1,3-dihydro-2H-indol-2-ones derivatives: Design, Synthesis, in vitro antibacterial, antifungal and antitubercular study

Article abstract of DOI:10.1016/j.ejmech.2011.09.023

1,3-dihydro-2H-indol-2-ones derivatives are reported to exhibit a wide variety of biodynamic activities such as antituberculer, anti HIV, fungicidal, antibacterial, anticonvulsant. These valid observations led us to synthesize some new indole-2-one deriva

Full text of DOI:10.1016/j.ejmech.2011.09.023

European Journal of Medicinal Chemistry 46 (2011) 5573e5579
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Original article
1,3-dihydro-2H-indol-2-ones derivatives: Design, Synthesis, in vitro antibacterial,
antifungal and antitubercular study
*
Tarunkumar Nanjibhai Akhaja, Jignesh Priyakant Raval
Department of Pharmaceutical Chemistry, Ashok & Rita Patel Institute of Integrated Study and Research in Biotechnology and Allied Sciences (ARIBAS),
New Vallabh vidyanagar, Gujarat 388121, India
a r t i c l e i n f o
a b s t r a c t
Article history:
1,3-dihydro-2H-indol-2-ones derivatives are reported to exhibit a wide variety of biodynamic activities
such as antituberculer, anti HIV, fungicidal, antibacterial, anticonvulsant. These valid observations led us
to synthesize some new indole-2-one derivative. Thus, herein we report synthesis of various 5-
substituted-3-[{5-(6-methyl-2-oxo/thioxo-4-phenyl-1,2,3,4 tetrahydro pyrimidin-5-yl)-1,3,4-thiadiazol-
2-yl}imino]-1,3-dihydro-2H-indol-2-one derivatives 4ael using one pot multicomponenteBiginelli
reaction via CaCl2 catalyst. Structures and purity of these compounds were confirmed by elemental, IR,
(1H & 13C) NMR and Mass spectral analysis. Newly synthesized compounds were also tested for their
in vitro anti-tubercular activity against Mycobacterium tuberculosis H37Rv, in vitro antibacterial activity
against selected human pathogens viz. Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae,
Salmonella typhi, Staphylococcus aureus, Staphylococcus pyogenus, Bacillus subtilis and antifungal activity
against Candida albicans, Aspergillus niger, Aspergillus clavatus strains.
Received 5 August 2011
Received in revised form
9 September 2011
Accepted 15 September 2011
Available online 22 September 2011
Keywords:
1,3-dihydro-2H-indol-2-ones
MCRs (Multicomponent Reactions)
Biginelli reaction
CaCl2 catalyst
in vitro antituberculer activity
Antimicrobial activity
Ó 2011 Elsevier Masson SAS. All rights reserved.
1. Introduction
extended by the variation of all three building blocks, allowing
access to a large number of multi-functionalized dihydropyr-
Drug discovery and development is a very laborious and costly
process involving synthesis and screening of diverse organic
compounds. In this regard, multicomponent reactions (MCRs) are
of increasing importance in the field of medicinal chemistry [1e3].
Currently, attention is put on speed, diversity, and efficiency in the
drug discovery process [4]. MCRs can provide products with the
diversity needed for the discovery of new lead compounds or lead
optimization employing combinatorial chemistry techniques. The
search and discovery for new MCRs on one hand [5], and the full
exploitation of already known multicomponent reactions on the
other hand, are therefore of considerable current interest. In 1893,
Pietro Biginelli has reported on the acid-catalyzed cyclo-
condensation reaction of ethylacetoacetate, benzaldehyde and
urea. The reaction was carried out by simply heating a mixture of
the three components dissolved in ethanol with a catalytic amount
of conc. HCl at reflux temperature. The product of this novel one-
pot, three-component synthesis that precipitated on cooling the
reaction mixture was identified correctly by Biginelli as dihy-
dropyrimidin-2-one [6]. The scope of this reaction was gradually
imidines of medicinal use [3b,7e10]. Dihydropyrimidines show
a diverse range of biological activities. They are known to possess
activities such as antiviral (nitractin) [11], anticancer [12], anti-
bacterial [13], analgesic and anti-inflammatory [13], as well as
efficacy as calcium channel modulators and
a1a-antagonists [14].
Thus development of methodologies for efficient lead structure
identification and for pharmacophore variation of dihydropyr-
imidines motif has always attracted the attention of pharmaceu-
tical industry [15]. Furthermore, certain compounds bearing 1,3-
dihydro-2H-indol-2-ones nucleus is used as a versatile lead mole-
cule for designing potential antivirals [16], antituberculars [17],
anticonvulsants [18] and anti-tumor therapeutic activities [19].
While, Schiff bases of 1,3-dihydro-2H-indol-2-ones and its deriva-
tives were reported for antibacterial [20], antifungal [20], anti-HIV
[21], anticonvulsant activities [22] and GAL3 receptor antagonists
[23].
Prompted by the biological properties of dihydropyrimidines
and 1,3-dihydro-2H-indol-2-ones nucleus, they were incorporated
with thiadiazoles [24] and schiff bases were synthesized. Moreover,
in the design of new drugs, the development of hybrid molecules
through the combination of different pharmacophores in one frame
may lead to compounds with interesting dual biological profiles,
which is being reflected in present work.
* Corresponding author. Tel.: þ91 2692 231894, þ91 2692 645801,
þ91 9427946892; fax: þ91 2692 229189.
E-mail address: drjpraval@yahoo.co.in (J.P. Raval).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.09.023

5574
T.N. Akhaja, J.P. Raval / European Journal of Medicinal Chemistry 46 (2011) 5573e5579
2. Chemistry
(oxo/thioxo)-4-phenyl-1,2,3,4-tetrahydropyrimidine-5-carbonyl
hydrazine-carbothioamide, which on heterocyclisation in presence
of conc. H2SO4 gave 5-(5-amino-1,3,4-thiadiazol-2-yl)-6-methyl-4-
phenyl-3,4-dihydropyrimidin-2(1H)-one/thione 3a/3b. Finally,
compounds 3a/3b on condensation with various 5-substituted
indoline-2,3-dione in acidic medium afforded the title compound
5-substituted-3-[{5-(6-methyl-2-oxo/thioxo-4-phenyl-1,2,3,4 tet-
rahydro pyrimidin-5-yl)-1,3,4-thiadiazol-2-yl}imino]-1,3-dihydro-
2H-indol-2-one 4ael. The purity of compounds was checked by TLC.
