Synthesis, characterization, molecular modeling, and potential antimicrobial and anticancer activities of novel 2-aminoisoindoline-1,3-dione derivatives

Article abstract of DOI:10.1016/j.bioorg.2016.03.003

In an effort to establish new drug candidates with improved antimicrobial and anticancer activities, we report here synthesis, molecular modeling, and in vitro biological evaluation of novel substituted N-amino phthalamide derivatives (3a-b, 4a-b, 5a-j, and 6). Structures of the newly synthesized compounds were described by IR, ¹H & ¹³CNMR and LC-MS spectral data. The novel compounds were evaluated for their antibacterial activity against four types of Gm+ve and two for Gm-ve types, and antifungal activity against three fungi microorganisms by well diffusion method. Of these novel compounds, Schiff bases showed mostly promising antibacterial activity compared to reference drugs. A successful step was done for explanation of their mode of action through molecular docking of most active molecules at DNA gyrase B enzyme and further were biologically tested. Moreover, the antiproliferative activity was tested against two human carcinoma cell lines (Human colon carcinoma (HCT-116) and human breast adenocarcinoma (MCF-7)) showing promising anticancer activity compared to doxorubicin drug. The data from structure-activity relationship (SAR) analysis revealed that the lypophilic properties of these compounds might be essential parameter for their activity and suggest that 2-amino phthalamide scaffold derivatives 5g and 5h exhibited good antimicrobial and anticancer activities and might used as leads for further optimization.

Full text of DOI:10.1016/j.bioorg.2016.03.003

Accepted Manuscript
Synthesis, Characterization, Molecular Modeling, and Potential Antimicrobial
and Anticancer Activity of Novel 2-Aminoisoindoline-1,3-dione Derivatives
Hany Emary Ali Ahmed, Hassan A. Abdel-Salam, Mohamed A. Shaker
PII:
S0045-2068(16)30022-0
DOI:
http://dx.doi.org/10.1016/j.bioorg.2016.03.003
YBIOO 1890
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
16 February 2016
8 March 2016
9 March 2016
Please cite this article as: H.E.A. Ahmed, H.A. Abdel-Salam, M.A. Shaker, Synthesis, Characterization, Molecular
Modeling, and Potential Antimicrobial and Anticancer Activity of Novel 2-Aminoisoindoline-1,3-dione
Derivatives, Bioorganic Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bioorg.2016.03.003
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Synthesis, Characterization, Molecular Modeling, and Potential Antimicrobial and
Anticancer Activity of Novel 2-Aminoisoindoline-1,3-dione Derivatives
1
,2*
3,4
3,5
Hany Emary Ali Ahmed , Hassan A. Abdel-Salam and Mohamed A. Shaker
1
Pharmacognosy and Pharmaceutical Chemistry Department, College of Pharmacy,
Taibah University, Al-Madinha Al-Munawaraha, Saudi Arabia.
2
Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Al-Azhar
University, Cairo, Egypt.
3
Pharmaceutics and Pharmaceutical Technology Department, College of Pharmacy,
Taibah University, Al-Madinha Al-Munawaraha, Saudi Arabia.
4
Department of Microbiology, Faculty of Pharmacy, Zagazig University, Zagazig 44519,
Egypt
5
Department of Pharmaceutics, Faculty of Pharmacy, Helwan University, Cairo, Egypt.
*
Corresponding author: Hany E. A. Ahmed
Tel: +966534391522
E-mail: jan_25_misr@yahoo.com
Running Title: Design and synthesis of novel 2-aminoisoindoline-1,3-dione compounds
Key words: 2-Aminoisoindoline-1,3-dione, sciff base, anticancer activity, structure-
selectivity relationships
1

Abstract
In an effort to establish new drug candidates with improved antimicrobial and anticancer
activities, we report here synthesis, molecular modeling, and in vitro biological evaluation of
novel substituted N-amino phthalamide derivatives (3a-b, 4a-b, 5a-j, and 6). Structures of
1
13
the newly synthesized compounds were described by IR, H & CNMR and LC-MS spectral
data. The novel compounds were evaluated for their antibacterial activity against four tpes of
Gm+ve and two for Gm-ve types, and antifungal activity against three fungi micro-organisms
by well diffusion method. Of these novel compounds, Schiff bases showed mostly promising
antibacterial activity compared to reference drugs. A successful step was done for
explanation of their mode of action through molecular docking of most active molecules at
DNA gyrase B enzyme and furthure were biologically tesed. Moreover, the antiproliferative
activity was tested against two human carcinoma cell lines (Human colon carcinoma (HCT-
116) and human breast adenocarcinoma (MCF-7)) showing promising anticancer activity
compared to doxroubcin drug. The data from structure-actvity relationship (SAR) analysis
revealed that the lypophilic properties of these compounds might be essential parameter for
their activity and suggests that 2-amino phthalamide scaffold derivatives 5g and 5h
exihibited good antimicrobial and anticancer activities and might used as leads for furthure
optimization.
2

1
. Introduction
The major of health problems faced by mankined at the present time are cancer and the
infectous diseases. The drug discovery research of antimicrobial agents, accompanied by
clinical development, has largely been conducted by many pharmaceutical companies. The
number of different classes of antibacterial [1, 2] and antifungal agents [3] have been
discovered. A major problem have been occurd by extensive use of antibiotics is the
appearance of multi-drug resistant microbial pathogens and stimulated the search for
discovery of new drugs [4]. Research efforts in cancer have led to much information that
allow to recognize new targets and molecular mechanisms to devolp a wide packages of
therapeutic agents. However, two properties of such marketed drugs are lacked, the first:
the optimal quantities of the drugs are not reached for the targets; and the second: they are
not effective enough in the tumor area [5]. Therefore, there is urgent need to search new
specific anticancer agents [6, 7] and to develop new safer, potent, and resistance-free
antimicrobial drugs [8]. Phthalamide ring (isoindoline-1,3-dione) represents a very
importantpreviliged substructurein organic synthesis forpreparing diverse biologically active
molecules [9-14].Typically, the most important pharmacological effects that have been
reported for phthalimide derivatives are anti-cancer [15], anti-microbial [16], anti-oxidant
[17], anti-inflammatory [18] and analgesic [19] activities.The chemical nature of
phthalimides(-CO-N(R)-CO-) look somehow hydrophobic and this increases their potential to
cross different biologicalmembranes in vivo [20].Hence, the chance to increase the
biological activity of phthalimide derivatives occurs through practical molecular hybridization
approach to introduce different pharmacophore subunits like sciff bases of aromatic
3

