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Article Cite This: J. Org. Chem. 2019, 84, 282−288

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Maximumins A−D, Rearranged Labdane-Type Diterpenoids with Four Different Carbon Skeletons from Amomum maximum Kai-Long Ji,† Yao-Yue Fan,† Zhan-Peng Ge,† Li Sheng,† You-Kai Xu,‡ Li-She Gan,§ Jing-Ya Li,† and Jian-Min Yue*,†

J. Org. Chem. 2019.84:282-288. Downloaded from pubs.acs.org by UNITED ARAB EMIRATES UNIV on 01/11/19. For personal use only.

†

State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Zhangjiang Hi-Tech Park, Shanghai 201203, China ‡ Key Laboratory of Tropical Plant Resources and Sustainable Use, Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, Menglun 666303, China § Institute of Modern Chinese Medicine, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China S Supporting Information *

ABSTRACT: Four highly rearranged labdane-type diterpenoids, maximumins A−D (1−4) possessing different new carbon skeletons, together with a biosynthetically related known analog 5 were isolated from Amomum maximum. The structures of new compounds with absolute configurations were characterized by spectroscopic and computational approaches. The plausible biogenetic pathways for 1−4 were proposed. These compounds showed moderate to weak activities against nuclear factor kappa B (NF-κB).

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INTRODUCTION

Amomum species are perennial herbs that are distributed mainly in the tropical area of Asia and Oceania. There are about 150 Amomum species globally, of which 24 species grow in the southern China.1 A. maximum, known as “hegu” in xishuangbanna of Yunnan province of China, has been used as folk medicine by Dai minority for the treatment of stomach disorders.2 The chemical components isolated from the different parts of this plant were reported to exhibit a wide spectrum of biological activities, including insecticidal and repellent activities,3 cytotoxicity,4 and NO inhibition.5 To further investigate the chemical components of this plant, four highly rearranged labdane-type diterpenoids, maximumins A− D (1−4), along with a known compound ottensinin (5) were isolated and characterized (Figure 1). It is noteworthy that compounds 1−3 are highly rearranged labdane diterpenoids sharing a 16(13 → 12)-abeo-γ-pyrone motif with compound 5 as their common hypothetically biosynthetic intermediate, of which compound 1 possessed a unique 6/6/6/6-fused ring system, and compounds 2 and 3 were the ring B highly modified labdane diterpenoids. Compound 4 was the first example of 13(12 → 17)-abeo-12,17-cyclolabdane diterpenoid featuring an unique subunit of rings C and D. Herein, we elaborate the isolation, characterization, biogenetic consideration, and bioactive evaluation of compounds 1−5.

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Figure 1. Chemical structures of 1−5

(calcd 317.2117) indicated a molecular formula C20H28O3 corresponding to seven double-bond equivalents (DBEs). The IR spectrum suggested the presence of hydroxyl (3436 cm−1) and γ-pyrone (1651 and 1608 cm−1)5,6 groups. Analysis of the 1H and 13C NMR data (Table 1) with the aid of DEPT and HSQC spectra revealed the presence of 20 carbon resonances that were attributed to three methyls, seven methylenes, four methines, and six quaternary carbons (two olefinic and one carbonyl). These functionalities accounted for three out of the seven DBEs, requiring the existence of a tetracyclic ring system for compound 1.

RESULTS AND DISCUSSION

Maximumin A (1) was obtained as colorless crystals (in MeOH). The HRESIMS ion peak at m/z 317.2118 [M + H]+ © 2018 American Chemical Society

Received: October 16, 2018 Published: December 9, 2018 282

DOI: 10.1021/acs.joc.8b02665 J. Org. Chem. 2019, 84, 282−288

Article

The Journal of Organic Chemistry Table 1. 1H and

13

C NMR Data of Compounds 1 and 2 1a

no. 1α 1β 2α 2β 3α 3β 4 5 6α 6β 7α 7β 8 9 10 11α 11β 12 13 14 15 16 17α 17β 18 19 20

