1,2,3-Triazole Tagged 3H-Pyrano[2,3-d]pyrimidine-6-carboxylate Derivatives: Synthesis, in Vitro Cytotoxicity, Molecular Docking and DNA Interaction Studies

Article abstract of DOI:10.1002/cbdv.201800101

A series of novel ethyl 2,7-dimethyl-4-oxo-3-[(1-phenyl-1H-1,2,3-triazol-4-yl)methyl]-4,5-dihydro-3H-pyrano[2,3-d]pyrimidine-6-carboxylate derivatives 7a – 7m were efficiently synthesized employing click chemistry approach and evaluated for in vitro cytot

Full text of DOI:10.1002/cbdv.201800101

Article Type: Full Paper
Abstract: A series of novel ethyl 2,7-dimethyl-4-oxo-3-((1-phenyl-1H-1,2,3-triazol-4-yl)methyl)-4,5-
dihydro-3H-pyrano[2,3-d]pyrimidine-6-carboxylate derivatives 7(a-m) were efficiently synthesized
employing click chemistry approach and evaluated for in vitro anti cancer activity against four tumor
cell lines: A549 (human lung adenocarcenoma cell line), HepG2 (Human hematoma), MCF-7 (Human
breast adenocarcinoma), and SKOV3 (human ovarian carcinoma cell line). Among the compounds
tested, the compounds 7a, 7b, 7f, 7l and 7m have shown potential and selective activity against
human lung adenocarcenoma cell line (A549) with IC50 ranging from 0.69 to 6.74 µM. Molecular
docking studies revealed that the compounds 7a, 7b, 7f, 7l and 7m are potent inhibitors of human
DNA topoisomerase-II and also showed compliance with stranded parameters of drug likeness. The
calculated binding constants, kb, from UV-Vis absorptional binding studies of 7a, 7l with CT-DNA
were 10.77 x 104, 6.48 x 104 respectively. Viscosity measurements revealed that the binding could be
surface binding mainly due to groove binding. DNA cleavage study showed that the 7a, 7l have the
potential to cleave pBR322 plasmid DNA without any external agents.
Keywords: Pyrano[2,3-d]pyrimidine, 1,2,3-triazoles, Anti cancer activity, Molecular docking, groove
binding
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Introduction
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Results and Discussion
A
series of ethyl 2,7-dimethyl-4-oxo-3-((1-phenyl-1H-1,2,3-triazol-4-yl)methyl)-4,5-dihydro-3H-
pyrano[2,3-d]pyrimidine-6-carboxylate derivatives 7(a-m) were synthesized in four steps (Scheme
1). In the first step, 4H-pyrans 4(a-d) were easily obtained via one pot reaction of malononitrile 3,
ethyl aceto acetate 2 and various substituted aromatic aldehydes 1(a-d) and catalytic amount of
piperidine in ethanol stirring for 15-30 min. Compounds 4(a-d) were used as precursors for the
synthesis of pyrano[2,3-d]pyrimidine-4-one derivatives 5(a-d) by using catalytic amount of sulphuric
acid in acetic anhydride under reflux for 10 min. In the third step, pyrano[2,3-d] pyrimidine-4-one
derivatives 5(a-d) were reacted with propargyl bromide in the presence of anhydrous potassium
carbonate in dry acetone under reflux for 4-5 h resulting in the formation of corresponding N-
propargylated pyrano[2,3-d]pyrimidine-4-one derivatives 6(a-d). These N-propargyl pyrano[2,3-
d]pyridines-4-one derivatives 6(a-d) were subjected to cycloaddition with substituted aromatic
azides and 1-azidohexane under click chemistry reaction conditions in the presence of copper(I) as
catalyst in 1:1 water/tert-butanol mixture for 12-14 h to result in novel ethyl 2,7-dimethyl-4-oxo-3-
((1-phenyl-1H-1,2,3-triazol-4-yl)methyl)-4,5-dihydro-3H-pyrano[2,3-d]
pyrimidine-6-carboxylate
derivatives 7(a-l) and ethyl 3-((1-hexyl-1H-1,2,3-triazol-4-yl)methyl)-2,7-dimethyl-4-oxo-4,5-dihydro-
3H-pyrano[2,3-d] pyrimidine-6-carboxylate 7m respectively in quantitative yields. The synthesized
1
3H-pyrano[2,3-d]pyrimidine-[1,2,3]triazole hybrids were well characterized by H NMR, 13C NMR, IR
and ESI-MS spectra.
In Vitro anticancer activity
All the synthesised pyrano[2,3-d]pyrimidine-6-carboxylate derivatives were evaluated for anticancer
activity in in vitro mode using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
assay[32] on the A549 (human lung adenocarcenoma cell line), HepG2 (Human hematoma cell line),
MCF-7 (Human breast adenocarcinoma cell line), and SKOV3 (human ovarian carcinoma cell line).
