The discovery and structure-activity relationships of nonpeptide, low molecular weight antagonists selective for the endothelin ETB receptor

The systematic modification of the ET_A selective N-(5-isoxazolyl)benzene-sulfonamide endothelin antagonists to give ET_B selective antagonists is reported. The reversal in selectivity was brought about by substitution of the 4-position with aryl and substituted aryl groups. Of all the aromatic substituents studied, the para-tolyl group gave rise to the most active and selective ET_B antagonist. Larger substituents caused a decrease in both ET_B activity and selectivity. A similar trend was observed by substitution at the 5-position of the N-(5-isoxazolyl)-2-thiophenesulfonamide ET_A receptor antagonists. The para-tolyl group was again found to be optimal for the ET_B activity and selectivity. The structural features that were found to be favorable for binding to the ET_B receptor, that is, the presence of a linear, conjugated π-system of definite shape and size, have been successfully incorporated into the design of ET_B selective polycyclic aromatic sulfonamides antagonists.     

Role of D-tryptophan for affinity of MEN 10207 tachykinin antagonist at NK2 receptors.

The role of the D-Trp residues in the sequence of the NK2-selective tachykinin antagonist, MEN 10207 (Asp-Tyr-D-Trp-Val-D-Trp-D-Trp-Arg-NH2). has been examined by replacement of each D-Trp with either the L-isomer or the residue naturally occurring in the same position of neurokinin A(4-10). The biological activity of the analogues thus obtained has been characterized, with special attention to the selectivity for the three tachykinin receptors and for the two subtypes of the NK2 receptor recently described. We conclude that the simultaneous presence of the three D-Trp residues of MEN 10207 is crucial both for affinity and for selectivity.     

Interaction of a substance P agonist and of substance P antagonists with lipid membranes. A thermodynamic analysis.

The molecular characteristics of the neuropeptide substance P (SP), its agonist [Sar9,Met-(O2)11]SP, and three of its antagonists [D-Arg1,D-Pro2,D-Trp7,9,Leu11]SP, [D-Arg1,D-Trp7,9,Leu11]SP, and [D-Pro2,D-Trp7,9]SP were investigated at the air/water interface and when bound to lipid monolayers and bilayers. Measurement of the Gibbs adsorption isotherm showed that the surface areas of SP and its agonist (240 +/- 5 A2 at biologically relevant concentrations) were distinctly larger than those of the antagonists (138 +/- 5 A2) [Seelig, A. (1990) Biochim. Biophys. Acta 1030, 111-118]. The surface activity of the peptides increased in the order [Sar9,Met(O2)11]SP less than SP less than [D-Pro2,D-Trp7,9]SP less than [D-Arg1,D-Trp7,9,Leu11]SP = [D-Arg1,D- Pro2,D-Trp7,9,Leu11]SP and correlated with the respective binding affinities to lipid membranes. The agonist did not insert into neutral and negatively charged bilayers or into densely packed lipid monolayers (at surface pressures greater than 31 mN/m). In contrast, the three antagonists gave rise to a strong binding both to neutral and to charged lipid monolayers and bilayers. The degree of binding was evaluated from the area increase of lipid monolayers upon peptide insertion, and the binding isotherms were analyzed in terms of the Gouy-Chapman theory. At the monolayer-bilayer equivalence pressure of approximately 32 mN/m, the binding can be described by a surface partition equilibrium with binding constants of (4.5 +/- 0.1) x 10(3) M-1 for [D-Pro2,D-Trp7,9]SP and (1.3 +/- 0.1) x 10(4) M-1 for both [D-Arg1,D-Trp7,9,Leu11]SP and [D-Arg1,D-Pro2,D-Trp7,9,Leu11]SP for pure palmitoyloleoylphosphatidylcholine (POPC) membranes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Some pharmacological properties of cyclic and linear analogs obtained by substituting each residue of an oxytocin antagonist with D-tryptophan.

