In vitro anticancer potentiality and molecular modelling study of novel amino acid derivatives based on N1,N3-bis-(1-hydrazinyl-1-oxopropan-2-yl) isophthalamide

Article abstract of DOI:10.1080/14756366.2019.1613390

A series of N¹,N³-bis (1-oxopropan-2-yl) isophthalamide-based derivatives 4–16 were prepared and their structures were confirmed by different spectral tools. The cytotoxic potentiality of novel compounds 4–16 was assessed by the MTT assay method on colon, lung and breast tumour cell lines. Compound 5 gave the most significant specificity anticancer activity with safety response on normal cell lines. In vitro enzyme assay and several apoptotic parameters were examined to elucidate the mode of action of compound 5. Molecular docking studies also were simulated to put insight and give better understanding to its structural features.

Full text of DOI:10.1080/14756366.2019.1613390

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
In vitro anticancer potentiality and molecular
modelling study of novel amino acid derivatives
based on N1,N3-bis-(1-hydrazinyl-1-oxopropan-2-yl)
isophthalamide
Asmaa F. Kassem, Gaber O. Moustafa, Eman S. Nossier, Hemat S. Khalaf,
Marwa M. Mounier, Suliman A. Al-Yousef & Sabry Y. Mahmoud
To cite this article: Asmaa F. Kassem, Gaber O. Moustafa, Eman S. Nossier, Hemat S. Khalaf,
Marwa M. Mounier, Suliman A. Al-Yousef & Sabry Y. Mahmoud (2019) Inꢀvitro anticancer
potentiality and molecular modelling study of novel amino acid derivatives based on N1,N3-bis-
(1-hydrazinyl-1-oxopropan-2-yl) isophthalamide, Journal of Enzyme Inhibition and Medicinal
Chemistry, 34:1, 1247-1258, DOI: 10.1080/14756366.2019.1613390
To link to this article: https://doi.org/10.1080/14756366.2019.1613390
© 2019 The Author(s). Published by Informa
UK Limited, trading as Taylor & Francis
Group.
Published online: 09 Jul 2019.
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2019, VOL. 34, NO. 1, 1247–1258
https://doi.org/10.1080/14756366.2019.1613390
RESEARCH PAPER
In vitro anticancer potentiality and molecular modelling study of novel amino acid
derivatives based on N1,N3-bis-(1-hydrazinyl-1-oxopropan-2-yl) isophthalamide
Asmaa F. Kassema, Gaber O. Moustafab, Eman S. Nossierc, Hemat S. Khalafd,e, Marwa M. Mounierf,
Suliman A. Al-Yousefg and Sabry Y. Mahmoudh
aDepartment of Chemistry of Natural and Microbial Products, Pharmaceutical and Drug Industries Research Division, National Research Centre,
b
c
Giza, Egypt; Department of Peptide Chemistry, Chemical Industries Research Division, National Research Centre, Cairo, Egypt; Department of
d
Pharmaceutical Medicinal Chemistry, Faculty of Pharmacy (Girls), Al-Azhar University, , Cairo, Egypt; Department of Chemistry, College of
Science and Arts, Jouf University, Al Qurayat, Saudi Arabia; Chemical Industries Research Division, Department of Photochemistry, National
e
f
Research Centre, Cairo, Egypt; Pharmaceutical and Drug Industries Research Division, Department of Pharmacognosy, National Research Centre,
g
Giza, Egypt; Department of Clinical Laboratory Science, College of Applied Medical Science, University of Hafr Al Batin, Hafr Al Batin, Saudi
h
Arabia; Department of Biology, College of Science, University of Hafr Al Batin, Hafr Al Batin, Saudi Arabia
ABSTRACT
ARTICLE HISTORY
A series of N1,N3-bis (1-oxopropan-2-yl) isophthalamide-based derivatives 4–16 were prepared and their
structures were confirmed by different spectral tools. The cytotoxic potentiality of novel compounds 4–16
was assessed by the MTT assay method on colon, lung and breast tumour cell lines. Compound 5 gave
the most significant specificity anticancer activity with safety response on normal cell lines. In vitro enzyme
assay and several apoptotic parameters were examined to elucidate the mode of action of compound 5.
Molecular docking studies also were simulated to put insight and give better understanding to its struc-
tural features.
Received 14 February 2019
Revised 22 April 2019
Accepted 23 April 2019
KEYWORDS
Isophthalamide; amino
acids; peptides; anticancer
activity; molecular docking
1. Introduction
structure13. Moreover, peptides comprise a major class of important
anticancer therapeutic agents14–16
.
Cancer is still the major health issue in most parts of the world and
the prominent cause of death world-wide1. According to World
Health Organization, cancer is the second leading cause of death
and was responsible for 8.8 million deaths in 20152. Cancer treat-
ment has been the first goal of pharmaceutical industries over the
last many decades3. Among the several treatment regimens avail-
able today, chemotherapy is most commonly used for treating
many kinds of cancers world-wide. Drug resistance and failure of
anti-tumour drugs are the main side effects and obstacles observed
in successful development of anticancer drugs. Therefore, there is a
vital need for the discovery of molecules which are endowed with
Heterocyclic rings have chemical structural similarity with
respect to the biologically active compounds within our body,
such as DNA, RNA, neurotransmitters and hormones, so they
become pivotal parts of bioactive molecules17. Heterocycles con-
taining 2-oxoindoline, 1,3,4-oxadiazole, and thiazole scaffolds II–IX
(Figure 1) exhibit significant antitumor activities via versatile and
different mechanisms18–25. So, a combination of small heterocyclic
molecules with a peptide motif (isophthalamide) can augment
pharmacokinetic properties of both the peptide and the small
molecules and provides more effective drug probes.
Depending on the previous observations and our program in
the development of peptide fragments with anticancer activ-
ity26–28, some isophthalamide based derivatives were synthesized
and evaluated for their cytotoxicity against human colon (HCT-
116), lung (A-549) and breast (MCF-7) cancerous cell lines. The
possible mechanisms of cytotoxicity action of the most active
compound were further studied through in vitro enzymatic assay,
DNA fragmentation and analysis of key proteins involved in the
apoptotic pathways.
excellent anticancer potency and lower side effects4,5
.
In the past years, biodegradable and biocompatible polymers have
become an attractive choice as drug delivery systems. Biopolymers or
biopeptides are an important class of low toxicity drug delivery vec-
tors and their role has been long recognized with controlled release
of drugs over long periods, easy conjugation for active targeting and
adjustable release of both hydrophobic and hydrophilic molecules6–8
.
Amongst this class of materials, amphiphilic polymers can successfully
transport a variety of molecules across mammalian cell membranes,
such as cancer therapy candidates and proteins9,10. The transport of
these polymers along different drugs to a particular site within cells
has shown a great improvement in the efficacy and accessibility of
therapeutics11,12. The parent polyamide, poly (L-lysine isophthalamide)
2. Experimental
2.1. Chemistry
(I) (Figure 1), was grafted with hydrophobic amino acids onto its pen- Melting points were determined in an “Electro Thermal” Digital
dant carboxylic acid groups to manipulate its amphiphilicity and melting point apparatus (Shimadzu, Tokyo, Japan) (model: IA9100).
