The metabolism of the anti-tumour agent 1(1-aziridinyl)- 2,4-dinitrobenzene (CB 1837).

1-(1-Aziridinyl)-2,4-dinitrobenzene (CB 1837) is the parent member of a series of nitrophenyla-ziridines having a highly specific action against the Walker tumour in the rat. CB 1837 was much less cytotoxic in vitro than was predicted on the basis of its antitumour activity in vivo, but its activity was enhanced in the presence of liver 9000-g supernatant and cofactors, which also converted it into a metabolite, 2-amino-1-(1-aziridinyl)-4-nitrobenzene. This metabolite was more active in the in vitro test than was CB 1837.CB 1837 is extensively metabolised in vivo by the rat, and the following urinary metabolites have been identified by mass spectrometry and quantitatively determined after administration of the tritium-labelled drug: 1-(2-chloroethyl)amino-2,4-dinitrobenzene and its 2-hydroxyethylamino analogue; 2,4-dinitroaniline: 2-amino-1-(1 -aziridinyl)-4-nitrobenzene,2-amino-1-(2-chloroethyl)amino-4-nitrobenzene and their respective N(2)-acetyl derivatives; and S-[N-(2,4-dinitrophenyl)-2-aminoethyl] mercapturic acid.     

Selective inhibition of ribonucleotide reductase by the monofunctional alkylating agent 5(1-aziridinyl)-2,4-dinitrobenzamide (CB 1954)

The monofunctional alkylating agent 5-(1-aziridinyl)-2,4-dinitrobenzamide (CB 1954) is a potent and selective inhibitor of the growth of the Walker carcinoma (ld₅₀ 0.002 $\text{μg}\text{ml}$). Growth inhibition by CB 1954 is accompanied by a rapid inhibition of DNA synthesis with little effect on RNA and protein biosynthesis. Interference with the biosynthesis of DNA by CB 1954 has been shown to be due to inhibition of ribonucleoside diphosphate reductase. The order of effectiveness of inhibition of the enzyme by CB 1954 in four cell lines parallels the tumour growth inhibitory activity. Furthermore, analogues of CB 1954 with methyl substitution on the amide group were less effective inhibitors of the growth of the Walker carcinoma and also less potent inhibitors of ribonucleotide reductase. Treatment of Walker cells with CB 1954 caused a rapid inhibition of enzyme activity and a corresponding fall in the levels of deoxyribonucleoside triphosphates which corresponded with the effect of the drug on DNA synthesis. A human bladder carcinoma has been shown to be particularly susceptible to growth inhibition by CB 1954. Measurement of the effect of CB 1954 on ribonucleotide reductase from biopsy specimens of human tumours could provide a basis for patient selection for therapy by this agent.     

Metabolic activation of the antitumor drug 5-(Aziridin-1-yl)-2,4-dinitrobenzamide (CB1954) by NO synthases

Nitric oxide synthases (NOSs) are flavohemeproteins that catalyze the oxidation of L-arginine to L-citrulline with formation of the signaling molecule nitric oxide (NO). In addition to their fundamental role in NO biosynthesis, NOSs are also involved in the formation of reactive oxygen and nitrogen species (RONS) and in the interactions with some drugs. 5-(Aziridin-1-yl)-2,4-dinitrobenzamide (CB1954) is a dinitroaromatic compound tested as an antitumor prodrug that requires reduction to the 2- and 4-hydroxylamines to be cytotoxic. Here, we studied the interaction of neuronal, inducible, and endothelial NOSs with CB1954. Our results showed that the three purified recombinant NOSs selectively reduced the 4-nitro group of CB1954 to the corresponding 4-hydroxylamine with minimal 2-nitroreduction. Little further two-electron reduction of the hydroxylamines to the corresponding 2- and 4-amines was observed. The reduction of CB1954 catalyzed by the neuronal NOS (nNOS) was inhibited by O 2 and a flavin/NADPH binding inhibitor, diphenyliodonium (DPI), but insensitive to the addition of the heme ligands imidazole and carbon monoxide and of l-arginine analogues. This reduction proceeded with intermediate formation of a nitro-anion free radical observed by EPR. Involvement of the reductase domain of nNOS in the reduction of CB1954 was confirmed by the ability of the isolated reductase domain of nNOS to catalyze the reaction and by the stimulating effect of Ca (2+)/calmodulin on the accumulation of 4- and 2-hydroxylamines. The recombinant inducible and endothelial NOS isoforms reduced CB1954 with lower activity but higher selectivity for the cytotoxic 4-hydroxylamine compared with nNOS. Finally, CB1954 did not modify the formation of l-citrulline and RONS catalyzed by nNOS. Our results show that all three NOS isoforms are involved in the nitroreduction of CB1954, with predominant formation of the cytotoxic 4-hydroxylamine derivative. This nitroreduction could be of interest for the selective activation of prodrugs by NOSs overexpressed in tumor cells.     

