NNC-711, a novel potent and selective gamma-aminobutyric acid uptake inhibitor: pharmacological characterization.

NNC-711 (1-(2-(((diphenylmethylene)amino)oxy)ethyl)-1,2,5,6-tetrahydro-3- pyridinecarboxylic acid hydrochloride) is a novel, potent and selective gamma-aminobutyric acid (GABA) uptake inhibitor. NNC-711 inhibited synaptosomal (IC50 = 47 nM), neuronal (IC50 = 1238 nM) and glial (IC50 = 636 nM) GABA uptake in vitro NNC-711 lacked affinity for other neurotransmitter receptor binding sites, uptake sites and ion channels examined in vitro. In vivo, NNC-711 was a potent anticonvulsant compound against rodent seizures induced by methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM) (ED50 (clonic) = 1.2 mg/kg i.p.), pentylenetetrazole (PTZ) (ED50 (tonic) = 0.72 mg/kg i.p., mouse; and ED50 (tonic) = 1.7 mg/kg, rat), or audiogenic (ED50 (clonic and tonic) = 0.23 mg/kg i.p.). At higher doses NNC-711 produced behavioral side effects characterized by inhibition of traction (ED50 = 23 mg/kg i.p.), rotarod (ED50 = 10 mg/kg i.p.) and exploratory locomotor activity (ED50 = 45 mg/kg i.p.) in the mouse. Following acute (3-h) in vivo pretreatment with NNC-711, behavioral tolerance developed to its motor impairing side effects (inhibition of traction, rotarod or exploratory locomotor activity) without corresponding tolerance to the anticonvulsant effects. These data suggest that NNC-711 will be useful for future in vitro and in vivo experiments to elucidate the role of the GABA uptake carrier in the central nervous system.     

4,5,6,7-Tetrahydroisothiazolo[5,4-c]pyridin-3-ol and related analogues of THIP. Synthesis and biological activity

The thio analogues of the GABA (gamma-aminobutyric acid) agonist THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol), the GABA uptake inhibitor THPO (4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridin-3-ol), and the glycine antagonist THAZ (5,6,7,8-tetrahydro-4H-isoxazolo[4,5-d]azepin-3-ol) have been synthesized and tested biologically on single neurons in the cat spinal cord and in vitro by using synaptic membrane preparations obtained from rat brains. In contrast to THIP, thio-THIP (4,5,6,7-tetrahydroisothiazolo[5,4-c]pyridin-3-ol, 5) was only a weak GABA agonist. Thio-THPO (4,5,6,7-tetrahydroisothiazolo[4,5-c]pyridin-3-ol, 10) was slightly weaker than THPO as an inhibitor of GABA uptake in vitro, and these two compounds were approximately equipotent in enhancing the inhibition of the firing of cat spinal neurons by GABA. Like THAZ and structurally related bicyclic isoxazole zwitterions, thio-THAZ (5,6,7,8-tetrahydro-4H-isothiazolo[4,5-d]azepin-3-ol, 15) was an antagonist at glycine receptors on cat spinal neurons. The I/U ratios, which reflect the ability of neutral amino acids to penetrate the blood-brain barrier (BBB), were calculated for 5 (I/U = 16), 10 (63), and 15 (200). These low I/U ratios, compared with the findings that THIP (I/U = 500 or 1500) and THPO (I/U = 2500) enter the brain after systemic administration, suggest that the thio analogues may penetrate the BBB very easily.     

