Search

Hyperactivation of mTOR kinase by mutations in the PI3K/mTOR pathway or by crosstalk with other mutant cancer drivers, such as RAS, is a feature of many tumors. Multiple allosteric inhibitors of mTORC1 and orthosteric dual inhibitors of mTORC1 and mTORC2 have been developed as anticancer drugs, but their clinical utility has been limited. To address these limitations, we have developed a novel class of "bi-steric inhibitors" that interact with both the orthosteric and the allosteric binding sites in order to deepen the inhibition of mTORC1 while also preserving selectivity for mTORC1 over mTORC2. In this report, we describe the discovery and preclinical profile of the development candidate RMC-5552 and the in vivo preclinical tool compound RMC-6272. We also present evidence that selective inhibition of mTORC1 in combination with covalent inhibition of KRAS

The discovery of reldesemtiv, a second-generation fast skeletal muscle troponin activator (FSTA) that increases force production at submaximal stimulation frequencies, is reported. Property-based optimization of high throughput screening hit 1 led to compounds with improved free exposure and in vivo muscle activation potency compared to the first-generation FSTA, tirasemtiv. Reldesemtiv demonstrated increased muscle force generation in a phase 1 clinical trial and is currently being evaluated in clinical trials for the treatment of amyotrophic lateral sclerosis.

To explore the feasibility of developing ligands targeted to the atypical C1 domains of protein kinase C ζ and ι, we have prepared diacylglycerol lactones substituted with hydrophilic groups on their side chains, which potentially could interact with the arginine residues that distinguish the atypical C1 domains of PKCζ and PKCι from typical C1 domains, and we have measured their binding to mutated versions of the C1b domain of PKCδ that incorporate one or more of these arginine residues. The most selective of the diacylglycerol lactones showed only a 10-fold reduction in binding affinity with the triple arginine mutant (N7R/S10R/L20R) compared to the wild-type, whereas phorbol 12,13-dibutyrate showed a 6000-fold loss of affinity. Molecular modeling confirms that these ligands are indeed able to interact with the arginine residues. Our results show that dramatic changes in selectivity can be obtained through appropriate substitution of diacylglycerol lactones.

We report herein the application of the phosphoramidate ProTide technology to improve the metabolism of the DNA methytransferase inhibitor, zebularine (Z). Zebularine is a riboside that must undergo a complex metabolic transformation before reaching the critical 2'-deoxyzebularine 5'-triphosphate (dZTP). Because 2'-deoxyzebularine (dZ) is not phosphorylated and therefore inactive, the ProTide strategy was employed to bypass the lack of phosphorylation of dZ and the inefficient reduction of zebularine 5'-diphosphate by ribonucleotide-diphosphate reductase required for zebularine. Several compounds were identified as more potent inhibitors of DNA methylation and stronger inducers of p16 tumor suppressor gene than zebularine. However, their activity was dependent on the administration of thymidine to overcome the potent inhibition of thymidylate synthase (TS) and deoxycytidine monophosphate (dCMP) deaminase by dZMP, which deprives cells of essential levels of thymidine. Intriguingly, the activity of the ProTides was cell line-dependent, and activation of p16 was manifest only in Cf-Pac-1 pancreatic ductal adenocarcinoma cells.

In this study, we report the SAR and characterization of two groups of isosorbide-based cholinesterase inhibitors. The first was based directly on the clinically used nitrate isosorbide mononitrate (ISMN) retention of the 5-nitrate group and introduction of a series of 2-carbamate functionalities. The compounds proved to be potent and selective inhibitors of human plasma butyrylcholinesterase ( huBuChE). In the second group, the nitrate ester was removed and replaced with a variety of alkyl and aryl esters. These generally exhibited nanomolar potency with high selectivity for BuChE over acetylcholinesterase (AChE). The most potent and selective compound was isosorbide-2-benzyl carbamate-5-benzoate with an IC 50 of 4.3 nM for BuChE and50000 fold selectivity over human erythrocyte AChE. Inhibition with these compounds is time-dependent, competitive, and slowly reversible, indicating active site carbamylation.

Diacylglycerol-lactone (DAG-lactone) libraries generated by a solid-phase approach using IRORI technology produced a variety of unique biological activities. Subtle differences in chemical diversity in two areas of the molecule, the combination of which generates what we have termed "chemical zip codes", are able to transform a relatively small chemical space into a larger universe of biological activities, as membrane-containing organelles within the cell appear to be able to decode these "chemical zip codes". It is postulated that after binding to protein kinase C (PKC) isozymes or other nonkinase target proteins that contain diacylglycerol responsive, membrane interacting domains (C1 domains), the resulting complexes are directed to diverse intracellular sites where different sets of substrates are accessed. Multiple cellular bioassays show that DAG-lactones, which bind in vitro to PKCalpha to varying degrees, expand their biological repertoire into a larger domain, eliciting distinct cellular responses.

Diacylglycerol lactones (DAG-lactones) are known to operate as effective agonists of protein kinase C (PKC), surpassing in potency the activity of natural diacylglycerol (DAG). Localization of activated PKC isozymes in the cell is determined in part by the different cellular scaffolds, the lipid composition of the specific membranes, and the targeting information intrinsic to the individual isoforms bound to DAG. This multifaceted control of diversity suggests that, to develop effective DAG-lactones capable of honing in on a specific cellular target, we need to gain a better understanding of the chemical space surrounding its binding site. Seeking to augment the chemical repertoire of DAG-lactone side chains that could steer the translocation of PKC to specific cellular domains, we report herein the effects of incorporating simple or substituted phenyl residues. A combined series of n-alkyl and phenyl substitutions were used to explore the optimal location of the phenyl group on the side chains. The substantial differences in binding affinity between DAG-lactones with identical functionalized phenyl groups at either the sn-1 or sn-2 position are consistent with the proposed binding model in which the DAG-lactone binds to the C1 domain of PKC with the acyl chain oriented toward the interior of the membrane and the alpha-alkylidene or alpha-arylalkylidene chains directed to the surface of the C1 domain adjacent to the lipid interface. We conclude that DAG-lactones containing alpha-phenylalkylidene side chains at the sn-2 position represent excellent scaffolds upon which to explore further chemical diversity.

