Monoterpene Indole Alkaloids from the Fruit of Tabernaemontana

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Monoterpene Indole Alkaloids from the Fruit of Tabernaemontana litoralis and Differential Alkaloid Composition in Various Fruit Components Yang Qu,† Razvan Simonescu,‡ and Vincenzo De Luca*,† †

Department of Biological Sciences and ‡Department of Chemistry, Brock University, 1812 Sir Isaac Brock Way, St. Catharines, Ontario L2S 3A1, Canada S Supporting Information *

ABSTRACT: Two new monoterpene indole alkaloids, isoakuammiline (1) and 18-hydroxypseudovincadifformine (2), and five known alkaloids, coronaridine (3), heyneanine (4), 3,19-oxidocoronaridine (5), tabersonine, and strictosidine, were identified from the fruit of Tabernaemontana litoralis. The structures of the alkaloids were determined using NMR and MS data analyses. While 18-hydroxypseudovincadifformine (2) showed a new hydroxylation pattern, isoakuammiline (1) revealed a novel skeleton for monoterpene indole alkaloids. In spite of the isolation of stemmadenine from the fruit tissues in other Tabernaemontana species, this vital biosynthetic precursor of iboga, aspidosperma, and pseudoaspidosperma skeletons was not found in T. litoralis.

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accounted for the four oxygen atoms of the formula C23H26N2O4 deduced from the mass spectrum. The indolic NH signal was absent in the 1H NMR and IR spectra, indicating a substitution or an imine-type structure. HSQC and HMBC data (Table 1, Figures S2 and S3, Supporting Information) showed five methylenes, two methines, a carbon of the imino group (δ 183.3), a tertiary carbon, two ester carbonyls (δ 170.9, 169.7), and two quaternary carbon atoms. The presence of the ethylidene side chain −C(20)C(19)H− C(18)H3 (H-18 δ 1.61, H-19 δ 5.38), characteristic of a number of corynanthe, strychnos, and akuammiline types of alkaloids, was apparent in the COSY spectrum (Figure S4, Supporting Information). The COSY data also revealed the presence of −NC(5)H2C(6)H2−, −NCH−, and −NCH2CH2CH− structural moieties. Therefore, the D-ring with the ethylidene side chain of compound 1 could be established, indicating that N-4 bonded a methine function corresponding to C-21 and a methylene group corresponding to C-3. From the HMBC data, cross-peaks were found for H-5 and H-6 with C-7, C-2, and C-21. Therefore, the C-2−C-21 bond of the C-ring of compound 1 was established. The isolated C-17 methylene showed cross-peaks with both ester carbonyls, as well as C-2, C-15, and C-16. Therefore, the quaternary C-16 is linked to C-7, C-15, the methoxycarbonyl, and C-17, which in turn is linked to the acetate moiety. Compound 1 is isomeric to the known MIA akuammiline (Scheme 1), possessing a C-2−C-3 bond4,5 rather than the C-

ith more than 3000 reported structures, monoterpene indole alkaloids (MIAs) are the largest and most diverse subgroup of alkaloids.1 Commonly found in plant families such as Apocynaceae, Rubiaceae, and Loganiaceae, many MIAs are shown to have important pharmaceutical properties. A few examples include the anticancer drugs vinblastine and vincristine from Catharanthus roseus (Madagascar periwinkle) and camptothecin from Camptotheca acuminata.2 The rearrangements of the highly malleable 10-carbon terpene moiety derived from secologanin result in a plethora of different MIA skeletons. In an effort to explore the MIA diversity of Tabernaemontana litoralis (Kunth) L. Allorge, the MIA profiles of seeds, arils, and capsules of the mature fruits (Figure 1) were analyzed. The respective tissues were extracted with MeOH, and the crude extracts were analyzed by LC-MS. The MIA profiles varied dramatically in fruit tissues (Figure 1), suggesting that different sets of enzymes and components of MIA biosynthesis are responsible for their formation in capsules, arils, and seeds. Strictosidine (m/z 531) found in arils and tabersonine (m/z 337) found in seeds were identified by comparing to authentic standards.3 MIAs 1−5 were purified using preparative TLC and were identified by 1H NMR, COSY, HMBC, and HSQC data. The 13C NMR chemical shifts were identified through HSQC and HMBC experiments. The 1H NMR data of compound 1 isolated from fruit capsules (Table 1 and Figure S1, Supporting Information) showed four aromatic resonances of an unsubstituted A-ring indole moiety (δ 7.25−7.47), a methyl singlet (δ 3.75) of a methoxycarbonyl side chain, and a methyl singlet (δ 2.08) of an acetate moiety. The methoxycarbonyl and acetate groups © XXXX American Chemical Society and American Society of Pharmacognosy

Received: May 6, 2016

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DOI: 10.1021/acs.jnatprod.6b00405 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Note

Figure 1. LC-MS chromatograms (280 nm) of alkaloid profiles of the capsules, arils, and crushed seed material of Tabernaemontana litoralis fruit.

