Several derivatives of 4,5-disubstituted imidazole, 2,4,5-trisubstituted pyrimidine, 2-substituted purine, thiazolo3,2-apurine, 1,3thiazino3,2-apurine, thiazolo2,3-ipurine, 1,3thiazino2,3-ipurine, ...and 6-substituted pyrazolo3,4-dpyrimidine were synthesized and tested as inhibitors of the xanthine oxidase enzyme. Of those, some 4-(acylamino)-5-carbamoylimidazoles and 2-thioalkyl-substituted purines exhibited very good inhibitory activity, being at least 500 times more effective than allopurinol. The ineffectiveness of 6-n-alkylpyrazolo3,4-dpyrimidines is imputable to the alkyl chain which could hinder the coordination with molybdenum according to the known mechanism for the binding of the inhibitor allopurinol; the effectiveness of imidazole derivatives, by contrast with the ineffectiveness of 4,5-diamino-2-(thioalkyl)-6-hydroxypyrimidines, indicates the relative importance of the five-membered ring in the interaction with the enzyme. Moreover, the marked effectiveness of the angularly-cyclized 1,3thiazino2,3-ipurinones, which constitute an interesting new class of inhibitors, together with the weak activity of linearly-cyclized derivatives, allowed us to characterize more precisely the lipophilic region of the enzyme facing the N(1)−C(2) positions of the substrate hypoxanthine.
The conformations of a set of phthalein derivatives with bacterial thymidylate synthase (TS) inibitory activity were investigated by
1H NMR spectra, performed at both room and low temperature, and by ...quantum chemical calculations. Since the crystal structure of the binary complex of phenolphthalein with the enzyme is known, we set out to study the conformation of various of its analogues in solution in order to observe the effects of the substituents on the phenolic rings, of the α-naphthol derivative and of the rigid analogue, fluorescein, and compare the results with the X-ray crystal structure studies. A relationship between the chemical shift of the proton on C4 (H4) of the phthalidic ring and the averaged angle formed by the phthalidic and the aromatic ring planes was found in which the most perpendicular conformations have the lowest H4 chemical shift values. At room temperature, the rotational freedom of all the studied compounds was similar, while at lower temperature the naphthol derivative assumed a partially blocked conformation. Finally, a qualitative relationship between the inhibitory properties of the compounds and their conformations is discussed.
1H NMR and quantum chemical calculations have been performed to study the conformations of a set of phthalein derivatives acting as thymidylate synthase inhibitors. The relationships between the conformational results and the biological activity of the compounds has also been proposed.
Thymidylate synthase (TS) is a well-recognized target for anticancer chemotherapy. Due to its key role in the sole de novo pathway for thymidylate synthesis and, hence, DNA synthesis, it is an ...essential enzyme in all life forms. As such, it has been recently recognized as a valuable new target against infectious diseases. There is also a pressing need for new antimicrobial agents that are able to target strains that are drug resistant toward currently used drugs. In this context, species specificity is of crucial importance to distinguish between the invading microorganism and the human host, yet thymidylate synthase is among the most highly conserved enzymes. We combine structure-based drug design with rapid synthetic techniques and mutagenesis, in an iterative fashion, to develop novel antifolates that are not derived from the substrate and cofactor, and to understand the molecular basis for the observed species specificity. The role of structural and computational studies in the discovery of nonanalog antifolate inhibitors of bacterial TS, naphthalein and dansyl derivatives, and in the understanding of their biological activity profile, are discussed.
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-naphtho2,3-
cfuran-1-one (
1–
5) and 3,3-bis(4-hydroxyphenyl)-1H,3H-naphtho1,8-c,dpyran-1-one (
6–
14) were synthesized to investigate the biological effect of halogen substitution on the inhibition and selectivity for the TS enzymes. Conformational properties 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 compounds 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
μM, 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 conformational effects are discussed in the context of selective recognition of the compounds investigated by the LcTS enzyme.
