Article pubs.acs.org/jnp
Bioactive Pentacyclic Triterpene Ester Derivatives from Alnus viridis ssp. viridis Bark Miroslav Novakovic,*,† Jasmina Nikodinovic-Runic,‡ Jovana Veselinovic,‡ Tatjana Ilic-Tomic,‡ Vera Vidakovic,§ Vele Tesevic,⊥ and Slobodan Milosavljevic⊥ †
Institute of Chemistry, Technology and Metallurgy, ‡Institute of Molecular Genetics and Genetic Engineering, §Institute for Biological Research “Sinisa Stankovic”, and ⊥Faculty of Chemistry, University of Belgrade, 11000 Belgrade, Serbia S Supporting Information *
ABSTRACT: Seven derivatives of pentacyclic triterpene acids (1−7) were isolated from the bark of Alnus viridis ssp. viridis using a combination of column chromatography and semipreparative HPLC. Compounds 1−3, 6, and 7 were determined to be new after spectroscopic data interpretation and were assigned as 27hydroxyalphitolic acid derivatives (1−3), a 27-hydroxybetulinic acid derivative (6), and a 3-epi-maslinic acid derivative (7), respectively. Pentacyclic triterpenoids with a C-27 hydroxymethyl group have been found in species of the genus Alnus for the first time. These compounds were subjected to cytotoxicity testing against a number of cancer cell lines. Also, selected pentacyclic triterpenoids were selected as potential inhibitors of topoisomerases I and IIα for an in silico investigation.
P
carbons C-3 and C-28 of the lupane skeleton seem to be important for this activity of the triterpenes.14 Betulinic acid exhibits potent antiviral as well as antitumor activity by decreasing the mitochondrial membrane potential and triggering the mitochondrial path to apoptosis in several different cancer cell lines, including melanoma.15,16 Betulinic acid and its derivatives have also been shown to be potent inhibitors of topoisomerases I and IIα by competing with DNA for topoisomerase binding, through direct interaction. Betulinic acid inhibits binary complex formation and the interaction of cellular topoisomerase I with damaged DNA. Although its efficacy is comparable to camptothecin, betulinic acid does not stabilize the “cleavable complex”.17,18 Employing structure− activity relationship studies, the pentacyclic skeleton and the carboxylic acid group were identified as important pharmacophores for topoisomerase inhibitory activity.19 Additionally, betulinic acid exhibits anti-HIV activity by inhibiting the maturation of the virus. Some derivatives (with ester groups at position C-3) are currently promising natural products lead compounds for anti-HIV drugs.20 Alnus viridis (Chaix) DC. ssp. viridis (green alder) was chosen for the present investigation since pentacyclic triterpenes are not the main compounds in the genus Alnus and they have been found in only a few Alnus species.21−23 A. viridis has been chemically investigated mostly for diarylheptanoids and other polyphenols.11 In the present study, seven derivatives of pentacyclic triterpenes (1−7) were isolated from the bark of
entacyclic triterpenes are a diverse group of natural products consisting of five rings and 30 carbon atoms. They can be found in their free form (sapogenins) or bound to sugars (saponins). Extracted from the bark, leaves, or the fruit wax of various plants1 such as Mediterranean Olea spp. or Chinese Zizyphus spp., pentacyclic triterpenes have been associated with multiple beneficial biological activities including prevention and treatment of metabolic and vascular diseases.2,3 Mediterranean diets, rich in olive oil consumption, are usually correlated with longevity and healthiness, due to the abundance of not only vitamins and phenolic substances but triterpenoids as well.4 Some of the best known representatives of the triterpenoids are oleanolic, ursolic, and maslinic acids.5,6 This type of secondary metabolite can also be found in the bark of Betula species (birches), from which betulin and betulinic acid have been isolated. Betulin was isolated in the 18th century by sublimation, and this approach is still in use today.7 In addition, bark triterpenoids were identified in other members of the family Betulaceae, including Corylus spp. (hazels), Caprinus spp. (hornbeams), and Alnus spp. (alders). Although the genus Alnus is better known for other phytochemicals such as diarylheptanoids, stilbenes, flavones, and catechins,8−13 it appears that pentacyclic triterpenoids are significant bark constituents.7 In folk medicine, birch bark (with 10−14% w/w betulin) has been used as an antiseptic and a treatment for a broad variety of diseases. Betulinic acid has been the most widely studied biologically active molecule of this type. Along with betulin and betulone, it was identified as the major antimycobacterial constituent in the bark of A. incana. The functionalities at © 2017 American Chemical Society and American Society of Pharmacognosy
