Crocrassins A and B: two novel sesquiterpenoids with an unprecedented carbon skeleton from Croton crassifolius

Abstract

Crocrassins A (1) and B (2), two novel sesquiterpenoids with a unique skeleton, were isolated from a Chinese medicinal plant Croton crassifolius. Their structures were assigned on the basis of detailed spectroscopic analyses (especially HRESIMS and 2D NMR), and their absolute configuration was established via single-crystal X-ray diffraction studies as well as chemical transformation.

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The genus Croton (Euphorbiaceae), composed of more than 1300 species globally, mainly grows in tropical and subtropical regions¹ with about 21 species distributed in the southern part of China.² It grows in sandy and wet soil and on damp areas near river banks. Several species have long been used in Chinese folk medicine to alleviate dysmenorrhea as a purgative, and to treat dyspepsia and malaria.³ Phytochemical investigations on genus Croton have been carried out and several types of compounds were obtained, including diterpenes, sesquiterpenes, triterpenes, alkaloids, flavanones, lignans, polyphenolic compounds, phenylbutanoids, and peptides.^1,3 Collectively, some compounds possess antimicrobial, anti-inflammatory, antibacterial, antimalarial, and cytotoxic activities.¹

Croton crassifolius Geisel, an undershrub of up to 30–50 cm in height, is mainly found in the southern parts of China, and is also distributed in Vietnam, Laos, and Thailand.⁴ The roots of C. crassifolius, known as “Jiguxiang” in traditional Chinese medicine, have been used for the treatment of snake bites, stomach ache, sternalgia, joint pain, as well as pharyngitis, jaundice, and rheumatoid arthritis for a long time.⁵ In previous studies, numerous clerodane-type diterpenes and sesquiterpenes were reported from this species.⁶ As a part of our continuing work on searching structurally interesting bioactive compounds from traditional herbal medicines, further phytochemical investigations on this plant resulted in the isolation of two novel sesquiterpenoids with an unprecedented carbon skeleton via the connection of a single carbon–carbon bond to form a cyclopropylcyclopentane (Fig. 1). This paper describes the isolation and structural elucidation of compounds 1 and 2 by extensive spectroscopic and single-crystal X-ray crystallographic analyses, as well as chemical transformation.

Fig. 1 Structures of crocrassin A (1) and crocrassin B (2).

The air-dried and powdered roots of C. crassifolius (9.5 kg) were exhaustively extracted with 95% EtOH at room temperature, and the residue (962 g) obtained by concentrating the EtOH extract in vacuo was suspended in water, which was then successively partitioned with EtOAc and n-BuOH. The EtOAc-soluble portion (731 g) was applied to silica gel CC eluted with the increasing polarities of petroleum ether–acetone mixtures (40 : 1 to 0 : 1) to afford combined fractions A–F. The fraction C was further separated by CC over silica gel and Sephadex LH-20 to yield 1 (4 mg) and 2 (2 mg).

Compound (1)‡§ was obtained as a colorless crystal, [α]²⁵_D + 60 (c 0.10, CHCl₃). It exhibited a quasimolecular ion peak at m/z 275.1616 corresponding to [M + Na]⁺ in the HRESIMS analysis, which provided the molecular formula C₁₅H₂₄O₃ (calcd for [M + Na]⁺ m/z 275.1618), indicating four degrees of unsaturation. The IR spectrum of 1 displayed characteristic absorption bands of a carboxylic acid group (1697 cm⁻¹),⁷ double bond (3072, 1642 cm⁻¹),⁸ and hydroxyl (3391 cm⁻¹) functionalities.

The analysis of the ¹H NMR data of 1 indicated the presence of two terminal olefinic protons at δ_H 4.64 (dd, J = 2.4, 1.8) and 4.79 (d, J = 2.4), an olefinic methyl at δ_H 1.67, three tertiary methyls at δ_H 1.20, 1.26, and 1.31, as well as signals with several complex multiplets from δ_H 1.20–2.5. The ¹³C NMR and DEPT spectra revealed 15 carbon resonances, which were determined to be four methyls, three methylenes (including one olefinic carbon), and four methines along with four quaternary carbons (including one carbonyl, one oxygenated sp³ carbon, and one olefinic carbon). From the number of carbon resonances in the ¹³C NMR, in combination with the molecular formula, it could be inferred that compound 1 could be a sesquiterpenoid. Moreover, deducting 2 degrees of unsaturation accounted for one carbonyl and one olefinic bond, and the remaining two degrees of unsaturation were indicative of the bicyclic ring system of 1.