δH (mult., J, Hz) 0.94 1.88 1.46 1.53 1.15 1.40

td dt m m td dt

(12.9, 3.2) (12.9, 3.6)

0.99 1.74 1.30 1.60 2.06

m dq (13.8, 3.3) qd (13.8, 3.3) m dt (12.9, 3.3)

δC

δH (mult., J, Hz)

δC

40.2

1.40 td (12.6, 3.9) 2.13 m 1.59 m (2H)

39.9

18.6 (13.2, 4.4) (13.2, 3.3)

1.56 m 2.59 dd (18.4, 8.0) 2.77 br d (18.4)

6.35 d (5.4) 7.71 d (5.4) 2.40 2.96 0.89 0.79 0.70

2a

d (18.5) d (18.5) s s s

41.8 33.4 55.9 20.2 42.3 72.0 53.6 38.7 18.3 123.2 178.5 115.5 154.8 162.6 37.9 33.6 21.7 14.1

1.10 td (13.5, 4.5) 1.47 m 1.29 1.65 1.44 1.68 2.17 2.67

dd (13.2, 6.4) m m m m t (8.9)

20.1 41.4 33.5 59.0 21.1 22.2

3.39 s (2H)

64.9 207.3 45.0 39.7

6.34 d (5.7) 7.72 d (5.7) 7.75 s

124.7 177.6 116.8 155.4 154.2

0.87 s 0.86 s 0.74 s

Figure 2. Single-crystal X-ray structures of 1 and 2 (ellipsoids shown at the 50% probability level).

determine its absolute configuration (5S, 8S, 9R, 10S) with the perfect Flack parameter [−0.01(9)].7 The structure of maximumin A (1) was thus established as a rearranged labdane diterpenoid possessing 6/6/6/6-fused rings system. Maximumin B (2), colorless crystals, had a molecular formula of C19H26O3 as determined by the HRESIMS ion peak at m/z 627.3660 [2 M + Na]+ (calcd 627.3662). The IR spectrum revealed the existence of ketone (1710 cm−1) and γpyrone (1657 and 1621 cm−1)5,6 groups. The 1H and 13C NMR data (Table 1) showed the typical signals assignable for a γ-pyrone ring [δH 6.34 d (J = 5.7), 7.72 d (J = 5.7), 7.75 s; δC 116.8, 124.7, 154.2, 155.4, 177.6]8 and a ketone group (δC 207.3). The multiple HMBC correlations (Figure S2) of H318/C-4, C-5, and C-19; H3-19/C-3; and H3-20/C-1, C-5, C-8, and C-10, along with the 1H−1H COSY correlations of H-1/ H-2/H-3 and H-5/H-6/H-7/H-8 constructed a 6/5-fused bicyclic scaffold for 2. The linkage of the γ-pyrone ring with the 6/5-fused bicyclic core was completed by the bridge of C9−C-11 bond as evidenced by the HMBC correlations of H-8/ C-9 and C-11, H2-11/C-12 and C-13, and H-16/C-11. The ROESY correlations (Figure S2) of H-8/H-1α and H-5 suggested that H-5 and H-8 were α-oriented, while the ROESY correlations of H3-20/H-1β, H-6β, and H3-19 showed that H319 and H3-20 were β-oriented. Finally, a single-crystal X-ray diffraction study of 2 (Figure 2) not only confirmed the above structural assignment but also determined its absolute configuration as 5S, 8S, 10S [Flack parameter: 0.03(13)].7 Compound 2 was thus assigned as an unprecedented 10(9 → 8)-abeo-labdane 17-norditerpnoid featuring a terminal γpyrone ring. Maximumin C (3) was obtained as colorless gum and had the molecular formula of C20H28O5 as deduced by the HRESIMS ion peak at m/z 347.1862 [M − H]− (calcd 347.1858), corresponding to seven DBEs. The IR spectrum revealed the presence of carboxylic acid (3404 cm−1, broad band; and 1727 cm−1), keto carbonyl (1715 cm−1), and γpyrone (1652 and 1608 cm−1)5,6 groups. Comparison of the 1 H and 13C NMR data of 3 (Table 2) with those of 2 showed many similarities around the ring A and γ-pyrone moiety, and the major differences were likely resulted from the absence of ring B in compound 3. Except for the rings A and γ-pyrone, the characteristic carbon resonances for a keto carbonyl (δC 206.8) and carboxylic carbon (δC 175.9) were observed. The abovementioned functional groups satisfied the requirement of seven

