Alkaloids from Pleiocarpa pycnantha: Pleiocarinine, a Bisindoline

Article Cite This: J. Nat. Prod. 2018, 81, 1193−1202

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Carbazole-, Aspidofractinine-, and Aspidocarpamine-Type Alkaloids from Pleiocarpa pycnantha Joseph T. Ndongo,*,†,‡ Joséphine N. Mbing,§ Aymeric Monteillier,⊥ Michel F. Tala,‡ Michael Rütten,∥ Daniel Mombers,∥ Muriel Cuendet,⊥ Dieudonné E. Pegnyemb,§ Birger Dittrich,∥ and Hartmut Laatsch*,‡ †

Department of Chemistry, Higher Teacher Training College, University of Yaoundé 1, P.O. Box 47, Yaoundé, Cameroon Institute of Organic and Biomolecular Chemistry, University of Göttingen, Tammannstrasse 2, D-37077 Göttingen, Germany § Department of Organic Chemistry, Faculty of Science, University of Yaoundé 1, P.O. Box 812, Yaoundé, Cameroon ⊥ School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Rue Michel-Servet 1, CH-1211 Genève 4, Switzerland ∥ Institute of Inorganic and Structural Chemistry, Heinrich-Heine University Düsseldorf, Universitätsstrasse 1, D-40225 Düsseldorf, Germany ‡

S Supporting Information *

ABSTRACT: Three new alkaloids, janetinine (1a), pleiokomenine A (2), and huncaniterine B (3a), and 13 known compounds, pleiomutinine (3b), huncaniterine A (3c), 1-carbomethoxy-β-carboline (4), evoxanthine (5), deformyltalbotine acid lactone (6), pleiocarpamine (7), N4-methyl-10hydroxygeissoschizol (8), spegatrine (9), neosarpagine (10), aspidofractinine (11), N1-methylkopsinin (12), pleiocarpine (13), and N1methylkopsinin-N4-oxide (14), were isolated from the stem bark of Pleiocarpa pycnantha. Janetinine (1a) is a carbazole alkaloid; in pleiokomenine A (2), two aspidofractinine-type alkaloids are bridged by a methylene unit in an unprecedented way, and huncaniterine B (3a) is a pleiocarpamine−aspidofractinine-type dimer. The structures and relative configurations of these compounds were elucidated on the basis of NMR and MS analyses. Their absolute configurations were defined by means of experimental and calculated ECD data, and additionally, the structures of 5 and 13 were determined by single crystal X-ray diffraction. Compounds 1a, 2, 3b, 4, 6, 9, and 12 displayed cancer chemopreventive properties through either quinone reductase induction (CD = 30.7, 30.2, 29.9, 43.5, and 36.7 μM for 1a, 4, 6, 9, and 12, respectively) and/or NF-κB inhibition with IC50 values of 13.1, 8.4, 9.4, and 8.8 μM for 2, 3b, 6, and 12, respectively.

T

Known compounds isolated in the present investigation are pleiomutinine (3b), huncaniterine A (3c), 1-carbomethoxy-βcarboline (4), evoxanthine (5), the indole alkaloids deformyltalbotine acid lactone (6), pleiocarpamine (7), N4-methyl-10hydroxygeissoschizol (8), spegatrine (9), and neosarpagine (10), and the monomeric and dimeric 2,3-dihydroindoles aspidofractinine (11), N1-methylkopsinin (12), pleiocarpine (13), and N1-methylkopsinin-N4-oxide (14) (Figure 1). Herein the isolation, structure determination, and biological activity of the new alkaloids, as well as the full 1H and 13C NMR assignments of deformyltalbotine acid lactone (6) and its first identification as a natural product, are described. The cancer chemopreventive activity of the crude extracts and isolated compounds was evaluated through quinone reductase (QR) induction and NF-κB inhibition activity, two well-established targets in cancer chemoprevention.5,6

he genus Pleiocarpa (Apocynaceae) is distributed in tropical Africa from Senegal to Tanzania and in Zimbabwe. The name Pleiocarpa is derived from the Greek words pleio (many, full of) and karpos (fruit).1 This genus, which is chemically still underexploited, incorporates 22 different scientific plant names, out of which only six are accepted species names. Preparations from the genus Pleiocarpa are used in traditional medicine for the treatment of gastrointestinal ailments, fever, malaria, pain, diabetes, and cancer.2 Plants of this genus are rich sources of indole and bisindole alkaloids. Previous studies on the root and bark extracts of Pleiocarpa pycnantha resulted in the isolation of a number of indole alkaloids, such as pycnanthine, pleiocarpamine (7), quebrachamine, macusine, and (−)-eburnamine.3,4 As part of a search for bioactive alkaloids, a new carbazole and two new bis(2,3-dihydroindole) alkaloids, namely, janetinine (1a), pleiokomenine A (2), and huncaniterine B (3a), respectively, were isolated from the stem bark of P. pycnantha (K. Schum.) Stapf collected in the Centre Region of Cameroon. © 2018 American Chemical Society and American Society of Pharmacognosy

Received: November 14, 2017 Published: April 17, 2018 1193

DOI: 10.1021/acs.jnatprod.7b00958 J. Nat. Prod. 2018, 81, 1193−1202

Journal of Natural Products

Article

Figure 1. Alkaloids 1a−14 isolated from P. pycnantha.

■

RESULTS AND DISCUSSION

OH and NH functions, while the UV spectrum with absorption maxima at 217, 232, 239, 250, 264, 300, and 345 nm was indicative of a carbazole chromophore (Figure S1, Supporting Information). ESIMS data showed an [M + H]+ ion at m/z 267, and ESIHRMS and 13C NMR data established the molecular formula as C17H18N2O. The 13C NMR and HSQC data showed a total of 17 carbon resonances, including a methyl, three methylene, and six methine groups, two tertiary carbons linked to the indolic nitrogen (corresponding to C-2 and C-13), and five quaternary carbon atoms (Table 1); four methines were due to the aromatic hydrogens of a 1,2disubstituted benzene moiety (δH 7.15−8.04); a one-proton singlet appeared in the aromatic region (δH 7.97, δC 118.2). One of the methylenes (δH 5.02, δC 58.3) appeared as a twoproton singlet, indicating an isolated spin system. The COSY spectrum (Figure 2) displayed the presence of NCH2CH2Cq

