Design and synthesis of novel carbazolo–thiazoles as potential anti-mycobacterial agents using a molecular hybridization approach

Abstract

Various substituted carbazolo–thiazoles (compounds 6a–6o) were synthesized in good yields using a molecular hybridization approach. The synthesized compounds were evaluated for their in vitro anti-tubercular activity against Mycobacterium tuberculosis H₃₇Rv strain at the National Institute of Allergy and Infectious Diseases (Bethesda, MD, USA). Among the tested series, compound 6c (minimum inhibitory concentration 21 μM) showed the most promising anti-mycobacterial activity. Brief structure–activity relationship studies showed that the electron-donating groups (OCH₃ and OH), particularly on the phenyl ring of the thiazole motif, had a positive correlation with the anti-mycobacterial activity. In addition, they displayed low cytotoxicity against a mammalian Vero cell line using the MTT assay, thereby having a high therapeutic index. This study shows the importance of molecular hybridization and the scope for the development of carbazole–thiazole compounds as potential anti-mycobacterial agents.

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1 Introduction

Tuberculosis is the leading cause of death from a single infectious agent and is responsible for more than three million deaths worldwide each year.¹ Despite half a century of anti-tubercular chemotherapy, there are still 8–10 million new cases of active tuberculosis each year and nearly two billion people are believed to harbor latent tuberculosis. Along with the spread of HIV infection,² tuberculosis is today a universal threat to health. India, China, the Russian Federation and South Africa have almost 60% of the world's cases of multi-drug resistant tuberculosis.³ It is estimated that more than 1000 people die each day from the disease.⁴ The combination of a long duration of treatment (6–9 months), the increased incidence of (multi- or extensive) drug resistance, co-morbidity with HIV-AIDS and a lack of investment in anti-infective drug discoveries has led to a situation in which the discovery, development and introduction of new treatments for tuberculosis has become critical.⁵ The current situation requires the re-engineering and repositioning of old drug families to develop new anti-mycobacterial treatments to achieve effective control of tuberculosis, including the resistant forms of TB.^6–8

Natural products or their direct derivatives play a crucial part in the modern day chemotherapy of tuberculosis.⁹ Promising natural anti-mycobacterial agents include the carbazole compounds (I–III) and carbazole alkaloids, such as clausine (IV) and micromeline (V), isolated independently from several sources¹⁰ (Fig. 1). Synthetic analogs of natural carbazole alkaloids show significantly improved in vitro as well as in vivo anti-mycobacterial activity against Mycobacterium tuberculosis H₃₇Rv.^11–15 In addition, thiazole conjugated with a wide range of heterocyclic moieties (VII–IX) has been reported to show potent anti-mycobacterial activity^16,17 (Fig. 1). In view of these facts, and as a part of our continuing studies in the area of anti-mycobacterial agents,^18–21 we aimed to synthesize biologically active hybrid pharmacophores (carbazole and thiazole) by integrating them into one molecular platform for biological evaluation. This approach was an attempt to investigate the possible synergistic influence of these structural hybridizations on the anticipated anti-mycobacterial activity in the hope of discovering a new lead molecule. We report here the synthesis and in vitro anti-mycobacterial activity of some novel carbazolo–thiazole hybrid molecules (6a–6o) against M. tuberculosis H₃₇Rv and their in vitro cytotoxicity profiles.

Fig. 1 Previously reported derivatives of carbazoles and thiazoles along with their anti-mycobacterial activities.^11–17 Compound 6c was the most promising anti-mycobacterial compound among the compounds synthesized in this work.

2 Chemistry

A novel series of thiazolyl-substituted carbazole hydrazine analogues (6a–6o) was synthesized using an efficient and versatile synthetic route (Scheme 1). It is clear that unique final steps were involved in the synthesis of the target compounds, which have structural variations on the hydrazine bond attached to the 3^rd position on the carbazole ring. The compounds, such as 9-methyl-carbazole (1), were prepared from commercially available carbazole by an N-methylation process using iodo-methane and sodium hydride. Compound 1 was subjected to a formylation reaction (Vilsmayer–Hack reaction) in the presence of POCl₃ and DMF to give 9-methyl-carbazole-3-carbaldehyde (2), which was further treated with thiosemicarbazide in the presence of methanol and catalytic glacial acetic acid to yield 1-(9-methyl-carbazol-6-yl methylene)thiosemicarbazide (3). The desired compounds 6a–6o were synthesized by a Hantzsch cyclo-condensation reaction of compound 3 with various appropriately substituted α-bromoaromatic/heteroaromatic ketones (5a–5o) in the presence of methanol.²² The structures of the key intermediate compound 3 and its corresponding thiazolyl-substituted carbazole hydrazine derivatives (6a–6o) were established on the basis of their physicochemical and spectral (IR, ¹H-NMR, ¹³C-NMR and HRMS) data.

Scheme 1 Synthetic outline of a novel series of thiazolyl-substituted carbazole hydrazine analogues 6(a–o). Reagents and conditions: (i) DMF, NaH, CH₃I, RT, 3h; (ii) POCl₃, DMF, 4 h reflux; (iii) thiosemicarbazide, AcOH, methanol, reflux, 2 h; (iv) Br₂, CHCl₃/AcOH, RT; (v) CuBr₂, EtOAc, CHCl₃, reflux, 12 h; (vi) compound 3, α-bromoaromatic/heteroaromatic ketones (a–o), methanol, reflux, 3 h.

3 Results and discussion

3.1 Spectral studies

All the newly synthesized compounds exhibited acceptable analyses of their anticipated structures, which are summarized in the experimental section. In general, the IR spectrum of compound 3 revealed typical absorption bands around 3373.23 cm⁻¹ for N–H (of the NH₂ group), 1619.92 cm⁻¹ for C=N and 1145.80 cm⁻¹ for the C=S groups. This structure was further substantiated from the ¹H-NMR spectrum of compound 3, which showed the presence of singlet signals around δ 11.38, 8.58, 8.21 and 3.89 ppm for the N–H proton of CH=N, the 4^th proton of the carbazole ring and the N–CH₃ protons, indicating its formation by a simple carbon–nitrogen bond formation process in the presence of thiosemicarbazide and acetic acid as a catalyst.²² The IR spectrum of compounds 6a–6o showed the disappearance of the typical peaks due to the NH₂ group (N–H Str.) and the appearance of moderately strong bands around 3319–3074 cm⁻¹ and 1629.47–1572.25 cm⁻¹, which are characteristic of the N–H and C=N groups, respectively. In the case of compounds 6i and 6j, the peaks appeared around 3454.28–3342.53 cm⁻¹ as a result of the OH group, while the prominent bands around 1721.35–1711.27 cm⁻¹ were attributed to C=O in compounds 6n and 6o.

The ¹H-NMR spectrum (400 MHz) of compounds 6a–6o recorded in DMSO-d₆ displayed some distinctive singlet signals around δ 12.59–8.38 ppm for N–H protons, δ 8.45–8.19 ppm for the CH=N proton at the 3^rd position of the carbazole motif and at δ 8.36–8.20 ppm for an aromatic proton at the 4^th position of the carbazole nucleus. The most informative singlet signal resonated between δ 7.92 and 7.20 ppm and was attributed to the aromatic proton (H-5) of the thiazole ring, indicating its formation through a cyclo-condensation process. This observation was consistent with similar previously reported compounds.²² The unique singlet signal resonating around δ 3.91–3.87 ppm indicated the presence of methyl protons (N–CH₃) on the 9^th position of the carbazole ring. Various aromatic/heteroaromatic proton signals were observed between δ 8.31 and 6.92 ppm.

