Parvifoline Derivatives as Tubulin Polymerization Inhibitors - Journal

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Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX

Parvifoline Derivatives as Tubulin Polymerization Inhibitors Edna M. Silva-García,† Carlos M. Cerda-García-Rojas,*,† Rosa E. del Río,‡ and Pedro Joseph-Nathan† †

Departamento de Química y Programa de Posgrado en Farmacología, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, Apartado 14-740, Mexico City 07000, Mexico ‡ Instituto de Investigaciones Químico-Biológicas, Universidad Michoacana de San Nicolás de Hidalgo, Ciudad Universitaria, Morelia, Michoacán 58030, Mexico J. Nat. Prod. Downloaded from pubs.acs.org by UNIV OF NEW ENGLAND on 03/18/19. For personal use only.

S Supporting Information *

ABSTRACT: A series of functionalized sesquiterpenoids derived from benzocyclooctene, including natural parvifoline (1), isoparvifoline (3), epoxyparvifoline (5), epoxyisoparvifoline (7), 8,12-oxyparfivoline (9), 8,14-oxyparvifoline (11), and the respective benzoyl derivatives 2, 4, 6, 8, 10, and 12, were prepared and tested for their inhibitory effect on the in vitro α,βtubulin polymerization process. The structural analysis and characterization of the new compounds 5−7 and 9−12 were achieved by 1D and 2D NMR spectroscopy, mass spectrometry, and X-ray diffraction analysis of 6, 7, and 9. Preparation of 9 and 12 involved molecular rearrangements of the epoxide group with transannular 1,5-hydride shifts. At 10 μM compounds 1, 5, and 8 inhibited the polymerization of the α,β-tubulin heterodimer by 24%, 49%, and 90% as compared to colchicine. These compounds were subjected to docking analysis that supported their interactions in a colchicine binding site located in the αtubulin subunit, in the pocket formed by Phe296, Pro298, Pro307, His309, Tyr312, Lys338, Thr340, Ile341, and Gln342. Competitive inhibition assays with colchicine were also performed for the three compounds, which supported their binding at the colchicine secondary site in α-tubulin. Also, evaluations of their cytotoxicity on MCF7 breast carcinoma, HeLa cervix carcinoma, and HCT 116 colon carcinoma cell lines were carried out and showed that 8 is active against the HeLa and HCT 116 cell lines with IC50 3.3 ± 0.2 and 5.0 ± 0.5 μM, respectively. he α,β-tubulin heterodimer is the basic structural unit of microtubules and one of the most studied targets for cancer therapy due to its significant role in cellular replication.1 The microtubule dynamic activity is based on polymerization and depolymerization processes, which can be modified by interaction with natural compounds and derivatives at specific sites in the protein, as has been the case for colchicine, taxanes, and vinca alkaloids. Thus, new substances that modify this process could in principle be included in biological studies and later in drug development protocols. Sesquiterpenoids constitute a large group of natural products that have demonstrated to induce antiproliferative activity,2 and in particular, some of them interfere with the microtubule cytoskeleton.3−5 Parvifoline (1) is a bicyclic sesquiterpenoid isolated in good yields from several species of the Asteraceae family, namely, Coreolepsis parvifolia,6 Perezia carpholepis,7 P. longifolia,8 and P. alamanii var. oolepis.9 Compound 1 and some analogues have been the subject of several synthetic protocols10−14 that have

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© XXXX American Chemical Society and American Society of Pharmacognosy

overcome the challenge of the eight-membered-ring formation. The chemical structure of parvifoline (1) shares some molecular features with colchicine, a representative inhibitor of tubulin polymerization.15 Both compounds have a mediumsize carbocyclic moiety attached to a benzene ring with electronegative atoms. In this work, a series of 12 functionalized benzocyclooctenes, including natural parvifoline (1) and the 11 derivatives 2−12 (Figure 1), were obtained and tested for their inhibitory effect on the α,β-tubulin polymerization process. The structural analysis and characterization of the seven new compounds 5−7 and 9−12 were achieved mainly by 1D and 2D NMR spectroscopy and X-ray diffraction analysis. Those molecules that inhibited the polymerization of the α,β-tubulin heterodimer were subjected to docking studies, which supported their interactions at a colchicine binding site. Received: October 26, 2018

A

DOI: 10.1021/acs.jnatprod.8b00860 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

epoxide. Subsequently, the oxygen atom attacks the tertiary C8 carbocation to form the tetrahydrofuran moiety of 9. Correspondingly, Figure 3 shows the transformation of

Figure 1. Formulas of parvifoline (1) and derivatives 2−12.

Figure 3. Proposed reaction mechanism for the formation of 12.

RESULTS AND DISCUSSION Preparation of Parvifoline Derivatives. Parvifoline (1)8 was obtained in good yields from the roots of Acourtia humboldtii, while compounds 2−4 and 8 were prepared as previously reported.16 In order to access a series of parvifoline derivatives, the new compounds 5−7 and 9−12 were obtained by epoxidation reactions, transposition of the epoxide groups, and esterification and hydrolysis reactions. Thus, the best yields of epoxides 5−7 were achieved by treatment of 1−3, respectively, with m-ClC6H4CO3H in CH2Cl2 under reflux, while compounds 9 and 12 were obtained by treatment of 5 and 8, respectively, with Et2O−BF3 in anhydrous benzene. To complete the series, compound 10 was prepared by esterification of 9 with benzoyl chloride in pyridine, while 11 was obtained by alkaline hydrolysis of 12 with KOH in MeOH. It is known that Et2O−BF3 efficiently promotes molecular rearrangements in carbocyclic structures.17 When epoxyparvifoline (5) was treated with this reagent, it underwent a rearrangement of the epoxide group to form the ether linkage between C-8 and C-12 of 9. As represented in Figure 2, this transformation involves a transannular 1,5-hydride shift18 from C-8 to C-13 with simultaneous opening of the BF3-activated

