/acs.jmedchem.7b00180 J. Med. Chem. 2017, 60, 3165−3186

Journal of Medicinal Chemistry

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Figure 1. Structures of compounds 1a and 1b and their biological properties.

Table 1. Structures of N-Propylpiperidine-4-carboxamide Derivatives 2−5 Obtained from Scaffold Hopping Modifications (Group 1 Compounds)

beyond the discovery stage.19−30 Therefore, there is still an unmet need to discover antiviral compounds as potential therapeutics for CHIKV infection. Results from our previous screening studies identified a novel series of thieno[3,2-b]pyrrole derivatives active against CHIKV and related alphaviruses.31 The initial structure−activity relationship (SAR) studies with variations at the C5 position of the thieno[3,2-b]pyrrole scaffold led to the discovery of compound 1a with modest antiviral activity (Figure 1). Specifically, the C5 piperidine carboxamide functionality on thieno[3,2-b]pyrrole 1a was found to be important in other bioisosteric analogues of thieno[3,2-b]pyrroles (i.e., indole) for anti-WEEV activities.32,33 Subsequent variations at the C2 and C6 positions resulted in a trisubstituted thieno[3,2-b]pyrrole 5carboxamide 1b as a more potent inhibitor against in vitro CHIKV infection in HEK 293T cells (Figure 1). Unfortunately, 1b has a short half-life in the presence of human liver microsomes (T1/2 = 2.91 min). Hence, we aim to expand our lead optimization studies based on 1a and its most potent derivative 1b with two goals in mind: (1) to retain the antiviral potency while ensuring low cytotoxicity and (2) to achieve

improved in vitro metabolic stability. Herein, we report the SAR and structure−metabolism relationship (SMR) studies of these analogues in order to deduce the necessary structural requirements for antiviral activity and optimal in vitro pharmacokinetic (PK) properties.

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RESULTS AND DISCUSSION Design and Synthesis of Analogues of Thieno[3,2b]pyrrole 1a and 1b. Several compound design strategies were employed to improve the in vitro metabolic stability of the lead compounds. Of these, scaffold hopping strategy was utilized due to concerns on the potential metabolic instability associated with the electron-rich 4H-thieno[3,2-b]pyrrole scaffold. 34 A series of target 5-carboxamide analogues possessing various monocyclic and bicyclic heterocyclic scaffolds including pyrrolo[2,3-d]thiazole were explored. On the basis of the results obtained in our previous work, the 4methoxybenzyl and amide moieties at the N4 and C5 positions respectively were retained to evaluate the impact of the core template on the toxicity, antiviral activity, and metabolic stability. The bromo substituent on the thiophene ring at the 3166



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Scheme 1. Preparation of Thieno[2,3-b]pyrroles 2a and 2ba

a Reagents and conditions: (a) 4-methoxybenzyl chloride, K2CO3, ACN (dry), reflux, >12 h (86% yield); (b) NaOH or KOH, EtOH/THF/H2O, 40 °C, 2−3 h (90−97% yield); (c) 4-(propylcarbamoyl)piperidin-1-ium trifluoroacetic salt, EDCI, DMAP, DIEA, DCM (dry), rt, >12 h (77−78% yield).

Scheme 2. Preparation of Pyrrolo[3,2-b]pyrrole 5-carboxamide 3 and Pyrrolo[2,3-d]imidazole 5-carboxamide 4a

a

Reagents and conditions: (a) Na, MeOH (dry), THF (dry), 40−50 °C, 48 h (quantitative yield); (b) 1 M TBAF, THF, reflux, 1 h (45% yield).

Scheme 3. Preparation of Pyrrole Derivatives 5a−ca

a

Reagents and conditions: (a) 4-methoxybenzyl chloride, NaOH, DCM, rt, >12 h (40% yield); (b) NBS, DCM (dry), rt, 4 h; (c) 4-methoxybenzyl chloride, K2CO3, ACN (dry), reflux, >12 h (79% yield); (d) NaOH or KOH, THF−H2O−EtOH, rt, >12 h (quantitative yield); (e) 4(propylcarbamoyl)piperidin-1-ium trifluoroacetic salt, EDCI, DMAP, DIEA, DCM (dry), rt, >12 h (51−73% yield).

carbonate to afford the desired esters 7a and 7b in good yield. Subsequent saponification gave the carboxylic acids 8a and 8b. Conversion of the corresponding carboxylic acids to the target carboxamides 2a and 2b was achieved using EDCImediated amide coupling reaction with n-propylpiperidine-4carboxamide. The piperidine was prepared as a salt form according to procedures reported previously.31 The syntheses of target carboxamides 2c−j and 3−4 were carried out in an analogous fashion starting from their respective ethyl esters. Of these target carboxamides, analogues 3 and 4 were obtained upon Boc deprotection of N-Boc pyrrolo[3,2-b]pyrrole 5carboxamide 9a and SEM deprotection of N-SEM pyrrolo[2,3d]imidazole 5-carboxamide 9b using freshly prepared NaOMe and commercially available TBAF respectively (Scheme 2). Pyrrole-modified analogues were prepared as shown in Scheme 3. Attempts to alkylate the intermediate 10a with 4methoxybenzyl chloride following procedures used to synthe-

C2 position was previously shown to be important in enhancing antiviral activity.31 Hence, a series of compounds bearing different core structures with similar spatial arrangements as the bromo substituent were synthesized and investigated. Table 1 summarizes the chemical structures of these analogues. The key precursors to the synthesis of the thieno[2,3b]pyrroles 2a and 2b are the thieno[2,3-b]pyrrole ethyl esters 6a and 6b, respectively. Starting from commercially available ethyl chloroacetate, preparation of ethyl esters 6a and 6b as well as other structurally similar ethyl esters possessing scaffolds such as pyrrolo[2,3-d]thiazole, furo[3,2-b]pyrrole, pyrrolo[3,2d]thiazole, pyrrolo[3,2-b]pyrrole, and pyrrolo[2,3-d]imidazole was carried out following procedures described previously.31 Scheme 1 summarizes the preparation of target thieno[2,3b]pyrroles 2a and 2b. Briefly, 6a and 6b were N-alkylated with 4-methoxybenzyl chloride in the presence of potassium 3167



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Table 2. Structures of Nondeuterated and Deuterated Thieno[3,2-b]pyrrole-5-carboxamides 13a−d (Group 2 Compounds)

size N-substituted ethyl esters 7a and 7b resulted in low yields of the desired product 11a despite the extended reaction time. The use of a stronger base, NaOH, gave the desired pyrrole 11a in moderate yield (40%). Subsequent hydrolysis and amide coupling reaction with n-propylpiperidine-4-carboxamide gave amide 5a. In the preparation of analogues 5b and 5c, bromination of ethyl 1H-pyrrole-2-carboxylate 10b was first carried out with 1.3 equiv of N-bromosuccinimide. A separable mixture of two isomeric compounds, C5 bromo 12a and C4 bromo 12b, and 4,5-dibromo substituted ester was obtained in a ratio of 2:4:1. Subsequently, the monobromo substituted esters were N-alkylated, saponified and coupled with npropylpiperidine-4-carboxamide to obtain the desired compounds 5b and 5c. In addition, a series of analogues of thieno[3,2-b]pyrrole 1a and 1b in which potential metabolically labile sites were removed or modified were also investigated. These sites include the benzylic position located at the N4 benzyl substituent and the α-methylene position of the n-propylamide. In an effort to improve metabolic stability, the n-propylamide was replaced with an isopropylamide. Deuterium replacement strategy was also employed by replacing the hydrogen atoms at these metabolically labile sites with deuteriums. The general chemical structures of these nondeuterated and deuterated-modified compounds are shown in Table 2. Following the procedures in Scheme 1, the carboxamides 13a−d were prepared via EDCImediated amide coupling reaction between deuterated or nondeuterated 2-bromo-4-(4-methoxybenzyl)-4H-thieno[3,2b]pyrrole-5-carboxylic acid and their respective deuterated or nondeuterated amine building blocks 15a,b or n-propylpiperidine-4-carboxamide. Starting from N-Boc isonipecotic acid, coupling reaction with deuterated or nondeuterated isopropylamine afforded amides 14a,b in moderate yields (Scheme 4). Subsequent deprotection of amides 14a,b to the amine building blocks 15a,b were effected using TFA. The other potential metabolically labile site is the methylene group at the α-position of the piperidine amide. In view of this, a series of thieno[3,2-b]pyrrole analogues possessing spiro-, bridged bicyclic-, or four- and five-membered ring derived amide moiety were explored with the aim of improving in vitro metabolic stability (Table 3). Following procedures described in Scheme 1, thieno[3,2-b]pyrroles 16a−j were obtained in 5− 50% yield upon EDCI-mediated amide coupling reaction between 2-bromo-4-(4-methoxybenzyl)-4H-thieno[3,2-b]pyrrole-5-carboxylic acid and their respective amine building blocks. The syntheses of these bridged bicyclic-, spiro-, four- or five-membered ring derived amines were carried out using similar procedures described in Scheme 4.

Scheme 4. Preparation of Nondeuterated and Deuterated Amines 15a,ba

a

Reagents and conditions: (a) CDI, DMAP, d7-isopropylamine hydrochloride, DMF, 40 °C, 1 h or EDCI, DMAP, isopropylamine, 0 °C to rt, >12 h (53−70% yield); (b) 30% TFA/DCM, rt, 2 h (74− 85% yield).

Other potential metabolically labile sites are the aromatic ring and 4-methoxy group on the N4 benzyl moiety, the C2 position of the thieno[3,2-b]pyrrole scaffold, and the terminal amide moiety on the C5 isonipecotamide group. To examine whether modifying or removing the metabolically labile moieties in these sites improves metabolic stability, a number of thieno[3,2-b]pyrroles (i.e., 17 and 19−21) reported in our previous work as well as N4 p-chlorobenzyl substituted thieno[3,2-b]pyrrole 18 and C2 o-fluorophenyl substituted thieno[3,2-b]pyrrole 5-carboxamide 22 were synthesized and evaluated for their in vitro metabolic stability (Table 4). Carboxamide 18 was prepared as in Scheme 1 except pchlorobenzyl chloride was used instead of p-methoxybenzyl chloride during the N-alkylation step. In the preparation of carboxamide 22, a Suzuki coupling reaction between thieno[3,2-b]pyrrole 1b and o-fluorophenylboronic acid was carried out following procedures described previously.31 Biological Evaluation of Analogues of Thieno[3,2b]pyrrole 1a and 1b. These analogues were evaluated for their cytotoxicities and antiviral activities against a luciferasetagged CHIKV isolate (i.e., CHIKV-IMT-Gluc) in assays as previously described.31 The assays were performed using serial dilutions of test compounds dissolved in DMEM supplemented with 10% fetal bovine serum (FBS). Compounds that displayed potent inhibitory activities against CHIKV-IMT-Gluc (i.e., EC50 < 10 μM) were selected for biological evaluation against in vitro wild-type (WT) CHIKV-IMT infection using immunofluorescence-based single cell quantification high content screening.31 Ribavirin was chosen as the positive control. In addition, the metabolic stabilities of these new analogues were also assessed for phase I oxidative metabolism in human liver microsomes (HLMs). The results from the screening of these compounds are summarized in Tables 5−7. 3168



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Table 3. Structures of Thieno[3,2-b]pyrrole-5-carboxamides 16a−j with Various C5 Amide Substitutions (Group 3 Compounds)

Table 4. Structures of Thieno[3,2-b]pyrrole Derivatives 17−22 (Group 4 Compounds)

Structure−Activity Relationship Studies. As shown in Table 5, the cytotoxic profile of group 1 compounds was similar or improved relative to the thieno[3,2-b]pyrrole 1a (CC50 = 18.8 μM). Shifting the sulfur atom in the thieno[3,2-b]pyrrole to other positions (i.e., compound 2a), replacing the thieno[3,2b]pyrrole of 1a with an indole ring (i.e., compound 2e), or replacing the sulfur atom with an oxygen atom (i.e., compound 2g) retained potency (EC50 = 6−9 μM) and showed improved cytotoxic profiles (CC50 = 23−37 μM). Interestingly, replacing the sulfur atom in thieno[3,2-b]pyrrole 1a with a nitrogen atom, i.e., compound 3, resulted in significant improvement in the cytotoxic profile (CC50 > 100 μM) with a slight reduction in antiviral activity. The insertion of a nitrogen atom into the thieno[3,2-b]pyrrole ring (i.e., compound 2c) led to an improvement in antiviral activity (EC50 = 4.56 μM) as compared to carboxamide 1a, with no apparent increase in cytotoxicity. The additional nitrogen atom may have contributed to the interaction of 2c with unknown target(s), possibly via hydrogen bonding, thereby leading to the observed improvement in potency. This may also explain the 2-fold loss in activity in pyrrolo[3,2-d]thiazole 2i (EC50 = 9.39 μM) when the sulfur and nitrogen atoms in compound 2c were switched on the thiazole ring. The replacement of the sulfur atom with a nitrogen atom in the thiazole ring of compound 2c (i.e., compound 4) resulted in significant loss of activity (EC50 = 42.8 μM). This may be due to the poor permeability of pyrrolo[2,3-d]imidazole 4 (CLogD < 2). These findings demonstrate that antiviral activity is strongly dependent on

the type of heteroatoms (i.e., sulfur, oxygen, or nitrogen atom) found on the five-membered ring fused to the pyrrole ring in the bicyclic scaffold as well as the spatial positions of these heteroatoms. A bromo substituent on the thiophene ring was previously shown to be essential for antiviral activity.31 A bromo substitution on the thieno[2,3-b]pyrrole template 2a (i.e., compound 2b) did not lead to much improvement in antiviral activity, although the cytotoxic profile improved (CC50 > 100 μM). Interestingly, compound 2d, a bromo derivative of the pyrrolo[3,2-d]thiazole 2c, displayed significantly enhanced potency, with an EC50 value in the low micromolar range (EC50 = 1.06 μM) and a selectivity index of 35, highlighting the significance of the C2 bromo substituent for antiviral activity. Likewise, compound 2h and compound 2j also resulted in a 3to 4-fold improvement in activity (EC50 ≈ 2 μM) as compared to compound 2g and compound 2i, respectively. However, bromo substitution on the indole ring (i.e., compound 2f) resulted in almost complete loss of activity. The choice of a monocyclic template was to improve metabolic stability of the lead compounds 1a and 1b by reducing the molecular weight and the CLogD value. Pyrrole 5a showed a 3-fold reduction in antiviral activity (EC50 = 25.7 μM) as compared to both lead compound 1a and indole 2e. On the contrary, the indole and pyrrole derivatives that were investigated by Sindac et al. showed comparable submicromolar anti-WEEV activities in the WEEV replicon assay.33 The bromo derivatives 5b and 5c displayed some improvements in the 3169



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Table 5. Cytotoxicity, Antiviral Activity, and Metabolic Half-Lives in HLMs of Group 1 Compounds

a

Concentration of compound required to achieve 50% reduction of viral infectivity in a HEK 293T cell model of CHIKV-IMT-Gluc infection, determined by the inhibition of Gaussia luciferase expression. The values are the mean ± SD from two independent experiments. EC50 values against 3170



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Table 5. continued WT CHIKV-IMT were calculated effective concentrations of compounds required to inhibit 50% CHIKV-IMT infectivity. The values are the mean ± SD from three independent experiments. bCC50 is defined as the compound’s concentration required for the reduction of cell viability by 50% as compared to the untreated control, determined by CellTiter-Glo luminescent assay after a 24 h exposure. cCalculated CLogD (Marvin Sketch, ChemAxon). CLogD = CLogP for nonionized compounds at pH = 7.4. dSelectivity index (SI) was calculated as (CC50 value)/(EC50 value). eHalflife in human liver microsomes incubation. Values are half-lives calculated from the equation T1/2 = ln(2)/slope, where the slope was ln(% detected) against time, plotted from the mean of at least n = 3 independent experiments. fEC50 was not measurable in the concentration range used. gCC50 was not measurable in the concentration range used.


a

Concentration of compound required to achieve 50% reduction of viral infectivity in a HEK 293T cell model of CHIKV-IMT-Gluc infection, determined by the inhibition of Gaussia luciferase expression. The values are the mean ± SD from two independent experiments. EC50 values against WT CHIKV-IMT were calculated effective concentrations of compounds required to inhibit 50% CHIKV-IMT infectivity. The values are the mean ± SD from three independent experiments. bCC50 is defined as the compound’s concentration required for the reduction of cell viability by 50% as compared to the untreated control, determined by CellTiter-Glo luminescent assay after a 24 h exposure. cCalculated CLogD (Marvin Sketch, ChemAxon). CLogD = CLogP for nonionized compounds at pH = 7.4. dSelectivity index (SI) was calculated as (CC50 value)/(EC50 value). eHalflife in human liver microsomes incubation. Values are half-lives calculated from the equation T1/2 = ln(2)/slope, where the slope was ln(% detected) against time, plotted from the mean of at least n = 3 independent experiments. fCC50 was not measurable in the concentration range used.

