Influence of 1-aminocyclohexane-1-carboxylic acid in position 2 or 3 of AVP and its analogues on their pharmacological properties.

In this study we describe the synthesis and some pharmacological properties of seven new analogues of arginine vasopressin (AVP) substituted in position 2 or 3 with 1-aminocyclohexane-1-carboxylic acid (Acc). All peptides were tested for the pressor, antidiuretic and uterotonic in vitro activities. The Acc3 modifications of AVP, dAVP, [d-Arg8]VP and [Cpa1]AVP have been found to be deleterious for interaction with all three neurohypophyseal hormone receptors, as judged from the several orders of magnitude decreased biological activities, whereas Acc2 substitution selectively altered the interaction with the receptors. Two of the new analogues, [Acc2]AVP and [Acc2, d-Arg8]AVP, are potent antidiuretic agonists. [Acc2]AVP exhibits moderate pressor agonistic activity and weak antiuterotonic properties. [Acc2, d-Arg8]AVP has been found to be a weak antagonist in the pressor and uterotonic tests. Another analogue - [Cpa1, Acc3]AVP - turned out to be a highly selective V2 agonist. This is an unexpected effect, as its parent peptide, [Cpa1]AVP is a very potent V1a receptor antagonist. This is the first Cpa1 modification to have resulted in V2 agonism enhancement. Besides providing useful information about structure-activity relationships, our results could open up new possibilities in the design of highly potent and selective V2 agonists.     

Analogs of arginine vasopressin modified in position 3 with (R)-alpha-hydroxymethylphenylalanine.

This study describes the synthesis and some pharmacological properties of three new analogs of arginine vasopressin (AVP) substituted in position 3 with (R)-alpha-hydroxymethylphenylalanine ([R]-HmPhe). All new peptides were tested for vasopressor and antidiuretic as well as uterotonic activity. None of the 3 analogs showed any pressor activity and their uterotonic activity was negligible. Only analog [Mpa1,(R)-HmPhe3]AVP exhibited significant antidiuretic activity.     

Analogues of arginine vasopressin modified in the N-terminal part of the molecule with enantiomers of N-methylphenylalanine.

Four new analogues of arginine vasopressin (AVP) substituted in positions 2 and 3 with all possible combinations of enantiomers of N-methylphenylalanine were synthesized and studied to assess the influence of N-methylation of the peptide bonds between the first three amino acids on the pharmacological properties of the resulting peptides. The next three analogues were designed to learn how the shortening of the peptide chain, by removal of one of the N-methylphenylalanine residues, would affect pharmacological properties of the resulting compounds. The activity of the analogues was tested in the in vitro uterotonic, pressor and antidiuretic tests. None of the prepared analogues displayed significant biological activity with the exception of [Me-d-Phe(2), Me-Phe(3)]AVP and [Me-d-Phe(2,3)]AVP, which showed low antiuterotonic activity (pA(2) = 6.6 and pA(2) = 6.4, respectively). Our results, while not impressive in terms of biological activity, may be helpful for designing potent and selective oxytocin antagonists.     

Influence of conformationally constrained amino acids replacing positions 2 and 3 of arginine vasopressin (AVP) and its analogues on their pharmacological properties

Synthesis of thirteen new analogues of arginine vasopressin (AVP) has been described. Amino acid residues at positions 2 and 3 of AVP, [3-mercaptopropionic acid (Mpa)(1)]AVP (dAVP), [Mpa(1),d-Arg(8)]VP (dDAVP) and [Mpa(1),Val(4),d-Arg(8)]VP (dVDAVP) were replaced with one amino acid residue using sterically constrained non-proteinogenic amino acids, 4-aminobenzoic acid (Abz), cis-4-aminocyclohexanecarboxylic acid (ach) or its trans-isomer (Ach). In the case of a potent V(1a) antagonist, [1-mercaptocyclohexaneacetic acid (Cpa)(1)]AVP, only one similar analogue has been prepared by replacing positions 2 and 3 with Abz. Unfortunately, all new peptides were inactive in bioassays for the pressor, antidiuretic and uterotonic in vitro activities in the rat.     

