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Journal of the Brazilian Chemical Society

Print version ISSN 0103-5053

J. Braz. Chem. Soc. vol.16 no.4 São Paulo July/Aug. 2005

http://dx.doi.org/10.1590/S0103-50532005000500014 

ARTICLE

 

Preparation and evaluation of a coumarin library towards the inhibitory activity of the enzyme gGAPDH from Trypanosoma cruzi

 

 

Joel Alvim Jr.I; Ricardo L. A. DiasI; Marcelo S. CastilhoII; Glaucius OlivaII; Arlene G. Corrêa*, I

IDepartamento de Química, Universidade Federal de São Carlos, 13565-905 São Carlos - SP, Brazil
IIInstituto de Física de São Carlos, Universidade de São Paulo, 13560-970 São Carlos - SP, Brazil

 

 


ABSTRACT

Chagas' disease, caused by Trypanosoma cruzi, is endemic in 15 countries in Latin America. In this work a library of 38 coumarins was prepared in solution phase and evaluated against T. cruzi glycolytic enzyme glyceraldehyde-3-phosphate-dehydrogenase (gGAPDH). The synthetic route was based on the Knoevenagel condensation of different 2-hydroxybenzaldehydes with Meldrum's acid or diethyl malonate, followed by O-alkylation and/or transesterification reactions. Among the prepared coumarins, the best values obtained to inhibit 50% of the enzymatic activity range from 80 to 130 µM.

Keywords: Trypanosoma cruzi, coumarin library, chalepin, gGAPDH


RESUMO

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 unsatisfactory2 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.3,4 Once intracellular amastigotes probably depend on glycolisis for ATP production, the inhibition of gGAPDH would prevent T. cruzi from being infective.2,5 For 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 screened6 and, among some other active coumarins, chalepin showed a promising IC50 value (64 µM).7 After 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 R2, 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.9,10

Pharmaceutical 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.11 That's why drug researchers are increasingly embracing natural-product-like libraries—moderate-size collections of complex compounds that are highly likely to exhibit interesting and useful types of biological activity.12

In order to contribute with the study of the structure-activity 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, a-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 acid14 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 NH4OAc 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-3-carboxylate 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 R3 or R2 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 R2 prevent inhibitors from binding unless R1 position is also full-filled. This result is unexpected since gGAPDH-chalepin crystallographic structure shows that R2 position should be directed to water molecule W348, which does not have an essential role in chalepin interaction profile.

Indeed, if compounds 1-7 bind in the same way as chalepin (Figure 1), explaining the structure-activity relationships becomes far from easy. In order to further investigate these results and thus generate useful data for the computer aided molecular design, modeling studies are being performed through docking protocol.

 

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. 1H and 13C 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 UV254 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 H2 as carrier and using a DB-5 column. Melting points were performed in Microquimica MQAPF - 301.

T. cruzi GAPDH activity

T. cruzi gGAPDH activity was determined according to a modification of a previously reported procedure.7 Reduced NADH was measured spectrophotometrically at 340 nm at 30 s interval. The reaction medium contained 50 mmol L-1 Tris-HCl pH 8.6 buffer, 1 mmol L-1 b-mercapto-ethanol, 30 mmol L-1 Na2HAsO4 2.5 mmol L-1 NAD+, 0.3 mmol L-1 glyceraldehyde-3-phosphate and 0.4-0.9 mg protein, in a total volume of 1000 µL. The reaction was initiated by addition of enzyme.

The specific activity (unit = U) of the enzyme was calculated as:

(U mg-1) = {(D absorbance / Dt) x volume of cell} / 6.22 x volume of enzyme x [enzyme]

where Dt = 0.5 min; volume of cell = 1.00 mL; NADH = 6.22 (mmol L-1); volume of enzyme = 0.005 mL; [enzyme] concentration of enzyme in mg mL-1.

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

7-hydroxy-2-oxo-2H-chromene-3-carboxylic acid (2a)

To a suspension of 2,4-dihydroxybenzaldehyde (1.3 g, 9.5 mmol) in water (21 mL) were added the Meldrum's acid (1.55 g, 10.4 mmol) and ammonium acetate (150 mg, 1.9 mmol). The suspension was stirred at room temperature for 1 h. The precipitate formed was filtered, washed with cold water (2 x 10 mL) and dried in vacuum to give 2a as a yellow solid (1.88 g, 96% yield). mp 283-284ºC; IR nmax/cm-1: 3129, 3037, 1712, 1684, 1617; 1H NMR (200 MHz, DMSO-d6) d 6.74 (s, 1H), 6.85 (d, J 8.2 Hz, 1H), 7.75 (d, J 8.2 Hz, 1H), 8.70 (s, 1H); 13C NMR (50 MHz, DMSO-d6) d 102.0, 110.8, 112.8, 114.3, 132.1, 149.4, 157.2, 158.0, 164.2, 164.5.

Coumarin-3-carboxylate derivatives (3a and 3b)

To a solution of coumarin 2a (50 mg, 0.24 mmol) in a saturated solution of NaHCO3 (250 µL) were added Adogen 464® (100 mg, 0.24 mmol) and a solution of 1-bromopropane (27 µL, 0.29 mmol) in dichloromethane (250 µL). The mixture was stirred for 72 h at 50ºC and, after cooling to room temperature dichloromethane (1 mL) and water (1 mL) were added. The organic layer was extracted with dichloromethane (4 x 2 mL), washed with water (3 mL), dried over Na2SO4 and the solvent was removed under reduced pressure. The material was purified by flash chromatography using 33% hexane in ethyl acetate as eluent, giving coumarins 3a (8.0 mg, 13% yield) and 3b (5.8 mg, 10% yield).

