Pharmacokinetics and toxicity of the novel oral demethylating agent zebularine in laboratory and tumor bearing dogs.

The purpose of this study was to determine the plasma pharmacokinetics (PK) and toxicity of zebularine, an oral cytidine analog with demethylating activity, in dogs. Plasma zebularine concentrations were determined by HPLC-MS/MS following an oral zebularine dose of 8 or 4 mg kg-1 . Plasma zebularine clearance was constant. Mean maximum concentration (Cmax ) was 23 ± 4.8 and 8.6 ± 1.4 µM following 8 and 4 mg kg-1 , respectively. Mean half-life was 5.7 ± 0.84 and 7.1 ± 2.1 following 8 and 4 mg kg-1 , respectively. A single 8 mg kg-1 dose was well tolerated. Daily 4 mg kg-1 treatment in three laboratory dogs resulted in grade 4 neutropenia (n = 3), grade 1 anorexia (n = 2) and grade 1 or 2 dermatologic changes (n = 2). All adverse events resolved with supportive care. A 4 mg kg-1 dose every 21 days was well tolerated. A follow-up dose escalation study is in progress with a lower starting dose.

Antitumor activity and biochemical effects of cyclopentenyl cytosine in mice.

Cyclopentenyl cytosine, a recently synthesized inhibitor of cytidine 5'-triphosphate synthesis, has marked antitumor activity. Treatment with 1 mg/kg i.p. on days 1-9 following inoculation with tumor produced 111-122% increased median life span in mice bearing L1210 leukemia, 73-129% increased median life span in mice bearing P388 leukemia, and 58-62% increased median life span in mice with B16 melanoma. A subline of L1210 selected for resistance to 1-beta-D-arabinofuranosylcytosine was more sensitive to cyclopentenyl cytosine than the parent tumor line. L1210 cell growth in cultures was greatly inhibited (greater than 90%) by 0.1 microM cyclopentenyl cytosine, but cells were protected from the growth inhibitory effects by cytidine (20 microM) and to a lesser extent by uridine or deoxycytidine. Exposure of cultured L1210 cells to 1 microM cyclopentenyl cytosine inhibited formation of [3H]cytidine nucleotides from [3H]uridine by 30% during the first 15 min of exposure to drug and by greater than 95% after 2 h of exposure. Treatment of mice bearing L1210 ascites with cyclopentenyl cytosine (1 mg/kg) produced rapid depletion of cytidine nucleotide pools in the tumor cells; these pools fell to 35% of control within 30 min. The effects of cyclopentenyl cytosine on nucleotide pools were tissue selective; the cytidine nucleotide pools of spleen, liver, kidney, and intestine were less sensitive than that of the L1210 ascites tumor. Cytidine nucleotide pools of spleen and liver were depleted by higher doses (10 mg/kg) of cyclopentenyl cytosine.

The transition from a pharmacophore-guided approach to a receptor-guided approach in the design of potent protein kinase C ligands.

The pharmacophore-guided approach used in the first phase of the design of novel protein kinase C (PKC) ligands was based on the study of the geometry of bioequivalent pharmacophores present in diacylglycerol (DAG) and in the more potent phorbol ester tumor promoters. A number of potent DAG lactones were generated by this approach, in which the glycerol backbone was constrained into various heterocyclic rings to reduce the entropic penalty associated with DAG binding. Based on the information provided by X-ray and NMR structures of the cysteine-rich, C1 phorbol ester/DAG binding domain, the DAG lactones were further modified to optimize their interaction with a group of highly conserved hydrophobic amino acids along the rim of the C1 domain. This receptor-guided approach culminated with the synthesis of a series of "super DAG" molecules that can bind to PKC with low nanomolar affinities. These compounds provide insight into the basis for PKC ligand specificity. Moreover, some of the compounds reviewed herein show potential utility as antitumor agents.

Mechanism of inhibition of DNA (cytosine C5)-methyltransferases by oligodeoxyribonucleotides containing 5,6-dihydro-5-azacytosine.

