Prenylated sulfonyl amides from the leaves of Glycosmis pentaphylla and their potential anti-proliferative and anti-inflammatory activities

1 Introduction

The genus Glycosmis belongs to the Rutaceae family, and many secondary metabolites with pharmacological and biological activities, such as alkaloids, flavonoids, and phenolic glycosides, are isolated from various parts of the Glycosmis plant (Yasir et al., 2019). Sulfur-containing amides are a special type of alkaloids, mainly derived from plants such as Glycosmis, and fungi, which are classified into four types (Hofer and Greger, 2000), including methylthiopropionic acid amides, methylsulfinylpropenoic acid amides, methylsulfonylpropenoic acid amides (MSPAAs), and methylthiocarbonic acid amides (Sutton et al., 1994). The structural characteristics of MSPAAs have a prenyloxy or geranyloxy in the para position of the benzene ring of the phenethylamide moiety. The structural diversity mainly comes from the oxidation of the geranyloxy side chain to form oxidation products such as hydroxyl, aldehyde, and ketone derivatives, or intramolecular cyclization to form dihydrofuranone rings containing ether and ketone functional groups (Yang et al., 2015; Greger et al., 1994; Hofer et al., 1995; Hofer et al., 2000; Hofer et al., 1998; Vajrodaya et al., 1998). Owing to their unique structure and biological activity, MSPAAs have attracted the attention of synthetic organic chemists, and the total synthesis of methylgerambullone has been completed (Moon et al., 2010).

Glycosmis pentaphylla (Retz.) DC. is an evergreen shrub native to tropical and subtropical regions of China, India, and other countries, and is used as traditional medicine to treat a variety of chronic diseases including cough, fever, inflammation, bronchitis, rheumatism, diabetes, cancer, and more (Khandokar et al., 2021). Previously, our research group performed phytochemical studies of G. pentaphylla leaves and isolated 18 previously undescribed sulphur-containing amides. Methylgerambullin has the strongest inhibitory effect on the proliferation of HepG2 cells, and its inhibitory effect on NO production is about 37 times that of the positive drug dexamethasone. Moreover, the antitumor mechanism of methylgerambullin is based on the activation of mitochondrial and endoplasmic reticulum stress signaling pathways and inhibition of AKT and STAT3 pathways (Nian et al., 2020; Wu et al., 2021). Taken together, these findings prompted us to isolate more sulphur-containing amides from G. pentaphylla leaves and evaluate their biological activities. Thus, six previously undescribed sulphur-containing amides were separated from the leaves of G. pentaphylla (Fig. 1). In addition, all isolated compounds were evaluated for their anti-inflammatory and anti-proliferative properties.

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Fig. 1

Structures of compounds 1–6.

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2 Materials and methods

3 Results and discussion

All isolated compounds are obtained as colorless oils and belong to the sulphur-containing amides through HR-ESI-MS data, which are the characteristic components of this plant. Moreover, two sets of resonance signals are observed in the ¹H and ¹³C spectrum of compounds 1–5 (Tables 1-3), indicating that these compounds are composed of two inseparable conformers. A comparison of NMR data of 1–4 with those of methyldambullin indicates that compounds 1–4 contain (E)-3-(methylsulfonyl)propenoic acid moiety and N-methyltyramine part. The rotation of the C—N amide bond is restricted due to the presence of the N-methyl group, resulting in two conformers, (a) s-cis and (b) s-trans, in which the ratio of the two conformers a/b ranges from 50 % to 75 % by ¹H NMR spectrum analysis. To facilitate structural analysis, we describe the structural identification of compounds 1–5 based on NMR data of conformer (a) (Greger et al., 1994).

