Isolation and structure elucidation of pyridine alkaloids from the aerial parts of the Mongolian medicinal plant Caryopteris mongolica Bunge

The seven pyridine alkaloids 1–7, the flavonoid acacetin (8), and L-proline anhydride (9) have been isolated from the aerial parts of the Mongolian medicinal plant Caryopteris mongolica Bunge. The structures of the natural products 1–9 have been assigned by MS, as well as IR, 1D NMR (1H, 13C, DEPT), and 2D NMR (COSY, HSQC, HMBC, NOESY) spectroscopic methods. The compounds 2 and 4–7 represent new chemical structures. Acacetin (8) and L-proline anhydride (9) have been obtained from C. mongolica for the first time.

Extraction and isolation of compounds. The air-dried and powdered aerial parts (12.5 kg) of Caryopteris mongolica were extracted three times at room temperature with 95% aqueous ethanol. The ethanol was removed and the residue treated with 2.5% HCl (pH = 1-2). The aqueous acidic solution was extracted successively with n-hexane and chloroform. Then, the residual aqueous solution was adjusted to pH = 9-10 by addition of aqueous NH 4 OH (25%), extracted with CHCl 3 and dried over Na 2 SO 4 . Finally, CHCl 3 was evaporated under reduced pressure to give 12.02 g of crude total alkaloid extract (0.096%).