activities of the cyclohexyl-4-carboxamide derivatives 16f and 16g. Figure 2 summarizes the structural requirements for antiviral activity which were described in the SAR study. Structure−Metabolism Relationship Studies. The results of the in vitro HLM metabolism studies of the analogues are expressed as metabolic half-life (T1/2) and summarized in Tables 5−7. It can be seen from Table 5 that various modifications of the scaffold resulted in significant variations in metabolic stabilities. Significant improvements in metabolic stability were observed when the overall lipophilicity (i.e., CLogD) and/or electron density of the central bicyclic ring is reduced, as evidenced by pyrrolo[2,3-d]thiazole 2c, pyrrolo[3,2-d]thiazole 2i, pyrrolo[3,2-b]pyrrole 3, and pyrrolo[2,3d]imidiazole 4. Of these, compounds 2c, 2i, and 3 showed 2- to 3-fold increase in their half-lives (T1/2 = 23−33 min) as compared to thieno[3,2-b]pyrrole 1a (T1/2 = 10.7 min). Likewise, in the case of pyrrolo[2,3-d]imidazole 4, the introduction of two nitrogen atoms on the five-membered ring fused to the pyrrole ring in the bicyclic scaffold led to

EC50 values. However, the overall antiviral activity was reduced as compared to all the compounds in the bicyclic series except pyrrolo[2,3-d]imidazole 4. The antiviral activities of group 2 compounds, i.e., compounds 13a−d, were comparable to that of the lead compound 1b (EC50 = 1−3 μM) as shown in Table 6. For group 3 compounds, the effects of various C5 amide substitutions on the thieno[3,2-b]pyrrole scaffold of the lead compound 1b were investigated. The antiviral activities of some analogues were reduced, e.g., compounds 16b and 16h, EC50 ≈ 6−10 μM, while others exhibited EC50 > 10 μM. Of these, thieno[3,2-b]pyrrole 5-carboxamides 16c and 16f were inactive. Interestingly, the antiviral activities of the isomers of bicyclo[3.3.1]nonyl derivatives 16c and 16d showed a large difference in activities (compound 16c, EC50 > 100 μM; compound 16d, EC50 = 14.0 μM) suggesting that the stereochemistry of the bicyclo[3.3.1]nonyl ring system plays an important role in affecting the binding of the molecule to the target(s). A similar observation was also made for the antiviral 3171



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a

Concentration of compound required to achieve 50% reduction of viral infectivity in a HEK 293T cell model of CHIKV-IMT-Gluc infection, determined by the inhibition of Gaussia luciferase expression. The values are the mean ± SD from two independent experiments. EC50 values against WT CHIKV-IMT were calculated effective concentrations of compounds required to inhibit 50% CHIKV-IMT infectivity. The values are the mean ± SD from three independent experiments. bCC50 is defined as the compound’s concentration required for the reduction of cell viability by 50% as compared to the untreated control, determined by CellTiter-Glo luminescent assay after a 24 h exposure. cCalculated CLogD (Marvin Sketch, ChemAxon). CLogD = CLogP for nonionized compounds at pH = 7.4. dSelectivity index (SI) was calculated as (CC50 value)/(EC50 value). eHalflife in human liver microsomes incubation. Values are half-lives calculated from the equation T1/2 = ln(2)/slope, where the slope was ln(% detected) against time, plotted from the mean of at least n = 3 independent experiments. fEC50 was not measurable in the concentration range used. gCC50 was not measurable in the concentration range used.

significant enhancement in metabolic stability (T1/2 = 76.2 min) as compared to thieno[3,2-b]pyrrole 1a. This could be explained by the reduced lipophilicity of the compound (CLogD = 1.99 vs 3.34) and/or the introduction of the

electronegative nitrogen atom, making the ring system less electron-rich and more resistant toward oxidation. On the contrary, changing the position of the sulfur atom (compound 2a) or replacing the thieno[3,2-b]pyrrole ring with an indole 3172



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Figure 2. Summary of the effects of various structural modifications on antiviral activity.

Table 8. Metabolic Half-Lives in HLMs of Thieno[3,2-b]pyrrole Derivatives 17−22 (Group 4 Compounds)

a

Calculated CLogD (Marvin Sketch, ChemAxon). CLogD = CLogP for nonionized compounds at pH = 7.4. bHalf-life in human liver microsomes incubation. Values are half-lives calculated from the equation T1/2 = ln(2)/slope, where the slope was ln(% detected) against time, plotted from the mean of at least n = 3 independent experiments.

In group 2 compounds, the effects of replacing the npropylamide in 1b with an isopropylamide and/or replacing the hydrogen atoms with deuteriums in compounds 1b and 13a on their metabolic stabilities were explored (Table 6). These compounds did not show improved metabolic stability (T1/2 = 3−5 min) as compared to 1b, suggesting that the abovementioned sites are not major sites of metabolism. Of the group 3 compounds, the exo-3-carboxamide-anti-9aminobicyclo[3.3.1]nonyl derivative, i.e., 16d, shows significant improvement in the half-lives (T1/2 = 45.1 min) as compared to the lead compound 1b (T1/2 = 2.91 min) despite the increase in lipophilicity in 16d as compared to that of 1b (CLogD = 5.71 vs 4.27). The metabolic half-lives of thieno[3,2-b]pyrrole derivatives 17−22 were assessed in phase I oxidative metabolism studies and summarized in Table 8. As shown in Table 8, the replacement of the terminal amide group at the C5 carboxamide in 1a with a ketone moiety (i.e., compound 17) did not result in improvement in its half-life. The replacement of the p-methoxy functionality on the N4 benzyl group in 1a with an electron-withdrawing group (i.e., Cl) did not improve the metabolic stability of the compound (T1/2 = 10.3 min). Of the C2 substituted thieno[3,2-b]pyrrole derivatives 19−22, compound 20 exhibited greater than 4-fold improvement in its metabolic half-life (T1/2 = 44.2 min) relative to 1a. Although 20 is more lipophilic than 1a (CLogD = 4.98 vs

ring (compound 2e) attenuated metabolic stability. This may be due to the increased lipophilicity (ClogD = 3.4−3.6). On the other hand, the metabolic stability was improved in the presence of mouse liver microsomes, i.e., ∼4-fold increase in half-life when the thieno[3,2-b]pyrrole ring of a similar compound was replaced with an indole ring as demonstrated by Sindac et al.32 Interestingly, the replacement of the sulfur atom with the more electronegative oxygen atom in 1a (compound 2g) led to poorer metabolic stability (T1/2 = 2.74 min), suggesting that the furan ring could be susceptible to oxidative metabolism. Remarkably, the bromo-substituted analog of pyrrolo[2,3-d]thiazole 2d showed a dramatic drop in metabolic stability (T1/2 = 2.99 min) as compared to its unsubstituted counterpart 2c. The electron-deficient nature of the carbon atom at the 2-thiazo position, due to the adjacent electron-withdrawing N, S, and Br atoms, coupled with the ease of displacement of bromine may result in high chemical and enzymatic susceptibility of the compound to nucleophilic attack, thus explaining its low in vitro metabolic stability. Interestingly, this observation was not reflected in the bromosubstituted analog of pyrrolo[3,2-d]thiazole 2j (T1/2 = 22.1 min). Pyrrole 5a was slightly less stable than 1a despite a significantly lower CLogD value (2.34 vs 3.34). This suggests that the monocyclic pyrrole ring is susceptible to microsomal metabolism. 3173



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Figure 3. Summary of the effects of various structural modifications on metabolic stability.

25 μM). As compared to pyrrolo[2,3-d]thiazoles 2c,d, the antiviral activities of the hybrid pyrrolo[2,3-d]thiazole derivatives which possess either a spiro- or bridged bicyclic-ring derived amide are poorer (EC50 = 5−13 μM vs 1−4 μM against CHIKV-IMT-Gluc infection) except 23a which contains the C5 isonipecotamide moiety. Compounds 23c,d and 23f−h also performed poorly against WT CHIKV-IMT infection (EC50 > 10 μM). The replacement of the hydrogen atom or bromine atom with a phenyl moiety at the C2 position in pyrrolo[2,3d]thiazole derivatives 2c, 23e, and 23g (i.e., compounds 23a, 23f, and 23h, respectively) resulted in significant improvement in metabolic half-lives (T1/2 = 35−42 min vs 3−28 min). In addition, the introduction of exo-3-carboxamide-anti-9-aminobicyclo[3.3.1]nonylcarboxamide at the C5 position of the pyrrolo[2,3-d]thiazole scaffold, i.e., compounds 23b−d, led to reasonably good metabolic stabilities (T1/2 = 27−52 min). Surprisingly, of these exo-3-carboxamide-anti-9-aminobicyclo[3.3.1]nonylpyrrolo[2,3-d]thiazole-5-carboxamides 23b−d, 23d exhibited the lowest metabolic half-life as compared to others (T1/2 = 27 min vs 45−52 min) despite bearing a phenyl moiety at the C2 position of the pyrrolo[2,3-d]thiazole scaffold. This could be due to the higher lipophilicity (CLogD = 6.04 vs 4−5) of compound 23d. In Vivo Pharmacokinetic Properties of Selected Bicyclic Heterocycles. On the basis of the general classification for in vitro intrinsic clearance (CLint) in which CLint < 30 (μL min−1 mg−1) indicates no risk for high first-pass metabolism in vivo, 30 < CLint < 92 (μL min−1 mg−1) indicates moderate risk, and CLint > 92 (μL min−1 mg−1) indicates high risk, thieno[3,2b]pyrrole 20 and pyrrolo[2,3-d]thiazole 23c possessing CLint of 31.4 μL min−1 mg−1 and 26.7 μL min−1 mg−1, respectively, indicate that both compounds exhibited low risk of first-pass metabolism in vivo (Table 10).35,36 Their favorable in vitro metabolic stabilities thus led us to investigate the in vivo PK properties of these two compounds. As shown in Table 10, both compounds exhibited low plasma clearance (i.e., CL < 27 mL min−1 kg−1), suggesting that these compounds are not readily excreted. On the other hand, both compounds demonstrated different apparent volume of distribution at steady state (Vss) in which thieno[3,2-b]pyrrole 20 has high Vss (i.e., Vss > 1.45 L/kg) while pyrrolo[2,3-d]thiazole 23c has low Vss (i.e., Vss < 0.725 L/kg), despite both compounds possessing similar lipophilicities (CLogD ≈ 5). Both compounds were absorbed very quickly through the gastrointestinal walls and into the systemic circulation (i.e., Tmax < 1 h) and exhibited

3.34), 20 is metabolically more stable than 1a, suggesting that the C2 phenyl group may have prevented the fused thiophene ring from metabolic attack. The installation of an electronwithdrawing group such as a fluorine atom onto the C2 phenyl group in 20 is to reduce its susceptibility toward oxidative metabolism. However, the C2 o-fluorophenyl substituted thieno[3,2-b]pyrrole 5-carboxamide 22 did not show an improved metabolic half-life (T1/2 = 35.5 min). Figure 3 summarizes the structural requirements for metabolic stability which were described in the SMR study. Inhibitory Properties of Groups 1−3 Compounds against CHIKV-IMT Infection. Analogues that exhibited potent activity against CHIKV-IMT-Gluc (EC50 < 10 μM) were selected for a secondary screen against WT CHIKV-IMT. This is to remove false positives from the CHIKV-IMT-Gluc screening. The results are summarized in Tables 5−7. The compounds tested in groups 1 and 2 showed comparable antiviral activities against both CHIKV-IMT-Gluc and WT CHIKV-IMT infections (EC50 = 1−8 μM) except for compound 2i which exhibited poorer activity against WT CHIKV-IMT infection (EC50 > 10 μM). Compounds possessing spiro- or bridged bicyclic-ring derived amides (i.e., 16b and 16h) in group 3 compounds performed more poorly against WT CHIKV-IMT infection as compared to CHIKV-IMT-Gluc, with EC50 > 10 μM. Design, Synthesis, and Biological Evaluation of Pyrrolo[2,3-d]thiazole Compounds. From the SAR studies above, a class of bicyclic heterocycles (i.e., pyrrolo[2,3-d]thiazoles 2c and 2d) that exhibited good antiviral activity (EC50 = 1−5 μM) against both CHIKV-IMT-Gluc and WT CHIKV-IMT was identified. Additionally, the SMR studies led to the discovery of the C2 phenyl substituted thieno[3,2-b]pyrrole 20 and the C5 exo-3-carboxamide-anti-9-aminobicyclo[3.3.1]nonylcarboxamide substituted thieno[3,2-b]pyrrole 16d which possess longer metabolic half-lives (i.e., T1/2 = 44−45 min) as compared to the lead compounds 1a and 1b (T1/2 = 3−10 min). Hence, a series of hybrid pyrrolo[2,3-d]thiazoles which included compounds possessing the C2 phenyl group and/or C5 exo-3-carboxamide-anti-9-aminobicyclo[3.3.1]nonyl amide was synthesized. The chemical structures and biological properties of these pyrrolo[2,3-d]thiazoles 23a−h are summarized in Table 9. The syntheses of these compounds were performed following procedures as previously described.31 The installation of the phenyl group and/or various ring derived amide moieties on the pyrrolo[2,3-d]thiazole in group 5 compounds resulted in moderate to low cytotoxicity (CC50 > 3174



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Table 9. Cytotoxicity, Antiviral Activity, and Metabolic Half-Lives in HLMs of Pyrrolo[2,3-d]thiazole-5-carboxamides 23a−h (Group 5 Compounds)

a

Concentration of compound required to achieve 50% reduction of viral infectivity in a HEK 293T cell model of CHIKV-IMT-Gluc infection, determined by the inhibition of Gaussia luciferase expression. The values are the mean ± SD from two independent experiments. EC50 values against WT CHIKV-IMT were calculated effective concentrations of compounds required to inhibit 50% CHIKV-IMT infectivity. The values are the mean ± SD from three independent experiments. bCC50 is defined as the compound’s concentration required for the reduction of cell viability by 50% as compared to the untreated control, determined by CellTiter-Glo luminescent assay after a 24 h exposure. cCalculated CLogD (Marvin Sketch, ChemAxon). CLogD = CLogP for nonionized compounds at pH = 7.4. dSelectivity index (SI) was calculated as (CC50 value)/(EC50 value). eHalflife in human liver microsomes incubation. Values are half-lives calculated from the equation T1/2 = ln(2)/slope, where the slope was ln(% detected) against time, plotted from the mean of at least n = 3 independent experiments. fEC50 was not measurable in the concentration range used. gCC50 was not measurable in the concentration range used.

Table 10. Pharmacokinetic Parameters of Thieno[3,2-b]pyrrole 20 and Pyrrolo[2,3-d]thiazole 23c after Intravenous or Oral Administration in Micea compdb

human CLintc

iv T1/2d

po T1/2d

iv CL,e Vssf

po Tmax,g Cmaxh

F (%)i

20 23c

31.4 26.7

2.07 ± 1.20 1.09 ± 0.24

2.37 ± 0.95 1.39 ± 0.37

18.6 ± 8.2, 2.45 ± 0.17 5.68 ± 0.40, 0.337 ± 0.035

0.667 ± 0.289, 720 ± 150 0.333 ± 0.144, 6700 ± 1460

22.0 ± 4.1 39.1 ± 17.2

Mouse PK: male C57BL/6 mice were dosed at 3 mg/kg iv and 10 mg/kg po. The data shown are the mean ± SD from three mice. bCompounds 20 and 23c were prepared as solution in 5% DMSO, 70% PEG400, and 25% water formulation. cHLM intrinsic clearance expressed in μL min−1 mg−1 microsomal proteins. dTerminal elimination half-life (h). ePlasma clearance expressed in mL min−1 kg−1. fVolume of distribution at steady state (L/kg). gThe po absorption expressed as Tmax (h). hThe po exposure expressed as Cmax (ng/mL). iOral bioavailability (%). a

moderate to high oral exposure (i.e., Cmax > 250 ng/mL) after a single 10 mg/kg oral dose in C57BL/6 mice. Of these,

pyrrolo[2,3-d]thiazole 23c has high oral exposure (Cmax = 6700 ng/mL) which is above its in vitro EC50 values against both 3175



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Figure 4. Plasma concentration−time profiles of compounds 20 and 23c dosed with either 3 mg/kg iv or 10 mg/kg po in C57BL/6 mice over 24 h. Plasma samples were collected at indicated time points after a single dosing in a mouse model. Concentrations were measured using HPLC−MS/MS methods. Plasma concentration at 24 h is not shown as it is below quantifiable limit of 0.5 ng/mL. (A) Plasma concentration−time profile of the thieno[3,2-b]pyrrole 20. (B) Plasma concentration−time profile of the pyrrolo[2,3-d]thiazole 23c.

Figure 5. Effects of thieno[3,2-b]pyrrole 20 and pyrrolo[2,3-d]thiazole 23c on viral RNA expression. The HEK 293T cells were infected with CHIKV-IMT at MOI = 0.1 and treated with serial dilutions of compounds 20 and 23c. Viral load was measured at 24 h postinfection using qRTPCR targeted against the E1 and nsP1 genes. Dose-dependent inhibition of viral RNA copy numbers upon post-treatment was observed in these compounds, and minimal cytotoxicity was observed at these concentrations. Data are the mean ± SEM and are representative of three independent experiments. Asterisk (∗) indicates p < 0.05 and double-asterisk (∗∗) indicates p < 0.01 in a two-tailed unpaired t test between treated samples and the untreated control.

difference in oral bioavailability (F = 39.1% vs 22.0%) as compared to compound 20. Inhibitory Properties of Selected Bicyclic Heterocycles against Viral RNA Synthesis. The positive-sense RNA genome in CHIKV encodes for the nonstructural proteins that are responsible for the production of new negative- and positivesense RNA strands in a CHIKV replication cycle.37 To investigate the effects of thieno[3,2-b]pyrrole 20 and pyrrolo[2,3-d]thiazole 23c against viral RNA production, qRT-PCR was performed on viral RNA isolated from IMT-infected cells treated with these compounds to detect nonstructural viral gene (nsP1) and structural viral gene (E1). Figure 5 shows that treatment with both compounds 20 and 23c resulted in a dosedependent reduction in viral load from 2.5 μM onward.

