Heterocycles 48. synthesis, characterization and biological evaluation of imidazo[2,1-b][1,3,4]thiadiazole derivatives as anti-inflammatory agents

Article abstract of DOI:10.3390/molecules23102425

Non-steroidal anti-inflammatory drugs (NSAIDs) are an important pharmacological class of drugs used for the treatment of inflammatory diseases. They are also characterized by severe side effects, such as gastrointestinal damage, increased cardiovascular r

Full text of DOI:10.3390/molecules23102425

molecules
Article
Heterocycles 48. Synthesis, Characterization and
Biological Evaluation of
Imidazo[2,1-b][1,3,4]Thiadiazole Derivatives as
Anti-Inflammatory Agents
Anamaria Cristina 1,2, Denisa Leonte 1, Laurian Vlase 3 , László Csaba Bencze 4, Silvia Imre 5,
Gabriel Marc 6 , Bogdan Apan 7, Cristina Mogosan 2,* and Valentin Zaharia 1,
*
,
1
Department of Organic Chemistry, Iuliu Hatieganu University of Medicine and Pharmacy,
,
Cluj-Napoca 400012, Romania; anamaria.cristina@umfcluj.ro (A.C.); hapau.denisa@umfcluj.ro (D.L.)
2
3
4
5
6
7
Department of Pharmacology, Physiology and Pathophysiology, Iuliu Ha,
Pharmacy, Cluj-Napoca 400349, Romania
Department of Pharmaceutical Technology and Biopharmaceutics, Iuliu Ha,
and Pharmacy, Cluj-Napoca 400012, Romania; laurian.vlase@umfcluj.ro
Biocatalysis and Biotransformation Research Group, Babe, -Bolyai University, Cluj-Napoca 400028, Romania;
cslbencze@yahoo.com
Department of Analytical Chemistry and Drug Analysis, Tîrgu Mure,
Târgu Mures 540139, Romania; silsta@yahoo.com
tieganu University of Medicine and
tieganu University of Medicine
s
s
University of Medicine and Pharmacy,
,
Department of Pharmaceutical Chemistry, Iuliu Hatieganu University of Medicine and Pharmacy,
,
Cluj-Napoca 400012, Romania; marc.gabriel@umfcluj.ro
Department of Pharmacology, Toxicology and Clinical Pharmacology, Iuliu Hatieganu University of
,
Medicine and Pharmacy, Cluj-Napoca 400349, Romania; apan_bogdan@yahoo.com
Correspondence: cmogosan@umfcluj.ro (C.M.); vzaharia@umfcluj.ro (V.Z.);
Tel.: +40-744-351-748 (C.M.); +40-264-594-148 (V.Z.)
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 1 September 2018; Accepted: 19 September 2018; Published: 21 September 2018
Abstract: Non-steroidal anti-inflammatory drugs (NSAIDs) are an important pharmacological
class of drugs used for the treatment of inflammatory diseases. They are also characterized
by severe side effects, such as gastrointestinal damage, increased cardiovascular risk and renal
function abnormalities. In order to synthesize new anti-inflammatory and analgesic compounds
with a safer profile of side effects, a series of 2,6-diaryl-imidazo[2,1-b][1,3,4]thiadiazole derivatives
5a
carrageenan-induced rat paw edema. Among all compounds, 5c showed better anti-inflammatory
activity compared to diclofenac, the standard drug, and compounds 5g 5i 5j presented a comparable
–l were synthesized and evaluated in vivo for their anti-inflammatory and analgesic activities in
,
,
antinociceptive activity to diclofenac. None of the compounds showed ulcerogenic activity.
Molecular docking studies were carried out to investigate the theoretical bond interactions between
the compounds and target, the cyclooxygenases (COX-1/COX-2). The compound 5c exhibited a higher
inhibition of COX-2 compared to diclofenac.
Keywords: imidazo[2,1-b][1,3,4]thiadiazole derivatives; anti-inflammatory activity; molecular
docking; antinociceptive activity
1. Introduction
Inflammation is a complex defensive process of the body which reflects the response of the
organism to various noxious stimuli. In the inflammatory process, the body tissues are affected by
heat, redness, swelling and pain [1]. Non-steroidal anti-inflammatory drugs (NSAIDs) are the most
Molecules 2018, 23, 2425; doi:10.3390/molecules23102425
www.mdpi.com/journal/molecules

Molecules 2018, 23, 2425
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widely used therapeutics for different diseases characterized by fever, pain and inflammation. It is well
known that NSAIDs inhibit prostaglandin-endoperoxide H synthase (PGHS) or cyclooxygenase (COX),
in a non-selective or selective manner. There are three isoforms of COX, the most studied being COX-1
and COX-2. COX-1 is the constitutive enzyme and is expressed in most tissues, being responsible
for the physiological production of prostaglandins, the protection of gastric mucosa, kidney function
and platelet aggregation. COX-2 is the inducible isoform and is the product of an immediate early
response gene in inflammatory cells [2–4]. Nevertheless, the long-term use of NSAIDs treatment is
often limited by gastrointestinal complications, such as ulcerations and hemorrhages that may result
from the suppression of physiological prostaglandins production in these tissues [2,5].
The ability of a selective COX-2 inhibitor to decrease prostaglandin production and acute
tissue inflammation at dosages that do not affect the stomach prostaglandin production has been
demonstrated. This fact suggests that COX-2 selective inhibitors may provide a safer therapeutic
alternative to non-selective NSAIDs [2,6].
Considering this, we proposed the synthesis of new derivatives that inhibit COX-2 in a higher
percentage than COX-1. To obtain this effect, we have chosen as reference molecule celecoxib, a COX-2
specific inhibitor. However, we have proposed the synthesis of compounds with COX-2 selective
inhibition, but not COX-2 specific inhibition because of the high cardiovascular risk. Thus, for the
comparison of the anti-inflammatory, analgesic and especially the ulcerogenic potential, we have
selected diclofenac as drug reference, which is a non-selective NSAID, but also the most active against
COX-2 compared to other NSAIDs carboxylic acids derivatives [6,7].
In recent years, imidazo[2,1-b][1,3,4]thiadiazole derivatives have been intensively studied
because of their wide range of biological activities. The imidazo[2,1-b][1,3,4]thiadiazole scaffold
makes up the core structure of pharmacological active molecules possessing antimicrobial [
8,
9],
anticonvulsant [10,11], diuretic [12], anticancer [13
anti-inflammatory [18,19] properties.
–15], antitubercular [16], analgesic [17] and
Encouraged by the above literature and in continuation of our research work concerning the
synthesis of diaryl-heterocyclic structures with anti-inflammatory and analgesic activities [20 22],
–
we proposed the synthesis of 2,6-diaryl-imidazo[2,1-b][1,3,4]thiadiazole derivatives. We used as
templates for our structures celecoxib and various imidazo[2,1-b][1,3,4]thiadiazoles (Figure 1)
with anti-inflammatory potential: 6-carbamic acid-2,6-dimethylimidazo[2,1-b][1,3,4]thiadiazoles [17],
6-(hetero)arylimidazo[2,1-b][1,3,4]thiadiazole-2-sulfonamides [23], 2-trifluoromethyl/sulfonamide-5,6-
diaryl substituted imidazo[2,1-b][1,3,4]thiadiazoles [19], 6-aryl-2-(6-methyl-benzofuran-3-ylmethyl)
imidazo[2,1-b][1,3,4]thiadiazoles [18], 2,6-diaryl-imidazo[2,1-b][1,3,4] thiadiazoles [24].
4' R
O
NH2
S
O
3
N
N
4
N
5
N
F3C
R4`
4"
R2
6
2
N
S
1
7a
7
R2= CF3
SO2NH2
R4`=H
OCH3
SCH3
R=H
OCH3
CH3
celecoxib
2-trifluoromethyl/sulfonamide-5,6-diaryl substituted
imidazo[2,1-b][1,3,4]thiadiazoles
3
N
4
N
5
R
4
6
3
2
N
7
S
1
8
6
O
R=Br, Cl, NO2
6-aryl-2-(6-methyl-benzofuran-3-ylmethyl)
imidazo[2,1-b][1,3,4]thiadiazoles
Figure 1. Structures of the literature lead compounds (Reproduced with permission from [Journal of
Saudi Chemical Society]; published by [Elsevier]; No. 4425891193269).

