Synthesis, Characterization, And antI-Cancer Activity of some new n′ (2-Oxoindolin-3-Ylidene)-2-Propylpentane Hydrazide-Hydrazones derivatives

Article abstract of DOI:10.3390/molecules200814638

Eight novel N′-(2-oxoindolin-3-ylidene)-2-propylpentane hydrazide-hydrazone derivatives 4a-h were synthesized and fully characterized by IR, NMR ( (1H-NMR and 13C-NMR), elemental analysis, and X-ray crystallography. The cyto-toxicity and in vitro anti-cancer evaluation of the prepared compounds have been assessed against two different human tumour cell lines including human liver (HepG2) and leukaemia (Jurkat), as well as in normal cell lines derived from human embryonic kidney (HEK293) using MTT assay. The compounds 3e, 3f, 4a, 4c, and 4e revealed promising anti-cancer activities in tested human tumour cells lines (IC50 values between 3 and 7 μM) as compared to the known anti-cancer drug 5-Fluorouracil (IC50 32-50 μM). Among the tested compounds, 4a showed specificity against leukaemia (Jurkat) cells, with an IC50 value of 3.14 μM, but this compound was inactive in liver cancer and normal cell lines.

Full text of DOI:10.3390/molecules200814638

Molecules 2015, 20, 14638-14655; doi:10.3390/molecules200814638
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molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis, Characterization, and Anti-Cancer Activity of Some
New N′-(2-Oxoindolin-3-ylidene)-2-propylpentane
hydrazide-hydrazones Derivatives
Ayman El-Faham 1,2,*, Muhammad Farooq *, Sherine N. Khattab , Nael Abutaha ,
3,
2
3
3
4
4,5
Mohammad A. Wadaan , Hazem A. Ghabbour and Hoong-Kun Fun
1
Department of Chemistry, College of Science, King Saud University, P.O. Box 2455,
Riyadh 11451, Saudi Arabia
2
3
4
Department of Chemistry, Faculty of Science, Alexandria University, P.O. Box 426,
Alexandria 21321, Egypt; E-Mail: Sh.n.Khattab@gmail.com
Department of Zoology, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451,
Saudi Arabia; E-Mails: nabutaha@ksu.edu.sa (N.A.); Wadaan@ksu.edu.sa (M.A.W.)
Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University,
P.O. Box 2457, Riyadh 11451, Saudi Arabia; E-Mails: ghabbourh@yahoo.com (H.A.G.);
hfun.c@ksu.edu.sa (H.-K.F.)
5
X-ray Crystallography Unit, School of Physics, Universiti Sains Malaysia, Penang 11800, Malaysia
*
Authors to whom correspondence should be addressed; E-Mails: aymanel_faham@hotmail.com (A.E.-F.);
fmuhammad@ksu.edu.sa (M.F.); Tel.: +966-114-673-195 (A.E.-F.);
Fax: +966-114-675-992 (A.E.-F.).
Academic Editor: Jean Jacques Vanden Eynde
Received: 5 July 2015 / Accepted: 6 August 2015 / Published: 13 August 2015
Abstract: Eight novel N′-(2-oxoindolin-3-ylidene)-2-propylpentane hydrazide-hydrazone
1
derivatives 4a–h were synthesized and fully characterized by IR, NMR ( H-NMR and
13C-NMR), elemental analysis, and X-ray crystallography. The cyto-toxicity and in vitro
anti-cancer evaluation of the prepared compounds have been assessed against two different
human tumour cell lines including human liver (HepG2) and leukaemia (Jurkat), as well as
in normal cell lines derived from human embryonic kidney (HEK293) using MTT assay.
The compounds 3e, 3f, 4a, 4c, and 4e revealed promising anti-cancer activities in tested human
tumour cells lines (IC50 values between 3 and 7 μM) as compared to the known anti-cancer
drug 5-Fluorouracil (IC50 32–50 μM). Among the tested compounds, 4a showed specificity