substituents and acyl moieties. Inspired by the presence of highly active reference
compounds (A-G) composed of phthalamide ring, Figure 1, and different side chain of
different properties with anticancer and antimicrobial activities profile, we have studied their
structures and introduced a new derivatives bearing the hot spots that might be responsible
for their activity [21-23]. Inspection of the reference thalidomide drug depicted in Figure 1 as
precursor led us to design and synthesize some new compounds containing mainly a
phthalimide core and enhanced by certain pharmacophores connected through nitrogen
atom of secondery or primary states, Figure 2. The main goal was to find new agents with
more efficacy to anticancer and antimicrobial effects with good pharmacokinetic profiles and
low host ctotoxicity. All the obtained new derivatives were tested as anticancer agents
(against two different cell lines) and anti-microbial agents (against nine microorganisms
belonging to G+, G− bacteria and fungi) using reported in vitro assay methods.
Figure 1
Figure 2
2
. Study rationale and design
Thalidomide reference drug 1 has a relatively simple chemical structure Figure 1, but
exhibits a multiple physiological activities (as privileged drug) in medicinal era. Its structure
consists of two connected essential cyclic imides sharing its activity. Since the mechanism
of action of thalidomide and its analogs is not fully clear [24] and the exact biological targets
4

for these substances has not been identified, the great look into the structure-activity
relationship of thalidomide and its analogs are mostly derived from molecular modelling and
continued research investigation [25-29].However, the information on SAR of thalidomide
and its derivatives is still in process so any trends had been tried are observed during
individual studies [30]. The objective of our search is modification of the side chain attached
to N atom of phthalimide that governs the anticancer activity by attaching different molecular
moieties. These substructures are ranging between secondery amine (-NH) or tertiary amine
(=N-) connected to aryl fragments through carbonyl, sulphonyl linker that affect total
hydrophobicity and polarity of resulted compounds, Figure 3.
Figure 3
3. Methods and Materials
3.1. General chemistry
All reagents were purchased from Sgma Aldrich Chemical Reagent Company and used
without further purification. Melting points were measured on capillary tube and were
uncorrected. IR spectra (in KBr pellets) were taken using Shimadzu FT-IR-8400S
spectrophotometer (Nishinokyo-Kuwabara-cho, Nakagyo-ku, Kyoto, Japan). 1H NMR and
13C NMR were recorded in DMSO-d6 and/or CDCl3 on a Jeol 500 MHz instrument using
TMS as internal standard (chemical shifts in d ppm). Mass spectra were recorded on a
Shimadzu PQ-5000 GC–MS apparatus. Solvent evaporation was performed under reduced
pressure using Buchan Rotatory Evaporator unless otherwise stated. TLC was performed
on precoated silica gel plates (60-F254, 0.2 mm), manufactured by EM Sciences, Inc, and
5

shortwave UV (254) nm was used to detect the UV absorbing compounds (CH2Cl2/EtOH
0:1).
1
General method of synthesis of 2-aminoisoindoline-1,3-dione (2)
In this procedure here, heating was avoided, which causes 2 to rearrange to the
phthalhydrazide [31]. To an ice-cold suspension of 14.7 g (0.1 mol) of phthalimide in 100 mL
of 95% ethanol at 10 °C, with stirring, 3.6 mL (0.11 mol) of 85% hydrazine was added
dropwise. A slightly exothermic reaction was observed, and the mixture was allowed to stir
at 5 °C for 2 h. The mixture was then diluted with 200 mL of ice-water, stirred, filtered,
washed with water, and dried in air to give 75% of pure product 2, mp 200 °C. 1H NMR (300
MHz, DMSO) δ 3.57 (brs, 2H, NH2), 7.73-7.85 (m, 4H, Phthalamide-H) ppm; 13C NMR (300
MHz, DMSO) δ 167.0, 134.5, 130.5, 123.4 ppm.
General method of synthesis of compounds (3a-b and 4a-b)
To a solution of the compound 2 (mLeq) in DMF(10 mL), TEA (0.5 mL) was added then acyl
or sulphonyl chloride derivatives (mLeq) was added with allowing the mixture to stir at 60 °C
temperature for 3 hours. The mixture was cooled and poured on 200 mL of ice-water,
stirred, filtered, washed with water, and dried in air to give the pure product.
N-(1,3-dioxoisoindolin-2-yl)-4-methylbenzenesulfonamide (3a)
This compound was obtained as white crystals; (1.5 g, 0.0048 mol), yield 78.5%, m.p. 112–
1
1
14 °C; IR (vmax/cm): 3285 (NH), 1780 and 1715 (2C=O), 1345 and 1150 (SO2); H NMR
(DMSO, 300 MHz, ppm): 1.78 (s, 1H, NH), 2.38 (s, 3H, CH3), 7.92-8.02 (m, 4H, H-Ar), 7.73-
1
3
7
.85 (m, 4H, H-phthalamide); C NMR (DMSO, 300 MHz, ppm): 169, 165, 162, 151, 148,
135, 134, 133, 130, 125, 124, 123, 111, 64, 15; HR-MS (ESI) m/z: calculated for
C15H12N2O4S [M + H]+: 316.051, found: 316.51, found: 316.53.
6

N-(1,3-dioxoisoindolin-2-yl)-4-isopropylbenzenesulfonamide (3b)
Pale yellow crystals; (0.93 g, 0.85 mol), yield 85.7%, m.p. 130–132 °C; IR (vmax/cm): 3280
1
(
NH), 1778 and 1720 (2C=O), 1340 and 1145 (SO2); H NMR (DMSO, 300 MHz, ppm): 1.25
(d, 6H, 2CH3), 2.65 (m, 1H, CH), 7.56-6.85 (m, 4H, H-Ar), 7.73-7.85 (m, 4H, H-
phthalamide); C13 NMR (DMSO, 300 MHz, ppm): 168, 165, 161, 150, 148, 138, 134, 133,
1
30, 125, 124, 123, 111, 64, 33, 24, 15; HR-MS (ESI) m/z: calculated for C17H16N2O4S [M
H]+: 344.082, found: 344.091.
+
N-(1,3-dioxoisoindolin-2-yl)furan-2-carboxamide (4a)
White crystals; (1.1 g, 0.004 mol), yield 69 %, m.p. 165–167 °C; IR (vmax/cm): 3230 (NH),
1
1
3
1
775 and 1701 (2C=O), 1685 (NH-C=O); H NMR (DMSO, 300 MHz, ppm): 7.40-6.65 (m,
13
H, furoyl-H), 7.94-7.74 (m, 4H, Phthalamide-H); C NMR (DMSO, 300 MHz, ppm): 163.6,
55.7, 146.7, 145.3, 134.9, 131.9, 124.3,116.9, 112.3; HR-MS (ESI) m/z: calculated for
C13H8N2O4 [M + H]+: 256.05, found: 256.08.
N-(1,3-dioxoisoindolin-2-yl)isonicotinamide (4b)
This compound was obtained as pale red crystals; (1.34 g, 0.005 mol), yield 81 %, m.p.
1
1
52–154 °C; IR (vmax/cm): 3410 (COOH), 1727 and 1617 (C=O), 1675 (NH-C=O); H NMR
(DMSO, 300 MHz, ppm): 8.7-8.1 (m, 4H, Pyridin-H), 7.92-7.2 (m, 4H, Phthalamide-H); C13
NMR (DMSO, 300 MHz, ppm): 163.6, 162.7, 150.5, 138.3, 134.9, 131.8, 124.1,121.6; HR-
MS (ESI) m/z: calculated for C14H9N3O3 [M + H]+: 267.06, found: 267.09.
General method of synthesis of compounds (5a-j)
To a solution of the compound 2 (mLeq) in ethanol (10 mL), the corresponding aromatic
aldhydeor sugar aldhyde (mLeq) was added and 0.5 mL of acetic acid (33%). The reaction
7

