A Novel Series of Highly Potent Small Molecule Inhibitors of

Article pubs.acs.org/jmc

A Novel Series of Highly Potent Small Molecule Inhibitors of Rhinovirus Replication Jinwoo Kim,† Yu Kyoung Jung,†,‡ Chonsaeng Kim,*,† Jin Soo Shin,† Els Scheers,# Joo-Youn Lee,§ Soo Bong Han,†,‡ Chong-Kyo Lee,† Johan Neyts,*,# Jae-Du Ha,† and Young-Sik Jung*,†,‡ †

Bio & Drug Discovery Division, Korea Research Institute of Chemical Technology, 141 Gajeongro, Yuseong, Daejeon 34114, Republic of Korea ‡ Department of Medicinal Chemistry and Pharmacology, University of Science and Technology, 217 Gajeongro, Yuseong, Daejeon 34113, Republic of Korea # Laboratory of Virology and Chemotherapy, Rega Institute for Medical Research, Department of Microbiology and Immunology, University of Leuven, B-3000 Leuven, Belgium § Korea Chemical Bank, Korea Research Institute of Chemical Technology, 141 Gajeongro, Yuseong, Daejeon 34114, Republic of Korea S Supporting Information *

ABSTRACT: Human rhinoviruses (hRVs) are the main causative pathogen for common colds and are associated with the exacerbation of asthma. The wide variety in hRV serotypes has complicated the development of rhinovirus replication inhibitors. In the current investigation, we developed a novel series of benzothiophene derivatives and their analogues (6−8) that potently inhibit the replication of both hRV-A and hRV-B strains. Compound 6g inhibited the replication of hRV-B14, A21, and A71, with respective EC50 values of 0.083, 0.078, and 0.015 μM. The results of a time-of-addition study against hRV-B14 and hRV-A16 and resistant mutation analysis on hRV-B14 implied that 6g acts at the early stage of the viral replication process, interacting with viral capsid protein. A molecular docking study suggested that 6g has a capsid-binding mode similar to that of pleconaril. Finally, derivatives of 6 also displayed significant inhibition against poliovirus 3 (PV3) replication, implying their potential inhibitory activities against other enterovirus species.

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INTRODUCTION Human rhinoviruses (hRVs) (genus Enterovirus, family Picornaviridae) are the major causative pathogen of upper respiratory illnesses, in particular, the common cold.1,2 hRVs have been commonly detected in children with respiratory symptoms, displaying varying seasonal distributions among extremely diverse species.3−5 Moreover, it has been demonstrated that hRV infection is associated with acute otitis media (AOM) development in infants.6−8 hRVs also cause several lower respiratory track symptoms and have been known to be responsible for the exacerbation of asthma, especially in infants, the elderly, and immunodeficient patients, accompanying airway obstruction and hyperresponsiveness.9−12 Also, it has been documented that hRV infection aggravates pneumonia or chronic obstructive pulmonary disease (COPD).13−15 More than 160 hRV strains with considerable genetic variations are known. They have been classified into hRV-A, hRV-B, and hRV-C subgroups.16 Their high diversity and © 2017 American Chemical Society

strain-specific interactions with antiserums have retarded the development of anti-hRV therapeutic agents.17,18 As is common in picornaviruses, hRVs contain a (+)-ssRNA strand in an icosahedral capsid composed of four different types of viral proteins (VP1−VP4).19 Viral RNA translation produces a single polyprotein, which is processed by 2A and 3C viral proteases to form the mature structural and nonstructural proteins required for the replication. The viral capsid has narrow ditches termed “canyons” around its 5-fold apexes. The majority of hRV-A and hRV-B species utilize intercellular adhesion molecule-1 (ICAM-1), a member of the immunoglobulin superfamily, as the host receptor.20−24 They employ the canyons for receptor binding, which provokes the capsid uncoating for cell entry.25 On the other hand, a minor group of hRVs utilizes sites close to the canyons for binding other Received: February 3, 2017 Published: June 5, 2017 5472

DOI: 10.1021/acs.jmedchem.7b00175 J. Med. Chem. 2017, 60, 5472−5492

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observed that ethyl 6-[{2-[(methylamino)carbonyl]-4pyridinyl}oxy]-benzo[b]thiophene-2-carboxylate (6a) displays antirhinoviral activity (Figure 2). In this preliminary study, 6a

receptors such as low-density lipoprotein receptor (LDL-R) and related proteins. The outer-canyon binding is known to trigger aggregation of other molecules for the capsid decomposition and host infection.26−28 Several hRV species contain a small molecule, presumably originating from the host cell, called the “pocket factor” in their canyons. The pocket factor stabilizes the viral capsid until proper receptor binding occurs.29 In spite of difficulties in dealing with the wide variety of subspecies, several compounds against rhinovirus replication have been developed. Capsid binding inhibitors such as pleconaril (WIN-63843) and vapendavir (BTA-798) bind strongly in the canyon and inhibit viral uncoating by stabilizing the viral capsid dynamics.30−34 On the other hand, enviroxime (LY122771-72) inhibit viral RNA replication by targeting viral 3A and related proteins.35 Rupintrivir (AG-7088) and 5 (AG7404) inhibit viral 3C protease, preventing viral polyprotein processing (Figure 1).36−38 However, to date, no drug has been approved for the treatment of rhinovirus infections.

Figure 2. Hit compound 6a from a HTS and its EC50 values against hRV-B14 and hRV-A21. EC50 values were measured by MTT assay in H1HeLa cell line. Shaded parts of 6a were modified in an optimization effort.

showed 50% effective concentrations (EC50 values) of 25 μM for hRV-B14 and 1.2 μM for hRV-A21. In an effort to optimize the antirhinoviral activity of this substance, we modified the 2carboxylic ester group, introduced substituents at the 3-position of the benzothiophene core, and modified the benzothiophene core structure and the N-methylpicolinamide moiety. Synthesis. Our initial target compounds for the optimization studies were derivatives of 6a. The synthetic routes for the variation on the benzo[b]thiophene substituents and ester moiety are described in Scheme 1 and Scheme 2. The 3-alkyl substituted ethyl benzo[b]thiophene-2-carboxylate core structures 15a−c were prepared from ethyl mercaptoacetate and corresponding 2-fluoro-4-methoxyaryl aldehydes or ketones through SNAr followed by the condensation. Demethylation of 6-methoxy group from 15a−c with BBr3 produced 16a−c. The phenolic hydroxyl groups in 16a−c were coupled with 4chloro-N-methylpicolinamide (10), which was obtained from the chlorination with SOCl2 and following amidation of picolinic acid,39 under neat conditions to generate 6a and its 3-alkyl derivatives 6b and 6c. Because direct transesterifications of 16a and 16b were not facile, the 2-carboxylic ester variants 16d−h were prepared from the corresponding carboxylic acids 17a and 17b, which were formed by hydrolysis of 16a and 16b with KOH. 16d−h were then coupled with 10 in neat condition to yield the ester derivatives 6d−h. Carboxylic acid derivative 6i was obtained from SNAr reaction between 17b and 10 in DMSO with Cs2CO3 as the base. The target isobutyl and benzyl esters 6j and 6k were obtained by treating 6i with 1ethyl-3-[3-(dimethylamino)-propyl]carbodiimide hydrochloride (EDCI·HCl), 4-dimethylaminopyridine (DMAP), and corresponding alcohols. The 2-carboxylamide derivatives 6l and 6m were obtained by amidation reactions of 6i with appropriate amines in the presence of EDCI·HCl and DIPEA. Synthetic routes for the preparation of other substituted benzo[b]thiophene analogues are depicted in Scheme 2. The 3amino derivative of the benzo[b]thiophene core 15n was prepared from 2-fluoro-4-methoxybenzonitrile and ethyl mercaptoacetate. Subsequent demethylation with BBr3, followed by coupling with 10 in neat condition, provided the 3amino derivative 6n. 6n was treated with tBuONO and HBF4 to form the diazonium intermediate, which was cyanated by CuCN and NaCN to yield the 3-cyano derivative 6o. The 3chloro derivative 17p, generated by following a reported procedure,40 was coupled with 10 using Cs2CO3 as the base and esterified to produce 6p. 7-Chloro analogue 6q was obtained in the same manner as for 6p using 17q, which was obtained by chlorination of 16a with N-chlorosuccinimide followed by the hydrolysis.

Figure 1. Representative known inhibitors 1−5 against hRVs and core structures of new 6-{[2-(methylcarbamoyl)pyridin-4-yl]oxy}benzo[b]thiophene-2-carboxylic ester and their derivatives 6−8.

Anti-hRV compounds with broad spectrum activities are required in order to overcome difficulties associated with the high mutation rate of hRV strains. In the study described below, we carried out high throughput screening (HTS) using a library containing 100,000 compounds to uncover novel small molecule inhibitors against hRV replication. This effort resulted in the identification of 6-{[2-(methylcarbamoyl)pyridin-4yl]oxy}benzo[b]thiophene-2-carboxylic ester and their derivatives 6−8 that have potent and selective antiviral activities against both hRV-A and hRV-B. The results of time-of-drugaddition studies revealed that these compounds act at the first, entry stage of the viral replication cycle.

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RESULTS AND DISCUSSION Hit Identification. Through HTS with phenotypic (cytopathogenic) assays on hRV-B14 in H1HeLa cells, we 5473



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Scheme 1. Variations on Ester Alkyl Moietya

Reagents and conditions: (a) ethyl mercaptoacetate, K2CO3, DMF, 25 °C, 16 h; (b) BBr3, CH2Cl2, −78 °C, 1 h then 25 °C, 23 h; (c) 10, neat, 150 °C, 48 h; (d) KOH, THF, H2O, 65 °C, 24 h; (e) H2SO4, R2OH, reflux, 20 h; (f) 10, Cs2CO3, DMSO, 120 °C, 15 h; (g) R2OH, EDCI·HCl, DIPEA, DMAP, CH2Cl2, 0 °C, 2 h then 25 °C, 30 h; (h) EtNH2, EDCI·HCl, DIPEA, H2O, CH2Cl2, 0 °C, 2 h then 25 °C, 20 h; (i) HNMe2·HCl, EDCI· HCl, DIPEA, CH2Cl2, 0 °C, 2 h then 25 °C, 20 h; (j) DMF, SOCl2, 45 °C then 75 °C, 72 h then CH3NH2, H2O, THF, 0 °C then 25 °C, 4 h.

a

Scheme 2. Variations on the Benzothiophene Substituenta

Reagents and conditions: (a) ethyl mercaptoacetate, K2CO3, DMF, 25 °C, 16 h; (b) BBr3, CH2Cl2, −78 °C, 1 h then 25 °C, 23 h; (c) 10, neat, 150 °C, 48 h; (d) tBuONO, HBF4, H2O, EtOH, 0 °C then 25 °C, 2 h then CuCN, NaCN, CH3CN, 25°C, 3 h; (e) N-chlorosuccinimide, THF, 30 °C, 6 h; (f) KOH, THF, H2O, 65 °C, 24 h; (g) 10, Cs2CO3, DMSO, 85 °C, 2 h then H2SO4, EtOH, reflux, 12 h.

a

Scheme 3. Variations on the Aryl Ether Substituenta

Reagents and conditions: (a) ArB(OH)2, Cu(OAc)2·H2O, pyridine, CH2Cl2, 25 °C, 12 h for 6r or ClCH2CH2Cl, 85 °C, 12 h for 6s−u; (b) 11, PhCl, 120 °C, 12 h; (c) KOH, THF, H2O, 65 °C, 12 h; (d) 13, Cs2CO3, DMSO, 100 °C, 15 h; (e) iPrOH, EDCI·HCl, DMAP, DMF, 0 °C then 25 °C, 15 h; (f) HOBt·H2O, EDCI·HCl, TEA, MeNH2, MeOH, CH2Cl2, 25 °C, 15 h. a

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Scheme 4. Variations on the Benzo[b]thiophene Core Structurea

a Reagents and conditions: (a) ethyl bromoacetate, K2CO3, DMF, 80 °C, 30 h; (b) KOH, THF, H2O, 65 °C, 10 h; (c) BBr3, CH2Cl2, −78 °C, 1 h then 10 °C, 11 h or −78 °C, 1 h then 25 °C, 23 h; (d) 10, Cs2CO3, DMSO, 120 °C, 10 h; (e) ethyl bromoacetate, K2CO3, acetone, 60 °C, 8 h; (f) DMFDMA, 90 °C, 48 h then AlCl3, CH2Cl2, 25 °C, 24 h; (g) NaH, MeI, DMF, 0 °C, 30 min then 25 C, 8 h; (h) H2SO4, EtOH, reflux, 24 h; (i) iPrOH, EDCI·HCl, DIPEA, DMAP, CH2Cl2, 0 °C, 10 min then 25 °C, 16 h; (j) isopropyl 2-isocyanoacetate, CuI, Cs2CO3, DMSO, 80 °C, 24 h; (k) 10, neat, 150 °C, 48 h.

Scheme 5. Synthesis of Naphthalene Derivativesa

Reagents and conditions: (a) 10, Cs2CO3, DMSO, 120 °C, 15 h; (b) ROH, H2SO4, reflux, 12 h; (c) PhOH, EDCI·HCl, DMAP, DMF, 0 °C, 2 h then 25 °C, 30 h; (d) 10, Cs2CO3, DMSO, 120 °C, 48 h then EtOH, H2SO4, reflux, 24 h. a

Scheme 3 describes synthetic routes for substances in which the N-methylpicolinamide moiety was varied. Phenyloxy, 4(methylcarbamoyl)phenoxy and 4-pyrydyloxy derivatives (6r− u) were obtained by Chen−Lam coupling of 16a or 16b with corresponding boronic acids in chlorinating solvents. Methyl picolinate analogue 6v was prepared by coupling 16b and methyl 4-chloropicolinate in chlorobenzene, albeit in low yield. The N-methyl-5-picolinamide derivative 6w was prepared by coupling 17b and N-methyl-5-fluoropicolinamide (13), which was prepared from the amidation of 5-fluoropicolinic acid with HOBt and EDCI·HCl. Subsequent esterification of 6w with EDCI and iPrOH provided the regioisomeric analogue 6x. Other analogues containing different heteroaromatic cores, such as benzofuran and indole, were prepared using the routes outlined in Scheme 4. The benzofuran and indole core

structure 19a and 19b were prepared from 2-hydroxy-4methoxybenzaldehyde and 3-methoxyaniline, respectively.41 19b was N-methylated with NaH and MeI to form Nmethylindole core 19c. 19a and 19c were demethylated with BBr3, hydrolyzed, and then coupled with 10 using Cs2CO3 as the base in DMSO solvent to obtain 22a and 22c, which were consequently esterified in corresponding alcohols to yield 7a and 7c, respectively. 3-Methylindole core structure with isopropyl ester (19d) was prepared from 2-chloro-4-methoxyacetophenone.42 Following demethylation and coupling with 10 in neat condition produced 7d, which was then selectively methylated with NaH and MeI to obtain 3-methyl-Nmethylindole derivative 7e. Substances containing naphthalene and quinoline cores were produced using the sequences shown in Scheme 5. 7-{[25475



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Substitution of 6-position with 4-pyridyloxy moiety (6u) quenched the activity significantly (entry 27). Increased hydrophobicity by varying the N-methylpicolinamide moiety into methyl picolinate (6v) also led to the loss of the activity, implying that the polar interaction through amide moiety is important (entry 28). When the N-methylpicolinamide group was linked with its 5-position, significant decrease in the effectiveness was observed (entry 29). Among other heteroarene derivatives tested, the benzofuran variant 7a was found not to be active against hRV-B14 and to have only moderate activities against hRV-A21 and hRV-A71 (entry 30). The N-methylindole counterpart 7c showed remarkable activities (EC50 = 0.82, 0.064, and 0.0071 μM for hRV-B14, hRV-A21, and hRV-A71, respectively), but it suffered from high cytotoxicity (CC50 = 10.3 μM) (entry 31). In contrast to observations made in studies of the benzothiophene candidates against hRV species, the activities were not improved with remaining cytotoxicity when a 3-methyl group was added to indole and N-methylindole core structure (entries 32 and 33). The activities of naphthalene derivatives 8a−h against the hRV strains were also investigated (Table 2). The activities of 2-naphthonate ester variants also seemed to be highly dependent on the steric bulkiness of the ester moiety. Among 2-carboxylic ester derivatives tested, ethyl and isopropyl ester (8b and 8c) showed noticeable activities against hRV-A species tested (8b, EC50 = 0.27 and 0.048 μM; 8c, EC50 = 0.48 and 0.019 μM against hRV-A21 and hRV-A71, respectively), while shorter or longer alkyl chain derivatives displayed only marginal effectiveness against hRV-B14. As the ester alkyl chain length became longer, increasing cytotoxicity tended to eclipse the effectiveness, as in the case of the benzothiophene derivatives (entries 3−6). The phenyl ester derivative 8e was found to be inactive against hRV-B14 and hRV-A21 (entry 7). The addition of 3-hydroxyl group on 8b suppressed the activity against hRVB14 (entry 8). Interestingly, 3-carboxylic ester regioisomer 8g showed slightly better activities against hRV strains tested while displaying lower activity against poliovirus 3 (vide infra) (entry 9). Hydrophilic quinoline analogue 8h was slightly effective against hRV-A71 and showed little activities against hRV-B14 and hRV-A21 (entry 10). In addition, we explored the antipoliovirus (sabin strain PV3) activity of the compounds obtained in this study, using pocapavir (V-073) as the reference compound (Tables 3 and 4).44−47 In general, substances with high antirhinoviral activities also tended to show remarkable activity against PV3, while 3methyl group on the bicyclic core seemed to be significant. Among alkyl ester variants of the benzothiophene derivative tested, ethyl and isopropyl ester variants having 3-methyl moiety on the benzothiophene core (6f and 6g) displayed significant activity against PV3, displaying EC50 values of 0.087 and 0.063 μM, respectively (Table 3, entries 8 and 9). Corresponding 2-carboxylic acid and 2-carboxamide derivatives were inactive against PV3 (entries 13−15). While the 3-amino derivative 6n had high cytotoxicity in the HeLa cell line, compounds with electron withdrawing substituents 6o−q yielded no or little activities against PV3 up to the cytotoxic concentrations (entries 16−19). Although the 3-methylbenzothiophene core structure itself showed slight effectiveness, substitution of N-methylpicolinamide with another arene moiety removed the activity, with the exception of Nmethylbenzamide candidate 6t, which suffered from the high cytotoxicity (entries 20−29). Benzofuran derivative 7a was still

