Synthesis and biological activities of new side chain and backbone cyclic bradykinin analogues.

A series of conformationally constrained cyclic analogues of the peptide hormone bradykinin (BK, Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg) was synthesized to check different turned structures proposed for the bioactive conformation of BK agonists and antagonists. Cycles differing in the size and direction of the lactam bridge were performed at the C- and N-terminal sequences of the molecule. Glutamic acid and lysine were introduced into the native BK sequence at different positions for cyclization through their side chains. Backbone cyclic analogues were synthesized by incorporation of N-carboxy alkylated and N-amino alkylated amino acids into the peptide chain. Although the coupling of Fmoc-glycine to the N-alkylated phenylalanine derivatives was effected with DIC/HOAt in SPPS, the dipeptide building units with more bulky amino acids were pre-built in solution. For backbone cyclization at the C-terminus an alternative building unit with an acylated reduced peptide bond was preformed in solution. Both types of building units were handled in the SPPS in the same manner as amino acids. The agonistic and antagonistic activities of the cyclic BK analogues were determined in rat uterus (RUT) and guinea-pig ileum (GPI) assays. Additionally, the potentiation of the BK-induced effects was examined. Among the series of cyclic BK agonists only compound 3 with backbone cyclization between positions 2 and 5 shows a significant agonistic activity on RUT. To study the influence of intramolecular ring closure we used an antagonistic analogue with weak activity, [D-Phe7]-BK. Side chain as well as backbone cyclization in the N-terminus of [D-Phe7]-BK resulted in analogues with moderate antagonistic activity on RUT. Also, compound 18 in which a lactam bridge between positions 6 and 9 was achieved via an acylated reduced peptide bond has moderate antagonistic activity on RUT. These results support the hypothesis of turn structures in both parts of the molecule as a requirement for BK antagonism. Certain active and inactive agonists and antagonists are able to potentiate the bradykinin-induced contraction of guinea-pig ileum.     

New agonist and antagonist analogues of bradykinin

Three new analogues of bradykinin (BK) have been tested for their agonistic and antagonistic actions on the rabbit jugular vein and the guinea pig ileum (B2 receptors), and six were studied on rabbit aorta strips (B1 receptors). Substitution of Gly4, Phe5, and Phe8 in BK with D-Trp gives analogues with a relative affinity lower than 1.0% as compared with BK. These analogues have no antagonistic properties on the rabbit jugular vein and on guinea pig ileum (B2 receptors). Substitution of Pro7 in des-Arg9-BK by Gly and by D-Ala give compounds that antagonise the effects of kinins on the rabbit aorta strips (B1-receptor system). These new antagonists are fairly potent with a pA2 value of 6.03 to 7.29 and seem competitive because the pA2--pA10 values approximate 0.95. These results suggest that the orientation of Phe8 is critical for the activation of B1 receptors by kinins.     

Efffect of noscapine,the antitussive opioid alkaloid,on bradykinin-induced smooth muscle contraction in the isolated ileum of the guinea-pig

Bradykinin (BK) and related kinins are autocoid peptides that play integral roles in many pathophysiological processes such as cough. In this study, the inhibitory effect of noscapine, the antitussive opioid alkaloid, on BK receptors, was tested in the guinea-pig ileum. Contractions of the isolated ileum of the guinea-pig in response to BK were inhibited by noscapine (10-1,000 nM) in a concentration-dependent manner. Concentration-response curves (CRCs) to BK were slightly shifted to the right with a concomitant decrease in the maximum effect. A pA2 value of 6.68 was calculated for noscapine. The slope of the Schild plot of the antagonism was found to be 0.56. Noscapine had no effect on contractions induced by KCl, acetylcholine, histamine, 5-hydroxy tryptamine or angiotensin II. In conclusion, noscapine has a specific antagonistic effect on BK receptors and the mode of inhibition was found to be non-competitive.     

Receptors for bradykinin and kallidin.