33.6 20.8 14.8

a Data were collected in CDCl3 at 500 MHz (1H) and 125 MHz (13C).

The gross structure of 1 was delineated by 2D NMR data analysis (Figure S1, Supporting Information (SI)), in which four spin-coupling structural fragments, as drawn with bold bond, were first identified by the 1H−1H COSY spectrum, and the connection of these fragments with the quaternary carbons and oxygen atoms was then achieved by HMBC spectrum. The HMBC correlation networks of H2-7/C-8; H-9/C-8; H3-18/C4, C-5, and C-19; H3-19/C-3 and C-4; and H3-20/C-1, C-5, C9, and C-10 allowed the establishment of rings A and B. The key HMBC correlations H2-11/C-12; H2-17/C-8, C-12, and C-16 furnished the six-membered ring C. In addition, a γpyrone of ring D was fused to ring C by the characteristic carbon resonances (δC 115.5, 123.2, 154.8, 162.6, 178.5),5 which was confirmed by HMBC correlations of H2-11/C-12 and C-13; H-14/C-13; and H-15/C-16; and the 1H−1H COSY correlation of H-14/H-15. The deshielded chemical shift of C-8 (δC 72.0) revealed that a hydroxy group was located at C-8. The relative configuration of 1 was mainly fixed by the ROESY spectrum (Figure S1), in which the correlations of H2β/H3-19; H-6β/H-17β; and H3-20/H-2β, H-11β, and H-17β indicated these protons were cofacial and arbitrarily assigned as β-orientation, while the H-5, H-9, and H3-18 were assigned as α-orientation by the ROESY correlations of H-5/H-7α and H9; and H-6α/H3-18. The key ROESY cross-peak of H-6β and H-17β suggested that the OH-8 took the equatorial bond and α-orientation. Finally, the quality crystals of 1 were obtained by the recrystallization in MeOH, and the subsequent X-ray crystallography study (Figure 2) enabled us to unambiguously 283

DOI: 10.1021/acs.joc.8b02665 J. Org. Chem. 2019, 84, 282−288

Article

The Journal of Organic Chemistry Table 2. 1H and

13

3a no. 1α 1β 2α 2β 3α 3β 4 5 6α 6β 7α 7β 8 9a 9b 10 11 12a 12b 13 14a 14b 15 16a 16b 17a 17b 18 19 20 OH-13

δH (mult., J, Hz) 1.44 1.58 1.41 1.55 1.16 1.38

m m m qt (13.1, 3.1) td (13.1, 3.1) m

4b δC 38.7 18.8 42.0

1.07 t (3.7) 1.61 m (2H)

35.4 53.3 20.5

2.71 m (2H)

46.6

2.25 d (13.5) 2.20 d (13.5)

1.06 2.54 1.52 1.65 1.19 1.46

td dt m qt td m

(13.5, 3.9) (13.5, 3.7)

δC 34.7 18.6

(13.5, 3.7) (13.5, 4.1)

1.14 dd (12.5, 1.4) 1.87 ddt (13.4, 7.0, 1.4) 1.49 m 2.35 ddd (19.5, 11,0, 7.0) 2.45 ddd (19.5, 6.1, 1.4)

