Synthesis and biological evaluation of new 2,4,6-trisubstituted pyrimidines and their N-alkyl derivatives

Article abstract of DOI:10.1016/j.bioorg.2018.10.068

A series of new 2,4,6-trisubstituted pyrimidines and their N-alkyl bromide derivatives were prepared based upon methoxy substituted azachalcones as the starting materials. All newly synthesized compounds were screened for their anti-proliferative, cytotoxic, antibacterial activities and DNA/protein binding affinity. In vitro cell proliferation inhibitory and cell cytotoxic effects of 2,4,6-trisubstituted pyrimidines (1–9) and their N-alkyl bromide derivatives (2a-c, 3a-c, 5a-c, 6a-c, 8a-c, 9a-c) were obtained with the help of the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) cell proliferation, LDH cytotoxicity detection, and microdilution assays. The antimicrobial activity for these compounds was also evaluated following the European Pharmacopoeia 8.0 protocol. The interactions of these compounds with DNA or bovine serum albumin were investigated by the spectrophotometric titration method. When the cytotoxic analysis and anticancer properties of the compounds were examined, most of the compounds significantly exhibited an anti-proliferative potency on cancer cells (IC₅₀ ～ 2–10 μg/mL) and caused a cytotoxic effect as low as control drugs, 5-fluorouracil, and cisplatin (～7–15%). Because the compound-DNA adducts are hyperchromic or hypochromic, they caused variations in their spectra. This situation shows they can be linked to DNA by the groove binding mode at a binding constant range of 2.0 × 104 and 2.4 × 105 M^?1. The antimicrobial screening results revealed that our new compounds for some human Gram(+) and Gram(?) pathogen bacteria showed remarkable activity with MIC values between <7.81 and 125 μg/mL. Overall, incorporation of alkyl chain to pyrimidines in the generation of N-alkyl bromides has resulted in showing differences in DNA/protein binding affinity, along with anti-proliferative and cytotoxic activity in favor of new compounds.

Full text of DOI:10.1016/j.bioorg.2018.10.068

Accepted Manuscript
Synthesis and Biological Evaluation of New 2,4,6-Trisubstituted Pyrimidines
and Their N-Alkyl Derivatives
Nuran Kahriman, Vildan Serdaroğlu, Kıvanç Peker, Ali Aydın, Asu Usta, Seda
Fandaklı, Nurettin Yaylı
PII:
S0045-2068(18)30743-0
DOI:
https://doi.org/10.1016/j.bioorg.2018.10.068
YBIOO 2602
Reference:
Bioorganic Chemistry
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Revised Date:
Accepted Date:
20 July 2018
19 October 2018
30 October 2018
Please cite this article as: N. Kahriman, V. Serdaroğlu, K. Peker, A. Aydın, A. Usta, S. Fandaklı, N. Yaylı, Synthesis
and Biological Evaluation of New 2,4,6-Trisubstituted Pyrimidines and Their N-Alkyl Derivatives, Bioorganic
Chemistry (2018), doi: https://doi.org/10.1016/j.bioorg.2018.10.068
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1
Synthesis and Biological Evaluation of New 2,4,6-Trisubstituted Pyrimidines and Their
N-Alkyl Derivatives
Nuran Kahriman1,*, Vildan Serdaroğlu1, Kıvanç Peker1, Ali Aydın2, Asu Usta3, Seda
Fandaklı1, Nurettin Yaylı4
1 Department of Chemistry, Faculty of Science, Karadeniz Technical University, 61080,
Trabzon-TURKEY
2 Department of Molecular Biology and Genetics, Gaziosmanpaşa University, 60250, Tokat-
TURKEY
3Department of Chemistry, Faculty of Arts and Sciences, Recep Tayyip Erdoğan University,
53100, Rize-TURKEY
4 Faculty of Pharmacy, Karadeniz Technical University, 61080, Trabzon-TURKEY
ABSTRACT
A series of new 2,4,6-trisubstituted pyrimidines and their N-alkyl bromide derivatives were
prepared based upon methoxy substituted azachalcones as the starting materials. All newly
synthesized compounds were screened for their anti-proliferative, cytotoxic, antibacterial
activities and DNA/protein binding affinity. In vitro cell proliferation inhibitory and cell
cytotoxic effects of 2,4,6-trisubstituted pyrimidines (1-9) and their N-alkyl bromide
derivatives (2a-c, 3a-c, 5a-c, 6a-c, 8a-c, 9a-c) were obtained with the help of the 3-[4,5-
dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) cell proliferation, LDH
cytotoxicity detection, and microdilution assays. The antimicrobial activity for these
compounds was also evaluated following the European Pharmacopoeia 8.0 protocol. The
interactions of these compounds with DNA or bovine serum albumin were investigated by the
spectrophotometric titration method. When the cytotoxic analysis and anticancer properties of
the compounds were examined, most of the compounds significantly exhibited an anti-
proliferative potency on cancer cells (IC50 ~2-10 µg/mL) and caused a cytotoxic effect as low
as control drugs, 5-fluorouracil, and cisplatin (~7-15 %). Because the compound-DNA
adducts are hyperchromic or hypochromic, they caused variations in their spectra. This
situation shows they can be linked to DNA by the groove binding mode at a binding constant
range of 2.0 x 10⁴ and 2.4 x 10⁵ M-1. The antimicrobial screening results revealed that our
new compounds for some human Gram(+) and Gram(-) pathogen bacteria showed remarkable
activity with MIC values between <7.81-125 µg/mL. Overall, incorporation of alkyl chain to
pyrimidines in the generation of N-alkyl bromides has resulted in showing differences in
DNA/protein binding affinity, along with anti-proliferative and cytotoxic activity in favor of
new compounds.
Keywords: 2,4,6-Trisubstituted pyrimidine, N-alkyl bromide, anti-proliferative, cytotoxic,
antibacterial activities and DNA/protein binding affinity
* Corresponding author. Tel: +90 462 377 42 76 fax: +90 462 325 31 96
e-mail: nuran_yayli@hotmail.com (N. Kahriman)

2
1. Introduction
Pyrimidine and derivatives are important subunits and they have been found in a variety of
natural products that are important for the synthesis of pharmaceutical and agrochemical
compounds. Pyrimidine ring system has extensive occurence in nature, including the alloxan,
alkaloids, nucleic bases, thiamine (vitaminB1), and numerous pharmacophores [1-3]. They
are also found in many synthetic compounds such as barbiturates and the HIV drug,
zidovudine [4]. Pyrimidine derivatives occupy an important place in medicinal chemistry with
the exhibited antimicrobial [5-6], anticancer [4-5, 7-8], antifungal [4], analgesic [9], antiviral
[5], anticonvulsant [10], anti-inflammatory [5], antitubercular [6], antifungal [4],
antileishmanial [11] and antimalarial activities [12-14].
Some of the of pyrimidine containing drugs, sulfadiazine, sulfamerazine, sulfamethazine, and
trimethoprim (antimicrobial), iodoxuridine, trifluoridine and zidovudine show that antiviral,
5-fluorouracil, ftorafur and piritrexim, isetionate (anticancer), flucytosine (antifungal) and
pyrimethamine (antimalarial) have been used clinically [15-17] (Figure 1).
-Figure 1-
Moreover, different conjugated pyrimidines have luminescence properties and therefore they
have been used in organic light emitting devices (OLED) and molecular wires. They also
perform as ligands in transition metal complexes sometimes forming supramolecular
assemblies. Due to the great importance of the pyrimidine moieties, a number of efficient
methods have been reported in literature for their synthesis [3, 5, 7, 11-14, 18-27].
Chalcones are the most used and very attractive bioactive starting materials for the synthesis
of substituted pyrimidines. Substituted chalcones and guanidene salts can easily react in basic
conditions according to 1,4-Michael addition and give 2,4,6-trisubstituted pyrimidines [5, 19-
20, 22, 11-14, 25, 28]. In recent years, N-alkyl substituted forms of heterocyclic compounds
including azachalcone, azaflavone, azaflavanone, pyridine, carbazole, indole, and
phenothiazine units have acquired much attention for their comprehensive inclusion in
chemistry, biology, and materials science [29-34]. The salt forms of these compounds have an
especially important role in agriculture, the food processing industry, clinics and medicinal
chemistry due to their high biological activity [30-35] because of the cation form of the
synthesised organic compounds.
Recently, there has been considerable interest for the synthesis of substituted pyrimidine due
to their broad biological activities. Therefore, there still is a need to synthesize a new series of
substituted pyrimidine and their N-alkylpyridinium salts to explore the pharmaceutical
relevance. Hence, the aim of our research was to synthesize a series of 2,4,6-trisubstituted
pyrimidines (1-9) and their 5, 10 and 15 carbon containing N-alkyl bromide derivatives (2a-c,
3a-c, 5a-c, 6a-c, 8a-c, 9a-c) and to screen for their antibacterial, DNA/protein binding affinity
and anticancer activities [4-5, 15-17, 20, 11-14, 24-25].
2. Results and discussion
2.1. Chemistry
The known sequential synthesis of the 2,4,6-trisubstituted pyrimidines (1-9) and their N-
alkylpyridinium bromides (2a-c, 3a-c, 5a-c, 6a-c, 8a-c, 9a-c) are depicted in Scheme 1.
-Scheme 1-

3
In the first part of the work, the designed pyrimidine compounds were synthesized by the
reaction which goes on either by the 1,2-addition and/or 1,4- addition (Michael addition) of
guanidine to the α, β-unsaturated carbonyl part of azachalcone, traced by cyclization to give
the corresponding 2-amino-4,6-disubstituted pyrimidines 1-9 [11-14, 20, 24-25]. In the
second step, the target N-alkylpyridinium bromides (2a-c, 3a-c, 5a-c, 6a-c, 8a-c, 9a-c) were
obtained by the bimolecular substitution reaction between pyridinyl nitrogen of pyrimidine
and alkyl bromide [30-35]. The purity of all the synthesized compounds was checked by TLC
and all synthetic compounds were purified by crystallization or column chromatography and
their structures were identified by spectroscopic methods.
FT-IR spectra of target compounds showed two bands in the regions of 1661-1605 cm-1 and
1368-1356 cm-1 which are characteristic for pyrimidine ring [20]. These bands emerged after
the intermolecular cyclization between chalcone and guanidine and own to –C=N- vibrations.
The disappearance of the carbonyl group absorption band at about 1680 cm-1 also supported
the formation of a pyrimidine structure. FT-IR spectra of pyrimidines and N-alkylated
pyrimidines gave bonds between 3457-3077 cm-1 and 3495-3179 cm-1, which provided more
evidence for the free and H-bonded -NH2 absorptions, respectively [20].
The 1H-NMR spectra of target compounds exhibited further support for the 2-
aminopyrimidine cycle, since they indicated a broad singlet for –NH2 at δ 5.2-5.4 ppm in
pyrimidines and δ 5.4-6.0 ppm in alkylated pyrimidines. The reason for the high field
chemical shift of alkylated compounds is their ionic form and 1H and 13C -NMR spectra of all
synthetic compound revealed the characteristic peaks at 7.5-8.2 ppm (1H) and 103.6-109.9
13
ppm (13C) for the H-5 and C-5 of pyrimidine ring [20]. All other carbon peaks in the C-
NMR spectra for the synthetic pyrimidine compounds were found in the range of 111.4-167.9
ppm.
LC-MS/MS spectra of all synthetic compounds were characterized by medium or low a
intensity molecular ions peaks at the appropriate m/z values. The main molecular ion peaks of
compounds 1-9 showed [M+Na]+, [M+1]+ or [methoxyphenyl-1]+ and alkylated pyrimidines
showed [M-79Br/81Br]+ fragments as the base peaks in the mass spectra.
In the second part of the study, we synthesized N-alkylpyridinium bromides containing 5, 10
and 15 carbon chains. As described above, the synthesis of these compounds was carried out
by SN2 reaction and the linking of the alkyl chain to the pyridine nitrogen was confirmed by
1
spectroscopic results. The observed triplet peak at about 4.7-5.1 ppm in H-NMR and the
carbon peak at about 61.8-62.8 ppm in 13C-NMR spectra of N-alkylpyridinium bromides were
typical to –N-CH2- chemical shifts and were evidence for the alkylation of compounds 1-9
due to the strong pKb value of pyridine (8.77) versus the pyrimidine (11.3) [16]. Compared to
pyridine, N-alkylation of pyrimidine is more difficult [16]. Also, the electron density of the
free amino group is decreasing by the mesomeric effect and in this case, it behaves as a poor
nucleophile. After alkylation, the chemical shifts of pyridine protons shifted to a low field.
1
For example, when the chemical shifts of compounds 2 and 2c in H-NMR spectra were
compared, it was clearly seen there was almost no change in chemical shifts of the pyrimidine
and methoxyphenyl protons, while the pyridine protons of 2c shifted to low field (δ (2/2c,
ppm)= 7.7/7.9 (H-5), 7.1/7.1 (H-3'), 7.5/7.5 (H-4'), 7.1/7.1 (H-5'), 7.9/7.9 (H-6'), 9.3/9.6 (H-
2''), 8.7/9.9 (H-4''), 7.4/8.3 (H-5''), and 8.4/9.0 (H-6'')). In addition to all the above, the
synthesis of 1a-c, 4a-c and 7a-c couldn’t be obtained because of the steric hindrance of
pyridine nitrogen of pyrimidines 1, 4 and 7 even under different reaction conditions. Example
studies are available in literature about this [28-29].

