Chemical synthesis, molecular modeling and pharmacophore mapping of new pyrrole derivatives as inhibitors of InhA enzyme and Mycobacterium tuberculosis growth

Article abstract of DOI:10.1007/s00044-019-02418-1

Abstract: Substituted phenylthiazolyl benzamide and pyrrolyl benzamide derivatives were developed using molecular hybridization technique to create novel lead antimycobacterial molecules used to fight against Mycobacteriumtuberculosis. The newly synthesized molecules have inhibited InhA, the enoyl-ACP reductase enzyme from the mycobacterial type II fatty acid biosynthetic pathway. Of these, compound 3b showed H-bonding interactions with Tyr158 and co-factor NAD⁺ that binds the active site of InhA. All the molecules were screened for in vitro antitubercular activity against M. tuberculosis H₃₇Rv, as well as some representative molecules as the inhibitors of InhA. Thirteen compounds exhibited good anti-TB activities (MIC = 1.6 μg/mL), but only few representative molecules showed the moderate InhA enzyme inhibition activity. [Figure not available: see fulltext.].

Full text of DOI:10.1007/s00044-019-02418-1

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-019-02418-1
ORIGINAL RESEARCH
Chemical synthesis, molecular modeling and pharmacophore
mapping of new pyrrole derivatives as inhibitors of InhA enzyme
and Mycobacterium tuberculosis growth
Shrinivas D. Joshi1 S. R. Prem Kumar1 Sonali Patil1 M. Vijayakumar1 Venkatarao H. Kulkarni1
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Mallikarjuna N. Nadagouda1 Aravind M. Badiger2 Christian Lherbet3 Tejraj M. Aminabhavi1
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Received: 12 March 2019 / Accepted: 1 August 2019
© Springer Science+Business Media, LLC, part of Springer Nature 2019
Abstract
Substituted phenylthiazolyl benzamide and pyrrolyl benzamide derivatives were developed using molecular hybridization
technique to create novel lead antimycobacterial molecules used to fight against Mycobacterium tuberculosis. The newly
synthesized molecules have inhibited InhA, the enoyl-ACP reductase enzyme from the mycobacterial type II fatty acid
biosynthetic pathway. Of these, compound 3b showed H-bonding interactions with Tyr158 and co-factor NAD+ that binds
the active site of InhA. All the molecules were screened for in vitro antitubercular activity against M. tuberculosis H37Rv, as
well as some representative molecules as the inhibitors of InhA. Thirteen compounds exhibited good anti-TB activities
(MIC = 1.6 μg/mL), but only few representative molecules showed the moderate InhA enzyme inhibition activity.
Supplementary information The online version of this article (https://
doi.org/10.1007/s00044-019-02418-1) contains supplementary
material, which is available to authorized users.
* Shrinivas D. Joshi
2
BDR Pharmaceuticals, Baroda, India
shrinivasdj@rediffmail.com
3
Laboratoire de Syntheseet Physico-chimie de Molecules d’Interet
Biologique, LSPCMIB, Universite de Toulouse, UPS, 118 Roote
de Narbonne, F-31062 Toulouse Cedex 9, France
1
Novel Drug Design and Discovery Laboratory, Department of
Pharmaceutical Chemistry, S.E.T’s College of Pharmacy, Sangolli
Rayanna Nagar, Dharwad, Karnataka, India

Medicinal Chemistry Research
Graphical Abstract
Basic core moiety and docked mode of all the synthesized compounds inside the proposed binding pocket of InhA with the
final selected pharmacophore model molecular alignment for InhA receptor ligands.
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Keywords Pyrroles Phenylthiazoles Tuberculosis M. tuberculosis H37Rv Enoyl-ACP reductase GALAHAD
Introduction
people along with other essential parts such as spine, kid-
ney, and brain if not controlled properly on time (WHO
2019).
Mycobacterium tuberculosis (M. tuberculosis) has been the
main widely spread contagious pathogen to cause tubercu-
losis (TB) among different strains of mycobacteria. As per
the latest WHO report, TB is one of the most deadly dis-
eases worldwide infecting nearly 10 million people with a
death toll of 1.30 million among the common people and
0.3 million resulting from the TB cases co-infected with
HIV. There is therefore an urgent need to fast-track the
death rate of yearly decline by at least 4–5% by 2020 to
achieve the milestones to eradicate the TB strategy. In
recent years, multidrug-resistant TB (MDR-TB) and the
emergence of rifampicin-resistant TB (RR-TB) have
become more prominent. Consequently, TB remains to be
the main public health threat attacking the lungs of the
At present, there is an urgent need to develop novel che-
motherapeutic agents to fight against TB, particularly multi-
drug and extensive drug resistance. Despite the availability of
abundant innovative drugs for the treatment (Fig. 1), yet the
drug-resistance still remains to be the challenging task.
Therefore, there is a pressing need for developing more
effective anti-TB agents with the activity against both MDR-
TB and latent TB, which would help to reduce combined drug
therapy. Recent trends towards targeted enzyme for testing
novel anti-TB agents is InhA (Joshi et al. 2014), which is an
enoyl acyl carrier protein reductase (ENR) from M. tubercu-
losis that is the main enzyme for type II fatty acid synthesis
(FAS II), which catalyzes the reduction of 2-trans-enoyl-ACP

Medicinal Chemistry Research
Fig. 1 Milestones in TB drug
development research.
Abbreviations: TB Tuberculosis,
MDR multi-drug resistant,
JATA Japan AntiTuberculosis
Association, NM4TB new
medicines for tuberculosis,
iM4TB innovative medicines for
tuberculosis
(acyl carrier protein) using NADH cofactor to give up NAD+
and reduced enoyl thioester-ACP substrate; this in turn, aids
the synthesis of mycolic acid that is the specific and essential
constituent of mycobacterial envelope present in the FAS
system of M. tuberculosis. InhA is an excellent target as
the FAS II system in bacteria is not present in the human
systems.
of which LL3858 is currently in pre-clinical stage used for
the treatment of TB (Arora et al. 2004).
In this perspective and in continuation of our ongoing
program of research to develop new antitubercular mole-
cules, herein we report the design and synthesis of new
pyrrole derivatives as direct inhibitors of enoyl ACP
reductase InhA (Fig. 2) (Joshi et al. 2016a, 2017, 2018),
which exhibited substantial anti-TB activities. On the basis
of versatile chemotherapeutic and pharmacological pro-
spective of thiazoles, pyrroles, and the importance of peptide
linkage, novel chemical scaffolds having phenyl thiazole,
pyrrole, and peptide linkages with both antimycobacterial
and antibacterial properties are developed in this research.
Even though there are several likely molecules having
important antimycobacterial potency that can act as inhibi-
tors of enoyl-ACP reductase enzyme, but after bedaquilin
and delamanid none of these have emerged as potential
drugs because these are still in their premature stage and
preclinical phase. Keeping in mind the above facts, we have
undertaken a much broader study on molecular modeling,
pharmacophore mapping and synthesis of direct inhibitors
of enoyl-ACP reductase containing pyrrolyl thiazoles and
pyrrolyl benzamides as the main core unit using in silico
methods involving the GALAHAD (pharmacophore based
on an automated computational alignment) technique along
with Surflex-docking studies to identify enoyl-ACP reduc-
tase as the probable target for these derivatives. Using these
Hantzsch et al. and others have reported earlier the
synthesis of thiazoles (Hantzsch and Weber 1887; Metzger
1984). Thiazole moiety is a part of many potent pharma-
cologically active drugs, such as sulfathiazole (antimicrobial
drug), tiazofurin (anticancer drug), and ritonavir (anti-
retroviral drug). Thiazole derivatives have various pharma-
cological and biological activities, such as antibacterial
(Hoshino et al. 2002; Dighe et al. 2011), antitubercular (anti-
TB) (Karuvalam et al. 2012; Shiradakar et al. 2007), anti-
fungal (Chimenti et al. 2007), anti-HIV (Masuda et al.
2005), and anti-inflammatory (Kalkhambkar et al. 2007).
Pyrrole is another nitrogen-containing heterocyclic moi-
ety present in plant (naturally occurring chlorophyll) as well
as animal kingdom (Hemin and vitamin-B12). Anti-
mycobacterial activity of BM212, an analog of pyrroles,
was first reported by Deidda et al. (1998). Almost imme-
diately after this discovery, a number of analogs of BM212
have been reported (Biava et al. 2006). Based on these
results, Lupin, Astra Zeneca, and many other companies
have synthesized a series of pyrrole-containing compounds

Medicinal Chemistry Research
Fig. 2 Some antitubercular
agents and our earlier reported
pyrrole derivatives (Joshi et al.
2016a, 2017, 2018) having
antitubercular and InhA
inhibition activities
approaches, we have performed quantitative pharmacophore
mapping method to identify the common functional groups
accountable for the specific ligand–receptor interactions
(Tropsha 2005) in conjunction with molecular-docking
analysis on phenyl thiazole and pyrrole benzamide deriva-
tives as the better InhA inhibitors and evaluated their bac-
tericidal as well as antimycobacterial activities.
It is well known that compounds with amino thiazole and
benzamide have displayed immpressive antimycobacterial
activities, inhibiting the bacteria by targeting InhA (Guardia
et al. 2016). Therefore, new antimycobacterial compounds
are designed in this study with pyrrolyl thiazole and pyrrole
benzamide nucleus by means of hybridization approach. In
this perspective, we report here the essential pharmaco-
phoric features derived from the basic structure of N-(4-
methylbenzoyl)-4-benzylpiperidine, containing carbonyl
H-bond acceptor and donor bridge between (4-benzylpi-
peridin-1-yl)(p-tolyl)methanone and 4-toluene, since they
are acting as hydrophobic moieties. Hence, (4-benzylpi-
peridin-1-yl)(p-tolyl)methanone and 4-toluene were mapped

