The Antifungal Effect of some 4-Chloro-6-Methoxy-N, N-Dimethylpyrimidin-2-Amine Derivatives Containing a Heterocyclic Compound on the Important types of Fungi

Jafar, Mahdi, Hadwan, and Alameri: The Antifungal Effect of some 4-Chloro-6-Methoxy-N, N-Dimethylpyrimidin-2-Amine Derivatives Containing a Heterocyclic Compound on the Important types of Fungi

Authors

INTRODUCTION

Pyrimidines are important member in the heterocyclic compounds. Pyrimidines exist in nature as a component of nuclear acid and isolated for the first time by Gabriel in 1899 Gabriel and Colman in 1899.1 The name pyrimidine was approved by the International Union of Pure and Applied Chemistry (IUPAC) for this heterocyclic ring system. Several hydro and oxo derivatives of the pyrimidine which are particularly important in biological systems are normally referred to by their nonsystematic names such as 2,4-(1H,3H)-pyrimidinedione2 (Uracil), 5-methyluracil3 (Thymine) and 4-aminopyrimidine-2-(1H)-one4 (Cytosine).2,3

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The pyrimidine nucleus also establishes the major part of vital molecules including vitamins such as thiamine,4 riboflavin,5 and folic acid.6 Derivatives of pyrimidine have a long and distinguished history extending from the days of their discovery as important constituents of nucleic acids to their current use in the chemotherapy of acquired immunodeficiency syndrome (AIDS).

Several pyrimidine derivatives exhibited significant antitumor activity e.g. hydrazine-pyrimidine-5-carbonitrile derivatives5 with inhibitory effects on the growth of a wide range of cancer cell lines generally at some cases at 10−7 M concentrations.7 2,4-Diamino-N4-6-diarylpyrimidnes6 were identified as blocked proliferation of tumor cell lines in vivo, especially duodenum cancer (DU145, IC50 = 0.23

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Azam et al.8 have synthesized pyrimidine bridged thiadiazole derivatives 5- {(4,6-disubstituted pyrimidine-2-yl) thio methyl}-N-phenyl-1,3,4, thiadiazol-2-amines which exhibited significant antitumor activity against human breast cancer MCF 7 cell line. However, moderate antioxidant activity was observed. Analogously, Amin and et al.9 synthesized three tetralin-6-yl pyrimidines found that they were active against liver cancer cell (Hep G2) with IC50=8.66 and 7.11 μg/ml respectively.

Several methods have been reported for the synthesis of various pyrimidines. One method was classic Biginelli reaction,10 many papers have been reported for the synthesis of pyrimidine derivatives.11-14 Monastrol, ethyl 4-(3-hydroxyphenyl)-6-methyl-2-sulfanylidene-3,4-dihydro-1H-pyrimidine-5-carboxylate was discovered by Prof. Mayer of Konstanz University, Germany,15 shown to inhibit the Kinesin Eg 5, a motor protein important for spindle bipolarity. Drugs that inhibit kinesins are being developed as anti-cancer agents with the hope that they will inhibit proliferation of tumor cells. Many substituted pyrimidine derivatives synthesized and evaluated against cancer and HIV showed different significant activity toward cancer cell in vitro.16-18 In continuous of our works to synthesized and evaluated some derivative of pyrimidine towards HIV, HCV, many type of bacteria, cyclin-dependent kinase 2 (CDK2) inhibitory activities and monastrol analogues,19-21 so we herein aim to study the activity of some heterocyclic rings like [1,2,3-thiadiazole, 1,2,3-selenadiazole, benzo[d]thiazol-2-ylthio)] and thiophenyl compounds against some fungi such as Aspergillus terreus and Aspergillus niger.

