Synthesis and biological activities of oxytocin and lysine vasopressin analogs containing glutamic acid gamma-hydrazide in position 4

Solution methods, using N-hydroxysuccinimide esters, were used to synthesize [Glu(NHNH2)4] oxytocin and [Glu(NHNH2)4, Lys8] vasopressin. In these analogs of neurohypophyseal hormones, the side-chain carboxamide function of a glutamine residue is formally replaced by a hydrazide group at position 4. The hormone analogs were assayed for uterototonic activity, milk ejection activity, antidiuretic activity, and rat pressor activity. The specific biological activities of the oxytocin and vasopressin analogs were decreased compared to the respective parent hormones in all assay systems.     

Titanium-carbon functionalities on an oxo surface defined by a calix [4] arene moiety and its redox chemistry

Lithiation of [p-Bu^t-calix[4]-(OMe)₂(OH)₂] (1), followed by reaction with TiCl₃(thf)₃ or TiCl₄(thf)₂, led to the corresponding titanium-calix[4]arene complexes [p-Bu^t-calix[4]-(OMe)₂(O)₂]TiCl] (2) and [p-Bu^t-calix[4]-(OMe)₂(O)₂]TiCl₂] (3), respectively. Reaction of 1 with TiCl₄(thf)₂ results in demethylation of the calix[4]arene and the obtention of [p-Bu^t-calix[4]-(OMe)₂(O)₃]TiCl] (4), whose hydrolysis led to [p-Bu^t-calix[4]-(OMe)(OH)₃] (6). The preparation of 6 can be carried out as a one-pot synthesis. Both 2 and 4 undergo alkylation reactions using conventional procedures, thus forming surprisingly stable organometallic species, namely [p-Bu^t-calix[4]-(OMe)₂(O)₂Ti(R)] (R = Me (7); CH₂Ph (8), p-MeC₆H₄ (9) and [p-Bu^t-calix[4]-(OMe)(O)₃Ti(R)] (R = Me (10); CH₂Ph (11); p-MeC₆H₄ (12)). Complexes 7 and 9 undergo a thermal oxidative conversion into 10 and 12, occurring with the demethylation of one of the methoxy groups. A solid state structural property of 9 and 12 has been revealed by X-ray analysis showing a self-assembly of the monomeric units into a columnar polymer, where the p-tolyl substituent at the metal functions as a guest group for an adjacent titanium-calixarene. Reductive alkylation of 3 with Mg(CH₂Ph)₂ gave 8 instead of forming the corresponding dialkyl derivative. Two synthetic routes have been devised for the synthesis of the Ti(III)-Ti(III) dimer [p-Bu^t-calix[4]-(OMe)(O)₃Ti]₂] (13): the reduction of 4 and the reaction of TiCl₃(thf)₃ with the lithiated form of 6. A very strong antiferromagnetic coupling is responsible for the peculiar magnetic behavior of 13. The proposed structures have been supported by the X-ray analyses of 4, 9, 12 and 13.     

An asymmetric conformation of 1,3,5-benzene tricarbonyl [Aib4OMe]3.

Molecules of 1,3,5-benzene tricarbonyl [Aib(4)OMe](3) do not possess any internal symmetry, neither exact nor approximate, in the crystalline state. The Aib(4)OMe moieties each form a 3(10)-helix with an appropriate pair of hydrogen bonds but the sense of rotation is right-handed for two of the helices and left-handed for the third one. The helices are not evenly positioned around the benzene ring, and their helix axes are inclined toward one side of the plane of the benzene ring, giving the molecule the shape of a shallow bowl with an irregular periphery. The molecules are largely surrounded by water and dimethyl sulfoxide (DMSO) solvent molecules that form hydrogen bonds with the CO and NH moieties that protrude from the surfaces of the peptide molecule. The space group is Cc with a = 23.618(4) A, b = 19.708(6) A, c = 17.939(7) A and beta = 100.09(3) degrees.     

Biologically active macromolecular forms of oxytocin. [8-Lysine]oxytocin as a suitable ligand.

