Journal Pre-proofs

Design, Synthesis, and Biological Evaluation of Benzo[d]imidazole-2-carbox-

amides as New Anti-TB Agents

Tejas M. Dhameliya, Kshitij I. Patel, Rishu Tiwari, Siva Krishna Vagolu,

Dulal Panda, Dharmarajan Sriram, Asit K. Chakraborti

PII: S0045-2068(20)31836-8
DOI: https://doi.org/10.1016/j.bioorg.2020.104538
Reference: YBIOO 104538

To appear in: Bioorganic Chemistry

Received Date: 12 July 2020

Revised Date: 17 October 2020
Accepted Date: 4 December 2020

Please cite this article as: T.M. Dhameliya, K.I. Patel, R. Tiwari, S. Krishna Vagolu, D. Panda, D. Sriram, A.K.
Chakraborti, Design, Synthesis, and Biological Evaluation of Benzo[d]imidazole-2-carboxamides as New Anti-
TB Agents, Bioorganic Chemistry (2020), doi: https://doi.org/10.1016/j.bioorg.2020.104538

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 Design, Synthesis, and Biological Evaluation of

Benzo[d]imidazole-2-carboxamides as New Anti-TB

Agents

Tejas M. Dhameliya,a Kshitij I. Patel,a Rishu Tiwari,b Siva Krishna Vagolu,c Dulal Panda,b

Dharmarajan Sriram,c and Asit K. Chakrabortia,d,*

aDepartment of Medicinal Chemistry, National Institute of Pharmaceutical Education and

Research (NIPER), Sector 67, S.A.S. Nagar 160 062, Punjab, India.
bDepartment of Biosciences & Bioengineering, Indian Institute of Technology Bombay, Mumbai

400 076, India.

cDepartment of Pharmacy, Birla Institute of Technology & Science – Pilani, Hyderabad Campus,

Jawahar Nagar, Hyderabad 500 078, India.

dPresent Address: Department of Chemistry, Indian Institute of Technology – Ropar, Rupnagar,

Punjab 140 001, India.

*Corresponding Author’s E-mail: akchakraborti@niper.ac.in; chakrabortiak@iitrpr.ac.in;

akchakraborti@rediffmail.com.

1
Graphical Abstract

Design, Synthesis, and Biological Evaluation of

Benzo[d]imidazole-2-carboxamides as New Anti-TB
Agents
Tejas M. Dhameliya,a Kshitij I. Patel,a Rishu Tiwari,b Siva Krishna Vagolu,c Dharmarajan Sriram,c
Dulal Panda,b and Asit K. Chakrabortia,d,*
aDepartment of Medicinal Chemistry, National Institute of Pharmaceutical Education and

Research (NIPER), Sector 67, S.A.S. Nagar, Punjab 160062, India.

bDepartment of Biosciences & Bioengineering, Indian Institute of Technology Bombay, Mumbai,

Maharashtra 400 076, India.

cDepartment of Pharmacy, Birla Institute of Technology & Science – Pilani, Hyderabad Campus,

Jawahar Nagar, Hyderabad, Telengana 500 078, India.

dPresent Address: Department of Chemistry, Indian Institute of Technology - Ropar, Rupnagar,

Punjab 140001, India.

*Corresponding Author: akchakraborti@niper.ac.in; chakrabortiak@iitrpr.ac.in;
akchakraborti@rediffmail.com.

N O

N HN R1

(L)
Benzo[d]imidazole-2-carboxamides
as new anti-TB chemotypes
13 Anti-mycobacterial Hits with MIC 0.78-6.25 µg/mL
Non-cytotoxic (RAW 264.7 cell lines)

The benzo[d]imidazole-2-carboxamide scaffold has been identified as novel anti-TB chemotype

with thirteen new hits synthesized through the new and environment friendly methodology.

2
Highlights

Design, Synthesis, and Biological Evaluation of

Benzo[d]imidazole-2-carboxamides as New Anti-TB
Agents
Tejas M. Dhameliya, † Kshitij I. Patel,† Rishu Tiwari, § Siva Krishna Vagolu,‡ Dharmarajan
Sriram,‡ Dulal Panda,§ and Asit K. Chakraborti†*
†Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and

Research (NIPER), Sector 67, S.A.S. Nagar 160 062, Punjab, India.
§Department of Biosciences & Bioengineering, Indian Institute of Technology Bombay, Mumbai

400 076, India.

‡Department of Pharmacy, Birla Institute of Technology & Science – Pilani, Hyderabad Campus,

Jawahar Nagar, Hyderabad 500 078, India.

*Corresponding Author: Tel: 91-(0)-172 229 2027; Fax: 91-(0)-172-2214692. E-mail:
akchakraborti@niper.ac.in; akchakraborti@rediffmail.com.

 Benzo[d]imidazole-2-carboxamide moiety has been designed as new anti-TB chemotype

through scaffold hopping.
 Twentyseven target compounds were synthesized using green synthetic protocol.
 Thirteen hits with MIC values of 0.78-6.25 µg/mL.
 All active compounds are non-cytotoxic (< 50% inhibition of RAW 264.7 cell lines at 50
µg/mL).
 The most potent compounds exhibited MIC values of 0.78 µg/mL with selectivity index >
60.
 The active compounds have favorable Lipniski parameters with high (10.66-10.77) LipE
values higher than that of empirically derived value of > 6 demonstrating their drug
likeliness.

3
Abstract

Tuberculosis is the leading cause of death globally among infectious diseases. Due to the

development of resistance of Mycobacterium tuberculosis to currently used anti-TB medicines and

the TB-HIV synergism the urgent need to develop novel anti-mycobacterial agents has been

realized. The drug-to-target path has been the successful strategy for new anti-TB drug

development. All the six drug candidates that have shown promise during the clinical trials and

some of these being approved for treatment against MDR TB are the results of phenotype screening

of small molecule compound libraries. In search of compounds belonging to novel pharmacophoric

class that could be subjected to whole cell assay to generate new anti-TB leads the

benzo[d]imidazole-2-carboxamide moiety has been designed as a novel anti-TB scaffold. The

design was based on the identification of the benzimidazole ring as a prominent substructure of

the FDA approved drugs, the structural analysis of reported anti-TB benzimidazoles, and the

presence of the C-2 carboxamido functionality in novel bioisoteric anti-TB benzothiazoles.

Twenty seven final compounds have been prepared via NH4Cl-catalyzed amidation of ethyl

benzo[d]imidazole-2-carboxylates, as the required intermediates, obtained through a green “all

water” one-pot synthetic route following a tandem N-arylation-reduction-cyclocondensation

procedure. All of the synthesised target compounds were assessed for anti-TB potential using

H37Rv ATCC27294 strain. Thirteen compounds were found with better MIC (0.78-6.25 µg/mL)

than the standard drugs and being non-cytotoxic nature (< 50% inhibition against RAW 264.7 cell

lines at 50 µg/mL). The compound 8e exhibited best anti-TB activity (MIC: 2.15 µM and

selectivity index: > 60) and a few others e.g., 8a, 8f, 8k and 8o are the next best anti-TB hits (MIC:

1.56 µg/mL). The determination and analysis of various physiochemical parameters revealed favorable

druglike properties of the active compounds. The compounds 8a-l and 8o, with MIC values of ≤ 6.25

4
μg/mL, have high LipE values (10.66-11.77) that are higher than that of the suggested value of >

6 derived from empirical evidence for quality drug candidates and highlight their therapeutic

potential. The highest LipE value of 11.77 of the best active compound 8e with the MIC of 0.78

μg/mL indicates its better absorption and clearance as a probable clinical candidate for anti-TB

drug discovery. These findings highlight the discovery of benzimidazole-2-carboxamides for

further development as new anti-TB agents.

5
Introduction

Tuberculosis (TB) has led to an estimated death of around 1.2 million among human immuno-

deficiency virus (HIV) positive people in 2018 [1] and is the second leading cause of deaths among

the infectious diseases [2]. Despite the advances in the understanding of Mycobacterium

tuberculosis (Mtb) since its discovery [3] it remains a challenging task to treat TB with its high

death toll. Various types of TB affecting kidney, brain, lungs, etc. are known but the mycobacteria

mainly affect the lungs because Mtb being aerobic mycobacteria acquires plenty of oxygen from

the lungs. Due to newly emerging types of TB such as multi-, extensively- and totally drug-

resistant TB i.e., MDR-TB, XDR-TB, and TDR-TB, respectively, the directly observed treatment

short therapy (DOTS) with the first line anti-TB drugs isoniazid (INH), rifampin (RIF), ethambutol

(EMB), and pyrazinamide (PZA), is inadequate to fight with this deadly disease [4-7]. Bedaquiline

recommended by United States Food and Drug Administration (USFDA) [8] and delamanid

approved by European Medical Agency (EMEA) [9] for treatment of MDR-TB have shown cases

of resistance [10]. However, bedaquiline and delamanid cause hepatotoxicity [11,12] and CNS

toxicity [13], respectively, apart from QT prolongation. The long-term treatment with bedaquiline

has been reported to be associated with cardiac arrhythmia as the serious side effects leading to

mortality [14]. Recently (August 2019), USFDA has approved pretomanid, a nitroimidazopyran

derivative, in conjunction with bedaquiline and linezolid for XDR-TB or MDR-TB to treat adult

TB patients [15].

However, DOTS (the commonly recommended protocol for treatment against TB) is not

effective in recovery of the tuberculotic patients due to the adaptive nature of Mtb. Because of the

worsening situation of tuberculosis, there has been strong need for development of new anti-

tubercular agents. The poor chemotherapeutics, cross-resistance, and scarcity of the effective anti-

6
TB drugs emphasize the necessity for search of novel anti-tuberculotic agents that might have

novel mode of action.

Herein we report compounds with the benzo[d]imidazole-2-carboxamide as the pharmacophoric

feature for novel anti-TB agents.

Design

Pharmacology (Selection of Screening Model)

The drug discovery program for TB involves two approaches: drug-to-target and target-to-drug

[16]. The former strategy i.e., drug-to-target has been proved to be successful as it led to the

discovery of bedaquiline (TMC207) (Figure 1) as highly potent and innovative anti-TB agent [17]

that has recently been approved as drug candidate with a hope to cure MDR-TB [8]. Several other

candidates such as the nitroimidazoles delamanid (OPC67683) [18,19] and pretomanid (PA-824)

[20], imidazopyridine amide (Q203) [21], diethylamine compound SQ109 [22], and

benzothiazinone (BTZ043) [23] that proved to be promising anti-TB drugs in the clinical trials and

some of them being approved (e.g., pretomanid and delamanid) for treatment of MDR TB are out

of the drug-to-target approach initiated by assessment of their anti-TB potential in whole-cell [24].

The strength and success story of phenotype screening is not limited to infectious diseases,

particularly TB, and extends to the discovery of various other therapeutics agents starting from

aspirin to the 1,4-dihydropyridines (e.g., nifedipine, nicardipine and nimodipine), verapamil, and

diltiazem, generically known as calcium blockers [25,26] to cholesterol absorption inhibitor

ezetimibe [27,28] now known as NPC1L1 cholesterol transporter. All these demonstrate the

phenotype screening, also known as classical/forward pharmacology or forward chemical biology,

to be a reliable tool and renewed approach for new drug discovery [29] in the early stage for

7
infectious diseases [30] especially in tuberculosis [31-33] as it is physiologically more relevant

and less artificial due to the use of intact cells and their native environments. Thus, in the present

effort in finding novel anti-TB agents it was planned to follow the phenotype screening path.

Insert Figure 1 here.

Chemistry (Synthetic Targets)

For selection of compounds that would be put to phenotype screening we focused in designing

a new pharmacophore for anti-TB activity. For this purpose, we took into consideration the various

structural diversity that are present in drug candidate molecules. We were attracted by the fact that

about 59% of the drug candidates possess N-containing heterocyclic ring [34] with the prominent

present of the fused ring system the benzimidazole moiety [35] being fifteenth in the list of FDA

approved drugs and pharmaceuticals [36]. Thus, we considered the benzimidazole moiety as the

core of our new anti-TB pharmacophore and were encouraged by literature reports on the anti-

mycobacterial activity of various benzimidazole-based compounds [37 – 47] (Figure 2). To design

a novel pharmacophore with the benzimidazole heterocyclic ring we analysed the structural

features of these benzimidazole derivatives in correlation with the anti-TB activity that revealed

the following:

i) The anti-TB benzo[d]imidazoles belong to C-2 alkyl/cycloalkyl/aryl substituted analogs

(motifs B-H) [38 – 47] except motif A [37] that bears the C-2 hetero (thio) substitution

(Figure 2).

ii) Among the C-2 alkyl/cycloalkyl/aryl substituted benzo[d]imidazoles (motifs B-H) the

1,2-disubstituted derivatives showed better anti-TB activity (e.g., motifs E and G) [44,

46].

