Chemical constituents of Clausena lenis

Abstract Phytochemical examination of Clausena lenis Drake (Rutaceae), collected in Thailand, led to the isolation of seven coumarins, four furoquinolines, two amides, and one flavonoid glycoside. Four of these compounds, one coumarine derivative named as gravelliferone A (3), two furoquinoline derivatives (kokusagenin A (8) and B (9)) and one amide, clausenalansamide H (13), are reported for the first time. Compound 3 was isolated from the root bark, compound 8 from the stem bark and compounds 9 and 13 from the leaves. The molecular structures of all isolated compounds were established by means of NMR experiments combined with mass spectrometry. Preliminary tests of the lipophilic stem bark extract against various human pathogenic bacteria strains revealed promising effects against Staphylococcus aureus ATCC 43300. Graphical Abstract


Introduction
The small palaeotropically distributed genus Clausena (Aurantioideae, Rutaceae) consists of twenty-five woody species occurring in various forest types (Molino 1994). In Thailand, eight taxa occur (Molino 1994). Several species, such as C. excavata Burm. f., C. harmandiana (Pierre) Guillaumin and C. anisata (Willd.) Hook. f. ex Benth., are widely used by local people for ethnomedicinal and other purposes (Albaayit et al. 2016;Mukandiwa et al. 2016aMukandiwa et al. , 2016b. Phytochemical studies on Clausena species revealed the preponderance of prenylated/geranylated coumarins and carbazole alkaloids with interesting bioactivities (Ito et al. 2000;Sripisut et al. 2012;Cao et al. 2018;Yan et al. 2019). Generally, prenylation of specialised metabolites seems to be common within rutaceous species and might be of chemotaxonomic importance for the subfamily Aurantioideae (Lukaseder et al. 2009;Sakunpak et al. 2013;Ma et al. 2018a). The studied species Clausena lenis occurs in the border region between China, Thailand and Burma. Phytochemical data are only available from individuals occurring in China (He et al. 2003a(He et al. , 2006Liu et al. 2019;Yan et al. 2019) so far. Herein we report four hitherto undescribed compounds from C. lenis and screening results of antibacterial activities.
The presence of bioactive Cand O-prenylated pyrano-and furocoumarins is quite common within Clausena species (Ouyang et al. 2016;Ma et al. 2018b;Wongthet et al. 2018) and was also reported for C. lenis (Liu et al. 2018). In contrast, furoquinoline derivatives (8-11) seem to be an extraordinary feature of this species, since there has been only one report of such compounds within the genus Clausena (He et al. 2003b). Within Aurantioideae, furoquinoline derivatives are reported only for Aegle marmelos (Linn.) Correa (Mohammed et al. 2016). They are also known to occur in Vepris (Kouam et al. 2018) and Melicope species (Chen et al. 2003;Rasamison et al. 2016), which both belong to the subfamily Toddalioideae.