The bioactivation of 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB1954)--II. A comparison of an Escherichia coli nitroreductase and Walker DT diaphorase.

A nitroreductase enzyme that has been isolated from Escherichia coli B is capable of bioactivating CB1954 [5-(aziridin-1-yl)-2,4-dinitrobenzamide] to a cytotoxic agent, a property shared with the mammalian enzyme Walker DT diaphorase [NAD(P)H dehydrogenase (quinone), EC 1.6.99.2] as isolated from Walker cells. In contrast to Walker DT diaphorase, which can only reduce the 4-nitro group of CB1954, the E. coli nitroreductase can reduce either (but not both) nitro groups of CB1954 to the corresponding hydroxylamino species. The two hydroxylamino species are formed in equal proportions and at the same rates. CB1954 is reduced much more rapidly by the E. coli nitroreductase than by Walker DT diaphorase. If the reduction of CB1954 was carried out in the presence of V79 cells (which are insensitive to CB1954) a large cytotoxic effect was evident. This cytotoxicity was only observed under conditions in which the E. coli nitroreductase or Walker DT diaphorase reduced the drug. It is proposed that E. coli B nitroreductase would be a suitable enzyme for antibody-directed enzyme prodrug therapy (ADEPT) in combination with CB1954.     

Crystal structure of human DT-diaphorase: a model for interaction with the cytotoxic prodrug 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB1954).

The crystal structure of human DT-diaphorase (NAD(P)H oxidoreductase (quinone); EC 1.6.99.2) has been determined to 2.3 A resolution. There are only minor differences in shape and volume between the active sites of the rat and human enzymes and in the hydrophobic environment in the vicinity of the substrate. The isoalloxazine ring of the bound FAD is more buried in the human structure. Molecular modeling was used to examine optimal positions for the antitumor prodrug CB1954 (5-(aziridin-1-yl)-2,4-dinitrobenzamide) in both the human and rat enzyme active sites. This suggests that the position of CB1954 in the active site of the human enzyme is very similar to that in the rat, although there are detailed differences in the predicted patterns of hydrogen bonding between side chains and the drug. Some of the differences are a consequence of the shift in position for the FAD molecule and may contribute to the observed differences in rate of the two-electron reduction of CB1954.     

The differences in kinetics of rat and human DT diaphorase result in a differential sensitivity of derived cell lines to CB 1954 (5-(aziridin-1-yl)-2,4-dinitrobenzamide)

DT diaphorase (NAD(P)H dehydrogenase (quinone), EC 1.6.99.2) isolated from Walker 256 rat carcinoma cells can convert CB 1954 (5-(aziridin-1-yl)-2,4-dinitrobenzamide) to a cytotoxic DNA interstrand cross-linking agent. This is achieved by reduction of the 4-nitro group of CB 1954 to produce the hydroxylamino species, a bioactivation which accounts for the much greater sensitivity of Walker cells to CB 1954 when compared with other cells which are unable to carry out this reduction (Knox et al., Biochem Pharmacol 37: 4661-4669 and 4671-4677, 1988). As predicted from their measured DT diaphorase activities a number of rat hepatoma and hepatocyte cell lines were also shown to be sensitive to CB 1954. However, no CB 1954-sensitive cell lines of human origin were found, although levels of DT diaphorase similar to those in the sensitive rat cells were present in these cells. The human cells were as sensitive as rat cells to the active form of CB 1954 (5-(aziridin-1-yl)-4-hydroxyla mino-2-nitrobenzamide). DT diaphorase, purified to homogeneity from human Hep G2 cells, did metabolize CB 1954 to this 4-hydroxylamino product, but the rate of CB 1954 reduction and thus production of the cytotoxic product, was much lower than that of purified Walker enzyme (ratio of Kcat = 6.4). In addition, CB 1954 could be considered an inhibitor of, rather than a substrate for, the human form of DT diaphorase. The purified rat and human DT diaphorases possessed otherwise similar biochemical and molecular properties. These findings explain the decreased sensitivity towards CB 1954 of human cell lines when compared to rat cell lines.     

