Fissisternoids A and B, two 2′,5′-quinodihydrochalcone-based meroterpenoid enantiomers with unusual carbon skeletons from Fissistigma bracteolatum

Abstract

Two pairs of meroterpenoid enantiomers, (±)-fissisternoids A (1) and B (2), along with their putative biogenetic precursor 3 were isolated from the branches and leaves of Fissistigma bracteolatum. Compound 1 represents an unprecedented meroterpenoid featuring a unique tricyclo [3,3,1,0^1′,5′] decane central framework, and compound 2 possesses a rare 6/6/5/4 tetracyclic carbon skeleton, both of which were derived from quinodihydrochalcone and monoterpenoid via a key [4 + 2] Diels–Alder cyclization and Prins reaction. Their structures and absolute configurations were established using spectroscopic data, X-ray diffraction, and electronic circular dichroism calculations. Compounds 1 and 2 exhibit anti-inflammatory activity (IC₅₀: 13.2 μM, 1; 9.8 μM, 2) via suppression of inflammatory cytokines IL-1β, IL-6, and iNOS.

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Introduction

The genus Fissistigma (Annonaceae) consists of more than 70 species, which are mainly distributed in tropical and subtropical regions in Asia.¹ Several species, such as F. oldhamii, F. polyanthum, and F. retusum, have been used as folk medicines for curing rheumatoid arthritis and inflammation-related diseases.² Phytochemical studies on Fissistigma were started in the 1980s, and flavonoids, alkaloids (aristolactam, phenanthrene, aporphine), sesquiterpenoids, and cyclopentenones have been reported from this genus.^3–5F. bracteolatum, an evergreen shrub, is grown mainly in Vietnam and southern China.⁶ As a traditional medicine, its root-bark is commonly used for the treatment of bone fracture, contusion, and for stopping bleeding.⁶ Previous work on F. bracteolatum has revealed the presence of a series of chalcone derivatives and several phenanthrene type alkaloids.^7,8 Nevertheless, four extraordinary meroterpenoids, fissistigmatins A–D, composed of a flavonoid and a sesquiterpene unit connected via a single C–C bond directly, were also isolated from F. bracteolatum.⁹

As a part of our exploration on metabolites with unique structures from traditional medicines,¹⁰ two novel meroterpenoids fissisternoids A and B (1 and 2) and a known dihydrochalcone (3) were isolated from F. bracteolatum (Fig. 1). Compound 1 possesses an unprecedented carbon skeleton with a rare tricyclo [3,3,1,0^1′,5′] decane core, formed by an uncommon oxidative quinodihydrochalcone and an acyclic monoterpenoid unit. The different coupling pattern of the same units formed compound 2 that features a specific 6/6/5/4 fused tetracyclic ring skeleton. These two isolates were deduced biosynthetically via a key [4 + 2] Diels–Alder cyclization and Prins reaction. To the best of our knowledge, the coupling modes of the two units to form complex ring systems such as 1 and 2 have never been reported. Herein, the isolation, structural elucidation, plausible biosynthetic pathway, and inflammatory activity of compounds 1 and 2 are described.

Fig. 1 Structures of compounds (+)-1/(−)-1 and (+)-2/(−)-2.

Results and discussion

Fissisternoid A (1) was obtained as a faint yellow crystal (CH₃OH). Its molecular formula was determined as C₂₇H₃₂O₆ by HR-ESI-MS (m/z 475.2081 [M + Na]⁺, calcd for C₂₇H₃₂O₆Na, 475.2091), indicating 12 degrees of unsaturation. The presence of absorption bonds for the hydroxyl (3401 cm⁻¹), carbonyl (1711, 1668 cm⁻¹), and benzene (1604, 1510 cm⁻¹) functionalities was observed in the IR spectrum. The ¹H NMR data of 1 showed typical signals for two methyl groups (δ_H 1.63, s, 3H; 1.80, d, J = 1.50, 3H), two methoxyl groups (δ_H 3.43, s, 3H; 4.18, s, 3H), a pair of exocyclic methylene protons (δ_H 4.80, 4.98, each, br s), an olefinic proton (δ_H 5.30, br s), and a mono-substituted benzene ring (δ_H 7.24, m, 2H; 7.16, 3H) (Table 1). The ¹H and ¹³C NMR data of 1, interpreted with the help of the HSQC spectrum, revealed the presence of 27 carbons, which were attributed to 4 methyls (including two methoxyls δ_C 60.6, 53.1), 5 methylenes (including one olefinic carbon δ_C 116.2), 8 methines (including six olefinic carbons δ_C 118.7, 128.4 × 2, 128.6 × 2, 125.9), and 10 quaternary carbons (including two carbonyls δ_C 193.8, 205.9; two oxygenated carbons δ_C 81.4, 81.7; six olefinic carbons δ_C 132.2, 149.2, 142.6, 135.8, 142.0) (Table 1). The above observations (including three double bonds, two carbonyls, and one benzene ring) accounted for nine out of the twelve unsaturations, suggesting 1 to be tricyclic.