These findings were further substantiated from the respective ¹³C-NMR spectra of the compounds. The characteristic ¹³C-NMR signals observed at around δ 148.72–141.12 and 110.21–101.48 ppm were assigned to carbons of the Schiff base (CH=N) and C-5 of the thiazole ring. A prominent carbon signal observed around δ 29.65–28.62 ppm indicated the presence of the N–CH₃ carbon in the title compounds. In case of compounds 6n and 6o, a characteristic signal appeared around δ 167.86–158.75 ppm as a result of the presence of the carbonyl carbon (C=O) of the coumarin ring, whereas various aromatic/heteroaromatic carbons resonated around δ 134.76–111.08 ppm.

3.2 Anti-mycobacterial activity

In vitro evaluation of the anti-mycobacterial activity of the newly synthesized compounds 3 and 6a–6o was carried out at the Infectious Disease Research Institute within the National Institute of Allergy and Infectious Diseases (NIAID) (Bethesda, MD, USA) screening program. The minimum inhibitory concentration (MIC) was determined against M. tuberculosis strain H₃₇Rv grown under aerobic conditions using a dual read-out (OD₅₉₀ and fluorescence) assay.^23–25 The assay was based on the measurement of the growth in liquid medium of a fluorescent reporter strain of H₃₇Rv, where the read-out was either the optical density or the fluorescence. The use of two read-outs minimizes the problems caused by compound precipitation or auto-fluorescence. The purpose of the screening program was to provide a resource whereby new experimental compounds can be tested for their capacity to inhibit the growth of virulent M. tuberculosis. Table 1 gives the results of the anti-mycobacterial activity tests. All the synthesized compounds showed an interesting activity profile, with MIC values ranging from 21 to 200 μM against the tested mycobacterial strain.

Table 1 Anti-mycobacterial activity and cytotoxicity data for a novel series of thiazolyl-substituted carbazole hydrazine analogues (3, 6a–6o)

Compound	Structure	MIC^a (μM)	IC50^b (μM)	IC90^c (μM)	Cytotoxicity IC50^d (μM)
a MIC = minimum inhibitory concentration at which M. tuberculosis H37Rv growth was completely inhibited.b IC50 = concentration at which growth is inhibited by 50%.c IC90 value = concentration at which growth is inhibited by 90%.d Cytotoxicity activity was determined on mammalian Vero cell line; ND = not determined.
3		ND	1.6	5.9	202.4
6a		>200	110	160	393.0
6b		32	35	62	321.5
6c		21	28	>50	220.8
6d		>200	>200	>200	389.6
6e		>200	>200	>200	421.5
6f		>200	61	170	268.5
6g		ND	ND	ND	ND
6h		>200	>200	>200	306.0
6i		31	39	>50	440.8
6j		37	41	>200	271.1
6k		>200	>200	>200	259.9
6l		>200	>200	>200	248.5
6m		>200	>50	>50	210.2
6n		>200	>25	>25	399.2
6o		>200	>200	>200	401.7
Rifampicin	—	0.0067	0.0037	0.007	—

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We also studied the effects of various aromatic/heteroaromatic substituents at the 4^th position of the thiazole ring, which was, in turn, attached to the 3^rd position of 9-methyl carbazole through a hydrazine linkage. Of these, compound 6c (MIC = 21 μM), with 3,4-dimethoxyl (OCH₃) groups on an aromatic nucleus, showed the most promising anti-mycobacterial activity with an IC₅₀ value of 28 μM. Compound 6b (MIC = 32 μM), with an unsubstituted aromatic ring, showed good inhibitory activity with an IC₅₀ value of 35 μM. The activity was considerably affected by substituents at position 4 on the thiazole nucleus. In general, electron-donating groups on the aromatic ring contribute greatly to the anti-mycobacterial activity, whereas electron-withdrawing substituents cause a decrease in activity. It was observed that compounds 6i (MIC = 31 μM) and 6j (MIC = 37 μM), with hydroxyl (OH) groups at the 2^nd and 4^th positions of the aromatic nucleus, displayed notable activity with IC₅₀ values of 39 and 41 μM, respectively. Compounds 6a, 6d, 6e, 6f, 6h, 6k and 6l, with either nitro- or halogen groups on the aromatic ring, were the least active with MIC values >200 μM. Replacing the substituted phenyl group at the 4^th position on the thiazole ring with heterocyclic groups such as thiophene (6m) and coumarin (6n and 6o) resulted in no significant change in the anti-mycobacterial activity, with MIC values >200 μM. In three instances we replaced the aromatic ring with thiophene (6m) and coumarin (6n and 6o) ring systems, which resulted in activity with an MIC value >200 μM.

3.3 Cytotoxic activity

The synthesized title compounds (3 and 6a–6o) were further screened for cytotoxicity (IC₅₀) in a mammalian Vero cell line. After 72 h of exposure, the viability of the cells was assessed on the basis of the cellular conversion of MTT into a formazan product using the Promega Cell Titer 96 non-radioactive cell proliferation assay (Table 1). The 16 derivatives tested showed IC₅₀ values ranging from 202.4 to 440.8 μM. None of the synthesized compounds displayed significant activity against the mammalian Vero cell line at concentrations <100 μM. Among the series tested, compounds 6a, 6b, 6e, 6h, 6i, 6n and 6o had a lower toxicity with IC₅₀ values >300 μM. These results are important, as compounds with increased cytoliability are attractive in the development of new chemical entities for the treatment of tuberculosis. This is primarily because the treatment of tuberculosis requires a lengthy course of drug treatment, leading to a number of side-effects. Thus there is a need for an agent with a high margin of safety.

4 Conclusion

We have reported the synthesis, spectral studies and preliminary in vitro anti-mycobacterial activity of a novel series of thiazolyl-substituted carbazole hydrazine analogues 6a–6o using a molecular hybridization approach. A structure activity–relationship study revealed that the electron-donating groups (OCH₃ and OH) on the phenyl ring of the thiazole moiety resulted in excellent anti-mycobacterial activity, whereas the electron-withdrawing substituents decreased the activity. Among the synthesized compounds, compound 6c (MIC = 21 μM) is a promising compound with good anti-mycobacterial activity. The title compounds were also assessed for their cytotoxic activity (IC₅₀) against the mammalian Vero cell line using the MTT assay. The results indicated that these compounds showed anti-tubercular activity at non-cytotoxic concentrations. These results indicate the importance of molecular hybridization and the development of carbazole–thiazole based lead candidates as potential anti-mycobacterial agents. The anti-mycobacterial activity can further be enhanced by slight modifications in the ring substituents and/or extensive additional functionalization, which warrants further investigation. We believe that the observed outcomes could contribute to global efforts toward the discovery of new lead compounds with improved anti-mycobacterial activity.

5 Experimental

5.1 Chemistry protocol

All the chemicals used were purchased from Sigma-Aldrich and Merck Millipore, South Africa. The commercially available chemicals 4-bromo-phenacyl bromide (5a) and phenacyl bromide (5b) were purchased from Sigma-Aldrich (South Africa). All the solvents, except those of laboratory-reagent grade, were dried and purified when necessary according to previously published methods. The progress of the reactions and the purity of the compounds were monitored by thin-layer chromatography (TLC) on pre-coated silica gel plates procured from E. Merck and Co. (Darmstadt, Germany) using ethyl acetate (10%) in dichloromethane as the mobile phase and iodine vapor as the visualizing agent.