A new cytotoxic, DNA interstrand crosslinking agent, 5-(aziridin-1-yl)-4-hydroxylamino-2-nitrobenzamide, is formed from 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954) by a nitroreductase enzyme in Walker carcinoma cells

Walker tumour cells in vivo or in vitro are exceptionally sensitive to the monofunctional alkylating agent 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954) (Cobb LM et al., Biochem Pharmacol 18: 1519-1527, 1969). CB 1954 forms DNA interstrand crosslinks in a time-dependent manner in Walker tumour cells but not in non-toxically affected Chinese hamster V79 cells [(Roberts JJ et al., Biochem Biophys Res Commun 140: 1073-1078, 1986)]. However, co-culturing Chinese hamster V79 cells with Walker cells in the presence of CB 1954 renders the hamster cells sensitive to CB 1954 and leads to the formation of interstrand crosslinks in their DNA, findings indicative of the formation by Walker cells of a diffusible toxic metabolite of CB 1954. A flavoprotein, of molecular weight 33.5 kDa as estimated by SDS-polyacrylamide gel electrophoresis, has been isolated from Walker cells and identified as a form of NAD(P)H dehydrogenase (quinone) (DT diaphorase, EC 1.6.99.2). This enzyme, in the presence of NADH or NADPH, catalyses the aerobic reduction of CB 1954 to 5-(aziridin-1-yl)-4-hydroxylamino-2-nitrobenzamide. This new compound can form interstrand crosslinks in the DNA of Chinese hamster V79 cells to which it is also highly toxic.     

The bioactivation of 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB1954)--I. Purification and properties of a nitroreductase enzyme from Escherichia coli--a potential enzyme for antibody-directed enzyme prodrug therapy (ADEPT).

A nitroreductase enzyme has been isolated from Escherichia coli B. This enzyme is an FMN-containing flavoprotein with a molecular mass of 24 kDa and requires either NADH or NADPH as a cofactor. Partial protein sequence analysis showed extensive homology with the "classical nitroreductase" of Salmonella typhimurium and a nitroreductase induced in Enterobacter cloacae. In common with the Salmonella enzyme, the E. coli B enzyme is capable of reducing nitrofurazone. The E. coli nitroreductase is also capable of reducing the anti-tumour agent CB1954 [5-(aziridin-1-yl)-2,4-dinitrobenzamide], a property shared with the mammalian enzyme DT diaphorase [NAD(P)H dehydrogenase (quinone)] as isolated from Walker cells. The reduction of CB1954 by the E. coli enzyme results in the generation of cytotoxic species. Both enzymes also share the properties of being able to reduce quinones and are both inhibited by dicoumarol. The nitroreductase is a more active enzyme against CB1954 (kcat = 360 min-1) than Walker DT diaphorase (kcat = 4 min-1) and also has a lower Km for NADH (6 vs 75 microM).     

The nitroreductase enzyme in Walker cells that activates 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954) to 5-(aziridin-1-yl)-4-hydroxylamino-2-nitrobenzamide is a form of NAD(P)H dehydrogenase (quinone) (EC 1.6.99.2)

A nitroreductase enzyme has been isolated from Walker 256 rat carcinoma cells which can convert 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954) to a cytotoxic DNA interstrand crosslinking agent by reduction of its 4-nitro group to the corresponding hydroxylamino species (Roberts JJ et al., Biochem Biophys Res Commun 140: 1073-1078, 1986; Knox RJ et al., Biochem Pharmacol 37: 4661-4669, 1988). The enzyme has now been identified as a form of NAD(P)H dehydrogenase (quinone) (DT diaphorase, menadione reductase (NMOR), phylloquinone reductase, quinone reductase, EC 1.6.99.2) by comparison of partial protein sequences, coenzymes, substrate and inhibitor specificities, and spectroscopic data. 2-Phenyl-5(4)-aminoimidazole-4(5)-carboxamide and 5(4)-aminoimidazole-4(5)-carboxamide were shown to be inhibitors of the isolated Walker cell enzyme. This observation could explain the reported antagonistic action of the aminoimidazole carboxamides to the antitumour effects of CB 1954.     

