Harry Ford

Synthesis of Conformationally Restricted Carbocyclic Nucleosides: The Role of the O(4′)‐Atom in the Key Hydration Step of Adenosine Deaminase

Conformationally restricted carbocyclic nucleosides with either a northern(N)-type conformation, i.e., N-type 2′-deoxy-methanocarba-adenosine 8 ((N)MCdAdo), or a southern(S)-type conformation, i.e.S-type 2′-deoxy-methanocarba-adenosine 9, ((S)MCdAdo), were used as substrates for adenosine deaminase (ADA) to assess the enzymes preference for a fixed conformation relative to the flexible conformation represented by the carbocyclic nucleoside aristeromycin (10). Further comparison between the rates of deamination of these compounds with those of the two natural substrates adenosine (Ado; 1) and 2′-deoxyadenosine (dAdo; 2), as well as with that of the conformationally locked nucleoside LNA-Ado (11), which, like the natural substrates, has a furanose O(4′) atom, helped differentiate between the roles of the O(4′) anomeric effect and sugar conformation in controlling the rates of deamination by ADA. Differences in rates of deamination as large as 10000 can be attributed to the combined effect of the O(4′) atom and the enzymes preference for an N-type conformation. The hypothesis proposed is that ADAs preference for N-type substrates is not arbitrary; it is rather the direct consequence of the conformationally dependent O(4′) anomeric effect, which is more efficient in N-type conformers in promoting the formation of a covalent hydrate at the active site of the enzyme. The formation of a covalent hydrate at the active site of ADA precedes deamination. A new and efficient synthesis of the important carbobicyclic template 14a, a useful intermediate for the synthesis of (N)MCdAdo (8) and other conformationally restricted nucleosides, is also reported.

Synthesis, Conformational Analysis, and Biological Activity of a Rigid Carbocyclic Analogue of 2′-Deoxy Aristeromycin Built on a Bicyclo[3.1.0]hexane Template #

Abstract A new chiral synthesis of the pseudosugar synthon (1R,2S,4R,5S)-1-[(benzyloxy)methyl]-2-tert-butyloxy-4-hydroxybicyclo[3.1.0]hexane (12) is reported. This compound was used as a template for the construction of carbocyclic nucleoside 4, a conformationally rigid analogue of 2′-deoxyaristeromycin. The X-ray structure and 1H NMR analysis confirmed the exclusive North [2′-exo (2E)] conformation of 4 which is vastly different from that of other non-rigid carbocyclic nucleosides. Compound 4 showed good in vitro antiviral activity against human cytomegalovirus and EBV with minimal cytotoxicity. #This manuscript is dedicated to Professor Yoshihisa Mizuno on the occasion of his 75th birthday.

Phase I clinical trial of continuous infusion cyclopentenyl cytosine.

Cyclopentenyl cytosine (CPE-C) is an investigational drug that is active against human solid tumor xenografts. The 5′-triphosphate of CPE-C inhibits CTP synthase, and depletes CTP and dCTP pools. We conducted a phase I clinical trial of CPE-C given as a 24-h continuous i. v. infusion every 3 weeks in 26 adults with solid tumors. The starting dose rate, 1 mg/m2 per h, was selected on the basis of both preclinical studies and pharmacokinetic data from two patients obtained after a test dose of 24 mg/m2 CPE-C as an i. v. bolus. Dose escalation was guided by clinical toxicity. A total of 87 cycles were given, and ten patients received four or more cycles. The mean CPE-C steady-state plasma levels (Cpss) increased linearly from 0.4 μM to 3.1 μM at dose levels ranging from 1 to 5.9 mg/m2 per h (actual body weight); the mean total body clearance was 146±38 ml/min per m2. CPE-C was eliminated by both renal excretion of intact drug and deamination to cyclopentenyl uracil in an apparent 2∶1 ratio. CTP synthase activity in intact bone marrow mononuclear cells was inhibited by 58% to 100% at 22 h compared to matched pretreatment samples at all CPE-C dose levels. When all data were combined, flux through CTP synthase was decreased by 89.6%±3.1% at 22h (mean ± SE,n=16), and remained inhibited by 67.6%±7.7% (n=10) for at least 24 h post-CPE-C infusion. Granulocyte and platelet toxicities were dose-dependent, and dose-limiting myelosuppression occurred during the initial cycle in two of three patients treated with 5.9 mg/m2 per h. Four of 11 patients (4 of 20 cycles) who received 4.7 mg/m2 per h CPE-C experienced hypotension 24–48 h after completion of the CPE-C infusion during their first (n=2), third (n=1) and sixth cycles (n=1), respectively. Two of these patients died with refractory hypotension despite aggressive hydration and cardiopulmonary resuscitation. One of 12 patients (28 total cycles) treated with 3.5 mg/m2 per h CPE-C experienced orthostatic hypotension during cycle 1, and this patient had a second episode of orthostatic hypotension at a lower dose (3.0 mg/m2per h). Hypotension was not seen in patients receiving ≤2.5 mg/m2 per h CPE-C. The occurrence of hypotension did not directly correlate with either CPE-C Cpss, CPE-U plasma levels, pretreatment cytidine plasma levels, baseline CTP synthase activity, or with the degree of enzyme inhibition during treatment. While the hypotension appeared to be dose-related, its unpredictable occurrence and the uncertainty concerning the mechanism preclude a recommendation of a tolerable dose for future studies.