We synthesized 10 analogs (1-10) derived from the sequence of [Pmp1,D-Trp2,Arg8]oxytocin, (parent antagonist or PA), (Pmp = beta,beta-pentamethylene-beta-mercaptopropionic acid) which is a potent antagonist (pA2 = 7.77) of the uterotonic effect of oxytocin (OT) in rats, as determined in our uterotonic assay. Eight of the following analogs were designed by replacement of each residue in the PA sequence, other than the residue at position 2, with D-tryptophan: Ac-D-Trp-D-Trp-Ile-Gln-Asn-Val-Pro- Arg-Gly-NH2, (1); [Pmp1,D-Trp(For)2,Arg8] OT, (2); [Pmp1,D-Trp2,D-Trp3,Arg8] OT, (3); [Pmp1,D-Trp2,D-Trp4,Arg8] OT, (4); [Pmp1,D-Trp2,D-Trp5,Arg8] OT, (5); Aaa-D-Trp-Ile-Gln-Asn-D-Trp-Pro-Arg- Gly-NH2, (6); [Pmp1,D-Trp2,D-Trp7,Arg8] OT, (7); [Pmp1,D-Trp2,D-Trp8] OT, (8); [Pmp1,D-Trp2,Arg8,D-Trp9] OT, (9); [Pmp1,D-Trp2,Arg8,D-Trp(For)9] OT, (10). To avoid free mercaptan groups, Val6 was chosen in analog 1 instead of Cys and Aaa1 (Aaa = 1-adamantaneacetic acid) in analog 6 instead of Pmp1. Of the linear analogs, 1 was inactive as an OT antagonist and 6 was a very poor antagonist, with a pA2 = 5.66, but it was more potent than Aaa-D-Trp-Ile-Gln-Asn-Val-Pro-Arg-Gly-NH2, which has a pA2 = 5.33, as we had previously reported. Analog 2, featuring D-Trp(For)2, pA2 = 7.37, was weaker than PA, indicating that the formyl group lowers potency. Analogs 3 and 4 were much weaker than PA, and analog 5 was inactive. Hence, other than at position 2, D-Trp is undesirable in the ring sequence of PA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Increased potency of antagonists of substance P having asparagine in position 6

The general structure of antagonists of substance P (SP) which was found with the development of Spantide and analogs based on Spantide served for further refinement. The antagonistic potency was tested in vitro on guinea pig ileum and taenia coli. It was unexpectedly found that introduction of Asn6 gave rise to a considerable increase in potency. The exchange of Gln6 for Asn6 entails the shortening of the side chain by one CH2 unit and seems slight for steric advantages and potency increase. The analog [D-Arg1,D-Cl2Phe5,Asn6,D-Trp7,9,Nle11]SP had pA2 values of 7.4 (ileum) and 8.0 (taenia coli). We then used this sequence as a new lead to introduce new changes, which were made in positions 1, 3, 5, 7 and 9. It was found that Arg1 is important, but Lys3 can be exchanged. The Pal3 derivative had pA2 values of 8.1 and 8.0 and the Nle3 counterpart had 7.7 and 7.4 D-Cl2Phe is an effective substituent in position 5. D-Trp in positions 7 and 9 were superior to other alternatives.     

Solid-phase synthesis of endothelin receptor antagonists

A new solid-phase synthesis for ET receptor antagonists suitable for automation is presented. A support bound 2-hydroxybutyric acid derivative was converted to the corresponding ether derivatives using 4-halo-2-methylsulfonylpyrimidines. Subsequent Suzuki coupling with various aryl boronic acids gave the desired antagonists in good yields and purities. Highly potent antagonists with excellent selectivity for ET(A) were obtained.     

1,3-Disubstituted-2-carboxy quinolones: highly potent and selective endothelin A receptor antagonists

The design, synthesis, and in vitro biological activity of a series of 2-carboxy quinolone antagonists selective for the endothelin A receptor are presented. Introduction of a second acid group in position 3 of the quinolone ring increases dramatically the selectivity for ET(A).     

