Selective cyclooxygenase inhibition and ulcerogenic liability of some newly prepared anti-inflammatory agents having thiazolo[4,5-d]pyrimidine scaffold

Article abstract of DOI:10.1016/j.bioorg.2019.102964

Novel candidates of thiazolo[4,5-d]pyrimidines (9a-l)were synthesized and their structures were elucidated by spectral and elemental analyses. All the novel derivatives were screened for their cyclooxygenase inhibitory effect, anti-inflammatory activity and ulcerogenic liability. All the new compounds exhibited anti-inflammatory activity, especially 1-(4-[7-(4-nitrophenyl)-5-thioxo-5,6-dihydro-3H-thiazolo[4,5-d]pyrimidin-2-ylideneamino]phenyl)ethanone (9g)was the most active derivative with 57%, 88% and 88% inhibition of inflammation after 1, 3 and 5h, respectively. Furthermore, this derivative 9g recorded higher anti-inflammatory activity than celecoxib which showed 43%, 43% and 54% inhibition after 1, 3 and 5h, sequentially. Moreover, the target derivatives 9a-l demonstrated moderate to high potent inhibitory action towards COX-2 (IC₅₀ = 0.87–3.78 μM), in particular, the derivatives 9e (IC₅₀ = 0.92 μM), 9g (IC₅₀ = 0.87 μM)and 9k (IC₅₀ = 1.02 μM)recorded higher COX-2 inhibitory effect than the selective COX-2 inhibitor drug celecoxib (IC₅₀ = 1.11 μM). The in vivo potent compounds (9e, 9g and 9k)caused variable ulceration effect (ulcer index = 5–12.25)in comparison to that of celecoxib (ulcer index = 3). Molecular docking was performed to the most potent COX-2 inhibitors (9e, 9g and 9k)to explore the binding mode of these derivatives with Cyclooxygenase-2 enzyme.

Full text of DOI:10.1016/j.bioorg.2019.102964

Accepted Manuscript
Selective Cyclooxygenase inhibition and ulcerogenic liability of some newly
prepared anti-inflammatory agents having thiazolo[4,5-d]pyrimidine scaffold
Rania B. Bakr, Amira A. Ghoneim, Amany A. Azouz
PII:
S0045-2068(19)30251-2
https://doi.org/10.1016/j.bioorg.2019.102964
102964
DOI:
Article Number:
Reference:
YBIOO 102964
Bioorganic Chemistry
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Accepted Date:
14 February 2019
8 April 2019
29 April 2019
Please cite this article as: R.B. Bakr, A.A. Ghoneim, A.A. Azouz, Selective Cyclooxygenase inhibition and
ulcerogenic liability of some newly prepared anti-inflammatory agents having thiazolo[4,5-d]pyrimidine scaffold,
Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.102964
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Selective Cyclooxygenase inhibition and ulcerogenic liability of some newly prepared anti-
inflammatory agents having thiazolo[4,5-d]pyrimidine scaffold
Rania B. Bakra,b*, Amira A. Ghoneimc,d , Amany A. Azouze
aDepartment of Pharmaceutical Chemistry, College of Pharmacy, Jouf University, Sakaka, Al Jouf-
2014, KSA.
bDepartment of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Beni-Suef University, Beni-
Suef-62514, Egypt.
cChemistry Department, Faculty of Science, Jouf University, P.O. box , 2014, Aljouf, Saudi Arabia
dChemistry Department, Faculty of Science, Zagazig University, Zagazig, Egypt.
eDepartment of Pharmacology and Toxicology, Beni-Suef University, Beni-Suef 62514, Egypt
*To whom correspondence should be addressed.
Rania B. Bakr, Ph.D.
Tel.: (002)-100-2324568, 00966583405023
Fax: (002)-082-2317958
E-mail address: raniabakr@ymail.com
Key words: Thiazolo[4,5-d]pyrimidine; Thiazolidine; Anti-inflammatory; Cox-2 inhibitors.

Abstract
Novel candidates of thiazolo[4,5-d]pyrimidines (9a-l) were synthesized and their structures were
elucidated by spectral and elemental analyses. All the novel derivatives were screened for their
cyclooxygenase inhibitory effect, anti-inflammatory activity and ulcerogenic liability. All the new
compounds exhibited anti-inflammatory activity, especially 1-(4-[7-(4-nitrophenyl)-5-thioxo-5,6-dihydro-
3H-thiazolo[4,5-d]pyrimidin-2-ylideneamino]phenyl)ethanone (9g) was the most active derivative with
57%, 88% and 88% inhibition of inflammation after 1, 3 and 5 h, respectively. Furthermore, this
derivative 9g recorded higher anti-inflammatory activity than celecoxib which showed 43%, 43% and
54% inhibition after 1, 3 and 5 h, sequentially. Moreover, the target derivatives 9a-l demonstrated
moderate to high potent inhibitory action towards COX-2 (IC50 = 0.87–3.78 µM), in particular, the
derivatives 9e (IC50 = 0.92 µM), 9g (IC50 = 0.87 µM) and 9k (IC50 = 1.02 µM) recorded higher COX-2
inhibitory effect than the selective COX-2 inhibitor drug celecoxib (IC50 = 1.11 µM). The in vivo potent
compounds (9e, 9g and 9k) caused variable ulceration effect (ulcer index = 5–12.25) in comparison to
that of celecoxib (ulcer index = 3). Molecular docking was performed to the most potent COX-2
inhibitors (9e, 9g and 9k) to explore the binding mode of these derivatives with Cyclooxygenase-2
enzyme.
2

1. Introduction
Cyclooxygenase enzyme (COX) is responsible for the formation of prostaglandins (PGs), and
thromboxanes (TXA2) from arachidonic acid [1, 2]. Cyclooxygenase enzyme exists in two distinct
isoforms, i) a constitutive form (COX-1) which is essential for the physiological production of (PGs) and
maintenance functions such as cytoprotection in the stomach, and ii) an inducible form (COX-2) which is
induced in inflammatory cells [3, 4]. Traditional non-steroidal anti-inflammatory drugs (NSAIDs) were
recorded to suppress both COX-1 and COX-2 producing adverse side effects as ulceration, and
gastrointestinal bleeding [5-7]. Coxibs were introduced in market as selective COX-2 inhibitors that
inhibit COX-2 isoform achieving the same anti-inflammatory effect as traditional NSAIDs with
minimized risk of ulcers [8, 9]. But some selective COX-2 inhibitors as valdecoxib exhibited
cardiovascular side effects as myocardial infarction and hypertension leading to their withdrawal from
markets [10, 11]. Thiazolidine derivatives revealed great attention due to their biological activity as
anticancer [12, 13], antimicrobial [14, 15], hypoglycemic [16, 17] and anti-inflammatory [18, 19]. Zarghi
et al [20] designed new derivatives of 2,3-diaryl-1,3-thiazolidin-4-ones which recorded COX-2 inhibitory
activity, in particular thiazolidino derivative 1 (Fig. 1) which was the most active derivative (IC50 = 0.12
µM), and selective COX-2 inhibitor (SI > 833). Moreover, this compound 1 was more COX-2 selective
than the standard drug celecoxib (SI > 403). In addition, (Z)-N-(3-chlorophenyl)-2-(4-((2,4-
dioxothiazolidin-5-ylidene)methyl)-2,6-dimethoxyphenoxy)acetamide (2) (Fig. 1) was detected to
suppress the iNOS activity (IC50 = 25.2 µM) and LPS-induced NO production (IC50 = 45.6 µM) in
RAW264.7 [21]. Furthermore, pyrimidine ring was recorded in literature as an important scaffold for anti-
inflammatory activity [22-24]. For example, compound 3 was prepared and evaluated for its anti-
inflammatory activity using carrageenan-induced paw oedema method in rats [25]. Results of this study
showed that this compound 3 exhibited percentage inhibition of oedema = 52.9% after 5 hours. Also,
3

