Factor J, an inhibitor of the classical and alternative complement pathway, does not inhibit esterolysis by factor D.

Factor J (FJ) is an inhibitor of the classical and alternative complement pathways. On the classical pathway factor J disrupts the C1 component, and on the alternative pathway, factor J disrupts the C3 convertase (C3b,Bb) by a direct interaction of FJ with the components C3b and Bb. The aim of this work was to verify whether FJ could have any effect on factor D proteolytic activity since previous experiments could not rule out an eventual inhibition by factor J on factor D enzymatic activity. For this purpose, the reactivity of serine proteinase factor D was determined by using two peptide thioester substrates, Z-Lys-SBzl.HCl and Z-Lys-Arg-SBzl.2HCl, in the presence and in the absence of factor J. Kinetic studies evidenced that FJ did not affect the enzymatic activity of factor D in any case.

Analgesic dipeptide derivatives. 4. Linear and cyclic analogues of the analgesic compounds arginyl-2-[(o-nitrophenyl)sulfenyl]tryptophan and lysyl-2-[(o-nitrophenyl)sulfenyl]tryptophan.

The syntheses of Trp(Nps)-Arg-OMe.HCl (15) [Trp(Nps) = 2-[(o-nitrophenyl)sulfenyl]tryptophan], its three stereoisomers, and their corresponding cyclic analogues are reported. The preparation of Trp(Nps)-Lys-OMe (19) and its cyclic analogue is also described. All these compounds have been designed as analogues of the analgesic dipeptide derivatives X-Trp(Nps)-OMe (1b, X = Arg; 2b, X = Lys). In the case of dipeptides containing Arg or D-Arg, the coupling reactions were achieved via the isobutyl chloroformate and N-methylmorpholine mediated mixed anhydride procedure, while in the case of the Lys analogue, the N,N-dicyclohexylcarbodiimide method was employed. Sulfenylation reactions were carried out with Nps-Cl in acidic media. Cyclization to the diketopiperazines was achieved by using acetic acid as catalyst. The antinociceptive effects of all these new Trp(Nps)-containing dipeptides were evaluated after icv administration in mice, and the effects were compared with those of 1b, 2b, Tyr-Arg (Kyotorphin), and Tyr-D-Arg. The most active compounds, 15 and 19, were found to exhibit a naloxone-reversible antinociceptive effect similar to those of 1b and 2b and approximately 50 and 12.5 times higher than those of Kyotorphin and its D isomer, respectively. Trp(Nps)-D-Arg-OMe.HC1, D-Trp(Nps)-Arg-OMe.HC1, and cyclo[Trp(Nps)-Arg].HC1 were also more effective than Kyotorphin (5, 10, and 10 times, respectively). In view of the structure-activity relationships obtained, several similarities between this series of Trp(Nps)-containing dipeptides and that of Kyotorphin analogues have emerged.

Analgesic dipeptide derivatives. 3. Synthesis and structure-activity relationships of o-nitrophenyl-modified analogues of the analgesic compound H-Lys-Trp(NPS)-OMe.