8
 iii) Though the compound of motif E exhibited the best anti-TB activity, the other two

compounds with comparable anti-TB potential are the 1,2-disubstituted analog of motif

G [46] and the C-2 cycloalkyl substituted under motif D [42] that bear electron

withdrawing substituents (e.g., NO2 and sulfonamide groups in motif G and carbamate

group in motif D).

iv) The presence of the electron withdrawing NO2 group in the compounds of motif A and

G that could be the major factor to attribute the anti-TB activity in these compounds,

however, could also be detrimental for further development as the nitro aryl features have

been considered being potentially mutagenic via DNA adduct formation and

methaemoglobinemia [48].

v) The devoid of cell viability studies in some of these cases (motifs A, F, and G) raises

concern whether the observed anti-TB activity could be due to the general cytotoxicity

of these compounds.

Insert Figure 2 here.

As a part of our on-going studies on further refinement of the anti-tubercular activity [49-52] we

previously identified benzo[d]thiazole-2-carboxamides (I-K, Figure 3) [53-55]. In further quest of

novel anti-TB scaffold we took into consideration the prevalence of the benzimidazole core in

FDA approved drugs and the above structural analysis of the reported anti-TB benzimidazoles we

planned to transpose the 1,2-disubstituted benzo[d]imidazole moiety of motif E and the C-2

carboxamide substitution pattern from the bioisosteric motifs I-K to design the benzo[d]imidazole-

2-carboxamide motif (L) as novel anti-TB scaffold following the scaffold hopping strategy

[50,51,53,54,56]. The newly designed hybrid scaffold L contains the 1,2-disubstituted

9
benzimidazole core and the C-2 carboxamido function/linkage (present in the first line anti-TB

drugs such as INH, RIF, and PZA) as the electron withdrawing group.

Insert Figure 3 here.

Results and discussion

Chemistry
The benzimidazole-2-carboxamide scaffold (L) can be constructed by the amidation of ethyl

benzo[d]imidazole-2-carboxylates with the required amines (Scheme 1) [53,54].

Insert Scheme 1 here.

The attempted cyclocondensation of various o-phenylenediamines and ethyl glyoxalate to obtain

the key starting material benzo[d]imidazole-2-carboxylate, as applicable to the reaction with o-

aminothiophenol [53,56], however led to the formation of quinoxaline-3-one rather than the

benzo[d]imidazole-2-carboxylate irrespective of the reaction conditions (Scheme 2) [57].

Insert Scheme 2 here.

We reasoned that the ease of simultaneous nucleophilic attack of the amino groups of o-

phenylenediamine to the aldehyde and ester carbonyl groups in ethyl glyoxalate drives the reaction

to afford the quinoxaline-3-ones as the sole products. Thus, it was anticipated that decreasing the

nucleophilic character of one of the amino groups of o-phenylenediamine through substitution with

a sterically hindered group would resist the simultaneous nucleophilic attack on both of the

carbonyl groups of ethyl glyoxalate and would result in the desired benzo[d]imidazole-2-

carboxylate. Thus, we designed an alternate synthetic route for the target benzo[d]imidazole-2-

carboxamides (Scheme 3) in which the cyclocondensation of N-phenyl-o-phenylene diamine (4)

with ethyl glyoxalate (5) gave the benzimidazole-2-carboxylate (6). The “all water” synthetic

strategy reported from this laboratory [58] was adopted that involves water-assisted N-arylation of

o-fluoro nitrobenzene 1 with anilines (2) to form the o-N-mono-arylaminonitroaniline (3) followed

10
by nitro group reduction to generate 4 and in situ cyclocondensation with ethyl glyoxalate (5) to

form the desired benzo[d]imidazole-2-carboxylates (6) in one-pot. The reaction of 1 with aniline

(2a) and 4-chloroaniline (2b) underwent successfully for the synthesis of the corresponding ethyl

N-aryl benzo[d]imidazole-2-carboxylates 6a and 6b in 80 and 63% yields, respectively.

Insert Scheme 3 here.

The synthesis of the desired benzimidazole-2-carboxamide from the benzimidazole-2-

carboxylate was planned following the NH4Cl-catalyzed amidation protocol [54]. Thus the

treatment of benzimidazole-2-carboxylates (6) with various substituted arylalkyl amines (7, 1

equiv) in the presence of NH4Cl (20 mol%) at 100 °C under neat conditions afforded the

corresponding N-arylalkyl amides in 67-94% (Table 1).

The NH4Cl-catalysed amidation reaction of ethyl benzimidazole-2-carboxylate was extended for

the reaction with various alicyclic amines 9 such as piperidine, morpholine, thiomorpholine, and

piperazines to from the corresponding cycloalkylamides in 65-96% yield (Table 2).

Insert Table 2 here.

Biological Evaluation
Determination of MIC (H37Rv)
For the estimation of minimum inhibitory concentration (MIC), the synthesized compounds (8,

10) were tested against Mtb H37Rv (ATCC27294 strain) [59]. The anti-TB activity of 8, 10 and the

currently used anti-TB medicines like isoniazid, rifampin, ethambutol, pyrazinamide and

ciprofloxacin (INH, R, E, Z, and Cfx, respectively) are provided in Table 3. The compounds having

the generalized structure 8 and 10 showed MICs from 0.78 to >25 µg/mL Among these, 8e was

identified as the most active anti-mycobacterial compound (MIC: 2.15 µM; approx. selectivity

index: 60), and was found to be superior to the anti-TB medicines E, Z and Cfx (Table 3).

Insert Table 3 here.

11
 It is observed that final compounds/carboxamides (8a-l) have better anti-TB activity than that of

the starting carboxylate, e.g., 8a is eight folds more active than the intermediate 6a. This could be

attributed due to the fact that the lipophilicity (cLogP, Table 3, Entries 1-2), estimated using in

silico methods, in the final carboxamides is increased from 3.87 to 5.44 with the incorporation of

the benzylamine group as it is an important parameter in crossing the lipophilic mycobacterial cell

wall [61]. Although a direct co-relationship between the MIC and cLogP of the compounds

couldn’t be established, the data under Table 3 reveals that the compounds (8 and 10) are more

lipophilic in comparison with INH, E, and Z allowing their passage through the mycolic acid-rich

cell wall.

While addressing the lipophilicity in targeting any pathogenic disease, the lipophilic efficiency

(LipE) is considered as one of the most significant parameters [62-65]. As the LiPE [66] links

potency with lipophilicity capturing both the values in a single parameter it is considered as an

important physicochemical parameter in drug design and discovery for qualitative assessment of

druglikeness of investigational compounds [67,68]. The LipE (MIC) offers means to compare

compounds of different potencies (pIC50s) and lipophilicities (LogP) and can be mathematically

calculated by subtracting Log(MIC) from cLogP. Generally, at a given concentration, a high pIC50

value reflects reduced risk of non-specific, off-target pharmacology and is a desirable attribute in

drug candidates.

Compounds with distinct anti-TB activity have been found to be more lipophilic than the inactive

compounds [69] and within a drug class lipophilic compounds exhibit better anti-TB activity than

their hydrophilic counterparts [70-73]. The in silico estimation of the various physiochemical

properties (Table 3) of the active benzo[d]imidazole-2-carboxamides found under this study

revealed these to possess favourable Lipinski parameters [74] that demonstrate the druglike

12
character of these novel benzimidazoles. Generally, at a given concentration, a high pIC50 value

reflects reduced risk of non-specific, off-target pharmacology and is a desirable attribute in drug

candidates. A higher LipE value of drug candidates indicates better absorption and clearance

suggesting a probable clinical candidate for drug discovery. In view of empirical evidence

suggesting quality drug candidates to have a high LiPE (> 6) [75], the higher LipE values (10.66-

11.77) of the compounds 8a-l and 8o, with MIC ≤ 6.25 μg/mL highlight their therapeutic potential

and in particular for 8e the MIC of 0.78 μg/mL and LipE of 11.77 indicate its better absorption

and clearance as a probable clinical candidate for anti-TB drug discovery.

Determination of Cytotoxicity
To eliminate the possibility of anti-tuberculotic potential of these compounds due to general

toxicity, the compounds with MIC ≤ 6.25 μg/mL were subjected for in vitro determination of

cytotoxicity against mouse leukemic monocyte macrophage (RAW 264.7) cell lines at 50 μg/mL.

The results obtained using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide)

(MTT) assay [76,77] has been summarized in Table 4. This study demonstrates the active anti-TB

hits to be non-cytotoxic with < 50% inhibition and 8e as the most potent compound with the

selectivity index (approximated) of > 64.

Insert Table 4 here.

Structure Activity Relationship (SAR)

Introduction of the lipophilic benzyl amine to the benzimidazole-2-carboxylate 6a (MIC 12.5

μg/mL) increases the activity by eight-fold in 8a (MIC 1.56 μg/mL) compared to the parent

compound. Upon incorporation of halogens such as fluorine and chlorine at ortho position of

phenyl ring, the anti-tubercular activity of 8b and 8g decreased slightly up to 3.125 μg/mL. Out of

the three tried difluoro substitutions such as 2,4-difluoro (8d), 3,4-difluoro (8e), and 2,5-difluoro

(8f), the highest anti-TB activity among these carboxamides has been observed with 8e (MIC of

13
0.78 μg/mL) bearing 3,4-difluoro substitutions devoid of any ortho steric clash. Four fold reduction

in anti-TB activity has been observed with incorporation of powerful electron withdrawing 4-

trifluoromethyl group in 8n (MIC 12.5 μg/mL) as compared to the parent compound 8a.

Incorporation of methyl at ortho position diminished the activity (8j, MIC 6.25 μg/mL) due to the

ortho steric clash of compared to the parent compound 8a. However, upon incorporation of

methoxy group at the same second position the activity is re-gained (8k, MIC 1.56 μg/mL) to the

same extent as that of parent compound 8a. However, this trend was found to be detrimental upon

incorporation of electron donating groups such as 2,4-di-methoxy (8l, MIC having 6.25 μg/mL)

and 3,4-di-methoxy (8m, MIC having >25 μg/mL) groups suggesting perhaps the presence of

strong electron releasing substituent at the 4/para positing of the phenyl ring of the amine would

be detrimental to ant-TB potential. The replacement of the phenylmethyl moiety of the

amide/amine part by alicyclic amines such as morpholine (10a, MIC 12.5 μg/mL), thiomorpholine

(10b, MIC 25 μg/mL), piperidine (10c, MIC >25 μg/mL), and substituted piperazines (10d-h), led

to a decrease of their anti-tuberculotic potential drastically, suggesting the necessity of the N-

arylalkyl groups of 8 for anti-mycobactericidal action. Increasing the size of the linker from n = 1

(8a, MIC 1.56 μg/mL) to n = 2 (8q, MIC 12.5 μg/mL), resulted decreased anti-TB activity of the

compounds suggesting that the ethyl spacer is detrimental to anti-TB potential of these compounds.

Incorporation of chlorine at para position of N-phenyl of benzimidazole decreases the anti-

mycobacterial activity (8s, MIC of >25 μg/mL) of parent compound 8q.

3. Conclusions

The benzimidazole-2-carboxamide moiety has been designed as novel anti-TB scaffold. The

synthesis of twenty-seven benzo[d]imidazole-2-carboxamides has been achieved through catalytic

amidation procedure from the ethyl benzo[d]imidazole-2-carboxylates. An “all-water” one-pot

14
methodology has been adopted for the construction of the ethyl benzo[d]imidazole-2-carboxylates,

used as key starting materials, through water-assisted tandem N-arylation-reduction-

cyclocondensation (with ethyl glyoxalate). All the final carboxamides were tested for anti-

mycobacterial activity in vitro against Mtb H37Rv (ATCC27294 strain). The compound 8e

exhibited the best anti-tubercular potential [MIC: 0.78 μg/mL (2.15 µM); selectivity (approx.)

index: > 64] and 8a, 8f, 8k and 8o are the next best compounds with MIC of 1.56 µg/mL. The

active anti-TB hits with MIC ≤ 6.25 µg/mL are non cytotoxic against RAW 264.7 cell lines. The

active compounds possess favourable physiochemical properties, specifically with respect to the

the LipE values (10.66 - 10.77) which are higher than that of the empirically derived value of > 6

for quality drug candidates demonstrating their drug likeliness. These results can be considered as

important start points amid the discovery of novel anti-mycobacterial chemotypes.