Novel class of potent 4-arylalkyl substituted 3-isoxazolol GABA(A) antagonists: synthesis,pharmacology, and molecular modeling

A number of analogues of the low-efficacy partial GABA(A) agonist 5-(4-piperidyl)-3-isoxazolol (4-PIOL, 5), in which the 4-position of the 3-isoxazolol ring was substituted by different groups, were synthesized and tested as GABA(A) receptor ligands. Substituents of different size and structural flexibility such as alkyl, phenylalkyl, diphenylalkyl, and naphthylalkyl were explored. Pharmacological characterization of the synthesized compounds was carried out using receptor binding assays and by electrophysiological experiments using whole-cell patch-clamp techniques. Whereas none of these compounds significantly affected GABA(B) receptor sites or GABA uptake, they did show affinity for the GABA(A) receptor site. While alkyl or benzyl substitution, compounds 7a-h, provided receptor affinities comparable with that of 5 (K(i) = 9.1 microM), diphenylalkyl and naphthylalkyl substitution, as in compounds 7m-t, resulted in a dramatic increase in affinity relative to 5. The 3,3-diphenylpropyl and the 2-naphthylmethyl analogues, compounds 7s and 7m, respectively, showed the highest affinities of the series (K(i) = 0.074 microM and K(i) = 0.049 microM). In whole-cell patch-clamp recordings from cultured cerebral cortical neurons, all of the tested compounds were able to inhibit the effect of the specific GABA(A) agonist isoguvacine (1), compounds 7m and 7s showing antagonist potency (IC(50) = 0.37 microM and IC(50) = 0.02 microM) comparable with or markedly higher than that of the standard GABA(A) antagonist 4 (IC(50) = 0.24 microM). Highly potent convulsant activity was demonstrated in mice with compounds 7m (ED(50) = 0.024 micromol/kg) and 7s (ED(50) = 0.21 micromol/kg) after intracerebroventricular administration, whereas no effects were found after subcutaneous administration. According to a previously proposed pharmacophore model for GABA(A) receptor agonists, a receptor cavity in the vicinity of the 4-position of the 3-isoxazolol ring in 4-PIOL exists. A molecular modeling study, based on compounds 7o,m,l,q,s, was performed to explore the dimensions and other properties of the receptor cavity. This study demonstrates the importance of the arylalkyl substituents in 7m and 7s and the considerable dimensions of this proposed receptor cavity.     

Novel 1-hydroxyazole bioisosteres of glutamic acid. Synthesis, protolytic properties, and pharmacology.

A number of 1-hydroxyazole derivatives were synthesized as bioisosteres of (S)-glutamic acid (Glu) and as analogues of the AMPA receptor agonist (R,S)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA, 3b). All compounds were subjected to in vitro pharmacological studies, including a series of Glu receptor binding assays, uptake studies on native as well as cloned Glu uptake systems, and the electrophysiological rat cortical slice model. Compounds 7a,b, analogues of AMPA bearing a 1-hydroxy-5-pyrazolyl moiety as the distal carboxylic functionality, showed only moderate affinity for [3H]AMPA receptor binding sites (IC(50) = 2.7 +/- 0.4 microM and IC(50) = 2.6 +/- 0.6 microM, respectively), correlating with electrophysiological data from the rat cortical wedge model (EC(50) = 280 +/- 48 microM and EC(50) = 586 +/- 41 microM, respectively). 1-Hydroxy-1,2,3-triazol-5-yl analogues of AMPA, compounds 8a,b, showed high affinity for [3H]AMPA receptor binding sites (IC(50) = 0.15 +/- 0.03 microM and IC(50) = 0.13 +/- 0.02 microM, respectively). Electrophysiological data showed that compound 8a was devoid of activity in the rat cortical wedge model (EC(50) > 1000 microM), whereas the corresponding 4-methyl analogue 8b was a potent AMPA receptor agonist (EC(50) = 15 +/- 2 microM). In accordance with this disparity, compound 8a was found to inhibit synaptosomal [3H]D-aspartic acid uptake (IC(50) = 93 +/- 25 microM), as well as excitatory amino acid transporters (EAATs) EAAT1 (IC(50) = 100 +/- 30 microM) and EAAT2 (IC(50) = 300 +/- 80 microM). By contrast, compound 8b showed no appreciable affinity for Glu uptake sites, neither synaptosomal nor cloned. Compounds 9a-c and 10a,b, possessing 1-hydroxyimidazole as the terminal acidic function, were devoid of activity in all of the systems tested. Protolytic properties of compounds 7a,b, 8b, and 9b were determined by titration, and a correlation between the pK(a) values and the activity at AMPA receptors was apparent. Optimized structures of all the synthesized ligands were fitted to the known crystal structure of an AMPA-GluR2 construct. Where substantial reduction or abolition of affinity at AMPA receptors was observed, this could be rationalized on the basis of the ability of the ligand to fit the construct. The results presented in this article point to the utility of 1-hydroxypyrazole and 1,2,3-hydroxytriazole as bioisosteres of carboxylic acids at Glu receptors and transporters. None of the compounds showed significant activity at metabotropic Glu receptors.     

