Cantharimide and Its Derivatives from the Blister Beetle Mylabris

Jul 26, 2016 - Eleven new monoterpenoids including three 1-methyl cantharimide-type derivatives (1–3), five 1,2-dimethyl cantharimide-type derivativ...
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Cantharimide and Its Derivatives from the Blister Beetle Mylabris phalerata Palla Yao-Bo Zeng,† Xiao-Ling Liu,† Yi Zhang,† Chuang-Jun Li,‡ Dong-Ming Zhang,*,‡ Yao-Zong Peng,§ Xing Zhou,† Hong-Fei Du,† Chun-Bing Tan,† Yu-Yu Zhang,† and Da-Jian Yang*,† †

Chongqing Academy of Chinese Material Medical, Chongqing, 400065, People’s Republic of China Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College (State Key Laboratory of Bioactive Substance and Function of Natural Medicines), Beijing, 100050, People’s Republic of China § School of Pharmaceutical Sciences, Southwest University, Chongqing, 400715, People’s Republic of China ‡

S Supporting Information *

ABSTRACT: Eleven new monoterpenoids including three 1methyl cantharimide-type derivatives (1−3), five 1,2-dimethyl cantharimide-type derivatives (4, 5, 7−9), and three 1hydroxymethyl-2-methyl cantharimide-type derivatives (10− 12), together with seven known cantharimides (6, 13−18), were isolated from Mylabis phalerata Palla. The planar structures and absolute configurations of compounds 1−14 were fully elucidated on the basis of spectroscopic analysis, ECD spectra, single-crystal X-ray diffraction analysis, and chemical methods. Compounds 6, 15, 16, and 18 were found to be potent inhibitors of HBV virus, with IC50 values of 62, 42, 58, and 19 μM.

T

he Chinese blister beetles Mylabis phalerata Palla and Mylabris cichorii Linnaeus of the family Meloidae were recorded in the Chinese Pharmacopoeia as traditional medicinal insects.1 They have been used for the treatment of rabies, dropsy, warts, impotence, and fever as traditional medicines for 2000 years in China.2 Cantharidin is a wellknown natural compound produced only by beetles of the families Meloidae and Oedemeridae.3 It has attracted the attention of synthetic chemists and pharmacologists due to its toxicity and antitumor activities especially in HepG2 (human hepatocellular carcinoma) cells,4 human pancreatic cancer cells,5 bladder cancer cells,6 lung cancer cells,7 and gastric cancer cells.8 The related compounds the cantharimides represent a rare class of natural compounds in which the anhydride oxygen atom in cantharidin is replaced by a nitrogen that is either unsubstituted or substituted, and their pharmacological activities have not been reported. In the course of a systematic search for unusual structural features and biologically active compounds from M. phalerata Palla, 11 new monoterpenoids, respectively named palasoninimides B−C (1−3) and cantharimides B, C, and E−J (4, 5, 7− 12), together with seven known analogue compounds (6, 13− 18) were obtained. Structurally, cantharimide derivatives can be classified into four types: 1-methyl-3,6-epoxycyclohexane-1,2dicarboximides, 1,2-dimethyl-3,6-epoxycyclohexane-1,2-dicarboximides, 1-hydroxymethyl-2-methyl-3,6-epoxycyclohexane1,2-dicarboximides, and bis[1,2-dimethyl-3,6-epoxycyclohexane-1,2-dicarboximidos]. The structures and configurations of all new compounds were determined on the basis of © XXXX American Chemical Society and American Society of Pharmacognosy

spectroscopic analysis, electronic circular dichroism (ECD) spectra, single-crystal X-ray diffraction analysis, and chemical methods. Furthermore, we also provide the X-ray diffraction analysis of the known compounds 13 and 14. All compounds were tested for their anti-HBV activities and cytotoxicity on HepG2.2.15 (human hepatocellular carcinoma), HCT-116 (human lung carcinoma), and BGC-823 (human gastric carcinoma) cell lines.

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RESULTS AND DISCUSSION Palasoninimide B (1) was obtained as a yellow oil. Its molecular formula of C14H20N2O4 was established on the basis of the HRESIMS ion at m/z 281.1493 [M + H]+. Comparison of the 1 H and 13C NMR data with those of palasoninimide A (13), previously isolated from the blister beetle Hycleus lunata,9 revealed that they share the same skeleton, with the sole difference being the presence of a side chain connected to N-8. The NMR spectra (Tables 1 and 2) showed signals for a methyl signal (C-6′: δC/H 23.2/2.00, s), three methylene signals (C-1′: δC/H 36.0/3.57, td, J = 6.4, 1.2 Hz; C-2′: δC/H 27.1/1.76, m; C3′: δC/H 35.5/3.16, td, J = 5.2, 4.8 Hz), an exchangeable proton signal (H-5′: δH 6.23, s), and a carbonyl resonance signal (C-4′: δC 170.0), along with the HRESIMS data, suggesting the side chain was N-methylbutanamide connected to N-8. This could also be confirmed by COSY correlations between H-1′ and HReceived: April 14, 2016