(Methylcarbamoyl)pyridin-4-yl]oxy}-2-naphthoic acid intermediate 28a was prepared by coupling 10 to 7-hydroxy-2naphtnoic acid 27a using Cs2CO3 as the base in DMSO solvent. The ester variants 8a−e were obtained by esterifying 28a, either with the aid of EDCI·HCl and DMAP or by refluxing it in the corresponding alcohol with catalytic H2SO4. 3-Hydroxy-2-naphthoic ester (8g), 3-naphthoic ester (8f), and quinoline-2-carboxylic ester (8h) variants were also prepared from corresponding carboxylic acids 27f−h.43 SAR Analysis. The antiviral activities of the benzothiophene and other heteroaromatic derivatives against hRV-B14, hRVA21, and hRV-A71 were assessed (Table 1) using pleconaril (1) as the reference compound. The general trends observed in this effort showed that the activities highly depend on the steric bulkiness of 3-substituent and 2-carboxylic ester on the benzo[b]thiophene or other heterocycle core, as well as the electronic nature of the heteroaromatic systems. The activities against three tested hRV species followed similar trends to each other, while hRV-B14 showed higher resistance against examined compounds. The hit compound 6a (entry 3) displayed notable activities against hRV-A21 and hRV-A71 (EC50 = 1.3 and 0.019 μM, respectively) but showed only marginal effectiveness toward hRV-B14 (EC50 = 58.4 μM) in the average of several tests. Notably, while the presence of a methyl group on the 3-position of benzo[b]thiophene core led to remarkable improvement in activities against hRV-B14, the extension of one more carbon displayed only marginal improvement, implying the key interaction between the 3position of heterocyclic system and the target protein (entries 4 and 5). While the isopropyl ester derivative from 6a (6e) also showed improvement in the activity against hRV-B14, the shorter methyl ester variant lost its effectiveness against hRVB14 and had only marginal activities against hRV-A21 and A71 (entries 6 and 7). When the ester moiety was varied from 3methylbenzo[b]thiophene analogue 6b, isopropyl ester derivative 6g seemed to have optimal steric bulkiness because linear 3-carbon or longer chain analogues showed significantly reduced activity compared to 6g (entries 8−12). Indeed, 6g displayed significant activities against the three tested hRV species (EC50 = 0.083, 0.078, and 0.015 μM for hRV-B14, hRVA21, and hRV-A71, respectively). 6g also displayed remarkable selectivity indices (SI > 200), comparable to those of pleconaril (1). The corresponding carboxylic acid derivative 6i was found to be ineffective (entry 13). Hydrophilic 2-carboxylamide analogues 6l and 6m did not show activity against all the hRVs tested up to the cytotoxic concentrations (entries 14 and 15). The activities of derivatives with other substituents on the benzo[b]thiophene core were also investigated. The 3-amine derivative 6n showed pronounced cytotoxicity and presented inactivity up to the CC50 (entry 16). The 3-cyano, 3-chloro, and 7-chloro derivatives 6o−q showed only moderate activity, suggesting the ineffectiveness of electron withdrawing substituents on the benzo[b]thiophene core (entries 17−19). The existence and direction of attachment of the Nmethylpicolinamide moiety at the 6-position of the benzo[b]thiophene core seemed to be important in governing antirhinovirus activity. When the (N-methylpicolinamid-4yl)oxy substituent on the 6-position of benzo[b]thiophene was replaced by hydroxy or methoxy moiety, the activities against tested hRVs were lost (entries 20−23). Phenyl variants 6r and 6s showed only marginal activities (entries 24 and 25). The pronounced toxicity of the N-methylbenzamide counterpart 6t eclipsed its effectiveness against hRV species (entry 26). 5476


compd

1 29 6a 6b 6c 6d 6e 6f 6g 6h 6 6k 6i 6l 6m 6n 6o 6p 6q 16a 15a 16b 15b 6r 6s 6t 6u 6v 6x 7a 7c 7d 7e

entry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33

pleconaril pocapavir H 3-Me 3-Et H H 3-Me 3-Me 3-Me 3-Me 3-Me 3-Me 3-Me 3-Me 3-NH2 3-CN 3-Cl 7-Cl H H 3-Me 3-Me H 3-Me 3-Me H 3-Me 3-Me H H 3-Me 3-Me

R1

4-PicNHMe 4-PicNHMe 4-PicNHMe 4-PicNHMe 4-PicNHMe 4-PicNHMe 4-PicNHMe 4-PicNHMe 4-PicNHMe 4-PicNHMe 4-PicNHMe 4-PicNHMe 4-PicNHMe 4-PicNHMe 4-PicNHMe 4-PicNHMe 4-PicNHMe H Me H Me Ph Ph 3-C6H4CONHMe 4-pyridinyl 4-PicOMe 5-PicNHMe 4-PicNHMe 4-PicNHMe 4-PicNHMe 4-PicNHMe

R2

S S S S S S S S S S S S S S S S S S S S S S S S S S S O NMe NH NMe

X

OEt OEt OEt OMe OiPr OMe OiPr OnPr OiBu OBn OH NHEt NMe2 OEt OEt OEt OEt OEt OEt OEt OEt OEt OEt OEt OEt OEt OiPr OEt OiPr OiPr OiPr

Y

hRV14 0.092 ± 0.014 >100 58.4 ± 3.9 1.7 ± 0.058 13.5 ± 2.1 >100 1.7 ± 0.23 2.0 ± 0.26 0.083 ± 0.029 2.9 ± 1.1 10.1 ± 0.4 >18.8 >100 >100 >42.5 >0.11 82.5 ± 7.4 11.7 ± 1.3 12.4 ± 1.3 >52.3 >70.9 >82.0 >100 68.6 ± 7.0 >100 3.4 ± 0.4 >42.4 >24.9 >7.6 >100 0.82 ± 0.28 >14.6 1.9 ± 0.2

H1HeLa CC50b(μM) 19.7 ± 4.3 >100 >100 50.9 ± 2.5 35.2 ± 10.7 >100 45.7 ± 8.1 10.8 ± 0.9 18.1 ± 0.64 37.4 ± 0.7 36.6 ± 0.1 18.8 ± 1.6 >100 >100 42.5 ± 4.8 0.11 ± 0.01 >100 46.9 ± 1.0 >100 52.3 ± 14.8 70.9 ± 14.2 82.0 ± 5.2 >100 >100 >100 5.1 ± 1.3 42.4 ± 5.6 24.9 ± 11.0 7.6 ± 1.3 >100 10.3 ± 2.1 14.6 ± 2.8 11.2 ± 2.6

Table 1. Inhibitory Effects of Benzo[b]thiophene and Heterocycle Derivatives against hRVsa

hRV21 0.073 ± 0.0006 2.0 ± 0.4 1.3 ± 0.1 0.29 ± 0.038 0.21 ± 0.064 75.6 ± 24.1 1.5 ± 0.1 0.25 ± 0.03 0.078 ± 0.003 0.33 ± 0.02 1.5 ± 0.1 0.55 ± 0.04 >100 >100 >42.5 >0.11 20.7 ± 2.7 1.7 ± 0.1 7.7 ± 0.7 >52.3 >70.9 >82.0 >100 51.1 ± 12.2 1.0 ± 0.0 0.085 ± 0.022 >42.4 >24.9 3.3 ± 0.1 66.0 ± 4.3 0.064 ± 0.012 2.5 ± 1.0 0.13 ± 0.03

hRV71 0.0094 ± 0.0046 0.25 ± 0.06 0.019 ± 0.009 0.34 ± 0.10 1.3 ± 0.1 4.6 ± 0.8 0.013 ± 0.000 1.6 ± 0.6 0.015 ± 0.001 0.064 ± 0.001 0.38 ± 0.00 1.4 ± 0.1 >100 >100 >42.5 >0.11 0.54 ± 0.06 2.7 ± 2.1 0.20 ± 0.01 8.9 ± 0.2 5.1 ± 1.1 >82.0 3.8 ± 1.5 2.8 ± 1.3 1.7 ± 0.1 0.15 ± 0.05 7.3 ± 0.4 15.0 ± 1.5 1.7 ± 0.4 1.4 ± 0.2 0.0071 ± 0.0068 1.9 ± 0.8 0.060 ± 0.005

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5.9

13

2.3 >1.5 160 5.8 86

>2.0 >100 60

>1.5 1.5

>4.8 28 >13

270 >50 >77 180 170 >1.3 31 43 230 110 24 34

hRV21

>1.2 4.0 >8.1

27 5.4 220 13 3.6

>1.7 30 2.6

e

210

hRV14

SId

>26 >36 >59 34 5.8 1.7 4.5 >71 1500 7.7 190

>190 17 >500 5.9 14

2100 >400 >5300 150 27 >22 3500 6.8 1200 580 96 13

hRV71

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inactive against PV3 (entry 30). 3-Methylindole derivatives 7d and 7e showed better activity against PV3 compared to its 3-H counterpart 7c (entries 31−33). Interestingly, naphthalene derivatives 8 generally displayed significant activity against PV3, showing similar trends with their antirhinovirus activities along the bulkiness of the ester moiety (Table 4). Among 2-naphthoate ester derivatives, 8b had outstanding activity against PV3 (EC50 = 0.0095 μM) with high selectivity index (SI = 1400). The addition of a 3-hydroxyl group decreased the effectiveness against PV3 (entry 8). Compared to 8b, the 3-carboxylic ester regioisomer 8g failed to retain high activity against PV3 (entry 9). The quinoline core structure variant 8h also showed marginal activity (entry 10). The overall results of the SAR analysis of compounds are summarized in Scheme 6. Mode of Action. A time-of-drug-addition study against hRV-B14 was performed in order to determine which step in the replication cycle is targeted by 6g. In this investigation, pleconaril (1) and rupintrivir (4) were used as representative capsid-binding (early stage) and 3C protease (late stage) inhibitors, respectively. The substances, in concentrations that are 6 times their EC50 values, were added at various time points during hRV-B14 infection process (Figure 3). The inhibitory effects of the compounds on hRV-B14 were determined by quantifying the number of infected cells using immunofluorescence staining of viral dsRNA with the anti-dsRNA antibody. As expected, pleconaril exhibited strong antiviral activity when added at early time points (−1 and 0 h) and its inhibitory effect was largely lost when it was added after the onset of infection. In contrast, rupintrivir retained strong activity until the +6 time point after the infection. Compound 6g displayed an activity profile that is similar to that of pleconaril, suggesting that it acts at the early stage of the viral infection. To investigate whether 6g confers similar effects against hRV-A species, a similar time-of-drug-addition experiment was performed using hRV-A16 because high titer virus was required for these assays (Figure 3B). First, antiviral activities of 6g, pleconaril, and rupintrivir against hRV-A16 were measured and EC50 values (average ± standard deviation) for each compound were found to be 0.23 ± 0.025, 0.35 ± 0.01, and 0.03 ± 0.001 μM, respectively. The results of the time-of-drug-addition experiment based on these EC50 values revealed that compound 6g displays a similar inhibition pattern against hRV-A16 with that of hRV-B14, suggesting that this compound acts at the early stage of both hRV-A and hRV-B infection. Resistant-Mutation Study. Drug-resistant hRV-B14 was isolated to investigate the molecular target of 6g. In the presence of 6g at concentrations exceeding the EC50 value, three separate lineages that replicated efficiently were obtained in parallel. After eight passages, RNAs from these viruses were purified and sequenced together with the RNA from original hRV-B14 to find resistant mutations. Interestingly, three 6gresistant viruses carried the same mutation in the viral capsid protein VP3-L25M (Figure 4) not in VP1, where most capsidbinding inhibitors bind.32,48−51 Together with the data from time-of-addition studies, this result suggests that 6g interacts with viral capsid and inhibits the entry of hRV-B14. Computational Study. To understand the nature of interactions between 6g and the viral capsid, a molecular modeling study was performed based on the X-ray co-crystal structure of hRV-B14 with capsid-binding pleconaril ligand (PDB 1NCQ) using Schrödinger Suite 2016-1 software (see the Experimental Section).51 The hRV-B14 protomer consists

a All data were obtained from at least three independent experiments and mean values ± standard deviations are listed. bCC50: cytotoxic concentration (μM) for 50% of cell death, measured by MTT assay in H1HeLa cell line. cEC50: effective concentration (μM) concentrations for 50% inhibition of each hRV species, measured by MTT assay in H1HeLa cell line. dSI: selectivity index, calculated by CC50/ EC50. eNot calculated because EC50 was higher than CC50.

Table 1. continued


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Table 2. Inhibitory Effects of Naphthalene Derivatives against hRVsa

EC50 (μM)c entry compd 1 2 3 4 5 6 7 8 9 10

1 23 8a 8b 8c 8d 8e 8f 8g 8h

R pleconaril pocapavir H H H H H OH CO2Et H

X

CO2Me CO2Et CO2(i-Pr) CO2(s-Bu) CO2C6H5 CO2Et H CO2Et

Y

H1HeLa CC50 (μM)

CH CH CH CH CH CH CH N

19.7 ± 4.3 >100 53.5 ± 9.6 20.8 ± 4.8 13.8 ± 3.6 16.0 ± 0.57 >100 >100 35.3 ± 0.8 >100

SId

b

hRV14

hRV21

0.092 ± 0.014 >100 11.0 ± 1.7 2.1 ± 0.5 1.8 ± 0.2 >16.0 >100 >100 2.1 ± 0.2 56.6 ± 4.0

0.073 ± 0.0006 2.0 ± 0.4 2.0 ± 0.1 0.27 ± 0.02 0.48 ± 0.00 0.41 ± 0.03 >100 0.41 ± 0.03 0.091 ± 0.016 44.3 ± 3.3

hRV71

hRV14

hRV21

hRV71

± ± ± ± ± ± ± ± ± ±

210 e 4.9 9.9 7.7

270 >50 27 77 29 39

2100 >400 33 430 730 1200 >12 >340 10000 >77

0.0094 0.25 1.6 0.048 0.019 0.013 8.27 0.29 0.0035 1.3

0.0046 0.06 0.1 0.011 0.000 0.000 4.73 0.01 0.0005 0.1

17 >1.8

>240 390 >2.3

All data were obtained from at least three independent experiments and mean values ± standard deviations are listed. bCC50: cytotoxic concentration (μM) for 50% of cell death, measured by MTT assay in H1HeLa cell line. cEC50: effective concentration (μM) concentrations for 50% inhibition of each hRV species, measured by MTT assay in H1HeLa cell line. dSI: selectivity index, calculated by CC50/EC50. eNot calculated because EC50 was higher than CC50. a

■

DISCUSSION On the basis of the time-of-drug-addition studies, mutation analysis, in silico experiments, and the observation of partial resistance in cross-mutational study, 6 was assumed to bind in the viral capsid in VP1 where it inhibits viral uncoating. The canyon in VP1 is composed of two compartments including a hydrophobic pocket composed of aliphatic residues and the other end having Asn219 anchor. The benzothiophene core of 6 seems to have significant hydrophobic interaction with the aliphatic residues in the hydrophobic pocket, as supported by SAR results in which polar variations displayed drastic reduction in the activity. On the other hand, the carbonyl oxygen of picolinamide scaffold of 6 is assumed to interact with Asn219 via hydrogen bonding with a water molecule. Although it has been reported that the effect of the hydrogen bonding network with Asn219 is less significant than the van der Waals interaction in the hydrophobic region for WIN compounds,52 remarkable reduction in activities in the absence (6r, 6s, and 6u) or weakened polarity of the picolinamide carbonyl group (6v) suggest its importance in the binding of 6. Significant variation on the anti-hRV activity by subtle change in ester alkyl chain seems to originate from the interaction with the hydrophobic pocket in the canyon. While methyl ester derivatives (6d and 6f) are assumed to leave empty space, 3or longer aliphatic ester alkyl chain variants are thought to collide with the hydrophobic residues. The mutation analysis suggested that 3-methyl moiety of 6g interacts with Leu25 on VP3 chain, which covers the VP1 canyon where capsid inhibitors bind. Notable differences in the activity along the variation on 3-position, especially against hRV-B14 and PV3, imply that this substrate−VP3 hydrophobic interaction is essential for the activity. While A150V mutation can prevent the pleconaril binding presumably by steric congestion in the aliphatic residue region, C199Y mutant can survive vapendavir treatment by reducing the space in the opposite site of the cavity.53 Compound 6g could overcome

of four viral coat proteins, VP1, VP2, VP3, and VP4, as represented in Figure 5A. VP1 contains a hydrophobic pocket in which an inhibitor molecule can bind. Some regions of the VP3 N-terminal coil, especially residues Ala24 and Leu25, cover the binding pocket in VP1. The result of the docking study suggested a binding model of 6g, in which it is bound to the same hydrophobic pocket in VP1 as is pleconaril (Figure 5B). The N-methylpicolinamide moiety, which makes hydrophobic interactions with Leu106, Tyr197, and Met221 in VP1, forms a nearly perpendicular geometry with respect to the benzothiophene core through the oxygen linker. The benzothiophene core participates in hydrophobic interactions with Ile104, Tyr128, Val188, and Val191, and a π−π staking interaction with Tyr152 in VP1. The isopropyl ester group binds in the hydrophobic pocket comprised of Ile130, Ala150, Val176, Phe186, and Met 224 in VP1. In particular, the 3-methyl group of the benzothiophene is oriented toward the VP3 N-terminal coil and participates in hydrophobic interactions with Pro174 in VP1 as well as Ala24 and Leu25 in VP3. Cross-Resistance Study. To gain further information about the binding mode of 6g and its potential applicability, cross-resistance studies were performed with two different hRV-B14 variants which are resistant to pleconaril (hRV-B14 VP1_A150V_E276K, hRV14plecres) or vapendavir (hRV-b14 VP1_C199Y, hRV14vapres), respectively (Table 5). Compound 5 (3C protease inhibitor)38 was used as a control. Whereas hRV14plecres showed a 20-fold resistance compared to the wildtype against pleconaril, only a 4-fold resistance was detected against 6g. Furthermore, hRV14vapres proved to be 792-fold resistant to vapendavir but was only 34-fold resistant to 6g. Combined with the data from time-of-addition and mutation studies, these results suggest that although 6g acts utilizing a similar mechanism to those of pleconaril and vapendavir, its interactions with the viral amino acid residues differ considerably from those of these substances. 5479



Article

Table 3. Inhibitory Effects of Benzo[b]thiophene and Heterocycle Derivatives against PV3a

entry

compd

R1

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33

1 29 6a 6b 6c 6d 6e 6f 6g 6h 6j 6k 6i 6l 6m 6n 6o 6p 6q 16a 15a 16b 15b 6r 6s 6t 6u 6v 6x 7a 7c 7d 7e

pleconaril pocapavir H 3-Me 3-Et H H 3-Me 3-Me 3-Me 3-Me 3-Me 3-Me 3-Me 3-Me 3-NH2 3-CN 3-Cl 7-Cl H H 3-Me 3-Me H 3-Me 3-Me H 3-Me 3-Me H H 3-Me 3-Me

R2

4-PicNHMe 4-PicNHMe 4-PicNHMe 4-PicNHMe 4-PicNHMe 4-PicNHMe 4-PicNHMe 4-PicNHMe 4-PicNHMe 4-PicNHMe 4-PicNHMe 4-PicNHMe 4-PicNHMe 4-PicNHMe 4-PicNHMe 4-PicNHMe 4-PicNHMe H Me H Me Ph Ph 3-C6H4CONHMe 4-pyridinyl 4-PicOMe 5-PicNHMe 4-PicNHMe 4-PicNHMe 4-PicNHMe 4-PicNHMe

X

S S S S S S S S S S S S S S S S S S S S S S S S S S S O NMe NH NMe

Y

HeLa CC50b (μM)

EC50 (μM)c PV3

SId PV3

OEt OEt OEt OMe OiPr OMe OiPr OnPr OiBu OBn OH NHEt NMe2 OEt OEt OEt OEt OEt OEt OEt OEt OEt OEt OEt OEt OEt OiPr OEt OiPr OiPr OiPr

25.9 ± 1.00 16.0 ± 10.5 >100 >100 16.7 ± 1.6 68.3 ± 4.9 45.0 ± 0.9 15.7 ± 0.5 9.6 ± 0.9 57.1 ± 23.1 28.2 ± 1.6 18.1 ± 1.3 >100 >100 44.4 ± 4.1 0.45 ± 0.03 >100 45.6 ± 0.2 >100 46.9 ± 10.2 44.4 ± 1.7 46.6 ± 11.3 >100 90.1 ± 1.8 47.6 ± 8.1 7.7 ± 0.2 43.9 ± 2.5 26.2 ± 5.2 12.9 ± 6.5 >100 46.1 ± 2.6 8.4 ± 1.0 8.5 ± 0.1

1.4 ± 0.1 0.080 ± 0.012 64.3 ± 11.7 0.093 ± 0.015 >16.7 >68.3 6.4 ± 3.3 0.087 ± 0.010 0.063 ± 0.015 0.71 ± 0.26 2.0 ± 0.3 0.34 ± 0.01 >100 >100 >44.4 >0.45 55.4 ± 6.7 >45.6 58.7 ± 9.3 >46.9 13.1 ± 3.1 9.4 ± 0.4 1.8 ± 0.0 >90.1 >47.6 0.38 ± 0.10 >43.9 >26.2 >12.9 >100 9.1 ± 5.1 0.072 ± 0.000 0.071 ± 0.001

19 200 >1.6 >1100

7.0 180 150 80 14 53

>1.8 >1.7 3.4 5.0 >56

20

5.1 120 120

All data were obtained from at least three independent experiments and mean values ± standard deviations are listed. bCC50: cytotoxic concentration (μM) for 50% of cell death, measured by MTT assay in HeLa cell line. cEC50: effective concentration (μM) concentrations for 50% inhibition of PV3, measured by MTT assay in HeLa cell line. dSI: selectivity index, calculated by CC50/EC50. a

analysis revealed that the activities highly depend on the steric bulkiness of the ester alkyl moiety, presence of 3-methyl group on the heteroarene core, and the electron richness of the heteroarene/naphthalene skeletons. N-Methylpicolinamide moiety, linked to the bicyclic core with ether linker, was found to be essential for the activity against hRVs. Among the novel compounds investigated, 6g was found to inhibit replication of the tested hRV species with EC50 values of 0.083, 0.078, and 0.015 μM against hRV-B14, hRV-A21, and hRV-A71, respectively, comparable to that of pleconaril. The results of time-of-addition experiments, in which 6g showed similar inhibition profile with that of pleconaril against both hRV-B14 and hRV-A16, and resistant mutation study, where only single point mutation VP3-L25M on the viral capsid protein was detected, implied that 6g act at the early stage of the viral infection, interacting with the viral capsid. The

those mutations by avoiding the resulting steric congestions from both sides. To compensate the shorter length of the picolinamide moiety, the “kink” oxygen seems to be shifted toward the Asn219 side of the canyon compared to the pivot of pleconaril. It drags the benzothiophene moiety to lessen the steric congestion with the aliphatic residues. The partial resistances in hRV14plecres and hRV14vapres seem to be due to the slight deformation of the cavity by larger residues than wild type.