The melting points of the synthesized compounds were determined using a Thermo Fisher Scientific (IA9000, UK) digital melting point apparatus and are uncorrected. The IR spectra were recorded on a Bruker Alpha FT-IR spectrometer (Billerica, MA, USA) using the ATR technique. The ¹H-NMR and ¹³C-NMR spectra were recorded on a Bruker AVANCE 400 (Bruker, Rheinstetten/Karlsruhe, Germany) spectrometer using CDCl₃ and/or DMSO-d₆. The chemical shifts are reported in δ ppm units with respect to TMS as the internal standard. HRMS spectra were recorded on an Autospec mass spectrometer with electron impact at 70 eV.

5.1.1 Synthesis of 9-methylcarbazole (1)²⁶. NaH (0.028 mol, 1.1 equiv.) was slowly and carefully added in portions to a constantly stirred solution of carbazole (5 g, 0.025 mol, 1 equiv.) in N,N-dimethylformamide (20 mL) maintained at 0 °C; stirring was continued for 30 min. Methyl iodide (4.34 g, 0.0306 mol, 1.2 equiv.) was then added and the reaction was stirred at room temperature for 5 h. The progress of the reaction was monitored by TLC. The reaction mixture was then poured into ice-cold water and the precipitated solid was filtered and washed with cold water to yield a white solid [compound (1)]. Yield, 93.33%; melting point, 86–88 °C. FTIR (ATR, ν_max, cm⁻¹): 3048.18 (Ar–H Str.), 2953.18 (C–H Str. of CH₃). ¹H-NMR (400 MHz, CDCl₃, δ ppm): 8.13 (d, J = 7.80 Hz, 2H, Ar–H), 7.50 (m, 2H, Ar–H), 7.43 (d, J = 8.20 Hz, 2H, Ar–H), 7.25 (m, 2H, Ar–H), 3.87 (s, 3H, N–CH₃). ¹³C-NMR (100 MHz, DMSO-d₆, δ ppm): 140.97, 125.63, 122.74, 120.27, 118.79, 108.38, 29.02 (N–CH₃).

5.1.2 Synthesis of 9-methyl-carbazole-3-carbaldehyde (2)²⁶. The Vilsmeier–Haack reagent was freshly prepared by the careful addition of POCl₃ (2 mL, 0.021 mol) in DMF (8 mL, 0.103 mol) at 0 °C with constant stirring. 9-Methyl carbazole (1, 5.0 g, 0.0238 mol, 1 equiv.) was added to this reagent with continuous stirring, maintaining the temperature at 0 °C for the initial 30 min, stirring at room temperature for 2 h and finally stirring at 60 °C for another 2 h. The reaction mixture was then poured into a sodium carbonate solution and stirring was continued at 90 °C for 2 h. After cooling to room temperature, the reaction mixture was suspended in water, extracted with dichloromethane (20 mL × three times) and the combined extracts were washed with water and dried over anhydrous sodium sulphate. The residue obtained after the in vacuo removal of dichloromethane was further recrystallized from ethanol to give the desired compound 2 as an off-white crystalline solid. Yield, 75%; melting point, 74–76 °C. FTIR (ATR, ν_max, cm⁻¹): 3022 (Ar–H Str.), 2993 (C–H Str. of CH₃), 1684.01 (C=O Str.). ¹H-NMR (400 MHz, DMSO-d₆, δ ppm): 10.09 (s, 1H, CHO), 8.15 (d, 1H, J = 7.76 Hz, Ar–H), 8.02 (dd, 1H, J = 8.52 Hz, ArH), 7.53 (t, 1H, Ar–H), 7.45 (dd, 2H, J = 8.28 Hz, Ar–H), 7.34 (t, 1H, Ar–H), 3.88 (s, 3H, N–CH₃). ¹³C-NMR (100 MHz, CDCl₃, δ ppm): 191.80 (C=O), 144.51, 141.70, 128.54, 127.24, 126.76, 123.83, 122.99, 120.65, 109.11, 29.38 (N–CH₃).

5.1.3 Synthesis of 1-(9-methyl-carbazol-6-yl methylene) thiosemicarbazide (3)²². A catalytic amount of glacial acetic acid (0.15 equiv.) was added to a constantly stirred solution of compound 2 (4.0 g, 0.0191 mol, 1 equiv.) and thiosemicarbazide (1.91 g, 0.021 mol, 1.1 equiv.) in anhydrous methanol (40 mL). The reaction mixture was refluxed for 4 h. After cooling to room temperature, the separated solid was filtered and washed with cold methanol to give the off-white crystalline solid of compound 3. Yield, 81%, melting point; 225–226 °C. FTIR (ATR, ν_max, cm⁻¹): 3373.23, 3261.31 (N–H Str. of NH₂), 3174.17 (N–H Str. of NH), 3036.85 (Ar–H), 1619.92 (C=N Str.), 1145.80 (C=S). ¹H-NMR (400 MHz, DMSO-d₆, δ ppm): 11.38 (s, 1H, NH), 8.58 (s, 1H, CH=N), 8.23 (d, 1H, J = 7.76, Ar–H), 8.21 (s, 1H, H-4 of carbazole), 8.15–7.98 (s, 2H, NH₂), 7.96 (dd, 1H, J = 8.60 Hz, Ar–H), 7.62 (d, 2H, J = 8.88 Hz, Ar–H), 7.49 (t, 1H, J = 8.10 Hz, Ar–H), 7.26 (t, 1H, J = 7.48 Hz, Ar–H), 3.89 (s, 3H, N–CH₃). ¹³C-NMR (100 MHz, DMSO-d₆, δ ppm): 176.92, 143.04 (CH=N), 141.06, 140.56, 125.60, 124.60, 121.67, 119.94, 118.76, 108.86, 28.62 (N–CH₃). HR ESI m/z: calculated for C₁₅H₁₄N₄S [M + H]⁺, 282.0939; found, 283.0931.

