Synthesis of new 4-(2,5-dimethylpyrrol-1-yl)/4-pyrrol-1-yl benzoic acid hydrazide analogs and some derived oxadiazole, triazole and pyrrole ring systems: A novel class of potential antibacterial, antifungal and antitubercular agents

Article abstract of DOI:10.1007/s00044-012-0112-0

A series of 4-(2,5-dimethylpyrrol-1-yl)/4-pyrrol-1-yl benzoic acid hydrazide analogs, some derived 1,3,4-oxadiazoles, 5-substituted-4-amino-1,2,4- triazolin-3-thione and 2,5-dimethyl pyrroles have been synthesized in good yields and structures of these compounds were established by IR, ¹H NMR, ¹³C NMR, Mass spectral and elemental analysis. These compounds were evaluated for their preliminary in vitro antibacterial, antifungal and antitubercular activities against Mycobacterium tuberculosis H₃₇Rv strain by broth dilution assay method. Twelve of these compounds displayed good antimicrobial activity, with a minimum inhibitory concentration (MIC) values 1-4 μg mL^-1. Several compounds 4, 8d, 9, 12c-d and 12f-h exhibited good in vitro antitubercular activity with MIC values 1-2 μg mL^-1. Further, some title compounds were also assessed for their cytotoxic activity (IC₅₀) against mammalian Vero cell lines and A₅₄₉ (lung adenocarcinoma) cell lines using MTT assay method. The results reveal that these compounds exhibit antitubercular activity at non-cytotoxic concentrations. Graphical Abstract: Synthesis, spectral studies and antibacterial, antifungal and antitubercular activities of a novel series of pyrrole derivatives are described [Figure not available: see fulltext.]

Full text of DOI:10.1007/s00044-012-0112-0

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:1073–1089
DOI 10.1007/s00044-012-0112-0
ORIGINAL RESEARCH
Synthesis of new 4-(2,5-dimethylpyrrol-1-yl)/4-pyrrol-1-yl benzoic
acid hydrazide analogs and some derived oxadiazole, triazole
and pyrrole ring systems: a novel class of potential antibacterial,
antifungal and antitubercular agents
•
S. D. Joshi Yogesh More H. M. Vagdevi
•
•
•
V. P. Vaidya G. S. Gadaginamath V. H. Kulkarni
•
Received: 23 December 2011 / Accepted: 22 May 2012 / Published online: 2 June 2012
Ó Springer Science+Business Media, LLC 2012
Abstract A series of 4-(2,5-dimethylpyrrol-1-yl)/4-pyr-
rol-1-yl benzoic acid hydrazide analogs, some derived
1,3,4-oxadiazoles, 5-substituted-4-amino-1,2,4-triazolin-3-
thione and 2,5-dimethyl pyrroles have been synthesized in
good yields and structures of these compounds were
Keywords Pyrroles ꢀ Acid hydrazide derivatives ꢀ
Antibacterial activity ꢀ Antitubercular activity ꢀ
Antifungal activity ꢀ Broth dilution assay method ꢀ
Cytotoxicity
1
established by IR, H NMR, 13C NMR, Mass spectral and
elemental analysis. These compounds were evaluated for
their preliminary in vitro antibacterial, antifungal and
antitubercular activities against Mycobacterium tubercu-
losis H37Rv strain by broth dilution assay method. Twelve
of these compounds displayed good antimicrobial activity,
with a minimum inhibitory concentration (MIC) values
1–4 lg mL-1. Several compounds 4, 8d, 9, 12c–d and
12f–h exhibited good in vitro antitubercular activity with
MIC values 1–2 lg mL-1. Further, some title compounds
were also assessed for their cytotoxic activity (IC50) against
mammalian Vero cell lines and A549 (lung adenocarci-
noma) cell lines using MTT assay method. The results
reveal that these compounds exhibit antitubercular activity
at non-cytotoxic concentrations.
Introduction
Tuberculosis (TB) is one of the dreadful infectious diseases
world-wide with diverse manifestations caused by Myco-
bacterium tuberculosis, which has been a scourge of
humanity for thousands of years and still remains one of
the prevalent health tribulations in the world (Dutt and
Stead, 1999). Today, TB is among the top five causes of
global mortality. The history of TB highlights man’s
struggle for existence against a disease that dates from
antiquity and is the story of failures and successes of
disaster and hope (Das, 2000). It has been estimated that
one-third of the world’s population is currently infected
with the Bacillus and about 30 million people would be
infected within next 20 years if proper control is not further
strengthened. In developing countries, where rates of both
infection and active disease have always been high, the
number of cases skyrocketed, so dramatically which led the
World Health Organization (WHO) to declare TB as a
global health emergency in 1993 and for the first time an
infectious disease achieved such a dubious distinction
(Martien et al., 2002; Rouhi, 1999). Approximately, 80 %
of TB cases are found in 23 countries; the highest rates of
incidence being found in Southeast Asia and Africa (Dye,
2002).
S. D. Joshi (&) ꢀ Y. More ꢀ G. S. Gadaginamath ꢀ
V. H. Kulkarni
Department of Pharmaceutical Chemistry, S.E.T’s College
of Pharmacy, S. R. Nagar, Dharwad, Karnataka, India
e-mail: shrinivasdj@rediffmail.com
H. M. Vagdevi
Department of Chemistry, Sahaydri Science College
(Autonomous), Shimoga, Karnataka, India
In addition, HIV-1 and HIV-2 infections, which impairs
the immune system and allows large number of people
already infected with the TB to progress to active disease.
The pathogenic synergy of tuberculosis with HIV enhances
V. P. Vaidya
Department of PG Studies and Research in Chemistry, School
of Chemical Sciences, Kuvempu University, Jnana Sahyadri,
Shankaraghatta, Karnataka, India
123

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Med Chem Res (2013) 22:1073–1089
the overall incidence of TB in HIV-positive patients by 50
times relative to the rate for HIV-negative individuals
(Maher et al., 2002).
containing pyrrole/2,5-dimethylpyrrole moieties and study
their antimicrobial, antitubercular activities followed by
cytotoxic activity.
Antitubercular drugs available at present for the treat-
ment were discovered in the period of 1945–1965. No new
drugs were synthesized during the last few decades (Desai
et al., 2001). Moreover, the recent outbreaks of multidrug-
resistant-tuberculosis (MDR-TB) and extensive drug-
resistant-tuberculosis (XDR-TB) have made the disease
hard to be treated (Sriram et al., 2005). This obstacle in the
treatment of tuberculosis and also the statistical facts about
its prevalence stress the strong need for searching and
synthesizing more potent and less resistant new therapeu-
tics with least side effects. The development of new
potential drugs would be one of the possible solutions to
treat various infectious diseases with multi-drug treatment
over a prolonged period of time.
Methods and materials
Chemistry
The synthetic strategies adopted to obtain the target com-
pounds are depicted in Schemes 1 and 2. The compound
4-(2,5-dimethylpyrrol-1-yl)benzoic acid hydrazide (2) was
chosen as starting compound to design several hydrazones
and oxadiazoles. Ethyl 4-aminobenzoate was prepared by
the reaction of 4-amino benzoic acid and absolute ethanol
in presence of HCl gas. Paal–Knorr condensation reaction
between ethyl 4-aminobenzoate and acetonyl acetone in
glacial acetic acid furnished ethyl 4-(2,5-dimethylpyrrol-1-
yl)benzoate (1). Nucleophilic reaction of hydrazine hydrate
with the ester 1 in ethanolic medium produced 4-(2,5-
dimethylpyrrol-1-yl)benzoic acid hydrazide (2). Reacting
the starting compound 2 with acetone resulted in the for-
mation of the corresponding 4-(2,5-dimethyl-pyrrol-1-yl)-
benzoic acid isopropylidene-hydrazide (3a). Condensation
of 2 with acetophenone, benzophenone and 4-aminoace-
tophenone yielded corresponding hydrazone derivatives
3b, 3c and 3d. The preparation of the 5-[4-(2,5-dimeth-
ylpyrrol-1-yl)phenyl]-1,3,4-oxadiazole-2-thiol (4) was
achieved by adopting a simple one-pot procedure that
involves reacting compound 2 with carbon disulphide
under strong basic conditions followed by acidification
with dil. HCl. Hydrazide 2 was converted into the corre-
sponding potassium dithiocarbazinate, which on cyclisation
with hydrazine hydrate afforded 4-amino-5-[4-(2,5-dim-
ethylpyrrol-1-yl)phenyl]-2,4-dihydro-1,2,4-triazole-3-thione
(5). Warming 2 with acetic anhydride afforded N-acetyl-4-
(2,5-dimethylpyrrol-1-yl)benzoic acid hydrazide (6). The
reaction of compound 2 with different aldehydes in alcohol
gave Schiff bases 7a–h. Cyclisation of 7a–h with acetic
anhydride afforded the desired 1,3,4-oxadiazole derivatives
8a–h. Further, 2 reacted with acetonyl acetone to afford
N-(2,5-dimethyl-pyrrol-1-yl)-4-(2,5-dimethylpyrrol-1-yl)
benzamide (9).
Pyrrole is one of the most ubiquitous heterocycles in the
plant and animal kingdom because of its participation as a
subunit of chlorophyll in plant cells and hemin and vitamin
B12 in animal cells. 2,5-Dimethylpyrrole compounds are
promising starting materials in drug research in view of
their various biological activities such as antibacterial
(Demirayak et al., 1999), antihypertensive (Demirayak
et al., 2004) and antitubercular (Biava et al., 1999). Several
macromolecular antibiotics having pyrrole structure were
isolated from biological sources and their activities were
defined (Jones and Bean, 1997; Jones, 1992).
Careful literature survey for functional groups which
could be considered as pharmacophores for the antituber-
cular activities revealed that the hydrazone moiety is
common among most of the antitubercular agents such as
saliniazid and verazide (Sriram et al., 2006). Several Schiff
bases, hydrazones and hydrazides of isoniazid have shown
good activity against tubercular, fungal and bacterial
infections (Desai et al., 1984; Shah et al., 1985; Vigorita
et al., 1999).
Lipophilicity is a key property that influences the ability
of a drug to reach the target by transmembrane diffusion
and to have a major effect on the biological activity. The
azole antituberculars are regarded as emerging class and
oxadiazoles known as lipophilic analogs similar to imida-
zoles are expected to increase log P. 1,3,4-Oxadiazoles are
utilized as pharmacophores because of their favourable
metabolic profile and ability to engage in hydrogen bond-
ing. Biological activities of oxadiazole and 3-acetyl-2,5-
disubstituted-2,3-dihydro-1,3,4-oxadiazoline ring systems
are well documented (Suresh Kumar et al., 2010; Khalil
et al., 1993).
As shown in Scheme 2, treatment of hydrazide 10 with
appropriate acyl chlorides in DMF produced the interme-
diates 11a–h through N-acylation which on cyclodehydration
with P2O5 in dry DMF gave 5-substituted-1,3,4-oxadiazoles
12a–h.
The structures of newly synthesized compounds were
assigned on the basis of their spectral and analytical data.
The physical data, FTIR, NMR and mass spectral data for
all the synthesized compounds are reported in experimental
protocols.