Molecules 2018, 23, 2425
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The validity of the synthesized molecules was assessed through preliminary in vivo
anti-inflammatory/analgesic screening and docking simulation, which highlighted the anti-inflammatory
potential of the compounds.
2. Results
2.1. Chemistry
The synthesis route of 2,6-substituted imidazo[2,1-b][1,3,4]thiadiazoles 5a
Scheme 1. The 6-(4-substituted phenyl)-2-(4-substituted phenyl)imidazo[2,1-b][1,3,4]thiadiazole
5a was achieved in a one-pot reaction by the condensation of 5-(4-substituted phenyl)-1,3,4-
thiadiazole-2-amine 3a with substituted phenacyl bromide 4a , under reflux in dry ethanol [19 25 26].
The 5-(4-substituted phenyl)-1,3,4-thiadiazole-2-amine 3a was previously obtained by the cyclization
, treated with thiosemicarbazide in the presence of phosphorus
–l, is illustrated in
–
l
–l
–l
,
,
–
l
of aromatic carboxylic acids 1a
–
l
2
oxytrichloride [9,20].
Scheme 1. The synthesis of 2,6-substituted imidazo[2,1-b][1,3,4]thiadiazoles 5a–l. (a) POCl3, reflux 2 h;
H2O, reflux 4 h; (b) EtOH, reflux 18 h.
The synthesized compounds were purified and physico-chemically characterized by melting
points (m.p.), infrared spectra (IR), nuclear magnetic resonance (1H-NMR, 13C-NMR and 19F-NMR)
and mass spectrometry (MS). The details of the synthetic procedures, physico-chemical analysis and
spectral data of the synthesized compounds are presented in the Materials and Methods section.
Three of the compounds, 5i
the compounds 5b 5f and 5h were reported in the literature possessing antibacterial (5f
antifungal (5b, 5f, 5h) [8,27] properties.
The compounds 5b 27 28], 5d [16,28], 5h [8,16,27], 5i [26], 5j [26], 5l [26] were physico-chemical
,
5j
,
5l were reported in literature as anticancer agents [26]. In addition,
,
,
5h) [ ] and
8
[
,
and spectral (IR, MS, 1H-NMR, 13C-NMR) characterized in the literature, while the compounds 5a [28]
and 5f [27,28] were incomplete characterized.
The IR spectra of the synthesized compounds 5a
bands at 3125–2922 cm−1
The 1H-NMR spectra of compounds 5a
imidazo[2,1-b][1,3,4]thiadiazole at 8.93–7.89 ppm. The signals appearing around
confirm the presence of OCH3 group from 5d, 5h, 5i, 5j, 5k, 5l compounds.
–l showed the characteristic =CH- stretching
.
–
l
displayed the characteristic singlet for C-5 proton of
4.06–3.19 ppm
δ
δ
The 13C-NMR spectra of the compounds 5a
5th position of imidazo[2,1-b][1,3,4]thiadiazole at
atoms from OCH3 group (5d 5h 5i 5j 5k
group (5c 5g 5k) were observed at
127.09–126.9 ppm. In the 19F-NMR spectra of the compounds 5c
5g, 5k, the signal of the -CF3 group appeared at δ −64.05–64.03 ppm.
–
δ
l
revealed the presence of the carbon atom from the
116.28–100.25 ppm. The aliphatic signals of carbon
,
,
,
,
, 5l) appeared at δ 56.82–55.33 ppm and those from CF3
,
,
δ
,

Molecules 2018, 23, 2425
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The MS spectra of the compounds 5b
,
5f and 5j with bromine atoms in their structure and of the
compounds 5e, 5f, 5g, 5h with chlorine atoms, presented characteristic peaks corresponding to the
two isotopes.
2.2. Anti-Inflammatory Activity
The 12 synthesized compounds 5a–l and diclofenac, as reference drug, were tested for their
in vivo anti-inflammatory activity by modified carrageenan-induced rat paw edema model, firstly
described by Winter et al. [29]. The results of anti-inflammatory potential are shown in Table 1.
Table 1. In vivo anti-inflammatory activity of synthesized compounds on carrageenan-induced paw
edema in rats.
Dose (mg/kg)
p.o.
Edema 1 h (mL) Edema 2 h (mL) Edema 3 h (mL) Edema 4 h (mL)
Compound
(%inhib.)
(%inhib.)
(%inhib.)
(%inhib.)
Negative control (vehicle)
-
1.38 ± 0.15
2.22 ± 0.24
3.05 ± 0.18
3.20 ± 0.25
1.33 ± 0.05
1.98 ± 0.20
2.52 ± 0.19
2.42 ± 0.24 *
5a
50
(3.50)
(10.79)
(17.63)
(24.40)
1.09 ± 0.26
1.95 ± 0.07
2.45 ± 0.15 *
2.38 ± 0.20 *
50
50
50
50
50
50
50
50
50
50
50
20
5b
5c
(20.87)
(12.37)
(19.92)
(25.60)
1.00 ± 0.14
1.52 ± 0.14 *
2.29 ± 0.04 *
2.32 ± 0.16 *
(27.50)
(31.63)
(24.95)
(27.53)
1.21 ± 0.14
1.95 ± 0.07
2.82 ± 0.04
2.84 ± 0.10
5d
5e
(12.55)
(12.29)
(7.81)
(11.05)
0.86 ± 0.14
1.85 ± 0.12
2.69 ± 0.13
2.64 ± 0.18
(37.88)
(16.72)
(11.90)
(17.41)
1.18 ± 0.18
1.93 ± 0.20
2.67 ± 0.26
2.66 ± 0.20
5f
(14.60)
(13.42)
(12.66)
(16.84)
1.23 ± 0.12
1.76 ± 0.15
2.66 ± 0.14
2.58 ± 0.15
5g
(10.86)
(20.84)
(12.99)
(19.45)
0.92 ± 0.17
2.09 ± 0.18
2.63 ± 0.14
2.03 ± 0.17 *
5h
5i
(33.78)
(6.00)
(13.86)
(28.05)
1.01 ± 0.16
1.97 ± 0.16
2.62 ± 0.26
2.35 ± 0.25 *
(26.90)
(11.39)
(14.19)
(26.59)
0.95 ± 0.15
1.81 ± 0.14
2.67 ± 0.19
2.32 ± 0.20 *
5j
(31.10)
(18.59)
(12.66)
(27.53)
1.27 ± 0.11
2.07 ± 0.09
2.59 ± 0.10
2.60 ± 0.19
5k
(8.08)
(7.05)
(15.34)
(18.61)
1.10 ± 0.07
1.99 ± 0.08
2.57 ± 0.06
2.74 ± 0.15
5l
(20.63)
(10.64)
(15.99)
(14.23)
1.21 ± 0.17
1.46 ± 0.16 *
1.95 ± 0.12 *
2.34 ± 0.10 *
Diclofenac (reference drug)
(12.30)
(34.33)
(36.03)
(26.96)
*
Statistically significant, p < 0.05; Values are expressed as mean
±
SEM; %inhib.—Edema inhibition; p.o.—per os
(oral administration).
When compared to control negative group, all the synthesized compounds (5a–l) administered
in doses of 50 mg/kg bw, by gavage, showed a reduction in edema volume. The group treated with
diclofenac (20mg/kg, bw), the non-selective NSAID, presented a significant reduction of edema
volume, especially at 2, 3 and 4 h after the λ-carrageenan administration. One hour after the
inflammation induction, compounds 5b (20.87%), 5c (27.50%), 5e (37.88%), 5h (33.78%), 5i (26.90%),
5j (31.10%), 5l (20.63%) showed better anti-inflammatory activity than diclofenac group, but the
results were not statistically significant. At 2 and 3 h after the inflammation occurred, the compounds
5c (31.63%, 24.95%) and 5b (19.92%) significantly decreased paw edema. Compounds 5a (24.40%),
5b (25.60%), 5c (27.53%), 5h (28.05%), 5i (26.59%) and 5j (27.53%) showed a significant edema inhibition
at 4 h after inflammation occurred and the compounds 5c, 5h, 5j had an anti-inflammatory activity
better than diclofenac (26.96%).

