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. 2024 Apr 1;15(5):1664–1674. doi: 10.1039/d4md00118d

Antitubercular activity of 2-mercaptobenzothiazole derivatives targeting Mycobacterium tuberculosis type II NADH dehydrogenase†

Pallavi Saha a, Shashikanta Sau b, Nitin Pal Kalia b,✉, Deepak K Sharma a,✉
PMCID: PMC11110738  PMID: 38784457

Abstract

Mycobacterium tuberculosis (Mtb) type II NADH dehydrogenase (NDH-2) transports electrons into the mycobacterial respiratory pathway at the cost of reduction of NADH to NAD+ and is an attractive drug target. Herein, we have synthesised a series of 2-mercaptobenzothiazoles (C1–C14) and evaluated their anti-tubercular potential as Mtb NDH-2 inhibitors. The synthesised compounds C1–C14 were evaluated for MIC90 and ATP depletion against Mtb H37Ra, M. bovis, and Mtb H37Rv mc2 6230. Compounds C3, C4, and C11 were found to be the active molecules in the series and were further evaluated for their MIC90 against Mtb-resistant strains and for their bactericidal potential against Mtb H37Rv mc26230. The Peredox-mCherry-expressing Mtb strain was used to examine whether C3, C4, and C11 possess NDH-2 inhibitory potential. Furthermore, cytotoxicity analysis against HepG2 displayed a safety index (SI) of >10 for C3 and C4. To get an insight into the mode of interaction at NDH-2, we have performed computational analysis of our active compounds.

Mycobacterium tuberculosis (Mtb) type II NADH dehydrogenase (NDH-2) transports electrons into the mycobacterial respiratory pathway at the cost of reduction of NADH to NAD+ and is an attractive drug target.graphic file with name d4md00118d-ga.jpg

Introduction

Tuberculosis (TB) is a complex, communicable disease arising from the infection of Mycobacterium tuberculosis (Mtb). In the pre-covid period, TB remained the leading cause of death from a single infectious agent. The timeline of the “End TB Strategy” by the WHO was delayed due to the outbreak of the COVID-19 pandemic. As a result, the advancement in the treatment of patients suffering from TB infection came to a halt, which included delayed diagnosis and failed access to TB treatment.1–3 Though TB is curable, the hardship in TB treatment is associated with the emergence of multi-drug resistant TB (MDR-TB) and extensively multi-drug resistant TB (XDR-TB).4–7Mtb can survive under extreme environmental conditions due to its metabolic plasticity, which allows it to remodel its metabolic pathway, fulfilling the energy need for survival, which, in turn, is responsible for the outbreak of drug-resistant strains.8–11

Although many anti-TB drugs are present in the market, the drug hunt is still going on for combating the drug-resistant Mtb strains. In 2012, the FDA approved bedaquiline (BDQ) for the treatment of drug-resistant TB. After the approval of BDQ, which acts by inhibiting the enzyme ATP-synthase of the oxidative phosphorylation pathway of Mtb, a drought of 40 years of no new anti-TB drug approval came to an end, and it marked a new direction to antitubercular drug discovery by highlighting the potential of the oxidative phosphorylation pathway as an essential drug target.12–15 NADH dehydrogenase is the first enzyme of the respiratory chain in the oxidative phosphorylation pathway. Electrons enter this metabolic pathway via NADH dehydrogenase, thereby oxidizing NADH to NAD+.16–19Mtb possesses two types of NADH dehydrogenase, type I and type II (ndh and ndhA). From a genetic knockout study of ndh and ndhA, the indispensable role of ndh in the unaltered functioning of the oxidative phosphorylation pathway has been reported. The other components of the respiratory system of Mtb do not transport sufficient electrons to meet up the need, resulting in severe ATP crises. Hence, targeting type II NADH dehydrogenase (NDH-2) can result in complete sterilization of the bacilli. Another benefit of targeting NDH-2 is that this enzyme is absent in mammalian systems, making it an attractive target for antibiotic development.20–22

Recently, Harbut et al. identified CBR-1825 (Fig. 1a) as a potent NDH-2 inhibitor.10 Murugesan et al. replaced the six-member saturated ring in CBR-1825 with a phenyl ring, resulting in 2-mercaptoquinazolinone amides (CBR-5992, Fig. 1b)23 with MIC values of 0.5 μg ml−1. Further exploring the substrate scope around the cyclohexyl amide side chain resulted in a decrease in activity.23 Most SAR studies were performed around the amide side chain because substituted 2-mercaptoquinazolinones are rarely available and difficult to synthesize. Unlike many antibacterial agents commonly used to treat mycobacterial infections, benzothiazole amides demonstrated bactericidal effects against Mtb.24a–d Viewed in this context, we replaced 2-mercaptoquinazolinone in CBR-5992 with 2-mercaptobenzothiazoles (Fig. 1c) and evaluated their antitubercular potential as Mtb NDH-2 inhibitors.

Fig. 1. (a and b) Previously reported NDH-2 inhibitors. (c) The target structure for antitubercular potential as an Mtb NDH-2 inhibitor.

Fig. 1

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Results and discussion

Chemistry

We have synthesized a series of 2-mercaptobenzothiazole derivatives C1–C14 (Scheme 1). We kept the N-cyclohexyl acetamide moiety unaltered as there is literature-reported evidence of the potential of this respective side chain.10,23 Precursor 2-bromo-N-cyclohexylacetamide (A) for the synthesis of the N-cyclohexyl acetamide side chain is formed by the reaction of bromoacetic acid and cyclohexylamine in the presence of EDC·HCl.25 2-mercaptobenzothiazoles (B) were synthesized by reacting 2-bromoanilines bearing the suitable substituent with potassium ethyl xanthate.26,27 The final compounds C1–C14 were formed by the condensation of A and B under appropriate reaction conditions.24d A variety of electron-donating groups (EDGs) and electron-withdrawing groups (EWGs) have been introduced to the core moiety to explore their effect on antitubercular activity (Table 1).

