Metabolism of 1alpha,25-dihydroxyvitamin D(3) in vitamin D receptor-ablated mice in vivo

The metabolism of 1alpha,25-dihydroxyvitamin D(3) was studied in vitamin D receptor-ablated mice following the administration of a physiological dose of 1alpha,25-dihydroxy-[26,27-(3)H]vitamin D(3). The degradation of 1alpha,25-dihydroxy-[26,27-(3)H]vitamin D(3) in the vitamin D receptor null mutant mice was substantially reduced compared to the wild-type control mice. At 24 h postadministration of radiolabeled 1alpha,25-dihydroxyvitamin D(3) more than 50% of the radioactivity was recovered unmetabolized, whereas in wild-type mice nearly all of the 1alpha,25-dihydroxy-[26,27-(3)H]vitamin D(3) was degraded. In wild-type mice three polar metabolites other than 1alpha,25-dihydroxyvitamin D(3) were detected and identified on straight-phase and reverse-phase high-performance liquid chromatography as 1alpha,24(R),25-trihydroxy-[26,27-(3)H]vitamin D(3), 1alpha,25(S),26-trihydroxy-[26,27-(3)H]vitamin D(3), and (23S, 25R)-1alpha,25-dihydroxy-[(3)H]vitamin D(3)-26,23-lactone. Only one metabolite was detected in the plasma and kidneys of vitamin D receptor null mutant mice at 3 h following an intrajugular dose of 1alpha,25-dihydroxy-[26,27-(3)H]vitamin D(3). This metabolite was clearly identified as 1alpha,25(S),26-trihydroxy-[26,27-(3)H]vitamin D(3) by comigration on two HPLC systems and periodate cleavage reaction. At 6, 12, and 24 h postinjection 1alpha,24(R), 25-trihydroxy-[26,27-(3)H]vitamin D(3) was also detected at low levels in plasma, kidneys, and liver of some but not all mutant mice. The presence of 25-hydroxyvitamin D(3)-24-hydroxylase mRNA in the kidneys of these vitamin D receptor null mutant mice was confirmed by ribonuclease protection assay.     

Design and synthesis of potent vitamin D receptor antagonists with A-ring modifications: remarkable effects of 2alpha-methyl introduction on antagonistic activity

Novel A-ring analogues of the vitamin D receptor (VDR) antagonist (3a), ZK-159222, and its 24-epimer (3b) were convergently synthesized. Preparation of the CD-ring portions with the side chains of 3a,b, followed by palladium-catalyzed cross-coupling with the A-ring enyne precursors (15a,b), (3S,4S,5R)- and (3S,4S,5S)-bis[(tert-butyldimethylsilyl)oxy]-4-methyloct-1-en-7-yne, afforded the 2alpha-methyl-introduced analogues (4a,b) and their 3-epimers (5a,b). The biological profiles of the hybrid analogues were assessed in terms of affinity for VDR, and antagonistic activity to inhibit HL-60 cell differentiation induced by the natural hormone, 1alpha,25-dihydroxyvitamin D(3). The analogue 4a showed an approximately fivefold higher antagonistic activity compared with 3a. The 2alpha-methyl introduction into 3a increased the receptor affinity, thereby enhancing VDR antagonism. This approach to design potent antagonists based on hybridization of structural motifs in the A-ring and in the side chain may prove to be valuable.     