Therefore, in view of the above facts and in continuation
of our search for biologically active pyrroles (Joshi et al.,
2008a, b, 2010), it was contemplated to synthesize
some novel hydrazones and 1,3,4-oxadiazole derivatives
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Med Chem Res (2013) 22:1073–1089
1075
CH3
N
R
b
CONHN=C
CH3
N
R'
a-d
3
CH3
R
R'
COOC2H5
CH3
3a
3b
3c
CH3
CH3
CH3
C6H5
C6H5
1
C6H5
3d
CH3
4-NH2 C6H4
a
CH3
CH3
N
O
N
c
N
N
CONHNH2
SH
CH3
CH3
4
2
CH3
H
N
N
d
N
S
N
h
CH3
NH2
5
CH3
CH3
N
H3C
e
N
CONHNHCOCH3
N
CONH
CH3
6
CH3
H3C
CH3
9
f
N
CONHN=CH
R
7a-h
CH3
R
g
CH3
COCH3
N
N
N
R
O
CH3
8a-h
R
R
e
f
3-NO2 C6H4
2-NO2 C6H4
a
b
C6H5
2-
4-
Cl C6H4
Br C6H4
c
d
g
h
4-OH C6H4
4-N(CH3)2 C6H4
2,6-Cl2 C6H3
Scheme 1 Synthetic route of 4-(2,5-dimethylpyrrol-1-yl)benzoic
acid hydrazide-derived hydrazones and oxadiazoles 3a–d, 4, 5, 6,
7a–h, 8a–h and 9. a Hydrazine hydrate, ethanol, reflux, 2 h, b ketone,
ethanol, reflux, 5 h, c potassium hydroxide, carbon disulphide,
ethanol reflux, 10 h, d potassium hydroxide, carbon disulphide,
ethanol, stir, 18 h, hydrazine hydrate, reflux, 7 h, e acetic anhydride,
warm, 2 h, f aromatic aldehyde, ethanol, reflux, 6 h, g acetic
anhydride, reflux, 4 h, h acetonyl acetone, ethanol, reflux, 5 h
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Med Chem Res (2013) 22:1073–1089
Scheme 2 Synthetic route of a
novel series of 4-(pyrrol-1-
yl)benzoic acid hydrazide-
derived 1,3,4-oxadiazoles
11a–h and 12a–h. a Acyl
chloride, DMF, reflux, 8 h,
b phosphorus pentoxide,
DMF, reflux, 4 h
N
CONHNH2
10
a
O
C
O
C
N
N
b
N
N
R
NH
NH
R
O
11a-h
12a-h
R
a C6H5
b 2-Br C6H4
c 4-Br C6H4
d 2-NO2 C6H4
e 3-NO2C6H4
f 4-NO2 C6H4
g 4-Cl C6H4
h 4-F, 3-CF3-C6H3
The synthetic strategies adopted to obtain the target
compounds are depicted in Schemes 1 and 2. IR, 1H NMR,
13C NMR and mass spectral data are in agreement with the
proposed structures of all newly synthesized compounds.
chromatography (TLC) was performed on precoated TLC
sheets of silica gel 60 F254 (Merck, Darmstadt, Germany),
visualizing by long- and short-wavelength ultraviolet (UV)
lamps.
Synthesis of ethyl 4-(2,5-dimethylpyrrol-1-yl)benzoate
Experimental
(1)
Chemicals used in the synthesis of the titled compounds
were purchased from Sigma-Aldrich, S. D. Fine-Chem
Limited and Spectrochem Pvt. Ltd. The melting points of
synthesized compounds were determined on Shital scien-
tific industries melting point apparatus and are uncorrected;
infra-red spectra were recorded on a Bruker spectropho-
A mixture of acetonyl acetone (13.69 g, 0.12 mol) and
ethyl 4-aminobenzoate (16.5 g, 0.1 mol) in glacial acetic
acid (100 mL) was refluxed for 1 h. The solvent was
removed under reduced pressure, residue thus obtained
was collected by filtration, washed with water, dried and
recrystallized from ethanol (yield 65 %). mp 87–88 °C.
1
tometer by using KBr pellets. H and 13C NMR spectra
were recorded on Bruker 300 MHz and Bruker AVANCE
III 500 MHz instruments using dimethylsulphoxide-d6
(DMSO-d6) as solvent and TMS as internal standard,
chemical shifts are expressed as d values (ppm). Mass
spectra (MS) were taken in JEOL GCMATE II GC-Mass
spectrometer and Schimadzu QP 20105 GC-Mass spec-
trometer. Microanalyses of compounds were also per-
formed on Leco Tru Spec CHNS Analyser for the
determination of percentage of C, H and N. All the new
compounds exhibited spectral data consistent with the
proposed structures and values of microanalysis are within
0.4 % of the theoretical values. Analytical thin-layer
Synthesis of 4-(2,5-dimethylpyrrol-1-yl)benzoic acid
hydrazide (2)
Compound 2 was synthesized by refluxing a mixture of ethyl
4-(2,5-dimethylpyrrol-1-yl)benzoate (1) (3.64 g, 15 mmol)
with hydrazine hydrate (10 mL) in absolute ethanol (10 mL)
for 3 h (monitored by TLC). The cooled mixture was poured
gradually onto crushed ice cubes with stirring. The mixture
was allowed to stand and solid was separated. It was filtered,
washed thoroughly with cold water, dried and recrystallized
from ethanol (yield 80 %). mp 170–172 °C; IR (KBr) 3,285,
3,195 (NH/NH2), 1,655 (amide C=O) cm-1 1H NMR
;
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Med Chem Res (2013) 22:1073–1089
1077
(300 MHz, DMSO-d6) d ppm: 1.95 (s, 6H, pyrrole methyls),
4.51 (s, 2H, NH2, disappeared on D2O exchange), 5.80 (s,
2H, pyrrole-C3 and C4–H), 7.33 (d, J = 8.5 Hz, 2H, bridging
phenyl-C3 and C5–H), 7.94 (d, J = 8.5 Hz, 2H, bridging
phenyl-C2 and C6–H), 10.00 (s, 1H, NH, disappeared on D2O
exchange); 13C NMR (400 MHz, DMSO-d6) d ppm: 165.18
(amide C=O), 140.60 (bridging phenyl-C4), 132.35 (bridg-
ing phenyl-C1), 128.54 (bridging phenyl-C2 and C6), 127.79
(bridging phenyl-C3 and C5), 127.50 (pyrrole-C2 and C5),
106.33 (pyrrole-C3 and C4), 12.85 (2CH3); MS m/z: found
229 [M?], 230 [M?1], 213, 209, 171, 94; calcd. 229.28 Anal
C13H15N3O.
(s, 2H, pyrrole-C3 and C4–H), 7.36–8.00 (m, 9 H, Ar–H),
10.91 (s, 1H, NH, disappeared on D2O exchange); 13C
NMR (300 MHz, DMSO-d6) d ppm: 170.62 (amide C=O),
156.20 (C=N–NH), 140.10 (bridging phenyl-C4), 132.20
(bridging phenyl-C1), 131.14 (phenyl-C1), 130.60 (phenyl-
C4), 129.02 (phenyl-C2 and C6), 128.60 (phenyl-C3 and
C5), 128.40 (bridging phenyl-C2 and C6), 128.12 (pyrrole-
C2 and C5), 127.90 (bridging phenyl-C3 and C5), 107.02
(pyrrole-C3 and C4), 17.63 (CH3), 10.10 (pyrrole-2CH3);
MS m/z: found 331 [M?], 332 [M?1], 286, 270, 214, 171,
154, 136, 94; calcd. 331.41 Anal C21H21N3O.
N’-(Benzylidene)-4-(1H-2,5-dimethylpyrrol-1-yl)
benzohydrazide (3c)
Synthesis of N0-(propan-2-ylidene)-4-(1H-2,5-
dimethylpyrrol-1-yl)benzohydrazide (3a)
(Yield 55 %). mp 252–254 °C; IR (KBr) 3,252 (NH),
1,646 (amide C=O) cm-1; 1H NMR (300 MHz, DMSO-d6)
d ppm: 2.10 (s, 6H, pyrrole-2CH3), 5.72 (s, 2H, pyrrole-C3
and C4––H), 7.42–8.56 (m, 14 H, Ar–H), 10.21 (s, 1H, NH,
disappeared on D2O exchange); MS m/z: found 393 [M?],
394 [M?1]; calcd. 393.48 Anal C26H23N3O.
A solution of 4-(2,5-dimethylpyrrol-1-yl)benzoic acid
hydrazide (2) (1.14 g, 5 mmol) in acetone (70 mL) was
refluxed for 1 h. Evaporation of solvent furnished a solid
which was recrystallized from ethanol (yield 78 %). mp
196–198 °C; IR (KBr) 3,214 (NH), 1,647 (amide
1
C=O) cm-1; H NMR (300 MHz, DMSO-d6) d ppm: 1.95
4-(2,5-Dimethylpyrrol-1-yl)benzoic acid[1-(4-
aminophenyl)ethylidene]hydrazide (3d)
(s, 6H, 2CH3), 1.97 (s, 6H, 2CH3), 5.81 (s, 2H, pyrrole-C3
and C4–H), 7.37 (d, J = 8.5 Hz, 2H, bridging phenyl-C3
and C5–H), 7.93 (d, J = 8.5 Hz, 2H, bridging phenyl-C2
and C6–H), 10.57 (s, 1H, amide NH, disappeared on D2O
exchange); 13C NMR (300 MHz, DMSO-d6) d ppm:
162.61 (amide C=O), 160.52 (C=N–NH), 140.76 (bridging
phenyl-C4), 133.19 (bridging phenyl-C1), 128.70 (bridging
phenyl-C2 and C6), 127.74 (bridging phenyl-C3 and C5),
127.53 (pyrrole-C2 and C5), 106.39 (pyrrole-C3 and C4),
25.10 (CH3), 18.08 (CH3) 12.91 (pyrrole-2CH3); FAB MS
m/z: found 270 [M??H], 271 [M??H?1], 242, 226, 198,
171, 165, 154, 136, 94; calcd. 269.34. Anal C16H19N3O.
(Yield 56 %). mp 248–250 °C; IR (KBr) 3,268 (NH),
1,652 (amide C=O) cm-1; 1H NMR (300 MHz, DMSO-d6)
d ppm: 1.40 (s, 3H, CH3), 2.02 (s, 6H, pyrrole-2CH3), 4.4
(s, 2H, NH2), 5.68 (s, 2H, pyrrole-C3 and C4–H), 7.22–8.68
(m, 14 H, Ar–H), 10.56 (s, 1H, NH, disappeared on D2O
exchange); MS m/z: found 346 [M?], 347 [M?1], 331,
252, 238, 224, 171, 154, 136, 94; calcd. 346.43 Anal
C21H22N4O.
Synthesis of 5-[4-(2,5-dimethylpyrrol-1-yl)phenyl]-
1,3,4-oxadiazole-2-thiol (4)
General procedure for the synthesis of 4-(2,5-
dimethylpyrrol-1-yl)benzoic acid(arylidene)hydrazides
(3b–3d)
Acid hydrazide 2 (0.687 g, 3 mmol) was dissolved in a
solution of potassium hydroxide (0.336 g, 6 mmol) in
water (2 mL) and ethanol (20 mL). Carbon disulphide
(2 mL) was then added while stirring and the reaction
mixture was refluxed for 10 h. The solvents were removed
under reduced pressure; the residue was treated with water
and then filtered. The filtrate was cooled, neutralized to pH
6 using dil. HCl and the separated product was filtered,
washed with water, dried and recrystallized from ethyl
acetate.
A mixture of 2 (3 mmol) and appropriate ketones (3 mmol)
in ethanol (20 mL) was refluxed for 5 h in the presence of
few drops of glacial acetic acid. The solvent was evapo-
rated and the product was poured onto cold water, filtered
and dried. The crude solid was recrystallized from aqueous
DMF to give the products 3b–d.
