Planta Med. 58(1992) 43
Indole Alkaloids with in vitro Antiproliferative Activity from the Ammoniacal Extract of Nauclea orientalis1 C. A. J. Erdel,neier2'5, U. Regenass3, T. Rali4, and 0. Sticher2 1
Part 3 in the series: Medicinal plants from Papua New Guinea'; for Part 2, see Ref. (2) Department of Pharmacy, Swiss Federal Institute of Technology (ETH) Zurich, CH-8092 Zurich, Switzerland Research Department, Pharmaceuticals Division, Ciba-Geigy Ltd., CH-4002 Base!, Switzerland Department of Chemistry, The University of Papua New Guinea, Port Moresby, Papua New Guinea Address for correspondence: Dr. Wilimar Schwabe Arzneimittel, Research and Development, D(W)-7500 Karlsruhe, Federal Republic Germany
Abstract Nine angustine-type alkaloids were isolated from ammoniacal extracts of Nauclea orientalis L. (Rubiaceae). Three of them, lO-hydroxyangustine and the two diastereoisomeric 3, 14-dihydroangustolines, have not been described in the literature thus far. The structures of the isolates were determined with spectroscopic methods, mainly 1D- and 2D-NMR spectroscopy.
The compounds were found to exhibit in vitro antiproliferative activity against the human bladder carcinoma T-24 cell line and against EGF (epidermal growth
factor)-dependent mouse epidermal keratinocytes. By using overpressure layer chromatography, it was shown that minor quantities of these alkaloids occur in dried Nauclea orientalis leaves. The use of ammonia in the ex-
traction process results in a significant increase in the
formation of angustine-type alkaloids from strictosamide-type precursors.
Key words
Nauclea orien talis, angustine-type alkaloids, antiproliferative activity, ammonia extraction, strictosamide-type precursors.
Introduction Nauclea orientalis L. (Rubiaceae) is a tree oc-
curring in Papua New Guinea, Indonesia, and Queensland (Australia). Villagers of the Central Province of Papua New Guinea use the bark and the leaves of this tree as a pain reliever for abdominal pains, animal bites, and wounds (1). Recently, we isolated a series of indole alkaloid glycosides from this plant species comprising strictosamide and 10hydroxystrictosamide as the major components (2). These glycosidic isolates were tested for antiproliferative activity, however they were found to be inactive.
On the other hand, alkaloids having an indolo-pyrido-naphthyridine ring system such as angustine, were reported from Nauclea species (3—5). It is proposed
that angustine-type alkaloids are produced from strictosidine- or strictosamide-type precursors during the extraction process with ammonia. We were prompted to investigate Nauclea orientalis for angustine-type alkaloids for two reasons: the biological potential of these compounds has not been studied. Structural similarity with the potent antineoplastic agent camptothecin suggested that angustine-type alkaloids might have cytotoxic potential. The further objective was to examine which individual alkaloids are found in an extract of Nauclea orientalis prepared with ammonia and whether such compounds originally occur in the dried plant material.
Here we describe the isolation, structure determination and in vitro antiproliferative activity of nine alkaloids having an indolo-pyrido-naphthyridine ring sys-
tem, obtained from an ammoniacal extract of Nauclea orientalis leaves.
Materials and Methods General experimental UV spectra were obtained on a Perkin-Elmer Lambda 3 spectrophotometer. IR spectra were recorded on a Perkin-Elmer 781 spectrometer. Optical rotation was measured on a Perkin-Elmer 141 polarimeter. El-mass spectra were recorded on a ZAB 2-SEQ spectrometer at 70 eV. The FAB-mass spectrum of 9 was recorded on a ZAB 2-SEQ spectrometer in the positive ion mode using 3-NOBA as matrix. One-dimensional NMR experiments as well as the reverse (1H-) detected 1H-13C short range correlation spectra (HMQC via direct coupling) (6) of angustine and 3,14-dihydroangustoline were performed with a Bruker AMX-300 spectrometer operating at 300 (1H) or 75.5 MHz (13C), with TMS as
internal standard (b 0 ppm). The reverse (1H-) detected 1F1-13C long range correlation spectrum (HMBC) (6) of angustine was recorded on a Bruker AMX-600 instrument. Bruker standard pulse sequences were used in all NMR experiments.
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Received: April 18, 1991
C. A. J. Erdelmeler et at
Planta Med. 58(1992)
Plant material The plant material was collected near Mariboi rubber estate 60km north of Port Moresby, Central Province of Papua New Guinea in September 1988 (7) and identified by Mr. Max Kuduk and Dr. M. Baltisberger. Herbarium specimens are deposited at Herbarium ZT, ETH, Zurich, Switzerland, as well as at
solid; liv A
Ill vh cm* 3419, 2926, 2855, 1658, 1612, 1454, 1408, 1327, 1262, 1043, 745; El-MS m/z (rel. mt. %): 287 (100), 272 (13), 257 (10), 229 (8), 183 (7), 144 (11), 129 (12), 115 (14); 1H-NMR see Table 1.
UPNG Herbarium, Port Moresby, P.N.G. and at National Herbarium in Lae, P.N.G.
Isolation N. oriental/s leaf material (1028 g) was lixiviated with ammonia (10%, 21). The resulting mass was then macerated with ethyl acetate (totally 121). Combined ethyl acetate extracts were evaporated to give 12.4 g residue. This residue was subjected to alkaloid liquid-liquid partitioning according to Phillipson et al. (3) resulting in a final, concentrated alkaloidal fraction of 1.95g. This fraction displayed significant growth inhibition (IC50 = 9.5 pg/ ml) for the human bladder carcinoma T-24 in vitro cell line (8) and was antiproliferative against EGF (epidermal growth factor)-dependent mouse epidermal keratinocytes (9) (IC50 = 8.1 pg/ml).
