• Computer-assisted analysis of structure-activity relationships of philanthotoxin analogues

      Huang, Yu-Ing; Callery, Patrick S.; Pou, Sovitj (1992)
      Discovery of several venom toxins from spiders and a digger wasp as potent glutamate receptor antagonists has provided new approaches for the investigation of glutamate receptors. Philanthotoxin-433, a wasp toxin and an allosteric inhibitor of nicotinic acetylcholine (nAch) receptors, and numerous analogues have been reported to be potent antagonists of quisqualate receptors. In this dissertation, molecular mechanics and semiempirical (AM1) calculations were performed on 28 philanthotoxin (PhTX) analogues. Conformational analyses were carried out by systematic/grid searches to determine energy minima. In the calculated preferred conformations, two intramolecular hydrogen bonds were found to stabilize the conformers; one hydrogen bond between the two amides and another between the aromatic ring {dollar}\pi{dollar}-cloud and the polyamine chain amino hydrogens. Antagonistic potencies against the quisqualate and nAch receptors were explained through a spatial occupancy model of the PhTX analogues. Quantitative correlations were determined by comparative molecular field analyses (CoMFA). These studies revealed that the quisqualate receptor antagonism observed for the PhTX analogues correlated well with their structural modifications at the butyryl, tyrosyl and polyamine moieties. No significant correlation between the nAch receptor antagonism and PhTX structures was found. A CoMFA model for quisqualate receptor antagonism was established and had a correlation coefficient of 0.898 and a predictive r{dollar}\sp2{dollar} of 0.614. The logarithm of the IC{dollar}\sb{lcub}50{rcub}{dollar} values provided the best descriptor for the biological data in these CoMFA studies. The relative contribution of steric and electrostatic fields to the CoMFA model was 64% and 36%, respectively. A spermine chain with a free terminal amino functionality was found to be optimal for quisqualate receptor antagonism while the tyrosyl and butyryl moieties were predicted to be better regions for structural modifications leading to new antagonists.
    • Polyamine analogues in anticancer drug design

      Li, Yanlong; Callery, Patrick S. (1996)
      The binding affinity of polyamines toward DNA makes polyamine analogues a potential source of new anticancer agents that can interact selectively with DNA. Moreover, the presence of an active polyamine transport system in tumor cells may offer additional selectivity of these polyamine analogues toward tumor cells. Based on this rationale, a group of alkylating spermine analogues, including a diaziridinylspermine (BAS), two methylated analogues of BAS, and a norspermine analogue of BAS, was designed, synthesized and assessed for their biological activities. Cytotoxicity induced by BAS was demonstrated to be associated both with the alkylating functional group, aziridine, and also with the polyamine backbone structure. These analogues degraded or cross-linked DNA at a concentration that was 1000-fold lower than that of L-phenylalanine nitrogen mustard (LPAM). Transport studies were carried out by determining the competitive inhibition by synthesized polyamine analogues of (3H) -spermidine uptake in L1210 murine leukemia cells. Comparative molecular field analysis (CoMFA) was used to extract information on the structural requirements of inhibitors of the polyamine transport system. BAS and three BAS analogues were predicted to be substrates of the polyamine transporter. In vivo studies were carried out in a murine L1210 model in female {dollar}\rm CD\sb2F\sb1{dollar} mice. BAS increased survival of mice implanted with L1210 cells longer than did thiotepa, a clinically effective anticancer drug, when the same dose schedule was applied. Preliminary in vivo studies in a human non-small cell lung tumor H522 model also showed that BAS was effective in reducing, or at least controlling, solid tumor growth. Further design efforts were focused on reducing the nonspecific toxic effects of BAS. It was found that methylated analogues of BAS and the norspermine analogue of BAS were more than ten times less toxic to mice than was BAS. The observation of in vivo activity against implanted L1210 cells suggests that BAS can be used as a lead compound in anticancer drug design.
    • The qualitative and quantitative assessment of aldoxime stability by thermospray mass spectrometry

