Exploiting the 4-hydrazinobenzoic acid moiety for the development of anticancer agents: Synthesis and biological profile

Article abstract of DOI:10.1016/j.bioorg.2020.104098

Thirteen 4-hydrazinobenzoic acid derivatives were elaborated and characterized by spectral analyses (NMR and MS). Evaluation of their in vitro cytotoxic activity showed that some of the targets demonstrated potent inhibitory effects against HCT-116 and MC

Full text of DOI:10.1016/j.bioorg.2020.104098

Journal Pre-proofs
Exploiting the 4-hydrazinobenzoic acid moiety for the development of anti-
cancer agents: synthesis and biological profile
Hatem A. Abuelizz, Hanem M. Awad, Mohamed Marzouk, Fahd A. Nasr,
Ahmed H. Bakheit, Ahmed M. Naglah, Nasser S. Al-shakliah, Rashad Al-
Salahi
PII:
S0045-2068(20)31395-X
DOI:
https://doi.org/10.1016/j.bioorg.2020.104098
YBIOO 104098
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
25 March 2020
26 May 2020
10 July 2020
Please cite this article as: H.A. Abuelizz, H.M. Awad, M. Marzouk, F.A. Nasr, A.H. Bakheit, A.M. Naglah, N.S.
Al-shakliah, R. Al-Salahi, Exploiting the 4-hydrazinobenzoic acid moiety for the development of anticancer
agents: synthesis and biological profile, Bioorganic Chemistry (2020), doi: https://doi.org/10.1016/j.bioorg.
2
020.104098
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Exploiting the 4-hydrazinobenzoic acid moiety for the development of
anticancer agents: synthesis and biological profile
a
b
b
c
Hatem A. Abuelizz , Hanem M. Awad , Mohamed Marzouk , Fahd A. Nasr , Ahmed H.
a,d
e,f
a
*a
Bakheit , Ahmed M. Naglah , Nasser S. Al-shakliah , Rashad Al-Salahi
a
b
c
d
e
f
Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, PO
Box 2457, Riyadh 11451, Saudi Arabia.
Department of Tanning Materials and Leather Technology, National Research Centre, 33 El-
Bohouth St. (Former El-Tahrir St.), Dokki, Cairo 12622, Egypt.
Medicinal Aromatic, and Poisonous Plants Research Center, College of Pharmacy, King Saud
University, PO Box 2457, Riyadh, 11451, Saudi Arabia.
Department of Chemistry, Faculty of Science and Technology, El-Neelain University, P.O.
Box 12702, Khartoum 11121, Sudan.
Department of Pharmaceutical Chemistry, Drug Exploration and Development Chair
(DEDC), College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia.
Peptide Chemistry Department, Chemical Industries Research Division, National Research
Centre, 33 El-Bohouth St. (Former El-Tahrir St.), Dokki, Cairo 12622, Egypt.
*
Corresponding author. E-mail: ralsalahi@ksu.edu.sa (R.Al-Salahi).
ABSTRACT
Thirteen 4-hydrazinobenzoic acid derivatives were elaborated and characterized by
spectral analyses (NMR and MS). Evaluation of their in vitro cytotoxic activity showed
that some of the targets demonstrated potent inhibitory effects against HCT-116 and MCF-
7
cancer cells. The IC values ranged between 21.3 ± 4.1 and 28.3 ± 5.1 µM, respectively,
50
whereas those of doxorubicin (reference drug) ranged between 22.6 ± 3.9 and 19.7 ± 3.1
µM, respectively. The active targets 6, 7 and 9 exhibited very weak cytotoxicity on normal
cells (RPE-1) and showed higher IC50 values against HCT-116 and MCF-7 cells in
comparison to doxorubicin. Furthermore, compounds 7, 9 and 10 inhibited the proliferation
of MCF-7 by the induction of apoptosis. The bioassay results in the regression plots
generated in 3D QSAR models were in agreement and correlated with the anticancer results

of the target molecules. The 4-hydazinobenzoic acid derivatives can be used as
cornerstones for further structural modifications as future anticancer agents.
Keywords: 4-Hydrazinobenzoic acid, MTT assay, Anticancer, Apoptosis, 3D QSAR
1
. Introduction
The development of chemotherapeutic agents is a fascinating challenge in the field of
pharmaceutical chemistry because cancer remains the most frequent cause of mortality,
particularly in the developed world. [1-5]. Currently, several anticancer agents (either alone
or in combination) have limited activity and their response rates still largely unimproved
in clinical trials. Although there have been major advances in chemotherapeutic therapy
and much has been done to study the biology of cancer, the goal of overcoming this
malignancy is a distant reality. Nowadays, most available chemotherapeutic agents are
non-selective; thus, exert their activities against both cancer and normal cells, specifically
those that rapidly proliferate. In addition, they have undesired toxic side effects. Thus, the
similarity between normal and cancer cells remains the main hurdle to the development of
an ultimate anticancer remedy [6-8].
Hydrazinobenzoic acid is a building block for the construction of many heterocyclic
compounds with a variety of pharmacological properties. For instance, 2-hydrazinobenzoic
acid reacted with N-cyanoimido(dithio)carbonates to afford 2-alkyloxy(thioalky)-
[1,2,4]triazolo[1,5-a]quinazolines. Some of the 1,2,4-triazoloquinazolines were found as
potent adenosine antagonist, antidiabetic, antioxidant, antihypertensive, antimicrobial,
antihistamine, and anti-inflammatory agents [9-16]. The 5-choro-[1,2,4]triazolo
quinazolines showed promising cytotoxicity against HepG2, melanoma (SK-MEL28), and
medulloblastoma (Daoy) cell lines [1]. A new series of 5-hydrazono-[1,2,4]triazolo[1,5-
a]quinazolines demonstrated potent anticancer activity against HeLa L929, MCF-7, and
Hep-G2 cells [7]. The novel bis-triazolo[1,5-a:4,3-c]quinazoline and 5-ethoxy-
[1,2,4]triazoloquinazoline were found to possess the highest cytotoxic effects against HCT-
1
16 and HepG2 carcinoma cells [17]. In our previous work, we incorporated a 4-
hydrazinobenzoic acid moiety with the triazole pharmacophore to afford the novel 1,2,4-
triazolebenzoic acid hybrid scaffolds with potent antiproliferative activity against HCT-
1
16 and MCF-7 human cancer cells [4].