The results of cytotoxic activity in vitro were expressed as the IC50 (µM) and doxorubicin was used as
positive control (Table 1). The structural diversity in all the derivatives was introduced by varying
substitution at 3rd position of the triazole ring and 4th position of pyran ring moiety intact. group. We
abserved that pyranopyrimidine, aromatic hydrocarbon and nitro group on triazole played an
important role. As shown in Table 1, most of the compounds were moderately active but, compound
7a, 7b, 7f, 7l and 7m showed better inhibition against A549 (IC50 0.69 µM), A549 (IC50 6.74 µM),
A549 (IC50 5.76 µM), A549 (IC50 1.85 µM) and A549 (IC50 3.62 µM) cell lines respectively. Structure
activity relationship studies revealed that the orientation of substituent on 1,2,3-triazole ring is not
only crucial but also required 3H-pyrano[2,3-d]pyrimidine along with a phenyl.
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Scheme 1. Synthesis of of 3H-pyrano[2,3-d]pyrimidine-[1,2,3]triazole hybrids.
Table 1. Anticancer activity of compounds 7(a-m)
IC50 in µM
Compound
A549
HepG2
MCF-7
SKOV3
7a
0.69 ± 0.02
6.74 ± 0.11
37.02 ± 0.09
>100
>100
71.41 ± 0.02
>100
96.46 ±0.79
>100
7b
80.18 ± 0.02
>100
7c
99.05 ± 0.01
>100
>100
7d
>100
>100
7e
22.12 ± 0.03
5.76 ± 0.01
82.43± 0.02
16.83 ± 0.02
>100
98.71 ± 0.01
>100
>100
81.06 ± 0.01
>100
7f
>100
7g
>100
96.46 ± 0.79
>100
90.16 ± 0.02
>100
7h
>100
7i
>100
>100
>100
7j
76.74 ± 0.11
1.85 ± 0.01
3.62 ± 0.02
0.14 ± 0.002
83.28 ± 0.08
>100
>100
>100
7l
>100
86.46 ± 0.79
>100
7m
69.85 ± 0.02
10.15 ± 0.003
>100
Doxorubicin
5.73 ± 0.06
16.37 ± 0.02
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Molecular docking studies
Exploration of molecular interaction of the title compounds was performed through molecular
docking studies using Discovery Studio 2.1 software. To study the molecular interaction of
compounds, crystallographic data of human DNA topoisomerase II (PDB: 3QX3) was retrieved from
Protein Data Bank. The compounds used in the molecular docking studies were 7a-c, 7f, 7h, 7l, 7m
and Doxorubicin on human DNA topoisomerase II has shown good dock score (LibDock). Dock score
and ligand interaction data of above compounds are shown in Table 2 (Table S1 in supplementary
material). The docking study of pyranopyrimidine-triazole derivative revealed the high docking
scores and binding affinities in the range of 200.959 to 216.394, as compared to Doxorubicin
(212.664) Figure 2 and (Figure S1 in supplementary information). Compound 7a was ranked with a
docking score of 203.217 which also showed good hydrogen bonding interactions. It also showed
some additional interactions with the binding site residues of target protein. The protein–ligand
interaction visualization of the compound 7a is shown in the Figure 3. This compound was docked
into the active site region making three hydrogen bond interactions with the residues ARG503 and
DG13. Two hydrogen bonds are formed with ARG503 and one with DG13. The NH1 of the residue
arginine503 interacted with O3 and N7 of the compound 7a (ARG503:NH1–7a:O3 and ARG503:NH1–
7a:N7) with hydrogen bond distances of 3.176 Å and 3.123 Å respectively. The N1 atom of the
nucleotide residue DG13 interacted with the N21 atom of the compound 7a with a hydrogen bond
distance of 3.101 Å. Also it formed some additional interactions with the nucleotide residue DG13.
The compound 7l was docked into the active site region making two hydrogen bond interactions
with the nucleotide residues DT9 and DC8 having dock score 200.959. One hydrogen bond was
formed by the nitrogen atom of DT9 interacted with the oxygen atom of the compound 7l (DT9:N3–
7l:O15) with a hydrogen bond distance of 2.942 Å. The second hydrogen bond interaction was
formed with the N4 atom of the nucleotide DC8 interacted with the N7 atom of the compound 7l
(DC8:N4–7l:N7) with a hydrogen bond distance of 2.611 Å. These results indicate that most of the
compounds bound within the binding site pocket of doxorubicin similar binding pattern with binding
site amino acid residues. The significant docking score on the target infers that these compounds are
promiscuous and can be potential leads against cancer.
