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Inhibitors of PDE4 (cAMP-specific phosphodiesterase) induce side effects, including nausea and emesis, that limit their therapeutic potential. We investigated the function of two catalytically active conformations of PDE4 (a low-affinity conformer detected by conventional cAMP hydrolytic activity and a high-affinity conformer detected by [3H]rolipram binding) in neuronal cells. We assessed enhancement of β-adrenoceptor-mediated cAMP accumulation in cortical neurons in vitro by eleven PDE4 inhibitors with diverse biochemical profiles. The compounds tested have a wide inhibition range of PDE4 catalytic activity and [3H]rolipram binding. Inhibition potency for PDE4 catalytic activity and [3H]rolipram binding for each compound was different. Potency in augmentation of cAMP correlated significantly with the inhibitory effect on [3H]rolipram binding, but not with that against PDE4 catalytic activity. On the other hand, the inhibitory effect on proliferation of T-lymphocytes of the same PDE4 inhibitors correlated both with inhibition of PDE4 catalytic activity and with inhibition of [3H]rolipram binding. These findings indicate that the high affinity PDE4 conformer exists at a high level in cortical neurons and is important in the regulation of cAMP. Furthermore, the relative contributions of the two PDE4 conformers in cell function may cause different PDE4 inhibitor effects on cortical neurons and T-lymphocytes. Keywords:: PDE4 inhibitor, rolipram, high-affinity rolipram binding site, neuron, T-lymphocyte

Prostaglandin (PG) D2, a major cyclooxygenase metabolite generated from immunologically stimulated mast cells, is known to induce activation and chemotaxis in eosinophils, basophils, and T helper 2 (Th2) lymphocytes via a newly identified PGD2 receptor, chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2). CRTH2 is hypothesized to play an important role in the outcome of allergic responses. However, the absence of selective CRTH2 antagonists has prevented the elucidation of the role of CRTH2 in pathogenesis of allergic diseases. We now report compounds discovered as selective CRTH2 antagonists, (2R*,4S*)-N-(1-benzoyl-2-methyl-1,2,3,4-tetrahydroquinolin-4-yl)-N-phenylisobutyramide (K117) and (2R*,4S*)-N-(1-benzoyl-2-methyl-1,2,3,4-tetrahydroquinolin-4-yl)-N-phenylcyclopropanecarboxamide (K604). K117 and K604 have inhibitory effects on human CRTH2 with Ki values of 5.5 and 11 nM, respectively. The effect of these compounds is CRTH2-specific with no cross-reactivity against 15 other receptors and four arachidonic acid-metabolizing enzymes. K117 and K604 has no effect on the basal Ca2+ level and inhibited the Ca2+ response induced by PGD2 in 293EBNA cells expressing human CRTH2. Also, K117 and K604 inhibit PGD2-induced human eosinophil chemotaxis with IC50 values of 7.8 and 42.2 nM, respectively, but they do not inhibit the CC-chemokine receptor 3 agonist eotaxin-induced chemotaxis. These results indicate that K117 and K604 are highly potent and selective antagonists for human CRTH2. These compounds have possibilities to become useful tools to explore CRTH2 functions in allergic diseases.

Due to the prevalence of allergic diseases such as bronchial asthma, allergic rhinoconjunctivitis and dermallergosis, efforts at the discovery of novel and effective medications for prevention and treatment of these conditions have been reinforced. Recently, it has been recognized that these allergic diseases are a chronic inflammatory disorder of the lower and upper airways and skin. In this article, we reviewed the recent development of the following new antiallergic therapies: anti-Th2 cytokine antibodies, decoy receptors, receptor antibodies, anti-IgE antibodies, anti-cell adhesion molecules antibodies, antisense oligonucleotides, keratinocyte modulators, inhibitors of phosphodiesterase 4, tachykinin receptor antagonists, and anti-histaminic drugs. Most of these new agents are aimed to inhibit various components of allergic inflammation. The future use of allergic disease therapies hold great promise and excitement.

