Phenethylthiazolylthiourea (PETT) compounds as a new class of HIV-1 reverse transcriptase inhibitors. 2. Synthesis and further structure-activity relationship studies of PETT analogs

Phenylethylthiazolylthiourea (PETT) derivatives have been identified as a new series of non-nucleoside inhibitors of HIV-1 RT. Structure-activity relationship studies of this class of compounds resulted in the identification of N-[2-(2-pyridyl)ethyl]-N'-[2-(5-bromopyridyl)]-thiourea hydrochloride (trovirdine; LY300046.HCl) as a highly potent anti-HIV-1 agent. Trovirdine is currently in phase one clinical trials for potential use in the treatment of AIDS. Extension of these structure-activity relationship studies to identify additional compounds in this series with improved properties is ongoing. A part of this work is described here. Replacement of the two aromatic moieties of the PETT compounds by various substituted or unsubstituted heteroaromatic rings was investigated. In addition, the effects of multiple substitution in the phenyl ring were also studied. The antiviral activities were determined on wild-type and constructed mutants of HIV-1 RT and on wild-type HIV-1 and mutant viruses derived thereof, Ile100 and Cys181, in cell culture assays. Some selected compounds were determined on double-mutant viruses, HIV-1 (Ile 100/Asn103) and HIV-1 (Ile100/Cys181). A number of highly potent analogs were synthesized. These compounds displayed IC50's against wild-type RT between 0.6 and 5 nM. In cell culture, these agents inhibited wild-type HIV-1 with ED50's between 1 and 5 nM in MT-4 cells. In addition, these derivatives inhibited mutant HIV-1 RT (Ile 100) with IC50's between 20 and 50 nM and mutant HIV-1 RT (Cys 181) with IC50's between 4 and 10 nM, and in cell culture they inhibited mutant HIV-1 (Ile100) with ED50's between 9 and 100 nM and mutant HIV-1 (Cys181) with ED50's between 3 and 20 nM.     

Nonpeptide angiotensin II antagonists derived from 1H-pyrazole-5-carboxylates and 4-aryl-1H-imidazole-5-carboxylates

Two series of potential angiotensin II antagonists derived from carboxyl-functionalized "diazole" heterocycles have been prepared and evaluated. Initially, a limited investigation of 4-arylimidazole-5-carboxylates led to 2-n-butyl-4-(2-chlorophenyl)-1-[[2'-(1H-tetrazol-5-yl)biphenyl-4-y l] methyl]-1H-imidazole-5-carboxylic acid (12b), which was found to be a highly potent antagonist of the rabbit aorta AT1 receptor (IC50 0.55 nM). In conscious, normotensive rats, 12b at 0.1 mg/kg iv inhibited the pressor response to AII by 88%, with a duration of > 6 h. More extensively studied was an isosteric series of 3-alkyl-4-[[2'-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]-1H-pyrazole -5- carboxylates bearing aryl, alkyl, or aralkyl substituents at N1. These compounds were available in highly regioselective fashion via condensation of a substituted hydrazine hydrochloride with a 2-(methoxyimino)-4-oxoalkanoate intermediate. In vitro, the most potent pyrazolecarboxylic acids had n-butyl at C3 and were substituted at N1 by such groups as 2,6-dichlorophenyl (19h), 2-(trifluoromethyl)phenyl (19k), benzyl (19t), and phenethyl (19u), all with IC50 values of 0.18-0.24 nM. Although less potent in the receptor assay, 3-n-propylpyrazolecarboxylic acids were at least as effective as their butyl counterparts in vivo. Several of the pyrazolecarboxylic acid derivatives demonstrated potent, long-lasting oral activity in rats. At 1 mg/kg po, the 1-benzyl-3-butyl (19t), 1-(2,6-dichlorophenyl)-3-propyl (19v), 3-propyl-1-(2,2,2-trifluoroethyl) (19y), and 1-benzyl-3-propyl (19z) analogues all gave > or = 75% inhibition of the AII pressor response in the rat model, with duration of action > 23 h.     