Considering the above facts and continuing our ongoing research dealing with the
chemistry of hydrazinobenzoic acid to elaborate new anticancer agents. The incorporation
of pharmacophoric templates into one molecule can have positively enhanced
chemotherapeutic potential [2, 4]. Herein, we incorporated 4-hydrazinobenzoic acid (1) as
free base with several isothiocyanates, aldehydes, and acid anhydrides to generate the
target compounds (2–13). Thereafter, their in vitro cytotoxicity was evaluated against
MCF-7 and HCT-116 cancer cell lines and normal human RPE-1 cells as well.
2
. Results and discussion
.1. Chemistry
-Hydrazinobenzoic acid hydrochloride was added in portions to a stirred solution of
2
4
dimethyl-N-cyanoimidodithiocarbonate to derive 4-hydrazinobenzoic acid as a free base in
yield of 45% and 4-(5-amino-3-(methylthio)-1,2,4-triazol-1-yl) benzoic acid. The later
structure was confirmed, characterized, and evaluated in our previous paper [4]. The free
base of 4-hydrazinobenzoic acid (1) was used as the starting material to produce the targets
2
2
–11 and 13 (Scheme 1). The reaction of 1 with several aldehydes afforded the hydrazones
–4 in a good yield. Whereas the hydrazinecarbothioamide derivatives 5–10 were obtained
from the reaction of 1 with different isothiocyanates, and the corresponding compounds 11
and 13 were resulted upon the treatment of 1 with appropriate acid anhydrides (Scheme 1).
Compound 12 was previously reported and characterized [18]. The chemical structures of
the compounds 1–11 and 13 were established by NMR and HREI-MS analyses (see
experimental data) and the aid of comparison with structural related compounds in
literature [4].

COOH
COOH
COOH
R2
Aldehydes
EtOH
RNCS
EtOH
H
H
N
N
(
1)
N
(
5-10)
NH
R
NH
NH
(
2-4)
H2N
R3
S
Acid anhydride
O
R1
CH COOH
3
R4
CH COOH
3
H
O
Acid
anhydrides
N
N
H
O
N
N
HOOC
O
COOH
N
(
11)
O
O
HN
COOH
(
12,13)
R : OH, R : methyl (2); R OH, R : methoxy (3); R : OH, R : nitro (4); R: methyl (5); R: ethyl (6); R: allyl (7); R:
1
2
1:
2
1
2
butyl (8); R: benzyl (9); R: phenethyl (10); R &R : H (12); R &R : Cl (13).
3
4
3
4
Scheme 1. Main synthetic routes for compounds 2-13
2
.2.
Biology
The targets 1–13 were evaluated for their cytotoxicity using the MTT assay. In the form of
cell viability, the in vitro cytotoxic activity was determined against two cancer cell lines
HCT-116 and MCF-7) and one normal cell line (RPE-1) in relative to doxorubicin as the
%
(
reference drug. As shown in Figures 1–3, all targets suppressed the cell lines in a dose-
dependent manner. A comparison study between the cytotoxic effect of each compound
and that of doxorubicin was performed to determine the efficacy of synthesized products
1
–13. Table 1 shows that compounds 7 and 10 (IC = 21.3 ± 4.1 and 21.8 ± 4.1 µM) had
5
0
significantly more potent cytotoxicity than doxorubicin (IC = 22.6 ± 3.9 µM) against HCT-
5
0
1
16 cells. However, compounds 6, 8 and 9 (IC = 25.6 ± 4.4, 24.1 ± 4.2 and 24.5 ± 4.9 µM)
5
0
exhibited good activity. In case of human MCF-7 cells, compounds 2, 4, 7, 9 and 10 (IC50
24.4 ± 2.9, 24.5 ± 4.6, 25.0 ± 4.6, 26.0 ± 4.3, and 23.5 ± 4.9 µM, respectively) showed high
cytotoxicity compared to that of doxorubicin (IC = 19.7 ± 3.1 µM). However, compounds
=
5
0
1
, 3 and 13 (IC = 34.6 ± 6.3, 30.9 ± 5.3 and 30.9 ± 5.6 µM) had slightly less cytotoxic effects.
5
0

