Anti-inflammatory, analgesic and COX-2 inhibitory activity of novel thiadiazoles in irradiated rats

Article abstract of DOI:10.1016/j.jphotobiol.2016.12.007

In this work, novel series of pyran, thiophene and thienopyrimidine derivatives based on 2-acetamide-thiadiazole scaffold were designed and synthesized for evaluation as selective COX-2 inhibitors in-vitro and investigated in-vivo as anti-inflammatory and analgesic agents against carrageenan-induced rat paw oedema model in irradiated rats, since its well-known that ionizing radiation plays an important role in exaggerating the inflammatory responses and in enhancing the release of inflammatory mediators in experimental animals. Toxicological studies were carried out to evaluate the ulcerogenic activity, acute toxicity and kidney and liver functions for the most potent compounds. In order to understand the binding mode of the synthesized compounds into the active site of COX-2, docking study was performed. Most of the tested compounds showed high inhibitory ability to COX-2. Among them, thiadiazole derivatives bearing thiophene and thienopyrimidine moieties were the most active derivatives, compound 26 showed extremely high selectivity index (SI) of >?555.5?μM which is nearly two folds better than celecoxib (>?277.7?μM), in addition to compounds 3, 16, 17, 21 and 26 with SI in the range of >?308.6- >?384.6?μM. The 4-chlorothieno[2.3-d]pyrimidine derivative of thiadiazole 21 showed the highest anti-inflammatory activity in this study having 24.49% of oedema compared to celecoxib (18.61%) in addition to compounds 17 and 26 with 24.70 and 25.40% of oedema, respectively, while the thiadiazol-2-acetamide derivative 2 was the most potent analgesic compound with the highest nociceptive threshold (85.72?g) very close to that of celecoxib (90.23?g). These compounds showed high safety margin on gastric mucosa with no ulceration effect. Also the most active in-vivo anti-inflammatory compounds 17, 21 and 26 were found to be non-toxic in experimental rats with normal kidney and liver functions. Docking study of the synthesized compounds showed similar orientation as celecoxib within the active site of COX-2 enzyme and similar ability to emerge deeply in the additional pocket and binding with Arg513 and His90 the key amino acids responsible for selectivity.

Full text of DOI:10.1016/j.jphotobiol.2016.12.007

Accepted Manuscript
Anti-inflammatory, analgesic and COX-2 inhibitory activity of
novel thiadiazoles in irradiated rats
Fatma A. Ragab, Helmi I. Heiba, Marwa G. El-Gazzar, Sahar M.
Abou-Seri, Walaa A. El-Sabbagh, Reham M. El-Hazek
PII:
DOI:
Reference:
S1011-1344(16)30718-7
doi: 10.1016/j.jphotobiol.2016.12.007
JPB 10671
To appear in:
Journal of Photochemistry & Photobiology, B: Biology
Received date:
Accepted date:
26 August 2016
10 December 2016
Please cite this article as: Fatma A. Ragab, Helmi I. Heiba, Marwa G. El-Gazzar, Sahar
M. Abou-Seri, Walaa A. El-Sabbagh, Reham M. El-Hazek , Anti-inflammatory, analgesic
and COX-2 inhibitory activity of novel thiadiazoles in irradiated rats. The address for
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ACCEPTED MANUSCRIPT
Anti-inflammatory, analgesic and COX-2 inhibitory activity of novel thiadiazoles in
irradiated rats.
Fatma A. Ragab1, Helmi I. Heiba2, Marwa G. El-Gazzar2*, Sahar M. Abou-Seri1, Walaa A.
El-Sabbagh2, Reham M. El-Hazek2
1Department of Pharmaceutical and Medicinal Chemistry, Faculty of Pharmacy, Cairo
University.
2Department of Drug Radiation Research, National Centre for Radiation Research and
Technology (NCRRT), Egyptian Atomic Energy Authority (EAEA), PO box 29, Nasr City,
Cairo, Egypt.
* Correspondence; e-mail: marwa.galal@eaea.org.eg
Abstract
In this work, novel series of pyran, thiophene and thienopyrimidine derivatives based on 2-
acetamide-thiadiazole scaffold were designed and synthesized for evaluation as selective
COX-2 inhibitors in-vitro and investigated in-vivo as anti-inflammatory and analgesic agents
against carrageenan-induced rat paw oedema model in irradiated rats, since its well-known
that ionizing radiation plays an important role in exaggerating the inflammatory responses
and in enhancing the release of inflammatory mediators in experimental animals.
Toxicological studies were carried out to evaluate the ulcerogenic activity, acute toxicity and
kidney and liver functions for the most potent compounds. In order to understand the binding
mode of the synthesized compounds into the active site of COX-2, docking study was
performed. Most of the tested compounds showed high inhibitory ability to COX-2. Among
them, thiadiazole derivatives bearing thiophene and thienopyrimidine moieties were the most
active derivatives, compound 26 showed extremely high selectivity index (SI) of >555.5 µM
which is nearly two folds better than celecoxib (>277.7 µM), in addition to compounds 3, 16,
17, 21 and 26
with SI in the range of >308.6- >384.6 µM. The 4-chlorothieno[2,3-
d]pyrimidine derivative of thiadiazole 21 showed the highest anti-inflammatory activity in
this study having 24.49% of oedema compared to celecoxib (18.61%) in addition to
compounds 17 and 26 with 24.70 and 25.40 % of oedema, respectively, while the thiadiazol-
2-acetamide derivative 2 was the most potent analgesic compound with the highest
nociceptive threshold (85.72g) very close to that of celecoxib (90.23g). These compounds
showed high safety margin on gastric mucosa with no ulceration effect. Also the most active
in-vivo anti-inflammatory compounds 17, 21 and 26 were found to be non-toxic in
experimental rats with normal kidney and liver functions. Docking study of the synthesized
compounds showed similar orientation as celecoxib within the active site of COX-2 enzyme
and similar ability to emerge deeply in the additional pocket and binding with Arg513 and
His90 the key amino acids responsible for selectivity.
Keywords: Thiadiazoles, COX-2 inhibitors, anti-inflammatory, analgesic, toxicological
studies, irradiated rats, docking.
1. Introduction
Non-steroidal anti-inflammatory drugs (NSAIDs) continue to be widely used group of
therapeutic agents, which inhibit both COX-1 and COX-2 with an extremely varying levels of
selectivity [1, 2]. Their clinical use as analgesics and anti-inflammatory agents is always
accompanied with adverse gastrointestinal disorders and the design of novel NSAIDs with an
advanced safety profile on GIT is a challenge in pharmaceutical industry. Since the discovery

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celecoxib, , researchers have focused on the synthesis of novel derivatives of this class which
reduce inflammation with fewer side effects [3, 4].
Inflammation is a complex biological response to harmful stimulus which may vary from a
localized response to a generalized one and is mediated by prostaglandins (PGs) [5]. The
biosynthesis of (PGs) is carried out by the bifunctional enzyme prostaglandin H2 synthase
(PGHS or cyclooxygenase, COX), which exhibits both cyclooxygenase and peroxidase
activities. There are three distinct COX isoforms: COX-1, the constitutive which is involved
in the regulation of physiological functions and production of cycloprotective prostaglandin
in GIT and maintaining platelet aggregation by production of proaggregatory thromboxane.
COX-2, the inducible form which is released in inflammatory cells in response to cytokines
such as tumor necrosis factor-α (TNF-α), interleukines, growth factors, and other
inflammatory mediators. COX-3, a third full active isoform, and two partial isoforms,
pCOX1a and b recently reported to be detected in the cerebral cortex and in human heart [6-
8].
Most studies on new anti-inflammatories have been focused on healthy population. However,
inflammatory processes have particular relevance in the context of cancer, as inflammation is
increasingly recognized as a contributor to cancer development and progression especially in
breast and prostate cancer [9]. Moreover, radiation therapy activates pro-inflammatory
cytokine production as part of a coordinated response designed to control damage and
promote tissue repair [10]. Pain management and controlling of inflammatory responses
should be concluded in protocols before starting of radiation treatment [11].
Carrageenan-induced paw oedema is an acute inflammatory model commonly used in
experimental rats [12]. Ionizing radiation has been shown to exaggerate the inflammatory
responses induced by this model due to enhancement of release of inflammatory mediators
through the cyclooxygenase and lipoxygenase pathways and also through the release of
reactive oxygen metabolites resulting from the interaction between radiation and water from
the cellular environment [13-15].
The common structure of COX-2 inhibitors includes two classes: tricyclic and non-tricyclic
compounds [16, 17], the tricyclic group consists of two aryl rings linked to a central
homocyclic, heterocyclic or fused ring moieties such as, thiophene, pyrazole, furanone,
isoxazole, cyclopentene and fused heterocyclics. One of the two aryl rings carries a
sulfonamide moiety which is deeply immerged into the additional hydrophilic pocket of
COX-2 enzyme and is capable of binding to the key amino acids His90 and Arg513
responsible for selectivity [16, 17]. In the non-tricyclics, the cyclic core is replaced by acyclic
centre such as olefinic, iminic, azo, urea, and a,b-unsaturated structures [18-20]. This
common pharmacophore presents a wide framework which allows medicinal chemists to
design novel selective COX-2 inhibitors with varying structures.
Pyran derivatives have recently attracted considerable attention due to their wide spectrum
ofbiological activity [21-24], Hyup et al. [25] introduced 2,3-diaryl benzopyrans as a part of
the vicinal diaryl heterocyclic family as a promising lead structure for selective COX-2
inhibition. Caturla et al [26] reported a new class of 2-phenylpyran-4-ones as selective COX-
2 inhibitors. Moreover, the anti-inflammatory activity of thiophene and thienopyrimidine
derivatives is well-documented in addition to their array of pharmacological activities [27-
30]. In 1995, Gierse et al.[31] introduced a new generation of selective COX-2 inhibitors that
include 5-bromo-2-(4-fluorophenyl)-3-(methylsulfonyl) thiophene (DuP-697). This new class
binds tightly to the COX-2 active cite and dissociate slowly showing a long lasting action.
Hence, different series of thiophene and thienopyrimidines have been synthesized with
optimal COX-2 inhibition [32].
The 1,3,4-thiadiazole ring is endowed with relatively high aromaticity and weak basicity due
to its sulfur inducible effect, the electron withdrawing effect of its nitrogen atoms are