Analogues of arginine vasopressin and its agonist and antagonist modified in the N-terminal part of the molecule with l-beta-homophenylalanine.

In continuation of our efforts to elucidate the role of positions 2 and 3 in arginine vasopressin (AVP) and its analogues, we designed and synthesized peptides modified in these positions with l-beta-homophenylalanine (beta-Hph). Two of them had just this single modification, the next two peptides are analogues of the V2 agonist, namely [3-mercaptopropionic acid (Mpa)1]AVP (dAVP). The last two compounds were designed by substitution of positions 2 or 3 of a potent V(1a) antagonist, [1-mercaptocyclohexaneacetic acid (Cpa)1]AVP, with beta-Hph. All the peptides were tested for their pressor and antidiuretic and uterotonic in vitro activities in the rat. All the activities tested have been found to be significantly decreased. Three analogues, i.e. [Mpa(1),beta-Hph2]AVP, [Cpa1,beta-Hph2]AVP, [Cpa1,beta-Hph3]AVP, turned out to be uterotonic antagonists with pA2 = 6.3 +/- 0.2, 6.3 +/- 0.1, 6.0 +/- 0.3 respectively. The last one exhibited antipressor properties also (pA2 = 6.4 +/- 0.1).     

Analogs of arginine vasopressin modified in the N-terminal part of the molecule with a conformationally constrained cis-peptide bond motif.

The present work is part of our studies aimed at clarifying the influence of steric constraints in the N-terminal part of arginine vasopressin (AVP) and its analogs on the pharmacological activity of the resulting peptides. We describe the synthesis of eight new analogs of AVP or [3-mercaptopropionic acid (Mpa)]AVP (dAVP) substituted at positions 2 and 3 or 3 and 4 with two diastereomers of 4-aminopyroglutamic acid. The steric constraints provided by this modification turned out, however, so strong that all the peptides were inactive in all of the bioassays (pressor, antidiuretic and uterotonic tests).     

Analogues of oxytocin antagonists bearing a ureido group in the amino acid side chain at position 4 or 5.

Substitution of the side chain carboxamido group at position 4 in the potent oxytocin antagonist (OTA) [ThiaPmp(1), D-Trp(2), Cys(6), Arg(8)]-OT, PA, in which ThiaPmp = beta,beta-(3-thiapentamethylene)-beta-mercaptopropionic acid, led to [Orn(Car)(4)]-PA, ([Cit(4)]-PA), which had uterotonic antagonistic activity equal to that of PA. The same modification at position 5, leading to [Cit(5)]-PA, resulted in antagonistic potency more than 10 times lower than that of PA. This paper also describes the same substitutions introduced in the highly potent OTA [Pen(6)]-PA (antioxytocic in vitro pA(2) = 8.72). Analogues of the general formula [U(4)-X(5)-Pen(6)]-PA, in which U = Lys, Orn, Dab, Dap or X = Orn, Dab or Dap, were synthesized by SPPS. Each of these analogues was carbamoylated by treatment with KCNO in DMF-H(2)O, yielding the corresponding U(Car)(4) or X(Car)(5) derivatives. In the uterotonic assay, the substitution with the ureido group at Gln(4) results in retention of high antagonistic potency, albeit somewhat lower than that of PA, e.g. [Orn(Car)(4), Pen(6)]-PA and [Dab(Car)(4), Pen(6)]-PA having pA(2) = 8.52 and pA(2) = 8.42 respectively. In the pressor assay, [Lys(Car)(4), Pen(6)]-PA and [Dab(Car)(4), Pen(6)]-PA were somewhat weaker antagonists of arginine vasopressin than [Pen(6)]-PA; [Dap(Car)(4), Pen(6)]-PA showed only a faint trace of pressor agonistic activity. The substitution with the ureido group at position 5 leads to a significant loss of OTA potency in the in vitro uterotonic assay. The [Orn(Car)(5), Pen(6)]-PA was the most potent of the series (pA(2) = 8.05). An interesting finding is that [Dap(Car)(5), Pen(6)]-PA is equipotent with its precursor [Dap(5), Pen(6)]-PA (potency in the uterotonic test in vitro, pA(2) = 7.71 and pA(2) = 7.68, respectively). Furthermore, neither [Dap(5), Pen(6)]-PA nor [Dap(5), Pen(6), Gly(9)]-PA exhibited activity in the antidiuretic or pressor assays. Although these last two analogues show some decrease in antioxytocin potency, they behave as pure oxytocin antagonists, which makes them attractive candidates for further studies on the development of potent and specific OTAs.     