CONTACT Gaber O. Moustafa
gosman79@gmail.com
Department of Peptide Chemistry, Chemical Industries Research Division, National Research Centre,
Cairo, Dokki, Egypt
ß 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

1248
A. F. KASSEM ET AL.
CN
O
N
H
H
O
NH
N
N
O
O
N
O
O
S
N
N
H
N
H
*
O
O
n
O
N
N
H
H
I
III
II
Poly (L-lysine iso-phthalamide)
SO2NH2
EGFR inhibitor
CDK-2 inhibitor
OH
O
O
NH
N
Cl
Cl
N
NH
N
H
N
N
N
Cl
O
S
O
O
N
H
N
VI
HIV
V
Thymidine phosphorylase inhibitor
c-Kit inhibitor
VEGFR-2 inhibitor
O
HN
CH3
O
HN
O
N
N
N
N
N
N
H
O
S
S
Cl
O
IX
VII
VIII
NF-kB, Nuclear factor-kappaB inhibitor
EGFR inhibitor
EGFR & B-raf V600E inhibitor
Lead Optimization
H
N
H
N
O
O
4-16
Figure 1. Design of newly synthesized isophthalamide based derivatives 4–16 concerning the chemical structures of poly (L-lysine isophthalamide) (I) and different het-
erocyclic motifs II–IX with various mechanisms of anticancer activity.
Elemental analysis was found within the acceptable limits of the S1: S-petroleum ether (b.p. 40–60ꢁC) (1:1); S2: S-petroleum ether
(b.p. 40–60 ꢁ C) (3:2); S3: S-petroleum ether (b.p. 40–60 ꢁC) (1:2) and
S4: butanol–water–acetic acid–pyridine (120:48:12:40). It is generally
known that basic reaction media enhance racemization. However,
under the reaction conditions employed in this work, especially
short reaction times and temperatures below (ꢁC), only negligible
racemization was observed.
calculated values (Micro-analytical Unit, NRC). Infrared spectra (KBr)
were recorded on a Nexus 670 FTIR Nicolet, Fourier Transform infra-
red spectrometer (Perkin Elmer, Hopkinton, MA, USA ). Proton
ꢀꢀ
nuclear magnetic resonance (1H-NMR) spectra were run in [d6]
DMSO on Jeol 270 MHz or 500MHz instruments (Joel Inc., Tokyo,
Japan). Chemical shifts d are given in ppm. Mass spectra were run
on a MAT Finnigan SSQ 7000 spectrometer (Shimadzu, Kyoto,
Japan; model: QP2010 ultra), using the electron impact technique
(EI). Analytical thin layer chromatography (TLC) was performed on
ꢀꢀ
2.1.1. Synthesis of L-Alanine methyl ester, isophthaloyl chloride (2)
The titled compound (2) prepared according to previously
silica gel aluminium sheets, 60 F254 (E. Merck ). The following
solvent systems (by volume) were used as eluents for the develop-
ment of the plates: S: chloroform–methanol–acetic acid (85:10:5); reported methods29–31
.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1249
2.1.2. Synthesis of isophthaloyl-bis-[L-Alanine methyl ester] (3)
A dichloromethane (DCM) solution of free L-Alanine methyl ester
(Mþ, 6), 531 (11), 487 (13), 419 (16), 389 (17), 371 (27), 362 (13), 346
(11), 317 (22), 308 (13), 288 (21), 160 (98), 143 (16), 132 (43), 129
(30 mmol, –20ꢁC) was added to a DCM solution of the isophthaloyl (39), 118 (20), 103 (100), 88 (23), 76 (74), 62 (28). Molecular formula
chloride (2) (–20ꢁC, 15mmol). And then the reacted mixture was (molecular weight): C30H26N8O6 (594.5). Calculated analysis: C, 60.60;
stirred for (3h, –20ꢁC), then for (24h at room temperature). It was H, 4.41; N, 18.85; Found: C, 60.64; H, 4.45; N, 18.82.
then washed with (water, 1 N sodium bicarbonate and 1 N potas-
N1-(1-(-2-(5-chloro-2-oxoindolin-3-ylidene)hydrazinyl)-1-oxopro-
hydrazinyl)-1-
sium hydrogen sulphate), followed by water again and then dried pan-2-yl)-N3-(1-(-2-(5-chloro-2-oxoindolin-3-ylidene)
over sodium sulphate. Next, the solvent was evaporated and the
obtained compound was solidified by petroleum ether (boiling
point 40–60 ꢁC). The solid was filtered off, dissolved in methanol
(MeOH) and precipitated by petroleum ether to give compound 3.
Dimethyl 2, 2’-(isophthaloyl-bis-(azanediyl)) dipropionate (3):
Yield: 75%; melting point: 132–134 ꢁC, IR (cmꢂ1): (KBr): ꢀ ¼ 3330
(NH stretching), 3060 (CH, aromatic), 2950 (CH, aliphatic), 1744
oxopropan-2-yl)isophthalamide (6)
Yield: 70%; melting point: 271–273 ꢁC, IR (cmꢂ1): (KBr): ꢀ ¼ 3393,
3239 (6NH), 2922 (CH aliphatic), 1698 (2CO) 1647 (2CO, amide),
1521 (2 C ¼ N). 1H-NMR (500 MHz, d, ppm, DMSO-d6): d ¼ 1.49–1.53
(m, 6H, 2CH3), 4.65 (m, 2H, CHCH3), 6.95–8.59 (m, 10H, Ar-H), 10.30
(s, 2H, NH, pyrrole ring), 11.35, 13.45 (s, 4H, 4NH). MS (EI, 70 eV):
m/z (%) ¼ 663 (Mþ, 12), 637 (19), 586 (24), 486 (21), 446 (83), 433
(31), 418 (48), 413 (16), 401 (21), 387 (24), 378 (58), 194 (52), 142
(13), 132 (14), 116 (18), 109 (20), 85 (100). Molecular formula
(molecular weight): C30H24Cl2N8O6: (663.47). Calculated analysis: C,
54.31; H, 3.65; Cl, 10.69; N, 16.89; Found: C, 54.36; H, 3.59; Cl,
10.61; N, 16.79.
(C ¼ O, ester), 1675, and 1610 (C ¼ O amide
I
and II,
respectively).1H-NMR (500 MHz, d, ppm, DMSO-d6): d ¼ 8.90, 8.85 (s,
2H, 2NH, D2O exchangeable, amide), 8.50–8.30 (4H, aromatic H),
4.35–4.25 (m, 2H, 2CH, a-L-Ala), 3.70–3.50 (s, 6H, 2OCH3), 1.50–1.44
(d, 6H, 2CH3, b-L-Ala). MS (EI, 70 eV): m/z (%) ¼ 336 (Mþ, 6.11), 222
(3.06), 78 (100), 63. Molecular formula (molecular weight):
C16H20N2O6 (336.3). Calculated analysis: C, 57.14; H, 5.99; N, 8.33;
Found: C, 57.10; H, 5.90; N, 8.8.
2.1.5. Synthesis of N1,N3-bis(1-(5-thioxo-4,5-dihydro-1,3,4-oxadia-
zol-2-yl)ethyl)isophthalamide (7)
A mixture of N1, N3-bis (1-hydrazinyl-1-oxopropan-2-yl) isophthala-
mide (3.3 g, 0.01 mol) and carbon disulphide (6 ml) in pyridine (10 ml)
and DMF (5 ml) was heated under reflux on water bath for 8 h. After
cooling, the solvent was evaporated under reduced pressure and
residue was triturated with an ice-water mixture and neutralized with
diluted HCl. the solid precipitate formed was filtered off and recrys-
tallized from ethanol to afford 7 as pale yellow crystals.
