Trisubstituted thieno[3,2-b]pyrrole 5-carboxamides as potent inhibitors of alphaviruses

Article

pubs.acs.org/jmc

Trisubstituted Thieno[3,2‑b]pyrrole 5‑Carboxamides as Potent

Inhibitors of Alphaviruses

Kuan-Chieh Ching,^†,‡Yiu-Wing Kam,^§Andres Merits,^∥Lisa F. P. Ng,*^,§,⊥and Christina L. L. Chai*^,†,‡,#

^†NUS Graduate School for Integrative Sciences and Engineering, Centre for Life Sciences, #05-01, 28 Medical Drive, Singapore

117456

^‡Department of Pharmacy, Faculty of Science, National University of Singapore, Block S4A, Level 3, 18 Science Drive 4, Singapore

117543

^§Singapore Immunology Network, A*STAR, 8A Biomedical Grove, Immunos Building, Level 4, Singapore 138648

^∥Institute of Technology, University of Tartu, Nooruse 1, Tartu, Estonia 50411

^⊥Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Block MD6, Centre for

Translational Medicine, 14 Medical Drive, #14-01T, Singapore 117599

^#Institute of Chemical and Engineering Sciences, A*STAR, 8 Biomedical Grove, Neuros Building, #07-01/02/03, Singapore 138665

S

* Supporting Information

ABSTRACT: Chikungunya virus (CHIKV) is a re-emerging vector-borne alphavirus and is transmitted to humans by Aedes

mosquitoes. Despite the re-emergence of CHIKV as an epidemic threat, there is no approved eﬀective antiviral treatment

currently available for CHIKV. Herein, we report the synthesis and structure−activity relationship studies of a class of thieno[3,2-

b]pyrroles and the discovery of a trisubstituted thieno[3,2-b]pyrrole 5-carboxamide 15c that exhibits potent inhibitory activity

against in vitro CHIKV infection. Compound 15c displayed low micromolar activity (EC₅₀value of ca. 2 μM) and limited

cytotoxic liability (CC50 > 100 μM) therefore furnishing a selectivity index of greater than 32. Notably, 15c not only controlled

viral RNA production, but eﬃciently inhibited the expression of CHIKV nsP1, nsP3, capsid, and E2 proteins at a concentration

as low as 2.5 μM. More importantly, 15c also demonstrated broad spectrum antiviral activity against other clinically important

alphaviruses such as O’nyong−nyong virus and Sindbis virus.

as they are either not aﬀordable¹²or not eﬀective when tested

INTRODUCTION

■

in the clinical setting.¹⁷In view of this, there is much interest in

Chikungunya virus (CHIKV) is an alphavirus that belongs to

the family of Togaviridae. CHIKV is transmitted by the Aedes

mosquitoes and causes Chikungunya fever (CHIKF) which

manifests acute symptoms such as abrupt high fever, skin rash,

the discovery of new drugs against CHIKV.¹⁸⁻²¹Some of the

lead compounds are natural products²²⁻²⁶and synthetic

molecules,²⁷⁻²⁹but none of these compounds has progressed

beyond the discovery stage. Taken together, there is a pressing

headache, back pain, and arthralgia that could become chronic

in some patients.¹⁻³The emergence of CHIKV in almost every

need to discover antiviral compounds as potential therapeutics

for CHIKV infection.

part of the world such as Indian Ocean islands, the Indian

subcontinent, Southeast Asia, Africa, and other nonendemic

In our preliminary studies, we examined the literature for

known inhibitors against related arboviruses. Arising from this

regions such as Europe and more recently in the Americas

presents a threat to human health.⁴⁻¹⁰The problem is further

exacerbated by the fact that there is no approved antiviral

survey, we synthesized a small library of 3 diﬀerent classes of

compounds, namely, the pyrazolines,³⁰thieno[3,2-b]pyrroles,³¹

and pyrimidinones/pyrimidinethiones,³²which were reported

treatment for CHIKV infection to date and that current

treatment only alleviates the disease symptoms.¹¹A number of

to be inhibitors of ﬂavivirus and alphaviruses. From the

known antiviral agents such as ribavirin and interferon-α,¹²

screening studies, the thieno[3,2-b]pyrrole family showed

Arbidol,¹³6-azauridine,¹⁴and mycophenolic acid¹⁵as well as

promising antiviral activity against CHIKV, and this paved

the antiparasitic agent chloroquine¹⁶have demonstrated

eﬀective and potent anti-CHIKV activity in vitro. However,

Received: July 6, 2015

these drugs are not in use for the treatment of CHIKV infection

A

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the way for the more extensive study reported here.

Thieno[3,2-b]pyrroles have been reported to be eﬀective

inhibitors, with a broad spectrum of antiviral activities against

RNA viruses such as Hepatitis C virus (HCV)^33,34and Western

Equine Encephalitis virus (WEEV).³¹Speciﬁcally, Peng et al.

discovered that a number of thieno[3,2-b]pyrrole carboxamides

possess potent antiviral activities against WEEV.³¹WEEV and

CHIKV, are arthropod-borne viruses in the Togaviridae

family,³⁵and they share a similar viral genome organization

and viral protein composition. Therefore, the thieno[3,2-

b]pyrroles may also be eﬀective inhibitors of the CHIKV.

However, prior to our discoveries described here, the activities

of the thieno[3,2-b]pyrroles against CHIKV have not been

reported. It is thus of interest to investigate the potential of

using thieno[3,2-b]pyrroles as the core pharmacophore for

CHIKV antiviral compounds development. In this study, we

have identiﬁed that selected thieno[3,2-b]pyrroles are inhibitors

of CHIKV and other alphaviruses in vitro. Moreover, structure−

activity relationship studies of these thieno[3,2-b]pyrroles

revealed the structural requirements for potent, yet safe

antiviral compounds.

using EDCI or CDI gave a series of 5-carboxamides 3, 4a−h

with the structures as shown in Table 1. The target amide 4h

was obtained in 41% yield by deprotection of the Boc-protected

piperazine carboxamide intermediate.

On the basis of the results of the initial biological screening

(see the Cytotoxicity and Anti-CHIKV Activity of the

Thieno[3,2-b]pyrrole Compounds section), substituted piper-

azine amides of carboxamide 4h and piperidine amides of

carboxamide 4f (Table 2) were synthesized from coupling of

Table 2. Structures of Piperazine 5-Carboxamide 6 and

Piperidine 5-Carboxamides 7 (Group 2 Compounds)

RESULTS AND DISCUSSION

■

Synthetic Chemistry. The initial target compounds for our

studies are the C-5 carboxylic ester, acid, and carboxamide

substituted thieno[3,2-b]pyrroles 1−4 (Table 1).

the intermediate 5-carboxylic acid 2 with the corresponding

amines, i.e., either 1-benzylpiperazine or appropriately sub-

stituted piperidines 8a−k.

Table 1. General Chemical Structures of Ester 1, 5-

Carboxylic Acid 2, and 5-Carboxamide Derivatives 3−4

(Group 1 Compounds)

The synthesis of the latter amines 8a−k is summarized in

Scheme 2. Brieﬂy, the coupling of the Boc-protected

isonipecotic acid 9 with the respective amines gave the amides

10a−i. Subsequent deprotection of carboxamides 10a−i to

piperidines 8a−i was eﬀected using TFA. For the synthesis of

piperidine derivatives 8j, a three step reaction sequence from

N-protected isonipecotic acid 9 was employed. In the ﬁrst step,

the acid 9 was converted to Weinreb amide 11 followed by

nucleophilic attack using nBuLi to form the ketone.

Deprotection of the Boc protecting group in 12 gave the

piperidine derivative 8j. The piperidine derivative 8k was

obtained from the esteriﬁcation of isonipecotic acid with

ethanol. With acid 2 and the piperidine derivatives 8a−k or 1-

benzylpiperazine in hand, a coupling reaction was carried out to

yield thieno[3,2-b]pyrrole amides 6, 7a, and 7c−l. Target

carboxamide 7b was obtained in 92% yield upon hydrolysis of

the ester 7a.

The synthesis of these compounds are summarized in

Scheme 1, starting from thieno[3,2-b]pyrrole ester 5, which was

synthesized following the procedures reported by Eras et al. and

Srinivasan et al.^36,37N-Alkylation of ester 5 with para-methoxy

benzyl chloride aﬀorded the desired ester 1 in moderate yield.

Saponiﬁcation of ester 1 gave the corresponding C-5 carboxylic

acid 2. With the acid in hand, coupling of the respective amines

Various C2 and/or C6 substituents of selected carboxamides

(i.e., compounds 6 and 7h) were installed to give target

compounds 13−16 with structures as shown in Table 3. To

access target compounds 13−16, fuctionalizations on the C2

and/or C6 positions of the thieno[3,2-b]pyrrole core structure

a

Scheme 1. General Synthetic Routes for the Synthesis of N-Substituted Ester 1, Carboxylic Acid 2, and Carboxamides 3−4

a

Reagents and conditions: (a) p-MeOBnCl, K₂CO₃, ACN, reﬂux, > 12 h (78% yield); (b) KOH, THF/EtOH/H₂O, reﬂux, > 12 h (86% yield); (c)

CDI, with or without DIEA, amines, THF, rt-45 °C, > 12 h or EDCI, with or without DIEA, amines, DCM, 0 °C-rt, > 12 h (31−69% yield); (d)

oxalyl chloride, DMF, DCM, rt, > 12 h followed by NH₄OH (aq), DCM, −10 °C, 30 min (83% yield over 2 steps).

B

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a

Scheme 2. Synthesis of the Piperidine Derivatives 8a−k for the Amide Coupling Reaction

a

Reagents and conditions: (a) di-tert-butyl pyrocarbonate, dioxane/water (2:1), rt, 3 h (67% yield); (b) SOCl₂, EtOH, reﬂux, 3 h (96% yield); (c)

DCC or EDCI, amines, DCM, 0 °C-rt, > 12 h (48−92% yield); (d) 30%TFA/DCM, rt, 1 h (72−99% yield); (e) NH₄Cl, EDCI, TEA, DMF, rt, 6 h

(59% yield); (f) EDCI, DIEA, O,N-dimethylhydroxylamine HCl, DCM/DMF, rt, > 12 h (44% yield); (g) nBuLi, THF, 0 °C-rt, 3 h (49% yield).

Table 3. Structures of C2 and C6 Substituted Carboxamide Derivatives 13−16 (Group 3 Compounds)

were ﬁrst carried out using the precursor, thieno[3,2-b]pyrrole

5.

illustrated in Scheme 4. In the ﬁrst and second routes, the

formyl functionalities on N-substituted esters 21a−b were

either reduced to the alcohol followed by O-benzylation to give

ethers 23a−b in 47−61% yield over 2 steps or converted to an

amino group through a one-pot reductive amination to give the

amines 24a−b in comparable yields. In the last synthetic route,

the ester 22a underwent a Suzuki coupling reaction with

phenylboronic acid to give 2-phenyl substituted ester 25. With

the esters 20a−b, 22a−b, and 23−25 in hand, hydrolysis and

amide coupling reactions were employed to aﬀord the desired

C2 and C6 substituted carboxamides 13a−e, 14b, 15a−c, and

16a. However, the target C6 amino substituted amide 14a was

obtained through a longer synthetic route which involved the

protection of the methyl amino group at the C6 position with a

Boc group followed by amide formation at the C5 position and

ﬁnally the deprotection of the Boc group.

Starting from thieno[3,2-b]pyrrole 5, C2 acetyl or benzoyl

intermediates 17a and 17b were obtained exclusively using the

Friedel−Crafts acylation reaction with either acetyl or benzoyl

chloride, respectively (Scheme 3). As formyl groups are useful

functionalities for further transformation, thieno[3,2-b]pyrrole

5 was subjected to the Vilsmeier−Haack reaction. In contrast to

the acetylation and benzoylation reactions, in this case, a

separable mixture of C2 formyl and C6 formyl substituted

esters 18a and 18b was obtained in a ratio of 1:1. For the

bromo functionalization of thieno[3,2-b]pyrrole 5, N-bromo-

succinimide was used to furnish a separable mixture of C2

bromo and C6 bromo substituted esters 19a and 19b in a ratio

of 4:1. With these C2 and C6 substituted intermediates 17−19

in hand, N-alkylation was carried out to aﬀord the desired N-

substituted carboxylates 20−22 (Scheme 3).

Target amides 15d−f and 16c−e were accessed via

carboxamide intermediates 26a−b by installing various amino

functionalities (i.e., benzylamine, n-propylamine, ( ) phenyl-

ethylamine) on either C2 or C6 position via a one-pot

In cases where further transformations of the formyl and

bromo functionalities are desired, the thieno[3,2-b]pyrrole

intermediates are accessed via three diﬀerent synthetic routes as

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Scheme 3. General Synthetic Routes for Formyl, Ketone, and Bromo Functionalizations of Thieno[3,2-b]pyrrole 5 and N-

a

Alkylation of Thieno[3,2-b]pyrroles 17−19

a

Reagents and conditions: (a) acetyl chloride or benzoyl chloride, AlCl₃, CS₂, rt, 15−17 h (17a, 43% yield and 17b, 49% yield); (b) POCl₃, DMF,

DCE, reﬂux, 2 h (18a, 52% yield, and 18b, 48% yield), (c) NBS, THF, rt, 1 h (19a, 81% yield, and 19b, 19% yield); (d) p-MeOBnCl, K₂CO₃, ACN,

reﬂux, > 12 h (72−98% yield).

a

Scheme 4. General Synthetic Routes for the Synthesis of N-Substituted Esters 23−25

a

Reagents and conditions: (a) NaBH₄, THF/MeOH (3:1), −40 °C, 1 h (90−92% yield); (b) BnBr, NaH, Bu₄NBr, THF, rt, 15−18 h (52−67%

yield); (c) ( ) α-phenylethylamine, NaCNBH₃, AcOH, MeOH, reﬂux, 18−22 h (48−60% yield); (d) PhB(OH)₂, Pd(OAc)₂, PPh₃, K₂CO₃, THF,

ethylene glycol, 60 °C, in sealed tube, 19 h (48% yield).

a

Scheme 5. General Synthetic Route for the Synthesis of Thieno[3,2-b]pyrroles, 15d−j, and 16b−e

a

Reagents and conditions: (a) amines, NaBH₄, Na₂SO₄, MeOH, rt-60 °C, 3 h or >12 h (26−96% yield); (b) NaBH₄, THF/MeOH (1:1), 0 °C, 3 h

(54−83% yield); (c) MeB(OH)₂or PhB(OH)₂, Pd(OAc)₂, PPh₃, Ag₂O, K₂CO₃, toluene/H₂O (19:1), 120 °C, in sealed tube, 36 h (10−33% yield);

(d) NH₂OH·HCl, DMF, 125 °C, > 12 h (30% yield).

reductive amination, while target amides 15g and 16b were

aﬀorded in good yields by converting the formyl group on

intermediate 26a−b to the alcohol via reduction (Scheme 5).

The formyl functionality in 26a was also converted to an oxime

via reaction with hydroxylamine followed by dehydration to

obtain the 2-cyano substituted carboxamide 15j. To access

compounds 15h and 15i, treatment of carboxamide 15c with

methyl- or phenyl-boronic acid under Suzuki coupling

conditions gave the respective 2-methyl and 2-phenyl

substituted carboxamides 15h and 15i.

Biological Assays. In the primary screening of the

compounds, the anti-CHIKV activities of the compounds

were measured using a luciferase-based reporter gene assay.³⁸

Synthetic compounds which showed potent inhibitory eﬀect

against CHIKV-Gluc infection were selected for validation.

Selected compounds were tested against wild-type CHIKV, and

antiviral activity was measured by immunoﬂuorescence-based

single-cell quantiﬁcation high content screening experiment.

