Synthesis And Biological Evaluation Of Amphiphilic Low Molecules-Cytarabine Prodrugs

1-β-D-Arabinofuranosylcytosine (cytarabine, Ara-C), a pyrimidine nucleoside analogue, predominantly used in the treatment of both acute and chronic myeloblastic leukemias. It is the cornerstone of induction therapy and consolidation therapy for AML. Cytarabine (Ara-C) as nucleoside analogues is inactive by itself and requires phosphorylation to the corresponding triphosphate (Ara-CTP) in vivo to exert its antineoplastic activity by inhibition of nucleic acid biosynthesis and rapidly deaminated by cytidine deaminase (CDA). At a molecular level, cytarabine anticancer mechanisms rely on sequential conversion from monophosphate, diphosphate, to triphosphate forms. Cytarabine triphosphate replaces deoxycytidine during DNA replication, which leads to cell arrest and failure to repair the DNA subsequently triggers apoptosis and blocks tumor growth. Cytarabine, like all nucleoside analogues (such as gemcitabine,5-fluorouracil, and fludarabine), suffers from several limitations. It has a low permeability in intestinal membrane and is rapidly deaminated to biologically inactive 1-β-D-arabinofuranosyluracil in intestinal and hepatic cells leading to a very low oral bioavailability. However, under intravenous or subcutaneous administration, it is also rapidly inactivated by systemic deamination which finally results in short plasma half-life (t1/2=2h). Consequently, cyatarabine must be administered in continuous infusion or on a complex schedule to maintain plasma levels and longer effective concentration in clinic application. In addition, the use of high-dose cytarabine has adverse risk for patients. Prodrug has been considered a chemistry-enabled drug delivery tool used to address shortcomings in bioavailability, efficacy or safety profiles of otherwise promising candidates.It has been reported that the modifications of cytarabine focused on increasing lipotropism and reducing inactive rate. The research purpose of this subject is overcoming using disorder of cytarabine, improving the lipotropism, increasing oral absorption and improving bioavailability.1.Synthesis and biological evaluation of a fatty acyl di-cytarabine prodrugHere, we developed a prodrug for acute myelogenous leukemia (AML) which could overcome the disadvantage of cytarabine such as short plasma half-life and rapid deamination to its inactive metabolite. The new drug-fatty acyl conjugate (Ara-R-Ara) has been synthesized from the hydrophilic anticancer drug cytarabine and hydrophobic fatty acyl suberoyl chloride via a hydrolyzable amido linkage. Suberoyl chloride has been conjugated to cytarabine for protecting the NH2 group from the enzymatic attach to exhibit longer blood retention half-life compared with the free drugs. A higher membrane permeability of Ara-R-Ara (Pam=17.71×10-6 cm/s) was obtained compared with pure cytarabine. In vitro cytotoxicity results showed that the new prodrug had a much lower IC50 and a higher cell inhibition rate compared with pure cytarabine for HL60 and K562 cells, indicating its effective therapy for leukemic cells.2. Synthesis and biological evaluation of a fatty acyl mono-cytarabine prodrugThe new cytarabine prodrug (LA-Ara) has been synthesized from the hydrophilic anticancer drug cytarabine and hydrophobic lauric acid via a hydrolyzable amido linkage. The chemical structure of the derivative was confirmed using 1H-NMR and MS. The stability experiments were performed in phosphate buffers of different pH values and simulated gastric fluid and simulated intestinal fluid. The chemical stability of LA-Ara was basically decreased with the decreasing pH of phosphate buffer. And the prodrug could maintain enough chemical stability in simulated gastric fluid and simulated intestinal fluid. Meanwhile, LA-Ara could self-assemble to form fiber structure in the water. The PAMPA assay results indicate that the permeability of prodrug is about 8.2 times of the cytarabine solution. In vitro cytotoxicity results showed that the new prodrug showed a higher cell inhibition rate compared with pure cytarabine for HL60 and K562 cells, indicating its effective therapy for leukemic cells. Wistar rats are used in vivo studies. Both free cytarabine solution and prodrug LA-Ara are administrated by oral lavage at single dose (15mg/kg body weight). At designated times, the blood samples are drawn from the sinus jugularis immediately separated by centrifugation. Acetonitrile is used to remove the endogenous proteins and the supernate is injected for analysis. The data for pharmacokinetics in rats are analyzed with DAS 2.0. The experimental results showed that LA-Ara was rapidly converted to cytarabine by ester enzymes in vivo and could maintain high concentration, while the concentration of LA-Ara was at lower. LA-Ara showed a higher bioavailability than cytarabine.To sum up, two new cytarabine prodrug molecules which contain different length hydrocarbon chain were synthesized for the first time. The higher membrane permeability of prodrugs were obtained compared with pure cytarabine. In vitro cytotoxicity results showed that the new prodrugs had higher cell inhibition rates compared with pure cytarabine for HL60 and K562 cells, indicating their effective therapy for leukemic cells. And LA-Ara could self-assemble to form fiber structure in the water. LA-Ara showed a higher bioavailability than cytarabine using oral lavage. The study above laid a theoretical foundation to research such amphiphilic prodrugs.