PNU 157706, a novel dual type I and II 5alpha-reductase inhibitor

PNU 157706 is a novel dual inhibitor of 5alpha-reductase (5alpha-R), the enzyme responsible for the conversion of testosterone (T) to 5alpha-dihydrotestosterone (DHT). Tested on a crude preparation of human or rat prostatic 5alpha-R, PNU 157706 caused enzyme inhibition with IC50 values of 20 and 34 nM, respectively, compared to the values of 32 and 58 nM shown by finasteride. Furthermore, PNU 157706 was highly potent in inhibiting human recombinant 5alpha-R type I and II isozymes, showing IC50 values of 3.9 and 1.8 nM and, therefore, it was several folds more potent than finasteride (IC50 values of 313 and 11.3 nM), particularly on the type I isozyme. PNU 157706 was shown to have no binding affinity for the rat prostate androgen receptor (RBA 0.009% that of DHT). In adult male rats, a single oral dose of 10 mg/kg of PNU 157706 caused a marked and longer lasting reduction of prostatic DHT than did finasteride (at 24 h inhibition by 89 and 47%, respectively). In prepubertal, T- or DHT-implanted castrated rats, PNU 157706, given orally for 7 days at the dose of 10 mg/kg/day, markedly reduced ventral prostate weight in T- but not in DHT-implanted animals, thus showing to be devoid of any anti-androgen activity. In adult rats treated orally for 28 days, PNU 157706 resulted markedly more potent (16-fold) than finasteride in reducing prostate weight, the ED50 values being 0.12 and 1.9 mg/kg/day, respectively. These results indicate that PNU 157706 is a promising, potent inhibitor of both type II and I human 5alpha-R with a very marked antiprostatic effect in the rat.     

trans-3-Benzyl-4-hydroxy-7-chromanylbenzoic acid derivatives as antagonists of the leukotriene B4 (LTB4) receptor

The SAR of a series of 2-(7-chromanyl)benzoic acids has been investigated with the aim of identifying potent and selective LTB4 receptor antagonists that maintain potency in complex biological fluids. We found optimal activity in derivatives with electron-withdrawing groups in the benzoic acid ring and with an unsubstituted C-3 benzyl group on the chromanol nucleus. While compounds containing a 3-(4-phenyl)benzyl chromanol substituent were potent LTB4 receptor antagonists, the increased lipophilicity imparted by the additional phenyl substituent led to decreased potency in the presence of plasma proteins. From among the potent compounds identified, CP-195543, the 5'-trifluoromethyl 3-benzyl chromanol, was selected for development.     

Synthesis and structure-activity relationships of carboxylated chalcones: a novel series of CysLT1 (LTD4) receptor antagonists

The synthesis and CysLT1 antagonistic activities of a new series of 2-, 3-, and 4-(2-quinolinylmethoxy)- and 3- and 4-[2-(2-quinolinyl)ethenyl]-substituted, 2'-, 3'-, 4'-, or 5'-carboxylated chalcones are described. Structure-activity relationship studies indicate a preference for the presence of a negatively charged (acidic) moiety, although in some cases nitrile or ester analogues also exhibit moderate activity. The quinoline moiety may be substituted at either the 3- or the 4-position. Replacement of this heterocycle by other aromatic groups results in compounds with comparable affinities [2-(7-chloroquinoline), 1-(1-methyl-2-benzimidazole), or 1-(2-benzothiazole)] or substantially lower activities [1-(1-ethoxyethyl)-2-benzimidazole, 2-naphthyl, or phenyl]. The quinoline and chalcone moieties may be connected by either an ethenyl or a methoxy spacer. The acidic moiety at the chalcone B ring may be attached to the 2'-, 3'-, 4'-, or 5'-position, for both the 3- and 4-substituted chalcones. There are no general patterns to specify which substitution positions gave the most potent compounds. The series contains several potent CysLT1 receptor antagonists, with K(D) values approaching the nanomolar range, as measured by the displacement of [3H]LTD4 from guinea pig lung membranes. Antagonism of LTD4-induced contraction of guinea pig ileum, the inhibition of antigen-induced contraction of guinea pig trachea in vitro, and the inhibition of LTD4-induced increase of vascular permeability in vivo are determined for chalcones with high CysLT1 receptor affinities (K(D) values below 0.1 microM). 2'-Hydroxy-4-(2-quinolinylmethoxy)-5'-(5-tetrazolyl)chalcone (14, VUF 4819) showed good activity in both in vitro and in vivo assays and has been selected for further evaluation.     