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responsible for its electron deficiency and susceptibility to nucleophilic attack, it is relatively
stable in aqueous acid solutions but can undergo ring cleavage with aqueous base. Thus, with
these structural properties, 1,3,4-thiadiazole derivatives are widely applied in medicinal
chemistry and displayed significant biological activities [33, 34].
Based on these facts, we decided to synthesize novel series of non-tricyclic COX-2 inhibitors
possessing a central azo acyclic core, the two aromatic rings are replaced by 1,3,4-
thiadiazole, and by either pyran, thiophene or thienopyrimidine. By using the structural
features of these biologically active moieties and by diverting the substituents, novel
inhibitors were synthesized. The aim is to study the SAR and the effect of thiadiazole ring on
the orientation and binding mode of pyran, thiophene and thienopyrimidine within COX-2
active site in order to reach the best inhibitory activity with higher selectivity index which
could lead to the discovery of new class of COX-2 inhibitors (Figure 1). These new
derivatives were screened using in-vitro COX inhibitory assay, the most potent candidates
were subjected to anti-inflammatory and analgesic testing. Toxicological studies were
performed by evaluation of their ulcerogenic activity, acute toxicity, and kidney and liver
functions in experimental rats. Molecular docking was carried out for all the synthesized
compounds into the active site of COX-2 to explore their binding interactions and their
accessibility to the additional pocket responsible for selectivity.
Figure 1. The designed synthesized compounds.
2. Material and Methods
2.1.Instruments
The melting points were taken in an open capillary tube on a Stuart melting point apparatus
(Stuart Scientific, Redhill, UK) and are uncorrected. The IR spectra of the compounds were
1
13
recorded on FT-IR Shimadzu spectrometer (Shimadzu, Tokyo, Japan). H-NMR and C-
NMR spectra were recorded on a Bruker AC-500 Ultra Shield NMR spectrometer (Bruker,
Flawil, Switzerland) at 500 MHz using TMS as an internal Standard and DMSO-d6 as
solvent. Mass spectra were run on HP Model MS-5988 (Hewlett Packard, Palo, Alto,
California, USA). Microanalyses were obtained on a Carlo Erba 1108 Elemental Analyzer
(Heraeus, Hanau, Germany), all values were within±0.4% of the theoretical values. The
purity of the compounds was checked by TLC on pre-coated SiO2 gel (HF254, 200 mesh)
aluminum plates (Merk, Darmstadt, Germany). A developing solvent system of
1
chloroform/methanol (8:2) was used and the spots were visualized in UV light. IR, H-NMR,
13C-NMR, Mass and elemental analysis were consistent with the assigned structures. All
reagents used were of AR grade and were purchased from Sigma (St. Louis, MO).
2.2.Chemistry
General procedure for the synthesis of compounds 3-5.
A mixture of 2-acetamide thiadiazole 2 (0.7 g, 0.004 mol), activated nitrile such as
malononitrile, ethyl cyanoacetate and/or ethyl acetoacetate (0.004 mol), sulphur (0.13 g,

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0.004 mol), ethanol (50 mL) and a catalytic amount of triethylamine (0.2 mL) were refluxed
for 6h. The reaction mixture was cooled, poured onto ice water and the precipitated solid was
collected by filtration, dried, and recrystallized from ethanol to give 3-5, respectively.
2-Amino-4-(5-thioxo-4,5-dihydro-1,3,4-thiadiazol-2-ylamino)thiophene-3-carbonitrile (3).
Yield, 85 %, mp 265–267ο C. IR (KBr, cm-1): 3330, 3229, 3200 (NH, NH2), 2200 (C≡N),
1
1626 (C=N), 1230 (C=S). H-NMR (DMSO-d6, ppm): 2.41 (s, 1H, NH, exchangeable with
D2O), 5.31 (s, 1H, CH-thiophene), 8.32, 9.71 (2s, 3H, NH, NH2, exchangeable with
D2O).13C-NMR (DMSO-d6, ppm):79.51 (C-CN), 104.21 (CH-thiophene), 115.32 (CN),
125.72 (C-thiophene), 151.41 (C-NH2), 156.64 (C-thiadiazole), 181.71 (C=S). MS m/z: 255
(M+). Analysis calculated for C7H5N5S3: C, 32.93; H, 1.97; N, 27.43, found: C, 32.83; H,
1.87; N, 27.33.
Ethyl 2-amino-4-(5-thioxo-4,5-dihydro-1,3,4-thiadiazol-2-ylamino)thiophene-3-carboxylate
(4).
Yield, 75 %, mp 199–201ο C. IR (KBr, cm-1): 3320, 3230, 3200 (NH, NH2), 2929, 2870 (CH
aliph.), 1720 (C=O), 1626 (C=N), 1230 (C=S). 1H-NMR (DMSO-d6, ppm): 1.23 (t, 3H, J=
3.5Hz, CH3 ethyl), 2.41 (s, 1H, NH, exchangeable with D2O), 4.32(q, 2H, J= 4.5Hz,CH2
ethyl), 5.71 (s, 1H, CH thiophene), 8.61, 10.70 (2s, 3H, NH, NH2, exchangeable with
D2O).13C-NMR (DMSO-d6, ppm): 14.3 (CH3 ethyl), 60.8 (CH2 ethyl), 103.8 (CH-thiophene),
124.1(C-thiophene), 124.6(C-thiophene), 156.6 (C-thiadiazole), 159.8 (C=O), 161.6 (C-NH2),
181.7 (C=S). MS m/z: 302 (M+). Analysis calculated for C9H10N4O2S3: C, 35.75; H, 3.33; N,
18.53, found: C, 35.65; H, 3.23; N, 18.43.
2-Methyl-4-(5-thioxo-4,5-dihydro-1,3,4-thiadiazol-2-ylamino)thiophene-3-ethylcarboxylate
(5).
Yield, 90 %, mp 200–202ο C. IR (KBr, cm-1): 3227, 3200 (NH), 2939, 2870 (CH aliph.),
1
1700 (C=O), 1626 (C=N), 1230 (C=S). H-NMR (DMSO-d6, ppm): 1.21 (t, 3H, J= 2.5Hz,
CH3 ethyl), 2.32 (s, 3H, CH3), 2.51 (s, 1H, NH, exchangeable with D2O), 4.21(q, 2H, J=
4.5Hz, CH2 ethyl), 5.35 (s, 1H, CH-thiophene), 8.32 (s, 1H, NH, exchangeable with
D2O).13C-NMR (DMSO-d6, ppm): 14.12 (CH3), 14.70 (CH3 ethyl), 60.82 (CH2 ethyl), 105.81
(CH-thiophene), 123.13(C-thiophene), 124.61(C-thiophene), 148.50 (C-CH3), 156.62(C-
thiadiazole), 160.61 (C=O), 181.70 (C=S). MS m/z: 301 (M+). Analysis calculated for
C10H11N3O2S3: C, 39.85; H, 3.68; N, 13.94. found: C, 39.65; H, 3.88; N,13.64.
General procedure for the preparation of Ethyl 2-amino-4-(4-substituted phenyl)-6-(5-thioxo-
4,5-dihydro-1,3,4-thiadiazol-2-ylamino)-4H-pyran-3-carboxylate (7-10) and 2-Amino-4-(4-
substituted phenyl)-6-(5-thioxo-4,5-dihydro-1,3,4-thiadiazol-2-ylamino)-4H-pyran-3-
carbonitrile (11-14).
A mixture of 2-acetamide thiadiazole 2 (0.7 g, 0.004 mol), 2-(4-substituted benzylidene)
cyanoacetate and/or 2-(4-substituted benzylidene) malononitrile 6a-h (0.004 mol) in ethanol

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(50 mL) and a catalytic amount of triethylamine (0.2 mL) were refluxed for 6h. The reaction
mixture was cooled, poured onto ice water and the precipitated solid was collected by
filtration, dried, and recrystallized from methanol to give 7-14, respectively.
Ethyl-2-amino-4-(4-chlorophenyl)-6-(5-thioxo-4,5-dihydro-1,3,4-thiadiazol-2-ylamino)-4H-
pyran-3-carboxylate (7).
Yield, 78 %, mp 270–272ο C. IR (KBr, cm-1): 3320, 3230, 3200 (NH, NH2), 3058 (CH
arom.), 2939, 2880 (CH aliph.), 1680 (C=O), 1626 (C=N), 1230 (C=S), 835 (C-Cl). 1H-NMR
(DMSO-d6, ppm): 1.32 (t, 3H, J= 5.2Hz, CH3 ethyl), 2.50 (s, 1H, NH, exchangeable with
D2O), 3.61 (d, 1H, J= 6.5Hz, CH-pyran), 4.14(q, 2H, J= 5.5Hz, CH2 ethyl), 4.71 (d, 1H, J=
5.1Hz, CH-pyran), 7.21,7.82 (2d, 4H, J= 9.2Hz, Ar-H), 8.72, 9.71 (2s, 3H, NH, NH2,
exchangeable with D2O).13C-NMR (DMSO-d6, ppm): 14.31 (CH3 ethyl), , 36.82 (CH-pyran),
61.84 (CH2 ethyl), 74.61 (CH- pyran), 75.80 (C- pyran), 128.13 (2C), 130.62 (2C), 142.22,
131.54 (C-Cl), 144.80 (C-thiadiazole), 162.17(C-pyran), 162.23 (C-NH2), 167.86 (C=O),
181.78 (C=S). MS m/z: 410 (M+), 412 (M+2). Analysis calculated for C16H15ClN4O3S2: C,
46.77; H, 3.68; N, 13.64, found: C, 46.67; H, 3.58; N, 13.74.
Ethyl 2-amino-4-(4-methoxyphenyl)-6-(5-thioxo-4,5-dihydro-1,3,4-thiadiazol-2-ylamino)-4H-
pyran-3-carboxylate (8).
Yield, 85 %, mp 159–161ο C. IR (KBr, cm-1): 3330, 3220, 3200 (NH, NH2), 3048 (CH
arom.), 2949, 2880 (CH aliph.), 1720 (C=O), 1627 (C=N), 1230 (C=S). 1H-NMR (DMSO-d6,
ppm): 1.32 (t, 3H, J= 6.2Hz, CH3 ethyl), 2.51 (s, 1H, NH, exchangeable with D2O), 3.54 (d,
1H, J= 5.5Hz, CH-pyran),3.75 (s, 3H, OCH3), 4.12(q, 2H, J= 5.5Hz, CH2 ethyl), 4.85 (d, 1H,
J= 6.1Hz, CH-pyran), 6.82,7.75 (2d, 4H, J= 7.5Hz, Ar-H), 8.12, 8.91 (2s, 3H, NH, NH2,
exchangeable with D2O).13C-NMR (DMSO-d6, ppm): 13.91 (CH3 ethyl), 36.72 (CH-pyran),
56.12 (OCH3), 61.92 (CH2 ethyl), , 73.63(CH- pyran), 75.81(C- pyran), 114.12 (2C), 130.60
(2C), 136.22, 156.81 (C-OCH3), 144.86 (C-thiadiazole), 161.91(C-pyran), 162.44 (C-NH2),
169.72 (C=O), 181.70 (C=S). MS m/z: 406 (M+). Analysis calculated for C17H18N4O4S2: C,
50.23; H, 4.46; N, 13.78, found: C, 50.13; H, 4.36; N, 13.68.
Ethyl-2-amino-4-(4-nitrophenyl)-6-(5-thioxo-4,5-dihydro-1,3,4-thiadiazol-2-ylamino)-4H-
pyran-3-carboxylate (9).
Yield, 85 %, mp 210–212ο C. IR (KBr, cm-1): 3320, 3230, 3200 (NH, NH2), 3058 (CH
arom.), 2939, 2880 (CH aliph.), 1690 (C=O), 1626 (C=N), 1230 (C=S). 1H-NMR (DMSO-d6,
ppm): 1.32 (t, 3H, J= 5.7Hz, CH3 ethyl), 2.52 (s, 1H, NH, exchangeable with D2O), 3.31 (d,
1H, J= 5.5Hz, CH-pyran), 4.26 (q, 2H, J= 6.5Hz, CH2 ethyl), 4.62 (d, 1H, J= 6.3Hz, CH-
pyran), 7.12,7.70 (2d, 4H, J= 9.5Hz, Ar-H), 8.12, 8.92 (2s, 3H, NH, NH2, exchangeable with
D2O).13C-NMR (DMSO-d6, ppm): 14.12 (CH3 ethyl), 36.42 (CH-pyran), 61.80 (CH2 ethyl),
74.62(CH-pyran), 75.81(C-pyran), 123.72 (2C), 127.61 (2C), 150.26,144.50 (C-NO2), 144.83