Highly potent cell differentiation-inducing analogues of 1alpha,25-dihydroxyvitamin D3: synthesis and biological activity of 2-methyl-1,25-dihydroxyvitamin D3 with side-chain modifications

Eight 2-methyl substituted analogues of 20-epi-22R-methyl-1alpha,25-dihydroxyvitamin D3 (5) and 20-epi-24,26,27-trihomo-22-oxa-1alpha,25-dihydroxyvitamin D3 (6: KH-1060) were convergently synthesized. Preparation of the CD-ring portions with modified side chains of 5 and 6, followed by palladium-catalyzed cross-coupling with the A-ring enyne synthons (20a-d), (3S,4S,5R)-, (3S,4R,5R)-, (3S,4S,5S)- and (3R,4R,5S)-3,5-bis[(tert-butyldimethylsilyl)oxy]-4-methyloct-1-en-7-yne, afforded two sets of four A-ring stereoisomers of 20-epi-2,22-dimethyl-1,25-dihydroxyvitamin D3 (7a-d) and 20-epi-24,26,27-trihomo-2-methyl-22-oxa-1,25-dihydroxyvitamin D3 (8a-d). The biological profiles of the hybrid analogues were assessed in terms of affinity for vitamin D receptor (VDR) and HL-60 cell differentiation-inducing activity in comparison with the natural hormone. The combined modifications of the A-ring at the 2-position and the side chain yielded analogues with high potency.     

Inhibition by prostacyclin and carbacyclins of endothelin-induced DNA synthesis in cultured vascular smooth muscle cells

Effects of prostacyclin and carbacyclins on endothelin-induced DNA synthesis were investigated in vascular smooth muscle cells. DNA synthesis was estimated by [3H]thymidine incorporation. Five carbacyclins used in this report were 5-[(1S, 5S, 6R, 7R)-7-hydroxy-6-[(E)-(S)-3-hydroxy-1-octenyl]bicyclo [3.3.0]oct-2-en-3-yl) pentanoic acid (TEI-7165), methyl 5-[(1S, 5S, 6R, 7R)-7-hydroxy-6-[(E)-(S)-3-hydroxy-1-octenyl]bicyclo[3.3.0]oct-2-en-3- yl]pentanoate (TEI-9090), 5-[(1S, 5S, 6R, 7R)-7-hydroxy-6-[(E)-(3S, 5S)-3-hydroxy-5-methyl-1-nonenyl]bicyclo[3.3.0]oct-2-en-3-yl)penta noic acid (TEI-9063), methyl 5-[(1S, 5S, 6R, 7R)-7-hydroxy-6-[(E)-(3S, 5S)-3-hydroxy-5-methyl-1- nonenyl]bicyclo[3.3.0]oct-2-en-3-yl)pentanoate (TEI-1324), 5-[(1S, 5S, 6R, 7R)-7-hydroxy-6-[(E)-(S)-4-hydroxy-4-methyl-1- octenyl]bicyclo[3.3.0]oct-2-en-3-yl] pentanoic acid (TEI-3356). Prostacyclin and the carbacyclins inhibited the endothelin-induced DNA synthesis within the nanomolar range. These results suggest that prostacyclin and carbacyclins are possibly effective in inhibiting the proliferation of vascular smooth muscle cells under some situations in vivo.     

Efficient synthesis and biological evaluation of all A-ring diastereomers of 1alpha,25-dihydroxyvitamin D3 and its 20-epimer

An improved synthesis of the diastereomers of 1alpha,25-dihydroxyvitamin D3 (1) was accomplished utilizing our practical route to the A-ring synthon. We applied this procedure to synthesize for the first time all possible A-ring diastereomers of 20-epi-1alpha,25-dihydroxyvitamin D3 (2). Ten-step conversion of 1-(4-methoxyphenoxy)but-3-ene (6), including enantiomeric introduction of the C-3 hydroxyl group to the olefin by the Sharpless asymmetric dihydroxylation, provided all four possible stereoisomers of A-ring enynes (3). i.e., (3R,5R)-, (3R,5S)-, (3S,5R)- and (3S,5S)-bis[(tert-butyldimethylsilyl)oxy]oct-1-en-7-yne, in good overall yield. Palladium-catalyzed cross-coupling of the A-ring synthon with the 20-epi CD-ring portion (5), (E)-(20S)-de-A,B-8-(bromomethylene)cholestan-25-ol, followed by deprotection, afforded the requisite diastereomers of 20-epi-1alpha,25-dihydroxyvitamin D3 (2). The biological profiles of the synthesized stereoisomers were assessed in terms of affinities for vitamin D receptor (VDR) and vitamin D binding protein (DBP). HL-60 cell differentiation-inducing activity and in vivo calcium-regulating potency in comparison with the natural hormone.     

