Novel hybrid-pyrrole derivatives: Their synthesis, antitubercular evaluation and docking studies

Article abstract of DOI:10.1039/c4ra14440f

Using novel hybrid molecules for the treatment of tuberculosis is one of the latest approaches. Keeping this concept in mind, thirty two hybrid compounds were synthesized, with pyrrole as one of the moieties, clubbed to coumarin, ibuprofen and isoniazid. The compounds were evaluated against Mycobacterium tuberculosis H₃₇Rv strain. Compounds 7e and 8e exhibited MIC of 3.7 and 5.10 μg mL^-1 and growth inhibition of 95% and 92%, respectively. These compounds were also active against single drug resistant bacterial strains. The compounds were devoid of cytotoxicity when tested against Vero African green monkey kidney cell line. Docking study was carried out on enoyl acyl carrier protein enzyme to provide some understanding into the mechanism of action of these compounds.

Full text of DOI:10.1039/c4ra14440f

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Novel Hybrid-Pyrrole Derivatives: Their Synthesis, Antitubercular Evaluation and
Docking Studies
Rikta Saha, Md. Mumtaz Alam, Mymoona Akhter*
Drug Design and Medicinal Chemistry Laboratory, Department of Pharmaceutical Chemistry,
Faculty of Pharmacy, Jamia Hamdard, New Delhi 110062, India. Ph: +91 11 26059688 x 5645
Docking on Mycobacterium InhA
Hybridization of the molecular fragments proved to be beneficial as revealed by the biological activity of the
synthesized compounds.

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DOI: 10.1039/C4RA14440F
Novel Hybrid-Pyrrole Derivatives: Their Synthesis,
Antitubercular Evaluation and Docking Studies
Cite this: DOI: xxxxxxxxx
Rikta Saha, Md. Mumtaz Alam, Mymoona Akhter*
Hybrid new molecules for the treatment of tuberculosis are one of the latest approaches. Keeping this
concept in mind, thirty two hybrid compounds were synthesized, with pyrrole as one of the moiety,
clubbed to coumarin, ibuprofen, isoniazid etc. The compounds were evaluated against Mycobacterium
tuberculosis H37Rv strain. Compounds 7e and 8e exhibited MIC of 3.7 and 5.10 ꢀg/mL and growth
inhibition of 95 and 92% respectively. These compounds were also active against single drug resistant
bacterial strains. The compounds were devoid of cytotoxicity when tested against Vero African green
monkey kidney cell line. Docking study was carried out on enoyl acyl carrier protein enzyme to provide
some understanding into the mechanism of action of these compounds.
Received xxxx,
Accepted xxxx
DOI: xxxxxxxxx
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1. Introduction
Pyrrole derivatives have been found to possess a wide spectrum of
activities8, 9 of which antiꢀTB is one of the prominent one10ꢀ12. Also,
coumarin containing compounds have been widely explored for
different activities like antibacterial, anticancer, antitubercular, etc.13ꢀ
15. Calanolide A, a natural product with coumarin nucleus is reported
Despite the enormous efforts have been made in the quest of new
drugs, tuberculosis (TB) still is one of the rapidly emerging and
deadly infections caused by the pathogen Mycobacterium
tuberculosis (Mtb). In the year 2013, 9 million new cases were
reported with 1.4 million fatal incidents1. The rampant bacterial to have antitubercular action at MIC of 3.13 ꢀg/mL16. Similarly
resistances viz. multiꢀdrug resistant (MDR) TB and extensively drugꢀ
ibuprofen (MIC 75 ꢀg/mL), naproxen (MIC 90 ꢀg/mL) and other
known antiꢀinflammatory agents are reported to possess good
antitubercular activity against Mtb H37Rv17. Since INH contains a
pyridine nucleus, large numbers of scientists have developed novel
agents bearing pyridine moiety for the treatment of TB18.
resistant (XDR) TB to existing antiꢀTB agents has become a
significant concern for the effective treatment of TB2, 3
.
Antitubercular agents can be divided into two classes: firstꢀline
drugs: isoniazid (INH), pyrazinamide (PZA), ethambutol (EMB),
rifampin (RIF), and streptomycin given for 6 months. If the
treatment fails due to bacterial drug resistance, second line drugs
such as pꢀamino salicylic acid (PAS), kanamycin, fluoroquinolones,
capreomycin, ethionamide, and cycloserine are used. These are
mostly either less effective or more toxic with serious sideꢀeffects.
Hence, it is quite essential to develop safe and costꢀeffective new
antitubercular agents.
Space for Fig. 2
Keeping in view the hybrid concept along with the antitubercular
potential of different moieties it was planned to synthesize hybrid
compounds (Fig. 2) that comprise the pyrrole nucleus with the
aforementioned fragments and their evaluation of antiꢀTB activity.
All the compounds were synthesized by conventional as well as
microwave method. Microwave reactions gave higher yields with
reduced reaction times. Whole cell assay of these compounds was
done by microplate alamar blue assay (MABA), against drug
sensitive and resistant strains. Compounds with more than 60%
growth inhibitory property were further selected for cytotoxicity
studies to figure out whether those are nonꢀtoxic or not.
These compounds are thought to act by inhibiting enoyl acyl carrier
protein enzyme (InhA target of INH). This was supported by
docking studies of the synthesized pyrroleꢀhybrids using GLIDE
module of Schrodinger. Since the Nꢀ(3,5ꢀdichlorophenyl)ꢀ5ꢀoxoꢀ1ꢀ
phenylpyrrolidineꢀ3ꢀcarboxamide is direct inhibitor of InhA which
forms a hydrogen bond with Tyr158, we focused to dock the most
potent compound in a similar manner.
Molecular hybridization of potential pharmacophore scaffold is
being used as valuable approach by medicinal chemists to design
new prototypes. These molecules effectively address resistance
problem and have an increased efficacy. The hybrid molecules are
generated by covalent addition of two active subunits either by (a) a
spacer or (b) fusion or (c) merging of compounds with desired
activities4.
Various hybrid derivatives have been synthesized and evaluated for
antitubercular activity, for example isoniazidꢀpyrazinamide5,
isoniazidꢀfluoroquinolone6 and pyrazoleꢀquinazoline7 (Fig. 1) and a
synergistic activity profile has been observed.
Space for Fig. 1
Keywords: Antitubercular agents, hybrid molecule, Mycobacterium
tuberculosis, Coumarin, Ibuprofen, microwave synthesis, pyrroles
Running title: Hybridꢀpyrrole as antitubercular agents
2. Results and discussion
Chemistry
The concept of hybridization is being widely used to synthesize
novel agents to target different diseases specially related to
development of resistance to existing drugs. Similar approach has
been used to develop novel antitubercular agents containing different
moieties linked together directly or through linkers. In this paper we
report the hybrid of pyrrole moiety with different moieties like
Drug Design and Medicinal Chemistry Laboratory, Department of
Pharmaceutical Chemistry, Faculty of Pharmacy, Jamia Hamdard,
New Delhi 110062, India. Ph: +91 11 26059688 x 5645
*Corresponding Author: Tel: +91 1126059688x5645; fax: +91
1126059663; Eꢀmail: mymoonaakhter@gmail.com
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isobutyl benzene, pyridyl, naphthyl, coumarin to explore their Preliminary antiꢀTB screening of all the synthesized compounds (7a-
potential as antitubercular agents. Thirty two different hybrid p & 8a-p) was done against drug sensitive strain of Mtb H37Rv
compounds (7a-p & 8a-p) reported herein were prepared by (ATCC 27294) by microplate alamar blue assay (MABA) as
following scheme 1. Step 1a describes the preparation of reported by Collins and Franzblau20. This methodology is simple,
intermediate hydrazides (3a-o) from different acids (1a-o). nonꢀtoxic, uses a thermally stable reagent and shows good
Primarily, corresponding aromatic acid (1a-o) and catalytic amount correlations with the proportional and BACTEC methods21. The
of concentrated HCl was refluxed in methanol to produce minimum inhibitory concentration (MIC) i.e. concentration of
corresponding ester (2a-o). The hydrazides (3a-o) were synthesized compounds required to completely inhibit the Mtb growth were
by refluxing ester (2a-o) with 99% hydrazine hydrate according to recorded. The MIC was calculated from dose response curve. The
the reported procedure19. Step 1b involves the preparation of compounds with more than 90 % inhibition of initial primary
coumarin based hydrazides (3p), where the ester obtained from 7ꢀ screening were further assayed for determination of MIC against
hydroxyꢀ4ꢀmethylꢀ2Hꢀchromenꢀ2ꢀonecorresponding ester (2p) by different Mtb clinical isolates (drug sensitive and resistant). The
refluxing with ethyl 2ꢀbromo acetate in presence of potassium pyrrole scaffold was used as the basis for the development of
carbonate as base. The starting material phenacyl bromide (4) was structural analogues, which yielded compounds 7a-p & 8a-p and
readily prepared from the acetophenone by treating with bromine at their activity data is presented in Table 1. The activity results
room temperature in diethyl ether. 1,4ꢀDiphenylbutaneꢀ1,4ꢀdione (5) indicate that thirteen compounds (7a, 7e, 7j, 7l, 7n, 7p, 8a, 8e, 8h,
was synthesized by refluxing bromo acetophenone 4 with zinc and 8j, 8l, 8n and 8p) which have different fragments show moderate to
pinch of iodine suspension in dry tetrahydrofuran. Following which
the ester was refluxed with hydrazine hydrate leading to the
formation of 3p. The starting material, 1ꢀphenylpentaneꢀ1,4ꢀdione
(6) was purchased from Sigma Aldrich. Finally, the title compounds
(7a-p & 8a-p) were produced by the nucleophilic attack of the most
good activity in the range of 60 to 95% inhibition at 50 ꢀg/mL
concentrations, while rest of the compounds show either a decreased
inhibition or are devoid of inhibition. The MIC values of the 2, 5ꢀdi
phenyl pyrrole analogs (7aꢀp) were found to be better as compared
to their 2ꢀmethyl 5ꢀphenyl analogues (8aꢀp) as the overall
basic nitrogen of hydrazides (3a-p) to the carbonyl group of the γꢀ hydrophobicity increased. Gratifyingly, compounds 7e and 8e with
diketone (5 & 6) following PaalꢀKnorr cyclocondensation. The Paalꢀ 4ꢀpyridoyl fragments were found to be most active with MIC value
Knorr condensation was carried out by conventional heating as well
as under microwave irradiation, however carrying the reaction by
conventional method needs a catalyst (pꢀtoluene sulfonic acid), and
takes longer time with lesser yields. The neat reagents were
irradiated for 7ꢀ15 minutes in the range of 100ꢀ130 oC, water
droplets appeared as reaction proceeded. The details are provided in
supplementary data.
of 3.7 and 5.10 ꢀg/mL and 95% and 92% growth inhibition
respectively comparable or better than other existing drugs e.g.
pyrazinamide 50 ꢀg/mL; cycloserine 50 ꢀg/mL22.