The planar bicyclic carbon skeleton of 1 was established by the 2D NMR experiments, including ¹H–¹H COSY, HMQC and HMBC spectra. The proton and protonated carbon signals in the NMR spectra of 1 were unequivocally assigned by the HMQC experiment. In the ¹H–¹H COSY spectrum, homonuclear coupling correlations from H₂-3 through H₂-2, H-1, H-5 to H-6, and then to H-7, suggested the presence of one key fragment (C-3/C-2/C-1/C-5/C-6/C-7). This structural fragment along with other isolated groups was connected by quaternary carbons according to the further examination of HMBC data (Fig. 2), especially correlations arising from tertiary methyl protons. The protons of H₂-3 (δ_H 1.68, 1.79) and H-5 (δ_H 2.34) showed significant HMBC correlations to C-4 (δ_C 81.6), which suggested that C-1, C-2, C-3, C-4, and C-5 constructed a five-membered carbon ring A. In the HMBC spectrum, two singlet methyls CH₃-13 (δ_H 1.20) and CH₃-14 (δ_H 1.31) together with the signals of two methines of C-6 (δ_H 0.97) and C-7 (δ_H 1.48) showed significant correlations to an sp³ quaternary carbon at δ_C 26.5 (C-8). These HMBC correlations established a cyclopropane ring B, as shown in Fig. 2. The connected position of the abovementioned two rings was established according to the following key correlations: in the ¹H–¹H COSY spectrum, H-5 correlated to H-6; and in the HMBC spectrum, H-1 correlated to C-6 and H-7 correlated to C-5. Thus, ring A and B were connected by a C–C σ-bond at C-5 and C-6. The C-4 position of 1 was substituted by a hydroxyl group based on the HMBC correlations of CH₃-12 (δ_H 1.26), H₂-3, and H-5 with the oxygenated quaternary carbon at δ_C 81.6. An isopropenyl group (δ_H 1.67, 4.64, and 4.79; δ_C 18.4, 111.8, and 147.0) was attached to the C-1 position (δ_C 55.4) because of the HMBC correlations of H-10a, H-10b, and CH₃-11 with C-1. The carboxylic acid group was assigned to C-7 by the HMBC correlation of H-7 with C-15 (δ_C 178.2). Therefore, the planar structure of 1 was constructed, as shown in Fig. 1.

Fig. 2 Key ¹H–¹H COSY and HMBC correlations for 1.

The relative stereochemistry of 1 was established by the interpretation of its NOESY spectrum (Fig. 3). In the NOESY spectrum, the cross peaks of H-6/H-7 and H-7/H₃-13 demonstrated that H-6, H-7, and H₃-13 were oriented on the same side of the cyclopropane ring B, while H₃-14 and COOH were oriented on the opposite side. Consequently, the cross peaks of H-5/H₃-11 and H-1/H₃-12 confirmed that H-1 and H₃-12 were on the same face of cyclopentane ring A, while H-5 was on the opposite face. The relative configuration between the conjoined bicyclic ring systems in 1 was defined by the observation of NOESY correlations between H-6 and H₃-12 and between H-5 and H₃-14, which could only be accommodated by the C-5R*/C-6S* relative configuration, as shown in Fig. 3.⁹

Fig. 3 Key NOESY correlations for 1.

After repeated attempts, a suitable single crystal of 1 was obtained from petroleum ether–acetone solution. The X-ray crystallographic data (CuKα) (Fig. 4) corroborated the planar structure and relative configuration of 1, which was elucidated via NMR data, and further allowed the assignment of its absolute configuration as 1R, 4S, 5R, 6S, 7R [the refined Flack parameter is 0.07(14)].¹⁰ Thus, the structure of 1 was established and it was named as crocrassin A.

Fig. 4 X-ray structure of compound 1.

Compound (2)¶ was obtained as a colorless gum with [α]²⁵_D + 80 (c 0.10, CHCl₃). Its molecular formula C₁₆H₂₆O₃ was determined on the basis of the HRESIMS at m/z 289.1775 [M + Na]⁺ (calcd 289.1774), indicating four degrees of unsaturation. It was 14 mass units heavier than that of 1. The IR spectrum of 2 indicated the presence of a hydroxyl group (3439 cm⁻¹) and a carbonyl group (1728 cm⁻¹). The similar ¹H and ¹³C NMR spectra revealed that 2 is an analogue of 1. A careful comparison of the NMR data for compounds 1 and 2 (Table 1) suggested that they had the same carbon skeleton including the cyclopropane and cyclopentane moieties, which were connected by a C–C σ-bond. The major difference between the ¹H NMR spectra of 2 and 1 was the presence of an additional oxymethyl group in 2 with the spectral data at δ_H 3.55 (s, 3H), which was further corroborated by the ¹³C NMR spectrum as an additional signal compared to 1 detected at δ_C 51.2. In addition, the ¹³C NMR spectrum of 2 indicated that the carbonyl resonance was moved upfield from δ_C 178.2 in 1 to δ_C 172.1 in 2, which demonstrated that the carboxylic acid group in 1 was esterified in 2. Further examination of the ¹H–¹H COSY, HSQC, HMBC, and NOESY spectra confirmed the structural and relative configuration of 2, as shown in Fig. 1.