A series of analogues of the analgesic dipeptide derivative H-Lys-Trp(NPS)-OMe has been designed to determine the influence of the (2-nitrophenyl)sulfenyl (NPS) moiety on the activity. The syntheses and antinociceptive effects of these analogues of general formula H-Lys-Trp(R)-OMe [R = phenylsulfenyl (PS) (9); R = (2-carbomethyoxyphenyl)sulfenyl (CmPS) (10); R = (4-nitrophenyl)sulfenyl (pNPS) (11); R = (2,4-dinitrophenyl)sulfenyl (DNPS) (12); R = [2-(acetylamino)-2-carbomethoxyethyl]sulfenyl (AacCmES) (13); R = [2-(acetylamino)phenyl]sulfenyl (AacPS) (17); R = tert-butylsulfenyl (t-BuS) (23); R = (2-carbomethoxyethyl)sulfenyl (CmES) (24)] are described. Reaction of Z-Lys(Z)-Trp-OMe (3) with PS-, CmPS-, pNPS-, DNPS-, and AacCmES-Cl afforded the corresponding 2-(sulfenyl)tryptophan derivatives, which on treatment with boron-tris(trifluoroacetate)/trifluoroacetic acid or trimethylsilyl iodide in acetonitrile (Me3SiI/CH3CN) provided 9-13, respectively. Sulfenylation of 3 with NPS-Cl gave Z-Lys(Z)-Trp(NPS)-OMe, which, on catalytic hydrogenation of the nitro group using 10% Pd/C followed by acetylation of the resulting amino function and removal of the protecting Z groups, gave 17. Condensation of 2-(tert-butylsulfenyl)- and 2-[(2-carbomethoxyethyl)sulfenyl]tryptophan methyl ester, obtained by reaction of methyl 3a-hydroxy-1,2,3,3a,8,8a-hexahydropyrrolo[2,3-b]indole-2-carboxyla te with the corresponding thiol, with Z-Lys(Z)-OSu afforded Z-Lys(Z)-Trp(t-BuS)-OMe and Z-Lys(Z)-Trp(CmES)-OMe, which on treatment with Me3SiI/CH3CN provided 23 and 24, respectively. Intracerebroventricular administration of 10 elicited a naloxone-reversible antinociceptive effect in mice similar to that of H-Lys-Trp(NPS)-OMe. No analgesia was however found with the phenylsulfenyl or acyclic sulfenyl substituted dipeptides 9, 11, and 17 or 13, 23, and 24. The Trp(DNPS)-containing analogue was neurotoxic. Structure-activity studies indicate that the role of the NPS and CmPS moieties could be related to the adoption of a preferential active conformation.

Boc-Trp-Orn(Z)-Asp-NH2 and derivatives: a new family of CCK antagonists.

The respective roles of the benzyloxycarbonyl group (Z) and of the N-terminal tripeptide moiety in the antagonist properties of the cholecystokinin CCK8 analogue Boc-[Nle28,Orn(Z)31]CCK27-33 (Marseigne et al. J. Med. Chem. 1988, 31, 966.) were studied with the following derivatives: Boc-[Nle28,Orn(X)31]CCK27-33, Boc[Nle28,Orn(X)31]CCK27-32, Boc-[Orn(X)31]CCK30-33, and Boc-[Orn(X)31]CCK30-32 (X = Z, Boc, H). These derivatives, the synthesis of eight of which is reported here, were tested for their abilities to inhibit the binding of [3H]pCCK8 to guinea pig pancreatic and brain membranes and for their potencies in stimulating amylase release from guinea pig pancreatic acini. None of the Z derivatives produced amylase secretion, but they competitively antagonized the stimulation induced by CCK8. The deletion of the N-terminal tripeptide and/or Phe-NH2(33) residue did not play a key role in the recognition of peripheral receptors and in the activity of these peptides, whereas replacement of the Z group by a Boc group slightly decreased the affinities of the compounds for both pancreatic and brain binding sites and their potencies as peripheral antagonists. Moreover, the tetrapeptide Boc-Trp-Orn(Boc)-Asp-Phe-NH2 behaved as a partial agonist and analogues in which the Z or Boc groups on the ornithine residue were removed were full agonists. Interestingly, the short peptide derivative Boc-Trp-Orn(Z)-Asp-NH2 displayed the same affinity (KI = 2.0 +/- 0.2 x 10(-7] and the same antagonist activity (pA2 = 6.63) as its parent compound Boc-[Nle28,Orn(Z)31]CCK27-33. This tripeptide could be an interesting tool for studying the structural relationships between peptide and non-peptide CCK antagonists.

beta-Turned dipeptoids as potent and selective CCK(1) receptor antagonists.