Space for Table 1
The compound 7e and 8e has also shown good activity against
rifampin (RMPꢀR1 & RMPꢀR2) and fluoroquinolone resistant (FQꢀ
R) Mtb strains; whereas it exhibited moderate activity against
isoniazid resistant (INHꢀR) strains with MIC value of >67.8 ꢀg/mL
(Table 2). As 7e and 8e both have activity against resistant strain,
these molecules can serve as lead for further generation of more
active compounds. The intracellular (macrophage) drug screening
assay evaluates intracellular drug effectiveness. The assay results
depicts that reduction in cell viability exhibited by compound 7e is
2.4 log reduction of CFU at 3.7 ꢀg/mL concentration; which is
comparable with the control drug INH. This is important because
Mtb can survive inside macrophages, which contributes to treatment
failure and disease relapse.
The structure of all the synthesized compounds (7a-p & 8a-p) was
established by analytical data. In general, IR peaks at 3268ꢀ3182
cmꢀ1 for NH and 1700ꢀ1640 cmꢀ1 for C=O confirmed that all the
1
derivatives contain amide bond. The analysis of H NMR spectra
(7a-p & 8a-p) showed the signals of the corresponding protons,
which were confirmed on the basis of their chemical shifts,
multiplicities and coupling constants. These spectra showed two
characteristic signals for CONH proton between 8.0 to 11.0 ppm and
CH=CH pyrrole protons at 6.2ꢀ6.8 ppm singlet type in case of 7a-p.
But CH=CH pyrrole protons of second series (8a-p) appeared as
doublet with different chemical shift values. The NH protons of all
compounds were confirmed by D2O exchange. In 13C NMR, peaks
were observed at 164ꢀ169 (C=O), 104ꢀ110 (pyrrole ring), further
confirming the desired structures. Remaining resonances were also
observed at their expected values. In MS (ESI) m/z was found at
M+H showing 100% base peak, which corresponds with actual
molecular weight of the compounds. The elemental analysis
confirmed the purity of the compounds, experimental values were
found within ± 0.4% of the theoretical values. The purification of all
compounds (7a-p & 8a-p) was achieved by chromatography using
gradient elution method (hexane and 0ꢀ30% ethyl acetate mixture),
and was confirmed by HPLC coupled with mass spectrometer. All
the compounds are more than 90% pure.
Space for Table 2 and Fig. 3
The cytotoxicity of compounds was assessed in Vero African green
monkey kidney cell line. Selectivity index is defined as TC50/MIC.
The compounds 7e and 8e are nonꢀtoxic with high selectivity index
(>27 and >19 respectively). The MBC is determined subsequent to
MIC testing by subꢀculturing diluted aliquots from wells that fail to
exhibit macroscopic growth. Since, the MBC value is close to MIC,
the compound 7e and 8e is bactericidal in nature (Table 1 and Fig.
3).
It is believed that compounds with an MIC ≤6.25 ꢀg/mL and an
SI>10 are interesting compounds, and are considered as excellent
leads23. These features make 7e and 8e as very promising antiꢀTB
agent.
Antitubercular activity
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active site. This hydrogenꢀbonding network is most important
Structure activity relationship (SAR)
Based on the above observation following structure activity feature among all the InhAꢀinhibitors identified to date. The
relationship could be established.
hydrogen bonds formed between Tyr158 and compound 7e clearly
supports that this compound might be acting through this
mechanism. 7e also shows van der waals interactions with the
hydrophobic residues Gly96, Met103, Phe149, Met155, Pro156,
Ala157, Met161, Pro193, Ala198, Ile215, and Leu218.
1. Replacement of one of the phenyl group with methyl group at
5ꢀposition in pyrrole ring resulted in decrease in activity as can
be seen from the differences in MIC of 7a-p and 8a-p, eg. MIC
of 7e is 3.7 ꢀg/mL whereas 8e is 5.10 ꢀg/mL
In silico pharmacokinetic property
2. In general, group with electropositive character attached to
amide link resulted in increase in activity, like change of phenyl
group to pyridyl group in 7e and 8e gave the most active
compounds with low MIC, GI of 95 and 92% & cell viability
100%. On the other hand, when the phenyl group is attached
with steric (+π) group (7b-d and 8b-d), the growth inhibitory
activity decreases. Moreover, introduction of OCH3 group in
phenyl ring (+σ and –π) favors the inhibitory property
Many drugs, at a late later stage of development, as well as lead
compounds fail due to adverse pharmacokinetic properties. It is
therefore important to incorporate ADME (adsorption, distribution,
metabolism and excretion) properties of above derivatives to
continue the further research. Most of the existing antiꢀTB drugs are
hydrophilic molecules, hence their low cellular penetration may be
contributing to the development of Mtb drug resistance 25. Therefore,
the development of hydrophobic molecules could increase the
cellular penetration of target tissues as it was hypothesize that they
are likely to pass easily through Mtb’s cell envelope 26. In addition to
their promising in vitro bactericidal activity against Mtb, all the
fragment based pyrrole derivatives have good physicochemical
properties that indicate great potential of these agents as orally
available compounds. They fulfill at least four of the five
physicochemical properties defined by the Lipinski ‘ruleꢀofꢀfive’,
which predicts aqueous solubility and intestinal permeability. All
compounds of the both series have <10 hydrogen bond acceptor, <5
hydrogen bond donor and molecular weights <500 gmolꢀ1. Details
are provided in supplementary data. In terms of lipophilicity,
compounds 7e, 8a-k and 8m-p show miLogP value of <5, while rest
of the compounds display miLogP values just above 5. The ease of
synthesis coupled with the promising physicochemical properties
signify that these compounds can be attractive leads for further
development as novel antiꢀTB agents.
3. Introduction of bulky groups like pꢀisobutyl benzene, or
coumarin or naphthyl group also increased the activity as
indicated by MIC of 6.25 ꢀg/mL for 7l, 7p and 8p, 12.5 ꢀg/mL
for 7j, 7n and 8l
4. Moreover, when tested against a panel of singleꢀdrug resistant
Mtb strains, derivatives 7e and 8e maintained the activity as for
the wild type, indicating that these derivatives may act with a
different mechanism of action when compared to the existing
drugs
Docking study
Since the most potent molecules share fragment of INH thus it was
assumed that the active compounds may act via a similar mechanism
as isoniazid but directly inhibiting the enzyme, therefore docking
studies of all the synthesized molecules was carried out on InhA.
The crystal structure of enoyl acyl carrier protein (2H7M) was used
for docking studies to get a preliminary idea about the interaction of
ligands with target. It was observed that some of the compounds
having docking scores more than ꢀ6.0 Kcal/mol were not good in in
vitro assay, which may be attributed to their high logP value. These
results suggest that these inhibitors do not yet have optimal
membrane permeability or are actively pumped out of the bacterial
cells by efflux pumps24. The visual inspection of the target ligand
interactions made it clear that the hydrogen bonding interaction with
Tyr 158 and NAD+ is important for activity as supported by the in
vitro results, compounds (7f, 7h, 7o and 8o) are inactive because
they do not show this interaction. Additionally extra interaction with
3. Conclusions
The hybrid design of compounds to develop potent antitubercular
agent was achieved successfully. Thirty two compounds were
synthesized and screened for antiꢀTB activity. Two compounds 7e
and 8e were active against Mtb at low MIC and also against rifampin
and fluoroquinoline resistance strains. The biological evaluation of
the synthesized compounds helped in identifying a hybrid of 4ꢀ
pyridyl with pyrrole moiety, as a lead molecule.
4. Experimental
Phe 149 has proven to be important for potency. Compounds lacking 4.1. Analysis and Instruments
All the chemicals were purchased from Sigma Aldrich, Spectrochem
Pvt. Ltd. and S.D. Fine Chemicals (India) and were used without
further purification. Tetrahydrofuran was dried over
π stacking with Phe 149 were found less active in in vitro studies
comparative to active ones. There may be some other factor for their
reduced activity in addition to this.
sodium/benzophenone prior to use. Anhydrous reactions were
performed under a positive pressure of inert nitrogen gas. Melting
points were determined on a digital melting point apparatus by the
open tube capillary method and are uncorrected. The thin layer
chromatography (TLC) plates (silica gel G) were used to check the
purity of commercial reagents used, desired product purity and to
monitor the reaction progress. Various solvent systems (ethyl
acetate: hexane (3:7) and methanol: chloroform (1:9)) were used to
run the TLC and spots were located under iodine vapors/UV light.
Space for Fig. 4(A), (B), (C) and (D)
A complete overview of receptorꢀinhibitor binding interactions of 7e
is illustrated in Fig. 4. The inhibitor fits the binding pocket of InhA
in the same manner as coꢀcrystalized ligand. The most active
compound 7e binds with the enzyme tightly to form a complex. The
oxygen of amide group and pyridyl nitrogen are connected through
hydrogen bond to 2’ꢀhydroxyl moiety of the nicotinamide ribose and
hydroxyl group of Tyr158, one of the catalytic residues in the InhA
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An infrared spectrum (IR) was recorded on a Bruker FTꢀIR zinc (1.04 g, 16.5 mmol) and pinch of iodine were added and
o
spectrometer using KBr as pellet. Elemental analyses were carried refluxed at 80 C for 3 h. After completion of reaction, zinc was
out on a Perkin–Elmer 2400 analyzer (USA) and were found within removed by filtration over celite. The organic filtrate was extracted
1
±0.4 % of the theoretical values. H NMR and 13C NMR spectra from water layer, dried over anhydrous sodium sulfate, concentrated
were recorded on a Bruker spectrometer.
under reduced pressure and purified by column chromatography.
Yield 40%, FTꢀIR (KBr pellet) cmꢀ1: 1674 (C=O str.); 1H NMR (400
MHz, CDCl3): δ (ppm) 8.03 (d, 4H, J = 7.6 Hz, aromaticꢀH), 7.57 (t,
2H, J = 6.8 Hz, aromaticꢀH), 7.47 (d, 4H, J = 7.6 Hz, aromaticꢀH),
3.46 (s, 4H, CH2).
HPLC and MS analysis
The purity of the compounds was measured by reversedꢀphase liquid
chromatography and a mass spectrometer (Agilent 1100 LC/MSD)
with a UV detector at λ = 214 and 254 nm and an electron spray
ionization (ESI) source. MS data were recorded using an Agilent
1100LC/MSD VL system (Phenomenex Gemini C18 column, 3×50
mm; 0.5 mL/min flow rate, acetonitrile/water binary solvent (95:5)).
UV detection was monitored at 214 and 254 nm. MS data was
acquired in positive mode scanning over the mass range of 50ꢀ1000
(attached in supporting information).