4. Experimental Section
4.1 General Chemistry

Reagents and solvents have been procured from Alfa Aesar, Aldrich, Fluka, Merck AG,

and S-D Fine Chemicals and used as such. 1H and 13C NMR spectra has been generated at 400 and

100 MHz respectively with tetra methyl silane as an internal standard and using deuteriated

solvents d-MeOD and d-CDCl3 as solvents on Bruker Advance DX spectrometer. For

characterization of the NMR spectra, following abbreviation: singlet (s); multiplet (m); doublet

(d); doublet of doublet (dd); triplet (t); quartet (q) and broad singlet (br s). MS has been determined

using the atmospheric pressure chemical ionization (APCI) mode with LCMS and on GCMS using

electron impact (EI) mode. IR spectra have been obtained using Perkin Elmer FTIR

spectrophotometer in the range of 4000-600 cm-1. Silica gel GF-254 was used for checking the

purity of the compounds and TLCs were visualized under UV (254 nm). Melting point apparatus

15
(Gupta Scientific) was used for recording the melting points. Buchi rotary evaporator has been

used for evaporation of solvent under reduced pressure. HPLC spectra of carboxamides have been

recorded using C18 column (4.6 × 250 mm, 5 micron), 70:30 of acetonitrile:water as eluent at 30

ºC by sampling of 20 µL with 1 mL/min (flow rate), and PDA detector. Elemental analyses of the

novel compounds have been obtained using organic element analyzer. The results of elemental

analysis summarized have been found in suitable range (± 0.4 % of calculated values).

General protocol for synthesis of ethyl benzo[d]imidazole-2-carboxylates

The mixture of 1 (141 mg, 1 mmol) and 2 (1 equiv) in water (2 mL) was stirred magnetically under

reflux (oil bath, 110 ℃) for 2 h (TLC shows complete consumption of starting materials). To this

mixture was added 2 N aq. HCl (2.5 mL, 5 equiv) and indium powder (287 mg, 2.5 equiv) and the

resultant mixture was stirred magnetically for 2 h. Next, 5 (204 mg, 2 mmol, 2 equiv) was added

to this reaction mixture and the magnetic stirring was continued for another 1 h. The reaction

mixture was cooled to rt and treated with solid NaHCO3 (~ 140 mg) to neutralize (ceasing of

effervescence) the acid. The reaction mixture was extracted with EtOAc (2 × 5 mL), the combined

EtOAc extracts were dried (anh MgSO4), and filtered through a cotton plug. The filtrate was

concentrated in vacuo and the crude product was subjected to column chromatography purification

on silica gel G (230–400) using (hexane:EtOAc, 85:15) as the eluent to afford analytically pure 6.

Ethyl 1-phenyl-1H-benzo[d]imidazole-2-carboxylate (6a): [78] Colorless solid (213 mg, 80%).

mp 256-260 °C; IR (KBr, cm-1) 3045, 2925, 2853, 1718, 1599, 1488, 1286, 1184, 1027; 1H NMR

(400 MHz, CDCl3) δ 8.00-7.98 (m, 1H), 7.61-7.56 (m, 3H), 7.43-7.36 (m, 4H), 7.19-7.16 (m, 1H),

4.39 (q, J = 7.1 Hz, 2H), 1.36 (t, J = 7.1 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ 159.1, 141.7,

141.3, 137.6, 136.5, 129.4, 129.1, 127.3, 126.0, 124.1, 121.8, 111.4, 62.2, 14.1.

16
Ethyl 1-(4-chlorophenyl)-1H-benzo[d]imidazole-2-carboxylate (6b): Pale yellow oil (189 mg,

63%). IR (KBr, cm-1) 3043, 2922, 2851, 1717, 1578, 1462, 1280, 1179, 1023; 1H NMR (400 MHz,

CDCl3) δ 8.00-7.98 (m, 1H), 7.56 (d, J = 8.6 Hz, 2H), 7.44-7.40 (m, 2H), 7.34 (d, J = 8.6 Hz, 2H),

7.17-7.15 (m, 1H), 4.41 (q, J = 7.1 Hz, 2H), 1.40 (t, J = 7.1 Hz, 3H); 13C NMR (100 MHz, CDCl3)

δ 159.1, 141.7, 141.0, 137.4, 135.2, 135.0, 129.7, 128.6, 126.3, 124.3, 121.9, 111.2, 62.4, 14.2.

HRMS (APCI-TOF) m/z: [M + H]+ Calcd for C16H14ClN2O2 301.0744; Found 301.0641.

General procedure for synthesis of the amides.

The ester (6a, 267 mg, 1 mmol) was treated with the amine (1 equiv) in the presence of NH4Cl

(20 mol%) under magnetic stirring at 100 ºC till consumption of 6a (TLC). The reaction mixture

was cooled at rt and extracted with EtOAc (4 × 5 mL). The combined EtOAc extracts were dried

(anh Na2SO4), filtered through a plug of cotton, and the filtrate was concentrated in vaccuo. The

crude product was subjected to column chromatography purification on silica gel G (60-120 mesh)

and eluting with hexane-EtOAc (70:30) to obtain the pure amide.

N-Benzyl-1-phenyl-1H-benzo[d]imidazole-2-carboxamide (8a): Pale yellow solid (307 mg,

94%). mp 156-160 °C; IR (KBr, cm-1) 3281, 3073, 2917, 1667, 1610, 1541, 1500, 1095, 860; 1H

NMR (400 MHz, CDCl3) δ 8.02 (s, 1H), 7.84-7.82 (m, 1H), 7.62-7.54 (m, 3H), 7.45-7.28 (m, 9H),

7.20-7.18 (m, 1H), 4.60 (d, J = 6.0 Hz, 2H); 13C NMR (100 MHz, MeOD) δ 159.6, 144.8, 141.0,

138.0, 136.9, 136.2, 129.2, 128.7, 128.2, 127.3, 127.0, 126.6, 125.1, 123.7, 120.0, 111.0, 42.7;

HRMS (ESI-TOF) m/z: [M + Na]+ Calcd for C21H17N3ONa 350.1269; Found 350.1260. HPLC

analysis: retention time = 7.725 min; peak area, 100%.

N-(2-Fluorobenzyl)-1-phenyl-1H-benzo[d]imidazole-2-carboxamide (8b): Pale yellow oil

(273 mg, 79%). IR (KBr, cm-1) 3078, 2941, 1721, 1515, 1487, 1267, 1071, 717; 1H NMR (400

MHz, CDCl3) δ 8.16-8.13 (m, 1H), 7.80 (d, J = 7.5 Hz, 1H), 7.57-7.51 (m, 3H), 7.40-7.31 (m, 5H),

17
7.25-7.21 (m, 1H), 7.15 (d, J = 7.6 Hz, 2H), 7.09-7.01 (m, 2H), 4.62 (d, J = 6.2 Hz, 2H); 13C NMR

(100 MHz, CDCl3) δ 162.3, 159.8, 158.5, 143.5, 141.0, 138.0, 136.6, 130.3, 130.2, 129.5, 129.4,

129.3, 129.0, 127.3, 125.3, 124.8, 124.7, 124.3, 124.3, 124.0, 120.6, 115.6, 115.4, 111.6, 37.3.

Anal. Calcd for C21H16FN3O Elemental Analysis: C, 73.03; H, 4.67; N, 12.17; Found: C, 73.05;

H, 4.68; N, 12.18. HPLC analysis: retention time = 5.808 min; peak area, 100%.

N-(4-Fluorobenzyl)-1-phenyl-1H-benzo[d]imidazole-2-carboxamide (8c): Pale yellow oil (283

mg, 82%). IR (KBr, cm-1) 3038, 2921, 1716, 1592, 1481, 1281, 1028, 699; 1H NMR (400 MHz,

CDCl3) δ 8.20 (br s, 1H), 7.83 (d, J = 7.8 Hz, 1H), 7.61-7.54 (m, 3H), 7.44-7.30 (m, 6H), 7.20 (d,

J = 7.8 Hz, 2H), 7.02 (t, J = 8.6 Hz, 2H), 4.56 (d, J = 6.0 Hz, 2H); 13C NMR (100 MHz, CDCl3) δ

163.5, 161.0, 158.4, 143.5, 141.0, 138.0, 136.6, 133.5, 133.5, 129.7, 129.7, 129.3, 129.0, 127.3,

125.3, 124.0, 120.5, 115.7, 115.5, 111.6, 42.6. Anal. Calcd for C21H16FN3O Elemental Analysis:

C, 73.03; H, 4.67; N, 12.17; Found: C, 73.00; H, 4.68; N, 12.20. HPLC analysis: retention time =

7.658 min; peak area, 100%.

N-(2,4-Difluorobenzyl)-1-phenyl-1H-benzo[d]imidazole-2-carboxamide (8d): Pale yellow

solid (247 mg, 68%). mp 96-98 °C; IR (KBr, cm-1) 3285, 3071, 2921, 1671, 1617, 1545, 1505,

1096, 861; 1H NMR (400 MHz, CDCl3) δ 8.03 (br s, 1H), 7.84 (d, J = 7.6 Hz, 1H), 7.59-7.55 (m,

3H), 7.43-7.35 (m, 5H), 7.18 (d, J = 7.6 Hz, 2H), 6.88-6.81 (m, 2H), 4.61 (d, J = 6.2 Hz, 2H); 13C

NMR (100 MHz, CDCl3) δ 158.5, 143.3, 141.0, 141.0, 138.0, 136.6, 131.2, 131.2, 131.1, 131.1,

129.3, 129.0, 127.2, 125.3, 124.0, 120.8, 120.8, 120.7, 120.7, 120.6, 111.6, 111.5, 111.3, 111.3,

104.2, 104.0, 36.7. Anal. Calcd for C21H15F2N3O Elemental Analysis: C, 69.41; H, 4.16; N, 11.56;

Found: C, 69.39; H, 4.12; N, 11.53. HPLC analysis: retention time = 7.042 min; peak area, 100%.

N-(3,4-Difluorobenzyl)-1-phenyl-1H-benzo[d]imidazole-2-carboxamide (8e): White solid

(243 mg, 67%). mp 110-112 °C; IR (KBr, cm-1) 3288, 2963, 1722, 1671, 1598, 1499, 1404, 1262,

18
799; 1H NMR (400 MHz, CDCl3) δ 8.24 (br s, 1H), 7.83 (d, J = 7.4 Hz, 1H), 7.59-7.56 (m, 3H),

7.44-7.38 (m, 4H), 7.21-7.17 (m, 2H), 7.15-7.11 (m, 2H), 7.08-7.07 (m, 1H), 4.54 (d, J = 6.3 Hz,

2H); 13C NMR (100 MHz, CDCl3) δ 158.5, 148.5, 143.3, 140.9, 138.0, 136.5, 134.9, 134.9, 134.8,

129.4, 129.3, 129.1, 129.0, 127.2, 125.4, 124.1, 123.8, 123.8, 123.8, 123.7, 120.5, 117.5, 117.3,

116.9, 116.8, 111.7, 111.5, 42.3. Anal. Calcd for C21H15F2N3O Elemental Analysis: C, 69.41; H,

4.16; N, 11.56; Found: C, 69.39; H, 4.13; N, 11.55. HPLC analysis: retention time = 6.875 min;

peak area, 100%.

N-(2,5-difluorobenzyl)-1-phenyl-1H-benzo[d]imidazole-2-carboxamide (8f): Pale yellow oil

(283 mg, 78%). 1H NMR (400 MHz, CDCl3) δ 8.14 (br s, 1H), 7.85 (d, J = 7.6 Hz, 1H), 7.61-7.54

(m, 3H), 7.44-7.35 (m, 4H), 7.19 (d, J = 7.7 Hz, 1H), 7.13-6.93 (m, 3H), 4.63 (d, J = 5.2 Hz, 2H);
13C NMR (100 MHz, CDCl3) δ 158.6, 157.5, 154.6, 143.2, 141.0, 138.0, 136.5, 129.3, 129.0,

127.2, 125.4, 124.1, 120.6, 116.5, 116.3, 115.5, 115.4, 111.6, 36.9; HRMS (ESI-TOF) m/z: [M +

K]+ Calcd for C21H15F2KN3OK 402.0820; Found 402.0811. HPLC analysis: retention time = 7.008

min; peak area, 100%.

