Utilization of cyanoacetohydrazide and 2-(1,3-dioxoisoindolin-2-yl) acetyl chloride in the synthesis of some novel anti-proliferative heterocyclic compounds

Article abstract of DOI:10.1080/00397911.2020.1786125

Owing to its high reactivity and commercial availability, 2-cyanoacetohydrazide can be utilized as a versatile and appropriate intermediate for synthesis of a broad variety of heterocyclic compounds. Thus, 2-cyanoacetohydrazide and 2-(1,3-dioxoisoindolin-2-yl) acetyl chloride were used as starting materials for construction of new heterocyclic compounds bearing 1,3-dioxoisoindoline moiety. The newly synthesized compounds were recognized by elemental analyses and spectral data (IR, ¹H-NMR, and ¹³C-NMR spectra). The synthesized compounds were screened for their anti-proliferative activity against two human epithelial cell lines; breast (MCF-7) and liver (HepG2) as well as to normal fibroblasts (WI-38). The data showed distinctly that compounds 1 and 12 presented promising in-vitro anti-proliferative activity against two cell lines (MCF-7 and HepG2) without harming normal fibroblasts.

Full text of DOI:10.1080/00397911.2020.1786125

Synthetic Communications
An International Journal for Rapid Communication of Synthetic Organic
Chemistry
ISSN: 0039-7911 (Print) 1532-2432 (Online) Journal homepage: https://www.tandfonline.com/loi/lsyc20
Utilization of cyanoacetohydrazide and 2-
(1,3-dioxoisoindolin-2-yl) acetyl chloride in
the synthesis of some novel anti-proliferative
heterocyclic compounds
Mohamed H. Hekal, Yasmeen M. Ali & Fatma S. M. Abu El-Azm
To cite this article: Mohamed H. Hekal, Yasmeen M. Ali & Fatma S. M. Abu El-Azm (2020):
Utilization of cyanoacetohydrazide and 2-(1,3-dioxoisoindolin-2-yl) acetyl chloride in the synthesis
of some novel anti-proliferative heterocyclic compounds, Synthetic Communications, DOI:
10.1080/00397911.2020.1786125
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V
R
SYNTHETIC COMMUNICATIONS
https://doi.org/10.1080/00397911.2020.1786125
Utilization of cyanoacetohydrazide and 2-(1,3-
dioxoisoindolin-2-yl) acetyl chloride in the synthesis of
some novel anti-proliferative heterocyclic compounds
Mohamed H. Hekal, Yasmeen M. Ali, and Fatma S. M. Abu El-Azm
Chemistry Department, Faculty of Science, Ain Shams University, Cairo, Egypt
ABSTRACT
ARTICLE HISTORY
Owing to its high reactivity and commercial availability, 2-cyanoace-
tohydrazide can be utilized as a versatile and appropriate intermedi-
ate for synthesis of a broad variety of heterocyclic compounds. Thus,
Received 23 May 2020
KEYWORDS
2
-cyanoacetohydrazide and 2-(1,3-dioxoisoindolin-2-yl) acetyl chloride
Benzimidazole derivatives;
benzoxazole derivatives; 2-
cyanoacetohydrazide; 2-(1,3-
dioxoisoindolin-2-yl) acetyl
chloride; 2-pyridone
derivatives
were used as starting materials for construction of new heterocyclic
compounds bearing 1,3-dioxoisoindoline moiety. The newly synthe-
sized compounds were recognized by elemental analyses and spec-
1
13
tral data (IR, H-NMR, and
C-NMR spectra). The synthesized
compounds were screened for their anti-proliferative activity against
two human epithelial cell lines; breast (MCF-7) and liver (HepG2) as
well as to normal fibroblasts (WI-38). The data showed distinctly that
compounds 1 and 12 presented promising in-vitro anti-proliferative
activity against two cell lines (MCF-7 and HepG2) without harming
normal fibroblasts.
GRAPHICAL ABSTRACT
O
CN
O
O
O
H
N
NH
X
NH
NH
N
HN
O
O
NC
Ar\
O
O
O
O
H
2
N
CN
H
N
NH
O
O
O
N
N
O
HN
NH
O
N
O
O
CH
3
Cl
O
CN
O
N
O
H
N
NH
S
R
CN
O
O
O
N
O
N
S
NH
O
O
N
O
CN
O
CN
O
O
O
O
O
H
N
NH
N
NH
CH3
H
N
NH
S
N
N
S
O
N
N
CH
3
O
Ph
CONTACT Mohamed H. Hekal
mohahekal2007@yahoo.com
Chemistry Department, Faculty of Science, Ain Shams
University, Cairo, Egypt
Supplemental data for this article can be accessed on the publisher’s website.
ß 2020 Taylor & Francis Group, LLC

