Synthesis and anti-inflammatory activity of sulfonamides and carboxylates incorporating trimellitimides: Dual cyclooxygenase/carbonic anhydrase inhibitory actions

Article abstract of DOI:10.1016/j.bioorg.2018.11.033

Trimellitimides 6–21 were prepared and investigated in vivo for anti-inflammatory and ulcerogenic effects and in vitro for cytotoxicity. They were subjected to in vitro cyclooxygenase (COX-1/2) and carbonic anhydrase inhibition protocols. Compounds 6–11 a

Full text of DOI:10.1016/j.bioorg.2018.11.033

Accepted Manuscript
Synthesis and anti-inflammatory activity of sulfonamides and carboxylates in-
corporating trimellitimides: dual cyclooxygenase/carbonic anhydrase inhibitory
actions
Alaa A.M. Abdel-Aziz, Andrea Angeli, Adel S. El-Azab, Mohammed E.A.
Hammouda, Magda A. El-Sherbeny, Claudiu T. Supuran
PII:
S0045-2068(18)31240-9
DOI:
https://doi.org/10.1016/j.bioorg.2018.11.033
YBIOO 2641
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
30 October 2018
18 November 2018
20 November 2018
Please cite this article as: A.A.M. Abdel-Aziz, A. Angeli, A.S. El-Azab, M.E.A. Hammouda, M.A. El-Sherbeny,
C.T. Supuran, Synthesis and anti-inflammatory activity of sulfonamides and carboxylates incorporating
trimellitimides: dual cyclooxygenase/carbonic anhydrase inhibitory actions, Bioorganic Chemistry (2018), doi:
https://doi.org/10.1016/j.bioorg.2018.11.033
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1
Synthesis and anti-inflammatory activity of sulfonamides and
carboxylates incorporating trimellitimides: dual
cyclooxygenase/carbonic anhydrase inhibitory actions
Alaa A.-M. Abdel-Aziza,b*, Andrea Angelic, Adel S. El-Azaba,d,
Mohammed E. A. Hammoudab, Magda A. El-Sherbenyb,e, Claudiu T. Supuranc*
Department of Pharmaceutical Chemistrya, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia
Department of Medicinal Chemistry b, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt
Università degli Studi di Firenzec, NEUROFARBA Dept., Sezione di Scienze Farmaceutiche, Via Ugo Schiff 6, 50019 Sesto Fiorentino,
Florence, Italy
Department of Organic Chemistryd, Faculty of Pharmacy, Al-Azhar University, Cairo 11884, Egypt
Department of Pharmaceutical Chemistrye, Faculty of Pharmacy, Delta University for Science and Technology, Gamasa City, Egypt
*Corresponding author
E-mail address: almoenes@ksu.edu.sa, alaa_moenes@yahoo.com (A.A.-M. Abdel-Aziz)
Address: Department of Pharmaceutical Chemistry, College of Pharmacy, P.O. Box 2457, King Saud
University, Riyadh-11451, Saudi Arabia
*Corresponding author
E-mail address: claudiu.supuran@unifi.it (C. T. Supuran)
Address: Università degli Studi di Firenze, NEUROFARBA Dept., Sezione di Scienze Farmaceutiche, Via Ugo
Schiff 6, 50019 Sesto Fiorentino, Florence, Italy

2
Synthesis and anti-inflammatory activity of sulfonamides and
carboxylates incorporating trimellitimides: dual
cyclooxygenase/carbonic anhydrase inhibitory actions
Alaa A.-M. Abdel-Aziza,b*, Andrea Angelic, Adel S. El-Azaba,d,
Mohammed E. A. Hammoudab, Magda A. El-Sherbenyb,e, Claudiu T. Supuranc*
Department of Pharmaceutical Chemistrya, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia
Department of Medicinal Chemistry b, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt
Università degli Studi di Firenzec, NEUROFARBA Dept., Sezione di Scienze Farmaceutiche, Via Ugo Schiff 6, 50019 Sesto Fiorentino,
Florence, Italy
Department of Organic Chemistryd, Faculty of Pharmacy, Al-Azhar University, Cairo 11884, Egypt
Department of Pharmaceutical Chemistrye, Faculty of Pharmacy, Delta University for Science and Technology, Gamasa City, Egypt
Abstract——Trimellitimides 6-21 were prepared and investigated in vivo for anti-inflammatory and
ulcerogenic effects and in vitro for cytotoxicity. They were subjected to in vitro cyclooxygenase (COX-1/2) and
carbonic anhydrase inhibition protocols. Compounds 6-11 and 18 exhibited anti-inflammatory activities and had
median effective doses (ED50) of 34.3–49.8 mg kg-1 and 63.6-86.6% edema inhibition relative to the reference
drug celecoxib (ED50: 33.9 mg kg-1 and 85.2% edema inhibition). Compounds 6-11 and 18 were weakly
cytotoxic at 10 M against 59 cell lines compared with the reference standard 5-fluorouracil (5-FU).
Compounds 6-11 had optimal selectivity against COX-2. The selectivity index (SI) range was > 200−490 and
was comparable to that for celecoxib [COX-2 (SI) > 416.7]. In contrast, compounds 12, 13, and 16-18 were
nonselective COX inhibitors with a selectivity index range of 0.92-0.25. The carbonic anhydrase inhibition
assay showed that sulfonamide incorporating trimellitimides 6-11 inhibited the cytosolic isoforms hCA I and
hCA II, and tumor-associated isoform hCA IX. They were relatively more susceptible to inhibition by
compounds 8, 9, and 11. The KI ranges were 54.1−81.9 nM for hCA I, 25.9–55.1 nM for hCA II, and 46.0–
348.3 nM for hCA IX. © 2018 Elsevier Science. All rights reserved.
Keywords: trimellitimide; benzenesulfonamides; anti-inflammatory activity; cytotoxic activity; COX-1/2
inhibition; carbonic anhydrase inhibition.

