Evaluation of farnesyl:protein transferase and geranylgeranyl:protein transferase inhibitor combinations in preclinical models.

Farnesyl:protein transferase (FPTase) inhibitors (FTIs) were originally developed as potential anticancer agents targeting the ras oncogene and are currently in clinical trials. Whereas FTIs inhibit the farnesylation of Ha-Ras, they do not completely inhibit the prenylation of Ki-Ras, the allele most frequently mutated in human cancers. Whereas farnesylation of Ki-Ras is blocked by FTIs, Ki-Ras remains prenylated in FTI-treated cells because of its modification by the related prenyltransferase, geranylgeranyl:protein transferase type I (GGPTase-I). Hence, cells transformed with Ki-ras tend to be more resistant to FTIs than Ha-ras-transformed cells. To determine whether Ki-ras-transformed cells can be targeted by combining an FTI with a GGPTase-I inhibitor (GGTI), we evaluated potent, selective FTIs, GGTIs, and dual prenylation inhibitors (DPIs) that have both FTI and GGTI activity. We find that in human PSN-1 pancreatic tumor cells, which harbor oncogenic Ki-ras, and in other tumor lines having either wild-type or oncogenic Ki-ras, treatment with an FTI/GGTI combination or with a DPI blocks Ki-Ras prenylation and induces markedly higher levels of apoptosis relative to FTI or GGTI alone. We demonstrate that these compounds can inhibit their enzyme targets in mice by monitoring pancreatic and tumor tissues from treated animals for inhibition of prenylation of Ki-Ras, HDJ2, a substrate specific for FPTase, and Rap1A, a substrate specific for GGPTase-I. Continuous infusion (72 h) of varying doses of GGTI in conjunction with a high, fixed dose of FTI causes a dose-dependent inhibition of Ki-Ras prenylation. However, a 72-h infusion of a GGTI, at a dose sufficient to inhibit Ki-Ras prenylation in the presence of an FTI, causes death within 2 weeks of the infusion when administered either as monotherapy or in combination with an FTI. DPIs are also lethal after a 72-h infusion at doses that inhibit Ki-Ras prenylation. Because 24 h infusion of a high dose of DPI is tolerated and inhibits Ki-Ras prenylation, we compared the antitumor efficacy from a 24-h FTI infusion to that of a DPI in a nude mouse/PSN-1 tumor cell xenograft model and in Ki-ras transgenic mice with mammary tumors. The FTI and DPI were dosed at a level that provided comparable inhibition of FPTase. The FTI and the DPI displayed comparable efficacy, causing a decrease in growth rate of the PSN-1 xenograft tumors and tumor regression in the transgenic model, but neither treatment regimen induced a statistically significant increase in tumor cell apoptosis. Although FTI/GGTI combinations elicit a greater apoptotic response than either agent alone in vitro, the toxicity associated with GGTI treatment in vivo limits the duration of treatment and, thus, may limit the therapeutic benefit that might be gained by inhibiting oncogenic Ki-Ras through dual prenyltransferase inhibitor therapy.

Statins suppress THP-1 cell migration and secretion of matrix metalloproteinase 9 by inhibiting geranylgeranylation.

Macrophages secrete matrix metalloproteinase 9 (MMP-9), an enzyme that weakens the fibrous cap of atherosclerotic plaques, predisposing them to plaque rupture and subsequent ischemic events. Recent work indicates that statins strongly reduce the possibility of heart attack. Furthermore, these compounds appear to exert beneficial effects not only by lowering plasma low-density-lipoprotein cholesterol but also by directly affecting the artery wall. To evaluate whether statins influence the proinflammatory responses of monocytic cells, we studied their effects on the chemotactic migration and MMP-9 secretion of human monocytic cell line THP-1. Simvastatin dose dependently inhibited THP-1 cell migration mediated by monocyte chemoattractant protein 1, with a 50% inhibitory concentration of about 50 nM. It also inhibited bacterial lipopolysaccharide-stimulated secretion of MMP-9. The effects of simvastatin were completely reversed by mevalonate and its derivatives, farnesylpyrophosphate and geranylgeranyl pyrophosphate, but not by ubiquinone. Additional studies revealed similar but more profound inhibitory effects with L-839,867, a specific inhibitor of geranylgeranyl transferase. However, alpha-hydroxyfarnesyl phosphonic acid, an inhibitor of farnesyl transferase, had no effect. C3 exoenzyme, a specific inhibitor of the prenylated small signaling Rho proteins, mimicked the inhibitory effects of simvastatin and L-839,867. These data supported the role of geranylgeranylation in the migration and MMP-9 secretion of monocytes.