Molecules 2018, 23, 2425
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2.3. Antinociceptive Activity
The antinociceptive activity was evaluated on a model of inflammatory pain induced with
-carrageenan in rats, by a modified method of Randall and Selitto [30 31]. The oral administration of
the synthesized compounds 5a in doses of 50 mg/kg bw, produced a good increase of nociceptive
λ
,
–l
threshold when compared to negative control group. The results are presented in Table 2.
Table 2. Nociceptive threshold after the oral administration of tested compounds (Randall-Selitto test).
Paw Pressure (g)
Group
Dose (mg/kg) p.o.
1 h
2 h
3 h
4 h
Negative control (vehicle)
-
94.17± 7.57
98.33 ± 5.27
78.33 ± 5.73
79.17 ± 3.75
87.50 ± 5.59
84.17 ± 2.39
79.17 ± 1.54
86.67 ± 6.54
87.50 ± 5.28
100.00 ± 8.56 *
92.50 ± 5.88
98.33 ± 4.94 *
103.33 ± 4.94 *
85.83 ± 3.01
85.83 ± 3.01
107.50 ± 4.61 *
64.17 ± 2.71
70.83 ± 4.54
70.00 ± 5.63
74.17 ± 2.39
64.17 ± 3.01
70.83 ± 3.96
72.50 ± 4.03
90.83 ± 6.38 *
76.67 ± 4.22
87.50 ± 4.43 *
90.83 ± 4.90 *
74.17 ± 1.54
75.00 ± 1.83
94.17 ± 5.83 *
44.17 ± 1.54
52.50 ± 1.71
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
50
50
50
50
50
50
50
50
50
50
50
50
20
107.5 ± 6.16
57.50 ± 4.03 *
58.33 ± 2.79 *
53.33 ± 2.11
122.50 ± 3.10 *
97.50 ± 5.28
113.33 ± 7.15
114.17 ± 4.55
125.83 ± 6.76 *
110.83 ± 7.00
130.83 ± 8.41 *
130.00 ± 8.46 *
111.67 ± 4.22
113.33 ± 4.01
124.17 ± 5.07 *
62.50 ± 3.10 *
63.33 ± 4.22 *
80.00 ± 5.16 *
65.83 ± 3.01 *
78.33 ± 3.33 *
77.50 ± 2.81 *
68.33 ± 1.67 *
68.33 ± 1.05 *
78.33 ± 4.77 *
Diclofenac (reference drug)
* Statistically significant, p < 0.05. Values are expressed as mean ± SEM; p.o.—per os (oral administration).
The induction of paw inflammation with
threshold as edema volume increased from 1 to 4 h. Compounds 5g
increase in the nociceptive threshold when compared to control negative group. At 4 h after the
intraplantar injection of -carrageenan, all the synthesized compounds, except 5a and 5d, presented a
λ-carrageenan produced a decrease in the nociceptive
,
5i, 5j showed a significant
λ
significant increase in nociception threshold and the compound 5g (80.00
antinociceptive action than diclofenac (78.33 ± 4.77 g).
±
5.16 g) induced a better
2.4. Ulcerogenic Activity
All the compounds were evaluated for their ulcerogenic side effect after a single intragastric
administration of 50 mg/kg bw in rats. After we isolated the stomach, we assessed the ulceration score
by examining the gastric mucosa for irritation, ulcerations and microhemorrhages by a magnifying
lens. The results are presented in Table 3.
Table 3. Ulcerogenic activity screening of the synthesized compounds.
Group
Dose (mg/kg) p.o.
Ulceration Score
Negative control (vehicle)
-
0.00 ± 0.00
0.33 ± 0.21
0.08 ± 0.08
0.08 ± 0.08
0.17 ± 0.11
0.17 ± 0.11
0.25 ± 0.17
0.17 ± 0.17
0.42 ± 0.20
0.17 ± 0.17
0.08 ± 0.08
0.00 ± 0.00
0.08 ± 0.08
2.58 ± 0.27 *
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
50
50
50
50
50
50
50
50
50
50
50
50
20
Diclofenac (reference drug)
* Statistically significant, p < 0.05. Values are expressed as mean ± SEM; p.o.—per os (oral administration).
All synthesized compounds have shown a reduced ulcerogenic activity, with the stomach
ulceration score between 0.00
±
0.00 and 0.42
±
0.20, when compared to diclofenac. The standard drug

Molecules 2018, 23, 2425
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showed a high score of 2.58
compared to negative control group. The compound 5k has shown a good safety profile, and none of
±
0.27, inducing a significant ulceration of the stomach mucosa when
the synthesized compounds induced significant ulceration.
2.5. Molecular Docking
The comparative molecular docking study of the compounds 5a–l to the cyclooxygenases, COX-1
and COX-2, was carried out in order to clarify the binding mode of the compounds and to evaluate
the affinity of the tested compounds to both isoforms. The results of the molecular docking study are
presented in Table 4.
Table 4. The selectivity of the tested compounds (5a–l) to the COX 1 and COX 2 enzymes expressed as
variation of the Gibbs free energy (∆G) and the consequent predicted inhibition constant (Ki).
COX-1
∆G (kcal/mol)
COX-2
∆G (kcal/mol)
Compound
KiCOX-2/KiCOX-
1
Ki (µM)
Ki (µM)
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
−7.75
−7.51
−7.25
−6.78
−7.18
−6.51
−6.37
−7.14
−7.81
−7.32
−6.57
−6.53
−7.85
2.09
3.13
4.85
10.72
5.46
16.91
21.42
5.84
1.88
4.31
15.28
16.35
1.76
−8.31
−8.36
−8.53
−7.29
−8.16
−6.23
−6.28
−8.16
−8.32
−8.21
−6.69
−6.81
−8.01
0.81
0.74
0.56
4.53
1.04
27.12
24.93
1.04
0.80
0.96
12.48
10.19
1.34
0.39
0.24
0.12
0.42
0.19
1.60
1.16
0.18
0.43
0.22
0.82
0.62
0.76
Diclofenac
∆G—Variation of the Gibbs free energy; Ki—Constant of inhibition.
Overall, our series of compounds have a higher affinity to COX-2, than COX-1. Some compounds
presented a higher affinity to COX-2, referred to diclofenac (5a 5b 5c 5e 5h 5i and 5j). Among them,
compounds 5c 5e 5h and 5j, seemed to have a higher selectivity for COX-2, expressed as a
,
,
,
,
,
,
,
KiCOX2/KiCOX1 low ratio.
The clustering analysis is reported in the literature as a method of removal of the false positive
results for the molecular docking study [32,33]. A compound with a high homogeneity in binding
to the protein can be considered as a true binder. Thus, a compound with a high homogeneity has
a high number of conformations in the cluster of the best binding pose and a low number of other
wandering conformations in other clusters, including few other conformations. Approaching the
idea in the opposite direction, a compound with many clusters resulted after the molecular docking
and with few poses in the same cluster can be considered as a false binder, having a high dispersion
between conformations given by the different docking runs with random beginning positions [32
,33].
The clustering analysis of the resulted bounds characteristic for the compounds 5a–l are presented in
Table 5.
All tested compounds 5a–l are predicted to bind to COX-1 in a homogenous manner. They have
at least half of the predicted conformations in the same two. A root mean square deviation (RMSD)
cluster with the conformation predicted to have the highest affinity. The same homogenous pattern in
binding is found in the side of COX-2 too, with some exceptions (5d and 5e), which both have a more
chaotic binding to the enzyme. Overall, compounds in our series have a maximum of three binding
clusters, suggesting homogenous binding to COX-1 and COX-2.

Molecules 2018, 23, 2425
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Table 5. Clustering analysis of the resulted poses from the molecular docking study to the COX-1 and
COX-2 enzymes.
COX-1
COX-2
Cluster of the Top Pose
Cluster of the Top Pose
Compound
Number of
Clusters
Number of
Clusters
Average ∆G
Conformations
Average ∆G
Conformations
(kcal/mol)
(kcal/mol)
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
50
47
50
34
38
42
49
28
45
25
28
50
31
−7.52
−7.48
−7.01
−6.60
−7.05
−6.42
−6.15
−6.97
−7.70
−6.16
−6.29
−6.32
−7.56
1
2
1
2
2
2
2
2
2
3
3
1
3
50
50
50
17
17
43
38
49
42
46
32
46
33
−8.29
−8.34
−8.26
−7.18
−8.11
−6.17
−6.19
−7.93
−8.29
−8.16
−6.52
−6.55
−7.81
1
1
1
2
2
3
3
2
1
2
3
2
2
Diclofenac
∆G—Variation of the Gibbs free energy.
The analysis of the binding pattern showed that some amino acid residues are important in the
formation of the hydrogen bond with the synthetic molecules. Based on the results we observed
that Tyr357 and Ser523 are the most important polar amino acids from COX-1 and Ser516, Arg106
and Tyr371 from COX-2. The hydrogen bonds and the interaction diagrams for the synthetized
compounds are shown in Table 6, and respectively in Figure 2 (interaction of 5c with COX-1) and
Figure 3 (interaction of 5c with COX-2).
Table 6. The hydrogen bonds predicted to take place between compounds 5a–l and COX-1 and
COX-2 respectively.
COX-1
COX-2
Ligand
Compound
Ligand
Protein
N/A
Protein
5a
N/A
N (imidazole)
O-CH3
N (thiadiazole)
N (thiadiazole)
CF3
Ser516
Trp373
Arg106
Arg106
Tyr371
N/A
Tyr357
N (thiadiazole)
5b
N (thiadiazole)
CF3
Tyr357
Ser532
N/A
5c
5d
N/A
N/A
5e
5f
N/A
N/A
N/A
N/A
N (imidazole)
N/A
Arg106
N/A
5g
5h
5i
5j
5k
5l
CF3
N/A
N (thiadiazole)
N/A
Ser532
N/A
Tyr357
N/A
Tyr387
N/A
Ser532
Tyr387
N/A
N/A
N (imidazole)
N (imidazole)
N (imidazole)
N/A
Arg106
Arg106
Arg106
N/A
N/A
Ser516
Tyr371
CF3
N/A
-COO
N/A
−
−
Diclofenac
-COO
N/A—Not available.

