INTRODUCTION:

Heterocyclic compounds offer a high degree of structural variety and are a vital part of the synthetic medicinal chemistry. Heterocyclic compounds have been enticing the medicinal chemist over decades as the most promising molecules to develop lead structures for the design of new drugs.

The chemistry of pyrimidines had been attracting widespread attention owing to its relationship with thymidine, cytosine and uracil. Triazolopyrimidines have been reported to exhibit antifungal activity¹ and some pyrimidine derivatives have proven to be effective against leshmaniasis.²Condensed pyrimidine derivatives have been widely synthesized and studied for their pharmacological actions. Thiazolo [3,2-α] pyrimidine derivatives being the bioisosteric analogues of purines have been investigated largely for their bioactivity.

They have been known to possess hypoglycemic and hypolipidemic potential.³ 1,2,3,4-tetrahydropyrimidine-2-thiones have shown close structural relationship to the clinically important dihydropyridine calcium channel blockers. The derivatives of 1,2,3,4-tetrahydropyrimidine-2-thiones have been reported to be calcium channel blockers,⁴ antitumor,⁵ antidepressant,⁶ antibacterial⁷ and antifungal.⁸

Rhodanine, a five-membered heterocyclic molecule containing a thiazole nucleus with thioxo group on second carbon and carbonyl group on fourth carbon has been constantly structurally modified and resulted in synthesis of compounds with a wide spectrum of pharmacological activities.⁹

Being optimistic by the above reports on condensed pyrimidines and rhodanine, in the present study, the synthesis of condensed pyrmidine derivatives by electrophilic addition of 2-Amino-4-aryl thiazole to 5-Arylidine-3-aryl rhodanines followed by subsequent cyclization was achieved. The synthesized bisthiazolo-pyrimidin-2-thione derivates were subjected to in vitro antifungal studies against Aspergillus niger and Fusarium oxysporium.

MATERIAL AND METHODS:

General Procedures:

Melting points were determined by open capillary method and are uncorrected. All chemicals used were reagent grade and were used as received without further purification.¹HNMR spectra were recorded at 400 MHZ on a Bruker AVANCE DPX (400 MHz) FT spectrometer in DMSO-d₆using TMS as internal standard. Mass spectra were recorded on a JEOL SX-102 mass spectrometer at 70ev. Elemental analyses were performed on a Coleman elemental analyzer. The reactions were monitored using preformed aluminium TLC plates (UV₂₅₄) and the plates were visualized in Iodine vapour. Column chromatography was carried out on silica gel (60–120 mesh, Merck chemicals).

VII a (Ar=Ar’’=C₆H₅), VII b (Ar= p-CH₃OC₆H₄; Ar’’=C₆H₅), VII c (Ar= p-ClC₆H₄; Ar’’=C₆H₅), VII d (Ar= m,p-(CH₃O)₂C₆H₃; Ar’’=C₆H₅), VII e (Ar= p-NO₂C₆H₄; Ar’’=C₆H₅), VII f (Ar’’= p-CH₃OC₆H₄; Ar=C₆H₅), VIIg (Ar’’= p-ClC₆H₄; Ar=C₆H₅), VII h (Ar’’= p-CH₃OC₆H₄; Ar= p-ClC₆H₄), VII i (Ar’’= p-CH₃OC₆H₄; Ar= m,p-(CH₃O)₂C₆H₃), VII j (Ar’’= p-CH₃OC₆H₄; Ar=p-NO₂C₆H₄)

Scheme 1

General procedure of synthesis of 2-Amino-4-aryl thiazole (V a-c):

A mixture of aromatic ketone (100mmol), thiourea (200 mmol), iodine (100mmol) and a few drops of 1,4-dioxane was thoroughly agitated to mixing and was heated at 80-90°C under reflux conditions for 8 h. The mass obtained was triturated with ether over a period of 12 h. The ether was then decanted and the solid left behind was washed with a 3 % w/v solution of sodium thiosulphate followed by washing with cold water. The residue obtained was dissolved in boiling water, filtered, cooled, and then rendered alkaline with ammonia solution. The resulting solid was washed and recrystallized from dimethyl sulfoxide-H₂O (1:1 v/v) and finally purified by silica gel column chromatography (benzene-MeOH, 8:2), yielding analytically pure V (a-c).

Synthesis of 3-Phenyl rhodanine (III):

In a continuously stirred ice-salt bath, 20mL of ammonia solution was added to CS₂ (48mmol). Aniline (20mmol) was added to this solution over a period of 30 min. The stirring was further continued for 3 h. The dithiocarbamate thus precipitated was allowed to stand overnight. It was filtered, washed with cold ether and dried by suction.