7-amino-5-(3,4,5-trimethoxyphenyl)-4-oxo-2-thioxo-1,2,3,4-tetrahydropyrido[2,3-d]pyrimidine-6-
carbonitrile (4) (Fig. 1) demonstrated better COX-2 inhibitory activity in a range (IC50 = 0.25 µM) than
the standard drug celecoxib (IC50 = 1.11 µM) [26].
In the view of the aforesaid studies and in continuation of our previous researches on the design of anti-
inflammatory agents [26-29], we have carried out synthesis of thiazolo[4,5-d]pyrimidine ring system
having both pyrimidine ring and thiazolidine moiety in one hybrid structure attempting to obtain more
potent and COX-2 selective anti-inflammatory agents with fewer gastric side effects.
Insert Fig.1 here
2. Results and discussion
2.1. Chemistry
A set of thiazolo[4,5-d]pyrimidine derivatives (9a-l) were synthesized using the reaction sequence
illustrated in Scheme 1. Accordingly, stirring 4-aminoacetophenone, 3-aminoacetophenone and/or
benzocaine (5a-c) with chloroacetyl chloride in dimethylformamide at room temperature yielded the
corresponding chloroacetamide derivatives 6a-c in high yields (54–78%), which upon cyclization with
ammonium thiocyanate in ethanol provided thiazolidinone derivatives 7a-c in 60-82% yield.
Condensation of the latter 7a-c with different aldehydes in glacial acetic acid and sodium acetate
produced 5-arylidenethiazolidin-4-one derivatives 8a-l which upon refluxing with thiourea in pyridine
afforded the target compounds 9a–l in (54–78%) yield. The chemical structures of all the novel
synthesized compounds were proved by IR, 1H NMR,13C NMR, mass spectra and elemental analyses.
Insert Scheme.1 here
2.2. Pharmacological screening
2.2.1. In vitro Cyclooxygenase inhibition assay
The target derivatives 9a-l were screened for their COX inhibitory activity by determining IC50 (the
concentration causing 50% Inhibition) using an enzyme immunoassay (EIA) kit for ovine COX enzyme.
COX-2 selectivity indexes (SI values) were recorded using celecoxib as a standard drug (Table 1). The
4

results showed that the target compounds (9a-l) demonstrated a moderate to weak potency towards COX-
1 (IC50 = 4.21–10.87 µM), and moderate to high potency towards COX-2 (IC50 = 0.87–3.78 µM) (Table
1). The target compounds 9e (IC50 = 0.92 µM, S.I. = 10.05), 9g (IC50 = 0.87 µM, S. I. = 8.68) and 9k (IC50
= 1.02 µM, S.I. = 8.06) showed higher COX-2 inhibitory effect and higher COX-2 selectivity than
celecoxib (IC50 = 1.11 µM, S.I. = 6.61). Furthermore, the target compound 9h (IC50 = 1.21 µM, S.I. =
7.26) demonstrated comparable COX-2 inhibition activity with better selectivity to that shown by
celecoxib (IC50 = 1.11 µM, S.I. = 6.61). On the other side, 4-nitrophenylthiazolo[4,5-d]pyrimidine
derivative 9g was the most potent inhibitor of COX-2 (IC50 = 0.87 µM), while the fluoro derivative 9e
was most COX-2 selective (COX-2 S.I. = 10.05). Structure activity relationship studies revealed that
derivatives incorporating electron withdrawing groups at the para position of phenyl ring attached to
pyrimidine moiety (9a-c, 9e, 9g and 9i-k) exhibited higher COX-2 inhibition activity than the candidates
with electron donating groups (9d and 9I). Furthermore, the acetyl group at position 4 on the phenyl ring
of compound 9e, 9g-h afforded higher COX-2 inhibitory activity than at position 3 of the new candidates
9a-d. In addition, compounds 9e, 9g-h having acetyl group at C4 of phenyl group showed higher COX-2
inhibitory activity than derivatives incorporating ethyl ester moiety 9i-l.
Insert Table1 here
2.2.2. In vivo anti-inflammatory activity
The novel thiazolo[4,5-d]pyrimidines were evaluated for their anti-inflammatory activity (9a-l) by using
the model of formalin-induced rat paw oedema in male rats. Each target compound was given orally (50
mg/kg) immediately before stimulating inflammation by formalin subcutaneous injection. The anti-
inflammatory activity was recorded according to change in the volume of paw after 1h, 3h and 5h from
formalin injection (Table 2). The obtained data demonstrated that compounds 9c, 9f, 9g and 9k (55%,
45%, 57% and 52%, respectively) revealed better anti-inflammatory activity than the standard drug
celecoxib (43%) after 1h. 1-(4-[7-(4-Nitrophenyl)-5-thioxo-5,6-dihydro-3H-thiazolo[4,5-d]pyrimidin-2-
ylideneamino]phenyl)ethanone (9g) was the most active candidate showing 88% inhibition of
5

inflammation after 3 h and 5 h. In addition, the trimethoxyphenylthiazolo[4,5-d]pyrimidine benzoic acid
ethyl ester (9l) showed the lowest activity between all the tested derivatives. Structure activity
relationship studies demonstrated that compounds incorporating electron withdrawing groups at the para
position of phenyl ring attached to pyrimidine moiety (9e-g and 9k) exhibited better oedema inhibition
than the candidates with electron donating groups (9d, 9h and 9l). Moreover, the acetyl group at position
4 on the phenyl ring of compound 9e-h afforded higher inhibitory activity than at position 3 of the new
candidates 9a-d. Furthermore, replacing the acetyl group at C4 of phenyl group with ethyl ester moiety
markedly reduced the anti-inflammatory activity, this is clear in comparing compounds 9e-h with 9i-I.
Insert Table 2here
2.2.3. Ulcerogenic liability
The most potent prepared compounds 9e, 9g and 9k were tested for their gastric ulcerogenic action in
male albino rats (Table 3). Ulcerogenic results of the tested derivatives were compared with celecoxib and
indomethacin. Celecoxib was chosen as a low ulcerogenic reference drug, which was reported to be about
seven folds less ulcerogenic than ibuprofen as traditional NSAID [27, 30]. From the obtained data, it is
clear that all the tested targets showed less ulcerogenic effect than indomethacin. 1-(4-[7-(4-
Fluorophenyl)-5-thioxo-5,6-dihydro-3H-thiazolo[4,5-d]pyrimidin-2-ylideneamino]phenyl)ethanone (9e)
(Ulcer index = 5) recorded the lowest ulcerogenic effect due to its high COX-2 selectivity index (COX-2
S.I. = 10.05).
Insert Table 3 here
2.3. Molecular docking study
The most potent COX-2 inhibitors 9e, 9g and 9k had been docked within COX-2 enzyme binding site to
demonstrate the mode of action of these target compounds. The X-ray crystal structure of COX-2 was
obtained from the protein data bank with code (PDB: ID 1CX2) and MOE.2010 software (Molecular
Operating Environment) was used for performing this study.
6