2
M. H. HEKAL ET AL.
O
O
N
O
N
O
NH
NH
O
O
O
O
NH
2
Thalidomide
(
Antineoplastic, Antileprotic)
Pomalidomide
Anti-angiogenic, immunomodulator)
(
O
O
O
O
N
S
O
O
N
NH
N
O
O
2 3
SO CH
O
NHAc
(Phosphodiesterase-4 inhibitors (PDE-4))
LASSBio 595
Apremilast
(
antiinflammatory)
O
O
N
S
O
N
O
S
LASSBio-468
(
Phosphodiesterase-4 inhibitor, antiinflammatory)
Figure 1. Several drugs bearing isoindoline-1,3-dione as a core moiety.
Introduction
Isoindoline-1,3-dione derivatives (phthalimides) act as a leading substructure in organic
[
1–4]
synthesis for preparing various biologically active molecules.
The most important
pharmacological effects that have been reported for phthalimide derivatives are anti-
[
5]
[6]
[7]
[8]
cancer, antimicrobial, anti-oxidant, anti-inflammatory, glucosidase inhibition,
[
9]
[10]
[11–13]
[14]
antimalarial, anti-angiogenic,
analgesic activities.
mentally hydrophobic, and this raises their potential to pass biological mem-
antitubercular activities,
The structure of isoindoline-1,3-dione (-CO-N(R)-CO-) is funda-
anticonvulsant
and
[
15]
[
16]
branes in vivo.
The phthalimide ring has been considered as a distinctive scaffold to design novel
lead drug compounds of diverse biological activities and applied for different diseases
like AIDS, leprosy, diabetes, tumor, convulsion, multiple myeloma, pain, inflammation,
[
17,18]
and bacterial infection.
The highly active reference compounds such as
Thalidomide (Antineoplastic, Antileprotic), Pomalidomide (Anti-angiogenic, immuno-
modulator), Apremilast (Phosphodiesterase-4 inhibitors (PDE-4)), LASSBio-468
(
Phosphodiesterase-4 inhibitor, Anti-inflammatory) and LASSBio 595 (Anti-inflamma-
[
19,20]
tory) bearing phthalimide moiety that are linked to various side chain
in Figure 1.
are shown
Inspection of the reference compounds which presented in Figure 1 drove us to
design and synthesize some novel compounds consisting mainly of a phthalimide sub-
unit and improved by certain pharmacophores. Therefore, to enhance the biological
activity of phthalimide derivatives, a molecular crossbreeding pathway was utilized to
insert different pharmacophore subunits like iminocoumarin, pyridone, 1,3-dithiolane,
1,3-dithialane and benzo- (oxazole and imidazole).