■

CONCLUSION

In summary, we identified and developed a novel series of compound 6−8 that potently inhibit the replication of hRV-A and hRV-B and evaluated their antiviral activities against hRVB14, hRV-A21, and hRV-A71. Several compounds displayed submicromolar EC50 values against the tested hRV species. SAR 5480



Article

Table 4. Inhibitory Effects of Naphthalene Derivatives against PV3.a

entry

compd

R

1 2 3 4 5 6 7 8 9 10

1 29 8a 8b 8c 8d 8e 8f 8g 8h

pleconaril pocapavir H H H H H OH CO2Et H

X

CO2Me CO2Et CO2(i-Pr) CO2(s-Bu) CO2C6H5 CO2Et H CO2Et

Y

HeLa CC50b (μM)

EC50 (μM)c PV3

SId PV3

CH CH CH CH CH CH CH N

25.9 ± 1.00 16.0 ± 10.5 33.8 ± 5.5 13.2 ± 0.1 8.7 ± 0.4 9.0 ± 0.1 >100 >100 34.1 ± 4.1 >100

1.4 ± 0.1 0.080 ± 0.012 0.044 ± 0.008 0.0095 ± 0.0007 0.010 ± 0.000 0.35 ± 0.00 60.1 ± 4.6 0.18 ± 0.13 0.41 ± 0.06 1.9 ± 0.1

19 200 770 1400 870 26 >1.7 >560 83 >53

All data were obtained from at least three independent experiments and mean values ± standard deviations are listed. bCC50: cytotoxic concentration (μM) for 50% of cell death, measured by MTT assay in HeLa cell line. cEC50: effective concentration (μM) concentrations for 50% inhibition of PV3, measured by MTT assay in HeLa cell line. dSI: selectivity index, calculated by CC50/EC50. a

Scheme 6. Overall Summary of SAR Analysis of 6−8 against Tested hRVs and PV3

Figure 3. Time-of-drug-addition. Compounds in concentrations that are 6 times their EC50 values were added to hRV-B14-infected cells (A) and hRV-A16-infected cells (B) at 1 h intervals, starting at 1 h before infection. At 9 h postinfection, virus-infected cells were visualized by staining with anti-dsRNA antibody and the percentage of infected cells was calculated. Cells are infected at 0 h. Error bars indicate the means ± standard deviations from three experiments. Asterisks indicate P values of 3 >10 721 >3

d

d

a

Pleconaril resistant hRV14 variant, hRV14plecres. bVapendavir resistant hRV14 variant, hRV14vapres. cEC50: effective concentrations (μM) for 50% inhibition of each hRV14 variant, measured by MTT assay in HeLa Rh cell line. dFR: fold resistance compared to hRV14 wild-type. e CC50: cytotoxic concentration (μM) for 50% cell death, measured by MTT assay in HeLa Rh cell line.

EXPERIMENTAL SECTION

Biology Assays. Cells, Viruses, and Chemicals. H1HeLa cells (ATCC CRL-1958) were purchased from ATCC and cultured in Minimum Essential Medium (MEM) supplemented with 10% fetal bovine serum. Human rhinovirus type 14 (ATCC VR-284), type 16 (ATCC VR-283), type 21 (ATCC VR-496), and type 71 (ATCC VR1181) were obtained from ATCC and expanded in H1HeLa cells. Human poliovirus 3 (ATCC VR-193) was purchased from ATCC and expanded in HeLa cells (ATCC CCL-2). Virus titers were measured using end-point dilution assay. Pleconaril (Sigma SML-0307) and rupintrivir (Santa Cruz sc-208317) were purchased and used as control compounds. Antiviral Assay. To test the antiviral activity of the compounds on human rhinoviruses, a cytopathic effect (CPE) reduction assay was performed using 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma) as previously described.54 H1HeLa cells were plated in 96-well plates 2 d before the assays were performed. Equal volumes of virus and compounds were added and incubated at 33 °C for 3 d in CO2 incubator. After removing the cell culture supernatant, MTT solution was added and the resulting mixture was incubated for 1 h at 37 °C. Formazan products were dissolved using organic solvent, and absorbances at 540 nm (main) and at 690 nm (reference) were measured using a microplate reader (Synergy H1, Biotek). Wells without viral infection and chemical treatment were considered as having a100% survival rate, and wells only infected with virus were

considered as having a 0% survival rate. The antiviral activities of the compound were calculated based on the survival rates of wells treated with compounds. Data were presented as means ± standard deviations from three experiments. To test antiviral activity of compounds on human poliovirus 3, similar experiments with methods used for human rhinoviruses with brief modification were performed. HeLa cells were used and incubated at 37 °C for 2 d in CO2 incubator. Time-of-Addition Assay. 6g or one of the control compounds (pleconaril and rupintrivir) was administered 1 h before virus infection for −1 h time points. Cells were infected with human rhinovirus type 14 or type 16 at 1 MOI and simultaneously treated with the compounds (0 h). Compounds were added at indicated time points. At 9 h postinfection, the cells were fixed with a 3:1 mixture of ice-cold methanol−acetone and stained with anti-dsRNA antibody (J2; English & Scientific Consulting Kft). Anti-mouse secondary antibody conjugated with Alexa Fluor 488 (Invitrogen) was used to stain the infected cells, followed by counterstaining with 4′,6-diamidino-2phenylindole (DAPI) (Thermo Scientific) for nuclei staining. Images were captured using Operetta system (PerkinElmer). Viral infection was quantified by dividing the number of infected cells by the total number of nuclei. Infection was calculated as a percentage of the virus control (not treated with compounds). Data were presented as means

Figure 5. Docking study of 6g in pleconaril-bound hRV-B14 crystal structure (PDB 1NCQ). (A) Cartoon representation of the hRV-B14 protomer (VP1 as a pink ribbon, VP2 as a green ribbon, VP3 as a cyan ribbon, and VP4 as an orange ribbon) in complex with the proposed binding mode of 6g (yellow ball and stick). (B) Docking mode of 6g (yellow ball and stick) and overlapped X-ray crystal structure of pleconaril (gray stick) in VP1. VP1 residues that interact with 6g are shown as sticks in pink, and VP3 residues are indicated by cyan sticks. For clarity, key interaction residues are visible in stick style and are labeled using the one-letter amino acid code and the important residue of VP3 is marked in a cyan dotted circle. 5482



Article

± standard deviations from three experiments. The difference between each time point and control was assessed by Student t test. Asterisks indicate P values of 95% pure, as determined by UPLC analysis conducted on Waters Acquity UPLC H-Class system with photodiode array (PDA) detector using a reverse-phase column with a linear H2O/CH3CN gradient system, 10−90% CH3CN in H2O. Ethyl 6-[(2-(Methylcarbamoyl)pyridin-4-yl)oxy]benzo[b]thiophene-2-carboxylate (6a). Step 1: Synthesis of Ethyl 6Methoxybenzo[b]thiophene-2-carboxylate (15a). 15a was obtained from the modified procedure of the reference.56 Ethyl mercaptoacetate

(7.80 g, 64.9 mmol) and K2CO3 (14.3 g, 104 mmol) were added to a solution of 2-fluoro-4-methoxybenzaldehyde (14a, 10.0 g, 59.5 mmol) in DMF (50 mL) and stirred at 25 °C for 16 h. The reaction mixture was diluted with Et2O (300 mL) and H2O (100 mL), and extracted with Et2O (50 mL × 3). The combined organic layer was dried over MgSO4, concentrated, and the residue was purified by SiO2 column chromatography (EtOAc/Hx = 1:19) to obtain 15a as white solid (13.01 g, 85%). 1H NMR (300 MHz, CDCl3) δ 7.96 (s, 1H), 7.73 (d, J = 8.8 Hz, 1H), 7.28 (d, J = 2.3 Hz, 1H), 7.02 (dd, J = 8.8, 2.3 Hz, 1H), 4.39 (q, J = 7.1 Hz, 2H), 3.88 (s, 3H), 1.40 (t, J = 7.1 Hz, 3H). LC/ MS (ESI) [M + H]+ = 237.0. Step 2: Synthesis of Ethyl 6-Hydroxybenzo[b]thiophene-2carboxylate (16a). BBr3 (1.0 M solution in CH2Cl2, 53 mL, 53 mmol) was added to a solution of 15a (5.00 g, 21.2 mmol) in CH2Cl2 (150 mL) dropwise at −78 °C. The mixture was stirred at −78 °C for 1 h and then at 25 °C for 23 h. The reaction was quenched by dropwise addition of EtOH (10 mL) and washed with satd aq NaHCO3. The aqueous layer was extracted with CH2Cl2 (50 mL × 3). The combined organic layer was dried over MgSO4, concentrated, and the residue was purified by SiO2 column chromatography (EtOAc/Hx = 1:19) to obtain 16a as white solid (3.01 g, 64%). 1H NMR (300 MHz, CDCl3) δ 7.97 (s, 1H), 7.73 (d, J = 8.7 Hz, 1H), 7.27 (d, J = 2.4 Hz, 1H), 6.96 (dd, J = 8.7, 2.3 Hz, 1H), 5.30 (s, 1H), 4.39 (q, J = 7.1 Hz, 2H), 1.41 (t, J = 7.1 Hz, 3H). LC/MS (ESI) [M + H]+ = 223.1. Step 3: Synthesis of Ethyl 6-[(2-(Methylcarbamoyl)pyridin-4yl)oxy]benzo[b]thiophene-2-carboxylate (6a). A mixture of 16a (3.00 g, 13.5 mmol) and 4-chloro-N-methylpicolinamide (10, 2.30 g, 13.5 mmol) was stirred at 150 °C for 48 h. The mixture was then cooled to 25 °C, dissolved in EtOH (50 mL), and diluted with EtOAc (500 mL) and H2O (200 mL). The aqueous layer was extracted with EtOAc (200 mL × 3). The combined organic layer was dried over MgSO4, concentrated, and the residue was purified by SiO2 column chromatography (EtOAc/Hx = 1:2) to obtain 6a (1.67 g, 35%) as white solid; mp 133−135 °C. 1H NMR (300 MHz, CDCl3) δ 8.43 (d, J = 5.5 Hz, 1H), 8.08 (s, 1H), 8.03 (br s, 1H), 7.92 (d, J = 8.7 Hz, 1H), 7.75 (d, J = 2.6 Hz, 1H), 7.59 (d, J = 2.1 Hz, 1H), 7.17 (dd, J = 8.7, 2.2 Hz, 1H), 7.02 (dd, J = 5.6, 2.6 Hz, 1H), 4.44 (q, J = 7.1 Hz, 2H), 3.02 (d, J = 5.1 Hz, 3H), 1.44 (t, J = 7.1 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 165.95, 164.38, 162.47, 153.02, 152.49, 149.85, 143.60, 136.43, 134.45, 129.82, 127.20, 118.96, 114.48, 114.28, 110.52, 61.72, 26.15, 14.33. IR (diamond) 3341, 2988, 2972, 1686, 1668, 1520, 1174 cm−1. HRMS (EI) m/z calcd for C18H16N2O4S [M+] 356.0831, found 356.0831. Ethyl 3-Methyl-6-{[2-(methylcarbamoyl)pyridin-4-yl]oxy}benzo[b]thiophene-2-carboxylate (6b). Step 1: Ethyl 6-Methoxy-3methylbenzo[b]thiophene-2-carboxylate (15b). Following the same procedure used to prepare 15a, 2-fluoro-4-methoxy benzaldehyde (14b, 10.0 g, 59.5 mmol), 1-(2-fluoro-4-methoxyphenyl)ethan-1-one (7.86 g, 65.4 mmol), and K2CO3 (24.7 g, 178 mmol) were used to obtain 15b (9.05 g, 61%) as white solid. 1H NMR (300 MHz, CDCl3) δ 7.70 (dd, J = 8.9, 0.5 Hz, 1H), 7.25 (d, J = 5.2 Hz, 1H), 7.03 (dd, J = 8.9, 2.4 Hz, 1H), 4.38 (q, J = 7.1 Hz, 2H), 3.89 (s, 3H), 2.73 (s, 3H), 1.41 (t, J = 7.1 Hz, 3H). LC/MS (ESI) [M + H]+ = 250.9. Step 2: Ethyl 6-Hydroxy-3-methylbenzo[b]thiophene-2-carboxylate (16b). Following the same procedure used to prepare 16a, 15b (5.00 g, 20.0 mmol), and BBr3 (12.5 g, 50.0 mmol) were used to obtain 16b (2.06 g, 44%) as white solid. 1H NMR (300 MHz, CDCl3) δ 7.72 (d, J = 8.8 Hz, 1H), 7.26 (d, J = 11.4 Hz, 4H), 6.99 (dd, J = 8.8, 2.3 Hz, 1H), 5.03 (s, 1H), 4.40 (q, J = 7.1 Hz, 2H), 2.75 (s, 3H), 1.43 (t, J = 7.1 Hz, 3H). LC/MS (ESI) [M + H]+ = 237.1. Step 3: Ethyl 3-Methyl-6-{[2-(methylcarbamoyl)pyridin-4-yl]oxy}benzo[b]thiophene-2-carboxylate (6b). Following the same procedure used to prepare 6a, 16b (3.00 g, 12.7 mmol), and 10 (2.17 g, 12.7 mmol) were used to obtain 6b (1.09 g, 23%) as yellow solid; mp 128− 130 °C. 1H NMR (300 MHz, CDCl3) δ 8.43 (d, J = 5.6 Hz, 1H), 8.02 (s, 1H), 7.89 (d, J = 8.8 Hz, 1H), 7.75 (d, J = 2.5 Hz, 1H), 7.56 (d, J = 2.2 Hz, 1H), 7.19 (dd, J = 8.8, 2.2 Hz, 1H), 7.02 (dd, J = 5.6, 2.6 Hz, 1H), 4.43 (q, J = 7.1 Hz, 2H), 3.03 (d, J = 5.1 Hz, 3H), 2.81 (s, 3H), 1.45 (t, J = 7.1 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 166.02, 164.40, 163.18, 153.20, 152.44, 149.82, 141.75, 140.48, 137.91, 127.49, 5483



Article

114.51, 114.26, 110.51, 52.60, 26.15. IR (diamond) 3328, 3093, 2921, 2853, 1718, 1665, 1514, 1248 cm−1. HRMS (EI) m/z calcd for C17H14N2O4S [M+] 342.0674, found 342.0676. Isopropyl 6-{[2-(Methylcarbamoyl)pyridin-4-yl]oxy}benzo[b]thiophene-2-carboxylate (6e). Step 1: Isopropyl 6-Hydroxybenzo[b]thiophene-2-carboxylate (16e). Following the same procedure used to prepare 16d, 17a (200. mg, 1.03 mmol), and a solution of 5% H2SO4 in iPrOH (20 mL) were used to obtain 16e (187 mg, 77%). 1H NMR (300 MHz, CDCl3) δ 7.98 (s, 1H), 7.74 (d, J = 8.7 Hz, 1H), 7.30 (d, J = 2.3 Hz, 1H), 6.99 (dd, J = 8.7, 2.3 Hz, 1H), 5.73 (s, 1H), 5.28 (hept, J = 6.2 Hz, 1H), 1.41 (d, J = 6.3 Hz, 6H). LC/MS (ESI) [M + H]+ = 237.1. Step 2: Isopropyl 6-{[2-(Methylcarbamoyl)pyridin-4-yl]oxy}benzo[b]thiophene-2-carboxylate (6e). Following the same procedure used to prepare 6a, 16e (100 mg, 0.423 mmol), and 10 (72.2 mg, 0.423 mmol) were used to obtain 6e (55.0 mg, 35%) as yellow oil. 1H NMR (300 MHz, CDCl3) δ 8.44 (d, J = 5.6 Hz, 1H), 8.06 (s, 1H), 8.02 (br s, 1H), 7.92 (d, J = 8.7 Hz, 1H), 7.77 (d, J = 2.5 Hz, 1H), 7.59 (d, J = 2.0 Hz, 1H), 7.17 (dd, J = 8.7, 2.2 Hz, 1H), 7.03 (dd, J = 5.6, 2.5 Hz, 1H), 5.29 (hept, J = 6.4 Hz, 1H), 3.03 (d, J = 5.1 Hz, 3H), 1.43 (d, J = 6.3 Hz, 6H). 13C NMR (126 MHz, CDCl3) δ 165.95, 164.39, 162.01, 152.93, 152.46, 149.84, 143.54, 136.45, 135.02, 129.58, 127.13, 118.90, 114.44, 114.26, 110.52, 69.50, 26.15, 21.93. IR (diamond) 3397, 3059, 2980, 2930, 1702, 1670, 1517, 1248 cm−1. HRMS (EI) m/ z calcd for C19H18N2O4S [M+] 370.0987, found 370.0986. Methyl 3-Methyl-6-{[2-(methylcarbamoyl)pyridin-4-yl]oxy}benzo[b]thiophene-2-carboxylate (6f). Step 1: 6-Hydroxy-3methylbenzo[b]thiophene-2-carboxylic Acid (17b). Following the same procedure used to prepare 17a, 16b (500 mg, 2.12 mmol), and KOH (238 mg, 4.23 mmol) were used to obtain 17b (390. mg, 83%). 1 H NMR (300 MHz, Acetone-d6) δ 7.80 (d, J = 8.8 Hz, 1H), 7.34 (s, 1H), 7.06 (dd, J = 8.8, 2.3 Hz, 1H), 2.73 (s, 3H). LC/MS (ESI) [M + H]+ = 209.2. Step 2: Methyl 6-Hydroxy-3-methylbenzo[b]thiophene-2-carboxylate (16f). Following the same procedure used to prepare 16d, 17b (150 mg, 0.720 mmol), and a solution of 5% H2SO4 in MeOH (20 mL) were used to obtain 16f (108 mg, 68%). 1H NMR (300 MHz, acetone-d6) δ 8.94 (s, 1H), 7.77 (d, J = 8.8 Hz, 1H), 7.33 (d, J = 2.2 Hz, 1H), 7.05 (dd, J = 8.8, 2.3 Hz, 1H), 3.87 (s, 3H), 2.70 (s, 3H). LC/MS (ESI) [M + H]+ = 223.0. Step 3: Methyl 3-Methyl-6-{[2-(methylcarbamoyl)pyridin-4-yl]oxy}benzo[b]thiophene-2-carboxylate (6f). Following the same procedure used to prepare 6a, 16f (100 mg, 0.450 mmol), and 10 (76.8 mg, 0.450 mmol) were used to obtain 6f (36.9 mg, 23%) as white solid; mp 148−150 °C. 1H NMR (300 MHz, CDCl3) δ 8.42 (d, J = 5.6 Hz, 1H), 8.02 (br s, 1H), 7.89 (d, J = 8.8 Hz, 1H), 7.74 (d, J = 2.6 Hz, 1H), 7.55 (d, J = 2.2 Hz, 1H), 7.18 (dd, J = 8.8, 2.2 Hz, 1H), 7.02 (dd, J = 5.6, 2.6 Hz, 1H), 3.95 (s, 3H), 3.02 (d, J = 5.1 Hz, 3H), 2.81 (s, 3H). 13C NMR (126 MHz, CDCl3) δ 165.99, 164.38, 163.54, 153.30, 152.46, 149.83, 141.78, 140.82, 137.81, 126.95, 125.48, 118.43, 114.43, 114.16, 110.48, 52.19, 26.15, 13.24. IR (diamond) 3405, 3054, 2953, 2914, 1678, 1672, 1520, 1274 cm−1. HRMS (EI) m/z calcd for C18H16N2O4S [M+] 356.0831, found 356.0829. Isopropyl 3-Methyl-6-{[2-(methylcarbamoyl)pyridin-4-yl]oxy}benzo[b]thiophene-2-carboxylate (6g). Step 1: Isopropyl 6Hydroxy-3-methylbenzo[b]thiophene-2-carboxylate (16g). Following the same procedure used to prepare 16d, 17b (150 mg, 0.720 mmol), and 5% H2SO4 in iPrOH (20 mL) were used to obtain 16g (52.1 mg, 29%). 1H NMR (300 MHz, CDCl3) δ 7.71 (d, J = 8.8 Hz, 1H), 7.25 (d, J = 2.3 Hz, 1H), 6.98 (dd, J = 8.8, 2.3 Hz, 1H), 5.33− 5.16 (m, 2H), 2.75 (s, 3H), 1.41 (d, J = 6.2 Hz, 6H). LC/MS (ESI) [M + H]+ = 251.0. Step 2: Isopropyl 3-Methyl-6-{[2-(methylcarbamoyl)pyridin-4yl]oxy}benzo[b]thiophene-2-carboxylate (6g). Following the same procedure used to prepare 6a, 16g (48.0 mg, 0.192 mmol), and 10 (32.8 mg, 0.192 mmol) were used to obtain 6g (33.2 mg, 45%) as offwhite solid; mp 80−82 °C. 1H NMR (300 MHz, CDCl3) δ 8.42 (d, J = 5.6 Hz, 1H), 8.02 (br s, 1H), 7.87 (d, J = 8.8 Hz, 1H), 7.75 (d, J = 2.5 Hz, 1H), 7.55 (d, J = 2.2 Hz, 1H), 7.17 (dd, J = 8.8, 2.2 Hz, 1H), 7.00 (dd, J = 5.6, 2.6 Hz, 1H), 5.28 (hept, J = 6.3 Hz, 1H), 3.02 (d, J = 5.1 Hz, 3H), 2.80 (s, 3H), 1.42 (d, J = 6.3 Hz, 6H). 13C NMR (126 MHz,