In order to establish if bradykinin (BK) and Lys-bradykinin (Lys-BK), alias kallidin, act on the same or on different receptors, experiments were performed on strips of cat terminal ileum and of rabbit aorta. The first preparation contains receptor B2 and the second has the newly identified receptor B1. The criterion used for establishing the identity of receptors for BK and Lys-BK in the cat ileum (receptor B2) was desensitization, while for the rabbit aorta (receptor B1) we measured the apparent affinity (pA2 value) of a competitive and specific inhibitor of BK, [Leu-OMe3,des-Arg9]-BK. Since cat ileum desensitized with BK or Lys-BK shows a significant decrease or a complete disappearance of the response to the other agent, while maintaining full sensitivity for histamine, and since the pA2 values of [Leu-OMe8,des-Arg9]-BK against BK and Lys-BK are identical in the rabbit aorta, we conclude that the two kinins act on the same types of receptors.     

Comparative agonist/antagonist responses in mutant human C5a receptors define the ligand binding site

The C terminus is responsible for all of the agonist activity of C5a at human C5a receptors (C5aRs). In this report we have mapped the ligand binding site on the C5aR using a series of agonist and antagonist peptide mimics of the C terminus of C5a as well as receptors mutated at putative interaction sites (Ile(116), Arg(175,) Arg(206), Glu(199), Asp(282), and Val(286)). Agonist peptide 1 (Phe-Lys-Pro-d-cyclohexylalanine-cyclohexylalanine-d-Arg) can be converted to an antagonist by substituting the bulkier Trp for cyclohexylalanine at position 5 (peptide 2). Conversely, mutation of C5aR transmembrane residue Ile(116) to the smaller Ala (I116A) makes the receptor respond to peptide 2 as an agonist (Gerber, B. O., Meng, E. C., Dotsch, V., Baranski, T. J., and Bourne, H. R. (2001) J. Biol. Chem. 276, 3394-3400). However, a potent cyclic hexapeptide antagonist, Phe-cyclo-[Orn-Pro-d-cyclohexylalanine-Trp-Arg] (peptide 3), derived from peptide 2 and which binds to the same receptor site, remains a full antagonist at I116AC5aR. This suggests that although the residue at position 5 might bind near to Ile(116), the latter is not essential for either activation or antagonism. Arg(206) and Arg(175) both appear to interact with the C-terminal carboxylate of C5a agonist peptides, suggesting a dynamic binding mechanism that may be a part of a receptor activation switch. Asp(282) has been previously shown to interact with the side chain of the C-terminal Arg residue, and Glu(199) may also interact with this side chain in both C5a and peptide mimics. Using these interactions to orient NMR-derived ligand structures in the binding site of C5aR, a new model of the interaction between peptide antagonists and the C5aR is presented.     

Novel approach to the design of synthetic radioiodinated linear V1A receptor antagonists of vasopressin.

We report the solid phase synthesis of six analogs of the potent and selective linear AVP vasopressor (V1a receptor) antagonist: Phaa1-D-Tyr(Et)2-Phe3-Gln4-Asn5-Lys6-Pro7-Arg-NH(8)2(A) (where Phaa = phenylacetyl) in which the Phaa1 residue is replaced by hydroxyphenylacetyl (HO-Phaa), hydroxyphenylpropionyl (HO-Phpa) and phenylpropionyl (Phpa) and the D-Tyr(Et)2 and Lys6 residues by D-Tyr(Me)2 and Arg6 substituents. The phenolic-containing peptides were synthesized to test the feasibility of using this approach for the design of high affinity selective ligands for AVP V1a receptors. The following analogs of A were synthesized: 11 [(HO)Phaa1]; 2. [(HO)Phaa1,D-Tyr(Me)2]; 3. [(HO)Phaa1,D-Tyr(Me)2, Arg6]; 4. [(HO)Phaa1,Arg6]; 5. [Phpa1]; 6. [(HO)Phpa1]. All six peptides were examined for agonistic and antagonistic potencies in vasopressor (V1a-receptor) and antidiuretic (V2-receptor) and in vitro oxytocic assays in rats. The affinities of the phenolic-containing peptides for hepatic V1a and uterine receptors were also determined. The phenolic-containing peptides all exhibit potent V1a antagonism. Their anti-V1a pA2 values range from 8.23 to 8.63 (the anti-V1a pA2 value of A = 8.69). Their inhibition constants (Ki in nM) range 0.4 to 1.0. They are weak antidiuretic agonists with activities ranging from 0.022 U/mg to 0.13 U/mg (A = 0.033 U/mg). They all exhibit OT antagonism in vitro. Their anti-OT pA2 values range from 7.28 to 7.71 (A = 7.62). All five phenolic compounds were iodinated using iodine chloride and tested in the same in vivo and in vitro assay system.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phase transfer catalysis in solid phase peptide synthesis. Preparation of cyclo[Xxx-Pro-Gly-Yyy-Pro-Gly] model peptides and their conformational analysis.

