An Iridoid Glucoside and the Related Aglycones from Cornus florida

Abstract

A new iridoid glucoside, cornusoside A (1), and four new natural product iridoid aglycones, cornolactones A–D (2–5), together with 10 known compounds were isolated from the leaves of Cornus florida. The structures of compounds 1–5 were established by interpretation of their spectroscopic data. Cornolactone B (3) is the first natural cis-fused tricyclic dilactone iridoid containing both a five- and a six-membered lactone ring. A biosynthesis pathway is proposed for cornolactones C (4) and D (5), the C-6 epimers of compounds 1–3.

The plant genus Cornus (dogwood) belongs to the family Cornaceae and consists of approximately 55 species distributed mainly in the northern hemisphere, eastern Asia, and eastern and northern parts of the United States.¹ This genus is a rich source of diverse iridoid glucosides, which have raised interest because of their wide range of promising bioactivities. These include antidiabetic,^2,3 antioxidant,⁴ anti-inflammatory,⁵ antiamnesic,⁶ and immunesuppressive effects.⁷

Cornus florida L., commonly known as flowering dogwood, is a tree native to eastern North America that has been traditionally used for the treatment of malaria.^8,9 Previous chemical investigations of C. florida have resulted in the isolation of a number of compounds including anthocyanins and other flavonoids, triterpenoids, and sterols.^9,10 The anthocyanins impart bright colors to several fruits and vegetables and possess anti-inflammatory,¹⁰⁻¹² antioxidative,^11,12 antineoplastic,^10,13 and antidiabetic activities.¹⁴

In the present study the chemical constituents of C. florida collected from Oxford, Mississippi, were investigated. A large-scale extraction of the leaves of C. florida yielded a new iridoid glucoside, cornusoside A (1), and four new natural product iridoid aglycones, cornolactones A–D (2–5). Cornolactone A (2) was previously reported as a synthetic intermediate in the enantioselective synthesis of semperoside A;¹⁵ however, this is the first report of this compound from a natural source. In addition, 10 known compounds were also isolated, which included five iridoids, two megastigmane compounds, and two ellagic acid derivatives, together with a flavonoid. The structures of the new compounds were assigned by detailed spectroscopic analysis and those of the known compounds by comparison with literature data. Cornusoside A (1) is one of a small number of C₁₀ iridoid glucosides with a ring-opening between C-1 and O-2 and a γ-lactone linkage between C-6 and C-11. Cornolactone B (3) is the first natural cis-fused tricyclic dilactone iridoid containing both a five- and a six-membered lactone ring. Herein are reported the isolation and structure elucidation of 1–5 and a possible biosynthetic pathway to cornolactones C (4) and D (5) as C-6 epimers of compounds 1–3.

A 90% aqueous ethanol extract of the dried leaves of C. florida (15 kg) was fractionated initially on silica gel (step gradient elution of hexane to EtOAc to MeOH). The 20% MeOH in EtOAc was then subjected to column chromatography on polymeric HP-20 (step gradient elution 10% Me₂CO in H₂O to 100% Me₂CO). The 20% Me₂CO fraction was then subjected to repeated fractionation on either polymeric HP-20ss, reversed-phase C₁₈, normal-phase silica gel, or molecular exclusion Sephadex LH-20 column chromatography followed by a series of HPLC separations on either a PRP-1 column or a C₈ or C₁₈ column to yield cornusoside A (1), cornolactones A–D (2–5), and the known compounds alternosides A,¹⁶ hastatoside,¹⁷ cornin,¹⁷ dihydrocornin,¹⁸ cornalternoside,¹⁶ lauroside A,¹⁹ (5S*,6R*)-9-hydroxymegastigm-7-en-3-one,²⁰ 3,3′-dimethyl-4-O-β-d-glucopyranosylellagic acid,²¹ 3,4,3′-trimethyl-4′-O-β-d-glucopyranosylellagic acid,²² and isoquercitrin.²³

