Design, synthesis, and docking study of new quinoline derivatives as antitumor agents

Article abstract of DOI:10.1002/ardp.201800355

New quinolines substituted with various heterocycles and chalcone moieties were synthesized and evaluated as antitumor agents. All the synthesized compounds were in vitro screened against 60 human cancer cell lines. Compound 13 showed the highest cytotoxi

Full text of DOI:10.1002/ardp.201800355

Received: 5 December 2018
Revised: 1 April 2019
Accepted: 3 April 2019
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DOI: 10.1002/ardp.201800355
F U L L P A P E R
Design, synthesis, and docking study of new quinoline
derivatives as antitumor agents
Eman E. Nasr1
Mohammed A. M. Massoud1
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Amany S. Mostafa1
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Magda A. A. El‐Sayed1,2
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1Pharmaceutical Organic Chemistry
Department, Faculty of Pharmacy, Mansoura
University, Mansoura, Egypt
Abstract
New quinolines substituted with various heterocycles and chalcone moieties were
synthesized and evaluated as antitumor agents. All the synthesized compounds were
in vitro screened against 60 human cancer cell lines. Compound 13 showed the
highest cytotoxicity toward 58 cell lines, exhibiting distinct growth inhibition values
(GI50) against the majority of them, including SR, HL‐60 (TB) strains (leukemia), and
MDA‐MB‐435 strains (melanoma), with GI50 values of 0.232, 0.260, and 0.300 µM,
respectively. It exhibited great selectivity toward cancer cell lines, with less toxic
effect against normal cells represented by skin fibroblast (BJ) and breast epithelial
cell lines (MCF‐10F). The enzyme inhibitory activity of compound 13 was evaluated
against topoisomerase 1 (Topo 1), epidermal growth factor receptor and vascular
endothelial growth factor receptor 2, where it displayed worthy Topo 1 inhibition
activity with an IC50 value of 0.278 µM compared with camptothecin as a reference
drug (IC50 0.224 µM). Docking studies were performed to investigate the recognition
profile of compound 13 with the Topo 1 enzyme binding site.
2Pharmaceutical Chemistry Department,
Faculty of Pharmacy, Horus University, New
Damietta, Egypt
Correspondence
Mohammed A. M. Massoud, Pharmaceutical
Organic Chemistry Department, Faculty of
Pharmacy, Mansoura University, Mansoura
35516, Egypt.
Email: massoudmam@yahoo.fr
K E Y W O R D S
antitumor activity, chalcones, docking, EGFR, quinolone, Topo 1, VEGFR2
compounds as bosutinib (B) (MDA‐MB‐231: IC50 = 0.25 µM),[11]
lenvatinib,[12,13] cabozantinib,[14] topotecan,[15] and irinotecan.[16]
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1
INTRODUCTION
Quinoline is an important ring system, found in many natural and
synthetic products that have diversity of applications.[1–3] Many
derivatives have various pharmacological activities, the majority of
which having antimalarial,[4] antibacterial,[5] antifungal,[6] anti‐inflam-
matory,[7] and anticancer activities.[8]
In addition, many heterocycle‐containing compounds proved to
have considerable anticancer activity. They may contain tetrazolo-
quinoline (C) (HL‐60: 70% inhibition at 100 µM),[17] tetrazole (D)
(Hep‐G2: IC50 = 2.07 µM) and (E) (Hep‐G2: IC50 = 1.65 µM),[18]
pyrazoloquinoline (F) (HCT‐116: IC50 = 2.3 µM),[19] or naphthopyran
nucleus in their structure (G) (PC‐3: IC50 = 43.6 µM)[20] and (H) (BT‐
20: 34% inhibition at 50 µM).[21] Besides, several chalcone derivatives
were well established to show antiproliferative activities (I) (MDA‐
MB‐468: IC50 = 0.12 µM)[22] (Figure 1).
The quinoline scaffold has a significant contribution in anticancer
drug development; since many derivatives had shown magnificent results
throughout different mechanisms of action varying between inhibition of
angiogenesis, apoptosis, disruption of cell migration, growth inhibition (GI)
by cell cycle arrest, or modulation of nuclear receptor responsiveness.[8,9]
Literature surveys have revealed the importance of quinoline
derivatives as potent anticancer agents, either from natural source
like camptothecin (A) (MCF7: IC50 = 0.23 µM),[10] or synthetic
Guided by these findings, and in a trial to develop new anticancer
therapeutic agents, we were encouraged to incorporate quinoline
moiety as a main scaffold with various heterocycles and chalcone
moieties to form new hybrid molecules.
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Arch Pharm Chem Life Sci. 2019;e1800355.
wileyonlinelibrary.com/journal/ardp © 2019 Deutsche Pharmazeutische Gesellschaft
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https://doi.org/10.1002/ardp.201800355

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FIGURE 1 Quinoline (a,b), tetrazoloquinolines (c), tetrazole (d,e), pyrazoloquinoline (f), naphthopyrane (g,h), and chalcone (i) containing
compounds with reported anticancer activity
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1.1
Rationale of the molecular design
at C3 of quinoline ring to afford the target hybrids. Furthermore,
quinoline nucleus at C2 and C3 was hybridized with pyrazole ring to
afford fused 1H‐pyrazolo[3,4‐b]quinoline nucleus substituted with
amino group which is easily converted into cyclic imides (Figure 2).
Our rationale depended mainly on molecular hybridization approach
that involved the combination of two or more pharmacophoric
moieties with relevant biological properties to obtain a new hybrid
compound with enhanced potency, efficacy, and safety.[23] This
approach allowed us to construct new quinoline‐hybrids which could
serve as effective anticancer agents, since quinoline nucleus was
hybridized with four heterocyclic moieties in addition to a chalcone
moiety. Embodying tetrazole moiety at N1 and C2 of quinoline ring
gave rise to the fused tetrazolo[1,5‐a]quinoline scaffold which in turn
was hybridized with pyrane, naphthopyrane, and chalcone moieties
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2
RESULTS AND DISCUSSION
Chemistry
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2.1
Our target compounds were obtained throughout three Schemes 1 to
3. Scheme 1 illustrates the synthetic pathways embraced for the
FIGURE 2 Rationale of molecular design of the target compounds

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SCHEME 1 Reagents and conditions: (a) CH3COONa, NH2OH, EtOH/H2O, stirring, rt, 2 hr, then reflux, 3 hr; (b) SOCl2, DMF, stirring, rt,
24 hr; (c) N2H4.H2O, abs. EtOH, reflux, 24 hr; (d) appropriate acid anhydride, glacial acetic acid, reflux, 3 hr; (e) appropriate phenol or
thiophenol, DMF, K2CO3, 85°C, 24 hr; (f) malononitrile, abs. EtOH, reflux 20 hr. DMF: dimethylformamide; rt: room temperature
SCHEME 2 Reagents and conditions: (a) NaN3, H2O, acetic acid, dimethyl sulfoxide, 40°C, 3 hr; (b) malononitrile, abs. EtOH, reflux, 12 hr;
(c) TBAB, abs. EtOH, reflux 12 hr; (d) N2H4.H2O or PhNHNH2, abs. EtOH, reflux, 20 hr. TBAB: tetrabutylammonium bromide