Synthesis and biological activity of angiotensin II analogues containing a Val-His replacement, Val psi[CH(CONH2)NH]His.

The dipeptide mimic Val psi[CH(CONH2)NH]His (4) was incorporated into angiotensin II (AII) analogues to provide an octapeptide saralasin derivative (29) as well as tetrapeptide analogue 19. Three C-terminal tetrapeptides (21, 25, and 28) were also prepared. All compounds were tested for their ability to displace 3H-AII from rabbit adrenal gland homogenate and as antagonists of AII and AI on guinea pig ileum. The octapeptide analogue 29 was 700 times less active than the parent peptide 30. All the C-terminal fragments 19, 21, 25, and 28 have no measurable AII antagonist activity. Of the four tetrapeptide fragments, only 21 showed any appreciable binding activity.

Cardiotonic agents. 1. Novel 8-aryl-substituted imidazo[1,2-a]- and -[1,5-a]pyridines and imidazo[1,5-a]pyridinones as potential positive inotropic agents.

Several 8-arylimidazo[1,2-a]pyridines, 8-arylimidazo[1,5-a]pyridines, and 8-arylimidazo[1,5-a]pyridinones were prepared and tested in vitro for potential cardiac inotropic and electrophysiological activity. Selected analogues were further tested in vivo in canine hemodynamic and cardiac electrophysiology models. Compounds having an imidazole substituent consistently showed activity. A pharmacophoric relationship between heterocycle-phenyl-imidazole and positive inotropic activity was noted. The significance of this relationship is discussed.

Piperazinylquinoxalines with central serotoninmimetic activity.

Regioselective syntheses of substituted 2-chloroquinoxalines and derived 2-(1-piperazinyl)quinoxalines are described. Selectivity in regards to serotonin reuptake blocking and serotoninmimetic activities of the piperazinylquinoxalines is reported. In general, introduction of a 6-substituent into the piperazinylquinoxaline enhanced serotonin reuptake blocking activity and diminished serotoninmimetic activity. Unsubstituted and 3-hydroxypiperazinylquinoxalines had primarily serotoninmimetic activity.

Synthesis, cardiac electrophysiology, and beta-blocking activity of novel arylpiperazines with potential as class II/III antiarrhythmic agents.

A series of novel arylpiperazines have been prepared in an attempt to incorporate both class II (beta-receptor blocking) and class III antiarrhythmic properties in a single molecule. The key step in the preparation of the new compounds involves a regioselective heterocyclic ring formation. All but four compounds significantly prolonged action potential duration in canine cardiac Purkinje fibers (class III activity). All but one of the compounds demonstrated beta-receptor affinity in a competitive binding assay and three had beta 1-receptor selectivity. Compared to sotalol, a reference class II/III agent, arylpiperazine 7a (4-[(methylsulfonyl)amino]-N-[(4- phenylpiperazin-2-yl)methyl]benzamide) demonstrated beta 1-selectivity and was 1 order of magnitude more potent in the in vitro class III and the beta 1-receptor screens. Compound 7a was evaluated further and found to be effective in preventing programmed electrical stimulation-induced arrhythmias in conscious dogs (class III activity) and against epinephrine-induced arrhythmias in halothane anesthetized dogs (class II activity).

Piperazinylpyrazines with central serotoninmimetic activity.

A series of 2-(1-piperazinyl)pyrazines was synthesized and evaluated for central serotonin-like activity. The most interesting member of the series, 6-chloro-2(1-piperazinyl)pyrazine (3a), had pharmacological properties characteristic of potent central serotoninmimetic activity and only weak peripheral serotoninmimetic action. Structural similarities between 3a and serotonin are discussed.

Piperazinylimidazo[1,2-a]pyrazines with selective affinity for in vitro alpha-adrenergic receptor subtypes.

