Synthesis and Antifungal Activities of Some Novel Pyrimidine Derivatives

Three series of new pyrimidine derivatives were synthesized and their antifungal activities were evaluated in vitro against fourteen phytopathogenic fungi. The results indicated that most of the synthesized compounds possessed fungicidal activities and some of them are more potent than the control fungicides. Preliminary SAR was also discussed.


Introduction
Phytopathogenic fungi that easily infect many crops are hard to control and risk resistance to the widely used commercial fungicides [1], therefore, there is a continuous need for new classes of antifungal agents.Natural compounds containing pyrimidine skeletons, such as vitamin B 1 [2,3], and nucleotide bases [4] play an important role in life science studies.Pyrimidine derivatives have attracted great interest due to their diverse biological activities.For example, Rashad [5] synthesized a series of 4-hydrazinopyrimidine derivatives with in vitro antimicrobial activity.Rotili [6] reported a novel series of diarylpyrimidine and dihydrobenzyloxopyrimidine hybrids endowed with high and wide-spectrum anti-HIV-1 activity both in cellular and enzyme assays.Different classes of pyrimidine derivatives were synthesized and screened for antitumor activity to give candidates in drug discovery [7,8].Pyrimidine derivatives have also occupied a prominent place in the field of agrochemicals because of their significant properties as fungicides in agriculture.To date, some important commercial pyrimidine fungicides, such as azoxystrobin [9,10], cyprodinil [11], pyrimethanil [12], and diflumetorim [13] OPEN ACCESS (Figure 1) have been used in agriculture.Encouraged by the numerous pharmacological activities of pyrimidine derivatives, we were prompted to develop some novel pyrimidine fungicides.Three series of new pyrimidine derivatives 1-4 were designed and synthesized.The synthetic routes are shown in Scheme 1.All of the new compounds were evaluated in vitro for their antifungal activities against fourteen phytopathogenic fungi and the results of preliminary bioassays were compared with those of some commercial agricultural fungicides: flumorph, dimethomorph, carbendazim, hymexazole and pyrimethanil (Figure 1), which were currently used in the field in China.The results indicated that most of the pyrimidine derivatives possessed certain fungicidal activities, and the preliminary SAR of these compounds was investigated.Ether analogues: Amine analogues:

Synthesis
Compounds 1a and 1b were prepared by an improved method using NaH and TBAB in freshly distilled DCM since they couldn't be obtained according to the method reported in the literature [14].Compounds 2a and 2b were obtained according to the similar method in literature [15] in the presence of KI.Using POCl 3 as chlorination reagent [16], the intermediate 4-chloro-2-isopropyl-6-methylpyrimidine was synthesized and used for the next substitution directly without purification in a small-scale synthesis.Reacting 4-chloro-2-isopropyl-6-methylpyrimidine with the corresponding amines in different solvents afforded 3a [17], 3b [18] and 3c [19], however, it was not necessary to use K 2 CO 3 in the preparation of 3a.In the preparation of compound 4a, the 4-morpholinecarbonyl chloride must be prepared freshly before use.Et 3 N and K 2 CO 3 were employed in preparation of 4b and 4c respectively, since there were more by-products when Et 3 N was used in preparing 4b while few impurities were noted while synthesizing 4c.After the 3,4,5-trimethoxybenzoic acid was ready, the condensation with 6-hydroxy-2-isopropyl-4-methylpyrimidine using EDCI as coupling reagent gave 4c.Most of the compounds were prepared is yields of over 50%.