Medicinal Chemistry Research
Fig. 3 Design concept used for
the synthesis of titled
compounds
with a carbonyl linkage (–CO–), which would allow pos-
sible interactions with the hydroxyl group of key residue
Tyr158 and NAD+ of InhA (Chollet et al. 2015) and
simultaneously hydrophobic moieties were mapped with o/
m/p-substituted phenyl analogs of thiazole and pyrrole
moieties as depicted in Fig. 3. These approaches were
considered for the synthesis of new molecules using Pall–
Knorr pyrrole synthesis (Joshi et al. 2016b). Hitherto, no
such molecules have been explored in the literature and
hence the reported compounds are novel.
triplet, (q) quartet, (dd) doublet of doublet, (brs) broad
singlet, (qu) quintet, and (m) multiplet.
Mass spectra (MS) were obtained on WATERS Q-Tof
premier mass spectrometer and Schimadzu QP 20105 GC-
mass spectrometer. Elemental analysis was done using
TRUSPEC CHN analyzer. Analytical thin-layer chromato-
graphy (TLC) was performed on precoated TLC sheets of
silica gel 60 F254 (Merck, Darmstadt, Germany) visualized by
long-wavelength and short-wavelength UV lamps. Chroma-
tographic purifications were performed on Merck aluminum
oxide (70–230 mesh) and Merck silica gel (70–230 mesh).
Purity of these compounds was confirmed by TLC analysis
and their structures were confirmed by spectral studies.
Experimental
Materials and methods
General procedure for the synthesis of N-4-(4-
substituted phenyl)thiazole-2-yl-4-(1H-pyrrol-1yl)
bezamides (3a–e)/N-4-(4-substituted phenyl)thiazol-
2-yl-4-(2,5-dimethyl-1H-pyrrol-1-yl)benzamides
(4a–f)
Melting points were determined on a Shital-digital pro-
grammable melting point apparatus and are not corrected.
Infrared spectra in KBr pellets were recorded on a Bruker
FTIR spectrophotometer. The 1H and 13C NMR spectra
were recorded on a Bruker AVANCE II at 400 and
100/75 MHz, respectively, using CDCl3 or DMSO-d6 as the
solvent; chemical shifts are expressed in parts per million
(δ ppm) relative to TMS. Abbreviations used to describe
peak patterns are: (b) broad, (s) singlet, (d) doublet, (t)
The 2-amino-4-(4-substituted phenyl)thiazoles (2a–f) (Pat-
tan et al. 2009) (0.0018 mol) and 4-(1H-pyrrolyl-1-yl)ben-
zoic acid (9a) (Joshi et al. 2018)/4-(2,5-dimethyl-1H-
pyrrolyl-1-yl)benzoic acid (9b) (Joshi et al. 2008)
(0.0019 mol) were dissolved, respectively in 50 mL of dry

Medicinal Chemistry Research
Scheme. 1 Synthesis of
substituted thiazolyl pyrrolyl
benzamides
dimethyl formamide. HBTU (0.87 g, 0.0023 mol) and DIEA
(0.93 mL, 0.0053 mol) were then added to the above mix-
tures and stirred for 24–30 h at ambient temperature. The
reaction was quenched by adding NaCl solution and the
mixture was extracted with ethyl acetate (3 × 50 mL). The
combined ethyl acetate layer was washed with 1 N HCl and
with a saturated sodium bicarbonate solution followed by
NaCl. The organic layer was dried over anhydrous sodium
sulfate and concentrated using a rotary flash evaporator.
Thus obtained residue was dried and purified by column
chromatography using petroleum ether:ethyl acetate (6:4)
mixture as the eluent to afford the compounds (3a–e) and
(4–f) (Scheme 1).
N-(4-phenyl-1,3-thiazol-2-yl)-4-(1H-pyrrol-1-yl)benzamide (3a)
Compound 3a was obtained as yellow solid (yield 75%). m.
p. 178–180 °C; FTIR (KBr): 3412, 3110, 1652, 1550 cm−1
.
1H NMR (400 MHz, DMSO-d6) δ ppm: 12.80 (1H, s, NH),
8.24 (2H, d, J = 2.3 Hz, H-16, H-20), 7.96 (2H, d, J =

Medicinal Chemistry Research
5.4 Hz, H-17, H-19), 7.81 (2H, d, J = 2.2 Hz, H-10, H-14),
7.68 (1H, s, H-5), 7.43-7.32 (3H, m, H-11, H-12, H-13),
7.26 (2H, d, J = 1.3 Hz, H-22, H-25), 6.33 (2H, d, J =
2.2 Hz, H-23, H-24). 13C NMR (75 MHz, DMSO-d6) δ
ppm: 165.32 (CO, C-8), 162.77 (C-2), 150.32 (C-4), 144.83
(C-15), 131.70 (C-9), 130.51(C-18), 128.98 (CH, C-11,C-
13), 128.73 (CH, C-16, C-20), 126.41 (CH, C-17, C-19),
126.26 (CH, C-10, C-14), 120.45 (CH, C-12), 118.02 (CH,
C-23, C-24), 111.20 (CH, C-22, C-25), 101.22 (CH, C-5);
MS (ESI): m/z = found 345.01 [M+] (calcd. 345.42); Anal.
Calcd. For C20H15N3OS: C, 69.54; H, 4.38; N, 12.17;
Found: C, 69.78; H, 4.62; N, 12.46.
N-[4-(4-nitrophenyl)-1,3-thiazol-2-yl]-4-(1H-pyrrol-1-yl)
benzamide (3d)
Compound 3d was obtained as yellow solid (yield 52%). m.
p. 194–196 °C; FTIR (KBr): 3398, 3147, 1603, 1513 cm−1
;
1H NMR (400 MHz, DMSO-d6) δ ppm: 12.62 (1H, s, NH),
8.22 (2H, d, J = 8.9 Hz, H-16, H-20), 8.09 (2H, d, J =
8.7 Hz, H-17, H-20), 7.67 (1H, s, H-5), 7.55 (2H, d, J =
2.4 Hz, H-10, H-14), 7.46 (2H, d, J = 1.8 Hz, H-11, H-13),
7.42 (2H, d, J = 2.1 Hz, H-24, H-25) 6.32 (2H, d, J =
2.0 Hz, H-23, H-26); 13C NMR (75 MHz, DMSO-d6) δ
ppm: 168.73 (CO, C-8), 149.93 (C-2), 146.50 (C-4), 143.32
(C-15), 141.23 (C-12), 139.92 (C-9), 131.09 (C-18), 129.59
(C-11, C-13), 126.94 (C-20, C-16), 125.22 (C-10, C-14),
124.76 (C-19, C-17), 118.10 (C-24, C-25), 110.33 (C-23, C-
26), 105.65 (C-5); MS (ESI): m/z = found 390.62 [M+]
(calcd. 390.42); anal. calcd. For C20H14N4O3S: C, 61.53; H,
3.61; N, 14.35; Found: C, 61.73; H, 3.91; N, 14.65.
N-[4-(4-bromophenyl)-1,3-thiazol-2-yl]-4-(1H-pyrrol-1-yl)
benzamide (3b)
Compound 3b was obtained as yellow solid (yield 70%).
m.p. 166–168 °C; FTIR (KBr): 3418, 3114, 1612,
1527 cm−1; 1H NMR (400 MHz, DMSO-d6) δ ppm: 12.81
(1H, s, NH), 8.24 (2H, d, J = 8.7 Hz, H-16, H-20), 7.91
(2H, d, J = 1.8 Hz, H-17, H-19), 7.76 (2H, d, J = 2.4 Hz,
H-10, H-16), 7.62 (1H, s, H-5), 7.54 (2H, d, J = 1.8 Hz,
H-11, H-13), 7.13 (2H, d, J = 35.4 Hz, H-23, H-26), 6.33
(2H, d, J = 2.2 Hz, H-24, H-25); 13C NMR (75 MHz,
DMSO-d6) δ ppm: 168.39 (CO, C-8), 164.33 (C-2),
148.55 (C-4), 142.81(C-15), 140.23 (C-12), 133.51 (C-9),
131.63 (C-18), 130.84 (CH, C-11, C-13), 129.99 (CH, C-
16, C-20), 127.96 (CH, C-10, C-14), 127.74 (CH, C-17,
C-19), 118.94 (CH, C-24, C-25), 109.23 (CH, C-23, C-
26), 102.34 (CH, C-5); MS (ESI): m/z = found 424.36 [M
+], 426.20 [M + 2] (calcd. 424.32); Anal. Calcd. For
C20H14BrN3OS: C, 56.61; H, 3.33; N, 9.90; Found: C,
57.01; H, 3.73; N, 10.10.
N-[4-(4-methoxyphenyl)-1,3-thiazol-2-yl]-4-(1H-pyrrol-1-yl)
benzamide (3e)
Compound 3e was obtained as yellow solid (yield 80%). m.
p. 186–188 °C; FTIR (KBr): 3415, 3114, 1652, 1526 cm−1
;
1H NMR (400 MHz, DMSO-d6) δ ppm: 12.76 (1H, s, NH),
8.26 (2H, d, J = 8.8 Hz, H-11, H-13), 7.91 (2H, d, J =
8.8 Hz, H-10, H-14), 7.80 (2H, d, J = 8.8 Hz, H-19, H-17),
7.56 (1H, s, H-5), 7.55 (2H, d, J = 2.2 Hz, H-20, H-16),
7.00 (2H, d, J = 27.0 Hz, H-23, H-26), 6.33 (2H, d, J =
2.1 Hz, H-24, H-25), 3.80 (3H, s, H-21, OCH3). 13C NMR
(75 MHz, DMSO-d6) δ ppm: 168.73 (CO, C-8), 165.25 (C-
2), 149.93 (C-4), 147.90 (C-15), 143.32 (C-9), 140.92
(C-18), 131.16 (C-12), 129.59 (C-11, C-13), 127.94 (C-16,
C-20), 126.22 (C-10, C-14), 119.10 (C-17, C-19), 118.87
(C-24, C-25), 111.33 (C-23, C-26), 106.65 (C-5), 60.80 (C-
21); MS (ESI): m/z = found 374.98 [M-1] (calcd. 375.45);
Anal. Calcd. For C21H17N3O2S: C, 67.18; H, 4.56; N,
11.19; Found: C, 67.46; H, 4.78; N, 11.35.
N-[4-(4-chlorophenyl)-1,3-thiazol-2-yl]-4-(1H-pyrrol-1-yl)
benzamide (3c)
Compound 3c was obtained as yellow solid (yield 69%). m.
p. 190–192 °C; FTIR (KBr): 3436, 3113, 1607, 1528 cm−1
;
1H NMR (400 MHz, DMSO-d6) δ ppm: 12.81 (1H, s, NH),
8.24 (2H, d, J = 8.7 Hz, H-16, H-20), 8.01 (2H, d, J =
8.4 Hz, H-10, H-14), 7.94 (2H, d, J = 2.6 Hz, H-17, H-19),
7.61 (1H, s, H-5), 7.55 (2H, d, J = 2.0 Hz, H-11, H-13),
7.36 (2H, d, J = 2.0 Hz, H-23, H-26), 6.33 (2H, d, J =
2.0 Hz, H-24, H-25); 13C NMR (75 MHz, DMSO-d6) δ
ppm: 168.33 (CO, C-8), 164.32 (C-2), 158.77 (C-4), 142.83
(C-15), 140.86 (C-12), 133.70 (C-9), 131.51 (C-18), 129.98
(CH, C-11, C-13), 128.73 (CH, C-16, C-20), 128.41 (CH,
C-10, C-14), 127.45 (CH, C-17, C-19), 119.00 (CH, C-24,
C-25), 111.39 (CH, C-23, C-26), 102.24 (C-5); MS (ESI):
m/z = found 380.02 [M+], 381.20 [M + 2] (calcd. 379.86);
anal. calcd. For C20H14ClN3OS: C, 63.24; H, 3.72;
N, 11.06; Found: C, 63.61; H, 4.02; N, 11.44.
4-(2,5-dimethyl-1H-pyrrol-1-yl)-N-(4-phenyl-1,3-thiazol-2-yl)
benzamide (4a)
Compound 4a was obtained as brown solid (yield 62%). m.
p. 154–156 °C; FTIR (KBr): 3320, 2920, 1606, 1516 cm−1
;
1H NMR (400 MHz, DMSO-d6) δ ppm: 12.89 (1H, s, NH),
8.07 (2H, d, J = 16.0 Hz, H-16, H-20), 7.98 (2H, d, J =
7.6 Hz, H-17, H-20), 7.80 (2H, d, J = 8.0 Hz,H-10, H-14),
7.70 (1H, s, H-5), 7.40–7.22 (3H, m, H-11, H-12, H-13),
5.85 (2H, d, J = 7.6 Hz, H-23, H-24), 2.02 (6H, s, H-26, H-
27-diCH3); 13C NMR (75 MHz, DMSO-d6) δ ppm: 168.73
(CO, C-8), 164.25 (C-2), 146.83 (C-4), 143.90 (C-15),
143.12 (C-12), 138.92 (C-9), 131.99 (C-18), 128.59 (CH,
C-11, C-14), 125.94 (CH, C-16, C-20), 124.29 (CH, C-10,