2.1.3. Synthesis of N1,N3-bis-(1-hydrazinyl-1-oxopropan-2-yl) iso-
phthalamide (4)
To a stirred methanolic solution (50 mL) of the corresponding
amino acid methyl ester (3) (1 mmol), hydrazine hydrate 99%
(0.35 mL, 10 mmol) was added. The mixture was then refluxed for
3 h, the solvent was evaporated and the obtained residue was tri-
turated with diethylether, filtered off, and precipitated from
MeOH/diethylether to give 4.
N1,N3-bis (1-(5-thioxo-4,5-dihydro-1, 3, 4-oxadiazol-2-yl) ethyl) iso-
phthalamide (7)
Yield: 75%; melting point: 232–234 ꢁC, IR (cmꢂ1): (KBr):ꢀ ¼ 3293
(2NH2), 3131 (4NH, amide), 2972 (CH aliphatic), 1650 (4CO), 1528
(2 C ¼ N). 1H-NMR (500 MHz, d, ppm, DMSO-d6): d ¼ 1.36–1.49 (m,
6H, 2CH3), 4.24 (s, 4H, 2NH2), 4.57, 5.38 (2q, 2H, 2CH–CH3),
7.24–8.25 (m, 4H, Ar-H), 8.74, 9.70 (s, 4H, 4NH). MS (EI, 70 eV): m/z
(%) ¼ 336 (Mþ, 16), 331 (10), 323 (25), 316 (23), 310 (35), 304 (69),
300 (25), 299 (15), 285 (24), 281 (19), 277 (11), 266 (21), 258 (31),
124 (13), 93 (12), 77 (18), 42 (100). Molecular formula (molecular
weight): C14H20N6O4 (336.3). Calculated analysis: C, 49.99; H, 5.99;
N, 24.99; Found: C, 49.92; H, 5.97; N, 24.88.
Yield: 80%; melting point: 132–134 ꢁC, IR (cmꢂ1): (KBr): ꢀ ¼ 3428,
3290 (4NH), 2919 (CH, aliphatic), 1635 (2CO), 1520 (2 C ¼ N), 1114,
1057 (2 C ¼ S). 1H-NMR (500 MHz, d, ppm, DMSO-d6): d ¼ 1.17–1.41
(m, 6H, 2CH3), 3.35–4.14 (m, 2H, CHCH3), 7.71–8.39 (m, 4H, Ar-H),
873, 9.25 (s, 4H, 4NH), 7.00 (s, 2H, NH, oxadiazole ring). MS (EI,
70 eV): m/z (%) ¼ 420 (Mþ, 5.5), 407 (10), 393 (10), 392 (16), 97
(19), 70 (100), 57 (59). Molecular formula (molecular weight):
C16H16N6O4S2: (420.07). Calculated analysis: C, 45.71; H, 3.84; N,
19.99; S, 15.25; Found: C, 45.75; H, 3.82; N, 19.89; S, 15.28.
2.1.6. General procedure for the synthesis of sugar hydrazone
(8, 9)
2.1.4. Synthesis of N1-(1-oxo-1-(-2-(2-oxoindolin-3-ylidene) hydra-
zinyl) propan-2-yl)-N3-(1-oxo-1-(-2-(2-oxoindolin-3-ylidene) hydra-
zinyl) propan-2-yl) isophthalamide (5) and N1-(1-(-2-(5-chloro-2-
oxoindolin-3-ylidene)hydrazinyl)-1-oxopropan-2-yl)-N3-(1-(-2-(5-
chloro-2-oxoindolin-3-ylidene)hydrazinyl)-1-oxopropan-2-yl)isoph-
thalamide (6)
A mixture of N1,N3-bis(1-hydrazinyl-1-oxopropan-2-yl) isophthala-
mide (3.3 g, 0.01 mol) in absolute ethanol (20 ml) containing glacial
acetic acid, the respective monosaccharide(3 g, 0.02 mol) in water
(2 ml). The mixture was heated under reflux for 3 h and the result-
ing solution was concentrated and left to cool to room tempera-
ture. The formed precipitate was filtered off, washed with water
and ethanol, then dried and recrystallized from ethanol to afford
8 and 9 as white crystals.
To a solution of compound bis (1-hydrazinyl-1-oxopropan-2-yl) iso-
phthalamide (4) (3.36 g, 0.01 mol) in DMF (10 ml) was added isatin
and/or chloroisatin (0.2 mol) refluxed for 4 h, cold poured on to
crushed ice. The product formed was filtered off and crystallized
from ethanol to give the target compounds.
N1,N3-bis (1-oxo-1-(-2-((2S, 3 R, 4 R)-2, 3, 4, 5-tetrahydroxypentyli-
dene) hydrazinyl) propan-2-yl) isophthalamide (8)
Yield: 60%; melting point: <300 ꢁC, IR (cmꢂ1): (KBr):ꢀ ¼ 3427
(OH), 3365, 3307 (2NH), 2922 (CH aliphatic), 1639 (4CO), 1527
N1-(1-oxo-1-(-2-(2-oxoindolin-3-ylidene) hydrazinyl) propan-2-yl)-
N3-(1-oxo-1-(-2-(2-oxoindolin-3-ylidene) hydrazinyl) propan-2-yl) iso-
phthalamide (5)
1
(2 C ¼ N). H-NMR (500 MHz, d, ppm, DMSO-d6): d ¼ 1.17–1.41 (m,6H,
Yield: 75%; melting point: 271–273 ꢁC, IR (cmꢂ1): (KBr): ꢀ ¼ 3391,
3235 (6NH), 2923 (CH aliphatic), 1698, 1647 (4CO), 1524 (2 C ¼ N).
1H-NMR (500 MHz, d, ppm, DMSO-d6): d ¼ 1.49–1.51 (m, 6H, 2CH3),
2CH3), 3.35 (m, 10H, H-5,50, H-4,3,2), 4.11 (m, 2H, 2OH), 4.45 (m,
4H, 4OH), 4.59 (m, 2H, CHCH3), 5.30 (m, 2H, 2OH), 7.62–8.52 (m,
6H, Ar-H þ H1),839, 8.80 (4H, 4NH). Molecular formula (molecular
4.64, (m, 2H, CHCH3), 6.92–8.49 (m, 10H, Ar-H), 10.00 (s, 2H, NH, pyr- weight): C25H37N5O12: (599.59). Calculated analysis: C, 50.08; H,
role ring), 11.25, 13.50 (s, 4H, 4NH). MS (EI, 70eV): m/z (%) ¼ 594 6.22; N, 11.69; Found: C, 50.14; H, 6.27; N, 11.54.