A key step in the predicted mechanism of enzymatic transfer of methyl groups from S-adenosyl-l-methionine (AdoMet) to cytosine residues in DNA is the transient formation of a dihydrocytosine intermediate covalently linked to cysteine in the active site of a DNA (cytosine C5)-methyltransferase (DNA C5-MTase). Crystallographic analysis of complexes formed by HhaI methyltransferase (M.HhaI), AdoMet and a target oligodeoxyribonucleotide containing 5-fluorocytosine confirmed the existence of this dihydrocytosine intermediate. Based on the premise that 5,6-dihydro-5-azacytosine (DZCyt), a cytosine analog with an sp3-hybridized carbon (CH2) at position 6 and an NH group at position 5, could mimic the non-aromatic character of the cytosine ring in this transition state, we synthesized a series of synthetic substrates for DNA C5-MTase containing DZCyt. Substitution of DZCyt for target cytosines in C-G dinucleotides of single-stranded or double-stranded oligodeoxyribonucleotide substrates led to complete inhibition of methylation by murine DNA C5-MTase. Substitution of DZCyt for the target cytosine in G-C-G-C sites in double-stranded oligodeoxyribonucleotides had a similar effect on methylation by M. HhaI. Oligodeoxyribonucleotides containing DZCyt formed a tight but reversible complex with M.HhaI, and were consistently more potent as inhibitors of DNA methylation than oligodeoxyribonucleotides identical in sequence containing 5-fluorocytosine. Crystallographic analysis of a ternary complex involving M.HhaI, S-adenosyl-l-homocysteine and a double-stranded 13-mer oligodeoxyribonucleotide containing DZCyt at the target position showed that the analog is flipped out of the DNA helix in the same manner as cytosine, 5-methylcytosine, and 5-fluorocytosine. However, no formation of a covalent bond was detected between the sulfur atom of the catalytic site nucleophile, cysteine 81, and the pyrimidine C6 carbon. These results indicate that DZCyt can occupy the active site of M.HhaI as a transition state mimic and, because of the high degree of affinity of its interaction with the enzyme, it can act as a potent inhibitor of methylation.

Batch and Pulsed Fed-Batch Cultures of Aspergillus flavipes FP-500 Growing on Lemon Peel at Stirred Tank Reactor.

Aspergillus flavipes FP-500 grew up on submerged cultures using lemon peel as the only carbon source, developing several batch and pulsed fed-batch trials on a stirred tank reactor. The effect of carbon source concentration, reducing sugar presence and initial pH on exopectinase and endopectinase production, was analyzed on batch cultures. From this, we observed that the highest substrate concentration favored biomass (X max) but had not influence on the corresponding specific production (q p) of both pectinases; the most acid condition provoked higher endopectinase-specific productions but had not a significant effect on those corresponding to exopectinases; and reducing sugar concentrations higher than 1.5 g/L retarded pectinase production. On the other hand, by employing the pulsed fed-batch operation mode, we observed a prolonged growth phase, and an increase of about twofold on endopectinase production without a significant raise on biomass concentration. So, pulsed fed-batch seems to be a good alternative for obtaining higher endopectinase titers by using high lemon peel quantities without having mixing and repression problems to the system. The usefulness of unstructured kinetic models for explaining, under a theoretic level, the behavior of the fungus along the batch culture with regard to pectinase production was evident.

Synthesis of two cyclopentenyl-3-deazapyrimidine carbocyclic nucleosides related to cytidine and uridine.

The cytosine analogue of neplanocin A, cyclopentenylcytosine (CPE-C, 3), has significant antitumor and antiviral activity commensurate with the drug's ability to produce a significant depletion of cytidine triphosphate (CTP) levels that result from the potent inhibition of cytidine triphosphate synthetase. Another important antitumor agent, previously identified as a potent inhibitor of the same enzyme, is 3-deazauridine (2). The synthesis of the cyclopentenyl nucleosides 3-deaza-CPE-C (5) and 3-deaza-CPE-U (6) was undertaken in order to investigate the effects of a modified 3-deaza pyrimidine aglycon moiety on the biological activity of the parent CPE-C. These compounds were synthesized via an SN2 displacement reaction on cyclopenten-1-ol methanesulfonate (10) by the sodium salt of the corresponding aglycon. In each case, separation and characterization of the corresponding N- and O-alkylated products was necessary before final removal of the blocking groups. The target compounds were devoid of in vitro antiviral activity against the HSV-1 and human influenza viruses. Although 3-deaza-CPE-C was nontoxic to L1210 cells in culture, 3-deaza-CPE-U displayed significant cytotoxicity against murine L1210 leukemia in vitro.

trans-2,3b,4,5,7,8b,9,10-Octahydronaphtho[1,2-c:5,6-c']dipyrazole, a new orally active antiallergic compound.

