Synthesis of N-benzyl-N-phenylthiophene-2-carboxamide analogues as a novel class of enterovirus 71 inhibitors

Abstract

A series of novel human enterovirus 71 inhibitors, N-benzyl-N-phenylthiophene-2-carboxamide analogues, were synthesized and their antiviral activities were evaluated in vitro. Most derivatives of this structure against EV71 had a low micromolar range in the RD (rhabdomyosarcoma) cell lines. The most potent compound 5a, N-(4-bromobenzyl)-N-(4-fluorophenyl)thiophene-2-carboxamide, showed low micromolar activity against EV71 (EC₅₀ = 1.42 μM) compared to the reference anti-EV71 drug enviroxime (EC₅₀ = 0.15 μM). Preliminary SAR studies revealed that the thiophene-2-carboxamide core is crucial for maintaining antiviral activity, and N-substituent phenyl groups largely influenced the anti-EV71 efficacy of this new class of potent antiviral agents.

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Introduction

The human enterovirus 71 (EV71), which belongs to the genus enterovirus of the picornaviridae family, was first isolated from the feces of infants suffering from encephalitis in 1969.^1,2 EV71 is one of the major causative agents of hand, foot and mouth disease (HFMD) that may progress to fatal encephalitis in infants and children.³ A mature EV71 virion consists of a single strand of positive-sense RNA and a symmetrical icosahedral virus capsid with four coat proteins VP1, VP2, VP3 and VP4. Most of the attachment and neutralization sites are densely clustered on VP1,⁴ and variations of capsid proteins VP1 to VP3 are responsible for the antigenic diversity among these types of enteroviruses.^5–7 Mostly, the symptoms of an EV71 infection are extremely similar to Coxsackie A16 (CA16), another main infection factor of HFMD, showing an herpangina infection and mild fever. Furthermore, EV71 infections are always associated with severe fatal central nervous system diseases, such as acute encephalitis, polio-like acute flaccid paralysis and neurogenic cardiopulmonary failure.^8–10 Generally, children and infants are considered to be relatively immunodeficient, hence, they are prone to EV71 infections that can be life threatening or even lethal, with high risks of morbidity and mortality.

From April 1998 a major outbreak of EV71 infection caused many severe cases and almost 80 fatalities in Taiwan¹¹ causing serious public health issues and highlighting the urgency and significance of developing novel anti-enteroviral agents. Because of the genetic heterogeneity and prevalent multiple serotypes, progress of antiviral agents against regional enterovirus infections is therefore very urgent.¹² To date there are no effective anti-EV71 agents available for preventing or treating enterovirus infection,^13,14 even though pleconaril (Fig. 1, 1), a candidate drug for inhibiting viral infection developed by Viropharma Inc in 1966, potently showed a broad spectrum activity against the enterovirus.¹⁵ Unfortunately, it has limited activity against EV71 and could not neutralize the cytopathic effect (CPE) in vitro assay.^16,17 Therefore, using the skeletons of pleconaril (Fig. 1), termed WIN compounds,¹² as templates for rational design, synthesis, modification and structure activity relationship (SAR) studies led to the development of many novel classes of significant anti-enteroviral compounds. Of these, there are only three kinds of distinguished small molecules, including pleconaril that possess anti-EV71 activity, as shown in Fig. 1 (compounds 1–3).^18–20

Fig. 1 The anti-enterovirus 71 compounds.

As a part of our continuous efforts to develop anti-virus agents,^21–24 herein, a series of novel small molecules with a thiophene-carboxamide core, different from the classical core structures of anti-EV71 compounds (as shown in Fig. 1),^{18–20,25,26} were developed and assayed for antiviral activity against EV71. Preliminary screenings showed that lead compound 5a has considerable potency relative to the drug compound enviroxime 4 and a broad spectrum of activity against both human rhinoviruses and enteroviruses,^27–31 which were tested at a low micromolar range (Fig. 2). Our SAR studies revealed that aryl substituent analogues at the para position, such as compound 5a, 5b and 5f, generally exhibited excellent efficacy against lethal EV71. These encouraging results prompted us to further ascertain this class of novel compounds and details of the investigation are described herein.

Fig. 2 General structure of enviroxime 4 and thiophene-carboxamide analogues.

Chemistry and synthesis

After considerable efforts in the discovery of anti-EV71 agents by compounds screening, we finally found some scaffolds that showed potent antiviral activities including these thiophene-carboxamide core compounds. Currently, we mainly focused on the modification of aromatic and heterocyclic moieties based on the peptide link to establish the requirements of optimum activity. Several methods were adopted to synthesize analogues in this series depending on the substitution pattern. Preparation of intermediate N-benzylaniline 8, outlined in Scheme 1, was done by the reductive amination process using substituted benzaldehydes 7 with anilines 6. Further trials found that an equivalent amount of acetic acid increased product yields dramatically.³² This was followed by the substitution reaction with aryl(het)carbonyl chlorides 9 under triethylamine and pyridine to produce the target compounds 5 and 10 in good to excellent yields (Scheme 1). Subsequently 10h and 10j were demethylated with BBr₃ under Ar atmosphere to give the phenol derivatives 10i and 10k, respectively.³³

Scheme 1 The synthetic route for the target compounds.

