Ceramide mediates nanovesicle shedding and cell death in response to phosphatidylinositol ether lipid analogs and perifosine.

Anticancer phospholipids that inhibit Akt such as the alkylphospholipid perifosine (Per) and phosphatidylinositol ether lipid analogs (PIAs) promote cellular detachment and apoptosis and have a similar cytotoxicity profile against cancer cell lines in the NCI60 panel. While investigating the mechanism of Akt inhibition, we found that short-term incubation with these compounds induced rapid shedding of cellular nanovesicles containing EGFR, IGFR and p-Akt that occurred in vitro and in vivo, while prolonged incubation led to cell detachment and death that depended on sphingomyelinase-mediated generation of ceramide. Pretreatment with sphingomyelinase inhibitors blocked ceramide generation, decreases in phospho-Akt, nanovesicle release and cell detachment in response to alkylphospholipids and PIAs in non-small cell lung cancer cell lines. Similarly, exogenous ceramide also decreased active Akt and induced nanovesicle release. Knockdown of neutral sphingomyelinase decreased, whereas overexpression of neutral or acid sphingomyelinase increased cell detachment and death in response to the compounds. When transferred in vitro, PIA or Per-induced nanovesicles increased ceramide levels and death in recipient cells. These results indicate ceramide generation underlies the Akt inhibition and cytotoxicity of this group of agents, and suggests nanovesicle shedding and uptake might potentially propagate their cytotoxicity in vivo.

A series of halogenated heterodimeric inhibitors of prostate specific membrane antigen (PSMA) as radiolabeled probes for targeting prostate cancer.

Prostate specific membrane antigen (PSMA) is a validated molecular marker for prostate cancer. A series of glutamate-urea (Glu-urea-X) heterodimeric inhibitors of PSMA were designed and synthesized where X = epsilon-N-(o-I, m-I, p-I, p-Br, o-Cl, m-Cl, p-Cl, p-F, H)-benzyl-Lys and epsilon-(p-I, p-Br, p-Cl, p-F, H)-phenylureido-Lys. The affinities for PSMA were determined by screening in a competitive binding assay. PSMA binding of the benzyllysine series was significantly affected by the nature of the halogen substituent (IC(50) values, Cl < I = Br << F = H) and the ring position of the halogen atom (IC(50) values, p-I < o-I << m-I). The halogen atom had little affect on the binding affinity in the para substituted phenylureido-Lys series. Two lead iodine compounds were radiolabeled with (123)I and (131)I and demonstrated specific PSMA binding on human prostate cancer cells, warranting evaluation as radioligands for the detection, staging, and monitoring of prostate cancer.

Specific ligands of the peripheral benzodiazepine receptor induce apoptosis and cell cycle arrest in human colorectal cancer cells.

The peripheral benzodiazepine receptor (PBR) has been implicated in growth control of various tumour models. Although colorectal cancers were found to overexpress PBR, the functional role of PBR in colorectal cancer growth has not been addressed to date. Using primary cell cultures of human colorectal cancers and the human colorectal carcinoma cell lines HT29, LS174T, and Colo320 DM we studied the involvement of PBR in the growth control and apoptosis of colorectal cancers. Both mRNA and protein expression of PBR were detected by RT-PCR and flow cytometry. Using confocal laser scanning microscopy and immunohistochemistry the PBR was localized in the mitochondria. The specific PBR ligands FGIN-1-27, PK 11195, or Ro5-4864 inhibited cell proliferation dose-dependently. FGIN-1-27 decreased the mitochondrial membrane potential, which indicates an early event in apoptosis. Furthermore, FGIN-1-27, PK 11195 or Ro5-4864 increased caspase-3 activity. In addition to their apoptosis-inducing effects, PBR ligands induced cell cycle arrest in the G(1)/G(0)-phase. Thus, our data demonstrate a functional involvement of PBR in colorectal cancer growth and qualify the PBR as a possible target for innovative therapeutic approaches in colorectal cancer.

Sar studies of piperidine-based analogues of cocaine. Part 3: oxadiazoles.