Aspergillus terreus was described as a filamentous fungus that linked with organic detritus decay in the soil rhizosphere,22 Additionally, A. terreus has been described as a human pathogen and can initiate cutaneous, and subcutaneous mycoses that disturb the nail bed,23 outer ear canal,24 and skin.25 Newly, A. terreus has been documented to be accompanying with postoperative osteomyelitis,26 endophthalmitis and peritonitis.27 In addition to Aspergillus niger which Genome sequencing of A. niger is significant because of its participation in formation citric acid as well as industrial enzymes, such as proteases and lipases. Additional properties of this species involve pathogens that initiate the decomposition of food and formation of secondary metabolites, such as aflatoxin.28,29

Experimental Section

General

Melting points are uncorrected and were measured on a strut melting point apparatus (England). NMR data were obtained on 500 MHz (1H) and (150.91 MHz (13C spectrometers (Bruker, Tehran Medical University) with DMSO-d6 solvent, with tetramethylsilane (TMS) as an internal standard, was used to record 1H-NMR spectra and 13C-NMR. Chemical shifts were reported in (δ ppm.) spectrometers. Elemental analyses were performed on a PE-2400 elemental analyzer; the C, H and N analysis were repeated duplicate. Microwave oven LG MOD MH7947S 1450- 1150 W. All solvents and reagents were obtained commercially from Aldrich, Fluka. Silica gel (0.040 – 0.063 mm) used for analytical silica gel TLC plates 60 F254 were purchased from Merck, compounds were detected by their absorption under UV light.

Experiment Chemistry
General procedure of O-nucleophilic displacement of chloride substituted in the pyrimidine heterocyclic ring.

A mixture of different nucleophilic compounds6,7 potassium carbonate (2.71 g, 19.65 mmol), potassium iodide (5.21 g, 19.6 mmol) and 4-chloro-6-methoxy-N, N-dimethylpyrimidin-2-amine 1 (0.5 g, 0.0026 mol) in (40 ml) of dry acetone and 2-3 drops of Aliquate 336 was refluxed for under microwave irradiation. The reaction was followed by TLC in chloroform until completion (40-45 min). Then the reaction mixture was cooled to room temperature, the precipitated salt was removed by filtration. The organic solvent was dried in vacuo together with the excess of compound 1 After that the product was separated from the traces of compound 1 on a Silica gel column (40 cm) with petroleum ether. Then, the product which was remained at upper the part of the column was eluted by chloroform.

4-(4-(1,2,3-selenadiazol-4-yl) phenoxy)-6-methoxy-N, N-dimethylpyrimidin-2-amine (2)

From 4-(1,2,3–seleniadiazol-4-yl) phenol compound 6 (0.87 g, 0.0039 mole)Yield 94%, color red, m.p = 87-88°C IR spectrum υ = cm-1 1591 (C=N), 1382 (C-N), 1030 (C-O-C), 1535 (N=N), 1490, 1450 (C=C) aromatic, 621 (C-Se): 1H-NMR (DMSO-d6) δ (ppm) 9.01 (s, 1H, Hseleniadiazole), 8.11-7.39 (m, 4H, Hphenoxy), 6.95 (s, 1H, Hprimidine), 3.78 (s, 3H, OMe), 3.08 (s, 6H, NMe2); 13C-NMR (DMSO-d6) δ (ppm) 171.63 (C4), 163.787 (C6), 162.06 (C2), 156.00 (C7), 130.03 (C10), 128.17 (C3’+C4’), 125.50 (C9), 122.84 (C11), 114.94 (C1’+C2’), 92.73 (C5), 52.45 (C-OMe), 36.41 (C-NMe2). Anal. calc. for C15H15N5O2Se (376.28 C, 47.88; H, 4.02; N, 18.61; Found: C, 47.48; H, 4.00; N, 18.01.

4-(4-(1,2,3-thiadiazol-4-yl) phenoxy)-6-methoxy-N, N-dimethylpyrimidin-2-amine (3)

From 4-(1,2,3–thiazol-4-yl) phenol compound 7 (0.69 g, 0.0039 mole). Yield 92%, color Pale yellow, m.p = 90-91°C IR spectrum υ = cm-1 1591 (C=N), 1380 (C-N), 1035 (C-O-C), 1537 (N=N), 1494, 1452 (C=C), 667 (C-S): 1H-NMR (DMSO-d6) δ (ppm) 9.02 (s, 1H, Hthiadiazole), 8.29-8.02 (m, 4H, Hphenoxy), 6.62 (s, 1H, Hprimidine), 3.90 (s, 3H, OMe), 3.20 (s, 6H, NMe2); 13C-NMR (DMSO-d6) δ (ppm) 171.22 (C4), 163.33 (C6), 162.28 (C2), 138.64 (C7), 136.98 (C10), 133.17 (C3’+C4’), 132.94 (C9), 128.54 (C11), 115.75 (C1’+C2’), 91.25 (C5), 53.43 (C-OMe), 36.95 (C-NMe2). Anal. calc. for C15H15N5O2S (329.38): C, 54.70; H, 4.59; N, 9.71; S, 9.73 Found: C, 54.40; H, 4.29; N, 9.56; S, 9.13.