N-(2-Chlorobenzyl)-1-phenyl-1H-benzo[d]imidazole-2-carboxamide (8g): Orange solid (264

mg, 73%). mp 108-110 °C; IR (KBr, cm-1) 3401, 3060, 2089, 1681, 1596, 1531, 1447, 746; 1H

NMR (400 MHz, CDCl3) δ 8.14 (br s, 1H), 7.85 (d, J = 7.6 Hz, 1H), 7.59-7.54 (m, 3H), 7.42-7.37

(m, 4H), 7.28-7.24 (m, 4H), 7.19 (d, J = 7.6 Hz, 1H), 4.71 (d, J = 6.3 Hz, 2H); 13C NMR (100

MHz, CDCl3) δ 157.4, 142.4, 140.0, 137.0, 135.6, 134.1, 133.3, 132.7, 128.9, 128.6, 128.2, 127.9,

126.2, 126.1, 124.2, 122.9, 119.6, 110.6, 40.7. Anal. Calcd for C21H16ClN3O Elemental Analysis:

C, 69.71; H, 4.46; N, 11.61; Found: C, 69.69; H, 4.45; N, 11.62. HPLC analysis: retention time =

8.358 min; peak area, 100%.

19
N-(3-Chlorobenzyl)-1-phenyl-1H-benzo[d]imidazole-2-carboxamide (8h): Pale yellow oil

(293 mg, 81%). 1H NMR (400 MHz, CDCl3) δ 8.56 (t, J = 6.1 Hz, 1H), 7.83-7.81 (m, 1H), 7.59-

7.56 (m, 3H), 7.46-7.43 (m, 2H), 7.41-7.34 (m, 3H), 7.25-7.20 (m, 4H), 4.56 (d, J = 6.2 Hz, 2H);
13C NMR (100 MHz, CDCl3) δ 158.6, 143.5, 141.0, 140.0, 138.0, 136.6, 134.5, 129.9, 129.3,

129.0, 127.9, 127.7, 127.3, 126.0, 125.4, 124.1, 120.6, 111.7, 42.7. Anal. Calcd for C21H16ClN3O

Elemental Analysis: C, 69.71; H, 4.46; N, 11.61; Found: C, 69.70; H, 4.47; N, 11.60. HPLC

analysis: retention time = 8.317 min; peak area, 100%.

N-(4-Chlorobenzyl)-1-phenyl-1H-benzo[d]imidazole-2-carboxamide (8i): Pale yellow oil (275

mg, 76%). 1H NMR (400 MHz, CDCl3) δ 8.17 (br s,1H), 7.70 (d, J = 7.8 Hz, 1H), 7.48-7.43 (m,

3H), 7.31-7.22 (m, 4H), 7.19-7.13 (m, 4H), 7.07 (d, J = 7.7 Hz, 1H), 4.42 (d, J = 6.1 Hz, 2H); 13C

NMR (100 MHz, CDCl3) δ 158.5, 143.5, 141.0, 138.0, 136.6, 136.3, 133.4, 129.3, 129.3, 129.0,

128.8, 127.3, 125.4, 124.0, 120.5, 111.6, 42.6. Anal. Calcd for C21H16ClN3O Elemental Analysis:

C, 69.71; H, 4.46; N, 11.61; Found: C, 69.70; H, 4.46; N, 11.60. HPLC analysis: retention time =

7.225 min; peak area, 100%.

N-(2-Methylbenzyl)-1-phenyl-1H-benzo[d]imidazole-2-carboxamide (8j): Pale yellow oil (236

mg, 76%). 1H NMR (400 MHz, CDCl3) δ 7.73-7.71 (m, 2H), 7.50-7.43 (m, 3H), 7.33 (d, J = 6.6

Hz, 2H), 7.29-7.22 (m, 3H), 7.12-7.08 (m, 4H), 4.49 (d, J = 5.7 Hz, 2H), 2.27 (s, 3H); 13C NMR

(100 MHz, CDCl3) δ 158.3, 143.5, 141.1, 138.0, 136.7, 136.6, 135.2, 130.6, 129.3, 128.9, 128.7,

127.9, 127.3, 126.3, 125.2, 123.9, 120.6, 111.6, 41.6, 19.1. Anal. Calcd for C22H19N3O Elemental

Analysis: C, 77.40; H, 5.61; N, 12.31. Found: C, 77.38; H, 5.63; N, 12.32. HPLC analysis:

retention time = 7.683 min; peak area, 100%.

N-(2-Methoxybenzyl)-1-phenyl-1H-benzo[d]imidazole-2-carboxamide (8k): Pale yellow solid

(300 mg, 84%). mp 110-112 °C; IR (KBr, cm-1) 3037, 2921, 1713, 1597, 1481, 1285, 1021, 699;

20
1H NMR (400 MHz, CDCl3) δ 8.17 (br s, 1H), 7.86 (d, J = 7.8 Hz, 1H), 7.60-7.52 (m, 3H), 7.44-

7.27 (m, 6H), 7.18 (d, J = 7.8 Hz, 1H), 6.95-6.91 (m, 2H), 4.62 (d, J = 6.1 Hz, 2H), 3.92 (s, 3H);
13C NMR (100 MHz, CDCl3) δ 158.2, 157.7, 143.9, 141.1, 138.0, 136.8, 129.8, 129.2, 129.0,

128.8, 127.3, 125.7, 125.1, 123.8, 120.6, 120.5, 111.6, 110.4, 55.4, 39.1. Anal. Calcd for

C22H19N3O2 Elemental Analysis: C, 73.93; H, 5.36; N, 11.76. Found: C, 73.90; H, 5.34; N, 11.77.

HPLC analysis: retention time = 7.042 min; peak area, 100%.

N-(2,4-Dimethoxybenzyl)-1-phenyl-1H-benzo[d]imidazole-2-carboxamide (8l): White solid

(318 mg, 82%). mp 177-179 °C; 1H NMR (400 MHz, CDCl3) δ 8.06-8.03 (m, 1H), 7.84 (d, J = 7.7

Hz, 1H), 7.60-7.53 (m, 3H), 7.43-7.32 (m, 5H), 7.23 (d, J = 8.2 Hz, 1H), 7.17 (d, J = 7.8 Hz, 1H),

6.49 (d, J = 2.4 Hz, 1H), 6.44 (dd, J = 2.4 Hz, 1H), 4.53 (d, J = 6.0 Hz, 2H), 3.89 (s, 3H), 3.82 (s,

3H); 13C NMR (100 MHz, CDCl3) δ 160.7, 158.8, 158.1, 144.0, 141.1, 138.0, 136.8, 130.6, 129.2,

128.8, 127.3, 125.0, 123.8, 120.5, 118.3, 111.5, 103.9, 98.6, 55.5, 38.6; HRMS (ESI-TOF) m/z:

[M + H]+ Calcd for C23H22N3O3 388.1661; Found 388.1655. HPLC analysis: retention time = 7.058

min; peak area, 100%.

N-(3,4-Dimethoxybenzyl)-1-phenyl-1H-benzo[d]imidazole-2-carboxamide (8m): White solid

(294 mg, 76%). mp 165-168 °C; IR (KBr, cm-1) 3419, 2929, 1682, 1517, 1091, 746; 1H NMR (400

MHz, CDCl3) δ 7.82-7.80 (m, 2H), 7.58-7.50 (m, 3H), 7.39-7.34 (m, 3H), 7.16 (d, J = 7.8 Hz, 1H),

6.82-6.71 (m, 4H), 3.86 (d, J = 1.0 Hz, 8H); 13C NMR (100 MHz, CDCl3) δ 158.5, 149.0, 147.7,

143.7, 141.0, 138.0, 136.7, 131.2, 129.3, 128.9, 127.2, 125.2, 123.9, 120.7, 120.5, 112.0, 111.6,

111.4, 55.9, 40.7, 35.5. Anal. Calcd for C23H21N3O3 Elemental Analysis: C, 71.30; H, 5.46; N,

10.85. Found: C, 71.33; H, 5.47; N, 10.84. HPLC analysis: retention time = 5.833 min; peak area,

100%.

21
1-Phenyl-N-(4-(trifluoromethyl)benzyl)-1H-benzo[d]imidazole-2-carboxamide (8n): Pale

yellow oil (312 mg, 79%). IR (KBr, cm-1) 3041, 2923, 1711, 1594, 1481, 1281, 1025, 700; 1H

NMR (400 MHz, CDCl3) δ 8.30 (br s, 1H), 7.68 (d, J = 7.3 Hz, 1H), 7.45-7.42 (m, 6H), 7.32-7.23

(m, 5H), 7.07-7.04 (m, 1H), 4.49 (d, J = 6.3 Hz, 2H); 13C NMR (100 MHz, CDCl3) δ 158.6, 143.4,

142.0, 140.9, 138.0, 138.0, 136.5, 129.4, 129.3, 129.0, 128.0, 127.3, 127.2, 126.0, 125.7, 125.6,

125.6, 125.5, 125.4, 124.1, 124.1, 121.8, 120.5, 111.7, 111.5, 42.8. Anal. Calcd for C22H16F3N3O

Elemental Analysis: C, 66.83; H, 4.08; N, 10.63; Found: C, 66.84; H, 4.09; N, 10.62. HPLC

analysis: retention time = 5.792 min; peak area, 100%.

1-Phenyl-N-(1-phenylethyl)-1H-benzo[d]imidazole-2-carboxamide (8o): Pale yellow oil (259

mg, 76%). IR (KBr, cm-1) 3039, 2922, 1709, 1589, 1479, 1279, 1071. 1H NMR (400 MHz, CDCl3)

δ 7.99 (d, J = 8.0 Hz, 1H), 7.83 (d, J = 7.7 Hz, 1H), 7.55-7.48 (m, 3H), 7.40-7.30 (m, 8H), 7.29-

7.23 (m, 1H), 7.14 (d, J = 7.8 Hz, 1H), 5.25-5.18 (m, 1H), 1.60 (d, J = 7.0 Hz, 3H); 13C NMR (100

MHz, CDCl3) δ 157.6, 143.7, 142.8, 141.0, 138.0, 136.6, 129.3, 128.9, 128.7, 127.4, 127.2, 126.2,

125.2, 123.9, 120.5, 111.6, 48.8, 22.1; HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C22H19N3ONa

364.1426; Found 3364.1417. HPLC analysis: retention time = 7.292 min; peak area, 100%.

N-(Benzo[d][1,3]dioxol-5-ylmethyl)-1-phenyl-1H-benzo[d]imidazole-2-carboxamide (8p):

Yellow solid (301 mg, 81%). 124-126 °C; IR (KBr, cm-1) 3436, 2068, 1634, 1536, 1499, 1259,

1034, 751; 1H NMR (400 MHz, CDCl3) δ 8.31 (br s, 1H), 7.82 (d, J = 7.6 Hz, 1H), 7.59-7.57 (m,

3H), 7.45-7.44 (m, 2H), 7.38-7.34 (m, 2H), 7.19 (d, J = 7.6 Hz, 1H), 6.82 (s, 1H), 6.76 (q, J = 7.4

Hz, 2H), 5.92 (s, 2H), 4.49 (d, J = 5.2 Hz, 2H); 13C NMR (100 MHz, CDCl3) δ 158.4, 147.9, 147.0,

143.7, 141.1, 138.0, 136.7, 131.6, 129.3, 129.0, 127.3, 125.2, 123.9, 121.4, 120.6, 111.6, 108.6,

108.3, 101.1, 43.2. Anal. Calcd for C22H17N3O3 Elemental Analysis: C, 71.15; H, 4.61; N, 11.31.

Found: C, 71.12; H, 4.60; N, 11.32. HPLC analysis: retention time = 11.267 min; peak area, 100%.

22
N-phenethyl-1-phenyl-1H-benzo[d]imidazole-2-carboxamide (8q): Pale yellow oil (283 mg,

83%). 1H NMR (400 MHz, CDCl3) δ 7.84-7.82 (m, 2H), 7.61-7.55 (m, 4H), 7.44-7.33 (m, 5H),

7.18 (d, J = 7.8 Hz, 1H), 6.85-6.82 (m, 1H), 6.79-6.78 (m, 2H), 3.64 (q, J = 6.9 Hz, 2H), 2.87 (t, J

= 7.2 Hz, 2H); 13C NMR (100 MHz, CDCl3) δ 158.5, 149.0, 147.7, 143.7, 141.0, 138.0, 136.7,

131.2, 129.3, 127.2, 125.2, 123.9, 120.7, 120.5, 111.9, 111.4, 40.8, 35.5. Anal. Calcd for

C22H19N3O Elemental Analysis: C, 77.40; H, 5.61; N, 12.31. Found: C, 77.38; H, 5.62; N, 12.33.