41.9 33.3 52.0 18.3 30.7 173.3 149.0

38.6 175.9 124.7

36.0 208.5 32.2

178.1 116.8

7.77 d (5.5) 7.80 s

155.9 154.5

d (16.6) d (16.6) s s s

δH (mult., J, Hz)

206.8 48.1

6.41 d (5.5)

3.46 3.38 0.87 0.90 1.02

seco-ring B labdane diterpenoid. The mutual ROESY correlations (Figure 3B) of H-2β/H3-19 and H3-20 and H3β/H3-19 indicated these protons were on the same side of ring A and assigned as β-orientation. The ROESY correlations of H3-18/H-3α and H-5 revealed that they were α-oriented. Although there were no obvious cotton effects observed at the region of 200−400 nm in the ECD spectrum of 3 (Figure S3) due likely to the chromophore of γ-pyrone group was far away from any of the chiral centers, it showed specific rotation value of +19.0. Further chiral HPLC analysis of compound 3 showed it was optically pure (Figure S4). The theoretical specific rotation calculation was thus applied to assign the absolute configuration of 3 (the details see Specific rotation calculation for compound 3 in SI).9,10 Both the experimental specific rotation (+19.0) and the calculated data (+32.2) for 3 showed positive signs (Table S3), suggesting that the absolute configuration of 3 was assigned as depicted (5S, 10R), which is consistent with the biosynthetic consideration (Scheme 1). Maximumin D (4) was obtained as an amorphous powder. The negative mode of HRESIMS gave a molecular ion peak at m/z 331.1909 [M − H]− (calcd 331.1909) corresponding to the molecular formula of C20H28O4 with seven DBEs. The IR spectrum indicated the presence of hydroxyl (3440 cm−1), lactone carbonyl (1804 cm−1), and α,β-unsaturated ketone (1664 and 1614 cm−1) functionalities. Analysis of the 1H and 13 C NMR data (Table 2) revealed the presence of three methyls (δH 0.88 s, 0.92 s, 1.09 s; δC 20.2, 21.6, 33.6), a carbonyl group (δC 175.0), and an α,β-unsaturated keto moiety (δC 149.0, 173.3, 208.5). These functionalities accounted for three out of seven DBEs, requiring four additional rings in compound 4. The 1 H−1 H COSY correlation sequences of H-1/H-2/H-3, H-5/H-6/H-7, and H-12/H-17, along with HMBC correlation networks of H2-7/ C-8 and C-9; H2-12/C-8, C-9, and C-11; H3-18/C-4, C-5, and C-19; H3-19/C-3 and C-4; and H3-20/C-1, C-5, C-9, and C-10 permitted the establishment of rings A−C (Figure 4A). The HMBC cross peaks of H2-14/C-13, C-15, and C-16; H2-16/C15; and H-17/C-13 and C-16 identified a γ-lactone ring D and attached it to the ring C via C-13−C-17 bond. In addition, a hydroxy group was located at C-13 by the deshielded chemical shift of C-13 (δC 77.4) and the HMBC correlations from OH13 to C-13 and C-14. Therefore, the planar architecture of 4 was established as a 13(12 → 17)-abeo-12,17-cyclolabdane diterpenoid featuring a unique subunit of rings C and D. Relative configuration of 4 was determined by a combination of ROESY spectrum and quantum chemical calculation of NMR data.11 In the ROESY spectrum (Figure 4B), the correlations of H3-20/H-6β and H3-19 revealed that these protons were cofacial and assigned arbitrarily as β-direction; and the correlations of H-5/H-7α and H 3 -18 were consequently indicated these protons were α-configured. However, the relative configurations of H-17 and OH-13 remained unassigned due to the rotational manner and the lack of reliable ROESY correlations. To solve this question, four theoretical stereoisomers 4a−4d (Figure 5) were taken into consideration, and their NMR calculations were accomplished by density functional theory (DFT) at the rmpw1pw91/631+g(d,p) level (the details see NMR calculation for compound 4 in SI). The calculated NMR data and experimental NMR data were then analyzed by the improved probability DP4+ method for isomeric compounds.12 The result showed that the stereoisomer 4a had much higher DP4+ probability score (94.55%) than those of 4b (0.00%), 4c