Molecules 2015, 20
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against leukaemia (Jurkat) cells, with an IC50 value of 3.14 μM, but this compound was
inactive in liver cancer and normal cell lines.
Keywords: valproic acid; isatin; hydrazide-hydrazone; anti-cancer activity
1
. Introduction
Hydrazide-hydrazone derivatives are molecules containing a highly reactive group (CO-NH-N=CH)
and considered to be a good candidate for development of a new drug [1]. Recently, hydrazide-hydrazones
have been considered to be of great interest in medicinal chemistry due to their diverse biological
properties, including anti-microbial [2–4], anti-mycobacterial [5,6], anti-convulsant [7], analgesic [8],
anti-inflammatory [9], anti-platelet [10], anti-tubercular[10–13], and anti-tumoral activities [14–19]. In
addition, hydrazide-hydrazones have been reported to elicit anti-cancer [18–21] and anti-HIV properties [22]
and they are therefore increasingly considered to be of great value in medicinal chemistry [16,23–27].
Because of the remarkable biological value of isatin as an important constituent of bioactive
compounds exhibiting caspase inhibitory [28,29], antibacterial, and antiproliferative activity [30], Schiff
bases of isatin derivatives have antismallpox [31] and GAL3 receptor antagonist capabilities [32]. In
addition, the analogous of isatin derivatives displayed inhibitory activity against eLF2 kinase activator [33],
TNF-α, CDK2 [34] and SARS protease [35]. Isatin also displays antiviral [36], anti-inflammatory,
analgesic [37], and anticonvulsant activities [38].
Valproic acid (VPA, 1) has an important biological value as a potent antiepileptic molecule [39–41],
as well as showing inhibition of angiogenesis both in vitro and in vivo [42–45]. VPA also acts as a
powerful histone deacetylase inhibitor [46,47] and induces differentiation and apoptosis in a variety of
malignant cells in vitro [48]. Clinical trials with VPA have focused on acute myeloid leukaemia and the
myelodysplastic syndromes. When it was used as mono therapy or in combination with all-trans retinoic
acid, which synergizes in vitro, VPA achieved hematologic improvement in a subset of patients [48].
As a continuation to our previously reported data [49–51], herein we designed eight isatin
hydrazide-hydrazone derivatives, considering some of the factors responsible for such activity, including
(
i) the presence of isatin moiety; (ii) the presence of the hydrazide-hydrazone functionality; and (iii)
valproic acid moiety (Figure 1).
Figure 1. Structure of the target product.

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All the prepared compounds were assessed against two different human tumour cell lines, including
human liver (HepG2) and leukaemia (Jurkat), as well as in normal cell lines derived from human
embryonic kidney (HEK293) using MTT assay.
2
. Results and Discussion
2
.1. Chemistry
Compound 2 was prepared following the reported method [49] and condensed with isatin derivatives
a–h in the presence of 2–3 drops of glacial HOAc and ethanol as the solvent to afford products 4a–h
3
(
Scheme 1). The structures of all synthesized compounds were in a good agreement with their spectral data.
Scheme 1. Synthesis of N′-(2-oxoindolin-3-ylidene)-2-propylpentanehydrazide Derivatives.
The IR spectra of the compounds 4a–g reveal absorption bands in the region 3195–3217 cm−1
−1
corresponding to the (NH), a band at 1720 and 1688 cm corresponding to the C=O group, and a peak
−1
around 1596 cm related to the C=N bond. The NMR of all the products 4a–g are in good agreement
with their structures (Figures S1–S7).
1
As a prototype H-NMR of 4g showed multiple peaks at δ 0.86–0.89, 1.27–1.29, 1.40–1.44,
1
.56–1.60 ppm, and a broad singlet at δ 2.51 ppm corresponding to the valporic acid moiety (2 CH
CH , and CH, respectively). Also, two triplet peaks were observed at δ 3.77 and 4.20 ppm corresponding
-CH Br), respectively. The observed peaks in the aromatic region at
δ 7.18 (t, 1H, Ar-H), 7.32 (d, 1H, Ar-H), 7.53 (t, 1H, Ar-H), and 7.88 (brs, 1H, ArH) are related to the
3
,
4
2
to the two methylene group (CH
2
2
1
3
isatin moiety, while the broad singlet peak at δ 12.28 ppm is related to the NH. The C-NMR of 4g
showed peaks at δ 14.5, 20.6, 29.9, 35.3, 39.7, 42.2, and 46.5 ppm, corresponding to the valporic acid
moiety and the two methylene groups, in addition to seven peaks related to the aromatic carbons and
imino function group. The two peaks at δ 169.8 and 176.0 ppm corresponded to the carbonyl groups of
the isatin moiety and the hydrazide group, respectively.

Molecules 2015, 20
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It is expected that compound 4g could adopt two different geometrical isomers (Z and E) as shown in
Figure 2A. Therefore, it is considered worthwhile to model the compounds using molecular mechanics
MM2 calculations. In addition, quantum chemical calculations were carried out with the GAUSSIAN 98
suite of programs. Geometry optimizations were carried out using the DFT level (B3LYP/6-31G **) of
theory to assess the relative stability of the Z and E isomeric species. Calculated relative energies of 4g
Z and E isomers are −3585.4860873 au and 3585.4635100 au, respectively. Computed energies indicate
the stability of the Z isomer over the E one by 0.0225773 au (14.1675 kcal/mol) (Figure 2B).
B
A
Figure 2. (A) The expected geometrical isomers, Z and E forms, of N′-(1-(2-bromoethyl)-2-
oxoindolin-3-ylidene)-2-propylpentane hydrazide; (B) The expected geometrical isomers,
the 3D structure of Z and E forms, of N′-(1-(2-bromoethyl)-2-oxoindolin-3-ylidene)-2-propyl
pentane hydrazide.
The X-ray single crystal structure determination of compound 4g (CCDC: 996592) confirmed that
the structure exists in the Z-conformer rather than the E-conformer (Figure 3). The details of data
collection and structure refinement are listed in Table 1, and the X-ray structures are shown in Figure 3.
Compound 4g crystallized from ethanol in the triclinic space group P-1. All bond lengths and angles are
in normal ranges [52].
In the crystal packing (Figure 4), the molecules are linked by intermolecular C17A—H17B···Br1B,
C17B—H17D···O2B and C18A—H18B···O2A hydrogen bonds (Table 2) into two-dimensional networks
parallel to the bc plane (Figure 4 and Table 2). The asymmetric unit contains two molecules of the
compound with disorder in one of the ethylene arms. The molecular structure of compound 4g is
composed of a 2-oxoindoline ring (C1/N1/C2-C8) which is linked with two side chains at N1 and C8.
The two molecules in the asymmetric unit are with Z configuration about the C8=N2 double bond
(
Figure 3). The Z configuration of 4a is stabilized with intramolecular hydrogen N3—H3N···O2 (Table 2).
The single bond N2—N3 is clearly characterized by the distances of 1.355 (3) Å and 1.361 (4) Å,
respectively, for molecules A and B. The double bond of C8=N2 is characterized by the distances of
1
.298 (3) Å and 1.283 (4) Å, for molecules A and B, respectively.