The synthesis of novel 4beta-aryl-1-methyl-3alpha-(3-substituted-1,2,4-oxadiazol-5-yl)piperidines, bioisosteres of ester (+)-1, is described. The synthesized oxadiazoles were evaluated for their affinity to the DAT and their ability to inhibit monoamine reuptake at the DAT, NET, and 5HTT. The results show that affinity to the DAT and ability to inhibit the reuptake at the DAT, NET, and 5HTT is a function of the size of the substituent in the oxadiazole ring. (+)-(3R,4S)-4beta-(4-Chlorophenyl)-1-methyl-3alpha-(3-methyl-1,2,4-oxadiazol-5-yl)piperidine [(+)-2a], which is structurally and pharmacologically most similar to the ester (+)-1 in this series, showed at least a 2-fold longer duration of action when compared to ester (+)-1.

Synthesis of N(1)-substituted analogues of (2R,4R)-4-amino-pyrrolidine-2,4-dicarboxylic acid as agonists, partial agonists, and antagonists of group II metabotropic glutamate receptors.

The chemical synthesis of a series of N(1)-substituted derivatives of (2R,4R)-4-aminopyrrolidine-2,4-dicarboxylic acid [(2R,4R)-APDC] as constrained analogues of gamma-substituted glutamic acids is described. Appropriate substitution of the N(1)-position results in agonist, partial agonist, or antagonist activity at mGluR2, mGluR3, and/or mGluR6.

Molecular modeling, structure--activity relationships and functional antagonism studies of 4-hydroxy-1-methyl-4-(4-methylphenyl)-3-piperidyl 4-methylphenyl ketones as a novel class of dopamine transporter inhibitors.

We previously disclosed the discovery of 4-hydroxy-1-methyl-4-(4-methylphenyl)-3-piperidyl 4-methylphenyl ketone (3) as a novel class of dopamine transporter (DAT) inhibitors and showed that (+/-)-3 has a significant functional antagonism against cocaine in vitro. Our previous preliminary structure-activity relationship study led to identification of a more potent DAT inhibitor [(+/-)-4] but this compound failed to show any significant functional antagonism. To search for more potent analogues than 3 but still displaying significant functional antagonism, further SARs, molecular modeling studies and in vitro pharmacological evaluation of this novel class of DAT inhibitors were performed. Sixteen new analogues were synthesized in racemic form and evaluated as DAT inhibitors. It was found that seven new analogues are reasonably potent DAT inhibitors with K(i) values of 0.041--0.30 and 0.052--0.16 microM in [(3)H]mazindol binding and inhibition of DA reuptake. Chiral isomers of several potent DAT inhibitors were obtained through chiral HPLC separation and evaluated as inhibitors at all the three monoamine transporter sites. In general, the (-)-isomer is more active than the (+)-isomer in inhibition of DA reuptake and all the (-)-isomers are selective inhibitors at the DAT site. Evaluation of cocaine's effect on dopamine uptake in the presence and absence of (+)-3 and (-)-3 showed that (-)-3 is responsible for the functional antagonism obtained with the original lead (+/-)-3. Out of the new compounds synthesized, analogue (+/-)-20, which is 8- and 3-fold more potent than (+/-)-3 in binding and inhibition of DA reuptake, appeared to have improved functional antagonism as compared to (+/-)-3.

Pharmacophore-based discovery of 3,4-disubstituted pyrrolidines as a novel class of monoamine transporter inhibitors.

3,4-Disubstituted pyrrolidines were discovered as a novel class of monoamine transporter inhibitors through 3-D database pharmacophore searching using a new pharmacophore model. The most potent analogue 12 has Ki values of 0.084 microM in [3H]mazindol binding, 0.20, 0.23, and 0.031 microM in inhibition of dopamine (DA), serotonin (SER), and norepinephrine (NE) reuptake, respectively. Functional antagonism testing in vitro showed that 11 and 12 are weak cocaine antagonists.

Rational design, synthesis, and biological evaluation of rigid pyrrolidone analogues as potential inhibitors of prostate cancer cell growth.

In view of its role in tumor promotion and signal transduction, protein kinase C (PKC) has proven to be an exciting target for cancer therapy. With the aid of molecular modeling, we rationally designed and stereoselectively synthesized a new class of rigidified pyrrolidone-based PKC activators. Pyrrolidone 15 was found to exhibit reasonable affinity for PKCdelta, with lower affinity for the other isozymes tested. Pyrrolidone 2 causes the dose-dependent induction of apoptosis in LNCaP prostate cancer cells. This apoptotic effect could be markedly potentiated by the use of LNCaP cells overexpressing the PKCalpha or delta isozymes.