The alkaloidal fraction (1.7 g) was further separated using repetitive lIP-MPLC on a 18.5mm ID. x 713 mm column, packed with Bondesil C15 (40 pm) and a mobile phase of methanol-acetonitrile-tetrahydrofuran-water (ST = 1.56, Ps 811), optimized according to the "PRISMA" system (10), at a flow rate of 1.5m1/min. A total of seven fractions (1—Vil) was obtained. Alkaloids of interest, showing intense bright yellow UV fluorescence on TLC and positive Dragendorif reaction, were found in fractions III (168mg), IV (47mg), V (92mg), and VII (170mg). These fractions were further separated with NP-HPLC employing a 16mm ID. x 250mm column, packed with Lichrosorb Si-60, 5pm, and using n-hexane-chloroform-ethanol-diethylamine (66: 28 : 5: 1) as mobile phase at a flow rate of 10 mI/mm. In this manner, from fraction VII, 51mg ofi (tr = 8.0 mm) and 5mg of2(tr= 10.8 mm), from fraction V. 28mgof3 (tr= 12.5 mm), 27mgof4(t= 15.5 mm), and
Compound 5: (0.00068%) yellow amorphous nm (log a): 388 (4.9), 364 (4.9), 250 (4.0), 203 (4.5);
Compound 6: (0.00195%) orange, crystalline
solid; laTh°: 0°; UV A ' nm (log a): 394 (4.4), 375 (4.4), 299 (3.9),
288 (3.9), 250 (4.1), 215 (4.4); III vcm: 3420, 2926, 2854, 1665, 1604, 1326, 1115, 747; El-MS ,n/z (rel. mt. %): 331 (100), 316 (64). 300(11), 288 (14), 286 (13), 157(16), 144 (13), 130 (13), 115 (14); 1H-NMR see Table 1; 13C-NMR see Table 3.
Compound 7: (0.00097) yellowish, amorphous
sohd; [aI°: — 172.5° (c 0.20, EtOH); liv A 2}' nm (log a): 395 (4.2),
372(4.2), 287 sh, 267(5.0), 217(5.6), 204(5.7); IRvcm: 3417, 3236, 2963, 2924, 2854, 1639, 1606, 1445, 1406, 1306, 746; ElMS m/z (rel. in %): 333(63), 315(60), 300(35), 169(53), 155(30), 143 (35), 115 (38), 43 (100); 1H-NMR see Table 2; 13C-NMR see Table 3. Compound 8: (0.00146%) yellowish, amorphous solid; [a]°: —301.4° (c 0.33, EtOH); UVA nm (log a): 396 (4.1),
373 (4.1), 294 sh, 265 (4.8), 220 sh, 204 (5.4); IR vicm: 3392, 3286, 2970, 2925, 2854, 1642, 1606, 1449, 1403, 1305, 1177, 743; El-MS in/z (rel. in %): 333 (17), 314 (7), 300 (4), 169 (5), 98 (4), 18 (100); 1H-NMR see Table 2; 13C-NMR see Table 3.
Compound 9: (0.00136%) orange, crystalline
nm (log a): 404 (4.1), 384 (4.1), 310 (3.6), 303 (3.6), 253 (3.8), 205 (4.3); IR v1cm1: 3417, 2923, 2854, 1629, 1605, 1401,1336,1213,835,744; FAB-MS m/z(rel. mt. %): 330 FM + H]t
solid; UV A
(33); 'H-NMR see Table 1.
Overpressure layer chromatography
(OPLC) 4mg of 5 (t. = 25.0 mm), and from fraction IV, further 3mg of 5 (tr = Instrument: Chrompres 10 (Laboratory Instru25.0 mm) and 20mg of 6 (tr = 27.5 mm) were obtained. Using the ments, Budapest, Hungary). The mobile phase was delivered with same NP-HPLC column with a mobile phase of n-hexane- a Lewa M3 pump (Lewa Herbert Ott, Leonberg. F.R.G.). Plate: 10 x chloroform-ethanol-diethylamine (50:42 : 7: 1), 10mg of 7 (tr 20cm aluminium-backed silica 60 F254 HPTLC (Merck, Darmstadt, 26.5 mm), 15mg of8 (t. = 31.4 mm), and 14mg of9(t. = 40.5 mm) F.R.G.). Plate-edge impregnation was performed in the usual manwere isolated from fraction III. ner with Impres II. Mobile phase: n-hexane-chloroform-ethanol-
Characterization of the isolates Compound 1: (0.00496%) orange, crystalline solid; UV A9JI' nm (log s): 399 (4.5), 379 (4.5), 305 (3.9), 294 (3.9),
254 (4.1), 216 sh, 205 (4.5); IR vcm1: 3420, 3233, 1642, 1602, 1529, 1399, 1324, 740; Ei-MS m/z(rel. i. %): 313(100), 298(27), 156 (26), 128 (27); 1H-NMR see Table 1; 13C-NMR see Table 3.
Compound 2: (0.00049%) orange, crystalline solid; UVAnm (logs) 395(4.3), 375(4.3), 287(4.0), 249 sh, 213 sh, 205 (4.5); IR vcm1: 3420, 2928, 2856, 1665, 1601, 1327, 1132,748,619; El-MS m/z(rel. mt. %): 315(100). 300 (28), 158(9), 122 (8); 'H-NMR see Table 1. Compound 3: (0.00272%) yellowish, amorphous solid; [aI°: — 296.7° (c 0.33, EtOH); UV A 'A nm (log a): 399 (3.6),
379 (3.6), 288 sh, 280 sh, 254 sh, 219(4.7), 206(4.7); IR vcm: 3237, 2924, 2853, 1643, 1605, 1439, 1401, 1325, 1305, 742; ElMS m/z (rel. in %): 315 (100), 300 (39), 286 (18), 169 (46), 117 (34); 1H-NMR see Table 2; 13C-NMR see Table 3.
Compound 4: (0.00263%) yellowish, amorphous
solid; UVA
nm (log a): 390(4.3), 375(4.3), 289 sh, 269(4.6), 222
(5.1), 204(5.0); IR vcm: 3262; 2966, 2927, 2875, 2854, 1634, 1599, 1442, 1400, 1324, 1302, 736; El-MS m/z (rel. mt. %): 317 (100), 302 (43), 288 (18), 169(42), 158(19), 143 (21), 115 (18), 91 (18); 1H-NMR see Table 2; 13C-NMR see Table 3.
diethylamine (83: 14: 2 : 1). Flow rate: 0.2 ml/min. Cushion pressure: 13 bar. Development time: 30 or 45mm, respectively (continuous development). A separation pathway of 17cm was used and the plate was preconditioned with n-hexane at a flow rate of 0.6 mi/mm. Samples: 50 pg of individual extracts were streaked in a 1 cm band using a Camag Linomat IV (Camag, Muttenz, Switzerland). Extracts were prepared with 1 g samples of Nanclea oriental/s dried leaves, using the classical extraction scheme with either
10% ammonia or 10% sodium carbonate foEowed by alkaloid liquid-liquid partitioning (3), or were obtained by extraction with
95% ethanol and subsequent alkaloid partitioning. Detection: Plates were viewed under 366 nm, angustine-type alkaloids are revealed by intense light-blue fluorescent spots.
