Preparation and Evaluation of a Coumarin Library Towards the Inhibitory Activity of the Enzyme gGAPDH from Trypanosoma cruzi

A doença de Chagas, causada pelo Trypanosoma cruzi, é endêmica em 15 países na América Latina. Neste trabalho uma coleção de 38 cumarinas foi preparada em solução e testada frente à enzima gliceraldeído-3-fostafo-desidrogenase (gGAPDH) do T. cruzi. A rota sintética foi baseada na condensação de Knoevenagel de diferentes 2-hidroxibenzaldeídos com ácido de Meldrum ou malonato de etila, seguido de O-alquilação e/ou reação de transesterificação. Dentre as cumarinas preparadas, os melhores resultados obtidos para inibir 50% da atividade catalítica da enzima foram entre 80 e 130 μM.


Introduction
Chagas' disease is endemic in 15 countries of Latin America, where 40 million people are at risk and 200,000 new cases are registered each year. 1 Owing to toxicity and lack of efficacy, most of the currently used compounds are unsatisfactory 2 and the design of novel classes of trypanocidal drugs has become urgent.In fact, the medical and economic problems caused by Chagas' disease explain why it has been selected by the World Health Organization for development of new or more effective treatments. 1 One promising approach to accomplish this task is through the selective inhibition of enzymes that participate in the glycolytic pathway of the parasite (Trypanosoma cruzi).Glyceraldehyde-3-phosphate dehydrogenase (gGAPDH), the sixth enzyme in the glycolytic pathway, plays an essential role in the control of glycolytic flux. 3,4nce intracellular amastigotes probably depend on glycolisis for ATP production, the inhibition of gGAPDH would prevent T. cruzi from being infective. 2,5For this reason it has been identified as a suitable target for the development of inhibitors.Another reason to select gGAPDH as a good target for inhibitors design comes from the fact that 95% deficiency of GAPDH in human erythrocytes does not cause any clinical symptoms.
In a continuous effort towards the development of specific, potent inhibitors of the T. cruzi gGAPDH, several natural products have been screened 6 and, among some other active coumarins, chalepin showed a promising IC 50 value (64 µM). 7After the structural characterization of the chalepin-gGAPDH complex by X-ray crystallography, 8 essential interaction sites could be identified and structural modifications in the coumarin ring proposed both by molecular modeling and de novo design (Figure 1).
Structure-based ligand design suggested a simplification of chalepin ring, through the substitution of 1,2-dimethyl-allyl moiety by a more polar group, thus replacing van der Waals contacts by hydrogen bonds, and additional moieties at R 2 , aiming again the increased hydrogen bond interactions.Recently, Montanari and coworkers.reported 3D QSAR studies on binding affinities of some natural and synthetic coumarins for T. cruzi gGAPDH. 9,10harmaceutical companies have now been using combinatorial chemistry for drug discovery for about a decade.Some of the earlier libraries they synthesized have been discredited for being poorly designed, impractically large, and structurally simplistic. 11That's why drug researchers are increasingly embracing natural-productlike libraries-moderate-size collections of complex compounds that are highly likely to exhibit interesting and useful types of biological activity. 12n order to contribute with the study of the structureactivity relationship, in this work a library containing 38 coumarin-3-carboxyesters was prepared in solution phase and evaluated against T. cruzi gGAPDH, following the structural modifications of chalepin shown in Figure 1.Coumarin-3-carboxamides and esters were reported as inhibitors of serine proteases, α-chymotrypsin (CT) and human leukocyte elastase (HLE).Serine proteases have been the focus of extensive study in terms of their vital roles in biological processes, their involvement in numerous diseases, and the development of suitable therapeutic inhibitors. 13

Results and Discussion
The synthetic route for the preparation of the coumarin derivatives was based on the Knoevenagel condensation of different 2-hydroxybenzaldehydes with Meldrum's acid 14 or diethyl malonate, 15 followed by O-alkylation and/ or transesterification or bromination reactions.The condensation of the 2,4-di-hydroxybenzaldehyde (1a) with Meldrum´s acid using catalytic amount of NH 4 OAc gave compound 2a that was O-alkylated to obtain coumarins 3a-c (Scheme 1).
Aldehydes 1a-e were reacted with diethyl malonate in the presence of piperidine to give ethyl coumarin-3carboxylate derivatives 4a-e that were O-alkylated with different alkyl bromides furnishing coumarins 5a-r (Scheme 2).Treatment of 2a and 4a with bromine in acetic acid gave compounds 2b and 6a, that were O-alkylated to furnish compounds 6b and 6c (Scheme 3).Some of coumarin derivatives 4 were submitted to transesterification reactions to form coumarins 7a-h (Scheme 4).In total, 38 coumarins were prepared (Table 1), submitted to inhibition assays and the observed inhibitory activities are shown in Table 2.
The prepared coumarins showed moderate inhibition profiles.The best values to inhibit 50% of the enzymatic activity range from 80 to 130 µM.One interesting point though was the unexpected behavior of compound 6b when compared to compounds 4d, 3c and 6c.According to inhibition assay results (compounds 4d and 6c) increased steric volume at R 3 or R 2 does not affect interaction profile significantly.On the other hand, when both substituents are present at the same time, deleterious effect on affinity is observed (compound 6b).
Another intriguing result comes from the comparison of compounds 5m, 5n and 5o.Inhibition assay results suggest that bulky substituents at R 2 prevent inhibitors from binding unless R 1 position is also full-filled.This result is unexpected since gGAPDH-chalepin crystallographic structure shows that