Results and discussion

A structure activity study was mainly performed by maintaining the three aromatic rings and varying the substituent groups with a bioisostere relationship. The carboxamide derivatives described herein were tested for anti-EV71 activity in a CPE reduction assay (EC₅₀) as well as undergoing a cytotoxicity evaluation (CC₅₀) in the RD cell lines under a standard procedure. These results are shown in Table 1 and compared to the reference anti-enterovirus drug enviroxime. As shown in Table 1, these compounds exhibit significant activities against human enterovirus 71. Initially the screened compound 5a demonstrated the highest level of activity against EV71 (EC₅₀ = 1.42 μM) with modest cytotoxicity. Enviroxime tested in this strain as a comparison showed an EC₅₀ value of 0.15 μM. It is very interesting to note that the replacement of thiophene in compound 5a with furan (5b) only produced imperceptible changes in potency (EC₅₀ = 1.50 μM) (Table 1, entries 1 and 2). However, replacement with a benzene ring (5c) led to a considerable loss in activity (entry 3). Introducing chloro on the 5-position of the thiophene ring (5d and 5e) resulted in a dramatically reduced or lost activity (entries 4 and 5), probably due to the fact that the interaction pocket is not large enough to occupy the shallow canyon space.^34–36 This observation was also supported by the binding mode of 5a with rhinoviruses (RV) protease (see Fig. 3 below). These significant results demonstrated that the five-membered aromatic heterocycle seems to play one of the important roles in maintaining the anti-EV71 activity. It is noted that the mono substituent of ortho (5n), para (10l)-position on the benzyl ring or 2,4-disubstituted (5h and 5m) also showed absolutely no activity, as shown in Table 1 (entries 8, 13, 14 and 26). For example, compound 10e and 10g with more electron-withdrawing cyano and a nitro group located at the para-position were found to have much less activity (entries 19 and 21). No substituted compound (5g) appears to have a lower EC₅₀ value than the corresponding disubstituted p-chloro (5l) and p-fluoro (10a) compounds (entries 7, 12 and 15). Electron-donating groups, such as methoxyl group (10h) at this position in this series of compounds, resulted in a double decrease in activity (EC₅₀ = 3.25 μM) than that of the lead compound (entry 22). Whereas the demethylated product (10i) with a more hydrophilic and polar hydroxyl group showed a dramatic decrease in activity against EV71 (entry 23). These observations provide remarkable evidence that the hydrophobic interaction and occupied space largely influence the anti-EV71 activity of these novel thiophene-carboxamide compounds. Next, the effect of substitution on the phenyl ring with respect to the antiviral activity was examined. Most compounds showed that introducing different groups on this ring also exhibited moderate to good potency, however, there was less of an effect on the activity for the benzyl ring. For example, compound 5f, 10b and 10f with no substituted or para bromo or chloro, had EC₅₀ values of 1.79 μM, 4.92 μM and 3.27 μM, respectively (entries 6, 16 and 20). In contrast the replacement of the fluoro group by a methyl group at the p-position resulted in a slightly higher efficacy with 5i and 5j (entries 9 and 10). More electron-withdrawing group such as trifluoromethyl at the 3-position (10d) resulted in a decreased activity against EV71 (EC₅₀ = 15 μM) compared to the methyl analogue (10c) at the same position (EC₅₀ = 5.38 μM) possibly due to the electronic effect (entries 17 and 18). Methoxyl substituted compound (10j) exhibited activity against EV71 with an EC₅₀ value up to 8.3 μM, while demethylation on the hydroxyl group (10k) resulted in a loss of activity (entries 24 and 25). Therefore, the interaction site appears to be a hydrophobic pocket VP1 that is located under the canyon floor.^37–39 In addition, prolonged linkage to the benzyl ring of compound 10m was without loss of activity (EC₅₀ = 8.54 μM), although almost 5-fold less than that of compound 5f (entries 6 and 27). This interesting discovery allows us to envision the binding interaction of virion with this novel class of molecules. Further SAR studies revealed that aryl substituent analogues at the para positions, such as compound 5a, 5b and 5f, generally exhibited a better efficacy against lethal EV71 than the ortho/meta positions compounds 10n–t. For example, in the case of compound 5a, when the fluoro group was changed from the para-position to the ortho or meta position of the phenyl ring (10n and 10o), decreased the antiviral activities (entries 1 vs. 28 and 29). Similar trends were also observed for analogues of compound 5f in which the para bromo on the phenyl ring of the benzyl group was moved to the ortho or meta position (10p and 10q). No obvious antiviral activities were observed (entries 3 vs. 30 and 31). For analogues of furan compounds 5b, 10r and 10s, which have both ortho (or meta) positions of the benzene ring and benzyl group substituted, the antiviral activities also decreased dramatically (entries 2 vs. 32 and 33). No activity was observed for the ortho substituted analogue (10t) of the carboxamide compound 5a (entries 1 vs. 34). Above all, the thiophene-2-carboxamide core compounds, especially 5a, are well qualified to serve as lead compounds for the further development of anti-EV71 agents (entry 1).

