PD 158771, a potential antipsychotic agent with D(2)/D(3) partial agonist and 5-HT(1A) agonist actions. I. Neurochemical effects.

The neurochemical effects of a novel dopamine (DA) D(2)-like and serotonin (5-HT) 5-HT(1A) agonist, PD 158771, are described. PD 158771 exhibited affinities for human D(2L), D(3) and D(4.2) receptors expressed in Chinese hamster ovary (CHO)-K1 cells with K(i) (nM) values of 5.2, 13.7 and 34.8 respectively. PD 158771 showed high affinity for cloned human 5-HT(1A) (K(i) = 2.6 nM) and rat hippocampal 5-HT(1A) receptors (K(i) = 3.5 nM). Weaker affinities were observed at alpha 1-adrenergic (K(i) = 43 nM), histamine H(1) (IC(50) = 30 nM), 5-HT(2A) (K(i) = 24.5 nM) and sigma (sigma) -1 binding sites (K(i) = 24.5 nM). In measures of in vitro functional activity, PD 158771 stimulated [(3)H]thymidine uptake in CHO p-5 cells transfected with hD(3) receptors with a maximal effect of 23% relative to quinpirole. In hD(2)L, the corresponding value was 60% with an EC(50) of 29 nM, again indicating partial DA agonist action of PD 158771. In vivo, PD 158771 produced a dose-related decrease in DA synthesis in the striatum and mesolimbic regions of rat brain treated with gamma-butyrolactone (GBL), indicating a DA autoreceptor agonist action. In animals not treated with GBL, PD 158771 produced a dose-related decrease in DA synthesis and extracellular DA. A decrease in 5-HT synthesis in several brain areas was observed consistent with an agonist response. Further support for DA autoreceptor agonist action is that PD 158771 produced a partial inhibition of the firing of substantia nigra zona compacta DA neurons, an effect reversed by haloperidol. In conclusion, PD 158771 exhibited affinities for DA and 5-HT receptors, appears to possess DA and 5-HT agonist actions; and it could provide improved antipsychotic profile with minimal side effects.

PD 158771, a potential antipsychotic agent with D2/D3 partial agonist and 5-HT(1A) agonist actions. II. Preclinical behavioral effects.

PD 158771 has been described in receptor binding and biochemical tests as a partial agonist at dopamine (DA) D2 and D3 receptors as well as an agonist at serotonin (5-HT)(1A) receptors. The present studies describe the profile of PD 158771 in rodent and primate behavioral tests. PD 158771 reduced spontaneous locomotor activity in mice (ED(50)=0.38 mg/kg, i.p.) and rats (ED(50) = 1.2 mg/kg, i.p. and 0.16 mg/kg, s.c.), and reduced amphetamine-stimulated locomotion in mice (ED(50) = 0.13 mg/kg, i.p.). At relatively higher doses up to 3 mg/kg, s.c. in rats, PD 158771 did not produce locomotor stimulation or induce stereotypy, indicating a lack of postsynaptic DA agonist activity. PD 158771 reduced apomorphine stimulated locomotion in rats at a dose 4.6-fold greater than those that reduced spontaneous locomotor activity, indicating weak postsynaptic DA antagonist actions; results consistent with a partial agonist profile. PD 158771 produced anxiolytic-like effects in the water-lick (Vogel) conflict test, effects possibly due to the 5-HT(1A) activity. However, PD 158771 was inactive in the water wheel behavioral despair model in rats, indicating lack of antidepressant properties. Similar to known antipsychotics, PD 158771 produced a potent and long-lasting inhibition of conditioned avoidance responding in squirrel monkeys. In contrast to standard antipsychotics, and similar to clozapine, PD 158771 did not cause catalepsy in rats at a dose 20-fold higher than the ED(50) dose for locomotor inhibition. PD 158771 also had a somewhat lower liability than haloperidol to produce extrapyramidal dysfunction in squirrel and cebus monkeys sensitized to the dystonic effects of haloperidol. The data indicate that PD 158771 is a DA partial agonist with weak intrinsic activity that selectively activates brain DA autoreceptors. PD 158771 produced behavioral effects consistent with potential antipsychotic and anxiolytic efficacy, and has an improved profile in the extrapyramidal side effect model when compared to certain currently available antipsychotic agents.