Cornusoside A (1) was isolated as a colorless gum. Its molecular formula, C₁₇H₂₆O₁₀, determined from the HRESIMS of the [M + Na]⁺ at m/z 413.1417, required five degrees of unsaturation. A preliminary analysis of the ¹H and ¹³C NMR data (Table 1) revealed two ester carbonyl groups (δ_C 172.8 and 169.0; IR 1730 cm^–1), an oxygenated methine [δ_H 5.03 (1H, t, J = 6.8 Hz, H-6); δ_C 83.6 (C-6)], an oxymethylene [δ_H 3.98 (1H, dd, J = 10.4, 3.6 Hz, H-1a), δ_H 3.38 (1H, m, H-1b); δ_C 66.7 (C-1)], a methoxy [δ_H 3.68 (3H, s); δ_C 52.7], and signals that could be attributed to a glucopyranosyl group. The coupling constant of the anomeric proton (δ_H 4.05, 1H, d, J = 7.8 Hz) suggested a β-configuration of the glucose. The presence of the β-d-glucopyranosyl group was confirmed by acid hydrolysis. These data led to the preliminary conclusion that 1 is an iridoid glucoside and accounted for three of the five double-bond equivalents, indicating the compound to be bicyclic.

Table 1. NMR Spectroscopic Data for Cornusoside A (1) and Cornolactone A (2)^a.

	1b		2c
position	δC, mult.	δH (J in Hz)	δC, mult.	δH (J in Hz)
1a	66.7, CH2	3.98, dd (10.4, 3.6)	61.4, CH2	3.87, dd (11.0, 4.0)
1b		3.38, m		3.58, dd (11.0, 9.0)
3	169.0, qC
4α	47.8, CH	3.94, d (5.6)	29.7, CH2	2.66, dd (18.8, 4.7)
4β				2.59, dd (18.8, 9.8)
5	45.5, CH	3.37, q (7.2)	40.2, CH	3.14, m
6	83.6, CH	5.03, t (6.8)	84.9, CH	5.00, t (6.0)
7α	40.6, CH2	1.99, dd (13.6, 6.0)	41.9, CH2	2.20, dd (14.0, 5.0)
7β		1.47, ddd (13.6, 11.6, 6.0)		1.44, ddd (14.0, 11.8, 5.5)
8	32.0, CH	1.90, m	32.8, CH	1.82, m
9	48.0, CH	1.83, m	50.6, CH	1.79, m
10	17.3, CH3	0.95, d (5.6)	17.6, CH3	1.02, d (5.6)
11	172.8, qC		178.3, qC
OMe	52.7, CH3	3.68, s
1′	103.0, CH	4.05, d (7.8)
2′	73.3, CH	2.92, td (8.0, 4.4)
3′	76.7, CH	3.10, m
4′	70.1, CH	3.03, m
5′	76.9, CH	3.07, d (3.9)
6a′	61.1, CH	3.64, m
6b′		3.43, m
OH-2′		4.72, d (4.3)
OH-3′		4.94, d (5.0)
OH-4′		4.93, d (5.5)
OH-6′		4.45, t (5.9)

^a1H NMR measured at 400 MHz, ¹³C NMR measured at 100 MHz.

^bMeasured in DMSO-d₆.

^cMeasured in CDCl₃.

The terpenoid portion of 1 was determined by detailed analysis of its NMR data (Figure 1). The observation of ¹H–¹H COSY correlations from H-6 to H-5 and H-7, H-7 to H-8, H-8 to H-9 and the methyl doublet H₃-10, and H-9 to H-5 established the presence of a cyclopentane ring with methyl substitution at C-8. An additional COSY correlation observed between H-9 and H-1a and H-1b of the oxygenated methylene group at C-1 together with an HMBC correlation from H-1′ to C-1 and from H-1 to C-1′ confirmed the connection of the β-d-glucopyranosyl group to C-1 and the connection of C-9 to C-1. Remaining to be assigned were two ester carbonyl carbons, C-3 (δ_C 169.0) and C-11 (δ_C 172.8), a methine, H-4 (δ_H 3.94; δ_C 47.8), and a methoxy group (δ_H 3.68; δ_C 52.7). HMBC correlations observed between H-6 (δ_H 5.03) and the ester carbonyl carbon at δ_C 172.8 (C-11) and from H-5 (δ_H 3.37) to both C-4 and C-11 established the presence of a γ-lactone ring. A COSY correlation observed between H-5 and H-4 and an HMBC correlation from H-4 to C-11 further supported this assignment. Finally, HMBC correlations observed from H-4, H-5, and the methoxy signal at δ_H 3.68 (OMe-3) to the remaining ester carbonyl carbon at δ_C 169.0 (C-3) established the connection of C-4 to C-3 and the presence of a methyl ester at C-3.