The aqueous P. pycnantha extract gave an orange precipitate with Dragendorff’s reagent, indicative of the presence of alkaloids. Extraction of alkaloids from the crude MeOH extract was carried out in a standard manner using the Stas−Otto method7,8 by partitioning the concentrated MeOH extract between acidic or basic aqueous phases, respectively, and EtOAc and subsequently n-BuOH. The alkaloids were separated by column chromatography on silica gel using a CH2Cl2/MeOH solvent system with increasing polarity and finally on Sephadex LH-20. The structures of the pure alkaloids were elucidated by a combination of NMR and MS methods. Janetinine (1a) was isolated as an optically active, pale yellowish, amorphous solid. The IR spectrum with absorption bands at 3450, 3380, and 3266 cm−1 indicated the presence of 1194



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Table 1. 1H and 13C NMR Data of Janetinine (1a) and Deformyltalbotine Acid Lactone (6)a 1ab position

δC

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

140.2 40.7

6c

δH (mult, J in Hz) 3.47 m 3.49 m

δC 134.2d 51.5 44.4 22.0d

124.0 123.8 120.9 120.0 127.1 111.9 142.0 23.9 128.5 121.5 58.3 20.3 53.2 125.5 118.2

8.04 7.15 7.37 7.48

configuration of 1a was defined by comparison of its experimental and computed electronic circular dichroism (ECD) curves (Figure 3). This was supported by the specific 20 rotation of 1a ([α]20 D +8.2) and 1b ([α]D +8.0), which also agreed with the positive sign of the calculated OR value. Pleiokomenine A (2) was isolated as a yellowish resin, which gave a pink spot on TLC after spraying with anisaldehyde/ sulfuric acid. The green color reaction with Ehrlich’s reagent and the UV spectrum with maxima at 207, 215, 262, and 304 nm were indicative of an indoline chromophore, while the IR spectrum showed an absorption band at 1724 cm−1, suggesting the presence of an ester functionality. ESIMS data showed an [M + H]+ ion at m/z 717, and ESIHRMS measurements established the molecular formula as C45H56N4O4. The 13C NMR data (Table 2) displayed, however, only 23 carbon resonances comprising two methyl, nine methylene, and five methine carbons, one carbonyl at δC 174.5, two tertiary carbons linked to nitrogen, and four quaternary carbons, so that a symmetric structure was assumed. The 1H NMR (Table 2) and COSY data showed the typical ABX pattern of a 1,2,4trisubstituted benzene ring, with 1H signals at δH 7.46 (d, J = 1.7 Hz), 6.85 (dd, J = 8.0, 1.7 Hz), and 6.31 (d, J = 8.0 Hz). In combination with HSQC and HMBC data, a singlet at δH 3.45 (δC 66.9) was interpreted as the signal of an isolated azamethine group; additionally, an N-methyl singlet at δH 2.66 (δC 31.2) and a methyl ester signal at δH 3.73 (δC 52.4) were evident. Furthermore, an isolated methylene group and N4−C-5−C-6−C-7, N-4−C-3−C-14−C-15−C-20, C-2−C-16− C-17−C-20, and C-2−C-18−C-19−C-20 partial structures were derived. Substructure searches in the Dictionary of Natural Products on DVD10 with these fragments indicated an aspidofractinine-type alkaloid.11 Therefore, on the basis of the MS and NMR data, 2 was assumed to be a dimeric aspidofractinine linked via the benzene rings by an isolated methylene group in a symmetrical fashion. The C-10−CH2−C10′ connection was further confirmed by the two- and threebond HMBC correlations from the methylene protons H2-22 at δH 3.74 to C-9,9′ at δC 122.3, C-10,10′ at δC 133.5, and C11,11′ at δC 127.7 (Figure 4). The NOE interactions (Figure 5) between H-9,9′ and H-11,11′ with H2-22 provided further support for the methylene group connecting two identical aspidofractinine units at C-10,10′. The relative configuration was established from coupling constants and extensive NOESY interpretations: correlations between H-16,16′/H2-18,18′ and H-16,16′/Me-N1,Me′-N1′, as well as interactions between H21,21′/H2-19,19′ (but not between H-16,16′/H2-6,6′), indicated that these proton groups were in the same spatial orientation as expected for N1-methylkopsinin (12) or the aspidofractinine moiety (see also Figure S3, Supporting Information). Compound 2 is therefore a bisindoline alkaloid constituted by connection of two N1-methylkopsinin (12) units via a methylene group; 2 was recently isolated from the stem bark of Pleiocarpa mutica by Champy, Beniddir, et al.35 and was named pleiokomenine A. The similarity between the ECD spectra of 2 and 12 indicated identical absolute configurations, as expected also for biosynthetic reasons; this was further confirmed by the DFT-calculated ECD data (Figure S4, Supporting Information). A possible biosynthetic pathway toward 2 is presented in Scheme 1. The sequence is initiated by a hydroxymethylation of N1-methylkopsinin (12) with formaldehyde.12 A Friedel−Crafts reaction of a second N1methylkopsinin (12) onto the intermediate i should lead to dimer ii, followed by deprotonation to 2.

d (8.2) t (8.2) t (8.2) d (8.2)

3.29 m 3.39 m

5.02 s 1.81 d (6.8) 4.74 q (6.8) 7.97 s

107.8 128.4 118.7 120.2 121.9 109.8 138.1 32.3d 36.0 54.9 168.9d 14.2 124.3 131.3 71.1

δH (mult, J in Hz) 4.09 m 3.15 3.54 2.73 2.88

dt (12.9, 6.1) brdd (12.3, 5.4) dd (15.6. 4.1) m

7.47 7.11 7.18 7.15

d (8.0) t (8.0) t (8.0) d (8.0)

1.30 2.40 3.64 5.18

ABX (12.9) brd (12.9) brdd (10.8, 5.7) d (5.7)

1.81 d (6.8) 5.71 q (7.1) 4.67 d (14.0) 5.04 d (14.0)

a

Assignments based on COSY, HSQC, and NOESY. bMethanol-d4, 600 (1H) and 125 MHz (13C). cCDCl3, 600 (1H) and 125 MHz (13C). d Derived from HMBC spectrum.

