Novel imidazo[2,1-b]-1,3,4-thiadiazoles as promising antifungal agents against clinical isolate of Cryptococcus neoformans

Article abstract of DOI:10.1016/j.ejmech.2015.03.021

We herein report the synthesis and in vitro antimicrobial evaluation of twenty five novel hybrid derivatives of imidazo [2,1-b]-1,3,4-thiadiazole containing chalcones (5a-o) and Schiff bases (6a-j) against three fungal strains (Candida albicans, Cryptococ

Full text of DOI:10.1016/j.ejmech.2015.03.021

Accepted Manuscript
Novel imidazo[2,1-b]-1,3,4-thiadiazoles as Promising Antifungal Agents Against
Clinical Isolate of C. Neoformans
Wesam S. Alwan, Rajshekhar Karpoormath, Mahesh B. Palkar, Harun M. Patel,
Rajesh A. Rane, Mahamadhanif S. Shaikh, Afsana Kajee, Koleka P. Mlisana
PII:
S0223-5234(15)00188-9
10.1016/j.ejmech.2015.03.021
EJMECH 7766
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 3 October 2014
Revised Date: 9 March 2015
Accepted Date: 11 March 2015
Please cite this article as: W.S. Alwan, R. Karpoormath, M.B. Palkar, H.M. Patel, R.A. Rane, M.S.
Shaikh, A. Kajee, K.P. Mlisana, Novel imidazo[2,1-b]-1,3,4-thiadiazoles as Promising Antifungal Agents
Against Clinical Isolate of C. Neoformans, European Journal of Medicinal Chemistry (2015), doi:
10.1016/j.ejmech.2015.03.021.
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ACCEPTED MANUSCRIPT
Graphical Abstract
Novel Imidazo[2,1-b]-1,3,4-thiadiazoles as Promising Antifungal Agents
Against Clinical Isolate of C. Neoformans
O
Ring C
Wesam S. Alwan, Mahesh B. Palkar, Rajesh A. Rane, Harun M. Patel, Mahamadhanif S.
a*.
Shaikh, Afsana Kajee, Koleka P. Mlisana, Rajshekhar Karpoormath
N
S
N
Abstract
Ring A
Ring B
N
Novel hybrids including chalcones (5a-o) and Schiff bases (6a-j) of
imidazo[2,1-b]-1,3,4-thiadiazole scaffold were synthesized and evaluated
against clinical isolate of C. Neoformans .
Designed analogues of Chalcones

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Novel imidazo[2,1-b]-1,3,4-thiadiazoles as Promising
Antifungal Agents Against Clinical Isolate of C.
Neoformans
a
a*
a
a
Wesam S. Alwan , , Rajshekhar Karpoormath , Mahesh B. Palkar , Harun M. Patel , Rajesh A.
a
a
a
b
Rane , Mahamadhanif S. Shaikh , Afsana Kajee , Koleka P. Mlisana
a
Department of Pharmaceutical Chemistry, Discipline of Pharmaceutical Sciences, College of Health
Sciences, University of KwaZulu-Natal, Westville Campus, Durban – 4000, South Africa.
b
Departemnt of Microbiology, National Health laboratory services (NHLS), Inkosi Albert Luthuli Central
Hospital (Durban, South Africa).
*
Corresponding author
E-mail: karpoormath@ukzn.ac.za, rvk2006@gmail.com
Tel no.: +27(0)312607179, +27721107207; Fax No.:+27(0)312607792
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Abstract
We herein report the synthesis and in vitro antimicrobial evaluation of twenty five novel hybrid
derivatives of imidazo [2,1-b]-1,3,4-thiadiazole containing chalcones (5a-o) and Schiff bases (6a-j)
against three fungal strains (Candida albicans, Cryptococcus neoformans and Aspergillus niger).
Most of the tested compounds displayed substantial anti-fungal activity with MICs ranging between
1
.56 - 100 µg/mL. Compounds 5a, 5b and 5n exhibited promising activity against Cryptococcus
neoformans at a MIC 1.56 µg/mL. In addition, compound 5n also demonstrated significant
antifungal activity against the clinical isolates of Cryptococcus neoformans at MIC 3.125 µg/mL.
However, moderate activity was observed for these compounds against four bacterial strains
(Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa) and
Mycobacterium tuberculosis (H Rv).
3
7
Keywords: Imdazo [2,1-b]-1,3,4-thiadiazole; Chalcones, Schiff bases; Antifungal activity;
Cryptococcosis; C. neoformans
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1. Introduction
In the recent years, mortality and morbidity rate of opportunistic fungal infections is exponentially
increasing and the number of fatal incidence due to fungi is becoming comparable with that of
tuberculosis and malaria [1]. This could be attributed to increase in the number of patients with
organ and stem cell transplantation, HIV/AIDS patients and other immune compromised patients
[2]. Furthermore, the primary organisms responsible for invasive fungal infections (e.g. Candida,
Cryptococcus and Aspergillus species) have developed drastic resistance. Patients with significant
immunosuppression frequently develop Cryptococcosis, which is caused by the encapsulated yeast
Cryptococcus neoformans and is responsible for serious clinical illnesses like lung infections, fungal
meningitis and encephalitis [3]. It spreads by gulping aerosolized spores which may enter the
pulmonary or the central nervous system [4]. Moreover, the virulence of C. neoforman depends
upon the strain resistance and the immune level of the host, which could be latent and may lead to a
permanent neurological injury [5-7]. A matter of grave concern in the treatment of fungal infections
is the availability of limited number of efficacious antifungal drugs (e.g. amphotericin B, 5-
fluorocytosine, fluconazole and voriconazole), which suffer from severe drawbacks such as; narrow
therapeutic spectrum, drug resistance, high toxicity and low bioavailability [8, 9] . Although the use
of a new generation of triazoles, the available polyenes in lipid formulations, the use of
echinocandins or the combination therapy have been introduced as alternatives in the last ten years,
but fungal infections still remains difficult to eradicate [10]. This necessitates urgent need to
discover and develop novel chemotype antifungal molecules.
The fused ring of imidazole with a 1,3,4-thiadiazole motif is a very important heterocyclic system
containing a bridgehead nitrogen atom known as imidazo[2,1-b]-1,3,4-thiadiazoles. Imidazo[2,1-b]-
1
,3,4 thiadiazoles are known to exhibit a diverse array of biological activities such as antibacterial
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[11-13], anti-fungal (I) [14], anti- tubercular [15], anticonvulsant (II) [16, 17], antihyperlipidemic
[18], anti-inflammatory (III), analgesic, antipyretic [19, 20], anti-cancer (IV), anthelmintic and anti
ambeic agents[21]. Yet the cellular biology and the interactions of these compounds with different
receptors and enzymes have not been widely studied [22]. Despite numerous attempts to develop
new structural prototype in the search for more effective antimicrobials, the imidazo[2,1-b]-1,3,4-
thiadiazole still remain as one of the most versatile scaffold, which could be further exploited to
develop hybrids as promising antimicrobial agents.
On the other hand, the concept of hybrid drugs has gained more attention wherein two or more
bioactive pharmacophores are linked covalently to have synergistic effect [23]. It is anticipated that
such approach may solve the problem of drug resistance by displaying dual drug action [24]. Using
this approach, several research groups have recently reported hybrid molecules by coupling
medicinally privileged motif chalcone with biologically important pharmacophores. For instance an
integration of coumarins with chalcones (V), led to hybrid compounds which displayed potent
antitumor activity [25]. Likewise integration of Isatins with chalcones (VI) exhibited more
efficacious activity than the commonly used chemotherapeutic drug cisplatin against the breast
cancer cell lines [26]. Similarly, fusion of bromopyrrole alkaloid with chalcones (VII)
demonstrated potent cytotoxicity and revealed that the integration of 4,5-dibromopyrrole moiety into
chalcones lead to significant improvement of cytotoxic profile of 4,5-dibromopyrrole [27].
Correspondingly, chalcones bearing 2,4-thiazolidinedione and benzoic acid moieties (VIII)
presented potential anti-bacterial activity against gram positive bacteria, particularly against
multidrug-resistant strains of clinical isolates [28]. Moreover, integration of 5-nitroisoquinolines
(IX) and Abacavir prodrugs (X) with Schiff bases enhanced the anti-malarial and anti-HIV scope of
these compounds [29, 30]. Equally, integrating isoniazid a well-known anti-TB drug with aryl
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hydrzone led to a hybrid (XI) which exhibited potent anti-tubercular activity [31]. Likewise,
nitrofurantion hybrid (XII) with anti-infective activity was discovered by hybridizing 5-nitrofuran
carboxyaldehyde motif with hydrazide feature [32] as illustrated in Figure 1. Therefore, in view of
the above facts and in continuation of our search on biologically active hybrid molecules, herein we
report the synthesis and spectral studies of novel chalcones (5a-o) and Schiff base (6a-j) hybrids of
imidazo[2,1-b]-1,3,4-thiadiazoles with their subsequent in vitro biological evaluation for
antibacterial, antifungal and anti-mycobacterial activity.
