Therapeutic approaches to the treatment of neuroinflammatory diseases.

Microglia cells are present in the central nervous system and respond quickly to pathogenic stimuli in order to protect the brain. When these immunological responses activate inappropriately or are prolonged, they can contribute to the neuronal damage observed in many neurodegenerative diseases. A variety of immune system modulators including complement proteins, inflammatory cytokines such IL-1 alpha, IL-1 beta, IL-3, IL-6, TNF-alpha, and S100 beta, colony-stimulating factor-1, coagulation proteins and matrix metalloproteases are made by both microglia and astrocytes. Additionally astrocytes, the predominant glial component of the brain, express cell-adhesion molecules, cytokine receptors and induce nitric oxide synthease. The pathophysiology of Alzheimer's disease, stroke, traumatic brain injury, and multiple sclerosis suggest that a large portion of the irreversible damage observed can be attributed to a neuroinflammatory mechanism. The immunomodulators of these diseases are reviewed and new agents within specific molecular mechanisms are presented and discussed.

Structure-activity relationship study of the inhibition of adrenal cortical 11 beta-hydroxylase by new metyrapone analogues.

Metyrapone, 2-methyl-1,2-di-3-pyridyl-1-propanone (1a), is a potent reversible inhibitor of the cytochrome P-450 11 beta-hydroxylase enzyme system (P-450(11) beta) of the adrenal cortex. The structural features of the metyrapone molecule have been systemically altered to determine the requirements necessary for inhibition of P-450(11) beta activity. Metyrapone and 14 analogues have been obtained or synthesized and evaluated as inhibitors using a crude, defatted bovine adrenal cortical mitochondrial preparation. The inhibition of P-450(11) beta activity with these derivatives demonstrated that (1) the A-ring phenyl derivatives 2a-d were better inhibitors than the respective dipyridyl analogues, (2) the ketone in the 1-position can be replaced by various functionalities without markedly reducing inhibition, and (3) at least one methyl group should be present in the 2-position to maintain inhibition. The observed inhibition of P450(11) beta activity with the metyrapone analogues suggest that A-ring phenyl metyrapone analogues 2a-d would be candidates for radioiodination and subsequently used as adrenal cortical imaging agents.

Synthesis and pharmacological evaluation of phenylacetamides as sodium-channel blockers.

The synthesis and structure-activity relationships of a series of phenylacetamides related to N-[3-(2,6-dimethyl-1-piperidinyl)propyl]-alpha-phenylbenzeneacetamide (1) (PD85639) acting at the voltage-dependent Na+ channel are described. All structural variations for this study were made in the phenylacetic acid portion of these molecules, and the compounds were synthesized by coupling the appropriately substituted phenylacetic acid derivative with 3-[1-(2,6-dimethyl)piperidinyl]-propanamine using standard methods of amide formation. Compounds were tested as inhibitors of [3H]batrachtoxinin binding in rat neocortical membranes and also as inhibitors of veratridine-induced Na+ influx in Chinese hamster ovary cells expressing type IIA Na+ channels. Diphenylacetic acid derivatives with halogenated aromatic rings (12-15) were very potent in both assays, while alkoxy and alkyl substitution did not affect activity (16 and 17). Selected compounds were tested as potential neuroprotective agents in two cell culture assays involving inhibition of veratridine-induced and hypoxia-induced lactate dehydrogenase release. Compound 15 was equipotent with flunarizine, a reference compound in both neuroprotection assays.

New and versatile approaches to the synthesis of CPP-related competitive NMDA antagonists. Preliminary structure-activity relationships and pharmacological evaluation.

Fourteen new CPP analogues have been prepared with methyl 1-(phenylmethyl) (+/-)-1,2-piperazinedicarboxylate 3 as a versatile synthetic intermediate. Derivatives were evaluated as NMDA ligands by their ability to displace [3H]CPP from rat cortical membranes. The binding affinity of various chain lengths at the N4-position of the CPP analogues, 5a, 5b, and 9a mimics the binding affinity observed for the acyclic derivatives AP6, AP8, and AP5. Analogue 9a, with a single methylene group in its phosphonate side chain, exhibited diminished affinity for the NMDA receptor when compared to the structurally similar piperidine compound CGS 19755. Replacement of the phosphonic acid moiety with monoionizable acidic groups such as a carboxylate or a phosphinate resulted in a reduction of binding affinity. An aryl spacer between the N4-nitrogen and the distal acidic group was detrimental to binding as was alkylation at the N1-position. Steric bulk, however, was better tolerated when a phenyl group was positioned alpha to the phosphonate, as seen with analogues 21 and 22.