A

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a structure similar to 1. The key difference was the substitution of a two-carbon chain at N-8 terminating with a primary carboxamide suggested by a methylene signal (δH 4.13, d, J = 2.8; δC 41.4) and a carbonyl signal at δC 170.6 (C-2′) in its NMR spectrum. These data were consistent with the elemental formula and degree of unsaturation of 2. Comparison of the NMR data of 3 (Tables 1 and 2) with those of 1 revealed that they share the same tricyclic scaffold. The NMR data of the side chain in 3 showed three methylene signals (C-1′: δC/δH 35.5/ 3.66; C-2′: δC/δH 30.5/1.77; C-3′: δC/δH 58.8/3.51) indicating the hydroxypropyl moiety at the N-8, which were consistent with the elemental formula and degree of unsaturation of 3. They ECD spectrum of compound 2 exhibited Cotton effects at 204 nm (Δε +2.76), 246 nm (Δε +1.26), and 279 nm (Δε −0.47), while that of compound 3 exhibited Cotton effects at 203 nm (Δε +2.02), 247 nm (Δε +1.18), and 279 nm (Δε −0.62), which were nearly identical to those of 13 (Figure 2), indicating the absolute configurations of 2 and 3 were the same as that of 13. Cantharimide B (4) was obtained as a yellow oil with a molecular formula of C13H19NO4, determined by HRESIMS. The NMR spectra of 4 exhibited characteristic signals, including signals for two methyl groups (6H, δH 1.14), two methylenes (2H, δH 1.65; 2H, δH 1.88), and two methines (2H, δH 4.47, br s) in its 1H NMR spectrum and five typical signals (δC 12.6, 24.6, 55.2, 85.1, 183.5) in the 13C NMR spectrum. Comparison of the 1H and 13C NMR data (Tables 1 and 2) with those of cantharimide A (15), previously isolated from M. phalerata Palla,10 showed the presence of the same skeleton. The structural differences between them were the side chain at N-8. The NMR data of the side chain in 4 showed three methylene signals (C-1′: δC/δH 37.2/3.55; C-2′: δC/δH 31.5/ 1.75; C-3′: δC/δH 60.4/3.55), which were similar to those of 3, indicating a hydroxypropyl moiety linked at N-8. These assignments were consistent with the elemental formula and degree of unsaturation obtained from the HRESIMS data of 4. Confirmation of the structure of 4 was achieved by an ammonolysis reaction of the cyclo-anhydride precursor. Treatment of cantharidin11 with 3-aminopropanol led to the generation of 4, which was confirmed as 3′-[(1S,2R,3S,6R)1,2-dimethyl-3,6-epoxycyclohexane-1,2-dicarboximide-8-yl]-hydroxypropane on the basis of NMR analysis and specific rotation. Furthermore, the absolute configuration of 4 was

2′ and H-3′ and the key HMBC correlations from H-1′ (δH 3.57) to C-7 (δC 181.7) and C-9 (δC 177.2), from H-3′ (δH 3.16) to C-2′ (δC 27.1) and C-4′ (δC 170.0), and from H-6′ (δH 2.00) to C-4′ (δH 170.0) (Figure 1). The ECD spectrum of 1 exhibited Cotton effects at 207 nm (Δε +4.24), 248 nm (Δε +1.84), and 281 nm (Δε −0.53) (Figure 2), quite similar to those of palasoninimide A (13), the absolute configuration of which was originally established by X-ray diffraction in our study (Figure 5), indicating the absolute configuration of 1 was 1S, 2R, 3S, 6R. Both palasoninimide C (2) and palasoninimide D (3) were obtained as yellow oils. On the basis of the HRESIMS and NMR data, their molecular formulas were determined as C11H14N2O4 and C12H17NO4, respectively. For compound 2, a direct comparison of the NMR data (Tables 1 and 2) with those of 1 suggested that 2 was a palasoninimide derivative with

Table 1. 1H NMR Data for Compounds 1, 5 (500 MHz), 2−4, and 6 (400 MHz) position 2 3 4 5 6 10 11 1′ 2′ 3′ 5′ 6′ a

1a δH (J in HZ)

2b δH (J in HZ)

2.41, 4.76, 1.75, 1.91, 1.60, 1.91, 4.63, 1.35,

s d (4.0) m m m m d (4.0) s

2.64, 4.69, 1.69, 1.99, 1.69, 1.72, 4.57, 1.37,

s d (5.2) m m m m d (4.4) s

3.57, 1.76, 3.16, 6.23, 2.00,

td (6.4, 1.2) m td (5.2, 4.8) s s

4.13, d (2.8)

3a δH (J in HZ) 2.41, 4.74, 1.77, 1.94, 1.59, 1.94, 4.64, 1.34,

s d (5.6) m m m m d (4.4) s

3.66, t (6.0) 1.77, m 3.51, t (5.2)

4b δH (J in HZ) 4.47, 1.65, 1.88, 1.65, 1.88, 4.47, 1.14, 1.14, 3.55, 1.75, 3.55,

br s m m m m br s s s m m m

5a δH (J in HZ) 4.51, 1.67, 1.79, 1.67, 1.79, 4.52, 1.11, 1.11, 3.52, 1.72, 3.12, 6.58, 1.97,

d (2.0) m m m m d (2.0) s s t (5.2) m t (5.2) s s

6b δH (J in HZ) 4.44, 1.62, 1.85, 1.62, 1.85, 4.44, 1.13, 1.13, 3.57, 3.57,

br s m m m m br s s s m m

In CDCl3. bIn CD3OD. B

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Table 2. 1H NMR Data for Compounds 11 (500 MHz), 7−10, 12, and 13 (400 MHz) position 2 3 4 5 6 10

a

7a δH (J in HZ) 4.55, 1.69, 1.81, 1.69, 1.81, 4.55, 1.13,

br s m m m m br s s

11 1′ 2′ 3′

1.13, s 3.80, t, (6.8) 2.52, t, (6.8)