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SYNTHETIC COMMUNICATIONS
3
CN
O
O
O
O
O
HN
NH
O
Cl
O
H
2
N
CN
Dioxane
N
+
N
N
H
stirring at r.t
O
(1)
Scheme 1. Synthesis of 2-cyano-N’-(2-(1,3-dioxoisoindolin-2-yl) acetyl) acetohydrazide (1).
Among diverse commercially obtainable substituted hydrazides, 2-cyanoacetohydrazide
can be considered as a versatile and appropriate intermediate for synthesis of a broad variety
of heterocyclic compounds. 2-Cyanoacetohydrazide can act as an ambident nucleophile (C-
[
21–26]
and N-nucleophile). In our literary survey,
we indicate that 2-cyanoacetohydrazide has
diverse functionality, which is appeared in two nucleophilic centers localized at carbon atom
of active methylene group and nitrogen atom of NH group, and also in two electrophilic
2
centers localized at carbon atoms of cyano and carbonyl groups. Hence, reactions of cyanoa-
cetohydrazide with several reactants (electrophiles and nucleophiles) are utilized in synthesis
[
27]
of a diversity of polyfunctional heterocyclic compounds of biological concern.
Results and discussion
As an aspect of our interest in the synthesis of a broad domain of heterocyclic
[
28–32]
systems with biological applications,
our present research is interested with the
exploitation of 2-cyanoacetohydrazide and 2-(1,3-dioxoisoindolin-2-yl) acetyl chloride as
starting materials for construction of new heterocyclic compounds bearing 1,3-dioxoi-
0
soindoline moiety. Thus, in the current study, 2-cyano-N -(2-(1,3-dioxoisoindolin-2-yl)
acetyl) acetohydrazide (1) was obtained via stirring 2-(1,3-dioxoisoindolin-2-yl) acetyl
chloride with 2-cyanoacetohydrazide in dioxane at room temperature for 30 min
(Scheme 1). The chemical structure of compound 1 was concluded from the study of its
spectroscopic data. Thus, the IR spectrum of compound 1 displayed t
at 3297,
NH
ꢀ
1
ꢀ1
ꢀ1
3
226 cm , t
at 1776, 1731 cm (vibrational coupling), t
at 1680 cm
C¼O
C¼O (amide)
ꢀ
1
and t
at 2263 cm . Furthermore, the existence of two singlet signals for CH pro-
2
CꢁN
tons of CH CN and N-CH CO at d 3.72 and 4.30 ppm, respectively, together with two
2
2
broad singlet signals for two NH protons at d 10.25 and 10.32 ppm beside the aromatic
1
13
protons in the H-NMR spectrum affirm the structure 1. Moreover, the C-NMR spec-
trum of the target compound 1 exhibited signals at 23.74 ppm (CH CN), 66.36 ppm
2
(
1
N–CH CO), 115.56 ppm (CꢁN), 123.29, 131.65, 134.68 ppm (aromatic carbons) and
2
61.23, 165.17, 167.37 ppm due to the presence of four CO groups which coincident
with the proposed structure. The formation of compound 1 could be illustrated based
on nucleophilic attack by nitrogen atom of the terminal amino group of 2-cyanoaceto-
hydrazide on the positively polarized carbonyl carbon of the acid chloride followed by
dehydrochlorination.
The functionalities in b-ketonitrile 1 made it beneficial precursor for construction of
new heterocyclic compounds bearing 1,3-dioxoisoindoline moiety as the cyano and car-
bonyl functions are suitably located to enable reactions with prevalent reagents.
Furthermore, the active methylene of b-ketonitrile 1 can participate in substitution and