Molecules 2015, 20
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Figure 3. ORTEP diagram of the asymmetric unit consisting of two symmetry-independent
molecules. The ellipsoids are drawn at the 50% probability level with hydrogen atoms
being shown as spheres of arbitrary radii. One of the molecules has disorder in the side
chain C11B—C13B.
Table 1. Details of data collection and structure refinement for compound 4g.
Compound
4g
Empirical formula
C
18
H24BrN
3
O
2
Formula weight
349.21
100
Temperature (K)
Wavelength (A)
Mo Kα radiation, λ = 0.71073 Å
Crystal system, Space group
Triclinic, P-1
Crystal
a (Å)
b (Å)
c (Å)
α (°)
Plate, yellow
8.8126 (11)
14.4104 (18)
14.479 (2)
92.002 (4)
β (°)
97.498 (4)
γ (°)
92.598 (4)
3
Volume (Å )
1819.6 (4)
−
3
Z, Dcalc (Mg·m )
F (000)
4, 1.275
816
Crystal size (mm)
0.58 × 0.41 × 0.12
θ Range for data (°)
Collection Limiting indices
Reflections collected/unique
restraints/parameters
2.33–28.11
−12 ≤ h ≤ 12, −20 ≤ k ≤ 20, −20 ≤ k ≤ 20
81,300/6824 [Rint = 0.136]
112/469
2
Goodness-of-fit on F
1.009
Final R indices[I > 2s(I)]
Absorption correction
Instrument
R1 = 0.053, wR2 = 0.135
Multi-scan, SADABS V2012/1 (Bruker AXS Inc., Madison, WI, USA)
Bruker APEX-II D8 venture diffractometer

Molecules 2015, 20
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Figure 4. Crystal packing showing intermolecular hydrogen bonds as dashed lines.
Table 2. Hydrogen-bond geometry (Å, °).
D—H···A
D—H
H···A
D···A
D—H···A
141 (3)
138 (3)
107.00
109.00
102.00
149.00
131.00
140.00
N3B−H3NB···O1B
N3A−H3NA···O1A 0.86 (3) 2.11 (3) 2.799 (3)
C10A−H10A···N2A
C10B−H10B···N2B
C17A−H17A···O1A
C17A−H17B···Br1B
0.81 (3) 2.10 (3) 2.779 (3)
0.9800 2.3700 2.815 (4)
0.9800 2.3600 2.833 (4)
0.9700 2.5700 2.930 (3)
0.9700 2.9100 3.779 (3)
0.9700 2.4100 3.139 (4)
0.9700 2.2900 3.096 (3)
i
ii
C17B−H17D···O2B
iii
C18A−H18B···O2A
i
ii
iii
Symmetry codes: ( ) x − 1, y, z; ( ) −x + 1, −y + 1, −z; ( ) −x + 1, −y + 1, −z + 1.
Reaction of valporic acid hydrazide 2 with N-acetylisatin 3h proceeds in a different way, where the
ring opening occurred to afford the product 4h. The IR and NMR data proved its structure in the open
form, due to the attack of the hydrazide group on C2 instead of C3 [53]. The IR spectrum of 4h reveals
−1
absorption bands in the region 3310 and 3223 cm corresponding to the NH, a band at 1720, 1709, 1688,
−1
1
and 1608 cm corresponding to the C=O and C=N group, respectively. H-NMR of 4h showed multiple
peaks at δ 0.86–0.89, 1.27–1.29, 1.40–1.44, 1.56–1.60, and a broad singlet peak at δ 2.21 ppm corresponding
to the valporic acid moiety (2 CH
is corresponding to the N-acetyl group. The observed peaks in the aromatic region at δ 7.28 (t, 1H), 7.68
t, 1H), 7.99 (d, 1H), and 8.17(d, 1H) ppm, are related to the phenyl ring, while the two singlet peaks at
3 2 2
, 2 CH , and 2CH , CH, respectively). The observed peak at δ 2.16 ppm
(
1
3
δ 10.03 and 10.72 ppm corresponded to the two NH. The C-NMR of 4h showed peaks at δ 14.6, 20.6,
9.9, 35.3, 43.8, and 46.5 ppm corresponding to the valporic acid moiety group, while the acetyl group
was observed at δ 25.0 ppm, in addition to six peaks related to the aromatic carbons at δ 121.5, 121.8,
23.8, 133.6, 135.9, and 140.3 ppm. The three peaks observed at δ 164.1, 169.6, and 174.7 ppm related
to the three CONH groups, while the peak observed at δ 193.0 ppm is related to the α-ketoamide group.
2
1
2.2. Anti-Cancer Activity
The cyto-toxicity activity was measured in vitro in HepG2, Jurkat, and HEK 293 cells using the MTT
1
-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan colorimetric assay. For comparison, 5-Fluorouracil