Propyl 7-propoxy-2-oxo-2H-chromene-3-carboxylate (3a).

mp 104-105 ºC; IR nmax/cm-1: 2964, 2928, 1777, 1603; 1H NMR (200 MHz, CDCl3) d 1.03 (t, J 7.0 Hz, 3H), 1.06 (t, J 7.0 Hz, 3H), 1.71-1.98 (m, 4H), 4.01 (t, J 6.5 Hz, 2H), 4.29 (t, J 6.7 Hz, 2H), 6.80 (d, J 2.4 Hz, 1H), 6.88 (dd, J 8.6 and 2.4 Hz, 1H), 7.49 (d, J 8.6 Hz, 1H), 8.49 (s, 1H); MS (IE): m/z 290 (M+), 248, 231, 206,189, 162 (100), 134, 105, 51.

Propyl 7-hydroxy-2-oxo-2H-chromene-3-carboxylate (3b)

mp 174-175 ºC; IR nmax/cm-1: 3443, 3273, 1741, 1709; 1H NMR (200 MHz, DMSO-d6) d 0.97 (t, J 7.4 Hz, 3H), 1.61-1.78 (m, 2H), 4.17 (t, J 6.5 Hz, 2H), 6.73 (s, 1H), 6.84 (d, J 8.3 Hz, 1H), 7.77 (d, J 8.3 Hz, 1H), 8.66 (s, 1H); MS (IE): m/z 248 (M+), 206, 189, 162 (100), 134, 105, 51.

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 K2CO3 (200 mg, 1.45 mmol), Me2SO4 (183 mg, 1.45 mmol), and the mixture was stirred under reflux for 5 h. After cooling to room temperature a saturated solution of NH4Cl (4 mL) was added and the product was extracted with ethyl acetate (4 x 4 mL). The organic layer was dried over Na2SO4 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 (18 mg, 32% yield). mp 123-124ºC; IR nmax/cm-1: 3057, 2953, 1746, 1699; 1H NMR (200 MHz, CDCl3) d 3.91 (s, 3H), 3.94 (s, 3H), 6.82 (d, J 2.3 Hz, 1H), 6.90 (dd, J 8.6 and 2.3 Hz, 1H), 7.51 (d, J 8.6 Hz, 1H), 8.55 (s, 1H); MS (IE): m/z 234 (M+), 203 (100), 176, 119, 76.

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.

Ethyl 7-hydroxy-2-oxo-2H-chromene-3-carboxylate (4a)

(1.94 g, 76% yield), mp 168-169 ºC (lit.16 mp 166-167 ºC); IR nmax/cm-1: 3550, 3470, 1739, 1617; 1H NMR (200 MHz, DMSO-d6) d 1.29 (t, J 7.1 Hz, 3H), 4.26 (q, J 7.1 Hz, 2H), 6.73 (s, 1H), 6.84 (d, J 8.3 Hz, 1H), 7.77 (d, J 8.3 Hz, 1H), 8.67 (s, 1H); 13C NMR (50 MHz, DMSO-d6) d 14.0, 60.7, 110.3, 112.0, 132.0, 149.3, 156.3, 162.8, 163.9; MS (IE): m/z 234 (M+), 206, 189, 178, 162 (100), 134, 105, 89, 77, 51.

Ethyl 8-hydroxy-2-oxo-2H-chromene-3-carboxylate (4b)

(350 mg, 45% yield), mp 174-175 ºC; IR nmax/cm-1: 3303, 3045, 2984, 1746, 1696, 1611; 1H NMR (200 MHz, CDCl3) d 1.32 (t, J 7.0 Hz, 3H), 4.30 (q, J 7.0 Hz, 2H), 7.17-7.26 (m, 2H), 7.28-7.39 (m, 1H), 8.69 (s, 1H); 13C NMR (50 MHz, CDCl3) d 14.0, 61.1, 117.7, 118.6, 120.0, 120.5, 124.7, 143.1, 144.3, 149.0, 155.9, 162.6; MS (IE): m/z 234 (M+), 206, 189, 162 (100), 134, 105, 89, 77, 51.

Ethyl 7,8-dihydroxy-2-oxo-2H-chromene-3-carboxylate (4c)

(560 mg, 70% yield), mp 233-234 ºC; IR nmax/cm-1: 3476, 3214, 2973, 1738, 1693, 1618, 1588; 1H NMR (200 MHz, CDCl3) d 1.30 (t, J 7.1 Hz, 3H), 4.26 (q, J 7.1 Hz, 2H), 6.87 (d, J 8.5 Hz, 1H), 7.27 (d, J 8.5 Hz, 1H), 8.63 (s, 1H); 13C NMR (50 MHz, CDCl3) d 14.3, 61.0, 111.4, 112.1, 113.5, 121.8, 132.0, 145.1, 150.2, 152.8, 156.6, 163.2; APCI/MS: m/z 249 ([M-H]-).

Ethyl 7-methoxy-2-oxo-2H-chromene-3-carboxylate (4d)

(406 mg, 68% yield), mp 132-133 ºC (lit.17 mp 120-125 ºC); IR nmax/cm-1: 3052, 2986, 1619, 1381, 1216; 1H NMR (200 MHz, CDCl3) d 1.40 (t, J 7.1 Hz, 3H), 3.90 (s, 3H), 4.40 (q, J 7.1 Hz, 2H), 6.82 (d, J 2.4 Hz, 1H), 6.90 (dd, J 8.6 and 2.4 Hz, 1H), 7.50 (d, J 8.6 Hz, 1H), 8.51 (s, 1H); 13C NMR (50 MHz, CDCl3) d 14.1, 55.8, 61.5, 100.2, 111.4, 113.4, 113.9, 130.6, 148.7, 157.0, 157.4, 163.2, 165.0; MS (IE): m/z 248 (M+), 203, 176 (100), 148, 119, 76.