The effects of a novel phosphodiesterase 7A and -4 dual inhibitor, YM-393059, on T-cell-related cytokine production in vitro and in vivo

YM-393059, (+/-)-N-(4,6-dimethylpyrimidin-2-yl)-4-[2-(4-methoxy-3-methylphenyl)-5-(4-methylpiperazin-1-yl)-4,5,6,7-tetrahydro-1H-indol-1-yl]benzenesulfonamide difumarate, is a novel phosphodiesterase (PDE) inhibitor that inhibited the PDE7A isoenzyme with a high potency (IC50=14 nM) and PDE4 with a moderate potency (IC50=630 nM). In a cell-based assay, YM-393059 was found to inhibit anti-CD3 antibody, Staphylococcal enterotoxin B, and phytohaemagglutinin-induced interleukin (IL)-2 production in mouse splenocytes with IC50 values ranging from 0.48 to 1.1 microM. It also inhibited anti-CD3 antibody-induced interferon (IFN)-gamma and IL-4 production in splenocytes with IC50 values of 1.8 and 2.8 microM, respectively. YM-393059's inhibition of anti-CD3 antibody-stimulated cytokine (IL-2, IFN-gamma, and IL-4) production was 20- to 31-fold weaker than that of YM976, a selective PDE4 inhibitor. However, orally administered YM-393059 and YM976 inhibited anti-CD3 antibody-induced IL-2 production equipotently in mice. In addition, YM-393059 inhibited lipopolysaccharide-induced tumor necrosis factor-alpha production in vivo more potently than IL-2 (ED50 values of 2.1 mg/kg and 74 mg/kg). In contrast to YM976, YM-393059 did not shorten the duration of alpha2-adrenoceptor agonist-induced sleep in mice, which is a model for the assessment of the typical side effects caused by PDE4 inhibitors, nausea and emesis. YM-393059 is a novel and attractive compound for the treatment of a wide variety of T-cell-mediated diseases.     

GABA-A agonists and GABA uptake inhibitors: Structure-activity relationships

Muscimol is a potent but non-selective GABA-A agonist. Structure-activity studies on the (S)- and (R)-forms of chiral muscimol analogues have disclosed a high degree of agonist stereoselectivity of the GABA-A receptors. THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol) is a specific GABA-A agonist, which has been the subject of clinical studies in different groups of patients. Even minor alterations of the structure of THIP result in substantial or complete loss of GABA-A agonist activity. 4-PIOL (5-(4-piperidyl)isoxazol-3-ol) shows in vivo GABA-A agonist activity on spinal neurones, whereas the in vitro pharmacological effects in brain tissue preparations are consistent with a GABA-A antagonist profile of 4-PIOL in the brain. Whereas nipetcotic acid and related GABA uptake inhibitors are substrates for the neuronal and glial transport carrier, the glia-selective GABA uptake inhibitors THPO (4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridin-3-ol) and probably also THAO (5,6,7,8-tetrahydro-4H-isoxazolo[4,5-c]azepin-3-ol) are not being transported by the glial uptake carrier. Introduction of the DPB (4,4-diphenyl-3-butenyl) or BEE (benzhydryl ethyl ether) substituents on the basic nitrogen atoms of GABA uptake inhibitors, including nipecotic acid and THPO, results in markedly more potent inhibitors. However, unlike THPO, N-DPB-THPO interacts non-selectively with neuronal and glial GABA uptake, and, in contrast to nipecotic acid, N-DPB-nipecotic acid (SKF-89976-A) has been shown not to be transported by the neuronal or glial GABA carriers. Whereas N-DPB- and N-BEE-GABA are weak inhibitors of synaptosomal GABA uptake, N-methylation of these compounds gives potent uptake inhibitors.     

