Design and synthesis of new pyranoquinolinone heteroannulated to triazolopyrimidine of potential apoptotic antiproliferative activity

Article abstract of DOI:10.1016/j.bioorg.2020.104392

Pyrano[3,2-c]quinoline derivatives have been synthesized and utilized to obtain various new hetero-annulated triazolopyrimidine, containing quinoline, pyran, 1,2,4-triazine and pyrimidine in good yields. Newly synthesized compounds have been characterized

Full text of DOI:10.1016/j.bioorg.2020.104392

Journal Pre-proofs
Design and synthesis of new pyranoquinolinone heteroannulated to triazolo-
pyrimidine of potential apoptotic antiproliferative activity
Mohamed Ramadan, Mohamed Abd El-Aziz, Yassin A. M. M. Elshaier,
Bahaa G.M. Youssif, Alan B. Brown, Hazem M. Fathy, Ashraf A. Aly
PII:
S0045-2068(20)31690-4
DOI:
https://doi.org/10.1016/j.bioorg.2020.104392
YBIOO 104392
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
30 July 2020
3 September 2020
15 October 2020
Please cite this article as: M. Ramadan, M. Abd El-Aziz, Y. A. M. M. Elshaier, B.G.M. Youssif, A.B. Brown,
H.M. Fathy, A.A. Aly, Design and synthesis of new pyranoquinolinone heteroannulated to triazolopyrimidine of
potential apoptotic antiproliferative activity, Bioorganic Chemistry (2020), doi: https://doi.org/10.1016/j.bioorg.
2
020.104392
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Design and synthesis of new pyranoquinolinone heteroannulated to
triazolopyrimidine of potential apoptotic antiproliferative activity
a*
b
c
Mohamed Ramadan , Mohamed Abd El-Aziz , Yassin A. M. M. Elshaier , Bahaa G.M.
d*
e
a
f*
Youssif , Alan B. Brown ; Hazem M. Fathy and Ashraf A. Aly
a
Organic Chemistry Department, Faculty of Pharmacy, Al-Azhar University, Assiut
b
Branch,71524 Assuit, Egypt; Medicinal Department, Faculty of Pharmacy, Minia
University, 61519 El Minia, Egypt; Organic and Medicinal Chemistry Department, Faculty
of Pharmacy, University of Sadat City, 32958 Menoufia, Egypt; Pharmaceutical Organic
c
e
Chemistry Department, Faculty of Pharmacy, Assiut University, Assiut, 71526, Egypt;
e
Chemistry Department, Florida Institute of Technology, Melbourne, FL 32901, USA;
Chemistry Department, Faculty of Science, Minia University, 61519 El-Minia, Egypt
f
Corresponding Authors: Prof Ashraf A. Aly, e-mail: ashrafaly63@yahoo.com; Dr Mohamed
Ramadan, e-mail: elbashamohammed@yahoo.com and Dr Bahaa G.M. Youssif, e-mail:
bgyoussif@ju.edu.eg.
1

Abstract
Pyrano[3,2-c]quinoline derivatives have been synthesized and utilized to obtain various new
hetero-annulated triazolopyrimidine, containing quinoline, pyran, 1,2,4-triazine and
pyrimidine in good yields. Newly synthesized compounds have been characterized by
spectral data and elemental analysis. Most of the synthesized compounds showed moderate to
weak antiproliferative activity on most cancer cell lines, especially leukemia and breast
cancer cell lines. The open chain formimidic acid ethyl ester is slightly more potent than
hetero-annulated systems. The most active compounds were further investigated for caspase
activation, Bax activation and Bcl-2 down regulation compared to doxorubicin as a standard,
and indeed exhibited mainly cell cycle arrest at the Pre-G1 and G2/M phases. The
transcription effects of 5a and 5b on the p53 were assessed and compared with the reference
doxorubicin. The results revealed an increase of 12-19 in p53 level compared to the test cells
and that p53 protein level of 5a and 5b was significantly inductive (991, and 639 pg / mL,
respectively) in relation to doxorubicin (1263 pg / mL)
Keywords: Pyrano[3,2-c]quinoline, triazolopyrimidine, formimidic acid , caspase, apoptosis,
antiproliferative.
2

Highlights






A series of pyrano[3,2-c]quinoline/triazolopyrimidine hybrids was made.
1
13
15
Products were characterized by IR, MS, and multinuclear NMR ( H, C, and N).
The products showed antiproliferative activity versus cancer cell lines.
Open chain formimidic esters were more potent than heteroannulated systems.
The most active compounds were also studied for caspase activation.
Compounds 5a and 5b showed an increase of 12-19 in p53 level compared to the test
cells.

The drug likeness profile of the synthesized compounds was predicted using the Swiss
ADME website.
3

1
. INTRODUCTION
Nitrogen containing heterocyclic compounds is a notable form of anticancer drug applicant
that strongly promotes cell apoptosis [1]. Many quinoline containing compounds have been
reported as potential antitumor agents [2,3]. Quinoline skeletal perform an important aspect
in anticancer drug improvement, as their derivatives show great results through different
operations such as growth inhibitors by cell cycle arrest, apoptosis, inhibition of
angiogenesis, disruption of cell migration, and modulation of nuclear receptor responsiveness
[4]. The fused pyranoquinoline moiety is an extremely common structural motif that occurs
in many naturally occurring or biologically active alkaloids, such as flindersine, atanine,
tabouensinium chloride, zanthoxylum simulans, oricine, verprisine, and araliopsis tabouensis
[5-7]. The unique biological activity of the pyranoquinoline derivatives has made these
compounds privileged targets in recent medicinal studies. For instance, compounds that
contain a pyranoquinoline nucleus have been frequently used for bactericidal and
bacteriolytic activities [8,9], acetylcholinesterase inhibition [10], antiallergenic [11], anti-
inflammatory [12], antimalarial [13], calcium-signalling inhibition [14], platelet aggregation
[6] and antitumor activities [15-18]. Recently, a pyranoquinoline nucleus has been employed
in the pharmacophores with potential medicinal features against various diseases, like
Alzheimer’s [19], venereal [5] and psychotropic [20].
The triazolo-pyrimidine moiety has been extensively used as a template in medicinal chemistry
for its diverse pharmacological properties. Due to its ability to bind to a wide range of enzymes
and receptors through various non-covalent interactions, triazolo-pyrimidines have been
extensively investigated and are known to have a versatile biological potential [22]. A series
of triazolopyrimidine derivatives have been found to be potentially useful as cyclin-dependent
kinase inhibitors [23] and have also shown antiproliferative activity on the cancer cell line [24].
Moreover, some triazolo-pyrimidines were reported to enhance the antitumor activity [25]. The
4

significance of the triazolo-pyrimidine moiety in the cytotoxic activity of certain derivatives
26] and their incorporation into many complex systems has been reported [21], in addition,
[
these derivatives proved to be active as anticancer agents [27]. In addition, triazolo-pyrimidine
derivatives were also found to induce apoptosis and/or reduce cell proliferation in different
solid tumors and several human cancer cell lines [28]. We reported the synthesis of ethyl 5,6-
dihydro-2,5-dioxo-6,9-disubstituted-2H-pyrano[3,2-c]quinoline-4-carboxylates I, Figure 1
from the reaction of equimolar amounts of 2,4(1H,3H)-quinolinediones and diethyl acetylene
dicarboxylate in absolute ethanol, containing catalytic triethylamine [29]. In previous work
with quinolones, Aly et al. synthesized various classes of 2-quinolones such as 2'-amino-2,5'-
dioxo-5',6'-dihydro-spiro(indoline-3,4'-pyrano[3,2-c]quinoline)-3'-carbonitriles II [30], 3-
(methylthio)-4-oxo-4,5-dihydrofuro[3,2-c]quinolone-2-carbonitriles
III
[31],
naphtho[2',3':4,5]furo[3,2-c]quinoline-6,7,12(5H)-trione derivatives IV (as ERK inhibitors
with efficacy in BRAF-mutant melanoma) [32], 2,3-bis-(4-hydroxy-2-oxo-1,2-
dihydroquinolin-3-yl)succinates, and arylmethylene-bis-3,3’-quinoline-2-ones V [33], and N-
2
,3-bis(6-substituted-4-hydroxy-2-oxo-1,2-dihydroquinolin-3-yl)naphthalene-1,4-diones VI
[34] , Figure 1.
5

