Molecules 2015, 20, 16620-16642; doi:10.

3390/molecules200916620
OPEN ACCESS

molecules
ISSN 1420-3049
[Link]/journal/molecules
Article

Design, Synthesis and Evaluation of Novel Phthalimide

Derivatives as in Vitro Anti-Microbial, Anti-Oxidant and
Anti-Inflammatory Agents
Phoebe F. Lamie 1,*, John N. Philoppes 1, Ahmed O. El-Gendy 2, Lucie Rarova 3 and Jiri Gruz 3

1
Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Beni Suef University,
Beni Suef 62514, Egypt; E-Mail: john_philoppes@[Link]
2
Department of Microbiology & Immunology, Faculty of Pharmacy, Beni Suef University,
Beni Suef 62514, Egypt; E-Mail: dr.ahmed_micro@[Link]
3
Laboratory of Growth Regulators & Department of Chemical Biology and Genetics,
Centre of the Region Haná for Biotechnological and Agricultural Research,
Institute of Experimental Botany ASCR & Palacký University, Šlechtitelů 27, 783 71 Olomouc,
The Czech Republic; E-Mails: [Link]@[Link] (L.R.); [Link]@[Link] (J.G.)

* Author to whom correspondence should be addressed; E-Mail: [Link]@[Link] or

[Link]@[Link]; Tel.: +20-82-266-76; Fax: +20-82-231-7958.

Academic Editor: Roman Dembinski

Received: 26 June 2015 / Accepted: 20 August 2015 / Published: 14 September 2015

Abstract: Sixteen new phthalimide derivatives were synthesized and evaluated for their
in vitro anti-microbial, anti-oxidant and anti-inflammatory activities. The cytotoxicity for all
synthesized compounds was also determined in cancer cell lines and in normal human cells.
None of the target derivatives had any cytotoxic activity. (ZE)-2-[4-(1-Hydrazono-ethyl)
phenyl]isoindoline-1,3-dione (12) showed remarkable anti-microbial activity. Its activity
against Bacillus subtilis was 133%, 106% and 88.8% when compared with the standard
antibiotics ampicillin, cefotaxime and gentamicin, respectively. Compound 12 also showed
its highest activities in Gram negative bacteria against Pseudomonas aeruginosa where
the percentage activities were 75% and 57.6% when compared sequentially with the standard
antibiotics cefotaxime and gentamicin. It was also found that the compounds 2-[4-(4-ethyl-3-
methyl-5-thioxo-1,2,4-triazolidin-3-yl)phenyl]isoindoline-1,3-dione (13b) and 2-[4-(3-methyl-
5-thioxo-4-phenyl-1,2,4-triazolidin-3-yl)phenyl]isoindoline-1,3-dione (13c) had anti-oxidant
activity. 4-(N'-{1-[4-(1,3-Dioxo-1,3-dihydro-isoindol-2-yl)-phenyl]-ethylidene}-hydrazino)-
benzenesulfonamide (17c) showed the highest in vitro anti-inflammatory activity of the
Molecules 2015, 20 16621

tested compounds (a decrease of 32%). To determine the mechanism of the anti-inflammatory

activity of 17c, a docking study was carried out on the COX-2 enzyme. The results confirmed
that 17c had a higher binding energy score (−17.89 kcal/mol) than that of the ligand celecoxib
(−17.27 kcal/mol).

Keywords: synthesis; phthalimides; anti-microbial; anti-oxidant; anti-inflammatory

1. Introduction

The most important biological activity properties that have been reported for phthalimide
(isoindoline-1,3-dione) derivatives 1 are anti-cancer [1], anti-microbial [2,3], anti-oxidant [4] and
anti-inflammatory [5]. According to the World Health Organization (WHO), infectious and parasitic
diseases are still the second cause of death worldwide. This is assumed to be due to resistance to the
anti-microbial agents used. There are a number of studies showing that compounds bearing a phthalimide
core may be a scaffold for designing new anti-microbial agents [2]. On the other hand, oxidation results
in free radicals which damage the cell via causing oxidative stress leading to inflammation [6].
A literature search of suitable nuclei of use as anti-oxidant and anti-inflammatory agents, revealed
phthalimide was one of these heterocyclic compounds [1,4,5]. The chemical core of phthalimides
(-CO-N(R)-CO-) shows they are hydrophobic and this increases their potential to cross biological
membranes in vivo [7]. To increase the biological activity of phthalimide derivatives, a molecular
hybridization approach was used to introduce different pharmacophore subunits such as pyrazoles,
diazoles, (oxo and thioxo) triazoles, benzo- (oxazoles, imidazoles and thiazoles) and compounds with the
azomethine group (Schiff basses). Earlier described synthons have been reported to have anti-microbial [8],
anti-oxidant [9] and anti-inflammatory [10–13] activities, apart from other pharmacological
actions like anti-convulsant [14], CNS depressant [15], anti-tumor [1,16], anti-proliferative [17] and
anti-pyretic [18] effects.
Inspection of the compounds 2–8 depicted in Figure 1 led us to design and synthesize some new
compounds containing mainly a phthalimide core and enhanced by certain pharmacophores. The aim
was to find new agents with anti-microbial, anti-oxidant and anti-inflammatory effects. All the obtained
new derivatives were tested as anti-microbial agents (G+, G− bacteria and fungi) and studied for their
anti-oxidant and anti-inflammatory activities using in vitro methods, (Figure 1).

Figure 1. Cont.
Molecules 2015, 20 16622

Figure 1. Representative examples of anti-microbial, anti-oxidant and anti-inflammatory

agents and structural rationalization of the newly designed compounds.

2. Results and Discussion

2.1. Chemical Synthesis

The synthetic procedures adopted to obtain the target compounds are depicted in Schemes 1–3.
Reaction of 2-(4-acetyl-phenyl)-isoindoline-1,3-dione (9), which was prepared according to the reported
method [19], with equimolar amounts of primary amine derivatives, namely 1-aminotriazole, 4-amino-
1,5-dimethyl-2-phenyl-1,2-dihydro-pyrazol-3-one (4-aminophenazone) and 5-amino-3-methyl-1-phenyl-
1H-pyrazole-4-carbonitrile in absolute ethanol at reflux temperature for 8 h to give isoindoline-1,3-dione
derivatives 10a–c. The structure of compounds 10a–c was investigated by elemental and spectral analyses.
The IR spectrum of 10c revealed a C≡N stretching band at ῡ 2212 cm−1. The 1H-NMR spectrum of 10b
exhibited three singlet peaks due to two CH3 and N-CH3 protons at δ 2.1, 2.5 and 2.63, respectively.
Moreover, 13C-NMR spectrum of 10c showed a peak at δ 115.04 corresponding to C≡N.
Molecules 2015, 20 16623

Compounds 11a–c were prepared by the reaction of 2-(4-acetyl-phenyl)-isoindoline-1,3-dione (9)

with o-amino derivatives namely, o-phenylenediamine, o-aminophenol and o-aminothiophenol in refluxing
absolute ethanol containing few drops of glacial acetic acid yielding 11a–c in good yields. Spectroscopic
data (IR, 1H-NMR, 13C-NMR and MS) and elemental analysis of compounds 11a–c confirmed their
structures. The 1H-NMR spectra of 11a–c exhibited D2O exchangeable singlet peaks at δ 3.35–4.45
due to the NH proton, confirming that 11a–c exists as cyclic benzimidazole, dihydrobenzoxazole
and dihydrobenzothiazole with isoindoline-1,3-dione. Another NH proton appeared in 11a at δ 7.65
corresponding to a benzimidazole that was exchanged with D2O (Scheme 1).
Ar

O
O
Ar'
N i
Ar

O
9 10a-c

ii

H
N
Ar
X
11a-c

Scheme 1. Synthesis of compounds 10a–c and compound 11a–c. Reagents and conditions:
(i) ArʹNH2, absolute ethanol, reflux, 8 h, (32%–44%); (ii) o-phenylenediamine, o-aminophenol
or o-aminothiophenol, absolute ethanol/glacial acetic acid, reflux, 12 h, (26%–46%).