3-hydroxy-quinazoline-2,4-dione as a useful scaffold to obtain selective Gly/NMDA and AMPA receptor antagonists

The synthesis and Gly/NMDA, AMPA and KA receptor binding activities of some 3-hydroxy-quinazoline-2,4-dione derivatives are reported. The binding data, together with functional antagonism studies, showed that the 3-hydroxy-quinazoline-2,4-dione moiety can be considered a useful scaffold to obtain selective Gly/NMDA and AMPA receptor antagonists. In fact, introduction of chlorine atom(s) on precise position(s) of the benzofused moiety yielded Gly/NMDA selective antagonists, while the presence of the 6-(1,2,4-triazol-4-yl) group shifted the affinity and selectivity towards the AMPA receptor.     

N-Methylbenzanilide derivatives as a novel class of selective V(1A) receptor antagonists.

During our efforts to develop a novel class of selective V(1A) receptor antagonists, the N-methylbenzanilide structure was applied to a 4,4-difluoro-1-benzazepine derivative, 4, which is a selective V(1A) receptor antagonist. Further structural modifications gave 16a with high V(1A) affinity and V(2)/V(1A) selectivity (K(i)=5.71 nM, V(2)/V(1A)=140) and potent V(1A) receptor antagonist activity (ID(50)=0.0080 mg/kg iv).     

Molecular basis for lysophosphatidic acid receptor antagonist selectivity

Recent characterization of lysophosphatidic acid (LPA) receptors has made possible studies elucidating the structure-activity relationships (SAR) for agonist activity at individual receptors. Additionally, the availability of these receptors has allowed the identification of antagonists of LPA-induced effects. Two receptor-subtype selective LPA receptor antagonists, one selective for the LPA1/EDG2 receptor (a benzyl-4-oxybenzyl N-acyl ethanolamide phosphate, NAEPA, derivative) and the other selective for the LPA3/EDG7 receptor (diacylglycerol pyrophosphate, DGPP, 8:0), have recently been reported. The receptor SAR for both agonists and antagonists are reviewed, and the molecular basis for the difference between agonism and antagonism as well as for receptor-subtype antagonist selectivity identified by molecular modeling is described. The implications of the newly available receptor-subtype selective antagonists are also discussed.     

New antagonists of LHRH. II. Inhibition and potentiation of LHRH by closely related analogues

Modifications of the previously described LHRH antagonists, [Ac-D-Nal(2)1, D-Phe(4Cl)2, D-Trp3, D-Cit6, D-Ala10]LHRH and the corresponding D-Hci6 analogue, have been made to alter the hydrophobicity of the N-terminal acetyl-tripeptide portion. Substitution of D-Trp3 with the less hydrophobic D-Pal(3) had only marginal effects on the antagonistic activities and receptor binding potencies of the D-Cit/D-Hci6 analogues, but it appeared to further improve the toxicity lowering effect of D-Cit/D-Hci6 substitution. Antagonists containing D-Pal(3)3 and D-Cit/D-Hci6 residues, i.e. [Ac-D-Nal(2)1, D-Phe(4Cl)2, D-Pal(3)3, D-Cit6, D-Ala10]LHRH (SB-75) and [Ac-D-Nal(2)1, D-Phe(4Cl)2, D-Pal(3)3, D-Hci6, D-Ala10]LHRH (SB-88), were completely free of the toxic effects, such as cyanosis and respiratory depression leading to death, which have been observed in rats with the D-Trp3, D-Arg6 antagonist and related antagonists. Replacement of the N-acetyl group with the hydrophilic carbamoyl group caused a slight decrease in antagonistic activities, particularly in vitro. Introduction of urethane type acyl group such as methoxycarbonyl (Moc) or t-butoxycarbonyl (Boc) led to analogues that showed LHRH-potentiating effect. The increase in potency induced by these analogues, e.g. [Moc-D-Nal(2)1, D-Phe(4Cl)2, D-Trp3, D-Cit6, D-Ala10]LHRH and [Boc-D-Phe1, D-Phe(4Cl)2, D-Pal(3)3, D-Cit6, D-Ala10]LHRH, was 170-260% and persisted for more than 2 h when studied in a superfused rat pituitary system.     

