Antonio Varnavas

Anthranilic acid derivatives: a new class of non-peptide CCK1 receptor antagonists.

Having successfully obtained new CCK(1) ligands holding appropriate groups on the anthranilic acid dimer used as molecular scaffold we were interested in increasing their micromolar affinity for the CCK(1) receptors by modifying the spatial relationship of the main pharmacophoric groups. Since, we have proposed simplified analogues reducing the anthranilic acid dimer to a monomer. In this stage of our research program we have prepared and tested on CCK receptors a series of N-substituted anthranilic acid derivatives keeping a Phe residue at the C-terminal site. The indole-2-carbonyl group imparts the best CCK(1) receptor binding affinity (compound 1: IC(50)=197.5 nM) while a sharp decrease in binding affinity is observed for the other indole containing derivatives. Moreover, in order to support the different binding behaviour observed for the synthesized compounds, a conformational investigation was carried out. Finally, on the basis of the main pharmacophoric groups of the obtained new lead compound (1) (coded VL-0395) a receptor binding hypothesis has been provided.

Anthranilic acid based CCK1 receptor antagonists: blocking the receptor with the same 'words' of the endogenous ligand.

The anthranilic acid diamides represent the more recent class of nonpeptide CCK(1) receptor antagonists. This class is characterized by the presence of anthranilic acid, used as a molecular scaffold, and two pharmacophores selected from the C-terminal tetrapeptide of CCK. The lead compound coded VL-0395, endowed with sub-micromolar affinity towards CCK(1) receptors, was characterized by the presence of Phe and 2-indole moiety at the C- and N-termini of anthranilic acid, respectively. Herein we describe the first step of the anthranilic acid C-terminal optimization using, instead of Phe, aminoacids belonging to the primary structure of CCK-8 and other not coded residues. Thus we demonstrate that the CCK(1) receptor affinity depends on the nature of the aminoacidic side chain as well as that the free carboxy group of the alpha-aminoacids is crucial for the binding. The R enantiomers of the most active compounds represent the eutomers of this class of antagonists confirming thus the stereo preference of the receptor. Moreover this SAR study demonstrates that the receptor binding pocket, that host the aminoacidic side chain, results much more tolerant respect to that accommodating the indole ring. As a result, an appropriate variation of the aminoacidic side chain could provide a better CCK(1) receptor affinity diorthosis.

Properties of methoxy(polyethylene glycol)-lipase from Candida rugosa in organic solvents

Abstract Lipase from Candida rugosa was modified with methoxy(polyethylene glycol)– p -nitrophenyl carbonate (NPC–mPEG). A total of 34 out of 35 lysine chains were substituted, and the modified lipase retained 98% of the hydrolytic activity of the non-modified enzyme. mPEG-lipase is perfectly soluble in organic solvent and efficiently catalyzes the esterification of lauric acid, with nearly 100% of yield over 6 h in toluene. Moreover, mPEG-lipase exhibits a preference for the S -isomer over the R -isomer in the stereoselective resolution of racemic naproxen.

Twenty years of non-peptide CCK1 receptor antagonists: all that glitters is not gold

During the last 20 years, pharmaceutical industry and academic efforts have led to several structurally unrelated classes of non-peptide cholecystokinin-1 receptor (CCK1-R) antagonists. Due to the lack of high resolution structure of CCK1-R and its peptide ligand, different strategies to design antagonists have been adopted. The rational design, based on conformational studies of the endogenous ligand, has provided the so-called ‘peptoid’ derivatives and conformationally restricted peptoids, whereas all of the other non-peptide antagonists derived by empirical and/or conventional approaches, including the chemical manipulation of natural products, disconnection strategies and the use of single key amino acids. All of these strategies include a final optimisation step of the obtained lead compound performed by chemical modifications. The CCK1-R antagonists, in addition to providing a better characterisation of the CCK1-R receptor subtype as pharmacological tools and improving the knowledge of the physiological and pathological role(s) of CCK, may possess therapeutic potential in humans. As the complex biological effects of CCK mediated by CCK1-R in the CNS are not yet completely established, the therapeutic potential of these antagonists at present is limited to the gastrointestinal system disorders. Up until now, loxiglumide and its R-enantiomer (dexloxiglumide) along with lintitript are the CCK1-R antagonists at the most advanced stage of clinical research in gastroenterology.

Synthesis of new anthranilic acid dimer derivatives and their evaluation on CCK receptors.

We have described previously an innovative bond disconnection strategy of asperlicin, a naturally occurring CCK receptor antagonist, leading to anthranilic acid dimer and tryptophan synthons. We have also demonstrated that when the tryptophan residue is connected to the C- or N-terminal sides of the anthranilic acid dimer, compounds with similar micromolar CCK-A receptor affinities are obtained. In order to investigate the binding effects of different N-terminal substitution, in this paper we describe a new series of anthranilic acid dimer derivatives, characterized by the presence of the tryptophan residue in the C-terminus of the dimer. Among the compounds synthesized, the N-1H-indol-3-propionyl derivative exhibited an improved, at the micromolar range, affinity for the CCK-A receptor in comparison to that of either, the N-unsubstituted derivative and asperlicin. The lead compound emerging from this key step of our investigation represents the new starting point for the development of a new class of CCK-A receptor ligands.