Olopatadine hydrochloride (olopatadine, 11-[(Z)-3-(dimethylamino)propylidene]-6,11-dihydrodibenz[b,e]oxepin-2-acetic acid monohydrochloride) is a novel antiallergic/histamine H1-receptor antagonistic drug that was synthesized and evaluated in our laboratories. Oral administration of olopatadine at doses of 0.03 mg/kg or higher inhibited the symptoms of experimental allergic skin responses, rhinoconjunctivitis and bronchial asthma in sensitized guinea pigs and rats. Olopatadine is a selective histamine H1-receptor antagonist possessing inhibitory effects on the release of inflammatory lipid mediators such as leukotriene and thromboxane from human polymorphonuclear leukocytes and eosinophils. Olopatadine also inhibited the tachykininergic contraction in the guinea pig bronchi by prejunctional inhibition of peripheral sensory nerves. Olopatadine exerted no significant effects on action potential duration in isolated guinea pig ventricular myocytes, myocardium and human ether-a-go-go-related gene channel. Olopatadine was highly and rapidly absorbed in healthy human volunteers. The urinary excretion of olopatadine accounted for not less than 58% and the contribution of metabolism was considerably low in the clearance of olopatadine in humans. Olopatadine is one of the few renal clearance drugs in antiallergic drugs. Olopatadine was shown to be useful for the treatment of allergic rhinitis and chronic urticaria in double-blind clinical trials. Olopatadine was approved in Japan for the treatment of allergic rhinitis, chronic urticaria, eczema dermatitis, prurigo, pruritus cutaneous, psoriasis vulgaris and erythema exsudativum multiforme in December, 2000. Ophthalmic solution of olopatadine was also approved in the United States for the treatment of seasonal allergic conjunctivitis in December, 1996 (Appendix: also in the European Union, it was approved in February 2002).

In rat striata, DOPA released is a causal factor for glutamate release and resultant delayed neuron death by four-vessel occlusion. Nanomolar DOPA cyclohexyl ester (CHE), a potent and relatively stable competitive DOPA antagonist, protects these events. We tried to clarify whether DOPA CHE protects these events in hippocampal CA1 pyramidal cell layers most vulnerable against ischemia. Five to 10 min ischemia caused slight to mild glutamate release in 10 min samples during microdialysis and mild to severe neuron death 96 h after reperfusion. DOPA and dopamine were under assay limit in this design, but were basally detected by 20 min sampling and released by 20 min ischemia. In 10 min samples, intrahippocampal perfusion of 100 nM DOPA CHE 10 min before ischemia for 70 min did not inhibit glutamate release by 10 min ischemia, while it abolished glutamate release and protected delayed neuron death by 5 min ischemia. DOPA CHE is neuroprotective under a mild ischemic condition in rat hippocampus CA1.

L-DOPA is probably a transmitter and/or modulator in the central nervous system (1). L-DOPA methyl ester (DOPA ME) is a competitive L-DOPA antagonist. However, it remains to be clarified whether there exist L-DOPAergic receptors. In Xenopus laevis oocytes injected with rat brain poly(A)+ RNA, L-DOPA induced small inward currents with ED50 of 2.2 mM at a holding potential of -70 mV. The currents were abolished by kynurenic acid or CNQX. Similar L-DOPA-currents were seen in oocytes co-injected with AMPA receptors, GluRs1,2,3 and 4. In brain membrane preparations, L-DOPA inhibited specific binding of [3H]-AMPA with IC50 of 260 microM. This inhibition was not modified by 200 microM ascorbic acid, an antioxidant. L-DOPA did not inhibit binding of [3H]-ligands of MK-801, kainate, DCKA and CGP39653. DOPA ME and L-DOPA cyclohexyl ester, a novel, potent and competitive antagonist (2), inhibited specific binding of [3H]-MK-801 with respective IC50 of 1 and 0.68 mM, but elicited no effect on that of the other [3H]-ligands. With low affinities, L-DOPA acts on AMPA receptors, while competitive antagonists act on NMDA ion channel domain. L-DOPAergic agonist and antagonist may not interact on ionotropic glutamate receptors. DOPA ME-sensitive L-DOPA recognition sites (1) seem to differ from glutamate receptors.

