Synthesis, in vivo anti-inflammatory, COX-1/COX-2 and 5-LOX inhibitory activities of new 2,3,4-trisubstituted thiophene derivatives

Article abstract of DOI:10.1016/j.bioorg.2020.103890

New series of thiophene derivatives were synthesized and evaluated for their in vivo anti-inflammatory activity using carrageenan-induced paw edema model. The most active in vivo anti-inflammatory compounds 5b, 11b, 14c, 18c, 19c and 20d were further evaluated for their in vitro COX-1/COX-2 and 5-LOX inhibitory activities. The in vitro assay results revealed that the N-(4-(4-chlorophenyl)-3-cyanothiophen-2-yl)-2-morpholinoacetamide (5b) possesses the highest selectivity toward COX-2 (IC₅₀ = 5.45 μM) with selectivity index value of 8.37 compared to celecoxib with COX-2 selectivity index value of 15.44. In addition, it showed acceptable 5-LOX inhibitory activity (IC₅₀ = 4.33 μM) compared to NDGA (IC₅₀ = 2.46 μM). Molecular modeling study was conducted to study the postulated binding of compound 5b into the active site of COX-2 and 5-LOX, and it revealed that 5b binds similarly to celecoxib and NDGA, respectively. Overall, the morpholinoacetamide-thiophene hybrid 5b could serve as a promising lead for further development of new potent anti-inflammatory agents that act as dual COX-2/5-LOX inhibitors.

Full text of DOI:10.1016/j.bioorg.2020.103890

Journal Pre-proofs
Synthesis, in vivo anti-inflammatory, COX-1/COX-2 and 5-LOX inhibitory
activities of new 2,3,4-trisubstituted thiophenes
Nermeen A. Qandeel, Ashraf K. El-Damasy, Maha H. Sharawy, Said M.
Bayomi, Nadia S. El-Gohary
PII:
S0045-2068(20)30389-8
DOI:
https://doi.org/10.1016/j.bioorg.2020.103890
YBIOO 103890
Reference:
To appear in:
Bioorganic Chemistry
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Revised Date:
Accepted Date:
18 February 2020
14 April 2020
25 April 2020
Please cite this article as: N.A. Qandeel, A.K. El-Damasy, M.H. Sharawy, S.M. Bayomi, N.S. El-Gohary,
Synthesis, in vivo anti-inflammatory, COX-1/COX-2 and 5-LOX inhibitory activities of new 2,3,4-trisubstituted
thiophenes, Bioorganic Chemistry (2020), doi: https://doi.org/10.1016/j.bioorg.2020.103890
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Synthesis, in vivo anti-inflammatory, COX-1/COX-2 and 5-LOX
inhibitory activities of new 2,3,4-trisubstituted thiophenes
Nermeen A. Qandeela, Ashraf K. El-Damasya,*, Maha H. Sharawyb, Said M.
Bayomia, Nadia S. El-Goharya,*
aDepartment of Medicinal Chemistry, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt
bDepartment of Pharmacology and Toxicology, Faculty of Pharmacy, Mansoura University, Mansoura,
35516, Egypt

Correspondence:
Ashraf K. El-Damasy, Department of Medicinal Chemistry, Faculty of Pharmacy, Mansoura University,
Mansoura 35516, Egypt, email: ph_karem2000@mans.edu.eg
Nadia S. El-Gohary, Department of Medicinal Chemistry, Faculty of Pharmacy, Mansoura University,
Mansoura 35516, Egypt, email: dr.nadiaelgohary@yahoo.com
Abstract: New series of thiophene derivatives (36 compounds) were synthesized and evaluated
for their in vivo anti-inflammatory activity using carrageenan-induced paw edema model. The
most active compounds 5b, 11b, 14c, 18c, 19c and 20d were further evaluated for their dual
COX-2 and 5-LOX inhibitory activities. The in vitro and in vivo assay results revealed that N-
(4-(4-chlorophenyl)-3-cyanothiophen-2-yl)-2-morpholinoacetamide (5b) possess the highest
selectivity towards COX-2 (IC50 = 5.45 μM) with selectivity index value of 8.37 being
comparable to celecoxib. In addition, 5b showed reasonable in vivo anti-inflammatory activity
compared to celecoxib. Moreover, it showed acceptable 5-LOX inhibitory activity (IC50 = 4.33
μM) compared to NDGA (IC50 = 2.46 μM). Molecular modeling study was conducted to study
the postulated binding of compound 5b into the active site of COX-2 and 5-LOX revealing that
it binds similarly to celecoxib and NDGA, respectively. Overall, the morpholinoacetamide
thiophene 5b could serve as a lead compound for further development of new potent dual COX-
2/5-LOX inhibitors as anti-inflammatory agents.
Keywords: Thiophenes, Anti-inflammatory, COX-1/COX-2, 5-LOX, Molecular modeling
- 1 -

1. Introduction
Inflammation is one of body’s defense mechanisms; it helps to protect the body against foreign
bodies, injuries and microbial infection. Inflammation is mediated by different inflammatory
mediators (tumor necrosis factor TNF-α and interleukins IL-1) and proinflammatory cytokines. It
may be acute or chronic, if a local acute inflammation is left uncontrolled; it may lead to chronic or
systemic serious inflammation disorders [1-3].
Arachidonic acid is one of main components of cell’s membrane phospholipids that is produced by
the action of phospholipase A2 on phospholipids. It acts as a normal substrate for different enzymes
such as: lipoxygenase enzyme (LOX) and cyclooxygenase enzyme (COX). LOX enzyme
metabolizes arachidonic acid producing leukotrienes, which are mediators in asthma and
inflammation. On the other hand, COX enzyme has three isoforms; COX-1 which is a constitutive
isoform of COX that is secreted normally in different sites such as gastric mucosa, platelets, uterine
epithelium and kidney [4-7]. COX-2 is the inducible isoform responsible for the release of
prostaglandins (prostaglandin E2 and prostaglandin b), prostacyclines and thromboxane, which are
responsible for induction of inflammation [8]. The third discovered isoform is COX-3 that is secreted
in the brain and inhibited by paracetamol [9,10].
It was found that inhibition of COX-1 by the non-selective non-steroidal anti-inflammatory drugs
(NSAIDs) like aspirin and ibuprofen promoted the appearance of gastrointestinal side effects,
including gastric ulceration and bleeding that highlighted the significance of the synthesis of selective
COX-2 inhibitors like celecoxib and its analogues [5,6]. However, prolonged use of selective COX-
2 inhibitors shifted the metabolism of arachidonic acid pathway toward LOX enzyme leading to
elevated levels of leukotrienes that exaggerates bronchoconstriction and increases airway
inflammation [11,12]. In this regard, extensive efforts were devoted for the identification of dual
COX-2 / 5-LOX inhibitors [13-16] that might be advantageous for the management of inflammation.
The quinoline bearing piperazine (A) is an example of dual COX-2 / LOX inhibitor with favorable
selectivity index (COX-2, IC50 = 1.13 µM, LOX, IC50 = 5.91 µM) with comparable anti-inflammatory
activity to celecoxib (COX-2, IC50 = 0.88 µM) and meclofenamate sodium (LOX, IC50 = 5.64 µM)
[17]. In addition, the substituted 1,3-dioxoisoindoline (B) [5] exerted a potent and selective COX-2
inhibitory activity. On the other hand, thiophene is one of the privileged scaffolds used for the
development of a number of potent anti-inflammatory agents [18-23]. Zileuton (C) is a
benzothiophene derivative with selective LOX inhibitory activity used in treatment of chronic asthma
- 2 -

[11]. Additionally, tetrasubstituted thiophene derivative (D) is a dual COX / LOX inhibitor reported
for its anti-inflammatory activity [24]. Apart from LOX inhibitors, compound (E) is another
thiophene derivative bearing the morpholinoacetamide moiety, was reported by Helal et al. [25] as a
potent anti-inflammatory agent surpassing the well-known drug indomethacin.
In order to expand the structural diversity of dual COX-2 / 5-LOX inhibition, a diverse set of
thiophene conjugates were designed adopting pharmacophore hybridization approach. Molecular
hybridization is a promising strategy widely used in medicinal chemistry for design of new active
compounds. It depends on combining two active chemical entities with different pharmacophores in
a single molecule which may lead to enhanced activities [26,27]. In our study, the substantial
thiophene core structure was conserved, and chloroacetamide and morpholino/(4-substituted
piperazin-1-yl)acetamide were incorporated to afford the new hybrids 4a,b and 5a,b-9a,b,
respectively. Moreover, a series of thiophene conjugates with the bicyclic quinazoline as isostere to
quinoline ring pharmacophore 10a,b and 11a,b or 1,3-dioxoisoindoline 13a,b were synthesized to
explore the impact of such structural modifications on the anti-inflammatory activity (Figure 1).
Furthermore, our chemical diversity was extended to include the combination of thiophene and 4-
oxobutanoic acid 12a,b, benzamides 14c,d and 15c,d, thiophene-2-carboxamides 16c,d or aliphatic
amides (acetamide 17c,d, propionamide 18c,d and butyramide 19c,d) (Figure 1). On the other hand,
benzenesulfonamide was reported as a substantial pharmacophore in various selective COX-2
inhibitors like celecoxib (F) [28]. From this perspective, we were motivated to design a new array of
thiophene
derivatives
containing
dibenzenesulfonamide
20c,d
and
21c,
and
monobenzenesulfonamide 22c,d and 23c (Figure 2). The designed new series of compounds were
synthesized and examined for their in vivo anti-inflammatory activity and in vitro COX-1/COX-2
and 5-LOX inhibitory activities. Detailed study of structure-activity correlation would pave the road
for synthesis and development of new more active members.
2. Results and discussion
2.1. Chemistry
The key starting materials, 2-amino-4-(4-substituted phenyl)thiophene-3-carbonitriles 3a-d for the
synthesis of the target thiophene derivatives have been prepared as illusrtated in Scheme 1. Treatment
of 2-(1-(4-substituted phenyl)ethylidene)malononitriles 2a-d [29-31] with elemental sulfur in
- 3 -

refluxing ethanol in the presence of diethylamine (DEA) produced the key building blocks, 2-
aminothiophene-3-carbonitriles 3a-d [29-31]. Referring to Scheme 2, the chloroaceatamide
derivatives 4a,b were prepared by stirring ortho aminonitriles 3a,b with chloroacetyl chloride in
chloroform in the presence of a catalytic amount of triethylamine (TEA). Nucleophilic substitution
of chloroacetamides 4a,b with morpholine or substituted piperazines in refluxing acetone in the
presence of TEA furnished morpholino-acetamides 5a,b and piperazinyl-acetamides 6a,b-9a,b,
respectively. On the other hand, the synthesis of quinazolinylthioacetamides 10a,b and 11a,b was
accomplished through reaction of chloroacetamide derivatives 4a,b with substituted 2-
mercaptoquinazolin-4-ones [32] in TEA/acetone under reflux condition. Fusion of ortho
aminonitriles 3a,b with succinic anhydride or phthalic anhydride yielded the 4-oxobutanoic acid
derivatives 12a,b and 2-(1,3-dioxoisoindolin-2-yl)thiophene-3-carbonitriles 13a,b, respectively.
With regard to Scheme 3, stirring ortho aminonitriles 3c,d with various aromatic acid chlorides in
pyridine gave the aromatic amides 14c,d-16c,d, whereas the aliphatic amides 17c,d-19c,d were
prepared through stirring ortho aminonitriles 3c,d with different aliphatic acid chlorides in
chloroform using a catalytic amount of TEA. Interestingly, applying the same conditions while
treating ortho aminonitriles 3c,d with 4-substituted benzenesulfonyl chloride afforded
disulfonamides 20c,d and 21c. Further, dissolution of the benzenesulfonamide derivatives 20c,d and
21c in methanol followed by addition of 4N NaOH in the presence of tetrahydrofuran (THF) as a
cosolvent yielded the monosulfonamide derivatives 22c,d and 23c.
2.2. Biological evaluation
2.2.1. In vivo anti-inflammatory screening
The evaluation of anti-inflammatory activity for all of the newly synthesized compounds was
carried out to determine the percentage inhibition (%I) of edema using in vivo rat carrageenan-
induced paw edema model [33]. Celecoxib was used as a reference drug in the positive control group.
The %I values were measured after 2h and 4h, and the results are listed in Table 1. The obtained data
revealed that compound 20d exhibited the highest %I of paw edema after 2h (66.52%) which is
comparable to that of celecoxib (66.19%). Additionally, compounds 18c and 19c exhibited high %I
of edema after 2h (63.99 and 62.66%, respectively), whereas compounds 4a, 5a, 6a, 8b, 9a, 10a,b,
11b, 13a,b, 14c,d, 15c,d, 16d, 17d and 21c showed moderate %I of edema after 2h (22.18-46.97%).
- 4 -