Structure elucidation of the new compounds
The molecular formula of 3 was determined as C 20 H 24 O 4 based both on the [M þ H] þ peak at m/z ¼ 329.1754 (calcd 329.1747 for C 20 H 25 O 4 ) and the [M þ Na] þ peak at m/ z ¼ 351.1575 (calcd 351.1567 for C 20 H 24 O 4 Na). The 1 H NMR spectra revealed three aromatic singlets at d H 7.52, 7.29 and 6.78 ppm lacking the typical doublet of ca J ¼ 10 Hz for H-3 and H-4 between 6.0-6.5 ppm (H-3) and 7.7-8.2 ppm (H-4) of coumarins (Szab o et al. 1985). This indicated (1) a substitution at one of these two positions and (2) the presence of a trisubstituted coumarin. Based on the aromatic protons being singlets only a 3,6,7-or 4,6,7-substitution pattern was possible. A NOESY crosspeak between the signals at d H 7.52 and 7.29 ppm eliminated the 4,6,7-type, and allowed the assignment of these resonances to H-4 and H-5, respectively. As substituents, one methoxyl (d H 3.89 ppm), one epoxyprenyl and one dimethylallyl group were identified. The epoxyprenyl sidechain is characterised by two methyl groups at d H 1.34 and 1.40 ppm which show HMBC crosspeaks to one quarternary oxygenated carbon at d C 58.9 and an oxymethine carbon at d C 63.4 ppm. These two carbons show additional long-range crosspeaks to the remaining CH 2 -protons at d H 2.77 & 2.98 ppm. The dimethylallyl group is characterised by its vinyl signals at d H /d C 6.17/145.6 ppm and d H /d C 5.08 & 5.09/112.1 ppm, a 6H-singlet at d H /d C 1.47/26.1 ppm and a quarternary carbon at d C 40.1 ppm. Analyses of NOESY and HMBC spectra resolved the subsitution pattern: in the NOESY spectra the methoxyl group showed a crosspeak to the singlet at d H 6.78 ppm (H-8), whereas the CH 2 -group of the epoxyprenyl moiety had a correlation to d H 7.29 ppm (H-5) and finally both methyl groups of the dimethylallyl sidechain gave crosspeaks to H-4. This pattern was also proved by relevant HMBC correlations, e.g., crosspeaks d H /d C 7.52/160.1 (H-4/C-2), d H /d C 7.52/128.4 (H-4/C-5) or d H /d C 2.77 & 2.98/ 128.5 (H-1 00 /C-5). Because no X-ray data were available the absolute stereochemistry of the epoxy substructure remains unclear. Therefore, the structure of compound 3 was elucidated as given in Figure 1 and denominated gravelliferone A. Significant HMBC and NOESY correlations of 3 are presented in Figure S1.
The molecular formula of 9 was determined as C  (Nunes et al. 2005). Additionally two aromatic singlet signals at d H 7.51 and 7.39 ppm were observed, suggesting the presence of a trisubstituted furoquinoline. This assumption was supported by the detection of two methoxyl groups (both d H 4.03 ppm) and one oxy-epoxyprenyl group, the latter characterised by two methyl groups, epoxycarbons at d C 58.3 and 61.4 ppm, and the oxymethylene signal at d H /d C 4.69 & 4.82/70.6 ppm. The substitution pattern was determined with the information extracted from 2 D NMR spectra, allowing complete signal assignment. The methoxyl groups showed NOESY crosspeaks to d H 7.51 ppm (H-5) and 7.39 ppm (H-8), locating them at positions C-6 and C-7, whereas H-3 (d H 6.96 ppm) gave a NOESY correlation to the OCH 2 -protons of the epoxyprenyl sidechain. Therefore, the structure of compound 9 was established as given in Figure 1 and denominated kokusagenin B.
Compound 8 had a molecular formula of C 18 H 19 NO 4 based on its HR-ESI-MS data at m/z ¼ 336.1223 [M þ Na] þ (calcd. 336.1206 for C 18 H 19 NO 4 Na) and showed 1 H NMR spectra similar to 9, with the only difference in the isoprenyl sidechain. The epoxide functionality was replaced by a double bond, characterised by the olefinic signal at d H /d C 5.64/119.3 ppm. NOESY correlations from the methoxyl groups to H-5 and H-8 and from the prenyl sidechain to H-3 revealed the same substitution pattern as for compound 9. Similar to 3, the absolute configuration of the oxirane remains unclear. Compound 8 was shown to be a new furoquinoline alkaloid as well, and was denominated kokusagenin A. The 1 H and 13 C NMR data of 8 and 9 are listed in Table S1.
Compound 13 was denominated as clausenalansamide H. This compound showed NMR spectra similar to those of clausenalansamide E (Shen et al. 2017) and gave also the same molecular formula of C 18 H 18 NO 2 Cl, calcd. from its [M þ Na] þ peak at m/ z ¼ 338.0917 (calcd 338.0918 for C 18 H 18 NO 2 ClNa). In the HMBC spectrum, a hydroxyl proton at d H 3.82 ppm showed a long-range correlation to the carbonyl carbon at d C 170.93 ppm, the oxymethin proton at d H 4.70 ppm gave a correlation to only a quarternary aromatic carbon at d C 137.92 ppm, whereas the chloromethin proton at d H 5.27 ppm had also long range crosspeaks to protonated aromatic carbons. These data confirmed 13 to be also a 3-hydroxy-4-chloro derivative, but compared to clausenalansamide E there are significant shift differences in the 1 H as well as in the 13 C spectra. So, the oxymethin resonances reported for clausenalansamide E are at d H /d C 4.86/ 72.6 ppm (for 13: 4.70/73.2 ppm) whereas those of the chloromethin are at d H /d C 5.04/ 62.5 ppm (for 13: 5.27/63.4 ppm). Therefore we assume that 13 is a diastereomer of clausenalansamide E with either R,Ror S,S-configuration. As we were also able to isolate the epoxide 12, it is hence likely that 13 was formed by addition of HCl, which is often present in traces in CHCl 3 used in the isolation process.

General experimental procedures
Technical details are provided in the supplementary material.

Plant material
The plant material was collected and identified by W. Aiyakool and N. Wongthet near Mueang Khong, Chiang Dao, Chiang Mai province, Thailand, in 2017. A voucher specimen (Aiyakool, W. No. 2017) was deposited at the Bangkok Forest Herbarium (BKF) in 10900 Bangkok, Thailand.

Extraction and isolation
A detailed description is provided in the supplementary material.

Conclusion
Thirteen lipophilic compounds, belonging to coumarins, furoquinolines, amides and one flavonoid glucoside, were isolated from root bark, stem bark and leaves of Clausena lenis. All these compounds were herein described for the first time for this plant species. Except the furoquinoline derivatives, the described compounds fit well to the array of compounds known from Clausena and related species from this subfamily. The antibacterial activity of the tested lipophilic extract from the stem bark suggests the presence of potential antibacterial agents but identification of these compounds was prevented by lack of plant material.