5.1.4 General procedure for the synthesis of 2-bromo-1-substituted phenylethanone 5(c–h)²⁷. Bromine (1.2 equiv.) was added to a cooled solution of appropriately substituted acetophenones 4(c–h) (1 equiv.) in chloroform/AcOH. The solution was stirred at room temperature for 3–5 h until TLC showed that all the starting materials had been consumed. The reaction mass was poured in ice-cold water and extracted three times with 10 mL of DCM. Anhydrous Na₂SO₄ was added to the combined organic layer, filtered and the excess solvent was removed in vacuo under reduced pressure. The resultant solid/liquid obtained was washed with hexane to yield compounds 5(c–h).
5.1.4.1 2-Bromo-1-(3,4-dimethoxyphenyl)ethanone (5c). Off-white solid. Yield, 93%. ¹H-NMR (400 MHz, CDCl₃, δ ppm): 7.61–7.58 (dd, 1H, J = 8.40 Hz, Ar–H), 7.52 (s, 1H, Ar–H), 6.90 (d, 1H, J = 8.36 Hz, Ar–H), 4.39 (s, 2H, CH₂Br), 3.94 (s, 3H, OCH₃), 3.92 (s, 3H, OCH₃).
5.1.4.2 2-Bromo-1-(4-chlorophenyl)ethanone (5d). Off-white solid. Yield, 80%. ¹H-NMR (400 MHz, CDCl₃, δ ppm): 7.91 (d, 2H, J = 8.52 Hz, Ar–H), 7.48 (d, 2H, J = 8.52 Hz, Ar–H), 4.65 (s, 2H, CH₂Br).
5.1.4.3 2-Bromo-1-(4-nitrophenyl)ethanone (5e). Yellow solid. Yield, 90%. ¹H-NMR (400 MHz, CDCl₃, δ ppm): 8.35 (d, 2H, J = 8.84 Hz, Ar–H), 8.16 (d, 2H, J = 8.88 Hz, Ar–H), 4.46 (s, 2H, CH₂Br).
5.1.4.4 2-Bromo-1-(4-fluorophenyl)ethanone (5f). White solid. Yield, 75%. ¹H-NMR (400 MHz, CDCl₃,δ ppm): 8.04 (m, 2H, Ar–H), 7.14–7.20 (m, 2H, Ar–H), 4.40 (s, 2H, CH₂Br).
5.1.4.5 2-Bromo-1-(p-tolyl)ethanone (5g). White solid. Yield, 78%. ¹H-NMR (400 MHz, CDCl₃, δ ppm): 7.88 (d, 2H, J = 8.28 Hz, Ar–H), 7.29 (d, 2H, J = 8.12 Hz, Ar–H), 4.42 (s, 2H, CH₂Br), 2.42 (s, 3H, CH₃).
5.1.4.6 2-Bromo-1-(3-nitrophenyl)ethanone (5h). Yellow solid. Yield, 65%. ¹H-NMR (400 MHz, CDCl₃, δ ppm): 8.82 (t, 1H, J = 7.86 Hz, Ar–H), 8.48 (dd, 1H, J = 8.08 Hz, Ar–H), 8.31–8.34 (m, 1H, Ar–H), 7.75–7.71 (t, 1H, J = 7.98 Hz, Ar–H), 4.47 (s, 2H, CH₂Br).

5.1.5 General procedure for the α-bromination of aromatic/heteroaromatic ketones 5(i–o)²⁸. A solution of appropriately substituted aromatic/heteroaromatic ketones 4(i–o) (0.003 mol, 1 equiv.) in chloroform (10 mL) was added dropwise over 30 min was added to a hot solution of copper(II) bromide (1.07 g, 0.007 mol, 2 equiv.) in ethyl acetate (10 mL). The reaction mixture was then refluxed for 12 h, cooled to room temperature and filtered through a Celite bed. The filtrate was washed with a saturated NaHCO₃/brine solution and dried over anhydrous Na₂SO₄. The resultant solution was concentrated under reduced pressure to give the desired compounds 5(i–o).
5.1.5.1 2-Bromo-1-(2-hydroxyphenyl)ethanone (5i). Yellow oil. Yield, 65%. ¹H-NMR (400 MHz, CDCl₃, δ ppm): 12.25 (s, 1H, OH), 7.75 (m, 2H, Ar–H) 7.54 (m, 2H, Ar–H), 4.44 (s, 2H, CH₂Br).
5.1.5.2 2-Bromo-1-(4-hydroxyphenyl)ethanone (5j). Yellow oil. Yield, 75%. ¹H-NMR (400 MHz, CDCl₃, δ ppm): 11.72 (s, 1H, OH), 7.94–7.90 (dd, 2H, J = 8.72–8.53 Hz, Ar–H), 6.95–6.91 (t, 2H, J = 8.78 Hz, Ar–H), 4.41 (s, 2H, CH₂Br).
5.1.5.3 2-Bromo-1-(3-chlorophenyl)ethanone (5k). Off-white solid. Yield, 67%. ¹H-NMR (400 MHz, CDCl₃, δ ppm): 7.95 (s, 1H, Ar–H), 7.86–7.82 (d, 1H, J = 7.76 Hz, Ar–H), 7.58–7.55 (dd, 1H, J = 8.00 Hz, Ar–H), 7.45–7.38 (m, 1H, Ar–H), 4.41 (s, 2H, CH₂Br).
5.1.5.4 2-Bromo-1-(3-fluorophenyl)ethanone (5l). White solid. Yield, 70%. ¹H-NMR (400 MHz, CDCl₃, δ ppm): 7.86–7.84 (d, 1H, J = 5.88 Hz, Ar–H), 7.76–7.75 (d, 2H, J = 6.28 Hz, Ar–H), 7.49–7.47 (d, 1H, J = 5.40 Hz, Ar–H), 4.42 (S, 2H, CH₂Br).
5.1.5.5 2-Bromo-1-(thiophen-2-yl)ethanone (5m). Yellow oil. Yield, 80%. ¹H-NMR (400 MHz, CDCl₃, δ ppm): 7.81–7.80 (dd, 1H, J = 4.00 Hz, Ar–H), 7.72–7.71 (dd, 1H, J = 5.00 Hz, Ar–H), 7.18–7.16 (dd, 1H, J = 7.76 Hz, Ar–H), 4.36 (s, 2H, CH₂Br).
5.1.5.6 3-(2-Bromoacetyl)-2H-chromen-2-one (5n). Yellow solid. Yield, 90%. ¹H-NMR (400 MHz, CDCl₃, δ ppm): 8.63 (s, 1H, Ar–H), 7.72–7.70 (d, 1H, J = 7.48 Hz, Ar–H), 7.41 (m, 3H, ArH), 4.75 (s, 2H, CH₂Br).
5.1.5.7 6-Bromo-3-(2-bromoacetyl)-2H-chromen-2-one (5o). Yellow solid. Yield, 90%. ¹H-NMR (400 MHz, CDCl₃, δ ppm): 8.52 (s, 1H, Ar–H), 7.82–7.80 (d, 1H, J = 7.24 Hz, Ar–H), 7.78–7.77 (d, 1H, J = 7.36 Hz, Ar–H), 7.30–7.28 (d, 1H, J = 8.84 Hz, Ar–H), 4.71 (s, 2H, CH₂Br).