Synthesis and biological activities of phenyl piperazine-based peptidomimetic growth hormone secretagogues

A new class of potent, orally active phenyl piperazine-based GH secretagogues have been discovered from attempts to mimic the arrangement of the phenyl substituent in the spiroindanyl piperidine and spiroindoline sulfonamide privileged structures of 4 and 1, respectively. The best of these compounds, 18 (EC50 = 2.8 nM) is nearly as potent as MK-0677 for releasing GH from rat pituitary cells.     

Tetrahydro-isoquinoline-based factor Xa inhibitors

Derivatives of (2-amidino-1,2,3, 4-tetrahydro-isoquinolin-7-yloxy)phenylacetic acid (TIPAC) were developed as inhibitors of factor Xa (fXa). The compounds are prepared using 15 synthetic steps on average. The most potent compounds (14, 17, 22-26) display inhibition constants of Ki = 21-55 nM but do not inhibit thrombin (Ki = 5->100 microM) and only weakly inhibit trypsin (Ki = 0.08-5 microM). They bear a second basic moiety, e.g., substituted 1-(iminomethyl)piperidines, which is linked to C-4 of the phenyl group of TIPAC via an oxygen atom. The inhibition constants of these compounds are almost independent of the size of the (iminomethyl)piperidine substituent. Due to the fact that fXa displays two cation binding sites, namely, the S1 and S4 sites, in principle two binding modes are conceivable for the novel dibasic fXa inhibitors. Molecular modeling experiments based on the X-ray structures of uninhibited fXa and the DX-9065a/fXa complex were carried out. The results taken together with the inhibition constants clearly favor one binding mode: the tetrahydro-isoquinoline fills the S1 pocket even better than the naphthalene moiety of DX-9065a, and the (iminomethyl)piperidine residues occupy the S4 site.     

Synthesis, structure, dopamine transporter affinity, and dopamine uptake inhibition of 6-alkyl-3-benzyl-2-[(methoxycarbonyl)methyl]tropane derivatives

A series of 6-alkyl-3 beta-benzyl-2-[(methoxycarbonyl)methyl]tropane analogues were synthesized and evaluated as cocaine binding site ligands at the dopamine transporter (DAT). The in vitro affinity (Ki) for the DAT of the 6-alkyl-3 beta-benzyl-2-[(methoxycarbonyl) methyl]tropane analogues was determined by inhibition of [3H]WIN 35,428 in rat caudate putamen tissue. The inhibition of dopamine uptake (IC50) was also measured for selected compounds which demonstrated moderate affinity for the dopamine transporter. The unsubstituted enantiopure analogues (-)-19a (Ki = 33 nM) and surprisingly (+)-20a (Ki = 60 nM) were found to be almost equipotent with the high-affinity binding components of cocaine and WIN 35,065-2 and exhibited slightly more potent dopamine uptake inhibition than both cocaine and WIN 35,065-2. In general, substitution at the 6-position of racemic 19a and 20a with alkyl groups was found to result in decreased activity relative to increased chain length of the substituent. The 3 beta-benzyl-2 beta-[(methoxycarbonyl)methyl]-6 beta-methyltropane (21b; Ki = 57 nM) was the only 6-alkyl derivative to exhibit moderately potent activity. The 6 beta-isomer 21b was 4-fold more potent than the 6 alpha-isomer 19b (Ki = 211 nM) and was nearly equipotent with (-)-19a and (+)-20a as well as with cocaine and WIN 35,065-2. The results of this study further demonstrate the steric constraints associated with the C(6)-C(7) methylene bridge of the tropane ring system for molecular recognition of cocaine analogues at the cocaine binding site(s) on the DAT.     