C NMR Data of Compounds 3 and 4

38.4 33.7 22.0 20.1

2.64 dd (18.0, 7.3) 2.13 dd (18.0, 3.1) 2.77 d (17.9) 2.59 d (17.9) 4.17 d (9.9) 3.97 d (9.9) 2.75 m 0.92 0.88 1.09 4.57

s s s s

77.4 41.3 175.0 75.8 51.2 33.6 21.6 20.2

a Data were collected in CDCl3 at 500 MHz (1H) and 125 MHz (13C). bData were collected in CDCl3 at 600 MHz (1H) and 125 MHz (13C).

DBEs for 3. In the HMBC spectrum (Figure 3A), the correlation networks from H2-7 to C-8 and from H2-17 to C-8,

Figure 3. Key 1H−1H COSY, HMBC (A) and ROESY (B) correlations of compound 3.

C-12, and C-13, together with the 1H−1H COSY correlations of H-5/H-6/H-7 allowed the connection of rings A and γpyrone via the carbon bridge of C-6 to C-8 and C-8−C-17 bond. The presence of a CH2COOH group was identified and attached to the C-10 at ring A by the key HMBC correlations of H2-9/C-5, C-10, and C-11; and H3-20/C-9. The gross structure of compound 3 was thus established as a rearranged 284

DOI: 10.1021/acs.joc.8b02665 J. Org. Chem. 2019, 84, 282−288

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The Journal of Organic Chemistry Scheme 1. Hypothetical Biosynthetic Pathways for Compounds 1−4

of 190−350 nm, the pattern of experimental ECD curve of 4 matched the calculated theoretical ones both in vacuum and in MeOH (Figure 6). The theoretical specific rotation of 4 was

Figure 4. Key 1H−1H COSY, HMBC (A) and ROESY (B) correlations of 4.

Figure 5. Four possible structures 4a−4d used for quantum chemical NMR calculation.

Figure 6. Experimental ECD curve of compound 4 (black line) and calculated ECD spectra of 4a in vacuum (red line) and in MeOH (blue line).

(5.45%), and 4d (0.00%) (Table S11), allowing the assignment of relative stereochemistry for compound 4 as depicted. To establish the absolute configuration for compound 4, the theoretical ECD spectrum of 4 was calculated using the TDDFT methodology at the B3LYP/6-311++G(2d,2p) level both in vacuum and a PCM solvent model of methanol (the detail see ECD calculation for compound 4 in SI). In the range

also calculated (+66.2) in vacuum, which was consistent in the positive sign with the experimental data (+28.3) for 4 (Table S14), further supporting the assignment for the absolute configuration of 4. The absolute configuration of 4 was thus assigned as 5S, 10S, 13S, 17R. The biosynthetic pathways for compounds 1−4 were proposed in Scheme 1. The biosynthetic precursor of 1−3 285