Ethyl 2-oxo-2H-chromene-3-carboxylate (4e)

(890 mg, 65% yield), mp 72-73 ºC (lit.16 mp 93-93 ºC); IR nmax/cm-1: 1779, 1617, 1607, 1567; 1H NMR (200 MHz, CDCl3) d 1.41 (t, J 7.1 Hz, 3H), 4.42 (q, J 7.1 Hz, 2H), 7.27-7.41 (m, 2H), 7.57-7.72 (m, 2H), 8.53 (s, 1H); 13C NMR (50 MHz, CDCl3) d 14.2, 61.9, 116.7, 117.8, 118.3, 124.8, 129.5, 134.3, 148.5, 155.1, 156.6, 163.0.; MS (IE): m/z 218 (M+) 190, 173, 146 (100), 118, 101, 89, 63.

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 Na2SO4 and evaporated under reduced pressure. The product was purified by successive recrystallization from hot ethyl acetate, then hexane was added.

Ethyl 7-acetyloxy-2-oxo-2H-chromene-3-carboxylate (5a)

(27 mg, 76% yield): mp 154-155 ºC; IR nmax/cm-1: 3087, 3060, 2981, 1756, 1700; 1H NMR (200 MHz, CDCl3) d 1.41 (t, J 7.1 Hz, 3H), 2.35 (s, 3H), 4.42 (q, J 7.1 Hz, 2H), 7.07-7.20 (m, 2H), 7.62 (d, J 8.3 Hz, 1H), 8.52 (s, 1H); MS (IE): m/z 276 (M+), 234, 189, 162 (100), 134, 105, 89, 76, 51.

Ethyl 8-acetyloxy-2-oxo-2H-chromene-3-carboxylate (5b)

(16 mg, 47% yield), mp 150-151 ºC; IR nmax/cm-1: 3045, 2984, 1760, 1695, 1620, 1577; 1H NMR (200 MHz, CDCl3) d: 1.40 (t, J 7.1 Hz, 3H), 2.42 (s, 3H), 4.41 (q, J 7.1 Hz, 2H), 7.25-7.55 (m, 3H), 8.51 (s, 1H); 13C NMR (50 MHz, CDCl3) d 14.1, 20.6, 62.1, 119.1, 124.5, 126.8, 127.6, 137.6, 148.0, 168.4; MS (IE): m/z 276 (M+), 234 (100), 188, 162, 134, 105, 76, 51.

Ethyl 7,8-bis(acetyloxy)-2-oxo-2H-chromene-3-carboxylate (5c)

(41 mg, 79% yield), mp 127-128 ºC; IR nmax/cm-1: 2998, 2940, 1774, 1723, 1578; 1H NMR (200 MHz, CDCl3) d 1.40 (t, J 7.1 Hz, 3H), 2.34 (s, 3H), 2.41 (s, 3H), 4.40 (q, J 7.1 Hz, 2H), 7.19 (d, J 8.6 Hz, 1H), 7.50 (d, J 8.6 Hz, 1H), 8.50 (s, 1H); 13C NMR (50 MHz, CDCl3) d 14.1, 20.2, 20.6, 62.1, 116.6, 118.0, 119.5, 126.5, 130.1, 147.3, 147.8, 148.4, 155.0, 162.6, 167.1, 167.4; MS (IE): m/z 334 (M+), 292, 250, 204 (100), 178, 120, 92, 79, 63.

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 Cs2CO3 (2 equiv. for each free hydroxyl group) and the corresponding bromide (2 equiv. for each free hydroxyl). The mixture was stirred at 50 ºC for 16 h. After cooling to room temperature, ethyl acetate was added and the organic layer was washed with saturated solution of NH4Cl. The solvent was dried over Na2SO4 and evaporated; the resulting products were purified by recrystallization from ethyl acetate or flash chromatography.

Ethyl 7-(allyloxy)-2-oxo-2H-chromene-3-carboxylate (5d)

(42 mg, 72% yield), mp 103-104 ºC; IR nmax/cm-1: 3055, 2985, 1756, 1618; 1H NMR (200 MHz, CDCl3) d 1.40 (t, J 7.1 Hz, 3H), 4.40 (q, J 7.1 Hz, 2H), 4.56-4.74 (m, 2H), 5.26-5.55 (m, 2H), 6.05 (ddt, J 17.1, 15.7 and 5.2 Hz, 1H), 6.82 (d, J 2.2 Hz, 1H), 6.91 (dd, J 8.7 and 2.2 Hz, 1H), 7.50 (d, J 8.7 Hz, 1H), 8.50 (s, 1H); 13C NMR (50 MHz, CDCl3) d 14.2, 61.7, 69.5, 101.20, 111.7, 114.11, 118.9, 130.7, 131.7, 148.9, 157.1, 157.4, 163.4, 164.0; MS (IE): m/z 274 (M+), 246, 229, 202 (100), 188, 160, 132, 105, 76, 50.

Ethyl 8-(allyloxy)-2-oxo-2H-chromene-3-carboxylate (5e)

(50 mg, 86% yield), mp 89-90 ºC; IR nmax/cm-1 : 1759, 1703, 1608, 1572, 1473; 1H NMR (200 MHz, CDCl3) d 1.41 (t, J 7.1 Hz, 3H), 4.41 (q, J 7.1 Hz, 2H), 4.71 (ddd, J 10.5, 3.0 and 1.5 Hz, 2H), 5.33 (ddd, J 10.5, 3.0 and 1.5 Hz, 2H), 5.45 (ddd, J 17.2, 3.0 and 1.5 Hz, 1H), 6.08 (ddt, J 17.2, 10.5 and 5.3 Hz, 1H), 7.11-7.30 (m, 3H), 8.49 (s, 1H); 13C NMR (50 MHz, CDCl3) d 14.2, 61.9, 70.2, 117.8, 118.5, 120.9, 124.5, 132.4, 145.2, 145.9, 148.7, 156.2, 163.0.; MS (IE): m/z 274 (M+), 246, 233, 205, 188, 177, 160, 133, 115, 105 (100), 76, 50; Anal. Calc. for C15H14O5: C, 65.69; H, 5.14, Found: C, 65.42; H, 4.82%.