Analogues of a potent oxytocin antagonist with truncated C-terminus or shorter amino acid side chain of the basic amino acid at position 8.

Twelve analogues were synthesized, their structure derived from modifications of [(S)Pmp1, D-Trp2, Pen6, Arg8]oxytocin, PA, in which (S)Pmp = beta,beta-(3-thiapentamethylene-beta-mercaptopropionic acid). PA is a potent antagonist of the uterotonic effect of oxytocin in the rat (uterotonic test in vitro, pA2 = 8.86) and in the baboon. Truncated analogues of PA from the C-terminus were systematically prepared ending in either the free acid or the amide, i.e. PA1-9 acid, PA1-8 acid, PA1-7 acid, PA1-6 acid, PA1-8 amide, PA1-7 amide and PA1-6 amide. PA1-8 amide was roughly as potent as PA in the rat uterotonic assay in vitro, and the shorter amides were only somewhat weaker antagonists. All four acid analogues were weaker antagonists than PA but still maintained rather high antagonistic potency. These findings suggest that, if these truncated acids form as metabolites in vivo, they may contribute to the overall biological effect of PA and their contribution should be taken into account. Furthermore, using these analogues, the radioimmunoassay measurements of PA may be standardized, as they may cross react with PA antibodies and interfere with the determination. In addition, five analogues were made by substituting Arg8 of PA with Lys, Orn8, Dab8, Dap8 and Cit8. All of these analogues maintained high potency as OTAs in the uterotonic assay, although their activity was only about 1.5-3 times lower than PA. The most potent analogue in the uterotonic assay, [Dap8]PA, pA2 = 8.53, had weak pressor activity (pA2 = 6.90) and no antidiuretic effect. The pressor activity was lower for all tested acids, and for PA1-6 acid it was even below the detection limit. Additionally, PA1-9 acid, PA1-7 acid and PA1-6 acid showed no antidiuretic activity. Hence, the PA1-6 acid is a potent OTA with pA2 = 8.27 and no measurable effect in the pressor or antidiuretic tests and thus it is a pure oxytocin antagonist. This fact makes it an attractive candidate for further studies on inhibition of OT biological effects and on preterm labour.     

Vasopressin receptor subtypes in dorsal hindbrain and renal medulla

We have investigated the ability of a series of synthetic vasopressin analogues and related peptides to compete with (3H)-arginine8 vasopressin for binding sites in rat renal medulla and dorsal hindbrain. In renal medulla, arginine8 vasopressin and deamino arginine8 vasopressin, a selective antidiuretic, were equipotent while two antagonists of the pressor action of arginine vasopressin were less potent. In the dorsal hindbrain, arginine8 vasopressin and the pressor antagonists were more potent than the synthetic antidiuretic. Potency profiles of these and other analogues suggest that the renal medulla and dorsal hindbrain vasopressin receptors represent different subtypes.     