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SCHEME 3 Reagents and conditions: (a,b) Appropriate aromatic ketone, DMF, alcoholic KOH 2%, stirring, rt, 24 hr; (c) N2H4.H2O, abs. EtOH,
reflux, 5 hr; (d) appropriate iso(thio)cyanate, THF, stirring, rt, 24 hr. DMF: dimethylformamide; rt: room temperature; THF: tetrahydrofuran
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preparation of compounds 5a–c, 6a–c and 7a–c. The starting
compound 1 was obtained using the method described by Meth‐
Cohn[24,25] via the treatment of acetanilide with the Vilsmeier–Haack
reagent. Condensation of 1 with hydroxylamine hydrochloride gave
rise to oxime 2.[2] Dehydration of 2 using thionyl chloride in the
presence of dimethylformamide (DMF) yielded nitrile 3 which was
refluxed with hydrazine hydrate in ethanol to give 4.[26] The reaction
of amino group in compound 4 with different acids anhydride
(succinic anhydride, maleic anhydride, and phthalic anhydride,
respectively) in the presence of glacial acetic acid yielded the cyclic
imide derivatives 5a–c. The reaction of 1 with appropriate phenol or
thiophenol in the presence of potassium carbonate and DMF yielded
6a–c which were refluxed with malononitrile in ethanol to give 7a–c
(Scheme 1).
2.2
Biological evaluation
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2.2.1
In vitro one‐dose anticancer screening
Out of the 27 synthesized compounds, 25 compounds were selected
by the National Cancer Institute (NCI) under the Developmental
Therapeutic Program (DTP) for evaluation of their anticancer activity
against 60 human tumor cell lines at a single dose of 10 µM. The
60‐cell panel is derived from nine different cancer types: leukemia,
lung, colon, melanoma, central nervous system, ovary, renal, breast,
and prostate cancers. The yield from single dose screening was
reported as a mean graph that was analyzed by the COMPARE
program.[29] Results for the most cytotoxic compounds 6b, 7a–c, 13
and 19f are listed in Table 1, expressed as GI% of cancer cells.
Analysis of data resulted from the primary assay proved that
compounds 13 and 19f displayed considerable antiproliferative
activity. Compounds 6b and 7a–c showed moderate to good activity
against leukemia cancer cell lines. Compound 13 exhibited high
selectivity and potency against CCRF‐CEM, HL‐60(TB) and RPMI‐
8226 (leukemia cancer cell lines), HCT‐116 (colon cancer cell line),
SF‐539 (CNS cancer cell line), MDA‐MB‐435 (melanoma cancer cell
line), OVCAR‐3 (ovarian cancer cell line), A498 (renal cancer cell
line), MDA‐MB‐468 (breast cancer cell line) that impressively
reached to 100% inhibition of tumor cell growth. It showed excellent
activity against other leukemia cell lines (K‐562, MOLT‐4, and SR)
with GI% values of 95.87, 89.37, and 96.12, respectively. Moreover,
compound 13 exhibited GI% of lung cancer cell lines with values
ranging from 46.36% to 88.68% with the best activity showed against
the NCI‐H460 cell line. It also showed good activity against prostate
cancer cell lines, in addition to the rest of cancer cell lines. The rest of
our synthesized compounds gave average % GI <12%.
Compound 8 was prepared through the reaction of 1 with sodium
azide according to the reported method.[27] Under Knoevenagel
condensation reaction compound 8 was reacted with malononitrile to
yield 2‐(tetrazolo[1,5‐a]quinolin‐4‐ylmethylene)malononitrile (9)
which was reacted via Michael cycloaddition reaction with the
appropriate β‐diketone (dimedone and acetylacetone), β‐ketoester
(ethyl acetoacetate) or the appropriate phenol in the presence of
tetrabutylammonium bromide (TBAB) as phase transfer catalyst to
give 10, 11a,b, 12a,b, and 13, respectively. The reaction of 9 with
hydrazine or phenylhydrazine afforded 14 and 15, respectively
(Scheme 2).
α,β‐Unsaturated carbonyl derivatives 16a,b and 17a,b were
synthesized via condensation of 8 with the appropriate aryl or
heteroaryl ketone. Hydrazone 18 was prepared by the reported
method[28] from 8. It was then reacted with the appropriate iso(thio)‐
cyanate to yield 19a–f, respectively (Scheme 3).

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TABLE 2 Five dose screening results of compound 13
TABLE 2 (Continued)
Panel/cell line
GI50 (µM)
TGI (µM)
LC50 (µM)
Panel/cell line
GI50 (µM)
TGI (µM)
LC50 (µM)
Leukemia
CCRF‐CEM
HL‐60(TB)
K‐562
MOLT‐4
RPMI‐8226
SR
BT‐549
1.61
1.83
0.466
>100
>100
>100
>100
>100
0.739
0.260
0.365
0.557
0.927
0.232
>100
16.7
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
T‐47D
MDA‐MB‐468
Note. GI50: molar concentration of the compound that inhibits 50% net
cell growth; LC50: molar concentration of the compound leading to 50%
net cell death; TGI: molar concentration of the compound resulting in
total growth inhibition.
Non‐small cell lung cancer
A549/ATCC
EKVX
HOP‐62
HOP‐92
NCI‐H226
NCI‐H23
NCI‐H322M
NCI‐H460
NCI‐H522
3.02
5.80
0.812
0.490
3.08
3.45
2.15
0.496
0.407
>100
>100
>100
>100
>100
>100
>100
>100
2.90
>100
>100
>100
>100
>100
>100
>100
>100
>100
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2.2.2
In vitro five‐dose anticancer screening
Based on the promising results obtained from single dose testing,
compound 13 was selected for further assay against the full 60‐cell
panel at five different concentrations (100, 10, 1.0, 0.1, and 0.001
µM). The anticancer activity was expressed according to three dose‐
response parameters namely, GI50, TGI, and LC50. Furthermore, a
mean graph midpoint (MG_MID) was calculated for each of the
aforementioned parameters, giving an averaged activity parameter
over all cell lines. The five‐dose assay results are shown in Table 2,
and graphically represented in Figure 1, as dose‐response curves
against nine different cancer panels.
Colon cancer
COLO 205
HCC‐2998
HCT‐116
HCT‐15
HT29
KM12
SW‐620
2.69
2.36
7.32
>1.00
>100
>100
>100
>100
>100
>100
>1.00
>100
>100
>100
>100
>100
0. 685
0.524
3.00
0.628
0.467
CNS cancer
SF‐268
SF‐295
SF‐539
SNB‐19
U251
Compound 13 showed significant anticancer activity against most
of the tested cancer cell lines with GI50 values in the range of 0.232–
18.7 µM. It was found to be highly sensitive against the leukemia SR
cell line (GI50 = 0.232 µM), HL‐60(TB) (GI50 = 0.260 µM), K‐562 (GI50
3.23
>100
>100
2.48
>100
>100
>100
>100
0.832
0.483
0.702
0.748
>100
>100
= 0.365 µM), non‐small cell lung cancer NCI‐H522 cell line (GI50
=
Melanoma
LOX
MALME‐3M
M14
MDA‐MB‐435
SK‐MEL‐2
SK‐MEL‐28
SK‐MEL‐5
UACC‐257
UACC‐62
0.407 µM), colon cancer SW‐620 cell line (GI50 = 0.467 µM), CNS
cancer SF‐539 cell line (GI50 = 0.483 µM), melanoma MDA‐MB‐435
0.966
0.852
0.948
0.300
0.616
4.04
0.610
9.09
0.451
>100
>100
>100
1.01
>100
>100
>100
7.83
>100
>100
>100
>100
>100
cell line (GI50 = 0.300 µM), ovarian cancer OVCAR‐3 cell line (GI50
=
0.440 µM), prostate cancer PC‐3 cell line (GI50 = 0.545 µM) and
breast cancer MDA‐MB‐468 cell line (GI50 = 0.466 µM). Furthermore,
it showed very good activity against other human cancer cell lines.
6.84
>100
>100
>100
>100
Ovarian cancer
IGROV1
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2.2.3
cells
In vitro cytotoxicity against human normal
0.820
0.440
18.7
2.76
2.85
>100
3.36
>100
>100
>100
46.6
>100
>100
>100
>100
>100
>100
>100
OVCAR‐3
OVCAR‐4
OVCAR‐5
OVCAR‐8
NCI/ADR‐RES
SK‐OV‐3
Cytotoxic selectivity of compound 13 toward cancer cell lines was
evaluated throughout screening against human normal cell lines,
represented by skin fibroblast cell line (BJ) and breast epithelial cell
0.534
2.05
>100
Renal cancer
786‐0
line (MCF 10F), using staurosporine as
a standard drug. The
1.28
0.783
1.71
5.74
1.40
0.990
4.80
0.725
>100
>100
>100
>100
6.59
>100
>100
>100
>100
>100
>100
>100
>1.00
>100
>100
>100
compound showed increased IC50 in both cell lines, compared to
the reference drug, indicating higher selectivity toward cancer cell
lines over normal cells (Table 3).
A498
ACHN
CAKI‐1
RXF 393
SN12C
TK‐10
UO‐31
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2.2.4
Epidermal growth factor receptor (EGFR),
vascular endothelial growth factor receptor 2
(VEGFR2), and topoisomerase 1 (Topo 1) enzyme
inhibition assay
Prostate cancer
PC‐3
0.545
2.77
>100
>100
>100
>100
DU‐145
Breast cancer
MCF7
MDA‐MB‐231/ATCC
HS 578T
0.471
0.976
1.51
>100
>100
49.3
>100
>100
>100
EGFR, VEGFR2, and Topo 1 were selected as specific targets to examine
the in vitro inhibitory activity of compound 13 against them; to inspect
the possible mechanism of action by which this compound may exert its
(Continues)