epoxyisoparvifoline benzoate (8) by treatment with Et2O− BF3, which also proceeds through a 1,5-hydride shift from C-8 to C-13 with epoxide ring opening. The oxygen atom, now at C-14, attacks the C-8 carbocation to assemble the transannular ether bond between C-14 and C-8, yielding 12. Structure Elucidation. The structures of the new compounds 5−7 and 9−12 followed from their physical and spectroscopic data, including 1D and 2D NMR studies, highresolution mass spectrometry, and X-ray diffraction analysis. The molecular formula of 12,13-epoxyparvifoline (5) was established as C15H20O2 by HRESIMS, showing a lithium adduct molecular ion at m/z 239.1623 (calcd as 239.1618 for [C15H20O2 + Li]+). Its 1H NMR data (Table 1) displayed distinctive signals for H-1 and H-4 as singlets at δH 6.55 and 6.86, respectively, while the signals for H-8, H-12, and the methylene at C-14 appeared at δH 2.99 (m), 2.74 (dd, J = 6.5 and 2.3 Hz), and 3.02 (d, J = 15.8 Hz) for H-14α and 3.22 (d, J = 16.4 Hz) for H-14β, respectively. The 13C NMR data (Table 2) confirmed the presence of the carbon atoms bearing the epoxide group at δC 63.5 (C-12) and 62.0 (C-13). COSY correlations (Figure S11, Supporting Information) were used to assign the hydrogen atoms attached to the C-9−C-8−C10−C-11−C-12 fragment, while the HMBC correlations (Figure S12, Supporting Information) between C-14 (δC 39.0) and H-4 (δH 6.86) and between C-14 and the methyl group attached to the epoxide ring H3-15 (δH 1.39) confirmed the epoxidation at C-12−C-13. The HRESIMS data of 12,13-epoxyparvifoline benzoate (6) also showed a lithium adduct molecular ion at m/z 337.1801 (calcd as 337.1798 for [C22H24O3 + Li]+). The 1H and 13C NMR spectra showed patterns similar to those of 5 (Tables 1 and 2). The H-1 and H-4 atoms were shifted at higher frequencies with respect to 5. They were shown as singlets at δH 6.87 and 7.03, respectively, while the signals for H-8, H-12, and the methylene at C-14 were detected at δH 3.06 (m), 2.77 (dd, J = 6.3 and 3.0 Hz), and 3.12 (d, J = 16.1 Hz) for H-14α and 3.28 (d, J = 16.4 Hz) for H-14β, respectively. The aromatic signals for the benzoyl moiety were located at δH 8.21 (2H, dd, J = 7.4, 1.3 Hz), 7.64 (1H, br t, J = 7.4 Hz), and 7.51 (2H, br t, J = 7.4 Hz) and at δC 164.8, 133.5, 130.1, 129.6, and

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Figure 2. Proposed reaction mechanism for the formation of 9. B



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Table 1. 1H NMR Data of Compounds 5−7 and 9−12 (300 MHz, CDCl3)a δH (ppm), mult. (J in Hz) hydrogen 1 4 7 8 9 10α 10β 11α 11β 12α 12β 13 14α 14β 15 HO 2′ 3′ 4′ 5′ 6′

5 6.55, 6.86, 2.16, 2.99, 1.32, 1.90, 1.16, 1.80, 0.68,

6

s s s m d (6.4) m m m m

6.87, 7.03, 2.15, 3.06, 1.35, 1.96, 1.22, 1.87, 0.76,

7

s s s m d (6.7) m m m m

2.74, dd (6.5, 2.3)

2.77, dd (6.3, 3.0)

3.02, 3.22, 1.39, 4.75,

3.12, d (16.1) 3.28, d (16.1) 1.43, s

d (16.4) d (16.4) s s

8.21, 7.51, 7.64, 7.51, 8.21,

6.65, 7.13, 2.21, 2.93, 1.29, 1.73, 1.34, 1.54, 1.18, 1.53, 1.83,

9

s s s m d (7.0) m m m m m m

3.76, s 1.52, s 4.81, s

10

11

12

6.67, s 6.83, s 2.18, s

6.98, s 6.99, s 2.15, s

6.45, s 6.90, s 2.25, s

6.82, s 7.07, s 2.24, s

1.68, 2.12, 1.89, 2.23, 1.80,

s m m m m

1.69, 1.92, 2.21, 2.30, 1.81,

s ddd (15.6, 4.7, 1.9) ddd (15.6, 7.0, 3.9) m m

4.20, 1.78, 2.80, 2.80, 1.00, 4.61,

dt (7.6, 2.5) m m m d (7.0) br s

4.23, 1.84, 2.89, 2.89, 1.03,

dt (7.6, 2.3) m m m d (7.0)

1.57, 1.70, 1.70, 1.41, 0.86, 1.50, 1.44, 1.82,

1.61, 1.78, 1.78, 1.49, 0.86, 1.58, 1.45, 1.90,

dd (7.4, 1.3) br t (7.4) br t (7.4) br t (7.4) dd (7.4, 1.3)

8.22, dd (7.4, 1.3) 7.52, br t (7.4) 7.64, br t (7.4) 7.52, br t (7.4) 8.22 dd (7.4, 1.3)

s m m m m m m m

4.94, s 1.15, d (7.0) 4.82, br s

s m m m m m m m

5.01, s 1.19, d (7.1) 8.23, dd (7.4, 1.3) 7.53, br t (7.4) 7.65, br t (7.4) 7.53, br t (7.4) 8.23 dd (7.4, 1.3)

a

Assigned by gCOSY, gNOESY, gHSQC, and gHMBC.

Table 2. 13C NMR Data of Compounds 5−7 and 9−12 (74.5 MHz, CDCl3)a

C-13. Likewise, the X-ray analyses of 7 and 9, also depicted in Figure 4, supported their structures and corroborated the 5 → 9 molecular rearrangement. The absolute configuration of 9, as shown in Figure 1, was verified by calculation of the Flack and Hooft X-ray parameters,19,20 which were small (0.1 and 0.07) for the correct enantiomer and much higher (0.9 and 0.93) for the inverted structure. In accordance with its X-ray structure, compound 7 showed its molecular ion at m/z 239.1638 (calcd as 239.1618 for [C15H20O2 + Li]+). Three distinctive 1H NMR signals appeared as singlets at δH 7.13, 6.65, and 3.76, which were assigned to H-4, H-1, and H-14. The phenol signal was observed at δH 4.81 (s), and that for H-8 was found at 2.93 (m). The HMBC correlations between C-3 (δC 127.4) and H1, H-8, and H-14 (Figure S20, Supporting Information) were used to confirm the position of the epoxide ring at C-13−C-14. Its 1H and 13C NMR spectra were assigned by COSY, gHSQC, and gHMBC experiments as listed in Tables 1 and 2. Compound 9 displayed a protonated molecular ion at m/z 233.1538 (calcd as 233.1536 for [C15H20O2 + H]+) also in line with its X-ray structure (Figure 4 and Table 3). The signals for aromatic H-1 and H-4 were observed at δH 6.67 (s) and 6.83 (s), respectively, while that for H-12 geminal to oxygen was shifted at higher frequencies (δH 4.20, dt, J = 7.6, 2.5 Hz) with respect to those found in 5 (δH 2.74) as expected for the rearrangement of epoxide to tetrahydrofuran (Figure 2). The H-14α and H-14β chemical shifts were similar, both close to δH 2.80, forming an ABX system with H-13, which was located at δH 1.78 (m). The HMBC correlations of C-8 (δC 84.6) with H-1 and the methyl group H3-9 (δH 1.69, s), as well as that of C-12 (δC 84.2) with the secondary methyl group H3-15 (δH 1.00, d, J = 7.0 Hz), supported the location of the ether bridge between C-8 and C-12. Compound 10 showed a sodium adduct molecular ion at m/ z 359.1626 (calcd as 359.1617 for [C22H24O3 + Na]+), in agreement with the incorporation of the benzoyl group, which

