Cycloartane- and Lanostane-Type Triterpenoids from the Resin of Parthenium argentatum AZ-2, a Byproduct of Guayule Rubber Production

Abstract

A total of 12 new cycloartane- and lanostane-type triterpenoids including 16-deoxyargentatin A (1), 16-deoxyisoargentatin A (2), 7-oxoisoargentatin A (3), 24-epi-argentatin H (4), 24-O-p-anisoylargentatin C (5), 24-O-trans-cinnamoylargentatin C (6), 16-dehydroargentatin C (7), 16,17(20)-didehydroargentatin C (8), isoargentatin C (9), isoargentatin H (10), 3-epi-quisquagenin (11), and isoquisquagenin (12) together with 10 known triterpenoids (13–22) were isolated from the resin of Parthenium argentatum AZ-2 obtained as a byproduct of Bridgestone guayule rubber production. The structures of new triterpenoids 1–12 and argentatin H (13), which has previously been characterized as its diacetate (23), were elucidated by extensive analysis of their spectroscopic data and chemical conversions, and the known compounds 14–22 were identified by comparison of their spectroscopic data with those reported. Of these, 13, 14, and 18 exhibited weak cytotoxic activity for several cancer cell lines.

Introduction

Guayule (Parthenium argentatum A. Gray, Asteraceae), a plant native to the southwestern United States and the Chihuahuan desert of northern Mexico,¹ is currently undergoing economic assessment as a reliable and sustainable source of natural rubber.² The outcome of this evaluation will determine the suitability of this plant as a commercially viable crop for arid land agriculture. Commercial processing of guayule for rubber involves extraction of the dried and chopped plant biomass with an acetone–hexane azeotrope, which results in an equal or larger amount of guayule resin,³ a byproduct with no current cost-effective commercial applications. It has been suggested that finding and/or developing high-value resin-based products could significantly ameliorate the manufacturing cost of guayule rubber.⁴ Previous studies on P. argentatum resin have resulted in the isolation of some major constituents including the cycloartane-type triterpenoids, argentatins A–C,⁵ and the sesquiterpenoids, guayulins A–D,⁶ whereas lanostane-type triterpenoids isoargentatins A and B, together with argentatins A–D, have been encountered in the roots of P. argentatum.⁷ Investigation of the resin of the hybrid plant, P. argentatum × Parthenium tomentosum, has afforded two pyridine alkaloids, guayulamines A and B,⁹ in addition to argentatins E–H, of which argentatins G and H were characterized as their diacetates.⁸ In addition to argentatins and guayulins, several fatty acid triglycerides have been isolated and characterized from the resins of some cultivars of P. argentatum.¹⁰

Guayule resin and some of its constituents are known to exhibit a variety of biological activities. The ability of guayule resin to protect wood from termite attack has been reported,¹¹ and subsequent studies have demonstrated moderate termite antifeedant activity of argentatin B.¹² Several argentatins have been reported to exhibit weak cytotoxic¹³ and antimicrobial activities.¹⁴ Importantly, despite its weak in vitro cytotoxic activity, argentatin A has recently been demonstrated to have promising in vivo antitumor activity in a mouse xenograft model of human colon cancer at high doses with no adverse toxicity.¹⁵

In our attempts to develop value-added products as a part of a collaborative USDA/NIFA-funded project on Sustainable Bio-economy for Arid Regions, we initiated a study to identify constituents of guayule resin with potential biological activity and/or those constituents which can be converted into their bioactive analogues. Herein, we report a detailed chemical investigation of resin samples obtained during guayule rubber processing by Bridgestone Americas Inc., which led to the isolation and characterization of 12 new and 10 known cycloartane- and lanostane-type triterpenoids, and evaluation of their cytotoxic activity.

Materials and Methods

General Experimental Procedures

Optical rotations were measured with a JASCO Dip-370 polarimeter using MeOH as the solvent. UV spectra were recorded with a Shimadzu UV 2601 spectrophotometer. Electronic circular dichroism (ECD) spectra were measured with a JASCO J-810 spectropolarimeter. One-dimensional (1D) and two-dimensional (2D) NMR spectra were recorded in CDCl₃ using the residual solvent as the internal standard on a Bruker AVANCE III 400 spectrometer at 400 MHz for ¹H NMR and 100 MHz for ¹³C NMR, respectively. The chemical shift values (δ) are given in parts per million (ppm), and the coupling constants (J values) are in Hz. Low-resolution MS spectra were recorded on a Thermo LQX mass spectrometer and high-resolution MS spectra on an Agilent G6224A TOF mass spectrometer. Analytical thin-layer chromatography (TLC) was carried out on silica gel 60 F₂₅₄ aluminum-backed TLC plates (Merck), and preparative TLC was performed on Analtech silica gel 500 μm glass plates. Compounds were visualized with short-wavelength UV (254 nm) and by spraying with anisaldehyde-sulfuric acid reagent and heating until the spots appeared. Silica gel flash chromatography was accomplished using 230–400 mesh silica gel. Sephadex LH-20 for gel-permeation chromatography was obtained from Amersham Biosciences. HPLC purifications were carried out using a Phenomenex Luna 5 μm C₁₈ (2) column (10 × 250 mm) for RP-C₁₈ chromatography and Econosil Si (10 μ) column (10 × 250 mm) for NP chromatography with a Waters Delta Prep system containing a PDA 996 detector. When required, MM2 energy minimizations of all possible conformers were carried out using Chem3D 15.0 from PerkinElmer, Inc. The resin samples (ID 2017-7-1-Res-45 and ID 2018-1-1-Res-25) extracted from P. argentatum line AZ-2¹⁶ used in this study were from the Bridgestone Americas Biorubber Process Research Center, Mesa, Arizona.

Isolation and Purification of Guayule Resin Constituents

Guayule resin (Bridgestone ID 2017-7-1-Res-45, 1.43 g) was first fractionated by solvent–solvent partitioning with 80% aq MeOH (100 mL) and hexanes (3 × 30 mL). The 80% aq MeOH fraction thus obtained was diluted with water to make it to 50% aq MeOH, which was then extracted with CHCl₃ (3 × 80 mL). Evaporation of each of these afforded hexanes (844 mg), CHCl₃ (570 mg), and 50% aq MeOH (5.6 mg) fractions. The hexanes fraction (844 mg) was further fractionated by gel-permeation chromatography on a column of Sephadex LH-20 (100 g). Elution with CH₂Cl₂/hexanes (4:1) afforded five fractions A1–A5. Of these, the major fraction A2 (288 mg) was subjected to silica gel (100 g) column chromatography and eluted with CHCl₃ to afford 18 (30.0 mg) and several minor fractions, which were further purified by RP-C₁₈ HPLC using aq. MeOH [gradient ranging from 85% MeOH–90% MeOH in H₂O to yield 1 (2.5 mg), 2 (1.0 mg), 13 (13.0 mg), 16 (9.1 mg), 19 (7.2 mg), and 21 (0.9 mg)]. The CHCl₃ fraction (570 mg) obtained from the above solvent–solvent partitioning was subjected to gel-permeation chromatography on Sephadex LH-20 (100 g). Elution with CH₂Cl₂/hexanes (4:1, v/v) afforded 10 fractions [B1 (61 mg), B2 (109 mg), B3 (210 mg), B4 (32 mg), B5 (98 mg), B6 (10 mg), B7 (31 mg), B8 (9 mg), B9 (25 mg), and B10 (42 mg)]. Of these, the major fractions with clear spots by TLC were further fractionated. Purification of fraction B2 (109 mg) by silica gel (100 g) column chromatography and elution with CHCl₃/MeOH (98:2, v/v) gave 18 (35.0 mg). Fraction B3 (210 mg) on further fractionation by silica gel (100 g) column chromatography and elution with CHCl₃/MeOH (98:2, v/v) afforded 14 (112.8 mg) and fractions containing several minor constituents which were further purified by RP-C₁₈ HPLC. Elution with a gradient of 85–95% aq MeOH (v/v) afforded 3 (1.5 mg), 5 (0.9 mg), 6 (1.0 mg), 8 (2.1 mg), 9 (2.1 mg), 15 (4.1 mg), 19 (3.6 mg), and 20 (2.3 mg). Fraction B5 (50.0 mg) was further purified by silica gel (50 g) column chromatography, and elution with CHCl₃/MeOH (98:2, v/v) afforded 13 (8.0 mg) and 16 (12.5 mg).