A series of 6- and 7-piperazinyl- and -piperidinylmethylbenzoxazinones with dopamine D4 antagonist activity: discovery of a potential atypical antipsychotic agent.

As part of a program to develop dopamine D4 antagonists for the treatment of schizophrenia, we discovered a series of 6- and 7-(phenylpiperazinyl)- and -(phenylpiperidinyl)methylbenzoxazinones through mass screening of our compound library. A structure-activity relationship SAR study was carried out involving substituents on the phenyl ring, and several selective D4 antagonists were identified. The 7-substituted benzoxazinones showed more activity in neurochemical and behavioral tests than the 6-substituted series. One of the most potent and selective compounds (26) was found to have potent activity in animal tests predictive of antipsychotic activity in humans after oral administration. This paper describes the SAR of the benzoxazinone series and the preclinical characterization of 26.

3-substituted GABA analogs with central nervous system activity: a review.

Gabapentin and Pregabalin are both 3-alkylated gamma-amino butyric acid (GABA) analogs. Gabapentin was designed as a lipophilic GABA analog and was first synthesized as a potential anticonvulsant and was launched in 1994 as add-on therapy for the treatment of epilepsy. In this review the discovery and development of gabapentin as an anticonvulsant are discussed. During human trials and while in clinical use, it became apparent that gabapentin induced some other potentially useful therapeutic effects in chronic pain states and behavioral disorders. A review of animal and clinical data relating to these other potential therapeutic utilities is presented. Pregabalin was identified after an investigation into other 3-substituted GABA analogs. It has since been shown to have a similar pharmacological profile to gabapentin with greater potency in preclinical models of pain and epilepsy. Studies of the mechanism(s) of action of these compounds are discussed. Work towards identifying new analogs of both gabapentin and pregabalin is also reviewed.

Novel antipsychotic-like effects on prepulse inhibition of startle produced by a neurotensin agonist.

Agonists of the neuropeptide neurotensin have been proposed as potential novel antipsychotics based on their ability to modulate neurotransmission in brain regions associated with schizophrenia. To test this hypothesis, we examined the effects of a neurotensin mimetic with improved metabolic stability in an animal model with strong predictive validity for antipsychotic activity. Subcutaneous injections of PD149163, a reduced amide neurotensin(8-13) mimetic, significantly antagonized the reduction of prepulse inhibition (PPI) of the rat startle reflex produced by amphetamine and by the phencyclidine analog dizocilpine. PD149163 had no significant effect on baseline PPI or on baseline startle amplitude and did not antagonize the reduction of PPI produced by the direct dopamine agonist apomorphine. These findings suggest that PD149163 has novel antipsychotic-like properties that are distinct from known members of both the "typical" and "atypical" families of antipsychotics.

Isoindolinone enantiomers having affinity for the dopamine D4 receptor.

PD 108635 (1) was identified as a potent dopamine D4 ligand and we wanted to replace the benzylic alcohol with a metabolically more stable moiety. Investigations led to the discovery of a series of isoindolinones having D4 affinity.

Pyrazolo[1,5-a]pyrimidine CRF-1 receptor antagonists.

A series of 3-phenylpyrazolo[1,5-a]pyrimidines was prepared and found to have affinity for the human CRF-1 receptor. The 3-dimensional structure of one of the most potent analogs in this series, 10d, was determined by X-ray crystallography and suggests the spatial requirements for potent CRF-1 receptor binding affinity in this series.

3-[[(4-Aryl-1-piperazinyl)alkyl]cyclohexyl]-1H-indoles as dopamine D2 partial agonists and autoreceptor agonists.