Antifungal Bioassay: Inhibitory Effects on Phytopathogenic Fungi
The fourteen phytopathogenic fungi chosen included Alternaria kukuchiana (AK), Alternaria mali (AM), Alternaria solani (AS), Botrytis cinerea (BC), Bipolaris maydi (BM), Cercospora arachidicola (CA), Gibberella zeae (GZ), Gibberella fujikuroi (GF), Macrophoma kuwatsukai (MK), Phytophthora infestans (Mont) de Bary (PI), Rhizoctonia solani (RS), Rhizoctonia cerealis (RC), Sclerotinia sclerotiorum (SS), Thanatephorus cucumeris (Frank), Domk (TC).All the fungi are typical and often occur in the Chinese agro-ecosystem.The antifungal activities of the eleven pyrimidine derivatives were investigated in vitro by poisoned food technique [20][21][22] at the concentration of 50 μg/mL.The commercial fungicides flumorph, dimethomorph, carbendazim, hymexazol and pyrimethanil were used as positive controls and they are widely used in field in China.The antifungal screening data are recorded in Table 1.As shown in Table 1, all of the new pyrimidine derivatives exhibited certain growth inhibition effects against most of the tested fungi, and the results provided useful information to study the structure-activity relationshipx for these new structures shown in Scheme 1.The inhibition of most compounds was equal to or higher than that of the positive controls, flumorph and dimethomorph (Figure 2).Especially, to PI, the activities of 1a, 3b, 3c, 4b (22.7%, 36.4%,36.4%,18.2%, respectively) were 1-3 times higher than dimethomorph (9.1%) which is widely used to prevent phytophthora infestans in filed [23,24].Thus, the findings demonstrate that the new synthesized pyrimidine derivatives represent a new structure skeleton for inhibiting PI.The activities of carbendazim and hymexazol were excellent (>50%) to most of tested fungi, however, they showed rather lower inhibition to BM, GZ, MK and PI than several new synthesized compounds (3b, 3c, 4b, et al.).The leading compound pyrimethanil showed excellent effect on most of the tested fungi, except on AM, GF GZ and PI (21.4%, 20%, 44.4%, and 22.7%).It exhibited parallel activity with 3b to GZ (44.4%), lower than 3b and 3c to PI (36.4% and 36.4%,respectively), at least 1-fold lower than 3b, 4b and 4c to GF (40%, 46.7% and 53.3%, respectively) and 2-fold lower to AM than 3b (50%).1a and 3b provided certain broad-spectrum to the tested fungi, and showed relatively higher activities, especially to TC (80.4% and 76.5% respectively) (Figure 3).The inhibition against the fungi except RS, GF, SS was significantly decreased when the (6-chloropyridin-3-yl)methyl of 1a was replaced by (6-chlorothiazole-3-yl) methyl in 1b, especially against TC, a 12-fold decrease in activity was observed, whereas no similar SAR was observed for 2a, 2b.Comparing pyrimethanil and compound 2 with compound 3, preliminary SAR presumed that 2-substituted pyrimidinamine was effective to Botrytis while 4-substituted pyrimidinamine was more potent to Cercospora, and the two classes possessed a certain degree of selectivity in inhibition to different genus.Compound 3b with a trifluoromethoxy on the 4-position of the phenyl ring was more effective than 3a on the tested fungi, except AS, CA, PI, SS.To a certain extent, the results indicated that -OCF 3 containing derivatives may have good activity because of their special atom size and their characteristics to form a H bond with the potential target.S. sclerotiorum (SS) is one of the most nonspecific, omnivorous and successful plant pathogens with an extensive host range.At least 361 species in 64 families of plants are susceptible to SS [25].Compounds 4b, 4c, 4d showed better fungicidal activities to SS (40%, 60%, 66.7%) than 4a (0%), which showed that fungicidal activity increased smoothly with increasing density of the electron cloud on the benzene ring.The inhibition of 4c was equal to that of pyrimethanil (66.7%), lower than carbendazim (73.3%) and hymexazole (86.7%).Encouragingly, all of the derivatives showed good activity to RS (>50%).

General
All reactions using air-or moisture-sensitive reagents were conducted under an inert nitrogen atmosphere.Anhydrous solvents were distilled prior to use.THF was distilled from sodium/benzophenone. DCM and CH 3 CN were distilled from CaH 2 and DMF was dried over P 2 O 5 .Silica (200-300 mesh) was used for column chromatography.The IR spectra were recorded on a Bruker VERTEX 70 FT-IR. 1H-NMR spectra were recorded on a 400 MHz spectrometer at 25 °C using TMS as an internal reference.Coupling constants (J) are reported in Hertz (Hz). 1 H-NMR splitting patterns are designated as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet).Melting points of the products were determined in open capillary tubes and uncorrected.Mass spectra were recorded with a JEOL MS-D 300 mass spectrometer.Elemental analysis was performed on a Carlo-1106 model automatic instrument.

N-cyclohexyl-2-isopropyl-6-methylpyrimidin-4-amine (3c).
To a solution of 4-chloro-2-isopropyl-6methylpyrimidine (0.17 g, 1 mmol) and cyclohexane (1.0 g, 10 mmol) in CH 3 CN (10 mL) was added powdered K 2 CO 3 (0.14 g, 1 mmol).The mixture was stirred at reflux for 7 h.After the solvent was removed in vacuo, the residue was dissolved in DCM and water, acidified by cold 0.5 N HCl aqueous to pH 7~7.5.The DCM layer was separated and the water layer was extracted with DCM for three times.The combined organic layer was dried over anhydrous MgSO 4 .The solvent was evaporated in vacuo to give 3c as a light-brown solid (0.16 g, 69%), m.p. 93-97 °C.IR (film, cm Preparation of 4-morpholinecarbonyl chloride.Triphogene (1.49 g, 5 mmol) was dissolved in DCM (150 mL), then a solution of morpholine (0.87 g, 10 mmol) and triethylamine (1.52 g, 15 mmol) in DCM (30 mL) was added dropwise slowly in a salt-ice bath.After the reaction was completed (monitored by Iodine smoked TLC), phosgene was blown away by N 2 , then the mixture was filtered and the filtrate was concentrated to give a light-brown oil (1.41 g, 94%, with a content of about 70%) which was used for the next step without further purification.

Conclusions
Eleven novel pyrimidine derivatives of three classes had been synthesized and identified.The bioactivity tests showed that most of them had antifungal activities.The antifungal activities of most of compounds were equal to or higher than those of the commercial fungicides flumorph and dimethomorph.Compounds 3b, 4b and 4c showed better activity than the lead compound, pyrimethanil to GF. Compound 3b was more potent to AM than pyrimethanil.Compounds 1a and 3b displayed certain broad-spectrum activity towards the tested fungi.Compounds 4a, 4b, 4c and 4d showed relatively moderate antifungal activity.The preliminary SAR can offer great help to design more active compounds as potent fungicides.

Table 1 .
The fungicidal activities of three series of pyrimidine derivatives.