Indole and benzimidazole derivatives as steroid 5alpha-reductase inhibitors in the rat prostate

A novel series of indole and benzimidazole derivatives were synthesized and evaluated for their inhibitory activity of rat prostatic 5alpha-reductase. Among these compounds, 4-?2-[1-(4,4'-dipropylbenzhydryl)indole-5-carboxamido]phenoxy?buty ric acid (15) and its benzimidazole analogue 25 showed potent inhibitory activities for rat prostatic 5alpha-reductase (IC50 values of 9.6+/-1.0 and 13+/-1.5 nM, respectively), with the potency very close to that of finasteride. Compound 30, in which the moiety between the benzene ring and amide bond was replaced by quinolin-4-one ring, showed almost equipotent activity (IC50= 19+/-6.2nM) with the correspondent amide derivative 13. This result was consistent with the previous observation that the coplanarity of this moiety might contribute to the potent inhibitory activity.     

Phosphorylation of triciribine is necessary for activity against HIV type 1

Triciribine (TCN) is a tricyclic nucleoside with known antineoplastic and antiviral activity. It is a potent and selective inhibitor of HIV-1 and HIV-2, including strains known to be resistant to AZT or TIBO. TCN is phosphorylated to its 5'-monophosphate (TCN-P) by intracellular adenosine kinase (AK), but is not converted to di- or triphosphates. We now report that 5'-phosphorylation is requisite for the activity of TCN against HIV-1. CEM cells incubated with TCN at concentrations ranging from 0.1 to 330 microM gave intracellular TCN-P concentrations from 27 to 775 microM, respectively. There was no difference in the amount of intracellular TCN-P detected in uninfected compared with HIV-1-infected CEM cells. The antiviral effect of TCN against HIV-1 was strongly antagonized by the AK inhibitor 5-iodotubercidin (ITu). In contrast, TCN and ITu only exhibited additive cytotoxicity. The 5'-deoxy analog of TCN, which cannot be phosphorylated, had no antiviral effect against HIV-1 at a concentration more than 100 times higher than the IC50 of TCN. Similarly, TCN was not active against HIV-1 in an AK-deficient cell line (AA-2) at concentrations shown to inhibit the virus by >95% in CEM cells. Consistent with its AK-deficient phenotype, this cell line phosphorylated TCN to only 3% of the extent observed in CEM cells. We conclude that TCN must be phosphorylated to TCN-P for activity against HIV-1.     

Halogenated mazindol analogs as potential inhibitors of the cocaine binding site at the dopamine transporter

A series of halogenated (F, Cl, Br, I), pyrimido and diazepino homologs of mazindol were prepared and evaluated for their ability to displace [3H]WIN 35,428 binding and to inhibit uptake of [3H]dopamine (DA) in rat striatal tissue. All of the compounds except for the 2'-chloro (6) and 2'-bromo (16) analogs of mazindol displaced [3H]WIN 35,428 binding and inhibited [3H]DA uptake more effectively than (R)-cocaine. Structure-activity studies indicated that best inhibition of [3H]WIN 35,428 binding occurred in the imidazo series with compounds containing one or two Cl or Br atoms in the 3'- or 4'-position of the free phenyl group. Replacement of the imidazo ring by a pyrimido or diazepino ring enhanced binding inhibition. The most potent inhibitors of [3H]WIN 35,428 binding and [3H]DA uptake were 6-(3'-chlorophenyl)-2,3,4,6-tetrahydropyrimido[2,1-alpha]isoind ol-6-ol (23; IC50 1.0 nM; 8 x mazindol) and 7-(3',4'-dichlorophenyl)-2,3,4,5-tetrahydro-7H-diazepino[2,1-alpha ]isoindol-7-ol (28; IC50 0.26 nM; 32 x mazindol), respectively. No significant differences was found between binding and uptake inhibition. Mazindol and the pyrimido and diazepino homologs 24 and 27 showed a selectivity for the DA uptake over the serotonin (5-HT) uptake site of 5-, 250-, and 465-fold, respectively, and displayed weak or no affinity for a variety of neurotransmitter receptor sites.     