4′

5.63, br s 5.89, br s

8a δH (J in HZ) 4.59, 1.70, 1.81, 1.70, 1.81, 4.59, 1.16,

br s m m m m br s s

9b δH (J in HZ) 4.46, 1.62, 1.88, 1.62, 1.88, 4.46, 1.14,

dd, (2.0, 1.2) m m m m dd, (2.0, 1.2) s

1.16, s 4.15, br s

1.14, s 4.31, s

6.00, br s 6.40, br s

2.14, s

10a δH (J in HZ) 4.58, 1.77, 1.82, 1.77, 1.82, 4.72, 3.74, 3.85, 1.22, 3.01,

d (4.0) m m m m d, (3.2) d (11.6) d (11.6) s s

11a δH (J in HZ) 4.60, 1.77, 1.84, 1.77, 1.84, 4.71, 3.77, 3.91, 1.25, 3.71, 1.74, 3.56,

d (4.0) m m m m br s d (11.6) d (11.6) s t (6.0) m t (5.6)

12a δH (J in HZ) 4.55, 1.67, 1.84, 1.67, 1.84, 4.63, 3.72, 3.92, 1.26, 3.69, 3.70,

d (3.2) m m m m br s d (11.6) d (11.6) s mc mc

13b δH (J in HZ) 2.54, 4.70, 1.68, 1.97, 1.68, 1.87, 4.56, 1.35,

s d (5.6) m m m m d (5.2) s

In CDCl3. bIn CD3OD. cOverlapped signals.

Figure 1. Key COSY and HMBC correlations of 1, 5, and 11.

Figure 3. Experimental ECD spectra of compounds 4−9 and 15.

Figure 2. Experimental ECD spectra of compounds 1−3 and 13.

confirmed by the ECD spectrum with Cotton effects at 208 nm (Δε +2.23) and 237 nm (Δε −0.53) (Figure 3). The molecular formula C15H22N2O4 of cantharimide C (5) was established by HRESIMS, with six indices of hydrogen deficiency. Comparison of the 1H and 13C NMR data (Tables 1 and 2) with those of cantharimide 4 showed they have the same skeleton with minor differences. The main differences between them were an additional methyl signal (C-6′: δC/H 23.1/1.97, s), three methylene signals (C-1′: δC/H 36.1/3.52, t, J = 5.2 Hz; C-2′: δC/H 27.1/1.72, m; C-3′: δC/H 35.7/3.12, t, J = 5.2), an exchangeable proton signal (H-5′: δH 6.58, s), and a carbonyl resonance (C-4′: δC 170.4) in 5. Key HMBC correlations from H-10 to C-1/C-6/C-7 and from H-11 to C-2/C-3/C-9 (Figure 1) confirmed the cantharimide framework of 5. Moreover,

Figure 4. Experimental ECD spectra of compounds 10−12 and 14.

COSY correlations of H-1′/H-2′/H-3′ and HMBC correlations of H-6′ to C-4′ and H-3′ to C-2′/C-4′/C-1′ (Figure 1) supported the presence of an N-methylbutanamide moiety, and its connection with N-8 was determined by the HMBC correlation of H-1′ to C-7/C-9 (Figure 1). The ECD spectrum of 5 exhibited ECD Cotton effects at 213 nm (Δε +2.40), 234 nm (Δε −1.17), and 252 nm (Δε −1.17), which were nearly identical to those of cantharimide 4 (Figure 2). Considering their biosynthetic origin, the absolute configuration of 5 should be the same as that of 4. C

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C CH CH CH2 CH2 CH C C CH3 55.7, 58.8, 83.5, 29.2, 24.9, 81.8, 184.9, 180.4, 16.2, C C CH CH2 CH2 CH C C CH2 CH3 CH2 CH2 53.8, 59.5, 84.0, 23.8, 24.2, 81.9, 181.3, 181.9, 59.9, 11.9, 41.9, 59.8, C C CH CH2 CH2 CH C C CH2 CH3 CH2 CH2 CH2 54.1, 59.3, 84.0, 23.9, 24.4, 82.2, 181.9, 182.3, 60.3, 11.9, 35.7, 30.5, 58.9, C C CH CH2 CH2 CH C C CH2 CH3 CH3 54.1, 58.9, 83.8, 23.8, 24.4, 82.1, 181.5, 181.6, 60.6, 11.8, 25.5, 55.2, C 55.7, C 85.2, CH 24.60, CH2 24.57, CH2 85.2, CH 183.2, C 182.8, C 12.72, CH3 12.76, CH3 43.2, CH2 201.8, C 27.0, CH3 C C CH CH2 CH2 CH C C CH3 CH3 CH2 C 54.1, 54.1, 84.3, 23.5, 23.5, 84.3, 180.5, 180.5, 12.0, 12.0, 41.2, 168.3, C C CH CH2 CH2 CH C C CH3 CH3 CH2 CH2 C 54.1, 54.1, 83.9, 23.9, 23.9, 83.9, 181.4, 181.4, 12.7, 12.7, 35.5, 33.8, 172.6, C C CH CH2 CH2 CH C C CH3 CH3 CH2 CH2 55.2, 55.2, 85.1, 24.5, 24.5, 85.1, 183.4, 183.4, 12.6, 12.6, 42.2, 59.3, C C CH CH2 CH2 CH C C CH3 CH3 CH2 CH2 CH2 C CH3 53.9, 53.9, 83.6, 23.6, 23.6, 83.6, 181.9, 181.9, 12.5, 12.5, 36.1, 27.1, 35.7, 170.4, 23.1, C C CH CH2 CH2 CH C C CH3 CH3 CH2 CH2 CH2

a

In CDCl3. bIn CD3OD.