CHIKV-IMT-Gluc and WT CHIKV-IMT infection (EC50 = 7− 12 μM). As shown in Figure 4, the plasma concentration of 23c reached nearly 10 000 ng/mL (approximately 17 μM) within the first hour after a single intravenous (iv) or oral (po) dose, while the plasma concentration of 20 only reached nearly 1000 ng/mL (approximately 2 μM). Interestingly, a longer terminal elimination half-life was observed in mouse after a single iv or po dose of 20 (T1/2 ≈ 2 h) as compared to dosing of 23c (T1/2 ≈ 1 h). This observation does not correlate with their CLint observed in HLM because the species utilized in these two studies (i.e., in vivo PK and in vitro metabolism studies) were different. Notably, the low clearance (CL) and high oral exposure (Cmax) in compound 23c resulted in a 2-fold 3176



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Table 11. Antiviral Activity of Thieno[3,2-b]pyrrole 20 and Ribavirin against Different Clinical Isolates of CHIKV as Well as Other Alphaviruses (ONNV and SINV) in HEK 293T Cells EC50 (μM)a species

virus (strain)

Chikungunya

O’nyong-nyong Sindbis

ribavirin

20

CHKV-IMT-Gluc CHIKV-SGP11-Gluc CHIKV-CNR20235-Gluc CHIKV-IMT Chad

3.30 2.26 2.18 4.03 3.24 1.65

± ± ± ± ± ±

0.13 0.13 0.11 0.87 0.20 0.18

16.2 1.89 1.97 20.0 18.4 5.12

± ± ± ± ± ±

1.6 0.24 0.20 4.5b 5.5b 2.00b

Calculated effective concentration of compound that is required to inhibit 50% virus infectivity. EC50 values are the average mean ± SD from three independent experiments. bEC50 values were obtained from our previous work.31 a

Table 12. Biological Evaluation of Thieno[3,2-b]pyrrole 20 and Ribavirin in Various Human Cell Lines ribavirin

20 cell line HEK 293T BJ

species human human

tissue/cell type kidney epithelium skin fibroblast

CC50 (μM) c

>100 >100c

a

b

EC50 (μM)

3.30 ± 0.13 5.07 ± 0.35

CC50 (μM) c

>100 >100c

a

EC50 (μM)b 16.2 ± 1.6 9.71 ± 0.40

a

CC50 is defined as the compound’s concentration required for the reduction of cell viability by 50% as compared to the untreated control, determined by CellTiter-Glo luminescent assay after a 24 h exposure. bConcentration of compound required to achieve 50% reduction of viral infectivity in a HEK 293T cell model of CHIKV-IMT-Gluc infection, determined by the inhibition of Gaussia luciferase expression. The values are the average mean ± SD from three independent experiments. cCC50 was not measurable in the concentration range used.

Figure 6. Pre- and post-treatment effects of thieno[3,2-b]pyrrole 20 on CHIKV. (A) HEK 293T cells were infected with CHIKV-IMT (MOI = 0.1) and then incubated for 1.5 h. Various concentrations of compound 20 were administered at 4 and 2 h before infection (samples 1−4) as well as at 0, 2, 6, 8, and 12 hpi (samples 5−9) with CHIKV-IMT. The treatment medium in samples 1 and 2 was removed prior to infection, and the cells were incubated in medium without the compound. Immunofluorescence high content screen was performed at 24 hpi. (B) Time-of-addition study: preand post-treatment of IMT-infected cells with compound 20. EC50 values of compound 20 obtained were determined from the dose−response curves in each sample (i.e., samples 3−9). Each bar represents the mean ± SD and is representative of at least three independent experiments. Mann−Whitney U test was used to analyze EC50 values obtained from different time points in comparison with the reference one (EC50 value corresponding to sample 5). (C) Immunofluorescence detection of alphavirus protein is used as an indication of CHIKV infection. Images are taken from stained infected cells that were treated with 5 μM 20 under various conditions as indicated in part A (samples 1−9). Cell nuclei were stained with DAPI (blue), and CHIKV infection is indicated by FITC (green) staining. Mock-infected cells were as shown as negative control. Images were captured with 60× magnification. Representative microscopic images per treatment conditions are illustrated. 3177



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stages as indicated by the increase in the EC50 values from times 2, 6, 8, and 12 hpi (samples 6−9 in Figure 6B). Notably, the increased EC50 values were not statistically different from the EC50 value observed at 0 h postinfection (sample 5). This strongly suggests that 20 could still be interfering with the early postentry (2 hpi) or late (6 hpi) replication phase of virus replication cycle. Figure 6C illustrates how treatment with 5 μM 20 at various time points prior to or after infection affects virus infection. A gradual increase in the number of CHIKV positive cells (in green) was observed from samples 6−9 when IMT-infected cells were treated at 2, 6, 8, and 12 hpi. In parallel, HEK 293T cells were treated with compound 20 at −4 and −2 hpi following which the cells were incubated in medium without the compound from 0 hpi onward (samples 1 and 2 in Figure 6A). However, this pretreatment resulted in poorer antiviral activity against CHIKV-IMT (EC50 = 14−15 μM). As shown in samples 1 and 2 in Figure 6C, there is almost negligible inhibition of CHIKV infection when pretreatment of 5 μM 20 was carried out. Overall, these studies show that 20 affects the replication phase of the CHIKV life cycle with an additional effect on the later steps, e.g., virus maturation.

Notably, greater than a 50-fold reduction in viral load is observed for IMT-infected cells that were treated with 5 μM thieno[3,2-b]pyrrole 20 as compared to untreated infected cells. Although there was a statistical difference in the viral load in IMT-infected cells treated with 5 μM, 10 μM, or 25 μM pyrrolo[2,3-d]thiazole 23c as compared to untreated infected cells, this difference is not large (only up to 5-fold reduction in viral load). Broad-Spectrum Antiviral Activities against Other Alphaviruses and Multiple CHIKV Isolates. In light of the good inhibitory activity of thieno[3,2-b]pyrrole 20 against CHIKV at low concentration (i.e., 5 μM), its breadth of antiviral activity was examined against other clinically important alphaviruses such as O’nyong nyong virus (ONNV), a close relative of CHIKV and Sindbis virus (SINV). A pan-alphavirus screen panel against these viruses was carried out using immunofluorescence-based high content screen. As shown in Table 11, 20 demonstrated broad-spectrum antiviral activity against these alphaviruses with EC50 values ranging between 1 and 4 μM. In parallel, luciferase reporter assays for 20 and ribavirin were performed in HEK 293T cell lines using luciferase-tagged CHIKV isolate IMT and analogous reporter viruses from the 2008 Singapore SGP11 isolate and the 2013 Caribbean CNR20235 isolate. On average, 20 demonstrated comparable activity against various clinical isolates, with EC50 values in the low micromolar range (i.e., EC50 = 2−4 μM). Interestingly, ribavirin was 8-fold less active against the IMT isolate than against the SGP11 and CNR20235 isolates, suggesting that 20 is more robust against infections from multiple CHIKV isolates as compared to ribavirin. Cytotoxic and Antiviral Activities in Other Cell Types. The antiviral activity against CHIKV infection in the BJ human fibroblast cell line was examined, since skin fibroblasts are the primary target cells for CHIKV replication in humans.38 In the study, BJ (ATCC CRL-2522) cells were infected with reporter virus CHIKV-IMT-Gluc at MOI 1 and treated with serial dilutions of 20 or ribavirin. In parallel, the cytotoxicity of 20 and ribavirin on BJ cells was determined using Cell-Titer Glo assay as previously described.31 20 not only performed equally well in both human cell lines (i.e., EC50 = 3−5 μM) but also demonstrated low cytotoxic liability in both mammalian cell lines (i.e., CC50 > 100 μM), giving selectivity indices (CC50/ EC50) greater than 20 (Table 12). The high selectivity indices of 20 in these human cell lines indicate that it is relatively nontoxic for human cells. Mechanism of Action Studies. To study and understand the stage at which thieno[3,2-b]pyrrole 20 targets in the CHIKV life cycle, a time-of-addition experiment was performed. Various concentrations of compound 20 were added to HEK 293T cells at different time points prior to infection (−4 and −2 h postinfection (hpi)), during virus adsorption (0 hpi), in the early postentry (2 hpi) and late stages of virus infection (6, 8, and 12 hpi) as indicated in Figure 6A. The end point of this study was at 24 hpi, and infectivity was assessed using immunofluorescence high content screen. As shown in Figure 6B, 20 demonstrated the most potent antiviral activity (EC50 ≈ 2.5 μM) when IMT-infected cells were treated at −4 and −2 hpi (samples 3 and 4) but the difference between these EC50 values and the EC50 value observed at 0 hpi (sample 5) is not statistically significant. Hence, this ruled out any effect of 20 on the early stages of the viral replication cycle including virus attachment/entry. In addition, the antiviral activity progressively reduced when 20 was added at postinfection

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CONCLUSIONS In this study, we report the optimization of the lead compounds 1a and 1b in light of their poor in vitro metabolic stabilities in HLMs. The structural requirements for antiviral activity and metabolic stability were also described in the SAR and SMR studies (Figures 2 and 3). Of these requirements, the heterocyclic scaffold plays a key role in both antiviral activity and metabolic stability. The SMR study suggests that the C2 position of the thieno[3,2-b]pyrrole scaffold and the piperidine amide at the C5 position of the thieno[3,2-b]pyrrole scaffold could be potential sites of metabolism. The installation of the phenyl group and the exo-3-carboxamide-anti-9-aminobicyclo[3.3.1]nonylcarboxamide moiety at the respective C2 and C5 positions of the thieno[3,2-b]pyrrole scaffold led to enhanced metabolic stability. From this study, thieno[3,2-b]pyrrole 20 and pyrrolo[2,3-d]thiazole 23c were identified to have improved in vitro metabolic stabilities (T1/2 = 44−52 min) in HLMs. A preliminary PK study determining the in vivo PK parameters of carboxamides 20 and 23c after a single iv or po dose was carried out to “validate” our in vitro approach to selecting compounds for future in vivo infection studies. Both carboxamides 20 and 23c showed low clearance and reasonably good oral bioavailability. Of these, 20 was found to inhibit viral RNA synthesis at concentrations as low as 5 μM in the qRTPCR studies. Additionally, 20 not only exhibits broad-spectrum antiviral activity against other alphaviruses such as ONNV and SINV and CHIKV isolates from different geographical locations (i.e., IMT, SGP11, CNR20235) but also retains its antiviral activity against CHIKV-IMT-Gluc infection (EC50 = 5.07 μM) in skin fibroblasts, highlighting its robust antiviral activity across different cell types. Further studies such as time-of-addition studies suggest that 20 targets the replication phase or the late stages of CHIKV replication cycle. All in all, our studies have identified two compounds with the potential for further development as antiviral drugs against CHIKV infection.

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EXPERIMENTAL SECTION

General. All reagents and solvents (of analytical and HPLC grades) were obtained from commercial sources and were used without further purification. Moisture sensitive or anhydrous reactions were performed under nitrogen atmosphere. PureSolv MD 4 solvent purification 3178



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degassed toluene (∼40 mL of solvent per mmol carboxamide) was added. The reaction mixture was stirred at 120 °C for 24 h before diluting with H2O. The aqueous suspension was extracted with EtOAc (3×) and the combined organic extracts were washed with brine (1×), dried with Na2SO4, and concentrated in vacuo to give a crude residue which was purified by semipreparative reversed-phase HPLC (C18, eluent MeOH/H2O) to afford the desired carboxamide. 1-[6-(4-Methoxybenzyl)-6H-thieno[2,3-b]pyrrole-5-carbonyl]-Npropylpiperidine-4-carboxamide (2a). Synthesized following general procedure A from 8a (200 mg, 0.7 mmol), EDCI (140 mg, 0.73 mmol), DMAP (17 mg, 0.14 mmol), 4-(propylcarbamoyl)piperidin-1ium trifluoroacetic salt (277 mg, 0.98 mmol), and DIEA (360 μL, 2.07 mmol). The crude material was purified by column chromatography (SiO2) using eluent 98% DCM, 2% MeOH. The product was isolated as a white solid (226 mg, 77%). Mp 183.0−185.0 °C; 1H NMR (400 MHz, CDCl3) δ 0.90 (t, J = 7.4 Hz, 3H), 1.43−1.61 (m, 4H), 1.70− 1.82 (m, 2H), 2.28 (m, 1H), 2.82−2.95 (m, 2H), 3.19 (q, J = 6.9 Hz, 2H), 3.75 (s, 3H), 4.38−4.52 (m, 2H), 5.29 (s, 1H), 5.78 (m, 1H), 6.50 (s, 1H), 6.78−6.84 (m, 3H), 6.92 (d, J = 5.3 Hz, 1H), 7.19 (d, J = 8.7 Hz, 2H); 13C NMR (100 MHz, CDCl3) δ 11.2, 22.7, 28.8, 41.2, 43.1, 44.6, 50.9, 55.2, 104.3, 114.0, 117.7, 119.5, 128.2, 128.6, 129.4, 130.2, 139.1, 159.3, 162.9, 173.9; ESI-TOF-HRMS (m/z) for C24H29N3O3S [M + H]+ 440.2008; found 440.2017. 1-[2-Bromo-6-(4-methoxybenzyl)-6H-thieno[2,3-b]pyrrole-5-carbonyl]-N-propylpiperidine-4-carboxamide (2b). Synthesized following general procedure A from 8b (150 mg, 0.41 mmol), EDCI (83 mg, 0.43 mmol), DMAP (10 mg, 0.08 mmol), 4-(propylcarbamoyl)piperidin-1-ium trifluoroacetic salt (175 mg, 0.62 mmol), and DIEA (215 μL, 1.23 mmol). The crude material was purified by column chromatography (SiO2) using eluent 98% DCM, 2% MeOH. The product was isolated as a white solid (166 mg, 78%). Mp 206.3−206.7 °C; 1H NMR (400 MHz, CDCl3) δ 0.92 (t, J = 7.4 Hz, 3H), 1.47− 1.65 (m, 4H), 1.75−1.88 (m, 2H), 2.34 (m, 1H), 2.88−3.02 (m, 2H), 3.17−3.27 (m, 2H), 3.79 (s, 3H), 4.39−4.51 (m, 2H), 5.24 (s, 2H), 5.64 (m, 1H), 6.45 (s, 1H), 6.84 (d, J = 8.7 Hz, 2H), 6.96 (s, 1H), 7.19 (d, J = 8.7 Hz, 2H); 13C NMR (100 MHz, CDCl3) δ 11.3, 22.8, 28.9, 41.3, 43.2, 44.6, 51.1, 55.3, 104.0, 106.1, 114.2, 120.7, 126.9, 127.9, 129.7, 129.8, 137.7, 159.6, 162.7, 173.9; ESI-TOF-HRMS (m/z) calcd for C24H28BrN3O3S [M + H]+ 520.1093; found 520.1101. 1-[4-(4-Methoxybenzyl)-4H-pyrrolo[2,3-d]thiazole-5-carbonyl]-Npropylpiperidine-4-carboxamide (2c). Synthesized following general procedure A from 8c (132 mg, 0.46 mmol), EDCI (93 mg, 0.48 mmol), DMAP (11 mg, 0.09 mmol), 4-(propylcarbamoyl)piperidin-1ium trifluoroacetic salt (196 mg, 0.69 mmol), and DIEA (240 μL, 1.38 mmol). The crude material was purified by column chromatography (SiO2) using 94−98% DCM, 2−6% MeOH. The product was isolated as a white solid (145 mg, 72%). Mp 166.7−168.3 °C; 1H NMR (400 MHz, CDCl3) δ 0.90 (t, J = 7.4 Hz, 3H), 1.28−1.58 (m, 4H), 1.60− 1.80 (m, 2H), 2.22 (m, 1H), 2.74−2.88 (m, 2H), 3.19 (q, J = 7.0 Hz, 2H), 3.74 (s, 3H), 4.20−4.40 (m, 2H), 5.52−5.64 (m, 3H), 6.45 (s, 1H), 6.78 (d, J = 8.7 Hz, 2H), 7.15 (d, J = 8.7 Hz, 2H), 8.60 (s, 1H); 13 C NMR (100 MHz, CDCl3) δ 11.2, 22.8, 28.6, 41.2, 43.0, 44.5, 48.2, 55.2, 101.9, 112.4, 113.9, 128.9, 129.3, 130.2, 152.4, 153.8, 159.1, 162.7, 173.8; ESI-TOF-HRMS (m/z) calcd for C23H28N4O3S [M + H]+ 441.1960; found 441.1957. 1-[2-Bromo-4-(4-methoxybenzyl)-4H-pyrrolo[2,3-d]thiazole-5carbonyl]-N-propylpiperidine-4-carboxamide (2d). Synthesized following general procedure A from 8d (86 mg, 0.24 mmol), EDCI (48 mg, 0.25 mmol), DMAP (6 mg, 0.05 mmol), 4-(propylcarbamoyl)piperidin-1-ium trifluoroacetic salt (96 mg, 0.34 mmol), and DIEA (130 μL, 0.75 mmol). The crude material was purified by column chromatography (SiO2) using eluent 98% DCM, 2% MeOH. The product was isolated as a white solid (73 mg, 59%). Mp 177.1−178.0 °C; 1H NMR (400 MHz, CDCl3) δ 0.89 (t, J = 7.4 Hz, 3H), 1.26− 1.42 (m, 2H), 1.49 (sext, J = 7.3 Hz, 2H), 1.61−1.73 (m, 2H), 2.23 (m, 1H), 2.73−2.84 (m, 2H), 3.13−3.22 (m, 2H), 3.74 (s, 3H), 4.12− 4.39 (m, 2H), 5.49 (s, 2H), 5.66 (m, 1H), 6.34 (s, 1H), 6.78 (d, J = 8.7 Hz, 2H), 7.12 (d, J = 8.7 Hz, 2H); 13C NMR (100 MHz, CDCl3) δ 11.2, 22.7, 28.6, 41.2, 42.9, 44.3, 48.2, 55.2, 101.8, 113.9, 114.8, 127.7,