2-({6-[(3R)-3-amino-3-methylpiperidine-1-yl]-1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydro-5H-pyrrolo[3,2-d]pyrimidine-5-yl}methyl)-4-fluorobenzonitrile (DSR-12727): a potent, orally active dipeptidyl peptidase IV inhibitor without mechanism-based inactivation of CYP3A

We report on the identification of 2-({6-[(3R)-3-amino-3-methylpiperidine-1-yl]-1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydro-5H-pyrrolo[3,2-d]pyrimidine-5-yl}methyl)-4-fluorobenzonitrile (DSR-12727) (7a) as a potent and orally active DPP-4 inhibitor without mechanism-based inactivation of CYP3A. Compound 7a showed good DPP-4 inhibitory activity (IC(50)=1.1 nM), excellent selectivity against related peptidases and other off-targets, good pharmacokinetic and pharmacodynamic profile, great in vivo efficacy in Zucker-fatty rat, and no safety concerns both in vitro and in vivo.     

Synthesis and biological evaluation of conformationally constrained analogs of the antitumor agents XK469 and SH80. Part 5

Conformational restriction of bioactive molecules offers the possibility of generating structures of increased potency. To this end, a synthesis has been achieved of (R,S)-2-[(8-chlorobenzofurano[2,3-b]quinolinyl)oxy]propionic acid (12a), a highly rigidified, polycyclic analog of 2-[4-[(7-chloro-2-quinoxalinyl)oxy]phenoxy]propionic acid (2a, XK469). Efforts to effect the same synthesis of the corresponding 8-bromo-derivative led to a mixture of intermediate, 8-chloro (9a), and 8-bromo-2-hydroxybenzofurano[2,3-b]quinoline (9b), generated by halogen-exchange, via an aromatic S(RN)1(A(RN)1) reaction of precursor, 8b, with pyridine hydrochloride. The presumption that conformational restriction of 1b-12a might enhance the antitumor potency of the latter has not been sustained. In fact, 12a proved to be significantly less active than 1b. However, it is apparent that virtually all of the spatial and steric properties of 12a, necessary for improved activity, including the disposition of the 2-oxypropionic acid side chain remain to be identified.     

In vivo and in vitro inhibition of rat liver vitamin D3-25-hydroxylase activity by 19-hydroxy-10(S),19-dihydrovitamin D3

Rats treated with varying amounts of 19-hydroxy-10(S),19-dihydrovitamin D3 prior to administration of physiologic doses of vitamin D3 exhibit normal intestinal calcium transport but are unable to mobilize bone calcium. In contrast, 19-hydroxy-10(R),19-dihydrovitamin D3 had no inhibitory activity. Circulating serum levels of 25-hydroxy[3H]vitamin D3 and 1 alpha, 25-dihydroxy[3H]vitamin D3 are markedly suppressed but not totally eliminated in animals predosed with 19-hydroxy-10(S),19-dihydrovitamin D3 before [3H]vitamin D3. Hepatic 25-hydroxy[3H]vitamin D3 levels were approximately equal in both 19-hydroxy-10(S),19-dihydroviotamin D3 treated and untreated rats. However, the rate of conversion of [3H]vitamin D3 to 25-hydroxyvitamin D3 in vivo is greatly reduced in the treated rats. The inhibitory vitamin analogue was also show to block hepatic microsomal 25-hydroxylation in vitro. These results indicate that 19-hydroxy-10(S),19-dihydrovitamin D3 is a specific inhibitor for a hepatic microsomal vitamin D3-25-hydroxylase system.     