Central receptor binding and cardiovascular effects of GABA analogues in the cat

Several structural analogues of GABA were shown to be inhibitors of GABAA receptor binding in membranes from cat cerebral cortex. These compounds were 3-aminopropanesulfonic acid (APS; IC50 = 0.04 microM), imidazoleacetic acid (IMA; IC50 = 0.4 microM), morpholinopropanesulfonic acid (MOPS; IC50 = 1.6 microM), 5-phenylpyrrolepropionic acid (PPP; IC50 = 15 microM), aminoethanethiosulfonic acid (AETS; IC50 = 22 microM), 3-amino-3-phenylpropionic acid (APP; IC50 = 35 microM), meta-aminobenzoic acid (MABA; IC50 = 58 microM) and urocanic acid (UCA; IC50 = 354 microM). The IC50 value for GABA was 0.03 microM. GABA, PPP, AETS, MABA and UCA were previously shown to reduce arterial pressure in the cat after intracerebroventricular infusion. In the present study MOPS (ED50 = 0.26 nmol/kg), APS (ED50 = 4.7 nmol/kg), APP (ED50 = 49 nmol/kg), and IMA (ED50 = 350 nmol/kg) were also found to be able to decrease blood pressure when infused into the fourth ventricle. All nine compounds reduced blood pressure to the same extent, but in some cases their relative potencies (ED50 values) exhibited significant differences. When the IC50 values for receptor binding were plotted against the ED50 values for the cardiovascular effects, no significant correlation emerged. This lack of a correlation does not necessarily imply that the reductions in blood pressure elicited by the drugs are not related to an activation of central GABAA receptors. Instead, it highlights the difficulties that are sometimes encountered in attempting to obtain quantitative measurements after intracerebroventricular infusion.     

Syntheses and antiinflammatory actions of 4,5,6,7-tetrahydroindazole-5-carboxylic acids.

A novel series of 1-aryl-4,5,6,7-tetrahydro-1H-indazole-5-carboxylic acids and 2-aryl-4,5,6,7-tetrahydro-2H-indazole-5-carboxylic acids were synthesized via condensation between a phenylhydrazine and a 2-(hydroxymethylene)cyclohexanone-4-carboxylate, and the antiinflammatory activity was determined. In the carrageenan edema test, 1-aryl-4,5,6,7-tetrahydro-1H-indazole-5-carboxylic acids exhibited fairly high antiinflammatory activity. However, the 2-aryl isomers were far less active than the former. The most active compound of the series was 1-phenyl-4,5,6,7-tetrahydro-1H-indazole-5-carboxylic acid, which had an ED50 value of 3.5 mg/kg.     

Glycine antagonists. Synthesis, structure, and biological effects of some bicyclic 5-isoxazolol zwitterions

The bicyclic 5-isoxazolol zwitterions 4,5,6,7-tetrahydroisoxazolo[4,3-c] pyridin-3-ol (3, iso-THPO), 5,6,7,8-tetrahydro-4H-isoxazolo [4,3-c]azepin-3-ol (12, iso-THAO), and 5,6,7,8-tetrahydro-4H-isoxazolo [3,4-c]azepin-3-ol (13, iso-THIA), which are structurally related to the glycine antagonist 5,6,7,8-tetrahydro-4H-isoxazolo[3,4-d]azepin-3-ol (iso-THAZ), have been synthesized and tested biologically. All of these compounds were glycine antagonists approximately equipotent with iso-THAZ during microelectrophoretic ejection near cat spinal neurons. In contrast to iso-THAZ, which also interacts with 4-aminobutyric acid (GABA) receptors in rat brains, neither 12 nor 13 show any significant affinities for GABA binding or uptake mechanisms in vitro. The glycine antagonist 3 was, however, shown also to be a moderately potent inhibitor of GABA uptake. The structure of 12 was established by an X-ray analysis. The bond lengths of the 5-isoxazolol anionic moiety of 12 are in agreement with a pronounced delocalization of the negative charge of this compound.     

