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*
a
Department 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
1
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 bioactivityguided 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 &
2
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,
13
C, 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 LH20 compound 1 was also isolated from other active subfractions Ac-13FII (4 mg), Ac3
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). MLFAc12 (121 mg) with 97 % of inhibition on catB was chromatographed over sephadex LH20 (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-ArgAMC (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
4
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.
5
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
6
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.
7
R4
4'
8
R1
6
A
O
C
R2
O
2
R6
2'
4
R5
B
R3
1 R1= OH, R2= OH, R3= O--Rha, R4= R5= R6= OH
2 R1= OH, R2= OH, R3= O--Glc, R4= R5= 67= OH
3 R1= OH, R2= OH, R3= O--Rha, R4=H, R5= R6= OH
4 R1= OH, R2= OH, R3= O-(4''-O-acetyl)--Rha, R4= R5= R6= OH
5 R1= OH, R2= OH, R3= R4= R5= R6= OH
6 R1= OH, R2= OH, R3=OH, R4=H, R5= R6= OH
7 R1= OH, R2= OH, R3= OH, R4=H, R5= OH, R6= H
8 R1= OH, R2= OH, R3= O--Glc, R4=H, R5= R6=OH
9 R1= OH, R2= OH, R3= O--Rha, R4= OH, R5=OCH3, R6= OH
10 R1= OAc, R2= R3=OAc, R4=H R5= R6= OAc
11 R1= OH, R2= OH, R3= H, R4=H R5= R6= OH
12 R1= OCH3, R2=OH, R3= OCH3, R4=H, R5=OCH3, R6=OH
13 R1= OCH3, R2=OH, R3= OCH3, R4=H, R5= R6= OCH3
14 R1= OCH3, R2= R3= OCH3, R4=H, R5= R6= OCH3
R4
4'
R1
A
O
C
6
4
R5
B
2
2'
R6
R3
R2
15 R1= OH, R2= OH, R3= -OH, R4=H, R5= R6=OH
16 R1= OH, R2= OH, R3= -OH, R4=H, R5= R6= OH
17 R1= OH, R2= OH, R3= O--Rha, R4= H, R5= R6= OH
18 R1= OAc, R2= OAc, R3= -OAc, R4= H, R5= R6= OAc
Figure S2. Chemical structures of flavonoids and derivatives. Rha: rhmamnopyranosyl;
Glc: glucopyranosyl.
8
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.
9
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.
10
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.
11
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.
12
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.
13
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.
14
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.
15
1.00
7.5
0.98
7.0
0.94
6.5
0.91
6.0
5.5
1.01
5.0
4.5
1.26
0.9242
0.9110
3.9322
3.8130
3.7133
3.6218
4.2069
4.2032
5.3691
6.3482
6.3444
6.1832
6.1782
7.4150
7.4100
7.3943
7.3893
7.3366
7.0839
7.0619
Methanol-d4
1.00
4.0
3.5
Chemical Shift (ppm)
3.31
3.0
2.5
2.0
1.5
1.0
0.5
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.
16
0
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.
17
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.
18
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.
19
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.
20
Figure S26. 1H NMR spectrum of epicatechin (16) acquired at 400 MHz in MeOD.
Figure S27. 1H NMR spectrum of catechin-3-O-α-rhamnopyranoside (17) acquired at
400 MHz in CD3OD.
21
Figure S28. HSQC of catechin-3-O-α-rhamnopyranoside (17) acquired at 400 MHz in
CD3OD.
Figure S29. HMBC of catechin-3-O-α-rhamnopyranoside (17) acquired at 400 MHz in
CD3OD.
22
Figure S30. 1H NMR spectrum of catechin pentaacetate (18) acquired at 400 MHz in
MeOD.
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