The ligand of COX-2 bromocelecoxib (S-58) had been redocked within the active site with a score
energy (S) = -11.93 kcal/mol. Arg513 and His90 amino acids interacted with -SO2 group via two
hydrogen bonding interactions with in a distance equal to 2.41 and 2.30 Å (Table 4).
Compound 9e showed higher docking score (S) = -13.76 kcal/mol than revealed by bromocelecoxib (S) =
-11.93 kcal/mol, in addition, this target 9e exhibited five hydrogen bonding interactions; i) Thiazole N
with Tyr355 (3.12 A°), ii) Pyrimidine N with Tyr355 (2.34 A°), iii) Pyrimidine N with Arg120 (2.47 A°),
iv) C=O with Tyr385 (2.76 A°), and v) C=O with Ser530 (2.74) (Fig. 2) (Table 4).
Insert Fig.2 here
In addition, the candidate 9g revealed one hydrogen interaction between His90 and the acetyl C=O with
energy score S = -12.65 kcal/mol (Fig. 3) (Table 4).
Insert Fig.3 here
Compound 9k recorded docking score (-13.21 Kcal/mol) forming two hydrogen bonding interactions: i)
C=O with His90 (2.77 A°), and ii) CH=N with Ser 530 (2.31 A°) (Fig. 4).
Insert Fig.4 here
Insert Table 4 here
3. Conclusion
Novel derivatives of thiazolo[4,5-d]pyrimidines (9a-l) were synthesized and screened for their
Cyclooxygenase inhibitory effect, ulcerogenic and anti-inflammatory activities. The results of this study
showed that the thiazolo[4,5-d]pyrimidine having a nitrophenyl moiety in position 7 and p-acetylphenyl
ring in position 2 (9g) was the most active candidate with edema inhibitory percent = 88% after three and
five hours, while the derivative having a fluorophenyl moiety in position 7 (9e) was the least ulcerogenic
(S.I= 5). In addition, these novel targets 9a-l were noticed to be selective inhibitors to COX-2 than COX-
1, in particular compounds 9e, 9g and 9k revealed better COX-2 inhibitory activity in a range (IC50 =
0.87-1.02 µM) than the standard drug celecoxib (IC50 = 1.11 µM). The most active candidates inhibiting
7

COX-2 (9e, 9g and 9k) were subjected to molecular modeling study within COX-2 binding site. Docking
studies showed that the target compounds (9e, 9g and 9k) exhibited better score energy S = -12.65- -13.76
than the cocrystallized ligand (S) = -11.93 kcal/mol and they also good fitted with the active site of COX-
2 enzyme. So docking studies confirmed the in vitro COX-2 assay since 9e, 9g and 9k exhibited better
COX-2 inhibitory activity than the standard drug. Therefore, mixing the thiazole scaffold with pyrimidine
moiety in one hybrid structure yields a drug design concept for the development of NSAIDs that have a
good anti-inflammatory activity with low ulcerogenic side effect.
4. Experimental
4.1. Chemistry
Melting points had been measured on a Thomas-Hoover capillary apparatus and are uncorrected. Infrared
1
(IR) spectra were measured on NaCl plates using a Nicolet 550 Series II Magna FT-IR spectrometer. H
NMR and 13C NMR spectra were recorded on a Bruker III 400 MHz for 1H and 100 MHz for 13C (Bruker
AG, Switzerland) with BBFO Smart Probe and Bruker 400 AEON Nitrogen-Free Magnet, Faculty of
Pharmacy, Mansoura University, Egypt in DMSO-d6 with TMS as the internal standard, where J
(coupling constant) values are estimated in Hertz (Hz) and chemical shifts were recorded in ppm on δ
scale. Mass spectra (MS) were detected on Hewlett Packard 5988 spectrometer. Microanalyses for C, H
and N were performed on Perkin-Elmer 2400 analyzer (Perkin-Elmer, Norwalk, CT, USA) at the Micro
analytical unit of Cairo University, Egypt and all compounds were within + 0.4% of the theoretical
values. All other chemicals, purchased from the Aldrich Chemical Company (Milwaukee, WI), had been
used without further purification. 2-Chloro-N-(aryl)acetamides (6b,c) [31] 2-(arylimino)thiazolidin-4-one
derivatives (7b,c) [32] and 2-arylimino-5-arylidenethiazolidin-4-one derivatives (8e-l) [28, 32] had been
synthesized as literature procedures.
4.1.1. N-(3-Acetyl-phenyl)-2-chloroacetamide (6a).
A mixture of 3-aminoacetophenone (5a) (1.35 gm, 0.01 mol), chloroacetyl chloride (0.8 mL, 0.01 mol)
and anhydrous potassium carbonate (1.38 gm, 0.01 mol) in dry dimethylformamide was stirred at room
8

temperature for 24 h. The reaction mixture was poured into ice-cold water and the separated product was
filtered, dried and crystallized from benzene to give 6a (1.5 g, 71%) as a buff powder: mp 188-200°C; IR
1
(film) 3447 (NH), 3043 (CH aromatic), 2957 (CH aliphatic), 1706, 1672 (2CO) cm-1; H NMR (DMSO-
d6) δ 2.56 (s, 3H, CH3), 4.28 (s, 2H, CH2), 7.47-7.51 (m, 1H, phenyl H-5), 7.69 (d, J = 7.5 Hz, 1H,
phenyl H-4), 7.86 (d, J = 7.5 Hz, 1H, phenyl H-6), 8.17 (s, 1H, phenyl H-2), 10.51 (s, 1H, NH, D2O
exchangeable); 13C NMR (DMSO-d6) δ 27.14 (CH3), 43.98 (CH2), 119.01 (acetylphenyl C-2), 124.31
(acetylphenyl C-4), 124.34 (acetylphenyl C-6), 129.76 (acetylphenyl C-5), 137.70 (acetylphenyl C-3),
139.29 (acetylphenyl C-1), 165.44 (NHC=O), 198.00 (C=O); EIMS (m/z) 211 (M+., 100%). Anal. Calcd
for C10H10ClNO2: C, 56.75; H, 4.76; N, 6.62. Found: C, 56.66; H, 4.60; N, 6.50.
4.1.2. 2-(3-Acetyl-phenylimino)thiazolidin-4-one (7a).
A solution of N-(3-acetyl-phenyl)-2-chloroacetamide (6a) (2.11 gm, 0.01 mol) and ammonium
thiocyanate (1.14 gm, 0.15 mol) in ethanol (25 mL) was refluxed for 7 h. After cooling, the precipitated
product was filtered off, and then recrystallised from acetic acid to give 7a (1.4 gm, 60%) as yellow
powder: mp 215-216°C; IR (film) 3445 (NH), 3042 (CH aromatic), 2959 (CH aliphatic), 1705, 1670
(2CO) cm-1; 1H NMR (DMSO-d6) δ 2.55 (s, 3H, CH3), 4.00 (s, 2H, CH2), 7.42-7.46 (m, 1H, phenyl H-5),
7.75 (d, J = 7.5 Hz, 1H, phenyl H-4), 7.97 (d, J = 7.5 Hz, 1H, phenyl H-6), 8.24 (s, 1H, phenyl H-2),
11.61 (s, 1H, NH, D2O exchangeable); 13C NMR (DMSO-d6) δ 25.10 (CH3), 40.23 (CH2), 120.11
(acetylphenyl C-2), 126.36 (acetylphenyl C-6), 127.06 (acetylphenyl C-4), 129.75 (acetylphenyl C-5),
138.69 (acetylphenyl C-3), 148.25 (acetylphenyl C-1), 163.40 (N=CH), 173.21 (NHC=O), 196.10 (C=O);
EIMS (m/z) 234 (M+., 100%). Anal. Calcd for C11H10N2O2S: C, 56.40; H, 4.30; N, 11.96. Found: C,
56.38; H, 4.55; N, 12.00.
4.1.3. General procedure for synthesis of 2-arylimino-5-arylidenethiazolidin-4-one derivatives (8a–d)
A mixture of thiazolidin-4-one derivatives 7a-c (0.005 mol), the aromatic aldehyde (0.005 mol) and
sodium acetate (0.01 mol) in acetic acid (20 ml) was refluxed for 18 hr. after cooling, the reaction mixture
9