HPLC analysis: retention time = 7.967 min; peak area, 100%.

N-(3,4-Dimethoxyphenethyl)-1-phenyl-1H-benzo[d]imidazole-2-carboxamide (8r): Pale

yellow oil (313 mg, 78%). 1H NMR (400 MHz, CDCl3) δ 7.96-7.93 (m, 1H), 7.83 (d, J = 7.9 Hz,

1H), 7.60-7.52 (m, 3H), 7.41-7.32 (m, 4H), 7.18 (d, J = 7.8 Hz, 1H), 3.88 (s, 6H), 3.65 (q, J = 6.9

Hz, 2H), 2.87 (q, J = 7.2 Hz, 2H); 13C NMR (100 MHz, CDCl3) δ 158.5, 149.0, 147.7, 143.8,

141.0, 137.9, 136.7, 131.2, 129.3, 128.9, 127.2, 125.2, 123.9, 120.7, 120.5, 112.0, 111.6, 111.4,

55.9, 55.9, 40.8, 35.4. Anal. Calcd for C24H23N3O3 Elemental Analysis: C, 71.80; H, 5.77; N,

10.47. Found: C, 71.79; H, 5.76; N, 10.48. HPLC analysis: retention time = 11.250 min; peak area,

100%.

N-Benzyl-1-(4-chlorophenyl)-1H-benzo[d]imidazole-2-carboxamide (8s): Yellowish solid

(304 mg, 84%). mp 123-125 °C; IR (KBr, cm-1) 3395, 3062, 2922, 1678, 1533, 1495, 1266, 1090,

745; 1H NMR (400 MHz, CDCl3) δ 8.04 (s, 1H), 7.80 (d, J = 7.3 Hz, 1H), 7.53 (d, J = 8.6 Hz, 3H),

7.40-7.27 (m, 9H), 7.15 (d, J = 7.4 Hz, 1H), 4.57 (d, J = 6.0 Hz, 2H); 13C NMR (100 MHz, CDCl3)

δ 158.3, 143.4, 141.1, 137.8, 137.5, 135.1, 134.9, 129.6, 128.8, 128.7, 128.0, 127.7, 125.5, 124.2,

120.7, 111.3, 43.4. Anal. Calcd for C21H16ClN3O Elemental Analysis: C, 69.71; H, 4.46; N, 11.61.

Found: C, 69.73; H, 4.48; N, 11.62. HPLC analysis: retention time = 7.950 min; peak area, 100%.

23
Morpholino(1-phenyl-1H-benzo[d]imidazol-2-yl)methanone (10a): Yellowish oil (240 mg,

78%). IR (KBr, cm-1) 3413, 2924, 1642, 1598, 1500, 1435, 1388, 758; 1H NMR (400 MHz, CDCl3)

δ 7.88-7.86 (m, 1H), 7.58-7.55 (m, 2H), 7.51 (d, J = 7.0 Hz, 1H), 7.46-7.44 (m, 2H), 7.38-7.33 (m,

3H), 3.70-3.66 (m, 6H), 3.57-3.54 (m, 2H); 13C NMR (100 MHz, CDCl3) δ 159.9, 145.2, 141.9,

135.6, 135.3, 129.8, 128.9, 126.1, 124.8, 123.6, 120.8, 110.9, 66.9, 66.6, 47.3, 42.4. Anal. Calcd

for C18H17N3O2 Elemental Analysis: C, 70.34; H, 5.58; N, 13.67. Found: C, 70.30; H, 5.56; N,

13.65. HPLC analysis: retention time = 4.692 min; peak area, 100%.

(1-Phenyl-1H-benzo[d]imidazol-2-yl)(thiomorpholino)methanone (10b): Yellowish oil (217

mg, 67%). IR (KBr, cm-1) 3434, 3058, 2961, 2927, 1730, 1650, 1597, 1500, 1447, 1212, 746; 1H

NMR (400 MHz, CDCl3) δ 7.90-7.88 (m, 1H), 7.60-7.53 (m, 4H), 7.48-7.46 (m, 2H), 7.36-7.35

(m, 2H), 3.97-3.95 (m, 2H), 3.86-3.83 (m, 2H), 2.64-2.60 (m, 2H), 2.57-2.54 (m, 2H); 13C NMR

(100 MHz, CDCl3) δ 160.2, 145.5, 141.8, 135.5, 135.2, 129.9, 129.0, 126.1, 124.7, 123.6, 120.8,

110.9, 49.5, 44.5, 28.1, 27.4. Anal. Calcd for C18H17N3OS Elemental Analysis: C, 66.85; H, 5.30;

N, 12.99. Found: C, 66.83 H, 5.29; N, 12.90. HPLC analysis: retention time = 4.750 min; peak

area, 100%.

(1-Phenyl-1H-benzo[d]imidazol-2-yl)(piperidin-1-yl)methanone (10c): Yellowish oil (229 mg,

75%). IR (KBr, cm-1) 3435, 2938, 1645, 1498, 1437, 1259, 1020, 761; 1H NMR (400 MHz, CDCl3)

δ 7.90-7.87 (m, 1H), 7.58-7.54 (m, 2H), 7.51-7.48 (m, 3H), 7.40-7.33 (m, 3H), 3.64-3.62 (m, 2H),

3.41-3.83 (m, 2H), 1.60-1.53 (m, 4H), 1.40-1.37 (m, 2H); 13C NMR (100 MHz, CDCl3) δ 160.0,

146.5, 142.1, 135.5, 134.9, 129.8, 128.8, 125.9, 124.4, 123.4, 120.7, 110.8, 47.9, 42.9, 26.3, 25.3,

24.3. Anal. Calcd for C19H19N3O Elemental Analysis: C, 74.73; H, 6.27; N, 13.76. Found: C,

74.70; H, 6.25; N, 13.78. HPLC analysis: retention time = 5.225 min; peak area, 100%.

24
(4-Methylpiperazin-1-yl)(1-phenyl-1H-benzo[d]imidazol-2-yl)methanone (10d): Pale yellow

oil (285 mg, 89%). 1H NMR (400 MHz, CDCl3) δ 8.16 (s, 1H), 7.92-7.90 (m, 1H), 7.59-7.54 (m,

5H), 7.38-7.35 (m, 2H), 4.14 (d, J = 7.1 Hz, 2H), 2.07 (s, 3H), 1.30-1.26 (m, 6H). 13C NMR (100

MHz, CDCl3) δ 171.2, 144.0, 142.3, 136.4, 133.7, 130.1, 128.1, 124.1, 123.7, 122.8, 120.6, 110.5,

60.4, 29.7, 21.1, 14.0. Anal. Calcd for Chemical Formula: C19H20N4O Elemental Analysis: C,

71.23; H, 6.29; N, 17.49. Found: C, 71.26; H, 6.30; N, 17.50. HPLC analysis: retention time =

4.440 min; peak area, 100%.

(4-(2-Methoxyphenyl)piperazin-1-yl)(1-phenyl-1H-benzo[d]imidazol-2-yl)methanone (10e):

Light brown solid (375 mg, 91%). mp 170-172 °C; IR (KBr, cm-1) 3401, 2916, 1642, 1594, 1524,

1496, 1243, 1021, 757; 1H NMR (400 MHz, CDCl3) δ 7.81 (d, J = 7 Hz, 1H), 7.51-7.47 (m, 2H),

7.43-7.40 (m, 3H), 7.28 (m, 2H), 6.98-6.94 (m, 1H), 6.86-6.78 (m, 4H), 3.82 (s, 2H), 3.78 (s, 3H),

3.69 (m, 2H), 2.94 (s, 2H), 2.84 (s, 2H); 13C NMR (100 MHz, CDCl3) δ 160.0, 152.2, 145.8, 142.1,

135.7, 135.1, 129.8, 128.9, 126.1, 124.6, 123.7, 123.5, 121.0, 120.9, 118.5, 111.3, 110.9, 55.4,

51.0, 50.5, 47.1, 42.2. Anal. Calcd for C25H24N4O2 Elemental Analysis: C, 72.80; H, 5.86; N,

13.58. Found: C, 72.79; H, 5.87; N, 13.60. HPLC analysis: retention time = 5.808 min; peak area,

100%.

(4-(4-Methoxyphenyl)piperazin-1-yl)(1-phenyl-1H-benzo[d]imidazol-2-yl)methanone (10f):

Brown solid (367 mg, 89%). mp 244-249 °C; 1H NMR (400 MHz, CDCl3) δ 7.92-7.90 (m, 1H),

7.60-7.56 (m, 2H), 7.53-7.48 (m, 3H), 7.40-7.37 (m, 3H), 6.90-6.84 (m, 4H), 3.89-3.86 (m, 2H),

3.82-3.79 (m, 5H), 3.06-3.04 (m, 2H), 2.96-2.93 (m, 2H); 13C NMR (100 MHz, CDCl3) δ 159.9,

154.5, 145.5, 145.1, 142.0, 135.7, 135.2, 129.8, 128.9, 126.1, 124.7, 123.5, 120.9, 119.1, 114.5,

110.9, 55.6, 51.4, 51.0, 47.0, 42.2. Anal. Calcd for Chemical Formula: C25H24N4O2 Elemental

25
Analysis: C, 72.80; H, 5.86; N, 13.58. Found: C, 72.78; H, 5.84; N, 13.56. HPLC analysis:

retention time = 3.842 min; peak area, 100%.

1-(4-(4-(1-Phenyl-1H-benzo[d]imidazole-2-carbonyl)piperazin-1-yl)phenyl)ethanone (10g):

Greenish yellow solid (293 mg, 69%). mp 182-185°C; 1H NMR (400 MHz, MeOD) δ 7.80-7.78

(m, 2H), 7.74-7.71 (m, 1H), 7.56-7.52 (m, 2H), 7.48-7.44 (m, 2H), 7.34-7.33 (m, 2H), 7.12 (d, J =

2.5 Hz, 2H), 6.91-6.85 (m, 2H), 3.72-3.70 (m, 2H), 3.54-3.51 (m, 2H), 3.29-3.27 (m, 2H), 3.15-

3.12 (m, 2H), 2.41 (s, 3H); 13C NMR (100 MHz, MeOD) δ 191.9, 154.2, 130.3, 129.9, 129.2,

129.2, 127.5, 126.0, 124.8, 123.9, 122.9, 122.9, 119.6, 111.6, 115.3, 113.7, 112.1, 110.9, 34.3,

30.8, 29.3, 28.7, 24.8. Anal. Calcd for Chemical Formula: C26H24N4O2 Elemental Analysis: C,

73.56; H, 5.70; N, 13.20. Found: C, 73.61; H, 5.69; N, 13.18. HPLC analysis: retention time =

4.767 min; peak area, 100%.

(4-Benzhydrylpiperazin-1-yl)(1-phenyl-1H-benzo[d]imidazol-2-yl)methanone (10h): Pale

yellow oil (373 mg, 79%). 1H NMR (400 MHz, CDCl3) δ 7.86-7.83 (m, 1H), 7.58-7.19 (m, 21H),

4.23 (m, 1H), 3.72-3.61 (m, 4H), 2.40-2.28 (m, 4H); 13C NMR (100 MHz, CDCl3) δ 159.8, 145.8,

142.0, 141.9, 135.7, 135.1, 130.1, 129.8, 128.6, 127.8, 127.2, 126.1, 124.1, 123.4, 120.8, 75.8,

52.0, 51.3, 47.1, 42.2. Anal. Calcd for C31H28N4O Elemental Analysis: C, 78.79; H, 5.97; N, 11.86.

Found: C, 78.76; H, 5.96; N, 11.84. HPLC analysis: retention time = 4.750 min; peak area, 100%.

Biological Evaluation

Determination of Minimum Inhibitory Concentration (MIC)

The protocol approved by NCCLS (National Committee for Clinical Laboratory Standards) for

estimation of minimum inhibitory concentration has been implemented for the determination of

susceptibility of compounds towards Mtb H37Rv ATCC 27294 strain [51]. Two-fold serial

dilutions in the range of 50.0, 25.0, 12.5, 6.25, 3.13, 1.56, 0.78, 0.4 µg/mL of carboxamides and

26
standard medicines have been made and treated with agar culture of Middlebrook 7H11 added

with oleic acid, albumin, dextrose, and catalase (OADC) growth supplements. To prepare the

inoculums of Mtb H37Rv ATCC 27294, Middlebrook 7H11 agar slants with OADC growth

supplements were used. Later it has been set to wet weight (1 mg/mL) in 0.05% of Tween 80,

saline diluted to 0.01 to yield ~ 107 colony forming units (CFU) per mL. A 5 μL of the suspension

was spiked into tubes of agar with ten times diluted compounds/mL and the tubes were incubated

at 37 ºC for four weeks and final results were recorded by performing the same experiment in

triplicates.