R1
CN
O
N
R2
O
O
O
O
R1
SCH3
R2
HN
CO Et
2
O
R2
N
O
H
N
O
III
NC
NH2
R1
II
I
R1
O
N
O
O
O
O
O
Ar
O
R2
R2
NH
O
OH
HN
O
OH
OH
HO
N
O
N
R1
R1
IV
V
VI
R2
R2
R2
Figure 1. Chemical structure of some previously reported Quinolones I-VI
In view of the important properties described above, the synthesis of heterocyclic molecules
containing these nuclei has become increasingly important in organic synthesis. Encouraged
by their potential clinical applications and in continuation of our work on bio-potent
heterocycles, our efforts are focused to design and synthesize newer biologically potent
heterocyclic systems via combination of the two therapeutically active moieties
pyranoquinoline and triazolopyrimidine together in a single scaffold. The cytotoxic activity
of the synthesized compounds against the NCI-60 cancer cell line panel was determined and
the most effective inhibitors were further investigated for Caspases 3, 8, 9 and Cytochrome
C, as well as the enzymatic activity on p53 as a possible plausible mechanism for their
anticancer efficacy.
6

2
. Results and discussion
2
.1.Chemistry
Synthesis of compounds 5a-e, 8a-e, and 9a-e was illustrated in Scheme 1. Equimolar
amounts of 1,6-disubstituted quinoline-2,4-(1H,3H)-diones 1a-e and 2-(4-
chlorobenzylidene) malonitrile 2 refluxed in absolute ethanol containing piperidine as a
basic catalyst, provided the pyranoquinoline derivatives 3a-e as yellow powders in excellent
yields. Elemental analysis and spectral data agreed well with the assigned structures 3a-e
(Scheme 1).
The IR spectrum of compound 3c, for example, showed characteristic absorption bands at
-1
max = 3387, and 2234 cm corresponding to the NH and the C≡N groups, respectively.
2
1
The H NMR spectrum of compound 3c displayed a characteristic singlet at δ 7.30 assigned
H
to NH -pyran and two singlets at δ 2.39 and 4.51 assigned to methyl group and CH-pyran,
2
H
1
3
respectively. The C NMR spectrum of 3c showed a characteristic signal at δ 191.61
C
assigned to C-CN and two carbon signals in the aliphatic region at δ 57.30 and 36.24
C
assigned to C-3 and C-4 of the pyran ring, respectively. The mass spectra and elemental
analyses indicated that the molecular weight is consistent with the molecular formula of the
target compounds.
Subsequent treatment of 3a-e with an excess of orthoformic esters 4a,b in the presence of
acetic anhydride afforded the desired formimidic acid esters 5a-e. The assignment of the
1
13
structures of 5a-e was established based on their spectral data (IR, H NMR, C NMR and
MS) and elemental analyses. For example, the IR spectrum of compound 5c showed
disappearance of the characteristic band of NH and appearance of absorption bands at 
2
maz
-1
1
=
3154, 2243 cm characteristic of C=H and C≡N, respectively. The H NMR spectrum
revealed absence of singlet at δ 7.30 characteristic of NH -pyran and appearance of
H
2
distinctive three sets of alkene and ethoxy signals at δH 8.94, 4.37 and 1.34 corresponding to
7

H-2b, H-2d and H-2e, respectively; the attached carbons appear at  159.65, 63.48 and
C
1
3.34 for C-2b, C-2d and C-2e, respectively, which confirmed the nucleophilic attack of the
lone pair of electrons of pyran-NH on orthoformic ester.
2
NH2
CN
NC CN
O
OH
R1
R2
R1
R2
EtOH, Pip.
reflux, 2h
+
N
R3
O
Cl
N
R3
O
Cl
1a-e
2
3a-e
reflux
OR
2O,
1
2
3
1
1
1
1
1
a= R = R =H, R = CH
3
Ac
RO OR
1
2
3
b= R = R , R = H
4
4
a= CH3
b=C H
1
2
3
c= R = CH , R = R = H
3
OR
2
5
1
2
3
d= R = Cl, R = R = H
N
1
2
3
e R = Br, R = R = H
CN
1
2
3
5
a= R = R =H, R = R = CH
O
3
1
2
3
R1
5b= R = R = R = H, R= CH
3
1
2
3
5
c= R = CH , R = R = H, R= C H
3 2 5
R2
N
R3
O
Cl
1
2
3
5
d= R = Cl, R = R = H, R= CH
3
H 2
1
2
3
5e R = Br, R = R = H, R= C H
2
5
5a-e
O
O
AcOH,
CONHN
6
7
3
NHNH2
reflux
CH
reflux
AcOH,
N
N
N
O
N
N
N
CH3
N
N
O
O
R1
R1
R2
8
a-e and 9a-e
R2
N
R
O
Cl
1
2
3
Cl a= R = R = H, R = CH3
N
R
O
3
3
1
2
3
b
=
R = R = R = H
1
2
3
8
a-e
9a-e
c= R = CH , R = R = H
3
1
2
3
d= R = Cl, R = R = H
1
2
3
e= R = Br, R = R = H
Scheme 1. Synthesis of the compounds 5a-e, 8a-e, and 9a-e.
The reaction mechanism presumably starts by proton abstraction by piperidine then Michael
addition and intramolecular cyclization, isomerization, followed by nucleophilic attack of the
lone pair of electrons of pyran-NH on orthoformic ester 4 to afford the corresponding products
2
5
a-e (Scheme 2).
8

N
C
N
OH
CN
Ar
O
O
CN
Ar
O
piperidine
HH
2
- H
N
H
1
O
N
H
O
N
H
O
N
H
O
OR
OR
CN
OR
H
NH
NH2
N
N
CN
Ar
CN
Ar
RO OR
CN
Ar
O
O
O
O
+
4
OR
ROH
Ar
-
-ROH
N
O
N
H
O
N
H
O
N
H
O
H
3
5
Scheme 2. Suggested mechanism for the formation of compounds 3a-e and 5a-e.
The new compounds 5a-e could enter cascade heterocyclization with acetohydrazide 6 or
furan-2-carbohydrazide 7 to form triazolopyrimidine hybrids 8a-e and 9a-e respectively, as
shown in Scheme 1. The cyclization was established by recording its IR, NMR and MS
spectra. As a representative example for series 8a-e, the IR spectrum of 8a showed the
absence of nitrile and carbonyl groups; this gives a strong indication of cyclization of 5a to
1
form 8a. Also, the H NMR spectrum of compounds 8c and 9c showed the absence of ethoxy
signals at δH 4.37 and 1.35 for H-2d and H-2e, respectively. In addition, they showed singlet
signals at δH 10.08 and 10.14 integrating for one proton of pyrimidine ring of compound 8c
and 9c, respectively. The smooth cyclization of 8a was further confirmed by characteristic
1
3
C NMR signals at  161.75 (C-2), 149.73 (C-14b), 144.02 (C-5), and 104.61 (C-14a).
C
Additionally, all five nitrogens can be observed: N-12 gives HMBC correlation with H-11,
N-1 and N-3 correlate with H-2a. N-4 and N-6 correlate with H-5. N-1 is differentiated from
1
N-3, and N-4 from N-6, based on hybridization. On the other hand, the H NMR spectrum of
9
c showed three singlets at δH 8.22, 8.05 and 7.19 corresponding to the furan ring protons H-
2
d, H-2b, and H-2c respectively, and showed only three nitrogens, two of which correlate
with H-5. Finally, the structure of the compound 8c and 9c was confirmed by their mass
spectra, which showed molecular ion peaks at m/z = 429 and 481 which are consistent with
the molecular formula C H ClN O and C H ClN O , respectively (Table 1).
23
16
5
2
26 16
5
3
9

5
3
N
6
N
4
N
2a
6a
2
7
1
4b
N
1
O
1
4a
9a
8
7a
9
7b
i
1
4
1
3a
10
o
1
1a N 13
O
p
Cl
11
m
H
12
8
c
Table 1. NMR spectroscopic assignments of compound 8c
1
COSY
Assignment
NH-12
HNMR (DMSO-d
6
)
1
1
8
2.05 (s; 1H)
0.08 (bs; 1H)
.73 (d, J = 1.6; 1H)
H-5
H-8
8.24, 3.09
8.73, 8.07
8.24
8
8
.24 (d, J = 8.2; 1H)
.07 (d, J = 8.5; 1H)
H-10
H-11
7
7
6
3
.85 (d, J = 7.9; 2H)
.74 (d, J = 7.8; 2H)
.44 (s; 1H)
7.74
7.85
H-m
H-o
H-14
.22 (s; 3H)
H-2a
3
.09 (s; 3H)
8.73
H-9a
HSQC:
HMBC:
Assignment
13
6
C NMR (DMSO-d )
6
.38, 4.33
C-13
1
1
1
1
1
1
1
64.0
61.75
60.49
58.17
49.73
44.02
41.11
3.22
C-2
10.08, 6.44
8.73, 8.07, 6.44
10.08
10.08
8.07, 3.09
8.73, 7.85, 7.74
C-6a
C-7a
C-14b
C-5
C-9
C-10
10.08
8
.24
1
38.72
8
.73, 7.74
C-i
1
1
1
1
1
1
1
1
1
1
3
38.58
37.58
36.72
32.04
31.88
23.96
19.47
16.26
07.17
04.61
7.39
8.73, 7.74
8.73, 8.24, 6.44
7.85, 6.44
7.74, 6.44
8.24,3.09
C-p
C-11a
C-m
C-o
C-8
C-11
C-7b
C-13a
C-14a
C-14
7.85
7.74
8.73
8.07
8.07
6.44
10.08, 6.44
7.85
6.44
2
1
1
1.90
2.59
5
3.09
8.73, 8.24, 3.09
3.22
C-9a
3.22
C-2a
HSQC
HMBC:
Assignment
NNMR (DMSO-d
6
)
2
2
2
1
63.5
3.22
N-3
N-6
N-4
N-1
43.9
26.0
49.6
10.08
10.08
3.22
1
47.4
8.07
N-12
1
0