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FIGURE 3 Five dose assay graph of compound 13 against the nine panel cancer cell lines at NCI
antitumor activity. Selection of such targets was based on the reported
inhibitory activity of many quinoline‐derived anticancer agents toward
one or more of these enzymes, such as camptothecin,[30] topotecan[15]
and irinotecan,[16] which exert their anticancer activity throughout
inhibition of Topo I. Besides, lenvatinib,[12,13] tivozanib[31] and cabozanti-
nib[14] are quinoline derivatives that proved to have considerable
inhibitory activity against VEGFR. Furthermore, many other quinoline‐
based derivatives[32] and quinoline isosteres[33] are well known to exhibit
anticancer activity via inhibition of EGFR. The tested compound showed
significant inhibition against Topo 1 with IC50 value of 0.278 µg/ml which
is comparable to that of the reference drug camptothecin (IC50 0.224 µg/
ml). On the other hand it displayed merely 60% of sorafenib activity
against VEGFR2 and about 30% of erlotinib activity against EGFR (Table
4). The results supported that Topo 1 could be a possible target for
compound 13 rather than VEGFR2 or EGFR kinases.
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2.3
Docking study
Molecular docking study of compound 13 was performed to rationalize
the promising Topo 1 inhibitory activity through inspection of the
potential binding affinity and binding mode with the active site. The
crystalline structure needed for determination of absolute configuration
of chiral C4 of pyran ring by X‐ray crystallography was not satisfying.
TABLE 4 In vitro enzyme inhibition of compound 13
TABLE 3 Cytotoxic activity (IC50) of compound 13 against human
normal cell lines
EGFR IC50
(µg/ml)
VEGFR2 IC50
(µg/ml)
Topo 1 IC50
(µg/ml)
Compounds
13
In vitro cytotoxicity IC50 (µg/ml)
0.721
0.218
–
0.412
–
0.278
–
Compounds
13
BJ
MCF‐10F
Erlotinib
Sorafenib
Camptothecin
79.91 2.14
25.88 0.61
32.92 0.73
27.95 0.75
0.243
–
–
Staurosporine
–
0.224

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(a) 2D binding of camptothecin with Topo 1 active site
(b) 3D docked conformation of camptothecin inside the
pocket of Topo 1 active site
FIGURE 4 Binding of camptothecin with Topo 1 active site (PDB code 1T8I). Topo 1: topoisomerase 1
Therefore, we proposed both R and S enantiomers to perform docking
study.
target for the designed compound for its antitumor activity owing to its
unique binding pattern to the active site.
Docking was carried out using the X‐ray crystal structure of Topo
1 with camptothecin, PDB entry 1T8I.[34] Binding of camptothecin to
the target pocket involved both the hydrogen bond formed between
the quinoline‐N atom with the conserved amino acid Arg364, and the
arene–arene interaction between quinoline ring with TGP, DC, and
deoxy adenine (DA; Figure 2). Docking of (R)‐enantiomer of
compound 13 demonstrated interesting binding modes similar to
that of camptothecin; with dockig score value of −5.9 kcal/mol;
through a hydrogen bond interaction that occurred between the
nitrogen atom of ligand‐CN group and the amino group hydrogen of
Arg364. Besides, a π–π stacking interaction was found to be formed
between the tetrazole ring and DA. Furthermore, an additional
interaction occurred with Lys425 residue in the enzyme pocket,
hence maintaining an improved binding pattern, comparable to that
of camptothecin. (S)‐Enantiomer of compound 13 shows weaker
binding affinity with Topo 1 active site in comparison to (R)‐
enantiomer, with dockig score value of −3.9 kcal/mol (Figure 3).
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4
EXPERIMENTAL
Chemistry
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4.1
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4.1.1
General
Melting points (°C) are uncorrected and were recorded using Fisher–John
melting point apparatus. Microanalyses were performed at the micro-
analytical unit, Cairo University. IR spectra were recorded on a Mattson
5000 FT‐IR spectrometer (υ in cm−1) in KBr disks at the Faculty of
Pharmacy, Mansoura University. The 1H‐NMR (500 MHz) and 13C‐NMR
(125 MHz) spectra were recorded on Jeol ECA‐500 II NMR spectrometer
in dimethyl sulfoxide (DMSO)‐d6 at the Faculty of Science, Mansoura
University. Chemical shifts in ppm are expressed in δ units using
tetramethylsilane (TMS) as internal standard. TLC technique was used for
determination of reaction times using silica gel plates 60 F245E (Merck
Merck KGaA, Darmstadt, Germany), the spots were visualized by UV
(366 nm). All reagents were purchased from the Sigma‐Aldrich Co. (St.
Louis, MO). Compounds 1–4 were synthesized in accordance with
method described in the literature.[24–26]
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3
CONCLUSION
The InChI codes of the investigated compounds are provided as
Supporting Information.
Molecular hybrids of quinoline were designed, synthesized and biologi-
cally evaluated for their anticancer activity via NCI protocol. Compound
13 was proved to have the most potent anticancer activity; with growth
inhibition (GI50) values <1.0 µM, exhibiting the highest selectivity toward
leukemia, breast cancer, and CNS cancer cell lines, while proved to be less
toxic against human normal cell lines. Furthermore, it showed significant
Topo 1 enzyme inhibitory activity, comparable to the standard drug
camptothecin. Docking study supported that Topo 1 could be a possible
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4.1.2
General procedure for synthesis of
compounds 5a–c
Mixture of 1H‐pyrazolo[3,4‐b]quinolin‐3‐amine (4) (1.84 g, 0.01 mol)
and the appropriate acid anhydride (0.01 mol) in glacial acetic acid

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(a) 2D binding of compound 13 (R)-enantiomer with
(b) 3D docked conformation of compound 13 (R)-
Topo 1 active site
enantiomer inside the pocket of Topo 1 active site
(d) 3D docked conformation of compound 13 (S)-
(c) 2D binding of compound 13 (S)-enantiomer with Topo 1
enantiomer inside the pocket of Topo 1 active site
active site
FIGURE 5 Binding mode of (R)‐ and (S)‐enantiomers of compound 13 in Topo 1 active site (PDB code 1T8I)
(30 ml) was refluxed for 3 hr. The reaction mixture was cooled to 0°C
and the solid product was filtered, washed with water, dried, and
crystallized from ethanol.
(d, J = 8.6 Hz, 1H, Ar–H), 8.12 (d, J = 8.2 Hz, 1H, Ar–H), 9.05 (s, 1H, Ar–H),
10.92 (s, 1H, NH); Anal. calcd. for C14H10N4O2 (266): C, 63.15; H, 3.79; N,
21.04%. Found: C, 63.25; H, 3.67; N, 21.23%.
1‐(1H‐Pyrazolo[3,4‐b]quinolin‐3‐yl)pyrrolidine‐2,5‐dione (5a)
Compound 5a was prepared by using succinic anhydride, yield (39%); mp
> 300°C; IR (cm–1): 3427 (NH), 1707 (C=O); 1H NMR: δ 2.61 (t, J = 6.0 Hz,
2H, CH2 pyrrolidine), 2.71 (t, J = 6.2 Hz, 2H, CH2 pyrrolidine), 7.45 (dd, J =
7.3, 7.4 Hz, 1H, Ar–H), 7.77 (dd, J = 7.2, 7.7 Hz, 1H, Ar–H), 7.94
1‐(1H‐Pyrazolo[3,4‐b]quinolin‐3‐yl)−1H‐pyrrole‐2,5‐dione (5b)
Compound 5b was prepared by using maleic anhydride, yield (45%);
mp > 300°C; IR (cm–1): 3422 (NH), 1702 (C=O); 1H NMR: δ 6.42 (d, J
= 12.0 Hz, 1H, CH pyrrole), 6.62 (d, J = 12.0 Hz, 1H, CH pyrrole), 7.47
(dd, J = 7.3, 7.4 Hz, 1H, Ar–H), 7.79 (dd, J = 8.1, 8.4 Hz, 1H, Ar–H),

