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To test whether a turnlike arrangement is involved in the bioactive conformation of CCK4 analogues upon CCK1 receptor recognition, we describe the preparation of two series of CCK4 derivatives, in which the central dipeptide Met-Asp has been replaced by recognized β-turn mimetics {(2S,5S,11bR)- and (2R,5R,11bS)-2-amino-5-carboxy-3-oxo-2,3,5,6,11,11b-hexahydro-1H-indolizino[8,7-b]indole (IBTM) and β-turn dipeptide, 2-oxo-7-thio-1-azabicyclo[4.3.0]nonane (BTD)}. The incorporation of the indolizinoindole IBTM type II β-turn mimetic is preferred over its type II‘ counterpart for efficient CCK1 receptor recognition, while BTD derivatives were completely inactive. The structure−conformation−activity relationship study in the IBTM series has shown some essential requirement of these CCK4 derivatives to favorably interact with CCK1 receptors:  (a) the adoption of turnlike conformations, (b) the presence of an l-Phe residue and a C-terminal carboxamide moiety, and (c) the indole ring of the IBTM skeleton. Moreover, the existence of π−π interactions between the phenyl ring of d-Phe residues and the indole ring of IBTM framework is detrimental for binding affinity. A series of potent and selective CCK1 receptor antagonists, exemplified by compounds 8a and 8b, emerges among these IBTM-containing derivatives.

A rational combination of site-directed mutagenesis studies, structure−activity relationships, and dynamic-based docking of pyridopyrimidine-derived CCK1R antagonists into a refined three-dimensional model of the CCK1R allowed us to identify the receptor residues and the ligand functional groups implicated in the molecular recognition process. Our results provided unambiguous evidence that the binding site of these antagonists is overlapping that of the C-terminal tetrapeptide of CCK. In particular, Asn333 and Arg336 residues of the CCK1R are essential for high-affinity binding of these ligands. Moreover, the 2-aryl group in the pyridopyrimidine derivatives shares the same binding pocket as the C-terminal Phe side chain of CCK. Our [pyridopyrimidine·CCK1R] complex model is consistent with previous suggestions concerning the molecular basis that governs functional activity and provides useful considerations about the high CCK1 versus CCK2 selectivity of our derivatives and could contribute to fine-tune the rational design of new molecules with optimized properties.

Starting from the structure of known β-lactam covalent human cytomegalovirus (HCMV) protease inhibitors and from the knowledge of the residues implicated in the active site of this enzyme, we designed a series of phenylalanine-derived 2-azetidinones bearing a 4-carboxylate moiety that could be apt for additional interactions with the guanidine group of the Arg165/Arg166 residues of the viral protease. Some compounds within this series showed anti-HCMV activity at 10−50 μM, but rather high toxicity. The presence of aromatic 1-acyl groups and a certain hydrophobic character in the region of the 4-carboxylate were stringent requirements for anti-HCMV activity. To go a step ahead into the search for effective HCMV medicines, we then envisaged a series of noncovalent inhibitors by simple deletion of the carbonyl group in the β-lactam derivatives to provide the corresponding azetidines. This led to low micromolar inhibitors of HCMV replication, with 17 and 27 being particularly promising lead compounds for further investigation, although their toxicity still needs to be lowered.

With the aim of studying the contribution of the β II turn conformation at the side chain of didemnins to the bioactive conformation responsible for their antitumoral activity, conformationally restricted analogues of aplidine and tamandarin A, where the side chain dipeptide Pro⁸-N-Me-d-Leu⁷ is replaced with the spirolactam β II turn mimetic (5R)-7-[(1R)-1-carbonyl-3-methylbutyl]-6-oxo-1,7-diazaspiro[4.4]nonane, were prepared. Additionally, restricted analogues, where the aplidine (pyruvyl⁹) or tamandarin A [(S)-Lac⁹] acyl groups are replaced with the isobutyryl, Boc, and 2-methylacryloyl groups, were also prepared. These structural modifications were detrimental to cytotoxic activity, leading to a decrease of 1−2 orders of magnitude with respect to that exhibited by aplidine and tamandarin A. The conformational analysis of one of these spirolactam aplidine analogues, by NMR and molecular modeling methods, showed that the conformational restriction caused by the spirolactam does not produce significant changes in the overall conformation of aplidine, apart from preferentially stabilizing the trans rotamer at the pyruvyl⁹−spirolactam amide bond, whereas in aplidine both cis and trans rotamers at the pyruvyl⁹−Pro⁸ amide bond are more or less equally stabilized. These results seem to indicate a preference for the cis form at that amide bond in the bioactive conformation of aplidine. The significant influence of this cis/trans isomerism upon the cytotoxicity suggests a possible participation of a peptidylprolyl cis/trans isomerase in the mechanism of action of aplidine.