Comparison of in vitro effects of the pure antiandrogens OH-flutamide, Casodex, and nilutamide on androgen-sensitive parameters

OBJECTIVES: A combination of flutamide (Eulexin) or nilutamide (Anandron) with a luteinizing hormone-releasing hormone (LHRH) agonist or orchiectomy is the only therapy demonstrated to prolong life in prostate cancer. Recently, the low 50-mg daily dose of Casodex, an analogue of the pure antiandrogen flutamide, was chosen for clinical studies on the basis that the compound was 5 to 10 times more potent than flutamide, as suggested by data obtained in the inappropriate intact rat model. The present study was designed to compare the in vitro antiandrogenic activity of OH-flutamide (OH-FLU), the active metabolite of flutamide, Casodex, and nilutamide. METHODS: The effect of the antiandrogens was tested on two androgen-sensitive parameters, namely proliferation of the SEM-107 clone of Shionogi mouse mammary tumor cells and secretion of the GCDFP-15 (gross cystic disease fluid protein 15 kDa) in T-47D and ZR-75-1 human breast cancer cells. RESULTS: The twofold stimulation of Shionogi cell proliferation caused by a 10-day exposure to 1 nM testosterone was competitively reversed by incubation with OH-FLU, Casodex, or nilutamide, at the respective IC50 values of 72, 243, and 412 nM. Moreover, the marked increase in GCDFP-15 release induced by 1 nM testosterone was blocked by OH-FLU. Casodex, or nilutamide at respective IC50 values of 29, 180, and 87 nM in T-47D cells and at 35, 142, and 75 nM in ZR-75-1 cells. Similar data were detected in 4-androstenedione-induced Shionogi cell proliferation and in dihydrotestosterone-induced GCDFP-15 secretion in T-47D cells. CONCLUSIONS: OH-FLU is 3.1- to 7.8-fold more potent than Casodex, as measured on two in vitro androgen-sensitive parameters, in agreement with our recent in vivo data obtained in the model of castrated rats supplemented with 4-androstenedione implants, in which threefold greater potency of flutamide was observed. The present data, as well as other data from the literature, strongly indicate the need to choose a more appropriate dose of Casodex for the treatment of prostate cancer.     

IgM response to a human Pneumocystis carinii surface antigen in HIV-infected patients with pulmonary symptoms

A series of eight new N-hydroxy-N'-aminoguanidine (HAG) Schiff bases [ArCH = NNHC(= NH)NHOH.tosylate] was synthesized as potential antitumor agents through the inhibition of the enzyme ribonucleotide reductase (EC 1.17.4.1). Five of the HAG derivatives (LK02 through LK06) were designed to contain an orthohydroxy group on the phenyl ring of ArCH = to increase the stability of the Schiff base formed. In addition, two compounds with a substituted quinoline [LK10; ArCH = (4-hydroxy-7-trifluoromethylquinolin-3-yl)methylene] or isoquinoline (LK11; ArCH = (5-nitroisoquinolin-1-yl)methylene] moiety were synthesized through multiple-step reactions involving reduction and/or oxidation. The IC50 values of the newly synthesized HAG Schiff bases were determined against human leukemic CCRF-CEM/0 cells in culture. The IC50 values of two previously reported HAG derivatives [ATL25; ArCH = (5-nitro-isoquinolin-1-yl)methylene] and [LW02; ArCH = 2-hydroxy-3-allyl-benzylidene)] were also determined for the first time against CCRF-CEM/0 cells. Among the compounds tested, LK11 was found to be the most potent (IC50, 2.95 +/- 0.1 microM) and the 4-methoxy-2-hydroxyphenyl derivative (LK02) to be the least potent (IC50, 121 +/- 16 microM). LK11 was about 15-fold more potent against CCRF-CEM/0 cells compared to the parent compound hydroxyguanidine sulfate (IC50, 46 +/- 7.1 microM) and was about 32 times more potent than LK10 (IC50, 97.6 +/- 0.9 microM). LK11 in combination was incubated in sequence with cytarabine (ara-C) at various molar ratios against CCRF-CEM/0 cells for 48 hr. The results were analyzed using both the isobologram and the median-effect methods.(ABSTRACT TRUNCATED AT 250 WORDS)     

