Enzymatic assays for 2,4-diamino-5-methyl-6-[(3,4,5-trimethoxyanilino)methyl]quinazoline, a promising new "nonclassical" antifolate

2,4-Diamino-5-methyl-6-[(3,4,5-trimethoxyanilino)-methyl]quinazoline (I) is a promising new "nonclassical" antifolate. Inhibition of dihydrofolate reductase from bacterial (Lactobacillus casei)and mammalian (beef liver) sources was employed to develop useful enzymatic assays for this compound. A linear relationship was obtained by plotting the I concentration versus 1/V. The resultant standard curves maintained linearity particularly well between the 30 and 70% control range, with a correlation coefficient of 0.99 for both enzyme systems. The two enzyme systems are characterized by differences in sensitivity, stability, and day-to-day variation. The ID50 for the beef liver reductase system was 1.6 X 10(-9) M (+/-0.03); for the L. casei system, it was 1.35 X 10(-8) M (+/- 0.2). The apparent advantage for the beef liver enzyme was offset somewhat by its relative instability and its higher day-to-day variability. Studies in mice demonstrated that these assays are suitable for pharmacokinetic studies in vivo. Such studies indicated that I has a serum t1/2 of 45 min in mice; a similar serum t1/2 (50 min) was estimated in studies with 14C-labeled I in position 6.     

Inhibition of metabolism of the 'nonclassical' antifolate, trimetrexate (2,4-diamino-5-methyl-6-[(3,4,5-trimethoxyanilino)methyl]quinazoline) by drugs containing an imidazole moiety

2,4-Diamino-5-methyl-6-[(3,4,5-trimethoxyanilino)methyl]quinazoline (TMQ; NSC No. 249008) is a 'nonclassical' antifolate which is advocated as an alternative to methotrexate. TMQ is rapidly and extensively demethylated by a cytochrome P-450-mediated oxidation to a weakly cytotoxic compound of increased polarity. In this study, the effects of clinically used imidazole drugs ketoconazole, miconazole, clotrimazole, cimetidine, etintidine, clonidine and cibenzoline on the rat hepatic microsomal demethylation of TMQ in vitro was investigated. The nitrogen-substituted imidazole drugs (ketoconazole, miconazole, and clotrimazole) were potent non-competitive inhibitors of TMQ metabolism with IC50 values obtainable at therapeutic doses (less than 2 microM). Cimetidine and etintidine were comparatively weak, competitive inhibitors of TMQ metabolism (IC50 greater than 300 microM) and clonidine and cibenzoline were even less inhibitory (IC50 greater than 1 mM). The nitrogen-substituted imidazole drugs have the potential to dramatically alter the pharmacokinetic and pharmacodynamic profile of TMQ as well as other drugs in vivo.     

Pneumocystis carinii and Toxoplasma gondii dihydrofolate reductase inhibitors and antitumor agents: synthesis and biological activities of 2,4-diamino-5-methyl-6-[(monosubstituted anilino)methyl] pyrido[2,3-d]pyrimidines.

Thirteen 2,4-diamino-5-methyl-6-[(monosubstituted anilino)methyl]pyrido[2,3-d]pyrimidines 5-17 were synthesized as potential Pneumocystis carinii (pc) and Toxoplasma gondii (tg) dihydrofolate reductase (DHFR) inhibitors and as antitumor agents. Compounds 5-17 were designed to investigate the structure-activity relationship of monomethoxy and monohalide substitution in the phenyl ring and N10-methylation of the C9-N10 bridge. The synthetic route to compounds 5-12 involved the reductive amination of a common intermediate, 2,4-diamino-5-methylpyrido[2, 3-d]pyrimidine-6-carbonitrile (18), with the appropriate anilines. N10-Methylation was achieved by reductive methylation. In contrast to previous reports of trimethoprim, the removal of methoxy and chloro groups from the phenyl ring in the 2, 4-diamino-5-methyl-6-[(substituted anilino)methyl]pyrido[2, 3-d]pyrimidine series generally did not decrease DHFR inhibitory activity. The monosubstituted phenyl analogues 5-12 were as potent against pcDHFR and tgDHFR as the previously reported disubstituted phenyl analogues. N10-Methylation generally resulted in a marginal increase in potency against both pcDHFR and tgDHFR. Compounds 5, 7, and 9 were evaluated and shown to inhibit the growth of T. gondii cells in culture at nanomolar concentrations. Compounds 6-8, 9, 11, and 16 were selected by the National Cancer Institute for evaluation in an in vitro preclinical antitumor screening program. All six compounds showed GI50 values in the 10(-7)-10(-9) M range in more than 20 cell lines.     