δC (ppm) carbon

5

6

7

9

10

11

12

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1′ 2′ 3′ 4′ 5′ 6′ CO

111.0 143.7 127.6 133.0 120.4 153.4 15.2 32.5 19.4 35.3 26.2 63.6 62.1 39.0 26.4

117.8 143.7 133.4 133.2 127.3 149.2 15.7 32.7 19.4 35.3 26.3 63.5 61.9 39.4 26.4 129.6 130.1 128.6 133.5 128.6 130.1 164.8

110.4 144.7 127.4 131.0 121.3 153.9 15.2 34.3 22.1 40.7 23.9 33.1 62.2 63.4 22.3

112.2 146.1 130.4 134.2 120.8 151.6 15.1 84.6 27.9 39.7 32.5 84.2 39.3 38.8 19.5

118.7 146.2 136.1 134.3 127.6 147.4 15.7 84.5 28.0 39.5 32.4 84.2 38.9 39.1 19.4 129.6 130.1 128.6 133.5 128.6 130.1 164.9

106.5 144.2 137.4 122.6 122.8 153.5 16.0 87.8 26.5 44.6 22.0 33.5 41.7 87.0 22.2

113.7 144.1 143.0 122.8 129.3 149.1 16.4 87.8 26.5 44.6 22.0 33.4 41.5 86.6 22.3 129.5 130.1 128.6 133.6 128.6 130.1 164.9

a

Assigned by gHSQC and gHMBC.

128.6. The 13C NMR data (Table 2) confirmed the presence of the carbon atoms bearing the epoxide group at δC 63.5 (C-12) and 61.9 (C-13), while the aromatic C-1, C-5, and C-6 were observed at δC 117.8, 127.3, and 149.2, respectively, in agreement with the esterification of the hydroxy group at C-6. The X-ray diffraction study of 6 (Figure 4 and Table 3) confirmed its structure and the α-oriented epoxide at C-12 and C



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7.52 (2H, br t, J = 7.4 Hz) and the shift at higher frequencies with respect to 9 of H-1 and H-4 atoms that were shown as singlets at δH 6.98 and 6.99, respectively. The 13C NMR data also displayed the pertinent changes for the ester moiety incorporation, in particular for C-1, C-3, C-5, and C-6, which were observed at δC 118.7, 136.1, 127.6, and 147.4, respectively. The remaining signal pattern was similar to that of 9, as is evident in Tables 1 and 2. Compounds 11 and 12 showed sodium and lithium adduct molecular ions at m/z 255.1361 (calcd as 255.1355 for [C15H20O2 + Na]+) and m/z 343.1894 (calcd as 343.1880 for [C22H24O3 + Li]+), respectively. The 1H NMR spectrum of 11 displayed three singlets at δH 6.90, 6.45, and 4.94 assigned to H-4, H-1, and H-14, respectively. The H-14 signal did not show a measurable coupling constant with H-13 since, according to a molecular model (Figure 4), the H-13−C13−C-14−H-14 dihedral angle was −93°. The signal for the aromatic methyl group was found at δH 2.25 (br s), while the 9- and 15-aliphatic methyl groups were located at δH 1.60 (s) and 1.15 (d, J = 7.0 Hz), respectively. The signals for the aliphatic hydrogen atoms of the C-10−C-11−C-12−C-13 fragment were located between δH 1.90 and 1.30, with the exception of H-11β, which resonated at δH 0.84 because of its spatial location close to the β-face of the aromatic ring, as shown in the molecular model of 11. The 1H NMR spectrum of the benzoyl derivative 12 resembled that of 11, although H1 is shifted from δH 6.45 to 6.98, due to the esterification of the phenolic group, while the signals of the benzoyl group were observed at δH 8.23 (2H, dd, J = 7.4, 1.3 Hz), 7.65 (1H, br t, J = 7.4 Hz), and 7.53 (2H, br t, J = 7.4 Hz). The gHMBC plot of 12 supports its structure, in particular the correlations of H-14 (δH 5.01) with C-2 (δC 144.1), C-3 (δC 143.0), C-8 (δC 87.3), C-12 (δC 33.4), and C-15 (δC 22.3), as well as the correlations of CH3-15 (δH 1.19) with C-12 (δC 33.4), C-13 (δC 143.0), and C-14 (δC 87.3). Tubulin Polymerization Inhibition. Parvifoline (1) and derivatives 2−12 were evaluated toward the dynamic instability of microtubules to explore their interactions with the α,β-tubulin heterodimer. Since 1 has some structural resemblance to colchicine, it would be expected that the compounds could inhibit the protein in the in vitro polymerization test. A variation of the described method21 was employed for this assay, which was carried out using porcine brain tubulin in a complex buffer containing guanosine 5′-triphosphate (GTP) and the compounds dissolved in DMSO. The mixtures of protein, GTP, and the compounds were incubated at 37 °C, and the α,β-tubulin polymerization was followed by variation of the absorbance (ΔA) at 450 nm for 60 min. Then, a depolymerizing stimulus (−20 °C) was applied followed by reincubation at 37 °C, measuring again the absorbance to evaluate repolymerization. Table 4 summarizes the results for the percentage of tubulin polymerization at 10 and 50 μM concentrations after 60 and 90 min. Graphics for the tubulin polymerization in the presence of compounds 1− 12 and that of colchicine are included in the Supporting Information (Figures S39−S57). The inhibitory effect of colchicine was evaluated in the polymerization experiments using different concentrations. It was observed that at 100 μM the amount of colchicine was sufficient to fully inhibit polymerization after 30 min, at 50 μM colchicine polymerization inhibition prevails by 94%, while at 10 and 5 μM the phases of polymerization22 could be observed more clearly with inhibition of 48% and 37%, respectively (Supporting