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(C-thiadiazole), 162.22(C-pyran), 162.40 (C-NH2), 167.70 (C=O), 181.71 (C=S). MS m/z:
421 (M+). Analysis calculated for C16H15N5O5S2: C, 45.60; H, 3.59; N, 16.62, found: C,
45.70; H, 3.49; N, 16.52.
Ethyl-2-amino-4-(4-(dimethylamino)phenyl)-6-(5-thioxo-4,5-dihydro-1,3,4-thiadiazol-2-
ylamino)-4H-pyran-3-carboxylate (10).
Yield, 79 %, mp 230–232ο C. IR (KBr, cm-1): 3320, 3220, 3200 (NH, NH2), 3048 (CH
arom.), 2929, 2870 (CH aliph.), 1720 (C=O), 1626 (C=N), 1230 (C=S). 1H-NMR (DMSO-d6,
ppm): 1.32 (t, 3H, J= 2.5Hz, CH3 ethyl), 2.51 (s, 1H, NH, exchangeable with D2O), 3.24 (s,
6H, 2CH3), 3.71 (d, 1H, J= 6.5Hz, CH-pyran), 4.22 (q, 2H, J= 5.9Hz CH2 ethyl), 4.81 (d, 1H,
J= 6.7Hz, CH-pyran), 6.82,7.84 (2d, 4H, J= 8.5Hz, Ar-H), 8.21, 8.93 (2s, 3H, NH, NH2,
exchangeable with D2O).13C-NMR (DMSO-d6, ppm): 14.12 (CH3 ethyl), 36.72 (CH-pyran),
41.30 (N-2CH3), 61.92 (CH2 ethyl), 73.62(CH- pyran), 75.82(C-pyran), 112.12 (2C), 126.61
(2C), 133.20, 148.51 (C-N(CH3)2), 144.84 (C-thiadiazole), 162.22(C-pyran), 162.43 (C-
NH2), 167.73 (C=O), 181.72 (C=S). MS m/z: 419 (M+). Analysis calculated for
C18H21N5O3S2: C, 51.53; H, 5.05; N, 16.69, found: C, 51.43; H, 5.15; N, 16.59.
2-Amino-4-(4-chlorophenyl)-6-(5-thioxo-4,5-dihydro-1,3,4-thiadiazol-2-yl amino)-4H-pyran-
3-carbonitrile (11).
Yield, 90 %, mp 230–232ο C. IR (KBr, cm-1): 3320, 3220, 3200 (NH, NH2), 3048 (CH
arom.), 2200 (C≡N), 1626 (C=N), 1230 (C=S), 825 (C-Cl). 1H-NMR (DMSO-d6, ppm): 2.56
(s, 1H, NH, exchangeable with D2O), 3.82 (d, 1H, J= 5.5Hz, CH pyran), 4.82 (d, 1H, J=
5.8Hz, CH-pyran), 7.26,7.83 (2d, 4H, J= 8.6Hz, Ar-H), 8.22, 8.91 (2s, 3H, NH, NH2,
exchangeable with D2O).13C-NMR (DMSO-d6, ppm): 26.70 (CH-pyran), 58.42(C-pyran),
74.62(CH-pyran), 119.26 (CN), 128.21 (2C), 130.62 (2C), 140.52, 131.72 (C-Cl), 144.84 (C-
thiadiazole), 159.61 (C-NH2), 162.73 (C-pyran), 181.72 (C=S). MS m/z: 363 (M+), 365
(M+2). Analysis calculated for C14H10ClN5OS2: C, 46.21; H, 2.77; N, 19.25, found: C, 46.11;
H, 2.87; N, 19.15.
2-Amino-4-(4-methoxyphenyl)-6-(5-thioxo-4,5-dihydro-1,3,4-thiadiazol-2-ylamino)-4H-
pyran-3-carbonitrile (12).
Yield, 79 %, mp 250–252ο C. IR (KBr, cm-1): 3330, 3230, 3200 (NH, NH2), 3048 (CH
arom.), 2939, 2870 (CH aliph.), 2190 (C≡N), 1626 (C=N), 1230 (C=S). 1H-NMR (DMSO-d6,
ppm): 2.51 (s, 1H, NH, exchangeable with D2O), 3.73 (s, 3H, OCH3), 3.92 (d, 1H, J= 5.8Hz,
CH- pyran), 4.72 (d, 1H, J= 5.2Hz, CH-pyran), 7.32,7.91 (2d, 4H, J= 9.8Hz, Ar-H), 8.32,
8.91 (2s, 3H, NH, NH2, exchangeable with D2O).13C-NMR (DMSO-d6, ppm): 26.81 (CH-
pyran), 55.30 (OCH3), 58.42(C-pyran), 74.85 (CH- pyran), 119.51 (CN), 114.22 (2C), 130.66
(2C), 134.52, 157.71 (C-OCH3), 144.82 (C-thiadiazole), 159.91 (C-NH2), 162.94 (C-pyran),
181.71 (C=S). MS m/z: 359 (M+). Analysis calculated for C15H13N5O2S2: C, 50.12; H, 3.65;
N, 19.48, found: C, 50.22; H, 3.55; N, 19.38.

ACCEPTED MANUSCRIPT
2-Amino-4-(4-nitrophenyl)-6-(5-thioxo-4,5-dihydro-1,3,4-thiadiazol-2-yl amino) -4H-pyran-
3-carbonitrile (13).
Yield, 88 %, mp 200–202ο C. IR (KBr, cm-1): 3320, 3220, 3200 (NH, NH2), 3038 (CH
arom.), 2200 (C≡N), 1626 (C=N), 1230 (C=S). 1H-NMR (DMSO-d6, ppm): 2.51 (s, 1H, NH,
exchangeable with D2O), 3.72 (d, 1H, J= 5.8Hz, CH-pyran), 4.82 (d, 1H, J= 5.2Hz, CH-
pyran), 7.22,7.81 (2d, 4H, J= 8.8Hz, Ar-H), 8.41, 8.81 (2s, 3H, NH, NH2, exchangeable with
D2O).13C-NMR (DMSO-d6, ppm): 26.71 (CH-pyran), 58.82(C-pyran), 75.62(CH-pyran),
119.81 (CN), 123.61 (2C), 126.62 (2C), 148.52, 144.54 (C-thiadiazole), 144.91 (C-NO2),
159.65 (C-NH2), 162.70 (C-pyran), 181.72 (C=S). MS m/z: 374 (M+). Analysis calculated
for C14H10N6O3S2: C, 44.91; H, 2.69; N, 22.45, found: C, 44.81; H, 2.59; N, 22.35.
2-Amino-4-(4-(dimethylamino)phenyl)-6-(5-thioxo-4,5-dihydro-1,3,4-thiadiazol -2-ylamino)-
4H-pyran-3-carbonitrile (14).
Yield, 92 %, mp 240–242ο C. IR (KBr, cm-1): 3330, 3220, 3200 (NH, NH2), 3048 (CH
arom.), 2939, 2870 (CH aliph.), 2190 (C≡N), 1626 (C=N), 1230 (C=S). 1H-NMR (DMSO-d6,
ppm): 2.52 (s, 1H, NH, exchangeable with D2O), 3.24 (s, 6H, 2CH3), 3.81 (d, 1H, J= 5.2Hz,
CH-pyran), 4.93 (d, 1H, J= 5.7Hz, CH-pyran), 6.82,7.41 (2d, 4H, J= 9.8Hz, Ar-H), 8.12,
8.82 (2s, 3H, NH, NH2, exchangeable with D2O).13C-NMR (DMSO-d6, ppm): 26.81 (CH-
pyran), 41.72 (2CH3), 58.41(C-pyran), 74.62(CH-pyran), 119.70 (CN), 112.22 (2C), 128.61
(2C), 132.55, 144.82 (C-thiadiazole), 148.71 (C-N(CH3)2), 154.22 (C-NH2), 163.80 (C-
pyran), 181.71 (C=S). MS m/z: 372 (M+). Analysis calculated for C16H16N6OS2: C, 51.59;
H, 4.33; N, 22.56, found: C, 51.49; H, 4.23; N, 22.46.
2-Chloro-N-(3-cyano-4-(5-thioxo-4,5-dihydro-1,3,4-thiadiazol-2-ylamino)
thiophen-2-
yl)acetamide (15).
A mixture of compound 3 (1.02 g, 0.004 mol) and chloroacetylchloride (0.9 mL, 0.008 mol)
in dimethylformamide was stirred for 8 h at room temperature, the reaction mixture was
poured onto ice water. The obtained solid was filtered, dried and recrystallized from ethanol
to give 15. Yield, 76 %, mp 200–202ο C. IR (KBr, cm-1): 3229, 3200 (NH), 2939, 2870 (CH
1
aliph.), 2200 (C≡N), 1680 (C=O), 1626 (C=N), 1230 (C=S), 825 (C-Cl). H-NMR (DMSO-
d6, ppm): 2.51 (s, 1H, NH, exchangeable with D2O), 4.22 (s, 2H, CH2-Cl), 5.72 (s, 1H, CH-
thiophene), 8.31, 8.92 (2s, 2H, 2NH, exchangeable with D2O).13C-NMR (DMSO-d6,
ppm):42.91 (CH2-Cl), 65.51(C-CN), 94.82 (CH-thiophene), 115.32 (CN), 125.72 (C-
thiophene), 162.81 (C-NH2), 156.73 (C-thiadiazole), 165.63 (C=O), 181.73 (C=S). MS m/z:
331 (M+), 333 (M+2). Analysis calculated for C9H6ClN5OS3: C, 32.58; H, 1.82; N, 21.11,
found: C, 32.48; H, 1.72; N, 21.21.
Ethyl-N-3-cyano-4-(5-thioxo-4,5-dihydro-1,3,4-thiadiazol-2-ylamino)-thiophen-2-yl-
formimidate (16).