Treatment of 9 with excess hydrazine hydrate 99% in dioxane at reflux temperature afforded
2-[4-(1-hydrazonoethyl)phenyl]isoindoline-1,3-dione (12, 45% yield). All collected data for compound
12 were in accord with the assumed structure. Thus, the absence of C=O group of the parent 9 and the
observation of forked peak at ῡ 3317 and 3235 cm−1 corresponding NH2 group in IR spectrum of 12
confirmed its structure. Further the 1H-NMR spectrum of 12 showed D2O exchangeable protons at
δ 5.79 corresponding to NH2 protons. Moreover, the absence of the C=O observed in the starting material
9 in 13C-NMR spectrum of 12 confirmed the predicted structure of 12.
Compound 12 was treated with isocyanate and/or isothiocyanate derivatives, e.g.,
p-chlorophenylisocyanate, ethylisothiocyanate and phenylisothiocyanate in dioxane at reflux temperature
to provide the corresponding oxo(or thioxo)triazole derivatives 13a–c rather than semicarbazide or
thiosemicarbazide analogues A. The structures of compounds 13a–c were investigated by elemental and
spectral analyses. The 13C-NMR spectra of 13a–c revealed peaks at δ 70.80–90.03 due to the CH3-C of
the triazole ring which is not present in the open form A.
The triazole ring was also assembled via reaction of compound 9 with a thiosemicarbazide derivative
yielding 14 rather than the open form B. The structure of compound 14 was established through,
spectroscopic and elemental analyses data. The 13C-NMR spectrum of 14 showed the presence of the
Molecules 2015, 20 16624

CH3-C moiety of the thioxotriazole ring at δ 66.80 that was absent in the starting material 9 or in the
thiosemicarbazide derivative B, thus confirming the putative structure 14.
Treatment of 9 with a molar equivalent of benzohydrazide derivative in dioxane at reflux temperature
for 7 h, gave Nʹ-{1-[4-(1,3-dioxoindolin-2-yl)phenyl]ethylidene}benzohydrazide (15) as a yellow solid
in 48% yield. The structure of compound 15 was confirmed by elemental and spectroscopic analyses.
The IR spectrum of 15 showed NH stretching at ῡ 3330 cm−1 apart from a C=O band at ῡ 1672 cm−1.
Moreover, the 1H-NMR spectrum of 15 revealed the presence of two singlet peaks at δ 2.56 and 2.64
due to the syn and anti CH3 groups around the CH3-C=N double bond. Also, in the mass spectrum of 15
the molecular ion [M+] (m/z 383) corresponding to the formula C23H17N3O3 was observed (Scheme 2).
S
NH2
N NH
Ar

//
N NH2
i iii
Ar 9

S
12
HN
NH
iv Ar
N
X R H
NH ii O 14
N NH
Ar // N NH
Ar
A
15

X
R
N
NH
Ar N
H

13a-c

X = O, S
R = 4-Cl-C6H4, C2H5, C6H5

Scheme 2. Synthesis of triazole derivatives 13a–c, 14 and compound 15. Reagents and
conditions: (i) NH2NH2, dioxane, reflux, 2 h, (24%); (ii) RNCX, dioxane, reflux, 5 h,
(39%–47%); (iii) NH2NHCSNH2, dioxane, reflux, 3 h, (33%); (iv) PhCONHNH2, dioxane,
reflux, 7 h, (48%).

Introducing a diazole moiety to compound 16 was achieved by heating 9 with an equimolar quantity
of ethylenediamine in refluxing dioxane for 4 h. The IR spectrum of 16 revealed the absence of the
C=O group of the parent compound 9. Moreover, aromatization of the diazole ring was confirmed from
the 1H-NMR spectrum that showed two CH diazole ring protons as doublets at δ 6.56 and 7.66 with a
coupling constant of 8.4 Hz. The mass spectrum of 16 exhibited a molecular ion peak [M+] at (m/z 303)
confirming its molecular formula C18H13N3O2.
Treatment of 9 with hydrochloride salt of phenylhydrazine, p-methanesulfonylphenylhydrazine or
p-aminosulfonylphenylhydrazine in a molar ratio (1:1) in refluxing absolute ethanol gave 17a–c in
excellent yields. All data for compounds 17a–c were consistent with the proposed structures. Thus, the
absence of the C=O group of the parent 9 in IR spectra of 17a–c and the appearance of NH (in 17a–c)
Molecules 2015, 20 16625

and NH2 (in 17c) stretching bands at ῡ 3428–3258 cm−1 confirmed the structures of 17a–c. Moreover,
the syn and anti CH3 protons were observed at δ 2.30–2.36 and 2.51–2.63. Further, the NH proton
singlet was recorded at δ 9.87–9.98 that was exchangeable with D2O confirmed structure of the 17a–c
(Scheme 3).

Rʹ = H, SO2CH3, SO2NH2

Scheme 3. Synthesis of compounds 16 and 17a–c. Reagents and conditions:

(i) NH2CH2CH2NH2, dioxane, reflux, 4 h, (26%); (ii) 4-Rʹ[Link], absolute ethanol,
reflux, 5–8 h, (49%–52%).

2.2. Biological Evaluation

2.2.1. Anti-Microbial Activity

Different species of microorganisms have varying degrees of susceptibility to antimicrobials. Further,

the pathogenic microbes may develop drug resistance to a particular type of antimicrobial agent on
prolonged use. Hence, the antimicrobial sensitivity tests are very useful to determine the level of
antimicrobial activity of a particular chemical compound on certain pathogenic microorganisms using
agar well diffusion method, (Figure 2).

Figure 2. Examples of some activities for compounds 11c, 12, 13a, 13b and 13c against
Bacillus subtilis.
Molecules 2015, 20 16626

Table 1. The zone of inhibition (mm) for test compounds *#.

Gram Positive Rods Gram Positive Cocci Gram Negative Rods Yeast
Bacillus Listeria inocua Mycobacterium Enterococcus Sarcina Staphylococcus aureus Escherichia coli Escherichia coli Psedomonas Proteus Candida
subtilis LMG 2710 pheli faecalis lutea LMG 3242 ATCC 25922 ATCC 5087 aeruginosa vulgaris albicans
10a 13 - - - - 14 - - - - 14
10b 13 15 14.5 13 - 15 - - - - 16
10c - 15 15 - - - - - 12 - 14
11a 15 13 13 16 - 14.5 13 - 13 - 14
11b 15 13 13 16.5 - 14 12 - 12 - 13
11c - 17 - - - 14.5 - - 13 - 20
12 26 17 23 17.5 25 16 17 17 25 - 21
13a 13 14 - 15.5 - 13 - - - - -
13b 13 15.5 13 14 14 13 - - - - 14
13c 14 16 16 13.5 17.5 13 - - 13 - 13
14 16 15 15.5 15 18 - - - 13 12 14
15 13 - - 13.5 - - - - 11 - 17.5
16 12 12 - 13 - - - - 12 - 18
17a 11 17 11 12 - - - - 11 12 16.5
17b 13 11 - 13 - - - - 12 - 12
17c - 16 - 13 - - - - 13 - 15
(a)
AMP 22 30 42 30 54 31 38 44 - - -
(b)
CTX 25 21 40 22 48 25 42 52 30 31 -
(c)
CN 28 31 36 20 40 24 34 40 36 39 -
FLU (d) - - - - - - - - - - 40
TIO (e) - - - - - - - - - - 36
* All synthesized compounds and control anti-microbial were dissolved in DMSO to give a final concentration of 10 mg/mL and then a 60 µL of each was inoculated into
cup in agar media. # Incubation temperature was 37 ± 1 °C for 24 h. (a) AMP: Ampicillin; (b) CTX: Cefotaxime; (c) CN: Gentamicin; (d) FLU: Fluconazole; (e) TIO: Tioconazole.
Molecules 2015, 20 16627

Table 2. MIC of samples against different microorganisms (µg/mL) *#.

