L-Enantiomers of transition state analogue inhibitors bound to human purine nucleoside phosphorylase

Human purine nucleoside phosphorylase (PNP) was crystallized with transition-state analogue inhibitors Immucillin-H and DADMe-Immucillin-H synthesized with ribosyl mimics of l-stereochemistry. The inhibitors demonstrate that major driving forces for tight binding of these analogues are the leaving group interaction and the cationic mimicry of the transition state, even though large geometric changes occur with d-Immucillins and l-Immucillins bound to human PNP.     

Structure-activity relationships for a collection of structurally diverse inhibitors of purine nucleoside phosphorylase

Values of inhibition constants, Ki, and concentrations required for 50% inhibition, IC50, for a collection of structurally diverse competitive inhibitors of calf spleen purine nucleoside phosphorylase have been determined employing inosine as substrate. These values have been employed to create predictive quantitative structure-activity relationships (QSAR) which link structure to values of Ki and IC50. These QSAR models have substantial power to predict values and the associated uncertainties for Ki and IC50 for unknown, structurally diverse inhibitors of purine nucleoside phosphorylase.     

An efficient and large-scale enantioselective synthesis of PNP405: a purine nucleoside phosphorylase inhibitor

An efficient and large-scale enantioselective synthesis of PNP405 (1), a purine nucleoside phosphorylase inhibitor, is described. This synthesis of 1 involved eight steps starting from o-fluorophenylacetic acid with a 21.6% overall yield and >99.5% enantiopurity. The key stereogenic center with (R)-configuration was created using Evans' asymmetric alkylation methodology. This synthesis also features the racemization-free reductive removal of the chiral auxiliary in 5 using sodium borohydride, protection of the gamma-cyano alcohol 6 as the trityl ether by a new water-assisted tritylation with trityl chloride and triethylamine or with trityl alcohol and catalytic trifluoroacetic acid, and an efficient one-pot cyclo-guanidinylation of 10 using cyanamide as the guanidinylating agent.     

Phosphate activation in the ground state of purine nucleoside phosphorylase

Phosphate and ribose 1-phosphate (R1P) bound to human purine nucleoside phosphorylase (PNP) have been studied by FTIR spectroscopy for comparison with phosphate bound with a transition state analogue. Bound phosphate is dianionic but exists in two distinct binding modes with similar binding affinities. The phosphate of bound R1P is also dianionic. Bound R1P slowly hydrolyzes to ribose and phosphate even in the absence of nucleobase. The C-OP bond is cleaved in bound R1P, the same as in the PNP-catalyzed reaction. Free R1P undergoes both C-OP and CO-P solvolysis. A hydrogen bond to one P-O group is stronger than those to the other two P-O groups in both the PNP.R1P complex and in one form of the PNP.PO4 complex. The average hydrogen bond strength to the PO bonds in the PNP.R1P complex is less than that in water but stronger than that in the PNP.PO4 complex. Hydrolysis of bound R1P may be initiated by distortion of the phosphate moiety in bound R1P. The unfavorable interactions on the phosphate moiety of bound R1P are relieved by dissociation of R1P from PNP or by hydrolysis to ribose and phosphate. The two forms of bound phosphate in the PNP.PO4 complex are interpreted to be phosphate positioned as the product in the nucleoside synthesis direction and as the reactant in the phosphorolysis reaction; their interconversion can occur by the transfer of a proton from one PO bond to another. The electronic structure of phosphate bound with a transition state analogue differs substantially from that in the Michaelis complexes.     

Alternative route towards the convergent synthesis of a human purine nucleoside phosphorylase inhibitor—forodesine HCl

Forodesine HCl is being investigated as a potential target for the control of T-cell proliferation. Herein we present an alternative route for the synthesis of the target molecule with addition of lactam to the lithiated deazahypoxanthine (generated in situ). The lactam was synthesized in five steps starting from l-pyroglutamic acid.     

Facile synthesis of 1-substituted c-norbenzomorphans

The olefinic-nitrile (4) undergoes a bromium ion-induced cyclisation to the tetrahydronaphthalene (5a) which on subsequent reduction and intramolecular nucleophilic displacement provides a facile route to 1-substituted C-norbenzomorphans.     

Facile synthesis of 2-substituted cyclopentenones

An efficient synthesis of 2-substituted cyclopentenones is reported. Key reactions are the cylopropanation of 2,2-dimethyl-3(2H)-furanone and subsequent conversion to the title compounds by oxidative fragmentation.     