1250
A. F. KASSEM ET AL.
N1,N3-bis (1-oxo-1-(-2-((2R, 3S, 4R)-2, 3, 4, 5-tetrahydroxypentyli- (s, 12H, 4OCH3), 4.55, 5.35 (2q, 2H, 2CHCH3), 7.00–8.42 (m, 10H,
dene) hydrazinyl) propan-2-yl) isophthalamide (9):
Ar-H), 8.50 (s, 2H, –N ¼ CH), 8.71,8.74 (2d, 2H, 2NH),11.32, 11.467
(2 s, 2H, 2NH). MS (EI, 70 eV): m/z (%) ¼ 632 (Mþ, 9.4), 607 (19),
592 (10), 582 (16), 54713), 558 (21), 547 (9), 493 (13), 488 (32), 462
Yield: 60%; melting point: < 300 ꢁC, IR (cmꢂ1): (KBr):ꢀ ¼ 3423
(OH),(3360, 3320 (2NH), 2922 (CH aliphatic), 1645 (4CO), 1531
1
(2 C ¼ N). H-NMR (500 MHz, d, ppm, DMSO-d6): d ¼ 1.18–1.42 (m,6H, (35), 390 (24), 318 (16), 300 (100), 206 (17), 76 (10), 40 (18).
2CH3),3.78 (m, 10H, H-5,50, H-4,3,2), 4.13 (m, 2H, 2OH), 4.45–4.52 Molecular formula (molecular weight): C32H32N6O8 (632.26).
(m, 4H, 4OH), 4.86 (m, 2H, CHCH3), 5.31 (m, 2H, 2OH), 7.62–8.52 Calculated analysis: C, 60.75; H, 5.74; N, 13.28; Found: C, 60.74; H,
(m, 6H, Ar-H þ H1), 872,8.83 (4H, 4NH). Molecular formula (molecu- 5.76; N, 13.38.
lar weight): C25H37N5O12: (599.59). Calculated analysis: C, 50.08; H,
6.22; N, 11.69; Found: C, 50.18; H, 6.24; N, 11.66.
2.1.8. General procedure for preparation of oxathiazolidin com-
pounds (14–16)
2.1.7. General procedure for preparation of Schiff’s base com-
pounds (10–13)
Thioglycolic acid (0.01 mol) was added to a well stirred solution of
Schiff’s bases 10–12 (0.01 mol) in dry benzene (20 ml), and then
A solution of bis hydrazide compound (3.36 g,0.01 mol) in absolute refluxed for 7–9 h. After completion of the reaction, excess solvent
ethanol (20 ml) containing glacial acetic acid (5 ml) was added dif- was evaporated under reduced pressure and the residue was neu-
ferent aromatic aldehydes (0.02 mol) the reaction mixture was tralized with cold dilute sodium bicarbonate solution, the formed
refluxed for 6–8 h. The formed precipitate was filtered off and product was filtered off, washed with water and then re-crystal-
crystallized form acetic acid to afford compounds 10–13.
N1,N3-bis-(1-(-2-(4-chlorobenzylidene) hydrazinyl)-1-oxopropan-2-
yl) isophthalamide (10):
lized from methanol to give compounds 14–16.
N1,N3-bis-(1-(2-(4-chlorophenyl)-4-oxothiazolidin-3-ylamino)-1-oxo-
propan-2-yl) isophthalamide (14)
Yield: 80%; melting point: 296–298 ꢁC, IR (cmꢂ1): (KBr): ꢀ ¼ 3332,
Yield: 65%; melting point: 218–220 ꢁC, IR (cmꢂ1): (KBr): ꢀ ¼ 3424,
3201 (4NH), 2983 (CH, aliphatic), 1639 (2CO), 1537 (2 C ¼ N). 1H- 3345 (4NH), 2925 (CH, aliphatic), 1724, 1677 (4CO). 1H-NMR
NMR (500 MHz, d, ppm, DMSO-d6): d ¼ 1.34–1.45 (m, 6H, 2CH3), (500 MHz, d, ppm, DMSO-d6): d ¼ 1.15, 1.29 (2d, 6H, 2CH3), 3.70,
4.55, 5.34 (2q, 2H, 2CH–CH3), 7.23–8.25 (m, 12H, Ar-H), 8.50 (s, 2H, 3.83 (s, 2H, S-CH2, thiazolidin ring), 4.42 (2 m, 2H, 2CH-CH3), 5.80,
–6.92–8.49 (m, 10H, Ar-H), 10.00 N ¼ CH), 8.72, 8.81 (2d, 2H, NH). 5.93 (2 s, 2H, 2 N-CH), 7.15–7.97 (m, 12H, Ar-H), 8.33, 8.75 (2d, 2H,
11.31, 11.40 (2 s, 2H, NH). MS (EI, 70 eV): m/z (%) ¼ 581 (Mþ, 5), 2NH). 10.29 (s, 2H, 2NH). MS (EI, 70 eV): m/z (%) ¼ 731 (Mþþ1,
575 (11), 523 (26), 519 (10), 494 (14), 488 (28), 460 (17), 425 (14), 0.23), 730 (Mþ, 1), 385 (1.4), 355 (9), 314 (2.5), 286 (2.5), 245 (11),
434 (31), 421 (40), 418 (15), 401 (24), 390 (39), 368 (100), 365 (21), 229 (7), 212 (22), 202 (14), 174 (23), 166 (13), 158 (15), 148 (18),
350 (31), 348 (15), 336 (16), 293 (12), 133 (10), 95 (20).Molecular 130 (17), 111 (30), 102 (18), 97 (100), 71 (53), 67 (16). Molecular
formula (molecular weight): C28H26Cl2N2O4 (581.14). Calculated ana- formula (molecular weight): C32H30Cl2N6O6S2 (729.65). Calculated
lysis: C, 57.84; H, 4.51; Cl, 12.19; N, 14.45; Found: C, 57.83; H, 4.52; analysis: C, 52.68; H, 4.14; Cl, 52.68 N, 11.52; S, 8.79; Found: C,
Cl, 12.09; N, 14.47.
52.69; H, 4.16; Cl, 52.69 N, 11.57; S, 8.76.
N1,N3-bis-(1-(-2-(4-fluorobenzylidene) hydrazinyl)-1-oxopropan-2-
yl) isophthalamide (11)
N1,N3-bis-(1-(2-(4-fluorophenyl)-4-oxothiazolidin-3-ylamino)-1-oxo-
propan-2-yl) isophthalamide (15)
Yield: 85%; melting point: 285–287 ꢁC, IR (cmꢂ1): (KBr): ꢀ ¼ 3332,
Yield: 64%; melting point: 168–170 ꢁC, IR (cmꢂ1): (KBr): ꢀ ¼ 3423,
3213 (4NH), 2981 (CH, aliphatic), 1645 (2CO), 1537 (2 C ¼ N). 1H- 3340 (4NH), 2921 (CH, aliphatic), 1721, 1679 (4CO). 1H-NMR
NMR (500 MHz, d, ppm, DMSO-d6): d ¼ 1.37–1.47 (m, 6H, 2CH3), (500 MHz, d, ppm, DMSO-d6): d ¼ 1.13, 1.28 (2d, 6H, 2CH3), 3.69,
4.56, 5.32 (2q, 2H, 2CH–CH3), 7.27–8.27 (m, 12H, Ar-H), 8.55 (s, 2H, 3.85 (s, 2H, S-CH2, thiazolidin ring), 4.42 (2 m, 2H, 2CH-CH3), 5.80,
–N ¼ CH), 8.74, 8.82 (2d, 2H, 2NH). 11.30, 11.42 (s, 2H, 2NH). MS 5.93 (2 s, 2H, 2 N-CH), 7.15–7.96 (m, 12H, Ar-H), 8.31,8.73 (2d, 2H,2
NH),10.27 (s, 2H, 2NH).13C-NMR (125 MHz, d, ppm, DMSO-d6):
(EI, 70 eV): m/z (%) ¼ 548 (Mþ, 6.4), 516 (20.5), 487 (10), 477 (37.9),
d ¼ 19.32 (2 CH3), 29.77 (2S-CH2), 49.75 (2 CH-CH3), 61.60 (N-CH),
463 (11.2), 433 (42), 419 (13), 406 (53), 394 (17), 394 (17), 376 (24),
115.69–158.00 (Ar-C), 166.03, 168.00, 172.14 (3 C ¼ O).MS (EI, 70 eV):
365 (75), 339 (100), 321 (59), 147 (19), 74 (63). Molecular formula
m/z (%) ¼ 696 (Mþ, 3.7), 601 (4), 578 (8), 461 (5.6), 414 (21), 382
(molecular weight): C28H26F2N6O4 (548.55). Calculated analysis: C,
(10), 366 (13), 351 (28), 333 (28), 324 (23), 294 (39), 285 (51), 281
(95), 267 (19), 256 (23), 130 (4), 80 (10), 78 (22), 56 (18), 40 (100).