Separately a solution of sodium chloroacetate was prepared by mixing cooled solution of chloroacetic acid (40mmol) in 15ml of water, followed by addition of anhydrous sodium carbonate until alkaline. The sodium chloroacetate solution was stirred and cooled to 5-10°C and the dithiocarbamate was added to it over 15 min. Stirring was continued while the temperature of flask raised to room temperature. The solid thus obtained was added to a mixture of 15ml of sulfuric acid and 7ml of water and heated at 90°C for 15 min. On cooling a pale yellow compound was obtained. This was purified by silica gel column chromatography (Benzene: MeOH, 6:4) to obtain pure compound (III).

General Procedure for synthesis of 5-Arylidine-3-Phenyl rhodanines (IV a-e):

A mixture of 3-Phenyl rhodanine (1mmol), different aromatic aldehyde (1mmol) and fused sodium acetate (1.1mmol) in glacial acetic acid was heated under reflux for 8 h. The reaction mixture was cooled and poured into water. The resulting solid was filtered, washed with water and recrystallised from ethanol. It was purified by silica gel column chromatography (Benzene: MeOH, 8:2) to the products IV (a-e).

General Procedure for synthesis of 3-Aryl-5-[aryl-(4-aryl-thiazol-2-yl-amino)-methyl]-2-thioxo-thiazolidin-4-one (VI a-j)

Equimolar mixture of 2-amino-4-aryl thiazole (100 mmol) and 5-arylidine-3-Phenyl rhodanine (100mmol) were dissolved in ethanol. The mixture was heated under reflux for 6-8 h. After the completion of the reaction the excess of the solvent was evaporated under reduced pressure. The residue obtained was purified by flash chromatography and crystallized with benzene-MeOH (8:2) to give pure compound VI (a-j).

General Procedure for synthesis of 1,4,8-Triaryl-1,4dihydro-bisthiazolo [3,2-a; 5'4'-e] pyrimidin-2-thione (VII a-j)

3-Aryl-5-[aryl-(4-aryl-thiazol-2-yl-amino)-methyl]-2-thioxo thiazolidin-4-one was dissolved in benzene and 10% H₂SO₄ was added to the solution. The mixture was heated for 3-4 h under reflux conditions. The mixture was cooled to room temperature resulting in the precipitation of a pale yellow crude product, which was purified by flash chromatography. The product was recrystallized with etahnol to give pure compound VII (a-j).

1,4,8-triphenyl-1,4-dihydro-bisthiazolo [3,2-a; 5'4'-e] pyrimidin-2-thione (VII a)

¹H NMR 400 MHz (DMSO d6): 6.46-7.30 (15H, m, ArH, aryl thiazole, aryl rhodanine), 5.73 (1H, s, -CH-thiazole), 3.5 (1H, s,-CH-pyrimidine). EIMS (M⁺): 455

1,8-diphenyl-4-(4’-methoxyphenyl)-1,4-dihydro-bisthiazolo [3,2-a; 5'4'-e] pyrimidin-2-thione (VII b)

¹H NMR 400 MHz (DMSO d6): 6.46-7.30 (14H, m, ArH, aryl thiazole, aryl rhodanine), 5.73 (1H, s,-CH-thiazole), 3.5 (1H, s,-CH-pyrimidine), 3.73 (3H, s, OCH₃-ArH). EIMS (M⁺): 485

1,8-diphenyl-4-(4’-chlorophenyl)-1,4-dihydro-bisthiazolo [3,2-a; 5'4'-e] pyrimidin-2-thione (VII c)

¹H NMR 400 MHz (DMSO d6): 6.46-7.30 (14H, m, ArH, aryl thiazole, ary rhodanine), 5.73 (1H, s, -CH-thiazole), 3.5 (1H, s, -CH-pyrimidine). EIMS (M⁺): 489

1,8-diphenyl-4-(3’,4’-dimethoxyphenyl)-1,4-dihydro-bisthiazolo [3,2-a; 5'4'-e] pyrimidin-2-thione (VII d)

¹H NMR 400 MHz (DMSO d6): 6.46-7.30 (13H, m, ArH, aryl thiazole, aryl rhodanine), 5.73 (1H, s,-CH- thiazole), 3.5 (1H, s, -CH- pyrimidine), 3.73 (6H, s, OCH₃ -ArH). EIMS (M⁺): 515