125.43, 118.37, 114.40, 114.17, 110.48, 61.29, 26.16, 14.35, 13.25. IR (diamond) 3392, 3062, 2922, 2853, 1710, 1673, 1525, 1250 cm−1. HRMS (EI) m/z calcd for C19H18N2O4S [M+] 370.0987, found 370.0988. Ethyl 3-Ethyl-6-{[2-(methylcarbamoyl)pyridin-4-yl]oxy}benzo[b]thiophene-2-carboxylate (6c). Step 1: Ethyl 3-Ethyl-6methoxybenzo[b]thiophene-2-carboxylate (15c). Following the same procedure for 15a, 1-(2-fluoro-4-methoxyphenyl)propan-1-one (14c, 274 mg, 1.63 mmol), ethyl mercaptoacetate (196 mg, 1.63 mmol), and K2CO3 (360 mg, 2.61 mmol) were used to obtain 15c (236 mg, 55%) as off-white solid. 1H NMR (300 MHz, CDCl3) δ 7.72 (d, J = 8.9 Hz, 1H), 7.26 (d, J = 2.2 Hz, 1H), 7.03 (dd, J = 8.9, 2.4 Hz, 1H), 4.38 (q, J = 7.1 Hz, 2H), 3.89 (s, 3H), 3.26 (q, J = 7.5 Hz, 2H), 1.41 (t, J = 7.1 Hz, 3H), 1.27 (t, J = 7.5 Hz, 3H). LC/MS (ESI) [M + H]+ = 265.2. Step 2: Ethyl 3-Ethyl-6-hydroxybenzo[b]thiophene-2-carboxylate (16c). Following the same procedure for 16a, 15c (200. mg, 0.756 mmol), and BBr3 (1.0 M solution in CH2Cl2, 1.89 mL, 1.89 mmol) were used to obtain 16c (142 mg, 75%) as off-white solid. 1H NMR (300 MHz, CDCl3) δ 7.74 (dd, J = 8.8, 1.2 Hz, 1H), 7.34−7.23 (m, 1H), 6.99 (d, J = 8.8 Hz, 1H), 5.35 (br s, 1H), 4.40 (q, J = 6.9 Hz, 2H), 3.28 (q, J = 7.4 Hz, 2H), 1.43 (t, J = 7.3 Hz, 3H), 1.29 (t, J = 7.4 Hz, 3H). LC/MS (ESI) [M + H]+ = 251.2. Step 3: Ethyl 3-Ethyl-6-{[2-(methylcarbamoyl)pyridin-4-yl]oxy}benzo[b]thiophene-2-carboxylate (6c). Following the same procedure used to prepare 6a, 16c (50.0 mg, 0.200 mmol), and 10 (34.1 mg, 0.200 mmol) were used to obtain 6c (33.2 mg, 43%) as white solid; mp 86−88 °C. 1H NMR (300 MHz, CDCl3) δ 8.43 (d, J = 5.6 Hz, 1H), 8.03 (br s, 1H), 7.90 (d, J = 8.8 Hz, 1H), 7.75 (d, J = 2.5 Hz, 1H), 7.56 (d, J = 2.2 Hz, 1H), 7.18 (dd, J = 8.8, 2.2 Hz, 1H), 7.03 (dd, J = 5.5, 2.6 Hz, 1H), 4.43 (q, J = 7.1 Hz, 2H), 3.33 (q, J = 7.5 Hz, 2H), 3.03 (d, J = 5.1 Hz, 3H), 1.44 (t, J = 7.1 Hz, 3H), 1.33 (t, J = 7.5 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 166.06, 164.41, 162.84, 153.13, 152.46, 149.81, 146.87, 142.18, 137.06, 127.09, 125.30, 118.40, 114.45, 114.32, 110.45, 61.27, 26.15, 20.80, 14.55, 14.31. IR (diamond) 3331, 3064, 2985, 2961, 2927, 2872, 1702, 1662, 1525, 1195 cm−1. HRMS (EI) m/z calcd for C20H20N2O4S [M+] 384.1144, found 384.1143. Methyl 6-{[2-(Methylcarbamoyl)pyridin-4-yl]oxy}benzo[b]thiophene-2-carboxylate (6d). Step 1: 6-Hydroxybenzo[b]thiophene-2-carboxylic Acid (17a). KOH (238 mg, 4.23 mmol) was added to a solution of 16a (500. mg, 2.12 mmol) in a 1:1 mixture of H2O and THF (10 mL). The mixture was stirred at 65 °C for 24 h, cooled to 25 °C, and concentrated to ∼50% of its original volume. The mixture was acidified to pH = 1−2 with 2 N aq HCl to form precipitate which was collected by filtration and washed with H2O (10 mL × 2) and then Et2O (10 mL). The remaining solid was dried under high-vacuum to obtain 17a as off-white solid (381 mg, 86%). 1H NMR (300 MHz, acetone-d6) δ 8.02 (s, 1H), 7.86 (d, J = 8.7 Hz, 1H), 7.40 (d, J = 2.2 Hz, 1H), 7.05 (dd, J = 8.7, 2.3 Hz, 1H). LC/MS (ESI) [M + H]+ = 195.1. Step 2. Methyl 6-hydroxybenzo[b]thiophene-2-carboxylate (16d). 17a (150 mg, 0.772 mmol) was taken into a solution of 5% H2SO4 in MeOH (20 mL) and the mixture was refluxed at 75 °C for 20 h. The mixture was then cooled to 25 °C, diluted with EtOAc (200 mL), washed with aq NaHCO3 (20 mL × 3) followed by brine (20 mL). The organic phase was dried over MgSO4, concentrated, and the residue was purified by SiO2 column chromatography (EtOAc/Hx = 1:6) to obtain 16d (138 mg, 86%) as off-white solid. 1H NMR (300 MHz, CDCl3) δ 8.00 (s, 1H), 7.76 (d, J = 8.7 Hz, 1H), 6.98 (dd, J = 8.7, 2.3 Hz, 1H), 5.16 (s, 1H), 3.95 (s, 3H). LC/MS (ESI) [M + H]+ = 209.0. Step 3. Methyl 6-{[2-(Methylcarbamoyl)pyridin-4-yl]oxy}benzo[b]thiophene-2-carboxylate (6d). Following the same procedure used to prepare 6a, 16d (35.0 mg, 0.168 mmol), and 10 (28.7 mg, 0.168 mmol) were used to obtain 6d (22.3 mg, 39%) as yellow solid; mp 128−130 °C. 1H NMR (300 MHz, CDCl3) δ 8.41 (dd, J = 5.6, 0.5 Hz, 1H), 8.06 (d, J = 0.8 Hz, 1H), 8.03 (br s, 1H), 7.91 (d, J = 8.7 Hz, 1H), 7.74 (dd, J = 2.6, 0.5 Hz, 1H), 7.57 (d, J = 2.3 Hz, 1H), 7.16 (dd, J = 8.7, 2.2 Hz, 1H), 7.01 (dd, J = 5.6, 2.6 Hz, 1H), 3.96 (s, 3H), 3.01 (d, J = 5.1 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 165.93, 164.37, 153.10, 152.49, 149.85, 143.62, 136.36, 133.87, 130.08, 127.26, 119.01, 5484



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CDCl3) δ 166.17, 164.53, 162.89, 153.25, 152.57, 149.93, 141.86, 140.25, 138.12, 128.23, 125.51, 118.44, 114.49, 114.28, 110.63, 69.18, 26.28, 22.11, 13.37. IR (diamond) 3394, 3059, 2980, 2924, 1702, 1675, 1525, 1253 cm−1. HRMS (EI) m/z calcd for C20H20N2O4S [M+] 384.1144, found 384.1144. n-Propyl 3-Methyl-6-{[2-(methylcarbamoyl)pyridin-4-yl]oxy}benzo[b]thiophene-2-carboxylate (6h). Step 1: n-Propyl 6-Hydroxy-3-methylbenzo[b]thiophene-2-carboxylate (16h). Following the same procedure used to prepare 16d, 17b (100 mg, 0.480 mmol), and a solution of 5% H2SO4 in nPrOH (10 mL) were used to obtain 16h (87.3 mg, 73%). 1H NMR (300 MHz, acetone-d6) δ 8.97 (s, 1H), 7.79 (d, J = 8.8 Hz, 1H), 7.33 (d, J = 2.1 Hz, 1H), 7.05 (dd, J = 8.8, 2.3 Hz, 1H), 4.26 (t, J = 6.5 Hz, 2H), 2.72 (s, 3H), 1.79 (tq, J = 7.4, 6.5 Hz, 2H), 1.04 (t, J = 7.4 Hz, 3H). LC/MS (ESI) [M + H]+ = 251.0. Step 2: n-Propyl 3-Methyl-6-{[2-(methylcarbamoyl)pyridin-4-yl]oxy}benzo[b]thiophene-2-carboxylate (6h). Following the same procedure used to prepare 6a, 16h (50.0 mg, 0.200 mmol), and 10 (34.1 mg, 0.200 mmol) were used to obtain 6h (18.3 mg, 24%) as yellow solid; mp 75−77 °C. 1H NMR (300 MHz, CDCl3) δ 8.42 (d, J = 5.5 Hz, 1H), 8.03 (br s, 1H), 7.88 (d, J = 8.8 Hz, 1H), 7.75 (d, J = 2.6 Hz, 1H), 7.55 (d, J = 2.2 Hz, 1H), 7.18 (dd, J = 8.8, 2.2 Hz, 1H), 7.01 (dd, J = 5.6, 2.6 Hz, 1H), 4.33 (t, J = 6.6 Hz, 2H), 3.02 (d, J = 5.1 Hz, 3H), 2.81 (s, 3H), 1.92−1.75 (m, 2H), 1.07 (t, J = 7.4 Hz, 3H). 13 C NMR (126 MHz, CDCl3) δ 166.03, 164.40, 163.26, 153.20, 152.44, 149.82, 141.79, 140.41, 137.93, 127.60, 125.44, 118.37, 114.40, 114.16, 110.50, 66.83, 26.16, 22.10, 13.24, 10.55. IR (diamond) 3392, 2959, 2919, 2848, 1707, 1670, 1520, 1192 cm−1. HRMS (EI) m/z calcd for C20H20N2O4S [M+] 384.1144, found 384.1144. 3-Methyl-6-{[2-(methylcarbamoyl)pyridin-4-yl]oxy}benzo[b]thiophene-2-carboxylic acid (6i). A mixture of 17b (1.00 g, 4.80 mmol), 10 (815 mg, 4.80 mmol), and Cs2CO3 (4.70 g, 14.4 mmol) in DMSO (15 mL) was stirred at 120 °C for 15 h. The mixture was then cooled to 25 °C, diluted with EtOAc (200 mL), and H2O (200 mL). The aqueous layer was acidified to pH = 4 with 2 N aq HCl and extracted with EtOAc (100 mL × 3). The combined organic layer was washed with brine (20 mL), dried over MgSO4, concentrated, and the residue was purified by SiO2 column chromatography (CH2Cl2/ MeOH = 9:1) to obtain 6i (869.6 mg, 56%) as white solid; mp 230− 232 °C. 1H NMR (300 MHz, acetone-d6) δ 8.52 (d, J = 5.6 Hz, 1H), 8.35 (br s, 1H), 8.09 (d, J = 8.8 Hz, 1H), 7.86 (d, J = 2.2 Hz, 1H), 7.62 (d, J = 2.6 Hz, 1H), 7.35 (dd, J = 8.8, 2.3 Hz, 1H), 7.17 (dd, J = 5.6, 2.6 Hz, 1H), 2.95 (d, J = 4.9 Hz, 3H), 2.83 (s, 3H). 13C NMR (126 MHz, DMSO-d6) δ 165.32, 164.04, 163.58, 152.58, 152.46, 150.45, 140.65, 139.14, 137.73, 128.68, 125.81, 118.57, 114.52, 114.39, 109.18, 25.90, 12.73. IR (diamond) 3354, 3344, 3339, 3062, 2919, 2908, 1683, 1649, 1525, 1229 cm−1. HRMS (EI) m/z calcd for C17H14N2O4S [M+] 342.0674, found 342.0672. Isobutyl 3-Methyl-6-{[2-(methylcarbamoyl)pyridin-4-yl]oxy}benzo[b]thiophene-2-carboxylate (6j). To a suspension of 6i (50.0 mg, 0.146 mmol) in CH2Cl2 (5 mL) was added EDCI·HCl (33.5 mg, 0.175 mmol) and DIPEA (37.7 mg, 0.292 mmol) sequentially at 0 °C. The mixture was stirred at 0 °C for 2 h. Then isobutanol (21.6 mg, 0.292 mmol) and DMAP (5.4 mg, 0.044 mmol) were added, and the reaction mixture was stirred at 25 °C for 30 h. The mixture was then diluted with CH2Cl2 (50 mL) and H2O (50 mL), and the aqueous layer was extracted with CH2Cl2 (20 mL × 3). The combined organic layer was dried over MgSO4, concentrated, and the residue was purified by SiO2 column chromatography (EtOAc:Hx = 1:6) to obtain 6j (6 mg, 10%) as white solid; mp 90−92 °C. 1H NMR (300 MHz, CDCl3) δ 8.41 (d, J = 5.6 Hz, 1H), 8.01 (br s, 1H), 7.87 (d, J = 8.8 Hz, 1H), 7.73 (d, J = 2.5 Hz, 1H), 7.54 (d, J = 2.2 Hz, 1H), 7.17 (dd, J = 8.8, 2.2 Hz, 1H), 7.00 (dd, J = 5.6, 2.6 Hz, 1H), 4.14 (d, J = 6.5 Hz, 2H), 3.01 (d, J = 5.1 Hz, 3H), 2.80 (s, 3H), 2.20−2.01 (m, 1H), 1.04 (d, J = 6.7 Hz, 6H). 13C NMR (126 MHz, CDCl3) δ 166.04, 164.38, 163.21, 153.23, 152.47, 149.81, 141.81, 140.40, 137.94, 127.64, 125.44, 118.37, 114.40, 114.15, 110.51, 71.27, 27.90, 26.15, 19.19, 13.24. IR (diamond) 3394, 2959, 2919, 2856, 1705, 1670, 1522, 1192 cm−1. HRMS (EI) m/z calcd for C21H22N2O4S [M+] 398.1300, found 398.1300.

