Synthesis Of Pyrazole Derivatives With Anti-virus And Anti-cancer Biological Activity

Pyrazole and their derivatives are important class of heterocyclic compounds.They occupy important position in medicinal and pesticide chemistry with wide range of bioactivities. As medicines, many of them display antifungal, antimicrobial, anti-HIV, antitubercular, anticancer, antiinflammatory, anticonvulsant, antidepressant, hypolipidemic, antiulcer, analgesic, immunotropic activities and are also known to act as thymidyalate synthase, poly(ADP-ribose) polymerase (PARP), and protein tyrosine kinase inhibitors. As pesticides, they are used as insecticides, fungicides, antibacterial agents, and antiviral agentssuch as TMV, CMV inhibitors. With growing application on their synthesis and bioactivity, there has been an enormous increase of interest among the biologists and chemists in recent years on the research of pyrazole derivatives.Tobacco mosaic virus (TMV) infection is very widely distributed, and can cause serious damage and large economic loss. It was found that in some fields 90-100% of the plants show mosaic by harvesting time. Regarding the unsatisfactory curatives (30-60% cure rate by common antiviral agent Ningnanmycin or Virus A) and economic loss of tobacco, it is of great necessary to develop highly efficient, novel environmental benign antiviral agent.In order to create new and high-efficient antiviral agent in agricuture, this paper hoped to use 1-substituted phenyl-3-methyl-4-formylpyrazole as the start material, designed and synthesized a series of pyrazole derivatives containing oxime ester and amide moiety (see figure), then the structures of the compounds are characterized by elemental analysis, IR, ¹H-NMR and ¹³C-NMR spectra. The title compounds are also tested in vitro and in vivo against pathogenic virus and cancer cells. Half-leaf method was used to determine curative efficacy in vivo of title compounds. The result showed that some compounds possessed good curative effect against TMV in vivo. The mechanism of action of the title compounds PS3-13 against TMV in vivo is also studies. Some conclusions were obtained: 1. The synthesis of title compounds l-substituted-5 substitutedphenylthio-4-pyrazolaldoxime ether derivatives (PS1)Starting from 1-substituted phenyl-3-methyl-4-formylpyrazole as raw materials, the intermediate M1, 1-substituted phenyl-3-methyl-4-formyl-5-chloropyrazole, was prepared by chlorination reaction with POCl₃. Thus, treatment of 1-substituted phenyl-3-methyl-4-formyl-5-chloropyrazole with substituted thiophenol afforded 1-substitutedphenyl-3-methyl-5-substitutedphenylthio-4-pyrazolaldehyde (M3).Reaction 1-substitutedphenyl-3-methyl-5-substitutedphenylthio-4-pyrazolaldehyde with hydroxylamine hydrochloride gave 9 1-substitutedphenyl-3-methyl-5-substitutedphenylthio-4-pyrazolaldoximes (M4). 11 the title compounds, 1-substituted -5-substitutedphenylthio-4-pyrazolaldoxime ether derivatives (PS1) were synthesized by the thioetherication reaction of l-substitutedphenyl-3-methyl-5-substituted phenylthio-4-pyrazolaldoximes (M4) with chloromethyl-heterocyclic compounds (C1-G3). A total of 11 compounds, of which 11 new compounds. The synthesized compounds were determied physical constants, their structures were clearly established by IR, ¹H NMR, ¹³C NMR and elemental analysis.2. The synthesis of title compounds 1-substituted-S-substitutedphenylsulfonyl-4-pyrazolaldoxime ether derivatives (PS2). 5 1-substituted-5-substitutedphenylsulfonyl-4-pyrazolaldoxime ether derivatives (PS2) were synthesized from the starting material 1-substitutedphenyl-3-methyl-5-substituted phenylthio-4-pyrazolaldoximes (M4) with chloromethyl-heterocyclic compounds (C1-G3) through etherication reaction. Thus, Oxidiation of potassium permanganate with PS1 in HOAc solution at room temperature to form 5 new compounds. The synthesized compounds was used to determine physical constants, and the structures of the title compounds were confirmed by IR, ¹H NMR, ¹³C NMR and elemental analysis.3. The synthesis of title compounds l-substituted-5-substitutedphenylsulfonyl-4-pyrazolaldoxime ester derivatives (PS4).14 the title compounds, 1-substituted-5-substitutedphenylthio-4-pyrazolaldoxime ester derivatives (PS3) were synthesized from the starting material 1-substitutedphenyl-3-methyl-5-substituted phenylthio-4-pyrazolaldoximes (M4) with acyl chloride. Thus, Oxidiation of potassium permanganate with PS3 in HOAc solution at room temperature to form 5 new compounds. The synthesized compounds was used to determine physical constants, and the structures of the title compounds were confirmed by IR, ¹H NMR, ¹³C NMR and elemental analysis.4. The synthesis of title compounds l-substituted-5-chloro-4- pyrazolecarbox-amide derivatives (PS5) and their pyrazolecarbonylurea derivatives (PS6).Starting from 1-substituted phenyl-3-methyl-4-formyl-chloropyrazole as raw materials, the intermediate M5, 1-substituted phenyl-3-methyl-5-chloropyrazole -4-carboxylic acid, was prepared by oxidation reaction with potassium permanganate. Thus, treatment of 1-substituted phenyl-3-methyl-5-chloropyrazole-4-carboxylic acid with SOCl₂ afforded 5 1-substituted phenyl-3-methyl-5-chloropyrazole-4-carbonyl chloride (M6) in refluex condition. Reaction 1-substituted phenyl-3-methyl-5-chloropyrazole-4-carbonyl chloride (M6) with 26% ammonia gave 5 1-substituted phenyl-3-methyl-5-chloro-4-pyrazolaldoximes (M7). 