4-[(1H-imidazol-4-yl) methyl] benzamidines and benzylamidines: novel antagonists of the histamine H3 receptor

A series of amidine substituted phenyl-, benzyl-, and phenethylimidazoles based on the known H3 agonist SK&F 91606 (4) has been synthesized and tested as ligands for the histamine H3 receptor. Insertion of a phenyl ring between the imidazole ring and the amidine moiety produces antagonists. The benzyl series was found to be the most potent and was further investigated. Compounds 9c and 18 (entries 5 and 12, Table 1) are potent ligands for the H3 receptor with K(i) values of 16 nM and 7.2 nM respectively. In vivo, both compounds were shown to be equipotent to thioperamide (2), the standard H3 antagonist.     

Structure-activity relationships of cyclic enediynes related to dynemicin A. I. Synthesis and antitumor activity of 9-acetoxy enediynes equipped with aryl carbamate moieties

A series of the 9-acetoxy enediyne compounds, 6a-k which were simplified from natural dynemicin A, and designed to be equipped with various aryl carbamate moieties, was synthesized and evaluated for DNA-cleaving ability, in vitro cytotoxicity, and in vivo antitumor activity. As a result of this study of the structure-activity relationships (SAR) with regard to the Rt substituent, both compounds 6a and 6f with the phenyl carbamate and 4-chlorophenyl carbamate moiety, respectively, were found to exhibit significant activity (T/C > 200%) against murine P388 leukemia in mice, in spite of having IC50 values in the micromolar range. In particular, compound 6f showed the most potent activity with a maximum T/C of 256% at a daily dosage of 4.0 mg/kg for four days. Furthermore, both compounds 6a and 6f were effective against Meth A sarcoma in mice and inhibited 71 and 77% of the tumor growth at 2.0 and 3.0 mg/kg dosages, respectively. In contrast to 6f, compound 6i possessing the 2-nitrophenyl carbamate moiety showed only a slight in vivo activity, while it had about one order of magnitude higher in vitro cytotoxicity than 6f. For the stereochemistry-activity relationships at the C9 position, the (9R*)-isomers of 6c, 6g, and 6j were found to show higher in vitro and in vivo potencies than the corresponding (9S*)-isomers.     

N-(2-Benzoylphenyl)-L-tyrosine PPARgamma agonists. 2. Structure-activity relationship and optimization of the phenyl alkyl ether moiety