Using as our lead structure a potent PKC ligand (1) that we had previously described, we investigated a series of branched DAG-lactones to optimize the scaffold for PKC binding affinity and reduced lipophilicity, and we examined the potential utility of select compounds as alpha-secretase activators. Activation of alpha-secretase upon PKC stimulation by ligands causes increased degradation of the amyloid precursor protein (APP), resulting in enhanced secretion of sAPPalpha and reduced deposition of beta-amyloid peptide (Abeta), which is implicated in the pathogenesis of Alzheimer's disease. We modified in a systematic manner the C5-acyl group, the 3-alkylidene, and the lactone ring in 1 and established structure-activity relationships for this series of potent PKC ligands. Select DAG-lactones with high binding affinities for PKC were evaluated for their abilities to lead to increased sAPPalpha secretion as a result of alpha-secretase activation. The DAG-lactones potently induced alpha-secretase activation, and their potencies correlated with the corresponding PKC binding affinities and lipophilicities. Further investigation indicated that 2 exhibited a modestly higher level of sAPPalpha secretion than did phorbol 12,13-dibutyrate (PDBu).

The constrained glycerol backbone of DAG-lactones, when combined with highly branched alkyl chains, has engendered a series of DAG-lactone ligands capable of binding protein kinase C (PK-C) with affinities that approximate those of phorbol esters. These branched chains not only appear to be involved in making important hydrophobic contacts with the protein (specific interactions) but also provide adequate lipophilicity to facilitate partitioning into the lipid-rich membrane environment (nonspecific interactions). With the idea of minimizing the nonspecific interactions without reducing lipophilicity, the present work explores the strategy of relocating lipophilicity from the side chain to the lactone “core”. Such a transfer of lipophilicity, exemplified by compounds 1 and 3, was conceived to allow the new hydrophobic groups on the lactone to engage in specific hydrophobic contacts inside the binding pocket without any expectation of interfering with the hydrogen-bonding network of the DAG-lactone pharmaco...

Preference for the northern (N) ring conformation of the ribose moiety of adenine nucleotide 3',5'-bisphosphate antagonists of P2Y(1) receptors was established by using a ring-constrained methanocarba (a bicyclo[3.1.0]hexane) ring as a ribose substitute (Nandanan et al. J. Med. Chem. 2000, 43, 829-842). We have now combined the ring-constrained (N)-methanocarba modification with other functionalities at the 2-position of the adenine moiety. A new synthetic route to this series of bisphosphate derivatives was introduced, consisting of phosphorylation of the pseudoribose moiety prior to coupling with the adenine base. The activity of the newly synthesized analogues was determined by measuring antagonism of 2-methylthio-ADP-stimulated phospholipase C (PLC) activity in 1321N1 human astrocytoma cells expressing the recombinant human P2Y(1) receptor and by using the radiolabeled antagonist [(3)H]2-chloro-N(6)-methyl-(N)-methanocarba-2'-deoxyadenosine 3',5'-bisphosphate 5 in a newly developed binding assay in Sf9 cell membranes. Within the series of 2-halo analogues, the most potent molecule at the hP2Y(1) receptor was an (N)-methanocarba N(6)-methyl-2-iodo analogue 12, which displayed a K(i) value in competition for binding of [(3)H]5 of 0.79 nM and a K(B) value of 1.74 nM for inhibition of PLC. Thus, 12 is the most potent antagonist selective for the P2Y(1) receptor yet reported. The 2-iodo group was substituted with trimethyltin, thus providing a parallel synthetic route for the introduction of an iodo group in this high-affinity antagonist. The (N)-methanocarba-2-methylthio, 2-methylseleno, 2-hexyl, 2-(1-hexenyl), and 2-(1-hexynyl) analogues bound less well, exhibiting micromolar affinity at P2Y(1) receptors. An enzymatic method of synthesis of the 3',5'-bisphosphate from the corresponding 3'-monophosphate, suitable for the preparation of a radiophosphorylated analogue, was explored.

The potency of nucleotide antagonists at P2Y(1) receptors was enhanced by replacing the ribose moiety with a constrained carbocyclic ring (Nandanan, et al. J. Med. Chem. 2000, 43, 829842). We have now synthesized ring-constrained methanocarba analogues (in which a fused cyclopropane moiety constrains the pseudosugar ring) of adenine and uracil nucleotides, the endogenous activators of P2Y receptors. Methanocarba-adenosine 5'-triphosphate (ATP) was fixed in either a Northern (N) or a Southern (S) conformation, as defined in the pseudorotational cycle. (N)-Methanocarba-uridine was prepared from the 1-amino-pseudosugar ring by treatment with beta-ethoxyacryloyl cyanate and cyclization to form the uracil ring. Phosphorylation was carried out at the 5'-hydroxyl group through a multistep process: Reaction with phosphoramidite followed by oxidation provided the 5'-monophosphates, which then were treated with 1,1'-carbonyldiimidazole for condensation with additional phosphate groups, The ability of the analogues to stimulate phospholipase C through activation of turkey P2Y(1) or human P2Y(1), P2Y(2), P2Y(4), P2Y(6), and P2Y(11) receptors stably expressed in astrocytoma cells was measured. At recombinant human P2Y(1) and P2Y(2) receptors, (N)-methanocarba-ATP was 138- and 41-fold, respectively, more potent than racemic (S)-methanocarba-ATP as an agonist. (N)methanocarba-ATP activated P2Y(11) receptors with a potency similar to ATP. (N)-Methanocarba-uridine 5'-triphosphate (UTP) was equipotent to UTP as an agonist at human P2Y2 receptors and also activated P2Y(4) receptors with an EC50 of 85 nM. (N)-Methanocarba-uridine 5'-diphosphate (UDP) was inactive at the hP2Y(6) receptor. The vascular effects of (N)-methanocarba-UTP and (N)-methanocarba-UDP were studied in a model of the rat mesenteric artery, The triphosphate was more potent than UTP in inducing a dilatory P2Y(4) response (pEC(50) = 6.1 +/- 0.2), while the diphosphate was inactive as either an agonist or antagonist in a P2Y(6) receptor-mediated contractile response. Our results suggest that new nucleotide agonists may be designed on the basis of the (N) conformation that favors selectivity for P2Y(1), P2Y(2), P2Y(4), and P2Y(11) receptors. (Less)

An approach to reduce the log P in a series of diacylglycerol (DAG)-lactones known for their high binding affinity for protein kinase C (PK-C) is presented. Branched alkyl groups with reduced lipophilicity were selected and combined with the replacement of the ester or lactone oxygens by NH or NOH groups. Compound 6a with an isosteric N-hydroxyl amide arm represents the most potent and least lipophilic DAG analogue known to date.