DOI: 10.1021/acs.joc.8b02665 J. Org. Chem. 2019, 84, 282−288

Article

The Journal of Organic Chemistry

fraction F3d (1.0 g) was separated over a RP-18 silica gel column (eluted with MeOH/H2O, from 3:2 to 9:1) and further purified by semipreparative HPLC (MeCN/H2O from 3:2 to 4:1) to yield compounds 1 (5.0 mg, tR = 15.0 min), 3 (3.0 mg, tR = 16.5 min), and 4 (1.5 mg, tR = 18.5 min), successively. Fraction F4 was treated initially by a silica gel CC (petroleum ether/ethyl acetate from 1:0 to 1:2) to afford subfractions F4a−F4c. The fraction F4b was then chromatographed on a Sephadex LH-20 gel column (EtOH) to afford compounds 2 (3.0 mg) and 5 (25.0 mg). Maximumin A (1). Colorless crystals; mp 203−205 °C; [α]17 D −67.9 (c 0.08, MeOH); UV (MeOH) λmax (log ε) 209 (3.96), 257 (3.99) nm; CD (MeOH) λmax (Δε) 210 (−2.22), 230 (+0.47), 258 (+0.94), 290 (−1.22) nm; IR (KBr) νmax 3436, 2945, 2926, 1651, 1608, 1566, 1444, 1392, 1368, 1260, 1234, 1199, 1037, 1018 cm−1; 1 H and 13C NMR data, see Table 1; (+)-ESIMS m/z 317.2 [M + H]+; (+)-HRESIMS m/z 317.2118 [M + H]+ (calcd for C20H29O3, 317.2117). Maximumin B (2). Colorless crystals; mp 106−108 °C; [α]18 D +146.0 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 205 (4.15), 249 (4.05) nm; CD (MeOH) λmax (Δε) 230 (−1.20), 288 (+4.81) nm; IR (KBr) νmax 2948, 2924, 1710, 1657, 1621, 1433, 1388, 1326, 1314, 1231, 1149, 1091, 981 cm−1; 1H and 13C NMR data, see Table 1; (+)-ESIMS m/z 627.4 [2 M + Na]+; (+)-HRESIMS m/z 627.3660 [2 M + Na]+ (calcd for C38H52O6Na, 627.3662). Maximumin C (3). Colorless gum; [α]18 D +19.0 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 204 (3.97), 249 (3.76) nm; IR (KBr) νmax 3404, 2930, 2867, 1727, 1715, 1652, 1608, 1447, 1392, 1327, 1204, 1143, 1035 cm−1; 1H and 13C NMR data, see Table 2; (+)-ESIMS m/ z 347.2 [M - H]−; (+)-HRESIMS m/z 347.1862 [M − H]− (calcd for C20H27O5, 347.1858). Maximumin D (4). White, amorphous powder; [α]18 D +28.3 (c 0.11, MeOH); UV (MeOH) λmax (log ε) 239 (3.96) nm; CD (MeOH) λmax (Δε) 200 (+4.03), 234 (+1.50), 316 (−0.90) nm; IR (KBr) νmax 3440, 2953, 2924, 2867, 1804, 1664, 1614, 1444, 1419, 1393, 1314, 1165, 1140, 1030 cm−1; 1H and 13C NMR data, see Table 2; (+)-ESIMS m/z 331.1 [M − H]−; (+)-HRESIMS m/z 331.1909 [M H]− (calcd for C20H27O4, 331.1909). X-ray Crystallographic Analysis for Compounds 1 and 2. The crystals of 1 and 2 were recrystallized from MeOH. X-ray analyses were carried out on a Bruker APEX-II CCD diffractometer with Cu Kα radiation (λ = 1.34139 Å). The acquisition parameters for 1 and 2 were provided in Tables S16 and S17, respectively, and crystallographic data for compounds 1 (deposition no. CCDC 1867979) and 2 (deposition no. CCDC 1867977) have been deposited at the Cambridge Crystallographic Data Center. Copies of the data can be obtained free of charge via www.ccdc.cam.ac.uk/ conts/retrieving.html. Specific Rotation Calculation for Compound 3. The calculation details are in Supporting Information (see Specific rotation calculation for compound 3, page S7). NMR Calculation for Compound 4. For all possible structures, conformational analyses were carried out via Monte Carlo searching using molecular mechanism with MMFF force field in the Spartan 14 program20 and reoptimized using DFT at the B3LYP/6-31G(d) level in vacuum by the Gaussian 09 program21 within an energy window of 2 kcal/mol. Conformers, whose relative Gibbs free energies in the range of 0−2 kcal/mol, were refined and considered for the next step gauge-independent atomic orbital (GIAO) calculations of 1H and 13C NMR chemical shifts using DFT at the rmpw1pw91/6-31+g(d,p) level in vacuum. The calculated 1H and 13C NMR data of the lowest energy conformers for each compound were averaged according to the Boltzmann distribution theory and relative Gibbs free energy (ΔG). The 1H and 13C NMR chemical shifts for TMS were also calculated by the same procedures and used as the reference. After calculation, the experimental and calculated 1H and 13C NMR data were analyzed by the improved probability DP4+ method.12 ECD Calculation for Compound 4. Theoretical ECD spectrum of 4 was calculated and compared with the corresponding experimental data. The energies, oscillator strengths, and rotational strengths of the first 60 electronic excitations were calculated using