[8-Lysine]oxytocin was synthesized on a solid support and possessed an oxytocic activity of 100 +/- 6 units mumol on the isolated rat uterus. The epsilon-carbamoyl, epsilon-3-carboxypropionyl and epsilon-3-carboxybutryl derivatives were prepared and had uterotonic activities of 400, 55 and 50 units/mumol respectively. [8-Lysine]oxytocin was coupled unambiguously through the epsilon-amino group to the carboxyl groups of carboxymethylated dextrans or epsilon-3-carboxypropionly-gelatin. The macromolecular oxytocins were water-soluble and retained signigicant oxytocic activity. [8-Lysine]oxytocin should prove a useful ligand for affinity chromatography of oxytocin-binding proteins.     

Synthesis of [D-Ala2,4'-125I-Phe3,Glu4]deltorphin and characterization of its delta opioid receptor binding properties.

Both [D-Ala2,Glu4]Deltorphin and [D-Ala2,4'-I-Phe3,Glu4]Deltorphin are highly selective ligands for delta, relative to mu, opioid receptors. Radiolabeled [D-Ala2, 4'-125I-Phe3,Glu4]Deltorphin ([125I]Deltorphin) was prepared with a specific activity of 2200 Ci/mmol from [D-Ala2, 4'-NH2-Phe3, Glu4]Deltorphin through a diazonium salt intermediate. The inhibition of [125I]Deltorphin binding to rat brain membranes by ligands selective for mu, delta, and kappa opioid receptors is consistent with binding by the radioligand to a single site having the properties of a delta opioid receptor. The results of these studies are in good agreement with those obtained by structurally different delta opioid receptor ligands. The similarity between the delta receptor site labeled by [125I]Deltorphin and those labeled by other delta receptor agonists, in contrast to differences seen by in vivo studies of their analgesic effects, is discussed.     

Effect of 48-hour infusion of the synthetic oxytocin antagonist, [1-beta-mercapto(beta-(CH2)5)1(OMe)Tyr2,Orn8]-oxytocin, on myometrial activity of pregnant sheep at 139-140 days of gestation.

Currently there is considerable interest in the actions of oxytocin antagonists on the pregnant myometrium. Few studies have been conducted involving long-term infusions of oxytocin antagonists to late-pregnant experimental animals. We set out to determine whether continuous infusion of an oxytocin antagonist ([1-beta-mercapto(beta-(CH2)5)1(OMe)Tyr2,Orn8]-oxytocin) would influence basal levels of myometrial activity of the contracture type and maternal prostaglandins in pregnant sheep. The antagonist was infused into a uterine vein at 80 micrograms.h-1 for 48 h starting at 139 days of gestational age. The antagonist significantly reduced total myometrial electromyogram activity and the frequency of contractures but did not change contracture duration. Antagonist infusion did not produce any significant alterations in maternal carotid or uterine vein 13,14-dihydro-15 keto prostaglandin F2 alpha concentrations. Contractures probably represent an intrinsic instability of the resting membrane potential of uterine muscle and these results suggest that oxytocin may play a role in regulating their frequency in sheep during the last third of gestation.     

Proteolytic conversion of arginine-vasopressin and oxytocin by brain synaptic membranes. Characterization of formed peptides and mechanisms of proteolysis