Furthermore, 4b, 5b, 6b, 7a,b, 9b, 11a, 17c, 19d, 20c and 23c displayed weak %I of edema after 2h
(2.23-18.48%). On contrary, compounds 8a, 12a,b, 16c, 18d and 22c,d augmented the inflammation
in rats with negative %I of edema after 2h ((-41.77)-(-8.40)%). Regarding the %I of edema after 4h,
compound 11b exhibited the highest %I of edema (71.63%) close to that of celecoxib (73.56%). In
addition, compounds 5b, 6b, 9b, 11a and 14c showed good %I of edema after 4h (58.77, 50.02,
50.71, 51.14 and 54.70%, respectively). While, compounds 4a,b, 5a, 7a,b, 8b, 9a, 10a,b, 12b, 13b,
14d, 15c,d, 16d, 18c, 19c, 20d and 21c showed moderate %I of edema after 4h (20.72-46.50%). The
other tested compounds exhibited weak %I of edema after 4h (0.84-17.93%). In agreement with the
obtained data after 2h, compounds 12a, 18d and 22c,d increased the inflammation in rats with
negative %I of edema after 4h ((-54.31)-(-5.14)%).
Structure-activity correletaion
Regarding chloroacetamide 4a,b, morpholino / (4-substituted piperazin-1-yl)acetamide 5a,b-9a,b,
(quinazolin-2-yl)thioacetamide 10a,b and 11a,b, 4-oxobutanoic acid 12a,b and 1,3-dioxoisoindoline
derivatives 13a,b
The chloroacetamide derivatives 4a,b showed reasonable %I of edema (4a: 46.50% and 4b: 39.26
after 4h) compared to celecoxib. Replacement of chlorine with morpholine ring 5a,b led to slight
increase in activity [34] with %I values of 40.98% after 4h for compound 5a and 58.77% after 4h for
compound 5b. Such improvement in activity may be attributed to extra hydrogen bonding interaction
of 5a,b with Tyr115 residue in the binding site of COX-2 enzyme. Moreover, replacement of
morpholine ring with substituted piperazines 6a,b-8a,b resulted in decreased activity with %I range
of -8.57-26.35% after 2h and 3.36-50.02% after 4h except for 4-methoxyphenylpiperazine
derivatives 9a,b (%I = 40.14% after 2h, 33.44% afer 4h for compound 9a, 11.55% after 2h and
50.71% after 4h for compound 9b) which might stem from the importance of 4-methoxyphenyl
moiety for binding with the enlarged hydrophobic pocket in COX-2 [5,35]. Additionally, conversion
of chloroacetamide moiety to thioacetamide with the presence of quinazoline ring led to slight
increase in activity, for example, the 3-(4-methylphenyl)quinazolines 10a and 10b exerted %I values
of 44.27 and 45.45% after 4h, respectively. Interestingly, the corresponding methoxyphenyl
congeners 11a and 11b exhibited remarkable anti-inflammatory activity with %I values of 51.14 and
71.63% after 4h, respectively. However, compound 11b showed greater increase in the anti-
inflammatory activity which may be attributed to higher number of hydrogen bond acceptor sites
- 5 -

(nO-N = 10). On the other hand, replacing the side chain of 5a,b-9a,b with 4-oxobutanoic acid 12a,b
diminished the anti-inflammatory activity (%I = -41.77 for compound 12a and -8.48 for compound
12b after 2h) which may be attributed to their structure similarity with arachidonic acid (with similar
terminal side chain carrying carboxylic acid group). Such similarity may induce inflammation by
activation of phospholipase A2 leading to increased release of inflammatory mediators (Figure 3).
Introduction of 1,3-dioxoisoindoline ring 13a,b led to slight decrease in anti-inflammatory activity
with %I values of 23.31 and 11.18% after 2h and 4h for compound 13a and 26.67 and 37.21% after
2h and 4h for compound 13b, respectively, which might be attributed to intramolecular hydrogen
bonding between carbonyl group of 1,3-dioxoisoindoline ring and nitrile group which led to less
interaction with the active site of COX enzyme.
Regarding ((un)substituted)benzamide 14c,d and 15c,d, thiophene-2-carboxamide 16c,d, acetamide
17c,d, propionamide 18c,d, butyramide 19c,d, dibenzenesulfonamide 20c,d and 21c, and
monobenzenesulfonamide derivatives 22c,d and 23c
Different aromatic and aliphatic amides were prepared. Generally speaking, it was found that
aliphatic amides are more potent (with %I values of -8.40-63.99%) than aromatic amides (with %I
values of -11.32-46.97%) after 2h. On contrary, aromatic amides showed higher anti-inflammatory
activity after 4h (with %I values of 4.40-54.70%) than aliphatic amides (with %I values of -6.48-
35.87%). Moreover, aliphatic side chain revealed that extending the side chain increases the
interaction with the hydrophobic pocket in the receptor due to increased lipophilicity, where the
butyramide derivatives 19c,d had higher %I values (19c, %I = 62.66 and 35.87% and 19d, %I = 7.61
and 17.93% after 2h and 4h, respectively) than propionamide derivatives 18c,d (18c, %I = 63.99 and
26.08% and 18d, %I = -8.40 and -5.14% after 2h and 4h, respectively) than acetamides 17c,d (17c:
%I = 16.30 and -6.48% and 19d: %I = 22.22 and 16.44% after 2h and 4h, respectively). On the other
hand, it was found that disulfonamide compounds 20c,d and 22c,d showed weak to diminished anti-
inflammatory activity, whereas compound 22d showed good activity with %I values of 66.52% after
2h and 27.56% after 4h. Generally, monosulfonamides 21c and 23c showed higher anti-inflammatory
activity than disulfonamides which might be due to less steric hindrance that would allow better
binding with the active site of COX-2 enzyme.
2.2.2. In vitro COX-1/COX-2 inhibition assay
- 6 -

The most active anti-inflammatory analogues (5b, 11b, 14c, 18c, 19c and 20d) with %I values of
54.70-71.63% were subjected to in vitro COX-1/COX-2 inhibition assay using celecoxib as a
reference drug. IC50 values of the tested compounds along with their selectivity index (SI) [36,37]
were illustrated in Table 2 and Figure 4. The obtained data disclosed that compounds 5b, 18c and
20d are the most active COX-2 inhibitors with IC50 values of 5.45, 3.28 and 9.33 µM, respectively
compared to their COX-1 IC50 values of 45.73, 5.70 and 15.77 µM, respectively. Additionally, 5b
had the highest SI value of 8.37 compared to that of celecoxib (SI = 15.44). Unexpectedly, compound
19c was proved to be more selective COX-1 inhibitor (IC50 = 3.88 µM) compared to COX-2 (IC50 =
32.16 µM).
2.2.3. In vitro 5-LOX inhibition assay
The most active anti-inflammatory compounds (5b, 11b, 14c, 18c, 19c and 20d) were evaluated for
their 5-LOX inhibitory activity. The IC50 values of the six compounds was determined and listed in
Table 2 and Figure 4. The results revealed that compounds 20d and 5b elicited good inhibitory
activity toward 5-LOX enzyme (IC50 = 1.18 and 4.33 µM, respectively) compared to
nordihydroguaiaretic acid (NDGA) (IC50 = 2.46 µM). In addition, compounds 14c, 18c and 19c
showed moderate ability to inhibit 5-LOX enzyme with IC50 values of 12.32, 12.96 and 9.92 µM,
respectively. Furthermore, compound 11b displayed the weakest 5-LOX inhibitory activity (IC50 =
26.08 µM).
3. In silico studies
3.1. Molecular modeling
3.1.1. Molecular modeling of COX-2 inhibitors
Molecular modeling was performed using the crystal structure of human COX-2 enzyme (code
5IKQ) [38]. The two-dimensional binding modes of celecoxib, 18c and 5b are depicted in Figure 5A,
B and C, respectively, and the types of binding interactions are listed in Table 3. The obtained data
showed that celecoxib binds to COX-2 through hydrogen bonding with Arg120 and arene-cation
interaction with Arg120 and Lys83. On the other hand, the most active COX-2 inhibitor 18c showed
high binding affinity toward Lys83 and Ser119 residues through the formation of two hydrogen
- 7 -

bonds. In addition, it binds to Arg120 by arene-cation interaction which mimics the binding mode of
celecoxib. In a similar fashion, the most selective COX-2 inhibitor 5b was engaged with Tyr115
through hydrogen bonding and arene-cation interaction with Arg120.
The docking of ligands inside the binding pocket of COX-2 was carried out [39] and the surface map
of the binding pocket was created, where celecoxib occupied the whole space lying into the groove
pocket (Figure 6A). Compounds 18c and 5b pocket alignments revealed similar pattern filling the
area of the active site with its bulkiness, forming favorable binding contacts (Figure 6B and C,
respectively).
In order to get insights about the structural requirements for anti-inflammatory activity, the flexible
alignment was conducted [40,41]. Results illustrated that the two most active COX-2 inhibitors 18c
and 5b are perfectly aligned (Figure 7).
Surface mapping comparison of celecoxib, 18c and 5b was performed to identify their surface
properties, and the resemblance in the position of hydrophobic and hydrophilic moieties that bind to
the active site of COX-2 (Figure 8A, B and C, respectively) [42], and it is obvious that there is
resemblance in the position of the hydrophobic moiety at the middle of the structure, including a
central aromatic ring, and in the presence of hydrophilic moiety at one side of the molecule, such as
sulfonamide group in celecoxib and cyano group in compounds 18c and 5b.
3.1.2. Molecular modeling of 5-LOX inhibitors
Regarding the inhibitory activity of compound 5b (IC50 = 4.33 µM) towards 5-LOX, molecular
modeling study was conducted using the crystal structure of human 5-LOX enzyme (code 3O8Y)
[43].
The 2D binding modes of NDGA and 5b were built (Figure 9). The types of binding interactions of
NDGA and 5b with 5-LOX enzyme are illustrated in Table 3. The obtained data underscored that
NDGA binds to 5-LOX through hydrogen bonding with Gln557 and Ile673 in addition to arene-
cation interaction with His372. On the other hand, compound 5b was involved in arene-cation
interaction with His372 and hydrogen bonding interaction with Asn425.
Based on the surface map of the binding pocket, NDGA occupied the whole space lying into the
groove pocket (Figure 10A). Compound 5b pocket docking showed similar pattern filling the active
site space (Figure 10B). Moreover, surface mapping comparison of NDGA and 5b revealed that there
is resemblance in the position of the hydrophobic moiety at the middle of both structures, including
- 8 -