Received: September 3, 2016 Published: April 3, 2017 1255
DOI: 10.1021/acs.jnatprod.6b00805 J. Nat. Prod. 2017, 80, 1255−1263
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Chart 1
A. viridis ssp. viridis, of which five (1−3, 6, 7) were characterized for the first time. The structures of these new compounds were established on the basis of their 1D and 2D NMR, HRESIMS, UV, and IR parameters. The cytotoxic activities of six of these compounds toward melanoma and lung cancer cells have been determined. Molecular docking studies have been performed for these six isolated compounds on both human DNA topoisomerases, given that these enzymes have been identified as targets of betulinic acid.19
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respectively. The aromatic regions of the 1H NMR spectra of 1 and 2 exhibited two systems, one 1,3,4-trisubstituted and the other 1,4-disubstituted. The first system was assigned to a caffeoyl group, and the second to a p-coumaroyl group. The important differences noted between the 1H NMR spectra of 1 and 2 were signals of a p-coumaroyl group, i.e., signals of protons from the double bond. In compound 1 this group showed the E configuration [δH 6.26 (H-7c) and δH 7.56 (H8c) and J ≈ 16.0 Hz], while in compound 2 it was Z [δH 5.72 (H-7c) and δH 6.86 (H-8c) and J ≈ 13.0 Hz]. The 13C NMR spectra of 1 and 2 (Table 1; Figures S3, S4, S7, and S8, Supporting Information) each showed 48 carbon signals, 25 aliphatic, three oxygenated, three carbonyl, and 17 aromatic and olefinic. The HMBC spectra revealed the caffeoyl group to be at position C-2 (Figure 1) in both 1 and 2 and the p-coumaroyl group at position C-3. The configuration at positions C-2 and C-3 for 1 and 2 was established as 2α,3β by NOESY analysis since the correlations H-2/H3-24 and H-3/H3-23 were found and by comparing the coupling constants (J2,3 ≈ 10.0 Hz according to the Karplus equation maximum for trans coplanar protons) and chemical shifts with literature data.24 In the NOESY spectra of 1 and 2, key correlations of H-5/H-9, H-9/ H-27a, H-13/H3-26, H-19/H-13, H-18/H-27b, H3-23/H-5, H323/H-9, H3-24/H3-25, and H3-25/H3-26 were observed. These data suggested that 1 and 2 are triterpenoid diester acids of the lupane type. This was also implied by comparison of data with those of 27-hydroxybetulonic acid recently isolated from Betula platyphylla var. japonica.24 Accordingly, compound 1 was assigned as 2-O-E-caffeoyl-3-O-E-p-coumaroyl-27-hydroxymethylalphitolic acid and 2 as 2-O-E-caffeoyl-3-O-Z-p-coumaroyl27-hydroxymethylalphitolic acid. Compound 3 was isolated as a white solid. The molecular formula was determined as C39H54O8 on the basis of the deprotonated molecular ion peak [M − H]− at m/z 649.37386 (calcd for C39H53O8, 649.37459) in the negative-mode HRESIMS. In the 1H and 13C NMR spectra (Figures S9− S12, Supporting Information) similar patterns of signals were observed to those for 1 and 2, except in the aromatic regions, where only the signals for an E-O-caffeoyl group were seen, whereas the coumaroyl signals were missing. This was confirmed with lower mass in the HRESIMS by 146 Da for 3 in comparison to 1 and 2. The HMBC correlations H-2/C-9c, H-7c/C-9c, H-2c, and H-6c/C-7c revealed the E-O-caffeoyl
RESULTS AND DISCUSSION