A series of 6-substituted amino analogs of 9-(2,3-dideoxy-2-fluoro-beta-D-threo-pentofuranosyl) purines (F-ddN) has been synthesized and characterized with the objective of finding compounds which might be superior to existing drugs for the treatment of HIV in the central nervous system. These compounds are intended to be more lipophilic than the currently approved anti-HIV drugs for better blood-brain barrier penetration. Subsequent adenosine deaminase (ADA)-catalyzed hydrolysis of these prodrugs in the brain is expected to produce the anti-HIV agent, 9-(2,3-dideoxy-2-fluoro-beta-D-threo-pentofuranosyl)hypoxanthine (F-ddI). The new compounds, synthesized from the corresponding 6-chloro analog, include F-ddN which contain methylamino, ethylamino, dimethylamino, hydroxylamino, methoxyamino, benzyloxyamino, hydrazino, and nitro substituents in the 6-position. The 6-nitro analog was isolated as an unexpected product during the preparation of the 6-chloro derivative. Among the analogs with anti-HIV activity, the ethylamino and dimethylamino compounds are ca. 100 times more lipophilic than ddI or F-ddI. As expected, 2'-fluoro substitution protects the compounds from acid-catalyzed glycosylic cleavage. Only the hydroxylamino and nitro analogs underwent any nonenzymatic hydrolysis at pH 1.0 or 7.4. This reaction, however, results in hydrolysis of the group in the 6-position rather than glycosylic bond cleavage. ADA catalyzes the hydrolysis of the 6-substituents at rates which vary from slightly slower (NO2, 1.7x) to much slower (NHEt, 5000x) than F-ddA. The 6-dimethylamino analog is the only compound which possesses anti-HIV activity (ED50 18 microM) without ADA hydrolysis. With the exception of the two inactive alkoxyamino compounds, the other prodrugs exhibited cellular protection in the HIV-1/PHA-PBM system with IC50 potencies of 7-40 microM.

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 III approximately IIIIV.

Conformationally constrained analogues of diacylglycerol (DAG) built on a racemic 5(-)[(acyloxy)-methyl]-5-(hydroxymethyl)tetrahydro-2-furanone template were shown previously to have excellent binding affinities for protein kinase C (PK-C). Since the interaction of PK-C with DAG is stereospecific, it was anticipated that PK-C would bind tightly to only one enantiomeric form of the compounds constructed with this new lactone template. Separation of enantiomers by chiral HPLC was discarded due to the ease with which acyl migration occurs in these class of compounds, and a total chiral synthesis was undertaken. Prior to chemical synthesis, the selection of the "correct" enantiomeric template was predicted by a molecular conformational analysis that compared the two enantiomers of DAG in their presumed "active" conformation with the two enantiomeric lactone templates. This presumed "active" conformation for DAG was derived from a previously developed pharmacophore model that uses the molecule of a potent phorbol diester as the ideal rigid template. The results from this analysis indicated that the "correct" lactone template corresponded to the inactive (R)-isomer of DAG. This analysis also predicted that the lactone template corresponding to the active (S)-DAG enantiomer would not fit adequately into the pharmacophore. The chiral syntheses of target compounds 2, 4, and 6, constructed on the selected, and presumably "correct" lactone template, were achieved from a common bicyclic intermediate (5R,8R,9R)-8,9-O-isopropylidene-2-keto-1,7-dioxaspiro[4.4]nonane (10) that was synthesized from commercially available 1,2:3,5-di-O-isopropylidene-alpha-D-threo-apiofuranose (7) by a very effective spirolactonization approach. On the basis of their ability to inhibit the binding of [3H-20]phorbol 12,13-dibutyrate (PDBU) to PK-C alpha, the enantiomeric ligands 2, 4, and 6 were twice as potent as the corresponding racemates. These results confirm that binding of these lactones is stereospecific and consistent with a binding mechanism similar to that of DAG.

The sugar moiety of nucleosides in solution is known to exist in a rapid dynamic equilibrium between extreme Northern and Southern conformations as defined in the pseudorotational cycle. In the present work, we describe how the bicyclo[3.1.0]hexane template fixes the ring pucker of 2‘-deoxy-methanocarba-nucleosides 1−5 and 12 to values corresponding to either one of these two extreme conformations that are typical of nucleosides. The syntheses of the fixed Northern conformers 1−5 were performed by Mitsunobu coupling of the heterocyclic bases with the chiral carbocyclic alcohol 6 [(1R,2S,4R,5S)-1-[(benzyloxy)methyl]-2-(tert-butyloxy)-4-hydroxybicyclo[3.1.0]hexane], while the synthesis of the Southern conformer, (S)-methanocarba-T (12), was reported earlier. Carbocyclic thymidine (carba-T, 13) was used as a reference, flexible carbocyclic nucleoside. Antiviral evaluation of these compounds revealed a very potent antiherpetic activity associated with the Northern thymidine analogue 2, which was more powerful...