2. Chemistry
The synthesis of a series of novel chalcones (5a-o) and Schiff base (6a-j) hybrids of imidazo[2,1-b]-
1
,3,4-thiadiazole was achieved through convenient and efficient synthetic route as outlined in
Scheme 1. Synthesis of the desired 2-substitutedphenyl-6-(4-bromophenyl)imidazo(2,1-b)1,3,4-
thiadiazole (3a-f) was carried out by the condensation of 2-amino-5-substituted phenyl-1,3,4-
thiadiazole (2a-f) with α-bromo ketone in DMF. Vilsmeier-Haack reaction of compounds (3a-f)
with phosphurs oxychloride and DMF yielded the corresponding derivatives 2-substituted
imidazo[2,1-b]- 1,3,4thiadiazole-5-carbaldehydes (4a-f) in good yields (85-90%). The aldehyde
th
functional group at the 5 position of the imidazo[2,1-b]1,3,4-thiadiazole nucleus was utilized to
perform Claisen-Schmidt reaction with different aryl/heteroaryl ketones in ethanolic NaOH (10%) to
afford corresponding chalcone derivatives (5a-o). Compounds (4a-f) were further reacted with
aliphatic cyclic amine using conventional method by refluxing in ethanol with catalytic amount of
glacial acetic acid for 24 h. This resulted in lower yields of Schiff bases (6a-j) (10-20%). Thus, in
order to improve the yield reactions were carried out under controlled microwave irradiation (CEM
Discover, Explorer-12 Hybrid, Microwave conditions: 25 min at 150 psi), which afforded the
corresponding Schiff base (6a-j) in good yields (44-83 %) [11, 27].
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3. Results and Discussion
Structures of compound (4a-f) and their corresponding final hybrid derivatives (5a-o and 6a-j) were
1
13
characterized based on their physicochemical and spectral (IR, H NMR, C NMR and MS,)
analysis. The analytical data of all the newly synthesized compounds along with their anticipated
structures are summarized in supporting information. The IR spectrum of compounds (4a-e)
-1
exhibited prominent and informative band, which appeared around 1757-1676 cm indicating the
presence of C=O (aldehydic carbonyl) group with confirming the formylation of compounds (3a-e)
1
by Vilsmeier-Haack reaction. This was further substantiated from H NMR spectra of compounds
(
4a-f), which exhibited a very distinct singlet peak resonating at δ10.14-10.09 ppm indicating the
presence of aldehydic (CHO) proton, thus confirming the formation of imidazo[2,1-b]-
,3,4thiadiazoles-5-carbaldehydes. The formation of title chalcone derivatives (5a-o) is evident from
their IR spectra, wherein the appearance of some prominent characteristic bands around 1674-1642
1
-1
-1
cm due to C=O stretch (chalcone), 1590-1406 cm for C=C stretch (α-β unsaturated carbons of
-1
1
chalcone) and 810.2-679 cm for C-Br Str. The H NMR spectra of compounds (5a-o) revealed two
distinctive doublets at δ 8.38-8.17 ppm and δ 8.09-8.03 ppm with coupling constant (J) of 14-16 Hz,
indicating the presence of α and β unsaturated protons of chalcones, while the various aromatic
protons appeared around δ 7.90-7.05 ppm.
The formation of imines (6a-j) was confirmed by IR spectra, where the disappearance of strong
-1
band around 1757-1676 cm of C=O group and appearance of characteristic imine (HC=N Str) band
-1
1
between 1671-1561 cm . Further, the H NMR spectra of Schiff base compounds (6a-j) displayed a
prominent singlet signal resonating around δ 8.64-7.97 ppm, which was attributed to imine proton
(CH=N), while the aromatic/heteroaromatic protons appeared as doublets/multiplet signals between
1
3
δ 8.14-6.96 ppm. In C NMR spectrum, it was observed that the most characteristic carbon signals
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(
CH , OCH , -CH=CH- and C=O) appeared at around δ 21.6, 55.6, 150-138.0 and 190.1-181.8 ppm
3
3
respectively, while the signals observed at around δ 121.5-114.4, 138.5-132.0, 145.9-138.3, 149.9-
1
1
7
45.7 ppm were assigned to C-5, C-7a, C-2, C-6. The aromatic carbon peaks appeared around δ
35.0-120.0 ppm, whereas the aliphatic cyclopropyl and/or cyclohexyl carbons resonated between δ
0.4-8.1 ppm. In addition, the formation of title compounds was also confirmed by recording their
respective mass spectra, which were in agreement with their expected molecular weights.
The Chalcone (5a-o) and Schiff base (6a-j) derivatives of imdazo[2,1-b]-1,3,4-thiadiazole scaffold
were evaluated for their in vitro antifungal activity against Candida albicans ATCC90028,
Cryptococcus neoformans ATCC6603 and Aspergillus niger ATCC16404, where Amphotericin B
was used as reference drug. The results (MIC values) of in vitro antifungal screening of the test
compounds are summarized in Table 1. However, a systematic analysis of the data as depicted in
Table 1 revealed that compounds 5a, 5b and 5n exhibited comparable antifungal activity (MICs =
1
.56 µg/mL), against Cryptococcus neoformans as the standard drug Amphotericin B (MIC = 1-2
µg/mL).
Compounds 5a, 5b and 5n exhibited most promising activity against C. neoformans at a MIC 1.56
µg/mL while compounds 5c, 5k and 5m were moderately active at a MIC 3.125 µg/mL. Compound
6
d (MIC = 12.5 µg/mL) showed moderate antifungal activity against C. albicans. Further, all the
synthesized compounds were tested against two well characterized clinical isolates of fungal strains
C. albicans and C. neoformans. The antifungal activity on the clinical isolates was carried out at the
Department of Microbiology, Inkosi Albert Luthuli Hospital, Durban, South Africa. Among the
tested series, compound 5n (MIC = 3.125 µg/mL) exhibited good antifungal activity against clinical
isolate of C. neoformans, while compounds 5a, 5i and 5k displayed moderate activity with MIC of
6
.25 µg/mL. In general, para and meta substitution on ring A with electron withdrawing (bromo and
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chloro), electron donating (methoxy) groups and presence of para bromo group on the phenyl group
of Ring B were observed to be beneficial feature for the antifungal activity. In case of Chalcones,
Ring C was tolerated to be unsubstituted phenyl and p-methyphenyl (p-CH -C H ) for antifungal
3
6
4
activity. The presence of heteroaryl group such as thiophene at ring C was not favored for the
antifungal activity. In case of Schiff bases, the presence of cyclic propyl or hexyl group exhibited
good antifungal activity. From literature, imidazo[2,1-b]-1,3,4-thiadiazoles derivatives were
reported for the antibacterial and anti-mycobacterial activity [13, 33, 34]. Hence, the synthesized
compounds were evaluated against Gram positive; Staphylococcus aureus ATCC25923, Bacillus
subtilis ATCC605 and Gram negative; Escherichia coli ATCC35218, Pseudomonas aeruginosa
ATCC27853] bacterial strain and M. tuberculosis H Rv strain. The antituburcular activity of these
3
7
compounds were carried out at National Institute of Allergy and Infectious Diseases (NIAID)
screening program, Bethesda, MD, USA [29-32]. All the synthesized compounds displayed
moderate or no activity against bacterial and mycobacterial strain as depicted in Table 1.
4. Conclusion
In conclusion, twenty five novel hybrids including chalcones (5a-o) and Schiff bases (6a-j) of
imidazo[2,1-b]-1,3,4-thiadiazole scaffold were synthesized and evaluated for their antifungal,
antitubercular and antibacterial activity. These synthesized hybrids displayed promising activity
against tested fungal strains, in particular for both the normal and clinical isolate of C. neoformans.
The chalcone (5a-o) derivatives exhibited significant antifungal activity when compared to the
Schiff bases (6a-j). The antifungal activity displayed by compounds 5a, 5b and 5n against
Cryptococcus neoformans indicates that these substituted hybrids can act as leads and can be further
exploited to develop potential antifungal agents. In addition, these active chalcones of
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imidazothiadiazole (5a-o) also displayed moderate activity (MIC >20 µg/mL) against M.
tuberculosis H Rv. The encouraging antifungal and anti-mycobacterial activity of synthesized
3
7
novel imidazo [2,1-b]-1,3,4-thiadiazole derivatives through modification of ring substituents and/or
additional functionalization indicated the potential for further research into the development
antifungal agents against the resistant clinical isolates.
