Letter Cite This: Org. Lett. XXXX, XXX, XXX−XXX
pubs.acs.org/OrgLett
Antibacterial Indole Alkaloids with Complex Heterocycles from Voacanga af ricana Cai-Feng Ding,†,‡,⊥ Hong-Xia Ma,§,⊥ Jing Yang,†,⊥ Xu-Jie Qin,† Guy S. S. Njateng,∥ Hao-Fei Yu,†,‡ Xin Wei,†,‡ Ya-Ping Liu,† Wan-Yi Huang,§ Zi-Feng Yang,§ Xin-Hua Wang,*,§ and Xiao-Dong Luo*,†,§ †
State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, People’s Republic of China ‡ Graduate University of the Chinese Academy of Sciences, Beijing 100049, People’s Republic of China § State Key Laboratory of Respiratory Disease, Guangzhou Medical University, Guangzhou 510120, People’s Republic of China ∥ Department of Biochemistry, Faculty of Science, University of Dschang, P.O. Box 67, Dschang, Cameroon S Supporting Information *
ABSTRACT: Voacafricines A and B, two unique monoterpenoid indole alkaloids each bearing five fused heterocycles, were obtained from the fruits of Voacanga af ricana. Their structures were elucidated by extensive spectroscopic methods and computational studies. A plausible biogenetic pathway was proposed from a common precursor, 19-epi-voacristine. Both compounds exhibited potent activity against Staphylococcus aureus and Salmonella typhi, and their activities were superior to those of the well-known antibacterial drugs berberine and fibrauretine.
B
isolated. Notably, their complex heterocyclic architectures involve a fused pyrrole/pyridine/pyrrolidine/piperidine/furan ring system, which might be biogenetically derived from a 6/5/ 7/6/6 iboga-type pentacyclic precursor, 19-epi-voacristine (also isolated as a major component in this experiment). Herein, we report the structural elucidation, a plausible biogenetic pathway, as well as antibacterial activities of these compounds. Voacafricine A (1, Figure 1) was found to have a molecular formula of C21H24N2O4 based on its 13C NMR spectral data (Table 1) and positive HRESIMS data (m/z 391.1633 [M + Na]+ (calcd 391.1628), and this formula suggests 11 degrees of
ecause antibiotics such as penicillin, daptomycin, and cefixime have been the first line of treatment for bacterial infections for a long time, they are commonly used, which has resulted in unexpected antibiotic resistance and numerous side effects.1 These problems can hopefully be solved by the development of plant-based antibiotics as an effective means of infection resistance. In recent years, numerous antimicrobials derived from plants have been reported. Emetine,2 quinine,3 and berberine4 have been used as antibiotic potentiators or antibiotic adjuvants in combination with conventional antibiotics for treating bacterial infections in clinical settings.5 Obviously, these successful practical examples indicate that plants have great promise in the development of new antibiotics. In Cote d’Ivore, Voacanga af ricana (Apocynaceae) is traditionally used to treat diarrhea, ulcers, carious teeth and microbial infections,6 and extracts of its seeds are applied to cure tooth decay and gonorrhea in Cameroon. Most applications of this plant are related to its antimicrobial activity.7 Previous pharmacological investigations have revealed that monoterpenoid indole alkaloids (MIAs) are the principal bioactive compounds responsible for its success as a traditional medicine.8 Chemical studies mainly focused on the MIAs9 due to their complex frameworks and varied biological activities.10 The fascinating chemical profile and traditional use of V. af ricana inspired us to search for novel antibacterial MIAs in this medicinal plant. Thus, we undertook investigations of the fruits of V. africana, and as a result, two MIAs with potent antibacterial activities against Staphylococcus aureus and Salmonella typhi were © XXXX American Chemical Society
Figure 1. Structures of compounds 1 and 2. Received: March 20, 2018
A
DOI: 10.1021/acs.orglett.8b00913 Org. Lett. XXXX, XXX, XXX−XXX
Letter
Organic Letters Table 1. 1H and 13C NMR Spectral Data of 1a and 2b in DMSO-d6 1a no. 2 3 5a 5b 6a 6b 7 8 9 10 11 12 13 14 15a 15b 16 17a 17b 18a 18b 19a 19b 20 21 22 OCH3 OH NH
δC 135.2 53.6 51.4 15.9 101.4 125.6 100.0 153.3 111.2 112.2 131.6 30.0 31.3 79.3 38.5 67.0 30.5 32.3 96.5 165.1 55.5
2b δH (J in Hz)
δC 135.7 52.7
3.29, overlap 4.30, td (12.0, 6.2) 3.83, dd (12.0, 5.1) 2.90, t (5.1) 2.81, m
50.5 16.0 101.3 125.4 99.8 153.2 111.0 112.2 131.6 29.2 39.4
6.85, d (2.3) 6.63, dd (8.8, 2.3) 7.15, d (8.8) 2.44, m 1.77, m 1.43, t (12.0)
78.8 37.9
2.94, d (6.0) 2.41, overlap 4.14, m 4.02, t (8.1) 2.09, m 1.74, dd (12.0, 8.1) 2.63, m 5.71, d (3.9)
67.4 41.2 74.8 99.0 165.7 55.4
3.68, s 10.61, s
a1
H and 13C NMR spectra were recorded at 400 and 100 MHz, respectively; respectively.