N0-(1-Methylbenzylidene)-4-(1H-2,5-dimethylpyrrol-1-yl)
benzohydrazide (3b)
(Yield 75 %). mp 255–257 °C; IR (KBr) 2,770 (SH),
1,615 (C=N) cm-1; 1H NMR (300 MHz, DMSO-d6) d ppm:
1.92 (s, 6H, pyrrole methyls), 3.43 (SH merged in HOD peak
of DMSO-d6), 5.84 (s, 2H, pyrrole-C3 and C4–H), 7.36 (d,
J = 8.5 Hz, 2H, bridging phenyl-C3 and C5–H), 7.94 (d,
J = 8.5 Hz, 2H, bridging phenyl-C2 and C6–H); 13C NMR
(Yield 65 %). mp 202–204 °C; IR (KBr) 3,224 (NH),
1,669 (amide C=O) cm-1; 1H NMR (300 MHz, DMSO-d6)
d ppm: 1.99 (s, 6H, pyrrole-2CH3), 2.38 (s, 3H, CH3), 5.82
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Med Chem Res (2013) 22:1073–1089
(300 MHz, DMSO-d6) d ppm: 175.24 (oxadiazole-C2),
158.48 (oxadiazole-C5), 141.64 (bridging phenyl-C4),
131.42 (bridging phenyl-C1), 128.24 (bridging phenyl-C2
and C6), 127.55 (bridging phenyl-C3 and C5), 127.21 (pyr-
role-C2 and C5), 107.98 (pyrrole-C3 and C4), 12.51 (2CH3);
MS m/z: found 272 [M??H], 273 [M??H?1], 239, 171, 94;
calcd. 271.34 Anal C14H13N3OS.
C4–H), 7.84 (d, J = 8.5 Hz, 2H, bridging phenyl-C3 and
C5–H), 8.54 (d, J = 8.5 Hz, 2H, bridging phenyl-C2 and
C6–H), 9.54 (s, 1H, amide NH, disappeared on D2O
exchange), 11.42 (s, 1H, NH, disappeared on D2O
exchange); 13C NMR (300 MHz, DMSO-d6) d ppm:
170.26 (acetyl-C=O), 164.24 (amide-C=O), 145.46
(bridging phenyl-C4), 130.85 (bridging phenyl-C1), 128.56
(bridging phenyl-C2 and C6), 127.23 (bridging phenyl-C3
and C5), 127.05 (pyrrole-C2 and C5), 107.25 (pyrrole-C3
and C4), 22.52 (CH3), 12.54 (2CH3); MS m/z: found 272
[M??H], 273 [M??H?1], 228, 171, 94; calcd. 271.31.
Anal C15H17N3O2.
Synthesis of 4-amino-5-[4-(2,5-dimethylpyrrol-1-
yl)phenyl]-2,4-dihydro-1,2,4-triazole-3-thione (5)
To a solution of potassium hydroxide (0.37 g, 6.7 mmol) in
absolute ethanol (30 mL) acid hydrazide 2 (0.687 g,
3 mmol), carbon disulphide (0.45 mL, 6 mmol) were
added and the reaction mixture was agitated for 18 h. To
the resulting solution anhydrous ether was added and the
precipitated potassium dithiocarbazinate was collected by
filtration, washed with ether and dried under vacuum. The
potassium salt was obtained in quantitative yield and was
used in the next step without further purification.
General procedure for the synthesis of N’-(arylidene)-4-
(1H-2,5-dimethylpyrrol-1-yl)benzohydrazide (7a–h)
Equimolar quantity of 4-(2,5-dimethylpyrrol-1-yl)benzoic
acid hydrazide (2) and different aromatic aldehydes was
refluxed in ethanol for 4–6 h in the presence of few drops
of glacial acetic acid. The solvent was evaporated and the
product was poured onto cold water, filtered and dried. The
crude solid was recrystallized from aqueous DMF to give
the products.
A suspension of the potassium salt, hydrazine hydrate
(1.5 mL) and water (1.0 mL) was refluxed for 7 h.
Hydrogen sulphide evolved and homogenous solution
resulted which was diluted with 50 mL water and sub-
sequent acidification with dilute acetic acid gave a white
precipitate which was filtered, washed with water and
recrystallized from aqueous DMF.
N’-(Benzylidene)-4-(1H-2,5-dimethylpyrrol-1-
yl)benzohydrazide (7a)
(Yield 75 %). mp 279–281 °C; IR (KBr) 3,292 (NH2),
3,122 (NH) cm-1; 1H NMR (300 MHz, DMSO-d6) d ppm:
1.89 (s, 6H, pyrrole methyls), 5.50 (s, 2H, NH2, disap-
peared on D2O exchange), 5.82 (s, 2H, pyrrole-C3 and C4–
H), 7.44 (d, J = 8.5 Hz, 2H, bridging phenyl-C3 and C5–
H), 8.22 (d, J = 8.5 Hz, 2H, bridging phenyl-C2 and
C6–H), 12.42 (s, 1H, NH, disappeared on D2O exchange);
13C NMR (300 MHz, DMSO-d6) d ppm: 166.22 (triazole-
C=S), 148.26 (triazole-C5), 143.52 (bridging phenyl-C4),
130.78 (bridging phenyl-C1), 128.88 (bridging phenyl-C2
and C6), 127.66 (bridging phenyl-C3 and C5), 127.11
(pyrrole-C2 and C5), 106.22 (pyrrole-C3 and C4), 13.87
(2CH3); MS m/z: found 286 [M??H], 287 [M??H?1],
270, 171, 94; calcd. 285.37. Anal C14H15N5S.
(Yield 60 %). mp 240–242 °C; IR (KBr) 3,230 (NH), 1,666
(amide C=O) cm-1; 1H NMR (300 MHz, DMSO-d6) d ppm:
2.02 (s, 6H, pyrrole-2CH3), 5.74 (s, 2H, pyrrole-C3 and C4–
H), 7.45–8.42 (m, 9H, Ar–H), 8.56 (s, 1H, –N=CH–Ar),
11.50 (s, 1H, amide NH, disappeared on D2O exchange); 13
C
NMR (300 MHz, DMSO-d6) d ppm: 168.20 (amide C=O),
152.38 (CH=N), 142.22 (bridging phenyl-C4), 133.34
(bridging phenyl-C2 and C6), 132.61 (phenyl-C4), 131.04
(phenyl-C1), 130.54 (phenyl-C2 and C6), 129.73 (C1), 128.88
(pyrrole-C2 and C5), 127.66 (phenyl-C3 and C5), 125.36 (C3
and C5), 108.26 (pyrrole-C3 and C4), 11.22 (pyrrole-2CH3);
MS m/z: found 317 [M?], 318 [M?1], 241, 171, 94; calcd.
317.38. Anal C20H19N3O.
N’-(2-Chlorobenzylidene)-4-(1H-2,5-dimethylpyrrol-1-
yl)benzohydrazide (7b)
Synthesis of N-acetyl-4-(2,5-dimethylpyrrol-1-
yl)benzoic acid hydrazide (6)
(Yield 58 %). mp 236–238 °C; IR (KBr) 3,244 (NH),
1,658 (amide C=O) cm-1; 1H NMR (300 MHz, DMSO-d6)
d ppm: 2.16 (s, 6H, pyrrole-2CH3), 5.74 (s, 2H, pyrrole-C3
and C4–H), 7.42–8.76 (m, 9H, Ar–H and –N=CH–Ar),
12.20 (s, 1H, amide NH, disappeared on D2O exchange);
13C NMR (300 MHz, DMSO-d6) d ppm: 170.99 (amide
C=O), 154.76 (CH=N), 141.54 (bridging phenyl-C4),
134.64 (phenyl-C6), 133.32 (phenyl-C1), 132.77 (phenyl-C4),
131.26 (bridging phenyl-C2 and C6), 130.54 (phenyl-C2),
Acid hydrazide 2 (0.687 g, 3 mmol) was warmed with acetic
anhydride (5 mL) for 2 h and the reaction mixture was left to
stand at room temperature. The deposited pale yellow solid
was filtered, washed and recrystallized from ethanol.
(Yield 65 %). mp 244–246 °C; IR (KBr) 3,384, 3,315
1
(NH), 1,685 (acetyl C=O), 1,655 (amide C=O) cm-1; H
NMR (300 MHz, DMSO-d6) d ppm: 1.92 (s, 6H, pyrrole
methyls), 1.96 (s, 3H, CH3), 5.90 (s, 2H, pyrrole-C3 and
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Med Chem Res (2013) 22:1073–1089
1079
129.53 (bridging phenyl-C1), 128.63 (pyrrole-C2 and C5),
127.43 (phenyl-C3 and C5), 126.86 (bridging phenyl-C3
and C5), 107.32 (pyrrole-C3 and C4), 10.32 (pyrrole-2CH3);
MS m/z: found 352 [M?], 353 [M?1], 317, 229, 171, 94;
calcd. 351.83. Anal C20H18ClN3O.
nitro-phenyl-C1), 133.46 (3-nitro-phenyl-C2), 132.17
(bridging phenyl-C2 and C6), 130.51 (3-nitro-phenyl-C3),
128.83 (bridging phenyl-C1), 128.03 (3-nitro-phenyl-C6),
127.58 (3-nitro-phenyl-C4), 124.34 (bridging phenyl-C3
and C5), 120.98 (pyrrole-C2 and C5), 106.56 (pyrrole-C3
and C4), 12.95 (pyrrole-2CH3); MS m/z: found 362 [M?],
363 [M?1], 317, 229, 171, 94; calcd. 362.38. Anal
C20H18N4O3.
N0-(4-Bromobenzylidene)-4-(1H-2,5-dimethylpyrrol-1-
yl)benzohydrazide (7c)
(Yield 55 %). mp 245–247 °C; IR (KBr) 3,260 (NH),
1,645 (amide C=O) cm-1; 1H NMR (300 MHz, DMSO-d6)
d ppm: 2.14 (s, 6H, pyrrole-2CH3), 5.72 (s, 2H, pyrrole-C3
and C4–H), 7.48–8.66 (m, 9H, Ar–H and –N=CH–Ar),
10.62 (s, 1H, amide NH, disappeared on D2O exchange);
13C NMR (300 MHz, DMSO-d6) d ppm: 170.24 (amide
C=O), 154.34 (CH=N), 143.33 (bridging phenyl-C4),
133.44 (phenyl-C3 and C5), 132.56 (bridging phenyl-C2
and C6), 131.74 (phenyl-C2 and C6), 130.42 (phenyl-C1),
130.22 (bridging phenyl-C1), 129.64 (pyrrole-C2 and C5),
129.56 (bridging phenyl-C3 and C5), 125.60 (phenyl-C4),
110.87 (pyrrole-C3 and C4), 10.44 (pyrrole-2CH3); FAB
MS m/z: found 397 [M??H], 398 [M??H?1], 317, 229,
171, 94; calcd. 396.28. Anal C20H18BrN3O.
N’-(2-Nitrobenzylidene)-4-(1H-2,5-dimethylpyrrol-1-
yl)benzohydrazide (7f)
(Yield 62 %). mp 229–231 °C; IR (KBr) 3,227 (NH), 1,630
(amide C=O), 1,535 (NO2) cm-1 1H NMR (300 MHz,
;
DMSO-d6) d ppm: 1.95 (s, 6H, pyrrole-2CH3), 5.78 (s, 2H,
pyrrole-C3 and C4–H), 7.50–8.86 (m, 8H, Ar–H and
–N=CH–Ar), 12.30 (s, 1H, amide NH, disappeared on D2O
exchange); 13C NMR (300 MHz, DMSO-d6) d ppm: 164.52
(amide C=O), 147.44 (CH=N), 144.26 (2-nitro-phenyl-C5),
142.87 (bridging phenyl-C4), 135.26 (2-nitro-phenyl-C1),
133.89 (2-nitro-phenyl-C2), 131.23 (bridging phenyl-C2 and
C6), 130.22 (2-nitro-phenyl-C3), 128.92 (bridging phenyl-
C1), 128.10 (2-nitro-phenyl-C6), 127.44 (2-nitro-phenyl-
C4), 124.78 (bridging phenyl-C3 and C5), 121.57 (pyrrole-C2
and C5), 107.44 (pyrrole-C3 and C4), 13.84 (pyrrole-2CH3);
MS m/z: found 362 [M?], 363 [M?1], 317, 229, 171, 94;
calcd. 362.38. Anal C20H18N4O3.