CH2 CH2 CH2 C CH3 36.0, 27.1, 35.5, 170.0, 23.2,

41.4, CH2 170.6, C

35.5, CH2 30.5, CH2 58.8, CH2

55.2, 55.2, 85.1, 24.6, 24.6, 85.1, 183.5, 183.5, 12.6, 12.6, 37.2, 31.5, 60.4, C CH CH CH2 CH2 CH C C CH3 53.4, 56.9, 80.6, 28.8, 24.3, 82.4, 182.3, 177.9, 16.5, C CH CH CH2 CH2 CH C C CH3 54.8, 57.6, 81.9, 29.2, 25.0, 83.7, 182.7, 178.4, 16.1, C CH CH CH2 CH2 CH C C CH3

4b δC 3a δC 2b δC 1a δC position

Table 3. 13C NMR Data for Compounds 1−13 (125 MHz)

5a δC

6b δC

7a δC

8a δC

9b δC

The molecular formulas of cantharimides D−G (6−9) were determined as C12H17NO4, C13H18N2O4, C12H16N2O4, and C13H17NO4, respectively, based on their HRESIMS spectra. Careful comparison of their 1H and 13C NMR data (Tables 1, 2, and 3) with those of 4 revealed that they differ only in the side chain at N-8. Compound 6 has been synthesized as an intermediate, but complete spectroscopic data have not been reported.12 Analysis of the NMR data of 6 (Tables 1 and 3) showed structural similarity to 4. The key difference was that the signals of a methylene (δH/δC 1.75/31.5) in 4 were not observed in 6, indicating a hydroxyethyl moiety was connected to N-8 in 6 instead of the hydroxypropyl moiety in 4. The structural differences between 7 and 5 were that the side chain signals of the methylene (C-2′: δC/H 27.1/1.72, m) and Nmethyl group (C-6′: δC/H 23.1/1.98, s) in 5 were absent and two N−H protons signals (δH 5.63, 5.89, br s) were observed in 7, suggesting the presence of a propionamide moiety in 7, which was consistent with its elemental formula and degree of unsaturation. The key difference between 8 and 7 was that the methylene signal (C-2′: δC/H 33.8/2.52) in 7 was not observed in 8, and the chemical shift of C-1′ was deshielded from 35.5 to 41.2 ppm, indicating the presence of a two-carbon primary amide moiety in 8. Analyses of the NMR data of 9 suggested it was a cantharimide derivative with structural similarity to 8. The side chain signals of two N−H protons in 8 were replaced by a methyl (C-3′: δC/δH 27.0/2.14, s) and a deshielded C-2′ resonance (δC 201.8) in 9, suggesting a propanone moiety at N-8. The ECD spectra of compounds 6−9 showed similar Cotton effects to those of 4 (Figure 3), which revealed that they had the same absolute configurations. Cantharimides H−J (10−12) were all obtained as yellow oils, and their molecular formulas of C11H15NO4, C13H19NO5, and C12H17NO5 were determined based on their HRESRMS data, respectively. Comparison of their NMR data (Tables 2 and 3) with those of the known compound 14 revealed that they share the same skeleton. Compound 10 showed five indices of hydrogen deficiency and 14 amu more than that of 14 and a methyl signal (δH/δC 3.01/25.5, s) in its NMR spectra, suggesting that the N-8 hydrogen in 14 was substituted by a methyl group in 10. Compound 11 possesses a hydroxypropyl moiety at N-8 instead of the methyl group in 10, which was supported by its NMR data, especially the HMBC correlations from H-1′ to C-7 and C-9 and from H-3′ to C-1′ and C-2′. Compound 12 possesses a hydroxyethyl moiety at N-8 instead

53.0, 56.4, 80.3, 28.4, 23.9, 82.0, 181.7, 177.2, 16.1,

Figure 5. X-ray ORTEP diagram of compound 13.

1 2 3 4 5 6 7 9 10 11 1′ 2′ 3′ 4′ 6′

10a δC

11a δC

12a δC

13b δC

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suggested that the 1,2-dimethylcantharimide type structure plays an important role in maintaining anti-HBV activities.

of a hydroxypropyl group in 11, which was supported by the methylene signal (C-2′: δC/H 30.5/1.74, m) in its NMR spectra and further confirmed by its COSY (Figure S57, Supporting Information) and HMBC correlations (Figure S59). Their absolute configurations were confirmed by comparison of their ECD Cotton effects (Figure 4) and their specific rotation values with those of 14, the absolute configuration of which was determined by X-ray diffraction with Cu Kα irradiation (Figure 6), suggesting the absolute configurations of 10−12 were the same as that of 14.