5.1.6 General procedure for the synthesis of thiazolyl-substituted carbazole hydrazine derivatives 6(a–o)²². A solution of carbazole thiosemicarbazide compound (3, 150 mg, 0.531 mol, 1 equiv.) and α-bromoaromatic/heteroaromatic ketones 5(a–o) (0.638 mol, 1.2 equiv.) in anhydrous methanol (10 mL) was refluxed for 3–5 h until TLC showed all the starting materials had been consumed. The residue was collected by filtration and was stirred in saturated NaHCO₃ solution for 30 min. The resultant solid was filtered, dried and purified by recrystallization with ethanol to give the desired compounds 6(a–o).
5.1.6.1 4-(4-Bromophenyl)-2-(2-((9-methyl-9H-carbazol-3yl)methylene)hydrazinyl)thiazole (6a). Brown solid. Yield, 62%; melting point, 262–264 °C. FTIR (ATR, ν_max, cm⁻¹): 3319.96 (N–H Str.), 3043.75 (Ar–H Str.), 2965.52 (C–H Str. of CH₃), 1618.20 (C=N Str.). ¹H-NMR (400 MHz, DMSO-d₆, δ ppm): 12.39 (s, 1H, NH), 8.38 (s, 1H, CH=N), 8.22 (s, 1H, H-4 of carbazole), 8.20 (s, 1H, Ar–H), 7.88–7.87 (dd, 1H, J = 8.56–8.60 Hz, Ar–H), 7.85–7.81 (d, 2H, J = 8.48 Hz, Ar–H), 7.67–7.59 (m, 4H, Ar–H), 7.52–7.50 (t, 1H, J = 7.60 Hz, Ar–H), 7.40 (s, 1H, H-5 of thiazole), 7.25–7.23 (t, 1H, J = 7.54 Hz, Ar–H), 3.90 (s, 3H, N–CH₃). ¹³C-NMR (100 MHz, DMSO-d₆, δ ppm): 168.05, 148.72 (CH=N), 142.60, 140.79, 140.58, 133.44, 131.05, 127.07, 125.67, 124.89, 123.08, 121.64, 119.99, 119.06, 118.83, 109.27, 103.69 (C-5 of thiazole), 28.69 (N–CH₃). HR ESI m/z: calculated for C₂₃H₁₇BrN₄S [M + H]⁺, 460.0357; found, 461.0359.
5.1.6.2 2-(2-((9-Methyl-9H-carbazol-3-yl) methylene)hydrazinyl)-4-phenylthiazole (6b). Brown solid. Yield, 58%, melting point, 246–248 °C. FTIR (ATR, ν_max, cm⁻¹): 3295.05 (N–H Str.), 3049.53 (Ar–H Str.), 2915.12 (C–H Str. of CH₃), 1620.14 (C=N Str.). ¹H-NMR (400 MHz, DMSO-d₆, δ ppm): 12.00 (s, 1H, NH), 8.37 (s, 1H, CH=N), 8.24 (s, 1H, H-4 of carbazole), 8.21 (s, 1H, Ar–H), 7.88–7.86 (d, 2H, J = 8.44 Hz, Ar–H), 7.67–7.60 (m, 2H, Ar–H), 7.52–7.48 (t, 2H, J = 7.72 Hz, Ar–H), 7.41–7.39 (t, 2H, J = 7.62 Hz, Ar–H), 7.32 (s, 1H, H-5 of thiazole), 7.25–7.23 (m, 2H, Ar–H), 3.90 (S, 3H, N–CH₃). ¹³C-NMR (100 MHz, DMSO-d₆, δ ppm): 168.37, 142.74 (CH=N), 141.09, 141.02, 134.76, 128.56, 127.44, 126.10, 125.47, 123.49, 122.09, 121.88, 120.43, 119.44, 109.71, 109.45, 103.18 (C-5 of thiazole), 55.99, 29.19 (N–CH₃), 18.51. HR ESI m/z: calculated for C₂₃H₁₈N₄S [M + H]⁺, 382.1252; found, 383.1253.
5.1.6.3 4-(3,4-Dimethoxyphenyl)-2-(2-((9-methyl-9H-carbazol-3-yl)methylene)hydrazinyl)thiazole (6c). Brown solid. Yield, 50%; melting point, 242–244 °C. FTIR (ATR, ν_max, cm⁻¹): 3074.36 (N–H), 3008.28 (Ar–H), 2930.41 (C–H Str. of CH₃), 1616.36 (C=N). ¹H-NMR (400 MHz, DMSO-d₆, δ ppm): 8.38 (s, 1H, NH), 8.31 (s, 1H, CH=N), 8.23 (s, 1H, H-4 of carbazole), 8.21–8.20 (d, 1H, J = 7.88 Hz, Ar–H), 7.89–7.87 (dd, 1H, J = 8.68–8.60 Hz, Ar–H), 7.67–7.63 (m, 2H, Ar–H), 7.52–7.48 (m, 1H, Ar–H), 7.43–7.41 (m, 2H, Ar–H), 7.27–7.25 (t, 1H, J = 7.52 Hz, Ar–H), 7.20 (s, 1H, H-5 thiazole), 7.00–6.98 (d, 1H, J = 8.36 Hz, Ar–H), 3.91 (s, 3H, N–CH₃), 3.82 (s, 3H, OCH₃), 3.78 (s, 3H, OCH₃). ¹³C-NMR (100 MHz, DMSO-d₆, δ ppm): 168.16, 148.68 (CH=N), 141.26, 126.12, 125.31, 123.56, 122.10, 121.89, 120.44, 119.57, 118.03, 111.79, 109.72, 101.48 (C-5 of thiazole), 55.51, 29.14 (N–CH₃). HR ESI m/z: calculated for C₂₅H₂₂N₄O₂S [M + H]⁺, 442.1463; found, 443.1464.
5.1.6.4 4-(4-Chlorophenyl)-2-(2-((9-methyl-9H-carbazol-3-yl)methylene)hydrazinyl)thiazole (6d). Brown solid. Yield, 48%, melting point, 236–238 °C. FTIR (ATR, ν_max, cm⁻¹): 3181.95 (N–H), 3054.74 (Ar–H), 2926.22 (C–H Str. of CH₃), 1592.71 (C=N). ¹H-NMR (400 MHz, DMSO-d₆, δ ppm): 12.08 (s, 1H, NH), 8.36 (s, 1H, CH=N), 8.22 (s, 1H, H-4 of carbazole), 8.20 (s, 1H, Ar–H), 7.89–7.85 (m, 3H, Ar–H), 7.67–7.64 (d, 1H, J = 8.60 Hz, Ar–H), 7.62–7.60 (d, 1H, J = 8.16 Hz, Ar–H), 7.51–7.49 (m, 3H, Ar–H), 7.37 (s, 1H, H-5 of thiazole), 7.26–7.22 (t, 1H, J = 7.36 Hz), 3.90 (s, 3H, N–CH₃). ¹³C-NMR (100 MHz, DMSO-d₆, δ ppm): 168.51, 149.14, 143.04 (CH=N), 141.23, 141.02, 133.54, 131.84, 128.58, 127.19, 126.12, 125.34, 123.53, 122.09, 121.87, 120.42, 119.49, 119.28, 109.71, 109.46, 104.03 (C-5 of thiazole), 56.00, 29.11 (N–CH₃), 18.49. HR ESI m/z: calculated for C₂₃H₁₇ClN₄S [M + H]⁺, 416.0862; found, 417.0865.
5.1.6.5 2-(2-((9-Methyl-9H-carbazol-3-yl)methylene)hydrazinyl)-4-(4-nitrophenyl)thiazole (6e). Brown solid. Yield, 55%; melting point, 236–238 °C. FTIR (ATR, ν_max, cm⁻¹): 3312.86 (N–H), 3046.57 (Ar–H), 2928.85 (C–H Str. of CH₃), 1621.16 (C=N). ¹H-NMR (400 MHz, DMSO-d₆, δ ppm): 12.16 (s, 1H, N–H), 8.38 (s, 1H, CH=N), 8.29–8.26 (d, 2H, J = 8.92 Hz, AR–H), 8.24 (s, 1H, H-4 of carbazole), 8.22–8.21 (d, 1H, J = 7.76 Hz, Ar–H), 8.13–8.11 (d, 2H, J = 8.9 Hz, AR–H), 7.88–7.86 (dd, 1H, J = 8.62 Hz, Ar–H), 7.70 (s, 1H, H-5 thiazole), 7.67–7.65 (d, 1H, J = 8.64, Ar–H), 7.63–7.61 (d, 1H, J = 8.16 Hz, Ar–H), 7.50–7.48 (t, 1H, J = 8.16 Hz, Ar–H), 7.27–7.25 (t, 1H, J = 7.50 Hz, Ar–H), 3.90 (s, 3H, N–CH₃). ¹³C-NMR (400 MHz, DMSO-d₆, δ ppm): 168.78, 148.52, 146.14, 143.30 (CH=N), 141.27, 141.03, 140.75, 126.29, 126.13, 125.26, 124.08, 123.55, 122.10, 121.88, 119.58, 119.29, 109.72, 109.47, 108.17 (C-5 of thiazole), 29.13 (N–CH₃). HR ESI m/z: calculated for C₂₃H₁₇N₅O₂S [M + H]⁺, 427.1103; found, 428.1107.