4
1
All the newly synthesized compounds were characterized by spectroscopic data including H,
13C, APT, COSY, ACD-NMR, FT-IR, LC-MS/MS, and elemental analysis. All data obtained
from the spectral analyses for all compounds were in full agreement with the proposed
structures.
N-alkylation of target 2-pyridinyl compounds (1a-c, 4a-c and 7a-c) were not obtained. This
could be the steric hindrance for the pyridine nitrogen at two positions as described in the
literature [32-33].
2.2. Biological Evaluation
2.2.1. Evaluation of Anticancer Properties of Molecules
Despite the intensive work on effective cancer treatment, 11 million people still have cancer
in the world, and 7 million people lose their lives due to cancer. Another important issue is
that 3 out of every 5 cancers occur in countries with lower and middle incomes and therefore
they are faced with a much higher degree of disease and economic burden. For this reason,
intensive studies could be designed to synthesise cheap, easily obtainable and effective
chemical molecules for the treatment of cancer in order to prevent deaths. Thus, we evaluated
the anticancer effects of the new synthesised 2,4,6-trisubstituted pyrimidines (1-9) and their 5,
10 and 15 carbon chain containing N-alkyl bromide derivatives (2a-c, 3a-c, 5a-c, 6a-c, 8a-c,
9a-c), using the MTT protocol. IC50 inhibition values generally used for the inhibition studies
and GI50, TGI and LC50 parameters were determined as suggested by NCI from
spectrophotometric data obtained from the MTT assay using cisplatin and 5FU as control
anticancer drugs. It was seen that compounds 1-9 were not sufficiently anti-proliferative on
lung cancer cell line A549 when compared with the control group, even at a high
concentration of 500 μg/mL (Table 1).
-Table 1-
Nevertheless, pyrimidines and their alkylated derivatives showed very strong anticancer
activity on C6 cell line (TGI 3.64-21.31 µg/mL and IC50 5.51-27.46 µg/mL for pyrimidines),
(TGI 1.01-12.16 µg/mL and IC50 1.01-17.48 µg/mL for alkyl derivatives) except compound 7
(Table 2).
-Table 2-
Many of the compounds also showed a very high anti-proliferative effect on HeLa cell line,
except compounds 1, 3, 7, and 9 (TGI 1.01-42.06 μg/mL and IC50 1.0-47.22 μg/mL for alkyl
derivatives) (TGI 5.80-18.07 μg/mL and IC50 8.86-34.64 μg/mL for pyrimidines). According
to the HT29 cell line investigations, synthesized compounds except 1, 3, and 7 exhibited
much better anti-proliferative performance than the positive controls (TGI value 1.19-14.33
μg/mL and IC50 value 1.14-11.71 μg/mL for N-alkyl bromide derivatives) (TGI value 8.01-
182.50 μg/mL and IC50 value 6.57-53.02 μg/mL for 2,4,6-trisubstituted pyrimidines) except
compounds 1, 3, and 7 (Table 2). The compounds 2, 9 (IC50 71.22 and 22.46 μg/mL,
respectively) and 2b-c, 3a-c, 5a-c, 6b-c, 8b-c, 9a-c (IC50 3.46-68.10 μg/mL), which were
effective in the MCF7 cell line, showed considerably high anticancer activities as shown in
Table 1. The alkyl derivatives 2b-c, 3b-c, 5a-c, 6b-c, 8b-c, and 9b-c further exhibited very
strong anticancer properties (IC50 5.57-39.48 μg/mL) on the A549 cancer cell line. It can be
easily seen that the compounds 2, 6, and 8 (IC50 22.50, 7.63, and 8.46 μg/mL, respectively)
and 2b-c, 3b-c, 5a-c, 6b-c, 8b-c, and 9b-c (IC50 6.01-35.60 μg/mL) indicated a remarkable
anti-proliferative effect against hepatocellular carcinoma cell line Hep2B. Anti-proliferative
effects of compounds 2, 4, 5, 6, and 8 (IC50 value 11.58-35.41 μg/mL) and 2a-c, 3a-c, 5a-c,
6b-c, 8a-c, and 9a-c (IC50 1.44-48.76 μg/mL) towards the FL cell line had similar behavior to

5
other cells. While the molecules that were found as anticancer effective according to the TGI
and IC50 values above, they were also examined in terms of LC50 values suggested by NCI
and the following results were obtained. The compounds 8 (LC50 222.34 μg/mL) for cervical
cancer, 3 for glioma cancer (LC50 305.89 μg/mL), 2, 4, 5, 6, 8, 9, 2a, 3a, 6a, 8a, and 9a for
colon cancer (LC50 values ranging from 125.09 μg/mL to >500 μg/mL), 2, 9, 2b-c, 3a, 5a-b,
8a-c, and 9a for breast cancer (LC50 120.20 μg/mL - >500 μg / mL), 2b, 5a-b, and 8b (LC50
449.23 μg/mL - >500 μg/mL) for lung cancer and 2, 8, 2b-c, 5a-b, 8b-c, and 9c (LC50 156.59
µg/mL - >500 µg/mL) for hepatocellular carcinoma are highly recommended for further
pharmacological studies since they emceed very high LC50 values. It showed that high LC50
values of compounds 2, 4, 5, 6, 8, 9, 3a, 5b, 8a-b, and 9a were not too lethal on normal cell
line when LC50 values of test molecules were examined on normal cell line (FL) (Tables 3
and 4).
-Table 3-
-Table 4-
For this reason, these molecules are pharmacologically valuable and may be candidates for
phase studies. When other effective molecules are examined, they need to be reformulated
because they have very low LC50 values and they also have low GI50 values. High lethal
concentration values indicate that the cytotoxic effects of the test substances are less and this
condition is desirable. Low GI50 and TGI values indicate that the cytocidal effects of the test
substances are greater, and again this is a desirable condition. When the anti-proliferative
properties of N-alkyl bromide derivatives were examined, the anti-proliferative properties of b
and c series were prominently increased except series a. This increase is nevertheless
important. It does bring undesirable cytotoxic characters, because when GI50 and TGI values
are examined (Tables 5 and 6), the effect of these molecules is extraordinarily high (GI50 and
TGI values 1-2 μg/mL) in any anticancer agent used in contemporary cancer clinics.
-Table 5-
-Table 6-
Due to this particular situation, we would like to see the full pharmacological potential of
these group molecules on the C6 and HeLa cell lines by conducting an ELISA BrdU assay,
which is a more sensitive measurement, in the next study. We did not have a positive control
that could help us with the nanomolar dose we used, so we had to evaluate the effect in and of
itself. When the ELISA BrdU and the MTT test results described below were evaluated
together, it was seen that the a, b and c series compounds were suitable for further testing in
both types of cancer. When the IC50 data of the test results were examined, except 6a and 9a,
a series compounds exhibited anticancer activity at the microgram level similar to 5-
fluorouracil (5FU) and cisplatin, which were control anticancer molecules, on C6 and HeLa
cells (Tables 5 and 6). When the GI50 values as given in Tables 5 and 6 were examined, b and
c series compounds showed more growth inhibition on C6 and HeLa cell lines than cisplatin
and 5FU at the nanogram level. According to the data given in Tables 7 and 8, the c series
compounds were more anti-proliferative than others.
-Table 7-
-Table 8-
In general, all compounds of the c series and only 9b of the b series exposed exceptional anti-
proliferative properties. ELISA BrdU measurement results also showed us that the test
compounds were not highly anti-proliferative to the normal cell line FL (GI50 values about
1000 ng/mL, TGI values about 3000 ng/mL, LC50 values about 7000 ng/mL) (Table 6). This
situation showed that the test compounds were cancer specific. The cytotoxic effects on the
cells as well as the anticancer activities of the tested compounds were determined by the
membrane damage measurement technique. When we evaluated the LDH activity
measurement results, it was found that the above-mentioned anticancer compounds 5, 8, 2a,

6
3a, and 6a for C6 cells, 3, 4, 7, 5b, 8b, and 9b for HeLa cells, 2a-b, 3b, 6a, and 9a-b for
A549 cells, 6a-b, 8a-b, and 9a for the Hep3B cells, 2a-b, 3a, 5a-b, 8b, and 9a-b for MCF7
cells, 2a, 3a, and 8b for HT29 cells, and 2a, 5a, and 8b for FL cells caused membrane
damage in the range of 5.57% - 18.37% at their IC50 concentration (Tables 7 and 9). These
values are very close to the cytotoxicity values % caused by our positive controls (5FU and
Cisplatin) (Table 8). The results of the pharmacological evaluation of the new molecules
described above on working cell lines largely meet the NCI criteria.
-Table 9-
2.2.2.Morphological Changes that Molecules Cause on Cells
Changes in cell morphology was found with the help of phase-contrast microscopy as a result
of incubation of 2,4,6-trisubstituted pyrimidines and their N-alkyl bromide derivatives after 24
hours at similar concentration ratios used in the above tests. The findings of this study are in
accord with the morphological changes observed in apoptotic cells such as cytoplasmic
extensions of bubble, pinhole, and pocket formation detected on phase-contrast microscopy
(Figures 2 and 3). At the end of the treatment with the test substances, cellular debrids
originating from some necrotic cells and some apoptotic cells were also found. Moreover, the
effect of newly synthesized compounds at low concentrations on cells were so small that it
didn’t change the normal appearance of the cells. However, cells tested at high concentrations
did not maintain normal morphology (Figures 2 and 3).
-Figure 2-
-Figure 3-
2.2.3.Evaluation of Antibacterial Effects of Molecules
The resistance of bacteria that cause non-healing infections to antibiotics is a major global
health problem nowadays [36]. For this reason, there is a need to develop and discover new
antimicrobial agents that are more effective against antibiotic-resistant bacteria. The study of
the antibacterial effects of some of the new compounds synthesized by our research group on
resistant bacteria that cause disease in the human body is very important in this regard. Those
that have MIC values of less than 125 μg/mL and less than our test molecules were evaluated
as antibacterial. This evaluation was made according to the MIC values of antimicrobial drugs
used as positive controls. Our test compounds showed highly antimicrobial activity against
the Gram (+) bacteria described below. When the MIC values of newly synthesized
compounds displayed on Gram (+) bacteria were examined, their antibacterial effects were
found to be more or similar to the SCF (sulbactam (30 µg) + cefoperazone (75 µg))antibiotic
used as a positive control against S. gordonii (NCTC 7870) for 2b-c, 3a-c, 5a, 6b-c, 8b-c, and
9a-c (<7.81 – 125 µg/mL), against S. mutans (ATCC 35668) for 1, 2, 3, 4, 5, 6, 8, 2a-c, 3a-c,
5a-c, 6b-c, 8a-c, and 9a-c, against S. aureus (MRSA ATCC 46300) for 2a-c, 3a-c, 5a-c, 6b-c,
8a-c, and 9a-c (<7.81-125 μg/ml), against S. aureus (MSSA ATCC 29213) for 1, 2, 2a-c, 3a-
c, 5a-c, 6bc, 8a-c, and 9a-c (<7.81 to 125 μg/ml), against S. aureus (ATCC 25923) for 2a-c,
3a-c, 5a-c, 6b-c, 8a-c, and 9a-c, against E. faecalis (ATCC 29212) for 2a-c, 3b-c, 5a-c, 6b-c,
8b-c, and 9b-c (<7.81 – 125 µg/mL) and against E. faecalis (VRE ATCC 19433) for 6, 9, 2c,
5b-c, 6b-c, and 8a (31.25 – 125 µg/mL) (Tables 10, 11 and 12).
-Table 10-
-Table 11-
-Table 12-
Our test compounds showed sufficient antimicrobial activities against the Gram (-) bacteria
mentioned below, but the effects of these antimicrobial activities were not as high as in Gram
(+) bacteria. When the MIC values of the newly synthesized compounds displayed on the

7
Gram (-) bacteria were examined, they had the same sensitivity as the SCF antibiotic used as a
positive control against A. actinomycetemcomitans (ATCC 33384) for compounds 2, 2a-c, 3a-
c, 5a-c, 6b-c, 8c, and 9b-c (<7.81–125 µg/mL), against E. coli (ESBL ATCC 35218) for
compounds 2b, 3b, 5b, 6b, 8b, and 9b (15.62–62.5 µg/mL) and against P. aeruginosa
(AGME ATCC 27853) for compounds 2b, 3b, 5b, 6b, and 9a (31.25–125 µg/mL) (Tables 10,
11 and 12). However, none of our new molecules have achieved a sufficiently strong
antibacterial effect on the E. coli ATCC 25922 strain. The results showed that when the
values of the in vitro antibacterial tests on compounds were evaluated, N-alkyl bromide
derivatives (a-c series) exhibited stronger antibacterial properties than their parent molecules,
2,4,6-trisubstituted pyrimidines (1-9). It is also interesting that both groups of molecules
exhibited antibacterial activities on relatively more Gram (+) bacteria. The most important
point is that the strong antimicrobial activities against resistant strains such as VRE, MRSA,
ESBL and AGME are much better and more desired than the control antibacterial drug SCF.
In general, it can be said that alkyl derivatives have an especially strong effect and are very
suitable for advanced pharmacological researches on some pathogen that cause disease in the
human body on both groups of molecules.
2.2.4.Characteristics of DNA / BSA Binding Properties of Molecules
Before advanced pharmacological testing of newly synthesized drug candidate molecules in
pharmaceutical chemistry, physical interactions with biomacromolecules such as DNA or
protein must be tried using various methods. Spectrophotometric techniques come first
because of the easy and high accuracy of the methods used for this purpose. The interaction of
candidate drug molecules with DNA or protein causes significant changes in the structure of
these biomolecules and can be observed with spectrophotometric techniques. These
significant spectral changes caused by candidate drug molecules on DNA and protein show up
as a hyperchromic or hypochromic effect on the absorption spectra [37]. If the increase or the
decrease in the absorption band occurs when DNA or protein is added at increasing
concentrations to the constant concentrations of the molecule tested, it is called a
hyperchromic effect and hypochromic effect, respectively. The hypochromic effect is usually
an indication that the complex is bound to the DNA by the electrostatic action or often
intercalated. In addition to this change in the spectra, the red or blue shift in the absorption
bands of the complexes may be a sign of the stability of the complex-DNA structure. The
DNA / BSA binding properties of newly synthesized chemicals synthesized were determined
using a UV-vis spectrophotometer. The interactions of these compounds with DNA are as
follows: In the UV-vis spectra of newly synthesized compounds, a single maximum
absorption peak was observed and on this peak, there was no clear bathochromic or
hypsochromic effect with the exception of compounds 3, 5 and 6. When CT-DNA is added in
increasing quantities to the reaction mixture, the reduction in the absorption intensity of
compounds 1 and 4 resulted in hypochromic effect and the increase in the absorption intensity
of compounds 2, 7, 8 and 9 caused hyperchromic appearance. Compounds 3, 5 and 6 also
caused redshift about 10 nm when they evaluated for their possible physical relationships with
DNA and showed hyperchromic properties, unlike the others. Likewise, when CT-DNA was
added in increasing amount to the reaction tube, compounds 2a, 2c, 3b, 5c, 6a, 6c, 8a, 9b, and
9c caused a hyperchromic effect, whereas compounds 2b, 3a, 3c, 5a, 5b, 6b, 8b, 8c, and 9a
from the same group showed a hypochromic action. The spectrum bands described below will
be the guide lines to predict possible interactions of newly synthesized drug candidate
molecules with BSA. The absorption spectra of alkyl derivatives, upon increasing the
concentration of BSA, showed gradually decreases in the peak intensities for compounds 2a,
2b, 3a, 5b and 8b and increases in the peak intensities for compounds 2c, 3b, 3c, 5c, 6b, 6c
and 8c.