Pharmacological and behavioral analysis of the effects of some bivalent ligand-based monoamine reuptake inhibitors.

Novel piperidine-based bivalent ligands were prepared in enantiomerically pure form and evaluated for their ability to inhibit reuptake of dopamine (DA), serotonin (5-HT), and norepinephrine (NE) into rat brain nerve endings (synaptosomes). In this study, we have succeeded in using (1) the length of the linking chain connecting the two piperidine-based monomer units and (2) the absolute configuration of the piperidine monomer as a means to tailor activity and selectivity at the three monoamine transporters tested. In this series, the bivalent ligand 16, comprised of two (+)-trans-piperidine units linked by a pentamethylene spacer, exhibits a combination of high DA transporter (DAT) and 5-HT transporter (SERT) activity (K(i) = 39 nM and 7 nM, respectively). Piperidine 16 is capable of reducing cocaine's locomotor effects in mice while not having any effect on locomotion when tested alone. Additionally, compound 16 (1-10 mg/kg) does not substitute for cocaine in drug discrimination studies in rats. However, the analogous bivalent ligand 15 comprised of two (-)-trans-piperidine units, which is SERT selective, was less effective in antagonizing cocaine's locomotor stimulant activity. The piperidine-based bivalent inhibitors described herein constitute a new class of monoamine reuptake inhibitors that exhibit varying levels of monoamine transporter activity and selectivity, and these ligands may serve as lead candidates in the discovery of new therapeutics to treat a range of neurological disorders including cocaine addiction.

Studies in polyphenol chemistry and bioactivity. 3.(1,2) stereocontrolled synthesis of epicatechin-4alpha,8-epicatechin, an unnatural isomer of the B-type procyanidins.

Oligomeric procyanidins containing 4alpha-linked epicatechin units are rare in nature and have hitherto not been accessible through stereoselective synthesis. We report herein the preparation of the prototypical dimer, epicatechin-4alpha,8-epicatechin (6), by reaction of the protected 4-ketones 11a,b with aryllithium reagents derived by halogen/metal exchange from the aryl bromides 26a,b. Removal of the 4-hydroxyl group from the resulting tertiary benzylic alcohols 27a,b was effected by tri-n-butyltin hydride and trifluoroacetic acid in a completely stereoselective manner, resulting in hydride delivery exclusively from the beta face. If benzyl was chosen for protection of the 3-hydroxyls, all protective groups could subsequently be removed in a single step by hydrogenolysis. tert-Butyldimethylsilyl groups, on the other hand, permitted selective deprotection of the 3-hydroxyls in preparation for their subsequent acylation with tri-O-benzylgalloyl chloride. Only monogalloylation at the "bottom" 3-hydroxyl took place when 28c was acylated under the previously reported conditions, reflecting the increased steric hindrance of the "top" 3-hydroxyl group in 28c compared with its 4beta,8-isomer 3. The preparation of compounds 14 and 17 containing phloroglucinol trimethyl ether in the 4alpha and 4beta linkages to epicatechin is also described. The 8-position of the bromine atom in 19, previously conjectured in analogy to the structurally characterized tetramethyl ether 20, was confirmed by transformation of both compounds into the common derivative 25.

Molecular modeling studies of the Akt PH domain and its interaction with phosphoinositides.

