Article Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX
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Structural Revision of Macropodumine A and Structure of 2‑Deoxymacropodumine A, Daphniphyllum Alkaloids with 11Membered Macrolactone Rings Jiahui Zhang,†,‡ Pei Cao,† Yuanliang Ma,† Xin Fang,† Jing Yang,† Yu Zhang,† Duozhi Chen,† Yucheng Gu,§ Yingtong Di,*,† and Xiaojiang Hao*,†
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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, People’s Republic of China ‡ Southwest University, Chongqing 400715, People’s Republic of China § Jealott’s Hill International Research Centre, Syngenta, Bracknell, Berkshire RG42 6EY, U.K. S Supporting Information *
ABSTRACT: An unusual Daphniphyllum alkaloid, 2-deoxymacropodumine A (1), possessing an 11-membered macrolactone ring, was obtained from an extract of the stems of Daphniphyllum angustifolium. The structure of 1 was elucidated by 1D and 2D NMR spectroscopic methods and chemical calculations. Based on a comparison of the experimental and calculated NMR data, the structure of macropodumine A (2′), an analogue of 1, was also revised.
D
aphniphyllum alkaloids (DAs) are a unique group of azapolycyclic natural products (>320 members) that are found in the genus Daphniphyllum (Daphniphyllaceae).1−3 Recently, DAs have attracted considerable interest due to their wide range of biological activities, including antitumor,4−6 antiviral,7 and nerve growth factor-regulating properties,8,9 and they have been considered promising targets for total syntheses.10−18 Among these compounds, 2-deoxymacropodumine A and macropodumine A are the only two Daphniphyllum alkaloids with 11-membered macrolactone moieties.19−21 Both compounds possess the rare 2,3,4,5,6,7hexahydroazulene-1,8-dione motif. Their structures had been established via detailed spectroscopic data analysis.19,20 In both of those papers, the authors assigned C-14 of 2-deoxymacropodumine A as a carbonyl carbon on the basis of the HMBC cross peak between H2-16 and C-14. However, the correlations of the atoms in the five-membered D-ring did not exclude the regioisomer with the carbonyl at C-9. Recently, in a continuing search for novel alkaloids from this genus,22−29 we isolated 2deoxymacropodumine A (1), possessing a 2,3,4,5,6,7-hexahydroazulene-1,4-dione moiety. The structure of macropodumine A, an analogue of 1, was revised from 2′ to 2 (Figure 1). Furthermore, a putative biosynthetic pathway for this type of alkaloid is proposed.
Figure 1. Structure of 2-deoxymacropodumine A (1) and originally proposed structures and revised structure of macropodumine A (2′ and 2): computed deviations in the 13C NMR chemical shifts (the size of the red sphere corresponds to the magnitude of the deviation, |ΔδC| > 10 ppm).