system (Innovative Technology) was employed to obtain anhydrous THF and DCM. Standard rotary evaporators were employed for removal of solvents under reduced pressure. Merck’s precoated silica gel plates were utilized in analytical thin layer chromatography (TLC), and the spots were visualized using UV light (254 nm) or staining with basic KMnO4, ninhydrin, or ceric ammonium molybdate (CAM) solution followed by heating. Flash chromatography on a glass column packed with Merck silica gel 60 (230−400 mesh) was employed to purify compounds. Shimadzu Ultra Fast Liquid Chromatograph Prominence series (LC-20AD) system with Phenomenex Gemini 5.0 μm C18 110 Å (150 mm × 10.0 mm) LC column at 254 nm was utilized to perform semipreparative HPLC purifications. HPLC separation was performed using gradient systems composed of methanol in water (70−90% v/v) or acetonitrile in water (5−90% v/v) and isocratic systems composed of methanol in water (80%, 85%, or 90% v/v). Solvent mixtures used for chromatography are always prepared as a vol/vol ratio. MPA100 automated melting point apparatus from Stanford Research Systems (SRS) was employed to determine the melting points. Shimadzu single quadrupole liquid chromatograph−mass spectrometer (LCMS-2020) was employed to generate ESI mass spectra of compounds using electrospray mode. Agilent Technologies 6210 time-of-flight LC/MS (negative and positive ionization modes) was employed to carry out high resolution electrospray mass analyses (HRESIMS) of compounds. Bruker Avance 400 MHz spectrometer was used for running the 1H NMR spectra (400 MHz) and 13C NMR spectra (100 MHz) with CDCl3, CD3OD, or DMSO-d6 as solvent. The unit ppm was assigned to chemical shifts and the residual protio-solvent signals were internally referenced as 1H NMR, CDCl3 (δ 7.26), CD3OD (δ 3.31), DMSO-d6 (δ 2.50); 13C NMR, CDCl3 (δ 77.0), CD3OD (δ 49.0), DMSO-d6 (δ 39.5). The δ-scale is relative to the residual solvent signal as an internal reference. The signals were described and abbreviated as s = singlet, d = doublet, t = triplet, q = quartet, quint = quintet, sext = sextet, m = multiplet, br = broad signal. Software MestReNova 6 (Mestrelab Research, 1994) was utilized to analyze the spectra. Analytical high-performance liquid chromatography (HPLC) was performed to determine the purity of the synthesized compounds used for biological testing. This technique was carried out on a Shimadzu Ultra Fast Liquid Chromatograph Prominence series (LC-20AD) system with Phenomenex Kinetex 2.6 μm C18 100 Å (150 mm × 4.60 mm) LC analytical column or CHIRALPAK IA 5.0 μm based on amylose tris(3,5-dimethylphenyl)carbamate (150 mm × 4.60 mm) LC analytical column at 254 nm. HPLC separation was performed using gradient systems that were composed of either acetonitrile in water (10−90% v/v) or methanol in water (30−90% v/v). All test compounds showed more than 95% purity. Marvin Sketch software version 5.11.2 (ChemAxon Ltd., 1998) was utilized to generate the CLogD values of the compounds at pH 7.4. Chemistry. Detailed experimental procedures of the intermediates are described in the Supporting Information. The data below are for the final compounds tested in the biological studies. General Synthetic Procedure A: Amide Coupling Reaction for the Synthesis of Carboxamides 2a−j, 5a−c, 13a−d, 16a−j, 18, 23b,c, 23e, and 23g. Carboxylic acid (1 equiv), EDCI (1.05 equiv), and DMAP (catalytic amount) or HOBt (1.03 equiv) were dissolved in DCM. The reaction mixture was stirred for 30 min at rt, after which a mixture of amine salt (1.2−1.5 equiv) and DIEA (2−3 equiv) in DCM (∼20 mL of solvent per mmol carboxylic acid) was added. The resulting mixture was stirred at rt overnight before washing with 5% citric acid (1×), saturated NaHCO3 (1×), H2O (1×), and brine (1×). The washed organic layer was dried over Na2SO4, filtered, and concentrated in vacuo to give a crude residue which was purified by column chromatography (SiO2, eluent PE/EtOAc or DCM/MeOH or Et2O) or purified by semipreparative reversed-phase HPLC (C18, eluent MeOH/H2O) to afford desired carboxamide. General Synthetic Procedure B: Suzuki Coupling Reaction for the Synthesis of Carboxamides 23a, 23d, 23f, and 23h. To a sealed tube were added carboxamide (1 equiv), PPh3 (30 mol %), Pd(OAc)2 (10 mol %), PhB(OH)2 (2 equiv), and K2CO3 (3 equiv) following which 3179



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1-[4-(4-Methoxybenzyl)-4H-pyrrolo[3,2-d]thiazole-5-carbonyl]-Npropylpiperidine-4-carboxamide (2i). Synthesized following general procedure A using 8g (71 mg, 0.25 mmol), 4-(propylcarbamoyl)piperidin-1-ium trifluoroacetic salt (87 mg, 0.29 mmol), DMAP (2.4 mg, 0.02 mmol), DIEA (85 μL, 0.49 mmol), and EDCI (49 mg, 0.26 mmol). After chromatography (SiO2, eluent 97−98% DCM, 2−3% MeOH), the product was obtained as a white solid (66 mg, 61%). Mp 190.3−192.2 °C; 1H NMR (400 MHz, CD3OD) δ 0.92 (t, J = 7.4 Hz, 3H), 1.45−1.67 (m, 4H), 1.69−1.87 (m, 2H), 2.46 (m, 1H), 2.89− 3.08 (m, 2H), 3.13 (t, J = 7.2 Hz, 2H), 3.78 (s, 3H), 4.25−4.55 (m, 2H), 5.31 (s, 2H), 6.79 (s, 1H), 6.90 (d, J = 8.7 Hz, 2H), 7.20 (d, J = 8.7 Hz, 2H), 8.60 (s, 1H); 13C NMR (100 MHz, CD3OD) δ 11.7, 23.6, 30.0, 42.1, 43.9, 52.7, 55.8, 103.3, 115.3, 129.2, 131.0, 132.8, 133.0, 146.5, 150.6, 161.4, 164.2, 176.9; ESI-TOF-HRMS (m/z) calcd for C23H28N4O3S [M + H]+ 441.1960; found 441.1959. 1-[2-Bromo-4-(4-methoxybenzyl)-4H-pyrrolo[3,2-d]thiazole-5carbonyl]-N-propylpiperidine-4-carboxamide (2j). Synthesized following general procedure A using 8h (90 mg, 0.25 mmol), 4(propylcarbamoyl)piperidin-1-ium trifluoroacetic salt (87 mg, 0.29 mmol), DMAP (2.4 mg, 0.02 mmol), DIEA (85 μL, 0.49 mmol), and EDCI (49 mg, 0.26 mmol). After chromatography (SiO2, eluent 97− 98% DCM, 2−3% MeOH), the product was obtained as a white solid (74 mg, 58%). Mp 195.3−197.3 °C; 1H NMR (400 MHz, CD3OD) δ 0.92 (t, J = 7.5 Hz, 3H), 1.52 (sext, J = 7.1 Hz, 2H), 1.56−1.89 (m, 4H), 2.48 (m, 1H), 2.94−3.08 (m, 2H), 3.13 (t, J = 7.2 Hz, 2H), 3.81 (s, 3H), 4.22−4.58 (m, 2H), 5.26 (s, 2H), 6.74 (s, 1H), 6.94 (d, J = 8.7 Hz, 2H), 7.24 (d, J = 8.7 Hz, 2H); 13C NMR (100 MHz, CD3OD) δ 11.7, 23.6, 30.0, 42.1, 44.0, 52.5, 55.8, 103.3, 115.5, 128.4, 131.4, 131.5, 132.0, 133.1, 143.9, 161.7, 164.0, 176.9; ESI-TOF-HRMS (m/z) calcd for C23H27BrN4O3S [M + H]+ 521.1045; found 521.1054. 1-[1-(4-Methoxybenzyl)-1,4-dihydropyrrolo[3,2-b]pyrrole-2-carbonyl]-N-propylpiperidine-4-carboxamide (3). NaOMe was prepared by introducing sodium metal (5.22 mg, 0.23 mmol, 4 equiv) into anhydrous MeOH (5 mL), and the mixture was stirred at 0 °C for 30 min. The freshly prepared NaOMe was then added dropwise to a mixture of N-Boc protected carboxamide 9a (31 mg, 0.06 mmol, 1 equiv) in anhydrous THF (5 mL). The reaction mixture was stirred at 40−50 °C for 2 days before diluting with H2O (5 mL), and the aqueous suspension was extracted using EtOAc (3 × 5 mL), and the combined organic extracts (15 mL) were washed with brine (1 × 6 mL), dried with Na2SO4, and concentrated in vacuo to give the desired product (25 mg, quant). Off-white solid; 1H NMR (400 MHz, CD3OD) δ 0.91 (t, J = 7.4 Hz, 3H), 1.39−1.58 (m, 4H), 1.63−1.78 (m, 2H), 2.39 (m, 1H), 2.84−3.00 (m, 2H), 3.06−3.19 (m, 2H), 3.72 (s, 3H), 4.31−4.47 (m, 2H), 5.24 (s, 2H), 5.94 (dd, J = 0.6, 3.0 Hz, 1H), 6.22 (s, 1H), 6.79 (d, J = 8.7 Hz, 2H), 6.87 (d, J = 3.0 Hz, 1H), 7.04 (d, J = 8.7 Hz, 2H), 7.80 (m, 1H); 13C NMR (100 MHz, CD3OD) δ 11.7, 23.7, 30.0, (42.1) 42.2, (44.1), 44.2, 50.2, 55.7, 91.0, 96.7, 114.9, 124.8, 126.9, 127.4, 129.7, 132.5, 135.3, 160.5, 166.5, (177.1), 177.1; ESI-TOF-HRMS (m/z) calcd for C24H30N4O3 [M + H]+ 423.2396; found 423.2396. 1-[4-(4-Methoxybenzyl)-1,4-dihydropyrrolo[2,3-d]imidazole-5carbonyl]-N-propylpiperidine-4-carboxamide (4). To a roundbottom flask was added N-SEM protected carboxamide 9b (22 mg, 0.04 mmol, 1 equiv) and THF (1 mL) followed by 1 M TBAF in THF (3 mL). The reaction mixture was refluxed for 1 h followed by the addition of deionized H2O (3 mL). Next, the mixture was extracted with Et2O (5 × 3 mL), and the organic layers were combined and washed with H2O (2 × 15 mL) and brine (1 × 15 mL). The washed organic fraction (15 mL) was then dried with Na2SO4, and concentrated in vacuo to give a crude residue which was purified by semipreparative reversed-phase HPLC (C18, eluent ACN in H2O (5− 90% v/v), 9.65 min run time, 2.0 mL/min, 30 °C, and 100 μL per injection) to afford the product as yellow sticky oil in 45% yield (7.4 mg). (Because of rotameric effects, broad signals are observed, and not all signals are observable.) 1H NMR (400 MHz, CD3OD) δ 0.91 (t, J = 7.4 Hz, 3H), 1.33−1.58 (m, 4H), 1.59−1.82 (m, 2H), 2.40 (m, 1H), 2.83−3.02 (m, 2H), 3.12 (t, J = 7.0 Hz, 2H), 3.76 (s, 3H), 4.05−4.45 (m, 2H), 5.39 (s, 2H), 6.51 (s, 1H), 6.87 (d, J = 8.6 Hz, 2H), 7.10 (d, J = 8.6 Hz, 2H), 8.63 (s, 1H); 13C NMR (100 MHz, DMSO-d6) δ 11.3,

129.3, 129.9, 134.9, 150.4, 159.2, 162.5, 173.7; ESI-TOF-HRMS (m/z) calcd for C23H27BrN4O3S [M + H]+ 521.1045; found 521.1051. 1-[1-(4-Methoxybenzyl)-1H-indole-2-carbonyl]-N-propylpiperidine-4-carboxamide (2e). Synthesized following general procedure A from 8k (53 mg, 0.19 mmol), EDCI (38 mg, 0.20 mmol), DMAP (4.5 mg, 0.04 mmol), 4-(propylcarbamoyl)piperidin-1-ium trifluoroacetic salt (73 mg, 0.26 mmol), and DIEA (65 μL, 0.38 mmol). The crude material was purified by column chromatography (SiO2) using eluent 98% DCM, 2% MeOH. The product was isolated as a white solid (33 mg, 41%). Mp 136.3−137.2 °C. (Because of rotameric effects, broad signals are observed, and not all signals are observable.) 1H NMR (400 MHz, CDCl3) δ 0.91 (t, J = 7.4 Hz, 3H), 1.30−1.58 (m, 4H), 1.60− 1.77 (m, 2H), 2.23 (m, 1H), 2.74−2.89 (m, 2H), 3.20 (q, J = 6.6 Hz, 2H), 3.75 (s, 3H), 5.43 (s, 2H), 6.59 (s, 1H), 6.77 (d, J = 8.6 Hz, 2H), 7.04 (d, J = 8.6 Hz, 2H), 7.14 (m, 1H), 7.26 (m, 1H), 7.42 (d, J = 8.3 Hz, 1H), 7.63 (d, J = 7.9 Hz, 1H); 13C NMR (100 MHz, CD3OD) δ 11.7, 23.6, 29.6, 42.1, 43.7, 47.7, 55.7, 105.2, 111.3, 115.1, 121.4, 122.6, 124.6, 128.0, 129.5, 131.7, 132.7, 139.2, 160.7, 165.3, 176.8; ESI-TOFHRMS (m/z) calcd for C26H31N3O3 [M + H]+ 434.2444; found 434.2456. 1-[5-Bromo-1-(4-methoxybenzyl)-1H-indole-2-carbonyl]-N-propylpiperidine-4-carboxamide (2f). Synthesized following general procedure A from 8l (200 mg, 0.56 mmol), EDCI (113 mg, 0.59 mmol), DMAP (14 mg, 0.11 mmol), 4-(propylcarbamoyl)piperidin-1ium trifluoroacetic salt (223 mg, 0.78 mmol), and DIEA (290 μL, 1.66 mmol). The crude material was purified by column chromatography (SiO2) using eluent 98% DCM, 2% MeOH. The product was isolated as a white solid (134 mg, 47%). Mp 222.9−223.9 °C. (Because of rotameric effects, broad signals are observed, and not all signals are observable.) 1H NMR (400 MHz, CDCl3) δ 0.91 (t, J = 7.4 Hz, 3H), 1.32−1.61 (m, 4H), 1.63−1.81 (m, 2H), 2.24 (m, 1H), 2.73−2.90 (m, 2H), 3.21 (q, J = 6.9 Hz, 2H), 3.75 (s, 3H), 5.37−5.46 (m, 3H), 6.52 (s, 1H), 6.78 (d, J = 8.7 Hz, 2H), 7.01 (d, J = 8.7 Hz, 2H), 7.29 (d, J = 8.8 Hz, 1H), 7.34 (dd, J = 1.5, 8.8 Hz, 1H), 7.75 (d, J = 1.5 Hz, 1H); 13 C NMR (100 MHz, CDCl3) δ 11.3, 22.8, 28.7, 41.2, 43.1, 47.1, 55.3, 103.1, 111.6, 113.5, 114.1, 124.0, 126.3, 128.1, 128.4, 129.9, 132.7, 136.2, 159.1, 162.8, 173.6; ESI-TOF-HRMS (m/z) calcd for C26H30BrN3O3 [M + H]+ 514.1528; found 514.1534. 1-[4-(4-Methoxybenzyl)-4H-furo[3,2-b]pyrrole-5-carbonyl]-N-propylpiperidine-4-carboxamide (2g). Synthesized following general procedure A using 8e (60 mg, 0.22 mmol), 4-(propylcarbamoyl)piperidin-1-ium trifluoroacetic salt (75 mg, 0.27 mmol), DMAP (2.7 mg, 0.02 mmol), DIEA (77 μL, 0.44 mmol), and EDCI (45 mg, 0.23 mmol). After chromatography (SiO2, eluent 98−99% DCM, 1−2% MeOH), the product was obtained as a white solid (52 mg, 56%). Mp 164.5−166.5 °C; 1H NMR (400 MHz, CDCl3) δ 0.91 (t, J = 7.3 Hz, 3H), 1.42−1.58 (m, 4H), 1.70−1.82 (m, 2H), 2.28 (m, 1H), 2.81− 2.95 (m, 2H), 3.21 (q, J = 6.8 Hz, 2H), 3.76 (s, 3H), 4.34−4.47 (m, 2H), 5.27 (s, 2H), 5.58 (m, 1H), 6.20−6.24 (m, 2H), 6.81 (d, J = 8.6 Hz, 2H), 7.11 (d, J = 8.6 Hz, 2H), 7.35 (d, J = 2.0 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 11.3, 22.8, 28.9, 41.2, 43.3, 44.8, 49.9, 55.3, 94.6, 98.8, 114.0, 126.7, 129.1, 129.7, 130.0, 146.1, 146.3, 159.1, 163.4, 173.9; ESI-TOF-HRMS (m/z) calcd for C24H29N3O4 [M + Na]+ 446.2056; found 446.2051. 1-[2-Bromo-4-(4-methoxybenzyl)-4H-furo[3,2-b]pyrrole-5-carbonyl]-N-propylpiperidine-4-carboxamide (2h). Synthesized following general procedure A using 8f (77 mg, 0.22 mmol), 4(propylcarbamoyl)piperidin-1-ium trifluoroacetic salt (75 mg, 0.27 mmol), DMAP (2.7 mg, 0.02 mmol), DIEA (77 μL, 0.44 mmol), and EDCI (45 mg, 0.23 mmol). After chromatography (SiO2, eluent 98− 99% DCM, 1−2% MeOH), the product was obtained as a white solid (67 mg, 60%). 1H NMR (400 MHz, CDCl3) δ 0.90 (t, J = 7.4 Hz, 3H), 1.41−1.59 (m, 4H), 1.71−1.83 (m, 2H), 2.31 (m, 1H), 2.83− 2.94 (m, 2H), 3.20 (q, J = 5.3 Hz, 2H), 3.77 (s, 3H), 4.32−4.43 (m, 2H), 5.21 (s, 2H), 5.78 (m, 1H), 6.14 (d, J = 0.6 Hz, 1H), 6.18 (d, J = 0.6 Hz, 1H), 6.82 (d, J = 8.7 Hz, 2H), 7.09 (d, J = 8.7 Hz, 2H); 13C NMR (100 MHz, CDCl3) δ 11.3, 22.8, 28.9, 41.2, 43.3, 44.8, 50.0, 55.3, 94.4, 100.9, 114.1, 125.3, 126.0, 129.2, 129.5, 129.6, 146.0, 159.3, 163.2, 173.7; ESI-TOF-HRMS (m/z) calcd for C24H28BrN3O4 [M + Na]+ 526.1140; found 526.1121. 3180