Regioselective syntheses of alkyl- and halogen-substituted piperazinylimidazo[1,2-a]pyrazines by novel oxidation-dehydration of [(beta-hydroxyalkyl)amino]pyrazines are described. Lanthanide shift reagent studies allowed correction of literature assignments of NMR chemical shifts and coupling constants for the imidazo[1,2-a]pyrazine ring system (e.g., J5,8 greater than J6,8). Equilibrium constants for displacement of specifically bound [3H]clonidine and [3H]prazosin from calf cerebral cortex homogenates in vitro are tabulated for reference and title compounds, and structure-affinity relationships for alpha 2- vs. alpha 1-adrenergic receptors are considered. Compound 2a, 8-(1-piperazinyl)imidazo[1,2-a]pyrazine, is equipotent with mianserin on the clonidine receptor (alpha 2) but ca. 70 times as selective as mianserin for this alpha 2-adrenergic receptor. Reduction of the imidazo ring (2,3-dihydro) lowers affinity for the alpha 2 receptor without affecting alpha 1-receptor affinity. Computer-assisted molecular modeling techniques are applied to the estimation of conformational energies of 2a and its 5-position isomer in relation to the semirigid molecule mianserin.

Synthesis and antiarrhythmic activity of novel 3-alkyl-1-[omega-[4-[(alkylsulfonyl)amino]phenyl]-omega- hydroxyalkyl]-1H-imidazolium salts and related compounds.

Novel 3-alkyl-1-[omega-[4-[(alkylsulfonyl)amino]phenyl]-omega-hydroxyalkyl]-1H -imidazolium salts were synthesized and investigated for their class III electrophysiological activity on isolated canine cardiac Purkinje fibers and ventricular muscle tissue. Structure-activity relationships are discussed for a series of 25 compounds. Compound 3, 1-[2-hydroxy-2-[4-[(methylsulfonyl)amino]phenyl]ethyl]-3-methyl-1H- imidazolium chloride, prolonged the functional refractory period in anesthetized dogs when given intraduodenally and was also effective in preventing reentrant ventricular tachycardia induced by programmed electrical stimulation when administered intravenously in anesthetized dogs 24 h after an acute myocardial infarction. Both enantiomers of 3 were synthesized. No enantioselectivity was found in the electrophysiological effects of 3.

An approach to peripheral vasodilator-beta-adrenergic blocking agents.

The syntheses of 2-phenyl- and 2-pyridyl-4-trifluoromethylimidazoles having a 3-tert-butylamino-2-hydroxypropoxy moiety attached to the aryl or heteroaryl substituent are described. Structure--activity relationships based on results from an evaluation of these compounds for antihypertensive, vasodilating, and beta-adrenergic blocking activities are discussed.

Heterocyclic analogues of benzamide antiarrhythmic agents.

A series of heterocyclic N-[(diethylamino)alkyl]arenamides related to acecainide was prepared and examined for antiarrhythmic activity. The compounds were synthesized from the corresponding known heterocyclic carboxylic acids or esters by using standard amide formation methods. The effects of the compounds on the electrophysiological properties of canine Purkinje fibers and ventricular muscle strips were determined. Most of the compounds showed effects consistent with weak class I activity. Two compounds, N-[2-(diethylamino)ethyl]-3,4,5-trimethyl-1H-pyrrole-2-carboxamide and N-[2-(diethylamino)ethyl]-1H-indole-2-carboxamide, displayed prolongation of the action potential duration and functional refractory period indicative of modest class III electrophysiological activity. Representative compounds were examined by using molecular modeling techniques. Compounds of differing activity classes displayed qualitatively different electrostatic potential maps.

Synthesis and class III antiarrhythmic activity of (phenylbut-2-enyl)ammonium salts. Effect of conformation on activity.

The syntheses of seven 4-(substituted phenyl)but-2-en(or yn)yl quaternary ammonium salts and four related tertiary amines are described. The Meerwein arylation reaction was the preferred synthetic method for the required intermediate 1-aryl-4-halo-2-butenes (15a-c, 18). In the case of 18, the trans stereochemistry of the Meerwein adduct of 2,3-dimethylbutadiene was established unambiguously by 2D NMR and X-ray studies. The title compounds represent conformationally restricted analogues of the class III antiarrhythmic agent clofilium (1) and exhibit comparable potency and efficacy in the in vitro evaluation using isolated canine Purkinje fibers. These results suggest that the alkylene chain in 1 is extended in the active conformation. Computer-aided conformational analysis (MM2) supports this conclusion. Selective catalytic hydrogen conditions were developed for the conversion of the unsaturated analogue 2 to clofilium (1) with minimal hydrogenolysis of the allylic quaternary ammonium moiety, thus completing a novel and efficient synthesis of this substance.