1,8-diphenyl-4-(4’-nitrophenyl)-1,4-dihydro-bisthiazolo [3,2-a; 5'4'-e] pyrimidin-2-thione (VII e)

¹H NMR 400 MHz (DMSO d6): 6.46-8.07 (14H, m, ArH, aryl thiazole, aryl rhodanine), 5.73 (1H, s, -CH- thiazole), 3.5 (1H, s,-CH- pyrimidine). EIMS (M⁺): 500

1,4-diphenyl-8-(4’’-methoxyphenyl)-1,4-dihydro-bisthiazolo [3,2-a; 5'4'-e] pyrimidin-2-thione (VII f)

¹H NMR 400 MHz (DMSO d6): 6.46-7.14 (14H, m, ArH, aryl rhodanine, aryl thiazole), 5.73 (1H, s, -CH- thiazole), 3.5 (1H, s, -CH- pyrimidine), 3.73 (3H, s, OCH₃-thiazole). EIMS (M⁺): 485

1,4-diphenyl-8-(4’’-chlorophenyl)-1,4-dihydro-bisthiazolo [3,2-a; 5'4'-e] pyrimidin-2-thione (VII g)

¹H NMR 400 MHz (DMSO d6): 6.46-7.19 (13H,m, ArH, aryl thiazole, aryl rhodanine), 5.73 (1H,s, -CH-thiazole), 3.5 (1H, s, -CH- pyrimidine), 3.73 (6H, s, OCH₃-ArH and thiazole). EIMS (M⁺): 515

1-phenyl-4-(4’-chlorophenyl)-8-(4’’-methoxyphenyl)-1,4-dihydro-bisthiazolo [3,2-a; 5'4'-e] pyrimidin-2-thione (VII h)

¹H NMR 400 MHz (DMSO d6): 6.46-7.15 (13H, m, ArH, aryl thiazole, aryl rhodanine), 5.73 (1H, s-CH-thiazole, 3.5 (1H,s,-CH- pyrimidine), 3.73 (3H, s,OCH₃-thiazole). EIMS (M⁺): 519

1-phenyl-4-(3’,4’-dimethoxyphenyl)-8-(4’’-methoxyphenyl)-1,4-dihydro-bisthiazolo [3,2-a; 5'4'-e] pyrimidin-2-thione (VII i)

¹H NMR 400 MHz (DMSO d6): 6.46-7.19 (12H, m, ArH, aryl thiazole, aryl rhodanine), 5.73 (1H, s,-CH-thiazole), 3.5(1H, s,-CH-pyrimidine), 3.73 (9H, s, OCH₃-thiazole, rhodanine and ArH). EIMS (M⁺): 545

1-phenyl-4-(4’-nitrophenyl)-8-(4’’-methoxyphenyl)-1,4-dihydro-bisthiazolo [3,2-a; 5'4'-e] pyrimidin-2-thione (VII j)

¹H NMR 400 MHz (DMSO d6): 6.46-8.07 (13H, m, ArH, aryl thiazole, aryl rhodanine), 5.73 (1H, s,-CH-thiazole, 3.5 (1H, s,-CH-pyrimidine), 3.73 (3H, s, OCH₃-thiazole). EIMS (M⁺): 530

Table 1: Analytical data of the newly synthesized compounds VII (a-j)

Compound No.	Yield %	M.P. °C	Molecular Formula	Found (Calcd.)%
Compound No.	Yield %	M.P. °C	Molecular Formula	C`	H	N
VII a	78	250-252	C₂₅H₁₇N₃S₃	65.9 (66.0)	3.7 (3.8)	9.2 (9.3)
VII b	81	212-214	C₂₆H₁₉N₃OS₃	64.3 (64.4)	3.9 (4.0)	8.6 (8.8)
VII c	80	205-206	C₂₅H₁₆ClN₃S₃	61.2 (61.3)	3.3 (3.4)	8.6 (8.7)
VII d	79	210-212	C₂₇H₂₁N₃O₂S₃	62.9 (63.0)	4.1 (4.2)	8.2 (8.3)
VII e	76	198-200	C₂₅H₁₆N₄O₂S₃	59.9 (60.0)	3.2 (3.3)	11.2 (11.3)
VII f	74	202-203	C₂₆H₁₉Cl N₃OS₃	64.3 (64.4)	3.9 (4.0)	8.6 (8.7)
VII g	75	215-216	C₂₇H₂₁N₃O₂S₃	62.8 (62.9)	4.1 (4.2)	8.2 (8.3)
VII h	79	180-182	C₂₆H₁₈Cl N₃O₃S₃	60.0 (60.1)	3.5 (3.7)	8.0 (8.1)
VII i	83	208-210	C₂₈H₂₃N₃O₃S₃	61.6 (61.7)	4.2 (4.3)	7.7 (7.8)
VII j	78	185-187	C₂₆H₁₈N₄O₃S₃	58.8 (58.9)	3.4 (3.5)	10.6 (10.7)

