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A series of indole cyclopropylmethylamines were found to be potent serotonin reuptake inhibitors. Nitrile substituents at the 5 and 7 positions of the indole ring gave high affinity for hSERT, and the preferred cyclopropane stereochemistry was determined to be (1S,2S)-trans. The cis-cyclopropanes had 20- to 30-fold less affinity than the trans, and the preferred cis stereochemistry was (1R,2S)-cis. Substitution of the indole N-1 position with methyl or ethyl groups gave a 10- to 30-fold decrease in affinity for hSERT, suggesting either a hydrogen-bonding interaction or limited steric tolerance in the region of the indole nitrogen. Compound (+)-12a demonstrated potent hSERT binding (Ki = 0.18 nM) in vitro and was more than 1000-fold less potent at hDAT, hNET, 5-HT1A, and 5-HT6. In vivo, (+)-12a produced robust, dose-dependent increases in extracellular serotonin in rat frontal cortex typical of a selective serotonin reuptake inhibitor. The maximal response produced by (+)-12a was similar to that of fluoxetine but at an approximately 10-fold lower dose.

Corticotropin releasing factor (CRF) is the primary regulator of the hypothalamus−pituitary−adrenal (HPA) axis, coordinating the endocrine, behavioral, and autonomic responses to stress. It has been postulated that small molecules that can antagonize the binding of CRF1 to its receptor may serve as a treatment for anxiety-related and/or affective disorders. Members within a series of 3,4-dihydro-1H-pyrido[2,3-b]pyrazin-2-ones, exemplified by compound 2 (IC50 = 0.70 nM), were found to be very potent antagonists of CRF1. Compound 8w showed high CRF1 receptor binding affinity and was examined further in vivo. The compound was efficacious in a defensive withdrawal model of anxiety in rats and had a long half-life and reasonable oral bioavailability in dog pharmacokinetic studies.

A growing body of evidence suggests that CRF1 receptor antagonism offers considerable therapeutic potential in the treatment of diseases resulting from elevated levels of CRF, such as anxiety and depression. A series of novel 1,2,3,7-tetrahydro-6H-purin-6-one and 3,7-dihydro-1H-purine-2,6-dione derivatives was synthesized and evaluated as corticotropin releasing factor-1 (CRF1) receptor antagonists. Compounds within this series, represented by compound 12d (IC50 = 5.4 nM), were found to be highly potent CRF1 receptor antagonists. In addition, compounds 12d and 12j were determined to be selective CRF1 antagonists. The synthesis, structure−activity relationships and pharmacokinetic properties of compounds within this series is presented.

A highly convergent synthesis was developed for the novel dopamine agonist dinapsoline (12) (Ghosh, D.; Snyder, S. E.; Watts, V. J.; Mailman, R. B.; Nichols, D. E. 8,9-Dihydroxy-2,3,7, 11b-tetrahydro-1H-naph[1,2,3-de]isoquinoline:  A Potent Full Dopamine D1 Agonist Containing a Rigid β-Phenyldopamine Pharmacophore. J. Med. Chem. 1996, 39 (2), 549−555). The crucial step in the new synthesis was a free radical-initiated cyclization to give the complete dinapsoline framework. The improved synthesis required half as many steps as the original procedure (Nichols, D. E.; Mailman, R.; Ghosh, D. Preparation of novel naphtho[1,2,3-de]isoquinolines as dopamine receptor ligands. PCT Int. Appl. WO 9706799 A1, Feb 27, 1997). One of the late-stage intermediates (11) was resolved into a pair of enantiomers. From there, the (R)-(+)-12 (absolute configuration by X-ray) of dinapsoline was identified as the active enantiomer. In unilateral 6-hydroxydopamine (6-OHDA)-lesioned rats, (+)-dinapsoline showed robust rotational behavior comparable to that of an external benchmark, trans-4,5,5a,6,7,11b-hexahydro-2-propyl-benzo[f]thieno[2,3-c]quinoline-9,10-diol, hydrochloride 18 (Michaelides, M. R.; Hong, Y. Preparation of heterotetracyclic compounds as dopamine agonists. PCT Int. Appl. WO 9422858 A1, Oct 13, 1994).