A second sample of the resin (Bridgestone ID 2018-1-1-Res-25, 116.0 g) was subjected to silica gel (3.5 kg) column chromatography. Elution with a solvent gradient of hexanes, hexanes/EtOAc (95:5 to 50:50, v/v), and EtOAc afforded 12 fractions [C1 (7.2 g), C2 (11.0 g), C3 (13.0 g), C4 (7.4 g), C5 (2.0 g), C6 (13.7 g), C7 (4.9 g), C8 (28.0 g), C9 (4.5 g), C10 (7.6 g), C11 (6.5 g), and C12 (10.0 g)]. Of these, only the fractions suspected to contain triterpenoids by TLC were further investigated. Fraction C4 (7.4 g) which contained a major compound when washed with hexanes (× 3) removed colored impurities, yielding 18 (6.18 g). A portion of the fraction C6 (2.0 g) was subjected to silica gel (60.0 g) column chromatography and eluted with CH₂Cl₂/acetone (98:2 to 90:10, v/v) to afford eight subfractions (C6-1 to C6-8). Further fractionation of C6-6 (400 mg) by silica gel (12 g) column chromatography and elution with CH₂Cl₂/acetone (99:1, v/v) yielded 19 (110 mg). Subfraction C6-7 (150 mg) was subjected to gel permeation chromatography on Sephadex LH-20 (4.5 g). Elution with hexanes/CH₂Cl₂ (1:1, v/v) afforded 4 (5.0 mg). Fraction C7 (4.9 g) was further fractionated by silica gel (150 g) column chromatography, and elution with CH₂Cl₂/acetone (98:2 to 90:10, v/v) gave nine subfractions [C7-1 (172 mg), C7-2 (430 mg), C7-3 (1.5 g), C7-4 (920 mg), C7-5 (165 mg), C7-6 (139 mg), C7-7 (344 mg), C7-8 (756 mg), and C7-9 (268 mg)]. A portion of the fraction C7-5 (15.0 mg) was separated by RP-C₁₈ HPLC with MeOH/H₂O (85:15, v/v, 3.0 mL/min) as the eluent to afford 10 (6.7 mg) and 13 (6.2 mg). Fraction C8 (28.0 g) was subjected to silica gel (900 g) column chromatography and elution with hexanes/EtOAc (98:2 to 0:100, v/v) gave 14 (8.0 g). Fraction C9 (3.3 g) was subjected to silica gel (100 g) column chromatography and eluted with CH₂Cl₂/i-PrOH (100:0 to 90:10, v/v) to afford seven subfractions [C9-1 (754 mg), C9-2 (500 mg), C9-3 (607 mg), C9-4 (280 mg), C9-5 (432 mg), C9-6 (570 mg), and C9-7 (190 mg)]. A portion of the fraction C9-4 (33.0 mg) was purified by RP-C₁₈ HPLC with MeOH/H₂O (85:15, v/v, 3.0 mL/min) as the eluent to afford, 11 (13.0 mg), 12 (2.0 mg), and 15 (16.0 mg).

Fraction C10 (7.3 g) on silica gel (220 g) column chromatography and elution with a gradient of CH₂Cl₂/iso-PrOH (98:1 to 90:10, v/v) afforded nine subfractions [C10-1 (1.0 g), C10-2 (1.8 g), C10-3 (818 mg), C10-4 (328 mg), C10-5 (1.2 g), C10-6 (550 mg), C10-7 (190 mg), C10-8 (543 mg), and C10-9 (656 mg)]. Of these, subfraction C10-5 (1.2 g) which was suspected to contain triterpenes was further fractionated on a column of RP-C₁₈ silica gel (36 g) and by elution with MeOH/H₂O (70:30 to 100:0, v/v) to provide 17 (820 mg). Subfraction C10-2 (200 mg) on further purification by RP-C₁₈ silica gel (6.0 g) column chromatography followed by RP-C₁₈ HPLC and elution with MeOH/H₂O (80:20, v/v, 3.0 mL/min) afforded 7 (3.2 mg) and 8 (3.0 mg). Subfraction C10-8 (533 mg) was further fractionated on a column of RP-C₁₈ silica gel (15 g) and by elution with MeOH/H₂O (70:30 to 100:0, v/v) to give four subfractions C10-8-1 (30.0 mg), C10-8-2 (11.0 mg), C10-8-3 (100 mg), and C10-8-4 (350 mg). Of these, C10-8-3 (24.5 mg) was separated by C₁₈ RP HPLC with MeOH/H₂O (80:20, v/v, 3.0 mL/min) as the eluent to afford 22 (20.7 mg).

16-Deoxyargentatin A [(20S,24R)-20,24-Epoxy-25-hydroxycycloartan-3-one] (1)

Amorphous colorless powder; [α]_D²⁵ + 21.3 (c 0.24, MeOH); UV (MeOH) λ_max(log ε): 200 (3.43) nm; ECD (MeOH) [θ] −2059 (295 nm); ¹H and ¹³C NMR data; see Table 1; positive HRESIMS m/z 439.3568 [MH – H₂O]⁺ (calcd for C₃₀H₄₇O₂, 439.3571).

Table 1. ¹H and ¹³C NMR Data of 1–3 in CDCl₃.

	1		2		3
position	δH	δC	δH	δC	δH	δC
1	1.82 (m)	33.4 CH2	1.63 (m)	36.0 CH2	2.40–2.50 (m)	34.2 CH2
	1.52 (m)
2	2.69 (dt, 6.5, 13.8)	37.5 CH2	2.57 (m)	34.6 CH2	2.97–3.05 (m)	35.1 CH2
	2.28 (ddd, 2.6, 4.2, 13.8)		2.37 (m)
3		216.7 C		217.9 C		218.2 C
4		50.2 C		47.4 C		47.1 C
5	1.68 (m)	48.5 CH	1.58 (m)	51.2 CH	1.65 (m)	51.6 CH
6	1.54 (m)	21.5 CH2	1.59 (m)	19.4 CH2	2.59–2.64 (m)	51.9 CH2
	0.92 (m)		0.91 (m)
7	2.05 (m)	25.9 CH2	2.03 (m)	26.3 CH2		198.4 C
	1.10 (m)		1.35 (m)
8	1.58 (m)	47.6 CH		134.9 C		138.3 C
9		20.8 C		133.0 C		164.0 C
10		25.7 C		36.9 C		49.4 C
11	1.36 (m)	26.8 CH2	2.02 (m)	21.1 CH2	1.66 (m)	18.6 CH2
	1.16 (m)				1.49 (m)
12	1.65–1.76 (m)	33.1 CH2	1.63–1.81 (m)	31.3 CH2	2.38 (m)	29.2 CH2
					2.28 (m)
13		46.1 C		45.0 C		49.4 C
14		48.7 C		49.9 C		47.0 C
15	1.28–1.39 (m)	35.1 CH2	1.22 (m)	30.5 CH2	2.07 (m)	43.8 CH2
					1.87 (m)
16	1.63–1.79 (m)	25.1 CH2	1.62–1.80 (m)	25.1 CH2	4.68 (dd, 8.0, 13.6)	72.4 CH
17	2.11 (dd, 6.6, 9.7)	55.1 CH	2.00 (m)	53.3 CH	2.25 (m)	53.7 CH
18	1.14 (s)	19.8 CH3	0.83 (s)	17.5 CH3	1.23 (s)	19.9 CH3
19	0.76 (d, 4.0)	29.8 CH2	1.09 (s)	18.5 CH3	1.08 (s)	27.3 CH3
	0.56 (d, 4.0)
20		84.9 C		85.0 C		86.4 C
21	1.22 (s)	26.1 CH3	1.22 (s)	25.6 CH3	1.26 (s)	26.3 CH3
22	1.83 (m)	38.4 CH2	1.79 (m)	38.2 CH2	2.20 (m)	37.3 CH2
	1.58 (m)		1.56 (m)		1.71 (m)
23	1.72–1.82 (m)	22.5 CH2	1.72–1.87 (m)	22.5 CH2	1.87–1.98 (m)	23.8 CH2
24	3.75 (dd, 7.0, 8.2)	84.0 CH	3.74 (dd, 7.1, 7.9)	83.9 CH	3.82 (t, 8.0)	84.3 CH
25		71.7 C		71.7 C		70.9 C
26	1.21 (s)	27.5 CH3	1.20 (s)	27.4 CH3	1.23 (s)	27.4 CH3
27	1.13 (s)	24.3 CH3	1.12 (s)	24.3 CH3	1.13 (s)	25.8 CH3
28	1.03 (s)	22.2 CH3	1.05 (s)	21.3 CH3	1.11 (s)	20.6 CH3
29	1.08 (s)	20.8 CH3	1.07 (s)	26.1CH3	1.06 (s)	27.7 CH3
30	0.91 (s)	19.5 CH3	0.90 (s)	24.5 CH3	1.10 (s)	18.9 CH3

16-Deoxyisoargentatin A [(20S,24R)-20,24-Epoxy-25-hydroxylanost-8-en-3-one] (2)

Amorphous colorless powder; [α]_D²⁵ + 41.6 (c 0.09, MeOH); UV (MeOH) λ_max (log ε) 252 (3.28) nm; ECD (MeOH) [θ] −883 (252 nm), +991 (262 nm); ¹H and ¹³C NMR data; see Table 1; positive HRESIMS m/z 439.3568 [MH – H₂O]⁺ (calcd for C₃₀H₄₇O₂, 439.3571).