Table 2: Antifungal activity of the synthesized compounds VII (a-j)

Compound	Average % Inhibition against
	A. *niger*			*F. oxysporium*
	1000 ppm	100 ppm	10 ppm	1000 ppm	100 ppm	10 ppm
VII a	78	60	48	75	42	35
VII b	80	63	52	82	41	49
VII c	81	69	55	78	65	36
VII d	75	56	32	72	52	31
VII e	79	35	26	72	31	24
VII f	74	58	49	70	35	26
VII g	89	81	61	85	75	67
VII h	92	76	65	90	59	42
VII i	76	48	35	73	51	32
VII j	72	42	33	73	65	42

RESULT AND DISCUSSION:

Chemistry:

In order to achieve the basic purpose of our study, the synthetic approach was confined to scheme 1 for obtaining the target condensed pyrimidine derivatives. A reaction of 3-Phenyl rhodanine with various aromatic aldehydes resulted in 5-Arylidine-3-Phenyl rhodanines IV(a-e), the prime reactant of the scheme, in good yield. 2-amino-4-arylthiazole V(a-c) was electrophilically added to compound IV(a-e) to obtain the target condensed pyrimidine-2-thione derivatives VII(a-j). The formula of the compounds VII (a-j) were confirmed by the elemental analyses and their structures were determined by IR, ¹HNMR and EIMS spectral data.

The IR spectra of all the compounds exhibited characteristic –C=S stretching vibration at 1275–1030 cm⁻¹. The ¹HNMR spectra of the compounds showed peaks of aryl protons at 6.46-7.30. A singlet at 5.73 ppm appeared due to the proton of the fused thiazole. The above peaks supported the formation of pyrimidine nucleus. MS of all compounds showed the molecular ion peak (M+) with low intensity and other peaks due to fragments that affirmed the expected structures (Table 1). Experiments were also performed to assess the antifungal activity of the compounds VII(a-j) against Aspergillus niger and Fusarium oxysporium using the disk diffusion method.

Microbiology:

The in vitro antifungal activity of the synthesized compounds was tested against Aspergillus niger and Fusarium oxysporium using the disk diffusion method where each disc contained either 10, 100 or 1000 ppm of the test compound. Briefly, nutrient agar in water was melted at 100°C and 20 mL of the molten agar was poured into different petri plates, and left on a flat surface to solidify. The diluted culture of each strain under study was pipetted into the agar plates. The surface of the agar plate was allowed to dry. Different concentrations of the test compound impregnated discs were applied to the surface of inoculated plates. The Petri plates were placed in an incubator and incubated at 37°C for 10-24 h. The petri plates were evaluated for the zone of inhibition of the fungal growth around the impregnated discs. All compounds were antifungal since they inhibited the growth of both the fungi viz; Aspergillus niger and Fusarium oxysporium in the range of 70-92% at 1000 ppm concentration. It was also evident from the results that the compounds with chloro or nitro substituted phenyl nucleus (VII c, e, g, h & j) were more detrimental to the growth of the fungi as compared to the other compounds. The antifungal screening data shows that are the screening compounds inhibited 70-92% mycelial growth of both test fungi at 1000ppm concentration but their activity decreased at lower concentration (100, 10ppm) the most active of these, VII b, VII c, VII g and VII h displayed fungicidal action equivalent to that of commercial fungicide Dithane M-45 at 1000ppm concentration and showed 24-65% growth inhibition even at 10ppm concentration (Table 2).

CONCLUSION:

The object of the present work was to combine the toxophoric qualities of 2-amino-4-aryl thiazole and 5-arylidene-3-aryl rhodanine moieties and synthesize some novelpyrimidin-2-thione derivatives and evaluate them for their potential fungicidal action against Aspergillus niger and Fusarium oxysporium. The study led to conclusion that the synthesized compounds were at par in antifungal potential with commercial fungicide Dithane M-45. Further studies need to be done in order to establish the statement obtained from the in vitro studies against the fungal strains.

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Received on 28.01.2020 Modified on 21.03.2020

Research J. Pharm. and Tech. 2021; 14(1):275-279.

DOI: 10.5958/0974-360X.2021.00049.4