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.

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 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.

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 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.

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.

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.

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.

Selective cytotoxicity of a system L specific amino acid nitrogen mustard.

The synthesis and characterization of DL-2-amino-7-bis[(2-chloroethyl)amino]-1,2,3,4-tetrahydro-2-naphthoic acid and DL-2-amino-5-bis[(2-chloroethyl)amino]-1,2,3,4-tetrahydro-2-napthoic+ ++ acid were accomplished. The correct assignment of the site of attachment of the bis(2-chloroethyl)amino side chain was ascertained by selective proton decoupling of the 13C NMR spectra performed on the corresponding nitrospirohydantoin precursors 2 and 3, which were obtained from the nitration of beta-tetralone hydantoin. The two target compounds 6 and 7 were designed as tumor-specific agents capable of being selectively transported into tumor cells by the leucine-preferring transport system (system L). Inhibition analysis of the initial rate of transport of the system L specific substrate 2-amino-bicyclo[2.2.1]heptane-2-carboxylic acid (BCH) by 6 and 7 indicated that the 7-substituted isomer 6 was an extremely potent competitive inhibitor of that transport system in murine L1210 leukemic cells (Ki = 0.2 microM). Evaluation of the selectivity of this compound indicated that it possessed enhanced in vitro antitumor activity and reduced myelosuppressive activity when compared to its prototype amino acid nitrogen mustard, L-phenylalanine mustard (L-PAM). In addition to being more selectively toxic to tumor cells, this compound differs from L-PAM in having a 2-3-fold shorter half-life (t1/2).

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.

Potential anti-AIDS drugs. 2',3'-Dideoxycytidine analogues.

5-Substituted 2',3'-dideoxycytidine analogues have been synthesized and evaluated in vitro for their capabilities to protect T4+ lymphocytes from the cytopathic effects of the HTLV-III/LAV (HIV) virus, the causative agent of acquired immunodeficiency syndrome (AIDS). These analogues were designed to be more lipophilic than 2',3'-dideoxycytidine (ddC) in order to enhance central nervous system penetration. Earlier reports had shown that ddC is a potent protective agent. When ddC is substituted at the 5-position with either methyl or bromo substituents, activity is completely abolished. However, when the substitution is fluoro (5-F-ddC), both activity and potency are retained. 2',3'-Dideoxy-5-azacytidine is also protective but more toxic than ddC or 5-F-ddC. In a different approach, an attempt was made to utilize ddCMP, ddTMP, and ddAMP as preformed nucleotides in order to circumvent the generally low level of phosphorylation achieved with dideoxynucleosides which function as relatively poor substrates for the cellular kinases. Only ddAMP is as active as its nucleoside precursor. Because ddAMP is not more active than ddA at low concentrations, it is possible that the active agent is ddA which is generated from ddAMP prior to cell entry.

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.

Conformationally constrained analogues of diacylglycerol (DAG). 17. Contrast between sn-1 and sn-2 DAG lactones in binding to protein kinase C.

In previous work, we have obtained potent protein kinase C (PK-C) ligands with low-namomolar binding affinities by constructing diacylglycerol (DAG) mimetics in which the sn-2 carbonyl of DAG was constrained into a lactone ring. An additional structural element that helped achieve high binding affinity was the presence of branched acyl or alpha-alkylidene chains. In the present study, the effects of similarly branched chains on a different lactone system, where the lactone carbonyl is now equivalent to the sn-1 carbonyl of DAG, are investigated. In this new lactone template, the two chiral centers must have the S-configuration for enzyme recognition. As with the sn-2 DAG lactones, the branched chains were designed to optimize van der Waals contacts with a group of conserved hydrophobic amino acids located on the rim of the C1 domain of PK-C. The acyl and alpha-alkylidene chains were also designed to be lipophilically equivalent (8 carbons each). Eight new compounds (7-14) representing all possible combinations of linear and branched acyl and alpha-alkylidene were synthesized and evaluated. The sn-1 DAG lactones were less effective as PK-C ligands than the sn-2 DAG lactones despite having a similar array of linear or branched acyl and alpha-alkylidene chains