In case of RPE-1 cells, compounds 3, 4, 8, 10 and 12 (IC = 60.8 ± 11.3, 56.2 ± 9.5, 62.3 ±
5
0
1
1
4.5, 62.5 ± 14.1 and 58.0 ± 11.9 µM) were more cytotoxic than doxorubicin (IC = 64.0 ±
5
0
3.2 µM), whereas 13 (IC = 64.2 ± 13.3 µM) was as cytotoxic as doxorubicin. Additionally,
5
0
the targets 6, 7 and 9 (IC = 85.7 ± 15.8, 75.2 ± 15.3 and 96.0 ± 17.5 µM) demonstrated
5
0
significantly less cytotoxic effects against RPE-1 cells and the remaining compounds had
slightly less cytotoxicity
Table 1. IC values of the target compounds according to the MTT assay
5
0
Cps
IC (µM) ± SD
Therapeutic Index (TI)
5
0
HCT-116
MCF-7
RPE-1
HCT-116
1.9
MCF-7
1.9
2.7
2.0
2.3
2.3
3.0
3.0
2.4
3.7
2.7
2.5
2.1
2.1
3.3
1
2
3
4
5
6
7
8
9
34.6 ± 5.9
31.2 ± 4.1
38.9 ± 5.9
29.3 ± 5.1
29.9 ± 4.6
25.6 ± 4.4
21.3 ± 4.1
24.1 ± 4.2
24.5 ± 4.9
21.8 ± 4.1
28.0 ± 4.1
33.3 ± 4.5
32.5 ± 5.1
22.6 ± 3.9
34.6 ± 6.3
24.4 ± 2.9
30.9 ± 5.3
24.5 ± 4.6
29.7± 5.5
28.3 ± 5.1
25.0 ± 4.6
26.2 ± 4.3
26.0 ± 4.3
23.5 ± 4.9
26.3 ± 4.9
28.2 ± 5.3
30.9 ± 5.6
19.7 ± 3.1
67.2 ± 12.5
66.8 ± 14.1
60.8 ± 11.3
56.2 ± 9.5
2.1
1.6
1.9
67.6 ± 14.7
85.7 ± 15.8
75.2 ± 15.3
62.3 ± 14.5
96.0 ± 17.5
62.5 ± 14.1
66.3 ± 13.1
58.0 ± 11.9
64.2 ± 13.3
64.0 ± 13.2
2.3
3.3
3.5
2.6
3.9
1
1
1
0
1
2
2.9
2.4
1.7
1
3
2.0
Doxorubicin
2.8
The therapeutic index (TI) was calculated to determine the safety of each compound
according to the following equation: (TI = IC in the normal cells / IC in the cancer
5
0
50
cells). As illustrated in Table 1, for MCF-7 cells, compound 9 had a higher TI (3.7) than
doxorubicin (3.3); however, compounds 2, 6, 7 and 10 had a slightly lower TIs than
doxorubicin, whereas 1, 3–5, 8 and 11- 13 had lower TIs than doxorubicin.

In case of HCT-116 cells, compounds 6, 7, 9, and 10 had higher TIs (3.3, 3.5, 3.9 and 2.9,
respectively) than doxorubicin (2.8); 8 had slightly lower TI (2.6) than doxorubicin; 1–5
and 11-13 had significantly less TIs than the reference drug. This suggests that 6, 7, 9 and
1
0 are more active anticancer candidate drugs against two human cancer cells in regard to
their safety and efficacy. Moreover, the results indicated that the targets 1–13 demonstrated
weak to high cytotoxicity against MCF-7 and HCT-116 cell lines with IC values ranging
5
0
in 23.5 ± 4.9 to 34.6 ± 6.3 and 21.3 ± 4.1 to 38.9 ± 5.9 µM, respectively (Table 1). The
insertion of isothiocyanate group significantly increased cytotoxicity as revealed by
compounds 6, 7, 9 and 10, which demonstrated significant TI values that ranged between
2
.7 and 3.9 against both cancer cell lines.
The condensation products with aldehydes (2–4) increased cytotoxicity in MCF-7 cells,
but did not increase cytotoxicity in HCT-116 cells as revealed by the structure-activity
relationship study The parent compound 1 showed the same activity against both cancer
cells; however, its transformed products 2, 5–11, 13 displayed higher activity against MCF-
7
and HCT-116 cells, respectively. In addition, compounds 3 and 12 exhibited a lower
activity against HCT-116 cells than 1. We noticed that the type of isothiocyanate in
compounds 5–10 and acid anhydride in 11–13 displayed varying cytotoxicity profiles
(Table 1).
Fig. 1. Dose dependent cytotoxic activities of compounds 1-13 against HCT-116 cancer
cells