Step 3: General procedure for the preparation of pyrrole
derivatives (7a-p & 8a-p):
Method I (Conventional method): An equimolar mixture of 1,4ꢀ
diphenylbutaneꢀ1,4ꢀdione (5, 10 mmol) or 1ꢀphenylpentaneꢀ1,4ꢀ
dione (6, 10 mmol), catalytic amount of pꢀtoluene sulfonic acid and
hydrazide derivatives (10 mmol) (3a-p) in absolute ethanol (30 mL)
were refluxed for 10ꢀ24 h. After completion of reaction, the residual
ethanol was evaporated to dryness and then purified by column
chromatography to yield 3a-p.
Microwave irradiation experiments
A
mono mode CEMꢀDiscover microwave reactor (CEM
Corporation, P.O. Box 200, Matthews, NC 28105) was used in the
standard configuration, including proprietary software. All the
experiments were carried out in sealed microwave process vials (10
mL) at the maximum power and designated temperature (100ꢀ130
oC). After completion of the reaction, the vial was cooled to 50 C
via airꢀjet cooling before it was opened.
Method II (Microwave irradiation method): To a dry 10 mL
microwave vial equipped with
a magnetic stir bar, 1,4ꢀ
diphenylbutaneꢀ1,4ꢀdione (5, 2 mmol) or 1ꢀphenylpentaneꢀ1,4ꢀdione
(6, 2 mmol) and hydrazide derivative (3a-p) (2 mmol) were added
and the vial was capped. The vial was shaken to mix the contents
and then heated in the microwave at 100ꢀ130 oC for 7–15 min (CEM
Discover reactor). Silica gel column chromatography (10 cm × 2 cm;
hexanes/ethyl acetate, 0ꢀ30%) gave 7a–p & 8a-p as solids in
satisfactory yield.
o
Step 1a: General procedure for the preparation of substituted
hydrazide (3a-o): To a solution of appropriate acids in ethanol, 2
drops of concentrated sulfuric acid was added and refluxed at 80 C
o
for 8ꢀ14 h. The reaction progress was monitored by TLC. After the
completion of ester formation, hydrazine hydrate (1.5 times, 90%)
was added and refluxed for 4ꢀ5 h. The residual solvent was
evaporated under reduced pressure; solid was filtered and washed
with iceꢀcold water. The crude mass was purified by recrystallization
from methanol. The purity was checked by TLC. After
recrystallization by methanol, the desired compounds 3a-o, were
isolated as solid (60ꢀ80%).
Spectral data
N-(2,5-Diphenyl-1H-pyrrol-1-yl)benzamide (7a). FTꢀIR (KBr pellet)
cmꢀ1: 3200 (N–H str.), 1648 (C=O str.), 1483 (C–O–N str.), 1294 (Cꢀ
N str.); 1H NMR (400 MHz, DMSOꢀd6): δ (ppm) 11.61 (bs, 1H, NH,
D2O exchangeable), 8.49 (d, 2H, J = 7.2 Hz, aromaticꢀH), 7.96ꢀ8.01
(m, 3H, aromaticꢀH), 7.72ꢀ7.77 (m, 2H, aromaticꢀH), 7.45ꢀ7.47 (m,
4H, J = 7.2 Hz, aromaticꢀH), 7.37ꢀ7.42 (m, 4H, aromaticꢀH), 6.34 (s,
2H, CH=CHꢀpyrrole); 13C NMR (100 MHz, DMSOꢀd6): δ (ppm)
164.80 (>C=O), 138.86, 135.26, 131.25, 129.94, 128.64, 128.07,
127.97, 127.50, 126.12, 126.66, 126.45, 107.35 (CH=CHꢀpyrrole);
MSꢀEI: m/z 339.2 (M+1); Anal Calcd for C23H18N2O: C, 81.63; H,
5.36; N, 8.29, Found C, 81.65; H, 5.40; N, 8.29.
Step 1b: Typical procedure for the preparation of 2-((4-Methyl-2-
oxo-2H-chromen-7-yl)oxy)acetohydrazide
(3p):
7ꢀHydroxyꢀ4ꢀ
methylꢀ2Hꢀchromenꢀ2ꢀone or 7ꢀhydroxyꢀ2Hꢀchromenꢀ2ꢀone (10
mmol) was dissolved in 100 mL dry acetone. To this, anhydrous
potassium carbonate (15 mmol), followed by ethyl 2ꢀbromoacetate
(15 mmol) was added and refluxed for 12 h. After cooling, the
organic layer was filtered and concentrated in vacuo. The mixture
was diluted with water (40 mL), extracted with CHCl3 (15 mL×3),
dried over anhydrous Na2SO4, concentrated under reduced pressure
and crystallized from methanol to give 2p.
To the solution of ester (2p) in absolute ethanol (100 mL), hydrazine
hydrate (15 mmol, 90%) was added and refluxed for 8ꢀ10 h. On
cooling, the precipitate obtained was filtered and washed with iceꢀ
cold water, dried and recrystallized from methanol as solid crystal
3p (70% yield).
N-(2,5-Diphenyl-1H-pyrrol-1-yl)-2-methylbenzamide (7b). FTꢀIR
1
(KBr pellet) cmꢀ1: 3208 (amide N–H), 1688 (C=O str.); H NMR
(400 MHz, DMSOꢀd6): δ (ppm) 11.01 (bs, 1H, NH, D2O
exchangeable), 7.92 (d, 1H, J = 7.2 Hz, aromaticꢀH), 7.65ꢀ768 (m,
6H, aromaticꢀH), 7.35ꢀ7.39 (m, 5H, aromaticꢀH), 7.20ꢀ7.22 (m, 2H,
aromaticꢀH), 6.42 (s, 2H, CH=CHꢀpyrrole), 2.36 (s, 2H, CH3); 13C
NMR (100 MHz, DMSOꢀd6): δ (ppm) 165.61(>C=O), 138.26,
135.27, 131.25, 129.64, 128.64, 128.07, 127.47, 127.05, 126.03,
126.69, 126.47, 107.48 (CH=CHꢀpyrrole), 20.02 (CH3); MSꢀEI: m/z
353.2 (M+1); Anal. Calcd for C24H20N2O: C, 81.79; H, 5.72, Found
C, 81.80, H, 5.75; N, 7.95.
Step 2: Synthesis of phenacyl bromide: phenacyl bromide was
prepared according to the reported procedure and used for the
preparation of 1,4ꢀdiphenylbutaneꢀ1,4ꢀdione27
N-(2,5-Diphenyl-1H-pyrrol-1-yl)-3-methylbenzamide (7c). FTꢀIR
(KBr pellet) cmꢀ1: 3209 (amide N–H), 1656 (C=O str.), 1584 (CꢀN
1
str.), 1477 (C–O–N str.); H NMR (400 MHz, DMSOꢀd6): δ (ppm)
Synthesis of 1,4-diphenylbutane-1,4-dione (5): To a solution of
11.36 (bs, 1H, NH, D2O exchangeable), 7.51ꢀ7.55 (m, 6H, aromaticꢀ
phenacyl bromide (3 g, 15 mmol) in dry tetrahydrofuran (30 mL),
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H), 7.31ꢀ7.35 (m, 6H, aromaticꢀH), 7.19ꢀ7.22 (m, 2H, aromaticꢀH), 2,4-Dichloro-N-(2,5-diphenyl-1H-pyrrol-1-yl)benzamide (7h). FTꢀ
6.43 (s, 2H, CH=CHꢀpyrrole), 2.28 (s, 2H, CH3); 13C NMR (100 IR (KBr pellet) cmꢀ1: 3208 (amide N–H), 1688 (C=O str.), 1628 (Cꢀ
1
N str.), 1392 (C–O–N str.), 1025 (CꢀCl str.); H NMR (400 MHz,
MHz, DMSOꢀd6): δ (ppm) 165.90 (>C=O), 138.08, 135.36, 132.81,
131.79, 131.64, 128.54, 128.38, 127.85, 127.15, 126.84, 124.25, DMSOꢀd6): δ (ppm) 8.68 (bs, 1H, NH, D2O exchangeable), 7.86ꢀ
107.46 (CH=CHꢀpyrrole), 20.84 (CH3); MSꢀEI: m/z 353.2 (M+1); 7.88 (m, 4H, aromaticꢀH), 7.44ꢀ7.47 (m, 6H, aromaticꢀH), 7.41ꢀ7.42
Anal Calcd for C24H20N2O: C, 81.79; H, 5.72; N, 7.96, found C, (m, 2H, aromaticꢀH), 6.60 (s, 2H, CH=CHꢀpyrrole); 13C NMR (100
81.82, H, 5.77; N, 7.96.
MHz, DMSOꢀd6): δ (ppm) 168.23 (>C=O), 135.74 (CꢀCl), 131.25,
129.04, 128.64, 128.55, 127.42, 127.00, 108.21 (CH=CHꢀpyrrole);
MSꢀEI: m/z 406.1 (M+1); Anal Calcd for C27H20N2O4: C, 67.83;
H,3.96; N, 6.88, Found C, 67.89, H, 3.96; N, 6.89.
N-(2,5-Diphenyl-1H-pyrrol-1-yl)-4-methylbenzamide (7d). FTꢀIR
(KBr pellet) cmꢀ1: 3209 (aromatic C–H str.), 1656 (C=O str.), 1584
1
(CꢀN str.), 1477 (C–O–N str.); H NMR (400 MHz, DMSOꢀd6): δ
(ppm) 8.89 (bs, 1H, NH, D2O exchangeable), 7.82 (d, 2H, J = 7.2 N-(2,5-Diphenyl-1H-pyrrol-1-yl)-2-phenoxyacetamide (7i). FTꢀIR
Hz, aromaticꢀH), 7.59ꢀ7.51 (m, 6H, aromaticꢀH), 7.33 (d, 2H, J = (KBr pellet) cmꢀ1: 3254 (amide N–H), 1693 (C=O str.), 1598 (CꢀN
1
7.2 Hz, aromaticꢀH), 6.95 (d, 4H, J = 6.8 Hz, aromaticꢀH), 6.43 (s,
2H, CH=CHꢀpyrrole), 2.36 (s, 3H, CH3); 13C NMR (100 MHz,
DMSOꢀd6): δ (ppm) 166.08 (>C=O), 140.14, 135.26, 131.25,
str.), 1491 (C–O–N str.); H NMR (400 MHz, DMSOꢀd6): δ (ppm)
8.78 (bs, 1H, NH, D2O exchangeable), 7.46 (d, 4H, J = 8.0 Hz,
aromaticꢀH), 7.32 (t, 4H, J = 8.0 Hz, aromaticꢀH), 7.25ꢀ7.29 (m, 2H,
129.64, 128.64, 128.07, 127.47, 127.05, 126.03, 126.69, 126.47, aromaticꢀH), 7.20 (t, 2H, J = 8.0 Hz, aromaticꢀH), 6.96ꢀ7.00 (m, 1H,
107.38 (CH=CHꢀpyrrole), 21.2 (CH3); MSꢀEI: m/z 353.2 (M+1); aromaticꢀH), 6.70 (d, 2H, J = 8.0 Hz, aromaticꢀH), 6.40 (s, 2H,
Anal. Calcd for C24H20N2O: C, 81.79; H, 5.72; N, 7.76, Found C, CH=CHꢀpyrrole), 4.45 (s, 2H, OCH2); 13C NMR (100 MHz,
81.82, H, 5.87; N, 7.96.