To improve our knowledge of the bioactive conformation of CCK(1) antagonists, we previously described that replacement of the alpha-MeTrp residue of dipeptoids with the (2S,5S, 11bR)-2-amino-3-oxohexahydroindolizino[8,7-b]indole-5-carbox ylate (IBTM) skeleton, a probed type II' beta-turn mimetic, led to restricted analogues (2S,5S,11bR,1'S)- and (2S,5S,11bR, 1'R)-2-(benzyloxycarbonyl)amino-5-[1'-benzyl-2'-(carboxy)ethyl]carbam oyl-3-oxo-2,3,5,6,11,11b-hexahydro-1H-indolizino[8,7-b]indole, 1a,b, showing high binding affinity and selectivity for CCK(1) receptors. In this report, we describe the synthesis and binding profile of new analogues of compounds 1 designed to explore the importance of the C-terminal residue and of the type of beta-turn on the receptor binding affinity and selectivity. Structure-affinity relationship studies show that a C-terminal free carboxylic acid and an S configuration of the Phe and betaHph residues are favorable for CCK(1) receptor recognition. Moreover, selectivity for this receptor subtype is critically affected by the beta-turn type. Thus, while compounds 15a and 16a, containing the (2S,5S,11bR)- and (2R,5R, 11bS)-IBTM frameworks, respectively, are both endowed with nanomolar affinity for CCK(1) receptors, restricted dipeptoid derivative 15a, incorporating the type II' IBTM mimetic, shows approximately 6-fold higher CCK(1) selectivity than analogue 16a, with the type II mimetic. From these results, we propose that the presence of a beta-turn-like conformation within the peptide backbone of dipeptoids could contribute to their bioactive conformation at the CCK(1) receptor subtype. Concerning functional activity, compounds 15a and 16a behave as CCK(1) receptor antagonists.

5-(Tryptophyl)amino-1,3-dioxoperhydropyrido[1,2-c]pyrimidine-based potent and selective CCK(1) receptor antagonists: structure-activity relationship studies on the central 1,3-dioxoperhydropyrido[1,2-c]pyrimidine scaffold.

To further define the pharmacophore of the potent and selective 5-(tryptophyl)amino-1,3-dioxoperhydropyrido[1,2-c]pyrimidine-based CCK(1) receptor antagonists the electronic and topographic properties of the central 1,3-dioxoperhydro-pyrido[1,2-c]pyrimidine scaffold have been modified. With this aim, the 1- and 3-oxo groups have been replaced by the thioxo- and deoxi-analogues, and the fused piperidine ring has been contracted to the corresponding pyrrolidine moiety. The results of the evaluation of the new analogues as CCK receptor ligands, in rat pancreas and cerebral cortex preparations, showed that, whereas replacement of oxygen with sulfur is allowed, reduction of the 1- or 3-oxo groups or the contraction of the fused piperidine ring lead to the complete loss of binding affinity at CCK(1) receptors. The thioxo-analogues 5a, 8a, 12a, and 12b showed functional CCK(1) antagonist activity, inhibiting the CCK-8-stimulated amylase release from pancreatic acinar cells. The 1-thioxo analogue 5a, with subnanomolar affinity (IC(50) = 0.09 x 10(-9) M), was found to be the most potent and selective compound within the family of 5-(tryptophyl)amino-1,3-dioxoperhydropyrido[1,2-c]pyrimidine-based CCK(1) antagonists.

5-(Tryptophyl)amino-1,3-dioxoperhydropyrido[1,2-c]pyrimidine-based potent and selective CCK(1)receptor antagonists: structure-activity relationship studies on the substituent at N2-position.

To establish structure-activity relationships a new series of analogues of the highly potent and selective CCK(1) receptor antagonist (4aS,5R)-2-benzyl-5-(N-Boc-tryptophyl)amino-1,3-dioxoperhydropyrido[1,2-c]-pyrimidine (1a) modified at N2-position of the central scaffold has been prepared and evaluated as CCK receptor ligands. With this aim the N2-benzyl group has been replaced by methyl, cyclohexyl, aromatic groups, 1-phenylethyl, and 1-carboxy-2-phenylethyl group. Then, substituents with different electronic and steric properties were introduced into different positions of the phenyl group of analogues 19a and 19b. The results of the CCK receptor binding and in vitro functional activity evaluation suggest the importance of the lipophilic character and an appropriate spatial orientation of the moiety linked at the N2-position of the 1,3-dioxoperhydropyrido[1,2-c]pyrimidine template for potent and selective binding and antagonist activity at CCK(1) receptor subtype. The 2-cyclohexyl and (2S)-1-naphthyl derivatives 18a and (2S)-20a have emerged as more potent and selective CCK(1) receptor antagonists than the lead compound 1a. Additionally, the results confirm the (4aS,5R)-stereochemistry at the central bicyclic skeleton as an essential structural requirement for potent binding to this receptor subtype.