The heterocyclization starts by nucleophilic attack of the hydrazide amino lone pair of electrons
of compounds 6 and 7 on the imine carbon of 5 with elimination of a molecule of alcohol,
followed by a second nucleophilic attack on the nitrile carbon, then intramolecular cyclization
of imine lone pair on carbonyl carbon with extrusion of a water molecule to afford compounds
8
a-e and 9a-e (Scheme 3).
OR
H H
N
O
H
N
O
O
N
H N
2
N
N
N
N
C
N
R
R
CN
R
CN
Ar
H
O
O
6,7
O
NH
Ar
Ar
N
H
O
N
H
O
N
H
O
H
5
N
OH
N
N
N
N
N
N
R
N
R
O
O
-
H O
2
Ar
Ar
N
H
O
N
O
H
8
,9
Scheme 3. Plausible mechanism for the formation of compounds 8a-e and 9a-e.
2
.2. Evaluation of biological activities
2
.2.1. Screening of antiproliferative activity by NCI
The target compounds 5a-e, 8a-e and 9a-e were selected by the National Cancer Institute [NCI,
Bethesda, MD, USA (http://www.dtp.nci.nih.gov.)] for evaluation at a single concentration of
1
0 µM towards a panel of sixty cancer cell lines of nine diverse tissues according to NCI
protocol [35, 36].The screening results were reported as the percent growth inhibition of treated
cells compared to untreated control cells. Results in Table 2 revealed that most compounds
showed weak to moderate activities on most cell lines especially, leukemia and breast cancer
cell lines. Moreover, the results revealed that, the open chain formimidic acid ethyl ester 5a-e
exhibited variable degrees of growth inhibitory activity towards a majority of the tested cancer
cell lines than the cyclized triazolo-pyrimidine derivatives 8a-e and 9a-e. Additionally, it is
1
1

obvious that the electronic effect on both position of substituent on the tested scaffolds had a
pronounced effect on their cytotoxic activity. Compound 5a with quinoline N-methyl (electron
donating group) showed more antiproliferative activity on a majority tested leukemia and
breast cancer cell lines than compounds 5b and 5d with unsubstituted or electron withdrawing
group respectively (Table 2). Compound 5a showed promising activities against SR, K-562,
MDA-MB-468, MDA-MB and MCF7 achieving 77.26, 80.38, 46.02, 48.75 and 58.30 % of
growth inhibition respectively; However, compound 5b and 5d showed weak cytotoxic
activities against the same cell lines. On the other hand, triazole-2-methyl derivatives 8a-e and
triazole-2-furan 9a-e were less potent than the open chain derivatives 5a-e. Exceptionally,
compound 8a showed moderate antiproliferative activity against RPMI-8226 and HOP-92
achieving 48.89 and 48.91% of growth inhibition respectively.
Table 2: Percentage growth inhibition (GI %) of in vitro subpanel tumor cell lines at 10 μM
concentration for compounds 5a-e, 8a-e, and 9a-e.
Subpanel
cancer cell
Lines
% Growth Inhibition (GI %) a
5
a
5b
5c
5d
5e
8a
8b
8c
8d
8e
9a
9b
9c
9d
9e
Leukemia
CCRF-CEM
HL-60(TB)
K-562
33.12 26.12
46.57 27.34
80.38 20.04
24.81 11.92
11.90 41.62
77.26 37.74
15.24 15.95 12.74 23.80
18.27 18.31 11.36 33.79
22.10 19.25 11.90 21.50
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
13.45
13.65
11.07
-
-
-
-
-
-
-
-
17.70
15.64
-
10.10
-
8.44
-
MOLT-4
RPMI-8226
SR
-
11.30
-
24.14
-
-
-
-
13.89
-
35.12 33.79 69.81 48.89 13.79 30.74
-
19.07 25.22 13.32 16.65
-
-
-
-
31.47
Non-small cell lung cancer
A549/ATCC
EKVX
36.35 25.15
10.05
-
-
13.62
-
-
-
-
-
-
-
-
-
-
-
-
-
-
14.54
-
-
-
-
-
-
-
-
-
-
26.02 51.18
35.37 50.04
20.83 62.30
18.04 18.32 30.46 22.72 13.90 21.29 11.84
9.01
2.21
-
HOP-62
25.26 19.40 29.26 15.39 12.72 13.67
38.58 38.91 49.25 48.91 25.85 34.83
-
-
-
-
-
-
-
HOP-92
12.06 21.53 29.77
31.91
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
-
34.54
15.58 18.36 21.07 13.11
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
19.67 23.92
14.08 24.70
20.12 14.92
13.70 12.90 14.63
-
-
-
-
-
-
-
12.01
-
1
2

NCI-H522
Colon cancer
COLO 205
HCC-2998
HCT-116
HCT-15
41.29 30.65
27.90 21.49 25.84 24.06 12.40 16.94
-
-
-
-
16.00 24.84
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
12.36
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
13.44
-
-
-
-
-
-
-
-
18.35 32.49
16.13 35.05
25.49 27.02
11.18
17.59 16.05 11.18 15.12
11.12
-
-
-
-
-
14.16
-
-
-
-
-
-
-
-
-
-
-
-
HT29
19.50 13.66
KM12
-
-
-
-
-
SW-620
16.61 26.37
15.40
CNS cancer
SF-268
20.19 25.67
23.11 12.07
13.62 34.54
13.62 24.20
12.61 19.18
14.31
-
-
-
-
-
16.67 14.31 15..06
-
-
-
-
-
11.6
-
-
-
-
-
-
-
-
-
-
11.68
-
-
-
-
-
-
-
-
-
-
SF-295
-
-
-
-
-
-
-
-
-
-
-
-
SF-539
24.08 16.41
13.43
22.73
-
-
13.5
-
-
13.58
-
SNB-19
11.79 15.26 20.47
U251
-
-
18.06
Melanoma
LOX IMVI
MALME-3
M14
12.60 23.26
14.18 22.51
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
13.17
-
-
-
-
-
-
-
-
-
-
11.02
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
18.24 16.08
12.02
-
-
-
-
25.06
14.84
10.92
-
12.90
-
-
-
-
-
-
-
-
-
-
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
Ovarian cancer
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
Renal cancer
A498
-
-
-
11.56 32.63
12.85 14.78
-
-
-
-
-
26.81 40.22
53.55
29.66 24.88
26.57 55.97
-
-
-
-
-
-
-
-
-
-
-
-
-
40.94
35.75
31.36
14.85
13.69
42.71
-
-
-
-
-
-
-
-
-
-
-
-
-
-
18.44 26.18
-
-
-
-
-
-
-
11.27
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
18.31
-
-
-
-
24.74 32.47
12.54 26.40
28.24 20.09
2.43
16.17
-
11.15
-
13.17
-
-
-
20.69
11.79
40.16
-
14.80
-
-
-
3.12
-
-
-
-
-
19.52 18.22
11.48
34.06 40.39
13.66 34.57
41.76 53.67
25.39 40.89
16.72 25.37
-
-
-
-
-
-
-
52.06
24.27
100.28
43.17
25.37
-
-
-
-
-
-
-
-
-
13.54 44.14
12.36 14.70
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
ACHN
-
-
-
CAKI-1
16.16
16.16 25.90 30.78
42.23
24.51
RXF 393
SN12C
-
-
-
-
-
-
26.55 12.64
19.90 20.64
-
22.14
-
-
-
-
-
TK-10
-
-
-
-
UO-31
36.13
65
35.49 15.14 45.81 20.92 35.49 37.11 39.27
33.15 15.14
Prostate cancer
PC-3
13.77 27.89
16.64
-
42.25
-
16.64 24.23 15.16
-
16.84
-
11.79
-
-
1
3