Conformationally constrained analogues of diacylglycerol (DAG) built on a 5(-)[(acyloxy)methyl]-5-(hydroxymethyl)tetrahydro-2-furanone template (1, Chart 1) were shown previously to bind tightly to protein kinase C alpha (PK-C alpha) in a stereospecific manner. These compounds, however, racemized readily through rapid acyl migration and lost biological potency. In order to circumvent this problem, the "reversed ester" analogues were designed as a new set of PK-C ligands. This reversal of the ester function produced some new DAG mimetics that are embedded in a C-4 doubly-branched heptono-1,4-lactone template. The reversed ester analogues were impervious to racemization, and their chemically distinct branches facilitated the enantiospecific syntheses of all targets. Compound 2, the simplest reversed ester analogue of 1 (Chart 1), exhibited a 3.5-fold reduction in binding affinity toward PK-C alpha which we attributed to the loss of a stabilizing gauche interaction that caused the ester branch in 2 to be more disordered than in the normal ester 1. However, conversion of the propanoyl branch of 2 into a propenoyl branch restored binding affinity (3 versus 5). As expected, the compounds bound to the enzyme with strict enantioselectivity (3 and 5 versus 4 and 6). Functionalization of the propenoyl-branched compounds as alpha-alkylidene lactones, in a manner which proved successful with the 5(-)[(acyloxy)methyl]-5-(hydroxymethyl)tetrahydro-2-furanone template (9 and 10), produced stable compounds with equivalent ultrapotent binding affinities for PK-C alpha (7 and 8). The additional incorporation of the propenoyl-branched carbonyl into a gamma-lactone ring was performed (11-14) not only to derive a possible additional entropic advantage but also to confirm the spatial disposition of this carbonyl function in the ligand-enzyme complex. Although no additional entropic advantage was derived, the high binding affinities displayed by compounds 11 and 12 helped to establish the correct orientation of the equivalent carbonyl group in PK-C-bound DAG. As expected, these DAG analogues activated PK-C alpha. The most potent agonist, compound 8, stimulated phosphorylation of the alpha-pseudosubstrate peptide, and in primary mouse keratinocytes it caused inhibition of binding of epidermal growth factor with an ED50 of approximately 1 microM. In contrast to the phorbol esters, compound 8 did not induce acute edema or hyperplasia in skin of CD-1 mice, and its pattern of downregulation with several PK-C isozymes was different from that of phorbol 12-myristate 13-acetate (PMA).

A series of 6-halo-(F-, Cl-, Br-, I-) and 6-alkoxy-(OMe-, OEt-) 9-(2,3-dideoxy-2-fluoro-beta-D-threopentofuranosyl) purines (F-ddN) have been synthesized and characterized with the objective of finding compounds which might be superior to existing drugs for the treatment of HIV in the central nervous system. These compounds, which contain lipophilic 6-substituents, were chosen as acid-stable prodrugs for the anti-HIV-active F-ddN, 9-(2,3-dideoxy-2-fluoro-beta-D-threo-pentofuranosyl) hypoxanthine (F-ddI), because of their potential to increase blood-brain-barrier penetration relative to F-ddI. All the new compounds were more lipophilic than the currently approved anti-AIDS drugs. Partition coefficient increases of 30- and 110-fold were achieved, relative to didanosine (ddI), for the 6-chloro- and 6-ethoxy analogues. 2'-Fluoro substitution abolished the pH 1, acid-catalyzed cleavage of the nucleoside glycosylic bond. However, pH 1, acid-catalyzed hydrolysis of the 6-fluoro substituent to produce F-ddI was observed to occur at a rate (t1/2 0.54 h) which was ca. 40-170 times faster than that of the other prodrugs. The utility of the F-ddNs as prodrugs for F-ddI depends upon their ability to act as substrates for adenosine deaminase. The relative rates of adenosine deaminase-catalyzed prodrug hydrolysis to F-ddI varied by a factor of > 25,000 with the 6-fluoro- and 6-ethoxy analogues reacting the fastest and slowest, respectively. All of the prodrugs possessed anti-HIV activity in the phytohemagglutinin-stimulated peripheral blood mononuclear cell test system and a qualitative correlation exists between prodrug anti-HIV activity and adenosine deaminase hydrolysis rates.

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.

Isosorbide-2-benzyl carbamate-5-benzoate is a highly potent and selective BuChE inhibitor. Meanwhile, isosorbide-2-aspirinate-5-salicylate is a highly effective aspirin prodrug that relies on the salicylate portion to interact productively with human BuChE. By integrating the salicylate group into the carbamate design, we have produced isosorbide-2-benzyl carbamate-5-salicylate, an inhibitor of high potency (150 pM) and selectivity for human BuChE over AChE (666000) and CES2 (23000). Modeling and mutant studies indicate that it achieves its exceptional potency because of an interaction with the polar D70/Y332 cluster in the PAS of BuChE in addition to pseudosubstrate interactions with the active site.

The synthesis, chemistry, biochemistry, and anti-HIV activity of a series of 1-(2,3-dideoxy-2-fluoro-beta-D-threopentofuranosyl)pyrimidines have been studied in an attempt to find useful anti-AIDS drugs. Synthesis is carried out via a 2,3-dideoxyribose intermediate which facilitates the preparation of analogues by removing the sugar 3'-hydroxyl group prior to, rather than after, condensation with a uracil or cytosine aglycon. The 2'-F-dd-uridine analogues 7a-d (with H, F, Cl, and CH3 substitution in the 5-position) as well as the 4-deoxy compound (12b) are nonprotective to ATH8 or CEM cells infected with HIV-1. In the corresponding cytidine series, the 5-chloro analogue (11) is inactive. However, 2'-fluoro-2',3'-dideoxyarabinosylcytosine, 10a, and its 5-fluoro analogue, 10b, are both active. While neither compounds is a potent as ddC or 5-F-ddC (2b), 10b gives complete protection against the cytopathic effects of HIV in both host cell lines. 2'-Fluoro substitution confers increased chemical and enzymatic stability on dideoxynucleosides. Even though dideoxy pyrimidine nucleosides are inherently more stable than the corresponding purine analogues toward acid-catalyzed cleavage of the glycosidic bond, 2'-fluoro substitution (10a) still increases stabilization relative to ddC (2b). No detectable deamination by partially purified cytidine deaminase is observed with the 2'-fluoro compounds 10a, 10b, or 11 under conditions which rapidly deaminate cytidine. A small amount of 2'-F-dd-ara-U (7a) is formed from 10a in monkey plasma after greater than 24 h of exposure. The octanol-water partition coefficients for the dideoxynucleosides in this study indicate their hydrophilic character, with log P values varying from -0.28 to -1.18.