The generation of phosphatidic acid in neutrophils stimulated by the chemotactic factor formylmethionyl-leucyl-phenylalanine (fMet-Leu-Phe) is inhibited by the diacylglycerol kinase inhibitor R59022. Superoxide generation produced by fMet-Leu-Phe, leukotriene B4, platelet-activating factor, or phorbol 12-myristate 13-acetate can be greatly increased in neutrophils pretreated with R59022. The potentiation occurs in the presence or absence of cytochalasin B and is evident in the absence of extracellular calcium. In addition, where the superoxide generated by fMet-Leu-Phe is not inhibited by the protein kinase C inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), the increase by R59022 is diminished by this compound. Unlike cytochalasin B, R59022 does not affect the increase in cytoskeletal actin produced by fMet-Leu-Phe or platelet-activating factor nor does it decrease the basal level. Furthermore, the basal intracellular concentration of free calcium, but not the rise produced by fMet-Leu-Phe or platelet-activating factor, is elevated by R59022. The data presented here suggest that the potentiation by R59022 of the oxidative burst is most likely mediated through protein kinase C.

Addition of pertussis toxin to rabbit neutrophils inhibits the fMet-Leu-Phe- induced increases in Na+ influx and in intracellular pH. In addition, pretreatment of the cells with the toxin inhibits the decrease in the levels of phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-phosphate and the enhanced production of phosphatidic acid produced by the chemotactic factor fMet-Leu-Phe. Furthermore, the fMet-Leu-Phe-induced changes in the phosphorylation of a 46-kDa protein and of several other proteins are also inhibited by the toxin. On the other hand, the phorbol 12-myristate 13-acetate (PMA)-induced increases in the phosphorylation of several proteins are not inhibited by the toxin. PMA, but not its inactive analogue 4 alpha-phorbol 12,13-didecanoate, was also found to stimulate Na+ influx and to increase the intracellular pH in rabbit neutrophils. These ionic effects, like those produced by fMet-Leu-Phe, are inhibited by amiloride. The stimulated Na+ influx and H+ efflux produced by the phorbol ester, on the other hand, are not inhibited by pertussis toxin. The results reported here suggest that the activity of the Na+/H+ antiport in neutrophils is regulated by protein kinase C; that the G-protein system, either directly or indirectly, is involved in the stimulus-response coupling sequence in these cells; and that the toxin acts at, or prior to, the steps responsible for the activation of phospholipase C, and it does not affect the sequence of reactions initiated by the activation of the protein kinase C.

The tumor co-promoter phorbol 12, myristate 13, acetate (PMA) has previously been shown to stimulate several of the characteristic functions (aggregation, degranulation, and the oxidative burst) of polymorphonuclear leukocytes (neutrophils). We describe here a novel feature of the action of PMA on neutrophils, namely its ability to inhibit the chemotactic factor-induced increased in the enzyme secretion and in the intracellular concentration of free calcium. The inhibition is maximal within 3 min of the addition of PMA and is concentration-dependent (IC50 = 8.5 ng/ml). The site of action of PMA is distal to the binding of the chemotactic factors. PMA inhibits both the release of intracellular calcium and the permeability changes to calcium induced by chemotactic factors, but does not affect the stimulation of the rate of influx of sodium produced by the same agents. The PMA analog 4 alpha-phorbol 12, 13-didecanoate, which lack tumorigenicity and the ability to activate the calcium- and phospholipid-dependent protein kinase (protein kinase C), does not inhibit any of the above fMet-Leu-Phe-stimulated neutrophil functions. The present results thus demonstrate that phorbol esters, either directly or indirectly, possibly through the activation of protein kinase C, inhibit the signal(s) responsible for the stimulated mobilization of calcium in rabbit neutrophils.

The addition of fMet-Leu-Phe, leukotriene B4, or arachidonic acid to rabbit neutrophils causes a rise in the level of intracellular free calcium as measured by the fluorescent dye quin-2. The calcium response is rapid and dose-dependent with an ED50 of 0.12 +/- 0.05 nM for leukotriene B4, 0.20 +/- 0.02 nM for fMet-Leu-Phe, and 320 +/- 30 nM for arachidonic acid. However, unlike fMet-Leu-Phe, leukotriene B4 at concentrations up to 70 nM does not cause a significant breakdown of any of the phosphoinositides or the generation of phosphatidic acid, arachidonic acid, or 1,2-diacylglycerol. The addition of arachidonic acid causes little (less than 20%) production of phosphatidic acid. Furthermore, the dose-response curve of the generation of phosphatidic acid by fMet-Leu-Phe is shifted to the right when compared with that for calcium mobilization. The results reported here indicate that, although similar in many respects, there are important qualitative differences between the mechanisms of action of these two chemotactic factors. They also show that calcium mobilization by leukotriene B4 is not mediated by the breakdown of any of the phosphoinositides, and calcium mobilization by leukotriene B4, arachidonic acid, and possibly fMet-Leu-Phe is not mediated by the generation of phosphatidic acid.

