Treatment of Vinca minor Leaves with Methyl ... - ACS Publications

Article Cite This: J. Nat. Prod. 2017, 80, 2905-2909

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Treatment of Vinca minor Leaves with Methyl Jasmonate Extensively Alters the Pattern and Composition of Indole Alkaloids Sara Abouzeid,†,⊥ Ulrike Beutling,‡ Frank Surup,§ Fatma M. Abdel Bar,⊥ Mohamed M. Amer,⊥ Farid A. Badria,⊥ Mahdi Yahyazadeh,† Mark Brönstrup,‡ and Dirk Selmar*,† †

Institute for Plant Biology, TU Braunschweig, 38106 Braunschweig, Germany Department of Chemical Biology and §Department of Microbial Drugs, Helmholtz Centre for Infection Research, Inhoffenstraße 7, 38124 Braunschweig, Germany ⊥ Pharmacognosy Department, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt ‡

S Supporting Information *

ABSTRACT: Alkaloids extracted from mature Vinca minor leaves were fractionated by preparative HPLC. By means of HRMS and NMR data, the main alkaloids were identified as vincamine, strictamine, 10-hydroxycathofoline, and vincadifformine. Upon treatment with methyl jasmonate (MeJA), the pattern and composition of the indole alkaloids changed extensively. While 10-hydroxycathofoline and strictamine concentrations remained unaltered, vincamine and vincadifformine levels showed a dramatic reduction. Upon MeJA treatment, four other indole alkaloids were detected in high quantities. Three of these alkaloids have been identified as minovincinine, minovincine, and 9-methoxyvincamine. Whereas minovincinine and minovincine are known to occur in trace amounts in V. minor, 9-methoxyvincamine represents a novel natural product. Based on the high similarities of vincamine and 9methoxyvincamine and their inverse changes in concentrations, it is postulated that vincamine is a precursor of 9methoxyvincamine. Similarly, vincadifformine seems to be converted first to minovincinine and finally to minovincine. Because MeJA treatment greatly altered the alkaloidal composition of V. minor, it could be used as a potential elicitor of alkaloids that are not produced under normal conditions.

J

that MeJA effectively enhanced indole alkaloid biosynthesis in developing seedlings of C. roseus and that the enhancement of alkaloid biosynthesis decreased with age of the seedlings.11 Other studies using C. roseus seedlings revealed a high variability of the MeJA response.6 The related impact of MeJA treatment on the expression of genes responsible for alkaloid biosynthesis varied drastically, in magnitude as well as in timing. Accordingly, it is difficult to correlate any putative changes in indole alkaloid contents with alterations in the expression of specific genes responsible for their biosynthesis.6 Moreover, when MeJA was applied to the detached leaves of C. roseus, the catabolism of alkaloids was strongly enhanced.9 These inconsistencies clearly illustrate that in forthcoming investigations, to elucidate any putative effect of MeJA on indole alkaloid biosynthesis, it is vital to consider the physiological status of the plants. In this way, the effects of MeJA could be segregated from the effects of endogenous jasmonates produced due to natural stress situations or due to aging and senescence processes.

asmonates are plant hormones that play key roles in signal transduction in many different responses to biotic or abiotic stress, including attacks by pathogens and herbivores or drought stress.1−4 In most cases, jasmonate signaling leads to altered expression of genes that encode various defense proteins or enzymes involved in the biosynthesis of natural products responsible for repelling herbivores or defending pathogens.5−7 Moreover, jasmonic acid (JA) is also relevant to the induction of leaf senescence and the corresponding catabolic processes.8−10 One of the most prominent examples of the impact of methyl jasmonate (MeJA) on secondary metabolism concerns the indole alkaloids in Catharanthus roseus.9,11−13 Since jasmonates are involved in the induction and regulation of many different metabolic processes, cause− effect relationships cannot be observed in most cases. Indeed, many, at first glance, contradictory findings are present in the literature dealing with indole alkaloids in C. roseus. For example, in hairy root cultures, the application of MeJA leads to a significant increase in the content of indole alkaloids and to a shift in their composition.12,13 In the same manner, also in mature C. roseus plants, the indole alkaloid content was increased by MeJA application.14 In contrast, Pan et al. showed that a MeJA treatment had no effect on the indole alkaloid accumulation in mature C. roseus leaves.15 Aerts et al. showed © 2017 American Chemical Society and American Society of Pharmacognosy