Figure 4. X-ray diffraction structures of 12,13-epoxyparvifoline benzoate (6), 13,14-epoxyparvifoline (7), and 8,12-oxyparvifoline (9) and molecular model of 8,14-oxyparvifoline (11).

was also consistent with the presence of 1H NMR signals at δH 8.22 (2H, dd, J = 7.4, 1.3 Hz), 7.64 (1H, br t, J = 7.4 Hz), and D



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Table 3. Crystal Data for Parvifoline Derivatives 6, 7, and 9 6 empirical formula fw cryst size (mm) cryst syst space group unit cell dimensions (Å) a b c volume (Å3) Z, ρ calculated (mg/mm3) absorp coeff (mm−1) F(000) θ range for data collection (deg) limiting indices collected reflns unique reflns completeness to θ (%) data/restraints/params goodness of fit on F2 final R indices [I > 2σ(I)] (%) largest diff peak and hole (e Å3) Flack and Hooft params inverted Flack and Hooft params

7

9

C22H24O3 336.41 0.30 × 0.30 × 0.20 orthorhombic P212121



5.9169(1) 8.0880(1) 38.6754(4) 1850.85(4) 4, 1.207 0.628 720 4.57 to 77.49 −6 ≤ h ≤ 7, −10 ≤ k ≤ 10, −48 ≤ l ≤ 48 69 095 [R(int) = 0.0001] 3933 99.7 3802/0/238 1.067 R1 = 3.10, wR2 = 8.51 0.127 and −0.124 −0.03(19) and −0.0(6) 1.03(19) and 1.00(5)

9.508(5) 10.278(3) 14.150(6) 1382.8(10) 4, 1.116 0.570 504 5.32 to 60.09 −1 ≤ h ≤ 10, 0 ≤ k ≤ 11, 0 ≤ l ≤ 14 1434 [R(int) = 0.0348] 1274 95.4 1169/0/170 1.056 R1 = 3.9, wR2 = 10.7 0.155 and −0.123

7.903(1) 9.777(1) 16.893(2) 1305.2(3) 4, 1.182 0.604 504 5.23 to 66.92 −9 ≤ h ≤ 8, −11 ≤ k ≤ 9, −19 ≤ l ≤ 19 9285 [R(int) = 0.0464] 2206 96.8 1870/0/161 1.039 R1 = 3.7, wR2 = 8.4 0.104 and −0.127 0.1(3) and 0.07(14) 0.9(3) and 0.93(14)

Table 4. Tubulin Polymerization Inhibition (%) of Colchicine and Compounds 1−12 polymerization inhibition at 10 μM

polymerization inhibition at 50 μM

compound

60 min

90 mina

60 min

90 mina

DMSO colchicineb 1 2 3 4 5 6 7 8 9 10 11 12

0.0 ± 5.5 100.0 ± 3.0 24.4 ± 3.7 11.8 ± 15.2 −45.8c ± 15.1 38.7 ± 25.4 48.7 ± 5.6 30.3 ± 6.2 30.3 ± 5.4 89.9 ± 15.5 40.2 ± 9.8 26.1 ± 15.8 38.7 ± 2.5 9.2 ± 4.1

0.0 ± 2.1 100.0 ± 3.5 51.0 ± 11.2 56.3 ± 7.5 58.1 ± 25.3 35.4 ± 10.5 46.9 ± 5.3 47.9 ± 8.1 8.3 ± 10.7 97.9 ± 15.3 43.7 ± 27.5 −10.4c ± 16.9 21.9 ± 15.1 2.1 ± 4.1

0.0 ± 5.5 100.0 ± 3.0 54.6 ± 2.0 46.2 ± 3.6 −13.3c ± 4.6 86.6 ± 13.9 33.6 ± 10.4 10.9 ± 10.5 19.3 ± 18.3 51.8 ± 18.3 66.3 ± 1.9 76.9 ± 9.5 76.1 ± 2.7 −3.4c ± 14.7

0.0 ± 2.1 100.0 ± 3.5 66.7 ± 8.7 58.9 ± 4.7 56.2 ± 11.4 105.7 ± 11.7d 44.7 ± 6.4 42.2 ± 8.8 36.9 ± 10.7 78.5 ± 18.6 67.0 ± 10.2 46.9 ± 20.9 62.5 ± 1.6 −5.7c ± 10.4

These values were measured in the repolymerization phase. bColchicine concentration was 10 μM in both cases. cNegative values indicate that the compound promoted polymerization. dValues above 100% indicate that the tested compound at the indicated concentration inhibited polymerization more efficiently than colchicine 10 μM.

a

Molecular Docking Analysis. Parvifoline (1) and those derivatives that inhibited tubulin polymerization more efficiently than 1 were subjected to a docking study using a described protocol.23 Thus, molecular structures of colchicine, parvifoline (1), and derivatives 5 and 8 were built and initially minimized using molecular mechanics force-field calculations followed by a Monte Carlo exploration to determine all possible conformers. The conformers within an energy interval of 3 kcal/mol were geometry optimized using the Hartree− Fock method with the 3-21G basis set to localize the global minimum structure which was reoptimized using density functional theory at the B3LYP/DGDZVP level of theory. The crystallographic file of tubulin was obtained from the RCSB

Information, Figure S39). The in vitro experiments carried out with parvifoline (1) reflected that at 10 μM the polymerization was favored during the first 30 min; however during the subsequent 30 min, the polymerization decreased substantially (Supporting Information, Figure S40). This effect was more evident in the repolymerization stage at minute 80. Compound 1 showed a much better inhibitory effect at 50 μM. The more active compounds at 10 μM were 5 and 8 (Supporting Information, Figures S44 and S47). The inhibitory effect of 5 at 10 μM was comparable to the inhibitory effect of 1 at 50 μM, while 8 showed an effect close to that of colchicine at 10 μM. E



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Figure 5. (a) Merging of ligands at the highest affinity binding site on α-tubulin subunit. (b) Docking interactions of colchicine with the amino acid residues at the binding site. (c) Docking interactions of parvifoline (1). (d) Docking interactions of 5. (e) Docking interactions of 8.