mixture was heated under reflux for 1.5 h then left to cool to room temperature. The solid
that separated out was filtered off, washed with ethanol, and dried.
(E)-2-((5-bromo-2-hydroxybenzylidene)amino)isoindoline-1,3-dione (5a)
Pale red crystals; (1.98 g, 0.0058 mol), yield 93.4 %, m.p. 188–190 °C; IR (vmax/cm): 3410
1
(
OH), 1727 and 1617 (C=O), 1590 (C=N); H NMR (DMSO, 300 MHz, ppm): δ 11.6 (s,1H,
OH), δ 9.3 (s,1H, C-H), 8.4-7.9 (m, 4H, phthalamid-H), 7.7 (s, 1H, Ar-H), 7.4 (d, 1H, J = 8.3
1
3
Hz, Ar-H), 6.9 (d, 1H, J = 8.3 Hz, Ar-H); C NMR (DMSO, 300 MHz, ppm): δ (160.9, 155.7,
50.6, 134.3, 132.3, 131.8, 129.8, 124.3, 118.6, 114.6, 113.3; HR-MS (ESI) m/z: calculated
for C15H9BrN2O3 [M + H]+: 343.98, found: 343.91.
E)-2-((3,5-dichloro-2-hydroxybenzylidene)amino)isoindoline-1,3-dione (5b)
1
(
Yellowish white crystals; (1.52 g, 0.0045 mol), yield 73 %, m.p. 212–214 °C; IR (vmax/cm):
1
3
310 (OH), 1725 and 1610 (C=O), 1590 (C=N); H NMR (CDCl3, 300 MHz, ppm): δ 11.8
(s,1H, OH), δ 9.6 (s,1H, C-H), 8.2-7.8 (m, 4H, Phthalamid-H), 7.4 (s, 1H, Ar-H), 7.1 (s, 1H,
1
3
Ar-H); C NMR (CDCl3, 300 MHz, ppm): δ 161.9, 154.7, 149.6, 132.3, 131.8, 131.6, 127.3,
1
24.6, 120.1; HR-MS (ESI) m/z: calculated for C15H8Cl2N2O3 [M + H]+: 335.14, found:
35.18.
3
(
E)-2-((3,4-dichlorobenzylidene)amino)isoindoline-1,3-dione (5c)
White crystals; (1.41 g, 0.0044 mol), yield 71.4 %, m.p. 216–218 °C; IR (vmax/cm): 1723
1
and 1620 (C=O), 1600 (C=O), 1585 (C=N); H NMR (DMSO, 300 MHz, ppm): δ 9.7 (s,1H,
C-H), 8.1-7.6 (m, 4H, phthalamid-H), 7.8 (s, 1H, Ar-H), 7.0 (d, 1H, J = 8.3 Hz, Ar-H), 6.8 (d,
1
3
1
H, J = 8.3 Hz, Ar-H); C NMR (DMSO, 300 MHz, ppm): δ (168.7, 164.7, 160.0, 150.0,
146.3, 133.2, 132.8, 133, 131.6, 124.9, 121.8, 117.1, 113.6; HR-MS (ESI) m/z: calculated
8

for C15H8ClN3O4 [M + H]+: 329.02; HR-MS (ESI) m/z: calculated for C15H8Cl2N2O2 [M +
H]+: 318.01, found: 318.06.
(E)-2-((2-chloro-5-nitrobenzylidene)amino)isoindoline-1,3-dione (5d)
Yellow crystals; (1.85 g, 0.0056 mol), yield 91 %, m.p. 204–205 °C; IR (vmax/cm): 1721 and
1
1
615 (C=O), 1596 (C=N), 1435 and 1342 (N=O); H NMR (CDCl3, 300 MHz, ppm): δ 9.8
(s,1H, C-H), 8.7 (s,1H, Ar-H), 7.9 (m, 4H, Phthalamide-H); 13C NMR (CDCl3, 300 MHz,
ppm): δ (162.9, 159.4, 150.7, 147.6, 133.5, 131.8, 126.3, 125.6, 123.3, 112.9, 109.3; HR-
MS (ESI) m/z: calculated for C15H8ClN3O4 [M + H]+: 330.02, found: 330.06.
(E)-2-((4-nitrobenzylidene)amino)isoindoline-1,3-dione (5e)
This compound was obtained as pale red crystals; (0.6 g, 63%), mp 179–180 °C; IR
1
(
vmax/cm): 1721 and 1615 (C=O), 1596 (C=N), 1435 and 1342 (N=O); H NMR (500 MHz,
CDCl3) δ 3.57 (brs, 2H, NH2), 7.73 (dd, 2H, J = 5.0, 3.0 Hz, aromatic Hs), 7.85 (dd, 2H, J =
.0, 3.0 Hz). HR-MS (ESI) m/z: calculated for CHClNO [M + H]+: 297.07, found: 297.12.
5
(E)-2-((3,4-dimethoxybenzylidene)amino)isoindoline-1,3-dione (5f)
This compound was obtained as pale red crystals; (0.6 g, 63%); with
1
mp 179–180 °C; IR (vmax/cm): 1714 and 1685 (C=O), 1596 (C=N); H NMR (500 MHz,
CDCl3) δ δ 9.1 (s,1H, C-H),7.9-7.8 (m, 4H, phthalamid-H), 7.5 (d, 1H, J = 8.6 Hz, Ar-H), 7.3
1
3
(
d, 1H, J = 8.6 Hz, Ar-H), 3.8 (s, 6H, OCH3); C NMR (CDCl3, 300 MHz, ppm): δ δ (164.9,
61.4, 152.7, 149.6, 135.3, 131.8, 126.3, 125.6, 123.3, 112.9, 109.3, 40.9; HR-MS (ESI)
m/z: calculated for C15H8Cl2N2O3 [M + H]+: 312.10, found: 310.15.
E)-2-((3,5-dibromo-2-hydroxybenzylidene)amino)isoindoline-1,3-dione (5g)
1
(
9