Pseudomonas Lactobacillus Listeria Staphylococcus Staphylococcus Candida
Compound Proteus Salmonella E. coli E. coli Sarcina Enterococcus Mycobacterium Bacillus
aeruginosa sakei innocua aureus aureus albicans
No. vulgaris typhi 25927 5087 lutea faecalis OS4 pheli subtilis
9027 LMG 2313 LMG 2710 LMG 3242 ATCC 43300 ATCC 60931
10b na na na na na na na na na na na na na na
11a na na na na na na 100 50 50 na 25 50 na na
11b na na na na na na na na 50 na na na na na
11c na na na na na na na na na na 100 na na 100
12 na na 100 na na 50 50 25 200 25 100 na na 100
13c 200 na 200 200 200 na 200 na 200 200 200 200 200 na
14 na na na na na na na na na na na na na na
16 na na na na na na na na na na na na na na
17a na na na na na na na na na na na na na 25
#
* All tested compounds and controls were used in a final concentration ranging from (200 µg/mL–0.7 µg/mL). Incubation temperature was 37 ± 1 °C for 24 h. na:
(not active >200 unit).
Molecules 2015, 20 16628

Candida albicans, Listeria innocua, Bacillus subtilis and Enterococcus faecalis were the
most susceptible microorganisms toward the majority of our synthetic chemical compounds, while
Escherichia coli, Proteus vulgaris and Sarcine lutea were the most resistant bacteria. Based on the
antimicrobial sensitivity determinations, all of our compounds showed marked activity against at least
two out of the six tested Gram positive bacteria, where the order of activity was as follows; compound 12
showed the highest activity followed by 13c, 13b, 14, (11a = 11b), 10b, 13a, 17a, (16 = 17b), 11c,
10c, 17c, 15 and finally 10a. Compounds 12, 13c and 13b were able to inhibit all six tested Gram positive
bacteria. Only compound 12 showed marked activity against Bacillus subtilis where the percentage
activities were 133%, 106% and 88.8% when compared with the standard antibiotics ampicillin,
cefotaxime and gentamicin, respectively. Activity against Gram negative bacteria was weak, and only
compound 12 was able to inhibit 3/4 tested organisms. Compound 12 also showed its highest activities
in Gram negative bacteria against Pseudomonas aeruginosa where the percentage activities were 75%
and 57.6% when compared with the standard antibiotics cefotaxime and gentamicin, respectively. The
order of activity against Gram negative bacteria was as follows; 12, 11a, 14, 11b, 17a, (11c = 13c = 17c),
(10c = 16 = 17b) and finally 15 while compounds 10b, 13a, 13b and 10a failed to show any activity
against any of the tested Gram negative bacteria at the tested concentrations. All compounds apart
from 13a showed activity against Candida albicans in the following order of activity: 12, 11c, 16, 15,
17a, 10b, 17c, (10a = 10 = 11a = 13b = 14), (11b = 13c) and 17b (Tables 1 and 2).

2.2.2. Anti-Oxidant Activity

Oxygen radical absorbance capacity (ORAC) is the ability of compounds to scavenge free peroxy
radicals in vitro. While 10a, 10c and 16 activities were below detection limits (<0.1), 13b and 13c
were 18 × (18.385 ± 0.857) and 14 × times (13.506 ± 0.819) more active than Trolox, the hydrophilic
derivative of vitamin E (Table 3).

Table 3. Oxygen radical absorbance capacity given as ratio between compound and Trolox
on an equimolar basis. Data are expressed as mean ± SD (n = 4).
Compound ORAC (Compound/Trolox)
10a <0.1
10b 0.112 ± 0.007
10c <0.1
11a 0.751 ± 0.081
11b 0.116 ± 0.004
11c 0.183 ± 0.01
12 8.116 ± 0.343
13a 1.169 ± 0.118
13b 18.385 ± 0.857
13c 13.506 ± 0.819
14 1.511 ± 0.045
15 0.192 ± 0.005
16 <0.1
17a 1.839 ± 0.035
17b 0.129 ± 0.005
17c 0.374 ± 0.007
Molecules 2015, 20 16629

2.2.3. Anti-Inflammatory Activity

The in vitro anti-inflammatory properties of the phthalimide derivatives were studied using ELISA in
pretreated Human Umbilical Vein Endothelial Cells (HUVEC) where these compounds could inhibit
NF-κB. E-selectin (ELAM) expression was induced by TNFα, which is indicative of NF-κB activation.
The observed reduction of ELAM expression on treatment of HUVECs with 30 µM or 50 µM of
phthalimide derivatives was significant only for 17c—p-aminosulfone derivative—(decrease of 32%).
Slight inhibition of ELAM expression and also cell viability was measured for 10b, 11c, 12, 13a, 13b,
13c, 14, 15, 17b. The results showed confirmed that the NF-κB pathway was targeted by the phthalimide
derivatives (Figure 3).

120
ELAM
Calcein
100
normalized number (%)

80

60

40

20

compound + TNFα

Figure 3. E-Selectin (ELAM) expression in TNFα -induced HUVECs compared to

cytotoxicity determined by Calcein AM. Curcumin (10 μM) was used as a positive control.

1 × 104 HUVECs/well were seeded into 96-well plates and grown to confluence. 50 µM of
phthalimide derivatives were added 30 min prior to application of 10 ng/mL TNFα for another 4 h. The
cells were then fixed and ELAM levels analysed by ELISA. At the same time, the compounds were
analysed by Calcein AM assay to monitor non-specific substance toxicity.
Molecules 2015, 20 16630

2.2.4. Cytotoxic Activity

Sixteen phthalimide derivatives (10a–c, 11a–c, 12, 13a–c, 14, 15, 16, 17a–c) were tested for
cytotoxicity in three different cancer cell lines derived from various tumours (CEM, MCF7, HeLa),
normal human fibroblasts (BJ) and human umbilical vein endothelial cells (HUVEC). Treatment of cancer
cell lines and fibroblasts with tested compounds for 72 h resulted in no loss of viability (data not shown).
After 24 h, the phthalimide derivatives showed no cytotoxicity towards HUVECs (data not shown).

2.3. Molecular Modeling

Compound 17c had the highest anti-inflammatory effects of all tested compounds. Given that the
most important enzymatic system in inflammation is the cyclooxygenase system [20], a molecular
modelling study was carried out to investigate the binding conformation for 17c and the active binding
site of the COX-2 enzyme. The molecular docking study was done using the crystal structure of COX-2
(protein database [PDB] entry: ID 3LN1] [21], with compound 17c (containing COX-2 pharmacophore
moiety –SO2NH2 and celecoxib (6) as the reference ligand. The modelling software which was used for
this study was the MOE version 2008.10 (Molecular Operating Environment-Montreal, QC, Canada) [22].
Celecoxib as the ligand used in this study, was flexibly docked to the binding site of the COX-2
enzyme, and the docking conformation that was represented by the lowest energy score value was selected.
The newly synthesized compound 17c was docked. The docking study showed that 17c occupied the
COX-2 binding site compared to celecoxib (6), (Figure 4).
The binding energy for compound 17c was −17.89 kcal/mol compared to −17.27 kcal/mol for
celecoxib (6). Moreover, compound 17c exhibited four hydrogen bonding interactions with Tyr341
(C=O), Arg106 (C=O), Arg499 (S=O), and Gln178 (NH) and amino acids with hydrogen bond lengths
(2.75, 2.77, 2.96, and 2.21 Å, respectively), (Table 4 and Figure 4).

Table 4. Molecular modelling data for compound 17c and celecoxib (6) during docking in
the COX-2 (PDB: ID3LN1) active site.
Compound No. E-Refine (kcal/mol) No. of Hydrogen Bonds Hydrogen Bonding Residues Distance (Å)
Tyr341 (C=O) 2.75
17c −17.89 4 Arg106 (C=O) 2.77
Arg499 (S=O) 2.96
Gln178 (NH) 2.21
Celecoxib (6) −17.27 2 Leu338 (NH) 2.63
Gln178 (NH) 2.55

3. Experimental Section

3.1. General Information

Melting points were determined on an Electrothermal digital melting point apparatus and are
uncorrected. (IR spectra were recorded on an R 435 spectrophotometer (Middlton, Madison West, WI,
USA) and values were reported in cm−1. 1H-NMR and 13C-NMR were carried out on Bruker Advance
III 400 MHz spectrophotometer (Bruker BioSpin AG, Fällanden, Switzerland) for 1H and 100 MHz for
Molecules 2015, 20 16631

13
C with BBFO Smart Probe and Bruker 400 AEON Nitrogen-Free Magnet, using TMS as an internal
standard and chemical shifts were recorded in ppm on δ scale, Faculty of Pharmacy, Beni Suef University,
Egypt. The electron impact (EI) mass spectra were recorded on a Hewlett Packard 5988 spectrometer
(Palo Alto, CA, USA), Microanalyses for C, H and N were carried out on Perkin-Elmer 2400 analyzer
(Perkin-Elmer, Norwalk, CT, USA) at the Micro analytical unit of Cairo University, Egypt, and all
compounds were within ±0.4% of the theoretical values. Thin-layer chromatography (TLC) was
performed on Merck (Darmstadt, Germany ) TLC aluminium sheets silica gel 60 F254 with detection by
UV quenching at 254 nm to follow the course of reactions and to check the purity of products. All
reagents and solvents were purified and dried by standard techniques.