a central aromatic ring, and in presence of polar moieties at peripheral parts of the molecules, such
as hydroxyl groups in NDGA, and cyano and morpholino groups in compound 5b (Figure 11A, B).
3.2. In silico simulation studies
In silico studies represent a useful tool in drug discovery process, since they are important in the
prediction of physicochemical properties, pharmacokinetics and toxicity of biologically active drugs
[44]. The most active compounds 5b, 11b, 14c, 18c, 19c and 20d were analyzed for prediction of
Lipinski’s rule [45] and Veber’s standards [46] applying ACD/I-Lab software [47]. Drug score values
were predicted applying Molsoft software [48]. Additionally, ADMET data were predicted applying
ADMET-SAR software [49].
3.2.1. ACD/I-Lab and Molsoft calculations
ACD/I-Lab software [47] was applied for determination of TPSA, Nrotb and the parameters of
Lipinski’s rule of the new compounds. Results (Table 4) demonstrated that the most active
compounds are under the acceptable standards of Lipinski’s rule, TPSA and Nrotb; therefore, they
are foreseen to exhibit good oral absorption and bioavailability in human.
The drug score combines cLogP, molecular weight, solubility, drug-likeness and toxicity risks in one
handy value that could be applied to judge the compound's overall potential to qualify for a drug. A
drug score value of 0.5 or more makes the compound a promising lead for development of safe and
efficient drugs [50]. The drug score values of the most active analogs and celecoxib were calculated
using Molsoft software [48] (Table 4), where compounds 5b and 11b showed good drug score values.
3.2.2. ADME-Tox Prediction
Over decades, properties affecting ADMET have become one of the most essential topics to
evaluate the effects that chemical compound may have on human beings. In silico studies have been
widely used instead to estimate these properties to avoid high-rate of drug withdrawal from market
[51,52].
For that, the blood–brain barrier penetration (BBB), human intestinal absorption (HIA), aqueous
solubility (LogS), cytochrome P450 2D6 substrate/inhibition (CYP2D6, one of the most essential
- 9 -

enzymes involved in drug metabolism), Ames toxicity, carcinogenicity and LD50 in rat model were
calculated for the most active compounds using ADMET-SAR, and results are listed in Table 5.
Referring to the predicted ADMET data of celecoxib and the most active compounds 5b, 11b,
14c, 18c, 19c and 20d, it was proved that they have comparable ADMET properties to that of
celecoxib. In addition, all of the most active members could cross BBB, and they are expected to be
absorbed by human intestine with high HIA values close to 1. The estimated LogS values indicated
that the most active compounds have good solubility values above -4 similarly to 80% of drugs in
the market, and they are anticipated to exhibit good oral absorption and distribution profile.
Furthermore, the most active compounds are predicted to be non-substrate/non-inhibitors of CYP2D6
which indicates the low possibility of drug-drug interaction upon their administration. Moreover,
toxicity tests disclosed that the most active compounds are predicted to be non-toxic in Ames test.
Also, the most active compounds are predicted to be non-carcinogenic. Additionally, they displayed
high LD50 doses in rat model close to that of celecoxib, confirming their safety.
4. Conclusion
In the current investigation, a diverse set of thiophene hybrids were designed, synthesized and
evaluated for their anti-inflammatory activity applying carrageenan-induced paw edema model. The
in vivo results proved that compounds 18c, 19c and 20d possess the highest %I with a range of 62.66-
71.63% compared to celecoxib. The in vitro COX-1/COX-2 inhibitory results highlighted that
compound 18c had the highest COX-2 inhibitory activity (IC50 = 3.28 µM), whereas 5b displayed
the highest selectivity towards COX-2 with SI value of 8.37. Moreover, compounds 20d and 5b
showed the highest 5-LOX inhibitory activity with IC50 values of 1.81 and 4.33 µM, respectively,
compared to NDGA with IC50 = 2.46 µM. Furthermore, docking results revealed that compounds
18c and 5b bind in a similar mode to celecoxib with COX-2, and that compound 5b mimics NDGA
in its binding with LOX enzyme. Overall, the morpholinoacetamide derivative 5b could serve as a
candidate for further development of new potent anti-inflammatory agents.
5. Experimental
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1
13
Melting points (°C) were determined by Stuart melting point apparatus (SMP30). H and C-
NMR spectra were recorded on Bruker Avance-III HD FT (400 MHz) at Faculty of Pharmacy,
Mansoura University, Egypt. Chemical shifts are expressed in δ ppm with reference to
tetramethylsilane. Mass spectral analysis was performed on Thermo Scientific GCMS model ISQ at
the Regional Center for Mycology and Biotechnology (RCMB), Al-Azhar University, Nasr City,
Egypt. Elemental microanalyses (% C, H, N) were achieved, and they were in agreement with the
expected structures within ±0.4% of the calculated values. Reaction times were controlled by TLC
plates (silica gel, 60F245 E, Merck) using chloroform/methanol or chloroform alone as elution
systems, and UV was utilized for spot visualization. Column chromatography was carried out on
silica gel 60 (0.063-0.200 mm, Merck), using isocratic and gradient elution systems. Reagents were
purchased from Sigma-Aldrich Co., USA, El-Gomhoria and El-Nasr Pharmaceutical and Chemical
Co., Egypt. In vivo anti-inflammatory screening was performed at Department of Pharmacology and
Toxicology, Faculty of Pharmacy, Mansoura University, Egypt. COX-1 / COX-2 and 5-LOX
enzyme assays were carried out at the Confirmatory Diagnostic Unit, VACSERA, Egypt.
5.1. Chemistry
5.1.1. Synthesis of 2-(1-(4-substituted phenyl)ethylidene)malononitriles 2a-d [29-31]
Malononitrile (2.64 g, 0.04 mol), ammonium acetate (3.70 g, 0.048 mol) and glacial acetic acid (2
mL) were added to a stirred solution of 4-substituted acetophenone 1a-d (0.02 mol) in benzene (30
mL). The mixture was refluxed for 9-12 h using Dean stark apparatus. The mixture was poured onto
ice and extracted with ethyl acetate. The organic layer was washed several times with brine solution
and dried over anhydrous sodium sulfate. The solvent was evaporated under vacuum and the obtained
residue was purified by column chromatography on silica gel using dichloromethane as eluent to
afford compounds 2a-d.
5.1.1.1. 2-(1-(4-Bromophenyl)ethylidene)malononitrile (2a) [30,31]
5.1.1.2. 2-(1-(4-Chlorophenyl)ethylidene)malononitrile (2b) [30,31]
5.1.1.3. 2-(1-(4-Fluorophenyl)ethylidene)malononitrile (2c) [30,31]
5.1.1.4. 2-(1-(4-Methylphenyl)ethylidene)malononitrile (2d) [30,31]
5.1.2. Synthesis of 2-amino-4-(4-substituted phenyl)thiophene-3-carbonitriles 3a-d [29-31]
- 11 -

Elemental sulfur (0.064 g, 0.002 mol) was added to a solution of compound 2a-d (0.002 mol) in hot
absolute ethanol (6 mL), the mixture was stirred for 5 min and DEA (0.15 mL) was then added
dropwise to the mixture over 10 min. The mixture was heated under reflux for 30-45 min and the
solvent was evaporated under vacuum. The obtained residue was purified by column chromatography
on silica gel using dichloromethane as eluent to afford compounds 3a-d.
5.1.2.1. 2-Amino-4-(4-bromophenyl)thiophene-3-carbonitrile (3a) [30,31]
5.1.2.2. 2-Amino-4-(4-chlorophenyl)thiophene-3-carbonitrile (3b) [30,31]
5.1.2.3. 2-Amino-4-(4-fluorophenyl)thiophene-3-carbonitrile (3c) [30,31]
5.1.2.4. 2-Amino-4-(4-methylphenyl)thiophene-3-carbonitrile (3d) [30,31]
5.1.3. Synthesis of 2-chloro-N-(3-cyano-4-(4-substituted phenyl)thiophen-2-yl)acetamides 4a,b
A mixture of compound 3a,b (0.002 mol), chloroacetyl chloride (0.68 g, 0.006 mol) and TEA (0.2
mL) in chloroform (6 mL) was stirred at room temperature for 18 h. The solvent was evaporated
under vacuum and the obtained residue was purified by column chromatography on silica gel using
dichloromethane as eluent to afford the desired compounds 4a,b.
5.1.3.1. N-(4-(4-Bromophenyl)-3-cyanothiophen-2-yl)-2-chloroacetamide (4a)
Yellowish white solid, yield (83%), m.p. 229-232 ºC. 1H-NMR (CDCl3): δ 4.36 (s, 2H, CH2), 6.98
(s, 1H, Thiophene-H), 7.49 (d, 2H, J = 8.8 Hz, Ar-H), 7.62 (d, 2H, J = 8.8 Hz, Ar-H), 9.50 (s, 1H,
NH). 13C-NMR: δ 42.0, 94.5, 115.2, 122.9, 129.0, 130.7, 132.1, 132.2, 137.7, 149.4, 163.5. MS m/z
(%): 360 (1.92, M++4), 359 (1.99, M++3), 358 (10.94, M++2), 357 (7.83, M++1), 356 (42.65, M+),
77 (100.00). Anal. Calcd (Found) for C13H8BrClN2OS (355.64): C, 43.91 (43.86); H, 2.27 (2.35); N,
7.88 (7.75).
5.1.3.2. 2-Chloro-N-(4-(4-chlorophenyl)-3-cyanothiophen-2-yl)acetamide (4b)
Orange white solid, yield (85%), m.p. 213-216 ºC. 1H-NMR (CDCl3): δ 4.36 (s, 2H, CH2), 6.97 (s,
1H, Thiophene-H), 7.46 (d, 2H, J = 8.8 Hz, Ar-H), 7.56 (d, 2H, J = 8.8, Ar-H), 9.51 (s, 1H, NH).
13C-NMR: δ 42.0, 94.7, 114.0, 115.2, 128.7, 129.3, 131.6, 134.8, 137.7, 149.2, 163.3. MS m/z (%):
313 (2.61, M++2), 312 (9.20, M++1), 311 (3.55, M+), 77 (100.00). Anal. Calcd (Found) for
C13H8Cl2N2OS (311.19): C, 50.18 (50.31); H, 2.59 (2.63); N, 9.00 (9.21).
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5.1.4. Synthesis of N-(3-cyano-4-(4-substituted phenyl)thiophen-2-yl)-2-morpholinoacetamides
5a,b and N-(3-cyano-4-(4-substituted phenyl)thiophen-2-yl)-2-(4-substituted piperazin-1-
yl)acetamides 6a,b-9a,b
A mixture of compound 4a,b (0.002 mol), morpholine (0.17 g, 0.002 mol) or substituted piperazine
(0.002 mol) and TEA (0.2 mL) in acetone (5 mL) was refluxed for 5-7 h. The solvent was evaporated
under vacuum and the obtained residue was purified by column chromatography on silica gel using
gradient elution system of dichloromethane : methanol to yield the desired compounds 5a,b-9a,b.
5.1.4.1.
N-(4-(4-Bromophenyl)-3-cyanothiophen-2-yl)-2-
morpholinoacetamide (5a)
The elution system used started from 100% dichloromethane till 95% dichloromethane : 5%
methanol.
Yellowish white solid, yield (60%), m.p. 139-140 ºC. 1H-NMR (CDCl3): δ 2.66 (s, 4H, Morpholine-
2CH2), 3.27 (s, 2H, COCH2), 3.79 (s, 4H, Morpholine-2CH2), 6.82 (s, 1H, Thiophene-H), 7.40 (d,
2H, J = 8.8 Hz, Ar-H), 7.51 (d, 2H, J = 8.4 Hz, Ar-H), 10.66 (s, 1H, NH). 13C-NMR: δ 53.8, 60.9,
67.0, 93.5, 114.4, 114.5, 122.8, 128.9, 132.2, 132.3, 137.3, 150.4, 167.5. MS m/z (%): 408 (1.62,
M++2), 407 (4.65, M++1), 406 (3.11, M+), 100 (100.00). Anal. Calcd (Found) for C17H16BrN3O2S
(406.30): C, 50.26 (50.36); H, 3.97 (3.99); N, 10.34 (10.39).
5.1.4.2.
N-(4-(4-Chlorophenyl)-3-cyanothiophen-2-yl)-2-morpholinoacetamide (5b)
The elution system used started from 100% dichloromethane till 95% dichloromethane : 5%
methanol.
1
White solid, yield (56%), m.p. 132-133 ºC. H-NMR (CDCl3): δ 2.77 (s, 4H, Morpholine-2CH2),
3.37 (s, 2H, COCH2), 3.90 (s, 4H, Morpholine-2CH2), 6.90 (s, 1H, Thiophene-H), 7.44 (d, 2H, J =
8.4 Hz, Ar-H), 7.54 (d, 2H, J = 8.4 Hz, Ar-H), 10.73 (s, 1H, NH). 13C-NMR: δ 53.8, 60.9, 67.0, 93.6,
114.4, 114.5, 128.6, 129.2, 131.9, 134.6, 137.3, 150.3, 167.4. MS m/z (%): 361 (15.60, M+), 100
(100.00). Anal. Calcd (Found) for C17H16ClN3O2S (361.85): C, 56.43 (56.71); H, 4.46 (4.62); N,
11.61 (11.50).
5.1.4.3.
2-(4-Acetylpiperazin-1-yl)-N-(4-(4-bromophenyl)-3-cyanothiophen-2-
yl)acetamide (6a)
The elution system used started from 100% dichloromethane till 93% dichloromethane : 7%
methanol.
- 13 -