A major pathway for HIV-1 resistance to nucleoside reverse transcriptase inhibitors (NRTIs) involves reverse transcriptase (RT) mutations that enhance ATP-dependent pyrophosphorolysis, which excises NRTIs from the end of viral DNA. We analyzed novel NRTIs for their ability to inhibit DNA synthesis of excision-proficient HIV-1 RT mutants. D-carba T is a carbocyclic nucleoside that has a 3′ hydroxyl on the pseudosugar. The 3′ hydroxyl group allows RT to incorporate additional dNTPs, which should protect D-carba TMP from excision. D-carba T can be converted to the triphosphate form by host cell kinases with moderate efficiency. D-carba T-TP is efficiently incorporated by HIV-1 RT; however, the next dNTP is added slowly to a D-carba TMP at the primer terminus. D-carba T effectively inhibits viral vectors that replicate using NRTI-resistant HIV-1 RTs, and there is no obvious toxicity in cultured cells. NRTIs based on the carbocyclic pseudosugar may offer an effective approach for the treatment of HIV-1 infections.

Glucocorticoids are used in the treatment of inflammatory bowel disease. A limitation to their use is that they undergo absorption from the GIT before reaching the colon causing severe systemic side effects. We report here on a novel prodrug approach to targeting corticosteroids to the colon. The design involves attaching a 21-ester group that suppresses absorption during transit to the colon. The prodrug is designed to be primed by colonic microflora liberating an amino ester that cyclizes releasing the steroid. One of the prodrugs 5b was as efficacious as prednisolone in the murine DSS model but did not cause thymic atrophy, a marker for systemic steroid effects.

2(1H)-Pyrimidinone riboside (zebularine, 1b) and its 5-fluoro (6b) and 2'-ara-fluoro (7b) analogues have been synthesized and evaluated in vivo as antitumor agents. Zebularine provides increase in life span (ILS) values of ca. 70% against intraperitoneal (ip) murine B16 melanoma and 50% against P388 leukemia. This compound is active when administered either ip or orally against ip or subcutaneously implanted L1210 leukemia, producing ILS values of about 100% at an optimum dose of 400 mg/kg. 1b is also active (60% ILS) against ara-C-resistant L1210. The analogous unsubstituted purine riboside nebularine (2) has modest activity against P388 leukemia (60% ILS). While 2'-ara-fluorozebularine (7b) is only marginally active (40% ILS) at high doses against L1210 leukemia, 5-fluoro analogue 6b is more active than zebularine and is ca. 100 times more potent. Although the activity of 6b is about the same as that of 1b against P388 leukemia, greater potency also is realized in this model. Zebularine is a strong inhibitor of cytidine deaminase, but in contrast to tetrahydrouridine, 1b is acid-stable. In an attempt to use this property to advantage in oral administration, 1b and ara-C have been orally coadministered to mice with ip L1210 leukemia. When zebularine is given in divided doses, up to a 2-fold increase in activity is realized, relative to treatment with the same dose of ara-C alone.

Selected acid-stable (2'-fluoro-2',3'-dideoxyarabinofuranosyl)adenine nucleosides containing methyl groups and other lipophilic functions at various positions in the adenine ring were prepared and evaluated as anti-HIV agents. The N6-methyl (1f), N6-benzoyl (1g), and 6-chloro (1i) analogues had modest activity, giving 30-50% protection to ATH8 cells infected with HIV. 2-Methyl (1d), 8-methyl (1h), and 2,N6-dimethyl (1e) substitution, as well as N1-oxide (21) formation, abolished the activity of the parent compound (1a). Several of these compounds, originally designed as agents for treating HIV in the central nervous system, were further investigated as substrates for adenosine deaminase (ADA). Kinetic experiments showed that ADA catalyzed the formation of the anti-HIV active inosine compound 1b from the N6-methyl analogue 1f in a quantitative manner. The anti-HIV activity of 1f and 1i was abolished when the ADA inhibitor, 2'-deoxycoformycin, was added to the test mixture. In contrast, the activity of 1f was significantly enhanced when ADA was added to the test system. These data indicate that 1f and 1i are prodrug forms of 1b in systems containing ADA.

Two ring-expanded analogues (compounds 2 and 3) of the anti-HIV fermentation product oxetanocin A (1) were synthesized from commercially available diacetone D-glucose. Antiviral testing against HIV in ATH8 cells revealed that the ring-expanded analogue 2 possessed a similar activity profile as oxetanocin A. Neither compound, however, was capable of providing full protection to the cells against HIV infection. The isomeric ring-expanded analogue 3 was totally devoid of anti-HIV activity. Molecular modeling suggested that while oxetanocin A and compounds 2 and 3 share a large common substructure with the potent anti-HIV drug, dideoxyadenosine (ddA), the extra hydroxymethyl substituent may contribute negatively to the binding of these molecules to a critical enzyme. The negative contribution may be less important in oxetanocin and isomer 2 than in isomer 3. From these studies it would appear that both oxetane and tetrahydrofuran rings are equivalent templates to support the adenine base in terms of anti-HIV activity.

Using a diacylglycerol-lactone (DAG-lactone) template previously developed in our laboratory as a scaffold with high binding affinity for C1 domains, we describe herein a series of novel DAG-lactones containing heterocyclic moieties (pyridines, quinolines, and indoles) as alpha-arylidene fragments. Some of the DAG-lactones obtained show selective binding to RasGRP3 as compared to PKCalpha by more than 2 orders of magnitude and possess subnanomolar affinities. Because activated C1 domains bound to their ligands (DAG or DAG-lactones) insert into membranes, the lipid composition of membranes (cellular, nuclear, and those of internal organelles) is an important determinant for specificity. Therefore, reaching a proper hydrophilic/lipophilic balance for these molecules is critical. This was achieved by carefully selecting partnering acyl fragments for the DAG-lactones with the appropriate lipophilicity. The results clearly show that the combination of chemical and physical properties in these molecules needs to be perfectly balanced to achieve the desired specificity.