White
crystals;
(0.6
g,
63%);
with
1
mp 179–180 °C; IR (vmax/cm): 3295 (OH), 1700 and 1698 (C=O), 1587 (C=N); ;H NMR
(500 MHz, CDCl3) δ 11.8 (s,1H, OH), 9.6 (s,1H, C-H), 8.01 (s,1H, Ar-H), 7.9 (s,1H, Ar-H),
1
3
7
1
.8 (m, 4H, Phthalamide-H); C NMR (CDCl3, 300 MHz, ppm): δ (161.1, 152.6, 146.3,
40.3, 133.5, 131.8, 129.3, 125.6, 125.6, 120.5, 122.7. HR-MS (ESI) m/z: calculated for
CHClNO [M + H]+: 425.89, found: 425.92.
(E)-2-((3-ethoxy-4-hydroxybenzylidene)amino)isoindoline-1,3-dione (5h)
Pale red crystals; (1.98 g, 0.0058 mol), yield 93.4 %, m.p. 188–190 °C; IR (vmax/cm): 3290
1
(
OH), 1711 and 1605 (C=O), 1583 (C=N); 1H NMR (CDCl3, 300 MHz, ppm): H NMR
(
DMSO, 300 MHz, ppm): δ 9.8 (s,1H, OH), δ 8.9 (s,1H, C-H), 7.9-7.8 (m, 4H, phthalamid-H),
1
3
7
.4 (s, 1H, Ar-H), 7.3 (d, 1H, J = 8.5 Hz, Ar-H), 6.8 (d, 1H, J = 8.5 Hz, Ar-H); C NMR
(
DMSO, 300 MHz, ppm): δ (169.7, 165.7, 162.0, 151.3, 147.7, 135.2, 134.8, 133, 130.6,
24.9, 123.8, 116.1, 111.6, 64.4, 15.1; HR-MS (ESI) m/z: calculated for C15H8ClN3O4 [M +
H]+: 312.10, found: 312.12.
1
(E)-2-((2-phenylpropylidene)amino)isoindoline-1,3-dione (5i)
Yellow crystals; (1.85 g, 0.0056 mol), yield 91 %, m.p. 204–205 °C; IR (vmax/cm): 1730 and
1
1
695 (C=O), 1586 (C=N); 1H NMR (CDCl3, 300 MHz, ppm): H NMR (DMSO, 300 MHz,
ppm): δ 9.5 (s,1H, C-H), 8.1-7.5 (m, 4H, phthalamid-H), 7.0-6.5 (m, 4H, Ar-H), 2.8 (d, 3H,
1
3
CH3), 3.6 (q, 1H, CH); C NMR (DMSO, 300 MHz, ppm): δ 169.5, 163.7, 160.0, 151.3,
46.3, 134.2, 132.8, 133, 110.2, 21.2; HR-MS (ESI) m/z: calculated for C15H8ClN3O4 [M +
H]+: 280.11, found: 280.14.
E)-2-((2,4-dichlorobenzylidene)amino)isoindoline-1,3-dione (5j)
1
(
10

Pale red crystals; (1.98 g, 0.0058 mol), yield 93.4 %, m.p. 188–190 °C; IR (vmax/cm): 1710
1
and 1680 (C=O), 1594 (C=N); 1H NMR (CDCl3, 300 MHz, ppm): H NMR (DMSO, 300
MHz, ppm): δ 9.7 (s,1H, C-H), 8.1-7.6 (m, 4H, phthalamid-H), 7.3 (s, 1H, Ar-H), 7.2 (d, 1H, J
1
3
=
1
8.6 Hz, Ar-H), 7.0 (d, 1H, J = 8.6 Hz, Ar-H); C NMR (DMSO, 300 MHz, ppm): δ (168.7,
64.7, 161.0, 150.0, 146.3, 134.2, 132.8, 133, 131.6, 124.9, 122.8, 117.1, 112.6; HR-MS
(ESI) m/z: calculated for C15H8ClN3O4 [M + H]+: 319.00, found: 319.05.
(E)-2-((2,3,4,5,6-pentahydroxyhexylidene)amino)isoindoline-1,3-dione (6)
Yellow crystals; (1.85 g, 0.0056 mol), yield 91 %, m.p. 204–205 °C; IR (vmax/cm): 3280
1
(
OH), 1750 and 1640 (C=O), 1610 (C=N); H NMR (CDCl3, 300 MHz, ppm): δ 8.9 (s,1H, C-
H), 8.1-7.7 (m, 4H, phthalamid-H), 5.5 (m, 1H, CH), 4.5–4.4 (m, 4H, 3OH+CH,
1
3
exchangeable with D2O), 3.5–3.8 (m, 5H, OH+CH, exchangeable with D2O); C NMR
DMSO, 300 MHz, ppm): δ 158.8, 147.1, 132.6, 131.8, 124.6, 75.4, 71.5, 71.4, 69.4, 60.7;
HR-MS (ESI) m/z: calculated for C15H8ClN3O4 [M + H]+: 326.02,found: 326.10.
(
3
.2. Biology
3.2.1. Antimicrobial screening
The microorganism inoculums were uniformly spread using sterile cotton swabs on a sterile
Petri dish malt extract agar (for fungi) and nutrient agar (for bacteria). One hundred cubic
millimeters of each sample was added to each well (10-mm-diameter holes were cut in the
agar gel, 20 mm apart from one another). The systems were incubated for 24–48 hr. at 37 C
(for bacteria) and at 28 C (for fungi). After incubation, the microorganism growth was
11

observed. Inhibition zones of the bacterial and fungal growth were measured in millimeters.
Tests were performed in triplicate [32, 33].
3.2.2. DNA gyrase ATPase assays
Gyrase B and ParE monomers have only modest ATPase activity which is enhanced in the
case of the holoenzymes and stimulated further by DNA. The phosphate released following
conversion of ATP into ADP can be detected by the addition of malachite green solution and
measured by monitoring the increase in absorbance at 600 nm. Topoisomerases were
purchased from Inspiralis Ltd. (Norwich, United Kingdom). For the Escherichia coli DNA
gyrase ATPase assay, the final assay composition was 10 nM DNA gyrase (a complex of
two GyrA and two GyrB subunits [the A2B2 complex]), 35 mM Tris, pH 7.5, 24 mM KCl, 2
mM MgCl2, 6.5% glycerol, 0.1 mg/ml bovine serum albumin (BSA), 2 mM dithiothreitol
(
DTT), 1 mM ATP, and 5% DMSO solution containing the compounds. For the E. coli
topoisomerase IV ATPase assay, the final assay composition was 10 nM topoisomerase IV
a complex two ParC and two ParE subunits), 40 mM HEPES–KOH, pH 7.6, 100 mM
(
potassium glutamate, 25 mM magnesium acetate, 10 g/ml single-stranded DNA, 0.2 mg/ml
BSA, 10 mM DTT, 0.5 mM ATP, and 5% DMSO solution containing the compounds. The
reactions were started by the addition of the ATP, and the reaction mixtures were allowed to
incubate at 30°C for 60 min. Reactions were stopped by adding malachite green solution
(0.034% malachite green, 10 mM ammonium molybdate, 1 M HCl, 3.4% ethanol, 0.01%
Tween 20). Color was allowed to develop for 5 min, and the absorbance at 600 nm was
measured spectrophotometrically. The half-maximum (50%) inhibitory concentration (IC50)
values were determined from the absorbance readings using no-compound and no-enzyme
controls. The values reported are the averages of at least four independent experiments.
12