Table 1 ¹H NMR (500 MHz) and ¹³C NMR (125 MHz) spectroscopic data of compounds 1 and 2 in CDCl₃

No.	1		2
	δ H (J in Hz)	δ C	δ H (J in Hz)	δ C
1	1.63, s	21.6	1.37, s	25.3
2		142.6		92.4
3	2.55, dd (12.0, 5.0)	46.7	2.33, m	53.0
4a	1.50, m	26.9	1.13, ddd (12.5, 11.5, 11.5)	30.4
4b	1.50, m		1.74, m
5	2.59, dd (4.0, 2.5)	41.7	1.99, t (9.7)	42.4
6		135.8		135.6
7	5.30, br s	118.7	5.28, d (6.5)	118.6
8a	2.01, m	28.8	2.33, m	29.7
8b	3.00, m		3.40, m
9a	4.80, brs	116.2	1.74, s	26.7
9b	4.98, brs
10	1.80, d (1.5)	21.6	1.48, s	20.4
1′		68.6		61.2
2′		193.8		88.6
3′		132.2		163.3
4′		149.2		132.6
5′		81.4		188.6
6′		81.7		78.1
7′		205.9		211.6
8′	2.79, m	45.0	2.83, m	42.6
9′a	2.80, m	30.9	2.93, m	30.4
9′b	2.91, m		3.04, m
10′		142.0		141.1
11′	7.24, m	128.4	7.29, d (7.0)	128.7
12′	7.16, m	128.6	7.18, t (7.0)	128.7
13′	7.16, m	125.9	7.22, t (7.0)	126.5
14′	7.16, m	128.6	7.18, t (7.0)	128.7
15′	7.24, m	128.4	7.29, d (7.0)	128.7
3′-OH	5.63, s		6.09, s
4′′-OCH3	4.18, s	60.6	3.74, s	60.1
6′′-OCH3	3.43, s	53.1	3.31, s	3.31

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In the HMBC spectrum, the correlations from CH₃-1/H-9 to C-2 and C-3, from H-3 to C-2, C-4, and C-9, from H-4 to C-2, C-5, and C-6, from H-5 to C-4 and C-6, from CH₃-10 to C-5 and C-7, and from H-8 to C-6 and C-7, together with the ¹H–¹H COSY correlations of H-3/H-4/H-5 and H-7/H-8 (Fig. 2) indicated the presence of unit I as an acyclic monoterpenoid moiety.^2,11 For the remaining part unit II, the HMBC correlations of methylenes H-8′ (δ_H 2.79, m, 2H) and H-9′ (δ_H 2.80, m; 2.91, m; 2H) with carbonyl C-7′ (δ_C 205.9) and aromatic carbon C-10′ (δ_C 142.0), and H-9′ with C-11′ (δ_C 128.4) and C-15′ (δ_C 128.4), assisted with the ¹H–¹H COSY correlation between H-8′ and H-9′ indicated the presence of the 3-phenylpropan-1-one moiety, which is a part of the framework of dihydrochalcone.¹² Dihydrochalcones have been widely reported from F. bracteolatum,⁷ thus, unit II was speculated as a dihydrochalcone. Apart from the acyclic monoterpenoid, two methoxyls, and 3-phenylpropan-1-one moiety, six extra carbons were observed in the ¹³C NMR spectrum. Thus, the remaining six carbon signals should be attributed to the other 6-membered aromatic ring (A) of dihydrochalcone, and the aromatic ring was highly oxidized with the chemical formula C₆O₅H₂, including two olefinic, one carbonyl, one quaternary and two oxygenated carbons. For ring A, the signal of a hydroxyl (3′-OH, δ_H 5.63, s) was observed, although 1 was dissolved in CDCl₃. The HMBC correlations from the hydroxyl 3′-OH to C-2′ (δ_C 193.8), C-3′ (δ_C 132.2), and C-4′ (δ_C 149.2) and from methoxyl (4′′-OCH₃) at δ_H 4.18 to C-4′ (Fig. 2) established a 3′-hydroxy-4′-methoxy-α,β unsaturated ketone moiety. The other methoxyl was attached to C-6′ (δ_C 81.7) according to the HMBC correlations of CH₃-6′′, H-7, and H-8 with C-6′ (Fig. 2), and the remaining chemical shift at δ_C 81.4 was of the oxygenated carbon C-5′ (δ_C 81.4) supported by the HMBC correlations of H-3and H-4 with C-5′. Thus, the fragment of ring A was deduced as shown in Fig. 2, and eventually, the dihydrochalcone for unit II was established.