ACCEPTED MANUSCRIPT
A solution of compound 3 (1.02 g, 0.004mol) in triethylortho formate (30 mL) containing 3
drops of acetic anhydride was refluxed for 8 h, the reaction mixture was cooled and then
poured onto ice water, the obtained solid was filtered, dried and recrystallized from methanol
to give 16. Yield, 86 %, mp 180–182ο C. IR (KBr, cm-1): 3240, 3200 (NH), 2949, 2870 (CH
1
aliph.), 2220 (C≡N), 1626 (C=N), 1230 (C=S). H-NMR (DMSO-d6, ppm): 1.25 (t, 3H, J=
6.1Hz, CH3 ethyl), 2.62 (s, 1H, NH, exchangeable with D2O), 3.71 (q, 2H, J= 6.6Hz, CH2
ethyl), 5.62 (s, 1H, CH-thiophene), 7.82 (s, 1H, N=CH), 8.61 (s, 1H, NH, exchangeable with
D2O).13C-NMR (DMSO-d6, ppm): 16.72 (CH3 ethyl), 61.81 (CH2 ethyl), 64.51(C-CN),
102.82 (CH-thiophene), 115.62 (CN), 124.71 (C-thiophene), 160.83 (C-NH2), 156.70 (C-
thiadiazole), 159.15(N=CH), 181.71 (C=S). MS m/z: 311 (M+). Analysis calculated for
C10H9N5OS3: C, 38.57; H, 2.91; N, 22.49, found: C, 38.47; H, 2.81; N, 22.39.
5-(4-Imino-3-(phenylamino)-3,4-dihydrothieno[2,3-d]pyrimidin-5-ylamino)-1,3,4-
thiadiazole-2(3H)-thione (17).
A mixture of 16 (1.2 g, 0.004mol) and phenyl hydrazine (0.004 mol) in ethanol (60 mL) was
heated under reflux for 8 h. The solid product was filtered on hot, dried, and recrystallized
from ethanol to give 17. Yield, 84 %, mp 160–162ο C. IR (KBr, cm-1): 3240, 3200 (NH),
1
3038 (CH arom.), 1626 (C=N), 1230 (C=S). H-NMR (DMSO-d6, ppm): 2.54 (s, 1H, NH,
exchangeable with D2O), 5.32 (s, 1H, CH-thiophene), 7.32-7.87 (m, 5H, Ar-H), 8.22 (s, 1H,
CH-pyrimidine), 8.52, 8.81, 10.85 (3s, 3H, 3NH, exchangeable with D2O).13C-NMR
(DMSO-d6, ppm): 103.63 (CH-thiophene), 113.41 (2C), 123.13, 129.72 (2C), 147.81, 117.86,
123.61, 146.14 (3C-thiophene), 147.12 (CH-pyrimidine), 156.26 (C=NH), 156.71 (C-
thiadiazole), 181.72 (C=S). MS m/z: 373 (M+). Analysis calculated for C14H11N7S3: C,
45.02; H, 2.97; N, 26.25, found: C, 45.12; H, 2.77; N, 26.15.
4-Imino-3-phenyl-5-(5-thioxo-4,5-dihydro-1,3,4-thiadiazol-2-ylamino)-3,4-
dihydrothieno[2,3-d]pyrimidin-2(1H)-one (18).
A mixture of compound 3 (1.02 g, 0.004 mol) and phenyl isocyanate (0.43 mL, 0.004 mol) in
ethanol (20 mL) was refluxed for 5 h. The reaction mixture was cooled and then poured onto
ice water, the solid obtained was filtered, dried and recrystallized from ethanol to give 18.
Yield, 74 %, mp 210–212ο C. IR (KBr, cm-1): 3240, 3200 (NH), 3038 (CH arom.), 1680
(C=O), 1626 (C=N), 1230 (C=S). 1H-NMR (DMSO-d6, ppm): 2.51 (s, 1H, NH, exchangeable
with D2O), 5.82 (s, 1H, CH-thiophene), 7.32-7.89 (m, 5H, Ar-H), 8.21, 8.85, 9.82
(3s, 3H, 3NH, exchangeable with D2O).13C-NMR (DMSO-d6, ppm): 97.63 (CH- thiophene),
128.12, 128.76 (2C), 129.14(2C), 132.82, 103.81, 123.64, 172.15 (3C-thiophene), 152.42
(C=O), 156.20 (C=NH), 156.71 (C-thiadiazole), 181.71 (C=S). MS m/z: 374 (M+). Analysis
calculated for C14H10N6OS3: C, 44.90; H, 2.69; N, 22.44, found: C, 44.70; H, 2.59; N, 22.34.
5-(5-Thioxo-4,5-dihydro-1,3,4-thiadiazol-2-ylamino)thieno[2,3-d]pyrimidin-4(3H)-one (19).
A solution of compound 3 (1.02 g, 0.004 mol) in formic acid (20 mL) was refluxed for 6 h,
the reaction mixture was cooled then poured onto ice water, the obtained solid was filtered,
dried and recrystallized from dioxane to give 19. Yield, 89 %, mp 198–200ο C. IR (KBr, cm-

ACCEPTED MANUSCRIPT
1
1): 3240, 3200 (NH), 3038 (CH arom.), 1720 (C=O), 1626 (C=N), 1230 (C=S). H-NMR
(DMSO-d6, ppm): 2.52 (s, 1H, NH, exchangeable with D2O), 5.81 (s, 1H, CH-thiophene),
8.14 (s, 1H, CH-pyrimidine), 8.57, 8.91 (2s, 2H, 2NH, exchangeable with D2O).13C-NMR
(DMSO-d6, ppm): 103.62 (CH-thiophene), 122.81, 133.65, 157.14 (3C-thiophene), 146.12
(CH-pyrimidine), 156.90 (C-thiadiazole), 162.73 (C=O), 181.71 (C=S). MS m/z: 283 (M+).
Analysis calculated for C8H5N5OS3: C, 33.91; H, 1.78; N, 24.72, found: C, 33.71; H, 1.68; N,
24.52.
5-(5-Thioxo-4,5-dihydro-1,3,4-thiadiazol-2-ylamino)thieno[2,3-d]pyrimidine-4(3H)-thione
(20).
A mixture of compound 19 (1.13 g, 0.004 mol) and phosphorus pentasulfide (0.43 mL, 0.004
mol) in pyridine (20 mL) was refluxed for 8 h, the reaction mixture was cooled, poured onto
ice water, then acidified with dil HCl. The solid obtained was filtered, dried and
recrystallized from ethanol to give 20. Yield, 93 %, mp 222–224ο C. IR (KBr, cm-1): 3240,
3200 (NH), 3038 (CH arom.), 1626 (C=N), 1230 (C=S). 1H-NMR (DMSO-d6, ppm): 2.42 (s,
1H, NH, exchangeable with D2O), 5.32 (s, 1H, CH-thiophene), 8.16 (s, 1H, CH-pyrimidine),
8.24, 9.12 (2s, 2H, 2NH, exchangeable with D2O).13C-NMR (DMSO-d6, ppm): 102.66 (CH-
thiophene), 118.82, 124.61, 147.13 (3C-thiophene), 145.15 (CH-pyrimidine), 156.90 (C-
thiadiazole), 180.14(C=S-pyrimidine), 181.76 (C=S-thiadiazole). MS m/z: 299 (M+).
Analysis calculated for C8H5N5S4: C, 32.09; H, 1.68; N, 23.39, found: C, 32.19; H, 1.78; N,
23.29.
5-(4-Chlorothieno[2,3-d]pyrimidin-5-ylamino)-1,3,4-thiadiazole-2(3H)-thione (21).
A solution of compound 19 (1.13 g, 0.004 mol) in thionyl chloride (15 mL) was refluxed for
3 h, the thionyl chloride was then removed by distillation and the obtained solid was washed
twice with methanol, dried and recrystallized from ethanol to give compound 21. Yield, 89
%, mp 266–268ο C. IR (KBr, cm-1): 3240, 3200 (NH), 3038 (CH arom.), 1626 (C=N), 1230
1
(C=S), 835 (C-Cl). H-NMR (DMSO-d6, ppm): 2.51 (s, 1H, NH, exchangeable with D2O),
5.92 (s, 1H, CH-thiophene), 8.15 (s, 1H, CH-pyrimidine), 8.24 [s, 1H, NH, exchangeable
with D2O].13C-NMR (DMSO-d6, ppm): 103.62 (CH- thiophene), 128.81, 141.62, 142.15 (3C-
thiophene), 151.40 (C-Cl), 156.92 (C-thiadiazole), 157.12 (CH-pyrimidine), 181.70 (C=S).
MS m/z: 301 (M+), 303(M+2). Analysis calculated for C8H4ClN5S3: C, 31.84; H, 1.34; N,
23.21, found: C, 31.74; H, 1.24; N, 23.31.
5-(4-Isothiocyanatothieno[2,3-d]pyrimidin-5-ylamino)-1,3,4-thiadiazole-2(3H)-thione (22).
A mixture of compound 21 (1.2 g, 0.004 mol) and ammonium thiocyanate (0.3 g, 0.004 mol)
was refluxed in dry acetone for 3 h. The reaction mixture was cooled and poured onto ice
water. The solid obtained was filtered, dried and recrystallized from ethanol to give 22. Yield,
90 %, mp 200–202ο C. IR (KBr, cm-1): 3240, 3200 (NH), 3038 (CH arom.), 2019 (N=C=S),
1
1626 (C=N), 1230 (C=S). H-NMR (DMSO-d6, ppm): 2.45 (s, 1H, NH, exchangeable with
D2O), 5.91 (s, 1H, CH-thiophene), 8.15 (s, 1H, CH- pyrimidine), 8.35 (s, 1H, NH,
exchangeable with D2O).13C-NMR (DMSO-d6, ppm): 102.62 (CH-thiophene), 124.82,