A new series of 6,11-dihydrodibenz[b,e]oxepin derivatives exerting both thromboxane synthase inhibitory (TXS-I) and thromboxane receptor antagonist (TXRA) activities is described. (-)-11-[(3-Pyridylmethyl)thio]-6,11-dihydrodibenz[b,e]oxepin -2- carboxylic acid [(-)-3] and (E)-11-[2-(3-pyridyl)ethylidene]-6,11-dihydrodibenz[b,e]oxepin+ ++-2- carboxylic acid methanesulfonate (11E) exhibited potent inhibitory effects on bovine platelet thromboxane synthase with IC50 values of 4.0 and 14 nM, respectively, and these derivatives also antagonized guinea pig platelet TXA2/PGH2 receptors with Ki values of 85 and 180 nM, respectively. Compound 11E exhibited the dual inhibitory activity in ex vivo experiments and demonstrated a significant protective effect in a rat acute renal failure model.

A new series of 11-substituted 6,11-dihydrodibenz[b,e]oxepin derivatives was synthesized and evaluated for antiallergic activity. Convenient methods for the preparation of sulfides from alcohols were developed. Structure-activity relationships are described. Compound 7, 11-[2-(dimethylamin)ethyl]thio-6,11-dihydrodibenz[b,e] oxepin-2-carboxylic acid hydrochloride, was the most potent in the rat passive cutaneous anaphylaxis test (ED50 = 0.92 mg/kg p.o.). It had a potent inhibitory effect on anaphylactic bronchoconstriction in guinea pigs (ED50 = 0.029 mg/kg p.o.) and H1 receptor antagonistic effect (Ki = 14 nM) with few central nervous system side effects. Additionally, an antagonistic effect against prostaglandin D2-induced contraction of isolated guinea pig trachea (pA2 = 5.73) was an attractive mechanism of action of the new antiallergic agent. Compound 7 was selected for further evaluation as KW-4994.

We employed an ex vivo [ 3 H]rolipram binding experiment to elucidate the mechanism of emetic activity of phosphodiesterase 4 inhibitors. In Suncus murinus (an insectivore used for evaluation of emesis), emetic potential as well as ability to occupy the high-affinity rolipram binding site in brain membrane fraction in vivo were determined for phosphodiesterase 4 inhibitors. In vitro , [ 3 H]rolipram bound to the membrane fraction of S. murinus brain with high affinity and its value was comparable to that for rat brain ( K d = 3.6 nM and 3.5 nM, respectively). The test compounds included denbufylline, rolipram, piclamilast, CDP840 and KF19514, each of which possessed similar affinities for the rolipram binding sites in both S. murinus and rat brain. In S. murinus , these compounds induced emesis via intraperitoneal administration. Their ED 50 values were as follows: denbufylline (1.4 mg/kg), rolipram (0.16 mg/kg), piclamilast (1.8 mg/kg), CDP840 (20 mg/kg), and KF19514 (0.030 mg/kg). In addition, these compounds occupied the high-affinity rolipram binding site in vivo as detected by dose-dependent reduction in capacity of ex vivo [ 3 H]rolipram binding in brain membrane fractions. A clear correlation was observed between dose required to induce emesis and that to occupy the high-affinity rolipram binding site for individual phosphodiesterase 4 inhibitors. We conclude that the emetic effect of phosphodiesterase 4 inhibitors is caused at least in part via binding to the high-affinity rolipram binding site in brain in vivo .

We explore stable potent competitive antagonists against L-DOPA. In anesthetized rats, DOPA cyclohexyl ester (DOPA CHE) (30-100 ng) microinjected in depressor sites of the nucleus tractus solitarii dose-dependently shifted the dose-response-curve for L-DOPA (18-300 ng) to the right, with DOPA CHE (100 ng)-induced slight reduction of the maximum response. DOPA methyl ester (DOPA ME) at 100 ng also produced competitive antagonism. Antagonistic activity of DOPA CHE (100 ng) was similar to that of DOPA ME (300 ng). DOPA CHE is suitable for the purpose of screening.

A new method for effecting the formation of a carbon-carbon single bond at the 11-position of 6, 11-dihydrodibenz[b, e]oxepins was developed. The reaction of 11-methoxydibenz[b, e]oxepins with active methylene compounds proceeded in the presence of TiCl4 under mild conditions to afford the correponding 11-substituted dibenzoxepins in moderate to excellent yield. This was considered to be a vinylogous reaction of ketals with active methylene compounds. The efficacy and some applications of this method are described.