Design, synthesis, and biological evaluation of 2,4-diamino-5-methyl-6-substituted-pyrrolo[2,3-d]pyrimidines as dihydrofolate reductase inhibitors.

2,4-diamino-5-methyl-6-(substituted-phenyl)thiopyrrolo[2,3-d]pyrimidines 4-11 were synthesized as dihydrofolate reductase (DHFR) inhibitors against opportunistic pathogens that afflict patients with AIDS. Synthesis was achieved from 2,4-diamino-5-methypyrrolo[2,3-d]pyrimidine and substituted phenylthiols under modified conditions reported by Gangjee et al. Some of these compounds were potent and selective against DHFR from both Toxoplasma gondii and Mycobacterium avium compared to mammalian DHFR. Compound 11 with a 1-naphthyl substituent is 16-fold more potent and equally selective against Toxoplasma gondii DHFR as the clinically used trimethoprim.     

Formulation of de novo substituent constants in correlation analysis: inhibition of dihydrofolate reductase by 2,4-diamino-5-(3,4-dichlorophenyl)-6-substituted pyrimidines.

A correlation equation based solely on de novo constants was formulated for 105 2,4-diamino-5-(3,4-dichlorophenyl)-6-substituted pyrimidines acting as inhibitors of dihydrofolate reductase. An equation with seven indicator variables gives a correlation with a correlation coefficient of 0.903 and a standard deviation of 0.229. The technique used is a modification of the Free-Wilson approach. The results indicate that correlation equations with fewer parameters than the theoretical required to account for all molecular changes may often be encountered. It is also shown that cross-product terms can be used to establish the significance of cooperative substituent effects.     

Synthesis of 2,4-diamino-6-[2'-O-(omega-carboxyalkyl)oxydibenz[b,f]azepin-5-yl]methylpteridines as potent and selective inhibitors of Pneumocystis carinii, Toxoplasma gondii, and Mycobacterium avium dihydrofolate reductase

Six previously undescribed N-(2,4-diaminopteridin-6-yl)methyldibenz[b,f]azepines with water-solubilizing O-carboxyalkyloxy or O-carboxybenzyloxy side chains at the 2'-position were synthesized and compared with trimethoprim (TMP) and piritrexim (PTX) as inhibitors of dihydrofolate reductase (DHFR) from Pneumocystis carinii (Pc), Toxoplasma gondii (Tg), and Mycobacterium avium (Ma), three of the opportunistic organisms known to cause significant morbidity and mortality in patients with AIDS and other disorders of the immune system. The ability of the new analogues to inhibit reduction of dihydrofolate to tetrahydrofolate by Pc, Tg, Ma, and rat DHFR was determined, and the selectivity index (SI) was calculated from the ratio IC(50)(rat DHFR)/IC(50)(Pc, Tg, or Ma DHFR). The IC(50) values of the 2'-O-carboxypropyl analogue (10), with SI values in parentheses, were 1.1 nM (1300) against Pc DHFR, 9.9 nM (120) against Tg DHFR, and 2.0 nM (600) against Ma DHFR. The corresponding values for the 2'-O-(4-carboxybenzyloxy) analogue (12) were 1.0 nM (560), 22 nM (21), and 0.75 nM (630). By comparison, the IC(50) and SI values for TMP were Pc, 13 000 nM (14); Tg, 2800 nM (65); and Ma, 300 nM (610). For the prototypical potent but nonselective inhibitors PTX and TMX, respectively, these values were Pc, 13 nM (0.26) and 47 nM (0.17); Tg, 4.3 nM (0.76) and 16 nM (0.50); Ma, 0.61 nM (5.4) and 1.5 nM (5.3). Thus 10 and 12 met the criterion for DHFR inhibitors that combine the high selectivity of TMP with the high potency of PTX and TMX.     