(a)

(b)

Figure 4. Cont.
Molecules 2015, 20 16632

(c)

(d)

Figure 4. Binding of celecoxib (6) and the most active compound 17c inside COX-2 active
site. (a) 2D Interaction for binding of celecoxib (6) inside COX-2 active site, the most
important amino acids are shown together with their respective numbers; (b) 3D interaction
of celecoxib (6); (c) 2D interaction for binding of the most active compound 17c inside
COX-2 active site, the most important amino acids are shown together with their respective
numbers; (d) 3D interaction of 17c.
Molecules 2015, 20 16633

3.1.1. General Procedure for the Synthesis of Compounds 10a–c

A mixture of 9 (2.65 g, 0.01 mol) and the appropriate primary amine (0.01 mol) was refluxed in
absolute ethanol (30 mL) for 8 h. The solid separated on hot was filtered, dried and crystallized
from dioxane.

(ZE)-2-{4-[1-(4H-1,2,4-Triazol-4-ylimino)ethyl]phenyl}isoindoline-1,3-dione (10a). Yield 32%; m.p.

222–224 °C; IR (KBr, cm−1): 1785 and 1714 (2C=O, isoindoline-1,3-dione); 1H-NMR (DMSO-d6)
δ 2.63 (s, 3H, CH3), 7.64–7.89 (m, 2H, Ar-H), 7.94 (s, 2H, Ar-H), 7.99–8.01 (m, 3H, Ar-H), 8.10–8.24
(m, 3H, Ar-H); 13C-NMR (DMSO-d6) δ 13.50, 124.03, 124.50, 127.54, 129.24, 131.98, 135.33, 136.28,
136.49, 158.80, 167.13; MS (m/z, %): 331 (M+, 1.77%), 64 (100%); Anal. Calcd. for C18H13N5O2: C,
65.25; H, 3.95; N, 21.14. Found: C, 65.03; H, 4.14; N, 21.30%.

(ZE)-2-{4-[1-(1,5-Dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-ylimino)ethyl]phenyl}isoindoline-
1,3-dione (10b). Yield 44%; m.p. 218–220 °C; IR (KBr, cm−1): 1784 and 1714 (2C=O, isoindoline-1,3-
dione), 1679 (C=O); 1H-NMR (DMSO-d6) δ 2.1 (s, 3H, CH3), 2.50 (s, 3H, CH3), 2.63 (s, 3H, CH3),
7.64–7.89 (m, 3H, Ar-H), 7.95–7.98 (m, 7H, Ar-H), 8.00–8.15 (m, 3H, Ar-H);13C-NMR (DMSO-d6)
δ 15.10, 16.50, 39.32, 122.80, 123.94, 127.29, 127.51, 128.38, 128.80, 129.24, 132.01, 133.29, 135.24,
137.20, 138.08, 150.20, 158.80, 160.70, 167.36; MS (m/z, %): 450 (M+, 2.40%), 64 (100%); Anal.
Calcd. for C27H22N4O3: C, 71.99; H, 4.92; N, 12.44. Found: C, 72.13; H, 4.85; N, 12.29%.

(ZE)-5-{1-[4-(1,3-Dioxoisoindolin-2-yl)phenyl]ethylidineamino}-3-methyl-1-phenyl-1H-pyrazole-4-
carbonitrile (10c). Yield 36%; m.p. 232–234 °C; IR (KBr, cm−1): 2212 (C≡N), 1785 and 1713 (2C=O,
isoindoline-1,3-dione);1H-NMR (DMSO-d6) δ 2.1 (s, 3H, CH3), 2.63 (s, 3H, CH3), 7.64 (d, 3H, J = 8 Hz,
Ar-H), 7.96–8.09 (m, 7H, Ar-H), 8.11 (d, 3H, J = 8 Hz, Ar-H); 13C-NMR(DMSO-d6) δ 11.60, 18.40,
92.51, 115.04, 123.80, 123.94, 124.04, 126.20, 127.54,129.25, 131.98, 132.20, 135.34, 136.30, 136.49,
150.20, 150.40, 158.40, 167.13; MS (m/z, %): 445 (M+, 1.47%), 70 (100%); Anal. Calcd. for C27H19N5O2:
C, 72.80; H, 4.30; N, 15.72. Found: C, 72.68; H, 4.42; N, 15.63%.

3.1.2. General Procedure for the Synthesis of Compounds 11a–c

A mixture of 9 (2.65 g, 0.01 mol) and the corresponding o-amino derivative (0.01 mol) was
refluxed in absolute ethanol (30 mL) containing a catalytic amount of glacial acetic acid (0.5 mL) for
12 h. The solid separated on hot was filtered, dried and crystallized from DMF.

2-[4-(2-Methyl-2,3-dihydro-1H-benzo[d]imidazol-2-yl)phenyl]isoindoline-1,3-dione (11a). Yield 46%;

m.p. 292–294 °C; IR (KBr, cm−1): 3447 (broad band, 2NH), 1785 and 1714 (2C=O, isoindoline-1,3-dione);
1
H-NMR (DMSO, d6) δ 2.09 (s, 3H, CH3), 3.35 (s, 1H, NH, D2O exchangeable), 7.22–7.46 (m, 3H,
Ar-H), 7.61–7.63 (m, 3H, Ar-H), 7.65 (s, 1H, NH, D2O exchangeable), 7.71, 7.72 (dd, 2H, J = 3.2 Hz,
3.2 Hz, Ar-H), 7.92, 8.01 (dd, 1H, J = 3.2 Hz, 3.2 Hz, Ar-H), 8.09–8.24 (m, 3H, Ar-H); 13C-NMR
(DMSO-d6) δ 26.30, 74.70, 112.91, 117.70, 120.20, 124.03, 127.34, 128.09, 129.75, 131.02, 132.30,
136.72, 169.08; MS (m/z, %): 355 (M+, 0.17%), 77 (100%); Anal. Calcd. for C22H17N3O2: C, 74.35; H,
4.82; N, 11.82. Found: C, 74.13; H, 4.62; N, 11.80%.
Molecules 2015, 20 16634

2-[4-(2-Methyl-2,3-dihydrobenzo[d]oxazol-2-yl)phenyl]isoindoline-1,3-dione (11b). Yield 26%; m.p.

229–231 °C; IR (KBr, cm−1): 3470 (NH), 1786 and 1713 (2C=O, isoindoline-1,3-dione); 1H-NMR
(DMSO-d6) δ 2.46 (s, 3H, CH3), 4.38 (s, 1H, NH, D2O exchangeable), 7.64 (d, 3H, J = 8 Hz, Ar-H),
7.99–8.00 (m, 6H, Ar-H), 8.11 (d, 3H, J = 8 Hz, Ar-H); 13C-NMR (DMSO-d6) δ 27.32, 98.21, 113.05,
114.01, 124.03, 126.90, 127.54, 128.20, 129.24, 131.99, 132.50, 135.33, 136.29, 136.50, 142.60,
167.13; MS (m/z, %): 356 (M+, 0.36%), 309 (100%); Anal. Calcd. for C22H16N2O3: C, 74.15; H, 4.53;
N, 7.86. Found: C, 74.31; H, 4.62; N, 7.80%.

2-[4-(2-Methyl-2,3-dihydrobenzo[d]thiazol-2-yl)phenyl]isoindoline-1,3-dione (11c). Yield 29%; m.p.

>300 °C; IR (KBr, cm−1): 3456 (NH), 1781 and 1714 (2C=O, isoindoline-1,3-dione); 1H-NMR
(DMSO-d6) δ 2.50 (s, 3H, CH3), 4.45 (s, 1H, NH,D2O exchangeable), 7.19 (t, 1H, J = 7.2 Hz, Ar-H),
7.28 (t, 1H, J = 7.2 Hz, Ar-H), 7.50–7.52 (m, 2H, Ar-H), 7.56–7.65 (m, 2H, Ar-H), 7.67–7.94 (m, 4H,
Ar-H), 7.98 (d, 2H, J = 8.4 Hz, Ar-H). 13C-NMR (DMSO-d6) δ 27.32, 66.81, 122.48, 123.96, 124.03,
127.53, 127.71, 127.76, 129.24, 129.36, 131.98, 135.25, 135.33, 136.28, 136.49, 167.13; MS (m/z, %):
372 (M+, 8.50%), 370 (100%); Anal. Calcd. for C22H16N2O2S: C, 70.95; H, 4.33; N, 7.52. Found: C,
70.73; H, 4.35; N, 7.31%.