Table 4. Anti-HBV Activities of Compounds 6, 15, 16, and 18a in Vitro (μM) HBeAg b

compound

CC50

6 15 16 18 ADVe

>420 280 ± 7 >320 >220 >400

IC50 62 42 58 19 290

c

± ± ± ± ±

HBsAg d

SI 2 4 3 2 6

6.7

IC50

SI

>210 >260 >160 >110 270 ± 9

Data are expressed as mean ± SD (n = 3). bCC50 = concentration of 50% cytotoxicity, 50% inhibition concentration against HepG2 2.2.15 cells. cIC50 = 50% effective concentration, 50% inhibition concentration against HBV virus. dSI = CC50/IC50. eAdefovir dipivoxil, an antiviral agent used as a positive control. a

In addition, compounds 1−18 were tested for cytotoxic activities against the HepG2.2.15, HCT-116, and BGC-823 cell lines, with cantharidin being used as the positive control, but none of them were active in vitro (IC50 > 10 μM).

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EXPERIMENTAL SECTION

General Experimental Procedures. Melting points (uncorrected) were determined on a Mettler Toledo MP50 melting point apparatus. Optical rotations were recorded with a Rudolph Autopol I polarimeter. UV spectra were recorded on a Shimadazu UV-3600 Plus spectrophotometer. ECD spectra were measured on a Jasco J-715 spectrometer. IR spectra were recorded on a Bruker Tensor 27. 1D and 2D NMR spectroscopic data were recorded on Mercury-400, Agilent DD2-500, and Bruker AV-III-500 spectrometers using TMS as an internal standard. HRESIMS were performed on a Micromass Auto Spec-Ultima ETOF, AccuTO FUS JMS-J100CS spectrometer. ESIMS were performed on an Agilent 1100 Series LC-MSD-Trap-SL spectrometer. Preparative HPLC was performed on a Shimadzu LC10A preparative liquid chromatograph with an Ultimate XB-C18 column (Welch, 21.2 × 250 mm). Column chromatography was performed on silica gel (100−200, 200−300, and 300−400 mesh; Qingdao Marine Chemical Inc.), SF-PRP gel (70−150 μm, Beijing Sun Flower and Technology Development Company), and Sephadex LH-20 (Pharmacia). Fractions were monitored by thin-layer chromatography (Qingdao Marine Chemical Inc.). Insect Material. The dried bodies of Mylabris phalerata Palla were collected in Guizhou, People’s Republic of China, in September 2013. The sample was identified by Ph.D. Rang-Yu Mo (Chongqing Academy of Chinese Material Medical), and a voucher specimen (SYM-241) has been deposited in the herbarium of Chongqing Academy of Chinese Material Medical, Chongqing, China. Extraction and Isolation. The dried bodies of M. phalerata Palla (13 kg) were extracted three times with 95% EtOH at room temperature over 7 days and concentrated at reduced pressure to obtain a crude extract, which was partitioned between H2O and CHCl3. The chloroform-soluble fraction was dried to yield 130 g of extract, which was chromatographed on a silica gel (100−200 mesh) column using cyclohexane−CHCl3−MeOH as the elution solvents (from 40:1:0 to 20:1:0 to 9:1:0 to 6:4:0 to 0:8:2 to 0:0:1) to obtain fractions 1−5. Fraction 3 (12 g) was chromatographed using silica gel (200−300 mesh) to afford five fractions (Fr3.1−Fr3.5) eluting with a step gradient of cyclohexane−CHCl3−MeOH solvents (1:0:0 to 9:1:0 to 6:4:0 to 0:1:0 to 0:6:4 to 0:0:1). Fr3.5 (4.6 g) was further separated into six fractions (Fr3.5.1−Fr3.5.6) by flash column chromatography on silica gel (300−400 mesh), eluting with solvents of PE−EtOAc (6:1). Fr3.5.3 (434 mg) was purified by preparative HPLC (Ultimate XB-C18, 20 mL/min) using a MeOH−H2O (40:60) solvent system to yield compounds 15 (10 mg) and 9 (14 mg). Fr3.5.4 (1.3 g) was

Figure 6. X-ray ORTEP diagram of compound 14.

Palasoninimide A (13) was obtained as colorless needles from MeOH. Its ion at m/z 204.2 [M + Na]+ was established by ESIMS, exhibiting 14 amu less than that of cantharimide A (15). Palasoninimide has been found in the blister beetle H. lunata,9 but its spectroscopic data and the configuration have not been reported. Here we report its NMR data and absolute configuration as 1S, 2R, 3S, 6R determined by X-ray diffraction with Cu Kα irradiation (Figure 5). Compound 14 was identified as the known 1-hydroxymethyl-2-methyl-3,6-epoxycyclohexane-1, 2-dicarboximide by comparison of its recorded and reported NMR and HRMS data,10 and its absolute configuration was determined as 1R, 2R, 3S, 6R by X-ray diffraction with Cu Kα irradiation (Figure 6). Identification of the known compounds 15−18 was performed by comparison of their NMR and HRMS data with data reported in the literature.10,13 Hepatitis B virus (HBV) is a partially double-stranded genomic DNA virus belonging to the family Hepadnaviridae. To discover effective antiviral natural products, compounds 1− 18 were assayed in the HepG2.2.15 cell line in vitro, using ELISA kits to detect the hepatitis B virus surface antigen secretion (HBsAg) and hepatitis B virus e antigen (HBeAg) in the cell culture medium, and ADV (adefovir dipivoxil, a frequently used clinical anti-HBV agent) was used as the positive control.14 Among them, compounds 6, 15, 16, and 18 exhibited inhibitory activities against the secretion of HBeAg with IC50 values of 62, 42, 58, and 19 μM, respectively. No toxicity was observed at effective concentrations, and they also showed weak anti-HBsAg effects (IC50 > 100 μM). In addition, the inhibitory effects of ADV on HBsAg and HBeAg were less potent than that on HBV DNA (Table 4). The above tests E

