Anisucoumaramide, a Bioactive Coumarin from Clausena anisum

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Anisucoumaramide, a Bioactive Coumarin from Clausena anisumolens Yun-Song Wang,† Bi-Tao Li,‡ Shi-Xi Liu,† Zheng-Qi Wen,‡ Jing-Hua Yang,*,† Hong-Bin Zhang,† and Xiao-Jiang Hao*,§ †

Key Laboratory of Medicinal Chemistry for Natural Resource, Ministry of Education, School of Chemical Science and Technology, Yunnan University, Kunming 650091, People’s Republic of China ‡ First Affiliated Hospital of Kunming Medical University, Kunming 650031, People’s Republic of China § State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, 132 Lanhei Road, Kunming 650201, People’s Republic of China S Supporting Information *

ABSTRACT: A new coumarin, anisucoumaramide (1), and a new δ-truxinate derivative, anisumic acid (2), were isolated from Clausena anisum-olens. Their structures were elucidated from extensive NMR and MS data. The absolute configurations of the coumarins were assigned using the experimental and calculated electronic circular dichroism data. Anisucoumaramide (1) represents the first example of a naturally occurring coumarin of which the terpenoidal side chain does not comply with the biosynthesis isoprene rule due to the presence of an unprecedented acetamido motif directly connected with the terpenoidal side chain. The δ-truxinate derivative was isolated from Clausena species for the first time. Compound 1 showed high selectivity for the MAO-B isoenzyme and inhibitory activity in the nanomolar range. Putative biosynthesis pathways toward 1 and 2 are proposed.

T

he genus Clausena (Rutaceae) is known for being a rich source of coumarins. Some species of this genus have been used in folk medicine to treat various diseases in Asia for a long time.1−13 Naturally occurring coumarins derived from Clausena have exhibited a variety of biological activities including antioxidant,8 hepatoprotective,9 anti-inflammatory,10 antibacterial and antifungal,4,8 neuroprotective,5 anti-HIV,11,12 and antimycobacterial,13 as well as potent inhibitory effects on Epstein−Barr virus early antigen activation induced by 12-Otetradecanoylphorbol-13-acetate in Raji cells.2 Clausena anisumolens Merr. is a perennial evergreen shrub or small tree that is mainly distributed in the Philippines, South China, and throughout Southeast Asia. The fresh fruits are edible, and the dried fruits are used for preventing phlegm formation and stopping coughing. The leaves and twigs have traditionally been used for the treatment of dysentery and arthritis in folk medicine.7 Earlier phytochemical investigations of this species have led to the discovery of new monoterpenoid coumarins.14−17 A reinvestigation of the minor constituents of the ethanol extract of C. anisum-olens afforded a new coumarin, anisucoumaramide (1), and a new 1,2,3,4-tetrasubstituted cyclobutane derivative, anisumic acid (2), as well as the known 8-methoxycapnolactone (3) and trans-4-hydroxycinnamic acid (4). Herein, the isolation, © 2017 American Chemical Society and American Society of Pharmacognosy

structural elucidation, and putative biosynthesis pathways toward the new compounds, as well as the inhibitory activity of 1 on human monoamine oxidase (MAO), are described.

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RESULTS AND DISCUSSION Compound 1 was isolated as a colorless oil, [α]21.5D −13 (c 1.5, MeOH). The molecular formula, C22H23NO7, was established by 13C NMR and HRFABMS data (414.1620 [M + Na]+, calcd for 414.1613), suggesting 12 indices of hydrogen deficiency. Analysis of the 13C NMR data (Table 1) and the HMQC spectrum shows that the 22 carbons comprise four aliphatic methylenes (including one oxygenated methylene), seven methines (six olefinic/aromatic carbons and one oxygenated aliphatic carbon), nine sp2 nonprotonated carbons (one amide carbonyl, two ester carbonyls, six olefinic/aromatic carbons), a methyl group, and a methoxy group. Strong UV bands at λmax 261 and 319 nm, an IR band at 1728 cm−1, and two AB coupling systems at δH 6.33 and 7.77 and δH 7.01 and 7.25 in the 1H NMR data (Table 1) indicated the presence of a 7,8dioxygenated coumarin nucleus.2 Comparison of the 1H and Received: May 2, 2016 Published: April 3, 2017 798