8
The binding constants (Kb) of these complexes with CT-DNA were revealed using
spectrophotometric techniques which can be applied easily in research laboratories with the
help of Benesi-Hildebrand equality shown below [38]: [DNA]/(εa–εf) = [DNA]/(εb–εf) +
1/Kb(εb–εf). The [DNA] symbol in this equation is the DNA concentration in the base pairs,
the symbols εa, εf and εb are the molar absorption coefficients of Aobserved/[complex], free
complex and complex-DNA solutions, respectively. Kb is the binding constant related to
compound able to bind to DNA and it can be calculated algebraically according to the slope
of a line between [DNA]/(εa-εf) and [DNA]. When the binding constants given in Table 13
were examined, it was seen that the Kb values of the newly synthesized 2,4,6-trisubstituted
pyrimidines were between 2.0 x 10⁴ and 8.9 x 10⁴ Mˉ¹. The binding constants of the
molecules in this group were ordered from small to large as follows: 2 > 7 > 9 > 3 > 1 > 5 > 8
> 6 > 4.
-Table 13-
When the binding constants of alkyl derivatives were examined in the same table, it was
understood that Kb values were 2.0 x 10⁴ to 2.4 x 10⁵ Mˉ¹ and their DNA binding constants
order was as 3c > 2a > 3b > 3a > 2b > 6a > 5b > 6b > 5a > 2c > 9a > 8c > 5c > 9b > 8a > 9c
> 6c > 8b. High Kb values indicate that the corresponding complexes strongly bind to DNA.
According to the CT-DNA binding studies performed with cisplatin and 5FU anticancer drugs
in the literature, the cisplatin binding constant was reported to be 5.73 × 10⁴ Mˉ¹ and the 5FU
binding constant to be 9.7 × 10⁴Mˉ¹ [39-40]. When we compared the DNA binding affinity of
these two anticancer drugs used clinically with the interest of the DNA binding of the tested
molecules, it was found that the newly synthesized molecules bound sufficiently tightly to the
DNA. When the data in Table 13 was examined, it reveals that the newly synthesized
molecules, especially compound 2a, 3a, 3b, and 3c, bound DNA much more strongly than
cisplatin and 5FU anticancer drugs.
3.
Conclusion
In the present paper, synthetic procedure of a new series of 2,4,6-trisubstitıted pyrimidines (1-
9) and their alkyl derivatives (2a-c, 3a-c, 5a-c, 6a-c, 8a-c, 9a-c) were described. A total of 27
compounds have been synthesized and only compounds 4, 7 and 9 are known in the literature
[17, 24] but no studies have been conducted on their anticancer and antimicrobial properties.
In addition, anti-proliferative, cytotoxic, antibacterial activities and DNA/protein binding
affinities for the all synthetic compounds (1-9, 2a-c, 3a-c, 5a-c, 6a-c, 8a-c, 9a-c) were
investigated. In general, many of the obtained compounds showed anti-proliferative effects on
tested cancer cell lines. Alkylated derivatives, especially all compounds of c series, have
exceptional efficacy. The anticancer results showed that the elongation of the alkyl chain has
a detrimental effect on cancer cell lines. On the other hand, all test compounds exhibited
highly antimicrobial activities against Gram (+) and Gram (-) bacteria but. Gram (+) bacteria
has the strongest activity. In particular, N-alkyl bromide derivatives (a-c series) exhibited
stronger antibacterial properties than their parent molecules 2,4,6-trisubstituted pyrimidines
(1-9). DNA binding affinities for all the synthetic compounds (1-9, 2a-c, 3a-c, 5a-c, 6a-c, 8a-
c, 9a-c) showed that all newly synthesized molecules bound DNA much stronger than
cisplatin and 5FU anticancer drugs The conclusion leads to these compounds being would be
useful for the development of new anticancer drugs.
4. Experimental
4.1. Materials and Equipment

9
All starting chemical reagents and solvents used in the synthesis, purification and biological
activity investigations were high grade commercial products purchased from Aldrich, Fluka,
Sigma, Merck, Amresco, Carlo-Erba, Lonza, Roche and used without further purification.
Thin-layer chromatography (TLC) and column chromatography were performed on Merck
precoated 60 Kieselgel F254 analytical aluminum acidic plates and silica gel 60 (0.040{0.063
1
13
mm), respectively. All reactions were monitored using TLC. H and C NMR spectra were
recorded on a Bruker 400 MHz NMR in CDCl3, CDCl3/CD3OD, CD3OD with tetramethyl-
silane (TMS) as an internal standard. The elemental analyses were performed by a Costech
ECS 4010 instrument. The mass spectral analyses were performed on a Micromass Quattro
LC-MS/MS spectrophotometer. Infrared spectra were obtained using a PerkinElmer 1600FT-
IR (4000-400 cm-1) spectrometer. Melting points were determined using a Stuart SMP10
apparatus.
4.2. Methods
The synthesis of known compounds i-ix were performed according to the previously reported
procedure [32, 42].
4.2.1. General procedure for synthesis of compounds 1-9
Methoxy substituted azachalcone (10 mmol), guanidine hydrochloride (10 mmol) and NaOH
(15 mmol) were mixed in a flask and 20 mL absolute ethanol was added to the flask. The
reaction mixture was refluxed for 6-12 h [5, 11-14, 19-20, 22, 25, 28] with the control of
progress by TLC examination and allowed to cool to room temperature after completion.
Water was added to the completed reactions and pyrimidine was precipated. The solid product
was washed with cold acetone and then water. The filtered product was dried with a freeze
dryer and the purity was checked with TLC. If necessary, it was purified by column
chromatography and the structure was confirmed by spectroscopic methods (1H, 1H-1H COSY
and 13C-APT NMR, LC-MS/MS and FT-IR) and elemental analysis. Numbering of atoms in 2
and 2a was given in Figure 4 as example for spectroscopic analysis of pyrimidines and
alkylated forms.
-Figure 4-
4.2.1.1. 4-(2-methoxyphenyl)-6-(pyridin-2-yl)pyrimidin-2-amine (1)
Yield: 69%. M.p.: 170-172 °C. Rf: 0.40. (Hexane/diethyl ether 1:2).
FT-IR (cm-1): 3297, 3169, 3004, 1626, 1604,1536, 1453, 1247, 751,745.
1H-NMR (400 MHz, CDCl3, ppm): =8.2 (s, 1H, H-5); = 7.0 (d, J= 8.1 Hz, H-3'); = 7.4 (t,
J=8.0 Hz, 1H, H-4'); = 7.1 (t, J=7.6 Hz, 1H, H-5'); = 7.8 (m, 1H, H-6'); = 8.7 (d, J=4.8
Hz, 1H, H-3''); = 7.3 (t, J= 7.6/4.8 Hz, 1H, H-4''); = 7.8 (m, 1H, H-5''); = 8.3 (d, J=8.0 Hz,
1H, H-6''); =5.2 (bs, 2H, NH2); 3.9 (s, 3H, -OCH3).
13C-NMR (100 MHz, CDCl3, ppm): 166.0, 163.7, 163.3, 157.7, 155.1, 149.4, 136.8, 131.1,
130.6, 127.4, 124.7, 121.7, 120.8, 111.4, 109.3, 55.7.
Poz. LC-MS/MS m/z (%): 301(100) [M+Na]+, 279 (30) [M+1]+.
Anal. cal. for C16H14N4O (278.31 g/mol): C 69.05, H 5.07, N 20.13, found: C 69.02, H 5.09,
N 20.12
4.2.1.2. 4-(2-methoxyphenyl)-6-(pyridin-3-yl)pyrimidin-2-amine (2)
Yield: 70%. M.p.: 96-98 °C. Rf: 0.47 (Hexane/diethyl ether 1:2).
FT-IR (cm-1): 3277, 3080, 1660, 1543, 1244, 1023, 754, 633.

10
1H-NMR (400 MHz, CDCl3, ppm): =7.7 (s, 1H, H-5); = 7.1 (d, J= 8.0 Hz, H-3'); = 7.5 (m,
1H, H-4'); = 7.1 (t, J=8.0 Hz, 1H, H-5'); = 7.9 (d, J=8.0 Hz, 1H, H-6'); = 9.3 (s, 1H, H-2'');
= 8.7 (dd, J=6.0 Hz, 1H, H-4''); = 7.4 (m, 1H, H-5''); = 8.4 (d, J=8.0 Hz, 1H, H-6''); =5.3
(bs, 2H, NH2); 3.9 (s, 3H, -OCH3).
13C-NMR (100 MHz, CDCl3, ppm): 166.0, 163.7, 163.3, 157.7, 155.1, 149. 4, 136.8, 131.1,
130.6, 127.4, 124.7, 121.7,120.8, 111.4, 109.3, 55.7.
Poz. LC-MS/MS m/z (%): 279 (100) [M+1]+.
Anal. cal. for C16H14N4O (278.31 g/mol): C 69.05, H 5.07, N 20.13, found: C 69.03, H 5.10,
N 20.12.
4.2.1.3. 4-(2-methoxyphenyl)-6-(pyridin-4-yl)pyrimidin-2-amine (3)
Yield: 68%. M.p.: 185-187 °C. Rf: 0.44 (Hexane/diethyl ether 1:2).
FT-IR (cm-1): 3310, 3141, 2987, 1648, 1578, 1525, 1245, 823, 744, 622.
1H-NMR (400 MHz, CDCl3, ppm): =7.7 (s, 1H, H-5); = 7.1 (d, J= 8.0 Hz, H-3'); = 7.5 (t,
J=8.0 Hz, 1H, H-4'); = 7.1 (t, J=8.0 Hz, 1H, H-5'); = 7.9 (d, J=8.0 Hz, 1H, H-6'); = 7.9 (d,
J=6.0 Hz, 2H, H-2''/6''); = 8.8 (d, J=6.0 Hz, 2H, H-3''/5''); =5.4 (bs, 2H, NH2); 3.9 (s, 3H, -
OCH3).
13C-NMR (100 MHz, CDCl3, ppm): 165.8, 163.5, 162.4, 157.7, 150.4, 145.3, 131.5, 130.7,
126.7, 121.2, 121.1, 111.6, 109.2 , 55.7.
Poz. LC-MS/MS m/z (%):106 (100) [Methoxyphenyl-1]+, 279 (80) [M+1]+.
Anal. cal. for C16H14N4O (278.31 g/mol): C 69.05, H 5.07, N 20.13, found: C 69.02, H 5.08,
N 20.11.
4.2.1.4. 4-(3-methoxyphenyl)-6-(pyridin-2-yl)pyrimidin-2-amine (4)
Yield: 74%. M.p.: 157-159 °C (lit. [28] 131-132oC). Rf: 0.53 (Hexane/diethyl ether 1:2).
FT-IR (cm-1): 3371, 3313, 3184, 2986, 1633, 1538, 1359, 1044, 774.
1H-NMR (400 MHz, CDCl3, ppm): =8.2 (s, 1H, H-5); = 7.4 (m, 1H, H-2'); = 7.1 (d, J=8.0
Hz, 1H, H-4'); = 7.4 (m,1H, H-5'); = 7.7 (d, J=8.0 Hz, 1H, H-6'); = 8.7 (d, J=4.8 Hz, 1H,
H-3''); = 7.4 (m, 1H, H-4''); = 7.8 (t, J=8.0 Hz, 1H, H-5''); = 8.4 (d, J=8.0 Hz, 1H, H-6'');
=5.2 (bs, 2H, NH2); 3.9 (s, 3H, -OCH3).
13C-NMR (100 MHz, CDCl3, ppm): 166.4, 164.7, 163.4, 159.9, 154.7, 149.4, 139.0, 136.9,
129.7, 124.9, 121.6, 119.8, 116.7, 112.1, 104.6, 55.4.
Poz. LC-MS/MS m/z (%): 279 (100) [M+1]+.
Anal. cal. for C16H14N4O (278.31 g/mol): C 69.05, H 5.07, N 20.13, found: C 69.04, H 5.10,
N 20.10
4.2.1.5. 4-(3-methoxyphenyl)-6-(pyridin-3-yl)pyrimidin-2-amine (5)
Yield: 73%. M.p.: 164-166 °C. Rf: 0.41 (Hexane/diethyl ether 1:3).
FT-IR (cm-1): 3385, 3263, 1636, 1556, 1527.
1H-NMR (400 MHz, CDCl3, ppm): =7.5 (s, 1H, H-5); = 7.7 (s, 1H, H-2'); = 7.1 (d, J=8.0
Hz, 1H, H-4'); = 7.4 (t, J=8.0 Hz,1H, H-5'); = 7.6 (d, J=8.0 Hz,1H, H-6'); = 9.3 (s, 1H, H-
2''); = 8.8 (d, J=4.8 Hz, 1H, H-4''); = 7.4 (t, J=4.4 Hz,1H, H-5''); = 8.4 (d, J=8.0 Hz, 1H,
H-6''); =5.2 (s, 2H, NH2); 3.9 (s, 3H, -OCH3).
13C-NMR (100 MHz, CDCl3, ppm): 166.6, 163.6, 163.5, 160.0, 150.9, 148.2, 138.6, 134.8,
133.4, 129.8, 123.7, 116.7, 115.5, 112.3, 104.2, 55.0.
Poz. LC-MS/MS m/z (%): 106 (100) [Methoxyphenyl-1]+, 279 (18) [M+1]+.
Anal. cal. for C16H14N4O (278.31 g/mol): C 69.05, H 5.07, N 20.13, found: C 69.04, H 5.08,
N 20.09.