unsaturation. Its IR spectrum indicated the presence of an amino group (3422 cm−1), a carbonyl group (1646 cm−1), and an aromatic ring (1595, 1487, and 1454 cm−1). The UV maxima at 219, 275, and 307 nm were similar to those of known MIAs.11 The 13C NMR spectrum (data shown in Table 1) of 1 showed 21 carbon signals, which on the basis of the DEPT and HSQC experiments included two quaternary carbons, including one carboxylic moiety at δC 165.1, three methine (one oxygenated at δC 96.5), and seven methylene (one oxygenated at δC 67.0). The other nine signals could be attributed to a 6-methoxyindole moiety11a (Figure 2) at δC 153.3 (s, C-10), 131.6 (s, C-13), 125.6 (s, C-8), 112.2 (d, C-12), 111.2 (d, C-11), 100.0 (d, C-9), 55.5 (q, 10-OCH3), 135.2 (s, C-2), and 101.4 (s, C-7), which are in agreement with the resonances in its 1H NMR spectrum at δH 10.61 (1H, s), 7.15 (1H, d, J = 8.8 Hz, H-12), 6.85 (1H, d, J = 2.3 Hz, H-9), 6.63 (1H, dd, J = 8.8, 2.3 Hz, H-11), and 3.68 (3H, s, 10-OCH3). The two characteristic signals at δC 51.4 and 53.6 were assigned to be the carbons bearing the nitrogen (C-5 and 3, respectively) based on the deshielding effects.12 In the 1H−1H COSY spectrum of 1, cross peaks from δH 3.83 and 4.30 (2H, H5) to 2.81 and 2.90 (2H, H-6) established an −NCH2CH2 fragment, which corresponded to −N4−C5−C6. This fragment was confirmed by the HMBC correlations from δH 2.81 and 2.90 (2H, H-6) to the indole skeleton carbons at δC 135.2 (C-2) and 125.6 (C-8) (Figure 2) and the correlation of 2.90 (H-6a)/6.85
b1
H and
13
δH (J in Hz) 3.36, overlap 3.23, d (11.3) 4.44, m 3.93, dd (11.8, 5.2) 2.97, dd (16.7, 5.2) 2.88, m
6.91, d (2.4) 6.69, dd (8.7, 2.4) 7.25, d (8.7) 2.53, overlap 2.51, overlap 1.94, d (13.9) 3.31, d (12.6) 2.51, overlap 4.15, t (8.5) 4.11, ddd (12.6, 8.5, 4.9) 2.27, dd (20.6, 12.6) 1.91, d (2.4) 6.14, s 3.75, s 5.79, s 10.53, s
C NMR data were recorded at 800 and 200 MHz,
Figure 2. 1H−1H COSY and key HMBC and NOE correlations of 1.