White solid, yield (60%), m.p. 175-176 ºC. 1H-NMR (CDCl3): δ 2.13 (s, 3H, CH3), 2.70 (t, J = 4.8
Hz, 4H, Piperazine-2CH2), 3.35 (s, 2H, COCH2), 3.64 (s, 2H, Piperazine-CH2), 3.78 (s, 2H,
Piperazine-CH2), 6.92 (s, 1H, Thiophene-H), 7.48 (d, 2H, J = 8.4 Hz, Ar-H), 7.60 (d, 2H, J = 8.0 Hz,
Ar-H), 10.62 (s, 1H, NH). 13C-NMR: δ 21.4, 41.5, 46.3, 53.2, 53.5, 60.4, 93.6, 114.5, 114.6, 122.8,
128.9, 132.2, 132.3, 137.2, 150.3, 167.2, 169.1. MS m/z (%): 449 (65.58, M++2), 448 (83.71, M++1),
447 (15.16, M+), 299 (100.00). Anal. Calcd (Found) for C19H19BrN4O2S (447.35): C, 51.01 (51.26);
H, 4.28 (4.10); N, 12.52 (12.41).
5.1.4.4.
2-(4-Acetylpiperazin-1-yl)-N-(4-(4-chlorophenyl)-3-cyanothiophen-2-
yl)acetamide (6b)
The elution system used started from 100% dichloromethane till 93% dichloromethane : 7%
methanol.
Yellowish white solid, yield (65%), m.p. 150-151 ºC. 1H-NMR (CDCl3): δ 2.14 (s, 3H, CH3), 2.72
(s, 4H, Piperazine-2CH2), 3.38 (s, 2H, COCH2), 3.65 (s, 2H, Piperazine-CH2), 3.80 (s, 2H,
Piperazine-CH2), 6.92 (s, 1H, Thiophene-H), 7.45 (d, 2H, J = 8.4 Hz, Ar-H), 7.56 (d, 2H, J = 8.4 Hz,
Ar-H), 10.62 (s, 1H, NH). 13C-NMR: δ 21.4, 41.6, 46.2, 53.1, 53.5, 60.3, 93.8, 106.3, 110.2, 114.6,
128.6, 129.3, 131.8, 134.6, 137.3, 150.2, 169.0. MS m/z (%): 405 (3.53, M++2), 404 (13.25, M++1),
403 (5.65, M+), 43 (100.00). Anal. Calcd (Found) for C19H19ClN4O2S (402.90): C, 56.64 (56.39); H,
4.75 (4.61); N, 13.91 (13.85).
5.1.4.5.
N-(4-(4-Bromophenyl)-3-cyanothiophen-2-yl)-2-(4-methylpiperazin-1-
yl)acetamide (7a)
The elution system used started from 100% dichloromethane till 95% dichloromethane : 5%
methanol.
1
White solid, yield (46%), m.p. 122-123 ºC. H-NMR (CDCl3): δ 1.76 (s, 3H, CH3), 2.85 (s, 2H,
COCH2), 3.54-3.78 (m, 4H, Piperazine-2CH2), 4.22-4.32 (m, 4H, Piperazine-2CH2), 7.31 (s, 1H,
Thiophene-H), 7.50 (d, 2H, J = 8.8 Hz, Ar-H), 7.55 (d, 2H, J = 8.8 Hz, Ar-H), 13.13 (s, 1H, NH).
MS m/z (%): 421 (14.76, M++2), 420 (17.26, M++1), 419 (100.00, M+). Anal. Calcd (Found) for
C18H19BrN4OS (419.34): C, 51.56 (51.68); H, 4.57 (4.81); N, 13.36 (13.55).
5.1.4.6.
N-(4-(4-Chlorophenyl)-3-cyanothiophen-2-yl)-2-(4-methylpiperazin-1-
yl)acetamide (7b)
The elution system used started from 100% dichloromethane till 95% dichloromethane : 5%
methanol.
- 14 -

Yellowish white solid, yield (41%), m.p. 77-79 ºC. 1H-NMR (CDCl3): δ 2.86 (s, 3H, CH3), 3.07-3.12
(m, 4H, Piperazine-2CH2), 3.35-3.43 (m, 2H, Piperazine-CH2), 3.48 (s, 2H, COCH2), 3.51-3.55 (m,
2H, Piperazine-CH2), 6.95 (s, 1H, Thiophene-H), 7.46 (d, 2H, J = 8.8 Hz, Ar-H), 7.55 (d, 2H, J = 8.8
Hz, Ar-H), 10.45 (s, 1H, NH). 13C-NMR: δ 29.7, 49.8, 53.9, 59.5, 89.7, 114.7, 115.2, 128.5, 129.4,
131.6, 134.9, 137.1, 149.4, 164.8. MS m/z (%): 377 (35.88, M++2), 376 (7.95, M++1), 375 (80.37,
M+), 42 (100.00). Anal. Calcd (Found) for C18H19ClN4OS (374.89): C, 57.67 (57.35); H, 5.11 (5.23);
N, 14.95 (14.86).
5.1.4.7.
N-(4-(4-Bromophenyl)-3-cyanothiophen-2-yl)-2-(4-ethylpiperazin-1-yl)acetamide
(8a)
The elution system used started from 100% dichloromethane till 97% dichloromethane : 3%
methanol.
Yellowish white solid, yield (60%), m.p. 134-135 ºC. 1H-NMR (CDCl3): δ 1.27 (t, 3H, J = 7.6 Hz,
CH2CH3), 2.60-3.15 (m, 10H, CH2CH3, Piperazine-4CH2), 3.38 (s, 2H, COCH2), 6.92 (s, 1H,
Thiophene-H), 7.50 (d, 2H, J = 8.4 Hz, Ar-H), 7.61 (d, 2H, J = 8.4 Hz, Ar-H), 10.68 (s, 1H, NH).
13C-NMR: δ 10.9, 52.2, 52.3, 52.4, 60.1, 93.4, 114.4, 114.8, 122.8, 128.9, 132.2, 132.3, 137.2, 150.5,
167.3. MS m/z (%): 435 (42.95, M++2), 434 (98.28, M++1), 433 (47.51, M+), 432 (100.00). Anal.
Calcd (Found) for C19H21BrN4OS (433.37): C, 52.66 (52.90); H, 4.88 (4.78); N, 12.93 (12.89).
5.1.4.8.
N-(4-(4-Chlorophenyl)-3-cyanothiophen-2-yl)-2-(4-ethylpiperazin-1-yl)acetamide
(8b)
The elution system used started from 100% dichloromethane till 97% dichloromethane : 3%
methanol.
White crystals, yield (45%), m.p. 76-77 ºC. 1H-NMR (CDCl3): δ 1.44 (t, 3H, J = 7.6 Hz, CH2CH3),
2.96-2.98 (m, 4H, Piperazine-2CH2), 3.06-3.11 (q, 2H, J = 8.0 Hz, CH2CH3), 3.39-3.41 (m, 4H,
Piperazine-2CH2), 3.54 (s, 2H, COCH2), 6.85 (s, 1H, Thiophene-H), 7.37 (d, 2H, J = 8.8 Hz, Ar-H),
13
7.46 (d, 2H, J = 8.8 Hz, Ar-H), 10.44 (s, 1H, NH). C-NMR: δ 9.1, 49.7, 51.4, 52.4, 59.5, 93.6,
114.6, 115.2, 128.5, 129.3, 131.6, 134.8, 137.0, 150.5, 166.1. MS m/z (%): 391 (1.77, M++2), 390
(7.65, M++1), 389 (5.58, M+), 70 (100.00). Anal. Calcd (Found) for C19H21ClN4OS (388.90): C 58.68
(58.57); H, 5.44 (5.67); N, 14.41 (14.72).
5.1.4.9.
N-(4-(4-Bromophenyl)-3-cyanothiophen-2-yl)-2-(4-(4-methoxyphenyl)piperazin-
1-yl)- acetamide (9a)
- 15 -