N0-(2,6-Dichlorobenzylidene)-4-(1H-2,5-dimethylpyrrol-1-
yl)benzohydrazide (7d)
(Yield 55 %). mp 234–236 °C; IR (KBr) 3,255 (NH),
1,638 (amide C=O) cm-1; 1H NMR (300 MHz, DMSO-d6)
d ppm: 2.10 (s, 6H, pyrrole-2CH3), 5.70 (s, 2H, pyrrole-C3
and C4–H), 7.24–8.72 (m, 8H, Ar–H and –N=CH–Ar),
11.90 (s, 1H, amide NH, disappeared on D2O exchange);
13C NMR (300 MHz, DMSO-d6) d ppm: 171.56 (amide
C=O), 153.44 (CH=N), 140.65 (bridging phenyl-C4),
135.64 (phenyl-C2 and C6), 133.80 (phenyl-C4), 132.54
(phenyl-C1), 131.44 (bridging phenyl-C2 and C6), 129.84
(pyrrole-C2 and C5), 129.54 (bridging phenyl-C1), 127.64
(phenyl-C3 and C5), 126.66 (bridging phenyl-C3 and C5),
108.42 (pyrrole-C3 and C4), 10.67 (pyrrole-2CH3); FAB
MS m/z: found 387 [M??H], 388 [M??H?1], 317, 229,
171, 94; calcd. 386.27. Anal C20H17Cl2N3O.
N0-(4-Hydroxybenzylidene)-4-(1H-2,5-dimethylpyrrol-1-
yl)benzohydrazide (7g)
(Yield 58 %). mp 256–258 °C; IR (KBr) 3,457 (OH), 3,268
(NH), 1,648 (amide C=O) cm-1 1H NMR (300 MHz,
;
DMSO-d6) d ppm: 1.93 (s, 6H, pyrrole-2CH3), 5.82 (s, 2H,
pyrrole-C3 and C4–H), 7.66–8.90 (m, 8H, Ar–H and
–N=CH–Ar), 11.38 (s, 1H, amide NH, disappeared on D2O
exchange), 12.22 (s, 1H, OH, disappeared on D2O
exchange); 13C NMR (300 MHz, DMSO-d6) d ppm: 165.62
(amide C=O), 148.63 (CH=N), 143.82 (bridging phenyl-C4),
143.56 (4-hydroxy-phenyl-C5), 136.54 (4-hydroxy-phenyl-
C1), 132.54 (4-hydroxy-phenyl-C2), 131.54 (bridging phe-
nyl-C2 and C6), 130.29 (4-hydroxy-phenyl-C3), 128.84
(bridging phenyl-C1), 128.16 (4-hydroxy-phenyl-C6),
127.22 (4-hydroxy-phenyl-C4), 123.84 (bridging phenyl-C3
and C5), 121.26 (pyrrole-C2 and C5), 107.54 (pyrrole-C3 and
C4), 13.84 (pyrrole-2CH3); MS m/z: found 334 [M?], 335
[M?1], 317, 229, 171, 94; calcd. 333.38. Anal C20H19N3O2.
N’-(3-Nitrobenzylidene)-4-(1H-2,5-dimethylpyrrol-1-
yl)benzohydrazide (7e)
(Yield 62 %). mp 225–227 °C; IR (KBr) 3,223 (NH),
1,659 (amide C=O), 1,532 (NO2) cm-1
;
1H NMR
(300 MHz, DMSO-d6) d ppm: 1.97 (s, 6H, pyrrole-2CH3),
5.84 (s, 2H, pyrrole-C3 and C4–H), 7.42–8.55 (m, 8H, Ar–
H and –N=CH–Ar), 12.25 (s, 1H, amide NH, disappeared
on D2O exchange); 13C NMR (300 MHz, DMSO-d6) d
ppm: 162.83 (amide C=O), 148.25 (CH=N), 145.50 (3-
nitro-phenyl-C5), 141.38 (bridging phenyl-C4), 136.17 (3-
N0-(4-N,N-Dimethylaminobenzylidene)-4-(1H-2,5-
dimethylpyrrol-1-yl)benzohydrazide (7h)
(Yield 65 %). mp 230–232 °C; IR (KBr) 3,274 (NH),
1,664 (amide C=O) cm-1; 1H NMR (300 MHz, DMSO-d6)
123

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Med Chem Res (2013) 22:1073–1089
d ppm: 1.95 (s, 6H, pyrrole-2CH3), 2.96 (s, 6H, 2CH3),
5.81 (s, 2H, pyrrole-C3 and C4–H), 7.52–8.46 (m, 8H, Ar–
H and –N=CH–Ar), 12.12 (s, 1H, amide NH, disappeared
on D2O exchange); MS m/z: found 361 [M?], 362 [M?1],
317, 229, 171, 94; calcd. 360.45. Anal C22H24N4O.
396 [M??H?1], 352, 317, 241, 171, 94; calcd. 393.87
Anal C22H20ClN3O2.
3-Acetyl-5-[4-(2,5-dimethylpyrrol-1-yl)phenyl]-2-(2-bromo
phenyl)-2,3-dihydro-1,3,4-oxadiazole (8c)
General procedure for the synthesis of 3-acetyl-5-[4-
(2,5-dimethylpyrrol-1-yl)phenyl]-2-substituted-2,3-
dihydro-1,3,4-oxadiazoles (8a–8h)
(Yield 55 %). mp 229–231 °C; IR (KBr) 1,666 (acetyl
1
C=O) cm-1; H NMR (300 MHz, DMSO-d6) d ppm: 2.06
(s, 6H, pyrrole-2CH3), 2.18 (s, 3H, acetyl-CH3), 5.70 (s,
2H, pyrrole-C3 and C4–H), 7.02 (s, 1H, oxadiazole-C2–H),
7.32–8.68 (m, 8H, Ar–H); 13C NMR (300 MHz, DMSO-
d6) d ppm: 171.42 (acetyl C=O), 154.88 (oxadiazole-C5),
141.78 (phenyl-C4 at C5 of oxadiazole), 140.26 (bridging
phenyl-C4), 132.52 (phenyl-C1 at C5 of oxadiazole), 131.44
(bridging phenyl-C1), 129.25 (bridging phenyl-C2 and C6),
127.48 (pyrrole-C2 and C5), 127.12 (phenyl-C2 and C6 at
C5 of oxadiazole), 126.26 (phenyl-C3 and C5 at C5 of
oxadiazole), 124.48 (bridging phenyl-C3 and C5), 110.88
(pyrrole-C3 and C4), 94.56 (oxadiazole-C2), 26.48 (CH3),
10.56 (pyrrole-2CH3); FAB MS m/z: found 439 [M??H],
440 [M??H?1], 396, 317, 241, 171, 94; calcd. 438.32.
Anal C22H20BrN3O2.
A mixture of 7 (3 mmol) and acetic anhydride (10 mL) was
refluxed for 4 h. After the reaction mixture attained room
temperature, excess acetic anhydride was decomposed by
water and the mixture was stirred for further 30 min. The
separated product was filtered, washed with water, dried and
recrystallized from ethanol to give the products.
3-Acetyl-5-[4-(2,5-dimethylpyrrol-1-yl)phenyl]-2-phenyl-
2,3-dihydro-1,3,4- oxadiazole (8a)
(Yield 62 %). mp 225–227 °C; IR (KBr) 1,650 (acetyl
C=O) cm-1; 1H NMR (300 MHz, DMSO-d6) d ppm: 1.95 (s,
6H, pyrrole-2CH3), 2.18 (s, 3H, acetyl-CH3), 5.81 (s, 2H,
pyrrole-C3 and C4–H), 7.09 (s, 1H, oxadiazole-C2–H),
7.52–7.82 (m, 9H, Ar–H); 13C NMR (300 MHz, DMSO-d6) d
ppm: 171.24 (acetyl C=O), 155.45 (oxadiazole-C5), 142.88
(phenyl-C4 at C5 of oxadiazole), 141.34 (bridging phenyl-
C4), 132.70(phenyl-C1 atC5 of oxadiazole), 131.22(bridging
phenyl-C1), 129.68 (bridging phenyl-C2 and C6), 129.36
(pyrrole-C2 and C5), 127.33 (phenyl-C2 and C6 at C5 of
oxadiazole), 126.34 (phenyl-C3 and C5 at C5 of oxadiazole),
122.44 (bridging phenyl-C3 and C5), 111.22 (pyrrole-C3 and
C4), 94.32 (oxadiazole-C2), 26.12 (CH3), 12.08 (pyrrole-
2CH3); FAB MS m/z: found 360 [M??H], 361 [M??H?1],
317, 241, 171, 94; calcd. 359.42. Anal C22H21N3O2.
3-Acetyl-5-[4-(2,5-dimethylpyrrol-1-yl)phenyl]-2-(2,6-
dichloro phenyl)-2,3-dihydro-1,3,4-oxadiazole (8d)
(Yield 51 %). mp 195–198 °C; IR (KBr) 1,606 (acetyl
1
C=O) cm-1; H NMR (300 MHz, DMSO-d6) d ppm: 1.91
(s, 6H, pyrrole-2CH3), 2.12 (s, 3H, acetyl-CH3), 5.82 (s,
2H, pyrrole-C3 and C4–H), 7.12 (s, 1H, oxadiazole-C2–H),
7.51–7.96 (m, 7H, Ar–H); 13C NMR (300 MHz, DMSO-
d6) d ppm: 170.25 (acetyl C=O), 155.42 (oxadiazole-C5),
141.52 (bridging phenyl-C4), 141.26 (phenyl-C4 at C5 of
oxadiazole), 132.88 (phenyl-C1 at C5 of oxadiazole),
131.56 (bridging phenyl-C1), 129.82 (bridging phenyl-C2
and C6), 128.28 (pyrrole-C2 and C5), 127.56 (phenyl-C2
and C6 at C5 of oxadiazole), 126.84 (phenyl-C3 and C5 at
C5 of oxadiazole), 125.36 (bridging phenyl-C3 and C5),
110.54 (pyrrole-C3 and C4), 93.86 (oxadiazole-C2), 27.24
(CH3), 12.46 (pyrrole-2CH3); FAB MS m/z: found 429
[M??H], 430 [M??H?1], 386, 317, 241, 171, 94; calcd.