Potent inhibitory GABAB receptors in stimulated guinea-pig taenia coli

gamma-Aminobutyric acid (GABA, half-maximal inhibitory concentration, IC50 = 9.9 microM) and (-)-baclofen (IC50 = 4.5 microM) but not 10(-4) M muscimol, exerted a presynaptic inhibitory effect on cholinergic transmission in field-stimulated guinea-pig taenia coli preparations, in vitro. The antagonism by 10(-5) M (1S, 9R)-bicuculline methiodide was not specific for GABA agonists. The results suggest that the GABA receptors involved were of type B.     

Quaternary and tertiary quinuclidine derivatives as inhibitors of choline uptake.

The uptake of choline into cholinergic neurons for acetylcholine (ACh) synthesis is by a specific, high-affinity, sodium- and temperature-dependent transport mechanism (HAChU). Of several quaternary quinuclidinol derivatives tested, the N-allyl derivative proved to be most potent. Though the methyl, ethyl, and isopropyl derivatives were less potent at comparable concentrations, at higher concentrations they also maximally inhibited HAChU. The benzyl, hydroxyethyl, and methoxyethyl derivatives failed to inhibit HAChU by greater than 50% at concentrations up to 100 microM. N-Allyl-3-quinuclidinol (NAQ) proved to be a specific inhibitor of HAChU (IC50 = 0.9 microM) and a poor inhibitor of both sodium-independent transport (IC50 = 680 microM) and choline acetyltransferase activity (Ki = 200 microM). The NAQ exhibited noncompetitive type inhibition compared with N-methyl-3-quinuclidinol, a competitive inhibitor of HAChU. Thus, substitution at the N-functional group not only alters potency, but may change the mechanism by which inhibition is produced. The optical isomers of NAQ and several derivatives were prepared and employed to examine the stereochemical selectivity for inhibition of choline uptake. The S(+)-isomer of NAQ (IC50 = 0.1 microM) had approximately 100-fold greater inhibitory activity for HAChU than the corresponding R(-)-isomer (IC50 = 10 microM). With all other quinuclidinols tested, the S(+)-isomers were also more potent than the corresponding R(-)-isomers. In an effort to obtain a tertiary inhibitor of HAChU that would be expected to cross the blood-brain barrier following peripheral administration, 3-biphenyl-3-quinuclidinol (BHQ) and 3-naphthyl-3-quinuclidinol (NHQ) were synthesized and evaluated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Monosaccharide-linked inhibitors of O(6)-methylguanine-DNA methyltransferase (MGMT): synthesis, molecular modeling, and structure-activity relationships.

A series of potential inhibitors of the human DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT) were synthesized, characterized in detail by NMR, and tested for their ability to deplete MGMT activity in vitro. The new compounds, omega-[O(6)-R-guan-9-yl]-(CH(2))(n)-beta-d-glucosides with R = benzyl or 4-bromothenyl and omega = n = 2, 4,. 12, were compared with the established inhibitors O(6)-benzylguanine (O(6)-BG), 8-aza-O(6)-benzylguanine (8-aza-BG), and O(6)-(4-bromothenyl)guanine (4-BTG), which exhibit in an in vitro assay IC(50) values of 0.62, 0.038, and 0.009 microM, respectively. Potential advantages of the glucosides are improved water solubility and selective uptake in tumor cells. The 4-BTG glucosides with n = 2, 4, 6 show moderate inhibition with an IC(50) of ca. 0.5 microM, while glucosides derived from BG and 8-aza-BG showed significantly poorer inhibition compared to the parent compounds. The 4-BTG glucosides with n = 8, 10, 12 were effective inhibitors with IC(50) values of ca. 0.03 microM. To understand this behavior, extensive molecular modeling studies were performed using the published crystal structure of MGMT (PDB entry: ). The inhibitor molecules were docked into the BG binding pocket, and molecular dynamics simulations with explicit water molecules were carried out. Stabilization energies for the interactions of specific regions of the inhibitor and individual amino acid residues were calculated. The alkyl spacer is located in a cleft along helix 6 of MGMT. With increasing spacer length there is increasing interaction with several amino acid residues which play an important role in the proposed nucleotide flipping mechanism required for DNA repair.     