The synthesis and antiallergic activity of a new heterocyclid steroidal molecule are described. Compound 1 was shown to inhibit the rat passive cutaneous anaphylaxis and its activity in this system was compared to that of disodium cromoglycate. It is orally active at a dose of 35 mg/kg (ED50) and its activity persists for up to 6 h for larger doses.

Potential central nervous system antitumor agents. Hydantoin derivatives.

Hydantoin derivatives of varying lipophilic character were prepared as nitrogen mustard carriers for CNS antitumor evaluation. Activity was studied in the murine ependymoblastoma brain tumor system. Multiple cures were observed for three of the four analogs examined. The compounds were also active in the intraperitoneal leukemia L1210 and P388 systems as well as in B16 melanoma and Lewis lung carcinoma.

Synthesis and biological activity of unsaturated carboacyclic purine nucleoside analogues.

Two new carboacyclic nucleoside analogues, 9-[4-hydroxy-3-(hydroxymethyl)-2-butenyl]adenine (6) and 9-[4-hydroxy-3-(hydroxymethyl)-2-butenyl]guanine (5), modeled on the unsaturated carbocyclic nucleoside analogue neplanocin A (2), have been synthesized and tested for antiviral activity against HSV-2 and, in the case of 6, for activity against influenza and in vitro inhibition of S-adenosylhomocysteine hydrolase. The synthesis was accomplished through the coupling of either adenine or the guanine precursor 2-amino-6-chloropurine (15) to the key intermediate 1-(benzyloxy)-2-[(benzyloxy)methyl]-4-chloro-2-butene (13). Debenzylation of the N-9 adenine adduct gave 6 directly, while the product of the debenzylation of the N-9 adduct of 15 when treated with sodium hydroxide gave the guanine analogue 5. The carboacyclic guanine analogue (5) exhibited significant antiviral activity against HSV-2 (VR = 1.5, MIC50 = 65.6 micrograms/mL), a level of activity that is superior to that of ara-A but inferior to that of acyclovir. The adenine analogue 6 was active against HSV-2 only at a very high dose; it was devoid of antiviral activity against influenza type A2, and it lacked inhibitory activity against S-adenosylhomocysteine hydrolase.

Dehydrogenase binding by tiazofurin anabolites.

Thiazole-4-carboxamide adenine dinucleotide (TAD) is the active anabolite of the new antitumor agent tiazofurin (NSC 286193). TAD is an analogue of NAD in which the nicotinamide ring has been replaced by a thiazole-4-carboxamide heterocycle. TAD putatively acts by inhibition of inosine monophosphate dehydrogenase (IMPd). In this study it is shown that TAD is a competitive inhibitor, with respect to NAD, of mammalian glutamate, alcohol, lactate, and malate dehydrogenases. TAD binds to these enzymes with 1-2 orders of magnitude less affinity than it binds to IMPd. Computer modeling studies suggest that dehydrogenase binding by TAD occurs at the regular cofactor site, the thiazole-4-carboxamide group mimicking the steric and hydrogen-bonding properties of the nicotinamide ring. Noncompetitive kinetics of TAD inhibition of the target enzyme IMPd are potentially due to a reverse order of addition of substrate and cofactor from that observed in the dehydrogenases studied here. The weaker binding of TAD to these dehydrogenases may be due to their inability to preserve a close sulfur-oxygen contact in the bound inhibitor.

Ara-tiazofurin: conservation of structural features in an unusual thiazole nucleoside.

Tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide, NSC 286193) is a C-glycosyl thiazole nucleoside with antitumor activity. Crystal structures of tiazofurin and its alpha,2'-deoxy and xylo analogues all show close contacts between the thiazole sulfur (S) and the furanose ring oxygen (O1'). These contacts have been interpreted in terms of an attractive intramolecular S-O interaction in the thiazole nucleosides. Ara-tiazofurin (2-beta-D-arabinofuranosylthiazole-4-carboxamide, ara-T) is the inactive arabinose analogue of tiazofurin. The crystal structure of ara-T is reported. This structure provides evidence for an attractive S-O interaction not seen in the other thiazole nucleosides. The conformation about the C-glycosyl bond in ara-T is such that close contacts are formed between the thiazole sulfur and both O1' and the 2'-hydroxyl oxygen O2'. This conformation is interpreted in terms of an additional attractive interaction between S and O2'. This interpretation is supported by comparison of the conformation of ara-T with those of other ara-nucleosides. These findings provide further evidence for an attractive S-O interaction in the thiazole nucleosides. Ara-T also demonstrates a second conformational feature found in these compounds: the carboxamide nitrogen remains cis to the thiazole nitrogen. Implications of these potentially constrained conformational features are discussed in terms of the mechanism of activity of tiazofurin.

Synthesis of 3-hydroxy-2- and -4-pyridone nucleosides as potential antitumor agents.

The ribo- and arabinofuranosyl nucleosides of antitumor active 2- and 4-pyridones 1a and 2a were prepared by direct condensation of the silylated bases with either 1-O-acetyl-2,3,5-tri-O-benzoyl-D-ribofuranose (4a) or 2,3,5-tri-O-benzyl-1-p-nitrobenzoyl-D-arabinofuranose (7) in the presence of trimethylsilyl triflate (Me3SiOTf). In the case of the arabinofuranosyl nucleosides, separation of the alpha and beta anomers was accomplished at the stage of O-benzyl-protected compounds (8b + 9b, and 10b + 11b) after chemical functionalization of the 3-hydroxy group of the pyridone aglycons with acetyl and benzyl groups, respectively. Deblocking of the protected ribo- and arabinofuranosyl nucleosides was performed by the standard methods. In vitro activity against P-388 cells in culture indicated that the 4-pyridone riboside 6d was the most active member of the series with a twofold lower ID50 than the parent pyridone 2a. However, this and all the other compounds tested in this series showed no activity against the in vivo model system of murine P-388 leukemia at doses ranging from 25 to 400 mg/kg qd 1-5.

Phosphonate-containing inhibitors of tyrosine-specific protein kinases.

Tyrosine-specific protein kinases (TPK) are important signal transducing enzymes involved in normal cellular growth and differentiation and have been implicated in the etiology of a number of human neoplastic processes. Efforts to develop agents which inhibit the function of these enzymes by interfering with the binding of substrate have been limited by the lack of detailed three-dimensional structural data. Many inhibitors of substrate binding share a common styrene nucleus 1 which has been postulated to function as a conformationally constrained analogue of tyrosine. In an effort to develop high-affinity compounds based on this hypothesis, a number of derivatives were synthesized in which either methylphosphonate (4a-c) or (hydroxymethyl)phosphonate (3a-c) were appended to the aromatic 4-position of styrene-containing moieties. The intent of this approach was to prepare hydrolytically stable analogues which expressed additional enzyme recognition features present during the phosphorylation of tyrosine itself. None of the analogues showed inhibitory activity up to the maximum concentration tested (1000 microM) when assayed against autophosphorylation of A-431-derived epidermal growth factor receptor (EGFR) or p56lck (autophosphorylation and transphosphorylation of rabbit muscle enolase). Additionally, a series of naphthalene-based inhibitors including (1-naphthalenylhydroxymethyl)phosphonic acid (14), its known 2-positional isomer 16, and sulfonate (19, 20) and phosphate derivatives (17, 18) were also tested under similar conditions. Only (2-naphthalenyl-hydroxymethyl)phosphonic acid (16) showed activity (IC50 = 250 microM in EGFR, in agreement with the reported literature value). These results suggest that the interaction of styrene-based inhibitors with the substrate binding domain of TPKS may not occur in a manner analogous to the interaction of tyrosine with this domain.