Scheme 1. Synthetic approach for 2-(benzo[d]thiazol-2-ylthio)-N-cyclohexylacetamide derivatives (C1–C14). aReagents and conditions: (a) cyclohexylamine (1 equiv.), bromoacetic acid (1 equiv.), EDC·HCl (1 equiv.), DIPEA (1 equiv.) DCM, room temperature, 12 h; (b) 2-bromoanilines (1 mmol), potassium ethyl xanthate (1.5 mmol), DMF, 120 °C, 4–8 h; (c) A (1 equiv.), B (1 equiv.), DMF, room temperature, 2–3 h. bStructures of compounds C1–C14 are presented in Table 1.

Scheme 1

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In vitro anti-TB activity of the synthesized compounds.

graphic file with name d4md00118d-u1.jpg
Comp. code Benzothiazole structure Mtb H37Ra MIC90 (μg ml−1) M. bovis (BCG) MIC90 (μg ml−1) Mtb H37Rv mc2 6230 MIC90 (μg ml−1) Clog P
C1 graphic file with name d4md00118d-u2.jpg 64 64 64 3.82
C2 graphic file with name d4md00118d-u3.jpg 32 16 32 4.31
C3 graphic file with name d4md00118d-u4.jpg 8 4 8 4.81
C4 graphic file with name d4md00118d-u5.jpg 4 4 4 5.24
C5 graphic file with name d4md00118d-u6.jpg 16 8 4 3.98
C6 graphic file with name d4md00118d-u7.jpg 32 64 16 4.55
C7 graphic file with name d4md00118d-u8.jpg 64 128 32 4.70
C8 graphic file with name d4md00118d-u9.jpg 32 128 64 4.77
C9 graphic file with name d4md00118d-u10.jpg 64 128 32 5.22
C10 graphic file with name d4md00118d-u11.jpg 16 32 16 3.66
C11 graphic file with name d4md00118d-u12.jpg 8 4 8 4.85
C12 graphic file with name d4md00118d-u13.jpg 64 64 64 5.64
C13 graphic file with name d4md00118d-u14.jpg 128 128 128 6.08
C14 graphic file with name d4md00118d-u15.jpg 128 64 128 4.99
CBR-5992 graphic file with name d4md00118d-u16.jpg 0.5 1 0.5 2.06
Isoniazid 0.015 0.06 0.06
Rifampicin 0.015 0.03 0.03
Clofazimine 0.25 0.25 0.25
Rotenone >64 >64 >64
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Minimum inhibitory concentration determination and structure–activity relationship

The synthesized compounds C1–C14 were screened for in vitro antitubercular activity as minimum inhibitory concentration (MIC) against the mycobacterium strains Mtb H37Ra, M. bovis (BCG), and Mtb H37Rv mc2 6230 using a liquid broth microdilution assay (Table 1). We observed that compounds C2–C4 with alkyl groups on the benzothiazole moiety showed a better antitubercular response than compound C1 with an unsubstituted benzothiazole core. Moving from C1 to C4, a decreasing trend of the MIC value with an increase in the Clog P value indicated the pivotal role that the optimal lipophilicity profile plays in establishing the antitubercular action. Antitubercular compounds with an optimal lipophilicity profile can easily penetrate the lipophilic bacterial cell wall, thereby hampering the cell wall's stability and function, ultimately ensuring cell death. Further, for all the antitubercular drug candidates with different modes of action, penetration of the bacterial cell wall is essential to reach the target site of action.28 Though lipophilicity was favoured, the activity was compromised in the presence of a bulky lipophilic naphthyl substituent (C14). Among electron-withdrawing substituted derivatives C5–C7, compound C5 with a 6-fluoro group (MIC = 4–16 μg ml−1) showed better anti-TB activities compared to those with 6-chloro (C6, MIC = 16–64 μg ml−1) and 6-bromo groups (C7, MIC = 32–128 μg ml−1). Moreover, other electron-withdrawing substituents, such as CF3 in C8, OCF3 in C9, and NO2 in C10, decreased the activity compared with compound C5. From the activity profile of C5, we could hypothesize that the fluorine atom interacted with mycobacterial effector sites. The lone pair of electrons on fluorine might increase binding affinity towards certain complementary fragments at the target site. Thus, the combination of fluorine's lipophilicity and dipole–dipole interactions at the target site could be responsible for the low MIC value of C5. In di-halo substituted derivatives (C11–C13), 4-bromo-6-fluoro substituted compound C11 has shown a considerable MIC of 4 μg ml−1. From the SAR study, we infer that electronic, steric, and lipophilicity profiles have some role to play in the inhibitory potential of the synthesized compounds. We have observed that none of the synthesized compounds showed anti-TB activities equivalent to CBR-5992.

Intracellular ATP depletion assay

NDH-2 is the first enzyme of the oxidative phosphorylation pathway. We may hypothesize that inhibition of NDH-2 would result in inhibition of ATP synthesis in Mtb. To validate our statement, we performed an intracellular ATP depletion assay of compounds C1–C14 in Mtb H37Ra, M. bovis (BCG), and Mtb H37Rv mc26230 (Table 2). From the ATP depletion assay, we observed that compounds C2–C4 with aliphatic substitution on benzothiazole showed lower ATP IC50 values as compared to compound C1 with an unsubstituted benzothiazole core and compounds C5–C13 with an electron-withdrawing group on the benzothiazole moiety. The lower ATP IC50 values of C2–C4 further emphasise the requirement of lipophilic groups. Among compounds C5–C13, C5 and C11 having a fluoro group on benzothiazole showed a better ATP depletion response, which complements their MIC90 values.

Intracellular ATP depletion assay of compounds C1–C14a.

Comp. code ATP IC50 (μg ml−1)
Mtb H37Ra M. bovis (BCG) Mtb H37Rv mc2 6230
C1 26.64 ± 1.37 33.18 ± 1.01 40.64 ± 0.21
C2 14.06 ± 1.55 14.39 ± 0.09 34.85 ± 1.02
C3 16.28 ± 1.62 13.44 ± 1.11 16.28 ± 1.19
C4 9.35 ± 1.12 9.89 ± 1.76 9.35 ± 1.11
C5 36.52 ± 1.83 19.67 ± 1.00 56.91 ± 0.25
C6 48.61 ± 1.03 32.85 ± 1.35 38.17 ± 1.60
C7 34.36 ± 1.42 31.76 ± 0.39 49.82 ± 1.24
C8 30.90 ± 0.3 19.71 ± 0.45 51.0 ± 0.08
C9 23.39 ± 0.88 30.43 ± 1.08 66.24 ± 1.94
C10 36.29 ± 1.03 34.43 ± 0.85 24.45 ± 0.95
C11 18.69 ± 1.44 13.50 ± 0.5 14.69 ± 1.34
C12 25.71 ± 1.32 39.84 ± 1.01 55.99 ± 1.12
C13 36.58 ± 1.04 40.80 ± 1.04 35.80 ± 1.36
C14 36.58 ± 0.05 37.96 ± 1.03 32.96 ± 1.06
CBR-5992 8.553 ± 0.05 4.988 ± 0.45 2.2258 ± 1.60
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a

Data are expressed as the mean ± SDs of triplicates.