Fig. 2 Key NMR correlations of 1.

The linking mode between units I and II was supported by HMBC correlations. The correlations of H-5 (δ_H 2.59, dd, J = 4.0, 2.5 Hz) with C-1′ and C-2′, H-4 (δ_H 1.5, m, 2H) with C-1′ and H-7 (δ_H 5.30, s) and H-8 (δ_H 2.01, 3.00, each, m, 2H) with C-6′ (Fig. 2) demonstrated the respective linkages of C-5–C-1′ and C-8–C-6′, to form the six membered ring B fused with ring A. In addition, the correlations of H-3 (δ_H 2.55, dd, J = 12.0, 5.0 Hz) with C-4′ and C-5′ and H-4 (δ_H 1.5, m, 2H) with C-5′ indicated the connection between C-3 and C-5′, to establish another six-membered ring C, which satisfied the remaining one degree of unsaturation for 1.

In the ROESY spectrum, the correlations of H-3 with H-8b (δ_H 3.00, m), H-7, and CH₃-10 and 5′-OH (Fig. 2) revealed that these protons were in a co-facial position of the cyclohexane ring C, and thus the C-6–CH-7–CH₂-8 moiety, 5′-OH and H-3 were randomly assigned to the α-configuration, while 6′′-OCH₃ and H-5 at both end carbons of the C-6–CH-7–CH₂-8 moiety were accordingly assigned to the β-orientation. Subsequently, the ROESY correlations of H-5/H-8′ (δ_H 2.79, m, 2H) indicated the same orientation of H-5 and 3-phenylpropan-1-one in ring B (Fig. 2). The ROESY correlations of H-3/5′-OH, H-3/H-9a, 5′-OH/H-9a, and CH₃-1/4′′-OCH₃ suggested that the 3′-hydroxy-4′-methoxy-α,β unsaturated ketone moiety was on the β-orientation of ring C. The β-orientation of 3-phenylpropan-1-one related to ring B and the β-orientation of the 3′-hydroxy-4′-methoxy-α,β unsaturated ketone moiety related to ring C established the configuration of C-1′ as shown in Fig. 2. Furthermore, X-ray crystallography data were used to determine its structure (Fig. 3, CDCC 2107685†). The X-ray diffraction analysis of 1 showed the space group P-1, indicating its racemic nature, as also evidenced by a small optical value ([α]²⁵_D +2.1). Compound 1 was separated via chiral HPLC to afford the optical enantiomers (+)-1 and (−)-1 (Fig. S1†). The stereochemistry of the enantiomers of 1 was further determined by comparison of their experimental ECD spectra with those predicted by time-dependent density functional theory (TDDFT) calculations at the B3LYP/6-31+G(d,p) level. As a result, the calculated ECD curves of 3R,5R,1′R,5′R,6′R-1 and 3S,5S,1′S,5′S,6′S-1 matched well with the experimental ones of (+)-1 and (−)-1, respectively (Fig. 4), and thus their absolute configurations were determined.

Fig. 3 X-ray ORTEP drawing of 1.

Fig. 4 Calculated and experimental ECD spectra of (+)-1 and (−)-1.