The eﬀects of selected compounds on CHIKV were quantitated

using biochemical methods including qRT-PCR and Western

blot analysis. In order to evaluate the spectrum of antiviral

activity of the compounds, antiviral assays were conducted

using other closely related alphaviruses such as O’nyong−

nyong virus (ONNV)³⁹and Sindbis virus (SINV).³⁵ONNV is

the most closely related virus to CHIKV alphaviruses. ONNV

causes human disease in Africa and Asia; hence, the

development of antiviral drugs against ONNV is also

considered important.³⁹

Cytotoxicity and Anti-CHIKV Activity of the Thieno[3,2-

b]pyrrole Compounds. Both cell viability (CellTiter-Glo

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luminescent cell viability assay; Promega) and antiviral assays

were performed using serial dilutions of test compounds

ranging from 0.01 μM to 100 μM where test compounds were

completely dissolved in DMEM supplemented with 10% fetal

bovine serum (FBS). There was no cytotoxicity or any

observable eﬀects for both uninfected cells and infected cells

when treated with 0.4% DMSO (highest concentration solvent

used in the screening) (Supporting Information). Ribavirin was

previously shown to demonstrate antiviral activity against

CHIKV replication by Briolant et al. and hence was selected as

a positive control in our in vitro antiviral studies.¹²

Table 5. Biological Results and the Calculated log D Values

of Group 2 Compounds (i.e., Thieno[3,2-b]pyrroles 6−7)

Structure−Activity Relationship Studies. The results from

the screening of thieno[3,2-b]pyrroles are summarized in

Tables 4, 5, and 6. Group 1 compounds required high

Table 4. Biological Results and the Calculated log D Values

of Group 1 Compounds (i.e., Thieno[3,2-b]pyrroles 1−4)

a

Calculated ClogD (Marvin Sketch, ChemAxon). If the compound

b

cannot be ionized at pH 7.4, ClogD = ClogP. Cell viability CC₅₀

values were determined by CellTiter-Glo luminescent assay after 24 h

of exposure of HEK 293T cells. CC₅₀is deﬁned as the compound’s

concentration required for the reduction of cell viability by 50% as

c

compared to the untreated control. EC₅₀values against CHIKV-Gluc

were determined by the inhibition of Gaussia luciferase expression in

the antiviral assay. The values are the mean SD from 2 independent

experiments. EC₅₀values against wild-type (WT) CHIKV-IMT were

calculated eﬀective concentrations of compounds required to inhibit

50% CHIKV-IMT infectivity. The values are the mean SD from 3

independent experiments. The positive control has EC₅₀values of 13.1

0.7 μM and 20.0 4.5 μM against CHIKV-Gluc and CHIKV-IMT,

respectively. Cell viability was above 80% for all EC₅₀concentrations,

d

showing minimal cytotoxicity. Selectivity index (SI) was calculated as

CC₅₀value/EC₅₀(against CHIKV-Gluc) value.

performed better (EC₅₀values ranging between 32 and 80

μM). Notably, cyclic amides show a range of EC₅₀values from

32 to 80 μM. The best activity is observed with piperazine

amide 4h (EC₅₀= 32.5 μM) among the Group 1 compounds.

In group 2 compounds, the eﬀect of substitution on the

piperazine ring of 4h and piperidine ring of 4f was further

explored. The presence of a benzyl substituent on the nitrogen

atom of the piperazine ring of 4h resulted in compound 6,

which showed a slight drop in activity (EC₅₀= 41.7 μM) as

compared to 4h. Interestingly, compound 7a which possesses

an ester moiety at the 4-position of the piperidine resulted in

signiﬁcant improvement in activity (EC₅₀= 13.1 μM) as

compared to the parent compound 4f. In contrast, the acid 7b

is inactive. This may be due to poor permeability (calculated

Log D value <1). Next, we investigated the eﬀect of various

amide substitutions on the 4-position of the piperidine ring of

4f. Of these amides, secondary amides were more active (EC₅₀

values ranging between 7 and 13 μM) as compared to the

primary amide 7c. Interestingly, of these secondary amides,

those amides possessing an aryl group (7d−f and 7l) were not

a

Calculated ClogD (Marvin Sketch, ChemAxon). If the compound

b

cannot be ionized at pH 7.4, ClogD = ClogP. Cell viability CC₅₀

values were determined by CellTiter-Glo luminescent assay after 24 h

of exposure of HEK 293T cells. CC₅₀is deﬁned as the compound’s

concentration required for the reduction of cell viability by 50% as

c

compared to the untreated control. EC₅₀values against CHIKV-Gluc

were determined by the inhibition of Gaussia luciferase expression in

the antiviral assay. The values are the mean SD from 2 independent

experiments. The positive control has an EC₅₀value of 13.1 0.7 μM

against CHIKV-Gluc. Cell viability was above 80% for all EC₅₀

concentrations, showing minimal cytotoxicity. Selectivity index (SI)

was calculated as CC₅₀value/EC₅₀value.

d

concentrations to cause 50% cell death (CC₅₀values >100

μM), suggesting that these compounds have low toxicities.

Comparison of the antiviral activities of ester 1, acid 2, and

amide 3 showed that amide 3 had the lowest EC₅₀values of

36.0 μM. Comparing 3 with other amides, we found that the

secondary amides 4a−c tested did not show any antiviral

activity (EC₅₀values >100 μM), while tertiary amides

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functionalities such as piperidine carboxamide and N-benzyl at

the, respective, C5 and N4 positions of thieno[3,2-b]pyrrole is

important for antiviral activities (i.e., EC₅₀ranging between 7

and 13 μM). These functionalities were also found to be

important in other bioisosteric analogues of thieno[3,2-

b]pyrroles (i.e., indole) for anti-WEEV activities as demon-

strated by Sindac et al.^40,41

Table 6. Biological Results and the Calculated log D Values

of Group 3 Compounds (i.e., Thieno[3,2-b]pyrroles 13−16)

For group 3 compounds, we investigated the eﬀects of

various C2 and C6 substitutions on the thieno[3,2-b]pyrrole

scaﬀold of amides 6 and 7h. Notably, amides 13a−e and 14a−b

required high concentrations to eﬀect 50% cell death,

suggesting that these compounds have low toxicities. Amides

13a, 13c, and 13e have similar calculated LogD values, but both

amides 13a and 13e have EC₅₀values >100 μM, suggesting that

the poor activity is related to the nature of the substituents.

Only two C-6 substituted compounds 14a and 14b were

available for investigation. Of the two, the C6 amino derivative

14a showed a 2-fold improvement in activity as compared to

the parent amide 6, while ether 14b was inactive. Interestingly,

C2 bromo (15c), methyl ether (15g), methyl (15h), phenyl

(15i), and cyano (15j) substituted thieno[3,2-b]pyrroles show

enhanced activities as compared to the amide 7h. Of these

compounds, amide 15c has the best selectivity index, i.e., > 32.

Both C2 cyano and phenyl substituted thieno[3,2-b]pyrroles

15i−j possess equally good cytotoxicity proﬁles as that of

compound 15c (i.e., CC₅₀> 100 μM). These studies suggest

that the lipophilicity of the substituent did not have a direct

eﬀect on cytotoxicity. However, C2 cyano substituted thieno-

[3,2-b]pyrrole 15j exhibited slightly weaker antiviral activity as

compared to the thieno[3,2-b]pyrroles which were substituted

with more lipophilic substituents (i.e., Ph, Me, and Br). No

direct relationship was observed between the electronegativity

of the substituent (i.e., electronegativity was CN > Br > Me or

Ph) and the antiviral activity or cytotoxicity. It was also

observed that thieno[3,2-b]pyrroles with similar substituents at

the C6 position (16a−b and 16d−e) show poorer activity as

compared to their corresponding C2 substituted amides (15c

and 15e−g).

a

Calculated ClogD (Marvin Sketch, ChemAxon). If the compound

b

cannot be ionized at pH 7.4, ClogD = ClogP. Cell viability CC₅₀

values were determined by CellTiter-Glo luminescent assay after 24 h

exposure of HEK 293T cells. CC₅₀is deﬁned as the compound’s

concentration required for the reduction of cell viability by 50% as

c

compared to the untreated control. EC₅₀values against CHIKV-Gluc

Antiviral Activity of Selected Thieno[3,2-b]pyrrole Com-

pounds against Wild-Type (WT) CHIKV-IMT Infection. The

most potent (EC₅₀values <10 μM) compounds (i.e., amides

7h−i, 13d, 15c, 15e, 15g−j, and 16a) from the CHIKV-Gluc

screen were selected for further study of the antiviral activity

against WT CHIKV, which originated from Reunion Island

(IMT). High content screen (Cellomics ArrayScan V) was used

to assess infectivity by determining the number of CHIKV

positive cells.^42,43No eﬀect was observed for infected cells

treated with 0.05% DMSO (highest concentration of the

solvent used in this screening) (Supporting Information). A

dose-dependent inhibition of CHIKV-IMT infection by these

carboxamides and ribavirin was observed. From their dose−

response curves, 50% eﬀective concentrations (EC₅₀) were also

determined (Tables 5 and 6). Although some compounds

(amides 13d, 15e, and 16a) show poorer activity (EC₅₀values

>10 μM) against CHIKV-IMT infection compared to their

activity against CHIKV-Gluc, other (amides 15c and 15g−i)

retained their potency, possessing EC₅₀values as low as 2 μM.

As shown in Figure 1, amides 15c and 15g show more than

60% inhibition of CHIKV-IMT infection at their 5 μM

concentrations compared to approximately 10% inhibition of

CHIKV-IMT infection by ribavirin, demonstrating the robust

antiviral activity of amides 15c and 15g at low micromolar

concentration.

were determined by the inhibition of Gaussia luciferase expression in

the antiviral assay. The values are the mean SD from 2 independent

experiments. EC₅₀values against wild-type (WT) CHIKV-IMT were

calculated eﬀective concentrations of compounds required to inhibit

50% CHIKV-IMT infectivity. The values are the mean SD from 3

independent experiments. The positive control has EC₅₀values of 13.1

0.7 μM and 20.0 4.5 μM against CHIKV-Gluc and CHIKV-IMT,

respectively. Cell viability was above 80% for all EC₅₀concentrations,

d

showing minimal cytotoxicity. Selectivity index (SI) was calculated as

CC₅₀value/EC₅₀(against CHIKV-Gluc) value.

toxic (CC₅₀values >100 μM) and gave a selectivity index >8,

while those amides with an alkyl group (7h−k) show some

cytotoxicity (CC₅₀values ranging between 18 and 30 μM).

Various structural modiﬁcations such as installing a methyl

group at the benzylic position (7e), substituting a bromo group

at the meta position of the benzene ring (7f), and increasing the

distance between the phenyl ring and amide moiety by one

carbon atom (7l) were made on amide 7d. However, not much

improvement in antiviral activity (7e−f and 7l) was observed.

Among the amides 7h−k, amide 7h exhibited the best activity

(EC₅₀= 7.00 μM). A 2-fold drop in activity was observed with

7g as compared to 7h suggesting that the amide substituent is

preferable to the ketone functionality for antiviral activity. In

brief, the antiviral results demonstrated that substitution with

F

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Figure 1. Dose-dependent inhibition of thieno[3,2-b]pyrroles 15c and 15g and ribavirin on CHIKV-IMT infectivity shown using a Cellomics high

content screen. HEK 293T cells were infected with WT CHIKV-IMT at MOI = 0.1, treated with serial dilutions of compounds 15c, 15g, and

ribavirin and ﬁxed at 24 h postinfection. (A) Immunoﬂuorescence detection of alphavirus protein is used as an indication of CHIKV infection.

Images are taken from stained infected cells that were treated with serial dilutions of ribavirin. Cell nuclei were stained with DAPI (blue), and

CHIKV infection is indicated by FITC (green) staining. Mock-infected cells were shown as the negative control. Images were captured with 60×

magniﬁcation. Representative microscopic images per treatment conditions are illustrated. (B) A dose−response inhibition of CHIKV-IMT infection

for the diﬀerent concentrations of thieno[3,2-b]pyrroles 15c and 15g and ribavirin are observed from their immunoﬂuorescence image. % infectivity

was calculated as follows: % infectivity = 100(% responder from infected cells after compound treatment at x μM/% responder from untreated

infected cells). Results are presented as the mean SEM. Data are representative of 3 independent experiments.

Inhibitory Properties of Selected Compounds against Viral

Protein Synthesis. To investigate the eﬀect of thieno[3,2-

b]pyrroles on CHIKV protein synthesis, Western blot analyses

of IMT-infected cells treated with compounds 15c and 15g

were carried out. A dose-dependent reduction of CHIKV

nonstructural proteins (i.e., nsP1 and nsP3) and structural

proteins (i.e., capsid and E2) for IMT-infected cells was

observed upon treatment with these compounds (Figure 2).

Compound 15c showed complete inhibition of CHIKV protein

synthesis at the lowest concentration (2.5 μM). Similarly,

treatment with 5 μM compound 15g showed a signiﬁcant

down-regulation of all the tested viral proteins. In contrast,

there was no inhibition of viral protein synthesis when infected

Figure 2. Eﬀects of thieno[3,2-b]pyrroles 15c and 15g and ribavirin on

viral protein expression. The HEK 293T cells were infected with WT

CHIKV-IMT at an MOI = 0.1 and treated with serial dilutions of

compounds 15c, 15g, and ribavirin. The expression of viral protein was

determined from cell lysates at 24 h postinfection using Western blot

analyses. Mock-infected (Mock) cell lysates were used as the negative

control. Lysates were subjected to SDS−PAGE gel and probed with

rabbit polyclonal antisera against nsP1, nsP3, capsid, or E2 proteins,

followed by secondary rabbit anti-IgG-HRP. Sizes of molecular weight

markers are indicated in the left part of the diagram. A dose-dependent

reduction of levels of CHIKV nsP1, nsP3, capsid, and E2 proteins are

observed upon post-treatment. Detection of β-actin expression served

as a loading control. Western blot images are representative of at least

2 independent experiments.

cells were treated with 5 μM of ribavirin (Figure 2). The

consistent expression levels of the “housekeeping gene” (β-

actin) between diﬀerent sample lanes indicated that the

concentrations of the test compounds did not aﬀect the levels

of host cellular proteins.

Inhibitory Properties of Selected Compounds against Viral

RNA Synthesis. In the early postentry stages of the CHIKV

replication cycle, the positive-sense RNA genome is used as a

template for the production of nonstructural proteins by host

cell translation machinery. These nonstructural proteins

combine to form a viral replicase to synthesize negative-sense

RNA.⁴⁴This negative-sense RNA is then used as a template to

synthesize more positive-sense RNA genomes used for

production on a larger amount of nonstructural proteins and

for production of infectious progeny viruses; in addition,

subgenomic RNA (or, in the case of CHIKV-Gluc, two

subgenomic RNAs) is made using negative-sense RNA as the

template. The subgenomic RNA is translated to produce

structural proteins. Postmodiﬁed structural proteins are

assembled with the newly synthesized positive-sense RNA to

form infectious progeny viruses that will infect other cells, and a

new replication cycle begins.⁴⁴In other words, any reduction in

the expression of viral protein or RNA production will produce

a chain of events that eventually results in a decrease of CHIKV

G

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Figure 3. Eﬀects of thieno[3,2-b]pyrroles 15c and 15g and ribavirin on viral RNA expression. The HEK 293T cells were infected with CHIKV-IMT

at an MOI = 0.1 and treated with serial dilutions of compounds 15c, 15g, and ribavirin. Viral load was measured at 24 h postinfection using qRT-

PCR targeted against the E1 and nsP1 genes. All compounds displayed dose−dependent inhibition of viral RNA copy numbers upon post-treatment,

and minimal cytotoxicity was observed at these concentrations. Data are the mean SEM and are representative of 3 independent experiments.

Asterisk (*) indicates a value of p < 0.05, (**) indicates a value of p < 0.01, and (***) indicates a value of p < 0.001 in a two-tailed unpaired t test

between treated samples and the untreated control.

infectivity. To investigate the eﬀect of thieno[3,2-b]pyrroles

against viral RNA production, qRT-PCR was performed on

viral RNA isolated from IMT-infected cells treated with

compounds 15c and 15g. Figure 3A and B shows that

treatment with these compounds resulted in a signiﬁcant dose-

dependent reduction in viral load from 2.5 μM or 5 μM

onward. Notably, a 100-fold reduction in viral load is observed

for IMT-infected cells that were treated with 2.5 μM compound

15c as compared to untreated infected cells.

Broad-Spectrum Antiviral Activities against Other Alpha-

viruses. A virus screen panel with other alphaviruses such as

ONNV and SINV using Cellomics high content screen was

carried out. As shown in Table 7, thieno[3,2-b]pyrrole 15c also

exhibited broad-spectrum antiviral activities (EC₅₀values ca. 1−

2 μM) against other alphaviruses such as ONNV and SINV,

which points toward a mechanism of action that possibly

involves a target that is common in the alphavirus life cycle. In

the case of WEEV antiviral activity, the mode of action of a

similar class of thieno[3,2-b]pyrroles was investigated by

Delekta et al. and was suggested to target an unknown host

factor that modulates cellular or viral gene expression, in part,

through suppression of cap-dependent translation.⁴⁵As such,

thieno[3,2-b]pyrrole 15c could be interacting with the same

target. If this is the case, this target also plays a crucial role in

the life cycle of other alphaviruses such as CHIKV, ONNV, and

SINV.

Altogether, compound 15c has the highest potential to be

developed as an anti-CHIKV compound for therapeutic use.