This study concerned the fragmentation of the nonapeptides arginine-vasopressin (AVP-(1-9)) and oxytocin (OXT-(1-9)) by proteolytic enzymes present in a brain synaptic membrane preparation. The peptides formed during digestion of arginine-vasopressin and oxytocin were isolated by high pressure liquid chromatography and chemically characterized by amino acid composition, NH2-terminal amino acid residues, and the presence of 14C radioactivity in tyrosine-2 and glycinamide-9. The major peptide fragments of arginine-vasopressin were [Cyt6]-AVP-(2-9), [Cyt6]-AVP-(3-9), [less than Glu4, Cyt6]-AVP-(4-9), and a peptide having the AVP-(4-8) sequence. The characterized fragments of oxytocin were [Cyt6]-OXT-(2-9), [Cyt6]-OXT-(3-9), [Cyt6]-OXT-(4-9), [less than Glu4, Cyt6]-OXT-(4-9), and [Cyt6] OXT-(5-9). Employing differentially 14C-labeled arginine-vasopressin and oxytocin, the proteolysis of the two peptides into fragments was followed with time. The results showed the sequential formation of peptide fragments by proteolytic cleavage from the NH2 terminus onward, demonstrating the action of an aminopeptidase-like enzyme. Arginine-vasopressin was converted significantly more rapidly by the amino-peptidase activity than oxytocin. In contrast to known brain aminopeptidases, the synaptic membrane-associated activity cleaved the nonapeptides without prior reduction of the disulfide bridge. From the present data it is concluded that aminopeptidases predominate in the proteolytic mechanism by which brain synaptic membranes convert arginine-vasopressin and oxytocin. The role of the proteolytic events and the significance of formed peptide fragments is discussed in view of the concept that arginine-vasopressin and oxytocin are precursors for neuropeptides in brain.     

The activation of human [Glu1]plasminogen by human single-chain urokinase

The activation of human [Glu1]plasminogen ([Glu1]Pg) by single-chain human urokinase (SCUKase) displays a substantial lag phase at physiological levels of [Glu1]Pg. Employing a monoclonal antibody that exhibits a high level of specificity for SCUKase, as compared to two-chain urokinase (TCUKase), we have demonstrated conclusively that during this lag phase a progressive loss of SCUKase occurs, most likely resulting from its conversion to TCUKase, in a reaction catalyzed by plasmin (HPm). The overall activation of [Glu1]Pg by SCUKase is inhibited by physiological levels of Cl- and stimulated by epsilon-amino caproic acid. Kinetic studies demonstrate that both these effects are based on first, the reaction of [Glu1]Pg with the TCUKase that is formed during the activation, and, second, the concomitant rate at which HPm is provided for the conversion of SCUKase to TCUKase. The results indicate that at physiological levels of [Glu1]Pg, its activation in the presence of SCUKase is regulated in one manner by the rate at which SCUKase is converted to TCUKase, in a process that is strongly influenced by physiological levels of Cl-. Finally, and importantly, we show that SCUKase possesses very little, if any, inherent ability to activate [Glu1]Pg at a rate that influences the kinetics of HPm generation under physiological conditions of [Glu1]Pg and Cl- concentrations.     

Vasopressin and oxytocin receptors on plasma membranes from rat mammary gland. Demonstration of vasopressin receptors by stimulation of inositol phosphate formation, and oxytocin receptors by binding of a specific 125I-labeled oxytocin antagonist, d(CH2)5(1)[Tyr(Me)2, Thr4,Tyr-NH2(9)]OVT