was traced back to a coexisting known labdane diterpenoid ottensinin (5),4 which was originated from labdadienyl PP ((+)-CPP).13 Compound 5 would undergo nucleophilic annulation to generate a key intermediate i, which was then transformed to compound 1 by involving two steps of chemical reactions. The protonated ottensinin (ii) would undergo Wagner−Meerwein 1,2-alkyl shift14 to form a key intermediate iii, which further underwent oxidation with decarboxylation procedures to produce compound 2 via the rearranged intermediate iv. Intermediate i was transformed to a key intermediate v via the concerted reactions of 1,2- and 1,3-alkyl shifts.14,15 The cleavage of cyclopropane ring of v would result in the formation of intermediate vi, which was further transformed to compound 3 by a sequence of oxidations. Compound 4 could be derived from 8,14-dihydro-8α,14αepoxypimaradiene,16,17 which would undergo Wagner−Meerwein 1,2-alkyl shift to form a key intermediate vii with a new carbon skeleton. The intermediate vii was then converted to intermediate viii, which was finally transformed to 4 via dehydration, cascade oxidations, and lactone formation. The inhibitory activities of compounds 1−5 against nuclear factor kappa B (NF-κB), a potential target for the regulation of the dysfunction of immunity and inflammation,18 were evaluated using Luciferase assay.19 Compounds 1−4 exhibited weak inhibition rates from 35% to 61% under a tested concentration of 20 μg/mL, and compound 5 showed a significant NF-κB inhibitory effect with an IC50 value of 7.99 ± 1.77 μΜ (Table S15).

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EXPERIMENTAL SECTION

General Experimental Procedures. Melting points were obtained with a SGW X-4 melting point apparatus and were uncorrected. Optical rotations were measured on an Autopol VI polarimeter. UV spectra were acquired on a Shimadzu UV-2550 spectrophotometer. CD spectra were collected on a JASCO J-810 spectrometer (0.8 mg/mL for 1, 0.5 mg/mL for 2, and 1 mg/mL for 3 and 4). IR spectra were determined on a Thermo IS5 infrared spectrometer with KBr disks. The NMR spectra were recorded on the Bruker AVANCE III 500 or Ascend 600 spectrometers with TMS as internal standard. The ESIMS and HRESIMS data were detected on a Waters-Micromass Q-TQF Ultima Global mass spectrometer. Semipreparative HPLC separation was conducted using a Waters 1525 binary pump system with a Waters 2489 UV detector and a YMC− Pack ODS-A column (250 × 10 mm, S-5 μm). Chiral HPLC analysis using the Lux Amylose-1 column (250 × 10 mm, S-5 μm) or the Lux Cellulose-1 column (250 × 10 mm, S-5 μm). Silica gel (300−400 mesh, Qingdao Maring Chemical Co. Ltd.), C18 reversed-phase (RP18) silica gel (20−45 μm, Fuji Silysia Chemical Ltd.), Sephadex LH20 gel (40−70 μm, Amersham BioSciences), and MCI gel (CHP20P, 75−150 μm, Mitsubishi Chemical Industries Ltd.) were used for column chromatography (CC). All solvents used for CC were of analytical grade (Shanghai Chemical Reagents Co. Ltd.), and solvents used for HPLC were of HPLC grade (J&K Scientific Ltd.). Plant Material. The roots of Amomum maximum were collected from Menglun town of Yunnan Province, China, in November 2015 and identified by You-Kai Xu, one of the authors. A voucher specimen (accession no. Amaxi-2015-YN-1Y) has been deposited in Shanghai Institute of Materia Medica, Chinese Academy of Sciences. Extraction and Isolation. Dried powder of the roots (5.0 kg) of A. maximum was extracted with 95% ethanol (RT) for three times to give a crude extract (300.0 g), which was then dissolved in water to give a suspension and partitioned with EtOAc. The EtOAc-soluble part (150.0 g) was fractionated on a MCI gel column (MeOH/H2O from 1:1 to 9:1) to give four fractions (F1−F4). Fraction F3 (10.0 g) was chromatographed over a silica gel column (petroleum ether/ acetone from 1:0 to 1:2) to obtain five subfractions (F3a−F3e). The 286