From the assay results of MIC90 and ATP IC50, compounds C3 and C4 from C2–C4 and C11 appeared to be the active compounds of the series. When the derivative strains of Mtb H37Rv mc26230 were exposed to ten two-fold serial dilutions of C3, C4 and C11 for 24 h, significant ATP depletion was observed (S1, ESI†).

Screening against drug-resistant TB strains

Compounds C3, C4 and C11 were tested against drug resistant strains of Mtb. We observed that the proposed NDH-2 inhibitors were equally potent against the isoniazid-resistant mc28243 strain, rifampicin-resistant mc28247 strain, and multidrug-resistant (isoniazid + rifampicin resistant) mc28259 strain (Table 3).

Minimum inhibitory concentration against drug resistant strains of Mtb.

Comp. code mc28243 MIC90 (μg ml−1) mc28247 MIC90 (μg ml−1) mc28259 MIC90 (μg ml−1)
C3 4 8 4
C4 4 4 4
C11 8 4 8
CBR-5992 0.125 0.250 0.125
Isoniazid >128 0.25 >128
Rifampicin 0.12 >128 >128
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Minimum bactericidal concentration determination

Next, we assessed whether C3, C4 and C11 are bactericidal. We have observed that C3, C4 and C11 displayed a dose-dependent bactericidal effect against Mtb H37Rv mc26230 and significantly reduced the bacterial load at 4× MIC after five days of exposure (Fig. 2) and therefore, are bactericidal in nature. CBR-5992, clofazimine (CLFZ) (targeting NDH-2), and bedaquiline (BDQ) (ATP synthase inhibitor) served as positive controls for comparing the killing efficacy of C3, C4 and C11. It was observed that both C3 and C4 showed comparable killing efficacy at 4× MIC with respect to CBR-5992 (Fig. 2).

Fig. 2. Bactericidal potency of C3, C4, and C11 against replicating Mtb H37Rv mc26230 strain. The first dotted line represents 90% bacterial killing (MBC90), while the second one represents 3log10 reduction in CFU w.r.t. the initial inoculum. ***Statistical difference (P < 0.001, Student's t-test) in the CFU number between Mtb H37Rv mc26230 treated with test compounds and inoculum size at the start of the experiment. Data are expressed as the mean ± SDs of triplicates for each concentration.

Fig. 2

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Peredox-mCherry experiment

The NAD(H) homeostasis is essential for maintaining the metabolic state of Mtb. The Peredox-mCherry reporter strain was used to examine the effect of Mtb inhibitors targeting NDH-2. The binding of inhibitors to the dehydrogenase enzyme restricts the conversion of the reduced NADH to oxidised NAD+, thereby leading to a surge in the NADH level. An increased level of NADH further binds with the circularly permuted T-sapphire, thereafter leading to a hike in green fluorescence as compared to no inhibition in the control. This indicates the inhibition of the enzyme by NDH-2 inhibitors. Thus, the proposed inhibitors of NDH-2 were expected to alter the NADH/NAD+ ratio. Hence, when the bacilli were exposed to C3, C4 and C11 for 24 h, significant inhibition of the activity of NDH-2 by C3 and C4 was observed when compared with CBR-5992 and a negative control (drug-free control, black bars) (Fig. 3). Other control drugs, CLFZ and rotenone (Rot) have failed to produce a significant change in the NADH/NAD+ ratio. CLFZ is itself reduced in the presence of the NDH-2 enzyme, thereby producing oxidative stress and faster oxidation of NADH to NAD+ with less or no change in the NADH/NAD+ ratio as compared to direct inhibitors of NDH-2. Rot, which is an inhibitor of type I NADH, partially alters the NADH/NAD+ ratio.

Fig. 3. Peredox-mCherry expressing Mtb strain responds to changes in the NADH : NAD+ ratio. The data are presented as mean ± SD in triplicates for each concentration of compounds. The significance difference was determined by using one way ANOVA followed by Dunnett's test.

Fig. 3

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Cell cytotoxicity study

The MTT assay was performed to evaluate the cytotoxic effect of C3, C4 and C11 against the HepG2 cell line. The cytotoxicity concentration (CC50) for all three compounds was in the range of 89–247 μg ml−1, as depicted by the graph (S2, ESI†) when log concentration values were plotted in the X-axis against the absorbance in the Y-axis. Further, it was noted that the safety index (SI) is >10 for C3 and C4. For C11, it was less than 10. Hence, it was concluded that C3 and C4 are safer than C11 (Table 4).

Cytotoxicity and selectivity index profile of the compoundsa.

Compound code CC50 (μg ml−1) Safety index (CC50/MIC90)
C3 247.2 ± 0.45 15.45
C4 423.2 ± 0.17 26.48
C11 89.41 ± 0.95 5.58
CBR-5992 48.83 ± 0.91 390.6
Clofazimine 49.02 ± 0.47 222.81
Tamoxifen 6.15 ± 0.55
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a

All the assays have been repeated in triplicate.

Molecular docking study

To visualize the probable mode of interaction of our synthesized compounds at the binding site, an in silico docking study was conducted post-synthesis and biological assays. We attempted to relate our biological assay results with the computational outcome. Since, no crystal structure of Mtb NDH-2 is reported in the literature, homology modelling was performed to develop the 3D protein structure using the Swiss-model. The protein structure with Uniprot ID: P95160 associated with the Mtb H37Rv strain was used as a template and the entire protein was placed inside the search space while performing docking, since no defined ligand binding pocket is known yet. The binding interactions of CBR-5992, C3, C4 and C11 are depicted in Fig. 4 and the binding interactions of all the synthesized compounds are included in the ESI† (S3, ESI†). The binding energy values (of CBR-5992, C3, C4 and C11) are mentioned in Table 5.