8-Amino-9-substituted guanines: potent purine nucleoside phosphorylase (PNP) inhibitors

A series of 8-amino-9-substituted guanines was synthesized and their activity evaluated against human purine nucleoside phosphorylase (PNP). All compounds were found to be potent inhibitors of human PNP (IC50s: 0.17-126 microM). They were also selectively cytotoxic to MOLT-4 lymphoblasts in the presence of a nontoxic amount (10 microM) of the PNP substrate, 2'-deoxyguanosine (GdR). The most potent of these analogs, 2,8-diamino-1,9-dihydro-9-(2-thienylmethyl)-6H-purin-6-one (8-amino-9-(2-thienylmethyl)guanine; PD 119,229) has an IC50 of 0.17 microM (Ki = 0.067 microM), significantly more potent than the known standard, 8-aminoguanosine (IC50 = 1.40 microM). Thus it represents the most potent PNP inhibitor known to date when tested without limiting the concentration of inorganic phosphate.     

Novel secoergoline derivatives inhibit both GABA and glutamate uptake in rat brain homogenates: synthesis, in vitro pharmacology, and modeling

Three of twelve secoergoline derivatives (Z ethyl 4-[(ethoxycarbonylmethyl)methylamino]-2-methyl-3-phenylpent-2-enoate, 8; ethyl 1,6-dimethyl-3-oxo-5-phenyl-1,2,3,6-tetrahydropyridine-2-carboxylate, 9; Z methyl 4-[(methoxycarbonylmethyl)methylamino)-2-methyl-3-phenylpent-2-enoate, 11), containing bioisosteric sequences of GABA and Glu, inhibited both GABA and Glu transport through cerebrocortical membranes specifically. Compounds 8, 9, and 11 appeared to be equipotent inhibitors of GABA and Glu transport with IC50 values between 270 and 1100 microM, whereas derivatives 1-7, 10, and 12 were without effects. In the presence of GABA and Glu transport-specific nontransportable inhibitors, inhibition of GABA and Glu transport by 8, 9, and 11 proceeded in two phases. The two phases of inhibition were characterized by IC50 values between 4 and 180 nM and 360-1020 microM and different selectivity sequences. These findings may indicate the existence of some mechanism possibly mediated by a previously unrecognized GABA-Glu transporter. Derivatives with the cis, but not the trans configuration of bulky ester groups (8 vs 7 and 11 vs 12) showed significant inhibitory effect (IC50 values of 270 microM vs >1000 microM and 1100 microM vs >1000 microM on GABA transport, respectively). The cis-trans selectivity can be explained by docking these secoergolines in a three-dimensional model of the second and third transmembrane helices of GABA transporter type 1.     

Synthesis and structure-activity relationship studies of 1,3-diarylprop-2-yn-1-ones: dual inhibitors of cyclooxygenases and lipoxygenases

