Ivan Votruba

Acyclic nucleotide analogues: synthesis, antiviral activity and inhibitory effects on some cellular and virus-encoded enzymes in vitro.

Several N-(S)-(3-hydroxy-2-phosphonylmethoxypropyl) (HPMP) and N-(2-phosphonylmethoxyethyl) (PME) derivatives of purine bases (adenine, guanine, 2-aminoadenine, 3-deazaadenine) and cytosine inhibit the growth of various DNA viruses. PME-derivatives (PMEA, PMEG and PMEDAP) are also active against retroviruses. Both types of nucleotide analogues undergo phosphorylation by cellular nucleotide kinases to their mono- and diphosphates. The phosphorylation with crude extracts of L-1210 cells is potentiated by an ATP-regenerating system. HPMPA is phosphorylated faster than PMEA with or without the ATP-regenerating system. The HPMP and PME analogues inhibit several virus-encoded target enzymes and their cellular counterparts: (1) HSV-1 DNA polymerase is inhibited by the diphosphates of the PME series; the virus-encoded enzyme is more sensitive than HeLa DNA pol alpha and beta. PMEApp terminates the growing DNA chain; it specifically replaces dATP. HPMPApp also acts as an alternative substrate of dATP, but, in contrast with PMEApp, it permits limited chain growth. (2) Diphosphates of both series inhibit HSV-1 ribonucleotide reductase; the greatest inhibition of CDP reduction to dCDP is exhibited by HPMPApp and PMEApp. The enzyme isolated from a PMEA-resistant HSV-1 mutant proved less sensitive to PMEApp, hydroxyurea and HPMPApp. (3) Diphosphates of PME derivatives efficiently inhibit AMV(MAV) reverse transcriptase. (4) The purine HPMP and PME analogues and, even more so, their monophosphate derivatives inhibit purine nucleoside phosphorylase from L-1210 cells.

6-(Het)aryl-7-Deazapurine Ribonucleosides as Novel Potent Cytostatic Agents

A series of novel 7-deazapurine ribonucleosides bearing an alkyl, aryl, or hetaryl group in position 6 and H, F, or Cl atom in position 7 has been prepared either by Pd-catalyzed cross-coupling reactions of the corresponding protected 6-chloro-(7-halogenated-)7-deazapurine ribonucleosides with alkyl- or (het)arylorganometallics followed by deprotection, or by single-step aqueous phase cross-coupling reactions of unprotected 6-chloro-(7-halogenated-)7-deazapurine ribonucleosides with (het)arylboronic acids. Significant cytostatic effect was detected with a substantial proportion of the prepared compounds. The most potent were 7-H or 7-F derivatives of 6-furyl- or 6-thienyl-7-deazapurines displaying cytostatic activity in multiple cancer cell lines with a geometric mean of 50% growth inhibition concentration ranging from 16 to 96 nM, a potency comparable to or better than that of the nucleoside analogue clofarabine. Intracellular phosphorylation to mono- and triphosphates and the inhibition of total RNA synthesis was demonstrated in preliminary study of metabolism and mechanism of action studies.

A novel and efficient one-pot synthesis of symmetrical diamide (bis-amidate) prodrugs of acyclic nucleoside phosphonates and evaluation of their biological activities

A novel and efficient method for the one-pot synthesis of diamide (bis-amidate) prodrugs of acyclic nucleoside phosphonates, starting from free phosphonic acids or phosphonate diesters is reported. The approach from phosphonate diesters via their bis(trimethylsilyl) esters is highly convenient, eliminates isolation and tedious purification of the phosphonic acids, and affords the corresponding bis-amidates in excellent yields (83-98%) and purity. The methodology has been applied to the synthesis of the potent anticancer agent GS-9219, and symmetrical bis-amidates of other biologically active phosphonic acids. Anti-HIV, antiproliferative, and immunomodulatory activities of the compounds are discussed including the bis-amidate prodrugs 14 and 17 that exhibited anti-HIV activity at submicromolar concentrations with minimal cytotoxicity.