Silica gel column chromatography followed by semipreparative reversed-phase HPLC separation of the fractions obtained from the CHCl3/CH3OH extract of the bark of A. viridis ssp. viridis led to the isolation of seven pentacyclic triterpene derivatives (1−7). Compounds 1 and 2 were isolated as brown solids. Their NMR, UV, IR, and MS data were very similar to each other, and thus these substances may be discussed together. The molecular formulas for both substances were determined as C48H60O10 from the deprotonated molecular ion peaks [M − H]− at m/z 795.41138 and 795.41162, respectively (calcd for C48H59O10, 795.41137) in the negative-mode HRESIMS. The IR spectra exhibited absorptions for carboxylic acid (3462 and 3407 cm−1, respectively) and conjugated carbonyl (1691 and 1698 cm−1, respectively) groups. The 1H NMR spectra of 1 and 2 (Table 1; Figures S1, S2, S5, and S6, Supporting Information) showed the presence of five methyl singlets, two oxygenated proton signals at δH 3.70 and 4.11 (d, J ≈ 12.5 Hz, compound 1) and δH 3.79 and 4.20 (d, J ≈ 12.5 Hz, compound 2) from a hydroxymethyl group, and two oxygenated ester proton signals (in each molecule) at δH 4.92 and 5.30 (1), and 4.88 and 5.21 (2). The characteristic chemical shift of a vinylic methyl at δH 1.69 (s) and olefinic proton signals at δH 4.58 (s) and 4.71 (s) for both 1 and 2 suggested the presence of an isopropenyl group in each compound. Olefinic proton signals at δH 6.0−6.2 and δH 7.4−7.6 were assigned to protons at the E double bond conjugated to a carbonyl group (compounds 1 and 2), while olefinic signals at δH 5.72 and δH 6.86 were characteristic for the protons from a Z double bond conjugated to a carbonyl (compound 2) (Table 1; Figures S1, S2, S5, and S6, Supporting Information). Coupling constants of 16.0 and 13.0 Hz were observed for the E and Z configuration of the double bonds, 1256
DOI: 10.1021/acs.jnatprod.6b00805 J. Nat. Prod. 2017, 80, 1255−1263
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Table 1. 1H and 13C NMR Data of Compounds 1−3 Recorded in CD3OD (500 MHz for 1H and 125 MHz for 13C) 1 position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 1cc 2c 3c 4c 5c 6c 7c 8c 9c 1pcd 2pc 3pc 4pc 5pc 6pc 7pc 8pc 9pc
δC 45.7 71.9 82.6 40.8 56.7 19.4 36.5 42.9 53.1 40.1 22.6 26.3 40.3 47.6 24.4 34.2 57.5 50.8 48.5b 152.1 31.8 38.2 29.0 18.2 18.4 17.2 61.1 180.7 110.4 19.8 127.8 115.3 146.8 149.7 116.6 123.2 147.5 115.1 169.1 127.2 131.4 117.0 161.3 117.0 131.4 147.3 115.1 169.6
2 δH
1.16 m, 2.16 dd (12.0; 4.5) 5.30 ddd (11.0; 10.0; 4.5) 4.92 d (10.0)
a
1.15 m 1.51 m, 1.57 m 1.50 m, 1.81 m 1.56 m 1.28 m, 1.40 m 0.88 dd (12.5; 3.5), 1.69 m 2.43 td (12.5; 2.5) 1.33 m, 1.88 m 1.44 m, 2.24 d (12.5) 1.75 m 3.02 m 1.41 1.47 0.93 1.01 1.08 1.00 3.70
m, 1.94 m m, 1.94 m s s s s d (12.5), 4.11 d (12.5)
4.58 brs, 4.71 brs 1.69 s 6.95 d (2.0)
6.72 6.85 7.43 6.09
d (8.5) dd (8.5; 2.0) d (16.0) d (16.0)
7.36 d (9.0) 6.72 d (9.0) 6.72 7.36 7.56 6.26
d d d d
(9.0) (9.0) (16.0) (16.0)
3
δC
δH
δC
δH
45.7 71.9 82.0 40.7 56.8 19.5 36.5 42.9 53.1 40.1 22.6 26.3 40.3 47.6 24.5 34.3 57.6 50.8 48.5b 152.2 31.9 38.2 29.0 18.0 18.3 17.3 61.1 180.7 110.3 19.8 127.8 115.2 146.9 149.7 116.6 123.3 147.5 115.1 168.9 127.8 133.8 116.0 160.1 116.0 133.8 146.3 116.7 168.2
1.12 m, 2.17 dd (12.0; 4.5) 5.21 ddd (11.0; 10.0; 4.5) 4.88 d (10.0)
45.7 74.3 81.3 41.2 57.0 19.6 36.6 42.9 53.2 40.0 22.6 26.4 40.3 47.6 24.5 34.3 57.6 50.9 48.5b 152.2 31.9 38.3 29.2 17.4 18.3 17.2 61.2 180.8 110.3 19.9 128.0 115.3 147.0 149.6 116.6 123.0 146.8 116.0 169.5
0.98 m, 2.11 dd (12.0; 4.5) 5.06 ddd (11.0; 10.0; 4.5) 3.23 d (10.0)
1.14 m 1.48 m, 1.56 m 1.49 m, 1.80 m 1.56 m 1.27 m, 1.41 m 0.90 m, 1.70 m 2.44 td (12.5; 2.5) 1.29 m, 1.87 m 1.44 m, 2.24 d (13.0) 1.75 m 3.03 m 1.39 1.44 0.92 0.86 0.99 1.06 3.79
m, 1.96 m m, 1.95 m s s s s d (12.5), 4.20 d (12.5)
4.58 brs, 4.71 brs 1.69 s 6.95 d (2.0)
6.69 6.76 7.46 6.11
d (8.5) dd (8.5; 2.0) d (16.0) d (16.0)
0.98 m 1.44 m, 1.56 m 1.48 m, 1.76 m 1.48 m 1.27 m, 1.40 m 0.85 m, 1.67 m 2.43 brt (13.0) 1.30 m, 1.87 m 1.42 m, 2.23 d (13.0) 1.73 m 3.03 m 1.39 1.43 1.04 0.86 1.03 0.98 3.78
m, 1.95 m m, 1.94 m s s s s d (12.5), 4.17 d (12.5)
4.57 brs, 4.70 brs 1.68 s 7.04 d (2.0)
6.77 6.94 7.56 6.28
d (8.5) dd (8.5; 2.0) d (16.0) d (16.0)
7.54 d (9.0) 6.70 d (9.0) 6.70 7.54 6.86 5.72
d d d d
(9.0) (9.0) (13.0) (13.0)
J values are given in parentheses. bSignal overlapped with the signal of solvent; value obtained from the HSQC spectrum. c“c”, caffeoyl. d“pc”, pcoumaroyl.