was poured into ice-cold water and the precipitated solid was filtered off and crystallized from
dimethylformamide to produce the target compounds 8a–l. Physical and spectral data are listed below.
4.1.3.1. 2-(3-Acetylphenylimino)-5-(4-fluorobenzylidene)thiazolidin-4-one (8a) Buff powder; Yield 62%;
mp 274–275◦C; IR (KBr) 3327 (NH), 3076 (CH aromatic), 2955 (CH aliphatic), 1763, 1675 (2CO) cm-1;
1H NMR (DMSO-d6) δ 2.56 (s, 3H, CH3), 7.52–7.57 (m, 4H, fluorophenyl H-3, H-5 and acetylphenyl H-
4, H-6), 7.61 (s, 1H, olefinic CH), 7.81–7.89 (m, 4H, fluorophenyl H-2, H-6 and acetylphenyl H-2, H-5),
13
11.87 (s, 1H, NH, D2O exchangeable); C NMR (DMSO-d6) δ 26.15 (CH3), 117.30 (fluorophenyl C-3,
C-5), 120.46 (acetylphenyl C-2), 122.58 (thiazolidine C-5), 126.22 (acetylphenyl C-6), 127.12
(acetylphenyl C-4), 127.15 (fluorophenyl C-2, C-6), 129.55 (acetylphenyl C-5), 130.12 (fluorophenyl C-
1), 138.72 (acetylphenyl C-3), 141.98 (benzylidene CH=), 148.95 (acetylphenyl C-1), 161.34
(fluorophenyl C-4), 163.21 (thiazolidine C-2), 168.10 (NHC=O), 197.11 (C=O); EIMS (m/z) 340 (M+.,
54%). Anal. Calcd for C18H13FN2O2S: C, 63.52; H, 3.85; N, 8.23. Found: C, 63.55; H, 3.81; N, 8.00.
4.1.3.2. 2-(3-Acetylphenylimino)-5-(4-chlorobenzylidene)thiazolidin-4-one (8b) Buff powder; Yield 65%;
◦
mp 292–293 C; IR (KBr) 3437 (NH), 3041 (CH aromatic), 2957 (CH aliphatic), 1761, 1677 (2CO) cm-1;
1H NMR (DMSO-d6) δ 2.55 (s, 3H, CH3), 7.10 (s, 1H, olefinic CH), 7.55–7.59 (m, 4H, chlorophenyl H-3,
H-5 and acetylphenyl H-4, H-6), 7.83-7.91 (m, 4H, chlorophenyl H-2, H-6 and acetylphenyl H-2, H-5),
13
12.10 (s, 1H, NH, D2O exchangeable); C NMR (DMSO-d6) δ 23.13 (CH3), 120.10 (acetylphenyl C-2),
122.18 (thiazolidine C-5), 126.23 (acetylphenyl C-6), 127.21 (acetylphenyl C-4), 127.56 (chlorophenyl C-
3, C-5), 128.82 (chlorophenyl C-2, C-6), 129.66 (acetylphenyl C-5), 132.65 (chlorophenyl C-4), 133.01
(chlorophenyl C-1), 138.54 (acetylphenyl C-3), 142.21 (benzylidene CH=), 148.21 (acetylphenyl C-1),
163.25 (thiazolidine C-2), 168.22 (NHC=O), 196.45 (C=O); EIMS (m/z) 356 (M+., 78%). Anal. Calcd for
C18H13ClN2O2S: C, 60.59; H, 3.67; N, 7.85. Found: C, 60.35; H, 3.50; N, 7.65.
4.1.3.3. 2-(3-Acetylphenylimino)-5-(4-nitrobenzylidene)thiazolidin-4-one (8c) Yellow powder; Yield
80%; mp 260–261◦C; IR (KBr) 3442 (NH), 3049 (CH aromatic), 2849 (CH aliphatic), 1789, 1680 (2CO)
10

1
cm-1; H NMR (DMSO-d6) δ 2.60 (s, 3H, CH3), 7.31 (s, 1H, olefinic CH), 7.54–7.68 (m, 4H, m, 4H,
nitrophenyl H-2, H-6 and acetylphenyl H-4, H-6), 7.82 (d, J = 8.2 Hz, 2H, acetylphenyl H-2, H-5), 8.10
(d, J = 8.2 Hz, 2H, nitrophenyl H-3, H-5), 12.06 (s, 1H, NH, D2O exchangeable); 13C NMR (DMSO-d6) δ
27.39 (CH3), 120.20 (acetylphenyl C-2), 122.16 (thiazolidine C-5), 126.41 (acetylphenyl C-6), 127.23
(acetylphenyl C-4), 127.46 (nitrophenyl C-3, C-5), 128.72 (nitrophenyl C-2, C-6), 129.79 (acetylphenyl
C-5), 132.64 (nitrophenyl C-4), 134.90 (nitrophenyl C-1), 138.51(acetylphenyl C-3), 142.00 (benzylidene
CH=), 148.93 (acetylphenyl C-1), 163.04 (thiazolidine C-2), 168.22 (NHC=O), 198.13 (C=O); EIMS
(m/z) 367 (M+., 100 %). Anal. Calcd for C18H13N3O4S: C, 58.85; H, 3.57; N, 11.44. Found: C, 58.50; H,
3.20; N, 11.49.
4.1.3.4. 2-(3-Acetylphenylimino)-5-(3,4,5-trimethoxybenzylidene)thiazolidin-4-one (8d) Yellow powder;
Yield 64%; mp >300◦C; IR (KBr) 3429 (NH), 3043 (CH aromatic), 2935 (CH aliphatic), 1715, 1671
(2CO) cm-1; 1H NMR (DMSO-d6) δ 2.57 (s, 3H, CH3), 3.74 (s, 3H, OCH3), 3.85 (s, 6H, 2OCH3), 6.81 (s,
1H, olefinic CH), 6.93 (s, 2H, trimethoxyphenyl H-2, H-6), 7.58–7.74 (m, 2H, and acetylphenyl H-4, H-
13
6), 8.02 (m, 2H, acetylphenyl H-2, H-5); 12.00 (s, 1H, NH, D2O exchangeable); C NMR (DMSO-d6) δ
27.24 (CH3), 56.33 (2OCH3), 60.69 (OCH3), 107.60 (trimethoxyphenyl C-2, C-6), 119.98 (acetylphenyl
C-2), 122.23 (thiazolidine C-5), 125.58 (acetylphenyl C-6), 127.84 (acetylphenyl C-4), 129.62
(trimethoxyphenyl C-1), 130.16 (acetylphenyl C-5), 131.61 (trimethoxyphenyl C-4), 138.07 (acetylphenyl
C-3), 142.85 (benzylidene CH=), 148.23 (acetylphenyl C-1), 153.64 (trimethoxyphenyl C-3, C-5),
163.02 (thiazolidine C-2), 168.92 (NHC=O), 196.08 (C=O); EIMS (m/z) 412 (M+. , 58%). Anal. Calcd
for C21H20N2O5S: C, 61.15; H, 4.89; N, 6.79.Found: C, 61.05; H, 4.65; N, 6.66.
4.1.4. General procedure for synthesis of 9a-l
A mixture of the appropriate 2-arylimino-5-arylidenethiazolidin-4-one derivatives (8a–l) (0.01 mol) and
thiourea (0.76 g, 0.01 mol) in the presence of few drops of piperidine was fused at 180 ºC for 5h. After
cooling, the product was poured onto ice and the solid was filtered off and recrystallized from dioxane to
afford compounds 9a-l. Physical and spectral data are listed below.
11