Molecules 2015, 20
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(5-FU) was used as a standard anti-cancer drug. Treatment with dimethylsulfoxide (DMSO) was used as
a control for the cancer cells.
The anti-cancer activity of the new prepared compounds against the HepG2 cell line revealed that
compounds 3a, 3h, 4a, 4b, 4f, 4g, and 4h showed no anti-cancer activity. Similarly, the starting
compounds 1 and 2 did not show promising anti-cancer activity in HepG2 cells. Compound 3e exhibited
higher potency against HepG2 cell line with IC50 = 2.82 ± 40.7 μM, which is much lower than the IC50 value
of 5-FU (32.15 ± 48.1 μM). Moreover, the results showed that compounds 3f, 4c, and 4e were also found to
be potent and selective against HepG2 with IC50 values of 3.13 ± 42.0, 4.39 ± 43.2, and 7.61 ± 44.6 μM,
respectively, which are also much lower than 5-FU (Figure 5 and Table 3).
The anti-cancer profile of newly synthesized compounds was also tested against leukaemia cells
(Jurkat cells, originated from human T lymphocytes). The compound 4c was most active, with an IC50 value
of just 2.90 ± 36.3 μM, which was much lower than the positive control (5-FU IC50 = 51.16 ± 47.4 μM).
Similarly, the compounds 3f, 4a, and 4c showed strong activity and IC50 values were much lower as
compared to 5-FU (Table 3 and Figure 5). The order of activity was 3f, 4a, 4e, and 3e in ascending
order (Table 3 and Figure 5). The compound 1 showed a weaker level of activity against Jurkat cells
(
IC50 79.35 ± 40.7 μM) and, indeed, this was much weaker than 5-FU (IC50 51.16 ± 47.4 μM).
Figure 5. Cont.

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Figure 5. Dose-dependent responses of hydrazide-hydrazone derivatives in three cell lines.
Each line represents the mean of three different experiments representing the % of mortality
as compared to mock treated control cells.
Table 3. In vitro cytotoxicity activity of the synthesized compounds in two cancer and one
normal cell line(s) as measured with MTT assay *.
IC50 (μM)
Compd. No.
HepG2
Jurkat
HEK293
1
2
69.74 ± 40.8 79.35 ± 40.7 62.98 ± 38.2
68.50 ± 37.1
N.A.
N.A.
N.A.
N.A.
16.73 ± 50.0
N.A.
3
a
e
3
2.82 ± 40.7 7.13 ± 46.0
3.13 ± 42.0 3.13 ± 42.0
3
f
N.A.
3
h
N.A.
N.A.
N.A.
N.A.
3.15 ± 43.3
N.A.
N.A.
4
a
N.A.
4
b
26.08 ± 31.3
4
c
4.39 ± 43.2 2.90 ± 36.3 4.77 ± 42.7
4
d
N.A.
N.A.
N.A.
N.A.
4
e
7.61 ± 44.6 3.19 ± 46.9
4
f
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
4
g
45.43 ± 48.9
N.A.
4
h
5
-FU
32.15 ± 48.1 51.16 ± 47.4
N.A. N.A.
N.A.
DMSO
N.A.
*
Data were expressed as the mean ± standard deviation (SD) of six independent experiments; N.A.: IC50 values
more than 100 μM was considered as No Activity.
Finally the cyto-toxicities of these compounds were screened in the normal human embryonic kidney
HEK293) cell line, in order to assess whether these compounds are active only in cancer cells or whether
(
they possess toxicity against normal cells as well. The results revealed that the compound 4c turned out
to be most toxic by disrupting the cell survival of normal cells (HEK 293) with IC50 4.77 ± 42.7 μM.
The compounds 3a, 4b, and 4g also possessed some level of cyto-toxicity against HEK 293 cells with
IC50 values 16.73 ± 50.0, 26.08 ± 31.3, and 45.43 ± 48.9 μM, respectively. The IC50 values of these