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.

Diacylglycerol (DAG) lactones have provided a powerful platform for structural exploration of the interactions between ligands and the C1 domains of protein kinase C (PKC). In this study, we report that DAG-dioxolanones, novel derivatives of DAG-lactones, exploit an additional point of contact (glutamine 27) in their binding with the C1b domain of PKC delta. Mutation of this point of contact to glutamate selectively impairs binding of the DAG-dioxolanones compared to that of the corresponding DAG-lactones (1200- to 3000-fold versus 35- to 55-fold, respectively). The differential response of this mutated C1b domain to the DAG-dioxolanones relative to the DAG-lactones provides a unique tool to probe the role of the C1b domain in PKC delta function, where the response to the DAG-lactones affords a positive control for retained function. Using this approach, we show that the C1b domain of PKC delta plays the predominant role in the translocation of PKC delta to the membrane in the presence of DAG.

Highly rigid and geometrically well-defined rods composed of ethynylene-substituted aromatic spacers [oligo(p-phenyleneethynylene), OPE] were incorporated as acyl moieties on diacylglycerol lactones (DAG-lactones) and investigated for their ability to bind to protein kinase C (PKC) and translocate PKC alpha and delta isoforms to plasma and internal membranes. The kinetics of PKC translocation were correlated with biological responses, viz. ERK phosphorylation, induction of IL-6 secretion, inhibition of cell proliferation, and induction of cellular attachment, that display very different time courses. Because OPE rods assemble through noncovalent forces and form stable films, they may influence the microdomain environment around the DAG-lactone membrane-binding site. A comparison of two DAG-lactones (1 and 10), one with two PE units (1) and the other with an equivalent flexible acyl chain (10) of matching lipophilicity, clearly demonstrated the effect of the rigid OPE chain in substantially prolonging the translocated state of both PKC alpha and delta.

Combining molecular dynamics (MD) in a hydrated phospholipid (DOPC) bilayer, a Monte Carlo search, and synthesis of locked nucleotide analogues, we discovered that the Southern conformation of the ribose is preferred for ligand recognition by the P2Y(6) receptor. 2'-Deoxy-(S)-methanocarbaUDP was found to be a full agonist of the receptor and displayed a 10-fold higher potency than that for the corresponding flexible 2'-deoxyUDP. MD results also suggested a conformational change of the second extracellular loop consequent to agonist binding.

4'-C-ethynyl-2'-deoxynucleosides belong to a novel class of nucleoside analogues endowed with potent activity against a wide spectrum of HIV viruses, including a variety of resistant clones. Although favorable selectivity indices were reported for several of these analogues, some concern still exists regarding the 3'-OH group and its role in cellular toxicity. To address this problem, we removed the 3'-OH group from 4'-C-ethynyl-2'-deoxycytidine (1a). This compound was chosen because of its combined high potency and low selectivity index. The removal of the 3'-OH was not straightforward; it required a different synthetic approach from the one used to synthesize the parent compound. Starting with glycidyl-4-methoxyphenyl ether, the target 4'-C-ethynyl-2',3'-dideoxycytidine analogue (rac-1h) was obtained after 13 steps. In a cellular assay, rac-1h was completely inactive (0.001-10 microM) against HIV(LAI), demonstrating the critical importance of the 3'-OH for antiviral activity. To determine whether the role of the 3'-OH was essential for the phosphorylation of the compound by cellular kinases or for inhibition of DNA polymerization, we synthesized and tested the 5'-triphosphate (rac-1h-TP) for its ability to inhibit HIV reverse transcriptase (RT). rac-1h-TP was slightly more potent than AZT-5'-triphosphate against wild-type HIV RT, suggesting that the role of the 3'-OH is crucial only for the activation of the drug by cellular kinases. The lipase-catalyzed resolution of rac-1h into ent-1h (beta-D-dideoxyribo) and ent-14 (beta-L-dideoxyribo) and the synthesis of the corresponding 5'-triphosphates established the stereochemical assignment based on HIV RT's preference for the beta-D-enantiomer, which was confirmed by assaying against the M184V variant, an RT mutant with a marked preference for incorporating nucleosides in the D-configuration.

The constrained glycerol backbone of DAG-lactones, when combined with highly branched alkyl chains, has engendered a series of DAG-lactone ligands capable of binding protein kinase C (PK-C) with affinities that approximate those of phorbol esters. These branched chains not only appear to be involved in making important hydrophobic contacts with the protein (specific interactions) but also provide adequate lipophilicity to facilitate partitioning into the lipid-rich membrane environment (nonspecific interactions). With the idea of minimizing the nonspecific interactions without reducing lipophilicity, the present work explores the strategy of relocating lipophilicity from the side chain to the lactone "core". Such a transfer of lipophilicity, exemplified by compounds 1 and 3, was conceived to allow the new hydrophobic groups on the lactone to engage in specific hydrophobic contacts inside the binding pocket without any expectation of interfering with the hydrogen-bonding network of the DAG-lactone pharmacophore. Surprisingly, both (E)-3 and (Z)-3 showed a significant decrease in binding affinity. From the molecular docking studies performed with the new ligands, we conclude that the binding pocket of the C1 domain of PK-C is sterically restricted and prevents the methyl groups at the C-3 position of the lactone from engaging in productive hydrophobic contacts with the receptor.