4.1.4.1.
1-{3-[7-(4-Fluorophenyl)-5-thioxo-5,6-dihydro-3H-thiazolo[4,5-d]pyrimidin-2-
ylideneamino]phenyl}ethanone (9a) Buff powder; Yield 60%; mp 297-298◦C; IR (KBr) 3429-3265
1
(2NH), 3053 (CH aromatic), 2922 (CH aliphatic), 1710 (CO) cm-1; H NMR (DMSO-d6) δ 2.56 (s, 3H,
CH3), 7.14–7.21 (m, 4H, fluorophenyl H-3, H-5 and acetylphenyl H-4, H-6), 7.30–7.41 (m, 4H,
fluorophenyl H-2, H-6 and acetylphenyl H-2, H-5), 11.57 (s, 1H, NH, D2O exchangeable), 12.26 (s, 1H,
NH, D2O exchangeable); 13C NMR (DMSO-d6) δ 22.85 (CH3), 84.56 (thiazolo[4,5-d]pyrimidin C-7a),
116.30 (fluorophenyl C-3, C-5), 122.32 (acetylphenyl C-2), 126.24 (acetylphenyl C-6), 127.02
(acetylphenyl C-4), 127.56 (fluorophenyl C-2, C-6), 129.72 (acetylphenyl C-5), 131.11 (fluorophenyl C-
1), 138.63 (acetylphenyl C-3), 148.95 (acetylphenyl C-1), 157.53 (thiazolo[4,5-d]pyrimidin C-7), 161.32
(fluorophenyl C-4), 163.92 (thiazolo[4,5-d]pyrimidin C-2), 164.23 (thiazolo[4,5-d]pyrimidin C-3a),
184.25 (C=S), 197.26 (C=O); EIMS (m/z) 396 (M+., 5.12%). Anal. Calcd for C19H13FN4OS2: C, 57.56;
H, 3.31; N, 14.13.Found: C, 57.50; H, 3.22; N, 14.00.
4.1.4.2.
1-{3-[7-(4-Chlorophenyl)-5-thioxo-5,6-dihydro-3H-thiazolo[4,5-d]pyrimidin-2-
ylideneamino]phenyl}ethanone (9b)
◦
White powder; Yield 54%; mp 288–289 C; IR (KBr) 3437-3211 (2NH), 3051 (CH aromatic), 2953 (CH
1
aliphatic), 1760 (2CO) cm-1; H NMR (DMSO-d6) δ 2.54 (s, 3H, CH3), 7.52–7.60 (m, 4H, chlorophenyl
H-3, H-5 and acetylphenyl H-4, H-6), 7.71-7.79 (m, 4H, chlorophenyl H-2, H-6 and acetylphenyl H-2, H-
5), 12.10 (s, 1H, NH, D2O exchangeable), 12.65 (s, 1H, NH, D2O exchangeable); 13C NMR (DMSO-d6) δ
22.92 (CH3), 85.01 (thiazolo[4,5-d]pyrimidin C-7a), 122.19 (acetylphenyl C-2), 126.33 (acetylphenyl C-
6), 127.12 (acetylphenyl C-4), 127.34 (chlorophenyl C-3, C-5), 128.7 (chlorophenyl C-2, C-6), 129.69
(acetylphenyl C-5), 132.65 (chlorophenyl C-4), 133.11 (chlorophenyl C-1), 138.62 (acetylphenyl C-3),
148.89 (acetylphenyl C-1), 157.36 (thiazolo[4,5-d]pyrimidin C-7), 163.15 (thiazolo[4,5-d]pyrimidin C-2),
164.52 (thiazolo[4,5-d]pyrimidin C-3a), 182.32 (C=S), 196.23 (C=O); EIMS (m/z) 412 (M+., 100%).
Anal. Calcd for C19H13ClN4OS2: C, 55.27; H, 3.17; N, 13.57. Found: C, 55.35; H, 3.20; N, 13.50.
12

4.1.4.3.
1-{3-[7-(4-Nitrophenyl)-5-thioxo-5,6-dihydro-3H-thiazolo[4,5-d]pyrimidin-2-
ylideneamino]phenyl}ethanone (9c)
◦
Yellow powder; Yield 72%; mp 295–296 C; IR (KBr) 3446-3232 (2NH), 3043 (CH aromatic), 2952 (CH
1
aliphatic), 1762 (CO) cm-1; H NMR (DMSO-d6) δ 2.61 (s, 3H, CH3), 7.63–7.81 (m, 4H, m, 4H,
nitrophenyl H-2, H-6 and acetylphenyl H-4, H-6), 7.84 (d, J = 8.2 Hz, 2H, acetylphenyl H-2, H-5), 8.16
(d, J = 8.2 Hz, 2H, nitrophenyl H-3, H-5), 11.93 (s, 1H, NH, D2O exchangeable), 12.11 (s, 1H, NH, D2O
exchangeable); 13C NMR (DMSO-d6) δ 22.89 (CH3), 85.23 (thiazolo[4,5-d]pyrimidin C-7a), 122.42
(acetylphenyl C-2), 123.51 (nitrophenyl C-3, C-5), 126.47 (acetylphenyl C-6), 127.16 (acetylphenyl C-4),
127.29 (nitrophenyl C-2, C-6), 129.73 (acetylphenyl C-5), 138.72 (acetylphenyl C-3), 141.53 (nitrophenyl
C-1), 147.61 (nitrophenyl C-4), 148.92 (acetylphenyl C-1), 157.01 (thiazolo[4,5-d]pyrimidin C-7),
163.22(thiazolo[4,5-d]pyrimidin C-2), 164.42 (thiazolo[4,5-d]pyrimidin C-3a), 182.09 (C=S), 196.15
(C=O); EIMS (m/z) 423 (M+., 4.07%). Anal. Calcd for C19H13N5O3S2: C, 53.89; H, 3.09; N, 16.54.
Found: C, 54.00; H, 3.20; N, 16.60.
4.1.4.4. 1-{3-(5-Thioxo-3-[7-(3,4,5-trimethoxyphenyl)-5,6-dihydro-3H-thiazolo[4,5-d]pyrimidin-2-
ylideneamino]phenyl}ethanone (9d)
◦
Yellow powder; Yield 78%; mp > 300 C; IR (KBr) 3422-3254 (2NH), 3061 (CH aromatic), 2931 (CH
1
aliphatic), 1760 (CO) cm-1; H NMR (DMSO-d6) δ 2.55 (s, 3H, CH3), 3.65 (s, 3H, OCH3), 3.69 (s, 6H,
2OCH3), 6.88 (s, 2H, trimethoxyphenyl H-2, H-6), 7.20–7.32 (m, 2H, and acetylphenyl H-4, H-6), 7.49-
7.61 (m, 2H, acetylphenyl H-2, H-5); 11.85 (s, 1H, NH, D2O exchangeable), 12.11 (s, 1H, NH, D2O
exchangeable); 13C NMR (DMSO-d6) δ 27.29 (CH3), 56.24 (2OCH3), 56.49 (OCH3), 83.85 (thiazolo[4,5-
d]pyrimidin C-7a), 106.58 (trimethoxyphenyl C-2, C-6), 122.13 (acetylphenyl C-2), 126.32 (acetylphenyl
C-6), 127.35 (acetylphenyl C-4), 129.01 (trimethoxyphenyl C-1), 129.63 (acetylphenyl C-5), 132.35
(trimethoxyphenyl C-4), 141.00 (acetylphenyl C-3), 146.96 (acetylphenyl C-1), 148.93 (thiazolo[4,5-
d]pyrimidin C-7), 153.26 (trimethoxyphenyl C-3, C-5), 163.29 (thiazolo[4,5-d]pyrimidin C-2), 164.52
13