Table 5. Docking Data for 1, 5, 8, and Colchicine in the Secondary Site at the α-Tubulin Subunit and Colchicine in the Primary Site between the α- and β-Tubulin Subunits optimized Edocka

kind of interaction

1

−7.88

5

−7.14

nonpolar hydrogen bond nonpolar polar hydrogen bond π−π interaction nonpolar polar hydrogen bond π−π interaction nonpolar polar hydrogen bond nonpolar

ligand

8

−9.19

colchicine at the secondary binding site on αtubulin

−7.07

colchicine at the primary binding site between the α- and β-tubulin

−8.99

amino acid residues with interaction Pro298, Phe296, Pro307, His309, Gly310, Lys311, Thr340, Ile341 Tyr312 (donor residue), Gln342 (acceptor residue) Pro298, Pro307, Ile341 Tyr312, Thr340 Lys311 (donor residue), Gln342 (acceptor residue) Phe296 Pro298, Pro307, His309, Tyr312, Thr340, Ile341, Gln342 Gly310 Lys311 (donor residue) Phe296 Phe296, Pro307, His309, Thr340, Ile341, Gln342 Pro298, Tyr312 Lys338 (donor residue) α-tubulin: Gly10, Gln11, Gln15, Asn101, Gly142, Gly143, Thr145; β-tubulin: Arg2, Thr36, Leu248, Lys254 α-tubulin: Ser140, Tyr224 α-tubulin: Ala12 (donor residue), Gly144 (donor residue), Glu183 (acceptor residue)

polar hydrogen bond a

Binding energy in kcal/mol determined with AutoDock v. 4.2.6.

of all possible interaction sites of each ligand with the protein. In the second step, the docking analysis was optimized and the interactions of 1, 5, 8, and colchicine with the protein were refined to explain the inhibitory effect observed in the in vitro analysis. The binding site with the highest affinity for 1, 5, and 8 was found on the surface of the α-tubulin subunit, in the

Protein Data Bank under the code 1JFF, which corresponds to Bos taurus α,β-tubulin heterodimer refined at 3.50 Å resolution.24 The docking analyses were carried out in two steps with the AutoDock program using colchicine as the control compound. The first step involved an initial scanning on the entire tubulin surface to complete a general exploration F



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pocket formed by turns 12, 13, 15, and 16 and coils 26 and 31 (Figure 5a) comprising a domain that includes Cys295, Phe296, Pro298, Cys305, Pro307, His309, Tyr312, Cys315, Cys316, Lys338, Thr340, Ile341, and Gln342. According to a literature search, colchicine has three binding sites.25,26 The primary and most studied site is located at the interphase between the α- and β-tubulin subunits,25 while for the two secondary sites, one resides in the α-tubulin subunit in the domain that includes four cysteine residues,26 namely, Cys295, Phe296, Cys305, Pro307, His309, Tyr312, Cys315, and Cys316, and the other one resides over the β-tubulin subunit around Val23, Asn26, Tyr36, His299, Ala233, Phe244, and Phe272.25 In view of our docking studies, parvifoline (1) and derivatives 5 and 8 mainly interact with tubulin in the colchicine secondary site located in the α-subunit and not in the main colchicine site between the two heterodimers. According to the binding energy of active compounds 1, 5, and 8, they interact with the tubulin α-subunit more strongly than colchicine at this binding site, although the binding energy for colchicine in its main binding site is much stronger than in the secondary site. Table 5 summarizes the docking energy for each compound, the kind of interactions with the protein, and the amino acids involved in the contacts. The interaction of colchicine in the tubulin α-subunit is shown in Figure 5b, while that for 1, 5, and 8 is represented in Figure 5c−e. A coordinate set of the docked models of the three sesquiterpenoids and colchicine appears in Figure S58 (Supporting Information), which shows the interaction in the same site located in the pocket formed by Cys295, Phe296, Pro298, Cys305, Pro307, His309, Tyr312, Cys315, Cys316, Lys338, Thr340, Ile341, and Gln342. In addition, it is important to emphasize the proximity of this pocket with the α-tubulin tail that plays a crucial role in the polymerization.27 Competitive Inhibition and Cytotoxicity Assays. With the aim to support the docking analysis, a competitive inhibition assay28 was performed using the three most active compounds, 1, 5, and 8, in the presence of colchicine, which is a natural fluorophore when interacting with tubulin. Interestingly, 1 and 5 showed a noticeable competitive effect when colchicine was added at 50 μM (Figure 6) but not at the usual concentration of 5 μM.28 These data are consistent with the competition occurring at a secondary binding site, once the first site has been saturated by colchicine. In the case of compound 8, an unusual fluorescence intensity effect was observed when colchicine was supplied at 5 and 50 μM (Figure 6). This result may perhaps be explained by a modification of the colchicine−tubulin complex at the main binding site when 8 binds to the secondary site. An abnormal behavior was also observed for this compound in the polymerization assays (Table 4), in which an evident drop in the inhibitory activity was observed when the concentration was increased from 10 μM to 50 μM. A possibility related to a more complex competition phenomenon, once the microtubules are being formed, is worth further exploration. To complement the tubulin polymerization inhibitory properties of 1, 5, and 8, an evaluation of their cytotoxicity on MCF7 breast carcinoma, HeLa cervix carcinoma, and HCT 116 colon carcinoma cell lines was achieved.29−31 As summarized in Table 6, compound 8 was active toward the HeLa and HCT 116 cell lines. In conclusion, the preparation and structural characterization of a systematic series of parvifoline derivatives permitted a study of their in vitro and in silico interaction

Figure 6. Competitive inhibition assays of 1, 5, and 8 with colchicine at (a) 5 μM and (b) 50 μM. The graphs represent the fluorescence values for each sample (F) divided by the fluorescence of the complex colchicine−tubulin (F0) at 60 min of interaction versus the competitor concentration. Paclitaxel and nocodazole were employed as negative and positive controls, respectively.