DOI: 10.1021/acs.jnatprod.6b00391 J. Nat. Prod. 2017, 80, 798−804

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Table 1. NMR Spectroscopic Data (500 MHz, Pyridine-d5) of Compounds 1 and 3 1 position 2 3 4 5 6 7 8 9 10 1′ 2′ 3′ 4′a 4′b 5′ 6′ 7′ 8′ 9′ 10′ 11′ 12′ OMe NHa NHb

δH (J in Hz) 6.33, 7.77, 7.25, 7.01,

d d d d

(9.5) (9.5) (8.6) (8.6)

4.70, d (6.2) 5.68, t (6.2) 2.39, 2.27, 5.08, 7.28,

dd (14.3, 5.0) dd (14.3, 7.8) m d (1.5)

2.88, t (7.0) 1.73, s 2.77, t (7.0) 3.95, s 8.32, br s 7.83, br s

3

δC, type 160.6, 113.6, 144.3, 123.6, 110.6, 155.3, 137.9, 114.3, 149.4, 66.2, 123.8, 136.5, 43.1,

C CH CH CH CH C C C C CH2 CH C CH2

80.0, 150.1, 133.6, 173.1, 21.2, 17.3, 33.5, 173.7, 61.1,

CH CH C C CH2 CH3 CH2 C CH3

HMBC C-2, C-2, C-4, C-5,

C-4, C-5, C-7, C-7,

NOESY

C-9 C-8, C-9, C-10 C-8, C-9, C-10 C-8, C-9, C-10

δH (J in Hz)

H-4 H-3, H-5 H-6, H-4

6.25, 7.62, 7.16, 6.84,

C-7, C-2′, C-3′, C-4′, C-10′ C-1′, C-4′, C-10′

H-10′, H-6 H-4′

4.69, d (6.2) 5.62, t (6.2)

C-2′, C-3′, C-5′, C-6′, C-10′

H-2′, H-5′

2.42, d (9.1)

C-3′, C-4′, C-6′, C-7′ C-5′, C-7′, C-8′, C-9′

H-10′, H-4′, H-6′ H-5′

5.00, m 7.02, d (1.6)

C-6′, C-7′, C-8′, C-11′, C-12′ C-2′, C-3′, C-4′ C-7′, C-9′, C-12′

H-11′ H-1′

1.83, s 1.90, s

C-8

d d d d

3.97, s

(9.4) (9.4) (8.6) (8.6)

δC, type 162.9, 114.9, 144.9, 124.7, 111.5, 154.7, 136.8, 115.2, 148.2, 67.2, 124.0, 138.0, 44.5,

C CH CH CH CH C C C C CH2 CH C CH2

80.8, 149.5, 130.9, 175.2, 12.0, 18.8,

CH CH C C CH3 CH3

62.8, CH3

13

C NMR data of compound 1 and 8-methoxycapnolactone (3), a known coumarin isolated from the same plant, indicated that these two compounds differed only in the side chain. The location of the side chain at C-7 was confirmed by the longrange C−H correlation between the methylene protons at δH 4.70 (H-1′) and the oxygenated carbon (δC 155.3, C-7). In the HMBC experiment (Figure 2), the three-bond cross-peak

Figure 2. 1H−1H COSY, key HMBC, and ROESY correlations for 1.

was identified from the NMR signals and IR band at 1730 cm−1.2,10 The EIMS spectra of 1 did not exhibit the expected molecular ion, although it showed characteristic 8-methoxycoumarin fragment ions at m/z 193 and 176, corresponding to the loss of the side chain (Scheme S1, Supporting Information).18 The successive loss of CO afforded fragments at m/z 164 and 148. Coumarins have been the subject of numerous mass spectrometric investigations due to their pharmacological relevance.19 The mass spectrometer employed in the present investigation was optimized using positive-ion EI, FAB, and ESI conditions. The positive-ion mode was found to be the most sensitive. The FAB mass spectrum of 1 exhibited a protonated molecular ion, [M + H]+, at m/z 414, and ions were observed at m/z 221, 207, and 115. The ESIMS exhibited the sodium adduct [M + Na]+ and protonated molecular ions [M + H]+ at m/z 436 and 414, respectively. Thus, the FAB and ESIMS data of 1 suggested the presence of a nitrogen atom. In the ESIMS data of compound 1, cleavage of the bond between the oxygen atom at C-7 and the side chain resulted in the formation of a stable 7-hydroxy-8-methoxycoumarin ion at m/z