Following this, the pharmacokinetic and pharmacodynamics

parameters of this new class of thieno[3,2-b]pyrrole-based

compounds will be optimized, and mouse models of CHIKV

infection can be employed to evaluate the compounds’ eﬃcacy

in mouse models. Compound 15c forms the basis for the

design of a next generation compound library that can be used

for the validation of antiviral activity.

CONCLUSIONS

■

In this study, we report the identiﬁcation of thieno[3,2-

b]pyrroles as potent inhibitors of CHIKV. The structural

requirements for this class of compounds were also described in

the SAR studies. Of these requirements, substitution with

tertiary isonipecotyl amide at the C5 position of the thieno[3,2-

b]pyrrole scaﬀold is important for potent activity, as

demonstrated by compound 7h. However, the toxicity proﬁle

of compound 7h was poor. This led to the investigation of the

C2 and C6 substituted derivatives of thieno[3,2-b]pyrrole

carboxamides 6 and 7h Among the compounds investigated,

C2 bromo substituted thieno[3,2-b]pyrrole (compound 15c)

which exhibited potent antiviral activity as well as a good

cytotoxicity proﬁle (i.e., SI > 32) was identiﬁed as a potential

Table 7. Antiviral Evaluation of the Thieno[3,2-b]pyrrole

15c and Ribavirin against Diﬀerent Alphaviruses, ONNV and

SINV

compound

15c

ribavirin

EC₅₀(μM)

18.4 5.5

a

virus

EC₅₀(μM)

ONNV

SINV

2.58 0.47

1.14 0.44

5.12 2.00

a

Calculated eﬀective concentration of compound that is required to

inhibit ONNV or SINV infectivity by 50%. EC₅₀values are the mean

SD from at least 3 independent experiments.

H

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Figure 4. Summary of the eﬀects of substitution at the C2, C6, and C5 positions of the thieno[3,2-b]pyrrole core structure on antiviral activities from

the respective studies.

were composed of methanol in water (75−90% v/v). Solvent mixtures

used for chromatography are always given as a vol/vol ratio. Melting

points were determined on a MPA100 automated melting point

apparatus from Stanford Research Systems (SRS). ESI and APCI mass

spectra were performed with electron spray or atmospheric-pressure

chemical ionization modes on an Applied Biosystems MDS SCIEX

API 2000 mass spectrometer. High resolution electrospray (HRE-

SIMS) techniques were carried out on an Agilent Technologies 6210

Time-of-ﬂight LC/MS (negative and positive ionization modes).

lead compound (Figure 4). Not only did compound 15c show

potent inhibition against CHIKV-Gluc, it was equally potent

(i.e., EC₅₀value ca. 2 μM) against WT CHIKV-IMT infection

from immunoﬂuorescence high content screening. Further

studies such as Western Blot analyses and qRT-PCR

demonstrated that compound 15c inhibits both protein

synthesis and viral RNA synthesis at as low as 2.5 μM

concentration, highlighting that compound 15c could be

interfering with some crucial pathway needed for RNA

production or expression of viral protein (i.e., nsP1, nsP3,

capsid, and E2). In addition, carboxamide 15c also exhibited

antiviral activity against other alphaviruses such as ONNV and

SINV. In summary, we have demonstrated that this class of

thieno[3,2-b]pyrroles exhibited novel antiviral activity against

CHIKV possibly via the inhibition of CHIKV infection after

virus entry but before/during RNA production. Further

mechanism of action studies will be carried out in the future

to identify the molecular targets of this class of thieno[3,2-

b]pyrroles.

1

NMR spectra were recorded at 400 MHz for H nuclei and at 100

MHz for ¹³C nuclei on a Bruker Ultra Shield spectrometer with

CDCl₃, (CD₃)₂CO, CD₃OD, or DMSO-d₆as solvent. The chemical

shifts are given in ppm and are internally referenced to residual protio-

solvent signals [¹H NMR, CDCl₃(δ 7.26), (CD₃)₂CO (δ 2.05),

CD₃OD (δ 3.31), DMSO-d₆(δ 2.50); ¹³C NMR, CDCl₃(δ 77.0),

(CD₃)₂CO (δ 29.8), CD₃OD (δ 49.0), DMSO-d₆(δ 39.5)]. Chemical

shifts are given using the δ-scale relative to the residual solvent signal

as an internal reference. The following abbreviations were used to

describe the signals: s = singlet, d = doublet, t = triplet, q = quartet,

quint = quintet, sext = sextet, m = multiplet, and br = broad signal.

The spectra were analyzed by a computer using the software

MestReNova 6 (Mestrelab research, 1994).

EXPERIMENTAL SECTION

The purity of the synthesized compounds used for biological testing

was determined by analytical high-performance liquid chromatography

(HPLC). Analytical HPLC was carried out on a Shimadzu Ultra Fast

Liquid Chromatograph Prominence series (LC-20AD) system with a

Phenomenex Kinetex 2.6 μm C18 100 Å (150 × 4.60 mm) LC

analytical column at 254 nm. The gradient or isocratic systems for

HPLC separation were composed of either acetonitrile in water (20−

90% v/v) or methanol in water (40−90% v/v) and were spiked with/

without 0.1% triﬂuoroacetic acid or triethylamine. All test compounds

displayed more than 95% purity. Elemental analysis was carried out

using a EuroEA3000 series CHNS Analyzer. The analytical results

obtained were within 0.4% of the theoretical values. CLogD values at

pH 7.4 were obtained from Marvin Sketch software, version 5.11.2

(ChemAxon Ltd., 1998).

Chemistry. Detailed experimental procedures of the intermediates

are described in the Supporting Information. The data below are for

the ﬁnal compounds tested in the biological studies.

General Synthetic Procedure A: Hydrolysis Reaction for the

Synthesis of 5-Carboxylic Acids 2 and 7b. To a solution of ester (1

equiv) in a mixture of THF/EtOH/H₂O was added KOH (3.5 equiv).

■

General. All reagents and solvents (of analytical and HPLC grades)

were purchased from Sigma-Aldrich or other commercial sources and

were used without further puriﬁcation. Reactions that were moisture

sensitive or using anhydrous solvents were performed under nitrogen

atmosphere. DMF, THF, DCM, and Et₂O were dried by the PureSolv

MD 4 Solvent Puriﬁcation System (Innovative Technology) prior to

use. The solvents were removed under reduced pressure using

standard rotary evaporators.

Analytical thin layer chromatography (TLC) was performed on

Merck’s precoated silica gel plates. Visualization was accomplished

with UV light (254 nm) or by staining with basic KMnO₄, ninhydrin,

or ceric ammonium molybdate (CAM) solution followed by heating.

Compounds were puriﬁed using ﬂash chromatography on a glass

column using Merck silica gel 60 (230−400 mesh) or a Buchi

Sepacore ﬂash chromatography system by gradient. Semipreparative

HPLC puriﬁcations were performed using a Shimadzu Ultra Fast

Liquid Chromatograph Prominence series (LC-20AD) system with a

Phenomenex Gemini 5.0 μm C18 110 Å (150 × 10.0 mm) LC column

at 254 nm. The gradient or isocratic systems for HPLC separation

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The reaction mixture was then reﬂuxed overnight, then diluted with

H₂O and THF and EtOH removed under reduced pressure. The

remaining aqueous suspension was washed 2× with Et₂O followed by

acidiﬁcation with 1 N HCl to pH 3−4 before extraction 3× with

EtOAc. The combined organic extracts were washed 1× with H₂O and

1× with brine, dried with Na₂SO₄, and concentrated in vacuo to aﬀord

the acid.

General Synthetic Procedure B: Amide Coupling Reaction for the

Synthesis of C5 Carboxamides 4, 6−7, 13−14, 15a−c, and 16a.

Carboxylic acid (1 equiv) and EDCI (1.05 equiv) were dissolved in

DCM, and the reaction mixture was cooled down to 0 °C and stirred

for 30 min. Alternatively, carboxylic acid (1 equiv) and CDI (1.05

equiv) were dissolved in THF, and the reaction mixture was warmed

up to 45 °C and stirred for 1 h. The reaction mixture was then added

to the respective amine or piperidium salt (1−2 equiv) with/without

DIEA (2−3 equiv). The reaction mixture was stirred overnight at rt

before washing 1× with 5% citric acid, 1× saturated NaHCO₃, 1× with

H₂O, and 1× with brine. The washed organic mixture was then dried

with Na₂SO₄and concentrated in vacuo to give a crude residue which

was puriﬁed using column chromatography (SiO₂, eluent: PE/EtOAc

or DCM/MeOH or DCM/EtOAc/MeOH) to aﬀord the desired

carboxamide.

General Synthetic Procedure C: Boc Deprotection Reaction for

the Synthesis of Carboxamides 4h and 14a. To a round-bottomed

ﬂask was added Boc-protected carboxamide (1 equiv) and 30−40%

TFA/DCM. The reaction mixture was stirred at rt for 1 h before

concentrating in vacuo to give a crude residue, which was diluted with

1 N NaOH. This aqueous suspension was extracted 3× with DCM,

and the combined organic extracts washed 1× with brine, dried with

Na₂SO₄, and concentrated in vacuo. The residue was puriﬁed by

column chromatography (SiO₂, eluent: DCM/MeOH/28%NH₃(aq)

or DCM/MeOH) to aﬀord the desired carboxamide.

General Synthetic Procedure D: Reductive Amination Reaction

for the Synthesis of C5 Carboxamides 15d−f and 16c−e. To a dried

round-bottomed ﬂask was added Na₂SO₄followed by a mixture of

carbaldehyde 26 (1 equiv) and respective amine (1.1−2 equiv) in

anhydrous MeOH. The reaction mixture was then stirred at 60 °C for

3 h or overnight before NaBH₄(0.5 equiv) was added. The reaction

mixture was left to stir at rt for a further 2 h before dilution with H₂O,

and MeOH was removed under reduced pressure. The aqueous layer

was extracted 3× with EtOAc. The combined organic extracts was

washed with 1× with brine, dried with Na₂SO₄, and concentrated in

vacuo to give a crude residue, which was puriﬁed by column

chromatography (SiO₂, eluent: PE/EtOAc or DCM/MeOH) to aﬀord

desired amine.

General Synthetic Procedure E: Reduction Reaction for the

Synthesis of Alochols 15g and 16b. To a mixture of carbaldehyde 26

(1 equiv) in a mixture of THF/MeOH (1:1) was added NaBH₄(0.5

equiv) at 0 °C. The reaction mixture was left to stir at 0 °C for 3 h

before dilution with EtOAc. The reaction mixture was washed 1× with

1 N HCl and 1× with brine, dried with Na₂SO₄, and concentrated in

vacuo to aﬀord the desired alcohol, which was puriﬁed by column

chromatography (SiO₂, eluent: PE/EtOAc or DCM/MeOH) to aﬀord

the desired amine.

General Synthetic Procedure F: Suzuki Coupling Reaction for the

Synthesis of Carboxamides 15h and 15i. To a sealed tube was added

carboxamide 15c (1 equiv), Pd(OAc)₂(10 mol %), PPh₃(30 mol %),

MeB(OH)₂or PhB(OH)₂(2 equiv), Ag₂O (3 equiv), and K₂CO₃(3

equiv). Degassed toluene/H₂O (19:1) was added, and the mixture was

heated to 120 °C and stirred for 36 h before diluting with H₂O. The

aqueous suspension was extracted 3× with EtOAc, and the combined

organic extracts were washed 1× with brine, dried with Na₂SO₄, and

concentrated in vacuo to give a crude residue, which was puriﬁed by

semipreparative reversed-phase HPLC (C18, eluent: MeOH/H₂O) to

aﬀord the desired carboxamide.

reduced pressure. The remaining aqueous suspension was extracted

with EtOAc (3 × 5 mL) and the combined organic extracts (15 mL)

washed with H₂O (2 × 8 mL) and brine (1 × 8 mL), dried with

Na₂SO₄, and concentrated in vacuo to give a crude residue, which was

puriﬁed by column chromatography (SiO₂, eluent: 95% PE, 5%

EtOAc) to obtain the desired N-p-MeOBn-thieno[3,2-b]pyrrole 1

(131 mg, 78%). Yellow solid; mp 49.4−50.4 °C; ¹H NMR (400 MHz;

CDCl₃) δ 1.35 (t, J = 7.0 Hz, 3H), 3.76 (s, 3H), 4.30 (q, J = 7.0 Hz,

2H), 5.68 (s, 2H), 6.79−6.83 (m, 2H), 6.86 (dd, J = 5.4, 0.6 Hz, 1H),

7.08−7.12 (m, 2H), 7.25 (d, J = 0.6 Hz, 1H), 7.29 (d, J = 5.4 Hz, 1H);

¹³C NMR (100 MHz, CDCl₃) δ 14.4, 49.9, 55.2, 60.1, 109.7, 110.7,

114.0, 122.3, 126.4, 128.2, 129.2, 130.2, 145.3, 158.9, 161.6; ESI-TOF-

HRMS (m/z) calcd for C₁₇H₁₇NO₃S [M + H]⁺316.1007; found

316.1009.

4-(4-Methoxybenzyl)-4H-thieno[3,2-b]pyrrole-5-carboxylic Acid

(2). Carboxylic acid 2 was prepared following general procedure A

using ester 1 (135 mg, 0.43 mmol) and KOH (84 mg, 1.50 mmol) in

EtOH (3 mL) and H₂O (1 mL). Carboxylic acid 2 was obtained in

86% yield (105 mg). White solid; mp 156.3−156.9 °C; ¹H NMR (400

MHz; CDCl₃) δ 3.76 (s, 3H), 5.68 (s, 2H), 6.78−6.83 (m, 2H), 6.86

(d, J = 5.4 Hz, 1H), 7.08−7.13 (m, 2H), 7.35 (d, J = 5.4 Hz, 1H), 7.38

(s, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 50.0, 55.2, 110.7, 111.7,

114.0, 122.7, 125.2, 128.3, 129.9, 130.5, 146.3, 159.0, 165.9; ESI-TOF-

HRMS (m/z) calcd for C₁₅H₁₃NO₃S [M-H]⁻286.0538; found

286.0537.

4-(4-Methoxybenzyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide (3).

To a solution of carboxylic acid 2 (250 mg, 0.87 mmol, 1 equiv) and

DMF (catalytic amount) in anhydrous DCM (5 mL) was added oxalyl

chloride (152 μL, 1.74 mmol, 2 equiv) dropwise at 0 °C. The reaction

mixture was stirred at rt for 15 h before concentration in vacuo to give

the crude acyl chloride. This acid chloride was dissolved in anhydrous

DCM (3 mL), following which 28% ammonium hydroxide solution

(786 uL, 5.66 mmol, 6.5 equiv) was added dropwise into the reaction

mixture at −10 °C. The reaction mixture was then stirred at −10 °C

for 30 min following which it was diluted with H₂O (5 mL) and

warmed to rt. The aqueous suspension was ﬁltered, and the resulting

residue was washed with H₂O (2 × 2 mL) and dried to aﬀord the

carboxamide 3 (206 mg, 83%). Yellow solid; mp 161.2−162.0 °C; ¹H

NMR (400 MHz; CDCl₃) δ 3.75 (s, 3H), 5.70 (s, 2H), 6.77−6.83 (m,

2H), 6.86 (d, J = 5.3 Hz, 1H), 6.89 (s, 1H), 7.10−7.17 (m, 2H), 7.25

(d, J = 5.3 Hz, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 49.9, 55.2, 105.2,

110.8, 113.9, 122.1, 128.3, 128.4, 128.5, 130.3, 144.7, 158.9, 163.8;

ESI-TOF-HRMS (m/z) calcd for C₁₅H₁₄N₂O₂S [M + H]⁺287.0854;

found 287.0855.

4-(4-Methoxybenzyl)-N-propyl-4H-thieno[3,2-b]pyrrole-5-carbox-

amide (4a). Carboxamide 4a was prepared following general

procedure B using carboxylic acid 2 (60 mg, 0.21 mmol), n-

propylamine (35 μL, 0.42 mmol), and EDCI (42 mg, 0.22 mmol).