ACCEPTED MANUSCRIPT
141.61, 143.15 (3C-thiophene), 137.82 (N=C=S), 142.74 (C-NCS), 156.98 (C-thiadiazole),
158.15 (CH-pyrimidine), 181.71 (C=S). MS m/z: 324 (M+). Analysis calculated for
C9H4N6S4: C, 33.32; H, 1.24; N, 25.90, found: C, 33.22; H, 1.14; N, 25.70.
General procedure for the preparation of 4-(4-imino-2-thioxo-5-(5-thioxo-4,5-dihydro-1,3,4-
thiadiazol-2-ylamino)-1,2-dihydrothieno[2,3-d]pyrimidin-3(4H)-yl) N-substituted
benzenesulfonamide (24-26).
A mixture of compound 3 (1.02 g, 0.004 mol) and the appropriate substituted 4-
isothiocyanato-benzenesulfonamide 23 a-c (0.004 mol) in dimethylformamide (20 mL) and a
catalytic amount of triethylamine (0.2 mL) was refluxed for 6h. The reaction mixture was
cooled, poured onto ice water and the precipitated solid was collected by filtration, dried, and
recrystallized from ethanol to give 24-26, respectively.
4-(4-imino-2-thioxo-5-(5-thioxo-4,5-dihydro-1,3,4-thiadiazol-2-ylamino)-1,2-
dihydrothieno[2,3-d]pyrimidin-3(4H)-yl)benzenesulfonamide (24).
Yield, 92 %, mp 200–202ο C. IR (KBr, cm-1): 3320, 3230, 3200 (NH, NH2), 3038 (CH
arom.), 1626 (C=N), 1230 (C=S), 1380, 1145 (SO2). 1H-NMR (DMSO-d6, ppm): 2.51 (s, 1H,
NH, exchangeable with D2O), 5.92 (s, 1H, CH-thiophene), 6.81, 7.92 (2d, 4H, J= 8.5Hz, Ar-
H), 8.15, 8.91, 9.24 (3s, 3H, 3NH, exchangeable with D2O), 11.15 (s, 2H, SO2NH2,
exchangeable with D2O). 13C-NMR (DMSO-d6, ppm): 103.62 (CH-thiophene), 118.81,
127.60, 159.14 (3C-thiophene), 126.72 (2C), 129.81 (2C), 136.15, 138.92, 156.15 (C=NH),
156.94 (C-thiadiazole), 179.82 (C=S pyrimidine), 181.71 (C=S). MS m/z: 469 (M+).
Analysis calculated for C14H11N7O2S5: C, 35.81; H, 2.36; N, 20.88, found: C, 35.71; H, 2.26;
N, 20.68.
4-(4-imino-2-thioxo-5-(5-thioxo-4,5-dihydro-1,3,4-thiadiazol-2-ylamino)-1,2-
dihydrothieno[2,3-d]pyrimidin-3(4H)-yl)-N-(pyridin-2-yl)benzenesulfonamide (25).
Yield, 86 %, mp 220–222ο C. IR (KBr, cm-1): 3230, 3200 (NH), 3048 (CH arom.), 1626
1
(C=N), 1230 (C=S), 1387, 1145 (SO2). H-NMR (DMSO-d6, ppm): 2.51 (s, 1H, NH,
exchangeable with D2O), 5.82 (s, 1H, CH-thiophene), 6.72, 7.24 (2d, 4H, J= 7.9Hz, Ar-H),
7.34-7.91 (m, 4H, CH pyridine), 8.15, 8.91, 9.32, 10.75 (4s, 4H, 4NH, exchangeable with
13
D2O). C-NMR (DMSO-d6, ppm): 102.61 (CH-thiophene), 108.15, 117.56, 137.81, 148.82,
153.53 (CH-pyridine), 122.82, 145.64, 159.15 (3C-thiophene), 126.70 (2C), 128.83 (2C),
135.17, 138.90, 156.42 (C=NH), 156.61 (C-thiadiazole), 179.80 (C=S pyrimidine), 181.73
(C=S). MS m/z: 546 (M+). Analysis calculated for C19H14N8O2S5: C, 41.74; H, 2.58; N,
20.50, found: C, 41.64; H, 2.48; N, 20.30.
4-(4-imino-2-thioxo-5-(5-thioxo-4,5-dihydro-1,3,4-thiadiazol-2-ylamino)-1,2-
dihydrothieno[2,3-d]pyrimidin-3(4H)-yl)-N-(quinoxalin-2-yl)benzene sulfonamide (26).
Yield, 84 %, mp 190–192ο C. IR (KBr, cm-1): 3240, 3200 (NH), 3038 (CH arom.), 1626
1
(C=N), 1230 (C=S), 1387, 1145 (SO2). H-NMR (DMSO-d6, ppm): 2.51 (s, 1H, NH,

ACCEPTED MANUSCRIPT
exchangeable with D2O), 5.84 (s, 1H, CH-thiophene), 6.88,7.25 (2d, 4H, J= 9.8Hz, Ar-H),
7.31-7.92 (m, 4H, CH-quinoxaline), 8.15 (s, 1H, CH-quinoxaline), 8.54, 8.91, 9.15, 10.72
(4s, 4H, 4NH, exchangeable with D2O). 13C-NMR (DMSO-d6, ppm): 103.61 (CH-thiophene),
117.82, 123.61, 159.15 (3C-thiophene), 124.70 (2C), 126.73 (2C), 135.16, 138.18, 125.80
(2C), 127.24 (2C), 135.15(2C), 137.13, 162.18(C, CH-quinoxaline), 156.42 (C=NH), 156.62
(C-thiadiazole), 179.80 (C=S-pyrimidine), 181.71 (C=S). MS m/z: 597 (M+). Analysis
calculated for C22H15N9O2S5: C, 44.21; H, 2.53; N, 21.09, found: C, 44.11; H, 2.33; N, 21.19.
2.3.Biological Evaluation
2.3.1. Materials and Animals
Male Wistar rats (150-180 g) aged (2-3 months) (used for paw oedema, ulcer and liver &
kidney function tests) and adult male swiss albino mice (25-35 g) aged (3-4 months) (used for
acute toxicity test) were purchased from the animal breeding unit of the National Research
Centre, Giza, Egypt, and acclimatized in the animal facility of the National Centre for
Radiation Research and Technology (NCRRT)-Atomic Energy Authority, Cairo, Egypt, for
one week before being used. Animals were housed at a temperature of 25 ± 5ºC, humidity of
60 ± 5% and 12/12-hour light-dark cycle. They were fed standard pellet diet obtained from
the National Research Centre, Dokki, Cairo and allowed free access to water ad libitum. The
study was conducted in accordance with the guidelines set by the European Economic
Community (EEC) regulations (Revised Directive 86/609/EEC) and approved by the Ethics
Committee at the Faculty of Pharmacy, Cairo University. An enzyme immunoassay (EIA) kit
(catalog number 560101, Cayman Chemical, Ann Arbor, MI, USA) was purchased and used
as manufacturer’s instructions.
2.3.2. Irradiation
Irradiation of animals was carried out using the facilities provided by National Centre for
Radiation Research and Technology (NCRRT), Atomic Energy Authority, Cairo, Egypt using
the gamma Cell-40 biological irradiator furnished with a Caesium137 (Cs 137) source
produced by the Atomic Energy of Canada (dose rate= 0.47 G/min). Animals were pre-
irradiated 24 hour before the experiment at a dose level of 4 Gy [35].
2.3.3. In-vitro COX-1/COX-2 enzyme inhibition assay
The in-vitro ability of the synthesized compounds and celecoxib to inhibit the COX-1 and
COX-2 isozymes was carried out using Cayman colorimetric COX (ovine) inhibitor
screening assay kit (kit catalog number 560101, Cayman Chemical, Ann Arbor, MI, USA)
according to the manufacturer's instructions. Celecoxib was used as a reference drug in this
screening assay. COX catalyzes the first step in the biosynthesis of arachidonic acid to PGH2.
The PGF2α produced from PGH2 by reduction with stannous chloride is measured by
enzyme immunoassay. All the synthesized compounds were dissolved in 100% DMSO to
prepare a stock concentration of 10mg/mL. Briefly, the enzyme COX-1 and COX-2 (10 μL),
heme (10 μL) and 10 μL of the tested drug solutions (100 μM) were added in duplicate to the

ACCEPTED MANUSCRIPT
supplied reaction buffer solution (950 μL, 0.1 M Tris–HCl, pH 8 containing 5 mM
ethylenediamine tetraacetate (EDTA) and 2 mM phenol).These solutions were incubated for a
period of 5 min at 37◦C after which 10 μL of AA (100 μM) solution were added and the COX
reaction was stopped by the addition of 50 μL of 1 M HCl after 2 min. This assay based on
competition between PGs and PGs acetylcholinesterase (AChE) conjugate (PG tracer) for a
limited amount of PG antiserum. The amount of PG tracer that it is able to bind to PG
antiserum was inversely proportional to concentration of PG in the well since the
concentration of PG tracer is held constant while the concentration of PGs varies. This
screening assay directly measures PGF2α produced by SnCl2 reduction of COX-derived
PGH2. This antibody PG complex binds to a mouse anti-rabbit monoclonal antibody that had
been previously attached to the well. The plate is washed to remove any unbound reagents
and then Ellman's reagent, which contains the substrate to acetylcholine esterase, is added to
the well. The product of this enzymatic reaction produces a distinct yellow colour that
absorbs at 410 nm. The intensity of this colour, determined spectrophotometrically, is
proportional to the amount of PG tracer bound to the well, which is inversely proportional to
the amount of PGs present in the well during the incubation: (Absorbance α [Bound PG
Tracer] α1/PGs). Percent inhibition was calculated by the comparison of compound treated
to various control incubations. The concentration of the test compound causing 50%
inhibition (IC50, μM) was calculated from the concentration-inhibition response curve.
2.3.4. Docking Study
All docking calculation and docking studies were carried out using Molecular Operating
Environment MOE version 2008.10. For this purpose, crystal structure of COX-2/SC-558 (a
selective inhibitor) complex (PDB codes: 1CX2) was obtained from the Protein Data Bank in
order to prepare the protein for docking studies. Docking procedure was followed using the
standard protocol implemented in MOE 2008.10 and the geometry of resulting complexes
was studied using the MOE’s Pose Viewer utility. The enzyme was prepared for docking as
follows: 1) The Co-crystallized ligand and water molecules were removed. 2) The enzyme
was 3D protonated, where hydrogen atoms were added at their standard geometry, the partial
charges were computed and the system was optimized. Flexible ligand- rigid receptor
docking of the most stable conformers was done with MOE-DOCK using triangle matcher as
placement method and London dG as a scoring function. The obtained poses were subjected
to force field refinement using the same scoring function. Thirty of the most stable docking
models for each ligand were retained with the best scored conformation.
2.3.5. In-vivo anti-inflammatory activity against carrageenan-induced rat paw oedema
pre-exposed to whole body gamma irradiation
The anti-inflammatory activity of twelve compounds of the most potent COX-2 inhibitors
resulting from in-vitro enzyme inhibition assay, compounds 2, 3, 7, 11, 14, 16, 17, 19, 21, 22,
24 and 26 was evaluated using in-vivo carrageenan-induced paw oedema model as reported
[36] using diclofenac and celecoxib as standards. A total of 90 Male Wister rats (150- 180 g
body weight) and aged (2-3 months) were used in this experiment. Rats were randomly
separated to 16 groups, each of 6 rats in labelled cages. Group 1 served as control