Fig. 2. Dose dependent cytotoxic activities of compounds 1-13 against MCF-7 cancer cells
Fig. 3. Dose dependent cytotoxic activities of compounds 1-13 against RPE-1 normal cells
2
.3.
To provide more evidence for the anticancer effect of promising compounds, Hoechst
3258 staining was carried out to investigate the nuclear morphological changes following
Apoptosis detection
3
compound treatment on MCF-7 cells. Control cells without treatment exhibited uniformly
light blue staining and intact cell membranes (Fig. 4). In contrast, cells treated with the IC50
of each compound for 24 h were low in numbers and stained bright blue because of
chromatin condensation. Moreover, quantitative analysis of the apoptotic effects of the

compounds on MCF-7 cells was conducted by flow cytometry using Annexin V-FITC and
PI double staining, which detect the phosphatidylserine externalization, as a consequence
of the initiation of apoptosis. After treatment with compounds 7, 9 and 10 (IC50 = 25, 26,
and 23.5 μM, respectively) for 24 h, the percentage of cells undergoing early apoptotic
events was significantly increased from 1.2 ± 0.5 % of the control to 17.3 ± 1.6, 10.1 ± 1.2
%
, and 6.3 ± 0.9, for compounds 7, 9, and 10, respectively. In the same manner, the
percentage of late apoptotic cells were increased to 3.6 ± 0.3 %, 3.1 ± 0.1 % and 5.9 ± 0.4
for compounds 7, 9, and 10, respectively in compare to control. However, no significant
%
differences were reported in the percent of necrotic deaths for all tested compounds which
indicated that induction of cell apoptosis reflected the antitumor effect of the compounds
(Fig. 5).

Fig. 4. Fluorescent images of Hoechst 33258 staining showing induced cell death. (a)
MCF-7 cells without treatment served as a control, (b-d) cells were treated for 24 h prior
to Hoechst staining compounds 7, 9 and 10 (25, 26 and 23.5, μM, respectively).
Cp.10
Control
Cp.7
Cp.9
Fig. 5. Compounds treatment induces apoptosis in MCF-7 cells. MCF-7 cells were treated with
compounds 7, 9 and 10 (25, 26 and 23.5 μM, respectively) for 24 h, then stained with Annexin

V-FITC/PI, and apoptosis was quantified using FACS analysis. Each quadrant represents cells
in different stages; A3 viable, A4 early apoptosis, A2 late apoptosis, and A1 necrosis.
2
.4.
3D QSAR analysis
Employing discovery Studio 4.5 to build 3D QSAR models of 4-hydrazinobenzoic acid
derivatives (1–13) based on their preliminary cytotoxicity results. The calculated pTI
values of the target compounds ranged between 5.45080 and 5.58846 (Table S1). The
model dataset was built with thirteen compounds with TIs that ranged from 1.6 to 3.9
(Table 1). The pTI values were determined from corresponding TI values using the formula
in the following equation: pTI = -log TI.
Discovery Studio 4.5 was divided the compounds in the preliminary set into a training set
and test set. For a perfect model of QSAR, the value of training set regression coefficient
2
2
(
R ) and test set regression coefficient (Q ) should be higher than 0.6 and 0.5, respectively.
2
In our study, the observed R comparable to the predicted training set response was 0.9352
2
and 0.9122, whereas the Q comparison of the test set was 0.6759 and 0.6696 for HCT-116
and MCF-7, respectively (Table 2S), which illustrated the acceptability of the QSAR
model. Figure 6 displays the plot of the pTI observed results compared to the predicted
results. The predicted pTI value and residual error of the thirteen compounds for that
QSAR model are summarized in Table S1. In addition, the iso-surfaces aligned compounds
of the 3D-QSAR model coefficients on van der Waals grids (Fig. 7b &d) and electrostatic
potential grids (Fig. 7a &c) are listed for HCT-116 and MCF-7 respectively. In the
electrostatic map, negative charge (high electron density) is sited in red contour regions
expecting high activity and partial positive charge (low electron density) is sited in blue
contour expecting high activity. In addition, in the steric map, the steric bulk areas (activity)
increase when the green contour is increased or yellow contour is decreased. In the maps,
the activity of the compounds increases with the increase in the positive charge and
decrease in the volume of compound as shown in Figure 7a–d. The summarized data
demonstrate that compound 9, the most potent antiproliferative agent (TI against HCT-116
=
3.9 and against MCF-7 = 3.7), has a benzylisothiocyanate substituent that could be

displayed a pivotal role in cytotoxicity profile. The 3D QSAR models fit the cytotoxic
activity well, which provides knowledge for further modification.
Fig. 6. Plot of experimental versus predicted (a) a human colon cancer and (b) a
breast cancer therapeutic index activities of training set and test set.

electrostatic potential
van der Waals grids
HCT-
16
1
MCF-
7
Fig. 7. 3D-QSAR model coefficients on van der Waals grids, Green represents positive
coefficients, yellow represents negative coefficients, (b) and (d). 3D-QSAR model
coefficients on electrostatic potential grids, Blue represents positive coefficients, red
represents negative coefficients, (a) & (c). For HCT-116, (a) & (b) and for MCF-7, (c) &
(d).