Table 1 Antiviral activity and cellular toxicity against EV-71^a

Entry	Compd	Ar (Het)	R^1	R^2	EC50^b (μM)	CC50^c (μM)	SI^d
a All data are obtained for at least three independent experiments.b EC50, concentration that inhibited the virus plaque formation by 50%.c CC50, concentration that inhibited cell growth by 50% compared with control cultures.d Selectivity Index (SI) was determined for the effective compounds dividing CC50 by IC50.e Compound 10m is specially referred as N-benzyl-N-(4-bromobenzyl)thiophene-2-carboxamide.f NA: no activity (EC50 > 100 μM).
1	5a	2-Thiophene	4-F	4-Br	1.42	11.17	7.9
2	5b	2-Furan	4-F	4-Br	1.50	57.19	38.1
3	5c	Phenyl	4-F	4-Br	NA^f	110.87	—
4	5d	5′-Cl-2-thiophene	4-F	4-Br	NA	17.8	0.1
5	5e	5′-Cl-2-thiophene	4-F	4 F	NA	130.01	—
6	5f	2-Thiophene	H	4-Br	1.79	66.35	37.1
7	5g	2-Thiophene	H	H	2.31	29.76	12.9
8	5h	2-Thiophene	4-F	2-Br-4-F	NA	22.34	—
9	5i	2-Thiophene	4-F	4-Cl	2.41	30.65	12.7
10	5j	2-Thiophene	4-CH3	4-Cl	2.28	36.27	15.9
11	5k	2-Thiophene	H	4-Cl	6.77	71.68	10.6
12	5l	2-Thiophene	4-Cl	4-Cl	4.61	30.92	6.7
13	5m	2-Thiophene	4-Cl	2-Br-4-F	NA	18.04	—
14	5n	2-Thiophene	4-Cl	2-Br	NA	114.08	—
15	10a	2-Thiophene	4-F	4-F	9.84	28.33	2.9
16	10b	2-Thiophene	4-Br	4-Br	4.92	18.51	3.8
17	10c	2-Thiophene	3-CH3	4-Br	5.38	24.67	4.6
18	10d	2-Thiophene	3-CF3	4-Br	15.0	18.72	1.3
19	10e	2-Thiophene	4-F	4-NO2	18.99	>224	>11.8
20	10f	2-Thiophene	4-Cl	4-Br	3.27	107.45	32.9
21	10g	2-Thiophene	4-F	4-CN	4.96	>237	>47.8
22	10h	2-Thiophene	4-F	4-OCH3	3.25	50.38	15.5
23	10i	2-Thiophene	4-F	4-OH	NA	78.50	—
24	10j	2-Thiophene	4-OCH3	4-Br	8.30	39.03	4.7
25	10k	2-Thiophene	4-OH	4-Br	NA	>206	—
26	10l	2-Thiophene	4-F	3-Br	NA	36.29	—
27	10m^e	2-Thiophene	Benzyl	4-Br	8.54	11.442	1.4
28	10n	2-Thiophene	3-F	4-Br	8.56	68.13	8.0
29	10o	2-Thiophene	2-F	4-Br	4.23	23.73	5.6
30	10p	2-Thiophene	H	2-Br	>100	>200	—
31	10q	2-Thiophene	H	3-Br	>50	88.4	—
32	10r	2-Furan	2-F	2-Br	>50	59.8	—
33	10s	2-Furan	3-F	3-Br	>100	>200	—
34	10t	2-Thiophene	3-F	3-Br	>100	>200	—
35	4	Enviroxime	—	—	0.15	>223.205	>1488

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Fig. 3 (A) Computer modeling of the complex structures of EV71 protease with 5a based on the published structure (PDB: 1CQQ) of RV protease with AG7088; (B) the surface type between 5a with EV71 protease (the oxygen atoms are shown in red and nitrogen atoms are shown in blue).

Because of the lack of a complex crystal structure of the protein and carboxamide compound, the binding mode of 5a was studied by the docking experiments into the EV71 protease. The docking experiments were carried out based on the published crystal structure of rhinoviruses (RV) protease (PDB: 1CQQ)^40,41 with AG7088. AutoDock 4.2 (ref. 42) was chosen to study the binding modes of 5a and the results showed a good binding score (−6.07 kcal mol⁻¹). Compound 5a showed a similar binding mode to that of AG7088 in the same protein pocket, with benzyl at P1, thiophene at P1′ and benzene at P2 (Fig. 3A). The surface type in Fig. 3B can clearly show the interaction of 5a with the protein. Especially, the phenyl ring group enters into the Leu127-Ser128 pocket forming a firm interaction. The benzyl group and thiophene enter into the pocket formed by Gly164-Asn165-Gly166-Phe170 and Phe25-Gly145-Cys147, respectively.

Conclusion

In summary, we have firstly developed an efficient synthesis of thiophene-carboxamides as novel potent anti-EV71 agents. According to the SAR, we successfully explored the efficacy of different substituents on the basis of the lead compound N-(4-bromobenzyl)-N-(4-fluorophenyl)thiophene-2-carboxamide (5a). Most of these serial compounds showed comparable anti-EV71 activity and a lower cell cytotoxicity than that of drug enviroxime. Further mechanistic studies on this new class of antiviral compounds are currently under way and will be reported in due course.

Experimental section

Unless otherwise noted, reagents and chemicals were obtained from commercial available suppliers and used without further purification. Dichloromethane (DCM) was distilled and dried over anhydrous CaCl₂ and methanol was freshly distilled from Na. All reactions were conducted in common conditions only when specially referred. Reaction progress was monitored by analytical thin-layer chromatography (TLC) and visualized by ultraviolet light (254 nm). ¹H NMR and ¹³C NMR spectra were detected by a Bruker Biospin AV400 (400 MHz) instrument. The chemical shifts were reported in parts per million (ppm) and were referenced to either tetramethylsilane or the solvent. Residual proton solvent signals for CDCl₃, δ 7.26 ppm, acetone, δ 2.05 ppm, respectively. ¹H NMR spectra were reported as follows: chemical shift (δ ppm), multiplicity (s = singlet, d = doublet, t = triplet, m = multiplet, br = broad), integration, coupling constant (Hz). ¹³C NMR spectra were reported as chemical shifts in ppm and multiplicity where appropriate. High resolution mass spectra were obtained from the Shanghai Mass Spectrometry Center. Melting points were determined by an X-4 Beijing Tech melting point apparatus and were uncorrected.

General procedure for reductive amination

4-Bromo benzaldehyde (100 mg, 1 eq.), 4-fluoroaniline (90.08 mg, 1.5 eq.) and acetic acid (48.68 mg, 1.5 eq.) were placed into a round-bottom flask (50 mL) sequentially, and 20 mL dry DCM were added and then stirred at room temperature for 8 h to yield the intermediate corresponding imine. The solvent was removed by a rotary evaporator under reduced pressure and then dissolved in anhydrous methanol (20 mL) and solid NaBH₄ (204.5 mg 10 eq.) were carefully added in an ice-bath, then allowed to stirr at room temperature until the raw material point disappeared. After evaporating the solvent, 20 mL NaOH (5% M) solution were added to quench the reaction mixture and DCM was used to extract the aqueous layer, then combined organic layers were dried by anhydrous Na₂SO₄, filtered and concentrated in a vacuum. The crude product was purified by silica gel column chromatography.