(H-9) (Figure 2) in the ROESY spectrum of 1. On the other hand, the correlations of δH 3.29 (H-3)/2.44/2.41, 2.94 in the 1 H−1H COSY spectrum were indicative of a −N4−C3−C14− C17− fragment (Figure 2). In addition, in its HMBC spectrum (Figure 2), the correlations of δH 3.29 (H-3), 4.30 (H-5), 2.44 (H-14) and 2.94 (H-17) with δC 79.3 (s) and of δH 2.94 (H-17) with δC 135.2 (C-2) constructed fused heterocycles C/D and suggested that these rings shared carbon C-16. In addition, a B
DOI: 10.1021/acs.orglett.8b00913 Org. Lett. XXXX, XXX, XXX−XXX
Letter
Organic Letters
calculated for the 14R,16S,20R,21S enantiomer of 1 agreed with the experimental curve (Figure 3), while that of the other
carboxylic group was located at C-16 based on the HMBC correlation of δH 2.94 (H-17) with δC 165.1(s, C-22) (Figure 2). In the 1H−1H COSY spectrum of 1, the correlations of δH 3.29 (H-3)/2.44/1.77 and 1.43/2.63/5.71 constructed the −N4−C3− C14−C15−C20−C21− fragment (Figure 2). Furthermore, a substituted piperidine ring (E) was established by the key HMBC correlations of δH 5.71 (H-21) with δC 51.4 (C-5) and 53.6 (C-3), and this ring shared the −N4−C3−C14− fragment with ring D. Then, the downfield proton at δH 5.71 (H-21) and the corresponding carbon at δC 96.5 (C-21) were proposed to be deshielded by the quaternary ammonium (N4) and an oxygen. Moreover, the proton spin system of δH 5.71 (H-21)/2.63 (H20)/1.74, 2.09 (H-19)/4.02, 4.14 (H-18) given by the 1H−1H COSY spectrum, together with the key HMBC correlations of δH 4.02 and 4.14 (2H, H-18) with δC 96.5 (C-21) revealed an ether bridge between C-18 and C-21, which formed the fused tetrahydrofuran ring (F). To satisfy its molecular formula, and balance the quaternary ammonium (N4), the carboxyl group (C22) should be present as a carboxylate ion to form a zwitterion; these charges caused noticeable deshielding of H-3, 5, 21 and 1713 (Table 1). In the ROESY experiment (Figure 2), the NOE correlations of H-20/H-21, H-20/H-17a, and H-21/H-5a suggested these protons were cofacial, and they were tentatively assigned to be α-oriented. In addition, the NOE correlations of H-14/H-17b and H-3/H-5b indicated that these protons were β-oriented, and the rigid molecular skeleton required the carboxylic group to be on the opposite face of ring D. Furthermore, the co-occurrence and biogenetic relationship of 1 and 19-epi-voacristine in the same plant (Scheme 1) allowed us to assign the configuration of compound 1 as either 14R,16S,20R,21S or 14S,16R,20S,21R. Unfortunately, all of our efforts to prepare a single crystal of 1 failed. Therefore, a powerful and reliable method of calculating theoretical electronic circular dichroism14 (ECD) spectra at the B3LYP/6-31G (d, p) level in methanol with a PCM model was applied to the conformational analysis of 1, and the calculated spectrum was compared to the experimental CD data. The curve
Figure 3. Experimental and calculated ECD spectra of 1.
possible configurations did not match as well (Figure S19). Furthermore, calculated 13 C NMR chemical shifts of 14R,16S,20R,21S were good in accordance with those of experiment (see Table S1). Thus, the absolute configuration of 1 was assigned. Voacafricine B (2) was assigned the molecular formula of C21H24N2O5 by negative HRESIMS analysis ([M − H]− m/z 383.1614, calcd 383.1612), which was 16 mass units more than that of 1. Comparison of the 13C and 1H NMR spectral data of 1 and 2 (Table 1) indicated that a methine carbon at δC 32.3 in 1 was replaced by a quaternary carbon at δC 74.8 in 2, and the corresponding proton appeared at δH 5.79 (−OH) in the spectrum of 2. These data, in conjunction with its molecular formula, suggested an additional hydroxyl moiety was present in 2, and this group was