DMSOꢀd6): δ (ppm) 167.88 (>C=O), 156.38, 133.62, 131.15,
129.64, 128.64, 128.07, 127.47, 127.05, 126.03, 126.69, 126.27,
122.83, 106.99 (CH=CHꢀpyrrole), 66.72 (OCH2); MSꢀEI: m/z 369.2
(M+1); Anal. Calcd. for C24H20N2O2: C, 78.24; H, 5.47; N, 7.60,
Found C, 78.26; H, 5.71; N, 7.61.
N-(2,5-Diphenyl-1H-pyrrol-1-yl)isonicotinamide (7e). FTꢀIR (KBr
pellet) cmꢀ1: 3254 (amide NꢀH), 1675 (C=O str.), 1287 (C–N str.);
1H NMR (400 MHz, DMSOꢀd6): δ (ppm) 11.75 (bs, 1H, NH, D2O
exchangeable), 8.58 (d, 2H, J = 2.8 Hz, aromaticꢀH), 7.99ꢀ8.07 (m,
1H, aromaticꢀCH), 7.51 (d, 2H, J = 2.8 Hz, aromaticꢀCH), 7.45 (d, N-(2,5-Diphenyl-1H-pyrrol-1-yl)-4-methoxybenzamide (7j). FTꢀIR
4H, J = 7.2 Hz, aromaticꢀCH), 7.24 (t, 3H, J = 7.2 Hz, aromaticꢀH), (KBr pellet) cmꢀ1: 3182 (amide NꢀH), 1678 (C=O str.), 1300 (CꢀN
1
7.14 (t, 2H, J = 7.2 Hz, aromaticꢀCH), 6.33 (s, 2H, CH=CHꢀ
str.); H NMR (300 MHz, DMSOꢀd6): δ (ppm) 8.27 (bs, 1H, NH,
pyrrole); 13C NMR (100 MHz, DMSOꢀd6): δ (ppm) 164.68 (>C=O), D2O exchangeable), 7.82 (d, 2H, J = 8.7 Hz, aromaticꢀH), 7.59ꢀ7.51
149.94, 138.86, 135.26, 131.25, 129.64, 128.64, 128.07, 127.47, (m, 6H, aromaticꢀH), 7.33 (t, 4H, J = 7.5 Hz, aromaticꢀH), 6.95 (d,
127.05, 126.03, 126.69, 126.47, 120.93, 107.38 (CH=CHꢀpyrrole); 2H, J = 8.7 Hz, aromaticꢀH), 6.42 (s, 2H, CH=CHꢀpyrrole), 3.80 (s,
3H, OCH3); 13C NMR (100 MHz, DMSOꢀd6): δ (ppm) 167.68
MSꢀEI: m/z 340.2 (M+1); Anal. Calcd for C22H17N3O: C, 77.86; H,
5.05; N, 12.38, Found C, 77.84; H, 5.45; N, 12.40.
(>C=O), 133.98, 131.25, 129.64, 128.64, 128.07, 127.47, 127.05,
126.03, 126.89, 126.38, 116.83, 107.22 (CH=CHꢀpyrrole), 58.7;
MSꢀEI: m/z 369.2 (M+1); Anal Calcd for C24H20N2O2: C, 78.24; H,
5.47; N, 7.60, Found C, 78.46; H, 5.77; N, 7.60.
2-Bromo-N-(2,5-diphenyl-1H-pyrrol-1-yl)benzamide (7f). FTꢀIR
(KBr pellet) cmꢀ1: 2947 (amide N–H), 1675 (C=O str.), 1511 (CꢀN
1
str.), 1291 (C–O–N str.); H NMR (400 MHz, DMSOꢀd6): δ (ppm)
11.42 (bs, 1H, NH, D2O exchangeable), 7.56 (d, 4H, J = 7.6 Hz, N-(2,5-Diphenyl-1H-pyrrol-1-yl)-2-phenylacetamide (7k). FTꢀIR
1
aromaticꢀH), 7.50 (t, 1H, J = 7.6 Hz, aromaticꢀH), 7.35 (t, 4H, J = (KBr pellet) cmꢀ1: 3271 (amide NꢀH), 1676 (C=O str.); H NMR
7.6 Hz, aromaticꢀH), 7.22ꢀ7.27 (m, 4H, aromaticꢀH), 6.92 (t, 1H, J =
7.6 Hz, aromaticꢀH), 6.33 (s, 2H, CH=CHꢀpyrrole); 13C NMR (100
(400 MHz, DMSOꢀd6):
exchangeable), 7.89ꢀ7.90 (m, 2H, aromaticꢀH), 7.92ꢀ7.94 (m, 5H,
δ (ppm) 8.17 (bs, 1H, NH, D2O
MHz, DMSOꢀd6): δ (ppm) 166.19 (>C=O), 135.71, 133.00, 131.49, aromaticꢀH), 7.56ꢀ7.52 (m, 4H, aromaticꢀH), 7.28ꢀ7.31 (m, 4H,
131.26, 128.71, 128.01, 127.83, 126.81, 119.42 (CꢀBr), 107.19 aromaticꢀH), 6.47 (s, 2H, CH=CHꢀpyrrole), 3.52 (s, 2H, CH2); 13C
(CH=CHꢀpyrrole); MSꢀEI: m/z 417.2 (M+1), 419.1 (M+2); Anal
Calcd for C23H17BrN2O: C, 66.20; H, 4.11; N, 6.71, Found C, 66.26,
H, 4.17; N, 6.77.
NMR (100 MHz, DMSOꢀd6): δ (ppm) 171.21 (>C=O), 136.22,
133.62, 131.15, 129.64, 128.64, 128.07, 127.55, 127.05, 126.61,
107.57 (CH=CHꢀpyrrole), 41.07 (OCH3); MSꢀEI: m/z 353.2 (M+1);
Anal Calcd for C28H22N2O2: C, 81.79; H, 5.72; N, 7.95, Found C,
81.76; H, 5.78; N, 7.96.
2-Chloro-N-(2,5-diphenyl-1H-pyrrol-1-yl)benzamide (7g). FTꢀIR
(KBr pellet) cmꢀ1: 3208 (amide NꢀH), 1698 (C=O str.), 1618 (CꢀN
str.), 1392 (C–O–N str.), 1020 (CꢀCl str.); 1H NMR (300 MHz,
DMSOꢀd6): δ (ppm) 8.23 (bs, 1H, NH, D2O exchangeable), 7.58 (d, (7l). FTꢀIR (KBr pellet) cmꢀ1: 3233 (aromatic C–H str.), 1669 (C=O
4H, J = 6.9 Hz, aromaticꢀH), 7.38ꢀ7.44 (m, 6H, aromaticꢀH), 7.35 str.), 1601 (CꢀN str.), 1354 (C–O–N str.); 1H NMR (400 MHz,
N-(2,5-Diphenyl-1H-pyrrol-1-yl)-2-(4-isobutylphenyl)propanamide
(d, 2H, J = 3 Hz, aromaticꢀH), 7.20 (d, 2H, J = 3 Hz, aromaticꢀH),
DMSOꢀd6): δ (ppm) 7.49 (bs, 1H, NH, D2O exchangeable), 7.29 (d,
6.45 (s, 2H, CH=CHꢀpyrrole); 13C NMR (100 MHz, DMSOꢀd6): δ 4H, J = 2.7 Hz, aromaticꢀH), 7.17ꢀ7.24 (m, 6H, aromaticꢀH), 6.82
(ppm) 167.08 (>C=O), 134.16 (CꢀCl), 133.76, 131.25, 129.64, (d, 2H, J = 7.8 Hz, aromaticꢀH), 6.71 (d, 2H, J = 7.8 Hz, aromaticꢀ
128.27, 127.41, 127.22, 126.03, 125.99, 125.47, 107.18 (CH=CHꢀ H), 6.25 (s, 2H, CH=CHꢀpyrrole), 3.35 (q, 1H, J = 7.2 Hz, CHꢀ
pyrrole); MSꢀEI: m/z 373.1 (M+1); Anal Calcd for C23H17ClN2O: C, CH3), 2.36 (d, 2H, J = 6.9 Hz, CH2ꢀCH), 1.77 (sep, 1H, J = 6.9 Hz,
74.30; H, 4.62; N, 7.51, Found C, 74.80; H, 4.71; N, 7.53.
CHꢀ(CH3)2), 1.28 (d, 3H, J = 7.2 Hz, CHꢀCH3), 0.82 (d, 6H, J = 6.9
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Hz, CHꢀ(CH3)2); 13C NMR (100 MHz, DMSOꢀd6): δ (ppm) 171.68
(>C=O), 140.12, 134.41, 132.51, 129.42, 128.64, 127.47, 126.03,
126.47, 125.19, 107.8 (CH=CHꢀpyrrole), 45.41 (CHꢀCH3), 44.84
(CH2ꢀCH), 29.05 {CHꢀ(CH3)2}, 22.18 {CHꢀ(CH3)2}, 15.65 (CHꢀ
CH3); MSꢀEI: m/z 423.2 (M+1); Anal. Calcd. for C29H30N2O: C,
82.43; H, 7.16; N, 6.63, Found C, 82.49; H, 7.23; N, 6.63.
101.61, 66.32 (OCH2), 18.09 (coumarin CH3); MSꢀEI: m/z 451.2
(M+1); Anal Calcd for C27H20N2O4: C, 74.30; H, 4.62; N, 6.22,
Found C, 74.36; H, 4.70; N, 6.23.
N-(2-Methyl-5-phenyl-1H-pyrrol-1-yl)benzamide (8a). FTꢀIR (KBr
pellet) cmꢀ1: 3284 (amide N–H), 1662 (C=O str.), 1600 (CꢀN str.),
1278 (C–O–N str.); 1H NMR (400 MHz, CDCl3): δ (ppm) 11.12 (bs,
1H, NH), 7.86 (d, 2H, J = 7.2 Hz, aromaticꢀH), 7.48 (t, 1H, J = 7.6
N-(2,5-Diphenyl-1H-pyrrol-1-yl)-2-(naphthalen-1-yl)acetamide
(7m). FTꢀIR (KBr pellet) cmꢀ1: 3218 (amide NꢀH), 1686 (C=O str.), Hz, aromaticꢀH), 7.37ꢀ7.42 (m, 4H, aromaticꢀH), 7.22 (t, 1H, J = 7.6
1628 (CꢀN str.), 1390 (C–O–N str.); H NMR (400 MHz, DMSOꢀ Hz, aromaticꢀH), 7.11 (t, 1H, J = 7.2 Hz, aromaticꢀH), 6.18 (d, 1H, J
1
= 3.6 Hz, pyrroleꢀH), 5.93 (d, 1H, J = 3.6 Hz, pyrroleꢀH), 2.14 (s,
3H, CH3); 13C NMR (100 MHz, CDCl3): δ (ppm) 166.50 (>C=O),
d6): δ (ppm) 10.96 (bs, 1H, NH, D2O exchangeable), 8.16 (m, 2H,
aromaticꢀH), 7.92 (m, 5H, aromaticꢀH), 7.83ꢀ7.87 (m, 5H, aromaticꢀ
H), 7.54ꢀ7.48 (m, 4H, aromaticꢀH), 7.18 (d, 1H, aromaticꢀH), 6.38 132.28, 131.94, 131.72, 130.73, 128.13, 127.95, 127.31, 126.62,
(s, 2H, CH=CHꢀpyrrole), 4.13 (s, 2H, CH2); 13C NMR (100 MHz, 125.94, 105.86, 104.88, 102.99, 10.80; ESIMS: m/z 277.2 (M+1);
Anal. Calcd for C18H16N2O: C, 78.24; H, 5.84; N, 10.14, Found C,
78.26; H, 5.94; N, 10.15.