Ethyl 7,8-bis(allyloxy)-2-oxo-2H-chromene-3-carboxylate (5f)

(43 mg, 69% yield), mp 91-92 ºC; IR nmax/cm-1: 3092, 2994, 1760, 1607; 1H NMR (200 MHz, CDCl3) d 1.40 (t, J 7.1 Hz, 3H), 4.39 (q, J 7.1 Hz, 2H), 4.61-4.80 (m, 4H), 5.21 (ddt, J 10.2, 2.0 and 1.7 Hz, 1H), 5.30 (ddd, J 6.6, 2.7 and 1.4 Hz, 1H), 5.37 (dd, J 2.8 and 1.4 Hz, 1H), 5.45 (ddt, J 17.2, 3.0 and 1.6 Hz, 1H), 5.95-6.25 (m, 2H), 6.90 (d, J 8.7 Hz, 1H), 7.29 (d, J 8.7 Hz, 1H), 8.46 (s, 1H); 13C NMR (50 MHz, CDCl3) d 14.2, 61.7, 70.0, 74.6, 110.4, 112.8, 114.7, 118.4, 118.8, 124.9, 132.0, 133.6, 149.0, 156.4, 157.0, 163.4; MS (IE): m/z 306, 302, 288, 257, 229, 210, 185, 170, 158, 129, 115, 103, 91, 77, 55 (100).

Ethyl 2-oxo-7-[3-(tetrahydro-2H-pyran-2-yloxy)propoxy]-2H-chromene-3-carboxylate (5g)

(62 mg, 77% yield), mp 83-84 ºC; IR nmax/cm-1: 2948, 2866, 1748, 1698, 1603; 1H NMR (200 MHz, CDCl3) d 1.40 (t, J 7.1 Hz, 3H), 1.50-1.90 (m, 6H), 2.03-2.20 (m, 2H), 3.44-3.65 (m, 2H), 3.75-4.03 (m, 2H), 4.18 (t, J 6.4 Hz, 2H), 4.40 (q, J 7.1 Hz, 2H), 4.56-4.67 (m, 1H), 6.83 (d, J 2.4 Hz, 2H), 6.89 (dd, J 8.6 and 2.4 Hz, 1H), 7.50 (d, J 8.6 Hz, 1H), 8.51 (s, 1H); 13C NMR (50 MHz, CDCl3) d 14.2, 19.5, 25.3, 29.3, 30.6, 61.6, 62.3, 63.5, 65.9, 98.9, 100.8, 111.5, 113.9, 130.6, 148.9, 157.1, 157.5, 163.4, 164.5; MS (IE): m/z 292, 234, 220, 206, 189, 162 (100), 134, 105, 76, 51; Anal. Calc. for C20H24O7: C, 63.82; H, 6.43, Found: C, 63.92; H, 6.36%.

Ethyl 2-oxo-8-[3-(tetrahydro-2H-pyran-2-yloxy)propoxy]-2H-chromene-3-carboxylate (5h)

(71 mg, 88% yield), viscous liquid, IR (film) nmax/cm-1: 2941, 2872, 1762, 1609; 1H NMR (200 MHz, CDCl3) d 1.41 (t, J 7.1 Hz, 3H), 1.45-1.65 (m, 4H), 1.66-1.90 (m, 2H), 2.10-2.25 (m, 2H), 3.42-3.57 (m, 1H), 3.58-3.72 (m, 1H), 3.77-3.89 (m, 1H), 3.90-4.02 (m, 1H), 4.19-4.30 (m, 2H), 4.41 (q, J 7.1 Hz, 2H), 4.56-4.65 (m, 1H), 7.10-7.30 (m, 3H), 8.49 (s, 1H); 13C NMR (50 MHz, CDCl3) d 14.1, 19.7, 25.3, 29.4, 30.6, 61.8, 62.5, 63.8, 66.6, 99.1, 117.2, 118.4, 118.5, 120.5, 124.6, 145.0, 146.5, 148.7, 156.2, 163.0; MS (IE): m/z 316, 278, 247, 234, 188, 162, 134, 129, 105, 85, 73 (100), 55.

Ethyl 2-oxo-7,8-bis[3-(tetrahydro-2H-pyran-2-yloxy)- propoxy]-2H-chromene-3-carboxylate (5i)

(87 mg, 81% yield), IR (film) nmax/cm-1: 2942, 2873, 1763, 1605; 1H NMR (200 MHz, CDCl3) d 1.39 (t, J 7.1 Hz, 3H), 1.46-1.90 (m, 12H), 2.00-2.27 (m, 4H), 3.41-3.76 (m, 4H), 3.77-4.05 (m, 4H), 4.24 (t, J 6.3 Hz, 4H), 4.39 (q, J 7.1 Hz, 2H), 4.55-4.60 (m, 1H), 4.61-4.69 (m, 1H), 6.93 (d, J 8.7 Hz, 1H), 7.29 (d, J 8.7 Hz, 1H), 8.46 (s, 1H); 13C NMR (50 MHz, CDCl3) d 14.2, 19.7, 25.3, 25.4, 29.5, 30.5, 30.6, 30.7, 61.6, 62.4, 62.5, 63.6, 64.2, 66.5. 71.3, 99.0, 99.1, 109.9, 112.6, 114.5, 124.8, 149.0, 149.4, 156.4, 157.5, 163.4; MS (IE): m/z 308, 281, 250, 204, 175, 143, 85 (100), 69, 57.