Table 6. Cytotoxicity of Derivative 8 on Cervix Carcinoma (HeLa) and Colon Carcinoma (HCT 116) Cell Lines IC50 (μM) compound

HeLa

HCT 116

8 colchicine

3.3 ± 0.2 0.011 ± 0.003

5.0 ± 0.5 0.040 ± 0.018

with α,β-tubulin. Although several compounds inhibited tubulin polymerization at different levels, the best effects were shown by (8R,12S,13R)-12,13-epoxyparvifoline (5) and (8R,13S,14R)-13,14-epoxyisoparvifoline benzoate (8). The inhibitory effect of the later was comparable to that of colchicine. According to the docking analysis, compounds 1, 5, and 8 inhibit polymerization of the α,β-tubulin heterodimer by interacting at the colchicine binding site close to the α-tubulin tail. Competitive inhibition assays of the three compounds with colchicine supported their binding at the colchicine secondary site in α-tubulin. Also, the evaluation of their cytotoxicity on three cancer cell lines was in agreement with the noticeable tubulin polymerization inhibitory properties of 8.

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EXPERIMENTAL SECTION

General Experimental Procedures. Melting points were determined on a Fisher-Johns apparatus and are uncorrected. Optical rotations were recorded in CHCl3 solutions on a PerkinElmer 341 polarimeter. IR spectra were obtained on a BUCK Scientific 500 spectrometer in CHCl3 solutions. 1D and 2D NMR spectra were measured at 300 MHz for 1H and 75.4 MHz for 13C on a Varian G



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chromatography using mixtures of hexanes−EtOAc. The fractions eluted with hexanes−EtOAc (9:1) yielded a white solid that was recrystallized from hexanes−CH2Cl2 to afford 9 (14 mg, 61%) as colorless prisms: mp 135−136 °C; [α]589 −3, [α]578 −3, [α]546 −4, [α]436 −9, [α]365 −18 (c 1.2, CHCl3); UV (EtOH) λmax (log ε) 220 (3.24), 282 (2.78) nm; IR (CHCl3) νmax 3588, 3303, 2970, 1499, 1454, 1286, 1108, 1090 cm−1; 1H and 13C NMR spectra, see Tables 1 and 2; EIMS m/z 232 [M]+ (47), 189 (31), 175 (100), 159 (30), 149 (27), 91 (19); HRESIMS m/z 233.1538 [M + H]+ calcd for [C15H20O2 + H]+ 233.1536. (8R,12S,13R)-8,12-Oxyparvifoline Benzoate (10). A solution of 9 (20 mg) in pyridine (0.5 mL) was treated with benzoyl chloride (20 μL). The mixture was heated on a steam bath for 3 h, poured over ice−H2O, and extracted with EtOAc. The organic layer was washed with 10% HCl, H2O, aqueous NaHCO3, and H2O, dried over anhydrous Na2SO4, filtered, and evaporated to dryness. The residue was chromatographed using mixtures of hexanes−EtOAc as the eluent. The fractions eluted with hexanes−EtOAc (19:1) gave 10 (20 mg, 70%) as colorless oil: [α]589 −9, [α]578 −9, [α]546 −10, [α]436 −19, [α]365 −33 (c 0.7, CHCl3); UV (EtOH) λmax (log ε) 225 (3.53) nm; IR (CHCl3) νmax 2971, 2927, 1731, 1601, 1265, 1074 cm−1; 1H and 13C NMR spectra, see Tables 1 and 2; EIMS m/z 336 [M]+ (11), 279 (5), 105 (100), 77 (30); HRESIMS m/z 359.1626 [M + Na]+ calcd for [C22H24O3 + Na]+ 359.1617. (8R,13S,14S)-8,14-Oxyisoparvifoline (11). A solution of 12 (36 mg) in CH3OH (3 mL) was treated with a solution of 10% KOH (0.2 mL). The mixture was refluxed for 30 min, concentrated under vacuum, poured over ice−H2O, and extracted with EtOAc. The organic layer was washed with H2O, dried over anhydrous Na2SO4, filtered, and evaporated to dryness. The residue was chromatographed using mixtures of hexanes−EtOAc as the eluent. The fractions eluted with hexanes−EtOAc (9:1) gave 11 (17 mg, 68%) as a colorless oil: [α]589 +18, [α]578 +19, [α]546 +23, [α]436 +51, [α]365 +113 (c 0.5, CHCl3); UV (EtOH) λmax (log ε) 221 (2.94), 280 (2.67) nm; IR (CHCl3) νmax 3603, 2927, 1450, 1248, 1168 cm−1; 1H and 13C NMR spectra, see Tables 1 and 2; EIMS m/z 232 [M]+ (31), 217 (9), 190 (100), 176 (11), 163 (12); HRESIMS m/z 255.1361 [M + Na]+ calcd for [C15H20O2 + Na]+ 255.1355. (8R,13S,14S)-8,14-Oxyisoparvifoline Benzoate (12). A solution of 8 (47 mg) in dried benzene (2.5 mL) was cooled at 0 °C and treated with Et2O−BF3 (50 μL). The mixture was stirred for 12 h at room temperature under anhydrous conditions. After workup as for 9, the residue was purified by column chromatography using mixtures of hexanes−EtOAc. Fractions eluted with hexanes−EtOAc (9:1) gave 12, which was further purified by preparative TLC developing with hexanes−EtOAc (9:1) (Rf 0.40) to yield the pure compound (16 mg, 34%) as a colorless oil: [α]589 +15, [α]578 +16, [α]546 +18, [α]436 +39, [α]365 +78 (c 0.58, CHCl3); UV (EtOH) λmax (log ε) 229 (3.35), 272 (2.72) nm; IR (CHCl3) νmax 2956, 2924, 1739, 1453, 1267, 1165 cm−1; 1H and 13C NMR spectra, see Tables 1 and 2; EIMS m/z 336 [M]+ (24), 294 (100), 105 (49), 77 (23); HRESIMS m/z 343.1894 [M + Li]+ calcd for [C22H24O3 + Li]+ 343.1880. Single-Crystal X-ray Diffraction. The data of 6 and 9 were collected on a Bruker D8 Venture diffractometer, while those of 7 were collected on a Bruker-Nonius CAD4 diffractometer using Cu Kα radiation (λ = 1.541 84 Å) at 293(2) K in the ω/2θ scan mode. The crystal data for compounds 6, 7, and 9 are summarized in Table 3. The structures were solved by direct methods using the SHELXS-97 program included in the WinGX v1.70.01 crystallographic software package. For the structural refinement, the non-hydrogen atoms were treated anisotropically, and the hydrogen atoms, included in the structure factor calculation, were refined isotropically. Crystallographic data (excluding structure factors) have been deposited at the Cambridge Crystallographic Data Centre. Copies of the data can be obtained free of charge on application to the CCDC, 12 Union Road, Cambridge CB2 IEZ, UK. Fax: +44-(0)1223-336033 or e-mail: [email protected]. The CCDC deposition numbers for 6, 7, and 9 are 1901572, 1901573, and 1901575, respectively. Tubulin Polymerization Experiments. Parvifoline (1) and derivatives 2−12 were dissolved in DMSO, 10 μL placed on 96-well