Determination of Cytotoxicity

The in vitro cytotoxicity of the anti-TB compounds (MIC ≤ 6.25 µg/mL) have been evaluated

using the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] MTT assay [76,77]

against growth inhibition of RAW 264.7 cells at 50 μg/mL concentration of benzo[d]imidazole-2-

carboxamides. The cell lines have been preserved at 37 ˚C in a humidified 5% CO2 incubator

(Thermo scientific). The adhered cells were detached followed by centrifugation to get the cell

pellet, which have been added with fresh media to count the cells using haemocytometer and plate

of 100 μL of media with cells ranging from 5,000-6,000 per well in a 96-well plate. The plate was

incubated overnight in the CO2 incubator for the cells to adhere and regain their shape. Later, cells

were treated with diluted media inoculated with 50 μg/mL of the test compounds to deduce the

percentage inhibition on normal cells followed by incubation of 48 h. Zero hour reading was noted

down with untreated cells and also control with 1% DMSO to subtract further from the 48 h

reading. Cells were treated by MTT dissolved in PBS (5 mg/mL) and incubated for 3-4 h at 37 ˚C.

The formazan crystals formed were dissolved in 100 μL of DMSO and the viability was measured

27
at 540 nm on a multimode reader (Spectra max). The values were further calculated for percentage

inhibition to record the cytotoxicity of the test compounds.

Approximation for Selectivity Index

The selectivity index is defined as the ratio of 50% inhibitory concentration (IC50) to minimum

inhibitory concentration (MIC99). Using MTT assay, the in vitro cell viability have been measured

at 50 µg/mL concentration of the potent anti-TB compounds with < 50% inhibition of cell lines

(Table 4). Thus, assuming IC50 for the target compounds ~ 50 µg/mL, the selectivity index has

been approximated.

Author Contributions

All authors contributed equally in this work.

Notes

The authors declare no competing financial interest.

Acknowledgments

T.M.D. and A.K.C. thank Department of Pharmaceuticals (DoP), New Delhi for financial

assistance. D.P. thanks the Department of Science and Technology (DST), India for partial support

by a grant (EMR/2014/001047). R.T. thanks University Grants Commission (UGC), New Delhi

for a research fellowship.

Appendix A. Supplementary data

Supplementary data related to this article can be found at https://doi.org/10.1016/j.bioorg.2020.

5. References
[1] World Health Organization (WHO). Global Tuberculosis Report; 2019.

28
 https://apps.who.int/iris/bitstream/handle/10665/329368/9789241565714-eng.pdf?ua=1

(accessed July 08, 2020).

[2] M. Raviglione, B. Marais, K. Floyd, K. Lönnroth, H. Getahun, G.B. Migliori, A.D. Harries,

P. Nunn, C. Lienhardt, S. Graham, J. Chakaya, K. Weyer, S. Cole, S.H. Kaufmann, A.

Zumla, Scaling up Interventions to Achieve Global Tuberculosis Control: Progress and

New Developments, Lancet 379 (2012) 1902–19013.

[3] The Nobel Prize. The Nobel Prize in Physiology or Medicine 1905.

https://www.nobelprize.org/prizes/medicine/1905/koch/facts/. (Accessed on July 08,

2020).

[4] P.S. Jadhavar, M.D. Vaja, T.M. Dhameliya, A.K. Chakraborti, Oxazolidinones as Anti-

tubercular Agents : Discovery, Development and Future Perspectives, Curr. Med. Chem.

22 (2015) 4379–4397.

[5] K. Dheda, C.E. Barry 3rd, G. Maartens, Tuberculosis, Lancet 387 (2015) 1211–1226.

[6] S. Tiberi, M. Muñoz-Torrico, R. Duarte, M. Dalcolmo, L. D’Ambrosio, G.B. Migliori,

New drugs and perspectives for new anti-tuberculosis regimens, Pulmonology 24 (2018)

86–98.

[7] H.M.A. Hameed, M.M. Islam, C. Chhotaray, C. Wang, Y. Liu, Y. Tan, X. Li, S. Tan, V.

Delorme, W.W. Yew, J. Liu, T. Zhang, Molecular Targets Related Drug Resistance

Mechanisms in MDR-, XDR-, and TDR-Mycobacterium tuberculosis Strains, Front. Cell.

Infect. Microbiol. 8 (2018) 1–21.

29
[8] SIRTURO® Is the First Medication for Pulmonary MDR-TB With a Novel Mechanism of

Action in Over 40 Years. URL: http://www.sirturo.com/ (accessed July 08, 2020).

[9] Otsuka Pharmaceutical Media Release. Otsuka Wins European Marketing Authorization

for Deltyba(TM) (delamanid)., (2015) 1–3.

http://www.otsuka.co.jp/en/company/release/2014/0430_01.html (accessed July 08,

2020).

[10] G. V Bloemberg, P.M. Keller, D. Stucki, A. Trauner, S. Borrell, T. Latshang, M. Coscolla,

T. Rothe, R. Hömke, C. Ritter, J. Feldmann, B. Schulthess, S. Gagneux, E.C. Böttger,

Acquired resistance to bedaquiline and delamanid in therapy for tuberculosis, New Eng. J.

Med. 373 (2015) 1986–1988.

[11] Highlights of Prescribing Information.,

https://www.accessdata.fda.gov/drugsatfda_docs/label/2013/204671s000lbl.pdf (accessed

July 08, 2020).

[12] M. V. Worley, S.J. Estrada, Bedaquiline: A novel antitubercular agent for the treatment of

multidrug-resistant tuberculosis, Pharmacotherapy. 34 (2014) 1187–1197.

[13] E. Harausz, H. Cox, M. Rich, C.D. Mitnick, P. Zimetbaum, J. Furin, QTc prolongation and

treatment of multidrug-resistant tuberculosis, Int. J. Tuberc. Lung Dis. 19 (2015) 385–391.

[14] L. Guglielmetti, M. Jaspard, L.D. Damien, M. Lachâtre, D. Marigot-Outtandy, C. Bernard,

N. Veziris, J. Robert, Y. Yazdanpanah, E. Caumes, M. Fréchet-Jachym, Long-term

outcome and safety of prolonged bedaquiline treatment for multidrug-resistant

tuberculosis. Eur. Respir. J. 49 (2017) 1601799.

30
[15] FDA approves new drug for treatment-resistant forms of tuberculosis that affects the lungs,

(2019). https://www.fda.gov/news-events/press-announcements/fda-approves-new-drug-

treatment-resistant-forms-tuberculosis-affects-lungs (accessed July 08, 2020).

[16] C. Sala, R.C. Hartkoorn, Tuberculosis drugs: new candidates and how to find more. Future

Microbiol 6 (2011) 617 – 633.

[17] K. Andries, P. Verhasselt, J. Guillemont, H.W. Gohlmann, J.M. Neefs, H. Winkler, J. Van

Gestel, P. Timmerman , M. Zhu, E. Lee, P. Williams, D. de Chaffoy, E. Huitric, S. Hoffner,

E. Cambau, C. Truffot-Pernot, N, Lounis, V. Jarlier, A diarylquinoline drug active on the

ATP synthase of Mycobacterium tuberculosis. Science 307 (2005) 223 – 227.

[18] M. Matsumoto, H. Hashizume, T. Tomishige, M. Kawasaki, H. Tsubouchi, H. Sasaki, Y.

Shimokawa, M. Komatsu, OPC-67683, a nitro-dihydro-imidazooxazole derivative with

promising action against tuberculosis in vitro and in mice, PLoS Med. 3 (2006) 2131-2144.

[19] H. Sasaki, Y. Haraguchi, M. Itotani, H. Kuroda, H. Hashizume, T. Tomishige, M.

Kawasaki, M. Matsumoto, M. Komatsu, H. Tsubouchi, Synthesis and antituberculosis

activity of a novel series of optically active 6-nitro-2,3-dihydroimidazo[2,1-b]oxazoles, J.

Med. Chem. 49 (2006) 7854-7860.

[20] C. K. Stover, P. Warrener, D.R. VanDevanter, D.R. Sherman, T.M. Arain, M.H.

Langhorne, S.W. Anderson, J.A. Towell, Y. Yuan, D.N. McMurray, B.N. Kreiswirth, C.E.

Barryk, W.R. Baker, A small-molecule nitroimidazopyran drug candidate for the treatment

of tuberculosis, Nat. 405 (2000) 962-966.

31
[21] K. Pethe, P. Bifani, J. Jang, S. Kang, S. Park, S. Ahn, J. Jiricek, J. Jung, H. K. Jeon, J.

Cechetto, T. Christophe, H. Lee, M. Kempf, M. Jackson, A. J. Lenaerts, H. Pham, V. Jones,

M. J. Seo, Y. M. Kim, M. Seo, J. J. Seo, D. Park, Y. Ko, I. Choi, R. Kim, S. Y. Kim, S.

Lim, S. A. Yim, J. Nam, H. Kang, H. Kwon, C. –T. Oh, Y. Cho, Y. Jang, J. Kim, A. Chua,

B. H. Tan, M. B. Nanjundappa, S. P. S. Rao, W. S. Barnes, R. Wintjens, J. R. Walker, S.

Alonso, S. Lee, J. Kim, S. Oh, T. Oh, U. Nehrbass, S. J. Han, Z. No, J. Lee, P. Brodin, S.-

N. Cho, K. Nam, J. Kim, Discovery of q203, a potent clinical candidate for the treatment

of tuberculosis. Nat. Med. 19 (2013) 1157-1160.

[22] M. Protopopova, C. Hanrahan, B. Nikonenko, R. Samala, P. Chen, J. Gearhart, L. Einck,

C. A. Nacy, Identification of a new antitubercular drug candidate, sq109, from a

combinatorial library of 1,2-ethylenediamines, J. Antimicrob. Chemother. 56 (2005) 968-

974.

[23] V. Makarov, G. Manina, K. Mikusova, U. Möllmann, O. Ryabova, B. Saint-Joanis, N.

Dhar, M.R. Pasca, S. Buroni, A.P. Lucarelli, A. Milano, E.De Rossi, M. Belanova, A.

Bobovska, P. Dianiskova, J. Kordulakova, C. Sala, E. Fullam, P. Schneider, J.D.

McKinney, P. Brodin, T. Christophe, S. Waddell, P. Butcher, J. Albrethsen, I.

Rosenkrands, R. Brosch, V. Nandi, S. Bharath, S. Gaonkar, R.K Shandil, V.

Balasubramanian, T. Balganesh, S. Tyagi, J. Grosset, G. Riccardi, S.T. Cole,

Benzothiazinones kill Mycobacterium tuberculosis by blocking arabinan synthesis,

Science 324 (2009) 801-804.

[24] B.E. Laughon, C.A. Nacy, Tuberculosis - drugs in the 2016 development pipeline. Nat.

Rev. Dis. Primers 3 (2017) 17015.

32
[25] D.J. Triggle, Calcium antagonists. History and perspective. Stroke 21 (1990) IV49–IV58.

[26] D.J. Triggle, Calcium channel antagonists: clinical uses – past, present and future.

Biochem. Pharmacol. 74 (2007) 1–9.

[27] M. Van Heek, C.F. France, D.S. Compton, R.L. McLeod, N.P. Yumibe, K.B. Alton, E.J.

Sybertz, H.R. Davis Jr., In vivo metabolism-based discovery of a potent cholesterol

absorption inhibitor, SCH58235, in the rat and rhesus monkey through the identification

of the active metabolites of SCH48461. J. Pharmacol. Exp. Ther. 283 (1997) 157–163.

[28] S.B. Rosenblum, T. Huynh, A. Afonso, H.R. Davis, N. Yumibe, J.W. Clader, D.A. Burnett,

Discovery of 1-(4-fluorophenyl)-(3R)-[3-(4-fluorophenyl)-(3S)-hydroxypropyl]-(4S)-(4 -

hydroxyphenyl)-2-azetidinone (SCH58235): a designed, potent, orally active inhibitor of

cholesterol absorption. J. Med. Chem. 41 (1998) 973–980.

[29] W. Zheng, N. Thorne, J.C. McKew, Phenotypic screens as a renewed approach for drug

discovery, Drug Disc. Today 18 (2013) 1067-1073.

[30] D.J. Payne, M.N. Gwynn, D.J. Holmes, D.L. Pompliano, Drugs for bad bugs: confronting

the challenges of antibacterial discovery. Nat. Rev. Drug Discov. 6 (2007) 29–40.