4
M. H. HEKAL ET AL.
O
O
O
CHO
OH
H
N
NH
O
N
HN
(
2)
EtOH/pip
O
O
CN
O
O
H
N
NH
N
OHC
EtOH/pip
N
N
(3)
CN
O
O
O
HN
NH
O
N
O
CN
N
CH
3
3
H C
N
N
O
O
O
(1)
N
H
N Cl
N
NH
NH
CH3
O
N
3
EtOH/CH COONa
O
N
(
4)
CH3
N
O
Ph
O
CN
O
O
H
N
NH
OEt
CH(OEt)3
N
Ac O
2
(5)
O
Scheme 2. Condensation of the b-ketonitrile 1 with some electrophilic reagents.
condensation reactions. Therefore, cyclocondensation of compound 1 with salicylalde-
hyde in ethanolic solution containing catalytic amount of piperidine yielded the 2-imino
coumarin derivative 2 (Scheme 2). The IR spectrum of compound 2 showed t
at
at
NH
ꢀ
1
ꢀ1
3
1
325, 3276 cm , t
at 1775, 1727 cm
(vibrational coupling), t
C¼O
C¼O (amide)
ꢀ
1
ꢀ1
684 cm , t
at 1640 cm and devoid of stretching absorption band for nitrile
C¼N
1
group. Moreover, H-NMR spectrum of the 2-imino coumarin derivative 2 showed
absence of methylene protons of CH CN group and presence of new singlet signal
2
assigned for C ¼ NH proton at d 8.45 ppm exchanged with D O, singlet at d 6.24 ppm
2
for HC -chromene proton beside signal for methylene protons of NCH at d 4.19, 4.25
4
2
as ABq signal with J ¼ 16.8 Hz. The higher coupling constant (J) is due to their exist-
13
ence as geminal protons of AB system. Likewise, C-NMR spectrum was in conformity
with the suggested structure. Similarly, b-ketonitrile 1 was subjected to react with 4-
(dimethylamino) benzaldehyde in ethanol containing a catalytic amount of piperidine to
produce the arylidene derivative 3 (Scheme 2).
The diazonium salt of 4-aminoantipyrine was subjected to a coupling reaction with com-
ꢂ
pound 1 in ethanolic sodium acetate solution at 0–5 C to afford the corresponding hydra-
1
zinyl derivative 4 (Scheme 2). Structure of compound 4 was supported by its IR, H-NMR
13
and C-NMR spectra. (cf. experimental)
-Cyano-N’-(2-(1,3-dioxoisoindolin-2-yl)acetyl)-3-ethoxyacrylohydrazide (5) was fur-
nished on reaction of compound 1 with triethyl orthoformate in the presence of
2

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SYNTHETIC COMMUNICATIONS
5
O
N
CN
O
N
CN
O
O
O
O
H
H
N
O
N
N
H
O
N
H
O
O
E- isomer
14%)
Z- isomer
(86%)
(
Figure 2. E/Z isomers of compound 5.
distilled acetic anhydride (Scheme 2). The suggested structure of 5 was confirmed by
spectroscopic data. Compound 5 exists as E/Z mixture in the ratio 14:86 as shown from
1
its HNMR spectrum which exhibits duplicates of triplets and quartets in addition to
two singlet signals for the olefinic proton at d 8.22, 8.34 ppm. The higher ratio of the Z-
isomer is due to formation of the intramolecular chelated H-bond in six-membered
ring which leads to increasing its stability, as shown in Figure 2.
1
3
Moreover, C-NMR spectrum of 5 affirmed the presence of ethoxy group carbons at
d 15.03 and 73.01 ppm.
The reactivity of acetohydrazide 1 toward b-diketones was also investigated.
Therefore, subjecting compound 1 to react with acetyl acetone and/or benzoyl acetone
in ethanolic solution containing catalytic amount of piperidine afforded the 2-pyridone
1
derivatives 6 and 7, respectively (Scheme 3). The HNMR spectrum of compound 6
supported its structure as it displayed signals at d 2.23 and 2.35 and 6.37 ppm attributed
to protons of CH and ¼CH
, respectively beside the absence of a singlet signal
for protons of CH CN group. Moreover, the C-NMR spectrum confirmed the exist-
3
pyridone
13
2
ence of two methyl groups at d 18.58, 20.83 ppm.
Refluxing of compound 1 with 2-((1,3-diphenyl-1H-pyrazol-4-yl) methylene) malono-
nitrile in dioxane containing a catalytic amount of piperidine gave 6-amino-2-oxopyri-
dine derivative 8 (Scheme 3). The structure of 2-pyridone derivative 8 was
unambiguously achieved from analytical and spectroscopic data. For example, the IR
ꢀ
1
spectrum of 8 exhibited bands for t
at 3340, 3211, 3200 cm , t
at 1773,
NH2, NH
C¼O
ꢀ
1
ꢀ1
1
1
717 cm and t
at 2215 cm . Moreover, H-NMR revealed the following signals
CꢁN
at d (ppm): 4.98 (s, 2H, NCH ), 7.71–7.97 (m, 14H, ArH þ 2H, NH ), 8.92 (s, 1H,
2
2
ArH), 11.25 (br s, 1H, NH, exchangeable with D O).
2
Compounds with active methylene moiety form carbanions in the presence of a base
to react with one carbon donor such as carbon disulfide to afford dithiocarboxylates
which can be transformed to ketene dithioacetals through its reaction with excess of the
alkylating reagent. Consequently, stirring of the cyanoacetyl acetohydrazide 1 with car-
bon disulfide in the presence of potassium hydroxide in dimethylformamide yielded the
nonisolable intermediate dipotassium dithiolate salt G which in situ underwent cyclode-
hydrobromination with 1,2-dibromoethane and/or 1,3-dibromopropane to yield dithio-
lane derivative 9 and dithiane derivative 10, respectively (Scheme 4). The
microanalytical and spectroscopic data were in conformity with the suggested structures
1
of compounds 9 and 10. For instance, H-NMR spectrum of compound 9 detected the
appearance of doublet-doublet signals at 3.58, 3.68 ppm for two SCH groups with
2