Figure 1.

Selected 2D NMR correlations for cornusoside A (1), and cornolactone A (2).

The relative configuration of 1 was determined by NOE correlations observed in a NOESY experiment and by scalar coupling (Figure 2). NOE correlations from H-5 to H-6 and H-9 together with correlations from H-7β to H-6 and H₃-10 indicated that H-5, H-6, H-9, and Me-10 are on the same side of the cyclopentane ring in a β-orientation. NOE correlations from H-8 to H-7α and H-1 confirmed the α-orientation of H-8 and the glucosylated side chain. A long-range W-coupling in the COSY spectrum between Me-10 and H-7α was consistent with the 1,2-diaxial arrangement of these two groups. Finally, the α-orientation of H-4 was indicated by a NOE correlation observed between H-4 and H-8. This was substantiated by a small coupling (5.6 Hz) observed between H-4 and H-5 that was similar to the coupling constants (ca. 5.0 Hz in both cases) of the previously reported C-1 to O-2 ring-opened iridoids gelsemiol²⁴ and borreriagenin.²⁵ The absolute configuration of 1 was determined based on biogenetic grounds in that nearly all iridoids found in Nature have a configuration of 5S and 9R and by analogy to the known co-isolated compounds that were found to have closely comparable NMR data and similar optical rotation values. Thus, the configuration of cornusoside A (1) was defined as 4S,5S,6S,8S,9R. Additional support for the absolute stereochemistry came from the isolation of cornolactone A (2), previously reported as a synthetic intermediate in the total synthesis of the iridoid semperoside A.¹⁵ Since this is the first report of 2 from a natural source, the isolation, structure elucidation, and full spectroscopic data are reported.

Figure 2.

Selected NOE correlations observed for cornusoside A (1) and cornolactone A (2).

Cornolactone A (2), isolated as a colorless gum, showed a [M + H]⁺ ion at m/z 171.1010 in the HRESIMS corresponding to the molecular formula C₉H₁₄O₃, requiring four degrees of unsaturation. An initial inspection of the NMR data revealed 2 to be similar to 1, except for the absence of the signals for the methine at C-4, the methyl ester at C-3, and the glucopyranosyl group and the appearance of signals for a diastereotopic methylene at δ_H 2.66 (1H, dd, J = 18.8, 4.7 Hz, H-4α) and δ_H 2.59 (1H, dd, J = 18.8, 9.8 Hz, H-4β). This suggested that 2 is an iridoid aglycone missing a C-3 methyl ester moiety. The observation of ¹H–¹H COSY correlations from both H₂-4 to H-5 and HMBC correlations from H₂-4 to C-5, C-6, and C-11 and from H-6 to C-11 further supported this assignment (Figure 1). The similarity of proton–proton coupling constants and ¹H and ¹³C NMR chemical shifts together with the NOESY spectrum of 2 showed the same relative configuration as 1 at the four chiral centers (Figure 2). Additional NOE correlations from H-4α to H-1b of the oxygenated methylene and from H-4β to H-5 confirmed this assignment. Compound 2 was found to have near-identical NMR data and a comparable optical rotation value ([α]¹⁸_D +3.2) (lit. [α]_D +14.7) to that of the reported synthetic compound.¹⁵