Received: May 15, 2017 Published: November 13, 2017 2905

DOI: 10.1021/acs.jnatprod.7b00424 J. Nat. Prod. 2017, 80, 2905−2909

Journal of Natural Products

Article

In contrast to the rich literature on the indole alkaloids in C. roseus, data on the impact of MeJA on the metabolism of the alkaloids in Vinca minor are not available. Similar to C. roseus, lesser periwinkle (V. minor), also a member of the Apocynaceae, contains large amounts and a wide spectrum of indole alkaloids. Thus far, more than 50 indole alkaloids have been extracted and isolated from lesser periwinkle.16−19 Although different studies have shown quite different compositions of indole alkaloids of V. minor, in all cases, the major alkaloid in the leaves was reported to be vincamine (1).16−19 Vincamine (1) has pronounced cerebrovasodilatory and neuroprotective activities. Accordingly, many remedies containing vincamine or its derivatives are on the market to treat memory disorders or to act as CNS stimulants.17,20 As reported for other alkaloids, the content of vincamine varies largely depending on growth and climatic conditions, the season, and putative stress situations.21 In this study, mature V. minor plants of defined physiological status were used to investigate the effect of MeJA treatment on the composition of alkaloids.

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RESULTS AND DISCUSSION Mature leaves of V. minor were freeze-dried, and the alkaloids were acid−base extracted. The analysis of this extract by LCMS (Figure 1a) revealed four main alkaloids, 1, 3, 4, and 5.

Figure 2. Metabolites isolated from Vinca minor within the scope of this study.

using carbon chemical shifts is hampered in cases where the compounds act as weak acids or bases. Surprisingly, the alkaloid pattern of V. minor leaves treated with MeJA was entirely different (Figure 1b). Whereas the concentrations of strictamine (3) and 10-hydroxycathofoline (4) were not affected by the alterations, vincamine (1) and vincadifformine (5) contents were strongly reduced in response to the MeJA treatment. At the same time, the production of four other indole alkaloids was strongly induced. Compounds 2, 6, and 7 were purified using semipreparative HPLC and analyzed by HR-MS, 1H, 13C, COSY, TOCSY, HSQC, and HMBC NMR data. Of these, 6 and 7 were identified as minovincinine and minovincine, respectively (Figure 2, Table 1, and Supporting Information). Both compounds have been described previously as minor alkaloids of V. minor.16−19 For 2, HR-MS data revealed a molecular formula of C22H28N2O4. 1H NMR and HSQC data (Table 2) showed the presence of three methyl groups, including two Omethyl singlets, seven methylenes, a nitrogenated methine, and three aromatic methine protons. The 13C NMR spectrum

Figure 1. LC-MS separation of the major indole alkaloids determined in Vinca minor. (A) Composition of indole alkaloids extracted from control plants. (B) Composition of V. minor plants treated with MeJA.

Table 1. Identification of Indole Alkaloids of V. minor Leaves by UHPLC-PDA-MS

These compounds were subsequently purified by semipreparative HPLC. HR-MS data were consistent with molecular formulas of C21H26N2O3 for 1, C20H22N2O2 for 3, C21H26N2O3 for 4, and C21H26N2O2 for 5. Utilizing 1H, 13C, COSY, TOCSY, ROESY, HSQC, and HMBC NMR data, the structures of the compounds were elucidated as vincamine (1), strictamine (3), 10-hydroxycathofoline (4), and vincadifformine (5) (Figures 1a and 2 and Supporting Information). Whereas the presence of vincamine, strictamine, and vincadifformine is in full accordance with the published data,16−19 10-hydroxycathofoline (4), which is known to occur in Vinca major,22 was isolated for the first time from V. minor. It is important to mention that the 1H and 13 C chemical shifts deviated significantly from literature data, probably due to the protonation of the basic nitrogen atoms by tetrafluoroacetic acid (TFA) or formic acid during HPLC fractionation. This illustrates that metabolite dereplication

tR (min)

UV (λmax) (nm)

MW

minovincinine (6)

7.87

220, 330

354

unknown minovincine (7)

9.35 9.99

220 221, 331

338 352

10-hydroxycathofoline (4) vincamine (1)