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Cantharimide F (8): yellow oil, [α]28 D +25 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 207 (3.25) nm; IR (KBr) νmax 3429, 3204, 2983, 2935, 1773, 1704, 1427, 1339, 1235, 1149, 1063, 999, 958, 926, 899, 849, 760, 555, 486 cm−1; ECD (c 1.07 mM, MeOH), λmax (Δε) 206 (+2.40), 237 (−0.91) nm; 1H and 13C NMR, Tables 2 and 3; HRESIMS m/z 253.1182 [M + H]+ (calcd for C12H17N2O4, 253.1183). Cantharimide G (9): transparent oil, [α]28 D +35 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 206 (3.14) nm; IR (KBr) νmax 2956, 2929, 1712, 1423, 1378, 1337, 1261, 1126, 1068, 1002, 899, 759, 556 cm−1; ECD (c 0.97 mM, MeOH), λmax (Δε) 208 (+2.69), 240 (−0.67) nm; 1H and 13C NMR, Tables 2 and 3; HRESIMS m/z 252.1231 [M + H]+ (calcd for C13H18NO4, 252.1230). Cantharimide H (10): yellow oil, [α]27 D 0 (c 0.09, MeOH); UV (MeOH) λmax (log ε) 208 (3.42) nm; IR (KBr) νmax 3448, 2957, 2928, 1771, 1698, 1441, 1385, 1221, 1107, 1056, 1000, 926, 816, 765, 574 cm−1; ECD (c 0.85 mM, MeOH), λmax (Δε) 206 (+1.72), 232 (−0.80), and 258 (−0.84) nm; 1H and 13C NMR, Tables 2 and 3; HRESIMS m/z 226.1068 [M + H]+ (calcd for C11H16NO4, 226.1074). Cantharimide I (11): yellow oil, [α]28 D +6 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 208 (3.47) nm; IR (KBr) νmax 3442, 2958, 2929, 1695, 1446, 1407, 1375, 1262, 1230, 1140, 1067, 1005, 765, 577, 437 cm−1; ECD (c 0.92 mM, MeOH), λmax (Δε) 204 (+2.90), 233 (−0.59), and 256 (−0.97) nm; 1H and 13C NMR, Tables 2 and 3; HRESIMS m/z 270.1330 [M + H]+ (calcd for C13H20NO5, 270.1336). Cantharimide J (12): yellow oil, [α]28 D +9 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 208 (3.14) nm; IR (KBr) νmax 3443, 2957, 2930, 1770, 1696, 1438, 1406, 1344, 1269, 1227, 1159, 1061, 1005, 929, 815, 764, 575 cm−1; ECD (c 1.05 mM, MeOH), λmax (Δε) 205 (+4.27), 235 (−0.35), and 255 (−0.68) nm; 1H and 13C NMR, Tables 2 and 3; HRESIMS m/z 256.1177 [M + H]+ (calcd for C12H18NO5, 256.1179). Palasoninimide A (13): colorless needles, mp 182−184 °C; [α]27 D +14 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 207 (3.35) nm; IR (KBr) νmax 3201, 3077, 2989, 1774, 1715, 1449, 1354, 1310, 1240, 1206, 1134, 1090, 1000, 958, 926, 877, 819, 763, 577, 452 cm−1; ECD (c 1.12 mM, MeOH), λmax (Δε) 204 (+2.84), 240 (+1.18), and 282 (−0.47) nm; 1H and 13C NMR, Tables 2 and 3; ESIMS m/z 204.2 [M + Na]+. (1R,2R,3S,6R)-1-Hydroxymethyl-2-methyl-3,6-epoxycyclohexane1,2-dicarboximide (14): colorless needles, mp 156−157 °C; [α]27 D +4 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 204 (2.75) nm; IR (KBr) νmax 3449, 3200, 2960, 2925, 2855, 1772, 1709, 1461, 1406, 1353, 1268, 1234, 1126, 1062, 998, 926, 895, 812, 765, 568, 463 cm−1; ECD (c 0.94 mM, MeOH), λmax (Δε) 203 (+2.31), 230 (−0.64), and 256 (−0.82) nm. Cantharimide A (15): colorless needles, mp 203−204 °C; [α]27 D +38 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 206 (2.91) nm; IR (KBr) νmax 3189, 3063, 2985, 1773, 1704, 1471, 1388, 1350, 1306, 1265, 1231, 1122, 993, 960, 924, 896, 840, 734, 540, 464 cm−1; ECD (c 1.10 mM, MeOH), λmax (Δε) 204 (Δε +1.68), and 237 (Δε −0.89) nm. X-ray Crystallographic Analysis of Compounds 13 and 14. Colorless crystals of 13 and 14 were obtained in MeOH. Single-crystal X-ray diffraction data were collected on an Agilent Gemini E diffractometer with Cu Kα radiation (λ = 1.5418 Å). The structures of 13 and 14 were solved by direct methods using Olex 2, and refinements were performed with Olex 2 using full-matrix leastsquares. All non-hydrogen atoms were refined anisotropically. Hydrogen atoms were refined and placed in idealized positions. Crystallographic data (excluding structure factors) for 13 and 14 have been deposited with the Cambridge Crystallographic Data Center: CCDC reference numbers 1472776 (for compound 13), 1453535 (14). Data of compounds 13 and 14 in CIF format can be obtained free of charge from the Cambridge Crystallographic Data Centre via https://summary.ccdc.cam.ac.uk/structure-summaryform. Crystallographic data for 13: orthorhombic, C9H11NO3, space group P212121, a = 5.3760(5) Å, b = 11.9366(11) Å, c = 13.1122(13) Å, α = β = γ = 90°, V = 841.43(14) Å3, Z = 4, T = 104.3 K, ρcalc = 1.430 mg/mm3, μ = 0.904 mm−1, and F(000) = 384. Crystal size = 0.250 × 0.230 × 0.200 mm3. Independent reflections: 1588 with Rint =