Table 1 ¹H (600 MHz) and ¹³C (150 MHz) NMR data for 1 and 2

no.	1^a		2^a
	δH, mult, (J in Hz)	δC	δH, mult, (J in Hz)	δC
a Measured in CDCl3.b Signal pattern unclear due to overlapping.
1	2.45 m	55.4 d	2.41 m	55.6 d
2a	1.81 m^b	28.1 t	1.79 m^b	28.3 t
2b	1.69 m^b		1.68 m^b
3a	1.79 m^b	41.8 t	1.78 m^b	41.8 t
3b	1.68 m^b		1.65 m^b
4		81.6 s		81.6 s
5	2.34 dd (10.8, 8.4)	48.0 d	2.27 dd (11.4, 8.4)	48.1 d
6	0.97 dd (10.8, 9.0)	39.4 d	0.90 dd (11.4, 9.6)	38.5 d
7	1.48 d (9.0)	28.3 d	1.48 d (9.6)	28.2 d
8		26.5 s		25.0 s
9		147.0 s		147.2 s
10a	4.79 d (2.4)	111.8 t	4.70 d (1.8)	111.0 t
10b	4.64 dd (2.4, 1.8)		4.58 d (1.8)
11	1.67 brs	18.4 q	1.67 brs	18.5 q
12	1.26 s	25.1 q	1.27 s	25.1 q
13	1.20 s	29.1 q	1.16 s	29.1 q
14	1.31 s	16.3 q	1.33 s	16.2 q
15		178.2 s		172.1 s
OMe			3.55 s	51.2 q

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Unfortunately, we failed to obtain a single crystal of 2 for determining its absolute configuration. To determine the absolute configuration of 2, compound 1 was methyl esterified using excess ethereal diazomethane in diethyl ether to give the compound 2. Thus, the absolute configuration of 2 was identical to that of 1 and was deduced as 1R, 4S, 5R, 6S, 7R. Finally, the structure of 2 was established, and it was named as crocrassin B.

To the best of our knowledge, crocrassins A (1) and B (2) are the first examples of a novel carbon skeleton via the connection of a single carbon–carbon bond to form a cyclopropylcyclopentane in the family of sesquiterpenoids.

Both 1 and 2 were evaluated for their cytotoxicity against four human cancer cell lines, HepG2, SGC-7901, K562, and K562/ADM, using the MTT method. Unfortunately, no significant activity was detected.

Acknowledgements

We are grateful to the National Natural Science Foundation of China (no. 21102065), the Natural Science Foundation of Gansu Province, China (no. 1208RJYA029) and the Foundational Research Funds for the Central Universities (no. lzujbky-2013-72 and no. lzujbky-2012-83) for the financial support of this research.

Notes and references

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Footnotes

† Electronic supplementary information (ESI) available: Experimental procedures, NMR, HRESIMS, and IR spectra of compounds 1 and 2. Crystallographic data for the structure of 1, CCDC 995584. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c4ra03798g

‡ Crocrassin A (1): colorless crystals; mp 113–115 °C; [α]²⁵_D + 60 (c 0.10, CHCl₃); IR (KBr) ν_max 3391, 3072, 2953, 2926, 1696, 1453, 1438, 1376, 923, 739 cm⁻¹; ¹H (600 MHz) and ¹³C NMR (150 MHz) data, see Table 1; HRESIMS m/z 275.1616 [M + Na]⁺ (calcd for C₁₅H₂₄O₃Na, 275.1618).

§ Crystal data for compound 1: C₁₅H₂₄O₃, M_r = 252.34, orthorhombic, a = 111.2310(5) Å, b = 17.3234(7) Å, c = 31.7240(19) Å, α = 90.00°, β = 90.00°, γ = 90.00°, V = 6172.2(5) ų, T = 293(2) K, space group P2₁2₁2₁, Z = 16, μ (CuKα) = 0.590 mm⁻¹, 39 759 reflections measured, 11 886 independent reflections (R_int = 0.0573). The final R₁ value was 0.0702 (I > 2σ(I)). The final wR(F²) value was 0.1736 (I > 2σ(I)). The final R₁ value was 0.0950 (all data). The final wR(F²) value was 0.2074 (all data). The goodness of fit on F² was 1.020.

¶ Crocrassin B (2): colorless gum; [α]²⁵_D + 80 (c 0.10, CHCl₃); IR (KBr) ν_max 3439, 2951, 1728, 1641, 1438, 1376, 928, 885 cm⁻¹; ¹H (600 MHz) and ¹³C NMR (150 MHz) data, see Table 1; HRESIMS m/z 289.1775 [M + Na]⁺ (calcd for C₁₆H₂₆O₃Na, 289.1774).

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