A series of macrocyclic diacylglycerol (DAG)-bis-lactones were investigated as extreme conformationally constrained analogues of DAG-lactones in order to seek more potent protein kinase C (PKC) ligands with higher binding affinities and less lipophilicity than previous compounds. The additional constraint achieved the desired objective as exemplified by the macrocyclic DAG-bis-lactone 57, which exhibited a 6-fold higher binding affinity for PKCalpha (K(i) = 6.07 nM) than the corresponding nonmacrocyclic 3-alkylidene DAG-lactone 6. A structure-activity relationship (SAR) analysis of the macrocyclic DAG-bis-lactones demonstrated a parabolic relationship between activity and lipophilicity, as well as a predilection for the Z-alkylidene isomers as the preferred ligands. Molecular docking studies revealed that macrocyclic DAG-bis-lactone 57 bound to the C1b domain of PKCalpha exclusively in the sn-1 binding mode in contrast to DAG-lactone 6, which showed both sn-1 and sn-2 binding modes. It is proposed that the high potency displayed by these macrocyclic DAG-bis-lactones results from a set of more favorable hydrogen bonding and hydrophobic interactions with PKCalpha as well as from a reduced entropy penalty due to conformational restriction.

A solid-phase method for the synthesis of diacylglycerol lactones as protein kinase C ligands was developed, and a small array of nine compounds were selected with the idea of testing this methodology and forecasting the reliability of the biological data as a preamble for the construction of large chemical libraries to be synthesized under the same conditions. The process started with the loading of 5-(hydroxymethyl)-5-[(4-methoxyphenoxy)methyl]-3,4,5-trihydrofuran-2-one (1) to a 3,4-dihydro-2H-pyran resin packed inside IRORI MacroKan reactors. The elements of diversity were introduced at the alpha-alkylidene (R(1)) and acyl (R(2)) positions using a set of three different aldehydes and three different acid chlorides, respectively. An LDA-mediated aldol condensation with R(1)CHO in the presence of ZnCl(2) followed by a DBU-catalyzed elimination of the triflate of the resulting aldol gave the alpha-alkylidene intermediates as mixtures of geometric isomers. Removal of the aryl-protecting group followed by acylation with R(2)COCl introduced the second element of diversity. Acid-assisted cleavage of the compounds from the resin afforded the final targets. The biological results obtained using the crude samples directly obtained from the resin compared well with those from pure materials, as the K(i) values between the two sets varied only by a factor between 1.5 and 3.7.

The conformationally locked nucleoside, (north)-methanocarbathymine (1a), is a potent and selective anti-herpes agent effective against herpes simplex type 1 (HSV1) and type 2 (HSV2) viruses. Hereby, we report on the synthesis and biological evaluation of a small set of 5-substituted pyrimidine nucleosides belonging to the same class of bicyclo[3.1.0]hexane nucleosides. Both the 5-bromovinyl (4) and the 5-bromo analogue (3) appeared to be exclusive substrates of HSV1 thymidine kinase (TK), contrasting with the 5-iodo analogue (2), which was significantly phosphorylated by the human cytosolic TK. The binding affinity constant and catalytic turnover for HSV1 TK were measured to assess the influence of the substitution on these parameters. In the plaque reduction and cytotoxicity assays, the 5-bromo analogue (3) showed good activity against HSV1 and HSV2 with less general toxicity than 1a. Against varicella-zoster virus (VZV), the north-locked 5-bromovinyl analogue (4) proved to be as potent as its conformationally unlocked 2'-deoxyriboside equivalent BVDU. The three compounds were also tested in vitro as prodrugs used in a gene therapy context on three osteosarcoma cell lines, either deficient in TK (TK(-)), nontransduced, or stably transduced with HSV1 TK. The 5-iodo compound (2, CC(50) 25 +/- 7 microM) was more efficient than ganciclovir (GCV, CC(50) 75 +/- 35 microM) in inhibiting growth of HSV1-TK transfected cells and less inhibitory than GCV toward TK(-) cells, whereas compound 3 inhibited transfected and nontransfected cell lines in a relatively similar dose-dependent manner.

The synthesis and biological evaluation of a bicyclo[3.1.0]hexene nucleoside designed as a conformational mimic of the anti-HIV agent stavudine (1, D4T) is described. The unsaturated methanocarbocyclic pseudosugar of N-MCD4T (2) was constructed from an iodo-substituted precursor by a DBU-catalyzed olefination reaction. Mitsunobu coupling with N(3)-benzoylthymine afforded the desired target after deprotection. Both D4T and N-MCD4T are in the North (N) hemisphere of the pseudorotational cycle but 70 degrees away from a perfect N (P = 0 degrees ) conformation toward the East and West hemispheres, respectively. Despite this large difference, the double bond reduces the puckering amplitude (nu(max)) of N-MCD4T to 6.81 degrees, and the superposition of both structures showed a RMS deviation of only 0.039 A. The combined structural analysis of P and nu(max) shows that while the value of P may differ substantially, the low nu(max) resolves the differences and becomes the dominant pseudorotational parameter. N-MCD4T is active against HIV-1 and HIV-2 in CEM, MT-2, and MT-4 cells, and while it is somewhat less potent than D4T, it also appears to be less toxic. The triphosphate (N-MCD4TTP) inhibits HIV reverse transcriptase with a 10-fold higher IC(50) than D4TTP. By virtue of its carbocyclic nature, N-MCD4T (2) is a more robust molecule stable to conditions that would cleave D4T.

The structures of N-hydroxylamides 1a and 1b, previously reported by Lee et al. in J. Med. Chem. 2001, 44, 4309-4312 as strong protein kinase C (PK-C) ligands, were incorrect and correspond instead to esters 2a and 2b, respectively. Here, we report the synthesis and complete characterization of 1a and 1b together with the associated biological activity in terms of PK-C binding affinity.

Diacylglycerol lactones (DAG lactones), analogous to highly potent diacylglycerols (DAGs) were synthesized to demonstrate the ability of PK-C to discriminate between two differential binding modes, sn-1 and sn-2. While both sn-1 and sn-2 binding modes are allowable in terms of hydrogen bonding, it has been found that in general, DAGs prefer to bind sn-1, while the corresponding analogous DAG lactones prefer to bind sn-2. However, this binding orientation can be directly influenced by the disposition and nature of the acyl substituent, particularly if it is highly branched. When the “binding driving force” (i.e., the larger branched acyl chain) is in the sn-2 position, a dramatic increase in binding affinity is observed in the DAG lactone as compared to its open chain DAG counterpart. As these analogous DAGs and DAG lactones have almost identical log P values, this difference in binding affinity is a direct result of the entropic advantage of constraining the glycerol backbone.