After chromatography (SiO₂, eluent: 99.5% DCM, 0.5% MeOH),

carboxamide 4a was obtained as a white solid (33 mg, 48%). mp

1

116.3−117.6 °C; H NMR (400 MHz; CDCl₃) δ 0.95 (t, J = 7.2 Hz,

3H), 1.59 (sext, J = 7.2 Hz, 2H), 3.35 (dt, J = 7.2, 6.2 Hz, 2H), 3.74 (s,

3H), 5.67 (s, 2H), 6.05 (br s, 1H), 6.70−6.81 (m, 3H), 6.86 (dd, J =

5.3, 0.5 Hz, 1H), 7.09−7.18 (m, 2H), 7.19 (d, J = 5.3 Hz, 1H); ¹³C

NMR (100 MHz, CDCl₃) δ 11.3, 23.0, 41.2, 49.7, 55.2, 103.2, 110.8,

113.9, 122.0, 127.2, 128.5, 130.4, 130.5, 143.9, 158.8, 162.2; ESI-TOF-

HRMS (m/z) calcd for C₁₈H₂₀N₂O₂S [M + H]⁺329.1324; found

329.1314. Anal. Calcd for C₁₈H₂₀N₂O₂S (MW = 328.43): C, 65.83%;

H, 6.14%; N, 8.53%; S, 9.76%. Found: C, 65.88%; H, 5.99%; N, 8.38%;

S, 9.44%.

N-Benzyl-4-(4-methoxybenzyl)-4H-thieno[3,2-b]pyrrole-5-car-

boxamide (4b). Carboxamide 4b was prepared following general

procedure B using carboxylic acid 2 (18 mg, 0.06 mmol), benzylamine

(11 μL, 0.10 mmol), and CDI (11 mg, 0.07 mmol). After

chromatography (SiO₂, eluent: 60% PE, 40% EtOAc), carboxamide

4b was obtained as a white solid (15 mg, 67%). mp 131.0−131.7 °C;

¹H NMR (400 MHz; CDCl₃) δ 3.76 (s, 3H), 4.58 (d, J = 5.6 Hz, 2H),

5.70 (s, 2H), 6.30 (br s, 1H), 6.76−6.82 (m, 3H), 6.88 (dd, J = 5.4, 0.5

Hz, 1H), 7.10−7.15 (m, 2H), 7.22 (d, J = 5.4 Hz, 1H), 7.24−7.37 (m,

5H); ¹³C NMR (100 MHz, CDCl₃) δ 43.4, 49.8, 55.2, 103.7, 110.8,

Ethyl 4-(4-Methoxybenzyl)-4H-thieno[3,2-b]pyrrole-5-carboxy-

late (1). Ester 5 (105 mg, 0.54 mmol, 1 equiv), 4-methoxybenzyl

chloride (73 μL, 0.54 mmol), and K₂CO₃(149 mg, 1.07 mmol) were

dissolved in CH₃CN (5 mL). The reaction mixture was reﬂuxed

overnight, then diluted with H₂O (5 mL) and CH₃CN removed under

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113.9, 122.0, 127.4, 127.5, 127.6, 128.4, 128.7, 130.0, 130.4, 138.4,

144.2, 158.9, 162.0; ESI-TOF-HRMS (m/z) calcd for C₂₂H₂₀N₂O₂S

[M + H]⁺377.1324; found 377.1317.

130.3, 143.0, 159.1, 163.1; ESI-TOF-HRMS (m/z) calcd for

C₁₉H₂₀N₂O₃S [M + H]⁺357.1273; found 357.1301.

(4-(4-Methoxybenzyl)-4H-thieno[3,2-b]pyrrol-5-yl) (piperazin-1-

yl)methanone (4h). Carboxamide 4h was prepared following general

procedure C using carboxamide 33 (75 mg, 0.16 mmol). After

chromatography (SiO₂, eluent: DCM/MeOH/28%NH₃(aq) =

100:10:1), pure carboxamide 4h was obtained as a colorless sticky

oil (24 mg, 41%). ¹H NMR (400 MHz; CDCl₃) δ 2.60−2.77 (m, 4H),

3.55−3.67 (m, 4H), 3.76 (s, 3H), 5.40 (s, 2H), 6.52 (s, 1H), 6.74−

6.83 (m, 2H), 6.89 (d, J = 5.3 Hz, 1H), 7.04−7.12 (m, 2H), 7.17 (d, J

= 5.3 Hz, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 46.1, 49.6, 55.3, 104.1,

110.6, 113.9, 121.9, 126.2, 128.7, 129.6, 130.3, 142.8, 159.1, 163.0;

ESI-TOF-HRMS (m/z) calcd for C₁₉H₂₁N₃O₂S [M + H]⁺356.1433;

found 356.1433.

4-(4-Methoxybenzyl)-N-(1-phenylethyl)-4H-thieno[3,2-b]pyrrole-

5-carboxamide (4c). Carboxamide 4c was prepared following the

representative general procedure B using carboxylic acid 2 (60 mg,

0.21 mmol), ( ) α-phenylethylamine (33 μL, 0.25 mmol), and EDCI

(42 mg, 0.22 mmol). After chromatography (SiO₂, eluent: 99.5%

DCM and 0.5% MeOH), carboxamide 4c was obtained as a white solid

1

(28 mg, 34%). mp 171.3−172.8 °C; H NMR (400 MHz; CDCl₃) δ

1.54 (d, J = 7.4 Hz, 3H), 3.73 (s, 3H), 5.24 (quint, J = 7.4 Hz, 1H),

5.63 (s, 2H), 6.23 (m, 1H), 6.72−6.80 (m, 3H), 6.85 (d, J = 5.3 Hz,

1H), 7.06−7.13 (m, 2H), 7.18 (d, J = 5.3 Hz, 1H), 7.25−7.38 (m,

5H); ¹³C NMR (100 MHz, CDCl₃) δ 21.9, 48.6, 49.7, 55.2, 103.6,

110.8, 113.9, 122.0, 126.1, 127.3, 127.4, 128.5, 128.6, 130.2, 130.3,

143.3, 144.0, 158.8, 161.4; ESI-TOF-HRMS (m/z) calcd for

C₂₃H₂₂N₂O₂S [M + H]⁺391.1480; found 391.1472. Anal. Calcd for

C₂₃H₂₂N₂O₂S (MW = 390.50): C, 70.74%; H, 5.68%; N, 7.17%; S,

8.21%. Found: C, 70.99%; H, 5.60%; N, 6.92%; S, 7.83%.

(4-Benzylpiperazin-1-yl)(4-(4-methoxybenzyl)-4H-thieno[3,2-b]-

pyrrol-5-yl)methanone (6). Carboxamide 6 was prepared following

general procedure B using carboxylic acid 2 (121 mg, 0.39 mmol), 1-

benzylpiperazine (80 μL, 0.46 mmol), and EDCI (77 mg, 0.40 mmol).

After chromatography (SiO₂, eluent: 30% PE, 70% EtOAc),

carboxamide 6 was obtained as a white solid (123 mg, 72%). mp

N,N-Diethyl-4-(4-methoxybenzyl)-4H-thieno[3,2-b]pyrrole-5-car-

boxamide (4d). Carboxamide 4d was prepared following general

procedure B using carboxylic acid 2 (60 mg, 0.21 mmol), diethylamine

(26 μL, 0.25 mmol), and EDCI (42 mg, 0.22 mmol). After column

chromatography (SiO₂, eluent: 99.5% DCM, 0.5% MeOH),

carboxamide 4d was obtained as a yellow sticky oil (22 mg, 31%).

¹H NMR (400 MHz; CDCl₃) δ 1.10 (t, J = 7.1 Hz, 6H), 3.41 (q, J =

7.1 Hz, 4H), 3.75 (s, 3H), 5.37 (s, 2H), 6.55 (s, 1H), 6.74−6.84 (m,

2H), 6.88 (d, J = 5.3 Hz, 1H), 7.04−7.13 (m, 2H), 7.14 (d, J = 5.3 Hz,

1H); ¹³C NMR (100 MHz, CDCl₃) δ 13.4, 49.7, 55.3, 102.6, 110.6,

113.9, 121.9, 125.7, 128.7, 130.3, 130.7, 142.4, 159.0, 163.8; ESI-TOF-

HRMS (m/z) calcd for C₁₉H₂₂N₂O₂S [M + H]⁺343.1480; found

343.1474.

1

84.0−85.2 °C; H NMR (400 MHz; CDCl₃) δ 2.10−2.40 (m, 4H),

3.46 (s, 2H), 3.59−3.75 (m, 4H), 3.79 (s, 3H), 5.40 (s, 2H), 6.51 (s,

1H), 6.78−6.84 (m, 2H), 6.91 (d, J = 5.3 Hz, 1H), 7.05−7.12 (m,

2H), 7.17 (d, J = 5.3 Hz, 1H), 7.23−7.39 (m, 5H); ¹³C NMR (100

MHz, CDCl₃) δ 49.6, 52.8, 55.3, 62.7, 104.3, 110.4, 114.0, 121.8,

126.3, 127.5, 128.4, 128.8, 129.3, 130.4, 142.9, 159.0, 162.9; ESI-TOF-

HRMS (m/z) calcd for C₂₆H₂₇N₃O₂S [M + H]⁺446.1902; found

446.1907.

Ethyl 1-(4-(4-Methoxybenzyl)-4H-thieno[3,2-b]pyrrole-5-

carbonyl)piperidine-4-carboxylate (7a). Carboxamide 7a was pre-

pared following general procedure B using carboxylic acid 2 (60 mg,

0.21 mmol), piperidine 8k (49 mg, 0.25 mmol), DIEA (73 μL, 0.42

mmol), and EDCI (42 mg, 0.22 mmol). After chromatography (SiO₂,

eluent: 98% DCM, 2% MeOH), carboxamide 7a was obtained as a

(4-(4-Methoxybenzyl)-4H-thieno[3,2-b]pyrrol-5-yl) (pyrrolidin-1-

yl)methanone (4e). Carboxamide 4e was prepared following general

procedure B using carboxylic acid 2 (60 mg, 0.21 mmol), pyrrolidine

(21 μL, 0.25 mmol) and EDCI (42 mg, 0.22 mmol). After

chromatography (SiO₂, eluent: 99.5% DCM, 0.5% MeOH),

carboxamide 4e was obtained as a colorless sticky oil (46 mg, 64%).

¹H NMR (400 MHz; CDCl₃) δ 1.65−1.93 (m, 4H), 3.42−3.65 (m,

1

yellow sticky oil (63 mg, 71%). H NMR (400 MHz; CDCl₃) δ 1.26

(t, J = 7.1 Hz, 3H), 1.35−1.55 (m, 2H), 1.70−1.89 (m, 2H), 2.47 (m,

1H), 2.90−3.05 (m, 2H), 3.75 (s, 3H), 4.14 (q, J = 7.1 Hz, 2H), 4.19−

4.38 (m, 2H), 5.38 (s, 2H), 6.52 (s, 1H), 6.74−6.82 (m, 2H), 6.91 (d,

J = 5.3 Hz, 1H), 7.03−7.12 (m, 2H), 7.17 (d, J = 5.3 Hz, 1H); ¹³C

NMR (100 MHz, CDCl₃) δ 14.2, 28.1, 41.0, 49.6, 55.2, 60.5, 103.9,

110.5, 113.9, 121.8, 126.2, 128.7, 129.6, 130.2, 142.7, 159.1, 163.0,

174.1; ESI-TOF-HRMS (m/z) calcd for C₂₃H₂₆N₂O₄S [M + H]⁺

427.1692; found 427.1677.

1-(4-(4-Methoxybenzyl)-4H-thieno[3,2-b]pyrrole-5-carbonyl)-

piperidine-4-carboxylic Acid (7b). Carboxylic acid 7b was prepared

following general procedure A using carboxylate 7a (14 mg, 0.03

mmol) and KOH (4.6 mg, 0.11 mmol) in THF (1 mL), EtOH (1

mL), and H₂O (1 mL). Carboxylic acid 7b was obtained in 92% yield

(12 mg). Yellow sticky oil; ¹H NMR (400 MHz; CDCl₃) δ 1.30−1.59

(m, 2H), 1.62−1.93 (m, 2H), 2.51 (m, 1H), 2.85−3.10 (m, 2H), 3.73

(s, 3H), 4.05−4.41 (m, 2H), 5.38 (s, 2H), 6.52 (s, 1H), 6.70−6.82 (m,

2H), 6.92 (d, J = 5.3 Hz, 1H), 7.00−7.11 (m, 2H), 7.18 (d, J = 5.3 Hz,

1H); ¹³C NMR (100 MHz, CDCl₃) δ 27.8, 40.7, 49.6, 55.2, 104.1,

110.5, 113.9, 121.8, 126.3, 128.7, 129.3, 130.2, 142.9, 159.1, 163.1,

179.3; ESI-TOF-HRMS (m/z) calcd for C₂₁H₂₂N₂O₄S [M-H]⁻

397.1222; found 397.1209.

1-(4-(4-Methoxybenzyl)-4H-thieno[3,2-b]pyrrole-5-carbonyl)-

piperidine-4-carboxamide (7c). Carboxamide 7c was prepared

following general procedure B using carboxylic acid 2 (60 mg, 0.21

mmol), piperidine 8i (61 mg, 0.25 mmol), DIEA (73 μL, 0.42 mmol),

and EDCI (42 mg, 0.22 mmol). After column chromatography (SiO₂,

eluent: DCM/EtOAc/MeOH = 1:1:0 to 0:1:0 to 19:0:1), the pure

carboxamide 7c was obtained (58 mg, 70%). White solid; mp 154.6−

156.6 °C; ¹H NMR (400 MHz; CDCl₃) δ 1.28−1.50 (m, 2H), 1.72−

1.91 (m, 2H), 2.34 (m, 1H), 2.78−3.00 (m, 2H), 3.75 (s, 3H), 4.28−

4.56 (m, 2H), 5.38 (s, 2H), 5.68 (br s, 2H), 6.52 (s, 1H), 6.75−6.85

(m, 2H), 6.91 (d, J = 5.3 Hz, 1H), 7.03−7.13 (m, 2H), 7.18 (d, J = 5.3

Hz, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 28.7, 42.6, 49.7, 55.3, 104.1,

110.5, 114.0, 121.8, 126.3, 128.8, 129.5, 130.3, 142.9, 159.1, 163.1,

4H), 3.74 (s, 3H), 5.50 (s, 2H), 6.68 (s, 1H), 6.73−6.83 (m, 2H), 6.85

(d, J = 5.3 Hz, 1H), 7.03−7.13 (m, 2H), 7.15 (d, J = 5.3 Hz, 1H); ¹³

C

NMR (100 MHz, CDCl₃) δ 24.2, 26.3, 46.1, 49.8, 55.2, 104.6, 110.7,

113.8, 121.9, 126.3, 128.5, 130.6, 131.1, 142.7, 158.9, 162.3; ESI-TOF-

HRMS (m/z) calcd for C₁₉H₂₀N₂O₂S [M + H]⁺341.1324; found

341.1318.

(4-(4-Methoxybenzyl)-4H-thieno[3,2-b]pyrrol-5-yl) (piperidin-1-

yl)methanone (4f). Carboxamide 4f was prepared following general

procedure B using carboxylic acid 2 (60 mg, 0.21 mmol), piperidine

(25 μL, 0.25 mmol), and EDCl (42 mg, 0.22 mmol). After column

chromatography (SiO₂, eluent: 99.5% DCM, 0.5% MeOH), pure

carboxamide 4f was obtained in 69% yield (51 mg); colorless sticky

1

oil; H NMR (400 MHz; CDCl₃) δ 1.30−1.54 (m, 4H), 1.54−1.63

(m, 2H), 3.48−3.65 (m, 4H), 3.75 (s, 3H), 5.39 (s, 2H), 6.51 (s, 1H),

6.75−6.82 (m, 2H), 6.87 (dd, J = 5.3, 0.5 Hz, 1H), 7.07−7.12 (m,

2H), 7.14 (d, J = 5.3 Hz, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 24.6,

26.0, 49.6, 55.3, 103.5, 110.6, 113.9, 121.9, 125.8, 128.7, 130.2, 130.3,

142.5, 159.0, 162.9; ESI-TOF-HRMS (m/z) calcd for C₂₀H₂₂N₂O₂S

[M + H]⁺355.1480; found 355.1476.

