Nepenthe-Like Indole Alkaloids with Antimicrobial ... - ACS Publications

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Letter Cite This: Org. Lett. 2018, 20, 4116−4120

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Nepenthe-Like Indole Alkaloids with Antimicrobial Activity from Ervatamia chinensis Hao-Fei Yu,†,‡,#,△ Xu-Jie Qin,†,△ Cai-Feng Ding,†,# Xin Wei,†,# Jing Yang,† Jie-Rong Luo,§ Lu Liu,†,∥ Afsar Khan,†,⊥ Lan-Chun Zhang,‡ Cheng-Feng Xia,¶ and Xiao-Dong Luo*,†,¶

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†

State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, P. R. China ‡ School of Pharmaceutical Sciences, Department of Zoology & Yunnan Key Laboratory of Pharmacology for Natural Products, Kunming Medical University, Kunming 650500, P. R. China § School of Mathematical Sciences, Zhejiang University, Hangzhou 310027, P. R. China ∥ Yunnan University of Traditional Chinese Medicine, Kunming 650500, P. R. China ⊥ Department of Chemistry, COMSATS Institute of Information Technology, Abbottabad 22060, Pakistan # University of the Chinese Academy of Sciences, Beijing 100049, P. R. China ¶ Key Laboratory of Medicinal Chemistry for Natural Resource, Ministry of Education and Yunnan Province, School of Chemical Science and Technology, Yunnan University, Kunming 650091, P. R. China S Supporting Information *

ABSTRACT: Two monoterpenoid indole alkaloid erchinines A (1) and B (2), possessing unique 1,4-diazepine fused with oxazolidine architecture and three hemiaminals, were isolated from Ervatamia chinensis. Their structures were elucidated on the basis of intensive spectroscopic analysis, and a plausible biosynthetic pathway from ibogaine was proposed. Both compounds exhibited significant antimicrobial activity against Trichophyton rubrum and Bacillus subtilis, and their activities were comparable to the first line antifungal drug griseofulvin and antibiotic cefotaxime.

N

displayed significant activity against Trichophyton rubrum and Bacillus subtilis. Herein, we report the isolation, structure elucidation, and antimicrobial activity of these compounds.7 Erchinine A (1)8 was assigned the molecular formula C20H24N2O4 (Figure 1) from a prominent pseudomolecular ion peak at m/z 357.1806 [M + H]+ in HRESIMS and represented 10 degrees of unsaturation. The UV spectrum showed absorption maxima at 231, 258, and 423 nm, which is

atural products play an important role in drug development, especially for antimicrobial drug discovery.1 Because of the widespread resistance and numerous side effects of antibiotics,2 there is an urgent need for new and improved antimicrobials with plant-based derivatives. Among natural products, monoterpene indole alkaloids (MIAs), an important class of N-heterocyclic secondary metabolites, have attracted the attention of a great deal of synthetic chemists and pharmacologists, due to their complex frameworks and pronounced bioactivities.3 Previous pharmacological investigations of MIAs showed significant antibacterial activities,4 and our phytochemical research has reported a series of novel MIAs.5 Thus, the fascinating properties of this group of natural products stimulated us to search novel compounds for antimicrobial drugs development. The plant Ervatamia chinensis (Apocynaceae), rich in MIAs, has been used to treat carbuncle abscess in folk medicine.6 Based on the chemical profile and traditional uses of E. chinensis, we undertook phytochemical investigation on roots of the plant. As a result, two indole alkaloids, characterized as a fused heterocyclic architecture with three unique hemiaminals (C-2, 3, and 6), were isolated. Interestingly, the two compounds © 2018 American Chemical Society

Figure 1. Structures of erchinines A (1) and B (2). Received: May 28, 2018 Published: June 21, 2018 4116

DOI: 10.1021/acs.orglett.8b01675 Org. Lett. 2018, 20, 4116−4120

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Organic Letters Table 1. 1H and 13C NMR Spectral Data of 1 and 2 (Acetone-d6, J in Hz) 1

2

no.

