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MACEUTICSciences
AL SCIENCES
Sudhakar
al. JOURNAL OF
World
Journal
ofYPharmacy
and
Pharmaceutical
SJIF Impact Factor 5.210
Volume 4, Issue 05, 1106-1116.
Research Article
ISSN 2278 – 4357
A CONVENIENT ONE-POT MULTI-COMPONENT SYNTHESIS OF
DIHYDROPYRIMIDO [4,5-B] QUINOLINETRIONES AND
EVALUATION OF THEIR ANTICANCER ACTIVITY
Suresh C. Jadhvar, Hanmant M. Kasraliker, Santosh V. Goswami and Sudhakar R.
Bhusare*
Department of chemistry, Dnyanopasak College, Parbhani-431401, MS, India.
Article Received on
25 Feb 2015,
ABSTRACT
Revised on 21 March 2015,
Accepted on 14 April 2015
dihydropyrimido[4,5-b] quinolinetriones by one-pot four-component
An effective protocol
was
developed for
the synthesis
of
reaction of a substituted salicylaldehyde, 3-chloro-4-fluroaniline,
dimedone and barbituric acid using ionic liquid [Msim]Cl as a catalyst.
*Correspondence for
Author
All the synthesized derivatives were evaluated for inhibition of cancer
Sudhakar R. Bhusare
cell. The initial assays indicated that some of the newly synthesized
Department of chemistry,
compounds displayed significantly good inhibition activities against
Dnyanopasak College,
human breast cancer cell (MCF7), cell lines compared with the control
Parbhani-431401, MS,
India.
(Adriamysin), which might be developed as novel lead scaffold for
potential anticancer agents.
KEYWORDS: anticancer activity, one-pot synthesis, salicylaldehyde, barbituric acid,
[Msim]Cl, dihydropyrimido[4,5-b] quinolinetriones.
INTRODUCTION
Nowadays one-pot multi-component reactions (MCRs) have gained great interest in synthetic
organic chemistry due to their advantages over typical multistep synthesis. MCRs are also
more environmentally benign and atom economic as they avoid time-consuming and
protection-deprotection steps. Therefore considering these points, the expansions of new
MCRs are most considerable in the fields of organic as well as medicinal chemistry.[1-3]
Pyrimidine derivatives have attracted great attention for their biological activity and
applications in medicinal chemistry such as chemotherapy of cancer and against human
immunodeficiency virus (HIV) infection and other viral diseases.[4-8] A variety of barbituric
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acid derivatives have been broadly applied as drugs for the treatment of cancer and
osteoporosis. Moreover there is widespread attention on barbituric acid derivatives due to
their previous medical applications.[9] Dihydropyridine moiety are mostly well known in
pharmacology as L-type calcium channel blockers.[10-11] The synthesis of pyrimido[4,5b]quinoline derivatives with a potentially broad range of biological and pharmacological
properties such as antioxidant[12] antitumor[13] and antiviral[14] activities. These derivatives can
be used to make biologically active drugs such as Quinine, Camptothecin, Chloroquine and
Luotonine-A.[15]
Herein we have developed an effective protocol for the synthesis of dihydropyrimido[4,5-b]
quinolinetriones using ionic liquid [Msim]Cl as catalyst under ambient temperature condition
(Scheme 1). All the synthesized derivatives were evaluated for inhibition of cancer cell.
R
CHO
NH2
O
OH
HN
+
R
1a-e
+
+
O
Cl O
2
F
3
OH
O
O
O
NH
[Msim]Cl
N
H
O
EtOH, RT
N
N
H
O
5-aj
4
Cl
F
Scheme 1
MATERIALS AND METHOD
All solvents were used as commercial anhydrous grade without further purification. The
column chromatography was carried out over silica gel (80–120 mesh). Melting points were
determined in open capillary tube and are uncorrected. 1H spectra were recorded on a Bruker
300 MHz spectrometer in CDCl3 solvent and TMS as an internal standard. 13C NMR spectra
were recorded on a Bruker-300 MHz spectrometer in CDCl3 solvent. Mass spectra were taken
on Polaris-Q Thermoscintific GC-MS.