Facile and regioselective synthesis of novel 2,4-disubstituted-5-fluoropyrimidines as potential kinase inhibitors

2,4-Disubstituted-5-fluoropyrimidine is a biologically active molecular core seen in various anticancer agents such as 5-fluorouracil (5-FU). As part of a programme aimed at discovering kinase inhibitors, routes to two series of novel compounds (5-fluoropyrimidine-2-carboxamides and 5-fluoropyrimidine-4-carboxamides) were successfully executed. For the first series, regioselective substitution at the 4-position of the pyrimidine with an amine (HNR¹R²) was achieved, followed by preparation of the amide at the 2-position. The route to the second series involved introduction of the methoxy protecting group at the 4-position, which allowed subsequent amine substitution to occur at the 2-position. The 4-amide substituent was finally introduced by direct conversion of the 4-methoxy into a 4-chloro group followed by transformation into an amide by palladium catalysis.     

Insight into catalytically relevant correlated motions in human purine nucleoside phosphorylase

The catalytic site of the homotrimeric enzyme human purine nucleoside phosphorylase enzyme (hPNP) features residue F200 and the 241-265 loop directly skirting the purine base and a residue belonging to the adjacent monomer, F159, immediately conterminous to the ribosyl moiety. Crystallographic B-factors of apo human purine nucleoside phosphorylase, and hPNP complexed with substrate/transition state (TS) analogues, show that residue E250 is the centroid of a highly mobile loop region. Furthermore, superimposition of apo hPNP and hPNP complexed with TS analogue Immucillin-H shows a tightening of the active site, caused by the ligand-dependent 241-265 loop rearrangement taking place upon substrate/inhibitor binding, suggesting a putative dynamic role of the loop in binding/catalysis. However, crystallographic structures reveal only average atomic positions, and more detailed information is needed to discern the dynamic behavior of hPNP. The Essential Dynamics (ED) method is used here to investigate the existence of correlated motions in hPNP and consequently proposes mutagenesis assays to estimate the relative importance of these motions in the phosphorolytic efficiency of the reaction catalyzed by hPNP. We compare the concerted motions obtained from multiple molecular dynamics simulations of apo and Michaelis complex of hPNP both in vacuo and in solution. The results of the principal component analysis for the apo hPNP indicate the existence of strong correlations predominantly in the vicinity of residue F159. However, for the Michaelis complex, concerted motions are seen mostly around both active site residue F200 and loop residue E250. Additionally, for a simulation depicting the relaxation of tight complexed hPNP with a TS analogue, toward its relaxed apo form (after removal of the TS analog), a combination of the apo hPNP and Michaelis complex motions is found, with prominent concerted modes centered around neighboring residues F159, F200, and E250. Finally, we probed the extent to which these concerted motions bear an intrinsic catalytic role by performing experimental site-directed mutagenesis on some residues, followed by kinetic analysis. The F159G and F200G mutants displayed a strong increase in K(M) and modest decrease in k(cat), suggesting that these concerted motions may provide dynamical roles in substrate binding and/or catalysis. However, further structural data for the hPNP mutants are needed to confirm our hypothesis.     

Immobilized purine nucleoside phosphorylase from Schistosoma mansoni for specific inhibition studies

The parasite Schistosoma mansoni (Sm) depends exclusively on the salvage pathway for its purine requirements. The enzyme purine nucleoside phosphorylase (PNP) is, therefore, a promising target for development of antischistosomal agents and an assay for screening of inhibitors. To enable this, immobilized SmPNP reactors were produced. By quantification of hypoxanthine by liquid chromatography, kinetic constants (K _M) for the substrate inosine were determined for the free and immobilized enzyme as 110 ± 6.90 μmol?L ^?1 and 164 ± 13.4 μmol?L ^?1 , respectively, indicating that immobilization did not affect enzyme activity. Furthermore, the enzyme retained 25 % of its activity after four months. Non-Michaelis kinetics for the phosphate substrate, and capacity for P_i-independent hydrolysis were also demonstrated, despite the low rate of enzymatic catalysis. Use of an SmPNP immobilized enzyme reactor (IMER) for inhibitor-screening assays was demonstrated with a small library of 9-deazaguanine analogues. The method had high selectivity and specificity compared with screening by use of the free enzyme by the Kalckar method, and furnished results without the need for verification of the absence of false positives.

Facile one-pot synthesis of 5-substituted hydantoins

5-Substituted and 5,5-disubstituted hydantoins are synthesised from the corresponding aldehydes or ketones, using a one-pot, gallium(III) triflate-catalysed procedure that is compatible with a range of substrates and solvents.     

Practical synthesis of novel purine analogues as Hsp90 inhibitors

The development of a straightforward synthesis of 4-amino-6-benzyl-6H-pyrrolo[3,4-d]pyrimidine and 7-amino-2-benzyl-2H-pyrazolo[4,3-d]pyrimidine derivatives allowed for the preparation of a small family of potential Hsp90 inhibitors. Some of the newly synthesized compounds showed Hsp90 inhibitory activity in preliminary biological assays.     