General procedure of S-nucleophilic displacement of chloride substituted in the pyrimidine heterocyclic ring.

A mixture of different nucleophilic compounds8,9 and 4-chloro-6-methoxy-N, N-dimethylpyrimidin-2-amine 1 (0.5 g, 0.0026 mol) in (30 ml) of dry DMF and few drops of conc. NaOH was refluxed for under microwave irradiation. The reaction was followed by TLC in chloroform until completion (30 min). Then the reaction mixture was cooled to room temperature, the precipitated salt was removed by filtration. The organic solvent was dried in vacuo together with the excess of compound 1, after that the product was separated from the traces of compound 1 on a Silica gel column (30 cm) with petroleum ether. Then, the product which was remained at upper the part of the column was eluted by chloroform.

4-methoxy-N, N-dimethyl-6-(phenylthio) pyrimidin-2-amine (4)

From sodium benzene thiolate compound 8 (0.51 g, 0.0039 mol). Yield 96%, color Pale yellow, m.p = 67-68°C IR spectrum υ = cm-1 1597 (C=N), 1382 (C-N), 1037 (C-O-C), 1450, 1419 (C=C), 621 (C-S): 1H-NMR (DMSO-d6) δ (ppm) 7.63-7.07 (m, 5H, Hphenyl), 6.82 (s, 1H, Hprimidine), 3.87 (s, 6H, NMe2), 3.16 (s, 3H, OMe); 13C-NMR (DMSO-d6) δ (ppm) 171.53 (C4), 163.70 (C6), 162.06 (C2), 137.26 (C1’), 132.88-124.19 (Caromatic), 92.22 (C5), 53.04 (C-OMe), 36.43 (C-NMe2). Anal. calc. for C13H15N3OS (261.34): C, 59.75; H, 5.79; N, 16.08; O, 6.12; S, 12.27 Found: C, 59.55; H, 5.09; N, 16.01; O, S, 12.07.

4-(benzo[d]thiazol-2-ylthio)-6-methoxy-N, N-dimethylpyrimidin-2-amine (5)

From benzo[d]thiazole-2-thiol compound 9 (0.65 g, 0.0039 mole). The yellow oil that was obtained is crystallized very slowly in ice bath. Yield 90%, color yellow, m.p = 112-113°C IR spectrum υ = cm-1 1593 (C=N), 1380 (C-N), 1033 (C-O-C), 1490, 1448 (C=C), 620 (C-S): 1H-NMR (DMSO-d6) δ (ppm) 8.02-8.31 (m, 4H, HBenzo), 6.62 (s, 1H, Hprimidine), 3.20 (s, 6H, NMe2), 3.09 (s, 3H, OMe); 13C-NMR (DMSO-d6) δ (ppm) 171.78 (C7), 164.55 (C6), 161.07 (C4), 185.89 (C2), 155.22 (C2’), 129.17 (C1’), 125.38 (C4’+C5’), 122.64 (C3’+C6’), 95.02 (C5), 35.54 (C-OMe), 36.85 (C-NMe2). Anal. calc. for C14H14N4OS2 (318.06): C, 52.75; H, 4.33; N, 17.44; S, 20.10 Found: C, 52.81; H, 4.43; N, 17.60; S, 20.14.

Antifungal activity Potato dextrose agar (PDA)

The culture was prepared by dissolving 40 gm. from (PDF) powder in amount of distilled water and completes the volume to 1000 ml, then it sterilized in autoclave at 121Cº and 15 pound /in2 pressure for 20 min. This medium was used for determination of optimum culture media for inhibition process.