DU-145
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
Breast cancer
MCF7
58.30 31.29
8.75 51.24
34.77 64.39
-
12.11
-
13.34 8.01
-
-
15.74 19.74
-
-
13.23 15.38
-
-
-
-
-
MDA-MB-
4
-
21.69
12.11 28.67 29.26
22.45
-
17.61
2
31/ATCC
HS 578T
BT-549
T-47D
-
-
-
2.87
4.72
-
-
-
-
-
-
-
34
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
18.64 23.12 152.73
152.73
15.06
14.23
11.83
34.91 39.11
13.97
-
10.40
3.08
13.97 24.47 26.35
15.22 10.76
-
-
MDA-MB-468 46.02 14.08
-
10%.
-
-
-
-
-
(
–): Weak activity GI
<
2
2
.2.2. In vitro anticancer activity
.2.2.1. Cell viability assay
Cell viability assay was carried out using human mammary gland epithelial cell line (MCF-
0A). MCF-10A cells were incubated with compounds 5a-e, 8a-e, and 9a-e for 4 days and 3-
4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to assess
1
(
cell viability [37, 38]. There were no cytotoxic effects in all compounds, and for most tested
compounds, more than 85% cell viability was reported at 50 μM.
2
.2.2.2. Cytotoxic activity evaluation of IC50
The tested compounds 5a-e, 8a-e, and 9a-e were evaluated for their antiproliferative activities
against four human cancer cell lines including pancreas cancer cell line (Panc-1), breast cancer
cell line (MCF-7), colon cancer cell line (HT-29) and epithelial cancer cell line (A-549) using
MTT assay [39, 40] and doxorubicin has been used as the reference compound. Graph Pad
Prism software (Graph Pad Software, San Diego, CA, USA) has been used to measure the
median inhibition concentration (IC ) for all compounds. Results were illustrated in Table 3.
5
0
Triazole-2-mehyl derivatives (8a-e) and triazole-2-furan (9a-e) showed weak to moderate
antiproliferative activity with IC ranging from 16.60 to 32.70 compared to doxorubicin IC
50
5
0
=
1.13µM, Table 3. Generally, formimidic acid ethyl ester 5a-e showed superior
antiproliferative activity compared to their triazolo-pyrimidine counterparts (8a-e) and (9a-e),
1
4

Table 3. For example, the unsubstituted formimidic acid ethyl ester derivative 5b showed an
average IC of 4.57 µM against the tested cell lines in comparison to triazole-2-methyl
5
0
derivative 8b or triazole-2-furan derivative 9b which showed average IC = 19.27 and 22.47
5
0
µM, respectively. The electronic effect of the substituents appeared in 5a-e derivatives,
electron withdrawing group Cl and Br in compounds 5d and 5e, respectively are much weaker
than unsubstituted or electron donating group in 5b and 5a, respectively. Moreover, the
position of the electron donating is important when we compare 5a and 5c, introduction of
electron donating group to quinoline nitrogen increase activity dramatically. The Formimidic
acid ethyl ester 5a (R , R = H, R = CH ) was the most potent among the synthesized
1
2
3
3
derivatives, with average IC value 2.85 µM against the four cell lines compared to the
5
0
reference doxorubicin (IC = 1.13 µM). (Table 3)
5
0
Table 3. Antiproliferative activity of compounds 5a-e, 8a-e and 9a-e and Doxorubicin
Cell
viability
%
Antiproliferative activity IC50± SEM (µM)
Comp.
A-549
MCF-7
Panc-1
HT-29
Average
(IC50)
5
a
91
84
86
89
85
87
82
2.9±0.5
4.7±0.4
7.7±0.4
10.9±0.8
8.8±3.2
30.4±3.2
19.2±1.5
2.2±0.08
4.3±0.8
7.9±0.6
10.5±1.1
8.5±2.7
30.5±2.7
18.9±1.2
3.1±0.2
4.6±0.6
7.6±0.8
10.6±0.8
8.7±3.1
30.7±3.1
19.2±2.2
3.2±0.2
4.7±0.4
7.9±0.8
10.4±1.2
8.2±3.4
31.2±3.4
19.8±2.4
2.850
4.575
7.775
10.60
8.300
30.700
19.275
5
b
5
c
5
d
5
8
e
a
8
b
8
c
90
15.7±2.5
15.6±2.9
15.8±1.9
15.8±1.6
15.725
8
d
90
79
92
96
16.9±0.3
32.5±0.2
24.5±2.6
22.3±2.5
16.8±1.6
32.1±0.1
23.6±2.2
22.9±1.8
16.6±1.5
32.4±0.2
24.6±2.9
22.5±2.3
16.9±1.1
32.9±0.6
24.8±1.4
22.2±1.4
16.800
32.474
24.375
22.475
8
e
9
a
9
b
9
c
89
18.2±0.6
17.9±0.3
18.8±0.5
18.9±0.8
18.475
9
9
d
e
91
89
--
29.4±3.6
25.5±2.6
1.21 ± 0.80
28.5±2.8
25.6±2.2
0.90± 0.62
32.6±3.5
25.6±2.9
1.41 ± 0.58
29.2±8.2
25.8±1.4
1.01 ± 0.82
29.925
25.375
1.136
Doxorubicin
1
5

2
.2.3. Activation of proteolytic caspases cascade
Caspase activation plays a crucial role in the initiation and effecting of the apoptotic process
41]. Among the caspases, caspase-3 is a chief player that cleaves multiple proteins in the cells,
[
causing apoptotic cell death [42]. Compounds 5a-c have been tested for caspase 3 activation
and compared with doxorubicin. The results revealed that compounds 5a-c showed an
improvement in caspase 3 levels by 4.7-5.9 fold compared with untreated control cells, and
that 5a was the most active compound with a remarkable over expression of caspase-3 level
(388.58 ± 4.20 pg/mL) compared to doxorubicin (503.2 ± 4.22 pg/mL) (Figure 2).
6
5
4
3
2
1
00
00
00
00
00
00
0
5
5
b
a
5c
Doxorubicin
Control
Figure 2. Caspase-3 level for compounds 5a-c and doxorubicin in human breast cancer cell
line (MCF-7)
Compound 5a was further investigated for activation of caspases 8 and caspases 9 and
cytochrome C to evaluate the effect of 5a on the intrinsic and extrinsic apoptotic pathway. It
was found that 5a increases the caspase levels 8 and 9 respectively by 5.47 and 13.79 fold,
relative to control cells, which suggests that intrinsic and extrinsic pathways are triggered with
greater effect on the intrinsic pathway as caspase 9 levels were higher [43] (Table 4).
1
6

Table 4. Effects of compounds 5a-c and doxorubicin on active Caspases 3, 8, 9 and
Cytochrome C in MCF-7 breast cancer cell line.
Compound
Number
Caspase-3
Caspase-8
Conc Fold
(ng/ml) change (ng/ml) change (ng/ml) change
Caspase-9
Conc Fold
Cytochrome C
Conc Fold
Conc
pg/ml)
388.58 ±4.20
310.47 ±2.19
345.92 ±3.92
Fold
change
5.92
(
5
a
0.93
5.47
12.83
13.79
0.548
11.91
5
b
4.73
5.27
--
--
--
--
--
--
5
c
--
--
--
--
--
--
Doxorubicin
Control
503.20 ±4.22
65.64
7.66
1
1.75
0.17
10.07
1
16.23
0.93
17.40
1
0.604
0.046
13.13
1
2
.2.4. Bax and Bcl-2 levels assay
Compounds 5a and 5b were further investigated for their effect on Bax and Bcl-2 levels using
doxorubicin as a guide against breast cancer cell line (MCF-7). The results show that
compounds 5a and 5b increased the Bax level by 29.21 and 20.84-fold greater than the control,
compared to doxorubicin 33.43-fold (Table 5). In addition, compounds 5a and 5d caused a
down-regulation of Bcl-2 protein level up to 4.68 and 2.61-fold less than the control compared
to doxorubicin 5.17-fold (Table 5).
Table 5. Bax and Bcl-2 levels for compounds 5a, 5b and Doxorubicin in MCF-7 breast cancer
cell line
Bax
Fold change Conc
Bcl-2
Fold change
Compound
Number
Conc
(
pg/ml)
(ng/ml)
5
a
2
41.30
72.14
29.21
1.085
4.68
5
b
1
20.84
33.43
1.943
0.983
2.61
5.17
Doxorubicin
Cont.
276.19
8.26
1
5.086
1.00
2
.2.5. Flow cytometric cell cycle analysis
Cell cycle analysis was performed in MCF-7 human breast cancer cell line treated with the
most active compound 5a.The percentage of cells in G0/G1 phase of the control was 53.71%
which reported a notable decrease to 34.71% upon treatment with compound 5a while the
1
7