Synthesis of a series of 4-(arylethynyl)-6-chloro-4-cyclopropyl-3,4-dihydroquinazolin-2(1H)-ones as novel non-nucleoside HIV-1 reverse transcriptase inhibitors.

As part of an ongoing effort to prepare novel non-nucleoside inhibitors of human immunodeficiency virus type-1 (HIV-1) reverse transcriptase (RT), a series of 4-(arylethynyl)-6-chloro-4-cyclopropyl-3,4-dihydroquinazolin -2(1H)-ones 4aa-l has been prepared. Target compounds 4a-e were synthesized via addition of various 1-lithio-2-(aryl)alkyne nucleophiles to a 1-protected-4-cyclopropylquinazolin-2(1H)-one (7), followed by deprotection. The 3-methyl compound 4aa was prepared in an analogous manner, with the 3-alkylation performed prior to deprotection. Alternatively, the target compounds 4f-l were prepared by addition of 1-lithio-2-(trimethylsilyl)acetylene to 7, followed by deprotection and subsequent palladium-catalyzed coupling with various aryl halides. By incorporating an aryl group onto the end of the 4-acetylene functionality, the requirement for a metabolically labile 3-methyl group on the dihydroquinazolinone nucleus has been eliminated. A number of the target compounds were shown to be potent inhibitors of HIV-1 RT. Compound 4a, which had exhibited the most favorable overall biological profile, was resolved via a four-step procedure to provide the enantiomers 13a and 13b. Compound 13a having the (-)-4(S) configuration was shown to be the active enantiomer and was selected as a candidate for further investigation.

Design of novel, potent, noncovalent inhibitors of thrombin with nonbasic P-1 substructures: rapid structure-activity studies by solid-phase synthesis.

Study of surface representations of the inhibitor-bound thrombin P-1 pocket revealed a lipophilic recess in this pocket which is not occupied by any known inhibitor. Solid-phase synthesis was used to generate benzylamides of D-diphenylAlaPro by aminolysis of Boc dipeptide Kaiser resin. The resulting amides inhibited thrombin in the range IC50 = 3-13,000 nM, and the structure-activity relationships and molecular modeling suggest a unique fit of the benzyl side chain into P-1 with the meta substituent occupying the recess.

Conformational requirements for histamine H2-receptor inhibitors: a structure-activity study of phenylene analogues related to cimetidine and tiotidine.

Two series of compounds related to cimetidine and tiotidine were synthesized as part of a study to evaluate the importance of conformational parameters in binding at histamine H2 receptors. The flexible methylthioethyl connecting chain was replaced by a conformationally restricting phenylene unit. These compounds were evaluated for antagonism of the dimaprit-stimulated chronotropic response in the guinea pig atrium and inhibition of histamine stimulated secretion of gastric acid in the dog. In both series, biological activity is markedly dependent on the m-phenylene regioisomers. Histamine H2-receptor activity is retained in both series; however, in the tiotidine series, gastric antisecretory activity is significantly improved. Regardless of the end group, N-cyanoguanidine 1,1-diamino-2-nitroethene or 3,4-diamino-1,2,5-thiadiazole 1-oxide, each 3 3-(2-guanidino-4-thiazolyl)phenyl analogue was ca. 8 and 90 times more potent intravenously than tiotidine and cimetidine, respectively. The electronic influences of the phenylene unit on biological activity were also evaluated. It was concluded that the geometric constraints imposed by the m-phenylene connecting element were more important than electronic factors in binding events at the histamine H2 receptor.

Cardiotonic agents. 2. (Imidazolyl)aroylimidazolones, highly potent and selective positive inotropic agents.

A series of 4-alkyl-1,3-dihydro-5-[(1H-imidazolyl)benzoyl]-2H-imidazol-2-ones 9 was synthesized and evaluated in vitro for positive inotropic and cyclic AMP phosphodiesterase inhibitory activity. A wide range of inotropic and enzyme-inhibitory potencies was observed, substitution on the imidazolyl moiety being the major determinant of activity. The 4-ethyl-5-[4-(1H-imidazol-1-yl)benzoyl] congener 9g exhibited the highest potency in vitro. Incorporation of a methyl group at the imidazolyl 2-position gave 9h, which was less potent but remarkably selective in vivo for positive inotropic effects over heart rate and hypotensive effects.