Table 1. 1H NMR (600 MHz), 13C NMRa (150 MHz), and HMBC Data for Isoakuammiline (1) (600 MHz, Acetone-d6) position

δC, type

2 3

183.3, C 48.2, CH2

5

57.8, CH2

6

38.4, CH2

7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 CO −OCH3 CH3COO CH3COO

68.4, 146.6, 120.3, 126.1, 127.3, 120.3, 153.0, 30.9,

C C CH CH CH CH C CH2

33.4, CH 59.0, C 66.4, CH2 12.6, 122.7, 134.3, 75.5, 170.9, 51.7, 20.1, 169.7,

CH3 CH C CH C CH3 CH3 C

δH (J in Hz)

HMBC

2.70, 3.00, 3.34, 3.49, 1.87, 2.90,

ddd (13.8, 4.3, 4.3) ddd (13.8, 11.5, 4.3) dd (12.7, 7.3) ddd (12.7, 12.7, 6.3) dd (13.4, 6.3) ddd (13.4,12.8, 7.3)

7.45, 7.25, 7.32, 7.47,

d (7.5) dd (7.4, 1.1) dd (7.6, 1.2) d (7.5)

13

2.09, ddd (10.1, 4.3, 4.3) 2.19, m 3.61, m

16

3.89, 5.23, 1.61, 5.38,

2, 16, CO 15, CO 19, 20

d d d q

(11.3) (11.3) (6.8) (6.8)

S8, Supporting Information) showed seven methylenes, three methines, two tertiary carbons, one ester carbonyl (δ 174.0), and one quaternary carbon atom. The UV absorption of compound 2 at λmax (MeOH) 227, 296, and 328 indicated an −indole nitrogen−CC− chromophore, which explained the two tertiary carbons corresponding to C-2 (δ 165.1) and C-16 (δ 95.6). The correlation of H-17 and C-16 in the HMBC spectrum, as well as the COSY correlations of H-17, H-14, and H-15, established the presence of the −C(16)C(17)H2C(14)HC(15)H2 structural moiety. Collectively, the presence of the pseudoaspidosperma skeleton was apparent. HRESIMS data showed that compound 2 had a molecular formula of C21H26N2O3, in which the methoxycarbonyl group accounted for two oxygen atoms. Consequently, the molecule contains a single hydroxy group. The significant deshielding of the resonance of the C-18 methylene group (δ 60.1) in the HSQC spectrum indicated the hydroxylation at C-18. Therefore, compound 2 was identified as the new 18-hydroxypseudovincadifformine. The NMR chemical shifts of compound 2 showed good agreement with those of the known pseudovincadifformine and 18,19-dihydroxy-, 19,20-dihydroxy-, and 20-hydroxypseudovincadifformine (pandoline).6−8 Therefore, the absolute configurations of C-3, C-14, and C-20 (Scheme 1) are likely consistent with those of the aforementioned compounds. This was confirmed by the similar specific rotations of 2 ([α]25D +184, c 0.1 in MeOH) compared to pseudovincadifformine.6,8 Similarly based on 1H NMR, COSY, HMBC, and HSQC data analyses, compound 3 isolated from seeds and compounds 4 and 5 isolated from fruit capsules were identified as coronaridine (Table S1), heyneanine (19S-hydroxycoronaridine, Table S2), and 3,19-oxidocoronaridine (Table S3), respectively. The respective NMR data agreed with previous reports from other Tabernaemontana species, but these three MIAs were reported for the first time in T. litoralis.9−12 In addition, HMBC correlation analyses for coronaridine, heyneanine, and 3,19-oxidocoronaridine in this study further supported previous identifications of these MIAs. In MIA biosynthesis, deglycosylation of the central precursor strictosidine afforded the labile aglycones that are further modified by various biosynthetic enzymes. Formation of the corynanthe-type MIA 4,21-dehydrogeissoschizine or its reduced form geissoschizine is the first critical branching point.13 Rearrangement of geissoschizine gives rise to a few major MIA

21 7, 21 8 2, 7, 8

3.90, d (2.3) 3.75, s 2.08, s

CO CH3COO

a13

C NMR values were extracted from HSQC and HMBC data.

