Novel quinoline derivatives carrying nitrones/oximes nitric oxide donors: Design, synthesis, antiproliferative and caspase-3 activation activities

Article abstract of DOI:10.1002/ardp.201800270

Novel quinoline derivatives carrying nitrones and oxime as nitric oxide donors were prepared and characterized using different spectroscopic techniques. Nitrones can release nitric oxide in larger amounts compared to corresponding oximes. Antiproliferativ

Full text of DOI:10.1002/ardp.201800270

Received: 15 September 2018
DOI: 10.1002/ardp.201800270
Revised: 21 October 2018
Accepted: 30 October 2018
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FULL PAPER
Novel quinoline derivatives carrying nitrones/oximes nitric
oxide donors: Design, synthesis, antiproliferative and
caspase-3 activation activities
Mahmoud S. Abdelbaset1
Mohamed Abdel-Aziz2
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Gamal El-Din A. Abuo-Rahma2
Mostafa H. Abdelrahman1
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Mohamed Ramadan1
Bahaa G. M. Youssif3,4
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1 Faculty of Pharmacy, Department of
Pharmaceutical Organic Chemistry, Al-Azhar
University, Assiut, Egypt
Abstract
Novel quinoline derivatives carrying nitrones and oxime as nitric oxide donors were
prepared and characterized using different spectroscopic techniques. Nitrones can
release nitric oxide in larger amounts compared to corresponding oximes. Anti-
proliferative screening results showed that the 2-benzylthioquinoline nitrones 6e and
6f and the 2-methylthio analogues 6g and 6h exhibited promising antiproliferative
activity especially against leukemia and colon cancer cell lines. Compounds 6c, 6e, and
6f exhibited higher potency as anticancer agents compared to doxorubicin, with IC50
ranging from 0.45 to 0.91 μM. Aremarkable overexpression of caspase-3 protein levels
was observed in cells treated with the tested compounds. Compound 6e exhibited
more pre-G1 apoptosis and cell cycle arrest at the G2/M phase than in other phases.
These results revealed that the tested compounds can cause programmed cell death
through overexpression of caspase 3, which may be attributed to the release of nitric
oxide.
2 Faculty of Pharmacy, Department of
Medicinal Chemistry, Minia University, Minia,
Egypt
3 Faculty of Pharmacy, Department of
Pharmaceutical Organic Chemistry, Assiut
University, Assiut, Egypt
4 Department of Pharmaceutical Chemistry,
College of Pharmacy, Jouf University, Sakaka,
Saudi Arabia
Correspondence
Prof. Mohamed Abdel-Aziz, Faculty of
Pharmacy, Department of Medicinal
Chemistry, Minia University, 61519 Minia,
Egypt.
Email: abulnil@hotmail.com
K E Y W O R D S
antiproliferative, apoptosis, caspase 3, nitric oxide, nitrones, oximes
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1
INTRODUCTION
explain the anticancer effect of quinoline derivatives including:
topoisomerase inhibition,[21–23] DNA intercalation,[24] protein kinases
Quinoline is a significant nucleus which has attracted the attention of
medicinal chemists due to its variable pharmacological activities. Easy
functionalization of various ring positions of quinoline makes it an
attractive synthetic building block for design and synthesis of new
drugs. Additionally, quinolines are considered as an interesting group
of compounds, many of which have widespread pharmacological
actions such as anti-microbial,[1–4] anti-inflammatory,[5–8] anti-
tubercular,[9–12] anti-convulsant,[13] antihypertensive,[14,15] antioxi-
dant,[16–19] and anticancer[20] activities. Several mechanisms can
inhibition,[25–27] tubulin polymerase inhibition,[28–31] and induction of
apoptosis.[32,33] Quinoline derivative I exhibited potent anticancer
activity against brain cancer cell line U87 with IC50 of 1.5 nM.[31]
Compound II exhibits remarkable anticancer activity against HL60
cancer cell line with IC50 of 0.68 μM (Figure 1).[33] Incorporation of
biologically active moieties into quinoline such as nitrones[34]
and oximes[35] may produce biologically active anticancer agents.
Compound III showed significant anticancer activity with IC50 of
31.42 μM against Hep-G2 cancer cell line (Figure 1).[34] Nitrones are a
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Arch Pharm Chem Life Sci. 2018;e1800270.
wileyonlinelibrary.com/journal/ardp
© 2018 Deutsche Pharmazeutische Gesellschaft
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https://doi.org/10.1002/ardp.201800270

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ABDELBASET ET AL.
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FIGURE 1 Structures of compounds I–III
class of compounds with various biological activities including anti-
inflammatory,[36] antioxidant,[37] anti-ischemic,[38] and antiprolifera-
tive[39] activities which may be attributed to nitric oxide release.
Alpha-phenyl N-tertiary-butyl nitrone (PBN) is the most famous
nitrone which is used as a spin trap and an antioxidant agent. Also,
PNB showed a potent anticancer activity and caused shrinkage
of cancer cells especially when administrated in early stage of
cancer.[40]
compounds were identified using different spectroscopic techniques
including 1H NMR, 13C NMR, mass spectroscopy, and elemental
analysis. Moreover, nitric oxide release was determined using Griess
method to evaluate the amount of NO released. The target compounds
were screened for their antiproliferative activity. Furthermore, the
IC50, caspase-3 activation assay, cell cycle analysis, and apoptosis
assay were established for the most active compounds of this
series.
Moreover, nitric oxide-releasing oximes have various potential
activities that depend on releasing of NO from their structures. These
activities include antihypertensive,[41] anti-inflammatory,[42] gastro-
protective,[42] and antiproliferative[43] activities. Several mechanisms
can explain the antiproliferative activity of nitrones and oximes, but the
most accepted one is through the release of nitric oxide and/or
induction of apoptosis.[44] NO may be converted inside the cell into
reactive nitrogen species (RNS) such as NO2 and N2O3, which targeted
P53 causing cytochrome-c release and caspases activation.[45]
Caspases are a group of vital mediators for apoptosis, but the most
critical one is caspase-3, which can activate death protease leading to
cleavage of many key cellular proteins.[46] 1,2,3-Triazole-substituted
N-alkyl nitrone IV was reported as antiproliferative agent with IC50 of
8.1 μM against PC-3 cancer and also recorded caspase-3 induction.[36]
Also, a disulfonyl derivative of phenyl-tert-butyl nitrone (OKN-007) V
was established as antiproliferaive agent leading to regression of
human glioma U87 through inhibition of caspases.[47] Based on the
above-mentioned promising aspects, the strategy of this work includes
gathering the two bioactive entities quinoline-nitrones or quinoline-
oximes in one compact structure for the purpose of synergism and
examined the prepared compounds as potential antiproliferative
agents. The presence of nitrone and oxime moieties may synergize
the antiproliferative activity of quinoline derivatives. The nitrones and
oximes are the source of NO and the accumulation of NO in cancer
cells subsequently will lead to apoptosis and arrest of cell cycle. The
substituent in position two of quinoline was designed to be with
different electronic and steric properties to investigate their effect
on the antiproliferative activity of these derivatives. The target
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2
RESULTS AND DISCUSSION
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2.1 Chemistry
The intermediates quinoline aldehydes 1a,b, 2a,b, 3a,b were synthe-
sized through reported methods in literature,[48–50] Scheme 1. The
2-alkylthioquinoline-3-carbaldhyde derivatives 4a–h were prepared
by stirring of compounds 1a or 1b with sodium sulfide in DMF, further
alkylation in the same reaction with different alkyl halides afforded the
intermediates 4a–h.[49] The structure of the new derivatives 4d–f was
confirmed by 1H NMR, mass spectroscopy, and elemental analysis.
1H NMR spectra of compounds 4d–f exhibited a common singlet signal
at δ 10.18–10.16 ppm related to −CHO. The aldehydic carbon
appeared at δ 191.9–191.6 ppm in 13C NMR spectrum. Additionally,
a characteristic singlet signal appeared at δ 8.75–8.80 ppm related to
H4 of quinoline. Furthermore, spectrum of compound 4d showed a
singlet signal at δ 4.56 ppm related to benzylic protons, which
confirmed by signal at δ 33.47 ppm in 13C NMR spectrum. Reaction of
2-chloroquinoline-3-carbaldhyde derivatives 1a,b with Na2S in DMF
followed by addition of propyl iodide afforded 2-isopropylquinoline-3-
carbaldhyde 4e,f. It is expected that the propyl iodide undergoes
rearrangement into the more stable isopropyl carbocation. The same
rearrangement occurred in a similar reaction of the propyl bromide in
the presence of mercuric salts.[51] 1H NMR spectra of compounds 4e,f
showed characteristic isopropyl pattern as a doublet at δ 1.43 and
1.42 ppm, and a septet at δ 4.18–4.25 ppm. 13C NMR spectrum of
compound 4e exhibited the characteristic signals for the isopropyl.