ANTICANCER ACTIVITY
The anti-cancer activity for these compounds was done in the Anti-cancer drug screening
facility (ACDF), Tata memorial centre, Aadvanced centre for treatment, research and
education in cancer (ACTREC). The in-vitro anti-cancer activity for the corresponding
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compounds and ADR (Adriamysin or doxorubicin) taken as a known drug, tested using SRB
(sulforhodamine B) assay protocol as exactly described by Skehan P. et al. Briefly, SRB is a
dye binds to the protein. The human breast cancer cell line MCF7 cultured in 96 well plate
treated with different concentrations of given compounds (10, 20, 40 and 80 µg/ml). After
treatment the cells were fixed in trichloroacetic acid and stained using sulforhodamine B
(0.4% wt/vol) prepared in 1% acetic acid for 30 minutes. Four washes with 1% acetic acid
were given to remove unbound dye. 10 mM unbuffered tris base was used to extract protein
bound dye and subjected for microtiter plate reader. The absorbance of dye was measured at
wavelength 565 nm. The absorbance is correlated with the net protein synthesis rate. 50%
inhibition of cell growth (GI50), 50% cell kill or lethal concentration (LC50) and 100%
(total) growth inhibition (TGI) was calculated. The GI50 value <10 µg/ml is considered to
demonstrate activity in case of pure compound. This experiment was done in triplicate and
the average values were plotted against % control growth versus drug concentrations.
EXPERIMENTAL
General
procedure
for
the
one-pot
synthesis
of
dihydropyrimido[4,5-b]
quinolinetriones: In a round-bottom flask, a dimedone (1 mmol), barbituric acid (1 mmol),
substituted salicylaldehyde (1 mmol) and 3-chloro-4-fluro aniline (1mmol) in solvent ethanol
(15 mL) were mixed and stirred at room temperature. Ionic liquid [Msim]Cl (10 mol%) was
added as catalyst and reaction mixture was stirred at room temperature for appropriate time
(Table 2). After the completion of reaction indicated by TLC (monitoring using petroleum
ether:ethyl acetate 8:2), the reaction mixture was diluted with 15 mL of water and extracted
with the ethyl acetate. The organic layer was dried over anhydrous Na2SO4, concentrated and
the resulting residue was purified by column chromatography using silica gel mesh 80-120 to
afford pure product.
10-(3-chloro-4-fluorophenyl)-8,9-dihydro-5-(2-hydroxy-3,5-diiodophenyl)-8,8-dimethyl
pyrimido[4,5-b]quinoline-2,4,6(1H,3H,5H,7H,10H)-trione (5b):
1
H NMR (300 MHz,
CDCl3): δ 9.98 (s, 2H, 2 x NH), 7.21-7.24 (m, 2H), 6.98-7.02 (m, 3H), 5.62 (s, 1H, CH), 5.38
(s, 1H, OH), 3.10 ( s, 2H, CH2), 2.78 (s, 2H, CH2), 1.39 (s, 6H, 2 x CH3 );
13
C NMR (300
MHz, CDCl3): δ 26.0, 34.8, 39.4, 46.1, 58.6, 72.8, 109.0, 114.7, 118.6 120.8 124.0, 128.2
130.9, 135.2, 139.4, 146.2, 151.8, 156.4, 159.5, 163.4, 165.2, 185.4; GC-MS, m/z: 733 (M+);
Anal. Calcd for C25H19ClFI2N3O4: C, 40.93; H, 2.61; N, 5.73; Found: C, 40.95; H, 2.65;N,
5.76.
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5-(5-bromo-2-hydroxyphenyl)-10-(3-chloro-4-fluorophenyl)-8,9-dihydro-8,8-dimethyl
pyrimido[4,5-b]quinoline-2,4,6(1H,3H,5H,7H,10H)-trione (5d):
1
H NMR (300 MHz,
CDCl3): δ 9.78 (s, 2H, 2 x NH), 7.12-7.18 (m, 3H), 6.92-6.96 (d, 1H, J = 4.6 Hz), 6.88-6.90
(m, 2H), 5.65 (s, 1H, CH), 5.30 (s, 1H, OH), 3.02 ( s, 2H, CH2), 2.67 (s, 2H, CH2), 1.35 (s,
6H, 2 x CH3 );
13
C NMR (300 MHz, CDCl3): δ 24.9, 33.5, 37.0, 44.5, 56.2, 73.5, 106.8,
113.8, 117.4 120.5 121.4, 126.0 130.2, 133.6, 138.9, 144.0, 152.4, 155.2, 158.0, 162.1,
164.0, 182.2 ; GC-MS, m/z: 560 (M+); Anal. Calcd for C25H20BrClFN3O4: C, 53.54; H,
3.59; N, 7.49; Found: C, 53.58; H, 3.57; N, 7.52.