Results and Discussion
A total of nine non-glycosidic mndole al-
kaloids were isolated from an ammoniacal extract of Nauclea orientalis leaves. The known angustine (1) was found to be the main constituent in the extract. It was identified from its spectroscopic data and by comparison of the data with the literature (3, 11, 12). On the basis of their simi-
lar spectral data, isolates 2, 5, 6 and 9, were assumed to have structures closely related to 1. The compounds 5 and 6
were identified by comparison of their spectral data with published data (11, 12, 13).
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44
Indole Alkaloids with in vitro Antiproliferative Activity from the Ammoniacal Extract of Nauclea orientalis
1
2
Compound No. 5
6
9
Planta Med. 58(1992) 45
Table 1 'H-NMR data of angustine-type alkaloids isolated from Nauclea orientalisa.
Proton at carbon: b
4.51 t (6.5)
4.51 t (6.6)
4.52 t (6.6)
4.51 t (6.6)
4.51 t (6.6)
6a'b
3.18t(8.0)
3.17t(6.6)
3.18t(6.6)
3.19t(6.6)
3.13t(6.6)
9
7.60 d (8.0)
7.61 d (7.9)
7.62 d (7.9)
7.61 d (8.0)
6.97 d (2.0)
10 11
7.13t(8.0)
7.16t(7.8)
7.17t(7.5)
7.16t(7.5)
—
7.30 t (8.0)
7.31 t (7.8)
7.31 t (7.5)
7.31 t (7.5)
6.90 dd (8.8,2.0)
12 14
7.46d(8.0) 7.16s 9.34s
7.44d(7.9) 6.93s 9.37s 1.36t(7.5)
7.44d(7.9) 6.76s 9.49s
7.46d(8.0) 7.22s 9.38s 1.67d(6.6)
7.30d(8.8) 7.17s 9.33s 5.62d(11.2)
—
—
—
5.45 q (6.6)
5.91 d (17.4) 7.25 dd (17.4,11.2)
17
—
5.61 d(12.0) — 5.89 d (18.0) 7.17 dd (18.0,12.0) 2.92 q (7.5)
18a 18b
19 20 21
—
—
7.36 d (5.5)
—
—
8.66s
8.44s
8.57d(5.5)
8.60s
8.67s
Table 2 'H-NMR data of 3,14-dihydroangustine-
Compound No.
3
4
5.01 d(br,— 12) 3.07 dt (12.5,3.9) 5.17 d (br, —13) 2.97 m
5.02d(br,— 12)
9
7.55 d (7.8)
7.55 d (7.5)
10
7.13t(7.8) 7.20t(7.8) 7.39d(7.8)
7.11 t(7.8)
2.87 dd (16.6, 12.5) 3.78 dd (16.0,3.9)
— 2•83b
9.14s 5.59d(11.2) 5.82d(17.5)
7
8
5.OOd(br,— 13) 3.07 dt (12.4,4.5) 5.18d (br, 12.4) 2.98 m 7.56 d (7.7)
4.96d(br— 13)
7.20t(7.8) 7.40d(7.5)
7.13t(7.7) 7.21t(7.7) 7.40d(7.7)
7.14t(7.8) 7.20t(7.8) 7.37d(7.8)
3.74 dd (16.5,3.9)
2.91 dd (16.3,13.2) 4.06 dd (16.3,3.7)
2.86 dd (16.5, 13.0) 3.76 dd (16.5,3.6)
9.lOs 1.28t(7.5)
9.14s 1.59d(6.7)
9.08s 1.51d(6.7)
type alkaloids isolated from Nauclea orientallsa.
Proton at carbon:
3
a 5b
6a,b
11
12
14 '4b 17 18a
'8b 19 21
3.07 dt (12.3,4.2) 5.16 d(br, —12) 2.97 m
2.98 dt (12.6,4.5) 5.13d (br, 12.6) 2.92 m 7.54 d (7.8)
—
—
—
6.92 dd (17.5, 11.2)
2.81 qb (7.5)
5.07 q (6.7)
5.20 q (6.7)
8.74s
8.48s
8.57s
8.75s
Spectra were measured at 300.13 MHz in CDCI3-CD3OD (4:1); data given in ppm relative to TMS; in brackets i-values in Hz. Signals overlapped.
In its El-mass spectrum, compound 2 showed a molecular ion at in/z 315, two mass units more than that of angustine (1), corresponding to a molecular formula C20H17N30. Its 1H-NMR spectrum was very similar to that of 1, except for the vinylic resonances which were absent. A triplet at ó = 1.36 ppm (H-18), accounting for three protons, and a quartet at 5 = 2.92 ppm (H-19) for two protons coupled to the protons at = 1.36 ppm, indicated that 2 bore an ethyl side chain at C-20. Compound 2 thus was identified as 18,19-dihydroangustine, for the first time reported here from a natural source.
As a further, unknown angustine-type compound, 9 was isolated. Its FAB-MS revealed a [M + 1] ion at ,n/z 330, analyzing for C20H15N302. The 1H-NMR spectrum of 9 again was very similar ro that of angustine (1). In con-
trast to the spectrum of 1, the 1H-NMR spectrum of 9 showed two signals, a doublet at 6 = 7.30 ppm (H-12) and a doublet of doublets at 6 = 6.90 ppm (H-li), for two ortho-
coupled aromatic protons. Additionally, the doublet of
doublets showed a meta-coupling (2.0 Hz) to a proton with resonance at 6= 6.97 ppm (H-9). This was consistent with a phenolic hydroxy group at C-loin the molecule of 9. Hence, isolate 9 was iO-hydroxyangustine, an alkaloid which has not been described in the literature thus far.