The molecular formula of 1 was deduced to be C₂₃H₃₅NO₆S through HR-ESI-MS (Fig. S9, Supplementary Information abbreviated SI) at m/z 476.20764 [M + Na]⁺ (calcd for C₂₃H₃₅NO₆SNa, 476.20773), which was composed of two conformers a and b in 2.5:1. In addition to the characteristic signals of (E)-3-(methylsulfonyl)propenoic acid moiety and N-methyltyramine part, compound 1 also contained 10 carbon signals, including three methyls [δ_C 28.6 (q), 25.2 (q), 24.4 (q)], two methylenes [δ_C 39.4 (t), 32.7 (t)], one oxygenated methylene [δ_C 66.2 (t)], one methine [δ_C 47.9 (d)], one oxygenated methine [δ_C 75.2 (d)], one sp³ quaternary carbon [δ_C 35.6 (s)], and one oxygenated sp³ tertiary carbon [δ_C 72.9 (s)]. Careful analysis of the 2D NMR (Fig. S4-S6, SI) yielded two structural fragments (A) –CH₂(4′′)CH₂(5′′)- and (B) –CH₂(1′′)(O)CH(2′′)CH(7′′)(O)-. HMBC correlations (Fig. 2) from CH₃-10′′ [δ_H 1.24 (s)] to δ_C 72.9 (s, C-3′′), 39.4 (t, C-4′′), and 47.9 (d, C-2′′), and from CH₃-8′′ [δ_H 0.97 (s)] and CH₃-9′′ [δ_H 1.00 (s)] to δ_C 75.2 (d, C-7′′), 35.6 (s, C-6′′), and 32.7 (t, C-5′′) suggested that two structural fragments A and B were connected to C-3′′ through C-4′′ and C-2′′, and C-6′′ through C-5′′ and C-7′′ respectively. Therefore, the remaining carbon signals could establish 2-oxymethylene-1, 4, 4-trimethylcyclohexane-1, 3-diol moiety. Moreover, HMBC correlations from CH₂-1′′ [δ_H 4.21] to δ_C 160.0 (s, C-6′) implied that this moiety was connected to C-6′. In ROESY spectrum (Fig. S7, SI), the correlation of H₃-10′′ with H₂-1′′ implied that 3′′–OH and H-2′′ were co-facial. The relative configuration of C-7′′ could not be determined due to the lack of useful ROESY correlations. Therefore, to further verify the relative configuration of C-7′′, two isomers (2′′S*, 3′′S*, 7′′R*)-1a and (2′′S*, 3′′S*, 7′′S*)-1b were subjected to NMR calculations with the DP4 + analysis. These results showed that (2′′S*, 3′′S*, 7′′R*)-1a (Figs. 4-5, Tables S3, S5, and S7-S8, Fig. 65, SI) was the most likely structure for 1 with a 100.00 % DP4 + probability based on all NMR data (Marcarino et al., 2020). This determined the relative configuration of 1. Compound 1 has a small optical rotation value and no cotton effect in the CD spectrum, indicating that they may be a racemic mixture, which was further resolved by chiral column [column: CHIRALPAK® IC 4.6 × 250 mm 5 µm; mobile phase: MeCN: H₂O (30:70); flow rate: 0.5 ml/min; detection: UV, 225 nm] to give a pair of enantiomer 1a and 1a′ at t_R 54.3 and 46.9 min respectively. By comparing the experimental ECD data with the calculated results, the absolute configurations of 1a/1a′ were assigned as (2′′S, 3′′S, 7′′R)-1a and (2′′R, 3′′R, 7′′S)- 1a′ (Fig. 6). Therefore, the structure of 1 was determined as shown in Fig. 1, and denoted as glycopentamide S.

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Fig. 2

Key HMBC and ¹H–¹H COSY correlations of compounds 1–6.

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Fig. 3

ROESY correlations for compounds 1, 2 and 6.

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Fig. 4

DP4 + analysis of two possible diastereomers (1a and 1b) for 1.

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Fig. 5

(A) Linear regression fitting of calculated ¹³C chemical shifts of two possible isomers of 1a and 1b with the experimental values (B) Deviation between calculated ¹³C chemical shifts of two possible isomers of 1a and 1b with the experimental values.