Metabolic pathways of N-methanocarbathymidine, a novel antiviral agent, in native and herpes simplex virus type 1 infected Vero cells

N-methanocarbathymidine ((N)-MCT), a thymidine analog incorporating a pseudosugar with a fixed Northern conformation, exhibits potent antiherpetic activity against herpes simplex virus types 1 (HSV-1) and 2 (HSV-2). This study contrasts the metabolic pathway of (N)-MCT and the well-known antiherpetic agent ganciclovir (GCV) in HSV-1-infected and uninfected Vero cells. Treatment of HSV-1 infected Vero cells immediately after viral infection with (N)-MCT profoundly inhibited the development of HSV-1 infection. Using standard plaque reduction assay to measure viral infection, (N)-MCT showed a potency greater than that of ganciclovir (GCV), the IC50s were 0.02 and 0.25 microM for (N)-MCT and GCV, respectively. (N)-MCT showed no cytotoxic effect on uninfected Vero cells (CC50>100 microM). Dose and time dependence studies showed high levels of (N)-MCT-triphosphate ((N)-MCT-TP), and GCV-triphosphate (GCV-TP) in HSV-1-infected cells incubated with (N)-MCT or GCV, respectively. In contrast, uninfected cells incubated with (N)-MCT showed elevated levels of (N)-MCT-monophosphate only, while low levels of mono, di- and triphosphates of GCV were found following incubation with GCV. Although the accumulation rate of (N)-MCT and GCV phosphates in HSV-1-infected cells were similar, the decay rate of (N)-MCT-TP was slower than that of GCV-TP. These results suggest that: (1) the antiviral activity of (N)-MCT against herpes viruses is mediated through its triphosphate metabolite; (2) in contrast to GCV, the diphosphorylation of (N)-MCT in HSV-1- infected cells is the rate limiting step; (3) (N)-MCT-TP accumulates rapidly and has a long half-life in HSV-1-infected cells; and (4) HSV-tk catalyzed the mono, and diphosphorylation of (N)-MCT while monophosphorylating GCV only. These results provide a biochemical rational for the highly selective and effective inhibition of HSV-1 by (N)-MCT.

A Rapid Microscale Method for the Determination of Partition Coefficients by HPLC

Abstract A rapid, reliable and simple microscale method for the determination of octanol-water partition coefficients has been developed and evaluated. Rapid solute partitioning and facile octanol-water phase separation is accomplished in a commercially available mixer-separator device. The relative concentration of solute in each phase is then measured directly by computer-based reverse phase HPLC. The procedure requires only 10 μg of sample, which need not be pure, and uses 1 ml or less of both n-octanol and pH 7.0 phosphate buffer. Log P values of 26 compounds, mainly nucleoside analogues with anti-HIV and antitumor activity, have been determined in the range 0.7 to -2.4 with a precision better than ±0.04 log units. For compounds with literature data available, measured log P values are in good agreement (better than ±0.2 log units) with those values obtained by traditional “shake-flask” methodology. The described method is applicable to both single compound analysis and simultaneous multiple compound ...