With the aim of reversing selectivity or antagonist/agonist functionality in the 5-(tryptophylamino)-1,3-dioxoperhydropyrido[1,2-c]pyrimidine-derived potent and highly selective CCK1 antagonists, a series of 4-benzyl and 4-methyl derivatives have been synthesized. Whereas the introduction of the benzyl group led, in all cases, to complete loss of the binding affinity, the incorporation of the methyl group gave a different result depending on the stereochemistry of the 1,3-dioxoperhydropyrido[1,2-c]pyrimidine scaffold. Thus, the introduction of the methyl group into the (4aS,5R)-diastereoisomers, giving a (4S)-configuration, produced a 3-fold increase in the CCK1 binding potency and selectivity. However, the same structural manipulation in the opposite (4aR,5S)-stereochemistry, leading to a (4R,4aR,5S)-configuration, produced reversal of the selectivity for CCK1 to the CCK2 receptors. The replacement of the Boc group at the tryptophan moiety by a 2-adamantyloxycarbonyl group also contributed to that reversal. The resulting compounds displayed moderate CCK2 antagonist activity in rat and human receptors, and a very small partial agonist effect on the production of inositol phosphate in COS-7 cells transfected with the wild-type human CCK2 receptor.

Compounds containing a novel tetracyclic ring system, a hybrid of tetrahydropyrrolo[2,3-b]indole and tetrahydroimidazo[1,2-a]indole, are synthesized via an acid-mediated stereospecific domino tautomerization of a tryptophan-derived α-amino nitrile. Characterization of these new compounds and preliminary studies on the reactivity of the tetracyclic heterocyclic system are reported.

To further define the pharmacophore of the potent and selective 5-(tryptophyl)amino-1,3-dioxoperhydropyrido[1,2-c]pyrimidine-based CCK1 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 CCK1 receptors. The thioxo-analogues 5a, 8a, 12a, and 12b showed functional CCK1 antagonist activity, inhibiting the CCK-8-stimulated amylase release from pancreatic acinar cells. The 1-thioxo analogue 5a, with subnanomolar affinity (IC50 = 0.09 × 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 CCK1 antagonists.

To establish structure−activity relationships a new series of analogues of the highly potent and selective CCK1 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 CCK1 receptor subtype. The 2-cyclohexyl and (2S)-1-naphthyl derivatives 18a and (2S)-20a have emerged as more potent and selective CCK1 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.

To improve our knowledge of the bioactive conformation of CCK1 antagonists, we previously described that replacement of the α-MeTrp residue of dipeptoids with the (2S,5S,11bR)-2-amino-3-oxohexahydroindolizino[8,7-b]indole-5-carboxylate (IBTM) skeleton, a probed type II‘ β-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]carbamoyl-3-oxo-2,3,5,6,11,11b-hexahydro-1H-indolizino[8,7-b]indole, 1a,b, showing high binding affinity and selectivity for CCK1 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 β-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 βHph residues are favorable for CCK1 receptor recognition. Moreover, selectivity for this receptor subtype is critically affected by the β-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 CCK1 receptors, restricted dipeptoid derivative 15a, incorporating the type II‘ IBTM mimetic, shows approximately 6-fold higher CCK1 selectivity than analogue 16a, with the type II mimetic. From these results, we propose that the presence of a β-turn-like conformation within the peptide backbone of dipeptoids could contribute to their bioactive conformation at the CCK1 receptor subtype. Concerning functional activity, compounds 15a and 16a behave as CCK1 receptor antagonists.

Analogues of the previously reported potent and highly selective CCK1 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 CCK1 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 CCK1 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.

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.