5. Experimental Section
The analytical grade (AR) chemicals and reagents procured from commercial suppliers (Merck and
Sigma-Aldrich) and used without further purification. The solvents except AR grade were purified
as per the literature methods when necessary. The progress of the reactions and the purity of the
synthesized compounds were monitored by thin layer chromatography using pre-coated silica gel
plates (Merck), UV light and/or Iodine vapors were used as visualization agents. Melting points
were determined in open capillaries using (Electrothermal 9300) digital melting point apparatus and
were uncorrected. The IR spectra were recorded on (Perkin Elmer 100) FT-IR spectrophotometer
1
13
with universal ATR sampling accessory. H and C NMR spectra were recorded on (Bruker
Advance IV) NMR spectrometer at 400 and 100 MHz respectively using CDCl and DMSO-d .
3
6
(
CEM Discover, Explorer-12 Hybrid) Microwave reactor was used to synthesize some Schiff base
derivatives.
.1 General procedure for the synthesis of 2-amino-5-substitutedphenyl 1,3,4-thiadiazole (2a-f)
5
The each substituted benzoic acid (6.001g, 0.05 mol), thiosemicarbazide (4.557 g, 0.05 mol) and
POCl (13 ml) were thoroughly stirred, mixed and heated at 75°C for 1 h with constant stirring.
3
After cooling to rt, water (40 ml) was slowly added. The reaction mixture was further refluxed for
4
h. After cooling, the mixture was basified to pH 8 by careful drop wise addition of 10% aqueous
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ammonia solution with constant stirring. The precipitate thus obtained was filtered and
recrystallized from ethanol: water mixture to yield the pure compounds (2a-e) [13].
5
.2 General procedure for the synthesis of 2-substitutedphenyl-6-(4-bromophenyl) imidazo(2,1-
b)1,3,4-thiadiazole (3a-f)
A mixture of equimolar quantities of 2-amino-5-substitutedphenyl 1,3,4-thiadiazole (2g, 0.0078
mol) and 4-bromo phenacylbromide (2.1g, 0.0078 mol) was heated for 12-16 h with constant stirring
in DMF (10 ml). The reaction mixture was poured onto crushed ice and the solid hydrobromide
separated was filtered, washed and dried. Neutralization of hydrobromide salt intermediate with
cold aqueous solution of sodium carbonate yielded the corresponding free bases (3a-f), which were
further purified by recrystallization from ethanol[11].
5
.3 General procedure for the synthesis of 2-substitutedphenyl-5-formyl-6-(4-bromophenyl)
imidazo(2,1-b)1,3,4-thiadiazole (4a-f)
Vilsmeier-Haack reagent was freshly prepared by the careful addition of phosphoryl chloride (2ml,
0
.021 mol) in DMF (8 ml, 0.103 mol) at 0 °C with constant stirring. Then an appropriately 2-
substitutedphenyl-6-(4-bromophenyl)imidazo(2,1-b)1,3,4-thiadiazole (2g, 0.0045 mol) was added to
the reagent with continuous stirring maintaining the temperature at 0 °C for initial 30 min and later
stirred at rt for 2 h, and finally at 60 °C for another 2 h. The reaction mixture was then poured in
sodium carbonate solution and stirring was continued at 90 °C for 2 h. After cooling to rt, the
reaction mixture was suspended into water, extracted with (20 ml) dichloromethane (3 times), and
the collective extracts were washed with water and dried over anhydrous sodium sulphate. The
residue obtained after the in-vacuo removal of dichloromethane was further recrystallized from
ethanol to afford the desired compound (4a-f) as colourless crystalline solid [35].
1
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5
.4
General
procedure
for
the
synthesis
of
3-(2-(2-substitutedphenyl)-6-(4-
bromophenyl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl)-1-substituted aryl/heteroaryl-prop-2-en-1-one
5a-o)
(
In a round bottom flask equipped with sealed mechanical stirrer, 10% sodium hydroxide solution (2
ml) and (10 ml) ethanol were constantly stirred in an ice-bath for 2 min. Then, the appropriately
substituted aryl/heteroaryl ketones (0.3 g, 0.002 mol) and compounds (4a-f) (1 g, 0.002 mol) were
slowly added to the above mixture and stirred for 30 min. The reaction mixture was further stirred
at rt for 6-10 h. The solid precipitate obtained was filtered, dried and recrystallized using ethanol to
yield the chalcone derivatives (5a-o) of imidazo [2,1-b][1,3,4]thiadiazoles [27].
5
.4.1 3-(2-(2-bromophenyl)-6-(4-bromophenyl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl)-1
phenylprop-2-en-1-one 5a.
-1
Yellow crystals; Yield 79%, mp. 231-233 °C; IR [ATR, νmax, cm ]: 3054.1(Ar C-H), 3001.4 (C=C-
1
H), 1723.8 (C=N), 1661.3 (C=O), 1590 (C=C), 687.2 (C-Br); H NMR [400 MHz, CDCl
3
, δ ppm]:
8
7
.37-8.33 (d, J = 15 Hz, 1H, H β), 8.09-8.07 (d, J = 15 Hz, 1H, Hα), 8.06-8.05 (m, 1H, Ar-H), 7.92-
.90 (m, 1H, Ar-H), 7.85-7.83 (m, 1H, Ar-H), 7.69-7.54 (m, 4H, Ar-H), 7.52-7.46 (m, 6H, Ar-H);
1
3
C NMR [101 MHz, CDCl , δ ppm]: 189.9, 160.3, 150.0, 149.4, 138.3, 134.8, 132.8, 132.6, 132.3,
3
1
1
5
32.1, 132.1, 131.8, 130.3, 130.2, 129.7, 129.7, 128.8, 128.6, 128.5, 128.2, 128.1, 123.2, 122.1,
21.5, 120.2; HRMS (EI) m/z calcd for C25
2 3
H15Br N OS: 562.9303; found: 562.9308.
.4.2 3-(2-(2-bromophenyl)-6-(4-bromophenyl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl)-1-p-
tolylprop-2-en-1-one 5b.
-1
Yellow crystals; Yield 56%, mp. 225-227 °C; IR [ATR, νmax, cm ]: 3057.8 (Ar C-H), 2923.8 (C=C-
1
H), 1728 (C=N), 1658.9 (C=O), 1587.3 (C=C), 687.2 (C-Br); H NMR [400 MHz, CDCl , δ ppm]:
3
8
.37-8.33 (d, J = 15 Hz, 1H, H β), 8.08-8.04 (d, J = 15, 1H, H α), 8.01-7.99 (m, 2H, Ar-H), 7.92-
1
1

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7
.83 (m, 2H, Ar-H), 7.69-7.63 (m, 4H, Ar-H), 7.56-7.44 (m, 2H, Ar-H), 7.31-7.29 (m, 2H, Ar-H),
1
3
2
1
1
.43 (s, 3H, Ar-CH3); C NMR [101 MHz, CDCl , δ ppm]: 189.4, 160.2, 149.8, 149.3, 143.6,
3
35.7, 134.8, 132.5, 132.4, 132.1, 131.8, 130.5, 130.3, 129.1, 129.6, 129.3, 129.1, 129.1, 128.6,
2 3
28.1, 127.8, 123.2, 122.1, 121.5, 120.4, 21.6; HRMS (EI) m/z calcd for C26H17Br N OS: 576.9459;
found: 576.9463.
5
2
.4.3 3-(2-(2-bromophenyl)-6-(4-bromophenyl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl)-1-(thiophen-
-yl)prop-2-en-1-one 5c.
-1
Yellow crystals; Yield 53%, mp. 263-265 °C; IR [ATR, νmax, cm ]: 3064.6 (Ar C-H), 2962.7 (C=C-
1
H), 1724.4 (C=N), 1647.3 (C=O), 1578.6 (C=C), 707.3(C-Br); H NMR [400 MHz, CDCl , δ ppm]:
3
8
.21-8.17 (d, J = 15 Hz, 1H, H β), 8.06-8.02 (d, J = 15, 1H, Hα), 8.89-7.61 (m, 8H, Ar-H), 7.56-7.52
1
3
(m, 1H, thiophene 3H), 7.47-7.42 (m, 1H, thiophene 4H), 7.17-7.15 (m, 1H, thiophene 5H);
C
3
NMR [101 MHz, CDCl , δ ppm]: 181.9, 160.3, 150.1, 149.4, 145.9, 134.8, 134.1, 133.7, 132.6,
1
32.3, 132.1, 131.8, 131.7, 130.3, 130.2, 129.7, 128.2, 128.1, 127.5, 123.3, 122.1, 121.3, 120.1;
HRMS (EI) m/z calcd for C H Br N OS : 568.8867; found: 568.8871.
2
3
13
2
3
2
5
.4.4 3-(6-(4-bromophenyl)-2-(2-chlorophenyl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl)-1-
phenylprop-2-en-1-one 5d.
