SUPPLEMENTARY MATERIAL

Evaluation of flavonols and derivatives as human cathepsin B inhibitor

Suelem D. Ramalhoa, Lorena R. F. de Sousaa, Marcela C. M. Burgera,

Maria Inês S. Limab, M. Fátima das G. F. da Silvaa, João B. Fernandesa and

Paulo C. Vieiraa*

aDepartment of Chemistry, Federal University of São Carlos, 13565-905 São Carlos,

SP, Brazil; bDepartment de Botany, Federal University of São Carlos, 13565-905 São

Carlos, SP, Brazil.

*Corresponding author: Email: dpcv@ufscar.br

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Evaluation of flavonols and derivatives as human cathepsin B inhibitor

Cathepsin B is a cysteine proteases involved in tumor progression and represent a

potential therapeutic target in cancer. Among the fiftheen evaluated extracts from

cerrado biome Myrcia lingua Berg. (Myrtaceae) extract demonstrated to be a

source of compounds with potential to inhibit cathepsin B. Using bioactivity-

guided fractionation, we have found flavonols as inhibitors and also some other

derivatives were obtained. From the evaluated compounds, myricetin (5) and

quercetin (6) showed the most promising results with IC50 of 4.9 and 8.2 μM,

respectively and mode of inhibition as uncompetitive on catB. The results

demonstrated polyhydroxylated flavonols as promising inhibitors of catB.

Keywords: flavonoids; cathepsin B; uncompetitive inhibitor.

Experimental

Chemistry

Bio-activity guided fractionation of the ethyl acetate fraction from Myrcia lingua Berg.

(Myrtaceae) leaves extracts resulted in the isolation of some active flavonols glycosides

such as myricetin 3-O-α-rhamnopyranoside (1) (Arot, Midiwo & Kraus, 1996),

myricetin 3-O-β-glucopyranoside (2) (Scharbert, Holzmann & Hofmann, 2004),

quercetin 3-O-α-rhamnopyranoside (3) (dos Santos, Schripsema & Kuster, 2005) and

myricetin O-(4”-O-acetyl)-α-rhamnopyranoside (4) (Mahmouda et al., 2001). In order

to have a better investigation on this class some other compounds previously isolated in

the laboratory of natural products were also evaluated on proteases as shown below.

Tamarixetin 3-O-α-rhamnopyranoside (9) (Son, Lee & Han, 2005) was isolated from

Esenbeckia grandiflora Mart. (Rutaceae); catechin-3-O-α-rhamnopyranoside (17)

(Ayres et al., 2008) was obtained from Vochysia thyrsoidea Pohl. (Vochysiaceae);

catechin (15) (Watanabe, 1998) and epicatechin (16) (Watanabe, 1998) were isolated

from Byrsonima coccolobifolia Kunth. (Malpighiaceae). Quercetin (6) (≥ 95 % - Sigma

337951), kaempferol (7) (≥ 97 % - Sigma 60010), quercetin 3-β-D-glucoside (8) (≥ 90

% - Sigma 17793) and luteolin (11) (≥ 98 % - Sigma L9283) were purchased from

Sigma Aldrich.

Some other derivatives were obtained as shown below. Myricetin (5) was obtained by

acid hydrolysis of compound 1 (Pizzolatti et al., 2003). Compound 6 and 15 were

acetylated with acetic anhydride in pyridine, resulting in quercetin pentaacetate (10) (de

Almeida et al., 2010) and catechin pentaacetate (18) (Basak, Mandal &

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Bandyopadhyay, 2003; de Almeida et al., 2010), respectively. Quercetin (6) was also

treated with diazomethane and gave the three corresponding methyl esters compounds

quercetin-3,7,4'-trimethyl ether (12) (Kaulich et al., 2003), quercetin-3,7,3',4'-

tetramethyl ether (13) (Sutthanut et al., 2007) and permethylated quercetin pentamethyl

ether (14) (Sutthanut et al., 2007) that were isolated using sephadex LH-20 column. All

derivatives were characterized by NMR spectra (1H, 13C, COSY, HSQC and HMBC)

and the obtained data were compared with those from the literature. 1D and 2D NMR

spectra were recorded on a Bruker DRX-400 NMR spectrometer (400 MHz).

Plant material

The leaves of Myrcia lingua Berg. (Myrtaceae) were collected in May 2011 at São

Carlos, São Paulo state, Brazil. A voucher specimen (8366) was deposited at the

herbarium of Botany Department (HUFSCar) at Federal University of São Carlos,

Brazil.

Bio-activity guided fractionation

The ethanolic extract of seven cerrado species were prepared by maceration of the air

dried and powdered parts of the plants. The crude extracts were obtained after dryness

of the solvents by rotavap under reduced pressure at 40 ºC. The extracts were suspended

in a solution containing ethanol/ water (1:3) and submitted to a liquid-liquid partition

with the organic solvents hexane and EtOAc, to yield the corresponding hexane, EtOAc

and hydroalcoholic new fractions. All the obtained extracts and fractions were

submitted to an enzymatic assay against catB and L.