a
axial) protons. Other characteristic signals in the 1H NMR spectrum, including singlets of olefinic protons at δH 4.57 and δH 4.70 (H2-29), doublets of a hydroxymethyl group at δH 3.78 and δH 4.12 (H2-27, J ≈ 12.5 Hz), and an allylic proton at δH 3.03 (H-19), were also visualized (Figures S9 and S10, Supporting Information). The same HMBC and NOESY correlations for the 27-hydroxylupane skeleton as in 1 and 2
group to be at C-2. A significantly lower chemical shift for the H-3 doublet (δH 3.23, J ≈ 10.0 Hz) than for H-3 in 1 and 2 indicated an OH group instead of an ester group at C-3 (Table 1). HMBC and COSY correlations confirmed this OH group by correlations between H-2/C-3 and H-2/H-3, respectively. The NOESY spectrum confirmed a 2α,3β configuration by the NOE correlations H-2/H3-24 and H-3/H3-23, and the coupling constant J2,3 ≈ 10.0 Hz confirmed the trans coplanar (both 1257
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2-O-E-caffeoylalphitolic acid, a known alphitolic acid derivative from Daphniphyllum oldhami.25 Compound 5 has been previously described as the 27-O-ester derivative of 27-hydroxybetulinic acid, known as 27-O-Ecaffeoylcylicodiscic acid, a significant antioxidant and cytotoxic compound.24,26 Compound 6 was isolated as a white solid. The elemental formula was determined as C39H52O7 on the basis of the protonated molecular ion peak [M + H]+ at m/z 633.37867 (calcd for C39H53O7, 633.37857) in the positive-mode HRESIMS, two protons less than 5. Like compounds 3, 4, and 5, compound 6 was found to possess an E-O-caffeoyl group. Its position was the same as in 5, since δH H2-27 was characteristic of the ester moiety in position C-27 (Table 2), and the HMBC correlation H-27a/C-9c matched the data of compound 5. In the 13C NMR spectrum of 6, one additional carbonyl besides C-28 and C-9c was noted at δC 221.5,
Figure 1. Characteristic HMBC correlations in compounds 1−3.
were found. All these observations confirmed 3 to be 2-O-Ecaffeoyl-27-hydroxymethylalphitolic acid. Compound 4 was isolated as a white solid. The 1H and 13C NMR spectra of 4 were similar to those for 3. The only difference was the occurrence of a C-27 methyl substituent in 4 instead of the 27-hydroxymethyl group in 3 (Figures S13 and S14, Supporting Information). Compound 4 was identified as
Table 2. 1H and 13C NMR Data of Compounds 6 and 7 Recorded in CD3OD (500 MHz for 1H and 125 MHz for 13C) 6
a
position
δC
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 1cb 2c 3c 4c 5c 6c 7c 8c 9c
41.0 35.1 221.3 48.3 55.9 20.9 36.1 42.7 52.7 38.4 23.0 26.9 40.6 47.1 25.4 33.9 57.5 50.9 48.4 152.0 31.8 38.1 27.6 21.4 17.2 16.9 64.2 180.3 110.5 19.8 127.8 115.3 147.0 149.9 116.7 123.2 147.2 115.4 169.5
7 δH
δC
1.54 m, 1.94 m 2.45 dt, 2.49 dt
1.52 m 1.51 m, 1.53 m 1.52 m, 1.65 m 1.54 m 1.31 m, 1.53 m 0.99 m, 1.81 m 2.59 brt (13.0)a 1.45 m, 1.87 m 1.30 m, 2.28 brd (13.0) 1.78 m 3.07 m 1.42 1.45 1.06 1.02 0.96 1.08 4.54
m, 1.96 m m, 1.97 m s s s s d (13.0), 4.70 d (13.0)
4.63 brs, 4.75 brs 1.73 s 7.04 d (2.0)
6.79 6.96 7.54 6.26
d (8.0) dd (8.0; 2.0) d (16.0) d (16.0)
39.4 72.0 77.8 40.9 49.6 19.3 34.0 43.2 48.8 39.8 24.7 123.6 145.5 40.1 29.0 24.2 47.8 42.9 47.4 31.8 35.0 34.0 29.3 22.5 17.0 17.9 26.6 182.0 33.7 24.1 128.0 115.3 147.0 149.7 116.6 123.0 147.0 115.8 169.1
δH 1.53 m, 1.62 m 5.29 ddd (12.0; 4.5; 2.5) 3.50 d (2.5) 1.34 m 1.44 m, 1.48 m 1.32 m, 1.55 m 1.83 m 1.94 m, 1.94 m 5.26 t (3.5)
1.07 m, 1.79 m 1.60 m, 2.02 td (13.5; 4.0) 2.86 dd (14.0; 4.0) 1.13 dd (4.5; 2.0), 1.70 m 1.21 1.55 1.01 0.96 1.08 0.83 1.20
m, 1.39 m m, 1.74 m s s s s s
0.91 s 0.94 s 7.04 d (2.0)
6.78 6.95 7.59 6.28
d (8.0) dd (8.0; 2.0) d (16.0) d (16.0)
J values are given in parentheses. b“c”, caffeoyl. 1258