(thiazolo[4,5-d]pyrimidin C-3a), 184.27 (C=S), 196.06 (C=O); EIMS (m/z) 468 (M+., 32.34%). Anal.
Calcd for C22H20N4O4S2: C, 56.40; H, 4.30; N, 11.96. Found: C, 56.55; H, 4.55; N, 12.00.
4.1.4.5.
1-{4-[7-(4-Fluorophenyl)-5-thioxo-5,6-dihydro-3H-thiazolo[4,5-d]pyrimidin-2-
ylideneamino]phenyl}ethanone (9e) Buff powder; Yield 58%; mp 291-292◦C; IR (KBr) 3411-3262
1
(2NH), 3051 (CH aromatic), 2932 (CH aliphatic), 1715 (CO) cm-1; H NMR (DMSO-d6) δ 2.54 (s, 3H,
CH3), 7.60 (d, J = 7.4 Hz, 2H, fluorophenyl H-3, H-5), 7.72–7.80 (m, 4H, fluorophenyl H-2, H-6 and
acetylphenyl H-2, H-6), 7.93 (d, J = 8.2 Hz, 2H, acetylphenyl H-3, H-5), 11.59 (s, 1H, NH, D2O
exchangeable), 12.13 (s, 1H, NH, D2O exchangeable); 13C NMR (DMSO-d6) δ 29.51 (CH3), 84.42
(thiazolo[4,5-d]pyrimidin C-7a), 115.70 (fluorophenyl C-2, C-6), 122.01 (acetylphenyl C-2, C-6), 126.39
(acetylphenyl C-3, C-5), 130.29 (fluorophenyl C-1), 130.68 (fluorophenyl C-3, C-5), 135.92
(acetylphenyl C-4), 153.21 (acetylphenyl C-1), 157.36 (thiazolo[4,5-d]pyrimidin C-7), 161.20
(fluorophenyl C-4), 163.87 (thiazolo[4,5-d]pyrimidin C-2), 168.17 (thiazolo[4,5-d]pyrimidin C-3a),
184.15 (C=S), 197.17 (C=O); EIMS (m/z) 396 (M+. , 8.98%). Anal. Calcd for C19H13FN4OS2: C, 57.56;
H, 3.31; N, 14.13.Found: C, 57.64; H, 3.53; N, 14.32.
4.1.4.6.
1-{4-[7-(4-Chlorophenyl)-5-thioxo-5,6-dihydro-3H-thiazolo[4,5-d]pyrimidin-2-
ylideneamino]phenyl}ethanone (9f)
◦
White powder; Yield 57%; mp 293–294 C; IR (KBr) 3435-3231 (2NH), 3048 (CH aromatic), 2951 (CH
1
aliphatic), 1759 (2CO) cm-1; H NMR (DMSO-d6) δ 2.54 (s, 3H, CH3), 7.48–7.55 (m, 4H, chlorophenyl
H-3, H-5 and acetylphenyl H-2, H-6), 7.89-7.95 (m, 4H, chlorophenyl H-2, H-6 and acetylphenyl H-3, H-
5), 12.10 (s, 1H, NH, D2O exchangeable), 12.65 (s, 1H, NH, D2O exchangeable); 13C NMR (DMSO-d6) δ
27.53 (CH3), 84.31 (thiazolo[4,5-d]pyrimidin C-7a), 115.67 (chlorophenyl C-3, C-5), 122.21
(acetylphenyl C-2, C-6), 126.37 (chlorophenyl C-2, C-6), 130.12 (chlorophenyl C-1), 130.59
(acetylphenyl C-4), 131.22 (chlorophenyl C-4), 135.86 (acetylphenyl C-3, C-5), 153.32 (acetylphenyl C-
1), 157.42 (thiazolo[4,5-d]pyrimidin C-7), 161.06 (thiazolo[4,5-d]pyrimidin C-2), 163.87 (thiazolo[4,5-
14

d]pyrimidin C-4a), 168.16 (C=O), 184.15 (C=S), 197.16 (C=O); EIMS (m/z) 412 (M+., 100%). Anal.
Calcd for C19H13ClN4OS2: C, 55.27; H, 3.17; N, 13.57. Found: C, 55.01; H, 3.32; N, 13.60.
4.1.4.7.
1-{4-[7-(4-Nitrophenyl)-5-thioxo-5,6-dihydro-3H-thiazolo[4,5-d]pyrimidin-2-
ylideneamino]phenyl}ethanone (9g)
◦
Yellow powder; Yield 68%; mp > 300 C; IR (KBr) 3449-3212 (2NH), 3065 (CH aromatic), 2949 (CH
aliphatic), 1757 (CO) cm-1; 1H NMR (DMSO-d6) δ 2.53 (s, 3H, CH3), 7.49–7.56 (m, 4H, nitrophenyl H-2,
H-6 and acetylphenyl H-2, H-6), 7.88-7.97 (m, 4H, nitophenyl H-3, H-5 and acetylphenyl H-3, H-5),
13
11.89 (s, 1H, NH, D2O exchangeable), 12.46 (s, 1H, NH, D2O exchangeable); C NMR (DMSO-d6) δ
26.84 (CH3), 85.32 (thiazolo[4,5-d]pyrimidin C-7a), 121.15 (phenyl C-2,C-6), 123.64 (nitrophenyl C-3,
C-5), 127.22 (phenyl C-4), 129.56 (nitrophenyl C-2, C-6), 130.88 (acetylphenyl C-3, C-5), 135.43
(nitrophenyl C-1), 149.35 (nitrophenyl C-4), 157.32 (acetylphenyl C-1), 162.52 (thiazolo[4,5-d]pyrimidin
C-7), 164.58 (thiazolo[4,5-d]pyrimidin C-3a), 168.12(thiazolo[4,5-d]pyrimidin C-2), 182.32 (C=S),
196.72 (C=O); EIMS (m/z) 423 (M+., 100%). Anal. Calcd for C19H13N5O3S2: C, 53.89; H, 3.09; N, 16.54.
Found: C, 53.50; H, 3.25; N, 16.50.
4.1.4.8.
1-{5-Thioxo-4-[7-(3,4,5-trimethoxyphenyl)-5,6-dihydro-3H-thiazolo[4,5-d]pyrimidin-2-
ylideneamino]phenyl}ethanone (9h)
◦
Yellow powder; Yield 69%; mp > 300 C; IR (KBr) 3432-3276 (2NH), 3053 (CH aromatic), 2930 (CH
1
aliphatic), 1758 (CO) cm-1; H NMR (DMSO-d6) δ 2.56 (s, 3H, CH3), 3.71 (s, 3H, OCH3), 3.73 (s, 6H,
2OCH3), 6.50 (s, 2H, trimethoxyphenyl H-2, H-6), 7.52 (d, J = 8.4 Hz, 2H, and acetylphenyl H-2, H-6),
7.74 (d, J = 8.4 Hz, 2H, acetylphenyl H-3, H-5); 11.54 (s, 1H, NH, D2O exchangeable), 12.45 (s, 1H, NH,
D2O exchangeable); 13C NMR (DMSO-d6) δ 21.53 (CH3), 56.26 (OCH3), 56.49 (OCH3), 84.5
(thiazolo[4,5-d]pyrimidin C-7a), 105.99 (trimethoxyphenyl C-2, C-6), 121.85 (acetylphenyl C-2, C-6),
129.23 (trimethoxyphenyl C-1), 130.24 (acetylphenyl C-4), 132.22 (trimethoxyphenyl C-4),
135.62(acetylphenyl C-3, C-5), 148.83 (thiazolo[4,5-d]pyrimidin C-7), 153.67 (trimethoxyphenyl C-3, C-
5), 157.22 (acetylphenyl C-1), 163.52 (thiazolo[4,5-d]pyrimidin C-2), 164.21(thiazolo[4,5-d]pyrimidin C-
15