DMSOꢀd6): δ (ppm) 168.81 (>C=O), 134.86, 133.15, 129.14,
128.34, 127.47, 126.11, 126.29, 125.22, 124.89, 107.10 (CH=CHꢀ
pyrrole), 41.56 (OCH2); MSꢀEI: m/z 403.1 (M+1); Anal Calcd for
C28H22N2O: C, 83.56; H, 5.51; N, 6.96, Found C, 83.59; H, 5.59; N,
6.97.
2-Methyl-N-(2-methyl-5-phenyl-1H-pyrrol-1-yl)benzamide (8b). FTꢀ
IR (KBr pellet) cmꢀ1: 3246 (amide N–H), 1658 (C=O str.), 1597 (Cꢀ
1
N str.), 1392 (C–O–N str.); H NMR (400 MHz, CDCl3): δ (ppm)
N-(2,5-Diphenyl-1H-pyrrol-1-yl)-2-(naphthalen-2-yloxy)acetamide
9.10 (bs, 1H, NH), 8.18ꢀ8.20 (m, 2H, aromaticꢀH), 7.87ꢀ7.89 (m,
(7n). FTꢀIR (KBr pellet) cmꢀ1: 3228 (amide NꢀH), 1690 (C=O str.), 3H, aromaticꢀH), 7.23ꢀ7.27 (m, 2H, aromaticꢀH), 7.15ꢀ7.16 (m, 2H,
1
1612 (CꢀN str.), 1389 (C–O–N str.); H NMR (400 MHz, DMSOꢀ aromaticꢀH), 6.23 (d, 1H, J = 3.6 Hz, pyrroleꢀH), 5.97 (d, 1H, J =
3.6 Hz, pyrroleꢀH), 2.38 (s, 3H, CH3), 2.16 (s, 3H, CH3); 13C NMR
d6): δ (ppm) 10.82 (bs, 1H, NH, D2O exchangeable), 8.16 (m, 3H,
(100 MHz, CDCl3): δ (ppm) 166.61 (>C=O), 142.67, 132.71,
aromaticꢀH), 7.92 (m, 4H, aromaticꢀH), 7.83 (m, 3H, aromaticꢀH),
7.57ꢀ7.44 (m, 5H, aromaticꢀH), 7.36 (m, 2H, aromaticꢀH), 6.40 (s, 131.61, 131.0, 129.04, 128.31, 127.94, 127.08, 126.94, 126.17,
2H, CH=CHꢀpyrrole), 4.65 (s, 2H, CH2); 13C NMR (100 MHz, 106.26, 105.13, 21.09, 10.81; ESIMS: m/z 291.2 (M+1); Anal Calcd
for C19H18N2O: C, 78.59; H, 6.25; N, 9.65, Found C, 78.62; H, 6.29;
N, 9.64.
DMSOꢀd6): δ (ppm) 168.08 (>C=O), 157.10, 133.26, 131.25,
132.04, 129.64, 128.64, 128.07, 127.47, 127.05, 126.03, 126.69,
126.47, 120.93, 118.29, 107.68 (CH=CHꢀpyrrole), 105.11, 67.23
(OCH2); MSꢀEI: m/z 419.2 (M+1); Anal Calcd for C28H22N2O2: C,
80.36; H, 5.30; N, 6.69, Found C, 80.45; H, 5.76, N, 6.69.
3-Methyl-N-(2-methyl-5-phenyl-1H-pyrrol-1-yl)benzamide (8c). FTꢀ
IR (KBr pellet) cmꢀ1: 3246 (amide N–H), 1657 (C=O str.), 1599 (Cꢀ
1
N str.), 1285 (C–O–N str.); H NMR (400 MHz, CDCl3): δ (ppm)
N-(2,5-Diphenyl-1H-pyrrol-1-yl)-2-(o-tolyl)acetamide (7o). FTꢀIR 8.72 (bs, 1H, NH), 7.89ꢀ7.90 (m, 2H, aromaticꢀH), 7.83 (s, 1H,
1
(KBr pellet) cmꢀ1: 3260 (amide N–H), 1675 (C=O str.); H NMR aromaticꢀH), 7.74ꢀ7.79 (m, 4H, aromaticꢀH), 7.20ꢀ7.21 (m, 2H,
aromaticꢀH), 6.23 (d, 1H, J = 3.6 Hz, pyrroleꢀH), 5.97 (d, 1H, J =
3.6 Hz, pyrroleꢀH), 2.38 (s, 3H, CH3), 2.14 (s, 3H, CH3); 13C NMR
(400 MHz, DMSOꢀd6): δ (ppm) 11.27 (bs, 1H, NH, D2O
exchangeable), 7.46 (d, 4H, J = 7.2 Hz, aromaticꢀH), 7.33ꢀ7.35 (m,
4H, aromaticꢀH), 7.06ꢀ7.16 (m, 5H, aromaticꢀH), 6.95ꢀ6.99 (m, 1H, (100 MHz, CDCl3): δ (ppm) 166.80 (>C=O), 140.66, 133.21,
aromaticꢀH), 6.34 (s, 2H, CH=CHꢀpyrrole), 3.45 (s, 2H, CH2), 131.13, 130.9, 129.00, 128.01, 127.99, 127.00, 126.49, 126.17,
2.25(s, 3H, CH3); 13C NMR (100 MHz, DMSOꢀd6): δ (ppm) 169.07
106.60, 105.30, 21.07, 10.79; ESIMS: m/z 291.14 (M+1); Anal.
Calcd for C19H18N2O: C, 78.59; H, 6.25; N, 9.65, Found C, 78.54; H,
6.28; N, 9.64.
(>C=O), 138.86, 135.26, 133.25, 129.64, 128.64, 128.07, 127.47,
127.05, 126.03, 126.69, 126.47, 106.48 (CH=CHꢀpyrrole), 39.5
(CH2), 18.94 (CH3); MSꢀEI: m/z 367.2 (M+1); Anal. Calcd. for
C25H22N2O: C, 81.94; H, 6.05; N, 7.64, Found C, 81.95; H, 6.11; N,
7.66.
4-Methyl-N-(2-methyl-5-phenyl-1H-pyrrol-1-yl)benzamide (8d). FTꢀ
IR (KBr pellet) cmꢀ1: 3236 (amide N–H), 1660 (C=O str.), 1605 (Cꢀ
1
N str.), 1297 (C–O–N str.); H NMR (400 MHz, CDCl3): δ (ppm)
N-(2,5-Diphenyl-1H-pyrrol-1-yl)-2-((4-methyl-2-oxo-2H-chromen-7- 8.72 (bs, 1H, NH), 7.58 (d, J = 8.0 Hz, 2H, aromaticꢀH), 7.36 (d,
yl)oxy)acetamide (7p). FTꢀIR (KBr pellet) cmꢀ1: 3216 (amide N–H), 2H, J = 8.0 Hz, aromaticꢀH), 7.23ꢀ7.27 (m, 2H, aromaticꢀH), 7.16ꢀ
1727 (C=O str.), 1689 (C=O str.), 1613 (CꢀN str.), 1391 (C–O–N 7.20 (m, 3H, aromaticꢀH), 6.23 (d, 1H, J = 3.6 Hz, pyrroleꢀH), 5.97
1
(d, 1H, J = 3.6 Hz, pyrroleꢀH), 2.37 (s, 3H, CH3), 2.12 (s, 3H, CH3);
str.); H NMR (400 MHz, DMSOꢀd6): δ (ppm) 11.54 (bs, 1H, NH,
D2O exchangeable), 7.57 (d, 4H, J = 8.4 Hz, coumarinꢀCH), 7.47 (d, 13C NMR (100 MHz, CDCl3): δ (ppm) 166.58 (>C=O), 142.67,
4H, J = 6.8 Hz, aromaticꢀH), 7.30 (t, 4H, J = 6.8 Hz, aromaticꢀH), 132.71, 131.61, 131.0, 129.04, 128.31, 127.94, 127.08, 126.94,
7.21 (d, 2H, J = 6.8 Hz, aromaticꢀH), 6.83 (d, 1H, J = 8.4 Hz, 126.17, 106.26, 105.13, 21.09, 10.81; ESIMS: m/z 291.2 (M+1);
coumarinꢀCH), 6.75 (s, 1H, coumarinꢀCH), 6.37 (s, 2H, CH=CHꢀ Anal Calcd for C19H18N2O: C, 78.59; H, 6.25; N, 9.65, Found C,
pyrrole), 6.21 (s, 1H, coumarinꢀCH), 4.62 (s, 2H, OCH2), 2.37 (s, 78.55; H, 6.27; N, 9.65.
3H, CH3); 13C NMR (100 MHz, DMSOꢀd6): δ (ppm) 166.77
N-(2-Methyl-5-phenyl-1H-pyrrol-1-yl)isonicotinamide (8e). FTꢀIR
(>C=O), 160.22 (>C=O), 154.35, 153.19, 135.22, 131.33, 128.86,
(KBr pellet) cmꢀ1: 3226 (amide N–H), 1665 (C=O str.), 1590 (CꢀN
126.78, 126.38, 113.85, 112.26, 111.59, 107.51 (CH=CHꢀpyrrole),
1
str.), 1276 (C–O–N str.); H NMR (400 MHz, CDCl3): δ (ppm)
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10.25 (bs, 1H, NH), 8.44 (2H, d, J = 5.6 Hz, aromaticꢀH), 7.40 (2H, 4-Methoxy-N-(2-methyl-5-phenyl-1H-pyrrol-1-yl)benzamide
d, J = 5.6Hz, aromaticꢀH), 6.14 (d, 1H, J = 3.2 Hz, pyrroleꢀH), 5.94 FTꢀIR (KBr pellet) cmꢀ1: 3211 (amide N–H), 1652 (C=O str.), 1604
(d, 1H, J = 3.2 Hz, pyrroleꢀH), 2.02 (s, 3H, CH3); 13C NMR (100
(8j).