The compounds 3, 4, 7, and 8 were identified as 3,14-dihydroangustine-type constituents. The 1HNMR spectra of these compounds show marked differences when compared to those of the angustine-type constituents. The latter group shows an olefinic singlet for the proton at C-i4 in the region 6 = 6.6—7.2 ppm, and two triplets for the methylene protons at C-5 and C-6, respectively. In the 3,14dihydro series, a broad doublet for H-3 appears in the region 6 = 4.9—5.0 ppm, and a doublet of doublets for each of the two geminal protons at C-14. In addition, in the 3,14-dihydro series, the two geminal protons at C-5 are not equiva-
lent, as opposed to the angustine-type compounds, and show markedly different chemical shifts, H5a at 5 = 2.9 3.1 ppm, and H-5b between 6 = 5.1 and 5.2 ppm. Corn-
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a Spectra were measured at 300.13 MHz in CDCI3-CD3OD (4:1); data given in ppm relative to TMS; in brackets J-values in Hz.
46 Planta Med. 58(1992)
C. A. J. Erdelmeier et al.
pound 3 displayed spectroscopic properties very close to data published for 3,14-dihydroangustine (11, 12).
N22
Isolate 4 showed similar spectral data as 3. Its El-mass spectrum revealed a molecular ion at m/z 317, two mass units higher than that of 3. The 1H-NMR spectrum of 4 was very similar to that of 3 except for a triplet at ó =
R'
________________________________ R2
name
H2c=cH—
H
angustine
2 5
H3c—cH2—
H
H
H
18,19 -dihydroangustine nauctefine
6
H3 C\ ,CH— HO
H
angustotine
9
HC=CH—
HO
lO-hydroxyangustine
R1
8 19
1
1.28 ppm
(J = 7.5 Hz, protons at C-18) and a quartet at
2.81 ppm (J = 7.5 Hz, protons at C-19), indicating an ethyl side chain in lieu of the vinylic group present in compound 3. Thus this constituent was identified as 3,14,18,19-tetrahydroangustine, previously not obtained from nature. The compounds 7 and 8 showed very similar spectroscopic data. Both displayed a molecular ion at m/z 333 in their El-mass spectra, accounting for a molecu-
data were very similar, though, not identical (Tables 2 and 3). In the 1H-NMR spectra of 7 and 8, prominent differences were observed for the signal of H-19 which was attached to one of the two asymmetric carbons in the molecule of these optical isomers. In the spectrum of 7, the H-19 quartet appeared at 6 = 5.07 ppm, whereas the respective proton in compound 8 resonated at 6 = 5.20 ppm, i.e. Aô = 0.13 ppm.
R
_________________________________________ name
R
3 4
18 19
I-f2c=cH— H3C—CH2—
3,11. -dihydroangustine 3,14, 18, 19-tetrahydrOangustine
In the 13C-NMR spectra of 7 and 8 the only significant difference is observed for the resonances of C-i 8 and C-i 9 (Table
3,14-dihydroangustoline, [e]0: —172.5°
3). The C-19 resonance in the spectrum of 7 is found at 6 = 67.2 ppm, shifted by Aô = 2ppm to higher frequencies when compared to the C-19 resonance in the spectrum of
3
7
,CH—
HO
HO
8
',CH—
isolate 8. Assuming that the alkaloids reported here are
3,11.-dihydroangustoline, [aID: —301.4°
formed from the strictosamide-type precursors recently described from Nauclea orientalis (2) which have 3a-H con-
H3c
Table 3 '3C-NMR spectral assignments of
Compound No.
C-Atom
2 3 5 6 7
8 9 10 11
12 13 14 15 16 17 18 19
20 21
22
3
4
6
7
8
127.2(H-6, H-14)
131.3'
132.2
137.1 (H-5, 1-1-14) 40.5(1-1-6) 19.3(1-1-5)
51.5 39.7 21.0 109.0 124.8 118.5 119.6 122.3 111.3 137.0 31.4 143.0 126.5 148.7** 120.2
51.7 39.8
127.5 137.4
132.0 51.4 39.6 20.9 108.8 126.4 118.4 119.6 122.2 111.3 136.9' 31.3 144.5 125.5
131.6 51.3 39.5 20.9 109.6 126.5 118.6 119.9 122.5 111.2 137.1'
149.1 23.1
149.3" 24.0 65.2
1b
115.0(H-5,H-6,H-9) 125.2(H-l0,H-12) 119.1(H-11) 119.8(H-12) 124.4(H-9) 111.5(H-10) 138.7(H-9,F-l.11) 94.1
139.8(H-17,H-21) 118.85(H-14,H-17) 149.3(H-21)
118.8
129.6(H-18b,H-21) 127.9(H-18,H-21) 146.4(H-17,H-19) 161.9
21.1 108.9 126.6 118.5 119.6 122.3 111.4 137.2
31.1 144.5
130.1
125.0 147.6 14.6 23.1
131.9'
136.4
149.5"
151.6 164.2
163.8
41.1 20.0 115.5
125.9 119.8 120.5 125.1 112.1 139.3 95.0 140.5 120.0 150.3 23.8 66.2 134.2 146.4 162.8
67.2 136.8' 150.0 163.7
31.4 142.7
124.6
136.8'
148.7" 163.4
75 MHz / solvent CDCI3-CD3OD (4: 1).
'H'3C long range correlations from HMBC experiment, delays optimized to observe J8 = 10 Hz. 'I" Assignments interchangeable within columns. §
Signals overlapped.
selected isolated alkaloidsa.
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lar formula of C20H19N302. As diastereoisomers, their NMR N
Indole Alkaloids with in vitro Antiproliferative Activity from the Ammoniacal Extract of Nauclea orientalis
Planta Med. 58(1992) 47
Fig. 1 Reverse (1H) detected 'H-'30 long range correlation spectrum of angustine 1 (pulse sequence optimized to observe c,H 10Hz; correlations of C-5 and 0-6 not shown(.