3.1.3. Synthesis of (ZE)-2-[4-(1-Hydrazonoethyl)phenyl]isoindoline-1,3-dione (12)

To a solution of compound 9 (2.65 g, 0.01 mol) in dioxane (30 mL), hydrazine hydrate 99.99%
(0.55 g, 0.011 mol) was added and the reaction mixture was heated at reflux temperature for 2 h. The
solid separated while hot was filtered, dried and crystallized from DMF. Yield 24%; m.p. >300 °C; IR
(KBr, cm−1): 3317, 3235 (NH2), 1785 and 1714 (2C=O, isoindoline-1,3-dione); 1H-NMR (DMSO-d6)
δ 2.23 (s, 3H, CH3), 5.79 (s, 2H, NH2, D2O exchangeable), 7.81–7.99 (m, 4H, Ar-H), 8.07–8.14 (m, 4H,
Ar-H); 13C-NMR (DMSO-d6) δ 14.46, 113.60, 125.67, 126.24, 127.13, 128.11, 132.20, 132.73, 150.82,
167.31; MS (m/z, %): 279 (M+, 11.71%), 370 (100%); Anal. Calcd. for C16H13N3O2: C, 68.81; H, 4.69;
N, 15.05. Found: C, 68.67; H, 4.55; N, 14.99%.

3.1.4. General procedure for the Synthesis of Compounds 13a–c

A mixture of 12 (2.79 g, 0.01 mol) and the corresponding isocyanate and/or isothiocyanate
derivative (0.01 mol) was refluxed in dioxane (30 mL) for 5 h. The solid separated while hot was
filtered, dried and crystallized from DMF/MeOH.

2-{4-[4-(4-Chlorophenyl)-3-methyl-5-oxo-1,2,4-triazolidin-3-yl]phenyl}isoindoline-1,3-dione (13a). Yield

47%; m.p. 254–256 oC; IR (KBr, cm−1): 3299, 3224 (2NH), 1785 and 1714 (2C=O, isoindoline-1,3-
dione), 1671 (C=O); 1H-NMR (DMSO-d6) δ 2.50 (s, 3H, CH3), 7.31 (d, 4H, J = 8.8 Hz, Ar-H), 7.34
(d, 4H, J = 8.8 Hz, Ar-H), 7.54–7.67 (m, 1H, Ar-H), 7.88–8.15 (m, 3H, Ar-H), 8.96 (s, 1H, NH, D2O
exchangeable), 11.54 (s, 1H, NH, D2O exchangeable); 13C-NMR (DMSO-d6) δ 27.70, 87.03, 118.17,
120.31, 120.55, 123.70, 125.86, 127.75, 128.90, 129.12, 130.70, 132.20, 139.20, 156.39, 167.31; MS
(m/z, %): 432.5 (M+, 0.7%), 104(100%); Anal. Calcd. for C23H17ClN4O3: C, 63.82; H, 3.96; N, 12.94.
Found: C, 64.02; H, 3.75; N, 13.11%.
Molecules 2015, 20 16635

2-[4-(4-Ethyl-3-methyl-5-thioxo-1,2,4-triazolidin-3-yl)phenyl]isoindoline-1,3-dione (13b). Yield 39%;

m.p. 247–249 °C; IR (KBr, cm−1): 3259, 3158 (2NH), 1782 and 1713 (2C=O, isoindoline-1,3-dione),
1216 (C=S); 1H-NMR (DMSO-d6) δ 1.13 (t, 3H, J = 6Hz, CH2CH3), 2.50 (s, 3H, CH3), 3.61 (q, 2H,
J = 6 Hz, CH2CH3), 7.88–7.98 (m, 5H, Ar-H), 8.06–8.23 (m, 3H, Ar-H), 9.15 (s, 1H, NH, D2O
exchangeable), 11.54 (s, 1H, NH, D2O exchangeable); 13C-NMR (DMSO-d6) δ 15.03, 37.13, 40.59,
90.03, 123.70, 125.59, 127.38, 128.63, 132.20, 133.06, 147.70, 167.31, 182.18; MS (m/z, %): 366
(M+, 0.5%), 104 (100%); Anal. Calcd. for C19H18N4O2S: C, 62.28; H, 4.95; N, 15.29. Found: C, 62.41;
H, 4.78; N, 15.33%.

2-[4-(3-Methyl-5-thioxo-4-phenyl-1,2,4-triazolidin-3-yl)phenyl]isoindoline-1,3-dione (13c). Yield 42%;

m.p. >300 °C; IR (KBr, cm−1): 3437, 3164 (2NH), 1785 and 1714 (2C=O, isoindoline-1,3-dione), 1217
(C=S); 1H-NMR (DMSO-d6) δ 2.50 (s, 3H, CH3), 7.15 (t, 1H, J = 7.2 Hz, Ar-H), 7.33 (t, 2H, J = 7.6 Hz,
Ar-H), 7,54 (d, 2H, J = 7.2 Hz, Ar-H), 7.88–7.99 (m, 4H, Ar-H), 8.06–8.19 (m, 4H, Ar-H), 9.45
(s, 1H, NH, D2O exchangeable), 11.54 (s, 1H, NH, D2O exchangeable); 13C-NMR (DMSO-d6) δ 14.44,
70.80, 123.92, 125.60, 127.36, 127.50, 132.02, 133.02, 133.5, 135.23, 137.63, 139.81, 147.49, 167.39,
179.39; MS (m/z, %): 414 (M+, 1.25%), 104 (100%); Anal. Calcd. for C23H18N4O2S: C, 66.65; H, 4.38;
N, 13.52. Found: C, 66.42; H, 4.17; N, 13.55%.

3.1.5. Synthesis of 2-[4-(3-Methyl-5-thioxo-1,2,4-triazolidin-3-yl)-phenyl]isoindole-1,3-dione (14)

To a solution of 9 (2.65g, 0.01 mol) in dioxane (30 mL), thiosemicarbazide (0.91 g, 0.01 mol) was
added. The reaction mixture was refluxed for 3 h. The solid separated on hot was filtered, dried and
crystallized from DMF/acetone. Yield 33%; m.p. >300 °C; IR (KBr, cm−1): 3417–3271 (3NH), 1781
and 1707 (2C=O, isoindoline-1,3-dione), 1222 (C=S); 1H-NMR (DMSO-d6) δ 2.35 (s, 3H, CH3), 7.48
(d, 2H, J = 8.4 Hz, Ar-H), 7.90, 7.91 (dd, 2H, J = 3.6 Hz, 2.8 Hz, Ar-H), 7.94, 7.96 (dd, 2H, J = 8 Hz,
2.8 Hz, Ar-H), 8.04 (s, 1H, NH, D2O exchangeable), 8.09 (d, 2H, J = 8 Hz, Ar-H), 8.35 (s, 1H, NH,
D2O exchangeable), 10.31 (s, 1H, NH, D2O exchangeable); 13C-NMR (DMSO-d6) δ 14.44, 66.80,
123.92, 127.36, 127.50, 132.02, 133.02, 135.23, 137.63, 167.39, 179.40; MS (m/z,%): 338 (M+, 0.22%),
153 (100%); Anal. Calcd. for C17H14N4O2S: C, 60.34; H, 4.17; N, 16.65. Found: C, 60.52; H, 4.17;
N, 16.84%.

3.1.6. Synthesis of (ZE)-Nʹ-{1-[4-(1,3-Dioxoindolin-2-yl)phenyl]ethylidene}benzohydrazide (15)

A mixture of 9 (2.65 g, 0.01 mol) and benzohydrazide (1.36 g, 0.01 mol) was refluxed in dioxane
(30 mL) for 7 h. The solid separated on hot was filtered, dried and crystallized from propanol. Yield
48%; m.p. 247–249 °C; IR (KBr, cm−1): 3330 (NH), 1781 and 1704 (2C=O, isoindoline-1,3-dione),
1672 (C=O); 1H-NMR (DMSO-d6) δ 2.56, 2.64 (2s, 3H, syn & anti CH3), 7.59–7.68 (m, 5H, Ar-H),
7.98–8.10 (m, 8H, Ar-H), 10.85 (s, 1H, NH, D2O exchangeable); 13C-NMR (DMSO-d6) δ 22.70, 123.95,
124.50, 127.30, 127.51, 128.41, 128.79, 129.40, 132.03, 133.11 135.25, 138.09, 147.79, 163.20, 167.36;
MS (m/z, %): 383 (M+, 4.05%), 105 (100%); Anal. Calcd. for C23H17N3O3: C, 70.05; H, 4.47; N, 10.96.
Found: C, 69.98; H, 4.25; N, 10.97%.
Molecules 2015, 20 16636

3.1.7. Synthesis of 2-[4-(2-Methyl-2H-imidazol-2-yl)phenyl]isoindole-1,3-dione (16)

To a solution of 9 (2.65 g, 0.01 mol) in dioxane (30 mL), ethylenediamine (0.6 g, 0.01 mol) was
added. The reaction mixture was refluxed for 4 h. The solution was allowed to cool; the solid separated
was filtered, dried and crystallized from ethanol 95%. Yield (26%); m.p. 215–217 °C; IR (KBr, cm−1):
1781 and 1704 (2C=O, isoindoline-1,3-dione); 1H-NMR (DMSO-d6) δ 2.62 (s, 3H, CH3), 6.56 (d, 1H,
J = 8.4Hz, Ar-H), 7.49–7.54 (m, 8H, Ar-H), 7.66 (d, 1H, J = 8.4Hz, Ar-H); 13C-NMR (DMSO-d6)
δ 27.32, 66.81, 124.03, 127.53, 128.58, 129.24, 131.98, 135.33, 136.28, 136.49, 167.13; MS (m/z, %):
303 (M+, 5.6%), 80 (100%); Anal. Calcd. for C18H13N3O2: C, 71.28; H, 4.32; N, 13.85. Found: C,
71.08; H, 4.27; N, 14.01%.