3
1. Introduction
Nonsteroidal anti-inflammatory drugs (NSAIDs) are extensively used in the treatment of inflammatory
disorders [1]. NSAIDs inhibit cyclooxygenase (COX-1/COX-2) isozymes, which convert arachidonic
acid to prostaglandins (inflammatory eicosanoids) [1-3]. The COX-1 isozyme maintains gastric and
renal integrity. Its inhibition accounts for the stomach and kidney side effects of NSAIDs [1b,2b,2c].
Cyclooxygenase (COX-2) isozyme produces the inflammatory mediator prostaglandin. Its inhibition is
responsible for the therapeutic effects of NSAIDs [1b,2a-b,2d-e]. A major challenge in the discovery
and development of pharmaceuticals is to develop a drug with anti-inflammatory properties but
without the toxic side effects associated with the currently used NSAIDs [1a,4,5].
COX-2 isozyme is overexpressed in human gastric, hepatocellular, breast, colon, lung, ovarian, and
prostate cancers [6-10]. COX-2 inhibitors may diminish the incidence and risk of these cancers.
Therefore, COX-2 isozyme is a potential cancer therapy target [6,8,9]. For example, celecoxib as
COX-2 inhibitor (Figure 1; A) has been investigated for its anti-inflammatory and antitumor activities
[7,9,11].
Carbonic anhydrases (CA) are zinc metalloenzymes (ZBGs) present in almost all living organisms
[12-14]. There are fifteen CA isoforms. Their major function is to catalyze the reversible hydration of
carbon dioxide to bicarbonate and protons. This process is involved in several crucial
pathophysiological pathways [12-14] like glaucoma [15], epilepsy [16], obesity [17], and cancers [18].
For this reason, it is challenging to find isoform-selective inhibitors. However, it has been reported
that various classes of inhibitors have these properties [19].
Certain CA inhibitors like celecoxib with antitumor or anti-inflammatory effects have dual COX-2 and
hCA II inhibitory activity [18,20-27]. Numerous studies on novel multitarget, polyfunctional
medications reported that they were used to treat bacterial infections, cancers, inflammation, and

4
neurological disorders [19b,20-22,24,26,28-30]. Hence, the synthesis of new dual COX and CA
inhibitors is a priority in drug discovery (Figure 1). Sulfonamides and carboxylates are versatile
inhibitors with multiple mechanisms (Figure 1; celecoxib A, B, C, and D) [31-33].
In addition, unique derivatives of cyclic imides incorporating sulfonamides, carboxylic acids, and
halides have received considerable attention because they presented with anti-inflammatory,
antitumor, and antihyperlipidemic properties [34-36]. We recently reported the synthesis and activity
of N-substituted cyclic imides as potent carbonic anhydrase and COX-2 inhibitors (Figure 1; B, C, and
D) [19b, 19c, 33a-c, 34b, 37-42].
Here, we report the synthesis of trimellitimide derivatives with sulfamoyl or carboxylate moieties. We
studied the in vivo anti-inflammatory and ulcerogenic activities and in vitro cytotoxicity of these zinc
metalloenzymes. Moreover, these compounds were tested against COX-1/2 and the CA isoforms
cytosolic hCA I and II, membrane-bound hCA IV, and tumor-associated hCA IX (Figure 1; E).
These cyclic imides (Figure 1; E) were designed and evaluated as COX-1/2 and CA inhibitors in order
to: (i) study their in vivo anti-inflammatory and ulcerogenic effects and in vitro cytotoxicity; (ii)
elucidate their molecular mechanisms using in vitro COX-1/2 and carbonic anhydrase (CA) inhibition
assays; (iii) describe the COX-1/COX-2 inhibition structure-activity relationships (SAR) with
molecules incorporating variously substituted trimellitimides; and (iv) compare the efficacies of 4-
aminoethylbenzenesulfonamide
and
4-aminomethylbenzenesulfonamide
with
those
of
benzenesulfonamide and benzoic acid in terms of inhibition of COX-1/2 and the carbonic anhydrase
isoforms CA I, II, IV, and IX in compounds incorporating the same scaffold (i.e., trimellitic acid and
its methyl ester).
2. Results and discussion
2.1. Chemistry