Glutamate is implicated in neuronal cell death. Exogenously applied DOPA by itself releases neuronal glutamate and causes neuronal cell death in in vitro striatal systems. Herein, we attempt to clarify whether endogenous DOPA is released by 10 min transient ischemia due to four-vessel occlusion during rat striatal microdialysis and, further, whether DOPA, when released, functions to cause glutamate release and resultant delayed neuronal cell death. Ischemia increased extracellular DOPA, dopamine, and glutamate, and elicited neuronal cell death 96 h after ischemic insult. Inhibition of striatal L-aromatic amino acid decarboxylase 10 min before ischemia increased markedly basal DOPA, tripled glutamate release with a tendency of decrease in dopamine release by ischemia, and exaggerated neuronal cell death. Intrastriatal perfusion of 10-30 nM DOPA cyclohexyl ester, a competitive DOPA antagonist, 10 min before ischemia, concentration-dependently decreased glutamate release without modification of dopamine release by ischemia. At 100 nM, the antagonist elicited a slight ceiling effect on decreases in glutamate release by ischemia and protected neurons from cell death. Glutamate was released concentration-dependently by intrastriatal perfusion of 0.3-1 mM DOPA and stereoselectively by 0.6 mM DOPA. The antagonist elicited no hypothermia during and after ischemia. Endogenously released DOPA is an upstream causal factor for glutamate release and resultant delayed neuronal cell death by brain ischemia in rat striata. DOPA antagonist has a neuroprotective action.

We explored l -DOPA esters with chemically bulky structures to find a potent stable competitive antagonist against l -DOPA, compared to DOPA methyl ester (DOPA ME). In anesthetized rats, DOPA cyclohexyl ester (DOPA CHE), DOPA cyclopentyl ester (DOPA CPE) and DOPA cyclopentyldimethyl ester (DOPA CPDME) at 1 μg microinjected into depressor sites of the nucleus tractus solitarii elicited or tended to elicit more marked antagonism against depressor responses to 60 ng l -DOPA, compared to DOPA ME. At 100 ng, DOPA CHE elicited the most potent antagonism. At 1 μg, duration of the antagonistic activity of DOPA CHE was approximately three times longer than that of DOPA ME. During microdialysis of the nucleus accumbens, conversion from DOPA CHE at 1 μΜ perfused via probes to extracellular l -DOPA was the lowest among these compounds and less than one half of that from DOPA ME. Binding studies showed that the recognition site for l -DOPA differs from ionotropic glutamatergic, dopaminergic D1 and D2 receptors. We recently found that l -DOPA evoked by transient ischemia may act as a DOPA CHE-sensitive causal factor for glutamate release and resultant neuronal cell death. DOPA CHE is the most potent, relatively stable competitive antagonist against l -DOPA and is a useful mother compound to develop neuroprotective drugs.

The histamine H1 receptor has an aspartate (Asp) residue in transmembrane helix 3 (TM3), which is well-conserved among biogenic amine receptors. The Asp residue is one of the most crucial amino acids for ligand binding. The tested histamine H1 receptor antagonists with tri- and tetracyclic structures were not selective for histamine H1 receptors and showed affinity for several other biogenic amine receptors. In contrast, KW-4679 ((Z)-11-[3-(dimethylamino)propylidene]-6,11-dihydrodibenz[b, e]oxepin-2-acetic acid hydrochloride), a tricyclic compound, was a selective histamine H1 receptor antagonist. [3H]KW-4679 had high affinity (Kd value of 2.5 +/- 0.12 nM) for wild-type human histamine H1 receptors. In the [3H]KW-4679 binding assay, replacement of Asp107 by alanine by site-directed mutagenesis greatly reduced the affinities (280-2100-fold) of tri- and tetracyclic compounds, whereas this mutation led to a comparatively small reduction (14-fold) in KW-4679 affinity. These results demonstrate that the tested tri- and tetracyclic histamine H1 receptor antagonists which have a tight interaction with the Asp residue are not selective for the histamine H1 receptor. Furthermore, the high selectivity of KW-4679 might be explained by a unique binding pocket, which consists of the Asp residue and other acceptor sites, in the histamine H1 receptor.

