Simultaneous, But Not Consecutive, Combination With Folinate Salts Potentiates 5-fluorouracil Antitumor Activity In Vitro and In Vivo

The combination of folinate salts to 5-fluoruracil (5-FU)-based schedules is an established clinical routine in the landscape of the colorectal cancer treatment. The aim of this study was to investigate the pharmacological differences between the sequential administration of folinate salts (1 h before, as in clinical routine) followed by 5-FU and the simultaneous administration of both drugs. Proliferation and apoptotic assays were performed on human colon cancer cells exposed to 5-FU, calcium (CaLV) or disodium (NaLV) levofolinate or their simultaneous and sequential combination for 24 and 72h. TYMS and SLC19A1 gene expression was performed with real time PCR. In vivo experiments were performed in xenografted nude mice, treated with 5-FU escalating doses and CaLV or NaLV alone or in simultaneous and sequential combination. The simultaneous combination of folinate salts and 5-FU was synergistic (NaLV) or additive (CaLV) in a 24h treatment in both cell lines. In contrast, the sequential combination of both folinate salts and 5-FU was antagonistic at 24 and 72h. The simultaneous combination of 5-FU and NaLV or CaLV inhibited TYMS gene expression at 24h, whereas the sequential combination reduced SLC19A1 gene expression. In vivo experiments confirmed the enhanced antitumor activity of the 5-FU+NaLV simultaneous combination with a good toxicity profile, whereas the sequential combination with CaLV failed to potentiate 5-FU activity. In conclusion, only the simultaneous, but not the consecutive, in vitro and in vivo combination of 5-FU and both folinate salt formulations potentiated the antiproliferative effects of the drugs

Fluorinated nucleosides as an important class of anticancer and antiviral agents

Fluorine-containing nucleoside analogs (NAs) represent a significant class of the US FDA-approved chemotherapeutics widely used in the clinic. The incorporation of fluorine into drug-like agents modulates lipophilic, electronic and steric parameters, thus influencing pharmacodynamic and pharmacokinetic properties of drugs. Fluorine can block oxidative metabolism of drugs and the formation of undesired metabolites by changing H-bonding interactions. In this review, we focus our attention on chemical fluorination reagents and methods used in the NAs field, including positron emission tomography radiochemistry. We briefly discuss both the cellular biology and clinical properties of FDA-approved and fluorine-containing nucleoside/nucleotide analogs in development as well as common resistance mechanisms associated with their use. Finally, we emphasize pronucleotide strategies used to improve therapeutic outcome of NAs in the clinic. </jats:p

Synthesis and Molecular Modeling Studies of Bicyclic Inhibitors of Dihydrofolate Reductase, Receptor Tyrosine Kinases and Tubulin

The results from this work are reported into two sections listed below: Synthesis: Following structural classes of compounds have been designed, synthesized and studied as inhibitors of pjDHFR, RTKs and tubulin: 1. 2,4-Diamino-6-(substituted-arylmethyl)pyrido[2,3-d]pyrimidines 2. 4-((3-Bromophenyl)linked)-6-(substituted-benzyl)-7H-pyrrolo[2,3-d]pyrimidin-2-amines 3. 6-Methyl-5-((substitutedphenyl)thio)-7H-pyrrolo[2,3-d]pyrimidin-2-amines A total of 35 new compounds (excluding intermediates) were synthesized, characterized and submitted for biological evaluation. Results from these studies will be presented in due course. Bulk synthesis of the potent lead compound 170 was carried out to facilitate in vivo evaluation. Docking Studies Docking studies were performed using LeadIT, MOE, Sybyl or Flexx for target compounds listed above and for other compounds reported by Gangjee et al. against the following targets: 1. Dihydrofolate reductase: human, P. carinii, P. jirovecii (pjDHFR) and T. gondii (tgDHFR) 2. Thymidylate synthase: human (hTS) and T. gondii (tgTS) 3. Receptor tyrosine kinases: VEGFR2, EGFR and PDGFR-β 4. Colchicine binding site of tublulin. Novel homology models were generated and validated for pjDHFR, tgDHFR, tgTS, PDGFR-β and the F36C L65P pjDHFR double mutant. The tgTS homology model generated in this study and employed to design novel inhibitors shows remarkable similarity with the recently published X-ray crystal structures. Docking studies were performed to provide a molecular basis for the observed activity of target compounds against DHFR, RTKs or tubulin. Results from these studies support structure-based and ligand-based medicinal chemistry efforts in order to improve potency and/or selectivity of analogs of the docked compounds against these targets. Novel topomer CoMFA models were developed for tgTS and hTS using a set of 85 bicyclic inhibitors and for RTKs using a set of 60 inhibitors reported by Gangjee et al. The resultant models could be used to explain the potency and/or selectivity differences for selected molecules for tgTS over hTS. Topomer CoMFA maps show differences in steric and/or electronic requirements among the three RTKs, and could be used, in conjuction with other medicinal chemistry approaches, to modulate the selectivity and/or potency of inhibitors with multiple RTK inhibitory potential. Drug design efforts that involve virtual library screening using these topomer CoMFA models in conjunction with traditional medicinal chemistry techniques and docking are currently underway