percentage of cells in the S phase were slightly reduced with compound 5a (31.35%) compared
to the control (37.42%) (Figures 3). The percentage of MCF-7 human breast cancer cell line
at the G2/M phase was apparently increased to 32.14% upon treatment with 5a compared to
the control (8.87%). Moreover, it was observed that the apoptotic cell percentage in the Pre-
G1 phase was increased from 1.93% for control untreated cell to 21.34% and 22.17% in cells
treated with 5a and doxorubicin, respectively (Figures 3 and 4). According to the above results,
compound 5a exhibited mainly cell cycle arrest at the Pre-G1 and G2/M phases. In addition,
the compound studied is not cytotoxic but antiproliferative which induces programmed cell
death and cell cycle arrest.
Apoptosis
25
20
15
10
5
0
5
a/MCF7
cont.MCF7
%
Total
%Early
%Late
%Necrosis
Apoptosis
Figure 3. Apoptosis induction analysis using Annexin V/PI for compound 5a
1
8

Figure 4. Cell cycle analysis and Apoptosis induction analysis of compound 5a on MCF-7.
.2.6. Effect of compounds 5a and 5b on p53 transcription
2
The transcription effects of 5a and 5b on p53 as a possible plausible mechanism for their
anticancer efficacy have been evaluated and compared to reference doxorubicin, (Table 6). The
results showed an increase of 12-19 in p53 relative to the test cells and that p53 protein levels
of 5a and 5b were significantly inductive (991 and 639 pg / mL, respectively) in relation to
doxorubicin (1263 pg / mL). A 19-fold increase in the amount of p53 compared with untreated
cells in the most active compound 5a.
1
9

Table 6. Effects of compounds 7a, 7c, 11 and doxorubicin on p53 in MCF-7 cell
line.
Compound
Number
p53
Conc. (pg/ml)
Fold change
19.33
5
a
991
639
5
b
12.40
Doxorubicin
Control
1263
51.65
24.45
1
2
.2.7. Drug likeness profile
Assessment of absorption, distribution, metabolism, and excretion (ADME) became an early
routine in drug discovery programs where the availability of computer models constitutes
valid alternatives to experiments. The drug likeness profile of the tested compounds 5a-e, 8a-
e and 9a-e were predicted using the Swiss ADME website [44]. The results of the drug
likeness profile of these compounds are shown in Tables 7,8. All the compounds were
predicted to have high oral absorption except 9e due to high molecular weight 546 and molar
refractivity 133. All the tested compounds showed no violation to Lipinski (Pfizer) filters
except one violation for compounds 8d, 8e (MlOGP  4.15), 9d and 9e (molecular weight 
5
00 [45] and no violations to Ghose except one violation for 5e and 8e, two violations for
a,c and three violations for 9d and 9e due to high WLOGP and molecular weight [46], no
9
violation for Veber (GSK) [47], Egan (Pharmacia) [48] and Muegge (Bayer) [49] filters. The
compounds were free from alerts for Pan Assay Interfering substances (PAINS) [50]. Total
polar surface area (TPSA) values for most compounds are 76.61-98.31, Table 6,7. This
consists of "good GIT absorption ". There is a correlation between the molecular weight of
compounds and their activity. In addition to low rigidity, this pattern highlighted low
molecular weight is favourable. Lipophilicity, together with the molecular weight and the
number of hydrogen bond donors and the number of hydrogen acceptors shown by these
compounds, plays the role of five.
2
0

Table 7: Molecular properties of compounds 5a-e and 8a-c predicted using Swiss ADME
website.
MOLECULE
MW
5a
405.83
5b
391.81
5c
419.86
5d
426.25
5e
484.73
8a
429.86
8b
415.83
8c
429.86
31
#
HEAVY ATOMS
29
28
30
29
30
31
30
#AROMATIC HEAVY ATOMS 16
16
16
16
16
25
25
25
FRACTION CSP3
0.14
3
0.1
0.17
4
0.1
0.14
4
0.13
1
0.09
1
0.13
1
#ROTATABLE BONDS
#H-BOND ACCEPTORS
#H-BOND DONORS
3
3
5
5
5
5
5
5
5
5
0
1
1
1
1
0
1
1
MR
111.58
76.61
3.68
3.42
4.13
2.69
4.53
3.69
-4.72
106.68
87.47
3.25
3.24
4.12
2.48
5.05
3.63
-4.54
1.14E-02
2.92E-05
116.46
87.47
3.59
3.97
4.81
2.9
111.69
87.47
3.42
3.86
4.77
2.96
5.69
4.14
-5.12
3.20E-03
7.50E-06
119.19
87.47
3.68
4.29
5.27
3.28
6.12
4.53
-5.68
1.02E-03
2.10E-06
117.8
74.31
3.58
3.76
4.22
3.99
3.59
3.83
-5.4
112.9
85.17
3.16
3.58
4.21
3.78
4.12
3.77
-5.22
2.48E-03
5.97E-06
117.86
85.17
3.4
TPSA
ILOGP
XLOGP3
3.94
4.52
3.99
4.64
4.1
WLOGP
MLOGP
SILICOS-IT LOG P
CONSENSUS LOG P
ESOL LOG S
5.97
4.25
-5.07
3.53E-03
8.42E-06
-5.52
1.30E-03
3.03E-06
ESOL SOLUBILITY (MG/ML) 7.71E-03
ESOL SOLUBILITY (MOL/L) 1.90E-05
1.69E-03
3.94E-06
ESOL CLASS
Moderately Moderately Moderately Moderately Moderately Moderately Moderately Moderately
soluble
-4.71
soluble
-4.75
soluble
-5.51
soluble
-5.39
soluble
-5.84
soluble
-5.01
soluble
-5.05
soluble
-5.43
ALI LOG S
ALI SOLUBILITY (MG/ML) 7.93E-03
6.96E-03
1.78E-05
1.30E-03
3.11E-06
1.72E-03
4.04E-06
7.01E-04
1.45E-06
4.17E-03
9.69E-06
3.67E-03
8.82E-06
1.60E-03
3.73E-06
ALI SOLUBILITY (MOL/L)
ALI CLASS
1.95E-05
Moderately Moderately Moderately Moderately Moderately Moderately Moderately Moderately
soluble
-6.84
soluble
-7.29
soluble
-8.05
soluble
-7.87
soluble
-8.45
soluble
-7.83
soluble
-8.28
soluble
-8.65
SILICOS-IT LOGSW
SILICOS-IT SOLUBILITY
5.91E-05
2.02E-05
3.71E-06
5.72E-06
1.70E-06
6.40E-06
2.19E-06
9.56E-07
(
MG/ML)
SILICOS-IT SOLUBILITY
MOL/L)
1.46E-07
5.16E-08
8.83E-09
1.34E-08
3.52E-09
1.49E-08
5.27E-09
2.22E-09
(
SILICOS-IT CLASS
Poorly
soluble
High
Poorly
soluble
High
Poorly
soluble
High
Poorly
soluble
High
Poorly
soluble
High
Poorly
soluble
High
Poorly
soluble
High
Poorly
soluble
High
GI ABSORPTION
BBB PERMEANT
No
No
No
Yes
Yes
No
Yes
-6.35
0
No
No
Yes
Yes
Yes
No
Yes
-6.39
0
No
No
No
Yes
Yes
No
Yes
-6.04
0
No
No
Yes
Yes
Yes
No
Yes
-6.16
0
No
No
Yes
Yes
Yes
No
Yes
-6.21
0
Yes
Yes
No
Yes
Yes
No
No
-6.25
0
No
Yes
No
Yes
Yes
No
No
-6.29
0
No
Yes
No
Yes
Yes
No
No
-6.12
0
PGP SUBSTRATE
CYP1A2 INHIBITOR
CYP2C19 INHIBITOR
CYP2C9 INHIBITOR
CYP2D6 INHIBITOR
CYP3A4 INHIBITOR
P
LOG K (CM/S)
LIPINSKI #VIOLATIONS
GHOSE #VIOLATIONS
VEBER #VIOLATIONS
EGAN #VIOLATIONS
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
2
1