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SCHEME 1 Synthesis of quinoline-3-carbaldhydes derivatives 2a,b, 3a,b, and 4a–h
1
These results are an evidence that this reaction occurs through SN
mechanism and formation of the most stable secondary carbocation to
form compounds 4e,f.
corresponding quinoline nitrones 6a–h using a procedure for
synthesis of similar nitrones.[34] 1H NMR spectra of nitrones 6a–h
showed a common singlet signal appearing at δ 10.26–10.00 ppm

The nitrones 6a–h were prepared as outlined in Scheme 2.
Reduction of nitrobenzene using ammonium chloride and zinc dust in
hot water afforded phenyl hydroxylamine 5.[52] Heating of the
appropriate quinoline-3-carbaldhyde 2a,b, 3a,b, 4c,d,g,h with
phenyl hydroxylamine 5 at 60°C in absolute ethanol afforded the
related to −N CH proton of the formed nitrone moiety. The
13C NMR spectra of nitrone derivatives 6a–h showed disappearance

of aldehydic carbonyl carbon and appearance of CH N carbon.
Also, the signals of the aromatic carbons appeared at their expected
chemical shifts.
SCHEME 2 Synthesis of quinoline nitrones 6a–h

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SCHEME 3 Synthesis of quinoline oximes 7a–l
The oxime derivatives 7a–l were prepared as outlined in Scheme 3.
Stirring at room temperature of quinoline aldhydes 2a,b, 3a,b, 4a–h
and hydroxylamine hydrochloride in ethanol using catalytic amount of
TEA afforded the target quinoline oximes 7a–l in a good yield.[35]
1H NMR spectra of compounds 8a–l showed a common singlet peak
appearing at δ 11.81–11.48 ppm related to −OH proton of oxime and a
common characteristic signal at δ 8.42–8.21 ppm related to −NCH
proton. The allylthio derivatives 7e,f showed the characteristic pattern
of allyl group. Also, the isopropylthio derivatives 7i,j exhibited the
characteristic pattern of isopropylthio group. The 13C NMR spectra of
oxime derivatives 7a–l showed disappearance of aldehydic carbon
released. To quantify amount of NO released, a standard curve was
made by measuring the change in absorbance of various concentration
of standard sodium nitrite solutions treated by the same way. The
results expressed as amount of NO released (mole/mole) are listed in
Table 1. Results revealed that nitrone and oxime containing molecules
are able to release NO at pH 7.4, where the NO-donating nitrones 6a–f
achieved maximum amount of NO released after 4 h. Compounds 6e
and 6f released the highest amount of NO among this group (0.587 and
0.506 mole/mole), respectively. Also, compounds 6c, 6d, 6g, and 6h
released a considerable amount of NO (0.441, 0.414, 0.433, and 0.418,
respectively). On the other hand, the amounts of NO released from
quinoline nitrones 6a–h are more than those released from their
corresponding oximes 7a–l (Figure 2).

and appearance of CH N carbon. Moreover, the aromatic carbons
appeared at their expected chemical shifts. The purity of the newly
prepared compounds was confirmed by elemental analysis; the results
are consistent with the molecular formula of the products. Mass
spectra of the target compounds are consistent with the calculated
ones.
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2.3 Biological evaluation
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2.3.1 Evaluation of in vitro antiproliferative activity
by NCI
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2.2 Measurement of nitric oxide release
The antiproliferative activity of quinolin nitrones 6a–h and quinolin
oximes 7a–l was evaluated according to the protocol of the drug
evaluation branch of the national cancer institute (NCI), Bethesda,
USA, for in vitro anticancer activity (http://www.dtp.nci.nih.gov).
Results for each tested compound were reported as the percentage of
growth of the treated cells when compared to the untreated control
cells (see supplementary data and Table 2).
The amount of NO released from quinoline nitrones and oximes was
measured quantitatively using modified Griess colorimetric
method.[53] This method is used for the indirect determination of
NO through spectrophotometric measurement of its stable decompo-
sition products NO2−. This reaction involves two steps; the first step is
diazotization reaction in which the NO-derived nitrosating agent,
dinitrogen trioxide (N2O3) generated from the auto-oxidation of NO,
reacts with sulfanilamide to produce a diazonium ion. The second step
is the coupling of diazonium ion with N-(1-naphthyl)ethylenediamine
dihydrochloride (NEDD) to form a colorimetric azo product that
absorbs strongly at λmax 546 nm. In order to evaluate thiol-induced NO
generation from the tested compounds, the NO-donating nitrones 6a–
h and oximes 7a–l, were incubated in aqueous phosphate buffer of pH
7.4 in the presence of excess N-acetylcysteine which act as a source of
thiols that are essential for release of NO from nitrones and oximes.
The signal intensity of the dye is proportional to the amount of NO
The quinolin nitrones 6c–h exhibited noteworthy antiproliferative
activity and growth inhibition percentage while the 2-hydroxyl
derivatives 6a,b exhibited weak antiproliferative. Moreover, the
2-methoxy analogue 6c showed complete cell death against leukemia
cancer cell line HL-60 and remarkable growth inhibition activity against
leukemia cancer cell line CCRF-CEM, K-562, MOLT-4, RPMI-8226, SR,
colon cancer cell line HCT-116, HCT-15, SW-62 and breast cancer cell
line MCF-7. Also, the 2-methoxy derivative 6d revealed a remarkable
growth inhibition activity against leukemia cancer cell line K-562 and
colon cancer cell line HCT-116. Moreover, 2-benzylthio derivative 6e