Medicinal Chemistry Research
C-14), 123.37 (CH, C-17, C-19), 119.20 (CH, C-23, C-24),
110.53 (CH, C-22, C-25), 105.85 (C-5), 13.45 (C-26,C-27-
diCH3); MS (ESI): m/z = found 373.97 [M+] (calcd.
373.47); anal. calcd. For C22H19N3OS: C, 70.75; H, 5.13; N,
11.25; Found: C, 70.98; H, 5.37; N, 11.47.
3.4 Hz, H-10, H-14), 8.11 (2H, d, J = 2.0 Hz, H-16, H-20),
7.56 (1H, s, H-5), 7.20 (2H, d, J = 4.0 Hz, H-17, H-19),
4.42 (2H, d, J = 1.2 Hz, H-24, H-25), 2.01 (6H, s, H-27, H-
28-diCH3). 13C NMR (75 MHz, DMSO-d6) δ ppm: 168.73
(CO, C-8), 164.25 (C-2), 146.83 (C-4), 143.90 (C-15),
143.12 (C-12), 138.92 (C-9), 131.99 (C-18), 128.59 (CH,
C-11, C-13), 125.94 (CH, C-16, C-20), 124.29 (CH, C-10,
C-14), 123.37 (CH, C-17, C-19), 119.20 (CH, C-24, C-25),
110.53 (CH, C-23, C-26), 105.96 (C-5), 13.45 (C-27,C-28-
diCH3); MS (ESI): m/z = found 418.00 [M+] (calcd.
418.47); anal. calcd. For C22H18N4O3S: C, 63.14; H, 4.34;
N, 13.39; Found: C, 63.44; H, 4.71; N, 13.49.
N-[4-(4-bromophenyl)-1,3-thiazol-2-yl]-4-(2,5-dimethyl-1H-
pyrrol-1-yl)benzamide (4b)
Compound 4b was obtained as brown solid (yield 70%).
m.p. 98–100 °C; FTIR (KBr): 3304, 2924, 1608, 1515 cm
1
−1. H NMR (400 MHz, DMSO-d6) δ ppm: 11.45 (1H, s,
NH), 8.14 (2H, d, J = 1.9 Hz, H-16, H-20), 7.89 (2H, d, J
= 8.3 Hz, H-17, H-19), 7.53 (2H, d, J = 2.0 Hz, H-10, H-
14), 7.54 (1H, s, H-5), 7.37 (2H, d, J = 3.2 Hz, H-11, H-
13), 5.94 (2H, d, J = 6.4 Hz, H-24, H-25), 2.01 (6H, s, H-
27, H-28-diCH3); 13C NMR (75 MHz, DMSO-d6) δ ppm:
168.66 (CO,C-8), 163.55 (C-2), 146.33 (C-4), 143.20 (C-
15), 142.02 (C-12), 138.32 (C-9), 130.65 (C-18), 129.11
(CH, C-11, C-13), 125.64 (CH, C-16, C-20), 123.27 (CH,
C-10, C-14), 121.37 (CH, C-17, C-19), 119.20 (CH, C-24,
C-25), 110.93 (CH, C-23, C-26), 105.55 (C-5), 13.65 (C-
27, C-28-diCH3); MS (ESI): m/z = found 452.17 [M+],
454.07 [M + 2] (calcd. 452.37); anal. calcd. For
C22H18BrN3OS: C, 58.41; H, 4.01; N, 9.29; Found: C,
58.61; H, 4.39; N, 9.42.
N-[4-(4-methoxyphenyl)-1,3-thiazol-2-yl]-4-(2,5-Dimethyl-
1H-pyrrol-1-yl)benzamide (4e)
Compound 4e was obtained as brown solid (yield 75%). m.
p. 104–106 °C; FTIR (KBr): 3244, 3100, 1669, 1541 cm−1
;
1H NMR (400 MHz, DMSO-d6) δ ppm: 12.86 (1H, s, NH),
8.09 (2H, d, J = 8.4 Hz, H-16, H-20), 7.87 (2H, d, J =
8.1 Hz, H-17, H-19), 7.68 (2H, d, J = 2.0 Hz, H-10, H-14),
7.60 (1H, s, H-5), 7.45 (2H, d, J = 4.9 Hz, H-11, H-13),
5.85 (2H, d, J = 7.0 Hz, H-24, H-25), 2.32 (3H, s, H-21,
OCH3), 2.00 (6H, s, H-27, H-28-diCH3); 13C NMR
(75 MHz, DMSO-d6) δ ppm: 165.93 (CO, C-8), 164.05 (C-
2), 150.83 (C-4), 142.90 (C-15), 132.12 (C-12), 131.30 (C-
9), 130.92 (C-18), 129.99 (CH, C-11, C-13), 128.69 (CH,
C-16, C-20), 125.94 (CH, C-10, C-14), 124.29 (CH, C-17,
C-19), 123.37 (CH, C-24, C-25), 106.32 (CH, C-23, C-26),
105.20 (C-5), 21.23 (C-21, OCH3), 13.08 (C-27,C-28,
diCH3); MS (ESI): m/z = found 387.44 [M+] (calcd.
387.14); anal. calcd. For C23H21N3OS: C, 71.29; H, 5.46; N,
10.84; Found: C, 71.62; H, 5.67; N, 11.04.
N-[4-(4-chlorophenyl)-1,3-thiazol-2-yl]-4-(2,5-dimethyl-1H-
pyrrol-1-yl)benzamide (4c)
Compound 4c was obtained as brown solid (yield 55%). m.
p. 162–164 °C; FTIR (KBr): 3312, 2923, 1607, 1516 cm−1
;
1H NMR (400 MHz, DMSO-d6) δ ppm: 12.91 (1H, s, NH),
8.26 (2H, d, H-16, H-20), 8.07 (2H, d, H-10, H-14), 7.81
(2H, d, H-17, H-19), 7.42 (1H, s, H-5), 7.12 (2H, d, H-11,
H-13), 5.85 (2H, s, H-24, H-25), 2.00 (6H, s, H-27, H-28-
diCH3). 13C NMR (75 MHz, DMSO-d6) δ ppm: 169.33 (CO,
C-8), 165.25 (C-2), 147.63 (C-4), 144.90 (C-15), 143.12 (C-
12), 139.92 (C-9), 130.99 (C-18), 128.99 (CH, C-11, C-13),
125.24 (CH, C-16, C-20), 124.69 (CH, C-10, C-14), 123.97
(CH, C-17, C-19), 118.20 (CH, C-24, C-25), 111.53 (CH, C-
23, C-26), 106.85 (C-5), 13.95 (C-27,C-28-diCH3); MS
(ESI): m/z = found 407.90 [M+], 409.12 [M + 2] (calcd.
407.92); anal. calcd. For C22H18ClN3OS: C, 64.78; H, 4.45;
N, 10.30; Found: C, 64.96; H, 4.71; N, 10.68.
N-[4-(3-bromophenyl)-1,3-thiazol-2-yl]-4-(2,5-dimethyl-1H-
pyrrol-1-yl)benzamide (4f)
Compound 4f was obtained as brown solid (yield 68%). m.
p. 84–86 °C; FTIR (KBr): 3452, 3118, 1673, 1523 cm−1; 1H
NMR (400 MHz, DMSO-d6) δ ppm: 12.88 (1H, s, NH),
8.07 (2H, d, J = 6.2 Hz, H-16, H-20), 7.99 (2H, d, J =
8.7 Hz, H-17, H-19), 7.72 (1H, s, H-5), 7.84–7.14 (4H, m,
H-14, H-12, H-10, H-13), 5.84 (2H, d, J = 6.8 Hz, H-24, H-
25), 2.01 (6H, s, H-27, H-28-diCH3); 13C NMR (75 MHz,
DMSO-d6) δ ppm: 166.33 (CO, C-8), 163.25 (C-2), 148.83
(C-4), 144.90 (C-15), 139.12 (C-12), 138.62 (C-9), 132.79
(C-18), 128.79 (CH, C-10, C-14), 125.75 (CH, C-16, C-20),
124.19 (C-11), 123.07 (CH, C-17, C-19), 120.20 (CH, C-
24, C-25), 108.85 (CH, C-23, C-26), 105.54 (C-5), 14.46
(C-27,C-28-diCH3); MS (ESI): m/z = found 452.26 [M+],
454.06 [M + 2] (calcd. 452.33); anal. calcd. For
C22H18BrN3OS: C, 58.41; H, 4.01; N, 9.29; Found: C,
58.78; H, 4.37; N, 9.37.
4-(2,5-Dimethyl-1H-pyrrol-1-yl)-N-[4-(4-nitrophenyl)-1,3-
thiazol-2-yl]benzamide (4d)
Compound 4d was obtained as brown solid (yield 65%). m.
p. 106–108 °C; FTIR (KBr): 3316, 3067, 1714, 1517 cm−1
;
1H NMR (400 MHz, DMSO-d6) δ ppm: 14.02 (1H, s, NH),
8.31 (2H, d, J = 2.0 Hz, H-11, H-13), 8.17 (2H, d, J =