428.31. Anal C22H19Cl2N3O2.
3-Acetyl-5-[4-(2,5-dimethylpyrrol-1-yl)phenyl]-2-(2-
chloro phenyl)-2,3-dihydro-1,3,4-oxadiazole (8b)
(Yield 58 %). mp 210–212 °C; IR (KBr) 1,656 (acetyl
1
C=O) cm-1; H NMR (300 MHz, DMSO-d6) d ppm: 2.02
(s, 6H, pyrrole-2CH3), 2.16 (s, 3H, acetyl-CH3), 5.74 (s,
2H, pyrrole-C3 and C4–H), 6.82 (s, 1H, oxadiazole-C2–H),
7.20–8.46 (m, 8H, Ar–H),; 13C NMR (300 MHz, DMSO-
d6) d ppm: 170.32 (acetyl C=O), 154.25 (oxadiazole-C5),
141.44 (phenyl-C4 at C5 of oxadiazole), 140.12 (bridging
phenyl-C4), 132.90 (phenyl-C1 at C5 of oxadiazole), 130.56
(bridging phenyl-C1), 129.67 (bridging phenyl-C2 and C6),
127.63 (pyrrole-C2 and C5), 127.38 (phenyl-C2 and C6 at
C5 of oxadiazole), 126.78 (phenyl-C3 and C5 at C5 of
oxadiazole), 124.22 (bridging phenyl-C3 and C5), 110.42
(pyrrole-C3 and C4), 93.12 (oxadiazole-C2), 27.54 (CH3),
11.88 (pyrrole-2CH3); FAB MS m/z: found 395 [M??H],
3-Acetyl-5-[4-(2,5-dimethylpyrrol-1-yl)phenyl]-2-(3-nitro
phenyl)-2,3-dihydro-1,3,4-oxadiazole (8e)
(Yield 53 %). mp 218–220 °C; IR (KBr) 1,648 (acetyl
C=O) cm-1; 1H NMR (300 MHz, DMSO-d6) d ppm: 1.98 (s,
6H, pyrrole-2CH3), 2.14 (s, 3H, acetyl-CH3), 5.82 (s, 2H,
pyrrole-C3 and C4–H), 7.02 (s, 1H, oxadiazole-C2–H),
7.80–8.00 (m, 8H, Ar–H); 13C NMR (300 MHz, DMSO-d6) d
ppm: 170.32 (acetyl C=O), 156.44 (oxadiazole-C5), 145.12
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Med Chem Res (2013) 22:1073–1089
1081
(phenyl-C4 at C5 of oxadiazole), 139.42 (bridging phenyl-
C4), 131.56(phenyl-C1 atC5 of oxadiazole), 130.24(bridging
phenyl-C1), 129.52 (bridging phenyl-C2 and C6), 127.24
(phenyl-C2 and C6 at C5 of oxadiazole), 121.24 (phenyl-C3
and C5 at C5 of oxadiazole), 120.46 (bridging phenyl-C3 and
C5), 116.48 (pyrrole-C2 and C5), 111.24 (pyrrole-C3 and C4),
95.24 (oxadiazole-C2), 27.44 (CH3), 12.02 (pyrrole-2CH3);
MS m/z: found 404 [M?], 405 [M?1], 362, 311, 239, 235,
171, 94; calcd. 404.42. Anal C22H20N4O4.
slowly quenched onto crushed ice with stirring and neu-
tralized with saturated sodium bicarbonate solution. The
solid which separated was filtered, washed with cold eth-
anol, dried and recrystallized from aqueous DMF to give
the products.
N-(4-Pyrrol-1-yl)benzoyl)benzoic acid hydrazide (11a)
(Yield 91 %). mp 291–294 °C; IR (KBr) 3,241 (NH),
3,135 (NH), 1,683 (amide C=O), 1,645 (amide
;
C=O) cm-1 1H NMR (300 MHz, DMSO-d6) d ppm:
Synthesis of N-(2,5-dimethyl-pyrrol-1-yl)-4-(2,5-
dimethylpyrrol-1-yl)benzamide (9)
6.36 (s, 2H, pyrrole-C3 and C4–H), 7.56–8.14 (m, 11H,
pyrrole-C2 and C5–H, Ar–H), 9.40 (s, 1H, CONH, dis-
appeared on D2O exchange), 10.61 (s, 1H, NHCO, dis-
appeared on D2O exchange); 13C NMR (300 MHz,
DMSO-d6) d ppm: 167.20 (C=O), 144.21 (bridging
phenyl-C4), 133.12 (phenyl-C1), 129.12 (phenyl-C4),
128.16 (bridging phenyl-C2 and C6), 127.30 (phenyl-C2
and C6), 124.02 (bridging phenyl-C1), 121.26 (phenyl-C3
and C5), 119.32 (bridging phenyl-C3 and C5), 118.90
(pyrrole-C2 and C5), 111.20 (pyrrole-C3 and C4); MS m/
z: found 307 [M??H], 229, 153, 143, 66; calcd. 305.33
Anal C18H15N3O2.
To a suspension of 4-(2,5-dimethylpyrrol-1-yl)benzoic acid
hydrazide (2) (0.687 g, 3 mmol) in ethanol (10 mL) were
added acetonyl acetone (0.684 g, 6 mmol) and glacial
acetic acid (1 mL) and the reaction mixture was heated on
a boiling water bath for 5 h. The reaction mixture was
concentrated to half of original volume and poured into
crushed ice (50 g). The separated solid was filtered, washed
with water, dried and recrystallized from ethanol.
(Yield 74 %). mp 236–238 °C; IR (KBr) 3,266 (NH),
1,672 (amide C=O) cm-1; 1H NMR (300 MHz, DMSO-d6)
d ppm: 1.88 (s, 6H, pyrrole methyls), 2.10 (s, 6H, pyrrole
methyls), 5.68 (s, 2H, dimethyl pyrrole-C3 and C4–H), 5.84
(s, 2H, pyrrole-C3 and C4–H), 7.78 (d, J = 8.5 Hz, 2H,
bridging phenyl-C3 and C5–H), 8.20 (d, J = 8.5 Hz, 2H,
bridging phenyl-C2 and C6–H), 12.16 (s, 1H, NH, disap-
peared on D2O exchange); 13C NMR (300 MHz, DMSO-d6)
d ppm: 164.64 (amide C=O), 144.64 (bridging phenyl-C4),
131.56 (bridging phenyl-C1), 128.26 (bridging phenyl-C2
and C6), 127.84 (bridging phenyl-C3 and C5), 127.55
(dimethyl pyrrole-C2 and C5), 127.15 (pyrrole-C2 and C5),
107.87 (pyrrole-C3 and C4), 105.54 (dimethyl pyrrole-C3 and
C4), 12.08 (2CH3), 11.44 (2CH3); MS m/z: found 308 [M?],
309 [M?1], 214, 171, 94; calcd. 307.39. Anal C19H21N3O.
N-(4-Pyrrol-1-yl)-2-bromo benzoyl)benzoic acid hydrazide
(11b)
(Yield 86 %). mp 268–270 °C; IR (KBr) 3,199 (NH),
1,605 (amide C=O) cm-1; 1H NMR (300 MHz, DMSO-d6)
d ppm: 6.34 (s, 2H, pyrrole-C3 and C4–H), 7.44–8.09 (m,
10H, pyrrole-C2 and C5–H, Ar–H), 9.35 (s, 1H, CONH,
disappeared on D2O exchange), 10.54 (s, 1H, NHCO, dis-
appeared on D2O exchange); 13C NMR (300 MHz, DMSO-
d6) d ppm: 167.52 (C=O), 143.80 (bridging phenyl-C4),
132.80 (2-bromo phenyl-C1), 129.20 (2-bromo phenyl-C4),
128.20 (bridging phenyl-C2 and C6), 127.52 (2-bromo
phenyl-C2 and C6), 124.22 (bridging phenyl-C1), 122.40
(2-bromo phenyl-C3 and C5), 119.12 (bridging phenyl-C3
and C5), 118.80 (pyrrole-C2 and C5), 111.40 (pyrrole-C3
and C4); MS m/z: found 385 [M??1] 229, 153, 143, 66;
calcd. 384.23 Anal C18H14BrN3O2.
Synthesis of 4-pyrrol-1-yl benzoic acid hydrazide (10)
(Joshi et al., 2008a) Ethyl 4-pyrrol-1-yl benzoate (3)
(3.22 g, 15 mmol) was refluxed with hydrazine hydrate
(10 mL) in absolute ethanol (10 mL) for 3 h. The reaction
mixture was cooled and the crystalline mass obtained was
recrystallized from ethanol (yield 74 %) mp 180–182 °C.
N-(4-Pyrrol-1-yl)-4-bromo benzoyl)benzoic acid
hydrazide (11c)
General procedure for the synthesis of
N0-(arylcarbonyl)-4-(1H-pyrrol-1-yl)benzohydrazide
derivatives (11a–h)
(Yield 80 %). mp 262–264 °C. IR (KBr) 3,138 (NH), 1,608
(amide C=O) cm-1 1H NMR (300 MHz, DMSO-d6) d
;
ppm: 6.30 (s, 2H, pyrrole-C3 and C4–H), 7.42–8.26 (m,
10H, pyrrole-C2 and C5–H, Ar–H), 9.50 (s, 1H, CONH,
disappeared on D2O exchange), 10.62 (s, 1H, NHCO, dis-
appeared on D2O exchange); MS m/z: found 385 [M??1]
229, 153, 143, 66; calcd. 384.23 Anal C18H14BrN3O2.
A mixture of 10 (1 mmol) and substituted acyl chlorides
(1.5 mmol) in 10 mL of dry DMF was refluxed for 7–8 h
(monitored by TLC). The reaction mixture was cooled and
123

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Med Chem Res (2013) 22:1073–1089
N-(4-Pyrrol-1-yl)-2-nitro benzoyl)benzoic acid hydrazide
(11d)
NHCO, disappeared on D2O exchange); MS m/z: found 392
[M? ? 1] 229, 153, 143, 66; calcd. 391.32 Anal
C19H13F4N3O2.
(Yield 76 %). mp 274–276 °C. IR (KBr) 3,181 (NH), 1,606
(amide C=O) cm-1 1H NMR (300 MHz, DMSO-d6) d
;
General procedure for the synthesis of 2-aryl-5-[4-(1H-
pyrrol-1-yl)phenyl]-1,3,4-oxadiazole derivatives
(12a–h)
ppm: 6.32 (s, 2H, pyrrole-C3 and C4–H), 7.52-8.46 (m,
10H, pyrrole-C2 and C5–H, Ar–H), 9.48 (s, 1H, CONH,
disappeared on D2O exchange), 10.40 (s, 1H, NHCO, dis-
appeared on D2O exchange); MS m/z: found 351 [M??1]
229, 153, 143, 66; calcd. 350.33 Anal C18H14N4O4.
To a suspension of 11 (1 mmol) in dry DMF (50 mL),
phosphorus pentoxide (2.0 g) was added portion wise with
stirring at room temperature and the reaction mixture was
refluxed for 4 h. The reaction mixture was cooled and then
filtered. The resulting solid that separated was washed with
water, dried and recrystallized from aqueous DMF to give
the products.
N-(4-Pyrrol-1-yl)-3-nitro benzoyl)benzoic acid hydrazide
(11e)
(Yield 80 %). mp 280–284 °C. IR (KBr) 3,181 (NH), 1,606
(amide C=O) cm-1 1H NMR (300 MHz, DMSO-d6) d
;
ppm: 6.32 ppm (s, 2H, pyrrole-C3 and C4–H), 7.62–8.24
(m, 10H, pyrrole-C2 and C5–H, Ar–H), 9.44 (s, 1H, CONH,
disappeared on D2O exchange), 10.64 (s, 1H, NHCO, dis-
appeared on D2O exchange); MS m/z: found 351 [M??1]
229, 153, 143, 66; calcd. 350.33 Anal C18H14N4O4.
2-Phenyl-5-[4-(1H-pyrrol-1-yl)phenyl]-1,3,4-oxadiazole
(12a)
(Yield 85 %). mp 284–286 °C; IR (KBr) 1,637
(C=N) cm-1 1H NMR (500 MHz, DMSO-d6) d ppm:
;
6.34 (dd, 2H, pyrrole-C3 and C5–H), 7.55 (dd, 2H, pyr-
role-C2 and C4–H), 7.65 (d, 2H, C3–H and C5–H), 7.88
(d, 2H, C2–H and C6–H of phenyl ring at oxadiazole),
8.15–8.20 (m, 5H, C2–H and C6–H, C3, C4 and C5–H of
phenyl ring at oxadiazole); 13C NMR (500 MHz, DMSO-
d6) d ppm: 164.40 (oxadiazole-C5), 164.10 (oxadiazole-
C2), 142.78 (bridging phenyl-C4), 132.50 (phenyl-C1 at C5
of oxadiazole), 129.89 (phenyl-C4 at C5 of oxadiazole),
128.75 (bridging phenyl-C2 and C6), 127.11 (phenyl-C2
and C6 at C5 of oxadiazole), 123.86 (bridging phenyl-C1),
120.21 (phenyl-C3 and C5 at C5 of oxadiazole), 119.91
(bridging phenyl-C3 and C5), 119.51 (pyrrole-C2 and C5),
111.9 (pyrrole-C3 and C4); FAB MS m/z: found 288
[M?], 211, 170, 153, 143, 66; calcd. 287.32 Anal
C18H13N3O.