Experimental
Unless otherwise noted, all commercially available reagents were purchased from Aldrich Chemical Co.Reagents and solvents were purified when necessary according to the usual procedures described in the literature. 1 H and 13 C NMR spectra were recorded on a Bruker ARX-200 (200 and 50 MHz respectively).The IR spectra refer to films and were measured on a Bomem M102 spectrometer.Mass Spectra were recorded on a Shimadzu GCMS-QP5000 or Mass Spectrometer QuatroLC-Micromass.Elemental analyses were performed on a Fisons EA 1108 CHNS-O.Analytical thin-layer chromatography was performed on a 0.25 µm film of silica gel containing fluorescent indicator UV 254 supported on an aluminum sheet (Sigma-Aldrich).Flash column chromatography was performed using silica gel (Kieselgel 60, 230-400 mesh, E. Merck).Gas chromatography was performed in a Shimadzu GC-17A with H 2 as carrier and using a DB-5 column.Melting points were performed in Microquimica MQAPF -301.

T. cruzi GAPDH inhibitory activity
The inhibitory activity was recorded using the same reaction medium described above.Absorbance was also read at 340 nm at 30 s interval.In each case, a control experiment was performed with 10% DMSO in the reaction medium.Inhibitory activity was calculated as follows, and the data presented in Table 2 are the means of 3 repetitions.% inhibitory activity = {(U mg -1 control -U mg -1 compound) / U mg -1 control} x 100

Methyl 7-methoxy-2-oxo-2H-chromene-3-carboxylate (3c)
To a solution of coumarin 2a (50 mg, 0.24 mmol) in dry acetone (2 mL) were added K 2 CO 3 (200 mg, 1.45 mmol), Me 2 SO 4 (183 mg, 1.45 mmol), and the mixture was stirred under reflux for 5 h.After cooling to room temperature a saturated solution of NH 4 Cl (4 mL) was added and the product was extracted with ethyl acetate (4 x 4 mL).The organic layer was dried over Na 2 SO 4 and the solvent was removed under reduced pressure.The product was purified by flash chromatography using 5% ethyl acetate in dichloromethane as eluent to give 3c (

Ethyl coumarin-3-carboxylate derivatives (4a-e)
To a solution of 2-hydroxybenzaldehydes 1a-e (3 mmol) in diethyl malonate (3 mmol) was added piperidine (10 drops), and the resulting solution was stirred for 30 minutes at room temperature.Then it was acidified with a solution of HCl 10%.The precipitate material was filtrated and washed with cold water.The desired products were purified by recrystallization from ethyl acetate or flash chromatography.

Acetylcoumarin derivatives (5a-c)
To a solution of hydroxycoumarin 4a-c (0.13 mmol) in pyridine (0.5 mL) was added acetic anhydride (3 equiv.for each free hydroxyl group) and the mixture was stirred for 3 h at room temperature.Ethyl acetate (4 mL) was added, the organic layer was washed with a solution of HCl 10% (3 x 3 mL), NaOH 10% (2 x 3 mL) and water (3 x 3 mL).The solvent was dried over Na 2 SO 4 and evaporated under reduced pressure.The product was purified by successive recrystallization from hot ethyl acetate, then hexane was added.

Ethyl 2-oxo-2H-chromene-3-carboxylate derivatives (5d-l)
To a stirred solution of coumarin 4a-c in dry DMF (4 mL mmol -1 ) were added Cs 2 CO 3 (2 equiv.for each free hydroxyl group) and the corresponding bromide (2 equiv.for each free hydroxyl).The mixture was stirred at 50 o C for 16 h.After cooling to room temperature, ethyl acetate was added and the organic layer was washed with saturated solution of NH 4 Cl.The solvent was dried over Na 2 SO 4 and evaporated; the resulting products were purified by recrystallization from ethyl acetate or flash chromatography.

Ethyl coumarin-3-carboxilates (5m-r)
To a solution of coumarin 5d-l (0.11 mmol) in methanol (2 mL) was added Amberlyst ® 15 (15% m/m) and the resulting mixture was stirred for 12 h at room temperature, then it was filtrated and washed with methanol (1 mL).The solvent was evaporated under reduced pressure and the product was purified by successive recrystallization.The product was dissolved in hot ethyl acetate, than hexane was added.

Coumarin-3-carboxylates (7a-i)
To a solution of coumarin 4b, 4d, 4e or 6b (0.12 mmol) in toluene (0.5 mL) was added ethylene glycol, 1,5pentanediol or iso-butyl alcohol (1.44 mmol) and PTSA (20% m/m).The reaction mixture was heated at 50 o C and stirred for 12 h.After cooling to room temperature the mixture was poured into water (2 mL) and the organic layer was extracted with ethyl acetate (3 x 4 mL).The combined organic layers were washed with saturated solution of NaCl (4 mL), dried over Na 2 SO 4 and evaporated under reduced pressure.

Figure 1 .
Figure 1.Proposed structural modifications in the chalepin structure.
To a solution of coumarin 6a (92 mg, 0.29 mmol) in dry acetone (2.0 mL), K 2 CO 3 (49 mg, 0.35 mmol) and Me 2 SO 4 (45 mg, 0.35 mmol) were added.The mixture was stirred under reflux for 5 h.After cooling to room temperature was added a saturated solution of NH 4 Cl (2 mL) and the product was extracted with ethyl acetate (4 x 4mL).The organic layer was dried over Na 2 SO 4 and the solvent was removed under reduced pressure.