Medicinal Chemistry Research
General procedure for the synthesis of N-(5-bromo-
4-(4-substitutedphenyl)-thiazol-2-yl)-4-(2,5-
dimethyl-1H-pyrrol-1-yl)benzamides (6a-e)/N-(5-
bromo-4-(4-substitutedphenyl)-thiazol-2-yl)-4-(1H-
pyrrol-1-yl)benzamides (7a-b)
d, J = 4.2 Hz, H-11, H-13), 5.88 (2H, s, H-25, H-26), 1.99
(6H, s, H-28, H-29-diCH3); 13C NMR (75 MHz, DMSO-d6)
δ ppm: 167.19 (CO, C-8), 166.58 (C-2), 144.01 (C-4),
134.01 (C-16), 131.38 (C-12), 128.16 (C-9), 127.95 (C-19),
127.78 (CH, C-10, C-14), 127.15 (CH, C-17, C-21), 125.45
(CH, C-18, C-20), 124.23 (CH, C-11, C-13), 122.32 (CH,
C-24, C-27), 108.69 (CH, C-25, C-26), 102.17 (C-5), 12.85
(C-28, C-29-diCH3); MS (ESI): m/z = found 531.19 [M+],
533.29 [M + 2], 535.09 [M + 4] (calcd. 531.27); anal.
calcd. For C22H17N3OSBr2: C, 49.74; H, 3.23; N, 7.91;
Found: C, 49.94; H, 3.57; N, 8.11.
The 4-(4-substituted phenyl)-5-bromo-2-aminothiazoles (5a–e)
(0.0018 mol) and 4-(2, 5-dimethyl-1H-pyrrol-1-yl)benzoic acid
(9b)/4(1H-pyrrol-1-yl)benzoic acid (9a) (0.0019 mol) were
dissolved, respectively in 50 mL of dry dimethyl formamide.
HBTU (0.87 g, 0.0023 mol) and DIEA (0.93 mL, 0.0053 mol)
were then added to the above mixtures and stirred for 24–30 h
at room temperature. The reaction was quenched by adding
NaCl solution and the mixture was extracted with ethyl acetate
(3 × 50 mL). The combined ethyl acetate layer was washed
with 1 N HCl and with a saturated sodium bicarbonate solution
followed by NaCl. The organic layer was dried over anhydrous
sodium sulfate and concentrated using a rotary flash eva-
porator. Thus obtained residue was dried and purified by col-
umn chromatography using petroleum ether:ethyl acetate (6:4)
mixture as the eluent to afford the compounds (6a–e) and
(7a–b) (Scheme 1).
N-[5-bromo-4-(4-chlorophenyl)-1,3-thiazol-2-yl]-4-(2,5-
dimethyl-1H-pyrrol-1-yl)benzamide (6c)
Compound 6c was obtained as red solid (yield 76%). m.p.
174–176 °C; FTIR (KBr): 3301, 2972, 1606 cm−1; 1H NMR
(400 MHz, DMSO-d6) δ ppm: 12.82 (1H, s, -NH), 8.04 (2H,
d, J = 8.7 Hz, H-17, H-21), 7.84 (2H, d, J = 8.7 Hz, H-10,
H-14), 7.57 (2H, d, J = 9.7 Hz, H-18, H-20), 7.17 (2H, d, J
= 9.4 Hz, H-11, H-13), 5.85 (2H, d, J = 7.3 Hz, H-25, H-
26), 2.01 (6H, s, H-28, H-29-diCH3); 13C NMR (75 MHz,
DMSO-d6) δ ppm: 167.29 (CO, C-8), 166.78 (C-2), 154.39
(C-4), 139.56 (C-16), 138.558 (C-12), 133.55 (C-9), 130.28
(C-19), 128.10 (CH, C-10, C-14), 128.00 (CH, C-17, C-21),
127.68 (CH, C-11, C-13), 124.43 (CH, C-18, C-20), 123.59
(CH, C-24, C-27), 109.09 (CH, C-25, C-26), 102.67 (C-5),
13.45 (C-28, C-29-diCH3); MS (ESI): m/z = found 486.82
[M+], 487.22 [M + 2], 490.08 [M + 4] (calcd. 486.81); anal.
calcd. For C22H17N3OSBrCl: C, 54.28; H, 3.52; N, 8.63;
Found: C, 54.46; H, 3.69; N, 8.86.
N-(5-bromo-4-phenyl-1,3-thiazol-2-yl)-4-(2,5-dimethyl-1H-
pyrrol-1-yl)benzamide (6a)
Compound 6a was obtained as red solid (yield 86%). m.p.
108–110 °C; FTIR (KBr): 3298, 3178, 1708, 1607 cm−1; 1H
NMR (400 MHz, DMSO-d6) δ ppm: 12.10 (1H, s, NH),
8.08 (2H, d, J = 8.4 Hz, H-17, H-21), 7.91 (2H, d, J =
3.7 Hz, H-18, H-20), 7.82 (2H, d, J = 6.7 Hz, H-10, H-14),
7.78–7.20 (3H, m, H-11, H-12, H-13), 5.77 (2H, s, H-24, H-
25), 2.00 (6H, s, H-27, H-28-diCH3); 13C NMR (75 MHz,
DMSO-d6) δ ppm: 168.19 (CO, C-8), 166.98 (C-2), 152.30
(C-4), 143.20 (C-16), 134.93 12 (C-12), 133.75 (C-9),
130.18 (C-19), 128.44 (CH, C-10, C-14), 128.15 (CH, C-
17, C-21), 127.78 (CH, C-11, C-13), 127.15 (CH, C-18, C-
20), 125.53 (CH, C-23, C-26), 108.69 (CH, C-24, C-25),
101.47 (C-5), 12.55 (C-27, C-28-diCH3); MS (ESI): m/z =
found 452.47 [M+], 454.10 [M + 2] (calcd. 452.37); anal.
calcd. For C22H18N3OSBr: C, 58.41; H, 4.01; N, 9.29;
Found: C, 58.69; H, 4.33; N, 9.52.
N-[5-bromo-4-(4-nitrophenyl)-1,3-thiazol-2-yl]-4-(2,5-
dimethyl-1H-pyrrol-1-yl)benzamide (6d)
Compound 6d was obtained as red solid (yield 80%). m.p.
164–166 °C; FTIR (KBr): 3304, 3150, 1664, 1600 cm−1
;
1H NMR (400 MHz, DMSO-d6) δ ppm: 10.79 (1H, s, NH),
8.14 (2H, d, J = 1.8 Hz, H-17, H-21), 8.10 (2H, d, J =
2.0 Hz, H-18, H-20), 7.85 (2H, d, J = 3.2 Hz, H-10, H-14),
7.74 (2H d, J = 6.9 Hz, H-11, H-13), 5.85 (2H, d, J =
2.4 Hz, H-25, H-26), 2.01 (6H, s, H-28, H-29-diCH3); 13C
NMR (75 MHz, DMSO-d6) δ ppm: 168.90 (NH-CO),
168.60 (C-2), 147.82 (C-4), 145.91 (C-16), 140.87 (C-12),
129.52 (C-9), 128.73 (C-19), 127.59 (CH, C-10, C-14),
126.27 (CH, C-17, C-21), 124.00 (CH, C-11, C-13), 123.19
(CH, C-18, C-20), 106.59 (CH, C-25, C-26), 13.54 (CH, C-
28, C-29-diCH3); MS (ESI): m/z = found 495.15 [M-2],
497.09 [M+] (calcd. 497.37); anal. calcd. For
C22H17N4O3SBr: C, 53.13; H, 3.45; N, 11.26; Found: C,
53.48; H, 3.76; N, 11.53.
N-[5-bromo-4-(4-bromophenyl)-1,3-thiazol-2-yl]-4-(2,5-
dimethyl-1H-pyrrol-1-yl)benzamide (6b)
Compound 6b was obtained as red solid (yield 78%). m.p.
138–140 °C; FTIR (KBr): 3297, 2969, 1667, 1607 cm−1; 1H
NMR (400 MHz, DMSO-d6) δ ppm: 12.7 (1H, s, NH), 8.10
(2H, d, J = 3.6 Hz, H-17, H-21), 7.77 (2H, d, J = 2.0 Hz, H-
18, H-20), 7.75 (2H, d, J = 1.9 Hz, H-10, H-14), 7.55 (2H,

Medicinal Chemistry Research
N-[5-bromo-4-(4-methoxyphenyl)-1,3-thiazol-2-yl]-4-(2,5-
dimethyl-1H-pyrrol-1-yl)benzamide (6e)
(C-12), 131.86 (C-9), 130.61 (C-19), 128.67 (C-10, C-14),
128.66 (CH, C-18, C-20), 126.89 (CH, C-11, C-13), 118.05
(C-18,C-20), 117.58 (CH, C-24,C-27), 110.39 (CH, C-25,
C-26), 97.09 (C-5); MS (ESI): m/z = found 502.87 [M+],
504.79 [M + 2], 506.09 [M + 4] (calcd. 503.21); anal.
calcd. For C20H13N3OSBr2: C, 47.74; H, 2.60; N, 8.35;
Found: C, 48.04; H, 2.70; N, 8.45.
Compound 6e was obtained as red solid (yield 90%). m.p.
178–180 °C; FTIR (KBr): 3307, 2927, 1668, 1609 cm−1; 1H
NMR (400 MHz, DMSO-d6) δ ppm: 10.74 (1H, s, NH),
8.00 (2H, d, J = 2.2 Hz, H-17, H-21), 7.84 (2H, d, J =
3.4 Hz, H-18, H-20), 7.67 (2H, d, J = 2.0 Hz, H-10, H-14),
7.35 (2H d, J = 2.3 Hz, H-11, H-13), 5.82 (2H, s, H-25, H-
26), 3.80 (3 H, s, OCH3), 2.00 (6H, s, H-28, H-29-diCH3);
13C NMR (75 MHz, DMSO-d6) δ ppm: 168.19 (CO, C-8),
166.38 (C-2), 133.01 (C-4), 131.45 (C-16), 130.38 (C-12),
128.21 (C-9), 127.95 (C-19), 127.78 (CH, C-10, C-14),
127.25 (CH, C-17, C-21), 124.13 (CH, C-11, C-13), 123.25
(CH, C-18, C-20), 120.89 (CH, C-10, C-14), 108.88 (CH,
C-25, C-26), 102.32 (C-5), 26.35 (C-22, OCH3), 13.35 (C-
28, C-29-diCH3); MS (ESI): m/z = found 482.38 [M+],
484.48 [M + 2] (calcd. 482.40); anal. calcd. For
C23H20N3O2SBr: C, 57.27; H, 4.18; N, 8.71; Found: C,
57.57; H, 4.35; N, 8.92.
Synthesis of 5-bromo-4-phenyl-2-(1H-pyrrol-1-yl)
thiazole (8)
The 5-bromo-4-phenylthiazole (0.01 mol) and 2,5-dime-
thoxytetrahydrofuran (0.012 mol) were refluxed in dry
acetic acid (15 mL) for 45 min, which was then poured into
ice cold water with stirring to yield 5-bromo-4-phenyl-2-
(1H-pyrrol-1-yl)thiazole (8) (Scheme 1).
5-bromo-4-phenyl-2-(1H-pyrrol-1-yl)-1,3-thiazole (8)
Compound 8 was obtained as white solid (yield 80%). m.p.
152–154 °C; FTIR (KBr): 3392, 2923, 1515 cm−1 1H
;
N-(5-bromo-4-phenyl-1,3-thiazol-2-yl)-4-(1H-pyrrol-1-yl)
benzamide (7a)
NMR (400 MHz, DMSO-d6) δ ppm: 7.98–7.71 (3H, m, H-
13, H-14, H-15), 7.52 (2H, d, J = 3.6 Hz, H-12, H-16), 7.32
(2H, d, J = 6.8 Hz, H-7, H-10), 7.26 (2H, d, J = 4.4 Hz, H-
8, H-9); 13C NMR (75 MHz, DMSO-d6) δ ppm: 163.73 C-
2), 162.25 (C-4), 135.83 (C-11), 129.90 (C-12), 128.12
(CH, C-13, C-15), 127.02 (CH, C-12, C-16), 123.99 (CH,
C-7, C-10), 108.59 (CH, C-8, C-9), 95.94 (C-5); MS (ESI):
m/z = found 307.14 [M + 2] (calcd. 305.19); anal. calcd.
For C13H9N2SBr: C, 51.16; H, 2.97; N, 9.18; Found: C,
51.39; H, 3.01; N, 9.36.
Compound 7a was obtained as Pale yellow solid (yield
82%). m.p. 178–180 °C; FTIR (KBr): 3290, 3137, 1681,
1
1608 cm−1; H NMR (400 MHz, DMSO-d6) δ ppm: 13.07
(1H, s, NH), 8.25 (2H, d, J = 3.2 Hz, H-18, H-20), 8.01
(2H, d, J = 8.5 Hz, H-17, H-21), 7.82 (2H, d, J = 8.1 Hz, H-
10, H-14), 7.66-7.21 (3H, m, H-11, H-12, H-13), 7.07 (2H,
d, J = 30.3 Hz, H-23, H-26), 6.33 (2H, d, J = 6.2 Hz, H-24,
H-25); 13C NMR (75 MHz, DMSO-d6) δ ppm: 168.19 (CO,
C-8), 166.76 (C-5), 151.11 (C-4), 143.06 (C-16), 133.08
(C-12), 131.06 (C-9), 130.11 (C-19), 128.77 (C-10, C-14),
128.46 (CH, C-17, C-21), 127.09 CH, (C-11, C-13), 119.05
(CH, C-18, C-20), 118.58 (CH, C-23, C-26), 111.39 (C-24,
C-25), 98.09 (C-5); MS (ESI): m/z = found 424.33 [M+],
425.33 [M + 2] (calcd. 424.32); anal. calcd. For
C20H14N3OSBr: C, 56.61; H, 3.33; N, 9.90; Found: C,
56.63; H, 3.39; N, 9.99.
General procedure for the synthesis of N-benzyl-4-
(2,5-dimethyl-1H-pyrrol-1-yl)benzamide (10)/4-(2,5-
dimethyl-1H-pyrrol-1-yl)-N-(substituted phenyl)
benzamides (11a–d)
Different aromatic amines (0.0018 mol) and 4-(2, 5-
dimethyl-1H-pyrrol-1-yl)benzoic acid (9b) (0.0019 mol)
were dissolved, respectively, in 50 mL of dry dimethyl
formamide. HBTU (0.87 g, 0.0023 mol) and DIEA
(0.93 mL, 0.0053 mol) were then added to the above mix-
ture and stirred for 24–30 h at the ambient temperature. The
reaction was quenched by adding NaCl solution and the
mixture was extracted with ethyl acetate (3 × 50 mL). The
combined ethyl acetate layer was washed with 1 N HCl and
with the saturated sodium bicarbonate followed by NaCl
solution. The organic layer was dried over an anhydrous
sodium sulfate and concentrated using rotary flash eva-
porator. Thus obtained residue was dried and purified by
column chromatography using petroleum ether:ethyl acetate
(6:4) mixture as the eluent to afford the desired compounds
(10) and (11a–d) (Scheme 2).
N-[5-bromo-4-(4-bromophenyl)-1,3-thiazol-2-yl]-4-(1H-
pyrrol-1-yl)benzamide (7b)
Compound 7b was obtained as pale yellow solid (yield
80%). m.p. 158–160 °C; FTIR (KBr): 3360, 3173, 1654,
1
1611 cm−1. H NMR (400 MHz, DMSO-d6) δ ppm: 12.61
(1H, s, NH), 7.85 (2H, d, J = 1.7 Hz, H-17, H-21), 7.84
(2H, d, J = 1.7 Hz, H-18, H-20), 7.56 (2H, d, J = 1.8 Hz, H-
10, H-14), 7.35 (2H, d, J = 2.2 Hz, H-11, H-13), 7.24 (2H,
d, J = 3.6 Hz, H-24, H-27), 6.17 (2H, d, J = 2.1 Hz, H-25,
H-26). 13C NMR (75 MHz, DMSO-d6) δ ppm: 167.07 (CO,
C-8), 164.05 (C-2), 151.02 (C-4), 144.06 (C-16), 132.08