Synthesis and stereochemical structure-activity relationships of 1,3-dioxoperhydropyrido[1,2-c]pyrimidine derivatives: potent and selective cholecystokinin-A receptor antagonists.

The synthesis and stereochemical structure--activity relationships of a new class of potent and selective non-peptide cholecystokinin-A (CCK-A) receptor antagonists based on the 1,3-dioxoperhydropyrido[1,2-c]pyrimidine skeleton are described. The most potent member of this series of eight diastereoisomers, (4aS,5R)-2-benzyl-5-[N-[(tert-butoxycarbonyl)-L-tryptophyl]-amino] - 1,3-dioxoperhydropyrido[1,2-c]pyrimidine, displays nanomolar CCK-A receptor affinity and higher than 8000-fold potency at the CCK-A than at the CCK-B receptor. As CCK-A antagonist, this compound inhibits the CCK-8-evoked amylase release from pancreatic acinar cells at a low concentration, similar to that of the typical antagonist Devazepide. Highly strict stereochemical requirements for CCK-A receptor binding and selectivity have been found. The L-Trp and the 4a,5-trans disposition of the bicyclic perhydropyrido[1,2-c]pyrimidine are essential for binding potency and selectivity.

Ketomethylene and (cyanomethylene)amino pseudopeptide analogues of the C-terminal hexapeptide of neurotensin.

A series of pseudopeptide analogues of the C-terminal hexapeptide of neurotensin (NT8-13), namely [Tyr11 psi[COCH2]Phe12]-, [Ile12 psi[COCH2]Phe13]-, and [Tyr11 psi[CH(CN)NH]Ile12]NT8-13 with different stereochemistries, has been synthesized and evaluated for its potency in displacing labeled NT from rat cortex membranes. Ketomethylene pseudohexapeptides were prepared from the corresponding Boc-protected ketomethylene dipeptide derivatives, previously formed, using different solid phase synthesis (SPS) conditions, while (cyanomethylene)amino analogues were directly prepared by SPS using Fmoc strategy. H-Arg-Arg-Pro-Tyr psi[COCH2]-Phe-Leu-OH was nearly as potent as NT8-13 and [Phe12]NT8-13 in binding to the receptor. Comparison of the affinities for the pseudohexapeptides, here reported, with those of the psi-[CH2NH] analogues indicates the importance of the CO group in the amide or surrogate linkage at 11-12 and 12-13 positions in the receptor binding process.

5-(Tryptophyl)amino-1,3-dioxoperhydropyrido[1,2-c]pyrimidine-based potent and selective CCK(1) receptor antagonists: structural modifications at the tryptophan domain.

Analogues of the previously reported potent and highly selective CCK(1) receptor antagonist (4aS, 5R)-2-benzyl-5-(N-Boc-tryptophyl)amino-1,3-dioxoperhydropyrido-[1, 2-c]pyrimidine (2a) were prepared to explore the structural requirements at the Boc-tryptophan domain for CCK(1) receptor affinity. Structural modifications of 2a involved the Trp side chain, its conformational freedom, the Boc group, and the carboxamide bond. Results of the CCK binding and in vitro functional activity evaluation showed three highly strict structural requirements: the type and orientation of the Trp side chain, the H-bonding acceptor carbonyl group of the carboxamide bond, and the presence of the Trp amino protection Boc. Replacement of this acid-labile group with 3, 3-dimethylbutyryl or tert-butylaminocarbonyl conferred acid stability to analogues 14a and 15a, which retained a high potency and selectivity in binding to CCK(1) receptors, as well as an in vivo antagonist activity against the acute pancreatitis induced by caerulein in rats. Oral administration of compounds 14a and 15a also produced a lasting antagonism to the hypomotility induced by CCK-8 in mice, suggesting a good bioavailability and metabolic stability.