MUEGGE #VIOLATIONS
BIOAVAILABILITY SCORE
PAINS #ALERTS
0
0
0
0
0
0
0
0
0.55
0.55
0.55
0.55
0.55
0.55
0.55
0.55
0
2
1
0
2
1
0
2
2
0
2
2
0
2
2
0
0
2
0
0
2
0
0
2
BRENK #ALERTS
LEADLIKENESS
#
VIOLATIONS
SYNTHETIC ACCESSIBILITY 4.15
4.08
4.29
4.08
4.19
3.86
3.75
3.86
Table 8: Molecular properties of compounds 8d,e and 9a-e predicted using Swiss ADME
website.
MOLECULE
MW
8D
450.28
8E
494.73
9A
481.89
9B
467.86
9C
481.89
9D
502.31
9E
546.76
#HEAVY ATOMS
31
31
35
34
35
35
35
#AROMATIC HEAVY
25
25
30
30
30
30
30
ATOMS
FRACTION CSP3
0.09
1
0.09
1
0.08
2
0.04
2
0.08
2
0.04
2
0.04
2
#ROTATABLE BONDS
#H-BOND ACCEPTORS
#H-BOND DONORS
5
5
6
6
6
6
6
1
1
0
1
1
1
1
MR
117.91
85.17
3.38
4.21
4.87
4.26
4.76
4.29
-5.81
6.90E-04
120.6
85.17
3.49
4.27
4.98
4.36
4.79
4.38
-6.13
3.68E-04
130.54
87.45
3.88
4.13
5.18
3.65
3.97
4.16
-5.93
5.64E-04
125.63
98.31
3.56
3.94
5.17
3.45
4.5
130.6
98.31
3.78
4.31
5.47
3.65
5.02
4.45
-6.05
4.34E-04
130.64
98.31
3.76
4.57
5.82
3.91
5.13
4.64
-6.34
2.32E-04
133.33
98.31
3.89
4.63
5.93
4.01
5.17
4.73
-6.65
1.23E-04
TPSA
ILOGP
XLOGP3
WLOGP
MLOGP
SILICOS-IT LOG P
CONSENSUS LOG P
ESOL LOG S
ESOL SOLUBILITY
4.12
-5.74
8.44E-04
(
MG/ML)
ESOL SOLUBILITY
MOL/L)
1.53E-06
7.44E-07
1.17E-06
1.80E-06
9.01E-07
4.62E-07
2.24E-07
(
ESOL CLASS
Moderately Poorly
Moderately
soluble
-5.67
Moderately
soluble
-5.7
Poorly
soluble
-6.09
Poorly
soluble
-6.36
Poorly
soluble
-6.42
soluble
-5.71
soluble
-5.77
ALI LOG S
ALI SOLUBILITY
8.81E-04
8.39E-04
1.02E-03
9.24E-04
3.93E-04
2.20E-04
2.08E-04
(
MG/ML)
ALI SOLUBILITY
MOL/L)
1.96E-06
1.70E-06
2.12E-06
1.98E-06
8.16E-07
4.39E-07
3.80E-07
(
ALI CLASS
Moderately Moderately Moderately
Moderately
soluble
-9.57
Poorly
soluble
-9.94
Poorly
soluble
-10.15
Poorly
soluble
-10.33
soluble
-8.86
soluble
-9.05
soluble
-9.12
SILICOS-IT LOGSW
SILICOS-IT SOLUBILITY
6.20E-07
4.41E-07
3.67E-07
1.26E-07
5.49E-08
3.56E-08
7.10E-11
Insoluble
2.55E-08
4.66E-11
Insoluble
(
MG/ML)
SILICOS-IT SOLUBILITY
MOL/L)
1.38E-09
8.91E-10
7.62E-10
2.69E-10
1.14E-10
(
SILICOS-IT CLASS
Poorly
soluble
High
Poorly
soluble
High
Poorly
soluble
High
Poorly
soluble
High
Poorly
soluble
High
GI ABSORPTION
BBB PERMEANT
PGP SUBSTRATE
CYP1A2 INHIBITOR
CYP2C19 INHIBITOR
High
No
Low
No
No
Yes
No
Yes
No
Yes
No
Yes
No
Yes
No
No
No
Yes
No
No
No
Yes
No
No
Yes
No
Yes
No
No
No
2
2

CYP2C9 INHIBITOR
CYP2D6 INHIBITOR
CYP3A4 INHIBITOR
Yes
No
No
-6.06
1
Yes
No
No
-6.29
1
Yes
No
No
-6.31
0
No
No
No
-6.36
0
No
No
No
-6.18
0
No
No
No
-6.12
1
No
No
No
-6.35
1
P
LOG K (CM/S)
LIPINSKI #VIOLATIONS
GHOSE #VIOLATIONS
VEBER #VIOLATIONS
EGAN #VIOLATIONS
MUEGGE
0
1
2
0
2
3
3
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
#
VIOLATIONS
BIOAVAILABILITY
SCORE
0.55
0.55
0.55
0.55
0.55
0.55
0.55
PAINS #ALERTS
0
0
2
0
0
2
0
0
2
0
0
2
0
0
2
0
0
2
0
0
2
BRENK #ALERTS
LEAD LIKENESS
#
VIOLATIONS
SYNTHETIC
3.76
3.77
4.11
4
4.11
4
4.01
ACCESSIBILITY
3
. Conclusion
Pyrano[3,2-c]quinoline derivatives have been synthesized and converted to heteroannulated
triazolo-pyrimidines, containing quinoline, pyran, 1,2,4-triazine and pyrimidine structures.
Most of the new compounds showed moderate to weak antiproliferative activity on most
cancer cell lines, especially leukemia and breast cancer cell lines. The open chain formimidic
acid ethyl ester is slightly more potent than heteroannulated systems. The most active
compounds were further investigated for caspase activation, Bax activation and Bcl-2 down
regulation compared to doxorubicin as a standard and exhibited cell cycle arrest at the Pre-G1
and G2/M phases. p53 assay results showed a 19-fold increase in the amount of p53
compared with untreated cells in the most active compound 5a.
Conflict of interest: The authors declare no conflict of interest.
2
3

4
4
. Experimental
.1. Chemistry
General details: See Appendix A
Starting materials
4
-Hydroxy-2-quinolones 1a-e [51, 52] and 2-(4-chlorobenzylidene) malononitrile 2 [53] were
synthesized according to reported literature.
.1.1. General method for synthesis of compounds 3a-e
4
To a solution of quinolone derivatives 1a-e (1 mmol) in dry ethanol (30 mL), 2-(4-
chlorobenzylidene) malononitrile 2 (0.188 g, 1 mmol) was added. The reaction mixture was
heated under reflux for 1–2 h (monitored by TLC). After reaction completion, the precipitated
solid was filtered, dried, and crystallized from methanol to afford compounds 3a-e.
4.1.1.1 2-Amino-4-(4-chlorophenyl)-6-methyl-5-oxo-5,6-dihydro-4H-pyrano[3,2-c]quinoline-3-
carbonitrile (3a) [52].
-1
Yield 0.312 g (86%); mp 280–282 °C, IR (KBr) υmax/cm 3388, 3300, 2964, 1636.
.1.1.2. 2-Amino-4-(4-chlorophenyl)-5-oxo-5,6-dihydro-4H-pyrano[3,2-c]quinoline-3-carbonitrile
3b) [51]
4
(
.
-1
Yield 0.310 g (89%); mp 310–312 °C, IR (KBr) υmax/cm 3399, 3322, 3164, 1686.
.1.1.3. 2-Amino-4-(4-chlorophenyl)-9-methyl-5-oxo-5,6-dihydro-4H-pyrano[3,2-c]quinoline-3-
carbonitrile (3c)
4
-
1
1
Yield 0.345 g (90%); mp 284–285 °C, IR (KBr) υmax/cm 3387, 2930, 2234, 1637. HNMR δ
H
1
1.72 (s; 1H; NH), 7.72 (bs, 1H; H-10), 7.42 (dd, J= 8.4, 1.7, 1H; H-8); 7.35 (d, J = 8.4, 2H;
H-m); 7.30 (bs, 2H; H-2a), 7.24 (d, J = 8.1, 1H; H-7), 7.23 (d, J = 8.5, 2H; H-o), 4.51 (s,1H;
2
4