The molecular formula of fissisternoid B (2) was deduced to be C₂₇H₃₂O₆ based on its HR-ESI-MS at m/z 475.2085 [M + Na]⁺ (calcd for C₂₇H₃₂O₆Na, 475.2091), which also required 12 degrees of unsaturation. By comparison of the ¹H NMR data of 2 with those of 1, the notable differences are the disappeared signals of the exocyclic terminal double bond, which was replaced with a singlet methyl group (δ_H 1.74, s). Besides, an additional oxygenated carbon at δ_H 92.4 was also observed. These data implied the exocyclic methylene in 1 to be oxygenated. The proposal was further confirmed by the HMBC correlations of CH₃-1 (δ_H 1.37, s) and H-3 (δ_H 2.33, m) with C-2 (δ_C 92.4) and C-9 (δ_C 20.4), and H-4 with C-2 (Fig. 5).

Fig. 5 Key NMR correlations of 2.

Detailed analysis of 2D NMR data for 2 suggested that its structure should be also established from the identical units of monoterpenoid (I) and quinodihydrochalcone (II) to those of 1. The same connectivities between 1 and 2via C-5–C-1′ and C-8–C-6′ for ring B were established by the key HMBC correlations from H-5 and H-4 to C-1′ and H-8 and H-7 to C-6′ (Fig. 5). The HMBC correlations of H-3 with C-2′ and C-3′, H-4 with C-2′, OH-3′ (δ_H 6.09, s) with C-2′, and C-3′, and C-4′, indicated the linkage of C-3–C-2′ in 2 to form ring C, which was distinct from that of 1 (C-3–C-5′). Additionally, the HMBC correlation of H-8 with carbonyl C-5′ (δ_C 188.6) (Fig. 5) indicated the nearby side of the carbonyl with H-8, and thus it was also suggested that it was carbonyl C-2′, but not C-5′ that participated in the Prins reaction to establish ring C. The C-2–O–C-2′ connectivity for ring D was demonstrated by the remaining one degree of unsaturation and the oxygenated carbon in the downfield region at C-2 (δ_C 92.4). Thus, the linking mode of 2 between units I and II was changed as shown in Fig. 5.

In the ROESY spectrum, the observed cross-peaks of H-5 and H-3 with H-8′ (δ_H 2.83, m, 2H) and H-3 with OH-3′ indicated that the protons of H-3 and H-5, the 3-phenylpropan-1-one group, and the chain of C-3′–OH were co-faced and assigned to be the β-side of ring C (Fig. 5). Furthermore, the ROESY correlations of CH₃-9/CH₃-10, CH₃-9/H-4a, and CH₃-10/H-4a revealed that the fractions of C-6–CH-7–CH₂-8 (ring B) and C-2–O (ring D) were on the same face and on the other side of ring C, and thus 6′′-OCH₃ should be situated at the β-side of ring B. Eventually, the structure of 2 should be deduced as shown in Fig. 5.

The small optical rotation value clearly suggested that compound 2 was also a pair of enantiomers. The subsequent chiral HPLC resolution of 2 yielded the anticipated enantiomers, (+)-2 and (−)-2 (Fig. S1†), which were opposite in terms of their CD curves. The experimental ECD spectra of (+)-2 and (−)-2 were also mirror images and agreed well with the calculated ECD spectra (Fig. 6). Finally, the absolute configurations of (+)-fissisternoid B and (−)-fissisternoid B were assigned as 3S,5R,1′R,2′R,6′R and 3R,5S,1′S,2′S,6′S, respectively.

Fig. 6 Calculated and experimental ECD spectra of (+)-2 and (−)-2.

