1-Phenyl-N-(benzothiazol-2-yl)methanimine derivatives as Middle East respiratory syndrome coronavirus inhibitors

Abstract

Middle East respiratory syndrome coronavirus (MERS-CoV) poses a serious threat to human health, and currently there are no effective or specific therapies available to treat it. Herein a series of 1-phenyl-N-(benzothiazol-2-yl)methanimine derivatives with inhibitory activity against MERS-CoV are described. The compound 4f with a 50% inhibition concentration value of 0.09 μM is a promising inhibitor that warrants further evaluation, towards the development of potential anti-MERS-CoV drugs.

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Introduction

Middle East respiratory syndrome coronavirus (MERS-CoV) is a novel zoonotic virus in the Middle East.^1,2 It first appeared in Saudi Arabia in June 2012,³ then spread to the rest of the Middle East and South Korea.⁴ MERS-CoV is primarily transmitted from animals to humans,⁵ but it can reportedly spread from human to human through close contact.^6,7 It can cause severe respiratory disease, with symptoms of fever, coughing, and dyspnea, and may contribute to severe gastrointestinal diseases and renal failure.⁸ As at 03 June 2020, the World Health Organization had reported 2494 laboratory-confirmed cases of MERS-CoV infection, including 858 confirmed deaths (an approximately 35% fatality rate) in over 27 countries.

MERS-CoV poses a severe threat to human health, as illustrated by the recent global coronavirus disease-2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2. Although some reported therapeutics for COVID-19 have recently been reported,^9–17 their curative effect is still very limited and there can be side effects.^18,19 Similarly, despite ongoing global efforts to develop small-molecule inhibitors,^20–26 peptide inhibitors,^27–30 neutralizing antibodies,^31–33 and vaccines^34–36 for the treatment and prevention of MERS-CoV, there is no effective approved specific antiviral therapy for MERS-CoV infection. Therefore, the development of specific antiviral drugs for the treatment of MERS-CoV is urgently needed.

Viral genomic studies indicate that MERS-CoV is an enveloped virus with positive-sense single-stranded RNA that belongs to lineage C in the genus Betacoronavirus of the family Coronaviridae under the Nidovirales order.³ It is the sixth coronavirus known to infect humans, and the first known lineage C Betacoronavirus associated with human infection.^37,38 The MERS-CoV genome is approximately 30 kilobases in size and contains more than 10 open reading frames (ORFs).³⁹ The large replicase ORF1a and ORF1b produce polyproteins PP1a and PP1ab, which are subsequently cleaved into 15 or 16 nonstructural proteins.^40–42 The region downstream of ORF1b encodes at least four main structural proteins: spike (S), membrane (M), envelope (E), and nucleocapsid (N).

The S protein serves as the critical surface-located trimeric glycoprotein of MERS-CoV and induces entry into host cells,⁴³ and it is composed of subunits S1 and S2. The receptor-binding domain on the S1 subunit attaches to dipeptidyl peptidase 4, which is a well-known target for diabetes.^44–49 The conformation of the S2 subunit then changes and a six-helix bundle fusion core is formed, which enables the formation of the viral envelope, facilitating tight host cell membrane binding for fusion and subsequent host cell entry.⁵⁰ Thus the S protein plays a pivotal role in mediating the entry of the virus into target host cells.

According to previous reports, MERS-CoV pseudovirus expressing S protein, which allows for single-cycle infection in cells expressing dipeptidyl peptidase 4, can be used to rapidly screen MERS-CoV entry inhibitors.⁵¹ In the current study, novel small-molecule inhibitors of entry events of pseudovirus expressing S protein were identified via high-throughput screening technology using a cell-based assay.

Materials and methods

Screening was performed using an orthogonal cocktail library⁵² composed of 48 000 compounds that had never been screened for anti-MERS-CoV activity (Fig. 1A). Compound library samples were orthogonally pooled as mixtures of 10 per well at 2 μg mL⁻¹ each, with duplicate representation for each compound (Fig. 1D). This bidirectional orthogonal pooling strategy enables greater screening efficiency and throughput when using large compound libraries. Using this approach, compound 1a was identified as a modest entry inhibitor (Fig. 1B) with a 50% inhibition concentration (IC₅₀) of 0.73 μM (Fig. 1C). Using 1a as a hit compound, a series of 1-phenyl-N-(benzothiazol-2-yl)methanimine derivatives were designed and synthesized, and structure–activity relationships and their inhibitory potencies against MERS-S pseudovirus were investigated.

Fig. 1 High-throughput screening output. (A) Data from the 48 000-compound orthogonal cocktail library primary screening experiments were plotted as inhibition of MERS-S pseudovirus infection in Huh-7 cells. Inhibition was calculated as 100% minus the percentage of luciferase readout of each well against the control well with no drug, i.e., 100% − Luc (compound)/Luc (control) × 100%. (B) The structure of the hit compound 1a. (C) Dose-dependent curve of inhibition activity and toxicity of compound 1a in a MERS-S pseudovirus assay. (D) Flow chart of the high-throughput screening and validation procedure.

Results and discussion

Four major regions were partitioned for diversification; the benzothiazole portion (blue), the linker region (pink), the imine portion (yellow), and the phenyl portion (green) (Fig. 1B). Compounds 1a–1g were synthesized first as outlined in Scheme 1. Commercially available aryl bromides were used as the starting material, and the corresponding intermediate amines 3a–3d were prepared using the Suzuki–Miyaura cross-coupling reaction.⁵³ Compounds 1a–1e were generated using the reaction of an intermediate amine with the corresponding aromatic aldehyde in the presence of AcOH as catalyst.⁵⁴ Compound 1g was generated by the reaction of 3a with 2-hydroxy-3-methoxybenzoyl chloride. Compound 1f was generated by a reduction reaction of 1a with NaBH₄.⁵⁵ Their IC₅₀ values against MERS-S pseudovirus and 50% cytotoxic concentration (CC₅₀) values are summarized in Table 1. The Schiff base structure of hit compound 1a was confirmed by comprehensive spectral data. The ¹H NMR spectrum depicted a sharp singlet peak at δ 8.67 parts per million (ppm) indicating the presence of azomethine proton (–CH=N–). Phenolic-OH proton and methoxy protons exhibited singlet peaks at δ 13.44 and δ 3.94 ppm respectively. A multiplet signal at δ 6.87–8.14 ppm was assigned to the aromatic protons. The ¹³C nuclear magnetic resonance spectrum of 1a revealed corresponding peaks in the range of δ 114.2–166.1 ppm which were assigned to azomethine (–CH=N–) carbon and aryl carbons. A peak at δ 55.2 ppm was identical to the methoxy carbon. The ¹H–¹H COSY spectrum demonstrated chemical shifts of three protons on ring C at δ 6.87–7.06 ppm, and two protons near the nitrogen atom side on ring B at δ 7.36–7.41 ppm. Correlation between the azomethine proton and the two protons near the nitrogen atom side on ring B was observed in the ¹H–¹H NOSEY correlation spectrum, indicating that the imine moiety of compound 1a is E-geometry. The HRMS (ESI) spectrum of 1a depicted a signal at m/z 383.0823, indicating a [M + Na]⁺ signal with the molecular formula C₂₁H₁₆N₂NaO₂S, which is concordant with the structure of 1a. Characteristic spectra and data are shown in the ESI.†

Scheme 1 Reagents and conditions. (a) 4-Aminophenylboronic acid pinacol ester or 5-aminopyridine-3-boronic acid pinacol ester, Pd(dppf)Cl₂·CH₂Cl₂, K₂CO₃, DMF, 70 °C, 16 h. (b) ArCHO, AcOH, MeOH, reflux, 2 h. (c) NaBH₄, MeOH, room temperature, 1 h. (d) 2-Hydroxy-3-methoxybenzoyl chloride, NaHCO₃, H₂O, Et₂O, room temperature, 12 h.

Table 1 Inhibitory activity of 1a–1g against MERS-S pseudovirus

Compound	X	Y	IC50^a	CC50^b
a IC50: 50% inhibitory concentration (μM).b CC50: 50% cytotoxic concentration (μM).c HRP: peptide mimics of heptad repeat domain in the MERS spike protein, which is responsible for conformational change during spike protein-mediated viral–host membrane fusion.
1a	S	CH	0.73 ± 0.66	>100
1b	O	CH	>10.0	>100
1c	NH	CH	>10.0	>100
1d	S	N	2.7 ± 0.19	>100
1e	—	—	>10.0	>100
1f	—	—	>10.0	>100
1g	—	—	>10.0	>100
HRP^c	—	—	2.0	>100

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Compounds with benzoxazole (1b) or benzimidazole (1c) motifs lost inhibitory activity, as did the compound without a phenyl group B (1e) in the linker region. Modest inhibitory activity was evident when the phenyl group B was replaced with a pyridyl group (1d). When the imine portion was reduced or replaced with amide (1f and 1g), inhibitory activity was markedly reduced. Modification was then focused on the phenyl ring C of the compound, and derivatives 1h–1m were designed and synthesized in a similar way as 1a. Their IC₅₀ values on MERS-S pseudovirus and CC₅₀ values are summarized in Table 2.

Table 2 Inhibitory activity of 1h–1u against MERS-S pseudovirus

Compound	Ar	IC50^a	CC50^b
a IC50: 50% inhibitory concentration (μM).b CC50: 50% cytotoxic concentration (μM).c HRP: peptide mimics of heptad repeat domain in the MERS spike protein, which is responsible for conformational change during spike protein-mediated viral–host membrane fusion.
1h		0.57 ± 0.11	>100
1i		0.52 ± 0.03	>100
1j		0.20 ± 0.05	>100
1k		0.40 ± 0.01	>100
1l		0.22 ± 0.03	>100
1m		0.34 ± 0.24	>100
1n		>10.00	>100
1o		0.14 ± 0.06	>100
1p		>10.00	>100
1q		6.52 ± 1.14	>100
1r		0.62 ± 0.31	>100
1s		>10.00	>100
1t		8.80 ± 0.01	>100
1u		>10.00	>100
HRP^c	—	2.00	>100

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All the derivatives demonstrated inhibitory activities with IC₅₀ values ranging from 0.20 μM to 0.57 μM, and no cell toxicity. Either changing the position of the hydroxyl group and methoxy group or the absence of these groups increased the activity slightly, the compounds containing 3-OH were more potent. Changing the phenyl ring C to a pyridine ring did not elicit significant changes in inhibitory activity.

