Mario Loriga

Discovery of potent pteridine reductase inhibitors to guide antiparasite drug development

Pteridine reductase (PTR1) is essential for salvage of pterins by parasitic trypanosomatids and is a target for the development of improved therapies. To identify inhibitors of Leishmania major and Trypanosoma cruzi PTR1, we combined a rapid-screening strategy using a folate-based library with structure-based design. Assays were carried out against folate-dependent enzymes including PTR1, dihydrofolate reductase (DHFR), and thymidylate synthase. Affinity profiling determined selectivity and specificity of a series of quinoxaline and 2,4-diaminopteridine derivatives, and nine compounds showed greater activity against parasite enzymes compared with human enzymes. Compound 6a displayed a Ki of 100 nM toward LmPTR1, and the crystal structure of the LmPTR1:NADPH:6a ternary complex revealed a substrate-like binding mode distinct from that previously observed for similar compounds. A second round of design, synthesis, and assay produced a compound (6b) with a significantly improved Ki (37 nM) against LmPTR1, and the structure of this complex was also determined. Biological evaluation of selected inhibitors was performed against the extracellular forms of T. cruzi and L. major, both wild-type and overexpressing PTR1 lines, as a model for PTR1-driven antifolate drug resistance and the intracellular form of T. cruzi. An additive profile was observed when PTR1 inhibitors were used in combination with known DHFR inhibitors, and a reduction in toxicity of treatment was observed with respect to administration of a DHFR inhibitor alone. The successful combination of antifolates targeting two enzymes indicates high potential for such an approach in the development of previously undescribed antiparasitic drugs.

Synthesis and in vitro antitumor activity of new quinoxaline derivatives

A series of novel 5,7-diamino-3-phenyl-2-benzylamino, 2-phenoxy, and 2-thiophenyl substituted quinoxalines has been designed, synthesized and evaluated for their in vitro antitumor activity towards cell lines of nine different types of human cancers. Some of these compounds exhibited inhibitory effects on the growth of a wide range of cancer cell lines generally at 10(-6) M, in some cases at 10(-7) M and 10(-8) M concentrations. Within this series the benzylamino quinoxaline derivatives 1b-7b were the most active, whereas compound 2c showed the highest MG_MD value (-5.66).

Synthesis of substituted 2-ethoxycarbonyl- and 2-carboxyquinoxalin-3-ones for evaluation of antimicrobial and anticancer activity

A series of variously substituted quinoxalin-3-ones bearing an ethoxycarbonyl or carboxy group in the C-2 position has been prepared and their structures proved by 1H NMR spectroscopy. The obtained compounds were investigated in vitro for antimicrobial and anticancer activities. Preliminary results showed a moderate activity against a few strains of bacteria but no significant anticancer and anti-HIV activity.

Preparation and biological evaluation of 6/7-trifluoromethyl(nitro)-, 6,7-difluoro-3-alkyl (aryl)-substituted-quinoxalin-2-ones: Part 3

A new series of quinoxalinones 6/7-trifluoromethyl or nitro- and 6,7-difluoro substituted bearing various side-chains (alkyl, halogenoalkyl, benzyl and phenyl groups) at C-3 of the ring system was synthesized and submitted to preliminary in vitro evaluation for antibacterial, antifungal, antimycobacterial, anticancer and anti-HIV activities. Results of these screenings showed that compounds 23-28 exhibited a good inhibition activity against various strains of Candida. Compound 24 showed also an interesting in vitro anticancer activity.

Quinoxalin-2-ones: Part 5: Synthesis and antimicrobial evaluation of 3-alkyl-, 3-halomethyl- and 3-carboxyethylquinoxaline-2-ones variously substituted on the benzo-moiety

A new series of 3-alkyl-, 3-trifluoromethyl-, 3-carboxyethyl- and 3-bromomethylquinoxaline-2-ones and 2,3-bis(bromomethyl)quinoxalines bearing Cl, CF3, morpholine on the benzo-moiety, were synthesised and submitted to a preliminary in vitro evaluation for antibacterial and anticandida activities. Results of the screening showed that compounds 9b, 14b and 19b (MIC=62.5 microg/ml) and 10b (MIC=15.6 microg/ml) were the most active against Vibrio alginolyticus.