A small, focused combinatorial library encompassing all possible permutations of acyl branched alkyl chains-small and large, saturated and unsaturated-was generated from the active diacylglycerol enantiomer (S-DAG) to help identify the analogue with the highest binding affinity (lowest Ki) for protein kinase C (PK-C) combined with the minimum lipophilicity (log P). The selected ligand (3B) activated PK-C more effectively than sn-1,2-dioctanoylglycerol (diC8) despite being 1.4 log units more hydrophilic. Compound 3B indeed represents the most potent, hydrophilic DAG ligand to date. With the help of a green fluorescent protein (GFP)-tagged PK-Calpha, 3B was able to translocate the full length protein to the membrane with an optimal dose of 100 microM in CHO-K1 cells, while diC8 failed to achieve translocation even at doses 3-fold higher. Molecular modeling of 3B into an empty C1b domain of PK-Cdelta clearly showed the existence of a preferred binding orientation. In addition, molecular dynamic simulations suggest that binding discrimination could result from a favorable van der Waals (VDW) interaction between the large, branched sn-1 acyl group of 3B and the aromatic rings of Trp252 (PK-Cdelta) or Tyr252 (PK-Calpha). The DAG analogue of 3B in which the acyl groups are reversed (2C) showed a decrease in binding affinity reflecting the capacity of PK-C to effectively discriminate between alternative orientations of the acyl chains.

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

The design of potent protein kinase C (PK-C) ligands with low nanomolar binding affinities was accomplished by the combined use of pharmacophore- and receptor-guided approaches based on the structure of the physiological enzyme activator, diacylglycerol (DAG). Earlier use of the former approach, which was based on the structural equivalence of DAG and phorbol ester pharmacophores, identified a fixed template for the construction of a semirigid "recognition domain" that contained the three principal pharmacophores of DAG constrained into a lactone ring (DAG-lactones). In the present work, the pharmacophore-guided approach was refined to a higher level based on the X-ray structure of the C1b domain of PK-Cdelta complexed with phorbol-13-O-acetate. A systematic search that involved modifying the DAG-lactone template with a combination of linear or branched acyl and alpha-alkylidene chains, which functioned as variable hydrophobic "affinity domains", helped identify compounds that optimized hydrophobic contacts with a group of conserved hydrophobic amino acids located on the top half of the C1 domain where the phorbol binds. The hydrophilic/hydrophobic balance of the molecules was estimated by the octanol/water partition coefficients (log P) calculated according to a fragment-based approach. The presence of branched alpha-alkylidene or acyl chains was of critical importance to reach low nanomolar binding affinities for PK-C. These branched chains appear to facilitate important van der Waals contacts with hydrophobic segments of the protein and help promote the activation of PK-C through critical membrane interactions. Molecular modeling of these DAG-lactones into an empty C1b domain using the program AutoDock 2.4 suggests the existence of competing binding modes (sn-1 and sn-2) depending on which carbonyl is directly involved in binding to the protein. Inhibition of epidermal growth factor (EGF) binding, an indirect PK-C mediated response, was realized with some DAG-lactones at a dose 10-fold higher than with the standard phorbol-12, 13-dibutyrate (PDBU). Through the National Cancer Institute (NCI) 60-cell line in vitro screen, DAG-lactone 31 was identified as a very selective and potent antitumor agent. The NCI's computerized, pattern-recognition program COMPARE, which analyzes the degree of similarity of mean-graph profiles produced by the screen, corroborated our principles of drug design by matching the profile of compound 31 with that of the non-tumor-promoting antitumor phorbol ester, prostratin. The structural simplicity and the degree of potency achieved with some of the DAG-lactones described here should dispel the myth that chemical complexity and pharmacological activity go hand in hand. Even as a racemate, DAG-lactone 31 showed low namomolar binding affinity for PK-C and displayed selective antitumor activity at equivalent nanomolar levels. Our present approach should facilitate the generation of multiple libraries of structurally similar DAG-lactones to help exploit molecular diversity for PK-C and other high-affinity receptors for DAG and the phorbol esters. The success of this work suggests that substantially simpler, high-affinity structures could be identified to function as surrogates of other complex natural products.

A series of substituted tetrahydrofurans with an embedded glycerol backbone carrying additional tetrahydrofuranylideneacetate or tetrahydrofuranylacetate motifs were grouped into four distinct templates (I-IV) according to stereochemistry. The compounds were designed to mimic three essential pharmacophores (C(3)-C=O, C(20)-OH and C(13)-C=O) of the phorbol esters according to a new, revised model. The tetrahydrofuran ring was constructed from glycidyl 4-methoxyphenyl ether, and the structures of the isomeric templates were assigned by NMR spectroscopy, including NOE. The binding affinity for protein kinase C (PKC) was assessed in terms of the ability of the ligands to displace bound [(3)H-20]phorbol 12, 13-dibutyrate (PDBU) from a recombinant alpha isozyme of PKC. Geometric Z- and E-isomers (1 and 3, respectively) containing a tetrahydrofuranylideneacetate motif were the most potent ligands with identical K(i) values of 0.35 microM. Molecular modeling studies of the four templates showed that the rms values when fitted to a prototypical phorbol 12,13-diacetate ester correlated inversely with affinities in the following order: I approximately IIIIIIV. These compounds represent the first generation of rigid glycerol templates seeking to mimic the binding of the C(13)-C=O of the phorbol esters. The binding affinities of the most potent compounds are in the same range of the diacylglycerols (DAGs) despite the lack of a phorbol ester C(9)-OH pharmacophore surrogate. This finding confirms that mimicking the binding of the C(13)-C=O pharmacophore of phorbol is a useful strategy. However, since the C(9)-OH and C(13)-C=O in the phorbol esters appear to form an intramolecular hydrogen bond that functions as a combined pharmacophore, it is possible the lack of this combined motif in the target templates restricts the compounds from reaching higher binding affinities.

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.