The elution system used started from 100% dichloromethane till 98% dichloromethane : 2%
methanol.
White crystals, yield (52%), m.p. 182-184 ºC. 1H-NMR (CDCl3): δ 2.93 (s, 4H, Piperazine-2CH2),
3.29 (s, 4H, Piperazine-2CH2), 3.40 (s, 2H, COCH2), 3.80 (s, 3H, CH3), 6.88 (d, 2H, J = 8.8 Hz, Ar-
H), 6.92 (s, 1H, Thiophene-H), 7.04 (d, 2H, J = 8.8 Hz, Ar-H), 7.50 (d, 2H, J = 6.8 Hz, Ar-H), 7.60
(d, 2H, J = 6.8 Hz, Ar-H). 13C-NMR: δ 51.5, 53.4, 55.6, 60.4, 93.5, 114.4, 114.6, 114.7, 119.2, 122.8,
128.9, 131.1, 132.2, 132.3, 137.2, 150.4, 166.7, 167.6. MS m/z (%): 513 (5.61, M++2), 512 (22.18,
M++1), 511 (12.11, M+), 70 (100.00). Anal. Calcd (Found) for C24H23BrN4O2S (511.44): C, 56.36
(56.49); H, 4.53 (4.62); N, 10.95 (10.89).
5.1.4.10.
N-(4-(4-Chlorophenyl)-3-cyanothiophen-2-yl)-2-(4-(4-methoxyphenyl)piperazin-
1-yl)-acetamide (9b)
The elution system used started from 100% dichloromethane till 98% dichloromethane : 2%
methanol.
Yellowish white crystals, yield (50%), m.p. 154-156 ºC.1H-NMR (CDCl3): δ 2.95 (s, 4H, Piperazine-
2CH2), 3.31 (s, 4H, Piperazine-2CH2), 3.41 (s, 2H, COCH2), 3.80 (s, 3H, CH3), 6.89 (d, 2H, J = 8.8
Hz, Ar-H), 6.91 (s, 1H, Thiophene-H), 7.05-7.08 (m, 2H, Ar-H), 7.44 (d, 2H, J = 8.4 Hz, Ar-H), 7.56
(d, 2H, J = 8.4, Ar-H). 13C-NMR: δ 51.6, 53.3, 55.6, 60.4, 93.6, 114.4, 114.6, 119.2, 119.3, 128.6,
129.2, 131.9, 134.6, 137.2, 140.3, 148.6, 150.4, 167.6. MS m/z (%): 469 (11.12, M++2), 468 (39.08,
M++1), 467 (34.24, M+), 466 (100.00, M+-1). Anal. Calcd (Found) for C24H23ClN4O2S (466.98): C,
61.73 (67.52); H, 4.96 (4.93); N, 12.00 (12.23).
5.1.5. Synthesis of N-(3-cyano-4-(4-substituted phenyl)thiophen-2-yl)-2-((6-nitro-4-oxo-3-(4-
substituted phenyl)-3,4-dihydroquinazolin-2-yl)thio)acetamides 10a,b and 11a,b
A mixture of compound 4a,b (0.002 mol), 2-mercapto-6-nitro-3-(4-substituted phenyl)quinazolin-
4(3H)-one [28] (0.002 mol) and TEA (0.2 mL) in acetone (6 mL) was refluxed for 5-7 h. The solvent
was evaporated under vacuum and the obtained solid was purified by column chromatography on
silica gel using dichloromethane as eluent to afford the desired compounds 10a,b and 11a,b.
5.1.5.1. N-(4-(4-Bromophenyl)-3-cyanothiophen-2-yl)-2-((3-(4-methylphenyl)-6-nitro-4-oxo-3,4-
dihydroquinazolin-2-yl)thio)acetamide (10a)
Yellow solid, yield (68%), m.p. 166-167 ºC. 1H-NMR (CDCl3): δ 2.40 (s, 3H, CH3), 4.00 (s, 2H,
CH2), 6.82 (s, 1H, Thiophene-H), 7.17 (d, 2H, J = 8.8 Hz, Ar-H), 7.31-7.33 (m, 4H, Ar-H), 7.46 (d,
- 16 -

2H, J = 8.4 Hz, Ar-H), 7.89 (d, 1H, J = 8.8 Hz, Quinazoline-H), 8.50 (dd, 1H, J = 2.8, 8.4 Hz,
Quinazoline-H), 9.03 (d, 1H, J = 2.8 Hz, Quinazoline-H), 10.21 (s, 1H, NH). 13C-NMR: δ 21.5, 35.4,
93.9, 114.6, 115.1, 118.5, 120.1, 122.9, 124.1, 128.5, 128.9, 129.2, 131.0, 131.6, 132.2, 135.0, 137.6,
141.7, 145.5, 150.1, 150.4, 160.0, 162.3, 165.6. MS m/z (%): 632 (4.65, M+), 365 (100.00). Anal.
Calcd (Found) for C28H18BrN5O4S2 (632.51): C, 53.17 (53.35); H, 2.87 (2.95); N, 11.07 (11.21).
5.1.5.2. N-(4-(4-Chlorophenyl)-3-cyanothiophen-2-yl)-2-((3-(4-methylphenyl)-6-nitro-4-oxo-3,4-
dihydroquinazolin-2-yl)thio)acetamide (10b)
Yellowish white solid, yield (73%), m.p. 243-244 ºC. 1H-NMR (CDCl3): δ 2.40 (s, 3H, CH3), 4.01
(s, 2H, CH2), 6.81 (s, 1H, Thiophene-H), 7.17 (d, 2H, J = 8.4 Hz, Ar-H), 7.30-7.34 (m, 4H, Ar-H),
7.39 (d, 2H, J = 8.4 Hz, Ar-H), 7.90 (d, 1H, J = 8.8, Quinazoline-H), 8.50 (dd, 1H, J = 2.8, 8.8 Hz,
Quinazoline-H), 9.03 (d, 1H, J = 2.8 Hz, Quinazoline-H), 10.21 (s, 1H, NH). 13C-NMR: δ 21.5, 35.4,
93.9, 114.6, 115.1, 120.1, 124.1, 128.5, 128.6, 129.2, 131.0, 131.6, 131.7, 133.1, 134.7, 137.6, 141.7,
145.5, 150.1, 150.4, 157.5, 160.0, 162.3, 165.6. MS m/z (%): 590 (40.47, M++2), 589 (13.57, M++1),
300 (100.00). Anal. Calcd (Found) for C28H18ClN5O4S2 (588.06): C, 57.19 (57.35); H, 3.09 (3.19);
N, 11.91 (11.79).
5.1.5.3.
N-(4-(4-Bromophenyl)-3-cyanothiophen-2-yl)-2-((3-(4-methoxyphenyl)-6-nitro-4-oxo-
3,4-dihydroquinazolin-2-yl)thio)acetamide (11a)
Yellowish white solid, yield (46%), m.p. 192-193 ºC. 1H-NMR (CDCl3): δ 3.82 (s, 3H, CH3), 4.00
(s, 2H, CH2 ), 6.82 (s, 1H, Thiophene-H), 7.00 (d, 2H, J = 8.8 Hz, Ar-H), 7.19 (d, 2H, J = 8.4 Hz,
Ar-H), 7.32 (d, 2H, J = 8.4 Hz, Ar-H), 7.46 (d, 2H, J = 8.8 Hz, Ar-H), 7.89 (d, 1H, J = 9.2,
Quinazoline-H), 8.50 (dd, 1H, J = 2.4, 9.2 Hz, Quinazoline-H), 9.03 (d, 1H, J = 2.4 Hz, Quinazoline-
H), 10.19 (s, 1H, NH). 13C-NMR: δ 35.5, 55.7, 93.9, 114.6, 115.1, 115.5, 120.1, 122.9, 124.1, 126.6,
128.5, 128.9, 129.2, 130.0, 132.2, 137.6, 142.5, 145.5, 150.1, 150.4, 160.1, 161.4, 162.7, 165.6. MS
m/z (%): 648 (39.89, M++2), 646 (52.00, M+), 560 (100.00). Anal. Calcd (Found) for
C28H18BrN5O5S2 (646.51): C, 51.86 (51.99); H, 2.80 (2.95); N, 10.80 (10.91).
5.1.5.4.
N-(4-(4-Chlorophenyl)-3-cyanothiophen-2-yl)-2-((3-(4-methoxyphenyl)-6-nitro-4-oxo-
3,4-dihydroquinazolin-2-yl)thio)acetamide (11b)
Yellowish white solid, yield (50%), m.p. 188-190 ºC. 1H-NMR (CDCl3): δ 3.82 (s, 3H, CH3), 4.01
(s, 2H, CH2), 6.81 (s, 1H, Thiophene-H), 7.01 (d, 2H, J = 8.8 Hz, Ar-H), 7.20 (d, 2H, J = 8.4 Hz, Ar-
H), 7.31 (d, 2H, J = 8.4 Hz, Ar-H), 7.39 (d, 2H, J = 8.8 Hz, Ar-H), 7.89 (d, 1H, J = 8.8, Quinazoline-
H), 8.49-8.52 (dd, 1H, J = 2.4, 8.8 Hz, Quinazoline-H), 9.03 (d, 1H, J = 2.4 Hz, Quinazoline-H),
- 17 -

13
10.20 (s, 1H, NH). C-NMR: δ 35.5, 55.7, 93.9, 114.6, 115.1, 115.5, 119.7, 120.1, 124.1, 126.6,
128.5, 128.6, 129.2, 130.0, 131.7, 134.7, 137.6, 145.5, 150.1, 150.4, 160.1, 161.4, 162.7, 165.5. MS
m/z (%): 606 (23.29, M++2), 604 (15.01, M+), 252 (100.00). Anal. Calcd (Found) for C28H18ClN5O5S2
(604.06): C, 55.68 (55.88); H, 3.00 (3.32); N, 11.59 (11.63).
5.1.6. Synthesis of 4-((3-cyano-(4-(4-substituted phenyl)thiophen-2-yl)amino)-4-oxobutanoic
acids 12a,b
Compound 3a,b (0.002 mol) was triturated with succinic anhydride (0.4 g, 0.01 mol) till complete
mixing, then the reactants were allowed to fuse together in silicon oil bath for 1-2 h. The obtained
solid was triturated with ice, filtered and purified by column chromatography on silica gel using
gradient elution system of dichloromethane : methanol to yield the desired compounds 12a,b.
5.1.6.1.
4-((4-(4-Bromophenyl)-3-cyanothiophen-2-yl)amino)-4-oxobutanoic acid (12a)
It was purified by column chromatography using 100% dichloromethane till 95% dichloromethane :
5% methanol.
White solid, yield (70%), m.p. 221-223 ºC. IR: 3267 (COOH), 2216 (CN), 1724 (COOH), 1694 (C
ONH). 1H-NMR (DMSO-d6): δ 2.57 (t, 2H, J = 8.0 Hz, COCH2), 2.80 (t, 2H, J = 8.0 Hz, CH2COOH),
7.30 (s, 1H, Thiophene-H), 7.54 (d, 2H, J = 8.8 Hz, Ar-H), 7.70 (d, 2H, J = 8.8 Hz, Ar-H), 11.88 (s,
1H, NH, D2O-exchangable), 12.26 (s, 1H, COOH, D2O-exchangable). 13C-NMR: δ 28.9, 30.3, 91.8,
115.2, 116.2, 122.1, 130.1, 132.2, 133.3, 136.9, 151.7, 171.4, 174.1. MS m/z (%): 381 (5.22, M++2),
380 (2.62, M++1), 379 (38.28, M+), 360 (100.00). Anal. Calcd (Found) for C15H11BrN2O3S (379.23):
C, 47.51 (47.72); H, 2.92 (2.85); N, 7.39 (7.50).
5.1.6.2.
4-((4-(4-Chlorophenyl)-3-cyanothiophen-2-yl)amino)-4-oxobutanoic acid (12b)
It was purified by column chromatography using 100% dichloromethane till 95% dichloromethane :
5% methanol.
White solid, yield (60%), m.p. 220-222 ºC. 1H-NMR (DMSO-d6): δ 2.57 (t, 2H, J = 6.8 Hz, COCH2),
2.80 (t, 2H, J = 6.8 Hz, CH2COOH), 7.30 (s, 1H, Thiophene-H), 7.56 (d, 2H, J = 8.8 Hz, Ar-H), 7.62
(d, 2H, J = 8.8 Hz, Ar-H), 11.87 (s, 1H, NH, D2O-exchangable), 12.25 (s, 1H, COOH, D2O-
exchangable). 13C-NMR: δ 28.9, 30.4, 91.9, 115.2, 116.3, 129.3, 129.8, 132.9, 133.5, 136.8, 151.7,
174.1, 171.4. MS m/z (%): 336 (4.78, M++1), 335 (10.07, M+), 334 (19.07, M+-1), 55 (100.00). Anal.
Calcd (Found) for C15H11ClN2O3S (334.78): C, 53.82 (53.59); H, 3.31 (3.64); N, 8.37 (8.59).
- 18 -