Relatively small cyclic peptides that contain functionalized side chains provide interesting model compounds for studying side chain-side chain interactions, peptide backbone flexibility (especially if X-Pro bonds are included), and as potential enzyme mimetics. In order to develop more efficient synthetic routes to compounds such as cyclo(Xxx-Pro-Gly-Yyy-Pro-Gly), using the Merrifield method, we have investigated several orthogonal solid phase synthesis strategies and contrasted the use of two solid phase peptide-resin cleavage techniques for preparing partially protected linear sequences. Phase transfer catalysis using tetrabutyl ammonium hydrogen sulfate in THF with saturated aqueous K2CO3 provides peptide acid salts in which most of the common protecting groups (Arg(NO2), Tyr(Bzl), Z-Lys, Lys(Boc), and Glu(tBu)) are not affected. Using 500 MHz proton NMR, peptides having a cyclo (L-L-Gly-L-L-Gly) sequence generally display two conformers in DMSO-d6 with the major isomer being the bis-cis conformer, while the minor form contains two beta turns. For peptides with a cyclo(D-L-Gly-L-L-Gly) sequence, the major conformer contains one cis and one trans X-Pro bond and one Type II beta turn, as previously predicted for related structure by Kopple and others.     

Competitive antagonists of bradykinin

The first sequence-related competitive inhibitors of the classic kinin in vitro (rat uterus guinea pig ileum) and in vivo (rat blood pressure) assays have been developed. Replacement of the proline residue at position 7 of bradykinin (BK) with a D-phenylalanine residue is the key modification which converts BK agonists into antagonists. [D-Phe7]-BK exhibits moderate (pA2 = 5.0) inhibition of BK activity on the guinea pig ileum but possesses weak BK-like myotropic activity on the isolated rat uterus and 2-4% of BK depressor potency in the rat blood pressure assay. The additional replacement of the phenylalanine residues at positions 5 and 8 of [D-Phe7]-BK with the isosteric beta-(2-thienyl)-alanine residue produces a potent antagonist of BK activity on the uterus (pA2 = 6.4), ileum (pA2 = 6.3), and in the rat blood pressure assay. The antagonism of BK action on smooth muscle is specific for kinins (BK, kallidin, Met-Lys-BK), but neither inhibitor antagonizes the smooth muscle activity of angiotensin or substance P. Inhibition is competitive and fully reversible.     