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7.96 (d, J = 8.7 Hz, 1H, Ar–H), 8.11 (d, J = 8.1 Hz, 1H, Ar–H), 9.09
(s, 1H, Ar–H); Anal. calcd. for C14H8N4O2 (264): C, 63.64; H, 3.05; N,
21.20%. Found: C, 63.48; H, 3.11; N, 21.13%.
2‐((2‐(4‐Chlorophenoxy)quinolin‐3‐yl)methylene)malononitrile
(7a)
Yield (44%); mp 219–220°C; IR (cm–1): 2227 (CN), 1618 (C=C), 1579
(C=N); 1H NMR: δ 7.39 (d, J = 8.9 Hz, 2H, Ar–H, phenyl), 7.57 (d, J =
8.7 Hz, 2H, Ar–H, phenyl), 7.60 (dd, J = 7.9, 7.9 Hz, 1H, Ar–H), 7.67 (d,
J = 8.4 Hz, 1H, Ar–H), 7.82 (dd, J = 8.2, 8.5 Hz, 1H, Ar–H), 8.11 (d, J =
8.2 Hz, 1H, Ar–H), 8.87 (s, 1 H, CH=C(CN)2), 9.06 (s, 1H, Ar–H); 13C
NMR: 85.80, 112.71, 113.72, 116.69, 124.07, 124.53, 126.35, 127.00,
129.43, 129.56, 133.39, 141.31, 146.96, 151.38, 155.87, 157.66;
Anal. calcd. for C19H10ClN3O (331.7): C, 68.79; H, 3.04; N, 12.67%.
Found: C, 68.66; H, 3.26; N, 12.54%.
2‐(1H‐Pyrazolo[3,4‐b]quinolin‐3‐yl)isoindoline‐1,3‐dione (5c)
Compound 5c was prepared by using phthalic anhydride, yield (33%);
mp > 300°C; IR (cm–1): 3425 (NH), 1722 (C=O); 1H NMR: δ 7.53 (dd, J
= 7.3, 7.4 Hz, 1H, Ar–H), 7.85 (dd, J = 7.3, 7.7 Hz, 1H, Ar–H), 7.95–
8.14 (m, 6H, Ar–H), 9.02 (s, 1H, Ar–H); 13C NMR: 111.68, 123.89,
124.11, 127.87, 129.58, 130.84, 131.12, 131.60, 132.84, 135.13,
148.11, 151.37, 151.55, 166.47; Anal. calcd. for C18H10N4O2 (314):
C, 68.79; H, 3.21; N, 17.83%. Found: C, 68.66; H, 3.12; N, 17.95%.
2‐((2‐(4‐(Trifluoromethyl)phenoxy)quinolin‐3‐yl)methylene)‐
malononitrile (7b)
|
4.1.3
General procedure for synthesis of
Yield (56%); mp 194–195°C; IR (cm–1): 2228 (CN), 1617 (C=C), 1585
(C=N); 1H NMR: δ 7.53–7.65 (m, 3H, Ar–H), 7.68 (d, J = 8.3 Hz, 1H, Ar–H),
7.83 (dd, J = 7.3, 7.5 Hz, 1H, Ar–H), 7.89 (d, J = 8.1 Hz, 2H, Ar–H, phenyl),
8.12 (d, J = 7.9 Hz, 1H, Ar–H), 8.88 (s, 1H, CH=C(CN)2), 9.09 (s, 1H, Ar–
H); 13C NMR: 86.34, 113.16, 114.17, 117.21, 123.39, 125.13, 125.99,
126.20, 126.52, 127.00, 127.49, 127.51, 129.92, 133.92, 141.90, 147.31,
156.13, 157.78; Anal. calcd. for C20H10F3N3O (365): C, 65.76; H, 2.76; N,
11.50%. Found: C, 65.55; H, 2.89; N, 11.62%.
compounds 6a–c
2‐Chloroquinoline‐3‐carbaldehyde (1) (1.91 g, 0.01 mol) was dis-
solved in DMF (25 ml), to which 4‐substituted phenol or thiophenol
(0.01 mol) and potassium carbonate (2.76 g, 0.02 mol) were added.
The reaction mixture was heated at 75°C overnight, then allowed to
cool, and poured on iced water. The precipitated solid was filtered
and washed with water, dried and recrystallized from methanol.
2‐(4‐Chlorophenoxy)quinoline‐3‐carbaldehyde (6a)
2‐((2‐((4‐Bromophenyl)thio)quinolin‐3‐yl)methylene)‐
malononitrile (7c)
Yield (58%); mp 141–142°C; IR (cm–1): 2860, 2730 (C–H, aldehyde),
1691 (C=O), 1590 (C=N); 1H NMR: δ 7.40 (d, J = 8.9 Hz, 2H, Ar–H), 7.53–
7.60 (m, 3H, Ar–H), 7.66 (d, J = 8.4 Hz, 1H, Ar–H), 7.80 (dd, J = 8.4, 8.5 Hz,
1H, Ar–H), 8.18 (d, J = 8.1 Hz, 1H, Ar–H), 8.95 (s, 1H, Ar–H), 10.49 (s, 1H,
CHO); 13C NMR: 120.07, 124.02, 124.98, 125.98, 126.90, 129.27,
129.56, 130.13, 133.17, 141.50, 147.41, 151.74, 159.90, 188.85; Anal.
calcd. for C16H10ClNO2 (283.71): C, 67.74; H, 3.55; N, 4.94%. Found: C,
67.66; H, 3.75; N, 4.74%.
Yield (66%); mp 190–191°C; IR (cm–1): 2230 (CN), 1615 (C=C), 1572
(C=N); 1H NMR: δ 7.55 (d, J = 8.4 Hz, 2H, Ar–H), 7.66 (dd, J = 7.8, 7.9
Hz, 1H, Ar–H), 7.70 (d, J = 8.4 Hz, 2H, Ar–H), 7.76 (d, J = 8.3 Hz, 1H,
Ar–H), 7.85 (dd, J = 8.2, 8.3 Hz, 1H, Ar–H), 8.08 (d, J = 7.8 Hz, 1H, Ar–
H), 8.84 (s, 1H, CH=C(CN)2), 8.85 (s, 1H, Ar–H); 13C NMR: 87.64,
112.17, 113.11, 122.88, 124.80, 125.13, 127.59, 127.85, 129.04,
129.15, 132.45, 133.07, 136.18, 138.31, 148.12, 157.55; Anal. calcd.
for C19H10BrN3S (392): C, 58.17; H, 2.57; N, 10.71%. Found: C,
58.34; H, 2.41; N, 10.64%.
2‐(4‐(Trifluoromethyl)phenoxy)quinoline‐3‐carbaldehyde (6b)
Yield (53%); mp 124–125°C; IR (cm–1): 2853, 2728 (C–H, aldehyde),
1695 (C=O), 1595 (C=N); 1H NMR: δ 7.58–7.63 (m, 3H, Ar–H), 7.69 (d, J
= 8.4 Hz, 1H, Ar–H), 7.82 (dd, J = 7.4, 7.9 Hz, 1H, Ar–H), 7.89 (d, J = 8.5
Hz, 2H, Ar–H), 8.21 (d, J = 8.0 Hz, 1H, Ar–H), 8.99 (s, 1H, Ar–H), 10.50 (s,
1H, CHO); 13C NMR: 120.64, 120.64, 123.26, 125.62, 125.97, 126.03,
126.29, 126.66, 127.43, 127.51, 127.55, 130.62, 133.74, 142.23, 147.79,
156.59, 159.99, 189.25; Anal. calcd. for C17H10F3NO2 (317): C, 64.36; H,
3.18; N, 4.41%. Found: C, 64.24; H, 3.29; N, 4.74%.
2‐(Tetrazolo[1,5‐a]quinolin‐4‐ylmethylene)malononitrile (9)
To a mixture of compound 8 (1.98 g, 0.01 mol) in absolute ethanol (30 ml)
malononitrile (2.64 g, 0.04 mol) was added. The reaction mixture was
refluxed for 20 h, allowed to cool. The precipitated solid was filtered,
washed with ethanol and dried to obtain the titled compound as light
green solid. Yield (80%); mp 200–201°C; IR (cm–1): 2236 (CN); 1H NMR: δ
7.84 (dd, J = 7.5, 7.7 Hz, 1H, Ar–H), 8.07 (dd, J = 7.6, 7.8 Hz, 1H, Ar–H),
8.18 (d, J = 8.0 Hz, 1H, Ar–H), 8.69 (s, 1H, CH=CC(CN)2), 8.74 (d, J = 8.4
Hz, 1H, Ar–H), 9.03 (s, 1H, Ar–H); 13C NMR: 87.15, 112.38, 113.40,
116.53, 117.20, 122.97, 128.93, 131.12, 131.31, 134.44, 136.07, 145.70,
153.12; Anal. calcd. for C13H6N6 (246): C, 63.41; H, 2.46; N, 34.13%.
Found: C, 63.38; H, 2.49; N, 34.11%.
2‐((4‐Bromophenyl)thio)quinoline‐3‐carbaldehyde (6c)[35]
Yield (43%), mp 84–85°C as recorded, 83.4–84.3°C as reported.
|
4.1.4
General procedure for the synthesis of
compounds 7a–c
|
4.1.5
General procedure for the synthesis of
To a mixture of compound 6a–c (0.01 mol) in absolute ethanol (30
ml), malononitrile (2.64 g, 0.04 mol) was added. The reaction mixture
was refluxed for 20 hr, and then allowed to cool. The precipitated
solid was filtered, dried and recrystallized from ethanol.
compounds 10, 11a,b, 12a,b and 13
A mixture of compound 9 (2.46 g, 0.01 mol) with either dimedone,
the appropriate 2,4‐dicarbonyl compound, the appropriate