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d2-1-[2-Bromo-4-(4-methoxybenzyl)-4H-thieno[3,2-b]pyrrole-5carbonyl]-N-propylpiperidine-4-carboxamide (13b). Synthesized following general procedure A using carboxylic acid 8m (60 mg, 0.16 mmol), 4-(propylcarbamoyl)piperidin-1-ium trifluoroacetic salt (56 mg, 0.20 mmol), DIEA (57 μL, 0.33 mmol), DMAP (2.0 mg, 0.02 mmol), and EDCI (33 mg, 0.17 mmol). After column chromatography (SiO2, eluent 98% DCM, 2% MeOH), the product was obtained in 92% yield (78 mg). White solid; mp 185.0−187.0 °C; 1H NMR (400 MHz, CDCl3) δ 0.92 (t, J = 7.4 Hz, 3H), 1.35−1.58 (m, 4H), 1.70− 1.85 (m, 2H), 2.25 (m, 1H), 2.80−2.97 (m, 2H), 3.21 (q, J = 6.9 Hz, 2H), 3.77 (s, 3H), 4.28−4.49 (m, 2H), 5.40 (m, 1H), 6.42 (d, J = 0.4 Hz, 1H), 6.81 (d, J = 8.7 Hz, 2H), 6.93 (d, J = 0.4 Hz, 1H), 7.07 (d, J = 8.8 Hz, 2H); 13C NMR (100 MHz, CDCl3) δ 11.3, 22.9, 28.8, 41.2, 43.3, 55.3, 103.8, 112.8, 113.9, 114.1, 122.0, 128.7, 128.9, 129.7, 140.4, 159.2, 162.8, 173.7; ESI-TOF-HRMS (m/z) calcd for C24H26D2BrN3O3S [M + H]+ 522.1218; found 522.1211. d7-1-[2-Bromo-4-(4-methoxybenzyl)-4H-thieno[3,2-b]pyrrole-5carbonyl]-N-isopropylpiperidine-4-carboxamide (13c). Synthesized following general procedure A using 2-bromo-4-(4-methoxybenzyl)4H-thieno[3,2-b]pyrrole-5-carboxylic acid (50 mg, 0.14 mmol), amine 15b (48 mg, 0.16 mmol), DIEA (48 μL, 0.27 mmol), DMAP (1.7 mg, 0.01 mmol), and EDCI (27 mg, 0.14 mmol). After column chromatography (SiO2, eluent 98% DCM, 2% MeOH), the product was obtained in 84% yield (61 mg). White solid; mp 183.8−185.8 °C; 1 H NMR (400 MHz, DMSO-d6) δ 1.27−1.46 (m, 2H), 1.52−1.69 (m, 2H), 2.30 (m, 1H), 2.78−2.98 (m, 2H), 3.70 (s, 3H), 4.05−4.30 (m, 2H), 5.34 (s, 2H), 6.59 (d, J = 0.3 Hz, 1H), 6.86 (d, J = 8.7 Hz, 2H), 7.12 (d, J = 8.7 Hz, 2H), 7.53 (s, 1H), 7.58 (br s, 1H); 13C NMR (100 MHz, DMSO-d6) δ 28.3, 41.5, 48.5, 54.9, 103.7, 110.9, 113.9, 115.1, 121.1, 128.8, 129.0, 129.8, 139.6, 158.7, 161.7, 172.6; ESI-TOF-HRMS (m/z) calcd for C24H21D7BrN3O3S [M + H]+ 527.1532; found 527.1531. d9-1-[2-Bromo-4-(4-methoxybenzyl)-4H-thieno[3,2-b]pyrrole-5carbonyl]-N-isopropylpiperidine-4-carboxamide (13d). Synthesized following general procedure A using carboxylic acid 8m (60 mg, 0.16 mmol), amine 15b (57 mg, 0.20 mmol), DIEA (57 μL, 0.33 mmol), DMAP (2.0 mg, 0.02 mmol), and EDCI (33 mg, 0.17 mmol). After column chromatography (SiO2, eluent 98% DCM, 2% MeOH), the product was obtained in 78% yield (67 mg). White solid; mp 174.1− 176.1 °C; 1H NMR (400 MHz, CDCl3) δ 1.38−1.56 (m, 2H), 1.68− 1.82 (m, 2H), 2.21 (m, 1H), 2.80−2.94 (m, 2H), 3.77 (s, 3H), 4.30− 4.45 (m, 2H), 5.19 (br s, 1H), 6.43 (d, J = 0.3 Hz, 1H), 6.81 (d, J = 8.7 Hz, 2H), 6.92 (d, J = 0.3 Hz, 1H), 7.07 (d, J = 8.7 Hz, 2H); 13C NMR (100 MHz, DMSO-d6) δ 28.3, 41.5, 54.9, 103.7, 110.9, 113.9, 115.1, 121.1, 128.8, 129.0, 129.7, 139.5, 158.7, 161.7, 172.6; ESI-TOF-HRMS (m/z) calcd for C24H19D9BrN3O3S [M + H]+ 529.1657; found 529.1645. (1R,5S,9r)-3-[2-Bromo-4-(4-methoxybenzyl)-4H-thieno[3,2-b]pyrrole-5-carbonyl]-N-propyl-3-azabicyclo[3.3.1]nonane-9-carboxamide (16a). Synthesized following general procedure A using 2bromo-4-(4-methoxybenzyl)-4H-thieno[3,2-b]pyrrole-5-carboxylic acid (60 mg, 0.16 mmol), amine 15c (64 mg, 0.20 mmol), DIEA (57 μL, 0.33 mmol), DMAP (2.0 mg, 0.02 mmol), and EDCI (33 mg, 0.17 mmol). After semipreparative reversed-phase HPLC (C18, eluent MeOH in H2O (75−88% v/v), 19.5 min run time, 2.0 mL/min, 30 °C, and 100 μL per injection), carboxamide 16a was obtained as the minor product in 21% yield (19 mg). Colorless sticky oil; (Because of rotameric effects, broad signals are observed, and not all signals are observable.) 1H NMR (400 MHz, CD3OD) δ 0.91 (t, J = 7.4 Hz, 3H), 1.03−1.72 (m, 6H), 1.72−1.92 (m, 2H), 2.02−2.38 (m, 2H), 2.50 (m, 1H), 3.17 (t, J = 7.1 Hz, 2H), 3.73 (s, 3H), 5.20−5.58 (m, 2H), 6.60 (d, J = 0.3 Hz, 1H), 6.81 (d, J = 8.8 Hz, 2H), 6.94 (d, J = 8.7 Hz, 2H), 7.14 (s, 1H); 13C NMR (100 MHz, CD3OD) δ 11.7, 20.2, 23.8, 27.1, 31.4, 42.2, 47.0, 50.4, 55.8, 105.9, 113.9, 115.2, 115.5, 123.6, 129.1, 130.6, 131.5, 142.3, 160.8, 165.1, 175.0; ESI-TOF-HRMS (m/z) calcd for C27H32BrN3O3S [M + H]+ 560.1406; found 560.1409. (1R,5S,9s)-3-[2-Bromo-4-(4-methoxybenzyl)-4H-thieno[3,2-b]pyrrole-5-carbonyl]-N-propyl-3-azabicyclo[3.3.1]nonane-9-carboxamide (16b). Carboxamide 16b was isolated as major product from above reaction mixture following semipreparative reversed-phase

22.3, 28.5, 41.8, 44.3, 46.5, 54.9, 93.6, 113.7, 118.3, 124.7, 128.7, 130.8, 138.5, 145.2, 158.4, 162.8, 173.5; ESI-TOF-HRMS (m/z) calcd for C23H29N5O3 [M + H]+ 424.2349; found 424.2346. 1-[1-(4-Methoxybenzyl)-1H-pyrrole-2-carbonyl]-N-propylpiperidine-4-carboxamide (5a). Synthesized following general procedure A from 8o (85 mg, 0.37 mmol), EDCI (75 mg, 0.39 mmol), DMAP (9 mg, 0.07 mmol), 4-(propylcarbamoyl)piperidin-1-ium trifluoroacetic salt (146 mg, 0.51 mmol), and DIEA (193 μL, 1.11 mmol). The crude material was purified by column chromatography (SiO2) using eluent 98% DCM, 2% MeOH. The product was isolated as an off-white solid (70 mg, 51%). Mp 143.5−144.2 °C; 1H NMR (400 MHz, CDCl3) δ 0.91 (t, J = 7.4 Hz, 3H), 1.30−1.44 (m, 2H), 1.51 (sext, J = 7.3 Hz, 2H), 1.66−1.76 (m, 2H), 2.23 (m, 1H), 2.77−2.86 (m, 2H), 3.20 (q, J = 6.6 Hz, 2H), 3.77 (s, 3H), 4.30−4.40 (m, 2H), 5.22 (s, 2H), 5.50 (m, 1H), 6.09 (m, 1H), 6.28 (m, 1H), 6.77−6.85 (m, 3H), 7.06 (d, J = 8.6 Hz, 2H); 13C NMR (100 MHz, CDCl3) δ 11.3, 22.8, 28.8, 41.2, 43.3, 44.3, 51.1, 55.3, 106.9, 112.6, 113.9, 125.0, 125.2, 128.8, 130.6, 159.0, 163.3, 173.9; ESI-TOF-HRMS (m/z) calcd for C22H29N3O3 [M + H]+ 384.2287; found 384.2307. 1-[5-Bromo-1-(4-methoxybenzyl)-1H-pyrrole-2-carbonyl]-N-propylpiperidine-4-carboxamide (5b). Synthesized following general procedure A from 8p (35 mg, 0.11 mmol), EDCI (23 mg, 0.12 mmol), DMAP (3 mg, 0.02 mmol), 4-(propylcarbamoyl)piperidin-1ium trifluoroacetic salt (45 mg, 0.16 mmol), and DIEA (60 μL, 0.34 mmol). The crude material was purified by column chromatography (SiO2) using eluent 98% DCM, 2% MeOH. The product was isolated as a white solid (26 mg, 51%). Mp 130.3−131.6 °C; 1H NMR (400 MHz, CDCl3) δ 0.89 (t, J = 7.4 Hz, 3H), 1.23−1.38 (m, 2H), 1.48 (sext, J = 7.4 Hz, 2H), 1.61−1.70 (m, 2H), 2.20 (m, 1H), 2.65−2.82 (m, 2H), 3.17 (q, J = 7.0 Hz, 2H), 3.75 (s, 3H), 4.17−4.29 (m, 2H), 5.33 (s, 2H), 5.62 (m, 1H), 6.15 (d, J = 3.9 Hz, 1H), 6.24 (d, J = 3.9 Hz, 1H), 6.79 (d, J = 8.7 Hz, 2H), 7.01 (d, J = 8.7 Hz, 2H); 13C NMR (100 MHz, CDCl3) δ 11.2, 22.8, 28.6, 41.1, 43.1, 44.4, 48.5, 55.2, 106.9, 109.9, 112.8, 113.9, 126.4, 128.5, 130.1, 158.9, 162.6, 173.8; ESI-TOF-HRMS (m/z) calcd for C22H28BrN3O3 [M + H]+ 462.1392; found 462.1381. 1-[4-Bromo-1-(4-methoxybenzyl)-1H-pyrrole-2-carbonyl]-N-propylpiperidine-4-carboxamide (5c). Synthesized following general procedure A from 8q (67 mg, 0.22 mmol), EDCI (44 mg, 0.23 mmol), DMAP (5 mg, 0.04 mmol), 4-(propylcarbamoyl)piperidin-1ium trifluoroacetic salt (94 mg, 0.33 mmol), and DIEA (115 μL, 0.66 mmol). The crude material was purified by column chromatography (SiO2) using eluent 98% DCM, 2% MeOH. The product was isolated as a white solid (74 mg, 73%). Mp 195−195.7 °C; 1H NMR (400 MHz, CDCl3) δ 0.90 (t, J = 7.4 Hz, 3H), 1.29−1.40 (m, 2H), 1.50 (sext, J = 7.4 Hz, 2H), 1.66−1.77 (m, 2H), 2.20 (m, 1H), 2.72−2.88 (m, 2H), 3.23−3.12 (m, 2H), 3.77 (s, 3H), 4.19−4.36 (m, 2H), 5.14 (s, 2H), 5.53 (m, 1H), 6.24 (d, J = 1.8 Hz, 1H), 6.76 (d, J = 1.8 Hz, 1H), 6.82 (d, J = 8.7 Hz, 2H), 7.07 (d, J = 8.7 Hz, 2H); 13C NMR (100 MHz, CDCl3) δ 11.3, 22.8, 28.7, 41.1, 43.2, 43.9, 51.3, 55.3, 94.5, 113.9, 114.0, 124.3, 125.7, 129.1, 129.5, 159.3, 161.9, 173.6; ESI-TOFHRMS (m/z) calcd for C22H28BrN3O3 [M + H]+ 462.1392; found 462.1402. N-Isopropyl-1-[4-(4-methoxybenzyl)-4H-thieno[3,2-b]pyrrole-5carbonyl]piperidine-4-carboxamide (13a). Synthesized following general procedure A using 2-bromo-4-(4-methoxybenzyl)-4H-thieno[3,2-b]pyrrole-5-carboxylic acid (40 mg, 0.11 mmol), amine 15a (37 mg, 0.13 mmol), DIEA (38 μL, 0.22 mmol), DMAP (1.3 mg, 0.01 mmol), and EDCI (22 mg, 0.12 mmol). After column chromatography (SiO2, eluent 20−30% PE, 70−80% EtOAc), the product was obtained in 46% yield (26 mg). White solid; mp 178.0−180.0 °C; 1H NMR (400 MHz, CDCl3) δ 1.14 (d, J = 6.6 Hz, 6H), 1.38−1.59 (m, 2H), 1.65−1.79 (m, 2H), 2.21 (m, 1H), 2.79−2.94 (m, 2H), 3.77 (s, 3H), 4.06 (m, 1H), 4.24−4.47 (m, 2H), 5.25 (m, 1H), 5.31 (s, 2H), 6.42 (s, 1H), 6.81 (d, J = 8.7 Hz, 2H), 6.92 (s, 1H), 7.06 (d, J = 8.7 Hz, 2H); 13 C NMR (100 MHz, CDCl3) δ 22.7, 28.7, 41.3, 43.2, 44.7, 49.7, 55.3, 103.8, 112.8, 113.9, 114.1, 122.1, 128.6, 128.9, 129.7, 140.4, 159.2, 162.8, 172.9; ESI-TOF-HRMS (m/z) calcd for C24H28BrN3O3S [M + H]+ 520.1093; found 520.1096. 3181