Molecules 2018, 23, 2425
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Figure 2. The top binding pose of compound 5c docked to the catalytic site of COX-1. In section (
the figure, compound 5c is depicted by segments, while the protein components are depicted in their
tertiary structure, colored distinctly. In the next sections ( ), the top binding pose of 5c is depicted
as in section ( ), visualized from both sides, with the NSAID binding pocket of the COX-1 illustrated
a) of
b,c
a
more realistically, using surfaces. Hydrogen bonds are depicted in red lines.
Figure 3. The top binding pose of compound 5c docked to the catalytic site of COX-2. In section (a) of
the figure, compound 5c is depicted by segments, while the protein components are depicted in their
tertiary structure, colored distinctly. In the next sections ( ), the top binding pose of 5c is depicted
as in section ( ), visualized from both sides, with the NSAID binding pocket of the COX-2 illustrated
b,c
a
more realistically, using surfaces. Hydrogen bonds are depicted in red lines.
The substituents from the para position of the both aromatic rings are defining for the interaction
between synthesized compounds in our series. These substituents influence the binding of the ligands
to the protein, not especially because of the bonds between them and the protein, but by influencing
the position of the ligand inside the pocket on the long axis of the ellipsoid which characterizes the
NSAID binding pocket. The substituent from the para position, especially if it has a large volume
(such as bromine 5b, 5f, 5j), will tend to be oriented to the distal end of the NSAID binding pocket.

Molecules 2018, 23, 2425
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This will influence the positioning of the heterocycle imidazo[2,1-b][1,3,4]-thiadiazole relative to Tyr387
in COX-1. The tyrosine residue is considered important in the literature reports, because it has a
pivotal role in the hydrophobic environment [34]. Depending on the size of the substituents on the
aromatic rings, it will lead to formation, or not, of a hydrogen bond with the nitrogen atoms from the
imidazo[2,1-b][1,3,4]-thiadiazole fragment. An interesting aspect observed in our series is the presence
of the CF3 substituent, which can form hydrogen bonds with Ser532 from COX-1 and Tyr371 from
COX-2 and could influence the orientation of the ligand in the binding pocket.
Paired sequence alignment between COX-1 and COX-2 brought an obvious similarity between
these two isozymes, with a low evolutionary distance. The evaluation of the similarity of the amino
acids after pairing was depicted underneath their alignment using graphical signs, depending on the
degree of similarity between the two amino acids (Figure 4).
Figure 4. Pairwise sequence alignment between COX-1 and COX-2. The amino acids present in the
catalytic site are depicted in red. Between the two proteins, large groups of identical amino acids
were found, and the specified pairing is marked with an asterisk (*). An intermediate pairing, such as
conservative substitutions between the two COX isozymes are depicted using a colon (:) or dot (.),
while the lack of similarity was depicted as white space. The 17 catalytic amino acids provided by
the blast analysis are depicted in red. The single difference in terms of amino acid substitution in the
catalytic site between the two enzymes is the presence of Ile86 in COX-1, while in COX-2, at the same
position, Leu86 can be found.
3. Discussion
Based on the molecules design reported in literature which is known to possess anti-inflammatory
activity by blocking the COX-1/COX-2 [13
,25], we synthesized and evaluated the anti-inflammatory and
analgesic activity of 2,6-diaryl-imidazo[2,1-b][1,3,4]thiadiazole substituted with various pharmacophores.

Molecules 2018, 23, 2425
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The synthesis of 5a
–
l
compounds was achieved by previously reported methods [19,25,26]. The structures
of the synthesized compounds were assessed based on their spectral analysis: IR, 1H-NMR, 13C-NMR,
19F-NMR and MS. In the IR spectrum, the establishment of synthesized structures 5a
by the absence of NH2 bands around 3200 ppm. In addition, in 1H-NMR spectra of 5a
–
l
was carried out
, we identified
–l
the presence of the characteristic signal of imidazole proton which confirms the formation of desired
compounds. In the 13C-NMR spectra, all signals of the carbon atoms of the aromatic rings are present
in the aromatic region and the aliphatic signals corresponding to the carbon atoms from the CF3 and
OCH3 groups are also present.
The MS spectra confirmed the structures of the synthesized compounds 5a
–l
by the presence of
characteristic molecular peak.
The physico-chemical and spectral results obtained for the compounds 5a
,
5b
,
5d 5f 5h 5i 5j 5l
,
,
,
,
,
are in accordance with those reported previously in the literature [8,16,26–28].
All synthesized compounds were evaluated in vivo for their anti-inflammatory, analgesic and
ulcerogenic potential and in silico (molecular modeling) for their interaction with cyclooxygenases.
The administration of carrageenan into the hind paw of rats, produces the cardinal signs of
inflammation: edema, erythema and hyperalgesia [35]. It is known that the peripheral inflammatory
response to carrageenan has a biphasic mechanism and if the edema inhibition is more evident in the
initial edematous phase, the compounds mechanism of action is due to the inhibition of histamine
and 5-hydroxytriptamine, followed by the inhibition of bradykinin release [
phase is attributed to an increase of prostaglandins in the damaged tissue [37 39]. The inflammatory
effect of -carrageenan is explained by its interaction with Toll-like receptor 4 which induces the
activation of Nuclear Factor B (NF B) and interleukin 8 (IL-8) through a pathway of innate immunity
mediated by B-cell CLL/lymphoma 10 [40]. NF- B is a major transcription factor for regulating
the expressions of proinflammatory enzymes and cytokines (iNOS, COX-2 and TNF- ) [41]. COX-2
is an inducible enzyme and is responsible for the elevated production of prostaglandins (E2 and
I2) during inflammation [ ]. The NSAIDs mechanism of action is correlated with the non-selective
inhibition of COX-1 and COX-2 isozymes [ ]. In the first phase of edema development, at 1 h after
the inflammation occurs, compounds 5b 5c 5d 5e 5f 5h 5i 5j and 5l presented an increased edema
inhibition compared to negative control group and better than diclofenac. Then, at 4 h after the
inflammation occurs, compounds 5a 5b 5c 5h 5i and 5j, have shown a significant inflammation
decrease when compared to negative control group. In these compounds, we can observe that the
significant reduction of edema volume was exceeded by those containing bromo (5b 5j), chloro (5h),
trifluoromethyl (5c) and methoxy (5h 5i 5j) substituents. The compound 5c seems to have the most
1,35,36]. The second
–
λ
κ
κ
κ
α
4
6
,
,
,
,
,
,
,
,
,
,
,
,
,
,
significant edema inhibition, at 2, 3 and 4 h after inflammation induction, when compared to control
negative group. The structure-activity relationship studies suggest that the presence of unsubstituted
phenyl inhibits both COX-1 and COX-2 and the presence of methoxy and halogen groups at the 4th
position of the 6-phenyl ring contributes for selective COX-2 inhibitory activity [19].
The molecular docking study was performed in order to compare the binding selectivity of the
tested compounds to the catalytic sites of COX-1 and COX-2 and to observe if there is a correlation
between in vivo and in silico studies of anti-inflammatory potential. Because of the closeness of
these protein structures, development of novel agents with selective inhibition against COX-2 is
challenging. Diclofenac binds to COX-1 and COX-2 through its carboxylate group hydrogen-bonded
to Tyr387 and Ser532, respectively Tyr371 and Ser516, amino acids residues which are important to its
anti-inflammatory activity [7].
The compound 5c had the smallest KiCOX-2/KiCOX-1 in our series, which can be interpreted as
having a higher selectivity to COX-2 instead of COX-1, compared to the other compounds. More than
that, compound 5c had a homogenous binding pattern to both catalytic sites of the enzymes, in terms
of the conformations found in the same 2 Å RMSD cluster. The para-substituents from both the
aromatic rings are important for the interaction of the compounds with the COX binding sites.