61.31; H, 4.78; F; 6.93, N, 15.32; Found: C, 61.32; H, 4.76; F; 6.95,
N, 15.34.
N1,N3-bis-(1-(-2-(4-methoxybenzylidene) hydrazinyl)-1-oxopropan- Molecular formula (molecular weight): C32H30N6O6S2 (696.16).
2-yl) isophthalamide (12)
Calculated analysis: C, 55.16; H, 4.34; F, 5.45; N, 12.06; S, 9.20;
Yield: 80%; melting point: 273–275 ꢁC, IR (cmꢂ1): (KBr): ꢀ ¼ 3332, Found: C, 55.19; H, 4.36; F, 5.49 N, 12.16; S, 9.25.
N1,N3-bis -(1-(2-(4-methoxyphenyl)-4-oxothiazolidin-3-ylamino)-1-
1
3205 (4NH), 2981 (CH, aliphatic), 1641 (2CO), 1533 (2C ¼ N). H-NMR
(500MHz, d, ppm, DMSO-d6): d ¼ 1.43–1.47 (m, 6H, 2CH3), 3.08 (s, oxopropan-2-yl) isophthalamide (16)
Yield: 65%; melting point: 242–244 ꢁC, IR (cmꢂ1): (KBr): ꢀ ¼ 3424,
6H, 2OCH3), 4.56, 5.34 (2q, 2H, 2CHCH3), 7.00–8.21 (m, 12H, Ar-H),
8.45 (s, 2H, -N¼ CH), 8.69, 8.78 (2d, 2H, 2NH), 11.30, 11.46 (2 s, 2H,
2NH). MS (EI, 70 eV): m/z (%) ¼ 572 (Mþ, 19%), 544 (28), 504 (25),
496 (19), 487 (22), 462 (16), 431 (36), 419 (25), 395 (33), 389 (22),
347 (24), 331 (24), 312 (100), 295 (34), 84 (64), 59 (77). Molecular for-
mula (molecular weight): C30H32N6O6 (572.62). Calculated analysis: C,
62.93; H, 5.63; N, 14.93; Found: C, 62.95; H, 5.64; N, 14.98.
3338 (4NH), 2923 (CH, aliphatic), 1725.1678 (4CO). 1H-NMR
(500 MHz, d, ppm, DMSO-d6): d ¼ 1.16, 1.30 (2d, 6H, 2CH3), 3.72,
3.76 (2 s, 6H, 2OCH3), 3.84–3.88 (s, 2H, S-CH2, thiazolidin ring),
4.46, 4.52 (2 M, 2H, 2CHCH3), 5.72, 5.79 (2 s, 2H, 2N-CH) 7.33–8.23
(m, 12H, Ar-H), 8.061, 8.68 (2d, 2H, 2NH). 1023 (s, 2H, 2NH). MS (EI,
70 eV): m/z (%) ¼ 721 (Mþþ1, 2.3), 721 (Mþ, 9.8), 710 (19), 687
N1,N3-bis-(1-(-2-(3, 4-dimethoxybenzylidene) hydrazinyl)-1-oxopro- (11), 659 (12), 650 (27), 627 (20), 593 (16), 589 (45), 564 (34), 535
pan-2-yl) isophthalamide (13)
(41), 503 (52), 477 (100), 465 (16), 422 (25), 381 (30), 353 (12), 336
Yield: 85%; melting point: 277–279 ꢁC, IR (cmꢂ1): (KBr): ꢀ ¼ 3330, (17), 236 (11), 97 (25), 73 (29). Molecular formula (molecular
3215 (4NH), 2985 (CH, aliphatic), 1647 (2CO), 1536 (2 C ¼ N). 1H- weight): C34H36N6O8S2 (720.20). Calculated analysis: C, 56.65; H,
NMR (500 MHz, d, ppm, DMSO-d6): d ¼ 1.43–1.46 (m, 6H, 2CH3), 3.08 5.03; N, 11.66; S, 8.90; Found: C, 56.62; H, 5.05; N, 11.68; S, 8.92.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1251
2.2. Biological evaluations
2.2.5. Enzyme-linked immunosorbent assay kit for tubulin
beta (TUBb)
2.2.1. In vitro cytotoxic activity
MCF-7 cells were inoculated at concentration 1.2–1.8 ꢃ 10,000
cells/well using DMEM (supplemented with 10% FBS and 1% peni-
cillin–streptomycin). Tested compound added for 18–24 h before
the enzyme assay for Tubulin. Avidin conjugated to HRP is added
to each microplate well and incubated. After TMB substrate solu-
tion is added, wells that contain TUBb, biotin-conjugated antibody
and enzyme-conjugated Avidin will exhibit a change in colour.
Sulphuric acid solution added to terminate enzymatic reaction.
Colour change is measured spectrophotometrically at a wave-
length of 450 nm 10 nm39.
Human colon carcinoma (HCT-116 cell line), human lung carcin-
oma (A-549), human breast carcinoma (MCF-7 cell line) and
human skin normal cell line (BJ-1) were obtained from Karolinska
Center, Department of Oncology and Pathology, Karolinska
Institute and Hospital, Stockholm, Sweden. IC50 values were per-
formed using SPSS computer program (SPSS for windows, statis-
tical analysis software package/version 9/1989 SPSS Inc.,
Chicago, IL).
The procedure was done in laminar air flow cabinet bio safety
class II level. Culturing and sub culturing were carried out accord-
ing to Thabrew et al.32. Doxorubicin was used as a positive con-
trol. DMSO used as negative control. Cell Viability Assay was done
according to Selim et al.33 as described by Mosmann et al.34. The
cells were seeded at concentration of 10 ꢃ 103 cells per well in
case of MCF-7, 20 ꢃ 103 cells/well in case of HCT-116 cell lines
using 96-well plates at 37 ꢁC. After 48-h incubation, the medium
was aspirated and 40 ll MTT salt (2.5 mg/ml) were added and fur-
ther incubated for 4 h. About 200 ll 10% sodium dodecyl sulphate
(SDS) was added. The absorbance was measured at 595 nm.