7-Oxoisoargentatin A [(16β,20S,24R)-20,24-Epoxy-16,25-dihydroxylanost-8-en-3,7-dione] (3)

Amorphous colorless powder; [α]_D²⁵ + 128.5 (c 0.14, MeOH); UV (MeOH) λ_max (log ε) 256 (3.72) nm; ECD (MeOH) [θ] −5597 (222 nm), +29,636 (256 nm), −5636 (341 nm); ¹H and ¹³C NMR data; see Table 1; positive HRESIMS m/z 509.3256 [M + Na]⁺ (calcd for C₃₀H₄₆O₅Na, 509.3238).

24-Epi-argentatin H [(16β,20R,24S)-16,24-Dihydroxycycloart-25-en-3-one] (4)

Amorphous colorless powder; [α]_D²⁵ + 52.0 (c 0.1, CHCl₃); UV (MeOH) λ_max(log ε) 276 (sh, 3.21) nm; ECD (MeOH) [θ] −1118 (297 nm); ¹H and ¹³C NMR data; see Table 2; positive HRESIMS m/z 479.3490 [M + Na]⁺ (calcd for C₃₀H₄₈O₃Na, 479.3496).

Table 2. ¹H and ¹³C NMR Data of 4–7 in CDCl₃.

	4		5		6		7
position	δH	δC	δH	δC	δH	δC	δH	δC
1	1.82 (m)	33.4 CH2	1.80 (m)	33.4 CH2	1.84 (m)	33.4 CH2	1.86 (m)	33.1 CH2
	1.54 (m)		1.52 (m)		1.51 (m)		1.53 (m)
2	2.69 (dt, 6.5, 13.6)	37.4 CH2	2.68 (dt, 6.5, 13.6)	37.5 CH2	2.70 (m)	37.5 CH2	2.70 (dt, 6.3, 14.0)	37.3 CH2
	2.28 (ddd, 2.6, 4.2, 14.0)		2.28 (ddd, 2.4, 4.2, 14.2)		2.28 (m)		2.31 (ddd, 2.6, 4.2, 14.0)
3		216.5 C		216.5 C		216.6 C		216.0 C
4		50.2 C		50.2 C		50.2 C		50.2 C
5	1.68 (m)	48.4 CH	1.67 (m)	48.4 CH	1.68 (m)	48.4 CH	1.73 (dd, 4.4, 12.2)	48.3 CH
6	1.56 (m)	21.4 CH2	1.54 (m)	21.4 CH2	1.55 (m)	21.4 CH2	1.59 (m)	20.8 CH2
	0.95 (m)		0.93 (m		0.93 (m)		0.95 (m)
7	1.37 (m)	26.1 CH2	1.31 (m)	26.4 CH2	1.31 (m)	26.4 CH2	1.33 (m)	26.2 CH2
	1.13 (m)		1.11 (m)		1.11 (m)		1.18 (m)
8	1.63 (m)	47.8 CH	1.62 m)	47.9 CH	1.64 (m)	48.0 CH	1.65 (dd, 4.4, 12.4)	47.4 CH
9		20.8 C		20.9 C		20.9 C		20.3 C
10		25.9 C		26.1 C		26.0 C		26.4 C
11	2.04 (m)	26.4 CH2	2.03 (m)	27.5 CH2	2.03 (m)	27.4CH2	2.15 (m)	26.2 CH2
	1.20 (m)		1.11 (m)		1.10 (m)		1.23 (m)
12	1.52–1.72 (m)	32.5 CH2	1.53–1.67 (m)	32.6 CH2	1.57–1.70 (m)	32.6 CH2	1.95 (m)	32.1 CH2
							1.23 (m)
13		45.3 C		45.4 C		45.4 C		42.0 C
14		46.6 C		46.6 C		46.6 C		45.3 C
15	2.02 (m)	47.3 CH2	1.96 (m)	48.0 CH2	1.98 (m)	48.0 CH2	2.05 (d, 18.7)	50.6 CH2
	1.35 (m)		1.31 (m)		1.33 (m)		1.99 (d, 18.7)
16	4.46 (dt, 5.2 7.5)	72.8 CH	4.27 (dt, 5.2, 7.8)	72.6 CH	4.35 (dt, 5.2, 7.8)	72.6 CH		221.3 C
17	1.63 (m)	56.8 CH	1.60 (m)	56.4 CH	1.64 (m)	56.4 CH	2.28 (d, 9.6)	62.0 CH
18	1.16 (s)	18.9 CH3	1.12 (s)	19.0 CH3	1.14 (s)	19.0 CH3	1.12 (s)	18.7 CH3
19	0.80 (d, 4.4)	29.8 CH2	0.78 (d, 4.4)	29.9 CH2	0.79 (d, 4.4)	29.9 CH2	0.83 (d, 4.4)	30.0 CH2
	0.57 (d, 4.4)		0.56 (d, 4.4)		0.57 (d, 4.4)		0.63 (d, 4.4)
20	1.63 (m)	27.2 CH	1.79 (m)	30.5 CH		30.5 CH	1.70 (m)	28.8 CH
21	0.93 (d, 6.2)	17.8 CH3	0.94 (d, 6.4)	18.0 CH3	0.95 (d, 6.4)	18.0 CH3	0.95 (d, 6.6)	18.2 CH3
22	1.79 (m)	30.8 CH2	2.02 (m)	26.4 CH2	2.01 (m)	26.5 CH2	1.96 (m)	32.1 CH2
	1.04 (m)		1.14 (m)		1.37 (m)		1.23 (m)
23	1.49–1.73 (m)	30.6 CH2	1.72–1.86 (m)	33.1 CH2	1.58–1.70 (m)	33.0 CH2	1.35–1.52 (m)	27.6 CH2
24	4.20 (dd, 3.6, 10.0)	72.9 CH	4.95 (dd, 3.2, 9.0)	80.9 CH	4.87 (dd, 3.2, 8.6)	80.8 CH	3.50 (dt, 11.0, 2.8)	75.9 CH
25		147.9 C		72.9 C		72.7 C		72.6 C
26	4.98 (brs)	110.2 CH2	1.26 (s)	25.6 CH3	1.24 (s)	25.5 CH3	1.20 (s)	26.5 CH3
	4.80 (brs)
27	1.71 (s)	18.2 CH3	1.27 (s)	25.9 CH3	1.24 (s)	25.9 CH3	1.15 (s)	23.4 CH3
28	1.08 (s)	20.8 CH3	1.07 (s)	20.8 CH3	1.07 (s)	20.8 CH3	1.09 (s)	20.7 CH3
29	1.02 (s)	22.1 CH3	1.02 (s)	22.2 CH3	1.02 (s)	22.1 CH3	1.04 (s)	22.2 CH3
30	0.87 (s)	19.9 CH3	0.82 (s)	20.0 CH3	0.84 (s)	20.0 CH3	1.14 (s)	20.0 CH3
1′				122.4 C		134.3 C
2′/6′			7.99 (d, 8.8)	131.7 CH	7.52 (m)	128.2 CH
3′/5′			6.91 (d, 8.8)	113.7 CH	7.38 (m)	128.9 CH
4′				163.5 C	7.38 (m)	134.3 CH
7′			3.85 (s)	55.5 CH3	7.71 (d, 15.6)	145.6 CH
8′				166.5 C	6.47 (d, 15.6)	117.8 CH
9′						167.3 C

24-O-p-Anisoylargentatin C [(16β,20R,24R)-24-p-Anisoyl-16,25-dihydroxycycloartan-3-one] (5)

Amorphous colorless powder; [α]_D²⁵ + 6.6 (c 0.08, MeOH); UV (MeOH) λ_max(log ε) 256 (4.09) nm; ECD (MeOH) [θ] −5628 (260 nm); ¹H and ¹³C NMR data; see Table 2; positive HRESIMS m/z 631.3989 [M + Na]⁺ (calcd for C₃₈H₅₆O₆Na, 631.3970).

24-O-trans-Cinnamoylargentatin C [(16β,20R,24R)-24-trans-Cinnamoyl-16,25-dihydroxycycloartan-3-one] (6)

Amorphous colorless powder; [α]_D²⁵ + 15.5 (c 0.09, MeOH); UV (MeOH) λ_max(log ε) 275 (4.09) nm; ECD (MeOH) [θ] −4438 (281 nm); ¹H and ¹³C NMR data; see Table 2; positive HRESIMS m/z: 627.4029 [M + Na]⁺ (calcd for C₃₉H₅₆O₅Na, 627.4020).

16-Dehydroargentatin C [(20R,24R)-24,25-dihydroxycycloartan-3,16-dione] (7)

Amorphous colorless powder; [α]_D²⁵ – 79.5 (c 0.12, MeOH); UV (MeOH) λ_max (log ε) 260 (sh, 1.54) nm; ECD (MeOH) [θ] −28196 (301 nm); ¹H and ¹³C NMR data; see Table 2; positive HRESIMS m/z 455.3509 [MH – H₂O]⁺ (calcd for C₃₀H₄₇O₃, 455.3520).