Medicinal Chemistry Research
Scheme. 2 Synthesis of substituted 2,5-dimethylpyrrolyl benzamides
N-benzyl-4-(2,5-dimethyl-1H-pyrrol-1-yl)benzamide (10)
11), 128.33 (CH, C-18, C-22), 128.03 (CH, C-8, C-10),
123.01 CH, (C-19, C-21), 121.36 (C-20), 109.66 (CH, C-2,
C-5), 106.94 (CH, C-3, C-4), 55.47 (C-19, OCH3), 13.39
(C-12, C-13-diCH3); MS (ESI): m/z = found: 321.06 [M +
H] (calcd. 320.39); anal. calcd. For C20H20N2O2: C, 74.98;
H, 6.29; N, 8.74; Found: C, 75.08; H, 6.41; N, 8.89.
Compound 10 was obtained as yellow solid (yield 90%). m.p.
158–160 °C; FTIR (KBr): 3299, 2921, 1635, 1607 cm−1; H
1
NMR (400 MHz, DMSO-d6) δ ppm: 9.18 (1H, s, NH), 8.03
(2H, d, J = 8.4 Hz, H-7, H-11), 7.39 (2H, d, J = 8.4 Hz, H-8,
H-10), 7.34–7.25 (3H, m, H-20, H-21, H-22), 7.24 (2H, d, J
= 4.8 Hz, H-19, H-23), 5.82 (2H, s, H-3, H-4), 4.52 (2 H, d, J
= 6.0 Hz, CH2), 1.98 (6H, s, H-12, H-13-diCH3); 13C NMR
(75 MHz, DMSO-d6) δ ppm: 166.15 (CO, C-15), 141.29 (C-
6), 138.98 (C-18), 133.76 (C-9), 128.76 (CH, C-7, C-11),
128.29 (CH, C-19, C-23), 128.01 (CH, C-8, C-10), 127.67
(CH, C-20, C-22), 126.25 (C-21), 122.25 (CH, C-2, C-5),
106.87 (CH, C-3, C4), 43.15 (C-17, CH2), 13.32 (C-12, C-13,
Pyrrole-diCH3); MS (ESI): m/z = found 303.46 [M-1] (calcd.
304.39); anal. calcd. For C20H20N2O: C, 78.92; H, 6.62; N,
9.20; Found: C, 79.04; H, 6.87; N, 9.44.
N-(2,6-dichlorophenyl)-4-(2,5-dimethyl-1H-pyrrol-1-yl)
benzamide (11b)
Compound 11b was obtained as light yellow solid (yield
72%). m.p. 104–106 °C; FTIR (KBr): 3381, 2922, 1696,
1
1604 cm−1; H NMR (400 MHz, DMSO-d6) δ ppm: 13.22
(1H. s, NH), 8.16 (2H, d, J = 40.0 Hz, H-1, H-11), 7.76 (2H,
d, J = 4.0 Hz, H-8,H-10), 7.41–7.30 (3H, m, H-19, H-20, H-
21), 5.83 (2H, s, H-3, H-4), 1.99 (6H, s, H-12, H-13-diCH3);
13C NMR (75 MHz, DMSO-d6) δ ppm: 167.36 (CO, C-8),
142.48 (C-6), 130.76 (C-17), 128.42 (C-9), 128.00 (CH, C-
7, C-11), 127.38 (CH, C-18, C-22), 126.91 (CH,C-8, C-10),
125.16 (CH, C-19, C-21), 124.26 (C-20), 111.91 (CH, C-2,
C-5), 106.61 (CH, C-3, C-4), 20.80, 13.30 (C-12, C-13-
diCH3); MS (ESI): m/z = found: 359.27 [M+], 361.15 [M +
2] (calcd. 359.25); anal. calcd. For C19H16N2OCl2: C, 63.52;
H, 4.49; N, 7.80; Found: C, 63.65; H, 4.62; N, 8.01.
4-(2,5-dimethyl-1H-pyrrol-1-yl)-N-(3-methoxyphenyl)
benzamide (11a)
Compound 11a was obtained as light yellow solid (yield
80%). m.p. 106–108 °C; FTIR (KBr): 3291, 2918, 1642,
1
1608 cm−1; H NMR (400 MHz, DMSO-d6) δ ppm: 10.35
(1H. s, NH), 8.07 (2H, d, J = 8.4 Hz, H-7, H-11), 7.50–7.28
(3H, m, H-20, H-21, H-22), 7.26 (2H, d, J = 8.0 Hz, H-8,
H-10), 6.71 (1H, s, H-18), 5.84 (2H, s, H-3, H-4), 3.76 (3H,
s, –OCH3), 2.01 (6H, s, H-12, H-13-diCH3). 13C NMR
(75 MHz, DMSO-d6) δ ppm: 165.44 (CO, C-15), 141.51
(C-6), 134.48 (C-17), 129.64 (C-9), 129.19 (CH, C-7, C-
N-(4-aminophenyl)-4-(2,5-dimethyl-1H-pyrrol-1-yl)
benzamide (11c)
Compound 11c was obtained as light yellow solid (yield
85%). m.p. 124–126 °C; FTIR (KBr): 3924, 2920, 1646,