Figure 1. Structures of compounds 1−4.

between OMe (δH 3.95) and C-8 at δC 137.9 indicated the location of the methoxy group at C-8. The E configuration of the C-2′/C-3′ double bond was deduced from the 13C NMR chemical shift value of C-10′ (δC 17.3) and HMBC analysis (Figure 2, Table 1). The NOE correlations between H-l′ and H-10′ and between H-2′ and H-4′ further supported this assignment. Additionally, the α,β-unsaturated γ-lactone moiety 799

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193, further confirming the presence of a 7,8-dioxygenated coumarin moiety. The fragment ion at m/z 222, corresponding to the side chain, originated from the loss of the coumarin nucleus. The characteristic fragment ion at m/z 205 indicated the loss of NH3. The subsequent loss of 28 mass units (CO) was evidenced by the ion at m/z 177. Observation of the 1H NMR high-field region at δH 2.2−2.9 in methanol-d4 is often complicated by overlapping signals. However, the signals can be shifted by pyridine-d5 as the solvent. Change of the NMR solvent produced a dramatic effect on the 1H NMR signals of 1. Two well-resolved sharp N−H proton signals at δH 8.32 and 7.83 (each 1H, br s) were observed in the 1H NMR spectra of 1. The difference between the 13C NMR spectra of 1 and 3 involved the presence of a characteristic acyl carbonyl signal at δC 173.3, two extra methylene carbon signals at δC 21.2 and 33.5 (Table 1), and the absence of the C-9′ methyl resonance at δC 12.0. Characteristic IR signals at 3450 cm−1 observed in 1 are indicative of the N− H stretching of the primary amide, and the strong bands at 1690 cm−1 are typical for the N−CO stretching region of amides. The 1H−1H COSY cross-peaks of H-9′/H-11′ suggested the linkage of C-9′ and C-11′. The HMBC crosspeaks from H-9′ to C-6′, C-7′, C-8′, C-11′, and C-12′ and from H-11′ to C-7′, C-9′, and C-12′ indicated the linkage of an acetamido unit to the terpenoidal side chain at C-9′, signals that are absent in the case of compound 3. Compound 1 was isolated as an oil, making it impossible to obtain a single crystal for X-ray diffraction analysis. The limited amount of 1 also made it challenging to obtain a derivative for further crystallographic analysis. Thus, the relative configuration was established by examining the cross-peaks in the ROESY spectrum. Correlations between H2-1′ and H3-10′ and between H-2′ and H-4′a/b indicated that the C-2′/C-3′ double bond was E-configured. Monoterpenoid coumarins isolated from Clausena species tend to have an E double bond in the C7 or C-8 side-chain moiety.2,5,6,10 The C-5′ absolute configurations of 1 and 3 were assigned by comparing the calculated electronic circular dichroism (ECD) spectra of the 5′R- and 5′S-enantiomers with the experimental spectra of both coumarins. The experimental ECD spectra of 1 and 3 exhibited negative Cotton effects at 204, 208, and 216 and at 205, 210, and 217 nm in methanol, respectively, indicating a 5′R configuration.10 The good agreement between the experimental and calculated ECD spectra of the (5′R)-enantiomers led to the unequivocal absolute configuration assignment (Figure 3). Therefore, the absolute configurations of 1 and 3 were identified as 5′R. Collectively, the data showed that the structure of anisucoumaramide (1) was as shown in Figure 1. The occurrence of coumarins featuring a C10 terpenoid moiety has been demonstrated in the genus Clausena and other genera;2,5,6,10,20−23 however, it is noteworthy that anisucoumaramide (1) represents the first example of a naturally occurring coumarin of which the terpenoidal side chain does not comply with the biosynthesis isoprene rule because an unprecedented acetamido motif is directly connected with a terpenoidal side chain. Anisucoumaramide (1) is possibly biosynthesized from 8-methoxycapnolactone (3), a coumarin occurring in the same plant. As shown in Scheme 1, the putative biosynthesis of 1 starts from a double-bond migration (C6′/C7′ to C7′/C9′) of 3. The enoyl moiety of 5 then undergoes decarboxylative Michael addition with malonyl CoA under radical conditions to yield the intermediate radical 6,24,25 which in turn is susceptible

Figure 3. Calculated ECD spectra for (5′R)- and (5′S)-enantiomers and experimental ECD spectra of 1 and 3 in MeOH.