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Figure 3. Receptor-ligand hydrogen bonds (green colour) and bumps (pink colour) of compounds 7a and 7l with active site residues of Human DNA
topoisomerase II (PDB: 3QX3).
Bioavailability and drug likeness screening
Different chemical descriptors for the pharmacokinetics properties were calculated to check the
compliance of studied compound with the standard range. For this the absorption level, blood-brain
barrier penetration, aqueous solubility, hepatotoxicity, cytochrome P450 2D6 binding. The
pharmacokinetic profiles of all the compounds under investigation were predicted by means of
Discovery Studio 2.1 ADMET models and result provided in Table S2 in the supplementary
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0
information. The aqueous solubility prediction (defined in water at 25 C) indicated that all the
compounds are soluble in water. All the compounds are found to be non- inhibitors of cytochrome
P450 2D6 (CYP2D6). The CYP2D6 enzyme is one of the important enzymes involved in drug
metabolism. During ADMET screening, the predictive hepatotoxicity was observed for the candidate
compounds in comparison to doxorubicin (toxic).
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The photo induced cleavage of super coiled pBR322 DNA by 7a, 7l was studied upon irradiation with
UV light at 365nm. The extent of cleavage was monitored by agarose gel electrophoresis. When
circular plasmid DNA is subjected to electrophoresis, relatively fast migration will be observed for
the intact super coiled form (Form I). If scission occurs on one strand (nicked), the super coiled will
relax to generate a slower-moving open circular form (Form II)[38]. If both strands are cleaved, a
linear form (Form III) that migrates between Form I and II will be generated. It was observed that the
compounds 7a, 7l promoted DNA cleavage upon irradiation. At 50 µM concentration, both were able
to partially transform supercoiled DNA (Form I) into open circular DNA (Form II) and to a much lesser
extent to linear DNA (Form III). At 100 µM concentration, both were able to completely transform
supercoiled DNA (Form I) into open circular DNA (Form II) and to a much lesser extent to linear DNA
(Form III). Both compounds behave as DNA photocleavers (Figure 6).
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Conclusions
In conclusion, we have synthesized a novel series of 1,2,3-triazole tagged 3H-pyrano[2,3-
d]pyrimidine-6-carboxylate derivatives by adopting click chemistry approach. In addition, all the
synthesized compounds were tested for their anticancer activity. Most of these compounds
exhibited significant cytotoxicity in the micromolar range against the human lung adenocarcenoma
cell line. Among those, 7a, 7b, 7f, 7l and 7m compounds have exhibited cytotoxicity with IC50 value
ranging from 0.69 to 6.74 µM in tested cell lines. Further, the DNA binding potentiality of these
active compounds 7a and 7l were evaluated through UV-vis spectroscopic titration studies. The
photo induced cleavage of super coiled pBR322 DNA by 7a, 7l were also studied. In addition,
Molecular docking of synthesized derivatives 7a-7c, 7f, 7h, 7l and 7m with human DNA
topoisomerase II revealed the LibDock score in the range of 200.959 to 216.394 showed compliance
with stranded parameters of drug likeness. Based on the above experimental results it is evident
that these compounds have the potential to be developed as a new class of cancer therapeutics.
Experimental Section
Chemistry
Melting points of all the compounds were recorded on Casia-Siamia (VMP-AM) melting point
apparatus and uncorrected. IR spectra were recorded on a Perkin –Elmer FT-IR 240-C spectrometer
1
using KBr optics. H-NMR spectra were recorded on Burker Avance 400 MHz in CDCl3 using TMS as
an internal standard. Electron impact (EI) and chemical ionization mass spectra were recorded on a
VG 7070 H instrument at 70 ev. All the reactions were monitored by thin layer chromatography (TLC)
on precoated M/s Merck silica gel 60 F254 (mesh); spots were visualized with UV light. Silica gel
(100-200 mesh) used for column chromatography was procured from Merck.
General procedure for the synthesis of 4H-pyrans 4(a-d)
A mixture of aromatic aldehyde 1(a-d) (5 mmol), ethyl acetoacetate 2 (5 mmol), malononitrile 3 (5
mmol) and the catalytic quantity of piperidine in ethanol (5 ml) was stirred at room temperature for
20-30 min. After completion of reaction (monitored by TLC) the separated product was filtered off,
washed with methanol.
General procedure for the synthesis of pyrano[2,3-d]pyrimidines 5(a-d)
4H-pyran 4(a-d) (5 mmol), acetic anhydride (5 ml), and concentrated sulfuric acid (0.5 mmol) were
mixed. The reaction mixture obtained was refluxed for 10 min, cooled to room temperature and
kept for 1 day. A precipitate formed was filtered off, washed with water (3×5 ml), ethanol (5 ml), and
0
light petroleum (5 ml). The product obtained was recrystallized from ethanol and dried at 120 C
until the weight became constant.