12 71 16$
$
7$
120
ic4)116 8$
132
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21
020
3$
13$ 134
15(
154
I
21Q
21
I
-150
174
p0 ppm
a
p
i
p
5
a
3
hH
figuration, and this stereochemistry at C-3 is maintained in the 3,14-dihydroangustines, the NMR data of compounds 7 and 8 indicate that these two isolated differ only in their stereochemistry at C-19. Attempts to determine the absolute configuration at C-19 in 7 and 8 were unsuccessful. Table 3 shows the 13C-NMR data of selected
isolates, mainly those obtained in major quantities. Complete and unambiguous carbon NMR assignments were possible for the main constituent angustine (1) with the use of 2D reverse detected proton-carbon correlation spectro-
scopy. A 1H-detected heteronuclear multiple-quantum coherence experiment via direct coupling (HMQC) allowed all protonated carbons of 1, and thus those for 6, to be assigned. The same experiment performed on compound 7 permitted assignment of protonated carbons for this isolate and by analogy for the compounds 3, 4, and 8. Unambiguous assignments were not possible for C-2, C-20, and C-17, C-21 in 3, as well as for C-13 and C-20 in 7, and C-13, C-20, and C-i 7, C-2 1 in 8. For the 1H-detected heteronuclear multiple-bond correlation (HMBC) spectrum (Fig. 1) of 1, the pulse sequence was optimized to observe couplings in the range of 10Hz. The cEI and 2c,H couplings observed in this spectrum (Table 3) afforded the necessary information to assign all resonances for non-protonated carbons of 1. The spectrum was obtained with a 10mg sample of 1 in a total acquisition time of 9.5 hours.
All isolated alkaloids displayed in vitro antiproliferative activity against both T-24 and EGF depen-
dent MK cells (Table 4). Thus the hypothesis was confirmed
that, as with the well-known and structurally related antineoplastic agent camptothecin, angustine-type alkaloids
would also exhibit cytotoxicity. Very recently, 19-0methylangustoline was isolated from Camptotheca acuminata and shown to be cytotoxic when tested against P-388 leukemia cells (13). The biological test results are shown in Table 4 and permit some structure-activity relations to be concluded. There is no major difference in activity between the angustine-type and 3,14-dihydroangustine-type isolates. Presence of a C2 side chain at C-20 is not essential for the activity. However, introduction of a hydroxy substituent in the side chain at C-20, as is the case with the compounds 6, 7, and 8, results in an apparent loss of activity. As can be seen from Table 4, the stereochemistry at C-i9 in isolates 7 and 8 is not crucial for the antiproliferative activity. Compounds 1 and 9 show activities of the same
magnitude as 5, 3, and 4, in the T-24 test system, though, significantly stronger inhibition of EGF-dependent MK cell growth. In regard to the second objective of this investigation, the question whether or not these alkaloids are mere artifacts or do occur in the dried plant material, vari-
ous extractions of N. orientalis were carried out and chromatographically compared. Since the conventional TLC separation of these indole alkaloids proved to be dif-
ficult, overpressure layer chromatography (OPLC) was
employed in order to be able to identify individual alkaloids in the extracts. For the OPLC separation the continuous de-
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ii$ 20$ 191
48 Planta Med. 58(1992)
C. A. I Erdelmeier et al.
Table 4 In vitro antiproliferative activity of isolated alkaloids against human bladder carcinoma cells (T.24) and against EGF-dependent mouse keratinocytes (MK).
T.24a
MK
Angustine-type Isolates
1
3.3 (10.5) (29.2) (16.4) (99.4) (10.3)
0.4 (1.3)
4.3 (13.6)
1.1 (3.5) 1.2 (3.8)
2 9.2 5 4.7 6 32.9 9 3.4
3.4 (10.8)
3.6 (12.5) 4.2 (12.7) 0.5 (1.5)
reasoned that, whenever angustine-type constituents are found, the respective 3,14-dihydro compounds will also be
present.
3,14-Dihydroangustine-type Isolates 3,14.Dihydroangustine 3,14,18,19.Tetrahydroangustine 3,14-Dihydroangustoline([a}D.172.5°) 3,14-Dihydroangustoline([a]D-301.4°)
the precursors of the latter compounds. It can thus be
3
4 7 8
5.0 (15.8) 12.4 (37.2) 14.3 (42.9)
6.6 (19.8) 6.1 (18.3)
lC58-values in g/ml (j.tmol); serial dilutions of isolates were tested, isolates which could be tested twice gave reproducible results.
It can be concluded that the alkaloids found in this study do occur in dried Nauclea orientalis leaves in minor quantities. When ammonia is used in the extraction, there is a significant artificial increase in the formation of 3,1 4-dihydroangustine-type and angustine-type alkaloids from glycosidic precursors of the strictosamide-type. Alkaloid glycosides of the strictosamide-series cannot be detected in ammoniacal extracts ofN. orientalis, however, are
clearly present in such extracts prepared without ammonia.
Acknowledgements This research was supported by a grant of the Swiss National Science Foundation. We wish to thank Dr. M. Baltis-
berger. ETH Zurich, for the preparation of the herbarium specimens and his assistance on the collecting trip. Our thanks are also due to Mr. Max Kuduk, University of Papua New Guinea for his val-
uable help in botanical matters. We gratefully acknowledge Ms. Claudia Oertel for technical assistance in the isolation work. We also thank Mr. H. Hafliger, ETH Zurich, for running the mass spectra and Dr. A. Wright for running 1D-NMR and 2D reverse detected 1H-13C short range correlation NMR spectra. Finally, we
wish to thank Dr. D. Moskau, Spectrospin AG, Fällanden, for measuring the 2D reverse detected 8H-13C long range correlation NMR spectrum of angustine.
References 1 Wolif-Eggert, R. (1977) Dissertation, Erlangen. 2 Erdelmeier, C. A. J., Wright, A. D., Orjala, J., Baumgartner, B., Hall, T., Sticher, 0. (1991) Planta Med. 57, 149—152. Fig. 2 Chromatograms of various extracts of Nauclea orientalis leaves, obtained by overpressure layer chromatography (OPLC). Continuous development for 45 mm (a) and 30 mm (b). Detection: Fluorescence at 366 nm. Samples were obtained by extraction of plant material, lixiviated with 10% ammonia (A) or with 10% sodium carbonate (B), followed by liquid-liquid partitioning. Samples C and D were obtained by extraction with 95% ethanol, and subjected to OPLC prior to (C) and after (D) liquid-liquid partitioning.