positioned at C-20 by the correlations of δH 5.79 (1H, s, −OH) with δC 39.4 (C-15), 41.2 (C-19), 74.8 (C20), and 99.0 (C-21). The 20-OH was assigned at same side of H-21, supported by NOE correlation of δH 5.79 (1H, s, −OH) with 6.14 (H-21), and hydroxyl substituent caused the signals of C-15, 19, and 21 in 2 to shift significantly downfield, relative to the corresponding signals in the spectrum of 1 (Table 1). The other fragments and the relative configuration of 2 were the same as those of 1 based on a thorough analysis of the HSQC, HMBC, 1 H−1H COSY, and ROESY spectra of 2 (Figure 4). Then, unlike that of 1, the chiral centers at C-20 and C-14 in 2 were determined to have been converted 20S and 14S, respectively, by the hydroxyl substituent. The deduction was further confirmed by comparing the experimental CD spectrum and the ECD data
Scheme 1. Proposed Biogenetic Pathway of 1 and 2 from 19epi-Voacristine
Figure 4. 1H−1H COSY and key HMBC and NOE correlations of 2. C
DOI: 10.1021/acs.orglett.8b00913 Org. Lett. XXXX, XXX, XXX−XXX
Letter
Organic Letters Author Contributions
calculated for 2 (see Figure S20), which use the same method as in 1. The biosynthesis of voacafricines A (1) and B (2) (Scheme 1) can be traced back to a common precursor, 19-epi-voacristine, which is the major indole alkaloid present in V. af ricana.15 19-epiVoacristine was dehydrated to afford 4, 20-didehydrovoacangine. Hydroxyl groups can be introduced to the C-18 and 20 positions of this compound to form a key dihydric alcohol intermediate (A). After that, an elimination of 20-OH accompanied by cleavage of the C-16/21 bond can generate the two double bonds at C-2/16 and C-20/21 (intermediate B). Then quaternary amines epoxide might be formed through the intramolecular addition of N-4 to C-16 and epoxied C-20/21 double bond (intermediate C). Furthermore, an intramolecular nucleophilic attack between 18-OH and C-21 can generate the complex pyrrole/pyridine/pyrrolidine/piperidine/furan heterocyclic system (intermediate D). Finally, the hydrolysis of intermediate D might afford 2, and reduction of 2 would then provide 1. The antibacterial activities of compounds 1 and 2 were evaluated against four bacterial strains. Compound 1 showed potent antibacterial activity against S. aureus with an MIC of 3.12 μg/mL, but 2 showed better antibacterial activity against S. aureus and S. typhi with MIC values of 3.12 and 0.78 μg/mL, respectively, and these values were compared to those of two famous antibiotics, berberine and fibrauretine (Table 2).
⊥
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS We are grateful to the Ministry of Science and Technology of China (SQ2017YFC170594-07) and the “Ten Thousand Plan,” a National High-level Talents Special Support Plan, for partial financial support.
■
Staphyloccus aureus
Escherichia coli
Salmonella typhi
Bacillus subtilis
1 2 berberine fibraurtine
3.12 3.12 6.25 25
>100 50 25 25
6.25 0.78 3.12 3.12
25 50 12.5 25
In conclusion, voacafricines A and B from V. af ricana represented an unprecedented 6/5/6/5/6/5 skeleton fused with five heterocycles. The proposed biosynthesis pathway try to explain the formation of fused multiple heterocycle reasonably. This research might provide an attractive architecture and promising plant-derived antimicrobial agents for further chemical and pharmacological investigations.
■
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.8b00913. Detailed experimental procedures and NMR data for compounds 1 and 2 (PDF)
■
REFERENCES