1
3
H-4), 2.39 (s, 3H; H-9a). C NMR δC 160.22 (C-5), 158.94 (C-2), 151.07 (C-10b), 143.40 (C-
i), 135.88 (C-6a), 132.46 (C-8), 131.21 (C-p), 131.08 (C-9), 129.28 (2C-o), 128.27 (2C-m),
1
21.22 (C-10), 119.61 (C-3a), 115.29 (C-7), 111.82 (C-10a), 108.97 (C-4a), 57.30 (C-3), 36.24
+
(
C-4), 20.62 (C-6b). MS m/z (%): 363/365 (M /M+2, 22/7), 286 (73), 252 (49), 77 (100). Anal.
Calcd for C H ClN O (363.80): C, 66.03; H, 3.88; N, 11.55. Found: C, 66.33; H, 3.99; N, 11.82.
2
20
14
3
4.1.1.4. 2-Amino-9-chloro-4-(4-chlorophenyl)-5-oxo-5,6-dihydro-4H-pyrano[3,2-c]quinoline-3-
carbonitrile (3d)
-
1
1
Yield 0.337 g (88%); mp 275–277 °C, IR (KBr) υmax/cm 3397, 3312, 2186, 1648. HNMR δ
H
1
1.93 (s; 1H; H-6), 7.950 (s, 1H; H-10), 7.64 (d, J = 8.8, 1H; H-8); 7.35 (d, J = 8.5, 2H; H-m);
.35 (d, J = 8.7, 1H; H-7), 7.37 (b, 2H; H-2a), 7.24 (d, J = 8.5, 2H; H-o), 4.52 (s,1H; H-4).
NMR δC 160.18 (C-5), 158.72 (C-2), 150.28 (C-10b), 143.05 (C-i), 136.56 (C-6a), 131.32
C-8), 131.19 (C-p), 129.37 (2C-o), 128.30(2C-m), 126.16 (C-9), 121.13 (C-10), 119.44 (C-
7
13C
(
3
a), 117.36 (C-7), 113.20 (C-10a), 110.04 (C-4a), 57.23 (C-3), 36.24 (C-4). MS m/z (%):
+
3
84/386 (M /M+2, 6/2), 272 (17), 75 (85), 66 (100). Anal. Calcd for C H Cl N O
(384.22):
19
11
2
3
2
C, 59.40; H, 2.89; N, 10.94. Found: C, 59.61; H, 2.63; N, 10.75.
4.1.1.5. 2-Amino-9-bromo-4-(4-chlorophenyl)-5-oxo-5,6-dihydro-4H-pyrano[3,2-c]quinoline-3-
carbonitrile (3e)
-
1
1
Yield 0.363 g (85%); mp 279–281 °C, IR (KBr) υmax/cm 3380, 3301, 2150, 1660. HNMR δ
H
1
1.92 (s; 1H; H-6), 8.20 (s, 1H; H-10), 7.75 (d, J = 7.5, 1H; H-8); 7.34 (m, 3H; H-m, 7); 7.25
1
3
(
m, 4H; H-o, 2a), 4.51 (s, 1H; H-4). CNMR δC 160.16 (C-5), 158.72 (C-2), 150.19 (C-10b),
1
43.06 (C-i), 136.87 (C-6a), 133.85 (C-8), 131.32 (C-p), 129.38 (2C-o), 128.31(2C-m), 124.12
(C-10), 119.44 (C-3a), 117.57 (C-7), 113.84 (C-9), 113.67 (C-10a), 110.00 (C-4a), 57.24 (C-
+
3
), 36.23 (C-4). MS m/z (%): 428/430 (M /M+2, 12/4), 361 (50), 316 (56), 74 (86), 66 (100).
2
5

Anal. Calcd for C H BrClN O (428.67): C, 53.24; H, 2.59; N, 9.80. Found: C, 53.43; H, 2.91;
2
19
11
3
N, 9.62.
4
.1.2. General method for synthesis of (5a-e)
A solution of compounds 3a-e (0.001 mol) and trimethyl orthoformate 4a or triethyl
orthoformate 4b (1 ml) containing 3 drops of acetic anhydride was refluxed for 6-8 h. The
reaction mixture was cooled and then poured onto ice water. The obtained solid was
recrystallized from methanol to afford the target compounds 5a-e.
4
.1.2.1. Methyl (E)-N-(4-(4-chlorophenyl)-3-cyano-6-methyl-5-oxo-5,6-dihydro-4H-pyrano-
[3,2-c]quinolin-2-yl) formimidate(5a)
-
1
1
Yield: 0.304 g (75%); mp 235–237 °C, IR (KBr) υmax/cm 2976, 2220, 1640. HNMR δ 8.97
H
(s, 1H; H-2b), 8.24 (dd, J = 8.0, 1.2, 1H; H-10), 7.74 (ddd, J= 8.6, 7.2, 1.4, 1H; H-8), 7.59 (d,
J = 8.4, 1H; H-7), 7.39 (m, 1H; H-9), 7.38 (d, J = 8.5, 2H; H-m), 7.34 (d, J = 8.7, 2H; H-o),
1
3
4
.79 (s, 1H; H-4), 3.93 (s, 3H; H-2d), 3.55 (s, 3H; H-6b). CNMR δC 162.72 (C-2b), 159.64
(C-5), 155.92 (C-2), 150.47 (C-10b), 141.48 (C-i), 138.79 (C-6a), 131.91 (C-8), 131.10 (C-p),
1
1
30.03 (2C-o), 128.43 (2C-m), 123.09 (C-10), 122.35 (C-9), 117.06 (C-3a), 114.89 (C-7),
12.58 (C-10a), 107.25 (C-4a), 82.18 (C-3), 54.94 (C-2d), 38.27 (C-4), 29.34 (C-6b). MS m/z
+
(
%): 405/407 (M /M+2, 50/20), 291 (48), 164 (56), 49 (100). Anal. Calcd for C H ClN O
22
16
3
3
(
405.84): C, 65.11; H, 3.97; N, 10.35; Found: C, 65.44; H, 3.70; N, 10.52
.1.2.2. Methyl (E)-N-(4-(4-chlorophenyl)-3-cyano-5-oxo-5,6-dihydro-4H-pyrano[3,2-c]-
quinolin-2-yl)formimidate (5b)
.
4
-
1
1
Yield: 0.281 g (70%); mp 241–243 °C, IR (KBr) υmax/cm 3178, 2210, 1617. HNMR δ 11.86
H
(
s, 1H; H-6), 8.95 (s, 1H; H-2b), 8.12 (d, J = 7.9, 1H; H-10), 7.59 (d”t”, J = 1.2, J = 7.7, 1H;
d ”t”
H-8), 7.39 (d, J = 8.6, 2H; H-m), 7.332 (d, J = 6.8, 1H; H-7), 7.330 (d, J = 8.5, 2H; H-o), 7.27
2
6

1
3
(
“t”, J = 7.6, 1H; H-9), 4.75 (s, 1H; H-4), 3.92 (s, 3H; H-2d). CNMR δC 162.23 (C-2), 159.81
(C-2b), 155.48 (C-5), 151.02 (C-10b), 137.48 (C-8), 131.42 (C-i), 131.03 (C-p), 131.01 (C-
6
1
a), 129.50 (2C-o), 127.99 (2C-m), 122.20 (C-9), 121.64 (C-3a), 116.66 (C-4a), 114.78 (C-7),
11.42 (C-10), 107.30 (C-10a), 81.94 (C-3), 54.45 (C-2d), 37.21 (C-4). MS m/z (%): 391/393
+
(
M /M+2, 12/4), 348 (18), 238 (66), 92 (100). Anal. Calcd for C H ClN O (391.81): C, 64.38;
21
14
3
3
H, 3.60; N, 10.72. Found: C, 64.77; H, 3.89; N, 11.05
.
4
.1.2.3. Ethyl (E)-N-(4-(4-chlorophenyl)-3-cyano-9-methyl-5-oxo-5,6-dihydro-4H-pyrano-
[3,2-c]quinolin-2-yl) formimidate (5c)
-
1
1
Yield: 0.318 g (76%); mp 252–253 °C, IR (KBr) υmax/cm 3154, 2940, 2243. HNMR δ 11.77
H
(s, 1H; H-6), 8.94 (s, 1H; H-2b), 7.89 (bs, 1H; H-10), 7.41 (dd, J= 8.7, 1.4; 1H; H-8), 7.38 (d,
J = 8.5; 2H; H-m), 7.31 (d, J = 8.5, 2H; H-o), 7.23 (d, J = 8.4, 1H; H-7), 7.330 (4.72 (s, 1H; H-
1
3
4
1
1
), 4.37 (q, J = 7.0, 2H; H-2d), 2.40 (s, 3H; H-9a), 1.34 (t, J = 7.1, 3H; H-2e). CNMR δC
61.80 (C-2), 159.65 (C-2b), 155.70 (C-5), 150.87 (C-10b), 141.16 (C-6a), 135.51 (C-8),
32.25 (C-9), 131.36 (C-i), 131.02 (C-p), 129.46 (2C-o), 127.98 (2C-m), 121.34 (C-3a), 116.74
(C-4a), 114.72 (C-7), 111.29 (C-10), 107.19 (C-10a), 81.35 (C-3), 63.48 (C-2d), 37.21 (C-4),
+
1
9.91 (C-9a), 13.34 (C-2e). MS m/z (%): 419/421 (M /M+2, 18/6), 308 (22), 252 (60), 77
(
100). Anal. Calcd for C H ClN O (419.87): C, 65.80; H, 4.32; N, 10.01. Found: C, 65.42; H,
23 18 3 3
4
.68; N, 10.28
.
4
.1.2.4.
Methyl
(E)-N-(9-chloro-4-(4-chlorophenyl)-3-cyano-5-oxo-5,6-dihydro-4H-
pyrano[3,2-c]quinolin-2-yl)formimidate (5d)
-
1
1
Yield: 0.310 g (73%); mp 258–260 °C, IR (KBr) υmax/cm 3190, 2251, 1649. HNMR δ 11.98
H
(s, 1H; H-6), 9.05 (s, 1H; H-2b), 8.20 (bs, 1H; H-10), 7.63 (dd, J= 8.8, 2.2, 1H; H-8), 7.38 (d,
J = 8.3, 2H; H-m), 7.329 (d, J = 8.5, 2H; H-o), 7.326 (d, J = 8.7, 1H; H-7), 4.75 (s, 1H; H-4),
1
3
3
.91 (s, 3H; H-2d). CNMR δC 162.69 (C-2), 159.58 (C-2b), 155.39 (C-5), 151.20 (C-10b),
2
7