      Jakubowski, Edward Michael, Jr.; Callery, Patrick S. (1991)
      The application of thermospray mass spectrometry to qualitative and quantitative chemical degradation studies was assessed. A cholinesterase reactivator drug, 2-pralidoxime (2-hydroxyiminomethyl-1-methylpyridinium chloride, 2-PAM), was elevated as a model compound. Extrapolation of the reported first order rate equations for 2-PAM degradation in solution to thermospray conditions predicted the percent breakdown that was observed in the thermospray system. Both apparent gas-phase and apparent solution-phase reactions were observed and the activation energies calculated from thermospray data were in close agreement with available literature values. Breakdown of 2-PAM was dependent on solution pH, temperature, and flow rate. This evidence supported a strong contribution from liquid-phase reactions. A thermal flow reactor, modeled after the thermospray probe, was developed. The reactor consisted of a directly heated, 30 cm long stainless steel tube (0.015 cm id) with a thermocouple to monitor temperature changes. The tube was electrically insulated from the rest of the liquid chromatography-mass spectrometry system by using non-conductive polymer HPLC tubing. Either a cation exchange column or a C{dollar}\sb{lcub}18{rcub}{dollar} column in series with the reactor was used to separate possible degradation products of 2-PAM. The characteristic conversion of 2-PAM to the corresponding 2-pyridone (in equilibrium with 2-hydroxy-1-methylpyridinium ion) was related to temperature and flow rate. Also shown was the incorporation of {dollar}\sp{lcub}18{rcub}{dollar}O-water into 2-pyridone as a function of reactor temperature. The {dollar}\sp{lcub}18{rcub}{dollar}O studies supported the accepted mechanism of 2-pyridone production from 2-PAM. Incorporation of {dollar}\sp{lcub}18{rcub}{dollar}O into fragments of other aldoximes was demonstrated under thermospray conditions. In general, the reactor demonstrated that solution-phase degradation can occur under simulated thermospray probe conditions. The reactor can also be used with a UV detector to analyze drug stability at elevated temperatures.
    • Synthesis and cytotoxic mechanism study of aziridinyl spermidine analogues

      Yuan, Zhi-Min; Callery, Patrick S. (1993)
      In this study, the three-membered alkylating moiety, aziridine, was chosen to replace a terminal amino group of the polyamine, spermidine, to generate N{dollar}\sp1{dollar}- and N{dollar}\sp8{dollar}-aziridinyl spermidine analogues. Cytotoxicity was tested in vitro against L1210 murine lymphoblastic leukemic and HL60 human leukemic cell lines. Aminoguanidine, a serum amine oxidase inhibitor, reduced the cell growth inhibitory effect of spermidine approximately 6- or 20-fold in HL60 or L1210 cells, respectively, but did not show any effect on the cytotoxicity induced by aziridinyl spermidines. N{dollar}\sp1{dollar}-Aziridinyl spermidine was slightly more potent than the N{dollar}\sp8{dollar}-isomer (IC{dollar}\sb{lcub}50{rcub}{dollar} values 0.15 {dollar}\mu{dollar}M vs 0.3 {dollar}\mu{dollar}M for L1210 cells and 0.14 {dollar}\mu{dollar}M vs 0.28 {dollar}\mu{dollar}M for HL60 cells). Assessment of cell viability with a flow cytometric technique using fluorescein diacetate and propidium revealed that both N{dollar}\sp{lcub}1{rcub}{dollar}- and N{dollar}\sp8{dollar}-aziridinyl spermidines were cytotoxic. Both aziridinyl spermidine analogues inhibited (H{dollar}\sp3{dollar}) -thymidine and (H{dollar}\sp3{dollar}) -uridine incorporation into L1210 cells in a dose-dependent fashion. This effect was observed after an exposure time as short as one hour. The fact that the cytotoxicity induced by these two aziridinyl derivatives of spermidine was potentiated by 24 hour pretreatment of L1210 cells with 100 {dollar}\mu{dollar}M {dollar}\alpha{dollar}-diflouromethylornithine (DFMO), and were prevented by coincubation with 3.7 {dollar}\mu{dollar}M exogenous spermidine suggests a mechanistic relationship of the aziridinyl spermidine analogues with the polyamine system. Under the same conditions, pretreatment with DFMO and coincubation with spermidine had no effect on the cytotoxicity induced by thiotepa. The cell growth inhibitory effect induced by DFMO (100 {dollar}\mu{dollar}M for 24 hours) was overcome by washing away the test compound and replenishment with 3.7 {dollar}\mu{dollar}M spermidine. However, the cytotoxicity induced by aziridinyl spermidines was not affected at all. These observations suggest that the cytotoxic effect of aziridinyl compounds is irreversible. The accumulation of both N{dollar}\sp1{dollar}- and N{dollar}\sp8{dollar}-aziridinyl spermidine increased proportionally with increasing extracellular concentrations. Enhancement of cellular accumulation of both aziridinyl compounds by DFMO pretreatment provided evidence to support the argument that the aziridinyl spermidine analogues might utilize the polyamine transport system to enter cells. Other evidence which strengthens this argument is the fact that both N- and N{dollar}\sp8{dollar}-aziridinyl spermidines inhibited the uptake of natural spermidine in a dose-dependent manner. The perturbation of polyamine biochemistry by the test compounds was characterized by their ability to deplete cellular putrescine, as well as spermidine and spermine. Finally, results from DNA alkylation studies showed the formation of aziridinyl spermidine-DNA adducts, suggesting that DNA might be one target of the test compounds. (Abstract shortened by UMI.)
    • Synthesis and evaluation of Aziridine-containing compounds as inhibitors of porcine kidney diamine oxidase