Effects of structural modifications of N-CPM-normorphine derivatives on agonist and antagonist activities in isolated organs

The agonistic and antagonistic properties of N-cyclopropylmethyl (N-CPM) morphine derivatives were observed in mouse vas deferens (MVD), longitudinal muscle of guinea pig ileum (GPI) and rabbit vas deferens (LVD). In MVD the K(e) values of the titled compounds (N-CPM-morphine, N-CPM-isomorphine, N-CPM-dihydromorphine, N-CPM-dihydroisomorpPhine, N-CPM-dihydromorphone and naltrexone) were measured for mu-, kappa- and delta-receptors using normorphine, ethylketocyclazocine (EKC) and D-Pen2-D-Pen5-enkephaline (DPDPE) as selective agonists on the receptors, respectively. For mu-receptors of MVD the tested compounds showed similar affinity. For kappa-receptors the non-iso-6-OH derivatives possessed much less affinity than the iso-derivatives. Similar difference could be observed for delta-receptors. The agonistic activities of these compounds in MVD were observed to be between 0-20% of the inhibition of muscle contractions. In GPI the compounds except naltrexone possessed strong agonistic activities effectively antagonized by nor-binaltorphimine (nor-BNI) (K(e) of nor-BNI was 0.23 nM) suggesting that they were strong kappa-receptor agonists. We investigated these agents in LVD too, which contains kappa-receptors, but they did not produce any agonist potencies. It raises the possibility that the kappa-receptor subtypes of LVD and MVD are different from the kappa-receptor subtype of GPI or the vasa deferentia contain much fewer kappa-receptors than GPI and the intrinsic activities of these compounds are too small to reach the 50% inhibition of the contractions.     

Conformational properties of a cyclic peptide bradykinin B2 receptor antagonist using experimental and theoretical methods.

The solution conformation of the cyclic peptide J324 (cyclo0,6-[Lys0,Glu6,D-Phe7]BK), an antagonist targeted at the bradykinin (BK) B2 receptor, has been investigated using experimental and theoretical methods. In order to gain insight into the structural requirements essential for BK antagonism, we carried out molecular dynamics (MD) simulations using simulated annealing as the sampling protocol. Following a free MD simulation we performed simulations using nuclear Overhauser enhancement (NOE) distance constraints determined by NMR experiments. The low-energy structures obtained were compared with each other, grouped into families and analyzed with respect to the presence of secondary structural elements in their backbone. We also introduced new ways of plotting structural data for a more comprehensive analysis of large conformational sets. Finally, the relationship between characteristic backbone conformations and the spatial arrangement of specific pharmacophore centers was investigated.     

Purification, structural characterization, and myotropic activity of a peptide related to des-Arg(9)-bradykinin from an elasmobranch fish, the little skate, Leucoraja erinacea

A bradykinin (BK)-related peptide was isolated from heat-denaturated plasma from an elasmobranch fish, the little skate, Leucoraja erinacea after incubation with porcine pancreatic kallikrein. The primary structure of the peptide (H-Gly-Ile-Thr-Ser-Trp-Leu-Pro-Phe-OH; skate BK) shows limited structural similarity to the mammalian B1 receptor agonist, des-Arg(9)-BK. The myotropic activities of synthetic skate BK, and the analog skate [Arg(9)]BK, were examined in isolated skate vascular and intestinal smooth muscle preparations. Skate BK produced a concentration-dependent constriction of the mesenteric artery (EC(50)=4.37x10(-8)M; maximum response=103.4+/-10.23% of the response to 60mM KCl) but the response to skate [Arg(9)]BK was appreciably weaker (response to 10(-6)M=73.0+/-23.4% of the response to 60mM KCl). Neither the first branchial gill arch nor the ventral aorta responded to either purified peptide. Skate BK also produced a concentration-dependent constriction of intestinal smooth muscle preparations (EC(50)=2.74x10(-7)M; maximum response 31.0+/-12.2% of the response to 10(-5)M acetylcholine). Skate [Arg(9)]BK was without effect on the intestinal preparation. The data provide evidence for the existence of the kallikrein-kinin system in a phylogenetically ancient vertebrate group and the greater potency of skate BK compared with the analog skate [Arg(9)]BK suggests that the receptor mediating vascular responses resembles the mammalian B1 receptor more closely than the B2 receptor.     