Given the results of 1h–1m, further structural extensions were investigated in the phenyl ring C area. A series of five-member aromatic heterocycles were introduced to replace the phenyl ring C. Derivatives 1o and 1r, substituted by a 3-thienyl group, exhibited marked inhibitory activity with respective IC₅₀ values of 0.14 μM and 0.62 μM. The introduction of a nitro group on the thiophene motif caused a slight reduction in inhibitory activity. Compound 1q exhibited modest inhibitory capacity, indicating that the substitution mode influenced activity. None of the compounds in this series exhibited any cytotoxicity.

Based on the above results, while retaining the 3-thienyl group at ring C position a structure–activity relationship investigation was conducted in which structural extensions at the benzothiazole part of this compound were assessed. As shown in Scheme 2, commercially available aryl bromides were used as starting materials, and the corresponding intermediate amines 3f–3u were prepared using Suzuki–Miyaura cross-coupling or a nucleophilic substitution reaction. The final compounds 4a–4p were readily generated via a similar method that has been previously outlined in Scheme 1. Their IC₅₀ values against MERS-S pseudovirus and their CC₅₀ values are summarized in Table 3.

Scheme 2 Reagents and conditions. (a) 4-Aminophenylboronic acid pinacol ester, Pd(dppf)Cl₂·CH₂Cl₂, K₂CO₃, DMF, 70 °C, 16 h. (b) Thiophene-3-carbaldehyde, AcOH, MeOH, reflux, 2 h. (c) For 4o: 4-aminophenol, t-BuONa, K₂CO₃, DMF, room temperature, 5 h; for 4p: 4-aminobenzenethiol, K₂CO₃, MeCN, reflux, 3 h.

Table 3 Inhibitory activity of 4a–4p against MERS-S pseudovirus

Compound	R	IC50^a	CC50^b
a IC50: 50% inhibitory concentration (μM).b CC50: 50% cytotoxic concentration (μM).c HRP: peptide mimics of heptad repeat domain in the MERS spike protein, which is responsible for conformational change during spike protein-mediated viral–host membrane fusion.
4a	—	>10.00	>100
4b	—	>10.00	>100
4c	—	>10.00	>100
4d	—	>10.00	>100
4e	7-F	1.67 ± 0.30	>100
4f	6-F	0.09 ± 0.01	>100
4g	5-F	1.00 ± 0.07	>100
4h	4-F	0.53 ± 0.02	>100
4i	6-OEt	>10.00	>100
4j	6-Me	>10.00	>100
4k	6-NHMe	>10.00	>100
4l	6-NMe2	>10.00	>100
4m	6-COOH	>10.00	>100
4n	6-CONHMe	>10.00	>100
4o	—	>10.00	>100
4p	—	>10.00	>100
HRP^c	—	2.00	>100

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Replacement of a benzothiazole unit (4a–4d) abolished the inhibitory activity (Table 3), further confirming that the benzothiazole motif played a pivotal role in inhibitory capacity. An atom of fluorine—the most electronegative of the halogens—was then introduced at different benzothiazole positions, and the derivatives obtained (4e–4h) exhibited diverse inhibitory capacity. Introduction of a fluorine atom at the 6-position of the phenyl ring A (4f) increased inhibition activity, with an IC₅₀ value of 0.09 μM, and introduction of a fluorine atom at another position resulted in modest inhibitory potency. The introduction of other groups such as alkoxy, alkyl, amino, and carboxyl groups (4i–4n) reduced inhibitory activity. The reduction in inhibitory capacity is most likely due to the steric effect of the bulky group. To increase the flexibility of the compounds, heteroatom was introduced between the benzothiazole group and phenyl ring B to obtain 4o and 4p, but unfortunately inhibitory activity was abolished. The CC₅₀ values of these compounds were greater than 100 μM, indicating no toxicity or at least lower toxicity to uninfected cells. The results of the study suggest that compound 4f has potential for the subsequent development of novel anti-MERS-CoV agents.

Conclusion

In summary, a series of new 1-phenyl-N-(benzothiazol-2-yl)methanimine derivatives was developed as MERS-CoV entry inhibitors via modification of a hit compound, which was obtained by screening an orthogonal cocktail library composed of 48 000 compounds. All of the compounds were assessed for their capacity to inhibit the entry of pseudovirus expressing MERS-CoV S protein. Structure–activity relationships indicated that benzothiazole was the best option for region A of the structure, and the presence of a phenyl ring B and an imine portion favored the retention of inhibitory activity against MERS-CoV. The introduction of a 6-member aromatic ring or a 3-thiophene ring at ring C position also favored inhibitory activity. Remarkably, compound 4f, with an IC₅₀ value of 0.09 μM, was more potent than the initial hit compound. In addition, none of these types of structures exhibited cytotoxicity. The present study demonstrates an approach for the structural modification of hit compounds, and a novel skeleton for new anti-MERS-CoV drug research. Further research on this series of derivatives is ongoing.

Experimental

Chemistry

Commercial reagents and solvents were purchased commercial suppliers and used without further purification. All non-aqueous reactions were under a nitrogen atmosphere and all non-aqueous reaction vessels were oven-dried. Flash column chromatography was performed using Qingdao Ocean silica gel (200–300) with the indicated eluents. All final compounds were characterized by their NMR and HRMS spectra, unless stated otherwise. ¹H NMR spectra were recorded at a spectrometer frequency of 400 MHz, and ¹³C NMR spectra at 100 MHz on Bruker Avance 400. Chemical shifts are reported in δ (ppm) using signals of tetramethylsilane (TMS) as the internal standard. High-resolution mass spectra (HRMS) were measured on a Bruker ESI-TOF high-resolution mass spectrometer. Melting points (mp) were uncorrected and were recorded on a Buchi B-54 melting point apparatus.

General procedure for the synthesis of compounds 3a–3d and 3f–3s

2a–2q (1 mmol), the corresponding pinacol borate (1.2 mmol) and Pd(dppf)Cl₂·CH₂Cl₂ (0.05 mmol) were suspended in DMF (6 mL) in the presence of 2 M K₂CO₃ (2 mL) and stirred at 70 °C overnight under nitrogen. The reaction mixture was diluted with H₂O (40 mL) and organics were extracted with EtOAc (3× 30 mL). The combined organic layers were washed with brine (30 mL), dried over MgSO₄, filtered and concentrated in vacuo to afford crude mixture. The crude mixture was then purified by flash column chromatography in a mixture of petroleum ether and ethyl acetate to yield the pure products 3a–3d and 3f–3s.

4-(Benzo[d]thiazol-2-yl)aniline (3a). Yellow solid; yield 60%.¹H NMR (400 MHz, DMSO-d₆) δ 8.01 (d, J = 7.9 Hz, 1H, ArH), 7.89 (d, J = 8.1 Hz, 1H, ArH), 7.76 (d, J = 8.4 Hz, 2H, ArH), 7.48–7.42 (m, 1H, ArH), 7.37–7.31 (m, 1H, ArH), 6.67 (d, J = 8.4 Hz, 2H, ArH), 5.88 (s, 2H, NH₂). ¹³C NMR (100 MHz, chloroform-d) δ 168.5, 154.3, 149.2, 134.7, 129.2, 126.1, 124.4, 124.0, 122.6, 121.4, 114.7.

4-(Benzo[d]oxazol-2-yl)aniline (3b). Light orange solid; yield 65%. ¹H NMR (400 MHz, DMSO-d₆) δ 7.86 (d, J = 8.3 Hz, 2H, ArH), 7.70–7.63 (m, 2H, ArH), 7.36–7.27 (m, 2H, ArH), 6.70 (d, J = 8.4 Hz, 2H, ArH), 5.96 (s, 2H, NH₂). ¹³C NMR (100 MHz, DMSO-d₆) δ 163.6, 152.5, 149.8, 142.1, 128.9 (2C), 124.3, 124.0, 118.7, 113.5 (2C), 112.7, 110.2.

4-(1H-Benzo[d]imidazol-2-yl)aniline (3c). Light orange solid; yield 70%. ¹H NMR (400 MHz, DMSO-d₆) δ 12.43 (s, 1H, imidazole), 7.87 (d, J = 8.2 Hz, 2H, ArH), 7.49 (s, 2H, ArH), 7.14–7.10 (m, 2H, ArH), 6.69 (d, J = 8.2 Hz, 2H, ArH), 5.58 (s, 2H, NH₂). ¹³C NMR (100 MHz, DMSO-d₆) δ 152.6, 150.5, 127.7 (2C), 121.2, 117.3, 113.5 (2C).

6-(Benzo[d]thiazol-2-yl)pyridin-3-amine (3d). Yellow solid; yield 70%. This intermediate is used directly in the next step without further purification.

4-(Thiazolo[4,5-b]pyridin-2-yl)aniline (3f). Yellow solid; yield 24%. ¹H NMR (400 MHz, chloroform-d) δ 8.66 (d, J = 3.6 Hz, 1H, ArH), 8.17 (d, J = 7.9 Hz, 1H, ArH), 8.00 (d, J = 8.4 Hz, 2H, ArH), 7.25–7.21 (m, 1H, ArH), 6.74 (d, J = 8.6 Hz, 2H, ArH), 4.09 (s, 2H, NH₂).

4-(4,5,6,7-Tetrahydrobenzo[d]thiazol-2-yl)aniline (3g). Light yellow solid; yield 62%. ¹H NMR (400 MHz, chloroform-d) δ 7.68 (d, J = 8.6 Hz, 2H, ArH), 6.67 (d, J = 8.5 Hz, 2H, ArH), 3.84 (s, 2H, NH₂), 2.83–2.75 (m, 4H, CH₂), 1.91–1.81 (m, 4H, CH₂). ¹³C NMR (100 MHz, chloroform-d) δ 164.2, 149.7, 146.8, 126.6 (2C), 126.3, 123.9, 113.8 (2C), 25.9, 22.6, 22.4, 22.1.