Nonclassical 5-substituted tetrahydroquinazolines as potential inhibitors of thymidylate synthase

Classical inhibitors of thymidylate synthase such as N^l0-propargyl-5,8-dideazafolic acid (1), N-(5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N-methylamino]-2-thenoyl)-L-glutamic acid (ZD1694, 2) and N-[2-amino-4-oxo-3,4-dihydro(pyrrolo[2,3-d]pyrintidin-5-yl)ethylbenzoyl]-L-glutamic acid (LY231514, 3) while potent, suffer from a number of potential disadvantages, such as impaired uptake due to an alteration of the active transport system required for their cellular uptake, as well as formation of long acting, non-effluxing polyglutamates via the action of folylpolyglutamate synthetase, which are responsible for toxicity. To overcome some of the disadvantages of classical inhibitors, there has been considerable interest in the synthesis and evaluation of nonclassical thymidylate synthase inhibitors, which could enter cells via passive diffusion. In an attempt to elucidate the role of saturation of the B-ring of non-classical, quinazoline antifolate inhibitors of thymidylate synthase, analogues 7-17 were designed. Analogues 13-17 which contain a methyl group at the 7-position, were synthesized in an attempt to align the methyl group in an orientation which allows interaction with tryptophan-80 in the active site of thymidylate synthase. The synthesis of these analogues was achieved via the reaction of guanidine with the appropriately substituted cyclohexanone-ketoester. These ketoesters were in turn synthesized via a Michael addition of the appropriate thiophenol with 2-carbethoxycyclohexen-1-one or 5-methyl-2-carbethoxycyclo-hexen-1-one to afford a mixture of diastereomers. The most inhibitory compound was the 3,4-dichloro, 7-methyl derivative 17 which inhibited the Escherichia coli and Pneumocystis carinii thymidylate syntheses 50% at 5 × 10⁵ M. Our results confirm the importance of the 7-CH₃ group and electron withdrawing groups on the aromatic side chain for thymidylate synthase inhibition.     

Facile synthesis of 4-substituted butenolides from furan

A variety of 4-substituted butenolides were prepared utilizing 2-acetoxyfuran as a key intermediate which can be easily synthesized by anodic oxidation of furan.     

Facile synthesis of chrysin-derivatives with promising activities as aromatase inhibitors

Flavones such as chrysin show structural similarities to androgens, the substrates of human aromatase, which converts androgens to estrogens. Aromatase is a key target in the treatment of hormone-dependent tumors, including breast cancer. Flavone-based aromatase inhibitors are of growing interest, and chrysin in particular provides a (natural) lead structure. This paper reports multicomponent synthesis as a means for facile modification of the chrysin core structure in order to add functional elements. A Mannich-type reaction was used to synthesize a range of mono- and disubstituted chrysin derivatives, some of which are more effective aromatase inhibitors than the benchmark compound, aminoglutethimide. Similarly, the reaction of chrysin with various isonitriles and acetylene dicarboxylates results in a new class of flavone derivatives, tricyclic pyrano-flavones which also inhibit human aromatase. Multicomponent reactions involving flavones therefore enable the synthesis of a variety of derivatives, some of which may be useful as anticancer agents.     

Synthesis of N-aryl spiro-sulfamides as potential glycogen phosphorylase inhibitors

A new C-glucosylated spiro-sulfamide has been prepared and evaluated toward glycogen phosphorylase inhibition. The synthesis was carried out successfully by nucleophilic displacement of 1-O-tosyl or 1-deoxy-1-iodo-α-d-gluco-hept-2-ulopyranose tetra-O-benzylated derivative using aryl amines, followed by the formation of the corresponding cyclic sulfamide.     

Designer gene therapy using an Escherichia coli purine nucleoside phosphorylase/prodrug system

Activation of prodrugs by Escherichia coli purine nucleoside phosphorylase (PNP) provides a method for selectively killing tumor cells expressing a transfected PNP gene. This gene therapy approach requires matching a prodrug and a known enzymatic activity present only in tumor cells. The specificity of the method relies on avoiding prodrug cleavage by enzymes already present in the host cells or the intestinal flora. Using crystallographic and computer modeling methods as guides, we have redesigned E. coli PNP to cleave new prodrug substrates more efficiently than does the wild-type enzyme. In particular, the M64V PNP mutant cleaves 9-(6-deoxy-alpha-L-talofuranosyl)-6-methylpurine with a kcat/Km over 100 times greater than for native E. coli PNP. In a xenograft tumor experiment, this compound caused regression of tumors expressing the M64V PNP gene.     

Facile synthesis of 4-substituted pyridines using grignard reagents

N-t-Butyldimethylsilylpyridinium triflate $\text{2}$ reacted with Grignard reagents at 4-position with almost complete regioselectivity ($\text{>}$99%) to afford the corresponding 1,4-dihydropyridine derivatives ($\text{3}$), which are easily oxidized by oxygen to give 4-substituted pyridines ($\text{4}$: 58 – 70%).