Selective Antiproliferative Withanolides from Species in the Genera

Article pubs.acs.org/jnp

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

Selective Antiproliferative Withanolides from Species in the Genera Eriolarynx and Deprea Sebastiań J. Castro,† Carina N. Casero,† Jose ́ M. Padroń ,*,‡ and Viviana E. Nicotra*,†

J. Nat. Prod. Downloaded from pubs.acs.org by AUCKLAND UNIV OF TECHNOLOGY on 05/09/19. For personal use only.

†

Facultad de Ciencias Químicas, Instituto Multidisciplinario de Biología Vegetal (IMBIV-CONICET), Universidad Nacional de Córdoba, Casilla de Correo 495, 5000 Córdoba, Argentina ‡ BioLab, Instituto Universitario de Bio-Orgánica “Antonio González” (IUBO-AG), Centro de Investigaciones Biomédicas de Canarias (CIBICAN), Universidad de La Laguna, C/Astrofísico Francisco Sánchez 2, 38206 La Laguna, Spain S Supporting Information *

ABSTRACT: Four new withanolides (2−5), together with 4β,7β,20-trihydroxy-1oxowitha-2,5,24-trienolide (1), were isolated from the aerial parts of Eriolarynx iochromoides. The antiproliferative activity of all compounds purified from E. iochromoides together with four withaphysalins and four physangulidines isolated previously from three Deprea species were evaluated against human solid tumor cell lines. Four withanolides showed antiproliferative activity comparable in potency to cisplatin. Selectivity toward cancer cells and interaction with P-glycoprotein of the active withanolides were evaluated.

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1) are described, as well as the antiproliferative activity of these compounds against human solid tumor cell lines. Withaphysalins and physangulidines from three Deprea species previously isolated by our group10 were evaluated as well, with the purpose of performing a preliminary structure−activity relationship analysis.

ithanolides belong to a group of naturally occurring C28-steroid lactones. These compounds are generally highly oxygenated, and this wealth of oxygen functions has led to many modifications of the carbocyclic part as well as of the side chain. The current classification of withanolides constitutes 22 types based on their structural differences. Withanolides have been isolated, largely but not exclusively, from genera belonging to the plant family Solanaceae,1 and they have captured interest mainly due to their structural diversity and significant biological activities.2−4 The genus Eriolarynx Miers is a small group of three species. Eriolarynx lorentzii (Dammer) Hunz. and E. iochromoides (Hunz.) Hunz., both arborescent, grow in the humid cloud forests of Sierra de Aconquija in northwestern Argentina, between 1000 and 2000 m high. The third species is E. fasciculata (Miers) Hunz., an endemic shrub from the dry, rocky slopes of the Andes of Bolivia (Departments of Chuquisaca, Cochabamba, Potosi,́ and Tarija), at an altitude of 2000−3000 m.1 A previous phytochemical study of E. lorentzii (syn. Acnistus lorentzii Dammer and Vassobia lorentzii (Dammer) Hunz.) resulted in the isolation of eight functionalized withanolides at the C-18 position, with various levels of oxidation (alcohol, aldehyde, and lactone), which included withaphysalins and their biogenetic precursors.5 The withaphysalins have been evaluated for several biological activities, including anti-inflammatory activity, induction of quinone reductase, and especially cytotoxic activity.6−9 Continuing a search for bioactive compounds, we evaluated the chemical content of E. iochromoides. Herein, details of the isolation and structural elucidation of compounds 1−5 (Figure © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION

The aerial parts of E. iochromoides were extracted with ethanol, and the extract was fractionated by several chromatographic techniques, finally obtaining 4β,7β,20-trihydroxy-1-oxowitha2,5,24-trienolide (1), which is already known, previously isolated from Acnistus australis,11 and four new withanolides (2−5). Compounds 1 and 2 are normal-type withanolides, while compounds 3−5 are withaphysalin-type withanolides (Figure 1). Compound 1 was reported by Kirson et al. 11 as (17S,20R,22R)-4β,7β,20-trihydroxy-1-oxowitha-2,5,24-trienolide. However, the orientation of the hydroxy group at C-7 and the orientation of the side chain were confirmed in 1 in this study via a NOESY experiment. The β orientation of the hydroxy group at C-7 was confirmed by the NOE observed between H-7 at δ 3.82 and the resonances corresponding to H9 at δ 1.61 and H-14 at δ 1.21, while the β orientation of the side chain was confirmed by the NOE observed between H3-18 Received: February 7, 2019

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DOI: 10.1021/acs.jnatprod.9b00117 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Figure 1. Compounds isolated from Eriolarynx iochromoides.

Table 1. 1H NMR Data of Compounds 1−5 in CDCl3a position

1

2

3 (18R/18S)

4

5

2 3 4 6 7 8 9 11a 11b 12a 12b 14 15a 15b 16a 16b 17 18 19 21 22 23a 23b 27 28 OCH2CH3 OCH2CH3 OCH3 OH-18

5.90 d (10.1) 6.71 dd (10.1,4.4) 4.59 d (4.3) 5.73 d (1.7) 3.82 brd (8.4) 1.42 m 1.61 m 1.82 m 1.53 m 2.00 m 1.32 dd (13.0, 3.3) 1.21 m 2.13 m 1.48 m 1.96 m 1.52 m 1.43 m 0.86 s 1.41 1.24 4.16 dd (13.3, 3.4) 2.36 t (14.2) 2.06 m 1.82 s 1.89 s

5.90 d (10.2) 6.71 dd (10.2,4.6) 4.59 d (3.3) 5.73 brs 3.76 d (7.9) 1.47 m 1.66 m 2.14 m 1.52 m 2.53 m 1.12 m 1.27 mb

5.88 d (10.0) 6.71 dd (10.0,4.3) 4.62 m 5.77 brs/5.84 brs 3.74 m/3.72 m 1.59 m/1.98 m 1.71 m/1.69 m

5.92 d (10.0) 6.73 dd (10.0,4.4) 4.62 brs 5.78 d (1.7) 3.82 brs 1.64 m 1.72 m 2.38 m 1.44 m 2.04 m 1.45 m 1.51 m 1.79 m 1.44 m 2.11 m 1.73 m 1.98 m 4.71 s 1.41 s 1.30 s 4.38 dd (13.0, 3.2) 2.37 t (13.0) 2.09 m 1.84 brs 1.88 brs

5.88 brd (10.2) 6.77 dd (10.2,4.3) 4.66 d (4.1) 5.59 d (2.0) 3.68 d (7.8) 1.24 m 1.71 m 2.13 m 1.75 m 4.46 brd (12.1) 1.51 m 1.46 m 1.62 m 1.41 m 1.78 m 1.46 m 1.93 m 4.72 s 1.31 s 1.37 s 4.58 dd (13.6, 3.2) 2.36 t (13.1) 1.98 m 1.83 brs 1.87 brs 3.85 m−3.28 m 1.11 t (7.1)

1.33 mb 3.61 m 1.43 s 1.40 s 4.24 dd (13.0, 3.5) 2.32 m 2.08 m 1.83 s 1.91 s

2.51 m/1.97 m 1.56 m/1.45 m 1.42 m/1.51 m

1.98 m/2.04 5.24 s/5.29 1.41 s/1.38 1.41 s/1.23 4.39 dd (13.4, 3.2)/4.51 2.33 m/2.42 1.98 m/2.12 1.79 brs 1.87 brs/1.88

m s s s dd (13.1, 2.2) m m brs

3.26 s 4.73 m

a

Chemical shifts (δ) downfield from TMS, J couplings (in parentheses) in Hz. run at 400.13 MHz. bAssignments may be interchanged.