3
. Conclusions
A series of 4-hydrazinobenzoic acid compounds were evaluated for their in vitro
cytotoxicity against MCF-7 and HCT-116 human cancer cell lines. The study revealed that
targets 6, 7, 9 and 10 demonstrated potent antiproliferative effects. The incorporation of an
isothiocyanate moiety into the 4-hydrazinobenzoic acid platform enhanced cytotoxicity.
The targets 6, 7 and 9 not only exhibited promising cytotoxicity against the tested cancer
cells, but showed very weak cytotoxic effects in normal cells, which implies that these can
be used as candidates for anticancer therapy. Moreover, compounds 7, 9 and 10 showed
antiproliferative activity by inducing apoptosis in MCF-7 cancer cells; thus, they are
worthy of further research for the development of novel anticancer drugs. In 3D QSAR
analysis, the predicted pTI values of the active compounds showed a significant correlation
with the experimental values from the generated regression and principal component
analysis plots. These discoveries strongly support the cytotoxicity results of compounds 6,
7
, 9 and 10.
4
. Experimental
4
.1. Chemistry
It is worth mentioning that the registration CAS numbers for the products 1–10 had already
been assigned. However, their chemical properties, and structural characterization are not
available in literature. In a basic medium, the free base 1 was resulted from addition of 4-
hydazinobenzoic acid HCl (1.1 mmol) in a portion-wise to stirred solution of dimethyl-N-
cyanoimidodithiocarbonate (1 mmol) in boiling ethanol (15 mL). A mixture of 1 (1.4
mmol) and aldehyde (1.5 mmol) or an appropriate isothiocyanate (1.5 mmol) was heated
in ethanol (15 mL) for 5-8 h. After cooling, the solid was filtered off, washed with water
and dried to afford the final products 2-10. When the parent 1 (2 or 1 mmol) was allowed
to react under reflux condition with pyromellitic dianhydride (1.1 mmol) or 4,5-
dichlorophthalic anhydride (1.1 mmol) in glacial acetic acid (10 mL) for 15 h, the
corresponding 11 and 13 were obtained after treatment the boiling solution with ice water.
4
.1.1. 4-Hydrazinylbenzoic acid (1)

1
CAS number: 619-67-0; pale yellow amorphous powder (45%); mp 222–224 °C; H
NMR (700 MHz, DMSO-d ): δ 12.38 (br s, 1H, -COOH), 9.46, 8.45 (each br s, 2H, -NH-
6
CS-NH-), 8.24 (m, 1H, Ar-NH), 7.68 (d, J = 7.8 Hz, 2H, H-2/6), 7.51 (s, 1H, -NH), 6.75
1
3
(
d, J = 7.8 Hz, 2H, H-3/5); C NMR (175 MHz, DMSO-d ): δ 168.1 (C-7), 156.3 (C-4),
6
1
1
31.3 (C-2/6), 117.8 (C-1), 110.3 (C-3/5); HRMS (EI), m/z Calcd for C7H8N2O2 (M)•+
52.0586, found 152.0613.
4
.1.2. (E)-4-(2-(2-Hydroxy-5-methylbenzylidene)hydrazinyl)benzoic acid (2)
1
CAS number: 1608619-21-1; white amorphous powder (70%); mp 271–273 °C; H
NMR (700 MHz, DMSO-d ): δ 12.33 (br s, 1H, -COOH), 10.83 (br s, 1H, -OH), 10.05 (br
6
s, 1H, -NH), 8.22 (s, 1H, -N=CH-), 7.83 (d, J = 8.2 Hz, 2H, H-2/6), 7.57 (br s, 1H, H-6'),
7
.02 (d, J = 8.2 Hz, 2H, H-3/5), 7.00 (br d, J = 8.3 Hz, 1H, H-4'), 6.79 (d, J = 8.2 Hz, 1H,
1
3
H-3'), 2.25 (s, 3H, Ar-CH ); C NMR (175 MHz, DMSO-d ): δ 167.6 (C-7), 154.1 (C-2'),
3
6
1
1
48.9 (C-4), 138.8 (C-7'), 131.8 (C-2/6), 130.9 (C-4'), 128.4 (C-5'), 127.2 (C-6'), 120.7 (C-
), 120.6 (C-1'), 116.3 (C-3'), 111.4 (C-3/5), 20.6 (Ar-CH ); HRMS (EI), m/z Calcd for
3
•+
C H N O (M) 270.1004, found 270.1053.
1
5
14
2
3
4
.1.3. (E)-4-(2-(2-Hydroxy-5-methoxybenzylidene)hydrazinyl)benzoic acid (3)
CAS number: 385406-90-6; white amorphous powder (72%); mp 265–267 °C; H
1
NMR (700 MHz, DMSO-d ): δ 12.33 (br s, 1H, -COOH), 10.86 (br s, 1H, -OH), 9.78 (br
6
s, 1H, -NH), 8.23 (s, 1H, -N=CH-), 7.83 (d, J = 8.8 Hz, 2H, H-2/6), 7.22 (d, J = 2.7 Hz,
1
3
1
H, H-6'), 7.04 (d, J = 8.8 Hz, 2H, H-3/5), 6.81 (m, 2H, H-3'/4'), 3.74 (s, 3H, OCH ); C
3
NMR (175 MHz, DMSO-d ): δ 167.7 (C-7), 152.8 (C-2'), 150.3 (C-5'), 148.9 (C-4), 138.1
6
(C-7'), 131.8 (C-2/6), 121.5 (C-1), 120.8 (C-1'), 117.3 (C-4'), 116.8 (C-3'), 111.4 (C-3/5),
•
+
1
2
10.4 (C-6'), 55.9 (OCH ); HRMS (EI), m/z Calcd for C H N O (M) 286.0954, found
3 15 14 2 4
86.1003.
.1.4. (E)-4-(2-(2-Hydroxy-5-nitrobenzylidene)hydrazinyl)benzoic acid (4)
CAS number: 850186-63-9; yellow amorphous powder (62%); mp 293–295 °C ; H
4
1
NMR (700 MHz, DMSO-d ): δ 12.38 (br s, 1H, -COOH), 11.64 (br s, 1H, -OH), 11.07 (br
6
s, 1H, -NH), 8.57 (br s, 1H, H-6'), 8.25 (s, 1H, -N=CH-), 8.07 (br d, J = 8.9 Hz, 1H, H-4'),