Cornolactone B (3) was isolated as a colorless gum. The molecular formula of cornolactone B (3), C₁₀H₁₂O₄, as determined from the HRESIMS of the [M + H]⁺ ion at m/z 197.0811, required five degrees of unsaturation. Analysis of the NMR data (Figure 3) revealed 3 to be similar to 2 except for the absence of the signals for the methylene group at C-4 and the addition of signals for an ester carbonyl carbon at δ_C 164.7 (C-3) and a methine δ_H 3.78 (1H, d, J = 9.6 Hz, H-4). Having accounted for all protons, carbons, and oxygens in the molecule and four of the five double-bond equivalents, as required by the molecular formula, this indicated that cornolactone B (3) is tricyclic. The only possible connection was between the C-1 oxygen and the ester carbonyl carbon C-3 to form a δ-lactone unit. The connection was confirmed with HMBC correlations observed between both H₂-1 (δ_H 4.47, 4.05) and H-4 (δ_H 3.80) to the ester carbonyl carbon at δ_C 170.2 (C-3). The relative configuration of 3 at C-5, C-6, C-8, and C-9 was determined to be identical to that of 1 and 2 by NOE correlations observed in a NOESY experiment and scalar coupling (Figure 4). The absence of any NOE correlations observed to or from H-4 made it difficult to assign the configuration at C-4. However, the presence of a large ¹H NMR coupling constant (J = 9.6 Hz) between H-4 and H-5 suggested the cis-relationship of these two protons and the β-orientation of H-4. This coupling constant is consistent with that observed for the cis-fused tricyclic iridoids semperoside (J = 10.5 Hz), 9-hydroxysemperoside (J = 11.4 Hz), and dihydrobrasoside (J = 10.5 Hz),²⁴ together with the dilactone compounds, asperuloside tetraacetate lactone (J = 9.8 Hz) and dihydroasperuloside tetraacetate lactone (J = 10.0 Hz), produced from the oxidation of the iridoid glucoside asperuloside.²⁶ Thus, the configuration of cornolactone B (3) was defined as 4S,5S,6S,8S,9R.

Figure 3.

Selected 2D NMR correlations for cornolactones B–D (3–5).

Figure 4.

Selected NOE correlations observed for cornolactones B–D (3–5).

Cornolactone C (4) was isolated as a colorless gum. The molecular formula of this compound, C₁₁H₁₆O₅, as determined from the HRESIMS of the [M + Na]⁺ ion at m/z 251.0884, required four degrees of unsaturation. The NMR data of 4 were similar to those of cornolactone B (3), except that the addition of a methoxy group (δ_H 3.80; δ_C 53.4) was shown and H-6 [δ_H 5.04, t (5.5)] was shifted upfield by 1.22 ppm (Table 2) as compared to that of 3. This suggested 4 is the product from methanolysis of the γ-lactone ring in 3. An HMBC correlation from the methoxy signal at δ_H 3.80 (OMe-11) to δ_C 169.2 (C-11) confirmed the presence of a methyl ester at C-11. Additional ¹H–¹H COSY and HMBC correlations (Figure 3) further supported this assignment. The relative configuration of 4 was determined by NOE correlations observed in a NOESY experiment and scalar coupling (Figure 4). NOE correlations from H-5 to H-4, H-7β, and H-9, together with correlations from H₃-10 to H-7β and H-9, established the cis-fusion of the cyclopenta[c]pyran skeleton with H-4, H-5, H-7β, and H-9 in the β-orientation. Finally, the α-orientation of H-6 was indicated by an NOE correlation observed from H-6 to H-7α and H-8 on the underside of the cyclopentane ring. Thus, the configuration of cornolactone C (4) was defined as 4R,5S,6R,8S,9R.

Table 2. NMR Spectroscopic Data for Cornolactones B–D (3–5) in CDCl₃^a.