at δ 0.86 and the signal corresponding to H-22 at δ 4.16 (Figure S1, Supporting Information). Compound 2 revealed a molecular formula of C28H38O7 via HRESIMS, with the 1H and 13C NMR data being closely comparable to those of 1 (Tables 1 and 2). The only differences between 2 and 1 were the absence of a signal corresponding to the H3-18 in the high-field region of the 1H NMR spectrum and the appearance of methylene carbinolic

resonances [δH 3.60 (m, 2H) and δC 58.8], suggesting the presence of an isolated C-18 hydroxymethylene group. Accordingly, the structure of 2 was elucidated as (17S,20R,22R)-4β,7β,18,20-tetrahydroxy-1-oxowitha-2,5,24trienolide. Compounds 3−5 were found to possess the characteristics of withaphysalin-type withanolides. Withaphysalins comprise a group that exhibit an oxygen bridge between C-18 and C-20; B


Journal of Natural Products Table 2.

a

13

Article

C NMR Data of Compounds 1−5 in CDCl3a

position

1

2

3 (18R/18S)

4

5

position

1

2

3 (18R/18S)

4

5

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

203.1 129.5 142.9 69.0 140.8 133.9 72.0 41.25 41.31 49.4 26.6 40.3 44.1 56.5 23.5 22.7

202.5 128.8 142.4 68.4 140.3 133.4 71.9 41.0 40.9 49.0 22.8d 34.8 54.4 54.8b 22.4c 25.8c

203.1/202.9 128.9 142.9 68.3 139.4/140.2 133.7/134.4 70.8/71.1 42.0/41.2 41.0/40.7 49.1/49.0 25.9d 34.7/37.0 57.3/59.0 55.7/53.9 25.3d 28.3d

202.5 128.9 142.4 68.5 140.1 133.4 71.7 41.4 40.6 48.9 25.6 36.9 55.4 53.5 25.5 29.2

203.2 128.9 144.7 67.8 141.0 132.8 70.5 44.9 41.3 48.9 26.0 35.0 56.2 56.5 26.4 28.9

17 18 19 20 21 22 23 24 25 26 27 28 OCH2CH3 OCH2CH3 OCH3

54.9 14.1 22.6 75.5 21.3 81.3 31.2 149.1 122.4 166.9 12.8 20.9

54.9b 58.8 22.2 75.4 21.5 80.7 31.7 148.8 122.2 166.8 12.2 20.4

56.4/56.7 101.5/103.2 21.7/22.2 85.0/85.1 21.7/24.7 80.5/80.4 31.2/32.2 148.5/149.7 122.0 166.4 12.3 20.2/20.3

55.6 110.2 22.3 84.5 24.1 81.0 32.2 148.1 122.3 166.1 12.5 20.4

56.6 107.5 22.7 86.2 20.9 80.3 31.3 149.5 122.4 167.4 12.8 20.8 64.7 15.5

Chemical shifts (δ) downfield from TMS; 100.03 MHz.

55.1

b−d

Assignments may be interchanged.

Figure 2. Compounds isolated from D. bitteriana (6 and 7), D. cuyacensis (8 and 9), and D. zamorae (10−13) for biological studies.

the withaphysalins. The absence of a signal for CH3-18 and the presence of two proton resonances at δ 5.24 (60%) and 5.29 (40%; Table 1) were indicative of a lactol functionality at C-18 and were assigned to the 18R and 18S epimers, respectively.5 The 13C NMR spectrum showed methine signals at δ 101.5 and 103.2 (C-18 R/S) and resonances at δ 57.3/59.0, δ 56.4/ 56.7, and δ 85.0/85.1 assigned at C-13, C-17, and C-20, respectively, thus confirming a five-membered lactol ring (Table 2). Accordingly, the structure of compound 3 was established as (17S,20R,22R)-18,20-epoxy-4β,7β,18α-trihydroxy-1-oxowitha-2,5,24-trien-26,22-olide/(17S,20R,22R)18,20-epoxy-4β,7β,18β-trihydroxy-1-oxowitha-2,5,24-trien26,22-olide.