Ethyl 2-oxo-7-[2-(tetrahydro-2H-pyran-2-yloxy)ethoxy]-2H-chromene-3-carboxylate (5j)

(63 mg, 80% yield), mp 93-94 ºC; IR nmax/cm-1: 2939, 2871, 1788, 1760, 1604; 1H NMR (200 MHz, CDCl3) d 1.40 (t, J 7.1 Hz, 3H), 1.50-1.90 (m, 6H), 3.49-3.60 (m, 1H), 3.79-3.94 (m, 2H), 4.05-4.15 (m, 1H), 4.23-4.28 (m, 2H), 4.39 (q, J 7.1 Hz, 2H), 4.69-4.72 (m, 1H), 6.85 (d, J 2.4 Hz, 1H), 6.93 (dd, J 8.6 and 2.4 Hz, 1H), 7.50 (d, J 8.6 Hz, 1H), 8.51 (s, 1H); 13C NMR (50 MHz, CDCl3) d 14.2, 19.3, 25.3, 30.4, 61.6, 62.2, 65.3, 68.2, 99.1, 101.1, 111.6, 114.0, 114.1, 130.6, 148.9, 157.4, 163.4, 164.4; MS (IE): m/z 362 (M+) 278, 248, 234, 206, 189, 162, 129, 105, 85 (100), 73, 55.

Ethyl 2-oxo-8-[2-(tetrahydro-2H-pyran-2-yloxy)ethoxy]-2H-chromene-3-carboxylate (5k)

(67 mg, 86% yield), IR (film) nmax/cm-1: 2942, 2872, 1761, 1609; 1H NMR (200 MHz, CDCl3) d 1.41 (t, J 7.1 Hz, 3H), 1.47-1.90 (m, 6H), 3.45-3.64 (m, 1H), 3.81-4.04 (m, 2H), 4.05-4.22 (m, 1H), 4.25-4.38 (m, 2H), 4.41 (q, J 7.1 Hz, 2H), 4.69-4.82 (m, 1H), 7.12-7.35 (m, 3H), 8.49 (s, 1H).; 13C NMR (50 MHz, CDCl3) d 14.1, 19.3, 25.3, 30.4, 61.8, 62.2, 65.5, 69.2, 99.0, 117.9, 118.4, 118.5, 120.0, 124.5, 145.2, 146.4, 148.6, 156.1, 163.0; MS (IE): m/z 316, 278, 247, 188, 162, 129, 105, 85, 73 (100), 55.

Ethyl 2-oxo-7,8-bis-[2-(tetrahydro-2H-pyran-2-yloxy)-ethoxy]-2H-chromene-3-carboxylate (5l)

(18 mg, 18% yield), viscous liquid, 1H NMR (200 MHz, CDCl3) d 1.39 (t, J 7.1 Hz, 3H), 1.45-1.92 (m, 12H), 3.42-3.61 (m, 2H), 3.76-3.95 (m, 4H), 3.96-4.19 (m, 2H), 4.25-4.47 (m, 6H), 4.64-4.80 (m, 2H), 6.95 (d, J 8.7 Hz, 1H), 7.29 (d, J 8.7 Hz, 1H), 8.46 (s, 1H); 13C NMR (50 MHz, CDCl3) d 14.2, 19.2, 19.3, 25.3, 25.4, 30.4, 30.5, 61.6, 62.1, 62.1, 65.6, 66.6, 68.9, 72.8, 98.8, 99.0, 110.6, 112.8, 114.7, 124.7, 135.5, 148.9, 149.4, 156.4, 157.1, 163.4. MS (IE): m/z 281, 251, 223, 179 (100), 151, 135, 121, 109, 95, 85, 67, 55.

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.

Ethyl 7-(3-hydroxypropoxy)-2-oxo-2H-chromene-3-carboxylate (5m)

(30 mg, 90% yield), mp 134-135 ºC; IR nmax/cm-1: 3524, 2956, 1757, 1620, 1608; 1H NMR (200 MHz, CDCl3) d 1.40 (t, J 7.1 Hz, 3H), 2.01-2.17 (m, 2H), 3.88 (t, J 5.9 Hz, 2H), 4.21 (t, J 6.0 Hz, 2H), 4.39 (q, J 7.1 Hz, 2H), 6.81 (d, J 2.4 Hz, 1H), 6.88 (dd, J 8.6 and 2.4 Hz, 1H), 7.48 (d, J 8.6 Hz, 1H), 8.48 (s, 1H); 13C NMR (50 MHz, CDCl3) d 14.2, 31.7, 59.2, 61.6, 65.9, 100.9, 111.5, 113.8, 116.6, 130.6, 148.9, 157.1, 157.5, 163.4, 164.5. MS (IE): m/z 292 (M+), 247, 234, 206, 189, 162 (100), 134, 105, 77, 51; Anal. Calc. for C15H16O6: C, 61.64; H, 5.52, Found: C, 61.20; H, 5.17%.

Ethyl 8-(3-hydroxypropoxy)-2-oxo-2H-chromene-3-carboxylate (5n)

(18 mg, 55% yield), mp 98-99ºC; IR nmax/cm-1: 3463, 2964, 1758, 1699, 1608, 1572; 1H NMR (200 MHz, CDCl3) d 1.40 (t, J 7.1 Hz, 3H), 2.05-2.21 (m, 2H), 2.26 (s, 1H), 3.93 (t, J 5.9 Hz, 2H), 4.27 (t, J 5.9 Hz, 2H), 4.40 (q, J 7.1 Hz, 2H), 7.12-7.30 (m, 3H), 8.46 (s, 1H); 13C NMR (50 MHz, CDCl3) d 14.1, 31.8, 59.4, 61.9, 67.0, 117.5, 118.3, 118.5, 120.8, 124.7, 145.0, 146.3, 148.7, 156.3, 163.0; MS (IE): m/z 292 (M+) 247, 234, 206, 188 (100), 162, 134, 105, 77, 51.