to oxidation, deprotonation, and amidation to give compound 1. Anisumic acid (2) was obtained as a white powder, [α]21.5D +13 (c 2, MeOH). Its molecular formula, C19H18O6, was determined on the basis of the molecular ion observed in negative-ion HRESI at m/z 341.1012 [M − H]− (calcd 341.1025), which corresponds to 11 indices of hydrogen deficiency. The UV spectrum of 2 in MeOH showed absorption maxima at 205, 230, 279, and 360 nm. The IR spectrum showed absorption bands for phenolic (3442, 1244 cm−1), carbonyl (1716 cm−1), and aromatic functionalities (1614, 1516, 720 cm−1). The resolution of overlapped proton signals was improved when the 1H NMR spectrum of 2 was recorded in pyridine-d5. Using HMQC data analysis, the 13C NMR and DEPT spectra resolved 19 carbon signals that were ascribed to one methoxy (δC 51.9), 12 methines [four sp3 methines, δC 48.6, 48.4, 46.3, 45.9, eight sp2 methines, 128.8 (×4), 116.5 (×4)], and six nonprotonated carbons (including four sp2 carbons, δC 132.8, 133.1, 158.1, 158.2, and two carbonyls, δC 173.9 and 175.6) (Table 2). In addition, 2 revealed 1H NMR signals in three distinct regions: aromatic protons ascribed to two 1,4disubstituted benzene rings [δH 7.51 (2H, d, J = 8.0 Hz), 7.46 (2H, d, J = 8.1 Hz), 7.16 (4H, d, J = 8.1 Hz)], four sp3 methines [δH 4.08, 3.97, 3.96, 3.94 (each 1H, m)], and a 800

DOI: 10.1021/acs.jnatprod.6b00391 J. Nat. Prod. 2017, 80, 798−804

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Scheme 1. Putative Biosynthesis Pathway toward Anisucoumaramide (1)

Table 2. NMR Spectroscopic Data (500 MHz, Pyridine-d5) of Compound 2 δH (J in Hz)

δC, type

HMBC

1 2 3

3.96, m 4.08, m 3.94, m

45.9, CH 48.6, CH 48.4, CH

4 1′ 2′ 3′ 4′ 5′ 6′ 1″ 2″ 3″ 4″ 5″ 6″ 7′ 8′ OMe

3.97, m

C-2, C-3, C-4, C-7′ C-1, C-3, C-4, C-1′, C-2′/6′, C-7′ C-2, C-4, C-1, C-1″, C-2″/6″, C-8′ C-1, C-2, C-3, C-8′

position

7.51, d (8.0) 7.16, d (8.1) 7.16, d (8.1) 7.51, d (8.0) 7.46, d (8.1) 7.16, d (8.1) 7.16, d (8.1) 7.46, d (8.1)

3.64, s

46.3, 133.1, 128.8, 116.5, 158.2, 116.5, 128.8, 132.8, 128.8, 116.5, 158.1, 116.5, 128.8, 175.6, 173.9, 51.9,

CH C CH CH C CH CH C CH CH C CH CH C C CH3

C-2, C-3′, C-4′ C-1′, C-2′, C-4′

Figure 4. 1H−1H COSY, key HMBC, and ROESY correlations of 2.

C-1′, C-4′, C-6′ C-2, C-4′, C-5′

an HMBC experiment based on the following key correlations: from H-1 to C-2, C-3, C-4, and C-7′; H-2 to C-1, C-3, C-4, C1′, C-2′, and C-7′; H-3 to C-2, C-4, C-1, C-1″, C-2″, and C-8′; and H-4 to C-1, C-2, C-3, and C-8′. This analysis produced four possible structures, A−D, based on the position of the hydroxycarbonyl and the methoxycarbonyl groups linked to the cyclobutane core (Scheme 2). The HMBC cross-peaks between the methoxy protons (δH 3.64) and C-8′ (δC 173.9) excluded the possibility of structures C and D. The analysis of the EIMS fragmentation pattern of 2 confirmed that only structure A was in accordance with the NMR and MS data (Figure 4). The successive fragmentation of the key ion of structure A at m/z 212 (55), 178 (100), 164 (80), 147 (87), and 131 (20) confirmed the 7′-COOH and 8′-COOMe groups. The HMBC cross-peaks between H-1 (δH 3.96) and C-7′ (δC 175.6) and from H-2 (δH 4.08) to C-1′, C-2′, and C-7′ (δC 175.6) supported the location of the hydroxycarbonyl group at C-1. On the basis of these data, the 2D structure of 2 was defined as 2,3-bis(4-hydroxyphenyl)-4-(methoxycarbonyl)cyclobutanecarboxylic acid as shown in Figure 1. Attempts at obtaining a single crystal for X-ray diffraction analysis failed, and the limited quantity of 2 made it difficult to produce a derivative for further crystallographic analysis. Thus,