4-(Benzo[d]imidazo[2,1-b]thiazol-2-yl)aniline (3h). Light yellow solid; yield 45%. ¹H NMR (400 MHz, chloroform-d) δ 7.82 (s, 1H, ArH), 7.70–7.65 (m, 3H, ArH), 7.57 (d, J = 8.0 Hz, 1H, ArH), 7.46–7.40 (m, 1H, ArH), 7.34–7.28 (m, 1H, ArH), 6.74 (d, J = 8.4 Hz, 2H, ArH), 3.74 (s, 2H, NH₂). ¹³C NMR (100 MHz, chloroform-d) δ 147.1, 146.7, 145.0, 131.3, 129.2, 125.4 (2C), 125.1, 123.6, 123.5, 123.3, 114.3 (2C), 111.4, 104.2.

4-(Imidazo[1,2-a]pyridin-2-yl)aniline (3i). Yellow solid; yield 56%. ¹H NMR (400 MHz, chloroform-d) δ 8.07 (d, J = 6.7 Hz, 1H, ArH), 7.76 (d, J = 8.5 Hz, 2H, ArH), 7.73 (s, 1H, ArH), 7.59 (d, J = 9.1 Hz, 1H, ArH), 7.15–7.09 (m, 1H, ArH), 6.77–6.70 (m, 3H, ArH), 3.75 (s, 2H, NH₂). ¹³C NMR (100 MHz, chloroform-d) δ 145.4, 145.3, 144.5, 126.2 (2C), 124.3, 123.3, 123.2, 116.2, 114.2 (2C), 111.0, 105.7.

4-(7-Fluorobenzo[d]thiazol-2-yl)aniline (3j). Yellow solid; yield 69%. ¹H NMR (400 MHz, DMSO-d₆) δ 7.80–7.73 (m, 3H, ArH), 7.50–7.42 (m, 1H, ArH), 7.24–7.18 (m, 1H, ArH), 6.69 (d, J = 8.3 Hz, 2H, ArH), 6.04 (s, 2H, NH₂). ¹³C NMR (100 MHz, DMSO-d₆) δ 169.0 (d, J = 1.5 Hz), 156.8 (d, J = 2.5 Hz), 156.3 (d, J = 246.1 Hz), 152.6, 129.1 (2C), 127.5 (d, J = 7.5 Hz), 120.1 (d, J = 16.2 Hz), 119.2, 118.0 (d, J = 3.3 Hz), 113.6 (2C), 109.9 (d, J = 18.7 Hz).

4-(6-Fluorobenzo[d]thiazol-2-yl)aniline (3k). White solid; yield 71%. ¹H NMR (400 MHz, chloroform-d) δ 7.94–7.90 (m, 1H, ArH), 7.85 (d, J = 7.8 Hz, 2H, ArH), 7.52 (d, J = 8.1 Hz, 1H, ArH), 7.20–7.14 (m, 1H, ArH), 6.73 (d, J = 8.9 Hz, 2H, ArH), 4.02 (s, 2H, NH₂). ¹³C NMR (100 MHz, chloroform-d) δ 167.2 (d, J = 3.2 Hz), 159.0 (d, J = 244.5 Hz), 149.8 (d, J = 1.8 Hz), 148.2, 134.5 (d, J = 11.1 Hz), 128.0 (2C), 122.6, 122.2 (d, J = 9.2 Hz), 113.8 (2C), 113.5 (d, J = 24.5 Hz), 106.7 (d, J = 26.7 Hz).

4-(5-Fluorobenzo[d]thiazol-2-yl)aniline (3l). White solid; yield 59%. ¹H NMR (400 MHz, DMSO-d₆) δ 8.01 (dd, J = 8.8, 5.4 Hz, 1H, ArH), 7.76 (d, J = 8.5 Hz, 2H, ArH), 7.72 (dd, J = 10.0, 2.6 Hz, 1H, ArH), 7.25–7.17 (m, 1H, ArH), 6.68 (d, J = 8.5 Hz, 2H, ArH), 5.98 (s, 2H, NH₂). ¹³C NMR (100 MHz, DMSO-d₆) δ 170.9, 161.3 (d, J = 240.0 Hz), 154.8 (d, J = 12.3 Hz), 152.4, 129.4 (d, J = 2.0 Hz), 128.8 (2C), 122.9 (d, J = 9.9 Hz), 119.8, 113.5 (2C), 112.3 (d, J = 24.7 Hz), 107.8 (d, J = 23.6 Hz).

4-(4-Fluorobenzo[d]thiazol-2-yl)aniline (3m). White solid; yield 61%. ¹H NMR (400 MHz, DMSO-d₆) δ 7.85 (dd, J = 7.6, 1.4 Hz, 1H, ArH), 7.77 (d, J = 8.6 Hz, 2H, ArH), 7.37–7.28 (m, 2H, ArH), 6.67 (d, J = 8.7 Hz, 2H, ArH), 5.99 (s, 2H, NH₂). ¹³C NMR (100 MHz, DMSO-d₆) δ 169.0, 154.3 (d, J = 252.5 Hz), 152.5, 142.2 (d, J = 13.2 Hz), 136.5 (d, J = 3.9 Hz), 129.0 (2C), 125.1 (d, J = 7.1 Hz), 119.5, 118.0 (d, J = 4.0 Hz), 113.5 (2C), 111.9 (d, J = 17.7 Hz).

4-(6-Ethoxybenzo[d]thiazol-2-yl)aniline (3n). Brown solid; yield 38%. ¹H NMR (400 MHz, chloroform-d) δ 7.88–7.82 (m, 3H, ArH), 7.31 (s, 1H, ArH), 7.04 (d, J = 8.9 Hz, 1H, ArH), 6.73 (d, J = 8.1 Hz, 2H, ArH), 4.09 (q, J = 6.8 Hz, 2H, CH₂), 3.96 (s, 2H, NH₂), 1.45 (t, J = 6.9 Hz, 3H, CH₃). ¹³C NMR (100 MHz, chloroform-d) δ 166.0, 156.6, 148.8, 148.7, 135.8, 128.8 (2C), 124.2, 122.9, 115.5, 114.8 (2C), 105.0, 64.1, 14.9.

4-(6-Methylbenzo[d]thiazol-2-yl)aniline (3o). Yellow solid; yield 60%. ¹H NMR (400 MHz, chloroform-d) δ 7.90–7.85 (m, 3H, ArH), 7.64 (s, 1H, ArH), 7.26–7.22 (m, 1H, ArH), 6.73 (d, J = 8.5 Hz, 2H, ArH), 3.97 (s, 2H, NH₂), 2.48 (s, 3H, CH₃). ¹³C NMR (100 MHz, chloroform-d) δ 166.1, 151.2, 142.9, 134.2, 134.0, 128.1, 127.8, 127.6, 126.3, 121.3, 118.9, 21.1.

2-(4-Aminophenyl)-N-methylbenzo[d]thiazol-6-amine (3p). Orange solid; yield 70%. ¹H NMR (400 MHz, chloroform-d) δ 7.82 (d, J = 8.5 Hz, 2H, ArH), 7.77 (d, J = 8.7 Hz, 1H, ArH), 6.98 (s, 1H, ArH), 6.77–6.70 (m, 3H, ArH), 3.91 (s, 2H, NH₂), 2.90 (s, 3H, CH₃). ¹³C NMR (100 MHz, chloroform-d) δ 162.7, 147.4, 146.0, 145.8, 135.7, 127.5 (2C), 123.6, 121.8, 113.8 (2C), 113.0, 100.8, 30.0.

2-(4-Aminophenyl)-N,N-dimethylbenzo[d]thiazol-6-amine (3q). Orange solid; yield 72%. ¹H NMR (400 MHz, chloroform-d) δ 7.85–7.81 (m, 3H, ArH), 7.10 (s, 1H, ArH), 6.93 (dd, J = 9.0, 2.5 Hz, 1H, ArH), 6.72 (d, J = 8.6 Hz, 2H, ArH), 3.92 (s, 2H, NH₂), 3.02 (s, 6H, CH₃). ¹³C NMR (100 MHz, chloroform-d) δ 163.9, 148.5, 148.5, 146.2, 136.6, 128.6 (2C), 124.6, 122.5, 114.8 (2C), 113.2, 103.3, 41.2.

2-(4-Aminophenyl)benzo[d]thiazole-6-carboxylic acid (3r). Yellow solid; yield 60%. ¹H NMR (400 MHz, DMSO-d₆) δ 12.99 (br, 1H, COOH), 8.62 (s, 1H, ArH), 8.00 (d, J = 8.5 Hz, 1H, ArH), 7.93 (d, J = 8.6 Hz, 1H, ArH), 7.79 (d, J = 8.3 Hz, 2H, ArH), 6.67 (d, J = 8.3 Hz, 2H, ArH), 6.04 (s, 2H, NH₂). ¹³C NMR (100 MHz, DMSO-d₆) δ 171.7, 167.0, 156.8, 152.7, 133.8, 129.1 (2C), 127.3, 126.3, 123.8, 121.3, 119.6, 113.5 (2C).

2-(4-Aminophenyl)-N-methylbenzo[d]thiazole-6-carboxamide (3s). Light yellow solid; yield 47%. ¹H NMR (400 MHz, DMSO-d₆) δ 8.51 (q, J = 4.2 Hz, 1H, –(C=O)NH–), 8.48 (s, 1H, ArH), 7.95–7.88 (m, 2H, ArH), 7.79 (d, J = 8.4 Hz, 2H, ArH), 6.68 (d, J = 8.6 Hz, 2H, ArH), 5.99 (s, 2H, NH₂), 2.82 (d, J = 4.4 Hz, 3H, CH₃). ¹³C NMR (100 MHz, DMSO-d₆) δ 170.4, 166.1, 155.6, 152.5, 133.6, 130.4, 129.0 (2C), 125.3, 121.1, 121.1, 119.7, 113.5 (2C), 26.4.