6
8
velopment technique was used to enhance resolution be-
tween components. Because of the typical long wave IJY fluorescence of the angustine-type compounds the detection was considerably facilitated. Fig. 2 shows a photograph of the OPLC separations of four different N. orientalis extracts, as 45 (Fig. 2a) and 30 mm (Fig. 2b) developments, respectively. The extract obtained with 10% ammonia and
subsequent alkaloid partitioning (A in Fig. 2) basically represents the extract prepared for the isolation and identification of individual alkaloids. All isolates of the angustine-
type series, angustine (1), 18,19-dihydroangustine (2),
Phillipson, J. D., Hemingway, S. H., Bisset, N. G., Houghton, P. J., Shellard, F. J. (1974) Phytochemistry 13. 973—978. Phillipson, J. D., Hemingway, S. R., Ridsdale, C. (1982) J. Nat. Prod. 45, 145—162. Zeches, M., Richard, B., Gueye-M'Bahia, L., Le Men-Olivier, L., Delaude, C. (1985) J. Nat. Prod. 48,42—46. Summers, M. F.. Marzilli, L. G., Bax, A. (1986) J. Am. Chem. Soc. 108,4285—4294. Baltisberger, M., Erdelmeier, C. A. J., Rali, T. (1989) Ber. Geobot. Inst. ETH 55, 252—259. Meyer, Th., Regenass, U., Fabbro, D., Alteri, E., ROsel, J.Müller, M., Carvatti, G., Matter, A. (1989) mt. J. Cancer 43, 851—856. Geissler, J. F., Traxier, P., Regenass, U., Murray, B. F., Roesel. J. L., Meyer, Th., McGlynn, E., Storni, A., Lydon, N. B. (1990) J. Biol. Chem. 265, 22255—22261.
° Nyiredy,
Sz., Dallenbach-Tölke, K., Sticher, 0. (1988) J. Planar
Chromatogr. 1, 336—342. Hotellier, F., Delaveau, P., Pousset, J. L. (1975) Phytochemistry 14, 1407— 1409. 12 13
Repke, D. B., Jahangir, Clark, R. D., Nelson, J. T., MacLean, D. B.
(1989) Tetrahedron 45, 2541—2550. Lin, L.-Z., Cordell, G. A. (1990) Phytochemistry 29, 2744—2746.
Downloaded by: University of British Columbia. Copyrighted material.
Angustine 18,19-Dihydroangustine Nauclefine Angustoline 10-Hydroxyangustine
nauclefine (5), angustoline (6), and lO-hydroxyangustine (9) are detectable in alkaloidal extracts prepared in the absence of ammonia (Fig. 2). However, the 3,14-dihydroangustine-type compounds do not display UV fluorescence and are not visible under these conditions. Since the 3,14dihydroangustine-type alkaloids easily transform into their angustine-type derivatives, they most probably represent
Indole Alkaloids with in vitro Antiproliferative Activity from the Ammoniacal Extract of Nauclea orientalis1 C. A. J. Erdel,neier2'5, U. Regenass3, T. Rali4, and 0. Sticher2 1
Part 3 in the series: Medicinal plants from Papua New Guinea'; for Part 2, see Ref. (2) Department of Pharmacy, Swiss Federal Institute of Technology (ETH) Zurich, CH-8092 Zurich, Switzerland Research Department, Pharmaceuticals Division, Ciba-Geigy Ltd., CH-4002 Base!, Switzerland Department of Chemistry, The University of Papua New Guinea, Port Moresby, Papua New Guinea Address for correspondence: Dr. Wilimar Schwabe Arzneimittel, Research and Development, D(W)-7500 Karlsruhe, Federal Republic Germany
Abstract Nine angustine-type alkaloids were isolated from ammoniacal extracts of Nauclea orientalis L. (Rubiaceae). Three of them, lO-hydroxyangustine and the two diastereoisomeric 3, 14-dihydroangustolines, have not been described in the literature thus far. The structures of the isolates were determined with spectroscopic methods, mainly 1D- and 2D-NMR spectroscopy.
The compounds were found to exhibit in vitro antiproliferative activity against the human bladder carcinoma T-24 cell line and against EGF (epidermal growth
factor)-dependent mouse epidermal keratinocytes. By using overpressure layer chromatography, it was shown that minor quantities of these alkaloids occur in dried Nauclea orientalis leaves. The use of ammonia in the ex-
traction process results in a significant increase in the
formation of angustine-type alkaloids from strictosamide-type precursors.
Key words
Nauclea orien talis, angustine-type alkaloids, antiproliferative activity, ammonia extraction, strictosamide-type precursors.
Introduction Nauclea orientalis L. (Rubiaceae) is a tree oc-
curring in Papua New Guinea, Indonesia, and Queensland (Australia). Villagers of the Central Province of Papua New Guinea use the bark and the leaves of this tree as a pain reliever for abdominal pains, animal bites, and wounds (1). Recently, we isolated a series of indole alkaloid glycosides from this plant species comprising strictosamide and 10hydroxystrictosamide as the major components (2). These glycosidic isolates were tested for antiproliferative activity, however they were found to be inactive.
On the other hand, alkaloids having an indolo-pyrido-naphthyridine ring system such as angustine, were reported from Nauclea species (3—5). It is proposed
that angustine-type alkaloids are produced from strictosidine- or strictosamide-type precursors during the extraction process with ammonia. We were prompted to investigate Nauclea orientalis for angustine-type alkaloids for two reasons: the biological potential of these compounds has not been studied. Structural similarity with the potent antineoplastic agent camptothecin suggested that angustine-type alkaloids might have cytotoxic potential. The further objective was to examine which individual alkaloids are found in an extract of Nauclea orientalis prepared with ammonia and whether such compounds originally occur in the dried plant material.
Here we describe the isolation, structure determination and in vitro antiproliferative activity of nine alkaloids having an indolo-pyrido-naphthyridine ring sys-
tem, obtained from an ammoniacal extract of Nauclea orientalis leaves.