[31] N. Aulner, A. Danckaert, J.E. Ihm, D. Shum, S.L. Shorte, Next-Generation Phenotypic

Screening in Early Drug Discovery for Infectious Diseases, Trends Parasitol. 35 (2019)

357-370.

[32] A. Koul, E. Arnoult, N. Lounis, J. Guillemont, K. Andries, The challenge of new drug

discovery for tuberculosis. Nature 469 (2011) 483–490.

33
[33] E.M. Grzelak, M.P. Choules, W. Gao, G. Cai, B. Wan, Y. Wang, J. B. McAlpine, J. Cheng,

Y. Jin, H. Lee, J.-W. Suh, G.F. Pauli, S.G. Franzblau, B.U. Jaki, S. Cho, Strategies in anti-

Mycobacterium tuberculosis drug discovery based on phenotypic screening, J. Antibiotics

72 (2019) 719–728.

[34] J.S. Carey, D. Laffan, M.T. Williams, Analysis of the Reactions Used for the Preparation

of Drug Candidate Molecules, Org. Biomol. Chem. 4 (2006) 2337–2347.

[35] S.D. Roughley, A.M. Jordan, The Medicinal Chemist’s Toolbox: An Analysis of Reactions

Used in the Pursuit of Drug Candidates, J. Med. Chem. 54 (2011) 3451–3479.

[36] E. Vitaku, D.T. Smith, J.T. Njardarson, Analysis of the Structural Diversity, Substitution

Patterns, and Frequency of Nitrogen Heterocycles among U.S. FDA Approved

Pharmaceuticals, J. Med. Chem. 57 (2014) 10257−10274.

[37] Z. Kazimierczuk, M. Andrzejewska, J. Kaustova, V. Klimešova, Synthesis and

antimycobacterial activity of 2-substituted halogenobenzimidazoles, Eur. J. Med. Chem.

40 (2005) 203–208.

[38] K. Gobis, H. Foks, M. Serocki, E. Augustynowicz-Kopeć, A. Napiórkowska, Synthesis

and evaluation of in vitro antimycobacterial activity of novel 1H-benzo[d]imidazole

derivatives and analogues, Eur. J. Med. Chem. 89 (2014) 13–20.

[39] K. Gobis, H. Foks, K. Suchan, E. Augustynowicz-Kopeć, A. Napiórkowska, K.

Bojanowski, Novel 2-(2-phenalkyl)-1H-benzo[d]imidazoles as antitubercular agents.

Synthesis, biological evaluation and structure-activity relationship, Biorg. Med. Chem. 23

(2015) 2112–2120.

34
[40] C. Raynaud, W. Daher, M.D. Johansen, F. Roquet-Banères, M. Blaise, O.K. Onajole, A.P.

Kozikowski, J.L. Herrmann, J. Dziadek, K. Gobis, L. Kremer, Active Benzimidazole

Derivatives Targeting the MmpL3 Transporter in Mycobacterium abscessus, ACS Infect.

Dis. 6 (2020) 324–337.

[41] K. Kumar, D. Awasthi, S.-Y. Lee, I. Zanardi, B. Ruzsicska, S. Knudson, P.J. Tonge, R. a

Slayden, I. Ojima, Novel trisubstituted benzimidazoles, targeting Mtb FtsZ, as a new class

of antitubercular Agents., J. Med. Chem. 54 (2011) 374–381.

[42] D. Awasthi, K. Kumar, S.E. Knudson, R.A. Slayden, I. Ojima, SAR studies on

trisubstituted benzimidazoles as inhibitors of Mtb FtsZ for the development of novel

antitubercular agents, J. Med. Chem. 56 (2013) 9756–9770.

[43] B. Park, D. Awasthi, S.R. Chowdhury, E.H. Melief, K. Kumar, S.E. Knudson, R.A.

Slayden, I. Ojima, Design, synthesis and evaluation of novel 2,5,6-trisubstituted

benzimidazoles targeting FtsZ as antitubercular agents, Bioorg. Med. Chem. 22 (2014)

2602–2612.

[44] N.S. Chandrasekera, T. Alling, M.A. Bailey, M. Files, J. V. Early, J. Ollinger, Y.

Ovechkina, T. Masquelin, P. V. Desai, J.W. Cramer, P.A. Hipskind, J.O. Odingo, T. Parish,

Identification of Phenoxyalkylbenzimidazoles with Antitubercular Activity, J. Med. Chem.

58 (2015) 7273–7285.

[45] R. V. Shingalapur, K.M. Hosamani, R.S. Keri, Synthesis and evaluation of in vitro anti-

microbial and anti-tubercular activity of 2-styryl benzimidazoles, Eur. J. Med. Chem. 44

(2009) 4244–4248.

35
[46] P.K. Ranjith, P. Rajeesh, K.R. Haridas, N.K. Susanta, T.N. Guru Row, R. Rishikesan, N.

Suchetha Kumari, Design and synthesis of positional isomers of 5 and 6-bromo-1-[(phenyl)

sulfonyl]-2-[(4-nitrophenoxy)methyl]-1H-benzimidazoles as possible antimicrobial and

antitubercular agents, Biorg. Med. Chem. Lett. 23 (2013) 5228–5234.

[47] V.K.A. Kalalbandi, J. Seetharamappa, U. Katrahalli, K.G. Bhat, Synthesis, crystal studies,

anti-tuberculosis and cytotoxic studies of 1-[(2E)-3-phenylprop-2-enoyl]-1H-

benzimidazole derivatives, Eur. J. Med. Chem. 79 (2014) 194–202.

[48] V. Purohit, A.K. Basu, Mutagenicity of nitroaromatic compounds, Chem. Res. Toxicol. 13

(2000) 673–692.

[49] P.S. Jadhavar, T.M. Dhameliya, M.D. Vaja, D. Kumar, J.P. Sridevi, P. Yogeeswari, D.

Sriram, A.K. Chakraborti, Synthesis, Biological Evaluation and Structure-Activity

Relationship of 2-Styrylquinazolones as Anti-tubercular Agents, Bioorg. Med. Chem. Lett.

26 (2016) 2663–2669.

[50] B. Tanwar, A. Kumar, P. Yogeeswari, D. Sriram, A.K. Chakraborti, Design, development

of new synthetic methodology, and biological evaluation of substituted quinolines as new

anti-tubercular leads, Bioorg. Med. Chem. Lett. 26 (2016) 5960–5966.

[51] S. Bhagat, M. Supriya, S. Pathak, D. Sriram, A.K. Chakraborti, α-

Sulfonamidophosphonates as new anti-mycobacterial chemotypes: Design, development

of synthetic methodology, and biological evaluation, Bioorg. Chem. 82 (2019) 246–252.

[52] P.S. Jadhavar, K.I. Patel, T.M. Dhameliya, N. Saha, M.D. Vaja, V.S. Krishna, D. Sriram,

A.K. Chakraborti, Benzimidazoquinazolines as new potent anti-TB chemotypes: Design,

36
 synthesis, and biological evaluation, Bioorg. Chem. 99 (2020)

10.1016/j.bioorg.2020.103774.

[53] P. Shah, T.M. Dhameliya, R. Bansal, M. Nautiyal, D.N. Kommi, P.S. Jadhavar, J.P.

Sridevi, P. Yogeeswari, D. Sriram, A.K. Chakraborti, N-Arylalkylbenzo[d]thiazole-2-

carboxamides as Anti-mycobacterial Agents: Design, New Methods of Synthesis and

Biological Evaluation, Med. Chem. Commun. 5 (2014) 1489–1495.

[54] S. Pancholia, T.M. Dhameliya, P. Shah, P.S. Jadhavar, J.P. Sridevi, P. Yogeshwari, D.

Sriram, A.K. Chakraborti, Benzo[d]thiazol-2-yl(piperazin-1-yl)methanones as new anti-

mycobacterial chemotypes: Design, synthesis, biological evaluation and 3D-QSAR

studies, Eur. J. Med. Chem. 116 (2016) 187–199.

[55] T.M. Dhameliya, R. Tiwari, A. Banerjee, S. Pancholia, D. Sriram, D. Panda, A.K.

Chakraborti, Benzo[d]thiazole-2-carbanilides as new anti-TB chemotypes: Design,

synthesis, biological evaluation, and structure-activity relationship, Eur. J. Med. Chem.

155 (2018) 364–380.

[56] K. Ablajan, W. Kamil, A. Tuoheti, S. Wan-Fu, An Efficient Three Component One-Pot

Synthesis of 5-Amino-7-aryl-7,8-dihydro-[1,2,4]triazolo[4,3-a]-pyrimidine-6-

carbonitriles, Molecules. 17 (2012) 1860–1869.

[57] T.M. Dhameliya, S.S. Chourasiya, E. Mishra, P.S. Jadhavar, P. V. Bharatam, A.K.

Chakraborti, Rationalization of benzazole-2-carboxylate versus benzazine-3-one/

benzazine-2,3-dione selectivity switch during cyclocondensation of 2-

aminothiophenols/phenols/anilines with 1,2-biselectrophiles in aqueous medium, J. Org.

Chem. 82 (2017) 10077–10091.

37
[58] D.N. Kommi, P.S. Jadhavar, D. Kumar, A.K. Chakraborti, “All-water” one-pot diverse

synthesis of 1,2-disubstituted benzimidazoles: hydrogen bond driven “Synergistic

Electrophile–Nucleophile dual activation” by water, Green Chem. 15 (2013) 798–810.

[59] P. Wayne, National Committee for Clinical Laboratory Standards. Antimycobacterial

susceptibility testing for Mycobacterium tuberculosis, tentative standard M24-T., 1995.

[60] Rifampin, Tuberculosis 88 (2008) 151–154.

[61] E.C. Hett, E.J. Rubin, Bacterial Growth and Cell Division: A Mycobacterial Perspective

Bacterial Perspective, J. Microbiol. Mol. Biol. Rev. 72 (2008) 126–156.

[62] J.J. Cui, M. Tran-Dube, H. Shen, M. Nambu, P.P. Kung, M. Pairish, L. Jia, J. Meng, L.

Funk, I. Botrous, M. Mctigue, N. Grodsky, K. Ryan, E. Padrique, G. Alton, S. Timofeevski,

S. Yamazaki, Q. Li, H. Zou, J. Christensen, B. Mroczkowski, S. Bender, R.S. Kania, M.P.

Edwards, Structure Based Drug Design of Crizotinib (Pf-02341066), a Potent and Selective

Dual Inhibitor of Mesenchymal-Epithelial Transition Factor (c-MET) Kinase and

Anaplastic Lymphoma Kinase (ALK), J. Med. Chem. 54 (2011) 6342–6363.

[63] T.W. Johnson, R.A. Gallego, M.P. Edwards, Lipophilic Efficiency as an Important Metric

in Drug Design, J. Med. Chem. 61 (2018) 6401–6420.

[64] S. Planken, D.C. Behenna, S.K. Nair, T.O. Johnson, A. Nagata, C. Almaden, S. Bailey,

T.E. Ballard, L. Bernier, H. Cheng, S. Cho-Schultz, D. Dalvie, J.G. Deal, D.M. Dinh, M.P.

Edwards, R.A. Ferre, K.S. Gajiwala, M. Hemkens, R.S. Kania, J.C. Kath, J. Matthews,

B.W. Murray, S. Niessen, S.T.M. Orr, M. Pairish, N.W. Sach, H. Shen, M. Shi, J. Solowiej,

K. Tran, E. Tseng, P. Vicini, Y. Wang, S.L. Weinrich, R. Zhou, M. Zientek, L. Liu, Y.

38
 Luo, S. Xin, C. Zhang, J. Lafontaine, Discovery of N-((3R,4R)-4-Fluoro-1-(6-((3-methoxy-

1-methyl-1H-pyrazol-4-yl)amino)-9-methyl-9H-purin-2-yl)pyrrolidine-3-yl)acrylamide

(PF-06747775) through Structure-Based Drug Design: A High Affinity Irreversible

Inhibitor Targeting Oncogenic EGFR Mutants, J. Med. Chem. 60 (2017) 3002–3019.

[65] K.D. Freeman-Cook, R.L. Hoffman, T.W. Johnson, Lipophilic efficiency: the most

important efficiency metric in medicinal chemistry, Future Med. Chem. 5 (2013) 113–115.

[66] T. Ryckmans, M. P. Edwards, V. A. Horne, M. Correia, D. R. Owen, L. R. Thompson, I.