3a), 182.26 (C=S), 196.43 (C=O); EIMS (m/z) 468 (M+., 19.88%). Anal. Calcd for C22H20N4O4S2: C,
56.40; H, 4.30; N, 11.96. Found: C, 56.35; H, 4.15; N, 11.82.
4.1.4.9.
Ethyl
4-[7-(4-fluorophenyl)-5-thioxo-5,6-dihydro-3H-thiazolo[4,5-d]pyrimidin-2-
ylideneamino]benzoate (9i) white powder; Yield 61%; mp 283-284◦C; IR (KBr) 3410-3259 (2NH), 3051
1
(CH aromatic), 2932 (CH aliphatic), 1719 (CO) cm-1; H NMR (DMSO-d6) δ 1.31 (t, J = 7.2 Hz, 3H,
CH3), 4.25 (q, J = 7.2 Hz, 2H, CH2), 7.13 (d, J = 7.8 Hz, 2H, fluorophenyl H-3, H-5), 7.82–7.98 (m, 4H,
fluorophenyl H-2, H-6 and benzoyl H-2, H-6), 8.03 (d, J = 8.6 Hz, 2H, benzoyl H-3, H-5), 11.93 (s, 1H,
NH, D2O exchangeable), 12.39 (s,1H, NH, D2O exchangeable); 13C NMR (DMSO-d6) δ 19.06 (CH3),
59.1 (CH2), 84 (thiazolo[4,5-d]pyrimidin C-7a), 115.6 (fluorophenyl C-2, C-6), 121.93 (benzoyl C-2, C-
6), 127.53 (fluorophenyl C-1), 129.21(fluorophenyl C-3, C-5), 130.61 (benzoyl C-4), 131.23 (benzoyl C-
3, C-5), 153.61(benzoyl C-1), 157.21 (thiazolo[4,5-d]pyrimidin C-7), 161.22 (fluorophenyl C-4), 163.25
(thiazolo[4,5-d]pyrimidin C-2), 164.32 (thiazolo[4,5-d]pyrimidin C-3a), 167.32 (C=O), 182.21 (C=S);
EIMS (m/z) 426 (M+., 70.91%). Anal. Calcd for C20H15FN4O2S2: C, 56.32; H, 3.55; N, 13.14. Found: C,
56.50; H, 3.60; N, 13.25.
4.1.4.10. Ethyl 4-[7-(4-chlorophenyl)-5-thioxo-5,6-dihydro-3H-thiazolo[4,5-d]pyrimidin-2-
ylideneamino]benzoate (9j)
White powder; Yield 59%; mp > 300◦C; IR (KBr) 3445-3265 (2NH), 3098 (CH aromatic), 2926 (CH
aliphatic), 1713 (CO) cm-1; 1H NMR (DMSO-d6) δ 1.31 (t, J = 7.2 Hz, 3H, CH3), 4.32 (q, J = 7.2 Hz, 2H,
CH2), 7.52–7.61 (m, 4H, chlorophenyl H-3, H-5 and benzoyl H-2, H-6), 7.65–7.98 (m, 4H, chlorophenyl
H-2, H-6 and benzoyl H-3, H-5), 11.71 (s, 1H, NH, D2O exchangeable), 12.30 (s, 1H, NH, D2O
exchangeable); 13C NMR (DMSO-d6) δ 13.51 (CH3), 59.25 (CH2), 84.62 (thiazolo[4,5-d]pyrimidin C-7a),
121.32 (benzoyl C-2, C-6), 127.22 (chlorophenyl C-2, C-6), 128.32 (chlorophenyl C-3, C-5), 129.65
(benzoyl C-4), 131.62 (benzoyl C-3, C-5), 133.21 (chlorophenyl C-1), 133.64 (chlorophenyl C-4), 153.26
(benzoyl C-1), 157.23 (thiazolo[4,5-d]pyrimidin C-7), 163.82 (thiazolo[4,5-d]pyrimidin C-2), 167.53
16

(thiazolo[4,5-d]pyrimidin C-3a), 164.35 (C=O), 182.41 (C=S); EIMS (m/z) 442 (M+., 15%). Anal. Calcd
for C20H15ClN4O2S2: C, 54.23; H, 3.41; N, 12.65. Found: C, 54.50; H, 3.62; N, 12.56.
4.1.4.11.
Ethyl
4-[7-(4-nitrophenyl)-5-thioxo-5,6-dihydro-3H-thiazolo[4,5-d]pyrimidin-2-
ylideneamino]benzoate (9k)
Yellow powder; Yield 69%; mp >300◦ C; IR (KBr) 3432 (2NH), 3093 (CH aromatic), 2972 (CH
aliphatic), 1721 (CO) cm-1; 1H NMR (DMSO-d6) δ 1.34 (t, J = 7.6 Hz, 3H, CH3), 4.33 (q, J = 7.6 Hz, 2H,
CH2), 7.73–7.80 (m, 4H, nitrophenyl H-2, H-6 and benzoyl H-2, H-6), 7.68 (d, J = 8.2 Hz, 2H, benzoyl
H-3, H-5), 8.27 (d, J = 8.2 Hz, 2H, nitrophenyl H-3, H-5), 11.36 (s, 1H, NH, D2O exchangeable), 12.38
13
(s, 1H, NH, D2O exchangeable); C NMR (DMSO-d6) δ 14.50 (CH3), 61.16 (CH2), 84.35 (thiazolo[4,5-
d]pyrimidin C-7a), 120.97 (benzoyl C-2,C-6), 121.98 (nitrophenyl C-3, C-5), 126.67 (benzoyl C-4),
129.25 (nitrophenyl C-2, C-6), 131.36 (benzoyl C-3, C-5), 141.35 (nitrophenyl C-1), 153.61 (nitrophenyl
C-4), 157.25 (benzoyl C-1), 163.29 (thiazolo[4,5-d]pyrimidin C-7), 164.01 (thiazolo[4,5-d]pyrimidin C-
3a), 165.12 (thiazolo[4,5-d]pyrimidin C-2), 165.63 (C=O), 182.31 (C=S); EIMS (m/z) 453 (M+., 100%).
Anal. Calcd for C20H15N5O4S2: C, 52.97; H, 3.33; N, 15.44. Found: C, 52.65; H, 3.62; N, 15.22.
4.1.4.12. Ethyl 4-[5-thioxo-7-(3,4,5-trimethoxyphenyl)-5,6-dihydro-3H-thiazolo[4,5-d]pyrimidin-2-
ylideneamino]benzoate (9l)
Yellow powder; Yield 69%; mp >300◦ C; IR (KBr) 3446 (2NH), 3071 (CH aromatic), 2932 (CH
aliphatic), 1725 (CO) cm-1; 1H NMR (DMSO-d6) δ 1.32 (t, J = 7.2 Hz, 3H, CH3), 4.30 (q, J = 7.2 Hz, 2H,
CH2), 3.75 (s, 3H, OCH3), 3.83 (s, 6H, 2OCH3), 6.81 (s, 2H, trimethoxyphenyl H-2, H-6), 7.93 (d, J = 8.6
Hz, 2H, benzoyl H-3, H-5), 8.11 (d, J = 8.6 Hz, 2H, benzoyl H-2, H-6), 11.35 (s, 1H, NH, D2O
exchangeable), 12.21 (s, 1H, NH, D2O exchangeable); 13C NMR (DMSO-d6) δ 14.66 (CH3), 56.51 (CH2),
60.67 (OCH3), 61.16 (OCH3), 84.32 (thiazolo[4,5-d]pyrimidin C-7a), 106.85 (trimethoxyphenyl C-2, C-
6), 121.63 (benzoyl C-2, C-6), 123.25 (benzoyl C-4), 129.30 (trimethoxyphenyl C-1), 131.01 (benzoyl C-
3, C-5), 132.12 (trimethoxyphenyl C-4), 148.83 (thiazolo[4,5-d]pyrimidin C-7), 153.67
(trimethoxyphenyl C-3, C-5), 157.64 (benzoyl C-1), 162.91 (thiazolo[4,5-d]pyrimidin C-2), 164.32
17