2−C-21 bond in 1. On the basis of the similarity, compound 1 with a novel MIA skeleton was named isoakuammiline. The 1H NMR data of compound 2, isolated from the fruit capsules (Table 2 and Figure S5, Supporting Information), showed the presence of an indolic NH (δ 9.28), four aromatic resonances of an unsubstituted A-ring indole moiety (δ 6.89− 7.32), and a methyl singlet (δ 3.72) of a methoxycarbonyl side chain. The COSY spectrum (Figure S6, Supporting Information) showed the presence of −NCH2CH2−, −NCHCHCH2CHCH2CH2, and −NCH2CHCH2CH2 structural units. HSQC and HMBC data (Table 2, Figures S7 and B



Note

Scheme 1

with similar mass were heyneanine and 18-hydroxypseudovincadifformine. It is possible that stemmadenine, as in many other MIA-producing species, is rapidly converted to the final iboga and aspidosperma skeletons. Interestingly, it was reported that only the fruits of T. donnell-smithii harvested in November contained appreciable amounts of stemmadenine, while the fruits harvested at other times of the year contained little or no stemmadenine.18 Developmental regulation may play a significant role in the biosynthetic destiny of stemmadenine. Drastic differences in MIA profile were noted among the fruit capsule, aril, and seeds of T. litoralis (Figure 1). Strictosidine is the major alkaloid in fruit arils; however in the capsule strictosidine has been converted to mainly iboga and pseudoaspidosperma alkaloids. In seeds, strictosidine is converted to both iboga and aspidosperma alkaloids, but the only major iboga alkaloid, coronaridine, is not substituted, whereas in fruit capsule coronaridine is oxidized to form heyneanine and 3,19-oxidocoronaridine. Although the biological significance is not determined in this study, such unique tissue-specific MIA distribution implies delicate control of specific sets of MIA synthesis enzymes in the fruits of T. litoralis. In summary, two new and five known MIAs were found in T. litoralis fruits. 18-Hydroxypseudovincadifformine (2) showed

skeletons including sarpagan, akuammiline, and strychnos types (Scheme 1). Formation of stemmadenine (strychnos type) leads to the second major branching point, as its rearrangement affords another three major MIA skeletons: iboga, aspidosperma, and pseudoaspidosperma types (Scheme 1).13 Because of the biosynthetic relationships, all MIAs that originate from strictosidine would possess the same C-15 stereogenic center that has been studied in a number of MIAs. For example in geissoschizine, H-15 is α-oriented relative to the indolic ring.14 It is highly possible that C-15 in isoakuammiline (1) would possess the same absolute configuration. With this assumption, the spatial restraint in the molecule would not allow the C-7−C-16−C-15 bridge to be above the tetracyclic ring system but rather extend beneath it (Scheme 1). However, we cannot rule out other possibilities for the absolute configuration. The MIA intermediate stemmadenine has been used as a biosynthetic intermediate for studying the formation of related MIAs in various plant cell culture systems.13,15 This vital MIA, from which three major MIA skeletons originate, was reported only from the fruits of T. donnell-smithii and the seeds of T. dichotoma.9,16 In addition, 16-epi-stemmadenine was reported in the fruits of T. heyneana.17 We were not able to isolate stemmadenine from T. litoralis, and the only two MIAs C