The structure of all the synthesized compounds was established by
IR, (1H & 13C) NMR and Mass spectral analysis. The result of
elemental analysis of the synthesized compounds was in agreement
with theoretical values.
Several improved procedures have been reported to carry out
Biginelli’s reactions using Lewis acid catalyst as well as protic acid
under classical reflux [25]. Other studies have focused on the use
of ionic liquids [26], microwave irradiation [27], combinatorial
techniques [28], use of boron compounds [29], Trimethylsilyl
chloride (TMSCl) [30] and heterogeneous catalysts viz. as tang-
stophosphoric acid [31], zeolite [32a,b], montmorillonite [33], ion-
exchange resins [34], and the also use of silica sulfuric acid [35],
Poly(p-phenylene ethynylene) (PPE) [36] etc. To the best of our
knowledge there have been relatively few reports available for
one-pot synthesis of dihydropyrimidinones using CaCl2 [37] as
catalyst. And in recent years, the development of more economical
and environmental friendly conversion processes is gaining
interest in the chemical community. Thus, herein, we report an
efficient, practical, environmentally benign and high yielding
method for Biginelli three component, one-pot synthesis of tet-
rahydropyrimidinones using CaCl2 [38] as catalyst for preparation
of compound 1a/1b.
3. Biological activity
3.1. in vitro evaluation of antimicrobial and antituberculosis
activity
The MICs of synthesized compounds were carried out by broth
microdilution method as described by Rattan [39]. Antibacterial
activity was screened against Escherichia coli (MTCC-443), Pseudo-
monas aeruginosa (MTCC-1688), Klebsiella pneumonia (MTCC-109),
Salmonella typhi (MTCC-98), Staphylococcus aureus (MTCC-96),
Staphylococcus pyogenus (MTCC-442) and Bacillus subtilis (MTCC-
441). Gentamycin, Ampicillin, Chloramphenicol, Ciprofloxacin,
Norfloxacin was used as a standard antibacterial agent. Antifungal
activity was screened against three fungal species C. albicans
(MTCC 227), Aspergillus niger (MTCC 282) and Aspergillus clavatus
(MTCC 1323). Nystatin and Griseofulvin was used as a standard
antifungal agent. The antimicrobial screening data are shown in
Tables 1 and 2.
The synthesis of title compound 4ael is outlined in Scheme 1. In
a typical experimental procedure a solution of b-ketoester, aldehyde
and urea/thiourea in ethanol was heated under reflux in the pres-
ence of catalystic amount of CaCl2 to give ethyl 6-methyl-4-phenyl-
2-(oxo/thioxo)-1,2,3,4-tetrahydropyrimidine-5-carboxylate 1a/1b,
followed by reaction with hydrazine hydrate in ethanol to give 6-
methyl-4-phenyl-2-oxo/thioxo-1,2,3,4-tetrahydropyrimidine-5-
carbohydrazide 2a/2b. Treatment of 6-methyl-4-phenyl-2-oxo/thi-
oxo-1,2,3,4-tetrahydropyrimidine-5-carbohydrazide 2a/2b with
ammonium thiocyanate in acidic medium afforded 6-methyl-2-
All MTCC cultures were collected from Institute of Microbial
Technology, Chandigarh and tested against known drugs. Muel-
lereHinton broth was used as nutrient medium to grow and
dilute the drug suspension for the test. Inoculum size for test
strain was adjusted to 108 CFU (Colony Forming Unit) per milli-
liter by comparing the turbidity. DMSO was used as diluents to
get desired concentration of drugs to test upon standard bacterial
strains.
C2H5O2C
NH2
CO2C2H5
CH3
(i)
HN
+
+
H2N
X
O
H
O
X
N
H
H3C
X = O, S
1a/1b
1a,
1b,
X=O
X=S;
(ii)
Table 1
Where
Antibacterial Activity (Minimal Inhibition Concentration, MICs,
m
g/mL).
S.py.
4a, R=H; X=S
4b, R=Br; X=S
4c, R=NO2; X=S
4d, R=F; X=S
Entry
R
X
E.c.
P.a
Kl.pn.
S.ty
S.a.
B.s.
CONHNH2
CH3
MTCC MTCC MTCC MTCC MTCC MTCC MTCC
4e,
R=I; X=S
HN
4f, R=Cl; X=S
4g, R=H; X=O
4h, R=Br; X=O
4i, R=NO2; X=O
443
1688 109
98
96
442
441
X
N
H
2a/2b
4a
4b
4c
4d
4e
4f
4g
4h
4i
H
S
S
S
S
S
S
O
O
O
O
O
O
500
200
62.5
62.5
250
200
500
250
200
200
100
62.5 100
250
100
250
250
100
62.5 100
250
200
1
100
50
25
10
200
200
100
200
250
100
100
250
200
250
250
200
100
250
200
1
200
100
100
200
100
200
100
200
200
500
100
250
100
200
200
0.5
100
50
50
10
200
100
100
100
200
100
500
100
200
100
250
200
0.5
100
50
50
10
Br
NO2
F
I
Cl
H
Br
NO2
F
I
Cl
4j,
R=F; X=O
4k, R=I; X=O
4l, R=Cl; X=O
2a,
2b,
X=O
X=S;
62.5
(iii)
200
200
250
200
200
200
200
500
100
200
62.5
250
200
0.25
250
50
50
10
O
R
62.5
O
N
N
N
N
S
4j
4k
4l
100
250
100
0.05
100
50
N
H
N
HN
250
200
0.05
100
50
25
S
R
NH2
HN
(iv)
O
CH3
X
N
Gentamycin
Ampicillin
Chloramphenicol
Ciprofloxacin
Norfloxacin
H
X
N
H
CH3
N
H
100
50
25
4a-l
3a/3b
25
10
3a,
3b,
X=O
X=S;
10
10
Scheme 1. Reaction Conditions. i) CaCl2/C2H5OH, reflux, 2 h; ii) NH2NH2.H2O/C2H5OH,
conc. H2SO4, reflux, 3 h; iii) 1 N HCl, NH4SCN, 8e10 h; conc. H2SO4, room temp., 2 h iv)
CH3OH/glacial CH3COOH, reflux, 45 min.