Ethyl 7,8-bis-(3-hydroxypropoxy)-2-oxo-2H-chromene-3-carboxylate (5o)

(33 mg, 80% yield), mp 100-101 ºC; IR nmax/cm-1: 3435, 3295, 2953, 2881, 1746, 1609. 1H NMR (200 MHz, CDCl3) d 1.39 (t, J 7.1 Hz, 3H), 2.02 (quint, J 5.7 Hz, 2H), 2.12 (quint, J 5.9 Hz, 2H), 2.92 (s, 2H), 3.88 (t, J 5.9 Hz, 2H), 3.94 (t, J 5.7 Hz, 2H), 4.27 (t, J 5.7 Hz, 2H), 4.29 (t, J 5.9 Hz, 2H), 4.39 (q, J 7.1 Hz, 2H), 6.94 (d, J 8.8 Hz, 1H), 7.30 (d, J 8.8 Hz, 1H), 8.44 (s, 1H); 13C NMR (50 MHz, CDCl3) d 14.2, 31.7, 32.5, 59.4, 59.9, 61.7, 67.0, 72.1, 109.9, 112.7, 114.4, 125.17, 134.8, 149.1, 156.5, 157.0, 163.1. APCI/MS: m/z 365 ([M-H]-). Anal. Calc. for C18H22O8: C, 59.01; H, 6.05, Found: C, 59.19; H, 5.84%.

Ethyl 7-(2-hydroxyethoxy)-2-oxo-2H-chromene-3-carboxylate (5p)

(24 mg, 76% yield), mp 138-139 ºC; IR nmax/cm-1: 3476, 2964, 1751, 1739, 1604; 1H NMR (200 MHz, CDCl3) d 1.40 (t, J 7.1 Hz, 3H), 2.18 (s, 1H), 4.03 (t, J 4.0 Hz, 2H), 4.19 (t, J 4.0 Hz, 2H), 4.39 (q, J 7.1 Hz, 2H), 6.83 (d, J 2.3 Hz, 1H), 6.92 (dd, J 8.6 and 2.3 Hz, 1H), 7.51 (d, J 8.6 Hz, 1H), 8.48 (s, 1H);. 13C NMR (50 MHz, CDCl3) d 14.2, 60.9, 61.7, 70.1, 101.1, 111.8, 113.8, 114.2, 130.7, 148.8, 157.1, 157.3, 163.3, 164.2; MS (IE): m/z 278 (M+) 232, 206, 189, 162 (100), 134, 105, 89, 77, 51.

Ethyl 8-(2-hydroxyethoxy)-2-oxo-2H-chromene-3-carboxylate (5q)

(20 mg, 47% yield), mp 115-116 ºC; IR nmax/cm-1: 3480, 3448, 2945, 1740, 1698; 1H NMR (200 MHz, CDCl3) d 1.41 (t, J 7.1 Hz, 3H), 2.74 (s, 1H), 4.03-4.14 (m, 2H), 4.19-4.28 (m, 2H), 4.41 (q, J 7.1 Hz, 2H), 7.13-7.32 (m, 3H), 8.48 (s, 1H); 13C NMR (50 MHz, CDCl3) d 14.2, 61.0, 62.0, 71.0, 117.4, 118.4, 118.5, 121.0, 124.7, 145.0, 146.2, 148.7, 156.2, 163.0; MS (IE): m/z 278 (M+), 247, 234, 206, 188 (100), 162, 134, 105, 89, 77, 51.

Ethyl 7,8-bis-(2-hydroxyethoxy)-2-oxo-2H-chromene-3-carboxylate (5r)

(10 mg, 82% yield), mp 131-132 ºC; IR nmax/cm-1: 3325, 3211, 2932, 1748, 1700, 1606; 1H NMR (200 MHz, DMSO-d6) d 1.30 (t, J 7.1 Hz, 3H), 3.70 (t, J 5.3 Hz, 2H), 3.78 (t, J 4.8 Hz, 2H), 4.09 (t, J 5.3 Hz, 2H), 4.19 (t, J 4.8 Hz, 2H), 4.28 (q, J 7.1 Hz, 2H), 5.74 (s, 2H), 7.18 (d, J 8.8 Hz, 1H), 7.63 (d, J 8.8 Hz, 1H), 8.69 (s, 1H); 13C NMR (50 MHz, DMSO-d6) d 14.0, 59.4, 60.2, 60.9, 70.9, 74.8, 110.7, 112.3, 113.6, 125.7, 134.1, 148.5, 149.3, 155.9, 156.9, 162.7; MS (IE): m/z 251, 223, 204 (100), 178, 150, 121, 92, 79, 65, 53.

Ethyl 8-bromo-7-hydroxy-2-oxo-2H-chromene-3-carboxylate (6a)

To a solution of coumarin 4a (570 mg, 2.41 mmol) in glacial acetic acid (16 mL) at 60 ºC was added slowly a solution of bromine (0.13 mL, 2.65 mmol) in acetic acid (1 mL). The mixture was stirred for 3 h, then the temperature was allowed to rise to room temperature and the precipitate was filtrated, washed with cool water and dried under vacuum giving coumarin 6a (365 mg, 49% yield). mp 270-271 ºC; IR nmax/cm-1: 3282, 1758, 1603, 1552; 1H NMR (200 MHz, DMSO-d6) d 1.31 (t, J 7.1 Hz, 3H), 4.28 (q, J 7.1 Hz, 2H), 7.01 (d, J 8.6 Hz, 1H), 7.76 (d, J 8.6 Hz, 1H), 8.69 (s, 1H), 11.89 (s, 1H); 13C NMR (50 MHz, DMSO-d6) d 14.0, 60.8, 96.2, 111.3, 112.6, 113.3, 130.4, 149.2, 153.6, 155.7, 160.6, 162.5; APCI/MS: m/z 313 ([M-H]- + 2), 311 ([M-H]-).