Synthesis and Significant Cytostatic Activity of 7-Hetaryl-7-deazaadenosines

A series of 7-aryl- and 7-hetaryl-7-deazaadenosines was prepared by the cross-coupling reactions of unprotected or protected 7-iodo-7-deazaadenosines with (het)arylboronic acids, stannanes, or zinc halides. Nucleosides bearing 5-membered heterocycles at the position 7 exerted potent in vitro antiproliferative effects against a broad panel of hematological and solid tumor cell lines. Cell cycle analysis indicated profound inhibition of RNA synthesis and induction of apoptosis in treated cells. Intracellular conversion to triphosphates has been detected with active compounds. The triphosphate metabolites showed only a weak inhibitory effect on human RNA polymerase II, suggesting potentially other mechanisms for the inhibition of RNA synthesis and quick onset of apoptosis. Initial in vivo evaluation demonstrated an effect of 7-(2-thienyl)-7-deazaadenine ribonucleoside on the survival rate in syngeneic P388D1 mouse leukemia model.

Inhibition of herpes simplex virus DNA polymerase by diphosphates of acyclic phosphonylmethoxyalkyl nucleotide analogues

The inhibition of HSV-1 DNA polymerase and HeLa DNA polymerases alpha and beta by diphosphoryl derivatives of acyclic phosphonylmethoxyalkyl nucleotide analogues was studied and compared with the inhibition by ACV-TP, araCTP, ddTTP and AZT-TP. In the series of phosphonylmethoxyethyl (PME-) derivatives of heterocyclic bases, the inhibitory effect of their diphosphates on HSV-1 DNA polymerase decreased in the order 2-amino-PMEApp (Ki = 0.03 microM) much greater than PMEGpp greater than PMEApp greater than PMETpp much greater than PMECpp much greater than n8z7PMEApp greater than PMEUpp. The diphosphate derivative of the antiherpes agent (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl) adenine (HPMPA) proved to be a relatively weak inhibitor of HSV-1 DNA polymerase (Ki = 1.4 microM). The inhibitors could be divided into three groups: (a) the diphosphoryl derivatives of acyclic nucleotide analogues (PME-type and HPMPA) and ACV-TP specifically inhibit HSV-1 DNA polymerase and DNA polymerase alpha and do not significantly inhibit DNA polymerase beta; (b) AZT-TP and ddTTP are effective only against DNA polymerase beta, and (c) araCTP inhibits all three enzymes. When dATP was omitted from the reaction mixture, the addition of HPMPApp stimulated DNA synthesis by HSV-1 DNA polymerase indicating that HPMPApp is an alternative substrate for in vitro DNA synthesis catalyzed by this enzyme.

Synthesis of carba-analogues of myoseverin by regioselective cross-coupling reactions of 2,6-dichloro-9-isopropylpurine

Abstract A series of 9-isopropylpurine derivatives bearing 4-methoxyphenyl, 4-methoxybenzyl, (4-methoxyphenyl)ethynyl and 2-(4-methoxyphenyl)ethyl groups in positions 2 and 6 were prepared as carba-analogues of antimitotic myoseverin. Cross-coupling reactions of 2,6-dichloro-9-isopropylpurine ( 1 ) with one equivalent of (4-methoxyphenyl)boronic acid or (4-methoxybenzyl)zinc chloride gave regioselectively the 6-substituted 2-chloropurines which were used for another cross-coupling reaction with a second equivalent of the organometallic reagent. The Sonogashira reaction of 1 with 4-(methoxyphenyl)ethyne gave 2,6-bis[(4-methoxyphenyl)ethynyl]-9-isopropylpurine that was hydrogenated to 2,6-bis[2-(4-methoxyphenyl)ethyl]-9-isopropylpurine. Regioselectivity of the couplings was proved by means of 1 H– 15 N HMBC experiments. 2,6-Bis[(4-methoxyphenyl)ethynyl]-9-isopropylpurine showed considerable cytostatic activity, while the other compounds were inactive.

Lasioglossins: Three Novel Antimicrobial Peptides from the Venom of the Eusocial Bee Lasioglossum laticeps (Hymenoptera: Halictidae)

