Cardioprotective effects of novel tetrahydroisoquinoline analogs of nitrobenzylmercaptopurine riboside in an isolated perfused rat heart model of acute myocardial infarction.

We have investigated the cardioprotective effects of novel tetrahydroisoquinoline nitrobenzylmercaptopurine riboside (NBMPR) analog nucleoside transport (NT) inhibitors, compounds 2 and 4, in isolated perfused rat hearts. Langendorff-perfused heart preparations were subjected to 10 min of treatment with compound 2, compound 4, or vehicle (control) followed by 30 min of global ischemia and 120 min of reperfusion. For determination of infarct size, reperfusion time was 180 min. At 1 microM, compounds 2 and 4 provided excellent cardioprotection, with left ventricular developed pressure (LVDP) recovery and end-diastolic pressure (EDP) increase of 82.9 +/- 4.0% (P<0.001) and 14.1 +/- 2.0 mmHg (P<0.03) for compound 2-treated hearts and 79.2 +/- 5.9% (P<0.002) and 7.5 +/- 2.7 mmHg (P<0.01) for compound 4-treated hearts compared with 41.6 +/- 5.2% and 42.5 +/- 6.5 mmHg for control hearts. LVDP recovery and EDP increase were 64.1 +/- 4.2% and 29.1 +/- 2.5 mmHg for hearts treated with 1 microM NBMPR. Compound 4 was the best cardioprotective agent, affording significant cardioprotection, even at 0.1 microM, with LVDP recovery and EDP increase of 76.0 +/- 4.9% (P<0.003) and 14.1 +/- 1.0 mmHg (P<0.03). At 1 microM, compound 4 and NBMPR reduced infarct size, with infarct area-to-total risk area ratios of 29.13 +/- 3.17 (P<0.001) for compound 4 and 37.5 +/- 3.42 (P<0.01) for NBMPR vs. 51.08 +/- 5.06% for control hearts. Infarct size was more effectively reduced by compound 4 than by NBMPR (P<0.02). These new tetrahydroisoquinoline NBMPR analogs are not only potent cardioprotective agents but are, also, more effective than NBMPR in this model.

Antisense anticancer oligonucleotide therapeutics.

Recent progress made in molecular biology, biotechnology, and genetics, especially in identifying, cloning, sequencing and characterization of normal and pathogenic genes, has led to the development of genetic therapy. Major efforts in the field can be summarized in two general approaches: gene therapy and antisense therapy. The second is to deliver to the target cells antisense molecules that target to mRNA with which they can hybridize and specifically inhibit the expression of pathogenic genes. Antisense oligonucleotides offer the possibility of specific, rational, genetic-based therapeutics. With encouraging results from preclinical and clinical studies of antisense oligonucleotides in the past decade, significant progress has been made in developing antisense therapy, with the first antisense drug now being approved for clinical use. In this article, we will discuss approaches to developing these drugs from preclinical to clinical settings. Of particular interest for the area of human cancer therapy, several cancer targets, including bcl-2, BCR-ABL, C-raf-1, Ha-ras, c-myc, PKC, PKA, p53 and MDM2, are reviewed as examples to illustrate the progress in this field and emphasize the importance of target selection and advanced antisense chemistry in the development of antisense therapy.

Cell cycle molecular targets in novel anticancer drug discovery.