Synthesis and pharmacological evaluation of 4a-phenanthrenamine derivatives acting at the phencyclidine binding site of the N-methyl-D-aspartate receptor complex.

A novel series of octahydrophenanthrenamines and their heterocyclic analogues have been synthesized as potential noncompetitive antagonists of the N-methyl-D-aspartate (NMDA) receptor complex. The compounds were evaluated for their affinity at the phencyclidine (PCP) binding site by determining their ability to displace [3H]TCP from crude rat brain synaptic membranes. A wide range of affinities were observed, with the most potent analogs possessing IC50's equivalent to that of the reference agent MK-801 (3, dizocilpine). NMDA antagonist activity was demonstrated by prevention of glutamate-induced accumulation of [45Ca2+] in cultured rat cortical neurons. Selected compounds were also studied in vivo to determine their ability to prevent the lethal effects of systemically injected NMDA in the mouse. In general, the SAR of the phenanthrenamine series may be summarized as follows: (a) for the amino group at C4a, NHMe > NH2 > NHEt >> NC5H10; (b) for the B-ring substitution, X = CH2 > S > O; (c) unsaturation of the C ring decreases receptor affinity; (d) cis-ring fusion between the B and C rings is desirable; (e) 6-hydroxy or 6-methoxy substitution of the phenanthrenamine system identified an additional hydrogen bonding interaction that substantially increased receptor affinity; (f) spiro analogues (such as 55, IC50 = 3400 nM), which altered the point of attachment of the C ring, caused a substantial reduction in PCP-site affinity. Molecules from this series were useful for refining a pharmacophore model consistent with previous models of the PCP site. In this model, the (R)-(+)-phenanthrenamine 13 superimposes closely onto MK-801 (3), and the angular 4a-amino group is believed to hydrogen bond with a putative receptor site atom. In the phenanthrenamine and thiaphenanthrenamine series, the (R)-(+)-enantiomers (9, 13, and 44) are more potent by approximately 5-10-fold than their corresponding (S)-(-)-enantiomers with respect to their affinity for the PCP site, their ability to prevent accumulation of [45Ca2+] in cultured neuronal cells, and their protection against the lethal effects of NMDA in mice. In general, there was no separation between the dose that prevented NMDA lethality and the dose that produced ataxia in mice, except in the case of the thiaphenanthrenamines 41 and 43. We have not yet obtained evidence that this small separation in activity offers a therapeutic advantage in the treatment of cerebral ischemia or other neurodegenerative disorders.

2-amino-4H-3,1-benzoxazin-4-ones as inhibitors of C1r serine protease.

A series of 2-amino-4H-3,1-benzoxazin-4-ones have been synthesized and evaluated as inhibitors of the complement enzyme C1r. C1r is a serine protease at the beginning of the complement cascade, and complement activation by beta-amyloid may represent a major contributing pathway to the neuropathology of Alzheimer's disease. Compounds such as 7-chloro-2-[(2-iodophenyl)-amino]benz[d][1,3]oxazin-4-one (32) and 7-methyl-2-[(2-iodophenyl)amino]benz[d][1,3]oxazin-4-one (37) show improved potency compared to the reference compound FUT-175. Many of these active compounds also possess increased selectivity for C1r compared to trypsin and enhanced hydrolytic stability relative to 2-(2-iodophenyl)-4H-3,1-benzoxazin-4-one (1).

Synthesis and pharmacological evaluation of hexahydrofluorenamines as noncompetitive antagonists at the N-methyl-D-aspartate receptor.