Fig. 4. Binding interactions of a) CBR-5992, b) C3, c) C4, and d) C11.

Fig. 4

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Binding affinity values of docked compounds.

Comp. code Binding affinity (Kcal mol−1) Interacting amino acid residues
CBR-5992 −7.9 PROA:53, THRA:181, ALAA:333, META:370, TRPA:396, HISA:400
C3 −6.9 LYSA:341, THRA:346, ALAA:349, ALAA:354, ARGA:359
C4 −8.0 ARGA:45, ALAA:119, ALAA:336
C11 −7.1 ARGA:45, LEUA:50, GLNA:52, ALAA:119
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A high binding affinity value (Table 5) is an indication of strong ligand binding affinity and stronger protein–ligand interaction at the binding site. Following the 2D and 3D interaction envisagement, a rudimentary idea about the nature of interactions at the binding site was generated. Most of the interactions in the binding site are hydrophobic alkyl and pi–alkyl interactions. C4 has the highest binding affinity value of all the synthesized derivatives. In C3, ALAA:354, ARGA:359 amino acid residues have participated in pi–alkyl interactions with the delocalized pi electron cloud of the benzothiazole core. The two methyl groups at the C-4 and C-6 positions of the benzothiazole ring system were involved in alkyl interactions with ALAA:349, ALAA:354, ARGA:359 residues. LYSA:341 has produced an alkyl interaction with the cyclohexyl ring of the amide side chain. In C4, the benzothiazole core was involved in pi–alkyl interactions with ARGA:45, ALAA:119 amino acid residues, similar to that observed in C3. ALAA:336 has participated in an additional alkyl interaction with the cyclohexyl ring. We observed that, though C4 possesses the highest binding affinity value, it has very few interactions at the ligand binding site. CBR-5992, which displayed weaker binding affinity than C4, has displayed superior inhibitory potential in biological evaluation. In CBR-5992, the side chain sulfur moiety was involved in pi–sulfur interactions with TRPA:396, HISA:400 amino acid residues. Introduction of a bromine atom at the C-4 position of C11 leads to an additional pi–cation interaction at the thiazole moiety, which is absent in C3 and C4.

From all the binding affinity values and the interacting amino acid residues of C1–C14, no consistent trend in the in silico analysis data and biological assay results was noted. Due to this disagreement, we assume that, so far, in vitro high throughput screening is the most adopted route by researchers for identification of Mtb NDH-2 inhibitors. More detailed information about the binding site is essential to bridge the variation between computational prediction and in vitro assay results.

Conclusions

NDH-2 emerged as a promising and safe target for anti-TB drug discovery, as the inhibition of this enzyme confirms the alteration in NAD(H) homeostasis in Mtb, resulting in depletion in the intracellular ATP level with no alteration in the human ETC function. Upon inhibiting NDH-2, the desired bactericidal response was achieved as Mtb does not possess any alternative energy metabolism pathway to meet the required energy needed for survival. In this work, we have synthesized a series of 2-mercaptobenzothiazole derivatives C1–C14 and performed in vitro studies and computational analysis to examine their antitubercular potential and NDH-2 inhibitory response. Compounds C3 and C4 are the potent compounds in the series, with safety index values higher than 10. C4 has the highest bactericidal response. Also, the Peredox-mCherry assay confirmed that NDH-2 is the site of action for C4, where it has a superior NDH-2 inhibitory profile compared to our standard reference CB4-5992. Also, we have reported the Mtb bactericidal response of our active compounds in monotherapy, which opens possibilities for future work on this scaffold to enhance the inhibitory response by making further modifications.

Experimental section

Materials and methods (chemistry)

All chemicals were purchased from Sigma-Aldrich, TCI Chemicals, SRL Chemicals, and Avra, and used as received. Molychem silica gel (60–120 mesh) was used for column chromatography, and thin-layer chromatography was performed on Merck pre-coated silica gel 60-F254 plates. All other chemicals and solvents were obtained from commercial sources and purified using standard methods.

Procedure for the synthesis of 2-bromo-N-cyclohexylacetamide (A)

An oven-dried round bottom flask was charged with EDC·HCl (1 mmol, 1 equiv.), bromoacetic acid (1 mmol, 1 equiv.), and cyclohexyl amine (1 mmol, 1 equiv.) in DCM. The reaction mixture was stirred at room temperature for 30 minutes. Next, DIPEA (0.5 equivalent) was added to the ongoing reaction and the reaction mixture was allowed to stir overnight. After completion of the reaction, the reaction mixture was extracted with ethyl acetate (15 ml × 3). The combined organic layers were washed with brine, dried over Na2SO4, and concentrated in a vacuum.

Procedure for the synthesis of benzo[d]thiazole-2-thiol derivatives (B)

An oven-dried round bottom flask was charged with 2-bromoaniline bearing the corresponding substituent (1 equiv., 5 mmol) and potassium ethyl xanthate (1.5 equiv.) in anhydrous DMF under a nitrogen atmosphere. The reaction mixture was stirred at 120 °C for 4–8 h under the mentioned inert atmosphere. Upon completion, the reaction mixture was cooled to room temperature and diluted with water (8 ml) and acidified to pH 3–4 by adding 1 N HCl. The formed suspension was subjected to uniform stirring for 30 minutes followed by filtration. The crude product (B) was collected by filtration under vacuum.

Procedure for the synthesis of 2-(benzo[d]thiazol-2-ylthio)-N-cyclohexylacetamide derivatives (C1–C14)

An oven-dried round bottom flask was charged with 2-bromo-N-cyclohexylacetamide (A) (1 mmol, 1 equiv.) and the corresponding substituted benzo[d]thiazole-2-thiol derivatives (B) (1 mmol, 1 equiv.) in DMF. The reaction mixture was stirred at room temperature for 2–4 h. After completion of the reaction, the reaction mixture was extracted with ethyl acetate (15 ml × 3). The combined organic layers were washed with brine, dried over Na2SO4, and concentrated in a vacuum. The residue was purified by column chromatography on silica gel to afford the desired products (C1–C14).