A series of arylpiperazines and tetrahydropyridines joined to indoles by semirigid cycloalkyl spacers were prepared. Target compounds were studied for their ability to bind to the DA D2 receptor in vitro and to inhibit dopamine synthesis and spontaneous locomotor activity in rats. Effects of tether length and relative stereochemistry were assessed for a series of 2-pyridylpiperazines. The cyclohexylethyl spacer was found to be the most active in the series. Further studies explored effects of changes in the arylpiperazine and indole portions of the molecule. From these studies trans-2-[[4-(1H-3-indolyl)cyclohexyl]ethyl]-4- (2-pyridinyl)piperazine (30a) was selected for further evaluation. It was characterized as a partial agonist of DA D2 receptors in vitro and caused decreases in dopamine synthesis and release as well as dopamine neuronal firing. Compound 30a was shown to be devoid of behavioral effects associated with postsynaptic DA D2 receptor activation. Furthermore, compound 30a was shown both to decrease DA synthesis and to inhibit Sidman avoidance responding in squirrel monkeys. These findings suggest that DA D2 partial agonists with the appropriate level of intrinsic activity can act to decrease dopamine synthesis and release and may have potential utility as antipsychotic agents.

Permeability of the blood-brain barrier to the neurotensin8-13 analog NT1.

Neurotensin (NT) has been suggested to be a neuropeptide with therapeutic potential. We used multiple-time regression analysis to measure the unidirectional influx constant (Ki) of a tritiated analog of NT8-13, NT1, with improved metabolic stability. The Ki of [3H]NT1 across the blood-brain barrier (BBB) was 5.12(10(-4)) ml/g-min and was decreased 66% by unlabeled NT1 system. The amount of NT1 crossing the BBB, 0.087% of the injected dose per gram of brain, is consistent with its exerting central effects after peripheral administration. The stable [3H]NT1 crossed the BBB in intact form as assessed by HPLC and completely crossed the endothelial cells that comprise the BBB as assessed by the capillary depletion method. The presence of a transport system could be important for the development of NT analogs.

Agonist properties of a stable hexapeptide analog of neurotensin, N alpha MeArg-Lys-Pro-Trp-tLeu-Leu (NT1).

The major signal transduction pathway for neurotensin (NT) receptors is the G-protein-dependent stimulation of phospholipase C, leading to the mobilization of intracellular free Ca2+ ([Ca2+]i) and the stimulation of cyclic GMP. We investigated the functional actions of an analog of NT(8-13), N alpha MeArg-Lys-Pro-Trp-tLeu-Leu (NT1), and other NT related analogs by quantitative measurement of the cytosolic free Ca2+ concentration in HT-29 (human colonic adenocarcinoma) cells using the Ca(2+)-sensitive dye fura-2/AM and by effects on cyclic GMP levels in rat cerebellar slices. The NT receptor binding affinities for these analogs to HT-29 cell membranes and newborn (10-day-old) mouse brain membranes were also investigated. Data obtained from HT-29 cell and mouse brain membrane preparations showed saturable single high-affinity sites and binding densities (Bmax) of 130.2 and 87.5 fmol/mg protein, respectively. The respective KD values were 0.47 and 0.39 nM, and the Hill coefficients were 0.99 and 0.92. The low-affinity levocabastine-sensitive site was not present (K1 > 10,000) in either membrane preparation. Although the correlation of binding between HT-29 cell membranes and mouse brain membranes was quite significant (r = 0.92), some of the reference agents had lower binding affinities in the HT-29 cell membranes. The metabolically stable compound NT1 plus other NT analogs and related peptides [NT, NT(8-13), xenopsin, neuromedin N, NT(9-13), kinetensin and (D-Trp11)-NT] increased intracellular Ca2+ levels in HT-29 cells, indicating NT receptor agonist properties. The effect of NT1 in mobilizing [Ca2+]i blocked by SR 48692, a non-peptide NT antagonist. Receptor binding affinities of NT analogs to HT-29 cell membranes were positively correlated with potencies for mobilizing intracellular calcium in the same cells. In addition, NT1 increased cyclic GMP levels in rat cerebellar slices, confirming the latter findings of its NT agonist action. These results substantiate the in vitro NT agonist properties of the hexapeptide NT analog NT1.