The addition of granulocyte-macrophage colony-stimulating factor (GM-CSF) to human neutrophils causes a rapid increase in the basal and fMet-Leu-Phe-stimulated Na+ influx and an increase in intracellular pH. The increase can be seen as early as 5 min after the addition of GM-CSF. Changes produced by GM-CSF are totally inhibited by amiloride and are significantly reduced in pertussis toxin-treated cells. The stimulation of the Na+/H+ exchange mechanism by GM-CSF inhibits further stimulation of this system with either fMet-Leu-Phe or phorbol 12-myristate 13-acetate. In addition, membrane preparations isolated from GM-CSF-treated neutrophils have higher basal and stimulated GTPase activities. The basal and the fMet-Leu-Phe- or platelet-activating factor-stimulated GTPase activities are reduced in pertussis toxin-treated cells. Cells pretreated with GM-CSF accumulate more radioactive phosphate than control cells, and this increase is diminished by pertussis toxin treatment. In addition, GM-CSF causes a rapid increase in the tyrosine phosphorylation levels of five proteins with molecular masses of 118 kDa, 92 kDa, 78 kDa, 54 kDa, and 40 kDa. These results clearly show that GM-CSF, on its own, can initiate several changes and that these changes are mediated in part by the pertussis toxin-sensitive guanine nucleotide regulatory protein.

Bone marrow-derived mouse mast cells were sensitized with monoclonal mouse IgE antibody and treated with cholera toxin (CT), which ADP-ribosylated the alpha-subunit of the stimulatory guanine nucleotide-binding regulatory protein Gs, prior to challenge with either antigen or thrombin. The CT treatment increased intracellular cAMP levels, but neither enhanced nor inhibited antigen-induced histamine release or arachidonate release. The same treatment of the sensitized bone marrow-derived mouse mast cells with CT markedly enhanced thrombin-induced histamine release without affecting arachidonate release. The CT treatment failed to affect antigen-induced and thrombin-induced generation of inositol trisphosphate and of diacylglycerol or mobilization of intracellular Ca2+. The results indicate that Gs in bone marrow-derived mouse mast cells is not involved in the transduction of the antigen-induced or thrombin-induced triggering signal to phospholipase C, which initiates the enhancement of phosphatidylinositol turnover. The enhancement of thrombin-induced histamine release by CT treatment with the observations that thrombin-induced histamine release was inhibited by pretreatment of the cells with pertussis toxin suggest that the involvement of a guanine nucleotide-binding regulatory protein in thrombin-induced biochemical events is an event distal to Ca2+ mobilization.

The addition of the calcium ionophore A23187 to rabbit neutrophils increases the amount of actin associated with the cytoskeleton regardless of the presence or absence of calcium in the incubation medium. In the presence of extracellular calcium, the effect of A23187 is biphasic with respect to concentration. The action of the ionophore is rapid, transient, and is inhibited by pertussis toxin, hyperosmolarity, and quinacrine. On the other hand, the addition of pertussis toxin or hyperosmolarity has small if any, effect on the rise in intracellular calcium produced by A23187. While quinacrine does not affect the fMet-Leu-Phe-induced increase in cytoskeletal actin and the polyphosphoinositide turnover, its addition inhibits completely the stimulated increase in Ca-influx produced by the same stimulus. The results presented here suggest that a rise in the intracellular concentration of free calcium is neither necessary nor sufficient for the stimulated increase in cytoskeletal-associated actin. A possible relationship between the lipid remodeling stimulated by chemoattractants and the increased cytoskeletal actin is discussed.

The ability of the major neutrophil-derived lipoxygenase metabolites of arachidonic acid to increase the rate of 45Ca influx in rabbit neutrophils was examined. The results obtained demonstrate that (5S),(12R)-dihydroxy-6,8,11,14-(cis,trans,trans,cis)-eicosatetraenoic acid (leukotriene B4) is the most active of the arachidonic acid metabolites. The activity of leukotriene B4 is highly stereospecific in that its three nonenzymatically derived isomers are essentially inactive. The omega-hydroxylation of leukotriene B4 results in a compound that is nearly as active as leukotriene B4 as far as its ability to stimulate calcium influx and neutrophil aggregation while being a much weaker secretagogue. The further conversion of leukotriene B4 into a dicarboxylic acid removes all detectable biological activity. 5,6-Oxido-7,9,11,14-eicosatetraenoic acid (leukotriene A4) methyl ester was also found to increase the rate of calcium influx, while the degradation products of native leukotriene A4 were essentially inactive. These results demonstrate that a close correlation exists between the ability of the various lipoxygenase products to alter calcium homeostasis in rabbit neutrophils and their biological activities.