The serine-threonine protein kinase Akt is a direct downstream target of phosphatidylinositol 3-kinase (PI3-K). The PI3-K-generated phospholipids regulate Akt activity via directly binding to the Akt PH domain. The binding of PI3-K-generated phospholipids is critical to the relocalization of Akt to the plasma membrane, which plays an important role in the process of Akt activation. Activation of the PI3-K/Akt signaling pathway promotes cell survival. To elucidate the structural basis of the interaction of PI3-K-generated phospholipids with the Akt PH domain with the objective of carrying out structure-based drug design, we modeled the three-dimensional structure of the Akt PH domain. Comparative modeling-based methods were employed, and the modeled Akt structure was used in turn to construct structural models of Akt in complex with selected PI3-K-generated phospholipids using the computational docking approach. The model of the Akt PH domain consists of seven beta-strands forming two antiparallel beta-sheets capped by a C-terminal alpha-helix. The beta1-beta2, beta3-beta4, and beta6-beta7 loops form a positively charged pocket that can accommodate the PI3-K-generated phospholipids in a complementary fashion through specific hydrogen-bonding interactions. The residues Lys14, Arg25, Tyr38, Arg48, and Arg86 form the bottom of the binding pocket and specifically interact with the 3- and 4-phophate groups of the phospholipids, while residues Thr21 and Arg23 are situated at the wall of the binding pocket and bind to the 1-phosphate group. The predicted binding mode is consistent with known site-directed mutagenesis data, which reveal that mutation of these crucial residues leads to the loss of Akt activity. Moreover, our model can be used to predict the binding affinity of PI3-K-generated phospholipids and rationalize the specificity of the Akt PH domain for PI(3,4)P2, as opposed to other phospholipids such as PI(3)P and PI(3,4,5)P3. Taken together, our modeling studies provide an improved understanding of the molecular interactions present between the Akt PH domain and the PI3-K-generated phospholipids, thereby providing a solid structural basis for the design of novel, high-affinity ligands useful in modulating the activity of Akt.

Pharmacophore-based discovery, synthesis, and biological evaluation of 4-phenyl-1-arylalkyl piperidines as dopamine transporter inhibitors.

Pharmacophore-based discovery, synthesis, and structure activity relationship (SAR) of a series of 4-phenyl-1-arylalkyl piperidines are disclosed. These compounds have been evaluated for their ability to inhibit reuptake of dopamine (DA) into striatal nerve endings (synaptosomes). The lead compound 5 and the most potent analogue 43 were found to have significant functional antagonism.

Novel monoamine transporter ligands reduce cocaine-induced enhancement of brain stimulation reward.

Six novel monoamine reuptake inhibitors were screened for their intrinsic effects on brain stimulation reward (BSR), as well as for their potential to reduce cocaine-induced reward-enhancement in that paradigm. Two of the compounds, nocaine-3B and 5-ara-74A (disubstituted piperidines) significantly reduced locus of rise (LOR), threshold measure of reward, at some doses. One compound, 1-RV-96A (a hybrid of the GBR and WIN-like agents) significantly reduced reward (increased LOR), but only at the highest dose tested. No effect of dose was found for MC9-20 (a GBR-like acyclic analogue of the N-bisarylmethoxyethyl-N'-phenylpropyl piperazine), nocaine-250B or 4-ara-42C (disubstituted piperidines). When cocaine (10 mg/kg, ip) and selected, hedonically neutral doses of novel compounds were combined, the following findings were obtained: MC9-20 (2.5 mg/kg, ip) showed a significant increase in cocaine-induced reward enhancement (0.2 log units or 53%). In contrast, nocaine-250B and 1-RV-96A (both at 10 mg/kg, ip) demonstrated a significant reduction (0.13 log units or 41%) in cocaine-induced reward enhancement (P<.01 and P<.05, respectively), as measured by changes in LOR. There were no differences in the maximum behavioral output (MAX) at either dose of each of the six drugs, or when selected doses were combined with cocaine. These results indicate that nocaine-250B and 1-RV-96A constitute two potential anticocaine compounds worthy of further behavioral and biochemical evaluation.

3-Deoxy-3-substituted-D-myo-inositol imidazolyl ether lipid phosphates and carbonate as inhibitors of the phosphatidylinositol 3-kinase pathway and cancer cell growth.

3-Modified D-myo-inositol imidazolyl ether lipid phosphates and a carbonate were synthesized and evaluated as inhibitors of P13-K and Akt. These data are presented along with IC50 values for the inhibition of the growth of three cancer cell lines.

Synthesis of 7,8-disubstituted benzolactam-V8 and its binding to protein kinase C.

7-Methoxy-8-decynyl-benzolactam-V8 4 is synthesized using a catalytic asymmetric alkylation reaction as a key step. This compound shows potent activity to three PKC isozymes tested (Ki =45.6, 91.1, and 121.3 nM to PKCalpha, delta, and epsilon, respectively), indicating that introduction of a suitable substituent at the 7-position of 8-decynyl-benzolactam-V8 only slightly reduces the PKC binding affinity.