Benzyl 3-Methyl-6-{[2-(methylcarbamoyl)pyridin-4-yl]oxy}benzo[b]thiophene-2-carboxylate (6k). Following the same procedure used to prepare 6j, 6i (50.0 mg, 0.146 mmol), EDCI·HCl (33.5 mg, 0.175 mmol), DIPEA (37.7 mg, 0.292 mmol), benzyl alcohol (31.6 mg, 0.292 mmol), and DMAP (5.4 mg, 0.044 mmol) were used to obtain 6k (12 mg, 19%) as white solid; mp 88−90 °C. 1H NMR (300 MHz, CDCl3) δ 8.43 (dd, J = 5.6, 0.5 Hz, 1H), 8.02 (br s, 1H), 7.89 (d, J = 8.8 Hz, 1H), 7.75 (d, J = 2.5 Hz, 1H), 7.55 (d, J = 2.2 Hz, 1H), 7.53− 7.33 (m, 5H), 7.19 (dd, J = 8.8, 2.2 Hz, 1H), 7.02 (dd, J = 5.6, 2.6 Hz, 1H), 5.42 (s, 2H), 3.03 (d, J = 5.1 Hz, 3H), 2.82 (s, 3H). 13C NMR (126 MHz, CDCl3) δ 166.00, 164.37, 162.91, 153.34, 152.47, 149.82, 141.90, 141.05, 137.85, 135.76, 128.64, 128.29, 128.03, 127.07, 125.51, 118.43, 114.43, 114.14, 110.54, 66.80, 26.15, 13.33. IR (diamond) 3495, 3344, 3059, 2922, 2853, 1705, 1668, 1518, 1190 cm−1. HRMS (EI) m/z calcd for C24H20N2O4S [M+] 432.1144, found 432.1145. 4-[2-(Ethylcarbamoyl)-3-methylbenzo[b]thiophen-6-yloxy]-Nmethylpicolinamide (6l). A solution of EDCI·HCl (27.2 mg, 0.175 mmol) in CH2Cl2 (2 mL) and DIPEA (37.7 mg, 0.292 mmol) were sequentially added to a suspension of 6i (50.0 mg, 0.146 mmol) in CH2Cl2 (5 mL) at 0 °C. The mixture was stirred at 0 °C for 2 h. Then aq EtNH2 (70%, 18.8 mg, 0.292 mmol) was added at 0 °C and the mixture was stirred at 25 °C for 20 h. After dilution with EtOAc (50 mL) and H2O (50 mL), the aqueous phase was extracted with EtOAc (20 mL × 3). The combined organic layer was dried over MgSO4, concentrated, and the resulting residue was purified by SiO2 column chromatography (CH2Cl2/MeOH = 9:1) to obtain 6l (22.1 mg, 41%) as white solid; mp 175−177 °C. 1H NMR (300 MHz, CDCl3) δ 8.42 (d, J = 5.5 Hz, 1H), 8.03 (br s, 1H), 7.83 (dd, J = 8.8, 0.5 Hz, 1H), 7.73 (d, J = 2.5 Hz, 1H), 7.53 (d, J = 2.2 Hz, 1H), 7.18 (dd, J = 8.8, 2.2 Hz, 1H), 7.01 (dd, J = 5.6, 2.6 Hz, 1H), 6.02 (s, 1H), 3.52 (qd, J = 7.3, 5.6 Hz, 2H), 3.02 (d, J = 5.1 Hz, 3H), 2.72 (s, 3H), 1.29 (t, J = 7.3 Hz, 4H). 13C NMR (126 MHz, CDCl3) δ 166.15, 164.42, 163.10, 152.57, 152.43, 149.81, 139.65, 138.36, 135.37, 130.98, 124.89, 118.55, 114.35, 114.27, 110.37, 35.10, 26.15, 14.91, 13.14. IR (diamond) 3376, 3331, 3064, 2969, 2930, 1657, 1644, 1512, 1248 cm−1. HRMS (EI) m/z calcd for C19H19N3O3S [M+] 369.1147, found 369.1134. 4-[2-(Dimethylcarbamoyl)-3-methylbenzo[b]thiophen-6-yloxy]N-methylpicolinamide (6m). Following the same procedure used to prepare 6l, 6i (50.0 mg, 0.146 mmol), EDCI·HCl (33.5 mg, 0.175 mmol), DIPEA (75.5 mg, 0.584 mmol), and dimethylamine hydrochloride (23.8 mg, 0.292 mmol) were used to obtain 6m (25.9 mg, 48%) as off-white solid; mp 177−179 °C. 1H NMR (300 MHz, CDCl3) δ 8.40 (d, J = 5.5 Hz, 1H), 8.00 (br s, 1H), 7.74 (d, J = 8.7 Hz, 1H), 7.68 (d, J = 2.6 Hz, 1H), 7.54 (d, J = 2.2 Hz, 1H), 7.16 (dd, J = 8.6, 2.2 Hz, 1H), 7.00 (dd, J = 5.6, 2.6 Hz, 1H), 3.12 (s, 6H), 3.00 (d, J = 5.1 Hz, 3H), 2.43 (s, 3H). 13C NMR (126 MHz, CDCl3) δ 166.43, 165.41, 164.45, 152.37, 151.66, 149.75, 140.56, 137.30, 131.43, 130.52, 124.06, 118.43, 114.50, 114.33, 110.12, 26.13, 12.79. IR (diamond) 3386, 3069, 2916, 2845, 1673, 1609, 1179 cm−1. HRMS (EI) m/z calcd for C19H19N3O3S [M+] 369.1147, found 369.1146. Ethyl 3-Amino-6-{[2-(methylcarbamoyl)pyridin-4-yl]oxy}benzo[b]thiophene-2-carboxylate (6n). Step 1: Ethyl 3-Amino-6methoxybenzo[b]thiophene-2-carboxylate (15n). Following the same procedure used to prepare 15a, 2-fluoro-4-methoxybenzonitrile (5.21 g, 34.5 mmol), ethyl mercaptoacetate (4.57 g, 38.0 mmol), and K2CO3 (9.54 g, 69.0 mmol) were used to obtain 15n (5.63 g, 65%) as yellow solid. 1H NMR (300 MHz, CDCl3) δ 7.53 (d, J = 8.8 Hz, 1H), 7.18 (d, J = 2.3 Hz, 1H), 6.99 (dd, J = 8.9, 2.3 Hz, 1H), 5.87 (s, 2H), 4.37 (q, J = 7.1 Hz, 2H), 3.91 (s, 3H), 1.41 (t, J = 7.1 Hz, 3H). LC/ MS (ESI) [M + H]− = 252.1. Step 2: Ethyl 3-Amino-6-hydroxybenzo[b]thiophene-2-carboxylate (16n). Following the same procedure used to prepare 16a, ethyl 3-amino-6-methoxybenzo[b]thiophene-2-carboxylate (15n, 251 mg, 1.00 mmol), and BBr3 (1.0 M solution in CH2Cl2, 2.5 mL, 2.5 mmol) were used to obtain 16n (176 mg, 74%) as yellow solid. 1H NMR (300 MHz, methanol-d4) δ 7.74 (d, J = 8.8 Hz, 1H), 7.05 (d, J = 2.2 Hz, 1H), 6.87 (dd, J = 8.8, 2.2 Hz, 1H), 4.30 (q, J = 7.1 Hz, 2H), 1.36 (t, J = 7.1 Hz, 3H). LC/MS (ESI) [M + H]+ = 237.7. Step 3: Ethyl 3-Amino-6-{[2-(methylcarbamoyl)pyridin-4-yl]oxy}benzo[b]thiophene-2-carboxylate (6n). Following the same proce5485



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16q (85.4 mg, 74%) as off-white solid. 1H NMR (300 MHz, CDCl3) δ 8.01 (s, 1H), 7.71 (d, J = 8.7 Hz, 1H), 7.16 (d, J = 8.7 Hz, 1H), 5.81 (s, 1H), 4.43 (q, J = 7.1 Hz, 2H), 1.44 (t, J = 7.1 Hz, 3H). LC/MS (ESI) [M − H]− = 254.8. Step 2: 7-Chloro-6-hydroxybenzo[b]thiophene-2-carboxylic Acid (17q). Following the same procedure used to prepare 17a, 16q (150 mg, 0.584 mmol), and KOH (65.6 mg, 1.17 mmol) were used to obtain 17q (123 mg, 92%) as off-white solid. 1H NMR (300 MHz, acetone-d6) δ 9.43 (br s, 1H), 8.09 (s, 1H), 7.85 (d, J = 8.6 Hz, 1H), 7.24 (d, J = 8.6 Hz, 1H), 6.17 (br s, 1H). LC/MS (ESI) [M − H]− = 226.7. Step 3: Ethyl 7-Chloro-6-{[2-(methylcarbamoyl)pyridin-4-yl]oxy}benzo[b]thiophene-2-carboxylate (6q). Following the same procedure used to prepare 6p, 17q (34.5 mg, 0.151 mmol), 10 (25.7 mg, 0.151 mmol), and Cs2CO3 (147 mg, 0.452 mmol) were used to obtain 6q (31.3 mg, 53%) as off-white solid; mp 168−170 °C. 1H NMR (300 MHz, CDCl3) δ 8.44 (d, J = 5.6 Hz, 1H), 8.10 (s, 1H), 8.03 (br s, 1H), 7.85 (d, J = 8.6 Hz, 1H), 7.66 (d, J = 2.6 Hz, 1H), 7.25 (d, J = 8.6 Hz, 1H), 7.01 (dd, J = 5.6, 2.6 Hz, 1H), 4.45 (q, J = 7.1 Hz, 2H), 3.02 (d, J = 5.1 Hz, 3H), 1.45 (t, J = 7.1 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 165.35, 164.31, 162.11, 152.51, 149.86, 147.87, 143.50, 137.41, 135.72, 130.38, 125.16, 120.65, 120.57, 113.87, 109.59, 61.95, 26.15, 14.30. IR (diamond) 3407, 3075, 2922, 2853, 1705, 1678, 1235 cm−1. HRMS (EI) m/z calcd for C18H15ClN2O4S [M+] 390.0441, found 390.0440. Ethyl 6-Phenoxybenzo[b]thiophene-2-carboxylate (6r). Pyridine (107 mg, 1.35 mmol) was added to a mixture of 16a (100 mg, 0.450 mmol), phenylboronic acid (82.3 mg, 0.675 mmol), and Cu(OAc)2· H2O (135 mg, 0.675 mmol) in CH2Cl2 (15 mL). The mixture was stirred at 25 °C for 12 h and diluted by adding satd aq NH4Cl (20 mL). The aqueous layer was extracted with CH2Cl2 (20 mL × 3). The combined organic layer was dried over MgSO4 and concentrated, and the residue was purified by SiO2 column chromatography (EtOAc/Hx = 1:6) to obtain 6r (12.5 mg, 9%) as colorless oil. 1H NMR (300 MHz, CDCl3) δ 8.03 (d, J = 0.8 Hz, 1H), 7.83 (d, J = 8.7 Hz, 1H), 7.48−7.35 (m, 3H), 7.25−7.05 (m, 4H), 4.42 (q, J = 7.1 Hz, 2H), 1.43 (t, J = 7.1 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 162.72, 157.18, 156.57, 143.73, 134.46, 132.72, 130.04, 129.98, 126.60, 124.01, 119.52, 117.79, 110.88, 61.52, 14.36. IR (diamond) 3062, 2980, 2932, 1705, 1588, 1211 cm−1. HRMS (EI) m/z calcd for C17H14O3S [M+] 298.0664, found 298.0664. Ethyl 3-Methyl-6-phenoxybenzo[b]thiophene-2-carboxylate (6s). Pyridine (101 mg, 1.27 mmol) was added to a mixture of 16b (100 mg, 0.423 mmol), phenylboronic acid (77.4 mg, 0.635 mmol), and Cu(OAc)2·H2O (127 mg, 0.635 mmol) in ClCH2CH2Cl (15 mL). The mixture was stirred at 85 °C for 12 h. The mixture was cooled to 25 °C and diluted by adding satd aq NH4Cl (20 mL). The aqueous layer was extracted with CH2Cl2 (20 mL × 3). The combined organic layer was dried over MgSO4 and concentrated, and the residue was purified by SiO2 column chromatography (EtOAc/Hx = 1:6) to obtain 6s (72.5 mg, 52%) as yellow oil. 1H NMR (300 MHz, CDCl3) δ 7.76 (d, J = 8.8 Hz, 1H), 7.43−7.31 (m, 3H), 7.20−7.03 (m, 4H), 4.37 (q, J = 7.1 Hz, 2H), 2.74 (s, 3H), 1.40 (t, J = 7.1 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 163.39, 157.30, 156.72, 141.81, 140.70, 136.00, 129.94, 125.96, 124.82, 123.90, 119.39, 117.21, 110.92, 61.09, 14.38, 13.26. IR (diamond) 3064, 2982, 2922, 2850, 1712, 1214 cm−1. HRMS (EI) m/z calcd for C18H16O3S [M+] 312.0820, found 312.0817. Ethyl 3-Methyl-6-[3-(methylcarbamoyl)phenoxy]benzo[b]thiophene-2-carboxylate (6t). Following the same procedure used to prepare 6s, 16b (50.0 mg, 0.212 mmol), [3-(methylcarbamoyl)phenyl]boronic acid (45.5 mg, 0.254 mmol), Cu(OAc)2·H2O (63.5 mg, 0.318 mmol), and pyridine (50.3 mg, 0.636 mmol) were used to obtain 6t (16.7 mg, 21%) as yellow solid; mp 94−96 °C. 1H NMR (300 MHz, CDCl3) δ 7.80 (dd, J = 8.9, 0.5 Hz, 1H), 7.54 (ddd, J = 7.7, 1.7, 1.1 Hz, 1H), 7.49−7.39 (m, 2H), 7.37 (dd, J = 2.3, 0.5 Hz, 1H), 7.19 (ddd, J = 8.1, 2.5, 1.1 Hz, 1H), 7.14 (dd, J = 8.8, 2.2 Hz, 1H), 6.23 (s, 1H), 4.40 (q, J = 7.1 Hz, 2H), 3.00 (d, J = 4.9 Hz, 3H), 2.77 (s, 3H), 1.43 (t, J = 7.1 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 167.38, 163.34, 157.26, 156.54, 141.80, 140.62, 136.72, 130.16, 126.34, 125.02,

dure used to prepare 6a, 16n (850 mg, 3.58 mmol), and 10 (611 mg, 3.58 mmol) were used to obtain 6n (75.8 mg, 6%) as yellow solid; mp 136−138 °C. 1H NMR (300 MHz, chloroform-d) δ 8.44 (d, J = 5.6 Hz, 1H), 8.02 (br s, 1H), 7.77 (d, J = 2.5 Hz, 1H), 7.70 (d, J = 8.7 Hz, 1H), 7.46 (d, J = 2.1 Hz, 1H), 7.13 (dd, J = 8.7, 2.2 Hz, 1H), 7.02 (dd, J = 5.6, 2.6 Hz, 1H), 5.95 (s, 2H), 4.39 (q, J = 7.1 Hz, 2H), 3.04 (d, J = 5.1 Hz, 3H), 1.42 (t, J = 7.1 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 165.92, 165.23, 164.30, 154.00, 152.36, 149.75, 147.88, 141.53, 129.14, 123.11, 117.57, 114.86, 114.47, 110.64, 99.65, 60.52, 26.20, 14.52. IR (diamond) 3431, 3376, 3315, 3072, 2930, 2853, 1670, 1656, 1514, 1282 cm−1. HRMS (EI) m/z calcd for C18H17N3O4S [M+] 371.0940, found 371.0939. Ethyl 3-Cyano-6-{[2-(methylcarbamoyl)pyridin-4-yl]oxy}benzo[b]thiophene-2-carboxylate (6o). Aqueous solution of HBF4 (48%, 125 μL) and tert-butylnitrite (132 mg, 1.28 mmol) were added sequentially to a suspension of 6n (52.6 mg, 0.142 mmol) in EtOH at 0 °C. The solution was stirred at 25 °C for 2 h. Then Et2O (10 mL) was added to the mixture to form yellow precipitate which was washed with Et2O (30 mL) to obtain the diazonium salt, which was suspended in CH3CN (1.0 mL). The suspension was added to a mixture of CuCN (17.9 mg, 0.2 mmol) and NaCN (9.8 mg, 0.2 mmol) in CH3CN (0.5 mL) dropwise. The resulting mixture was stirred at 25 °C for 3 h and diluted with EtOAc (30 mL) and H2O (30 mL). The aqueous phase was extracted with EtOAc (10 mL × 3). The combined organic layer was dried over MgSO4 and concentrated, and the residue was purified by SiO2 column chromatography (EtOAc/Hx = 1:2) to obtain 6o (7.2 mg, 13%) as yellow solid; mp 166−168 °C. 1H NMR (300 MHz, CDCl3) δ 8.49 (d, J = 5.5 Hz, 1H), 8.15 (d, J = 8.8 Hz, 1H), 8.03 (br s, 1H), 7.76 (d, J = 2.5 Hz, 1H), 7.64 (d, J = 2.2 Hz, 1H), 7.37 (dd, J = 8.8, 2.2 Hz, 1H), 7.07 (dd, J = 5.5, 2.6 Hz, 1H), 4.55 (q, J = 7.1 Hz, 2H), 3.04 (d, J = 5.1 Hz, 3H), 1.51 (t, J = 7.1 Hz, 3H). 13 C NMR (126 MHz, CDCl3) δ 165.45, 164.31, 160.08, 154.60, 152.82, 150.21, 142.17, 141.34, 135.72, 126.17, 120.84, 115.00, 114.25, 112.83, 110.83, 110.35, 63.22, 26.32, 14.25. IR (diamond) 3415, 3323, 3083, 2985, 2935, 1710, 1670, 1512, 1198 cm−1. HRMS (EI) m/z calcd for C19H15N3O4S [M+] 381.0783, found 381.0785. Ethyl 3-Chloro-6-{[2-(methylcarbamoyl)pyridin-4-yl]oxy}benzo[b]thiophene-2-carboxylate (6p). A mixture of 3-chloro-6hydroxybenzo[b]thiophene-2-carboxylic acid (17p, 100 mg, 0.438 mmol),40 10 (74.6 mg, 0.438 mmol), and Cs2CO3 (428 mg, 1.31 mmol) in DMSO (2 mL) was stirred at 85 °C for 2 h. The mixture was then cooled to 25 °C and diluted with CH2Cl2 (50 mL) and H2O (50 mL). The aqueous layer was extracted with 5% EtOH in CH2Cl2 (20 mL × 3). The combined organic layer was washed with H2O (10 mL), dried over MgSO4, and concentrated, and the residue was taken into 3% H2SO4 in EtOH (3 mL) and refluxed at 80 °C for 12 h. It was then cooled to room temperature, basified with NaHCO3, and diluted with EtOAc (30 mL) and H2O (30 mL). The aqueous layer was extracted with EtOAc (10 mL × 3), and the combined organic layer was dried over MgSO4 and concentrated, and the residue was purified by SiO2 column chromatography (EtAOc/Hx = 1:4) to obtain 6p (17 mg, 13%) as off-white solid; mp 139−141 °C. 1H NMR (300 MHz, CDCl3) δ 8.45 (d, J = 5.6 Hz, 1H), 8.10−7.97 (m, 2H), 7.76 (d, J = 2.5 Hz, 1H), 7.55 (d, J = 2.2 Hz, 1H), 7.28−7.24 (m, 1H), 7.04 (dd, J = 5.6, 2.6 Hz, 1H), 4.47 (q, J = 7.1 Hz, 2H), 3.04 (d, J = 5.1 Hz, 3H), 1.46 (t, J = 7.1 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 165.64, 164.28, 161.00, 154.20, 152.59, 149.95, 139.99, 134.70, 127.01, 126.39, 125.80, 119.45, 114.61, 114.10, 110.66, 61.91, 26.16, 14.27. IR (diamond) 3402, 2985, 2924, 2850, 1691, 1668, 1509, 1285 cm−1. HRMS (EI) m/z calcd for C18H15ClN2O4S [M+] 390.0441, found 390.0439. Ethyl 7-Chloro-6-{[2-(methylcarbamoyl)pyridin-4-yl]oxy}benzo[b]thiophene-2-carboxylate (6q). Step 1: Ethyl 7-Chloro-6hydroxybenzo[b]thiophene-2-carboxylate (16q). N-Chlorosuccinimide (66.1 mg, 0.495 mmol) was added to a solution of 16a (100 mg, 0.450 mmol) in THF (2 mL). The mixture was stirred at 30 °C for 6 h then diluted with EtOAc (50 mL) and H2O (50 mL). The aqueous layer was extracted with EtOAc (20 mL × 3). The combined organic layer was dried over MgSO4 and concentrated, and the residue was purified by SiO2 column chromatography (EtOAc/Hx = 1:5) to obtain 5486