(4-(4-Methoxybenzyl)-4H-thieno[3,2-b]pyrrol-5-yl) (morpholino)-

methanone (4g). Carboxamide 4g was prepared following general

procedure B using carboxylic acid 2 (18 mg, 0.06 mmol), morpholine

(9 μL, 0.10 mmol), and CDI (11 mg, 0.07 mmol). After

chromatography (SiO₂, eluent: 60% PE, 40% EtOAc), pure

carboxamide 4g was obtained as a colorless sticky oil (15 mg, 0.04

1

mmol, 67%). H NMR (400 MHz; CDCl₃) δ 3.45−3.52 (m, 4H),

3.60−3.68 (m, 4H), 3.76 (s, 3H), 5.40 (s, 2H), 6.52 (d, J = 0.4 Hz,

1H), 6.77−6.83 (m, 2H), 6.92 (dd, J = 5.3, 0.4 Hz, 1H). 7.04−7.10

(m, 2H), 7.19 (d, J = 5.3 Hz, 1H); ¹³C NMR (100 MHz, CDCl₃) δ

45.7, 49.6, 55.3, 66.8, 104.3, 110.5, 113.9, 121.9, 126.5, 128.6, 129.0,

K

DOI: 10.1021/acs.jmedchem.5b01047

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

176.0; ESI-TOF-HRMS (m/z) calcd for C₂₁H₂₃N₃O₃S [M + H]⁺

398.1538; found 398.1539.

and EDCI (42 mg, 0.22 mmol). After column chromatography (SiO₂,

eluent: 96% DCM, 4% MeOH), pure carboxamide 7h was obtained

(55 mg, 60%). White solid; mp 182.6−184.2 °C; ¹H NMR (400 MHz;

CDCl₃) δ 0.91 (t, J = 7.3 Hz, 3H), 1.35−1.60 (m, 2H), 1.51 (sext, J =

7.3 Hz, 2H), 1.66−1.85 (m, 2H), 2.24 (m, 1H), 2.78−2.98 (m, 2H),

3.17−3.25 (m, 2H), 3.76 (s, 3H), 4.30−4.55 (m, 2H), 5.37 (s, 2H),

5.46 (br s, 1H), 6.52 (s, 1H), 6.76−6.85 (m, 2H), 6.89 (d, J = 5.3 Hz,

1H), 7.05−7.12 (m, 2H), 7.16 (d, J = 5.3 Hz, 1H); ¹³C NMR (100

MHz, CDCl₃) δ 11.3, 22.8, 28.8, 41.1, 43.3, 49.6, 55.3, 104.0, 110.5,

114.0, 121.9, 126.2, 128.7, 129.6, 130.2, 142.7, 159.1, 163.0, 173.7;

ESI-TOF-HRMS (m/z) calcd for C₂₄H₂₉N₃O₃S [M + H]⁺440.2008;

found 440.2006; Anal. Calcd for C₂₄H₂₉N₃O₃S (MW = 439.57): C,

65.58%; H, 6.65%; N, 9.56%; S, 7.29%. Found: C, 65.54%; H, 6.55%;

N, 9.26%; S, 7.10%.

N-Cyclopropyl-1-(4-(4-methoxybenzyl)-4H-thieno[3,2-b]pyrrole-

5-carbonyl)piperidine-4-carboxamide (7i). Carboxamide 7i was

prepared following general procedure B using carboxylic acid 2 (80

mg, 0.28 mmol), piperidine 8f (94 mg, 0.33 mmol), DIEA (47 μL,

0.56 mmol), and EDCI (56 mg, 0.29 mmol). After column

chromatography (SiO₂, eluent: 98% DCM, 2% MeOH), pure

carboxamide 7i was obtained (91 mg, 75%). White solid; mp

218.0−219.3 °C; ¹H NMR (400 MHz; CDCl₃) δ 0.43−0.53 (m, 2H),

0.72−0.82 (m, 2H), 1.32−1.53 (m, 2H), 1.68−1.82 (m, 2H), 2.20 (m,

1H), 2.70 (m, 1H), 2.78−2.97 (m, 2H), 3.77 (s, 3H), 4.28−4.52 (m,

2H), 5.37 (s, 2H), 5.55 (br s, 1H), 6.51 (s, 1H), 6.75−6.84 (m, 2H),

6.84 (d, J = 5.3 Hz, 1H), 7.03−7.11 (m, 2H), 7.17 (d, J = 5.3 Hz, 1H);

¹³C NMR (100 MHz, CDCl₃) δ 6.7, 22.6, 28.7, 43.0, 49.6, 55.3, 104.0,

N-Benzyl-1-(4-(4-methoxybenzyl)-4H-thieno[3,2-b]pyrrole-5-

carbonyl)piperidine-4-carboxamide (7d). Carboxamide 7d was

prepared following general procedure B using carboxylic acid 2 (32

mg, 0.11 mmol), piperidine 8a (38 mg, 0.11 mmol), DIEA (69 μL,

0.39 mmol), and EDCl (23 mg, 0.12 mmol). After column

chromatography (SiO₂, eluent: PE/EtOAc = 2:3 to 1:4), pure

carboxamide 7d was aﬀorded (27 mg, 49%). White solid; mp

141.0−143.0 °C; ¹H NMR (400 MHz; CDCl₃) δ 1.40−1.53 (m, 2H),

1.70−1.82 (m, 2H), 2.29 (m, 1H), 2.80−2.91 (m, 2H), 3.73 (s, 3H),

4.32−4.49 (m, 2H), 4.44 (d, J = 5.5 Hz, 2H), 5.37 (s, 2H), 5.77 (br s,

1H), 6.52 (s, 1H), 6.76−6.82 (m, 2H), 6.90 (d, J = 5.3 Hz, 1H), 7.04−

7.10 (m, 2H), 7.17 (d, J = 5.3 Hz, 1H), 7.22−7.38 (m, 5H); ¹³C NMR

(100 MHz, CDCl₃) δ 28.7, 43.2, 43.5, 49.6, 55.3, 104.0, 110.5, 114.0,

121.9, 126.3, 127.6, 127.7, 128.7, 128.8, 129.6, 130.2, 138.2, 142.8,

159.1, 163.1, 173.6; ESI-TOF-HRMS (m/z) calcd for C₂₈H₂₉N₃O₃S

[M + H]⁺488.2008; found 488.2010.

1-(4-(4-Methoxybenzyl)-4H-thieno[3,2-b]pyrrole-5-carbonyl)-N-

(1-phenylethyl)piperidine-4-carboxamide (7e). Carboxamide 7e was

prepared following general procedure B using carboxylic acid 2 (60

mg, 0.21 mmol), piperidine 8d (87 mg, 0.25 mmol), DIEA (73 μL,

0.42 mmol), and EDCI (42 mg, 0.22 mmol). After column

chromatography (SiO₂, eluent: 98% DCM, 2% MeOH), pure

carboxamide 7e was obtained (71 mg, 68%). White solid; mp

137.5−139.5 °C; ¹H NMR (400 MHz; CDCl₃) δ 1.31−1.61 (m, 2H),

1.46 (d, J = 7.0 Hz, 3H), 1.61−1.76 (m, 2H), 2.22 (m, 1H), 2.69−2.98

(m, 2H), 3.70 (s, 3H), 4.22−4.58 (m, 2H), 5.10 (quint, J = 7.0 Hz,

1H), 5.33 (s, 2H), 6.04 (m, 1H), 6.48 (s, 1H), 6.73−6.81 (m, 2H),

6.86 (d, J = 5.3 Hz, 1H), 7.01−7.09 (m, 2H), 7.14 (d, J = 5.3 Hz, 1H),

7.20−7.55 (m, 5H); ¹³C NMR (100 MHz, CDCl₃) δ 21.6, 28.5, 28.6,

42.9, 48.4, 49.5, 55.1, 103.8, 110.5, 113.9, 121.8, 125.9, 126.2, 127.2,

128.6, 129.6, 130.0, 142.6, 143.1, 159.0, 162.9, 172.8; ESI-TOF-HRMS

(m/z) calcd for C₂₉H₃₁N₃O₃S [M + H]⁺502.2164; found 502.2149.

N-(3-Bromobenzyl)-1-(4-(4-methoxybenzyl)-4H-thieno[3,2-b]-

pyrrole-5-carbonyl)piperidine-4-carboxamide (7f). Carboxamide 7f

was prepared following general procedure B using carboxylic acid 2

(60 mg, 0.21 mmol), piperidine 8c (103 mg, 0.25 mmol), DIEA (73

μL, 0.42 mmol), and EDCl (42 mg, 0.22 mmol). After column

chromatography (SiO₂, eluent: 98% DCM, 2% MeOH), the pure

110.5, 114.0, 121.9, 126.2, 128.7, 129.6, 130.3, 142.8, 159.1, 163.0,

175.1; ESI-TOF-HRMS (m/z) calcd for C₂₄H₂₇N₃O₃S [M + H]⁺

438.1851; found 438.1853.

N-(Cyclopropylmethyl)-1-(4-(4-methoxybenzyl)-4H-thieno[3,2-b]-

pyrrole-5-carbonyl)piperidine-4-carboxamide (7j). Carboxamide 7j

was prepared following general procedure B using carboxylic acid 2

(80 mg, 0.28 mmol), piperidine 8g (99 mg, 0.33 mmol), DIEA (47 μL,

0.56 mmol), and EDCI (56 mg, 0.29 mmol). After column

chromatography (SiO₂, eluent: 98% DCM, 2% MeOH), pure

carboxamide 7j was obtained (117 mg, 94%). White solid; mp

172.0−173.0 °C; ¹H NMR (400 MHz; CDCl₃) δ 0.17−0.23 (m, 2H),

0.48−0.55 (m, 2H), 0.94 (m, 1H), 1.35−1.55 (m, 2H), 1.70−1.87 (m,

2H), 2.27 (m, 1H), 2.82−2.95 (m, 2H), 3.12 (dd, J = 7.1, 5.4, Hz, 2H),

3.76 (s, 3H), 4.32−4.52 (m, 2H), 5.38 (s, 2H), 5.49 (br s, 1H), 6.53

(s, 1H), 6.76−6.83 (m, 2H), 6.89 (d, J = 5.3 Hz, 1H), 7.06−7.12 (m,

2H), 7.17 (d, J = 5.3 Hz, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 3.3,

10.6, 28.8, 43.2, 44.2, 49.6, 55.2, 103.9, 110.5, 113.9, 121.8, 126.2,

128.7, 129.6, 130.2, 142.7, 159.0, 163.0, 173.6; ESI-TOF-HRMS (m/z)

calcd for C₂₅H₂₉N₃O₃S [M + H]⁺452.2008; found 452.2007.

N-Isopentyl-1-(4-(4-methoxybenzyl)-4H-thieno[3,2-b]pyrrole-5-

carbonyl)piperidine-4-carboxamide (7k). Carboxamide 7k was

prepared following general procedure B using carboxylic acid 2 (80

mg, 0.28 mmol), piperidine 8h (104 mg, 0.33 mmol), DIEA (47 μL,

0.56 mmol), and EDCI (56 mg, 0.29 mmol). After column

chromatography (SiO₂, eluent: 98% DCM, 2% MeOH), pure

carboxamide 7k was obtained (83 mg, 64%). White solid; mp

111.0−113.0 °C; ¹H NMR (400 MHz; CDCl₃) δ 0.92 (d, J = 6.6 Hz,

6H), 1.33−1.50 (m, 2H), 1.38 (dt, J = 7.7, 7.3 Hz, 2H), 1.60 (m, 1H),

1.69−1.81 (m, 2H), 2.23 (m, 1H), 2.78−2.94 (m, 2H), 3.26 (dt, J =

7.5, 5.9 Hz, 2H), 3.76 (s, 3H), 4.31−4.47 (m, 2H), 5.30−5.44 (m,

3H), 6.52 (d, J = 0.4 Hz, 1H), 6.75−6.84 (m, 2H), 6.89 (dd, J = 5.3,

0.4 Hz, 1H), 7.04−7.12 (m, 2H), 7.16 (d, J = 5.3 Hz, 1H); ¹³C NMR

(100 MHz, CDCl₃) δ 22.4, 25.9, 28.8, 37.8, 38.5, 43.3, 49.6, 55.3,

104.0, 110.5, 114.0, 121.9, 126.2, 128.7, 129.6, 130.2, 142.7, 159.1,

163.0, 173.6; ESI-TOF-HRMS (m/z) calcd for C₂₆H₃₃N₃O₃S [M +

H]⁺468.2321; found 468.2317.

1

carboxamide 7f was aﬀorded (68 mg, 58%). Colorless sticky oil; H

NMR (400 MHz; CDCl₃) δ 1.30−1.55 (m, 2H), 1.69−1.85 (m, 2H),

2.28 (m, 1H), 2.77−2.93 (m, 2H), 3.72 (s, 3H), 4.30−4.49 (m, 2H),

4.39 (d, J = 5.8 Hz, 2H), 5.36 (s, 2H), 5.93 (m, 1H), 6.51 (s, 1H),

6.75−6.82 (m, 2H), 6.90 (d, J = 5.3 Hz, 1H), 7.03−7.10 (m, 2H),

7.13−7.23 (m, 3H), 7.33−7.45 (m, 2H); ¹³C NMR (100 MHz,

CDCl₃) δ 28.7, 42.8, 43.1, 49.6, 55.3, 104.0, 110.5, 114.0, 121.8, 122.7,

126.2, 126.3, 128.7, 129.5, 130.2, 130.3, 130.6, 130.6, 140.7, 142.8,

159.1, 163.0, 173.7; ESI-TOF-HRMS (m/z) calcd for C₂₈H₂₈BrN₃O₃S

[M + H]⁺566.1113; found 566.1106.

1-(1-(4-(4-Methoxybenzyl)-4H-thieno[3,2-b]pyrrole-5-carbonyl)-

piperidin-4-yl)pentan-1-one (7g). Carboxamide 7g was prepared

following general procedure B using carboxylic acid 2 (80 mg, 0.28

mmol), piperidine 8j (80 mg, 0.28 mmol), DIEA (145 μL, 0.84

mmol), and EDCI (56 mg, 0.29 mmol). After column chromatography

(SiO₂, eluent: 99% DCM, 1% MeOH), the pure carboxamide 7g was

1

obtained (70 mg, 58%). Yellow oil; H NMR (400 MHz; CDCl₃) δ

0.91 (t, J = 7.4 Hz, 3H), 1.22−1.39 (m, 4H), 1.48−1.58 (m, 2H),

1.66−1.80 (m, 2H), 2.40 (t, J = 7.4 Hz, 2H), 2.49 (m, 1H), 2.81−3.00

(m, 2H), 3.76 (s, 3H), 4.25−4.47 (m, 2H), 5.38 (s, 2H), 6.51 (d, J =

0.4 Hz, 1H), 6.73−6.81 (m, 2H), 6.91 (dd, J = 5.3, 0.4 Hz, 1H), 7.03−

7.10 (m, 2H), 7.17 (d, J = 5.3 Hz, 1H); ¹³C NMR (100 MHz, CDCl₃)

δ 13.8, 22.3, 25.7, 27.5, 40.2, 48.4, 49.6, 55.2, 103.9, 110.5, 113.9,

121.8, 126.2, 128.7, 129.6, 130.2, 142.8, 159.1, 163.0, 211.6; ESI-TOF-

HRMS (m/z) calcd for C₂₅H₃₀N₂O₃S [M + H]⁺439.2055; found

439.2046.

1-(4-(4-Methoxybenzyl)-4H-thieno[3,2-b]pyrrole-5-carbonyl)-N-

propylpiperidine-4-carboxamide (7h). Carboxamide 7h was prepared

following general procedure B using carboxylic acid 2 (60 mg, 0.21

mmol), piperidine 8e (71 mg, 0.25 mmol), DIEA (73 μL, 0.42 mmol),

1-(4-(4-Methoxybenzyl)-4H-thieno[3,2-b]pyrrole-5-carbonyl)-N-

phenethylpiperidine-4-carboxamide (7l). Carboxamide 7l was

prepared following general procedure B using carboxylic acid 2 (60

mg, 0.21 mmol), piperidine 8b (87 mg, 0.25 mmol), DIEA (424 μL,

0.73 mmol), and EDCI (42 mg, 0.22 mmol). After column

chromatography (SiO₂, eluent: 98% DCM, 2% MeOH), pure

carboxamide 7l was obtained (60 mg, 57%). White solid; mp

L

DOI: 10.1021/acs.jmedchem.5b01047

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

150.1−152.1 °C; ¹H NMR (400 MHz; CDCl₃) δ 1.25−1.50 (m, 2H),

1.53−1.75 (m, 2H), 2.16 (m, 1H), 2.62−2.92 (m, 2H), 2.80 (t, J = 6.8

Hz, 2H), 3.49 (q, J = 6.8 Hz, 2H), 3.74 (s, 3H), 4.17−4.50 (m, 2H),

5.35 (s, 2H), 5.57 (m, 1H), 6.49 (s, 1H), 6.73−6.81 (m, 2H), 6.88 (d,

J = 5.3 Hz, 1H), 7.03−7.11 (m, 2H), 7.12−7.38 (m, 6H); ¹³C NMR

(100 MHz, CDCl₃) δ 28.6, 35.5, 40.4, 43.0, 49.6, 55.2, 103.9, 110.5,

113.9, 121.8, 126.2, 126.5, 128.6, 128.6, 128.7, 129.6, 130.1, 138.7,

142.7, 159.0, 162.9, 173.7; ESI-TOF-HRMS (m/z) calcd for

C₂₉H₃₁N₃O₃S [M + H]⁺502.2164; found 502.2153.