Synthesis of 4-(benzo[d]thiazol-2-yloxy)aniline (3t)

4-Aminophenol (2 mmol) was dissolved in DMF (5 mL) followed by the addition of t-BuONa (2.4 mmol) and stirred at room temperature for 1 h. Then 2a (2 mmol) and K₂CO₃ (2.4 mmol) were added and stirred for another 4 h. The reaction mixture was diluted with H₂O (40 mL) and organics were extracted with EtOAc (3× 30 mL). The combined organic layers were washed with brine (30 mL), dried over MgSO₄, filtered and concentrated in vacuo to afford crude mixture. The crude mixture was then purified by flash column chromatography in a mixture of petroleum ether and ethyl acetate to yield the pure product 3t. Yellow oil, yield 80%. ¹H NMR (400 MHz, chloroform-d) δ 7.73 (d, J = 8.1 Hz, 1H, ArH), 7.63 (d, J = 8.0 Hz, 1H, ArH), 7.40–7.34 (m, 1H, ArH), 7.25–7.21 (m, 1H, ArH), 7.12 (d, J = 8.7 Hz, 2H, ArH), 6.71 (d, J = 8.7 Hz, 2H, ArH), 3.74 (s, 2H, NH₂). ¹³C NMR (100 MHz, chloroform-d) δ 173.6, 149.4, 147.2, 145.0, 132.2, 126.2, 123.8, 121.8 (2C), 121.6, 121.3, 115.9 (2C).

Synthesis of 4-(benzo[d]thiazol-2-ylthio)aniline (3u)

4-Aminobenzenethiol (2 mmol) and 2a (2 mmol) was dissolved in MeCN (5 mL) followed by the addition of K₂CO₃ (2.4 mmol) and stirred at reflux for 3 h. Upon completion, the reaction was allowed to cold to room temperature. The reaction mixture was diluted with H₂O (40 mL) and organics were extracted with EtOAc (3× 30 mL). The combined organic layers were washed with brine (30 mL), dried over MgSO₄, filtered and concentrated in vacuo to afford crude mixture. The crude mixture was then purified by flash column chromatography in a mixture of petroleum ether and ethyl acetate to yield the pure product 3u. White solid; yield 50%. ¹H NMR (400 MHz, chloroform-d) δ 7.84 (d, J = 8.2 Hz, 1H, ArH), 7.62 (d, J = 8.0 Hz, 1H, ArH), 7.50 (d, J = 8.4 Hz, 2H, ArH), 7.41–7.35 (m, 1H, ArH), 7.25–7.19 (m, 1H, ArH), 6.74 (d, J = 8.4 Hz, 2H, ArH), 4.02 (s, 2H, NH₂). ¹³C NMR (100 MHz, chloroform-d) δ 172.4, 153.3, 148.0, 136.6 (2C), 134.4, 125.0, 122.8, 120.6, 119.7, 115.6, 114.9 (2C).

General procedure for the synthesis of compounds 1a–1d, 1h–1u and 4a–4p

The catalytic amount of acetic acid was added to a solution of 3a–3d, 3f–3u (0.44 mmol) and the corresponding aromatic aldehyde (0.53 mmol) in MeOH (4 mL), the mixture was stirred at reflux for 2 h. The resulting precipitate was isolated by suction filtration and washed with MeOH (8 mL) to give the pure products 1a–1d, 1h–1u and 4a–4p.

(E)-2-(((4-(Benzo[d]thiazol-2-yl)phenyl)imino)methyl)-6-methoxyphenol (1a). Orange solid; yield 63%. Mp 175–176 °C. ¹H NMR (400 MHz, chloroform-d) δ 13.44 (s, 1H, OH), 8.67 (s, 1H, –CH=N–), 8.14 (d, J = 8.2 Hz, 2H, ArH), 8.07 (d, J = 8.2 Hz, 1H, ArH), 7.90 (d, J = 7.9 Hz, 1H, ArH), 7.52–7.48 (m, 1H, ArH), 7.41–7.36 (m, 3H, ArH), 7.06–6.99 (m, 2H, ArH), 6.93–6.87 (m, 1H, ArH), 3.94 (s, 3H, CH₃). ¹³C NMR (100 MHz, chloroform-d) δ 166.1, 162.3, 153.1, 150.5, 149.3, 147.5, 134.0, 131.2, 127.7, 125.4, 124.2, 123.0, 122.2, 120.8, 120.6, 118.0, 117.7, 114.2, 55.2. HRMS (ESI) m/z: calculated for C₂₁H₁₆N₂NaO₂S [M + Na]⁺: 383.0825, found: 383.0823.

(E)-2-(((4-(Benzo[d]oxazol-2-yl)phenyl)imino)methyl)-6-methoxyphenol (1b). Orange solid; yield 55%. Mp 218–219 °C. ¹H NMR (400 MHz, chloroform-d) δ 13.35 (s, 1H, OH), 8.69 (s, 1H, –CH=N–), 8.31 (d, J = 8.3 Hz, 2H, ArH), 7.80–7.76 (m, 1H, ArH), 7.61–7.57 (m, 1H, ArH), 7.42 (d, J = 8.2 Hz, 2H, ArH), 7.38–7.34 (m, 2H, ArH), 7.06–7.01 (m, 2H, ArH), 6.94–6.88 (m, 1H, ArH), 3.95 (s, 3H, CH₃). ¹³C NMR (100 MHz, chloroform-d) δ 162.7, 161.5, 150.6, 149.9, 149.8, 147.5, 141.2, 127.9 (2C), 124.6, 124.2, 123.7, 123.0, 120.8 (2C), 119.0, 118.0, 117.8, 114.3, 109.6, 55.2. HRMS (ESI) m/z: calculated for C₂₁H₁₆N₂NaO₃ [M + Na]⁺: 367.1053, found: 367.1041.

(E)-2-(((4-(1H-Benzo[d]imidazol-2-yl)phenyl)imino)methyl)-6-methoxyphenol (1c). Red solid; yield 36%. Mp > 220 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 13.10 (s, 1H, OH), 12.94 (s, 1H, imidazole), 9.06 (s, 1H, –CH=N–), 8.27 (d, J = 8.2 Hz, 2H, ArH), 7.68 (d, J = 7.5 Hz, 1H, ArH), 7.60 (d, J = 8.2 Hz, 2H, ArH), 7.54 (d, J = 7.5 Hz, 1H, ArH), 7.28 (d, J = 7.5 Hz, 1H, ArH), 7.23–7.20 (m, 2H, ArH), 7.15 (d, J = 7.7 Hz, 1H, ArH), 6.95–6.91 (m, 1H, ArH), 3.84 (s, 3H, CH₃). ¹³C NMR (100 MHz, DMSO-d₆) δ 163.9, 150.7, 150.6, 149.0, 147.9, 143.9, 135.0, 128.6, 127.6 (2C), 123.9, 122.6, 122.0 (2C), 121.7, 119.3, 118.8, 118.7, 115.8, 111.3, 55.9. HRMS (ESI) m/z: calculated for C₂₁H₁₇N₃NaO₂ [M + Na]⁺: 366.1213, found: 366.1198.

(E)-2-(((6-(Benzo[d]thiazol-2-yl)pyridin-3-yl)imino)methyl)-6-methoxyphenol (1d). Yellow solid; yield 64%. Mp > 220 °C. ¹H NMR (400 MHz, chloroform-d) δ 13.00 (s, 1H, OH), 8.71 (s, 1H, –CH=N–), 8.61 (d, J = 2.4 Hz, 1H, ArH), 8.43 (d, J = 8.4 Hz, 1H, ArH), 8.09 (d, J = 8.2 Hz, 1H, ArH), 7.96 (d, J = 8.0 Hz, 1H, ArH), 7.74 (dd, J = 8.4, 2.5 Hz, 1H, ArH), 7.54–7.49 (m, 1H, ArH), 7.44–7.41 (m, 1H, ArH), 7.09–7.03 (m, 2H, ArH), 6.96–6.90 (m, 1H, ArH), 3.95 (s, 3H, CH₃). ¹³C NMR (100 MHz, chloroform-d) δ 167.5, 163.8, 153.3, 150.4, 148.7, 147.5, 144.6, 142.3, 135.2, 127.6, 125.3, 124.7, 123.2, 122.5, 121.0, 120.3, 118.1, 117.9, 114.7, 55.2. HRMS (ESI) m/z: calculated for C₂₀H₁₅N₃NaO₂S [M + Na]⁺: 384.0777, found: 384.0786.

(E)-N-(4-(Benzo[d]thiazol-2-yl)phenyl)-1-phenylmethanimine (1h). Yellow solid; yield 22%. Mp 200–202 °C. ¹H NMR (400 MHz, chloroform-d) δ 8.51 (s, 1H, –CH=N–), 8.14 (d, J = 8.5 Hz, 2H, ArH), 8.08 (d, J = 8.2 Hz, 1H, ArH), 7.96–7.93 (m, 2H, ArH), 7.91 (d, J = 8.0 Hz, 1H, ArH), 7.52–7.47 (m, 4H, ArH), 7.41–7.36 (m, 1H, ArH), 7.32 (d, J = 8.4 Hz, 2H, ArH). ¹³C NMR (100 MHz, chloroform-d) δ 166.6, 160.1, 153.4, 153.2, 134.9, 134.0, 130.8, 130.2, 128.0 (2C), 127.8 (2C), 127.6 (2C), 125.3, 124.1, 122.1, 120.6, 120.5 (2C). HRMS (ESI) m/z: calculated for C₂₀H₁₄N₂NaS [M + Na]⁺: 337.0770, found: 337.0752.

(E)-2-(((4-(Benzo[d]thiazol-2-yl)phenyl)imino)methyl)phenol (1i). Light yellow solid; yield 60%. Mp 205–207 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 12.81 (s, 1H, OH), 9.06 (s, 1H, –CH=N–), 8.19–8.17 (m, 3H, ArH), 8.07 (d, J = 8.1 Hz, 1H, ArH), 7.71 (d, J = 7.5 Hz, 1H, ArH), 7.60 (d, J = 8.4 Hz, 2H, ArH), 7.58–7.54 (m, 1H, ArH), 7.47–7.44 (m, 2H, ArH), 7.03–6.99 (m, 2H, ArH). ¹³C NMR (100 MHz, DMSO-d₆) δ 166.6, 164.3, 160.3, 153.6, 150.7, 134.5, 133.7, 132.6, 131.1, 128.4 (2C), 126.7, 125.5, 122.8, 122.4 (2C), 122.3, 119.3, 119.3, 116.7. HRMS (ESI) m/z: calculated for C₂₀H₁₅N₂OS [M + H]⁺: 331.0900, found: 331.0902.