Novel analogues of arginine vasopressin containing α‐2‐indanylglycine enantiomers in position 2

Continuing our efforts to obtain potent and selective analogues of AVP we synthesized and pharmacologically evaluated ten new compounds modified at position 2 with α‐2‐indanylglycine or its D ‐enantiomer (Igl or D ‐Igl, respectively). All the peptides were tested for pressor, antidiuretic, and in vitro uterotonic activities. We also determined the binding affinity of these compounds to human OT receptor. The Igl² substitution resulted in a significant change of the pharmacological profile of the peptides. The new analogues were moderate or potent OT antagonists (pA₂ values ranging from 7.19 to 7.98) and practically did not interact with V_1a and V₂ receptors. It is worth emphasizing that these new peptides were exceptionally selective. On the other hand, the D ‐Igl² substituted counterparts turned out to be weak antagonists of the pressor response to AVP and displayed no antidiuretic activity. Some of the results were unexpected, e.g. dual activity in the rat uterotonic test in vitro: the D ‐Igl peptides showed a strong antioxytocic potency (pA₂ values ranging from 7.70 to 8.20) at low concentrations and full agonism at high concentrations. The results provided useful information about the SAR of AVP analogues. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Antagonists of oxytocin featuring replacement with modified beta-mercaptopropionic acids at position 1.

Twenty analogues were synthesized of [Pmp1, D-Trp2, Arg8]oxytocin, PA, (Pmp = beta,beta-pentamethylene-beta-mercaptopropionic acid), a potent antagonist of the uterotonic effect of oxytocin in the rat (uterotonic test in vitro, pA2 = 7.77) and in the baboon. Systematic substitution of Pmp1 was made with beta-mercaptopropionic acids featuring replacement of the 4-methylene group of the cyclohexyl ring of Pmp with isosteric O, S, NH or with C=O. Since the more hydrophilic NH and C=O substitutions showed a sharply decreased antagonistic potency (rat uterotonic test in vitro), additional modifications were made to reduce their hydrophilicity. Acylation of the NH group with various acyl groups, and ketalization or thioketalization of C=O with more or less bulky substituents led to a partial restoration of potency, the N-carbamyl- and the 2-mercapto-2-adamantaneacetyl analogues being equipotent with PA. Internal cyclization by amidation of the NH-group with Gly-9, resulted in a bicyclic analogue, (cyclo 1-9)[(HN)Pmp1, Gly9]PA which was equipotent with PA. When Pen-6 was introduced into the bicyclic derivative instead of Cys-6, to reduce the flexibility of the rings, the resulting (cyclo 1-9)[(HN)Pmp1, Pen6, Gly9]PA had somewhat better potency (pA2 = 8.17) in the uterotonic test and no detectable activity in the antidiuretic assay. In the case of substitution of PA with beta,beta-(3-thiapentamethylene)-beta-mercaptopropionic acid, (S)Pmp, there was also an increase in inhibitory potency in the uterotonic test (pA2 = 8.08): the analogue had extremely weak antidiuretic activity. To establish the importance of the steric effects of the Pen-6 substitution, analogues [Pen6]PA and [(S)Pmp1, Pen6]PA were made and found to be very potent, with a pA2 of 8.72 and 8.86, respectively. The high potency of the latter analogue and its extremely weak action in the diuretic assay makes it an attractive candidate for studies on the inhibition of the biological effects of oxytocin and for the prevention of preterm labour.     

New bradykinin analogues substituted in positions 7 and 8 with sterically restricted 1-aminocyclopentane-1-carboxylic acid.

A sterically constrained non-coded amino acid, 1-aminocyclopentane-1-carboxylic acid (Apc), was introduced in position 7 or 8 of the bradykinin (BK) B(2) receptor antagonist, [D-Arg(0), Hyp(3), Thi(5, 8), D-Phe(7)]BK, previously synthesized by Stewart's group. This modification is believed to reduce the flexibility of the peptides, thereby forcing the peptide backbone and side chains to adopt specific orientations. Apc substitution was combined with acylation of the N-terminus with 1-adamantaneacetic acid (Aaa). The activity of four new analogues was assayed in isolated rat uterus and in rat blood pressure tests. The results clearly demonstrated that the Apc residue inserted in position 7 led to a reduction of antagonistic properties in the rat uterus assay or even restored the agonism in the blood pressure test, whereas Apc at position 8 enhanced antagonistic potency in both the tests. In both cases, acylation of the N-terminus led to the enhancement of the antagonistic potency. On the basis of these findings, new potent and selective B(2) blockers might be designed.     