Materials and Methods General experimental UV spectra were obtained on a Perkin-Elmer Lambda 3 spectrophotometer. IR spectra were recorded on a Perkin-Elmer 781 spectrometer. Optical rotation was measured on a Perkin-Elmer 141 polarimeter. El-mass spectra were recorded on a ZAB 2-SEQ spectrometer at 70 eV. The FAB-mass spectrum of 9 was recorded on a ZAB 2-SEQ spectrometer in the positive ion mode using 3-NOBA as matrix. One-dimensional NMR experiments as well as the reverse (1H-) detected 1H-13C short range correlation spectra (HMQC via direct coupling) (6) of angustine and 3,14-dihydroangustoline were performed with a Bruker AMX-300 spectrometer operating at 300 (1H) or 75.5 MHz (13C), with TMS as
internal standard (b 0 ppm). The reverse (1H-) detected 1F1-13C long range correlation spectrum (HMBC) (6) of angustine was recorded on a Bruker AMX-600 instrument. Bruker standard pulse sequences were used in all NMR experiments.
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Received: April 18, 1991
C. A. J. Erdelmeler et at
Planta Med. 58(1992)
Plant material The plant material was collected near Mariboi rubber estate 60km north of Port Moresby, Central Province of Papua New Guinea in September 1988 (7) and identified by Mr. Max Kuduk and Dr. M. Baltisberger. Herbarium specimens are deposited at Herbarium ZT, ETH, Zurich, Switzerland, as well as at
solid; liv A
Ill vh cm* 3419, 2926, 2855, 1658, 1612, 1454, 1408, 1327, 1262, 1043, 745; El-MS m/z (rel. mt. %): 287 (100), 272 (13), 257 (10), 229 (8), 183 (7), 144 (11), 129 (12), 115 (14); 1H-NMR see Table 1.
UPNG Herbarium, Port Moresby, P.N.G. and at National Herbarium in Lae, P.N.G.
Isolation N. oriental/s leaf material (1028 g) was lixiviated with ammonia (10%, 21). The resulting mass was then macerated with ethyl acetate (totally 121). Combined ethyl acetate extracts were evaporated to give 12.4 g residue. This residue was subjected to alkaloid liquid-liquid partitioning according to Phillipson et al. (3) resulting in a final, concentrated alkaloidal fraction of 1.95g. This fraction displayed significant growth inhibition (IC50 = 9.5 pg/ ml) for the human bladder carcinoma T-24 in vitro cell line (8) and was antiproliferative against EGF (epidermal growth factor)-dependent mouse epidermal keratinocytes (9) (IC50 = 8.1 pg/ml).
The alkaloidal fraction (1.7 g) was further separated using repetitive lIP-MPLC on a 18.5mm ID. x 713 mm column, packed with Bondesil C15 (40 pm) and a mobile phase of methanol-acetonitrile-tetrahydrofuran-water (ST = 1.56, Ps 811), optimized according to the "PRISMA" system (10), at a flow rate of 1.5m1/min. A total of seven fractions (1—Vil) was obtained. Alkaloids of interest, showing intense bright yellow UV fluorescence on TLC and positive Dragendorif reaction, were found in fractions III (168mg), IV (47mg), V (92mg), and VII (170mg). These fractions were further separated with NP-HPLC employing a 16mm ID. x 250mm column, packed with Lichrosorb Si-60, 5pm, and using n-hexane-chloroform-ethanol-diethylamine (66: 28 : 5: 1) as mobile phase at a flow rate of 10 mI/mm. In this manner, from fraction VII, 51mg ofi (tr = 8.0 mm) and 5mg of2(tr= 10.8 mm), from fraction V. 28mgof3 (tr= 12.5 mm), 27mgof4(t= 15.5 mm), and
Compound 5: (0.00068%) yellow amorphous nm (log a): 388 (4.9), 364 (4.9), 250 (4.0), 203 (4.5);
Compound 6: (0.00195%) orange, crystalline
solid; laTh°: 0°; UV A ' nm (log a): 394 (4.4), 375 (4.4), 299 (3.9),
288 (3.9), 250 (4.1), 215 (4.4); III vcm: 3420, 2926, 2854, 1665, 1604, 1326, 1115, 747; El-MS ,n/z (rel. mt. %): 331 (100), 316 (64). 300(11), 288 (14), 286 (13), 157(16), 144 (13), 130 (13), 115 (14); 1H-NMR see Table 1; 13C-NMR see Table 3.
Compound 7: (0.00097) yellowish, amorphous
sohd; [aI°: — 172.5° (c 0.20, EtOH); liv A 2}' nm (log a): 395 (4.2),
372(4.2), 287 sh, 267(5.0), 217(5.6), 204(5.7); IRvcm: 3417, 3236, 2963, 2924, 2854, 1639, 1606, 1445, 1406, 1306, 746; ElMS m/z (rel. in %): 333(63), 315(60), 300(35), 169(53), 155(30), 143 (35), 115 (38), 43 (100); 1H-NMR see Table 2; 13C-NMR see Table 3. Compound 8: (0.00146%) yellowish, amorphous solid; [a]°: —301.4° (c 0.33, EtOH); UVA nm (log a): 396 (4.1),
373 (4.1), 294 sh, 265 (4.8), 220 sh, 204 (5.4); IR vicm: 3392, 3286, 2970, 2925, 2854, 1642, 1606, 1449, 1403, 1305, 1177, 743; El-MS in/z (rel. in %): 333 (17), 314 (7), 300 (4), 169 (5), 98 (4), 18 (100); 1H-NMR see Table 2; 13C-NMR see Table 3.
Compound 9: (0.00136%) orange, crystalline
nm (log a): 404 (4.1), 384 (4.1), 310 (3.6), 303 (3.6), 253 (3.8), 205 (4.3); IR v1cm1: 3417, 2923, 2854, 1629, 1605, 1401,1336,1213,835,744; FAB-MS m/z(rel. mt. %): 330 FM + H]t
solid; UV A
(33); 'H-NMR see Table 1.