(2-Benzoyl-4-(4-methoxybenzyl)-4H-thieno[3,2-b]pyrrol-5-yl)(4-

benzylpiperazin-1-yl)methanone (13a). Carboxamide 13a was

prepared following general procedure B using carboxylic acid 30b

(77 mg, 0.20 mmol), 1-benzylpiperazine (41 μL, 0.23 mmol), and

EDCI (39 mg, 0.20 mmol). After chromatography (SiO₂, eluent: 30%

PE, 70% EtOAc), pure carboxamide 13a was aﬀorded (79 mg, 74%).

Yellow solid; mp 90.6−92.6 °C; ¹H NMR (400 MHz; CDCl₃) δ 1.70−

2.50 (br m, 4H), 3.33 (s, 2H), 3.39−3.61 (m, 4H), 3.70 (s, 3H), 5.26

(s, 2H), 6.43 (s, 1H), 6.68−6.78 (m, 2H), 6.94−7.02 (m, 2H), 7.13−

7.28 (m, 5H), 7.31 (s, 1H), 7.35−7.53 (m, 3H), 7.68−7.76 (m, 2H);

¹³C NMR (100 MHz, CDCl₃) δ 49.7, 52.7, 55.2, 62.7, 103.7, 114.1,

EDCI (26 mg, 0.14 mmol). After chromatography (SiO₂, eluent: 30%

PE, 70% EtOAc), pure carboxamide 13e was obtained as a yellow

1

sticky oil (38 mg, 52%). H NMR (400 MHz; CDCl₃) δ 2.00−2.35

(m, 4H), 3.35 (s, 2H), 3.48−3.62 (m, 4H), 3.70 (s, 3H), 4.48 (s, 2H),

4.60 (s, 2H), 5.28 (s, 2H), 6.40 (s, 1H), 6.67−6.75 (m, 2H), 6.76 (s,

1H), 6.96−7.03 (m, 2H), 7.13−7.30 (m, 10H); ¹³C NMR (100 MHz,

CDCl₃) δ 49.5, 52.9, 55.2, 62.9, 67.6, 71.5, 104.3, 110.2, 114.0, 122.0,

127.2, 127.7, 127.9, 128.3, 128.4, 128.7, 129.0, 129.1, 130.3, 137.6,

137.9, 141.6, 142.0, 159.0, 162.8; ESI-TOF-HRMS (m/z) calcd for

C₃₄H₃₅N₃O₃S [M + H]⁺566.2477; found 566.2471.

(4-Benzylpiperazin-1-yl)(4-(4-methoxybenzyl)-6-(((1-

phenylethyl)amino)methyl)-4H-thieno[3,2-b]pyrrol-5-yl)methanone

(14a). Carboxamide 14a was prepared following general procedure C

using carboxamide 32 (50 mg, 0.07 mmol). After chromatography

(SiO₂, eluent: 97.5% DCM, 2.5% MeOH), pure carboxamide 14a was

aﬀorded as a yellow sticky oil (39 mg, 63%). (Because of rotameric

eﬀects, broad signals are observed, and not all signals are observable.)

¹H NMR (400 MHz; (CD₃)₂CO) δ 1.35 (d, J = 6.6 Hz, 3H), 2.08−

2.42 (br m, 2H), 3.03−3.25 (br m, 2H), 3.35 (s, 2H), 3.57 (s, 2H),

3.72−3.88 (m, 4H), 5.23−5.35 (m, 2H), 6.82−6.88 (m, 2H), 7.05−

7.11 (m, 2H), 7.13 (d, J = 5.3 Hz, 1H), 7.18−7.35 (m, 9H), 7.38−7.43

(m,2H); ¹³C NMR (100 MHz, (CD₃)₂CO) δ 24.7, 43.7, 49.8, 53.3,

53.4, 55.6, 58.3, 63.2, 111.5, 114.8, 116.6, 122.6, 126.9, 127.6, 127.7,

127.9, 128.2, 129.0, 129.1, 129.7, 129.8, 131.5, 139.2, 142.6, 146.7,

160.2, 163.2; ESI-TOF-HRMS (m/z) calcd for C₃₆H₃₉N₃O₂S [M +

H]⁺579.2794; found 579.2783.

(6-((Benzyloxy)methyl)-4-(4-methoxybenzyl)-4H-thieno[3,2-b]-

pyrrol-5-yl)(4-benzylpiperazin-1-yl)methanone (14b). Carboxamide

14b was prepared following general procedure B using carboxylic acid

30d (29 mg, 0.07 mmol), 1-benzylpiperazine (15 μL, 0.09 mmol), and

EDCI (15 mg, 0.08 mmol). After chromatography (SiO₂, eluent: 97%

DCM, 3% MeOH), carboxamide 14b was obtained (30 mg, 74%).

(Because of rotameric eﬀects, broad signals are observed, and not all

signals are observable.) Yellow sticky oil; ¹H NMR (400 MHz;

CDCl₃) δ 1.83−2.60 (br m, 2H), 2.82−3.58 (br m, 3H), 3.33 (s, 2H),

3.78 (s, 3H), 4.52 (s, 2H), 4.53 (s, 2H), 5.28 (br s, 2H), 6.76−6.83

(m, 2H), 6.92 (d, J = 5.3 Hz, 1H), 7.02−7.09 (m, 2H), 7.18 (d, J = 5.3

Hz, 1H), 7.20−7.42 (m, 10H); ¹³C NMR (100 MHz, CDCl₃) δ 49.5,

52.7, 55.3, 62.8, 64.0, 72.3, 110.3, 113.5, 114.1, 122.1, 126.2, 127.2,

127.6, 127.8, 128.0, 128.3, 128.3, 128.8, 129.0, 130.1, 137.6, 138.1,

142.1, 159.2, 162.5; ESI-TOF-HRMS (m/z) calcd for C₃₄H₃₅N₃O₃S

[M + H]⁺566.2477; found 566.2472.

1-(2-Benzoyl-4-(4-methoxybenzyl)-4H-thieno[3,2-b]pyrrole-5-

carbonyl)-N-propylpiperidine-4-carboxamide (15a). Carboxamide

15a was prepared following general procedure B using carboxylic

acid 30b (100 mg, 0.26 mmol), piperidine 8e (87 mg, 0.31 mmol),

DIEA (89 μL, 0.51 mmol), and EDCI (51 mg, 0.27 mmol). After

chromatography (SiO₂, eluent: 99% DCM, 1% MeOH), carboxamide

15a was obtained as a yellow solid (65 mg, 47%). (Because of

rotameric eﬀects, broad signals are observed, and not all signals are

observable.) mp 209.9−211.5 °C; ¹H NMR (400 MHz; CDCl₃) δ 0.91

(t, J = 7.4 Hz, 3H), 1.22−1.60 (m, 2H), 1.51 (sext, J = 7.3 Hz, 2H),

1.70−2.00 (m, 2H), 2.26 (m, 1H), 2.72−3.03 (m, 2H), 3.21 (q, J = 6.6

Hz, 2H), 3.77 (s, 3H), 3.97−4.28 (br m, 2H), 5.34 (s, 2H), 5.48 (m,

1H), 6.53 (s, 1H), 6.75−6.90 (m, 2H), 7.00−7.15 (m, 2H), 7.38 (s,

1H), 7.43−7.63 (m, 3H), 7.73−7.87 (m, 2H); ¹³C NMR (100 MHz,

CDCl₃) δ 11.3, 22.8, 28.7, 41.2, 43.1, 49.8, 55.3, 103.5, 114.1, 118.4,

128.4, 128.9, 129.0, 129.2, 131.9, 134.3, 138.4, 141.5, 142.8, 159.4,

162.1, 173.5, 188.9; ESI-TOF-HRMS (m/z) calcd for C₃₁H₃₃N₃O₄S

[M + H]⁺544.2270; found 544.2255.

118.3, 127.3, 128.3, 128.3, 128.8, 128.9, 128.9, 129.0, 129.3, 131.8,

134.1, 137.4, 138.4, 141.5, 142.8, 159.2, 161.9, 188.8; ESI-TOF-HRMS

(m/z) calcd for C₃₃H₃₁N₃O₃S [M + H]⁺550.2164; found 550.2168.

1-(5-(4-Benzylpiperazine-1-carbonyl)-4-(4-methoxybenzyl)-4H-

thieno[3,2-b]pyrrol-2-yl)ethanone (13b). Carboxamide 13b was

prepared following general procedure B using carboxylic acid 30a

(46 mg, 0.14 mmol), 1-benzylpiperazine (29 μL, 0.17 mmol), and

EDCI (28 mg, 0.15 mmol). After chromatography (SiO₂, eluent:

97.5% DCM, 2.5% MeOH), the pure carboxamide 13b was aﬀorded

1

(58 mg, 86%). Yellow solid; mp 128.7−129.7 °C; H NMR (400

MHz; CDCl₃) δ 1.87−2.49 (br m, 4H), 2.54 (s, 3H), 3.41 (s, 2H),

3.47−3.75 (m, 4H), 3.79 (s, 3H), 5.38 (s, 2H), 6.48 (d, J = 0.4 Hz,

1H), 6.78−6.85 (m, 2H), 7.04−7.12 (m, 2H), 7.22−7.38 (m, 5H),

7.52 (d, J = 0.4 Hz, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 26.5, 49.6,

52.6, 55.3, 62.6, 103.9, 114.2, 115.3, 127.6, 128.2, 128.5, 128.8, 129.3,

129.6, 133.7, 141.6, 143.8, 159.3, 162.0, 191.2; ESI-TOF-HRMS (m/z)

calcd for C₂₈H₂₉N₃O₃S [M + H]⁺488.2008; found 488.2008.

(4-Benzylpiperazin-1-yl)(4-(4-methoxybenzyl)-2-phenyl-4H-

thieno[3,2-b]pyrrol-5-yl)methanone (13c). Carboxamide 13c was

prepared following general procedure B using carboxylic acid 30g (33

mg, 0.09 mmol), 1-benzylpiperazine (19 μL, 0.11 mmol), and EDCI

(18 mg, 0.10 mmol). After chromatography (SiO₂, eluent: 99% DCM,

1% MeOH), carboxamide 13c was obtained as a yellow sticky oil (33

1

mg, 71%). H NMR (400 MHz; CDCl₃) δ 2.10−2.40 (m, 4H), 3.44

(s, 2H), 3.60−3.72 (m, 4H), 3.78 (s, 3H), 5.41 (s, 2H), 6.50 (s, 1H),

6.75−6.83 (m, 2H), 7.04−7.13 (m, 2H), 7.15 (s, 1H), 7.21−7.42 (m,

8H), 7.54−7.63 (m, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 49.6, 52.9,

55.3, 62.8, 104.5, 106.5, 114.0, 121.2, 125.6, 127.4, 127.5, 128.4, 128.7,

128.9, 129.2, 130.3, 135.4, 143.1, 145.0, 159.1, 162.8; ESI-TOF-HRMS

(m/z) calcd for C₃₂H₃₁N₃O₂S [M + H]⁺522.2215; found 522.2213.

(4-Benzylpiperazin-1-yl)(4-(4-methoxybenzyl)-2-(((1-

phenylethyl)amino)methyl)-4H-thieno[3,2-b]pyrrol-5-yl)methanone

(13d). Carboxamide 13d was prepared following general procedure B

using carboxylic acid 30e (63 mg, 0.15 mmol), 1-benzylpiperazine (31

μL, 0.18 mmol), and EDCI (30 mg, 0.16 mmol). After

chromatography (SiO₂, eluent: 70% PE, 30% EtOAc), carboxamide

1

13d was obtained (54 mg, 63%). Yellow sticky oil; H NMR (400

MHz; CDCl₃) δ 1.30 (d, J = 6.6 Hz, 3H), 2.07−2.28 (m, 4H), 3.37 (s,

2H), 3.53−3.64 (m, 4H), 3.70 (d, J = 13.5 Hz, 1H), 3.72 (s, 3H), 3.78

(d, J = 13.5 Hz, 1H), 3.79 (q, J = 6.6 Hz, 1H), 5.26 (d, J = 15.3 Hz,

1H), 5.31 (d, J = 15.3 Hz, 1H), 6.41 (s, 1H), 6.61 (s, 1H), 6.71−6.77

(m, 2H), 6.98−7.04 (m, 2H), 7.16−7.35 (m, 10H); ¹³C NMR (100

MHz, CDCl₃) δ 24.3, 47.3, 49.5, 53.0, 55.2, 56.8, 62.9, 104.5, 108.7,

113.9, 120.7, 126.7, 127.0, 127.2, 128.3, 128.5, 128.6, 129.1, 130.4,

137.6, 141.9, 145.0, 145.8, 159.0, 162.9; ESI-TOF-HRMS (m/z) calcd

for C₃₆H₃₉N₃O₂S [M + H]⁺579.2794; found 579.2788.

(2-((Benzyloxy)methyl)-4-(4-methoxybenzyl)-4H-thieno[3,2-b]-

pyrrol-5-yl)(4-benzylpiperazin-1-yl)methanone (13e). Carboxamide

13e was prepared following general procedure B using carboxylic acid

30c (54 mg, 0.13 mmol), 1-benzylpiperazine (28 μL, 0.16 mmol), and

1-(2-Acetyl-4-(4-methoxybenzyl)-4H-thieno[3,2-b]pyrrole-5-car-

bonyl)-N-propylpiperidine-4-carboxamide (15b). Carboxamide 15b

was prepared following general procedure B using carboxylic acid 30a

(100 mg, 0.30 mmol), piperidine 8e (104 mg, 0.36 mmol), DIEA (106

μL, 0.61 mmol), and EDCI (61 mg, 0.32 mmol). After

chromatography (SiO₂, eluent: 99% DCM, 1% MeOH), carboxamide

15b was obtained as a white solid (129 mg, 89%). (Because of

rotameric eﬀects, broad signals are observed, and not all signals are

observable.) mp 203.1−204.7 °C; ¹H NMR (400 MHz; CDCl₃) δ 0.92

(t, J = 7.4 Hz, 3H), 1.21−1.58 (m, 2H), 1.52 (sext, J = 7.2 Hz, 2H),

M

DOI: 10.1021/acs.jmedchem.5b01047

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

1.65−1.89 (m, 2H), 2.25 (m, 1H), 2.54 (s, 3H), 2.78−2.97 (m, 2H),

3.14−3.26 (m, 2H), 3.78 (s, 3H), 3.93−4.73 (br m, 2H), 5.37 (s, 2H),

5.41 (br s, 1H), 6.50 (s, 1H), 6.78−6.90 (m, 2H), 7.03−7.17 (m, 2H),

7.51 (s, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 11.3, 22.9, 26.5, 28.7,

41.2, 43.1, 49.7, 55.3, 103.6, 114.2, 115.4, 128.4, 128.8, 129.4, 134.0,

141.4, 143.7, 159.4, 162.1, 173.5, 191.2; ESI-TOF-HRMS (m/z) calcd

for C₂₆H₃₁N₃O₄S [M + H]⁺482.2114; found 482.2111.

1-(2-Bromo-4-(4-methoxybenzyl)-4H-thieno[3,2-b]pyrrole-5-car-

bonyl)-N-propylpiperidine-4-carboxamide (15c). Carboxamide 15c

was prepared following general procedure B using carboxylic acid 30h

(1.14 g, 3.11 mmol), piperidine 8e (1.06 g, 3.74 mmol), DIEA (1.09

mL, 6.23 mmol), and EDCI (631 mg, 3.27 mmol). After

chromatography (SiO₂, eluent: PE/EtOAc/formic acid = 30:70:1 to

10:90:1), carboxamide 15c was obtained as a white solid (372 mg,

23%). The remaining material in the crude mixture was unreacted

carboxylic acid 30h. mp 182.2−183.2 °C; ¹H NMR (400 MHz;

CDCl₃) δ 0.92 (t, J = 7.4 Hz, 3H), 1.32−1.58 (m, 2H), 1.52 (sext, J =

7.4 Hz, 2H), 1.69−1.89 (m, 2H), 2.25 (m, 1H), 2.78−2.97 (m, 2H),

3.11−3.29 (m, 2H), 3.77 (s, 3H), 4.19−4.51 (m, 2H), 5.32 (s, 2H),

5.38 (br s, 1H), 6.42 (s, 1H), 6.73−6.88 (m, 2H), 6.93 (s, 1H), 7.03−

7.13 (m, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 11.3, 22.9, 28.8, 41.2,

43.3, 49.7, 55.3, 103.8, 112.8, 113.9, 114.1, 122.0, 128.7, 128.9, 129.8,

140.4, 159.2, 162.8, 173.6; ESI-TOF-HRMS (m/z) calcd for

C₂₄H₂₈BrN₃O₃S [M + H]⁺520.1093; found 520.1070.