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characteristic for a ketone carbonyl group. Differences in the chemical shifts δH for H-2, H3-23, and H3-24 and δC for C-2 and C-4 in comparison to 5, together with the HMBC correlations H2-2, H-1a, H3-23, H3-24/C-3, indicated a keto carbonyl group at position C-3 (Table 2; Figures S21−S24, Supporting Information). Other signals in the NMR spectra were characteristic for a lupane type skeleton, like 1−5. All these data were similar to those of 27-hydroxybetunolic acid, a known constituent of B. platyphylla var. japonica.24 However, 27-O-E-caffeoylbetunolic acid was found for the first time in this investigation. Compound 7 was isolated as a white solid. The molecular formula was found to be C39H54O7, according to the deprotonated molecular ion peak [M − H]− at m/z 633.37989 (calcd for C39H53O7, 633.37968) in the negative HRESIMS. Similar to compounds 1−4, signals of the E-Ocaffeoyl group were recognized in the 1H and 13C NMR spectra of 7 (Table 2; Figures S25−S28, Supporting Information). The same HMBC correlations as in 1−4 and the chemical shift of H-2 supported the presence of an E-O-caffeoyl moiety at C-2. From the seven methyl group singlets observed, the triplet at δH 5.26, and the absence of the signals for protons from the exocyclic double bond found in 1−6, a skeleton different from 1−6 (lupanes) was proposed. Additionally, HMBC correlations H-12/C-9, C-18 and H3-27/C-13, together with the NOE correlations H-12/H-11, H-18, confirmed the Δ12,13 double bond. These data referred to an oleanane triterpene. Investigation of the configuration at C-3 of 7 led to a 3α configuration. The known 2-O-E-caffeoylmaslinic acid27 possesses a 2α,3β configuration, while 3-epi-maslinic acid possesses a 2α,3α arrangement.28 In compound 7, a 2α,3α configuration was determined according to the coupling constant J2,3 ≈ 2.5 Hz and the strong NOE correlations between H3-23, H3-24/H3 and between H3-24, H3-25/H-2. The coupling constant J2,3 ≈ 2.5 Hz corresponded to a torsion angle H-2/H-3 of about 60° according to the Karplus equation, which means that the OH group is axial, i.e., in an α position. In 1−4 and 2-O-Ecaffeoylmaslinic acid (which is 2α,3β),27 the coupling constant observed was J2,3 ≈ 10.0 Hz, which corresponded to a 3β configuration. Additionally, NOE correlations H3-27/H-19, H21, H-22 were consistent with the cis junction of the D and E rings, characteristic for maslinic acid and an oleanane skeleton. Hence, compound 7 was assigned as 2-O-E-caffeoyl-3-epimaslinic acid, and it has been isolated for the first time. Given that previously characterized triterpenoids have exhibited cytotoxic effects against cancer cell lines,15−20,29−32 the cytotoxicity of six triterpenoids (1−4, 6, and 7) was determined. Using the MTT assay, included were a human lung fibroblast normal cell line (MRC5) and two human cancer cell lines, non-small-cell lung carcinoma (A549) and melanoma (A375). This cytotoxicity study revealed that the isolated derivatives inhibited cell proliferation in a concentrationdependent manner. The IC50 values of the pentacyclic triterpene derivatives isolated and betulinic acid obtained after 48 h of cell exposure are presented in Table 3. On the basis of the cytotoxicity screening results, betulinic acid was less effective in inhibiting the growth of A549 human lung carcinoma and A375 melanoma cells in comparison to the new pentacyclic triterpene derivatives. IC50 values obtained for betulinic acid within this study were in agreement with the literature.33−35 Compound 2 was the most active among the compounds tested against both cancer cell lines, with IC50 values of 3.8 and 5.0 μM for A375 and A549 cells, respectively.