C-3, C-3″, C-4″ C-1″, C-2″, C-4″ C-1″, C-3″, C-4″, C-6″ C-3, C-4″, C-5″

C-8′

methoxy group (δH 3.64). The 1H−1H COSY spectrum also showed five separate spin systems (shown in bold in Figure 4) that comprised two 1,4-disubstituted benzene moieties and four contiguous sp3 methines. Thus, the direct connections between C-1, C-2, C-3, and C-4 implied the presence of a cyclobutane moiety. The two carbonyl carbons and eight aromatic carbons represented 10 indices of hydrogen deficiency. The remaining index of hydrogen deficiency further supports that 2 possesses a four-membered ring system. Analyses of 1D and 2D NMR data indicated that 2 was a cyclobutane derivative, possibly assembled by two phenylpropanoid subunits: part A and part B (Figure 4). The connection of parts A and B was inferred by 801

DOI: 10.1021/acs.jnatprod.6b00391 J. Nat. Prod. 2017, 80, 798−804

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Scheme 2. Possible Structures and EIMS Fragmentation Analysis of 2

the relative configurations of the stereogenic centers in 2 were assigned by analysis of the ROESY correlations of the cyclobutyl and aromatic proton signals (Figure 4). The ROESY spectrum of 2 showed NOE correlations between H2 (δH 4.08) and H-6′ (δH 7.51)/H-2″ (δH 7.46) and correlations between H-1/H-2′ (δH 7.51) and H-3/H-2′ (δH 7.51). No diagnostic NOE correlation was observed among the four mutually coupled proton signals of the cyclobutane moiety. Therefore, the key NOE correlations suggested that H-1 and H-3 occupied the face of the cyclobutane moiety opposite that of H-2 and H-4, thus confirming the 1,2-trans-3,4trans relative configuration of the cyclobutane moiety.26 Considering the remaining possible δ- or μ-truxinic-type structures, only the former conformed to these conditions. The deshielded methoxy resonance (δH 3.64) also accounted for the adjacent trans-oriented aromatic groups on the cyclobutane ring.27,28 Thus, the relative configuration of compound 2 was established, and the compound was named anisumic acid (2). Compound 2 is a new δ-truxinate derivative possessing a cyclobutane core. This δ-truxinate derivative was isolated from Clausena species for the first time. The relative configuration of the cyclobutyl unit is in accord with those of recently reported δ-truxinic acid derivatives.27,28 Compound 2 is similar to monomethyl 3,3′,4,4′-tetrahydroxy-δ-truxinate, a δtruxinate derivative isolated from Lysimachia clethroides.28 The difference is that the latter possesses 1,3,4-trisubstituted aromatic rings instead of the para-substituted rings in compound 2. Cyclobutane-containing organic compounds, including natural products and/or drugs, present an intriguing group of metabolites with a variety of biological activities and may serve as potential drug leads or provide new ideas for the study of enzymatic mechanisms and/or organic synthesis.29,30 It is generally presumed that cyclobutane derivatives originate from the coupling of two phenylpropenoids. Thus, compound 2 may originate from (E)-4-hydroxycinnamic acid (4), which was isolated from the same plant (Scheme 3). A key intermolecular [2+2] cycloaddition of 4 formed the anti-head-to-head dimer (8), followed by monoesterification to afford compound 2. (E)4-Hydroxycinnamic acid (4) is also a biogenetically significant