depending on the oxidation state of C-18, a lactol or lactone ring may result. The hemiacetal arrangement is characterized by the absence of the methyl-18 1H NMR singlet at a lowfrequency chemical shift, and by the appearance of two methine carbinolic resonances, indicating the presence of R/S mixtures of epimeric hemiacetals at C-18.12 The HRESIMS of withaphysalin 1 (3) showed a molecular ion [M + Na]+ at m/z 507.2387, corresponding to an elemental formula of C28H36NaO7. Inspection of the 1D and 2D NMR spectroscopic data indicated that compound 3 possesses rings A−C as well as a side-chain closely related to those of compounds 1 and 2, suggesting that the structural difference could be restricted to the additional five-membered lactol ring typical of C



Article

Table 3. In Vitro Antiproliferative Activity of Compounds 1−13 against Human Solid Tumor Cell Linesa compound

A549

HBL-100

HeLa

SW1573

T-47D

WiDr

BJ-hTERT

1 2 3 4 5 6 7 8 9 10 11 12 13 cisplatin

>10 >10 >10 3.1 ± 0.6 2.8 ± 0.1 >10 >10 >10 >10 >10 >10 >10 >10 4.9 ± 0.2

4.8 ± 0.3 >10 >10 3.6 ± 0.4 3.2 ± 0.3 >10 >10 >10 >10 >10 >10 >10 >10 1.9 ± 0.2

5.3 ± 0.8 >10 >10 3.2 ± 0.7 2.5 ± 0.03 >10 >10 >10 >100 >10 >10 3.3 ± 0.2 >10 1.8 ± 0.5

4.1 ± 1.0 >10 >10 3.3 ± 0.6 2.4 ± 0.9 >10 >10 >10 >10 >10 >10 4.4 ± 1.9 >10 2.7 ± 0.4

>10 >10 >10 3.9 ± 1.0 4.7 ± 1.0 >10 >10 >10 >10 >10 >10 6.4 ± 0.9 >10 >10

>10 >10 >10 >10 6.1 ± 1.9 >10 >10 >10 >10 >10 >10 >10 >10 >10

>10 n.t. >10 >10 >10 n.t. n.t. n.t. n.t. >10 n.t. 5.3 ± 1.7 n.t. >10

a

Expressed as GI50 and standard deviation. Values are given in micromolarity and determined as means of two to three experiments.

The similarity in the 1H and 13C NMR chemical shifts of compounds 3−5 indicated that they possess the same 1-oxo2,5-diene-4β,7β-dihydroxy substitution pattern in rings A and B, the same side chain, and the same withaphysalin typical characteristics. The only differences between 3, 4, and 5 were observed in the vicinity of the C-18 carbon. The NMR spectra of compound 4 showed the signals of H-18 and C-18 at δ 4.71 and δ 110.2, respectively. In addition, the signals at δH 3.26 (s, 3H) and δC 55.1 suggested the presence of an OCH3-18 substituent. The location of the methoxy group was confirmed by an HMBC correlation between the signal of CH3O and C18 (Figure S13, Supporting Information), and the β orientation of the methoxy group was established by the NOE observed between H-18 and H-11b at δ 1.44 (Figure S18, Supporting Information). Regarding compound 5, its 1H NMR spectrum showed a signal at δ 4.72 assigned at H-18 and three signals corresponding to an ethoxy group at δ 3.85 (m, 1H), 3.28 (m, 1H), and 1.11 (t, 7.1 Hz, 3H). The signals at δ 107.5 (C-18), 64.7 (OCH2CH3), and 15.5 (OCH2CH3) observed in the 13C NMR spectrum and the HMBC correlation between H-18 and (OCH2CH3) agreed with the ethoxy group substitution at C-18 (Figure S17, Supporting Information). The α orientation of the ethoxy group was determined from the NOE observed between H-18 and H-8 (Figure S18, Supporting Information). HRMS measurements agreed with the proposed molecular formulas of 4 and 5. Thus, the structures of compounds 4 and 5 were established as (17S,20R,22R)-18,20-epoxy-4β,7β-dihydroxy-18β-methoxy-1oxowitha-2,5,24-trien-26,22-olide and (17S,20R,22R)-18,20epoxy-18α-ethoxy-4β,7β-dihydroxy-1-oxowitha-2,5,24-trien26,22-olide, respectively. The ease with which hemiacetals such as 3 react with the alcohols is well documented in the literature and suggests the artifactual nature of compounds 4 and 5.4,13 The antiproliferative activity against cancer cell lines of withaphysalins has been widely reported.14 Previous cytotoxic activity studies and structural activity have indicated that the cytotoxicity depends mainly on the substitution pattern of the A and B rings, with the presence of both an α,β-unsaturated ketone in ring A and a 5β,6β-epoxy or 5-ene system being important for the activity.9,12,15,16 In view of the previous reports regarding the bioactivities of withanolides, the in vitro antiproliferative activity was determined for the withanolides (1−5) isolated in this study. As a model for the biological tests, a panel of six representative human solid tumor cell lines