δC a

δHb (J, Hz)

δCc

δHd (J, Hz)

2 3 5a 5b 6 7 8 9 10 11 12 13 14 15a 15b 16 17a 17b 18 19a 19b 20 21 10-OCH3 2-OH 2-OCH3

91.7 95.5 54.2

4.77, d (4.2) 3.14, dd (12.6, 5.4) 3.90, d (12.6) 5.79, d (5.4) 6.95, d (2.6) 7.23, dd (8.9, 2.6) 7.18, d (8.9) 1.75, m 1.87, ddd (13.8, 9.8, 4.6) 1.04, overlap 2.19, dt (11.6, 2.4) 1.09, t (11.6) 1.04, overlap 0.93, t (7.3) 1.59, m 1.45, m 1.48, m 3.31, br s 3.79, s 5.23, s

96.8 96.1 54.6

40.2 55.1 56.6

4.77, d (3.8) 3.14, dd (12.7, 5.4) 3.65, d (12.7) 5.86, d (5.4) 6.97, s 7.28, overlap 7.28, overlap 1.74, m 1.86, m 1.02, overlap 2.13, dt (11.7, 2.5) 1.08, t (11.7) 1.02, overlap 0.91, t (7.3) 1.55, m 1.44, overlap 1.44, overlap 3.18, br s 3.81, s

51.1

3.02, s

80.9 201.0 120.7 105.5 154.0 127.6 112.4 156.0 30.9 30.4 42.8 19.6 11.9 28.2 39.7 54.4 55.9 -

81.5 201.0 122.7 105.3 154.9 128.7 113.7 157.8 31.3 31.0 43.6 20.0 12.5 28.8

a

800 MHz. b200 MHz. c600 MHz. d150 MHz.

consistent with the absence of a characteristic indolic −NH signal in the 1H NMR spectrum of 1. In its 1H−1H COSY spectrum, cross signals of δH 5.79 (H-6) with 3.14 (Ha-5) and δH 2.19 (H-16) with 3.31 (H-21) indicated two fragments of C-6/5 and C-16/21 and then established the linkage of C5−C6−N1− C2−C16−C21. Signals at δC 54.4 (C-21) and 54.2 (C-5) were assigned to carbons linked to nitrogen (N-4), which were supported by the HMBC correlations of δH 3.14, 3.90 (2H, H-5) with δC 54.4 (C-21). Then, ring C with a 1,4-diazepane partial structure was constructed. Furthermore, correlations of δH 3.90 (Hb-5) and 3.31 (H-21) with δC 95.5 (C-3) in its HMBC spectrum assigned another carbon attached to N4. Meanwhile, the HMBC correlation of δH 5.79 (H-6) with δC 95.5 (C-3), together with the downfield chemical shift of C-3, and a remaining oxygen atom in the molecule suggested an ether linkage between C-3 and C-6 to form a 1,3-oxazolidine ring D in the structure.11 Notably, the fused rings C and D sharing C-5, 6, and N4 built the framework of 7-oxa-1,5-diazabicyclo[4.2.1]nonane with unique one hemiaminal (C-2) and two hemiaminal ethers (C-3 and 6). In the 1H−1H COSY spectrum, the correlations of δH 4.77 (H-3)/1.75/1.09 and 1.04/2.19/3.31 assigned the fragment N4−C3−C14−C17−C16−C21 (Figure 2) to form a piperidine ring (E). On the other hand, the correlations of δH 4.77 (H-3)/1.75/ 1.04 and 1.87/1.48 assigned the fragment of N4−C3−C14−C15− C20, combined with the HMBC correlations of δH 1.48 (H-20) and 1.87 (H-15a) to δC 54.4 (C-21) (Figure 2), established another fused piperidine ring (F). In addition, the 1H−1H COSY correlations of δH 1.48 (H-20)/1.45 and 1.59/0.93 (3H) placed the ethyl group at C-20 (Figure 2). Therefore, the planar structure of 1 was assigned with a 6/5/7/5/6/6 ring system,

somewhat reminiscent of alkaloids possessing a pseudoindoxyl chromophore. 9 The IR spectrum displayed absorptions attributable to hydroxyl (3430 cm−1) and carbonyl (1705 cm−1) groups and a benzene ring (1630 and 1493 cm−1). The 1H, 13C NMR, and DEPT spectra of 1 (Table 1) displaying signals at δC 201.0 (s, C-7), 156.0 (s, C-13), 154.0 (s, C-10), 127.6 (d, C-11), 120.7 (s, C-8), 112.4 (d, C-12), 105.5 (d, C-9), 91.7 (s, C-2), 55.9 (q, 10-OCH3) and δH 7.23 (1H, dd, J = 8.9, 2.6 Hz, H-11), 7.18 (1H, d, J = 8.9 Hz, H-12), 6.95 (1H, d, J = 2.6 Hz, H-9), 3.79 (s, 10-OCH3) represented a 1,2,5trisubstituted indolin-3-one unit.10 Besides the 5-methoxyoxygenindole moiety constituting rings A/B, compound 1 possessed six methine carbons at δC 95.5, 80.9, 54.4, 42.8, 39.7, and 30.9, four methylenes at δC 54.2, 30.4, 28.2, and 19.6, and one methyl at δC 11.9. In the HMBC spectrum of 1 (Figure 2),