ACCEPTED MANUSCRIPT
inflammation and rest groups were exposed to whole body γ-irradiation (4 Gy) [35] (24 h
prior to carrageenan sub-plantar injection (0.1 ml of 1% soln.) of each rat. Each tested
compound was dissolved in 5% tween 80 and administered intraperitoneally in a dose of 100
mg/kg.b.wt. 1 h before induction of oedema by carrageenan. The initial volume (Vi) of
induced oedema was measured using a water digital plethysmometer, LE 7500 (Panlab,
HARVARD Apparatus, Spain) by immersing the paw till the level of tibiotarsic articulation
into the container of the plethysmometer and the displacement volume (in mL) was measured
by two platinum electrodes introduced beforehand into the container. The rat’s foot pad
became oedematous soon after the injection of carrageenan and the paw volume (Vf) was
measured again 3 h after carrageenan injection. The increase in paw volume was calculated
as percentage of oedema compared to the basal paw volume according to the formula:
% of oedema = [(Vf - Vi) / Vi] x 100
2.3.6. In-vivo analgesic activity against carrageenan-induced rat hyperalgesia pre-
exposed to whole body gamma irradiation
Analgesic activity of the same twelve potent compounds 2, 3, 7, 11, 14, 16, 17, 19, 21, 22, 24
and 26 was evaluated in hyperalgesic rats using the Carrageenan-induced hyperalgesia
method according to Randall and Selitto [37] using diclofenac and celecoxib as standards. A
total of 96 Male Wister rats (150- 180 g body weight) and aged (2-3 months) were used in
this experiment. Rats were randomly separated to 16 groups, each of 6 rats in labelled cages.
Group 1 served as normal, group 2 served as control inflammation and groups from 3-16
were exposed to whole body γ-irradiation (4 Gy) [35] 24 h prior to carrageenan injection (0.1
ml of 1% soln.). Each tested compound was dissolved in 5% tween 80 and administered
intraperitoneally in a dose of 100 mg/kg.b.wt. 1 h before carrageenan injection. The
nociceptive threshold of the hind paw injected with carrageenan was quantified with an
anlgesimeter (Ugo Basile, Comerio, Varese, Italy) 3 h after carrageenan injection. The force,
in grams, applied to the paw was increased at a constant rate until the rat withdraws its paw.
The pressure was immediately removed and the force required to elicit the end-point response
was recorded.
2.4.Toxicological studies
2.4.1. Ulcerogenic liability
The ulcerogenic effect of the same twelve potent compounds having the highest anti-
inflammatory activity and the standards diclofenac and celecoxib were evaluated by the
reported method [38]. To measure gastric ulceration, a total of 90 adult male Wister rats
(150- 180 gm body weight) and aged (2-3 months) were divided into 15 groups of six animals
each. Animals were kept under standard laboratory conditions and fasted 24 h prior to
administration of the tested compounds. The tested compounds were administered orally in a
dose of 100 mg.kg.bwt. The animals sacrificed 4 h after drug treatment [38]. The stomachs
were removed and examined with an eye lens for the presence of ulcers and erosions. Ulcer

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index was calculated according to the method of Kulkarni [39] using the following scores
involving number and severity of ulcers:
0.0= normal colored stomach 0.5= red coloration
streaks. 2.0= ulcers with area >3mm2 but ≤5 mm2.
1.0= spot ulcer
1.5= hemorrhagic
3.0= ulcers with area >5mm2.
Ulcer index (UI) = (UN+US+UP) * 10-1
Where, UN= average no. of ulcers per animal. US= average of severity scores. UP=
percentage of animals with ulcers per group.
The presence of any one of these criteria was considered to be evidence of ulcerogenic
activity.
2.4.2. Acute Toxicity Study
The approximate 50% lethal dose (ALD50) of the most representative promising compounds
(17, 21 and 26) was determined. Adult Swiss male albino mice (3-4 months) weighing 25- 35
g were obtained from the animal breeding unit of the National Research Centre, Giza, Egypt.
The study was conducted in accordance with the guidelines set by the European Economic
Community (EEC) regulations (Revised Directive 86/609/EEC) and approved by the Ethics
Committee at the Faculty of Pharmacy, Cairo University. A total of 40 mice were taken and
separated to 10 mice groups in labelled cages. The tested compounds were injected
intraperitoneally (i.p.) at different dose levels (100, 200, 500 and 1000 mg/kg. b.wt) .
Animals were kept under observation for 24- 48 h during which any mortality in each group
was recorded. All the animals had free access to food and water after drug administration.
After 24 hrs, they were sacrificed by cervical dislocation. From the data obtained, the
ALD50 was calculated by the method of Smith [40].
2.4.3. Liver and kidney functions estimation
This study was carried on adult male wistar rats (150- 180g), 36 rats were randomly divided
into 6 groups, 6 rats in each group, group 1 received vehicle (5% tween 80) and served as
control group, groups 2 & 3 received diclofenac and celecoxib (50 mg/ kg. i.p.) and groups
from 4 to 6 received the promising compounds (17, 21 and 26; 100 mg/ kg. i.p.) [according to
pilot studies]. All animals were injected with the selected vehicle or drug for 2 consecutive
days [41]; blood samples were collected 4 hours from the last dose of vehicle or drug. Blood
samples were allowed to clot for 45 min at room temperature and serum was separated by
centrifugation at 3000 rpm for 15 min and analyzed for assessment of liver and kidney
functions such as serum glutamic- pyruvic transaminase (SGPT) and serum glutamatic-
oxaloacetic transaminase (SGOT) by reported method of Reitman and Frankel [42], and
serum creatinine (SCr) by Schirmeister et. al. method [43], respectively.

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2.5.Statistical Analysis
All values were expressed as means ± S.E. Data was analyzed using one-way ANOVA
followed by Tukey-Kramer multiple comparison test. The p value was considered significant
at P < 0.05. Graphpad software instat (version 6) was used to carry out these statistical tests.
3. Results, discussion and SAR findings
3.1.Chemistry
The synthetic pathways adopted to obtain in good yields the target compounds 3-26 are
outlined in schemes 1 and 2. For the exploration of structure activity relationship of 1,3,4-
thiadiazole based compounds as selective COX-2 inhibitors, different synthetic strategies
were done by varying the substituents on the reported 2-acetamide thiadiazole derivative 2
[44] which was obtained from monoacetylation of the reported 5-amino-3H-
[1,3,4]thiadiazole-2-thione 1 [45] via simple synthetic methods.
Following Gewald’s thiophene synthesis, the thiophene derivatives 3-5 were synthesized
through reaction of 2 with activated nitrile (malononitrile, ethyl cyanoacetate and/or ethyl
acetoacetate) in the presence of elemental sulfur [46]. Treatment of 2-acetamide thiadiazole 2
with the appropriate 2-(4-substituted benzylidene) cyanoacetate and/or 2-(4-substituted
benzylidene) malononitrile [47] in ethanol yielded the corresponding pyran derivatives 7-14
following the reported method [48]. (scheme 1).
Scheme 1. Synthetic pathways for compounds 3-5, 7-14.
2-Chloroacetamido thiophene derivative 15 was obtained by stirring compound 3 with
chloroacetyl chloride in DMF. While, the ethyl formamidate thiophene derivative 16 was
prepared by refluxing compound 3 in triethylorthoformate in the presence of a few drops of
acetic anhydride, followed by cycloaddition of phenyl hydrazine yielding the 3-
(phenylamino)-thieno[2,3-d]pyrimidine derivative 17. Refluxing compound 3 with phenyl
isocyanate in ethanol yielded the thieno[2,3-d]pyrimidin-2(1H)-one derivative 18 by
nucleophilic substitution followed by intramolecular cyclization. (scheme 2)
Formylation of compound 3 was carried out by heating with formic acid to give the
thieno[2,3-d]pyrimidin-4(3H)-one derivative 19. Compound 19 was further subjected to
thionation with phosphorus pentasulfide in pyridine to afford the thieno[2,3-d] pyrimidine-
4(3H)-thione derivative 20, and chlorination with thionyl chloride yielding the 4-
chlorothieno[2,3-d]pyrimidine derivative 21. Compound 21 was then converted to the 4-
isothiocyanatothieno[2,3-d]pyrimidine derivative 22 through reaction with ammonium
thiocyanate in dry acetone. On the other hand, refluxing compound 3 with the appropriate
substituted 4-isothiocyanato benzenesulfonamide 23a-c prepared by the reported method [49]
in DMF yielded the thieno[2,3-d] pyrimidin-3(4H)-yl) N-substitutedbenzenesulfonamide
derivatives 24-26, respectively. (scheme 2).