E.c. ¼ E. coli (MTCC-443); P.a. ¼ P. aeruginosa (MTCC-1688); Kl.pn. ¼ Kl. pneumoniae
(MTCC-109); S.ty. S. typhi (MTCC-98); S.a. S. aureus (MTCC-96);
S.py. ¼ S. pyogenus (MTCC-442); B.s. ¼ B. subtilis (MTCC-441).
¼
¼

T.N. Akhaja, J.P. Raval / European Journal of Medicinal Chemistry 46 (2011) 5573e5579
5575
Table 2
J ¼ 7.0) & 3.78 (q, 2H, J ¼ 7.1) ppm which proved the synthetic
nucleus 1a/1b. Formation of 6-methyl-4-phenyl-2-oxo/thioxo-
1,2,3,4-tetrahydropyrimidine-5-carbohydrazide 2a/2b showed
broad stretching bands around 3200 & 3420 cmꢀ1 for NH2 and NH,
Antifungal Activity (Minimal Fungicidal Concentration, MICs, mg/mL).
Entry
C. albicans
A. niger
A. clavatus
MTCC 227
MTCC 282
MTCC 1323
in 1H NMR spectrum a singlet at
d 4.80, 8.90 ppm for 2a & d 4.87,
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
>1000
500
250
100
>1000
500
250
500
250
100
500
500
100
100
>1000
500
250
500
250
100
500
500
250
100
>1000
500
250
>1000
500
200
>1000
500
8.93 ppm for 2b were accounted for NH2 and NH, while triplet-
quatert pattern of ethyl chain vanished. Also disappearance of
these NH and NH2 signals in 3a/3b with 1H NMR proved that the
ring closure is resulted in the formation of 1,3,4-thiadiazole ring
from 2a/2b. Which is further evident from IR spectra, which dis-
played broad stretching vibration at 3084 cmꢀ1 due to the aromatic
ring (ArH) and absorption band at 1579 cmꢀ1 due to C]N group
while at 1063 cmꢀ1 is due to thiadiazole ring. Condensation of
compounds 3a/3b with various 5-substituted indoline-2,3-dione
produces the final 1,3-dihydro-3H-indol-3-one derivatives 4ael.
Position & numbers of protons have been recorded in 1H NMR
spectrum, which favors multiplet of aromatic protons at
>1000
>1000
100
>1000
500
100
Nystatin
Greseofulvin
100
100
500
100
d
7.00e8.90, while range for proton of iminebase (eCH]) is
observed between
d
4.54e4.59. The 13C NMR and Mass spectral
in vitro antituberculosis activity of all the newly synthesized
compounds against Mycobacterium tuberculosis H37Rv strain was
determined by using LowensteineJensen medium (conventional
method) as described by Rattan [39] and the observed MIC of
compounds are presented in Table 3.
data of compound 4ael is given in the experimental section.
4.2. Biological results
The literature survey revealed that introduction of electron-
withdrawing groups at positions 5, 6, and 7 greatly increased
activity from that of 1,3-dihydro-2H-indol-2-one, with substitution
at the 5th position being most favorable. This is not surprising, as
Ce5 substitution has previously been associated with increased
biological activity for a range of indole-based compounds [40,41]
and the presence of substituted aromatic ring at 3rd position has
been reported to be associated with antimicrobial properties
[42,43]. The various substituent at 3rd position of the isatin which
were reported, were various substituted phenyl ring moieties
[44,45] heterocyclic rings [46e48] and aliphatic system [49]. These
observations led to the conception that a series of some different
novel schiff bases of 5-(5-amino-1,3,4-thiadiazol-2-yl)-6-methyl-4-
phenyl-3,4-dihydropyrimidin-2(H)-one/thione using different 5-
substituted indoline-2,3-dione.
From in vitro antibacterial activity data, it is confirmed that
compounds containing strong electron withdrawing (fluorine
group) i.e. 4d & 4j exhibited excellent activity against all microbial
strains, while compounds 4b & 4h exhibited comparable activity
against gram positive strains, while compound 4c & 4i are found to
be highly active against gram negative strains as compared to
standard antibiotic ampicillin.
From in vitro antifungal activity data, it is found that compound
4d & 4f is displaying highest activity against all fungal strains, while
compounds 4c & 4i are showing somewhat less activity compare to
compounds 4d & 4i. But overall, all the compounds have displayed
significant antibacterial and antifungal activity. In general, the
order of antibacterial activity of the substituents at the 5th position
of 1H-indole-2,3-diones is F > NO2>Br > Cl > H]I and also due to
presence of sulphur atom at positione2 in the compounds 4aef is
responsible for better activity compared to oxygen atom (at posi-
tione2) for compounds 4gel. The in vitro antibacterial and anti-
fungal screening results are summarized in Tables 1 and 2.
The encouraging results from the antibacterial and antifungal
studies impelled us to go for preliminary screening of synthesized
compounds against M. tuberculosis H37Rv, which is summarized in
Table 3. Compound 4j containing 5-flouro substituent on indolone
ring with oxygen atom on tetrahydropyrimidine nucleus showed
4. Results and discussion
4.1. Analytical results
A series of 1,3-dihydro-2H-indol-2-ones derivatives has been
synthesized in good yields by using the synthetic route as outlined
in Scheme 1. IR, (1H & 13C) NMR and mass spectral data are in well
agreement with the proposed structures of all newly synthesized
compounds.
Here, in novel synthetic research work, an effort has been made
to undertake the synthesis of 1a/1b ethyl 6-methyl-4-phenyl-2-
(oxo/thioxo)-1,2,3,4-tetrahydropyrimidine-5-carboxylate via Bigi-
nelli’s reaction through one step process. Purity and yield of
compounds 1a/1b was excellent without any tedious workup
procedure and also mass spectrum gives molecular ion peak at m/z
260.3 for 1a and m/z 276.2 for 1b respectively. And IR spectrum
displayed stretching vibration at 1735, 1700 and 1652 cmꢀ1 for
ethyl ester (COOC2H5) and secondary amine (NH) functional
groups, while 1H NMR spectrum showed singlet of amine at
7.20 and13.53 multiplet of aromatic ring at 7.15e7.42, singlet of
single proton of cyclized heterocyclic ring at range 5.05 and
1.15 (t, 3H,
d 2.24,
d
d
triplet-quartet patterns of ethyl chain is recorded at
d
Table 3
Antitubercular Activity (Minimal Inhibition Concentration, MICs, mg/mL).