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Fig. 6

Calculated and experimental ECD spectra of 1, 2, 5 and 6.

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The molecular formula of 2 was deduced to be C₂₃H₃₃NO₅S through HR-ESI-MS (Fig.S21, SI) at m/z 458.19687 [M + Na]⁺ (calcd for C₂₃H₃₃NO₅SNa, 458.19716), which was composed of two conformers a and b in 2.3:1. Careful comparison of the NMR data of compounds 1 and 2 revealed that compound 2 was the absence of one oxygenated tertiary carbon and methylene, but had one more tri-substituted double bond. It was speculated that compound 2 might be the dehydration product of 1. However, through careful analysis of the 2D NMR (Fig. S17-S18, SI), two structural fragments (A) –CH(4′′)CH₂(5′′)CH(6′′)(O)- and (B) –CH₂(1′′)(O)CH(2′′)- were obtained which were completely different from the dehydration product of 1. In the same way, HMBC correlations from CH₃-10′′ [δ_H 1.73 (s)] to δ_C 135.2 (s, C-3′′), 121.4 (d, C-4′′), and 50.8 (d, C-2′′), and from CH₃-8′′ [δ_H 1.06 (s)] and CH₃-9′′ [δ_H 0.94 (s)] to δ_C 74.9 (d, C-6′′), 38.4 (s, C-7′′), and 50.8 (d, C-2′′) constructed 5- oxymethylene-4,6,6-trimethylcyclohex-3-en-1-ol moiety, which was connected to C-6′ by HMBC correlation from CH₂-1′′ [δ_H 4.26, 4.02] to δ_C 159.3 (s, C-6′). The relative configuration of 2 was determined by interpreting the ROESY spectrum (Fig. 3, Fig. S19, SI), where the correlation of H-2′′ with H-6′′ indicated that these protons were co-facial. NMR calculations further confirmed the relative configuration of 2 (Tables S4 and S6, SI). At the B3LYP/6–31 + G(d) level, the absolute configuration of 2 was determined by the ECD calculations using the TDDFT/ECD method. The calculated ECD curve of (2′′S, 6′′R)-2 was in good agreement with the experimental ECD spectrum (Fig. 6). Therefore, the absolute configuration of 2 was shown in Fig. 1, and denoted as glycopentamide T.

The molecular formula of 3 was deduced to be C₂₂H₃₃NO₆S through HR-ESI-MS (Fig. S31, SI) at m/z 462.19199 [M + Na]⁺ (calcd for C₂₂H₃₃NO₆SNa, 462.19208), which was composed of two conformers a and b in 2.6:1. Its ¹H and ¹³C NMR data (Fig. 23–25, SI) were similar to those of methyldambullin (Greger et al., 1994), except for the presence of a 1,1-dimethoxyethyl group at C-4′′ [δ_H 1.72 (2H, m), 4.34 (1H, m), 3.30 (6H, s); δ_C 32.0 (t), 105.9 (d), 2 × 53.6 (q)] in 3 instead of a prenyl group at C-4′′ in methyldambullin. Thus, compound 3 was the oxidation product of the double bond Δ^{6′′(7′′)} of methyldambullin, resulting in the loss of three carbon atoms. These result was further supported by HMBC correlations (Fig. 27, SI) from CH₃O– [δ_H 3.30(s)] to δ_C 105.9 (d, C-6′′) and from H₂-4′′ [δ_H 2.10] to δ_C 105.9 (d, C-6′′), and ¹H–¹H COSY of –CH(6′′)CH₂(5′′)CH₂(4′′)-. Moreover, the configuration of double bond Δ^{2′′(3′′)} was assigned as E based on ROESY correlation of Me-10′′/H₂-1′′. Thus, the structure of 3 was determined as shown in Fig. 1, and denoted as glycopentamide U.