Scheme 3. Putative Biosynthesis Pathway toward Anisumic Acid (2)

constituent for 7-O-isoprenylcoumarins. The (E)- and (Z)-phydroxycinnamic acids are the precursors for all known rutaceous coumarins that are oxygenated at C-7.31 Coumarins have drawn considerable attention in recent years as an important group of organic compounds that exhibits significant biological activities associated with neurological disorders. Both natural and synthesized coumarin analogues showed potent MAO-B inhibitory activity, particularly for the 3-, 4-, and 7-substituted coumarin analogues.32−34 Thus, the potential inhibitory effects of the new coumarin 1 were evaluated on human recombinant monoamine oxidase (hMAO) isoforms. The inhibitory effects were assessed by measuring the production of H2O2 from p-tyramine using the Amplex Red MAO assay kit with selegiline and iproniazide as reference drugs. The IC50 values and MAO-B selectivity indices for the inhibitory effects of both the new compound and reference inhibitors were calculated (Table 3). Compound 1 inhibited MAO-B with an IC50 value of 143.65 ± 0.90 nM (MAO-B selectivity index >696), but it was inactive at 100 μM to the MAO-A, demonstrating that compound 1 shows high selectivity for the MAO-B isoenzyme and inhibitory activity in the nanomolar range. 802

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Computational Methods. The geometries of compounds 1 and 3 were calculated using the DFT (B3LYP) method at the 6-31G(d) level by Gaussian 09.35 The ECD computation was performed using timedependent density-functional theory at the same level with the PCM solvent model for MeOH. The ECD spectra were generated by the program GaussView using a Gaussian band shape with 0.333 eV exponential half-width from dipole-length dipolar and rotational strengths. Bioassay Methods. The effects of 1 on the hMAO isoform enzymatic activity were evaluated by measuring the effects on the production of H2O2 from p-tyramine using a fluorimetric method. Selegiline and iproniazide served as reference inhibitors. Briefly, the study medium comprised 0.1 mL of Na3PO4 buffer (0.05 M, pH 7.4), various concentrations of compound 1 or reference compounds, and adequate amounts of recombinant hMAO-A or hMAO-B (SigmaAldrich) required to oxidize (in the control group) 165 pmol of ptyramine/min (hMAO-A, 1.1 μg of protein; specific activity: 150 nmol of p-tyramine oxidized to p-hydroxyphenylacetaldehyde/min/mg protein; hMAO-B, 7.5 μg of protein; specific activity: 22 nmol of ptyramine transformed/min/mg protein). This mixture was incubated for 15 min at 37 °C in a flat-black-bottom 96-well microtest plate placed in the dark multimode microplate reader chamber. After this incubation period, the reaction was initiated by adding 200 μM Amplex Red reagent, 1 U/mL horseradish peroxidase (HRP), and 1 mM p-tyramine as a common substrate for both hMAO-A and hMAOB. The production of H2O2 catalyzed by MAO isoforms was detected using Amplex Red reagent, a nonfluorescent probe reacting with H2O2 in the presence of HRP. The fluorescent product resorufin was quantified at 37 °C in a multidetection microplate fluorescence reader with excitation at 545 nm and emission at 590 nm for 15 min. The specific fluorescence emission was calculated after subtraction of the background activity. Control experiments were carried out simultaneously by replacing the test drugs (compound 1 and reference inhibitors) with appropriate dilutions of the vehicles. In addition, the possible capacity of the above-mentioned test drugs to directly react with Amplex Red reagent was determined by adding these drugs to solutions containing only the Amplex Red reagent in a sodium phosphate buffer.36,37

Table 3. MAO-A and MAO-B Inhibitory Activity Results for Compound 1 and Reference Compounds compound

MAO-A IC50

MAO-B IC50

1 selegiline iproniazide

− 67.25 ± 1.02 μM 6.56 ± 0.76 μM

143.65 ± 0.90 nM 19.60 ± 0.86 nM 7.54 ± 0.36 μM

a

SIb >696 3431 0.87

Inactive at 100 μM (highest concentration tested). Each IC50 value is the mean ± SEM from three experiments. bSI: MAO-B selectivity index = IC50(MAO-A)/IC50 (MAO-B). a