11
4.2.1.6. 4-(3-methoxyphenyl)-6-(pyridin-4-yl)pyrimidin-2-amine (6)
Yield: 59%. M.p.: 154-156 °C. Rf: 0.52 (Hexane/diethyl ether 1:2).
FT-IR (cm-1): 3502, 3324, 3077, 1632, 1560, 1531, 1260, 778.
1H-NMR (400 MHz, CDCl3, ppm): =7.5 (s, 1H, H-5); = 7.6 (bs, H-2'); = 7.1 (d, J=8.0 Hz,
1H, H-4'); = 7.4 (t, J=8.0 Hz, 1H, H-5'); = 7.6 (d, J=8.0 Hz, 1H, H-6'); = 7.9 (d, J=8.0 Hz,
2H, H-2''/6''); = 8.8 (d, J=8.0 Hz, 2H, H-3''/5''); =5.3 (s, 2H, NH2); 3.9 (s, 3H, -OCH3).
13C-NMR (100 MHz, CDCl3, ppm): 167.3, 164.0, 164.0, 160.5, 150.7, 145.6, 139.0, 130.3,
121.6, 119.0, 117.1, 112.8, 104.9, 55.9.
Poz. LC-MS/MS m/z (%):106 (100) [Methoxyphenyl-1]+ , 279 (23) [M+1]+.
Anal. cal. for C16H14N4O (278.31 g/mol): C 69.05, H 5.07, N 20.13, found: C 69.07, H 5.08,
N 20.11.
4.2.1.7. 4-(4-methoxyphenyl)-6-(pyridin-2-yl)pyrimidin-2-amine (7)
Yield: 61%. M.p.: 208-210 °C (lit. [28] 199-200 oC). Rf: 0.47 (Hexane/diethyl ether 1.5:2.5).
FT-IR (cm-1): 3326, 3185, 1643, 1606, 1561, 1531, 1360, 1256, 1231, 787.
1H-NMR (400 MHz, CDCl3, ppm): =8.1 (s, 1H, H-5); = 8.2 (d, J=8.0 Hz, 2H, H-2'/6'); =
7.0 (d, J=8.0 Hz, 2H, H-3'/5'); = 8.7 (d, J=4.8 Hz, 1H, H-3''); = 7.4 (t, J=8.0 Hz, 1H, H-4'');
= 7.8 (t, J=8.0 Hz, 1H, H-5''); = 8.3 (d, J=8.0 Hz, 1H, H-6''); =5.3 (s, 2H, NH2); 3.9 (s,
3H, -OCH3).
13C-NMR (100 MHz, CDCl3, ppm): 166.3, 164.4, 163.4, 161.9, 154.9, 149.3, 137.1, 129.8,
128.9, 125.0, 121.5, 114.3, 103.7, 55.6.
Poz. LC-MS/MS m/z (%):109 (100) [Methoxybenzene+1] +, 279 (70) [M+1]+.
Anal. cal. for C16H14N4O (278.31 g/mol): C 69.05, H 5.07, N 20.13, found: C 69.04, H 5.09,
N 20.12.
4.2.1.8. 4-(4-methoxyphenyl)-6-(pyridin-3-yl)pyrimidin-2-amine (8)
Yield: 77%. M.p.: 161-163 °C. Rf: 0.50 (Hexane/diethyl ether 1:3).
FT-IR (cm-1): 3457, 3328, 3210, 3188, 1644, 1540, 1515, 1364, 1240, 1023, 803.
1H-NMR (400 MHz, CDCl3, ppm): =7.5 (s, 1H, H-5); = 8.1 (d, J=8.0 Hz, 2H, H-2'/6'); =
7.1 (d, J=8.0 Hz, 2H, H-3'/5'); = 9.3 (d, J=2.0 Hz, 1H, H-2''); = 8.8 (dd, J=4.8 Hz, 1H, H-
4''); = 7.5 (t, J=8.0 Hz, 1H, H-5''); = 8.4 (d, J=8.0 Hz, 1H, H-6''); =5.2 (s, 2H, NH2); 3.9
(s, 3H, -OCH3).
13C-NMR (100 MHz, CDCl3, ppm): 166.1, 163.6, 163.3, 161.9, 151.1, 148.4, 134.5, 133.5,
130.0, 128.9, 123.7, 114.3, 103.5, 55.4.
Poz. LC-MS/MS m/z (%):106 (100) [Methoxyphenyl-1]+, 279 (12) [M+1]+.
Anal. cal. for C16H14N4O (278.31 g/mol): C 69.05, H 5.07, N 20.13, found: C 69.04, H 5.10,
N 20.11.
4.2.1.9. 4-(4-methoxyphenyl)-6-(pyridin-4-yl)pyrimidin-2-amine (9)
Yield: 67%. M.p.: 206-208 °C (lit. [11] 128-130 oC). Rf: 0.45(Hexane/diethyl ether 1.5:2.5).
FT-IR (cm-1): 3326, 3188, 1643, 1606, 1581, 1561, 1531.
1H-NMR (400 MHz, CDCl3, ppm): =7.5 (s, 1H, H-5); = 8.1 (d, J=8.8 Hz, 2H, H-2'/6'); =
7.0 (d, J=8.8 Hz, 2H, H-3'/5'); = 7.9 (d, J=6.4 Hz, 2H, H-2''/6''); = 8.8 (d, J=6.4 Hz, 2H, H-
3''/5''); =5.2 (s, 2H, NH2); 3.9 (s, 3H, -OCH3).
13C-NMR (100 MHz, CDCl3, ppm): 166.4, 163.2, 162.1, 150.2, 145.5, 129.4, 128.7, 121.2,
114.3, 103.7, 55.4.
Poz. LC-MS/MS m/z (%): 279 (100) [M+1]+.
Anal. cal. for C16H14N4O (278.31 g/mol): C 69.05, H 5.07, N 20.13, found: C 69.03, H 5.09,
N 20.12.

12
4.2.2.General procedure for synthesis of compounds 2a-c, 3a-c, 5a-c, 6a-c, 8a-c, 9a-c
Pyrimidines (2, 3, 5, 6, 8, 9) as starting materials (~5. 0 mmol for each) were dissolved in 15
mL of acetonitrile separately. 1-Bromopentane (a series), 1-bromodecane (b series), and 1-
bromopentadecane (c series), 5.0 mmol each) were added to reaction flasks one by one for
every pyrimidines. All solutions were refluxed separately for 12-22 h [31-36]. On completion
of the reaction, followed by TLC examination, the mixtures cooled to room temperature. The
resulting lemon yellow coloured precipitates were filtered and washed first with hexane, then
with ethyl acetate. Also some compounds were obtained in oily form. The purities of dried
products were checked with TLC and structures were identified by spectroscopic methods.
4.2.2.1. 3-[2-amino-6-(2-methoxyphenyl)pyrimidin-4-yl]-1-pentylpyridinium bromide (2a)
Yield: 69%. Oil. Rf :0.35 (Ethyl acetate/ methanol 1:1).
FT-IR (cm-1): 3443, 3322, 3184, 3075, 2931, 2863, 1638, 1599, 1572, 1540, 1247, 756.
1H-NMR (400 MHz, CDCl3, ppm): =7.9 (s, 1H, H-5); = 7.1 (d, J= 8.0 Hz, H-3'); = 7.5 (t,
J=8.0 Hz, 1H, H-4'); = 7.1 (t, J=8.0 Hz, 1H, H-5'); = 7.9 (d, 8.0 Hz, 1H, H-6'); = 9.6 (s,
1H, H-2''); = 9.8 (d, J=6.0 Hz , 1H, H-4''); = 8.3 (t, J=6.0 Hz, 1H, H-5''); = 9.0 (d, J=8.0
Hz, 1H, H-6''); = 5.1 (t, J=8.0 Hz, 2H, H-1'''); = 2.1 (m, 2H, H-2'''); = 1.5-1.4 (m, 4H, H-
3'''- 4'''); = 0.9 (t, J=8.0 Hz, 3H, H-5'''); =5.4 (s, 2H, NH2); 4.0 (s, 3H, -OCH3).
13C-NMR (100 MHz, CDCl3, ppm): 166.9, 163.3, 158.0, 156.8, 146.1, 142.7, 142.3, 138.3,
131.3, 130.8, 128.7, 125.7, 121.1, 111.7, 109.0, 62.8, 56.1, 31.7, 28.0, 22.1, 13.8.
Poz. LC-MS/MS m/z (%): 349 (100) [M-79Br/81Br]+, 350 (25) [M-79Br/81Br+1]+.
Anal. cal. for C21H25BrN4O (429.36 g/mol): C 58.75, H 5.87, N 13.05, found: C 58.79, H
5.89, N 13.03.
4.2.2.2. 3-[2-amino-6-(2-methoxyphenyl)pyrimidin-4-yl]-1-decylpyridinium bromide (2b)
Yield: 80%. M.p.: 146-148 °C. Rf : 0.60 (Ethyl acetate/ methanol 2:1).
FT-IR (cm-1): 3449, 3322, 3183, 3073, 2923, 2853, 1638, 1599, 1569, 1540, 1249, 750.
1H-NMR (400 MHz, CDCl3, ppm): =7.9 (s, 1H, H-5); = 7.0 (d, J= 8.0 Hz, H-3'); = 7.4 (t,
J=8.0 Hz, 1H, H-4'); = 7.1 (t, J=8.0 Hz, 1H, H-5'); = 7.9 (d, J=8.0 Hz, 1H, H-6'); = 9.7 (s,
1H, H-2''); = 9.6 (d, J=6.0 Hz,1H, H-4''); = 8.3 (t, J=8.0 Hz, 1H, H-5''); = 9.0 (d, J=8.0
Hz, 1H, H-6''); = 5.1 (t, J=8.0 Hz, 2H, H-1'''); = 2.1 (m, 2H, H-2'''); = 1.3-1.2 (m,14H, H-
3'''- 9'''); = 0.9 (t, J=6.5 Hz, 3H, H-10'''); =5.6 (s, 2H, NH2); 4.0 (s, 3H, -OCH3).
13C-NMR (100 MHz, CDCl3, ppm): 163.3, 166.8, 158.0, 156.8, 145.8, 142.8, 142.4, 138.1,
132.2, 130.8, 128.7, 125.8, 121.0, 111.7, 109.0, 62.7, 56.1, 32.0, 31.8, 29.4, 29.3, 29.2, 29.1,
26.1, 22.6, 14.1.
Poz. LC-MS/MS m/z (%): 419 (100) [M-79Br/81Br]+, 420 (85) [M-79Br/81Br+1]+.
Anal. cal. for C26H35BrN4O (499.49 g/mol): C 58.75, H 5.87, N 13.05, found: C 58.80, H
5.88, N 13.01.
4.2.2.3. 3-[2-amino-6-(2-methoxyphenyl)pyrimidin-4-yl]-1-pentadecylpyridinium
bromide
(2c)
Yield: 55%. M.p.: 153-155 °C. Rf: 0.52 (Ethyl acetate/ methanol 2:1).
FT-IR (cm-1): 3451, 3323, 3183, 3073, 3003, 2920, 2851, 1639, 1599, 1571, 1540, 1250, 749.
1H-NMR (400 MHz, CDCl3, ppm): =7.9 (s, 1H, H-5); = 7.1 (d, J= 8.0 Hz, H-3'); = 7.5 (t,
J=8.0 Hz, 1H, H-4'); = 7.1 (t, J=8.0 Hz, 1H, H-5'); = 7.9 (d, J=8.0 Hz 1H, H-6'); = 9.6 (s,
1H, H-2''); = 9.9 (d, J=6.0 Hz 1H, H-4''); = 8.3 (t, J=8.0 Hz, 1H, H-5''); = 9.0 (d, J=8.0
Hz, 1H, H-6''); = 5.2 (t, J=8.0 Hz, 2H, H-1'''); = 2.1 (m, 2H, H-2''');= 1.5-1.3 (m, 24H, H-
3'''-14'''); = 0.9 (t, J=8.0 Hz, 3H, H-15'''); =5.4 (s, 2H, NH2); 4.0 (s, 3H, -OCH3).