Entry
M. tuberculosis H37Rv
MTCC e 200
% Inhibition
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
500
250
62.5
25
1000
62.5
1000
500
62.5
50
98
98
99
99
98
99
98
98
99
99
98
98
98
99
better activity (50
mg/ml) and compounds 4c, 4f and 4i showed
1000
100
40
good activity (50e62.5
mg/ml) which is attributed due to 5-nitro, 5-
Rifampicin
Isoniazid
chloro substituents of 2-thioxo-tetrahydropyrimidine indolone
nucleus and 5-nitro substituents of 2-oxo-tetrahydropyrimidine
0.20

5576
T.N. Akhaja, J.P. Raval / European Journal of Medicinal Chemistry 46 (2011) 5573e5579
indolone nucleus respectively, where as compound 4d which is
having inductively electron withdrawing but mesomerically elec-
tron releasing sulphur atom with 5-flouro substituent on indolone
5.1.1.3. Preparation of 5-(5-amino-1, 3, 4-thiadiazol-2-yl)-6-methyl-
4-phenyl-3, 4-dihydropyrimidin-2 (1H)-one/thione 3a/3b. Title
compound 3a/3b is obtained using reported procedure using 1 N
HCl, ammonium thiocynate and conc. H2SO4 [50].
ring showed better activity (25 mg/ml) compared to other analoges.
Thus, further developmental studies to acquire more information
about structure-activity relationships are in progress in our
laboratories.
Spectral data for 3a: Pale yellow solid, yield 72%; mp 165e167 ꢁC;
MS: m/z [288.05]þ; 1H- NMR [400 MHz,
d, ppm, DMSO-d6] 8.90,
5.90 (1H ꢂ 2, s, eNH), 7.56 (2H, s, eNH2), 6.99e7.36 (4H, m,
aromatic), 5.65 (1H, s, eCH]),1.98 (3H, s, eCH3). 13C-NMR
5. Experimental section
[100 MHz, d, ppm, DMSO-d6]: 17.20 (C1), 109.12e145.06
(C2,C5,C8,C9,C10,C11,C12,C13), 157.70 (C6), 162.28 (C7), 170.16 (C4).
Anal. Calcd. for C13H13N5OS (287.34): C, 54.34; H, 4.56; N, 24.37;
Found: C, 54.28; H, 4.21; N, 24.07%.
5.1. Chemistry
All the melting points were taken in open capillaries and are
uncorrected. The purity of compounds was checked routinely by
TLC (0.5 mm thickness) using silica gel-G coated aluminium plates
(Merck) and spots were visualized by exposing the dry plates in
iodine vapours. Mass spectra were scanned on a Shimadzu LCMS
2010 spectrometer. IR spectra (nmax in cmꢀ1) were recorded on
Shimadzu FTIR spectrophotometer using KBr technique. 1H & 13C
NMR spectra on a Bruker’s WM 400 FT MHz NMR instrument using
DMSO-d6 as solvent and TMS as internal reference (chemical shifts
Spectral data for 3b: Dark yellow solid, yield 65%; mp
150e152 ꢁC; MS: m/z [304.15]þ; 1H- NMR [400 MHz,
d, ppm,
DMSO-d6] 1.90, 11.50 (1H ꢂ 2, s, eNH), 2.16 (3H, s, eCH3), 5.25 (1H,
s, eCH]), 6.96 (2H, s, eNH2), 7.05e7.40 (4H, m, aromatic). 13C-NMR
[100 MHz,
d, ppm, DMSO-d6]: 16.80 (C1), 110.56e141.08
(C2,C5,C8,C9,C10,C11,C12,C13), 157.30 (C6), 160.17 (C7), 173.52 (C4).
Anal. Calcd. for C13H13N5S2 (303.41): C, 51.46; H, 4.32; N, 23.08;
Found: C, 51.12; H, 3.91; N, 22.74%.
in
d
ppm). The elemental analysis (C, H, and N) of compounds was
5.1.1.4. Preparation of 5-substituted-3-[{5-(6-methyl-2-oxo/thioxo-
4-phenyl-1,2,3,4 tetrahydro pyrimidin-5-yl)-1,3,4-thiadiazol-2-yl}
performed on Carlo Erba e 1108 elemental analyzer.
imino]-1,3-dihydro-2H-indol-2-one
4ael. Compounds
3a/3b
5.1.1. General procedure
5.1.1.1. Preparation of ethyl 6-methyl-4-phenyl-2-(oxo/thioxo)-
1,2,3,4-tetrahydropyrimidine-5-carboxylate
(0.01 mol) and 5-substituted indoline-2,3-dione (0.01 mol) were
dissolved in methanol(10 mL) in presence of catalytic amount of
glacial acetic acid and reflux for 45 min. A progress of reaction was
monitored using TLC. After completion of reaction, crude mass was
allowed to cool and poured on crushed ice. Final product obtained
was filtered, washed with cold ether (20e30 ml), dried and purified
by crystallization in THF:water (60:40).
The compounds 4ael were prepared in the same fashion
using appropriate 5-substituted indoline-2,3-diones and 3a/3b
(Scheme 1).
1a/1b
[37]. Title
compounds 1a/1b was prepared using reported procedure [32b,37]
with minor modifications. Urea/thiourea (0.5 mol), ethyl-
acetoacetate (0.75 mol) and benzaldehyde (0.75 mol) were mixed
in ethanol (25 ml). Catalytic amount of CaCl2 (0.020 mol) was added
to the reaction mixture and was refluxed for 2 h. White/yellowish
precipitates was obtained during reflux. The progress of reaction
was monitored by TLC. After completion of reaction, the stirring
was stopped, and the reaction mass was cooled and treated with
crushed ice. Almost pure product obtained as white/cream solid
was filtered and dried. And further crystallized using methanol:
water (60: 40), yield: 1a, 85% & 1b, 90%, mp: 1a, 201 ꢁC & 1b, 218 ꢁC.