Ethyl 8-bromo-7-methoxy-2-oxo-2H-chromene-3-carboxylate (6b)

To a solution of coumarin 6a (92 mg, 0.29 mmol) in dry acetone (2.0 mL), K2CO3 (49 mg, 0.35 mmol) and Me2SO4 (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 NH4Cl (2 mL) and the product was extracted with ethyl acetate (4 x 4mL). The organic layer was dried over Na2SO4 and the solvent was removed under reduced pressure. The product was purified by flash chromatography eluting with 10% ethyl acetate in dichloromethane and gave 6b (58 mg, 60% yield). mp 189-190 ºC; IR nmax/cm-1: 1757, 1609, 1543; 1H NMR (200 MHz, CDCl3) d 1.40 (t, J 7.1 Hz, 3H), 4.03 (s, 3H), 4.38 (q, J 7.1 Hz, 2H), 6.93 (d, J 8.7 Hz, 1H), 7.55 (d, J 8.7 Hz, 1H), 8.46 (s, 1H); 13C NMR (50 MHz, CDCl3) d 14.2, 57.0, 61.8, 99.4, 108.5, 112.7, 115.1, 129.5, 148.3, 153.6, 155.9, 161.2, 162.9; MS (IE): m/z 328 (M+ + 2), 326 (M+), 282, 280, 254, 256, 228, 226,199, 197, 147, 103, 88, 75 (100), 53; Anal. Calc. for C13H11BrO5: C, 47.73; H, 3.39, Found: C, 48.05; H, 3.63%.

Methyl 8-bromo-7-methoxy-2-oxo-2H-chromene-3-carboxylate (6c)

To a solution of coumarin 2a (70 mg, 0.34 mmol) in glacial acetic acid (2.3 mL) at 60 ºC was added slowly a solution of bromine (0.16 mL, 0.37 mmol) in acetic acid (0.15 mL) and the mixture was stirred for 3 h. The heating and the stirring were removed; the precipitate was filtrated, washed with cool water and dried under vacuum. The crude product 2b (50 mg) was dissolved in dry acetone (1.5 mL) and K2CO3 (145 mg, 1.05 mmol) and Me2SO4 (132 mg, 1.05 mmol) were added. The mixture was stirred under reflux for 5 h. After cooling to room temperature was added a saturated solution of NH4Cl (4 mL) and the product was extracted with ethyl acetate (4 x 4mL). The organic layer was dried over Na2SO4 and the solvent was removed under reduced pressure. The product was purified by flash chromatography eluting with 10% ethyl acetate in dichloromethane, giving 6c (24 mg, 45% yield). IR nmax/cm-1: 1753, 1710, 1612 971, 804; 1H NMR (200 MHz, DMSO-d6) d 3.83 (s, 3H), 4.02 (s, 3H), 7.25 (d, J 8.8 Hz, 1H), 7.96 (d, J 8.8 Hz, 1H), 8.78 (s, 1H); 13C NMR (50 MHz, CDCl3) d 52.7, 57,0, 99.4, 108.6, 112.7, 114.7, 129.7, 148.9, 153.6, 155.8, 161.3, 163.6; MS (IE): m/z 314 (M+ + 2), 312 (M+), 282, 280, 256, 254, 197, 195, 184, 182, 147, 103, 75 (100), 59.

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, 1,5-pentanediol or iso-butyl alcohol (1.44 mmol) and PTSA (20% m/m). The reaction mixture was heated at 50 ºC 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 Na2SO4 and evaporated under reduced pressure.

2-Hydroxyethyl 8-hydroxy-2-oxo-2H-chromene-3-carboxylate (7a)

(21 mg, 65% yield), mp 199-200 ºC; IR nmax/cm-1: 3532, 3453, 3103, 1746, 1609; 1H NMR (200 MHz, DMSO-d6) d 3.70 (dd, J 5.5 and 4.9 Hz, 2H), 4.26 (t, J 4.9 Hz, 2H), 4.93 (t, J 5.5 Hz, 1H), 7.17-7.26 (m, 2H), 7.26-7.37 (m, 1H), 8.74 (s, 1H), 10.40 (s, 1H); 13C NMR (50 MHz, DMSO-d6) d 59.1, 67.0, 117.5, 118.8, 120.2, 120.8, 125.0, 143.4, 144.6, 149.3, 156.1, 162.7; MS (IE): m/z 250 (M+), 220, 189 (100), 162, 134, 105, 77, 51.

2-Hydroxyethyl 7-methoxy-2-oxo-2H-chromene-3-carboxylate (7b)

(15 mg, 50% yield), mp 172-173 ºC; IR nmax/cm-1: 3309, 1740, 1618, 1560; 1H NMR (200 MHz, DMSO-d6) d 3.69 (t, J 5.2 Hz, 2H), 3.90 (s, 3H), 4.24 (t, J 5.2 Hz, 2H), 5.74 (s, 1H), 6.50-7.10 (m, 2H), 7.82 (d, J 9.0 Hz, 1H), 8.76 (s, 1H); 13C NMR (50 MHz, DMSO-d6) d 56.5, 59.2, 66.9, 100.6, 111.6, 113.4, 113.7, 131.9, 149.6, 156.6, 157.3, 162.9, 165.1; MS (IE): m/z 264 (M+), 234, 203 (100), 176, 148, 133, 119, 76, 50.