13
13C-NMR (100 MHz, CDCl3, ppm): 167.1, 163.4, 158.0, 156.7, 146.1, 142.6, 142.2, 138.3,
132.2, 130.8, 128.6, 125.8, 121.1, 111.7, 109.0, 62.8, 56.1 32.0, 31.9, 29.7, 29.6, 29.5, 29.4,
29.1, 26.1, 22.7, 14.1.
Poz. LC-MS/MS m/z (%): 489 (100) [M-79Br/81Br]+, 490 (95) [M-79Br/81Br +1]+.
Anal. cal. for C31H45BrN4O (569.63 g/mol): C 65.37, H 7.96, N 9.84, found: C 65.32, H 7.99,
N 9.81.
4.2.2.4. 4-[2-amino-6- (2-methoxyphenyl)pyrimidin-4-yl] -1-pentylpyridinium bromide (3a)
Yield: 55%. M.p.: 82-84 °C. Rf: 0.39 (Ethyl acetate/ methanol 2:1).
FT-IR (cm-1): 3324, 3205, 2956, 2932, 2863, 1637, 1581, 1541, 1450, 1360, 1247,1014, 769.
1H-NMR (400 MHz, CDCl3/CD3OD, ppm): =7.9 (s, 1H, H-5); = 7.0 (d, J= 8.0 Hz, H-3');
= 7.5 (t, J=8.0 Hz, 1H, H-4'); = 7.1 (t, J=8.0 Hz, 1H, H-5'); = 7.9 (bs, 1H, H-6'); = 8.6 (d,
J=5.6 Hz, 2H, H-2''/6''); = 9.3 (d, J=5.6 Hz, 2H, H-3''/5''); = 4.9 (t, J=8.0 Hz, 2H, H-1'''); =
2.0 (m, 2H, H-2'''); = 1.4 (bs, 4H, H-3'''- 4'''); = 0.9 (t, J=8.0 Hz, 3H, H-5'''); =5.6 (s, 2H,
NH2); 3.9 (s, 3H, -OCH3).
13C-NMR (100 MHz, CDCl3/CD3OD, ppm): 167.1, 163.5, 157.8, 157.1, 153.3, 145.0, 132.2,
130.5, 125.6, 125.4, 121.0, 111.6, 109.7, 61.8, 55.7, 31.3, 28.0, 22.0, 13.6.
Poz. LC-MS/MS m/z (%): 349 (100) [M-79Br/81Br]+.
Anal. cal. for C21H25BrN4O (429.36 g/mol): C 58.75, H 5.87, N 13.05, found: C 58.80, H
5.88, N 13.09.
4.2.2.5. 4-[2-amino-6- (2-methoxyphenyl)pyrimidin-4-yl] -1-decylpyridinium bromide (3b)
Yield: 75%. M.p.: 88-90 °C. Rf: 0.38 (Ethyl acetate/ methanol 2:1).
FT-IR (cm-1): 3322, 3193, 2923, 2853, 1637, 1540, 1447, 1248, 1017, 754.
1H-NMR (400 MHz, CDCl3/CD3OD, ppm): =7.9 (s, 1H, H-5); = 7.1 (d, J= 8.0 Hz, H-3');
= 7.5 (t, J=8.0 Hz, 1H, H-4'); = 7.1 (t, J=8.0 Hz, 1H, H-5'); = 7.9 (d, J=8.0 Hz 1H, H-6');
= 8.7 (d, J=6.8 Hz, 2H, H-2''/6''); = 9.3 (d, J=6.8 Hz, 2H, H-3''/5''); = 4.9 (t, J=8.0 Hz, 2H,
H-1'''); = 2.0 (m, 2H, H-2''');= 1.4-1.3 (m,14H, H-3'''- 9'''); = 0.9 (t, J=8.0 Hz, 3H, H-10''');
=5.7 (s, 2H, NH2); 4.0 (s, 3H, -OCH3).
13C-NMR (100 MHz, CDCl3/CD3OD, ppm): 166.9, 163.4, 158.0, 157.1, 153.1, 145.1, 132.4,
130.7, 125.4, 125.3, 121.1, 111.6, 109.0, 61.9, 55.9, 31.8, 29.4, 29.3, 29.2, 29.0, 26.1, 22.6,
14.0.
Poz. LC-MS/MS m/z (%): 420 (100) [M-79Br/81Br+1]+.
Anal. cal. for C26H35BrN4O (499.49 g/mol): C 58.75, H 5.87, N 13.05, found: C 58.87, H
5.84, N 13.04.
4.2.2.6. 4-[2-amino-6- (2-methoxyphenyl)pyrimidin-4-yl] -1-pentadecylpyridinium bromide
(3c)
Yield: 50%. M.p.: 101-103 °C. Rf: 0.42 (Ethyl acetate/ methanol 1:1).
FT-IR (cm-1): 3434, 3316, 3198, 3039, 2921, 2849, 1636, 1541, 1247, 750.
1H-NMR (400 MHz, CDCl3, ppm): =7.9 (s, 1H, H-5); = 7.0 (d, J= 8.0 Hz, H-3'); = 7.5 (t,
J=8.0 Hz, 1H, H-4'); = 7.1 (t, J=8.0 Hz, 1H, H-5'); = 7.9 (d, J=8.0 Hz, 1H, H-6'); = 8.6 (d,
J=8.0 Hz, 2H, H-2''/6''); = 9.6 (d, J=8.0 Hz, 2H, H-3''/5''); = 5.0 (t, J=8.0 Hz, 2H, H-1'''); =
2.1 (m, 2H, H-2'''); = 1.3-1.2 (m, 24H, H-3'''-14'''); = 0.9 (t, J=8.0 Hz, 3H, H-15'''); =5.5 (s,
2H, NH2); 4.0 (s, 3H, -OCH3).
13C-NMR (100 MHz, CDCl3, ppm): 166.9, 163.5, 158.1, 157.0, 153.2, 145.4, 132.4, 130.8,
125.4, 125.2, 121.1, 111.6, 109.9, 61.8, 55.9, 31.9, 29.68, 29.67, 29.66, 29.65, 29.64, 29.63,
29.59, 29.50, 29.35, 29.08, 26.12, 22.68, 14.1.
Poz. LC-MS/MS m/z (%): 489 (100) [M-79Br/81Br]+, 490 (98) [M-79Br/81Br +1]+.

14
Anal. cal. for C31H45BrN4O (569.63 g/mol): C 65.37, H 7.96, N 9.84, found: C 65.33, H 8.1,
N 9.83.
4.2.2.7. 3-[2-amino-6-(3-methoxyphenyl)pyrimidin-4-yl]-1-pentylpyridinium bromide (5a)
Yield: 58%. Oil. Rf: 0.47 (Ethyl acetate/ methanol 1:1).
FT-IR (cm-1): 3329, 3209, 2931, 1634, 1548,1368, 1267, 1221, 791, 681.
1H-NMR (400 MHz, CDCl3/CD3OD, ppm): =7.9 (s, 1H, H-5); = 7.7 (s, 1H, H-2'); = 7.1
(d, J=8.0 Hz, 1H, H-4'); = 7.5 (t, J=8.0 Hz, 1H, H-5'); = 7.8 (d, J=8.0 Hz, 1H, H-6'); = 9.9
(s, 1H, H-2''); = 9.2 (d, J=8.0 Hz, 1H, H-4'') = 8.2 (t, J=8.0 Hz, 1H, H-5''); = 9.1 (d, J=8.0
Hz, 1H, H-6''); = 4.9 (t, J=8.0 Hz, 2H, H-1'''); = 2.1 (m, 2H, H-2'''); = 1.5-1.4 (m, 4H, H-
3'''- 4'''); = 1.0 (t, J=6.4 Hz, 3H, H-5'''); =5.8 (s, 2H, NH2); 4.0 (s, 3H, -OCH3).
13C-NMR (100 MHz, CDCl3/CD3OD, ppm): 167.6, 163.7, 160.2, 158.1, 145.0, 143.5, 143.0,
138.2, 137.9, 130.1, 128. 6, 120.3, 117.7, 112.5, 104.7, 62.7, 55.9, 31.8, 28.3, 22.3, 13.9.
Poz. LC-MS/MS m/z (%): MW= 428 (79Br)/430 (81Br) g/mol; 349 (100) [M-79Br/81Br]+.
Anal. cal. for C21H25BrN4O (429.36 g/mol): C 58.75, H 5.87, N 13.05, found: C 58.77, H
5.91, N 13.01.
4.2.2.8. 3-[2-amino-6-(3-methoxyphenyl)pyrimidin-4-yl]-1-decylpyridinium bromide (5b)
Yield: 78%. M.p.: 79-81 °C. Rf: 0.48 (Ethyl acetate/ methanol 1:1).
FT-IR (cm-1): 3495, 3306, 3176, 2924, 2854, 1625, 1570, 1543, 1463, 1367, 1233, 1040, 788.
1H-NMR (400 MHz, CDCl3/CD3OD, ppm): =7.9 (s, 1H, H-5); = 7.7 (s, 1H, H-2'); = 7.0
(d, J=8.0 Hz, 1H, H-4'); = 7.4 (t, J=8.0 Hz, 1H, H-5'); = 7.8 (d, J=8.0 Hz, 1H, H-6'); = 9.9
(s, 1H, H-2''); = 9.2 (d, J=8.0 Hz, 1H, H-4'') = 8.1 (t, J=8.0 Hz, 1H, H-5''); = 9.0 (d, J=8.0
Hz, 1H, H-6''); = 4.9 (t, J=8.0 Hz, 2H, H-1'''); = 2.0 (m, 2H, H-2'''); = 1.4-1.2 (m, 14H, H-
3'''- 9'''); = 0.9 (t, J=8.0 Hz, 3H, H-10'''); =5.8 (s, 2H, NH2); 3.9 (s, 3H, -OCH3).
13C-NMR (100 MHz, CDCl3/CD3OD, ppm): 167.3, 163.5, 160.0, 157.9, 144.7, 143.3, 142.8,
137.9, 137.7, 130.0, 128.4, 120.1, 117.5, 112.3, 104.4, 62.5, 55.8, 31.9, 31.8, 29.5, 29.4, 29.2,
29.1, 26.1, 22.6, 14.0.
Poz. LC-MS/MS m/z (%): 419 (100) [M-79Br/81Br]+, 420 (89) [M-79Br/81Br+1]+.
Anal. cal. for C26H35BrN4O (499.49 g/mol): C 58.75, H 5.87, N 13.05, found: C 58.77, H
5.90, N 13.07.
4.2.2.9. 3-[2-amino-6-(3-methoxyphenyl)pyrimidin-4-yl]-1-pentadecylpyridinium
bromide
(5c)
Yield: 50%. M.p.: 74-75 °C. Rf: 0.42 (Ethyl acetate/ methanol 1:1).
FT-IR (cm-1): 3334, 3206, 2921, 2851, 1633, 1569, 1570, 1548, 1508, 1465, 1223, 790, 681.
1H-NMR (400 MHz, CDCl3/CD3OD, ppm): =7.9 (s, 1H, H-5); = 7.7 (s, 1H, H-2'); = 7.0
(d, J=8.0 Hz, 1H, H-4'); = 7.4 (t, J=8.0 Hz, 1H, H-5'); = 7.7 (d, J=8.0 Hz, 1H, H-6'); = 9.9
(s, 1H, H-2''); = 9.2 (d, J=8.0 Hz, 1H, H-4'') = 8.1 (t, J=8.0 Hz, 1H, H-5''); = 9.0 (d, J=6.0
Hz, 1H, H-6''); = 4.9 (t, J=8.0 Hz, 2H, H-1'''); = 2.0 (m, 2H, H-2'''); = 1.4-1.2 (m, 24H, H-
3'''- 14'''); = 0.9 (t, J=8.0 Hz, 3H, H-10'''); =5.7 (bs, 2H, NH2); 3.9 (s, 3H, -OCH3).
13C-NMR (100 MHz, CDCl3/CD3OD, ppm): 167.3, 163.4, 159.9, 157.9, 144.7, 143.2, 142.7,
138.0, 137.7, 129.8, 128.3,120.0, 117.4, 112.2, 104.4, 62.5, 55.6, 31.8, 29.57, 29.56, 29.53,
29.48, 29.41, 29.27, 29.23, 28.95, 26.01, 22.56, 14.0.
Poz. LC-MS/MS m/z (%): 489 (100) [M-79Br/81Br]+, 490 (97) [M-79Br/81Br +1]+.
Anal. cal. for C31H45BrN4O (569.63 g/mol): C 65.37, H 7.96, N 9.84, found: C 65.41, H 7.93,
N 9.80.

15
4.2.2.10. 4-[2-amino-6-(3-methoxyphenyl)pyrimidin-4-yl]-1-pentylpyridinium bromide (6a)
Yield: 55%. M.p.: 148-150 °C. Rf: 0.37 (Ethyl acetate/ methanol 2:1).
FT-IR (cm-1): 3327, 3207, 3116, 2957, 2933, 2868, 1638, 1599, 1542, 1362, 1264, 1217,
1029.
1H-NMR (400 MHz, CDCl3/CD3OD, ppm): =7.8 (s, 1H, H-5); = 7.6 (s, 1H, H-2'); = 7.1
(d, J=8.0 Hz, 1H, H-4'); = 7.4 (t, J=8.0 Hz, 1H, H-5'); = 7.7 (d, J=8.0 Hz, 1H, H-6'); = 8.8
(d, J=8.0 Hz, 2H, H-2''/6''); = 9.2 (d, J=8.0 Hz, 2H, H-3''/5''); = 4.7 (t, J=8.0 Hz, 2H, H-1''');
= 2.1 (m, 2H, H-2'''); = 1.38-1.37 (m, 4H, H-3'''- 4'''); = 0.9 (t, J=8.0 Hz, 3H, H-5'''); =6.0
(s, 2H, NH2); 3.9 (s, 3H, -OCH3).
13C-NMR (100 MHz, CDCl3/CD3OD, ppm): 167.9, 163.8, 160.0, 158.3, 153.1, 144.8, 137.7,
129.9, 125.6, 119.4, 117.3, 112.3, 105.2, 61.8, 55.4, 31.2, 28.0, 21.9, 13.5.
Poz. LC-MS/MS m/z (%): 349 (100) [M-79Br/81Br]+.
Anal. cal. for C21H25BrN4O (429.36 g/mol): C 58.75, H 5.87, N 13.05, found: C 58.79, H
5.89, N 13.06.
4.2.2.11. 4-[2-amino-6-(3-methoxyphenyl)pyrimidin-4-yl]-1-decylpyridinium bromide (6b)
Yield: 75%. M.p.: 125-127 °C. Rf: 0.40 (Ethyl acetate/ methanol 1:1).
FT-IR (cm-1): 3364, 3288, 3181, 3004, 2922, 2851, 1630, 1540, 1449, 1354, 1266, 1042, 835,
777.
1H-NMR (400 MHz, CDCl3/CD3OD, ppm): =7.8 (s, 1H, H-5); = 7.7 (s, 1H, H-2'); = 7.1
(d, J=8.0 Hz, 1H, H-4'); = 7.4 (t, J=8.0 Hz, 1H, H-5'); = 7.7 (d, J=8.0 Hz, 1H, H-6'); = 8.8
(d, J=8.0 Hz, 2H, H-2''/6''); = 9.1 (d, J=8.0 Hz, 2H, H-3''/5''); = 4.7 (t, J=8.0 Hz, 2H, H-1''');
= 2.0 (m, 2H, H-2'''); = 1.4-1.2 (m, 14H, H-3'''- 9'''); = 0.9 (t, J=8.0 Hz, 3H, H-10'''); =6.0
(s, 2H, NH2); 3.9 (s, 3H, -OCH3).
13C-NMR (100 MHz, CDCl3/CD3OD, ppm): 167.8, 163.7, 160.0, 158.3, 153.0, 144.8, 137.7,
129.9, 125.6, 119.8, 117.3, 112.3, 105.2, 61.8, 55.4, 31.7, 31.5, 29.3, 29.2, 29.0, 28.9, 26.0,
22.5, 28.0, 13.8.
Poz. LC-MS/MS m/z (%): 419 (100) [M-79Br/81Br]+.
Anal. cal. for C26H35BrN4O (499.49 g/mol): C 58.75, H 5.87, N 13.05, found: C 58.78, H
5.88, N 13.04.
4.2.2.12. 4-[2-amino-6-(3-methoxyphenyl)pyrimidin-4-yl]-1-pentadecylpyridinium bromide
(6c)
Yield: 55%. M.p.: 205-207 °C. Rf: 0.48 (Ethyl acetate/ methanol 1:1).
FT-IR (cm-1): 3366, 3267, 3179, 2918, 2849, 1639, 1604, 1541, 1359, 1271, 1030, 786.
1H-NMR (400 MHz, CDCl3, ppm): =7.6 (s, 1H, H-5); = 7.6 (s, 1H, H-2'); = 7.0 (d, J=8.0
Hz, 1H, H-4'); = 7.4 (t, J=8.0 Hz, 1H, H-5'); = 7.7 (d, J=8.0 Hz, 1H, H-6'); = 8.5 (d, J=8.0
Hz, 2H, H-2''/6''); = 9.4 (d, J=8.0 Hz, 2H, H-3''/5''); = 4.9 (t, J=8.0 Hz, 2H, H-1'''); = 2.0
(m, 2H, H-2'''); = 1.3-1.2 (m, 24H, H-3'''- 14'''); = 0.9 (t, J=8.0 Hz, 3H, H-15'''); =5.6 (s,
2H, NH2); 3.9 (s, 3H, -OCH3).
13C-NMR (100 MHz, CDCl3, ppm): 167.4, 163.5, 160.0, 157.9, 152.5, 145.1, 137.4, 129.9,
125.4, 119.9, 117.7, 112.1, 105.2, 61.8, 55.7, 31.9, 31.7, 29.4, 29.63, 29.62, 29.59, 29.58,
29.54, 29.4, 29.3 29.0, 26.1, 22.6, 14.1.
Poz. LC-MS/MS m/z (%): 489 (100) [M-79Br/81Br]+, 490 (95) [M-79Br/81Br+1]+.
Anal. cal. for C31H45BrN4O (569.63 g/mol): C 65.37, H 7.96, N 9.84, found: C 65.39, H 7.99,
N 9.88.
4.2.2.13. 3-[2-amino-6-(4-methoxyphenyl)pyrimidin-4-yl]-1-pentylpyridinium bromide (8a)
Yield: 56%. M.p.: 243-245 °C. Rf: 0.48 (Ethyl acetate/ methanol 1:2).