Folate antagonists. 15. 2,3-Diamino-6-(2-naphthylsulfonyl)quinazoline and related 2,4-diamino-6-[(phenyl and naphthyl)sulfinyl and sulfonyl]quinazolines, a potent new class of antimetabolites with phenomenal antimalarial activity.

Oxidation of an array of 2,4-diamino-6-(arylthio)quinazolines provided the corresponding arylsulfinyl and arylsulfonyl analogues. A variety of these nonclassical analogues of methotrexate exhibited suppressive antimalarial activity superior to that of the parent thioquinazolines against drug-sensitive lines of Plasmodium berghei in mice and P. gallinaceum in chicks, and several displayed potent prophylactic activity against P. gallinaceum. The sulfinyl- and sulfonylquinazolines also retained antimalarial effects against chloroquine-, cycloguanil-, and DDS-resistant lines of P. berghei in mice and against chloroquine- and pyrimethamine-resistant strains of P. falciparum in owl monkeys. Coadministration of one of the most active of these compounds, 2,4-diamino-6-(2-naphthylsulfonyl)-quinazoline (35), with sulfadiazine to monkeys infected with P. falciparum of P. vivax led to greatly enhanced activity and prevented the development of quinazoline resistance.     

2,4-Diamino-5-benzylpyrimidines and analogues as antibacterial agents. 10. 2,4-Diamino-5-(6-quinolylmethyl)- and -[(tetrahydro-6-quinolyl)methyl]pyrimidine derivatives. Further specificity studies

A series of 18 2,4-diamino-5-[(1,2,3,4-tetrahydro-6-quinolyl)methyl]pyrimidines has been prepared by the condensation of 2,4-diamino-5-(hydroxymethyl)pyrimidine with 1,2,3,4-tetrahydroquinolines in acidic medium. Several derivatives were catalytically aromatized; others were synthesized from these by routine aromatic substitution or by condensations of (anilinomethyl)pyrimidines to give quinolinylmethyl analogues. Compounds with 4-methyl-8-methoxy substitution are closely related to trimethoprim (1a) in structure and are excellent inhibitors of bacterial dihydrofolate reductase, with activity at least equivalent to that of 1a. The highest degree of inhibition was achieved with the rigid aromatic series, but greater specificity was accomplished among the tetrahydroquinoline derivatives. This was directly related to N-1 substitution of 4-methyl-8-methoxy derivatives. The spatial relationships around N-1 and protonation at this site may both affect selectivity. Such compounds also had excellent broad-spectrum in vitro antibacterial activity.     

Synthesis of N-{4-[(2,4-diamino-5-methyl-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)thio]benzoyl}-L-glutamic acid and N-{4-[(2-amino-4-oxo-5-methyl-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)thio]benzoyl}-L-glutamic acid as dual inhibitors of dihydrofolate reductase and thymidylate synthase and as potential antitumor agents