16,17(20)-Didehydroargentatin C [(20R,24R)-24,25-Dihydroxycycloart-17-en-3,16-dione] (8)

Amorphous colorless powder; [α]_D²⁵ – 94.7 (c 0.40, MeOH); UV (MeOH) λ_max (log ε) 257 (3.95) nm; ECD (MeOH) [θ] −16953 (259 nm), −6852 (345 nm); ¹H and ¹³C NMR data; see Table 3; positive HRESIMS m/z 493.3297 [M + Na]⁺ (calcd for C₃₀H₄₆O₄Na, 493.3289).

Table 3. ¹H and ¹³C NMR Data of 8–10 in CDCl₃.

	8		9		10
position	δH	δC	δH	δC	δH	δC
1	1.87 (m)	33.2 CH2	1.96 (m)	35.9 CH2	1.97 (m)	35.6 CH2
	1.54 (m)		1.60 (m)		1.60 (m)
2	2.71 (dt, 6.4, 14.0)	37.3 CH2	2.57 (ddd, 7.1, 11.4, 15.8)	34.6 CH2	2.57 (ddd, 7.1, 11.4, 15.8)	34.6 CH2
	2.31 (ddd, 2.4, 4.0, 14.0)		2.38 (ddd, 3.6, 6.7, 15.8)		2.38 (ddd, 3.6, 6.7, 15.8)
3		216.1C		217.8 C		217.7 C
4		50.2 C		47.4 C		47.4 C
5	1.72 (4.6, 12.2)	48.2 CH	1.58 (m)	51.2 CH	1.58 (m)	51.2 CH
6	1.60 (m)	21.2 CH2	1.55–1.67 (m)	19.4 CH2	1.54–1.66 (m)	19.3 CH2
	0.96 (m)
7	1.36 (m)	25.9 CH2	2.04–2.16 (m)	26.2 CH2	2.00–2.18 (m)	26.1 CH2
	1.15 (m)
8		45.6 CH		134.6 C		134.7 C
9		20.2 C		133.6 C		133.6 C
10		26.3 C		36.9 C		36.9 C
11	2.18 (m)	26.4 CH2	1.95–2.07 (m)	20.7 CH2	1.95–2.15 (m)	20.7 CH2
	1.31 (m)
12	2.08 (m)	30.8 CH2	1.67–1.80 (m)	30.9 CH2	1.63–1.76 (m)	30.9 CH2
13		48.4 C		44.5 C		44.5 C
14		42.3 C		47.7 C		47.8 C
15	2.22 (m)	51.2 CH2	1.95 (m)	43.1 CH2	1.93 (m)	43.0 CH2
	2.03 (m)		1.60 (m)		1.65 (m)
16		209.8 C	4.52 (dt, 5.6, 7.3)	73.1 CH	4.50 (dt, 5.5, 7.7)	73.0 CH
17		142.3 C	1.53 (m)	55.1 CH	1.52 (m)	55.1 CH
18	1.33 (s)	23.8 CH3	0.87 (s)	16.6 CH3	0.86 (s)	16.5 CH3
19	0.85 (d, 4.4)	29.8 CH2	1.11 (s)	18.6 CH3	1.11 (s)	18.6 CH3
	0.62 (d, 4.4)
20		151.2 C	1.91 (m)	26.8 CH	1.83 (m)	31.3 CH
21	1.88 (s)	20.2 CH3	0.93 (d, 6.4)	18.1 CH3	0.94 (d, 6.4)	18.6 CH3
22	3.27 (m)	31.4 CH2	1.57 (m)	26.1 CH2	1.62 (m)	32.3 CH2
	2.21 (m)		1.35 (m)		1.07 (m)
23	1.45–1.72 (m)	30.0 CH2	1.66–1.84 (m)	31.3 CH2	1.74 (m)	32.1 CH2
					1.45 (m)
24	3.16 (brd, 10.0)	76.2 CH	3.57 (dd, 2.4, 11.8)	75.1 CH	4.05 (dd, 4.6, 8.6)	77.2 CH
25		72.3 C		73.1 C		147.9 C
26	1.13 (s)	23.4 CH3	1.20 (s)	26.8 CH3	4.94 (brs)	110.8 CH2
					4.81 (brs)
27	1.14 (s)	26.1 CH3	1.14 (s)	22.9 CH3	1.72 (s)	17.8 CH3
28	1.09 (s)	20.7 CH3	1.07 (s)	26.2 CH3	1.07 (s)	26.1 CH3
29	1.04 (s)	22.1 CH3	1.05 (s)	21.3 CH3	1.05 (s)	21.3 CH3
30	0.98 (d, 0.8)	21.0 CH3	0.85 (s)	25.1 CH3	0.84 (s)	25.2 CH3

Isoargentatin C [(16β,20R,24R)-16,24,25-Trihydroxylanost-8-en-3-one] (9)

Amorphous colorless powder; [α]_D²⁵ + 63.4 (c 0.20, MeOH); UV (MeOH) λ_max (log ε) 250 (sh, 3.07) nm; ECD (MeOH) [θ] −842 (316 nm); ¹H and ¹³C NMR data; see Table 3; positive HRESIMS m/z 497.3623 [M + Na]⁺ (calcd for C₃₀H₅₀O₄Na, 497.3602).

Isoargentatin H [(16β,20R,24R)-16,24-Dihydroxylanosta-8,25-dien-3-one] (10)

Amorphous colorless powder; [α]_D²⁵ + 60 (c 0.1, CHCl₃); UV (MeOH) λ_max (log ε) 200 (2.67) nm; ECD (MeOH) [θ] +336 (291 nm); ¹H and ¹³C NMR data; see Table 3; positive HRESIMS m/z 439.3559 [MH – H₂O]⁺ (calcd for C₃₀H₄₇O₂, 439.3571).

3-Epi-quisquagenin [(3α,16β,20S,24R)-20,24-Epoxy-cycloartan-3,16,25-triol] (11)

Amorphous colorless powder; [α]_D²⁵ + 34.6 (c 0.1, MeOH); UV (MeOH) λ_max(log ε) 202 (sh, 2.90) nm; ECD (MeOH) [θ] −321 (202 nm); ¹H and ¹³C NMR data; see Table 4; positive HRESIMS m/z 475.3781 [MH]⁺ (calcd for C₃₀H₅₁O₄, 475.3787).

Table 4. ¹H and ¹³C NMR Data of 11–13 in CDCl₃.

	11		12		13
position	δH	δC	δH	δC	δH	δC
1	2.24 (m)	37.6 CH2	1.71 (m)	35.1 CH2	1.81 (m)	33.4 CH2
	1.66 (m)		1.22 (m)		1.52 (m)
2	1.91 (m)	28.5 CH2	1.64 (m)	28.1 CH2	2.68 (m)	37.5 CH2
	1.62 (m)				2.27 (m)
3	3.45 (m)	76.9 CH	3.21 (m)	78.9 CH		216.6 C
4		39.5 C		38.5 C		50.2 C
5	1.80 (m)	41.2 CH	1.03 (m)	50.4 CH	1.67 (m)	48.4 CH
6	1.48 (m)	21.2 CH2	1.67 (m)	18.2 CH2	1.53 (m)	21.4 CH2
	0.75 (m)				0.93 (m)
7	1.85 (m)	27.3 CH2	2.05 (m)	20.7 CH2	2.03 (m)	26.4 CH2
	0.98 (m)				1.14 (m)
8	1.64 (m)	48.0 CH		134.5 C	1.65 (m)	47.9 CH
9		19.4 C		133.7 C		20.9 C
10		25.8 C		37.0 C		26.0 C
11	1.32 (m)	25.7 CH2	2.08 (m)	26.4 CH2	1.36 (m)	25.9 CH2
	1.06 (m)				1.10 (m)
12	1.76 (m)	33.2 CH2	1.75 (m)	31.6 CH2	1.56–1.68 (m)	32.5 CH2
	1.65 (m)
13		46.2 C		47.6 C		45.3 C
14		46.7 C		45.2 C		46.7 C
15	1.98 (m)	48.5 CH2	1.90 (m)	43.4 CH2	1.99 (m)	47.6 CH2
	1.47 (m)		1.78 (m)		1.35 (m)
16	4.57 (q)	73.6 CH	4.61 (q)	74.0 CH	4.44 (dt, 4.8, 8.0)	72.6 CH
17	2.11 (d, 7.7)	55.5 CH	2.02 (d, 7.7)	54.8 CH	1.59 (m)	56.8 CH
18	1.26 (s)	25.3 CH3	1.05 (s)	18.7 CH3	1.15 (m)	19.0 CH3
19	0.54 (d, 4.0)	30.4 CH2	0.97 (s)	18.9 CH3	0.79 (d, 4.0)	29.9 CH2
	0.34 (d, 4.0)				0.56 (d, 4.0)
20		87.3 C		87.1 C	1.82 (m)	31.0 CH
21	1.40 (s)	20.9 CH3	1.30 (s)	25.8 CH3	0.92 (d, 6.4)	18.3 CH3
22	2.01 (m)	26.6 CH2	2.17 (m)	37.8 CH2	1.71 (m)	32.1 CH2
	1.15 (m)		1.68 (m)		1.43 (m)
23	1.92 (m)	23.8 CH2	1.91 (m)	24.2 CH2	1.72 (m)	33.0 CH2
					1.40 (m)
24	3.83 (t, 7.5)	84.5 CH	3.81 (t, 7.7)	84.3 CH	4.02 (dd, 3.2, 9.2)	77.2 CH
25		70.9 C		70.9 C		148.0 C
26	1.11 (s)	26.0 CH3	1.11 (s)	26.0 CH3	4.92 (brs)	110.7 CH2
					4.79 (brs)
27	1.22 (s)	27.4 CH3	1.22 (s)	27.8 CH3	1.71 (s)	17.8 CH3
28	0.86 (s)	20.3 CH3	0.83 (s)	25.2 CH3	1.07 (s)	20.8 CH3
29	0.86 (s)	21.1 CH3	0.80 (s)	15.4 CH3	1.02 (s)	22.1 CH3
30	0.93 (s)	26.1 CH3	0.98 (s)	27.9 CH3	0.86 (s)	20.8 CH3