Note

mL min−1; 8.3−10.0 min 99% A, 1% B at 0.3 mL min−1. The mass spectrometer was operated at a capillary voltage of 3.1 kV, cone voltage of 48 V, desolvation gas flow of 600 L h−1, desolvation temperature of 350 °C, and source temperature of 150 °C. The optical rotations were measured using an Autopol IV automatic polarimeter (Rudolph Research Analytical, Hackettstown, NJ, USA) at 25 °C. Plant Material. T. litoralis fruits were collected on the campus of University of Hawaii at Manoa, Honolulu, HI, USA, in December 2015, and the species was identified by Dr. Gerald Carr at Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR, USA. The herbarium voucher 632988 was deposited at Bernice Pauahi Bishop Museum (Herbarium Pacificum collection), HI, USA. Extraction and Isolation. Capsules, arils, and seeds of 1.5 kg of fresh fruits were separated, crushed, and extracted with MeOH at 4 °C for 2 days. The extract was dried under vacuum and separated on TLC (silica gel 60 F254, Merck, Darmstadt, Germany) with solvent system (SS) 1, followed by one of the three SS 2−4. MIA 1, 2, 4, and 5 were isolated from fruit capsules, and MIA 3 was isolated from seeds. SS4, SS2, SS2, SS2, and SS3 were used to isolate MIA 1−5, respectively. SS1: toluene/EtOAc/MeOH, 15:4: 2 (v/v/v); SS2: toluene/acetone/ MeCN, 8:1:2 (v/v/v); SS3/toluene/acetone/MeOH, 2:1:1 (v/v/v); SS4: 100% MeOH. Isoakuammiline (1): yellow oil, 0.2 mg; [α]25D +82 (c 0.01, MeOH); UV λmax (MeOH) 223, 275; Rf (SS1) 0.14, Rf (SS4) 0.31; IR (dry film) νmax 2921, 2850, 1732, 1625, 1605, 1461 cm−1; EIMS m/z 394 [M+] (3), 366 (41), 335 (18), 269 (12), 227 (14), 208 (50), 167 (41), 152 (100), 105 (23); CIDMS m/z 395 [MH+], 367, 353, 335, 323, 284; HREIMS m/z 394.1888 (calcd for C23H26N2O4, 394.1893). 18-Hydroxypseudovincadifformine (2): yellow, amorphous solid, 1 mg; [α]25D +184 (c 0.1, MeOH); UV λmax (MeOH) 227, 296, 328; Rf (SS1) 0.39, Rf (SS2) 0.08; IR (dry film) νmax 3369, 2921, 1732, 1673, 1607, 1465 cm−1; EIMS m/z 354 [M+] (24), 167 (21), 157 (22), 140 (100), 138 (25), 132 (18), 126 (16), 115 (12), 69 (13); HREIMS m/z 354.1938 (calcd for C21H26N2O3, 354.1945). Coronaridine (3): light yellow, amorphous solid, 1 mg; [α]25D −15 (c 0.1, MeOH); UV λmax (MeOH) 227, 285, 293; Rf (SS1) 0.65, Rf (SS2) 0.59; IR (dry film) νmax 3350, 2925, 2850, 1727, 1460 cm−1; EIMS m/z 338 [M+] (92), 323 (28), 253 (12), 214 (27), 136 (58), 124 (36), 111 (48), 119 (39), 97 (67), 95 (50), 85 (68), 83 (65), 71 (85), 69 (71), 57 (100), 55 (61); HREIMS m/z 338.1975 (calcd for C21H26N2O2, 338.1994). Heyneanine (4): light yellow, amorphous solid, 1 mg; [α]25D −8 (c 0.1, MeOH); UV λmax (MeOH) 225, 285, 293; Rf (SS1) 0.50, Rf (SS2) 0.14; IR (dry film) νmax 3347, 2921, 2850, 1727, 1460 cm−1; EIMS m/z 354 [M+] (19), 336 (17), 214 (15), 153 (100), 152 (53), 110 (28), 109 (38), 90 (83), 89 (56), 65 (55); HREIMS m/z 354.1938 (calcd for C21H26N2O3, 354.1945). 3,19-Oxidocoronaridine (5): yellow oil, 1 mg; [α]25D +15 (c 0.1, MeOH); UV λmax (MeOH) 222, 283, 293; Rf (SS1) 0.43, Rf (SS3) 0.63; IR (dry film) νmax 3340, 2925, 2860, 1727, 1460 cm−1; EIMS m/z 352 [M+] (100), 337 (15), 308 (21), 270 (39), 229 (32), 214 (74), 182 (16), 168 (20), 167 (21), 154 (33), 138 (18), 127 (15), 94 (20); HREIMS m/z 352.1792 (calcd for C21H24N2O3, 352.1787).