5
The lead molecules used to design and synthesize the COX-2 inhibitors and CAIs were sulfanilamides
incorporating different imide moieties [33a-b, 37-42]. Here, we prepared compounds incorporating
carboxylate and sulfonamide moieties. The syntheses of trimellitimide derivatives incorporating both
sulfamoyl and carboxylate 6-11 are depicted in Scheme 1. Trimellitic acid anhydride 1 or 2 was heated
with 4-aminobenzenesulfonamide (3), 4-(aminomethyl)benzenesulfonamide (4), or 4-(2-
aminoethyl)benzenesulfonamide (5) in acetic acid in the presence of anhydrous sodium acetate [33a-b,
34-41]. The imides 6-11 were generated in reasonable yields (Scheme 1).
Compounds 12-21 had carboxylic acid and methyl carboxylate moieties and were synthesized with
aromatic amines like 4-aminobenzoic acid, ethyl 4-aminobenzoate, aniline, benzylamine, and
phenethylamine and the trimellitic acid anhydrides 1 and 2 to give the corresponding acid imides 12-
21 (Scheme 2) [43,44]. The structures of the isolated products 6-11 and 12-21 were verified from their
spectral analyses.
2.2. Biological activity
2.2.1. In vivo anti-inflammatory studies
Compounds 6-21 and the reference drug celecoxib were subjected to in vivo anti-inflammatory activity
screening with a conventional rat carrageenan-induced foot paw edema model. The percent edema
inhibition was measured after 2 h of carrageenan treatment (Table 1) at which time the maximum
inhibition of carrageenan-induced edema was attained [5,45].
Most of the tested compounds reduced edema by 20.8−86.6% (Table 1). A major exception was
compound 15, which was relatively latent and mitigated edema by only 10.5%. The carboxylic acid
derivatives were generally more active than their corresponding methyl ester derivatives. For example,
compounds 6, 7, and 8 showed the highest anti-inflammatory activities with 79.0%, 83.2%, and 86.6%
edema reduction, respectively. These efficacies were comparable to that for celecoxib (85.2% edema
inhibition). Conversion of the carboxylic acids 6, 7, and 8 into their corresponding methyl esters 9, 10,

6
and 11 slightly decreased their anti-inflammatory activity levels (66.3%, 70.9%, and 75.3% edema
inhibition, respectively). The dual carboxylic acid compound 12 showed higher anti-inflammatory
activity than its ester analogs 13-15 (50.8%, 29.4%, 38.6%, and 10.5% edema inhibition, respectively).
Compound 18, which incorporates one carboxylic moiety at the 5-position of the trimellitimide core,
presented with 63.6% edema inhibition. In contrast, its ester derivative (compound 21) provided only
22.6% edema inhibition. The trimellitimides incorporating sulfonamide fragments (compounds 6-11)
had the greatest anti-inflammatory activity levels among all tested products. Compounds 7 and 8 were
the most potently anti-inflammatory (83.2% and 86.6% edema reduction, respectively). These
products had similar efficacies to celecoxib (85.2% edema reduction). The trimellitimide-like
compound 18 incorporating a phenethyl moiety showed higher anti-inflammatory activity (63.6%
edema inhibition) than their corresponding analogs compound 16 (31.0% edema reduction) and
compound 17 (35.3% edema reduction). Isoindole-1,3-diones containing a sulfonamide moiety like
compounds 6 and 9 had stronger anti-inflammatories potency than those with carboxylic or ester
fragments at the same positions (compounds 12, 13, and 14). These results indicate that the
sulfonamide moiety is strongly influential at the COX receptor binding site. The anti-inflammatory
activities of compounds 6, 7, 8, 10, 11, 18, and celecoxib were studied using three graded doses as
shown in Table 2.
2.2.2. Ulcerogenicity
The ulcerogenic activity levels of compounds 6, 7, 8, 10, 11, 18, celecoxib, and diclofenac were
determined by a previously reported method [5,46]. Compounds 6, 7, and 8 were as ulcerogenic as
celecoxib but less ulcerogenic than diclofenac. On the other hand, compounds 10 and 11 showed
negligible ulcerogenic activity relative to compound 18, which had the highest ulcerogenic effect of all
tested compounds (Table 3).

7
2.2.3. Cytotoxicity
In vitro cytotoxicity evaluations of certain compounds are shown in Table 4. These substances were
selected by the National Cancer Institute (Bethesda, MD, USA) on the basis of the structural variations.
Compounds 6-11 and 18 were used in single doses of 10 µM in a full NCI 59 cell line panel assay. The
source tissues were the tumor subpanels leukemia, non-small cell lung, colon, CNS, melanoma,
ovarian, renal, prostate, and breast cancers [28,35b,47]. Table 4 presents the % tumor cell growth
inhibition (GI %) caused by the test compounds. The positive cytotoxic effects (PCE) of compounds
6-11 and 18 at 10 M were 4/59, 1/59, 3/59, 7/59, 3/59, 4/55, and 2/55, respectively, while 5-
fluorouracil (5-FU) had a PCE of 55/59 (Table 4).
2.2.4. COX-1/2 inhibition
The molecular mechanisms of compounds 6-21 were evaluated in terms of COX-1 and COX-2
inhibition using an ovine COX-1/COX-2 assay kit (Catalog No. 560101, Cayman Chemicals Inc., Ann
Arbor, MI, USA). The IC50 (M) and the selectivity indices (SI; IC50 (COX-1) / IC50 (COX-2)) are
listed in Table 5 [3,34,48]. The COX-1/COX-2 assay showed that the IC50 values of celecoxib for
COX-1 and COX-2 were >50 M and 0.12 M, respectively, and the SI of COX-2 was > 416.7. Table
5 shows that molecules incorporating a benzenesulfonamide fragment like compounds 6-11 are
extremely potent COX-2 inhibitors (IC50  0.10−0.25 M and SI  490−> 200). This efficacy is
comparable to that for their COX-1 inhibition (IC50  37.2−49.0 M). On the other hand, compounds
12-21, which lack any sulfonamide moiety, are nonselective COX-1/2 inhibitors and their SI range is
0.25−1.11.
The IC50 values of the most potent inhibitors, compounds 6, 7, 8, 9, 10, and 11 were 0.14 M, 0.13
M, 0.10 M, 0.25 M, 0.25 M, and 0.2 M, respectively. Their SIs were 265.7, 346.9, 200, 200,
490.0, and 250.0, respectively. These values are comparable to those for celecoxib (IC50 0.12 M;