Synthesis and biological activities of angiotensin II and Sarmesin analogues containing cyclohexylalanine

Analogues of angiotensin II with cyclohexylalanine (Cha) at position 4 or 8, and analogues of the competitive (type II) angiotensin antagonist [Sar1,Tyr(Me)4]ANG II (Sarmesin) with Cha at position 8, have been prepared by the solid phase method and purified by reversed-phase HPLC. Analogues of ANG II with Cha at position 8 in which the position 1 residue was substituted with sarcosine (Sar) or amino-isobutyric acid (Aib) or was deleted (Des), were slowly reversing (Type I) antagonists with "pA2" values in the rat isolated uterus assay of approximately 8.5. The additional substitution of Tyr(Me) for Tyr at position 4 of these peptides gave reversible competitive (Type I/II) antagonists with pA2 values of 6.7, 5.8, and less than 5, while substitution of Phe for Tyr gave pA2 values of 7.4, 6.7, and less than 5, respectively. All 19 peptides synthesized in this study had low intrinsic agonist activity in the rat isolated uterus assay except for the type I antagonists [Sar1, Cha8]ANG II (7%), [Aib1, Cha8]ANG II (12%) and [Des1, Cha8]ANG II (20%). These data illustrate that the substitution of Cha at position 8 of ANG II analogues produces potent antagonists; however, Type I antagonists retain significant agonist activity whereas Type I/II antagonists do not. In contrast, substitution of Cha at position 4 in a variety of ANG II analogues resulted in severely diminished biological activity, illustrating that the presence of an aromatic ring quadrupole at position 4 is obligatory for receptor binding and activity.     

Conversion of A3 adenosine receptor agonists into selective antagonists by modification of the 5'-ribofuran-uronamide moiety

The highly selective agonists of the A(3) adenosine receptor (AR), Cl-IB-MECA (2-chloro-N(6)-(3-iodobenzyl)-5'-N-methylcarboxamidoadenosine), and its 4'-thio analogue, were successfully converted into selective antagonists simply by appending a second N-methyl group on the 5'-uronamide position. The 2-chloro-5'-(N,N-dimethyl)uronamido analogues bound to, but did not activate, the human A(3)AR, with K(i) values of 29 nM (4'-O) and 15 nM (4'-S), showing >100-fold selectivity over A(1), A(2A), and A(2B)ARs. Competitive antagonism was demonstrated by Schild analysis. The 2-(dimethylamino)-5'-(N,N-dimethyl)uronamido substitution also retained A(3)AR selectivity but lowered affinity.     

Orn5]URP acts as a pure antagonist of urotensinergic receptors in rat cortical astrocytes

Cultured rat astrocytes, which express functional urotensin II (UII)/UII-related peptide (URP) receptors (UT), represent a very suitable model to investigate the pharmacological profile of UII and URP analogs towards native UT. We have recently designed three URP analogs [D-Trp4]URP, [Orn5]URP and [D-Tyr6]URP, that act as UT antagonists in the rat aortic ring bioassay. However, it has been previously reported that UII/URP analogs capable of inhibiting the contractile activity of UII possess agonistic activity on UT-transfected cells. In the present study, we have compared the ability of URP analogs to compete for [125 I]URP binding and to modulate cytosolic calcium concentration ([Ca2+]c) in cultured rat astrocytes. All three analogs displaced radioligand binding: [D-Trp4]URP and [D-Tyr6]URP interacted with high- and low-affinity sites whereas [Orn5]URP only bound high-affinity sites. [D-Trp4]URP and [D-Tyr6]URP both induced a robust increase in [Ca2+]c in astrocytes while [Orn5]URP was totally devoid of activity. [Orn5]URP provoked a concentration-dependent inhibition of URP- and UII-evoked [Ca2+]c increase and a rightward shift of the URP and UII dose-response curves. The present data indicate that [D-Trp4]URP and [D-Tyr6]URP, which act as UII antagonists in the rat aortic ring assay, behave as agonists in the [Ca2+]c mobilization assay in cultured astrocytes, whereas [Orn5]URP is a pure selective antagonist in both rat aortic ring contraction and astrocyte [Ca2+]c mobilization assays.     

Substance P and antagonists. Surface activity and molecular shapes.