5.1.7. Synthesis
of
2-(1,3-dioxoisoindolin-2-yl)-4-(4-substituted
phenyl)thiophene-3-
carbonitriles 13a,b
Compound 3a,b (0.002 mol) was triturated with phthalic anhydride (0.59 g, 0.01 mol) till complete
mixing, then the reactants were allowed to fuse together in silicon oil bath for 1-2 h. The obtained
solid was purified by column chromatography on silica gel using dichloromethane as eluent to
produce the desired compounds 13a,b.
5.1.7.1.
4-(4-Bromophenyl)-2-(1,3-dioxoisoindolin-2-yl)thiophene-3-carbonitrile (13a)
White solid, yield (60%), m.p. 206-208 ºC. IR: 2225 (C≡N), 1731 (C=O). 1H-NMR (DMSO-d6): δ
7.63 (d, 2H, J = 8.8 Hz, Ar-H), 7.71 (d, 2H, J = 8.8 Hz, Ar-H), 7.99-8.01 (m, 2H, Ar-H), 8.07 (s, 1H,
Thiophene-H), 8.08-8.10 (m, 2H, Ar-H). 13C-NMR: δ 96.4, 108.2, 113.9, 122.7, 124.7, 125.5, 130.1,
131.7, 132.5, 135.9, 139.1, 142.7, 165.7. MS m/z (%): 411 (17.29, M++2), 410 (79.23, M++1), 409
(20.44, M+), 76 (100.00). Anal. Calcd (Found) for C19H9BrN2O2S (409.26): C, 55.76 (55.59); H, 2.22
(2.53); N, 6.85 (6.83).
5.1.7.2.
4-(4-Chlorophenyl)-2-(1,3-dioxoisoindolin-2-yl)thiophene-3-carbonitrile (13b)
White solid, yield (65%), m.p. 186-188 ºC. 1H-NMR (DMSO-d6): δ 7.63 (d, 2H, J = 8.8 Hz, Ar-H),
7.71 (d, 2H, J = 8.8 Hz, Ar-H), 7.99-8.01 (m, 2H, Ar-H), 8.06 (s, 1H, Thiophene-H), 8.08-8.10 (m,
2H, Ar-H). 13C-NMR: δ 108.2, 113.9, 124.7, 125.5, 129.6, 129.8, 131.7, 132.2, 134.0, 135.9, 139.0,
142.7, 165.7. MS m/z (%): 364 (32.72, M+-1), 259 (100.00). Anal. Calcd (Found) for C19H9ClN2O2S
(364.74): C, 62.56 (62.75); H, 2.49 (2.68); N, 7.68 (7.62).
5.1.8. Synthesis
((un)substituted)benzamides
phenyl)thiophen-2-yl)thiophene-2-carboxamides 16c,d
of
N-(3-cyano-4-(4-substituted
phenyl)thiophen-2-yl)-4-
14c,d and 15c,d, and
N-(3-cyano-4-(4-substituted
A mixture of compound 3c,d (0.002 mol) and 4-(un)substituted benzoyl chloride (0.008 mol) or
thiophene-2-carbonyl chloride (1.17 g, 0.008 mol) in pyridine (3 mL) was stirred at room temperature
for 12-18 h. The reaction mixture was poured onto ice, and the precipitated solid was filtered. The
products were then purified by column chromatography on silica gel using dichloromethane as eluent
to afford the desired compounds 14c,d-16c,d.
5.1.8.1.
N-(3-Cyano-4-(4-fluorophenyl)thiophen-2-yl)benzamide (14c)
White solid, yield (50%), m.p. 190-192 ºC. 1H-NMR (CDCl3): δ 6.92 (s, 1H, Thiophene-H), 7.19 (d,
2H, J = 8.8 Hz, Ar-H), 7.60 (d, 2H, J = 8.8 Hz, Ar-H), 7.63 (d, 2H, J = 8.8 Hz, Ar-H), 7.69 (t, 1H, J
- 19 -

= 8.8 Hz, Ar-H), 7.80 (d, 2H, J = 8.8 Hz, Ar-H ), 9.09 (s, 1H, NH). 13C-NMR: δ 93.6, 114.4, 114.8,
115.9, 116.2, 127.4, 129.1, 129.2, 129.3, 129.6, 131.3, 133.4, 137.6, 150.8, 163.8, 164.1. MS m/z
(%): 323 (2.29, M++1), 322 (7.64, M+), 77 (100.00). Anal. Calcd (Found) for C18H11FN2OS (322.36):
C, 67.07 (67.28); H, 3.44 (3.65); N, 8.69 (8.79).
5.1.8.2.
N-(3-Cyano-4-(4-methylphenyl)thiophen-2-yl)benzamide (14d)
Yellowish white solid, yield (65%), m.p. 169-170 ºC. 1H-NMR (CDCl3): δ 2.34 (s, 3H, CH3), 6.84
(s, 1H, Thiophene-H), 7.20 (d, 2H, J = 8.8 Hz, Ar-H), 7.46 (d, 2H, J = 8.8 Hz, Ar-H), 7.51 (d, 2H, J
= 8.8 Hz, Ar-H), 7.59 (t, 1H, J = 8.0 Hz, Ar-H), 7.90 (d, 2H, J = 8.8 Hz, Ar-H), 9.00 (s, 1H, NH).
13C-NMR: δ 21.3, 93.8, 113.9, 115.1, 127.3, 127.4, 129.3, 129.7, 130.6, 131.4, 133.4, 138.5, 138.7,
150.6, 163.8. MS m/z (%): 319 (7.00, M++1), 318 (26.86, M+), 77 (100.00). Anal. Calcd (Found) for
C19H14N2OS (318.39): C, 71.68 (71.82); H, 4.43 (4.59); N, 8.80 (8.65).
5.1.8.3.
N-(3-Cyano-4-(4-fluorophenyl)thiophen-2-yl)-4-methylbenzamide (15c)
Yellowish white solid, yield (60%), m.p. 187-188 ºC. 1H-NMR (CDCl3): δ 2.42 (s, 3H, CH3), 7.34-
7.36 (m, 2H, Ar-H), 7.38-7.42 (m, 3H, Ar-H, Thiophene-H), 7.63-7.69 (m, 2H, Ar-H), 7.90 (d, 2H,
13
J = 8.8 Hz, Ar-H), 11.86 (s, 1H, NH). C-NMR: δ 21.6, 95.6, 115.3, 116.2, 116.4, 117.2, 128.9,
129.6, 129.7, 130.1, 130.2, 130.6, 137.6, 143.6, 151.5, 161.3, 163.8, 165.9. MS m/z (%): 336 (21.35,
M+), 287 (100.00). Anal. Calcd (Found) for C19H13FN2OS (336.38): C, 67.84 (67.72); H, 3.90 (3.98);
N, 8.33 (8.45).
5.1.8.4.
N-(3-Cyano-4-(4-methylphenyl)thiophen-2-yl)-4-methylbenzamide (15d)
Yellowish white solid, yield (55%), m.p. 167-170 ºC. 1H-NMR (CDCl3): δ 2.43 (s, 3H, CH3), 2.49
(s, 3H, CH3), 6.90 (s, 1H, Thiophene-H), 7.29 (d, 2H, J = 8.8 Hz, Ar-H), 7.38 (d, 2H, J = 8.8 Hz, Ar-
H), 7.55 (d, 2H, J = 8.8 Hz, Ar-H), 7.88 (d, 2H, J = 8.8 Hz, Ar-H), 9.06 (s, 1H, NH). 13C-NMR: δ
21.3, 21.7, 93.5, 113.9, 115.2, 127.3, 127.4, 128.6, 128.9, 129.7, 129.9, 130.7, 138.5, 144.3, 150.8,
163.7. MS m/z (%): 332 (8.08, M+), 91 (100.00). Anal. Calcd (Found) for C20H16N2OS (332.42): C,
72.26 (72.54); H, 4.85 (4.69); N, 8.43 (8.75).
5.1.8.5.
N-(3-Cyano-4-(4-fluorophenyl)thiophen-2-yl)thiophene-2-carboxamide (16c)
White crystals, yield (40%), m.p. 186-188 ºC. 1H-NMR (CDCl3): δ 6.90 (s, 1H, Thiophene-H), 7.16-
7.20 (m, 2H, Ar-H), 7.22-7.25 (dd, 1H, J = 4, 4.8 Hz, Thiophene-H), 7.62-7.63 (m, 2H, Ar-H), 7.72-
7.74 (dd, 1H, J = 1.2, 3.8 Hz, Thiophene-H), 7.80 (d, 1H, J = 3.8 Hz, Thiophene-H ), 8.90 (s, 1H,
NH). 13C-NMR: δ 93.7, 114.6, 114.8, 115.8, 115.9, 116.1, 116.2, 128.4, 128.5, 129.2, 129.3, 130.1,
- 20 -