8
COX-2 (SI) > 416.7). Trimellitimides with both sulfonamide and carboxylic groups like compounds 6-
8 (COX-2 (SI) 265.7−490.0) are more potent than their corresponding ester derivatives like
compounds 9-11 (COX-2 (SI) > 200.0−> 250.0). These molecules may interact differently with the
COX-2 binding pocket. The presence of two carboxylic groups (compound 12) results in nonselective
inhibition with relatively higher COX-1 inhibitory activity (IC50
11.6 M) and lower COX-2
inhibitory activity (IC50 30.4 M). Introduction of two ester groups (compound 15) diminished
COX-1/2 inhibition (IC50 > 50 M). Compound 18 has one carboxylic group and the N-phenethyl
substituent. It had very low COX-2 inhibition (IC50 40.1 M) and very high COX-1 inhibition (IC50
9.9 M) relative to its analogs 8 and 11 ((SI) > 490.0 and > 250.0, respectively). Therefore, the
sulfonamide fragment is essential for binding with the COX-2 receptor pocket. Compounds 14, 19, 20,
and 21 were the weakest COX-1/2 inhibitors (SI = 1.02 > 1.11).
The structure-activity relationships (SAR) for COX-1/2 inhibition with compounds 6-21 (Table 5)
indicated the following. (i) COX-2 may have been inhibited by 6-11. Their IC50 (M) range was 0.10-
0.25, which is comparable to that for celecoxib (IC50 0.12 M). (ii) Both the 5-carboxylic acid and
the 5-methyl ester moieties of the isoindole-1,3-dione derivatives incorporating benzenesulfonamide
(compounds 6-11) are potentially effective COX-2 inhibitors but the non-sulfonamide derivatives 12-
21 are not. Therefore, the sulfonamide fragment (SO2NH2) is necessary for COX-2 inhibition. (iii) The
trimellitimide with a 4-(2-aminoethyl)benzenesulfonamide moiety (compound 8; IC50 0.1 M) had
the strongest COX-2 inhibition activity. On the other hand, the derivatives with 4-
(aminomethyl)benzenesulfonamide and 4-aminobenzenesulfonamide moieties (compounds 7 (IC50 =
0.13 M) and 6 (IC50 0.14 M), respectively) demonstrated the importance of the spacer group for
COX-2 inhibition. (iv) Replacement of the sulfonamide fragments (-SO2NH2) in compounds 6, 7, and
8 (IC50 0.14 M, 0.13 M, and 0.10 M, respectively) with hydrogen atoms in compounds 16, 17,

9
and 18 increased COX-1 inhibition (IC50
35.9 M, 35.9 M, and 9.9 M, respectively) and
decreased COX-2 inhibition (IC50 40.1−44.0 M). (v) Conversion of the sulfonamide fragment (-
SO2NH2) into a carboxylic acid (-COOH) or ethyl carboxylate (-COOCH2CH3) moiety incorporating
the same 5-carboxylphthalimide scaffold (compounds 12 and 13) increased COX-1 inhibition (IC50
10.0 M and 30.0 M, respectively) and decreased COX-2 inhibition (IC50 30.4 M and 32.5 M,
respectively). (vi) Other non-sulfonamide and non-carboxylic trimellitimides had significantly
different activity levels. Compounds 19, 20, and 21 were much weaker inhibitors than compounds 6-
11. In brief, the most potent trimellitimide scaffolds were the 5-carboxyphthalimides and 5-
methylcarboxyphthalimides 6-11. They had higher selective COX-2 inhibition levels (SI = >
200−490.0) than celecoxib (SI = > 416.7).
2.2.5. Carbonic anhydrase inhibition
Compounds 6-21 and the reference standard acetazolamide (AAZ) were screened for their CA
inhibitory activity by a stopped-flow CO2 hydrase assay [49]. The isoforms hCA I and II (cytosolic),
hCA IV, and the tumor-associated hCA IX were tested. The results are shown in Table 6.
The following structure–activity relationship (SAR) was inferred from the inhibition data shown in
Table 6.
i) The slow cytosolic isoform hCA I was inhibited by a medium-high nanomolar range (KI =
54.1−276.5 nM) of the sulfonamides. The linker between the trimellitimide scaffold and the
sulfonamide in 6-11 modulated the inhibition profile. The methylene chain in compound 7
did not significantly affect the inhibition profile. However, further chain elongation as in
compound 8 doubled KI. For compounds 9-11, the length of the methylene linker was
critical because one carbon atom in the spacer reduced inhibition efficacy by five times.
Compounds 12-21 did not inhibit this isoform.