Conformationally locked nucleoside analogues. Synthesis of dideoxycarbocyclic nucleoside analogues structurally related to neplanocin C.

The glycon moiety of nucleosides in solution is known to exist in a rapid dynamic equilibrium between extreme northern and southern conformations as defined by the pseudorotation cycle. The concept of preparing rigid nucleoside analogues with the glycon conformation locked in one of these two extremes was tested with the synthesis of some cyclopropane-fused dideoxycarbocyclic nucleosides, similar to the well-known class of anti-HIV active dideoxynucleosides. The new compounds described here are dideoxynucleoside analogues of the fermentation product neplanocin C (6) which exhibits a typical northern geometry for its 6-oxabicyclo[3.1.0]hexane pseudosugar moiety. However, in view of the lability of the epoxide ring in this system, the equivalent cyclopropane-fused bicyclo[3.1.0]hexane system was used instead to prepare the corresponding dideoxynucleoside analogues bearing all the common bases [(+/-)-9-13]. Due to the well-documented preference of unrestricted bicyclo[3.1.0]hexane systems to exist exclusively in a boat conformation, the resulting nucleosides are structurally locked in a typical northern conformation similar to that of neplanocin C. The locked northern conformation in these nucleosides remained unchanged in solution in the 20-80 degrees C temperature range according to variable temperature 1H NMR studies. For the synthesis of these compounds, racemic trans-1-[(benzyloxy)methyl]-4-hydroxybicyclo[3.1.0]hexane [(+/-)-18] was prepared by a samarium-promoted cyclopropanation reaction with the antecedent cyclopentenol. All of the bases were incorporated under Mitsunobu conditions and converted to the desired final products following a standard methodology. Anti-HIV evaluation revealed that only the adenosine analogue (+/-)-9 possessed enough activity to warrant resolution into its optical antipodes. This was realized by chiral HPLC chromatography to give the individual enantiomers (-)-32 and (+)-33. Adenosine deaminase was used to identify isomer (+)-33 as the enantiomer with the "natural" configuration which was solely responsible for the observed biological activity and toxicity of (+/-)-9. It is possible that the exclusive northern conformation adopted by these nucleosides reduces their substrate affinity for the various activating kinases, except in the case of the adenosine analogue.

Protein kinase C. Modeling of the binding site and prediction of binding constants.

A detailed examination of the mode of binding of phorbol esters to protein kinase C led to a model of the phorbol binding site in the enzyme. The efficacy with which various synthetic diacylglycerol analogs and ribonolactones are able to bind to this site was determined by means of semiempirical quantum mechanical calculations using PM3, and an estimate of the binding energy was made in each case. Sixteen synthetic analogs of 1,2-diacylglycerol and two natural products were studied, and their calculated energies of binding to this model were correlated with the measured Ki values. The binding energies calculated for this receptor model, together with solubility and entropy considerations, allow prediction through regressive fit of free energies of binding which correlate very well with the measured binding constants.

cycloSal-Pronucleotides of 2'-fluoro-ara- and 2'-fluoro-ribo-2',3'- dideoxyadenosine as a strategy to bypass a metabolic blockade.

Novel, lipophilic cycloSal triesters 4a-c and 5a-c were synthesized, respectively, from the ara- and ribo-configurated 2'-fluorinated-2', 3'-dideoxyadenosines 2 and 3. The cycloSal phosphotriesters were used as tools to study the effects of the two different sugar pucker conformations induced by two opposite configurations of the fluorine substituent at C2' of the dideoxyribose moiety. F-ara-ddA (2) is known to be an active anti-HIV agent, whereas the ribo-analogue 3 is inactive. Hydrolysis studies with the triester precursors 4a-c and 5a-c showed selective formation of the monophosphates of 2 and 3. The lipophilicity of the triester prodrugs was considerably increased by the cycloSal mask with respect to ddA (1), F-ara-ddA (2), and F-ribo-ddA (3). Phosphotriesters 4 and 5 proved to be completely resistant to ADA and AMPDA deamination. In parallel experiments, ribo-nucleoside 3 showed a 50-fold faster deamination rate relative to the ara-analogue 2. Against HIV in CEM cells, the phosphotriesters 4 proved to be 10-fold more potent than the parent nucleoside 2. Furthermore, the prodrugs 4 were active against MSV-induced transformation of C3H/3T3 fibroblasts, while 2 was inactive. More interestingly, the ribo-configurated phosphotriesters 5, prepared from the inactive F-ribo-ddA (3), showed a level of anti-HIV activity that was even higher than that of F-ara-ddA (2). Our findings clearly prove that the application of the cycloSal-pronucleotide concept to F-ribo-ddA (3) overcomes a metabolic blockade in the formation of the corresponding monophosphate.