A series of 11-[2-(1-benzimidazolyl)ethylidene]-6,11-dihydrodibenz[b,e]oxep in-2- carboxylic acid derivatives and related compounds were synthesized and found to be potent TXA2/PGH2 receptor antagonists. Each compound synthesized was tested for its ability to displace [3H]U-46619 binding from guinea pig platelet TXA2/PGH2 receptors. Structure-activity relationship studies revealed that the following key elements were required for enhanced activities: (1) an (E)-2-(1-benzimidazolyl)ethylidene side chain in the 11-position of the dibenzoxepin ring system and (2) a carboxyl group in the 2-position of the dibenzoxepin ring system. The studies also indicated that the TXA2/PGH2 receptor binding affinities of this series of compounds in guinea pig platelet were poorly correlated with those in human platelet. Introduction of substituent(s) to the benzimidazole moiety was effective and sodium (E)-11-[2-(5,6-dimethyl-1-benzimidazolyl)ethylidene]- 6,11-dihydrodibenz[b,e]oxepin-2-carboxylate monohydrate (57) recorded the highest affinity for human platelet TXA2/PGH2 receptor with a K(i) value of 1.2 +/- 0.14 nM. It demonstrated potent inhibitory effects on U-46619-induced guinea pig platelet aggregation (in vitro and ex vivo) and human platelet aggregation (in vitro). Compound 57, now designated as KW-3635, is a novel, orally active, and specific TXA2/PGH2 receptor antagonist with neither TXA2/PGH2 receptor agonistic nor TXA2 synthase inhibitory effects. It is now under clinical evaluation.

(Z)-11-[3-(Dimethylamino)propylidene]-2-(methoxycarbonyl)methyl-6, 11- dihydrodibenz[b,e]oxepin-9-acrylic acid (5) was prepared for application to the radioimmunoassay of KW-4679 (1, (Z)-11-[3-(dimethylamino)propylidene]-6,11-dihydrodibenz[b,e ] oxepin-2-acetic acid hydrochloride). The acrylic acid moiety in the 9-position of 5 was employed for coupling with an amino group of bovine serum albumin (BSA) to provide 17. Subsequently, the conjugate 17 was treated with aqueous NaOH to hydrolyze the terminal methoxycarbonyl group in the 2-position of the BSA conjugated 5. Antiserum raised against the antigenic BSA-conjugate 4 finally obtained was specific for 1.

We examined the binding of [3H]U-46619, a thromboxane A2 agonist, to human and guinea pig platelets and the binding of [3H]SQ 29,548, a thromboxane A2 antagonist, to human, rat and guinea pig platelets. KW-3635 (sodium (E)-11-[2-(5,6-dimethyl-1- benzimidazolyl)ethylidene]-6,11-dihydrodibenz[b,e]oxepin-2-c arboxylate monohydrate) concentration-dependently inhibited the [3H]U-46619 binding to human and guinea pig platelets with inhibition constants of 1.2 nM and 2.7 nM, respectively. KW-3635 also potently inhibited the [3H]SQ 29,548 binding to human and guinea pig platelets with inhibition constants of 1.9 nM and 3.2 nM, respectively. In contrast, KW-3635 was less active against thromboxane A2/prostaglandin H2 receptors in rat platelets with an inhibition constant of 97 nM. KW-3635 at 10(-5) M did not antagonize various receptors including prostaglandin E2, prostaglandin I2 and neurotransmitters. In addition, 10(-5) M KW-3635 did not alter the prostaglandin D2-induced cAMP accumulation in EBTr cells. KW-3635 was inactive towards thromboxane synthase, cyclooxygenase and prostaglandin I2 synthase up to 10(-5) M. KW-3635 slightly inhibited 5-lipoxygenase with an IC50 value of 71 microM. These data indicate that KW-3635 is a potent and selective non-prostanoic thromboxane A2 antagonist, and it can recognize the species differences in thromboxane A2/prostaglandin H2 receptors.

New methods for the preparation of multi-functionalized-6,11-dihydrodibenz[b,e]oxepins were developed. The structural requirements of KW-4994 (1), a promising orally active antiallergic agent, were defined. A carboxyl group at C-2 was critical for enhanced antiallergic activity of 1. The introduction of bromine atom at C-9 of 1 could elongate the duration of the action of the parent. Antiplatelet activity, a new pharmacological property of this series of compounds, was observed in one of the derivatives of 1.