12 of 16
NASR ET AL.
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3‐substituted phenol or α‐naphthol (0.01 mol) and TBAB (0.8 g,
0.0025 mol) in absolute ethanol (30 ml) was heated under reflux for
12 hr. The reaction mixture was allowed to cool to room temperature
where the precipitated solid was filtered, dried and crystallized from
ethanol to give the target compounds.
6.46 (d, J = 2.3 Hz, 1H, Ar–H), 6.96 (d, J = 8.5 Hz, 1H, Ar–H), 7.08
(s, 2H, NH2), 7.82 (dd, J = 7.1, 7.5 Hz, 1H, Ar–H), 7.95 (dd, J = 8.4, 8.5
Hz, 1H, Ar–H), 8.23 (s, 1H, Ar–H), 8.27 (d, J = 7.7 Hz, 1H, Ar–H), 8.59
(d, J = 8.2 Hz, 1H, Ar–H), 9.76 (s, 1H, OH); 13C NMR: 37.61, 52.78,
102.44, 110.66, 112.13, 116.22, 120.55, 123.75, 128.48, 129.42,
129.52, 129.57, 130.16, 131.18, 146.39, 149.44, 157.66, 161.23;
Anal. calcd. for C19H12N6O2 (356.34): C, 64.04; H, 3.39; N, 23.58%.
Found: C, 64.23; H, 3.78; N, 23.34%.
2‐Amino‐7,7‐dimethyl‐5‐oxo‐4‐(tetrazolo[1,5‐a]quinolin‐4‐yl)‐
5,6,7,8‐tetrahydro‐4H‐chromene‐3‐carbonitrile (10)
Yield (75%); mp 265–266°C; IR (cm–1): 3414, 3327 (NH2), 2189 (CN),
1666 (C=O); 1H NMR: δ 0.83 (s, 3H, CH3), 1.04 (s, 3H, CH3), 2.00 (d, J
= 16.1 Hz, 1H, CH2 cyclohex‐2‐enone), 2.28 (d, J = 16.2 Hz, 1H, CH2
cyclohex‐2‐enone), 2.48 (d, J = 15.1 Hz, 1H, CH2 cyclohex‐2‐enone),
2.63 (d, J = 17.7 Hz, 1H, CH2 cyclohex‐2‐enone), 4.79 (s, 1H, CH
pyran), 7.24 (s, 2H, NH2), 7.81 (dd, J = 7.2, 7.6 Hz, 1H, Ar–H), 7.94 (dd,
J = 8.0, 8.3 Hz, 1H, Ar–H), 8.22 (s, 1H, Ar–H), 8.27 (d, J = 7.8 Hz, 1H,
Ar–H), 8.58 (d, J = 8.3 Hz, 1H, Ar–H); 13C NMR: 26.49, 28.67, 31.81,
33.97, 49.92, 54.57, 109.59, 116.20, 119.53, 123.72, 127.88, 128.50,
129.24, 129.42, 130.89, 131.16, 146.17, 159.63, 164.27, 196.11;
Anal. calcd. for C21H18N6O2 (386): C, 65.27; H, 4.70; N, 21.75%.
Found: C, 65.44; H, 4.59; N, 21.37%.
2,7‐Diamino‐4‐(tetrazolo[1,5‐a]quinolin‐4‐yl)−4H‐chromene‐3‐
carbonitrile (12b)
Compound 12b was prepared by using 3‐aminophenol, yield (69%);
mp 262–263°C; IR (cm–1): 3403, 3353 (NH2), 2182 (CN); 1H NMR: δ
5.17 (s, 1H, CH pyran), 5.29 (s, 2H, NH2), 6.18 (dd, J = 2.1, 8.3 Hz, 1H,
Ar–H), 6.25 (d, J = 2.1 Hz, 1H, Ar–H), 6.78 (d, J = 8.3 Hz, 1H, Ar–H),
7.00 (s, 2H, NH2), 7.81 (dd, J = 7.5, 7.9 Hz, 1H, Ar–H), 7.94 (dd, J = 7.9,
8.3 Hz, 1H, Ar–H), 8.18 (s, 1H, Ar–H), 8.25 (d, J = 7.8 Hz, 1H, Ar–H),
8.59 (d, J = 8.3 Hz, 1H, Ar–H); 13C NMR: 37.58, 52.86, 100.13,
106.90, 110.74, 116.16, 120.74, 123.78, 128.40, 128.92, 129.32,
129.41, 129.82, 130.08, 131.03, 146.46, 149.37, 149.41, 161.42;
Anal. calcd. for C19H13N7O (355): C, 64.22; H, 3.69; N, 27.59%.
Found: C, 64.12; H, 3.88; N, 27.45%.
5‐Acetyl‐2‐amino‐6‐methyl‐4‐(tetrazolo[1,5‐a]quinolin‐4‐yl)‐
4H‐pyran‐3‐carbonitrile (11a)
Compound 11a was prepared by using acetylacetone, yield (65%); mp
240–241°C; IR (cm–1): 3414, 3327 (NH2), 2189 (CN), 1669 (C=O); 1H
NMR: δ 2.19 (s, 3H, CH3), 2.33 (s, 3H, COCH3), 5.08 (s, 1H, CH pyran),
7.09 (s, 2H, NH2), 7.81 (dd, J = 7.6, 7.9 Hz, 1H, Ar–H), 7.95 (dd, J = 8.1,
8.3 Hz, 1H, Ar–H), 8.15 (s, 1H, Ar–H), 8.25 (d, J = 7.7 Hz, 1H, Ar–H),
8.59 (d, J = 8.3 Hz, 1H, Ar–H); 13C NMR: 19.18, 30.30, 36.32, 54.35,
112.57, 116.14, 119.54, 123.78, 128.31, 128.54, 129.36, 129.45,
130.57, 131.15, 146.38, 157.31, 159.57, 197.69; Anal. calcd. for
C18H14N6O2 (346): C, 62.42; H, 4.07; N, 24.27%. Found: C, 62.29; H,
4.13; N, 24.47%.
2‐Amino‐4‐(tetrazolo[1,5‐a]quinolin‐4‐yl)−4H‐benzo[h]‐
chromene‐3‐carbonitrile (13)
Compound 13 was prepared by using α‐naphthol, yield (57%); mp
288–289°C; IR (cm–1): 3475, 3377 (NH2), 2187 (CN); 1H NMR: δ 5.56
(s, 1H, CH pyran), 7.26 (d, J = 8.6 Hz, 1H, Ar–H), 7.36 (s, 2H, NH2),
7.55 (d, J = 8.6 Hz, 1H, Ar–H), 7.58 (dd, J = 7.3, 7.5 Hz, 1H, Ar–H),
7.67 (dd, J = 7.2, 7.6 Hz, 1H, Ar–H), 7.83 (dd, J = 7.7, 7.8 Hz, 1H, Ar–
H), 7.87 (d, J = 8.1 Hz, 1H, Ar–H), 7.96 (dd, J = 7.6, 8.4 Hz, 1H, Ar–H),
8.29 (d, J = 7.9 Hz, 1H, Ar–H), 8.33 (d, J = 8.4 Hz, 1H, Ar–H), 8.36 (s,
1H, Ar–H), 8.59 (d, J = 8.3 Hz, 1H, Ar–H); 13C NMR: 38.51, 52.94,
114.79, 116.20, 120.34, 120.90, 122.73, 123.70, 123.75, 125.61,
126.67, 126.95, 127.67, 128.45, 128.83, 129.50, 129.59, 130.92,
131.29, 133.08, 143.52, 146.42, 161.07; Anal. calcd. for C23H14N6O
(390): C, 70.76; H, 3.61; N, 21.53%. Found: C, 70.56; H, 3.45; N,
21.33%.
Ethyl 6‐amino‐5‐cyano‐2‐methyl‐4‐(tetrazolo[1,5‐a]quinolin‐4‐
yl)‐4H‐pyran‐3‐carboxylate (11b)
Compound 11b was prepared by using ethylacetoacetate, yield
(73%); mp 222–223°C; IR (cm–1): 3417, 3325 (NH2), 2187 (CN), 1663
(C=O); 1H NMR: δ 0.90 (t, J = 7.1 Hz, 3H, CH3CH2COO), 2.38 (s, 3H,
CH3), 3.89 (m, 2H, CH2COO), 4.95 (s, 1H, CH pyran), 7.14
(s, 2H, NH2), 7.81 (dd, J = 7.6, 7.8 Hz, 1H, Ar–H), 7.94 (dd, J = 7.9,
8.3 Hz, 1H, Ar–H), 8.15 (s, 1H, Ar–H), 8.26 (d, J = 7.9 Hz, 1H, Ar–H),
8.59 (d, J = 8.3 Hz, 1H, Ar–H); 13C NMR: 13.60, 18.55, 36.35, 53.96,
60.16, 103.78, 116.15, 119.49, 123.75, 128.38, 128.87, 129.24,
129.37, 130.88, 131.12, 146.42, 159.10, 159.49, 165.16; Anal. calcd.
for C19H16N6O3 (376): C, 60.63; H, 4.28; N, 22.33%. Found: C, 60.44;
H, 4.35; N, 22.57%.
4‐(Tetrazolo[1,5‐a]quinolin‐4‐ylmethylene)−4H‐pyrazole‐3,5‐
diamine (14)
A mixture of compound 9 (2.46 g, 0.01 mol) and hydrazine hydrate
(99%, 1 g, 0.02 mol) in absolute ethanol (30 ml) was heated under
reflux for 20 hr. The reaction mixture was cooled to room
temperature. The titled compound precipitated as green crystalline
solid which was filtered and washed with ethanol. Yield (43%); mp
271–272°C; IR (cm–1): 3307, 3385 (NH2), 1566 (C=N); 1H NMR: δ
7.49 (dd, J = 7.7, 7.9 Hz, 1H, Ar–H), 7.78 (dd, J = 8.2, 8.3 Hz, 1H, Ar–
H), 7.99 (d, J = 8.7 Hz, 1H, Ar–H), 8.14 (d, J = 8.2 Hz, 1H, Ar–H), 8.44
(s, 1H, CH=C), 8.95 (s, 1H, Ar–H); 13C NMR: 116.06, 121.72, 123.79,
123.83, 124.43, 128.17, 128.67, 128.74, 128.87, 129.34, 130.21,
146.26; Anal. calcd. for C13H10N8 (278): C, 56.11; H, 3.62; N, 40.27%.
Found: C, 56.23; H, 3.44; N, 40.12%.
2‐Amino‐7‐hydroxy‐4‐(tetrazolo[1,5‐a]quinolin‐4‐yl)‐
4H‐chromene‐3‐carbonitrile (12a)
Compound 12a was prepared by using resorcinol, yield (60%); mp
285–286°C; IR (cm–1): 3522 (OH), 3461, 3339 (NH2), 2186 (CN); 1H
NMR: δ 5.26 (s, 1H, CH pyran), 6.40 (dd, J = 2.3, 8.5 Hz, 1H, Ar–H),