(thiazolo[4,5-d]pyrimidin C-3a), 166.65 (C=O), 179.34 (C=S); EIMS (m/z) 498 (M+., 15.32%). Anal.
Calcd for C23H22N4O5S2: C, 55.41; H, 4.45; N, 11.24. Found: C, 55.50; H, 4.30; N, 11.31.
4.2. Pharmacological studies
4.2.1. In vitro COX-1/COX-2 inhibition assay:
The inhibition of ovine COX-1/COX-2 was measured using an enzyme immuno assay (EIA) kit as a
reported procedure (27). Various concentrations of the tested compounds and positive control (celecoxib)
were incubated with the enzymes for a period of 5 min at 25 ◦C. After the incubation period and the
addition of colorimetric substrate and arachidonic acid, absorbance was measured at 590 nm using plate
reader.
4.2.2. Formalin induced rat paw edema
The newly prepared target compounds 9a–l were evaluated for their in vivo anti-inflammatory activity by
the use of formalin-induced paw edema method in male rats (32). Wister albino male rats were divided
into fourteen groups of four animals each. The first group was given a vehicle 0.5 %
carboxymethylcellulose (CMC) and considered as a control. Celecoxib was administered orally to the
second group as a reference standard in (50 mg/kg) dose. The rest of the groups were given orally the
tested target compounds 9a–l in a dose of (50 mg/kg). Thickness of the left hind paw of each rat was
measured in millimeters, at the beginning of the experiment. Induction of paw edema was performed by
subcutaneous injection of formalin 2.5% (0.05 ml) into the right hind paw of each rat, one hour after
administration of vehicle, test compounds or celecoxib. Paw thickness of each rat was measured after 1, 3
and 5 h of formalin injection using plethysmometer. Then the change in thickness and % inhibition of
paw edema were calculated.
4.2.3. Ulcerogenic liability study
18

The most potent COX-2 inhibitors (9e, 9g and 9k) as well as celecoxib were tested for their ulcerogenic
liability using the previously reported method [27]. The ulcerogenic effects of compounds 9e, 9g, 9k,
celecoxib, and indomethacin were evaluated. Twenty four rats were used in this study, divided into 6
groups and fasted for 18 h before drug administration. The control group received the vehicle, while other
groups received test compounds, celecoxib or indomethacin at a dose of 50 mg/kg, then animals were fed
after 2 h. Rats were given the specified dose orally for three successive days. Rats were sacrificed after 2
h of the last dose, then the stomach of each rat was removed and opened along the greater curvature for
determination of the ulcer number and ulcer index.
4.2.4. Molecular docking
The crystal structures of bromocelecoxib bound at COX-2 isoform (Protein Data Bank; PDB: ID 1CX2)
[28]. Docking was performed using London dG force and sophistication of the results was done using
force field energy. Preparation of the synthesized compounds for docking was attained via their 3D
structure built by Molecular Operating Environment (MOE, Version 2005.06, Chemical Computing
Group Inc., QC, Canada). Definite procedures were in use before docking which include: 3D protonation
of the structures, running conformational analysis using systemic search, selecting the least energetic
conformer and applying the same docking protocol used with ligands. Docking for the synthesized
compounds was applied. Amino acid interactions and the hydrogen bond lengths were summarized in
(Table 4).
4.2.5. Statistical analysis
The presented data are mean ± SD, and the statistical analysis was performed using one way ANOVA
followed by Dunnett multiple comparisons test. Differences were considered significant at p < 0.05.
Statistical analysis was performed using SPSS for Windows (SPSS, Inc., Chicago, IL).
Acknowledgement
The authors appreciate Jouf University, KSA, for funding this work through a research grant (Project no.
39/222).
19

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Figure, schemes and table captions
Fig. 1: Chemical Structures of some reported thiazolidine derivatives (1, 2) and pyrimidine derivatives (3,
4) with anti-inflammatory activity.
Fig.2: Binding of the 9e inside COX-2. (A) 2D of the binding mode of 9e inside the active site of COX
resulting from docking, the most important amino acids are shown It forms five H bonds with Tyr355,
Arg120, Tyr385 and Ser530; (B) 3D binding of 9e.
23

Fig.3: Binding of the 9g inside COX-2. (A) 2D of the binding mode of 9g inside the active site of COX
resulting from docking, the most important amino acids are shown it forms one H bonds with His90 (B)
3D binding of 9g.
Fig.4: Binding of the 9k inside COX-2. (A) 2D of the binding mode of 9k inside the active site of COX
resulting from docking, the most important amino acids are shown It forms two H bonds with His90, and
Ser530; (B) 3D binding of 9k.
Scheme 1. Synthesis of the compounds 9a-l. Reagents and conditions: a) ClCH2COCl, DMF, K2CO3,
stirring, 24h; b) NH4SCN, C2H5OH, reflux, 7h; c) ArCHO, CH3COOH, CH3COONa, 18h; d) NH2CSNH2,
C6H5N, 180 ºC, 5h.
Table 1. In vitro COX-1 and COX-2 inhibition of the novel candidates 9a-l and celecoxib.
Table 2. Results of the anti-inflammatory activity of control, celecoxib and the novel targets 9a-l.
Table 3. Ulcerogenic effect of the targets 9e, 9g, 9k, indomethacin and celecoxib.
Table 4: Molecular modeling data for compounds 9e, 9g, 9k and S-58 during docking in the active site of
COX-2 enzyme (PDB:ID 1CX2).
24

H3CO2S
O
S
MeO
O
NH
H
N
S
Cl
N
O
O
O
OMe
(2)
F
(1)
N
NH2
OMe
OMe
MeO
N
O
CN
HN
NO2
(3)
S
N
H
N
NH2
(4)
H
N
N
S
N
NH
S
Ar
(9a-l)
R
25

(A)
(B)
26

(A)
(B)
(A)
(B)
27

O
NH2
O
Cl
HN
a
b
NH
N
S
R
R
R
(6a-c)
(7a-c)
(5a-c)
5a, R = 3-COCH3
5b, R = 4-COCH3
5c, R = 4-COOC2H5
c
O
Ar
H
N
N
S
HN
N
N
S
d
NH
S
R
Ar
(8a-l)
(9a-l)
R
R
Ar
R
Ar
9h 4-COCH3
9i 4-COOC2H5
3,4,5-(OCH3)3-C6H2
4-F-C6H4
4-Cl-C6H4
4-(NO2)-C6H4
3,4,5-(OCH3)3-C6H2
9a
9b
9c
9d
9e
9f
3-COCH3
3-COCH3
3-COCH3
3-COCH3
4-COCH3
4-COCH3
4-COCH3
4-F-C6H4
4-Cl-C6H4
4-NO2-C6H4
3,4,5-(OCH3)3-C6H2
4-F-C6H4
4-Cl-C6H4
9j
9k 4-COOC2H5
9l 4-COOC2H5
4-COOC2H5
9g
4-NO2-C6H4
Scheme 1. Synthesis of the compounds 9a-l. Reagents and conditions: a) ClCH2COCl, DMF, K2CO3,
stirring, 24h; b) NH4SCN, C2H5OH, reflux, 7h; c) ArCHO, CH3COOH, CH3COONa, 18h; d) NH2CSNH2,
C6H5N, 180 ºC, 5h.
28

IC50 (µM)a
COX-1 (µm IC50)
Compound Number
COX-2 S.I.
COX-2 (µm IC50)
9a
4.21
4.65
6.32
4.21
9.25
10.87
7.55
8.78
5.25
7.21
8.23
8.52
7.34
1.45
1.65
1.28
2.52
0.92
3.78
0.87
1.21
1.83
2.03
1.02
2.13
1.11
2.90
2.81
4.94
1.67
10.05
2.88
8.68
7.26
2.87
3.55
8.06
4.00
6.61
9b
9c
9d
9e
9f
9g
9h
9i
9j
9k
9l
Celecoxib
aIC50 represents the compound concentration that causes 50% suppression of COX-1 or COX-2.
bSelectivity index (COX-1 IC50/COX-2 IC50).
29