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TABLE 1 Amount of NO released from synthesized compounds 6a–h and 7a–l
Amount of NO released (mole/mole)
Compound
1 h
2 h
3 h
4 h
5 h
6 h
6a
6b
6c
6d
6e
6f
0.117 0.014
0.123 0.029
0.159 0.009
0.189 0.032
0.239 0.007
0.179 0.021
0.210 0.022
0.191 0.027
0.098 0.012
0.134 0.025
0.123 0.027
0.131 0.014
0.141 0.003
0.149 0.010
0.215 0.032
0.171 0.012
0.111 0.011
0.178 0.014
0.177 0.033
0.179 0.022
0.121 0.025
0.118 0.012
0.204 0.010
0.210 0.021
0.311 0.013
0.278 0.026
0.298 0.020
0.274 0.015
0.133 0.034
0.166 0.032
0.147 0.023
0.165 0.019
0.179 0.019
0.206 0.017
0.211 0.014
0.199 0.023
0.153 0.015
0.198 0.006
0.198 0.005
0.201 0.020
0.260 0.028
0.286 0.015
0.318 0.029
0.390 0.023
0.456 0.027
0.344 0.021
0.368 0.039
0.377 0.037
0.172 0.039
0.197 0.029
0.201 0.027
0.187 0.012
0.211 0.015
0.255 0.023
0.233 0.034
0.240 0.026
0.248 0.018
0.206 0.009
0.201 0.019
0.288 0.006
0.301 0.022
0.307 0.015
0.441 0.033
0.414 0.022
0.58 0.034
0.506 0.024
0.433 0.013
0.418 0.014
0.191 0.018
0.230 0.016
0.290 0.044
0.255 0.010
0.277 0.039
0.296 0.025
0.282 0.015
0.273 0.019
0.261 0.019
0.218 0.021
0.232 0.005
0.312 0.029
0.109 0.019
0.166 0.011
0.207 0.039
0.205 0.019
0.246 0.017
0.287 0.023
0.180 0.021
0.212 0.012
0.102 0.033
0.136 0.025
0.202 0.017
0.214 0.022
0.223 0.018
0.208 0.012
0.232 0.036
0.211 0.035
0.101 0.009
0.197 0.018
0.202 0.027
0.189 0.016
0.097 0.020
0.155 0.024
0.113 0.024
0.199 0.016
0.180 0.018
0.212 0.014
0.111 0.015
0.101 0.033
0.084 0.015
0.112 0.023
0.173 0.018
0.189 0.011
0.160 0.021
0.153 0.037
0.186 0.026
0.141 0.013
0.067 0.021
0.176 0.030
0.144 0.008
0.101 0.015
6g
6h
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
showed the most promising results in this series, compound 6e
revealed a complete cell death against melanoma cancer cell line MDA-
MB-435 and ovarian cancer cell line OVCAR-3. A remarkable growth
inhibition activity was recorded for 6e against leukemia cancer cell line
CCRF-CEM, HL-60, MOLT-4, RPMI-8226, non-small cell lung cancer
cell line HOP-62, HOP-92, colon cancer cell line HCT-116, HCT-15,
HT-29, KM-12, SW-620, CNS cancer cell line SF-539, SNB-75, U251,
melanoma cancer cell line LOX IMVI, M-14, SK-MEL-5, UACC-62,
ovarian cancer cell line OVCAR-8, NCI/ADR-RES, renal cancer cell line
SN12C and breast cancer cell line MCF-7. Furthermore, the other
2-benzylthio derivative 6f revealed remarkable growth inhibition
activity against leukemia cancer cell line RPMI-8226, colon cancer cell
line HCT-116, HCT-15, HT-29, SW-620, melanoma cancer cell line
LOX IMVI, MDA-MB-435, ovarian cancer cell line OVCAR-3 and
breast cancer cell line MCF-7. Similarly, the 2-methylthio derivative 6g
showed complete cell death against leukemia cancer cell line
HL-60 and remarkable growth inhibition activity against leukemia
cancer cell line CCRF-CEM, K-562, MOLT-4, RPMI-8226, SR, colon
cancer cell line HCT-116, HCT-15, SW-620, CNS cancer cell line
SF-539, melanoma cancer cell line M-14, ovarian cancer cell line
FIGURE 2 Amount of NO released from nitrone 6e and corresponding oxime 7g

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ABDELBASET ET AL.
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TABLE 2 Growth inhibition percentage of compounds 6c–h
Growth inhibition percentage
Panel
Cell line
CCRF-CEM
HL-60
6c
6d
6e
6f
6g
6h
Leukemia
86.86
118.47
86.52
70.51
85.84
88.52
19.57
35.58
89.82
81.04
30.00
15.98
71.61
65.78
26.97
43.95
59.86
60.44
57.83
26.91
37.17
13.76
62.72
55.12
40.88
71.41
53.40
19.72
66.46
17.25
30.86
60.11
18.25
27.52
84.93
32.80
4.88
88.84
85.88
59.63
72.79
84.89
Nd
64.95
28.77
52.31
46.29
79.87
Nd
89.45
113.36
84.92
88.03
85.48
87.95
32.13
46.79
89.61
81.33
19.84
−0.47
81.14
80.25
32.72
53.75
61.86
82.39
54.13
47.51
48.53
33.14
60.50
71.49
54.75
75.06
51.10
29.69
69.12
50.62
44.24
Nd
K-562
MOLT-4
RPMI-8226
SR
Non-small lung cancer
Colon cancer
HOP-62
HOP-92
HCT-116
HCT-15
HT-29
74.46
80.88
90.93
88.52
90.00
74.98
77.34
98.62
88.58
87.33
81.03
93.09
124.09
82.43
68.69
112.04
87.65
92.11
73.90
88.24
33.79
38.66
85.85
71.74
83.88
59.79
77.14
22.64
45.26
57.67
69.07
70.78
70.78
20.27
43.62
68.49
64.34
56.74
48.36
69.32
34.22
53.25
85.19
44.26
47.93
28.43
71.63
31.28
37.18
27.37
48.88
25.05
22.26
5.13
KM-12
9.5
SW-620
SF-539
50.13
10.20
20.45
4.55
CNS cancer
Melanoma
SNB-75
U 251
LOX IMVI
M 14
33.33
13.22
3.10
MDA-MB-435
SK-MEL-5
UACC-62
OVCAR-3
OVCAR-8
NCI/ADR-RES
SN12C
3.82
23.36
3.44
45.26
36.72
22.43
23.88
51.39
42.40
Ovarian cancer
7.36
18.87
9.86
Renal cancer
Breast cancer
MCF7
41.41
Nd, not detected.
NCI/ADR-RES and breast cancer cell line MCF-7. In addition,
compound 6h exhibited remarkable growth inhibition activity against
leukemia cancer cell line K562, colon cancer cell line HCT-116 and
SW-620. On the other hand, all the screened quinolone oximes 7a–l
showed moderate to weak antiproliferative activity on all tested
cancer cell lines. Also, leukemia, colon cancer cell lines HCT-116,
HCT-15, HT-29, KM-12, SW-620 and breast cancer cell line MCF7 are
the most affected types by tested nitrones (see the Supporting
Information).
greater than the corresponding methylthio analogue. Moreover,
results indicated that substitution of quinoline ring in position two
with hydroxyl group can drastically decrease the antiproliferative
activity of quinoline nitrones.
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2.3.2 Cytotoxic activity using MTT assay and
evaluation of IC50
According to the above-mentioned results, it is obvious that
quinoline nitrones exhibited higher antiproliferative activity than
corresponding quinoline oximes. Moreover, the substitution of
quinoline nitrones at position two with benzylthio or methylthio
groups increases the antiproliferative activity greatly. On the other
hand, anticancer activity will be abolished when the position two of
quinoline is occupied by hydroxyl group. It is obvious that the presence
of 2-benzylthio derivatives can increase the antiproliferative activity
Cell viability assay
Cell viability assay was carried out for selected most active quinoline
nitrones 6c, 6e–g using human mammary gland epithelial cell line
(MCF-10A). Compounds were treated with MCF-10A cells for 4 days
and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) assay was applied to evaluate the viability of cells. Tested
compounds were revealed to be non-toxic where exhibiting more than
91% MCF-10A cell viability.