Two novel classical antifolates N-{4-[(2,4-diamino-5-methyl-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)thio]benzoyl}-L-glutamic acid 3 and N-{4-[(2-amino-4-oxo-5-methyl-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)thio]benzoyl}-L-glutamic acid 4 were designed, synthesized, and evaluated as antitumor agents. Compounds 3 and 4 were obtained from 2,4-diamino-5-methylpyrrolo[2,3-d]pyrimidine 7 and 2-amino-4-oxo-5-methylpyrrolo[2,3-d]pyrimidine 12, respectively, in a concise three-step sequence. Compound 3 is the first example, to our knowledge, of a 2,4-diamino classical antifolate that has potent inhibitory activity against both human dihydrofolate reductase (DHFR) and human thymidylate synthase (TS). Compound 4 was a dual DHFR-TS inhibitor against the bifunctional enzyme derived from Toxoplasma gondii (tg). Further evaluation of the mechanism of action of 3 implicated DHFR as its primary intracellular target. Both 3 and 4 were folylpolyglutamate synthetase (FPGS) substrates. Compound 3 also inhibited the growth of several human tumor cell lines in culture with GI50 < 10(-8) M. This study shows that the pyrrolo[2,3-d]pyrimidine scaffold is conducive to dual DHFR-TS and tumor inhibitory activity, and the potency is determined by the 4-position substituent.     

Folate antagonists. 13. 2,4-Diamino-6-](alpha,alpha,alpha-trifluoro-m-tolyl)thio]quinazoline and related 2,4-diamino-6-[(phenyl- and naphthyl)thio]quinazolines, a unique class of antimetabolites with extraordinary antimalarial and antibacterial effects.

An array of nonclassical thioquinazoline analogues (VIII) of methotrexate was prepared by cyclization of the requisite 2-amino-5-(arylthio)benzonitrile with chloroformamidine hydrochloride (28--79%). The aminonitrile precursors were obtained by SnCl2-HCl reduction (28--99%) of the corresponding 2-nitro-5-(arylthio)benzonitriles, which were synthesized by the condensation of the appropriate 5-chloro-2-nitrobenzonitriles with various arylthiols (36--83%). Many of the thioquinazolines (VIII) showed suppressive antimalarial activity comparable with or superior to chloroquine, cycloguanil, and pyrimethamine against drug-sensitive lines of Plasmodium berghei in mice and Plasmodium gallinaceum in chicks, and several displayed potent prophylactic activity with P. gallinaceum. Moreover, the thioquinazolines retained potent antimalarial effects against chloroquine-, cycloguanil-, pyrimethamine- and DDS-resistant lines of P. berghei in mice and against chloroquine- and pyrimethamine-resistant strains of Plasmodium falciparum in owl monkeys. The most active compound, namely, 2,4-diamino-6-[alpha,alpha,alpha-trifluoro-m-tolyl)thio]quinazoline, was designated for preclinical toxicological studies. Numerous substances exhibited in vitro activity against a broad spectrum of pathogenic bacteria at concentrations of less than 0.25 microgram/mL. The thioquinazolines also prove to be potent folate antagonists, causing 50% inhibition of Streptococcus faecalis R (ATCC 8043) at drug concentrations ranging from 0.2 to 2.0 ng/mL. Structure--activity relationships are discussed.     

The behavioral profile of the potent and selective mGlu5 receptor antagonist 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine (MTEP) in rodent models of anxiety.

Previous reports have demonstrated the anxiolytic effect of the potent and systemically active metabotropic glutamate subtype 5 (mGlu5) receptor antagonist 2-methyl-6-(phenylethynyl)pyridine (MPEP) in rodents. Here, we present evidence for the anxiolytic activity of a novel mGlu5 receptor antagonist, 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine (MTEP), in rats and compare its profile to the benzodiazepine receptor agonist diazepam. MTEP occupied mGlu5 receptors in a dose-dependent manner with essentially full receptor occupancy at the highest dose tested (10 mg/kg, i.p.). At doses appropriate for mGlu5 receptor-mediated effects, MTEP significantly reduced fear-potentiated startle and increased punished responding in a modified Geller-Seifter conflict model consistent with an anxiolytic-like profile. In both models, the magnitude of the anxiolytic-like response was similar to that seen with diazepam. In contrast, MTEP decreased unpunished responding to a lesser extent than diazepam and had no effect on rotarod performance when administered either alone or in combination with ethanol. Repeated dosing with MTEP in this model eliminated the increase in punished responding observed with acute dosing. The present results suggest that mGlu5 receptor antagonists lack the side effects seen with benzodiazepines, such as sedation and ethanol interaction, and provide insight into a possible role for mGlu5 receptor antagonists in the modulation of mood disorders.     