DOI: 10.1021/acs.joc.8b02665 J. Org. Chem. 2019, 84, 282−288

Article

The Journal of Organic Chemistry

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the TDDFT methodology at the B3LYP/6-311++G(2d,2p) level both in vacuum and PCM solvent model of MeOH. The ECD spectra were simulated by the overlapping Gaussian function (σ = 0.3 eV),22 in which velocity rotatory strengths of the first 21 exited states for simulation of ECD curve in vacuum and 15 exited states for simulation of ECD curve in MeOH were adopted, respectively. To get the conformationally averaged ECD spectra, the simulated spectra of the lowest energy conformers were averaged according to the Boltzmann distribution theory and their relative Gibbs free energy (ΔG). NF-κB Luciferase Assay. HEK293 with stable NF-κB expression cell line was used for the luciferase assay.19 First, cells were seeded into 96-well plates and incubated for 24 h. The cells were then treated with different concertrations of compounds, following by stimulation with 10 ng/mL TNF-α. After incubation for 6 h, the luciferase substrate was added to each well, and the released luciferin signal was detected using an EnVision microplate reader. IC50 values were derived from a nonlinear regression model (curvefit) based on a sigmoidal dose−response curve (variable slope) and computed using Graphpad Prism version 5.02 (Graphpad Software). PS-341 was used as the positive control.23

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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.joc.8b02665. The key 2D NMR correlations of 1 and 2; the experimental ECD spectra of 1−3; the chiral HPLC analysis of 3; the specific rotation calculation of 3; the NMR and ECD calculations of 4; the crystallographic data of 1 and 2; and the 1D and 2D NMR, MS and IR spectra of 1−5 (PDF) Crystal data for 1 in CIF format (CIF) Crystal data for 2 in CIF format (CIF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Jian-Min Yue: 0000-0002-4053-4870 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support from the National Natural Science Foundation (no. 21532007) of the P. R. China is highly acknowledged.

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REFERENCES

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DOI: 10.1021/acs.joc.8b02665 J. Org. Chem. 2019, 84, 282−288

Article

The Journal of Organic Chemistry (22) Stephens, P. J.; Harada, N. ECD Cotton Effect Approximated by the Gaussian Curve and Other Methods. Chirality 2010, 22, 229− 233. (23) Sunwoo, J. B.; Chen, Z.; Dong, G.; Yeh, N.; Bancroft, C. C.; Sausville, E.; Adams, J.; Elliott, P.; Waes, C. V. Novel Proteasome Inhibitor PS-341 Inhibits Activation of Nuclear Factor-κB, Cell Survival, Tumor Growth, and Angiogenesis in Squamous Cell Carcinoma. Clin. Cancer Res. 2001, 7, 1419−1428.

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DOI: 10.1021/acs.joc.8b02665 J. Org. Chem. 2019, 84, 282−288