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TABLE 3 IC50 values of compounds 6c, 6e–g, and doxorubicin
against RPMI-8226 and HCT-116 cell lines
Leukemia cell RPMI-8226 was treated with compounds 6c, 6e–g
and doxorubicin (0.81, 0.45, 0.79, 1.35, and 0.91 μM), respectively,
then cytochrome c and caspase-3 concentrations were evaluated.
Results in Table 4 indicated that a remarkable overexpression of
both cytochrome c and caspase-3 protein levels were observed in
treated cells. The overexpression of caspase-3 by 2-benzylthioqui-
noline-nitrones 6e and 6f were 577.9 and 626.9 pg/mL higher than
that of doxorubicin 565.1 pg/mL. On the other hand, the methoxy
IC50 (μM) SEM
Compound number
Against RPMI-8226
0.81 0.11
Against HCT-116
1.32 0.05
2.10 0.12
2.98 0.17
0.98 0.05
0.41 0.03
6c
6e
0.45 0.03
6f
0.79 0.06
6g
1.35 0.08
quinoline–nitrone 6c and methylthio derivative 6g showed
a
Doxorubicin
0.91 0.1
comparable induction of caspase-3 (538.8 and 513 pg/mL, respec-
tively) compared to doxorubicin, but less than that obtained by
benzylthio derivatives 6e and 6f. Also, overexpression of cyto-
chrome c by 2-benzylthioquinoline-nitrones 6e was 0.6665 pg/mL
higher than that of doxorubicin 0.6042 pg/mL. The most active
compound 6e causes overexpression of both cytochrome c and
caspase-3 protein levels in leukemia cell RPMI-8226 about 12- and
13-fold, respectively, higher than control. So, the above results may
be considered as a suggestion that apoptosis may be attributed to
over-expression of both cytochrome c and caspase-3 induced by the
tested compounds.
Evaluation of IC50
The most active compounds from NCI results, compounds 6c, 6e–g
were selected to evaluate cytotoxicity using MTT assay and IC50 value
was evaluated for each compound against RPMI-8226 leukemia and
HCT-116 colon cancer cell lines. Doxorubicin was used as a reference
drug and all of IC50 values in micromolar (μM) are listed in Table 3.
The results in Table 3 showed that all of the tested compounds
showed potent antiproliferative activity against RPMI-8226 leukemia
cancer cell line compared to doxorubicin. Compounds 6c, 6e, and 6f
exhibited remarkable antiproliferative activity with IC50 values of 0.81,
0.45, and 0.79 μM, respectively. Moreover, compound 6g showed a
comparable activity to doxorubicin with IC50 value of 1.35 μM. These
results indicated that 2-benzylthioquinoline-nitrones 6e and 6f
exhibited the highest activity among the tested compounds and the
lowest IC50 values rather than 2-methoxy and 2-methylthioquinoline
nitrones 6c and 6g, respectively. Concerning HCT-116 colon cancer
cell line, all of the tested compounds showed a comparable activity to
doxorubicin with IC50 values of 1.32, 2.10, 2.98, and 0.98 μM,
respectively. Also, the 2-methylthioquinoline-nitrone 6g exhibited the
highest antiproliferative activity among all tested compounds against
colon cancer with IC50 of 0.98 μM.
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2.3.4 Cell cycle analysis and apoptosis
Cell cycle analysis was performed for the most active compound 6e
(0.45 and 0.90 μM) and doxorubicin (0.91 μM) as standard drug against
leukemia RPMI-8226 cell line. In accordance of IC50 concentration
(0.45 μM), the percentages of cells of RPMI-8226 cell in G0/G1 phase
of the cell cycle in the control was 46.89% which recorded a
noteworthy decrease to 23.59 and 25.18% upon treatment with
compound 6e and doxorubicin, respectively (Figures 3 and 5). These
percentages were markedly increased at the G2/M phase to 44.13%
and 44.06% for compound 6e and doxorubicin, respectively, due to
accumulation of cells at this phase. Furthermore, it is obvious that the
apoptotic cell percentage was increased from 2.34% for control
untreated leukemia RPMI-8226 cell to 18.64% and 22.17% in treated
cells with compound 6e and doxorubicin, respectively (Figures 3 and
5). The results indicated that the percentage of the late apoptosis is
more than that of early apoptosis which a good evidence for
irreversible apoptosis caused by compounds 6e (Figures 4 and 5). In
accordance of IC50 concentration duplicate (0.90 μM), the same results
|
2.3.3 Apoptotic proteins activation assay
Cytochrome c and caspases concentration in the cell can be used as
valuable markers for apoptosis induction. Quinoline–nitrones 6c, 6e–g
were evaluated as cytochrome c and caspase-3 activators against
RPMI-8226 leukemia cancer cell line and the results are listed in Table 4.
TABLE 4 Cytochrome c and caspase-3 levels for compounds 6c, 6e–g, doxorubicin, and control untreated leukemia RPMI-8226 cell
Cytochrome c
Caspase-3
Compound number
Conc (pg/mL)
0.4435
Fold change
4.30
Conc (pg/mL)
538.8
Fold change
11.60707
12.44938
13.50495
11.05127
12.17363
1.0000
6c
6e
0.6665
6.47
577.9
6f
0.4769
4.63
626.9
6g
0.5051
4.98
513
Doxorubicin
Cont
0.6042
5.86
565.1
0.1035
1.00
46.42

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FIGURE 3 Cell cycle analysis on RPMI-8226 treated with compound 6e and doxorubicin
in cell cycle phases were observed which reflect that changes occurred
during different phases are dose dependent.
NO released after 4 h. In general, nitrones can release larger amount
than their corresponding oximes. Antiproliferative activity screening of
the target compounds showed that the nitrone derivatives are more
active than the oxime analogues. The quinolin nitrones 6c–h exhibited
promising antiproliferative activity and growth inhibition percentage
against most of the tested cell lines while the quinoline oximes 7a–l
exhibit weak activity. The IC50 was determined for compounds 6c,
6e–g against RPMI-8226 leukemia and HCT-116 colon cancer cell
lines. Compounds 6c, 6e, 6f exhibited more potency than standard
drug doxorubicin against RPMI-8226 leukemia cancer cell line with
IC50 value of 0.81, 0.45, 0.79, and 0.91 μM, respectively. Moreover, all
of the tested compounds showed a comparable activity to doxorubicin
with IC50 value of 1.32, 2.10, 2.98, and 0.98 μM, respectively, against
HCT-116 colon cancer cell line. Furthermore, the quinoline nitrones
6c, 6e–g were evaluated as caspase-3 activators against RPMI-8226
leukemia cancer cell line where a remarkable overexpression of
caspase-3 protein level was observed in treated cells. Also, compound
According to the above results, it is clear that the compound 6e
exhibited pre-G1 apoptosis and cell cycle arrest at G2/M phase.
Moreover, it is obvious that the tested compounds are not cytotoxic
but antiproliferative causing programmed cell death and cell cycle
arrest.
|
3
CONCLUSIONS
Two nitric oxide donating series, quinolone nitrones and quinoline
oxime derivatives were prepared and identified by different spectro-
scopic techniques including 1H NMR, 13C NMR, mass spectrometry,
and elemental microanalysis. Nitric oxide release measurements using
modified Griess colorimetric method revealed that the target nitrone
and oxime are able to release NO at pH 7.4 with maximum amount of
FIGURE 4 Percentage of apoptosis and necrosis for compounds 6e and doxorubicin on RPMI-8226 cells

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FIGURE 5 Cell cycle analysis and apoptosis induction analysis using Annexin V/PI of compound 6e, doxorubicin and control untreated
RPMI8226 cell
|
6e exhibited pre-G1 apoptosis and cell cycle arrest at G2/M phase
{apoptosis is occurred in pre-G1 phase and the proportion of cells (cell
accumulation) is the highest in G2/M phase than other phases}. It is
obvious that there is a direct relationship between nitric oxide release,
potency as antiproliferative agents and caspase-3 protein overpro-
duction. The nitric oxide donating 2-benzylthioquinoline nitrones 6e
and 6f; the 2-methylthioquinoline nitrones 6g and 6h represent novel
highly potent antiproliferative agents that require further in vivo
studies.
4
EXPERIMENTAL
|
4.1 Chemistry
|
4.1.1 General
Chemicals and solvents used in the preparation of the target
compounds are of commercial grade and purchased from Sigma–
Aldrich, Alfa Aesar, Merck, Fluka and El-Nasr pharmaceutical chemicals