3.1.8. General Procedure for the Synthesis of Compounds 17a–c

A mixture of 9 (2.65 g, 0.01 mol) and the corresponding phenyl hydrazine hydrochloride derivative
(0.01 mol) was heated at reflux temperature in absolute ethanol (30 mL) for 5–8 h (monitored by TLC).
The solid separated on hot was filtered, dried and crystallized from dioxane.

2-{4-[1-(Phenyl-hydrazono)-ethyl]-phenyl}-isoindole-1,3-dione (17a). Yield 49%; m.p. 185–187 °C;

IR (KBr, cm−1): 3355 (NH), 1783 and 1711 (2C=O, isoindoline-1,3-dione); 1H-NMR (DMSO-d6)
δ 2.30, 2.63 (2s, 3H, syn & anti CH3), 7.26–7.45 (m, 3H, Ar-H), 7.47 (d, 1H, J = 8.4 Hz, Ar-H), 7.64
(d, 1H, J = 8.8 Hz, Ar-H), 7.93–7.97 (m, 4H, Ar-H), 7.99 (m, 3H, Ar-H), 8.11 (d, 1H, J = 8.8 Hz, Ar-H),
9.87 (s, 1H, NH, D2O exchangeable); 13C-NMR (DMSO-d6) δ 13.27, 113.34, 119.49, 124.03, 127.53,
128.58, 129.24, 131.98, 132.01, 135.33, 136.28, 139.35, 150.80, 167.49; MS (m/z, %): 355 (M+, 9.26%),
250 (100%); Anal. Calcd. for C22H17N3O2: C, 74.35; H, 4.82; N, 11.82. Found: C, 74.28; H, 4.69;
N, 12.03%.

2-(4-{1-[(4-Methanesulfonyl-phenyl)-hydrazono]-ethyl}-phenyl)-isoindole-1,3-dione (17b). Yield 51%;

m.p. 208–210 °C; IR (KBr, cm−1): 3330 (NH), 1783 and 1714 (2C=O, isoindolne-1,3-dione);1H-NMR
(DMSO-d6) δ 2.36, 2.63 (2s, 3H, syn & anti CH3), 3.12 (s, 3H, SO2CH3), 7.42 (d, 1H, J = 8.8 Hz,
Ar-H), 7.50 (d, 1H, J = 8.2 Hz, Ar-H), 7.64 (d, 2H, J = 8.8 Hz, Ar-H), 7.75 (d, 1H, J = 8.2 Hz, Ar-H),
7.96–8.01 (m, 5H, Ar-H), 8.11 (d, 2H, J = 8.4 Hz, Ar-H), 9.98 (s, 1H, NH, D2O exchangeable);
13
C-NMR (DMSO-d6) δ 13.77, 44.73, 112.88, 123.92, 126.42, 127.52, 127.56, 129.15, 130.20, 132.01,
132.10, 135.22, 136.26, 150.26, 167.44; MS (m/z, %): 433 (M+, 58.21%), 163 (100%); Anal. Calcd.
for C23H19N3O4S: C, 63.73; H, 4.42; N, 9.69. Found: C, 63.58; H, 4.66; N, 9.73%.

4-(Nʹ-{1-[4-(1,3-Dioxo-1,3-dihydro-isoindol-2-yl)-phenyl]-ethylidene}-hydrazino)-benzenesulfonamide
(17c). Yield 52%; m.p. 265–267 °C; IR (KBr, cm−1): 3428–3258 (NH2 and NH), 1784 and 1711
(2C=O, isoindoline-1,3-dione); 1H-NMR (DMSO-d6) δ 2.34, 2.51 (2s, 3H, syn & anti CH3), 7.1 (s, 2H,
NH2, D2O exchangeable), 7.36 (d, 2H, J = 8.8 Hz, Ar-H), 7.49 (d, 2H, J = 8.8 Hz, Ar-H), 7.68 (d, 2H,
J = 8.8 Hz, Ar-H), 7.96–8.12 (m, 6H, Ar-H), 9.88 (s, 1H, NH, D2O exchangeable); 13C-NMR (DMSO-d6)
δ 13.65, 112.56, 123.92, 126.32, 127.57, 127.71, 131.93, 132.03, 134.21, 135.22, 138.89, 142.89,
148.90, 167.47; MS (m/z, %): 434 (M+, 32.06%), 88 (100%); Anal. Calcd. for C22H18N4O4S: C, 60.82;
H, 4.18; N, 12.90. Found: C, 60.69; H, 4.17; N, 12.73%.
Molecules 2015, 20 16637

3.2. Biological Testing

3.2.1. Antimicrobial Sensitivity Test Using Agar Diffusion Method (Cup Technique)

The cup plate method is an accepted method when test samples diffuse from the cup through an agar
layer in a Petri dish or plate to such an extent that the growth of added microorganisms is completely
restricted to a circular area or zone around the cavity containing the solution of an antibiotic substance [23].
The anti-microbial activity was expressed as zone diameter in millimetres, which is measured by a
ruler [24].

Microorganisms

Gram-positive organisms used in the study were Staphylococcus aureus LMG 3242, Sarcina lutea,
Enterococcus faecalis OS4, Listeria innocua LMG 2710, Mycobacterium pheli and Bacillus subtilis.
Gram-negative organisms used were Escherichia coli ATCC 5087, Escherichia coli ATCC 25922,
Proteus vulgaris and Pseudomonas aeruginosa. The yeast used was Candida albicans.

Standard Antimicrobials

Gentamicin (CN), Cefotaxime (CTX), Ampicillin (AMP), Fluconazole (FLU) and Tioconazole (TIO)
were all were prepared in concentration of 10 mg/mL.

Preparation of Sample Stock Solutions

All test samples (10a–c, 11a–c, 12, 13a–c, 14, 15, 16, 17a–c) were dissolved in DMSO to give
a final concentration of 10 mg/mL.

Experimental Procedure

Each overnight culture of the tested microorganisms was mixed with Muller Hinton agar media to
give a final concentration of 1% microorganism (about 0.5 McFarland) and poured into sterile Petri
dishes in a fixed amount of 20 mL under aseptic conditions. A sterile cork borer was used to prepare
cups of 10 mm diameter. Test samples and standard drugs with volumes of 60 μL were introduced into
the cups using a micropipette. All the plates were kept at room temperature for effective diffusion of the
test drug and standard and incubated at 37 ± 1 °C for 24 h. The presence of inhibition zones around the cup
indicated antibacterial activity. The diameter of the zone of inhibition was measured and recorded [25]. The
percentage inhibition activities of tested compounds relative to standard antibiotics were calculated by
applying this equation:
The percentage inhibition = (Zone of inhibition of tested compound − cup diameter)/
(Zone of inhibition of standard antibiotic − cup diameter) × 100
Molecules 2015, 20 16638

Determination of Minimum Inhibitory Concentration (MIC) Using Agar Dilution Method According
to Clinical Laboratory Standards Institute (CLSI)

For each sample, different concentrations were diluted with Muller Hinton agar to give a final
concentration ranging from (200 µg/mL–0.7 µg/mL). DMSO was used as negative control plate. All
bacterial isolates were subcultured on Brain Heart Infusion agar (B.H.I.A.) and incubated at 37 °C for
24 h [26,27]. Three colonies of each microorganism were suspended in 5 mL saline, and the suspension
was adjusted to 0.5 McFarland standards and then diluted 10-fold with saline to give an organism
suspension of (1 × 106 to 5 × 106 CFU/mL). This suspension was then further diluted by putting 1 mL
suspension in 9 mL saline to give a final suspension volume of 1 × 105 to 5 × 105 CFU/mL. A multiple
inoculator was used to inoculate the prepared agar plates. A 100 μL (i.e., 104 CFU) of the prepared
inoculums were put in the well of a multi-inoculator, where each inoculation time by multi-inoculator
gave about 10 μL of prepared inoculums per plate (i.e., 103 CFU). Each experiment was performed in
duplicate. All plates were incubated at 35 °C for 48 h. The results were recorded in terms of MIC,
which is the lowest concentration of antimicrobial/antifungal agent causing almost complete inhibition
of growth or giving no visible growth.