General procedure for the preparation of amides

Formylchloride (0.45 mmol, 1.5 eq.) was added in one portion to a solution of the amine (0.3 mmol, 1 eq.) with dry dichloromethane (20 mL), then Et₃N (3 mmol, 10 eq.) and pyridine (0.45 mmol, 1.5 eq.) were injected into the reaction mixture forming white smoke rapidly. The solution was stirred overnight at room temperature, the solvent evaporated and any residual Et₃N, then diluted with H₂O, was extracted with DCM, dried by anhydrous Na₂SO₄, filtered, concentrated in a vacuum and purified with silica gel column chromatography.

N-(4-Bromobenzyl)-N-(4-fluorophenyl)thiophene-2-carboxamide (5a). White solid (87% yield), mp 116.8–118.9 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.41 (d, J = 8.3 Hz, 2H), 7.34 (dd, J = 4.4, 1.5 Hz, 1H), 7.15 (d, J = 8.2 Hz, 2H), 7.07–6.98 (m, 4H), 6.86–6.79 (m, 2H), 4.97 (s, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 163.55, 162.49, 161.07, 138.17, 138.13, 137.47, 135.94, 132.82, 131.66, 131.28, 130.95, 130.86, 130.79, 126.84, 121.72, 116.87, 116.64, 54.08. HRMS (ESI) calcd for C₁₈H₁₃BrFNOSH [M + H]⁺ 389.9964, found 389.9955.

N-(4-Bromobenzyl)-N-(4-fluorophenyl)furan-2-carboxamide (5b). White solid (89% yield), mp 110.5–112.2 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.41 (d, J = 8.3 Hz, 2H), 7.34 (d, J = 1.0 Hz, 1H), 7.15 (d, J = 8.3 Hz, 2H), 7.07–6.94 (m, 4H), 6.23 (dd, J = 3.5, 1.7 Hz, 1H), 5.88 (d, J = 2.3 Hz, 1H), 4.96 (s, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 163.26, 160.79, 159.13, 146.64, 144.77, 138.07, 138.05, 135.84, 131.64, 130.81, 130.25, 130.16, 121.73, 117.03, 116.65, 116.42, 111.17, 53.50. HRMS (ESI) calcd for C₁₈H₁₃BrFNO₂H [M + H]⁺ 374.0192, found 374.0189.

N-(4-Bromobenzyl)-N-(4-fluorophenyl)benzamide (5c). White solid (89% yield), mp 119.8–121.8 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.42 (d, J = 8.1 Hz, 2H), 7.32–7.14 (m, 7H), 6.84 (d, J = 6.3 Hz, 4H), 5.04 (s, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 170.59, 162.20, 159.74, 139.16, 139.14, 136.24, 135.47, 131.69, 130.39, 129.89, 129.51, 129.42, 128.65, 127.90, 121.59, 116.22, 116.00, 53.33. HRMS (ESI) calcd for C₂₀H₁₅BrFNOSH [M + H]⁺ 384.0399, found 384.0397.

N-(4-Bromobenzyl)-5-chloro-N-(4-fluorophenyl)thiophene-2-carboxamide (5d). Yellow solid (75% yield), mp 79.7–82.5 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.41 (d, J = 7.6 Hz, 2H), 7.19–6.95 (m, 6H), 6.69 (d, J = 12.5 Hz, 2H), 4.93 (s, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 163.80, 161.31, 137.41, 137.38, 136.63, 135.65, 135.43, 132.75, 131.71, 131.14, 131.05, 130.81, 126.19, 121.86, 117.14, 116.91, 54.13. HRMS (ESI) calcd for C₁₈H₁₂BrClFNOSH [M + H]⁺ 423.9574, found 423.9565.

N-(4-Fluorobenzyl)-5-chloro-N-(4-fluorophenyl)thiophene-2-carboxamide (5e). Yellow solid (79% yield), mp 78.6–82.7 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.21 (dd, J = 8.5, 5.5 Hz, 2H), 7.10–7.03 (m, 2H), 7.026.94 (m, 4H), 6.72–6.65 (m, 2H), 4.95 (s, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 161.30, 161.25, 137.41, 137.37, 136.55, 135.57, 132.68, 132.47, 132.44, 131.21, 131.12, 130.92, 130.84, 126.17, 117.07, 116.85, 115.55, 115.34, 53.97. HRMS (ESI) calcd for C₁₈H₁₂ClF₂NOSH [M + H]⁺ 364.0371, found 364.0366.

N-(4-Bromobenzyl)-N-phenylthiophene-2-carboxamide (5f). White solid (83% yield), mp 122.2–124.5 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.40 (d, J = 8.4 Hz, 2H), 7.38–7.30 (m, 4H), 7.17 (d, J = 8.3 Hz, 2H), 7.08–7.03 (m, 2H), 6.78 (dd, J = 4.9, 4.0 Hz, 1H), 6.72 (dd, J = 3.8, 1.1 Hz, 1H), 5.00 (s, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 162.52, 142.29, 137.94, 136.19, 132.55, 131.56, 131.06, 130.75, 129.73, 129.05, 128.55, 126.78, 121.55, 54.09. HRMS (ESI) calcd for C₁₈H₁₄BrNOSH [M + H]⁺ 372.0058, found 372.0049.

N-Benzyl-N-phenylthiophene-2-carboxamide (5g). White solid (86% yield), mp 101.8–104.7 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.37–7.23 (m, 9H), 7.09–7.04 (m, 2H), 6.78 (dd, J = 4.9, 3.9 Hz, 1H), 6.72 (dd, J = 3.8, 0.9 Hz, 1H), 5.06 (s, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 162.46, 142.52, 138.26, 137.18, 132.42, 130.89, 129.60, 129.11, 128.94, 128.44, 128.39, 127.50, 126.74, 54.67. HRMS (ESI) calcd for C₁₈H₁₅NOSH [M + H]⁺ 294.0953, found 294.0943.