10
ii) The dominant physiological isoform hCA II was inhibited by compounds 6-11 in the medium
nanomolar range (KI = 25.9 – 82.9 nM). The methylene linker was not essential for
inhibition profile modulation. Esterification of the carboxylic groups in compounds 9-11
nearly doubled (compounds 10 and 11) and tripled (compound 9) inhibition efficacy
against this isoform. Compounds 12-21 did not inhibit hCA II.
iii) The membrane-bound hCA IV was inhibited by compounds 6-11 in the high nanomolar to
micromolar range (KI = 316.6 nM-7.0 μM). Compounds 9-11 with trimellitimide
carboxylic ester scaffolds showed better inhibition profiles than compounds 6-8 with
trimellitimide carboxylic acid scaffolds. The methylene linker for this isoform is critical in
determining inhibition potency. One carbon atom in the spacer between the trimellitimide
scaffold and the sulfonamide resulted in the highest inhibition efficacy. The hCA IV
isoform was not inhibited by compounds 12-21.
iv) The second membrane-bound, tumor-associated isoform, hCA IX, was inhibited in the high
nanomolar to micromolar range by all compounds with sulfonamide groups (6-11) except
9, which inhibited it in the medium nanomolar range (KI = 46.0 nM). Unlike hCA IV, the
introduction of a linker in compounds 6-11 had various effects on the hCA IX inhibition
profile. Inhibition potency decreased with a one-carbon methylene linker (compound 7, KI
= 2,857.7 nM; compound 10, KI = 1,271.3 nM). In contrast, further chain elongation as in
compounds 8 and 11 increased inhibition potency (KI = 348.3 nM (8); KI = 185.6 nM (11)).
Compounds 12-21 did not inhibit this isoform.

11
3. Conclusion
The trimellitimide compounds 6-21 were synthesized and screened for anti-inflammatory activity and
cytotoxicity. They were also evaluated for the inhibition of COX-1/COX-2 and the carbonic anhydrase
isoforms hCA I, hCA II, hCA IV, and hCA IX. The results showed that benzenesulfonamide
derivatives were stronger selective COX-2 inhibitors than the non-sulfonamide products incorporating
the same scaffolds. Compounds 6-11 were the most potent COX-2 inhibitors (IC50 = 0.14 M, 0.13
M, 0.10 M, 0.25 M, 0.25 M, and 0.20 M) and were comparable to celecoxib (IC50 = 0.12 M).
Only trimellitimides bearing sulfonamide moieties inhibited the cytosolic isoforms hCA I and hCA II.
Therefore, these are candidates for preclinical evaluation in the treatment of glaucoma and other eye
diseases involving these enzymes. Moreover, the tumor-associated hCA IX isoform was susceptible to
inhibition by compounds 8, 9 and 11 whose KI were in the range of 46.0–348.3 nM.
4. Experimental
4.1. Chemistry
Melting points (uncorrected) were recorded on a Barnstead 9100 electrothermal melting apparatus
(APS Water Services Corporation, Van Nuys, CA, USA). IR spectra were recorded on a FT-IR Perkin-
1
Elmer spectrometer (PerkinElmer Inc., Waltham, MA, USA). The H NMR and 13C NMR were
measured in DMSO-d6 and CDCl3, respectively, on Bruker 500 and 125 MHz instruments,
respectively (Bruker, Billerica, MA, USA), using TMS as an internal standard. Chemical shifts were
reported in  ppm. Mass spectra were recorded on an Agilent 6320 Ion Trap mass spectrometer
(Agilent Technologies, Santa Clara, CA, USA). C, H, and N were analyzed at the Research Centre of
College of Pharmacy, King Saud University, Saudi Arabia. The results were within ± 0.4% of the
theoretical values. Compounds 12, 13, 16, 17, and 18 were prepared according to a previously reported
procedure [43,44,50].

12
4.1.1. General procedure for the synthesis of trimellitimides 6-8 (Scheme 1)
Benzenesulfonamide (5.0 mmol), anhydrous sodium acetate (0.69 g, 5.0 mmol), and the
appropriate acid anhydrides (5.0 mmol) in glacial acetic acid (15 mL) were heated with refluxing for
24 h. After cooling the reaction mixture, the precipitate was filtered, washed with water, dried, and re-
crystallized using the appropriate solvents.
4.1.1.1. 1,3-Dioxo-2-(4-sulfamoylphenyl)isoindoline-5-carboxylic acid (6)

M.P. > 300 C, 87% yield (CH3OH); IR (KBr, cm-1) : 3400 (OH), 1775, 1730 (C=O), 1300,
1
1210 (O=S=O); H NMR (500 MHz, DMSO-d6):  7.50 (s, 2H), 7.69−7.71 (d, 2H, J = 8.5 Hz),
7.99−8.01 (d, 2H, J = 8.5 Hz), 8.07−8.08 (d, 1H, J = 8.0 Hz), 8.32 (s, 1H), 8.42−8.43 (d, 1H, J = 7.5
Hz); 13C NMR (125 MHz, DMSO-d6): 123.98, 124.30, 126.86, 127.97, 132.33, 134.92, 135.10,
136.02, 138.11, 143.85, 166.44, 166.47; C15H10N2O6S: m/z (346).
4.1.1.2. 1,3-Dioxo-2-(4-sulfamoylbenzyl)isoindoline-5-carboxylic acid (7)

M.P. > 300 C, 83% yield (CH3CH2OH); IR (KBr, cm-1) : 3451 (OH), 1784, 1711 (C=O),
1360, 1190 (O=S=O); 1H NMR (500 MHz, DMSO-d6):  4.88 (s, 2H), 7.35 (s, 2H), 7.52-7.54 (d, 2H,
J = 8.0 Hz), 7.78-7.79 (d, 2H, J = 8.5 Hz), 8.01-8.03 (d, 1H, J = 8.0 Hz), 8.25 (s, 1H), 8.37-8.39 (d,