1-(4-(4-Methoxybenzyl)-2-((propylamino)methyl)-4H-thieno[3,2-

b]pyrrole-5-carbonyl)-N-propylpiperidine-4-carboxamide (15d).

Carboxamide 15d was prepared following general procedure D using

carbaldehyde 26a (80 mg, 0.17 mmol), n-propylamine (16 μL, 0.19

mmol), and NaBH₄(3.2 mg, 0.08 mmol) in anhydrous MeOH (5

mL). After chromatography (SiO₂, eluent: DCM/MeOH = 1:0 to

23:2), carboxamide 15d was obtained as a yellow sticky oil (52 mg,

96%). ¹H NMR (400 MHz; CDCl₃) δ 0.90 (t, J = 7.4 Hz, 3H), 0.91 (t,

J = 7.4 Hz, 3H), 1.33−1.60 (m, 6H), 1.65−1.80 (m, 2H), 2.23 (m,

1H), 2.62 (t, J = 7.2 Hz, 2H), 2.78−2.93 (m, 2H), 3.19 (q, J = 7.2 Hz,

2H), 3.75 (s, 3H), 3.96 (s, 2H), 4.26−4.53 (m, 2H), 6.46 (s, 2H), 5.53

(m, 1H), 6.46 (s, 1H), 6.73−6.84 (m, 3H), 7.02−7.12 (m, 2H); ¹³C

NMR (100 MHz, CDCl₃) δ 11.3, 11.7, 22.8, 23.0, 28.8, 41.1, 43.3,

49.5, 49.6, 50.9, 55.2, 104.3, 108.6, 113.9, 120.7, 128.4, 128.6, 130.3,

141.9, 145.7, 159.0, 163.1, 173.7; ESI-TOF-HRMS (m/z) calcd for

C₂₈H₃₈N₄O₃S [M + H]⁺511.2743; found 511.2732.

1-(4-(4-Methoxybenzyl)-2-(((1-phenylethyl)amino)methyl)-4H-

thieno[3,2-b]pyrrole-5-carbonyl)-N-propylpiperidine-4-carboxa-

mide (15e). Carboxamide 15e was prepared following general

procedure D using carbaldehyde 26a (80 mg, 0.17 mmol), ( ) α-

phenylethylamine (24 μL, 0.19 mmol), and NaBH₄(3.2 mg, 0.08

mmol) in anhydrous MeOH (5 mL). After chromatography (SiO₂,

eluent: DCM/MeOH = 99:1 to 24:1), carboxamide 15e was obtained

as a yellow sticky oil (26 mg, 27%). The remaining materials in the

crude mixture were the unreacted carbaldehyde 26a and alcohol 15g.

¹H NMR (400 MHz; CDCl₃) δ 0.91 (t, J = 7.4 Hz, 3H), 1.36 (d, J =

2H), 1.60−1.85 (m, 2H), 2.25 (m, 1H), 2.80−2.95 (m, 2H), 3.21 (dt,

J = 7.0, 6.4 Hz, 2H), 3.76 (s, 3H), 3.84 (s, 2H), 3.98 (s, 2H), 4.32−

4.50 (m, 2H), 5.35 (s, 2H), 5.43 (m, 1H), 6.49 (s, 1H), 6.76 (s, 1H),

6.78−6.84 (m, 2H), 7.04−7.12 (m, 2H), 7.22−7.38 (m, 5H); ¹³C

NMR (100 MHz, CDCl₃) δ 11.3, 22.9, 28.8, 41.1, 43.4, 48.8, 49.5,

52.6, 55.3, 104.4, 108.8, 114.0, 120.8, 127.0, 128.2, 128.4, 128.5, 128.6,

139.9, 141.9, 145.7, 159.0, 163.1, 173.7; ESI-TOF-HRMS (m/z) calcd

for C₃₂H₃₈N₄O₃S [M + H]⁺559.2743; found 559.2732.

1-(2-(Hydroxymethyl)-4-(4-methoxybenzyl)-4H-thieno[3,2-b]-

pyrrole-5-carbonyl)-N-propylpiperidine-4-carboxamide (15g). Car-

boxamide 15g was prepared following general procedure E using

carbaldehyde 26a (30 mg, 0.06 mmol) and NaBH₄(1.2 mg, 0.03

mmol). After column chromatography (SiO₂, eluent: 97% DCM, 3%

MeOH), carboxamide 15g was obtained as a yellow solid in 83% yield

(25 mg). mp 120.8−122.8 °C; ¹H NMR (400 MHz; CDCl₃) δ 0.88 (t,

J = 7.4 Hz, 3H), 1.30−1.58 (m, 2H), 1.48 (sext, J = 7.2 Hz, 2H), 1.58−

1.76 (m, 2H), 2.19 (m, 1H), 2.70−2.90 (m, 2H), 3.12−3.20 (m, 2H),

3.72 (s, 3H), 4.20−4.42 (m, 2H), 4.73 (s, 2H), 5.24 (s, 2H), 5.76 (m,

1H), 6.44 (s, 1H), 6.72−6.80 (m, 3H), 6.98−7.05 (m, 2H); ¹³C NMR

(100 MHz, CDCl₃) δ 11.3, 22.8, 28.7, 41.1, 43.1, 49.4, 55.2, 61.0,

104.2, 108.8, 113.9, 121.5, 128.6, 128.8, 130.1, 141.7, 145.7, 159.0,

163.0, 173.8; ESI-TOF-HRMS (m/z) calcd for C₂₅H₃₁N₃O₄S [M +

H]⁺470.2114; found 470.2112.

1-(4-(4-Methoxybenzyl)-2-methyl-4H-thieno[3,2-b]pyrrole-5-car-

bonyl)-N-propylpiperidine-4-carboxamide (15h). Carboxamide 15h

was prepared following general procedure F using carboxamide 15c

(30 mg, 0.06 mmol), Pd(OAc)₂(1.3 mg, 0.0.01 mmol), PPh₃(4.6 mg,

0.02 mmol), MeB(OH)₂(6.9 mg, 0.12 mmol), Ag₂O (40 mg, 0.17

mmol), and K₂CO₃(24 mg, 0.17 mmol). After semipreparative

reversed-phase HPLC (conditions: MeOH in H₂O (80% v/v), 12 min

run time, 2.0 mL/min, 30 °C, and 100 μL per injection), carboxamide

15h was obtained as a yellow sticky oil (2.5 mg, 10%). The remaining

materials in the crude mixture were the unreacted carboxamide 15c

1

and carboxamide 7h. H NMR (400 MHz; CDCl₃) δ 0.92 (t, J = 7.2

Hz, 3H), 1.33−1.58 (m, 4H), 1.70−1.82 (m, 2H), 2.24 (m, 1H), 2.52

(s, 3H), 2.80−2.97 (m, 2H), 3.15−3.27 (m, 2H), 3.76 (s, 3H), 4.34−

4.47 (m, 2H), 5.33 (s, 2H), 5.39 (br s, 1H), 6.45 (s, 1H), 6.59 (s, 1H),

6.75−6.83 (m, 2H), 7.03−7.11 (m, 2H); ¹³C NMR (100 MHz,

CDCl₃) δ 11.3, 16.9, 22.9, 28.9, 41.2, 43.5, 49.5, 55.3, 104.5, 108.8,

114.0, 120.1, 127.8, 128.6, 130.5, 141.6, 142.4, 159.0, 163.3, 173.8;

ESI-TOF-HRMS (m/z) calcd for C₂₅H₃₁N₃O₃S [M + Na]⁺476.1984;

found 476.1985.

1-(4-(4-Methoxybenzyl)-2-phenyl-4H-thieno[3,2-b]pyrrole-5-car-

bonyl)-N-propylpiperidine-4-carboxamide (15i). Carboxamide 15i

was prepared following the general procedure F using carboxamide

15c (30 mg, 0.06 mmol), Pd(OAc)₂(1.3 mg, 0.0.01 mmol), PPh₃(4.6

mg, 0.02 mmol), PhB(OH)₂(14 mg, 0.12 mmol), Ag₂O (40 mg, 0.17

mmol), and K₂CO₃(24 mg, 0.17 mmol). After semipreparative

reversed-phase HPLC (conditions: MeOH in H₂O (85−90% v/v), 12

min run time, 2.0 mL/min, 30 °C, and 100 μL per injection),

carboxamide 15i was obtained as a yellowish white solid (9.9 mg,

33%). The remaining materials in the crude mixture were the

unreacted carboxamide 15c and carboxamide 7h. ¹H NMR (400 MHz;

DMSO-d₆) δ 0.76 (t, J = 7.4 Hz, 3H), 1.22−1.40 (m, 2H), 1.34

(sextet, J = 7.4 Hz, 2H), 1.48−1.63 (m, 2H), 2.27 (m, 1H), 2.75−2.89

(m, 2H), 2.93−2.97 (m, 2H), 3.63 (s, 3H), 4.03−4.29 (m, 2H), 5.33

(s, 2H), 6.58 (s, 1H), 6.75−6.84 (m, 2H), 7.06−7.14 (m, 2H), 7.18−

7.26 (m, 1H), 7.31−7.40 (m, 2H), 7.55−7.62 (m, 2H), 7.67 (m, 1H),

7.71 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ 11.3, 22.3, 28.4, 40.1,

41.6, 48.5, 54.9, 104.0, 108.0, 113.9, 120.2, 124.9, 127.4, 128.8, 129.0,

129.4, 130.1, 134.8, 142.3, 143.1, 158.6, 161.8, 173.4; ESI-TOF-HRMS

(m/z) calcd for C₃₀H₃₃N₃O₃S [M + Na]⁺538.2140; found 538.2137.

1-(2-Cyano-4-(4-methoxybenzyl)-4H-thieno[3,2-b]pyrrole-5-car-

bonyl)-N-propylpiperidine-4-carboxamide (15j). To a round-bot-

tomed ﬂask was added carboxamide 26a (20 mg, 0.04 mmol, 1 equiv)

and hydroxylamine hydrochloride (12 mg, 0.17 mmol, 4 equiv),

followed by anhydrous DMF (2 mL). The reaction mixture was heated

at 125 °C and stirred overnight. Next, the reaction mixture was cooled

to rt before diluting with EtOAc (2 mL). The organic mixture was

washed with H₂O (2 × 2 mL) and brine (1 × 2 mL), dried with

6.6 Hz, 3H), 1.40−1.60 (m, 2H), 1.51 (sext, J = 7.2 Hz, 2H), 1.65−

1.80 (m, 2H), 2.24 (m, 1H), 2.78−2.95 (m, 2H), 3.20 (dt, J = 7.2, 6.0

Hz, 2H), 3.76 (s, 3H), 3.76 (d, J = 14.2 Hz, 1H), 3.84 (d, J = 13.4 Hz,

1H), 3.85 (q, J = 6.6 Hz, 1H), 4.31−4.50 (m, 2H), 5.30 (d, J = 15.2

Hz, 1H), 5.35 (d, J = 15.2 Hz, 1H), 5.50 (m, 1H), 6.48 (s, 1H), 6.67

(s, 1H), 6.75−6.85 (m, 2H), 7.02−7.10 (m, 2H), 7.20−7.39 (m, 5H);

¹³C NMR (100 MHz, CDCl₃) δ 11.3, 22.8, 24.2, 28.8, 41.1, 43.3, 47.2,

49.5, 55.2, 56.8, 107.4, 108.7, 113.9, 120.7, 126.7, 127.0, 128.4, 128.5,

128.6, 130.3, 141.8, 145.0, 145.9, 159.0, 163.1, 173.7; ESI-TOF-HRMS

(m/z) calcd for C₃₃H₄₀N₄O₃S [M + H]⁺573.2899; found 573.2901.

1-(2-((Benzylamino)methyl)-4-(4-methoxybenzyl)-4H-thieno[3,2-

b]pyrrole-5-carbonyl)-N-propylpiperidine-4-carboxamide (15f).

Carboxamide 15f was prepared following general procedure D using

carbaldehyde 26a (80 mg, 0.17 mmol), benzylamine (21 μL, 0.19

mmol), and NaBH₄(3.2 mg, 0.05 mmol) in anhydrous MeOH (5

mL). After chromatography (SiO₂, eluent: DCM/MeOH = 99:1 to

24:1), carboxamide 15f was obtained as a yellow sticky oil (28 mg,

30%). The remaining materials in the crude mixture were unreacted

1

carbaldehyde 26a and alcohol 15g. H NMR (400 MHz; CDCl₃) δ

0.92 (t, J = 7.4 Hz, 3H), 1.33−1.60 (m, 2H), 1.50 (sext, J = 7.3 Hz,

N

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Na₂SO₄, and concentrated in vacuo to a crude residue which was

puriﬁed by semipreparative reversed-phase HPLC (C18, eluent:

MeOH in H₂O (75−90% v/v), 12 min run time, 2.0 mL/min, 30

°C and 100 μL per injection) to aﬀord pure carboxamide 15j as a

yellowish white solid in 30% yield (5.8 mg). The remaining material in

the crude mixture was the unreacted carboxamide 26a. (Because of

rotameric eﬀects, broad signals are observed, and not all signals are

1-(4-(4-Methoxybenzyl)-6-(((1-phenylethyl)amino)methyl)-4H-

thieno[3,2-b]pyrrole-5-carbonyl)-N-propylpiperidine-4-carboxa-

mide (16d). Carboxamide 16d was prepared following general

procedure D using carbaldehyde 26b (23 mg, 0.05 mmol), ( ) α-

phenylethylamine (13 μL, 0.10 mmol), and NaBH₄(0.9 mg, 0.03

mmol) in anhydrous MeOH (3 mL). After chromatography (SiO₂,

eluent: PE/EtOAc = 3:7 to 0:1), carboxamide 16d was obtained as a

yellow sticky oil (13 mg, 46%). The pure compound is a mixture of

isomers. (Because of rotameric eﬀects, broad signals are observed, and

1

observable.) H NMR (400 MHz; DMSO-d₆) δ 0.82 (t, J = 7.4 Hz,

3H), 1.14−1.88 (m, 4H), 1.39 (sextet, J = 7.2 Hz, 2H), 2.32 (m, 1H),

2.71−3.07 (m, 4H), 3.71 (s, 3H), 5.36 (s, 2H), 6.73 (s, 1H), 6.82−

6.90 (m, 2H), 7.10−7.18 (m, 2H), 7.74 (m, 1H), 8.22 (s, 1H); ¹³C

NMR (100 MHz, DMSO-d₆) δ 11.3, 22.3, 28.2, 40.1, 41.4, 48.8, 55.0,

103.3, 105.3, 114.0, 115.6, 122.5, 126.2, 129.0, 129.2, 134.9, 138.9,

158.6, 160.9, 173.3; ESI-TOF-HRMS (m/z) calcd for C₂₅H₂₈N₄O₃S

[M + H]⁺465.1960; found 465.1954.

1

not all signals are observable.) H NMR (400 MHz; CDCl₃) δ 0.78−

0.98 (m, 3H), 1.30−1.40 (m, 3H), 1.40−1.58 (m, 2H), 3.09−3.29 (m,

2H), 3.42−3.90 (m, 6H), 5.05−5.57 (m, 3H), 6.68−6.85 (m, 2H),

6.85−6.89 (m, 1H), 7.00−7.10 (m, 2H), 7.13−7.44 (m, 6H); ¹³C

NMR (100 MHz, CDCl₃) δ 11.3, 22.8, 24.1, 28.5, 41.1, 42.7, 42.9,

43.4, 49.5, 49.6, 55.3, 57.4, 110.4, 113.9, 114.1, 126.2, 126.3, 126.9,

126.9, 128.4, 128.5, 128.8, 130.2, 141.6, 159.1, 159.2, 162.6, 163.6,

173.5, 173.7; ESI-TOF-HRMS (m/z) calcd for C₃₃H₄₀N₄O₃S [M +

H]⁺573.2899; found 573.2871.

1-(6-Bromo-4-(4-methoxybenzyl)-4H-thieno[3,2-b]pyrrole-5-car-

bonyl)-N-propylpiperidine-4-carboxamide (16a). Carboxamide 16a

was prepared following general procedure B using carboxylic acid 30i

(19 mg, 0.05 mmol), EDCI (10 mg, 0.05 mmol), DIEA (54 μL, 0.30

mmol), and piperidine 8e (17 mg, 0.06 mmol) in DCM (1 mL). After

chromatography (SiO₂, eluent: PE/EtOAc = 3:7 to 1:4), carboxamide

16a was obtained as a white solid (15 mg, 57%). The pure compound

is a mixture of isomers. (Because of rotameric eﬀects, broad signals are

1-(6-((Benzylamino)methyl)-4-(4-methoxybenzyl)-4H-thieno[3,2-

b]pyrrole-5-carbonyl)-N-propylpiperidine-4-carboxamide (16e).