A number of potential molecular targets for novel anticancer drug discovery have been identified in cell cycle control mechanisms. Prominent among these are the regulatory proteins, cyclins and their effector counterparts the cyclin dependent kinases (CDKs). Aberrant expression of these proteins, particularly cyclins involved in the G1 phase of the cell cycle, namely the D and E cyclins, has been associated with a variety of human cancers, including breast and colorectal cancer, B-lymphoma, prostate and non-small cell lung cancer. Inhibition of CDK kinase activity has turned out to be the most productive strategy for the discovery and design novel anticancer agents specifically targeting the cell cycle. Other potentially useful cell cycle areas for exploration include cyclin-CDK interactions, Cdc25 activation of cyclin-CDK complexes, ubiquitin-mediated proteolysis of cyclins, cell cycle check point kinases like Chk1, and recently identified oncogenic cell cycle-related aurora and polo-like kinases. Potent specific inhibitors have been identified that bind to the ATP site of CDKs, mainly cyclin B-CDK1, cyclin A-CDK2, and cyclin D-CDK4 complexes, and inhibit kinase activity. X-ray crystallographic data of CDKs, and their complexes with inhibitors have played a major role in the success of drug discovery efforts. Combinatorial chemistry, highthroughput screening, functional genomics and informatics have also contributed. CDK inhibitors currently under investigation include flavopiridol, olomoucine, roscovitine, puvalanol B, the dihydroindolo[3,2-d][1]benzazepinone kenpaullone, indirubin-3 -monoxime and novel diaminothiazoles such as AG12275. The anticancer therapeutic potential of CDK inhibitors has been demonstrated in preclinical studies, and Phases I and II clinical trials in cancer patients are currently underway.

Molecular targets in cancer drug discovery: cell-based profiling.

The phrase "molecular target-based drug discovery" usually implies an in vitro biochemical assay or battery of assays. One portion of the U.S. National Cancer Institute's drug discovery program, to the contrary, examines molecular targets for cancer therapy in a cell-based format. That approach has a number of significant limitations, but it has produced databases of significant utility on the activities and structures of tested compounds, as well as on molecular characteristics of the cell types used for testing.

Novel anticancer drug discovery.

There is at present, much optimism about the possibility of finding selective anticancer drugs that will eliminate the cytotoxic side effects associated with conventional cancer chemotherapy. This hope is based on uncovering many novel molecular targets that are 'cancer-specific', which will allow the targeting of cancer cells while normal cells are spared. Thus far, encouraging results have been obtained with several of these novel agents at the preclinical level, and clinical trials have begun. These targets are involved at one level or more in tumor biology, including tumor cell proliferation, angiogenesis and metastasis. Novel targets for which advances are being made include the following: growth factor receptor tyrosine kinases such as the epidermal growth factor receptor and HER-2/neu (proliferation); the vascular endothelial growth factor receptor and the basic fibroblast growth factor receptor (angiogenesis); the oncogenic GTP-binding protein Ras (especially agents targeting Ras farnesylation, farnesyltransferase inhibitors) (proliferation); protein kinase C (proliferation and drug resistance); cyclin-dependent kinases (proliferation); and matrix metalloproteinases and angiogenin (angiogenesis and metastasis). Less explored, but potentially useful targets include the receptor tyrosine kinase platelet-derived growth factor receptor, mitogen-activated protein kinase cascade oncogenes such as Raf-1 and mitogen-activated protein kinase kinase, cell adhesion molecules such as integrins, anti-apoptosis proteins such as Bcl-2, MDM2 and survivin, and the cell life-span target telomerase.

A protein expression database for the molecular pharmacology of cancer.

In the last six years, the Developmental Therapeutics Program (DTP) of the US National Cancer Institute (NCI) has screened over 60,000 chemical compounds and a larger number of natural product extracts for their ability to inhibit growth of 60 different cancer cell lines representing different organs of origin. Whereas inhibition of the growth of one cancer cell type gives no information on drug specificity, the relative growth inhibitory activities against 60 different cells constitute patterns that encode detailed information on mechanisms of action and resistance (as reviewed in Boyd and Paull, Drug Devel. Res. 1995, 34, 19-109 and Weinstein et al., Science 1997, 275, 343-349). In order to correlate the patterns of activity with properties of the cells, we and other laboratories are characterizing the cells with respect to a large number of factors at the DNA, mRNA, and protein levels. As part of that effort, we have developed a two-dimensional gel electrophoresis (2-DE) protein expression database covering all 60 cell types (Buolamwini et al., submitted). Here we present analyses of the correlations among protein spots (i) in terms of their patterns of expression and (ii) in terms of their apparent relationships to the pharmacology of a set of 3989 screened compounds. The correlations tend to be stronger for the latter than for the former, suggesting that the spots have more robust signatures in terms of the pharmacology than in terms of expression levels. Links to pertinent databases and tools of analysis will be updated progressively at http:@www.nci.nih.gov/intra/lmp/jnwbio.htm and http:@epnwsl.ncifcrf.gov:2345/dis3d/dtp.++ +html.