(A549, HBL-100, HeLa, SW1573, T-47D, and WiDr) was selected using the SRB assay of the US-NCI.17 In addition, two withaphysalins isolated from Deprea bitteriana (6 and 7), two 13,14-seco withaphysalins isolated from D. cuyacensis (8 and 9), and four physangulidines isolated from D. zamorae10 (10− 13) were evaluated. Figure 2 shows the chemical structure of compounds 6−13. The results of the antiproliferative tests (Table 3) are expressed as 50% growth inhibition (GI50) and were calculated according to the NCI.18 In this evaluation, the anticancer drug cisplatin was used as a reference compound to gain insight on the potency of the molecules under study. Figure 1 (S19, Supporting Information) displays the GI50 data reported in Table 3 as a GI50 range plot for cisplatin and compounds 1−13 for an overall view. Overall, the most active compounds showed potency comparable to that of cisplatin. These substances comprising compounds 1, 4, 5, and 12 caused significant growth inhibition with GI50 values below 10 μM in most cell lines (Table 3). Besides tumor cells, compounds 1, 3−5, 10, and 12 were tested against the nontumor BJ-hTERT cell line, in order to determine their selectivity. The results (Table 3 and Figure S19, Supporting Information) indicated that all compounds but 12 were 2−10 times more selective toward the cancer cells used. From the antiproliferative data obtained, the following structure−activity relationship information can be inferred. When considering the subset of withanolides 1−5, it was observed that the acetal framework favors the antiproliferative activity (4, 5 > 1). Interestingly, a hydroxy group substituent at C-18 (2) produces a prominent decrease in activity, which is reverted in part with the presence of a hemicetal group (3 > 2). Acetals 4 and 5 are more active than hemicetal 3. The differences in activity could be attributed to the lipophilicity of the compounds, with the ClogP values for 3−5 being −0.07, 0.54, and 0.93, respectively. The data indicated that a hydroxy substituent at C-4 is essential and clearly marks the differences in activity between withanolides 1−5 and withaphysalin 6−9. Physangulidines comprise a group of withanolides scarcely explored regarding their biological activities. To our knowledge, the only reports refer to physangulidines A−C, which have shown significant antiproliferative activity against several cancer cell lines.19,20 This could result from the fact that they possess an α,β-unsaturated keto carbonyl group in ring A and a 5β,6β-epoxy group in ring B, which would be the key D



Article

Table 4. Antiproliferative Activities (GI50 values) of compounds 1, 4, 5, and 12 and Tubulin-Interacting Drugs Using the SW1573 and SW1573/Pgp Cell Linesa −verapamil SW1573 1 4 5 12 PTX VB