Three novel structurally related pentadecapeptides, named lasioglossins, were isolated from the venom of the eusocial bee Lasioglossum laticeps. Their primary sequences were established as H‐Val‐Asn‐Trp‐Lys‐Lys‐Val‐Leu‐Gly‐Lys‐Ile‐Ile‐Lys‐Val‐Ala‐Lys‐NH2 (LL‐I), H‐Val‐Asn‐Trp‐Lys‐Lys‐Ile‐Leu‐Gly‐Lys‐Ile‐Ile‐Lys‐Val‐Ala‐Lys‐NH2 (LL‐II) and H‐Val‐Asn‐Trp‐Lys‐Lys‐Ile‐Leu‐Gly‐Lys‐Ile‐Ile‐Lys‐Val‐Val‐Lys‐NH2 (LL‐III). These lasioglossins exhibited potent antimicrobial activity against both Gram‐positive and Gram‐negative bacteria, low haemolytic and mast cell degranulation activity, and a potency to kill various cancer cells in vitro. The lasioglossin CD spectra were measured in the presence of trifluoroethanol and sodium dodecyl sulfate solution and indicated a high degree of α‐helical conformation. NMR spectroscopy, which was carried out in trifluoroethanol/water confirmed a curved α‐helical conformation with a concave hydrophobic and convex hydrophilic side. To understand the role of this bend on biological activity, we studied lasioglossin analogues in which the Gly in the centre of the molecule was replaced by other amino acid residues (Ala, Lys, Pro). The importance of the N‐terminal part of the molecule to the antimicrobial activity was revealed through truncation of five residues from both the N and C termini of the LL‐III peptide. C‐terminal deamidation of LL‐III resulted in a drop in antimicrobial activity, but esterification of the C terminus had no effect. Molecular modelling of LL‐III and the observed NOE contacts indicated the possible formation of a bifurcated H‐bond between hydrogen from the Lys15 CONH peptide bond and one H of the C‐terminal CONH2 to the Ile11 oxygen atom. Such interactions cannot form with C‐terminal esterification.

Transport of 9-(2-phosphonomethoxyethyl)adenine across plasma membrane of HeLa S3 cells is protein mediated.

9-(2-Phosphonomethoxyethyl)adenine (PMEA) is an acyclic adenine nucleotide analog which exhibits potent and selective antiviral activity against herpesviruses and retroviruses. The study of [14C]PMEA uptake in HeLa S3 cells has shown that intracellular levels of the drug plateau after 1 h. Transport across the plasma membrane is saturable (concentration at half-maximal saturation [Kt], 0.39 microM; maximum rate of uptake [Vmax], 1.72 pmol/min.10(6) cells), and it can operate against the concentration gradient. Its significant dependence on temperature and on cellular density has been demonstrated. Following the treatment of cells with proteases, PMEA uptake strongly decreases. The transport process is considerably specific, since only a few phosphonate analogs act effectively as competitive inhibitors. Of these, 9-(2-phosphonomethoxyethyl)-2,6-diaminopurine (Ki = 0.24 microM) is the most efficient. Also, natural nucleotides competitively inhibit PMEA transport, depending on the nature of the nucleobase (thymine = adenine > guanine > cytosine < uracil) and on the position and number of phosphate groups. Nucleosides and nucleobases do not interfere with PMEA uptake. Cellular transport of adenosine and thymidine or uptake of AMP and ATP via conjugated activity of ectonucleotidases and nucleoside transporters is not affected by PMEA. By using vectorial labeling of plasma membrane proteins with Na125I combined with affinity chromatography, a 50-kDa protein which may mediate cellular transport of PMEA has been identified.

SYNTHESIS AND CYTOSTATIC ACTIVITY OF NUCLEOSIDES AND ACYCLIC NUCLEOSIDE ANALOGUES DERIVED FROM 6-(TRIFLUOROMETHYL)PURINES

Abstract Glycosylation and alkylation of 6-(trifluoromethyl)purine by several protected halogenoses or hydroxyalkyl chlorides afforded regio- and stereoselectively the 9-β-nucleosides or 9-alkylated purine derivatives in good yields. Deprotection of these intermediates gave a series of nucleoside (β-D-ribofuranosyl, 2-deoxy-β-D-ribofuranosyl and β-D-arabinofuranosyl) and acyclonucleoside (2,3-dihydroxypropyl and (2-hydroxyethyl)oxymethyl) derivatives of 6-(trifluoromethyl)purine. While the ribofuranosyl derivative 1 showed significant cytostatic activity, the other derivatives were inactive.

Covalent analogues of DNA base-pairs and triplets. Part 2: synthesis and cytostatic activity of bis(purin-6-yl)acetylenes,-diacetylenes and related compounds.

The title bis(purin-6-yl)acetylenes, -diacetylenes, -ethylenes and -ethanes were prepared as covalent base-pair analogues starting from 6-ethynylpurines and 6-iodopurines by cross-coupling and homo-coupling reactions and hydrogenations. The bis(purin-6-yl)acetylenes and -diacetylenes exhibited significant cytostatic activity in vitro (IC(50)=0.4-1.0 micromol/l).