The source of fungi isolates

The fungi which include Aspergillus terreus, Aspergillus niger were obtained from the unit of isolation and classification of fungi isolates /Department of Biology /College of Science /University of Babylon.

Method of holes

Antifungal bioassays were performed on petri dish plates contain 20 ml of standard PDA agar. After the mycelia colonies were developed, 3 wells of 0.5 cm in diameter were made 1cm from the rim. The antibiotic was (1 ml) to add to the wells and the plates were incubated at 25Cº for 5d. Growth inhibition zone were observed. The percentage inhibition of radial mycelia growth of the test fungus was calculated. Stability.

The stability of antibiotic compounds was monitored by using FT-IR spectrum for each compound (one measure every one week). The spectrum was obtained for compounds that stored at refrigerator (2-8Cº). Any change in the FT-IR spectrum is interpreted as a change in the structure of the corresponding compound.

Solubility

The solvents listed in Table 3 and synthesized compounds were used without further purification. The water used was distilled, deionized, and filtered (0.2µm). The temperature was kept with 25 ± 0.02°C.

Toxicity

The toxicity of synthesized compounds was studied via determination the lethal dose (LD50). Lethal dose (LD50) is represents the quantity of an ingested material that kills fifty percent of a test sample in the study. It is expressed in milligrams of material per kilogram of body weight. A suitable method of Miller and Tainter30 is used; as described by Agina et al.31

Statistical analysis

Data analysis was established using SPSS 21 for Windows (SPSS Inc., Chicago, IL, USA). Data were evaluated by one-way analysis of variance (ANOVA) followed by LSD test.

RESULT AND DISCUSSION

The pyrimidine derivative compounds are widely used in different application fields It is anti-HIV drugs that some substituted pyrimidine derivatives exhibited a potent activity against the fatal disease AIDS such as Riplivirin and Etravrin.32-35 These molecules prompted us to synthesize new models of substituted pyrimidines having potential substituents, aiming to optimize the antibiotic activity as well as evaluation their activity against other harmful diseases, so 4-chloro-6-methoxy-N,N-dimethylpyrimidin-2-amine 1 has been selected as a precursor for the synthesis of new pyrimidine derivatives. Herein the reaction occurs is nucleophilic substitution with O- and S-nucleophiles (phenoxides and thiophens ions), bearing some heterocyclic rings like 1,2,3-seleniadiazole, 1,2,3-thiadiazole and benzothiazole, which are formed in situ in the reactions of phenols and thiophenyl in bases with chlorinated pyrimidine derivative 1, has been reported in (scheme 1).

Treatment of 1 with 4-(1,2,3-selenadiazol-4-yl) phenol or 4-(1,2,3-thiadiazol-4-yl) phenol in refluxing DMF with few drops of conc. NaOH afforded, via nucleophilic displacements of the chlorine group, 2 and 3 in 94 and 92% yield, respectively. Similarly, reaction of 1 with sodium benzenethiolate and benzo[d]thiazole-2-thiol afforded compound 4 and 5 in 96 and 90% yield in refluxing dry acetone and Aliquite 336 as catalyst working as surfactants (Scheme 1).

Scheme 1

Schematic representation of synthetic procedure for compounds 1-9.

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The structures of 2-5 were determined from the1H, and13C NMR spectra. The methyl protons (NMe2) (6H) appeared as singlets at the range d = 3.20-2.08 ppm, while H-5 of the pyrimidine ring resonated at the range d = 6.62-6.95 ppm. The1H NMR spectrum of all compounds showed a doublet of doublets of doublets (ddd) at d = 7.07-8.31 ppm with J constant between for all of them (J = 7.1-7.5 Hz). The1H NMR spectrum of 2 showed a singlet at d = 9.01 ppm assigned to the proton in 1,2,3-sleniadiazole heterocyclic ring, whereas the singlet at d = 9.02 ppm attributed to the proton in 1,2,3-thiadiazole heterocyclic ring for compound 3.