5.1.1.4.1. 3-{[5-(6-methyl-4-phenyl-2-thioxo-1,2,3,4-tetrahydrop-
yrimidin-5-yl)-1,3,4-thiadiazol-2-yl]imino}-1,3-dihydro-2H-indol-2þ-
one 4a. Grey solid, Yield 70%, mp.210e212 ꢁC; MS: m/z [432.08] ;
IR [nmax, cmꢀ1, KBr]: 3459, 3088, 2970 (CeH, aromatic), 3331, 3167
(NH), 1724 (C]O), 1633 (C]N, iminebase), 1660 (C]S), 1467 (CeH,
aliphatic), 1463 (C]C, aromatic), 1377 (C]N), 1273, 1185 (CeSeC),
5.1.1.2. Preparation
of
6-methyl-4-phenyl-2-oxo/thioxo-1,2,3,4-
tetrahydropyrimidine-5-carbohydrazide 2a/2b. 1a/1b (0.01 mol) in
ethanol (20 ml), hydrazine hydrate (0.01 mol) was added followed
by the addition of a catalytic amount of conc. H2SO4 and allowed to
stir for 3 h at 75 ꢁC. Yellowish precipitates were obtained during
reflux. A progress of reaction was monitored using TLC. After
completion of reaction, crude mass was allowed to cool and poured
on crushed ice. Product obtained as yellowish precipitate, was
filtered and dried. Purification was done by crystallization using
ethanol.
725, 759, 695, 662 (CeH, deformation). 1H- NMR [400 MHz,
d, ppm,
DMSO-d6]: 2.05, 8.00, 13.76 (1H ꢂ 3, s, NH), 2.26 (3H, s, CH3), 4.59
(1H, s, eCH ¼ ), 7.23e7.86 (9H, m, AreH). 13C NMR [100 MHz,
d,
ppm, DMSO-d6]: 16.74 (C31), 62.92 (C17), 117.11 (C11), 119.20e132.15
(C12,C13,C14,C15,C16,C21,C25,C26,C27,C28,C29),140.60 (C10),143.22 (C24),
150.30 (C8), 156.60 (C2), 158.27 (C5), 164.90 (C7), 173.70 (C19). Anal.
Calcd. for C21H16N6OS2 (432.52): C, 58.31; H, 3.73; N, 19.43. Found:
C, 58.03; H, 3.22; N, 18.90%.
5.1.1.4.2. 5-bromo-3-{[5-(6-methyl-4-phenyl-2-thioxo-1,2,3,4-
tetrahydropyrimidin-5-yl)-1,3,4-thiadiazol-2-yl]imino}-1,3-dihydro-
2H-indol-2-one 4b. Yellowish solid, Yield 72%, mp. 199e200 ꢁC;
MS: m/z [511.99]þ; IR [nmax, cmꢀ1, KBr]: 3447, 3078, 2988 (CeH,
aromatic), 3344, 3167 (NH), 1739 (C]O), 1466 (CeH, aliphatic),
1460 (C]C, aromatic), 1366 (C]N), 1283, 1185 (CeSeC), 1658 (C]
S), 1643 (C]N, iminebase), 733, 759, 688, 662 (CeH, deformation),
Spectral data of 2a: Yellowish solid, yield 82%; mp 190 ꢁC; MS: m/
z [247.05]þ; IR [nmax, cmꢀ1, KBr]: 1H- NMR [400 MHz,
d, ppm,
DMSO-d6] 2.37 (3H, s, eCH3), 4.80 (2H, s, eNH2), 5.40 (1H, s,
eCH]), 6.51, 8.90 (1H ꢂ 2, s, eNH), 7.11e7.39 (5H, m, aromatic).
13C-NMR [100 MHz,
d, ppm, DMSO-d6]: 17.90 (C1), 58.87 (C4),
115.10e146.90 (C3,C7,C8,C9,C10,C11,C12), 152.62 (C2), 154.73 (C5),
167.56 (C6). Anal. Calcd. for C12H14N4O2 (246.27): C, 58.53; H, 5.73;
N, 22.75. Found: C, 58.21; H, 5.52; N, 22.39%.
610 (CeBr). 1H- NMR [400 MHz,
(1H ꢂ 3, s, NH), 2.25 (3H, s, CH3), 4.55 (1H, s, eCH ¼ ), 7.15e8.15 (8H,
m, AreH). 13C NMR [100 MHz,
, ppm, DMSO-d6]: 16.94 (C31), 63.92
d, ppm, DMSO-d6]: 2.23, 8.24, 13.73
Spectral data of 2b: Yellowish solid, yield 85%; mp 195 ꢁC; MS:
d
m/z [263.45]þ; IR [nmax, cmꢀ1, KBr]: 1H- NMR [400 MHz,
d
, ppm,
(C17), 117.60e133.15 (C11,C12,C13,C14,C15,C16,C21,C25,C26,C27,C28,C29),
140.10 (C10), 143.72 (C24), 149.90 (C8), 156.90 (C2), 158.35 (C5),
162.30 (C7), 173.65 (C19). Anal. Calcd. for C21H15BrN6OS2 (511.42): C,
49.32; H, 2.96; N, 16.43. Found: C, 49.12; H, 3.74; N, 16.08%.
5.1.1.4.3. 3-{[5-(6-methyl-4-phenyl-2-thioxo-1,2,3,4-tetrahydrop-
yrimidin-5-yl)-1,3,4-thiadiazol-2-yl]imino}-5-nitro-1,3-dihydro-2H-
indol-2-one 4c. Yellow solid, Yield 74%, mp. 202e204 ꢁC; MS: m/z
DMSO-d6] 1.89, 4.87, 8.93,11.96 (1H ꢂ 4, s, eNH), 2.30 (3H, s, eCH3),
5.46 (1H, s, eCH]), 7.20e7.42 (5H, m, aromatic). 13C-NMR
[100 MHz, d, ppm, DMSO-d6]: 17.60 (C1), 60.40 (C4), 110.34e148.13
(C3,C7,C8,C9,C10,C11,C12), 162.12 (C2), 167.42 (C6), 176.29 (C5). Anal.
Calcd. for C12H14N4OS (262.33): C, 54.94; H, 5.38; N, 21.36. Found: C,
54.71; H, 5.01; N, 21.00%.