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were determined with a JASCO P-1020 polarimeter. UV spectra were measured on a Shimadzu UV-2401PC spectrophotometer. ECD spectra were acquired on a Chirascan instrument. IR spectra were obtained on a Bio-Rad FTS-135 infrared spectrophotometer. 1D and 2D NMR spectra were obtained at 500 and 125 MHz for 1H and 13C, respectively, on a Bruker DRX-500 spectrometer with tetramethylsilane as an internal standard. MS data were recorded on a VG Autospec-3000 mass spectrometer. Commercially available silica gel (100−200 mesh or 200−300 mesh, Qingdao Haiyang Chemical Co.), Lobar LiChroprep RP-18 (40−63 μm, Merck), and Sephadex LH-20 (Pharmacia) were used for open-column chromatography. All the solvents were distilled prior to use. Plant Material. The leaves and twigs of Clausena anisum-olens Merr. were collected in Hekou County of Yunnan Province, People’s Republic of China, in May 2003 and identified by Prof. De-Ding Tao of the Kunming Institute of Botany. A voucher specimen (No. 02041705) was deposited in the State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences. Extraction and Isolation. The powdered leaves and twigs of C. anisum-olens Merr. (22.5 kg) were repeatedly extracted with 90% aqueous EtOH (3 × 80 L) at room temperature. The extract was concentrated under reduced pressure to give a brown syrup, which was partitioned into H2O (15 L) and extracted successively with petroleum ether (5 × 10 L), EtOAc (5 × 10 L), and n-BuOH (5 × 10 L); the extracts were kept separately. The EtOAc extract (110.5 g) was subjected to silica gel column chromatography, eluting with petroleum ether−EtOAc (4:1, 2:1, 1:1, 2:3), EtOAc, EtOAc−MeOH (8:2, 7:3, 6:4, 1:1), and finally MeOH to afford nine fractions (I−IX). Fraction II (18 g) was resubjected to silica gel column chromatography and Pharmadex LH-20 (MeOH) to give compound 4 (9 mg). Fraction III (25.8 g) was resubjected to silica gel column chromatography, Pharmadex LH-20 (MeOH), and RP C18 to yield compounds 2 (3 mg) and 3 (52 mg). Fraction IV (4.8 g) was resubjected to silica gel column chromatography, Pharmadex LH-20 (MeOH), and RP C18 to afford compound 1 (2 mg). Anisucoumaramide (1): colorless oil; [α]21.5D −13 (c 1.5, MeOH); UV (MeOH) λmax 208, 215, 261, and 319 nm; ECD (c 0.44 mM, MeOH) λmax (Δε) 204 (−5.3), 208 (−5.3), 216 (−2.2) nm; IR (KBr) νmax 3450, 2925, 2856, 1730, 1728, 1690, 1621 cm−1; 1H and 13C NMR data see Table 1; HRFABMS [M + Na]+ m/z 414.1620 (calcd for C22H23NO7, 414.1613); FABMS m/z 414 [M + H]+ (95), 207 (100), 115 (74); ESIMS m/z 436 [M + Na]+, 414 [M + H]+, 222, 205, 193, 187, 177, 159, 131. Anisumic Acid (2): white powder; [α]21.5D +13 (c 3, MeOH); UV (MeOH) λmax 205, 230, 279, and 360 nm; IR (KBr) νmax 3442, 2955, 1716, 1614, 1516, 1244 cm−1; 1H and 13C NMR data see Table 2; HRESIMS [M − H]− m/z 341.1012 (calcd for C19H18O6, 341.1025); EIMS m/z 342 [M]+ (10), 316 (1), 306 (27), 263 (7), 237 (27), 212 (55), 178 (100), 164 (80), 147 (87), 131 (20). 8-Methoxycapnolactone (3): colorless semisolid; UV (MeOH) λmax 204 and 319 nm; ECD (c 0.22 mM, MeOH) λmax (Δε) 205 (−8.1), 210 (−5.0), 217 (−9.2) nm; 1H and 13C NMR data see Table 1; EIMS m/z 356 [M]+, 192, 163, 147, 97.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b00391. HRESIMS and 1D and 2D NMR spectra of compounds 1 and 2; proposed MS fragmentation pathway of 1 (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Tel: +86 871 65033715. E-mail: [email protected]. *Tel: +86 871 65219684. E-mail: [email protected]. ORCID

Jing-Hua Yang: 0000-0001-5835-2445 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (Grant Nos. 21462048, 21162038, 21262040, and 21662040) and the China Scholarship Council (CSC) Fund No. 201508535020, and Grant No. 2007PY01-23. We are grateful to the High Performance Computing Center of Yunnan University for providing the calculation resources. 803

DOI: 10.1021/acs.jnatprod.6b00391 J. Nat. Prod. 2017, 80, 798−804

Journal of Natural Products

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Article

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DOI: 10.1021/acs.jnatprod.6b00391 J. Nat. Prod. 2017, 80, 798−804