2.2.6. Measurement of DNA fragmentation using DPA assay
DNA fragmentation of the cells was assayed, as previously
described40,41. Briefly, the cells were lysed with 20 mM EDTA, 0.5%
(v/v) Triton X-100, and 5 mM Tris (pH 8.0) for 15 min on ice. The
cells were then centrifuged for 20 min, at 27,000ꢃg, to separate
intact chromatin from DNA fragments. The amount of DNA was
measured by using a diphenylamine reagent. The optical density
is measured at 600 nm.
2.3. Molecular docking studies
2.2.2. In vitro enzyme inhibition assay
The molecular modelling of the compound 5 was carried out
using molecular operating environment (MOE, 10.2008) software42.
The X-ray crystallographic structure of EGFR co-crystallized with
erlotinib as an inhibitor (PDB ID: 1M17)43 was downloaded from
the protein data bank. The receptor was prepared for docking
study using Protonate 3D protocol in MOE with default options
followed by water molecules removal. The co-crystalized ligand
was used to define the active site for docking. Docking setup was
first validated by re-docking of the co-crystallized ligand in the
vicinity of the active site of the receptor. The validated setup was
then used in predicting the ligand–receptor interactions at the
active site for compound 5.
The in vitro enzyme inhibition assessment for compound 5 was
performed at confirmatory diagnostic unit, Vacsera, Egypt. The
screening achieved profiling of the compound 5 against a range
of four protein kinases [EGFR, VEGFR-2, CDK-2 and c-kit] by ELISA
assay method using staurosporine as a standard according to the
previously reported methods28.
2.2.3. Effect of compound 5 on the level of Bax/BCL-2/p53
Bax protein levels were evaluated according to Onur et al.
35
.
Monoclonal antibody specific to Bax captured on the plate is
added. After incubation, Strep avid in conjugated to Horseradish
peroxidase (HRP) is added. The reaction is terminated by the add-
ition of acid and optical density of the colour produced measured
at 450 nm.
3. Results and discussion
3.1. Chemistry
BCL-2 in the samples and standards were estimated according
to Barbareschi et al. 36. A biotin-conjugated antibody was added
followed by streptavidin-HRP. The reaction is then terminated by
addition of acid and absorbance was measured at 450 nm.
Human p53 present in the sample or standard binds to anti-
bodies adsorbed to the microwells. A biotin-conjugated is added.
After incubation and dispense of unbound biotin-conjugated
streptavidin HRP is added. The reaction is terminated by addition
In our previous studies of amino acid and peptide derivatives,
gave a good antimicrobial properties, anti-inflammatory, analgesic
agents44–48 and anticancer activities26–28,49, as well as, pyrazolo-
derivatives as anticancer agents50. Thus, our study aims to conju-
gate L-alanine and isophthalic acid. The novel candidates 3–16
were synthesized based on N1,N3-bis-(1-hydrazinyl-1-oxopropan-2-
yl) isophthalamide (4) which may be expected to possess promis-
ing anticancer properties. Accordingly, a rational design, synthesis
and structural characterization of N1, N3-bis (1-hydrazinyl-
1-oxopropan-2-yl) isophthalamide, were synthesized via synthetic
peptide coupling methods, (in solution). The compound N1,N3-bis-
(1-hydrazinyl-1-oxopropan-2-yl) isophthalamide (4) was synthe-
sized by the reaction of isophthalic acid (1) with thionyl chloride,
to give isophthaloyl chloride (2), while compound 2 was then
coupled with a free L-Alanine methyl ester to give dimethyl 2,2’-
(isophthaloyl-bis-(azanediyl)) dipropionate (3). IR of compound 3
showed absorption bands at 3330 cmꢂ1 due to NH in addition to
absorptions of carbonyl groups at 1744 cmꢂ1 (C ¼ O ester). 1H-
NMR of ester 3 revealed two signals in the region d 3.7–3.5 of
2CH3(CH3 ester) in addition to the D2O exchangeable signals of
amidic. Hydrazinolysis of dimethyl 2,2’-(isophthaloyl-bis-(azanediyl))
of acid and absorbance is measured at 450 nm37
.
2.2.4. Human caspase-7 (CASP-7) estimation
The micro ELISA plate provided in this kit pre-coated with CASP7-
specific antibody. A biotinylated CASP7 antibody and Avidin–HRP
conjugate was added. Aspire the excess components. The sub-
strate solution was added wells that contain CASP7, biotinylated
detection antibody and Avidin–HRP conjugate will appear blue in
colour. The colour turns yellow followed the addition of sulphuric
acid solution. The optical density (OD) was measured at a wave-
length of 450 nm 2 nm38.

1252
A. F. KASSEM ET AL.
Cl
O
C
O
O
H
N
H
N
S
Cl
ClH . H2N
Cl
HO
OH
Cl
OCH3
NH2
O
O
O
O
O
O
O
O
O
O
(1)
(3)
(2)
TEA
O
O
H
N
H
N
NH2NH2 . H2O
H2N
N
H
N
H
O
(4)
Scheme 1. Synthetic routes for N1,N3-bis-(1-hydrazinyl-1-oxopropan-2-yl) isophthalamide (4).
O
O
O
H
N
H
N
N
N
Cl
N
H
N
H
HN
O
O
O
O
O
NH
(5)
H
N
H
O
N
N
N
N
H
N
H
HN
O
O
(6)
NH
O
O
DMF/ Reflux
O
Cl
Cl
O
DMF/ Reflux
O
N
H
N
H
O
O
H
N
H
H2N
N
NH2
S
N
H
N
H
E t O H /
Pyridine / DMF/
Reflux
(4)
CS2
HO
O
O
O
EtOH/
A c O H
HO
OH
OH
S
HO
HO
O
O
H
O
A
H
N
HN
NH
c
N
N
O
N
OH
OH
H
O
O
H
H
N
O
O
O
N
N
N
(7)
N
N
H
H
HO
HO
O
O
O
HO
OH
OH
(9)
OH
OH
H
N
H
N
N
N
N
H
N
H
HO
HO
HO
O
O
HO
(8)
OH
OH
OH
OH
HO
Scheme 2. Synthetic routes for N1,N3- bis-(1-hydrazinyl-1-oxopropan-2-yl) isophthalamide derivatives 5–9.
dipropionate (3) with hydrazine hydrate led to the corresponding N1,N3-bis-(1-hydrazinyl-1-oxopropan-2-yl) isophthalamide (4) was
hydrazide 4 in 75% yield (Scheme 1). 1H-NMR of compound 4 reacted with the respective monosaccharide in ethanol containing
exhibited the D2O exchangeable signals at d 9.70 and 8.74 in add- glacial acetic acid which produced sugar hydrazone derivatives 8
1
ition to the signals of 2NH2 at d 4.24. The mass spectrum of and 9 (Scheme 2). H-NMR spectra for 8 and 9 exhibited the char-
hydrazide 4 showed a characteristic peaks equal to its molecular acteristic signals of CH, –N ¼ CH, at d 7.50, characteristic multiplets
weight and base beak at m/z ¼ 336 and42, respectively.
at the range d 4.11–5.31 (8 OH of sugar derivatives) and dupletes
at d 1.17–1.41 (2CH3, L-Ala).