N-(2-Bromo-4-fluorobenzyl)-N-(4-fluorophenyl)thiophene-2-carboxamide (5h). White solid (67% yield), mp 87.1–88.7 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.50 (dd, J = 8.6, 6.1 Hz, 1H), 7.36 (dd, J = 5.0, 1.1 Hz, 1H), 7.23 (dd, J = 8.2, 2.6 Hz, 1H), 7.10–6.97 (m, 5H), 6.91–6.81 (m, 2H), 5.17 (s, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 163.64, 162.88, 162.63, 161.16, 160.38, 137.83, 137.80, 137.29, 133.00, 132.11, 132.07, 131.80, 131.71, 131.42, 130.96, 130.87, 126.88, 124.14, 124.04, 120.06, 119.82, 116.82, 116.59, 115.12, 114.91, 53.11. HRMS (ESI) calcd for C₁₈H₁₂BrF₂NOSH [M + H]⁺ 407.9869, found 407.9850.

N-(4-Chlorobenzyl)-N-(4-fluorophenyl)thiophene-2-carboxamide (5i). White solid (80% yield), mp 108.0–109.2 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.34 (dd, J = 4.2, 1.2 Hz, 1H), 7.30–7.18 (m, 4H), 7.07–6.99 (m, 4H), 6.87–6.79 (m, 2H), 4.98 (s, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 163.55, 162.49, 161.07, 138.17, 138.14, 137.49, 135.43, 133.56, 132.81, 131.26, 130.96, 130.87, 130.45, 128.70, 126.82, 116.85, 116.62, 54.01. HRMS (ESI) calcd for C₁₈H₁₃ClFNOSH [M + H]⁺ 346.0469, found 346.0461.

N-(4-Chlorobenzyl)-N-(p-tolyl)thiophene-2-carboxamide (5j). White solid (83% yield), mp 111.6–114.2 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.31 (dd, J = 4.9, 1.1 Hz, 1H), 7.26–7.21 (m, 4H), 7.14 (d, J = 8.0 Hz, 2H), 6.93 (d, J = 8.2 Hz, 2H), 6.82–6.76 (m, 2H), 4.98 (s, 2H), 2.38 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 162.54, 139.55, 138.66, 137.98, 135.79, 133.31, 132.65, 131.06, 130.45, 130.36, 128.81, 128.57, 126.77, 54.07, 21.26. HRMS (ESI) calcd for C₁₉H₁₆ClFNOSH [M + H]⁺ 342.0719, found 342.0719.

N-(4-Chlorobenzyl)-N-phenylthiophene-2-carboxamide (5k). White solid (88% yield), mp 87.4–89.5 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.38–7.30 (m, 4H), 7.24 (d, J = 2.7 Hz, 3H), 7.20 (dd, J = 4.9, 3.9 Hz, 1H), 7.09–7.02 (m, 2H), 6.78 (dd, J = 4.9, 3.9 Hz, 1H), 6.73 (dd, J = 3.8, 1.1 Hz, 1H), 5.01 (s, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 162.59, 142.23, 137.86, 135.63, 134.92, 133.91, 133.39, 132.67, 131.15, 130.43, 129.74, 129.07, 128.61, 128.09, 126.80, 54.06. HRMS (ESI) calcd for C₁₈H₁₄ClNOSH [M + H]⁺ 328.0563, found 328.0557.

N-(4-Chlorobenzyl)-N-(4-chlorophenyl)thiophene-2-carboxamide (5l). White solid (86% yield), mp 79.4–82.3 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.37–7.30 (m, 3H), 7.26–7.19 (m, 4H), 7.02–6.95 (m, 2H), 6.86–6.81 (m, 2H), 4.99 (s, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 162.50, 140.75, 137.38, 135.31, 134.89, 134.43, 133.86, 133.60, 132.86, 131.32, 130.40, 130.34, 129.98, 128.74, 128.08, 126.92, 53.94. HRMS (ESI) calcd for C₁₈H₁₃Cl₂NOSH [M + H]⁺ 362.0168, found 362.0164.

N-(2-Bromo-4-fluorobenzyl)-N-(4-chlorophenyl)thiophene-2-carboxamide (5m). Yellow solid (69% yield), 85.8-87.3 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.49 (dd, J = 8.6, 6.0 Hz, 1H), 7.37 (dd, J = 4.9, 1.2 Hz, 1H), 7.33–7.27 (m, 2H), 7.24 (dd, J = 8.2, 2.6 Hz, 1H), 7.06–6.97 (m, 3H), 6.90–6.83 (m, 2H), 5.16 (s, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 162.89, 162.62, 160.40, 140.50, 137.26, 134.50, 132.93, 132.02, 131.98, 131.64, 131.55, 131.40, 130.28, 129.90, 126.93, 124.03, 123.94, 120.11, 119.87, 115.13, 114.92, 53.06. HRMS (ESI) calcd for C₁₈H₁₂BrClFNOSH [M + H]⁺ 423.9568, found 423.9563.

N-(2-Bromobenzyl)-N-(4-chlorophenyl)thiophene-2-carboxamide (5n). White solid (70% yield), mp 96.5–99.8 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.48 (dd, J = 10.7, 8.4 Hz, 2H), 7.37 (d, J = 4.8 Hz, 1H), 7.28 (dd, J = 7.5, 5.5 Hz, 3H), 7.12 (t, J = 7.1 Hz, 1H), 7.06 (d, J = 8.6 Hz, 2H), 6.92–6.82 (m, 2H), 5.20 (s, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 162.63, 140.76, 137.41, 135.88, 134.32, 132.86, 131.29, 130.22, 130.19, 129.82, 129.14, 127.76, 126.91, 123.88, 53.93. HRMS (ESI) calcd for C₁₈H₁₃BrClNOSH [M + H]⁺ 405.9663, found 405.9659.