Compounds 1 and 2 represent a pair of unprecedented meroterpenoids with unusual 6/6/6 and 6/6/5/4 polycyclic core systems, respectively. The meroterpenoids were both formed from an acyclic monoterpenoid (unit I) and a 2′,5′-quinodihydrochalcone (unit II) moiety. The acyclic monoterpenoid moiety, (Z)-β-ocimene, as a diene for Diels–Alder cycloaddition, was identified from genus Fissistigma.² The highly oxygenated 2′,5′-quinodihydrochalcones have rarely been reported from the same genus,³ and its precursor of 2,6-dihydroxy-4-methoxydihydrochalcone¹³3 was isolated by us. Thus, the biosynthetic pathways of compounds 1 and 2 should be proposed as shown in Scheme 1. Initially, precursor 3 would undergo oxidation and methylation processes to generate the intermediate unit II. The [4 + 2] Diels–Alder reaction between II (Δ^1′(6′)) and (Z)-β-ocimene (Δ^{5(6), 7(8)}) established the connectivity of C-5–C-1′ and C-8–C-6′ for ring B of III-a and b, and thus 6′′-OCH₃ and the 3-phenylpropan-1-one moiety of both 1 and 2 should be at the same side according to the Diels–Alder reaction mode.¹⁴ Moreover, the Prins reaction of the remaining alkene (Δ²⁽³⁾) for (Z)-β-ocimene with the carbonyls of C-2′ and C-5′ for 2′,5′-quinodihydrochalcone establishes the connectivity of C-3–C-2′ (IV-c and d) and C-3–C-5′ (IV-a and b), respectively.¹⁵ On one hand, the 2′-hydroxyl group in intermediates IV-c and d attacks the carbocation C-2 to construct the oxetane moiety for the D ring to form compound 2. On the other hand, IV-a and b would undergo the dehydrogenation reaction to form the Δ²⁽⁹⁾-double bond for compound 1.

Scheme 1 Plausible biosynthetic pathways of 1 and 2.

The racemic mixtures 1 and 2 were evaluated for their inhibitory effects on NO production in LPS-activated RAW 264.7 cells. MTT results revealed that the cell viabilities were over 90% under the treatment of 1 and 2 at concentrations up to 30 μM (Table S5†). Furthermore, compounds 1 and 2 displayed NO production inhibitory activity with smaller IC₅₀ values of 13.2 and 9.8 μM, respectively, compared with that of the positive control N^G-monomethyl-L-arginine (L-NMMA, IC₅₀ 26.8 μM) (Table S5†). Since inflammatory cytokines such as IL-1β, TNF-α, IL-6, and iNOS play important roles in inflammation,¹⁶ we detected the release of these inflammatory cytokines in LPS-induced RAW264.7 cells through qRT-PCR analysis. Expectedly, compounds 1 and 2 dose-dependently (7.5, 15, 30 μM) inhibited the release levels of IL-1β, TNF-α, and IL-6 in mRNA expression, and exhibited a weak inhibitory effect on TNF-α compared with the LPS group (Fig. 7). These studies revealed that these enantiomers exhibited anti-inflammatory activity via the suppression of excessive inflammatory cytokines in LPS-activated RAW264.7 cells.

Fig. 7 Inhibitory effects of 1 and 2 on the LPS-induced release of pro-inflammatory cytokines (IL-1β, IL-6, iNOS, and TNF-α) by qRT-PCR analysis. ^###p < 0.001, compared with the control group. *p < 0.05, *p < 0.01, ***p < 0.001, compared with the LPS-treated group.

Conclusions

In conclusion, fissisternoids A (1) and B (2), the first two unprecedented examples with polycyclic carbon skeletons presumably biosynthesized from 2′,5′-quinodihydrochalcone and an acyclic monoterpenoid, along with their possible precursor 3, were isolated from the traditional Chinese medicine F. bracteolatum. In our search for natural products to be a potential lead as a therapeutic agent for inflammatory diseases,¹⁷ racemic mixtures 1 and 2 exhibited anti-inflammatory activity via inhibiting the release of IL-1β, IL-6 and iNOS at mRNA levels. The structural novelty and complexity together with their bioactivity might attract the interest of pharmacologists and synthetic chemists.