The addition of oxytocin to minces of rat mammary gland preincubated with (3H)myo-inositol stimulated the formation of inositol phosphate (IP) in both lactating and regressed glands. Stimulation was about 4 times greater in regressed tissue, consistent with an oxytocin effect on myoepithelial cells, which are enriched relative to epithelial cells during regression. The stimulation of IP formation was agonist specific, as shown with several oxytocin analogs. Arginine vasopressin (AVP), however, was more than twice as potent as oxytocin in stimulating IP formation in regressed tissue. Both V1- and V2-selective AVP receptor antagonists inhibited the stimulation of IP formation by oxytocin. The V1-selective antagonist was about 10 times more inhibitory than the V2-selective antagonist. [3H]AVP was bound to plasma membranes from the mammary gland of the lactating rat with an apparent Kd of about 0.7 nM and Bmax of 54.6 fmol/mg protein. These values were comparable with those found for AVP receptors of kidney plasma membranes. Our results suggest that the stimulation of IP formation in rat mammary gland by oxytocin occurs through occupancy of AVP, and not oxytocin, receptor sites. A second aspect of these studies was to determine if a recently developed iodinated antagonist of oxytocin-induced uterine contractions could be used as a specific probe for oxytocin receptors in the rat mammary gland. Under steady state conditions, [125I]d(CH2)5(1)[Tyr(Me)2,Thr4,Tyr-NH2(9)]OVT was bound to a single class of independent binding sites in mammary gland plasma membrane from lactating rats with an apparent Kd of 65 pM and Bmax of 225 fmol/mg protein. Noniodinated antagonist had an affinity about 150 times less than the monoiodinated form. The affinity of binding sites for AVP was 10 times greater than the noniodinated antagonist and 2.4 times greater than oxytocin. In view of the presence of AVP receptors in mammary tissue, these findings suggested that the iodinated antagonist binds to AVP receptors. However, comparison of the binding of iodinated antagonist to plasma membranes from the lactating mammary gland with kidney medulla and liver, target sites for AVP, showed that binding was specific for the mammary gland and hence oxytocin receptors. The concentration of oxytocin receptors in mammary gland, as determined by [125I]d(CH2)5(1)[Tyr(Me)2,Thr4,Tyr-NH2(9)]OVT binding, was 4 times greater than the concentration of high-affinity AVP receptors, as determined by [3H]AVP binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding of [3H]oxytocin to cells isolated from the mammary gland of the lactating rat.

More than 90 percent of the cells isolated from the mammary gland of lactating rats with 0.1 percent collagenase were viable by dye exclusion. Myoepithelial cells comprised about one-third of the mammary cells and appeared to be morphologically intact in electron micrographs. [(3)H]Oxytocin-binding activity was localized in an enriched myoepitheial cell fraction obtained by density gradient centrifugation of the isolated cells. The amount of [(3)H] oxytocin bound at 20 degree C and pH 7.6 was proportional to the concentration of oxytocin and the number of cells, reaching a steady state by 40 min. About 0.45 fmol of oxytocin were bound per 10(6) cells. There was a single class of independent binding sites with an apparent K(d), estimated from equilibrium conditions, of 5 nM. This value agrees within experimental error with the value calculated from the ratio of reverse to forward rate constants (5.8 x 10(-4)s(-1) and 2.2 x 10(5) M(-1)s(-1), respectively), consistent with a single-step model for the interaction of oxytocin with binding sites on the cells. Erythrocytes bound only 3.5 percent of the amount of oxytocin bound by an equal number of mammary cells. Oxytocin analogues competed with [(3)H]oxytocin for binding sites in the following order: [deamino]oxytocin > [4-threonine]oxytocin > oxytocin > [O- methyltyrosine]oxytocin > [8-lysine]vasopressin; [lysine]-bradykinin and [4-proline]oxytocin were not inhibitory in the dose ranges tested. These results demonstrate that isolated mammary cells possess oxytocin receptors with properties comparable to those found in broken mammary cell preparations.     

Photoaffinity labelling of the oxytocin receptor in plasma membranes from rat mammary gland

Plasma membranes from rat mammary gland containing a high concentration of [3H]oxytocin binding sites (2.8 pmol/mg protein) were used for photoaffinity labelling experiments. Competitive binding experiments show that these receptors bind with high affinity the specific oxytocin agonist [Thr4, Sar7]oxytocin and the analogue of 1-deamino-[8-lysine]vasopressin containing a photoreactive azidobenzoyl group (Abz) at the side chain of lysine. The tritium-labelled (50 Ci/mol) photoreactive analogue incorporated into a membrane protein with an apparent relative molecular mass of 65,000 +/- 3000 Da (n = 16). The labelling of this protein was completely suppressed by an excess of oxytocin.     

A reversible radioligand specific for the ETB receptor: [125I]Tyr13-Suc-[Glu9,Ala11,15]-endothelin-1(8- 21), [125I]IRL 1620.