Pharmacological evaluation of IQM-95,333, a highly selective CCKA receptor antagonist with anxiolytic-like activity in animal models.

1. The pyridopyrimidine derivative IQM-95,333 ((4aS,5R)-2-benzyl-5-[N alpha-tert-butoxicarbonyl)L-tryptophyl] amino-1,3dioxoperhydropyrido[1,2-c]pyrimidine), a new non-peptide antagonist of cholecystokinin type A (CCKA) receptors, has been evaluated in vitro and in vivo in comparison with typical CCKA and CCKB receptor antagonists, such as devazepide, lorglumide, L-365,260 and PD-135,158. 2. IQM-95,333 displaced [3H]-CCK-8S binding to CCKA receptors from rat pancreas with a high potency in the nanomolar range. Conversely, the affinity of this new compound at brain CCKB receptors was negligible (IC50 > 10 microM). IQM-95,333 was a more selective CCKA receptor ligand than devazepide and other CCKA receptor antagonists. 3. Like devazepide, IQM-95,333 was a more potent antagonist of CCK-8S- than of CCK-4-induced contraction of the longitudinal muscle from guinea-pig ileum, suggesting selective antagonism at CCKA receptors. 4. IQM-95,333 and devazepide were also potent inhibitors of CCK-8S-stimulated amylase release from isolated pancreatic acini, a CCKA receptor-mediated effect. The drug concentrations required (IC50s around 20 nM) were higher than in binding studies to pancreas homogenates. 5. Low doses (50-100 micrograms kg-1, i.p.) of IQM-95,333 and devazepide, without any intrinsic effect on food intake or locomotion, blocked the hypophagia and the hypolocomotion induced by systemic administration of CCK-8S, two effects associated with stimulation of peripheral CCKA receptors. 6. IQM-95,333 showed an anxiolytic-like profile in the light/dark exploration test in mice over a wide dose range (10-5,000 micrograms kg-1). Typical CCKA and CCKB antagonists, devazepide and L-365,260 respectively, were only effective within a more limited dose range. 7. In a classical conflict paradigm for the study of anxiolytic drugs, the punished-drinking test, IQM-95,333, devazepide and L-365,260 were effective within a narrow dose range. The dose-response curve for the three drugs was biphasic, suggesting that other mechanisms are operative at higher doses. 8. In conclusion, IQM-95,333 is a potent and selective CCKA receptor antagonist both in vitro and in vivo with an anxiolytic-like activity in two different animal models, which can only be attributed to blockade of this CCK receptor subtype.

Ketomethylene and methyleneamino pseudopeptide analogues of insect allatostatins inhibit juvenile hormone and vitellogenin production in the cockroach Blattella germanica.

Metabolic studies on insect allatostatins have suggested that the dipeptide Leu-Tyr may be a target for endopeptidases. In order to increase resistance to degradation, methyleneamino psi [CH2NH] and ketomethylene psi [COCH2] peptide bond surrogates have been introduced at the position Leu3-Tyr4 of the allatostatin Asp-Arg-Leu-Tyr-Ser-Phe-Gly-Leu-amide (BLAST-2), and Leu3-Phe4 of [Phe4]BLAST-2, respectively. Assays of inhibition of juvenile hormone (JH) synthesis in vitro by corpora allata from the cockroach Blattella germanica showed that both analogues were similarly active to the respective model peptides. The methyleneamino analogue was further tested in vivo as an inhibitor of JH synthesis, and in vivo and in vitro as an inhibitor of vitellogenin production by the fat body of B. germanica. The analogue was less active than BLAST-2 when tested in vitro, but more active than it when tested in vivo.

Antinociceptive effects in rodents of the dipeptide Lys-Trp (Nps) and related compounds.