6
M. H. HEKAL ET AL.
CH3
H C
CN
3
O
O
O
O
N
NH
O
N
EtOH/pip
(
6)
O
CH3
Ph
CN
CN
O
O
N
O
O
O
O
HN
NH
NH
O
Ph
EtOH/pip
O
N
N
(7)
O
O
(1)
NC
Ar\
H N
CN
2
CN
O
O
N
Ar\
NH
O
CN
N
Dioxane/pip
Ph
N
\
(
8)
Ar =
O
N
Ph
Scheme 3. Synthesis of some novel 2-pyridone derivatives from the cyanoacetohydrazide 1.
J ¼ 16.8, 17.7 Hz (the protons of each methylene group are not magnetically equivalent
and they undergo germinal coupling as the two CH groups are in a cyclic system. This
2
is evidenced from the higher coupling constant values). (cf. experimental)
Otherwise, treatment of the dipotassium dithiolate salt G in situ with methyl iodide
yielded the ketene S,S-dithioacetal 11 (Scheme 4). The structure of compound 11 was
clarified from the elemental analysis and spectral data. Thus, the IR spectrum displayed
ꢀ1
the appearance of absorption bands at 3272, 2203 and 1776, 1721, 1694 cm for NH,
1
CꢁN, and C ¼ O groups, respectively. As well, the H-NMR spectrum exhibited a sing-
let signal at d 2.61 ppm for six protons of two identical S-methyl protons, a signal for
the NCH protons at d 4.30 ppm in addition to two broad singlet signals for two NH
2
protons at d 10.44 and 10.52 ppm disappeared with D O.
2
Afterwards, the reactivity of the ketene S,S-dithioacetal 11 toward some nitrogen
nucleophiles was examined. Consequently, refluxing compound 11 with bidentate nucle-
ophiles such as o-phenylenediamine and/or o-aminophenol in absolute ethanol yielded
the corresponding benzimidazole derivative 12 and benzoxazole derivative 13, respect-
1
13
ively (Scheme 5). Structures 12 and 13 were achieved from H-NMR and C-NMR
spectra which revealed the absence of the two SCH groups. (cf. Experimental).
3
Regrettably, refluxing of the ketene S,S-dithioacetal 11 with hydrazine hydrate in
dioxane afforded sulfur-free compound which was identified as 2,3-dihydrophthalazine-