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mg, 52%) as off-white solid. 1H NMR (300 MHz, CDCl3) δ 7.53 (d, J = 8.7 Hz, 1H), 7.46 (d, J = 1.0 Hz, 1H), 7.07 (s, 1H), 6.93 (dd, J = 8.7, 2.3 Hz, 1H), 4.42 (q, J = 7.1 Hz, 2H), 3.86 (s, 3H), 1.42 (t, J = 7.1 Hz, 3H). LC/MS (ESI) [M + H]+ = 220.7. Step 2: 6-Methoxybenzofuran-2-carboxylic Acid (20a). Following the same procedure used to prepare 17a, 19a (600. mg, 2.91 mmol) and KOH (327 mg, 5.82 mmol) were reacted at 65 °C for 10 h to obtain 20a (491 mg, 88%). 1H NMR (300 MHz, acetone-d6) δ 7.66 (d, J = 8.7 Hz, 1H), 7.58 (s, 1H), 7.20 (d, J = 1.7 Hz, 1H), 6.98 (dd, J = 8.7, 2.3 Hz, 1H), 5.68 (br s, 1H), 3.91 (s, 3H). LC/MS (ESI) [M + H]+ = 192.6. Step 3: 6-Hydroxybenzofuran-2-carboxylic Acid (21a). Following the same procedure used to prepare 16a, 21a (450 mg, 2.34 mmol) and BBr3 (1.0 M in CH2Cl2, 5.9 mL, 5.9 mmol) were reacted at −78 °C for 1 h then at 10 °C for 11 h to obtain 21a (286 mg, 69%). 1H NMR (300 MHz, acetone-d6) δ 7.61 (d, J = 8.6 Hz, 1H), 7.57 (d, J = 1.0 Hz, 1H), 7.05 (d, J = 2.2 Hz, 1H), 6.95 (dd, J = 8.5, 2.1 Hz, 1H). LC/MS (ESI) [M + H]+ = 176.8. Step 4: Ethyl 6-{[2-(Methylcarbamoyl)pyridin-4-yl]oxy}benzofuran-2-carboxylate (7a). 10 (27.3 mg, 0.160 mmol) and Cs2CO3 (156 mg, 0.480 mmol) were sequentially added to a suspension of 6-hydroxybenzofuran-2-carboxylic acid (21a, 28.5 mg, 0.160 mmol) in DMSO (5 mL) and the resulting mixture was stirred at 120 °C for 10 h, cooled to 25 °C, and diluted with EtOAc (20 mL) and H2O (20 mL). The aqueous phase was acidified to pH = 4 and extracted with 5% iPrOH in EtOAc (10 mL × 3). The combined organic layer was washed with H2O (5 mL), dried over MgSO4, and concentrated to obtain crude 6-{[2-(methylcarbamoyl)pyridin-4yl]oxy}benzofuran-2-carboxylic acid (22a). The crude 22a was dissolved in 5% H2SO4 in EtOH (2.0 mL). After refluxing at 85 °C for 24 h, the reaction mixture was cooled to 25 °C and diluted with EtOAc (30 mL) and aq NaHCO3 (30 mL). The aqueous phase was extracted with EtOAc (10 mL × 3). The combined organic layer was dried over MgSO4 and concentrated, and the residue was purified by SiO2 column chromatography (EtOAc/Hx = 1:2) to obtain 7a (32.8 mg, 60%) as yellow crystal; mp 98−100 °C. 1H NMR (300 MHz, CDCl3) δ 8.41 (d, J = 5.6 Hz, 1H), 8.01 (br s, 1H), 7.73 (s, 1H), 7.71 (d, J = 6.8 Hz, 1H), 7.55 (d, J = 1.0 Hz, 1H), 7.34 (d, J = 1.6 Hz, 1H), 7.08 (dd, J = 8.5, 2.1 Hz, 1H), 7.01 (dd, J = 5.6, 2.6 Hz, 1H), 4.46 (q, J = 7.1 Hz, 2H), 3.01 (d, J = 5.1 Hz, 3H), 1.44 (t, J = 7.1 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 165.97, 164.34, 159.21, 156.11, 153.72, 152.49, 149.82, 146.87, 124.69, 124.05, 117.59, 114.41, 113.53, 110.38, 105.07, 61.64, 26.13, 14.32. IR (diamond) 3360, 3067, 2990, 2924, 2856, 1731, 1662, 1522, 1221 cm−1. HRMS (EI) m/z calcd for C18H16N2O5 [M+] 340.1059, found 340.1061. Isopropyl 1-Methyl-6-{[2-(methylcarbamoyl)pyridin-4-yl]oxy}-1Hindole-2-carboxylate (7c). Step 1: Ethyl (3-Methoxyphenyl)glycinate (24). 19b and the intermediate 24 were prepared by following the reference procedure.41 3-Methoxyaniline (12.0 g, 97.4 mmol) was added to K2CO3 (20.2 g, 146 mmol) in acetone (100 mL) and refluxed at 60 °C for 1 h. Ethyl bromoacetate (1.79 g, 107 mmol) was added at 60 °C, and the mixture was further stirred at 60 °C for 7 h. After cooling to 25 °C, the reaction mixture was filtered through Celite and the filtrate was concentrated under reduced pressure. The residue was purified by SiO2 column chromatography (EtOAc/Hx = 1:10) to obtain ethyl (3-methoxyphenyl)glycinate (24) as the intermediate (13.9 g, 68%). 1H NMR (300 MHz, CDCl3) δ 7.12 (t, J = 8.1 Hz, 1H), 6.34 (d, J = 7.2 Hz, 1H), 6.25 (d, J = 8.0 Hz, 1H), 6.18 (s, 1H), 4.33 (s, 1H), 4.27 (q, J = 7.1 Hz, 2H), 3.91 (s, 2H), 3.79 (s, 3H), 1.32 (t, J = 7.1 Hz, 3H). LC/MS (ESI) [M + H]+ = 210.9. Step 2: Ethyl 6-methoxy-1H-indole-2-carboxylate (19b). 24 (13.9 g, 66.4 mmol) was taken into N,N-dimethylformamide dimethyl acetal (15.8 g, 133 mmol), and the reaction mixture was stirred at 90 °C for 48 h. The crude mixture was concentrated under reduced pressure, and the residue was purified by SiO2 column chromatography (EtOAc/Hx = 1:4) to obtain ethyl (E)-3-(dimethylamino)-2-[(3methoxyphenyl)amino)acrylate (860 mg, 5%) as the intermediate. AlCl3 (430. mg, 3.22 mmol) was added to a solution of the intermediate in CH2Cl2 (250 mL), and the reaction mixture was stirred at 25 °C for 24 h. The reaction mixture was filtered, and the

121.96, 117.53, 117.35, 111.57, 61.15, 26.88, 14.36, 13.24. IR (diamond) 3318, 3064, 2922, 2853, 1705, 1638, 1522, 1219 cm−1. HRMS (EI) m/z calcd for C20H19NO4S [M+] 369.1035, found 369.1024. Ethyl 6-[(Pyridin-4-yl)oxy]benzo[b]thiophene-2-carboxylate (6u). Following the same procedure used to prepare 6s, 16a (100 mg, 0.450 mmol), 4-pyridylboronic acid (110.6 mg, 0.900 mmol), Cu(OAc)2 (122.6 mg, 0.675 mmol), and pyridine (107 mg, 1.35 mmol) were used to obtain 6u (12.1 mg, 9%) as off-white solid; mp 81−83 °C. 1H NMR (300 MHz, CDCl3) δ 8.52 (d, J = 6.4 Hz, 1H), 8.08 (d, J = 0.8 Hz, 1H), 7.93 (d, J = 8.7 Hz, 1H), 7.60 (d, J = 2.1 Hz, 1H), 7.19 (dd, J = 8.7, 2.2 Hz, 1H), 6.91 (d, J = 6.4 Hz, 1H), 4.44 (q, J = 7.1 Hz, 1H), 1.45 (t, J = 7.1 Hz, 2H). 13C NMR (126 MHz, CDCl3) δ 164.59, 162.60, 153.46, 151.70, 143.67, 136.30, 134.41, 129.97, 127.17, 119.13, 114.20, 112.58, 61.86, 14.46. IR (diamond) 3059, 2985, 2924, 2853, 1699, 1575, 1243 cm−1. HRMS (EI) m/z calcd for C16H13NO3S [M+] 299.0616, found 299.0618. Methyl 4-{[2-(Ethoxycarbonyl)benzo[b]thiophen-6-yl]oxy}picolinate (6v). Methyl 4-chloropicolinate (11, 109 mg, 0.635 mmol) was added to a solution of 16b (100 mg, 0.423 mmol) in chlorobenzene (0.65 mL). The mixture was stirred at 120 °C for 12 h, cooled to 25 °C, diluted with EtOAc (30 mL), and washed with aq NaHCO3. The aqueous phase was extracted with EtOAc (10 mL × 3), and the combined organic layer was dried over MgSO4 then concentrated. The residue was purified by SiO2 column chromatography (EtOAc/Hx = 1:4) to obtain 6v (10.1 mg, 6%) as white solid; mp 129−131 °C. 1H NMR (300 MHz, CDCl3) δ 8.63 (d, J = 5.6 Hz, 1H), 7.91 (d, J = 8.8 Hz, 1H), 7.70 (d, J = 2.5 Hz, 1H), 7.57 (d, J = 2.2 Hz, 1H), 7.19 (dd, J = 8.8, 2.2 Hz, 1H), 7.07 (dd, J = 5.6, 2.5 Hz, 1H), 4.43 (q, J = 7.1 Hz, 2H), 4.00 (s, 3H), 2.82 (s, 3H), 1.44 (t, J = 7.1 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 165.65, 165.31, 163.12, 153.03, 151.49, 150.10, 141.79, 140.45, 138.03, 127.68, 125.51, 118.27, 115.12, 114.18, 113.71, 61.33, 53.06, 14.35, 13.25. IR (diamond) 3468, 3070, 2922, 2848, 1718, 1683, 1586, 1277 cm−1. HRMS (EI) m/z calcd for C19H17NO5S [M+] 371.0827, found 371.0824. Isopropyl 3-Methyl-6-{[6-(methylcarbamoyl)pyridin-3-yl]oxy}benzo[b]thiophene-2-carboxylate (6x). Step 1: 3-Methyl-6-[6(methylcarbamoyl)pyridin-3-yloxy]benzo[b]thiophene-2-carboxylic Acid (6w). Following the same procedure used to prepare 6i, 17b (577 mg, 2.77 mmol), 5-fluoro-N-methylpicolinamide (13, 427 mg, 2.77 mmol), and Cs2CO3 (2.7 g, 8.31 mmol) were reacted at 100 °C for 15 h to obtain 6w (378 mg, 40%). 1H NMR (300 MHz, DMSO-d6) δ 8.71 (q, J = 4.6 Hz, 1H), 8.47 (d, J = 2.6 Hz, 1H), 8.05 (d, J = 9.0 Hz, 1H), 8.02 (d, J = 9.4 Hz, 1H), 7.82 (d, J = 2.3 Hz, 1H), 7.58 (dd, J = 8.6, 2.9 Hz, 1H), 7.31 (dd, J = 8.9, 2.3 Hz, 1H), 2.82 (d, J = 4.9 Hz, 3H), 2.72 (s, 3H). LC/MS (ESI) [M + H]+ = 342.6. Step 2: Isopropyl 3-Methyl-6-{[6-(methylcarbamoyl)pyridin-3yl]oxy}benzo[b]thiophene-2-carboxylate (6x). Following the same procedure used to prepare 6j, 6w (100 mg, 0.292 mmol), EDCI·HCl (61.6 mg, 0.321 mmol), DMAP (7.1 mg, 0.058 mmol), iPrOH (35 mg, 0.58 mmol), and DMF as the solvent (0.6 mL) were used at 25 °C for 15 h to obtain 6x (76 mg, 68%) as white solid; mp 125−127 °C. 1H NMR (300 MHz, DMSO-d6) δ 8.71 (br s, 1H), 8.48 (d, J = 2.6 Hz, 1H), 8.06 (dd, J = 8.6, 2.2 Hz, 2H), 7.84 (d, J = 2.2 Hz, 1H), 7.59 (dd, J = 8.6, 2.8 Hz, 1H), 7.33 (dd, J = 8.9, 2.3 Hz, 1H), 5.15 (hept, J = 6.2 Hz, 1H), 2.82 (d, J = 4.8 Hz, 3H), 2.74 (s, 3H), 1.34 (d, J = 6.2 Hz, 6H). 13C NMR (126 MHz, CDCl3) δ 164.42, 162.73, 156.11, 154.91, 144.99, 141.78, 140.12, 139.08, 137.31, 127.73, 125.40, 125.27, 123.40, 117.40, 112.51, 69.01, 26.12, 21.98, 13.23. IR (diamond) 3355, 3091, 2971, 2927, 2882, 1681, 1660, 1533, 1232 cm−1. HRMS (EI) m/z calcd for C20H20N2O4S [M+] 384.1144, found 384.1142. Ethyl 6-{[2-(Methylcarbamoyl)pyridin-4-yl]oxy}benzofuran-2-carboxylate (7a). Step 1: Ethyl 6-Methoxybenzofuran-2-carboxylate (19a). Ethyl bromoacetate (1.06 g, 6.32 mmol) was added to a mixture of 2-hydroxy-4-methoxybenzaldehyde (18, 1.00 g, 6.02 mmol) and K2CO3 (1.66 g, 12.0 mmol) in DMF (10 mL) and stirred at 80 °C for 30 h. The reaction mixture was diluted with EtOAc (50 mL) and H2O (50 mL). The aqueous layer was extracted with EtOAc (50 mL × 3), dried over MgSO4, and concentrated, and the residue was purified by SiO2 column chromatography (EtOAc/Hx = 1:20) to obtain 19a (643 5487



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filtrate was washed with H2O (60 mL × 3). The organic layer was dried over Na2SO4 and concentrated, and the residue was purified by SiO2 column chromatography (EtOAc/Hx = 1:4) to obtain 19b (345 mg, 49%). 1H NMR (300 MHz, CDCl3) δ 8.89 (br s, 1H), 7.57 (d, J = 8.9 Hz, 1H), 7.19 (s, 1H), 6.85 (d, J = 7.8 Hz, 2H), 4.12 (q, J = 7.1 Hz, 2H), 3.88 (s, 3H), 1.43 (t, J = 7.1 Hz, 3H). LC/MS (ESI) [M + H]+ = 219.8. Step 3: Ethyl 6-Methoxy-N-methylindole-2-carboxylate (19c). NaH (60% in mineral oil, 145 mg, 3.61 mmol) was added to a solution of 19b (660 mg, 3.01 mmol) at 0 °C, and the reaction mixture was stirred at 0 °C for 30 min. Iodomethane (512 mg, 3.61 mmol) was added dropwise, and the reaction mixture was stirred at 25 °C for 8 h. Aqueous NH4Cl (100 mL) was added to the reaction mixture, and the aqueous layer was extracted with EtOAc (50 mL x 3). The combined organic layer was dried over Na2SO4 and concentrated, and the residue was purified by SiO2 column chromatography (EtOAc/Hx = 1:20) to obtain 19c (581 mg, 83%). 1H NMR (300 MHz, CDCl3) δ 7.56 (d, J = 8.7 Hz, 1H), 7.27 (d, J = 3.1 Hz, 1H), 6.84 (dd, J = 8.7, 2.1 Hz, 1H), 6.78 (s, 1H), 4.38 (q, J = 7.1 Hz, 2H), 4.06 (s, 3H), 3.92 (s, 3H), 1.42 (t, J = 7.1 Hz, 3H). LC/MS (ESI) [M + H]+ = 234.0. Step 4: 6-Methoxy-N-methylindole-2-carboxylic Acid (20c). Followed the same procedure used to prepare 17a, 19c (100 mg, 0.429 mmol) and KOH (120. mg, 2.15 mmol) were reacted at 65 °C for 10 h to obtain 20c (60.0 mg, 68%). 1H NMR (300 MHz, DMSOd6) δ 7.44 (d, J = 8.6 Hz, 1H), 6.94 (d, J = 1.8 Hz, 1H), 6.87 (s, 1H), 6.70 (dd, J = 8.6, 2.2 Hz, 1H), 4.02 (s, 3H), 3.82 (s, 3H). LC/MS (ESI) [M + H]+ = 205.9. Step 5: 6-Hydroxy-N-methylindole-2-carboxylic acid (21c). Followed the same procedure used to prepare 16a, 20c (60.0 mg, 0.293 mmol) and BBr3 (1.0 M in CH2Cl2, 580 μL, 0.58 mmol) were reacted at −78 °C for 1 h then at 10 °C for 11 h to obtain 21c (15.0 mg, 27%). 1H NMR (300 MHz, DMSO-d6) δ 9.51 (s, 1H), 7.45 (d, J = 8.6 Hz, 1H), 7.09 (s, 1H), 6.76 (s, 1H), 6.67 (d, J = 8.8 Hz,1 H), 3.90 (s, 3H). LC/MS (ESI) [M − H]− = 189.9. Step 6. Isopropyl 6-{[2-(methylcarbamoyl)pyridin-4-yl]oxy}-Nmethylindole-2-carboxylate (7c). Following the same procedure to prepare 22a, 21c (35.0 mg, 0.183 mmol), 10 (32.0 mg, 0.183 mmol), and Cs2CO3 (176 mg, 0.543 mmol) were used to obtain crude 1methyl-6-{[2-(methylcarbamoyl)pyridin-4-yl]oxy}-1H-indole-2-carboxylic acid (22c). To a solution of the crude 22c in CH2Cl2 (10 mL) was added EDCI·HCl (46.4 mg, 0.242 mmol) at 0 °C. After 10 min stirring at 0 °C, iPrOH (26.4 mg, 0.440 mmol) and DMAP (2.7 mg, 0.022 mmol) were added sequentially and the mixture was stirred at 25 °C for 16 h. The mixture was then diluted with EtOAc (50 mL) and H2O (30 mL), and the aqueous phase was extracted with EtOAc (20 mL × 3). The combined organic layer was dried over MgSO4 and concentrated, and the residue was purified by SiO2 column chromatography (EtOAc/Hx = 1:4) to obtain 7c (5.0 mg, 9%) as colorless oil. 1H NMR (300 MHz, DMSO-d6) δ 8.77 (br s, 1H), 8.52 (d, J = 5.6 Hz, 1H), 7.82 (d, J = 8.6 Hz, 1H), 7.56 (s, 1H), 7.40 (d, J = 2.5 Hz, 1H), 7.32(s, 1H), 7.18 (dd, J = 5.6, 2.6 Hz, 1H), 6.99 (dd, J = 8.6, 2.0 Hz, 1H), 5.17 (sep, J = 6.2 Hz, 1H), 4.00 (s, 3H), 2.79 (d, J = 4.8 Hz, 3H), 1.36 (d, J = 6.2 Hz, 6H). 13C NMR (126 MHz, CDCl3) δ 166.91, 164.69, 161.61, 152.41, 151.78, 149.76, 140.21, 129.70, 124.36, 123.93, 114.83, 114.19, 110.42, 110.28, 102.52, 68.33, 32.04, 26.25, 22.14. IR (diamond) 3394, 3062, 2980, 2935, 1707, 1671, 1530, 1224 cm−1. HRMS (EI) m/z calcd for C20H21N3O4 [M+] 367.1532, found 367.1528. Isopropyl 3-Methyl-6-[3-(methylcarbamoyl)phenoxy]-1H-indole2-carboxylate (7d). Step 1: Isopropyl 6-Methoxy-3-methyl-1Hindole-2-carboxylate (19d). 19d was prepared by following the reference procedure.42 DMSO used in this reaction was degassed by using three cycles of freeze−thawing under high vacuum. A solution of 1-(2-chloro-4-methoxyphenyl)ethan-1-one (25, 1.49 g, 8.05 mmol) in DMSO (5 mL) and a solution of isopropyl 2-isocyanoacetate (1.00 g, 8.86 mmol) in DMSO (5 mL) were sequentially added to a mixture of CuI (1.54 g, 8.06 mmol) and Cs2CO3 (5.25 g, 16.1 mmol) in DMSO (20 mL). The mixture was stirred at 80 °C for 24 h, cooled to 25 °C, and diluted with EtOAc (300 mL) and H2O (300 mL). The aqueous layer was extracted with EtOAc (50 mL × 3). The combined organic