Synthesis of pyrimidin-2-one nucleosides as acid-stable inhibitors of cytidine deaminase.

One of the problems encountered in the use of tetrahydrouridine (THU, 2) and saturated 2-oxo-1,3-diazepine nucleosides as orally administered cytidine deaminase (CDA) inhibitors is their acid instability. Under acid conditions these compounds are rapidly converted into inactive ribopyranoside forms. A solution this problem was sought by functionalizing the acid-stable but less potent CDA inhibitor 1-beta-D-ribofuranosyl-2(1H)-pyrimidinone (1) with the hope of increasing its potency to the level achieved with THU. The selection of the hydroxymethyl substituent at C-4, which led to the synthesis of 4-(hydroxymethyl)-1-beta-D-ribofuranosyl-2(1H)-pyrimidinone (10), 3,4-dihydro-4-(hydroxymethyl)-1-beta-D-ribofuranosyl-2(1H)-pyrimidinone (7), and 3,4,5,6-tetrahydro-4-(dihydroxymethyl)-1-beta-D-ribofuranosyl-2(1H)-p yrimidinone (28) was based on the transition-state (TS) concept. The key intermediate precursor, 4-[(benzoyloxy)methyl]-1-(2,3,5-tri-O-benzoyl-beta-D-ribofuranosyl)-2(H) -pyrimidinone (24), was obtained via the classical Hilbert-Johnson reaction between 2-methoxy-4-[(benzoyloxy)methyl]pyrimidine (20) and 2,3,5-tri-O-benzoyl-1-D-ribofuranosyl bromide (21). Deprotection of 24 afforded compound 10, while its sodium borohydride reduction products afforded compounds 7 and 28 after removal of the blocking groups. Syntheses of 3,4-dihydro-1-beta-D-ribofuranosyl-2(1H)-pyrimidinone (9) and 3,6-dihydro-1-beta-D-ribofuranosyl-2(1H)-pyrimidinone (8), which lack the hydroxymethyl substituent, was accomplished in a similar fashion. The new compounds bearing the hydroxymethyl substituent were more acid stable than THU, and their CDA inhibitory potency, expressed in terms of Ki values, spanned from 10(-4) to 10(-7) M in a manner consistent with the TS theory. Compound 7, in particular, was superior to its parent 1 and equipotent to THU (Ki = 4 X 10(-7) M) when examined against mouse kidney CDA. The superior acid stability of this compound coupled to its potent inhibitory properties against CDA should provide a means of testing oral combinations of rapidly deaminated drugs, viz. ara-C, without the complications associated with the acid instability of THU.

Furanose-pyranose isomerization of reduced pyrimidine and cyclic urea ribosides.