The noncompetitive (PCP) site of the N-methyl-D-aspartate (NMDA) receptor complex has been implicated in a number of pathologies, including the etiology of ischemic stroke. Recent testing has shown that cis-1,2,3,4,9,9a-hexahydro-N-methyl-4aH-fluoren-4a-amine (1), a rigid analog of PCP, is a potent antagonist at this site (IC50 = 30 nM for displacement of [3H]TCP). On the basis of this finding, a number of derivatives encompassing variations in stereochemistry, amine substitution and position, aromatic and aliphatic ring substitution, and heteroatom ring substitution have been prepared to explore the structure-activity relationships around this ring system. All compounds were evaluated for their PCP receptor affinity; potent compounds were also tested in vitro (cultured neurons) and in vivo (prevention of NMDA-induced lethality in mice). The present hexahydrofluorenamines demonstrated a wide range of potencies, with optimal affinity concentrated in analogs containing a heteroatom (sulfur) in the B ring (IC50 of 11 nM versus [3H]TCP for 16b), methyl substitution on the amine, and R stereochemistry at the 4a position. No significant improvement in affinity was seen with aromatic ring substitution. Aliphatic ring substitution, large amine substituents, and alterations in the position of amine substitution on the ring system resulted in a loss of potency. To explore the effect of simultaneous hydrogen bonding with a putative receptor atom from two directions, the 2-hydroxymethyl derivatives were prepared. This substitution resulted in a loss in receptor binding affinity. Molecular modeling, X-ray, and NMR studies have been used to determine an optimal conformation of the hexahydrofluoreneamines at the receptor site.

Binding of the A1-selective adenosine antagonist 8-cyclopentyl-1,3-dipropylxanthine to rat brain membranes.

8-Cyclopentyl-1,3-dipropylxanthine (PD 116,948) is a very potent, very A1-selective adenosine antagonist, with a Ki of 0.46 nM in 3H-CHA binding to A1 receptors in rat whole brain membranes and 340 nM in 3H-NECA binding to A2 receptors in rat striatal membranes. Its 740-fold A1-selectivity is the highest reported for an adenosine antagonist. 3H-PD 116,948 (117 Ci/mmol) was prepared by reduction of the diallyl analog. 3H-PD 116,948 bound to a single site in rat whole brain membranes, with a Bmax of 46 pmol/g wet weight and Kd of 0.42 nM. Nonspecific binding was extremely low, amounting to about 3% of total binding under standard conditions and less than 1% when higher tissue concentrations were used. Affinities of compounds for inhibition of 3H-PD 116,948 binding were highly consistent with an A1 adenosine receptor. Antagonists were equally potent in 3H-PD 116,948 binding and in 3H-CHA binding, while agonists were consistently about 12-fold more potent in 3H-CHA binding. Hill coefficients were 1.0 for antagonists and about 0.65 for agonists. 3H-PD 116,948 should be a useful antagonist ligand for adenosine A1 receptors.

PD 115,199: an antagonist ligand for adenosine A2 receptors.

PD 115,199, N-[2-(dimethylamino)ethyl]-N-methyl-4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3- dipropyl-1H-purin-8-yl)benzenesulfonamide, was found to have high affinity for the A2 adenosine receptor labeled by 3H-NECA in rat striatal membranes (Ki 15.5 nM). Unlike other potent adenosine antagonists, which always showed some degree of selectivity for the A1 receptor, PD 115,199 had equal affinity at A1 and A2 receptors (Ki in 3H-CHA binding to A1 receptors 13.9 nM). 3H-PD 115,199 (126 Ci/mmol) was prepared by reduction of the diallyl analog, and binding experiments were performed with 0.5 nM 3H-PD 115,199 at 25 degrees C in rat striatal membranes. By nonlinear least-squares analysis of the concentration-inhibition curve for the highly A1-selective adenosine antagonist PD 116,948 (8-cyclopentyl-1,3-dipropylxanthine), it could be demonstrated that about 11% of specific 3H-PD 115,199 binding was to A1 receptors, and the remainder to A2 receptors. A 20 nM concentration of PD 116,948 was included in subsequent experiments to eliminate the A1 component of binding. The remaining binding had a Kd of 2.6 nM and Bmax of 56 pmol/g wet weight. Specific binding was about 79% of total binding. Affinities of compounds in the 3H-PD 115,199 assay were consistent with binding to a high-affinity A2 receptor: antagonists were consistently about three times more potent in 3H-PD 115,199 binding than in 3H-NECA binding, whereas agonists were consistently about fivefold less potent.(ABSTRACT TRUNCATED AT 250 WORDS)

Substituted 2-benzothiazolamines as sodium flux inhibitors: quantitative structure-activity relationships and anticonvulsant activity.