Molecules 2015, 20
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compounds in HEK cells, however, are much higher compared to their activity in cancer cells (Table 3
and Figure 5). The compounds 3e, 3f, 4a, and 4e showed no activity in HEK 293 cells, but strong activity
in liver (HepG2) and leukaemia (Jurkat) cancer cells, suggesting that these compounds are potent and
potentially viable anti-cancer molecules.
3
3
3
. Experimental Section
.1. Chemistry
.1.1. Materials
The solvents used were of HPLC reagent grade. Melting points were determined with Melting points
were obtained in open capillary tubes using a MEL-Temp melting point apparatus (Sigma-Aldrich
Chemie GmbH, 82024 Taufkirchen, Germany) and are uncorrected and are uncorrected. Infrared (IR)
spectra were recorded on a Perkin-Elmer 1600 series Fourier transform instrument (PerkinElmer Life
and Analytical Sciences, Shelton, CT, USA) as KBr pellets. Nuclear Magnetic resonance spectra
(1
H-NMR and C-NMR spectra) were recorded on 400 MHz JEOL spectrometer (JEOL Ltd., Tokyo,
13
Japan) at room temperature. Chemical shifts are reported in parts per million (ppm) and are referenced
relative to residual solvent (e.g., CHCl at δ 7.26 ppm for CDCl , DMSO at δ 2.50 ppm for DMSO-d ).
3
3
6
Spin multiplicities are represented by the following signals: singlet (s), broad singlet (br s), doublet (d),
broad doublet (br d), doublet of doublets (dd), triplet (t), doublet of triplets (dt), and multiplet (m).
Elemental analyses were performed on a Perkin-Elmer 2400 elemental analyzer (PerkinElmer Inc.,
Waltham, MA USA), and the values found were within ±0.3% of the theoretical values. Follow-up of
the reactions and checks of the purity of the compounds was done by TLC on silica gel-protected
aluminum sheets (Type 60 GF254, Merck Millipore, Billerica, MA, USA) and the spots were detected by
exposure to UV-lamp (Company Seven, Montpelier, MD, USA) at λ 254 nm for a few seconds. The
compounds were named using Chem. Draw Ultra version 11, Cambridge soft Corporation. Crystallographic
data for the structure reported in this paper have been deposited at the Cambridge Crystallographic Data
Center and allocated with the deposition numbers: CCDC 996592 for compound 4g. CCDC 996592
contains the supplementary crystallographic data for this paper. These data can be obtained free of charge
via http://www.ccdc.cam.ac.uk/conts/retrieving.html (or from the CCDC, 12 Union Road, Cambridge
CB2 1EZ, UK; Fax: +44 1223 336033; E-mail: deposit@ccdc.cam.ac.uk).
3
.1.2. Synthesis of Valproic Hydrazide 2
The product was prepared according to the reported procedure [50] and obtained as white needle
−1 1
crystals, in 85%, yield; mp 123–124 °C; IR (KBr): 3284 (NH), 1631 (CO, amide) cm ; H-NMR (CDCl
3
)
δ (ppm): 0.86 (t, J = 4.4 Hz, 6H, 2CH ), 1.14–1.39 (m, 6H, 3CH ), 1.53–1.604 (m, 2H, CH ), 1.95–2.01
3
2
2

Molecules 2015, 20
m, 1H, CH), 3.95 (brs, 2H, NH
14647
1
3
(
2
), 7.05 (s, 1H, NH). C-NMR (CDCl
3
) δ (ppm): 14.11, 20.86, 35.05,
4
5.61, 177.06.
3
.1.3. Synthesis of Compounds 3e–h
Compounds 3e–h were prepared following the reported procedure [54]. The entire prepared compounds
were in a good agreement with the reported data.
3
.1.4. General Method for Preparation of N′-(2-Oxoindolin-3-ylidene)-2-propylpentane Hydrazide
Derivatives 4a–h
A solution of valproic hydrazide 2 (316 mg, 2 mmol) in ethanol (20 mL) was added to a solution of
substituted isatin 3a–h (1 mmol) in ethanol (20 mL), and glacial acetic acid (2 drops); the reaction mixture
was refluxed for 3–4 h. The product was separated out on cooling, filtered, and recrystallized from ethanol
or ethylacetate to afford N′-(2-oxoindolin-3-ylidene)-2-propylpentanehydrazide derivatives 4a–h.
(
Z) N′-(2-Oxoindolin-3-ylidene)-2-propylpentane Hydrazide 4a
The product was obtained as yellow solid, in 89% yield; mp 175–176 °C. IR (KBr): 3194 (NH), 1720
−
1 1
(
CO), 1663 (C=N), 1602 (CO) cm ; H-NMR (DMSO-d
m, 4H, 2 CH ), 1.40–1.42 (m, 2H, CH ), 1.56–1.58 (m, 2H, CH
H, Ar-H), 7.06 (brs, 1H, Ar-H), 7.38 (brs, 1H, ArH), 8.02 (brs,1H, ArH), 10.80 (s, 1H, NH), 11.08
6
) δ (ppm): 0.85-0.86 (m, 6H, 2 CH
3
), 1.26–1.27
(
2
2
2
), 2.51 (brs, 1H, CH), 6.90 (d, J = 4.6 Hz,
1
1
3
(
s, 1H, NH); C-NMR (DMSO-d
44.3, 165.3, 169.80, 172.5. Anal. Calcd for C16
H, 7.44; N, 14.78.
6
) δ (ppm): 14.5, 20.6, 35.1, 39.8,111.2, 115.9, 122.2, 126.6, 133.1,
1
H
21
N
3
O
2
: C, 66.88; H, 7.37; N, 14.62; found: C, 66.70;
(
Z) N′-(5-Bromo-2-oxoindolin-3-ylidene)-2-propylpentane Hydrazide 4b
The product was obtained as yellow solid, in 86% yield; mp 172–173 °C; IR (KBr): 3222 (NH), 1729
−1 1
(
CO), 1663 (C=N), 1609 (CO) cm ; H-NMR (DMSO-d
6
): δ (ppm): 0.85–0.89 (m, 6H, 2 CH
3
), 1.27–1.29
(
m, 4H, 2 CH ), 1.40–1.43 (m, 2H, CH ), 1.57–1.59 (m, 2H, CH
2
2
2
), 2.50 (brs, 1H, CH), 6.90 (brs, 1H,
1
3
Ar-H), 7.54 (brs, 1H, ArH), 8.02 (brs, 1H, ArH), 10.91 (s, 1H, NH), 11.31 (s, 1H, NH); C-NMR