Synthesis and antiarrhythmic activity of novel 3-alkyl-1-[omega-[4-[(alkylsulfonyl)amino]phenyl]-omega- hydroxyalkyl]-1H-imidazolium salts and related compounds. 2.

Novel analogues of the class III antiarrhythmic agent 1-[2-hydroxy-2-[4-[(methylsulfonyl)amino]phenyl]ethyl]-3-methyl-1H- imidazolium chloride, 1 (CK-1649), were prepared and investigated for their class III electrophysiological activity on isolated canine cardiac Purkinje fibers and ventricular muscle tissue. Structure-activity relationships are discussed for a series of 11 compounds. One compound, N-[4-[1-hydroxy-2-(4,5-dihydro-2-methyl-1H-imidazol-1- yl)ethyl]phenyl]methanesulfonamide hydrochloride, 9, was comparable in activity to 1 in vitro and prolonged the functional refractory period in anesthetized dogs when given intraduodenally. Unlike 1, compound 9 was ineffective at preventing ventricular tachycardia induced by programmed electrical stimulation in anesthetized dogs 24 h after an acute myocardial infarction.

Synthesis and cardiac electrophysiological activity of N-substituted-4-(1H-imidazol-1-yl)benzamides--new selective class III agents.

The synthesis and cardiac electrophysiological activity of 18 N-substituted imidazolylbenzamides or benzene-sulfonamides are described. Compounds 6a,d,f-k and 11 exhibited potency in the in vitro Purkinje fiber assay comparable to that of N-[2-(diethylamino)ethyl]-4- [(methylsulfonyl)amino]benzamide (1, sematilide), a potent selective class III agent which is undergoing clinical trials. These data indicate that the 1H-imidazol-1-yl moiety is a viable replacement for the methylsulfonylamino group for producing class III electrophysiological activity in the N-substituted benzamide series. N-[2-(Diethylamino)ethyl]-4-(1H-imidazol-1-yl)benzamide dihydrochloride (6a) was further studied in two in vivo models of reentrant arrhythmias and showed potency and efficacy comparable to those of 1.

Cardiotonic agents. 6. Histamine analogues as potential cardiovascular selective H2 agonists.

Twenty-six alkyl and aralkyl histamine analogues were prepared as potential cardiotonic agents. Compounds were designed to allow interaction with a putative secondary aryl binding site at the H2 receptor, the presence of which was inferred from the structure of cyprohepatadine, which is known to have H2-antagonist properties. The compounds were examined for inotropic activity in ferret papillary muscle. Potent inotropic activity was generally found in N-alkyl- and N,N-dialkylimidazole-4-ethanamines, whereas N-(amidoalkyl)imidazole-4-ethanamines and N-alkylimidazole-4-propanamines were at best weakly active. Five compounds were examined in screens designed to assess hemodynamic effects and gastric acid secretion in vivo. Two of these compounds, alpha-(3-phenyl-2-transpropenyl)-1H-imidazole-4-ethanamine and N-heptyl-1H-imidazole-4-ethanamine, showed positive inotropic activity with minimal effects on heart rate and mean arterial pressure in vivo; however, both compounds were found to stimulate gastric acid secretion. These results demonstrate that selectivity between various H2-receptor-mediated activities can be obtained with substituted histamine analogues.

A series of potent HIV-1 protease inhibitors containing a hydroxyethyl secondary amine transition state isostere: synthesis, enzyme inhibition, and antiviral activity.

A series of HIV-1 protease inhibitors containing a novel hydroxyethyl secondary amine transition state isostere has been synthesized. The compounds exhibit a strong preference for the (R) stereochemistry at the transition state hydroxyl group. Molecular modeling studies with the prototype compound 11 have provided important insights into the structural requirements for good inhibitor-active site binding interaction. N-Terminal extension of 11 into the P2-P3 region led to the discovery of 19, the most potent enzyme inhibitor in the series (IC50 = 5.4 nM). 19 was shown to have potent antiviral activity in cultured MT-4 human T-lymphoid cells. Comparison of analogs of 19 with analogs of 1 (Ro31-8959) demonstrates that considerably different structure-activity relationships exist between these two subclasses of hydroxyethylamine HIV-protease inhibitors.