Correlation between circular dichroism data and biological activities of 2,5 substituted enkephalin analogues

The relative structural rigidity of enkephalin analogues characterized by the molar ellipticity data obtained from the circular dichroism spectra of peptides was correlated with the opioid agonist activities of compounds displayed in isolated, electrically stimulated longitudinal muscle strip of guinea-pig ileum and mouse vas deferens preparations. It was found that the so called μ receptors modelled by guinea-pig ileum preferred the analogues with high capacity to exist in folded form, whilst the so called δ receptors (mouse vas deferens) accepted flexible ligands as readily as rigid ones.     

Gamma Ray Irradiation of the Vasoactive Peptide Bradykinin Reveals a Residue- and Position-Dependent Structural Modification

In this work the effect of radical species generated by gamma ray irradiation of aqueous solution upon structure of vasoactive peptide bradykinin (BK, RPPGFSPFR) was investigated. Increasing doses of 1–15 kGy Co⁶⁰ gamma radiation were applied to BK solutions and a progressive degradation of its structure in a non-linear mode was observed. Two main peptide derivatives generated by these treatments were isolated and characterized through a combined amino acid analysis and daughter ion scanning mass spectrometry approach. Notably, it was observed that only the Phe residue located at position 8 and not 5 of BK was oxidized by reactive hydroxyl radical species given rise to Tyr⁸-BK and m-Tyr⁸-BK analogues. Comparative circular dichroism (CD) experiments of these peptides revealed that BK presents greater conformational similarity to Tyr⁸-BK than to m-Tyr⁸-BK. These results are in agreement with the biological potencies of these compounds measured in rat uterus and guinea pig ileum muscle contractile experiments. In summary, gamma irradiation of BK solutions revealed a residue- and surprisingly, position-structural modification effect of reactive radicals even in small peptides. Also of value for peptide chemistry field, the approach of applying controlled strong electromagnetic radiation in solution seems to be an alternative and unique strategy for generating, in some cases, peptides derivatives with uncommon structures and valuable for their further therapeutic potential evaluations.     

Modified melanocortin tetrapeptide Ac-His-dPhe-Arg-Trp-NH at the arginine side chain with ureas and thioureas.

The Ac-His-dPhe-Arg-Trp-NH2 tetrapeptide is a nonselective melanocortin agonist and replacement of Arg in the tetrapeptide with acidic, basic or neutral amino acids results in reduced potency at the melanocortin receptor (MCR) isoforms (MC1R and MC3-5R). To determine the importance of the positive charge and the guanidine moiety for melanocortin activity, a series of urea- and thiourea-substituted tetrapeptides were designed. Replacement of Arg with Lys or ornithine reduced agonist activity at the mouse mMC1 and mMC3-5 receptors, thus supporting the hypothesis that the guanidine moiety is important for receptor potency, particularly at the MC3-5 receptors. The Arg side chain-modified tetrapeptides examined in this study include substituted phenyl, naphthyl, and aliphatic urea and thiourea residues using a Lys side-chain template. These ligands elicit full-agonist pharmacology at the mouse MCRs examined in this study.     

Metabolism of bradykinin analogs by angiotensin I converting enzyme and carboxypeptidase N.

Bradykinin (BK) analogs such as Lys-Lys-BK, des-Arg9-BK and [Leu8]des-Arg9-BK were poor substrates for angiotensin I converting enzyme (ACE), and analogs containing D-Phe7 residues, or a pseudopeptide C-terminal bond, were completely resistant. However, many of these analogs were metabolized by carboxypeptidase N (CPN) including Lys-Lys-BK, [Tyr8(OMe)]BK and D-Phe7-containing analogs, with Km and Vmax values comparable to those for BK. The only analogs completely resistant to both ACE and CPN were the B2 agonist [Phe8 psi(CH2NH)Arg9]BK, the B2 agonist D-Arg[Hyp3,D-Phe7,Phe8 psi(CH2NH)Arg9]BK, and the B1 agonist [D-Phe8]des-Arg9-BK. These data indicate an important role for plasma CPN and vascular CPN-like activity in the metabolism of the widely used ACE-resistant/D-Phe7-containing antagonists of B2 kinin receptors.     