N-(4-Fluorobenzyl)-N-(4-fluorophenyl)thiophene-2-carboxamide (10a). White solid (81% yield), mp 93.5–95.6 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.33 (dd, J = 4.3, 1.9 Hz, 1H), 7.28–7.20 (m, 2H), 7.07–6.92 (m, 6H), 6.86–6.80 (m, J = 2H), 4.99 (s, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 163.53, 162.42, 161.09, 161.05, 138.17, 138.13, 137.61, 132.75, 132.72, 131.21, 131.02, 130.94, 130.88, 130.80, 126.82, 116.80, 116.58, 115.49, 115.28, 53.91. HRMS (ESI) calcd for C₁₈H₁₃F₂NOSH [M + H]⁺ 330.0759, found 330.0755.

N-(4-Bromobenzyl)-N-(4-bromophenyl)thiophene-2-carboxamide (10b). White solid (77% yield), mp 125.3–127.1 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.49–7.44 (m, 2H), 7.41 (d, J = 8.4 Hz, 2H), 7.38–7.33 (m, 1H), 7.15 (d, J = 8.4 Hz, 2H), 6.95–6.89 (m, 2H), 6.83 (d, J = 3.1 Hz, 2H), 4.97 (s, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 162.40, 141.33, 137.42, 135.83, 132.97, 132.77, 131.69, 131.27, 130.71, 130.62, 126.92, 122.43, 121.77, 53.94. HRMS (ESI) calcd for C₁₈H₁₃Br₂NOSH [M + H]⁺ 449.9157, found 449.9151.

N-(4-Bromobenzyl)-N-(m-tolyl)thiophene-2-carboxamide (10c). White solid (85% yield), mp 92.6–94.8 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.40 (d, J = 8.3 Hz, 2H), 7.31 (d, J = 4.4 Hz, 1H), 7.24–7.14 (m, 4H), 6.91 (s, 1H), 6.86–6.72 (m, 3H), 4.97 (s, 2H), 2.31 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 162.43, 142.18, 139.87, 137.99, 136.30, 132.59, 131.51, 131.08, 130.72, 129.44, 129.43, 129.34, 126.77, 126.13, 121.47, 54.15, 21.31. HRMS (ESI) calcd for C₁₉H₁₆BrNOSH [M + H]⁺ 386.0214, found 386.0209.

N-(4-Bromobenzyl)-N-(3-(trifluoromethyl)phenyl)thiophene-2-carboxamide (10d). White solid (68% yield), mp 122.7–124.8 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.62 (d, J = 7.7 Hz, 1H), 7.47–7.39 (m, 4H), 7.36–7.34 (m, 1H), 7.20–7.13 (m, 3H), 6.84–6.80 (m, 2H), 5.02 (s, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 162.49, 142.94, 137.12, 135.62, 132.84, 132.64, 131.76, 131.40, 130.63, 130.30, 126.90, 125.74, 125.71, 125.18, 125.15, 121.88, 54.00. HRMS (ESI) calcd for C₁₉H₁₃BrF₃NOSH [M + H]⁺ 439.9932, found 439.9927.

N-(4-Fluorophenyl)-N-(4-nitrobenzyl)thiophene-2-carboxamide (10e). Yellow solid (83% yield), mp 142.5–145.2 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.17 (d, J = 8.7 Hz, 2H), 7.48 (d, J = 8.7 Hz, 2H), 7.37 (dd, J = 4.9, 1.3 Hz, 1H), 7.06 (d, J = 6.4 Hz, 4H), 6.90–6.83 (m, 2H), 5.11 (s, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 144.33, 133.13, 131.63, 130.76, 130.67, 129.66, 126.95, 123.84, 117.12, 116.89, 54.21. HRMS (ESI) calcd for C₁₈H₁₃BrFN₂O₃SH [M + H]⁺ 357.0704, found 357.0698.

N-(4-Bromobenzyl)-N-(4-chlorophenyl)thiophene-2-carboxamide (10f). White solid (82% yield), mp 103.6–105.3 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.40 (d, J = 8.3 Hz, 2H), 7.36–7.27 (m, 3H), 7.15 (d, J = 8.3 Hz, 2H), 7.02–6.95 (m, 2H), 6.85–6.80 (m, 2H), 4.97 (s, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 162.43, 140.79, 137.45, 135.87, 134.40, 132.77, 131.69, 131.27, 130.73, 130.33, 129.98, 126.92, 121.76, 53.98. HRMS (ESI) calcd for C₁₈H₁₃BrClNOSH [M + H]⁺ 405.9663, found 405.9651.

N-(4-Cyanobenzyl)-N-(4-fluorophenyl)thiophene-2-carboxamide (10g). White solid (73% yield), mp 132.6–138.8 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.60 (d, J = 8.2 Hz, 2H), 7.42 (d, J = 8.2 Hz, 2H), 7.36 (dd, J = 4.8, 1.3 Hz, 1H), 7.09–7.01 (m, 4H), 6.89–6.81 (m, 2H), 5.07 (s, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 163.59, 162.66, 161.11, 142.34, 138.14, 138.11, 137.06, 133.05, 132.40, 131.57, 130.76, 130.68, 129.50, 126.95, 118.69, 117.06, 116.83, 111.56, 54.46. HRMS (ESI) calcd for C₁₉H₁₃FN₂OSH [M + H]⁺ 337.0806, found 337.0801.

N-(4-Fluorophenyl)-N-(4-methoxybenzyl)thiophene-2- carboxamide (10h). White solid (71% yield), mp 69.8–75.1 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.34–7.29 (m, 1H), 7.18 (d, J = 8.6 Hz, 2H), 7.05–6.96 (m, 4H), 6.85–6.77 (m, 4H), 4.96 (s, 2H), 3.78 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 163.47, 162.30, 159.07, 138.29, 138.26, 137.93, 132.58, 131.12, 131.03, 131.01, 130.46, 129.05, 126.76, 116.66, 116.43, 113.81, 55.23, 54.02. C₁₉H₁₆FNO₂SH [M + H]⁺ 342.0964, found 342.0957.