Figure 2. COSY, selected HMBC, and NOE correlations of janetinine (1a).

and NCH(CH3)Cq partial structures, which were shown by the HMBC data to correspond to N-3−C-4−C-14−C-15 and N3−C-19(C-18)−C-20, respectively, of an olivacine skeleton.9 The assignment of the singlet at δH 7.97 (δC 118.2) to H-21 in 1a was confirmed by the three-bond HMBC correlations from this hydrogen to C-8, C-15, and C-19. This was supported by the NOE interactions between H-21 and H-9 and between H18 and H-19. The isolated methylene must be connected to an oxygen to account for its downfield shifts (δH 5.02, δC 58.3) and was linked, therefore, with C-16. This assignment was supported by the three-bond HMBC correlations from H2-17 to C-2 (δC 140.2) and C-15 (δC 128.5), as well as the NOE correlation between H2-17 and H-14a (Figure 2). Comparison of the 1H and 13C NMR data showed a similarity to those of janetine (1b),9 except for replacement of the C-16 methyl signal by the singlet of a hydroxymethyl group. The absolute R 1195



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Figure 3. ECD spectra of janetinine (1a); green: experimental in methanol, blue: calculated for (R)-1a.

A with B via C-12′ and the isolated N-methylene group C-22′ (δH 3.81/3.99, δC 41.7), forming the dimeric indoline alkaloid pleiomutinine (3a). Despite identical HMBC correlations, the pleiocarpamine moiety in 3a showed strong shift differences with respect to 3b and 3c for C-3,5,14,19, and C-21 (Table 2), suggesting that the remaining oxygen atom is present as an N4-oxide of unit B. On the basis of these considerations, the structure of huncaniterine B was elucidated as 3a; it is an isomer of the co-occurring pleiomutinin-N4′-oxide huncaniterine A (3c). The relative configuration of 3a was determined by NOESY correlations. Cross signals between H3-18′/21′ indicated the endo-orientation of Me-18′. Further correlations between H5′b/16′a, H-5′b/17′a, H-15′a/19′, H-15′b/21′, H3-18′/15′b, and H3-18′/H-22′a showed that these hydrogens were in the same spatial orientation as in monomeric cycloaspidospermidine. The NOESY correlations of H-19 to H-21a, and Me-18 to H15, established the Z configuration of the ethylidene side chain, while correlations from H-3 to Me-18′ and H-5a, as well as from H-16 to H-14a and H-15, indicated that these protons were cofacial oriented in the pleiocarpamine unit. On the basis of molecular modeling and for biosynthetic reasons, the NOESY correlations of H-22′b with H-9, H-5a, and H-16a, and H-11′ with H-14b and H-16, established the geometry of bridging the two alkaloid monomers as found in pleiomutinine (3b) and in the N-oxide 3c. The NOE association between H16 and H-11′ and additionally the AB signal of CH2-22′ confirmed the 16S* configuration.15 The absolute configuration of 3a was determined by comparison of the experimental and calculated ECD spectra and found to be identical with those of the pleiomutinins 3b and 3c (Figure S8, Supporting Information). Deformyltalbotine acid lactone (6) was isolated as a yellowish resin. The IR spectrum showed bands at 3380 and 1735 cm−1 indicating the presence of NH and ester carbonyl functionalities, while the UV spectrum showed indole absorption bands at 224, 275, 286, and 290 nm (Figure S9, Supporting Information). The molecular formula was estab-

The new compound huncaniterine B (3a) showed a UV spectrum reminiscent of an alkylaniline/hydroindole as well, with absorption maxima at 205, 221, 264, and 290 nm (Figure S6, Supporting Information). ESIMS of 3a showed a protonated molecular ion [M + H]+ at m/z 631, and ESIHRMS (m/z 631.3615) established the molecular formula as C40H47N4O3. The 13C NMR spectrum confirmed the number of carbon atoms as in the molecular formula (Table 2), and the HSQC spectra of 3a distinguished three methyl, 12 methylene, and 13 methine groups, four tertiary carbons linked to the nitrogen, and eight quaternary carbon atoms. The 1H NMR spectrum (Table 2) displayed a 1,2,3trisubstituted benzene moiety with 1H signals at δH 7.21 (d, J = 7.9 Hz), 6.62 (t, J = 7.9 Hz), and 7.39 (d, J = 7.9 Hz). The COSY and HMBC data showed an isolated azamethine H-21′ (δH 4.09, δC 72.7), and the fragments N-4′−C-5′−C-6−C-7′, N-4′−C-3′−C-14′−C-15′ and C-2′−C-19′−(C-18′)−C-20′ indicated a cycloaspidospermidine unit. Analysis of the 2D NMR data confirmed this substructure, unit A, in 3a (Figure 6). The 1H and 13C NMR signals at δH 7.10 (d, J = 7.6 Hz), 6.68 (t, J = 7.6 Hz), 6.93 (t, J = 7.6 Hz), and 6.11 (d, J = 7.6 Hz) indicated an additional 1,2-disubstituted benzene moiety. A further azamethine signal at δH 5.18 (d, J = 4.8 Hz) is characteristic for H-16 of pleiocarpamine (7)13 and bisindolines containing this structure, such as the co-occurring pleiomutinine (3b, δH 5.02, J = 4.8 Hz; δC 57.7) and huncaniterine A (3c, δH 5.06, J = 4.4 Hz; δC 57.2).14 The carbonyl resonance at δC 171.3 and the HMBC-correlated methoxy group at δH 3.74 (δC 53.0) indicated the presence of an ester function, which was confirmed by the IR absorption maximum at 1732 cm−1. HMBC and COSY data (Figure 6) showed, in addition to an N-4−C-21−C-20 fragment and an ethylidene side chain (δMe‑18 1.75, δH‑19 5.63), the presence of N-4−C-5−C-6−C-7 and N4−C-3−C-14−C-15−C-16−N-1 substructures. The threebond HMBC correlations from H-18 to C-20 and from H-19 to C-15 and C-21 indicated the connection of the ethylidene group with C-20, confirming a pleiocarpamine unit B in 3a. Three-bond HMBC correlations from H-11′ and H2-22′ to C-2 of the second fragment B indicated the connection of units 1196



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Table 2. 1H and 13C NMR Data of Pleiokomenine A (2), Huncaniterine B (3a), Pleiomutinine (3b),a and Huncaniterine A (3c) 2b position

δC

2 3a 3b 5a 5b 6a 6b 7 8 9 10 11 12 13 14a 14b 15a 15b 16 17a 17b 18a 18b 19a 19b 20 21a 21b 22 COOMe COOMe NMe 2′ 3′a 3′b 5′a 5′b 6′a 6′b 7′ 8′ 9 10 11 12′ 13′ 14′a 14′b 15′a 15′b 16′a 16′b 17′a 17′b 18′a 18′b 19′a 19′b 20′

70.7 46.9 50.5 33.7 57.3 137.4 122.3 133.5 127.7 108.2 148.9 15.9 34.3 41.6 32.4 23.9 33.8 30.1 66.9 41.4 174.5 52.4 31.2 70.7 46.9 50.5 33.7 57.3 137.4 122.3 133.5 127.7 108.2 148.9 15.9 34.3 41.6 32.4 23.9 33.8 30.1