NASR ET AL.
13 of 16
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(E)‐5‐Imino‐1‐phenyl‐4‐(tetrazolo[1,5‐a]quinolin‐4‐ylmethy-
lene)‐4,5‐dihydro‐1H‐pyrazol‐3‐amine (15)
Ar–H), 8.82 (s, 1H, Ar–H), 9.10 (d, J = 15.9 Hz, 1H, CH=CH); Anal.
calcd. for C21H14ClN7O (415.8): C, 60.65; H, 3.39; N, 23.58%. Found:
C, 60.48; H, 3.55; N, 23.42%.
To a mixture of compound 9 (2.46 g, 0.01 mol) in absolute ethanol (30
ml), phenylhydrazine (1.47 g, 0.015 mol) was added and the mixture
was heated under reflux for 20 hr. The formed precipitate was
filtered and dried to give the titled compound as a yellow solid. Yield
(66%); mp 210–211°C; IR (cm–1): 3260 (NH), 1556 (C=N); 1H NMR: δ
6.86 (dd, J = 7.1, 7.3 Hz, 1H, Ar–H), 7.24 (d, J = 7.5 Hz, 2H, Ar–H), 7.3
(dd, J = 7.3, 8.4 Hz, 2H, Ar–H), 7.80 (dd, J = 8.1, 8.2 Hz, 1H, Ar–H),
7.91 (dd, J = 8.4, 8.5 Hz, 1H, Ar–H), 8.30 (d, J = 7.6 Hz, 1H, Ar–H),
8.41 (s, 1H, Ar–H), 8.57 (s, 1H, CH=C), 8.59 (d, J = 8.3 Hz, 1H), 11.10
(s, 1H, NH); 13C NMR: 112.60, 116.15, 119.99, 121.10, 124.42,
125.28, 128.32, 128.58, 129.11, 129.27, 129.53, 130.58, 144.43,
146.25; Anal. calcd. for C19H14N8 (354): C, 64.40; H, 3.98; N, 31.62%.
Found: C, 64.69; H, 3.87; N, 31.33%.
(E)‐1‐(5‐Methyl‐1‐(4‐nitrophenyl)‐1H−1,2,3‐triazol‐4‐yl)‐3‐(tet-
razolo[1,5‐a]quinolin‐4‐yl)prop‐2‐en‐1‐one (17b)
Yield (20%); mp > 300°C; IR (cm–1): 1663 (C=O), 1592 (C=N); 1H
NMR: δ 2.75 (s, 3H, CH3), 7.91 (dd, J = 7.1, 7.6 Hz, 1H, Ar–H), 8.05–
8.11 (m, 3H, Ar–H), 8.17 (d, J = 15.9 Hz, 1H, CH=CH), 8.31 (d, J = 7.5
Hz, 1H, Ar–H), 8.55 (d, J = 9.0 Hz, 2H, Ar–H), 8.71 (d, J = 8.3 Hz, 1H,
Ar–H), 8.87 (s, 1H, Ar–H), 9.16 (d, J = 15.9 Hz, 1H, CH=CH); Anal.
calcd. for C21H14N8O3 (426): C, 59.15; H, 3.31; N, 26.28%. Found: C,
59.25; H, 3.41; N, 26.35%.
|
4.1.7
General procedure for synthesis of
compounds 19a–f
|
4.1.6
General procedure for synthesis of
To a solution of compound 18[28] (2.12 g, 0.01 mol) in tetrahydrofur-
an (THF) (50 ml), the appropriate iso(thio)cyanate (0.012 mol) was
added. The mixture was allowed to stir overnight. The formed
precipitate was filtered and washed with THF, dried and crystallized
from ethanol.
compounds 16a,b and 17a,b
A solution of tetrazolo[1,5‐a]quinoline‐4‐carbaldehyde (8) (1.98 g,
0.01 mol) in DMF (10 ml) was added gradually to a solution of
4‐substituted acetophenone or 1‐(1‐(4‐substituted phenyl)‐5‐methyl‐
1H‐1,2,3‐triazol‐4‐yl)ethanone (0.01 mol) in alcoholic KOH (2%, 25
ml). The mixture was stirred for 24 hr, then filtered, dried and
crystallized from DMF.
N‐Phenyl‐2‐(tetrazolo[1,5‐a]quinolin‐4‐ylmethylene)hydrazi-
necarboxamide (19a)
Yield (40%); mp 246–247°C; IR (cm–1): 3370 (NH), 1691 (C=O), 1570
(C=N); 1H NMR: δ 7.07 (dd, J = 7.3, 7.3 Hz, 1H, Ar–H), 7.37 (dd, J =
7.6, 7.9 Hz, 2H, Ar–H), 7.70 (d, J = 7.7 Hz, 2H, Ar–H), 7.86 (dd, J = 7.4,
7.5 Hz, 1H, Ar–H), 8.00 (dd, J = 7.5, 7.5 Hz, 1H, Ar–H), 8.25 (d, J = 7.8
Hz, 1H, Ar–H), 8.51 (s, 1H, CH=N), 8.66 (d, J = 8.2 Hz, 1H, Ar–H), 8.92
(s, 1H, Ar–H), 9.27 (s, 1H, NH), 11.38 (s, 1H, NH); 13C NMR: 116.38,
119.60, 120.07, 122.81, 123.98, 128.55, 128.64, 129.31, 129.78,
131.65, 133.04, 138.79, 146.16, 152.76; Anal. calcd. for C17H13N7O
(331): C, 61.62; H, 3.95; N, 29.59%. Found: C, 61.53; H, 3.88; N,
29.78%.
3‐(Tetrazolo[1,5‐a]quinolin‐4‐yl)−1‐(p‐tolyl)prop‐2‐en‐1‐one
(16a)
Yield (23%); mp > 300°C; IR (cm–1): 1656 (C=O), 1586 (C=N); 1H
NMR: δ 2.41 (s, 3H, CH3), 7.44 (d, J = 8.0 Hz, 2H, Ar–H), 7.85 (dd, J =
7.6, 7.6 Hz, 1H, Ar–H), 8.01–8.04 (m, 4H), 8.22 (d, J = 7.9 Hz, 1H, Ar–
H), 8.65 (d, J = 8.3 Hz, 1H, Ar–H), 8.78 (d, J = 16.5 Hz, 1H, CH=CH),
8.8 (s, 1H, Ar–H); Anal. calcd. for C19H14N4O (314): C, 72.60; H, 4.49;
N, 17.82%. Found: C, 72.55; H, 4.57; N, 17.63%.
1‐(4‐Methoxyphenyl)−3‐(tetrazolo[1,5‐a]quinolin‐4‐yl)prop‐2‐
en‐1‐one (16b)
N‐(3‐Chlorophenyl)−2‐(tetrazolo[1,5‐a]quinolin‐4‐ylmethylene)‐
hydrazinecarboxamide (19b)
Yield (19%); mp > 300°C; IR (cm–1): 1659 (C=O), 1583 (C=N); 1H
NMR: δ 3.90 (s, 3H, OCH3), 7.19 (d, J = 8.8 Hz, 2H, Ar–H), 7.87 (dd, J
= 7.4, 7.9 Hz, 1H, Ar–H), 8.01–8.07 (m, 2H), 8.14 (d, J = 8.8 Hz, 2H,
Ar–H), 8.25 (d, J = 7.8 Hz, 1H, Ar–H), 8.67 (d, J = 8.3 Hz, 1H, Ar–H),
8.81 (s, 1H, Ar–H), 8.82 (d, J = 15.5 Hz, 1H, CH=CH); 13C NMR: 55.67,
114.44, 116.39, 120.76, 123.87, 127.42, 128.64, 130.03, 130.07,
130.14, 130.80, 132.57, 136.30, 136.42, 146.26, 163.57, 187.20;
Anal. calcd. for C19H14N4O2 (330): C, 69.08; H, 4.27; N, 16.96%.
Found: C, 69.23; H, 4.15; N, 16.88%.
Yield (45%); mp 252–253°C; IR (cm–1): 3271 (NH), 1709 (C=O), 1587
(C=N); 1H NMR: δ 7.12 (d, J = 9.1 Hz, 1H, Ar–H), 7.39 (dd, J = 8.1, 8.1
Hz, 1H, Ar–H), 7.66 (d, J = 8.1 Hz, 1H, Ar–H), 7.86 (dd, J = 7.4, 7.5 Hz,
1H, Ar–H), 7.90 (s, 1H), 8.00 (dd, J = 8.0, 8.1 Hz, 1H, Ar–H), 8.25 (d, J
= 7.9 Hz, 1H, Ar–H), 8.52 (s, 1H, CH=N), 8.65 (d, J = 8.3 Hz, 1H, Ar–
H), 8.94 (s, 1H, Ar–H), 9.41 (s, 1H, NH), 11.48 (s, 1H, NH); 13C NMR:
116.37, 117.91, 118.84, 119.85, 122.37, 123.93, 128.56, 129.13,
129.82, 130.34, 131.69, 133.05, 133.39, 140.44, 146.22, 152.65;
Anal. calcd. for C17H12ClN7O (365.7): C, 55.82; H, 3.31; N, 26.81%.
Found: C, 55.77; H, 3.54; N, 26.92%.
(E)‐1‐(1‐(4‐Chlorophenyl)−5‐methyl‐1H−1,2,3‐triazol‐4‐yl)‐3‐
(tetrazolo[1,5‐a]quinolin‐4‐yl)prop‐2‐en‐1‐one (17a)
Yield (25%); mp 290–291°C; IR (cm–1): 1661 (C=O), 1593 (C=N); 1H
NMR: δ 2.65 (s, 3H, CH3), 7.77 (s, 4H, Ar–H), 7.87 (dd, J = 7.6, 7.7 Hz,
1H, Ar–H), 8.05 (dd, J = 7.6, 7.9 Hz, 1H, Ar–H), 8.11 (d, J = 15.9 Hz,
1H, CH=CH), 8.27 (d, J = 8.0 Hz, 1H, Ar–H), 8.67 (d, J = 8.3 Hz, 1H,
N‐(4‐Chlorophenyl)−2‐(tetrazolo[1,5‐a]quinolin‐4‐ylmethylene)‐
hydrazinecarboxamide (19c)
Yield (33%); mp 268–269°C; IR (cm–1): 3298 (NH), 1704 (C=O), 1591
(C=N); 1H NMR: δ 7.38 (d, J = 9.0 Hz, 2H, Ar–H), 7.71 (d, J = 8.9 Hz,