10.49

354

12.14

208, 245, 312 220, 270

13.62 15.17

220, 270 221, 270

322 384

19.77

221, 330

338

compound

strictamine (3) 9-methoxyvincamine (2) vincadifformine (5)

2906

354

ESI+ (m/z) (rel int %) 355 (100), 323 (59) 339 (100) 353 (100), 321 355 (100) 355 (100), 337 (60) 323 (100) 385 (100), 367 339 (100), 307 (83)

refs 36, 37 37, 38 22 39, 40 41 23 39



Article

Table 2. NMR Spectroscopic Data (700 MHz for 1H, 175 MHz for 13C) of 9-Methoxyvincamine (2) TFA Salt in CDCl3 pos.

δC, mult.

2 3

122.6, C 60.0, CH

5

51.6, CH2

6

18.0, CH2

7 8 9 10 11 12 13 14 15 16 17

105.1, C 117.6, C 154.7, C 101.5, CH 124.8, CH 103.8, CH 136.5, C 81.4, C 43.4, CH2 35.8, C 22.9, CH2

18

18.2, CH2

19

44.5, CH2

20

21

δH, mult. (J, Hz)

COSY

4.47, br s 3.61, dt (12.58, 6.2); 3.72, m

3.37, m; 3.26, m

6.58, d (8.2) 7.14, dd (8.2, 8.2) 6.71, d (8.2)

5b, 6a, 6b 5a, 6a, 6b 6b 5a, 5b, 6a

NOESY 5, 15, 20 3

6 6

3, 21

3, 14, 16, 17 16, 20 3, 16, 19

28.7, CH2

17 17 18 18 19b 19a 20, 21

17, 17, 21 18 18 19b 19a 3, 20

7.4, CH3

2.57, dq (14.7, 7.4) 0.98, t (7.4)

20, 21 20, 20

20, 21 15, 17, 20

3.87, s 3.90, s

2, 7, 8 2, 7

8, 9, 12 9, 13 8, 10

1.85, td (14.2, 3.6); 1.66, m 1.62, m 2.18, m 2.94, m; 3.30, br d (10.8) 1.61, m;

22 173.3, C 23 54.8, CH3 955.3, CH3 OMe

2, 7, 16, 17, 19, 20 19 7, 3

11 10, 12 11

2.27, m

C HMBC

3, 17 3, 15, 16, 17, 21 3, 15, 16, 21 20, 16

Figure 3. Postulated conversion of indole alkaloids in Vinca minor in response to stress induced by MeJA treatment.

22 9

of such conversions could be provided by classical pulse-chase experiments using 13C- or 14C-labeled alkaloids, which could be generated by feeding the corresponding labeled precursors (tryptophan) to V. minor plants before MeJA treatment. Jasmonic acid is a part of numerous signal transduction chains, including the elicitation of phytoalexins,26,27 the enhancement of protective secondary metabolites in response to various environmental factors such as insect attacks7 or abiotic stress situations,28 and the induction of senescence.8,10 Accordingly, the application of MeJA will induce many different metabolic events, which cannot be attributed to only one unique stress situation. Moreover, this elicitation is strongly affected by the physiological status of the leaves. Notwithstanding, elicitation generally leads to qualitative as well as quantitative changes in the content of bioactive secondary metabolites.29−31 In general, these alterations are due to related changes in the expression of the enzymes responsible for the biosynthesis.6,12,25,32 These coherences are nicely documented by the large differences in the alkaloid contents (vincamine and total indole alkaloids) in hairy root cultures of V. minor treated with different combinations of growth regulators (e.g., H2O2) and various inhibitors of the enzymes involved in the biosynthesis of indole alkaloids (e.g., cyclo-oxygenase).33 However, the authors did not analyze the entire alkaloid pattern. The complexity of the signal-transducing effects of jasmonic acid is vividly demonstrated by the putative contradictory findings in C. roseous. Whereas in hairy root cultures it leads to a significant