16
FT-IR (cm-1): 3461,3367, 3188, 2952, 1651, 1572, 1547, 1509, 1369, 1247, 1018, 823.
1H-NMR (400 MHz, CDCl3/CD3OD, ppm): =7.9 (s, 1H, H-5); = 8.2 (d, J=8.0 Hz, 2H, H-
2'/6'); = 7.0 (d, J=8.0 Hz, 2H, H-3'/5'); = 9.9 (s, 1H, H-2''); = 9.1 (d, J=8.0 Hz, 1H, H-4'');
= 8.1 (t, J=8.0 Hz, 1H, 5''); = 8.9 (d, J=8.0 Hz, 1H, 6''); = 4.9 (t, J=8.0 Hz, 2H, H-1'''); =
2.1 (m, 2H, H-2'''); = 1.40-1.38 (m, 4H, H-3'''- 4'''); = 0.9 (t, J=8.0 Hz, 3H, H-5'''); =5.7 (s,
2H, NH2); 3.9 (s, 3H, -OCH3).
13C-NMR (100 MHz, CDCl3/CD3OD, ppm): 166.7, 163.3, 162.2, 157.3, 144.5, 143.1, 142.7,
137.9, 129.3, 128.4, 128.3,114.1, 103.5, 62.3, 55.4, 28.0, 22.0, 13.7.
Poz. LC-MS/MS m/z (%): 349 (100) [M-79Br/81Br]+.
Anal. cal. for C21H25BrN4O (429.36 g/mol): C 58.75, H 5.87, N 13.05, found: C 58.80, H
5.84, N 13.08.
4.2.2.14. 3-[2-amino-6-(4-methoxyphenyl)pyrimidin-4-yl]-1-decylpyridinium bromide (8b)
Yield: 70%. M.p.: 197-199 °C. Rf: 0.40 (Ethyl acetate/ methanol 1:2).
FT-IR (cm-1): 3387, 3284, 3190, 3067, 2922, 2853, 1605, 1590, 1505, 1367, 1247, 1173,
1029, 806.
1H-NMR (400 MHz, CDCl3/CD3OD, ppm): 7.9 (s, 1H, H-5); = 8.2 (d, J=8.0 Hz, 2H, H-
2'/6'); = 7.0 (d, J=8.0 Hz, 2H, H-3'/5'); = 10.0 (s, 1H, H-2''); = 9.1 (d, J=8.0 Hz, 1H, H-
4''); = 8.1 (t, J=8.0 Hz, 1H, 5''); = 9.0 (d, J=8.0 Hz, 1H, 6''); = 4.9 (t, J=8.0 Hz, 2H, H-1''');
= 2.0 (m, 2H, H-2'''); = 1.38-1.23 (m, 14H, H-3'''- 9'''); = 0.8 (t, J=8.0 Hz, 3H, H-10''');
=5.6 (s, 2H, NH2); 3.9 (s, 3H, -OCH3).
13C-NMR (100 MHz, CDCl3/CD3OD, ppm): 167.0, 163.4 ,162.3, 157.4, 144.5, 143.1, 142.7,
138.1, 129.2, 128.5, 128.3, 114.1, 103.6, 62.4, 55.4, 31.8, 31.7, 29.3, 29.2, 29.1, 28.9, 26.0,
22.5, 13.9.
Poz. LC-MS/MS m/z (%): 419 (100) [M-79Br/81Br]+, 420 (88) [M-79Br/81Br+1]+.
Anal. cal. for C26H35BrN4O (499.49 g/mol): C 58.75, H 5.87, N 13.05, found: C 58.78, H
5.90, N 13.10.
4.2.2.15. 3-[2-amino-6-(4-methoxyphenyl)pyrimidin-4-yl]-1-pentadecylpyridinium bromide
(8c)
Yield: 54%. M.p.: 210-212 °C. Rf: 0.35 (Ethyl acetate/ methanol 1:2).
FT-IR (cm-1): 3387, 3284, 3187, 3073, 2916, 2850, 1605, 1546, 1505, 1368, 1246, 1172,
1029, 807.
1H-NMR (400 MHz, CDCl3/CD3OD, ppm): =8.0 (s, 1H, H-5); = 8.3 (d, J=8.0 Hz, 2H, H-
2'/6'); = 7.0 (d, J=8.0 Hz, 2H, H-3'/5'); = 10.1 (s, 1H, H-2''); = 9.2 (d, J=8.0 Hz, 1H, H-
4''); = 8.1 (t, J=8.0 Hz, 1H, 5''); = 9.0 (d, J=8.0 Hz, 1H, 6''); = 5.0 (t, J=8.0 Hz, 2H, H-1''');
= 2.1 (m, 2H, H-2'''); = 1.38-1.23 (m, 24H, H-3'''-14'''); = 0.9 (t, J=8.0 Hz, 3H, H-15''');
=5.4 (s, 2H, NH2); 3.9 (s, 3H, -OCH3).
13C-NMR (100 MHz, CDCl3/CD3OD, ppm): 167.0, 163.4, 162.3, 157.4, 144.5, 143.1, 142.7,
138.1, 129.2, 128.5, 128.3, 114.1, 103.6, 62.4, 55.3, 31.8, 29.7, 29.6, 29.5, 29.4, 29.3, 29.2,
29.0, 28.7, 28.1, 26.0, 22.7, 14.0.
Poz. LC-MS/MS m/z (%): 489 (100) [M-79Br/81Br]+, 490 (96) [M-79Br/81Br+1]+.
Anal. cal. for C31H45BrN4O (569.63 g/mol): C 65.37, H 7.96, N 9.84, found: C 65.42, H 7.94,
N 9.88.
4.2.2.16. 4-[2-amino-6-(4-methoxyphenyl)pyrimidin-4-yl]-1-pentylpyridinium bromide (9a)
Yield: 62%. M.p.: 187-189 °C. Rf: 0.48 (Ethyl acetate/ methanol 1:1).
FT-IR (cm-1): 3421, 3274, 3187, 2927, 1580, 1537, 1510, 1362, 1240, 1173, 1024, 812, 785.

17
1H-NMR (400 MHz, CDCl3/CD3OD, ppm): =7.7 (s, 1H, H-5); = 8.1 (d, J=8.0 Hz, 2H, H-
2'/6'); = 7.0 (d, J=8.0 Hz, 2H, H-3'/5'); = 8.7 (d, J=8.0 Hz, 2H, H-2''/6''); = 9.1 (d, J=8.0
Hz, 2H, H-3''/5''); = 4.7 (t, J=8.0 Hz, 2H, H-1'''); = 2.0 (m, 2H, H-2'''); = 1.4.-1.3 (m, 4H,
H-3'''- 4'''); = 0.9 (t, J=8.0 Hz, 3H, H-5'''); =5.7 (s, 2H, NH2); 3.8 (s, 3H, -OCH3).
13C-NMR (100 MHz, CDCl3/CD3OD, ppm): 167.6, 163.7, 162.4, 157.9, 153.3, 144.8, 129.1,
128.6, 125.4, 114.3, 104.6, 61.9, 55.4, 31.2, 28.0, 22.0, 13.6.
Poz. LC-MS/MS m/z (%): 349 (100) [M-79Br/81Br]+.
Anal. cal. for C21H25BrN4O (429.36 g/mol): C 58.75, H 5.87, N 13.05, found: C 58.80, H
5.88, N 13.08.
4.2.2.17. 4-[2-amino-6-(4-methoxyphenyl)pyrimidin-4-yl]-1-decylpyridinium bromide (9b)
Yield: 80%. M.p.: 217-219 °C. Rf: 0.50 (Ethyl acetate/ methanol 1:2).
FT-IR (cm-1): 3452, 3325, 3210, 2925, 2852, 1621, 1545, 1512, 1361, 1246, 1171, 1024, 821.
1H-NMR (400 MHz, CDCl3/CD3OD, ppm): =7.7 (s, 1H, H-5); = 8.1 (d, J=8.0 Hz, 2H, H-
2'/6'); = 7.0 (d, J=8.0 Hz, 2H, H-3'/5'); = 8.7 (d, J=8.0 Hz, 2H, H-2''/6''); = 9.1 (d, J=6.4
Hz, 2H, H-3''/5''); = 4.7 (t, J=8.0 Hz, 2H, H-1'''); = 2.0 (m, 2H, H-2'''); = 1.3.-1.2 (m, 14H,
H-3'''- 9'''); = 0.8 (t, J=8.0 Hz, 3H, H-5'''); =5.7 (s, 2H, NH2); 3.9 (s, 3H, -OCH3).
13C-NMR (100 MHz, CDCl3/CD3OD, ppm): 167.5, 163.7, 162.4, 157.9, 153.3, 144.7, 129.0,
128.5, 125.5, 114.2, 104.5, 61.8, 55.3, 31.7, 31.5, 29.3, 29.2, 29.1, 28.9, 26.0, 22.5, 13.9.
Poz. LC-MS/MS m/z (%): 420 (100) [M-79Br/81Br+1]+.
Anal. cal. for C26H35BrN4O (499.49 g/mol): C 58.75, H 5.87, N 13.05, found: C 58.77, H
5.84, N 13.07.
4.2.2.18. 4-[2-amino-6-(4-methoxyphenyl)pyrimidin-4-yl]-1-pentadecylpyridinium bromide
(9c)
Yield: 55%. M.p.: 181-183 °C. Rf: 0.48 (Ethyl acetate/ methanol 1:2).
FT-IR (cm-1): 3425, 3281, 3193, 2916, 2849, 1606, 1539, 1511, 1364, 1242, 1176, 1026, 812.
1H-NMR (400 MHz, CDCl3/CD3OD, ppm): =7.5 (s, 1H, H-5); = 8.1 (d, J=8.0, 2H, H-2'/6');
= 7.0 (d, J=8.0 Hz, 2H, H-3'/5'); = 8.7 (d, J=6.0 Hz, 2H, H-2''/6''); = 9.1 (d, J=6.0 Hz, 2H,
H-3''/5''); = 4.7 (t, J=8.0 Hz, 2H, H-1'''); = 2.0 (m, 2H, H-2'''); = 1.3-1.2 (m, 24H, H-3'''-
14'''); = 0.8 (t, J=8.0 Hz, 3H, H-15'''); =5.7 (s, NH2); 3.9 (s, 3H, -OCH3).
13C-NMR (100 MHz, CDCl3/CD3OD, ppm): 167.7, 163.8, 162.4, 158.0, 153.5, 144.8, 129.0,
128.7, 125.6, 114.3, 104.6, 61.9, 55.4, 31.8, 31.6, 29.58, 29.55, 29.54, 29.49, 29.40, 29.27,
29.26, 29.25, 28.92, 26.05, 22.57, 13.9.
Poz. LC-MS/MS m/z (%): 489 (100) [M-79Br/81Br]+, 490 (94) [M-79Br/81Br+1]+.
Anal. cal. for C31H45BrN4O (569.63 g/mol): C 65.37, H 7.96, N 9.84, found: C 65.42, H 8.0,
N 9.86.
4.2.3. Pharmacology
4.2.3.1. Preparation of cell culture
The anticancer potential of the compounds were investigated on cancerous HeLa (ATCC®
CCL2™), HT29 (ATCC® HTB38™), MCF7 (ATCC® HTB22™), A549 (ATCC®
CCL185™), C6 (Rat brain glioma, ATCC® CCL-107™), and Hep3B (ATCC® HB8064™)
and normal FL cells (ATCC® CCL62™). The cell lines were cultured in a cell medium
(Dulbecco’s modified eagle’s or RPMI 1640) enriched with 10% (v/v) fetal bovine serum and
2% (v/v) Penicillin-Streptomycin (10,000 U/mL). First, old medium was removed out of the
flask while cells had reached approximately 80% confluence. Next, cells were taken from the
flasks surface using trypsin-EDTA solution and then subjected to centrifugation. Following,
the cell pellet was suspended with fresh media and was inoculated into wells.