Thirty-two aryl-substituted 2-benzothiazolamines have been tested for their ability to modulate sodium flux in rat cortical slices. A QSAR analysis, applied to these derivatives, showed a trend toward increasing potency as sodium flux inhibitors with increasing lipophilicity, decreasing size, and increasing electron withdrawal of the benzo ring substituents. Additionally, 4- or 5-substitution of the benzo ring was found to decrease potency. The combination of increased lipophilicity, small size, and electron withdrawal severely limited which groups were tolerated on the benzo ring, thus suggesting that the optimal substitution patterns have been prepared within this series. Nine of these compounds were potent inhibitors of veratridine-induced sodium flux (NaFl). These nine compounds also proved to be anticonvulsant in the maximal electroshock (MES) assay. Fourteen additional 2-benzothiazolamines demonstrated activity in the MES screen, yet exhibited no activity in the NaFl assay. These derivatives may be interacting at the sodium channel in a manner not discernible by the flux paradigm, or they may be acting by an alternative mechanism in vivo.

Inhibition of bone resorption by difluoromethylene diphosphonate in organ culture.

A newly synthesized diphosphonate, difluoromethylene diphosphonate (F2MDP), was studied for its effects on bone resorption, as measured by the release of previously incorporated 45Ca. F2MDP (10 microM to 1000 microM) effectively inhibited both unstimulated and parathyroid hormone-stimulated resorption, and the amount of 45Ca release decreased with time. Dichloromethylene diphosphonate (Cl2MDP) and ethane-1-hydroxyl-1, 1-diphosphonate (EHDP) inhibited resorption to similar extents with two exceptions: At concentrations of 10 microM and 100 microM, F2MDP was more effective than EHDP and less effective than Cl2MDP. No greater inhibition was observed when bones had been stimulated with PTH prior to the addition of F2MDP. In addition, bones treated with F2MDP only during the first half of the incubation period exhibited reductions in the amount of 45Ca released during the second half similar to that observed when F2MDP was continuously in the medium, indicating a prolonged effect. Morphologic alterations of osteoclasts suggestive of cell degeneration were observed in F2MDP-treated bones, which were similar to those observed in bones treated with Cl2MDP and EHDP. Due to the presence of fluorine, F2MDP may be useful as an experimental tool to investigate the mode of action of all diphosphonates, in addition to its possible use as a therapeutic agent for diseases of increased bone resorption.

Specific binding of the novel Na+ channel blocker PD85,639 to the alpha subunit of rat brain Na+ channels.

The local anesthetic-like Na+ channel-blocking drug [3H]PD85639 [alpha-([4-3H]phenyl)-N-[3-(2,6-dimethyl-1-piperizinyl)-alpha-prop yl] [4-3H]benzeneacetamide] binds specifically to receptor sites on Na+ channels in intact synaptosomes and synaptosomal membranes, purified and reconstituted Na+ channels, and type IIA Na+ channel alpha subunits expressed in the transfected Chinese hamster ovary cell line CNaIIA-1. No specific binding was observed in nontransfected CHO-K1 cells, confirming the specificity of binding to Na+ channels. Two classes of binding sites that differed in affinity and dissociation rate were observed in all three preparations. In synaptosomes, the high affinity sites had Kd values of 3-20 nM and a Bmax of approximately 0.2 pmol/mg, whereas the low affinity sites had Kd values of 0.4-20 microM and a Bmax of approximately 5 pmol/mg. Binding of PD85,639 was inhibited by the local anesthetics tetracaine, bupivacaine, and mepivacaine at concentrations in the same range as those that inhibit Na+ channels. Tetracaine did not affect the dissociation rate of PD85,639, consistent with competitive binding of these two drugs at the same receptor site. In contrast, binding of PD85,639 was unaffected by the anticonvulsants phenytoin and carbamazepine, which also inhibit Na+ channels. Veratridine and batrachotoxin, which bind at neurotoxin receptor site 2 on Na+ channels, inhibited specific PD85,639 binding completely. PD85,639 accelerated dissociation of specifically bound batrachotoxin, consistent with an indirect allosteric interaction between these two compounds. Thus, like local anesthetics, PD85,639 inhibits binding of batrachotoxin by an allosteric mechanism. The results indicate that PD85,639 binds specifically to a local anesthetic receptor site on the Na+ channel alpha subunit that is allosterically linked to neurotoxin receptor site 2. PD85,639 may be a useful molecular probe of this important drug receptor site on the Na+ channel.

Adrenal cortical 11 beta-hydroxylase and side-chain cleavage enzymes. Requirement for the A- or B-pyridyl ring in metyrapone for inhibition.