Carboxamide 16e was prepared following general procedure D using

carboaldehyde 26b (60 mg, 0.13 mmol), benzylamine (28 μL, 0.26

mmol), and NaBH₄(2.4 mg, 0.06 mmol) in anhydrous MeOH (3

mL). After chromatography (SiO₂, eluent: DCM/MeOH = 49:1 to

24:1), carboxamide 16e was obtained as a yellow sticky oil (18 mg,

26%). The pure compound is a mixture of isomers. The remaining

materials in the crude mixture were the unreacted carbaldehyde 26b

and alcohol 16b. (Because of rotameric eﬀects, broad signals are

1

observed, and not all signals are observable.) H NMR (400 MHz;

CDCl₃) δ 0.91 (t, J = 7.4 Hz, 3H), 1.43−1.57 (m, 2H), 1.80−1.93 (m,

2H), 2.18 (m, 1H), 3.13−3.27 (m, 2H), 3.63−3.80 (m, 3H), 5.13−

5.55 (m, 3H), 6.75−6.86 (m, 2H), 6.93−7.00 (m, 1H), 7.03−7.14 (m,

2H), 7.15−7.21 (m, 1H); ¹³C NMR (100 MHz; CDCl₃) δ 11.3, 22.9,

28.4, 28.9, 30.3, 41.2, 41.6, 42.8, 43.4, 47.0, 50.2, 50.3, 55.4, 55.5, 89.2,

89.9, 110.8, 110.8, 114.0, 114.3, 123.4, 123.5, 126.0, 126.2, 128.1,

128.7, 129.0, 129.0, 129.3, 129.5, 140.9, 141.5, 159.3, 159.4, 160.9,

161.8, 173.5, 173.9; ESI-MS (m/z) calcd for C₂₄H₂₈BrN₃O₃S [M +

H]⁺518.1113; found 518.1107.

1

observed, and not all signals are observable.) H NMR (400 MHz;

CDCl₃) δ 0.86−0.96 (m, 3H), 1.44−1.63 (m, 4H), 2.11 (m, 1H),

3.12−3.25 (m, 2H), 3.60−3.88 (m, 7H), 5.05−5.60 (m, 3H), 6.74−

6.85 (m, 2H), 6.88−6.97 (m, 1H), 7.02−7.13 (m, 2H), 7.15−7.21 (m,

1H), 7.21−7.40 (m, 5H); ¹³C NMR (100 MHz, CDCl₃) δ 11.3, 22.8,

28.5, 41.1, 42.5, 43.4, 44.4, 44.5, 49.5, 53.2, 55.3, 110.3, 110.3, 113.9,

114.1, 115.8, 116.0, 121.6, 121.8, 126.1, 126.3, 126.8, 126.8, 126.9,

127.5, 128.3, 128.3, 128.8, 130.1, 130.3, 140.3, 141.6, 142.1, 159.1,

159.2, 162.6, 163.6, 173.4, 173.7; ESI-TOF-HRMS (m/z) calcd for

C₃₂H₃₈N₄O₃S [M + H]⁺559.2743; found 559.2728.

Biology. Cells and Viruses. HEK 293T cells were cultured in

DMEM supplemented with 10% FBS. Full-length CHIKV (La

Reunion isolate) infectious clones tagged with Gaussia luciferase

(Gluc) gene (CHIKV-Gluc) were produced from pSP6-ICRES1-2SG

plasmid using similar standard molecular biology techniques as those

previously described by Tsetsarkin et al.³⁸WT CHIKV-IMT used in

this study were originally isolated from two French patients returning

from Reunion Island during the 2006 CHIKF outbreak.⁴⁶WT

O’nyong−nyong virus (ONNV) used in this study was originally

isolated from a patient in Chad.⁴⁷CHIKV and ONNV were kindly

1-(6-(Hydroxymethyl)-4-(4-methoxybenzyl)-4H-thieno[3,2-b]-

pyrrole-5-carbonyl)-N-propylpiperidine-4-carboxamide (16b). Car-

boxamide 16b was prepared following general procedure E using

carbaldehyde 26b (23 mg, 0.05 mmol) and NaBH₄(0.9 mg, 0.03

mmol). After column chromatography (SiO₂, eluent: PE/EtOAc = 3:7

to 0:1), carboxamide 16b was obtained as a yellow solid in 54% yield

(13 mg). The pure compound is a mixture of isomers. (Because of

rotameric eﬀects, broad signals are observed, and not all signals are

1

observable.) mp 115.8−117.8 °C; H NMR (400 MHz; CDCl₃) δ

0.75−0.97 (m, 3H), 1.39−1.55 (m, 2H), 1.58−1.80 (m, 2H), 2.19 (m,

1H), 3.10−3.25 (m, 2H), 3.70−3.80 (m, 3H), 4.55−4.72 (m, 2H),

5.10−5.60 (m, 3H), 6.74−6.84 (m, 2H), 6.84−6.94 (m, 1H), 7.01−

7.11 (m, 2H), 7.14−7.20 (m, 1H); ¹³C NMR (100 MHz, CDCl₃) δ

11.3, 22.8, 28.6, 41.1, 41.2, 42.8, 43.2, 49.6, 49.6, 55.3, 57.0, 57.4,

110.4, 110.5, 113.9, 114.2, 116.2, 116.5, 121.6, 122.0, 126.0, 126.3,

127.2, 127.9, 128.8, 129.6, 129.8, 141.6, 142.1, 159.2, 159.3, 162.5,

163.3, 173.5, 173.8; ESI-TOF-HRMS (m/z) calcd for C₂₅H₃₁N₃O₄S

[M + Na]⁺492.1933; found 492.1918.

́

provided by Dr. Hugues Tolou (Groupe d’Etude en Preventologie

(GEP), France). WT Sindbis virus (SINV) was provided by National

University of Singapore (NUS). Virus stocks (CHIKV-IMT, CHIKV-

Gluc, ONNV, and SINV) used for in vitro studies were prepared via

numerous passages in Vero-E6 cells, precleared by centrifugation, and

titered before storage at −80 °C.⁴⁸

1-(4-(4-methoxybenzyl)-6-((propylamino)methyl)-4H-thieno[3,2-

b]pyrrole-5-carbonyl)-N-propylpiperidine-4-carboxamide (16c).

Carboxamide 16c was prepared following general procedure D using

carbaldehyde 26b (60 mg, 0.13 mmol), n-propylamine (13 μL, 0.15

mmol, 1.2 equiv), NaBH₄(2.4 mg, 0.06 mmol) and Na₂SO₄in

anhydrous MeOH (5 mL). After chromatography (SiO₂, eluent:

DCM/MeOH = 1:0 to 23:2), carboxamide 16c was obtained as a

yellow sticky oil (30 mg, 47%). The pure compound is a mixture of

isomers. (Because of rotameric eﬀects, broad signals are observed, and

not all signals are observable.) ¹H NMR (400 MHz; CDCl₃) δ 0.90 (t,

J = 7.4 Hz, 6H); 1.35−1.55 (m, 4H), 2.17 (m, 1H), 2.48−2.67 (m,

2H), 3.10−3.25 (m, 2H), 3.68−3.84 (m, 5H), 5.05−5.70 (m, 3H),

6.73−6.84 (m, 2H), 6.85−6.93 (m, 1H), 7.00−7.10 (m, 2H), 7.12−

7.19 (m, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 11.3, 11.3, 11.5, 11.5,

21.8, 22.1, 22.8, 22.8, 28.6, 28.8, 41.1, 41.2, 42.9, 43.5, 44.1, 44.3, 49.8,

49.9, 50.1, 55.3, 110.6, 112.7, 113.6, 114.0, 114.2, 122.6, 123.1, 126.5,

128.2, 128.7, 128.9, 129.1, 129.5, 141.4, 141.8, 159.3, 162.9, 163.3,

173.6, 173.9; ESI-TOF-HRMS (m/z) calcd for C₂₈H₃₈N₄O₃S [M +

H]⁺511.2743; found 511.2729.

Cell Viability Assay. The cytotoxicity of candidate compounds was

assessed using CellTiter-Glo luminescent cell viability assay (Promega,

USA). HEK 293T cells were seeded (3 × 10⁴per well) into 96-well

plate and incubated overnight. The cells were treated with serial

dilutions of library compounds with concentrations between 0.01 μM

and 100 μM in triplicates. The eﬀect of ﬁnal DMSO concentration was

examined concurrently. Dilution series was prepared by diluting

compound stocks with DMEM (pH 6.8) supplemented with 10% FBS.

The untreated controls received only cells and medium, while blank

controls received only the medium. Twenty-four hours postincubation,

plates were equilibrated to room temperature for 30 min, and 100 μL

of CellTiter-Glo reagent was added. Luminometric readout was

measured using a GloMax-Multi detection system (Promega, USA) 1

h postincubation. CC₅₀values (i.e., concentration at which there is

50% viable cells) of these compounds were calculated from the dose−

response cell viability curves.

O

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Journal of Medicinal Chemistry

Article

Antiviral Assay (CHIKV-Gluc Assay). HEK 293T cells were seeded

into 96-well plates at densities of 3 × 10⁴cells/well, incubated

overnight, and infected with CHIKV-Gluc stock overlay at a

Multiplicity of Infection (MOI) of 0.1. After the infected cells were

incubated for 1.5 h, the virus overlay was removed, and each

compound was screened at various concentrations between 0.01 μM

and 100 μM in triplicate. The eﬀect of ﬁnal DMSO concentration was

examined concurrently. The untreated controls were cells infected

with the virus but without treatment with the compound, while

negative controls were mock-infected cells without both virus and

treatment with the compound. At 24 h postinfection, 50 μL of

conditioned media from each well was transferred to a new plate, and

50 μL of 20 μM XenoLight RediJect coelenterazine h diluted in PBS

supplemented with 5 mM NaCl was added. The luminometric readout

was then measured using a GloMax-Multi detection system (Promega,

USA). EC₅₀values (i.e., concentration causing 50% reduction of

infectivity) of these compounds were calculated from the dose−

response antiviral curves.

Antiviral Assay (Cellomics High Content Screen). Cell seeding,

virus infection with WT CHIKV-IMT (MOI = 0.1), SINV (MOI =

0.1), or ONNV (MOI = 0.1) and compound seeding were carried out

in a similar manner as previously described in the antiviral assay

(CHIKV-Gluc Assay) section. The eﬀect of ﬁnal DMSO concentration

was examined concurrently. The cells were ﬁxated with 4%

paraformaldehye for 30 min at room temperature, permeabilized

with PBS supplemented with 0.2% Tween 20, and incubated for 10

min at room temperature. Cells were then stained with antialphavirus

antibody (Santa Cruz Biotechnology) diluted in PBS for 1 h at 37 °C.

This was followed by incubation with goat antimouse secondary

antibody conjugated to FITC for 1 h at 37 °C. Cell nuclei were stained

with DAPI. Images were acquired and analyzed quantitatively by the

Cellomics ArrayScan VTI HCS Reader.⁴³

Western Blot Analysis. Cell seeding, virus infection with WT

CHIKV-IMT (MOI = 0.1), and compound seeding were carried out in

a similar manner as previously described in the antiviral assay

(CHIKV-Gluc Assay) section. CHIKV infected cells were harvested

and washed once with PBS and incubated for 5 min with RIPA lysis

and extraction buﬀer (Thermo Scientiﬁc) and protease inhibitors

(Roche). Protein concentrations were quantiﬁed using a DC protein

assay (Bio-Rad), and absorbance readings were taken at 750 nm using

a Magellan microplate reader.⁴⁹Lysates of virus-infected cells were

boiled for 5 min at 100 °C in Laemmli buﬀer supplemented with 10%

β-mercaptoethanol. Proteins were separated by 10% SDS−PAGE, and

transferred to nitrocellulose membranes at 180 mA for 1.5 h in transfer

buﬀer (24 mM Tris, 77 mM glycine, and 20% methanol). Membranes

were blocked for 1 h in blocking buﬀer (5% (w/v) skim milk in Tris-

buﬀered saline containing 0.05% Tween 20), followed by overnight

incubation at 4 °C with either rabbit polyclonal antisera against nsP1,

nsP3, capsid, or E2 proteins⁵⁰or mouse anti-β actin antibody

(BioLegend) diluted in blocking buﬀer. Horseradish peroxidase-

conjugated antirabbit lgG or antimouse lgG secondary antibodies were

then added and incubated for 1 h at room temperature, followed by

chemiluminescence detection using ECL Plus detection reagents

(Amersham Biosciences). Blots were exposed to ﬁlms (Pierce, Thermo

Scientiﬁc) and developed.

Viral RNA Extraction and qRT-PCR. Cell seeding, virus infection

with WT CHIKV-IMT (MOI = 0.1), and compound seeding were

carried out in a similar manner as previously described in the antiviral

assay (CHIKV-Gluc Assay) section. Viral RNA was extracted from 140

μL of culture, using QIAmp Viral RNA Kit (QIAGEN). Viral load

quantiﬁcation was performed by qRT-PCR using a QuantiTect Probe

RT-PCR kit (QIAGEN) modiﬁed from a previously described method

to detect for CHIKV nsP1⁵¹and E1 RNA.⁵²qRT-PCR reaction mixes

were done in 12.5 μL of reaction volume and performed in Applied

Biosystems (ABI) 7900HT Fast Real-Time PCR System in 384-well

plates, with the following conditions: (a) 30 min reverse transcription

at 50 °C; (b) 15 min PCR initial activation at 95 °C; (c) 2-step cycling

which consists of 15 s denaturation at 94 °C followed by 60 s combine

annealing and extension at 55 °C (for nsP1) or 60 °C (for E1); 45

cycles. Viral load was estimated from a standard curve generated using

serial dilutions of synthetic RNA transcripts complementary to the

nsP1 region or corresponding to the E1 coding region as described

previously by Her et al.^51,52

Compound Libraries and Reference Compounds. All compounds

obtained from the synthesized compound library were dissolved in

dimethyl sulfoxide (DMSO) and stored as 100 mM, 50 mM, and 25

mM stock solutions depending on their solubility in DMSO. Ribavirin

was purchased from Tokyo Chemical Industry (TCI). This compound

was also dissolved in DMSO and stored as 100 mM stock.

Data Analysis. All data from the antiviral and cell viability assays

were normalized using untreated infections and noninfected cell

cultures (in antiviral assays) or untreated and reagent background

samples (in cell viability assays), which were set as 100% and 0%

values, respectively. CC₅₀values (in cell viability assays) and EC₅₀

values (in antiviral assays) were determined by ﬁtting the results from

dose−response studies into asymmetric (ﬁve parameter) curves with

GraphPad Prism 5.03 software (Graphpad Prism, 1995). All data from

qRT-PCR were analyzed with ABI 7900HT SDS, version 2.4 (ABI

7900HT SDS).

ASSOCIATED CONTENT

* Supporting Information

■

S

The Supporting Information is available free of charge on the

ACS Publications website at DOI: 10.1021/acs.jmed-

chem.5b01047.

Detailed synthetic procedures and characterization data

of the intermediate compounds, NMR spectra of the ﬁnal

compounds, and biological data (PDF)

AUTHOR INFORMATION

Corresponding Authors

■

*(L.F.P.N.) E-mail: lisa_ng@immunol.a-star.edu.sg.

*(C.L.L.C.) E-mail: phacllc@nus.edu.sg or christina_chai@ices.

a-star.edu.sg.

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

We thank the Agency for Science Technology and Research

(A*STAR, grant number JCO 1231BFG046) and National

University of Singapore (NUS, grant number R-148-000-146-

133) for ﬁnancial support of this project. We also wish to

acknowledge M. L. Ng (Department of Microbiology, National

University of Singapore) and H. Tolou (Groupe d’Etude en

́

Preventologie, Villenave d’Ornon, France) for providing the

SINV and ONNV isolates.

ABBREVIATIONS USED

■

EDCI, 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide;

CDI, carbonyldiimidazole; DIEA, N,N-diisopropylethylamine;

TFA, triﬂuoroacetic acid; DCC, N,N′-dicyclohexylcarbodii-

mide; TEA, triethylamine; NBS, N-bromosuccinimide; ACN,

acetonitrile; THF, tetrahydrofuran; DMF, dimethylformamide;

DCM, dichloromethane; PE, petroleum ether; DCE, dichloro-

ethane; Boc, tert-butoxycarbonyl; Gluc, Gaussia luciferase;

FITC, ﬂuorescein isothiocyanate; DAPI, 4′,6-diamidino-2-

phenylindole

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Trisubstituted thieno[3,2-b]pyrrole 5-carboxamides as potent inhibitors of alphaviruses

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