Synthesis, chiral chromatographic separation, and biological activities of the enantiomers of 10,10-dimethylhuperzine A.

(+/-)-10,10-Dimethylhuperzine A (2, DMHA) has been synthesized, and its enantiomers have been separated using chiral HPLC. (-)-DMHA inhibits AChE with a Ki value approaching that of (-)-huperzine A, whereas (+)-DMHA shows no AChE inhibitory activity. On the other hand, both enantiomers are equally potent against glutamate-induced neurotoxicity when tested in neurons.

Studies in polyphenol chemistry and bioactivity. 2. Establishment of interflavan linkage regio- and stereochemistry by oxidative degradation of an O-alkylated derivative of procyanidin B2 to (R)-(-)-2,4-diphenylbutyric acid.

The assignment of interflavan bond regio- and stereochemistry in oligomeric proanthocyanidins has in the past relied on empirical spectroscopic techniques which are influenced by the conformation of the C rings. Only recently was the 4,8-regiochemistry of procyanidin B2 (3b) firmly established by 2-dimensional NMR methods. We describe herein the proof of 4beta-stereochemistry in 3b by oxidative degradation of the derivative 3d bearing differential (O-benzyl and O-methyl) protecting groups in its "top" and "bottom" epicatechin moieties, to (R)-(-)-2,4-diphenylbutyric acid. The key elements of the degradative process are (1) removal of the C-3 alcohol functions through a modified Barton deoxygenation employing hypophosphorous acid as the reducing agent; (2) deprotection of the "top" unit by hydrogenolysis, followed by exhaustive aryl triflate formation with N,N-bis(trifluoromethanesulfonyl)aniline and DBU in DMF; (3) hydrogenolytic deoxygenation of the "top" unit over Pearlman's catalyst with concomitant scission of the O-C2 bond; (4) selective oxidation of the "bottom" unit with NaIO4/RuCl3. The hitherto unreported absolute configuration of (-)-2,4-diphenylbutyric acid was established as R by X-ray crystal structure analysis of the (R)-(+)-alpha-methylbenzylamine salt. As a corollary, the selectivity of hydrogenolytic and solvolytic reactions of epicatechin-derived tetrasulfonates has been investigated.

Chemical synthesis and pharmacology of 6- and 7-hydroxylated 2-carbomethoxy-3-(p-tolyl)tropanes: antagonism of cocaine's locomotor stimulant effects.

In our efforts to identify molecules that might act as cocaine antagonists or cocaine partial agonists, efforts were made to further capitalize on our earlier finding regarding the ability of a 7-methoxylated pseudococaine analogue to act as a weak cocaine functional antagonist. Herein, a series of the 6- and 7-hydroxylated WIN analogues possessing a boat or chair conformation of the tropane ring were prepared and tested for their ability to displace [(3)H]mazindol binding and to inhibit high-affinity monoamine uptake into rat brain nerve endings. These 6- and 7-hydroxylated WIN analogues were readily prepared by use of a classical Willstätter synthesis to construct an appropriately functionalized tropane ring followed by use of a Suzuki coupling reaction to introduce the aryl group at position 3. Reduction of the resulting tropene by use of SmI(2) or by catalytic hydrogenation followed by deprotection delivered the final target compounds. Some of these compounds were found to retain considerable affinity as inhibitors of the dopamine transporter (DAT) and the norepinephrine transporter (NET), but they were less potent inhibitors of the serotonin transporter (SERT). None of the compounds of the present series revealed any substantial potency difference in [(3)H]mazindol binding versus [(3)H]DA uptake, and failed to show "cocaine antagonism" when tested for their ability to prevent cocaine's inhibition of DA transport. However, one of these hydroxylated WIN analogues, namely 12b, which possesses nanomolar potency at the DAT and NET and micromolar potency at the SERT, when tested in vivo, was found capable of attenuating cocaine's locomotor activity (AD(50) = 94 mg/kg). Taken together, this work provides further support for our hypothesis that drugs that lack the ability to inhibit transport by all three monoaminergic transporters may exhibit "partial" cocaine-like properties, but act as cocaine antagonists. Consequently, it may prove valuable to examine the behavioral activity of other 6- and 7-substituted tropanes in animal behavioral paradigms in the search for a cocaine medication.