Tran, M. F. Tutt, T. Young, Rapid assessment of a novel series of selective CB2 agonists

using parallel synthesis protocols: A Lipophilic Efficiency (LipE) analysis. Bioorg. Med.

Chem. Lett. 19 (2009) 4406−4409.

[67] M.P. Edwards, D.A. Price. Role of physicochemical properties and ligand lipophilicity

efficiency in addressing drug safety risks. Ann. Rep. Med. Chem. 45 (2010) 381–391.

[68] P.D. Leeson, B. Springthorpe (November 2007). The influence of drug-like concepts on

decision-making in medicinal chemistry. Nat. Rev. Drug Discov. 6 (2007) 881–90.

[69] S. Ekins, J.S. Freundlich, I. Choi, M. Sarker, C. Talcott, Computational databases, pathway

and cheminformatics tools for tuberculosis drug discovery. Trends Microbiol. 19 (2011)

65–74.

[70] J. Mao, H. Yuan, Y. Wang, B. Wan, M. Pieroni, Q. Huang, R.B. van Breemen, A.P.

Kozikowski S.G. Franzblau, From serendipity to rational antituberculosis drug discovery

of mefloquine-isoxazole carboxylic acid esters. J. Med. Chem. 52 (2009) 6966–6978.

39
[71] A. Lilienkampf, M. Pieroni, S.G. Franzblau, W.R. Bishai, A.P. Kozikowski, Derivatives

of 3-isoxazolecarboxylic acid esters - a potent and selective compound class against

replicating and nonreplicating Mycobacterium tuberculosis. Curr. Top. Med. Chem. 12

(2012) 729–734.

[72] A. Lilienkampf, M. Pieroni, B. Wan, Y. Wang, S. G. Franzblau, A.P. Kozikowski,

Rational design of 5-phenyl-3-isoxazolecarboxylic acid ethyl esters as growth inhibitors of

Mycobacterium tuberculosis. A potent and selective series for further drug development.

J. Med. Chem. 53 (2010) 678–688.

[73] M. Pieroni, S.K. Tipparaju, S. Lun, Y. Song, A.W. Sturm, W.R. Bishai, A.P. Kozikowski.

Pyrido[1,2-a]benzimidazole-based agents active against tuberculosis (TB), Multidrug-

Resistant (MDR) TB and extensively drug-resistant (XDR) TB. ChemMedChem 6 (2011)

334–342.

[74] C.A. Lipinski, F. Lombardo, B.W. Dominy, P.J. Feeney, Experimental and computational

approaches to estimate solubility and permeability in drug discovery and development

settings. Adv. Drug Deliv. Rev. 23 (1997) 3–25.

[75] https://en.wikipedia.org/wiki/Lipophilic_efficiency#:~:text=LiPE%20allows%20capturin

g%20both%20values,ranking%20series%20and%20individual%20compounds (accessed

on October 17, 2020).

[76] T. Mosmann, Rapid colorimetric assay for cellular growth and survival: application to

proliferation and cytotoxicity assays. J. Immunol. Methods 65 (1983) 55–63.

40
[77] J. van Meerloo, G.J.L. Kaspers, J. Cloos, Cell sensitivity assays: The MTT assay, in: I.A.

Cree (Ed.), Cancer Cell Culture. Methods in Molecular Biology (Methods and Protocols),

Humana Press, 731 (2011) 237–245.

[78] J.H. Musser, T.T. Hudec, K. Bailey, A Simple One-Step Synthesis of Alkyl Benzazol-2-

carboxylates, Synth. Comm. 14 (1984) 947–953.

FIGURE CAPTIONS
Figure 1. Recently approved anti-TB drugs and novel chemical entities (NCEs) in TB pipeline.
MICs have been provided in the parenthesis.

Figure 2. Benzo[d]imidazoles reported as potent anti-TB lead molecules. The reported MIC values
converted into µg/mL have been provided in parenthesis.

Figure 3. Benzo[d]imidazole-2-carboxamide (L) as novel anti-TB scaffold.

TABLES

Table 1. The NH4Cl-catalysed catalyzed amidation of 6 with various arylalkylamines to from 8.a

R1
R1
R3
N O
+ R2 n NH2 NH4Cl (20 mol%) R3
N O neat, 100 ºC N HN n
N O (7)
(6a/b) (8) R2

Yield
Time of 8
Entry R1 n R2 R3 Product
(h)
(%)b

41
 1 H 1 H H 8a 0.5 94
2 H 1 2-F H 8b 1.5 79
3 H 1 4-F H 8c 1.5 82
4 H 1 2,4-di-F H 8d 2 68
5 H 1 3,4-di-F H 8e 2 67
6 H 1 2,5-di-F H 8f 1 78
7 H 1 2-Cl H 8g 2 73
8 H 1 3-Cl H 8h 1 81
9 H 1 4-Cl H 8i 1 76
10 H 1 2-Me H 8j 2 69
11 H 1 2-OMe H 8k 0.5 84
12 H 1 2,4-di-OMe H 8l 0.5 82
13 H 1 3,4-di-OMe H 8m 2 76
14 H 1 4-CF3 H 8n 1 79
15 H 1 H Me 8o 1.5 76
16 H 1 3,4-methylenedioxy H 8p 0.5 81
17 H 2 H H 8q 0.5 83
18 H 2 3,4-di-OMe H 8r 1.5 78
19 Cl 1 H H 8s 1 84
aEthyl 1-phenyl-1H-benzo[d]imidazole-2-carboxylate (6) (1 mmol) was treated with the
arylalkyl amines (7) (1 mmol, 1 equiv) in presence of NH4Cl (20 mol%) at 100 ºC under neat
conditions. bThe isolated yield of the corresponding carboxamides 8.

Table 2. The NH4Cl-catalysed catalyzed amidation of 6a with various alicyclic amines 9 to form

the cycloalkylamides (10).

42
 HN NH4Cl (20 mol%) N O
+
N O Z neat, 100 ºC N N
N O (9) (10)
Z
(6a)

Yield of
Entry Z R1 Product Time (h)
10 (%)b

1 O - 10a 0.5 78
2 S - 10b 1 67
3 CH2 - 10c 0.5 75
4 NR1 Me 10d 1 89
5 NR1 2-OMe-C6H4 10e 1.5 91
6 NR1 4-OMe-C6H4 10f 1.5 89
7 NR1 4-COMe-C6H4 10g 2 69
8 NR1 CH(C6H4)2 10h 2 79
aEthyl 1-phenyl-1H-benzo[d]imidazole-2-carboxylate (6a) (1 mmol) was treated with different
alicyclic amines (9) (1 mmol, 1 equiv) in the presence of NH4Cl (20 mol%) at 100 ºC under neat
conditions for the indicated time period. bThe isolated yield of the corresponding cycloalkylamides
10.
Table 3. Anti-mycobacterial activity, cLogP and cLipE values of the synthesized compounds and

MIC of a few standard anti-TB drugs.

Compd MICa cLipE Compd MICa cLipE

cLogPb cLogPb
No. (μg/mL) (MIC)c No. (μg/mL) (MIC)c

6a 12.5 3.87 8.77 8q 12.5 5.58 10.48

8a 1.56 5.44 11.25 8r >25 5.23 ND

8b 3.125 5.59 11.10 8s >25 6.17 ND

8c 6.25 5.59 10.79 10a 12.5 2.65 7.55

43
 8d 3.125 5.73 11.24 10b 25 3.39 7.99

8e 0.78 5.66 11.77 10c >25 3.68 ND

8f 1.56 5.73 11.54 10d >25 3.21 ND

8g 3.125 6.16 11.67 10e >25 4.67 ND

8h 6.25 6.16 11.36 10f >25 4.67 ND

8i 6.25 6.16 11.36 10g >25 4.39 ND

8j 6.25 5.90 11.10 10h 12.5 6.28 11.18

8k 1.56 5.37 11.18 INH 0.098 -0.668 6.34

8l 6.25 5.46 10.66 R 0.197 3.719d 10.42

8m >25 ND ND E 1.56 0.118 5.92

8n 12.5 6.33 11.23 Z 6.25 -0.676 4.53

8o 1.56 5.76 11.57 Cfx 1.56 -0.725 5.08

8p 25 5.41 10.01

aCalculated using ChembioDraw Ultra 12.0. b99% inhibition of growth of Mtb H37Rv (ATCC

27294 strain). ccLipE calculated using formula cLipE = Log(MIC in gm/mL)-cLogP. dData taken

from ref [60]. ND: Not determined.

Table 4. In vitro cell viability against RAW 264.7 cells lines for compounds with MIC ≤ 6.25

μg/mL.

Compd. MIC RAW 264.7

Entry MIC (μM)a
Code (μg/mL)a (% Inhibition)b

1 8a 1.56 4.77 23.32

2 8b 3.125 9.05 32.17

44
 3 8c 6.25 18.10 27.64

4 8d 3.125 8.60 33.65

5 8e 0.78 2.15 34.89

6 8f 1.56 4.29 26.12

7 8g 3.125 8.64 43.65

8 8h 6.25 17.27 56.7

9 8i 6.25 17.27 18.9

10 8j 6.25 18.31 60.34

11 8k 1.56 4.36 34.2

12 8l 6.25 16.13 23.12

13 8o 1.56 4.57 20.77

aMIC determined against Mtb H37Rv strain. b% inhibition at 50 μg/mL concentration determined

against RAW 264.7 cells lines.

45
 O N
O

N
O
F3CO
Br Delamanid (OPC67683) N
(MIC 0.006 µg/mL) NO2
O
N
O N NO2

Ref 18-19
F3CO
N

2006
N

Re

20
OH

f1
O

ef
R

00
7
20
Pretomanid (PA824)

20
05
Approved (MIC 0.13 µg/mL)
Bedaquiline (TMC207) Anti-TB drugs
(MIC 0.004-0.07 µg/mL) and
NCEs in TB R
23 ef O
21 N
20 O
ef

H 3C H 3C
09
13
R

N
Ref 22
20

2005
F3C S O
H 3C NH HN
SQ109
(MIC 0.7-1.56 µM or NO2 BTZ043
0.23-0.51 µg/mL) (MIC 0.001 µg/mL)
Cl O
N N
N H
OCF3
N
Q203
(MIC 2.7 nM or 1.5 µg/mL)

Figure 1. Recently approved anti-TB drugs and novel chemical entities (NCEs) in TB
pipeline. MICs have been provided in the parenthesis.

46
 OnBu

N NH O
NH

R
N
NH
NH Cl Cl
N MIC: 0.8 µg/mL
R MIC
NEt2 1 µM (0.39 µg/mL)

Ref 39
MIC: 0.75 µg/mL C

2
NMe2 0.06 µg/mL

Re

-4
40
f3
O-4-F-C6H4 0.63 µg/mL
D

ef
B

R
N H N
H Ref 37 Ref 43 N
Br N NO2 N
S A N E Br
N Re MIC: 52 ± 2 nM
f4
47

Br MIC: 2 µM 4 (0.02 µg/mL)

ef

H F
R

(0.89 µg/mL)
Ref 45
G Cl

NO2
O N
Cl
N HN
F Br
N O
MIC: 1.6 µg/mL O MIC: >7.25 µg/mL
S N
N
F O

Br
MIC: 0.0625 µg/mL

Figure 2. Benzo[d]imidazoles reported as potent anti-TB lead molecules. The reported MIC
values converted in to µg/mL have been provided in parenthesis.

47
 S O S O

Cl N HN CF3 N N

F F N
O
MIC 2.3 µM MIC 2.18 µM
I J

N O
Br S O O
E N HN R1 K
N
N F3C N HN O
H N
O
(52 ± 2nM) (L) MIC 1.9 µM

Figure 3. Benzo[d]imidazole-2-carboxamide (L) as novel anti-TB scaffold.

SCHEMES

N NHR1 N OEt
1
N O N + NH2R
O

Scheme 1. Synthetic strategy for the construction of the newly designed scaffold L.

O H H
NH2 N O N O
OEt
+ H +
NH2 N OEt N
R O R R
(Not f ormed) Only product

Scheme 2. The cyclocondensation of substituted o-phenylene diamine with ethyl glyoxalate.

48
 NH2 O
OEt
H
NO2 R1 NO2 R1 NH2 R1 O N O
(2) In/HCl (aq) (5)
Water, ref lux, 2h Water, 1 h N OEt
F N N
2h H H
1 3 4

R1
6a, R1 = H; 80%
6b, R1 = Cl; 63%

Scheme 3. “All-water” one-pot synthesis of benzo[d]imidazole-2-carboxylates.

49
Declaration of interests

☒ The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.

☐The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests:

50