Tetrahydrouridine (THU, 2) and other fully reduced cyclic urea ribofuranosyl nucleosides undergo a rapid, acid-catalyzed isomerization to their more stable ribopyranosyl form. This isomerization is characterized by a change in spectral properties and by a greater than 10-fold decrease in potency for those nucleosides that act as potent inhibitors of cytidine deaminase in their ribofuranose form. 1-(beta-D-Ribopyranosyl)hexahydropyrimidin-2-one (7) was synthesized and used in conjunction with its furanose isomer 6 as a model compound for more extensive 1H and 13C NMR, mass spectral, and kinetic studies of this isomerization. The 0.4 delta upfield shift and 4-Hz increase in the J1',2' coupling constant for the pyranose anomeric proton in the 1H NMR spectrum is indicative of a pyranose beta-CI conformation in which the aglycon and C-2' and C-4' hydroxyls are equatorial. The mass spectra of trimethylsilylated pyranose nucleosides also show a characteristic large shift in the m/z 204-217 abundance and the appearance of two new rearrangement ions at M-133 and M-206. For furanose 6 the rate of isomerization is pH and temperature dependent with pyranose 7 predominating by a factor of 6-9 equilibrium. At pH 1 and 37 degrees C, furanose 6 has an initial half-life of less than 12 min. Accordingly, this isomerization may explain the observed lack of enhanced ara-C levels in studies evaluating the oral administration of an ara-C and THU combination to species with an acidic stomach content.

Conformationally constrained analogues of diacylglycerol. 13. Protein kinase C ligands based on templates derived from 2,3-dideoxy-L-erythro(threo)-hexono-1,4-lactone and 2-deoxyapiolactone.

In the present investigation, the last two possible modes of generating conformationally semirigid diacylglycerol (DAG) analogues embedded into five-membered ring lactones as templates III and IV are investigated. The first two templates studied in previous investigations corresponded to 2-deoxyribonolactone (template I) and 4,4-disubstituted gamma-butyrolactone (template II), with the latter producing potent protein kinase C (PK-C) ligands with low nanomolar binding affinities. The templates reported in this work correspond to 2,3-dideoxy-L-erythro- or -threo-hexono-1,4-lactone (template III) and 2-deoxyapiolactone (template IV). Compounds constructed with the dideoxy-L-erythro- or -threo-hexono-1,4-lactone template were synthesized stereospecifically from tri-O-acetyl-L-glucal and L-galactono-1,4-lactone, respectively. Compounds constructed with the 2-deoxyapiolactone template were synthesized stereoselectively from di-O-isopropylidene-alpha-D-apiose. Inhibition of the binding of [3H]phorbol-12,13-dibutyrate to PK-C alpha showed that only the threo-isomer, 5-O-tetradecanoyl-2,3-dideoxy-L-threo-hexono-1,4-lactone (2) was a good PK-C ligand (Ki = 1 microM). The rest of the ligands had poorer affinities with Ki values between 10 and 28 microM. With these results, the order of importance of five-membered ring lactones as competent templates for the construction of semirigid DAG surrogates with effective PK-C binding affinity can be established as II >> I approximately III > IV.

Molecular modeling and site-directed mutagenesis studies of a phorbol ester-binding site in protein kinase C.

The protein kinase C (PKC) binding site used by PKC activators such as phorbol esters and diacylglycerols (DAGs) has been characterized by means of molecular modeling and site-directed mutagenesis studies. Based upon a NMR-determined solution structure of the second cysteinerich domain of PKC alpha, molecular modeling was used to study the structures of the complexes formed between the PKC receptor and a number of PKC ligands, phorbol esters, and DAGs. Site-directed mutagenesis studies identified a number of residues important to the binding of phorbol esters to PKC. Analysis of the molecular modeling and mutagenesis results allows the development of a binding model for PKC ligands for which the precise binding nature is defined. The calculated hydrogen bond energies between the protein and various ligands in this binding model are consistent with their measured binding affinities. The binding site for phorbol esters and DAGs is located in a highly conserved, hydrophobic loop region formed by residues 6-12 and 20-27. For the binding elements in phorbol esters, the oxygen at C20 contributes most to the overall binding energy, and that at C3 plays a significant role. The oxygen atom at C12 is not directly involved in the interaction between phorbol esters and PKC. Our results also suggest that the oxygens at C9 and C13 are involved in PKC binding, while the oxygen at C4 is of minimal significance. These results are consistent with known structure-activity relationships in the phorbol ester family of compounds. Comparisons with the X-ray structure showed that although the X-ray data support the results for oxygens at C3, C12, and C20 of phorbol esters, they suggest different roles for oxygens at C4, C9, and C13. Several factors which may contribute to these discrepancies are discussed.