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General procedure for Synthesis of N-propargyl pyrano[2,3-d]pyrimidine-4-one derivatives 6(a-d)
Compound 5(a-d) (0.02 mol) was dissolved in dry acetone (25 ml), anhydrous potassium carbonate
(0.04 mol) was added. Then, propargyl bromide (0.03 mol) was added slowly while stirring. The
reaction mixture was refluxed for 1-2 hours. After completion of reaction (monitored by TLC), the
solvent was removed under reduced pressure and the residue was diluted with ice cold distilled
water and extracted with chloroform, the organic layer was dried over anhydrous Na2SO4 and
concentrated to get the product 6(a-d). The crude product was purified by column chromatography.
Ethyl
2,7-dimethyl-4-oxo-3-(prop-2-yn-1-yl)-5-(p-tolyl)-4,5-dihydro-3H-pyrano[2,3-d]pyrimidine-6-
carboxylate (6c)
0
1
White solid, Yield: 90%, M.p. 140–142 C, H NMR (400 MHz, CDCl3): 7.22 (d, J = 8.1 Hz, 2H, Ar-H);
7.06 (d, J = 7.8 Hz, 2H, Ar-H); 4.98 (dd, J = 16.8, 3.2 Hz, 2H, CH2); 4.49 (s, 1H, H of pyran ring); 4.06 (q,
J = 7.1 Hz, 2H, CH2); 2.63 (s, 3H, CH3); 2.49 (s, 3H, CH3); 2.27 (s, 3H, CH3); 2.26 (s, 1H, CH); 1.18 (t, J
= 7.1 Hz, 3H, CH3); 13C NMR (100 MHz, CDCl3): 166.2; 160.9; 158.6; 158.4; 158.1; 140.7; 136.4;
128.9; 128.4; 108.6; 101.9; 76.5; 73.0; 60.5; 36.6; 33.2; 22.5; 21.1; 18.8; 14.0.
General procedure for synthesis of
ethyl 2,7-dimethyl-4-oxo-3-((1-phenyl-1H-1,2,3-triazol-4-
yl)methyl)-4,5-dihydro-3H-pyrano[2,3-d] pyrimidine-6-carboxylate derivatives 7(a-m)
Compound 6(a-d) (3 mmol) and substituted aromatic azides/1-azidohexane (3 mmol) were
suspended in 12 ml of a 1:1 water/tert-butanol mixture. Aqueous solution of Sodium ascorbate (0.3
mmol) was added, followed by the addition of aqueous solution of copper (II) sulfate pentahydrate
(0.03 mmol). The heterogeneous mixture was stirred vigorously overnight, at which point it cleared
and TLC analysis indicated complete consumption of the reactants. The reaction mixture was diluted
with 50 ml of water and cooled in ice, and the precipitate was collected by filtration. After being
washed with cold water (2- 25 ml), the precipitate was dried under vacuum to afford pure product as
a powder.
Ethyl 2,7-dimethyl-3-((1-(4-nitrophenyl)-1H-1,2,3-triazole-4-yl)methyl)-4-oxo-5phenyl-4,5-dihydro-3H
pyrano [2,3-d]pyrimidine-6-carboxylate (7a)
0
Yellow solid, Yield: 91%, M.p.135–137 C, IR (KBr) cm-1 3088, 2980, 1709, 1672, 1559, 1428, 1244.
1H NMR (400 MHz, CDCl3): 8.41 (d, J = 4.4 Hz, 2H, Ar-H); 8.16 (s, 1H, =CH of triazole ring); 7.88 (d, J =
8.6 Hz, 2H, Ar-H); 7.34 (d, J = 8.6 Hz, 2H, Ar-H); 7.30 – 7.21 (m, 2H, Ar-H); 7.18 (t, J = 7.3 Hz, 1H, Ar-H);
5.35 (s, 2H, N-CH2); 4.99 (s, 1H, H of pyran ring); 4.07 (q, J = 7.1 Hz, 2H, CH2); 2.84 (s, 3H, CH3); 2.49
(s, 3H, CH3); 1.17 (t, J = 7.1 Hz, 3H, CH3). 13C NMR (100 MHz, CDCl3): 166.1; 161.9; 161.7; 158.5;
147.4; 146.9; 143.7; 143.1; 140.8; 128.6; 128.2; 125.5; 122.4; 122.2; 120.6; 108.3; 101.9; 60.6; 39.9;
37.1; 23.3; 18.8; 14.1. MS (ESI) (m/z) (M+1) calculated for C27H24N6O6, 528.18, found 529.30.