(E)-N-(4-(Benzo[d]thiazol-2-yl)phenyl)-1-(3-methoxyphenyl)methanimine (1j). Light yellow solid; yield 45%. Mp 118–119 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.67 (s, 1H, –CH=N–), 8.15–8.13 (m, 3H, ArH), 8.06 (d, J = 8.1 Hz, 1H, ArH), 7.57–7.54 (m, 3H, ArH), 7.48–7.42 (m, 4H, ArH), 7.14 (d, J = 8.2 Hz, 1H, ArH), 3.84 (s, 3H, CH₃). ¹³C NMR (100 MHz, DMSO-d₆) δ 166.8, 161.8, 159.5, 153.8, 153.6, 137.2, 134.4, 130.4, 130.0, 128.3 (2C), 126.6, 125.4, 122.7, 122.3, 122.0 (2C), 121.9, 118.1, 112.7, 55.2. HRMS (ESI) m/z: calculated for C₂₁H₁₇N₂NaOS [M + H]⁺: 345.1056, found: 345.1071.

(E)-3-(((4-(Benzo[d]thiazol-2-yl)phenyl)imino)methyl)benzene-1,2-diol (1k). Red solid; yield 26%. Mp > 220 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 9.01 (s, 1H, –CH=N–), 8.19–8.13 (m, 3H, ArH), 8.07 (d, J = 8.1 Hz, 1H, ArH), 7.59 (d, J = 8.2 Hz, 2H, ArH), 7.57–7.54 (m, 1H, ArH), 7.49–7.45 (m, 1H, ArH), 7.15 (d, J = 6.9 Hz, 1H, ArH), 6.99 (d, J = 6.8 Hz, 1H, ArH), 6.84–6.80 (m,1H, ArH). ¹³C NMR (100 MHz, DMSO-d₆) δ 166.6, 164.7, 153.6, 150.5, 149.4, 145.6, 134.5, 131.1, 128.4 (2C), 126.7, 125.5, 122.9, 122.8, 122.4 (2C), 122.3, 119.4, 119.3, 118.9. HRMS (ESI) m/z: calculated for C₂₀H₁₄N₂NaO₂S [M + Na]⁺: 369.0668, found: 369.0654.

(E)-5-(((4-(Benzo[d]thiazol-2-yl)phenyl)imino)methyl)-2-methoxyphenol (1l). Yellow solid; yield 54%. Mp 214–215 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 9.40 (br, 1H, OH), 8.52 (s, 1H, –CH=N–), 8.14–8.12 (m, 3H, ArH), 8.05 (d, J = 8.1 Hz, 1H, ArH), 7.57–7.52 (m, 1H, ArH), 7.47–7.44 (m, 2H, ArH), 7.39–7.35 (m, 3H, ArH), 7.06 (d, J = 8.3 Hz, 1H, ArH), 3.85 (s, 3H, CH₃). ¹³C NMR (100 MHz, DMSO-d₆) δ 166.9, 161.3, 154.3, 153.6, 151.3, 146.8, 134.4, 129.9, 128.9, 128.2 (2C), 126.6, 125.4, 122.9, 122.7, 122.3, 121.9 (2C), 113.7, 111.6, 55.6. HRMS (ESI) m/z: calculated for C₂₁H₁₇N₂O₂S [M + H]⁺: 361.1005, found: 361.0986.

(E)-N-(4-(Benzo[d]thiazol-2-yl)phenyl)-1-(pyridin-3-yl)methanimine (1m). Yellow solid; yield 17%. Mp 151–153 °C. ¹H NMR (400 MHz, chloroform-d) δ 9.05 (s, 1H, –CH=N–), 8.73 (d, J = 4.7 Hz, 1H, ArH), 8.55 (s, 1H, ArH), 8.32 (d, J = 8.0 Hz, 1H, ArH), 8.15 (d, J = 8.1 Hz, 2H, ArH), 8.08 (d, J = 8.2 Hz, 1H, ArH), 7.91 (d, J = 8.0 Hz, 1H, ArH), 7.52–7.48 (m, 1H, ArH), 7.46–7.42 (m, 1H, ArH), 7.41–7.37 (m, 1H, ArH), 7.33 (d, J = 8.1 Hz, 2H, ArH). ¹³C NMR (100 MHz, chloroform-d) δ 167.3, 158.0, 154.2, 153.6, 152.3, 151.1, 135.1, 135.0, 131.8, 131.6, 128.6 (2C), 126.4, 125.2, 123.9, 123.2, 121.6, 121.5 (2C). HRMS (ESI) m/z: calculated for C₁₉H₁₄N₃S [M + H]⁺: 316.0903, found: 316.0893.

(E)-N-(4-(Benzo[d]thiazol-2-yl)phenyl)-1-(furan-3-yl)methanimine (1n). Light yellow solid; yield 60%. Mp 170–172 °C. ¹H NMR (400 MHz, chloroform-d) δ 8.44 (s, 1H, –CH=N–), 8.12 (d, J = 8.3 Hz, 2H, ArH), 8.07 (d, J = 8.1 Hz, 1H, ArH), 7.92–7.90 (m, 2H, ArH), 7.54–7.46 (m, 2H, ArH), 7.40–7.37 (m, 1H, ArH), 7.27 (d, J = 8.3 Hz, 2H, ArH), 6.97 (s, 1H, ArH). ¹³C NMR (100 MHz, chloroform-d) δ 166.6, 153.5, 153.2, 151.7, 146.2, 143.5, 134.0, 130.1, 127.5 (2C), 125.3, 124.9, 124.1, 122.0, 120.6, 120.4 (2C), 106.8. HRMS (ESI) m/z: calculated for C₁₈H₁₃N₂OS [M + H]⁺: 305.0743, found: 305.0755.

(E)-N-(4-(Benzo[d]thiazol-2-yl)phenyl)-1-(thiophen-3-yl)methanimine (1o). Light yellow solid; yield 58%. Mp 162–164 °C. ¹H NMR (400 MHz, chloroform-d) δ 8.51 (s, 1H, –CH=N–), 8.12 (d, J = 8.2 Hz, 2H, ArH), 8.07 (d, J = 8.1 Hz, 1H, ArH), 7.90 (d, J = 7.9 Hz, 1H, ArH), 7.85 (s, 1H, ArH), 7.71 (d, J = 5.0 Hz, 1H, ArH), 7.51–7.47 (m, 1H, ArH), 7.41–7.36 (m, 2H, ArH), 7.29 (d, J = 8.2 Hz, 2H, ArH). ¹³C NMR (100 MHz, chloroform-d) δ 167.6, 155.0, 154.5, 154.3, 140.6, 135.1, 131.2, 130.9, 128.6 (2C), 126.9, 126.3, 126.0, 125.1, 123.1, 121.6, 121.5 (2C). HRMS (ESI) m/z: calculated for C₁₈H₁₃N₂S₂ [M + H]⁺: 321.0515, found: 321.0513.

(E)-N-(4-(Benzo[d]thiazol-2-yl)phenyl)-1-(1H-pyrrol-3-yl)methanimine (1p). White solid; yield 55%. Mp > 220 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 11.41 (s, 1H, pyrrole), 8.48 (s, 1H, –CH=N–), 8.13 (d, J = 8.0 Hz, 1H, ArH), 8.08 (d, J = 8.1 Hz, 2H, ArH), 8.04 (d, J = 8.1 Hz, 1H, ArH), 7.56–7.52 (m, 1H, ArH), 7.47–7.43 (m, 2H, ArH), 7.30 (d, J = 8.1 Hz, 2H, ArH), 6.91 (s, 1H, ArH), 6.59 (s, 1H, ArH). ¹³C NMR (100 MHz, DMSO-d₆) δ 167.1, 156.4, 155.6, 153.6, 134.3, 129.0, 128.2 (2C), 126.6, 125.3, 125.3, 123.0, 122.6, 122.2, 121.7 (2C), 120.5, 106.2. HRMS (ESI) m/z: calculated for C₁₈H₁₄N₃S [M + H]⁺: 304.0903, found: 304.0894.

(E)-N-(4-(Benzo[d]thiazol-2-yl)phenyl)-1-(thiophen-2-yl)methanimine (1q). Light green solid; yield 57%. Mp 184–185 °C. ¹H NMR (400 MHz, chloroform-d) δ 8.62 (s, 1H, –CH=N–), 8.12 (d, J = 8.1 Hz, 2H, ArH), 8.07 (d, J = 8.2 Hz, 1H, ArH), 7.90 (d, J = 8.0 Hz, 1H, ArH), 7.59–7.46 (m, 3H, ArH), 7.41–7.35 (m, 1H, ArH), 7.32 (d, J = 8.2 Hz, 2H, ArH), 7.18–7.14 (m, 1H, ArH). ¹³C NMR (100 MHz, chloroform-d) δ 167.6, 154.2, 153.7, 153.7, 142.6, 135.0, 133.0, 131.3, 131.1, 128.6 (2C), 127.9, 126.4, 125.1, 123.1, 121.7 (2C), 121.6. HRMS (ESI) m/z: calculated for C₁₈H₁₂N₂NaS₂ [M + Na]⁺: 343.0334, found: 343.0333.

(E)-N-(4-(Benzo[d]thiazol-2-yl)phenyl)-1-(5-nitrothiophen-3-yl)methanimine (1r). Yellow solid; yield 74%. Mp 213–215 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.70 (s, 1H, –CH=N–), 8.60 (s, 1H, ArH), 8.42 (s, 1H, ArH), 8.16–8.14 (m, 3H, ArH), 8.06 (d, J = 8.2 Hz, 1H, ArH), 7.57–7.53 (m, 1H, ArH), 7.48–7.43 (m, 3H, ArH). ¹³C NMR (100 MHz, DMSO-d₆) δ 166.7, 155.4, 153.6, 153.1, 152.1, 138.7, 138.1, 134.4, 130.8, 128.3 (2C), 127.2, 126.7, 125.5, 122.8, 122.3, 122.1 (2C). HRMS (ESI) m/z: calculated for C₁₈H₁₂N₃O₂S₂ [M + H]⁺: 366.0365, found: 366.0360.