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ABDELBASET ET AL.
|
companies. Chemicals and solvents were used without purification.
Thin layer chromatography (TLC) was used for monitoring chemical
reactions and was carried out using silica gel 60 F254 precoated sheet
20 × 20 cm, layer thickening 0.2 mm (E, Merck, Germany), and were
visualized using UV-lamp at 254 nm. Melting points (mp) were
determined using Stuart electrochemical melting point apparatus
(Stuart Scientific, England) and were uncorrected. 1H NMR spectra
were carried out on 400 MHz Bruker spectrometer, using TMS as an
internal reference. Chemical shift (δ) values are given in parts per
million (ppm) relative to DMSO-d6 (2.5) and coupling constants (J) in
Hertz (Hz). Splitting patterns are designated as follows: s, singlet; d,
doublet; t, triplet; q, quartet; dd, doublet of doublet; m, multiplet.
13C NMR spectra were carried out on Bruker spectrometer (100 MHz).
Shimadzu GC MS and LC-MS/MS Agilent Technologies, 6420
TripleQuad apparatus, Faculty of Pharmacy, Minia University, were
used for mass spectroscopy. Vario EL III German CHN Elemental
analyzer model was used for elemental analysis. The intermediate
compounds were prepared as reported: 1a,b,[49] 2a,b,[48,54] 3a,b,[50]
and 5.[52]
MS: m/z calcd.: 231.07, found: 231.93. Anal. calcd. for C13H13NOS
(231.07): C, 67.50; H, 5.66; N, 6.06. Found: C, 67.41; H, 5.89; N, 6.23.
2-(Isopropylthio)-6-methylquinoline-3-carbaldehyde 4f
Yellowish white powder; yield: 71%; mp: 98–99°C, 1H NMR (400 MHz,
DMSO-d6) δ: 10.17 (s, 1H, CHO), 8.75 (s, 1H, Ar-H), 7.84–7.73 (m, 3H,
Ar-H), 4.25–4.24. (m, 1H, −CH(CH3)2), 2.49(s, 3H, −CH3), 1.42 (d,
J = 6.8 Hz, 6H, −CH(CH3)2). 13C NMR (101 MHz, DMSO-d6) δ: 191.6,
157.7, 147.8, 144.5, 136.4, 135.9, 128.7, 127.6, 127.5, 124.6, 33.9,
23.1, 21.4. GC MS: m/z calcd.: 245.09, found: 245.80. Anal. calcd. for
C14H15NOS (245.09): C, 68.54; H, 6.16; N, 5.71. Found: C, 68.76; H,
6.34; N, 5.78.
|
4.1.3 General procedure for the synthesis of
quinoline nitrones 6a–h
A mixture of substituted quinoline aldhydes 2a,b, 3a,b, 4c,d,g,h
(0.001 mole) and phenylhydroxylamine 5 (0.001 mol) in absolute
ethanol (5 mL) was heated in water bath at 60°C for 6 h. After cooling
the formed precipitate was filtered off, washed, dried, and crystallized
from methanol to give the corresponding nitrones[34] 6a–h.
The InChI codes of the investigated compounds together with
some biological activity data are provided as Supporting Information.
N-((2-Hydroxyquinolin-3-yl)methylene)aniline oxide 6a
Yellow powder; yield: 79%; mp: 115–117°C, 1H NMR (400 MHz,
DMSO-d6) δ: 12.17 (s, 1H, OH), 10.06 (s, 1H, NCH), 8.49 (s, 1H, Ar-H),
7.88 (dd, J = 8.0, J = 4.0 Hz, 2H, Ar-H), 7.80 (d, J = 8.4 Hz, 1H, Ar-H),
7.59–7.55 (m, 4H, Ar-H), 7.33 (d, J = 8.0 Hz, 1H, Ar-H), 7.25–7.21 (m,
1H, Ar-H). 13C NMR (101 MHz, DMSO-d6) δ: 160.9, 149.0, 139.2,
136.9, 132.2, 130.6, 129.9, 129.8, 128.2, 123.1, 122.8, 121.7, 119.6,
115.6. GC MS: m/z calcd.: 264.09, found [M+H]+: 265.01. Anal. calcd.
for C16H12N2O2 (264.09): C, 72.72; H, 4.58; N, 10.60. Found: C, 72.89;
H, 4.72; N, 10.48.
|
4.1.2 General procedure for the synthesis of
2-alkylthio-6-substituted-quinoline-3-carbaldehyde
4a–h
To a solution of compounds 1a,b (0.01 mol) in DMF (10 mL), sodium
sulfide (1.7 g, 0.015 mole) was added and stirred overnight at room
temperature (monitored by TLC). After the reaction completion, alkyl
halide (0.01 mol) was added and stirred for further 1 h. The reaction
mixture was poured into crushed ice and the precipitated solid
obtained was filtered, washed with water, dried, and crystallized from
methanol/water. Structures of compounds 4a,[55] 4b,[56] 4c,[49] 4g,[49]
and 4h[56] were confirmed by melting point as reported in literature.
N-((2-Hydroxy-6-methylquinolin-3-yl)methylene)aniline
oxide 6b
2-(Benzylthio)-6-methylquinoline-3-carbaldehyde 4d
Yellow powder; yield: 77%; mp: 111–113°C, 1H NMR (400 MHz,
Yellowish white powder; yield: 85%; mp: 107–109°C, 1H NMR
(400 MHz, DMSO-d6) δ: 10.16 (s, 1H, CHO), 8.79 (s, 1H, Ar-H), 7.95–
7.92 (m, 1H, Ar-H), 7.86 (s, 1H, Ar-H), 7.77 (d, J = 8.5 Hz, 1H, Ar-H),
7.52 (d, J = 7.6 Hz, 2H, Ar-H), 7.31–7.29 (m, 2H, Ar-H), 7.23 (d,
J = 7.0 Hz, 1H, Ar-H), 4.56 (s, 2H, −CH2), 2.51(s, 3H, −CH3). 13C NMR
(101 MHz, DMSO-d6) δ: 191.9, 156.9, 147.6, 145.3, 138.6, 136.5,
136.1, 129.8, 128.8, 128.7, 127.7, 127.4, 127.3, 124.8, 33.8, 21.4.
GC MS: m/z calcd.: 293.09, found: 293.23. Anal. calcd. for
DMSO-d6) δ: 12.10 (s, 1H, OH), 10.00 (s, 1H, N CH), 8.48 (s, 1H,