Conformational analysis of antineoplastic antifolates: the crystal structure of trimetrexate and the aminopterine derivative 4-[N-[(2,4-diamino-6-pteridinyl)methyl]amino]benzoic acid

The crystal structures of trimetrexate (TMQ) (2,4-diamino-5-methyl-6-[(3,4,5-trimethoxyanilino)methyl]quinazoli ne) and 4-[N-[(2,4-diamino-6-pteridinyl)methyl]amino]benzoic acid (PMAB) were determined to examine their conformational features with respect to the enzyme-bound form of methotrexate (MTX). TMQ and MTX are antineoplastic drugs that act by inhibiting the enzyme dihydrofolate reductase. The molecular conformation of TMQ is extended with the trimethoxyanilino ring twisted 89 degrees from the quinazoline plane, and the molecular conformation of PMAB is completely planar. The geometry of the 2,4-diaminopteridine and 2,4-diaminoquinazoline rings are sensitive to protonation, and both TMQ and PMAB have geometries characteristic to a free base. TMQ crystallizes as a dimethyl sulfoxide hydrate. The quinazoline ring forms an antiparallel stacking arrangement in the lattice and forms a network of N...O hydrogen bonds with the solvent molecules. In PMAB there are both pteridine-benzoic acid (N...O) hydrogen bonds and pteridine-pteridine (N...N) hydrogen bonds. Although the molecular conformation of TMQ and PMAB differ from enzyme-bound MTX, rotational energy barriers calculated using CAMSEQ indicate that they can adopt a similar conformation to that seen for MTX complexed with dihydrofolate reductase. These energy calculations show that PMAB is quite flexible and further suggest that the 5-methyl in TMQ reduces its conformational flexibility in a different manner than the N(10)-methyl in MTX. These structural data also show that full geometry optimization and proper parameterization of electronic effects at N(10) are required to accurately represent antifolate conformational preferences for enzyme binding.     

Discovery of 5-[[4-[(2,3-dimethyl-2H-indazol-6-yl)methylamino]-2-pyrimidinyl]amino]-2-methyl-benzenesulfonamide (Pazopanib), a novel and potent vascular endothelial growth factor receptor inhibitor

Inhibition of the vascular endothelial growth factor (VEGF) signaling pathway has emerged as one of the most promising new approaches for cancer therapy. We describe herein the key steps starting from an initial screening hit leading to the discovery of pazopanib, N(4)-(2,3-dimethyl-2H-indazol-6-yl)-N(4)-methyl-N(2)-(4-methyl-3-sulfonamidophenyl)-2,4-pyrimidinediamine, a potent pan-VEGF receptor (VEGFR) inhibitor under clinical development for renal-cell cancer and other solid tumors.     

Synthesis and biological evaluation of 2,4-diamino-6-(arylaminomethyl)pyrido[2,3-d]pyrimidines as inhibitors of Pneumocystis carinii and Toxoplasma gondii dihydrofolate reductase and as antiopportunistic infection and antitumor agents