The molecular properties of substance P (SP) (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met amide) and three of its antagonists were derived by measuring the Gibbs adsorption isotherm, providing information on the surface activity, the molecular shape, and the pK values of the different molecules. The following three antagonists were investigated: [D-Arg1,D-Pro2,D-Trp7,9,Leu11]SP, ANT I; [D-Arg1,D-Trp7,9,Leu11]SP, ANT II and [D-Pro2,D-Trp7,9]SP, ANT III. SP is only moderately surface active. The amino acid substitutions lead, however, to an increased surface activity of the antagonists. From the concentration dependence of the surface activity it was possible to quantify the packing characteristics of the individual neuropeptides. SP shows cross-sectional areas of 300 +/- 5 A2 to 240 +/- 5 A2 (pH 5 to 8, 154 mM NaCl) at concentrations below 10(-5) M, i.e., in the physiological concentration range, indicating a folded SP conformation. Upon increasing the packing density to concentrations larger than 10(-5) M the surface area was only half as large (148 +/- 5 A2 to 124 +/- 3 A2) suggesting now a relatively extended conformation of the SP molecule with its long molecular axis perpendicular to the air/water interface. In contrast, the three antagonists were characterized by surface areas of 147 +/- 3 A2 to 126 +/- 3 A2 which were almost independent of concentration. The antagonists thus adopt a relatively extended conformation in the whole concentration range measured. This is further supported by computer modelling which shows that the antagonists are motionally restricted and can adopt neither a bent nor a alpha-helical conformation. The surface activity of the neuropeptides was dependent on the pH of the solution. At low peptide concentrations (about 10(-6) M) it was possible to resolve and determine the pK values of all individual charged amino acid side chains. The pK values observed for the neuropeptides were about two pK units lower than those of the free amino acids in solution. The pK shifts of the neuropeptides at the air/water interface are explained in terms of the Gouy-Chapman theory. SP and its antagonists bind to lipid bilayers in the order of their surface activity. While the binding of SP is mainly due to electrostatic interactions, hydrophobic peptide-lipid interactions contribute to the binding of the antagonists.     

Interactions of substance P antagonists with serotonin in the mouse spinal cord

Administered intrathecally (IT) to mice, the putative substance P antagonist [D-Pro2,D-Trp7,9-substance P (DPDT) blocked substance P- and serotonin-induced reciprocal hindlimb scratching with ID50 values of 4.6 (2.9-6.9) and 3.0 (1.9-4.8) micrograms, respectively. The duration of this antagonistic effect was 90-120 min. In contrast, DPDT did not block bombesin-, somatostatin-, glycine- or glutamate-induced scratching. These data indicate that DPDT is an effective antagonist of serotonin-induced behaviors in the mouse spinal cord. Phenoxybenzamine (IT) also blocked substance P- and serotonin-induced scratching. Its onset of action was more rapid for serotonin than for DPDT implying differences in agonist-induced receptor activation. Methysergide (IT) blocked serotonin-induced scratching [ID50 = 0.7 (0.3-1.5) micrograms], but not substance P-induced scratching. Similar to DPDT, [D-Arg1,D-Trp7,9,Leu11]-substance P, [des-Arg1,D-Pro2, D-Trp7,9]-substance P(2-11) and [D-Pro4,D-Trp7,9]-substance P(4-11) blocked substance P and serotonin-induced scratching. In contrast, [D-Pro2,D-Phe7,D-Trp9]-substance P and [D-Pro4,D-Trp7,9,10]-substance P(4-11) blocked only substance P-induced scratching. Thus, some, but not all putative substance P antagonists may also be behavioral antagonists of serotonin in the mouse spinal cord.     

X-ray crystallographic structure-based design of selective thienopyrazole inhibitors for interleukin-2-inducible tyrosine kinase

Beginning with a screening hit, unique thienopyrazole-indole inhibitors of Itk (interleukin-2-inducible tyrosine kinase) were designed, synthesized, and crystallized in the target kinase. Although initial compounds were highly active in Itk, they were not selective. Increasing the steric bulk around a tertiary alcohol at the 5-indole position dramatically improved selectivity toward Lyk and Syk, but not Txk. Substitutions at the 3- and 4-indole positions gave less active compounds that remained poorly selective. A difluoromethyl substitution at the 5-position of the thienopyrazole led to a highly potent and selective compound. Phenyl at this position reduced activity and selectivity while pushing the side-chains of Lys-391 and Asp-500 away from the binding pocket. Novel and selective thienopyrazole inhibitors of Itk were designed as a result of combining structure-based design and medicinal chemistry.     