Effect of Halogen Substitutions on dUMP to Stability of Thymidylate Synthase/dUMP/mTHF Ternary Complex Using Molecular Dynamics Simulation

The stability of the thymidylate synthase (TS)/2-deoxyuridine-5-monophosphate (dUMP)/5,10-methylene-5,6,7,8-tetrahydrofolate (mTHF) ternary complex formation and Michael addition are considered as important steps that are involved in the inhibition mechanism of the anticancer prodrug 5-fluorouracil (5-FU). Here, the effect of three different halogen substitutions on the C-5 position of the dUMP (XdUMPs = FdUMP, CldUMP, and BrdUMP), the normal substrate, on the stability of the TS/dUMP and TS/dUMP/mTHF binary and ternary complexes, respectively, was investigated via molecular dynamics simulation. The simulated results revealed that the stability of all the systems was substantially increased by mTHF binding to the catalytic pocket. In the ternary complex, a much greater stabilization of the dUMP and XdUMPs through electrostatic interactions, including charge–charge and hydrogen bond interactions, was found compared to mTHF. An additional unique hydrogen bond between the substituted fluorine of FdUMP and the hydroxyl group of the TS Y94 residue was observed in both the binary and ternary complexes. The distance between the S^– atom of the TS C146 residue and the C6 atom of dUMP, at <4 Å in all systems, suggested that a Michael addition with the formation of a S–C6 covalent bond potentially occurred, although the hydrogen atom on C6 of dUMP is substituted by a halogen atom. The MM/PBSA binding free energy revealed the significant role of the bridging waters around the ligands in the increased binding affinity (∼10 kcal/mol) of dUMP/XdUMP, either alone or together with mTHF, toward TS. The order of the averaged binding affinity in the ternary systems was found to be CldUMP ≈ FdUMP > dUMP > BrdUMP, suggesting that CldUMP could be a potent candidate TS inhibitor, the same as FdUMP (the metabolite form of 5-FU)

Radiochemical synthesis of 18F-radiolabelled ProTides for Positron Emission Tomography

Positron Emission Tomography (PET) is a highly sensitive imaging technique used in cancer diagnosis, treatment planning and monitoring of therapy response. [18F] is an optimal PET label considering its half-life (110 min) and imaging resolution. One of the major challenges in [18F]-PET research is the installation of the weakly nucleophilic [18F]fluoride into a precursor molecule to access novel [18F]-tracers. Fluorinated nucleosides represent an important class of diagnostic probes for PET imaging as well as anticancer and antiviral therapeutic agents. However drug resistance still represents a major problem. The ProTide approach is a strategy to synthesize prodrugs of the nucleoside monophosphates which overcome their main resistance mechanisms. The challenge of the project is the [18F]-fluorination (hot fluorination) of ProTides which may be potential new PET imaging agents and could thus represent a model system to visualize pharmaceutical effects and bioactivation of ProTides directly in vivo. The pro-nucleotide multistep synthetic chemistry has been applied for the synthesis of ProTides. The [18F]-radiolabeling of the precursor molecules was performed in an Eckert & Ziegler automated synthetic Modular Lab placed into a shielded hot cell. The radioactive reaction mixtures were analyzed by radio HPLC, and radio TLC. Two different approaches have been followed to access two chemically distinct radiolabelled ProTides. The 3’-[18F]FLT ProTide was synthesised via a late stage [18F]fluorination of ad hoc synthesised precursor molecules (Figure 1). Figure 1: Radiochemical synthesis of 18F- FLT ProTides The 2’-[18F]FIAU ProTide was synthesised via an early stage [18F]fluorination approach (Figure 2). Figure 2: Radiochemical synthesis of 18F-FIAU ProTides These radiolabelled probes could provide evidence for the in vivo behaviour of this class of compounds by answering key questions about their metabolism and uptake directly. In addition, the project focused on the synthesis of two novel classes of non-radiolabelled fluorinated ProTides. A series of uridine based ProTides (FIAU ProTides) and a series of coumarin based FLT ProTides have been synthesised and evaluated for their antiviral activity and fluorescent properties respectively