δH (mult, J in Hz) 3.25 3.30 3.42 3.46 1.72 2.74

m m m m dt (15.0, 9.7) m

7.46 d (1.7) 6.85 dd (8.0, 1.7) 6.31 d (8.0) 1.60 1.93 1.38 1.58 2.96 1.45 2.77 1.48 1.49 1.32 1.40

m m m m dd (10.8, 8.3) m m m m m m

3.45 s

3ac δC 70.9 71.0 64.4 33.6 46.5 134.1 122.4 120.4 128.6 110.8 146.8 22.1 30.7

3bb

δH (mult, J in Hz) 3.86 m 3.72 3.93 2.07 2.43

7.10 6.68 6.93 6.11

δC 67.3 54.1

m m m m

48.1 27.5 46.1 135.0 120.3 117.9 126.9 109.2 146.8 26.4

d (7.6) t (7.6) t (7.6) d (7.6)

2.71 m 3.12 m 3.77 m

30.6

δH (mult, J in Hz) 3.19 brs 2.88 brd (14.2, 4.2) 3.19, 3.20 br ABM (∼14) 1.17 d (14.6) 2.44 dt (14.2, 4.1)

6.89 6.58 6.88 6.03

d (7.5) t (7.5) t (7.5) d (7.5)

1.85 m 3.12 m 3.53 m

3cc δC

δH (mult, J in Hz)

67.3 54.0

3.13 m

48.1 27.3 46.1 135.2 120.3 117.9 126.8 109.0 146.9 26.8 30.7

2.87 3.17 1.12 2.44

brd (13.1, 4.1) m m m

6.88 6.56 6.87 6.02

d (7.5) t (7.5) t (7.5) d (7.5)

1.78 m 3.07 m 3.54 m

58.4 171.3

5.18 d (4.8)

57.7 170.4

5.05 d (4.8)

57.2 170.9

5.06 d (4.4)

13.0

1.75 d (7.0)

12.3

1.62 d (6.8)

12.3

1.62 d (6.7)

126.6

5.63 q (7.0)

118.5

5.39 q (6.8)

117.9

5.37 q (6.7)

130.7 69.0

3.64 d (15.0) 5.55 d (15.0)

135.7 53.7

3.11 m 4.53 dt (13.2, 2.8)

136.8 53.7

3.07 d (12.3) 4.48 d (12.3)

3.74 s 3.73 s 2.66 s 3.25 3.30 3.42 3.46 1.72 2.74

m m m m dt (15.0, 9.7) m

7.46 d (1.7) 6.85 dd (8.0, 1.7) 6.31 d (8.0) 1.60 1.93 1.38 1.58 2.96

m m m m dd (10.8, 8.3)

1.45 2.77 1.48 1.49 1.32 1.40

m m m m m m

53.0 81.6 49.2 56.4 35.9 59.0 134.0 124.1 119.1 131.0 119.2 149.6 20.3

3.74 s

3.38 3.62 3.56 3.79 1.71 2.51

52.5

2.39 q (7.3)

34.6

55.0 35.1 57.8 132.1 123.1 118.0 129.8 120.4 148.2 19.5

7.21 d (7.9) 6.62 t (7.9) 7.39 d (7.9)

10.9

22.4

80.1 47.7

m m m m m m

1.95 2.00 1.65 1.84 1.69 2.25 1.72 2.32 0.58

25.6

51.9

m m m m m m m m d (7.3)

ddd (13.6, 9.9, 4.2) m m t (10.7) m dd (15.3, 7.5)

7.44 d (7.5) 6.51 t (7.5) 7.15 d (7.5)

10.8 51.3

2.18 q (7.3)

21.7 33.9

44.0

1197

3.02 3.66 3.16 3.76 1.87 2.37

1.89 1.95 1.55 1.76 1.61 2.07 1.61 2.12 0.51

25.2

45.0

3.68 s

m m m ddd (12.9, 7.6, 4.9) m m m m d (7.3)

51.8

3.69 s

79.6 65.3

3.73 3.74 3.79 4.09 2.06 2.33

68.8 34.2 57.2 131.7 122.7 117.4 129.9 120.9 148.4 20.1

m m m m m m

7.13 d (7.5) 6.45 t (7.5) 7.17 d (7.5)

10.4

1.91 2.13 1.53 1.72 1.54 2.06 1.52 2.13 0.52

50.9

2.22 d (7.3)

25.5 21.8 32.9

m m m m m m m m d (7.3)

45.3



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Table 2. continued 2b

a

position

δC

21′ 22′a 22′b COOMe′ COOMe′ NMe′

66.9

3.45 s

δH (mult, J in Hz)

174.5 52.4 31.2

3.73 s 2.66 s

3ac

3bb

δC

δH (mult, J in Hz)

δC

72.7 41.7

4.09 s 3.81 d (12.5) 3.89 d (12.5)

71.2 41.3

δH (mult, J in Hz) 3.96 s 3.56 d (11.8) 3.63 d (11.8)

3cc δC 87.7 41.2

δH (mult, J in Hz) 3.92 brs 3.57 m 3.63 m

Assignments based on COSY, HSQC, and NOESY. bCDCl3, 600 (1H) and 125 MHz (13C). cMethanol-d4, 600 (1H) and 125 MHz (13C).

Figure 4. COSY and selected HMBC correlations of pleiokomenine A (2).

Figure 6. COSY, selected HMBC, and NOE correlations of huncaniterine B (3a).

lished as C19H20N2O2 from the ESI HR mass spectrum and the 13 C NMR data. The 1H NMR spectrum (Table 1) showed a 1,2-disubstituted benzene moiety with four one-proton signals, three methine protons (δH 3.64, 4.09, and 5.18), and one olefinic methyl doublet. The 13C NMR and HSQC data showed a total of 19 carbon signals, including a methyl, four sp3 methylenes, five sp2 and three sp3 methines, an ester carbonyl at δC 168.9, two tertiary carbons linked to the indolic nitrogen (corresponding to C-2 and C-13), and three quaternary sp2

Figure 5. DFT-calculated main conformer of pleiokomenine A (2) with selected NOESY correlations as red/blue stereo view. For clarity, only the left part of the symmetrical molecule is depicted.