3.2.3. Cytotoxic screening
Human colon carcinoma (HCT-116) and human breast adenocarcinoma (MCF-7) cell lines
were obtained from the American Type Culture Collection (ATCC, Rockville, MD). The cells
were grown on RPMI-1640 medium supplemented with 10% inactivated fetal calf serum and
5
0 µg/ml gentamycin. The cells were maintained at 37 ºC in a humidified atmosphere with
% CO2 and were subcultured two to three times a week. Potential cytotoxicity of the
5
compounds was evaluated on tumor cells using the method of Gangadevi and Muthumary
34]. The cells were grown as monolayers in growth RPMI-1640. The monolayers of 104
cells adhered at the bottom of the wells in a 96-well microtiter plate incubated for 24 h at 37
ºC in a humidified incubator with 5% CO . The monolayers were then washed with sterile
phosphate buffered saline (0.01 M pH 7.2) and simultaneously the cells were treated with
00 µL from different dilutions of tested sample in fresh maintenance medium and incubated
[
2
1
at 37 ºC. A control of untreated cells was made in the absence of tested sample. Positive
controls containing doxroubcin drug was also tested as reference drug for comparison. Six
wells were used for each concentration of the test sample. Every 24 hours the observation
under the inverted microscope was made. The number of the surviving cells was determined
by staining the cells with crystal violet [35, 36] followed by cell lysing using 33% glacial
acetic acid and read the absorbance at 590 nm using microplate reader (SunRise, TECAN,
Inc, USA) after well mixing. The absorbance values from untreated cells were considered as
1
00% proliferation. The number of viable cells was determined using microplate reader as
previously mentioned before and the percentage of viability was calculated as [1-
ODt/ODc)]x100% where ODt is the mean optical density of wells treated with the tested
sample and ODc is the mean optical density of untreated cells. The relation between
(
13

surviving cells and drug concentration is plotted to get the survival curve of each tumor cell
line after treatment with the specified compound. The 50 % inhibitory concentration (IC50),
the concentration required to cause toxic effects in 50% of intact cells, was estimated from
graphic plots.
3
.3. Molecular modeling
The new synthesized compounds were docked into the crystal structure of E. coli
topoisomerase II DNA gyrase B (PDB code 1KZN). AutoDock 3.0 (38) and MOE softwares
[
37] were used for all docking calculations. The AutoDockTools [38] package was employed
to generate the docking input files and to analyze the docking results. A grid box size of
0×90×90 points with a spacing of 0.375 Å between the grid points was generated that
9
covered almost the entire protein surface. Ligands were fully flexibly docked. All non-polar
hydrogens and crystallographic water molecules were removed prior to the calculations. The
docking grid was centered on the mass center of the bound colorobiocin. In each case, 100
docked structures were generated using genetic algorithm searches. A default protocol was
applied with an initial population of 50 randomly placed conformations, a maximum number
of 2.5×105 energy evaluations, and a maximum number of 2.7×104 generations. A mutation
rate of 0.02 and a crossover rate of 0.8 were used. Heavy atom comparison root mean
square deviations (RMSD values) were calculated and initial ligand binding modes were
plotted. Protein-ligand interaction plots were generated using MOE. Quantum mechanical
calculations and surface molecular orbitals were generated by simulation module in MOE
software.
4. Results and Discussion
4
.1. Chemistry
14

The synthetic routes adopted to obtain the target compounds 2, 3a-b, 4a-b, 5a-j, and 6
derivatives were diagramed in Schemes 1-4. The structures of target compounds were
confirmed by IR, NMR and MS spectral data. As depicted in Figure 4, N-aminophthalamide
2
was synthesized using the reported procedure [31].
Figure 4
Hydrazinolysis of commercially available N-phthalamide ring 1 in absolute ethanol carefully
to give N-aminophthalamide 2 on very low temperature to inhibit rearrangement to
phthalazine. The amino compound was subsequently reacted in DMF and TEA as base with
p-substituted toluenesulphonyl chloride to obtain N-phthamide sulphonamides 3a-b.
Moreover, compound 2 was also acylated with heteroaromatic acid chlorides, furoyl and
isonicotinic acid chlorides halogenated to afford N-phthalamide amides 4a-b, Figure 5.
Figure 5
Figure 5
In addition, schiff base derivatives 5a-j were prepared through the condensation of the
primary amine 1 with corresponding substituted aromatic Aldhydes in ethanol and drops of
glacial acetic acid, Figure 6.
Figure 6
15

Figure 6
Condensation of amine compound 1 with D-sugar glucose provided target compound 6,
Figure 7.
Figure 7
4
.2. Biological screening
4
.2.1. Antimicrobial screening
All the newly synthesized compounds were screened for their antibacterial and antifungal
activities via agar diffusion well method [32]. The minimum inhibitory concentrations (MIC)
were determined by serial dilution method [33]. The activity of the synthesized compounds
was tested against four Gram-positive bacteria including Streptococcus pneumoniae (RCMB
010010), Bacillus subtilis (RCMB 010067), Staphylococcus aureus (RCMB 000106), and
Methicillin-Resistant Staphylococcus aureus (MRSA 2658 RCMB), two Gram-negative
bacteria including Escherichia coli (RCMB 010052), and Salmonella typhimurium (RCMB
000106), three fungi including Aspergillus fumigatus (RCMB 02568), Geotricum candidum
(RCMB 05097), and Candida albicans (RCMB 05036). Ampicillin, gentamicin, ciprofloxacin,
amphotericin B, vancomycin were used as control drugs [39, 40]. The observed inhibition
zone (IZ) and minimum inhibitory concentration (MIC) antimicrobial data of the compounds
and the reference drugs are given in Table 1-2 and Figure 8. The data showed that most of
the tested compounds exhibited appreciable bacterial and fungal inhibition activity compared
16

to reference drugs with inhibition zone around 20 mm. In general, the synthesized
compounds have tendency to be effective against the Gram-negative having IZ value more
than 20 mm. the acyl derivatives (3a-b and 4a-b) showed mild inhibitory activity against all
tested of microorganisms. In contrast, schiff bases 5a-j exihibited good inhibitory activity
with equal or greater than 20 mm IZ especially Gm+ve ( S.P and B.S), Gm-ve, and A.F and
G.C fungal types. Among the those compounds 2e-2j were found to be more effective
against E. coli by IZ range 21 to 24 mm and MIC 0.49 to 3.9 µg/ml and against S.T by IZ 22-
26 mm. The order of antimicrobial activity is explained by; 5a-j is more potent than 3, 4 ,
and 6 derivatives compounds. Compounds 5g and 5h exhibited especially the most potent
inhibitory activity against Gm+ve and Gm-ve types with MIC range of 0.49 to 4.1. Only 5h
one showed antifungal activity over the others. Generally, the investigation of antimicrobial
activity of the novel derivatives showed them more potent than reference drugs against gm-
ve bacteria and mild activity towards gm+ve and fungi, and mycobacterium strain.
Table 1
Figure 8
Table 2
4
.2.2. Inhibition of DNA gyrase in vitro activity
Based upon the published data upon the importance of DNA gyrase enzyme as bacterial
target, we decided to test only compounds 5d-h and 6 for their ability to inhibit the ATPase
17

