Daniele D’Alonzo

Synthesis and evaluation of folate-based chlorambucil delivery systems for tumor-targeted chemotherapy.

The development of tumor-targeting drug delivery systems, able to selectively transport cytotoxic agents into the tumor site by exploiting subtle morphological and physiological differences between healthy and malignant cells, currently stands as one of the most attractive anticancer strategies used to overcome the selectivity problems of conventional chemotherapy. Owing to frequent overexpression of folate receptors (FRs) on the surface of malignant cells, conjugation of cytotoxic agents to folic acid (FA) via suitable linkers have demonstrated to enhance selective drug delivery to the tumor site. Herein, the chemical synthesis and biological evaluation of two novel folate-conjugates bearing the anticancer agent chlorambucil (CLB) tethered to either an aminoether (4,7,10-trioxa-1,13-tridecanediamine) or a pseudo-β-dipeptide (β-Ala-ED-β-Ala) linker is reported. The two drug delivery systems have been prepared in high overall yields (54% and 34%) through straightforward and versatile synthetic routes. Evaluation of cell specificity was examined using three leukemic cell lines, undifferentiated U937 (not overexpressing FRs, FR(-)), TPA-differentiated U937 (overexpressing FRs, FR(+)), and TK6 (FR(+)) cells. Both conjugates exhibited high specificity only to FR(+) cells (particularly TK6), demonstrating comparable antitumor activity to CLB in its free form. These data confirm the reliability of folate-based drug delivery systems for targeted antitumor therapy; likewise, they lay the foundations for the development of other folate-conjugates with antitumor potential.

Highly Stereoselective de Novo Synthesis of l-Hexoses

An efficient and general de novo synthetic route to enantiomerically pure L-hexoses has been accomplished starting from the heterocyclic homologating agent 5,6-dihydro-1,4-dithiin-2-yl[(4-methoxybenzyl)oxy]methane and methyl alpha,beta-isopropylidene-L-glycerate. The sugar scaffold was constructed by an acid-catalyzed domino reaction, which enabled selective preparation of either methyl 2,3-dideoxy-alpha-L-threo-hex-2-enopyranosides or 1,6-anhydro-2,3-dideoxy-beta-L-threo-hex-2-enopyranose as key intermediates. The subsequent double bond functionalization by syn or anti dihydroxylation reactions allowed introduction of the remaining stereogenic centers, leading to desired orthogonally protected L-hexopyranosides with a high degree of diastereoselectivity and with very good overall yields. These and previous results (based on the use of the corresponding L-erythro epimers) contribute to make our approach general and place it among the few methods able to synthesize the whole series of the rare L-hexoses.

A combined fermentative-chemical approach for the scalable production of pure E. coli monophosphoryl lipid A

Lipid A is the lipophilic region of lipopolysaccharides and lipooligosaccharides, the major components of the outer leaflet of most part of Gram-negative bacteria. Some lipid As are very promising immunoadjuvants. They are obtained by extraction from bacterial cells or through total chemical synthesis. A novel, semisynthetic approach to lipid As is ongoing in our laboratories, relying upon the chemical modification of a natural lipid A scaffold for the fast obtainment of several other lipid As and derivatives thereof. The first requisite for this strategy is to have this scaffold available in large quantities through a scalable process. Here, we present an optimized fed-batch fermentation procedure for the gram-scale production of lipid A from Escherichia coli K4 and a suitable phenol-free protocol for its purification. A study for regioselective de-O-phosphorylation reaction was then performed to afford pure monophosphoryl lipid A with an attenuated endotoxic activity, as evaluated by cytokine production in human monocytic cell line THP-1 in vitro. The reported method for the large-scale obtainment of monophoshoryl lipid A from the fed-batch fermentation broth of a recombinant strain of E. coli may permit the access to novel semisynthetic lipid A immunoadjuvant candidates.

Highly Stereoselective Synthesis of Lamivudine (3TC) and Emtricitabine (FTC) by a Novel N-Glycosidation Procedure.

The combined use of silanes (Et3SiH or PMHS) and I2 as novel N-glycosidation reagents for the synthesis of bioactive oxathiolane nucleosides 3TC and FTC is reported. Both systems (working as anhydrous HI sources) were devised to act as substrate activators and N-glycosidation promoters. Excellent results in terms of chemical efficiency and stereoselectivity of the reactions were obtained; surprisingly, the nature of the protective group at the N4 position of (fluoro)cytosine additionally influenced the stereochemical reaction outcome.

N-Butyl-l-deoxynojirimycin (l-NBDNJ): Synthesis of an Allosteric Enhancer of α-Glucosidase Activity for the Treatment of Pompe Disease

The highly stereocontrolled de novo synthesis of l-NBDNJ (the unnatural enantiomer of the iminosugar drug Miglustat) and a preliminary evaluation of its chaperoning potential are herein reported. l-NBDNJ is able to enhance lysosomal α-glucosidase levels in Pompe disease fibroblasts, either when administered singularly or when coincubated with the recombinant human α-glucosidase. In addition, differently from its d-enantiomer, l-NBDNJ does not act as a glycosidase inhibitor.