Procedure for the synthesis of CBR-5992

An oven-dried round bottom flask was charged with anthranilamide (1 equiv., 5 mmol) and potassium ethyl xanthate (1.5 equiv.) in anhydrous DMF under a nitrogen atmosphere. The reaction mixture was stirred at 120 °C for 4 h under the mentioned inert atmosphere. Upon completion, the reaction mixture was cooled to room temperature and diluted with water (8 ml) and acidified to pH 3–4 by adding 1 N HCl. The formed suspension was subjected to uniform stirring for 30 minutes followed by filtration. The crude product was collected by filtration under vacuum.

In the next step, an oven-dried round bottom flask was charged with 2-bromo-N-cyclohexylacetamide (A) (1 mmol, 1 equiv.) and the vacuum dried product of the previous step (1 mmol, 1 equiv.) in DMF. The reaction mixture was stirred at room temperature for 2 h. After completion of the reaction, the reaction mixture was extracted with ethyl acetate (15 ml × 3). The combined organic layers were washed with brine, dried over Na2SO4, and concentrated in a vacuum. The residue was purified by column chromatography on silica gel to afford the desired product.

N-Cyclohexyl-2-((4-oxo-3,4-dihydroquinazolin-2-yl)thio)acetamide (CBR-5992)

Colourless solid, yield 80%. 1H NMR (DMSO-d6,500 MHz) δ 12.68 (s, 1H), 8.17 (d, J = 8 Hz), 7.79–7.76 (m, 1H), 7.49 (d, J = 8.5 Hz, 1H), 7.44–7.41 (m, 1H), 3.93 (s, 2H), 3.56–3.50 (m, 1H), 1.74–1.71 (m, 2H), 1.68–1.65 (m, 2H), 1.55–1.52 (m, 1H), 1.26–1.14 (m, 5H); 13C NMR (DMSO-d6, 125 MHz) δ 166.4, 161.6, 155.9, 148.8, 135.1, 126.5, 126.3, 126.2, 120.4, 48.4, 34.6, 37.7, 25.6, 24.9.

2-(Benzo[d]thiazol-2-ylthio)-N-cyclohexylacetamide (C1)

Colourless solid, yield 89%. 1H NMR (DMSO-d6,500 MHz) δ 8.21 (d, J = 8 Hz, 1H), 8.03–8.01 (m, 1H), 7.83–7.81 (m, 1H), 7.49–7.43 (m, 1H), 7.38–7.35 (m, 1H), 4.19(s, 2H), 3.58–3.52 (m, 1H), 1.76–1.66 (m, 4H), 1.55–1.52 (m, 1H), 1.25–1.17 (m, 5H); 13C NMR (DMSO-d6, 125 MHz) δ 166.8, 166.6, 153.1, 136.2, 126.9, 124.9, 122.3, 121.5, 48.4, 37.3, 32.6, 25.6, 24.8. HRMS (ESI-TOF) m/z: [M + H]+ calcd. for C15H18N2OS2: 307.4500; found: 307.0939.

N-Cyclohexyl-2-((6-methylbenzo[d]thiazol-2-yl)thio)acetamide (C2)

Colourless, yield 85%. 1H NMR (DMSO-d6, 500 MHz) δ 8.21 (d, J =7.5 Hz, 1H), 7.81 (s, 1H), 7.70 (d, J = 8.5 Hz, 1H), 7.29 (d, J = 8.5 Hz, 1H), 4.09 (s, 2H), 3.54 (m, 1H), 2.41 (s, 1H), 1.74–1.72 (m, 2H), 1.69–1.65 (m, 2H), 1.55–1.52 (m, 1H), 1.30–1.13 (m, 5H); 13C NMR (DMSO-d6, 125 MHz) δ 165.7, 165.4, 151.2, 135.3, 134.6, 128.2, 121.9, 121.0, 48.4, 37.3, 32.6, 25.6, 24.8, 21.4. HRMS (ESI-TOF) m/z: [M + H]+ calcd. for C16H21N2OS2: 321.1095; found: 321.1089.

N-Cyclohexyl-2-((4,6-dimethylbenzo[d]thiazol-2-yl)thio)acetamide (C3)

Colourless solid, yield 83%. 1H NMR (DMSO-d6,500 MHz) δ 8.19 (d, J = 8 Hz), 7.61 (s, 1H), 7.12 (s, 1H), 4.07 (s, 2H), 3.57–3.53 (m, 1H), 2.56 (s, 3H), 2.37 (s, 3H), 1.76–1.73 (m, 2H), 1.69–1.65 (m, 2H), 1.55–1.52 (s, 1H), 1.27–1.12 (m, 5H); 13C NMR (DMSO-d6, 125 MHz) δ165.8, 164.0, 154.4, 135.1, 134.3, 130.5, 128.7, 119.2, 48.5, 37.3, 32.7, 25.6, 24.9, 21.4, 18.3. HRMS (ESI-TOF) m/z: [M + H]+ calcd. for C17H24N2OS2: 335.1252; found: 335.1250.

N-Cyclohexyl-2-((6-isopropylbenzo[d]thiazol-2-yl)thio)acetamide (C4)

Colourless solid, yield 78%. 1H NMR (DMSO-d6,500 MHz) δ 8.21 (d, J= 7.5 Hz, 1H), 7.84 (d, J = 1.5 Hz, 1H), 7.23 (d, J = 8.5 Hz, 1H), 7.36 (d, J = 1.5, 4.25 Hz, 1H), 4.09 (s, 1H), 3.581–3.510 (m, 1H), 3.03–2.98 (m, 1H), 1.75–1.72 (m, 2H), 1.67–1.65 (m, 1H), 1.55–1.52 (m, 1 H), 1.30–1.12 (m, 12H); 13C NMR (DMSO-d6, 125 MHz) δ165.65, 165.57, 151.43, 145.62, 135.39, 125.73, 121.15, 119.36, 48.45, 37.32, 33.90, 32.66, 25.61, 24.82, 24.49. HRMS (ESI-TOF) m/z: [M + H]+ calcd. for C18H25N2OS2: 349.1408; found: 349.1402.