5.1.6.6 4-(4-Fluorophenyl)-2-(2-((9-methyl-9H-carbazol-3-yl)methylene)hydrazinyl)thiazole (6f). Dark brown solid. Yield, 60%, melting point, 228–229 °C. FTIR (ATR, ν_max, cm⁻¹): 3180 (NH), 3047.51 (Ar–H), 2926.67 (C–H Str. of CH₃), 1624.30 (C=N). ¹H-NMR (400 MHz, DMSO-d₆, δ ppm): 11.69 (s, 1H, N–H), 8.37 (s, 1H, CH=N), 8.23 (s, 1H, H-4 of carbazole), 8.22–8.20 (d, 1H, J = 7.76 Hz, Ar–H), 7.91–7.85 (m, 3H, Ar–H), 7.67–7.64 (d, 1H, J = 8.59 Hz, Ar–H), 7.62–7.60 (d, 1H, J = 8.28 Hz, Ar–H), 7.51–7.47 (t, 1H J = 7.72 Hz, Ar–H), 7.28 (s, 1H, H-5 of thiazole), 7.26–7.24 (m, 3H, Ar–H), 3.90 (s, 3H, N–CH₃). ¹³C-NMR (100 MHz, DMSO-d₆, δ ppm): 168.47, 160.35, 149.20, 143.06 (CH=N), 141.23, 141.01, 131.24, 127.52, 127.44, 126.13, 125.33, 123.53, 122.08, 121.87, 120.41, 119.48, 119.28, 115.51, 115.29, 109.70, 109.45, 102.98 (C-5 of thiazole), 29.12 (N–CH₃). HR ESI m/z: calculated for C₂₃H₁₇FN₄S [M + H]⁺, 400.1158; found, 401.1156.
5.1.6.7 2-(2-((9-Methyl-9H-carbazol-3-yl)methylene)hydrazinyl)-4-(p-tolyl)thiazole (6g). Off-white solid. Yield, 60%, melting point, 252–254 °C. FTIR (ATR, ν_max, cm⁻¹): 3109.40 (N–H), 3048.24 (Ar–H), 2917.32 (C–H Str. of CH₃), 1613.95 (C=N). ¹H-NMR (400 MHz, DMSO-d₆, δ ppm): 12.30 (s, 1H, N–H), 8.38 (s, 1H, CH=N), 8.24 (s, 1H, H-4 of carbazole), 8.22–8.21 (d, 1H, J = 7.68 Hz, Ar–H), 7.89–7.88 (dd, 1H, J = 8.56 Hz, Ar–H), 7.86–7.85 (d, 2H, J = 8.08 Hz, Ar–H), 7.76–7.74 (d, 1H, J = 8.60 Hz, Ar–H), 7.67–7.65 (d, 1H, J = 8.24 Hz, Ar–H), 7.63–7.61 (t, 1H, J = 7.52 Hz, Ar–H), 7.26–7.24 (m, 3H, Ar–H), 7.21 (s, 1H, H-5 of thiazole) 3.90 (s, 3H, N–CH3), 2.32 (s, CH₃). ¹³C-NMR (100 MHz, DMSO-d₆, δ ppm): 168.28, 141.26 (CH=N), 141.02, 136.89, 131.67, 129.15, 128.96, 127.37, 126.12, 125.50, 125.31, 123.56, 122.11, 121.88, 120.43, 119.56, 119.28, 109.72, 109.46, 102.38 (C-5 of thiazole), 29.13 (N–CH₃), 20.78 (Ar–CH₃). HR ESI m/z: calculated for C₂₄H₂₀N₄S [M + H]⁺, 396.1409; found, 397.1409.
5.1.6.8 2-(2-((9-Methyl-9H-carbazol-3-yl)methylene)hydrazinyl)-4-(3-nitrophenyl)thiazole (6h). Yellow solid. Yield, 68%, melting point, 220–222 °C. FTIR (ATR, ν_max, cm⁻¹): 3107.31 (N–H), 3085.87 (Ar–H), 2925.61 (C–H Str. of CH₃), 1572.25 (C=N). ¹H-NMR (400 MHz, DMSO-d₆,δ ppm): 8.68 (s, 1H, NH), 8.36 (s, 1H, CH=N), 8.31–8.29 (d, 1H, J = 7.84 Hz, Ar–H), 8.22–8.21 (s, 1H, H-4 of carbazole), 8.19 (d, 1H, J = 7.72 Hz, Ar–H), 8.14–8.12 (dd, 1H, J = 8.04 Hz, Ar–H), 7.88–7.85 (dd, 1H, J = 8.56–8.60 Hz, Ar–H), 7.71–7.60 (m, 4H, Ar–H), 7.57 (s, 1H, H-5 of thiazole), 7.51–7.47 (t, 1H, J = 7.56 Hz, Ar–H), 7.26–7.22 (t, 1H, J = 7.44 Hz, Ar–H), 3.89 (s, 3H, N–CH3). ¹³C-NMR (100 MHz, DMSO-d₆, δ ppm): 169.14, 148.24, 148.07, 142.99 (CH=N), 141.19, 141.01, 136.33, 131.50, 130.15, 126.10, 125.44, 123.53, 122.09, 121.88, 120.40, 119.86, 119.42, 119.26, 109.68, 109.43, 105.70 (C-5 of thiazole), 30.64, 29.11 (N–CH₃). HR ESI m/z: calculated for C₂₃H₁₇N₅O₂S [M + H]⁺, 427.1103; found, 428.1106.
5.1.6.9 2-(2-(2-((9-Methyl-9H-carbazol-3-yl)methylene)hydrazinyl)thiazol-4-yl)phenol (6i). Brown solid. Yield, 65%, melting point, 245–247 °C. FTIR (ATR, ν_max, cm⁻¹): 3342.53 (OH), 3116.66 (NH), 3057.16 (Ar–H), 2894.84 (C–H Str. of CH₃), 1621.58 (C=N). ¹H-NMR (400 MHz, DMSO-d₆, δ ppm): 12.59 (s, 1H, N–H), 8.41 (s, 1H, CH=N), 8.29 (s, 1H, H-4 of carbazole), 8.23–8.21 (d, 1H, J = 7.68 Hz, Ar–H), 7.91–7.89 (dd, 1H, J = 8.64–8.72 Hz, Ar–H), 7.80–7.78 (dd, 1H, J = 7.84–7.80 Hz, Ar–H), 7.69–7.66 (d, 1H, J = 8.64 Hz, Ar–H), 7.63–7.61 (d, 1H, J = 8.20 Hz, Ar–H), 7.52–7.47 (t, 1H, J = 7.30 Hz, Ar–H), 7.40 (s, 1H, H-5 of thiazole), 7.27–7.23 (t, 1H, J = 7.48 Hz, Ar–H), 7.20–7.18 (t, 1H, J = 8.42 Hz, Ar–H), 6.92–6.88 (m, 2H, Ar–H), 5.10 (s, 1H, OH), 3.91 (s, 3H, N–CH₃). ¹³C-NMR (100 MHz, DMSO-d₆, δ ppm): 167.67, 156.65, 155.13, 141.12 (CH=N), 141.04, 129.20, 127.19, 126.16, 124.98, 123.71, 122.09, 121.87, 120.43, 119.91, 119.35, 119.11, 116.70, 109.75, 109.51, 103.70 (C-5 of thiazole), 30.66, 29.15 (N–CH₃). HR ESI m/z: calculated for C₂₃H₁₈N₄OS [M + H]⁺, 398.1201; found, 399.1203.
5.1.6.10 4-(2-(2-((9-Methyl-9H-carbazol-3-yl)methylene)hydrazinyl)thiazol-4-yl)phenol (6j). Brown solid. Yield, 55%, melting point, 284–285 °C. FTIR (ATR, ν_max, cm⁻¹): 3454.28 (OH), 3124.46 (N–H), 3059.35 (Ar–H), 2940.97 (C–H Str. of CH₃), 1629.47 (C=N). ¹H-NMR (400 MHz, DMSO-d₆, δ ppm): 12.40 (s, 1H, N–H), 8.37 (s, 1H, CH=N), 8.23 (s, 1H, H-4 of carbazole), 8.21 (s, 1H, Ar–H), 7.88–7.86 (d, 1H, J = 8.56 Hz, Ar–H), 7.65–7.59 (m, 4H, Ar–H), 7.52–7.50 (t, 1H, J = 7.76 Hz, Ar–H), 7.26–7.24 (t, 1H, J = 7.42 Hz, Ar–H), 7.05 (s, 1H, H-5 of thiazole), 6.80–6.74 (d, 2H, J = 8.56 Hz, Ar–H), 4.66 (s, OH), 3.91 (s, 3H, N–CH₃). ¹³C-NMR (100 MHz, DMSO-d₆, δ ppm): 157.68, 141.80 (CH=N), 141.57, 129.41, 127.51, 126.62, 125.86, 124.12, 122.64, 122.43, 120.93, 119.79, 115.81, 110.21, 109.97, 100.80 (C-5 of thiazole), 29.65 (N–CH₃). HR ESI m/z: calculated for C₂₃H₁₈N₄OS [M + H]⁺, 398.1201; found, 399.1204.