Ar-H), 7.88–7.87 (m, 2H, Ar-H), 7.58–7.57 (m, 4H, Ar-H), 7.41 (d,
J = 8.0 Hz, 1H, Ar-H), 7.24 (d, J = 7.4 Hz, 1H, Ar-H), 2.37 (s, 3H, CH3).
13C NMR (101 MHz, DMSO-d6) δ: 160.8, 149.0, 137.3, 136.7, 133.5,
132.2, 130.6, 129.8, 129.2, 128.3, 122.7, 121.7, 119.5, 115.6, 20.8. GC
MS: m/z calcd.: 278.11, found [M+H]+: 279.13. Anal. calcd. for
C
17H14N2O2 (278.11): C, 73.37; H, 5.07; N, 10.07. Found: 73.68; H,
5.19; N, 10.23.
C
18H15NOS (293.09): C, 73.69; H, 5.15; N, 4.77. Found: C, 73.51;
H, 5.28; N, 4.90.
N-((2-Methoxyquinolin-3-yl)methylene)aniline oxide 6c
Yellow powder; yield: 75%; mp: 99–101°C, 1H NMR (400 MHz,
DMSO-d6) δ: 10.26 (s, 1H, NCH), 8.50 (s, 1H, Ar-H), 8,01 (d, J = 8.0 Hz,
1H, Ar-H), 7.93–7.91 (m, 2H, Ar-H), 7.80 (d, J = 8.0 Hz, 1H, Ar-H),
7.76–7.72 (m, 1H, Ar-H), 7.62–7.57 (m, 3H, Ar-H), 7.51–7.48 (m, 1H,
Ar-H), 4.10 (s, 3H, OCH3). 13C NMR (101 MHz, DMSO-d6) δ: 158.8,
149.2, 146.1, 136.3, 131.7, 130.7, 129.8, 129.6, 127.4, 126.9, 125.5,
125.0, 122.0, 116.0, 54.4. GC MS: m/z calcd.: 278.11, found [M+H]+:
2-(Isopropylthio)quinoline-3-carbaldehyde 4e
Yellowish white powder; yield: 74%; mp: 100–102°C, 1H NMR
(400 MHz, DMSO-d6) δ: 10.49 (s, 1H, CHO), 8.29 (s, 1H, Ar-H), 7.85–
7.11 (m, 4H, Ar-H), 4.41–4.10 (m, 1H, −CH(CH3)2), 1.50 (d, J = 6.8 Hz,
6H, −CH(CH3)2). 13C NMR (101 MHz, DMSO-d6) δ: 191.7, 157.9,
147.5, 144.6, 136.6, 136.0, 129.0, 127.6, 127.1, 124.8, 34.0, 23.1. GC

ABDELBASET ET AL.
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279.00. Anal. calcd. for C17H14N2O2 (278.11): C, 73.37; H, 5.07; N,
10.07. Found: C, 73.60; H, 5.13; N, 10.29.
7.79–7.74 (m, 2H, Ar-H), 7.62–7.59 (m, 4H, Ar-H), 2.69 (s, 3H, SCH3),
2.47 (s, 3H, CH3). 13C NMR (101 MHz, DMSO-d6) δ: 156.9, 149.2,
146.0, 136.2, 133.8, 133.2, 130.9, 129.9, 128.4, 128.0, 127.5, 125.2,
122.9, 122.0, 21.4, 13.3. LC-MS/MS: m/z calcd.: 308.10, found:
308.90. Anal. calcd. for C18H16N2OS (308.10): C, 70.10; H, 5.23; N,
9.08. Found: C, 70.38; H, 5.31; N, 9.31.
N-((2-Methoxy-6-methylquinolin-3-yl)methylene)aniline
oxide 6d
Yellow powder; yield: 71%; mp: 97–99°C, 1H NMR (400 MHz, DMSO-

d6) δ: 10.15 (s, 1H, N CH), 8.45 (s, 1H, Ar-H), 7.91–7.89 (m, 2H, Ar-H),
7.74 (s, 1H, Ar-H), 7.68 (d, J = 8.4 Hz, 1H, Ar-H), 7.59–7.56 (m, 4H, Ar-
H), 4.06 (s, 3H, OCH3), 2.46 (s, 3H, CH3). 13C NMR (101 MHz, DMSO-
d6) δ: 158.4, 149.2, 144.5, 135.7, 134.7, 133.5, 130.7, 129.8, 128.5,
127.4, 126.7, 124.9, 121.8, 115.7, 54.3, 21.1. GC MS: m/z calcd.:
292.12, found [M+H]+: 293.06. Anal. calcd. for C18H16N2O2 (292.12):
C, 73.95; H, 5.52; N, 9.58. Found: C, 74.12; H, 5.70; N, 9.35.
|
4.1.4 General procedure for the synthesis of
quinoline oximes 7a–l
A mixture of appropriate quinoline aldhydes 2a,b, 3a,b, and 4a–h
(0.001 mole), hydroxylamine hydrochloride (0.001 mol) and TEA
(0.002 mol) in absolute ethanol (10 mL) was stirred at room tempera-
ture for 1 h. The reaction mixture was poured into crushed ice and the
formed precipitate was filtered off, washed, dried, and crystallized
from methanol/water to give the corresponding oximes[35] 7a–l.
N-((2-(Benzylthio)quinolin-3-yl)methylene)aniline oxide 6e
Yellow powder; yield: 73%; mp: 100–102°C, 1H NMR (400 MHz,

DMSO-d6) δ: 10.13 (s, 1H, N CH), 8.39 (s, 1H, Ar-H), 8.05 (d,
J = 8.0 Hz, 1H, Ar-H), 7.99 (d, J = 8.0 Hz, 1H, Ar-H), 7.90–7.88 (m, 2H,
Ar-H), 7.84–7.81 (m, 1H, Ar-H), 7.58–7.54 (m, 6H, Ar-H), 7.34–7.30
(m, 2H, Ar-H), 7.26–7.23 (m, 1H, Ar-H), 4.69 (s, 2H, −CH2). 13C NMR
(101 MHz, DMSO-d6) δ: 157.1, 149.2, 147.3, 138.1, 134.1, 132.0,
130.9, 129.9, 129.8, 129.7, 128.9, 127.8, 127.7, 127.6, 126.9, 125.5,
122.8, 122.0, 34.2. GC MS: m/z calcd.: 370.11, found: 370.12. Anal.
calcd. for C23H18N2OS (370.11): C, 74.57; H, 4.90; N, 7.56. Found: C,
74.13; H, 5.24; N, 7.89.
2-Hydroxyquinoline-3-carbaldehyde oxime 7a
Yellowish white powder; yield: 90%; mp: 157–159°C, 1H NMR
(400 MHz, DMSO-d6) δ: 12.02 (s, 1H, OH), 11.50 (s, 1H, OH), 8.28 (s,

1H, N CH), 8.22 (s, 1H, Ar-H), 7.76 (d, J = 8.0 Hz, 1H, Ar-H), 7.53–
7.49 (m, 1H, Ar-H), 7.32 (d, J = 8.0 Hz, 1H, Ar-H), 7.21–7.17 (m, 1H, Ar-
H). 13C NMR (101 MHz, DMSO-d6) δ: 161.1, 143.7, 139.2, 134.7,
131.3, 129.1, 124.6, 122.7, 119.4, 115.5. LC-MS/MS: m/z calcd.:
188.06, found [M+H]+: 189.00. Anal. calcd. for C10H8N2O2 (188.06): C,
63.82; H, 4.28; N, 14.89. Found: C, 63.65; H, 4.35; N, 15.04.
N-((2-(Benzylthio)-6-methylquinolin-3-yl)methylene)aniline
oxide 6f
Yellow powder; yield: 68%; mp: 103–105°C, 1H NMR (400 MHz,
2-Hydroxy-6-methylquinoline-3-carbaldehyde oxime 7b
Yellowish white powder; yield: 87%; mp: 152–154°C, 1H NMR
(400 MHz, DMSO-d6) δ: 11.94 (s, 1H, OH), 11.48 (s, 1H, OH), 8.21 (s,