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Ethyl
3-((1-(3,4-dichlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-2,7-dimethyl-4-oxo-5-phenyl-4,5-
dihydro-3H-pyrano[2,3-d]pyrimidine-6-carboxylate (7d)
0
Yellow solid, Yield: 85%, M.p. 143–145 C, IR (KBr) cm-1 3085, 2930, 1710, 1672, 1559, 1429, 1245,
1H NMR (400 MHz, CDCl3): 8.00 (s, 1H, =CH of triazole ring); 7.75 (d, J = 7.3 Hz, 1H, Ar-H); 7.57 (d, J =
7.3 Hz, 1H, Ar-H); 7.55 – 7.25 (m, 5H, Ar-H); 7.20 (s, 1H, Ar-H); 5.32 (s, 2H, N-CH2); 4.99 (s, 1H, H of
pyran ring); 4.07 (q, J = 7.1 Hz, 2H, CH2); 2.82 (s, 3H, CH3); 2.49 (s, 3H, CH3); 1.17 (t, J = 7.1 Hz, 3H,
CH3). MS (ES+) (m/z) (M+1) calculated for C27H23Cl2N5O4, 551.11, found 552.
Ethyl
2,7-dimethyl-4-oxo-5-phenyl-3-((1-(o-tolyl)-1H-1,2,3-triazol-4-yl)methyl)-4,5-dihydro-3H-
pyrano[2,3-d] pyrimidine-6-carboxylate (7e)
0
Yellow solid, Yield: 82 %, M.p.120–122 C, IR (KBr) cm-1 3072, 2929, 1705, 1670, 1557, 1423, 1238.
1H NMR (400 MHz, CDCl3): 7.99 (s, 1H, =CH of triazole ring); 7.51 (d, J = 8.4 Hz, 2H, Ar-H); 7.34 (d, J =
7.2 Hz, 2H, Ar-H); 7.30 – 7.30 (m, 3H, Ar-H); 7.23 (d, J = 10.9 Hz, 1H, Ar-H); 7.18 (d, J = 7.2 Hz, 1H, Ar-
H); 5.35 (s, 2H, N-CH2); 4.99 (s, 1H, H of pyran ring); 4.07 (q, J = 7.1 Hz, 2H, CH2); 2.83 (s, 3H, CH3);
2.49 (s, 3H, CH3); 2.42 (s, 3H, CH3); 1.17 (t, J = 7.1 Hz, 3H, CH3). MS (ESI) (m/z) (M+1) calculated for
C28H27N5O4, 497.21, found 498.10.
Ethyl 3-((1-(4-methoxy-2-nitrophenyl)-1H-1,2,3-triazol-4-yl)methyl)-2,7-dimethyl-4-oxo-5-phenyl-4,5-
dihydro-3H-pyrano[2,3-d]pyrimidine-6-carboxylate (7f)
Yellow solid, Yield: 86%, M.p.125–126 0C, IR (KBr) cm-1 3023, 2977, 1709, 1672, 1541, 1428, 1244. 1H
NMR (400 MHz, CDCl3): 8.05 (t, J = 7.1 Hz, 1H, Ar-H); 7.86 (s, 1H, =CH of triazole ring); 7.51 (d, J = 4.6
Hz, 1H, Ar-H); 7.38 – 7.33 (m, 1H, Ar-H); 7.32 (d, J = 7.3 Hz, 2H, Ar-H); 7.23 (d, J = 7.6 Hz, 2H, Ar-H);
7.16 (t, J = 7.1 Hz, 1H, Ar-H); 5.18 (s, 2H, N-CH2); 4.96 (s, 1H, H of pyran ring); 4.06 (q, J = 7.1 Hz, 2H,
CH2); 3.59 (s, 3H, OCH3); 2.87 (s, 3H, CH3); 2.49 (s, 3H, CH3); 1.15 (t, J = 7.1 Hz, 3H, CH3). MS (ESI)
(m/z) (M+1) calculated for C28H26N6O7, 558.19, found 559.
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Ethyl
3-((1-(3,4-dichlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-2,7-dimethyl-4-oxo-5-(p-tolyl)-4,5-
dihydro-3H-pyrano[2,3-d]pyrimidine-6-carboxylate (7g)
Yellow solid, Yield: 81%, M.p.135–136 0C, IR (KBr) cm-1 3050, 2977, 1710, 1674, 1559, 1428, 1241. 1H
NMR (400 MHz, CDCl3): 8.07 (s, 1H, =CH of triazole ring); 7.84 (s, 1H, Ar-H); 7.58 (t, J = 11.5 Hz, 1H,
Ar-H); 7.41 (d, J = 8.8 Hz, 1H, Ar-H); 7.22 (d, J = 7.9 Hz, 2H, Ar-H); 7.07 (d, J = 7.7 Hz, 2H, Ar-H); 5.23
(d, J = 10.2 Hz, 2H, N-CH2); 4.95 (s, 1H, H of pyran ring); 4.07 (q, J = 7.1 Hz, 2H, CH2); 2.82 (s, 3H, CH3);
2.47 (s, 3H, CH3); 2.28 (s, 3H, CH3); 1.19 (t, J = 5.6 Hz, 3H, CH3). MS (ESI) (m/z) (M+1) calculated for
C28H25Cl2N5O4, 565.13, found 566.42.