Table 3. Cytotoxicity IC50 Data (μM) of Pentacyclic Triterpene Derivatives from Alnus viridis ssp. viridis on Various Cancer Cell Lines Determined by a MTT Assay cell line compound 1 2 3 4 6 7 BAb cisplatin
MRC5 7.8 10.0 18.5 7.9 9.7 7.9 32.8 3.3
± ± ± ± ± ± ± ±
0.2 0.3 0.5 0.2 0.4 0.3 0.6 0.1
A375 6.3 3.8 11.5 6.3 7.7 11.8 43.7 4.1
± ± ± ± ± ± ± ±
0.1 0.1 0.4 0.3 0.2 0.6 0.7 0.2
A549
SIa A375
SIa A549
± ± ± ± ± ± ± ±
1.25 2.66 1.60 1.25 1.25 0.67 0.75 0.80
1.25 2.00 1.20 0.50 0.63 1.66 0.75 1.32
6.3 5.0 15.4 15.8 15.5 4.7 43.7 2.5
0.1 0.2 0.3 0.5 0.4 0.1 0.5 0.1
a
SI, selectivity indices defined as ratio between IC50 values for noncancerous cells and cancer cells. bBA, betulinic acid.
Moreover, it showed the best selectivity, with a selectivity index (SI) of 2.66 toward A375 and 2.00 toward A549 cells (Table 3). Compound 7 exhibited high activity and selectivity against A549 cells, whereas 1, 4, and 6 exhibited significant activity toward A375 cells, with modest selectivity. To explore the type of cell death induced by the isolated compounds, flow cytometric analysis of annexin V-FITC and propidium iodide (PI) stained cells was performed. Given that compound 2 showed the highest cytotoxicity toward two cancer cell lines, it was selected for examination of its mechanism of action, while betulinic acid was used as a positive control. Flow cytometric analysis of annexin V/PI double staining of treated A549 cells revealed the induction of apoptosis in human lung carcinoma cells (Figure 3). It was shown that the cytotoxic effect of 2 was more pronounced toward cancer cells compared to noncancerous cells. Exposure of A549 cells to compound 2 resulted in a significantly elevated number of early apoptotic cells compared to both betulinic acid-treated and nontreated cells. The percentages of A549 cells in early apoptosis were 12% for compound 2, 5.7% for betulinic acid, and 2.3% for the nontreated control (Table 4). These results suggest that compound 2 induced apoptosis in the A549 lung cancer cell line at low micromolar concentrations after 24 h of incubation. Betulinic acid treatment of A375 cells increased the percentage of apoptotic cells (Figure 2). Apoptotic cells were present in small amounts in the untreated control (3.25%), whereas after treatment with betulinic acid at IC50, 8% of apoptotic melanoma cells were observed (including early and late apoptosis). This is consistent with literature data showing that betulinic acid induces apoptosis in human melanoma cells.36,37 Betulinic acid and its derivatives exert potent antitumor activities by triggering the mitochondrial pathway of apoptosis in several different cancer cell lines. Betulinic acid directly induces loss of the mitochondrial transmembrane potential associated with inhibition of topoisomerases to cause cell death of various cancerous cells. It can also modulate the expression levels of different Bcl-2 family proteins. The treatment of neuroblastoma, glioblastoma, and melanoma cells with betulinic acid resulted in upregulation of the proapoptotic Bcl-2 family protein Bax.38 It was also shown that this triterpene is cytotoxic toward human gastric carcinoma (EPG85-257) and human pancreatic carcinoma (EPP85-181), both drug-sensitive and drug-resistant cell lines.39 1259
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Figure 2. Induction of apoptosis by compound 2 and betulinic acid (BA) in A549 cells. After treatment with IC50 concentration of compound 2 and betulinic acid for 24 h, A549 cells were stained with annexin V and propidium iodide with nontreated cells used as a control. The figure is a representative profile of at least three experiments.