N-Cyclohexyl-2-((6-fluorobenzo[d]thiazol-2-yl)thio)acetamide (C5)

Colourless solid, yield 88%. 1H NMR (DMSO-d6,500 MHz) δ 8.21 (d, J = 8 Hz, 1H), 7.97 (dd, J = 9 Hz, 3 Hz, 1H), 7.83 (q, J = 5 Hz, 1H), 7.34 (td, J = 9 Hz, 2.5 Hz, 1H), 4.11 (s, 2H), 3.57–3.51 (m, 1H), 1.75–1.72 (m, 2H), 1.69–1.65 (m, 2H), 1.55–1.53 (m, 1H), 1.27–1.13 (m, 5H); 13C NMR (DMSO-d6, 125 MHz) δ 166.6, 149.9, 136.5 (d, JF–C = 3.12 Hz), 122.5 (d, JF–C = 9.6 Hz), 115.0 (d, JF–C = 24.25 Hz), 109.1, 108.9, 48.5, 37.4, 32.7, 25.6, 24.8; 19F NMR (DMSO-d6, 471 MHz,) δ −117.18 to −117.23 (m, 1F). HRMS (ESI-TOF) m/z: [M + H]+ calcd. for C15H18FN2OS2: 325.0845; found:325.0840.

2-((6-Chlorobenzo[d]thiazol-2-yl)thio)-N-cyclohexylacetamide (C6)

Colourless solid, yield 86%. 1H NMR (DMSO-d6,500 MHz) δ 8.22 (d, J = 8 Hz, 1H), 8.19 (d, J = 2.5 Hz, 1H), 7.80 (d, J = 9 Hz, 1H), 7.50 (dd, J = 8.75 Hz, 2.5 Hz,1H), 4.12, (s, 2H), 3.93–3.52 (m, 1H), 1.75–1.72 (m, 2H), 1.69–1.66 (m, 2H), 1.55–1.53 (m, 1H), 1.28–1.14 (m, 5H); 13C NMR (DMSO-d6, 125 MHz) δ 168.2, 165.5, 151.9, 136.8, 129.3, 127.3, 122.5, 122.0, 48.3, 37.4, 32.6, 25.6, 24.8. HRMS (ESI-TOF) m/z: [M + H]+ calcd. for C15H18ClN2OS2: 341.0549; found: 341.0545.

2-((6-Bromobenzo[d]thiazol-2-yl)thio)-N-cyclohexylacetamide (C7)

Colourless solid, yield 85%. 1H NMR (DMSO-d6, 500 MHz) δ 8.32 (d, J = 2 Hz, 1H), 8.23 (d, J = 8 Hz, 1H), 7.75 (d, J = 8.5 Hz, 1H), 7.62 (dd, J = 6.5 Hz, 2 Hz, 1H), 4.11 (s, 2H), 3.57–3.51 (m, 1H), 1.75–1.72 (m, 2H), 1.69–1.65 (m, 2H), 1.55–1.53 (m, 1H), 1.27–1.14 (m, 5H); 13C NMR (DMSO-d6, 125 MHz) δ = 168.3, 165.5, 152.1, 137.2, 130.0, 124.9, 122.9, 117.3, 48.5, 37.3, 32.6, 25.6, 24.8. HRMS (ESI-TOF) m/z: [M + H]+ calcd. for C15H17BrN2OS2: 385.0044; found: 385.0039.

N-Cyclohexyl-2-((6-(trifluoromethyl)benzo[d]thiazol-2-yl)thio)acetamide (C8)

Colourless solid, yield 86%. 1H NMR DMSO-d6, 500 MHz) δ 8.56 (s, 1H), 8.25 (d, J= 7.5 Hz, 1H), 7.97 (d, J = 8.5 Hz, 1H), 7.78 (dd, J = 1.5 Hz, 8.5 Hz, 1H), 4.16 (s, 2H), 3.56–3.54 (m, 1H), 1.76–1.73 (m, 2H), 1.69–1.66 (m, 2H), 1.55–1.52 (m, 1H), 1.29–1.12 (m, 5H); 13C NMR (DMSO-d6, 125 MHz) δ = 171.8, 165.4, 155.3, 135.8, 125.9, 125.1, 124.9, 123.8 (q, JF–C = 15 Hz), 121.9, 120.4 (q, JF–C = 14.5 Hz), 48.5, 37.4, 32.6, 25.6, 24.8; 19F NMR (DMSO-d6, 471 MHz) δ = −79.71 (s, 3F). HRMS (ESI-TOF) m/z: [M + H]+ calcd. for C16H18F3N2OS2: 375.0813; found: 375.0810.

N-Cyclohexyl-2-((6-(trifluoromethoxy)benzo[d]thiazol-2-yl)thio)acetamide (C9)

Colourless solid, yield 84%. 1H NMR (DMSO-d6, 500 MHz) δ 8.24 (d, J = 8 Hz, 1H), 8.19 (d, J = 1.5 Hz, 1H), 7.89 (d, J = 8.5 Hz, 1H), 7.48–7.46 (m, 1H), 4.13 (s, 2H), 3.58–3.53 (m, 1H), 1.75–1.72 (m, 2H), 1.69–1.65 (m, 2H), 1.55–1.52 (m, 1H), 1.29–1.12 (m, 5H); 13C NMR (DMSO-d6, 125 MHz) δ 169.2, 165.5, 151.9, 145.2, 136.4, 122.4, 120.6 (d, JF–C = 2.04 Hz), 120.6, 115.6, 48.5, 37.4, 32.7, 25.7, 24.8; 19F NMR (DMSO- d6, 471 MHz) δ −58.05 (s, 3F). HRMS (ESI-TOF) m/z: [M + H]+ calcd. for C16H18F3N2OS2: 391.0762; found: 391.0760.

N-Cyclohexyl-2-((6-nitrobenzo[d]thiazol-2-yl)thio)acetamide (C10)

Yellow solid, yield 85%. 1H NMR (DMSO-d6,500 MHz) δ 9. 23 (d, J = 2 Hz, 1H), 8.32–8.27 (m, 2H), 7.97 (d, J = 8.5 Hz, 1H), 4.20 (s, 2H), 3.58–3.53 (m, 1H), 1.76–1.74 (m, 2H), 1.70–1.67 (m, 2H), 1.60–1.54 (m, 1H), 1.28–1.4 (m, 5); 13C NMR (DMSO-d6, 125 MHz) δ 175.0, 165.3, 156.9, 144.0, 136.0, 122.5, 121.5, 119.4, 48.6, 37.6, 32.7, 25.6, 24.9. HRMS (ESI-TOF) m/z: [M + H]+ calcd. for C15H17Br F3N3O3S2: 352.0790; found:352.0783.