The condensation of the bis (1-hydrazinyl-1-oxopropan-2-yl)
isophthalamide (4) with isatin or chloroisatin took place through
The condensation of the hydrazide 4 with 4-chlorobenzalde-
refluxing in DMF to produced hydrazide substituted candidates 5 hyde or 4-fluorobenzaldehyde took place through refluxing in
and 6 (Scheme 2). 1H-NMR of hydrazide candidates 5 and 6 in ethanol containing glacial acetic acid. This produced substituted
highly yield (75% and 70%), respectively. showed the characteristic hydrazides 10 and 11 (Scheme 3). 1H-NMR of hydrazones 10 and
signals of NH, pyrrole ring, at 10.00 and 10.30, respectively, as 11 illustrated the characteristic signal of NH in the range d
well as signals of NH in the range d 13.50–11.25. The mass spec- 8.72–8.81, as well as signal of -N ¼ CH at 8.50 and 8.55, respect-
trum of hydrazide derivatives 5 and 6 displayed a characteristic ively. The mass spectrum of 10 and 11 revealed a characteristic
peaks equal to its molecular weights at m/z ¼ 594 and 663, peaks equal to its molecular weights at m/z ¼ (581 and 548) and
respectively. On the contrary, hydrazide 4 was reacted with car- base beak at m/z ¼ (368 and 339), respectively. On the contrary,
bon disulphide in pyridine and DMF which produced oxadiazole- N1,N3-bis (1-hydrazinyl-1-oxopropan-2-yl) isophthalamide (4) was
derivative
7 (Scheme 2). The structure of oxadiazole 7 is reacted with 4-Anisaldehyde or Veratraldehyde to give the corre-
established under the basis of their spectral data. In addition to, sponding hydrazide derivatives 12 and 13, respectively

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1253
Cl
Cl
F
F
S
S
S
S
N
O
N
O
N
O
N
O
NH
HN
NH
HN
H
H
H
H
O
O
O
O
N
N
N
N
O
O
O
F
O
(14)
(15)
Cl
Cl
F
HO
O
HO
O
HS
HS
N
N
O
N
N
O
NH
HN
NH
HN
H
H
H
H
N
N
N
N
O
O
O
O
O
O
F
(10)
(11)
Cl
H
O
O
O
H
O
O
H
N
H
H2N
N
NH2
N
H
N
O
H
O
O
O
(4)
O
H
O
H
O
O
O
O
O
O
N
N
O
N
O
N
NH
HN
H
H
NH
HN
H
N
H
N
N
N
O
O
O
O
O
(12)
O
O
(13)
O
HO
HS
O
S
S
N
O
N
O
NH
HN
H
N
H
N
O
O
O
O
(16)
Scheme 3. Synthetic routes for N1,N3-bis-(1-hydrazinyl-1-oxopropan-2-yl) isophthalamide derivatives 10–16.
3.2. Biological evaluations
3.2.1. In vitro anticancer activity
(Scheme 3), and its structures were proven under the basis of
their spectral data.
The compounds 10–12 were reacted with 2-mercaptoacetic
acid in ethanol containing glacial acetic acid to give thiazolidine
derivatives 14–16, respectively (Scheme 3). 1H-NMR spectra
showed characteristic singles at the range d 3.69–3.88 (CH2 of
thiazolidine ring), 4.42 (2CH of a-L-Ala), and the mass spectrum of
14 and 15 exhibited a characteristic peaks equal to its molecular
weights at m/z ¼ (731 and 696) and base beak at m/z ¼ (97 and
40), respectively.
Cancer is ranked as the second cause global lifelessness; therefore,
there is a need for developing an antitumor candidate with min-
imal side effects to increase the efficacy of chemotherapy. In this
study thirteen compounds were preliminary screened for their
cytotoxicity utilizing three human carcinoma cell lines, namely
human colon carcinoma (HCT-116), lung carcinoma (A-549) and
human breast carcinoma (MCF-7) (Figure 2). The compounds
which revealed percentage of inhibition higher than 70% were

1254
A. F. KASSEM ET AL.
Cytotoxicity on HCT-116, A-549 and MCF-7 cell lines
120
100
80
60
40
20
0
HCT-116
A-549
MCF-7
4
5
6
7
8
9
10 11 12 13 14 15 16
Figure 2. In vitro cytotoxicity of compounds 4–16, against human colon tumour (HCT-116), lung tumor (A-549) and human breast tumour (MCF-7) cell lines at concen-
tration 100 mM. Each result is a mean of 3 replicate and values are represented as % inhibition ( standard error).
promising cytotoxic activity against tested cell lines revealed low
Table 1. IC50 values (concentration required to diminish 50% of the cell) of
active compounds possessing ꢄ 70% cytotoxicity on colon carcinoma (HCT-116), cytotoxicity on normal BJ-1 cells with % inhibition 32.6 which
lung carcinoma (A-549) and breast carcinoma (MCF-7) cell lines.
makes it interesting candidate for further biological evaluation.
IC50 (mean SD) (mM)a
Compd. no.
HCT-116
A-549
MCF-7
3.2.2. Structure–activity relationship
5
7
12
14
0.014 0.22
0.040 1.43
–
0.061 2.12
0.041 1.91
0.049 1.30
0.014 0.70
0.073 2.91
0.051 3.10
0.031 1.51
0.045 2.20
–
–
Depending on the previous results, it was noticed that the open
chain isophthalamide derivative 4 exhibited very weak cytotoxic
activities against HCT-116, A-549 and MCF-7 cell lines (% inhibition
¼ 2.3, 0.9 and 1.8, respectively). Cyclization with 2-oxoindoline
moiety gave the most potent derivative 5 against the three tested
cell lines (IC50 ¼ 0.014 0.22, 0.040 1.43 and 0.014 0.70 mM,
respectively) in comparison with the reference drug, doxorubicin
(IC50 ¼ 0.065 1.00, 0.049 1.30 and 0.045 2.20 mM, respectively).
On the one hand, substitution of 2-oxoindoline with chlorine
atom at p-5 in compound 6 displayed drastic decrease in the
activity towards all tested cell lines (% inhibition ¼ 33.1, 48.4 and
19.6, respectively). On the other hand, cyclization with 1,3,4-oxo-
diazole scaffold in compound 7 increased the sensitivity only
0.054 1.80
0.065 1.00
Doxorubicin
aValues of IC ( standard error) are calculated using SPSS statistical program.
% of cytotoxicity on BJ-1
120
100
80
60
40
20
0
towards MCF-7 cell line (% inhibition
0.073 2.91 mM). A dramatic drop in the anticancer effect was
¼
77.5 and IC50
¼
5
7
12
14
detected upon attachment of isophthalamide with 2,3,4,5-tetrahy-
Figure 3. cytotoxic percentage upon normal skin human cell line (BJ-1) at con-
centration 100mM of those compounds gave ꢄ 70% cytotoxicity over the three droxypentyl sugars in 8 and 9. Insertion of phenyl ring substituted
tumour cell lines. Each result is a mean of 3 replicate and values are represented
as % inhibition ( standard error).
at p-4 with electron withdrawing groups as Cl or F in compounds
10 and 11, or with electron donating group as methoxy in com-
pound 12 showed variable and noticeable increase in the cyto-
Table 2. Inhibitory evaluation of compound 5 against EGFR, VEGFR-2, CDK-2
and c-Kit kinases.
toxic activity, while substitution with two methoxy groups at p-3,4
led to reduction in the activity due to steric hindrance in com-
pound 13. The 4-oxothiazolidine derivative linked at p-2 to 4-
IC50 (mean SD) (mM)
chlorophenyl 14, exhibited excellent anticancer potency (IC50
¼
Kinase
5
Staurosporine
0.054 1.80, 0.041 1.91and 0.031 1.51 mM, respectively) and it
displayed higher percentage of inhibition at 100 lM than the
4-fluorophenyl and 4-methoxyphenyl congeners 15 and 16.