Role of cyclooxygenase-1-mediated prostacyclin synthesis in endothelium-dependent vasoconstrictor activity of porcine interlobular renal arteries

This study aimed to determine whether PGI(2) would be evoked by the endogenous endothelial B(2) receptor agonist bradykinin (BK) in the porcine interlobular renal artery and, if so, to determine how it would influence the vasomotor reaction, and the specific cyclooxygenase (COX) isoform(s) involved in its synthesis. The production of the PGI(2) metabolite 6-keto-PGF(1α) was analyzed with HPLC-mass spectroscopy, while vasomotor reaction to PGI(2) or BK was determined with isometric force measurement. Results showed that BK evoked an increase in the production of 6-keto-PGF(1α), which was abolished by endothelial denudation that removed COX-1 expression, or was reduced by COX-1 inhibition. Interestingly, PGI(2) evoked a potent contraction, which was prevented by antagonizing thromboxane-prostanoid (TP) receptors and was not enhanced by antagonizing the vasodilator PGI(2) (IP) receptors. The IP receptor agonists MRE-269 and iloprost did not induce any relaxation. Moreover, iloprost, which is also a PGI(2) analog, caused a contraction, which was sensitive to TP receptor antagonism, but was to a significantly lesser extent than that of PGI(2). Indeed, IP receptors were not detected by RT-PCR or Western blotting in the vessel. Following nitric oxide synthase (NOS) inhibition, BK also evoked an endothelium-dependent contraction, which was blocked by TP receptor antagonism. In addition, inhibition of COX-1 (but not COX-2) impeded the vasoconstrictor activity of BK and expedited the relaxation induced by the agonist in NOS-intact vessels. These results demonstrate that in the porcine interlobular renal artery BK evokes endothelial COX-1-mediated PGI(2) synthesis, which mainly leads to the activation of TP receptors and a vasoconstrictor response, possibly due to a scarcity of vasodilator activity mediated by IP receptors. Also, our data suggested that the effect of a PGI(2) analog on TP receptors could be reduced compared with that of PGI(2) due to modified structure as with iloprost.     

Identification of secretin,vasoactive intestinal peptide and glucagon binding sites: from chimaeric receptors to point mutations

We have identified two basic residues that are important for the recognition of secretin and vasoactive intestinal peptide (VIP) by their respective receptors. These two peptides containing an Asp residue at position 3 interacted with an arginine residue in transmembrane helix 2 (TM2) of the receptor, and the lysine residue in extracellular loop 1 (ECL1) stabilized the active receptor conformation induced by the ligand. The glucagon receptor possesses a Lys instead of an Arg in TM2, and an Ile instead of Lys in ECL1; it markedly prefers a Gln side chain in position 3 of the ligand. Our results suggested that, in the wild-type receptor, the Ile side chain prevented access to the TM2 Lys side chain, but oriented the glucagon Gln(3) side chain to its proper binding site. In the double mutant, the ECL1 Lys allowed an interaction between negatively charged residues in position 3 of glucagon and the TM2 Arg, resulting in efficient receptor activation by [Asp(3)]glucagon as well as by glucagon.     