Isoquisquagenin [(3β,16β,20S,24R)-20,24-epoxy-lanost-8-en-3,16,25-triol] (12)

Amorphous colorless powder; [α]_D²⁵ + 21.3 (c 0.2, MeOH); UV (MeOH) λ_max (log ε) 264 (sh, 2.39) nm, λ_max (log ε) 205 (sh, 2.60) nm; ECD (MeOH) [θ] +1181 (205 nm); ¹H and ¹³C NMR data; see Table 4; positive HRESIMS m/z 475.3773 [MH]⁺ (calcd for C₃₀H₅₁O₄, 475.3787).

Argentatin H [(16β,20R,24R)-16,24-Dihydroxycycloart-25-en-3-one] (13)

Colorless amorphous solid; [α]_D²⁵ + 31.2 (c 0.88, MeOH); UV (MeOH) λ_max (log ε) 275 (sh, 3.89) nm; ECD (MeOH) [θ] −1697 (296 nm); ¹H and ¹³C NMR data; see Table 4; positive HRESIMS m/z 479.3494 [M + Na]⁺ (calcd for C₃₀H₄₈O₃Na, 479.3496).

Preparation of (R)- and (S)-MTPA Esters of 4, 7, and 10

Each triterpenoid (4, 7, or 10; 1.0 mg) was dissolved in pyridine-d₅ (0.5 mL), and the solution was transferred into a dry NMR tube. (S)-(−)-α-Methoxy-α-(trifluoromethyl)-phenylacetyl chloride [(S)-MTPA chloride] (5.0 μL) was added to the NMR tube immediately under a stream of N₂ and was shaken carefully to mix the sample and MTPA chloride. The NMR tube was allowed to stand at 25 °C for 1 h to afford the (R)-MTPA ester derivatives (4a, 7a, and 10a). Another portion of 4, 7, or 10 (1.0 mg) in pyridine-d₅ was reacted in a second NMR tube with (R)-(+)-α-methoxy-α-(trifluoromethyl)-phenylacetyl chloride [(R)-MTPA chloride] (5.0 μL) at 25 °C for 1 h to afford the (S)-MTPA ester derivatives (4b, 7b, and 10b). ¹H NMR data of 4a (400 MHz, pyridine-d₅): δ 5.718 (m, 1H, H-24), 5.191 (brs, 1H, H-26a), 5.011 (brs, 1H, H-26b), 2.234 (m, 1H, H-20), 2.102 (m, 1H, H-23a), 1.811 (m, 1H, H-23b), 1.800 (s, 3H, H₃-27), 1.754 (m, 1H, H-22b), 1.136 (m, 1H, H-22a), 1.001 (d, J = 6.2 Hz, 3H, H₃-21); ¹H NMR data of 4b (400 MHz, pyridine-d₅): δ 5.678 (m, 1H, H-24), 5.076 (brs, 1H, H-26a), 4.952 (brs, 1H, H-26b), 2.286 (m, 1H, H-20), 2.124 (m, 1H, H-23a), 1.884 (m, 1H, H-23b), 1.763 (m, 1H, H-22a), 1.660 (s, 3H, H₃-27), 1.402 (m, 1H, H-22b), 1.049 (d, J = 6.2 Hz, 3H, H₃-21).; ¹H NMR data of 7a (400 MHz, pyridine-d₅): δ 5.620 (m, 1H, H-24), 2.312 (d, J = 7.8 Hz, 1H, H-17), 1.920 (m, 2H, H₂-23), 1.800 (m, 1H, H-20), 1.500 (s, 6H, H₃-26, H₃-27), 1.400 (m, 2H, H₂-22), 0.930 (d, J = 5.6 Hz, 3H, H₃-21); ¹H NMR data of 7b (400 MHz, pyridine-d₅): δ 5.630 (m, 1H, H-24), 2.420 (d, J = 7.9 Hz, 1H, H-17), 2.050 (m, 2H, H₂-23), 1.970 (m, 1H, H-20), 1.700 (m, 2H, H₂-22), 1.440 (s, 3H, H₃-26), 1.430 (s, 3H, H₃-27), 1.080 (d, J = 6.7 Hz, 3H, H₃-21); ¹H NMR data of 10a (400 MHz, pyridine-d₅): δ 5.699 (m, 1H, H-24), 5.140 (brs, 1H, H-26a), 5.010 (brs, 1H, H-26b), 2.680 (m, 1H, H-20), 2.022 (m, 2H, H₂-23), 1.782 (m, 2H, H₂-22), 1.669 (s, 3H, H₃-27), 1.069 (d, J = 6.2 Hz, 3H, H₃-21); ¹H NMR data of 10b (400 MHz, pyridine-d₅): δ 5.751 (m, 1H, H-24), 5.232 (brs, 1H, H-26a), 5.051 (brs, 1H, H-26b), 2.258 (m, 1H, H-20), 1.999 (m, 2H, H₂-23), 1.802 (s, 3H, H₃-27), 1.776 (m, 2H, H₂-22), 1.062 (d, J = 6.2 Hz, 3H, H₃-21).

Hydrolysis of 5 and 6

A solution of the triterpene ester 5 or 6 (0.1 mg) in MeOH (0.1 mL) containing Na₂CO₃ (0.1 mg) was stirred at 25 °C for 2 h (TLC control). TLC [silica gel, CHCl₃/MeOH (95:5)] and HPLC [RP C₁₈, MeOH/H₂O (85:15), t_R = 24.1 min)] comparison of the resulting product with an authentic sample confirmed the hydrolysis product to be identical to argentatin C (17).

Acetylation of Argentatin H (13)

To a solution of 13 (2.2 mg) in anhydrous pyridine (0.5 mL) was added Ac₂O (0.5 mL), and the mixture was stirred at 25 °C for 6 h (TLC control), after which it was poured into ice/water (10.0 mL) and extracted with EtOAc (3 × 10.0 mL). The EtOAc extracts were combined and washed with H₂O (3 × 5.0 mL), dried over anhydrous Na₂SO₄, and evaporated under reduced pressure. The crude product thus obtained was separated by silica gel (1.0 g) column chromatography. Elution with CHCl₃/MeOH (99:1, v/v) afforded the acetylated product (2.3 mg), which was identified as argentatin H diacetate (23) by comparison of its ¹H and ¹³C NMR data with those reported.⁸

Reduction of Argentatin A (14) to 3-Epi-quisquagenin (11) and Quisquagenin (15)

To a stirred solution of 14 (25.0 mg) in MeOH (10.0 mL) at 0 °C was added NaBH₄ (3.0 mg), and stirring was continued at this temperature until the disappearance of the starting material (TLC control). The reaction was then quenched with ice-cold water (10.0 mL), concentrated under reduced pressure, and extracted with EtOAc (3 × 10.0 mL). The EtOAc extracts were combined and washed with H₂O (3 × 5.0 mL), dried over anhydrous Na₂SO₄, and evaporated under reduced pressure. The crude product (25.0 mg) thus obtained was separated by RP-HPLC (MeOH–H₂O; 82.5:17.5) to afford major [21.5 mg, 85%; t_R 30.5 min; [α]_D²⁵ + 47.4 (c 0.2, MeOH)] and minor [1.5 mg, 6%; t_R 34.5 min; [α]_D + 34.2 (c 0.1, MeOH)] products as white solids. These were identified as quisquagenin (15) and 3-epi-quisquagenin (11), respectively, by comparison of their ¹H and ¹³C NMR data with those of the natural products obtained above.

Reduction of Isoargentatin A (16) to Isoquisquagenin (12)

To a stirred solution of isoargentatin A (16, 4.0 mg) in MeOH (2.0 ml) at 0 °C was added NaBH₄ (1.0 mg), and the reaction mixture was stirred at 0 °C until the disappearance of the starting material (TLC control). The reaction mixture was quenched with ice-cold water (5.0 mL), concentrated under reduced pressure, and extracted with EtOAc (3 × 5.0 mL). The EtOAc extracts were combined and washed with H₂O (3 × 2.0 mL), dried over anhydrous Na₂SO₄, and evaporated under reduced pressure to afford isoquisquagenin (12) (4.0 mg, 99%) as a white solid; [α]_D²⁵ + 21.6 (c 0.15, MeOH); ¹H and ¹³C NMR data were identical to those of the natural product obtained above.