Synthesis and biological characterization of novel hybrid 7-[[2-(4-phenyl-piperazin-1-yl)-ethyl]-propyl-amino]-5,6,7,8-tetrahydro-naphthalen-2-ol and their heterocyclic bioisosteric analogues for dopamine D2 and D3 receptors

In a recent preliminary communication we described the development of a series of hybrid molecules for the dopamine D2 and D3 receptor subtypes. The design of these compounds was based on combining pharmacophoric elements of aminotetralin and piperazine molecular fragments derived from known dopamine receptor agonist and antagonist molecules. Molecules developed from this approach exhibited high affinity and selectivity for the D3 receptor as judged from preliminary [(3)H]spiperone binding data. In this report, we have expanded our previous finding by developing additional novel molecules and additionally evaluated functional activities of these novel molecules in the [(3)H]thymidine incorporation mitogenesis assay. The binding results indicated highest selectivity in the bioisosteric benzothiazole derivative N6-[2-(4-phenyl-piperazin-1-yl)-ethyl]-N6-propyl-4,5,6,7-tetrahydro-benzothiazole-2,6-diamine (14) for the D3 receptor whereas the racemic compound 7-([2-[4-(2,3-dichloro-phenyl)-piperazin-1-yl]-ethyl]-propyl-amino)-5,6,7,8-tetrahydro-naphthalen-2-ol (10c) showed the strongest potency. Mitogenesis studies to evaluate functional activity demonstrated potent agonist properties in these novel derivatives for both D2 and D3 receptors. In this regard, compound 7-[[4-(4-phenyl-piperazin-1-yl)-butyl]-prop-2-ynyl-amino]-5,6,7,8-tetrahydro-naphthalen-2-ol (7b) exhibited the most potent agonist activity at the D3 receptor, 10 times more potent than quinpirole and was also the most selective compound for the D3 receptor in this series. Racemic compound 10a was resolved; however, little separation of activity was found between the two enantiomers of 10a. The marginally more active enantiomer (-)-10a was examined in vivo using the 6-OH-DA induced unilaterally lesioned rat model to evaluate its activity in producing contralateral rotations. The results demonstrated that in comparison to the reference compound apomorphine, (-)-10a was quite potent in inducing contralateral rotations and exhibited longer duration of action.     

Practical stereoselective synthesis of (2,3,4, 5-tetrahydro-1H-benzo[b]azepin-5-yl)acetic acid

Highly enantioselective asymmetric hydrogenation of readily accessible olefins, (E)- and (Z)-[1-(toluene-4-sulfonyl)-1,2,3, 4-tetrahydro-1H-benzo[b]azepin-5-ylidene]acetic acid (4a and 4b, respectively) and [1-(toluene-4-sulfonyl)-2, 3-dihydro-1H-benzo[b]azepin-5-yl]acetic acid (4c), is presented as an efficient and straightforward route to (R)-[1-(toluene-4-sulfonyl)-2,3,4, 5-tetrahydro-1H-benzo[b]azepin-5-yl]acetic acid [(R)-1] which is a key intermediate for the synthesis of non-peptide AVP V2-agonist. Hydrogenation of carboxylic acid 4c gave (R)-1 in quantitative yield and 85% ee using Ru(OAc)2[(S)-H8-BINAP], a Ru(II) complex of a partially hydrogenated BINAP (H8-BINAP), as a catalyst. When (R)-1 of 76% ee was transformed into the corresponding isopropylamide 6, pure enantiomer (R)-6 was obtained in 75% yield by recrystallization from MeOH.     