Mercury 300 spectrometer from CDCl3 solutions using tetramethylsilane as internal reference. Chemical shift values are reported in ppm, and coupling constants (J) are in Hz. LRMS were measured on a Varian Saturn 2000 GC-MS system, while HRESIMS data were recorded on a Waters Synapt G2 spectrometer at the Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO, USA. Silica gel 230−400 mesh (Merck) was used for column chromatography. Plant Material. Specimens of Acourtia humboldtii (Less.) B.L.Turner were collected during the flowering stage on September 30, 2008, near km 63 of the Morelia−Carapan federal road no. 15, N 19°44.45′ W 101°40.99′ at 2285 m above sea level. A voucher specimen (No. 227798) was deposited at the Herbarium of Instituto ́ Pátzcuaro, Michoacán, de Ecologia,́ A. C., Centro Regional del Bajio, Mexico, where Prof. Jerzy Rzedowski identified the plant material. Isolation of Parvifoline (1). Air-dried roots of A. humboldtii (300 g) were extracted with hexanes (1 L) for 5 days at room temperature. Filtration and evaporation of the extract afforded a brown viscous oil (41 g). A portion of the extract (5 g) was chromatographed over silica gel using hexanes−EtOAc (19:1) as eluent. Fractions of 20 mL were collected, monitored by TLC, and analyzed by 1H NMR spectroscopy. Parvifoline (1) was isolated as colorless crystals, mp 89−90 °C (50 mg, 1%) [lit.7 89−90 °C]. (8R,12S,13R)-12,13-Epoxyparvifoline (5). A solution of 1 (140 mg) in CH2Cl2 (8 mL) was treated with m-ClC6H4CO3H (140 mg). The mixture was refluxed for 3 h, poured over ice−H2O, and extracted with CH2Cl2. The organic layer was washed with H2O, aqueous NaHCO3, and H2O, dried with anhydrous Na2SO4, filtered, and evaporated to dryness. The product was purified by column chromatography using mixtures of hexanes−EtOAc as the eluent. Compound 5 was obtained in the fractions eluted with hexanes− EtOAc (19:1) (120 mg, 80%) as a colorless oil: [α]589 −62, [α]578 −65, [α]546 −74, [α]436 −132, [α]365 −225 (c 0.5, CHCl3); UV (EtOH) λmax (log ε) 221 (3.03), 281 (2.53) nm; IR (CHCl3) νmax 3588, 3399, 2956, 2921, 1277, 1156 cm−1; 1H and 13C NMR spectra, see Tables 1 and 2; EIMS m/z 232 [M]+ (45), 215 (39), 176 (100), 147 (36); HRESIMS m/z 239.1623 [M + Li]+ calcd for [C15H20O2 + Li]+ 239.1618. (8R,12S,13R)-12,13-Epoxyparvifoline benzoate (6). A solution of 2 (65 mg) in CH2Cl2 (4 mL) was treated with m-ClC6H4CO3H (65 mg) under reflux for 2 h and processed as above. The residue was purified by column chromatography with hexanes−EtOAc (49:1) as the eluent. Fractions 70−85 yielded a white solid, which was recrystallized from CHCl3−hexanes to give 6 (39 mg, 57%) as white prisms: mp 75−77 °C; [α]589 −47, [α]578 −50, [α]546 −56, [α]436 −99, [α]365 −163 (c 1.9, CHCl3); UV (EtOH) λmax (log ε) 201 (4.59), 224 (4.12) nm; IR (CHCl3) νmax 2977, 2939, 1743, 1509, 1462, 1273 cm−1; 1H and 13C NMR spectra, see Tables 1 and 2; EIMS m/z 336 [M]+ (5), 319 (8), 279 (10), 105 (100), 77 (26); HRESIMS m/z 337.1801 [M + H]+ calcd for [C22H24O3 + H]+ 337.1798. (8R,13S,14R)-13,14-Epoxyisoparvifoline (7). A solution of 3 (200 mg) in CH2Cl2 (10 mL) was treated with m-ClC6H4CO3H (200 mg) as in the case for 5. The crude product was chromatographed using mixtures of hexanes−EtOAc as the eluent. The fractions eluted with hexanes−EtOAc (19:1) gave a white solid, which was recrystallized using CHCl3−hexanes to yield 7 (150 mg, 70%) as colorless prisms: mp 134−135 °C; [α]589 +37, [α]578 +39, [α]546 +44, [α]436 +71 (c 1.5, CHCl3); UV (EtOH) λmax (log ε) 222 (2.82), 280 (2.27) nm; IR (CHCl3) νmax 3590, 3383, 2959, 2925, 1618, 1502, 1232, 1157 cm−1; 1 H and 13C NMR spectra, see Tables 1 and 2; EIMS m/z 232 [M]+ (24), 215 (12), 189 (100), 176 (23), 159 (26); HRESIMS m/z 239.1638 [M + Li]+ calcd for [C15H20O2 + Li]+ 239.1618. (8R,12S,13R)-8,12-Oxyparvifoline (9). A solution of 5 (23 mg) in dried benzene (1.2 mL) was cooled to 0 °C and treated with Et2O− BF3 (25 μL). The mixture was stirred for 12 h at room temperature under anhydrous conditions, poured over ice−H2O, and extracted with EtOAc. The organic layer was washed with H2O, aqueous NaHCO3, and H2O, dried with anhydrous Na2SO4, filtrated, and evaporated under vacuum. The residue was purified by column H