transient breaks in the double helix. Eukaryotic type I topoisomerases are monomeric enzymes that modulate the topology by creating transient single-stranded breaks in the DNA. Eukaryotic type II topoisomerases function as homodimers. Topoisomerase II unwinds, unknots, and untangles the genetic material by generating transient double-stranded breaks in the DNA. Under normal conditions, topoisomerase−DNA covalent complexes are temporary and found at very low levels. Stabilization of these complexes generates DNA lesions, inducing cell arrest and apoptosis.40 However, human DNA topoisomerases (topo I and topo II) are expressed at different levels in different cancer types.41−43 For this reason the inhibition of human DNA topoisomerases is a promising anticancer treatment target. Hence, the effects of the isolated compounds were investigated on the structures of topo I and II. The binding energies of the studied compounds to the active site of topoisomerase I and topoisomerase IIα from applied docking score functions are presented in Tables S2 and S4 (Supporting Information), respectively. Identified hydrogen bonds between ligands and amino acids from the active site of the enzyme are presented in Tables S3 and S5 (Supporting Information), respectively. The best calculated positions for all studied compounds inside the active site of the two enzymes are presented in Figure 3. Two-dimensional representations of interactions of the compounds studied with amino acids inside the binding pocket of the enzyme are presented in Figures S29−S42 (Supporting Information). Molecular docking simulations against human topoisomerases I and IIα were performed, resulting in a correlation between the observed cytotoxic activity (Table 3) and the in silico molecular docking scores of the compounds. The binding affinity of a ligand to the active site of the enzyme can be estimated with score values obtained with the application of scoring functions from the molecular docking method. According to the results from the molecular docking studies for both topoisomerases I and IIα, compound 1 had the highest MolDock score, while betulinic acid had the lowest (Tables S2 and S4, Supporting Information). Similarly, the highest Rerank score value for both topoisomerases was obtained for compound 6. The lowest Rerank score value pertains to compound 1 for topoisomerase I and 3 for topoisomerase IIα. The pose energy score value determines the internal energy of a ligand. According to the obtained results, the highest energy of the studied ligand for both topoisomerase activities was obtained for 1. According to ligand efficiency parameters (LE1 and LE3) for the topoisomerase I activity,
Figure 3. Best calculated positions for all pentacyclic triterpenoids studied inside the active site of topoisomerase I (A) and topoisomerase IIα (B).
Table 4. FACS Analysis of AnnexinV/PI Staining of the A549 Cell Line Treated with Compound 2 and Betulinic Acid treatment
annexin V
annexin V/PI
PI
2 BAa nontreated
12.0 ± 0.2 5.7 ± 0.2b 2.3 ± 0.3
0.3 ± 0.5 2.3 ± 0.1 1.0 ± 0.1
1.0 ± 0.2 1.3 ± 0.4 0.7 ± 0.2
a
BA, betulinic acid (positive control). bValues expressed as % of total cell populations calculated from three independent experiments and are expressed as means ± SD.
Betulinic acid and its derivatives have been reported to inhibit topoisomerases I and IIα with the pentacyclic skeleton and carboxylic acid group identified as important pharmacophores for topoisomerase inhibitory activity.17−19 For cellular processes such as replication, recombination, and transcription, topoisomerases are essential enzymes that relax DNA supercoiling because they modulate the DNA topology by generating 1260
DOI: 10.1021/acs.jnatprod.6b00805 J. Nat. Prod. 2017, 80, 1255−1263
Journal of Natural Products
Article
were obtained. Prior to the investigation of biological activity, the purity of all compounds was checked by HPLC-DAD at 280 nm and by NMR spectroscopy. The purity was 98% for compounds 2, 4, and 6 and 99% for compounds 1, 3, and 7, while compound 5 could not be purified sufficiently for the further investigation of bioactivity. 2-O-E-Caffeoyl-3-O-E-p-coumaroyl-27-hydroxymethylalphitolic acid (1): brown solid; [α]22 D −168.5 (c 1.0, CH3OH); UV (CH3OH) λmax (log ε) 222 (4.28), 235 (sh) (4.17) 250 (3.87), 297 (4.44), 314 (4.47) nm; IR (KBr) νmax 3462, 2950, 1691, 1632, 1605, 1516, 1448, 1377, 1271, 1171, 1117, 1016, 984, 884, 831 cm−1; 1H NMR and 13C NMR data, see Table 1; HRESIMS m/z 795.41138 [M − H]− (calcd for C48H60O10 − H, 795.41137). 2-O-E-Caffeoyl-3-O-Z-p-coumaroyl-27-hydroxymethylalphitolic acid (2): brown solid; [α]22 D +39.3 (c 1.0, CH3OH); UV (CH3OH) λmax (log ε) 221 (4.33), 236 (sh) (4.16), 252 (4.00), 298 (4.34), 316 (4.37) nm; IR (KBr) νmax 3407, 2946, 1698, 1628, 1605, 1515, 1448, 1390, 1271, 1169, 1117, 1022, 985, 887, 854 cm−1; 1H NMR and 13C NMR data, see Table 1; HRESIMS m/z 795.41162 [M − H]− (calcd for C48H60O10 − H, 795.41137). 