A series of 2,4-diamino-6-(arylaminomethyl)pyrido[2,3-d]pyrimidines were synthesized and evaluated as inhibitors of Pneumocystis carinii (pc), Toxoplasma gondii (tg), and rat liver (rl) dihydrofolate reductase (DHFR) and as inhibitors of the growth of tumor cell lines in culture. Compounds 4-15 were designed as part of a continuing effort to examine the effects of substitutions at the 5-position, in the two-atom bridge, and in the side chain phenyl ring on structure-activity/selectivity relationships of 2,4-diaminopyrido[2,3-d]pyrimidines against a variety of DHFRs. Reductive amination of the common intermediate 2,4-diaminopyrido[2,3-d]pyrimidine-6-carbonitrile 16 with the appropriate anilines afforded the target compounds 4-12. Nucleophilic substitution or reductive methylation afforded the N10-methyl target compounds 13-15. As predicted, compounds 4-15 were, in general, less potent against all three DHFRs compared to the corresponding 2,4-diamino-5-methyl analogues previously reported; however, the greater decrease in potency against rlDHFR compared to pcDHFR and tgDHFR resulted in appreciable selectivity toward pathogenic DHFRs from different pathogens. The 2',5'-dichloro analogue 8 showed selectivity ratios (IC(50) against rlDHFR/IC(50) against pcDHFR or tgDHFR) of 15.7 and 23 for pcDHFR and tgDHFR, respectively. Thus, the selectivity of 8 for pcDHFR is higher than the first line clinical agent trimethoprim (TMP). In a P. carinii cell culture study, analogue 8 exhibited 88% cell growth inhibition at a concentration of 10 muM and afforded marginal effects in an in vivo study in the T. gondii mouse model. Selected compounds were evaluated in the National Cancer Institute (NCI) in vitro preclinical antitumor screening program and inhibited the growth of tumor cells in culture at micromolar to submicromolar concentrations and were selected for evaluation in a NCI in vivo hollow fiber assay.     

Piperazine-based CCR5 antagonists as HIV-1 inhibitors. II. Discovery of 1-[(2,4-dimethyl-3-pyridinyl)carbonyl]-4- methyl-4-[3(S)-methyl-4-[1(S)-[4-(trifluoromethyl)phenyl]ethyl]-1-piperazinyl]- piperidine N1-oxide (Sch-350634), an orally bioavailable, potent CCR5 antagonist

Truncation of the original piperidino-2(S)-methyl piperazine lead structure 2, from a family of muscarinic antagonists, gave compound 8 which has improved selectivity for the HIV-1 co-receptor CCR5 over muscarinic receptors. Further optimization for pharmacokinetic properties afforded Sch-350634 (1), a prototypical piperazine-based CCR5 antagonist, which is a potent inhibitor of HIV-1 entry and replication in PBMCs. The title compound (1) has excellent oral bioavailability in rat, dog, and monkey.     

Synthesis, calcium channel antagonist activity, and anticonvulsant activity of 3-ethyl 5-methyl 1,4-dihydro-2-[(2-hydroxyethoxy) methyl]-6-methyl-4-(2,3-dichlorophenyl)-3,5-pyridinedicarboxylate coupled to a 1-methyl-1,4-dihydropyridyl-3-carbonyl chemical delivery system

3-Ethyl 5-methyl 1,4-dihydro-2-[(2-hydroxyethoxy) methyl]-6-methyl-4-(2,3-dichlorophenyl)-3,5-pyridinedicarboxylate (13), a bioisostere of amlodipine, was prepared by the reaction of ethyl 4-(2-hydroxyethoxy)acetoacetate (11) with methyl 2-(2,3-dichlorobenzylidene)acetoacetate (12) and NH4OAc. Compound 13 was elaborated to the target product 3-ethyl 5-methyl 1,4-dihydro-2-[2- [(1-methyl-1,4-dihydropyridyl-3-carbonyloxy)ethoxy]methyl]- 6-methyl-4-(2,3-dichlorophenyl)-3,5-pyridinedicarboxylate (16). The C-2 CH2OCH2CH2OH compound (13, IC50 = 6.56 x 10(-9) M) was about 44-fold more active as a calcium channel antagonist than the reference drug nimodipine (IC50 = 1.49 x 10(-8) M), but 4-fold less potent than felodipine (IC50 = 1.45 x 10(-9) M). Compound 16, possessing the 1-methyl-3-pyridylcarbonyloxy chemical delivery system moiety is a slightly less potent calcium channel antagonist (IC50 = 2.99 x 10(-8) M) than the parent compound 13. Compounds 13, 16, felodipine and nimodipine are highly lipophilic (Kp = 227, 344, 442 and 187, respectively). The C-2 CH2OCH2CH2OH compound (13) exhibited equipotent anticonvulsant activity to nimodipine in the maximal electroshock (MES) anticonvulsant screen. Unlike nimodipine, 13 provided modest protection in the subcutaneous metrazol (scMet) anticonvulsant screen. In contrast, compound 16 was inactive in both the MES and scMet screens.     