Article

half-area ELISA plates at final concentrations of 10, 50, and 100 μM. The experiments were carried out using porcine brain α,β-tubulin 97% (Cytoskeleton, Inc.) with a described protocol.21,23 Freshly reconstituted solutions of the protein in a tubulin general buffer at pH 6.9, consisting of 80.0 mM piperazine-N,N′-bis(2-ethanesulfonic acid) sesquisodium salt, 2.0 mM MgCl2, 0.5 mM ethylene glycol-bis(βaminoethyl ether)-N,N,N′,N′-tetraacetic acid, and 1.0 mM guanosine 5′-triphosphate to give a final tubulin concentration of 2 mg/mL, were added, in aliquots of 100 μL at 0 °C, to the wells having the compounds to be tested. The plate was transferred to a BioTek EL808 IU microplate reader preheated (30 min) at 37 °C. The tubulin polymerization was monitored by measuring the absorbance at 450 nm every minute for 60 min, followed by application of a depolymerizing stimulus by cooling the plate at −20 °C for 15 min and reincubation at 37 °C for 40 min. DMSO (10 μL) was used as negative control and colchicine as positive control. The reading at time zero was subtracted from subsequent readings to obtain Δabsorbance. All assays were carried out in triplicate, and graphs were prepared in GraphPad Prism (GraphPad Software). The results were evaluated using an analysis of variance followed by a Tukey’s test, p < 0.05. Competitive Inhibition Assays. Parvifoline (1) and derivatives 5 and 8, as well as paclitaxel and nocodazole as controls, were dissolved in DMSO. Aliquots of 10 μL were placed on 96-well half-area ELISA plates to provide final concentrations of 10, 20, and 50 μM. Freshly reconstituted solutions of porcine brain α,β-tubulin 97% in the tubulin general buffer as described above for the tubulin polymerization experiments, with the exception of guanosine 5′-triphosphate, were added to each well in aliquots of 100 μL at 0 °C to give a final tubulin concentration of 1 mg/mL. The plate was transferred to a BioTek Synergy HTX reader preheated (30 min) at 37 °C. The fluorescence (F) was monitored at 350 nm for excitation and 430 nm for emission every minute for 30 min. Aliquots (10 μL) of colchicine were added to obtain final concentrations of 5 and 50 μM, and the fluorescence was monitored for 60 min. A DMSO control and the complex colchicine−tubulin control (F0) were also included in the experiments. Normalized F/F0 values after 60 min of interaction versus concentration28 are plotted in Figure 6. Cytotoxicity Assays. The cytotoxic activity of 1, 5, and 8 was evaluated according to described methodologies.29,30 The compounds were screened in vitro against MCF7 breast carcinoma, HeLa cervix carcinoma, and HCT 116 colon carcinoma cell lines. All cell lines were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum and were cultured at 37 °C in an atmosphere of 5% CO2 in air (100% humidity). The cells were harvested at log phase of their growth cycle and were treated in triplicate with various concentrations of 1, 5, or 8 (0.5−120 μM) and incubated at 37 °C in a humidified atmosphere of 5% CO2 for 72 h. The cell concentrations were determined by the sulforhodamine B method.31 Results are expressed as the concentration that inhibits 50% control growth after the incubation period (IC50). The values were estimated from a semilog plot of the drug concentration (μM) against the percentage of growth inhibition. Colchicine was included as a positive control. Docking Analysis. Molecular structures for colchicine, 1, 5, and 8 were built and minimized using MMFF force-field calculations as implemented in the Spartan’04 program (Wavefunction, Inc.) followed by exploration of the conformational space using the Monte Carlo method and HF 3-21G energy minimization. The global minimum was DFT optimized at the B3LYP/DGDZVP level of theory employing the Gaussian 03W program revision C.02 (Gaussian, Inc.). Docking analyses between the optimized conformer of each compound and the α,β-tubulin heterodimer were carried out with the AutoDock program v. 4.2.6, and molecular visualizations were carried out with AutoDock Tools 4.2 (The Scripps Research Institute, La Jolla, CA, USA) and Pymol 1.3 for Windows. The crystallographic file of the protein was obtained from the RCSB Protein Data Bank under the code 1JFF.24 Computer simulations were done on an Intel Core i7-2670QM CPU at 2.20 GHz, 8 Gb of RAM, and a NVIDIA GeForce GT video card at 550 MB. In the

preliminary surface scanning, a grid box size of 103 × 61 × 63 Å in the x, y, and z dimensions was set, centering the coordinates at x = 18.4, y = −1.7, and z = 4.6 Å. The search parameters using the Lamarckian genetic algorithm (LGA) were 100 LGA runs, a population size of 150, a maximum number of energy evaluations of 2.5 × 106, and a maximum number of generations of 2.7 × 104. In the refinement step, a smaller box size (41 × 41 × 41 Å) in the x, y, and z dimensions was set, centering the coordinates at x = 45.9, y = −15.9, and z = 12.2 Å to cover the best binding modes taking into account the lowest binding energy (Edock) and the highest number of interactions. Docking analysis of colchicine was also carried out with the AutoDock program v. 4.2.6 and refined for the primary site located in the interphase between the α- and β-tubulin subunits (Edock = −8.99 kcal/mol) and for the secondary site located at the α-tubulin subunit (Edock = −7.07 kcal/mol) as indicated in Table 5. A reported binding energy in the primary site was estimated as −9.88 kcal/mol,32 using the Extra Precision program in the Glide docking mode (Schrödinger, Inc.).

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b00860. 1

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H, 13C, COSY, NOESY, HSQC or HETCOR, and HMBC spectra of parvifoline derivatives and graphs of tubulin polymerization in the presence of colchicine and compounds 1−12; minimum energy molecular structures and Cartesian coordinates of 1−12 (PDF)

AUTHOR INFORMATION

Corresponding Author

*(C. M. Cerda-Garcı ́a-Rojas) E-mail: [email protected]. Tel: +52 55 5747 4035. Fax: +52 55 5747 7137. ORCID

Edna M. Silva-García: 0000-0003-3921-4009 Carlos M. Cerda-García-Rojas: 0000-0002-5590-7908 Rosa E. del Río: 0000-0001-8932-552X Pedro Joseph-Nathan: 0000-0003-3347-3990 Author Contributions

Taken in part from the Ph.D. thesis of Edna M. Silva-Garcı ́a. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Dedicated to Dr. Rachel Mata, Universidad Nacional Autónoma de México, Mexico City, Mexico, for her pioneering work on bioactive natural products. We thank CONACYTMexico (Grant No. CB2014-241053-Q) for financial support. E.M.S.G. is grateful to CONACYT-Mexico for scholarship 407118. We are grateful to Professor J. Rzedowski, Instituto de Ecologı ́a, A. C., Centro Regional del Bajı ́o, Pátzcuaro, Michoacán, Mexico, for identifying the plant material. We acknowledge Professor R. Pereda-Miranda and Dr. M. C. Fragoso-Serrano, Facultad de Quı ́mica, Universidad Nacional Autónoma de México, for their valuable support in performing the cytotoxicity assays.

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REFERENCES

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