-1
Yellow crystals; Yield 62%, mp. 223-225 °C; IR [ATR, νmax, cm ]: 3064.5(Ar C-H), 2601.3 (C=C-
1
H), 1714.6 (C=N), 1654.7 (C=O), 1586 (C=C), 687.5 (C-Br); H NMR [400 MHz, CDCl
3
, δ ppm]:
8
7
1
1
5
.34-8.30 (d, J = 16 Hz, 1H, H β), 8.07-8.05 (d, J = 15, 1H, H α), 8.04-7.98 (m, 3H, Ar-H), 7.66-
1
3
3
.52 (m, 5H, Ar-H), 7.51-7.46 (m, 5H, Ar-H); C NMR [101 MHz, CDCl , δ ppm]: 189.9, 159.1,
50.1, 149.5, 138.3, 132.8, 132.8, 132.7, 132.4, 132.1, 132.1, 131.4, 131, 131, 130.2, 128.6, 128.6,
28.4, 128.2, 128.2, 127.6, 124.5, 123.2, 121.3, 120.2; HRMS (EI) m/z calcd for C H BrClN OS:
25
15
3
18.9808; found: 518.9812.
1
2

ACCEPTED MANUSCRIPT
`
5
.4.5 3-(6-(4-bromophenyl)-2-(2-chlorophenyl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl)-1-
p-tolylprop-2-en-1-one 5e.
-1
Yellow crystals; Yield 78%, mp. 236-238 °C; IR [ATR, νmax, cm ]: 3054.6 (Ar C-H),2599.9 (C=C-
1
H), 1731.4 (C=N), 1658.6 (C=O), 1587.7 (C=C), 756.9 (C-Br); H NMR [400 MHz, CDCl
3
, δ ppm]:
8
7
.34-8.30 (d, J = 16 Hz, 1H, H β), 8.06-8.05 (d, J = 15, 1H, H α), 8.03-7.98 (m, 3H, Ar-H), 7.66-
1
3
.60 (m, 5H, Ar-H),7.52-7.48 (m, 2H, Ar-H), 7.30-7.28 (d, 2H, Ar-H), 2.42 (s, 3H, Ar-CH3); C
NMR [101 MHz, CDCl , δ ppm]: 189.4, 159.0, 149.9, 149.4, 143.6, 135.8, 132.4, 132.1, 132.1,
3
1
1
5
2
31.4, 131, 131, 130.1, 130.1, 129.3, 129.3, 128.6, 128.6, 127.8, 127.8, 127.6, 127.6, 123.2, 121.4,
20.3. 21.7; HRMS (EI) m/z calcd for C H BrClN OS: 532.9964; found: 532.9968.
2
6
17
3
.4.6 3-(6-(4-bromophenyl)-2-(2-chlorophenyl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl)-1-(thiophen-
-yl)prop-2-en-1-one 5f.
-1
Yellow crystals; Yield 86%, mp. 260-263 °C; IR [ATR, νmax, cm ]: 3062.4(Ar C-H), 2585.1 (C=C-
1
H), 1899.3 (C=N), 1646.9 (C=O), 1584.5 (C=C), 725.8 (C-Br); H NMR [400 MHz, CDCl
3
, δ ppm]:
8
.23-8.19 (d, J = 15 Hz, 1H, H β),8.09-8.07 (d, J = 15, 1H, Hα), 8.06-8.04 (m, 1H, Ar-H), 7.89-7.88
(m, 1H, Ar-H),7.67-7.63 (m, 1H, Ar-H), 7.55-7.52 (m, 2H, thiophene 3H,4H), 7.20-7.18 (m, 1H
1
3
thiophene, 5H); C NMR [101 MHz, CDCl , δ ppm]: 181.9, 159.1, 150.2, 145.8, 133.6, 132.8,
3
1
1
5
32.4, 132.1, 131.8, 131.6, 131.4, 131, 130.2, 129.7, 129.7, 128.2, 128.2, 127.6, 127.5, 125, 123.3,
21.1, 120.1; HRMS (EI) m/z calcd for C23 OS : 524.9372; found: 524.9376.
.4.7 3-(6-(4-bromophenyl)-2-phenylimidazo[2,1-b][1,3,4]thiadiazol-5-yl)-1-phenylprop-2-en-1-
H
13BrClN
3
2
one 5g.
-1
Yellow crystals; Yield 53%, mp. 263-265 °C; IR [ATR, νmax, cm ]: 3064 (Ar C-H), 1655.3 (C=O),
1
1
8
572.5 (C=C), 690 (C-Br); H NMR [400 MHz, CDCl , δ ppm]: 8.29-8.25 (d, J = 15 Hz, 1H, H β),
3
.03-8.00 (d, J = 15, 1H, Hα), 7.96-7.89 (m, 4H, Ar-H), 7.60-7.56 (m, 5H, Ar-H), 7.51-7.40 (m, 5H,
1
3

ACCEPTED MANUSCRIPT
`
1
3
Ar-H); C NMR [101 MHz, CDCl , δ ppm]: 190.0, 162.7, 149.8, 148.6, 138.4, 132.7, 132.2, 132.2,
3
1
1
5
32.2, 132.1, 132.1, 130.1, 130.1, 130.1, 129.5, 129.5 , 128.7, 128.7, 128.4, 128.4, 128.3, 126.9,
23.1, 121.6, 120.3; HRMS (EI) m/z calcd for C25 OS: 485.0197; found: 485.0201.
.4.8 3-(6-(4-bromophenyl)-2-phenylimidazo[2,1-b][1,3,4]thiadiazol-5-yl)-1-p-tolylprop-2-en-1-
H16BrN
3
one 5h.
-1
Yellow crystals; Yield 80%, mp. 244-246 °C; IR [ATR, νmax, cm ]: 3033.1 (Ar C-H), 1655.6
1
(
C=O), 1582.9 (C=C), 688.8 (C-Br); H NMR [400 MHz, CDCl
3
, δ ppm]: 8.38-8.34 (d, J = 15 Hz,
1
7
H, H β),8.08-8.04 (d, J = 15, 1H, Hα), 8.02-7.92 4 (m, 4H, Ar-H), 7.67-7.66 (m, 3H, Ar-H), 7.62-
1
3
.60 (m, 4H, Ar-H), 7.37-7.35 (m, 2H, Ar-H), 2.48 (s, 3H, Ar-CH3); C NMR [101 MHz, CDCl , δ
3
ppm]: 189.5, 162.6, 148.5, 143.6, 135.8, 132, 132, 131.9, 131.9, 130.5, 130.5, 130.1, 130.1, 129.5,
29.46, 129.41, 128.6, 128.6, 127.9, 127.9, 126.9, 126.9, 123.1, 121.6, 120.5, 21.7; HRMS (EI) m/z
calcd for C H BrN OS: 499.0354; found: 499.0358.
1
2
6
18
3
5
2
.4.9 3-(6-(4-bromophenyl)-2-phenylimidazo[2,1-b][1,3,4]thiadiazol-5-yl)-1-(thiophen-2-yl)prop-
-en-1-one 5i.
-1
Yellow crystals; Yield 66%, mp. 267-269 °C; IR [ATR, νmax, cm ]: 3074.7 (Ar C-H), 1642.6 (C=O),
1
1
8
575.6 (C=C), 685.3 (C-Br); H NMR [400 MHz, CDCl , δ ppm]: 8.21-8.17 (d, J = 15 Hz, 1H, H β),
3
.04-8.01 (d, J = 15, 1H, H α), 7.97-7.95 (m, 2H, Ar-H), 7.90-7.88 (m, 1H, Ar-H),7.69-7.68 (m, 1H,
Ar-H), 7.62-7.59 (m, 3H, Ar-H), 7.58-7.57 (m, 2H, thiophene 3H,4H), 7.22-7.20 (m, 1H, thiophene
1
3
5
1
1
H); C NMR [101 MHz, CDCl , δ ppm]: 181.8, 162.7, 149.8, 148.6, 145.8, 133.6, 132.3, 132.3,
3
32.2, 132.2, 132.1, 131.5, 131.5, 130.1, 130.1, 129.8, 129.5, 129.5, 128.3, 127.6, 126.9, 123.2,
21.4; HRMS (EI) m/z calcd for C H BrN OS : 490.9762; found: 490.9766.
2
3
14
3
2
1
4

ACCEPTED MANUSCRIPT
`
5
.4.10 3-(6-(4-bromophenyl)-2-(4-chlorophenyl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl)-1-
phenylprop-2-en-1-one 5j.