18
4.2.3.2. Cell proliferation assay (MTT assay)
A cell suspension containing approximately 1 × 104 cells in 100 µL was seeded into the wells
of 96-well culture plates. The cells were treated with the compounds and control drug, cis-
platin and 5 fluorouracil (5FU), dissolved in sterile DMSO (max 0.5% of DMSO) at final
concentrations of 1.96, 3.91, 7.81, 15.625, 31.25, 62.5, 125, and 250 µg/mL at 37°C with 5%
CO2 for overnight. The final volume of the wells was set to 200 µL by medium. Cell
proliferation assay was evaluated by MTT (yellow tetrazolium MTT (3-(4,5-
dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) methods. Briefly, An MTT stock
solution (5 mg of MTT/mL of distilled water) was filtered and kept for at -20°C until use. The
cells were exposed to a MTT reagent (consisting of one parts of MTT stock solutions and nine
parts of fresh RPMI 1640 without phenol red) for 4 h to form MTT formazan dye followed by
the dye dissolved in DMSO with Sorenson’s buffer for 30 min at room temperature and then
the plate was measured at 560 nm, with 690 nm as a reference interval, using a microplate
reader. Each experiment was repeated at least three times for each cell line.
4.2.3.3. Cytotoxic activity assay
The cytotoxicity of the compounds, cisplatin and 5 fluorouracil on cells was determined
through a Lactate Dehydrogenase Assay Kit according to the manufacturer’s instructions.
Approximately 5 x 103 cells in 100 µL were placed into 96-well plates as triplicates and
treated with 25, 50, 75, and 100 µg/mL concentrations of test compounds at 37°C with 5%
CO2 for 24 h. LDH activity was obtained by determining absorbance at 492 - 630 nm using a
microplate reader. The cytotoxicity assay results were noted as the percent cytotoxicity
according to the following formula: % Cytotoxicity = [(Experimental Value - Low Control /
High Control - Low Control) x 100].
4.2.3.4. BrdU Cell Proliferation Assay (BCPA)
A Cell suspension containing 5x103 cells in 100 µL was pipeted into wells of 96-well cell
culture plates (COSTAR, Corning, USA). Test compounds were dissolved in sterile DMSO.
DMSO amount was adjusted to 0.5 %. The cells were treated with test compounds at final
concentrations of 1000, 2000, 4000, 6000, 8000, 10000, 15000, and 20000 ng/mL. Cell
controls and solvent controls were treated with supplemented DMEM and sterile DMSO
respectively. The final volume of the wells was adjusted to 200 µL by supplemented DMEM.
The cells then were incubated at 37 °C with 5 % CO2 for overnight. The antiproliferative
activity of the compounds was determined using BrdU Cell proliferation ELISA Kit (Roche,
USA), a calorimetric immunoassay based on BrdU incorporation into the cellular DNA,
according to manufacturer’s protocol. Briefly, cells were exposed to BrdU labeling reagent
for 4 h followed by fixation in FixDenat solution for 30 min at room temperature. Then, cells
were cultured with 1:100 diluted anti-BrdU-POD for 1.30 h at room temperature, substrate
solution was added to each well and BrdU incorporation was measured at 450-650 nm using a
microplate reader (Rayto, China). Each experiment was repeated at least three times for each
cell line.
4.2.3.5. Microdilution assay
MIC values of the compounds against bacterial strains were determined on the basis of a
micro-well dilution method. To determine the minimal inhibitory concentration (MIC) values,
inocula of bacteria were prepared using 12 h broth cultures and suspensions were adjusted to
0.5 McFarland standard turbidity. Each substance dissolved in dimethyl sulfoxide (DMSO)

19
and serial twofold dilutions were made in a concentration range from 7.81–1000 µg/mL in
microplate wells containing nutrient broth. Growth of microorganisms was determined
visually after incubation for 24 h at 35 °C. The lowest concentration at which there was no
visible growth (turbidity) was taken as the MIC.
4.2.3.6. DNA binding studies
To find the interaction of the compounds with calf thymus DNA and to calculate the binding
constants ( ), UV–Visible absorption spectroscopy technique was used. A 2.5 mg calf
thymus DNA was dissolved in 10.0 mL Tris–HCl buffer (20 mM Tris–HCl, 20 mM NaCl, pH
7.0) and stabile during one week in the refrigerator. The concentration of calf thymus DNA
was obtained spectrophotometrically with help of ε value of 6600 M−1 cm−1 at 260 nm. After
dissolving the calf thymus DNA fibers in Tris–HCl buffer, the purity of this solution was
checked from the absorbance ratio A260/A280. The calf thymus DNA solution at A260/A280
ratio was equal to 1.87, implying that the DNA was pure enough. These compounds were
diluted with Tris–HCl buffer to obtain 25 µM concentrations. Test compounds in the solutions
were incubated at room temperature for nearly 30 min before the process. The UV-visible
spectral studies were performed in a mixed solvent system (1/9 DMSO/Tris–HCl buffer)
using eight points and the fine structure is observed by UV-visible absorption. The UV
absorption titrations were conducted by keeping the concentration of these compounds fixed
while varying the CT-DNA concentrations (6.5-800 μM). Absorption spectra were recorded
by using 1-cm-path quartz cuvettes at room temperature. To evaluate the interaction of the
compounds with BSA was also used a UV–Visible absorption spectroscopy technique. A 2.5
mg BSA was dissolved in 10.0 mL in Tris–HCl buffer (5 mM Tris–HCl, 10 mM NaCl at pH
7.4) and stored in the refrigerator. The UV–Visible absorption spectra of the BSA solutions
(6.5-800 μM) in the presence of a conserved concentration of the compounds (25 μM) were
scanned in the wavelength range from 300 to 550 nm.
4.2.3.7. Calculation of IC50 and three dose response parameters
IC50 value is a concentration that inhibits half of the cells in vitro. The half maximal
inhibitory concentration (IC50) of the test and control compounds was calculated using XLfit5
or Excel spreadsheet and represent in µM at 95% confidence intervals. The proliferation assay
results were expressed as the percent inhibition according to the following formula: %
Inhibition = [1 - (Absorbance of Treatments / Absorbance of DMSO) x 100]. Three dose
response parameters (GI50, TGI, LC50) were calculated according to the following formulas
using the absorbance measurements of time zero (Tz), control growth (C), and test growth in
the presence of drug (Ti). Growth inhibition of 50 % (GI50) was calculated from [(Ti-Tz)/(C-
Tz)] x 100 = 50, which is the drug concentration resulting in a 50% reduction in the net
growth increase in control cells during the drug incubation. The drug concentration resulting
in total growth inhibition (TGI) was calculated from Ti = Tz. The LC50 indicating a net loss of
cells following treatment was calculated from [(Ti-Tz)/Tz] x 100 = -50.
Acknowledgements
This study was supported by grants from Karadeniz Technical University and the
Scientific and Technological Research Council of Turkey (TÜBİTAK-114R025).
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Scheme 1. Sequential synthesis of 2,4,6-trisubstituted pyrimidines (1-9) and their N-
alkylpyridinium bromides (2a-c, 3a-c, 5a-c, 6a-c, 8a-c, 9a-c).

24
Figure Captions:
Figure 1. Structure of some drugs bearing pyrimidine core
Figure 2. Effect of A-2c, B-2a, C-5b and D-6b on the morphologies of C6 cell lines.
Exponentially growing cells were incubated overnight with various concentrations of
these molecules at 37 °C. Control cells were treated with only DMSO. All
measurements were 100 μm.
Figure 3. Effect of A-2a and B-6a on the morphologies of HeLa cell lines. Exponentially
growing cells were incubated overnight with various concentrations of these molecules
at 37 °C. Control cells were treated with only DMSO. All measurements were 100 μm.
Figure 4. Numbering of atoms on compounds 2 and 2a.
Figure 1.
A-2c
B-2a

25
C-5b
D-6b
Figure 2.
A-2a
B-6a
Figure 3.
Figure 4.

26
Table 1. GI50, TGI, LC50 and IC50 values for 1-9 and 2a-c, 3a-c, 5a-c, 6a-c, 8a-c, 9a-c against MCF7, A549, and
Hep3B
Compound
(µg/mL)
MCF7
TGI
>500
A549
LC50
Hep3B
TGI
>500
23.08
>500
GI50
2.64
2.52
LC50
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
IC50
>500
71.22
334.35 136.68
98.99 213.49
>500
307.11 130.24
>500 317.05
>500
22.46
>500
9.67
8.55
63.31
4.46
6.71
9.75
10.47
5.52
319.02
3.46
5.65
40.29
9.45
8.84
68.10
5.43
GI50
15.78
5.92
TGI
IC50
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
39.48
7.20
>500
7.96
6.40
16.11
33.21
5.57
>500
6.18
GI50
4.40
3.20
3.44
LC50
>500
>500
>500
>500
IC50
>500
22.50
>500
92.82
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
63.64
>500
72.08
40.68
449.23
>500
31.13
>500
37.22
32.01
>500
>500
52.64
>500
77.38
66.32
1
2
3
4
5
6
7
8
78.95
1.65 466.56
1.65 122.83
>500
1.46 440.76
>500
>500
>500
>500
>500
>500
>500
>500
>500
50.67
8.21
2.68 101.04
3.68 119.39
1.80
21.34
>500 111.37
2.56
3.46
1.71
7.73
>500
8.73
84.87
>500
>500
7.63
>500
8.46
1.38
1.33
1.83
4.51
2.65
2.67
2.81
1.83
2.55
2.57
2.68
2.23
>500
>500
24.90
>500
9.94 222.59
8.73 120.20
14.99
26.15
7.25
5.68
2.91
31.94
3.11
2.81
4.26
5.35
2.60
32.41
2.77
2.70
40.75
4.09
3.14
33.24
3.54
3.29
9
2.24 125.67
4.75 127.20
>500 112.85
>500 121.17
>500
2a
2b
2c
3a
3b
3c
5a
5b
5c
6a
6b
6c
8a
8b
8c
9a
9b
9c
3.44
3.44
6.77
2.22
3.24
3.77
4.13
2.72
3.69
2.51
3.10
4.89
4.36
3.64
16.98
12.89 197.47
16.68
12.73
>500
6.01
9.32
14.49
26.23
7.77
>500
6.58
8.04
>500
35.60
70.30
4.56
6.81
>500
45.07
50.81
>500
9.05
7.07
>500
6.09
9.40
>500
60.42
71.36
10.03 280.29
10.75 304.59
19.61
42.00
6.13
14.66 227.85
26.74
7.85
>500
6.65
8.10
>500
36.42
>500
69.07
>500
49.06
47.86
>500
>500
5.61
1.97 419.32
41.01
>500
35.75
37.58
>500
>500
6.83
6.40
1.56
2.32
3.51
2.57
2.73
2.97
2.09
2.08
3.54
5.74
43.23
5.82
>500
31.70
8.40
>500
23.97
7.51
>500
9.43
9.72 238.47
9.03 123.53
13.89 212.63
13.72
74.75
5.54
5.81
>500
52.70
67.66
>500
10.44
9.35
4.90 171.61
>500 162.50
2.72
2.63
8.27
86.81
8.17
8.98
5.68
8.39
9.11 156.59
Table 2. GI50, TGI, LC50 and IC50 values for 1-9 and 2a-c, 3a-c, 5a-c, 6a-c, 8a-c, 9a-c against C6, HeLa, and HT29
Compound
C6
HeLa
HT29
(µg/mL)
GI50
3.98
3.60
1.53
1.73
3.29
3.21
>500
5.83
7.75
2.30
1.00
1.00
1.60
1.00
1.00
1.00
1.00
1.00
4.92
1.00
1.00
2.73
1.00
1.00
3.94
1.00
1.00
TGI
12.51
8.25
4.78
3.64
8.13
8.19
>500
16.53
21.31
4.86
1.02
1.02
3.00
1.01
1.01
1.01
1.01
1.01
12.16
1.03
1.02
5.88
1.01
1.01
9.00
1.01
1.02
LC50
IC50
GI50
>500
8.67
>500
2.47
6.29
7.23
>500
4.71
>500
5.91
1.00
1.00
10.40
1.00
1.00
1.00
1.00
1.00
21.13
1.00
1.00
8.48
1.00
1.00
6.01
1.00
1.00
TGI LC50
IC50
>500
21.86
>500
8.86
27.72
23.40
>500
34.64
>500
17.63
1.00
1.00
24.23
1.01
1.04
1.05
1.04
1.07
47.22
1.04
1.02
24.22
1.03
1.02
19.06
1.00
1.02
GI50
1.43
TGI
>500
8.01
>500
11.49
24.09
9.18
427.61 >500
13.98 >500
182.50 >500
9.47
2.34
1.19
11.87
1.27
2.81
3.85
5.21
5.47
14.33
4.01
2.47
7.43
4.44
6.41
9.69
4.75
10.96
LC50
>500
320.22 6.57
>500
>500
>500
>500
IC50
413.34
101.72 19.85
32.34 11.96
305.89 10.86
>500
>500
49.20
>500
30.49
97.23
65.20
>500
222.34
>500
43.28
1.09
1
2
3
4
5
6
7
8
18.20
>500
5.80
18.21
17.72
>500
18.07
>500
13.30
1.01
2.12
1.30
1.32
1.73
1.99
1.73
1.68
1.72
2.53
1.38
1.02
3.23
1.04
1.57
1.93
2.31
2.46
2.76
1.90
1.42
2.30
1.95
2.53
2.42
2.15
2.77
79.78
6.70
13.74
7.18
90.31
9.11
53.02
20.95
40.02
44.11
>500
86.64
96.53
20.14
1.66
1.62
13.11
1.22
1.07
1.52
1.44
1.32
50.13
1.78
1.54
22.78
1.21
1.34
33.82
1.35
1.62
5.51
12.20
12.66
>500
24.65
27.46
7.01
1.06
1.06
4.26
1.01
1.00
1.03
1.02
1.01
17.48
1.08
1.04
8.42
1.01
1.02
12.94
1.02
1.06
9
233.38 8.07
9.41
3.50
183.98 10.48
4.49
10.67
15.89
25.70
24.68
>500
20.13
10.25
2a
2b
2c
3a
3b
3c
5a
5b
5c
6a
6b
6c
8a
8b
8c
9a
9b
9c
2.21
1.14
1.01
1.13
21.03
1.01
52.55
1.24
1.20
2.69
3.62
4.81
5.11
11.71
3.65
2.24
1.01
1.50
1.02
1.84
1.03
1.63
1.02
1.86
42.06
1.02
97.81
1.60
1.01
1.42
18.90
1.01
56.86
1.44
125.09 6.38
27.98
41.42
340.78 8.05
23.77 4.37
276.91 9.31
4.02
5.82
1.01
1.38
13.72
1.01
45.77
1.12
1.02
1.40