The ethyl acetate fraction (MLFAc) from M. lingua Berg. leaves containing 12.0 g

showed significant inhibition on catB at the concentration 125 µg/mL. MLFAc was

fractionated on silica gel 60 column 230-400 Mesh (6.0 x 22.0 cm; 2:8 hexane/acetate)

affording 13 fractions that were subsequently evaluated on both enzymes. From the

obtained results fractions MLFAc-12 (121 mg) and MLFAc-13 (3.2 g) were selected for

further isolation process. MLFAc-13 with 95 % of inhibition on catB was

chromatographed over sephadex LH-20 (4.0 x 65 cm; MeOH isocratic) and 6

subfractions were obtained. Active subfraction Ac-13D (16.5 mg) after

chromatographic separations by sephadex LH-20 (1.5 x 47 cm; MeOH isocratic) yielded

subfraction Ac-13DIV, identified as compound 1 (2.1 mg). Using sephadex LH-20

compound 1 was also isolated from other active subfractions Ac-13FII (4 mg), Ac-

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13EIII (2 mg) being one of the major compounds on the evaluated fractions. Fraction

Ac-13D also afforded the subfraction Ac-13DIII (4.5 mg), which was chromatographed

over sephadex LH-20 (1.5 x 47 cm; MeOH isocratic) and yielded compound 2 (1.7 mg).

Fraction Ac-13B (45 mg) was chromatographed over sephadex LH-20 (2.0 x 140 cm;

MeOH isocratic) affording 5 fractions. The obtained active fraction Ac-13BII (15.3 mg)

was refractioned using sephadex LH-20 (1.5 x 47 cm; MeOH isocratic) and 3

subfractions were obtained. HPLC separation of the active subfraction Ac-13BIIC (6.5

mg) was performed using C-18 Phenomenex Luna 10 µm column (1.0 x 25 cm) at a

flow-rate of 4.0 mL/ min with isocratic elution using as mobile phase acetonitrile/H2O

(20:80, v/v) and monitoring at 240 and 350 nm to give compound 3 (0.8 mg). MLFAc-

12 (121 mg) with 97 % of inhibition on catB was chromatographed over sephadex LH-

20 (2.0 x 140 cm; MeOH:CH2Cl2 (1:1)) affording 5 fractions. The active subfraction Ac-

12E (17 mg) was chromatographed over sephadex LH-20 (1.5 x 47.0 cm; MeOH

isocratic) affording 4 fractions. HPLC separation of the active subfraction Ac-12EII (8.4

mg) was performed using the same conditions above described to give compound 4 (1.5

mg). Isolation procedures were carried out on silica gel 60 (Merck, 230-400 mesh),

sephadex LH-20 and analytical thin-layer chromatography (TLC) were performed on

pre-coated aluminum silica 60 F254 (Merck).

Kinectic measurements

All commercially available chemicals, catB (Sigma C8571) and catL from human liver

(Sigma C6854) were purchased from Aldrich Chemical Co. and Sigma. Kinetic

measurements were carried out in a fluorimeter Molecular Devices Spectra MAX M3.

Inhibitory activity was measured using the synthetic fluorometric substrate Z-Phe-Arg-

AMC (benzyloxycarbonyl-phenylalanyl-arginine 4-methyl-7-coumarylamide) at the

concentration of 185 μM for catB (Km 123 μM) and 10 μM for catL (Km 1.2 μM). CatB

was assayed at 62 nM and catL was assayed at 55 nM. The enzyme was activated

during 5 min with DTE and acetate buffer (pH 5.5) at 37 ºC, and then the reaction

mixture was incubated during 5 min with the sample. The experiments were carried out

in triplicate (in 96-well black plates) and the final volume of the reaction mixture was

200 μL, kept under stirring (λex 355 nm, and λem 460 nm). All inhibitors were screened

against catL and B at initial concentration of 100 μM. Control assays were performed

without inhibitor (negative control) and in the presence of the irreversible inhibitor for

cysteine peptidase, E-64 (positive control). Values of IC50 were determined by rate

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measurements with at least six inhibitor concentrations. The inhibition type and Ki were

determined at the same experimental conditions. All kinetic parameters were

determined by nonlinear regression employing the SigmaPlot 12.0 enzyme kinetics

module and the type of inhibitor was established by Lineweaver-Burk plots of 1/V

versus 1/S at different inhibitor concentrations.

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Table S1. Inhibitory activities of flavonoids towards catL and B

Compound IC50 (μM)

Cathepsin L Cathepsin B

1 114.5 ± 10.3 37.2 ± 3.0

2 ND 17.2 ± 1.4

3 - 193.4 ± 32.9

4 ND 22.4 ± 3.2

5 23.9 ± 2.1 4.9 ± 0.5

6 26.3 ± 1.8 8.2 ± 0.9

7 28.4 ± 2.3 15.0 ± 1.1

8 115.0 ± 8.6 -

9 - -

10 47.2 ± 2.9 -

11 79.2 ± 4.1 36.2 ± 4.1

12 - -

13 - -

14 - -

15 179.0 ± 15.9 174.2 ± 15.4

16 - -

17 - -

18 - -

E-64b 0.027 ± 0.004 0.037 ± 0.004

Note: The values represent means of three individual experiments ± SE. bE-64 as

reference positive control. ND: not determined. Empty space represented as (-) means

values ˃ 250 μM. * P < 0.05, Student’s t-test analysis.