Suc-[Glu9,Ala11,15]-endothelin(ET)-1(8-21), IRL 1620, is a linear ET-analog specific for the ET-isopeptide-nonselective ETB receptor. The radio-iodinated analog, [125I]IRL 1620, showed a single class of saturable binding to the ETB receptors in porcine lung membranes with a Kd of 18 pM and a Bmax of 930 fmol/mg protein, which are almost comparable to the values obtained with [125I]ET-3 (6 pM and 900 fmol/mg protein). In competitive binding assays with [125I]IRL 1620, unlabeled ET-1, ET-3, IRL 1620 and [monoiodo-Tyr13]-IRL 1620 showed almost identical displacement curves with Ki of 8 to 16 pM. However, [125I]IRL 1620 was dissociated from the binding sites by addition of an excess amount (100 nM) of any of these unlabeled peptides, each with the same t1/2 of 100 min. This was in marked contrast to [125I]ET-3 which was hardly dissociated from the binding sites.     

3H]desGly-NH2(9)-d(CH2)5[D-Ileu2,Ileu4]AVP: an AVP V2 receptor antagonist radioligand

Binding characteristics of the selective V₂ antagonist radioligand [³H]desGly-NH₂⁹-d(CH₂)₅[D-Ileu²,Ileu⁴]AVP to rat kidney were determined. Binding was specific, saturable and reversible. The peptide bound to a single class of high-affinity binding sites with B_max 69.4±6.8 fmol/mg protein and K_D 2.8±0.3 nM. AVP and other related peptides displaced [³H]desGly-NH₂⁹-d(CH₂)₅[D-Ileu²,Ileu⁴]AVP binding. The order of potency of inhibition was desamino-D-AVP > AVP > d(CH₂)₅[D-Ileu²,Ileu⁴]AVP > oxytocin > d(CH₂)₃[Tyr(Me)²]AVP > d(CH₂)₅[sarcosine⁷]AVP, which is typical of a selective V₂ radioligand. Autoradiographic localization of [³H]desGly-NH₂⁹-d(CH₂)₅[D-Ileu²,Ileu⁴]AVP binding sites in kidney showed dense binding in the inner and outer medulla with less binding in the cortex, which is consistent with known renal V₂ receptor distribution.     

Structural studies with weak oxytocin antagonists

Summary [Aib³,Thr⁵]OT, [Aib³,Thr(OMe)⁵]OT, [Aib³,Orn⁸]OT, [Thr(OMe)⁵,Orn⁸]OT and [Phe²,Thr(OMe)⁵,Orn⁸]OT were synthesized by solid-phase techniques. From the biological properties of these peptides, it seems that the simultaneous replacement of positions 3 and 5 of oxytocin with Aib and Thr(OMe) results in an analogue devoid of antagonistic activity in comparison with the singly substituted compounds. Simultaneous Orn⁸ substitution does the same in the case of the Aib³ analogue and even leads to agonistic activity in the case of the Thr(OMe)⁵ analogue. Replacement of Tyr² by Phe², e.g. [Phe²,Thr(OMe)⁵,Orn⁸]OT, again favors the appearance of minor antagonistic potency.     

Design and synthesis of potent tyr(OMe)5-gonadotropin-releasing hormone (GnRH) analogues with modifications at positions 6, 9 and 10

We have recently reported the synthesis and the conformational properties of some Gonadotropin-releasing hormone (GnRH) analogues in which the tyrosine residue at position 5 is substituted with tyrosine-O-methyl (Keramida et al., Let. Pept. Sci., 3 (1996) 257/Matsoukas et al., Eur. J. Med. Chem., 32 (1997) 927). The analogue [Tyr-(OMe)⁵]-GnRH was found to exert a lower degree of desensitization than the native GnRH peptides in terms of pituitary gonadotropin (GTH) release in goldfish. Compared to GnRH, however, [Tyr-(OMe)⁵]-GnRH exerted a lower GTH-release potency in cultured goldfish pituitary fragments, and was bound with a lower binding affinity to the rat pituitary GnRH receptors. In order to increase the potency of [Tyr-(OMe)⁵]-GnRH, we have synthesized a group of GnRH peptides containing Tyr-(OMe)⁵ in combination with other substitutions at positions 6, 9 and 10 and we have estimated their binding affinity for the rat pituitary receptors and gonadotropin (GTH) release potency in the goldfish pituitary. A selected number of these analogues was also tested for their ability to induce ovulation in seabass. The important structural modifications that increased the binding and gonadotropic activity of [Tyr(OMe)⁵]-GnRH in vitro and in vivo were found to include the replacement of the proline at position 9 with azetidine, glycine amide terminus with an alkyl amide group and Gly⁶ residue with hydrophilic D-amino acids such as D-Arg⁶. Overall, the findings provide SAR information on a group of novel GnRH peptides that can be also used to induce ovulation in teleosts.     