Molecules 2015, 20
14648
(
DMSO-d
6
) δ (ppm): 14.6, 20.6, 35.1, 39.8, 112.9, 117.5, 122.2, 128.7, 135.1, 144.3, 166.3, 170.80,
: C, 52.47; H, 5.50; N, 11.47; found: C, 52.66; H, 5.61; N, 11.23.
1
72.5. Anal. Calcd for C16
H20BrN
3
O
2
(
Z) N′-(5-Chloro-2-oxoindolin-3-ylidene)-2-propylpentane Hydrazide 4c
The product was obtained as yellow solid, in 88% yield; mp 154–156 °C; IR (KBr): 3233 (NH), 1720
−1 1
(
CO), 1689 (C=N), 1620 (CO) cm ; H-NMR (DMSO-d
m, 4H, 2 CH ), 1.40–1.43 (m, 2H, CH ), 1.57–1.59 (m, 2H, CH
.42 (m, 1H, ArH), 8.2 (brs, 1H, ArH), 10.91 (s, 1H, NH), 11.35 (brs, 1H, NH); C-NMR (DMSO-d
δ (ppm): 14.6, 20.6, 35.3, 39.7, 112.8, 118.5, 122.0, 126.2, 134.1, 145.3, 165.3, 170.80, 172.5. Anal.
Calcd for C16 : C, 59.72; H, 6.26; N, 13.06; found: C, 59.55; H, 6.18; N, 13.33.
6
) δ (ppm): 0.87–0.93 (m, 6H, 2 CH
3
), 1.26–1.29
(
2
2
2
), 2.51 (brs, 1H, CH), 6.91 (m, 1H, Ar-H),
1
3
7
6
)
H
20ClN
3
O
2
(
Z) N′-(5-Fluoro-2-oxoindolin-3-ylidene)-2-propylpentane Hydrazide 4d
The product was obtained as yellow solid in 89% yield; mp 180–182 °C; IR (KBr): 3181 (NH), 1721
−1 1
(
CO), 1686 (C=N), 1630 (CO) cm ; H-NMR (DMSO-d
m, 4H, 2 CH ), 1.40–1.43 (m, 2H, CH ), 1.57–1.59 (m, 2H, CH
.24 (m, 1H, ArH), 8.2 (brd, 1H, ArH), 10.81 (s, 1H, NH), 11.23 (brs, 1H, NH); C-NMR (DMSO-d
δ (ppm): 14.6, 20.6, 35.3, 39.7, 112.8, 118.5, 119.8, 124.2, 134.1, 140.3, 162.3, 169.80, 172.5. Anal.
Calcd for C16 : C, 62.94; H, 6.60; N, 13.76; found: C, 63.13; H, 6.77; N, 13.52.
6
) δ (ppm): 0.86–0.89 (m, 6H, 2 CH
3
), 1.26–1.27
(
2
2
2
), 2.50 (brs, 1H, CH), 6.88 (m, 1H, Ar-H),
1
3
7
6
)
H
20FN
3
O
2
(
Z) N′-(1-Methyl-2-oxoindolin-3-ylidene)-2-propylpentane Hydrazide 4e
The product was obtained as yellow solid, in 89% yield; mp 154–156 °C; IR (KBr): 1720 (CO), 1689
−1 1
(
C=N), 1620 (CO) cm ; H-NMR (DMSO-d
CH ), 1.40–1.43 (m, 2H, CH ), 1.56–1.59 (m, 2H, CH
m, 2H, Ar-H), 7.46 (m, 1H, ArH), 8.20 (brs, 1H, ArH), 11.23 (brs, 1H, NH); C-NMR (DMSO-d )
6
) δ (ppm): 0.85–0.87 (m, 6H, 2 CH
3
), 1.24–1.29 (m, 4H,
2
2
2
2
), 2.50 (brs, 1H, CH), 3.18 (s, 3H, CH
3
), 7.08–7.14
1
3
(
6

