Design, Synthesis, and Characterization of Novel sn-1 Heterocyclic DAG-Lactones as PKC Activators.

DAG-lactones represent useful templates for the design of potent and selective C1 domain ligands for PKC isozymes. The ester moiety at the sn-1 position, a common feature in this template, is relevant for C1 domain interactions, but it represents a labile group susceptible to endogenous esterases. An interesting challenge involves replacing the ester group of these ligands while still maintaining biological activity. Here, we present the synthesis and functional characterization of novel diacylglycerol-lactones containing heterocyclic ring substituents at the sn-1 position. Our results showed that the new compound 10B12, a DAG-lactone with an isoxazole ring, binds PKCα and PKCε with nanomolar affinity. Remarkably, 10B12 displays preferential selectivity for PKCε translocation in cells and induces a PKCε-dependent reorganization of the actin cytoskeleton into peripheral ruffles in lung cancer cells. We conclude that introducing a stable isoxazole ring as an ester surrogate in DAG-lactones emerges as a novel structural approach to achieve PKC isozyme selectivity.

NMR study on the stabilization and chiral discrimination of sulforaphane enantiomers and analogues by cyclodextrins.

Sulforaphane (SFN), a phytochemical isolated from broccoli, is an important antitumoral compound with additional beneficial effect on other important diseases. However, the chemical instability of SFN has hampered its clinical use. In order to circumvent this problem, we report the first comparative study on the inclusion complexes of SFN and SFN homologues with different cyclodextrins by NMR spectroscopy. From this study it has been shown that α-CD is the most indicated cyclodextrin for the stabilization of SFN and SFN homologues, and that the highest affinity constant is that of the isothiocyanate obtained from the wasabi. Furthermore, the study of the inclusion complexes of α-CD and the non-natural SFN and analogues with S absolute configuration at sulfur shows for the first time that α-CD is able to discriminate between the two enantiomers, with the natural R enantiomers forming the inclusion complexes with higher affinity.

Exploring the influence of indololactone structure on selectivity for binding to the C1 domains of PKCα, PKCε, and RasGRP.

C1 domain-containing proteins, such as protein kinase C (PKC), have a central role in cellular signal transduction. Their involvement in many diseases, including cancer, cardiovascular disease, and immunological and neurological disorders has been extensively demonstrated and has prompted a search for small molecules to modulate their activity. By employing a diacylglycerol (DAG)-lactone template, we have been able to develop ultra potent analogs of diacylglycerol with nanomolar binding affinities approaching those of complex natural products such as phorbol esters and bryostatins. One current challenge is the development of selective ligands capable of discriminating between different protein family members. Recently, structure-activity relationship studies have shown that the introduction of an indole ring as a DAG-lactone substituent yielded selective Ras guanine nucleotide-releasing protein (RasGRP1) activators when compared to PKCα and PKCε. In the present work, we examine the effects of ligand selectivity relative to the orientation of the indole ring and the nature of the DAG-lactone template itself. Our results show that the indole ring must be attached to the lactone moiety through the sn-2 position in order to achieve RasGRP1 selectivity.

Studies on the diastereoselective oxidation of 1-thio-ß-D-glucopyranosides: synthesis of the usually less favoured R(S) sulfoxide as a single diastereoisomer.

A detailed study on the diastereoselective oxidation of 1-thio-ß-D-glucopyranosides is reported. It has been shown that the sense and the degree of stereochemical outcome of the oxidation are highly dependent on the substituent of the sulfur and on the protective group of the C2-OH. In the case of thioglycosides with a bulky aglycone, the mesylation of C2-OH has a significant effect on the stereochemical outcome of the oxidation, affording the usually less favoured RS sulfoxide as a single diastereoisomer. The absolute configuration of the final sulfinyl glycosides was ascertained by NMR analysis and corroborated by X-ray crystallography.

Sulforaphane homologues: Enantiodivergent synthesis of both enantiomers, activation of the Nrf2 transcription factor and selective cytotoxic activity.