3.4 3.0 2.1 4.1 1.5 0.9

± ± ± ± ± ±

0.1 0.5 0.3 1.5 0.5 0.3

+verapamil

SW1573/Pgp

Rf

± ± ± ± ± ±

0.9 1.1 1.1 1.1 128 2388

3.2 3.3 2.3 4.5 196 2051

0.2 0.5 0.05 0.4 53 682

SW1573 4.2 3.5 3.9 5.5 1.6 0.8

± ± ± ± ± ±

0.7 0.4 0.01 1.2 0.2 0.2

SW1573/Pgp

Rf

± ± ± ± ± ±

0.8 0.7 0.4 0.5 2.6 1.3

3.2 2.3 1.7 2.7 4.2 1.0

0.03 0.1 0.2 0.8 0.9 0.5

Values are given in micromolarity and represent the mean values of at least two independent experiments ± standard deviation. PTX, paclitaxel; VB, vinblastine.

a

Preparative TLC separations were performed according to previously described procedures.24 Plant Material. The aerial parts of Eriolarynx iochromoides were collected in Tafí del Valle, Tucumán, Argentina, in March 2005. A voucher specimen was deposited at the Museo Botánico Córdoba (CORD), Universidad Nacional de Córdoba (Barboza et al. 4391). The plant material was identified by Gloria E. Barboza (IMBIVCONICET, Córdoba, Argentina). Extraction and Isolation. The dried and pulverized aerial parts of E. iochromoides (130 g) were extracted successively using EtOH at room temperature, and the solvent was evaporated under reduced pressure. The resulting residue (12.7 g) was partitioned with solvents of increasing polarity according to procedures previously described.10 The residue obtained by partition with CH2Cl2 (5.00 g) was chromatographed initially on a Sephadex LH-20 column, using MeOH as an eluent. Fractions with similar TLC profiles were then combined and reduced to a fraction of 2.6 g. This fraction was subjected to column chromatography with hexane−EtOAc mixtures of increasing polarity (100:00 to 0:100), yielding four fractions containing withanolides (fractions I−IV). Fraction I (200 mg) was separated by column chromatography with hexane-EtOAc mixtures of increasing polarity to give two mixtures that were further fractionated by preparative TLC (CH2Cl2-MeOH), yielding compounds 4 (5.3 mg) and 5 (6.4 mg). Fraction II (270 mg) was separated by column chromatography with CH2Cl2/MeOH mixtures of increasing polarity (100:00 to 90:10) to give a mixture that was fractionated by TLC with CH2Cl2/MeOH to yield compound 4 (14.6 mg). Fraction III (310 mg) was subjected to silica gel 60 G CC. Elution with CH2Cl2/ MeOH mixtures of increasing polarity (100:0−90:10) afforded an epimeric mixture of compound 3 (45 mg). Fraction IV (200 mg) was applied to a silica gel 60 G column using CH2Cl2/MeOH mixtures of increasing polarity (100:0−93:07) to afford compound 1 (30 mg) and one impure fraction (19 mg), which was fractionated by TLC with CH2Cl2/MeOH, to yield compound 2 (1.5 mg). Fraction V (400 mg) was an epimeric mixture of compound 3. All new compounds were determined to be >95% pure by 1H NMR spectroscopy. [(17S,20R,22R)-4β,7β,18,20-Tetrahydroxy-1-oxowitha-2,5,24-trienolide] (2). White amorphous powder; [α]D21 +57 (c 0.10, acetone); λmax(log ε): 208 (3.77) nm. IR (dry film): νmax 3414, 2974, 2939, 1687 cm−1. 1H and 13C NMR data: see Tables 1 and 2. HRESIMS m/ z [M + Na]+: 509.2494 (calcd for C28H38NaO7: 509.2510). Withaphysalin 1 [(17S,20R,22R)-18,20-epoxy-4β,7β,18α-trihydroxy-1-oxowitha-2,5,24-trien-26,22-olide/(17S,20R,22R)-18,20epoxy-4β,7β,18β-trihydroxy-1-oxowitha-2,5,24-trien-26,22-olide] (3). White amorphous powder; [α]D21 +25.7 (c 0.21, acetone); λmax (log ε): 210 (3.72) nm. IR (dry film): νmax 3420, 2937, 1690, 1385 cm−1. 1 H and 13C NMR data: see Tables 1 and 2. HRESIMS m/z [M + Na]+: 507.2387 (calcd for C28H36NaO7: 507.2353). 18β-O-Methyl-withaphysalin 1 [(17S,20R,22R)-18,20-epoxy-4β,7βdihydroxy-18β-methoxy-1-oxowitha-2,5,24-trien-26,22-olide] (4). White amorphous powder; [α]D21 +88.73 (c 0.11, acetone); λmax (log ε): 202.5 (3.68) nm. IR (dry film): νmax 3390, 2940, 2878, 1684 cm−1. 1H and 13C NMR data: see Tables 1 and 2. HRESIMS m/z [M + Na]+: 521.2481 (calcd for C29H38NaO7: 521.2510).