2-((4-Bromo-6-fluorobenzo[d]thiazol-2-yl)thio)-N-cyclohexylacetamide (C11)

Colourless solid, yield 82%. 1H NMR (DMSO-d6, 500 MHz) δ 8.22 (d, J = 7.5 Hz, 1H), 8.02 (dd, J = 8 Hz, 2.5 Hz, 1H), 7.73 (dd, J = 8.5 Hz, 2.5 Hz, 1H), 4.11 (s, 1H), 3.99 (s, 1H), 1.76–1.65 (m, 4H), 1.55–1.53 (m, 1H), 1.27–1.22 (m, 5H); 13C NMR (CDCl3, 125 MHz) δ 167.9 (d, JF–C = 2.5HZ), 167.1, 159.35(d, JF–C = 248.75HZ), 147.5 (d, JF–C = 2.5HZ), 136.4 (d, JF–C = 7.5HZ), 118.3 (d, JF–C = 27.5HZ), 114.4 (d, JF–C = 12 HZ), 107.1 (d, JF–C = 27.5HZ), 48.8, 36.9, 32.9, 25.4, 24.8; 19F NMR (DMSO-d6, 471 MHz) δ −115.64 (t, 1F). HRMS (ESI-TOF) m/z: [M + H]+ calcd. for C15H17BrFN2OS2: 402.9950; found: 402.9942.

2-((4-Bromo-6-(trifluoromethyl)benzo[d]thiazol-2-yl)thio)-N-cyclohexylacetamide (C12)

Colourless solid, yield 84%. 1H NMR (DMSO-d6,500 MHz) δ 8.61–8.60 (m, 1H), 8.26 (d, J = 8 Hz, 1H), 8.06 (d, J = 1 Hz, 1H), 4.18 (s, 2H), 3.57–3.54 (m, 1H), 1.78–1.75 (m, 2H), 1.70–1.66 (m, 1H), 1.56–1.53 (m, 1H), 1.27–1.14 (m, 5H); 13C NMR (CDCl3, 125 MHz) δ 172.1, 166.8, 152.5, 136.1, 128.0, 127.7, 126.8 (q, JF–C = 2.5 Hz), 124.2, 122.1, 118.0 (q, JF–C = 3.75 Hz), 114.8, 48.9, 36.6, 32.9, 25.4, 24.9; 19F NMR (CDCl3, 471 MHz) δ −61.52 (s, 3F). HRMS (ESI-TOF) m/z: [M + H]+ calcd. for C16H17Br F3N2OS2: 452.9918; found: 9910.

2-((4-Bromo-6-(trifluoromethoxy)benzo[d]thiazol-2-yl)thio)-cyclohexylacetamide (C13)

Colourless solid, yield 81%. 1H NMR (DMSO-d6,500 MHz) δ 8.26–8.23 (m, 2H), 7.82–7.81 (m, 1H), 4.41 (s, 2H), 3.59–3.52 (m, 1H), 1.77–1.74 (m, 2H), 1.69–1.65 (m, 2H), 1.60–1.53 (m, 1H), 1.27–1.12 (m, 5H); 13C NMR (DMSO-d6, 125 MHz) δ 170.8, 166.3, 158.3, 150.2, 136.6, 123.8, 119.5, 115.1, 114.1, 48.7, 37.6, 32.7, 25.6, 24.9; 19F NMR (DMSO-d6, 471 MHz) δ −57.18 (s, 3F). HRMS (ESI-TOF) m/z: [M + H]+ calcd. for C16H17BrF3N2O2S2: 468.9867; found: 468.9859.

N-Cyclohexyl-2-(naphtho[1,2-d]thiazol-2-ylthio)acetamide (C14)

Colourless solid, yield 86%. 1H NMR (CDCl3,500 MHz) δ 8.71–8.69 (m, 1H), 8.36–8.35 (m, 1H), 8.15 (s, 1 H), 7.76–7.70 (m, 2H), 7.26 (s, 1 H), 4.06 (s, 2H), 3.84–3.77 (m, 1H), 1.88–1.86 (m, 2H), 1.60–1.51 (m, 3H), 1.35–1.27 (m, 2H), 1.13–1.00 (m, 3H); 13C NMR (CDCl3, 125 MHz) δ 166.96, 165.54, 148.55, 131.93, 130.27, 127.92, 127.82, 127.96, 123.96, 122.07, 119.69, 48.60, 36.75, 32.77, 25.39, 24.59. HRMS (ESI-TOF) m/z: [M + H]+ calcd. for C19H21N2OS2: 357.1095; found: 357.1090.

Materials and methods (biology)

Mtb H37Ra was procured from ATCC, USA. Mtb mc26230, mc28243, and mc28259 were a kind gift from Prof. William Jacob Jr. (Albert Einstein College of Medicine, USA). Isoniazid, rifampicin, and catalase were procured from Himedia Labs. Bovine serum albumin (BSA) and DMSO were purchased from Sigma-Aldrich (St Louis, MO, USA). 7H9 Middlebrook broth and 7H10 Middlebrook agar were procured from BD Difco Laboratories, USA.

Bacteria growth conditions

The synthesized molecules were screened in vitro for determining the MIC against Mtb H37Ra ATCC 25177 and derivative BSL2 strains of Mtb H37Rv, i.e. Mtb mc26230, Mtb mc28243 (isoniazid resistant), Mtb mc28247 (rifampicin resistant), and Mtb mc28259 (MDR-both isoniazid and rifampicin resistant). The bacteria were cultured in Middlebrook (BD Difco Laboratories) 7H9 liquid broth medium supplemented with 0.2% glycerol, 0.05% Tween 80, and 10% albumin–dextrose–saline (ADS) and were maintained at 37 °C in the presence of 5% CO2, while the derivative strains of H37Rv were cultured using the above-mentioned media except Tween 80 (tyloxapol was used with the same concentration) supplemented with l-methionine (50 mg L−1), l-leucine (50 mg L−1), l-arginine (200 mg L−1), and d-pantothenic acid (24 mg L−1) as supplements.