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= 7.4 Hz, 3H), 1.20−1.53 (m, 6H), 1.69−1.79 (m, 2H), 1.81−1.92 (m, 2H), 2.05 (m, 1H), 2.98 (q, J = 6.1 Hz, 2H), 3.59−3.76 (m, 4H), 5.64 (s, 2H), 6.83 (d, J = 8.7 Hz, 2H), 7.01 (s, 1H), 7.16 (d, J = 8.7 Hz, 2H), 7.49 (s, 1H), 7.69 (m, 1H), 8.08 (m, 1H); 13C NMR (100 MHz, DMSO-d6) δ 11.3, 22.4, 28.4, 31.5, 40.0, 43.2, 47.4, 48.6, 55.0, 104.5, 111.8, 113.8, 115.3, 121.0, 128.6, 129.6, 130.5, 140.7, 158.5, 160.6, 174.6; ESI-TOF-HRMS (m/z) calcd for C25H30BrN3O3S [M + Na]+ 556.1068; found 556.1066. 2-Bromo-4-(4-methoxybenzyl)-N-[4-(propylcarbamoyl)cyclohexyl]-4H-thieno[3,2-b]pyrrole-5-carboxamide, trans (16g). Carboxamide 16g was isolated as major product from above reaction mixture following semipreparative reversed-phase HPLC. The title compound was obtained as a white solid in 24% yield (21 mg). 1H NMR (400 MHz, DMSO-d6) δ 0.83 (t, J = 7.4 Hz, 3H), 1.32−1.61 (m, 6H), 1.65−1.79 (m, 2H), 1.80−1.95 (m, 2H), 2.24 (m, 1H), 3.01 (q, J = 6.0 Hz, 2H), 3.68 (s, 3H), 3.87 (m, 1H), 5.62 (s, 2H), 6.82 (d, J = 8.7 Hz, 2H), 7.07 (s, 1H), 7.16 (d, J = 8.7 Hz, 2H), 7.50 (s, 1H), 7.63 (m, 1H), 8.00 (m, 1H); 13C NMR (100 MHz, DMSO-d6) δ 11.3, 22.4, 25.2, 28.7, 40.1, 40.5, 45.9, 48.6, 55.0, 104.8, 111.7, 113.7, 115.2, 120.9, 128.6, 129.7, 130.5, 140.6, 158.5, 161.0, 174.2; ESI-TOF-HRMS (m/z) calcd for C25H30BrN3O3S [M + Na]+ 554.1089; found 554.1087. 6-[2-Bromo-4-(4-methoxybenzyl)-4H-thieno[3,2-b]pyrrole-5-carbonyl]-N-propyl-6-azaspiro[2.5]octane-1-carboxamide (16h). Synthesized following general procedure A using 2-bromo-4-(4-methoxybenzyl)-4H-thieno[3,2-b]pyrrole-5-carboxylic acid (100 mg, 0.27 mmol), amine 15g (102 mg, 0.33 mmol), DIEA (96 μL, 0.55 mmol), HOBt (38 mg, 0.28 mmol), and EDCI (55 mg, 0.29 mmol). The pure product was obtained in 21% yield (31 mg) after column chromatography (SiO2) using 100% Et2O followed by 99% DCM, 1% MeOH as the eluent. White solid; 1H NMR (400 MHz, CD3OD) δ 0.79 (m, 1H), 0.93 (t, J = 7.5 Hz, 3H), 1.08 (m, 1H), 1.15−1.62 (m, 7H), 3.09−3.19 (m, 2H), 3.37 (m, 1H), 3.46−3.80 (m, 6H), 5.33 (s, 2H), 6.53 (d, J = 0.4 Hz, 1H), 6.82 (d, J = 8.7 Hz, 2H), 7.03 (d, J = 8.7 Hz, 2H), 7.22 (d, J = 0.4 Hz, 1H); 13C NMR (100 MHz, CD3OD) δ 11.8, 18.1, 23.8, 27.7, 28.0, 29.6, 37.3, 42.5, 50.5, 55.8, 105.2, 113.5, 115.1, 115.5, 123.4, 129.9, 130.2, 131.4, 142.1, 160.9, 164.8, 173.3; ESI-TOF-HRMS (m/z) calcd for C26H30BrN3O3S [M + Na]+ 568.1068; found 568.1069. (S)-1-[2-Bromo-4-(4-methoxybenzyl)-4H-thieno[3,2-b]pyrrole-5carbonyl]-N-propylpyrrolidine-2-carboxamide (16i). Synthesized following general procedure A using 2-bromo-4-(4-methoxybenzyl)4H-thieno[3,2-b]pyrrole-5-carboxylic acid (80 mg, 0.22 mmol), amine 15h (71 mg, 0.26 mmol), DIEA (76 μL, 0.44 mmol), DMAP (2.6 mg, 0.02 mmol), and EDCI (44 mg, 0.23 mmol). After column chromatography (SiO2, eluent 97% DCM, 3% MeOH), the product was obtained in 50% yield (55 mg). Colorless sticky oil. (Because of rotameric effects, broad signals are observed, and not all signals are observable.) 1H NMR (400 MHz, CDCl3) δ 0.87 (t, J = 7.4 Hz, 3H), 1.47 (sext, J = 7.2 Hz, 2H), 1.58−2.50 (m, 4H), 3.07−3.27 (m, 2H), 3.53−3.68 (m, 2H), 3.75 (s, 3H), 4.63 (m, 1H), 5.30−5.54 (m, 2H), 6.62 (br s, 1H), 6.79 (d, J = 8.7 Hz, 2H), 6.89 (s, 1H), 7.03 (d, J = 8.7 Hz, 2H); 13C NMR (100 MHz, CD3OD) δ 11.7, 23.6, 26.2, 31.0, 42.2, (42.3), 50.7, 51.9, 55.7, 62.1, 106.8, 115.0, 115.7, 123.6, 130.0, 131.0, 131.4, 142.2, 160.8, 164.6, 174.5, (174.6); ESI-TOF-HRMS (m/z) calcd for C23H26BrN3O3S [M + H]+ 506.0936; found 506.0943. 5-[2-Bromo-4-(4-methoxybenzyl)-4H-thieno[3,2-b]pyrrole-5-carbonyl]-N-propyl-5-azaspiro[2.3]hexane-1-carboxamide (16j). Synthesized following general procedure A using 2-bromo-4-(4-methoxybenzyl)-4H-thieno[3,2-b]pyrrole-5-carboxylic acid (60 mg, 0.16 mmol), crude amine 15i (56 mg), DIEA (57 μL, 0.33 mmol), DMAP (2.0 mg, 0.02 mmol), and EDCI (33 mg, 0.17 mmol). The pure product was obtained in 17% yield (14 mg) after column chromatography (SiO2) using 100% Et2O followed by 98% DCM, 2% MeOH as the eluent. Yellow solid. (Because of rotameric effects, broad signals are observed, and not all signals are observable.) 1H NMR (400 MHz, CD3OD) δ 0.93 (t, J = 7.4 Hz, 3H), 1.12 (m, 1H), 1.23 (m, 1H), 1.53 (sext, J = 7.3 Hz, 2H), 1.81 (m, 1H), 3.03−3.23 (m, 2H), 3.74 (s, 3H), 4.01−4.42 (m, 4H), 5.55 (s, 2H), 6.74 (s, 1H), 6.82 (d, J = 8.7 Hz, 2H), 7.05 (d, J = 8.1 Hz, 2H), 7.13 (s, 1H); 13C NMR (100

HPLC. The title compound was obtained as a yellow sticky oil in 48% yield (44 mg). 1H NMR (400 MHz, CD3OD) δ 0.92 (t, J = 7.5 Hz, 3H), 1.24 (m, 1H), 1.37−2.32 (m, 9H), 2.41 (m, 1H), 3.11−3.21 (m, 2H), 3.33−3.68 (m, 2H), 3.73 (s, 3H), 3.96 (m, 1H), 4.38 (m, 1H), 5.18−5.55 (m, 2H), 6.54 (s, 1H), 6.80 (d, J = 8.7 Hz, 2H), 6.93 (d, J = 8.7 Hz, 2H), 7.12 (d, J = 0.3 Hz, 1H), 7.82 (m, 1H); 13C NMR (100 MHz, CD3OD) δ 11.8, 20.7, 23.7, 31.9, 32.4, 33.0, 33.5, 42.1, 44.7, 46.9, 50.4, 50.5, 55.7, 105.6, 113.7, 115.1, 115.5, 123.5, 129.1, 130.6, 131.4, 142.1, 160.7, 165.1, 175.9; ESI-TOF-HRMS (m/z) calcd for C27H32BrN3O3S [M + Na]+ 580.1245; found 580.1261. 2-Bromo-4-(4-methoxybenzyl)-N-[(1R,3s,5S,9r)-3(propylcarbamoyl)bicyclo[3.3.1]nonan-9-yl]-4H-thieno[3,2-b]pyrrole-5-carboxamide (16c). Synthesized following general procedure A using 2-bromo-4-(4-methoxybenzyl)-4H-thieno[3,2-b]pyrrole5-carboxylic acid (100 mg, 0.27 mmol), amine 15d (111 mg, 0.33 mmol), DIEA (95 μL, 0.55 mmol), HOBt (38 mg, 0.28 mmol), and EDCI (55 mg, 0.29 mmol). After column chromatography (SiO2, eluent Et2O), the pure carboxamide 16c was afforded as the minor product in 7% yield (10 mg). White solid; 1H NMR (400 MHz, DMSO-d6) δ 0.83 (t, J = 7.5 Hz, 3H), 1.22−1.78 (m, 10H), 1.89−2.05 (m, 2H), 2.23−2.36 (m, 2H), 2.65 (m, 1H), 3.00 (q, J = 6.7 Hz, 2H), 3.50 (m, 1H), 3.69 (s, 3H), 5.62 (s, 2H), 6.84 (d, J = 8.7 Hz, 2H), 7.16 (s, 1H), 7.19 (d, J = 8.7 Hz, 2H), 7.50 (s, 1H), 7.63 (m, 1H), 7.75 (m, 1H); 13C NMR (100 MHz, DMSO-d6) δ 11.3, 14.9, 22.4, 27.0, 28.7, 30.4, 33.0, 34.5, 48.5, 53.1, 55.0, 105.2, 111.7, 113.8, 115.3, 120.9, 128.6, 129.7, 130.4, 140.6, 158.5, 161.2, 175.6; ESI-TOF-HRMS (m/z) calcd for C28H34BrN3O3S [M + Na]+ 596.1381; found 596.1380. 2-Bromo-4-(4-methoxybenzyl)-N-[(1R,3s,5S,9s)-3(propylcarbamoyl)bicyclo[3.3.1]nonan-9-yl]-4H-thieno[3,2-b]pyrrole-5-carboxamide (16d). Carboxamide 16d was isolated as major product from above reaction mixture following column chromatography. The title compound was obtained as a white solid in 24% yield (38 mg). Mp 172.5−174.5 °C; 1H NMR (400 MHz, DMSO-d6) δ 0.84 (t, J = 7.3 Hz, 3H), 1.10−1.31 (m, 3H), 1.34−1.45 (m, 2H), 1.55−1.65 (m, 2H), 1.71−1.85 (m, 3H), 1.96−2.10 (m, 4H), 2.62 (m, 1H), 2.99 (q, J = 6.8 Hz, 2H), 3.69 (s, 3H), 4.06 (m, 1H), 5.59 (s, 2H), 6.82 (d, J = 8.7 Hz, 2H), 7.09 (s, 1H), 7.17 (d, J = 8.7 Hz, 2H), 7.53 (d, J = 0.3 Hz, 1H), 7.65 (m, 1H), 7.73 (m, 1H); 13C NMR (100 MHz, DMSO-d6) δ 11.3, 14.4, 22.3, 26.0, 27.5, 29.9, 34.6, 40.1, 46.0, 48.6, 55.0, 105.2, 111.8, 113.7, 115.2, 120.9, 128.7, 129.7, 130.4, 140.6, 158.5, 161.5, 174.9; ESI-TOF-HRMS (m/z) calcd for C28H34BrN3O3S [M + H]+ 574.1562; found 574.1556. 1-[2-Bromo-4-(4-methoxybenzyl)-4H-thieno[3,2-b]pyrrole-5-carbonyl]-N-propylazetidine-3-carboxamide (16e). Synthesized following general procedure A using 2-bromo-4-(4-methoxybenzyl)-4Hthieno[3,2-b]pyrrole-5-carboxylic acid (100 mg, 0.27 mmol), amine 15e (102 mg, 0.33 mmol), DIEA (96 μL, 0.55 mmol), HOBt (38 mg, 0.28 mmol), and EDCI (55 mg, 0.29 mmol). After column chromatography (SiO2, eluent 97−99% DCM, 1−3% MeOH), the product was obtained in 43% yield (58 mg). White solid; mp 195.8− 197.8 °C. (Because of rotameric effects, broad signals are observed, and not all signals are observable.) 1H NMR (400 MHz, CDCl3) δ 0.93 (t, J = 7.4 Hz, 3H), 1.54 (sext, J = 7.2 Hz, 2H), 3.18−3.32 (m, 3H), 3.77 (s, 3H), 4.29−4.37 (m, 2H), 4.38−4.46 (m, 2H), 5.50 (m, 1H), 5.58 (s, 2H), 6.61 (d, J = 0.4 Hz, 1H), 6.81 (d, J = 8.7 Hz, 2H), 6.88 (d, J = 0.4 Hz, 1H), 7.11 (d, J = 8.7 Hz, 2H); 13C NMR (100 MHz, CDCl3) δ 11.3, 22.8, 34.1, 41.6, 50.0, 55.2, 105.8, 114.0, 114.1, 114.5, 122.5, 126.5, 128.4, 130.0, 141.6, 159.0, 163.2, 171.0; ESI-TOFHRMS (m/z) calcd for C22H24BrN3O3S [M + Na]+ 514.0599; found 514.0596. 2-Bromo-4-(4-methoxybenzyl)-N-[4-(propylcarbamoyl)cyclohexyl]-4H-thieno[3,2-b]pyrrole-5-carboxamide, cis (16f). Synthesized following general procedure A using 2-bromo-4-(4-methoxybenzyl)-4H-thieno[3,2-b]pyrrole-5-carboxylic acid (60 mg, 0.16 mmol), amine 15f (59 mg, 0.20 mmol), DIEA (57 μL, 0.33 mmol), DMAP (2.0 mg, 0.02 mmol), and EDCI (33 mg, 0.17 mmol). After semipreparative reversed-phase HPLC (C18, eluent MeOH in H2O (75−90% v/v), 13 min run time, 2.0 mL/min, 30 °C, and 100 μL per injection), carboxamide 16f was obtained as the minor product in 5% yield (4 mg). White solid; 1H NMR (400 MHz, DMSO-d6) δ 0.83 (t, J 3182



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MHz, CD3OD) δ 11.7, 16.6, 23.8, 24.3, 25.0, 42.4, 50.7, 55.7, 107.4, 115.0, 115.2, 115.7, 123.9, 128.1, 129.4, 131.6, 143.1, 160.7, 165.0, 172.8; ESI-TOF-HRMS (m/z) calcd for C24H26BrN3O3S [M + Na]+ 538.0776; found 538.0773. 1-{1-[4-(4-Methoxybenzyl)-4H-thieno[3,2-b]pyrrole-5-carbonyl]piperidin-4-yl}pentan-1-one (17). The experimental procedure and characterization data of this compound were reported previously.31 1-[4-(4-Chlorobenzyl)-4H-thieno[3,2-b]pyrrole-5-carbonyl]-Npropylpiperidine-4-carboxamide (18). Synthesized following general procedure A using carboxylic acid 8n (50 mg, 0.17 mmol), 4(propylcarbamoyl)piperidin-1-ium trifluoroacetic salt (58 mg, 0.21 mmol), DIEA (60 μL, 0.34 mmol), and EDCI (35 mg, 0.18 mmol). After chromatography (SiO2, eluent 97% DCM, 3% MeOH), the product was obtained as a white solid (20 mg, 26%). Mp 194.5−195.9 °C; 1H NMR (400 MHz, CDCl3) δ 0.92 (t, J = 7.3 Hz, 3H), 1.40− 1.62 (m, 4H), 1.71−1.83 (m, 2H), 2.29 (m, 1H), 2.79−3.00 (m, 2H), 3.13−3.29 (m, 2H), 4.31−4.52 (m, 2H), 5.42 (s, 2H), 5.46 (m, 1H), 6.57 (s, 1H), 6.84 (d, J = 5.3 Hz, 1H), 7.07 (d, J = 8.3 Hz, 2H), 7.18 (d, J = 5.3 Hz, 1H), 7.23 (d, J = 8.3 Hz, 2H); 13C NMR (100 MHz, CDCl3) δ 11.3, 22.9, 28.9, 41.2, 43.2, 44.6, 49.5, 104.4, 110.3, 122.2, 126.8, 128.6, 128.7, 129.5, 133.3, 136.6, 142.8, 162.8, 173.7; ESI-TOFHRMS (m/z) calcd for C23H26ClN3O2S [M + H]+ 444.1513; found 444.1524. 1-[4-(4-Methoxybenzyl)-2-methyl-4H-thieno[3,2-b]pyrrole-5-carbonyl]-N-propylpiperidine-4-carboxamide (19). The experimental procedure and characterization data of this compound were reported previously.31 1-[4-(4-Methoxybenzyl)-2-phenyl-4H-thieno[3,2-b]pyrrole-5-carbonyl]-N-propylpiperidine-4-carboxamide (20). The experimental procedure and characterization data of this compound were reported previously.31 1-[2-Cyano-4-(4-methoxybenzyl)-4H-thieno[3,2-b]pyrrole-5-carbonyl]-N-propylpiperidine-4-carboxamide (21). The experimental procedure and characterization data of this compound were reported previously.31 1-[2-(2-Fluorophenyl)-4-(4-methoxybenzyl)-4H-thieno[3,2-b]pyrrole-5-carbonyl]-N-propylpiperidine-4-carboxamide (22). To a sealed tube was added carboxamide 1b (30 mg, 0.06 mmol, 1 equiv), PPh3 (4.6 mg, 0.02 mmol, 30 mol %), Pd(OAc)2 (1.3 mg, 0.006 mmol, 10 mol %), o-FPhB(OH)2 (16 mg, 0.12 mmol, 2 equiv), Ag2O (40 mg, 0.17 mmol, 3 equiv), and K2CO3 (24 mg, 0.17 mmol, 3 equiv) following which degassed 2 mL of toluene/H2O (19:1) was added. The reaction mixture was stirred at 120 °C for 24 h before diluting with H2O (2 mL). The aqueous suspension was extracted with EtOAc (3 × 2 mL) and the combined organic extracts (8 mL) were washed with brine (1 × 8 mL), dried with Na2SO4, and concentrated in vacuo to give a crude residue which was purified by semipreparative reversedphase HPLC (conditions, MeOH in H2O (80% v/v), 20 min run time, 2.0 mL/min, 30 °C, and 100 μL per injection) to afford the product as a yellow solid (8.1 mg, 26%). 1H NMR (400 MHz, DMSO-d6) δ 0.83 (t, J = 7.1 Hz, 3H), 1.27−1.48 (m, 4H), 1.55−1.72 (m, 2H), 2.35 (m, 1H), 2.84−3.04 (m, 4H), 3.70 (s, 3H), 4.05−4.42 (m, 2H), 5.41 (s, 2H), 6.68 (s, 1H), 6.86 (d, J = 8.7 Hz, 2H), 7.16 (d, J = 8.7 Hz, 2H), 7.23−7.40 (m, 3H), 7.68−7.81 (m, 3H); 13C NMR (100 MHz, DMSO-d6) δ 11.3, 22.3, 28.4, 40.1, 41.6, 48.5, 54.9, 103.6, 110.8 (d, 3 JCF = 7 Hz), 113.9, 116.4 (d, 2JCF = 22 Hz), 121.5 (d, 4JCF = 5 Hz), 122.3, 122.5, 125.1 (d, 5JCF = 3 Hz), 128.2 (d, 5JCF = 3 Hz), 128.8, 128.9 (d, 3JCF = 8 Hz), 130.1 (d, 3JCF = 8 Hz), 135.6 (d, 4JCF = 4 Hz), 141.7, 158.1 (d, 1JCF = 248 Hz), 158.7, 161.7, 173.4; ESI-TOF-HRMS (m/z) calcd for C30H32FN3O3S [M + H]+ 534.2227; found 534.2233. 1-[4-(4-Methoxybenzyl)-2-phenyl-4H-pyrrolo[2,3-d]thiazole-5carbonyl]-N-propylpiperidine-4-carboxamide (23a). Synthesized following general procedure B using carboxamide 2d (26 mg, 0.05 mmol), Pd(OAc)2 (1.3 mg, 0.005 mmol), PPh3 (4.6 mg, 0.02 mmol), PhB(OH)2 (14 mg, 0.12 mmol), and K2CO3 (24 mg, 0.17 mmol). After semipreparative reversed-phase HPLC (conditions, MeOH in H2O (80% v/v), 15 min run time, 2.0 mL/min, 30 °C, and 100 μL per injection), the product was obtained as a yellow solid (9.2 mg, 36%). 1 H NMR (400 MHz, CD3OD) δ 0.91 (t, J = 7.4 Hz, 3H), 1.21−1.57 (m, 4H), 1.57−1.79 (m, 2H), 2.37 (m, 1H), 2.78−2.98 (m, 2H), 3.12