Differential effects of the nonpeptide neurotensin antagonist, SR 48692, on the pharmacological effects of neurotensin agonists.

In in vitro studies, SR 48692, a nonpeptide neurotensin receptor antagonist, inhibited the binding of [3H] or [125I]neurotensin to membrane preparations from 10-day-old mouse brains and from HT-29 cells with Ki values of 3.9 and 8.6 nM, respectively. SR 48692 also antagonized the neurotensin-induced mobilization of intracellular calcium in HT-29 cells, in agreement with previous findings. In rat cerebellar slices SR 48692 blocked the increase in cyclic GMP levels evoked by neurotensin in a dose-dependent manner. In vivo, SR 48692 antagonized the increase in rat brain mesolimbic dopamine turnover induced by the systemically active neurotensin peptide, EI [(N-Me)Arg-Lys-Pro-Trp-tert-Leu-Leu]. No effects on dopamine turnover of either EI or SR 48692 were observed in the striatum. SR 48692 did not antagonize the EI-induced decreases in mouse body temperature and spontaneous locomotor activity (LMA) or the decreases in LMA induced by ICV-administered neurotensin. Although other explanations are possible, these findings support the hypothesis that a subtype of the NT receptor may mediate the locomotor and hypothermic actions of this peptide and that it is different from the NT receptor that is involved in dopamine turnover.

Studies of the active conformation of a novel series of benzamide dopamine D2 agonists.

Analogs of dopamine D2 agonist 11 were prepared in which a rigid trans decalin ring system was used to mimic various conformations of 11. The four rigid analogs where compared for their ability to bind to the DA D2 receptor and to inhibit forskolin-stimulated cAMP formation, a measure of DA agonist activity. Of the four rigid analogs of compound 11, only compound 12b had significant activity in both assays. Molecular modeling studies of 12a-d showed each had a single conformation with regard to the distance between the benzamide aryl-centroid and the 4-nitrogen atom of the pyridylpiperazine. Compound 12b was shown to have a greater distance between these functionalities (11.8 A) as compared to the other isomers (9.8-10.4 A). The distance between these two functionalities in 12b was similar to that of a conformer of 11 which has an extended conformation. This suggest that 11 is likely in an extended conformation when bound to the DA D2 receptor.

Structure-activity and conformational studies of a series of modified C-terminal hexapeptide neurotensin analogues.

Neurotensin (NT), is a linear tetradecapeptide (pGlu1-Leu2-Tyr3-Glu4-Asn5- Lys6-Pro7-Arg8-Arg9-Pro10-Tyr11-Ile12-Leu13) that has been found in the central nervous system and peripheral tissues and appears to have a variety of physiological properties. A C-terminal hexapeptide analogue [N alpha Me-Arg-Lys-Pro-Trp-Tle-Leu, (1) Tle = tert-leucine] has recently been reported to have high affinity for the NT receptor and appears to possess central activity after systemic administration. In an effort to probe the structure-activity and conformational properties of the dipeptide, Pro-Trp for binding and functional activity, these residues have been substituted with several natural and unnatural amino acids. Some of these analogues have binding affinities similar to compound 1, while in other cases, such as D-amino acid substitutions, the peptides had negligible binding affinity. In general, the Pro10 position seems more tolerant of substitution by amino acids that favor a reverse turn, rather than those that favor an extended conformation. The Trp11 position accepted extra steric bulk more readily than conformational constraints.

Enhancement of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) toxicity by acetohydroxamic acid analogues of 3-nitropyrazole in vitro.