Conformational studies of neurohypophyseal hormones analogues with glycoconjugates by NMR spectroscopy

Two glycosylated peptides have been studied using NMR spectroscopy supported by molecular modeling. Peptide I is an oxytocin (OT) analogue in which glutamine 4 was replaced by serine with attached α‐d ‐mannose through the oxygen β atom, whereas peptide II is a lysine‐vasopressin (LVP) analogue with lysine 8 side chain modified by the attachment of glucuronic acid through an amide bond. Both peptides exhibit very weak uterotonic effect and are less susceptible to proteolytic degradation than the mother hormones. Additionally, peptide II reveals very weak pressor and antidiuretic activities. Our results have shown that the conformational preferences of glycosylated analogues are highly similar to those of their respective mother hormones. OT glycosylated analogue (I) exhibits a 3,4 β‐turn characteristic of OT‐like peptides, and vasopressin‐glycosylated analogue (II) exhibits β‐turns typical of vasopressin‐like peptides. Therefore, the lack of binding of the glycosylated analogues to the receptors can be attributed to a steric interference between the carbohydrate moieties and the receptors. We also consider this to be the reason of the very low activity of the analyzed glycopeptides. We expect that results from these studies will be helpful in designing new OT‐like and vasopressin‐like drugs. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Investigation of mechanism of desmopressin binding in vasopressin V2 receptor versus vasopressin V1a and oxytocin receptors: molecular dynamics simulation of the agonist-bound state in the membrane-aqueous system

The vasopressin V2 receptor (V2R) belongs to the Class A G protein-coupled receptors (GPCRs). V2R is expressed in the renal collecting duct (CD), where it mediates the antidiuretic action of the neurohypophyseal hormone arginine vasopressin (CYFQNCPRG-NH2, AVP). Desmopressin ([1-deamino, 8-D]AVP, dDAVP) is strong selective V2R agonist with negligible pressor and uterotonic activity. In this paper, the interactions responsible for binding of dDAVP to vasopressin V2 receptor versus vasopressin V1a and oxytocin receptors has been examined. Three-dimensional activated models of the receptors were constructed using the multiple sequence alignment and the complex of activated rhodopsin with Gt(alpha) C-terminal peptide of transducin MII-Gt(alpha) (338-350) prototype (Slusarz, R.; Ciarkowski, J. Acta Biochim Pol 2004 51, 129-136) as a template. The 1-ns unconstrained molecular dynamics (MD) of receptor-dDAVP complexes immersed in the fully hydrated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) membrane model was conducted in an Amber 7.0 force field. Highly conserved transmembrane residues have been proposed as being responsible for V2R activation and G protein coupling. Molecular mechanism of the dDAVP binding has been suggested. The internal water molecules involved in an intricate network of the hydrogen bonds inside the receptor cavity have been identified and their role in the stabilization of the agonist-bound state proposed.     

Design of novel bicyclic analogues derived from a potent oxytocin antagonist.