(t, J = 7.0 Hz, 2H), 3.74 (s, 3H), 4.07−4.46 (m, 2H), 5.57 (s, 2H), 6.63 (s, 1H), 6.84 (d, J = 8.7 Hz, 2H), 7.14 (d, J = 8.7 Hz, 2H), 7.40− 7.53 (m, 3H), 7.98−8.06 (m, 2H); 13C NMR (100 MHz, CD3OD) δ 11.7, 23.7, 29.8, 42.1, 43.8, 49.1, 55.8, 104.0, 114.5, 115.2, 127.2, 129.5, 130.1, 130.2, 131.2, 131.5, 135.8, 155.5, 160.9, 164.6, 169.5, 176.8; ESI-TOF-HRMS (m/z) calcd for C29H32N4O3S [M + H]+ 517.2273; found 517.2283. 4-(4-Methoxybenzyl)-N-[(1R,3s,5S,9s)-3-(propylcarbamoyl)bicyclo[3.3.1]nonan-9-yl]-4H-pyrrolo[2,3-d]thiazole-5-carboxamide (23b). Synthesized following general procedure A using carboxylic acid 8c (23 mg, 0.08 mmol), amine 15d (32 mg, 0.10 mmol), DIEA (28 μL, 0.16 mmol), DMAP (1.0 mg, 0.01 mmol), and EDCI (16 mg, 0.08 mmol). After column chromatography (SiO2, eluent 97−98% DCM, 2−3% MeOH), the product was afforded in 42% yield (17 mg). White solid; mp 98.8−100.8 °C; 1H NMR (400 MHz, DMSO-d6) δ 0.84 (t, J = 7.3 Hz, 3H), 1.06−1.33 (m, 3H), 1.40 (sext, J = 7.1 Hz, 2H), 1.55− 1.68 (m, 2H), 1.70−1.89 (m, 3H), 1.92−2.12 (m, 4H), 2.64 (m, 1H), 3.00 (q, J = 5.8 Hz, 2H), 3.68 (s, 3H), 4.09 (m, 1H), 5.72 (s, 2H), 6.81 (d, J = 8.7 Hz, 2H), 7.15−7.23 (m, 3H), 7.67 (m, 1H), 7.74 (m, 1H), 9.01 (s, 1H); 13C NMR (100 MHz, DMSO-d6) δ 11.3, 14.5, 22.3, 26.0, 27.5, 29.9, 34.6, 40.1, 46.0, 47.6, 55.0, 103.5, 111.8, 113.7, 128.8, 129.5, 130.6, 154.3, 155.0, 158.5, 161.4, 175.0; ESI-TOF-HRMS (m/z) calcd for C27H34N4O3S [M + H]+ 495.2430; found 495.2433. 2-Bromo-4-(4-methoxybenzyl)-N-[(1R,3s,5S,9s)-3(propylcarbamoyl)bicyclo[3.3.1]nonan-9-yl]-4H-pyrrolo[2,3-d]thiazole-5-carboxamide (23c). Synthesized following general procedure A using carboxylic acid 8d (48 mg, 0.13 mmol), amine 15d (53 mg, 0.16 mmol), DIEA (45 μL, 0.26 mmol), DMAP (1.2 mg, 0.01 mmol), and EDCI (26 mg, 0.14 mmol). After semipreparative reversed-phase HPLC (conditions, MeOH in H2O (80−90% v/v), 12 min run time, 2.0 mL/min, 30 °C, and 100 μL per injection), the product was obtained as a white solid (14 mg, 19%). 1H NMR (400 MHz, CD3OD) δ 0.93 (t, J = 7.4 Hz, 3H), 1.20−1.30 (m, 2H), 1.35 (m, 1H), 1.52 (sext, J = 7.4 Hz, 2H), 1.70−1.80 (m, 4H), 1.92 (m, 1H), 2.05−2.20 (m, 4H), 2.69 (m, 1H), 3.14 (q, J = 5.8 Hz, 2H), 3.71 (s, 3H), 4.13 (m, 1H), 5.67 (s, 2H), 6.78 (d, J = 8.8 Hz, 2H), 6.94 (s, 1H), 7.12 (d, J = 8.8 Hz, 2H), 7.51 (m, 1H), 7.92 (m, 1H); 13C NMR (100 MHz, CD3OD) δ 11.7, 15.6, 23.7, 27.4, 29.4, 31.3, (37.0), 37.0, (42.2), 42.3, (48.0), 48.1, 49.4, 55.7, 104.6, 114.9, 116.7, 129.8, 130.4, 131.5, 137.6, 152.6, 160.7, 164.1, 178.9; ESI-TOF-HRMS (m/z) calcd for C27H33BrN4O3S [M + H]+ 575.1515; found 575.1527. 4-(4-Methoxybenzyl)-2-phenyl-N-[(1R,3s,5S,9s)-3(propylcarbamoyl)bicyclo[3.3.1]nonan-9-yl]-4H-pyrrolo[2,3-d]thiazole-5-carboxamide (23d). Synthesized following general procedure B using carboxamide 23c (24 mg, 0.04 mmol), Pd(OAc)2 (0.9 mg, 0.004 mmol), PPh3 (3.3 mg, 0.01 mmol), PhB(OH)2 (10 mg, 0.09 mmol), and K2CO3 (18 mg, 0.13 mmol). After semipreparative reversed-phase HPLC (conditions, MeOH in H2O (90% v/v), 11 min run time, 2.0 mL/min, 30 °C, and 50 μL per injection), the product was obtained as an off-white solid (5.7 mg, 24%). 1H NMR (400 MHz, CDCl3) δ 0.92 (t, J = 7.4 Hz, 3H), 1.29−1.60 (m, 5H), 1.72−2.29 (m, 9H), 2.58 (m, 1H), 3.13−3.29 (m, 2H), 3.73 (s, 3H), 4.22 (m, 1H), 5.57 (m, 1H), 5.84 (s, 2H), 6.17 (m, 1H), 6.75 (s, 1H), 6.79 (d, J = 8.7 Hz, 2H), 7.36 (d, J = 8.7 Hz, 2H), 7.39−7.51 (m, 3H), 7.97−8.06 (m, 2H); 13C NMR (100 MHz, CDCl3) δ 11.3, 14.5, 22.8, 26.5, 28.3, 30.1, 36.3, 41.2, 45.8, 48.5, 55.2, 102.2, 113.3, 113.8, 126.4, 128.9, 129.3, 130.1, 130.7, 134.5, 155.1, 159.0, 161.3, 168.8, 175.7; ESI-TOF-HRMS (m/z) calcd for C33H38N4O3S [M + H]+ 571.2743; found 571.2738. (1R,5S,9s)-3-[2-Bromo-4-(4-methoxybenzyl)-4H-pyrrolo[2,3-d]thiazole-5-carbonyl]-N-propyl-3-azabicyclo[3.3.1]nonane-9-carboxamide (23e). Synthesized following general procedure A using carboxylic acid 8d (61 mg, 0.17 mmol), amine 15c (65 mg, 0.20 mmol), DIEA (58 μL, 0.33 mmol), DMAP (2.0 mg, 0.02 mmol), and EDCI (34 mg, 0.18 mmol). After semipreparative reversed-phase HPLC (C18, eluent MeOH in H2O (70−90% v/v), 12.5 min run time, 2.0 mL/min, 30 °C, and 100 μL per injection), the product was obtained in 32% yield (30 mg). White solid. (Because of rotameric effects, broad signals are observed, and not all signals are observable.) 1 H NMR (400 MHz, CD3OD) δ 0.92 (t, J = 7.4 Hz, 3H), 1.17−1.94 (m, 8H), 1.96−2.29 (m, 2H), 2.40 (m, 1H), 3.07−3.21 (m, 2H), 3.28−3.53 (m, 2H), 3.74 (s, 3H), 3.87 (m, 1H), 4.37 (m, 1H), 5.34− 3183



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outbreak.42 WT O’nyong-nyong virus (ONNV) used in this study was originally isolated from a patient in Chad.43 CHIKV and ONNV were kindly provided by Dr. Hugues Tolou (Groupe d’Etude en Préventologie (GEP), France). WT Sindbis virus (SINV) was provided by National University of Singapore (NUS). Virus stocks used for in vitro studies were prepared via numerous passages in BHK21 or VeroE6 cells followed by purification and ultracentrifugation. Virus stocks were titered using standard plaque assays in VeroE6 cells before storage at −80 °C.44 Cytotoxicity and Antiviral Assays. Cell viability assay, antiviral assay (CHIKV-IMT-Gluc, CHIKV-SGP11-Gluc, or CHIKVCNR20235-Gluc assay), and Cellomics high content screen were performed as previously described.31 Viral RNA Extraction and qRT-PCR. Cell seeding, virus infection with WT CHIKV-IMT (MOI = 0.1), and compound seeding were carried out in a similar manner as previously described in the antiviral assay (CHIKV-IMT-Gluc assay). Extraction of viral RNA and quantification of viral load were performed as previously described.31 Human Liver Microsomal Stability Assay. Synthesized compounds were tested for HLM metabolic stability by Pharmaron Beijing Co., Ltd., In Vitro ADME Laboratory (China). In Vivo Pharmacokinetics Study. Compound 20 or 23c were tested for in vivo pharmacokinetics by Pharmaron Beijing Co., Ltd., PK and Bioanalytical Laboratory (China). Compound Libraries and Reference Compounds. All compounds obtained from the synthesized compound library and ribavirin were dissolved in DMSO (Sigma-Aldrich) and stored as 100 mM, 50 mM, and 25 mM stock solutions depending on their solubility in DMSO. Ribavirin was purchased from Tokyo Chemical Industry (TCI). Data Analysis. All data from the cell viability and antiviral assays were normalized using untreated and reagent background samples (in cell viability assay) or untreated infections and noninfected cell cultures (in antiviral assays), which were set as 100% and 0% values, respectively. CC50 values in cell viability assay and EC50 values in antiviral assays were determined by fitting the results from dose− response studies into asymmetric (five-parameter) curves with GraphPad Prism 5.03 software (GraphPad Prism, 1995). All data from qRT-PCR were analyzed with ABI 7900HT SDS version 2.4 (ABI 7900HT SDS).

5.66 (m, 2H), 6.57 (s, 1H), 6.80 (d, J = 8.8 Hz, 2H), 6.96 (d, J = 8.8 Hz, 2H); 13C NMR (100 MHz, CD3OD) δ 11.8, 20.7, 23.7, 31.7, 32.4, 32.9, 33.7, 42.1, 44.7, 46.8, 50.4, 55.7, 104.1, 115.1, 116.6, 129.4, 129.6, 131.2, 136.9, 151.9, 160.8, 164.6, 175.8; ESI-TOF-HRMS (m/z) calcd for C26H31BrN4O3S [M + H]+ 561.1358; found 561.1355. (1R,5S,9s)-3-[4-(4-Methoxybenzyl)-2-phenyl-4H-pyrrolo[2,3-d]thiazole-5-carbonyl]-N-propyl-3-azabicyclo[3.3.1]nonane-9-carboxamide (23f). Synthesized following general procedure B using carboxamide 23e (30 mg, 0.05 mmol), Pd(OAc)2 (1.2 mg, 0.005 mmol), PPh3 (4.2 mg, 0.02 mmol), PhB(OH)2 (13 mg, 0.11 mmol), and K2CO3 (22 mg, 0.16 mmol). After semipreparative reversed-phase HPLC (conditions, MeOH in H2O (90% v/v), 10 min run time, 2.0 mL/min, 30 °C, and 50 μL per injection), the product was obtained as an off-white solid (11 mg, 35%). (Because of rotameric effects, broad signals are observed, and not all signals are observable.) 1H NMR (400 MHz, CDCl3) δ 0.92 (t, J = 7.4 Hz, 3H), 1.22 (m, 1H), 1.35−2.24 (m, 9H), 2.27 (m, 1H), 3.12−3.71 (m, 4H), 3.74 (s, 3H), 4.02 (m, 1H), 4.50 (m, 1H), 5.54−5.76 (m, 3H), 6.50 (s, 1H), 6.76 (d, J = 8.7 Hz, 2H), 7.12 (d, J = 8.7 Hz, 2H), 7.34−7.50 (m, 3H), 7.93−8.05 (m, 2H); 13C NMR (100 MHz, CDCl3) δ 11.4, 19.4, 22.9, 30.8, 32.2, 41.1, 46.3, 48.2, 55.2, 103.2, 112.9, 113.8, 126.3, 128.4, 128.6, 128.9, 129.8, 130.7, 134.7, 154.3, 158.9, 162.9, 167.6, 172.9; ESI-TOF-HRMS (m/z) calcd for C32H36N4O3S [M + H]+ 557.2586; found 557.2584. 6-[2-Bromo-4-(4-methoxybenzyl)-4H-pyrrolo[2,3-d]thiazole-5carbonyl]-N-propyl-6-azaspiro[2.5]octane-1-carboxamide (23g). Synthesized following general procedure A using carboxylic acid 8d (50 mg, 0.14 mmol), amine 15g (50 mg, 0.16 mmol), DIEA (47 μL, 0.27 mmol), DMAP (1.6 mg, 0.01 mmol), and EDCI (27 mg, 0.14 mmol). After column chromatography (SiO2, eluent 10−25% PE, 75− 90% EtOAc), the product was afforded in 55% yield (41 mg). White solid; mp 103.2−105.2 °C. (Because of rotameric effects, broad signals are observed, and not all signals are observable.) 1H NMR (400 MHz, CD3OD) δ 0.78 (m, 1H), 0.93 (t, J = 7.4 Hz, 3H), 1.00−1.80 (m, 8H), 3.05−3.21 (m, 2H), 3.31 (m, 1H), 3.40−3.86 (m, 6H), 5.47 (s, 2H), 6.55 (s, 1H), 6.83 (d, J = 8.7 Hz, 2H), 7.07 (d, J = 8.7 Hz, 2H); 13C NMR (100 MHz, CD3OD) δ 11.8, 18.1, 23.8, 27.6, 27.9, 29.4, 37.2, 42.5, 55.8, 103.6, 115.2, 116.4, 129.3, 130.2, 131.2, 136.8, 151.8, 161.0, 164.3, 173.3; ESI-TOF-HRMS (m/z) calcd for C25H29BrN4O3S [M + H]+ 547.1202; found 547.1200. 6-[4-(4-Methoxybenzyl)-2-phenyl-4H-pyrrolo[2,3-d]thiazole-5carbonyl]-N-propyl-6-azaspiro[2.5]octane-1-carboxamide (23h). Synthesized following general procedure B using carboxamide 23g (41 mg, 0.07 mmol), Pd(OAc)2 (1.7 mg, 0.007 mmol), PPh3 (5.9 mg, 0.02 mmol), PhB(OH)2 (18 mg, 0.15 mmol), and K2CO3 (31 mg, 0.22 mmol). After semipreparative reversed-phase HPLC (conditions, MeOH in H2O (85% v/v), 11 min run time, 2.0 mL/min, 30 °C, and 50 μL per injection), the product was obtained as a yellow solid (15 mg, 36%). Mp 112.5−114.5 °C; 1H NMR (400 MHz, CDCl3) δ 0.75 (m, 1H), 0.94 (t, J = 7.4 Hz, 3H), 0.98−1.36 (m, 4H), 1.54 (sext, J = 7.3 Hz, 2H), 1.77−2.02 (m, 2H), 3.13−3.32 (m, 2H), 3.37−3.69 (m, 4H), 3.76 (s, 3H), 5.61 (d, J = 14.7 Hz, 1H), 5.67 (d, J = 14.7 Hz, 1H), 5.80 (m, 1H), 6.44 (s, 1H), 6.78 (d, J = 8.7 Hz, 2H), 7.22 (d, J = 8.7 Hz, 2H), 7.36−7.52 (m, 3H), 7.95−8.07 (m, 2H); 13C NMR (100 MHz, CDCl3) δ 11.4, 17.9, 23.1, 26.7, 27.4, 28.3, 36.4, 41.5, 48.1, 55.3, 102.4, 112.8, 113.9, 126.3, 128.1, 128.9, 129.6, 129.8, 130.8, 134.7, 154.1, 159.0, 162.9, 167.5, 170.3; ESI-TOF-HRMS (m/z) calcd for C31H34N4O3S [M + H]+ 543.2430; found 543.2426. Biology. Cells and Virus. HEK 293T cells and BJ (ATCC CRL2522) human fibroblast cells were cultured in Dulbecco’s modified Eagle medium (DMEM; HyClone), supplemented with 10% fetal bovine serum (FBS; HyClone). All cell lines were maintained at 37 °C with 5% CO2. Full-length CHIKV (La Réunion isolate) infectious clone tagged with Gaussia luciferase (Gluc) gene (CHIKV-IMT-Gluc) was produced from pSP6-ICRES1-2SG plasmid using similar standard molecular biology techniques as previously described by Tsetsarkin et al.39 CHIKV infectious clones tagged with Gluc from Gaussia princeps were produced from Singapore isolate (SGP11)40 and the Caribbean isolate (CNR20235)41 using a similar technique for producing CHIKV-IMT-Gluc. WT CHIKV-IMT used in this study were originally isolated from Réunion Island during the 2006 CHIKF