T.N. Akhaja, J.P. Raval / European Journal of Medicinal Chemistry 46 (2011) 5573e5579
5577
[477.07]þ; IR [nmax, cmꢀ1, KBr]: 3454, 3098, 2980 (CeH, aromatic),
3334, 3177 (NH), 1729 (C]O), 1668 (C]S), 1623 (C]N, iminebase),
1574 (N]O), 1468 (C]C, aromatic), 1450 (CeH, aliphatic), 1283,
1195 (CeSeC), 1371 (C]N), 723, 749, 690, 652 (CeH, deformation).
151.30 (C8), 152.05 (C19), 155.20 (C2), 157.85 (C5), 162.90 (C7), Anal.
Calcd. for C21H16N6O2S (477.52): C, 60.56; H, 3.87; N, 20.18. Found:
C, 60.10; H, 3.42; N, 20.03%.
5.1.1.4.8. 5-bromo-3-{[5-(6-methyl-2-oxo-4-phenyl-1,2,3,4-tetra-
hydropyrimidin-5-yl)-1,3,4-thiadiazol-2-yl]imino}-1,3-dihydro-2H-
indol-2-one 4h. Orange solid, Yield 79%, mp. 250e252 ꢁC; MS: m/z
[494.02]þ; IR [nmax, cmꢀ1, KBr]: 3174, 2969, 2880 (CeH, aromatic),
1712, 1724 (C]O), 1633 (C]N, iminebase), 1478 (CeH, aliphatic),
1448 (C]C, aromatic), 1263, 1205 (CeSeC), 1377 (C]N), 743, 769,
1H- NMR [400 MHz,
d
, ppm, DMSO-d6]: 2.07, 8.33, 13.70 (1H ꢂ 3, s,
NH), 2.27 (3H, s, CH3), 4.54 (1H, s, eCH ¼ ), 7.25e8.51 (8H, m,
AreH). 13C NMR [100 MHz,
, ppm, DMSO-d6]: 16.80 (C31), 65.35
(C17), 117.95 (C11), 122.50e144.15 (C12,C13,C14,C15,C16,C21,C25,C26
d
,
C27,C28,C29), 143.34 (C24), 147.95 (C10), 150.55 (C8), 155.63 (C2),
159.12 (C5), 164.20 (C7), 174.15 (C19). Anal. Calcd. for C21H15N7O3S2
(477.52): C, 52.82; H, 3.17; N, 20.53. Found: C, 52.33; H, 3.02; N,
20.13%.
5.1.1.4.4. 5-fluoro-3-{[5-(6-methyl-4-phenyl-2-thioxo-1,2,3,4-tet-
rahydropyrimidin-5-yl)-1,3,4-thiadiazol-2-yl]imino}-1,3-dihydro-2H-
indol-2-one 4d. Yellowish solid, Yield 78%, mp. 205e206 ꢁC; MS: m/z
[550.07]þ; IR [nmax, cmꢀ1, KBr]: 3465, 3105, 2970 (CeH, aromatic),
3345, 3187 (NH), 1734 (C]O), 1658 (C]S), 1623 (C]N, iminebase),
1468(CeH, aliphatic),1448(C]C, aromatic),1371(C]N),1321(CeF),
1268,1205 (CeSeC), 723, 757, 690, 655 (CeH, deformation).1H- NMR
670, (CeH, deformation), 630 (CeBr). 1H- NMR [400 MHz,
DMSO-d6]: 2.25 (3H, s, CH3), 4.55 (1H, s, eCH]), 6.15, 8.28 (1H ꢂ 3,
s, NH), 7.22e8.14 (8H, m, AreH). 13C NMR [100 MHz,
, ppm, DMSO-
d6]: 15.10 (C31), 60.15 (C17), 118.40e133.95 (C11,C12,C13,C14,C15,C16
d, ppm,
d
,
C21,C25,C26,C27,C28,C29), 141.22 (C10), 142.68 (C24), 151.45 (C19),
152.90 (C8), 155.80 (C2), 158.52 (C5), 163.40 (C7). Anal. Calcd. for
C21H15BrN6O2S (495.35): C, 50.92; H, 3.05; N,16.97. Found: C, 50.71;
H, 2.82; N, 16.58%.
5.1.1.4.9. 3-{[5-(6-methyl-2-oxo-4-phenyl-1,2,3,4-tetrahydropyri-
midin-5-yl)-1,3,4-thiadiazol-2-yl]imino}-5-nitro-1,3-dihydro-2H-in-
dol-2-one 4i. Yellow color, Yield 75%, mp. 253e254 ꢁC; MS: m/z
[461.09]þ; IR [nmax, cmꢀ1, KBr]: 3318, 2945 (NH), 3129, 2989, 2855
(CeH, aromatic), 1720, 1749 (C]O), 1630 (C]N, iminebase), 1574
(N]O), 1454 (CeH, aliphatic), 1448 (C]C, aromatic), 1368 (C]N),
1273, 1175 (CeSeC), 723, 769, 690, (CeH, deformation). 1H- NMR
[400 MHz, d, ppm, DMSO-d6]: 2.23 (3H, s, CH3), 2.34, 8.18, 13.74
(1H ꢂ 3, s, NH), 4.57 (1H, s, eCH]), 7.28e8.10 (8H, m, AreH).13C NMR
[100 MHz, d, ppm, DMSO-d6]: 16.30 (C31), 65.05 (C17), 111.06e142.68
(C11,C12,C13,C14,C15,C16,C21,C25,C26,C27,C28,C29), 145.33 (C24), 150.13
(C8), 153.95 (C10), 155.23 (C2), 157.98 (C5), 164.25 (C7), 174.15 (C19).
Anal. Calcd. for C21H15FN6OS2 (450.51): C, 55.99; H, 3.36; N, 18.65.
Found: C, 55.86; H, 3.12; N, 18.43%.