N-(4-Pyrrol-1-yl)-4-nitro benzoyl)benzoic acid hydrazide
(11f)
(Yield 58 %). mp 278–280 °C. IR (KBr) 3,195 (NH), 1,693
(amide C=O), 1,607 (amide C=O) cm-1
;
1H NMR
(300 MHz, DMSO-d6) d ppm: 6.34 (s, 2H, pyrrole-C3 and
C4–H), 7.52 (s, 2H, pyrrole-C2 and C5–H), 7.78–8.42 (m,
8H, Ar–H), 9.46 (s, 1H, CONH, disappeared on D2O
exchange), 10.80 (s, 1H, NHCO, disappeared on D2O
exchange); MS m/z: found 351 [M??1] 229, 153, 143, 66;
calcd. 350.33 Anal C18H14N4O4.
N-(4-Pyrrol-1-yl)-4-chloro benzoyl)benzoic acid hydrazide
(11g)
(Yield 90 %). mp 262–264 °C; IR (KBr) 3,191 (NH), 1,605
(amide C=O) cm-1; 1H NMR (300 MHz, DMSO-d6) d ppm:
6.30 (s, 2H, pyrrole-C3 and C4–H), 7.42 (s, 2H, pyrrole-C2
and C5–H), 7.82–8.46 (m, 8H, Ar–H), 9.44 (s, 1H, CONH,
disappeared on D2O exchange), 10.56 (s, 1H, NHCO, dis-
appeared on D2O exchange); MS m/z: found 341 [M??1]
229, 153, 143, 66; calcd. 339.78 Anal C18H14ClN3O2.
2-(2-Bromophenyl)-5-[4-(1H-pyrrol-1-yl)phenyl]-1,3,4-
oxadiazole (12b)
(Yield 88 %). mp 264–266 °C; IR (KBr) 1,618
(C=N) cm-1; 1H NMR (500 MHz, DMSO-d6) d ppm: 6.32
(dd, 2H, pyrrole-C3 and C5–H), 7.51 (dd, 2H, pyrrole-C2
and C4–H), 7.72 (d, 2H, C3–H and C5–H), 7.78–8.06 (m,
6H, C2–H and C6–H, C3, C4, C5, and C6–H of phenyl ring
at oxadiazole); 13C NMR (500 MHz, DMSO-d6) d ppm:
163.70 (oxadiazole-C5), 154.88 (oxadiazole-C2), 142.79
(bridging phenyl-C4), 133.37 (bridging phenyl-C1), 130.02
(phenyl-C1 at C5 of oxadiazole), 129.05 (phenyl-C2 and C6
at C5 of oxadiazole), 128.79 (bridging phenyl-C2 and C6),
127.96 (phenyl-C4 at C5 of oxadiazole), 119.96 (phenyl-C3
and C5 at C5 of oxadiazole), 119.48 (bridging phenyl-C3
and C5), 118.77 (pyrrole-C2 and C5), 111.90 (pyrrole-C3
N-(4-Pyrrol-1-yl)-4-fluoro-3-trifluoromethyl
benzoyl)benzoic acid hydrazide (11h)
(Yield 57 %). mp 272–274 °C; IR (KBr) 3,195 (NH),
1,610 (amide C=O) cm-1; 1H NMR (300 MHz, DMSO-d6)
d ppm: 6.34 (s, 2H, pyrrole-C3 and C4–H), 7.46 (s, 2H,
pyrrole-C2 and C5–H), 7.78–8.56 (m, 7H, Ar–H), 9.56 (s,
1H, CONH, disappeared on D2O exchange), 10.82 (s, 1H,
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Med Chem Res (2013) 22:1073–1089
1083
and C4); MS m/z: found 367 [M??1], 287, 211, 170, 153,
143, 66; calcd. 366.21 Anal C18H12BrN3O.
(bridging phenyl-C4), 142.88 (bridging phenyl-C1), 138.61
(phenyl-C4 at C5 of oxadiazole), 129.66 (bridging phenyl-C2
and C6), 129.47 (phenyl-C1 at C5 of oxadiazole), 129.01
(phenyl-C2 and C6 at C5 of oxadiazole), 124.26 (phenyl-C3
and C5 at C5 of oxadiazole), 119.49 (bridging phenyl-C3 and
C5), 119.02 (pyrrole-C2 and C5), 111.75 (pyrrole-C3 and C4);
MS m/z: found 332 [M?], 287, 211, 170, 153, 143, 66, calcd.
332.31. Anal C18H12N4O3.
2-(4-Bromophenyl)-5-[4-(1H-pyrrol-1-yl)phenyl]-1,3,4-
oxadiazole (12c)
(Yield 76 %). mp 258–260 °C; IR (KBr) 1,605
(C=N) cm-1; 1H NMR (300 MHz, DMSO-d6) d ppm: 6.33
(d, 2H, pyrrole-C2 and C5–H), 7.54 (d, 2H, pyrrole-C3 and
C4–H), 7.83 (d, 2H, C3–H and C5–H), 7.87 (d, 2H, C2–
H and C6–H of phenyl ring at oxadiazole), 8.09 (d, 2H, C2–
H and C6–H), 8.19 (d, 2H, C3–H and C5–H of phenyl ring
at oxadiazole); MS m/z: found 367 [M?], 287, 211, 170,
153, 143, 66, calcd. 366.21. Anal C18H12BrN3O.
2-(4-Chlorophenyl)-5-[4-(1H-pyrrol-1-yl)phenyl]-1,3,4-
oxadiazole (12g)
(Yield 86 %). mp 254–256 °C; IR (KBr) 1,601 (C=N)
1
cm-1; H NMR (500 MHz, DMSO-d6) d ppm: 6.35 (dd,
2H, pyrrole-C3 and C4–H), 7.50 (s, 2H, pyrrole-C2 and
C5–H), 7.54 (d, 1H, bridging phenyl-C6–H), 7.74 (d, 1H,
bridging phenyl-C2–H), 7.87 (d, 1H, C5–H of phenyl ring
at oxadiazole), 7.95 (d, 1H, C3–H of phenyl ring at oxa-
diazole), 8.00 (d, 1H, C6–H of phenyl ring at oxadiazole),
8.11 (d, 1H, C2–H of phenyl ring at oxadiazole), 8.19
(d, 2H, C3–H and C5–H); MS m/z: found 323 [M?1], 170,
153, 143, 139, 112, 66, calcd. 321.76. Anal C18H12ClN3O.
2-(2-Nitrophenyl)-5-[4-(1H-pyrrol-1-yl)phenyl]-1,3,4-
oxadiazole (12d)
(Yield 70 %). mp 270–272 °C. IR (KBr) 1,605
(C=N) cm-1; 1H NMR (500 MHz, DMSO-d6) d ppm: 6.30
(dd, 2H, pyrrole-C3 and C4–H), 7.48 (dd, 2H, pyrrole-C2
and C5–H), 7.72 (d, 2H, C6–H and C2–H), 7.78 (s, 2H, C3–
H and C5–H), 8.06–8.09 (m, 4H, C3, C4, C5, and C6–H of
phenyl ring at oxadiazole); MS m/z: found 332 [M?], 211,
170, 153, 143, 66, calcd. 332.31. Anal C18H12N4O3.
2-[4-Fluoro-3-(trifluoromethyl)phenyl]-5-[4-(1H-pyrrol-1-
yl)phenyl]-1,3,4-oxadiazole (12h)
(Yield 56 %) mp 266–268 °C; IR (KBr) 1,614
(C=N) cm-1; 1H NMR (500 MHz, DMSO-d6) d ppm: 6.33
(dd, 2H, pyrrole-C3 and C5–H), 7.52 (dd, 2H, pyrrole-C2
and C4–H), 7.69–8.33 (m, 7H, C2, C3, C5, C6–H and C2, C5,
C6–H of phenyl ring at oxadiazole); 13C NMR (500 MHz,
DMSO-d6) d ppm: 165.39 (trifluoromethyl-C), 164.00
(oxadiazole-C5), 163.70 (oxadiazole-C2), 142.86 (bridging
phenyl-C4), 120.10 (bridging phenyl-C1), 135.32 (phenyl-
C1 at C5 of oxadiazole), 129.08 (bridging phenyl-C2 and
C6), 128.79 (phenyl-C2 and C6 at C5 of oxadiazole), 127.96
(phenyl-C4 at C5 of oxadiazole), 119.95 (phenyl-C3 and C5
at C5 of oxadiazole), 119.48 (bridging phenyl-C3 and C5),
119.03 (pyrrole-C2 and C5), 111.90 (pyrrole-C3 and C4);
MS m/z: found 373 [M?], 211, 170, 164, 143, 66, calcd.
373.30. Anal C19H11F4N3O.
2-(3-Nitrophenyl)-5-[4-(1H-pyrrol-1-yl)phenyl]-1,3,4-
oxadiazole (12e)
(Yield 85 %). mp 274–276 °C; IR (KBr) 1,604
(C=N) cm-1; 1H NMR (500 MHz, DMSO-d6) d ppm: 6.32
(dd, 2H, pyrrole-C3 and C4–H), 7.51 (dd, 2H, pyrrole-C2
and C5–H), 7.71 (d, 2H, C3–H and C5–H), 7.87 (t, 1H, C5–
H of phenyl ring at oxadiazole), 8.03 (d, 2H, C2–H and C6–
H), 8.37 (dd, 1H, C6–H of phenyl ring at oxadiazole), 8.46
(dd, 1H, C4–H of phenyl ring at oxadiazole), 8.75 (t, 1H,
C2–H of phenyl ring at oxadiazole); MS m/z: found 332
[M?], 287, 211, 170, 153, 143, 66, calcd. 332.31. Anal
C18H12N4O3.
2-(4-Nitrophenyl)-5-[4-(1H-pyrrol-1-yl)phenyl]-1,3,4-
oxadiazole (12f)
Biological activities
(Yield 64 %). mp 268–270 °C; IR (KBr) 1,619 (C=N)
cm-1; 1H NMR (500 MHz, DMSO-d6) d ppm: 6.32 (dd, 2H,
pyrrole-C3 and C4–H), 7.51 (dd, 2H, pyrrole-C2 and C5–H),
7.71 (d, 2H, C3–H and C5–H), 8.02 (d, 2H, C2–H and
C6–H), 8.16 (dd, 2H, C2–H and C6–H of phenyl ring at
oxadiazole), 8.38 (dd, 2H, C3–H and C5–H of phenyl ring at
oxadiazole); 13C NMR (500 MHz, DMSO-d6) d ppm:
165.41 (oxadiazole-C5), 164.86 (oxadiazole-C2), 149.92
In vitro evaluation of antimicrobial activity
The minimum inhibitory concentration (MIC) determina-
tion of the tested compounds (2, 3a–d, 4, 5, 6, 7a–h, 8a–h,
9, 11a–h, 12a–h) was carried out simultaneously in com-
parison with ciprofloxacin, norfloxacin against Gram-
positive (Staphylococcus aureus, Streptococcus faecalis,
Bacillus subtilis) and Gram-negative bacteria (Klebsiella
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Med Chem Res (2013) 22:1073–1089
pneumoniae, Escherichia coli, Pseudomonas aeruginosa)
by broth microdilution method (Goto et al., 1981). The
antifungal activity was assayed against yeasts (Candida
tropicalis, Saccharomyces cerevisiae) and moulds (Asper-
gillus niger). Serial dilutions of the test compounds and
reference drugs were prepared in Mueller–Hinton broth for
bacteria and Sabouraud Liquid Medium for fungi. Drugs
(10 mg) were dissolved in DMSO (1 mL). Further pro-
gressive dilutions were done to obtain final concentrations
radioactive cell proliferation assay (Gundersen et al.,
2002). The IC50 values are mean SEM of three inde-
pendent experiments and are presented in Table 2.