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b00180. Detailed synthetic procedures and characterization data of the intermediate compounds and NMR spectra of the final compounds (PDF) Molecular formula strings (CSV)

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AUTHOR INFORMATION

Corresponding Author

*Phone: +65-66011061. E-mail: [email protected]. ORCID

Christina L. L. Chai: 0000-0002-9199-851X Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank the Agency for Science Technology and Research (A*STAR, Grant JCO 1231BFG046), Ministry of Education, Singapore, and National University of Singapore (Grant R-148-000-146-133) for financial support of this project. The authors also thank A. Merits (Institute of Technology, University of Tartu) for providing the infectious 3184



Article

acute infection and chloroquine treatment. Vector-Borne Zoonotic Dis. 2008, 8, 837−840. (18) Delogu, I.; Pastorino, B.; Baronti, C.; Nougairede, A.; Bonnet, E.; de Lamballerie, X. In vitro antiviral activity of arbidol against Chikungunya virus and characteristics of a selected resistant mutant. Antiviral Res. 2011, 90, 99−107. (19) Ahola, T.; Couderc, T.; Ng, L. F. P.; Hallengard, D.; Powers, A.; Lecuit, M.; Esteban, M.; Merits, A.; Roques, P.; Liljestrom, P. Therapeutics and vaccines against Chikungunya virus. Vector-Borne Zoonotic Dis. 2015, 15, 250−257. (20) Kaur, P.; Chu, J. J. H. Chikungunya virus: an update on antiviral development and challenges. Drug Discovery Today 2013, 18, 969− 983. (21) Rashad, A. A.; Mahalingam, S.; Keller, P. A. Chikungunya virus: emerging targets and new opportunities for medicinal chemistry. J. Med. Chem. 2014, 57, 1147−1166. (22) Abdelnabi, R.; Neyts, J.; Delang, L. Antiviral strategies against Chikungunya virus. Methods Mol. Biol. 2016, 1426, 243−253. (23) Pohjala, L.; Utt, A.; Varjak, M.; Lulla, A.; Merits, A.; Ahola, T.; Tammela, P. Inhibitors of alphavirus entry and replication identified with a stable Chikungunya replicon cell line and virus-based assays. PLoS One 2011, 6, e28923. (24) Kaur, P.; Thiruchelvan, M.; Lee, R. C. H.; Chen, H. X.; Chen, K. C.; Ng, M. L.; Chu, J. J. H. Inhibition of Chikungunya virus replication by harringtonine, a novel antiviral that suppresses viral protein expression. Antimicrob. Agents Chemother. 2013, 57, 155−167. (25) Cruz, D. J. M.; Bonotto, R. M.; Gomes, R. G. B.; da Silva, C. T.; Taniguchi, J. B.; No, J. H.; Lombardot, B.; Schwartz, O.; Hansen, M. A. E.; Freitas, L. H. Identification of novel compounds inhibiting Chikungunya virus-induced cell death by high throughput screening of a kinase inhibitor library. PLoS Neglected Trop. Dis. 2013, 7, e2471. (26) Gigante, A.; Canela, M. D.; Delang, L.; Priego, E. M.; Camarasa, M. J.; Querat, G.; Neyts, J.; Leyssen, P.; Perez-Perez, M. J. Identification of [1,2,3]triazolo[4,5-d]pyrimidin-7(6H)-ones as novel inhibitors of Chikungunya virus replication. J. Med. Chem. 2014, 57, 4000−4008. (27) Hwu, J. R.; Kapoor, M.; Tsay, S. C.; Lin, C. C.; Hwang, K. C.; Horng, J. C.; Chen, I. C.; Shieh, F. K.; Leyssen, P.; Neyts, J. Benzouracil-coumarin-arene conjugates as inhibiting agents for Chikungunya virus. Antiviral Res. 2015, 118, 103−109. (28) Bourjot, M.; Delang, L.; Nguyen, V. H.; Neyts, J.; Gueritte, F.; Leyssen, P.; Litaudon, M. Prostratin and 12-O-tetradecanoylphorbol 13-acetate are potent and selective inhibitors of Chikungunya virus replication. J. Nat. Prod. 2012, 75, 2183−2187. (29) Varghese, F. S.; Thaa, B.; Amrun, S. N.; Simarmata, D.; Rausalu, K.; Nyman, T. A.; Merits, A.; McInerney, G. M.; Ng, L. F.; Ahola, T. The antiviral alkaloid berberine reduces Chikungunya virus-induced mitogen-activated protein kinase signaling. J. Virol. 2016, 90, 9743− 9757. (30) Nothias-Scaglia, L. F.; Pannecouque, C.; Renucci, F.; Delang, L.; Neyts, J.; Roussi, F.; Costa, J.; Leyssen, P.; Litaudon, M.; Paolini, J. Antiviral activity of diterpene esters on Chikungunya virus and HIV replication. J. Nat. Prod. 2015, 78, 1277−1283. (31) Ching, K. C.; Kam, Y. W.; Merits, A.; Ng, L. F.; Chai, C. L. Trisubstituted thieno[3,2-b]pyrrole 5-carboxamides as potent inhibitors of alphaviruses. J. Med. Chem. 2015, 58, 9196−9213. (32) Sindac, J. A.; Yestrepsky, B. D.; Barraza, S. J.; Bolduc, K. L.; Blakely, P. K.; Keep, R. F.; Irani, D. N.; Miller, D. J.; Larsen, S. D. Novel inhibitors of neurotropic alphavirus replication that improve host survival in a mouse model of acute viral encephalitis. J. Med. Chem. 2012, 55, 3535−3545. (33) Sindac, J. A.; Barraza, S. J.; Dobry, C. J.; Xiang, J.; Blakely, P. K.; Irani, D. N.; Keep, R. F.; Miller, D. J.; Larsen, S. D. Optimization of novel indole-2-carboxamide inhibitors of neurotropic alphavirus replication. J. Med. Chem. 2013, 56, 9222−9241. (34) Venable, J. D.; Cai, H.; Chai, W.; Dvorak, C. A.; Grice, C. A.; Jablonowski, J. A.; Shah, C. R.; Kwok, A. K.; Ly, K. S.; Pio, B.; Wei, J.; Desai, P. J.; Jiang, W.; Nguyen, S.; Ling, P.; Wilson, S. J.; Dunford, P. J.; Thurmond, R. L.; Lovenberg, T. W.; Karlsson, L.; Carruthers, N. I.;

clones from various CHIKV isolates and M. L. Ng (Department of Microbiology, National University of Singapore) and H. Tolou (Groupe d’Etude en Préventologie, Villenave d’Ornon, France) for providing the SINV and ONNV isolates.

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ABBREVIATIONS USED DMSO, dim ethyl sulfoxide; EDCI, 1-ethyl-3-(3dimethylaminopropyl)carbodiimide; DIEA, N,N-diisopropylethylamine; DMAP, 4-dimethylaminopyridine; HOBt, hydroxybenzotriazole; ACN, acetonitrile; THF, tetrahydrofuran; DMF, dimethylformamide; DCM, dichloromethane; PE, petroleum ether; TBAF, tetrabutylammonium fluoride; TFA, trifluoroacetic acid; CDI, carbonyldiimidazole; Gluc, Gaussia luciferase; Boc, tert-butoxycarbonyl; SEM, 2-(trimethylsilyl)ethoxymethyl; FITC, fluorescein isothiocyanate; DAPI, 4′,6diamidino-2-phenylindole

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REFERENCES

(1) Her, Z.; Kam, Y. W.; Lin, R. T. P.; Ng, L. F. P. Chikungunya: a bending reality. Microbes Infect. 2009, 11, 1165−1176. (2) Couderc, T.; Lecuit, M. Chikungunya virus pathogenesis: from bedside to bench. Antiviral Res. 2015, 121, 120−131. (3) Sam, I. C.; Kummerer, B. M.; Chan, Y. F.; Roques, P.; Drosten, C.; AbuBakar, S. Updates on Chikungunya epidemiology, clinical disease, and diagnostics. Vector-Borne Zoonotic Dis. 2015, 15, 223−230. (4) Schwartz, O.; Albert, M. L. Biology and pathogenesis of Chikungunya virus. Nat. Rev. Microbiol. 2010, 8, 491−500. (5) Pulmanausahakul, R.; Roytrakul, S.; Auewarakul, P.; Smith, D. R. Chikungunya in Southeast Asia: understanding the emergence and finding solutions. Int. J. Infect. Dis. 2011, 15, e671−676. (6) Parola, P.; de Lamballerie, X.; Jourdan, J.; Rovery, C.; Vaillant, V.; Minodier, P.; Brouqui, P.; Flahault, A.; Raoult, D.; Charrel, R. N. Novel Chikungunya virus variant in travelers returning from Indian Ocean islands. Emerging Infect. Dis. 2006, 12, 1493−1499. (7) Weaver, S. C.; Lecuit, M. Chikungunya virus and the global spread of a mosquito-borne disease. N. Engl. J. Med. 2015, 372, 1231− 1239. (8) Staples, J. E.; Fischer, M. Chikungunya virus in the Americas what a vectorborne pathogen can do. N. Engl. J. Med. 2014, 371, 887− 889. (9) Morrison, T. E. Re-emergence of Chikungunya virus. J. Virol. 2014, 88, 11644−11647. (10) Rougeron, V.; Sam, I. C.; Caron, M.; Nkoghe, D.; Leroy, E.; Roques, P. Chikungunya, a paradigm of neglected tropical disease that emerged to be a new health global risk. J. Clin. Virol. 2015, 64, 144− 152. (11) Higgs, S. Chikungunya virus: a major emerging threat. VectorBorne Zoonotic Dis. 2014, 14, 535−536. (12) Thiberville, S. D.; Moyen, N.; Dupuis-Maguiraga, L.; Nougairede, A.; Gould, E. A.; Roques, P.; de Lamballerie, X. Chikungunya fever: epidemiology, clinical syndrome, pathogenesis and therapy. Antiviral Res. 2013, 99, 345−370. (13) Briolant, S.; Garin, D.; Scaramozzino, N.; Jouan, A.; Crance, J. M. In vitro inhibition of Chikungunya and Semliki Forest viruses replication by antiviral compounds: synergistic effect of interferonalpha and ribavirin combination. Antiviral Res. 2004, 61, 111−117. (14) Rada, B.; Dragun, M. Antiviral action and selectivity of 6azauridine. Ann. N. Y. Acad. Sci. 1977, 284, 410−417. (15) Khan, M.; Dhanwani, R.; Patro, I. K.; Rao, P. V. L; Parida, M. M. Cellular IMPDH enzyme activity is a potential target for the inhibition of Chikungunya virus replication and virus induced apoptosis in cultured mammalian cells. Antiviral Res. 2011, 89, 1−8. (16) De Lamballerie, X.; Ninove, L.; Charrel, R. N. Antiviral treatment of Chikungunya virus infection. Infect. Disord.: Drug Targets 2009, 9, 101−104. (17) De Lamballerie, X.; Boisson, V.; Reynier, J. C.; Enault, S.; Charrel, R. N.; Flahault, A.; Roques, P.; Le Grand, R. On Chikungunya 3185



Article

Edwards, J. P. Preparation and biological evaluation of indole, benzimidazole, and thienopyrrole piperazine carboxamides: potent human histamine H4 antagonists. J. Med. Chem. 2005, 48, 8289−8298. (35) Gising, J.; Belfrage, A. K.; Alogheli, H.; Ehrenberg, A.; Akerblom, E.; Svensson, R.; Artursson, P.; Karlen, A.; Danielson, U. H.; Larhed, M.; Sandstrom, A. Achiral pyrazinone-based inhibitors of the Hepatitis C virus NS3 protease and drug-resistant variants with elongated substituents directed toward the S2 pocket. J. Med. Chem. 2014, 57, 1790−1801. (36) Houston, J. B. Utility of in vitro drug metabolism data in predicting in vivo metabolic clearance. Biochem. Pharmacol. 1994, 47, 1469−1479. (37) Solignat, M.; Gay, B.; Higgs, S.; Briant, L.; Devaux, C. Replication cycle of Chikungunya: a re-emerging arbovirus. Virology 2009, 393, 183−197. (38) Couderc, T.; Chretien, F.; Schilte, C.; Disson, O.; Brigitte, M.; Guivel-Benhassine, F.; Touret, Y.; Barau, G.; Cayet, N.; Schuffenecker, I.; Despres, P.; Arenzana-Seisdedos, F.; Michault, A.; Albert, M. L.; Lecuit, M. A mouse model for Chikungunya: young age and inefficient type-I interferon signaling are risk factors for severe disease. PLoS Pathog. 2008, 4, e29. (39) Tsetsarkin, K.; Higgs, S.; McGee, C. E.; De Lamballerie, X.; Charrel, R. N.; Vanlandingham, D. L. Infectious clones of Chikungunya virus (La Réunion isolate) for vector competence studies. Vector-Borne Zoonotic Dis. 2006, 6, 325−337. (40) Her, Z. S.; Malleret, B.; Chan, M.; Ong, E. K. S.; Wong, S. C.; Kwek, D. J. C.; Tolou, H.; Lin, R. T. P.; Tambyah, P. A.; Renia, L.; Ng, L. F. P. Active infection of human blood monocytes by Chikungunya virus triggers an innate immune response. J. Immunol. 2010, 184, 5903−5913. (41) Teo, T. H.; Her, Z.; Tan, J. J.; Lum, F. M.; Lee, W. W.; Chan, Y. H.; Ong, R. Y.; Kam, Y. W.; Leparc-Goffart, I.; Gallian, P.; Renia, L.; de Lamballerie, X.; Ng, L. F. Caribbean and La Réunion Chikungunya virus isolates differ in their capacity to induce proinflammatory Th1 and NK cell responses and acute joint pathology. J. Virol. 2015, 89, 7955−7969. (42) Bessaud, M.; Peyrefitte, C. N.; Pastorino, B. A. M.; Tock, F.; Merle, O.; Colpart, J. J.; Dehecq, J. S.; Girod, R.; Jaffar-Bandjee, M. C.; Glass, P. J.; Parker, M.; Tolou, H. J.; Grandadam, M. Chikungunya virus strains, Reunion Island outbreak. Emerging Infect. Dis. 2006, 12, 1604−1606. (43) Bessaud, M.; Peyrefitte, C. N.; Pastorino, B. A. M.; Gravier, P.; Tock, F.; Boete, F.; Tolou, H. J.; Grandadam, M. O’nyong-nyong virus, Chad. Emerging Infect. Dis. 2006, 12, 1248−1250. (44) Kam, Y. W.; Lee, W. W. L.; Simarmata, D.; Harjanto, S.; Teng, T. S.; Tolou, H.; Chow, A.; Lin, R. T. P.; Leo, Y. S.; Renia, L.; Ng, L. F. P. Longitudinal analysis of the human antibody response to Chikungunya virus infection: implications for serodiagnosis and vaccine development. J. Virol. 2012, 86, 13005−13015.

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Structural Optimizations of Thieno[3,2-b]pyrrole

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