Cytotoxicity Assay

The tetrazolium-based colorimetric (MTT) assay was employed for evaluation of cytotoxic activity of samples against the sentinel cancer cell lines, human non-small cell lung (NCI-H460), human CNS glioma (SF-268), and human breast (MCF-7). Compounds were also tested versus human prostate adenocarcinoma (PC-3M), human metastatic breast adenocarcinoma (MDA-MB-231), and normal human lung fibroblast (WI-38) cells as described previously.¹⁷ Doxorubicin and dimethyl sulfoxide (DMSO) were used as positive and negative controls, respectively.

Results and Discussion

Characterization of Compounds

The resin of the P. argentatum line AZ-2 obtained as a byproduct from Bridgestone Americas, Inc. rubber production was fractionated by solvent–solvent partitioning, size permeation chromatography, and silica gel chromatography. The resulting fractions were further purified by preparative TLC and/or HPLC to afford 19 triterpenoids. Nine of these were identified as argentatin A (14),^5a quisquagenin (15),¹⁸iso-argentatin A (16),^7a argentatin C (17),^5a argentatin B (18),^5a argentatin D (19),^5a,19 3-epi-argentatin D (20),^5a,19iso-argentatin B (21),^7a and cyclofoetigenin A (22)²⁰ by comparison of their ¹H and ¹³C NMR spectroscopic data with those reported for these compounds. It should be noted that 3-epi-argentatin D (20) has been incorrectly referred as isoargentatin D.²¹ In order to avoid similar confusions, here, we provide systematic names for all new compounds based on their triterpenoid skeletons (Figure 1).

Figure 1.

Structures of triterpenoids 1–22 from guayule resin and diacetylargentatin H (23).

Triterpenoid 1 was determined to have the molecular formula C₃₀H₄₈O₃ from its HRESIMS and NMR data and indicated 7 degrees of unsaturation. The ¹H NMR spectrum of 1 (Table 1) exhibited seven singlet methyls [δ_H 0.91 (H₃-30), 1.03 (H₃-28), 1.08 (H₃-29), 1.13 (H₃-27), 1.14 (H₃-18), 1.21 (H₃-26), and 1.22 (H₃-21)], an oxygenated methine [δ_H 3.75 (d, J = 7.0, 8.2 Hz, H-24)], and a pair of doublets [δ_H 0.56 (d, J = 4.0 Hz, H-19) and 0.76 (d, J = 4.0 Hz, H-19)], typical of geminal methylene protons of a tetra-substituted cyclopropane ring, characteristic of a cycloartane-type triterpenoid.²² The ¹³C NMR spectrum of 1 (Table 1) assigned with the help of HSQC and HMBC data (Figure 2 and Figures S4 and S5 in the Supporting Information) and comparison of these data with those of argentatin A (14)^7a indicated that the only difference between 1 and 14 is that the oxygenated methine (CHOH-16) signal of 14 was replaced by a methylene signal [δ_C 25.1 (CH₂-16)] in 1. The signal due to CH₂-15 in the ¹³C NMR spectrum of 1 displayed a high-field shift compared to that of 14 due to the absence of the β-effect of substituent perturbations by the OH group at C-16.²³ Thus, the structure of 1 was determined as 16-deoxyargentatin A [(20S,24R)-20,24-epoxy-25-hydroxycycloartan-3-one].

Figure 2.

Key HMBC correlations of 1, 3, 4, 7, 8, 10, and 13 and key NOESY correlation of 8.

The HRESIMS and ¹H and ¹³C NMR spectroscopic data of triterpenoid 2 were consistent with the molecular formula C₃₀H₄₈O₃, indicating 7 degrees of unsaturation. In its ¹H NMR spectrum, 2 exhibited eight singlet methyl signals characteristic of lanostane-type triterpenoids^7a bearing a 19-methyl, 8(9)-ene and a side-chain tetrahydrofuran moiety. Comparison of the ¹H NMR data of 2 (Table 1) with those of isoargentatin A (16) which was found to co-occur in this resin sample indicated that unlike 16, which has two oxygenated methine protons, 2 contained only one of these protons [δ_H 3.74 (dd, J = 7.1, 7.9 Hz)] suggesting that 2 is probably the 16-deoxygenated analogue of 16. Analysis of the ¹³C NMR spectrum of 2 with the help of HSQC and HMBC data and comparison of the ¹³C NMR data with those of isoargentatin A (16)^7a confirmed that the major structural differences between 2 and 16 are due to the substituents in ring D. These data also suggested that the oxygenated methine moiety (CHOH-16) of 16 was replaced by a methylene moiety [δ_C 25.1, CH₂-16] in 2. On the basis of the foregoing evidence, the structure of 2 was determined as 16-deoxyisoargentatin A [(20S,24R)-20,24-epoxy-25-hydroxylanost-8-en-3-one].

The molecular formula of triterpenoid 3 was determined as C₃₀H₄₆O₅ on the basis of its HRESIMS and NMR data, indicating 8 degrees of unsaturation. The ¹H NMR spectrum of 3 (Table 1) exhibited signals due to two oxygenated methines [δ_H 3.82 (dd, J = 8.0 Hz, H-24), 4.68 (dd, J = 8.0), 13.6 Hz, H-16] similar to isoargentatin A (16) in addition to eight singlet methyl signals characteristic of lanostane-type triterpenoids bearing a 19-methyl, 8(9)-ene, and a side-chain tetrahydrofuran ring.^7a However, its ¹³C NMR spectrum (Table 1) indicated the presence of signals due to two carbonyl carbons [δ_C 218.2 (C-3), 198.4 (C-7)] compared to 16, which contains only the C-3 carbonyl group. The second carbonyl of 3 was located at C-7 by the HMBC correlations of H₃-19 [δ_H 1.08 (s)]/C-9 (δ_C 164.0) and H₃-30 [δ_H 1.10 (s)]/C-8 (δ_C 138.3). Based on the above evidence, the structure of 3 was determined as 7-oxoisoargentatin A [(16β,20S,24R)-20,24-epoxy-16,25-dihydroxylanost-8-en-3,7-dione].

Triterpenoid 4 was determined to have the molecular formula C₃₀H₄₈O₃ by the analysis of its HRESIMS and NMR data, indicating 7 degrees of unsaturation. The ¹H NMR spectrum (Table 2) of 4 showed six methyl signals, two oxygenated methines, a pair of olefinic methylene signals, and a pair of doublets due to geminal protons of a tetra-substituted cyclopropane moiety typical of cycloartane-type triterpenoids. The ¹³C NMR spectrum of 4 (Table 2) exhibited 30 signals including those due to carbonyl [δ_C 216.5 (C-3)], olefinic [δ_C 147.9 (C-25) and 110.2 (C-26)], and oxygenated [δ_C 72.8 (CH-16) and 72.9 (CH-24)] carbons. These data indicated that 4 has the same carbon skeleton as argentatin C (17),^7b and HMBC correlation analysis of 4 (Figure 2) confirmed that its planar structure is the same as that of argentatin H (13),⁸ both of which are cometabolites of guayule resin (see below). The β-orientation of 16-OH in 4 was supported by the NMR chemical shifts of C-16 and H-16 and the ¹H NMR coupling pattern of H-16, which is similar to that of 13. The forgoing suggested 4 differed from argentatin H (13) only due to the configuration of the OH group at C-24. The absolute configuration of the chiral center at C-24 of 4 was determined to be S by the application of the modified Mosher’s ester method (Figure 3).²⁴ Therefore, the structure of 4 was determined as 24-epi-argentatin H [(16β,20R,24S)-16,24-dihydroxycycloart-25-en-3-one].

Figure 3.

Δδ values [Δδ values (in ppm) = δ_S – δ_R] obtained for (S)- and (R)-MTPA esters of 4, 7, and 10.