5.1.6.11 4-(3-Chlorophenyl)-2-(2-((9-methyl-9H-carbazol-3-yl)methylene)hydrazinyl)thiazole (6k). Brown solid. Yield, 48%, melting point, 238–240 °C. FTIR (ATR, ν_max, cm⁻¹): 3215.93 (N–H), 3052.00 (Ar–H), 2936.70 (C–H Str. of CH₃), 1593.86 (C=N). ¹H-NMR (400 MHz, DMSO-d₆, δ ppm): 12.29 (s, 1H, N–H), 8.37 (s, 1H, CH=N), 8.31 (s, 1H, H-4 of carbazole), 8.22–8.21 (d, 1H, J = 7.56 Hz, Ar–H), 7.92 (s, 1H, H-5 of thiazole), 7.88–7.85 (dd, 1H, J = 8.72 Hz, Ar–H), 7.84–7.82 (d, 1H, J = 7.76 Hz, Ar–H), 7.67–7.65 (d, 1H, J = 8.64 Hz, Ar–H), 7.63–7.61 (d, 1H, J = 8.24 Hz, Ar–H), 7.52–7.44 (m, 3H, Ar–H), 7.36–7.34 (m, 1H, Ar–H), 7.26–7.23 (t, 1H, Ar–H), 3.91 (s, 3H, N–CH₃). ¹³C-NMR (100 MHz, DMSO-d₆, δ ppm): 141.76 (CH=N), 141.57, 133.94, 130.97, 126.61, 125.70, 124.51, 124.06, 122.64, 120.93, 119.78, 110.21, 109.96 (C-5 of thiazole), 29.65 (N–CH₃). HR ESI m/z: calculated for C₂₃H₁₇ClN₄S [M + H]⁺, 416.0862; found, 417.0864.
5.1.6.12 4-(3-Fluorophenyl)-2-(2-((9-methyl-9H-carbazol-3-yl)methylene)hydrazinyl)thiazole (6l). Brown solid. Yield, 45%, melting point, 235–237 °C. FTIR (ATR, ν_max, cm⁻¹): 3187.06 (N–H), 3046.62 (Ar–H), 2929.54 (C–H Str. of CH₃), 1590.14 (C=N). ¹H-NMR (400 MHz, DMSO-d₆, δ ppm): 12.24 (s, 1H, NH), 8.37 (s, 1H, CH=N), 8.22 (s, 1H, H-4 of carbazole), 8.21 (s, 1H, Ar–H), 7.88–7.86 (dd, 1H, J = 8.58 Hz, Ar–H), 7.73–7.71 (d, 1H, J = 7.84 Hz, Ar–H), 7.67–7.61 (m, 3H, Ar–H), 7.50–7.46 (m, 2H, Ar–H), 7.44 (s, 1H, H-5 of thiazole), 7.26–7.23 (t, 1H, J = 7.18 Hz, Ar–H), 7.15–7.10 (t, 1H, J = 8.45 Hz, Ar–H), 3.91 (s, 3H, N–CH₃). ¹³C-NMR (100 MHz, DMSO-d₆, δ ppm): 168.47, 163.70, 142.93 (CH=N), 141.22, 141.02, 130.59, 130.51, 126.10, 125.38, 123.50, 122.10, 121.89, 121.48, 120.43, 119.49, 119.26, 112.09, 111.86, 109.71, 109.45, 104.69 (C-5 of thiazole), 29.13 (N–CH₃). HR ESI m/z: calculated for C₂₃H₁₇FN₄S [M + H]⁺, 400.1158; found, 401.1159.
5.1.6.13 2-(2-((9-Methyl-9H-carbazol-3-yl)methylene)hydrazinyl)-4-(thiophen-2-yl)thiazole (6m). Brown solid. Yield, 45%, melting point, 241–243 °C. FTIR (ATR, ν_max, cm⁻¹): 3152 (N–H) 3055.87 (Ar–H), 2928.05 (C–H Str. of CH₃), 1621.29 (C=N). ¹H-NMR (400 MHz, DMSO-d₆, δ ppm): 12.19 (s, 1H, NH), 8.37 (s, 1H, CH=N), 8.22–8.20 (d, 1H, J = 7.84 Hz, Ar–H), 8.19 (s, 1H, H-4 of carbazole), 7.87–7.84 (dd, 1H, J = 8.44–8.48 Hz, Ar–H), 7.66–7.64 (d, 1H, J = 8.24 Hz, Ar–H), 7.62–7.60 (d, 1H, J = 8.60 Hz, Ar–H), 7.51–7.45 (m, 3H, Ar–H), 7.26–7.22 (m, 1H, Ar–H), 7.15 (s, 1H, H-5 of thiazole), 7.09–7.07 (m, 1H, Ar–H), 3.90 (s, 3H, N–CH₃). ¹³C-NMR (100 MHz, DMSO-d₆, δ ppm): 168.2, 145.1, 143.0 (CH=N), 141.2, 141.0, 138.9, 127.8, 126.1, 125.3, 123.1, 122.0, 121.8, 120.4, 119.5, 119.2, 109.7, 101.5 (C-5 of thiazole), 29.13 (N–CH₃). HR ESI m/z: calculated for C₂₁H₁₆N₄S₂ [M + H]⁺, 388.0816; found, 389.0814.
5.1.6.14 3-(2-(2-((9-Methyl-9H-carbazol-3-yl)methylene)hydrazinyl)thiazol-4-yl)-2H-chromen-2-one (6n). Yellow solid. Yield, 67%, melting point, 267–269 °C. FTIR (ATR, ν_max, cm⁻¹): 3164.52 (N–H), 3015.73 (Ar–H), 2871.89 (C–H Str. of CH₃), 1721.35 (C=O), 1604.68 (C=N). ¹H-NMR (400 MHz, DMSO-d₆, δ ppm): 8.52 (s, 1H, N–H), 8.35 (s, 1H, CH=N), 8.23 (s, 1H, H-4 of carbazole), 8.20 (s, 1H, H-4 of coumarin), 7.86–7.84 (dd, 1H, J = 8.60 Hz, Ar–H), 7.83–7.81 (dd, 1H, J = 7.80 Hz, Ar–H), 7.75 (s, 1H, H-5 of thiazole), 7.64–7.58 (m, 3H, Ar–H), 7.50–7.48 (m, 4H, Ar–H), 7.26–7.22 (t, 1H, J = 7.48 Hz, Ar–H), 3.91 (s, 3H, N–CH₃). ¹³C-NMR (100 MHz, DMSO-d₆, δ ppm): 158.75 (C=O), 152.22, 150.22, 143.81 (CH=N), 143.32, 142.92, 141.24, 138.09, 132.61, 131.64, 129.13, 128.73, 126.13, 125.25, 124.80, 123.55, 122.07, 120.38, 119.29, 117.77, 116.04, 112.91, 110.21 (C-5 of thiazole), 29.09 (N–CH₃), 18.47. HR ESI m/z: calculated for C₂₆H₁₈N₄O₂S [M + Na]⁺, 450.1150; found, 473.1153.
5.1.6.15 6-Bromo-3-(2-(2-((9-methyl-9H-carbazol-3-yl)methylene)hydrazinyl)thiazol-4-yl)-2H-chromen-2-one (6o). Yellow solid. Yield, 67%; melting point, 267–269 °C. FTIR (ATR, ν_max, cm⁻¹): 3201.01, 3119.73 (N–H), 3042.34 (Ar–H), 2929.74, 1711.27 (C=O), 1627.14 (C=N). ¹H-NMR (400 MHz, DMSO-d₆, δ ppm): 12.11 (s, 1H, N–H), 8.45 (s, 1H, CH=N), 8.36 (s, 1H, H-4 of carbazole), 8.24 (s, 1H, H-4 of coumarin), 8.21–8.19 (d, 1H, J = 7.64 Hz, Ar–H), 8.10–8.09 (d, 1H, J = 7.31 Hz, Ar–H), 7.87–7.84 (dd, 1H, J = 8.64 Hz, Ar–H), 7.79 (s, 1H, H-5 of thiazole), 7.75–7.72 (dd, 1H, J = 8.72–8.88 Hz, Ar–H), 7.65–7.63 (d, 1H, J = 8.64 Hz, Ar–H), 7.61–7.59 (d, 1H, J = 8.32 Hz, Ar–H), 7.51–7.47 (t, 1H, J = 7.54 Hz, Ar–H), 7.40–7.38 (d, 1H, J = 8.76 Hz, Ar–H), 7.22–7.26 (t, 1H, J = 7.52 Hz, Ar–H), 3.89 (s, 3H, N–CH₃). ¹³C-NMR (100 MHz, DMSO-d₆, δ ppm): 167.86 (C=O), 158.27, 151.22, 143.88 (CH=N), 141.24, 136.56, 133.57, 130.58, 126.10, 125.27, 123.54, 122.08, 121.86, 120.40, 119.51, 118.06, 116.32, 111.08, 109.69 (C-5 of thiazole), 29.11 (N–CH₃). HR ESI m/z: calculated for C₂₆H₁₇BrN₄O₂S [M + Na]⁺, 528.0256; found, 551.0253.