Medicinal Chemistry Research
1
1607 cm−1; H NMR (400 MHz, DMSO-d6) δ ppm: 10.00
Table 1 In vitro evaluation of antitubercular activity (MIC values in
µg/mL) and enzyme inhibition values (results are expressed as % InhA
inhibition)
(1H, s, NH), 8.09 (2H, d, J = 8.4 Hz, H-7, H-11), 8.06 (2H,
d, J = 12.0 Hz, H-8, H-10), 7.46 (2H, d, J = 6.4 Hz, H-19,
H-21), 7.41 (2H, d, J = 2.8 Hz, H-18, H-22), 5.85 (2H, d, J
= 5.6 Hz, H-3, H-4), 4.95 (2H, s, NH2), 2.00 (6H, s, H-12,
H-13-diCH3); 13C NMR (75 MHz, DMSO-d6) δ ppm:
165.30 (CO, C-8), 145.73 (C-6), 141.48 (C-17), 134.42 (C-
9), 129.48 (CH, C-7, C-11), 129.18 (CH, C-18, C-22),
128.96 (CH, C-8, C-10), 128.23 (CH, C-19, C-21), 120.96
(C-20), 113.13 (CH, C-2, C-5), 106.89 (CH, C-3, C-4),
29.36, 13.36 (C-12, C-13-diCH3); MS (ESI): m/z = found:
305.42 [M+] (calcd. (305.38); anal. calcd. For C19H19N3O:
C, 74.73; H, 6.27; N, 13.76; Found: C, 74.93; H, 6.47;
N, 13.92.
Comp.
MIC values (µg/mL)
% Inhibition at 50 µM
(M. tuberculosis H37Rv)
3a
25
NIa
25
3b*
1.6
3c
3.12
1.6
NTb
3d*
30
3e*
1.6
NTb
NTb
NTb
38
4a
50
4b
1.6
4c
1.6
4d
12.5
1.6
NTb
4e
38
N-(2-aminophenyl)-4-(2,5-dimethyl-1H-pyrrol-1-yl)
benzamide (11d)
4f
1.6
NTb
NTb
NTb
NTb
NTb
NT
NTb
NTb
NTb
12
6a
12.5
1.6
6b*
Compound 11d was obtained as light yellow solid (yield
85%). m.p. 134–136 °C; FTIR (KBr): 3223, 2972, 1647,
6c*
1.6
6d
3.12
1.6
1
1608 cm−1; H NMR (400 MHz, DMSO-d6) δ ppm: 9.77
6e*
(1H, s, NH), 8.12 (2H, d, J = 8.0 Hz, H-7, H-11), 7.70 (2H,
d, J = 3.2 Hz, H-8, H-10), 7.69–7.30 (4H, m, H-19, H-20,
H-21), 7.18 (1H, d, J = 7.6 Hz, H-22), 5.84 (2H, s, H-3, H-
7a
12.5
6.25
1.6
7b
4), 4.95 (2H, s, -NH2), 2.01 (6H, s, H-12, H-13-diCH3); 13
C
8
10
3.12
12.5
1.6
NMR (75 MHz, DMSO-d6) δ ppm: 165.13 (CO, C-15),
143.78 (C-6), 141.37 (C-17), 134.13 (C-20), 128.46 (CH,
C-7, C-11), 128.23 (CH, C-18, C-22), 128.02 (CH, C-8, C-
10), 127.31 (CH, C-19, C-21), 127.09 (C-20), 116.52 (CH,
C-2, C-5), 106.90 (CH, C-3, C-4), 13.40 (CH, C-12, C-13-
diCH3); MS (ESI): m/z = found: 305.19 [M+] (calcd.
305.38); anal. calcd. For C19H19N3O: C, 74.73; H, 6.27; N,
13.76; Found: C, 74.90; H, 6.44; N, 13.98.
11a
NTb
11b
31
11c
1.6
30
11d
3.12
3.12
6.25
–
12
Pyrazinamide
Streptomycin
Triclosan
–
–
>99
Astrisk indicates the compounds used for Pharmacophore mapping
aNI stands for no inhibition
bNT stands for not tested
Biological activity
In vitro evaluation of antitubercular studies
prevented the color change from blue to pink. Table 1
shows the anti-TB activity data expressed in MIC.
All the compounds were tested for inhibition of M. tuber-
culosis strain H37Rv using the Microplate Alamar Blue
Assay (MABA) method described earlier (Franzblau et al.
1998). The 96 wells plate received 100 mL of Middlebrook
7H9 broth and serial dilution of compounds were made
directly on the plate with drug concentrations of 0.2, 0.4,
0.8, 1.6, 3.125, 6.25,12.5, 25, 50, and 100 µg/mL. Plates
were covered and sealed with parafilm and incubated at
37 °C for 5 days. Then, 25 mL of freshly prepared 1:1
mixture of alamar blue reagent and 10% Tween 80 were
added to the plate and incubated for 24 h. A blue color in
the well was interpreted as no bacterial growth, while the
appearance of pink color was scored as the growth. The
MIC value (defined as the lowest drug concentration)
In vitro evaluation of antibacterial activity
MIC determination of the tested compounds was investi-
gated side-by-side by comparison with ciprofloxacin against
Gram-positive (S. aureus) and Gram-negative bacteria
(E. coli) using the broth microdilution method (Goto et al.
1981; Villanova 1985). Serial dilutions of the test com-
pounds and reference drugs were done in Mueller–Hinton
agar. Drugs (10 mg) were dissolved in dimethylsulfoxide
(DMSO, 1 mL). Further progressive dilutions with the
molten Mueller–Hinton agar were performed to obtain the
required concentrations of 0.2, 0.4, 0.8, 1.6, 3.125, 6.25,

Medicinal Chemistry Research
12.5, 25, 50, and 100 µg/mL. The tubes were then inocu-
lated with 105 cfu/mL (colony forming unit/mL) and incu-
bated at 37 °C for 18 h. MIC represents the lowest
concentration of the tested compound that yielded no visible
growth on the plate. To ensure that solvent did not affect
bacterial growth the control experiment was performed with
the test medium supplemented with DMSO using the same
dilutions as used for the molecules in the experiment, but
DMSO did not show any effect on the microorganisms in
the studied concentration range. Table 2 summarizes the
antibacterial activity data expressed in MIC values.
MTT-based cytotoxicity activity
Cellular conversion of MTT [3-(4,5-dimethylthiazo-2-yl)-
2,5-diphenyl-tetrazolium bromide] into a formazan product
(Mosmann 1983) was performed to evaluate cytotoxic
activity (IC50) of some of the compounds against A549
(lung adenocarcinoma) MV cell-lines up to concentration of
50 mg/mL using Promega Cell Titer 96 non-radioactive cell
proliferation assay (Gundersen et al. 2002) using cisplatin
as the positive control. The IC50 values given in Table 2 are
the averages SEM of three independent measurements.
Enzyme inhibition studies
Table 2 In vitro evaluation of antibacterial activity (MIC values in µg/
mL) and MTT-based cytotoxicity activity of selected compounds
against human lung cancer cell lines A549 and MV cell line
(IC50 in µg/mL)
InhA expression and purification
The production and purification of InHA-6xHis protein
from a protease-deficient strain of E. coli (BL21) trans-
formed with pHAT5/InhA plasmid were performed. A 1 mL
of bacteria was grown in 100 mL of Lysogeny broth (LB)
medium containing ampicillin (100 µg/mL) and 2% glucose
at 37 °C. After 4 h, the solution was re-diluted in 1 L of the
same medium and re-grown at 37 °C. After attaining proper
concentration (OD595 = 0.6–0.8), the culture was cen-
trifuged at 3300 g factor for 10 min at 4 °C and bacteria
were suspended in LB medium containing ampicillin
(100 µg/mL). Protein expression was induced for overnight
incubation in 1 mM isopropyl-β-D-galactopyranoside
(IPTG) at 20 °C. Cells were harvested by centrifugation at
6000×g for 30 min at 4 °C. Dry pellet was kept at −80 °C
for several months and purification was done with Ni-NTA
Agarose from QIAGEN following the manufacturer’s pro-
tocol. The purified recombinant protein was applied to PD-
10 desalting columns (GE Healthcare, Piscataway, NJ)
equilibrated with PIPES 30 mM pH 6.8 and 150 mM NaCl
to remove the imidazole. Samples were analyzed using
SDS–PAGE and Coomassie blue staining and then stored at
4 °C for a short-time at −80 °C with 20% glycerin for long-
term storage (Menendez et al. 2011).
Compound
IC50 (µM)a
Gram
negative
Gram
positive
c
MV
A549
E. coli
(µg/mL)
S. aureus
(µg/mL)
cell-linesb
3a
–
–
25
25
3b
220 0.8
218 0.7 25
50
216 0.2 12.5
3.12
6.25
3.12
3.12
25
3c
–
–
3d
212 0.6
3e
–
–
–
–
12.5
50
4a
–
4b
–
25
3.12
1.6
4c
214 0.2
216 0.5 25
25
214 0.4 25
4d
–
–
6.25
1.6
4e
217 0.3
4f
–
–
–
–
–
–
–
–
–
–
25
1.6
6a
–
50
6.25
1.6
6b
–
12.5
12.5
25
6c
–
3.12
3.12
3.12
12.5
6.25
3.12
12.5
3.12
3.12
3.12
6.25
2
6d
–
6e
–
25
7a
–
50
7b
–
50
8
–
50
10
218 0.3
–
216 0.4 50
50
InhA activity inhibition
11a
–
11b
210 0.3
216 0.4
219 0.2
–
214 0.4 50
216 0.7 25
211 0.3 25
2
Triclosan and NADH were obtained from Sigma-Aldrich
(Bangalore, INDIA). Stock solutions of the selected com-
pounds were prepared in DMSO such that the final con-
centration of this co-solvent was constant at 5% (v/v) in the
final volume of 1 mL for all the kinetic reactions. Kinetic
assays were performed using trans-2-dodecenoyl-coenzyme
A (DDCoA) and wild type InhA as previously described
(Menendez et al. 2012). Briefly, the reactions were performed
at 25 °C in an aqueous buffer (30 mM PIPES and 150 mM
NaCl, pH 6.8) containing 250 µM cofactor (NADH), 50 µM
substrate (DDCoA) and the test compound (at 50 µM).
11c
11d
Ciprofloxacin
INH
>450
>450
aCytotoxicity is expressed as IC50 which is the concentration of
compound reducing by 50% of the optical density of treated cells with
respect to untreated cells using MTT assay. Values are the means
SEM of three independent experiments
bMammalian Vero cell-lines (NCCS-Pune, India)
cA549 (lung adenocarcinoma) cell-lines (NCCS-Pune, India)

Medicinal Chemistry Research
Reactions were initiated by adding InhA (100 nM final) and
NADH oxidation was monitored at the fixed wavelength of
340 nm. Inhibitory activity of each derivative was expressed
as % inhibition of InhA activity (initial velocity of the reac-
tion) with respect to control reaction without the inhibitor.
These results are shown in Table 1.
set to carry out pharmacophore hypothesis and modeling
studies. All the ligands were aligned in two steps. In the first
step, six compounds (3b, 3d, 3e, 6b, 6c, and 6e labeled with
asterisks shown in Table 1) were chosen to perform the
pharmacophore hypothesis; the genetic algorithm was used
to create the conformers for all the compounds. The mole-
cules that were selected to create pharmacophore hypothesis
were found to be exceptionally active. GALAHAD module
was used for flexible alignment of all the selected ligands
that are completely independent of template with a popu-
lation size of 45 and a maximum generation value of 50
with molecules requiring hitting of 4. Twenty pharmaco-
phore models were generated and analyzed on the basis of
fitness scores and percentage of aligned molecule (Tripose,
MO 2014). The experimentally obtained anti-TB activity of
each molecule was denoted as minimal inhibitory con-
centration (MIC) against M. tuberculosis and MIC values
were used in pharmacophore analysis.
GALAHAD formed a set of likely hypotheses by means
of the flexible alignment (Table 3, 20 models). SPECIFI-
CITY is a logarithmic indicator of the expected dis-
crimination for each query. The actual number of hits is
given in N_HITS column and the values in FEATS column
indicate the total number of features in the model query.
The next five columns are model score components from
the genetic algorithm. Pareto is a Pareto rank of each model,
where all the models have a Pareto rank of zero. This means
none of the models are superior to any other when using all
the four criteria in columns 5–8 (ENERGY, STERICS,
H_BOND, and MOL_QRY). ENERGY calculated is the
total energy of the model; STERICS is a steric overlap for
the model; H_BOND, pharmacophoric concordance and
MOL_QRY are in agreement between the query tuplet and
pharmacophoric tuplets for ligands as a group. Last four
columns are the scores for individual ligands within each
model. Thus, in this case, every cell in each of the last four
columns contains a list of five values.
For rigid alignment of the left over ligands in a data set
(in the second step), we need to choose one best template
model and choice of the model from the obtained 20 models
in the first stage based on the model needed to “hit” all the
six active compounds. The model required to have high
sterics with low energy and pharmacophoric features. We
have constructed the scatter plot (ENERGY vs. STERICS
vs. MOL_QRY) to visualize the Pareto surface and selected
the best pharmacophore model (Fig. 4). Taking into account
the ENERGY, STERICS, and MOL_QRY criteria, the best
model is depicted in the graph, where ENERGY is ration-
ally low and STERICS score is high. Among the measured
models, MODEL_18 (denoted with a black circle in Fig. 4)
has the best position as it fulfills all the three criteria and has
improved Specificity, N hits and Feats values (Caballero
2010; Zhao et al. 2010).
Computational details
General method
The crystal structure of enoyl acyl carrier protein reductase
InhA in complex with N-(4-methylbenzoyl)-4-benzylpiper-
idine (PDB ID 2NSD, 1.9 Å X-ray resolution) was retrieved
from the Brookhaven Protein Database (PDB http://www.
rcsb.org/pdb). In the current condition, biopolymer and each
molecule in the data set were energetically minimized by
employing the Tripos force field (Clark et al. 1989). Powell
optimization method (Powell 1977), Amber7FF9902 (bio-
polymer) and MMFF94 (molecules) charges (NB cut-off
9.0 as well as dielectric constant 4.0) with a convergence
criterion set at 0.001 kcal/mol Å. The pharmacophore
models were generated and analyzed using GALAHAD
(Genetic Algorithm with Linear Assignment of Hypermo-
lecular Alignment of Datasets) module. All calculations
were performed on a commercially available SYBYL-X
2.0 software package (Tripos Associates, St. Louis, MO,
USA) (Tripose, USA 2012).
Docking protein–ligand interactions is a significant
approach in current drug discovery research. If the com-
position of binding site is identified, subsequently modeling
procedures will afford important insights into such inter-
actions and in several affirmative cases, one can validate the
hits from the virtual screening. In this process,
pharmacophore-based 3D searching also proved to be
beneficial (Guner, CA 2000). It is categorized in terms of
subgroups of two, three or four features, and the preparation
with particular characteristics, which are essential for
molecule binding in addition to spatial connection among
them gives a fast and flexible tool to perform these searches.
The GALAHAD (Richmond et al. 2006) fit implemented in
SYBYL 2.0 program was also employed in the building of
pharmacophore hypothesis for the synthesized compounds,
It is a distinctive technique, since it does not require any
template structure, but allows for instant and efficient
creation of partial-coverage models with multiple partial
match constraints (Shepphird and Clark 2006).
Alignment and pharmacophore generation
In this study, a sum of 24 phenyl thiazole and pyrrolyl
benzamide antimycobacterial molecules were used as data