The13C NMR spectra of 2-5 contained similar resonance signals of the pyrimidine carbons ring C2 - C6, as well as the methoxy and NMe2 carbons. Carbon atoms of methoxy and NMe2 pyrimidine ring of compounds 2-5 resonated at the ranges δ = 62.4 -53.8 ppm and δ = 36.9-36.4 ppm, respectively. In the13C NMR spectrum of 2, d = C-2, C-4, C-5 and C-6 resonated at d = 162.0, 171.0, 92.7 and 163.7 ppm, respectively. The13C NMR of compounds 2 and 3 characterized by the presence of the down-field signals at d = 130.0 and 122.6 and 136.9 and 128.5 ppm were assigned to two carbons atoms in heterocyclic rings, respectively.

The structure of compound 3 was confirmed in13C NMR were all similar carbons atom apereas at same regions (C-NMe, C-OMe, C5 and C6) as in the above, multiplet signals resonated at d = 137.2-124.1 for aromatic carbons. Similarly, the carbon atoms of compound 4 characterized by the presence of C1’, C2’, (C4’-C5’), (C3’-C6’) = 129.1, 155.2, 125.3 and 122.6 ppm, respectively. The IR spectrum of compound 2 showed υ = cm-1 1591 (C=N), 1382 (C-N), 1030 (C-O-C), 1535 (N=N), 1490-1450 (C=Caromatic) and 621 (C-SeHeterocyclic). The IR spectrum of compound 3 showed υ = cm-1 1591 (C=N), 1380 (C-N), 1033 (C-O-C), 1537 (N=N), 1494-1452 (C=Caromatic) and 667 (C-SHeterocyclic). But IR spectrum of compound 4 showed υ = cm-1 1597 (C=N), 1382 (C-N), 1037 (C-O-C), 1537 (N=N), 1450-1419 (C=Caromatic) and 621 (C-S). Furthermore compound 5 exhibited absorption bands at υ = cm-1 1593 (C=N), 1380 (C-N), 1033 (C-O-C), 1537 (N=N), 1490-1448 (C=Caromatic) and 620 (C-SHeterocyclic) all these dates were confirmed the functional groups that make up the overall structure of the molecules.

In addition to all these compounds were confirmed by micro elemental analysis that proved compounds formation.

Table 1

Effect of antibiotic compound (µM) on fungus growth (cm) inhibition zone rate.

Compound type Fungal type1234Fluconazole
5010020050100200501002005010020050100200
Aspergillus niger6.37*5.55*4.89*2.932.671.324.61*4.61*3.98*3.083.602.983.233.143.00
Aspergillus terreus4.52*4.09*3.56*1.601.320.983.363.122.542.962.841.652.332.452.77

[i] *The mean difference is significant at the 0.05 level compared to Fluconazole.

Table 2

Stability and toxicity of the antibiotic compound.

Compound type Parameter1234
Stability (month)More than six monthMore than five monthMore than six monthMore than six month
Toxicity (LD50)220 mg/kg220 mg/kg210 mg/kg190 mg/kg
Table 3

Solubility of the compounds (g of antibiotic/kg of solvent) in different solvents at 25°C temperature.

Compound type Solvent1234
2-propanol117119105115
Water23252527
Figure 1

The inhibition zone with most significant results.

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Antifungal activity

The results were based upon the scale developed by Arora and Bhardwaj.36 The zone of inhibition and the result of drug sensitivity were described as below.

The results showed activity against A. terreus with different zone of inhibition, respectively, with the three concentrations of 50 μM, 100 μM and 200 μM respectively. 2 compound showed activity against with A. terreus 0.98 cm of zone of inhibition with the concentration of 200 Mm Table 1. We also tested these compounds against A. niger the antimicrobial activity 2.96, 2.84 and 1.65 with 50 μM, 100 μM and 200 μM respectively. The results of Table 2 show the stability and toxicity of antibiotic compound; while the results of Table 3 elucidate the solubility of compounds (g of antibiotic/kg of solvent) in different solvents at 25°C temperature. Figure 1 shows the inhibition zone with most significant results.

From the observation of the antimicrobial (AM) activity of the compounds the bioactivities of a series of pyrimidine derivatives are as follows and we can correlate a good structure function relationship amongst them.

ACKNOWLEDGEMENT

None.

Notes

[2] Conflicts of interest CONFLICT OF INTEREST All authors have none to declare.

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