[3H]Arachidonic acid is released after stimulation of rabbit neutrophils with fMet-Leu-Phe or platelet-activating factor (PAF). The release is rapid and dose-dependent, and is inhibited in phorbol 12-myristate 13-acetate (PMA)-treated rabbit neutrophils. The protein kinase C (PKC) inhibitor 1-(5-isoquinoline-sulphonyl)-2-methylpiperazine (H-7) prevents this inhibition. In addition, PMA increases arachidonic acid release in H-7-treated cells stimulated with fMet-Leu-Phe. [3H]Arachidonic acid release, but not the rise in the concentration of intracellular Ca2+, is inhibited in pertussis-toxin-treated neutrophils stimulated with PAF. The diacylglycerol kinase inhibitor R59022 increases the concentration of diacylglycerol and potentiates [3H]arachidonic acid release in neutrophils stimulated with fMet-Leu-Phe. This potentiation is not inhibited by H-7. These results suggest several points. (1) A rise in the intracellular concentration of free Ca2+ is not sufficient for arachidonic acid release in rabbit neutrophils stimulated by physiological stimuli. (2) A functional pertussis-toxin-sensitive guanine nucleotide regulatory protein and/or one or more of the changes produced by phospholipase C activation are necessary for arachidonic acid release produced by physiological stimuli. (3) Agents that stimulate PKC potentiate arachidonic acid release, and this potentiation is not inhibited by H-7. These agents produce their actions in part by direct membrane perturbation.

The addition of the platelet-activating factor (PAF) to neutrophils causes an increase in cytoskeletal actin, a rise in the intracellular concentration of free calcium, release of arachidonic acid, and the synthesis of PAF. The PAF synthesis in human neutrophils stimulated by PAF is greatly potentiated by the human granulocyte-macrophage colony-stimulating factor. Incubation of human neutrophils with the tumor copromoter phorbol 12-myristate 13-acetate (PMA) for 3 min prior to the addition of the stimulus inhibits all these responses produced by PAF. The inhibition is prevented when the cells are incubated with protein kinase C inhibitors such as 1-(5-isoquinolinesulfonyl)-2-methylpiperazine for 5 min prior to the addition of PMA. The rise in the intracellular concentration of free calcium in human neutrophils stimulated with leukotriene B4 is also inhibited by PMA, and this inhibition is prevented by protein kinase C inhibitors such as staurosporine. Unlike PMA, the inactive ester 4 alpha-phorbol 12,13-didecanoate has no inhibitory effect on the stimulated rise in the intracellular concentration of free calcium. The binding of either PAF or leukotriene B4 to intact cells is inhibited by PMA. The most important finding of the present studies is that PMA interferes with the binding of PAF and leukotriene B4 to their respective receptors. Whether PMA inhibits the binding of these lipid mediators by activating protein kinase C or by perturbing the membrane directly remains to be elucidated.

Platelet-activating factor (l-O-alkyl-2-acetyl-sn-glycerol-3-phosphocholine; PAF) enhances the release of newly synthesized PAF as measured by [3H]acetate incorporation into PAF in human neutrophils. The response was dose-dependent, rapid, transient, and inhibitable by the PAF antagonist BN-52021. The non-metabolizahle bioactive PAF analogue (C-PAF) but not lyso-PAF enhances the release of newly synthesized PAF. Newly synthesized PAF was also released after stimulation of these cells with fMet-Leu-Phe. The human granulocyte-macrophage colony-stimulating factor potentiates the stimulated release of PAF. The intracellular calcium chelator BAPTA inhibits the rise of [Ca2+]iand the release of PAF but not the Na+/H+antiport activity. PAF release, but not the rise in the intracellular concentration of free calcium, was inhibited in pertussis toxin-treated neutrophils stimulated with PAF. The release of PAF in pertussis toxin-treated cells was also inhibited in cells stimulated with fMet-Leu-Phe or opsonized zymosan. These results suggest that functional pertussis toxin-sensitive guanine nucleotide regulatory protein and/or one or more of the changes produced by phospholipase C activation are necessary for PAF release produced by physiological stimuli. It appears that PAF release requires a coordinated action of receptor-coupled G-proteins, calcium, and other parameters.