Medicinal Chemistry Research
At the end, all the molecules were aligned with the use of
pharmacophore (MODEL_18) as a template using GALA-
HAD’s template procedure (Table 4). Pharmacophore and
steric bitmaps were formed for each molecule and (com-
pressed) the count vectors were then created for the band.
Subsequently, the post-processing action in GALAHAD
involves taking the genetic algorithm results and producing
the final models together with their alignments, scoring of
the models, and displaying their rank. In GALAHAD, the
needed frame of reference is generated via post processing
using hypermolecular alignment program linear assignment
for molecular dataset alignment (LAMDA) (Tripose,
MO 2014).
Surflex-docking
To generate and score the putative protein–ligand com-
plexes according to their calculated binding affinities at
the active site of ENR with ligands, molecular modeling
was carried out using the Surflex-dock module of one
more superior version of SYBYL software (X 2.0). Such
docking approach aligns the ligand to a “protomol” also
idealized ligand in the active site of the target. Surflex-
Dock, which uses observed scoring function and a
patented search engine (Jain 1996, 2003) was engaged for
molecular modeling of the training set in addition to the
test set compounds into the active site of the crystal
structure of ENR catalytic core.
Results and discussion
Chemistry
All the reactions were carried out as per Schemes 1 and 2.
The Pall–Knorr pyrrole synthesis involves the reaction of
amine with diketone viz., 2,5-dimethoxytetrahydrofuran/
2,5-hexane dione, which is the general method for the
synthesis of pyrrole ring as used for compounds (3a–e),
(4a–f), (6a–e), (7a,b), 8, 9, 10 and (11a–d).
In Scheme 1, 2-amino-4-(substituted phenyl)thiazoles
(2a–f) were synthesized as described earlier in which
4-substituted acetophenones (1a–f) were used as the start-
ing materials.
The reaction of 2-amino-4-(4-substituted phenyl)thia-
zoles (2a–f) with 4-(1H-pyrrolyl-1-yl)benzoic acid/4-(2,5-
dimethyl-1H-pyrrolyl-1-yl)benzoic acid, respectively using
HBTU as a coupling agent and DIEA as a base in DMF
medium to give N-4-(4-substituted phenyl)thiazole-2-yl-4-
(1H-pyrrol-1yl)bezamides (3a–e)/N-4-(4-substituted phe-
nyl)thiazol-2-yl-4-(2,5-dimethyl-1H-pyrrol-1-yl)benza-
mides (4a–f). The reaction of 4-(4-substituted phenyl)-5-
bromo-2-aminothiazoles (5a–e) with 4-(2, 5-dimethyl-1

Medicinal Chemistry Research

Medicinal Chemistry Research

Medicinal Chemistry Research
Fig. 4 Pareto scatter plot (energy
vs. sterics) from 20 models,
black circled model_18 was
selected for further alignment of
all compounds
Table 4 GALAHAD score for all aligned molecules using
MODEL_18
H-pyrrol-1-yl)benzoic acid/4(1H-pyrrol-1-yl)benzoic acid
in DMF medium using HBTU as a coupling agent and
MIC
ENERGY
STERICS
H_BOND
MOL_QRY
DIEA as
a
base to give N-(5-bromo-4-(4-sub-
stitutedphenyl)-thiazol-2-yl)-4-(2,5-dimethyl-1H-pyrrol-1-
yl)benzamides (6a–e)/N-(5-bromo-4-(4-substitutedphenyl)-
thiazol-2-yl)-4-(1H-pyrrol-1-yl)benzamides (7a,b). The
reaction of 5-bromo-4-phenylthiazole with 2,5-dimethox-
ytetrahydrofuran in refluxing acetic acid yielded 5-bromo-4-
phenyl-2-(1H-pyrrol-1-yl)thiazole (8).
10
11a
4a
4b
4c
25
5.49
14.82
4.39
171.40
548.10
0.20
0.90
0.27
0.27
12.5
50
2109.50
5686.60
5859.60
5081.40
3957.20
2804.20
730.70
41.90
41.90
41.90
59.10
41.90
41.90
58.80
83.10
58.80
182.40
0.90
0.16
1.6
15.99
7.72
0.16
1.6
0.16
4d
4e
12.5
25
8.56
0.16
8.91
0.16
Further, compounds 10 and (11a–d) (Scheme 2) were
prepared by reacting 4-(2,5-dimethyl-1H-pyrrol-1-yl)ben-
zoic acid (9) with substituted aromatic amines in the pre-
sence of diisopropyl ethylamine (DIEA) and 2-(1H-
benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexa-
fluorophosphate (HBTU) as a catalyst. The synthesized
compounds were isolated in pure form using column
chromatography. The prepared derivatives were confirmed
by spectral analysis.
4f
1.6
5.97
0.16
6a
6b
6c
12.5
1.6
5.12
35.43
58.04
35.43
14.43
0.27
6.96
5808.20
3783.60
5259.30
419.80
1.6
5.08
6d
11b
6e
3.12
1.6
120.46
6.64
1.6
5.54
6288.10
406.10
215.20
83.10
83.10
0.00
58.04
58.04
58.04
0.00
7a
7b
8
12.5
6.25
1.6
14.04
6.84
1764.90
1.30
−1.21
13.24
21.86
11.34
9.23
1
FTIR, H NMR, 13C NMR, and mass spectral data were
11c
11d
3a
3b
3c
12.5
12.5
25
814.50
17.70
1.20
0.27
481.60
0.27
found to be in agreement as per the projected structures of
all the molecules.
477.10
83.10
83.10
83.10
215.20
384.50
58.04
58.04
58.04
58.04
58.04
1.6
2866.80
2539.30
3722.10
4634.50
FTIR spectrum of 4b showed an absorption band at
3304 cm−1 associated with the secondary amine (–NH)
group and an absorption band of carbonyl group appeared at
3.12
1.6
10.69
10.50
16.58
3e
3d
1.6
1
1608 cm−1. The H NMR spectrum showed a characteristic

Medicinal Chemistry Research
Fig. 5 a Final selected
pharmacophore model and
b molecular alignment for InhA
receptor ligands (total of six
compounds) containing acceptor
atoms (green), positive nitrogen
atom (red) and
hydrophobes (cyan)
Fig. 6 Docked mode of all the
synthesized compounds inside
the proposed binding pocket
of InhA
triplet signal for two protons of pyrrole at C3 and C4 posi-
tions and a singlet thiazole proton at C5 position with the δ
values of 5.94–5.82 and 7.54 ppm, respectively. The signals
of aromatic protons (doublet, doublet of doublet, and mul-
tiplet) appeared between δ 7.53 and 8.14 ppm. In the 13C
NMR spectrum of compound 4b, C = O carbon resonated at

Medicinal Chemistry Research
Fig. 7 a Docked mode of
2NSD_ligand; b Inside the
proposed binding pocket of
InhA; c 3D docked view of the
2NSD_ligand. Binding site
residues; cyan colored Tyr 158
amino acid, green colored
co-factor NAD+ and the
molecule is colored according to
atom type
δ of 168.66 ppm. The signals at δ of 121.37–146.33 ppm
was due to aromatic carbons. Similarly, compound 6b
showed an absorption band at 3297 cm−1 associated with
the secondary amine (–NH) group, while the absorption
FTIR spectra of compounds 10 and (11a–d) showed
absorption bands at 3223–3381 cm−1 owing to the NH
group (2° amine) stretching and carbonyl group as strong
bands in the region of 1635–1696 cm−1, confirming the
1
1
band of carbonyl group appeared at 1667 cm−1. In the H
formation of compounds 10 and (11a–d). In the H NMR
NMR spectrum, two protons of pyrrole at C3 and C4 posi-
tions appeared as characteristic singlet with the δ value
5.88 ppm. The signals shown between δ 7.50 and δ
8.10 ppm were attributed to aromatic protons, while the
singlet signal at 2.01 confirms six pyrrolyl dimethyl protons
at C2 and C5 positions. The 13C NMR spectrum showed the
signal at δ of 167.19 ppm, corresponds to the carbonyl
group. All the aromatic carbons resonated in the expected
values of δ of 124.23–134.01 ppm.
spectra, the resonating signals of two protons of pyrrole at
C3 and C4 positions are seen as triplet with the δ values
ranging from 5.82 to 5.85 ppm. Aromatic protons
appeared as doublet/doublet of doublet/doublet of triplet/
triplet/triplet of doublet/multiplet around δ value of 6.79
and 8.12 ppm. The 13C NMR spectra showed character-
isitic signals of carbonyl carbon atom in the range of δ
values of 165.13–167.36 ppm, whereas the remaining
aromatic carbons appeared in the range of
δ