DMSO-d6) δ: 10.03 (s, 1H, N CH), 8.36 (s, 1H, Ar-H), 7.90–7.87 (m,

3H, Ar-H), 7.79 (s, 1H, Ar-H), 7.60–7.56 (m, 3H, Ar-H), 7.54–7.52 (m,
2H, Ar-H), 7.33–7.22 (m, 4H, Ar-H), 4.66 (s, 2H, −CH2), 2.49 (s, 3H,
−CH3). 13C NMR (101 MHz, DMSO-d6) δ: 156.0, 145.9, 138.1, 136.4,
134.1, 133.6, 130.9, 129.9, 129.8, 128.7, 128.4, 128.0, 127.6, 127.5,
125.4, 122.8, 122.0, 121.5, 34.1, 21.4. m/z GC MS: m/z calcd.: 384.13,
found: 384.09. Anal. calcd. for C24H20N2OS (384.13): C, 74.97; H,
5.24; N, 7.29. Found: C, 74.63; H, 5.38; N, 7.40.
1H, N CH), 8.19 (s, 1H, Ar-H), 7.53 (s, 1H, Ar-H), 7.34 (d, J = 8.4 Hz,
1H, Ar-H), 7.22 (d, J = 8.4 Hz, 1H, Ar-H), 2.33 (s, 3H, CH3). 13C NMR
(101 MHz, DMSO-d6) δ: 161.0, 143.8, 137.3, 134.4, 132.6, 131.7,
128.5, 124.5, 119.4, 115.5, 20.8. LC-MS/MS: m/z calcd.: 202.07,
found [M+H]+: 203.00. Anal. calcd. for C11H10N2O2 (202.07): C, 65.34;
H, 4.98; N, 13.85. Found: C, 65.70; H, 5.12; N, 14.13.
2-Methoxyquinoline-3-carbaldehyde oxime 7c
N-((2-(Methylthio)quinolin-3-yl)methylene)aniline oxide 6g
Yellow powder; yield: 69%; mp: 93–94°C, 1H NMR (400 MHz, DMSO-
Yellowish white powder; yield: 89%; mp: 147–149°C, 1H NMR
(400 MHz, DMSO-d6) δ: 11.63 (s, 1H, OH), 8.52 (s, 1H, NCH), 8.30 (s,
1H, Ar-H), 7.96 (d, J = 8.0 Hz, 1H, Ar-H), 7.78 (d, J = 8.4 Hz, 1H, Ar-H),
7.70–7.66 (m, 1H, Ar-H), 7.46–7.43 (m, 1H, Ar-H), 4.04 (s, 3H, −OCH3).
13C NMR (101 MHz, DMSO-d6) δ: 159.4, 146.3, 143.3, 134.8, 130.8,
128.8, 126.7, 125.2, 125.1, 117.7, 54.1. LC-MS/MS: m/z calcd.:
202.07, found [M+H]+: 203.00. Anal. calcd. for C11H10N2O2 (202.07):
C, 65.34; H, 4.98; N, 13.85. Found: C, 65.11; H, 5.16; N, 14.19.

d6) δ: 10.11 (s, 1H, N CH), 8.42 (s, 1H, Ar-H), 8.03 (d, J = 7.6 Hz, 1H,
Ar-H), 7.92–7.89 (m, 3H, Ar-H), 7.81–7.77 (m, 1H, Ar-H), 7.63–7.60
(m, 3H, Ar-H), 7.58–7.54 (m, 1H, Ar-H), 2.72 (s, 3H, SCH3). 13C NMR
(101 MHz, DMSO-d6) δ: 158.1, 149.2, 147.41, 133.6, 131.9, 131.0,
130.0, 129.7, 127.9, 127.7, 126.7, 125.3, 123.0, 122.0, 13.4. GC MS:
m/z calcd.: 294.08, found [M+H]+: 295.01. Anal. calcd. for
C17H14N2OS (294.08): C, 69.36; H, 4.79; N, 9.52. Found: C, 69.02;
H, 4.56; N, 9.67.
2-Methoxy-6-methylquinoline-3-carbaldehyde oxime 7d
Yellowish white powder; yield: 88%; mp: 154–156°C, 1H NMR

N-((6-Methyl-2-(methylthio)quinolin-3-yl)methylene)-
anilineoxide 6h
Yellow powder; yield: 66%; mp: 90–92°C, 1H NMR (400 MHz, DMSO-
(400 MHz, DMSO-d6) δ: 11.60 (s, 1H, OH), 8.39 (s, 1H, N CH), 8.28 (s,
1H, Ar-H), 7.68–7.65 (m, 2H, Ar-H), 7.49 (d, J = 8.8 Hz, 1H, Ar-H), 4.01
(s, 3H, −OCH3), 2.43 (s, 3H, −CH3). 13C NMR (101 MHz, DMSO-d6) δ:
158.9, 144.7, 143.4, 134.3, 134.2, 132.7, 127.6, 126.8, 125.0, 117.6,
d6) δ: 10.00 (s, 1H, NCH), 8.37 (s, 1H, Ar-H), 7.90–7.88 (m, 2H, Ar-H),

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ABDELBASET ET AL.
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54.0, 21.3. LC-MS/MS: m/z calcd.: 216.09, found [M+H]+: 217.10.
Anal. calcd. for C12H12N2O2 (216.09): C, 66.65; H, 5.59; N, 12.96.
Found: C, 66.91; H, 5.70; N, 13.24.
1H, Ar-H), 7.96 (d, J = 8.0 Hz, 1H, Ar-H), 7.86 (d, J = 8.4 Hz, 1H, Ar-H),
7.75–7.71 (m, 1H, Ar-H), 7.54–7.50 (m, 1H, Ar-H), 4.27–4.20 (m, 1H,
−CH−(CH3)2), 1.43 (d, J = 6.8 Hz, 6H, −CH−(CH3)2). 13C NMR
(101 MHz, DMSO-d6) δ: 157.6, 147.6, 145.0, 134.2, 131.1, 128.9,
127.7, 126.3, 125.4, 125.3, 35.1, 23.2. LC-MS/MS: m/z calcd.: 246.08,
found [M+H]+: 247.00. Anal. calcd. for C13H14N2OS (246.08): C, 63.39;
H, 5.73; N, 11.37. Found: C, 63.04; H, 5.51; N, 11.58.
2-(Allylthio)quinoline-3-carbaldehyde oxime 7e
Yellowish white powder; yield: 86%; mp: 143–145°C, 1H NMR

(400 MHz, DMSO-d6) δ: 11.80 (s, 1H, OH), 8.42 (s, 1H, N CH), 8.37
(s, 1H, Ar-H), 7.97 (d, J = 8.0 Hz, 1H, Ar-H), 7.89 (d, J = 8.4 Hz, 1H, Ar-H),
7.76–7.72 (m, 1H, Ar-H), 7.53 (t, J = 7.8 Hz, 1H, Ar-H), 6.03–5.96 (m, 1H,
2-(Isopropylthio)-6-methylquinoline-3-carbaldehyde oxime 7j
Yellowish white powder; yield: 83%; mp: 139–141°C, 1H NMR

−CH2−CHCH2), 5.39 (d, J = 16.8 Hz, 1H, −CH2−CH CH2), 5.13 (d,

J = 10.0 Hz, 1H, −CH2−CHCH2), 4.03 (d, J = 7.2 Hz, 2H, −CH2−CHCH2).
13C NMR (101 MHz, DMSO-d6) δ: 156.6, 147.3, 145.2, 134.8, 134.3,
131.2, 128.8, 127.7, 126.5, 125.6, 125.2, 118.6, 32.8. LC-MS/MS: m/z
calcd.: 244.07, found: 244.90. Anal. calcd. for C13H12N2OS (244.07): C,
63.91; H, 4.95; N, 11.47. Found: C, 64.24; H, 5.12; N, 11.80.
(400 MHz, DMSO-d6) δ: 11.74 (s, 1H, OH), 8.31 (s, 1H, N CH), 8.30 (s,
1H, Ar-H), 7.76 (d, J = 8.8 Hz, 1H, Ar-H), 7.71 (s, 1H, Ar-H), 7.57–7.52
(m, 1H, Ar-H), 4.25–4.18 (m, 1H, −CH−(CH3)2), 2.46 (s, 3H, CH3), 1.42
(d, J = 7.8 Hz, 6H, −CH−(CH3)2). 13C NMR (101 MHz, DMSO-d6) δ:
156.4, 146.29, 145.1, 135.8, 133.6, 133.1, 127.5, 125.4, 125.2, 35.0,
23.2, 21.4. LC-MS/MS: m/z calcd.: 260.10, found [M+H]+: 261.00.
Anal. calcd. for C14H16N2OS (260.10): C, 64.58; H, 6.19; N, 10.76.
Found: C, 64.51; H, 6.40; N, 10.89.
2-(Allylthio)-6-methylquinoline-3-carbaldehyde oxime 7f
Yellowish white powder; yield: 84%; mp: 144–146°C, 1H NMR