substitution patterns for active withanolides. In the present study, for compounds 10−13, the antiproliferative activity was tested. The four physangulidines tested have different substitution patterns in rings A and B, and only physangulidine F showed significant activity. These results suggest that the type of side chain present in the physangulidines does not affect the resultant activity and that a 1-oxo-2,4-diene-6βhydroxy substitution in ring A and B plays a key role in mediating antiproliferative activity. From the set of withanolides described in this work, compounds 1, 4, 5, and 12 were tested for their interaction with P-glycoprotein (P-gp). This protein is a transporter belonging to the ATP-binding cassette family that functions as a biological barrier by extruding toxins and xenobiotics out of the cell.21 As a model to test the effect of P-gp overexpression in the compounds tested, a cell line based assay was used. For this purpose, one wild type cell line (SW1573) and its P-gp overexpressing variant (SW1573/Pgp) were employed.22 Additionally, compounds were tested against both cell lines in the presence or absence of 10 μM verapamil (a known P-gp and CYP3A4/5 inhibitor).23 The standard microtubuleinteracting drugs, paclitaxel (PTX) and vinblastine (VB), were used as reference drugs in this assay. For a better comparison of the data, the resistance factor (Rf) was defined for a given compound as the ratio of GI50 values in the P-gp overexpressing line to those in the wild type cell line. Table 4 shows the experimental GI50 values obtained after 48 h of exposure compounds to wild type and P-gp-overexpressing SW1573 cells, and in the presence (+) or absence (−) of verapamil. All compounds showed low Rf values, denoting no effect of P-gp on their biological activity. Overall, the results indicate that the test compounds were not found to be substrates for P-gp.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a JASCO P-1010 polarimeter. The UV spectra were obtained using a Shimadzu-260 spectrophotometer, and IR spectra were produced using a Thermo Scientific Nicolet iN10 FT-IR Microscope. NMR spectra were recorded on a Bruker AVANCE II AV-400 instrument operating at 400.13 MHz for 1H and 100.63 MHz for 13C, while 2D spectra (COSY, HSQC, HMBC, and NOESY) were obtained using standard Bruker software. Chemical shifts are given in parts per million (δ) downfield from the TMS internal standard. HRESIQTOFMS were determined on a Micro TOF II mass spectrometer (Bruker Daltonics, Bellerica, MA, USA). Chromatographic separations were performed by column chromatography on silica gel 60 (0.063−0.200 mm) and Sephadex LH-20 and preparative TLC on silica gel 60 F254 (0.2 mm thick) plates. E