Characterisation of the actions of group I metabotropic glutamate receptor subtype selective ligands on excitatory amino acid release and sodium-dependent re-uptake in rat cerebrocortical minislices

In this study we have tested the effects of a wide range of metabotropic glutamate receptor ligands on (i) depolarisation-evoked efflux of pre-accumulated d-[3H]aspartic acid (d-[3H]asp) from rapidly superfused rat cerebrocortical minislices, and (ii) Na+-dependent uptake of d-[3H]asp into cerebrocortical tissue. Transient elevations in extracellular K+ produced concentration-dependent increases in d-[3H]asp efflux. A submaximally effective concentration (50 mm) was used in all subsequent experiments. The broad-spectrum mGlu receptor agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid [(1S,3R)-ACPD; EC50 17.8 microm], the group I mGlu-selective agonist (S)-3,5-dihydroxyphenylglycine [(S)-3,5-DHPG; EC50 0.5 microm] and the mGlu5 receptor subtype-selective agonist (RS)-2-chloro-5-hydroxyphenylglycine [(RS)-CHPG; EC50 7.3 microm] all concentration-dependently potentiated high K+-evoked d-[3H]asp efflux in the absence of effects on basal outflow of radiolabel. At concentrations selective for mGlu1 receptors, the antagonists (RS)-1-aminoindan-1,5-dicarboxylic acid [(RS)-AIDA; 10-300 microm]; (+)-2-methyl-4-carboxyphenylglycine [LY367385; 1-100 microm] and 7-hydroxyiminocyclopropan[b]chromen-1a-carboxylate ethyl ester [CPCCOEt, 1-30 microm] all failed to inhibit responses to (S)-3,5-DHPG. However, the broad-spectrum mGlu receptor antagonist (S)-alpha-methyl-4-carboxyphenylglycine [(S)-MCPG; IC50 88.5 microm] together with the recently described mGlu5-selective antagonists, 2-methyl-6-(phenylethynyl)-pyridine (MPEP; IC50 0.6 microm), 6-methyl-2-(phenyl-azo)-3-pyridinol (SIB-1757; IC50 4.4 microm) and (E)-2-methyl-6-(2-phenylethenyl)pyridine (SIB-1893; IC50 3.1 microm), at mGlu5-selective concentrations, all powerfully and concentration-dependently inhibited (S)-3,5-DHPG-evoked responses. Two selective excitatory amino acid (EAA) uptake inhibitors, l-trans-2,4-pyrrolidine dicarboxylate (l-trans-2,4-PDC; IC50 229 microm) and dl-threo-beta-benzyloxyaspartate (dl-TBOA; IC50 665 microm) both inhibited the Na+-dependent uptake of d-[3H]asp into cerebrocortical minislices. Importantly, none of the mGlu ligands utilized in the present study significantly inhibited d-[3H]asp uptake at concentrations shown to potentiate K+-evoked efflux. These data demonstrate for the first time that mGlu5 ligands modulate extracellular EAA concentrations by a direct effect on mGlu5-type autoreceptors on EAA nerve terminals as they evoke clear changes in EAA release in the absence of any effects on EAA uptake. Selective mGlu5 receptor antagonists that show high potency and good central bioavailability may provide novel classes of neuroprotective agents for the treatment of brain disorders associated with abnormal EAAergic neurotransmission.     

Bovine Brain Coated Vesicles Contain Adenosine A1 Receptors. Presence of Adenylate Cyclase Coupled to the Receptor

Clathrin-coated vesicles purified from bovine brain express adenosine A1 receptor binding activity. N6-Cyclohexyl[3H]adenosine [( 3H]CHA), an agonist for the A1 receptor, binds specifically to coated vesicles. High and low agonist affinity states of the receptor for the radioligand [3H]CHA with KD values of 0.18 and 4.4 nM, respectively, were detected. The high purity of coated vesicles was established by assays for biochemical markers and by electron microscopy. Binding competition experiments using agonists (N6CHA, N-cyclopentyladenosine, 5'-(N-ethylcarboxamido)adenosine, and N6-[(R)- and N6-[(S)-phenylisopropyl]adenosine) and antagonists (theophylline, 3-isobutyl-1-methylxanthine, and caffeine) confirmed the typical adenosine A1 nature of the binding site. This binding site presents stereospecificity for N6-phenylisopropyladenosine, showing 33 times more affinity for N6-[(R)- than for N6-[(S)-phenylisopropyl]adenosine. The specific binding of [3H]CHA in coated vesicles is regulated by guanine nucleotides. [3H]CHA specific binding was decreased by 70% in the presence of the hydrolysis-resistant GTP analogue guanyl-5-yl-imidodiphosphate. Bovine brain coated vesicles present adenylate cyclase activity. This activity was modulated by forskolin and CHA. The results of this study support the evidence that adenosine A1 receptors present in coated vesicles are coupled to adenylate cyclase activity through a Gi protein.     