additionally confirmed the presence of eight further carbons including a methoxycarbonyl, a hemiaminal carbon, three quaternary carbons, an oxygenated tertiary carbon, and two nitrogenated tertiary carbons. The NMR data were closely related to those of vincamine (1), with the key difference of the replacement of H-9 (Figure 2) in 1 with an oxygenated tertiary carbon (δC 154.7) in 2. An additional O-methyl (δH 3.89, δC 55.3) group showed an HMBC correlation to C-9. In addition, the splitting pattern of the aromatic protons H-10, H-11, and H-12 indicated that they are contiguous and ortho coupled as J10,11 = J11,12 = 8.2 Hz. Therefore, the structure of 2 was defined as 9-methoxyvincamine. A closely related compound, 11methoxyvincamine, was reported earlier from V. minor,23,24 but 9-methoxyvincamine was isolated for the first time as a natural product. Based on the structural similarities of vincamine (1) and 9methoxyvincamine (2) and their inverse changes in concentrations, it is postulated that 1 may be a precursor for 2 (Figure 3). In the same manner, it could be deduced that vincadifformine (5) is converted first to minovincinine (6) and subsequently to minovincine (7) (Figure 3). Because of the similarities to other modifications of secondary metabolites,12,25 it is likely that these reactions are catalyzed by cytochrome P450 enzymes. An unequivocal confirmation for the occurrence 2907



Article

six plants were cut from their roots and shock frozen and freeze-dried in liquid nitrogen. Analytical Methods. For determination of alkaloids, 300 mg of freeze-dried leaves was ground to a fine powder using a ball mill (RetschMM200). Then a sample was extracted with methanol (3 × 2 mL) in an ultrasonic bath at 50 °C for 30 min. After centrifugation (10 min at 5000g), the combined MeOH extracts were evaporated using a Zymark Turbo Vap evaporator at 50 °C. The dried MeOH extract was further extracted with 2 mL and subsequently with 1 mL of 3% HCl (twice) in an ultrasonic bath at 50 °C for 30 min. After centrifugation (10 min at 5000 rpm), the combined aqueous extracts were cooled at 4 °C and treated with 25% NH4OH to reach pH 8−9. Alkaloids were extracted with CHCl3 (3 × 2 mL). Combined CHCl3 extracts were dried (constant weight) and dissolved in MeOH (1 mL). Before performing LCMS analysis, samples were filtered (0.45 μm, Spartan). The separation was performed on a Kinetex C18-column (1.7 μm, 100 Å, 150 × 2.1 mm) from Phenomenex, using a gradient system. Solvent A was H2O with 0.1% formic acid, and solvent B was MeCN with 0.1% formic acid. The gradients used were 0 min, 1% B; 5 min, 15% B; 42 min, 30% B; and 50 min, 100% B. The flow rate was 300 μL/min. The overall runtime was 60 min at a column temperature of 40 °C. Extraction and Isolation of Pure Compounds from Treated Plants. Ten grams of freeze-dried aerial parts was extracted as previously mentioned to obtain approximately 150 mg of crude alkaloids. The alkaloids were dissolved in MeOH and separated by HPLC. A: MeCN; B: NH4OAc (15 mM), containing 0.2% Et3N, adjusted with HOAc to pH 4. The gradients used were 0 min, 15% A, 85% B; 7 min, 20% A, 80% B; 42 min, 40% A, 60% B; 47 min, 80% A, 20% B; 50 min, 15% A, 85% B; and 60 min, 15% A, 85% B. The flow rate was 3 mL/min. The injection volume was 100 μL by manual injection. Alkaloids were monitored using a photo diode array (PDA) detector at 254, 280, and 330 nm. Fractions were manually collected and combined according to the observed peaks. Seven fractions were collected (Figure S20, Supporting Information). Fraction 1 afforded compound 6 with a retention time tR = 16.8 min; fraction 2, compound 4 at tR = 21.8 min; fraction 3, compound 1 at tR = 25.9 min; fraction 4, compound 7 at tR = 29.4 min; fraction 5, compound 2 at tR = 30.2 min; fraction 6, compound 3 at tR = 35.1 min; and fraction 7, compound 5 at tR = 39.3 min. Due to the presence of NH4OAc and Et3N, the exact weight of the substances could not be determined at this stage of isolation. Thus, to eliminate the NH4OAc and Et3N and for further purification the freeze-dried samples were dissolved in MeOH and again purified by HPLC. Solvent A: MeCN 0.1% formic acid or TFA; B: H2O with 0.1% formic acid or TFA. The gradients used were 0 min, 15% A; 5 min, 20% A; 42 min, 40% A; 45 min, 80% A. The flow rate was 3.5 mL/min. After freeze-drying, the alkaloids were weighed (approximate yields compound 6 = 3 mg, 4 = 3 mg, 1 = 3 mg, 7 = 1.5 mg, 2 = 3 mg, 3 = 2 mg, 5 = 2 mg) and analyzed. Vincamine TFA salt (1): UV (MeOH) λmax 220, 269 nm; 1H and 13 C NMR see Table Sa, Supporting Information; HRESIMS m/z 355.2024 ([M + H]+, calcd for C21H27N2O3, 355.2016). 9-Methoxyvincamine TFA salt (2): colorless oil, [α]20D +117 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 224 nm (3.63), 268 nm (3.06) nm; 1H and 13C NMR see Table 2; HRESIMS m/z 385.2130 ([M + H]+, calcd for C22H29N2O4, 385.2122). Strictamine TFA salt (3): UV (MeOH) λmax 220, 270 nm; 1H and 13 C NMR see Table Sb, Supporting Information; HRESIMS m/z 323.1757 ([M + H]+, calcd for C20H23N2O2, 323.1745). 10-Hydroxycathofoline TFA salt (4): UV (MeOH) λmax 208, 312 nm; 1H and 13C NMR see Table Sc, Supporting Information; HRESIMS m/z 355.2023 ([M + H]+, calcd for C21H27N2O3, 355.2016). Vincadifformine TFA salt (5): UV (MeOH) λmax 221, 293, 330 nm; 1 H and 13C NMR see Table Sd, Supporting Information; HRESIMS m/z 339.2072 ([M + H]+, calcd for C21H27N2O2, 339.2067). Minovincinine TFA salt (6): UV (MeOH) λmax 220, 290, 330 nm; 1 H NMR (DMSO-d6, 700 MHz) δH 9.45 (s, N1−H), 7.20 (d, J = 7.5 Hz, 9-H), 7.07 (dd, J = 7.5, 7.5 Hz, 11-H), 7.01 (d, J = 7.5 Hz, 12-H), 6.79 (dd, J = 7.5, 7.5 Hz, 10-H), 3.18 (q, J = 6.5 Hz, 19-H), 3.05 (m, 3-