(400 MHz, DMSO-d6) δ: 11.77 (s, 1H, OH), 8.35 (s, 1H, N CH), 8.30
(s, 1H, Ar-H), 7.78 (d, J = 8.4 Hz, 1H, Ar-H), 7.70 (s, 1H, Ar-H), 7.56 (dd,
2-(Methylthio)quinoline-3-carbaldehyde oxime 7k
J = 8.6, J = 4.0 Hz, 1H, Ar-H), 6.02–5.97 (m, 1H, −CH2−CH CH2), 5.39
Yellowish white powder; yield: 86%; mp: 137–139°C, 1H NMR


(d, J = 16.8 Hz, 1H, −CH2−CHCH2), 5.12 (d, J = 10.0 Hz, 1H,
(400 MHz, DMSO-d6) δ: 11.81 (s, 1H, OH), 8.40 (s, 1H, N CH), 8.37 (s,


−CH2−CH CH2), 4.01 (d, J = 7.2 Hz, 2H, −CH2−CH CH2), 2.46 (s,
3H, CH3). 13C NMR (101 MHz, DMSO-d6) δ: 155.4, 146.0, 145.2, 135.9,
134.4, 134.1, 133.18, 127.5, 127.5, 125.5, 125.2, 118.5, 32.8, 21.4. LC-
MS/MS: m/z calcd.: 258.08, found: 258.90. Anal. calcd. for C14H14N2OS
(258.08): C, 65.09; H, 5.46; N, 10.84. Found: C, 64.89; H, 5.31; N, 10.97.
1H, Ar-H), 7.96 (d, J = 8.0 Hz, 1H, Ar-H), 7.89 (d, J = 8.4 Hz, 1H, Ar-H),
7.75–7.71 (m, 1H, Ar-H), 7.53–7.50 (m, 1H, Ar-H), 2.65 (s, 3H, SCH3).
13C NMR (101 MHz, DMSO-d6) δ: 157.8, 147.5, 145.2, 134.4, 131.1,
128.8, 127.7, 126.3, 125.4, 125.2, 13.4. LC-MS/MS: m/z calcd.:
218.05, found: 218.90. Anal. calcd. for C11H10N2OS (218.05): C,
60.53; H, 4.62; N, 12.83. Found: C, 60.17; H, 4.88; N, 13.01.
2-(Benzylthio)quinoline-3-carbaldehyde oxime 7g
Yellowish white powder; yield: 85%; mp: 161–163°C, 1H NMR
6-Methyl-2-(methylthio)quinoline-3-carbaldehyde oxime 7l
(400 MHz, DMSO-d6) δ: 11.78 (s, 1H, OH), 8.42 (s, 1H, N CH), 8.35
Yellowish white powder; yield: 87%; mp: 131–133°C, 1H NMR


(s, 1H, Ar-H), 7.97 (d, J = 7.6 Hz, 2H, Ar-H), 7.79–7.75 (m, 1H, Ar-H),
7.56–7.51 (m, 3H, Ar-H), 7.35–7.19 (m, 3H, Ar-H), 4.60 (s, 2H, CH2).
13C NMR (101 MHz, DMSO-d6) δ: 156.8, 147.2, 145.4, 138.5, 135.2,
131.2, 129.8, 128.8, 127.7, 127.4, 126.5, 125.6, 125.0, 34.2. GC MS:
m/z calcd.: 294.08, found [M+H]+: 295.07. Anal. calcd. for C17H14N2OS
(294.08): C, 69.36; H, 4.79; N, 9.52. Found: C, 69.12; H, 4.95; N, 9.84.
(400 MHz, DMSO-d6) δ: 11.77 (s, 1H, OH), 8.35 (s, 1H, −CH N), 8.28
(s, 1H, Ar-H), 7.78 (d, J = 8.8 Hz, 1H, Ar-H), 7.70 (s, 1H, Ar-H), 7.57–
7.55 (m, 1H, Ar-H), 2.63 (s, 3H, SCH3), 2.46 (s, 3H, CH3). 13C NMR
(101 MHz, DMSO-d6) δ: 156.6, 146.1, 145.2, 135.7, 133.7, 133.1,
127.5, 125.4, 125.1, 21.4, 13.4. GC MS: m/z calcd.: 232.07, Found:
232.10. Anal. calcd. for C12H12N2OS (232.07): C, 62.04; H, 5.59; N,
12.96. Found: C, 61.87; H, 5.70; N, 13.24.
2-(Benzylthio)-6-methylquinoline-3-carbaldehyde oxime 7h
Yellowish white powder; yield: 81%; mp: 164–166°C, 1H NMR
|
4.2 Nitric oxide release measurement

(400 MHz, DMSO-d6) δ: 11.75 (s, 1H, OH), 8.33 (s, 1H, N CH), 8.30 (s,
1H, Ar-H), 7.86 (d, J = 8.4 Hz, 1H, Ar-H), 7.70 (s, 1H, Ar-H), 7.59 (d,
J = 8.4 Hz, 1H, Ar-H), 7.51–7.49 (m, 2H, Ar-H), 7.32–7.28 (m, 2H, Ar-
H), 7.24–7.20 (m, 1H, Ar-H), 4.57 (s, 2H, CH2), 2.47 (s, 3H, CH3).
13C NMR (101 MHz, DMSO-d6) δ: 155.7, 145.9, 145.5, 138.5, 135.9,
134.5, 133.3, 129.8, 128.8, 127.5, 127.5, 127.4, 125.6, 125.0, 34.2,
21.4. GC MS: m/z calcd.: 308.10, found [M+H]+: 309.01. Anal. calcd.
for C18H16N2OS (308.10): C, 70.10; H, 5.23; N, 9.08. Found: C, 70.48;
H, 5.16; N, 9.19.
The nitrite calibration curve in addition to the absorbance of the tested
compounds were measured on Spectronic Genesys UV-VIS spectro-
photometer connected to an IBM computer loaded with the Winspec
application software (Milton Roy, USA) (see Appendix A, Supporting
Information).
|
4.3 Biological screening
|
4.3.1 The NCI-60 anticancer drug screen
2-(Isopropylthio)quinoline-3-carbaldehyde oxime 7i
Yellowish white powder; yield: 82%; mp: 141–143°C, 1H NMR
The methodology of the NCI for primary anticancer assay was
performed at 60 human tumor cell lines panel derived from nine

(400 MHz, DMSO-d6) δ: 11.76 (s, 1H, OH), 8.41 (s, 1H, N CH), 8.33 (s,

ABDELBASET ET AL.
13 of 14
|
neoplastic diseases, according to the protocol of the Drug Evaluation
Branch, National Cancer Institute, Bethesda (see Appendix A,
Supporting Information).
ORCID
Gamal El-Din A. Abuo-Rahma
http://orcid.org/0000-0003-3908-
1832
Bahaa G. M. Youssif
http://orcid.org/0000-0002-6834-6548
|
4.3.2 Cell culture protocol
Cell line cells were obtained from American Type Culture Collection,
cells was cultured using DMEM (Invitrogen/Life Technologies)
supplemented with 10% FBS (Hyclone), 10 μg/mL of insulin (Sigma),
and 1% penicillin–streptomycin. All of the other chemicals and
reagents were from Sigma or Invitrogen (see Appendix A, Supporting
Information).
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How to cite this article: Abdelbaset MS, Abdel-Aziz M,
Abuo-Rahma GEA, Abdelrahman MH, Ramadan M, Youssif
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