Caffeine, aminoimidazolecarboxamide and dicoumarol, inhibitors of NAD(P)H dehydrogenase (quinone) (DT diaphorase), prevent both the cytotoxicity and DNA interstrand crosslinking produced by 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954) in Walker cells

A form of NAD(P)H dehydrogenase (quinone) (DT diaphorase, menadione reductase (NMOR), phylloquinone reductase, quinone reductase, EC 1.6.99.2) has been isolated from Walker 256 rat carcinoma cells. This enzyme can convert 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954) to a cytotoxic DNA interstrand crosslinking agent by reduction of its 4-nitro group to the corresponding hydroxylamino species (Knox et al. Biochem Pharmacol, 37: 4661-4669 and 4671-4677, 1988). 2-Phenyl-5(4)-aminoimidazole-4(5)-carboxamide and AICA [5(4)-aminoimidazole-4(5)-carboxamide] have previously been reported to be antagonists of the anti-tumour effects of CB 1954. We have shown that both these compounds are inhibitors of the above enzyme and that AICA protects against both the cytotoxicity and the formation of DNA interstrand crosslinks, produced by CB 1954 in Walker cells. Similarly, known inhibitors of NAD(P)H dehydrogenase (quinone) such as dicoumarol, also reduced the cytotoxicity and DNA-interstrand crosslinking of CB 1954 in Walker cells. Caffeine was shown to be a novel inhibitor of NAD(P)H dehydrogenase (quinone) and also elicited the above protective effects. All of the above inhibitors were also shown to potentiate the toxic effects of menadione against the Walker cell. This quinone is known to be detoxified by NAD(P)H dehydrogenase (quinone) and thus emphasises the ability of these compounds to inhibit this enzyme within the cell.     

Molecular interaction of the antagonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1- (2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide with the CB1 cannabinoid receptor

N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (SR141716; 1) is a potent and selective antagonist for the CB1 cannabinoid receptor. Using the AM1 molecular orbital method, conformational analysis of 1 around the pyrazole C3 substituent identified four distinct conformations designated Tg, Ts, Cg, and Cs. The energetic stability of these conformers followed the order Tg > Cg > Ts > Cs for the neutral (unprotonated) form of 1 and Ts > Tg > Cs > Cg for its piperidine N-protonated form. Unified pharmacophore models for the CB1 receptor ligands were developed by incorporating the protonated form of 1 into the superimposition model for the cannabinoid agonists 4-[4-(1,1-dimethylheptyl)-2-hydroxyphenyl]perhydro-2alpha,6beta-dihydroxynaphthalene (CP55244; 2) and the protonated form of (R)-[2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl](1-naphthalenyl)methanone (WIN55212-2; 3) reported previously (Shim et al. In Rational Drug Design Symposium Series; Parrill, A. L., Reddy, M. R., Eds.; American Chemical Society: Washington, DC, 1999; pp 165-184). Values of K(i) for 1 and a series of 31 structural analogues were determined from radioligand binding analyses by competitive displacement of [3H]CP55940 from cannabinoid receptors in a rat brain membrane preparation. Comparative molecular field analysis (CoMFA) was employed to construct three-dimensional (3D)-quantitative structure-activity relationship (QSAR) models for this data set as unprotonated species assuming the Tg, Cg, and Ts conformers and for the protonated species assuming the Ts, Tg, and Cs conformers. Values of the conventional r2 and cross-validated r2 (r(cv)2) associated with these CoMFA models exceeded the threshold for statistical robustness (r2 > or = 0.90) and internal predictive ability (r(cv)2 > or = 0.50) in each of these six cases except for the protonated species assuming the Tg conformer (i.e., r2 = 0.97; r(cv)2 = 0.36). Results from conformational analyses, superimposition models, and 3D-QSAR models suggest that the N1 aromatic ring moiety of 1 dominates the steric binding interaction with the receptor in much the same way as does the C3 alkyl side chain of cannabinoid agonists and the C3 aroyl ring of the aminoalkylindole agonists. We also determined that several of the conformers considered in this study possess the proper spatial orientation and distinct electrostatic character to bind to the CB1 receptor. We propose that the unique region in space occupied by the C5 aromatic ring of 1 might contribute to conferring antagonist activity. We further propose that the pyrazole C3 substituent of 1 might contribute to conferring either neutral antagonist or inverse agonist activity, depending upon the interaction with the receptor.     