Table 2. 1H NMR (600 MHz), 13C NMRa (150 MHz), and HMBC Data for 18-Hydroxypseudovincadifformine (2) (600 MHz, Acetone-d6) position

δC, type

2 3 5

165.1, C 66.3, CH 50.8, CH2

6

44.4, CH2

7 8 9 10 11 12 13 14 15

55.3, 137.4, 121.4, 120.0, 127.4, 109.3, 143.8, 36.6, 32.8,

C C CH CH CH CH C CH CH2

16 17

95.6, C 26.3, CH2

18

60.1, CH2

19

39.3, CH2

20 21

30.1, CH 54.8, CH2

CO −OCH3 NH

167.3, C 50.0, CH3

δH (J in Hz)

HMBC

3.01, d (3.9) 2.76, ddd (11.6, 8.1, 4.2) 2.94 m 1.72, dd (11.3, 4.2) 2.00 m

3, 6, 7 3, 8 2, 5, 7, 8

7.32, 6.89, 7.17, 7.06,

11, 13 8, 12 9, 13 8, 10

d (7.4) dd (7.5, 1.0) dd (7.6, 1.1) d (7.7)

1.47, m 1.50, m 1.88, ddd (13.4, 6.5, 5.2)

8, 17

3, 14, 17, 21 3, 14, 17, 19, 20, 21

2.41, dd (14.5, 11.4) 2.56, ddd (14.5, 3.5, 1.3) 3.64, m 3.64, m 1.67, ddd (19.9, 6.5, 6.5) 1.81, ddd (19.9, 7.9, 6.5) 1.97 m 2.89, d (5.6) 2.93, d (5.6)

2, 3, 14, 16 2, 3, 16 19, 20

3.72, s 9.28, s

CO

15, 18, 20, 21 15, 18, 20, 21 3, 19 3, 19, 20

a13

C NMR values were extracted from HSQC and HMBC data.

a novel pattern of hydroxylation on the pseudoaspidosperma skeleton. More interestingly is the identification of isoakuammiline. It bears a similar overall structure to akuammiline; however the D-ring is inverted, resulting in the formation of a C-2−C-21 bond (Scheme 1). The isoakuammiline skeleton possessing C-2−C-21 and C-7−C-16 bonds represents a novel class in MIA structures.

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EXPERIMENTAL SECTION

General Experimental Procedures. NMR spectra were recorded on a Bruker Avance AV I 600 digital NMR spectrometer with a 14.1 T Ultrashield Plus magnet using TOPSPIN 2.1 software for data acquisition and analysis on a Windows 7 workstation. The 1D spectra were acquired with a sweep width of 20.5 ppm with an FID size of 32k points for protons. Acetone-d6 (99.8% pure, Cambridge Isotope Laboratories) was used as the solvent using the internal reference of 1H = 2.05 ppm. The 2D spectra were acquired with 2048 points and 256 increments, and they were processed with 1024 × 1024 points. The H−C coupling constants in the HSQC, edHSQC, and HMBC were set to 145 Hz, and the long-range coupling constant in the HMBC was set to 10 or 6 Hz. LC-MS was performed using Acquity UPLC systems (Waters, Milford, MA, USA) equipped with a BEH C18 column (2.1 × 50 mm, particle size 1.7 μm), a photodiode array detector, and a mass spectrometer. The solvent systems for alkaloid analysis were as follows: solvent A, MeOH/MeCN/5 mM NH4OAc at 6:14:80; solvent B, MeOH/MeCN/5 mM NH4OAc at 24:64:10. The following linear elution gradient was used: 0−0.5 min 99% A, 1% B at 0.3 mL min−1; 0.5−0.6 min 99% A, 1% B at 0.4 mL min−1; 0.6−8.0 min 1% A, 99% B at 0.4 mL min−1; 8.0−8.3 min 99% A, 1% B at 0.4

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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.6b00405. 1

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H NMR spectrum and COSY, HSQC, HMBC correlations of compounds 1−5 (PDF)

AUTHOR INFORMATION

Corresponding Author

*Tel: (905)688-5550, ext. 4554. Fax: (905)688-1855. E-mail: [email protected]. D



Note

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. T. Ranker at Department of Botany of University of Hawaii at Manoa for collecting the fruit samples. We also thank Dr. L. Qiu at Department of Chemistry at Brock University for performing the mass spectrometry experiments. This work was supported by a Natural Sciences and Engineering Research Council of Canada Discovery Grant (V.D.L.) and by a Tier 1 Canada Research Chair in Plant Biotechnology (V.D.L.).

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REFERENCES

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Monoterpene Indole Alkaloids from the Fruit of Tabernaemontana

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