An information-intensive approach to the molecular pharmacology of cancer.

Since 1990, the National Cancer Institute (NCI) has screened more than 60,000 compounds against a panel of 60 human cancer cell lines. The 50-percent growth-inhibitory concentration (GI50) for any single cell line is simply an index of cytotoxicity or cytostasis, but the patterns of 60 such GI50 values encode unexpectedly rich, detailed information on mechanisms of drug action and drug resistance. Each compound's pattern is like a fingerprint, essentially unique among the many billions of distinguishable possibilities. These activity patterns are being used in conjunction with molecular structural features of the tested agents to explore the NCI's database of more than 460,000 compounds, and they are providing insight into potential target molecules and modulators of activity in the 60 cell lines. For example, the information is being used to search for candidate anticancer drugs that are not dependent on intact p53 suppressor gene function for their activity. It remains to be seen how effective this information-intensive strategy will be at generating new clinically active agents.

Ibogaine and a total alkaloidal extract of Voacanga africana modulate neuronal excitability and synaptic transmission in the rat parabrachial nucleus in vitro.

Ibogaine is a natural alkaloid of Voacanga africana that is effective in the treatment of withdrawal symptoms and craving in drug addicts. As the synaptic and cellular basis of ibogaine's actions are not well understood, this study tested the hypothesis that ibogaine and Voacanga africana extract modulate neuronal excitability and synaptic transmission in the parabrachial nucleus using the nystatin perforated patch-recording technique. Ibogaine and Voacanga africana extract dose dependently, reversibly, and consistently attenuate evoked excitatory synaptic currents recorded in parabrachial neurons. The ED50 of ibogaine's effect is 5 microM, while that of Voacanga africana extract is 170 micrograms/ml. At higher concentrations, ibogaine and Voacanga africana extract induce inward currents or depolarization that are accompanied by increases in evoked and spontaneous firing rate. The depolarization or inward current is also accompanied by an increase in input resistance and reverses polarity around 0 mV. The depolarization and synaptic depression were blocked by the dopamine receptor antagonist haloperidol. These results indicate that ibogaine and Voacanga africana extract 1) depolarize parabrachial neurons with increased excitability and firing rate; 2) depress non-NMDA receptor-mediated fast synaptic transmission; 3) involve dopamine receptor activation in their actions. These results further reveal that the Voacanga africana extract has one-hundredth the activity of ibogaine in depressing synaptic responses. Thus, ibogaine and Voacanga africana extract may produce their central effects by altering dopaminergic and glutamatergic processes.

Application of the electrotopological state index to QSAR analysis of flavone derivatives as HIV-1 integrase inhibitors.

PURPOSE: A QSAR study based on electrotopological state (E-state) indices was conducted for a series of flavone HIV-1 integrase inhibitors to guide drug design. METHODS: E-state indices formulated to encode electronic and topological information for each skeletal atom in a molecule (Kier and Hall Pharm. Res. 7:801-807 (1990)) were calculated using the Molconn-X program, and partial least squares (PLS) multivariate regression was used to derive QSAR models. RESULTS: Predictive models with correlation coefficients (r2) of 0.98 (3 PLS components) and 0.99 (5 PLS components) and corresponding cross-validated correlation coefficients (c.v. r2) of 0.51 and 0.73, were obtained for inhibition of cleavage and integration, respectively, with one molecule omitted from the analysis. CONCLUSIONS: E-state indices at C6, C3', C5', C5, and O4 were found to be more important for prediction of activity than those for any of the other 12 flavone skeletal atoms that are common to the molecules in the data set.

Three-dimensional quantitative structure-activity relationship (QSAR) of HIV integrase inhibitors: a comparative molecular field analysis (CoMFA) study.

We present the results from a comparative molecular field analysis (CoMFA) of a set of flavone analogs that inhibit HIV-1 integrase-mediated cleavage (3'-processing step) and integration (strand transfer step) in vitro. The results indicate a strong correlation between the inhibitory activity of these flavones and the steric and electrostatic fields around them. CoMFA quantitative structure-activity relationship models with considerable predictive ability (cross-validated r2 as high as 0.8) were obtained.