131.0, 133.3, 133.6, 135.7, 137.6, 150.4, 158.4, 164.2. MS m/z (%): 329 (11.21, M++1), 328 (60.13,
M+), 111 (100.00). Anal. Calcd (Found) for C16H9FN2OS2 (328.38): C, 58.52 (58.31); H, 2.76 (2.54);
N, 8.53 (8.71).
5.1.8.6.
N-(3-Cyano-4-(4-methylphenyl)thiophen-2-yl)thiophene-2-carboxamide (16d)
White crystals, yield (50%), m.p. 197-198 ºC. 1H-NMR (CDCl3): δ 2.33 (s, 3H, CH3), 6.18 (s, 1H,
Thiophene-H), 7.12-7.14 (dd, 1H, J = 4.0, 4.8 Hz, Thiophene-H), 7.20 (d, 2H, J = 8.8 Hz, Ar-H),
7.45 (d, 2H, J = 8.8 Hz, Ar-H), 7.62-7.63 (dd, 1H, J = 1.2, 3.8 Hz, Thiophene-H), 7.70-7.71 (dd, 1H,
J = 1.2, 3.8 Hz, Thiophene-H ), 8.85 (s, 1H, NH). 13C-NMR: δ 21.3, 93.7, 114.1, 115.1, 127.3, 128.4,
129.7, 129.9, 130.6, 133.1, 135.8, 138.5, 138.7, 150.2, 158.4. MS m/z (%): 325 (8.97, M++1), 324
(22.86, M+), 111 (100.00). Anal. Calcd (Found) for C17H12N2OS2 (324.42): C, 62.94 (62.72); H, 3.73
(3.91); N, 8.64 (8.85).
5.1.9. Synthesis of N-(3-cyano-4-(4-substituted phenyl)thiophen-2-yl)acetamides 17c,d, N-(3-
cyano-4-(4-substituted phenyl)thiophen-2-yl)propionamides 18c,d and N-(3-cyano-4-(4-
substituted phenyl)thiophen-2-yl)butyramides 19c,d
A mixture of compound 3c,d (0.002 mol), acetyl chloride (0.63 g, 0.008 mol) or propionyl chloride
(0.74 g, 0.008 mol) or butyryl chloride (0.85 g, 0.008 mol) and TEA (0.5 mL) in chloroform (6 mL)
was stirred at room temperature for 10-14 h. The solvent was evaporated and the obtained residue
was purified by column chromatography on silica gel using dichloromethane as eluent to produce the
desired compounds 17c,d , 18c,d and 19c,d, respectively.
5.1.9.1.
N-(3-Cyano-4-(4-fluorophenyl)thiophen-2-yl)acetamide (17c)
Yellowish white crystals, yield (50%), m.p. 247-248 ºC. 1H-NMR (CDCl3): δ 2.36 (s, 3H, CH3), 6.85
(s, 1H, Thiophene-H), 7.14-7.19 (m, 2H, Ar-H), 7.57-7.60 (m, 2H, Ar-H), 8.58 (s, 1H, NH). 13C-
NMR: δ 29.7, 98.0, 114.2, 114.9, 115.9, 116.1, 129.2, 129.3, 137.4, 141.2, 171.5. MS m/z (%): 261
(7.34, M++1), 260 (22.37, M+), 43 (100.00). Anal. Calcd (Found) for C13H9FN2OS (260.29): C, 59.99
(59.69); H, 3.49 (3.72); N, 10.76 (10.89).
5.1.9.2.
N-(3-Cyano-4-(4-methylphenyl)thiophen-2-yl)acetamide (17d)
1
White solid, yield (45%), m.p. 207-208 ºC. IR: 3464 (NH), 2225 (C≡N), 1690 (C=O). H-NMR
(CDCl3): δ 2.24 (s, 3H, CH3), 2.33 (s, 3H, CH3), 6.74 (s, 1H, Thiophene-H), 7.18 (d, 2H, J = 8.8 Hz,
Ar-H), 7.41 (d, 2H, J = 8.8 Hz, Ar-H), 9.09 (s, 1H, NH). 13C-NMR: δ 21.3, 23.2, 92.5, 113.7, 115.2,
127.3, 129.4, 129.6, 130.7, 138.4, 150.9, 167.4. MS m/z (%): 257 (9.82, M++1), 256 (25.36, M+), 43
- 21 -

(100.00). Anal. Calcd (Found) for C14H12N2OS (256.32): C, 65.60 (65.87); H, 4.72 (4.50); N, 10.93
(10.75).
5.1.9.3.
N-(3-Cyano-4-(4-fluorophenyl)thiophen-2-yl)propionamide (18c)
Reddish brown crystals, yield (54%), m.p. 179-180 ºC. 1H-NMR (CDCl3): δ 1.24 (t, 3H, J = 7.6 Hz,
CH2CH3), 2.47-2.52 (q, 2H, J = 7.6 Hz, CH2CH3), 6.75 (s, 1H, Thiophene-H), 7.05-7.09 (m, 2H, Ar-
H), 7.47-7.51 (m, 2H, Ar-H), 8.49 (s, 1H, NH). 13C-NMR: δ 9.1, 29.5, 92.6, 114.1, 114.9, 115.9,
116.1, 129.2, 129.3, 137.6, 150.8, 170.9. MS m/z (%): 274 (4.16, M+), 57 (100.00). Anal. Calcd
(Found) for C14H11FN2OS (274.31): C, 61.30 (61.63); H, 4.04 (4.25); N, 10.21 (10.45).
5.1.9.4.
N-(3-Cyano-4-(4-methylphenyl)thiophen-2-yl)propionamide (18d)
Yellowish white solid, yield (45%), m.p. 176-177 ºC. 1H-NMR (CDCl3): δ 2.24 (t, 3H, J = 7.6 Hz,
CH2CH3), 2.43 (s, 3H, CH3), 2.54-2.60 (q, 2H, J = 7.6 Hz, CH2CH3), 6.58 (s, 1H, Thiophene-H),
7.26 (d, 2H, J = 8.8 Hz, Ar-H), 7.31 (d, 2H, J = 8.8 Hz, Ar-H), 12.21 (s, 1H, NH). 13C-NMR: δ 9.3,
21.3, 30.0, 112.3, 115.2, 129.4, 129.7, 133.4, 137.4, 138.6, 149.5, 167.6, 171.4. MS m/z (%): 271
(8.36, M++1), 270 (3.16, M+), 57 (100.00). Anal. Calcd (Found) for C15H14N2OS (270.35): C, 66.64
(66.82); H, 5.22 (5.46); N, 10.36 (10.52).
5.1.9.5.
N-(3-Cyano-4-(4-fluorophenyl)thiophen-2-yl)butyramide (19c)
Reddish brown crystals, yield (39%), m.p. 200-202 ºC. 1H-NMR (CDCl3): δ 1.05 (t, 3H, J = 7.2 Hz,
CH2CH2CH3), 1.80-1.86 (m, 2H, CH2CH2CH3), 2.52 (t, 2H, J = 7.2 Hz, CH2CH2CH3), 6.83 (s, 1H,
Thiophene-H), 7.14-7.18 (m, 2H, Ar-H), 7.56-7.60 (m, 2H, Ar-H), 8.71 (s, 1H, NH). 13C-NMR: δ
13.7, 18.7, 38.1, 92.5, 114.1, 114.9, 115.9, 116.1, 129.2, 129.3, 137.4, 150.9, 170.3. MS m/z (%):
289 (2.28, M++1), 288 (10.83, M+), 71 (100.00). Anal. Calcd (Found) for C15H13FN2OS (288.34): C,
62.48 (62.71); H, 4.54 (4.69); N, 9.72 (9.98).
5.1.9.6.
N-(3-Cyano-4-(4-methylphenyl)thiophen-2-yl)butyramide (19d)
1
White crystals, yield (53%), m.p. 172-173 ºC. H-NMR (CDCl3): δ 1.04 (t, 3H, J = 7.2 Hz,
CH2CH2CH3), 1.77-1.87 (m, 2H, CH2CH2CH3), 2.42 (s, 3H, CH3), 2.52 (t, 2H, J = 7.2 Hz,
CH2CH2CH3), 6.83 (s, 1H, Thiophene-H), 7.27 (d, 2H, J = 8.8 Hz, Ar-H), 7.51 (d, 2H, J = 8.8 Hz,
Ar-H), 8.83 (s, 1H, NH). 13C-NMR: δ 13.7, 18.7, 21.3, 38.1, 92.5, 113.6, 115.2, 127.3, 128.9, 129.6,
130.8, 138.4, 150.9, 170.4. MS m/z (%): 284 (12.81, M+), 71 (100.00). Anal. Calcd (Found) for
C16H16N2OS (284.38): C, 67.58 (67.71); H, 5.67 (5.84); N, 5.63 (5.86).
- 22 -

5.1.10. Synthesis
of
N-((4-substituted
phenyl)sulfonyl)-N-(3-cyano-4-(4-substituted
phenyl)thiophen-2-yl)benzenesulfonamides 20c,d and 21c
A mixture of compound 3c,d (0.002 mol), 4-substituted benzenesulfonyl chloride (0.01 mol) and
TEA (0.5 mL) in chloroform (4 mL) was stirred at room temperature for 16-18 h. The solvent was
evaporated and the obtained residue was purified by column chromatography on silica gel using
dichloromethane as eluent to afford the desired compounds 20c,d and 21c.
5.1.10.1. 4-Bromo-N-((4-bromophenyl)sulfonyl)-N-(3-cyano-4-(4-fluorophenyl)thiophen-2-yl)-
benzenesulfonamide (20c)
Brown crystals, yield (55%), m.p. 150-152 ºC. 1H-NMR (CDCl3): δ 7.17-7.21 (m, 2H, Ar-H), 7.46
(s, 1H, Thiophene-H), 7.53-7.57 (m, 2H, Ar-H), 7.77-7.80 (m, 4H, Ar-H), 7.93-7.96 (m, 4H, Ar-H).
13C-NMR: δ 107.3, 112.4, 114.6, 116.2, 116.4, 125.1, 128.7, 129.5, 129.6, 130.6, 130.8, 132.8, 136.7,
141.5, 142.5, 161.9, 164.5. MS m/z (%): 656 (18.23, M+), 98 (100.00). Anal. Calcd (Found) for
C23H13Br2FN2O4S3 (656.36): C, 42.09 (42.39); H, 2.00 (2.32); N, 4.27 (4.52).
5.1.10.2.
4-Bromo-N-((4-bromophenyl)sulfonyl)-N-(3-cyano-4-(4-methylphenyl)thiophen-
2-yl)benzenesulfonamide (20d)
Reddish brown crystals, yield (60%), m.p. 190-193 ºC. 1H-NMR (CDCl3): δ 2.33 (s, 3H, CH3), 7.20
(d, 2H, J = 8.8 Hz, Ar-H ), 7.36 (s, 1H, Thiophene-H), 7.37 (d, 2H, J = 8.8 Hz, Ar-H), 7.68 (d, 4H, J
13
= 8.8 Hz, Ar-H), 7.85 (d, 4H, J = 8.8 Hz, Ar-H). C-NMR: δ 21.31, 112.6, 114.7, 124.6, 127.5,
129.7, 129.8, 130.6, 130.8, 132.8, 136.7, 139.3, 142.1, 142.7. MS m/z (%): 656 (5.65, M++4), 654
(49.03, M++2), 652 (3.16, M+), 433 (100.00). Anal. Calcd (Found) for C24H16Br2N2O4S3 (652.40): C,
44.19 (44.28); H, 2.47 (2.31); N, 4.29 (4.36).
5.1.10.3.
N-(3-Cyano-4-(4-fluorophenyl)thiophen-2-yl)-4-methoxy-N-((4-methoxyphen-
yl)sulfonyl)benzenesulfonamide (21c)
Reddish brown crystals, yield (55%), m.p. 183-184 ºC. 1H-NMR (DMSO-d6): δ 3.91 (s, 6H, 2OCH3),
7.24-7.26 (m, 4H, Ar-H), 7.37-7.41 (m, 2H, Ar-H), 7.61-7.65 (m, 2H, Ar-H), 7.86-7.89 (m, 4H, Ar-
13
H), 8.09 (s, 1H, Thiophene-H). C-NMR: δ 56.5, 113.1, 113.9, 116.5, 116.7, 128.2, 128.6, 129.4,
129.5, 130.1, 130.2, 130.3, 131.6, 139.9, 143.3, 165.1. MS m/z (%): 558 (8.08, M+-1), 123 (100.00).
Anal. Calcd (Found) for C25H19FN2O6S3 (559): C, 53.75 (53.91); H, 3.43 (3.74); N, 5.01 (5.29).
5.1.11. Synthesis of 2-((4-substituted phenyl)sulfamoyl)-4-(4-substituted phenyl)thiophene-3-
carboxylic acids 22c,d and 23c
- 23 -