1
40.85 (C-6a), 136.16 (C-8), 131.49 (C-i), 131.08 (C-p), 129.54 (2C-o), 128.01 (2C-m), 126.06
(C-9), 121.36 (C-3a), 116.77 (C-4a), 116.58 (C-7), 112.66 (C-10), 108.39 (C-10a), 81.72 (C-
+
3
7
), 54.36 (C-2d), 37.20 (C-4). MS m/z (%): 426/428 (M /M+2, 78/26), 382 (32), 314 (100),
5 (87). Anal. Calcd for C H Cl N O (426.25): C, 59.17; H, 3.07; N, 9.86. Found: C, 59.54; H,
21
13
2
3
3
3.20; N, 9.68.
4
.1.2.5. Ethyl (E)-N-(9-bromo-4-(4-chlorophenyl)-3-cyano-5-oxo-5,6-dihydro-4H-pyrano-
[3,2-c] quinolin-2-yl)formimidate (5e)
-
1
1
Yield: 0.339 g (70%); mp 266–268 °C, IR (KBr) υmax/cm 3223, 2890, 2235, 1669. HNMR
δH 11.98 (s, 1H; NH-6), 9.00 (s, 1H; H-2b), 8.29 (d, J = 1.9, 1H; H-10), 7.75 (dd, J = 8.8, 2.2,
1
4
H; H-8), 7.39 (d, J = 8.5, 2H; H-m), 7.33 (d, J = 8.5, 2H; H-o), 7.28 (d, J = 8.8, 1H; H-7),
1
3
.75 (s, 1H; H-4), 4.37 (q, J = 7.1, 2H; H-2d), 1.35 (t, J = 7.1, 3H; H-2e). CNMR δC 162.71
(C-2b), 160.02 (C-5), 156.12 (C-2), 150.59 (C-10b), 141.35 (C-i), 136.99 (C-6a), 134.21 (C-
8
1
), 131.94 (C-p), 129.97 (2C-o), 128.46 (2C-m), 124.59 (C-10), 117.45 (C-3a), 117.06 (C-7),
14.20 (C-9), 113.58 (C-10a), 108.83 (C-4a), 81.77 (C-3), 63.92 (C-2d), 37.65 (C-4), 13.81
+
(
C-2e). MS m/z (%):484/486 (M /M+2, 10/10), 428 (14), 318 (100), 316 (74). Anal. Calcd for
C H BrClN O (484.73): C, 54.51; H, 3.12; N, 8.67. Found: C, 54.32; H, 3.36; N, 8.74
.
22
15
3
3
4
.1.3. General method for the synthesis of compounds (8a-e)
A mixture of formimidate derivatives 5a-e (0.001 mol) and acetohydrazide 6 (0.001 mol) in
acetic acid (30 mL) was refluxed overnight. The mixture was cooled, and the solvent was
removed under reduced pressure to afford the target compounds 8a-e and purified by
crystallization from acetic acid.
4
.1.3.1. 14-(4-Chlorophenyl)-2,12-dimethyl-12,14-dihydro-13H-[1,2,4]triazolo[1'',5'':1',6']-
pyrimido[5',4':5,6]pyrano[3,2-c]quinolin-13-one (8a)
2
8

-
1
1
Yield: 0.310 g (73%); mp 235–237 °C, IR (KBr) υmax/cm 3167, 1677. HNMR δ 10.01 (bs,
H
1
8
H; H-5), 8.90 (d, J = 8.1, 1H; H-8), 8.36 (“t”, J = 7.8, 1H; H-10), 8.18 (d, J = 8.6, 1H; H-11),
.12 (“t”, J = 7.6, 1H; H-9), 7.87 (d, J = 8.0; 2H, H-m), 7.69 (d, J = 8.0, 2H; H-o), 6.38 (s, 1H;
1
3
H-14), 4.33 (s, 3H; H-12a), 3.20 (s, 3H; H-2a). CNMR δC 165.06 (C-13), 161.27 (C-2),
1
1
60.66 (C-6a), 155.88 (C-7a), 149.56 (C-14b), 143.83 (C-5), 141.02 (C-11a), 139.28 (C-p),
38.37 (C-i), 136.25 (C-10), 132.02 (2C-m), 131.82 (2C-o), 127.75 (C-9), 125.39 (C-8), 117.72
(C-7), 116.06 (C-7b), 109.91 (C-13a), 104.94 (C-14a), 38.36 (C-14), 32.70 (C-12a), 12.50 (C-
+
2
a). MS m/z (%):429/431 (M /M+2, 56/18), 318 (100), 111 (16), 75 (58). Anal. Calcd for
C H ClN O (429.86): C, 64.27; H, 3.75; N, 16.29. Found: C, 63.94; H, 3.90; N, 16.60.
23
16
5
2
4
.1.3.2.
14-(4-Chlorophenyl)-2-methyl-12,14-dihydro-13H-[1,2,4]triazolo[1'',5'':1',6']-
pyrimido[5',4':5,6]pyrano[3,2-c]quinolin-13-one (8b)
-
1
1
Yield: 0.337 g (78%); mp 245–247 °C, IR (KBr) υmax/cm 3315, 3100, 1663. HNMR δ 12.05
H
(
s, 1H; NH), 10.06 (s, 1H; H-5), 8.90 (d, J = 7.9, 1H; H-8), 8.35 (d”t”, J = 1.0, J = 7.8, 1H;
d ”t”
H-10), 8.18 (“t”, J = 7.6, 1H; H-9), 8.12 (d, J = 8.4; 1H, H-11), 7.84 (d, J = 8.6, 2H; H-o), 7.72
1
3
(
d, J = 8.5, 2H; H-m), 6.42 (s, 1H; H-14), 3.21 (s, 3H, H-2a). CNMR δC 164.74 (C-13),
1
1
1
1
61.67 (C-2), 160.50 (C-6a), 158.04 (C-7a), 149.68 (C-14b), 143.95 (C-5), 138.61 (C-i),
38.50 (C-p), 136.69 (C-10), 136.10 (C-11a), 132.04 (2C-o), 131.82 (2C-m), 129.01 (C-9),
24.83 (C-8), 119.37 (C-11), 115.99 (C-7b), 108.78 (C-13a), 104.58 (C-14a), 37.41 (C-14),
+
2.54 (C-2a). MS m/z (%): 415/417 (M /M+2, 42/14), 304 (92), 111 (34), 75 (100). Anal.
Calcd for C H ClN O (415.84): C, 63.54; H, 3.39; N, 16.84. Found: C, 63.67; H, 3.60; N, 16.67.
2
22
14
5
4
.1.3.3. 14-(4-Chlorophenyl)-2,9-dimethyl-12,14-dihydro-13H-[1,2,4]triazolo[1'',5'':1',6']-
pyrimido[5',4':5,6]pyrano[3,2-c]quinolin-13-one (8c)
-
1
1
Yield: 0.337 g (78%); mp 250–253 °C, IR (KBr) υmax/cm 3324, 3175, 1656. HNMR δ 10.08
H
(bs, 1H; H-5), 8.73 (d, J = 1.6, 1H; H-8), 8.24 (d, J = 8.2, 1H; H-10), 8.07 (d, J = 8.5, 1H; H-
2
9