Ethyl 3-((1-(4-acetylphenyl)-1H-1,2,3-triazol-4-yl)methyl)-2,7-dimethyl-4-oxo-5-(p-tolyl)-4,5-dihydro-
3H-pyrano[2,3-d]pyrimidine-6-carboxylate (7h)
Yellow solid, Yield: 85%, M.p.142–143 0C, IR (KBr) cm-1 2977, 2927, 1710, 1674, 1558, 1428, 1241. 1H
NMR (400 MHz, CDCl3): 8.16 (s, 1H, =CH of triazole ring); 8.11 (d, J = 6.4 Hz, 2H, Ar-H); 7.81 (d, J = 8.6
Hz, 2H, Ar-H); 7.27 (d, J = 8.1 Hz, 2H, Ar-H); 7.08 (d, J = 8.1 Hz, 2H, Ar-H); 5.38 (s, 2H, N-CH2); 4.95 (s,
1H, H of pyran ring); 4.07 (q, J = 7.1 Hz, 2H, CH2); 2.83 (s, 3H, CH3); 2.66 (s, 3H, CH3); 2.47 (s, 3H, CH3);
2.28 (s, 3H, CH3); 1.17 (t, J = 7.1 Hz, 3H, CH3). MS (ESI) (m/z) (M+1) calculated for C30H29N5O5, 539.22,
found 540.10.
Ethyl 2,7-dimethyl-3-((1-(4-nitrophenyl)-1H-1,2,3-triazol-4-yl)methyl)-4-oxo-5-(p-tolyl)-4,5-dihydro-
3H-pyrano[2,3-d]pyrimidine-6-carboxylate (7i)
Yellow solid, Yield: 90%, M.p.132–133 0C, IR (KBr) cm-1 3093, 2981, 1712, 1671, 1558, 1429, 1241. 1H
NMR (400 MHz, CDCl3): 8.41 (d, J = 8.9 Hz, 2H, Ar-H); 8.20 (s, 1H, =CH of triazole ring); 7.91 (d, J = 8.9
Hz, 2H, Ar-H); 7.22 (d, J = 7.8 Hz, 2H, Ar-H); 7.08 (d, J = 7.5 Hz, 2H, Ar-H); 5.38 (s, 2H, N-CH2); 4.95 (s,
1H, H of pyran ring); 4.07 (q, J = 7.1 Hz, 2H, CH2); 2.83 (s, 3H, CH3); 2.47 (s, 3H, CH3); 2.28 (s, 3H, CH3);
1.19 (t, J = 5.8 Hz, 3H, CH3). MS (ESI) (m/z) (M+1) calculated for C28H26N6O6, 542.19, found 543.47.
Ethyl
2,7-dimethyl-3-((1-(4-nitrophenyl)-1H-1,2,3-triazol-4-yl)methyl)-4-oxo-5-(3,4,5-
trimethoxyphenyl)-4,5-dihydro-3H-pyrano[2,3-d]pyrimidine-6-carboxylate (7j)
Yellow solid, Yield: 88%, M.p.202–203 0C, IR (KBr) cm-1 3095, 2971, 1716, 1672, 1559, 1425, 1233. 1H
NMR (400 MHz, CDCl3): 8.42 (d, J = 9.1 Hz, 2H, Ar-H); 8.16 (s, 1H, =CH of triazole ring); 7.89 (d, J = 9.1
Hz, 2H, Ar-H); 6.55 (s, 2H, Ar-H); 5.39 (s, 2H, N-CH2); 4.96 (s, 1H, H of pyran ring); 4.13 (q, J = 7.1 Hz,
2H, CH2); 3.81 (s, 6H, OCH3); 3.79 (s, 3H, OCH3); 2.84 (s, 3H, CH3); 2.49 (s, 3H, CH3); 1.22 (t, J = 7.1 Hz,
3H, CH3). 13C NMR (100 MHz, CDCl3): 166.1; 161.9; 158.9; 158.5; 152.9; 147.4; 143.7; 140.8; 139.3;
137.0; 125.6; 122.3; 120.6; 108.4; 105.6; 101.7; 60.7; 60.7; 56.1; 39.9; 37.2; 23.3; 18.8; 14.2. MS (ESI)
(m/z) (M+1) calculated for C30H30N6O9, 618.21, found 619.54.