[400 MHz, d, ppm, DMSO-d6]: 2.25 (3H, s, CH3), 4.55 (1H, s, eCH]),
5.93, 8.03 (1H ꢂ 3, s, NH), 7.20e8.56 (8H, m, AreH). 13C NMR
5.1.1.4.5. 5-iodo-3-{[5-(6-methyl-4-phenyl-2-thioxo-1,2,3,4-tetr-
ahydropyrimidin-5-yl)-1,3,4-thiadiazol-2-yl]imino}-1,3-dihydro-2H-
indol-2-one 4e. Cream solid, Yield 70%, mp. 215e218 ꢁC; MS: m/z
[557.98]þ; IR [nmax, cm-1, KBr]: 3454, 3088, 2980 (CeH, aromatic),
3334, 3163 (NH), 1739 (C]O), 1668 (C]S), 1633 (C]N, iminebase),
1478 (C]C, aromatic), 1463 (CeH, aliphatic), 1370 (C]N), 1264,
1185 (CeSeC), 733, 749, 685, 652 (CeH, deformation), 550 (CeI).
[100 MHz, d, ppm, DMSO-d6]: 15.00 (C31), 58.95 (C17), 117.60 (C11),
123.76e144.58 (C12,C13,C14,C15,C16,C21,C24,C25,C26,C27,C28,C29), 148.95
(C10), 150.05 (C19), 151.35 (C8), 155.90 (C2), 158.68 (C5), 163.50 (C7).
Anal. Calcd. for C21H15N7O4S (461.45): C, 54.66; H, 3.28; N, 21.25.
Found: C, 54.13; H, 2.82; N, 21.10%.
5.1.1.4.10. 5-fluoro-3-{[5-(6-methyl-2-oxo-4-phenyl-1,2,3,4-tetra-
hydropyrimidin-5-yl)-1,3,4-thiadiazol-2-yl]imino}-1,3-dihydro-2H-
indol-2-one 4j. Cream color, Yield 78%, mp. 214e216 ꢁC; MS: m/z
[435.10]þ; IR [nmax, cmꢀ1, KBr]: 3244, 2922 (NH), 3115, 2979, 2852
(CeH, aromatic), 1724, 1701 (C]O), 1647 (C]N, iminebase), 1473
(CeH, aliphatic), 1453 (C]C, aromatic), 1368 (C]N), 1313 (CeF),
1290, 1222 (CeSeC), 782, 758, 699, (CeH, deformation). 1H- NMR
1H- NMR [400 MHz,
NH), 2.25 (3H, s, CH3), 4.56 (1H, s, eCH]), 7.24e8.16 (8H, m, AreH).
13C NMR [100 MHz,
, ppm, DMSO-d6]: 15.60 (C31), 59.95 (C17),
d, ppm, DMSO-d6]: 2.05, 8.43, 13.72 (1H ꢂ 3, s,
d
91.66e138.18 (C11, C12,C13,C14,C15,C16,C21,C25,C26,C27,C28,C29), 140.95
(C10), 143.33 (C24), 150.53 (C8), 155.30 (C2), 158.46 (C5), 163.35 (C7),
173.85 (C19). Anal. Calcd. for C21H15IN6OS2 (558.42): C, 45.17; H,
2.71; N, 15.05. Found: C, 44.90; H, 2.58; N, 14.80%.
[400 MHz, d, ppm, DMSO-d6]: 2.27 (3H, s, CH3), 4.57 (1H, s, eCH]),
5.92, 8.26 (1H ꢂ 3, s, NH), 7.22e7.86 (8H, m, AreH). 13C NMR
5.1.1.4.6. 5-chloro-3-{[5-(6-methyl-4-phenyl-2-thioxo-1,2,3,4-tet-
rahydropyrimidin-5-yl)-1,3,4-thiadiazol-2-yl]imino}-1,3-dihydro-2H-
indol-2-one 4f. Pale yellow solid, Yield 70%, mp. 210e212 ꢁC; MS:
m/z [466.04]þ; IR [nmax, cmꢀ1, KBr]: 3444, 3098, 2969 (CeH,
aromatic), 3350, 3177 (NH), 1735 (C]O), 1643 (C]N, iminebase),
1659 (C]S), 1478 (C]C, aromatic), 1465 (CeH, aliphatic), 1378 (C]
N), 1267, 1205 (CeSeC), 728, 765, 687, 662 (CeH, deformation), 715
[100 MHz, d, ppm, DMSO-d6]: 15.85 (C31), 57.05 (C17),110.18e146.88
(C11,C12,C13,C14,C15,C16,C21,C24,C25,C26,C27,C28,C29), 150.28
(C19),
151.43 (C8), 154.60 (C10), 155.37 (C2), 158.48 (C5), 163.58 (C7). Anal.
Calcd. for C21H15FN6O2S (434.35): C, 58.06; H, 3.48; N, 19.34. Found:
C, 57.86; H, 3.26; N, 19.03%.
5.1.1.4.11. 5-iodo-3-{[5-(6-methyl-2-oxo-4-phenyl-1,2,3,4-tetrahy
dropyrimidin-5-yl)-1,3,4-thiadiazol-2-yl]imino}-1,3-dihydro-2H-in
dol-2-one 4k. Reddish pink color, Yield 70%, mp. 218e220 ꢁC; MS:
m/z [442.45]þ; IR [nmax, cmꢀ1, KBr]: 3332, 2922 (NH), 3154, 2998,
2890 (CeH, aromatic), 1729, 1749 (C]O), 1643 (C]N, iminebase),
1472 (CeH, aliphatic), 1468 (C]C, aromatic), 1371 (C]N), 1293,
1176 (CeSeC), 743, 749, 670, (CeH, deformation), 556 (CeI). 1H-
(CeCl). 1H- NMR [400 MHz,
2.42, 8.16, 13.77 (1H ꢂ 3, s, NH), 4.55 (1H, s, eCH]), 7.22e7.83 (8H,
m, AreH). 13C NMR [100 MHz,
, ppm, DMSO-d6]: 16.55 (C31), 59.90
d, ppm, DMSO-d6]: 2.27 (3H, s, CH3),
d
(C17), 92.00e137.05 (C11,C12,C13,C14,C15,C16,C21,C25,C26,C27,C28,C29),
141.30 (C10),143.10 (C24),150.40 (C8),155.48 (C2),159.10 (C5),164.60
(C7), 174.10 (C19). Anal. Calcd. for C21H15ClN6OS2 (466.97): C, 54.01;
H, 3.24; N, 18.00. Found: C, 53.83; H, 3.01; N, 17.82%.