(1) (a) Singh, R.; Sripada, L.; Singh, R. Mitochondrion 2014, 16, 50−54. (b) Vanetten, H. D.; Mansfield, J. W.; Bailey, J. A.; Farmer, E. E. Plant Cell 1994, 6, 1191−1192. (2) Sands, M.; Torres, J. Trans. R. Soc. Trop. Med. Hyg. 1977, 71, 454− 455. (3) Wolf, R.; Baroni, A.; Greco, R.; Donnarumma, G.; Ruocco, E.; Tufano, M. A.; Ruocco, V. Ann. Clin. Microbiol. Antimicrob. 2002, 1, 5. (4) Zuo, G. Y.; Li, Y.; Han, J.; Wang, G. C.; Zhang, Y. L.; Bian, Z. Q. Molecules 2012, 17, 10322−10330. (5) Wright, G. D. Trends Microbiol. 2016, 24, 862−871. (6) Tan, P. V.; Penlap, V. B.; Nyasse, B.; Nguemo, J. D. B. J. Ethnopharmacol. 2000, 73, 423−428. (7) (a) Glick, S. D.; Gallagher, C. A.; Hough, L. B.; Rossman, K. L.; Maisonneuve, I. M. Brain Res. 1992, 588, 173−176. (b) Popik, P.; Layer, R. T.; Skolnick, P. Pharmacol Rev. 1995, 47, 235−253. (8) Federici, E.; Palazzino, G.; Nicoletti, M.; Galeffi, C. Planta Med. 2000, 66, 93−95. (9) (a) Chen, H. M.; Deng, Y.; Song, C. X.; Dai, Y.; Hu, J.; Kang, M.; Dan, X. M.; Li, H. X. Chin. J. Exp. Tradit. Med. Form. 2015, 21, 216−222. (b) Chen, H. M.; Yang, Y. T.; Li, H. X.; Cao, Z. X.; Dan, X. M.; Mei, L.; Guo, D. L.; Song, C. X.; Dai, Y.; Hu, J. Nat. Prod. Rep. 2016, 30, 1145− 1149. (c) Kikura-Hanajiri, R.; Maruyama, T.; Miyashita, A.; Goda, Y. Yakugaku Zasshi 2009, 129, 975−982. (10) (a) 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. (b) Kuehne, M. E.; Reider, P. J. J. Org. Chem. 1985, 50, 1464−1467. (c) Sundberg, R. J.; Cherney, R. J. J. Org. Chem. 1990, 55, 6028−6037. (d) Qin, X. J.; Zhao, Y. L.; Song, C. W.; Wang, B.; Chen, Y. Y.; Liu, L.; Li, Q.; Li, D.; Liu, Y. P.; Luo, X. D. Nat. Prod. Bioprospect. 2015, 5, 185−193. (e) 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. (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) Feng, T.; Cai, X. H.; Li, Y.; Wang, Y. Y.; Liu, Y. P.; Xie, M. J.; Luo, X. D. Org. Lett. 2009, 11, 4834−4837. (h) Feng, T.; Li, Y.; Liu, Y. P.; Cai, X. H.; Wang, Y. Y.; Luo, X. D. Org. Lett. 2010, 12, 968−971. (i) Harada, M.; Asaba, K. N.; Iwai, M.; Kogure, N.; Kitajima, M.; Takayama, H. Org. Lett. 2012, 14, 5800−5803. (j) Pan, Z. Q.; Qin, X. J.; Liu, Y. P.; Wu, T.; Luo, X. D.; Xia, C. F. Org. Lett. 2016, 18, 654−657. (k) 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. (l) Chen, Y. Y.; Yang, J.; Yang, X. W.; Khan, A.; Liu, L.; Wang, B.; Zhao, Y. L.; Liu, Y. P.; Ding, Z. T.; Luo, X. D. Tetrahedron Lett. 2016, 57, 1754−1757. (11) (a) Prakash Chaturvedula, V. S.; Sprague, S.; Schilling, J. K.; Kingston, D. G. I. J. Nat. Prod. 2003, 66, 528−531. (b) Kam, T. S.; Sim, K. M. J. Nat. Prod. 2002, 65, 669−672. (c) Sheludko, Y.; Gerasimenko, I.; Kolshorn, H.; Stöckigt, J. J. Nat. Prod. 2002, 65, 1006−1010. (12) Feng, T.; Cai, X. H.; Liu, Y. P.; Li, Y.; Wang, Y. Y.; Luo, X. D. J. Nat. Prod. 2010, 73, 22−26. (13) Qin, X. J.; Zhao, Y. L.; Lunga, 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. (14) (a) Zhang, Y. L.; Ge, H. M.; Zhao, W.; Dong, H.; Xu, Q.; Li, S. H.; Li, J.; Zhang, J.; Song, Y. C.; Tan, R. X. Angew. Chem., Int. Ed. 2008, 47,
Table 2. Minimum Inhibitory Concentration (MIC, μg/mL) of the Tested Samples against Four Bacterial Strains samples
C.-F.D., H.-X.M., and J.Y. contributed equally to this work.
Notes
AUTHOR INFORMATION
Corresponding Authors
*Phone/fax: +86-871-65223177. E-mail:
[email protected]. *Phone/fax: +86-871-65223177. E-mail:
[email protected]. cn. ORCID
Ya-Ping Liu: 0000-0002-2164-2489 Xiao-Dong Luo: 0000-0002-6768-5679 D
DOI: 10.1021/acs.orglett.8b00913 Org. Lett. XXXX, XXX, XXX−XXX
Letter
Organic Letters 5823−5826. (b) Zhao, S. D.; Shen, L.; Luo, D. Q.; Zhu, H. J. Curr. Org. Chem. 2011, 15, 1843−1862. (c) Specht, K. M.; Nam, J.; Ho, D. M.; Berova, N.; Kondru, R. K.; Beratan, D. N.; Wipf, P., Jr; Pascal, R. A.; Kahne, D. J. Am. Chem. Soc. 2001, 123, 8961−8966. (15) Perera, P.; Samuelsson, G.; van Beek, T. A.; Verpoorte, R. Planta Med. 1983, 47, 148−150.
E
DOI: 10.1021/acs.orglett.8b00913 Org. Lett. XXXX, XXX, XXX−XXX