Intracerebroventricular administration of the synthetic dipeptide derivative Lys-Trp (Nps) (LTN) elicits a potent and naloxone-sensitive antinociceptive effect in mice and in rats using heat and electrical current respectively as the noxious stimuli. LTN does not induce analgesia by directly acting on opioid receptors but the peptidase inhibiting activity of the new compound may account in part for the behavioral effect. LTN produces also a marked decrease in the met-enkephalin content of the periaqueductal gray suggesting a possible enkephalin releasing property. Structure-activity studies with different analogs of LTN indicate that replacement of Lys by other basic amino acids results also in compounds with a potent antinociceptive effect whereas replacement by neutral or acidic amino acids leads to a complete loss of activity.

Prolonged antinociceptive activity of pseudodipeptide analogues of Lys-Trp(Nps) and Trp(Nps)-Lys.

Peptide bond substitution in the molecules of Lys-Trp(Nps) (LTN) and Trp(Nps)-Lys (TNL) by an aminomethylene and ketomethylene bond, respectively, afforded pseudodipeptides with analgesic activity. The new compounds Lys psi(CH2NH)-Trp(Nps)-OMe (LTNAM) and Trp(Nps)psi(COCH2)(R,S)-Lys (TNLKM) induced a dose-dependent and naloxone-reversible analgesia following intracerebroventricular (ICV) administration to mice. The antinociceptive effects were longer lasting compared to those induced by the parent compounds. The pseudodipeptides protected Met-enkephalin degradation by rat striatal slices and, combined with an ineffective dose of the opioid peptide, induced analgesia. LTNAM and TNLKM were as potent as LTN to inhibit brain aminopeptidase in vitro and ex vivo. An increased resistance to proteolysis of the pseudodipeptides may explain their prolonged analgesic activity.

2,5-Diketopiperidine derivatives as non-peptide ligands for cholecystokinin receptors.

Easily accessible 2,5-diketopiperidines have been used as templates for the construction of Trp-Phe and Trp-Asp-Phe-NH2 mimics. The cycle [L-Trp psi[COCH2]-L-Phe] analogue 1a has shown to possess significant and selective affinity for CCKA receptors.

Analgesic dipeptides. VI.: Synthesis and structure-activity relationships of N-terminal modified analogues of the analgesic compounds H-Xaa-Trp(Nps)-OMe (Xaa=Lys, Orn, Arg).

In order to determine the influence of the N-terminal amino group of the dipeptide derivatives H-Xaa-Trp(Nps)-OMe[Xaa = Lys (2a), Orn (2b), Arg (2c)] on their antinociceptive effects, the syntheses of their corresponding deaminated, acetylated and dimethylated analogues have been achieved. Deamino and dimethyl analogues of 2a,b,6a,b, and 18a,b were prepared by coupling the corresponding N omega-Z- and N omega-Z-N alpha-Me2 amino acids with H-Trp-OMe, using the DCC/HOSu method, followed by sulfenylation of the resulting compounds and removal of the Z groups. Guanidylation of 6b and 18b provided the arginine analogues 6c and 18c, respectively. Ac-Xaa-Trp(Nps)-OMe [Xaa = Lys (11a), Orn (11b) were synthesized by acetylation of H-Xaa(Z)-Trp(Nps)-OMe with acetic anhydride, in the presence of 4-dimethylaminopyridine, and subsequent removal of the Z groups. Coupling of Ac-Arg-OH.HC1 with H-Trp-OMe, using the DCC/HOSu procedure, followed by sulfenylation of the resulting 8:3 diastereomeric mixture of L,L and L,D dipeptides afforded Ac-ambo-Arg-Trp(Nps)-OMe 11c+11d. The antinociceptive effects of 6a-c, 11a-d, and 18 a-c were evaluated after i.c.v. administration in mice. The N alpha-acetyl dipeptides 11 were found to exhibit a naloxone-reversible antinociceptive effects comparable with those of 2, while N-deaminated and N,N-dimethylated analogues were inactive.

Analgesic dipeptide derivatives. II. Synthetic dipeptides containing a C-terminal 2-(2-nitrophenylsulfenyl) tryptophan residue.

The syntheses of dipeptide derivatives of the general formula X-Trp(Nps)-OR (R = H or Me; X = Lys, Gly, Ala, Leu, Ser, Glu, His, Arg, Orn, Dpr, Gpr and Har) are described. The analgesic activities of Gpr- and Har-containing dipeptides have been evaluated and, in the light of these results, the influence of the side chain length of the basic amino acid on the analgesic effect is studied.

