Lucia Lassiani

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.

Effects of isomeric aryldimethyltriazenes on lewis lung carcinoma growth and metastases in mice

The effects of the ortho and para isomers of aryldimethyltriazeno carboxylic and benzensulfonic acids and amides have been examined in mice bearing subcutaneous Lewis lung carcinoma. The toxicity of these compounds varies widely and does not correlate with the effects on tumor progression. The growth of subcutaneous primary tumor is unaffected or only marginally inhibited by all the tested compounds, while a marked depression in the formation of spontaneous lung metastasis is observed, with the exception of the sodium salt of the ortho benzensulfonic acid. The compounds showing the greatest activity on metastasis are the hydrosoluble salts of the ortho and para carboxylic acid and the para sulfonamido derivative, indicating that, in addition to the activity reported for the potassium salt of the para carboxylic acid, also its ortho isomer and the para sulfonamido derivative possess selective and pronounced antimetastatic effects. No correlation can be found between the reported effects on tumor progression and physicochemical parameters of the triazenes tested.

Mechanism of the antileukemic effects of 1-p-carboxamidophenyl3,3-dimethyltriazene and its in vitro metabolites☆

DM-CONH2, a dimethyltriazene active in prolonging the survival time of mice bearing TLX5 lymphoma, requires metabolic activation by liver homogenate supernatant and cofactors in order to exert in vitro cytotoxic effects on the same tumor cells, as determined by in vivo bioassay of their viability. From the examination of the metabolites produced during these in vitro experiments, it is found that in vitro cytotoxicity is attributable to the generation of MM-CONH2 by oxidative N-demethylation of DM-CONH2. Also the generation of DM-COO is observed, although this compound is not cytotoxic in vitro. The in vivo effects of DM-CONH2 and CM-COOK on TLX5 lymphoma are not caused exclusively by cytotoxic effects of the drugs, since they are evident also when no reduction in the number or viability of peritoneal tumor cells is evident, whereas these parameters are significantly reduced by MM-CONH2. The increase in survival time of mice bearing TLX5 lymphoma caused by the dimethyltriazenes used appears to be caused by the drugs without being subjected to metabolic activation, with a mechanism different from cytotoxicity for tumor cells.

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.

Effects of an inducer and an inhibitor of hepatic metabolism on the antitumor action of dimethyltriazenes.

SummaryTo investigate the role of monomethyltriazenes as the active metabolites of antitumor dimethyltriazenes, the in vivo simultaneous treatment with an inducer (phenobarbital, PB) or an inhibitor (carbon tetrachloride, CCl4) of hepatic drug metabolism was examined in mice bearing Lewis lung carcinoma. Treatment with PB or CCl4 with the dosage and schedules employed proved to be effective in markedly modifying the N-demethylation of the three dimethyltriazenes tested, as had been determined in vitro. No unambiguous increase by PB, or decrease by CCl4, which might theoretically be expected if metabolic conversion to monomethyltriazenes was involved, was observed for the antitumor and antimetastatic activity of dimethyl-triazenes. At the same time, a difference was noted between the effects on primary tumors and those on metastases. These data support the view that generalizations on the relevance of monomethyltriazenes as the active metabolites responsible for the antitumor and antimetastatic activity of dimethyltriazenes may not be valid.