layer was washed with brine (20 mL), dried over MgSO4, and concentrated, and the residue was purified by SiO2 column chromatography (EtOAc/Hx = 1:9) to obtain 19d (380 mg, 19%). 1 H NMR (300 MHz, CDCl3) δ 8.61 (s, 1H), 7.51 (d, J = 8.6 Hz, 1H), 7.26 (s, 1H), 6.85−6.72 (m, J = 8.3 Hz, 2H), 5.28 (sep, J = 6.2 Hz, 1H), 3.85 (s, 3H), 2.57 (s, 3H), 1.39 (d, J = 6.3 Hz, 6H). LC/MS (ESI) [M + H]+ = 247.8. Step 2: Isopropyl 6-Hydroxy-3-methyl-1H-indole-2-carboxylate (26d). Following the same procedure used to prepare 16a, 19d (200 mg, 0.809 mmol) and BBr3 (1.0 M solution in CH2Cl2, 2.4 mL, 2.4 mmol) were used to obtain 26d (110 mg, 60%). 1H NMR (300 MHz, CDCl3) δ 8.49 (s, 1H), 7.50 (d, J = 8.6 Hz, 1H), 6.77 (d, J = 1.9 Hz, 1H), 6.71 (dd, J = 8.6, 2.2 Hz, 1H), 5.35−5.20 (m, 1H), 4.98 (s, 1H), 2.56 (s, 3H), 1.39 (d, J = 6.2 Hz, 6H). LC/MS (ESI) [M + H]+ = 233.8 Step 3: Isopropyl 3-Methyl-6-[3-(methylcarbamoyl)phenoxy]-1Hindole-2-carboxylate (7d). Following the same procedure used to prepare 6a, 26d (50.0 mg, 0.214 mmol) and 10 (36.5 mg, 0.214 mmol) were used to obtain 7d (19.7 mg, 25%) as yellow oil. 1H NMR (300 MHz, CDCl3) δ 8.96 (s, 1H), 8.36 (d, J = 5.5 Hz, 1H), 8.02 (br s, 1H), 7.73 (d, J = 1.9 Hz, 1H), 7.66 (d, J = 8.7 Hz, 1H), 7.07 (d, J = 1.9 Hz, 1H), 6.96 (dd, J = 5.4, 2.3 Hz, 1H), 6.87 (dd, J = 8.7, 2.0 Hz, 1H), 5.29 (sep, J = 6.2 Hz, 1H), 3.00 (d, J = 5.1 Hz, 3H), 2.61 (s, 3H), 1.40 (d, J = 6.3 Hz, 6H). 13C NMR (126 MHz, CDCl3) δ 166.69, 164.63, 161.96, 152.22, 152.00, 149.62, 136.14, 126.58, 124.68, 122.44, 120.04, 114.11, 113.88, 110.34, 103.53, 68.40, 26.15, 22.12, 9.99. IR (diamond) 3339, 3064, 2980, 2930, 1691, 1665, 1530, 1227 cm−1. HRMS (EI) m/z calcd for C20H21N3O4 [M+] 367.1532, found 367.1528. Isopropyl 1,3-Dimethyl-6-[2-(methylcarbamoyl)pyridin-4-yloxy]1H-indole-2-carboxylate (7e). To a solution of 7d (10.0 mg, 0.0272 mmol) in DMF (0.5 mL) was added NaH (60% in mineral oil, 1.3 mg, 0.033 mmol) at 0 °C. The mixture was stirred at 0 °C for 30 min. To the mixture was added a solution of MeI (3.9 μL, 0.027 mmol) in DMF (0.5 mL) dropwise at 0 °C and, the resulting mixture was stirred at 25 °C for 8 h and diluted with EtOAc (30 mL) and H2O (30 mL), and the aqueous layer was extracted with EtOAc (10 mL × 5). The combined organic layer was then washed with brine (10 mL), dried over MgSO4, and concentrated, and the residue was purified by SiO2 colum chromatography (EtOAc/Hx = 1:1) to obtain 7e (5.6 mg, 54%) as yellow solid; mp 117−119 °C. 1H NMR (300 MHz, CDCl3) δ 8.39 (d, J = 5.6 Hz, 1H), 8.03 (br s, 1H), 7.74 (d, J = 2.5 Hz, 1H), 7.71 (d, J = 8.6 Hz, 1H), 7.08 (d, J = 1.9 Hz, 1H), 6.98 (dd, J = 5.6, 2.6 Hz, 1H), 6.89 (dd, J = 8.6, 2.0 Hz, 1H), 5.40−5.26 (m, 1H), 3.98 (s, 3H), 3.03 (d, J = 5.1 Hz, 3H), 2.61 (s, 3H), 1.45 (d, J = 6.3 Hz, 6H). 13C NMR (126 MHz, CDCl3) δ 166.83, 164.57, 162.30, 152.27, 151.96, 149.61, 139.06, 126.24, 125.15, 122.48, 120.60, 113.98, 113.73, 110.29, 102.10, 68.17, 32.32, 26.11, 22.18, 10.90. IR (diamond) 3357, 3064, 2980, 2932, 1694, 1673, 1528, 1095 cm−1. HRMS (EI) m/z calcd for C21H23N3O4 [M+] 381.1689, found 381.1691. Methyl 7-{[2-(Methylcarbamoyl)pyridin-4-yl]oxy}]-2-naphthoate (8a). Step 1: 7-{[2-(Methylcarbamoyl)pyridin-4-yl]oxy}-2-naphthoic Acid (28a). Following the same procedure used to prepare 6i, 7hydroxy-2-naphthoic acid (27a, 615 mg, 3.27 mmol), 10 (558 mg, 3.27 mmol), and Cs2CO3 (3.20 g, 9.81 mmol) were used to obtain 28a (960 mg, 91%). 1H NMR (300 MHz, CDCl3) δ 7.80 (dd, J = 8.9, 0.5 Hz, 1H), 7.54 (ddd, J = 7.7, 1.7, 1.1 Hz, 1H), 7.49−7.39 (m, 2H), 7.37 (dd, J = 2.3, 0.5 Hz, 1H), 7.19 (ddd, J = 8.1, 2.5, 1.1 Hz, 1H), 7.14 (dd, J = 8.8, 2.2 Hz, 1H), 6.23 (s, 1H), 4.40 (q, J = 7.1 Hz, 2H), 3.00 (d, J = 4.9 Hz, 3H), 2.77 (s, 3H), 1.43 (t, J = 7.1 Hz, 3H). LC/MS (ESI) [M + H]+ = 370.2 Step 2: Methyl 7-{[2-(Methylcarbamoyl)pyridin-4-yl]oxy}]-2naphthoate (8a). Two to three drops of H2SO4 were added to a suspension of 28a (50.0 mg, 0.155 mmol) in MeOH (5 mL), and the mixture was refluxed at 75 °C for 12 h. The mixture was then cooled to 25 °C and diluted with EtOAc (30 mL) and H2O (50 mL). The aqueous phase was neutralized to pH = 7−8 and extracted with EtOAc (20 mL × 3). The combined organic layer was dried over MgSO4 and concentrated, and the residue was purified by SiO2 column chromatography (EtOAc/Hx = 1:2) to obtain 8a (38.1 mg, 73%) as 5488



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yellow solid; mp 106−108 °C. 1H NMR (300 MHz, CDCl3) δ 8.54 (s, 1H), 8.42 (dd, J = 5.6, 0.5 Hz, 1H), 8.08 (dd, J = 8.6, 1.7 Hz, 1H), 8.02 (br s, 1H), 7.98−7.90 (m, 2H), 7.76 (d, J = 2.1 Hz, 1H), 7.63 (d, J = 2.4 Hz, 1H), 7.35 (dd, J = 8.9, 2.4 Hz, 1H), 7.03 (dd, J = 5.6, 2.6 Hz, 1H), 3.99 (s, 3H), 3.01 (d, J = 5.1 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 166.92, 165.91, 164.39, 152.49, 152.06, 149.85, 133.40, 133.29, 130.50, 130.41, 128.48, 128.17, 125.29, 122.65, 118.67, 114.51, 110.57, 52.35, 26.14. IR (diamond) 3257, 3059, 2953, 1710, 1660, 1530, 1279 cm−1. HRMS (EI) m/z calcd for C19H16N2O4 [M+] 336.1110, found 336.1110. Ethyl 7-{[2-(Methylcarbamoyl)pyridin-4-yl]oxy}-2-naphthoate (8b). Following the same procedure used to prepare 8a, 28a (50.0 mg, 0.155 mmol) and EtOH (5 mL) as the solvent were used to obtain 8b (34.7 mg, 64%) as off-white solid; mp 105−107 °C. 1H NMR (300 MHz, CDCl3) δ 8.56 (s, 1H), 8.44 (d, J = 5.6 Hz, 1H), 8.12 (d, J = 1.7 Hz, 1H), 8.04 (br s, 1H), 8.00−7.91 (m, 2H), 7.78 (d, J = 2.5 Hz, 1H), 7.65 (d, J = 2.4 Hz, 1H), 7.36 (dd, J = 8.9, 2.4 Hz, 1H), 7.04 (dd, J = 5.6, 2.6 Hz, 1H), 4.46 (q, J = 7.1 Hz, 2H), 3.03 (d, J = 5.1 Hz, 3H), 1.46 (t, J = 7.1 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 166.44, 165.93, 164.40, 152.49, 152.04, 149.84, 133.40, 133.26, 130.49, 130.30, 128.83, 128.10, 125.33, 122.57, 118.63, 114.52, 110.58, 61.26, 26.14, 14.38. IR (diamond) 3392, 3064, 3019, 2940, 1710, 1670, 1285 cm−1. HRMS (EI) m/z calcd for C20H18N2O4 [M+] 350.1267, found 350.1263. Isopropyl 7-{[2-(Methylcarbamoyl)pyridin-4-yl]oxy}-2-naphthoate (8c). Following the same procedure used to prepare 8a, 28a (50.0 mg, 0.155 mmol) and iPrOH (5 mL) as the solvent were used to obtain 8c (27.0 mg, 48%) as colorless oil. 1H NMR (300 MHz, CDCl3) δ 8.53 (s, 1H), 8.42 (dd, J = 5.6, 0.5 Hz, 1H), 8.08 (dd, J = 8.6, 1.7 Hz, 1H), 8.01 (br s, 1H), 7.99−7.89 (m, 2H), 7.77 (dd, J = 2.6, 0.5 Hz, 1H), 7.64 (s, 1H), 7.35 (dd, J = 8.9, 2.4 Hz, 1H), 7.03 (dd, J = 5.6, 2.5 Hz, 1H), 5.32 (hept, J = 6.2 Hz, 1H), 3.01 (d, J = 5.1 Hz, 3H), 1.42 (d, J = 6.3 Hz, 6H). 13C NMR (126 MHz, CDCl3) δ 165.95, 165.92, 164.41, 152.48, 152.02, 149.84, 133.38, 133.22, 130.49, 130.21, 129.24, 128.03, 125.37, 122.50, 118.60, 114.52, 110.59, 68.73, 26.14, 22.00. IR (diamond) 3396, 3059, 2980, 2932, 1707, 1670, 1528, 1232 cm−1. HRMS (EI) m/z calcd for C21H20N2O4 [M+] 364.1423, found 364.1421. sec-Butyl 7-{[2-(Methylcarbamoyl)pyridin-4-yl]oxy}-2-naphthoate (8d). Following the same procedure used to prepare 8a, 28a (50.0 mg, 0.155 mmol) and sBuOH (5 mL) as the solvent were used to obtain 8d (23.2 mg, 40%) as colorless oil. 1H NMR (300 MHz, CDCl3) δ 8.56−8.51 (m, 1H), 8.42 (dd, J = 5.6, 0.5 Hz, 1H), 8.09 (dd, J = 8.6, 1.7 Hz, 1H), 8.02 (br s, 1H), 7.98−7.89 (m, 2H), 7.77 (dd, J = 2.6, 0.5 Hz, 1H), 7.64 (d, J = 2.4 Hz, 1H), 7.35 (dd, J = 8.9, 2.4 Hz, 1H), 7.02 (dd, J = 5.6, 2.6 Hz, 1H), 5.17 (h, J = 6.3 Hz, 1H), 3.01 (d, J = 5.1 Hz, 3H), 1.89−1.68 (m, 2H), 1.39 (d, J = 6.3 Hz, 3H), 1.01 (t, J = 7.5 Hz, 3H); 13C NMR (126 MHz, CDCl3) δ 166.07, 165.96, 164.41, 152.49, 152.02, 149.84, 133.40, 133.23, 130.49, 130.18, 129.26, 128.05, 125.39, 122.51, 118.63, 114.51, 110.60, 73.24, 29.00, 26.14, 19.61, 9.80. IR (diamond) 3394, 3064, 2969, 2924, 2847, 1710, 1673, 1530, 1227 cm−1. HRMS (EI) m/z calcd for C22H22N2O4 [M+]: 378.1580, found: 378.1579. Phenyl 7-{[2-(Methylcarbamoyl)pyridin-4-yl]oxy}-2-naphthoate (8e). Following the same procedure used to prepare 6j, 28a (50.0 mg, 0.155 mmol), EDCI·HCl (29.7 mg, 0.155 mmol), DMAP (5.7 mg, 0.047 mmol) and phenol (29.1 mg, 0.310 mmol) were used to obtain 8e (27.0 mg, 44%) as white solid; mp 77−79 °C. 1H NMR (300 MHz, CDCl3) δ 8.75 (s, 1H), 8.46 (d, J = 5.6 Hz, 1H), 8.24 (dd, J = 8.6, 1.7 Hz, 1H), 8.13−7.96 (m, 3H), 7.80 (d, J = 2.5 Hz, 1H), 7.70 (d, J = 2.4 Hz, 1H), 7.48 (dd, J = 8.3, 7.3 Hz, 2H), 7.42 (dd, J = 8.9, 2.4 Hz, 1H), 7.36−7.27 (m, 3H), 7.07 (dd, J = 5.6, 2.6 Hz, 1H), 3.03 (d, J = 5.1 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 165.90, 165.04, 164.34, 152.48, 152.24, 150.97, 149.84, 133.59, 133.42, 131.24, 130.60, 129.56, 129.53, 128.42, 127.90, 126.00, 125.54, 123.03, 121.72, 118.73, 114.63, 110.62, 26.16. IR (diamond) 3392, 3062, 2935, 1731, 1673, 1528, 1187 cm−1. HRMS (EI) m/z calcd for C24H18N2O4 [M+] 398.1267, found 398.1269. Ethyl 3-Hydroxy-7-{[2-(methylcarbamoyl)pyridin-4-yl]oxy}-2naphthoate (8f). Following the same procedure used to prepare 7a

from 21a, ethyl 3,7-dihydroxy-2-naphthoate (27g, 204 mg, 1.00 mmol), 10 (171 mg, 1.00 mmol) and Cs2CO3 (977 mg, 3.00 mmol) were reacted in DMSO at 120 °C for 48 h then in EtOH with catalytic amount of H2SO4 at 85 °C for 24 h to obtain 8f (119 mg, 33%) as yellow solid; mp 137−139 °C. 1H NMR (300 MHz, CDCl3) δ 10.58 (s, 1H), 8.46 (d, J = 0.6 Hz, 1H), 8.43 (dd, J = 5.6, 0.6 Hz, 1H), 8.03 (s, 1H), 7.79 (d, J = 5.4 Hz, 1H), 7.78 (s, 1H), 7.53 (d, J = 2.3 Hz, 1H), 7.39 (s, 1H), 7.29 (dd, J = 8.9, 2.5 Hz, 1H), 7.03 (dd, J = 5.6, 2.6 Hz, 1H), 4.52 (q, J = 7.2 Hz, 2H), 3.03 (d, J = 5.1 Hz, 3H), 1.50 (t, J = 7.1 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 166.51, 165.72, 164.39, 153.44, 152.52, 149.88, 136.49, 132.10, 130.81, 130.29, 127.77, 127.63, 126.40, 121.02, 117.08, 114.72, 110.81, 61.22, 26.15, 14.40. IR (diamond) 3233, 3062, 2972, 2930, 1710, 1662, 1469, 1282 cm−1. HRMS (EI) m/z calcd for C20H18N2O4 [M+] 350.1267, found 350.1269. Ethyl 6-{[2-(Methylcarbamoyl)pyridin-4-yl]oxy}-2-naphthoate (8g). Following the same procedure used to prepare 8f, 6-hydroxy2-naphthoic acid (27g, 29.0 mg. 0.154 mmol), 10 (26.2 mg, 0.154 mmol), and Cs2CO3 (151 mg, 0.462 mmol) were used to obtain 8g (22.1 mg, 41%) as white solid; mp 132−134 °C. 1H NMR (300 MHz, CDCl3) δ 8.64−8.62 (m, 1H), 8.43 (d, J = 5.5 Hz, 1H), 8.11 (dd, J = 8.6, 1.7 Hz, 1H), 8.06−7.96 (m, 2H), 7.81 (d, J = 9.0 Hz, 1H), 7.79 (d, J = 2.7 Hz, 1H), 7.54 (d, J = 2.4 Hz, 1H), 7.30 (dd, J = 8.9, 2.4 Hz, 1H), 7.04 (dd, J = 5.6, 2.6 Hz, 1H), 4.46 (q, J = 7.1 Hz, 2H), 3.01 (d, J = 5.1 Hz, 3H), 1.46 (t, J = 7.1 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 169.64, 166.12, 164.45, 156.53, 152.41, 149.79, 149.76, 135.69, 131.63, 128.98, 127.27, 123.77, 118.48, 115.32, 114.31, 111.97, 110.35, 61.99, 26.13, 14.21. IR (diamond) 3405, 3225, 3064, 2988, 2901, 1675, 1638, 1514, 1211 cm−1. HRMS (EI) m/z calcd for C20H18N2O5 [M+] 366.1216, found 366.1217. Ethyl 7-{[2-(Methylcarbamoyl)pyridin-4-yl]oxy}quinoline-2-carboxylate (8h). Following the same procedure used to prepare 8f, 7hydroxyquinoline-2-carboxylic acid (27h, 80.0 mg, 0.423 mmol), 10 (86.2 mg, 0.508 mmol), and Cs2CO3 (413 mg, 1.269 mmol) were used to obtain 8h (27.0 mg, 18%) as yellow solid; mp 117−119 °C. 1H NMR (300 MHz, chloroform-d) δ 8.46 (dd, J = 5.6, 0.6 Hz, 1H), 8.34 (dd, J = 8.7, 0.7 Hz, 1H), 8.19 (d, J = 8.5 Hz, 1H), 8.02 (br s, 1H), 7.96 (d, J = 8.9 Hz, 1H), 7.91 (d, J = 2.4 Hz, 1H), 7.82 (dd, J = 2.6, 0.5 Hz, 1H), 7.45 (dd, J = 8.9, 2.4 Hz, 1H), 7.11 (dd, J = 5.5, 2.5 Hz, 1H), 4.56 (q, J = 7.1 Hz, 2H), 3.02 (d, J = 5.1 Hz, 3H), 1.48 (t, J = 7.1 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 165.17, 165.06, 164.23, 155.79, 152.72, 150.02, 149.18, 148.59, 137.13, 129.85, 126.91, 122.96, 120.81, 118.97, 115.39, 111.60, 62.37, 26.16, 14.35. IR (diamond) 3373, 3064, 3027, 2938, 1705, 1670, 1528, 1285 cm−1. HRMS (EI) m/z calcd for C19H17N3O4 [M+] 351.1219, found 351.1220. 4-Chloro-N-methylpicolinamide (10). 10 was prepared by following the reference procedure.39 2-Picolinic acid (7.5 g, 61 mmol) was added slowly to a solution of DMF (1 mL) in SOCl2 (25 mL) at 45 °C, and the reaction mixture was refluxed at 72 °C. After 36 h, an additional portion of DMF (1 mL) was added and the reaction mixture was stirred at 72 °C for 36 h further. The reaction mixture was cooled to 25 °C and concentrated under reduced pressure. Remaining SOCl2 was removed by adding toluene (10 mL) and evaporating the solvents under reduced pressure three times. The residue was taken into THF (100 mL) and cooled to 0 °C, and 40% aq CH3NH2 (13.2 mL) was added slowly. After stirring at 20 °C for 4 h, the reaction mixture was concentrated to 20% of its orginal volume, diluted with EtOAc, washed with H2O and brine, dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by SiO2 column chromatography (EtOAc/Hx = 1:2) to obtain 10 as yellow solid (4.07 g, 39%). 1H NMR (300 MHz, CDCl3) δ 8.46 (d, J = 5.2 Hz, 1H), 8.23 (d, J = 1.8 Hz, 1H), 7.97 (br s, 1H), 7.45 (dd, J = 5.2, 2.0 Hz, 1H), 3.06 (d, J = 5.1 Hz, 3H). LC/MS (ESI) [M + H]+ = 170.6 5-Fluoro-N-methylpicolinamide (13). 1-Hydroxybenzotriazole hydrate (1.15 g, 21.3 mmol), triethylamine (3.59 g, 35.5 mmol), EDCI· HCl (2.72 g, 14.2 mmol), and 2 N CH3NH2 in methanol (11.0 mL) were added sequentially to a solution of 5-fluoropicolinic acid (1.00 g, 7.09 mmol) in CH2Cl2 (10 mL) and the reaction mixture was stirred at 25 °C for 15 h. The reaction was quenched by adding water (60 5489



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mL), and the aqueous layer was extracted with CH2Cl2 (60 mL × 2). The combined organic layer was washed brine, dried over Na2SO4, and concentrated, and the residue was purified by SiO2 column chromatography (EtOAc/Hx = 1:4) to obtain 13 as yellow solid (578 mg, 53%). 1H NMR (300 MHz, CDCl3) δ 8.37 (d, J = 2.8 Hz, 1H), 8.23 (dd, J = 8.7, 4.7 Hz, 1H), 7.84 (br s, 1H), 7.54−7.50 (m, 1H), 3.02 (d, J = 5.1 Hz, 3H). LC/MS (ESI) [M + H]+ = 154.4.