RESULTS AND DISCUSSION
Initially in our research, a model reaction of 5-bromo salicylaldehyde, dimedone, barbituric
acid and 3-chloro-4-fluro aniline was carried out in different solvents using 10 mol% of
catalyst [Msim]Cl at room temperature. In this optimization study, ethanol was found to be an
excellent solvent over other solvents such as methanol, water, acetonitrile, dichloromethane
and n-hexane in terms of shorter reaction times and product yields (Table 1, Entry 2). The
results are summarized in Table 1 (Table 1, Entries 1-6). In the solvent methanol, product 5d
was obtained in good yield 72% within 7 h (Table 1, Entry 1). In the water and acetonitrile,
the reaction afforded 32 and 68% product yield (Tabel 1, Entries 3 and 4 respectively). In the
solvent dichloromethane and n-hexane, the corresponding product 5d was obtained in lower
yield with increased reaction time (Table 1, Entries 5 and 6, respectively). Afterwards, we
investigated the effect of catalytic concentration on the model reaction in solvent ethanol.
Initially at the catalytic concentration of 5 mol % ionic liquid [Msim]Cl, reaction was
completed with extended reaction time and afforded 74% product yield (Table 1, Entry 7).
By increasing the catalyst concentration from 10 to 15 mol%, it was observed that at a 15 mol
% catalyst concentration, no yield improvements were obtained (Table 1, Entry 8). As a
result, we selected 10 mol % of catalyst [Msim]Cl in solvent ethanol as the optimum
conditions for the one-pot synthesis of dihydropyrimido[4,5-b] quinolinetriones at room
temperature condition. To establish the significance of [Msim]Cl as catalyst, a reaction was
carried out in the absence of catalyst observing that only 22% yield of corresponding product
was obtained after 20 hours (Table 1, Entry 9).
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Table 1: Screening of the solvent and catalyst concentration for synthesis of
dihydropyrimido[4,5-b] quinolinetrionea
Entry Catalyst (mol%)
Solvent
Time (h)
Yieldb (%)
1
[Msim]Cl (10 mol%)
MeOH
7
72
2
[Msim]Cl (10 mol%)
EtOH
5
92
[Msim]Cl (10 mol%)
H2O
14
32
3
4
[Msim]Cl (10 mol%)
CH3CN
8
68
5
[Msim]Cl (10 mol%)
DCM
11
39
6
[Msim]Cl (10 mol%)
n-Hexane
9
42
7
[Msim]Cl (5 mol%)
EtOH
8
74
8
[Msim]Cl (15mol%)
EtOH
5.2
90
9
EtOH
20
22
a
Conditions: Dimedone (1 mmol), barbituric acid (1 mmol), 5-bromosalicylaldehyde (1
mmol) and 3-chloro-4fluro anilines (1mmol), [Msim]Cl (mol %), Solvent (10 mL) at room
temperature. Reaction was monitored by thin layer chromatography. bIsolated yield
Optimistic by these delightful results, we have screened a variety of substituted
salicylaldehydes for the synthesis of corresponding dihydropyrimido[4,5-b] quinolinetrione
derivatives. We observed that all products are obtained with excellent yields (Table 2, Entries
1-10).
Table
2:
One-pot
quinolinetriones
Sr.No.
Multi-component
Synthesis
of
Dihydropyrimido[4,5-b]
a
Products (5a-j)
Time (h)
M.P.