Flow cytometric quantitation of nucleoside transporter sites on human leukemic cells.

Quantitation of equilibrative, nitrobenzylthioinosine (NBMPR) sensitive (es) nucleoside transporters on blast cells isolated from patients with acute myeloblastic leukemia is useful in predicting intracellular accumulation of the antileukemic nucleoside drug, cytosine arabinoside. We previously reported the synthesis of a fluorescein-labeled ligand for the es nucleoside transporter, 5-(SAENTA-x2)-fluorescein. This paper reports the synthesis of 5-(SAENTA-x8)-fluorescein in which the linkage between fluorescein and nucleoside ligand has been increased from 2 atoms to 8 atoms. This new ligand had a sixfold increase in affinity (Kd 0.9 +/- 0.1 nM) as well as an 86% increase in the cell associated fluorescence output compared to its prototype 5-(SAENTA-x2)-fluorescein. The fluorescence signal arising from 5-(SAENTA-x8)-fluorescein specifically bound to freshly isolated and cultured leukemic myeloblasts was converted to molecules of equivalent soluble fluorescein (MESF) using standardized fluorescein microbeads and compared with the number of es nucleoside transporter sites assayed concurrently by [3H]NBMPR equilibrium binding analysis. A high correlation between the two assays was observed (r = 0.98), which enabled the cell-bound fluorescence output of 5-(SAENTA-x8)-fluorescein to be expressed in numbers of es nucleoside transporter sites per cell. The improved properties of 5-(SAENTA-x8)-fluorescein over those of its prototype molecule make it a suitable reagent for flow cytometric quantitation of nucleoside transporter expression on leukemic cells isolated from patient samples.

Synthesis and antinociceptive activity of 1-[2-(pyridyl)ethyl] and 1-[2-(dihydropyridyl)ethyl] analogues of fentanyl.

The syntheses and antinociceptive activities of all three isomeric 1-[2-(pyridyl)ethyl]-4-(propionanilido)-piperidine isosteres (11a-c) of fentanyl (1) are described. The 2- (11a), 3- (11b) and 4-pyridyl (11c) isomers exhibited 10, 2 and 0.2 times the antinociceptive activity of fentanyl, respectively. The ED50 values for 11a, 11b, 11c and fentanyl in the rat 4% NaCl-induced writhing test were 0.00023, 0.00085, 0.0087 and 0.0021 mg/kg sc, respectively. The 3-pyridyl (11b) and 4-pyridyl (11c) compounds were further elaborated to the 6-phenyl-1,6-dihydropyridine (12), C-2 H, Me, n-Bu and Ph 1,2-dihydropyridine (13a-d) analogues having a phenoxycarbonyl substituent on the dihydropyridine ring nitrogen. The most active compound in this series was 1-(2-[3-(1-phenoxycarbonyl-6-phenyl-1,6-dihydropyridyl)ethyl ])-4- (propionanilido)piperidine (12), which provided a 58% inhibition of writhing at a dose of 0.4 mg/kg sc. 1-(2-[4-(1-phenoxycarbonyl-1,2-dihydropyridyl)ethyl])-4- (propionanilido)piperidine (13a) exhibited an ED50 of 1.3 mg/kg sc, indicating a decrease in antinociceptive activity of about a 100 fold relative to the parent 4-pyridyl compound (11c). The dihydropyridine analogues 12 and 13 exhibit substantial antinociceptive activity relative to meperidine (ED50 = 0.6 mg/kg sc). The muscular rigidity effect induced by the pyridine compounds (11a-c) at a dose of 4 mg/kg sc, was not illicited by the dihydropyridine analogues at the same dose, or at a high dose of 40 mg/kg sc (13a). Compounds 12 and 13 may therefore be useful lead compounds for the development of more useful 4-anilidopiperidines if the antinociceptive activity can be dissociated from the muscular rigidity effect.

Synthesis and antinociceptive activity of 2- and 3-methyl derivatives of 4-(pyridyl) isosteres of meperidine.