2-Hydroxyethyl 2-oxo-2H-chromene-3-carboxylate (7c)

(16 mg, 59% yield), mp 135-136 ºC; IR nmax/cm-1: 3505, 3304, 1752, 1609; 1H NMR (200 MHz, CDCl3) d 3.92-3.99 (m, 2H), 4.46-4.53 (m, 2H), 7.32-7.43 (m, 2H), 7.61-7.73 (m, 2H), 8.57 (s, 1H). 13C NMR (50 MHz, CDCl3) d 60.7, 67.5, 116.8, 117.8, 125.0, 129.6, 134.5, 149.2, 155.1; MS (IE): m/z 204, 173 (100), 146, 118, 101, 89, 63.

5-Hydroxypentyl 8-hydroxy-2-oxo-2H-chromene-3-carboxylate (7d)

(18 mg, 53% yield), mp 121-123 ºC; 1H NMR (200 MHz, DMSO-d6) d 1.35-1.55 (m, 4H), 1.62-1.68 (m, 2H), 2.08 (s, 1H), 4.24 (t, J 6.5 Hz, 2H), 4.40 (t, J 5.0 Hz, 1H), 7.15-7.27 (m, 2H), 7.28-7.40 (m, 1H), 8.68 (s, 1H); 13C NMR (50 MHz, DMSO-d6) d 21.8, 27.8, 31.9, 60.4, 65.0, 118.6, 120.0, 120.4, 124.7, 144.3, 144.7, 148.9; MS (IE): m/z 278, 247, 234, 188 (100), 162, 134, 105, 89, 77, 51.

5-Hydroxypentyl 7-methoxy-2-oxo-2H-chromene-3-carboxylate (7e)

(24 mg, 65% yield), mp 81-82 ºC; IR nmax/cm-1: 3497, 2940, 2857, 1762; 1H NMR (200 MHz, CDCl3) d 1.44-1.91 (m, 6H), 3.68 (t, J 6.3 Hz, 2H), 4.35 (t, J 6.5 Hz, 2H), 6.81 (d, J 2.3 Hz, 1H), 6.89 (dd, J 8.8 and 2.3 Hz, 1H), 7.51 (d, J 8.6 Hz, 1H), 8.50 (s, 1H); 13C NMR (50 MHz, CDCl3) d 22.2, 28.3, 32.2, 56.0, 62.6, 65.6, 100.3, 111.6, 113.6, 130.7, 149.0, 157.6, 163.6, 165.2; MS (IE): m/z 306 (M+), 276, 220, 203 (100), 176, 148, 133, 119, 76, 55.

5-Hydroxypentyl 2-oxo-2H-chromene-3-carboxylate (7f)

(17 mg, 52% yield), mp 82-84 ºC; IR nmax/cm-1: 3397, 2948, 2863, 1777, 1761; 1H NMR (200 MHz, CDCl3) d 1.40-1.73 (m, 4H), 1.74-1.90 (m, 2H), 3.69 (t, J 6.1 Hz, 2H), 4.37 (t, J 6.5 Hz, 2H), 7.25-7.42 (m, 2H), 7.57-7.72 (m, 2H), 8.52 (s, 1H); 13C NMR (50 MHz, CDCl3) d 22.2, 28.3, 32.1, 62.6, 65.9, 116.7, 117.8, 118.4, 124.8, 129.5, 134.3, 148.6, 155.2, 163.0; MS (IE): m/z 246, 218, 191, 173 (100), 146, 118, 101, 89, 63; Anal. Calc. for C15H16O5: C, 65.21; H, 5.84, Found: C, 64.46; H, 5.55%.

Isobutyl 7-methoxy-2-oxo-2H-chromene-3-carboxylate (7g)

(16 mg, 50% yield), mp 110-111 ºC; IR nmax/cm-1: 2958, 2886, 1755, 1688; 1H NMR (200 MHz, CDCl3) d 1.03 (d, J 6.7 Hz, 6H), 1.99-2.19 (m, 1H), 3.91 (s, 3H), 4.12 (d, J 6.7 Hz, 1H), 6.81 (d, J 2.3 Hz, 1H), 6.89 (dd, J 8.7 and 2.3 Hz, 1H), 7.50 (d, J 8.7 Hz, 1H), 8.48 (s, 1H); MS (IE): 276 (M+), 220, 203 (100), 176, 148, 119, 76, 50.

Isobutyl 8-bromo-7-methoxy-2-oxo-2H-chromene-3-carboxylate (7h)

(17 mg, 40% yield), mp 167-168 ºC; IR nmax/cm-1: 1749, 1611, 1543; 1H NMR (200 MHz, CDCl3) d 1.04 (d, J 6.7 Hz, 6H), 2.00-2.20 (m, 1H), 4.05 (s, 3H), 4.13 (d, J 6.6 Hz, 1H), 6.94 (d, J 8.8 Hz, 1H), 7.56 (d, J 8.8 Hz, 1H), 8.46 (s, 1H); MS (IE): m/z 355 (M++1), 353 (M+-1), 299, 297, 282, 280 (100), 255, 253, 198, 196, 103, 88, 75, 57; Anal. Calc. for C15H15BrO5: C, 50.72; H, 4.26, Found: C, 50.42; H, 3.93%.

 

Acknowledgments

The authors are grateful to CAPES, CNPq, FAPESP and IFS/OPCW for financial support.

 

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Received: October 5, 2004
Published on the web: May 18, 2005

 

 

FAPESP helped in meeting the publication costs of this article.
* e-mail: agcorrea@power.ufscar.br