Design, Synthesis And Anti-Tumor Activities Of Azacytidine Analogue

5-Azacytidine（5-aza-CR） and 5-aza-2’-deoxycytidine（5-aza-CdR,Decitabine） are known DNA methyltransferase inhibitors and have been approved for the treatment of myelodysplastic syndrome（MDS）.Although both agents are characterized by significant cytotoxicity at high concentrations,they have been thought to mediate their therapeutic effects in MDS at low concentrations through the inhibition of DNA methyltransferase.Aberrant DNA methylation in the promoter region can silence the expression of many genes.Some tumor suppressor genes have been found to be silenced due to DNA hypermethylation and they can be reactivated by DNA demethylation.Treatments of malignant cells with 5-aza-CR or 5-aza-CdR have been found to be associated with reversal of specific gene suppression.Due to the ribonucleoside structure of 5-aza-CR and 5-aza-CdR,both compounds are incorporated into DNA or RNA and cause substantial cytotoxicity.The structure of 4-Amino-1,3,5-triazin-2（1 H）-one moiety is capable of cleavage to form N-aminoiminomethyl-N’-β-D-（2-deoxy）-ribofuranosyl-urea in alkaline and neutral which is a inactive product.We have synthesized a series of 5-aza-CR analogues with introduction of a methyl,an ethyl or a phenyl group into 6 position of 5-aza-CR and/or with a replacement of O by S or with a replacement of ribofuranosyl moiety by pyranosyl sugars or disaccharides.Fifty-two 5-azacytidine analogues, including 4-amino-6-alkyl-1-pyranosyl/ribofuranosyl-1,3,5-triazin-2（1 H）-ones,6-amino-4-alkyl/ -aryl-1-pyranosyl/ribofuranosyl-1,3,5-triazin-2（1 H）-ones and 4-amino-6-alkyl-1,3,5-triazin-2-y1-1 -thio-pyranosides/ribofuranosides were synthesized.The antiproliferative activities as well as their differentiation effects of these compounds alone and in combination with all-trans retinoic acid （ATRA） were investigated in human leukemia HL-60 and MCF-7 cells.The inhibitory effects of these compounds on DNA methyltransferase is under investigation.The structures of all compounds were confirmed by ¹H-NMR,MS and IR.The configuration of nucleoside was readily deduced from the trans-diaxial coupling between H-1’ and H-2’ of pyranose in ¹H-NMR spectra.The mannopyranosyl/rhamnopyranosyl nucleosides wereα-configuration nucleosides.The residues wereβ-configuration nucleosides.The target compounds were synthesized by three different methods in the process.As a result, it was shown that the trimethylsilylational method was fit for the synthesis of N3-,S-,and 6-methyl-N₁-nucleosides.However,isocyanate procedure was more suitable for S-nucleoside.In addition,it is possible that N-aminoimino -methyl-N’-P-D-pyranosyl-urea cyclization was used for the synthesis of 6-larger substituted groups-N₁-nucleoside.Meanwhile,the regioselectivity of products which were attained by the trimethylsilylational method was discussed simply.Vorbrüggen coupling of N-trimethylsilyl-6-（trimethylsilyl）oxy-1,3,5-triazin-2-amine with acylated sugars and 4-methyl-N-trimethylsilyl-6-（trimethylsilyl）oxy-1,3,5-triazin-2-amine with 1-0 -acetyl-2,3,5-tri-O-benzoyl-β-D-ribose in anhydrous acetonitrile with SnCl₄ or trimethylsilyltriflate （TMSOTf） catalysis gave acylated N₁-nucleosides.However when the coupling procedure was applied to 4-Methyl-N-trimethylsilyl-6-（trimethylsilyl）oxy-1,3,5-triazin-2-amine and acetylated pyranoses in the presence of SnCl₄（2eq ）,acylated N₃-isomers were obtained as the main products. It was observed that N₁-isomers were predominant products while a weaker Lewis acid TMSOTf （2eq ） was used as the catalyst.N3-Nucleosides were predominant products even though TMSOTf was used as catalyst.While ethyl and phenyl were introduced to 6 position the steric hindrance turned to be a major factor determining the regioselectivity.The acylated S-nucleosides which were kinetically controlled products were predominant products during couplings of with acylated sugars in anhydrous acetonitrile with SnCU or TMSOTf catalysis.It was obtained C1 conformation mannopyranoside and 1C conformation rhamnopyranoside formed when vorbriiggen coupling of 4-alkyl/aryl -N-trimethylsilyl-6-（trimethylsilyl）oxy-1,3,5 -triazin-2-amine with the 1C conformation acetylated mannose or C1 conformation acetylated rhamnose because of antianomeric effect.However,the rentention of conformation of 4-amino-6-alkyl-1,3,5-triazin-2-yl-1-thio-mannopyranoside/rhamnopyranoside were obtained when vorbrüggen coupling of 4-alkyl-N-trimethylsilyl-6-（trimethylsilyl）thio-1,3,5-triazin-2-amine with the 1C conformation acetylated mannose or C1 conformation acetylated rhamnose for anomeric effect.Compounds GG-07,GG-30 and GG-50 had a lower GI₅₀ value of 18.5μM,1.7μM and 1.48μM respectively.Other compounds did not inhibit 50.0%cell growth at concentrations less than 50μM.Comparing the antiproliferative effects of 5-aza-CR,it was found that there were lower growth inhibition effects when the replacement of ribofuranosyl moiety with pyranosyl sugars or disaccharides.The replacement of 5-aza-CR into bioisostere ribofuranosyl S-nucleoside and the replacement of H with methyl,ethyl,phenyl group significantly decreased the antiproliferative effect also. All of these compounds alone did not induce NBT differentiation but the treatment of ATRA in combination with compound GG-02,GG-04 or GG-38 induced a significant increase in differentiation in HL-60 and NB-4 cells.The treatment of ATRA in combination with compound GG-16,GG-37,GG-39 or GG-48 induced a moderate growth inhibiton effect and the ratio of growth inhibiton rate was increased from 38.7%into above 50.0%comparing with 0.1μM ATRA alone.