The respective cis- and trans-[3-Me,4-(pyridyl)] diastereoisomers of 4-(2-pyridyl)- (8a and 8b), 4-(3-pyridyl)- (8d and 8e) and 4-(4-pyridyl)-1,3-dimethyl-4-ethoxycarbonylpiperidines (8h and 8i) were synthesized for evaluation as 3-methyl substituted isosteres of meperidine. Alkylation of ethyl 2-, 3- or 4-pyridylacetate (7) with N-(2-chloroethyl)-N-(2-chloropropyl)methylamine (6) afforded the respective 3-methyl substituted compounds 8a and 8b, 8d and 8e or 8h and 8i, together with the corresponding 2-methyl substituted compounds 8c (only the trans-isomer was obtained), 8f and 8g, or 8j and 8k. Antinociceptive test results, acquired using the 4% sodium chloride assay in rats, indicated that a cis-3-methyl substituent usually enhanced antinociceptive potency slightly, whereas a trans-3-methyl substituent lowered activity 3-4 fold relative to the parent 3-unsubstituted compounds 3b-d, at a dose of 2 mg/kg sc. A trans-2-methyl substituent (8g and 8k), like a cis-methyl substituent (8a, 8d and 8h), also maintained or provided a small increase in antinociceptive activity. Trans-1,2-Dimethyl-4-ethoxycarbonyl-4-(3-pyridyl)piperidine (8g) and cis-1,3-dimethyl-4-ethoxycarbonyl-4-(4-pyridyl)piperidine (8h) were the most active antinociceptives producing a 66% inhibition of writhing at a dose of 2 mg/kg sc, relative to the reference drug meperidine (ED50 = 0.6 mg/kg sc).

Synthesis and antinociceptive activity of 4-pyridyl and -dihydropyridyl analogues of meperidine and ketobemidone.

The synthesis and antinociceptive activity of 4-pyridylpiperdines (7 and 10) in which the phenyl group pf meperidine (1) or ketobemidone (3) is replaced by 2'-, 3'- or 4'-pyridyl is described. All were active in a rat writhing test, and the results showed that the point of attachment of the pyridyl ring to the 4-position was an important determinant of the activity; the relative potency order was 3'-pyridyl greater than 4'-pyridyl greater than 2'-pyridyl in each of the two series. The most potent compound (half the potency of meperidine) was 4-ethoxycarbonyl-4-(3'-pyridyl)-l-methylpiperidine (7b). This compound, and the corresponding 4'-isomer (7c), were further elaborated to provide 1'-phenyl, 1',6'-dihydropyridine (11), and 1'-phenyl, methyl or n-butyl. 1',2'-dihydropyridine (12) analogues containing an acyl function on the ring nitrogen. The most active compound in this series was 4-[4'-(1'-phenoxycarbonyl-2'-n-butyl-l',2'-dihydropyridyl)]- 4- ethoxycarbonyl-l-methylpiperdine (12k). Though less potent than the parent pyridyl compound (7c), it induced 70% inhibition in the writhing test at a dose of 8 mg/kg sc. [The ED50 for meperidine was 0.6 mg/kg sc.]

Synthesis and antinociceptive activity of 7-aryloxysulfonyl piperidines.

Compounds having structural relationships with meperidine (14) were synthesised and examined for antinociceptive activity (rat writhing test). The compounds comprised a group of 7-aryloxysulfonyl-2,7-diazabicyclo[4.1.0]hept-4-enes (8), 1,2,5,6-tetrahydropyridylidene- (9) and piperidylidene-2-aryloxysulfonamides (10) bearing N-substituents (R1 = H,Me,8-10), 3-substituents (R2 = Me, n-Bu, Ph, 8) and 6-substituents (R2 = H, Me, n-Bu, Ph, 9-10). The relative order of potency in the test was generally 10 greater than 9 greater than 8. Replacement of the R2 hydrogen substituent of 8-10d-g by a Me, n-Bu or Ph substituent generally enhanced activity. 1-Methyl-6-phenylpiperidylidene-2-phenoxysulfonamide (10g) was the most active antinociceptive agent in the series, providing 79.6% inhibition of writhing after a 50 mg kg sc dose.