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18α-O-ethyl-withaphysalin 1 [(17S,20R,22R)-18,20-epoxy-18αethoxy-4β,7β-dihydroxy-1-oxowitha-2,5,24-trien-26,22-olide] (5). White amorphous powder; [α]D21 +70 (c 0.2, acetone); λmax (log ε) 207 (3.76) nm. IR (dry film): νmax 3387, 2921, 2851, 1687, 1380 cm−1. 1H and 13C NMR data: see Tables 1 and 2. HRESIMS m/z [M + Na]+: 535.2646 (calcd for C30H40NaO7: 535.2666). Biological Activity Assays. All starting materials were commercially available research-grade chemicals and used without further purification. RPMI 1640 medium was purchased from Flow Laboratories (Irvine, UK); fetal calf serum (FCS) from Gibco (Grand Island, NY, USA); trichloroacetic acid (TCA) and glutamine from Merck (Darmstadt, Germany); and penicillin G, streptomycin, dimethyl sulfoxide (DMSO) and sulforhodamine B (SRB) from Sigma (St Louis, MO, USA). Cells, Culturing, and Plating. The human solid tumor cell lines A549 (lung), HBL-100 (breast), HeLa (cervix), SW1573 (nonsmall cell lung) and its P-gp overexpressing variant (SW1573/Pgp), T-47D (breast), and WiDr (colon) were used in this study. These cell lines were a kind gift from Prof. Godefridus J. Peters (VU Medical Center, Amsterdam, The Netherlands). The human fibroblast cell line BJhTERT was a gift from Dr. Raimundo Freire (Unidad de Investigación HUC, Tenerife, Canary Islands). Cells were maintained in 25 cm2 culture flasks in RPMI 1640 supplemented with 5% FBS and 2 mM L-glutamine in a 37 °C, 5% CO2, 95% humidified air incubator. Exponentially growing cells were trypsinized and resuspended in an antibiotic-containing medium (100 units penicillin G and 0.1 mg of streptomycin per mL). Single cell suspensions were counted with Moxi Z. After counting, dilutions were made to give the appropriate cell densities for inoculation onto 96-well microtiter plates. Cells were inoculated in a volume of 100 μL per well at densities of 2500 (A549, HBL-100, HeLa, and SW1573) or 5000 (SW1573/Pgp, T-47D, WiDr, and BJ-hTERT) cells per well, based on their doubling times. In Vitro Antiproliferative Tests. The antiproliferative activity of the reported compounds was tested against the human solid tumor cell lines (A549, HBL-100, HeLa, SW1573, SW1573/Pgp, T-47D, WiDr) and the human fibroblast cell line BJ-hTERT kindly provided by Dr. G. J. Peters (The Netherlands) and Dr. Raimundo Freire (Canary Islands), respectively. In all cell experiments, the exposure time to the compounds was 48 h, and the results are expressed as GI50 (dose that produces 50% growth inhibition). Cisplatin was used as a reference drug for antiproliferative tests, and verapamil was used as a P-gp transport inhibitor. Paclitaxel and vinblastine were used as positive controls in SW1573/Pgp experiments. The cell culture medium containing verapamil was prepared by adding the final concentration of 10 μM verapamil. Briefly, cell line suspensions were counted with Moxi Z and diluted to reach the appropriate cell densities (A549, HBL-100, HeLa, and SW1573, 2500 cells/well; SW1573/Pgp, T-47D, WiDr, and BJhTERT, 5000 cells/well). Then, cells were seeded onto 96-well microtiter plates and placed in the incubator. After 24 h, compounds 1−13 were added to the cells at serial dilutions (0.01−100 μM) and incubated for an additional 48 h. Then, the SRB assay was performed.17 The optical density of each well was measured at 530 and 620 nm with a PowerWave XS microplate reader, and the GI50 values for each compound were calculated according to NCI formulas.18

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Article

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Sebastián J. Castro: 0000-0001-5342-6660 José M. Padrón: 0000-0002-7488-1774 Viviana E. Nicotra: 0000-0002-5255-4721 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by grants from CONICET and SeCyT-UNC. S.J.C. thanks CONICET (Argentina) for a fellowship. We thank F. Chiarini for the photography of E. iochromoides and G. Bonetto for NMR assistance.

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.9b00117. NMR spectra of compounds 2−5. The relevant NOE correlations of compounds 1, 4, and 5 (PDF) F



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