Absorption and metabolism of 5-(4-nitrophenyl)-2,4-pentadienal (Spydust)

5-(4-Nitrophenyl)-2,4-pentadienal (NPPD) was recently alleged to have been used as a tracking agent to monitor the activities of U.S. citizens in the Soviet Union. In order to better assess human risk from possible exposure to this compound the absorption and metabolism of [14C]NPPD has been investigated in male F344 rats. These studies have revealed that NPPD was readily and quantitatively absorbed from the gastrointestinal tract, distributed throughout the tissues, metabolized, and rapidly excreted primarily in urine. NPPD was sparingly absorbed following dermal administration of 0.01, 0.1, and 1.0 mg/cm2. The amount absorbed increased as the dose increased but the percentage of the dose absorbed decreased as the dose increased, e.g., 50% (5 of 10 micrograms applied) of the low dose was absorbed but only 5% (50 of 100 micrograms applied) of the high dose was absorbed. The material absorbed after dermal administration was rapidly excreted and the distribution and metabolism of the dermally administered compound was no different from oral administration. A total of five metabolites, 4-nitrocinnamic acid, 4-acetamidobenzoic acid, 4-nitrohippuric acid, 4-acetamidocinnamic acid, and 4-nitrobenzoic acid, were identified by cochromatography with authentic standards and comparison of UV spectra. These metabolites are formed by oxidative metabolism of the pentadienal side chain, reduction of the nitro group, and/or conjugation of the resulting amino group with acetate or carboxylic acid with glycine.     

Hepatic nitroreduction, toxicity and toxicokinetics of the anti-tumour prodrug CB 1954 in mouse and rat

5-(Aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954), a promising anti-tumour compound, is associated with clinical hepatotoxicity. We have previously demonstrated that human liver preparations are capable of endogenous 2- and 4-nitroreduction of CB 1954 to generate highly potent cytotoxins. The present study initially examined the in vitro metabolism of CB 1954 in S9 preparations of several non-clinical species and strains. The CD-1 nu/nu mouse and Sprague-Dawley rat were subsequently chosen for further assessment of in vivo metabolism and hepatotoxicity of CB 1954, as well as the mechanisms that may be involved. Animals were administered the maximum tolerated dose (MTD). At 562 micromol/kg, the mouse exhibited transaminase elevation and centrilobular hepatocyte injury. Moreover, thiol adducts as well as hepatic glutathione depletion paralleled temporally by maximal nitroreduction were observed. The rat had a much lower MTD of 40 micromol/kg and showed signs of gastro-intestinal disturbances. In contrast to mouse, peri-portal damage and biliary changes were observed in rat without any alterations in plasma biomarkers or hepatic glutathione levels. Immunohistochemical analysis did not reveal any correlation between the location of injury and expression of cytochrome P450 reductase and NAD(P)H quinone oxidoreductase 1, two enzymes implicated in the bioactivation of this drug. In conclusion, the present study showed that following administration of CB 1954 at the respective MTDs, hepatotoxicity was observed in both mouse and rat. However, the degree of sensitivity to the drug and the mechanisms of toxicity involved appear to be widely different between CD-1 nu/nu mice and Sprague-Dawley rats.     

Synthesis and structure-activity relationships of amide and hydrazide analogues of the cannabinoid CB(1) receptor antagonist N-(piperidinyl)- 5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (SR141716)

Analogues of the biaryl pyrazole N-(piperidinyl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (SR141716; 5) were synthesized to investigate the structure-activity relationship (SAR) of the aminopiperidine region. The structural modifications include the substitution of alkyl hydrazines, amines, and hydroxyalkylamines of varying lengths for the aminopiperidinyl moiety. Proximity and steric requirements at the aminopiperidine region were probed by the synthesis of analogues that substitute alkyl hydrazines of increasing chain length and branching. The corresponding amide analogues were compared to the hydrazides to determine the effect of the second nitrogen on receptor binding affinity. The N-cyclohexyl amide 14 represents a direct methine for nitrogen substitution for 5, reducing the potential for heteroatom interaction, while the morpholino analogue 15 adds the potential for an additional heteroatom interaction. The series of hydroxyalkyl amides of increasing chain length was synthesized to investigate the existence of additional receptor hydrogen binding sites. In displacement assays using the cannabinoid agonist [(3)H](1R,3R,4R)-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-4-(3-hydroxypropyl) cyclohexan-1-ol (CP 55 940; 2) or the antagonist [(3)H]5, 14 exhibited the highest CB(1) affinity. In general, increasing the length and bulk of the substituent was associated with increased receptor affinity and efficacy (as measured in a guanosine 5'-triphosphate-gamma-[(35)S] assay). However, in most instances, receptor affinity and efficacy increases were no longer observed after a certain chain length was reached. A quantitative SAR study was carried out to characterize the pharmacophoric requirements of the aminopiperidine region. This model indicates that ligands that exceed 3 A in length would have reduced potency and affinity with respect to 5 and that substituents with a positive charge density in the aminopiperidine region would be predicted to possess increased pharmacological activity.     