3.2.2. Anti-Oxidant Testing

Determination of Oxygen Radical Absorbance Capacity (ORAC)

Oxygen radical absorbance capacity (ORAC) was determined as described previously [28]. Briefly,
100 μL of 500 nM fluorescein and 25 μL of diluted solutions of tested compounds were pipetted into
each working well of a microplate (96 well) preincubated at 37 °C. 25 μL of 250 mM AAPH was
then added and the microplate was shaken for 5 s in a fluorometer Infinite 200 (Tecan, Mannedorf,
Switzerland). The fluorescence (Ex. 485 nm, Em. 510 nm) was read every 2 min for 60 min. The net area
under the curve was used to calculate ORAC which was expressed as a ratio between tested compound
and trolox on an equimolar basis.

3.2.3. Anti-Inflammatory Testing

Cell Culture

Stock solutions (10 mmol/L) of tested compounds were prepared by dissolving an appropriate
quantity of each substance in DMSO. Dulbecco’s modified Eagle’s medium (DMEM, RPMI 1640
medium), foetal bovine serum (FBS), L-glutamine, penicillin and streptomycin were purchased from
Sigma (St. Louis, MO, USA). Calcein AM was obtained from Molecular Probes (Life Technologies,
Carlsbad, CA, USA).
The screening cell lines (T-lymphoblastic leukaemia cell line CEM, breast carcinoma cell line
MCF7, cervical carcinoma cell line HeLa and human fibroblasts BJ) were obtained from the American
Type Culture Collection (Manassas, VA, USA). CEM cell line were cultured in RPMI 1640 medium
and the others in DMEM medium (Sigma); both media supplemented with 10% foetal bovine serum,
2 mmol/L L-glutamine, 10,000 U penicillin and 10 mg/mL streptomycin. The cell lines were maintained
Molecules 2015, 20 16639

under standard cell culture conditions at 37 °C and 5% CO2 in a humid environment. Cells were
subcultured twice or three times a week using the standard trypsinization procedure.
Human Umbilical Vein Endothelial Cells (HUVEC) were cultured in ECGM medium (Endothelial
Cell Growth Medium, Provitro, Berlin, Germany), supplemented with 10% foetal bovine serum
(Sigma-Aldrich, Munich, Germany). Cells were maintained under standard cell culture conditions at
37 °C and 5% CO2 in a humid environment. Cells were subcultured twice or three times a week using the
standard trypsinization procedure. HUVECs were a kind gift from Prof Jitka Ulrichová (Medical Faculty,
Palacky University, Olomouc, The Czech Republic).

CD62E (E-Selectin, ELAM)-induction Assays

Each well of the 96-well plates were coated with collagen G by applying 200 µL for 15 min at 37 °C.
Outer wells (A1-A12, H1-H12, 1-H1 and A12-H12) contained only 200 µL/well medium and served
as an evaporation barrier. 1 × 104 HUVECs (Human Umbilical Vein Endothelial Cells) were seeded in
each of the other wells in 200 µL medium and grown for 48 h to optimal confluence. Increasing
concentrations of compounds were then added to the HUVEC-containing wells in triplicates, and the
cells were incubated for 30 min, after which 10 ng/mL TNFα was added per well to stimulate NF-κB,
and thus ELAM. After another 4 h incubation, the levels of ELAM in each of the HUVEC-containing
wells were determined by enzyme-linked activity assays (ELISAs) as described below.

Cell-Surface ELISA ELAM

Cells were washed once with PBS and fixed with 0.1% glutaraldehyde (Sigma-Aldrich), for 15 min
at room temperature. The cells were then washed 3 times with 200 µL per well PBS/0.05% Tween 20,
blocked with 200 µL/well 5% BSA/PBS for 1 h, and washed again 3 times with 200 µL per well
PBS/0.05% Tween 20. Anti-ELAM-antibody (clone BBA-1, R & D Systems, Minneapolis, MN, USA)
diluted 1:5000 in 0.1% BSA/PBS (100µL per well) was then added for 1 h at room temperature and
washed 5 times with 200 µL per well PBS/0.05% Tween 20. Subsequently, goat anti mouse-HRP antibody
(Sigma-Aldrich) diluted 1:10000 in 0.1% BSA/PBS (100 µL per well) was applied and the cells were
incubated for 1 h in the dark at room temperature and, after decanting, washed 5 times with 200 µL
per well PBS/0.05% Tween 20. The HRP-activity of the cells in each of the wells was estimated using
Fast-OPD (o-phenylenediamine dihydrochloride) (Sigma-Aldrich) assay as described [29] and absorbance
was measured at OD492nm in a plate reader (Tecan).

3.2.4. Cytotoxicity Testing

For the ELAM expression assay the toxicity of tested compounds was assessed in HUVECs by
Calcein AM (Molecular Probes, Invitrogen, Karlsruhe, Germany) cytotoxicity assays in 96-well microtiter
plates [30]. 20 µL portions of each of the compound concentrations were added in triplicate to the cells,
which were then incubated at 37 °C in an atmosphere containing 5% CO2 for 4 h, after which Calcein
AM solution was added for 1 h according to the manufacturer’s instructions. The fluorescence of viable
cells was quantified using a Fluoroskan Ascent instrument (Lab-systems, Vantaa, Finland) reader and on
the basis of triplicate experiments the cytotoxic concentrations were calculated. Cytotoxicity of tested
Molecules 2015, 20 16640

compounds was determined also in HUVEC after 24 h and in CEM, MCF7, HeLa and BJ after 72 h by
Calcein AM assays as described above.

Molecular Modeling and Docking

The celecoxib-COX-2 crystal structure was obtained from (PDB: ID 3LN1) [21]. Docking of the ligand
was carried out. The root mean square deviation was 0.50 Å. Docking was performed using London
dGforce. Force field energy was used to refine the results. The most active compound 17c was docked
by MOE after preparation of the selected compound through its 3D protonation and selecting the least
energetic conformer. The same docking method used for both ligand and 17c. Amino acid interactions
and hydrogen bond lengths were measured and summarized in (Table 4).

4. Conclusions

The tested compounds in this study were evaluated against Gram positive, Gram negative and Fungi.
The most active phthalimide derivative as an anti-microbial agent was hydrazonoethyl-phenylisoindoline-
1,3-dione 12. Most phthalimide derivatives showed antimicrobial activity with the exception of 10a and
10b and 13a and 10 b on Gram negative bacteria, in addition to 13a which had no action on Candida
albicans. None of the tested phthalimide derivatives had any cytotoxic activity. Two, 13b and 13c
(ethyl and phenyl isothiocyanate derivatives) had anti-oxidant activity. Phthalimide derivatives had mild
anti-inflammatory activity in vitro albeit a strong inhibition of E-selectin by 17c.

Acknowledgments

Phoebe F. Lamie would like to express her deepest thanks to all the members of the NMR center at
Beni Suef University, Beni Suef, Egypt for their efforts in analyzing all the prepared compounds. All
cytotoxic, anti-inflammatory and anti-oxidant assays were financed by a Czech Ministry of Education
grant from the National Program for Sustainability I (LO1204). We thank Jitka Ulrichová for the kind
gift of HUVEC cells and Olga Hustáková for excellent technical assistance.

Author Contributions

P.F.L. proposed the subject and carry out the synthesis, purification and characterization of the
compounds. J.N.P. conceived the study and participated in its design, results, discussion, and coordination.
A.O.E.-G. carried out the anti-microbial screening. L.R. and J.G. carried out the anti-inflammatory,
anti-oxidants and cytotoxic assay.

Conflicts of Interest

The authors have no conflict of interest to declare.