1α, 25-Dihydroxy-26, 26, 26, 27, 27, 27-hexafluorocholesterol was synthesized by two procedures. Cholenic acid was converted to the 1α-hydroxy-24-sulfone derivative and hexafluoroacetone was reacted with the lithiated 24-sulfone. Subsequent removal of the sulfonyl group afforded the hexafluoro-1α, 25-dihydroxy compound. An alternative process consists of construction of the hexafluoro-25-hydroxy part on the side chain followed by the introduction of a hydroxy group at the 1α-position. The hexafluoro derivative was converted to the vitamin D form, 1α, 25-dihydroxyhexafluorovitamin D3, via the corresponding 5, 7-diene.

Reaction of 3, 6β-diacetoxycholest-2-ene (2) with cesium fluoroxysulfate gave 6β-acetoxy-2α-fluorocholestan-3-one (3), which was successfully converted to 2α-fluorovitamin D3 (10). A single dose of 500 ng of 10 produced an intestinal calcium transport response and a bone calcium mobilization response in vitamin D-deficient rats equivalent to those of vitamin D3.

1α-Fluorovitamin D3 has been synthesized from 6β-acetoxy-5α-cholest-1-en-3-one (2) via 1α-fluorocholesterol (11). The key reaction was the trans diaxial ring opening of the 1β, 2β-epoxide 5 with potassium hydrogen difluoride in ethyleneglycol by heating. The resulting 1α-fluoro-2β, 3β-diol was subjected to regeneration of the 5-ene function and reductive removal of the 2β-hydroxyl group by Barton's method to give 1α-fluorocholesterol (11). Transformation of 11 into 1α-fluorovitamin D3 led to revision of the previously reported stereochemical assignment of 1α, 25-difluorovitamin D3.

Radiolabeled 25,26-dihydroxyvitamin D3 was prepared in vitro by using chicken kidney homogenates and in vivo in rats from [23,24-3H]-25-hydroxyvitamin D3. These compounds were mixed with synthetic (25S)- and (25R)-25,26-dihydroxyvitamin D3, converted to the corresponding (+)-alpha-methoxy-alpha-trifluoromethylphenylacetyl esters, and subjected to high-performance liquid chromatography that separates the derivatized epimers. The radiolabeled 25,26-dihydroxyvitamin D3 derivatives were a 1:1 mixture of the 25S and 25R isomers. Similarly unlabeled 25,26-dihydroxyvitamin D3 isolated from the plasma of rats given large amounts of vitamin D3 was shown to be a 1:1 mixture of the S and R isomers. Therefore, naturally occurring 25,26-dihydroxyvitamin D3 is a mixture of the 25R and 25S isomers and not just the S isomer reported previously.

1α-Fluorovitamin D3 has been synthesized from 6β-acetoxy-5α-cholest-1-en-3-one (2) via 1α-fluorocholesterol (11). The key reaction was the trans diaxial ring opening of the 1β, 2β-epoxide 5 with potassium hydrogen difluoride in ethyleneglycol by heating. The resulting 1α-fluoro-2β, 3β-diol was subjected to regeneration of the 5-ene function and reductive removal of the 2β-hydroxyl group by Barton's method to give 1α-fluorocholesterol (11). Transformation of 11 into 1α-fluorovitamin D3 led to revision of the previously reported stereochemical assignment of 1α, 25-difluorovitamin D3.

trans Diaxial opening with potassium hydrogen difluoride of the 1β, 2β-epoxy-3β-ol derived from cholenic acid gave the 1α-fluoro-2β, 3β-diol derivative. Construction of the 25-hydroxy cholesterol side chain and deoxygenation of the 2β-hydroxy group afforded 1α-fluoro-3β, 25-dihydroxycholest-5-ene, which was transformed into 1α-fluoro-25-hydroxyvitamin D3. A single dose of 1.3μg of 1α-fluoro-25-hydroxyvitamin D3 produced neither an intestinal calcium transport response nor a bone calcium mobilization response in vitamin D-deficient rats. In the same rats, 50ng of 25-hydroxyvitamin D3 produced a marked response. In contrast, 1α-fluoro-25-hydroxyvitamin D3 was 30 times more active than 25-hydroxyvitamin D3 in binding to the chick intestinal receptor for 1α, 25-dihydroxyvitamin D3. Addition of 1α-fluoro-25-hydroxyvitamin D3 to human promylocytic leukemia cells resulted in strong phagocytic activity.