Yield (%)b
6
210-212
92
5
241-243
84
OH
O
O
NH
1
N
N
H
O
Cl
5a
F
I
I
O
OH
O
NH
2
N
N
H
O
Cl
F
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5b
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World Journal of Pharmacy and Pharmaceutical Sciences
Br
Br
OH
O
O
NH
3
N
N
H
O
5
231-233
86
5.3
263-265
87
5.4
221-224
95
6
272-274
89
5.5
198-200
80
Cl
5c
F
Br
OH
O
O
NH
4
N
N
H
O
Cl
5d
F
OCH3
OH
O
O
NH
5
N
N
H
O
Cl
5e
F
Cl
OH
O
O
NH
6
N
N
H
O
Cl
5f
F
O2N
OH
O
O
NH
7
N
N
H
O
Cl
F
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5g
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World Journal of Pharmacy and Pharmaceutical Sciences
Cl
Cl
OH
O
O
NH
8
N
O
N
H
6
255-257
84
5.30
215-217
91
5.70
281-283
85
Cl
5h
F
Br
OCH3
OH
O
O
NH
9
N
N
H
O
Cl
5i
F
I
OH
O
O
NH
10
N
N
H
O
Cl
5j
Conditions: Dimedone (1 mmol), barbituric acid (1 mmol), substituted salicylaldehyde (1
F
a
mmol) and 3-chloro-4fluro anilines (1mmol), [Msim]Cl (10 mol %), EtOH (10 mL) at room
temperature. Reaction was monitored by thin layer chromatography.
b
Isolated yield
The anti-cancer activity for these compounds was done in the Anti-cancer drug screening
facility (ACDF), Tata memorial centre, Aadvanced centre for treatment, research and
education in cancer (ACTREC). The in-vitro anti-cancer activity for the corresponding
compounds and ADR (Adriamysin or doxorubicin) taken as a known drug, tested using SRB
(sulforhodamine B) assay protocol as exactly described by Skehan P. et al. Briefly, SRB is a
dye binds to the protein. The human breast cancer cell line MCF7 cultured in 96 well plate
treated with different concentrations of given compounds (10, 20, 40 and 80 µg/ml). After
treatment the cells were fixed in trichloroacetic acid and stained using sulforhodamine B
(0.4% wt/vol) prepared in 1% acetic acid for 30 minutes. Four washes with 1% acetic acid
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were given to remove unbound dye. 10 mM unbuffered tris base was used to extract protein
bound dye and subjected for microtiter plate reader. The absorbance of dye was measured at
wavelength 565 nm. The absorbance is correlated with the net protein synthesis rate. 50%
inhibition of cell growth (GI50), 50% cell kill or lethal concentration (LC50) and 100%
(total) growth inhibition (TGI) was calculated. The GI50 value <10 µg/ml is considered to
demonstrate activity in case of pure compound. This experiment was done in triplicate and
the average values were plotted against % control growth versus drug concentrations.
Different evidence has demonstrated the ability of quinolines as potential MCF-7 human
breast cancer cells. All differentiation-inducing quinolines caused growth suppression in
MCF-7 cells. The mechanism of action of the differentiation-inducing quinolines has been
proposed to involve strong suppression of E2F1 (cell line) that inhibits growth by preventing
cell cycle progression and fosters differentiation by creating a permissive environment for
cell differentiation. A series of new dihydropyrimido[4,5-b] quinolinetriones were tested for
antiproliferative activity in vitro against human breast cancer cell lines. All the compounds
exhibited antiproliferative activity comparable to Adrimycine. Indeed suresh et al., showed
that another thioxoquinoline analog was active on human MCF7 cell line. A novel
intercalating compound of a quinoline series also showed cytotoxic effect in a concentration
and time-dependent manner. Cell cycle analysis and titrated assays revealed that this
compound affects the cell cycle progression by arresting the
nuclear condensation and changes in the expression levels of
phase. DNA fragmentation,
LC 50, TGI, and GI50
confirmed the activation of apoptosis. Other evidence has demonstrated that these kinds of
structures, like the well known quinoline analog, suppress cell signaling through Ras
molecular pathway, inhibiting PKC activity. The effect of this compound is dose-dependent
on MCF-7 cancer cell lines. When we tested quinoline derivatives for breast cancer cell lines,
it is observed that all the compounds are active and acts as anticancer agents. (Table 3)
Table3: Anticancer activity of dihydropyrimido[4,5-b] quinolinetriones against human
breast cancer cells
% control growth
Drug concentration (µg/ml)
Experiment 1
10
20
40
80
Q1 68.3 63.2 48.4 21.3
Q2 85.2 79.4 64.1 38.2
Q3 83.3 77.3 61.1 36.6
Q4 75.7 69.8 53.7 27.4
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10
69.7
85.3
83.2
75.6
Experiment 2
20
40
80
59.4 50.3 27.8
77.2 67.6 45.8
75.6 65.9 43.9
68.4 58.1 35.8
10
65.4
83.2
80.3
73.9
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Experiment 3
20
40
80
57.2 52.3 33.1
78.4 68.3 45.3
76.3 66.3 42.1
65.7 61.1 40.2
10
67.8
84.5
82.3
75.0
Average values
20
40
80
59.9 50.3 27.4
78.3 66.6 43.1
76.4 64.4 40.9
67.9 57.6 34.4
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Sudhakar et al.