	3		4		5
position	δC, mult.	δH (J in Hz)	δC, mult.	δH (J in Hz)	δC, mult.	δH (J in Hz)
1a	68.8, CH2	4.47, dd (12.2, 4.8)	69.3, CH2	4.27, dd (12.0, 5.6)	68.6, CH2	4.18, dd (11.6, 4.0)
1b		4.05, dd (12.2, 8.0)		4.00, dd (11.6, 6.0)		4.08, dd (12.0, 3.2)
3	164.7, qC		169.0, qC		174.0, qC
4	45.7, CH	3.80, d (9.6)	50.4, CH	3.58, d (6.6)	32.4, CH2	2.57, d (5.2)
5	42.3, CH	3.35, dt (9.5, 6.4)	46.1, CH	2.76, dt (11.6, 7.2)	42.6, CH	2.39, m
6	85.1, CH	5.04, t (5.5)	77.4, CH	3.82, ddd (10.2, 7.4, 6.0)	77.8, CH	3.71, ddd (10.2, 7.8, 5.5)
7α	41.2, CH2	2.36, dd (14.8, 6.8)	43.2, CH2	2.12, td (11.7, 6.0)	42.6, CH2	2.01, m
7β		1.68, ddd (14.8, 9.4, 5.6)		1.38, dt (12.1, 10.2)		1.27, dt (12.1, 11.2)
8	34.2, CH	2.04, m	33.9, CH	1.78, m	33.1, CH	1.79, m
9	43.1, CH	2.10, m	42.8, CH	2.20, ddd (11.7, 9.4, 6.3)	43.2, CH	1.97, m
10	19.4, CH3	1.12, d (6.7)	18.9, CH3	1.07, d (6.3)	18.7, CH3	1.03, d (6.4)
11	170.2, qC		169.2, qC
OMe			53.4, CH3	3.80, s

^a1H NMR measured at 400 MHz, ¹³C NMR measured at 100 MHz.

Cornolactone D (5) was isolated as a colorless gum. The molecular formula, C₉H₁₄O₃, was determined from the HRESIMS of the [M + H]⁺ ion at m/z 193.0834 (calcd 193.0835) and required three degrees of unsaturation. An initial inspection of the NMR data revealed that 5 is similar to 4, except for the absence of the signals for the methine at C-4 and the methyl ester at C-3 and the appearance of a methylene group at δ_H 2.57 (2H, d, J = 5.2 Hz, H-4). This suggested that 5 did not have a C-11 methyl ester unit. The observation of ¹H–¹H COSY correlations from H₂-4 to H-5 and HMBC correlations from H₂-4 to C-3, C-5, and C-9, together with a correlation from H-6 to C-4, confirmed this assignment (Figure 3). The similarity of proton–proton coupling constants and ¹H and ¹³C NMR chemical shifts together with a NOESY spectrum of 5 showed the same relative configuration as 4 at the four chiral centers (Figure 4). Thus, the configuration of cornolactone D (5) was defined as 5R,6R,8S,9R.

Compounds 1–5 and cornin were evaluated for cell growth inhibitory activities against human embryonic stem cells (BG02) and human breast cancer cell lines (MCF-7 and MDA-MB-231). No cytotoxicity was observed for any of the compounds at 100 μM except for slight cytotoxicty being observed for compound 5 against the MDA-MB-231 cell line. The compounds were also examined for agonistic activity against peroxisome proliferator-activated receptor γ (PPARγ), but no activity was observed.

Cornusoside A (1) is one of a small number of C₁₀ iridoid glucosides where the δ-lactone is ring-opened between the C-1 and O-2 positions and contains a γ-lactone linkage between C-6 and C-11. Other examples reported include gelsemiol 3-O-β-d-glucoside,²⁴ gelsemiol 6′-trans-caffeoyl-1-glucoside,²⁷ and verbenabrasides A and B.²⁸ Cornolactone B (3) is the first natural cis-fused tricyclic dilactone iridoid containing both a five- and a six-membered lactone ring. Interestingly, cornolactones C (4) and D (5) have an opposite configuration at C-6 compared to that of compounds 1–3. This suggests that rather than cleavage of the γ-lactone in 3 by methanolysis at C-11, to give the C-6 epimer of 4 (6-epi-4) with retention of configuration, an alternative biosynthetic pathway is necessary (Figure 5). A plausible pathway leading to inversion of configuration at C-6 could occur through the S_N2 hydrolysis of 3 at C-6, followed by esterification to give cornolactone C (4) or decarboxylation to give cornolactone D (5). Alternatively, cornolactones C (4) and D (5) could also have been formed from the reduction followed by oxidation of the co-isolated iridoid cornin (6). Previously it has been shown that reduction of 6 with NaBH₄ gives approximately a 1:1 mixture of both epimers at C-6.¹⁸ It is also conceivable that 6-epi-4 could undergo a lactonization reaction to give cornolactone B (3) and could provide an explanation of why no 6-epi-4 was isolated.

Figure 5.

Plausible biosynthetic route to 3 and potential pathways to inversion of configuration at C-6 in 4 and 5.