Eleven new analogues were synthesized by modification of the potent oxytocin antagonist (OTA) [(S)Pmp(1), D-Trp(2), Pen(6), Arg(8)]-Oxytocin, or PA (parent antagonist), in which (S)Pmp = beta,beta-(3-thiapentamethylene)-beta-mercapto-propionic acid. By internal acylation of Lys, Orn, L-1,4-diaminobutyric acid (Dab), L-1,3-diaminopropionic acid (Dap) at position 4 with the C-terminal Gly of the peptide tail, we prepared cyclo-(4-9)-[Lys(4), Gly(9)]-PA (pA(2) = 8.77 +/- 0.27), 1, and cyclo-(4-9)-[Orn(4), Gly(9)]-PA (pA(2) = 8.81 +/- 0.25), 3, which are equipotent with PA (pA(2) = 8.68 +/- 0.18) in the rat uterotonic assay and cyclo-(4-9)-[Dab(4), Gly(9)]-PA, 4, cyclo-(4-9)-[Dap(4), Gly(9)]-PA, 5, and cyclo-(4-9)-[Pmp(1), Lys(4), Gly(9)]-PA, 2, which were weaker OTAs. Neither 1 nor 3 had activity as agonists or antagonists in the antidiuretic assay. In the pressor assay, both analogues 1 and 3, with pA(2) = 7.05 +/- 0.10 and pA(2) = 6.77 +/- 0.12, respectively, are somewhat weaker antagonists than PA (pA(2) = 7.47 +/- 0.35) showing significant gain in specificity. The [desamido(9)] PA-ethylenediamine monoamide, 6, and the dimer ([desamido(9)]-PA)(2) ethylenediamine diamide, 7, had lower potency in the uterotonic assay than PA. Additionally, we synthesized cyclo-(1-5)-[(HN)Pmp(1), Asp(5)]-PA, 8, inactive in all tests, which suggests that the intact Asn(5) side chain may be critical in the interaction of the OTAs with the oxytocin (OT) receptor. Similarly, cyclo-(5-9)-[Dap(5), Gly(9)]-PA, 9, had very low uterotonic potency. Two derivatives of PA truncated from the C-terminus were internally cyclized to Lys(4), giving rise to cyclo-(4-8)-desGly-NH(2)(9)[Lys(4), Arg(8)]-PA, 10 (pA(2) = 8.35 +/- 0.20), which maintains the high potency of PA and has no activity in the rat antidiuretic assay, and in the rat pressor assay it is about ten times weaker (pA2 = 6.41 +/- 0.15) than PA (pA2 = 7.47 +/- 0.35), thus showing gains in specificity, and to cyclo-(4-7)-desArg-Gly-(NH)(2)(8-9)[Lys(4), Pro(7))-PA, 11, which has much weaker potency than PA. Synthesis of cyclo-(4-6)-desPro-Arg-Gly-(NH)(2)(7-9)[Lys(4)]-PA failed.     

In vivo potentiation of corticotropin releasing factor activity by vasopressin analogues

The ability of the neurohypophyseal hormones and related synthetic peptides to potentiate the action of synthetic ovine corticotropin releasing factor (CRF-41) was examined using male rats whose endogenous CRF release was blocked with chlorpromazine, morphine and nembutal. CRF potency was clearly related to the pressor activity of the analogues. However, the threshold dose for eliciting a significant corticosterone response with the neurohypophyseal hormones was greater than with CRF-41. When arginine vasopressin (AVP) was coadministered with CRF-41 at subthreshold doses of both peptides, a significant corticosterone response was observed. When the neurohypophyseal hormone analogues were compared with regard to intrinsic CRF bioactivity, it was observed that an L-basic residue in sequence position 8 is necessary for high intrinsic activity. With one exception, l-Deamino-(9-D-Ala) arginine vasopressin, the ability to potentiate the effect of CRF-41 was related to the intrinsic CRF potency of the analogues. These results support previous reports of in vitro potentiation of CRF-41 by AVP and point out the complexity of CRF-neurohypophyseal hormone interaction in vivo.     

Stimulation of inorganic-phosphate incorporation into phosphatidylinositol in rat thoracic aorta mediated through V1-vasopressin receptors.

Vasopressin stimulates the incorporation of [32P]Pi into phosphatidylinositol but not into other phospholipids in rat thoracic aorta strips. The relative abilities of three vasopressin analogues to stimulate phosphatidylinositol labelling in rat aorta are similar to their relative pressor potencies in vivo and to their relative potencies in stimulating the metabolism of rat hepatocytes, but very different from their relative antidiuretic potencies. The vasopressor antagonist [1-(beta-mercapto-beta, beta-cyclopentamethylenepropionic acid),8-arginine]vasopressin competitively inhibits [Arg8]vasopressin-stimulated phosphatidylinositol labelling in rat aorta with a pA2 of 8.1. It is concluded that the Ca2+-mobilizing vasopressin receptors (V1-receptors) of the rat aorta stimulate phosphatidylinositol metabolism, probably by enhancing phosphatidylinositol breakdown.     