13
13
1H, J = 7.5 Hz); C NMR (125 MHz, DMSO-d6): 41.20, 123.72, 124.14, 126.42, 128.30, 132.57,
135.42, 135.87, 136.82, 140.73, 143.68, 166.28, 167.41, 167.44; C16H12N2O6S: m/z (360).
4.1.1.3. 1,3-Dioxo-2-(4-sulfamoylphenethyl)isoindoline-5-carboxylic acid (8)

M.P. > 300 C, 89% yield (CH3OH); IR (KBr, cm-1) : 3398 (OH), 1778, 1718 (C=O), 1330,
1210 (O=S=O); 1H NMR (500 MHz, CDCl3/DMSO-d6):  3.00-3.03 (t, 2H, J = 7.5 Hz), 3.84-3.87 (t,
2H, J = 7.5 Hz), 7.23 (s, 2H), 7.36-7.37 (d, 2H, J = 8.0 Hz), 7.72-7.75 (t, 3H, J = 8.0 Hz), 8.26 (s, 1H),
8.30-8.32 (d, 1H, J = 7.5 Hz); 13C NMR (125 MHz, CDCl3/DMSO-d6): 34.07, 38.83, 122.68,
123.86, 126.26, 129.42, 131.54, 132.41, 135.21, 142.70, 142.86, 167.92, 168.07; C17H14N2O6S: m/z
(374).
4.1.1.4. Methyl 1,3-dioxo-2-(4-sulfamoylphenyl)isoindoline-5-carboxylate (9)

M.P. 283-285 C, 76% yield (CH3OH/CH2Cl2); IR (KBr, cm-1) : 3180 (NH), 1776, 1711
(C=O), 1333, 1199 (O=S=O); 1H NMR (500 MHz, CDCl3/DMSO-d6):  3.95 (s, 3H), 7.62-7.63 (d,
2H, J = 8.5 Hz), 7.70 (s, 1H), 7.98-8.00 (d, 2H, J = 8.5 Hz), 8.05-8.06 (d, 2H, J = 6.5 Hz), 8.39 (s,
1H), 8.43-8.45 (d, 1H, J = 7.5 Hz); 13C NMR (125 MHz, DMSO-d6): 53.03, 118.81, 124.29, 126.91,
127.21, 132.14, 134.74, 135.18, 135.88, 135.97, 143.81, 164.95, 165.94, 169.21; C16H12N2O6S: m/z
(360).

14
4.1.1.5. Methyl 1,3-dioxo-2-(4-sulfamoylbenzyl)isoindoline-5-carboxylate (10)
M.P. 216-218 C, 79% yield (CH3OH); IR (KBr, cm-1) : 3231 (NH), 1790, 1736 (C=O), 1309,
1119 (O=S=O); 1H NMR (500 MHz, DMSO-d6):  3.92 (s, 3H), 4.86 (s, 2H), 7.21 (s, 2H), 7.46-7.47
(d, 2H, J = 8.0 Hz), 7.78-7.80 (d, 2H, J = 8.0 Hz), 7.96-7.98 (dd, 1H, J = 8.0, 6.0 Hz), 8.30 (s, 1H),
8.36-8.38 (dd, 1H, J = 7.5, 6.5 Hz); 13C NMR (125 MHz, DMSO-d6): 41.23, 53.06, 123.94, 123.96,
126.47, 128.34, 132.37, 135.44, 135.57, 135.69, 140.24, 143.84, 164.99, 166.96; C17H14N2O6S: m/z
(374).
4.1.1.6. Methyl 1,3-dioxo-2-(4-sulfamoylphenethyl)isoindoline-5-carboxylate (11)

M.P. 182-184 C, 81% yield (CH3CH2OH); IR (KBr, cm-1) : 3222 (NH), 1792, 1732 (C=O),
1
1343, 1119 (O=S=O); H NMR (500 MHz, CDCl3/DMSO-d6):  3.00-3.03 (t, 2H, J = 7.5 Hz), 3.86-
3.89 (t, 2H, J = 7.5 Hz), 3.91 (s, 3H), 7.34-7.37 (t, 3H, J = 8.0 Hz), 7.74-7.75 (d, 3H, J = 8.0 Hz),
7.91-7.93 (d, 1H, J = 8.0 Hz), 8.23 (s, 1H), 8.32-8.34 (d, 1H, J = 7.5 Hz); 13C NMR (125 MHz,
CDCl3/DMSO-d6): 33.98, 38.75, 53.10, 123.67, 123.81, 126.16, 126.33, 129.91, 131.46, 135.42,
135.60, 142.42, 142.85, 165.04, 167.29; C18H16N2O6S: m/z (388).