27
Table 3. GI50, TGI, LC50 and IC50 values for 1-9 and 2a-c, 3a-c, 5a-c, 6a-c, 8a-c, 9a-c against FL
Compound
FL
Compounds
FL
(µg/mL)
(µg/mL)
GI50
5.83
3.86
3.01
3.38
3.14
2.83
8.29
3.40
4.98
4.80
2.90
1.68
4.32
1.09
1.36
TGI
>500
23.36
LC50
>500
>500
IC50
>500
22.96
108.91
31.34
GI50
2.88
3.08
1.06
3.85
1.25
1.55
4.48
3.52
1.79
4.70
1.39
1.85
TGI
7.86
9.99
1.45
>500 >500
2.21
3.23
23.30 >500
13.02 187.22 12.88
4.03
49.89 >500
LC50
55.29
110.52 9.90
IC50
7.80
1
2
3
4
5
6
7
8
9
2a
2b
2c
3a
3b
3c
5a
5b
5c
6a
6b
6c
8a
8b
8c
9a
9b
9c
115.61 >500
10.94
1.44
>500
2.19
3.20
22.98
32.27
13.66
11.74
>500
36.57
>500
392.98 13.48
343.44 11.58
>500
>500
16.36
23.52
>500
35.41
331.06
34.66
8.84
3.68
43.37
1.57
358.00 >500
27.74
4.00
48.76
2.64
4.19
35.43
8.93
3.71
44.45
1.58
2.64
>500
90.53
27.88
>500
11.75
21.31
2.66
4.22
17.86
29.35
2.62
Table 4. IC50 (µg/mL) of positive controls in cell lines
HeLa
HT29
A549
MCF7
C6
Hep3B
FL
Cisplatin
5FU
50.29
61.59
40.39
65.19
60.49
69.79
63.79
74.19
33.08
54.30
48.69
62.89
52.79
59.09
Table 5. GI50, TGI, LC50 and IC50 values for 2a, 3a, 5a, 6a, 8a, 9a against C6, HeLa, and FL*
Compound
GI50
TGI
LC50
IC50
(µg/mL)
C6
HeLa FL
C6
HeLa FL
C6
HeLa FL
C6
HeLa FL
17.5
15.1
9.3
37.9
250.2 4.07 51.1
15.2 3.22 18.5
5.45 43.2
183.1 33.66 141.7 >500 >500 58.9
>500 74.49 296.3 >500 >500 108.2 >500 43.17
29.6 8.56 46.02 67.5 51.80 23.5 35.53 7.62
179.1 26.26
2a
3a
5a
114.1 >500 5.66 >500 >500 >500 >500 >500 >500 >500 >500 >500
6a
25.6
72.8
4.14 71.2
>500 19.61 >500 >500 >500 121.9 192.3 15.62
8a
32.9
104.3 2.72 275.2 >500 17.14 >500 >500 >500 >500 >500 11.39
9a
*These parameters were determined by ELISA BrdU Assay
Table 6. GI50, TGI, LC50 and IC50 values for 2b-c, 3b-c, 5b-c, 6b-c, 8b-c, 9b-c against C6, HeLa, and FL*
Compound
(ng/mL)
GI50
HeLa
289
37
943
213
810
83
1
1
36
92
TGI
HeLa
860
154
2075
676
1844
309
4
LC50
HeLa
3158
1111
4996
2746
4641
1683
61
IC50
HeLa
1469
356
2614
1254
2402
644
12
C6
247
FL
1602
633
C6
748
1002
1971
511
696
318
1
6
234
11
350
6
FL
C6
2822
3177
4583
2215
2620
1732
4
82
1436
158
1849
82
FL
C6
1187
1386
2364
936
1144
662
2
16
510
30
722
16
FL
3586
1704
3861
2738
4879
2836
3610
2423
2862
1267
3624
2950
8766
5342
11179
6167
10387
6354
7730
5802
6743
4635
7392
7529
3262
1504
3400
2568
4614
2661
3421
2245
2671
1026
3474
2670
2b
3b
5b
6b
8b
9b
2c
3c
5c
6c
8c
9c
373
922
156
231
85
1
2
60
3
1505
1311
2438
1364
1798
1105
1320
422
3
44
8
154
334
280
6
1186
1747
1636
75
366
686
600
15
96
2
73
2
1881
1275
*These parameters were determined by ELISA BrdU Assay

28
Table 7. % Cytotoxicity of 2a-c, 3a-c, 5a-c, 6a-c, 8a-c, 9a-c at IC50 concentrations against A549, Hep3B,
MCF7, C6, HeLa, HT29, and FL
A549
15.52
15.64
23.43
17.29
14.63
19.25
23.50
21.03
25.46
14.82
25.97
16.78
18.49
26.09
23.50
15.96
14.19
24.07
Hep3B
19.57
22.80
21.28
16.53
19.82
18.56
19.63
17.73
18.05
7.60
14.12
16.28
12.41
11.27
19.38
8.42
MCF7
12.79
13.68
16.47
8.80
17.10
18.11
15.39
15.52
24.76
17.16
18.56
24.64
17.35
15.96
16.66
5.57
C6
HeLa
26.92
24.33
26.22
19.39
23.25
19.32
22.67
18.37
24.45
23.25
22.74
20.90
27.61
14.44
20.14
19.32
14.33
19.51
HT29
15.40
22.55
20.58
15.48
22.61
20.71
21.41
21.66
22.04
21.60
18.87
19.51
20.90
16.91
22.55
24.45
25.97
23.56
FL
Compound
11.96
30.08
27.68
15.65
22.49
29.07
21.41
27.55
25.71
14.32
26.16
27.80
22.11
23.06
29.32
28.75
25.78
23.38
14.76
18.68
22.29
20.84
16.53
18.68
15.39
24.83
20.52
24.13
19.32
16.47
16.35
25.14
18.18
23.56
26.22
18.94
2a
2b
2c
3a
3b
3c
5a
5b
5c
6a
6b
6c
8a
8b
8c
9a
9b
9c
21.91
21.03
13.87
16.02
Table 8. % Cytotoxicity of positive controls at IC50 concentrations
HeLa
9.85
8.83
HT29
11.23
7.91
A549
8.63
9.19
MCF7
10.71
7.69
C6
9.04
10.01
Hep3B
8.46
9.67
FL
8.33
8.44
Cisplatin
5FU
Table 9.
%
Cytotoxicity of 1-9 at IC50
concentrations against C6, HeLa, and HT29
C6
HeLa
19.95
26.60
12.54
10.64
22.99
21.47
9.56
HT29
20.71
25.08
24.76
20.39
22.10
20.91
21.16
24.07
17.29
Compound
20.52
25.39
19.63
19.89
10.39
22.30
18.56
15.83
23.05
1
2
3
4
5
6
7
8
9
21.16
22.36

29
Table 10. Minimum-inhibitory concentrations (MIC, in µg/mL) of 1-9
Microorganisms
E. faecalis VRE
ATCC 19433
E. faecalis
ATCC 29212
S. aureus
ATCC 25923
S. aureus MSSA
ATCC 29213
S. aureus MRSA
ATCC 46300
E. coli
ATCC 25922
E. coli ESBL
ATCC 35218
P. aeruginosa AGME
ATCC 27853
S. mutans
1
2
3
4
5
6
7
8
9
KCN
SCF
500
1000
1000
1000
500
125
250
500
125
NE
250
>1000
500
>1000
1000
125
>1000
1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
125
>1000
>1000
>1000
>1000
>1000
>1000
>1000
62.5
>1000
>1000
>1000
>1000
>1000
>1000
>1000
62.5
>1000
>1000
>1000
>1000
>1000
>1000
>1000
500
>1000
>1000
>1000
>1000
>1000
>1000
>1000
125
>1000
>1000
>1000
>1000
>1000
>1000
>1000
NE
NE
NE
NE
NE
NE
NE
62.5
250
125
>1000
>1000
>1000
>1000
>1000
125
NA
>1000
>1000
>1000
>1000
125
1000
>1000
>1000
>1000
62.5
250
15.62
31.25
250
500 NE
125
ATCC 35668
S. gordonii
500
>1000
125
>1000
1000
>1000
>1000
>1000
1000
>1000
>1000
>1000
>1000
>1000
>1000
1000 NE
1000 NE
125
NCTC 7870
A. actinomycetemcomitans
ATCC 33384
500
62.5
SCF, sulbactam (30 µg) + cefoperazone (75 µg), as a positive control
KCN, potassium cyanide, as a negative control, NE, No effect NA, Not available
Table 11. Minimum-inhibitory concentrations (MIC, in µg/mL) of 2a-c, 3a-c, 5a-c
Microorganisms
E. faecalis VRE
ATCC 19433
E. faecalis
2a
2b
2c
3a
3b
3c
5a
5b
5c
KCN
SCF
250
1000
62.5
500
250
>1000
1000
31.25
62.5
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
250
125
62.5
62.5
62.5
250
<7.81
<7.81
<7.81
<7.81
>1000
15.62
31.25
<7.81
<7.81
<7.81
62.5
62.5
250
125
<7.81
<7.81
<7.81
<7.81
>1000
15.62
62.5
62.5
125
62.5
125
<7.81
<7.81
<7.81
<7.81
>1000
31.25
125
<7.81
<7.81
<7.81
<7.81
>1000
1000
62.5
250
ATCC 29212
S. aureus
ATCC 25923
S. aureus MSSA
ATCC 29213
S. aureus MRSA
ATCC 46300
E. coli
ATCC 25922
E. coli ESBL
ATCC 35218
P. aeruginosa AGME
ATCC 27853
S. mutans
ATCC 35668
S. gordonii
NCTC 7870
A. actinomycetemcomitans
ATCC 33384
<7.81
62.5
62.5
125
<7.81
62.5
<7.81
15.62
250
NA
250
>1000
1000
>1000
<7.81
15.62
62.5
500
>1000
>1000
>1000
<7.81
31.25
125
15.62
31.25
250
250
1000
1000
31.25
62.5
125
250
500
>1000
15.62
62.5
>1000
<7.81
>1000
31.25
<7.81
250
<7.81
62.5
<7.81
>1000
<7.81
125
125
125
<7.81
31.25
62.5
SCF, sulbactam (30 µg) + cefoperazone (75 µg), as a positive control
KCN, potassium cyanide, as a negative control, NE, No effect NA, Not available

30
Table 12. Minimum-inhibitory concentrations (MIC, in µg/mL) of 6a-c, 8a-c, 9a-c
Microorganisms
E. faecalis VRE
ATCC 19433
E. faecalis
6a
6b
6c
8a
8b
8c
9a
9b
9c
KCN SCF
1000
31.25
62.5
125
>1000
>1000
500
1000
1000 NE
250
500
250
<7.81
<7.81
<7.81
<7.81
>1000
15.62
125
31.25
125
250
125
<7.81
15.62
62.5
15.62
250
500
125
125
125
250
500
125
125
62.5
250
<7.81
250
15.62 NE
62.5
250
ATCC 29212
S. aureus
15.62 NE
31.25 NE
<7.81 NE
>1000 NE
>1000 NE
>1000 NE
31.25 NE
<7.81 NE
125 NE
ATCC 25923
S. aureus MSSA
ATCC 29213
S. aureus MRSA
ATCC 46300
E. coli
ATCC 25922
E. coli ESBL
ATCC 35218
P. aeruginosa AGME
ATCC 27853
S. mutans
ATCC 35668
S. gordonii
NCTC 7870
A. actinomycetemcomitans
ATCC 33384
250
<7.81
<7.81
>1000
>1000
>1000
31.25
62.5
31.25
62.5
500
31.25
<7.81
>1000
>1000
>1000
<7.81
<7.81
125
<7.81
<7.81
>1000
15.62
250
NA
250
<7.81
>1000
62.5
250
1000
1000
>1000
31.25
500
15.62
31.25
250
500
1000
62.5
250
250
<7.81
31.25
<7.81
<7.81
<7.81
500
<7.81
125
125
125
250
31.25
250
15.62
62.5
SCF, sulbactam (30 µg) + cefoperazone (75 µg), as a positive control
KCN, potassium cyanide, as a negative control, NE, No effect NA, Not available
Table 13. The binding constants (Kb) of these compounds
No
1
2
3
4
5
6
7
No
2a
2b
2c
3a
3b
3c
5a
5b
5c
No
6a
6b
6c
8a
8b
8c
9a
9b
9c
Kb (Mˉ¹)
6.3 x 104
8.9 x 104
6.5 x 104
2.0 x 104
5.8 x 104
3.3 x 104
8.2 x 104
4.8 x 104
6.6 x 104
Kb (Mˉ¹)
1.9 x 10⁵
9.5 x 104
4.7 x 104
9.9 x 104
1.4 x 10⁵
2.4 x 10⁵
5.9 x 104
8.0 x 104
4.0 x 104
Kb (Mˉ¹)
9.4 x 104
7.9 x 104
2.2 x 104
3.1 x 104
2.0 x 104
4.4 x 104
4.5 x 104
3.5 x 104
2.6 x 104
8
9