New acylated bradykinin analogues: effect on rat blood pressure and rat uterus.

It was previously reported that acylation of the N-terminus of several known B(2) antagonists with various types of bulky acyl groups consistently improved their antagonistic potency in the rat blood pressure assay. On the other hand, earlier results seem to suggest that the effects of acylation on the contractility of isolated rat uterus depend substantially on the chemical character of the acyl group, as it was observed that this modification may either change the range of antagonism or even transform it into agonism. Bearing all this in mind, three new analogues of bradykinin were designed by modifying the moderately potent B(2) antagonist, previously synthesized by Stewart's group, D-Arg-Arg-Pro-Hyp-Gly-Thr-Ser-D-Phe-Thi-Arg. New analogues were obtained by acylation of the N-terminus of the above peptide with succinic acid, 12-aminododecanoic acid and 4-aminobenzoic acid in order to confirm whether either the positive or the negative charge on the N-terminal end of the peptide is responsible for the transformation of activity. The activity of analogues was assessed on blood pressure and in uterotonic in vitro tests. The modifications proposed either preserved or increased the antagonistic potency in the rat blood pressure test. On the other hand, the three substituents, depending on their chemical character, differently influenced the interaction with the rat uterine receptors. The results may be of value in the design of new B(2) agonists and antagonists.     

Design, synthesis and biological activity of new neurohypophyseal hormones analogues conformationally restricted in the N-terminal part of the molecule. Highly potent OT receptor antagonists

In this study we present the synthesis and some pharmacological properties of fourteen new analogues of neurohypophyseal hormones conformationally restricted in the N-terminal part of the molecule. All new peptides were substituted at position 2 with cis-1-amino-4-phenylcyclohexane-1-carboxylic acid (cis-Apc). Moreover, one of the new analogues: [cis-Apc(2), Val(4)]AVP was also prepared in N-acylated forms with various bulky acyl groups. All the peptides were tested for pressor, antidiuretic, and in vitro uterotonic activities. We also determined the binding affinity of the selected compounds to human OT receptor. Our results showed that introduction of cis -Apc(2) in position 2 of either AVP or OT resulted in analogues with high antioxytocin potency. Two of the new compounds, [Mpa(1),cis-Apc(2)]AVP and [Mpa(1),cis-Apc(2),Val(4)]AVP, were exceptionally potent antiuterotonic agents (pA(2) = 8.46 and 8.40, respectively) and exhibited higher affinities for the human OT receptor than Atosiban (K (i) values 5.4 and 9.1 nM). Moreover, we have demonstrated for the first time that N -terminal acylation of AVP analogue can improve its selectivity. Using this approach, we obtained compound Aba[cis-Apc(2),Val(4)]AVP (XI) which turned out to be a moderately potent and exceptionally selective OT antagonist (pA(2) = 7.26).     

Synthesis and biological evaluation of oxytocin analogues containing L-alpha-t-butylglycine [Gly(Bu t)] in positions 8 or 9

We report the solid phase synthesis and some pharmacological properties of seventeen new oxytocin (OT) analogues. Basic modification at positions 8 and/or 9 (introduction of L-alpha-t-butylglycine [Gly(Bu(t))]) was combined with D-Cys(6), D-Tyr(Et)(2), Mpa(1) or Pen(1) modifications and their various combinations. We also present properties of two previously reported re-synthesized analogues ([Gly(Bu(t))(8)]OT and [Mpa(1), Gly(Bu(t))(8)]OT). The analogues were tested for rat uterotonic activity in vitro, in the rat pressor assay and for binding affinity to human OTR.