Scheme 1. Proposed Biosynthetic Pathway to Pleiokomenine A (2)

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(11),22 N1-methylkopsinin (12),23 pleiocarpine (13),23 and N1methylkopsinin N4-oxide (14),24 were isolated and identified by comparison of observed and literature data. In addition the structures of evoxanthine (5) and pleiocarpine (13) were analyzed by single-crystal X-ray diffraction, as their solid-state structures were not known (Figures S14 and S15, Supporting Information). The cancer chemopreventive activity of the alkaloids was tested by measuring QR induction and NF-κB inhibition as described in a previous paper.15 The crude extracts of P. pycnantha were screened at 20 μg/mL for their QR induction and NF-κB inhibition activities. The crude MeOH extract doubled the QR activity, while alkaloid-enriched fractions inhibited 54% of NF-κB activity. Therefore, the isolated compounds from both extracts were further tested in both assays (Table 3). Deformyltalbotine acid lactone (6) and N1-

carbon atoms. Analysis of the COSY and HSQC data revealed, in addition to an ethylidene side chain, the presence of N-4− CH2−CH2−C-7 and −CH−CH2−CH−CH partial structures. The 2J and 3J HMBC correlations from H-16 to C-2, C-14, C15, and the ester carbonyl C-17 indicated the connection of the C-3−C-14−C-15−C-16 partial structure with N1, while the two-bond HMBC correlations from CH2-6 to C-5 and C-7 indicated the connection of the N-4−C-5−C-6 fragment to C-7 via C-6. The cross-peaks from Me-18 to C-19 and C-20 were consistent with the attachment of the ethylidene side chain at C-20. The remaining C-21 oxymethylene group (δH 4.67/5.04; δC 71.1) correlated with C-15, C-17, C-19, and C-20 and was, therefore, attached to C-17 and C-20, resulting in the lactone 6. The NOE correlations between H3-18/15 and H3-18/14α (1.81/2.40) and between H-19/21 established the Z configuration of the ethylidene side chain. The relative configurations of the remaining stereogenic centers were determined through coupling constants and analysis of NOESY correlations (Figure 7). The NOE between

Table 3. Biological Activity of Compounds Isolated from P. pycnantha QR induction compound janetinine (1a) pleiokomenine A (2) pleiomutinine (3b) 1-carbomethoxy-β-carboline (4) deformyltalbotine acid lactone (6) spegatrine (9) N1-methylkopsinin (12) 4′-bromoflavonec parthenolide

a

NF-κB

CD (μM)

IC50 (μM)

30.7 ± n.d.b n.d. 30.2 ± 29.9 ± 43.5 ± 36.7 ± 19.2 ±

>50 13.1 ± 2.4 8.4 ± 2.4 >50 9.4 ± 0.6 >50 8.8 ± 1.7

4.8

6.1 4.1 4.7 5.9 2.4

0.4 ± 0.1

a

CD = concentration required to double the QR activity. bn.d. not determined because of toxicity. c4′-Bromoflavone was used as positive control for QR induction and parthenolide for NF-kB activity.

Figure 7. COSY, selected HMBC, and NOE correlations of deformyltalbotine acid lactone (6).

methylkopsinin (12) were the only compounds able to both double the QR activity and inhibit TNF-α induced NF-κB activity, making them particularly interesting as potential cancer chemopreventive agents. Furthermore, janetinine (1a), 1carbomethoxy-β-carboline (4), and spegatrine (9) doubled the QR activity at concentrations ranging from 30 to 44 μM, while pleiokomenine A (2) and pleiomutinine (3b) inhibited TNF-α induced NF-κB activity. Pleiokomenine A (2) and pleiomutinine (3b) were toxic toward Hepa1c1c7, and their QR-inducing activity could therefore not be determined. No active compound showed toxicity on HEK293 cells.

H-15 and H-3 indicated their syn-facial orientation. The small coupling constant of 5.7 Hz between H-15 and H-16 suggested their cis orientation (calculated dihedral angle 55°). This was further supported by the strong NOE cross signal between H15/H16 (calculated distance 2.43 Å for 15,16-cis, 3.03 Å for 16-epi isomers), which is of similar intensity to that between H15 and Hα-14 (distance 2.56 Å), but stronger than the signal between H-15 and Hβ-14 (distance 3.04 Å; see Figure S12, Supporting Information). We analyzed additionally the configuration by DFT calculation of the 13C NMR data of the C-16 epimers, which clearly favored the (15S*,16S*) isomer (Figure S10, Supporting Information). Comparison of experimental and calculated ECD spectra (Figure S11, Supporting Information) finally gave the absolute (15S,16S) configuration as depicted in structure 6; this also agreed well with the NOE between H-12 and H-16, which are 3.28 Å apart in the (16R) epimer, but closer together (2.54 Å) in (16S)-6 (Figure S13, Supporting Information). Although 6 was obtained previously by alkaline deformylation and lactonization of talbonine,16 only selected 1H NMR, but no 13 C NMR, data were reported. This compound is described here for the first time as a natural product. Additional compounds, pleiomutinine (3b),15 huncaniterine A (3c),14 1-carbomethoxy-β-carboline (4),17 evoxanthine (5),18 pleiocarpamine (7),13 N4-methyl-10-hydroxygeissoschizol (8),19 spegatrine (9),20 neosarpagine (10),21 aspidofractinine

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a PerkinElmer polarimeter, model 241, at the sodium D line (λ = 589 nm). UV/vis spectra were recorded on a JASCO V650 spectrometer (JASCO Labor- and Datentechnik Deutschland GmbH, Gross-Umstadt, Germany). ECD spectra were recorded on a JASCO J-810 spectrometer equipped with a JASCO ETC-505S/PTC423S temperature controller. IR data were acquired on a Jasco FT/IR4100 type A instrument (JASCO Labor- and Datentechnik Deutschland GmbH). The NMR spectra were measured on a Bruker AMX 300 (300.135 MHz) (Bruker BioSpin GmbH, Rheinstetten, Germany), a Varian Unity 300 (300.145 MHz), and a Varian Inova 600 (599.740 MHz) spectrometer (Varian Deutschland GmbH, Darmstadt, Germany). Chemical shifts (δ) were referenced to CH3OH at δH 3.30 and at δC 49.0 and to CHCl3 at δH 7.24 and at δC 77.0. ESIHRMS spectra were acquired on a micrOTOF 10237 (Bruker Daltonik, Bremen, Germany). Open-column chromatography (CC) was done 1199