The molecular formula of 4 was deduced to be C₂₄H₃₅NO₅S through HR-ESI-MS (Fig. S40, SI) at m/z 450.23120 [M + H]⁺ (calcd for C₂₄H₃₆NO₅S, 450.23087), which was composed of two conformers a and b in 2.6:1. By comparing with the NMR data (Fig. S32-34, SI) of methyldambuline and methylgerambullin, it was found that the substituent pattern of 4 on the benzene ring was different from that of methyldambuline and methylgerambullin (Greger et al., 1994). The para-substitution pattern of the benzene ring of methyldambullin and methylgerambullin was replaced by the 1,2,4-trisubstitution benzene ring of 4. HMBC correlations (Fig. S36, SI) from H-4′ [δ_H 6.76] and MeO [δ_H 3.83] to δ_C 151.3 (s, C-5′), from H-1′′ [δ_H 4.55] to δ_C 148.3 (s, C-6′), and ROESY correlations (Fig. S38, SI) from MeO to H-4′ confirmed the methoxyl and geranyloxy were attached to C-5′ and C-6′, respectively. Moreover, the configuration of double bond Δ^{2′′(3′′)} was established as E through ROESY correlation of Me-10′′ with H₂-1′′. Thus, the structure of 4 was determined as shown in Fig. 1, and denoted as glycopentamide V.

The molecular formula of 5 was deduced to be C₂₃H₃₅NO₅S through HR-ESI-MS (Fig. S48, SI) at m/z 460.21243 [M + Na]⁺ (calcd for C₂₃H₃₅NO₅SNa, 460.21282), which was composed of two conformers a and b in 1:1. The NMR data of compound 5 (Fig. S41-43, SI) were similar to those of glycopentamide B (Nian et al., 2020), except that glycopentamide B had a trans double bond Δ¹⁽²⁾ [δ_H 6.80 (d, J = 15.0 Hz), δ_C 133.3; δ_H 6.95 (d, J = 15.0 Hz), δ_C 139.8], while 5 had two methylene signals [δ_H 3.14 (t, J = 7.2 Hz), δ_C 51.4; δ_H 2.47 (t, J = 7.2 Hz), δ_C 27.0], indicating that 5 was the saturated product of the double bond Δ¹⁽²⁾ of glycopentamide B. This deduction was further confirmed by HMBC correlations (Fig. S45, SI) from H₂-2 [δ_H 2.47] and H₂-3 [δ_H 3.14] to δ_C 171.7 (s, C-1), and ¹H–¹H COSY of –CH₂(2)CH₂(3)-. The absolute configuration of 5 was assigned as 6′S based on the ECD calculations. Thus, the structure of 5 was determined as shown in Fig. 1, and denoted as glycopentamide W.