Q5
Q6
Q7
Q8
Q9
Q10
ADR
65.7
73.2
88.4
80.6
70.7
78.2
5.7
60.7
66.4
82.3
74.4
65.8
72.1
4.1
45.3
51.5
67.4
58.5
50.8
55.7
-0.8
World Journal of Pharmacy and Pharmaceutical Sciences
19.2
25.3
40.3
33.6
23.2
31.3
-29.9
66.7
73.6
87.8
80
72.4
78.4
1.4
57.4
65.8
79.4
72.3
62.3
70.4
5.0
47.8
55.9
70.3
62.6
53.2
60.1
-2.2
24.9
33.1
47.9
40.2
30.8
38.3
-31.8
63.2
70.9
85.5
78.4
67.4
76.3
1.2
54.4
62.7
80.6
70.3
59.4
68.4
6.2
49.3
58.3
70.7
67.4
55.4
63.9
2.5
30.5
37.8
47.6
44.2
35.3
42.1
-36.4
Drug concentrations µg/ml calculated from graph
MCF7
LC 50
TGI
Q1
>80
>80
Q2
>80
>80
Q3
>80
>80
Q4
>80
>80
Q5
>80
79.2
Q6
>80
>80
Q7
>80
>80
Q8
>80
>80
Q9
>80
>80
Q10
>80
>80
ADR
>80
43.7
GI50
65.2
72.5
87.2
79.6
70.1
77.6
2.8
57.5
64.9
80.7
72.3
62.5
70.3
5.1
47.4
55.2
69.4
62.8
53.1
59.9
-0.2
GI50
43.2
68.96
64.5
55.04
39.68
51.20
72.32
62.88
47.52
59.52
<10
Growth inhibition of 50 % (GI50) calculated from [(Ti-Tz)/(C-Tz)] x 100 = 50,
drug concentration resulting in a 50% reduction in the net protein increase
TGI
Drug concentration resulting in total growth inhibition (TGI) will calculated from
Ti = Tz
LC50 Concentration of drug resulting in a 50% reduction in the measured protein at the
end of the drug treatment as compared to that at the beginning) indicating a net
loss of 50% cells following treatment is calculated from [(Ti-Tz)/Tz] x 100 = -50.
MCF7 of 5c
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1114
24.8
32.0
45.2
39.3
29.7
37.2
-32.7
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World Journal of Pharmacy and Pharmaceutical Sciences
CONCLUSION
In conclusion, we have developed a convenient method for the synthesis of
dihydropyrimido[4,5-b] quinolinetriones indoles by one-pot four-component reaction of
dimedone, barbituric acid, substituted salicylaldehyde and 3-chloro-4-fluro aniline in solvent
ethanol in presence of ionic liquid [Msim]Cl as catalyst. This modified protocol offers
increased performance over the many conventional methods. The delightful features of this
protocol are use of environmentally benign catalyst, easy work up and excellent yields of
corresponding products. All the synthesized derivatives were evaluated for their anticancer
activities. The initial assays indicated that some of the newly synthesized compounds
displayed significantly good inhibition activities against human breast cancer cell (MCF7),
cell lines compared with the control (Adriamysin), which might be developed as novel lead
scaffold for potential anticancer agents.
ACKNOWLEDGEMENTS
We are thankful to Dr. P. L. More, Principal, Dr. W. N. Jadhav, Dnyanopasak College,
Parbhani and Dr. Balasaheb Chavan, Principal, Yogeshwari Mahavidyalaya, Ambajogai for
providing necessary facilities to the research work. We are also thankful to Tata Memorial
Centre Advanced Centre for Treatment Research and Education in Cancer (ACTREC), Navi
Mumbai for providing anticancer activity.
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