Experimental section

General experimental procedures

Optical rotations were measured using a JASCO P-1020 polarimeter in MeOH. UV spectra were recorded on a UV-2450 visible spectrophotometer (Shimadzu, Tokyo, Japan). IR spectra were acquired using a Nicolet iS10 microscope spectrometer in MeOH (Thermo Scientific, USA). All of the nuclear magnetic resonance (NMR) spectra were recorded using a Bruker AVIII-500 NMR instrument (¹H: 500 MHz, ¹³C: 125 MHz) (Bruker, Karlsruhe, Germany), and tetramethylsilane (TMS) was used as an internal standard. High-resolution electrospray ionization mass spectra (HRESIMS) were recorded using an AB Sciex Triple-TOF 6600 (AB SCIEX, Framingham, MA, USA). Preparative HPLC was carried out on an LC-120 system (Separation, Beijing, China) equipped with a YMC-pack RP-C₁₈ column (250 mm × 20 mm i.d., 5 μm, YMC, Tokyo, Japan) with a flow rate of 3 mL min⁻¹, detected using a binary channel UV detector at 210 and 310 nm. Analytical HPLC was performed with an Agilent 1260 Series instrument equipped with a DAD detector using a Shim-pack VP-ODS column (4.6 × 250 mm; Agilent Technologies, Palo Alto, CA, USA). MCI (Mitsubishi, Tokyo, Japan) and RP-C₁₈ silica (40–63 μm, Fuji, Tokyo, Japan) were used for column chromatography (CC). All solvents used were of analytical grade (TJshield Fine Chemicals Co., Ltd, Tianjin, China).

Plant material

The branches and leaves of F. bracteolatum were collected from the Yunnan province of China in May 2019. The plant material was identified by Prof. Cheng-Ming Dong (Henan University of Chinese Medicine). A voucher specimen (no. 201905) was deposited in the Department of Natural Product Chemistry, School of Pharmacy, Henan University of Chinese Medicine.

Extraction and isolation

The branches and leaves of F. bracteolatum (10.0 kg) were chopped and refluxed three times with 95% EtOH. The EtOH extract was concentrated under reduced pressure to obtain the extract (800 g), which then was dissolved in water (4.0 L) and successively partitioned with petroleum ether (PE, 4.0 L × 4), CH₂Cl₂ (4.0 L × 4), EtOAc (4.0 L × 4), and n-butanol to afford FrA–FrD.

The CH₂Cl₂ extract (FrB, 180 g) was subjected to D101 macroporous resin CC using EtOH/H₂O in step gradients (v/v 30%, 60%, 90% and 100%) as an eluent. The 60% MeOH fraction (50.2 g) was chromatographed by silica gel CC eluted with CH₂Cl₂–MeOH (100 : 0 → 0 : 100) to afford 7 fractions FrB1–FrB7. FrB1 (11.3 g) was chromatographed by MCI CC using a gradient elution of MeOH–H₂O (v/v 20, 40, 60, 80, 100%) to yield six subfractions (FrB1a–FrB1e). FrB1d (4.3 g) was subjected to Sephadex LH-20 eluted with MeOH to afford FrB1d1–FrB1d9. FrB1d2 (0.5 g) was further subjected to reversed-phase C₁₈ CC (40–63 μm, 40 mm × 250 mm) eluted with MeOH/H₂O (v/v, 50–100%) to afford FrB1d2a–FrB1d2l. FrB1d2g (60.2 mg) was separated by pre-HPLC (10 mm × 250 mm, 5 μm, YMC, Tokyo, Japan) using 45–100% MeCN/H₂O (v/v, 50 min) as the mobile phase to yield compound 1 (4.8 mg, t_R 38.8 min). FrB1d2l (43.2 mg) was subjected to pre-HPLC (v/v, 40–100% MeCN/H₂O) to yield compound 2 (10.2 mg, t_R 33.1 min).

Fissisternoids A (±1): faint yellow crystal (CH₃OH); [α]²⁵_D +2.1 (c 0.1, MeOH), UV (MeOH) λ_max (log ε) 204 (4.01), 298 (3.78) nm; IR (MeOH) ν_max 3401, 2926, 1711, 1668, 1604, 1509, 1379, 1251, 1102, 1034, 830 cm⁻¹. ¹H and ¹³C NMR data (Table 1); HRESIMS m/z 475.2081 [M + Na]⁺ (calcd for C₂₇H₃₂O₆Na, 475.2091).

(+)-1: [α]²⁵_D +68.5 (c 0.1, MeOH), ECD (MeOH) λ_max (Δε): 220.0 (+1.0), 254.0 (−0.2), 288.0 (+1.8), 322.0 (−3.2) nm; (−)-1: [α]²⁵_D −70.3 (c 0.1, MeOH), ECD (MeOH) λ_max (Δε): 218.0 (−1.3), 253.0 (+0.2), 287.0 (−3.1), 322.0 ( + 5.2) nm.