Experimental Section

General Experimental Procedures

Optical rotations were measured on a JASCO P-2000 polarimeter (c g/100 mL) equipped with a halogen lamp (589 nm) and a 1 mL microcell. IR spectra were recorded on a Thermo Electronic Corporation Nicolet IR-100 spectrophotometer. All NMR spectra were acquired with a Varian MercuryPlus 400 spectrometer using solvent signals (DMSO-d₆: ¹H, δ_H 2.50; ¹³C, δ_C 39.52; CDCl₃: ¹H, δ_H 7.24 ppm; ¹³C, δ_C 77.23 ppm) as references. Short- and long-range ¹H–¹³C correlations were determined with gradient-enhanced inverse-detected HSQC and HMBC experiments, respectively. NOE correlations were detected with NOESY experiments with a 0.5 s mixing time. The HRESIMS were obtained using an Agilent 6220 series TOF mass spectrometer. HPLC was performed on a Shimadzu LC-20AT instrument with a Shimadzu SPD-M20A UV/vis photodiode detector and a Shimadzu ELSD-LTII detector.

Plant Material

The leaves of Cornus florida L. were collected from a one-mile radius around Timber Lake, Oxford, Mississippi, during the spring and summer of 2011 by M.T.H. Voucher specimens are kept in the Hamann Laboratory at the University of Mississippi, School of Pharmacy, Oxford, MS (Cf2011).

Extraction and Purification Procedures

The leaves of C. florida (15.0 kg, dry weight) were extracted with 90% aqueous ethanol and dried under reduced pressure to give a crude extract (900 g). A portion of this crude extract (400 g) was separated on a silica gel column (20 × 70 cm) using a stepwise gradient of hexanes/EtOAc (100:0, 80:20, 50:50, and 0:100, v/v, each 3 L) and EtOAc/MeOH mixtures (80:20, 60:40, 50:50, and 0:100, v/v, each 3 L) to afford eight fractions. Fraction E (220 g) was then chromatographed on HP-20 (8 × 50 cm) using a stepwise gradient of acetone/water (10:90, 20:80, 50:50, 60:40, 80:20, and 100:0, v/v, each 2 L) to give six subfractions (Fr. E₁–E₆). Fraction E₂ (40 g) was chromatographed on a preparative C₁₈ reversed-phase MPLC column (20 × 250 mm; 20–35% CH₃OH/H₂O over 40 min, 35–65% CH₃OH/H₂O over 20 min; flow rate: 12 mL/min) to afford eight subfractions (Fr. E_2a–E_2h). Fraction E_2d (4.0 g) was chromatographed on a preparative C₁₈ reversed-phase HPLC column (Shim-pak RP-C₁₈ column; 5 μm; 20 × 250 mm; 8–45% CH₃CN/H₂O over 120 min, 7 mL/min) to yield eight subfractions (Fr. E_2d-1–E_2d-8). Fraction E_2d-4 (50 mg) was purified by C₈ reversed-phase HPLC (Polar-C₈; 5 μm; 10 × 250 mm; 15–45% CH₃OH/H₂O over 90 min, 5 mL/min) to yield cornusoside A (1, 5.0 mg, t_R 76.2 min). Fraction E_2d-6 (832 mg) was subjected to silica gel column chromatography using CH₂Cl₂/MeOH (90:10 to 0:100, v/v) to afford nine fractions (Fr. E_2d-6-a–Fr. E_2d-h). Fraction E_2d-6-a (320 mg) was further purified by C₈ reversed-phase HPLC (Polar-C₈; 5 μm; 10 × 250 mm; 5–25% CH₃CN/H₂O over 70 min, 5 mL/min) to yield two fractions. The first fraction (60 mg) was purified on a polymeric HPLC column (Hamilton PRP-1; 5 μm; 20 × 250 mm; 10–40% CH₃CN/H₂O over 50 min, 7 mL/min) to yield cornolactone C (4, 8.0 mg, t_R 41.5 min). The second fraction (40 mg) was chromatographed on a polymeric HPLC column (Hamilton PRP-1; 5 μm; 20 × 250 mm; 15–45% CH₃CN/H₂O over 70 min, 7 mL/min) to yield cornolactone B (3, 4.0 mg, t_R 37.9 min). Cornolactones A (2, 15.0 mg) and D (5, 12 mg) were purified from the remaining crude extract (500 g) using a similar procedure (see Supporting Information).