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(4) Morikawa, S.; Hiroi, S.; Kase, T. Detection of Respiratory Viruses in Gargle Specimens of Healthy Children. J. Clin. Virol. 2015, 64, 59− 63. (5) Morikawa, S.; Kohdera, U.; Hosaka, T.; Ishii, K.; Akagawa, S.; Hiroi, S.; Kase, T. Seasonal Variations of Respiratory Viruses and Etiology of Human Rhinovirus Infection in Children. J. Clin. Virol. 2015, 73, 14−19. (6) Chonmaitree, T.; Alvarez-Fernandez, P.; Jennings, K.; Trujillo, R.; Marom, T.; Loeffelholz, M. J.; Miller, A. L.; McCormick, D. P.; Patel, J. A.; Pyles, R. B. Symptomatic and Asymptomatic Respiratory Viral Infections in the First Year of Life: Association With Acute Otitis Media Development. Clin. Infect. Dis. 2015, 60, 1−9. (7) Heikkinen, T.; Chonmaitree, T. Importance of Respiratory Viruses in Acute Otitis Media. Clin. Microbiol. Rev. 2003, 16, 230−241. (8) Seppälä, E.; Sillanpäa,̈ S.; Nurminen, N.; Huhtala, H.; Toppari, J.; Ilonen, J.; Veijola, R.; Knip, M.; Sipilä, M.; Laranne, J.; Oikarinen, S.; Hyö ty, H. Human Enterovirus and Rhinovirus Infections are Associated with Otitis Media in a Prospective Birth Cohort Study. J. Clin. Virol. 2016, 85, 1−6. (9) Aab, A.; Wirz, O.; van de Veen, W.; Söllner, S.; Stanic, B.; Rückert, B.; Aniscenko, J.; Edwards, M. R.; Johnston, S. L.; Papadopoulos, N. G.; Rebane, A.; Akdis, C. A.; Akdis, M. Human Rhinoviruses Enter and Induce Proliferation of B Lymphocytes. Allergy 2017, 72, 232−243. (10) Gern, J. E. How Rhinovirus Infections Cause Exacerbations of Asthma. Clin. Exp. Allergy 2015, 45, 32−42. (11) Message, S. D.; Laza-Stanca, V.; Mallia, P.; Parker, H. L.; Zhu, J.; Kebadze, T.; Contoli, M.; Sanderson, G.; Kon, O. M.; Papi, A.; Jeffery, P. K.; Stanciu, L. A.; Johnston, S. L. Rhinovirus-Induced Lower Respiratory Illness is Increased in Asthma and Related to Virus Load and Th1/2 Cytokine and IL-10 Production. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 13562−13567. (12) Papadopoulos, N. G.; Sanderson, G.; Hunter, J.; Johnston, S. L. Rhinoviruses Replicate Effectively at Lower Airway Temperatures. J. Med. Virol. 1999, 58, 100−104. (13) Kim, H.-C.; Choi, S.-H.; Huh, J.-W.; Sung, H.; Hong, S. B.; Lim, C.-M.; Koh, Y. Different Pattern of Viral Infections and Clinical Outcomes in Patient with Acute Exacerbation of Chronic Obstructive Pulmonary Disease and Chronic Obstructive Pulmonary Disease with Pneumonia. J. Med. Virol. 2016, 88, 2092−2099. (14) Mallia, P.; Message, S. D.; Gielen, V.; Contoli, M.; Gray, K.; Kebadze, T.; Aniscenko, J.; Laza-Stanca, V.; Edwards, M. R.; Slater, L.; Papi, A.; Stanciu, L. A.; Kon, O. M.; Johnson, M.; Johnston, S. L. Experimental Rhinovirus Infection as a Human Model of Chronic Obstructive Pulmonary Disease Exacerbation. Am. J. Respir. Crit. Care Med. 2011, 183, 734−742. (15) Annamalay, A. A.; Jroundi, I.; Bizzintino, J.; Khoo, S.-K.; Zhang, G.; Lehmann, D.; Laing, I. A.; Gern, J.; Goldblatt, J.; Mahraoui, C.; Benmessaoud, R.; Moraleda, C.; Bassat, Q.; Le Souëf, P. Rhinovirus C is Associated with Wheezing and Rhinovirus A is Associated with Pneumonia in Hospitalized Children in Morocco. J. Med. Virol. 2017, 89, 582−588. (16) Palmenberg, A. C.; Spiro, D.; Kuzmickas, R.; Wang, S.; Djikeng, A.; Rathe, J. A.; Fraser-Liggett, C. M.; Liggett, S. B. Sequencing and Analyses of All Known Human Rhinovirus Genomes Reveal Structure and Evolution. Science 2009, 324, 55−59. (17) Oberste, M. S.; Nix, W. A.; Maher, K.; Pallansch, M. A. Improved Molecular Identification of Enteroviruses by RT-PCR and Amplicon Sequencing. J. Clin. Virol. 2003, 26, 375−377. (18) Price, W. H. The Isolation of a New Virus Associated with Respiratory Clinical Disease in Humans. Proc. Natl. Acad. Sci. U. S. A. 1956, 42, 892−896. (19) Rossmann, M. G.; Arnold, E.; Erickson, J. W.; Frankenberger, E. A.; Griffith, J. P.; Hecht, H.-J.; Johnson, J. E.; Kamer, G.; Luo, M.; Mosser, A. G.; Rueckert, R. R.; Sherry, B.; Vriend, G. Structure of a Human Common Cold Virus and Functional Relationship to Other Picornaviruses. Nature 1985, 317, 145−153. (20) Colonno, R. J.; Condra, J. H.; Mizutani, S.; Callahan, P. L.; Davies, M. E.; Murcko, M. A. Evidence for the Direct Involvement of

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S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b00175. UPLC purity of each compound examined and characterization data for compounds 6−8, including the spectral copies of 1H and 13C NMR spectra (PDF) Molecular formula strings of compounds 6−8 (CSV)

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AUTHOR INFORMATION

Corresponding Authors

*For Y.-S.K.J.: E-mail: [email protected] Phone: (+82)-42860-7135. *For C.K.: E-mail: [email protected]. *For J.N.: E-mail: [email protected]. ORCID

Soo Bong Han: 0000-0002-7831-1832 Young-Sik Jung: 0000-0001-9492-6848 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by Korea Research Institute of Chemical Technology (grant no. KK1503-C00 and SI1507) and National Research Foundation of Korea (grant no. NRF2016R1C1B2009585). We thank the Korea Chemical Bank for providing the chemical library with which this work was conducted.

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ABBREVIATIONS USED hRV, human rhinovirus; VP, viral capsid protein; PV3, poliovirus 3; COPD, chronic obstructive pulmonary disease; AOM, acute otitis media; HTS, high-throughput screening; SNAr, nucleophilic aromatic substitution; EDCI, 1-ethyl-3-[3(dimethylamino)-propyl]carbodiimide; DMAP, 4-dimethylaminopyridine; DMFDMA, N,N-dimethylformamide dimethyl acetal; SI, selectivity index; FR, fold resistance; MEM, minimum essential medium; MTT, 3-(4,5-dimethylthiazol2yl)-2,5-diphenyltetrazolium bromide; CPE, cytopathic effect; MMFF, Merck molecular force field; TLC, thin layer chromatography

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REFERENCES

(1) Jacobs, S. E.; Lamson, D. M.; St. George, K.; Walsh, T. J. Human Rhinoviruses. Clin. Microbiol. Rev. 2013, 26, 135−162. (2) Rollinger, J. M.; Schmidtke, M. The Human Rhinovirus: Humanpathological Impact, Mechanisms of Antirhinoviral Agents, and Strategies for Their Discovery. Med. Res. Rev. 2011, 31, 42−92. (3) Lee, W.-M.; Kiesner, C.; Pappas, T.; Lee, I.; Grindle, K.; Jartti, T.; Jakiela, B.; Lemanske, R. F., Jr.; Shult, P. A.; Gern, J. E. A Diverse Group of Previously Unrecognized Human Rhinoviruses Are Common Causes of Respiratory Illnesses in Infants. PLoS One 2007, 2, e966. 5490



Article

the Rhinovirus Canyon in Receptor Binding. Proc. Natl. Acad. Sci. U. S. A. 1988, 85, 5449−5453. (21) Greve, J. M.; Davis, G.; Meyer, A. M.; Forte, C. P.; Yost, S. C.; Marlor, C. W.; Kamarck, M. E.; McClelland, A. The Major Human Rhinovirus Receptor is ICAM-1. Cell 1989, 56, 839−847. (22) Olson, N. H.; Kolatkar, P. R.; Oliveira, M. A.; Cheng, R. H.; Greve, J. M.; McClelland, A.; Baker, T. S.; Rossmann, M. G. Structure of a Human Rhinovirus Complexed with its Receptor Molecule. Proc. Natl. Acad. Sci. U. S. A. 1993, 90, 507−511. (23) Papi, A.; Johnston, S. L. Rhinovirus Infection Induces Expression of Its Own Receptor Intercellular Adhesion Molecule 1 (ICAM-1) via Increased NF-κB-Mediated Transcription. J. Biol. Chem. 1999, 274, 9707−9720. (24) Staunton, D. E.; Merluzzi, V. J.; Rothlein, R.; Barton, R.; Marlin, S. D.; Springer, T. A. A Cell Adhesion Molecule, ICAM-1, is the Major Surface Receptor for Rhinoviruses. Cell 1989, 56, 849−853. (25) Reisdorph, N.; Thomas, J. J.; Katpally, U.; Chase, E.; Harris, K.; Siuzdak, G.; Smith, T. J. Human Rhinovirus Capsid Dynamics is Controlled by Canyon Flexibility. Virology 2003, 314, 34−44. (26) Hofer, F.; Gruenberger, M.; Kowalski, H.; Machat, H.; Huettinger, M.; Kuechler, E.; Blass, D. Members of the Low Density Lipoprotein Receptor Family Mediate Cell Entry of a Minor-group Common Cold Virus. Proc. Natl. Acad. Sci. U. S. A. 1994, 91, 1839− 1842. (27) Rankl, C.; Kienberger, F.; Wildling, L.; Wruss, J.; Gruber, H. J.; Blaas, D.; Hinterdorfer, P. Multiple Receptors Involved in Human Rhinovirus Attachment to Live Cells. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 17778−17783. (28) Verdaguer, N.; Fita, I.; Reithmayer, M.; Moser, R.; Blaas, D. Xray Structure of a Minor Group Human Rhinovirus Bound to a Fragment of its Cellular Receptor Protein. Nat. Struct. Mol. Biol. 2004, 11, 429−434. (29) Lewis, J. K.; Bothner, B.; Smith, T. J.; Siuzdak, G. Antiviral Agent Blocks Breathing of the Common Cold Virus. Proc. Natl. Acad. Sci. U. S. A. 1998, 95, 6774−6778. (30) De Palma, A. M.; Vliegen, I.; De Clercq, E.; Neyts, J. Selective Inhibitors of Picornavirus Replication. Med. Res. Rev. 2008, 28, 823− 884. (31) Diana, G. D.; Rudewicz, P.; Pevear, D. C.; Nitz, T. J.; Aldous, S. C.; Aldous, D. J.; Robinson, D. T.; Draper, T.; Dutko, F. J. Picornavirus Inhibitors: Trifluoromethyl Substitution Provides a Global Protective Effect against Hepatic Metabolism. J. Med. Chem. 1995, 38, 1355−1371. (32) Feil, S. C.; Hamilton, S.; Krippner, G. Y.; Lin, B.; Luttick, A.; McConnell, D. B.; Nearn, R.; Parker, M. W.; Ryan, J.; Stanislawski, P. C.; Tucker, S. P.; Watson, K. G.; Morton, C. J. An Orally Available 3Ethoxybenzisoxazole Capsid Binder with Clinical Activity against Human Rhinovirus. ACS Med. Chem. Lett. 2012, 3, 303−307. (33) Patick, A. K. Rhinovirus Chemotherapy. Antiviral Res. 2006, 71, 391−396. (34) Shih, S.-R.; Chen, S.-J.; Hakimelahi, G. H.; Liu, H.-J.; Tseng, C.T.; Shia, K.-S. Selective Human Enterovirus and Rhinovirus Inhibitors: An Overview of Capsid-Binding and Protease-Inhibiting Molecules. Med. Res. Rev. 2004, 24, 449−474. (35) Wikel, J. H.; Paget, C. J.; DeLong, D. C.; Nelson, J. D.; Wu, C. Y. E.; Paschal, J. W.; Dinner, A.; Templeton, R. J.; Chaney, M. O. Synthesis of Syn and Anti Isomers of 6-[[(Hydroxyimino)phenyl]methyl]-1-[(1-methylethyl)sulfonyl]-1H-benzimidazol-2-amine. Inhibitors of Rhinovirus Multiplication. J. Med. Chem. 1980, 23, 368−372. (36) Dragovich, P. S.; Prins, T. J.; Zhou, R.; Webber, S. E.; Marakovits, J. T.; Fuhrman, S. A.; Patick, A. K.; Matthews, D. A.; Lee, C. A.; Ford, C. E.; Burke, B. J.; Rejto, P. A.; Hendrickson, T. F.; Tuntland, T.; Brown, E. L.; Meador, J. W.; Ferre, R. A.; Harr, J. E. V.; Kosa, M. B.; Worland, S. T. Structure-Based Design, Synthesis, and Biological Evaluation of Irreversible Human Rhinovirus 3C Protease Inhibitors. 4. Incorporation of P1 Lactam Moieties as l-Glutamine Replacements. J. Med. Chem. 1999, 42, 1213−1224. (37) Lacroix, C.; George, S.; Leyssen, P.; Hilgenfeld, R.; Neyts, J. The Enterovirus 3C Protease Inhibitor SG85 Efficiently Blocks Rhinovirus

Replication and Is Not Cross-Resistant with Rupintrivir. Antimicrob. Agents Chemother. 2015, 59, 5814−5818. (38) Patick, A. K.; Brothers, M. A.; Maldonado, F.; Binford, S.; Maldonado, O.; Fuhrman, S.; Petersen, A.; Smith, G. J.; Zalman, L. S.; Burns-Naas, L. A.; Tran, J. Q. In Vitro Antiviral Activity and SingleDose Pharmacokinetics in Humans of a Novel, Orally Bioavailable Inhibitor of Human Rhinovirus 3C Protease. Antimicrob. Agents Chemother. 2005, 49, 2267−2275. (39) Bankston, D.; Dumas, J.; Natero, R.; Riedl, B.; Monahan, M.-K.; Sibley, R. A Scaleable Synthesis of BAY 43−9006: A Potent Raf Kinase Inhibitor for the Treatment of Cancer. Org. Process Res. Dev. 2002, 6, 777−781. (40) Higa, T.; Krubsack, A. J. Oxidations by Thionyl Chloride. VI. Mechanism of the Reaction with Cinnamic Acids. J. Org. Chem. 1975, 40, 3037−3045. (41) Cruz, M. d. C.; Jiménez, F.; Delgado, F.; Tamariz, J. Regioselective and Versatile Synthesis of Indoles via Intramolecular Friedel-Crafts Heteroannulation of Enaminones. Synlett 2006, 2006, 0749−0755. (42) Cai, Q.; Li, Z.; Wei, J.; Ha, C.; Pei, D.; Ding, K. Assembly of Indole-2-Carboxylic Acid Esters Through a Ligand-Free CopperCatalysed Cascade Process. Chem. Commun. 2009, 7581−7583. (43) Sakai, N.; Tamura, K.; Shimamura, K.; Ikeda, R.; Konakahara, T. Copper-Catalyzed [5 + 1] Annulation of 2-Ethynylanilines with an N,O-Acetal Leading to Construction of Quinoline Derivatives. Org. Lett. 2012, 14, 836−839. (44) National Research Council. Exploring the Role of Antiviral Drugs in the Eradication of Polio: Workshop Report; The National Academies Press: Washington, DC, 2006. (45) Buontempo, P. J.; Cox, S.; Wright-Minogue, J.; DeMartino, J. L.; Skelton, A. M.; Ferrari, E.; Albin, R.; Rozhon, E. J.; Girijavallabhan, V.; Modlin, J. F.; O’Connell, J. F. SCH 48973: A Potent, Broad-Spectrum, Antienterovirus Compound. Antimicrob. Agents Chemother. 1997, 41, 1220−1225. (46) McKinlay, M. A.; Collett, M. S.; Hincks, J. R.; Oberste, M. S.; Pallansch, M. A.; Okayasu, H.; Sutter, R. W.; Modlin, J. F.; Dowdle, W. R. Progress in the Development of Poliovirus Antiviral Agents and Their Essential Role in Reducing Risks That Threaten Eradication. J. Infect. Dis. 2014, 210, S447−S453. (47) Oberste, M. S.; Moore, D.; Anderson, B.; Pallansch, M. A.; Pevear, D. C.; Collett, M. S. In Vitro Antiviral Activity of V-073 against Polioviruses. Antimicrob. Agents Chemother. 2009, 53, 4501−4503. (48) Chapman, M. S.; Minor, I.; Rossmann, M. G.; Diana, G. D.; Andries, K. Human Rhinovirus 14 Complexed with Antiviral Compound R 61837. J. Mol. Biol. 1991, 217, 455−463. (49) Smith, T. J.; Kremer, M. J.; Luo, M.; Vriend, G.; Arnold, E.; Kamer, G.; Rossmann, M. G.; McKinlay, M. A.; Diana, G. D.; Otto, M. J. The Site of Attachment in Human Rhinovirus 14 for Antiviral Agents that Inhibit Uncoating. Science 1986, 233, 1286. (50) Zhang, A.; Nanni, R. G.; Li, T.; Arnold, G. F.; Oren, D. A.; Jacobo-Molina, A.; Williams, R. L.; Kamer, G.; Rubenstein, D. A.; Li, Y.; Rozhon, E.; Cox, S.; Buontempo, P.; O’Connell, J.; Schwartz, J.; Miller, G.; Bauer, B.; Versace, R.; Pinto, P.; Ganguly, A.; Girijavallabhan, V.; Arnold, E. Structure Determination of Antiviral Compound SCH 38057 Complexed with Human Rhinovirus 14. J. Mol. Biol. 1993, 230, 857−867. (51) Zhang, Y.; Simpson, A. A.; Ledford, R. M.; Bator, C. M.; Chakravarty, S.; Skochko, G. A.; Demenczuk, T. M.; Watanyar, A.; Pevear, D. C.; Rossmann, M. G. Structural and Virological Studies of the Stages of Virus Replication That Are Affected by Antirhinovirus Compounds. J. Virol. 2004, 78, 11061−11069. (52) Hadfield, A. T.; Oliveira, M. A.; Kim, K. H.; Minor, I.; Kremer, M. J.; Heinz, B. A.; Shepard, D.; Pevear, D. C.; Rueckert, R. R.; Rossmann, M. G. Structural Studies on Human Rhinovirus 14 Drugresistant Compensation Mutants. J. Mol. Biol. 1995, 253, 61−73. (53) Heinz, B. A.; Rueckert, R. R.; Shepard, D. A.; Dutko, F. J.; McKinlay, M. A.; Fancher, M.; Rossmann, M. G.; Badger, J.; Smith, T. J. Genetic and Molecular Analyses of Spontaneous Mutants of Human 5491



Article

Rhinovirus 14 that are Resistant to an Antiviral Compound. J. Virol. 1989, 63, 2476−2485. (54) Kang, H.; Kim, C.; Kim, D.-e.; Song, J.-H.; Choi, M.; Choi, K.; Kang, M.; Lee, K.; Kim, H. S.; Shin, J. S.; Kim, J.; Han, S.-B.; Lee, M.Y.; Lee, S. U.; Lee, C.-K.; Kim, M.; Ko, H.-J.; van Kuppeveld, F. J. M.; Cho, S. Synergistic Antiviral Activity of Gemcitabine and Ribavirin Against Enteroviruses. Antiviral Res. 2015, 124, 1−10. (55) Lacroix, C.; Querol-Audí, J.; Roche, M.; Franco, D.; Froeyen, M.; Guerra, P.; Terme, T.; Vanelle, P.; Verdaguer, N.; Neyts, J.; Leyssen, P. A Novel Benzonitrile Analogue Inhibits Rhinovirus Replication. J. Antimicrob. Chemother. 2014, 69, 2723−2732. (56) Kolasa, T.; Gunn, D. E.; Bhatia, P.; Woods, K. W.; Gane, T.; Stewart, A. O.; Bouska, J. B.; Harris, R. R.; Hulkower, K. I.; Malo, P. E.; Bell, R. L.; Carter, G. W.; Brooks, C. D. W. Heteroarylmethoxyphenylalkoxyiminoalkylcarboxylic Acids as Leukotriene Biosynthesis Inhibitors. J. Med. Chem. 2000, 43, 690−705.

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