Results and discussion
Synthetic and spectral studies
of 1, 2, 4, 8, 16, 31.25, 62.5, 125, 250 and 500 lg mL-1
.
In the IR spectrum of 2, broad stretching bands around
3,285 and 3,195 cm-1 were due to amine/amide NH while
strong stretching band at 1,655 cm-1 was assigned to
The tubes were inoculated with 105 cfu mL-1 (colony
forming unit mL-1) and incubated at 37 °C for 18 h. The
MIC was the lowest concentration of the tested compound
that yields no visible growth on the plate. To ensure that
the solvent had no effect on the bacterial growth, a control
was performed with the test medium supplemented with
DMSO at the same dilutions as used in the experiments and
DMSO had no effect on the microorganisms in the con-
centrations studied.
1
amide carbonyl. The H NMR spectrum of 2 showed a
singlet at d 1.95 which was accounted for two methyl
groups on pyrrole ring. A singlet at d 4.51 was assigned to
NH2 group which disappeared on D2O exchange. The C3
and C4 protons of pyrrole ring appeared as singlet at d 5.80.
Four protons of phenyl moiety resonated as two doublets at
d 7.94 and 7.33. A broad singlet appeared at d 10.0 was
accounted for NH which vanished on D2O exchange. The
mass spectrum of 2 displayed a molecular ion peak at m/
z 229 which confirmed its molecular weight.
In vitro anti-M. tuberculosis assay
The preliminary antitubercular screening for test com-
pounds (2, 3a–d, 4, 5, 6, 7a–h, 8a–h, 9, 11a–h, 12a–h) was
carried for M. tuberculosis H37Rv, the MIC of each drug
was determined by broth dilution assay method (Suling
et al., 2000; Yajko et al., 1995) and is defined as the lowest
concentration of drug, which inhibits C99 % of bacterial
population present at the beginning of the assay. A frozen
culture in Middlebrook 7H9 broth supplemented with 10 %
albumin–dextrose–catalase and 0.2 % glycerol was thawed
and diluted in broth to 105 cfu mL-1 for M. tuberculosis
and used as the inoculum. In the assay, U-tubes were used
to accommodate compounds in 1–500 lg mL-1 dilutions.
Each test compound was dissolved in DMSO and then
diluted in broth at twice the desired concentration. The
final concentration of DMSO in the assay medium was
1.3 %. Each U-tube was then inoculated with 0.05 mL of
standardized culture and then incubated at 37 °C for
21 days. The growth in the U-tubes was compared with
visibility against positive control (without drug), negative
control (without drug and inoculum) and with a standard
drug isoniazid. The tests were carried out in triplicate.
Disappearance of NH2 stretching band in the IR spec-
trum of 3a confirmed the formation of hydrazone. A
stretching band at 3,214 cm-1 was due to amide NH, while
strong stretching band at 1,647 cm-1 was related to amide
1
carbonyl. The H NMR spectrum of 3a showed two sing-
lets at d 1.95 and 1.97 which were accounted for four
methyl groups. The C3 and C4 protons of pyrrole ring
appeared as singlet at d 5.81. The four protons of phenyl
moiety resonated as two doublets at d 7.93 and 7.37. A
singlet appeared at d 10.57 was accounted for NH which
vanished on D2O exchange. The 13C NMR data of 3a also
supported the structure which displayed a peak at d 12.91
due to two methyl carbons of pyrrole. Peaks at d 18.08 and
25.10 were due to two methyl groups. The C=N resonance
appeared at d 160.52. The FAB mass spectrum of 3a
showed a molecular ion peak (M??H) at m/z 270 which
confirmed its molecular weight.
1
Lack of H NMR resonances observed with NH and
1
NH2 functions in the H NMR spectrum of 4 proved that
ring closure starting from 2 resulted in the formation of
1,3,4-oxadiazole ring. This was further substantiated by the
13C NMR data of 4 which showed a peak at d 175.24 and
158.48 due to C2 and C5 carbons of oxadiazole. Mass
spectrum of 4 displayed a molecular ion base peak at
m/z 272 which confirmed its molecular weight.
Cytotoxic activity
The cellular conversion of MTT [3-(4,5-dimethylthiazo-2-
yl)-2,5-diphenyl-tetrazolium bromide] into a formazan
product (Mosmann, 1983) was used to evaluate cytotoxic
activity (IC50) of some synthesized compounds (4, 5, 6,
11a–h and 12a–h) against mammalian Vero cell lines and
The IR spectrum of 5 exhibited stretching bands at 3,292
and 3,122 cm-1 due to NH/NH2. The 1H NMR spectrum of
this compound showed a singlet at d 12.42 and 5.50 which
were accounted for NH and NH2, respectively. 13C NMR
spectrum of 5 showed signals at d 166.22 and 148.26 due to
C=S of triazole and C5 carbon of triazole, respectively. The
A
549 (lung adenocarcinoma) cell lines up to concentrations
of 62.5 lg mL-1 using the Promega Cell Titer 96 non-
123

Med Chem Res (2013) 22:1073–1089
1085
mass spectrum of 5 displayed a molecular ion peak at
m/z 286 which confirmed its molecular weight.
were accounted for four protons of pyrrole ring. Two
doublets were observed at d 7.65 and 7.88 which were
assigned to two protons of C3 and C5 of phenyl ring and
two protons of C2 and C6 of phenyl ring at oxadiazole,
respectively. Multiplets between d 8.15 and 8.20 were
assigned to two protons of C2 and C6 of phenyl ring and
three protons of C3, C4 and C5 of phenyl ring at oxadiazole,
respectively. Formation of oxadiazoles was further con-
firmed by their 13C NMR and mass spectra. In 13C NMR
spectrum of 12a, we have observed most characteristic
signals appeared at d 164.10 and 164.40 for C2 and C5
carbons of oxadiazole, respectively. The signals appeared
at around d 119.51 and 111.91 C3, C4, C2 and C5 carbons of
1
The three singlets at d 1.96, 9.54 and 11.42 in the H
NMR spectrum of 6 were assigned to CH3, amide NH and
NH protons, respectively. The structure of 6 was further
confirmed by the 13C NMR data which displayed signals at
d 22.52, 164.24 and 170.26 due to CH3, benzamide and
acetyl carbonyl carbons, respectively. The mass spectrum
exhibited the molecular ion peak at m/z 272 which con-
firmed its molecular weight.
Hydrazones 7a–h obtained from hydrazide 2 showed
carbonyl amide stretching bands in 1,600–1,660 cm-1
1
region and N–H bands in 3,250–3,460 cm-1 region. H
NMR and 13C NMR data were also in agreement with the
pyrrole ring and carbons of Ph groups at
d
1
formation of hydrazones. H NMR spectra of 7a showed
120.21–142.78 ppm. The mass spectrum of 12a showed a
molecular ion peak at m/z 288 which corresponds to its
molecular weight.
two singlets at d 8.56 and 11.50 which were attributed to
the N=CH and NH protons, respectively.
IR spectra of 8a–h had different characteristics as they
showed no NH stretching bands and only C=O bands at
around 1,650 cm-1 which were attributed to the C=O
stretching of acetyl group. Two singlets at around d 7.00
and 2.16 in the 1H NMR spectra of 8a–h were attributed to
the O–CHR–N resonance of the oxadiazolines ring and
acetyl CH3, respectively. The formation of the oxadiazo-
lines was further supported by the 13C NMR data of
compounds 8a–h. While the N=CH carbon of compounds
in hydrazone structures were observed at around d 148–154
in their 13C NMR spectra, the same carbon atoms (OCHR–
N) were observed at around d 93–95 after cyclisation of
oxadiazoline ring. In the 13C NMR spectrum of 8a, this
signal was observed at d 94.32 due to the sp3 hybridization.
The 1H NMR spectrum of 9 showed singlet at d 2.10 due
to methyl protons of pyrrole, while singlet at d 5.68 was
due to two protons of pyrrole. The mass spectrum of 9
showed a molecular ion peak at m/z 308 which confirmed
its molecular weight.
Antimicrobial activity
The results of antimicrobial activities of the synthesized
compounds against selected three Gram-positive, three
Gram-negative bacteria, yeasts, moulds and M. tuberculo-
sis H37Rv are illustrated in Tables 1 and 2, respectively.
The antimicrobial and antifungal activity results are
expressed in MIC values along with the activity of cipro-
floxacin, norfloxacin and fluconazole for comparison.
The antimicrobial screening data revealed that all the
compounds showed moderate to significant microbial
inhibition. Compounds 2, 3a–d, 4, 5, 6, 7a–h, 8a–h and 9
(Scheme 1) showed antimicrobial activity at MIC values of
2–500 lg mL-1. Compounds showed enhanced activity
against Gram-positive bacteria than the Gram-negative
bacteria. Compounds 4, 8d and 9 were found to be more
active than the others at an MIC value of 2 lg mL-1
against Staphylococcus aureus. The synthesized com-
pounds showed antimicrobial activity with MIC values
between 4 and 62.5 lg mL-1 against B. subtilis. Com-
pounds exhibited lower potencies against Streptococcus
faecalis. All the compounds exhibited moderate activity
against K. pneumoniae. Compounds showed lower activity
against E. coli and P. aeruginosa. Compounds 8a–h which
have 2,3-dihydro-1,3,4-oxadiazole ring were more active
than the starting Schiff bases 7a–h against some of the test
microorganisms.
Evidence for the structure 11a was obtained by the IR
spectrum which showed the presence of absorption bands
at 3,241, 3,135 cm-1 and 1,683 cm-1, 1,645 cm-1 which
were due to amide –NH and carbonyl stretching frequen-
1
cies. The H NMR spectrum of 11a showed a singlet at d
6.36 which was accounted for C3 and C4 protons of pyrrole.
The multiplets between d 7.56 and 8.14 were assigned for
two protons of C2 and C5 protons of pyrrole and nine
protons of aromatic rings, respectively. Two broad peaks at
d 9.40 and 10.61 were attributed to –NHCO and –CONH
protons, respectively. The FAB mass spectrum of 11a
showed a molecular ion peak (M??H) at m/z 307 which
confirmed its molecular weight. Disappearance of –NH
stretching bands in the IR spectrum of 12a confirmed the
formation of oxadiazole ring. The C=N stretching band
Compounds 11a–h and 12a–h (Scheme 2) showed
antimicrobial activity at MIC values of 1–500 lg mL-1
.