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from two methyls, eight sp3 methylenes, five sp3 methines, an sp3 quaternary carbon, two carbonyls (δC 199.8 and 213.2), a
RESULTS AND DISCUSSION Compound 1, isolated as a colorless oil, showed a molecular formula of C21H27NO4 based on its 13C NMR and HREIMS data (m/z 357.1944 [M]+, calcd 357.1940), indicating nine indices of hydrogen deficiency. A total of 21 carbon signals © XXXX American Chemical Society and American Society of Pharmacognosy
Special Issue: Special Issue in Honor of Susan Horwitz Received: March 20, 2018
A
DOI: 10.1021/acs.jnatprod.8b00232 J. Nat. Prod. XXXX, XXX, XXX−XXX
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fragments and the nitrogen atom, four quaternary carbons (C5, C-8, and C-13), and the three carbonyls (C-1, C-9, and C10) were assigned via the HMBC data. The correlations from H2-7 and H2-19 to C-4 and from Me-21 to C-4, C-5, and C-6 suggested the presence of a dimethyloctahydroindolizinyl unit (rings A and B). Ring C was found to have a cyclohept-2-one unit based on the HMBC correlations from H-2 to C-13, H-18 to C-1, and Me-21 to C-8. The connection of partial fragments b and c was verified by the HMBC correlations from H-12 and H-17 to the ester carbonyl (δC 172.1), which was assigned as C-10. In addition, the HMBC correlations from H2-14(9) and H-15 to C-8, C-9(14), and C-13 led to the construction of the cyclopent-2-one unit (ring D) and the 11-membered macrolactone moiety (ring E). However, the data were not sufficient to determine the position of the carbonyl group in ring D. Thus, two possible 2D structures of 1 were tentatively established (1-A and 1-B) with different cyclopentenone moieties, as shown in Figure 2. Because the methylene group being positioned at C-9 (in 1A) or C-14 (in 1-B) would significantly change its distance from Me-21, a ROESY experiment was performed. The β orientations of H-2, H-4, H-6, Me-20, and Me-21 in 1 were established by analysis of its ROESY data. Owing to a lack of sufficient ROE correlations, neither the position of the carbonyl group in ring D nor the orientation of H-15 could be determined; thus, four possible isomers of 1 were considered, and they were named 9-oxo,15Hβ (1a), 9oxo,15Hα (1b), 14-oxo,15Hβ (1c), and 14-oxo,15Hα (1d), respectively. These structures (1a−1d) were then optimized at the B3LYP/6‑3111++G(d) level, as shown in Figure 3, in
diagnostic ester carbonyl (δC 172.1), and two nonprotonated olefinic carbons (δC 141.7 and 166.0) were observed in the 13C NMR data (Table 1). Two methylene groups (δC 52.6 and Table 1. 1H (600 MHz) and 13C (150 MHz) NMR Spectroscopic Data of 1 Recorded in Pyridine-d5 (δ in ppm) position
δC
1 2 3
199.8, C 48.6, CH 19.8, CH2
4 5 6 7
70.2, 50.9, 47.9, 52.6,
8 9 10 11
141.7, C 213.2, C 172.1, C 31.5, CH2
12
21.0, CH2
13 14
166.0, C 31.3, CH2
15 16
42.0, CH 28.7, CH2
17
59.9, CH2
18 19
33.7, CH 51.7, CH2
20 21
17.3, CH3 20.8, CH3
CH C CH CH2
δH (J in Hz) − 1.92, 1.63, 1.81, 2.03, − 2.05, 2.24, 2.54, − − − 2.24, 2.28, 1.63, 2.35, − 2.53, 3.07, 2.47, 1.68, 2.70, 3.93, 4.43, 3.38, 1.49, 2.72, 0.85, 1.50,
m m m br d (9.0) m overlap dd (9.0, 5.4)
overlap m overlap m dd (18.6, 6.9) dd (18.6, 2.4) m m overlap dd (12.0, 6.0) td (12.0, 3.6) m m overlap d (4.8) s
51.7) and a methine (δC 70.2) were determined to be bound to nitrogen atoms, while a methylene group (δC 59.9) carried an oxygen atom. The presence of three carbonyl groups and an olefinic functionality indicated that compound 1 has a pentacyclic ring system. The 2D structure of 1 was defined by analysis of its 2D NMR data. The COSY and HSQC correlations revealed the presence of the three structural fragments (a−c), drawn with bold bonds in Figure 2. The linkages between these three Figure 3. Four possible structures of 1 (1a−1d) and its key ROESY correlations (dashed red line with double arrow) and proton spin− spin coupling constants (dashed black line).
which the distances between the protons near Me-21 and C-15 were calculated. In 1a and 1b, the distances between H3-21 and H-14 were 5.01 and 4.83 Å, respectively, whereas in 1c and 1d, the distances between Me-21 and H-9 were 2.49 and 2.50 Å, respectively. Considering that no ROE correlations were observed between these groups in 1, the carbonyl being located at C-9 was in agreement with the experimental results. Moreover, the ROESY interaction between H-14α and H17α indicated that H-15 was β-oriented, which was fully consistent with the distance calculated for isomer 1a.