2-O-E-Caffeoyl-27-hydroxymethylalphitolic acid (3): white solid; [α]22 D −19.0 (c 1.0, CH3OH); UV (CH3OH) λmax (log ε) 218 (4.06), 233 (sh) (3.89), 244 (3.90), 298 (4.02), 329 (4.13) nm; IR (KBr) νmax 3377, 2945, 1696, 1630, 1605, 1559, 1516, 1452, 1387, 1272, 1177, 1119, 1042, 983, 881, 857 cm−1; 1H NMR and 13C NMR data, see Table 1; HRESIMS m/z 649.37386 [M − H]− (calcd for C39H54O8 − H, 649.37459). 27-O-E-Caffeoylbetunolic acid (6): white solid; [α]22 D −10.0 (c 1.0, CH3OH); UV (CH3OH) λmax (log ε) 218 (4.11), 249 (3.89), 298 (4.07), 328 (4.18) nm; IR (KBr) νmax 3463, 2947, 2871, 1693, 1634, 1605, 1518, 1453, 1383, 1273, 1174, 1116, 1020, 982, 886, 856 cm−1; 1 H NMR and 13C NMR data, see Table 2; HRESIMS m/z 633.37867 [M + H]+ (calcd for C39H52O7 + H, 633.37857). 2-O-E-Caffeoyl-3-epi-maslinic acid (7): white solid; [α]22 D +26.0 (c 1.0, CH3OH); UV (CH3OH) λmax (log ε) 216 (4.15), 234 (3.93), 244 (3.93), 300 (4.05), 329 (4.16) nm; IR (KBr) νmax 3430, 2930, 2862, 1695, 1631, 1557, 1518, 1455, 1386, 1273, 1159, 1117, 1060, 1018, 988, 813 cm−1; 1H NMR and 13C NMR data, see Table 2; HRESIMS m/z 633.37989 [M − H]− (calcd for C39H54O7 − H, 633.37968). Cytotoxicity Assay. Cytotoxic activities of the triterpenoids, as well as betulinic acid and cisplatin (Sigma-Aldrich, Munich, Germany), were measured using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay.45 The following cell lines (obtained from ATCC) were used in this study: human melanoma (A375), human lung carcinoma (A549), and noncancerous human lung fibroblasts (MRC5). The cells were plated in a 96-well flatbottomed plate at a concentration of 1 × 104 cells per well, grown in a humidified atmosphere of 95% air and 5% CO2 at 37 °C, and maintained as monolayer cultures in RPMI-1640 medium supplemented with 100 μg/mL streptomycin, 100 U/mL penicillin, and 10% (v/v) fetal bovine serum (FBS). After 24 h of incubation, the medium containing increasing concentrations of each test compound (2.5; 5.0; 7.5; 10.0; 12.5; 25.0; 50.0 μg/mL) was added to the cells. Compounds were dissolved in DMSO and filter-sterilized (0.2 μm, EMD Millipore, Billerica, MA, USA) to prepare stock solutions (50 mg/mL) prior to usage in the cell-culture experiments. Control cultures received the solvent DMSO, and blank wells contained 200 μL of growth medium. After 48 h of further incubation with each test compound, cell viability was determined using an MTT assay. The percentage viability values were plotted against the log of concentration, and a sigmoidal dose− response curve was calculated by nonlinear regression analysis using Graphpad Prism software, version 5.0, for Windows (Graphpad Software, San Diego, CA, USA). Cytotoxicity is expressed as the concentration of the compound that inhibited growth by 50% (IC50). Flow Cytometric Analysis (Annexin V-FITC/Propidium Iodide Apoptosis Assay). Apoptosis of noncancerous and cancer cell lines was detected by flow cytometry. Briefly, the cells were seeded in six-well plates (2.5 × 105 per well) and incubated for 24 h at 37 °C and in 5% CO2. Cells were then treated with the concentration of each compound that caused 50% cell death (IC50 concentration values) for 24 h, treated with trypsin, and washed in PBS (phosphate buffer
compounds 6 and 4 are identified as ligands with the highest efficiency, while the lowest values were obtained for compound 2. According to ligand efficiency parameters (LE1 and LE3) for the topoisomerase IIα activity, 4 and 1 were identified as ligands with the highest efficiency, while the least promising results were obtained for 2. The number of hydrogen bonds and length and energy of the hydrogen bonds formed between ligand and enzyme have an important role in the ligand’s effect on the investigated activity. The H-bond value represents the total energy of hydrogen bonds between the ligand and the amino acids in the active site of the enzyme. The H-bond parameter values for topoisomerase I demonstrated that interactions were the greatest for 1 and 4, while for topoisomerase IIα they were greatest for 3 and 6. It can be deduced that all pentacyclic triterpene derivatives investigated in this study have a higher potential to inhibit human topoisomerases than betulinic acid, which is a known topoisomerase inhibitor.19 Recently, a number of semisynthetic betulinic acid analogues were reported to possess strong inhibitory effects on topoisomerases I and II, in addition to cytotoxic activity against cancer cells compared to betulinic acid.44
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EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were measured on a Rudolph Research Analytical Autopol IV automatic polarimeter. UV spectra were recorded on a GBC Cintra UV/vis spectrometer. IR spectra were obtained on a Thermoscientific Nicolet 6700 FT-IR spectrometer. NMR spectra were recorded on a Bruker Avance III 500 spectrometer at 500.26 MHz for 1H and 125.80 MHz for 13C, with CD3OD as a solvent. HRESIMS data were obtained on an Agilent Technologies 6210 TOF LC/MS system. For column chromatography (CC) silica gel 60 (SiO2;