7
.86 (d, J = 8.3 Hz, 2H, H-2/6), 7.09 (d, J = 8.3 Hz, 2H, H-3/5), 7.05 (d, J = 8.9 Hz, 1H,
1
3
H-3'); C NMR (175 MHz, DMSO-d ): δ 167.7 (C-7), 161.5 (C-2'), 148.7 (C-4), 140.6 (C-
6
7
'), 134.5 (C-5'), 131.8 (C-2/6), 125.5 (C-4'), 122.5 (C-1), 121.5 (C-1'), 121.3 (C-3'), 116.9
•+
(
C-6'), 111.7 (C-3/5); HRMS (EI), m/z Calcd for C H N O (M) 301.0699, found
14 11 3 5
3
01.0743.
.1.5. 4-(2-(Methylcarbamothioyl)hydrazinyl)benzoic acid (5)
4
CAS number: 855294-99-4; white amorphous powder (76%); mp 256–258 °C
1
(
DMF); H NMR (700 MHz, DMSO-d ): δ 12.37 (br s, 1H, -COOH), 9.41, 8.44 (each br s,
6
2
H, -NH-CS-NH-), 8.51 (m, 1H, Ar-NH), 7.79 (d, J = 8.7 Hz, 2H, H-2/6), 6.69 (d, J = 8.7
1
3
Hz, 2H, H-3/5), 2.80 (s, 3H, -NHCH ); C NMR (175 MHz, DMSO-d ): δ 182.9 (>C=S),
3
6
1
67.7 (C-7), 152.7 (C-4), 131.3 (C-2/6), 121.4 (C-1), 111.9 (C-3/5), 31.2 (-NH-CH3);
•+
HRMS (EI), m/z Calcd for C H N O S (M) 225.0572, found 225.0623.
9
11
3
2
4
.1.6. 4-(2-(Ethylcarbamothioyl)hydrazinyl)benzoic acid (6)
CAS number: 474819-57-3; white amorphous powder (70%); mp 242–244 °C; H
1
NMR (700 MHz, DMSO-d ): δ 12.37 (br s, 1H, -COOH), 9.36, 8.43 (each br s, 2H, -NH-
6
CS-NH-), 8.19 (m, 1H, Ar-NH), 7.79 (d, J = 8.8 Hz, 2H, H-2/6), 6.69 (d, J = 8.8 Hz, 2H,
1
3
H-3/5), 3.47 (q, J = 7.0 Hz, 2H, H-1'), 1.04 (t, J = 7.0 Hz, 3H, H-2'); C NMR (175 MHz,
DMSO-d ): δ 181.9 (>C=S), 167.7 (C-7), 152.7 (C-4), 131.3 (C-2/6), 121.4 (C-1), 112.0 (C-
6
•+
3
/5), 38.6 (C-1'), 15.1 (C-2'); HRMS (EI), m/z Calcd for C H N O S (M) 239.0728,
10 13 3 2
found 239.0773.
4
.1.7. 4-(2-(Allylcarbamothioyl)hydrazinyl)benzoic acid (7)
CAS number: 855202-41-4; pale yellow amorphous powder (80%); mp 220–222 °C;
1
H NMR (700 MHz, DMSO-d ): δ 12.38 (br s, 1H, -COOH), 9.49, 8.48 (each br s, 2H, -
6
NH-CS-NH-), 8.34 (m, 1H, Ar-NH), 7.79 (d, J = 8.3 Hz, 2H, H-2/6), 6.70 (d, J = 8.3 Hz,
1
3
2
H, H-3/5), 5.81 (m, 1H, H-2'), 5.03 (m, 2H, H-3'a,3'b), 4.1 (m, 2H, H-1'); C NMR (175
MHz, DMSO-d ): δ 182.5 (>C=S), 167.7 (C-7), 152.6 (C-4), 135.6 (C-2'), 131.3 (C-2/6),
6
1
21.4 (C-1), 115.5 (C-3'), 112.0 (C-3/5), 45.9 (C-1'); HRMS (EI), m/z Calcd for
•+
C H N O S (M) 251.0728, found 251.0743.
1
1
13
3
2