C-6 functionalized analogs of 25-hydroxyvitamin D3 and 1alpha,25-dihydroxyvitamin D3: synthesis and binding analysis with vitamin D-binding protein and vitamin D receptor.

In this article, we describe the development of a general synthetic strategy to functionalize the C-6 position of vitamin D3 and its biologically important metabolites, i.e. 25-hydroxyvitamin D3 (25-OH-D3) and 1alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3]. We employed Mazur's cyclovitamin D method to synthesize vitamin D3 analogs with several functionalities at the C-6 position. In addition, we synthesized 6-(3-hydroxypropyl) and 6-[(2-bromoacetoxy)propyl] derivatives of 25-OH-D3 15 and 16, respectively, and 6-(3-hydroxypropyl) derivative of 1,25(OH)2D3 17. Competitive binding assays of 15-17 with human serum vitamin D-binding protein showed that all these analogs specifically bound to this protein, although with significantly lower affinity than the 25-OH-D3, the strongest natural binder, but with comparable affinity with 1,25(OH)2D3, the hormone. On the other hand, 6-[3-hydroxypropyl], 1alpha,25-dihydroxyvitamin D3 17 did not show any specific binding for recombinant nuclear vitamin D receptor. These results indicated that the region containing the C-6 position of the parent seco-steroid [1,25(OH)2D3] may be an important recognition marker towards vitamin D receptor binding. Information, delineated in this article, will be important for evaluating structure-activity relationship in synthetic analogs of vitamin D and its metabolites.     

Comparison of the nuclear and cytosolic forms of the Ah receptor from Hepa 1c1c7 cells: charge heterogeneity and ATP binding properties.

2-[125I]iodo-7,8-dibromo-p-dioxin ([125I]Br2DpD) and 2-[125I]iodo-3-azido-7,8-dibromo-p-dioxin ([125I]N3Br2-DpD) are both capable of binding to the Ah receptor (AhR) with a high degree of specificity in cultured Hepa 1c1c7 cells. After incubation with either [125I]N3Br2DpD or [125I]Br2DpD Hepa 1c1c7 cytosolic and high salt nuclear extracts were analyzed by sucrose density gradient analysis with the following results: (i) With both radioligands an approximately 9 S form of the AhR was observed in cytosolic extracts. (ii) Nuclear extracts labeled with [125I]N3Br2DpD revealed both approximately 6 S and approximately 9 S forms of the AhR. (iii) In contrast, analysis of nuclear extracts labeled with [125I]Br2DpD revealed only an approximately 6 S form of the AhR. The approximately 9 S [125I]N3Br2DpD-labeled AhR was preferentially extracted with 100 mM KCl from a nuclear fraction and mixed with monoclonal antibody 8D3, an anti-90-kDa heat shock protein antibody. Monoclonal antibody 8D3 was able to bind to the approximately 9 S nuclear form of the AhR and caused the receptor to sediment as a heavier complex on sucrose density gradients. This would indicate that the AhR can reside in the nucleus bound to 90-kDa heat shock protein. The [125I]N3Br2DpD-labeled approximately 6 S peak fractions were collected and subjected to denaturing two-dimensional gel electrophoresis. A comparison of [125I]N3Br2DpD-labeled cytosolic (9 S) AhR preparations with the nuclear (6 S) AhR by 2-D gel electrophoresis was performed. The cytosolic form of the AhR was present in the apparent pI range of 5.2-5.7; the nuclear form focused between 5.5 and 6.2. The [125I]N3Br2DpD-labeled nuclear extracts were incubated with ATP-agarose and 43% of the photoaffinity-labeled AhR bound to the affinity gel. In contrast, approximately threefold lower binding of [125I]N3Br2DpD-labeled receptor was obtained when GTP-, AMP-, or ADP-agarose was used. Only 2% of the [125I]N3Br2DpD-labeled cytosolic AhR was able to bind to ATP-agarose. These results suggest that after the AhR translocates into the nucleus the following biochemical changes occur: (i) The sedimentation value for the AhR changes from an approximately 9 S to an approximately 6 S species. (ii) The AhR attains the ability to bind with specificity to ATP. (iii) The AhR undergoes a shift to a more basic pI.     