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NASR ET AL.
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2H, Ar–H), 7.82 (dd, J = 7.8, 8.0 Hz, 1H, Ar–H), 7.96 (dd, J = 8.4, 8.5
Hz, 1H, Ar–H), 8.21 (d, J = 7.5 Hz, 1H, Ar–H), 8.48 (s, 1H, CH=N), 8.62
(d, J = 8.2 Hz, 1H, Ar–H), 8.89 (s, 1H, Ar–H), 9.33 (s, 1H, NH), 11.40 (s,
1H, NH); 13C NMR: 116.46, 119.95, 121.22, 124.01, 126.49, 128.51,
128.65, 129.22, 129.34, 129.87, 131.80, 133.43, 137.91, 146.26,
152.81; Anal. calcd. for C17H12ClN7O (365.7): C, 55.82; H, 3.31; N,
26.81%. Found: C, 55.74; H, 3.54; N, 26.77%.
plating densities ranging from 5000 to 40,000 cells/well depending
on the replication time of individual cell lines. After cell inoculation,
the microtiter plates were incubated at 37°C, 95% air, 5% CO2, and
100% relative humidity for 24 hr before addition of experimental
compound. Two plates of each cell line were fixed in situ with
trichloroacetic acid, to signify a measurement of the cell population
for each cell line at the time of compound addition (Tz).
The experimental compounds were dissolved in DMSO at 400‐
fold the desired final maximum test concentration and put away
frozen before utilization. At the time of compound addition, an
aliquot of frozen concentrate was defrosted and diluted to double
the wanted final maximum test concentration with total medium
containing 50 mg/ml gentamicin extra four, 10‐fold, or 1/2log serial
dilutions were made to afford a total of five drug concentrations in
addition to control. Aliquots of 100 µl of these distinctive compound
dilutions were added to the proper microtiter wells already contain-
ing 100 µl of medium, bringing about the required final drug
concentrations. More details of this evaluation technique and the
integral data which is encoded by the activity pattern for all cell lines
were reported elsewhere.[36]
2‐(Tetrazolo[1,5‐a]quinolin‐4‐ylmethylene)‐N‐(p‐tolyl)hydrazi-
necarboxamide (19d)
Yield (55%); mp 252–253°C; IR (cm–1): 3264 (NH), 1689 (C=O), 1578
(C=N); 1H NMR: δ 2.28 (s, 3H, CH3), 7.16 (d, J = 8.2 Hz, 2H, Ar–H),
7.57 (d, J = 8.3 Hz, 2H, Ar–H), 7.85 (dd, J = 7.2, 7.6 Hz, 1H, Ar–H),
7.99 (dd, J = 8.1, 8.2 Hz, 1H, Ar–H), 8.24 (d, J = 7.8 Hz, 1H, Ar–H),
8.49 (s, 1H, CH=N), 8.64 (d, J = 8.3 Hz, 1H, Ar–H), 8.91 (s, 1H, Ar–H),
9.16 (s, 1H, NH), 11.32 (s, 1H, NH); 13C NMR: 20.40, 116.35, 119.65,
120.07, 123.97, 128.52, 129.09, 129.19, 129.74, 131.60, 131.68,
132.83, 136.23, 146.14, 152.77; Anal. calcd. for C18H15N7O (345): C,
62.60; H, 4.38; N, 28.39%. Found: C, 62.51; H, 4.44; N, 28.24%.
N‐(Naphthalen‐2‐yl)‐2‐(tetrazolo[1,5‐a]quinolin‐4‐ylmethy-
lene)hydrazinecarboxamide (19e)
|
4.2.2
In vitro cytotoxic screening against normal
Yield (40%); mp 271–272°C; IR (cm–1): 3301 (NH), 1691 (C=O), 1,573
(C=N); 1H NMR: δ 7.46–7.61 (m, 2H, Ar–H), 7.71 (dd, J = 7.7, 7.9 Hz,
1H, Ar–H), 7.76 (d, J = 8.2 Hz, 1H, Ar–H), 7.85 (dd, J = 7.7, 7.8 Hz, 1H,
Ar–H), 7.95–8.10 (m, 3H, Ar–H), 8.22 (d, J = 7.8 Hz, 1H, Ar–H), 8.48
(s, 1H, Ar–H), 8.61 (d, J = 8.5 Hz, 1H, Ar–H), 8.67 (d, J = 8.3 Hz, 1H,
Ar–H), 8.81 (s, 1H, CH=N), 9.70 (s, 1H, Ar–H), 11.53 (s, 1H, NH);13C
NMR: 116.38, 117.42, 118.60, 120.39, 121.94, 123.94, 124.10,
125.74, 125.92, 126.13, 126.25, 128.33, 128.52, 129.70, 131.22,
131.76, 133.58, 133.71, 134.11, 145.66, 153.31; Anal. calcd. for
C21H15N7O (381): C, 66.13; H, 3.96; N, 25.71%. Found: C, 66.27; H,
3.87; N, 25.54%.
cell lines (MTT [3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5‐
diphenyl tetrazolium bromide] assay)[37]
Cell lines and reagents: The skin fibroblast cell line (BJ) and the
breast epithelial cell line (MCF‐10F) were obtained from The
American Type Culture Collection. RPMI‐1640 medium, staurospor-
ine, MTT, and DMSO (Sigma‐Aldrich, St. Louis, MO), fetal bovine
serum (Gibco, UK).
The assay depends on the ability of mitochondrial dehydrogenases in
viable cell to cleave the tetrazolium ring of MTT (yellow color) yielding
purple formazan crystals which are dissolved in acidified isopropanol
where the resulting purple solution is spectrophotometrically measured.
An increase or decrease in cell number results in a simultaneous change
in the amount of formazan created, demonstrating the degree of
cytotoxicity caused by the test compound. Cells were cultured in fresh
growth medium and incubated at 37°C for 24 hr. After that, cells were
treated with (0.39, 1.56, 6.25, 25, and 100 μg/ml) concentrations of the
tested compound and incubated for 24 hr. MTT solution was then added
in an amount equivalent to 10% of the culture medium volume and
incubated for further 4 hr; after which the resulting formazan crystals
were dissolved by adding an amount of acidified isopropanol equal to the
original culture medium volume. The absorbance was measured spectro-
photometrically at a wavelength of 570 nm using ROBONIK TM P2000
Eliza plate reader, Robonik India Pvt. Ltd, Maharashtra.
N‐(4‐Nitrophenyl)‐2‐(tetrazolo[1,5‐a]quinolin‐4‐ylmethylene)‐
hydrazinecarbothioamide (19f)
Yield (44%); mp 269–260°C; IR (cm–1): 3238 (NH), 1599 (C=C), 1556
(C=N); 1H NMR: δ 7.85 (dd, J = 7.6, 7.8 Hz, 1H, Ar–H), 8.00 (dd, J =
7.6, 7.8 Hz, 1H, Ar–H), 8.17 (d, J = 9.1 Hz, 2H, Ar–H), 8.23 (d, J = 7.9
Hz, 1H, Ar–H), 8.31 (d, J = 9.1 Hz, 2H, Ar–H), 8.64 (d, J = 8.3 Hz, 1H,
Ar–H), 8.71 (s, 1H, CH=N), 8.98 (s, 1H, Ar–H), 10.78 (s, 1H, NH); 13C
NMR: 116.45, 119.53, 123.80, 124.13, 128.66, 129.99, 130.07,
130.63, 132.14, 135.96, 143.66, 145.04, 146.20, 175.61; Anal. calcd.
for C17H12N8O2S (392): C, 52.03; H, 3.08; N, 28.56%. Found: C,
52.20; H, 3.19; N, 28.47%.
|
4.2
Biology
|
|
Enzyme inhibition assay
4.2.1
Preliminary in vitro cytotoxic screening
4.2.3
The human tumor cell lines of the cancer screening panel were grown
in supplemented RPMI 1640 medium which contains 2 mM L‐
glutamine and 5% fetal bovine serum. For a typical screening test,
cells were inoculated into 96‐well microtiter plates in 100 µl. The
Topo 1 in vitro enzyme assay
The inhibitory activities of compounds 13 and camptothecin toward
Topo 1 enzyme were evaluated using enzyme‐linked immunosorbent
assay (ELISA) technique with purified human DNA Topo 1 ELISA kit.