Molecules 2015, 20
14649
δ (ppm): 14.5, 20.6, 27.6, 35.3, 39.7, 115.3, 119.6, 122.7, 129.2, 131.1, 134.8, 164.0, 169.80, 171.5.
Anal. Calcd for C17 : C, 67.75; H, 7.69; N, 13.94; found: C, 67.63; H, 7.78; N, 14.21.
H
23
N
3
O
2
(
Z) N′-(1-Benzyl-2-oxoindolin-3-ylidene)-2-propylpentane Hydrazide 4f
The product was obtained as yellow solid, in 83% yield; mp 170–172 °C; IR (KBr): 1718 (CO), 1686
−
1 1
(
CH
m, 2H, Ar-H), 7.33–7.39 (m, 6H, ArH), 8.20 (brs, 1H, ArH), 11.28 (brs, 1H, NH); C-NMR (DMSO-d
δ (ppm): 14.6, 20.6, 35.3, 39.7, 43.2, 115.4, 122.8, 122.9, 126.8, 127.8, 128.1, 129.2, 129.3, 136.8, 144.2,
64.2, 170.5. Anal. Calcd for C23 : C, 73.18; H, 7.21; N, 11.13; found: C, 73.41; H, 7.40; N, 10.94.
C=N), 1620 (CO) cm ; H-NMR (DMSO-d
6
) δ (ppm): 0.86-0.89 (m, 6H, 2 CH
3
), 1.27–1.29 (m, 4H, 2
2
), 1.40–1.44 (m, 2H, CH ), 1.56–1.60 (m, 2H, CH
2
2
), 2.51 (brs, 1H, CH), 4.96 (s, 2H, CH
2
), 7.00–7.20
13
(
6
)
1
H N O
27 3 2
(
Z)-N′-(1-(2-Bromoethyl)-2-oxoindolin-3-ylidene)-2-propylpentane Hydrazide 4g
The product was obtained as yellow crystals, in 83% yield; mp 90–91 °C; IR (KBr): 1700 (CO), 1686
−
1 1
(
C=N), 1608 (CO) cm ; H-NMR (DMSO-d
CH ), 1.40–1.44 (m, 2H, CH ), 1.56–1.60 (m, 2H, CH
CH -CH Br), 4.20 (t, J = 6.4 Hz, 2H, CH -CH Br), 7.18 (t, J = 8.4 Hz, 1H, Ar-H), 7.32 (d, J = 8.0 Hz,
H, Ar-H), 7.53 (t, J = 8.0 Hz, 1H, Ar-H), 7.88 (brs, 1H, ArH), 12.28 (brs, 1H, NH); C-NMR (DMSO-d
δ (ppm): 14.5, 20.6, 29.9, 35.3, 39.7, 42.2, 46.5, 110.9, 119.7, 122.7, 123.2, 134.1, 142.9, 164.0, 169.80,
76.0. Anal. Calcd for C18 : C, 54.83; H, 6.13; N, 10.66; found: C, 55.12; H, 6.23; N, 10.90.
6
) δ (ppm): 0.86–0.89 (m, 6H, 2 CH
3
), 1.27–1.29 (m, 4H,
2
2
2
2
), 2.51 (brs, 1H, CH), 3.77 (t, J = 6.8 Hz, 2H,
2
2
2
2
1
3
1
6
)
1
H
24BrN
3
O
2
N-(2-(2-Oxo-2-(2-(2-propylpentanoyl)hydrazinyl)acetyl)phenyl) Acetamide 4h