Cornusoside A (1):

colorless gum; [α]¹⁸_D −13.8 (c 0.01, MeOH); UV (MeOH) λ_max (log ε) 230 (3.4) nm; IR (KBr) ν_max 3355, 2922, 1730, 1073, 1020 cm^–1; ¹H and ¹³C NMR (400 MHz, d₆-DMSO), see Table 1; HRESIMS m/z 413.1417 [M + Na]⁺ (calcd for C₁₇H₂₆O₁₀Na, 413.1418).

Cornolactone A (2):

colorless gum; [α]¹⁸_D +3.3 (c 0.2, MeOH); UV (MeOH) λ_max (log ε) 237 (3.2) nm; IR (KBr) ν_max 3355, 2922, 1730, 1073, 1020 cm^–1; ¹H and ¹³C NMR (400 MHz, CDCl₃), see Table 1; HRESIMS m/z 171.1010 [M + H]⁺ (calcd for C₉H₁₅O₃, 171.1016).

Cornolactone B (3):

colorless gum; [α]¹⁸_D −7.5 (c 0.02, MeOH); UV (MeOH) λ_max nm 243 (3.1) nm; IR (KBr) ν_max 2944, 1658, 1023 cm^–1; ¹H and ¹³C NMR (400 MHz, CDCl₃), see Table 2; HRESIMS m/z 197.0811 [M + H]⁺ (calcd for C₁₀H₁₃O₄, 197.0808).

Cornolactone C (4):

colorless gum; [α]¹⁸_D +13.3 (c 0.03, MeOH); UV (MeOH) λ_max (log ε) 241 (3.1) nm; IR (KBr) ν_max 3337, 2952, 2937, 1733, 1650, 1017 cm^–1; ¹H and ¹³C NMR (400 MHz, CDCl₃), see Table 2; HRESIMS m/z 215.0884 [M + Na]⁺ (calcd for C₁₁H₁₆O₅Na, 215.0890).

Cornolactone D (5):

colorless gum; [α]¹⁸_D +18.5 (c 0.02, MeOH); UV (MeOH) λ_max (log ε) 244 (2.8) nm; IR (KBr) ν_max 3401, 2952, 1733, 1089, 1068 cm^–1; ¹H and ¹³C NMR (400 MHz, CDCl₃), see Table 2; HRESIMS m/z 193.0834 [M + Na]⁺ (calcd for C₉H₁₄O₃Na, 193.0835).

Acid Hydrolysis and Sugar Analysis

Cornusoside A (1) (2 mg) was hydrolyzed by using 1 M HCl (0.4 mL) at 100 °C for 2 h under argon and neutralized with Amberlite IR400. After drying under reduced pressure, the residue was dissolved in pyridine (0.4 mL) containing l-cysteine ethyl ester hydrochloride (2 mg) and heated at 60 °C for 1 h. A 0.4 mL solution of 3,5-dichlorophenyl isothiocyanate (2 mg) in pyridine was added to the mixture, which was heated at 60 °C for 1 h. The reaction mixture was directly analyzed by analytical HPLC on a Shim-pak RP-C₁₈ column, 5 μm, 4.6 × 250 mm, by eluting with a gradient of 30–80% CH₃CN in H₂O with 0.02% HCOOH for 40 min and subsequent washing of the column with 100% CH₃CN at a flow rate 0.8 mL/min. In the acid hydrolysate of 1, d-glucose was confirmed by comparison of the retention times of their derivatives with those of l-glucose and d-glucose derivatives prepared in the same way, which showed retention times of 34.8 and 34.0 min, respectively.

Supporting Information Available

Full isolation procedures and 1D and 2D NMR spectroscopic data of 1–5 are available including ¹H, ¹³C, COSY, HSQC, HMBC, and NOESY. This material is available free of charge via the Internet at http://pubs.acs.org.

The authors declare no competing financial interest.

Funding Statement

National Institutes of Health, United States