function of DNA gyrase in in vitro biochemical assays. Purified, recombinant enzyme from
Escherichia coli were used. Novobiocin, a competitive inhibitor of the ATPase activity of the
bacterial type II topoisomerases [41], was included as a control. Data is reported in Table 3.
The assay showed the ability of some tested compounds to inhibit the enzyme with IC50
range 0.56 to 52.8 µM. The most active compound was 5g with aromatic ring having two
bromine and phenolic OH atoms. Modification of substitution with methoxy and phenolic OH
groups as in 5h one still keep the activity (IC50 1.21 µM). By removing of phenolic OH
substituent as in 5d and 5f compounds exifibited weaker potency compared to reference
drug novobiocin. Changing the side chain connected to amino of Phthalamide to more polar
moiety. like in compound 6 led to loss of activity. Thus, compounds 5g and 5h are
considered as potent inhibitors of gyrase in enzyme assays.
Table 3
4.2.3. Cytotoxic screening
The in vitro cytotoxic activity was performed by MTT assay [36, 42] against four human
carcinoma cell lines: Human colon carcinoma (HCT-116) and human breast
adenocarcinoma (MCF-7) cell lines. Doxorubicin was used as a positive control which has
high cytotoxic activity. The inhibitory effects of compounds on the growth of the two cell
lines are shown in Table 4. Most of compounds showed comparable cytotoxicity towards
HCT-116 than MCF-7 to reference drug. The order of compound activity was 5g-h > 5a-b >
3a-b > 4a-b > 5j-I > 6. Only two compounds 5b,g,h displayed good activity against both cell
lines (IC50 9.73, 912, and 10.2 µg/mL respectively) while 4a-b and 3a-b compounds
18

exhibited weaker activity. Thus, although this inhibition is still not sufficient, we feel that
further research would be able to improve the inhibitory activity, Figure 9.
Table 4
Figure 9
4.3. Computational studies and SAR analysis
4.3.1. Docking studies
Different biological targets found in microorganisms are considered of medicinally interest.
Because of the pharmacological profile as primary investigation of growth inhibition of
microorganisms of test compounds and structural similarities for reference ligands, we
decided to run molecular modeling studies for most active compounds for SAR analysis.
Docking simulations were performed as primary step to gain insight into the plausible
mechanism of antibacterial activity of target compounds. E. coli topoisomerase II DNA
gyrase B (responsible for the super-coiling activity of DNA in bacteria) (PDB code: 1KZN;
resolution 2.30 A°) was used by dock module implemented in MOE software[37, 43] have
been reported as good target for studying inhibitory activity against these bacteria [44]. For
that reason the most active compounds 4g was docked into active site of E. coli
topoisomerase II DNA gyrase B complexed with the natural inhibitor clorobiocin. The least
energy binding mode of poses has been studied. The binding pocket of such target
composed of different hydrophobic amino acids residues including Pro79, IIe78, Val157,
Val43, Val120, and Val46 and polar residues formed of Asp73, Glu50, Gly77, Asn46, Arg70,
and Arg136 surrounding and stabilizing the endogenous ligand. The bound clorobiocin drug
displayed specific interaction including hydrogen bonding interactions with Asp73 (1.911 A°),
19

Thr165 (2.109 A°), Asn46 (2.034 A°), Arg76, and Arg136 (2.071 A°), Figure 10. The results
of docking experiment is presented in Figure 10. The binding map of the compound in the
pocket was explained through four different fragments, 2-C=O of phthalamide, OH, and Br.
These fragments formed stable hydrogen bonding interactions with panel of corresponding
pocket residues Asn46 (2.28 A°), Thr165 (2.95 A°), Gly77 (21.41 A°), Asp73 (2.9 A°), Asp49
(2.7 A°), Glu50, Arg136, and Arg76. The types of formed hydrogen bonds are displayed
either direct between structural fragments and corresponding residues or indirect through
water molecules. The phthalamide ring is considered one of the milestones of activity of
compound 4g by contribution in the binding through hydrophobic interaction to different
residues Val167, Val43, and Val120 which contributes considerably to the strength of
binding interactions. This analysis was agreed with published work [45, 46]. The substitution
at aromatic ring affects positively the antimicrobial and cytotoxic activity of most of
compounds especially those of electron withdrawing property like halogens, nitro, and
hydroxyl groups. This was also observed in previous studies [47-49] and this was clear with
derivatives, 5a-j. The Br derivatives were in most cases highly active being clearly more
effective than chloro with agreement with previous studies [50]. Our results are compatible
with previous published work [51, 52] offering an in-case pharmacophore model formed of
essential pharmacophore points (two terminal hydrophobic/aromatic moieties with central
hydrogen bonding moiety) that are consistant with the novel amino isoindoline structures.
Figure 10
4.3.2. Molecular descriptors-based SAR analysis
20

Quantum Mechanical calculations were done for SAR analysis of some represented
molecules (3a; sulphonamide derivative, 4a; acyl derivative, 5g; Schiff derivative, and 6;
sugar derivative) using MOPAC quantum engine implemented in MOE software [37]. The
results are showed in Figure 11a-d. It is quite clear that the nature of linker between the
phthalamide scaffold and terminal aromatic ring is different from 3a passing to 6
compounds. Moreover, the substituents around the terminal aromatic ring plays an
important rule in activity by affecting electron density destribuation around the ring. In
general, the introduction of groups to the target scaffold appears to contribute positively to
the antimicrobial activity especially and aided in understanding SAR of synthesized
compounds. Quantum calculations confirmed these findings. The density distributions of
HUMO and LUMO surfaces are highlighted in Figure 11 between representative molecules
having different activity profile. In addition, 2D molecular structural descriptors were
calculated including solubility, Van Der Waals surface area, total polar surface area, partition
coeffecient, Lipiniski hydrogen bond acceptor, and molecular weight for topological
molecular analysis. The values among the different compounds are somehow variable and
contribute to different activity profile, Table 5.
Figure 11
Table 5
5
. Conclusion
21

This study has identified new 2-amino phthalamide derivatives having potential dual
antimicrobial and anticancer activities. Fifteen compounds were obtained and tested against
panel of nine microorganisms belonging to Gm+ve, Gm-ve, and fungi type. The results
exihibited their ability of most derivatives to inhibit their growth with higher inhibition zones
and lower MIC values compared to reference drugs. Among these compounds, a group of
Schiff bases with great LogP values and hence more lipophilicity showed good and
promising antbacterial activity. The mode of action of these compounds was studied through
molecular modeling against specific bacterial target enzyme, DNA gyrase which is
considered a prominent target for potent antibiotics, and exihibited the good interaction to
the enzyme at the ATP binding pocket. The obtained data was confirmed with DNA gyrase
activity testing for the most potent compounds. In addition, their cytotoxic activity against two
cell lines was measured and the results showed that they have mostly prevent the cell
growth with lower IC50 values. It is conceivable that the notion of such results can be
utilized in phthalamide-based antimicrobial and anticancer compound design
.
Acknowledgments
The authors would like to express their appreciation to the Deanship of Scientific Research
at Taibah University, Al-Madinah Al-Munawarah, Saudi Arabia.
22