5.2 In vitro anti-mycobacterial evaluation

The in vitro anti-mycobacterial activity of the synthesized compounds 6a–6o was determined at the Infectious Disease Research Institute within the NIAID screening program. The activity was assessed against M. tuberculosis H₃₇Rv grown under aerobic conditions using a dual read-out (OD₅₉₀ and fluorescence) assay procedure.^23–25 The test compounds were prepared as 10-point two-fold serial dilutions in DMSO and diluted into 7H9-Tw-OADC medium in 96-well plates with a final DMSO concentration of 2%. The highest concentration of the compounds was 200 μM and the compounds were soluble in DMSO at 10 μM. For compounds with limited solubility, the highest concentration was 50× less than the stock concentration, e.g. 100 μM for 5 mM DMSO stock solution and 20 μM for 1 μM DMSO stock. For potent compounds, the assays were repeated at lower starting concentrations. Each plate included assay controls for the background (medium/DMSO only, no bacterial cells), zero growth (100 μM rifampicin) and maximum growth (DMSO only), as well as a rifampicin dose–response curve. The plates were inoculated with M. tuberculosis and incubated for 5 days. The growth was measured by OD₅₉₀ and fluorescence (Ex₅₆₀/Em₅₉₀) using a BioTek Synergy4 plate reader. The growth was calculated separately for OD₅₉₀ and RFU. The MIC was calculated on the basis of a 10-point dose–response curve plotted as percentage growth. The MIC was defined as the minimum concentration at which growth was completely inhibited and was calculated from the inflection point of the fitted curve to the lower asymptote (zero growth). In addition, dose–response curves were generated using the Levenberg–Marquardt algorithm and the concentrations that resulted in 50 and 90% inhibition of growth were determined (IC₅₀ and IC₉₀, respectively).

5.3 Cytotoxicity studies: MTT assay

Mammalian Vero cells were cultured in Dulbecco's Modified Eagle Medium containing 2 μM Na₂CO₃ supplemented with 10% v/v fetal bovine serum. The cells were incubated at 37 °C under 5% CO₂ and 95% air in a humidified atmosphere until confluent and then diluted with phosphate-buffered saline to 106 cells per mL. Stock solutions were prepared in dimethyl sulfoxide (DMSO) and further dilutions were made with fresh culture medium. The concentration of DMSO in the final culture medium was 1%, which had no effect on the cell viability. In a transparent 96-well plate, serially diluted stock solutions were placed at 37 °C for 72 h. The medium was then removed and the monolayer was washed twice with 100 μL of warm Hanks' balanced salt solution. A 100 μL volume of warm medium and 20 μL of freshly made MTS-PMS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium and phenyl methasulfazone] (Promega) were added to each well, then the plates were incubated for 3 h and the absorbance determined at 490 nm using a plate reader. The same experimental conditions were used for all the compounds and the analysis was repeated three times for each cell line.²⁹

Acknowledgements

We thank the Department of Pharmaceutical Chemistry, Discipline of Pharmaceutical Sciences, College of Health Sciences, University of KwaZulu-Natal, South Africa for their constant support, encouragement and financial assistance. The authors are grateful to the National Institute of Allergy and Infectious Diseases (NIAID), Bethesda, MD, USA for anti-mycobacterial screening and to Mr Dilip Jagjivan (UKZN, South Africa) for his assistance with the NMR experiments.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4ra11752b

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