ortho-HalogenNaphthaleinsasSpecificInhibitorsof Lactobacillus casei ThymidylateSynthase.ConformationalPropertiesand BiologicalActivity Stefano Ghelli, a,y Marcella Rinaldi, b Daniela Barlocco, c Arianna Gelain, c Piergiorgio Pecorari, b Donatella Tondi, b Giulio Rastelli b and Maria Paola Costi b, * a Dipartimento di Chimica, Universita’ degli Studi di Modena e Reggio Emilia, Via Campi 183 41100, Modena, Italy b Dipartimento di Scienze Farmaceutiche, Universita’ degli Studi di Modena e Reggio Emilia, Via Campi 183, 41100 Modena, Italy c Istituto Chimico Farmaceutico, Universita ` degli Studi di Milano, Viale Abruzzi 42, 20131 Milan, Italy Received 3 July 2002; revised 1 October 2002; accepted 18 October 2002 Abstract—Thymidylate synthase (TS) (EC 2.1.1.45), an enzyme involved in the DNA synthesis of both prokaryotic and eukaryotic cells, is a potential target for the development of anticancer and antinfective agents. Recently, we described a series of phthalein and naphthalein derivatives as TS inhibitors. These compounds have structures unrelated to the folate (Non-Analogue Antifolate Inhibitors, NAAIs) and were selective for the bacterial versus the human TS (hTS). In particular, halogen-substituted molecules were the most interesting. In the present paper the halogen derivatives of variously substituted 3,3-bis(4-hydroxyphenyl)-1H,3H- naphtho[2,3-c]furan-1-one (1–5) and 3,3-bis(4-hydroxyphenyl)-1H,3H-naphtho[1,8-c,d]pyran-1-one (6–14) were synthesized to investigate the biological effect of halogen substitution on the inhibition and selectivity for the TS enzymes. Conformational prop- erties of the naphthalein series were explored in order to highlight possible differences between molecules that show species-specific biological profile with respect to non species-specific ones. With this aim, the conformational properties of the synthesized com- pounds were investigated by NMR, in various solvents and at different temperatures, and by computational analysis. The apparent inhibition constants (K i ) for Lactobacillus casei TS (LcTS) were found to range from 0.7 to 7.0 mM, with the exception of the weakly active iodo-derivatives (4, 10, 13); all] the compounds were poorly active against hTS. The di-halogenated compounds 7, 8, 14 showed the highest specificity towards LcTS, their specificity index (SI) ranging between 40 and > 558. The di-halogenated 1,8-naphthalein derivatives (7–10) exhibited different conformational properties with respect to the tetra-haloderivatives. Though a clear explanation for the observed specificity by means of conformational analysis is difficult to find, some interesting confor- mational effects are discussed in the context of selective recognition of the compounds investigated by the LcTS enzyme. # 2003 Elsevier Science Ltd. All rights reserved. Introduction Thymidylate synthase (TS) (EC 2.1.1.45), an enzyme involved in the DNA synthesis of both prokaryotic and eukaryotic cells, is a potential target for the develop- ment of antibacterial, antimycotic, anticancer and, recently, antiviral agents. 1 6 TS catalyzes the conversion of deoxyuridine monophosphate (dUMP) to deoxy- thymidine monophosphate (dTMP) by a reductive methylation involving N 5 ,N 10 -methylentetrahydrofolate (mTHF) as cofactor. 1,2 In the absence of dTMP formation via Thymidine Kinase (TK) (salvage pathway), the reaction catalyzed by TS is the rate-limiting step in the DNA synthesis, for it is the sole de novo pathway for the synthesis of thymidine triphosphate (dTTP). Inhibi- tion of TS would therefore prevent cell multiplication. Compounds structurally related to the substrate (dUMP) or to the cofactor (mTHF) have been devel- oped as classical antimetabolites. 3 A huge number of derivatives have been tested and some are on clinical trial or on the market for anticancer therapy, such as FdUMP (5-fluoro-2 0 -deoxyuridine-5 0 -monophosphate) and the folate analogues ZD1694, BW1843U89 (Fig. 1). These compounds prevent the substrate or the cofactor from binding the TS active site and, as a consequence, they block the synthesis of dTMP. Nevertheless, the 0968-0896/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved. PII: S0968-0896(02)00541-2 Bioorganic & Medicinal Chemistry 11 (2003) 951–963 *Corresponding author. Tel.: +39-059-205-5134; fax: +39-059-205- 5131; e-mail: costimp@mail.unimo.it y Present address: Spin, Via Tamagno 3, Rubiera, 42048 Reggio Emilia, Italy.