increase in the content of indole alkaloids and to a shift in their composition,12,13 it seems to have no effect on the indole alkaloid accumulation in mature C. roseus leaves.14 Moreover, the response of developing seedlings of C. roseus to MeJA varies strongly.11,6 Schluttenhofer et al. tried to explain these contradictions by the involvement of the WRKY transcription factors,34 which are key regulators of many processes in plants, including responses to drought stress, salt stress, and also exposure to jasmonates.35 The authors showed that the MeJArelated induction of strictosidine synthase, the key enzyme of indole alkaloid biosynthesis, is correlated with corresponding changes in the expression of WRKYs. However, as the WRKYs also regulate various stress-related temporary downstream steps in modifications of the main alkaloidal structures, the diverse responses may interfere, resulting in a complex physiological situation.34 Many further approaches are required to elucidate the observed changes in the pattern of indole alkaloids in V. minor. In summary, we postulate that MeJA treatment induces oxidative enzymes in V. minor, as suggested by the shifts in alkaloid distribution from vincamine (1) to 9-methoxyvincamine (2) and from vincadifformine (5) first to minovincinine (6) and finally to minovincine (7). The identification of the enzymes invoved appears to be an attractive topic for further studies. In addition, the discovery of 9-methoxyvincamine (2) as a novel natural product further implies that even well-known, extensively studied plants such as V. minor might be an auspicious source for the generation of novel phytochemical drugs, when grown under defined stress conditions.