An aqueous solution of 4N NaOH (10 mL) was added to methanolic solution of compound 20c,d or
21c (0.002 mol). THF was then added dropwise until both solutions became miscible, and the reaction
mixture was stirred for 1-2 h. The solvent was evaporated under vacuum, and the obtained residue
was washed with 1N HCl then extracted with dichloromethane. The organic layer was washed several
times with brine solution, dried over anhydrous sodium sulfate and evaporated under vacuum. The
obtained product was purified by column chromatography on silica gel using gradient elution system
of dichloromethane : methanol : glacial acetic acid (starting from 100% dichloromethane then 99%
dichloromethane : 1% methanol and finally 97% dichloromethane : 2% methanol : 1% glacial acetic
acid) to produce the desired compounds 22c,d and 23c, respectively.
5.1.11.1. 2-((4-Bromophenyl)sulfamoyl)-4-(4-fluorophenyl)thiophene-3-carboxylic acid (22c)
Brown crystals, yield (39%), m.p. 63-65 ºC. IR: 3447 (COOH), 1722 (COOH). 1H-NMR (CDCl3): δ
6.95 (s, 1H, Thiophene-H), 7.11-7.16 (m, 2H, Ar-H), 7.46-7.50 (m, 2H, Ar-H), 7.70 (d, 2H, J = 8.4
Hz, Ar-H), 7.80 (d, 2H, J = 8.4 Hz, Ar-H). MS m/z (%): 457 (24.02, M++1), 55 (100.00). Anal. Calcd
(Found) for C17H11BrFNO4S2 (456.31): C, 44.75 (44.61); H, 2.43 (2.65); N, 3.07 (3.27).
5.1.11.2.
(22d)
2-((4-Bromophenyl)sulfamoyl)-4-(4-methylphenyl)thiophene-3-carboxylic
acid
Green crystals, yield (43%), m.p. 109-112 ºC. 1H-NMR (CDCl3): δ 2.39 (s, 3H, CH3), 6.95 (s, 1H,
Thiophene-H), 7.25 (d, 2H, J = 8.8 Hz, Ar-H), 7.41 (d, 2H, J = 8.8 Hz, Ar-H), 7.69 (d, 2H, J = 8.4
Hz, Ar-H), 7.79 (d, 2H, J = 8.4 Hz, Ar-H), 7.92 (s, 1H, NH). MS m/z (%): 452 (75.84, M+), 450
(25.12, M+-2), 236 (100.00). Anal. Calcd (Found) for C18H14BrNO4S2 (452.34): C, 47.80 (47.98); H,
3.12 (3.24); N, 3.10 (3.35).
5.1.11.3.
(23c)
4-(4-Fluorophenyl)-2-((4-methoxyphenyl)sulfamoyl)thiophene-3-carboxylic acid
1
Brown crystals, yield (40%), m.p. 43-45 ºC. H-NMR (DMSO-d6): δ 3.82 (s, 3H, OCH3), 6.57 (s,
1H, Thiophene-H), 7.10 (d, 2H, J = 8.4 Hz, Ar-H), 7.27-7.32 (m, 3H, Ar-H, NH), 7.54-7.58 (m, 3H,
Ar-H, COOH), 7.74 (d, 2H, J = 8.4 Hz, Ar-H). 13C-NMR: δ 57.6, 113.3, 113.8, 115.6, 116.5, 116.7,
128.2, 128.7, 129.4, 129.5, 130.1, 130.2, 130.3, 132.3, 139.9, 142.8, 164.6. MS m/z (%): 407 (53.45,
M+), 85 (100.00). Anal. Calcd (Found) for C18H14FNO5S2 (407.44): C, 53.08 (53.27); H, 3.46 (3.61);
N, 3.44 (3.59).
5.2. Biological screening
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5.2.1. In vivo anti-inflammatory screening
Male Sprague Dawley rats weighing 150-200 g with average age of 6–7 weeks were obtained
from Holding Company for Biological Products and Vaccines (VACSERA), Giza, Egypt. Rats were
kept at temperature and light-controlled environment. All experimental procedures were in
compliance with the guidelines described by the Ethics Committee of Faculty of Pharmacy,
Mansoura University, Mansoura, Egypt.
Rats were deprived from food with free access to water for 16 h before experiment. The tested
compounds were prepared as suspension in 0.5% carboxymethyl cellulose (CMC), and celecoxib
(Adweya company) was used as a reference drug. The in vivo anti-inflammatory activity was assessed
using the rat model of carrageenan-induced paw edema to produce local tissue swelling and
inflammation. Edema was induced by subplantar injection of 100ꢀμL of 1% freshly prepared solution
of carrageenan (Sigma-Aldrich) in distilled water into the right-hind paws of each rat of all the groups
[53].
Celecoxib and the compounds under investigation were given orally to rats by oral gavage, and the
doses of compounds were calculated equimolar with celecoxib (2.622 X 10-5 mol/kg of rat). The
thickness (mm) of the right-hind paw of each animal was measured at three time intervals (0, 2 and
4 h). The percentage edema (%E) at each time point and the percentage inhibition of edema (%I) of
each group were calculated as follows:
%E = PTt ― PT0
PTt
PT0 and PTt are the paw thickness (mm) measured before and after carrageenan subplantar injection,
respectively.
ED
%I = [1- ( )] × 100
EC
ED and EC are the mean percentage edema of the treated and control group [54], respectively.
Prism version 6.01 for windows (Graphpad Software Inc., San Diego, CA, USA) was used for
statistical analysis.
5.2.2. In vitro COX-1/COX-2 inhibition assay
The colorimetric COX-1/COX-2 inhibition assay kit (kit catalog number 560101, Cayman
Chemical, Ann Arbor, MI, USA) was used following the manufacturer’s instructions to test the
ability of the test compounds and celecoxib to inhibit COX-1/COX-2 isozymes [2,36,55].
- 25 -

5.2.3. In vitro 5-LOX inhibition assay
The 5-LOX inhibition assay kit (kit catalog number 760700, Cayman Chemical, Ann Arbor, MI,
USA) was used following the manufacturer’s instructions to test the ability of the test compounds
and NDGA to inhibit 5-LOX enzyme [56,57].
5.3. Molecular modeling
Merck molecular force field 94 (MMFF94) (RMS gradient of 0.01 kcal/Å mol) implemented in
MOE 2009.10 [58,59] was used to understand the behavior of anti-inflammatiry compounds 18c and
5b. Additionally, flexible alignment was done utilizing MOE/MMFF94 flexible alignment tool [60].
Molecular modeling calculations were performed utilizing “molecular operating environment (MOE)
version 2009.11” Chemical Computing Group Inc. software [58]. The most stable conformer of
newly synthesized ‘global-minima’ analogues was docked into the binding site of COX-2 enzyme
which is in complex with mefenamic acid, code 5IKQ and 5-LOX enzyme, code 3O8Y, respectively.
It was attained from the research collaboratory for structural bioinformatics Protein Data Bank. The
hydrogens were added, then enzyme structure was refined by addition of the missed bonds and
protein potential fixation where the constraints on the enzyme were gradually removed and
minimized until the root mean square deviation (RMSD) gradient was 0.01 kcal/mol Å. The active
site of the enzyme was identified by removal of mefenamic acid. The three-dimensional structures
of the two most active COX-2 inhibitors 5b and 18c were built utilizing the builder interface of the
MOE software. Conformational analysis of 5b and 18c was carried out utilizing MMFF94 force field
with root mean square (RMS) gradient of 0.01 kcal/mol Å. The flexible alignments of 5b and 18c
was carried out utilizing the flexible alignment tool of the program adjusting the energy cut off to
15 kcal/mol, and RMSD tolerance to 0.5. The surfaces and maps tool of the MOE program was
utilized to generate molecular surface maps. In the molecular surface maps, regions colored pink
have potential hydrogen bonding ability, regions colored blue are mild polar, and green ones are the
hydrophobic regions [38,61].
Acknowledgments
- 26 -

We would like to express our gratitude to Mahmoud M. Samaha and Kareem M. Saad,
Department of Pharmacology and Toxicology, Faculty of Pharmacy, Mansoura University, for their
help during the conduction of the in vivo anti-inflammatory screening.
Supplementary data
Supplementary data associated with this article is available.
List of captions
Figure 1. The structures of certain reported anti-inflammatory compounds and the designed target
compounds 4a,b, 5a,b-9a,b, 10a,b, 11a,b, 13a,b and 14c,d-19c,d.
Figure 2. The design of new thiophene dibenzenesulfonamides 20c,d and 21c and thipohene
monobenzenesulfonamides 22c,d and 23c.
Figure 3. Flexible alignment of compound 12a (blue) and arachidonic acid (violet) showing their
structural similarity.
Figure 4. In vitro COX-1/COX-2 and 5-LOX inhibitory activities of compounds 5b, 11b, 14c, 18c,
19c and 20d.
Figure 5. 2D binding mode and residues involved in the interaction of (A) celecoxib, (B) the most
active COX-2 inhibitor 18c and (C) the most selective COX-2 inhibitor 5b in the COX-2 binding
pocket.
Figure 6. The aligned conformations of (A) celecoxib (space filled orange), (B) the most active
COX-2 inhibitor 18c occupying the COX-2 binding pocket (space filled blue) and (C) the most
selective COX-2 inhibitor 5b occupying the COX-2 binding pocket (space filled pink).
Figure 7. Flexible alignment of the most active COX-2 inhibitor 18c (blue) and the most selective
COX-2 inhibitor 5b (pink).
Figure 8. (A) Surface map for celecoxib in pocket side, (B) Surface map for the most active COX-2
inhibitor 18c in pocket side and (C) Surface map for the most selective COX-2 inhibitor 5b in pocket
side. Hydrophilic (pink), mild polar (blue), hydrophobic (green).
Figure 9. 2D Binding mode and residues involved in the recognition of (A) NDGA and (B) 5-LOX
inhibitor 5b in the 5-LOX binding pocket.
Figure 10. The aligned conformations of (A) NDGA (space filled red). (B) 5-LOX inhibitor 5b
occupying the 5-LOX binding pocket (space filled yellow).
Figure 11. (A) Surface map for NDGA in pocket side. (B) Surface map for 5b in pocket side.
Hydrophilic (pink), mild polar (blue), hydrophobic (green).
- 27 -

Table 1. The percentage inhibition (%I) of carrageenan-induced paw edema by the newly
synthesized compounds
Table 2. Results of in vitro COX-1/COX-2 and 5-LOX inhibition assay.
Table 3. The types of binding interactions of the most active COX-2 inhibitor 18c, the most selective
COX-2 inhibitor 5b and celecoxib with COX-2 as well as the types of binding interactions of
compound 5b and NDGA with 5-LOX.
Table 4. Calculated Veber’s and Lipiniski’s rule parameters, and drug score values for the most
active compounds.
Table 5. Predicted ADME-Tox data.
Scheme 1. Reaction conditions and yields: a) NH4OAc, Gl. acetic acid, Benzene, 82 ºC, 9-12 h, 65-
80% b) Sulfur, DEA, Ethanol, 90 ºC, 30-45 min, 50-55%.
Scheme 2. Reaction conditions and yields: a) Chloroacetyl chloride, Et3N, Chloroform, rt, 18 h, 83-
85%, b) Morpholine or piperazines (for 5a,b-9a,b) or substituted 2-mercaptoquinazolin-4-one (for
10a,b and 11a,b), Acetone, 60 ºC, 5-7 h, 41-73%, c) Succinic anhydride (for 12a,b) or phthalic
anhydride (for 13a,b), fusion at 130-140 ºC, 1-2 h, 60-70%.
Scheme 3. Reaction conditions and yields: a) Aromatic acid chorides, Pyridine, rt, 12-18 h, 40-65%,
b) Aliphatic acid chorides (for 17c,d-19c,d) or 4-substituted benzenesulfonyl chloride (for 20c,d and
21c), TEA, Chloroform, rt, 10-18 h, 39-60%, c) 4N NaOH, THF, Methanol, rt, 1-2 h, 39-43%.
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