Effectors of the activation of human [Glu1]plasminogen by human tissue plasminogen activator

The activation of human [Glu1]plasminogen [( Glu1]Pg) by human recombinant (rec) two-chain tissue plasminogen activator (t-PA) is inhibited by Cl-, at physiological concentrations, and stimulated by epsilon-aminocaproic acid (EACA), as well as fibrin(ogen). Chloride functions as a result of its binding to [Glu1]Pg, with a Ki of approximately 9.0 mM, thereby rendering [Glu1]Pg a less effective substrate for two-chain rec-t-PA. EACA stimulates the activation in Cl-(-)containing solutions, with a Ka of approximately 4.0 mM, primarily by reversal of the Cl-(-)inhibitory effect. Fibrinogen appears to exert its stimulatory properties mainly through effects on the enzyme, two-chain rec-t-PA, with a Ka of approximately 3.7 microM in activation systems containing physiological levels of Cl-. Analysis of the results of this paper reveals that normal plasma components, Cl- and fibrinogen, exert major regulatory roles on the ability of [Glu1]Pg to be activated by two-chain rec-t-PA, in in vitro systems. The presence of Cl- inhibits the stimulation of [Glu1]Pg activation that would normally occur in the presence of fibrinogen, a result of possible importance to the observation that some degree of systemic fibrinogenolysis accompanies therapeutic use of tissue plasminogen activator.     

Role of Asn(2) and Glu(7) residues in the oxidative folding and on the conformation of the N-terminal loop of apamin

The X-ray structure of [N-acetyl]-apamin has been solved at 0.95 A resolution. It consists of an 1-7 N-terminal loop stabilized by an Asn-beta-turn motif (2-5 residues) and a helical structure spanning the 9-18 residues tightly linked together by two disulfide bonds. However, neither this accurate X-ray nor the available solution structures allowed us to rationally explain the unusual downfield shifts observed for the Asn(2) and Glu(7) amide signals upon Glu(7) carboxylic group ionization. Thus, apamin and its [N-acetyl], [Glu(7)Gln], [Glu(7)Asp], and [Asn(2)Abu] analogues and submitted to NMR structural studies as a function of pH. We first demonstrated that the Glu(7) carboxylate group is responsible for the large downfield shifts of the Asn(2) and Glu(7) amide signals. Then, molecular dynamics (MD) simulations suggested unexpected interactions between the carboxylate group and the Asn(2) and Glu(7) amide protons as well as the N-terminal alpha-amino group, through subtle conformational changes that do not alter the global fold of apamin. In addition, a structural study of the [Asn(2)Abu] analogue, revealed an essential role of Asn(2) in the beta-turn stability and the cis/trans isomerization of the Ala(5)-Pro(6) amide bond. Interestingly, this proline isomerization was shown to also depend on the ionization state of the Glu(7) carboxyl group. However, neither destabilization of the beta-turn nor proline isomerization drastically altered the helical structure that contains the residues essential for binding. Altogether, the Asn(2) and Glu(7) residues appeared essential for the N-terminal loop conformation and thus for the selective formation of the native disulfide bonds but not for the activity.     