A series of acetohydroxamic acid derivatives of 3-nitropyrazole were synthesized and evaluated for their ability to potentiate (chemosensitization) the activity of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) against EMT-6 mouse mammary tumor cells in vitro. The compounds were designed to test the hypothesis that the chemosensitizing activity of the analogues would be proportional to the rate of isocyanate formation via a Lossen rearrangement, in part a function of the leaving group at the N terminus of each acetohydroxamate. Substitution of acetohydroxamic acid side chains at the N-1 position of the parent 3-nitropyrazole resulted in compounds which were preferentially toxic to cells treated under hypoxic conditions, and which were capable of enhancing the toxicity of CCNU in hypoxia. As was observed for cytotoxicity, the enhancement of CCNU toxicity by these sensitizing agents was significantly reduced under aerobic treatment conditions. A strong correlation was established between hypoxic toxicity and chemosensitizing potency. The activity of the analogue, however, was not proportional to their excepted rates of Lossen rearrangement. Nevertheless, several potent chemosensitizing compounds were identified; some of which were 10-50 X's more potent on a molar basis than Misonidazole, the reference chemosensitizing compound.

Radiosensitization by acetohydroxamic acid derivatives of 3-nitropyrazole.

A series of acetohydroxamic acid derivatives of 3-nitropyrazole were synthesized and evaluated as radiation sensitizing agents in vitro to test the hypothesis that any increase in sensitizing efficiency over that predicted from electron affinity considerations would be proportional to the rate of isocyanate (R--N = C = O) liberation subsequent to a Lossen rearrangement. EMT-6/Ro cells were exposed to the drugs for 1 h prior to irradiation under aerobic and hypoxic conditions. Sensitizer enhancement ratios (SER) were determined for each compound, and corresponding C1.6 values were plotted as a function of reduction potential (E 1/2). Substitution of acetohydroxamates at the N-1 position of the parent nitropyrazole produced a series of compounds with sensitizing potentials exceeding (9- to 50-fold) those predicted based on their electron affinities. While the current studies do not rule out isocyanate involvement in the enhanced sensitization, they suggest that the enhanced sensitizing ability was not directly proportional to the rate of the Lossen rearrangement. The data suggest that the addition of an acetohydroxamic acid side chain can effectively enhance the sensitizing ability of electron-affinic compounds in excess of that associated with redox potential.

Preliminary evaluation of isocyanate-generating nitroheterocycles as chemosensitizers, radiosensitizers and hypoxic cell cytotoxic agents.

Nitro-compounds containing an acetylated acetohydroxamic acid side chain in the N-1 position of a 5-membered ring nitrogen heterocycle have been synthesized. These compounds, which can generate isocyanates via a Lossen rearrangement, were evaluated in order to test the hypothesis that they may be effective radiation and chemosensitizing agents by nature of their isocyanate-associated carbamoylating potential. Evaluation of one such compound, DJW-77 (1(O-Acetyl-Acetohydroxamic acid)-3-nitropyrazole) as a radiation sensitizer, chemosensitizer and hypoxic cell toxin is reported. In vitro DJW-77 demonstrates a potent selective cytotoxicity toward hypoxic EMT-6 tumor cells, is an effective potentiator of CCNU toxicity and is comparable to MISO with respect to its radiation-sensitizing potential. The activity of the drug is eliminated under aerobic conditions. To test the hypothesis that the activity of DJW-77 is related to isocyanate generation, the non-acetylated analog of DJW-77 (which does not directly undergo the Lossen rearrangement) and the parent 3-nitropyrazole ring structure were evaluated. Neither compound enhanced CCNU toxicity, and on an equimolar basis were inferior to DJW-77 as radiation sensitizers. While the non-acetylated analog was cytotoxic to hypoxic cells, relative to DJW-77 this activity was substantially reduced. These studies indicate that the addition of a side chain capable of generating an isocyanate can enhance the cytotoxicity and sensitizing activity of nitroheterocycles.