The HRESIMS and ¹H and ¹³C NMR spectroscopic data of 5 were consistent with the molecular formula C₃₈H₅₆O₆, suggesting 11 degrees of unsaturation and indicating that it contained eight carbons more than that of a typical cycloartane or lanostane triterpenoid. The ¹H NMR spectrum of 5 (Table 2) exhibited the signals characteristic of a p-anisoyl group [δ_H 6.91 (2H, d, J = 8.8 Hz, H-3′/5′), 7.99 (2H, d, J = 8.8 Hz, H-2′/6′), 3.85 (3H, s, 4′-OMe)] besides those of a cycloartane-type triterpenoid including seven methyls, two oxygenated methines, and a pair of signals of a tetra-substituted cyclopropane moiety. The ¹³C NMR spectrum of 5 (Table 2) analyzed with the help of HSQC and HMBC data exhibited thirty signals resembling those of argentatin C (17)^7a and six additional signals [δ_C 166.5 (C-8′), 164,5 (C-7′), 131.7 (CH-2′/6′), 122.4 (C-1′), 113.7 (CH-3′/5′), and 55.5 (CH₃-8′)], confirming the presence of a p-anisoyl substituent in 5. Careful comparison of the ¹³C NMR data of 5 with those of argentatin C (17)^7b suggested that the chemical shift of C-24 of 5 had moved to downfield (2.5–3.0 ppm) compared to that of 17, indicating that the p-anisoyl group is attached to the OH group at C-24.²⁵ The configuration at C-24 of 5 was deduced to be R, same as that of 17 by comparison of their ¹³C NMR chemical shift data. This was confirmed by the hydrolysis (NaCO₃/MeOH) of 5, affording a product identical (¹H and ¹³C NMR) to argentatin C (17). Based on foregoing information, compound 5 was identified as 24-O-p-anisoylargentatin C [(16β,20R,24R)-24-p-anisoyloxy-16,25-dihydroxycycloartan-3-one].

Compound 6 was determined to have the molecular formula C₃₉H₅₆O₅ by the analysis of its HRESIMS and NMR data, indicating 12 degrees of unsaturation. The ¹H and ¹³C NMR spectroscopic data of 6 (Table 2) were almost identical to those of 5 except for the signals due to the substituent acyl group, suggesting that 6 is also a 24-O-acylated analogue of argentatin C (17). This acyl substituent of 6 was determined to be a trans-cinnamoyl group by its characteristic ¹H NMR [δ_H 6.47 (1H, d, J = 15.6 Hz, H-8′), 7.71 (1H, d, J = 15.6 Hz, H-7′), 7.52 (2H, m, H-2’/6′), 7.38 (3H, m, H-3′/5′ and H-4′)] and ¹³C NMR [δ_C 167.3 (C-9′), 145.6 (CH-7′), 134.3 (CH-4′ and C-1′), 128.9 (CH-3’/5′), 128.2 (CH-2’/6′), and 117.8 (CH-8′)] data. Hydrolysis (NaCO₃/MeOH) of 6 afforded argentatin C (17), confirming the identity of 6 as 24-O-trans-cinnamoylargentatin C [(16β,20R,24R)-24-trans-cinnamoyloxy-16,25-dihydroxycycloartan-3-one].

Triterpenoid 7 exhibited a [MH – H₂O]⁺ ion at m/z 455.3509 in its HRESIMS, consistent with the molecular formula C₃₀H₄₈O₄, indicating 7 degrees of unsaturation. The ¹H NMR data of 7 suggested it to be an analogue of argentatin C (17) as it contained signals due to seven methyls in addition to the tetra-substituted cyclopropane moiety (Table 2). Complete analysis of the ¹³C NMR spectrum of 7 with the help of HMBC data (Figure 2) suggested that it contained signals due to two ketone carbonyls [δ 216.0 (C-3) and 221.3 (C-16)] besides those typical of the triterpenoid skeleton. Comparison of these data with those of argentatin C (17) located the additional carbonyl group at C-16, indicating that the CHOH moiety of 17 at this position has undergone oxidation to a C=O moiety. The signal due to C-24 of 7 appeared at 75.9 ppm in its ¹³C NMR spectrum similar to that of 17, indicating that 7 has the same R configuration at C-24 as argentatin C (17). This was confirmed by the application of the modified Mosher’s ester method (Figure 3).²⁴ Thus, the structure of 7 was determined as 16-dehydroargentatin C [(20R,24R)-24,25-dihydroxycycloartan-3,16-dione].

The HRESIMS and ¹³C NMR spectroscopic data of 8 were consistent with the molecular formula C₃₀H₄₆O₄ and indicated 8 degrees of unsaturation. The ¹H NMR spectrum of 8 (Table 4) exhibited signals due to seven methyl groups and a pair of signals characteristic of geminal methylene protons of a tetra-substituted cyclopropane ring, suggesting that 8 is an argentatin C-type triterpenoid. These ¹H NMR data also indicated that the signal due to H₃-21 that usually appears at δ_H 0.92–0.95 as a doublet (J = 6.2–6.6 Hz) in these triterpenoids has moved downfield to δ_H 1.88 and appeared as a singlet in 8, suggesting that this methyl group is attached to a sp² carbon of a double bond. The ¹³C NMR spectrum of 8 analyzed with the help of HMBC data (Figure 2) showed the presence of signals due to two ketone carbonyls (δ_C 221.3 and 209.8) and a double bond (δ_C 142.3 and 151.2) besides those typical of the triterpenoid skeleton. The additional degree of unsaturation in 8 compared to 16-dehydroargentatin C (7) was attributable to this double bond and the low-field shift of the C-16 carbonyl carbon from δ_C 221.3 to 209.8 ppm in its ¹³C NMR spectrum indicated that this double bond is in conjugation with C-16 carbonyl moiety. Analysis of the HMBC data confirmed the presence of a 17(20)-en-16-one moiety in 8 (Figure 2). The ¹³C NMR signal due to C-24 of 8 appeared at δ_C 76.2 ppm, almost the same as that of argentatin C (17), suggesting that 8 has the same R configuration at C-24 as in 17. The configuration of the C-17(20) double bond was assigned as Z by the NOESY correlation between H₃-18 (δ_H 1.33) and H₃-21 (δ_H 1.88) (Figure 2). Based on the foregoing evidence, the structure of 8 was determined as 16,17(20)-didehydroargentatin C [(20R,24R)-24,25-dihydroxycycloart-17(20)-en-3,16-dione].

The molecular formula of triterpenoid 9 was determined as C₃₀H₅₀O₄ by its HRESIMS and ¹³C NMR spectroscopic data and indicated 6 degrees of unsaturation. The ¹H NMR spectrum of 9 (Table 3) exhibited signals due to eight methyl groups. These data and the absence of typical signals due to the cyclopropane moiety in its ¹H NMR spectrum suggested that 9 is a lanostane-type triterpenoid.^7b The ¹³C NMR spectrum of 9 showed 30 signals including those due to eight methyls, a ketone carbonyl, two olefinic carbons, and three oxygenated carbons (Table 3). The NMR signals due to the side-chain moiety were almost identical to those of argentatin C (17).^7b These data suggested that in 9, the cyclopropane moiety of 17 is replaced with CH₃-19 and 8,9-ene moieties, resulting in a lanostane-type triterpenoid. Therefore, the structure of 9 was determined as isoargentatin C [(16β,20R,24R)-16,24,25-trihydroxylanost-8-en-3-one].

Triterpenoid 10 was determined to have the molecular formula C₃₀H₄₈O₃ by the analysis of its HRESIMS and NMR data, indicating 7 degrees of unsaturation. The ¹H and ¹³C NMR spectroscopic data of 10 (Table 3) analyzed with the help of HMBC data (Figure 2) resembled closely to those of isoargentatin C (9) but contained a side chain with a gem-disubstituted olefin moiety [δ_H 4.81 (brs, 1H) and 4.94 (brs, 1H); δ_C 110.8 and 147.9] bearing a methyl group [δ_H 1.71 (s, 3H); δ_C 17.8] instead of the terminal iso-propanol group in 9. These data and comparison of their molecular formulae suggested that 10 may be the 25,26-ene analogue resulting from isoargentatin C (9) by the loss of a molecule of water. This was supported by the HMBC correlations of H-26 (δ_H 4.94 and 4.81)/CH₃-27 (δ_C 17.8) and H-26/CH-24 (δ_C 77.2) (Figure 2). The ¹³C NMR chemical shift of CH-24 (δ_C 77.2 ppm) was almost the same as that of argentatin H (13), suggesting R configuration for C-24. This was further confirmed by the application of the modified Mosher’s ester method (Figure 3).²⁴ Triterpenoid 10 was thus identified as isoargentatin H [(16β,20R,24R)-16,24-dihydroxylanosta-8,25-dien-3-one].

The HRESIMS and ¹³C NMR spectroscopic data of triterpenoids 11 and 12 were consistent with the molecular formula C₃₀H₅₀O₄ and indicated 6 degrees of unsaturation. The ¹H and ¹³C NMR spectra of 11 (Table 4) exhibited signals due to seven methyls, three oxygenated methines, and a tetra-substituted cyclopropane moiety, characteristic of cycloartane-type triterpenoids.²² The presence of three oxygenated methine signals at δ_C 84.5 (C-24), 76.9 (C-3), and 73.6 (C-16) in the ¹³C NMR spectrum of 11 suggested that it has a structure closely related to that of quisquagenin (15) [δ_C 84.5 (C-24), 78.7 (C-3), and 73.4 (C-16)].¹⁸ These data suggested that 11 and 15 had close structural similarities except for the orientation of the OH group at C-3. This was confirmed by the reduction (NaBH₄/MeOH) of the corresponding C-3 ketone, argentatin A (14), affording 15 (85% yield) and 11 (6% yield) having [α]_D, ¹H, and ¹³C NMR data identical to the natural products encountered in this work. This product distribution can be explained by taking into consideration the reducing agent (NaBH₄) approaching mainly from the less sterically hindered α face of the molecule (Figure 4). Thus, compound 11 was identified as 3-epi-quisquagenin [(3α,16β,20S,24R)-20,24-epoxy-cycloartan-3,16,25-triol].

