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

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 Kahriman^1,*, Vildan Serdaroğlu¹, Kıvanç Peker¹, Ali Aydın², Asu Usta³, Seda

Fandaklı¹, Nurettin Yaylı⁴

¹Department of Chemistry, Faculty of Science, Karadeniz Technical University, 61080,

Trabzon-TURKEY

²Department 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

⁴Faculty 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^-1and

1368-1356 cm^-1which 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^-1also supported

the formation of a pyrimidine structure. FT-IR spectra of pyrimidines and N-alkylated

pyrimidines gave bonds between 3457-3077 cm^-1and 3495-3179 cm^-1, which provided more

evidence for the free and H-bonded -NH₂absorptions, respectively [20].

The ¹H-NMR spectra of target compounds exhibited further support for the 2-

aminopyrimidine cycle, since they indicated a broad singlet for –NH₂at 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 S_N2 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 pK_bvalue 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. IC₅₀inhibition values generally used for the inhibition studies

and GI₅₀, TGI and LC₅₀parameters 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 IC₅₀5.51-27.46 µg/mL for pyrimidines),

(TGI 1.01-12.16 µg/mL and IC₅₀1.01-17.48 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 IC₅₀1.0-47.22 μg/mL for alkyl

derivatives) (TGI 5.80-18.07 μg/mL and IC₅₀8.86-34.64 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 IC₅₀value 1.14-11.71 μg/mL for N-alkyl bromide derivatives) (TGI value 8.01-

182.50 μg/mL and IC₅₀value 6.57-53.02 μg/mL for 2,4,6-trisubstituted pyrimidines) except

compounds 1, 3, and 7 (Table 2). The compounds 2, 9 (IC₅₀71.22 and 22.46 μg/mL,

respectively) and 2b-c, 3a-c, 5a-c, 6b-c, 8b-c, 9a-c (IC₅₀3.46-68.10 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 (IC₅₀5.57-39.48 μg/mL) on the A549 cancer cell line. It can be

easily seen that the compounds 2, 6, and 8 (IC₅₀22.50, 7.63, and 8.46 μg/mL, respectively)

and 2b-c, 3b-c, 5a-c, 6b-c, 8b-c, and 9b-c (IC₅₀6.01-35.60 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 (IC₅₀value 11.58-35.41 μg/mL) and 2a-c, 3a-c, 5a-c,

6b-c, 8a-c, and 9a-c (IC₅₀1.44-48.76 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 IC₅₀values above, they were also examined in terms of LC₅₀values suggested by NCI

and the following results were obtained. The compounds 8 (LC₅₀222.34 μg/mL) for cervical

cancer, 3 for glioma cancer (LC₅₀305.89 μg/mL), 2, 4, 5, 6, 8, 9, 2a, 3a, 6a, 8a, and 9a for

colon cancer (LC₅₀values ranging from 125.09 μg/mL to >500 μg/mL), 2, 9, 2b-c, 3a, 5a-b,

8a-c, and 9a for breast cancer (LC₅₀120.20 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 (LC₅₀156.59

µg/mL - >500 µg/mL) for hepatocellular carcinoma are highly recommended for further

pharmacological studies since they emceed very high LC₅₀values. 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 LC₅₀values 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 LC₅₀values and they also have low GI₅₀values. High lethal

concentration values indicate that the cytotoxic effects of the test substances are less and this

condition is desirable. Low GI₅₀and 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 GI₅₀and TGI values

are examined (Tables 5 and 6), the effect of these molecules is extraordinarily high (GI₅₀and

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 IC₅₀data 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 GI₅₀values 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 (GI₅₀values about

1000 ng/mL, TGI values about 3000 ng/mL, LC₅₀values 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 IC₅₀concentration (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 A_observed/[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 F₂₅₄analytical 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] 131-132^oC). 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 × 10⁴cells 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%

CO₂for 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 10³cells 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%

CO₂for 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 5x10³cells 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 % CO₂for 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⁻¹cm⁻¹at 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 IC₅₀and 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 LC₅₀indicating 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, LC₅₀and IC₅₀values 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

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

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

IC₅₀

>500

22.50

>500

92.82

>500

63.64

>500

72.08

40.68

449.23

>500

31.13

>500

37.22

32.01

>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

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

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

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

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

5.61

1.97 419.32

41.01

>500

35.75

37.58

>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, LC₅₀and IC₅₀values 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

1.60

1.00

4.92

1.00

2.73

1.00

3.94

1.00

TGI

12.51

8.25

4.78

3.64

8.13

8.19

>500

16.53

21.31

4.86

1.02

3.00

1.01

12.16

1.03

1.02

5.88

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

10.40

1.00

21.13

1.00

8.48

1.00

6.01

1.00

TGI LC₅₀

IC₅₀

>500

21.86

>500

8.86

27.72

23.40

>500

34.64

>500

17.63

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

IC₅₀

413.34

101.72 19.85

32.34 11.96

305.89 10.86

>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

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, LC₅₀and IC₅₀values for 1-9 and 2a-c, 3a-c, 5a-c, 6a-c, 8a-c, 9a-c against FL

Compound

FL

Compounds

FL

(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

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

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, LC₅₀and IC₅₀values 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, LC₅₀and IC₅₀values 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

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 IC₅₀concentrations 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 IC₅₀concentrations

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

500

125

250

500

125

NE

250

>1000

500

>1000

1000

125

>1000

1000

>1000

125

>1000

62.5

>1000

62.5

>1000

500

>1000

125

>1000

NE

62.5

250

125

>1000

125

NA

>1000

125

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 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

250

125

62.5

250

<7.81

>1000

15.62

31.25

<7.81

62.5

250

125

<7.81

>1000

15.62

62.5

125

62.5

125

<7.81

>1000

31.25

125

<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

125

<7.81

62.5

<7.81

15.62

250

NA

250

>1000

1000

>1000

<7.81

15.62

62.5

500

>1000

<7.81

31.25

125

15.62

31.25

250

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

<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

500

1000

1000 NE

250

500

250

<7.81

>1000

15.62

125

31.25

125

250

125

<7.81

15.62

62.5

15.62

250

500

125

250

500

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

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

>1000

31.25

62.5

31.25

62.5

500

31.25

<7.81

>1000

<7.81

125

<7.81

>1000

15.62

250

NA

250

<7.81

>1000

62.5

250

1000

>1000

31.25

500

15.62

31.25

250

500

1000

62.5

250

<7.81

31.25

<7.81

500

<7.81

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 (K_b) 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

Article Doi

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

DOI: 10.1016/j.bioorg.2018.10.068

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

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

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