Synthesis and biological activity of novel 4,4-difluorobenzazepine derivatives as non-peptide antagonists of the arginine vasopressin V1A receptor

To find potent and selective antagonists of the arginine vasopressin (AVP) V1A receptor, optimization studies of compounds structurally related to (Z)-N-{4'-[(4,4-difluoro-5-carbamoylmethylidene-2,3,4,5-tetrahydro-1H-1-benzazepin-1-yl)carbonyl]phenyl}carboxamide were performed. The synthesis and pharmacological properties of these compounds are described. We first investigated the effect of the carboxamide moiety, and found that a 2-methylfuran-3-carbonyl group at this position increased V1A binding affinity and selectivity for the V1A receptor versus the V2 receptor. The amino group of the 5-carbamoylmethylidene moiety was also examined, and a 4-piperidinopiperidino group was found to be optimal at this position. The hemifumarate of compound 12l (YM218) was shown to exhibit potent binding affinity, V1A receptor selectivity, and in vivo antagonist activity.     

Discovery of N-propylurea 3-benzylpiperidines as selective CC chemokine receptor-3 (CCR3) antagonists

The discovery of novel and selective small molecule antagonists of the CC Chemokine Receptor-3 (CCR3) is presented. Simple conversion from a 4- to 3-benzylpiperidine gave improved selectivity for CCR3 over the serotonin 5HT(2A) receptor. Chiral resolution and exploration of mono- and disubstitution of the N-propylurea resulted in several 3-benzylpiperidine N-propylureas with CCR3 binding IC(50)s under 5 nM. Data from in vitro calcium mobilization and chemotaxis assays for these compounds ranged from high picomolar to low nanomolar EC(50)s and correlated well with antagonist binding IC(50)s.     

Spinal actions of substance P analogues on cardiovascular responses in the rat: a structure-activity analysis

Ten substance P (SP) analogues were tested for their effects on mean arterial pressure and heart rate following intrathecal administration in the pentobarbital anaesthetized rat. The 10 analogues are [D-Pro4,D-alpha Npa7,9,10]SP(4-11) (A-I), (D-alpha Npa7,9,10]SP (A-II), [D-Trp7,9,10]SP (A-III), [D-Pro4,D-Npa7,9,Phe11]SP(4-11) (A-IV), [D-Pro4,D-beta Npa7,D-alpha Npa9,D-Phe11]SP(4-11) (A-V), [D-Pro4,Lys6,D-Trp7,9,10,Phe11]SP(4-11) (A-VI), [D-Pro4,D-Trp7,9,10,Phe11]SP(4-11) (A-VII), [D-Pro4,D-Trp7,9,10,Trp11]SP(4-11) (A-VIII), [D-Trp7,9,10,Trp11]SP (A-IX), and [D-Pro4,D-Phe7,9,10,Phe11]SP(4-11) (A-X). At 6.5 nmol, the analogues containing the amino acid D-Npa (A-I, A-II, A-IV, and A-V) or D-Phe (A-X) in positions 7, 9, or 10 of SP or its C-terminal octapeptide are devoid of the long-lasting cardio- and vaso-depressor effects, which are otherwise seen with analogues containing the amino acid D-Trp (A-III, A-VI, A-VII, A-VIII, and A-IX) in the same positions. Some of the analogues containing D-Npa maintain the initial hypotensive effect seen with SP while the analogue containing D-Phe produces only a small hypertensive response. The 10 analogues when tested at a dose that failed to alter basal mean arterial pressure and heart rate did not block the cardiovascular responses elicited by SP and no cross desensitization was observed between SP and these analogues. It appears that these SP analogues exert cardiovascular effects in the rat spinal cord probably without interacting with SP receptors.