NMR [400 MHz, d, ppm, DMSO-d6]: 2.26 (3H, s, CH3), 4.55 (1H, s,
eCH]), 6.15, 8.15 (1H ꢂ 3, s, NH), 7.24e8.08 (8H, m, AreH). 13C
5.1.1.4.7. 3-{[5-(6-methyl-2-oxo-4-phenyl-1,2,3,4-tetrahydropyri-
midin-5-yl)-1,3,4-thiadiazol-2-yl]imino}-1,3-dihydro-2H-indol-2þ-one
4g. Yellow solid, Yield 74%, mp. 214e216 ꢁC; MS: m/z [416.11] ; IR
NMR [100 MHz, d, ppm, DMSO-d6]: 16.15 (C31), 58.70 (C17),
91.80e137.58 (C11,C12,C13,C14,C15,C16,C21,C25,C26,C27,C28,C29), 141.42
(C10), 143.52 (C24), 150.10 (C8), 155.28 (C2), 158.90 (C5), 163.55 (C7),
173.85 (C19). Anal. Calcd. for C21H15IN6O2S (542.35): C, 46.51; H,
2.79; N, 15.50. Found: C, 46.27; H, 2.58; N, 15.26%.
5.1.1.4.12. 5-chloro-3-{[5-(6-methyl-2-oxo-4-phenyl-1,2,3,4-tet-
rahydropyrimidin-5-yl)-1,3,4-thiadiazol-2-yl]imino}-1,3-dihydro-2H-
indol-2-one 4l. Reddish color, Yield 70%, mp. 225e227 ꢁC; MS: m/z
[450.07]þ; IR [nmax, cmꢀ1, KBr]: 3334, 2936 (NH), 3174, 3018, 2867
(CeH, aromatic), 1730, 1749 (C]O), 1619 (C]N, iminebase), 1470
(C]C, aromatic), 1448 (CeH, aliphatic), 1359 (C]N), 1276, 1195
[
nmax, cmꢀ1, KBr]: 3334, 2912 (NH), 3154, 2988, 2840 (CeH,
aromatic), 1701, 1729 (C]O), 1623 (C]N, iminebase), 1478 (C]C,
aromatic), 1450 (CeH, aliphatic), 1371 (C]N), 1283, 1195 (CeSeC),
741, 759, 678, (CeH, deformation). 1H- NMR [400 MHz,
d
, ppm,
DMSO-d6]: 2.28 (3H, s, CH3), 4.57 (1H, s, eCH]), 6.13, 8.08 (1H ꢂ 3,
s, NH), 7.25e7.89 (9H, m, AreH). 13C NMR [100 MHz,
, ppm, DMSO-
d6]: 15.74 (C31), 60.92 (C17), 120.10e131.15 (C11,
C12,C13,C14,C15,C16,C21,C25,C26,C27,C28,C29), 141.60 (C10), 142.72 (C24),
d

5578
T.N. Akhaja, J.P. Raval / European Journal of Medicinal Chemistry 46 (2011) 5573e5579
(CeSeC), 733, 759, 690, (CeH, deformation), 710 (CeCl). 1H- NMR
6. Conclusion
[400 MHz, d, ppm, DMSO-d6]: 2.25 (3H, s, CH3), 4.54 (1H, s, eCH]),
6.13, 8.22 (1H ꢂ 3, s, NH), 7.25e7.85 (8H, m, AreH). 13C NMR
Tetrahydropyrimidinyl-1,3,4-thiadiazolylimino-1,3-dihydro-
2H-indol-2-ones derivatives 4ael were synthesized and charac-
terized for their structure elucidation. Various chemical and spec-
tral data supported the structures of newly synthesized
compounds. The Biginelli’s reaction for preparation of tetrahy-
dropyrimidinones derivatives 1a/1b was efficiently carried out
using CaCl2 as catalyst. Compounds 4d and 4j showed significant
antibacterial and antifungal activity. While the compound 4d dis-
played promising antitubercular activity compared to standards.
Thus, present library model can be used to design the new ligand of
this class for their antimicrobial and antitubercular activity.
[100 MHz, d, ppm, DMSO-d6]: 15.05 (C31), 59.05 (C17), 91.80e137.18
(C11,C12,C13,C14,C15,C16,C21,C25,C26,C27,C28,C29), 141.10 (C10), 143.30
(C24), 150.70 (C8), 151.90 (C19), 155.28 (C2), 157.98 (C5), 164.88 (C7).
Anal. Calcd. for C21H15ClN6OS2 (542.35): C, 54.01; H, 3.24; N, 18.00.
Found: C, 53.78; H, 3.08; N, 17.95%.
5.2. Biological activity
5.2.1. in vitro evaluation of antimicrobial activity
The antibacterial screening results are summarized in Tables 1
and 2 The MICs of synthesized compounds were carried out by
broth Microdilution method. DMSO was used as diluents to get
desired concentration of drugs to test upon standard bacterial
strains. Serial dilutions were prepared in primary and secondary
screening. The control tube containing no antibiotic was imme-
diately subcultured (before inoculation) by spreading a loopful (1
loop) evenly over a quarter of plate of medium suitable for the
growth of the test organism and put for incubation at 37 ꢁC
overnight. The tubes were then incubated overnight. The MIC of
the control organism was read to check the accuracy of the drug
concentrations. The lowest concentration inhibiting growth of the
organism was recorded as the MIC. All the tubes not showing
visible growth (in the same manner as control tube described
above) was subcultured and incubated overnight at 37 ꢁC. The
amount of growth from the control tube before incubation (which
represents the original inoculum) was compared. Subcultures
might show: similar number of colonies indicating bacteriostatic;
a reduced number of colonies indicating a partial or slow bacte-
ricidal activity and no growth if the whole inoculum has been
killed. The test included a second set of the same dilutions inoc-
ulated with an organism of known sensitivity. Each synthesized
Acknowledgements
The author (J.P. Raval) wishes to express his thanks to Chairman-
(CVM), Charutar Vidya Mandal, Director-SICART & Director-ARIBAS
for providing necessary research facilities. Dr. Dhanji Rajani,
Microcare laboratories, Surat for extending help for assessment of
biologicalactivity. We arealsothankfulto Mr. PriyakantR. Raval&Dr.
Kishor R. Desai, Director, Shri C. G. Bhakta Insititute of Biotechnology,
Uka e Tarsadia University, Bardoli for research advice.
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