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2, respectively; ESI m/z 631 [M + H]+; ESIHRMS m/z 631.3615 [M + H]+ (calcd 631.3643 for C40H47N4O3). Deformyltalbotine acid lactone (6): yellowish oil; UV (MeOH) λmax (log ε) 224 (4.31), 275 (3.79), 286 (3.78), 290 (3.75) nm; ECD (c 6.5 × 10−5 mol/L, MeOH) λmax (Δε) 235 (−17.58), 268 (1.76), 281 (1.02), 289 (1.01), 310 (−1.06) nm; IR (neat) νmax 3380, 1735 cm−1; 1H and 13C NMR data, see Tables 1 and 2, respectively; ESI m/ z 309 [M + H]+; ESIHRMS m/z 309.1587 [M + H]+ (calcd 309.1598 for C19H21N2O2). Computational Methods. The Merck Molecular Force Field program (MMFF, SPARTAN’14) was used to analyze the conformer distribution for compounds 1−3a and 6. After geometry optimizations with HF/3-21G, the conformers in an energy range of 40 kJ/mol above the global minimum were further optimized within a window of 20 kJ/mol using wB97X-D/6-31G* [= wB97X-D/6-31G(d)]. On the basis of these geometries, the energies and the ECD spectra were calculated using the RwB97X-D functional and the 6-311G(d,p) basis set with Gaussian 09.25 For the OR calculations, RwB97X-D/6311G(d,p) [polar = optrot CPHF = RdFreq] was applied on geometries optimized as described above, and NMR shifts were calculated with EDF2/6-31G* using SPARTAN’14, on the basis of the wB97X-D/6-31G* geometries. X-ray Structure Determinations. Crystals for both evoxanthine (5) and pleiocarpine (13) were selected using a polarization microscope. Only small and thin plates could be found for 5, whereas cubic crystals of high quality could be obtained for 13. Data collection was carried out on a Huber Type 512 four-circle diffractometer equipped with a Bruker APEX I CCD detector. The X-ray source of the diffractometer was an Incoatec second-generation Microsource, which is mounted off-focus to give a beam size of 170 μm. Data integration was carried out with the SAINT software, version 8.37A.26 To correct for the different crystal volume that was irradiated at each time, an empirical correction for absorption (and other effects) was carried out using the program SADABS, version 2015/1.27 SADABS also provided the merged hkl files in both cases. In the case of evoxanthine (5) in space group P21/c all reflections were merged, whereas for pleiocarpine (13) in space group P21 Friedel pairs were not merged for subsequent refinement. After structure solution with dual-space direct methods28 that confirmed the result of the earlier structure determinations by noncrystallographic methods in both cases, least-squares structure refinement made use of aspherical scattering factors of the invariom database.29 Input files for invariom refinement were generated with the preprocessor program INVARIOMTOOL.30 The charge-density software XDLSM,31 which relies on the Hansen/Coppens multipole model,32 was used for that purpose. Both structures could be considered of good quality, although the data to parameter ratio for pleiocarpine (13) was comparably low and the absolute configuration could not be determined from anomalous dispersion effects due to the molybdenum radiation employed. Full crystallographic details (crystal data, data collection, and structure refinement details) are contained in the cif files [CCDC entries 1565860 for evoxanthine (5) and 1565889 for pleiocarpine (13)]. Tables with crystallographic details are given in the Supporting Information Quinone Reductase Induction Assay. QR induction was evaluated as described elsewhere.33 Briefly, Hepa1c1c7 cells (ATCC, Rockville, MD, USA) were seeded at 1750 cells/well in 96-well plates and incubated at 37 °C with 5% CO2 for 24 h in MEM-α (Thermo Fisher Scientific, Waltham, MA, USA) enriched with 10% fetal bovine serum (Biowest, Nuaillé, France), 100 U/mL penicillin, and 100 μg/ mL streptomycin (Thermo Fisher Scientific). Cells were then treated with samples for 48 h, and QR induction was determined using the well-established NADPH-dependent menadiol-mediated reduction of 3-(4,5-dimethylthiazo-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to blue formazan reaction. The absorbance was measured at 595 nm after 5 min of incubation using a Cytation 3 multimode plate reader (Biotek, Winooski, VT, USA). The cell viability was determined by crystal violet staining, and the absorbance was measured at 595 nm. The concentrations required to double the QR activity (expressed as CD) and cytotoxicity (expressed as IC50) were calculated using