References

1. Kamal, A.; Bolla, N.R.; Srikanth, P.S.; Srivastava, A.K. Naphthalimide derivatives with therapeutic
characteristics: A patent review. Expert Opin. Ther. Pat. 2013, 23, 299–317.
Molecules 2015, 20 16641

2. Amin, K.M.; El-masry, A.H.; Mohamed, N.A.; Awad, G.E.A.; Habib, B.S. Synthesis, characterization
and anti-microbial activity of some novel isoindole-1,3-dione derivatives. Der Pharm. Chem. 2013,
5, 97–108.
3. El-Gaby, M.S.A.; Zahran, M.A.; Ismail, M.M.F.; Ammar, Y.A.A. A novel Synthesis of
dibenzo[c,f]chromenes, dibenzo[c,h]chromenes and benzo[7,8]chromeno[3,4-f]isoindoles as
anti-microbial agents. IlFarmaco 2000, 55, 227–232.
4. Rajasekaran, S.; Rao, G.K.; Pai, S.; Ranjan, A. Design, synthesis, anti-bacterial and in vitro anti-oxidant
activity of substituted 2H-benzopyran-2-one derivatives. Int. J. ChemTech Res. 2011, 3, 555–559.
5. Pophale, R.A.; Deodhar, M.N. Synthesis and evaluation of novel phthalimide derivatives as
analgesic and anti-inflammatory agents. Der Pharm. Chem. 2010, 2, 185–193.
6. Bhatnagar, A.; Sharma, P.K.; Kumar, N.; Upadhyay, A. Synthesis and in vitro evaluation of
anti-oxidant and anti-inflammatory activity of 3-[4,5-dihydro-(5-substituted phenyl)-1H-pyrazol-3-
yl]-2H-chromen2-one derivatives. Pharm. Chem. J. 2012, 46, 482–487.
7. Bansode, T.N.; Shelke, J.V.; Dongre, V.G. Synthesis and antimicrobial activity of some new
N-acyl substitutedphenothiazines. Eur. J. Med. Chem. 2009, 44, 5094–5098.
8. Siddiqui, N.J.; Idrees, M.; Khati, N.T.; Dhond, M.G. Synthesis and anti-microbial activities of
some new pyrazoles, oxadiazoles and isoxazole bearing benzofuran moiety. S. Afr. J. Chem. 2013,
66, 248–253.
9. Anthony, P.; Bashir, N.; Parveen, R. Regioselectivesynthesisof 1,4-disubstituted 1,2,3-bistriazoles
and their anti-fungal and anti-oxidant evaluation. Asian J. Biomed. Pharm. Sci. 2014, 4, 9–13.
10. Bosquesi, P.L.; Melo, T.R.F.; Vizioli, E.O.; Santos, J.L.; Chung, M.C. Anti-inflammatory drug
design using a molecular hybridization approach. Pharmaceuticals 2011, 4, 1450–1474.
11. Kaur, J.; Bhardwaj, A.; Huang, Z.; Knaus, E.E. N-1 and C-3 substituted indole Schiff bases as
selective COX-2 inhibitors: synthesis and biological evaluation. Bioorg. Med. Chem. 2012, 22,
2154–2159.
12. Kaplancikli, Z.A.; Altintop, M.D.; Ozdemir, A.; Turan, Z.G.; Khan, S.I.; Tabanca, N. Synthesis
and biological evaluation of some hydrazine derivatives as anti-inflammatory agents. Lett. Drug
Des. Discov. 2012, 9, 310–315.
13. Kushwaha, T.A. Synthesis of novel 2-benzylbenzo[d]thzole-6-sulfonamide derivatives as potential
anti-inflammatory agent. J. Chem. Pharm. Sci. 2014, 7, 34–38.
14. Shiradkar, M.R.; Ghodake, M.; Bothara, K.G.; Bhandari, S.V.; Nikalje, A.; Akula, K.C.; Desai, N.C.;
Burange, P.J. Synthesis and anti-convulsant activity of clubbed thiazolidinone-barbituric acid and
thiazolidinone–triazole derivatives. ARKIVOC 2007, XIV, 58–74.
15. Moffet, R.S. Central Nervous System Depressants. VII.1 pyridylcoumarins. J. Med. Chem. 1964,
7, 446–449.
16. Mavrova, A.T.; Wesselinova, D.; Tsenov, Y.A.; Denkova, P. Synthesis, cytotoxicity and effects of
some 1,2,4-triazole and 1,3,4-thiadiazole derivatives on immunocompetent cells. Eur. J. Med. Chem.
2009, 44, 63–69.
17. Pertino, M.W.; Verdugo, V.; Theoduloz, C.; Hirschmann, G.S. Synthesis and anti-proliferative
activity of some novel triazole derivatives from dehydroabietic acid. Molecules 2014, 19, 2523–2535.
18. Sharma, V.; Shrivastava, B.; Bhatia, R.; Bachwani, M.; Bachwani, M.; Khandelwal, R.; Ameta, J.
Exploring potential of 1,2,4-triazole: A brief review. Pharmacol. Online 2011, 1, 1192–1222.
Molecules 2015, 20 16642

19. Lamie, P.F. Synthesis and anti-microbial activity of some novel isoindoline-1,3-dione derivatives.
J. Adv. Chem. 2014, 8, 1660–1666.
20. Abdellatif, K.R.; Chowdhury, M.A.; Dong, Y.; Velázquez, C.; Das, D.; Suresh, M.R.; Knaus, E.E.
Diazen-1-ium-1,2-diolated nitric oxide donor ester prodrugs of 5-(4-hydroxymethylphenyl)-1-(4-
aminosulfonylphenyl)-3-trifluromethyl-1H-pyrazole and its methanesulfonyl analog: Synthesis,
biological evaluation and nitric oxide release studies. Bioorg. Med. Chem. 2008, 16, 9694–9698.
21. RCSB Protein Data Bank. Available online: [Link] (accessed on
24 August 2015).
22. Abdellatif, K.R.; Huang, Z.; Chowdhury, M.A.; Kaufman, S.; Knaus, E.E. A diazen-1-ium-1,2-
diolated nitric oxide donor ester prodrug of 3-(4-hydroxymethylphenyl)-4-(4-methanesulfonylphenyl)-
5H-furan-2-one: Synthesis, biological evaluation and nitric oxide release studies. Bioorg. Med.
Chem. Lett. 2011, 21, 3951–3956.
23. Seeley, H.W.; Van, D.P.J. A Laboratory Manual of Microbiology, 2nd ed.; Sons and Co: Bombay,
India, 1975; pp. 55–80.
24. Ahmed, S.H.; Amin, M.A.; Saafan, A.E.; El-Gendy, A.O.; El-Gendy, A.O.; Islam, M. Measuring
susceptibility of Candida albicans biofilms towards antifungal agents. Der Pharm. Lett. 2013, 5,
376–383.
25. El-Gendy, A.O.; Essam, T.M.; Amin, M.A.; Ahmed, S.H.; Nes, I.F. Clinical Screening for
Bacteriocinogenic Enterococcus faecalis Isolated from Intensive Care Unit Inpatient in Egypt.
J. Microb. Biochem. Technol. 2012, 4, 161–167.
26. Sader, H.S.; Flamm, R.K.; Jones, R.N. Antimicrobial activity of daptomycin tested against
Gram-positive pathogens collected in Europe, Latin America, and selected countries in the
Asia-Pacific Region. Diagn. Microbiol. Infect. Dis. 2013, 75, 417–422.
27. Tarek, N.; Hassan, H.M.; AbdelGhani, S.M.; Radwan, H.O.; El-Gendy, A.O. Comparative chemical
and anti-microbial study of nine essential oils obtained from medicinal plants growing in Egypt.
Beni Suef Univ. J. Basic Appl. Sci. 2014, 3, 149–156.
28. Ou, B.; Hampsch-Woodill, M.; Prior, R.L. Development and validation of improved oxygen
radical absorbance capacity assay using fluorescein as the fluorescent probe. J. Agric. Food. Chem.
2001, 49, 4619–4626.
29. Gridling, M.; Stark, N.; Madlener, S.; Lackner, A.; Popescu, R.; Benedek, B.; Diaz, R.; Tut, F.M.;
Nha Vo, T.P.; Huber, D.; et al. In vitro anti-cancer activity of two ethno-pharmacological healing plants
from Guatemala Pluchea odorata and Phlebodiumdecumanum. Int. J. Oncol. 2009, 34, 1117–1128.
30. Madlener, S.; Svacinova, J.; Kitner, M.; Kopecky, J.; Eytner, R.; Lackner, A.; Vo, T.P.; Frisch, R.;
Grusch, M.; de Martin, R.; et al. In vitro anti-inflammatory and anti-cancer activities of extracts of
Acalypha alopecuroidea (Euphorbiaceae). Int. J. Oncol. 2009, 35, 881–891.

Sample Availability: Samples of the compounds 9, 12, 14, 15, 17a–c are available from the authors.

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