Synthesis of Fmoc-protected 4-carboxydifluoromethyl-L-phenylalanine: A phosphotyrosyl mimetic of potential use for signal transduction studies

Abstract 4-(Carboxymethyl)phenylalanine ( 2 ) and its α,α-difluoro homologue 4-(carboxydifluoromethyl)phenylalanine ( 3 ) have been described as phosphotyrosyl mimetics. Herein we report the synthesis of N-Fmoc 4-( O -tert-butyl carboxymethyl)phenylalanine ( 4 ) and N-Fmoc 4-( O -tert-butyl carboxydifluoromethyl)phenylalanine ( 5 ) in high enantiomeric purity. These analogues bear orthogonal protection suitable for the preparation of inhibitors directed against a variety of signal transduction pathways, including SH2 and PTP domains and protein-tyrosine phosphatases.

Determination of 2'-β. -fluoro-2', 3' -dideoxyadenosine, an experimental anti-AIDS drug, in human plasma by high-performance liquid chromatography

Abstract 2′-β-Fluoro-2′,3′-dideoxyadenosine (F-ddA, lodenosine) is an experimental anti-AIDS drug currently being evaluated in a Phase I clinical trial. A simple and specific HPLC method with UV detection, suitable for use in clinical studies, has been developed to determine both F-ddA and its deaminated catabolite, 2′-β-fluoro-2′,3′-dideoxyinosine (F-ddI) in human plasma. After inactivation of plasma HIV by 0.5% Triton X-100, the compounds of interest are isolated and concentrated using solid-phase extraction. Processed samples are separated by use of a pH 4.8 buffered methanol gradient on a reversed-phase phenyl column. The method has a linear range of 0.05–5 μg/ml (0.2–20 μ M ) and intra-assay precision is better than 8%. Analyte recovery is quantitative and plasma protein binding is minimal. In addition, drug and metabolite levels measured in Triton-treated human plasma remain stable for at least 5 months when samples are stored frozen without further treatment. Compound concentrations determined after samples are processed and then frozen for up to 1 month before analysis are also unchanged.

LESSONS FROM THE PSEUDOROTATIONAL CYCLE : CONFORMATIONALLY RIGID AZT CARBOCYCLIC NUCLEOSIDES AND THEIR INTERACTION WITH REVERSE TRANSCRIPTASE

Abstract Two conformationally locked carba-AZT nucleoside 5′-triphosphates built on a rigid bicyclo[3.1.0]hexane template showed exclusive Northern (2E) and Southern (3E) conformations, respectively. Inhibition of reverse transcriptase (RT) occurred selectively with the Northern carba-AZX triphosphate.

Chemistry and Anti-HIV Activity of 2′-β-Fluoro-2′,3′-dideoxyguanosine

Abstract The 2′-β-fluoro analogue of 2′,3′-dideoxyguanosine has been prepared by two synthetic routes. This compound and two analogues have anti-HIV activity in at least two of three host cell systems used (ATH8, CEM, PBL). These compounds, as well as their ddGuo parents, have been characterized with regard to their acid-stabilities, octanol-water partition coefficients, and enzyme substrate properties for adenosine deaminase and purine nucleoside phosphorylase. F-ddGuo analogues are less potent but more stable than their non-fluorinated parent compounds.

Reverse phase HPLC determination and murine pharmacokinetics of arabinosyl-5-azacytosine

Abstract A sensitive and specific reverse phase HPLC assay has been developed to measure the new antitumor agent arabinosyl-5-aza-cytosine (ara-AC) in biological fluids at concentrations as low as 50 ng/ml (0.2 μM). This assay also detects arabinosyl-N-formyl-guanylurea (AGU-CHO), the initial hydrolytic metabolite of ara-AC. 2′-Deoxy-5-azacytidine, an analogue with similar chemical stability, is used as an internal standard. Chromatographically interfering plasma ribosides are removed by solid phase extraction on a phenyl boronic acid cartridge. Separation of ara-AC, AGU-CHO and internal standard is then accomplished isocratically (1% CH3CN in 10 mM pH 6.8 phosphate buffer) on fully carbon loaded and end-capped C8 and C18 columns connected in tandem. The compounds of interest are detected by UV absorption at 240 nm and total analysis time is 20 min. This assay has been used to determine bolus dose plasma kinetics in male BDF1 mice given 200 mg/kg ara-AC as a tail vein injection. Plasma elimination of the ...