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The CF3 substituent from 5c formed hydrogen bonds with Ser532 from COX-1 and Tyr371 from COX-2.
The interaction with the serine amino acid residue from COX-1 is important to COX-1 inhibition [42].
The antinociceptive activity evaluated in a model of inflammatory pain showed an increase in pain
threshold for all tested compounds when compared to negative control group. Compounds 5g (para-Cl
and unsubstituted phenyl), 5i (para-OCH3 and unsubstituted phenyl) and 5j (para-OCH3 and para-Br)
presented a significant increase in pain threshold compared to negative control group. Analgesic
activity studies revealed that compounds with unsubstituted phenyl and with para-bromophenyl and
para-fluorophenyl exhibited good analgesic activities [43,44].
Most of the NSAIDs are associated with gastric side effects at the anti-inflammatory, analgesic and
antipyretic dosage. The inhibition of COX-1 is associated with gastric injury. Diclofenac, a non-selective
NSAID, when administered by gavage (20 mg/kg bw) had the highest ulceration score among all tested
compounds, even if it is considered the most active against COX-2 among several other carboxylic
acid-containing NSAIDs [7]. The ulceration risk of all synthesized compounds was significantly
reduced when compared to diclofenac group, giving them a good safety profile.
4. Materials and Methods
4.1. Chemistry
4.1.1. Reagents and Solvents
All the commercially available chemicals necessary for the synthesis were purchased from
Sigma-Aldrich Chemie GmbH (Steinheim, Germany), TCI Europe N.V. (Zwijndrecht, Belgium) or
Merck KgaA (Darmstadt, Germany) and were used as supplied, without further purification.
4.1.2. Analytical Methods
Chemical reactions were monitored by thin layer chromatography (TLC) on pre-coated silica
gel 60F254 sheets from Merck (Darmstadt, Germany), and dichloromethane and a mixture of
dichloromethane: acetone 25:1 v/v was used as elution system. The spots were viewed in UV light at
254 nm. The compounds were recrystallized from ethanol or a mixture of ethanol: water. Preparative
chromatographic purifications were performed using Merck Kieselgel 60 Å column chromatography
and dichloromethane and a mixture of dichloromethane: acetone 25:1 v/v as eluent.
Melting points were determined using open capillary tube method, with an Electrothermal IA
9000 digital apparatus (Bibby Scientific Limited, Staffordshire, UK), and were uncorrected.
The MS spectra were recorded using an Agilent 1100 Ion Trap mass spectrometer (Agilent
Technologies, Santa Clara, CA, USA) operating at 70 eV. 1H-NMR, 13C-NMR and 19F-NMR spectra were
recorded on Bruker Avance DRX-600 spectrometer (Billerica, MA, USA) operating at 600 MHz, 151 MHz
and 565 MHz, in trifluoroacetic acid (TFA)-d6 and acetone-d6 (external standard), dimethylsulfoxide
(DMSO)-d6, deuterated chloroform (CDCl3), dimethylformamide (DMF)-d6, acetic acid-d6 and as
solvents and tetramethylsilane (TMS) as internal standard. The spectral data are mentioned in
the Materials and Methods section, using the following abbreviations for peak patterns: s-singlet,
d-doublet, dd-double doublet, t-triplet, q-quartet.
The FT-IR spectra were performed on a Jasco FT/IR 470 Plus spectrometer (Easton, MD, USA)
using the ATR technique. The IR spectra were recorded between 4000 and 400 cm−1 wavelengths at
4 cm−1 resolution.
4.1.3. Synthesis of Imidazo[2,1-b][1,3,4]thiadiazole Derivatives
Synthesis of 5-(4-substituted phenyl)-1,3,4-thiadiazole-2-amine 3a–l
0.1 M of aromatic carboxylic acid 1a–l was refluxed with 0.2 M of thiosemicarbazide 2 and 5 mL
of phosphorous oxytrichloride for 2 h. After 2 h the mixture was cooled and diluted with 10 mL of
water and again refluxed for additional 4 h. Then the mixture was filtered hot and the filtrate was

Molecules 2018, 23, 2425
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neutralized with a solution of 10% potassium hydroxide. The formed precipitate was filtered off and
recrystallized from absolute ethanol.
General procedure for the synthesis of 6-(4-substituted phenyl)-2-(4-substituted phenyl)imidazo[2,1-
b][1,3,4]thiadiazole 5a–l
To a solution of 5-(4-substituted phenyl)-1,3,4-thiadiazole-2-amine 3a–l (0.01 mol) in 10 mL
absolute ethanol, the corresponding phenacyl bromide was added (0.02 mol). The mixture was
refluxed for 18 h or more, depending of the reaction progress, monitored by TLC. After the reaction
was complete, the mixture was filtered and the solid hydrobromide was collected. The precipitate
was neutralized by aqueous sodium carbonate solution, filtered and purified by recrystallization or by
column chromatography to remove unwanted products. The purification method is indicated below
for each individual compound.
2,6-Diphenylimidazo[2,1-b][1,3,4]thiadiazole (5a) [28]: Yield 53% (0.146 g); purified by column
◦
chromatography, eluent dichloromethane: acetone 25:1 v/v; m.p. 200–202 C; cristaline white powder;
FT-IR (solid state,
8.53 (s, 1H, CH-5 imidazo[2,1-b][1,3,4]thiadiazole), 7.93 (dd, J = 7.6, 2H, CH-2, CH-6), 7.88 (d,
J = 7.2 Hz, 2H, CH-2’, CH-6’), 7.62–7.53 (m, 3H, CH-4, CH-3’, CH-5’), 7.40 (t, J = 7.7 Hz, 2H,
CH-3, CH-5), 7.28 (t, J = 7.4 Hz, 1H, CH-4’); 13C-NMR (151 MHz, DMSO-d6, CDCl3)
151.13 (C,
υ
cm−1): 3094, 3049, 3026 (CH aromatic); 1H-NMR (600 MHz, DMSO-d6, CDCl3)
δ
δ
C-2 imidazo[2,1-b][1,3,4]thiadiazole), 135.85 (C, C-8 imidazo[2,1-b][1,3,4]thiadiazole), 134.54 (C, C-6
imidazo[2,1-b][1,3,4]thiadiazole), 123.85 (C, C-1), 121.82 (C, C-1’), 119.87 (C, C-4), 119.43 (C, C-3’,
C-5’), 118.61 (C, C-3, C-5), 117.36 (C, C-2’, C-6’), 116.65 (C, C-4’), 114.85 (C, C-2, C-6), 100.25 (C, C-5
imidazo[2,1-b][1,3,4]thiadiazole); ESI+-MS: m/z 278.2 [M + H]+ (calcd. 278.3 for C16H11N3S + H+).
6-(4-Bromophenyl)-2-phenylimidazo[2,1-b][1,3,4]thiadiazole (5b
240–242 ◦C; yellow powder; FT-IR (solid state,
(600 MHz, TFA-d6, acetone-d6)
2H, CH-2, CH-6), 7.23 (m, J = 7.9, 3.8 Hz, 4H, CH-2’, CH-3’, CH-5’ CH-6’), 7.08 (t, J = 7.5 Hz, 1H,
CH-4), 6.99 (t, J = 7.7 Hz, 2H, CH-3, CH-5); 13C-NMR (151 MHz, TFA-d6, acetone-d6)
157.60 (C,
)
[27,28]: Yield 40% (0.140 g); m.p.
1
υ
cm−1): 3095, 3071, 3051 (CH aromatic); H-NMR
δ
7.89 (s, 1H, CH-5 imidazo[2,1-b][1,3,4]thiadiazole), 7.41 (d, J = 8.5 Hz,
δ
C-2 imidazo[2,1-b][1,3,4]thiadiazole), 157.33 (C, C-8 imidazo[2,1-b][1,3,4]thiadiazole), 156.89 (C, C-6
imidazo[2,1-b][1,3,4]thiadiazole), 132.47 (C, C-1), 132.06 (CH, C-3’, C-5’), 131.56 (CH, C-4), 130.44 (C,
C-1’), 128.99 (CH, C-3, C-5), 128.62 (CH, C-2’, C-6’), 125.75 (CH, C-2, C-6), 125.52 (C, C-4’), 115.86 (C,
C-5 imidazo[2,1-b][1,3,4]thiadiazole); ESI+-MS: m/z 356.2 ([M + H]+, 79Br), 358.2 ([M + H]+, 81Br) (calcd.
for C16H10BrN3S + H+: 356.1 79Br, 358.0 81Br).
2-Phenyl-6-(4-(trifluoromethyl)phenyl)imidazo[2,1-b][1,3,4]thiadiazole (5c): Yield 38% (0.130 g);
◦
purified by column chromatography, eluent dichloromethane; m.p. 218–220 C; yellow powder;
FT-IR (solid state,
υ δ 8.21
cm−1): 3107, 3084 (CH aromatic); 1H-NMR (600 MHz, TFA-d6, acetone-d6)
(s, 1H, CH-5 imidazo[2,1-b][1,3,4]thiadiazole), 7.83 (t, J = 8.0 Hz, 2H, CH-2, CH-6), 7.71 (m, J = 8.4 Hz,
4H, CH-2’, CH-3’, CH-5’, CH-6’), 7.55 (t, J = 8.5 Hz, 1H, CH-4), 7.48–7.42 (m, 2H, CH-3, CH-5);
13C-NMR (151 MHz, TFA-d6, acetone-d6)
δ 172.26 (C, C-2 imidazo[2,1-b][1,3,4]thiadiazole), 161.09 (C,
C-8 imidazo[2,1-b][1,3,4]thiadiazole), 147.89 (C, C-6 imidazo[2,1-b][1,3,4]thiadiazole), 140.75 (C, C-10),
136.89 (q, J2C-F = 34.7 Hz, C, C-40), 133.22 (CH, C-3, C-5), 132.23 (CH, C-4), 132.04 (C, C-1), 130.74 (q,
J
3C-F =3 Hz, CH, C-3’, C-5’), 130.19 (CH, C-2, C-6), 129.76 (CH, C-2’, C-6’), 126.9 (q, J1C-F = 272 Hz, C,
CF3), 115.65 (C, C-5 imidazo[2,1-b][1,3,4]thiadiazole); 19F-NMR (565 MHz, TFA-d6, acetone-d6):
(s, -CF3); ESI+-MS: m/z 346.3 [M + H]+ (calcd. 346.3 for C17H10F3N3S + H+).
δ -64.05
6-(4-Methoxyphenyl)-2-phenylimidazo[2,1-b][1,3,4]thiadiazole (5d) [16,28]: Yield 27% (0.080 g); m.p.
◦
1
232–234 C; gray powder; FT-IR (solid state,
MHz, DMF-d6) 8.93 (s, 1H, CH-5 imidazo[2,1-b][1,3,4]thiadiazole), 8.08 (d, J = 6.9 Hz, 2H, CH-2,
CH-6), 7.98 (d, J = 8.8 Hz, 2H, CH-2’, CH-6’), 7.73–7.64 (m, 3H, CH-3, CH-4, CH-5) 7.10 (d,
J = 8.8 Hz, 2H, CH-3’, CH-5’), 3.87 (s, 3H, CH3); 13C-NMR (151 MHz, DMF-d6)
164.03 (C, C-2
imidazo[2,1-b][1,3,4]thiadiazole), 160.46 (C, C-4’), 144.50 (C, C-8 imidazo[2,1-b][1,3,4]thiadiazole
υ
cm−1): 3107, 3069 (CH aromatic); H-NMR (600
δ
δ