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1
The synthesized compounds were characterized by IR, H-NMR,13C-NMR and mass spectra
and were in conformity with the assigned structures as listed in the material and methods
section.
Scheme 2. Synthetic pathways for compounds 15-18, 19-22 and 24-26.
3.2.Biological activity
3.2.1. In-vitro COX-1/COX-2 enzyme inhibition assay
All the target compounds were evaluated for their ability to inhibit COX-1 and COX-2
using an ovine COX-1/COX-2 assay kit (Catalog No. 560101, Cayman Chemicals Inc.,
Ann Arbor, MI, USA). The concentration causing 50% enzyme inhibition (IC50 µM) was
determined as well as the selectivity index (SI values) which is defined as IC50 (COX-
1)/IC50(COX2). In the assay system, we selected two commercially available drugs,
diclofenac, a potent non-selective COX inhibitor, and celecoxib, a potent selective COX-2
inhibitor as reference drugs. The IC50 values of diclofenac on COX-1 and COX-2 were 0.25
and 0.26 µM respectively, with low SI (0.96 µM). The IC50 values of celecoxib on COX-1
and COX-2 were determined to be >50 and 0.18 µM respectively, with high SI (>277.77
µM). The results showed that most of the synthesized compounds showed no activity on
COX-1 with IC50 values >50 µM, among them only three compounds 2, 11 and 19 were
active on COX-1, with IC50 values 23.5, 40.1and 25.3 µM, respectively. With respect to
activity on COX-2, some of the tested compounds showed potent COX-2 inhibition with
IC50 values ranging from 0.09-0.36 µM) compared to celecoxib. The most potent
compounds in this study were 3, 7, 14, 16, 17, 21, 22, 24 and 26 having similar or even
higher SI than celecoxib ranging from 231.48-555.55 µM as listed in Table 1.
As aforementioned, the aim of the present study was to obtain selective COX-2 inhibitors, so
it is worthy to understand the SAR of the synthesized compounds. The design of the target
compounds relies on the synthesis of several distinct novel compounds based on 2-acetamido
thiadiazole scaffold. The activity of unsubstituted 2-amino-thiadiazole ring was explored by
testing compound 1 the starting material which has no activity on both COX enzymes, while,
upon addition of acetamide group at position 2, significant COX-1/COX-2 activity was
observed but with lower selectivity index as compound 2 (COX-1/IC50= 23.5, COX-2/IC50=
0.366; SI=64.2 µM).
Introduction of thiophene ring in compounds 3-5, 15 and 16 showed COX-2 inhibition
depending on the substitution at position 2 and 3 of the thiophene ring, , the 2-amino-3-
carbonitrile substitution resulted in potent COX-2 activity with high selectivity index in
compound 3 (IC50= 0.15; SI>333.3 µM), the 2-ethyl formamidate-3-carbonitrile substitution
resulted in high COX-2 activity and high selectivity index (IC50= 0.198; SI=384.61 µM) as in
compound 16, while, the 2-amino-3-ethylcarboxylate, the 2-methyl-3-ethyl-carboxylate and
2-chloracetamide-3-carbonitrile substitution in compounds 4, 5 and 15 showed no activity on
both COX enzymes.

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The COX-2 inhibitory activity and the selectivity indices of the pyran derivatives 7-14
varies according to the substituent at position 3 of the pyran ring and the para-substitution
on the aromatic ring at position 4. the para-chloro-ethylcarboxylate 7 (IC50= 0.171;
SI>292.3 µM) showed potent COX-2 inhibition with extremely high SI, while, the para-
chloro-carbonitrile 11 derivative (IC50= 0.257; SI=156.03 µM) showed similar COX-2
activity but with lower SI. In addition, N-dimethyl-carbonotrile 14 derivative (IC50= 0.168;
SI>297.6 µM) showed significant COX-2 activity with high SI. Other pyran derivatives
have no activity on both COX enzymes.
Considering the thieno[2,3-d]pyrimidine derivatives 17-22, SAR is discussed according to
the type and position of the substituent on the pyrimidine ring., the 3-phenylamino-4-imino
derivative 17 (IC50= 0.151; SI>331.1 µM) showed increased COX-2 activity with high SI,
while, the 2-oxo-3-N-phenyl-4-imino derivative 18 showed no activity on both COX
enzymes. Upon removing the substituents at position 2 and 3 and only introducing carbonyl
group at position 4 in compound 19, the SI is highly affected and decreased to be 121.05
µM. The nature of the substituent at position 4 of the pyrimidine ring affected the COX-2
activity, where, the 4-thioxo derivative 20 displayed diminished activity on both enzymes.
While, COX-2 activity is restored upon chlorination in compound 21 and slightly decreased
in the 4-isothiocyanato derivative 22 (IC50= 0.162, 0.216; SI=308.6, 231.48 µM,
respectively). Introduction of sulfonamide group at position 3 of the pyrimidine was very
successful especially for the sulfanilamide derivative 24 (IC50= 0.156; SI>320.51 µM) and
the sulfaquinoxaline derivative 26 (IC50= 0.09; SI>555.55 µM) derivative which was the
most potent and selective COX-2 inhibitors in this study. Generally, introduction of
thieno[2,3-d]pyrimidine with sulfonamide moiety on thiadiazole ring led to the most potent
and selective compounds in this study, also, we can observe the crucial inhibitory ability of
either thiophene and pyran substituents on thiadiazole ring may be by allowing a better
orientation of the compounds within the additional pocket responsible for selectivity of
COX-2 enzyme.
Finally, it could be noted from the above results that 1,3,4-thiadiazole bearing thiophene,
pyran or thieno[2,3-d]pyrimidine moieties are promising scaffolds to develop potent selective
COX-2 inhibitors.
Table 1. In-vitro COX-1/COX-2 enzyme inhibition assay.
3.2.2. Docking Study
In order to further interpret the mechanism of interaction and the binding mode of the
synthesized compounds within COX-2 active site, docking study was performed. Docking of
all the new compounds together with the reference drugs celecoxib and diclofenac into the
crystal structure of COX-2 enzyme catalytic domain in complex with the celecoxib analogue
SC-558 [PDB ID code 1CX2] was performed using Molecular Operating Environment MOE
version 2008.10. The most stable docking pose was selected according to the best scored
conformation predicted by the MOE scoring function, the docking results are presented in

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Table 2, where, the docking scores, amino acids interactions and bond lengths within the
active site are listed for all the compounds.
The COX active site consists of three distinct sites, the carboxylate site at entrance which is
composed of three hydrophilic residues Arg120, Tyr 355 and Glu524 arranged in a way to
form H-bond network. This entrance leads to a long hydrophobic channel that deeply extends
into the catalytic domain. The main difference between the two COX isoforms is that in
COX-2, this hydrophobic channel forks to a primary hydrophobic pocket and side pocket.
The primary pocket is defined by the amino acids Tyr385, Trp87, Phe518 and Ser530 which
is the site of NSAIDS binding. While, the side pocket is located above Arg120 and this
pocket is bordered by Val523 and contains the main residues responsible for COX-2
selectivity His90 and Arg513. This structural difference between the two COX isoform is the
result of exchange of the relatively bulky isoleucine (Ile) at position of 523 in COX-1 with
the less bulky Val residue at the same position in COX-2, this modification allows the access
to an additional side pocket responsible for selectivity [6, 16, 18, 19].
To validate the docking protocol, the co-crystallized ligand was re-docked into COX-2 active
site and the root mean square deviation (RMSD) is calculated and found to be 0.209 Å with
docking score (S= -13.01 kcal/mol). Celecoxib was docked into the active site of COX-2, it
showed similar orientations as reported [56], where, the toluene moiety is towards the
primary hydrophobic pocket, the trifluoromethyl group is near Arg120, while, the phenyl
sulfonamide moiety is inserted in the side pocket where the SO2 group H-bonded with His90
and Arg513 with bond lengths 2.90 and 1.56 Å, respectively and docking score (S= -11.03
kcal/mol). On the other hand, docking of diclofenac revealed that it could only access to the
primary hydrophobic pocket, where, its carboxylate group binds to Ser530 and Tyr385 as
reported (Table 2).
Considering docking of the synthesized compounds, there is strong correlation between the
results of in-vitro binding assay with that of docking study. The most potent compounds 3, 7,
14, 16, 17, 21, 22, 24 and 26 showed the best docking scores in the range of -11.49 to -9.54
kcal/mol. By closer observation of the binding mode and SAR of the synthesized compounds,
in the thiophene containing compounds 3-5, 15 and 16, the 1,3,4-thiadiazole bearing
thiophene-2-Amino-3-carbonitrile moiety 3 showed similar orientation as celecoxib, the
introduced CN group was able to bind to Arg513 and His90, similarily, the 2-formamidate
moiety of compound 16 was able to enter deeply into the selectivity pocket and binds to
Arg513. On the other hand, no binding to the selectivity pocket was observed for compounds
4, 5 and 15 which were inactive in the previous study.
In the pyran series 7-14, the most selective candidates with higher SI were compounds 7 and
14, the introduced p-chlorophenyl and the carboxylate group of 7 were able to interact with
Arg513, the p-N-dimethylphenyl and carbonitrile moieities bonded with Arg513 in addition
to another H-bond with Val523 in case of compound 14, the bulky N-dimethyl group pushed
the compound into the selectivity pocket. (Figures 2-4; Table 2). In case of compound 11, the
less bulky carbonitrile group failed to enter the selectivity pocket and resulted in a different
orientation which explains the low SI in the previous experiment.
In the thieno[2,3-d]pyrimidine series 17-22, the bulkiness and the position of substitutions on
pyrimidine ring greatly affected the orientation of the compounds within the active site, the 3-
phenylamino substitution in 17 was oriented in a way to bind through arene-cation interaction