Figure 2. Key 1H−1H COSY (bold lines) and HMBC (red arrows) correlations of 1 and its two possible isomers. B
DOI: 10.1021/acs.jnatprod.8b00232 J. Nat. Prod. XXXX, XXX, XXX−XXX
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and the maximum deviation was 6.5 ppm. The average |ΔδC| of 2′ was 8.7 ppm, and the maximum was 23.8 ppm (as shown in the Supporting Information). Therefore, based on the above data, the structure of macropodumine A should be revised from 2′ to 2. The relatively rare structure of this compound contains an unprecedented skeleton with a sterically congested cyclopentenone ring with numerous fused substituents, including a cycloheptenone unit and an 11-membered lactone fragment. Presumably, this congested core controls the molecular conformation and increases the flexibility of the medium-size bridged ring system, which prevented the use of NMR data to elucidate the substitution pattern and identify the correct isomer, and this may account for the previous misinterpretation of the subtleties of the 1D and 2D NMR signals involving C-9 and C-14. A putative biosynthetic pathway for 2-deoxymacropodumine A (1) is proposed as shown in Scheme 1. Compound 1 seems
To further confirm the position of the carbonyl group in the structure of 1, the 13C NMR shifts of 1a and 1c were calculated at the mpw1pw91/6‑31G(d,p) level. The calculated chemical shifts of 1a (Table 2) were in good agreement with the Table 2. Experimental and Calculated 13C NMR Data (δ, ppm) for the Original and Revised Structures of 2deoxymacropodumine A original structure (1′) no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 LADb MADc
revised structure (1)
exptl
calcd
|ΔδC|a
calcd
|ΔδC|
197.7 48.3 20 69.9 50.9 47.8 51.9 141.5 213.5 172.3 31.5 20.4 166.3 31.2 41.8 28.7 59.7 33.7 51.7 17.4 20.3
197.1 51.0 21.5 66.2 61.9 58.5 64.6 134.9 195.1 171.8 39.4 37.3 184.1 44.4 54.3 25.7 62.0 36.8 51.6 18.6 29.7
0.6 2.7 1.5 3.7 11.0 10.7 12.7 6.6 18.4 0.5 7.9 16.9 17.8 13.2 12.5 3.0 2.3 3.1 0.1 1.2 9.4
193.2 49.8 22.1 66.9 53.0 50.1 49.4 139.6 209.3 168.1 35.4 20.9 169.9 33.9 43.9 31.6 58.4 37.3 49.9 18.0 20.6
4.5 1.5 2.1 3.0 2.1 2.3 2.5 1.9 4.2 4.2 3.9 0.5 3.6 2.7 2.1 2.9 1.3 3.6 1.8 0.0.6 0.3
18.4 7.5
Scheme 1. Putative Biosynthesis Pathway for 1
6.5 2.6
to be derived from 22-nor-calyciphylline A-type alkaloids with an appropriate leaving group at C-13. Cleavage of the C-1−C8 bond and subsequent formation of the C-1−C-13 bond would lead to an expansion of ring C, and formation of the Δ8(13) double bond of ring D would generate intermediate i. Subsequently, the 11-membered macrolactone (ring E) could be constructed by the oxidative cleavage of the Δ9(10) double bond and insertion of an oxygen atom into the C-10−C-17 bond. The cytotoxicity of compound 1 against HeLa, MCF-7, A549, MGC-803, and COLO-205 human cancer cell lines in vitro has been evaluated using the MTT method.30 Compound 1 exhibited moderate cytotoxicity against HeLa cells, with an IC50 of 3.89 μM.