purified by preparative HPLC (Ultimate XB-C18, 20 mL/min) using a MeOH−H2O (30:70) solvent system to afford compound 13 (33 mg). Fr3.5.5 (363 mg) was separated by preparative HPLC (Ultimate XBC18, 20 mL/min) using a MeOH−H2O (45:55) solvent system to afford compound 16 (18 mg). Fr3.5.6 (1.4 g) was separated by preparative HPLC (Ultimate XB-C18, 20 mL/min) using a MeOH−H2O (30:70) solvent system to afford compounds 3 (4 mg), 10 (6 mg), 6 (36 mg), and 4 (6 mg) and further purified by using a CH3CN−H2O (30:70) solvent system to afford compounds 17 (17 mg) and 18 (12 mg). Fraction 5 (18 g) was separated into four fractions (Fr5.1−Fr 5.4) by using SF-PRP gel with a step gradient of MeOH−H2O (20:80−100:0). Fr5.1 (4.7 g) was further separated into five fractions (Fr5.1.1−Fr5.1.5) by flash chromatography using CHCl3−acetone−MeOH (5:3:2). Fr5.1.1 (350 mg) was purified by Sephadex LH-20 column chromatography with MeOH as the mobile phase to yield compound 5 (30 mg). Fr5.1.2(450 mg) was separated by preparative HPLC (Ultimate XB-C18, 20 mL/min) using a MeOH−H2O (25:75) solvent system to afford compounds 8 (37 mg), 14 (8 mg), 1 (6 mg), and 2 (2 mg). Fr5.1.3 (116 mg) was purified by preparative HPLC (Ultimate XB-C18, 20 mL/min) using a MeOH−H2O (20:80) solvent system to afford compound 11 (2 mg). Fr5.1.4 (260 mg) was purified by preparative HPLC (Ultimate XB-C18, 20 mL/min) using a MeOH−H2O (25:75) solvent system to afford compounds 12 (38 mg) and 7 (9 mg). Palasoninimide B (1): yellow oil, [α]27 D +18 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 208 (3.52) nm; IR (KBr) νmax 3277, 2959, 2927, 2858, 1699, 1549, 1447, 1406, 1377, 1261, 1065, 1014, 927, 861, 800, 584 cm−1; ECD (c 1.07 mM, MeOH), λmax (Δε) 207 (+4.24), 248 (+1.84), and 281 (−0.53) nm; 1H and 13C NMR, Tables 1 and 3; HRESIMS m/z 281.1493 [M + H]+ (calcd for C14H21N2O4, 281.1496). Palasoninimide C (2): yellow oil, [α]27 D +16 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 207 (3.35) nm; IR (KBr) νmax 3431, 3201, 2961, 2929, 1709, 1426, 1334, 1282, 1199, 1128, 1072, 996, 926, 861, 747, 545, 478 cm−1; ECD (c 0.96 mM, MeOH), λmax (Δε) 204 (+2.76), 246 (+1.26), and 279 (−0.47) nm; 1H and 13C NMR, Tables 1 and 3; HRESIMS m/z 239.1025 [M + H]+ (calcd for C11H15N2O4, 239.1026). Palasoninimide D (3): yellow oil, [α]27 D +19 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 208 (3.52) nm; IR (KBr) νmax 3457, 2961, 2934, 1770, 1700, 1445, 1406, 1374, 1266, 1196, 1141, 1068, 1000, 926, 858, 760, 548, 435 cm−1; ECD (c 1.04 mM, MeOH), λmax (Δε) 203 (+2.02), 247 (+1.18), and 279 (−0.62) nm; 1H and 13C NMR, Tables 1 and 3; HRESIMS m/z 240.1231 [M + H]+ (calcd for C12H18NO4, 240.1230). Cantharimide B (4): yellow oil, [α]28 D +37 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 208 (3.47) nm; IR (KBr) νmax 3456, 2962, 2933, 1769, 1697, 1443, 1407, 1375, 1277, 1237, 1208, 1138, 1071, 997, 899, 749, 555 cm−1; ECD (c 0.98 mM, MeOH), λmax (Δε) 208 (+2.23), 237 (−0.53) nm; 1H and 13C NMR, Tables 1 and 3; HRESIMS m/z 254.1388 [M + H]+ (calcd for C13H20NO4, 254.1387). Cantharimide C (5): yellow oil, [α]27 D +34 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 208 (3.55) nm; IR (KBr) νmax 3295, 2964, 2931, 1769, 1697, 1544, 1442, 1408, 1374, 1277, 1238, 1160, 1073, 999, 927, 899, 750, 555 cm−1; ECD (c 1.08 mM, MeOH), λmax (Δε) 213 (+2.40), 234 (−1.17), and 252 (−1.17) nm; 1H and 13C NMR, Tables 1 and 3; HRESIMS m/z 295.1662 [M + H]+ (calcd for C15H23N2O4, 295.1652). Cantharimide D (6): yellow oil, [α]27 D +38 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 207 (3.26) nm; IR (KBr) νmax 3453, 2981, 2932, 1770, 1697, 1435, 1407, 1339, 1268, 1234, 1208, 1153, 1061, 996, 961, 927, 899, 856, 554 cm−1; ECD (c 1.05 mM, MeOH), λmax (Δε) 209 (+1.41), 236 (−1.14) nm; 1H and 13C NMR, Tables 1 and 3; HRESIMS m/z 240.1236 [M + H]+ (calcd for C12H18NO4, 240.1230). Cantharimide E (7): transparent oil, [α]25 D +30 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 207 (3.30) nm; IR (KBr) νmax 3427, 3205, 2961, 2929, 1698, 1444, 1406, 1332, 1234, 1132, 1066, 1002, 926, 900, 763, 555, 436 cm−1; ECD (c 1.13 mM, MeOH), λmax (Δε) 206 (+0.62), 234 (−0.36) nm; 1H and 13C NMR, Tables 2 and 3; HRESIMS m/z 267.1338 [M + H]+ (calcd for C13H19N2O4, 267.1339). F