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Ethyl
3-((1-(3,4-dichlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-2,7-dimethyl-4-oxo-5-(3,4,5-
trimethoxyphenyl)-4,5-dihydro-3H-pyrano[2,3-d]pyrimidine-6-carboxylate (7k)
Yellow solid, Yield: 85%, M.p.199–200 0C, IR (KBr) cm-1 2975, 2942, 1711, 1672, 1559, 1424, 1236. 1H
NMR (400 MHz, CDCl3): 8.84 (s, 1H, Ar-H); 8.25 (s, 1H, =CH of triazole ring); 7.95 (t, J = 9.1 Hz, 2H, Ar-
H); 7.88 (t, J = 9.1 Hz, 2H, Ar-H); 6.45 (s, 2H, Ar-H); 5.38 (s, 2H, N-CH2); 4.81 (s, 1H, H of pyran ring);
4.07 (q, J = 7.1 Hz, 2H, CH2); 3.68 (s, 6H, OCH3); 3.34 (s, 3H, OCH3); 2.65 (s, 3H, CH3); 2.41 (s, 3H, CH3);
1.15 (t, J = 7.1 Hz, 3H, CH3). MS (ESI) (m/z) (M+1) calculated for C30H29Cl2N5O7, 641.14, found 642.
Ethyl
2,7-dimethyl-3-((1-(4-nitrophenyl)-1H-1,2,3-triazol-4-yl)methyl)-4-oxo-5-(thiophen-2-yl)-4,5-
dihydro-3H-pyrano[2,3-d]pyrimidine-6-carboxylate (7l)
Yellow solid, Yield: 89%, M.p.136–137 0C, IR (KBr) cm-1 3087, 2973, 1715, 1672, 1557, 1429, 1224. 1H
NMR (400 MHz, CDCl3): 8.42 (d, J = 8.8 Hz, 2H, Ar-H); 8.26 (s, 1H, =CH of triazole ring); 7.93 (d, J = 8.9
Hz, 2H, Ar-H); 7.12 (d, J = 4.8 Hz, 1H, Ar-H); 6.93 (dd, J = 14.3, 10.4 Hz, 1H, Ar-H); 6.87 (d, J = 4.8 Hz,
1H, Ar-H); 5.40 (s, 2H, N-CH2); 5.23 (s, 1H, H of pyran ring); 4.16 (q, J = 7.1 Hz, 2H, CH2); 2.86 (s, 3H,
CH3); 2.48 (s, 3H, CH3); 1.23 (t, J = 7.1 Hz, 3H, CH3). 13C NMR (100 MHz, CDCl3): 165.9; 161.8; 158.9;
158.8; 155.9; 147.5; 143.7; 142.9; 140.8; 126.6; 125.5; 125.1; 124.2; 122.6; 120.6; 108.1; 101.3; 60.8;
39.9; 31.7; 23.4; 18.8; 14.1. MS (ESI) (m/z) (M+1) calculated for C25H22N6O6S, 534.13, found 535.20.
Ethyl
3-((1-hexyl-1H-1,2,3-triazol-4-yl)methyl)-2,7-dimethyl-4-oxo-5-phenyl-4,5-dihydro-3H-
pyrano[2,3-d] pyrimidine-6-carboxylate (7m)
Yellow solid, Yield: 81%, M.p.114–116 0C, IR (KBr) cm-1 3021, 2927, 1713, 1665, 1555, 1433, 1216. 1H
NMR (400 MHz, CDCl3): 7.57 (s, 1H, =CH of triazole ring); 7.33 (d, J = 7.3 Hz, 2H, Ar-H); 7.22 (d, J =
13.0 Hz, 2H, Ar-H); 7.17 (t, J = 7.2 Hz, 1H, Ar-H); 5.27 (s, 2H, N-CH2); 4.97 (s, 1H, H of pyran ring); 4.26
(dt, J = 11.4, 6.6 Hz, 2H, CH2); 4.07 (q, J = 7.1 Hz, 2H, CH2); 2.80 (s, 3H, CH3); 2.48 (s, 3H, CH3); 1.86 –
1.78 (m, 2H); 1.32 – 1.22 (m, 10H); 1.17 (t, J = 7.1 Hz, 3H, CH3); 0.88 (t, J = 6.8 Hz, 3H, CH3). MS (ESI)
(m/z) (M+1) calculated for C29H37N5O4, 519.20, found 520.20.