References
1] P.C. Appelbaum, P.A. Hunter, The fluoroquinolone antibacterials: past, present and
future perspectives, International journal of antimicrobial agents, 16 (2000) 5-15.
2] P. Ball, Quinolone generations: natural history or natural selection?, The Journal of
antimicrobial chemotherapy, 46 (2000) 17-24.
3] V.T. Andriole, Current and future antifungal therapy: new targets for antifungal therapy,
International journal of antimicrobial agents, 16 (2000) 317-321.
4] J.M. Frere, Beta-lactamases and bacterial resistance to antibiotics, Molecular
microbiology, 16 (1995) 385-395.
5] J.L. Arias, Drug targeting strategies in cancer treatment: an overview, Mini reviews in
medicinal chemistry, 11 (2011) 1-17.
6] A.D. Settimo, F.D. Settimo, A.M. Marini, G. Primofiore, S. Salerno, G. Viola, L.D. Via,
[
[
[
[
[
[
S.M. Magno, Synthesis, DNA binding and in vitro antiproliferative activity of
purinoquinazoline, pyridopyrimidopurine and pyridopyrimidobenzimidazole derivatives as
potential antitumor agents, European journal of medicinal chemistry, 33 (1998) 685-696.
[7] S.M. Sondhi, J. Singh, R. Rani, P.P. Gupta, S.K. Agrawal, A.K. Saxena, Synthesis, anti-
inflammatory and anticancer activity evaluation of some novel acridine derivatives,
European journal of medicinal chemistry, 45 (2010) 555-563.
[
8] G. Devasahayam, W.M. Scheld, P.S. Hoffman, Newer antibacterial drugs for a new
century, Expert opinion on investigational drugs, 19 (2010) 215-234.
9] A. Couture, E. Deniau, P. Grandclaudon, C. Hoarau, A New Approach to
Isoindolobenzazepines. A Simple Synthesis of Lennoxamine, Tetrahedron, 56 (2000) 1491-
499.
[
1
23

[
10] C. Hoarau, A. Couture, E. Deniau, P. Grandclaudon, A Versatile Synthesis of Poly- and
Diversely Substituted Isoindolin-1-ones, Synthesis, 5 (2000) 655-660.
11] A. Couture, E. Deniau, P. Grandclaudon, C. Hoarau, Total Syntheses of Taliscanine,
Velutinam, and Enterocarpam II, The Journal of Organic Chemistry, 63 (1998) 3128-3132.
12] N. Guzior, M. Bajda, J. Rakoczy, B. Brus, S. Gobec, B. Malawska, Isoindoline-1,3-dione
[
[
derivatives targeting cholinesterases: Design, synthesis and biological evaluation of
potential anti-Alzheimer’s agents, Bioorganic & medicinal chemistry, 23 (2015) 1629-1637.
[13] N.S. El-Gohary, M.I. Shaaban, Synthesis, Antimicrobial, Antiquorum-Sensing, and
Cytotoxic Activities of New Series of Isoindoline-1,3-dione, Pyrazolo[5,1-a]isoindole, and
Pyridine Derivatives, Archiv der Pharmazie, 348 (2015) 666-680.
[
14] A. Gupta, P. Singh, B. Kamble, A. Kulkarni, M.J.N. Chandrasekar, Synthesis, Docking
and Biological Evaluation of Some Novel 5-bromo-2- (5-aryl-1,3,4-thiadiazol-2-yl)isoindoline-
,3-dione Derivatives Targeting ATP-binding Site of Topoisomerase II Letters in Drug
Design & Discovery, 13 (2016) 668-675.
15] A. Kamal, N.R. Bolla, P.S. Srikanth, A.K. Srivastava, Naphthalimide derivatives with
therapeutic characteristics: a patent review, Expert opinion on therapeutic patents, 23
2013) 299-317.
16] K.M. Amin, A.H. El-masry, N.A. Mohamed, G.A. Awad, B.S. Habib, Synthesis,
1
[
(
[
characterization and antimicrobial activity of some novel isoindole-1,3-dione derivatives, Der
Pharma Chemica, 5 (2013) 97-108.
[
17] M.S. el-Gaby, M.A. Zahran, M.M. Ismail, Y.A. Ammar, A novel synthesis of
dibenzo[c,f]chromenes, dibenzo[c,h]chromenes and benzo[7,8]chromeno[3,4-f]isoindoles as
antimicrobial agents, Farmaco (Societa chimica italiana : 1989), 55 (2000) 227-232.
24

[
18] S. Rajasekaran, R. Krishna, P. Sanjay, A. Ranjan, Design, Synthesis, Antibacterial and
invitro Antioxidant activity of substituted 2H-Benzopyran-2-one derivatives, 3 (2011) 555-
59.
19] R.A. Pophale, M.N. Deodhar, Synthesis and evaluation of novel phthalimide derivatives
as analgesic and antiinflammatory agents, Der Pharma Chemica 2(2010) 185-193.
20] T.N. Bansode, J.V. Shelke, V.G. Dongre, Synthesis and antimicrobial activity of some
new N-acyl substituted phenothiazines, European journal of medicinal chemistry, 44 (2009)
094-5098.
21] M.H. Norman, D.J. Minick, G.C. Rigdon, Effect of linking bridge modifications on the
5
[
[
5
[
antipsychotic profile of some phthalimide and isoindolinone derivatives, Journal of medicinal
chemistry, 39 (1996) 149-157.
[22] X. Collin, J. Robert, G. Wielgosz, G. Le Baut, C. Bobin-Dubigeon, N. Grimaud, J. Petit,
New anti-inflammatory N-pyridinyl(alkyl)phthalimides acting as tumour necrosis factor-alpha
production inhibitors, European journal of medicinal chemistry, 36 (2001) 639-649.
[
23] V. Ravi, a facile synthesis of biologically active phthalimides & its analogues-a study.
International science congress association, Indore-452005 (MP), India., 2013.
24] T. Latif, N. Chauhan, R. Khan, A. Moran, S.Z. Usmani, Thalidomide and its analogues
in the treatment of Multiple Myeloma, Experimental hematology & oncology, 1 (2012) 27.
25] G.W. Muller, L.G. Corral, M.G. Shire, H. Wang, A. Moreira, G. Kaplan, D.I. Stirling,
[
[
Structural modifications of thalidomide produce analogs with enhanced tumor necrosis
factor inhibitory activity, Journal of medicinal chemistry, 39 (1996) 3238-3240.
[26] H.W. Man, L.G. Corral, D.I. Stirling, G.W. Muller, Alpha-fluoro-substituted thalidomide
analogues, Bioorganic & medicinal chemistry letters, 13 (2003) 3415-3417.
25