DOI: 10.1021/acs.jnatprod.6b00332 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

0.0214. The final R1 = 0.0364 and wR2 = 0.0929 [I > 2σ(I)]. The final R1 = 0.0373 and wR2 = 0.0945 (all data). Flack parameter = 0.02(19). Crystallographic data for 14: orthorhombic, C10H13NO4, space group P212121, a = 7.33508(19) Å, b = 7.7336(2) Å, c = 16.8499(4) Å, α = β = γ = 90°, V = 955.84(4) Å3, Z = 4, T = 100.3 K, ρcalc = 1.468 mg/mm3, μ = 0.961 mm−1, and F(000) = 448. Crystal size = 0.55 × 0.55 × 0.45 mm3. Independent reflections: 1790 with Rint = 0.0160. The final R1 = 0.0318 and wR2 = 0.0855 [I > 2σ(I)]. The final R1 = 0.0321 and wR2 = 0.0857 (all data). Flack parameter = 0.02(19). Preparation of 4. A mixture of cantharidin (Sigma, 71.4 mg, 0.364 mmol), 3-aminopropanol (Sigma, 50.5 mg, 0.437 mmol), and triethylamine (26 mL) was heated at 150 °C for 5 h in a sealed tube. After being cooled to room temperature, the reaction mixture was diluted with EtOAc, and the aqueous layer was acidified with HCl and extracted with EtOAc (3 × 50 mL). The organic layer was dried over MgSO4 and concentrated to give a product (50 mg, yellow oil), and the product was purified by reversed-phase HPLC (Welch, C18, 21.2 × 250 mm, 15 mL/min, 5 μm) using MeOH−H2O, 35:65, solvent system to afford compound 4 (23 mg, tR = 10 min). Synthetic 1 4: yellow oil, [α]28 D +35 (c 0.1, MeOH); H NMR (400 MHz, CD3OD) δ 1.14 (6 H, s, 1-Me, 2-Me), 1.65 (2 H, m, H-4, H-5), 1.88 (2 H, m, H-4, H-5), 4.47 (2 H, td, J = 2.4, 0.8 Hz, H-3, H-6), 3.54 (4 H, m, H-1′, H-3′), 1.75 (2 H, m, H-2′); FT-MS m/z 276.12029 [M + Na]+. Anti-HBV Assay. The anti-HBV activity and cytotoxicity of compounds 1−18 were evaluated on the HepG 2.2.15 cell lines, which were stably transfected with the HBV genome using Lipofectamine 2000 reagent (Invitrogen). The HepG 2.2.15 cells were cultured in DMEM medium (Gibco) with 10% (v/v) fetal bovine serum (Gibco), 100 μg/mL G418 (Gibco), 100 IU/mL penicillin (Gibco), and 100 IU/mL streptomycin (Gibco) in a 37 °C incubator with 5% CO2 for 24 h. The cultured cells were treated with test compounds that were diluted by the culture medium for an additional 96 h. The culture media were collected, and the anti-HBV inhibition activity was assayed by the enzyme-linked immunosorbent assay (ELISA; Autobio Diagnostics Co., Ltd.) according to the manufacturer’s instructions. Adefovir dipivoxil was used as the positive control.14 Cytotoxicity Assay. The cytotoxicity was assessed using the MTT method on the HepG2 2.2.15, HCT-116, and BGC-823 cell lines.15 The tumor cells was treated with serial dilutions of the test compounds ranging from 1.00 to 100 μg/mL for 72 h; then MTT (400 μg/mL) was added. Four hours later, the MTT was removed and replaced with DMSO (100 μL) and the absorbance was measured at 540 nm with an automatic plate reader (Thermo Labsysterms Multiskan MK3).

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(cstc2014yykfC10004). We appreciate the Academy of Military Medical Sciences for their excellent technical assistance in the antiviral activity assay.

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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.6b00332. 1D and 2D NMR, HRESIMS, ESIMS, IR, ECD spectra and X-ray ORTEP diagrams (PDF) Crystallographic data for compounds 13 and 14 (ZIP)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (D.-M. Zhang). *Tel: (86) 023-89029081. Fax: 023-89029008. E-mail: [email protected] (D.-J. Yang). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This project was supported financially by the Application and Development Project of Chongqing, China G

DOI: 10.1021/acs.jnatprod.6b00332 J. Nat. Prod. XXXX, XXX, XXX−XXX