EGFR
0.78 1.25
3.17 1.00
6.24 1.21
2.85 0.04
1.00 1.12
0.08 1.10
0.11 1.13
0.60 1.00
VEGFR-2
CDK-2
c-kit
IC50: compound concentration required to inhibit the enzyme activity by 50%,
SEM ¼ standard error mean; each value is the mean of three values.
3.2.3. In vitro enzymatic assays
Based on observing the results of in vitro cytotoxicity studies of
the newly synthesized isophthalamide derivatives, the highly
active compound 5 was selected for evaluation of its inhibitory
activities versus a panel of four different kinases: epidermal
growth factor receptor (EGFR), vascular endothelial growth factor
receptor-2 (VEGFR-2), cyclin-dependent kinase-2 (CDK-2) and c-kit
(receptor tyrosine kinase type III) using staurosporine as multitar-
get inhibitor.
From IC50 values depicted in Table 2, it was noticed that com-
pound 5 potently inhibited EGFR rather than the reference (IC50
0.78 1.25 and 1.00 1.12 lM, respectively). In contrast, compound
5 seemed to have weak inhibitory activities against VEGFR-2, CDK-
2 and c-Kit kinases with IC50 values ranging from 2.85 0.04
further assessed for determination of their median growth inhibi-
tory concentration (IC50) and doxorubicin was used as the refer-
ence drug as shown in Table 1. Compounds 5, 7, 12 and 14
showed moderate to excellent % of inhibition ranging from 72.5
to 98.3 towards the three tested cancer cell lines.
Concerning IC50 data, it was observed that only the com-
pounds 5 and 14 exhibited higher potency against all tested cell
lines in comparison with the standard drug.
¼
Furthermore, the active compounds 5, 7, 12 and 14 were also
screened for their cytotoxic effects on human normal skin fibro-
blasts cell line BJ-1 at 100 lM to test their safety on normal cells
(Figure 3). It was clear that the oxoindoline derivative 5 with the to 6.24 1.21 lM.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1255
Figure 4. quantitative analysis of Bax, BCL-2 and Bax/BCL-2 ratio and p53 for compound 5 compared to doxorubicin, where Y-axis represents the protein levels
(ng/ml).
3.2.4. Effect of compound 5 on the level of Bax, BCL-2, p53, CASP- active and promising chemotherapeutic agent. Accordingly, the
inhibitory evaluation of compound 5 was carried out against dif-
ferent human proteins which expressed in most of tumour cells
especially breast carcinoma namely, EGFR, VEGFR-2, CDK-2 and c-
Kit, to validate and specify the mechanism of action or proteins
responsible for the anticancer activity.
Consequently, molecular docking was performed with the aim of
explaining the promising EGFR inhibitory activity of compound 5
through investigating its binding mode and interaction with the key
amino acids (hot spots) in the active site of the EGFR. Docking simu-
lations were done using Molecular Operating Environment software
10.2008 (MOE), Chemical Computing Group Inc., Montreal, Quebec,
Canada42. The X-ray crystallographic structure of EGFR (pdb code:
1M17)43 was downloaded from the protein data bank with its ligand,
erlotinib. The docking protocol was validated by re-docking the co-
crystallized ligand, erlotinib into the EGFR binding pocket, followed
by docking of our compound 5 in the same binding site.
7, tubulin polymerization (TubB) and % of DNA fragmentation:
According to our results, compound 5, which has the highest activ-
ity on all tested cancer cells including breast carcinoma cells, was
considered the safest one among other promising compounds on
normal skin cells with weak effect of cytotoxicity (32.6%). Therefore,
compound 5 was chosen to elucidate its possible apoptotic mode
of action on breast carcinoma cells through investigation of its
effect on Bax, BCL-2, Bax/BCL2 ratio, P53, CASP-7, Tubulin polymer-
ization (TubB) and % of DNA fragmentation.
Apoptosis of cancer cells is regulated by the pro-apoptotic Bax,
the anti-apoptotic Bcl-2 proteins and the tumour suppressor gene
p5351. Exposure of MCF-7 cells to compound 5 at IC50 for 24 h
resulted in significant up-expression of p53 and Bax, with conse-
quent down-expression in the level of BCL-2 compared to the
control (Figure 4). As a result, the tested compound 5 showed sig-
nificant elevation in Bax/BCL-2 ratio, which supports its ability to
elevate the therapeutic response in MCF-7 cells.
The obtained results are pictured in Figures 6 and revealed that
the isophthalamide derivative bearing 2-oxoindoline moiety 5,
formed two hydrogen bonding with the same amino acid Met769
as erlotinib, through the N atom and the carbonyl oxygen localized
in its oxoindoline moiety (distance: 1.93 and 2.43 Å, respectively).
There are two vital apoptotic pathways, the death receptor
(extrinsic) and the mitochondrial (intrinsic) pathways52. The two
pathways focus on the same terminal, which was initiated by the
activation of CASP-7 and results in DNA fragmentation53. In the cur-
rent study, compound 5 significantly elevated the Bax/BCL-2 ratio. Moreover, the other 2-oxoindoline moiety exhibited a third hydro-
Therefore, the subsequent step was to investigate the level of gen bond acceptor between its carbonyl oxygen and the sidechain
active CASP-7, which is the key executer of apoptosis. Treatment of of Arg817 (distance: 2.74 Å). Additionally, a hydrophobic interaction
MCF-7 cells with compound 5 produced a significant increase in (arene–arene interaction) with EGFR binding site was introduced
the level of active CASP-7. Moreover, compound 5 showed remark- through the central phenyl moiety of compound 5 and Phe699.
This helped us to find an explanation for the excellent binding
able % of DNA fragmentation and moderate effect on microtubule-
polymer mass in comparison with colchicine (Figure 5). In affinity between compound 5 and EGFR, as presence of two 2-
conclusion, compound 5 showed potent pro-apoptotic effect by oxoindoline moieties in its structure let it to get good fitting with
induction of the intrinsic mitochondrial pathway of apoptosis.
the binding site.
3.3. Molecular docking studies
4. Conclusion
Cytotoxic activity of all synthesized derivatives has been realized In the current study, novel isophthalamide based derivatives incor-
by using in vitro assay and proved that compound 5 is the most porated to different hetercycles 4–16 were synthesized using

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A. F. KASSEM ET AL.
Figure 5. Graphical representation for Caspase-7 concentration (ng/ml) in treated breast cancer cells with compound 5 and untreated compared to doxorubicin and
for % DNA fragmentation and IC50 (ng/ml) on tubuline B in comparison with colchicine.
effect upon different apoptotic parameters in breast cancer cells.
The docking studies also support the results concluded from the
enzyme assay and the anti-proliferation screening. It was clear
that our ligand 5 that bearing 2-oxoindoline moiety may be con-
sidered as a suitable lead for further development of anticancer
drugs in future.
Acknowledgement
This work was funded by the University of Hafr Al Batin, College
of Applied Medical Science, Clinical Laboratory Science
Department, and was funded by the National Research Center at
Dokki, Cairo, Egypt, through the Research Groups Program Grant
no. 11010339.
Disclosure statement
The authors declare no conflict of interest.
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