a
Numbers in bold italics highlight the discrepancies between the calculated and experimental values over 10 ppm (|ΔδC | > 10 ppm). b LAD = largest absolute deviation. cMAD = mean absolute deviation, computed as (1/n)∑ni |δcalcd − δexptl|.
experimental data; the average difference was 2.6 ppm, and the maximum deviation was 6.5 ppm. Notably, the average |ΔδC| of 1c was 7.5, and the maximum was 18.4 ppm. Therefore, the structure of compound 1 with the 2,3,4,5,6,7-hexahydroazulene-1,4-dione moiety was the most plausible, which further confirmed the structure of 1 as shown. Furthermore, comparison of the NMR data of 1 (recorded in CDCl3) with those of 2-deoxymacropodumine A (1′)19 indicated a high degree of structural similarity between these compounds. Macropodumine A (2′) was the other Daphniphyllum alkaloid of this type, also harboring a C-14 instead of a C-9 carbonyl group (Figure 1). However, in the raw data of macropodumine A, no clear ROE correlation between H-9 and H3-21 was observed, which should be present if the assigned C-14 carbonyl group is correct. Likewise, correlations of H-9 with H-11 or H3-21 were not present in the ROESY spectrum (recorded in pyridine-d5) of compound 1. Thus, we proposed that, in the structure of macropodumine A (2), the methylene group should be at C-14, and the remaining carbonyl is located at the C-9 position. To further support this assignment, the 13C NMR shifts of 2 and 2′ were calculated using the same method as that used for 1. The average difference between the calculated and experimental chemical shifts of 2 was 2.5 ppm,
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EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were obtained on a Horiba SEPA-300 polarimeter. UV spectra were recorded using a Shimadzu UV-2401A spectrophotometer. A Tenor 27 spectrophotometer was used to record the IR spectra of KBr pellets. An API QSTAR time-of-flight spectrometer was used to collect the HREIMS data. Bruker AVANCEIII-600 spectrometers with TMS as an internal standard were used to acquire the 1D and 2D NMR spectra. Column chromatography was performed using silica gel (200−300 mesh and 300−400 mesh, Qingdao Marine Chemical, Inc., Qingdao, P. R. China). Plant Material. The aerial parts of D. angustifolium were collected in November 2013 from Jinfo mountain in Chongqing municipality, People’s Republic of China. The plant samples were identified by Prof. Hong-Ping Deng of the School of Life Sciences, Southwest University.
C
DOI: 10.1021/acs.jnatprod.8b00232 J. Nat. Prod. XXXX, XXX, XXX−XXX
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Extraction and Isolation. The air-dried stems of D. angustifolium (20 kg) were powdered and extracted three times with MeOH at room temperature. The crude extract was dissolved in 0.1% aqueous tartaric acid, and the solution was extracted with EtOAc to remove the nonalkaloid components. After the pH of the aqueous solution was adjusted to 10.0 with saturated Na2CO3, the water-soluble materials were partitioned with CHCl3. The CHCl3-soluble materials (100.6 g) were subjected to normal-phase silica gel column chromatography (100−200 mesh; CHCl3/MeOH, 1:0 to 0:1) to yield five fractions (Fr.1−Fr.5). Fr.3 (CHCl3/MeOH, 20:1 to 10:1, 26.3 g) was separated by medium-pressure chromatography (RP-18 column) with a MeOH/H2O gradient (10, 20, 30, 40, 50, 80, and 100%) to afford crude alkaloids (Fr.3A, 20−50%, 3.8 g). Fr.3A was chromatographed on a Sephadex LH-20 gel column (CHCl3/MeOH, 1:1) to give pure total alkaloids (Fr.3A1, 815 mg). Fr.3A1 was separated by silica gel column chromatography with a CHCl3/acetone gradient (10:1 to 8:1) into two fractions: Fr.3A1A (263 mg) and Fr.3A1B (117 mg). Fraction 3A1A was separated by silica gel column chromatography with a CHCl3/MeOH gradient (50:1 to 10:1) to afford three fractions. Fr.3A1A2 (CHCl3/MeOH, 40:1, 4 mg) was successfully purified on a Sephadex LH-20 column (MeOH) to afford compound 1 (3.6 mg). 2-Deoxymacropodumine A (1): colorless oil; [α]25 D −21 (c 0.3, MeOH); UV (MeOH) λmax (log ε) 233 (3.52), 205 (3.45) nm; IR (KBr) νmax (cm−1) 3434, 2925, 2854, 1731, 1704, 1661, 1641, 1459, 1382, 1308, 1258; 1H and 13C NMR data, Table 1; HREIMS m/z 357.1944 [M + H]+ (calcd for C23H46NO4, 357.1940).