-1
Yellow crystals; Yield 64%, mp. 247-250 °C; IR [ATR, νmax, cm ]: 3063.5 (Ar C-H), 1656.9 (C=O),
1
1
8
7
1
1
572.6 (C=C), 679.3 (C-Br); H NMR [400 MHz, CDCl
3
, δ ppm]: 8.29-8.25 (d, J = 15 Hz, 1H, H β),
.07-8.05 (d, J = 15, 1H, H α), 8.04-8.00 (m, 2H, Ar-H), 7.90-7.88 (d, J =8.56 Hz, 2H, Ar-H), 7.62-
1
3
.56 (m, 5H, Ar-H), 7.54-7.50 (m, 4H, Ar-H); C NMR [101 MHz, CDCl , δ ppm]: 190, 161.4,
3
49.9, 148.7, 138.5, 138.3, 132.8, 132.8, 132.2, 132.2, 132.1, 132.1, 130.1, 129.9, 129.9, 128.9,
28.9, 128.7, 128.7, 128.4, 128.3, 128.1, 123.2, 121.6, 120.5; HRMS (EI) m/z calcd for
C H BrClN OS: 518.9808; found: 518.9812.
25
15
3
5
.4.11
3-(6-(4-bromophenyl)-2-(4-chlorophenyl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl)-1-p-
tolylprop-2-en-1-one 5k.
-1
Yellow crystals; Yield 50%, mp. 246-248 °C; IR [ATR, νmax, cm ]: 2915.3 (Ar C-H), 1658.5 (C=O),
1
1
8
586.7 (C=C), 810.2 (C-Br); H NMR [400 MHz, CDCl , δ ppm]: 8.29-8.25 (d, J = 15 Hz, 1H, H β),
3
.03-7.99 (d, J = 15, 1H, Hα), 7.98-7.96 (d, J =8.12 Hz, 2H, Ar-H), 7.90-7.87 (d, J = 8.60 Hz, 2H,
Ar-H), 7.62-7.60 (m, 4H, Ar-H), 7.56-7.54 (d, J =8.48 Hz, 2H, Ar-H), 7.33-7.31(d, J =8.08 Hz, 2H,
13
Ar-H), 2.44 (s, 3H, Ar-CH3); C NMR [101 MHz, CDCl , δ ppm]: 189.5, 161.3, 149.6, 148.2,
3
1
43.7, 138.4, 135.8, 132.3, 132.1, 132.1, 131.8, 130.1, 130.1, 129.8, 129.4, 129.4, 129.2, 129, 128.5,
1
28.3, 128. 127.8, 123.1, 121.6, 120.6, 21.6; HRMS (EI) m/z calcd for C H BrClN OS: 532.9964;
2
6
17
3
found: 532.9968.
5
.4.12
3-(6-(4-bromophenyl)-2-(4-chlorophenyl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl)-1-
(thiophen-2-yl)prop-2-en-1-one 5l.
-1
Yellow crystals; Yield 45%, mp. 266-268 °C; IR [ATR, νmax, cm ]: 3341.2 (Ar C-H), 1645.4
1
(
C=O), 1406 (C=C), 719.2 (C-Br); H NMR [400 MHz, CDCl , δ ppm]: 8.17-8.13 (d, J = 15 Hz,
3
1
5

ACCEPTED MANUSCRIPT
`
1
H, H β), 8.04-8.00 (d, J = 15, 1H, Hα), 7.91-7.87 (m, 3H, Ar-H), 7.69-7.62 (m, 5H, Ar-H), 7.59-
1
3
7
.55 (m, 2H, thiophene 3H,4H), 7.22-7.20 (t, J = 8.68 Hz , 1H, thiophene 5H); C NMR [101 MHz,
, δ ppm]: 181.8, 161.4, 149.9, 148.4, 145.7, 138.5, 133.6, 132.2, 132.1, 132.1, 131.8, 131.5,
30.1, 129.9, 129.5, 129.3, 128.3, 128, 127.9, 127.5, 123.3, 121.44, 120.4; HRMS (EI) m/z calcd for
OS : 524.9372; found: 524.9376.
3-(6-(4-bromophenyl)-2-(4-methoxyphenyl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl)-1-
phenylprop-2-en-1-one 5m.
CDCl
3
1
C
23
H
13BrClN
3
2
5
.4.13
-1
Pale Yellow crystals; Yield 52%, mp. 229-231 °C; IR [ATR, νmax, cm ]: 2964.7 (Ar C-H), 1652.6
1
(
C=O), 1563.8 (C=C), 818.8 (C-Br); H NMR [400 MHz, CDCl , δ ppm]: 8.32-8.28 (d, J = 15 Hz,
3
1
H, H β), 8.08-8.04 (m, 2H, Ar-H), 8.04-8.00 (d, J = 15, 1H, H α), 7.89-7.87 (d, J =8.88 Hz, 2H, Ar-
1
3
H), 7.64-7.50 (m, 7H, Ar-H), 7.06-7.03 (d, J = 8.84 Hz, 2H, Ar-H), 3.92 (s, 3H, Ar-OCH3);
C
3
NMR [101 MHz, CDCl , δ ppm]: 190.1, 162.8, 162.5, 149.5, 148.5, 138.4, 132.7, 132.4, 132, 132,
1
5
5
30.1, 130.1, 128.7, 128.7, 128.6, 128.6, 128.5, 128.5, 128.4, 128.4, 123, 122.3, 121.5, 120.1, 114.9,
5.6; HRMS (EI) m/z calcd for C H BrN O S: 515.0303; found: 515.0308.
2
6
18
3
2
.4.14
3-(6-(4-bromophenyl)-2-(4-methoxyphenyl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl)-1-p-
tolylprop-2-en-1-one 5n.
-1
Yellow crystals; Yield 72%, mp. 252-254 °C; IR [ATR, νmax, cm ]: 2939.2 (Ar C-H), 1654.6
1
(
C=O), 1581.9 (C=C), 764.3 (C-Br); H NMR [400 MHz, CDCl
3
, δ ppm]: 8.32-8.28 (d, J = 15 Hz,
1
H, H β), 8.03-8.00 (d, J = 13 Hz, 1H, H α), 7.99-7.98 (d, J = 5.76 Hz, 2H, Ar-H), 7.89-7.87 (d, J
=
8.7 Hz, 2H, Ar-H), 7.64-7.59 (q, J = 4.2 Hz, 4H, Ar-H), 7.33-7.31 (d, J = 8.0 Hz, 2H, Ar-H), 7.06-
1
3
7
.04 (d, J = 4.84 Hz, 2H, Ar-H), 3.9 (s, 3H, Ar-OCH3), 2.46 (s, 3H, Ar-CH3); C NMR [101 MHz,
CDCl , δ ppm]: 189.6, 162.7, 149.3, 143.5, 135.9, 132, 132, 131.7, 131.7, 130.1, 130.1, 130.1,
3
1
6

ACCEPTED MANUSCRIPT
`
1
29.4, 129.4, 129.1, 129.1, 128.5, 128.5, 128.1, 128.1, 123, 122.4, 121.5, 120.3, 114.9, 55.6, 21.7;
HRMS (EI) m/z calcd for C H BrN O S: 529.0460; found: 529.0464.
2
7
20
3
2
5
.4.15
3-(6-(4-bromophenyl)-2-(4-methoxyphenyl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl)-1-
(thiophen-2-yl)prop-2-en-1-one 5o.
-1
Orange crystals; Yield 57%, mp. 257-259 °C; IR [ATR, νmax, cm ]: 3063.7 (Ar C-H), 1674 (C=N),
1
1
647 (C=O), 1579.9 (C=C), 716.5 (C-Br); H NMR [400 MHz, CDCl , δ ppm]: 8.19-8.16 (d, J = 15
3
Hz, 1H, H β), 8.03-7.99 (d, J = 15 Hz, 1H, H α), 7.90-7.87 (m, 3H, Ar-H), 7.64-7.59 (m, 5H, Ar-H),
.22-7.19 (m, 1H, thiophene 3H), 7.07-7.05 (d, J = 8.76 Hz, 2H, thiophene 4H,5H), 3.9 (s, 3H Ar-
7
1
3
OCH3); C NMR [101 MHz, CDCl , δ ppm]: 181.9, 162.8, 162.5, 149.6, 148.6, 145.8, 133.6,
3
1
1
5
32.4, 132, 131.8, 131.5, 130.1, 129.5, 128.5, 128.4, 128.3, 127.7, 123.1, 122.3, 121.3, 120, 114.9,
14.4, 55.6; HRMS (EI) m/z calcd for C24 : 520.9867; found: 520.9871.
.5 General procedure for the synthesis of 6-(4-bromophenyl)-2-(substitutedphenyl)imidazo[2,1-
3 2 2
H16BrN O S
b][1,3,4]thiadiazol-5-yl)methylene)cyclosubstitutedamine (6a-j)
Compounds (4a-f) (0.2 g, 0.0003 mol) and aliphatic cyclic amine (0.025g, 0.0003 mol) in ethanol (5
ml) and catalytic amount of glacial acetic acid were transferred into a 10 mL microwave tube kitted
with mechanical stirrer. The reaction mixture was irradiated with microwave radiations for 20-30
min at 150 psi pressure. The completion of reaction was monitored by TLC using ethyl acetate and
hexane (1:3). The solid thus obtained was filtered, dried and recrystallized using the suitable solvent
to afford the Schiff base derivatives (6a-j) of imidazo [2, 1-b][1,3,4]thiadiazoles.