The molecular formula of 6 was deduced to be C₂₃H₃₃NO₅S through HR-ESI-MS (Fig. S58, SI) at m/z 458.19693 [M + Na]⁺ (calcd for C₂₃H₃₃NO₅SNa, 458.19716). Interestingly, a comparison of the NMR data of 1–5 with that of 6 (Fig. S50-52, SI) showed that 6 displayed only one set of NMR signals, indicating that 6 lacked the s-cis and s-trans conformers. A comparison of its NMR data with those of methyldambullin and methylgerambullin implied that they possessed para-geranyloxy phenyl group (Greger et al., 1994). Apart from the abovementioned signals, the remaining signals included two methyls [δ_C 43.1 (q), 35.1(q)], two oxygenated methines [δ_C 73.3 (d); 71.8 (d)], two methylenes [δ_C 55.0 (t); 56.3 (t)], and one carbonyl [δ_C 168.2 (s)]. Detailed analysis of ¹³C NMR data of compounds 1–6 suggested that the two methyls were connected to N atom and sulfonyl respectively. Two structural fragments (A) –CH₂(1′)CH(2′)(O)- and (B) –CH₂(3)CH(2)(O)- were obtained by careful analysis of the 2D NMR (Fig. S53-55, SI). HMBC correlations from –NCH₃ [δ_H 3.03 (s)] to δ_C 55.0 (t, C-1′) and 168.2 (s, C-1), and from H-2 [δ_H 4.83] to δ_C 71.8 (d, C-2′), 168.2 (s, C-1) and 56.3 (t, C-3) suggested that two structural fragments A and B were connected to N through C-1′ and C-1, and connected by ether bond through C-2 to C-2′. Therefore, 4-methyl-2-((methylsulfonyl)methyl) morpholin-3-one moiety could be established. HMBC correlations from H-2′ [δ_H 5.12] to δ_C 129.2 (d, C-4′, 8′) confirmed the morpholin-3-one moiety was connected to C-3′. The relative configuration of 6 was determined by interpreting the ROESY spectrum (Fig. S56, SI), where the correlation of H-2′ with H₂-3 indicated that these protons were co-facial and H-2′ and H-2 were assigned trans. The absolute configuration of 6 was determined by comparing the experimental and calculated ECD spectra. Consequently, the absolute configuration of 6 was defined as (2R, 2′S)-6. Therefore, the absolute configuration of 6 was shown in Fig. 1, and denoted as glycopentamide X.

The inhibitory effect of these compounds on HepG2 cell proliferation was determined by CCK-8 method, and their anti-inflammatory effects were assessed by inhibiting the activity of NO production stimulated by lipopolysaccharide in mouse macrophage RAW 264.7 cells (Table S9). As a result, only compound 4 significantly inhibited lipopolysaccharide-induced NO production in mouse macrophage RAW 264.7 cells with the IC₅₀ value of 0.55 µM (dexamethasone as a positive control, IC₅₀: 9.24 µM). The remaining compounds were inactive showing IC₅₀ values in excess of 20 μM. Moreover, compounds 3 and 4 exhibited different anti-proliferative activities against HepG-2 with IC₅₀ values ​​of 11.52 and 9.41 µM, respectively (cisplatin as a positive control, IC₅₀: 5.96 µM). The other compounds showed no obviously anti-proliferative activities with IC₅₀ values in excess of 20 μM. Based on previous research and our results (Nian et al., 2020), the geranyloxy group at C-6′ of these methylsulfonylpropenoic acid amides might be crucial for the anti-proliferative and anti-inflammatory activities.

4 Conclusions

Sulfur-containing amides are a special type of alkaloids, and mainly isolated from the genus Glycosmis. This compounds showed novel structure and exhibited diversity bioactivity, like antimicrobial, cytotoxic, antitrypanosomal, antiviral activities. In this paper, six previously undescribed sulfur-containing amides with structural diversities were isolated from the leaves of G. pentaphylla. It is noteworthy that 6 represents the first example of sulfur-containing amide possessing a morpholin-3-one moiety. Moreover, Compound 4 had the strongest proliferative activity on HepG2 cells and had a significant inhibitory effect on NO production. The relationship between structure and activity indicates that the geranyloxy group at at C-6′ plays an important role in the anti-inflammatory and anti-proliferative activities of these alkaloids. These findings indicate that among the active ingredients of G. pentaphylla that exert anti-inflammatory and anticancer activities, in addition to the flavonoids reported in the previous literature (Shoja et al., 2015; Shoja et al., 2016), sulfur-containing amides also play an important role. These findings could explain the traditional use of G. pentaphylla to treat liver cancer and inflammatory diseases and provide a scientific basis for traditional medicine knowledge.

CRediT authorship contribution statement

Wenli Xie: Investigation, Formal analysis, Writing – original draft. Hefeng Nian: Investigation, Formal analysis. Xueni Li: Formal analysis. Jing Xu: Formal analysis. Yu Chen: Formal analysis. Zhinan Mei: Supervision, Writing – review & editing. Guangzhong Yang: Supervision, Writing – review & editing.