15
4.1.2. General procedure for the synthesis of trimellitimides 12-21 (Scheme 2)
The appropriate aromatic amines (5.0 mmol), anhydrous sodium acetate (0.69 g, 5.0 mmol),
and an acid anhydride (5.0 mmol) in glacial acetic acid (15 mL) were heated with refluxing for 6-12 h.
After cooling the reaction mixture, the precipitate was filtered, washed with water, dried, and re-
crystallized using the appropriate solvents.
4.1.2.1. 4-(5-(Methoxycarbonyl)-1,3-dioxoisoindolin-2-yl)benzoic acid (14)

M.P. 295-297 C, 80% yield (CH3CH2OH); IR (KBr, cm-1) : 3404 (OH), 1791, 1745 (C=O);
1H NMR (500 MHz, CDCl3/DMSO-d6):  3.95 (s, 3H), 7.58-7.95 (d, 2H, J = 8.5 Hz), 8.07-8.10 (q,
3H, J = 7.5, 5.0 Hz) 8.36-8.37 (d, 1H, J = 5.0 Hz), 8.43-8.45 (d, 1H, J = 7.5 Hz); 13C NMR (125 MHz,
DMSO-d6): 53.18, 124.07, 124.36, 126.94, 130.29, 130.63, 132.32, 135.38, 135.75, 135.81, 135.92,
165.05, 166.06, 167.09; C17H11NO6: m/z (M+1, 326).
4.1.2.2. Methyl 2-(4-(ethoxycarbonyl)phenyl)-1,3-dioxoisoindoline-5-carboxylate (15)

M.P. 151-152 C, 95% yield (CH3OH/CH2Cl2); IR (KBr, cm-1) : 1798, 1756 (C=O); 1H NMR
(500 MHz, CDCl3/DMSO-d6):  1.35-1.38 (t, 3H, J = 7.5 Hz), 3.95 (s, 3H), 4.32-4.36 (q, 2H, J = 7.0
Hz), 7.60-7.61 (d, 2H, J = 8.0 Hz), 8.06-8.11 (q, 3H, J = 8.5, 7.5 Hz), 8.39 (s, 1H), 8.44-8.45 (d, 1H, J
13
= 8.0 Hz); C NMR (125 MHz, CDCl3/DMSO-d6): 14.53, 53.13, 61.21, 124.18, 124.31, 126.90,

16
129.70, 130.11, 132.25, 135.30, 135.82, 135.94, 136.08, 164.99, 165.40, 165.96; C19H15NO6: m/z
(353).
4.1.2.3. Methyl 1,3-dioxo-2-phenylisoindoline-5-carboxylate (19)

1
M.P. 207-209 C, 94% yield (CH2Cl2); IR (KBr, cm-1) : 1787, 1726 (C=O); H NMR (500
MHz, CDCl3):  4.03 (s, 3H), 7.43-7.48 (q, 3H, J = 7.5, 6.5 Hz), 7.53-7.56 (t, 2H, J = 7.5 Hz), 8.05-
13
8.06 (d, 1H, J = 7.5 Hz), 8.50-8.51 (d, 1H, J = 7.5 Hz), 8.61 (s, 1H); C NMR (125 MHz, CDCl3):
52.94, 123.85, 124.88, 126.49, 128.37, 129.21, 131.41, 131.99, 135.06, 135.77, 136.01, 165.15,
166.34, 166.36; C16H11NO4: m/z (281).
4.1.2.4. Methyl 2-benzyl-1,3-dioxoisoindoline-5-carboxylate (20)

1
M.P. 105-107 C, 96% yield (CH2Cl2); IR (KBr, cm-1) : 1777, 1718 (C=O); H NMR (500
MHz, CDCl3):  4.00 (s, 3H), 4.88-4.89 (d, 2H, J = 5.0 Hz), 7.24-7.32 (m, 3H), 7.42-7.43 (d, 2H, J =
13
7.5 Hz), 7.88-7.90 (m, 1H), 8.36-8.38 (m, 1H), 8.44 (s, 1H); C NMR (125 MHz, CDCl3): 41.91,
52.87, 123.41, 124.45, 127.99, 128.70, 128.74, 132.36, 135.45, 135.62, 136.02, 165.20, 167.05,
167.10; C17H13NO4: m/z (295).
4.1.2.5. Methyl 1,3-dioxo-2-phenethylisoindoline-5-carboxylate (21)

17

1
M.P. 117-118 C, 93% yield (CH2Cl2); IR (KBr, cm-1) : 1778, 1717 (C=O); H NMR (500
MHz, CDCl3):  3.00-3.03 (t, 2H, J = 7.5 Hz), 3.95-3.98 (t, 2H, J = 7.5 Hz), 4.00 (s, 3H), 7.21-7.30
13
(m, 5H), 7.90-7.91 (d, 1H, J = 8.0 Hz), 8.40-8.41 (d, 1H, J = 7.5 Hz), 8.46 (s, 1H); C NMR (125
MHz, CDCl3):  34.49, 39.56, 52.85, 123.28, 124.30, 126.73, 128.59, 128.83, 132.28, 135.35, 135.37,
135.55, 137.79, 165.22, 167.15, 167.19; C18H15NO4: m/z (309).
4.2. Biological evaluation
4.2.1. Anti-inflammatory activity
The anti-inflammatory activity was evaluated with an in vivo carrageenan-induced rat paw edema
model [45]. This assay is conventional for acute inflammation determinations. Rats were divided into
six groups with five rats per group. Each was injected intraperitoneally with equimolar doses
(equivalent to 0.170 M kg-1) of the test compounds and the reference drug (celecoxib). One hour after
drug administration, the rats were subcutaneously injected with 0.05 mL of a 1% carrageenan solution
in saline (prepared 24 h before use) into the subplantar tissue of the right hind paw. The left hind paw
of each rat received a subplantar injection of 0.05 mL normal saline. Two hours after the injections,
the rats were killed by cervical dislocation and both of their hind paws were excised at the tibiotarsic
articulation and weighed. The weight difference between the right and left paws was recorded for each
rat. The percentage weight increases of the carrageenan-injected paws relative to the control paws
were calculated. The percentage reductions in edema relative to the control group were used as activity
measures. Compounds 6, 7, 8, 10, 11, 18, and celecoxib were tested in three graded doses to determine
their ED50.
4.2.2. Potential ulcerogenicity
Ulcerogenicity was determined according to the method of Adami et al. [46]. The rats were divided
into four groups with five rats each. Compounds 6, 7, 8, 10, 11, 18, celecoxib, and diclofenac sodium