Figure 2. Key 2D NMR correlations of 1.

correlations of δH 5.23 (−OH) and 2.19 (1H, dt, H-16) with δC 91.7 (hemiaminal carbon) supported an indole carbon (C-2) unit,10 positioned the hydroxyl, and assigned a methine (C-16) adjacent to C-2. Similarly, correlations of δH 5.79 (H-6) with δC 91.7 (C-2) and 156.0 (C-13) assigned the tertiary carbon (C-6) connected to N1 of the indole ring (Figure 2), which was 4117

DOI: 10.1021/acs.orglett.8b01675 Org. Lett. 2018, 20, 4116−4120

Letter

Organic Letters

erchinine A were elucidated as 2R*,3S*,6S*,14R*,16R*,20S*,21S*. To further determine the absolute configuration of 1, the electronic circular dichroism (ECD) curves for the possible stereostructure were calculated using the time-dependent density functional theory (TD-DFT) at the B3LYP/6-31G (d, p) level. The final calculated ECD spectrum was obtained by Boltzmann averaging of the ECD spectra of the conformers.13 The experimental ECD spectrum of 1 displayed a positive Cotton effect at 244 and 342 nm and negative Cotton effects at 270 and 424 nm. The calculated ECD spectrum of 2R,3S,6S,14R,16R,20S,21S was consistent with the experimental ECD spectrum (Figure 4). Thus, the absolute configuration of 1 was established.

incorporating a 7-oxa-1,5-diazabicyclo[4.2.1]nonane scaffold within the complex fused polyheterocyclic skeleton (Figure 2). The relative configuration of 1 was established on the basis of the ROESY spectrum. In the molecular model, NOE correlation of δH 7.18 (H-12)/5.79 (H-6) required H-6 at the plane of the indole ring, which caused a deshielding effect on H-6 and shielding effect on C-6. NOE correlations of δH 5.79 (H-6)/4.77 (H-3) and 4.77 (H-3)/3.14 (Ha-5) placed those protons at the same side of the oxazolidine ring D, and then Hb-5 was at the opposite side (Figure 3), which was consistent with the coupling

Figure 3. Key NOE correlations of 1.

constants of Ha-5 (dd, J = 12.6, 5.4 Hz) and Hb-5 (d, J = 12.6 Hz). Furthermore, NOE correlations of δH 3.90 (Hb-5)/3.31 (H-21)/5.23 (−OH) and 3.31 (H-21)/2.19 (H-16), placed them at the upper side of ring C. The rigid structure of 1 needed the boat/boat conformation for the fused rings E/F and allowed H-14 to be contradicted with H-21 in the ring system, which was further supported by the NOE correlation of δH 1.75 (H-14)/ 4.77 (H-3). The correlations of δH 2.19 (H-16)/1.48 (H-20) suggested that both protons were cofacial, while the ethyl group (C-18/19) was at the other side. Furthermore, due to the cooccurrence of 1 and ibogaine from the same plant and biogenetic relationship of them (Scheme 1), the absolute configuration at C-14, 16, 20, and 21 in 1 should be kept consistence with ibogaine.11,12 Then, the configurations of chiral carbons in

Figure 4. Experimental ECD spectra of 1, 2, and calculated ECD for 1.

The molecular formula of compound 214 was determined as C21H26N2O4 by HRESIMS (m/z 371.1965 [M + H]+), which was 14 mass units more than that of 1. The 1H and 13C NMR spectral data (Table 1) of 2 were similar to those of 1, except for the replacement of the hydroxyl at C-2 in 1 by a methoxyl in 2, which was further supported by the correlation of δH 3.02 (−OCH3) with δC 96.8 (C-2) in the HMBC spectrum of 2. Just like compound 1, the 2-OCH3 was assigned to be β-orientation of the indole ring supported by NOE correlations of δH 3.02 (2OCH3)/3.65 (Hb-5)/3.18 (H-21)/2.13 (H-16) in its ROESY spectrum. Other parts and configuration of 2 were identical to those of 1 by detailed analysis of 1D and 2D NMR spectra of 1 and 2. Moreover, the same absolute configuration was proposed for 2 by the well matched ECD Cotton effects of 2 and 1 (Figure 4). The biosynthetic pathway of 1 and 2 could be traced back to ibogaine, a major constituent of Ervatamia plants (Scheme 1). First, oxidation of a C-2/7 double bond and C-6 of the precursor might yield oxo-ibogaine derivative A, which could further undergo an oxidative cleavage of C6−C7 to afford the key aldehyde intermediate B. After the formation of a hemiaminal ether between N1 and C-6 (intermediate C), the N4 might be converted to iminium followed with dehydrogenation (intermediate D). Then, another hemiaminal ether was formed between the hydroxyl and iminium to afford erchinine A (1), and 2 could be produced by 2-OH methylation of 1. Compounds 1 and 2 were evaluated for their antimicrobial activities against four bacterial strains and one fungal strain following the protocols described previously.15 Both the compounds exhibited a potent antibacterial activity against B.