N-(4-Fluorophenyl)-N-(4-hydroxybenzyl)thiophene-2-carboxamide (10i). White solid (89% yield), mp 172.7–175.3 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.33 (dd, J = 4.9, 1.3 Hz, 1H), 7.12 (d, J = 8.5 Hz, 2H), 7.05–6.98 (m, 4H), 6.85–6.80 (m, 2H), 6.75 (d, J = 8.5 Hz, 2H), 5.63 (s, 1H), 4.95 (s, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 163.53, 162.50, 155.46, 138.20, 137.65, 132.80, 131.19, 131.10, 131.01, 130.57, 128.77, 126.81, 116.71, 116.48, 115.42, 54.18. HRMS (ESI) calcd for C₁₈H₁₄FNO₂SH [M + H]⁺ 328.0803, found 328.0799.

N-(4-Bromobenzyl)-N-(4-methoxyphenyl)thiophene-2-carboxamide (10j). Yellow solid (76% yield), mp 101.8–104.6 °C ¹H NMR (400 MHz, CDCl₃) δ 7.40 (d, J = 8.4 Hz, 2H), 7.32 (dd, J = 4.9, 1.2 Hz, 1H), 7.17 (d, J = 8.4 Hz, 2H), 6.97–6.92 (m, 2H), 6.87–6.80 (m, 4H), 4.95 (s, 2H), 3.83 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 162.56, 159.57, 137.78, 136.30, 134.74, 132.84, 131.54, 131.21, 130.88, 130.31, 126.76, 121.52, 114.81, 55.48, 54.17. HRMS (ESI) calcd for C₁₉H₁₆BrNO₂SH [M + H]⁺ 402.0163, found 402.0158.

N-(4-Bromobenzyl)-N-(4-hydroxyphenyl)thiophene-2-carboxamide (10k). White solid (88% yield), mp 203.1–205.7 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.40 (d, J = 8.4 Hz, 2H), 7.33 (dd, J = 5.0, 1.2 Hz, 1H), 7.16 (d, J = 8.4 Hz, 2H), 6.93–6.86 (m, 3H), 6.85–6.77 (m, 3H), 5.44 (s, 1H), 4.95 (s, 2H). ¹³C NMR (100 MHz, acetone) δ 163.37, 159.02, 139.88, 138.77, 135.38, 133.56, 132.72, 132.52, 132.29, 131.79, 128.08, 122.10, 117.56, 54.91. HRMS (ESI) calcd for C₁₈H₁₄BrNO₂SH [M + H]⁺ 388.0001, found 387.9997.

N-(3-Bromobenzyl)-N-(4-fluorophenyl)thiophene-2-carboxamide (10l). White solid (89% yield), mp 111.4–113.4 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.45–7.37 (m, 2H), 7.35 (dd, J = 4.8, 1.3 Hz, 1H), 7.23–7.14 (m, 2H), 7.05 (d, J = 6.5 Hz, 4H), 6.86–6.81 (m, 2H), 4.99 (s, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 163.58, 162.50, 161.10, 139.23, 138.17, 138.14, 137.40, 132.90, 131.84, 131.33, 130.94, 130.84, 130.11, 127.58, 126.85, 122.55, 116.89, 116.66, 54.11. HRMS (ESI) calcd for C₁₈H₁₃BrFNOSH [M + H]⁺ 389.9964, found 389.9960.

N-Benzyl-N-(4-bromobenzyl)thiophene-2-carboxamide (10m). Colorless viscous oil (70% yield). ¹H NMR (400 MHz, CDCl₃) δ 7.52–7.44 (m, 3H), 7.40–7.24 (m, 6H), 7.15 (d, J = 7.9 Hz, 2H), 6.96 (dd, J = 5.2, 4.0 Hz, 1H), 4.71 (s, 2H), 4.65 (s, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 165.12, 137.41, 136.28, 135.68, 131.98, 129.72, 129.01, 128.76, 127.81, 127.67, 127.08, 121.59, 110.00. HRMS (ESI) calcd for C₁₉H₁₆BrNOSH [M + H]⁺ 386.0214, found 386.0206.

N-(4-Bromobenzyl)-N-(3-fluorophenyl)thiophene-2-carboxamide (10n). Colorless viscous oil (81% yield). ¹H NMR (400 MHz, CDCl₃) δ 7.44–7.38 (m, 2H), 7.36–7.33 (m, 1H), 7.32–7.25 (m, 1H), 7.17 (d, J = 8.4 Hz, 2H), 7.10–7.03 (m, 1H), 6.89–6.77 (m, 4H), 4.99 (s, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 164.18, 162.48, 161.70, 143.85, 143.76, 137.47, 135.86, 132.61, 131.68, 131.26, 130.89, 130.79, 130.62, 126.92, 124.93, 124.90, 121.73, 116.34, 116.12, 115.74, 115.54, 53.98. MS (ESI) m/z, 390.00.

N-(4-Bromobenzyl)-N-(2-fluorophenyl)thiophene-2-carboxamide (10o). White solid (67% yield), mp 86.8–88.7 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.42–7.37 (m, 2H), 7.36–7.31 (m, 2H), 7.17 (d, J = 8.4 Hz, 2H), 7.14–7.06 (m, 2H), 7.01 (td, J = 8.0, 1.8 Hz, 1H), 6.91 (dd, J = 3.8, 0.8 Hz, 1H), 6.82 (dd, J = 5.0, 3.8 Hz, 1H), 5.29 (d, J = 11.1 Hz, 1H), 4.65 (d, J = 11.1 Hz, 1H). ¹³C NMR (100 MHz, CDCl₃) δ 162.92, 159.74, 137.17, 135.74, 132.14, 131.53, 131.12, 131.02, 130.82, 130.49, 130.41, 126.82, 124.96, 124.92, 121.69, 117.21, 117.01, 109.99, 53.08. MS (ESI) m/z, 390.08.