5
.5.1 2-(2-bromophenyl)-6-(4-bromophenyl)imidazo[2,1-b][1,3,4]thiadiazol-5-
yl)methylene)cyclopropanamine 6a.
-1
Yellow crystals; Yield 45%, mp. 204-206 °C; IR [ATR, νmax, cm ]: 3136.9 (Ar C-H), 3064.7 (C=C-
1
H), 1561.4 (CH=N), 749.6 (C-Br); H NMR [400 MHz, CDCl , δ ppm]: 8.06 (s,1H,CH=N), 7.84-
3
1
7

ACCEPTED MANUSCRIPT
`
7
.82 (m, 1H, Ar-H), 7.74-7.70 (m, 3H, Ar-H), 7.53-7.45 (d, J= 8.25 Hz, 2H, Ar-H ), 7.43-7.39 (m,
1
3
1
H, Ar-H), 7.37-7.35 (m, 1H, Ar-H), 1.71 (br s, 4 H, Aliphatic-H), 1.23 (s, 1H, Aliphatic-H); C
, δ ppm]: 159.5, 146.5, 145.7, 134.3, 132.8, 132.2, 132.2, 132.2, 131.8,
31.7, 130.7, 127.9, 127.9, 126.7, 122, 121.5, 109.3, 29.6, 10.2, 10.2; HRMS (EI) m/z calcd for
S: 499.9306; found: 499.9310.
.5.2 2-(2-bromophenyl)-6-(4-bromophenyl)imidazo[2,1-b][1,3,4]thiadiazol-5-
yl)methylene)cyclohexanamine 6b.
NMR [101 MHz, CDCl
3
1
20 2 4
C H14Br N
5
-1
Orange crystals; Yield 60%, mp. 197-199 °C; IR [ATR, νmax, cm ]: 3063.3 (Ar C-H), 2852.8 (C=C-
1
H), 1639.1 (CH=N), 752.5 (C-Br); H NMR [400 MHz, CDCl , δ ppm]: 8.64 (s, 1H, CH=N), 7.94-
3
7
1
.92 (m, 3H, Ar-H), 7.74-7.72 (m, 1H, Ar-H), 7.57-7.55 (m, 2H, Ar-H ), 7.39-7.37 (m, 1H, Ar-H),
.81-1.77 (m, 4H, Aliphatic-H), 1.65-1.60 (m, 3H, Aliphatic-H), 1.38-1-30 (m, 4H, Aliphatic-H);
1
3
3
C NMR [101 MHz, CDCl , δ ppm]: 160.1, 147.2, 146.2, 134.3, 132.6, 132.3, 132.1, 132, 131.5,
1
30.7, 130.5, 130.2, 128, 127.9, 122.6, 122.1, 121.9, 70.4, 34.4, 25.6, 25.1, 24.65, 24.60; HRMS
(
EI) m/z calcd for C H Br N S: 541.9775; found: 541.9779.
23 20 2 4
5
.5.3 6-(4-bromophenyl)-2-(2-chlorophenyl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl)
methylene)cyclopropanamine 6c.
-1
Yellow crystals; Yield 50%, mp. 189-191 °C; IR [ATR, νmax, cm ]: 3136.8 (Ar C-H), 3068.2 (C=C-
1
H), 1648.5 (CH=N), 750 (C-Br); H NMR [400 MHz, CDCl
3
, δ ppm]: 8.06 (s, 1H, CH=N), 8.00-
7
2
.98 (m, 1H, Ar-H), 7.72-7.69 (d, J = 8.48 Hz, 2H, Ar-H), 7.53-7.45 (m, 3H, Ar-H), 7.43-7.39 (m,
1
3
3
H, Ar-H), 1.77 (s, 4H, Aliphatic-H), 1.23 (s, 1H, Aliphatic-H); C NMR [101 MHz, CDCl , δ
ppm]: 158.2, 146.7, 145.8, 132.8, 132.6, 132.1, 132.1, 132.1, 131.8, 131, 130.9, 128.6, 128.6, 127.4,
1
4
26.7, 121.5, 109.2, 29.7, 11.2, 11.2; HRMS (EI) m/z calcd for C H BrClN S: 455.9811; found:
20 14 4
55.9815.
1
8

ACCEPTED MANUSCRIPT
`
5
.5.4 6-(4-bromophenyl)-2-(2-chlorophenyl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl)
methylene)cyclohexanamine 6d.
-1
Yellowish green crystal; Yield 70%, mp. 188-190 °C; IR [ATR, νmax, cm ]: 3056.4 (Ar C-H),
1
2
849.8 (C=C-H), 1637.9 (CH=N), 747.2 (C-Br); H NMR [400 MHz, CDCl
3
, δ ppm]: 8.68 (s, 1H,
CH=N), 8.14-8.00 (m, 1H, Ar-H), 7.95-7.93 (d, J = 8.25 Hz, 2H, Ar-H), 7.57-7.53 (m, 2H, Ar-H),
.45-7.40 (m, 3H, Ar-H), 1.85-1.78 (m, 4H, Aliphatic-H), 1.66-1.61 (t, J = 11.48 Hz, 3H, Aliphatic-
7
1
3
H), 1.39-1.34 (m, 4H, Aliphatic-H); C NMR [101 MHz, CDCl , δ ppm]: 158.7, 147.9, 147.3,
3
1
3
5
46.2, 132.7, 132.6, 132.1, 132.1, 131.4, 131.2, 130.9, 130.2, 128.6, 128.6, 127.4, 122.6, 121.8 70.4,
4.4, 25.7, 25.7, 25.7, 24.5; HRMS (EI) m/z calcd for C H BrClN S: 498.0281; found 498.0285.
2
3
20
4
.5.5 6-(4-bromophenyl)-2-phenylimidazo[2,1-b][1,3,4]thiadiazol-5- yl)methylene)
cyclopropanamine 6e.
-1
Yellowish green crystals; Yield 83%, mp. 190-192 °C; IR [ATR, νmax, cm ]: 3117.5 (Ar C-H), 2923
1
(
C=C-H), 1671.4 (CH=N), 760.1 (C-Br); H NMR [400 MHz, CDCl , δ ppm]: 8.02 (s, 1H, CH=N),
3
7
.87 – 7.84 (d, J = 8.40 Hz, 2H, Ar-H), 7.70-7.68 (d, J = 8.56 Hz, 2H, Ar-H), 7.52-7.49 (m, 5H, Ar-
1
3
H), 1.64 (br s, 3H, Aliphatic-H), 1.23-120 (m, 2H, Aliphatic-H); C NMR [101 MHz, CDCl
3
, δ
ppm]: 161.7, 145.5, 145.4, 132.8, 131.8, 131.8, 131.7, 131.7, 130.1, 130.1, 129.3, 129.3, 129.3,
1
4
5
26.7, 126.6, 121.4, 109.5, 32.5, 8.1, 8.1; HRMS (EI) m/z calcd for C H BrN S: 422.0201; found
20 15 4
22.0205.
.5.6 6-(4-bromophenyl)-2-(4-chlorophenyl)imidazo[2,1-b][1,3,4]thiadiazol-5- yl)methylene)
cyclopropanamine 6f.
-1
Yellow crystals; Yield 52%, mp. 187-189 °C; IR [ATR, νmax, cm ]: 3056 (Ar C-H), 2847.6 (C=C-
1
H), 1640.3 (CH=N), 761.3(C-Br); H NMR [400 MHz, CDCl
3
, δ ppm]: 8.01 (s, 1H, CH=N), 7.80-
7
.78 (d, J = 8.60 Hz, 2H, Ar-H), 7.69-7.67 (d, J = 8.56 Hz, 2H, Ar-H), 7.52-7.46 (m, 4H, Ar-H),
1
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1
3
1
.61 (br s, 4H, Aliphatic-H), 1.22 (s, 1H, Aliphatic-H); C NMR [101 MHz, CDCl , δ ppm]: 160.4,
3
1
1
5
45.7, 145.3, 137.9, 132.6, 131.8, 131.8, 131.6, 129.6, 129.6, 128.6, 128.6, 127.9, 127.9, 126.6,
21.5, 109.5, 50.8, 30.9, 30.9; HRMS (EI) m/z calcd for C20
4
H14BrClN S: 455.9811; found 455.9815.
.5.7 6-(4-bromophenyl)-2-(4-chlorophenyl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl)methylene)
cyclohexanamine 6g.