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EXPERIMENTAL SECTION

General Experimental Procedures. High-resolution LC-MS analysis was performed using a Bruker Maxis HD UHR-TOF mass spectrometer with an Apollo II ion funnel ESI electrospray source. A UHPLC system (Ultimate3000RS from Dionex/Thermo) was used for separation. Optical rotations were determined using a PerkinElmer 241 spectrometer, and UV spectra were recorded using a Shimadzu UV-2450 UV−vis spectrophotometer. The HPLC system consisted of a Young Lin quaternary pump, vacuum degasser, column compartment, and diode array detector with a Midas Spark Holland autosampler. Separation was performed on a VP 250/10 Nucleosil 100-5 μ RP-18 semipreparative column. NMR spectra were recorded using a Bruker Avance III 700 spectrometer with a 5 mm TCI cryoprobe (1H 700 MHz, 13C 175 MHz) and an Avance III 500 (1H 500 MHz, 13C 125 MHz) spectrometer. Plant Material. Vinca minor mature plants at the blooming stage were purchased from a commercial market garden (Brennecke GmbH, Braunschweig, Germany). The individual plants were transferred into pots (15 cm in diameter) containing a soil−sand mixture (3:1), prepared from commercially available substrate (Floragard, Oldenburg, Germany) and sand. During the acclimation phase, the soil moisture of all pots was adjusted to 25−30%, and the temperature range during the experiment was 15−20 °C. Plants were grown in the garden of the Institute of Plant Biology of TU Braunschweig. Plants were kept under rain shelter to prevent water input from the rain. Application of Methyl Jasmonate. For application of MeJA, plants were taken out of the growth area and transferred in an open area at about 20 m distance from the control plants and sprayed with MeJA solution (0.5 mM, containing 0.2% Triton X) on both sides of the leaves, until liquid dripped from the leaves (15 mL/plant). MeJA was applied two times during the experiment (on days 1 and 4). Treated plants were harvested after 9 days. Sampling. Arial parts of experimental plants were cut from their roots. Leaves were separated from the stems. The plant material was shock frozen in liquid nitrogen and stored at −20 °C until further extraction and analyses. For preparative work, above-ground parts of 2908



Article

Prospects; Naeem, M., Aftab, T., Khan, M. M. A., Eds.; Springer International Publishing: Cham, 2017; p 47. (15) Pan, Q.; Chen, Y.; Wang, Q.; Yuan, F.; Xing, S.; Tian, Y.; Zhao, J.; Sun, X.; Tang, K. Plant Growth Regul. 2010, 60, 133−141. (16) D’Amelio, F. S., Sr.; Mirhom, Y. W.; Williamson, Y. V.; Schulbaum, P. L.; Krueger, E. B. Planta Med. 2012, 78, PF4. (17) Hasa, D.; Perissutti, B.; Dall’Acqua, S.; Chierotti, M. R.; Gobetto, R.; Grabnar, I.; Cepek, C.; Voinovich, D. Eur. J. Pharm. Biopharm. 2013, 84, 138−144. (18) Proksa, B.; Grossmann, E. Phytochem. Anal. 1991, 2, 74−76. (19) Taylor, W. I. In The Alkaloids: Chemistry and Physiology; Manske, R. H. F., Ed.; Academic Press: New York, 1965; Vol. 8; Chapter 12, pp 269−285. (20) Vas, A.; Gulyas, B. Med. Res. Rev. 2005, 25, 737−757. (21) Boyadzhiev, L.; Mecheva, D.; Yordanov, B. C. R. Acad. Bulg. Sci. 2002, 55, 12−49. (22) Balsevich, J.; Constabel, F.; Kurz, W. G. W. Planta Med. 1982, 44, 91−93. (23) Štrouf, O.; Trojánek, J. Collect. Czech. Chem. Commun. 1964, 29, 447−456. (24) Szabó, L.; Kalaus, G.; Szántay, C. Nat. Prod. Lett. 1996, 8, 237− 240. (25) Giddings, L. A.; Liscombe, D. K.; Hamilton, J. P.; Childs, K. L.; DellaPenna, D.; Buell, C. R.; O’Connor, S. E. J. Biol. Chem. 2011, 286, 16751−16757. (26) Tamogami, S.; Rakwal, R.; Kodama, O. FEBS Lett. 1997, 412, 61−64. (27) Konan, Y. K. F.; Kouassi, K. M.; Kouakou, K. L.; Koffi, E.; Kouassi, K. N.; Sekou, D.; Kone, M.; Kouakou, T. H. Int. J. Agron. 2014, 2014, 1−11. (28) Ahmad, P.; Rasool, S.; Gul, A.; Sheikh, S. A.; Akram, N. A.; Ashraf, M.; Kazi, A. M.; Gucel, S. Front. Plant Sci. 2016, 7, 813. (29) Poulev, A.; O’Neal, J. M.; Logendra, S.; Pouleva, R. B.; Timeva, V.; Garvey, A. S.; Gleba, D.; Jenkins, I. S.; Halpern, B. T.; Kneer, R.; Cragg, G. M.; Raskin, I. J. Med. Chem. 2003, 46, 2542−2547. (30) Wojakowska, A.; Muth, D.; Narozna, D.; Madrzak, C.; Stobiecki, M.; Kachlicki, P. Metabolomics 2013, 9, 575−589. (31) Sampaio, B. L.; Edrada-Ebel, R.; Da Costa, F. B. Sci. Rep. 2016, 6, 29265. (32) Rischer, H.; Oresic, M.; Seppänen-Laakso, T.; Katajamaa, M.; Lammertyn, F.; Ardiles-Diaz, W.; Van Montagu, M. C.; Inzé, D.; Oksman-Caldentey, K. M.; Goossens, A. Proc. Natl. Acad. Sci. U. S. A. 2006, 103, 5614−5619. (33) Verma, P.; Khan, S. A.; Mathur, A. K.; Shanker, K.; Lal, R. K. Appl. Biochem. Biotechnol. 2014, 173, 663−672. (34) Schluttenhofer, C.; Pattanaik, S.; Patra, B.; Yuan, L. BMC Genomics 2014, 15, 502. (35) Phukan, U. J.; Jeena, G. S.; Shukla, R. K. Front. Plant Sci. 2016, 7, 760. (36) Das, B.; Biemann, K.; Chatterjee, A.; Ray, A. B.; Majumder, P. L. Tetrahedron Lett. 1966, 7, 2483−2486. (37) Plat, M.; Fellion, E.; Le Men, J.; Janot, M. Ann. Pharm. Fr. 1962, 20, 899−906. (38) Laforteza, B. N.; Pickworth, M.; Macmillan, D. W. C. Angew. Chem., Int. Ed. 2013, 52, 11269−11272. (39) Akhgari, A.; Laakso, I.; Seppänen-Laakso, T.; Yrjönen, T.; Vuorela, H.; Oksman-Caldentey, K. M.; Rischer, H. A. Molecules 2015, 20, 22621−22634. (40) Kovácǐ k, V.; Kompiš, I. Collect. Czech. Chem. Commun. 1969, 34, 2809−2818. (41) Mokrý, J.; Kompiš, I.; Spiteller, G. Collect. Czech. Chem. Commun. 1967, 32, 2523−2531.