4
.1.8. 4-(2-(Butylcarbamothioyl)hydrazinyl)benzoic acid (8)
CAS number: 475079-88-0; white amorphous powder (70%); mp 239–241 °C; H
1
NMR (700 MHz, DMSO-d ): δ 12.38 (br s, 1H, -COOH), 9.35, 8.42 (each br s, 2H, -NH-
6
CS-NH-), 8.15 (m, 1H, Ar-NH), 7.79 (d, J = 8.6 Hz, 2H, H-2/6), 6.69 (d, J = 8.6 Hz, 2H,
H-3/5), 3.44 (t, J = 7.4 Hz, 2H, H-1'), 1.46 (quintet, J = 7.4 Hz, 2H, H-2'), 1.22 (sixtet, J =
1
3
7
.4 Hz, 2H, H-3'), 0.86 (t, J = 7.4 Hz, 3H, H-4'); C NMR (175 MHz, DMSO-d ): δ 182.1
6
(>C=S), 167.7 (C-7), 152.7 (C-4), 131.3 (C-2/6), 121.4 (C-1), 112.0 (C-3/5), 43.4 (C-1'),
•+
3
2
1.4 (C-2'), 19.8 (C-3'), 14.3 (C-4'); HRMS (EI), m/z Calcd for C H N O S (M)
12 17 3 2
67.1041, found 267.1073.
4
.1.9. 4-(2-(Benzylcarbamothioyl)hydrazinyl)benzoic acid (9)
1
CAS number: 509109-88-0; white amorphous powder (82%); mp 236–238 °C; H
NMR (700 MHz, DMSO-d ): δ 12.39 (br s, 1H, -COOH), 9.56, 8.53 (each br s, 2H, -NH-
6
CS-NH-), 8.76 (m, 1H, Ar-NH), 7.80 (d, J = 8.5 Hz, 2H, H-2/6), 7.27 (m, 4H, H-3'/5', 2'/6'),
1
3
7
.21 (br t, J = 7 Hz, 1H, H-4'), 6.73 (d, J = 8.5 Hz, 2H, H-3/5), 4.72 (s, 2H, CH -7'); C
2
NMR (175 MHz, DMSO-d ): δ 182.9 (>C=S), 167.7 (C-7), 152.7 (C-4), 140.0 (C-1'), 131.3
6
(C-2/6), 128.5 (C-3'/5'), 127.6 (C-2'/6'), 127.1 (C-4'), 121.4 (C-1), 112.0 (C-3/5), 46.9 (Ar-
•+
CH ); HRMS (EI), m/z Calcd for C H N O S (M) 301.0885, found 301.0921.
2
15 15
3
2
4
.1.10. 4-(2-(Phenethylcarbamothioyl)hydrazinyl)benzoic acid (10)
CAS number: 502432-82-8; white amorphous powder (80%); mp 252–254 °C; H
1
NMR (700 MHz, DMSO-d ): δ 12.38 (br s, 1H, -COOH), 9.46, 8.45 (each br s, 2H, -NH-
6
CS-NH-), 8.24 (m, 1H, Ar-NH), 7.80 (d, J = 8.7 Hz, 2H, H-2/6), 7.25 (br t, J = 7.4 Hz, 2H,
H-3'/5'), 7.18 (m, 3H, H-2'/6',4'), 6.69 (d, J = 8.7 Hz, 2H, H-3/5), 3.65 (t, J = 7.6 Hz, 2H,
1
3
H-8'), 2.81 (t, J = 7.6 Hz, 2H, H-7'); C NMR (175 MHz, DMSO-d ): δ 182.2 (>C=S), 167.7
6
(C-7), 152.6 (C-4), 139.7 (C-1'), 131.3 (C-2/6), 129.1 (C-3'/5'), 128.8 (C-2'/6'), 126.5 (C-
4
'), 121.5 (C-1), 112.1 (C-3/5), 45.3 (C-8'), 35.3 (C-7'); HRMS (EI), m/z Calcd for
•+
C H N O S (M) 315.1041, found 315.1083.
1
6
17
3
2
4
.1.11. 4,4'-((1,3,5,7-Tetraoxo-5,7-dihydropyrrolo[3,4-f]isoindole-2,6(1H,3H)diyl)bis-
(azane-diyl)) dibenzoic acid (11)

1
Brown amorphous powder (52%); mp >300 °C; H NMR (700 MHz, DMSO-d ): δ
6
1
8
2.52 (br s, 2H, 2 x -COOH), 9.27 (br s, 2H, 2 x -NH), 8.43 (s, 2H, H-4'/8'), 7.80 (d, J =
1
3
.5 Hz, 4H, H-2/6, 2''/6''), 6.91 (d, J = 8.5 Hz, 4H, H-3/5, 3''/5''); C NMR (175 MHz,
DMSO-d ): δ 167.5 (C-7/7''), 165.2 (C-1',3',5',7'), 150.9 (C-4/4''), 135.8 (C-3'a,4'a,7'a,8'a),
6
1
31.5 (C-2/6,2''/6''), 122.4 (C-1,1''), 118.9 (C-4'/8'), 112.1 (C-3/5,3''/5''); HRMS (EI), m/z
•+
Calcd for C H N O (M) 486.0812, found 486.0863.
2
4
14
4
8
4
4
.1.12. 4-(1,3-dioxoisoindolin-2-yl)amino)benzoic acid (12): The data given in [18].
.1.13. 4-((5,6-Dichloro-1,3-dioxoisoindolin-2-yl)amino)benzoic acid (13)
1
Yellow amorphous powder (68%); mp >300 °C ; H NMR (700 MHz, DMSO-d ): δ
6
1
2.50 (br s, 1H, -COOH), 9.18 (br s, 1H, -NH), 8.31 (s, 2H, H-4'/7'), 7.78 (d, J = 8.8 Hz,
1
3
2
H, H-2/6), 6.87 (d, J = 8.8 Hz, 2H, H-3/5); C NMR (175 MHz, DMSO-d ): δ 167.5 (C-
6
7
), 165.1 (C-1',3'), 150.9 (C-4), 138.3 (C-5'/6'), 131.4 (C-2/6), 130.2 (C-3'a/7'a), 126.3 (C-
4
'/7'), 122.2 (C-1), 111.9 (C-3/5); HRMS (EI), m/z Calcd for C H Cl N O (M)
1
5
8
2
2
4
•+3
49.9861, found 249.9901.
.
4
4
.2.
Biology
.2.1. In-vitro cell cultures and MTT cytotoxicity assay
The detailed methodology was reported by Abuelizz et collaborators [4]. All
experiments were conducted in triplicate and repeated on three different days. All values
are represented as mean ± SD. IC values were determined by Probit analysis by SPSS Inc
5
0
Probit analysis (IBM Corp., Armonk, NY, USA).
4
.2.2. Hoechst 33258 nuclear staining
The hoechst nuclear staining was done according to the reported protocol [19]. In brief,
5
MCF-7 cells were seeded in 12-well plates (1 × 10 cells/well) and incubated with the IC50
values of compounds 7, 9 and 10 (25, 26 and 23.5 μM, respectively) for 24 h. Cells were
then washed twice with cold phosphate-buffered saline (PBS), fixed with 4%
paraformaldehyde, permeabilized by cold methanol, and stained with Hoechst 33258