Interactions between potential anti-tumour 2,5-bis(1-aziridinyl)-1,4-benzoquinone derivatives and glutathione: reductive activation, conjugation and DNA damage

The interaction between glutathione and potential anti-tumour 3,6-disubstituted 2,5-bis(1-aziridinyl)-1,4-benzoquinone (BABQ) derivatives has been studied using u.v. spectrophotometry and h.p.l.c. The formation of BABQ-glutathione adducts was demonstrated in vitro for the BABQ parent compound (TW13), triaziquone (2,3,5-tris(1-aziridinyl)-1,4-benzoquinone) and for BABQ derivatives containing halogen substituents. The clinically-used BABQ derivative diaziquone (AZQ; 2,5-bis(1-aziridinyl)-3,6-bis(ethoxycarbonylamino)-1,4-benzoquinon e) did not react with glutathione. TW13 and triaziquone markedly inactivated bacteriophage M13-DNA in the presence of glutathione. This inactivation is probably produced by reductive activation of the BABQ derivative to a DNA-alkylating semiquinone radical. However, formation of bulky glutathione adducts decreases reactivity to DNA. Halogen-substituted BABQ derivatives react rapidly with glutathione to form adducts. This appeared to prevent DNA alkylation by these compounds. Comparison of these results with in vivo and in vitro activity against tumour models (L1210) suggests that in vivo halogen-substituted BABQ derivatives are efficiently inactivated by glutathione conjugation. The differences between the halogen-substituted BABQ derivatives on the one hand and TW13 and triaziquone on the other hand are probably caused by a difference in reaction mechanism with glutathione. From the viewpoint of drug design, halogen-substituted BABQ derivatives are expected to be inactive anti-tumour agents, in spite of high reactivity and activity in tumour models in vitro.     

N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (SR141716A) interaction with LYS 3.28(192) is crucial for its inverse agonism at the cannabinoid CB1 receptor

In superior cervical ganglion neurons, N-(piperidiny-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (SR141716A) competitively antagonizes the Ca(2+) current effect of the cannabinoid (CB) agonist (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone (WIN55212-2), and behaves as an inverse agonist by producing opposite current effects when applied alone. In contrast, in neurons expressing CB1 with a K-->A mutation at residue 3.28(192) (i.e., K3.28A), SR141716A competitively antagonizes the effects of WIN55212-2, but behaves as a neutral antagonist by producing no current effects itself. Receptor modeling studies suggested that in the CB1 inactive (R) state, SR1417A16A stabilizes transmembrane helix 6 in its inactive conformation via aromatic stacking with F3.36/W6.48. In this binding site, SR141716A would exhibit higher affinity for CB1 R due to a hydrogen bond between the SR141716A C3 substituent and K3.28(192), a residue available to SR141716A only in R. To test this hypothesis, a "mutant thermodynamic cycle" was constructed that combined the evaluation of SR141716A affinity at WT CB1 and K3.28A with an evaluation of the wild-type CB1 and K3.28A affinities of an SR141716A analog, 5-(4-chlorophenyl)-3-[(E)-2-cyclohexylethenyl]-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole (VCHSR), that lacks hydrogen bonding potential at C3. Binding affinities suggested that K3.28 is involved in a strong interaction with SR141716A in WT CB1, but does not interact with VCHSR. Thermodynamic cycle calculations indicated that a direct interaction occurs between the C3 substituent of SR141716A and K3.28 in WT CB1. Consistent with these results, VCHSR acted as a neutral antagonist at WT CB1. These results support the hypothesis that hydrogen bonding of the SR141716A C3 substituent with K3.28 is responsible for its higher affinity for the inactive R state, leading to its inverse agonism.