9 the title compounds, 1-substituted-5-chloro-4-pyrazole-carboxamide derivatives (PS5) were synthesized by the amidiation reaction of 1-substitutedphenyl-3-methyl- 5-chloro-4-pyrazolcarbonyl chloride (M6) with aromatic amines (G5-NH2). Reaction of 1-substituted-5-chloro-4-pyrazolecarboxamide (M7) with grass chloride, followed by carbonylurea reaction with fluoroaromatic amine (G5-NH2) to yield 10 pyrazolecarbonylurea derivatives (PS6). A total of 19 compounds, of which 19 new compounds. The synthesized compounds were determied physical constants, their structures were clearly established by IR, ¹H NMR, ¹³C NMR and elemental analysis.5. The synthesis of title compounds l-substituted-5-substitutedphenylthio-4-pyrazolyl-methylamino derivatives (PS8).5 the title compounds, l-substituted-5-substitutedphenylthio-4-pyrazolylmethyl-amino derivatives (PS8) were synthesized from the starting material 1-substituted phenyl-3-methyl-5-substitutedphenylthio-4-formylpyrazole (M3) with 4-trifluoro -methyaniline. Thus, reduction of NaBH₄ with 1-substitutedphenyl-3 -methyl-5-substituted phenylthio-4-pyrazoleimine (M10) in EtOH solution at room temperature to form 5 new compounds. The synthesized compounds was used to determine physical constants, and the structures of the title compounds were confirmed by IR, ¹H NMR, ¹³C NMR and elemental analysis.6. The anticancer activities of title compounds.The antitumor activites of the title compounds were assayed by the MTT method. It was found that these compounds exhibit moderate to good activities against PC3, Bcap37 and BGC-823 cell lines in vitro. The target compounds can strongly inhibit PC3 cells, with IC₅₀ value of 7.06-46.81μM. The IC₅₀ value of title compounds to Bcap37 and BGC-823 cell lines were 1.77-206.18μM and 7.75-38.35μM, respectively. Using TRYPAN BLUE and AO/EB stained fluorescent microscope, the compound PS6-7 couldn't induce apoptosis of PC3 cancer cells, causing significant reduction of cell death number.7. The anti-TMV activities of title compounds.Half-leaf method was used to determine curative effects in vivo of 40 title compounds. The result showed that title compounds PS3-1, PS 1-1, PS3-2, PS3-6, PS3-8, PS3-7, PS3-12, PS3-13, PS5-4, PS5-5 and PS5-8 possessed good curative effect against TMV in vivo, with values of 47.8, 47.0, 40.4, 50.0, 46.4, 47.7, 62.0, 60.0, 44.0, 46.5 and 46.0% at 500μg/mL, respectively. Among these compounds, PS3-12, PS3-13 were much more active against TMV than the other ones, with the curative rate of 62.0, 60.0%, which is equivalent to Ningnanmycin (56-58%) against TMV at 500μg/mL. Choosing fourty new compounds was used to test protective and inactivation effects.It can be observed that the title compounds PS1-1, PS3-2, PS1-7, PS3-8,PS3-12,PS3-13,PS3-14 possess potential inactivation bioactivities, with values of 81.4, 74.1, 80.6, 72.1, 74.6, 73.7, 73.9% at 500μg/mL, respectively. Among these compounds, PS1-1, PS1-7 is much more active against TMV than the other ones, with the inactivation rate of 81.0, 80.0%, which is little lower than Ningnanmycin (95-98%) against TMV at 500μg/mL. The other compounds exhibited weak to moderate inactivation bioactivities, with the inactivation rate of below to 70%. The data also indicate that title compounds PS5-7, PS5-5, PS5-2, PS3-6, PS3-10, PS1-7 show good protection activity against TMV of up to 50.0, 45.7, 48.4, 40.9, 43.5% and 59.0% at 500μg/mL. While the title compounds PS5-7, PS5-5, PS5-2, PS3-6, PS3-10 have a relatively lower protection activity than PS5-7. Primary study on the anti-TMV action mechanism of PS3-12 was inoculated TMV dealt with the tobacco plants using anti-TMV biochemical research. The results showed that their PAL enzyme, peroxidase enzyme, SOD enzyme, and control within a certain amount of time have relevance. SOD activity and chlorophyll content were increased in the tobacco plant treated by PS3-12. PS3-12 induced PAL and POD activity enhanced, different from the action of Ningnanmycin. Gene expression of Nicotiana tobacum treated by PS3-12. PS3-12 can induce up-regulation of PR-1a gene and acquire SAR and possess antiviral activity.