N-(2-Bromobenzyl)-N-phenylthiophene-2-carboxamide (10p). Colorless viscous oil (77% yield). ¹H NMR (400 MHz, CDCl₃) δ 7.41–7.33 (m, 2H), 7.22–7.12 (m, 5H), 7.05–7.00 (m, 2H), 6.99–6.95 (m, 1H), 6.71–6.64 (m, 2H), 5.11 (s, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 162.69, 142.32, 137.94, 136.19, 132.78, 132.68, 131.19, 130.02, 129.68, 128.98, 128.86, 128.55, 127.70, 126.89, 123.77, 54.28. MS (ESI) m/z, 372.00.

N-(3-Bromobenzyl)-N-phenylthiophene-2-carboxamide (10q). White solid (85% yield), mp 96.1–97.7 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.34 (t, J = 1.6 Hz, 1H), 7.27–7.19 (m, 4H), 7.19–7.16 (m, 1H), 7.11 (d, J = 7.7 Hz, 1H), 7.04–6.99 (m, 1H), 6.98–6.94 (m, 2H), 6.66–6.60 (m, 2H), 4.90 (s, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 162.49, 142.28, 139.56, 137.93, 132.62, 131.77, 131.18, 130.68, 130.10, 129.81, 129.00, 128.65, 127.54, 126.89, 122.50, 54.12. MS (ESI) m/z, 372.00.

N-(2-Bromobenzyl)-N-(2-fluorophenyl)furan-2-carboxamide (10r). White solid (68% yield), mp 104.8–105.6 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.54 (dd, J = 7.7, 1.0 Hz, 1H), 7.45 (d, J = 7.9 Hz, 1H), 7.35–7.21 (m, 3H), 7.14–7.00 (m, 4H), 6.24 (d, J = 1.8 Hz, 1H), 6.07 (s, 1H), 5.46 (d, J = 10.8 Hz, 1H), 4.94 (d, J = 10.8 Hz, 1H). ¹³C NMR (100 MHz, CDCl₃) δ 159.78, 159.50, 157.29, 146.68, 144.81, 135.75, 132.63, 130.67, 130.42, 130.11, 130.03, 129.64, 129.51, 129.11, 127.62, 124.68, 124.64, 123.98, 116.78, 116.58, 116.54, 111.20, 52.53. MS (ESI) m/z, 374.08.

N-(3-Bromobenzyl)-N-(3-fluorophenyl)furan-2-carboxamide (10s). Yellow viscous oil (67% yield). ¹H NMR (400 MHz, CDCl₃) δ 7.44 (t, J = 1.6 Hz, 1H), 7.41–7.36 (m, 1H), 7.34–7.24 (m, 2H), 7.22–7.12 (m, 2H), 7.05–7.03 (m, 1H), 6.86–6.76 (m, 2H), 6.25 (dd, J = 3.6, 1.7 Hz, 1H), 6.07 (d, J = 3.6 Hz, 1H), 4.99 (s, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 164.07, 161.60, 159.09, 146.54, 144.85, 139.02, 131.66, 130.84, 130.67, 130.58, 130.11, 127.36, 124.15, 124.12, 122.55, 117.30, 115.61, 115.41, 115.39, 115.20, 111.27, 53.40. MS (ESI) m/z, 374.00.

N-(3-Bromobenzyl)-N-(3-fluorophenyl)thiophene-2-carboxamide (10t). Colorless viscous oil (71% yield). ¹H NMR (400 MHz, CDCl₃) δ 7.44 (t, J = 1.6 Hz, 1H), 7.41–7.36 (m, 1H), 7.34 (dd, J = 4.8, 1.2 Hz, 1H), 7.32–7.26 (m, 1H), 7.24–7.20 (m, 1H), 7.18–7.14 (m, 1H), 7.09–7.04 (m, 1H), 6.89–6.78 (m, 4H), 5.00 (s, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 164.18, 162.52, 161.70, 143.81, 143.72, 139.15, 137.37, 136.10, 135.46, 132.73, 131.68, 131.35, 130.95, 130.86, 130.17, 128.55, 127.40, 126.96, 124.91, 124.87, 122.58, 116.32, 116.10, 115.81, 115.61, 54.00. MS (ESI) m/z, 390.08.

Antiviral activity

The antiviral activity of the compounds was calculated by an MTS-based CPE reduction assay, which compares the optical density of infected compound-treated cells with uninfected compound-free cells.¹⁹ Briefly, 5 × 10⁵ RD cells (rhabdomyosarcoma) were grown in 96-well plates, then serial dilutions of the compounds and 100 TCID50 of the EV71 virus were added. After incubation at 37 °C for 24–36 h (until complete CPE was observed in the virus infected and compounds free control (VC)), cell viability was measured using the MTS/PMS method (Promega, Leiden, The Netherlands). The absorbance at 498 nm was recorded using a TecanGENiosmicroplate reader (Tecan, Switzerland). CPE values were calculated and the 50% effective concentration (EC₅₀) was defined as the concentration of compound that inhibited the virus-induced cytopathic effect formation by 50% and was calculated using the software Calcusyn.⁴³ Each experiment was repeated at least three times.

Acknowledgements

We are grateful to the NSFC (81172935, 81373255), the Key Scientific Research Project of the Ministry of Education of China (313040), the Hubei Province’s Outstanding Medical Academic Leader Program, the National Mega Project on Major Drug Development (2011ZX09401-302) and the Fundamental Research Funds for the Central Universities (2014306020201, 2042014kf0204) for support of this research.

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Footnotes

† Electronic supplementary information (ESI) available: NMR spectra of 5a–n and 10a–t. See DOI: 10.1039/c5ra07286g

‡ These two authors contributed equally to this work.

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