      Wu, Yi-Ying; Callery, Patrick S. (1994)
      Diamine oxidase is an enzyme that catalyzes the oxidative deamination of diamines. Putrescine and cadaverine are among the best substrates of porcine kidney diamine oxidase. In this study, a variety of diamines with structures that are related to putrescine and cadaverine, namely N-(3-aminopropyl)aziridine, N-(4-aminobutyl)aziridine, N-(5-aminopentyl)aziridine, N-(6-aminohexyl)aziridine, N-(7-aminoheptyl)aziridine and the rigid analogue, N-(p-aminomethylbenzyl)aziridine have been synthesized and their interactions with diamine oxidase evaluated. These aziridinyl-containing compounds were synthesized in three steps from the condensation of aziridine with bromonitriles to yield aziridinylnitriles. Reduction of the nitriles with LiAlH{dollar}\sb4{dollar} provided the aziridinyl amines. These aziridinyl derivatives were relatively stable in aqueous buffer solutions at 37{dollar}\sp\circ{dollar}C. All of the compounds contain an aziridine ring as an alkylating group to inactivate the enzyme. Calculated IC{dollar}\sb{lcub}50{rcub}{dollar} values ranged from 0.01 to 1.7 mM. The activity of these inhibitors correlated with the separation length of the two nitrogen atoms of the diamine analogues. Four-carbon and five-carbon chain analogues were more potent than the other analogues tested. 3-Aziridinylpropylhydrazine, which contains an aziridine ring as well as a hydrazine group, was synthesized by reduction of the hydrazone formed between 3-aziridinylpropanal and hydrazine. This bifunctional inhibitor showed an IC{dollar}\sb{lcub}50{rcub}{dollar} value of 0.0085 {dollar}\mu{dollar}M which classifies it as one of the most potent inhibitors of diamine oxidase reported. All of the inhibitors showed time-dependent irreversible inhibition of diamine oxidase, which suggests that the formation of an enzyme-inhibitor complex is a reversible step, and a subsequent irreversible step leads to the inactivation of the enzyme. The inhibition pattern depends on the length of the inter-nitrogen distance in the analogues. Evaluation of the kinetics of inhibition of putrescine deamination indicated that the analogues of up to 5 or 6 carbons were competitive inhibitors. The 7-carbon analogue, N-(7-aminoheptyl)aziridine, showed mixed-competitive inhibition and a high IC{dollar}\sb{lcub}50{rcub}{dollar} value. The observed switching of the inhibitory mechanism is consistent with an additional binding region in the active site of diamine oxidase which can be accessed by longer-chained, lower-affinity inhibitors.