The adrenal cortical enzyme systems, 11 beta-hydroxylase, P-450 11 beta, and the side-chain cleavage complex, P-450 scc, differ only in their cytochrome P-450s. Structural modifications of metyrapone, an inhibitor of cytochrome P-450 enzyme systems, have been made to determine the requirement for the A- or B-pyridyl ring for inhibition of P-45011 beta and P-450 scc activities. Three new analogs of metyrapone (A-phenylmetyrapone, B-phenylmetyrapone and diphenylmetyrapone) were synthesized and evaluated as inhibitors using a crude, defatted bovine adrenal cortical mitochondrial preparation. Characterization of the mitochondrial preparation demonstrated: enhancement of both activities by the addition of 15.0 microM adrenodoxin, the addition of 1% ethanol decreased both activities less than 10%, and the apparent Km of deoxycorticosterone for P-45011 beta was 6.8 microM and the apparent Km of cholesterol for P-450 scc was 21.6 microM. Inhibition of P-45011 beta and P-450 scc activities with these compounds demonstrated: the B-pyridyl ring of metyrapone is required for inhibition of both activities whereas requirement for the A-ring is less stringent, and the four metyrapone analogs were more selective inhibitors of P-45011 beta activity. These studies suggest that the A-phenyl metyrapone analog is a good candidate for further development of a selective adrenocortical radiopharmaceutical.

Preparation of an autolysis-resistant interleukin-1 beta converting enzyme mutant.

We describe the expression, purification, and characterization of human interleukin-1 beta converting enzyme (ICE) containing an affinity tag and modified to resist autoproteolysis. The point mutation Asp381 to Glu was added to eliminate the major site of autolytic degradation while maintaining catalytic activity, and an N-terminal polyhistidine tag was added in place of the ICE pro-region to facilitate purification. N-His (D381E) ICE was expressed in Escherichia coli and purified by nickel-chelating Sepharose and size-exclusion chromatography (SEC). The enzyme was stabilized greater than 80-fold against autolytic degradation relative to wild-type N-His ICE. SDS-PAGE analysis with silver-staining revealed no impurities, and 85% of the protein was catalytically active as determined by titration with a novel titrant, PD 163594 (3-[2-(2-benzyloxycarbonylamino-3-methylbutyrylamino)prop ionylamino]-4- oxo-5-(2-oxo-2H-chromen-7-yloxypentanoic acid). An oxidized adduct of ICE with glutathione, formed by disulfide rearrangement with oxidized glutathione to inhibit and stabilize the enzyme during purification, was rapidly reduced upon exposure to 5 mM DTT. One mole of glutathione was released per mole of active enzyme. Of the nine cysteines in ICE, eight were present in their reduced form in the glutathione adduct. N-His (D381E) ICE cleaved Ac-YVAD-Amc with the Michaelis-Menten parameters K(M) = 14 microM and Kcat = 0.7 s-1, values essentially identical to those reported for enzyme from natural sources.

Benzenesulfonamide derivatives of 2-substituted 4H-3,1-benzoxazin-4-ones and benzthiazin-4-ones as inhibitors of complement C1r protease.

A series of 2-sulfonyl-4H-3,1-benzoxazinones was prepared that inhibit C1r protease in vitro. Several compounds were found to be selective for C1r verses the related serine protease trypsin. Selected compounds demonstrated functional activity in a hemolysis assay.

An alpha-mercaptoacrylic acid derivative is a selective nonpeptide cell-permeable calpain inhibitor and is neuroprotective.

Overactivation of calcium-activated neutral protease (calpain) has been implicated in the pathophysiology of several degenerative conditions, including stroke, myocardial ischemia, neuromuscular degeneration, and cataract formation. Alpha-mercaptoacrylate derivatives (exemplified by PD150606), with potent and selective inhibitory actions against calpain, have been identified. PD150606 exhibits the following characteristics: (i) Ki values for mu- and m-calpains of 0.21 microM and 0.37 microM, respectively, (ii) high specificity for calpains relative to other proteases, (iii) uncompetitive inhibition with respect to substrate, and (iv) it does not shield calpain against inactivation by the active-site inhibitor trans-(epoxysuccinyl)-L-leucyl-amido-3-methylbutane, suggesting a nonactive site action for PD150606. The recombinant calcium-binding domain from each of the large or small subunits of mu-calpain was found to interact with PD150606. In low micromolar range, PD15O6O6 inhibited calpain activity in two intact cell systems. The neuroprotective effects of this class of compound were also demonstrated by the ability of PD150606 to attenuate hypoxic/hypoglycemic injury to cerebrocortical neurons in culture and excitotoxic injury to Purkinje cells in cerebellar slices.