NASR ET AL.
15 of 16
|
TABLE 5 In silico physicochemical properties of compound 13 in comparison to camptothecin
Compounds
13
Log P
3.65
2.08
MW
n‐HBA
n‐HBD
n‐RB
TPSA
Log S
−3.79
−3.01
Lipiniski’s violation
390.41
348.36
7
6
1
1
1
1
170.15
148.23
0
0
Camptothecin
Note. Log P: Compound’s hydrophobicity; MW: molecular weight; n‐HBA: number of hydrogen bond acceptors; n‐HBD: number of hydrogen bond donors;
n‐RB: number of rotatable bonds; TPSA: topological polar surface area; Log S: solubility parameter.
Ninety‐six‐well plates were precoated by antibody specific to Topo 1
enzyme. One hundred microliter of standards or test sample were
added to the appropriate wells and incubated for 2 hr at 37°C. One
hundred microlite of biotin‐conjugated antibody was added to each
well and incubated for 1 hr at 37°C. Avidin conjugated to horseradish
peroxidase (HRP‐avidin) (100 μl) was added and the wells were
incubated for 1 hr at 37°C. 3,3′,5,5′‐Tetramethylbenzidine (TMB)
substrate solution (90 μl) was added to the wells and they were
incubated for 15−30 minutes at 37°C. Enzyme‐substrate reaction
was terminated by addition of stop solution (50 μl) to each well and
the color change was measured by the determination the optical
density of each well using a microplate reader set to wavelength 450
nm. The concentration of Topo 1 was then determined by using
standard curve.
software to identify ligand–receptor interactions and to predict its
binding affinity and binding mode with the active site.
|
4.4
In silico prediction of physicochemical
properties
Theoretical prediction of physicochemical properties for both
compound 13 and the reference drug camptothecin was performed,
using pkCSM online software.[38] Results in Table 5 revealed that
compound 13 complies with Lipinski’s rule of five with no violations,
suggesting that it has drug‐likeness compared with camptothecin,
and hence it would be a promising candidate as a lead structure for
future design of new anticancer agents.
ACKNOWLEDGMENTS
EGFR‐TK and VEGFR2 in vitro enzyme assay
Ninety‐six‐well plates were precoated with substrate peptide (poly
(Glu:Tyr 4:1) solution in case of EGFR‐TK inhibition assay and biotin‐
gastrin precursor (Tyr87) peptide for VEGFR2 inhibition assay). The
enzyme was transferred from –80°C and allowed to thaw on ice. The
enzyme reaction was conducted in kinase assay buffer (240 mM 4‐(2‐
hydroxyethyl)‐1‐piperazineethanesulfonic acid pH 7.5, 20 mM
MgCI2, 20 mM MnCI2, 12 μM Na3VO4). The prediluted test sample
(10 µl) and diluted solution of purified enzyme (20 µl) were added to
each reaction well. Adenosine‐5′‐triphosphate disodium salt solution
(50 µl, 5 mmol/l) was added to initiate the reaction, incubated at
37°C for 1 hr, and then the plates were washed with phosphate‐
buffered saline (PBS) three times. Prediluted monoclonal phospho-
tyrosine antibody in PBS (100 μl) were added to each well. After
incubation for another 1 hr at 37°C, the wells were washed with PBS
three times. The reaction was visualized by the addition of TMB.
After incubation at room temperature for 15 minutes, the reaction
was terminated by adding the stop solution and read spectro-
photometrically at 450 nm. The enzyme concentration was then
determined by using standard curve.
The authors wish to credit the support of National Cancer Institute
(NCI), developmental therapeutics program (DTP) of the United
States for performing the screening of compounds.
CONFLICT OF INTERESTS
The authors declare that there are no conflict of interests.
ORCID
Amany S. Mostafa
http://orcid.org/0000-0003-1909-1072
http://orcid.org/0000-0001-9599-9248
Magda A. A. El‐Sayed
Mohammed A. M. Massoud
http://orcid.org/0000-0002-3347-3483
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