V
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SYNTHETIC COMMUNICATIONS
7
O
O
O
HN
NH
O
CN
CN
O
O
N
H
N
NH
S
(
1)
Br
Br
N
S
O
(
9)
CS2 / KOH DMF
O
O
O
CN
O
S
O
O
O
H
CN
H
N
N
N
NH
S
H
Br
Br
S
N
N
K
S
K
(10)
O
O
(G)
O
CN
O
O
H
N
NH
S
3
CH I
N
S
(
11)
O
Scheme 4. Treatment of the dipotassium dithiolate salt G with some alkyl halides.
O
CN
O
O
H
N
NH
NH
N
HN
(12)
O
NH
NH
2
2
O
CN
EtOH
NH2
OH
O
O
O
O
O
O
H
N
NH
O
H
2
N
NH
2
H
NH
2
(
11)
N
HN
NH
O
Fusion
EtOH
NH2.
(13)
Dioxane NH
2
H
2
O
O
O
O
(16)
2
H N
O
O
O
N
O
O
H
NH
NH
NH
NH
2 2
NH2.H O
N
NH
(1) or
O
Dioxane
N
S
(15)
O
(14) O
O
Scheme 5. Reactivity of the ketene S,S-dithioacetal 11 towards some nitrogen nucleophiles.
[
33]
1
,4-dione 14 rather than the respective pyrazole derivative 15
(Scheme 5).
The phthalazine-1,4-dione 14 was also obtained upon hydrazinolysis of the cyanoacetyl
acetohydrazide 1 in refluxing dioxane. The phthalazine-1,4-dione 14 structure was proven
from comparison (TLC, IR, mp) with an authentic sample prepared from reaction of
39
[
]
phthalic anhydride with hydrazine hydrate in refluxing dioxane
(Scheme 5).

8
M. H. HEKAL ET AL.
Table 1. Effect of the newly synthesized compounds on the viability of HepG2, MCF7 and WI-
8 cells.
3
a
IC50 (mM)
Compd. No.
MCF-7
HepG2
WI-38
1
2
3
4
5
6
7
8
9
1
1
1
1
2.53 ± 4.6
26.97 ± 1.9
79.91 ± 5.0
22.09 ± 1.7
64.87 ± 4.3
36.23 ± 2.8
39.48 ± 1.7
45.64 ± 0.4
58.9 ± 4.0
53.31 ± 4.0
38.4 ± 3.5
4.59 ± 0.3
17.29 ± 4.1
4.17 ± 0.2
3.56 ± 3.6
11.26 ± 1.3
75.81 ± 4.1
21.45 ± 1.7
91.78 ± 5.0
25.89 ± 1.9
25.47 ± 4.5
34.81 ± 0.3
68.2 ± 4.4
56.04 ± 3.4
40.0 ± 3.6
5.88 ± 0.2
15.73 ± 2.8
4.50 ± 0.3
96.44 ± 4.5
56.4 ± 4.3
72.4 ± 4.8
45.23 ± 2.9
29.5 ± 2.2
23.2 ± 1.2
49.2 ± 3.9
91.31 ± 5.1
42.2 ± 3.3
39.1 ± 2.9
44.6 ± 4.2
70.02 ± 4.6
64.29 ± 1.8
6.72 ± 0.5
0
1
2
3
DOX
DOX: doxorubicin.
a
IC50 ((mM)): 1–10 (very strong), 11–20 (strong), 21–50 (moderate), 51–100 (weak), above 100 (non-cytotoxic).
By the same way, reaction of the ketene S,S-dithioac etal 11 with formamide yielded
isoindoline-1,3-dione (phthalimide) 16 which was also compared with an authentic sam-
40
[
]
ple prepared from fusion of phthalic anhydride with urea (TLC, IR, mp)
.
The structure of the obtained isoindoline-1,3-dione 16 was elucidated using analytical
1
13
and spectroscopic data (IR, H-NMR, and C-NMR) (Scheme 5).
In vitro anti-proliferative activity
The in vitro anti-proliferative activity of the novel synthesized compounds was evaluated
against breast (MCF7) and human liver (HepG2) cancer cell lines as well as to normal
fibroblasts (WI-38) and compared to the activity of doxorubicin as a standard drug
(Table 1; Figure 3). The report showed that doxorubicin had an IC50 of ꢃ4–7 mM
against all cells investigated with no discrimination between cancer and normal cells.
The novel cyanoacetyl acetohydrazide derivative 1 displayed promising anti-proliferative
activity against cancer cell lines with IC50 of ꢃ2.5–3.5 mM which had much higher
effective anti-proliferative activity than the activity of doxorubicin. Likewise, the novel
benzimidazole derivative 12 showed almost equal activity to doxorubicin against cancer
cell lines with IC50 of ꢃ4.5–5.8 mM. Compounds 1 and 12 were safe to the normal
fibroblasts with IC50 at ꢃ70–96 mM. Compounds 1 and 12 showed very strong activity,
this is due to the presence of two NH groups in 1 and four NH groups in 12 which
may be added to any unsaturated moiety in DNA or forming hydrogen bonds with
either one of the nucleobases of the DNA and causes it damage. On the other hand,
compound 2 exhibited strong cytotoxic activity against HepG2 cell line with IC50
(11.26 ± 1.3) lM, while compound 13 exhibited strong cytotoxic activity against MCF7
cell line with IC50 (17.29 ± 4.1) lM. Moderate activity toward HepG-2 and MCF-7 cell
lines was observed with compounds 4, 6–8 and 11.
0
0
[34]
2
-C-Cyano-2 -deoxy-1-b-D-arabinopentofuranosylcytosine
(CNDAC)
is
a
nucleoside analogue with a novel mechanism of action that is being assessed in