(CꢀN str.), 1283 (C–O–N str.); 1H NMR (400 MHz, CDCl3): δ (ppm)
MHz, CDCl3): δ (ppm) 165.12 (>C=O), 149.92, 138.54, 132.52, 10.91 (bs, 1H, NH), 7.84 (d, 2H, J = 8.4 Hz, aromaticꢀH), 7.39 (d,
131.34, 130.46, 128.03, 126.79, 126.41, 120.80, 106.60, 105.55, 2H, J = 7.2 Hz, aromaticꢀH), 7.19 (t, 2H, J = 7.2 Hz, aromaticꢀH),
10.68; ESIMS: m/z 278.2 (M+1); Anal Calcd for C17H15N3O: C, 7.08 (d, 1H, J = 7.2 Hz, aromaticꢀH), 6.85 (d, 2H, J = 8.4 Hz,
73.63; H, 5.45; N, 15.15, Found C, 73.66; H, 5.65; N, 15.20.
aromaticꢀH), 6.15 (d, 1H, J = 3.2 Hz, pyrroleꢀH), 5.90 (d, 1H, J =
3.2 Hz, pyrroleꢀH), 3.76 (s, 3H, OCH3), 2.11 (s, 3H, CH3); 13C NMR
(100 MHz, CDCl3): δ (ppm) 160.38 (>C=O), 157.21, 127.31,
126.39, 125.86, 124.25, 122.92, 121.60, 120.85, 118.89, 108.32,
100.79, 99.80, 49.98, 5.97; ESIMS: m/z 307.2 (M+1); Anal Calcd
for C19H18N2O2: C, 74.49; H, 5.92; N, 9.14, Found C, 74.49; H, 5.98;
N, 9.15.
2-Bromo-N-(2-methyl-5-phenyl-1H-pyrrol-1-yl)benzamide (8f). FTꢀ
IR (KBr pellet) cmꢀ1: 3218 (amide N–H), 1672 (C=O str.), 1596 (Cꢀ
1
N str.), 1292 (C–O–N str.), 748 (CꢀBr str.); H NMR (400 MHz,
CDCl3): δ (ppm) 8.19 (bs, 1H, NH), 7.56ꢀ7.59 (m, 1H, aromaticꢀH),
7.38 (d, 2H, J = 7.2 Hz, aromaticꢀH), 7.24ꢀ7.35 (m, 6H, aromaticꢀ
H), 6.19 (d, 1H, J = 4.0 Hz, pyrroleꢀH), 5.99 (d, 1H, J = 3.2 Hz,
pyrroleꢀH), 2.28 (s, 3H, CH3); 13C NMR (100 MHz, CDCl3): δ
(ppm) 166.2 (>C=O), 134.41, 133.22, 131.69, 131.42, 130.89,
N-(2-Methyl-5-phenyl-1H-pyrrol-1-yl)-2-phenylacetamide (8k). FTꢀ
IR (KBr pellet) cmꢀ1: 3244 (amide N–H), 1670 (C=O str.), 1600 (Cꢀ
1
129.14, 127.96, 127.72, 127.11, 126.59, 119.25, 106.5, 105.39, 10.2; N str.), 1348 (C–O–N str.); H NMR (400 MHz, CDCl3): δ (ppm)
ESIMS: m/z 355.2, 357 (M+1, M+2); Anal Calcd for C18H15BrN2O: 8.17 (bs, 1H, NH), 7.24ꢀ7.38 (m, 6H, aromaticꢀH), 7.12ꢀ7.19 (m,
C, 60.86; H, 4.26; N, 7.89, Found C, 60.88; H, 4.31; N, 7.89.
4H, aromaticꢀH), 6.14 (d, 1H, J = 3.6 Hz, pyrroleꢀH), 5.89 (d, 1H, J
= 3.6 Hz, pyrroleꢀH), 3.52 (s, 2H, CH2), 2.01 (s, 3H, CH3); 13C
NMR (100 MHz, CDCl3): δ (ppm) 169.97 (>C=O), 132.99, 130.47,
128.92, 128.85, 127.86, 126.13, 126.5, 106.79, 105.6, 41.0, 10.1;
ESIMS: m/z 291.1 (M+1); Anal. Calcd for C19H18N2O: C, 78.59; H,
5.92; N, 9.65, Found C, 78.62; H, 5.92; N, 9.65.
2-Chloro-N-(2-methyl-5-phenyl-1H-pyrrol-1-yl)benzamide (8g). FTꢀ
IR (KBr pellet) cmꢀ1: 3210 (amide N–H), 1651 (C=O str.), 1603 (Cꢀ
1
N str.), 1282 (C–O–N str.), 1027 (CꢀCl str.); H NMR (400 MHz,
CDCl3): δ (ppm) 11.01 (bs, 1H, NH), 7.86 (d, J = 7.2 Hz, 2H,
aromaticꢀH), 7.42 (d, 2H, J = 6.4 Hz, aromaticꢀH), 7.22 (s, 2H,
aromaticꢀH), 7.12 (d, 1H, J = 6.0 Hz, aromaticꢀH), 6.88 (d, 2H, 2-(4-Isobutylphenyl)-N-(2-methyl-5-phenyl-1H-pyrrol-1-
J=7.2 Hz, aromaticꢀH), 6.17 (s, 1H, pyrroleꢀH), 5.92 (s, 1H, pyrroleꢀ yl)propanamide (8l). FTꢀIR (KBr pellet) cmꢀ1: 3227 (amide N–H),
1666 (C=O str.), 1591 (CꢀN str.), 1281 (C–O–N str.); 1H NMR (400
H), 2.12 (s, 3H, CH3); 13C NMR (100 MHz, CDCl3): δ (ppm) 165.92
(>C=O), 162.23, 132.27, 131.99, 130.83, 129.26, 127.95, 126.56, MHz, CDCl3): δ (ppm) 8.61 (bs, 1H, NH), 7.83ꢀ7.84 (m, 2H,
125.87, 123.85, 113.35, 105.78, 104.80, 10.98; ESIMS: m/z 311 aromaticꢀH), 7.34ꢀ7.36 (m, 3H, aromaticꢀH), 7.28 (d, 2H, J = 8.0
(M+1), 312 (M+2); Anal. Calcd for C19H18N2O: C, 69.57; H, 4.86; Hz, aromaticꢀH), 7.11 (d, 2H, J = 8.0 Hz, aromaticꢀH), 6.05 (d, 1H,
N, 9.01, Found C, 69.58; H, 4.84; N, 9.00.
J = 3.6 Hz, pyrroleꢀH), 5.90 (d, 1H, J = 3.6 Hz, pyrroleꢀH), 3.58 (q,
1H, J = 6.0 Hz, CHꢀCH3), 2.44 (d, 2H, J = 6.8 Hz, CHꢀCH2ꢀPh),
2.12 (s, 3H, CH3), 1.85 (d, 1H, J = 6.0 Hz, CH), 1.48 (d, 3H, J = 9.6
Hz, CH3), 0.90 (d, 6H, J = 6.0 Hz, CHꢀ(CH3)2); 13C NMR (100
MHz, CDCl3): δ (ppm) 176.11 (>C=O), 141.71, 134.69, 132.3,
131.22, 130.16, 129.60, 127.08, 107.13 (CH near the Ph of pyrrole),
106.72 (CH near the methyl of pyrrole), 45.40 (CHꢀCH3), 39.98
(CH2ꢀCH), 29.15 [CHꢀ(CH3)2], 22.12 [CHꢀ(CH3)2], 17.41 (CHꢀCH3),
10.72 (CH3); ESIMS: m/z 360.3 (M+1); Anal. Calcd for C24H28N2O:
C, 79.96; H, 7.83; N, 7.77, Found C, 79.98, H, 7.87; N, 7.78.
2,4-Dichloro-N-(2-methyl-5-phenyl-1H-pyrrol-1-yl)benzamide (8h).
FTꢀIR (KBr pellet) cmꢀ1: 3216 (amide N–H), 1672 (C=O str.), 1584
(CꢀN str.), 1290 (C–O–N str.), 1048 (CꢀCl str.); 1H NMR (400 MHz,
CDCl3): δ (ppm) 8.47 (s, 1H, NH), 7.30ꢀ7.35 (s, 1H, aromaticꢀH),
7.20ꢀ7.25 (m, 2H, J = 8.0 Hz, aromaticꢀH), 6.19 (d, 1H, J = 3.6 Hz,
pyrroleꢀH), 5.88 (d, 1H, J = 3.6 Hz, pyrroleꢀH), 2.20 (s, 3H, CH3);
13C NMR (100 MHz, CDCl3): δ (ppm) 164.58 (>C=O), 137.30,
132.87, 131.62, 131.39, 130.63, 130.46, 130.40, 129.79, 127.88,
127.50, 127.09, 126.54, 106.53, 105.45, 11.02; ESIMS: m/z 345.0,
346 (M+1, M+2); Anal. Calcd for C18H14Cl2N2O: C, 62.62; H, 4.09;
N, 8.11, Found C, 62.71; H, 4.10; N, 8.11.
N-(2-Methyl-5-phenyl-1H-pyrrol-1-yl)-2-(naphthalen-1-yl)acetamide
(8m). FTꢀIR (KBr pellet) cmꢀ1: 3211 (amide N–H), 1665 (C=O str.),
1
1596 (CꢀN str.), 1317 (C–O–N str.); H NMR (400 MHz, CDCl3): δ
N-(2-Methyl-5-phenyl-1H-pyrrol-1-yl)-2-phenoxyacetamide (8i). FTꢀ
IR (KBr pellet) cmꢀ1: 3187 (amide N–H), 1685 (C=O str.), 1596 (Cꢀ
N str.), 1233 (C–O–N str.), 1079 (CꢀOꢀC str.); H NMR (400 MHz,
(ppm) 8.19 (bs, 1H, NH), 7.57–7.45 (m, 4H, aromaticꢀH), 7.31ꢀ7.32
(m, 2H, aromaticꢀH), 7.06ꢀ7.20 (m, 6H, aromaticꢀH), 6.13 (d, 1H, J
= 3.6 Hz, pyrroleꢀH), 5.90 (d, 1H, J = 3.6 Hz, pyrroleꢀH), 4.11 (s,
2H, CH2), 2.11 (s, 3H, CH3); 13C NMR (100 MHz, CDCl3): δ (ppm)
169.12 (>C=O), 136.76, 132.69, 131.22, 130.6, 130.3, 129.16,
127.53, 126.84, 106.09, 105.21, 41.5, 10.8; ESIMS: m/z 341.2
(M+1); Anal. Calcd for C23H20N2O: C, 81.15; H, 5.92; N, 8.23,
Found C, 81.19, H, 5.96; N, 8.23.