Reported is an enantiodivergent approach for the synthesis of both enantiomers of sulforaphane (SFN) homologues with different chain lengths between the sulfinyl sulfur and the isothiocyanate groups and different substituents on the sulfinyl sulfur. The homologues were designed in order to unravel the effect of all the diversity elements included in sulforaphane's structure. The key step of the approach is the diastereoselective synthesis of both sulfinate ester epimers at sulfur, using as single chiral auxiliary the sugar derived diacetone-d-glucose. The approach allows the first synthesis of both enantiomers of 5-methylsulfinylpentyl isothiocyanate, and the biologically important 6-methylsulfinylhexyl isothiocyanate (6-HITC) found in Japanese horseradish, wasabi (Wasabia japonica). The ability of the synthesized compounds as inductors of phase II detoxifying enzymes has been studied by determining their ability to activate the cytoprotective transcription factor Nrf2. The cytotoxic activity of all the synthesized compounds against human lung adenocarcinoma (A549) and foetal lung fibroblasts (MRC-5) is also reported.

Structural determinants of efficacy at A3 adenosine receptors: modification of the ribose moiety.

We have found previously that structural features of adenosine derivatives, particularly at the N6- and 2-positions of adenine, determine the intrinsic efficacy as A3 adenosine receptor (AR) agonists. Here, we have probed this phenomenon with respect to the ribose moiety using a series of ribose-modified adenosine derivatives, examining binding affinity and activation of the human A3 AR expressed in CHO cells. Both 2'- and 3'-hydroxyl groups in the ribose moiety contribute to A3 AR binding and activation, with 2'-OH being more essential. Thus, the 2'-fluoro substitution eliminated both binding and activation, while a 3'-fluoro substitution led to only a partial reduction of potency and efficacy at the A3 AR. A 5'-uronamide group, known to restore full efficacy in other derivatives, failed to fully overcome the diminished efficacy of 3'-fluoro derivatives. The 4'-thio substitution, which generally enhanced A3 AR potency and selectivity, resulted in 5'-CH2OH analogues (10 and 12) which were partial agonists of the A3 AR. Interestingly, the shifting of the N6-(3-iodobenzyl)adenine moiety from the 1'- to 4'-position had a minor influence on A3 AR selectivity, but transformed 15 into a potent antagonist (16) (Ki = 4.3 nM). Compound 16 antagonized human A3 AR agonist-induced inhibition of cyclic AMP with a K(B) value of 3.0 nM. A novel apio analogue (20) of neplanocin A, was a full A3 AR agonist. The affinities of selected, novel analogues at rat ARs were examined, revealing species differences. In summary, critical structural determinants for human A3 AR activation have been identified, which should prove useful for further understanding the mechanism of receptor activation and development of more potent and selective full agonists, partial agonists and antagonists for A3 ARs.

Design, synthesis, and biological evaluation of aryloxyethyl thiocyanate derivatives against Trypanosoma cruzi.

As a continuation of our project aimed at the search for new and safe chemotherapeutic and chemoprophylactic agents against American trypanosomiasis (Chagas' disease), several drugs structurally related to 4-phenoxyphenoxyethyl thiocyanate (4) were designed, synthesized, and evaluated as antiproliferative agents against the parasite responsible for this disease, the hemoflagellated protozoan Trypanosoma cruzi. This thiocyanate derivative was previously shown to be an effective and potent agent against T. cruzi proliferation. Several drugs possessing thiocyanate groups proved to be effective growth inhibitors of T. cruzi growth. Among the designed compounds, it is important to point out the extremely potent activity shown by 11, 23, 38, 53, 90, 99, and 117 against the epimastigote forms of the parasite. All of them exhibited IC(50) values in the low micromolar range, and these values were comparable with those presented by our lead drug 4 and ketokonazole, a well-known antiparasitic agent. The activity displayed by the nitrogen-containing derivative 90 was very promising with IC(50) values of 3.3 microM. Several other thiocyanate derivatives also proved to be very potent inhibitors of the multiplication of T. cruzi epimastigotes, such as compounds 28, 33, 43, 48, 56, 61, 66, 71, 76, and 124. Compound 43 resulted in being a promising drug because it was also very effective against amastigotes, the clinically more relevant form of the parasite. This compound was 3-fold more potent than 4, while 11 showed nearly the same activity as our lead drug against intracellular T. cruzi. It was very surprising that the experimental juvenoid 124, although fairly devoid of activity against epimastigotes, was very effective against intracellular amastigotes growing in myoblasts. The rest of the designed compounds showed a broad degree of inhibitory action, from moderately active drugs to drugs almost devoid of antiparasitic activity. Compound 43 is an interesting example of an effective antichagasic agent that presents excellent prospectives not only as a lead drug but also to be used for further in vivo studies.