Existence of both neuropeptide Y, Y1 and Y2 receptors in pig spleen: evidence using subtype-selective antagonists in vivo

The effects of the first selective, non-peptide, NPY Y2 receptor antagonist (S)-N2-[[1-[2-[4-[(R,S)-5,11-dihydro-6(6h)-oxodibenz[b,e]azepin-11-yl]-1-piperazinyl]-2-oxoethyl]cyclopentyl]acetyl]-N-[2-[1,2-dihydro-3,5 (4H)-dioxo-1,2-diphenyl-3H-1,2,4-triazol-4-yl]ethyl]-argininamid (BIIE0246) were studied on splenic vascular responses evoked in the pig in vivo. BIIE0246 abolished the splenic vasoconstrictor response to the NPY Y2 receptor agonist N-acetyl[Leu25Leu31]NPY(24-36), but did not affect the response to the NPY Y1 receptor agonist [Leu31Pro34]NPY, which in turn was abolished by the selective NPY Y1 receptor antagonist (2R)-5-([amino(imino)methyl]amino)-2-[(2,2-diphenylacetyl)amino]-N-[(IR)-1-(4-hydroxyphenyl)ethyl]-pentanamide (H 409/22). Furthermore, the PYY-evoked splenic vasoconstrictor response was partially antagonized by BIIE0246 and subsequently almost abolished by the addition of H 409/22. It is concluded that BIIE0246 exerts selective (vs the NPY Y1 receptor) NPY Y2 receptor antagonism, and thus represents an interesting tool for classification of NPY receptors, in vivo. In addition, evidence for NPY Y2 receptor mediated vasoconstriction was presented. Furthermore, both NPY Y1 and Y2 receptors are involved in the splenic vasoconstrictor response to PYY.     

Discovery and stereoselective synthesis of the novel isochroman neurokinin-1 receptor antagonist 'CJ-17,493'

A novel central nervous system (CNS) selective neurokinin-1 (NK(1)) receptor antagonist, (2S,3S)-3-[(1R)-6-methoxy-1-methyl-1-trifluoromethylisochroman-7-yl]-methylamino-2-phenylpiperidine 'CJ-17,493' (compound (+)-1), was synthesized stereoselectively using a kinetic resolution by lipase-PS as a key step. Compound (+)-1 displayed high and selective affinity (K(i)=0.2 nM) for the human NK(1) receptor in IM-9 cells, potent activity in the [Sar(9), Met(O(2))(11)]SP-induced gerbil tapping model (ED(50)=0.04 mg/kg, s.c.) and in the ferret cisplatin (10mg/kg, i.p.)-induced anti-emetic activity model (vomiting: ED(90)=0.07 mg/kg, s.c.), all levels of activity comparable with those of CP-122,721. In addition, compound (+)-1 exhibited linear pharmacokinetics rather than the super dose-proportionality of CP-122,721 and this result provides a potential solution for the clinical issue observed with CP-122,721.     