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), 142.45 (C, C-6 imidazo[2,1-b][1,3,4]thiadiazole), 132.88 (C, C-1), 130.03 (CH, C-4), 129.45 (CH,
C-3, C-5), 127.39 (CH, C-2’, C-6’), 127.03 (CH, C-2, C-6), 123.49 (C, C-1’), 114.75 (C, C-5
imidazo[2,1-b][1,3,4]thiadiazole), 110.51 (C, C-3’, C-5’), 55.40 (C, OCH3); ESI+-MS: m/z 308.3 [M + H]+
(calcd. 308.3 for C17H13N3OS + H+).
2-(4-Chlorophenyl)-6-phenylimidazo[2,1-b][1,3,4]thiadiazole (5e): Yield 32% (0.100 g); m.p. 220–222
◦C; gray powder; FT-IR (solid state,
υ
cm−1): 3125, 3096 (CH aromatic); 1H-NMR (600 MHz,
TFA-d6, acetone-d6)
7.87 (d, 2H, CH-2’, CH-6’), 7.77–7.72 (m, 5H, CH-3, CH-4, CH-5, CH-3’, CH-5’); 13C-NMR
(151 MHz, TFA-d6, acetone-d6) 168.84 (C, C-2 imidazo[2,1-b][1,3,4]thiadiazole), 145.47 (C, C-8
δ 8.42 (s, 1H, CH-5 imidazo[2,1-b][1,3,4]thiadiazole), 8.10 (d, 2H, CH-2, CH-6),
δ
imidazo[2,1-b][1,3,4]thiadiazole), 142.94 (C, C-6 imidazo[2,1-b][1,3,4]thiadiazole), 141.72 (C, C-4), 133.06
(C, C-1), 131.82 (CH, C-3, C-5), 131.32 (CH, C-3’, C-5’), 130.02 (CH, C-2, C-6), 127.38 (CH, C-2’, C-6’),
127.07 (C, C-1’), 126.63 (C, C-4’), 112.58 (CH, C-5 imidazo[2,1-b][1,3,4]thiadiazole); ESI+-MS: m/z 312.6
([M + H]+, 35Cl), 314.6 ([M + H]+, 37Cl) (calcd. for C16H10ClN3S + H+: 312.8 35Cl, 314.8 37Cl).
6-(4-Bromophenyl)-2-(4-chlorophenyl)imidazo[2,1-b][1,3,4]thiadiazole (5f) [27
,
28]: Yield 77% (0.230
◦
g); purified by column chromatography, eluent dichloromethane; m.p. 256–258 C; gray powder;
FT-IR (solid state,
8.11 (s, 1H, CH-5 imidazo[2,1-b][1,3,4]thiadiazole), 7.77 (d, J = 8.4 Hz, 2H, CH-2, CH-6), 7.57 (d,
J = 8.3 Hz, 2H, CH-2’, CH-6’), 7.45 (d, J = 8.4 Hz, 2H, CH-3, CH-5), 7.41 (d, J = 8.3 Hz, 2H, CH-3’, CH-5’);
13C-NMR (151 MHz, TFA-d6, acetone-d6)
168.82 (C, C-2 imidazo[2,1-b][1,3,4]thiadiazole), 145.55 (C,
υ
cm−1): 3119, 3071, 3047 (CH aromatic); 1H-NMR (600 MHz, TFA-d6, acetone-d6)
δ
δ
C-8 imidazo[2,1-b][1,3,4]thiadiazole), 142.94 (C, C-6 imidazo[2,1-b][1,3,4]thiadiazole), 140.58 (C, C-4),
134.55 (CH, C-3’, C-5’), 131.65 (CH, C-3, C-5), 129.78 (CH, C-2’, C-6’), 128.54 (CH, C-2, C-6), 127.62 (C,
C-1), 126.72 (C, C-1’), 125.64 (C, C-4’), 116.28 (CH, C-5 imidazo[2,1-b][1,3,4]thiadiazole); ESI+-MS: m/z
390.2 ([M + H]+, 79Br), 392.2 ([M + H]+, 81Br) (calcd. for C16H9BrClN3S + H+: 390.6 79Br, 392.6 81Br).
2-(4-Chlorophenyl)-6-(4-(trifluoromethyl)phenyl)imidazo[2,1-b][1,3,4]thiadiazole (5g): Yield 54% (0.200
g); purified by column chromatography, eluent dichloromethane; m.p. 228–230 ◦C; yellow powder;
FT-IR (solid state,
8.20 (s, 1H, CH-5 imidazo[2,1-b][1,3,4]thiadiazole), 7.77 (d, J = 7.0 Hz, 2H, CH-3’, CH-5’),
7.69–7.67 (m, 4H, CH-2, CH-3, CH-5, CH-6), 7.43 (d, J = 6.7 Hz, 2H, CH-2’, CH-6’); 13C-NMR
(151 MHz, TFA-d6, acetone-d6) 170.88 (C, C-2 imidazo[2,1-b][1,3,4]thiadiazole), 147.89 (C, C-8
υ
cm−1): 3104, 2922 (CH aromatic); 1H-NMR (600 MHz, TFA-d6, acetone-d6)
δ
δ
imidazo[2,1-b][1,3,4]thiadiazole), 144.66 (C, C-6 imidazo[2,1-b][1,3,4]thiadiazole), 141.73 (C, C-4) 136.89
0
(q, J2C-F = 34.7 Hz, C, C-4 ), 133.63 (CH, C-3, C-5), 132.03 (C, C-1), 131.92 (CH, C-2, C-6), 130.11 (q, J3C-F
= 3 Hz, CH, C-30, C-50), 129.81 (CH, C-20, C-60 ), 128.77 (C, C-10), 127.07 (q, J1C-F = 272 Hz, C, CF3),
115.71 (CH, C-5 imidazo[2,1-b][1,3,4]thiadiazole); 19F-NMR (565 MHz, TFA-d6, acetone-d6):
δ -64.05 (s,
-CF3); ESI+-MS: m/z 380.2 ([M + H]+, 35Cl), 382.2 ([M + H]+, 37Cl) (calcd. for C17H9ClF3N3S + H+: 380.7
35Cl, 382.7 37Cl).
2-(4-Chlorophenyl)-6-(4-methoxyphenyl)imidazo[2,1-b][1,3,4]thiadiazole (5h) [
8,16,27]: Yield 44%
(0.150 g); purified by column chromatography, eluent dichloromethane: acetone 25:1 v/v; m.p.
◦
1
228–230 C; gray powder; FT-IR (solid state,
MHz, TFA-d6, acetone-d6) 8.02 (s, 1H, CH-5 imidazo[2,1-b][1,3,4]thiadiazole), 7.75 (d, J = 8.2 Hz, 2H,
CH-2’, CH-6’), 7.51 (d, J = 8.4 Hz, 2H, CH-2, CH-6), 7.42 (d, J = 8.2 Hz, 2H, CH-3’, CH-5’), 7.02 (d,
J = 8.4 Hz, 2H, CH-3, CH-5), 3.84 (s, 3H, CH3); 13C-NMR (151 MHz, TFA-d6, acetone-d6)
168.55 (C,
υ
cm−1): 3119, 3086 (CH aromatic); H-NMR (600
δ
δ
C-2 imidazo[2,1-b][1,3,4]thiadiazole), 162.39 (C, C-4’), 145.02 (C, C-8 imidazo[2,1-b][1,3,4]thiadiazole),
142.82 (C, C-6 imidazo[2,1-b][1,3,4]thiadiazole), 141.17 (C, C-4), 131.67 (CH, C-3, C-5), 129.82 (CH, C-2’,
C-6’), 129.24 (CH, C-2, C-6), 126.90 (C, C-1), 120.45 (C, C-1), 117.03 (CH, C-3’, C-5’), 111.94 (CH, C-5
imidazo[2,1-b][1,3,4]thiadiazole), 56.82 (C, OCH3); ESI+-MS: m/z 342.6 ([M + H]+, 35Cl), 344.6 ([M + H]+
,
37Cl) (calcd. for C17H12ClN3OS + H+: 342.8 35Cl, 344.8 37Cl).
2-(4-Methoxyphenyl)-6-phenylimidazo[2,1-b][1,3,4]thiadiazole (5i) [26]: Yield 74% (0.230 g); m.p.
◦
198–200 C; yellow powder; FT-IR (solid state,
υ
cm−1): 3127, 3071 (CH aromatic); 1H-NMR (600 MHz,