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(3) Chattopadhyay, A. K.; Hanessian, S. Chem. Rev. 2017, 117, 4104−4146. (4) Zhang, H.; Yang, S. P.; Fan, C. Q.; Ding, J.; Yue, J. M. J. Nat. Prod. 2006, 69, 553−557. (5) Zhang, C. R.; Liu, H. B.; Feng, T.; Zhu, J. Y.; Geng, M. Y.; Yue, J. M. J. Nat. Prod. 2009, 72, 1669−1672. (6) Liu, S.; Zhang, J. H.; Di, Y. T.; Dong, J. Y.; Hao, X. Nat. Prod. Res. 2018, 32, 2165. (7) Xu, J. B.; Zhang, H.; Gan, L. S.; Han, Y. S.; Wainberg, M. A.; Yue, J. M. J. Am. Chem. Soc. 2014, 136, 7631−7633. (8) Morita, H.; Ishioka, N.; Takatsu, H.; Shinzato, T.; Obara, Y.; Nakahata, N.; Kobayashi, J. Org. Lett. 2005, 7, 459−462. (9) Saito, S.; Yahata, H.; Kubota, T.; Obara, Y.; Nakahata, N.; Kobayashi, J. Tetrahedron 2008, 64, 1901−1908. (10) Tantillo, D. J. Org. Lett. 2016, 18, 4482−4484. (11) Shi, H. Y.; Michaelides, I. N.; Darses, B.; Jakubec, P.; Nguyen, Q. N. N.; Paton, R. S.; Dixon, D. J. J. Am. Chem. Soc. 2017, 139, 17755−17758. (12) Liu, Y. M.; Li, F.; Wang, Q.; Yang, J. Tetrahedron 2017, 73, 6381−6385. (13) Coussanes, G.; Bonjoch, J. Org. Lett. 2017, 19, 878−881. (14) Shao, H.; Bao, W.; Jing, Z. R.; Wang, Y. P.; Zhang, F. M.; Wang, S. H.; Tu, Y. Q. Org. Lett. 2017, 19, 4648−4651. (15) Li, J.; Zhang, W. H.; Zhang, F.; Chen, Y.; Li, A. J. Am. Chem. Soc. 2017, 139, 14893−14896. (16) Chen, Y.; Zhang, W. H.; Ren, L.; Li, J.; Li, A. Angew. Chem., Int. Ed. 2018, 57, 952−956. (17) Chen, X. M.; Zhang, H. J.; Yang, X. K.; Lv, H. Q.; Shao, X. R.; Tao, C.; Wang, H. F.; Cheng, B.; Li, Y.; Guo, J. J.; Zhang, J.; Zhai, H. B. Angew. Chem., Int. Ed. 2018, 57, 947−951. (18) Zhang, W. H.; Ding, M.; Li, J.; Guo, Z. C.; Lu, M.; Chen, Y.; Liu, L. C.; Shen, Y. H.; Li, A. J. Am. Chem. Soc. 2018, 140, 4227− 4231. (19) Li, Z. Y.; Chen, P.; Xu, H. G.; Peng, S. Y.; Yang, Y. M.; Zhao, Z. Z.; Guo, Y. W. Chin. J. Chem. 2008, 26, 348−352. (20) Zhang, W.; Guo, Y. W.; Krohn, K. Chem. - Eur. J. 2006, 12, 5122−5127. (21) In the original paper, compound 1′ was named as 2deoxymacropodumine A. However, by comparison of the structure of 1′ with that of 2′, the 2-H is retained and only the O atom is lost. Therefore, we used “deoxy” to replace “dehydroxy” in this paper as per and editor’s suggestion. (22) Zhang, Y.; Di, Y. T.; Zhang, Q.; Mu, S. Z.; Tan, C. J.; Fang, X.; He, H. P.; Li, S. L.; Hao, X. Org. Lett. 