[27] C.M. Avila, N.C. Romeiro, G.M. da Silva, C.M. Sant'Anna, E.J. Barreiro, C.A. Fraga,
Development of new CoMFA and CoMSIA 3D-QSAR models for anti-inflammatory
phthalimide-containing TNFalpha modulators, Bioorganic & medicinal chemistry, 14 (2006)
6874-6885.
[
28] E.R. Lepper, S.S. Ng, M. Gutschow, M. Weiss, S. Hauschildt, T.K. Hecker, F.A. Luzzio,
K. Eger, W.D. Figg, Comparative molecular field analysis and comparative molecular
similarity indices analysis of thalidomide analogues as angiogenesis inhibitors, Journal of
medicinal chemistry, 47 (2004) 2219-2227.
[
29] T. Noguchi, H. Fujimoto, H. Sano, A. Miyajima, H. Miyachi, Y. Hashimoto, Angiogenesis
inhibitors derived from thalidomide, Bioorganic & medicinal chemistry letters, 15 (2005)
509-5513.
30] G.W. Muller, R. Chen, S.Y. Huang, L.G. Corral, L.M. Wong, R.T. Patterson, Y. Chen, G.
5
[
Kaplan, D.I. Stirling, Amino-substituted thalidomide analogs: potent inhibitors of TNF-alpha
production, Bioorganic & medicinal chemistry letters, 9 (1999) 1625-1630.
[
31] S.K. Dey, D.A. Lightner, 1,1'-bipyrroles: synthesis and stereochemistry, J Org Chem, 72
2007) 9395-9397.
32] D.A. Vanden-Berghe, A.J. Vlientinck, screening Methods for Antibacterial and Antiviral
(
[
Agents from Higher Plants, Screening Methods for Antibacterial and Antiviral Agents from
Higher Plants, In: Hostettmann K, editor. Methods in Plant Biochemistry VI. Assays for
Bioactivity, Academic Press Inc, San Diego, United States, 1991.
[33] J.G. Cappuccino, N. Sherman, Microbiology: A Laboratory Manual, Benjamin-
Cummings Pub Co, Microbiology: A Laboratory Manual, California, 1999.
26

[34] A. Klancnik, S. Piskernik, B. Jersek, S.S. Mozina, Evaluation of diffusion and dilution
methods to determine the antibacterial activity of plant extracts, Journal of microbiological
methods, 81 (2010) 121-126.
[
35] J.M. V Gangadevi, Preliminary studies on cytotoxic effect of fungal taxol on cancer cell
lines, African Journal of Biotechnology, 6 (2007) 1382-1386.
36] T. Mosmann, Rapid colorimetric assay for cellular growth and survival: application to
proliferation and cytotoxicity assays, J Immunol Methods, 65 (1983) 55-63.
37] M. Molecular Operating Environment (MOE) Chemical Computing Group , Quebec,
Canada. 2012; http://www.chemcomp.com. Accessed on 30/02/2013, DOI.
38] G.M. Morris, D.S. Goodsell, R.S. Halliday, R. Huey, W.E. Hart, R.K. Belew, A.J. Olson,
[
[
[
Automated docking using a Lamarckian genetic algorithm and an empirical binding free
energy function, Journal of Computational Chemistry, 19 (1998) 1639-1662.
[39] M.I.C. Atta-ur-Rahman, William J. Thomsen, Bioassay Techniques for Drug
Development Bioassay Techniques for Drug Development . Harwood Academic Publishers,
Amsterdam, The Netherlands.2001.
[
40] J.A. Smania, F.D. Monache, E.F.A. Smania, R.S. Cuneo, Antibacterial Activity of
Steroidal Compounds Isolated from Ganoderma applanatum (Pers.) Pat.
Aphyllophoromycetideae) Fruit Body., International Journal of Medicinal Mushrooms, 1
1999) 325-330.
(
(
[41] A. Sugino, N.P. Higgins, P.O. Brown, C.L. Peebles, N.R. Cozzarelli, Energy coupling in
DNA gyrase and the mechanism of action of novobiocin, Proceedings of the National
Academy of Sciences of the United States of America, 75 (1978) 4838-4842.
27

[
42] M.C. Alley, D.A. Scudiero, A. Monks, M.L. Hursey, M.J. Czerwinski, D.L. Fine, B.J.
Abbott, J.G. Mayo, R.H. Shoemaker, M.R. Boyd, Feasibility of drug screening with panels of
human tumor cell lines using a microculture tetrazolium assay, Cancer research, 48 (1988)
589-601.
[43] A.A. Abdel-Aziz, Y.A. Asiri, M.H. Al-Agamy, Design, synthesis and antibacterial activity
of fluoroquinolones containing bulky arenesulfonyl fragment: 2D-QSAR and docking study,
European journal of medicinal chemistry, 46 (2011) 5487-5497.
[
44] C.P. Kaushik, K. Lal, A. Kumar, S. Kumar, Synthesis and biological evaluation of amino
acid-linked 1,2,3-bistriazole conjugates as potential antimicrobial agents, Med Chem Res,
3 (2014) 2995-3004.
45] N. Zidar, T. Tomašić, R. Šink, V. Rupnik, A. Kovač, S. Turk, D. Patin, D. Blanot, C.
2
[
Contreras Martel, A. Dessen, M. Müller Premru, A. Zega, S. Gobec, L. Peterlin Mašič, D.
Kikelj, Discovery of Novel 5-Benzylidenerhodanine and 5-Benzylidenethiazolidine-2,4-dione
Inhibitors of MurD Ligase, Journal of medicinal chemistry, 53 (2010) 6584-6594.
[46] M. Brvar, A. Perdih, M. Renko, G. Anderluh, D. Turk, T. Solmajer, Structure-Based
Discovery of Substituted 4,5′-Bithiazoles as Novel DNA Gyrase Inhibitors, Journal of
medicinal chemistry, 55 (2012) 6413-6426.
[
47] L.C. Tavares, J.J. Chiste, M.G. Santos, T.C. Penna, Synthesis and biological activity of
nifuroxazide and analogs. II, Bollettino chimico farmaceutico, 138 (1999) 432-436.
48] A. Masunari, L.C. Tavares, A new class of nifuroxazide analogues: synthesis of 5-
[
nitrothiophene derivatives with antimicrobial activity against multidrug-resistant
Staphylococcus aureus, Bioorganic & medicinal chemistry, 15 (2007) 4229-4236.
28

[
49] L.C. Tavares, T.C. Penna, A.T. Amaral, Synthesis and biological activity of nifuroxazide
and analogs, Bollettino chimico farmaceutico, 136 (1997) 244-249.
50] L. Cronenberger, B. Dolfin, H. Pacheco, [Correlation between antifungal properties of
[
substituted salicylic aldehydes and chemical displacement of the phenolic proton], Comptes
rendus hebdomadaires des seances de l'Academie des sciences. Serie D: Sciences
naturelles, 269 (1969) 1334-1337.
[51] T. Aboul-Fadl, F.A. Bin-Jubair, O. Aboul-Wafa, Schiff bases of indoline-2,3-dione (isatin)
derivatives and nalidixic acid carbohydrazide, synthesis, antitubercular activity and
pharmacophoric model building, European journal of medicinal chemistry, 45 (2010) 4578-
4586.
[52] T. Aboul-Fadl, H.A. Abdel-Aziz, M.K. Abdel-Hamid, T. Elsaman, J. Thanassi, M.J.
Pucci, Schiff Bases of Indoline-2,3-dione: Potential Novel Inhibitors of Mycobacterium
Tuberculosis (Mtb) DNA Gyrase, Molecules (Basel, Switzerland), 16 (2011) 7864.
29