Quinoxaline chemistry. Part 16. 4-Substituted anilino and 4-substituted phenoxymethyl pyrrolo[1,2-a]quinoxalines and N-[4-(pyrrolo[1,2-a]quinoxalin-4-yl)amino and hydroxymethyl]benzoyl glutamates. Synthesis and evaluation of in vitro biological activity

Twenty eight pyrrolo[1,2-a]quinoxalines bearing at position 4 various substituents related to the moieties present in classical and non classical antifolic agents were prepared and evaluated in vitro for antiproliferative activity. In an in vitro screening performed at NCI, several compounds emerged as potent antiproliferative agents at concentrations ranging between 10 and 100 microM. Interestingly, some of these compounds proved active also against bovine and murine DHFR (Farmaco 53 (1998) 480). More recently, a compound of classical antifolate type has been reported to be a potent inhibitor of hDHFR in vitro (Farmaco 58 (2003) 51). We then synthesized new derivatives that, in our hands, were endowed with in vitro antiproliferative activities as low as 3.4 microM against a panel of cell lines derived from hematological and solid tumours. In addition, a complete screening of cytotoxicity, antiretroviral HIV-1 and antimicrobial activity has been carried out.

Quinoxaline chemistry: Part 13: 3-carboxy-2-benzylamino-substituted quinoxalines and N -[4-[(3-carboxyquinoxalin-2-yl) aminomethyl]benzoyl]- L -glutamates: synthesis and evaluation of in vitro anticancer activity

Among a new series of 28 3-carboxy or carbethoxy quinoxalines bearing a substituted benzylamino or N-[4-(aminomethyl)benzoyl]glutamate group on position 2 of the ring and various substituents at C-6, 7 positions, 21 were selected at the National Cancer Institute for evaluation of their in vitro anticancer activity. The results obtained seem to confirm that the carboxy or carbethoxy group on position 3 is not helpful, with a few exceptions, for the anticancer activity.

Synthesis and evaluation for biological activity of 3-alkyl and 3-halogenoalkyl-quinoxalin-2-ones variously substituted. Part 4

A new series of 3-isopropyl-, 3-trifluoromethyl- and 3-bromomethylquinoxaline-2-ones variously substituted on the benzo-moiety were synthesized and submitted to a preliminary in vitro evaluation for antibacterial, antifungal and anti-HIV activities. Furthermore, all compounds were also tested for cytotoxicity. Results of the screening showed that compound 10 exhibits moderate antimicrobial activity against Staphylococcus aureus (MIC = 33 microM), and that 25 and 26 showed interesting cytotoxicity versus mock-infected MT-4 cells. All the other compounds were inactive.

Quinoxaline chemistry. Part 11. 3-Phenyl-2[phenoxy- and phenoxymethyl]-6(7) or 6,8-substituted quinoxalines and N-[4-(6(7)-substituted or 6,8-disubstituted-3-phenylquinoxalin-2-yl)hydroxy or hydroxymethyl] benzoylglutamates. Synthesis and evaluation of in vitro anticancer activity and enzymatic inhibitory activity against dihydrofolate reductase and thymidylate synthase.

Twenty-four out of twenty-nine quinoxalines were selected at the National Cancer Institute, Bethesda, Md, USA, for in vitro anticancer screening. Among these, 10 derivatives exhibited high values of percent tumor growth inhibition at a concentration of 10(-4) M in all cancer cell lines. Four of these compounds maintained these values at 10(-5) M, whereas a certain number exhibited significant values of percent inhibition at the most diluted concentrations (10(-8)-10(-6) M). Inhibitory activity against dihydrofolate reductase (DHFR) (bovine and rat liver) was determined for the most active compounds. This test showed that this type of quinoxaline exhibited an appreciable activity in comparison with the previously described aza analogues. In the other test (Lactobacillus casei, thymidylate synthase (TS), human HTS) no or poor activity was detected in both series of compounds.

Quinoxaline chemistry: Part 9: Quinoxaline analogues of trimetrexate (TMQ) and 10-propargyl-5,8-dideazafolic acid (CB 3717) and its precursors: synthesis and evaluation of in vitro anticancer activity

Eighteen quinoxalines bearing a methyleneanilino or methyleneaminobenzoylglutamate group on position 6 of the ring and various lipophilic substituents on positions 2 and 3 were prepared in order to discover if their structural analogy with both trimetrexate (TMQ) and 10-propargyl-5,8-dideazafolic acid (CB 3717) might display in vitro anticancer activity. Among these, 12 compounds were selected at the National Cancer Institute, Bethesda, MD, USA; they exhibited moderate (4b,d,i,l,m and 8) to strong (4f,h and 5a,e) cell-growth inhibition at a concentration of 10(-4) M. Interesting selectivities were also recorded between 10(-8) and 10(-6) M. These analogues proved to be less potent inhibitors of tumor cells than other classical and non-classical antifolate analogues previously described by us.