Demethylierung in vivo und in vitro von Methixen (9-[(N-C14-Methyl-3-piperidyl)methyl]-thioxanthen-hydrochlorid)

Zusammenfassung 1. Es ist die Demethylierungsrate von N-Methyl-C¹⁴-signiertem Tremaril® (basisch substituiertes Thioxanthen) an inkubierten Leberschnitten, in der isolierten perfundierten Rattenleber sowie im Belastungsversuch durch Messung der C¹⁴O₂-Bildung bestimmt worden. Die für Ratten erhaltenen Werte betragen — je nach Versuchsanordnung — 0,1–0,8 Mol/g Leber-Trockengewicht/Std und sind rund zehnmal kleiner als die entsprechenden Demethylierungsraten von Dimethylaminoantipyrin (DMAA).2. Die an Schnitten von Menschenleber bestimmte Demethylierungsrate (0,05 Mol/g/Std) ist ca. 15mal kleiner als der für Rattenleber bestimmte Wert, wogegen DMAA nur etwa dreimal langsamer demethyliert wird. Leberschnitte beider Species oxydieren Methylenglykol und Formiat gleich rasch (ca. 25 Mol/g/Std).3. In Schnitten von Rattenleber überwiegt die Sulfoxydbildung (4–5 Mol/g/Std) die Demethylierung um ca. das Sechsfache. Während sich unverändertes Tremaril in den inkubierten Gewebsschnitten um einen Faktor von ca. 40 (Leber) bzw. bis über 100 (Gehirn) anreichert, wird Tremarilsulfoxyd, der hauptsächliche Metabolit, weitgehend in das suspendierende Medium abgegeben.Mit 4 TextabbildungenMarkenname: Tremaril Wander.     

3-[(2R)-Amino-2-phenylethyl]-1-(2,6-difluorobenzyl)-5-(2-fluoro-3-methoxyphenyl)- 6-methylpyrimidin-2,4-dione (NBI 42902) as a potent and orally active antagonist of the human gonadotropin-releasing hormone receptor. Design, synthesis, and in vitro and in vivo characterization

Further structure-activity relationship studies of a series of substituted uracils at the 1, 3, and 5 positions resulted in the discovery of several potent antagonists of the human gonadotropin-releasing hormone receptor. Uracils bearing a side chain derived from phenylglycinol at the 3-position were shown to be orally bioavailable in monkeys. 3-[(2R)-Amino-2-phenylethyl]-1-(2,6-difluorobenzyl)-5-(2-fluoro-3-methoxyphenyl)-6-methylpyrimidin-2,4-dione (R-13b, NBI 42902) displayed subnanomolar binding affinity (K(i) = 0.56 nM) and was a potent functional antagonist (IC(50) = 3.0 nM in Ca(2+) flux assay) at the human GnRH receptor. It also bound to the monkey GnRH receptor with high affinity (K(i) = 3.9 nM). In addition, R-13bhad good plasma exposure in cynomolgus monkeys after oral administration, with a C(max) of 737 ng/mL and an AUC of 2392 ng/mL.h at a 10 mg/kg dose. Moreover, oral administration of R-13b to castrated male cynomolgus monkeys resulted in a significant decrease in serum levels of luteinizing hormone. These results demonstrate that compounds from this series of uracils are potent GnRH antagonists with good oral bioavailability and efficacy in nonhuman primates.