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Acetic acid) δ 8.28 (s, 1H, CH-5 imidazo[2,1-b][1,3,4]thiadiazole), 7.94 (d, J = 7.7 Hz, 2H, CH-2, CH-6),
7.82 (d, J = 6.7 Hz, 2H, CH-2’, CH-6’), 7.47 (t, J = 6.6 Hz, 2H, CH-3’, CH-5’), 7.39 (t, J = 6.4 Hz, 1H, CH-4’),
7.12 (d, J = 7.9 Hz, 2H, CH-3, CH-5), 3.91 (s, 3H, OCH3); 13C-NMR (151 MHz, Acetic acid)
δ 166.00 (C,
C-2 imidazo[2,1-b][1,3,4]thiadiazole), 164.57 (C, C-4), 146.96 (C, C-8 imidazo[2,1-b][1,3,4]thiadiazole),
145.30 (C, C-6 imidazo[2,1-b][1,3,4]thiadiazole), 133.28 (C, C-1’), 130.54 (CH, C-3’, C-5’), 130.25 (C,
C-2’, C-6’), 129.90 (C, C-1), 126.97 (C, C-2, C-6), 123.68 (CH, C-4’), 116.39 (C, C-3, C-5), 111.73 (CH,
C-5 imidazo[2,1-b][1,3,4]thiadiazole), 56.62 (C, OCH3); ESI+-MS: m/z 308.4 [M + H]+ (calcd. 308.3 for
C17H13N3OS + H+).
6-(4-Bromophenyl)-2-(4-methoxyphenyl)imidazo[2,1-b][1,3,4]thiadiazole (5j) [26]: Yield 74% (0.280
g); purified by column chromatography, eluent dichloromethane: acetone 25:1 v/v; m.p. 224–225 ◦C;
yellow powder; FT-IR (solid state,
8.79 (s, 1H, CH-5 imidazo[2,1-b][1,3,4]thiadiazole), 7.90 (d, 2H, CH-2, CH-6), 7.84 (d, 2H, CH-3’,
CH-5’), 7.63 (d, 2H, CH-3, CH-5), 7.16 (d, 2H, CH-2’, CH-6’), 3.86 (s, 3H, OCH3); 13C-NMR (151
MHz, DMSO-d6) 162.22 (C, C-2 imidazo[2,1-b][1,3,4]thiadiazole), 161.40 (C, C-4), 144.50 (C, C-8
υ
cm−1): 3107, 3084 (CH aromatic); 1H-NMR (600 MHz, DMSO-d6)
δ
δ
imidazo[2,1-b][1,3,4]thiadiazole), 144.07 (C, C-6 imidazo[2,1-b][1,3,4]thiadiazole), 133.14 (C, C-1’),
131.64 (CH, C-3’, C-5’), 128.48 (CH, C-2’, C-6’), 126.62 (CH, C-2, C-6), 121.94 (C, C-1), 120.18 (C, C-4’),
115.05 (CH, C-3, C-5), 111.06 (CH, C-5 imidazo[2,1-b][1,3,4]thiadiazole), 55.64 (C, OCH3); ESI+-MS: m/z
386.1 ([M + H]+, 79Br), 388.1 ([M + H]+, 81Br) (calcd. for C16H9BrClN3S + H+: 386.2 79Br, 388.2 81Br).
2-(4-Methoxyphenyl)-6-(4-(trifluoromethyl)phenyl)imidazo[2,1-b][1,3,4]thiadiazole (5k): Yield 59%
◦
(0.220 g); m.p. 228–230 C; yellow powder; FT-IR (solid state,
1H-NMR (600 MHz, TFA-d6, acetone-d6)
8.16 (s, 1H, CH-5 imidazo[2,1-b][1,3,4]thiadiazole), 7.80
(d, J = 8.8 Hz, 2H, CH-2, CH-6), 7.66-7.69 (m, 4H, CH-2’, CH-3’, CH-5’, CH-6’), 7.01 (d, J = 8.8 Hz,
2H, CH-3, CH-5), 3.84 (s, 3H, OCH3); 13C-NMR (151 MHz, TFA-d6, acetone-d6)
171.58 (C, C-2
υ
cm−1): 3087, 3024 (CH aromatic);
δ
δ
imidazo[2,1-b][1,3,4]thiadiazole), 167.09 (C, C-8 imidazo[2,1-b][1,3,4]thiadiazole), 147.68 (C, C-6
imidazo[2,1-b][1,3,4]thiadiazole), 141.32 (C, C-4), 136.86 (q, J2C-F = 34.7 Hz, C, C-4’), 132.86 (C, C-2, C-6),
132.14 (C, C-1), 130.10 (q, J3C-F = 3 Hz, C, C-3’, C-5’), 129.79 (C, C-2’, C-6’), 127.09 (q, J1C-F = 270.3 Hz, C,
CF3), 123.39 (C, C-10), 118.84 (C, C-3, C-5), 115.59 (CH, C-5 imidazo[2,1-b][1,3,4]thiadiazole), 58.65 (C,
OCH3); 19F-NMR (565 MHz, TFA-d6, acetone-d6):
δ
-64.03 (s, -CF3); ESI+-MS: m/z 376.4 [M + H]+ (calcd.
376.3 for C18H12F3N3OS + H+).
2,6-Bis(4-methoxyphenyl)imidazo[2,1-b][1,3,4]thiadiazole (5l) [26]: Yie◦ld 60% (0.200 g); purified
by column chromatography, eluent dichloromethane; m.p. 242–244 C; yellow powder; FT-IR
1
(solid state,
υ
cm−1): 3124, 3047 (CH aromatic); H-NMR (600 MHz, CDCl3)
δ
7.94 (s, 1H, CH-5
imidazo[2,1-b][1,3,4]thiadiazole), 7.82 (d, J = 8.8 Hz, 2H, CH-2, CH-6), 7.77 (d, J = 8.7 Hz, 2H, CH-2’,
CH-6’), 7.03 (d, J = 8.8 Hz, 2H, CH-3, CH-5), 6.98 (d, J = 8.7 Hz, 2H, CH-3’, CH-5’), 3.89 (s, 3H, CH3),
3.83 (s, 3H, OCH3); 13C-NMR (151 MHz, CDCl3)
δ 164.73 (C, C-2 imidazo[2,1-b][1,3,4]thiadiazole),
163.21 (C, C-4), 160.50 (C, C-4’), 150.09 (C, C-8 imidazo[2,1-b][1,3,4]thiadiazole) 143.69 (C, C-6
imidazo[2,1-b][1,3,4]thiadiazole), 128.78 (CH, C-2’, C-6’), 126.97 (CH, C-2, C-6), 124.74 (C, C-1), 120.95
(C, C-1’), 114.98 (CH, C-3’, C-5’), 114.63 (CH, C-3, C-5), 108.42 (CH, C-5 imidazo[2,1-b][1,3,4]thiadiazole),
55.59 (C, OCH3), 55.33 (C, OCH3). ESI+-MS: m/z 338.4 [M + H]+ (calcd. 338.3 for C17H13N3OS + H+).
4.2. Pharmacology
4.2.1. Animals
For the evaluation of the biological potential of the synthesized compounds, 14 groups of female
Charles River Wistar (Crl:WI) rats (n = 6) weighing 180–220 g were purchased from the Practical
Skills and Experimental Medicine Centre of the ”Iuliu Ha,
Pharmacy (Cluj-Napoca, Romania). The animals were housed in polycarbonate type IV-S open-top
cages (Tecniplast, Italy) and kept under controlled room temperature (22
2 ◦C; relative humidity
45% 10%,) with a 12/12 h light/dark cycle. The animals were fed with standard pelleted diet
tieganu” University of Medicine and
±
±