Fissisternoids B (±2): faint yellow oil; [α]²⁵_D +1.5 (c 0.1, MeOH), UV (MeOH) λ_max (log ε) 276 (3.21) nm; IR (MeOH) ν_max 1683, 1635, 1496, 1455, 1438, 1386, 1329, 1303, 1278, 1187, 1121, 1085, 1043, 930, 827, 768, 701; ¹H and ¹³C NMR data (Table 1); HRESIMS m/z 475.2085 [M + Na]⁺ (calcd for C₂₇H₃₂O₆Na, 475.2091).

(+)-2: [α]²⁵_D +45.0 (c 0.1, MeOH), ECD (MeOH) λ_max (Δε): 256 (+3.6), 290 (−12.4), 328.7 (+7.4) nm; (−)-2: [α]²⁵_D −46.5 (c 0.1, MeOH), ECD (MeOH) λ_max (Δε): 255 (−5.2), 290 (+17.4), 329 (−10.2) nm.

Quantum chemical ECD calculations

The relative configurations of 1 and 2 were established initially according to their ROESY NMR spectra. Their conformers were first identified using the Spartan software using the MMFF94S force field. Then, on the basis of the Boltzmann distribution, the selected conformers were further optimized at the B3LYP/6-31+G* level in MeOH, and TDDFT ECD calculations were performed using the Gaussian 09 program package at the B3LYP/6-31+G(d,p) level with a CPCM model. The ECD curves of different conformers were simulated using SpecDis and weighted according to the Boltzmann distribution after UV correction.

Measurement of NO release

The MTT assay was first used to evaluate RAW264.7 cell viability as previously described.¹⁷ Briefly, the cells were plated in 96-well plates (5 × 10³ cells per well) for 18 h and then incubated with compounds 1 and 2 at various concentrations with or without LPS (1.0 μg mL⁻¹). Eighteen hours later, the prepared MTT solution (20 μL, 5 mg mL⁻¹) was added, and the cells were incubated for another 4 h. After the formazan that formed was fully dissolved in DMSO (150 μL per well), the absorbance was read at 570 nm on a microplate reader. The viability of the RAW264.7 cells for the control group (with DMSO only) is defined as 100%.

NO production was determined by the level of accumulated nitrite in cell culture supernatants using Griess reagent from Beyotime Institute of Biotechnology (Jiangsu, China). Briefly, RAW264.7 cells (8 × 10⁵ cells per mL) were incubated in 96-well plates and pretreated with test samples for 1 h, followed by incubation with 1 μg mL⁻¹ LPS stimulation for 18 h. Then, the culture supernatant was mixed with an equal volume of the Griess reagent. The absorbance of the mixture was read at 540 nm using a microplate reader. Nitrite concentrations were calculated using sodium nitrate as a standard. N^G-Monomethyl-L-arginine (L-NMMA) was used as a positive control in the experiments.

Quantitative real-time PCR

The methods for the total RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR) have been described previously.¹⁸ Total RNA was extracted from spleens or cultured cells using TRIzol according to the manufacturer's instructions (Invitrogen Corp.). Subsequently, RNA was reverse transcribed into cDNA using HiScript II Reverse Transcriptase (Vazyme, Nanjing, China). Real-time quantitative PCR was performed using real-time reverse transcription (RT)-PCR (Applied Biosystems, CA, USA) based on general fluorescence detection by SYBR Green. For mRNA transcript analysis, gene-specific values were normalized to the Actb gene.

Statistical analysis

Statistical analysis of the data was performed using the Prism Demo Prism 4.0 software. Statistical analysis data were expressed as mean ± SD. Values were analyzed by one-way analysis of variance (ANOVA) using the SPSS version 12.0 software. P < 0.05 was considered statistically significant.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This research work was financially supported by the National Natural Science Foundation of China (82003606), the Scientific and Technological Key Project in Henan Province (212102311093), the Key Scientific Research Projects of Universities of Henan Province (21A360002), and the Scientific Research Nursery Project (MP2020-29).

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: X-ray crystallographic data of compound 1, and 1D NMR, 2D NMR, HRESIMS, UV, and IR spectra of 1 and 2. CCDC 2107685. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/d1qo01404h

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