1
CDCl3): δ (ppm) 8.85 (bs, 1H, NH), 7.25ꢀ7.36 (m, 7H, aromaticꢀH),
7.04 (t, 1H, J = 7.2 Hz, aromaticꢀH), 6.85 (d, 2H, J = 8.0 Hz,
aromaticꢀH), 6.24 (d, 1H, J = 3.6 Hz, pyrroleꢀH), 6.02 (d, 1H, J =
3.6 Hz, pyrroleꢀH), 4.60 (s, 2H, OCH2), 2.14 (s, 3H, CH3); 13C NMR
(100 MHz, CDCl3): δ (ppm) 167.23(>C=O), 156.37, 132.81, 131.31,
130.56, 129.41, 128.05, 127.09, 126.46, 122.06, 114.16, 106.68,
105.51, 66.51, 10.88; ESIMS: m/z 306.2 (M+1); Anal Calcd for
C19H18N2O2: C, 74.49; H, 5.92; N, 9.14, Found C, 74.59; H, 5.97; N,
9.15.
N-(2-Methyl-5-phenyl-1H-pyrrol-1-yl)-2-(naphthalen-2-
yloxy)acetamide (8n). FTꢀIR (KBr pellet) cmꢀ1: 3245 (amide N–H),
1692 (C=O str.), 1628 (CꢀN str.), 1394 (C–O–N str.), 1063 (CꢀOꢀC
1
str.); H NMR (400 MHz, CDCl3): δ (ppm) 11.02 (bs, 1H, NH),
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7.29ꢀ7.36 (m, 6H, aromaticꢀH), 7.24ꢀ7.25 (m, 4H, aromaticꢀH), inhibited and was calculated from the dose response curve at zero
7.03ꢀ7.08 (m, 2H, aromaticꢀH), 6.45 (d, 1H, J = 3.6 Hz, pyrroleꢀH), growth.
5.88 (d, 1H, J = 3.6 Hz, pyrroleꢀH), 4.66 (s, 2H, OCH2), 2.12 (s, 3H, % inhibition = [1 ꢀ (growth index of test sample/growth index of
CH3); 13C NMR (100 MHz, CDCl3): δ (ppm) 170.11 (>C=O),
control)] × 100.
156.09, 136.12, 131.22, 130.43, 129.29, 127.13, 126.64, 118.19,
Intracellular activity assay
106.3 (CH near the methyl of phenyl), 105.12 (CH near the methyl
of pyrrole), 70.01 (OCH2), 10.31 (CH3); ESIMS: m/z 357.2 (M+1);
Anal. Calcd for C23H20N2O2: C, 77.51; H, 5.66; N, 7.86, Found C,
77.55, H, 5.68; N, 7.85.
Murine J774 macrophages were infected with a luminescent strain of
H37Rv (which constitutively expresses luxABCDE) at a multiplicity
of infection of 1. After 18 h, extracellular bacteria were removed by
washing and subsequently test and positive control (INH) were
added. Infected macrophages were incubated in the presence of
compound for 4 days at 1 and 10 times of MIC (as determined in
aerobic culture in liquid medium). Bacteria were harvested from
macrophages by lysis with 0.1% SDS, inoculated into growth media
and allowed to grow aerobically for 5 days, where the amount of
bacteria present was determined by luminescence. All assays were
conducted in triplicate; each assay included a positive control (INH)
and a negative control (DMSO). The baseline of infection was
determined by harvesting bacteria from macrophages at 0 day before
compound addition and plating for CFU determination in triplicate.
The intracellular activity = [Log CFU Day 4 Compound] ꢀ [Log
CFU Day 4 DMSO]
N-(2-Methyl-5-phenyl-1H-pyrrol-1-yl)-2-(o-tolyl)acetamide
(8o).
FTꢀIR (KBr pellet) cmꢀ1: 3249 (amide N–H), 1674 (C=O str.), 1601
(CꢀN str.), 1348 (C–O–N str.); 1H NMR (400 MHz, CDCl3): δ (ppm)
7.54 (bs, 1H, NH), 7.06ꢀ7.36 (m, 7H, aromaticꢀH), 7.02 (t, 2H, J =
8.0 Hz, aromaticꢀH), 6.14 (d, 1H, J = 3.6 Hz, pyrroleꢀH), 5.92 (d,
1H, J = 3.6 Hz, pyrroleꢀH), 3.66 (s, 2H, OCH2), 3.16 (s, 3H, CH3),
2.09 (s, 3H, CH3); 13C NMR (100 MHz, CDCl3): δ (ppm) 169.07
(>C=O), 136.76, 132.69, 131.3, 131.22, 130.61, 130.37, 129.94,
129.61, 127.3, 126.41, 106.34, 105.2, 39.5 (CH2), 18.94 (CH3),
10.78 (CH3); ESIMS: m/z 305.2 (M+1); Anal. Calcd for C20H20N2O:
C, 78.92; H, 6.62; N, 9.20, Found C, 78.95; H, 6.68; N, 9.20.
2-((4-Methyl-2-oxo-2H-chromen-7-yl)oxy)-N-(2-methyl-5-phenyl-
1H-pyrrol-1-yl)acetamide (8p). FTꢀIR (KBr pellet) cmꢀ1: 3208
(amide N–H ), 1711(C=O str.), 1682 (C=O str.), 1612 (CꢀN str.),
Activity against resistant isolates
1
The activity of compounds against five resistant isolates of Mtb
strains under aerobic conditions was assessed by determining the
minimum inhibitory concentration (MIC) of compounds. Strains
tested were two isoniazid resistant strains (INHꢀR1 and INHꢀR2),
two rifampicin resistant strains (RIFꢀR1 and RIFꢀR2) and a
fluoroquinolone resistant strain (FQꢀR1). The assay is based on
measurement of growth in liquid medium of each strain where the
readout is optical density (OD).
INHꢀR1 was derived from H37Rv and is a katG mutant (Y155* =
truncation). INHꢀR2 is strain ATCC35822. RIFꢀR1 was derived
from H37Rv and is an rpoB mutant (S522L). RIFꢀR2 is strain
ATCC35828. FQꢀR1 is a fluoroquinoloneꢀresistant strain derived
from H37Rv and has an unidentified mutation.
1391 (C–O–N str.); H NMR (400 MHz, CDCl3): δ (ppm) 8.87 (bs,
1H, NH), 7.76 (1H, d, J = 8.4 Hz, aromaticꢀH), 7.06ꢀ7.36 (m, 7H,
aromaticꢀH), 7.02 (t, 2H, J = 8.4 Hz, aromaticꢀH), 6.99 (1H, dd, J =
8.4 Hz, J = 2.0 Hz, aromaticꢀH), 6.08 (d, 1H, J = 3.6 Hz, pyrroleꢀH),
6.08 (s, 1H, coumarinꢀH), 5.93 (d, 1H, J = 3.6 Hz, pyrroleꢀH), 4.70
(s, 2H, OCH2), 2.28 (s, 3H, CH3), 2.10 (s, 3H, CH3); 13C NMR (100
MHz, CDCl3): δ (ppm) 170.21 (>C=O), 160.89 (C=O of coumarin),
159.09 (CꢀO), 131.76, 130.46, 129.09, 127.78, 117.44, 108.03 (CH
near the Ph of pyrrole), 106.21 (CH near the methyl of pyrrole),
104.73, 68.20 (OCH2), 19.53 (coumarin CH3), 10.18 (pyrrole CH3);
ESIMS: m/z 389.2 (M+1); Anal. Calcd for C23H20N2O4: C, 71.12; H,
5.19; N, 7.21, Found C, 71.16; H, 5.25; N, 7.22.
4.2. Antitubercular activity
All the synthesized compounds were tested through in vitro against
Mycobacterium tuberculosis H37Rv (Mtb H37Rv) in
The MIC of compound was determined by measuring bacterial
growth after 5 days in the presence of compounds. Compounds were
prepared as 10ꢀpoint twoꢀfold serial dilutions in DMSO and diluted
into 7H9ꢀTwꢀOADC medium in 96ꢀwell plates with a final DMSO
concentration. Plates were inoculated with Mtb and incubated for 5
days; growth was measured by OD590. To calculate the MIC, the 10ꢀ
point dose response curve was plotted as % growth and fitted to the
Gompertz model using GraphPad Prism 5. In addition dose response
curves were generated using the LevenbergꢀMarquardt algorithm
and the concentrations that resulted in 50% and 90% inhibition of
growth were determined (IC50 and IC90 respectively).
a high
throughput screen using procedure adapted from the microplate
alamar blue assay. The experiment was carried out in 96ꢀwell
microplates 7H9ꢀTweenꢀOADC medium by measuring bacterial
growth after 5 days in the presence of test compounds. Test
compounds were prepared as tenꢀfold serial dilutions in DMSO.
Compounds 7a–p & 8a-p and reference drugs were dissolved in
DMSO at a concentration of 5 mg/mL and refrigerated until used.
Plates were inoculated with Mtb H37Rv ATCC 27294, covered and
o
sealed with parafilm and incubated at 37 C for five days. Testing
was conducted in duplicate and the following controls were taken i)
medium only (sterility control); ii) organism in medium (negative
control); and iii) reference compounds (positive control). The growth
was measured by OD590 and fluorescence (Ex 560/Em 590) using a
BioTek™ Synergy 4 plate reader. To calculate the MIC, the 10ꢀpoint
dose response curve was plotted as % growth and fitted to the
Gompertz model using GraphPad Prism 5. The MIC was defined as
the minimum concentration at which growth was completely
4.3. Evaluation of Cytotoxicity
A drug cytotoxicity control plate assay (MTT cell proliferation) was
conducted using Vero African green monkey kidney cell line to
confirm that concentrations utilized for testing were not toxic to that
cell line. The cells were plated in flatꢀbottom 96ꢀwell plates cultured
for 72 h in a controlled atmosphere and nonꢀadherent cells were
washed by gentle flushing with RPMI 1640 supplemented with 10%
fetal bovine serum. Compounds were prepared as 10ꢀpoint threeꢀfold
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serial dilutions in DMSO. The highest concentration of compound
tested was 100 ꢀg/mL where compounds were soluble in DMSO.
The plates were incubated for additional 2 days after addition of test
compounds and MTT reagent was added. The cell viability was
measured on the basis of absorbance in each well and using positive
control drug and medium as blank. Each plate included staurosporine
as a control.
Notes and references
1. D. J. Payne, M. N. Gwynn, D. J. Holmes and D. L. Pompliano,
Nature Reviews. Drug Discovery, 2007, 6, 29ꢀ40.
2. M. Berry and O. M. Kon, Eur. Respir. Rev., 2009, 18, 195ꢀ197.
3. J. C. Palomino and A. Martin, J. Antimicrob. Chemother., 2013,
68, 275ꢀ283.
4. F. W. Muregi and A. Ishih, Drug Dev Res, 2010, 71, 20ꢀ32.
5. A. Imramovsky, S. Polanc, J. Vinsova, M. Kocevar, J. Jampilek,
Z. Reckova and J. Kaustova, Bioorg. Med. Chem., 2007, 15,
2551ꢀ2559.
6. A. V. Shindikar and C. L. Viswanathan, Bioorg. Med. Chem.
Lett., 2005, 15, 1803ꢀ1806.