18
were administered orally to the rats in equimolar doses (equivalent to 0.170 M kg-1) daily for 5 d. The
animals were sacrificed by ether overdose 10 h after the last treatment. The stomachs were removed,
rinsed with saline, opened along the greater curvature, and inspected under a hand lens (x10
magnification). The number and total length of the ulcers for each animal were recorded. The means
were calculated and taken as the ulcer indices.
4.2.3. In vitro COX-1/COX-2 inhibition assay
The colorimetric COX (ovine) inhibitor screening assay kit (catalog no. 560101, Cayman Chemicals
Inc., Ann Arbor, MI, USA) was used according to the manufacturer’s instructions to determine the
relative abilities of the test compounds and reference drugs to inhibit COX-1/COX-2 isozymes
[3,34,48].
4.2.4 Carbonic anhydrase inhibition
The SX.18MV-R stopped-flow instrument (Applied Photophysics, Oxford, UK) was used to assay the
inhibition of various CA isozymes [49]. Phenol Red (0.2 mM) was the indicator. The absorbance was
557 nm. Hepes 10 mM (pH 7.4) was the buffer and 0.1 M Na2SO4 or 0.1 M NaClO4 was used to
maintain a constant ionic strength. At this concentration, these anions are not inhibitory [53]. The CA-
catalyzed CO2 hydration reaction then proceeded for 5−10 s. Saturated aqueous CO2 solutions at 25 °C
were used as substrates. Stock 10-mM inhibitor solutions were prepared using a 1:1 v/v DMSO:water
solvent mixture. Dilutions up to 0.01 nM were prepared using the aforementioned assay buffer. At
least seven inhibitor concentrations were used to determine the inhibition constant. Inhibitor- and
enzyme solutions were mixed and preincubated for 10 min at room temperature before the assay to
allow the E-I complex to form. Triplicate experiments were conducted for each inhibitor
concentration. The values reported in the present study are the means of these measurements. The

19
inhibition constants were obtained by the nonlinear least-squares method using the Cheng-Prusoff
equation as reported earlier [39,42]. They represent the means of ≥ 3 different determinations.
Acknowledgments
The authors extend their appreciation to the Deanship of Scientific Research at King Saud University
for funding the work through the research group project No. RGP-163.
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25
Figure Legends
Figure 1. Reported examples of selective COX-2 inhibitors and CA inhibitors (A-D) and the designed trimellitimides
(E).
Table 1. Results of anti-inflammatory activity of the tested compounds against carrageenan induced rat paw edema
in rats.
Table 2. Results of anti-inflammatory activity of compounds 6, 7, 8, 11, 18, and celecoxib against carrageenan
induced rat paw edema in rats at three graded doses.
Table 3. Ulcerogenic potential of the tested compounds 6, 7, 8, 11, 18, and celecoxib in mice.
Table 4. Antitumor activity of trimellitimides derivatives 6-11 and 18 presented as growth inhibition percentages
(GI %) over 59 subpanel tumor cell lines.
Table 5. In vitro cyclooxygenase (COX-1/COX-2) enzyme inhibition assay and calculated selectivity indices.
Table 6: In vitro CA I, II, IV and IX inhibition with compounds 6-21 and acetazolamide (AAZ) as standard, by a
stopped-flow, CO2 hydrase assay [49].
Scheme 1: Synthesis of trimellitimides incorporating N-benzenesulfonamides 6-11.
Scheme 2: Synthesis of trimellitimides incorporating aromatic amines 12-21.

26
Figure 1. Reported examples of selective COX-2 inhibitors and CA inhibitors (A-D) and the designed trimellitimides (E).

27
Compound No.
Mean %a
increase in
% Inhibition of
paw oedema
Compound No.
Mean %a
increase in
% Inhibition of
paw oedema
Table 1. Results of anti-inflammatory activity of the tested compounds against carrageenan induced rat paw edema in rats.

28
paw weight
±SEMb
from control
group
paw weight
±SEMb
from control
group
15
16
6
7
38.7±0.5
30.9±0.4
79.0±0.7
83.2±0.6
152.3±1.1 10.5±0.2
127.3±0.9 31.0±0.1
8
17
26.6±0.3
86.6±0.8
119.3±1.0 35.3±0.4
9
18
19
62.1±0.6
53.6±0.3
66.3±0.4
70.9±0.4
67.1±0.5
63.6±0.5
10
146.1±1.2 20.8±0.1
134.5±1.1 27.1±0.3
11
12
20
21
45.5±0.7
90.7±0.6
75.3±0.6
50.8±0.3
142.7±1.3 22.6±0.1
13
14
130.2±1.4 29.4±0.1
113.3±1.1 38.6±0.3
27.3±0.4
85.2±0.3
Celecoxib
aResult of control group (%): 166.5±1.5. bSignificant difference from control and celecoxib-treated group using unpaired Student’s ‘‘t’’
test p< 0.05.

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