Effects of conformational constraint in 2- and 8-cycloleucine analogues of oxytocin and [1-penicillamine] oxytocin examined by circular dichroism and biosassay

The analogues of oxytocin and [1-penicillamine]oxytocin, containing a cycloleucine (Cle) residue in position 2 or 8, were investigated by means of circular dichroism measurements in different solvents, and the results examined in terms of their biological activities. A cycloleucine residue in position 2 substantially reduces the free conformational space of the hormone 20-membered ring moiety (including the disulfide group), and stabilizes a conformation which is close to one of the possible conformations of oxytocin and involves a -turn. In position 8, the Cle residue affects the conformation of the Tyr² side chain, apparently forcing it away from the space above the 20-membered disulfide ring. However, it does not appear that the Cle residue has any significant effect on the overall backbone conformation of the hormone. The steric effect of the penicillamine residue in position 1 on the conformation of the disulfide group and Tyr² side chain from previous investigations is further confirmed. The synthesis and biological potency of [1-penicillamine, 8-cycloleucine]oxytocin is described. This analogue exhibits a strong inhibitory effect on the uterotonic activity of oxytocinin vitro. It also inhibited the vasopressor response to vasopressin.     

Synthesis, characterization and cytotoxic activity of substituted benzyl iminoether Pt(II) complexes of the type cis- and trans-[PtCl2{E-N(H)=C(OMe)CH2-C6H4-p-R}2] (R=Me, OMe, F). X-ray structure of trans-[PtCl2{E-N(H)=C(OMe)CH2-C6H4-p-F}2

New substituted benzyl iminoether derivatives of the type cis- and trans-[PtCl(2){E-N(H)C(OMe)CH(2)-C(6)H(4)-p-R}(2)] (R=Me (1a, 2a), OMe (3a, 4a), F (5a, 6a)) have been synthesized and characterized by elemental analyses, FT-IR spectroscopy and NMR techniques. The iminoether ligands are in the E configuration, which is stable in solution and in the solid state, as confirmed by the (1)H NMR data. Complex trans-[PtCl(2){E-N(H)C(OMe)CH(2)-C(6)H(4)-p-F}(2)] (6a) was also characterized by an X-ray diffraction study. Complexes 1a-6a have been tested against a panel of human tumor cell lines in order to evaluate their cytotoxic activity. cis-Isomers were significant more potent than the corresponding trans-isomers against all tumor cell lines tested; moreover, complexes 1a and 5a showed IC(50) values from about 2-fold to 6-fold lower than those exhibited by cisplatin, used as reference platinum anticancer drug.     

Design and Synthesis of Potent Tyr(OMe)5-Gonadotropin-Releasing Hormone (GnRH) Analogues with Modifications at Positions 6, 9 and 10

Summary We have recently reported the synthesis and the conformational properties of some Gonadotropin-releasing hormone (GnRH) analogues in which the tyrosine residue at position 5 is substituted with tyrosine-O-methyl (Keramida et al., Let. Pept. Sci., 3 (1996) 257/Matsoukas et al., Eur. J. Med. Chem., 32 (1997) 927). The analogue [Tyr-(OMe)⁵]-GnRH was found to exert a lower degree of desensitization than the native GnRH peptides in terms of pituitary gonadotropin (GTH) release in goldfish. Compared to GnRH, however, [Tyr-(OMe)⁵]-GnRH exerted a lower GTH-release potency in cultured goldfish pituitary fragments, and was bound with a lower binding affinity to the rat pituitary GnRH receptors. In order to increase the potency of [Tyr-(OMe)⁵]-GnRH, we have synthesized a group of GnRH peptides containing Tyr-(OMe)⁵ in combination with other substitutions at positions 6, 9 and 10 and we have estimated their binding affinity for the rat pituitary receptors and gonadotropin (GTH) release potency in the goldfish pituitary. A selected number of these analogues was also tested for their ability to induce ovulation in seabass. The important structural modifications that increased the binding and gonadotropic activity of [Tyr(OMe)⁵]-GnRH in vitro and in vivo were found to include the replacement of the proline at position 9 with azetidine, glycine amide terminus with an alkyl amide group and Gly⁶ residue with hydrophilicd-amino acids such asd-Arg⁶. Overall, the findings provide SAR information on a group of novel GnRH peptides that can be also used to induce ovulation in teleosts.