With the aim of investigating the specificity of 1,8-naphthalein derivatives towards
Lactobacillus casei thymidylate synthase with respect to human Thymidylate Synthase two series of variously substituted derivatives of 3,3-bis (4-hydroxyphenyl)-
1H,
3H-naphtho2,3-
cfuran-1-one and 3,3-bis(4-hydroxyphenyl)-
1H,
3H-naphtho1,8-
c,dpyran-1-one were synthesized. The conformational properties of the compounds were studied through
1H NMR and quantum chemical calculations.
A new set of phthalein derivatives stemming from the lead compound, phenolphthalein, were designed to specifically complement structural features of a bacterial form of thymidylate synthase ...(Lactobacillus casei, LcTS) versus the human TS (hTS) enzyme. The new compounds were screened for their activity and their specificity against TS enzymes from different species, namely, L. casei (LcTS), Pneumocystis carinii (PcTS), Cryptococcus neoformans (CnTS), and human thymidylate synthase (hTS). Apparent inhibition constants (K i) for all the compounds against LcTS were determined, and inhibition factors (IF, ratio between the initial rates of the enzymatic reaction in the presence and absence of each inhibitor) against each of the four TS species were measured. A strong correlation was found between the two activity parameters, IF and K i, and therefore the simpler IF was used as a screening factor in order to accelerate biological evaluation. Compounds 5b, 5c, 5ba, and 6bc showed substantial inhibition of LcTS while remaining largely inactive against hTS, illustrating for the first time remarkable species specificity among TSs. Due to sequence homology between the enzymes, several compounds also showed high activity and specificity for CnTS. In particular, 3-hydroxy-3-(3-chloro-4-hydroxyphenyl)-6-nitro-1H,3H-naphtho1,8-c,dpyran-1-one (6bc) showed an IF < 0.04 for CnTS (K i = 0.45 μM) while remaining inactive in the hTS assay at the maximum solubility concentration of the compound (200 μM). In cell culture assays most of the compounds were found to be noncytotoxic to human cell lines but were cytotoxic against several species of Gram-positive bacteria. These results are consistent with the enzymatic assays. Intriguingly, several compounds also had selective activity against Cr. neoformans in cell culture assay. In general, the most active and selective compounds against the Gram-positive bacteria were those designed and found in the enzyme assay to be specific for LcTS versus hTS. The original lead compound was least selective against most of the cell lines tested. To our knowledge these compounds are the first TS inhibitors selective for bacterial TS with respect to hTS.
The chloro substituted 3,3‐di‐(4′‐hydroxyphenyl)‐1‐oxo‐1H,3H‐naphtho1,8‐cdpyran was synthesized in a 40/60 mixture of C6 or C7 substituted isomers, respectively. The two isomers were separated by ...hplc. The X‐ray crystal structure of the mixture was obtained. Both the mixture and the single isomers were tested against Lactobacillus Casei thymidylate synthase. The X‐ray analysis clearly revealed co‐crystallization of the two isomeric species. The apparent Ki of the mixture was 0.8 muM, while those of the C6 and C7 substituted isomers were 0.42 and 0.52 muM, respectively, thus showing that the position of the chlorine in the naphthalene ring was not critical for enzymatic activity.
Abstract
The chloro substituted 3,3‐di‐(4′‐hydroxyphenyl)‐1‐oxo‐1
H
,3
H
‐naphtho1,8‐
cd
pyran was synthesized in a 40/60 mixture of C
6
or C
7
substituted isomers, respectively. The two isomers were ...separated by hplc. The X‐ray crystal structure of the mixture was obtained. Both the mixture and the single isomers were tested against
Lactobacillus Casei
thymidylate synthase. The X‐ray analysis clearly revealed co‐crystallization of the two isomeric species. The apparent Ki of the mixture was 0.8 mu
M
, while those of the C
6
and C
7
substituted isomers were 0.42 and 0.52 mu
M
, respectively, thus showing that the position of the chlorine in the naphthalene ring was not critical for enzymatic activity.
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