A group of 1,3-diarylprop-2-yn-1-ones (13, 17, 23, 26 and 27) possessing a C-3 p-SO2Me COX-2 pharmacophore were designed, synthesized and evaluated as potential dual inhibitors of cyclooxygenase-1/2 (COX-1/2) and 5/15-lipoxygenases (5/15-LOX) that exhibit vivo antiinflammatory and analgesic activities. Among this class of compounds, 3-(4-methanesulfonylphenyl)-1-(4-fluorophenyl)prop-2-yn-1-one (13h) was identified as a potent and selective inhibitor of COX-2 (COX-2 IC50 = 0.1 microM; SI = 300), being 5-fold more potent than rofecoxib (COX-2 IC50 = 0.5 microM; SI > 200). In a rat carrageenan-induced paw edema assay 13h exhibited moderate antiinflammatory activity (26% inhibition of inflammation) at 3 h after administration of a 30 mg/kg oral dose. A related dual COX-1/2 and 5/15-LOX inhibitor 3-(4-methanesulfonylphenyl)-1-(4-cyanophenyl)prop-2-yn-1-one (13g, COX-1 IC50 = 31.5 microM; COX-2 IC50 = 1.0 microM; SI = 31.5; 5-LOX IC50 = 1.0 microM; 15-LOX IC50 = 3.2 microM) exhibited more potent antiinflammatory activity (ED50 = 90 mg/kg), being superior to the reference drug aspirin (ED50 = 129 mg/kg). Within this group of compounds 3-(4-methanesulfonylphenyl)-1-(4-isopropylphenyl)prop-2-yn-1-one (13e) emerged as having an optimal combination of in vitro COX-1/2 and 5/15-LOX inhibitory effects (COX-1 IC50 = 9.2 microM; COX-2 IC50 = 0.32 microM; SI = 28; 5-LOX IC50 = 0.32 microM; 15-LOX IC50 = 0.36 microM) in conjunction with a good antiinflammatory activity (ED50 = 35 mg/kg) compared to the reference drug celecoxib (ED50 = 10.8 mg/kg) when administered orally. A molecular modeling study where 13e was docked in the COX-2 binding site indicated the C-1 p-i-Pr group was positioned within a hydrophobic pocket (Phe205, Val344, Val349, Phe381 and Leu534), and that this positioning of the i-Pr group facilitated orientation of the C-3 p-SO2Me COX-2 pharmacophore such that it inserted into the COX-2 secondary pocket (His90, Arg513, Ile517 and Val523). A related docking study of 13e in the 15-LOX binding site indicates that the C-3 p-SO2Me COX-2 pharmacophore was positioned in a region closer to the catalytic iron site where it undergoes a hydrogen bonding interaction with His541 and His366, and that the C-1 p-i-Pr substituent is buried deep in a hydrophobic pocket (Ile414, Ile418, Met419 and Ile593) near the base of the 15-LOX binding site.     

GABA uptake inhibitors. Design, molecular pharmacology and therapeutic aspects

In the mid seventies a drug design programme using the Amanita muscaria constituent muscimol (7) as a lead structure, led to the design of guvacine (23) and (R)-nipecotic acid (24) as specific GABA uptake inhibitors and the isomeric compounds isoguvacine (10) and isonipecotic acid (11) as specific GABAA receptor agonists. The availability of these compounds made it possible to study the pharmacology of the GABA uptake systems and the GABAA receptors separately. Based on extensive cellular and molecular pharmacological studies using 23, 24, and a number of mono- and bicyclic analogues, it has been demonstrated that neuronal and glial GABA transport mechanisms have dissimilar substrate specificities. With GABA transport mechanisms as pharmacological targets, strategies for pharmacological interventions with the purpose of stimulating GABA neurotransmission seem to be (1) effective blockade of neuronal as well as glial GABA uptake in order to enhance the inhibitory effects of synaptically released GABA, or (2) selective blockade of glial GABA uptake in order to increase the amount of GABA taken up into, and subsequently released from, nerve terminals. The bicyclic compound (R)-N-Me-exo-THPO (17) has recently been reported as the most selective glial GABA uptake inhibitor so far known and may be a useful tool for further elucidation of the pharmacology of GABA transporters. In recent years, a variety of lipophilic analogues of the amino acids 23 and 24 have been developed, and one of these compounds, tiagabine (49) containing (R)-nipecotic acid (24) as the GABA transport carrier-recognizing structure element, is now marketed as an antiepileptic agent.     