on silica gel 60 (0.063−0.20 mm), and PTLC was performed on silica gel P/UV254 (both obtained from Macherey-Nagel, Dü r en, Germany). Size exclusion chromatography was performed on Sephadex LH-20 (Lipophilic Sephadex, Amersham Biosciences, Ltd., purchased from Sigma-Aldrich Chemie, Steinheim, Germany). Plant Material. Plant material was collected from Mont-Kalla in the Centre Region of Cameroon on March 2014 and identified botanically by Victor Nana (National Herbarium, Yaoundé, Cameroon). A voucher specimen (23188 SFR Cam) is deposited in the National Herbarium in Yaoundé, Cameroon. Extraction and Isolation. The stem bark of P. pycnantha (1.5 kg) was air-dried, crushed into small pieces, and then powdered and extracted with MeOH (3 × 4 L, room temperature, 48 h). The concentrated MeOH extract (104 g) was partitioned between EtOAc and 5% HCl. The aqueous phase was basified to pH 8−9 with saturated Na2CO3 and then successively extracted with EtOAc and nBuOH, to yield 3.8 and 7.1 g of crude alkaloids, respectively. The EtOAc extract was subjected to CC over silica gel and eluted with CH2Cl2/MeOH (gradient of increasing polarity, 50:1 to 3:1, v/v), to produce four major fractions (I−IV) on the basis of TLC composition. Fraction II (0.6 g) was applied to a Sephadex LH-20 gel column (MeOH) to yield janetinine (1a, 4 mg), pleiomutinine (3b, 65 mg), 1carbomethoxy-β-carboline (4, 7 mg), and pleiocarpine (13, 35 mg). Fraction III (0.6 g) was first subjected to a Sephadex LH-20 gel column (MeOH), affording four subfractions (IIIa−IIId). Fraction IIIb (100 mg) was subjected to CC on silica gel and eluted with CH2Cl2/ MeOH (15:1 to 3:1, v/v) to give N1-methylkopsinin (12) (8 mg) and subfraction IIIb-2 (28 mg), which was further subjected to PTLC using 10% MeOH in CH2Cl2 to give pleiokomenine A (2, 4 mg) and N1-methylkopsinin N4-oxide (14, 2 mg). Fraction IV (0.4 g) was also subjected to a Sephadex LH-20 gel column (MeOH) to yield huncaniterine B (3a, 5 mg) and huncaniterine A (3c, 7 mg). The n-BuOH alkaloid extract was fractionated by a Sephadex LH-20 gel column (MeOH) to give four fractions (I−IV). Fraction II (2.0 g) was separated by silica gel (CH2Cl2/MeOH, from 30:1 to 3:1, v/v) to afford four subfractions (IIa−IId). Fraction IIb (0.6 g) was subjected to C 18 CC (MeOH/H2 O, from 4:6 to 8:1, v/v) to yield pleiocarpamine (7, 10 mg) and evoxanthine (5, 3.2 mg). Fraction IIc (0.5 g) was first subjected to silica gel (CH2Cl2/MeOH, from 15:1 to 2:1, v/v) and then purified by Sephadex LH-20 (MeOH) to give N4-methyl-10-hydrogeissoschizol (8, 56 mg) and aspidofractinine (11, 5.2 mg). Fraction III (1.5 g) was separated by silica gel (CH2Cl2/ MeOH, from 25:1 to 3:1, v/v) to afford spegatrine (9, 8 mg) and two subfractions (IIIa, IIIb). Fraction IIIa (21 mg) was purified by preparative TLC, yielding again pleiomutinine (3b, 10 mg) and deformyltalbotine acid lactone (6, 2.5 mg). Fraction IIIb (70 mg) was purified by silica gel CC (CH2Cl2/MeOH, from 10:1 to 5:1, v/v) to yield neosarpagine (10, 2 mg). Janetinine (1a): pale yellow, amorphous solid; [α]20 D +8.2 (c 0.8, MeOH); mp 231−233 °C; UV (MeOH) λmax (log ε) 217 (4.74), 232 (4.40), 239 (4.39), 250 (4.23), 264 (4.17), 289 (4.05), 300 (4.05), 328 (3.82), 345 (3.71) nm; ECD (c 7.5 × 10−5 mol/L, MeOH) λmax (Δε) 225 (−1.26), 239 (1.78), 250 (0.63), 262 (1.25), 300 (−1.45) nm; IR (neat) νmax 3450, 3380, 3266, 2915, 1421 cm−1; 1H and 13C NMR data, see Tables 1 and 2, respectively; ESI m/z 267 [M + H]+; ESIHRMS m/z 267.1496 [M + H]+ (calcd 267.1492 for C17H19N2O). Pleiokomenine A (2): yellowish oil; [α]20 D −13.8 (c 0.9, MeOH); UV (MeOH) λmax (log ε) 207 (4.75), 215 (4.61), 262 (4.19), 304 (3.93) nm; ECD (c 2.2 × 10−5 mol/L, MeOH) λmax (Δε) 220 (−9.71), 244 (2.17), 265 (9.97), 317 (−2.84) nm; IR (neat) νmax 2940, 1724 cm−1; 1H and 13C NMR data, see Tables 1 and 2, respectively; ESI m/z 717 [M + H]+; ESIHRMS m/z 717.4385 [M + H]+ (calcd 717.4374 for C45H57N4O4). Huncaniterine B (3a): brownish solid; [α]20 D +16 (c 0.8, MeOH); mp 180−182 °C; UV (MeOH) λmax (log ε) 205 (4.97), 221 (4.83), 264 (4.32), 290 (4.15); ECD (c 3.2 × 10−5 mol/L, MeOH) λmax (Δε) 220 (−35.07), 222 (−33.54), 235 (−3.36), 241 (−4.16), 263 (25.28), 282 (1.11), 287 (−0.75), 302 (2.64), 317 (−0.87), 334 (2.04) nm; IR (neat) νmax 2962, 1732 cm−1; 1H and 13C NMR data, see Tables 1 and 1200



Article

(5) Cuendet, M.; Oteham, C. P.; Moon, R. C.; Pezzuto, J. M. J. Nat. Prod. 2006, 69, 460−463. (6) Aggarwal, B. B.; Sung, B. Cancer Discovery 2011, 1, 469−471. (7) Stas, J. S. Bull. Acad. R. Med. Belg 1851, 11, 304−312. (8) Otto, F. J. Liebigs Ann. Chem. 1856, 100, 39. (9) Azoug, M.; Loukaci, A.; Richard, B.; Nuzillard, J. M.; Moreti, C.; Zèches-Hanrot, M.; Le Men-Olivier, L. Phytochemistry 1995, 39, 1223−1228. (10) Dictionary of Natural Products on DVD; CRC Press: Boca Raton, Fl, USA, 2017. (11) Yap, W. S.; Gan, C. Y.; Sim, K. S.; Lin, S. H.; Low, Y. Y.; Kam, T. S. J. Nat. Prod. 2016, 79, 230−239. (12) Dewick, P. M. Medicinal Natural Products: A Biosynthetic Approach, 3rd ed.; John Wiley & Sons: Chichester, UK, 2009; pp 162− 165. (13) Keawpradub, N.; Houghton, P. J.; Eno-Amooquaye, E.; Burke, P. J. Planta Med. 1997, 63, 97−101. (14) Mohamad, K.; Suzuki, T.; Baba, Y.; Zaima, K.; Matsuno, Y.; Hirasawa, Y.; Mukhtar, M. R.; Awang, K.; Hadi, H. A.; Morita, H. Heterocycles 2007, 74, 969−976. (15) Ndongo, J. T.; Ngo Mbing, J.; Feussi Tala, M.; Monteillier, A.; Pegnyemb, D. E.; Cuendet, M.; Laatsch, H. Phytochemistry 2017, 144, 189−196. (16) Pinar, M.; Hanaoka, M.; Hesse, M.; Schmid, H. Helv. Chim. Acta 1971, 54, 15−43. (17) Achenbach, H.; Biemann, K. J. Am. Chem. Soc. 1965, 87, 4177− 4181. (18) Paris, R. R.; Stambouli, A. C. R. Hebd. Seances Acad. Sci. 1958, 247, 2421−2423. (19) Rapoport, H.; Windgassen, R. J., Jr.; Hughes, N. A.; Onak, T. P. J. Am. Chem. Soc. 1960, 82, 4404−4414. (20) Orazl, O. O.; Corral, R. A.; Stoichevich, M. E. Can. J. Chem. 1966, 44, 1523−1529. (21) Pillay, P. P.; Rao, D. S.; Rao, S. B. J.S.I.R 1960, 19B, 135−137. (22) Djerassi, C.; Budzikiewicz, H.; Owellen, R. J.; Wilson, J. M.; Kump, W. G.; Le Count, D. J.; Battersby, A. R.; Schmid, H. Helv. Chim. Acta 1963, 46, 742−751. (23) Kump, I. W. G.; Schmid, H. Helv. Chim. Acta 1961, 44, 1503− 1516. (24) Chen, J.; Yang, M.-L.; Zeng, J.; Gao, K. Phytochem. Lett. 2014, 7, 156−160. (25) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X., Hratchian, H. P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Montgomery, J. A.; Peralta, J. E.; Ogliaro, F.; Bearpark, M.; Heyd, J. J.; Brothers, E.; Kudin, K. N.; Staroverov, V. N.; Kobayashi, R.; Normand, J.; Raghavachari, K.; Rendell, A.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Rega, N.; Millam, J. M.; Klene, M.; Knox, J. E.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Zakrzewski, V. G.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Dapprich, S.; Daniels, A. D.; Farkas, O.; Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.; Fox, D. J. Gaussian 09W, Version 7.0; Gaussian: Wallingford, CT, 2009. (26) Bruker. APEX2 (Version 2014.11-0) and SAINT (Version 8.37A); Bruker AXS Inc.: Madison, WI, USA, 2013. (27) Sheldrick, G. M. SADABS version 2015/1; University of Goettingen, 2015. (28) Sheldrick, G. M. Acta Crystallogr., Sect. A: Found. Adv. 2015, A71, 3−8. (29) Dittrich, B.; Hübschle, C. B.; Pröpper, K.; Dietrich, F.; Stolper, T.; Holstein, J. J. Acta Crystallogr., Sect. B: Struct. Sci., Cryst. Eng. Mater. 2013, B69, 91−104. (30) Hübschle, C. B.; Luger, P.; Dittrich, B. J. Appl. Crystallogr. 2007, 40, 623−627.