Antidipsogenic effects of eel bradykinins in the eel Anguilla japonica

A peptide with bradykinin (BK)-like immunoreactivity was isolated from an incubate of heat-denatured eel plasma with porcine pancreatic kallikrein. The purified peptide had the following amino acid sequence: Arg-Arg-Pro-Pro-Gly-Ser-Trp-Pro-Leu-Arg. This decapeptide, named eel [Arg(0)]BK, was identical to two previously identified BK homologs from cod and trout. High conservation of the BK sequence among distant teleost species suggests an important function in this vertebrate group. Bolus intra-arterial injections of eel [Arg(0)]BK, BK, and [Arg(0)]-des-Arg(9)-BK (1-10 nmol/kg) caused significant (P < 0.05) inhibition of drinking in seawater-adapted eels. The potency of the inhibition was ranked in the following order: [Arg(0)]BK > [Arg(0)]-des-Arg(9)-BK = BK. The BK peptides also produced an immediate, transient increase followed by a sustained increase in arterial blood pressure and an initial decrease followed by an increase in heart rate. Strong tachyphylaxis occurred for the cardiovascular effect but not for the antidipsogenic effect. The order of the potency of the cardiovascular actions, [Arg(0)]BK > BK > [Arg(0)]-des-Arg(9)-BK, was different from that of the antidipsogenic action. Slow infusions of eel [Arg(0)]BK in the dose range 1-1,000 pmol x kg(-1) x min(-1) produced concentration-dependent inhibition of drinking without changes in arterial pressure, plasma osmolality, and hematocrit. At the infusion rate of >100 pmol x kg(-1) x min(-1), plasma concentrations of angiotensin II, a potent dipsogenic hormone in eels, increased, suggesting an interaction of the kallikrein-kinin and renin-angiotensin systems. In mammals, BK is dipsogenic and vasodepressor, so that our data demonstrate opposite effects on fluid and cardiovascular regulation of BK in the eel and suggest a new physiological role for the kallikrein-kinin system in teleost fish.     

Discovery of 6-[5-(4-fluorophenyl)-3-methyl-pyrazol-4-yl]-benzoxazin-3-one derivatives as novel selective nonsteroidal mineralocorticoid receptor antagonists

In the course of our study on selective nonsteroidal mineralocorticoid receptor (MR) antagonists, a series of novel benzoxazine derivatives possessing an azole ring as the core scaffold was designed for the purpose of attenuating the partial agonistic activity of the previously reported dihydropyrrol-2-one derivatives. Screening of alternative azole rings identified 1,3-dimethyl pyrazole 6a as a lead compound with reduced partial agonistic activity. Subsequent replacement of the 1-methyl group of the pyrazole ring with larger lipophilic side chains or polar side chains targeting Arg817 and Gln776 increased MR binding activity while maintaining the agonistic response at the lower level. Among these compounds, 6-[1-(2,2-difluoro-3-hydroxypropyl)-5-(4-fluorophenyl)-3-methyl-1H-pyrazol-4-yl]-2H-1,4-benzoxazin-3(4H)-one (37a) showed highly potent in vitro activity, high selectivity versus other steroid hormone receptors, and good pharmacokinetic profiles. Oral administration of 37a in deoxycorticosterone acetate-salt hypertensive rats showed a significant blood pressure-lowering effect with no signs of antiandrogenic effects.     

Synthesis and structural studies of N-p-toluensulfonyl cyclodipeptides

In view of the chemical and structural interest of cyclopeptides bearing an electron withdrawing substituent directly bonded at the amide nitrogen atom, the two N-p-toluensulfonyl (N-tosyl) derivatives cyclo[-Phe(Tos)-D-Phe-] (I) and cyclo[-Phe(Tos)-D-Pro-] (II) have been synthesized and their stereochemistry defined. The molecular structure of I, as determined by x-ray diffraction analysis, is reported together with 1H-nmr parameters indicating the preferred rotameric conformation in chloroform solution. The N-tosyl group alters the geometry of the cyclodipeptide ring by lengthening both the N-C bonds departing from the tosylated nitrogen and reducing the corresponding ring angle. The 6-membered peptide ring adopts an unusual "sofa" conformation with the Tos-Phe C alpha atom deviating 0.230(3) A out of the mean plane of the other five ring atoms. One of the two S-O bonds forms a planar system that involves the tosylated nitrogen and the corresponding amide carbonyl. In the crystal, both the benzylic side chains are folded over the heterocyclic ring, whereas in chloroform solution, the benzylic side chain of the D-Phe prefers an extended conformation.