-1
Yellowish green crystal; Yield 61%, mp. 238-240 °C; IR [ATR, νmax, cm ]: 3147.2 (Ar C-H),
1
2
848.3 (C=C-H), 1652 (CH=N), 730.9(C-Br); H NMR [400 MHz, CDCl , δ ppm]: 8.62 (s, 1H,
3
CH=N), 7.93-7.90 (d, J = 8.64 Hz, 2H, Ar-H), 7.86-7.84 (d, J = 8.52 Hz, 2H, Ar-H), 7.57-7.55 (d, J
8.56 Hz, 2H, Ar-H ), 7.48-7.46 (d, J = 8.52 Hz, 2H, Ar-H), 1.83-1.79 (m, 4H, Aliphatic-H), 1.67-
=
1
3
1
1
1
4
5
.62 (m, 3H, Aliphatic-H), 1.41-1.30 (m, 4H, Aliphatic-H); C NMR [101 MHz, CDCl , δ ppm]:
3
61, 147.1, 146.2, 138, 132.5, 132, 131.5, 131.1, 130.5, 130.1, 129.8, 129.1, 128.6, 128.3, 127.7,
4
22.68, 122.2, 70.4, 34.4, 25.7, 25.7, 25.7, 24.5; HRMS (EI) m/z calcd for C23H20BrClN S:
98.0281; found 498.0285.
.5.8 6-(4-bromophenyl)-2-(4-methoxyphenyl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl)methyl
yl)methylene)cyclopropanamine 6h.
-1
Yellowish green crystal; Yield 62%, mp. 201-203°C; IR [ATR, νmax, cm ]: 3008.4 (Ar C-H), 2934.1
1
(
C=C-H), 1605.4 (CH=N), 722 (C-Br); H NMR [400 MHz, CDCl , δ ppm]: 7.97 (s, 1H, CH=N),
3
7
.96-7.77 (d, J = 8.80 Hz, 2H, Ar-H), 7.68-7.66 (d, J = 8.44 Hz, 2H, Ar-H), 7.51-7.49 (d, J = 8.52
Hz, 2H, Ar-H), 6.99-6.96 (d, J = 8.76 Hz, 2H, Ar-H), 3.86 (s, 3H, Ar-OCH ), 2.14 (s, 1H, Aliphatic-
, δ ppm]: 162.4, 161.6, 145.3, 145.1,
41.7, 133.9, 132.9, 131.8, 131.6, 129.2, 128.3, 126.5, 122.7, 121.2, 114.7, 114.5, 109.4, 55.5. 34.2,
0.6, 10.6; HRMS (EI) m/z calcd for C H BrN OS: 452.0306; found 452.0310.
3
1
3
H), 1.80 (br s, 4H, Aliphatic-H); C NMR [101 MHz, CDCl
3
1
1
2
1
17
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5
.5.9
6-(4-bromophenyl)-2-(4-iodophenyl)imidazo[2,1-b][1,3,4]thiadiazol-5-
yl)methylene)cyclopropanamine 6i.
-1
Yellowish green crystal; Yield 57%, mp. 203-205°C; IR [ATR, νmax, cm ]: 3138.8 (Ar C-H), 2961.2
1
(
C=C-H), 1581.2 (CH=N), 729.8 (C-Br); H NMR [400 MHz, CDCl , δ ppm]: 8.00 (s, 1H, CH=N),
3
7
.85-7.83 (d, J = 8.48 Hz, 2H, Ar-H), 7.69-7.67 (d, J= 8.48 Hz, 2H, Ar-H), 7.58-7.56 (d, J = 8.48
Hz, 2H, Ar-H ),7.52-7.50 (d, J = 8.48 Hz, 2H, Ar-H), 1.76 (br s, 4H, Aliphatic-H)1.23 (s, 1H,
1
3
Aliphatic-H); C NMR [101 MHz, CDCl
31.8, 131.6, 129.6, 129.1, 129.1, 128, 126.6, 121.5, 109.5, 98.3, 30.9, 10.3, 10.3; HRMS (EI) m/z
calcd for C H BrIN S: 547.9167; found 547.9171.
3
, δ ppm]: 160.7, 145.7, 145.2, 138.5, 138.5, 132.6, 131.8,
1
2
0
14
4
5
.5.10 6-(4-bromophenyl)-2-(4-iodophenyl)imidazo[2,1-b][1,3,4]thiadiazol-5-
yl)methylene)cyclohexanamine 6j.
-1
Pale green crystal; Yield 44%, mp. 239-241°C; IR [ATR, νmax, cm ]: 2918.3 (Ar C-H), 2848.3
1
(
C=C-H), 1635.6 (CH=N), 764.7(C-Br); H NMR [400 MHz, CDCl
3
, δ ppm]: 8.62 (s, 1H, CH=N),
7
.93-7.91 (d, J = 8.48 Hz, 2H, Ar-H), 7.84-7.82 (d, J = 8.48 Hz, 2H, Ar-H), 7.61-7.60 (d, J = 8.42
Hz, 2H, Ar-H ), 7.56-7.54 (2H, Ar-H), 1.86-1.79 (m, 4H, Aliphatic-H) 1.67-1.62 (m, 3H, Aliphatic-
1
3
H) 1.41-1.21(m, 4H, Aliphatic-H); C NMR [101 MHz, CDCl , δ ppm]: 161.2, 147, 146.4, 145.2,
3
1
3
5
38.5, 138.4, 132.5, 132, 131.4, 130.4, 130.1, 129.5, 128.3, 128.2, 122.6, 122.2, 98.4, 70.4, 34.4,
4
0.9, 29.7, 25.7, 24.5; HRMS (EI) m/z calcd for C23H20BrIN S: 589.9637; found 589.9641.
.6 Biological activity
5
.6.1 In vitro evaluation of antimicrobial activity
The chalcone (5a-o) and Schiff base (6a-j) derivatives of imidazo[2,1-b]- 1,3,4 thiadiazole were
further assessed for antimicrobial activity against panel of bacterial and fungal strains by following
earlier reported MIC assay method using resazurin dye [36-38].
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5
.6.2 Microorganism used
Standard cultures of two gram +ve [Staphylococcus aureus ATCC25923, Bacillus subtilis
ATCC6051], two gram –ve [Escherichia coli ATCC35218, Pseudomonas aeruginosa ATCC27853],
three fungal strains [Candida albicans ATCC90028, Cryptococcus neoformans ATCC66031 and
Aspergillus niger ATCC16404] and two clinical isolates of [Candida albicans and Cryptococcus
neoformans] were used for the antibacterial and antifungal activity respectively. Culturing and
subculturing (one day prior to testing) of these microorganisms was carried out at the department of
microbiology, Inkosi Albert Luthuli hospital, Durban, South Africa. Subcultering of these
microorganisms were used in this assay.
5
.6.3 In vitro evaluation of antitubercular activity
The Anti-TB activity of the synthesized compounds was determined by measuring bacterial growth
after 5 d in the presence of test compounds. Compounds were prepared as 10-point two-fold serial
dilutions in DMSO and diluted into 7H9-Tw-OADC medium in 96-well plates with a final DMSO
concentration of 2%. The highest concentration of compound was 200 µM where compounds were
soluble in DMSO at 10 mM. For compounds with limited solubility, the highest concentration was
5
0X less than the stock concentration e.g. 100 µM for 5 mM DMSO stock, 20 µM for 1 mM DMSO
stock. For potent compounds, assays were repeated at lower starting concentrations. Each plate
included assay controls for background (medium/DMSO only, no bacterial cells), zero growth (100
µM rifampicin) and maximum growth (DMSO only), as well as a rifampicin dose response curve.
Plates were inoculated with M. tuberculosis and incubated for 5 days: growth was measured by
OD590 and fluorescence (Ex 560/Em 590) using a BioTek™ Synergy 4 plate reader. Growth was
calculated separately for OD590 and RFU [39-41].
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Acknowledgment
The authors sincerely thank College of Health Science, University of KwaZulu-Natal, Durban,
South Africa and the National Research Foundation (NRF) for funding this project. We are also
grateful to National Health laboratory services (NHLS), Inkosi Albert Luthuli Central Hospital
(Durban, South Africa) for performing the antimicrobial activity as well as the Infectious Disease
Research Institute (IDRI), National Institute of Allergy and Infectious Diseases (NIAID) screening
program, Bethesda, MD, USA for carrying out the antitubercular testing. The authors would also
like to acknowledge Mr. Dilip Jagjivan (School of Chemistry and Physics, UKZN, South Africa) for
NMR spectral data.
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References
[
[
[
[
1] G.D. Brown, D.W. Denning, S.M. Levitz, Science, 336 (2012) 647.
2] D.P. Kontoyiannis, R.E. Lewis, Lancet, 359 (2002) 1135-1144.
3] K. Tripathi, V. Mor, N.K. Bairwa, M. Del Poeta, B.K. Mohanty, Front Microbiol, 3 (2012) 187.
4] R. Velagapudi, Y.P. Hsueh, S. Geunes-Boyer, J.R. Wright, J. Heitman, Infect Immun, 77 (2009)
4
345-4355.
[
[
(
[
[
[
[
[
(
[
(
[
5] C.C. Lai, C.K. Tan, Y.T. Huang, P.L. Shao, P.R. Hsueh, J Infect Chemother, 14 (2008) 77-85.