Compound 12h was found to be more active than the others
at an MIC value of 1 lg mL-1 against B. subtilis. The
synthesized compounds showed antimicrobial activity with
MIC values between 8 and 62.5 lg mL-1 against Strep-
tococcus faecalis. Compounds exhibited lower potencies
against K. pneumoniae. Compounds showed good activity
1
appeared at 1,637 cm-1. The H NMR spectrum of 12a
showed two doublet of doublet at d 6.34 and 7.55 which
123

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Med Chem Res (2013) 22:1073–1089
Table 1 In vitro antimicrobial activity
Compounds
MIC values (lg mL-1
Gram-positive organisms
)
Gram-negative organisms
Yeasts
Sa
Sf
Bs
Kp
Ec
Pa
Sc
Ct
An
2
62.5
31.25
62.5
62.5
31.25
2
62.5
31.25
31.25
31.25
62.5
4
62.5
16
16
31.25
62.5
4
62.5
500
500
500
500
500
500
62.5
500
500
62.5
500
500
500
500
[500
500
500
500
500
500
16
500
500
500
500
500
62.5
500
500
62.5
500
[500
500
500
[500
500
500
500
500
500
8
62.5
62.5
62.5
62.5
62.5
8
62.5
62.5
62.5
62.5
62.5
62.5
8
3a
31.25
3b
500
62.5
3c
500
62.5
3d
500
62.5
4
62.5
2
5
16
16
31.25
16
31.25
8
500
4
2
4
6
31.25
31.25
16
31.25
62.5
31.25
31.25
16
500
62.5
62.5
31.25
31.25
31.25
31.25
31.25
16
62.5
62.5
31.25
16
7a
62.5
31.25
7b
62.5
8
7c
16
16
16
31.25
31.25
31.25
31.25
16
8
500
8
16
7d
16
500
4
4
7e
31.25
62.5
16
31.25
31.25
16
500
16
16
7f
[500
62.5
16
16
7g
16
16
7h
31.25
62.5
16
31.25
62.5
31.25
31.25
2
62.5
31.25
62.5
31.25
31.25
16
31.25
31.25
31.25
16
8a
62.5
16
8b
31.25
31.25
62.5
8
8c
16
8
8
16
8d
2
4
4
4
8e
31.25
31.25
31.25
31.25
2
31.25
31.25
31.25
31.25
16
16
16
16
16
16
16
16
16
16
16
16
16
16
8
62.5
62.5
500
62.5
500
500
31.25
16
62.5
500
62.5
500
500
62.5
16
31.25
31.25
31.25
31.25
16
8
8
8f
500
8
8
8g
31.25
31.25
62.5
8
8
8h
31.25
31.25
16
9
4
11a
62.5
62.5
62.5
62.5
62.5
62.5
62.5
62.5
31.25
31.25
31.25
31.25
31.25
31.25
31.25
31.25
\5
62.5
62.5
31.25
31.25
62.5
31.25
31.25
31.25
31.25
31.25
16
[500
[500
[500
[500
[500
[500
[500
[500
[500
[500
[500
[500
[500
[500
[500
[500
B1
31.25
31.25
31.25
31.25
31.25
31.25
31.25
16
31.25
31.25
16
11b
8
11c
16
16
8
16
11d
31.25
16
16
31.25
31.25
31.25
31.25
31.25
8
11e
16
31.25
11f
16
16
31.25
11g
16
16
8
11h
8
8
4
12a
8
8
31.25
31.25
31.25
31.25
31.25
31.25
8
31.25
31.25
16
12b
8
8
8
4
12c
4
4
8
4
8
12d
16
4
4
4
31.25
31.25
31.25
31.25
16
12e
31.25
16
8
8
8
31.25
12f
4
4
4
31.25
12g
16
4
4
4
4
2
–
–
12h
8
1
2
2
4
4
Ciprofloxacin
Norfloxacin
\5
B1
B1
B1
B1
[5
[5
–
–
\5
\5
B1
–
–
123

Med Chem Res (2013) 22:1073–1089
1087
Table 1 continued
Compounds
MIC values (lg mL-1
)
Gram-positive organisms
Gram-negative organisms
Yeasts
Sa
Sf
Bs
Kp
Ec
Pa
Sc
Ct
An
Fluconazole
–
–
–
–
–
–
B1
B1
B1
MIC is expressed in lg mL-1; Gram-positive bacteria: Sa Staphylococcus aureus ATCC 11632, Sf Streptococcus faecalis ATCC 14506, Bs
Bacillus subtilis ATCC 60511; Gram-negative bacteria: Kp K. pneumoniae ATCC 10031, Ec E. coli ATCC 10536, Pa P. aeruginosa ATCC
10145; Yeasts: Sc Saccharomyces cerevisiae (ATCC 9763) and Ct C. tropicalis (ATCC 1369), mould: An A. niger (ATCC 6275)
against E. coli and P. aeruginosa. Compounds 12a–
h which have oxadiazole ring were more active than the
starting compounds 11a–h. Compounds 12c, 12d, 12f, 12g
and 12h which have electron-withdrawing substituents on
phenyl ring were found to be the most active compounds
against the tested bacteria. The majority of compounds
exhibited good inhibitory activity against Gram-positive
bacteria, moulds and yeasts than Gram-negative bacteria.
activities than electron-withdrawing nitro group. The sig-
nificant activity of compound 8d may be attributed due to
the presence of an additional electron-withdrawing group
in it. Furthermore, the derivatives having 1,3,4-oxadiazoles
12a–h have more pronounced antimycobacterial potency
compared to its precursor compounds 11a–h having
(–CONH–NHCO–) moiety. The high bioactivity of com-
pounds 12a–h makes them suitable hits for additional in
vitro and in vivo evaluations, to develop new antimyco-
bacterial drugs with potential use in the treatment of
tuberculosis.
Antitubercular activity
The tested compounds (Scheme 1) showed activities
against mycobacteria with MIC values ranging from 2 to
62.5 lg mL-1 (Table 2). Preliminary examination of the
antimycobacterial activity revealed that compounds 8a–h,
containing the 1,3,4-oxadiazoline ring and acetyl group,
showed better activity against M. tuberculosis H37Rv
and compounds 4, 8d and 9 exhibited highest activity
(MIC 2 lg mL-1). The compounds 11a–h and 12a–h
(Scheme 2) showed activities against mycobacteria with
MIC values ranging from 1 to 8 lg mL-1 (Table 2).
Compounds 12c, 12d, 12f and 12g showed antitubercular
activity at MIC value of 2 lg mL-1. Compound 12h
showed the highest activity at MIC value of 1 lg mL-1
when compared with first-line drug isoniazid (MIC =
0.25 lg mL-1).
It is a well-known fact that the biological activity
associated with the hydrazone compounds is attributed due
to the presence of the active pharmacophore (–CONH–
N=C–). Hence many hydrazone compounds 7 containing
the active moiety (–CONH–N=C–) showed good anti-
bacterial/antitubercular activities. The interesting observa-
tion is that the conversion of the (–CONH–N=C–) moiety
in active compounds 7a–h, into 1,3,4-oxadiazoles 8a–h,
strengthened their activities. The presence of electron-
withdrawing groups on the aromatic ring increases the
antibacterial and antitubercular activities as evidenced by
compounds 8b–d. Compounds 8b–d and 8e–f have the
electron-withdrawing groups but compounds 8b–d are
more active than the compounds 8e–f which indicates the
fact that the electron-withdrawing halo group is more
effective in improving the antibacterial and antitubercular
Cytotoxic activity
Some compounds 4, 5, 6, 11a–h and 12a–h were further
examined for toxicity (IC50) in mammalian Vero cell lines
and A549 (lung adenocarcinoma) cell lines up to 62.5
lg mL-1 concentrations. After 72 h of exposure, viability
was assessed on the basis of cellular conversion of MTT
into a formazan product using the Promega Cell Titer 96
non-radioactive cell proliferation assay and results are
summarized in Table 2. Among the 13 derivatives tested,
showed IC50 values ranging from 175.4 to 268.2 lM
against mammalian Vero cell lines. All the compounds did
not show significant activity against mammalian Vero cell
line at concentrations \100 lM. Among the test com-
pounds, 1,3,4-oxadiazole derivatives 12a–h showed infe-
rior toxicity with IC50 values of [250 lM against both
mammalian Vero cell lines and A549 (lung adenocarci-
noma) cell lines. A comparison of the substitution pattern
on pyrrole nucleus demonstrated that 2,5-dimethyl substi-
tuted analogs were more cytotoxic than the 2,5-unsubsti-
tuted pyrrole analogs. These results are important as these
compounds with their increased cytoliability are much
attractive in the development of new chemical entities for
the treatment of TB. This is primarily due to the fact that
the disease is chronic by its nature, the therapy needs to be
continued for at least about 1–2 years in most of the cases
and the need for an agent with a high margin of safety
becomes a primary concern. The IC50 for the compound
12h was found to be 268.2 lM against the mammalian
Vero cell lines tested.
123

1088
Med Chem Res (2013) 22:1073–1089
Conclusion
Table 2 Cytotoxicity and antimycobacterial activities of pyrrole
derivatives
Compounds MIC values (lg mL-1
M. tuberculosis H37Rv
)
IC50 (lM)
The synthesis of novel pyrrole derivatives were described,
which were also evaluated for their preliminary in vitro
antibacterial, antifungal and antitubercular activities
against M. tuberculosis H37Rv strain by broth dilution
assay method. Further, some title compounds were also
assessed for their cytotoxic activity (IC50) against mam-
malian Vero cell lines and A549 (lung adenocarcinoma) cell
lines using MTT assay method. The results indicated that
these compounds exhibit antitubercular activity at non-
cytotoxic concentrations. Due to the better activity against
tested microorganisms and mycobacteria, compounds 4,
8d, 9 and 12h have been selected for further development
and studies to acquire more information about structure–
activity relationships are in progress in our laboratories. In
summary, we have identified a novel series of substituted
pyrrole derivatives which may be developed into potential
class of antitubercular and antimicrobial agents.
MV cell linesc A5d49
2
62.5
NT
NT
NT
3a
31.25
NT
3b
31.25
NT
NT
3c
31.25
NT
NT
3d
31.25
NT
NT
4
2
261.2 0.9
160.1 1.1
172.5 1.3
NT
254 0.9
170 0.7
180 0.5
NT
5
4
6
4
7a
31.25
7b
16
NT
NT
7c
16
NT
NT
7d
16
NT
NT
7e
62.5
NT
NT
7f
62.5
NT
NT
7g
31.25
NT
NT
Acknowledgments We thank Shri. H. V. Dambal, President,
S. E. T’s College of Pharmacy, Dharwad, Karnataka, India, for pro-
viding necessary facilities. We are thankful to Dr. A. M. Godbole for
his valuable suggestions and appreciate the co-operation of Mr. Uttam
More in the preparation of this manuscript. We are grateful to Dr.
K. G. Bhat, Maratha Mandal’s Dental College, Hospital and Research
Centre, Belgaum, Karnataka, India for providing the facilities for
antibacterial and antitubercular activities. We also wish to thank
Director, SAIF, Indian Institute of Technology, Chennai, India and
Director, USIC, Karnataka University Dharwad, India for providing
NMR and Mass spectral data.
7h
31.25
NT
NT
8a
16
190.2 1.2
200.6 0.8
195.1 0.7
220.5
198 0.4
206 1.3
195 1.6
0.8 224 0.7
197 0.4
182 2.1
190 1.9
186 0.9
225 1.3
215 0.8
201 0.7
201 0.9
205 1.3
203 2.3
198 2.2
185 0.9
200 0.7
256 1.6
252 1.3
264 0.9
267 0.4
256 0.8
258 0.4
256 1.3
270 1.6
[450
8b
8
8c
8
8d
2
8e
31.25
188.4 1.2
175.4 2.3
187.2 1.7
175.4 1.5
230.1 0.6
200.4 0.7
198.2 1.5
185.6 1.2
182.3 1.4
194.7 1.3
190.1 0.9
179.3 0.9
196.2 0.5
250.2 0.7
258.2 1.5
260.1 1.6
266.2 1.8
251.4 1.6
257.6 1.2
253.1 1.9
268.2 0.6
[450
8f
31.25
8g
16
16
2
8h
9
11a
11b
11c
11d
11e
11f
11g
11h
12a
12b
12c
12d
12e
12f
12g
12h
Isoniazid
8
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