V
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SYNTHETIC COMMUNICATIONS
9
100
90
80
70
60
50
40
30
20
10
0
MCF-7
HepG2
WI-38
1
2
3
4
5
6
7
8
9
10 11 12 13 DOX
Figure 3. Cytotoxic activity of the tested compounds on different cell lines.
Scheme 6. Mechanism of the antitumor action of CNDAC.

1
0
M. H. HEKAL ET AL.
NC
NC
NC
NH
HN
O
NH
N
OH
NH
N
O-P-P-P
O
O
O
O
O
O
N
N
N
O
O
O
(
1)
incorporation
into DNA
DNA
O
DNA
O
BASE
CN
BASE
CN
O
O
O
O
NC
NC
P O
O
P O
O
N
O
NH
O
HO
N
O
N
O
O
N
N
O
O
DNA elongation
DNA
O
BASE
CN
O
O
CN
HO
P
O
O
O
H
P O
O
DNA
N
O
O
O
N
O
P
O
O
DNA
O
BASE
CN
DNA
N
O
CN
N
O
O
P O
O
O
N
DNA chain is
terminated
O
N
O
Cell cycle is blocked
in the G phase
2
Scheme 7. Mechanism of the antitumor action of compound 1.
clinical experiments. Incorporation of CNDAC triphosphate into DNA and extension
during replication leads to single-strand breaks directly caused by b-elimination.
These breaks, or the lesions that emerge from more processing, cause cells to
[
35]
arrest in G . The electron withdrawing effect
2
of the cyano group at the arabinose
2
0
0
-b-position rises the acidity of the 2 -a proton and facilitates a b-elimination
reaction involving an oxygen of the phosphate group at the 3 -b position that leads
0