Molecules 2015, 20
14650
The product was obtained as yellowish white solid, in 83% yield; mp 180–181 °C; IR (KBr): 3310
−
1 1
(
NH), 3223(NH), 1720 (CO), 1709 (CO), 1686 (C=N), 1608 (CO) cm ; H-NMR (DMSO-d
6
) δ (ppm):
), 1.56–1.60 (m, 2H, CH ),
), 2.21 (brs, 1H, CH), 7.28 (t, J = 8.0 Hz, 1H, Ar-H), 7.68 (t, J = 8.0 Hz, 1H, Ar-H),
.99 (d, J = 7.3 Hz, 1H, Ar-H), 8.17(d, J = 8.1 Hz, 1H, ArH), 10.03 (s, 1H, NH), 10.64 (s, 1H, NH),
0
2
7
1
1
.86–0.89 (m, 6H, 2 CH
3
), 1.27–1.29 (m, 4H, 2 CH
2
), 1.40–1.44 (m, 2H, CH
2
2
.16 (s, 3H, CH
3
1
3
0.72 (s, 1H, NH); C-NMR (DMSO-d
6
) δ (ppm): 14.6, 20.6, 25.0, 29.9, 35.3, 43.8, 46.5, 121.5, 121.8,
: C, 62.23; H, 7.25;
23.8, 133.6, 135.9, 140.3, 164.1, 169.6, 174.7, 193.0. Anal. Calcd for C18
H
25
N
3
O
4
N, 12.10; found: C, 62.55; H, 7.42; N, 12.38.
.2. X-ray Crystallography
Single crystals were obtained by slow evaporation from ethanol. A good crystal with a suitable size
3
was selected for X-ray diffraction analysis. Data were collected on a D8 Venture area diffractometer
equipped with graphite monochromatic MoK/α radiation (λ = 0.71073 Å) at 100 K. Cell refinement and
data reduction were done by Bruker SAINT (Bruker, Madison, WI USA); the program used to solve
structure and refine structure is SHELXS-97 [55]. The final refinement was performed by full-matrix
2
least-squares techniques with anisotropic thermal data for non-hydrogen atoms on F . All the hydrogen
atoms were placed in calculated positions and constrained to ride on their parent atoms. Multi-scan
absorption correction was applied by use of SADABS software [56].
3
.3. Biology
3
.3.1. Cell Culture and Cell Viability Assay
The stock concentration of the entire compound in DMSO was 10 mM and this concentration was
used to prepare the working dilution. The final DMSO concentration used in the experiments was ≤0.5%
as the working concentration. Human liver cancer cell lines (HepG2), human leukemia cell line (Jurkat),
and human embryonic kidney cell line (HEK293) cells were cultured in high glucose Dulbecco’s Modified
Eagle Medium (DMEM: Life technologies cat #11995073) supplemented with 10% Fetal bovine serum
(
FBS: Life technologies cat #16000044) in a humidified incubator with 5% CO
cells were seeded in each well of a 24-well cell culture plate and were allowed to adhere and grow for
4 h. The Jurkat cells grow in suspension so they were cultured and allowed to grow overnight before
2
at 37 °C. Around 2 ×103
2
the addition of compounds. The compounds in serial dilutions (1, 3, 15, 45, 100, and 300 μM) were
added after 24 h of culture and the cells were cultured for another 24 h at 37 °C. The cell viability was
determined in each experiment using MTT 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan colorimetric
assay. Briefly, the treated or untreated cells were trypsenized and centrifuged, and the resulting pellet was
re-suspended in 100 μL of DMEM serum-free medium and incubated at 37 °C for 2 h. After incubation,
20 μL of MTT solution (5 mg/mL in PBS: Sigma Aldrich cat #M2003) was added to each well and further
incubated for 2 h. The plate was centrifuged at 40,000 rpm for 10 min then the medium was removed
from each well and 2-propanol containing 0.04 M HCl was added to dissolve the formazan produced in
the cells. The optical density of the formazan product in solution was measured with a microplate reader
at 540 nm. The experiment was conducted in triplicate. Data were calculated as percent of cell viability
by the following formula:

Molecules 2015, 20
cell viability = (Mean absorbance in test wells/Mean absorbance in control wells) × 100
14651
%
(1)
The IC50 values were calculated from the means of six different concentrations by Bio Data Fit 1.02
using the software Bio Tool Kit (Version 300; Chang Bioscience Inc., Castro Valley, CA, USA) and
online program http://ic50.tk/index.html.
3.3.2. Statistics
All data were analyzed using Origin (Version 6.1052; Origin Lab Corp Northampton, MA, USA).
One-way ANOVA analysis of variance and student T TEST was used to compare different experimental
groups, and data were considered statistically significant for p values less than 0.05.
4
. Conclusions
In conclusion, the screening results from human cancer and normal cell lines suggested that isatin
derivatives were remarkably influenced by various substituents on the isatin ring and at the N-terminal
of the hydrazide-hydrazone moiety. The data revealed that replacement of N-hydrogen at position 1 of
the isatin moiety 3a by methyl and benzyl of the targeted compound (3e and 3f) noticeably enhanced the
activity against the selected two cell lines, HepG2 and Jurkat, for compound 3f. The presence of the
valproic acid moiety as hydrazide-hydrazone derivatives 4a make the compound more targeted towards
Jurakt cells in vitro. Introducing the chlorine atom to the molecule 4c, meanwhile, increased the reactivity
more significantly than with 4a and 3a towards the three cell lines HepG2, Jurkat, and HEK293.
Furthermore, the methyl group in the same analogous compound 4e increased the reactivity towards the
two cell lines HepG2 and Jurkat more than the benzyl group 4f. The rest of the compounds had no effect
on the three cell lines. Further studies to assess the effect of more derivatives on various cancer cell
biomarkers are currently underway in our lab.
Supplementary Materials
Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/20/08/14638/s1.
Acknowledgments
The authors extend their sincere appreciation to the Deanship of Scientific Research at King Saud
University for funding this work through research group project no. RGP-234 (Saudi Arabia).
Author Contributions
Ayman El-Faham and Sherine Khattab carried out the synthesis and designed the proposed methods
and analyzed the data statistically together. Hazem Ghabbour and Hoong-Kun Fun carried out X-ray
method and the characterization; Muhammad Farooq, Nael Abutaha, and Mohammad Wadaan carried
out all the biological activities studies. All authors read and approved the final manuscript.
Conflicts of Interest
The authors declare no conflict of interest.

Molecules 2015, 20
14652
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