Annexin II is the membrane receptor that mediates the rapid actions of 1alpha,25-dihydroxyvitamin D(3)

1alpha,25-Dihydroxyvitamin D(3) has been shown to exert its effects by both genomic (minutes to hours) and rapid (seconds to minutes) mechanisms. The genomic effects are mediated by interaction with the nuclear vitamin D receptor. We show that the vitamin D analog, [(14)C]-1alpha,25-dihydroxyvitamin D(3) bromoacetate, is specifically bound to a protein (molecular weight 36 kDa) in the plasma membrane of rat osteoblastlike cells (ROS 24/1). The plasma membrane protein labeled with the bromoacetate analog was identified as annexin II by sequence determination and Western blot. Partially purified plasma membrane proteins (PI 6.9-7.4) and purified annexin II exhibited specific and saturable binding for [(3)H]-1alpha, 25-dihydroxyvitamin D(3). Antibodies to annexin II inhibited [(14)C]-1alpha,25-dihydroxyvitamin D(3) bromoacetate binding to ROS 24/1 plasma membranes, immunoprecipitated the ligand-protein complex, and inhibited 1alpha,25-dihydroxyvitamin D(3)-induced increases in intracellular calcium in ROS 24/1 cells. The results indicate that annexin II may serve as a receptor for rapid actions of 1alpha, 25-dihydroxyvitamin D(3).     

New bisabolane sesquiterpenes and coumarin from Leontopodium longifolium

From the roots of Leontopotium longifolium, three new bisabolane sesquiterpenes, rel-(1S,4R,5S,6R)-4,5-diacetoxy-6-[(R)-1,5-dimethylhexa-3,5-dienyl]-3-methylcyclohex-2-enyl (Z)-2-methylbut-2-enoate (1), rel-(1S,4R,5S,6R)-4,5-diacetoxy-6-[(R)-5-hydroxy-1,5-dimethylhex-3-enyl]-3-methylcyclohex-2-enyl (Z)-2-methylbut-2-enoate (2), rel-(1R,2S,4R,5S)-4-acetoxy-2-[(R)-5-hydroxy-1,5-dimethylhex-3-enyl]-5-methylcyclohexyl (Z)-2-methylbut-2-enoate (3), and a new coumarin, 2,3-dihydro-5-hydroxy-2-(1-methylethenyl)-7H-pyrano[2,3-g][1,4]benzodioxin-7-one (4) together with nine known compounds have been isolated. The structures of these compounds were established by spectroscopic methods. Compounds 1 and 2 exhibited moderate cytotoxic activities against human promyelocytic leukemia (HL-60) cells.     

Syntheses and evaluation of antioxidant activity of sydnonyl substituted thiazolidinone and thiazoline derivatives

3-Aryl-4-formylsydnone 4'-phenylthiosemicarbazones (3a-d) and 3-aryl-4-formylsydnone thiosemicarbazones (3e-h), which are precursors of 3-aryl-4-heterocyclic sydnones, are prepared by the condensation of 3-aryl-4-formylsydnones (1a-d) with 4'-phenylthiosemicarbazide (2a) and thiosemicarbazide (2b), respectively. The thiosemicarbazones 3 reacted with cyclic reagents such as ethyl chloroacetate (4a), ethyl 2-chloroacetoacetate (4b) and 2-bromoacetophenone (4c) to produce heterocyclic substituted sydnone derivatives 5-7 that possess 4-oxo-thiazolidine and thiazoline groups. The antioxidant activity of synthesized compounds 5a-7h was evaluated. Among these compounds, 4-methyl-2-[(3-arylsydnon-4-yl-methylene)hydrazono]-2,3-dihydro-thiazole-5-carboxylic acid ethyl ester (6e-h) and 4-phenyl-2-[(3-arylsydnon-4-yl-methylene)hydrazono]-2,3-dihydro-thiazoles (7e-h) exhibit the potent DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging activity, comparable to that of vitamin E.