Results and discussion
After isolation from the aerial parts of Caryopteris mongolica Bunge, the structures of the compounds 1-9 ( Fig. 1) were elucidated by a combination of mass spectrometry and 2D NMR spectroscopy ( 1 H and 13 C NMR data, see: Tables 1, 2, 3). The assignment of the NMR is based on the 1D and 2D NMR spectra (Supplementary Information).  (Table 1): three signals for the aromatic pyridine protons, two broad singlets for the exocyclic methylene group, a doublet of doublets for CH-O, two signals for the two methylene protons at C-6, and a broad line at 3.2 ppm for the OH group. In the 13 C NMR spectrum, nine resonances appeared: five signals for the carbon atoms of the pyridine ring and four signals for the CH-O, methylene, and C=CH 2 moieties. Based on the HSQC and HMBC spectra, an unambiguous assignment of the NMR signals was achieved. Particularly, the long-range HMBC correlations of C-4a with H-1, H-3, H-5, and H-6, as well as of C-8 with H-1, H-4, H-5, H-6, and olefinic H-9 confirmed the bicyclic pyridine framework of 1. Compound 1 has been isolated previously from Caryopteris tangutica 13 . Based on the spectroscopic data which are in agreement with those reported in the literature 20, 21 , compound 1 was assigned as 7-methylene-6,7-dihydro-5H-cyclopenta[c]pyridin-5-ol.  (Table 1): three signals for the pyridine protons, a doublet of doublets for CH-O, two doublets of doublets for the two methylene protons at C-6, a singlet at 1.53 ppm for the isolated methyl group, and the signals characteristic for the ethoxy group. Based on all NMR data and supported by long-range HMBC correlations in analogy to those of 1, the framework was confirmed. The strong NOE correlation between the protons H-5 and H-9 (Me) indicated a cisarrangement for the hydroxy and the ethoxy group. The 2D NMR correlations supporting the stereochemical assignment for compound 2 are shown in the Supplementary Information (Figures S12-S17-1). Compound 2 has been identified as (5S*,7R*)-7-ethoxy-6,7-dihydro-7-methyl-5H-cyclopenta[c]pyridin-5-ol and represents a new compound obtained from C. mongolica for the first time. However, it cannot be excluded that compound 2 has been formed by electrophilic addition at 1 on treatment with ethanol/HCl during the isolation process.  Table 1). The 13 C NMR spectrum of 3 Table 1. 1 H and 13 C NMR data of the compounds 1-3 in CDCl 3 , (δ in ppm, J in Hz). www.nature.com/scientificreports/  www.nature.com/scientificreports/ displays a characteristic peak at 206.0 ppm for the C=O group, five carbon signals for the pyridine ring, one signal for the methylene group, and the signals for the CH-Me moiety at 31.5 and 21.2 ppm ( Table 1). The assignment of the signals was confirmed by the 2D NMR spectra. The NMR data of compound 3 are in agreement with those previously reported in the literature 14,15 . Thus, 3 has been assigned as 7-methyl-6,7-dihydro-5H-cyclopenta[c] pyridin-5-one (aucubinine B) (3). Compound 3 was obtained from C. mongolica for the first time.
The polycyclic pyridine alkaloids 4 and 5 were obtained as pure compounds and as a mixture ( Figure S51). They show the same ESI-MS-peak at m/z = 296.2 [M + H] + corresponding to the molecular formula C 18 H 17 NO 3 . The 1 H NMR and 13 C NMR signals of 4 and 5 are clearly different from each other ( Table 2). Their structures have been assigned based on the 2D NMR spectra.
A series of longe-range HMBC correlations has been used to confirm the connectivity of the structural moieties in compound 4 (Fig. 2). The signals of the pyridine protons showed interactions with the neighboring carbon atoms: the doublet of H-4 with the carbonyl C-5 signal at δ = 205.50 ppm and the singlet of H-1 with the quaternary C-11a at δ = 58.90 ppm. Moreover, cross peaks of H-5a and H-6 with C-5; of H-13 (Me) and H-6 with C-11a; of H-11 with C-11a, C-5a, C-6, C-7a, C-10, C-10a, and C-11b; of H-7, H-6, and H-9 with C-7a; and of H-7, H-9, and H-14 (Me) with C-10a suggest the polycyclic skeleton for compound 4 with a C-6-O-C-11 bridge. The stereochemical arrangement of cyclic moieties in 4 has been determined by NOESY measurements. A positive NOE correlation between the protons H-5a and H-13 of the methyl group confirms the cis-configuration of the annulated five-membered ring (Fig. 3). Based on the extensive NMR analysis, compound 4 was identified as (5aR*,6S*,10S*,11R*,11aR*)-10,11a-dimethyl-6,7,9,10,11,11a-hexahydro-5H-6,11-epoxycyclopenta [6,7] (Table 2): one singlet and two doublets for the pyridine protons, two olefinic protons, three methine groups, one methylene group, and two singlets for the two methyl groups. Due to the H-D exchange, the hydroxyl group gives no signal. The 13 C spectrum exhibits signals for 18 carbon atoms ( Table 2): five pyridine carbon atoms, four olefinic carbon atoms, two methyl groups, three methine groups, one methylene group, two quaternary carbon atoms, and one signal for a carbonyl group at δ = 204.76 ppm. The molecular structure of compound 5 has been secured by COSY, HSQC, HMBC, and NOESY measurements in order to confirm the stereochemistry (Fig. 3).
We also performed detailed NMR studies of the compounds 6 and 7. While compound 6 was isolated as a pure substance, compound 7 was obtained only as a mixture, either together with the compounds 6, 1, and 2, or together with the compounds 1 and 2 (Supplementary Information). All isolated fractions of either pure 6 or mixtures containing 7 showed the same ESI-MS peak at m/z = 293.2 ([M + H] + ) that corresponds to a molecular formula of C 18 H 16 N 2 O 2 . However, we obtained two different sets for the 1 H NMR and 13 C NMR signals of both compounds (Table 3). Thus, we were able to assign the structure for 6 and for the by-product 7 which has the same molecular formula but a different constitution (see below).
Compound 7 is a colorless solid and was obtained as a small by-product during the extraction of compound 6. The ESI-MS (m/z = 293.2 [M + H] + ) corresponds to the molecular formula C 18 H 16 N 2 O 2 . The 1 H NMR spectrum of 7 exhibited 11 signals for 16 protons (Table 3): four doublets and two singlets for the six pyridine protons, one doublet for the methine proton, two doublets of methylene bridge, and two singlets for the two methyl groups. The 13 C NMR spectrum showed signals for 18 carbon atoms (Table 3): ten carbon atoms of the two pyridine rings, one carbonyl group (202.52 ppm), one methine group (64.12 ppm), three quaternary carbon atoms, one methylene group (57.36 ppm), and two methyl groups.
Compound 8 (23.6 mg) was obtained as colorless needles. The ESI-MS (m/z = 285.1 [M + H] + ) corresponds to the molecular formula C 16 H 12 O 5 . The 1 H NMR spectrum shows signals for 10 protons including seven aromatic protons and a methoxy group at δ 3.88 ppm. The 13 C NMR spectrum exhibits signals for 16 carbon atoms: seven aromatic methine (CH) groups, six aromatic carbon atoms, one olefinic carbon atom, a methoxy group at 55.94 ppm, and a carbonyl group at 181.20 ppm. The assignment was additionally supported by 2D NMR spectroscopy. Thus, the structure of compound 8 was confirmed as 5,7-dihydroxy-4'-methoxyflavone (acacetin) 8,13,16,17 . This natural product was isolated for the first time from the aerial parts of C. mongolica. 1  The 1 H NMR spectrum displays six proton signals for six methylene groups and two methine protons. The 13 C NMR spectrum exhibits five signals for six methylene groups, two methine groups and two carbonyl groups. Therefore, compound 9 has been assigned as octahydro-5H,10H-dipyrrolo[1,2-a:1' ,2'-d]pyrazine-5,10-dione (L-proline anhydride). Compound 9 was isolated for the first time from the aerial parts of C. mongolica. 1
Most likely, the cyclopenta[c]pyridine framework of the compounds 1-3 derives from an 11-nor-iridoid compound by condensation with ammonia 14,15 . The novel compounds 4-7 would be generated by subsequent condensations of the cyclopenta[c]pyridine. Compounds 4 and 5 could be formed by fusion of the cyclopenta[c]pyridine with an 11-nor-iridoid, while compounds 6 and 7 would be generated by dimerization of two cyclopenta[c] pyridines. However, a formation of the cyclopenta[c]pyridine skeleton on treatment with ammonia during the extraction process cannot be ruled out completely. Compound 2 might have been formed by electrophilic addition of ethanol at compound 1 during the extraction. According to database search (SciFinder and Reaxys), the compounds 2, 4, 5, 6, and 7 (Fig. 6) represent new chemical structures.