2009, 11, 5414−5417. (23) Yang, T. Q.; Di, Y. T.; He, H. P.; Zhang, Q.; Zhang, Y.; Hao, X. Helv. Chim. Acta 2011, 94, 397−403. (24) Zhang, Y.; Di, Y. T.; He, H. P.; Li, S. F.; Lu, Y.; Gong, N. B.; Hao, X. J. Eur. J. Org. Chem. 2011, 2011, 4103−4107. (25) Tan, C. J.; Wang, Y. H.; Di, Y. T.; He, H. P.; Mu, S. Z.; Li, S. F.; Zhang, Y.; Hao, X. J. Tetrahedron Lett. 2012, 53, 2588−2591. (26) Cao, M. M.; He, H. P.; Gu, Y. C.; Zhang, Q.; Li, X. N.; Zuo, G. Y.; Di, Y. T.; Yuan, C. M.; Li, S. L.; Zhang, Y.; Hao, X. Nat. Prod. Bioprospect. 2013, 3, 29−32. (27) Cao, M. M.; Wang, L.; Zhang, Y.; He, H. P.; Gu, Y. C.; Zhang, Q.; Li, Y.; Yuan, C. M.; Li, S. L.; Di, Y. T.; Hao, X. J. Fitoterapia 2013, 89, 205−209. (28) Zhang, Q.; Zhang, Y.; Yang, T. Q.; Di, Y. T.; Hao, X. RSC Adv. 2013, 3, 9658−9661. (29) Tan, C. J.; Di, Y. T.; Hao, X. J. Chin. Tradit. Herbal Drugs 2015, 46, 2989−2991. (30) Mosmann, T. J. Immunol. Methods 1983, 65, 55−63.
ASSOCIATED CONTENT
* Supporting Information S
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b00232. 1
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H and 13C NMR, HSQC, HMBC, 1H−1H COSY, NOESY, IR, UV, and HREIMS spectra of 1, together with DFT calculation details (PDF)
AUTHOR INFORMATION
Corresponding Authors
*Tel. (X.-J. H.): +86-871-6522-3070. E-mail:
[email protected]. ac.cn. *Tel. (Y.-T. D.): + 86-871-6522-3263. E-mail:
[email protected]. ac.cn. ORCID
Yucheng Gu: 0000-0001-8562-344X Xiaojiang Hao: 0000-0001-9496-2152 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We gratefully acknowledge the National Natural Science Foundation of China (21432010, 31770392, and 81573323), Technological Leading Talent Project of Yunnan (2015HA020), and Central Asian Drug Discovery and Development Center of Chinese Academy of Sciences (CAM201402 and CAM201302) for their financial support. The Syngenta postgraduate studentship awarded to J.-H.Z. (2014−2016) is also acknowledged.
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REFERENCES
(1) Kobayashi, J.; Kubota, T. Nat. Prod. Rep. 2009, 26, 936−962. (2) Wu, H. F.; Zhang, X. P.; Ding, L. S.; Chen, S.; Yang, J.; Xu, X. Planta Med. 2013, 79, 1589−1598. D
DOI: 10.1021/acs.jnatprod.8b00232 J. Nat. Prod. XXXX, XXX, XXX−XXX