Biology
Anticancer activity
Cytotoxicity of formulations was assessed using MTT assay to determine the cell viability according
to a method described by Hansen et al. The assay is based on the reduction of MTT by the
mitochondrial dehydrogenase of viable cells into purple formazan crystals which gets dissolved in
DMSO and read at 570 nm. Briefly, 1x104 exponentially growing cells were seeded into each 96 well
plate (counted by Trypan blue exclusion dye method) allowed to grow to 60-70% confluence then
compounds were added to the culture medium with the final concentrations ranging from of 0.1, 1,
5 and 10 µM and along with controls (negative without compound) and positive (Doxorubicin)
incubated for 24 hours CO2 incubator at 37 0C with a 90% humidified atmosphere and 5% CO2. Then
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the media of the wells were replaced with 90µl of fresh serum free media and 10 µl of MTT (5mg/mL
0
of PBS), plates were incubated at 37 C for 2 h, thereafter the above media was discarded allow to
0
dry for 30minutes. Add 100 µl of DMSO in each well at 37 C for 5 min. The purple formazan crystals
were dissolved and immediately read absorbance at 570 nm was measured using Spectra Max plus
384 UV-Visible plate readers (Molecular Devices, Sunnyvale, CA, USA). IC50 values were determined
by the probit analysis software package of MS-excel. % Cell viability (from control) versus
concentration.
DNA interaction studies
Concentrated CT-DNA stock solution was prepared in Tris HCl buffer ( 5mM Tris HCl/50 mM NaCl in
water at pH = 7.5) and the concentration of DNA solution was determined by UV absorbance at 260
nm. The molar absorption coefficient was taken as 6600 M-1 cm-1. Solution of CT-DNA in Tris HCl
buffer gave a ratio of UV absorption at 260 nm and 280 nm A260/A280 of ca. 1.8-1.9, indicating that
the DNA was sufficiently free of protein. All stock solutions were stored at 4 0C and wre used within
four days. The DNA binding experiments were done in Tris-HCl buffer using 1mM 5% v/v
DMSO/Water solution of active compounds 7a, 7l. Absorption titration experiments were performed
by maintaining a constant compound concentration (2µM) and varying the DNA concentration (0–10
μM) in the buffer. The active compounds 7a, 7l exhibited maximum absorption at around 210 nm.
After each addition of DNA to the respected compound, the resulting solution was allowed to
equilibrate at 250C for 5min, after which, the change in absorbance was recorded. The intrinsic
binding constant, Kb, was determined from the spectral data using the Wolfe–Shimmer Equation[39]
,
[DNA]/(εa–εf) = [DNA]/(εb–εf) + 1/Kb (εb–εf), where [DNA] = Concentration of DNA in base pairs, εa
= the apparent extinction coefficient = (Aobsd/[Compound]), εf = the extinction coefficient for free
compound, εb = the extinction coefficient for free compound in the fully bound form. In plots of
[DNA]/(εa−εf) versus [DNA] Kb is given by the ratio of slope to the intercept.
Viscosity measurements were performed with an Ostwald Viscometer at room temperature.
The concentration of DNA was 20μM, compounds concentration varied from 0 to 100 μM and the
flow times were measured with a digital timer and each sample was measured three times for
accuracy, and an average flow time was calculated. Data was presented as (η/ηo)1/3 versus
[compound]/ [DNA], where η is the viscosity of DNA in the presence of complex and ηo that of DNA
alone. Viscosity values were calculated from the observed flow time of DNA containing solutions (t)
corrected for that buffer alone (5 mM Tris–HCl/50 mM NaCl) (to), η = (t – to).
For gel electrophoresis experiments TE-buffer (10mMTris-HCl and 1 mM Na2EDTA), TAE-buffer
(pH 8.0; 40 mM Tris-base, 20 mM acetic acid, 1 mM EDTA) were used. TE-buffer (10mMTris-HCl and
1 mM Na2EDTA) used for dilution of pBR322 DNA. Super coiled pBR322 DNA (0.1 g/μL) was treated
with 50 μM and 100 μM of title derivatives were irradiated at 365 nm for 1 h. A loading buffer
containing 25% bromophenol blue, 0.25% xylene cyanol, and 30% glycerol (2 μL) was added. The
samples were then analyzed by 0.8% agarose gel electrophoresis at 50 V for 2 h. The gel was stained
with 2 μL (from 1 mg/100 μL) of ethidium bromide[40] and photographed under UV light. The gels
were viewed with a gel documentation system and photographed using a CCD camera.
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Supplementary Material
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/MS-numbe
Acknowledgements
We gratefully acknowledge DST-FIST Program of India (SR/FST/CSI-213/2010) for financial support.
One of the author S K Boda. thanks to UGC-New Delhi for Research Fellowship. We also thank the
Director, CFRD-OU, Hyderabad for providing spectral analysis facilities.
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