Synthesis and inhibitory activities against enkephalin degrading aminopeptidase of H-Trp(Nps)-Lys-OMe analogues bearing chelating groups.

With the aim of increasing the inhibitory potency of the analgesic dipeptide H-Trp(Nps)-Lys-OMe against enkephalin-degrading aminopeptidases, the following derivatives bearing chelating groups at the N-terminus have been synthesized: Ac-Trp(Nps)-Lys-OMe (3), HS(CH2)nCO-Trp(Nps)-Lys-OMe [n = 1 (4), n = 2 (5)], MeOCO(CH2)n-Trp(Nps)-Lys-OMe [n = 1 (6), n = 2 (7)] and analogues in which the N alpha-amino group has been replaced by a methoxycarbonyl group (8) and a bidentate hydroxamate function (9), respectively. The inhibitory activities of all these compounds and the S-protected derivatives EtNHCOS(CH2)nCO-Trp(Nps)-Lys-OMe [n = 1 (16), n = (17)] against the mentioned enzyme, isolated from rat striatum, are compared with those of the parent dipeptide 2 and bestatin. All the new derivatives showed, in general, inhibitory potencies of the same order of magnitude as compound 2.

Solid phase synthesis of a fully active analogue of cholecystokinin using the acid-stable Boc-Phe (p-CH2) SO3H as a substitute for Boc-Tyr(SO3H) in CCK8.

Substitution of the -OSO3H group in the sulfated-tyrosine by the non-hydrolyzable-CH2SO3H group was the first described modification of the sulfate ester that does not affect CCK8 activity. In addition to its capacity to mimic the sulfated tyrosine residue, the amino acid Phe(p-CH2SO3Na) was shown to be stable in acidic media, including HF containing mixtures. The synthesis of Boc-Phe(p-CH2SO3Na)-OH in racemic and resolved forms and its introduction into the sequence of CCK8 by solid phase using standard Boc/benzyl synthesis conditions and BOP as coupling reagent is now reported. The two CCK8 analogues containing the L- or the D-Phe(p-CH2SO3Na) residue, obtained in satisfactory yields, were separated by HPLC and the stereochemistry of Phe(p-CH2SO3Na) residue in each peptide was established by NMR spectroscopy and confirmed by a separate solid phase synthesis in which the pure L isomer was used. Both CCK8 analogues displayed high affinities for peripheral and central receptors (KI approximately 1 nM) and proved to be full agonists in the stimulation of pancreatic amylase secretion. The "stabilized-CCK8 peptide", easily prepared by solid phase, could replace the native peptide in biochemical and pharmacological studies. Moreover the modified amino acid Phe (p-CH2SO3Na) could also be used in solid phase synthesis to prepare a wide variety of CCK analogues and more generally, peptides analogues containing the acid-labile O-sulfated tyrosine.

Entry to new conformationally constrained amino acids. First synthesis of 3-unsubstituted 4-alkyl-4-carboxy-2-azetidinone derivatives via an intramolecular N(alpha)-C(alpha)-cyclization strategy.

A systematic study on the base-assisted intramolecular alkylation of N-benzyl-N-chloroacetyl amino acid derivatives is described. This study resulted in the first concise and versatile route to the preparation of 3-unsubstituted 4-alkyl-4-carboxy-2-azetidinones, to be included into the scarce family of beta-lactams with quaternary centers at the C(4) position. Particularly noteworthy is that the intramolecular N(alpha)-C(alpha)-cyclization of Phe and Leu derivatives afforded the corresponding beta-lactam derivatives with moderate enantioselectivity (up to 56%). It is suggested that, in these particular cases, the cyclization reaction proceeds by way of planar enolate intermediates, which possess dynamic chirality. The described sequence of reactions, that is compatible with commonly used protecting moieties for the alpha-carboxy group, cannot be applied to dipeptides, since the cyclization to the six-membered 2,5-diketopiperazine ring occurs preferentially.