N. Kahriman, et al.
BioorganicChemistry99(2020)103805
DNA/BSA binding properties of the newly synthesized compounds were
determined by using UV–Vis spectrophotometer, and properties of
binding species as well as binding constants were explained below. A
single maximum absorption peak was observed in the spectrum of the
compounds obtained by UV–Vis studies, and no clear redshifts or
blueshifts on this peak were observed except for compounds 3, 5, and 6.
When CT-DNA was added to increase amounts to the reaction medium,
the absorption intensities of compounds 2, 7, and 8 were reduced re-
sulting in hypochromic effect, and the increase in the absorption in-
tensity of compounds 1, 4, and 9 caused hyperchromic appearance.
Evaluation of compounds 3, 5, and 6 for their possible interaction with
DNA exhibited hyperchromic properties, while compounds 3 and 5
caused redshift about 10 nm and compound 6 exhibited blueshift about
8 nm. Likewise, when CT-DNA was added in increasing amounts to the
reaction mixture, compounds 2c, 3c, 3a, 5c, and 8a caused hyper-
chromic effect, whereas compounds 2a, 2b, 3b, 5a, 5b, 6a, 6b, 6c, 8b,
8c, 9a, 9b, and 9c from the same group chemicals exhibited hypo-
chromic feature. Investigating the possible interactions of the newly
synthesized drug candidate molecules with BSA gives us important in-
formation about their transport pathway in blood since BSA is the
standard molecule used as a carrier for many drugs and metabolites. For
this reason, the spectrum bands described below will be guiding the
prediction of the BSA-molecule relationship. According to the absorp-
tion spectra of 5, 10 and 15 carbons containing N-alkyl bromide deri-
vatives upon increasing the concentration of BSA, compounds 2b, 2c,
3b, 3c, 5c, 6c, and 8c caused hyperchromic effect, whereas only
compound 18c displayed hypochromic effect. These changes suggest
that the compounds interact with BSA.
3. Conclusion
In summary, a new series of 2-amino-4-aryl-6-pyridopyrimidines (1-
9) and their N-alkyl bromide derivatives bearing different length
carbon chains (2a-c, 3a-c, 5a-c, 6a-c, 8a-c, 9a-c) were effectively
synthesized in good yields, and characterized by using spectral tech-
niques. In total, 27 compounds have been obtained whereof only
compound 7, 8, and 9 are already known in the literature [28,31].
Although those mentioned compounds being known in the literature,
there is no study revealing their antimicrobial and anticancer proper-
ties. In this work, the biological activities -anti-proliferative, cytotoxic,
antibacterial properties and DNA/protein binding affinities- of the
synthetic compounds (1-9, 2a-c, 3a-c, 5a-c, 6a-c, 8a-c, 9a-c) were
explored. The majority of the synthetic compounds displayed sig-
nificant anti-proliferative effects on the tested cancer cell lines. Espe-
cially, b and c series of alkylated derivatives showed a remarkable
impact. The anticancer results revealed that the alkyl chain prolonga-
tion has a deleterious effect on cancer cell lines. On the other hand, the
results of antimicrobial activities showed that the majority of the
compounds were more effective against Gram (+) bacteria than Gram
(−) bacteria. In particular, N-alkyl bromide derivatives (especially a-c
series on G (+) and b series on G (−)) exhibited stronger antibacterial
properties than their parent molecules (2-amino-4-aryl-6-pyridopyr-
imidines) (1-9). The evaluation of the synthetic compounds (1-9, 2a-c,
3a-c, 5a-c, 6a-c, 8a-c, 9a-c) for their DNA binding affinities showed
that the new compounds; particularly compound 1 and 8, formed a
stronger bond with DNA than the 5FU anticancer drug although the
binding constants between new compounds and DNA were not higher
but similar when compared to cisplatin. The findings show strong evi-
dence for new investigations involving analogous compounds and the
methods to explore mechanistic aspects of anticancer activity.
In our laboratory, absorbance data obtained by the spectro-
photometric method was used to derive the binding constants (Kb),
showing the affinity of the complex to DNA, of the newly synthesized
complexes with the aid of the following the Wolfe–Shimmer equation
[51]: [DNA]/(εa–εf) = [DNA]/(εb–εf) + 1/Kb(εb–εf), [DNA] is the
concentration of DNA in the base pairs, and εa, εf and εb are the molar
absorptivities of Aobserved/[complex], free complex and complex-DNA
solutions, respectively. Kb is the binding constant that is related to af-
finity between the complex and DNA and can be calculated algebrai-
cally from the line graph drawn between [DNA]/(εa-εf) and [DNA] as
the slope. According to Table 11, the Kb values of pyrimidines were
between 1.2 × 103 and 7.9 × 104 M−1 and Kb values of N-alkyl de-
rivatives respectively were between 0.4 × 103 and 5.5 × 104 M−1. The
binding constants of the pyrimidines and alkyl derivatives were ordered
from small to large in their own groups as 1 > 8 > 6a > 9a > 6b >
6c=9c > 5c > 5a > 2b > 8c > 5 > 6 > 8b > 3b > 2=9 > 3c >
7 > 2c > 4 > 3a > 2a > 9b > 5b > 3 > 8a. The Kb values of
the compounds-DNA adduct are mostly consistent with the IC50 data
obtained using the MTT assay. Generally, the N-alkyl bromide deriva-
tive compounds exhibited equal DNA binding activity comparable to
that of pyrimidines. Among other mechanisms, their DNA binding
properties can help explain the effects of N-alkyl bromide derivatives
against cell proliferation. The high Kb values suggest that the com-
plexes were strongly bound to DNA. To compare, the Kb values of two
important anticancer drugs (cisplatin and 5FU) were also examined and
it was shown that these anticancer drugs had similar Kb values with
newly synthesized compounds. While the binding constants of our
compounds were 0.4 × 103 to 7.9 × 104 M−1, the binding constants of
the cisplatin and 5FU were reported as 5.73 × 104 M−1 and 9.7 × 104
M−1, respectively, according to CT-DNA binding studies using these
strongly than the 5FU anticancer drug although the binding constants
between new compounds and DNA were not very high but similar when
compared with cisplatin.
4. Experimental
The materials and equipment used in this study are given in sup-
plementary information. The procedure of the synthesis of the new
compounds designed for this study and pharmacological experiments
that include the preparation of cell culture, cell proliferation assay
(MTT assay), cytotoxic activity assay, BrdU cell proliferation assay
(BCPA), microdilution assay, and DNA binding studies are provided in
supplementary information. The calculation of IC50 and three dose re-
sponse parameters were explained in the supplementary information.
All spectral data, melting points, retention factors and elemental ana-
lysis results of the obtained compounds were also given in supplemental
files.
Acknowledgements
This study was supported by grants from Karadeniz Technical
University and the Scientific and Technological Research Council of
Turkey (TÜBİTAK-114R025).
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://
References
[1] S. Kumar, S.M. Lim, K. Ramasamy, M. Vasudevan, S.A.A. Shah, Synthesis, molecular
docking and biological evaluation of bis-pyrimidine Schiff base derivatives, Chem.
M. Ceylan, Synthesis of chalcone-imide derivatives and investigation of their an-
ticancer and antimicrobial activities, carbonic anhydrase and acetylcholinesterase
enzymes inhibition profiles, Arch. Physiol. Biochem. 124 (2018) 61–68, https://doi.
10