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with Arg513 in addition to binding to His90 through the thiadiazole ring, increasing the
COX-2 activity. (Figure 5; Table 2). In compounds 21 and 22, the 4-chloro and the 4-
Isothiocyanate substituents bind to Arg513 through arene-cation interaction in addition to
binding of the pyrimidine ring to His90 (Figures 6 and 7; Table 2). On the other hand, the
selectivity of the sulfonamide derivatives 24 and 26 was explained by their higher binding
affinity within the active site of COX-2 through SO2 group which was pushed more deep
within the side pocket resulting in stronger bonds with Arg513 and His90 with shorter bond
length (Figures 8 and 9; Table 2).
Generally, it was interesting to note that the docking study helped to more understand the
mechanism of binding of the synthesized compounds within the active site of COX-2. The
most potent compounds showed similar orientation as celecoxib and showed high liability to
enter the additional side pocket. The compounds that showed low SI were deeply immerged
into the primary hydrophobic pocket in similar manner as traditional NSAIDS.
Table 2. Binding scores, amino acid interactions and bond length of the synthesized
compounds within the active site of COX-2.
3.2.3. In-vivo anti-inflammatory activity against carrageenan-induced rat paw oedema
pre-exposed to whole body gamma irradiation
The most potent COX-2 inhibitors resulting from in-vitro enzyme inhibition assay,
compounds 2, 3, 7, 11, 14, 16, 17, 19, 21, 22, 24 and 26 were subjected to in-vivo anti-
inflammatory assay using standard carrageenan-induced rat paw oedema model pre-exposed
to 4 Gy of whole body gamma irradiation 24 h prior to carrageenan sub-plantar injection.
However, cytokines production post irradiation is time-dependent and peaking usually
achieved at 4–24 hrs after irradiation [50, 51]. The results are listed in Table 3 and are
expressed as the percentage of oedema compared to basal paw volume. From the results we
can conclude that the inflammatory response produced by carrageenan in irradiated rats was
significantly higher than that induced in non-irradiated animals. This response is attributed to
the increased levels of prostaglandins as well as lysosomal enzymes as a result of disruption
of the cell membranes due to radiation exposure caused by direct interaction of cellular
membranes with gamma-rays or by the action of the free radicals produced by ionizing
radiation on the cellular membranes [52, 53]. Treatment of irradiated inflamed rats with the
tested compounds showed significant decrease in the percentage of oedema in the range
between 24.49-62.50%. The most potent compound in this study was the thiadiazole
derivative bearing the 4-chlorothieno[2,3-d]pyrimidine moiety 21 having 24.49% of oedema
compared to celecoxib (18.61%). Similarly, potent anti-inflammatory activity and non-
significant difference from celecoxib was observed for the 3-phenylamino thieno[2,3-
d]pyrimidine 17 and the sulfonamide derivative 26 with percentages of oedema of 24.70 and
25.40 %, respectively. Also compounds 14 and 16 showed potent anti-inflammatory activity
and significant decrease in the percentage of oedema to 40.08-41.30%, respectively. These
results were in agreement with the results of in-vitro enzyme inhibition assay indicating the
crucial anti-inflammatory role of introduction of either thiophene and pyran moieties to the

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thiadiazole scaffold, as well as incorporation of the bicyclic thieno[2,3-d]pyrimidine moiety
and the sulfonamide substitution on the 1,3,4-thiadiazole ring.
3.2.4. In-vivo analgesic activity against carrageenan-induced rat hyperalgesia pre-
exposed to whole body gamma irradiation
The most potent COX-2 inhibitors resulting from in-vitro enzyme inhibition assay,
compounds 2, 3, 7, 11, 14, 16, 17, 19, 21, 22, 24 and 26 were subjected to in-vivo analgesic
assay using standard carrageenan-induced hyperalgesia model pre-exposed to 4 Gy of whole
body gamma irradiation 24 h prior to carrageenan sub-plantar injection. The nociceptive
threshold defined as the maximum force, in grams, applied at a constant rate until the rat
withdraws its paw, was quantified with an analgesimeter 3 h after carrageenan injection and
presented in Table 3. Since whole body γ-irradiation led to significant increment in the paw
volume and based on the sensitization theory of Randall and Selitto, the first to make use of
the knowledge that inflammation increases sensitivity to pain [37], it was expected that upon
exertion of mechanical hyperalgesia on the inflamed paw of irradiated rats, a significant
decrement in the nociceptive threshold would be observed. In the present study the
nociceptive threshold recorded in the inflamed irradiated rats did not differ significantly from
that observed in the inflamed group. This was in accordance to the study of Kereskenyiova
and Smajda who reported the possible underlying mechanism beyond this response showing
that ionizing radiation would exert an analgesic effect mediated by the release of endogenous
opioids [54]. The results showed that, celecoxib exhibited significantly high nociceptive
threshold (90.23 g) when compared to the control group. All the tested compounds showed
relatively high analgesic activities with nociceptive thresholds significantly different from
control group in the range of 42.31-85.72 g. The most potent analgesic compound in this
study was the thiadiazol-2-acetamide derivative 2 having the highest nociceptive threshold
(85.72 g) very close to that of celecoxib. Also the pyran derivatives 11and 14, thiophene
schiff”s base derivative 16, thieno[2,3-d]pyrimidine 19 and the sulfonamide derivative 24
showed potent analgesic activities with nociceptive thresholds significantly different from
control group of 73.41, 62.12, 62.81, 61.50 and 61.21g, respectively. These results were also
in agreement with the results of in-vitro enzyme inhibition assay underlining the analgesic
activity of 1,3,4-thiadiazole-2-acetamide, sulfonamide substituted thiadiazole and 1,3,4-
thiadiazole bearing pyran, thiophene or thieno[2,3-d]pyrimidine moieties.
Table 3. In-vivo anti-inflammatory, analgesic.
3.3.Toxicological studies
3.3.1. Ulcerogenic liability
The ulcerogenic potential of the most potent COX-2 inhibitors 2, 3, 7, 11, 14, 16, 17, 19, 21,
22, 24 and 26 was evaluated and the ulcer index was calculated as reported [38, 39]. The
ulcerogenic liability was compared to celecoxib and the classical NSAID diclofenac. From
the data listed in Table 4, it was observed that celecoxib as selective COX-2 inhibitor showed
no ulceration effect (UI=0.0± 0.0), similarly, compounds 3, 7, 14, 16, 17, 21, 22 and 26

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showed extremely high safety margin on gastric mucosa with no ulceration effect (UI ranges
0.0-0.083), these results were in agreement with their high SI found in COX inhibitory assay.
Furthermore, compounds 2, 11, 19 and 24 caused gastric ulceration effects of (UI=
0.50±0.033; 0.52±0.042; 0.45± 0.04 and 0.10± 0.004, respectively) in the experimental
animals, these compounds showed low SI in the in-vitro binding assay due to non-selective
inhibition of both COX enzymes. But interestingly, their observed ulcerogenic liabilities were
less than that of diclofenac (UI=2.37± 0.09). In case of compound 24, although it showed no
activity on COX-1 and relatively high SI, slight ulcers were observed in experimental
animals. This behavior could be interpreted from the docking study, the sulfonamide group
was able to bind to Tyr355 in the carboxylate site where binds NSAIDS.
In conclusion, the potential value of the tested compounds as anti-inflammatory agents is
their extremely high safety margin on gastric mucosa than diclofenac. These findings support
the objective of the present work to present novel 1,3,4-thiadiazole bearing pyran, thiophene
or thieno[2,3-d]pyrimidine moieties as selective COX-2 inhibitors with diminished gastric
injuries.
Table 4. Ulcerogenic liability in rats.
3.3.2. Acute Toxicity Study
The most potent selective COX-2 inhibitors and anti-inflammatory compounds resulted from
the previous experiments 17, 21 and 26 were selected to study their acute toxicity in rats. The
approximate 50% lethal dose (ALD50) was determined according to Smith’s method as
presented in Table 5. The results revealed that the tested compounds were relatively non-
toxic in experimental rats showing ALD50>1000 mg/kg.
3.3.3. Liver and kidney functions estimation
After determining the acute toxicity of the most potent compounds resulted from the previous
experiments 17, 21 and 26. They were further studied for their renal and hepatotoxic effect.
Their effect on biochemical parameters (serum enzymes and serum creatinine) was studied.
As shown in Table 5, activities of the liver enzymes serum glutamate oxaloacetate
transaminase (SGOT) and serum glutamate pyruvate transaminase (SGPT) of compounds 17,
21 and 26 almost remain the same with respect to the normal values. The histopathological
studies of the liver samples of these compounds do not show any significant pathological
changes in comparison to standard drug celecoxib. No hepatocyte necrosis or degeneration
was seen in any of the samples.
On the other hand, serum creatinine (SCr) is the most sensitive biochemical marker employed
in the diagnosis of renal damage. Therefore, marked increase in serum creatinine is an
indication of functional damage to the kidney [55].
None of the tested compounds showed significant change in the mean values of creatinine in
serum of rats when compared with the standard drug celecoxib. According to these
indicators, the tested compounds 17, 21 and 26 are therefore, not nephrotoxic in rats.

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Table5. In-vivo acute toxicity, kidney and liver Function Parameters.
4. Conclusion
The present study reported the design and synthesis of novel series of selective COX-2
inhibitors based on 1,3,4-thiadiazole moiety. The synthesized compounds were evaluated for
their COX-1/COX-2 inhibitory activity in-vitro. Compounds 3, 7, 14, 16, 17, 21, 22, 24 and
26 were found to be potent and selective COX-2 inhibitors (IC50= 0.09-0.366 µM) and were
inactive against COX-1 (IC50> 50 µM). SAR was discussed in terms of the enzyme inhibitory
activity and was supported by molecular docking simulations and analysis of the binding
modes of the new inhibitors within COX-2 active site. In addition, the most potent COX-2
inhibitors were assessed for their anti-inflammatory, analgesic activities and toxicological
studies in-vivo. Interestingly, the most potent compounds in this study having the highest
nociceptive threshold very close to that of celecoxib was the thiadiazol-2-acetamide
derivative 2, pyran derivatives 14 and 11 and the sulfonamide derivative 24. While, the
thiophene, thienopyrimidine derivatives 17 and 21 and the sulfonamide derivative 26 showed
the highest anti-inflammatory activity compared to the reference drug celecoxib, they also
had high safety margin on gastric mucosa with no ulceration effect and they were found to be
non-toxic in experimental rats with normal kidney and liver function profiles. These results
suggested a contributory role of 1,3,4-thiadiazole derivatives in designing potent and
selective COX-2 inhibitors with diminished gastric injuries.
Acknowledgments
The authors are kindly appreciative to the staff members of gamma irradiation unit at the
National Center for Radiation Research and Technology (NCRRT) for their kind cooperation
in carrying out the experimental irradiation.
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Figure 1. The designed synthesized compounds.
Scheme 1. Synthetic pathways for compounds 3-5, 7-14.

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Scheme 2. Synthetic pathways for compounds 15-18, 19-22 and 24-26.
Figure 2. 3D binding interactions of 3 within the active site amino acids of COX-2, hydrogen bonds
are shown as magenta lines (left panel); 2D binding interactions of 7 within the active site amino acids
of COX-2 (right panel).

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Figure 3. 3D binding interactions of 7 within the active site amino acids of COX-2, hydrogen bonds
are shown as magenta lines (left panel); 2D binding interactions of 7 within the active site amino acids
of COX-2 (right panel).
Figure 4. 3D binding interactions of 14 within the active site amino acids of COX-2, hydrogen bonds
are shown as magenta lines (left panel); 2D binding interactions of 14 within the active site amino
acids of COX-2 (right panel).