Scheme 1. Proposed Biosynthetic Pathway to 1 and 2

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DOI: 10.1021/acs.orglett.8b01675 Org. Lett. 2018, 20, 4116−4120

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Organic Letters Table 2. Minimum Inhibitory Concentrations (MIC, μg/mL) of Compounds 1 and 2 against Bacteria and Fungusa samples

1

2

berberine

fibraurtine

control

Bacillus subtilis Salmonella typhi Escherichia coli Staphyloccus aureus Trichophyton rubrum

0.78 25 50 >100 12.5

0.78 12.5 6.25 100 6.25

12.5 3.12 25 6.25 12.5

25 3.12 25 25 25

0.39 0.39 0.39 0.39 6.25

a

Control: cefotaxime for bacteria and griseofulvin for T. rubrum.

subtilis, which was roughly comparable to the first line antibiotic cefotaxime and much better than the plant-derived antibacterial drugs, berberine and fibraurtine (Table 2). Additionally, compound 2 showed equal bioactivity to antifungal drug griseofulvin against T. rubrum. It is noteworthy that the infection induced by the most spread dermatophyte T. rubrum was difficult to cure, which indicates that commonly generating reinfection by other microbials might become life threatening.16 Therefore, the discovery of potent natural compounds might be attractive to chemists and biologists, which could be leads for controlling B. subtilis or/and T. rubrum infections after further chemical and pharmacological evaluation.