(E)-N-(4-(Benzo[d]thiazol-2-yl)phenyl)-1-(thiazol-4-yl)methanimine (1s). Golden solid; yield 69%. Mp 173–174 °C. ¹H NMR (400 MHz, chloroform-d) δ 8.93 (s, 1H, ArH), 8.72 (s, 1H, –CH=N–), 8.17–8.10 (m, 3H, ArH), 8.07 (d, J = 8.2 Hz, 1H, ArH), 7.91 (d, J = 8.0 Hz, 1H, ArH), 7.53–7.47 (m, 1H, ArH), 7.42–7.36 (m, 3H, ArH). ¹³C NMR (100 MHz, chloroform-d) δ 167.5, 154.5, 154.2, 154.1, 153.7, 153.6, 135.1, 131.7, 128.6 (2C), 126.4, 125.2, 123.2, 122.3, 121.7 (2C), 121.6. HRMS (ESI) m/z: calculated for C₁₇H₁₁N₃NaS₂ [M + Na]⁺: 344.0287, found: 344.0275.

(E)-N-(4-(Benzo[d]thiazol-2-yl)phenyl)-1-(thiazol-5-yl)methanimine (1t). Yellow solid; yield 72%. Mp 180–182 °C. ¹H NMR (400 MHz, chloroform-d) δ 8.95 (s, 1H, ArH), 8.71 (s, 1H, –CH=N–), 8.29 (s, 1H, ArH), 8.13 (d, J = 8.2 Hz, 2H, ArH), 8.07 (d, J = 8.2 Hz, 1H, ArH), 7.91 (d, J = 8.0 Hz, 1H, ArH), 7.53–7.47 (m, 1H, ArH), 7.41–7.37 (m, 1H, ArH), 7.32 (d, J = 8.2 Hz, 2H, ArH). ¹³C NMR (100 MHz, chloroform-d) δ 167.3, 157.0, 154.2, 153.0, 151.3, 147.8, 138.3, 135.0, 131.9, 128.6 (2C), 126.4, 125.2, 123.2, 121.7, 121.6 (3C). HRMS (ESI) m/z: calculated for C₁₇H₁₂N₃S₂ [M + H]⁺: 322.0467, found: 322.0471.

(E)-N-(4-(Benzo[d]thiazol-2-yl)phenyl)-1-(thiazol-2-yl)methanimine (1u). Light green solid; yield 64%. Mp 193–195 °C. ¹H NMR (400 MHz, chloroform-d) δ 8.75 (s, 1H, –CH=N–), 8.15 (d, J = 8.1 Hz, 2H, ArH), 8.08 (d, J = 8.2 Hz, 1H, ArH), 8.03 (d, J = 3.1 Hz, 1H, ArH), 7.91 (d, J = 8.0 Hz, 1H, ArH), 7.55 (d, J = 3.0 Hz, 1H, ArH), 7.53–7.47 (m, 1H, ArH), 7.42–7.36 (m, 3H, ArH). ¹³C NMR (100 MHz, chloroform-d) δ 167.2, 166.9, 154.2, 153.8, 152.1, 144.8, 135.1, 132.5, 128.7 (2C), 126.4, 125.3, 123.2, 123.0, 121.9 (2C), 121.7. HRMS (ESI) m/z: calculated for C₁₇H₁₁N₃NaS₂ [M + Na]⁺: 344.0287, found: 344.0271.

(E)-N-(4-(Thiazolo[4,5-b]pyridin-2-yl)phenyl)-1-(thiophen-3-yl)methanimine (4a). Light yellow solid; yield 32%. Mp 176–178 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.72–8.68 (m, 2H, ArH and –CH=N–), 8.65 (d, J = 8.0 Hz, 1H, ArH), 8.27 (s, 1H, ArH), 8.20 (d, J = 8.1 Hz, 2H, ArH), 7.73–7.69 (m, 2H, ArH), 7.50–7.45 (m, 4.7 Hz, 1H, ArH), 7.42 (d, J = 8.1 Hz, 2H, ArH). ¹³C NMR (100 MHz, DMSO-d₆) δ 170.1, 164.2, 156.5, 154.9, 148.4, 140.3, 133.1, 131.8, 129.7, 128.4 (2C), 128.3, 128.0, 125.4, 122.0 (2C), 120.3. HRMS (ESI) m/z: calculated for C₁₇H₁₁N₃NaS₂ [M + Na]⁺: 344.0287, found: 344.0289.

(E)-N-(4-(4,5,6,7-Tetrahydrobenzo[d]thiazol-2-yl)phenyl)-1-(thiophen-3-yl) methanimine (4b). Khaki solid; yield 60%. Mp 150–151 °C. ¹H NMR (400 MHz, chloroform-d) δ 8.49 (s, 1H, –CH=N–), 7.91 (d, J = 8.2 Hz, 2H, ArH), 7.82 (s, 1H, ArH), 7.69 (d, J = 5.1 Hz, 1H, ArH), 7.41–7.37 (m, 1H, ArH), 7.22 (d, J = 8.2 Hz, 2H, ArH), 2.87–2.79 (m, 4H, CH₂), 1.94–1.86 (m, 4H, CH₂). ¹³C NMR (100 MHz, chloroform-d) δ 163.1, 153.5, 151.9, 150.4, 139.7, 130.8, 129.6, 128.0, 126.1 (2C), 125.8, 124.9, 120.3 (2C), 25.9, 22.7, 22.4, 22.0. HRMS (ESI) m/z: calculated for C₁₈H₁₇N₂S₂⁺[M + H]⁺: 325.0828, found: 325.0814.

(E)-N-(4-(Benzo[d]imidazo[2,1-b]thiazol-2-yl)phenyl)-1-(thiophen-3-yl) methanimine (4c). Khaki solid; yield 38%. Mp 162–163 °C. ¹H NMR (400 MHz, chloroform-d) δ 8.54 (s, 1H, –CH=N–), 7.98 (s, 1H, ArH), 7.90 (d, J = 8.1 Hz, 2H, ArH), 7.81 (s, 1H, ArH), 7.73–7.68 (m, 2H, ArH), 7.62 (d, J = 8.1 Hz, 1H, ArH), 7.49–7.43 (m, 1H, ArH), 7.40–7.32 (m, 2H, ArH), 7.28 (d, J = 8.1 Hz, 2H, ArH). ¹³C NMR (100 MHz, chloroform-d) δ 153.0, 150.1, 147.1, 146.4, 139.9, 131.1, 130.6, 129.3, 129.2, 125.7, 125.2, 125.0 (2C), 124.9, 123.8, 123.4, 120.3 (2C), 111.6, 105.6. HRMS (ESI) m/z: calculated for C₂₀H₁₄N₃S₂ [M + H]⁺: 360.0624, found: 360.0609.

(E)-N-(4-(Imidazo[1,2-a]pyridin-2-yl)phenyl)-1-(thiophen-3-yl)methanimine (4d). Light yellow solid; yield 34%. Mp 203–205 °C. ¹H NMR (400 MHz, chloroform-d) δ 8.53 (s, 1H, –CH=N–), 8.10 (d, J = 6.8 Hz, 1H, ArH), 7.98 (d, J = 8.5 Hz, 2H, ArH), 7.85 (s, 1H, ArH), 7.80 (s, 1H, ArH), 7.70 (d, J = 5.1 Hz, 1H, ArH), 7.62 (d, J = 9.1 Hz, 1H, ArH), 7.40–7.36 (m, 1H, ArH), 7.28 (d, J = 8.3 Hz, 2H, ArH), 7.19–7.13 (m, 1H, ArH), 6.79–6.74 (m, 1H, ArH). ¹³C NMR (100 MHz, chloroform-d) δ 153.1, 150.6, 144.7, 144.5, 139.8, 130.5, 129.2, 125.8 (2C), 125.7, 124.9, 124.5, 123.6, 120.3 (2C), 116.5, 111.4, 106.9. HRMS (ESI) m/z: calculated for C₁₈H₁₄N₃S [M + H]⁺: 304.0903, found: 304.0897.

(E)-N-(4-(7-Fluorobenzo[d]thiazol-2-yl)phenyl)-1-(thiophen-3-yl)methanimine (4e). Light yellow solid; yield 38%. Mp 166–167 °C. ¹H NMR (400 MHz, chloroform-d) δ 8.50 (s, 1H, –CH=N–), 8.12 (d, J = 8.5 Hz, 2H, ArH), 7.88–7.85 (m, 2H, ArH), 7.71 (d, J = 5.1 Hz, 1H, ArH), 7.48–7.40 (m, 2H, ArH), 7.30 (d, J = 8.5 Hz, 2H, ArH), 7.13–7.09 (m, 1H, ArH). ¹³C NMR (100 MHz, chloroform-d) δ 167.6 (d, J = 1.7 Hz), 156.1 (d, J = 250.1 Hz), 156.0 (d, J = 2.5 Hz), 154.2, 153.8, 139.5, 130.2, 129.5, 127.7 (2C), 126.2 (d, J = 7.3 Hz), 125.9, 124.9, 121.1 (d, J = 16.5 Hz), 120.5 (2C), 117.9 (d, J = 3.5 Hz), 109.5 (d, J = 18.7 Hz). HRMS (ESI) m/z: calculated for C₁₈H₁₂FN₂S₂ [M + H]⁺: 339.0420, found: 339.0418.

(E)-N-(4-(6-Fluorobenzo[d]thiazol-2-yl)phenyl)-1-(thiophen-3-yl)methanimine (4f). Light yellow solid; yield 52%. Mp 187–189 °C. ¹H NMR (400 MHz, chloroform-d) δ 8.50 (s, 1H, –CH=N–), 8.08 (d, J = 8.3 Hz, 2H, ArH), 8.00 (dd, J = 8.8, 4.8 Hz, 1H, ArH), 7.86 (s, 1H, ArH), 7.70 (d, J = 4.9 Hz, 1H, ArH), 7.60–7.56 (m, 1H, ArH), 7.43–7.39 (m, 1H, ArH), 7.29 (d, J = 8.3 Hz, 2H, ArH), 7.25–7.19 (m, 1H, ArH). ¹³C NMR (100 MHz, chloroform-d) δ 166.3 (d, J = 3.3 Hz), 159.4 (d, J = 245.7 Hz), 154.1, 153.5, 149.8 (d, J = 1.8 Hz), 139.5, 135.0 (d, J = 11.1 Hz), 130.1, 129.8, 127.4 (2C), 125.9, 124.9, 122.9 (d, J = 9.2 Hz), 120.5 (2C), 113.9 (d, J = 24.6 Hz), 106.8 (d, J = 26.8 Hz). HRMS (ESI) m/z: calculated for C₁₈H₁₂FN₂S₂ [M + H]⁺: 339.0420, found: 339.0431.