Medicinal Chemistry Research
Fig. 8 a Docked mode of
compound 3d; b 3D docked
view of compound 3d. Binding
site residues; green colored Tyr
158 amino acid, orange colored
co-factor NAD+ and the
molecule is colored according to
atom type; c Inside the proposed
binding pocket of InhA
106.61–144.73 ppm. Additionally, electron impact ioni-
zation (EI-MS) spectra that exhibited the molecular ion
[M+] also confirmed the structures of the synthesized
compounds.
activities against mycobacteria with the MIC values ranging
from 1.6 to 50 µg/mL. Compounds 3b, 3d, 3e, 4b, 4c, 4e,
4f, 6b, 6c, 6e, 8. 11b, and 11c inhibited mycobacterial
growth very effectively compared to others in the series
with a MIC value of 1.6 µg/mL.
Antimycobacterial and cytotoxicity studies
The inclusion of bulky groups or halogen atoms
increased the lipophilicity of the compound, while the
mycobacterial cell wall is especially lipophilic, whose
contributions of these lipophilic substituents played a sig-
nificant role. Additionally, we have investigated the
potential toxicity of eight selected pyrrole derivatives (3b,
3d, 4c, 4e, 10, 11b, 11c, and 11d) towards the mammalian
All the synthesized molecules were evaluated by deter-
mining minimal inhibitory concentration (MIC) on
M. tuberculosis H37Rv strain (MTCC300) (Table 1). Pyr-
azinamide and streptomycin were used as reference drugs
for comparison. The evaluated compounds exhibited

Medicinal Chemistry Research
Fig. 9 a Docked mode of
compound 6b; b 3D docked
view of compound 6b. Binding
site residues; green colored Tyr
158 amino acid, orange colored
co-factor NAD+ and the
molecule is colored according to
atom type; c Inside the proposed
binding pocket of InhA
Vero cell-lines and A549 (lung adenocarcinoma) cell-lines
up to MIC values of 62.5 µg/mL were investigated. These
tested molecules showed a modest cytotoxicity compared to
the standard INH (see Table 2).
Table 1. All the evaluated compounds gave related activities
against InhA ranging between 12% and 38% of inhibition at
50 mM with the lowest values for compounds 10 and 11d
and the highest for compounds 4c and 4e.
InhA inhibition assay
Antibacterial activities
Twelve synthetic molecules were analyzed in vitro as
potential InhA inhibitors from M. tuberculosis at 50 mM
using triclosan as the reference drug by applying the gen-
erally accepted concepts and the results are displayed in
Antibacterial activity was also carried out for all the com-
pounds against both Gram-positive bacteria (S. aureus-
MTCC096) and Gram-negative bacteria (E. Coli-
MTCC443). The antimicrobial results have shown that all

Medicinal Chemistry Research
Fig. 10 a Hydrophobic amino
acids surrounded to compounds
3d (green color) and 6b (cyan
color). b Hydrophilic amino
acids surrounded to compounds
3d and 6b
the compounds showed a moderate to good microbial
inhibition in the range of MIC values of 1.6–50 µg/mL.
These results suggest that the compounds are highly active
towards Gram-positive bacteria S. aureas than the Gram-
negative bacteria E. coli. The compounds 4c, 4e, 4f, and 6b
have shown the highest activity against E. coli (MIC value
of 1.6 µg/mL).
as one positive nitrogen atom (CONH linkage). The phar-
macophore model clearly exhibits the importance of
hydrophobic phenyl, thiazole, and pyrrole rings with polar
oxygen to confirm the anti-TB activity. The linking chain is
also vital for exhibiting the activity.
Molecular docking
Pharmacophore results
The active pocket was measured to be the location where
2NSD_ligand complexes with enoyl-ACP reductase in
2NSD. The 2NSD_ligand was re-docked to get its interac-
tions and direction at the active site for the evaluation with
other synthesized compounds (Fig. 6a, b). Docking studies
with Surflex-Dock showed that the compound 3d reside in
the same binding site as that of 2NSD_ligand. The
2NSD_ligand has shown two H-bond interactions, the
oxygen of carbonyl group makes two hydrogen bonds
(Fig. 7a–c) with that of OH of the active site of NAD+
ribose (2.06 Å) and with OH of the active site TYR158
(1.87 Å). The molecules 3d and 6b also bind in the similar
manner i.e., oxygen atom of carbonyl group is responsible
for H-bonding interactions, one bonding interaction with
amino acid Tyr 158 and one more with co-factor NAD+
(Figs. 8a–c and 9a–c). Figure 10a, b exhibits hydrophobic
and hydrophilic amino acids surrounded around molecules
3d and 6b.
In view of extensive applications of pharmacophore mod-
eling in drug design research, the pharmacophore model
was generated with six most active molecules with a MIC
value of 1.6 µg/mL. Generally, the GALAHAD provides a
total of 20 pharmacophore models ranked as per their fitness
function. The generated models were ranked on the basis of
energy profile, specificity value and Pareto rankings. As a
result, the top-ranked MODEL_18 between all the
20 selected was used to understand the structural needs of
the considered compounds, and was also taken into account
for the final rigid alignment of the left over molecules. The
description resulted from the final MODEL_18 are dis-
played in Fig. 5a, b, which contains five hydrophobes
centered on two benzene rings, pyrrole ring as well as
thiazole ring, two acceptor atoms center on oxygen atom
(CONH linkage) and nitrogen atom (thiazole ring), as well

Medicinal Chemistry Research
PMF scoree G scoref Chem scoreg
−250.959 −46.922
Table 5 Surflex dock scores
(kcal/mol) of pyrrole derivatives
Compounds C scorea Crash scoreb Polar scorec D scored
4PI
3a
9.25
7.62
6.93
6.61
6.47
6.14
5.29
4.96
4.92
4.88
4.82
4.55
4.26
4.26
4.18
4.03
3.97
3.81
3.61
3.44
3.34
3.29
3.28
3.19
2.95
2.25
−0.93
−0.53
−1.08
−1.47
−1.83
−3.16
−2.01
−2.17
−0.94
−4.37
−1.86
−0.95
−2.16
−3.48
−4.01
−4.11
−2.20
−3.36
−3.10
−3.09
−4.95
−3.22
−3.48
−3.68
−4.81
−4.67
1.54
1.18
0.00
1.48
1.15
0.67
0.92
0.01
0.00
0.00
0.01
1.92
0.77
0.00
0.01
0.02
0.39
0.00
0.00
0.39
0.02
1.11
0.03
0.00
0.87
0.01
−150.083 −63.091
−125.987 −84.693
−128.036 −37.925
−136.790 −71.223
−138.107 −86.509
−127.964 −81.663
−136.362 −58.543
−138.049 −46.821
−95.662 −70.793
−160.854 −41.194
−133.654 −36.897
−115.813 −58.893
−152.168 −64.038
−141.933 −46.565
−168.982 −46.808
−159.142 −39.039
−132.946 −66.200
−121.191 −70.110
−112.664 −63.417
−118.026 −82.290
−167.251 −35.635
−136.719 −81.659
−144.721 −69.152
−137.692 −47.965
−150.398 −70.340
−143.402 −70.116
−250.274 −43.687
−230.226 −33.826
−255.580 −43.699
−269.607 −45.489
−279.021 −42.267
−253.306 −40.722
−234.920 −37.495
−204.471 −36.643
−312.924 −41.741
−205.954 −35.242
−181.923 −35.745
−235.404 −41.954
−258.198 −40.697
−295.678 −44.407
−296.074 −44.544
−264.862 −40.865
−252.681 −39.696
−269.857 −36.774
−266.253 −38.665
−304.477 −45.662
−243.112 −46.527
−286.852 −42.124
−250.815 −40.751
−288.449 −46.516
−272.019 −38.299
10
3c
7a
11d
3d
11c
8
3e
11a
TCL
11b
4e
6d
6a
4c
4b
4a
4d
6b
3b
6c
4f
7b
6e
aCScore (Consensus Score) integrates a number of popular scoring functions for ranking the affinity of
ligands bound to the active site of a receptor and reports the output of total score
bCrash-score revealing the inappropriate penetration into the binding site. Crash scores close to 0 are
favorable. Negative numbers indicate penetration
cPolar indicating the contribution of polar interactions to the total score
dD-score for charge and van der Waals interactions between the protein and the ligand
ePMF-score indicating Helmholtz free energies of interactions for protein–ligand atom pairs (potential of
mean force, PMF)
fG-score showing hydrogen bonding, complex (ligand–protein), and internal (ligand–ligand) energies
gChem-score points for H-bonding, lipophilic contact, and rotational entropy along with an intercept term
The molecules displayed consensus score in the range
of 7.62–2.25, demonstrating the requirement of all forces
of interaction between the ligands and the enoyl ACP
reductase enzyme. Charge and van der Waals interactions
among the protein and molecules varied from −168.98 to
−95.66. Helmholtz free energies of interactions for
protein–ligands atom pairs ranged between −86.50 and
−35.63 and its H-bonding, complex (ligand–protein), and
internal (ligand–ligand) energies ranged from −304.47 to
−181.92, while those values ranging from −46.92 to
−33.82 indicate the ligands due to the presence of H-
bonding, lipophilic contact, and rotational entropy. These
scores suggest that the molecules selectively bind to the
InhA in agreement with the reference 2NSD_ligand
(Table 5).
Conclusions
New pyrrole derivatives have been synthesized that dis-
played interesting activities against InhA. These compounds
were tested for inhibition of M. tuberculosis growth and
antimicrobial activities against S. aureus and E. coli.
Interestingly, molecules 3b, 3d, 3e, 4b, 4c, 4e, 4f, 6b, 6c,
6e, 8, and 11c showed the best activities with a MIC value
of 1.6 µg/mL compared to other compounds. The potent

Medicinal Chemistry Research
antimicrobial activities of the most active compounds were
accompanied with a comparatively no evidence of cyto-
toxicity, indicating their nontoxic behavior. In addition,
some compounds possess moderate InhA inhibition
activities.
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drug–receptor-binding interactions. On the other hand,
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the GALAHAD Module of SYBYL 2.0 molecular model-
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optimized pharmacophore model (MODEL_18) developed
in this study showed the superior statistical parameters
during the entire validation process. We believe that results
of this study are useful as the guiding principles for
designing and developing some putative new direct InhA
inhibitors/antitubercular agents based on the suitable struc-
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Acknowledgements The authors acknowledge the financial support
from Indian Council of Medical Research, New Delhi [ICMR letter
Ref no. BIC/12(13)2014 dated 13-02-2017 (IRIS Cell no. 2014-
2676)]. We are grateful to Dr. H.V. Dambal, President, S.E.T’s Col-
lege of Pharmacy, Dharwad, India for providing the facilities. We
thank Dr. K.G. Bhat of Maratha Mandal’s Dental College, Hospital
and Research Centre, Belgaum, India for providing antitubercular and
cytotoxic activities. Authors are grateful to the Director, SAIF, Panjab
University, Chandigarh, Panjab, and IIT-Kanpur, India for providing
some NMR and mass spectral data.
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Kokai Tokkyo Koho CODEN; JKXXAF JP 2002322054 A2
20021108. Chem Abstr 137:346134
Conflict of interest The authors declare that they have no conflict of
interest.
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continuous differentiable function tuned to compute binding
affinities. J Comput Aided Mol Des 10:427–440
Publisher’s note: Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
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