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P. K.; Yang, X. W.; Song, C. W.; Cheng, G. G.; Liu, L.; Chen, Y. Y.; Liu, Y. P.; Luo, X. D. Tetrahedron 2015, 71, 4372−4378. (5) (a) Ding, C. F.; Ma, H. X.; Yang, J.; Qin, X. J.; Njateng, G. S. S.; Yu, H. F.; Wei, X.; Liu, Y. P.; Huang, W. Y.; Yang, Z. F.; Wang, X. H.; Luo, X. D. Org. Lett. 2018, 20, 2702−2706. (b) Pan, Z. Q.; Qin, X. J.; Liu, Y. P.; Wu, T.; Luo, X. D.; Xia, C. F. Org. Lett. 2016, 18, 654−657. (c) Bao, M. F.; Yan, J. M.; Cheng, G. G.; Li, X. Y.; Liu, Y. P.; Li, Y.; Cai, X. H.; Luo, X. D. J. Nat. Prod. 2013, 76, 1406−1412. (d) Yang, X. W.; Yang, C. P.; Jiang, L. P.; Qin, X. J.; Liu, Y. P.; Shen, Q. S.; Chen, Y. B.; Luo, X. D. Org. Lett. 2014, 16, 5808−5811. (e) Li, X. N.; Zhang, Y.; Cai, X. H.; Feng, T.; Liu, Y. P.; Li, Y.; Ren, J.; Zhu, H. J.; Luo, X. D. Org. Lett. 2011, 13, 5896−5899. (f) Cai, X. H.; Bao, M. F.; Zhang, Y.; Zeng, C. X.; Liu, Y. P.; Luo, X. D. Org. Lett. 2011, 13, 3568−3571. (g) Cai, X. H.; Du, Z. Z.; Luo, X. D. Org. Lett. 2007, 9, 1817−1820. (h) Cai, X. H.; Tan, Q. G.; Liu, Y. P.; Feng, T.; Du, Z. Z.; Li, W. Q.; Luo, X. D. Org. Lett. 2008, 10, 577−580. (i) Wei, X.; Jiang, L. P.; Guo, Y.; Khan, A.; Liu, Y. P.; Yu, H. F.; Wang, B.; Ding, C. F.; Zhu, P. F.; Chen, Y. Y.; Zhao, Y. L.; Chen, Y. B.; Wang, Y. F.; Luo, X. D. Nat. Prod. Bioprospect. 2017, 7, 413−419. (6) (a) Levi, M. S.; Borne, R. F. Curr. Med. Chem. 2002, 9, 1807−1818. (b) Luo, X. G.; Zhao, S. J.; Wang, J.; Chen, H. S. Zhongyaocai 2002, 25, 756−758. (7) Air-dried roots of E. chinensis (30 kg) were extracted with 90% aqueous MeOH under reflux, and then the solvent was concentrated under reduced pressure. The residue was partitioned between EtOAc and a 0.5% HCl solution. The aqueous acidic layer was adjusted to pH 9−10 with 10% ammonia and partitioned with EtOAc to obtain the alkaloid-rich mixture. The total-alkaloid extract (42 g) was divided into give 10 fractions (Fr-1 to Fr-10). The Fr-2 (1.5 g) was chromatographed on a reversed-phase C-18 column eluting with MeOH/H2O (30:70 → 100:0) and further purified by Sephadex LH-20 and semipreparative HPLC to afford erchinines A (1.0 mg) and B (4.0 mg). (8) Erchinine A, amorphous powder, [α]25 D (−92, 0.04, MeOH); UV (MeOH) λmax (log ε) nm 196 (3.96), 231 (4.24), 258 (3.76), and 423 (3.34); IR (KBr) νmax 3430, 2926, 1705, 1630, 1493, 1048 cm−1; 1H and 13 C NMR spectroscopic data, see Table 1; ESIMS m/z 357 [M + H]+; HRESIMS m/z 357.1806 [M + H]+ (calcd for C20H25N2O4, 357.1814). (9) Cheng, G. G.; Li, D.; Hou, B.; Li, X. N.; Liu, L.; Chen, Y. Y.; Lunga, P. K.; Khan, A.; Liu, Y. P.; Zuo, Z. L.; Luo, X. D. J. Nat. Prod. 2016, 79, 2158−2166. (10) Nge, C. E.; Gan, C. Y.; Lim, K. H.; Ting, K. N.; Low, Y. Y.; Kam, T. S. Org. Lett. 2014, 16, 6330−6333. (11) Tang, B. Q.; Wang, W. J.; Huang, X. J.; Li, G. Q.; Wang, L.; Jiang, R. W.; Yang, T. T.; Shi, L.; Zhang, X. Q.; Ye, W. C. J. Nat. Prod. 2014, 77, 1839−1846. (12) Wenkert, E.; Cochran, D. W.; Gottlieb, H. E.; Hagaman, E. W.; Filho, R. B.; Abreu Matos, F. J.; Lacerda, M. M. M. I. Helv. Chim. Acta 1976, 59, 2437−2442. (13) Smith, S. G.; Goodman, J. M. J. Am. Chem. Soc. 2010, 132, 12946−12959. (14) Erchinine B, amorphous powder, [α]25 D (−42, 0.03, MeOH); UV (MeOH) λmax (log ε) nm 201 (3.68), 229 (3.66), and 426 (2.72); IR (KBr) νmax 2926, 1709, 1630, 1493, 1032 cm−1; 1H and 13C NMR spectroscopic data, see Table 1; ESIMS m/z 371 [M + H]+; HRESIMS m/z 371.1965 [M + H]+ (calcd for C21H27N2O4, 371.1971). (15) Farooq, U.; Khan, S.; Naz, S.; Khan, A.; Khan, A.; Ahmed, A.; Rauf, A.; Bukhari, S. M.; Khan, S. A.; Kamil, A.; Riaz, N.; Khan, A. R. Chin. J. Nat. Med. 2017, 15, 944−949.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.8b01675.

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Detailed experimental procedures and NMR data for compounds 1 and 2 (PDF)

AUTHOR INFORMATION

Corresponding Author

*Phone/fax: +86-871-65223177. E-mail: [email protected]. ORCID

Xiao-Dong Luo: 0000-0002-6768-5679 Author Contributions △

H.-F.Y. and X.-J.Q. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are grateful to the National Key Research and Development Program of China (2017YFC1704007), and the “Ten Thousand Plan”, a National High-Level Talents Special Support Plan for partial financial support.

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REFERENCES

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Organic Letters (16) Warrilow, A. G. S.; Parker, J. E.; Price, C. L.; Garvey, E. P.; Hoekstra, W. J.; Schotzinger, R. J.; Wiederhold, N. P.; Nes, W. D.; Kelly, D. E.; Kelly, S. L. Antimicrob. Agents Chemother. 2017, 61, e00333−17.

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