7. U. Pandit and A. Dodiya, Medicinal Chemistry Research, 2013,
22, 3364ꢀ3371.
8. V. Padmavathi, C. Prema Kumari, B. C. Venkatesh and A.
Padmaja, Eur. J. Med. Chem., 2011, 46, 5317ꢀ5326.
9. K. M. Hilmy, M. M. Khalifa, M. A. Hawata, R. M. Keshk and A.
A. elꢀTorgman, Eur. J. Med. Chem., 2010, 45, 5243ꢀ5250.
10. M. Biava, G. C. Porretta, G. Poce, A. De Logu, R. Meleddu, E.
De Rossi, F. Manetti and M. Botta, Eur. J. Med. Chem., 2009,
44, 4734ꢀ4738.
11. S. D. Joshi, H. M. Vagdevi, V. P. Vaidya and G. S.
Gadaginamath, Eur. J. Med. Chem., 2008, 43, 1989ꢀ1996.
12. A. Kamal, S. Prabhakar, M. Janaki Ramaiah, P. Venkat Reddy,
C. Ratna Reddy, A. Mallareddy, N. Shankaraiah, T. Lakshmi
Narayan Reddy, S. N. Pushpavalli and M. PalꢀBhadra, Eur. J.
Med. Chem., 2011, 46, 3820ꢀ3831.
4.4. Computational work
The protein crystal structure of Mtb ENR (InhA) (PDB ID 2H7M)
with coꢀcrystallized ligand was downloaded from protein data bank.
The protein was prepared using the “protein preparation tool” of
Schrödinger. After adding hydrogen atoms, bond orders and formal
charges were checked following which minimization was performed
with the rootꢀmeanꢀsquareꢀdisplacement (RMSD) cutoff of 0.30Å. A
grid file was created surrounding the area in the cavity that contains
coꢀcrystallized
ligand
[Nꢀ(3,5ꢀdichlorophenyl)ꢀ5ꢀoxoꢀ1ꢀ
phenylpyrrolidineꢀ3ꢀcarboxamide] and the grid was defined as an
enclosing box with 15Å in all three dimensions. Three dimensional
coordinates of the all the ligand (7a-p and 8a-p), their isomeric,
ionization and tautomeric states were generated using the LigPrep
module of Maestro 9.4 (LigPrep with default setting, Schrödinger).
The poses were selected based on hydrophobic, hydrogen bond and
chargeꢀcharge interactions. Special care was also taken to identify
poses that may involve binding modes of the molecules in the
hydrophobic cavity. The cutoff distance for hydrogen bond, van der
waals interaction, and electrostatic interaction is 3.20 Å (angle >90ꢀ
180°), 4.20 Å, and 4.00 Å respectively.
13. X. M. Peng, G. L. Damu and C. Zhou, Curr Pharm Des, 2013,
19, 3884ꢀ3930.
14. H. Li, X. Wang, G. Xu, L. Zeng, K. Cheng, P. Gao, Q. Sun, W.
Liao and J. Zhang, Bioorg. Med. Chem. Lett., 2014, DOI:
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Biol. Drug Des., 2012, 80, 929ꢀ936.
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1997, 41, 1004ꢀ1009.
Prediction of pharmacokinetic property (ADME)
Pharmacokinetic properties of title compounds (7a-p and 8a-p) were
calculated using Molinspiration online property calculation program.
Polar surface area (TPSA), miLogP, number of rotatable bonds (nꢀ
ROTB), molecular volume (MV), number of hydrogen donor (nꢀOH
and NH) and acceptor atoms (nꢀO and N atom) and violations of
Lipinski’s rule of five of titled compounds (7a-p and 8a-p) are
presented in Table 4.
Software used: (a) Schrödinger Suite 2013 Protein Preparation
Wizard; LLC, New York, NY, Glide 5.9 Schrödinger, LLC; New
York, NY, 2013, LigPrep, version 2.6, Schrödinger, LLC, New
York, NY, 2013. (b) Molinspiron property calculation program
21. R. S. Reis, I. Neves, Jr., S. L. Lourenco, L. S. Fonseca and M. C.
Lourenco, J. Clin. Microbiol., 2004, 42, 2247ꢀ2248.
22. Tuberculosis, 2008, 88, 85ꢀ169.
23. I. Orme, Antimicrob. Agents Chemother., 2001, 45, 1943ꢀ1946.
24. X. He, A. Alian, R. Stroud and P. R. Ortiz de Montellano, J.
Med. Chem., 2006, 49, 6308ꢀ6323.
Acknowledgements
This work was financially supported by University Grant
Commission, New Delhi, India to Rikta Saha. We gratefully
acknowledge the TAACF (Tuberculosis Antimicrobial Acquisition
Coordinating Facility, National Institute of Allergy and Infectious
Diseases (Contract No. HHSN272201100009I), USA for carrying 25. G. J. Nuermberger E, Eur. J. Clin. Microbiol. Infect. Dis., 2004,
23, 243ꢀ255.
out the biological activity.
26. A. Kumar, G. Patel and S. K. Menon, Chem. Biol. Drug Des.,
2009, 73, 553ꢀ557.
27. R. M. Cowper and L. H. Davidson, Org. Synth., 1939, 19, 24.
Conflict of interest
The authors report no declarations of interest.
† Supplementary data: Physicochemical properties, calculated
ADMET properties, 1Hꢀ, 13CꢀNMR and purity data of the
compounds can be found online.
This journal is © The Royal Society of Chemistry xxxx
J. Name., xxxx, 00, xxx | 9

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ARTICLE
DOI: 10.1039/C4RA14440F
Scheme 1 Synthetic route of hybridꢀpyrrole analogues 7a-p & 8a-p
Figure legends
Figure 1. Structures of different hybrid drugs showing antitubercular
activity
Figure 2. Compounds represented with antitubercular activity and
portions highlighted have been used to design new hybrid
compounds
Figure 3. Dose response plot for minimum bactericidal
concentration (MBC) determination
Figure 4. (A) Reference ligand binding pose (B) Surface of the
binding pose between reference ligand and InhA (C) Docked
conformation of the most potent inhibitor 7e with the crystal
structure of a cofactor NAD+ and Tyr 158 in InhA (D) Surface of
the binding pose which shows the interactions between InhA site and
compound 7e
Table captions
Table 1 Preliminary antitubercular activity of 7a-p & 8a-p against
Mtb H37Rv
Table 2 Cytotoxicity, minimum bactericidal concentration (MBC),
drug sensitive and single drugꢀresistant (SDR) antitubercular
activities of 7e & 8e
This journal is © The Royal Society of Chemistry xxxx
J. Name., xxxx, 00, xxx | 10

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DOI: 10.1039/C4RA14440F
7e
8e
Fig. 3 Dose response plot for minimum bactericidal concentration (MBC) determination (7e & 8e); ___130 µg/mL; ____65
µg/mL; ____4.4 µg/mL; ___3.25 µg/mL; ___ DMSO; ….Lower limit of detection; _ _ _upper limit of detection; MBC of
rifampin is 0.78 µg/mL

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DOI: 10.1039/C4RA14440F
85x55mm (300 x 300 DPI)

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DOI: 10.1039/C4RA14440F
67x40mm (300 x 300 DPI)

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85x51mm (300 x 300 DPI)

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Table 1 Preliminary antitubercular activity of 7a-p & 8a-p against Mtb H37Rv
DOI: 10.1039/C4RA14440F
7
8
MICa, b GIc % Cell viabilityd
MICa,b GIc % Cell viabilityd
ID
R
Docking Score
ID
R
Docking Score
7a
25
70
26
100
ND
ND
-5.017
-4.585
-5.393
8a
50
70
26
100
ND
ND
-6.412
7b
7c
>100
>100
8b
8c
>100
>100
-5.956
-6.496
NS
NS
7d
>100
22
ND
-7.164
8d
>100
22
ND
-6.045
7e
7f
3.70
>100
62.5
95
NS
52
100
ND
ND
-5.873
-5.741
-6.164
8e
8f
5.10
>100
>100
92
NS
42
100
ND
ND
-6.832
-6.150
-6.337
7g
8g
7h
7i
>100
>100
12.5
NS
28
ND
ND
90
-5.469
-6.697
-6.102
-4.653
8h
8i
12.5
>100
25
62
28
95
-6.463
-6.305
-7.034
-4.369
ND
100
ND
7j
75
8j
65
7k
>100
NS
ND
8k
>100
NS
7l
6.25
70
100
-4.528
8l
12.5
60
90
-4.565
7m
7n
7o
>100
12.5
40
80
ND
97
-4.355
-6.502
-3.058
8m
8n
8o
>100
25
40
75
ND
100
ND
-5.181
6.514
-3.840
O
>100
NS
ND
>100
NS
7p
6.25
80
97
-6.076
8p
6.25
85
100
-6.097
RIF
PYZ
0.06
50
98
85
ND
ND
ND
ND
INH
PC
0.4
~125
94
-
ND
ND
ND
-8.907
a, minimum inhibitory concentrations (MIC) are the highest concentration which prevent the complete growth of inoculum; b, µg/mL; c, growth
inhibition (GI) of Mtb H37Rv at 50 µg/mL; d, percent viability of cells at 100 µg/mL; Docking score calculated as –Kcal/mol; ND, not determined;
NS, Not significant; PC, N-(3,5-dichlorophenyl)-5-oxo-1-phenylpyrrolidine-3-carboxamide which is used as reference molecule in the docking study

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Table 2 Cytotoxicity, minimum bactericidal concentration (MBC), drug sensitive and single drug-resistant (SDR) antitubercularVaiecwtivAritticielesOonflin7ee & 8e
TC50
Strain
H37Rv
MIC
3.7
IC50
2.71
IC90 DMOI:B1C0.*1039/C4RA14440F
3.72
>67.8
>67.8
1.12
INH-R1
INH-R2
RMP-R1
RMP-R2
FQ-R1
>67.8
>67.8
1.25
>67.8
>67.8
0.78
> 100
7e
4.4
2.44
2.84
4.06
2.40
1.62
2.61
LORA
>67.8
>67.8
>67.8
Intracellular macrophage (3.7 and 37.0 µg/mL) 2.4 (Log reduction of CFU)
H37Rv
INH-R1
INH-R2
RMP-R1
RMP-R2
FQ-R1
5.10
>55.4
>55.4
14.9
4.92
>55.4
>55.4
14.4
5.16
>55.4
>55.4
19.9
>100
8e
7.8
30.4
21.32
20.77
0.86
30.4
28.5
27.7
LORA
1.94
1.27
Intracellular macrophage (5.10 and 51.0 µg/mL) 2.4 (Log reduction of CFU)
H37Rv
INH-R1
INH-R2
0.4-0.6
>200
0.12
110
95
0.21
130
110
INH
>200
All concentration are in µg/mL; selectivity index (SI) = TC50/MIC; *, MBC of Rifampin is 0.78 µg/mL