6] B.J. Park, K.A. Wannemuehler, B.J. Marston, N. Govender, P.G. Pappas, T.M. Chiller, AIDS, 23
2009) 525-530.
7] K. Voelz, R.C. May, Eukaryot Cell, 9 (2010) 835-846.
8] T.S. Harrison, Clin Infect Dis, 48 (2009) 1784-1786.
9] R.E. Lewis, Mayo Clin Proc, 86 (2011) 805-817.
10] T.F. Patterson, Lancet, 366 (2005) 1013-1025.
11] G. Mazzone, F. Bonina, G. Puglisi, R.R. Arrigo, C. Cosentino, G. Blandino, Farmaco Sci, 37
1982) 685-700.
12] Y. Luo, S. Zhang, Z.-J. Liu, W. Chen, J. Fu, Q.-F. Zeng, H.-L. Zhu, Euro J Med Chem, 64
2013) 54-61.
13] B. Chandrakantha, A.M. Isloor, P. Shetty, H.K. Fun, G. Hegde, Euro J Med Chem, 71 (2014)
3
16-323.
[14] B. Chaturvedi, N. Tiwari, Nizamuddin, Ind. J. Chem, 28B (1989) 358–361.
[15] V.P. Arya, F. Fernandes, V. Sudarsanam, Ind. J. Chem, 10 (1972) 598-601.
[16] I.T. Barnish, P.E. Cross, R.P. Dickinson, B. Gadsby, M.J. Parry, M.J. Randall, I.W. Sinclair, J
Med Chem, 23 (1980) 117-121.
[
(
[
[
17] I.A. Khazi, C.S. Mahajanshetti, A.K. Gadad, A.D. Tarnalli, C.M. Sultanpur, Arzneimitt, 46
1996) 949-952.
18] H.M. Patel, M.N. Noolvi, A. Goyal, B.S. Thippeswamy, Eur J Med Chem, 65 (2013) 119-133.
19] E. Abignente, F. Arena, E. Luraschi, C. Saturnino, E. Marmo, M. Cazzola, F. Rossi, E. Lampa,
Farmaco Sci, 40 (1985) 190-199.
20] A.K. Gadad, M.B. Palkar, K. Anand, M.N. Noolvi, T.S. Boreddy, J. Wagwade, Bioorg Med
Chem, 16 (2008) 276-283.
[
2
4

ACCEPTED MANUSCRIPT
`
[21] A. Marin, N. Valls, F.J. Berenguer, M.T. Alonso, A. Ramon Martinez, M. Mercedes Martinez,
J. Elguero, Farmaco, 47 (1992) 63-75.
[
22] I.A.M. Khazi, A.K. Gadad, R.S. Lamani, B.A. Bhongade, Tetrahedron, 67 (2011) 3289-3316.
23] C. Viegas-Junior, A. Danuello, V. da Silva Bolzani, E.J. Barreiro, C.A. Fraga, Curr Med Chem,
[
1
4 (2007) 1829-1852.
[24] C. Lazar, A. Kluczyk, T. Kiyota, Y. Konishi, J Med Chem, 47 (2004) 6973-6982.
[25] K.V. Sashidhara, A. Kumar, M. Kumar, J. Sarkar, S. Sinha, Bioorg Med Chem Lett, 20 (2010)
7
205-7211.
[
26] C. Karthikeyan, V.R. Solomon, H. Lee, P. Trivedi, Biomed Prevent Nutr, 3 (2013) 325-330.
27] R.A. Rane, N.U. Sahu, S.D. Gutte, A.A. Mahajan, C.P. Shah, P. Bangalore, Eur J Med Chem,
[
6
3 (2013) 793-799.
[
28] X.F. Liu, C.J. Zheng, L.P. Sun, X.K. Liu, H.R. Piao, Eur J Med Chem, 46 (2011) 3469-3473.
29] P. Rathelot, P. Vanelle, M. Gasquet, F. Delmas, M.P. Crozet, P. Timon-David, J. Maldonado,
[
Eur J Med Chem, 30 (1995) 503-508.
30] D. Sriram, P. Yogeeswari, N.S. Myneedu, V. Saraswat, Bioorg Med Chem Lett, 16 (2006)
127-2129.
31] I.V. Drapak, B.S. Zimenkovsky, R.B. Lesyk, D.V. Atamanyuk, U.B. Tchulovska, I.O.
Nektegajev, Farm. Zh, 21 (2008) 57-62.
[
2
[
[32] M. Maaland , L. Guardabassi, Vet Microbiol, 151 (2011) 396-399.
[33] S.G. Alegaon, K.R. Alagawadi, P.V. Sonkusare, S.M. Chaudhary, D.H. Dadwe, A.S. Shah,
Bioo Med Chem Lett, 22 (2012) 1917-1921.
[
[
[
[
34] A.K. Gadad, M.N. Noolvi, R.V. Karpoormath, Bioorg Med Chem, 12 (2004) 5651-5659.
35] K.R. Alagawadi, S.G. Alegaon, Arab J Chem, 4 (2011) 465-472.
36] C.M. Mann, J.L. Markham, J App Microbiol, 84 (1998) 538-544.
37] A. Espinel-Ingroff, A. Fothergill, M. Ghannoum, E. Manavathu, L. Ostrosky-Zeichner, M.
Pfaller, M. Rinaldi, W. Schell, T. Walsh, J Clin Microbiol, 43 (2005) 5243-5246.
[38] M. Jameel, M. Islamuddin, A. Ali, F. Afrin, M. Ali, Comp Altern Med, 14 (2014) 98.
[39] J. Ollinger, M.A. Bailey, G.C. Moraski, A. Casey, S. Florio, T. Alling, M.J. Miller, T. Parish,
A, PLoS One, 8 (2013) 60531.
40] P. Carroll, L.J. Schreuder, J. Muwanguzi-Karugaba, S. Wiles, B.D. Robertson, J. Ripoll, T.H.
Ward, G.J. Bancroft, U.E. Schaible, T. Parish, PLoS One, 5 (2010) 9823.
[
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`
[41] A. Zelmer, P. Carroll, N. Andreu, K. Hagens, J. Mahlo, N. Redinger, B.D. Robertson, S. Wiles,
T.H. Ward, T. Parish, J. Ripoll, G.J. Bancroft, U.E. Schaible, J Antimicrob Chemother, 67 (2012)
948-1960.
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List of captions
Tables
Table 1. Anti-fungal, antibacterial and anti-tubercular activity of a novel series of chalcone (5a-o)
and Schiff base (6a-j) analogues of imidazo[2,1-b]-1,3,4thiadiazole
Figures
Fig. 1. Design of various chalcone (5a-o) and Schiff base (6a-j) analogues of imidazo[2,1-b]-1,3,4-
thiadiazole by molecular hybridization approach
Schemes
Scheme 1. The synthetic outline for the synthesis of novel series of chalcone (5a-o) and Schiff base
(6a-j) analogues of imidazo[2,1-b]-1,3,4-thiadiazole; Reagents and conditions: (a)
Thiosemicarbazide, POCl3, reflux, 4 h, basify 40% NH4OH; (b) Thiosemicarbazide, FeCl3, Sodium
citrate, citric acid, reflux, 1 h, basify, 40% NH4OH; (c) 4-Bromo phenacylbromide, DMF, heating,
1
1
3
2-16 h; (d) DMF, POCl3, 0 °C, stir, 90 °C, 6 h, Na2CO316 h; (e) Aryl/Heteroaryl ketones, Ethanol,
0% NaOH, stir, RT, 6-10 h; (f) Cyclopropylamine or Cyclohexylamine, glacial acetic acid MW 20-
0 min at 150 W.
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Table 1. Anti-fungal, antibacterial and anti-tubercular activity of a novel series of chalcone (5a-o) and Schiff base (6a-j) analogues of imidazo[2,1-b]-1,3,4thiadiazole.
a
a,c
a
a,c
a
a
a
a
a
b
C. albicans
C. albicans
C. neoformans
C. neoformans
A. niger
S. aureus
B. subtilis
E. coli
P. aeruginosa
M. tuberculosis
Code
Structure
ATCC90028
(Clinical Isolate)
ATCC66031
(Clinical Isolate) ATCC16404 ATCC25923 ATCC6051 ATCC35218
ATCC27853
H Rv
37
5
a
200
>200
1.56
1.56
6.25
12.5
>200
>200
200
100
>200
>200
>200
>200
>200
>200
>20
>20
5b
200
>200
5
c
>200
>200
>200
>200
3.125
12.5
>200
>200
>200
>200
>200
>200
>200
100
>200
>200
>20
>20
5d
12.5
12.5
5e
50
>200
50
100
>200
200
>200
>200
>200
>20
5
f
50
>200
>200
200
25
200
200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>20
>20
5g
>200
1