Figure 4.

Chemical conversion of isoargentatin A (16) to isoquisquagenin (12) and argentatin A (14) to 3-epi-quisquagenin (11) and quisquagenin (15).

The ¹H and ¹³C NMR data of 12 (Table 4) closely resembled those of quisquagenin (15)¹⁸ except for the absence of signals due to the cyclopropane moiety. Instead, 12 contained an additional methyl group [δ_H 0.97 (s, H₃-19); δ_C 18.9 (C-19)] and a tetra-substituted double bond [δ_C 134.5 (C-8) and 133.7 (C-9)], suggesting that it is a lanostane-type triterpenoid.^7a The presence of signals due to three oxygenated methines [δ_H 4.61 (q), 3.81 (t, J = 15.2 Hz), and 3.21 (m); δ_C 84.3 (CH), 78.9 (CH), 74.0 (CH)] in its ¹H and ¹³C NMR spectra and comparison of these data with those of isoargentatin A (16) indicated that 12 contained a CHOH moiety at C-3 instead of carbonyl moiety in 16. This was confirmed by the reduction (NaBH₄/MeOH) of C-3 carbonyl group of 16, affording 12 in 99% yield. Formation of 12 with a β-OH at C-3 as the sole product is known for related 3-keto steroids²⁶ and can be explained by taking into account the reducing agent (NaBH₄) approaching from the less sterically hindered α face of the molecule (Figure 4). Thus, compound 12 was identified as isoquisquagenin [(3β,16β,20S,24R)-20,24-epoxy-lanost-8-en-3,16,25-triol]. A literature search revealed that a compound with the gross structure as in 12 has been reported previously²⁷ but with unspecified stereochemistry.

Triterpenoid 13 exhibited a [M + Na]⁺ ion at m/z 479.3494 in its HRESIMS, consistent with the molecular formula C₃₀H₄₈O₃, indicating 7 degrees of unsaturation. The ¹H and ¹³C NMR spectra of 13 (Table 4) analyzed with the help of HSQC data were very similar to those of 24-epi-argentatin H (4) obtained above, suggesting that 13 is the 24-epimer of 4 and should be identical to argentatin H. Argentatin H has previously been characterized as its diacetate (23)⁸ but with undefined absolute stereochemistry at C-24. Acetylation (Ac₂O/pyridine) of 13 afforded its diacetate with ¹H and ¹³C NMR data identical to those reported for argentatin H diacetate (23).⁸ Since the absolute configuration at C-24 of 4 was determined to be S (see above and Figure 3), argentatin H should have R absolute configuration at this position. Thus, argentatin H was identified as (16β,20R,24R)-16,24-dihydroxycycloart-25-en-3-one (13). Careful inspection of the ¹³C NMR data for 4 and 13 suggested that the γ-effect of the chiral center C-20 can be used to distinguish the stereochemistry of C-24. The R isomer (13) has weaker γ-effect, which results in C-20 (δ_C 31.0) and C-24 (δ_C 77.2) at lower-field compared to those of the S isomer (4), which appeared at δ_C 27.2 ppm for C-20 and δ_C 72.9 ppm for C-24. These differences may be utilized in future studies to determine the stereochemistry of C-24 of triterpenoids with similar side chains.

Cytotoxic Activity

Previous reports of cytotoxic activity of cycloartane and lanostane types of triterpenoids including argentatins prompted us to evaluate 1–22 for their cytotoxic activity in a panel of three sentinel human cancer cell lines MCF-7 (breast adenocarcinoma), NCI-H460 (non-small cell lung), and SF-268 (CNS glioma), two metastatic cancer cell lines MDA-MB-231 (breast adenocarcinoma) and PC-3M (prostate adenocarcinoma), and normal lung epithelial cells (WI-38). Initially, a test concentration of 35 μM was selected based on recently reported in vitro cytotoxic activity for argentatin A (14).¹³ Cytotoxicity assay data revealed that at this concentration most of the triterpenoids showed weak to moderate inhibition (Table S1 in the Supporting Information). Among these, argentatin H (13), argentatin A (14), and argentatin B (18) were selected for further evaluation based on their activities and availability. Their IC₅₀ (concentration required to kill 50% of cells) data (Table 5) suggested that 13 and 18 were more cytotoxic than 14 to all cancer and normal cells tested, but 14 had moderate selectivity for all cancer cell lines compared to normal cells. It was also found that the cytotoxic activity (IC₅₀ = 31.9–35.0 μM) of argentatin A (14) for the cell lines tested for 72 h is slightly better than the activity (IC₅₀ = 43.2–61.3 μM) reported for 14 for a series of colon cancer cell lines.¹³ It is also noteworthy that the cytotoxic activity of argentatin B (18) is ca. 2-fold higher than that of argentatin A (14) for some cancer cell lines, suggesting the possible effect of structure on cytotoxic activity of these triterpenoids. We are currently investigating the effect of structural modification of major triterpenoid constituents of guayule resin on their biological activities, and outcomes of these studies will be reported in due course.

Table 5. Cytotoxicity (IC₅₀) Data for 13, 14, and 18 against Cancer Cell Lines and Normal Cells^a.

	Cell linesb
Compound	PC-3M	NCI-H460	MCF-7	SF-268	MDA-MB-231	WI-38
13	21.8 ± 2.7	19.6 ± 1.3	15.2 ± 0.7	24.3 ± 0.3	22.3 ± 0.5	19.0 ± 0.7
14	31.9 ± 0.7	31.2 ± 0.0	32.8 ± 1.2	35.0 ± 0.2	32.9 ± 0.1	41.4 ± 2.8
18	13.5 ± 1.7	17.5 ± 0.9	23.1 ± 0.4	31.2 ± 1.7	32.0 ± 0.8	21.0 ± 2.9
Doxorubicin	0.25 ± 0.02	0.05 ± 0.01	0.32 ± 0.09	0.45 ± 0.07	0.67 ± 0.11	0.80 ± 0.10

^aResults are expressed as IC₅₀ values in μM. Doxorubicin and DMSO were used as positive and negative controls.

^bKey: PC-3M = metastatic human prostate adenocarcinoma; NCI-H460 = human non-small cell lung cancer; MCF-7 = human breast cancer; SF-268 = human CNS cancer (glioma), MDA-MB-231 = human metastatic breast adenocarcinoma; WI-38 = normal human lung fibroblast cells.

In conclusion, detailed investigation of the resin of P. argentatum AZ-2, a byproduct of guayule rubber production, led to the isolation, identification, and cytotoxicity evaluation of 12 new and 10 known cycloartane- and lanostane-type triterpenoids. The cytotoxic activity of argentatin A (14), together with its recently demonstrated promising antitumor activity in a mouse xenograft model of human colon cancer with no adverse toxicity,¹⁵ warrants further investigation of cycloartane-type triterpenoids for their development as potential anticancer agents. Furthermore, the relative cytotoxic activities associated with argentatin H (13), argentatin A (14), and argentatin B (18) suggest the possibility of structural modification of major triterpenoids of guayule resin into value-added products with potential anticancer activity.

Glossary

Abbreviations

ECD

electronic circular dichroism

EtOAc

ethyl acetate

HMBC

heteronuclear multibond correlation

HPLC

high-performance liquid chromatography

HRESIMS

high-resolution electrospray ionization mass spectroscopy

HSQC

heteronuclear single quantum coherence

MS

mass spectroscopy

MTT

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

NMR

nuclear magnetic resonance

NOESY

nuclear Overhauser effect spectroscopy

NP

normal phase

PDA

photodiode array

RP

reverse phase

TLC

thin-layer chromatography

UV

ultraviolet

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.1c01714.

• Spectroscopic data including 1D and 2D NMR of new compounds 1–13 (PDF)

This project was supported by the USDA-NIFA (grant # 2017-68005-26867; Dr. Kimberly Ogden, PI) and Hatch projects 1005072 and 1020652 to A.A.L.G and I.M. Any opinions, findings, conclusions, or recommendations expressed in this publication/work are those of the authors and do not necessarily reflect the view of the U.S. Department of Agriculture. We thank Dr. Lijiang Xuan, Shanghai Institute of Material Medica, Chinese Academy of Sciences, P. R. China for HRMS data.

The authors declare the following competing financial interest(s): A.A.L.G. and I.M. have disclosed financial interests in Sun Pharma Advanced Research Co. Ltd., India (A.A.L.G.), Regulonix, LLC. USA (A.A.L.G.), Teva Pharmaceuticals Works Ltd., Hungary (I.M.) and the University of Debrecen, Hungary (I.M.) that are unrelated to the subject of the research presented here. All other authors declare no competing financial interests.