Synthesis of Novel CYP1 Activated Heterocyclic Anticancer Prodrugs
The cytochrome P450 superfamily of enzymes are critical in the metabolism of endogenous and exogenous substrates. CYP1A1 and CYP1B1 have been found to be over-expressed in tumour cells whilst undetected or present in very low levels in corresponding normal tissue. This presented a novel target for the development of anti-cancer prodrugs, which would remain non-toxic until undergoing metabolism to toxic species by CYP1 enzymes over-expressed at tumour sites. The chalcones have been shown to exhibit effective anti-cancer prodrug activity, but are labile to photoisomerisation reactions converting the potent trans isomer to the less toxic cis isomer. Several heterocyclic ring systems were incorporated across the α,β-unsaturated moiety of the chalcones to produce rigid structures, eliminating the possibility of photoisomerisation occurring whilst maintaining the substituted phenyl groups in a trans like geometry. Lead compounds were identified using an in vitro MTT screening assay against a panel of tumour cell lines characterised for their constitutive or inducible CYP1 expression. These were the MDA 468, MCF7 and MDA 231 cell lines. The non-tumour MCF10A cell line which has no basal CYP1 expression was used as the control. A library of eighteen 3,5-diarylpyrazoles were synthesised. The lead pyrazole DMU 10107 (3-(2,3,4-trimethoxyphenyl)-5-(3,4-methylenedioxyphenyl)pyrazole) gave an IC50 value of 8μM towards the MDA 468 cell line. The MCF7 cells, TCDD induced and non-induced gave IC50 values of 10μM each. Although the pyrazoles showed plausible tumour toxicity, an investigation into six membered pyrimidine heterocycles was undertaken in an attempt to obtain enhanced cytotoxicities than those observed from the five membered pyrazoles. Therefore, a library of fifteen 2-amino-4,6-diarylpyrimidines was synthesised. The lead amino-pyrimidine DMU 10212 (2-amino-4-(2,4-dimethoxyphenyl)-6-(3,4-methylenedioxyphenyl)pyrimidine) showed significant cytotoxicity towards the MDA 468 cell line with an IC50 value of 0.01μM. Notable IC50 values of 0.3μM and 0.07μM were also observed towards the MCF7 and MCF7 cells induced with TCDD. The important toxicity seen from the 2-amino-4,6-diarylpyrimidines prompted the investigation of the 2-position of the pyrimidine ring, and to assess the tumour toxicities of the synthesised compounds. The 2-amino-4,6-diarylpyrimidines were converted to produce 4,6-diarylpyrimidones by a one-step conversion reaction using sodium nitrate. The pyrimidone DMU 10313 (4-(2-methoxyphenyl)-6-(3,4-methylenedioxyphenyl)pyrimidin-2-one) showed high toxicity with an IC50 value of 0.07μM towards the MDA 468 cells and IC50 values of 1.8μM and 0.5μM 3 towards the MCF7 and MCF7 cells induced with TCDD. A library of nine 2-morpholino-4,6-diarylpyrimidines was synthesised. The lead compound DMU 10405 (4-(2,4-dimethoxyphenyl)-6-(4-methoxyphenyl)-2-morpholinopyrimidine) gave an IC50 value of 10μM towards the MDA 468 cells. DMU 10600 (4-(2,4-dimethoxyphenyl)-6-(3,4-methylenedioxyphenyl)-2-dimethylethylenediaminopyrimidine), showed an IC50 value of 7μM towards the MDA 468 cells and an identical IC50 value of 10μM towards the MCF7 and MCF7 cells treated with TCDD. DMU 10700 (2-methyl-4-(2,4-dimethoxyphenyl)-6-(3,4-methylenedioxyphenyl)pyrimidine), a substituted pyrimidine based on the phenyl substitutions of DMU 10212 gave an IC50 value of 2.5μM towards the MDA 468 cells. DMU 10800 (4-(2,4-dimethoxyphenyl)-6-(3,4-methylenedioxyphenyl)pyrimidine), also based on the phenyl substitutions of DMU 10212 showed an IC50 value of 0.08μM towards the MDA 468 cells and equal IC50 values of 0.2μM against the MCF7 and MCF7 cells induced with TCDD. All lead compounds did not show toxicity towards the non-tumour MCF10A cell line. DMU 10212 was selected as the overall lead compound due to the significant tumour toxicities recorded, and for the non-toxicity observed towards the MCF10A cells. Inhibition studies using the known CYP1 inhibitor α-naphthoflavone (α-NF) were conducted to show that DMU 10212 was a substrate of the CYP1 enzymes. The resulting data showed that the cytotoxicity of DMU 10212 was completely eliminated suggesting CYP1 enzymes play an activating role in the cytotoxic effect of DMU 10212. LCMS metabolism studies using isolated CYP1 isoforms were performed showing that DMU 10212 is metabolised to produce four metabolites (M1, M2, M3 and M4), determined from their individual retention times and molecular masses. The metabolites of DMU 10212 were also found to be generated at a greater rate with CYP1A1 than CYP1B1. Metabolite structures were proposed as CYP1 enzyme reactions are known. The metabolite M2 was synthesised and was identified to be an authentic metabolite of DMU 10212 via LCMS and co-elution studies. Screening of M2 against the tumour cells gave an IC50 value of 0.6μM towards the MDA 468 cells, and IC50 values of 0.6μM and 1μM against the MCF7 and MCF7 cells induced with TCDD. In conclusion, DMU 10212, a novel CYP1 activated anticancer prodrug with selective high toxicity towards tumour cells has been identified.
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