Molecules 2018, 23, 2425
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(Cantacuzino Institute, Bucharest, Romania) and received water ad libitum. Food was withdrawn
24 h before experiments. All animal procedures were performed in accordance with the EEC Directive
63/2010, which regulates the care and use of laboratory animals for scientific purposes and were
approved by The Sanitary-Veterinary and Food Safety Directorate from Cluj (66/06.06.2017).
The animals were divided into groups (n = 6) and 12 synthesized compounds were solubilized in
0.5% Tween 80 solution and administered in dose of 50 mg/kg bw by gavage to the corresponding
groups. The negative control group received the vehicle (0.5% Tween 80 solution, by gavage) and the
control positive group was treated with diclofenac sodium (Gerot Lannach GmbH, Lannach, Austria)
(20 mg/kg bw, by gavage) as reference drug.
4.2.2. Anti-Inflammatory Activity
Anti-inflammatory activity was determined by
Acute inflammation was induced 1 h after the intragastric administration of substances, by intraplantar
injection of 100 L of 1% -carrageenan (Sigma Aldrich, St. Louis, MO, USA) saline solution into left
hind paw of the rats. The paw volume (mL) was measured plethysmometrically (Ugo Basile 7140,
Varese, Italy) at 0, 1, 2, 3 and 4 h after -carrageenan injection. Edema volume and the percentage of
edema inhibition were expressed as follows:
λ-carrageenan-induced rat paw edema test [29,45].
µ
λ
λ
Edema volume (mL) = Vt − V0
Inhibition of edema (%) = [1 − (Et/Ec) × 100]
where V0 is the mean paw volume before
(1)
(2)
λ
-carrageenan intraplantar injection, Vt is the mean paw
volume at ”t” hours, Et is mean edema volume in treated animals and Ec is mean edema volume in the
control group.
4.2.3. Antinociceptive Activity
The nociceptive withdrawal threshold was assessed by Randall-Selitto test [30,46]. For this
experiment, to reduce the number of animals, the same groups of rats previously tested for
anti-inflammatory potential were used. In this model of inflammatory pain, the pain threshold
of the inflamed hind paw of the rats was determined at 0, 1, 2, 3 and 4 h after the λ-carrageenan
intraplantar injection, using an analgesimeter (Ugo Basile 37215, Varese, Italy). The analgesimeter
applied a linearly increased force (grams) until the animal produced a response characterized by
removal of the paw or noises, interpreted as mechanical hypernociception. The instrument recorded
the maximal amount of pressure (grams) withstood by rats at each time interval.
4.2.4. Ulcerogenic Activity
Ulcerogenic activity of the synthesized compounds after a single oral administration of 50 mg/kg
bw was evaluated and scored by the method of Cioli et al. [47] adapted by Assarzadeh et al. [44].
To test the ulcerogenic activity, all 14 groups (n = 6) were fasted for 24 h prior to drug administration
and housed in mesh floor grid cages to avoid the coprophagy. They had access to water ad libitum.
At 6 h after the substance administration, all animals were sacrificed under deep anesthesia, then
their stomachs were removed, opened along the great curvature and rinsed with saline solution 0.9%.
The gastric mucosa was inspected under magnifying lens (2×) to assess the incidence of redness and
spot ulcers. The mucosal damage was evaluated according to the following score: 0.5: redness; 1.0:
spot ulcers; 1.5: hemorrhagic streaks; 2.0: ulcers > 3 but
ulceration score was calculated by the difference between the mean score of each treated group and
the mean score of control group. Diclofenac (20 mg/kg bw) was used as reference drug.
≤
5; and 3.0: ulcers > 5. The gastric mucosal

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4.2.5. Statistical Data Analysis
Statistics were performed with one-way analysis of variance (ANOVA), followed by Dunnett’s
multi-comparison test. All data are presented as mean ± standard error or mean (SEM), p < 0.05 was
considered to be significant.
4.3. Molecular Docking
In order to understand the mechanism of action of the synthesized compounds and to know the
nature of interactions between the compounds and the active sites of COX, we performed a molecular
docking study. Because no three-dimensional crystallographic structures of COX-1 and COX-2 isolated
from Rattus norvegicus are available in the Protein Data Bank (www.rcsb.org), these structures were
built by homology modeling using Swiss-Model [48]. The primary amino acid sequences of both
proteins were retrieved from Uniprot (www.uniprot.org). The construction of the chimeric COX-1
was made using the Q63921 sequence and the PDB three-dimensional structure 1CQE as template,
with 96% coverage and 85.94% identity, referred to as the primary amino acid sequence. The chimeric
COX-2 structure was built using the P35355 sequence and the 4RRX PDB structure, with which it has
97% coverage and 96.08% identity. The sequence alignment between both Q63921 and P35355 primary
amino acid sequences and the corresponding templates 1CQE, respectively 4RRX used for homology
modeling are presented in Supplementary Materials (Figures S1 and S2).
The ligands and the constructed chimeric structures COX-1 and COX-2 were further handled
to prepare them for the molecular docking study as targets using AutoDock Tools 1.5.6 [49].
The preparation followed the general protocol, represented by the protonation of amines,
the deprotonation of carboxylic acids and the addition of the Gasteiger partial charges [50].
The search space was set as cubic for both enzymes, with edges set equal to 50 Å, resulting in
125.000 Å3, with a spacing set to 0.375 Å. The Cartesian coordinates of the center of the search space
were set to x = 27.805, y = 35.244, z = 207.085 for COX-1, respectively x = 26.131, y =
−
40.62, z =
−
16.423
for COX-2. Search space was defined relative to the template structures, to include the crystallized
inhibitors found in the templates used in modeling. In addition, the sequences of the built COX-1 and
COX-2 chimeric structures were analyzed using blast [51], to obtain information about the substrate
binding sites and to ensure the inclusion of the catalytic residues into the docking search space.
For each compound, 50 conformations were generated using AutoDock 4.2 [49]. The results of
the docking study were ranked in descending order, according to the calculated binding energies (
and grouped in clusters with 2 Å RMSD tolerance of the atoms’ coordinates.
∆G)
The binding interactions between ligands and targets were depicted using UCSF Chimera
1.10.2 [52]. Sequence alignment between FASTA sequences was performed using Clustal Omega [53].
Diclofenac was docked as reference.
5. Conclusions
In the present study we report the synthesis, characterization and anti-inflammatory/analgesic
evaluation of 2,6-diaryl-imidazo[2,1-b][1,3,4]thiadiazole derivatives 5a
–
l
.
The spectral analysis
1
(IR, H-NMR, 13C-NMR, 19F-NMR and MS) confirmed the structures of the desired compounds.
The anti-inflammatory activity of these compounds was evaluated in vivo, in a model of acute
inflammation induced by λ-carrageenan in rats; molecular docking studies were made to elucidate the
potential mechanism of anti-inflammatory activity. The results of the present study showed that in vivo
anti-inflammatory studies are in accordance with in silico studies and the compound 5c which showed
the best anti-inflammatory activity, presented the highest COX-2 inhibition. Compounds 5g, 5i and 5j
presented significant analgesic properties. All compounds showed significantly lower ulcerogenic risk
than the reference drug, diclofenac. These compounds are good candidates for further investigations
regarding their potential use in therapy for the treatment of pain and inflammatory diseases.

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Supplementary Materials: The following are available online, Figure S1: Alignment of amino acids in COX-1,
Figure S2: Alignment of amino acids in COX-2.
Author Contributions: Conceptualization, V.Z., C.M., A.C.; Methodology, V.Z., A.C.; Investigation: A.C., D.L.,
V.L., L.C.B., S.I.; Molecular Docking: G.M.; Formal analysis B.A., G.M., A.C.; Writing-Original Draft Preparation,
A.C.; Writing-Review & Editing, V.Z., C.M., D.L.; Supervision, V.Z., C.M.
Funding: This research was funded by Iuliu Hat,ieganu University of Medicine and Pharmacy, Cluj-Napoca,
Romania [7690/35/15.04.2016] and [5200/32/01.03.2017].
Acknowledgments: Structures “2-trifluoromethyl/sulfonamide-5,6-diaryl substituted imidazo[2,1-b][1,3,4]thiadiazoles”
and “6-aryl-2-(6-methyl-benzofuran-3-ylmethyl) imidazo[2,1-b][1,3,4]thiadiazoles” from Figure 1 were reprinted
from Journal of Saudi Chemical Society, 26, Bhongade, B.A.; Talath, S.; Gadad, R.A.; Gadad, A.K., Biological
activities of imidazo[2,1-b][1,3,4]thiadiazole derivatives: A review, S463–S475, 2016, with permission from Elsevier.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 5a–l are available from the authors.
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