In vitro determination of the minimum inhibitory concentration (MIC)

To determine the MIC of the synthesized compounds, the liquid broth micro-dilution method was used in 96-well round bottom microtiter plates (Corning, USA) as mentioned in ref. 29. Briefly, the bacterial cultures were grown to a mid-log phase (i.e. OD600nm range of 0.4–0.6) which were equivalent to 1.0 × 108 CFU ml−1. The final inoculum was ready by diluting it to an OD600 of 0.005 (1.0 × 106 CFU ml−1). The test compounds were two-fold serially diluted in a volume of 100 μl media in each well. Finally, 100 μl of mycobacterial strains were dispensed into each well of the plate and were incubated at 37 °C for 10 days. After completion of the incubation period, the plates were read visually and the minimum concentration of the compound displaying no turbidity was considered as the MIC. In this assay, isoniazid and rifampicin were kept as standard positive controls.

Determination of the minimum bactericidal concentration (MBC)

To determine the MBC90 of the compounds, the bacterial inoculum was taken from the mid log phase (i.e. OD600nm range of 0.4–0.6) and was adjusted at an OD 600 nm of 0.005 as per ref. 30. The bacteria were exposed to different concentrations of the compounds for 5 days at 37 °C. After 5 days of incubation, the bacteria were 10-fold serially diluted and spotted on 7H10 agar plates to determine the colony forming units (CFU), and the agar plates were incubated for 3 to 4 weeks. A reduction of 90% bacterial population as compared to the initial inoculum was considered as the MBC90. Clofazimine and BDQ were used as positive controls.

Determination of intracellular ATP depletion

The BacTiter-Glo™ microbial cell viability assay (Promega, USA) was used to quantify the ATP inside the bacteria as per ref. 31. Here, the bacteria were harvested from the mid log phase and adjusted to an OD: 600 nm of 0.05. The compounds were added with the desired concentration into each well and 100 μl of cultures were dispensed in 96-well flat bottom white opaque microtiter plates (Corning, USA). The plates were incubated at 37 °C for 24 h. Further, 25 μl of the BacTiter-Glo reagent was added to each well and kept for 12 min inside the incubator. The luminescence reading was taken using a BioTek Cytation 5 multi-mode reader and the data were analyzed using GraphPad Prism 8 software. In this assay, BDQ was used as a standard drug.

Fluorimetric based determination of the NADH/NAD+ ratio

The NADH/NAD+ determination assay was performed using the Mtb strain transformed with pMV762-Peredox-mCherry. The Mtb-Peredox-mCherry strain was grown in a suitable 7H9 medium supplemented with required supplements to a log phase (OD600nm 0.4–0.6). The compounds were dispensed into each well as per the desired concentration and 200 μl of the bacterial suspension was directly inoculated per well in 96-well black clear flat bottom plates (Corning, USA). The plates were incubated at 37 °C for 24 h and the spectra were measured using a BioTek Cytation 5 multi-mode reader having excitation wavelengths of 400 nm and 587 nm and emission was taken at 510 nm and 615 nm, respectively.32,33 The ratio (green/red) was plotted using GraphPad Prism 8 software to determine the NADH/NAD+ ratio in the bacterial cells. In this assay, clofazimine was used as a control.

Determination of cytotoxicity using the HepG2 cell line

The cell cytotoxicity of the compounds was evaluated using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay in the human hepatocellular carcinoma HepG2 cell line (ATCC HB-8065) in DMEM glucose supplemented with 10% foetal bovine serum (FBS), streptomycin (100 μg ml−1), penicillin (75 μg ml−1), HEPES (10 mM), and l-glutamine (2 mM) at 37 °C. Cells were seeded in a 96-well flat bottom plate (Tarsons) at a density of 1 × 104 cells per well and incubated for 24 h (37 °C; 5% CO2). Ten two-fold serial dilutions of the test compounds were made with a range from 1000–0.48 μg ml−1. MTT was added at a concentration of 2.5 mg ml−1 dissolved in phosphate buffer saline. Cell viability was determined using a BioTek Cytation 5 Cell Imaging multi-mode reader by measuring the absorbance of the reduced formazan dissolved in DMSO at 570 nm. Cytotoxicity was reported as CC50, the concentration that causes a 50% reduction in cell viability, and data were analysed using GraphPad prism 8.34 Tamoxifen was used as a standard in this assay.

Determination of the selectivity index (SI)

The selectivity index of the test compounds was calculated by the ratio of cytotoxicity (CC50) to the biological activity (MIC) of a compound. For a compound to be in the therapeutic window, the SI should be >10.35

Materials and methods (molecular docking study)

The structure of type II NADH:quinone oxidoreductase Ndh of Mtb, strain ATCC 25618/H37Rv was used as template (GMQC: 0.93) for homology modelling (Uniprot ID: P95160). The structural refinement of the ligands was done using AutoDock 1.5.7 prior docking. Docking was performed using AutoDock Vina 1.1.2.36 The input is provided in the form of a receptor, a ligand and a configuration file consisting information on the size and centers of the grid box, while the output is in the form of a list of poses ranked by a score that corresponds to the predicted binding energy (ΔG, kcal mol−1). The exhaustiveness was set at 8, the number of binding modes was set at 9 and the energy of all the binding poses was fixed at 3 kcal mol−1. Docking of the library of compounds (CBR-5992, C1–C14) against the homology modelled macromolecular structure of Mtb Ndh-2 was done following blind docking where the entire protein was placed inside the search place. All the bound small molecules were manually removed before docking. After the ligands were docked against the receptor proteins, the best docked poses of all the compounds and the interactions with the binding residues of the proteins were visualized using BIOVIA Discovery Studio Visualiser v21.1.0.20298.

Author contributions

Pallavi Saha and Shashikanta Sau have contributed equally to this work. Deepak K. Sharma and Nitin Pal Kalia contributed to the design and implementation of the research.

Conflicts of interest

There are no conflicts to declare.

Supplementary Material

MD-015-D4MD00118D-s001

Acknowledgments

The authors PS and SS thank IIT (BHU), NIPER Hyderabad and the Ministry of Education, India for providing a teaching assistant fellowship. Deepak K. Sharma and Nitin Pal Kalia acknowledge SERB-CRG 2023/003559.

†

Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4md00118d

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