(Sigma-Aldrich, St. Louis, MO, USA) for 30 min in the dark. The nuclear morphological
changes of apoptotic cells were observed with a fluorescence microscope (Zeiss Axio
Observer D1 Zeiss, Germany).
4
.2.3. FITC annexin V / PI fluorescence analysis for apoptosis quantification
The annexin V-PI apoptosis measurement was carried out in accordance to the
6
manufacturer’s protocol (Invitrogen, CA, USA). Briefly, MCF-7 cells (1 × 10 cells/well)
were treated with the IC50 values of compounds 7, 9 and 10 (25, 26 and 23.5 μM,
respectively) for 24 h. Thereafter, both untreated and treated MCF-7 cells were washed
twice with ice-cold PBS and re-suspended in 100 µL of 1X binding buffer. To the
resuspended solution, 5 μL of each FITC-annexin-V and PI were added. After 15 min
incubation in the dark, a binding buffer (400 μL) was added to each tube and apoptosis was
quantified by flow cytometry (Cytomics FC 500; Beckman Coulter, CA, USA) [20].
4
.3.
QSAR model
In the HCT-116 model, ten compounds (80%) as a training set and three compounds (20%)
as tests were used for QSAR modeling. The test compounds were selected by the Diverse
Molecules protocol in Discovery Studio 4.5 as the external test subset. Compounds 1, 3,
and 10 were the chosen test compounds. In the MCF-7 model, by the same protocol, ten
compounds (80%) as a training set and three compounds (20%) as tests were selected for
QSAR modeling. The pTI of the compounds was calculated from pTI = -log TI. In
Discovery Studio, CHARMm was chosen to assign partial charges to the ligands during
the assignment of a force field. In addition, the potentials of electrostatics and van der
Waals were treated as separate terms. A + le point change was employed as an electrostatic
potential probe, whereas the effect of the solvent was mimic by the use of the constant of
distance-dependent dielectrics. In regard to the potential of van der Waals, a 1.73 Å radius
carbon atom was used as a probe. An energy grid descriptor was used to build a model of
partial least squares (PLS). The Create 3D QSAR Model protocol was used to build QSAR
2
models in Discovery Studio 4.5. For a perfect model of QSAR, the value of R (training
2
set regression coefficient) and Q (test set regression coefficient) must be greater than 0.6

and 0.5, respectively [21, 22]. In addition, the values of the Pearson-R value must be higher
than 0.5 and root mean square error (RMSE) must be less than 0.5 [23-25].
Acknowledgments
The authors wish to thank the Deanship of Scientific Research at King Saud University for
funding this work through the research group No. RG-1435-068.
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Highlights
•
A series of 4-Hydrazinylbenzoic derivatives were synthesized.
•
All derivatives were evaluated for in vitro anticancer activity against HCT-116
and MCF-7 cancer cells
•The active targets 6, 7 and 9 exhibited very weak cytotoxicity on normal cells
(
RPE-1) and showed higher IC values against HCT-116 and MCF-7 cells in
5
0
relation to doxorubicin.
•
Compounds 7, 9 and 10 inhibited the proliferation of MCF-7 by the induction of
apoptosis.
•
The bioassay results in the regression plots generated in 3D QSAR models were
in agreement and correlated with the anticancer results of the target molecules.
.

Graphical abstract
The 4-hydrazinobenzoic acid derivatives were elaborated and evaluated of their in
vitro cytotoxicity. Compounds 7, 9 and 10 showed potent inhibitory effects against
HCT-116 and MCF-7 cancer cells giving IC ranged between 21.3 ± 4.1 and 28.3
5
0
±
5.1 µM in comparison to doxorubicin. The active targets 7 and 9 exhibited very
weak cytotoxicity on normal cells (RPE-1) and inhibited the proliferation of MCF-
7
by the induction of apoptosis.
Cp.10
Control
Cp.7
Cp.9