Synthesis of gastrin antagonists, analogues of the C-terminal tetrapeptide of gastrin, by introduction of a beta-homo residue

A series of analogues of Boc-Trp-Leu-Asp-Phe-NH2, a potent gastrin agonist, were synthesized by introducing a beta-homo residue in the sequence. These compounds were tested in vivo on acid secretion, in the anesthetized rat, and for their ability to inhibit binding of labeled gastrin to its receptors on gastric mucosal cells. These analogues behaved as gastrin antagonists. The most potent compounds in this series were Boc-Trp-Leu-beta-homo-Asp-NHCH2C6H5 (10) (IC50 = 1 microM, ED50 = 0.2 mg/kg), Boc-Trp-Leu-beta-homo-Asp-NHCH2CH2C6H5 (11) (IC50 = 0.75 microM, ED50 = 0.5 mg/kg), Boc-Trp-Leu-beta-homo-Asp-Phe-NH2 (12) (IC50 = 1.5 microM, ED50 = 0.1 mg/kg), and Boc-Trp-Leu-beta-homo-Asp-D-Phe-NH2 (13) (IC50 = 2 microM, ED50 = 0.1 mg/kg). We could demonstrate the importance of the region of the peptide bond between leucine and aspartic acid and of the structure of the C-terminal dipeptide Asp-Phe-NH2, for exhibiting biological activity on acid secretion.     

Synthesis and anti-HIV-1 activity of 4,5,6,7-tetrahydro-5-methylimidazo[4,5,1-jk][1,4]benzodiazepin-2(1H)-on e (TIBO) derivatives. 2

In the first paper of this series a new structure with anti-HIV-1 activity was disclosed and analogues were synthesized to explore the structure-activity relationship of changes in the substituent (R) attached at the N-6 position of 9. This study describes the syntheses and anti-HIV-1 testing of analogues with variations of the five-membered urea ring of the 4,5,6,7-tetrahydro-5-methylimidazo[4,5,1-jk] [1,4]benzodiazepin-2(1H)-one (TIBO) structures. Although many different rings were synthesized to replace the cyclic urea of TIBO, most were found to be inactive in inhibiting the replication of the HIV-1 virus in MT-4 cells. The exceptions were replacement of the urea oxygen with sulfur or selenium to give the corresponding thio- or selenoureas. These were found to be more active than the oxygen counterparts. A small series of analogues was synthesized and tested which allowed direct comparison of urea and thiourea derivatives. Without exception, the latter were always more active than the former. The most active compound of this series (8d) was found to inhibit the HIV-1 virus with an IC50 of 0.012 microM which is comparable to that of AZT.     

Pharmacological and biochemical studies with three metabolites of nomifensine

Three major metabolites (M1, M2, M3) of nomifensine (8-amino-1,2,3,4-tetrahydro-2-methyl-4-phenyl-isoquinoline) are formed by hydroxylation and methoxylation of the phenyl ring. They were compared with nomifensine 1. in various psychopharmacological tests in vivo, carried out in mice after oral or i.p. treatment and 2. in neurochemical in vitro studies, measuring inhibition of noradrenaline (NA), dopamine (DA), and serotonin (5-HT) uptake in rat brain synaptosomes. M1 (4'-hydroxy-nomifensine) was the most active metabolite, while M2 and M3 had little or no effect in pharmacological tests. M1 reversed reserpine hypothermia in doses greater than 2.5 mg/kg, antagonized tetrabenazine catalepsy (ED50 68 mg/kg) and reversed oxotremorine hypothermia (ED50 33 mg/kg). In these tests nomifensine was also active, being about 3-10 times more potent than M1. In contrast to nomifensine M1 had also serotoninergic activity, potentiating both phenelzine-induced twitching (ED50 11 mg/kg) and the anticonvulsant effect of 5-hydroxytryptophan. Moreover, M1 prolonged the hexobarbital sleeping time in doses greater than 10 mg/kg, prevented nicotine-induced convulsions (ED50 58 mg/kg) and reduced the oxotremorine tremor (ED50 59 mg/kg). The LD50 of M1 was 1100 mg/kg orally. In vitro M1 was equipotent with nomifensine in inhibiting DA uptake (IC50 1.5 x 10(-7) M) and twice as active in inhibiting NA uptake (IC50 1.1 x 10(-8) M). In contrast to nomifensine M1 was also a potent inhibitor of 5-HT uptake (IC50 3.3 x 10(-7) M). M2 and M3 were less active than M1 in all experiments.