C-terminal anthranoyl-anthranilic acid derivatives and their evaluation on CCK receptors.

A series of C-terminal anthranoyl-anthranilic acid derivatives arising from a strict bond disconnection approach of asperlicin were synthesized and examined for their CCK receptor affinities. These compounds represent the second step of our investigation directed toward the search for alternative substructures of asperlicin as a starting point for the development of a new class of CCK ligands. The obtained micromolar affinities for CCK-A rather than CCK-B receptor confirm that the anthranilic acid dimer represents a useful template for the development of selective CCK-A receptor ligands.

Improved activity in acidic media of immobilized lysozyme

Polyethylene glycols (PEGs) of various chain length were used tocrosslink lysozyme onto an insoluble support such as oxirane. A very high degree of modification and noinactivation of lysozyme were obtained with PEG 20000, but enzymatic activity increased up to 20 times at pH 3.0, at which point the activity of the native enzyme was lower when using Leuconos tok oenus as a macromolecular substrate.

New anthranilic acid based antagonists with high affinity and selectivity for the human cholecystokinin receptor 1 (hCCK1-R).

The anthranilic acid diamides represent the most recent class of nonpeptide CCK(1) receptor (CCK(1)-R) antagonists. Herein we describe the second phase of the anthranilic acid C-terminal optimization using nonproteinogenic amino acids containing a phenyl ring in their side chain. The Homo-Phe derivative 2 (VL-0797) enhanced 12-fold the affinity for the rat CCK(1)-R affinity and 15-fold for the human CCK(1)-R relative to the reference compound 12 (VL-0395). The eutomer of 2 (6) exhibited a nanomolar range affinity toward the human CCK(1)-R and was at least 400-fold selective for the CCK(1)-R over the CCK(2)-R. Molecular docking in the modeled CCK(1)-R and its validation by site-directed mutagenesis experiments showed that the 6 binding site overlaps that occupied by the C-terminal bioactive region of the natural agonist CCK. Owing to their interesting properties, new compounds provided by this study represent a solid basis for further advances aimed at synthesis of clinically valuable CCK(1)-R antagonists.

2D-QSAR and 3D-QSAR/CoMFA analyses of the N-terminal substituted anthranilic acid based CCK1 receptor antagonists: ‘Hic Rhodus, hic saltus’

A research is presented on quantitative structure-activity relationship (QSAR) studies on the more recent class of non-peptidic CCK(1) receptor antagonists. Our results suggest that the balance of hydrophobicity and volume dependent polarizability term plays a key role in the antagonism of CCK(1) receptor. The size of the substitution of ligands at particular position which induce steric fit is crucial as well as their hydrophobic contribution. Indicator variables were used after the best model was found to account for the usual structural features. The CoMFA results show a good variance explanation and the best self-predictivity is slightly lower than 60% with both leave-one-out and random-group methods. The CoMFA molecular fields showed the importance of steric hindrance of the substituent. From the GRIND models it can be deduced that the shape differences of the molecules are secondary in the regulation of the activity, or better, that their polar substituents are capable of occupying the same zones of the space in the most of the cases.

Antineoplastic action ofp-(3-methyl-1-triazeno)benzoic acid potassium salt, a monomethyl derivative of the antimetastatic compound DM-COOK

SummaryThe antitumor and antimetastatic effects ofp-(3-methyl-1-triazeno)benzoic acid potassium salt (MM-COOK) as compared with those of the parent 3,3-dimethyl derivative (DM-COOK) were examined using Lewis lung carcinoma, MCa mammary carcinoma of the CBA mouse and TLX5 lymphoma. Similarly to DM-COOK, MM-COOK reduces metastasis formation and significantly prolongs the survival of mice bearing the Lewis lung carcinoma when given at a daily dose corresponding to one-half that of DM-COOK. Unlike DM-COOK, MM-COOK exhibits significant cytotoxicity to metastatic foci and pronounced inhibition of primary tumor development. MM-COOK also causes cytotoxic effects on TLX5 lymphoma cell growing in the peritoneal cavity, even when used at low doses. The antimetastatic effects observed in mice bearing MCa mammary carcinoma are unrelated to the inhibition of primary tumor growth and are more likely due to the selection of clones endowed with lower metastatic ability. It appears that MM-COOK exhibits the same antineoplastic activity as DM-COOK, but the former does so at a lower daily dose and produces interesting cytotoxic effects other than those reflecting its antimetastatic properties. It thus seems to be a valid alternative to DM-COOK, in view of the possible introduction of newer aryltriazenes into clinical use.