H′), 2.84 (m, 5-H′), 2.54−2.60 (m, 27-H, 21-H), 2.45 (m, H-5′), 2.33 (dt, J = 10.5 Hz, 3.0 Hz, 3-H′′), 1.85 (m, 6-H′), 1.72 (m, 14-H′), 1.55−1.46 (m, 14-H′′, 6-H′′, 15-H′), 1.41 (m, 15-H′′), 0.73 (d, J = 6.5, 18-H3); 13C NMR (DMSO-d6, 175 MHz) δC 167.7 (C-22), 166.5 (C-2), 143.8 (C-13), 136.7 (C-8), 127.2 (C-11), 120.4 (C-9), 119.9 (C-10), 109.9 (C-12), 91.3 (C-16), 67.4 (C-21), 64.9 (C-19), 55.0 (C7), 50.9 (C-5), 50.4 (C-23), 50.2 (C-3), 46.0 (C-6), 42.1 (C-20), 25.7 (C-17), 25.6 (C-15), 21.4 (C-14), 18.0 (C-18); HRESIMS m/z 355.2023 ([M + H]+, calcd for C21H27N2O3, 355.2016). Minovincine TFA salt (7): UV (MeOH) λmax 221, 293, 331 nm; 1H and 13C NMR see Table Sf, Supporting Information; HRESIMS m/z 353.1869 ([M + H]+, calcd for C21H25N2O3, 353.1860).

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b00424. 1 H and 13C NMR spectra of compounds 1−7 and 2D NMR spectra of 2 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: + (49)-531-391-5881. Fax: + (49)-531-391-8180. E-mail: [email protected]. ORCID

Sara Abouzeid: 0000-0001-5843-7772 Frank Surup: 0000-0001-5234-8525 Dirk Selmar: 0000-0003-2331-3168 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The Egyptian Ministry of Higher Education and Scientific Research is acknowledged for the fellowship support to S.A. F.S. thanks Prof. M. Stadler for generous support and C. Kakoschke for help with NMR measurements.

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REFERENCES

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