Table S2. Ki values of flavonoids on catB

Compound Ki (µM) Inhibition type

5 11.3 Uncompetitive

6 9.0 Uncompetitive

7 11.4 Uncompetitive

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Figure S1. Screening of crude extracts towards catB and L. Ethanolic extract (E); stems

(C); leaves (F); roots (R); Drimys brasiliensis (DB); Memora axillaris (MA); Tabebuia

ochracea (TO); Erythroxylum suberosum (ES); Vochysia tucanorum (VT); Dalbergia

miscolobium (DM); Myrcia lingua (ML). Negative control (acetate buffer and DMSO).

The values represent means of three individual experiments ± SE. ns P ≥ 0.05, Student’s

t-test analysis.

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Figure S2. Chemical structures of flavonoids and derivatives. Rha: rhmamnopyranosyl;

Glc: glucopyranosyl.

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Figure S3. Ki graphics of compounds 5 (A), 6 (B) and 7 (C) on catB. Kinetics

measurements were conducted in the presence of increasing concentration of inhibitors.

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Figure S4. 1H NMR spectrum of myricetin 3-O-α-rhamnopyranoside (1) acquired at 400 MHz in MeOD.

Figure S5. HSQC of myricetin 3-O-α-rhamnopyranoside (1) acquired at 400 MHz in MeOD.

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Figure S6. HMBC of myricetin 3-O-α-rhamnopyranoside (1) acquired at 400 MHz in MeOD.

Figure S7. 1H NMR spectrum of myricetin 3-O-β-glucopyranoside (2) acquired at 400 MHz in MeOD.

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Figure S8. HSQC of myricetin 3-O-β-glucopyranoside (2) acquired at 400 MHz in MeOD.

Figure S9. HMBC of myricetin 3-O-β-glucopyranoside (2) acquired at 400 MHz in MeOD.

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Figure S10. 1H NMR spectrum of quercetin 3-O-α-rhamnopyranoside (3) acquired at 400 MHz in DMSO-d6.

Figure S11. HSQC of quercetin 3-O-α-rhamnopyranoside (3) acquired at 400 MHz in DMSO-d6.

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Figure S12. 1H NMR spectrum of myricetin O-(4”-O-acetyl)-α-rhamnopyranoside (4) acquired at 400 MHz in DMSO-d6.

Figure S13. HSQC of myricetin O-(4”-O-acetyl)-α-rhamnopyranoside (4) acquired at 400 MHz in DMSO-d6.

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Figure S14. HMBC of myricetin O-(4”-O-acetyl)-α-rhamnopyranoside (4) acquired at 400 MHz in DMSO-d6.

Figure S15. 1H NMR spectrum of myricetin (5) acquired at 400 MHz in MeOD.

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Figure S16. 1H NMR spectrum of tamarixetin 3-O-α-rhamnopyranoside (9) acquired at 400 MHz in MeOD.

Figure S17. HSQC of tamarixetin 3-O-α-rhamnopyranoside (9) acquired at 400 MHz in MeOD.

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Figure S18. 1H NMR spectrum of quercetin pentaacetate (10) acquired at 400 MHz in Acetone-d6.

Figure S19. 1H NMR spectrum of quercetin-3, 7, 4'-trimethyl ether (12) acquired at 400 MHz in DMSO-d6.

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Figure S20. HMBC of quercetin-3, 7, 4'-trimethyl ether (12) acquired at 400 MHz in DMSO-d6.

Figure S21. 1H NMR spectrum of quercetin-3, 7, 3', 4'-tetramethyl ether (13) acquired at 400 MHz in DMSO-d6.

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Figure S22. HMBC of quercetin-3, 7, 3', 4'-tetramethyl ether (13) acquired at 400 MHz in DMSO-d6.

Figure S23. 1H NMR spectrum of permethylated quercetin pentamethyl ether (14) acquired at 400 MHz in DMSO-d6.

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Figure S24. HMBC of permethylated quercetin pentamethyl ether (14) acquired at 400 MHz in DMSO-d6.

Figure S25. 1H NMR spectrum of catechin (15) acquired at 400 MHz in MeOD.

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Figure S26. 1H NMR spectrum of epicatechin (16) acquired at 400 MHz in MeOD.

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Figure S27. 1H NMR spectrum of catechin-3-O-α-rhamnopyranoside (17) acquired at 400 MHz in CD3OD.

Figure S28. HSQC of catechin-3-O-α-rhamnopyranoside (17) acquired at 400 MHz in CD3OD.

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Figure S29. HMBC of catechin-3-O-α-rhamnopyranoside (17) acquired at 400 MHz in CD3OD.

Figure S30. 1H NMR spectrum of catechin pentaacetate (18) acquired at 400 MHz in MeOD.

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