GraphPad Prism 6.05 (GraphPad Software, Inc.). Each compound was tested in triplicate, and three independent experiments were performed. 4′-Bromoflavone was used as positive control. NF-κB Inhibition Assay. NF-κB inhibitory activity and cell viability were determined as described elsewhere.34 Briefly, HEK293/ NF-κB-luc cells (Panomics, Fremont, CA, USA) were incubated for 1 h with 2.5 μM Cell Tracker Green CMFDA (Thermo Fisher Scientific), a fluorescent dye used to quantify cell viability, and seeded in 96-well plates (104 cells/well). After an overnight incubation, cells were treated with the samples and stimulated for 5 h with 20 ng/mL of TNF-α (Sigma-Aldrich, Saint Louis, MO, USA). Cell lysis was performed by the addition of reporter lysis buffer (Promega, Madison, WI, USA). Fluorescence was read on a Cytation 3 multimode plate reader (Biotek), followed by addition of luciferase assay reagent (Promega) and reading of the luminescence signal. The luminescence signal of each well was normalized with its fluorescence signal in order to limit the influence of cell toxicity in luminescence signal intensity. IC50 values were calculated using GraphPad Prism 6.05. Each compound was tested in duplicate and in three independent experiments. Parthenolide was used as positive control.

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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.7b00958. Crystallographic data for 5 (CIF) Crystallographic data for 13 (CIF) Additional figures and tables (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Tel: +237 690224780. Fax: +237 222234496. E-mail: thierry. [email protected]. *Tel: +49 551 3933211. Fax: +49 551 399660. E-mail: [email protected]. ORCID

Joseph T. Ndongo: 0000-0003-0222-7949 Birger Dittrich: 0000-0002-4306-5918 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the Alexander von Humboldt Foundation through a Georg-Forster Fellowship (3.4-KAM/1155628) to J.T.N. and was also supported by the TWAS (The World Academic of Science, No. 13-174 RG/ CHE/AF/AC_G; UNESCO FR: 3240277733) to D.E.P. The authors would like to thank Dr. H. Frauendorf and Dr. M. John for MS and NMR measurements. We are also indebted to Mrs. F. Lissy and Mr. F. Borlat for technical assistance and to Mr. V. Nana (National Herbarium of Cameroon) for the botanical identification.

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REFERENCES

(1) WCSP World Checklist of Selected Plant Families, Royal Botanic Gardens, Kew, http://apps.kew.org/wcsp/qsearch.do. 2013. Accessed May 18, 2016. (2) Omoyeni, O. A.; Hussein, A. A.; Iwuoha, E.; Green, I. R. Phytochem. Rev. 2017, 16, 97−115. (3) Gorman, A. A.; Schmid, H. Monatsh. Chem. 1967, 98, 1554− 1566. (4) Gorman, A.; Dastoor, N.; Hesse, M.; Von Philipsborn, W.; Renner, U.; Schmid, H. Helv. Chim. Acta 1969, 52, 33−55. 1201



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(31) Volkov, A.; Macchi, P.; Farrugia, L. J.; Gatti, C.; Mallinson, P.; Richter, T.; Koritsánszky, T. XDLSM; University at Buffalo, NY, USA; University of Milano, Italy; University of Glasgow, UK; CNRISTM, Milano, Italy; Middle Tennessee State University, TN, USA, 2006. (32) Hansen, N.; Coppens, P. Acta Crystallogr., Sect. A: Cryst. Phys., Diffr., Theor. Gen. Crystallogr. 1978, A34, 909−921. (33) Kang, Y. H.; Pezzuto, J. M. Methods Enzymol. 2004, 382, 380− 414. (34) Tran, T. V. A.; Malainer, C.; Schwaiger, S.; Hung, T.; Atanasov, A. G.; Heiss, H. E.; Dirsch, V. M.; Stuppner, H. J. Ethnopharmacol. 2015, 159, 36−42. (35) Obiang, E. O. N.; Genta-Jouve, G.; Gallard, J.-F.; Kumulungui, B.; Mouray, E.; Grellier, P.; Evanno, L.; Poupon, E.; Champy, P.; Beniddir, M. A. Org. Lett. 2017, 19, 6180−6183.

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NOTE ADDED AFTER ASAP PUBLICATION After ASAP publication of this paper April 17, 2018, the authors realized that their alkaloid 2 is identical with pleiokomine A, whose structure was elucidated very recently by Obiang, E. O. N.; Genta-Jouve, G.; Gallard, J.-F.; Kumulungui, B.; Mouray, E.; Grellier, P.; Evanno, L.; Poupon, E.; Champy, P.; Beniddir, M. A. Org. Lett. 2017, 19, 6180-6183. To avoid confusion, the name pleiokomine A, given by Chiang, Beniddir, et al., was substituted for the authors’ previous designation. The corrected version was reposted on May 17, 2018.

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Alkaloids from Pleiocarpa pycnantha: Pleiocarinine, a Bisindoline

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