(E)-N-(4-(5-Fluorobenzo[d]thiazol-2-yl)phenyl)-1-(thiophen-3-yl)methanimine (4g). Khaki solid; yield 51%. Mp 183–185 °C. ¹H NMR (400 MHz, chloroform-d) δ 8.49 (s, 1H, –CH=N–), 8.09 (d, J = 8.5 Hz, 2H, ArH), 7.85 (dd, J = 3.0, 1.2 Hz, 1H, ArH), 7.81 (dd, J = 8.8, 5.1 Hz, 1H, ArH), 7.73 (dd, J = 9.6, 2.5 Hz, 1H, ArH), 7.70 (d, J = 5.1 Hz, 1H, ArH), 7.40 (dd, J = 5.1, 2.9 Hz, 1H, ArH), 7.28 (d, J = 8.5 Hz, 2H, ArH), 7.17–7.13 (m, 1H, ArH). ¹³C NMR (100 MHz, chloroform-d) δ 169.0, 160.9 (d, J = 243.2 Hz), 154.1, 154.1 (d, J = 12.1 Hz), 153.6, 139.5, 130.1, 129.8, 129.3 (d, J = 1.9 Hz), 127.5 (2C), 125.9, 124.9, 121.2 (d, J = 9.9 Hz), 120.5 (2C), 112.7 (d, J = 25.0 Hz), 108.2 (d, J = 23.6 Hz). HRMS (ESI) m/z: calculated for C₁₈H₁₂FN₂S₂ [M + H]⁺: 339.0420, found: 339.0423.

(E)-N-(4-(4-Fluorobenzo[d]thiazol-2-yl)phenyl)-1-(thiophen-3-yl)methanimine (4h). Yellow solid; yield 36%. Mp 153–154 °C. ¹H NMR (400 MHz, chloroform-d) δ 8.50 (s, 1H, –CH=N–), 8.15 (d, J = 8.6 Hz, 2H, ArH), 7.86 (dd, J = 2.9, 1.1 Hz, 1H, ArH), 7.70 (dd, J = 5.1, 0.9 Hz, 1H, ArH), 7.66 (dd, J = 8.0, 0.8 Hz, 1H, ArH), 7.41 (dd, J = 5.0, 2.6 Hz, 1H, ArH), 7.36–7.29 (m, 1H, ArH), 7.28 (d, J = 8.6 Hz, 2H, ArH), 7.19 (ddd, J = 10.4, 8.1, 0.8 Hz, 1H, ArH). ¹³C NMR (100 MHz, chloroform-d) δ 167.1, 154.8 (d, J = 257.2 Hz), 154.2, 153.7, 142.0 (d, J = 13.4 Hz), 139.5, 136.6 (d, J = 3.6 Hz), 130.1, 129.6, 127.8 (2C), 125.9, 124.9, 124.7 (d, J = 7.1 Hz), 120.5 (2C), 116.3 (d, J = 4.3 Hz), 111.0 (d, J = 18.0 Hz). HRMS (ESI) m/z: calculated for C₁₈H₁₂FN₂S₂ [M + H]⁺: 339.0420, found: 339.0417.

(E)-N-(4-(6-Ethoxybenzo[d]thiazol-2-yl)phenyl)-1-(thiophen-3-yl)methanimine (4i). Yellow solid; yield 32%. Mp 181–183 °C. ¹H NMR (400 MHz, chloroform-d) δ 8.51 (s, 1H, –CH=N–), 8.06 (d, J = 8.2 Hz, 2H, ArH), 7.93 (d, J = 8.9 Hz, 1H, ArH), 7.85 (s, 1H, ArH), 7.70 (d, J = 4.8 Hz, 1H, ArH), 7.41 (s, 1H, ArH), 7.34 (s, 1H, ArH), 7.29 (d, J = 8.2 Hz, 2H, ArH), 7.08 (d, J = 9.4 Hz, 1H, ArH), 4.11 (q, J = 6.9 Hz, 2H, CH₂), 1.47 (t, J = 6.9 Hz, 3H, CH₃). ¹³C NMR (100 MHz, chloroform-d) δ 164.0, 156.0, 153.9, 152.9, 147.7, 139.6, 135.3, 130.3, 129.9, 127.2 (2C), 125.9, 124.9, 122.5, 120.4 (2C), 115.0, 103.9, 63.1, 13.8. HRMS (ESI) m/z: calculated for C₂₀H₁₆N₂NaOS₂ [M + Na]⁺: 387.0596, found: 387.0613.

(E)-N-(4-(6-Methylbenzo[d]thiazol-2-yl)phenyl)-1-(thiophen-3-yl)methanimine (4j). Golden solid; yield 37%. Mp 191–192 °C. ¹H NMR (400 MHz, chloroform-d) δ 8.50 (s, 1H, –CH=N–), 8.10 (d, J = 8.3 Hz, 2H, ArH), 7.94 (d, J = 8.3 Hz, 1H, ArH), 7.85 (s, 1H, ArH), 7.72–7.68 (m, 2H, ArH), 7.43–7.38 (m, 1H, ArH), 7.32–7.27 (m, 3H, ArH), 2.50 (s, 3H, CH₃). ¹³C NMR (100 MHz, chloroform-d) δ 166.6, 155.0, 154.2, 152.3, 140.6, 135.3, 135.2, 131.3, 131.0, 128.4 (2C), 127.9, 126.9, 125.9, 122.6, 121.5 (2C), 121.4, 21.6. HRMS (ESI) m/z: calculated for C₁₉H₁₅N₂S₂ [M + H]⁺: 335.0671, found: 335.0678.

(E)-N-Methyl-2-(4-((thiophen-3-ylmethylene)amino)phenyl)benzo[d]thiazol-6-amine (4k). Yellow solid; yield 59%. Mp 200–201 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.67 (s, 1H, –CH=N–), 8.23 (s, 1H, ArH), 7.99 (d, J = 8.2 Hz, 2H, ArH), 7.72 (d, J = 8.8 Hz, 1H, ArH), 7.70–7.68 (m, 1H, ArH), 7.65–7.64 (m, 1H, ArH), 7.35 (d, J = 8.2 Hz, 2H, ArH), 7.04 (s, 1H, ArH), 6.81 (d, J = 8.8 Hz, 1H, ArH), 6.11 (q, J = 5.0 Hz, 1H, NH), 2.75 (d, J = 4.9 Hz, 3H, CH₃). ¹³C NMR (100 MHz, DMSO-d₆) δ 160.0, 155.7, 152.9, 148.4, 145.0, 140.4, 136.7, 132.6, 131.0, 127.8, 127.4 (2C), 125.4, 122.9, 121.8 (2C), 114.3, 100.3, 29.9. HRMS (ESI) m/z: calculated for C₁₉H₁₆N₃S₂ [M + H]⁺: 350.0780, found: 350.0757.

(E)-N,N-Dimethyl-2-(4-((thiophen-3-ylmethylene)amino)phenyl)benzo[d]thiazol-6-amine (4l). Orange solid; yield 60%. Mp 177–178 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.68 (s, 1H, –CH=N–), 8.24 (s, 1H, ArH), 8.02 (d, J = 8.6 Hz, 2H, ArH), 7.83 (d, J = 9.1 Hz, 1H, ArH), 7.71–7.69 (m, 1H, ArH), 7.65–7.64 (m, 1H, ArH), 7.36 (d, J = 8.5 Hz, 2H, ArH), 7.32 (s, 1H, ArH), 7.01 (d, J = 9.0 Hz, 1H, ArH), 3.00 (s, 6H, CH₃). ¹³C NMR (100 MHz, DMSO-d₆) δ 161.1, 155.8, 153.1, 148.7, 145.3, 140.4, 136.6, 132.7, 130.8, 127.9, 127.5 (2C), 125.4, 122.8, 1218 (2C), 113.3, 103.1, 40.5. HRMS (ESI) m/z: calculated for C₂₀H₁₈N₃S₂⁺[M + H]⁺: 364.0937, found: 364.0948.

(E)-2-(4-((Thiophen-3-ylmethylene)amino)phenyl)benzo[d]thiazole-6-carboxylic acid (4m). Yellow solid; yield 86%. Mp: >220 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 13.16 (br, 1H, COOH), 8.77 (s, 1H, –CH=N–), 8.68 (s, 1H, ArH), 8.26 (s, 1H, ArH), 8.15 (d, J = 8.1 Hz, 2H, ArH), 8.12–8.06 (m, 2H, ArH), 7.71–7.69 (m, 1H, ArH), 7.66–7.64 (m, 1H, ArH), 7.40 (d, J = 8.1 Hz, 2H, ArH). ¹³C NMR (100 MHz, DMSO-d₆) δ 170.4, 166.9, 156.4, 156.4, 154.6, 140.3, 134.5, 133.1, 129.8, 128.6 (2C), 127.9, 127.5, 127.5, 125.4, 124.3, 122.4, 121.9 (2C). HRMS (ESI) m/z: calculated for C₁₉H₁₃N₂O₂S₂ [M + H]⁺: 365.0413, found: 365.0428.

(E)-N-Methyl-2-(4-((thiophen-3-ylmethylene)amino)phenyl)benzo[d]thiazole-6- carboxamide (4n). Yellow solid; yield 72%. Mp: >220 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.69 (s, 1H, –CH=N–), 8.61–8.55 (m, 2H, ArH and –(C=O)NH–), 8.26 (s, 1H, ArH), 8.15 (d, J = 8.5 Hz, 2H, ArH), 8.09 (d, J = 8.5 Hz, 1H, ArH), 7.98 (d, J = 8.5 Hz, 1H, ArH), 7.72–7.68 (m, 1H, ArH), 7.66–7.65 (m, 1H, ArH), 7.40 (d, J = 8.5 Hz, 2H, ArH), 2.84 (d, J = 4.4 Hz, 3H, CH₃). ¹³C NMR (100 MHz, DMSO-d₆) δ 169.2, 166.0, 156.4, 155.2, 154.5, 140.3, 134.4, 133.0, 131.5, 129.9, 128.5 (2C), 127.9, 125.6, 125.4, 122.2, 121.9 (2C), 121.6, 26.4. HRMS (ESI) m/z: calculated for C₂₀H₁₅N₃NaOS₂ [M + Na]⁺: 400.0549, found: 400.0565.