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R
SYNTHETIC COMMUNICATIONS
11
0
to single strand break that affords a DNA molecule lacking a 3 -hydroxyl, which
blocks its repair by ligation and leads to suppression of the cell cycle at the G2
phase (Scheme 6).
The suggested mechanism of DNA interaction with compound 1 may be similar to
the mechanism of DNA interaction with CNDAC and it is represented in Scheme 7.
Experimental
All melting points were measured on a Griffin and Georgy melting-point apparatus
(Griffin & Georgy Ltd., Wembley, Middlesex, UK) and are uncorrected. IR spectra were
recorded on Pye Unicam SP1200 spectrophotometer (Pye Unicam Ltd., Cambridge,
1
UK) by using the KBr wafer technique. H-NMR spectra were determined on a Varian
Gemini 300 MHz on Bruker Avance III using tetramethylsilane as an internal standard
1
3
(
chemical shifts in d scale), while C NMR spectra were run at 100 MHz. Elemental
analyses were carried out at the Microanalytical Unit, Faculty of Science, Ain Shams
University, using a Perkin-Elmer 2400 CHN elemental analyzer (Waltham, MA), and
satisfactory analytical data (±0.4) were obtained for all compounds. The homogeneity of
the synthesized compounds was controlled by thin layer chromatography (TLC), using
aluminum sheet silica gel F254 (Merck). The antitumor activities were performed at
Micro analytical Center of Mansoura University, Egypt.
2-Cyano-N’-(2-(1,3-dioxoisoindolin-2-yl) acetyl) acetohydrazide (1). A mixture of 2-
(
(
1,3-dioxoisoindolin-2-yl) acetyl chloride (2.2 g, 10 mmol) and 2-cyanoacetohydrazide
0.99 g, 10 mmol) was stirred in dioxane (20 ml) at room temperature for 30 min. The
deposited solid on stirring was filtered off, dried and recrystallized from EtOH/dioxane
ꢀ
1
mixture to give 1 as white crystals; mp: 258–260˚C, yield: 80%. IR (KBr, ꢀ, cm ):
3
1
297, 3226 (NH), 3058 (CH aromatic), 2977, 2923 (CH aliphatic), 2263 (CꢁN), 1776,
2
1
731, 1680 (C¼O). H-NMR (300 MHz, DMSO-d ) d (ppm): 3.72 (s, 2H, CH CN), 4.30
6
2
(s, 2H, NCH ), 7.85-7.93 (m, 4H, ArH), 10.25 (br s, 1H, NH, exchangeable with D O),
2
2
13
1
0.32 (br s, 1H, NH, exchangeable with D O). C-NMR (100 MHz, DMSO-d ) d
2
6
(ppm): 23.74, 66.36, 115.56, 123.29, 131.65, 134.68, 161.23, 165.17, 167.37. Anal. Calc.
for C H N O (286.24): C, 54.55; H, 3.52; N, 19.57. Found: C, 54.23; H, 3.44;
1
3
10 4 4
N, 19.50.
Pharmacological activity
Cytotoxicity assay
The cytotoxic activity of sixteen compounds was tested against two human tumor cell
lines namely: mammary gland (breast) MCF-7 and hepatocellular carcinoma (liver)
HePG-2 in addition to normal fibroblasts (WI-38). The cell lines were obtained from
the ATCC via the Holding Company for Biological Products and Vaccines (VACSERA,
Cairo, Egypt). Doxorubicin was used as a standard anticancer drug for comparison. The
reagents used were RPMI-1640 medium, MTT, DMSO and Doxorubicin (Sigma Co., St.
Louis, MO, USA), and Fetal Bovine Serum (GIBCO, Paisley, UK). The different cell
[
36,37]
lines
mentioned above were used to determine the inhibitory effects of compounds
on cell growth using the MTT assay. This colorimetric assay is based on the conversion

1
2
M. H. HEKAL ET AL.
of the yellow tetrazolium bromide (MTT) to a purple formazan derivative by mitochon-
drial succinate dehydrogenase in viable cells. The cells were cultured in RPMI-1640
medium with 10% fetal bovine serum. Antibiotics added were 100 units/mL penicillin
ꢂ
and 100 mg/mL streptomycin at 37 C in a 5% CO incubator. The cell lines were
seeded
2
[
38]
4
ꢂ
in a 96-well plate at a density of 1.0 ꢄ 10 cells/well at 37 C for 48 h under
5
% CO incubator. After incubation the cells were treated with different concentration
2
of compounds and incubated for 24 h. After 24 h of drug treatment, 20 mL of MTT solu-
tion at 5 mg/mL was added and incubated for 4 h. Dimethyl sulfoxide (DMSO) in vol-
ume of 100 mL was added into each well to dissolve the purple formazan formed. The
colorimetric assay is measured and recorded at absorbance of 570 nm using a plate
reader (EXL 800, BioTech, Winoosky, VT).
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