We previously reported the identification of (2S)-((2-benzoylphenyl)amino)-3-?4-[2-(5-methyl-2-phenyloxazol-4-y l)e thoxy]phenyl?propanoic acid (2) (PPARgamma pKi = 8.94, PPARgamma pEC50 = 9.47) as a potent and selective PPARgamma agonist. We now report the expanded structure-activity relationship around the phenyl alkyl ether moiety by pursuing both a classical medicinal chemistry approach and a solid-phase chemistry approach for analogue synthesis. The solution-phase strategy focused on evaluating the effects of oxazole and phenyl ring replacements of the 2-(5-methyl-2-phenyloxazol-4-yl)ethyl side chain of 2 with several replacements providing potent and selective PPARgamma agonists with improved aqueous solubility. Specifically, replacement of the phenyl ring of the phenyloxazole moiety with a 4-pyridyl group to give 2(S)-((2-benzoylphenyl)amino)-3-?4-[2-(5-methyl-2-pyridin-4-yloxazol+ ++- 4-yl)ethoxy]phenyl?propionic acid (16) (PPARgamma pKi = 8.85, PPARgamma pEC50 = 8.74) or a 4-methylpiperazine to give 2(S)-((2-benzoylphenyl)amino)-3-(4-?2-[5-methyl-2-(4-methylpiperazin+ ++- 1-yl)thiazol-4-yl]ethoxy?phenyl)propionic acid (24) (PPARgamma pKi = 8.66, PPARgamma pEC50 = 8.89) provided two potent and selective PPARgamma agonists with increased solubility in pH 7.4 phosphate buffer and simulated gastric fluid as compared to 2. The second strategy took advantage of the speed and ease of parallel solid-phase analogue synthesis to generate a more diverse set of phenyl alkyl ethers which led to the identification of a number of novel, high-affinity PPARgamma ligands (PPARgamma pKi's 6.98-8.03). The combined structure-activity data derived from the two strategies provide valuable insight on the requirements for PPARgamma binding, functional activity, selectivity, and aqueous solubility.     

Uptake of L-glutamate into synaptic vesicles: competitive inhibition by dyes with biphenyl and amino- and sulphonic acid-substituted naphthyl groups

The specificity of the vesicular L-glutamate carrier was characterized using dyes with biphenyl and amino- and sulphonic acid substituted naphthyl groups, structurally similar to the specific vesicular L-glutamate inhibitor Evans Blue. The dye Trypan Blue was the most potent inhibitor; the IC50 value was determined to be 49 nM. Naphthol Blue Black, Reactive Blue 2, Benzopurpurin 4B, Ponceau SS, Direct Blue 71 and Acid red 114 were also highly potent inhibitors with IC50 values from 330 to 1670 nM (series 1). The dyes were competitive inhibitors of vesicular glutamate uptake, and acted therefore on the glutamate transporter. Their IC50 values for the vesicular uptake of gamma-aminobutyric acid (GABA) were all higher than 20 microM. They had no effect on synaptosomal uptake of glutamate. Furthermore, we have also found several other dyes with IC50 values for the vesicular uptake of glutamate ranging between 1 and 30 microM and for gamma-aminobutyric acid higher than 50 microM (series 2). The most potent inhibitor Trypan Blue contains a biphenyl group, linked by azo groups to side chains containing sulphonic, amino and/or hydroxyl groups coupled to a naphthalene ring system. Trypan Blue and Evans Blue are by molecular mechanics, shown to have planar structures with conjugated double bonds throughout the structure. The other dyes, which were less effective, had phenyl and/or naphthalene groups linked by an azo group. We have also tested a series of amino and/or hydroxyl naphthalene di-/sulphonic acids that correspond to the side chains of the most potent dyes, but they had no effect on glutamate nor on gamma-aminobutyric acid uptake. We conclude that the inhibitory action of these compounds is strictly dependent of the complete molecule.     

Synthesis of 3 beta-aryl-8-azabicyclo[3.2.1]octanes with high binding affinities and selectivities for the serotonin transporter site

A novel entry to tropane analogs of cocaine was developed based on the reaction of rhodium-stabilized vinylcarbenoids with pyrroles. These analogs were tested in binding to dopamine, serotonin (5-HT), and norepinephrine transporters in membranes from rat striatum and frontal cortex. In all the analogs, the aryl group at the 3 position was directly bound to the tropane ring and an ethyl ketone moiety was present at the 2 position. By appropriate modification of the aryl and nitrogen substituents, highly potent and 5-HT selective tropanes were prepared. The most potent and selective compound was 3 beta-[4-(1-methylethenyl)phenyl]-2 beta-propanoyl-8-azabicyclo[3.2.1]octane (13b) which had a Ki of 0.1 nM at 5-HT transporters and was 150 times more potent at 5-HT vs dopamine transporters and almost 1000 times more potent at 5-HT vs norepinephrine transporters.