(E)-N-(4-(Benzo[d]thiazol-2-yloxy)phenyl)-1-(thiophen-3-yl)methanimine (4o). White solid; yield 18%. Mp 95–97 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.66 (s, 1H, –CH=N–), 8.21 (s, 1H, ArH), 7.94 (d, J = 8.0 Hz, 1H, ArH), 7.73–7.67 (m, 2H, ArH), 7.66–7.61 (m, 1H, ArH), 7.49 (d, J = 8.4 Hz, 2H, ArH), 7.46–7.41 (m, 1H, ArH), 7.38–7.30 (m, 3H, ArH). ¹³C NMR (100 MHz, DMSO-d₆) δ 171.9, 155.8, 152.1, 149.9, 148.6, 140.4, 132.4, 131.8, 127.8, 126.5, 125.4, 124.2, 122.4 (2C), 122.2, 121.7 (2C), 121.1. HRMS (ESI) m/z: calculated for C₁₈H₁₃N₂OS₂ [M + H]⁺: 337.0464, found: 337.0474.

(E)-N-(4-(Benzo[d]thiazol-2-ylthio)phenyl)-1-(thiophen-3-yl)methanimine (4p). White solid; yield 20%. Mp 109–111 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.66 (s, 1H, –CH=N–), 8.27 (s, 1H, ArH), 7.94 (d, J = 8.0 Hz, 1H, ArH), 7.87–7.80 (m, 3H, ArH), 7.72–7.68 (m, 1H, ArH), 7.67–7.63 (m, 1H, ArH), 7.48–7.43 (m, 1H, ArH), 7.41–7.31 (m, 3H, ArH). ¹³C NMR (100 MHz, DMSO-d₆) δ 169.7, 156.8, 154.0, 153.5, 140.2, 136.6 (2C), 134.8, 133.1, 127.9, 126.4, 125.4, 125.1, 124.4, 122.7 (2C), 121.7, 121.3. HRMS (ESI) m/z: calculated for C₁₈H₁₃N₂S₃ [M + H]⁺: 353.0235, found: 353.0232.

Synthesis of (E)-2-((benzo[d]thiazol-2-ylimino)methyl)-6-methoxyphenol (1e)

The catalytic amount of acetic acid was added to a solution of 3e (0.5 mmol) and 2-hydroxy-3-methoxybenzaldehyde (0.6 mmol) in MeOH (4 mL), the mixture was stirred at reflux for 2 h. The resulting precipitate was isolated by suction filtration and washed with MeOH to give the pure product 1e. Orange solid; yield 32%. Mp 170–172 °C. ¹H NMR (400 MHz, chloroform-d) δ 12.44 (s, 1H, OH), 9.26 (s, 1H, –CH=N–), 7.96 (d, J = 8.2 Hz, 1H, ArH), 7.83 (d, J = 8.0 Hz, 1H, ArH), 7.51–7.45 (m, 1H, ArH), 7.39–7.33 (m, 1H, ArH), 7.13 (d, J = 7.9 Hz, 1H, ArH), 7.06 (d, J = 8.0 Hz, 1H, ArH), 6.95–6.89 (m, 1H, ArH), 3.93 (s, 3H, –CH₃). ¹³C NMR (100 MHz, chloroform-d) δ 167.9, 166.4, 151.1, 150.5, 147.5, 133.7, 125.7, 124.3, 124.2, 122.0, 120.7, 118.3, 117.4, 115.8, 55.3. HRMS (ESI) m/z: calculated for C₁₅H₁₂N₂NaO₂S [M + Na]⁺: 307.0512, found: 307.0509.

Synthesis of 2-(((4-(benzo[d]thiazol-2-yl)phenyl)amino)methyl)-6-methoxy-phenol (1f)

NaBH₄ (0.84 mmol) was added to a suspension of 1a (0.42 mmol) in MeOH (6 mL) and stirred at room temperature for 1 h. Upon completion, MeOH was removed under reduced pressure. The crude mixture was then purified by flash column chromatography in a mixture of petroleum ether and ethyl acetate to yield the pure product 1f. Light yellow solid; yield 66%. Mp 187–188 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.79 (s, 1H, OH), 8.00 (d, J = 7.4 Hz, 1H, ArH), 7.89 (d, J = 8.1 Hz, 1H, ArH), 7.78 (d, J = 8.4 Hz, 2H, ArH), 7.46–7.43 (m, 1H, ArH), 7.35–7.31 (m, 1H, ArH), 6.88–6.84 (m, 2H, ArH and NH), 6.82–6.80 (m, 1H, ArH), 6.73–6.68 (m, 3H, ArH), 4.30 (d, J = 5.9 Hz, 2H, CH₂), 3.80 (s, 3H, CH₃). ¹³C NMR (100 MHz, DMSO-d₆) δ 168.0, 153.9, 151.6, 147.3, 143.8, 133.7, 128.6, 126.2, 125.5, 124.3, 121.8, 121.7, 120.0, 120.0, 118.6, 111.9, 110.4, 55.8, 40.8. HRMS (ESI) m/z: calculated for C₂₁H₁₉N₂O₂S [M + H]⁺: 363.1162, found: 363.1190.

Synthesis of N-(4-(benzo[d]thiazol-2-yl)phenyl)-2-hydroxy-3-methoxybenzamide (1g)

2-Hydroxy-3-methoxybenzoyl chloride (0.53 mmol) was dissolved in Et₂O (0.5 mL) and then added dropwise to a stirring solution of 3a (0.44 mmol) and NaHCO₃ (0.88 mmol) in Et₂O (1 mL) and H₂O (0.5 mL) at 0 °C. Then the reaction was allowed to warm to room temperature and stirred for 12 h. The resulting precipitate was isolated by suction filtration and washed with H₂O (2 mL), 2 N HCl (2 mL) and H₂O (2 mL) sequentially to yield the pure product 1g. White solid; yield 14%. Mp 215–216 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 11.33 (s, 1H, OH), 10.60 (s, 1H, –(C=O)NH–), 8.13–8.09 (m, 3H, ArH), 8.04 (d, J = 8.2 Hz, 1H, ArH), 7.93 (d, J = 8.4 Hz, 2H, ArH), 7.53 (d, J = 7.8 Hz, 2H, ArH), 7.47–7.42 (m, 1H, ArH), 7.18 (d, J = 8.0 Hz, 1H, ArH), 6.96–6.90 (m, 1H, ArH), 3.84 (s, 3H, CH₃). ¹³C NMR (100 MHz, DMSO-d₆) δ 167.0, 166.8, 153.6, 148.4, 148.3, 141.1, 134.3, 128.4, 127.9 (2C), 126.6, 125.3, 122.6, 122.2, 120.9 (2C), 120.1, 118.6, 118.0, 115.5, 56.0. HRMS (ESI) m/z: calculated for C₂₁H₁₆N₂NaO₃S [M + Na]⁺: 399.0774, found: 399.0774.

Plasmid and pseudovirus production

To produce MERS spike protein pseudotyped HIV virions, we constructed a MERS spike protein (MERS-S) expression plasmid. The codon-optimized gene of full-length S protein of MERS-CoV (Genebank: AFS88936), with replacement of the N-terminal signal peptide (aa 1–17) with CD5 signal sequence, were synthesized (Inovogen, China) and insertion into pcDNA3.1 vector, verified by sequencing. A HIV backbone vector pNL4-3. Luc.R-E- was used for pseudovirus package. 293T cells were co-transfected with MERS spike protein expression plasmid plus pNL4.3LucR-E- plasmid using X-tremeGENE DNA HP Transfection Reagent (Roche) according the manufacturer's instructions.²⁹ At 72 h post-transfection, supernatants were harvested and filtered through 0.45 μm filter, the filtered supernatant was further concentrated 10 folds by ultracentrifugation with 100 kDa cut-off, aliquoted and stored at −80 °C as stock vial. The p24 level of pseudovirus stock solution was determined by ELISA kit according to the manual (ab218268, Abcam).

Compound library and HTS screening

Compound library samples were orthogonally pooled as mixtures of 10 compounds per well at 2 μg mL⁻¹ each, with duplicate representation for each compound. This bidirectional orthogonal pooling strategy allows for greater screening efficiency and throughput for large compound libraries. The pooling of compound library was prepared as described previously.⁵²

Test compounds were freshly prepared from initial dimethyl sulfoxide (DMSO) stocks, 80 cocktails per plate. Huh-7 cells were then trypsinized, counted and seeded at a final concentration of 104 cells per well. Plates were placed overnight in a CO₂ incubator. The following day, samples were then added with an automatic liquid handler (FX from Beckman–Coulter). Finally, the pseudotyped virus, thawed and diluted immediately prior to use in cell culture medium, was added under a BSL-2 hood using Hydra (Thermo Fisher Scientific). The final concentration of DMSO in all wells was maintained at 2%. The plates were incubated at 37 °C in a humidified CO₂ incubator for 48 h. Bright-Glo substrate (Promega) was added directly to each well and cell lysis was allowed to proceed in the dark for 5 min.

Cytotoxic effect of compounds was assayed by MTT method which evaluated the reduction product of MTT by cellular succinate dehydrogenase as an indicator of cell liability. Toxicity was calculated as 100% minus percentage of OD_@570 readout each well against the control well with no drug, 100% − OD_@570 (compound)/OD_@570 (control) × 100%.

Luciferase activity was measured using the Envision microplate luminometer (PerkinElmer). Inhibition was calculated as 100% minus percentage of luciferase readout each well against the control well with no drug, 100% − Luc (compound)/Luc (control) × 100%.

Measurement of IC₅₀ and CC₅₀

For determining CC₅₀ and IC₅₀, compounds were serially diluted and assayed as described for the HTS screening, CC₅₀ and IC₅₀ were determined by regression analysis using 4 parameter regression.

Conflicts of interest

There are no conflicts of interest to declare.

Acknowledgements

This work was supported by the National Key Research and Development Program of China (2018YFC1200604). The pNL4.3.Luc.R-.E- and MERS-S expression plasmid was kindly gifted by Dr Shibo Jiang and Dr Lu Lu from Fudan University.

Notes and references

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: 10.1039/d0ra08442e

‡ These authors contributed equally to this work.

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