Overpressure layer chromatography
(OPLC) 4mg of 5 (t. = 25.0 mm), and from fraction IV, further 3mg of 5 (tr = Instrument: Chrompres 10 (Laboratory Instru25.0 mm) and 20mg of 6 (tr = 27.5 mm) were obtained. Using the ments, Budapest, Hungary). The mobile phase was delivered with same NP-HPLC column with a mobile phase of n-hexane- a Lewa M3 pump (Lewa Herbert Ott, Leonberg. F.R.G.). Plate: 10 x chloroform-ethanol-diethylamine (50:42 : 7: 1), 10mg of 7 (tr 20cm aluminium-backed silica 60 F254 HPTLC (Merck, Darmstadt, 26.5 mm), 15mg of8 (t. = 31.4 mm), and 14mg of9(t. = 40.5 mm) F.R.G.). Plate-edge impregnation was performed in the usual manwere isolated from fraction III. ner with Impres II. Mobile phase: n-hexane-chloroform-ethanol-
Characterization of the isolates Compound 1: (0.00496%) orange, crystalline solid; UV A9JI' nm (log s): 399 (4.5), 379 (4.5), 305 (3.9), 294 (3.9),
254 (4.1), 216 sh, 205 (4.5); IR vcm1: 3420, 3233, 1642, 1602, 1529, 1399, 1324, 740; Ei-MS m/z(rel. i. %): 313(100), 298(27), 156 (26), 128 (27); 1H-NMR see Table 1; 13C-NMR see Table 3.
Compound 2: (0.00049%) orange, crystalline solid; UVAnm (logs) 395(4.3), 375(4.3), 287(4.0), 249 sh, 213 sh, 205 (4.5); IR vcm1: 3420, 2928, 2856, 1665, 1601, 1327, 1132,748,619; El-MS m/z(rel. mt. %): 315(100). 300 (28), 158(9), 122 (8); 'H-NMR see Table 1. Compound 3: (0.00272%) yellowish, amorphous solid; [aI°: — 296.7° (c 0.33, EtOH); UV A 'A nm (log a): 399 (3.6),
379 (3.6), 288 sh, 280 sh, 254 sh, 219(4.7), 206(4.7); IR vcm: 3237, 2924, 2853, 1643, 1605, 1439, 1401, 1325, 1305, 742; ElMS m/z (rel. in %): 315 (100), 300 (39), 286 (18), 169 (46), 117 (34); 1H-NMR see Table 2; 13C-NMR see Table 3.
Compound 4: (0.00263%) yellowish, amorphous
solid; UVA
nm (log a): 390(4.3), 375(4.3), 289 sh, 269(4.6), 222
(5.1), 204(5.0); IR vcm: 3262; 2966, 2927, 2875, 2854, 1634, 1599, 1442, 1400, 1324, 1302, 736; El-MS m/z (rel. mt. %): 317 (100), 302 (43), 288 (18), 169(42), 158(19), 143 (21), 115 (18), 91 (18); 1H-NMR see Table 2; 13C-NMR see Table 3.
diethylamine (83: 14: 2 : 1). Flow rate: 0.2 ml/min. Cushion pressure: 13 bar. Development time: 30 or 45mm, respectively (continuous development). A separation pathway of 17cm was used and the plate was preconditioned with n-hexane at a flow rate of 0.6 mi/mm. Samples: 50 pg of individual extracts were streaked in a 1 cm band using a Camag Linomat IV (Camag, Muttenz, Switzerland). Extracts were prepared with 1 g samples of Nanclea oriental/s dried leaves, using the classical extraction scheme with either
10% ammonia or 10% sodium carbonate foEowed by alkaloid liquid-liquid partitioning (3), or were obtained by extraction with
95% ethanol and subsequent alkaloid partitioning. Detection: Plates were viewed under 366 nm, angustine-type alkaloids are revealed by intense light-blue fluorescent spots.
Results and Discussion
A total of nine non-glycosidic mndole al-
kaloids were isolated from an ammoniacal extract of Nauclea orientalis leaves. The known angustine (1) was found to be the main constituent in the extract. It was identified from its spectroscopic data and by comparison of the data with the literature (3, 11, 12). On the basis of their simi-
lar spectral data, isolates 2, 5, 6 and 9, were assumed to have structures closely related to 1. The compounds 5 and 6
were identified by comparison of their spectral data with published data (11, 12, 13).
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44
Indole Alkaloids with in vitro Antiproliferative Activity from the Ammoniacal Extract of Nauclea orientalis
1
2
Compound No. 5
6
9
Planta Med. 58(1992) 45
Table 1 'H-NMR data of angustine-type alkaloids isolated from Nauclea orientalisa.
Proton at carbon: b
4.51 t (6.5)
4.51 t (6.6)
4.52 t (6.6)
4.51 t (6.6)
4.51 t (6.6)
6a'b
3.18t(8.0)
3.17t(6.6)
3.18t(6.6)
3.19t(6.6)
3.13t(6.6)
9
7.60 d (8.0)
7.61 d (7.9)
7.62 d (7.9)
7.61 d (8.0)
6.97 d (2.0)
10 11
7.13t(8.0)
7.16t(7.8)
7.17t(7.5)
7.16t(7.5)
—
7.30 t (8.0)
7.31 t (7.8)
7.31 t (7.5)
7.31 t (7.5)
6.90 dd (8.8,2.0)
12 14
7.46d(8.0) 7.16s 9.34s
7.44d(7.9) 6.93s 9.37s 1.36t(7.5)
7.44d(7.9) 6.76s 9.49s
7.46d(8.0) 7.22s 9.38s 1.67d(6.6)
7.30d(8.8) 7.17s 9.33s 5.62d(11.2)
—
—
—
5.45 q (6.6)
5.91 d (17.4) 7.25 dd (17.4,11.2)
17
—
5.61 d(12.0) — 5.89 d (18.0) 7.17 dd (18.0,12.0) 2.92 q (7.5)
18a 18b
19 20 21
—
—
7.36 d (5.5)
—
—
8.66s
8.44s
8.57d(5.5)
8.60s
8.67s
Table 2 'H-NMR data of 3,14-dihydroangustine-
Compound No.
3
4
5.01 d(br,— 12) 3.07 dt (12.5,3.9) 5.17 d (br, —13) 2.97 m
5.02d(br,— 12)
9
7.55 d (7.8)
7.55 d (7.5)
10
7.13t(7.8) 7.20t(7.8) 7.39d(7.8)
7.11 t(7.8)
2.87 dd (16.6, 12.5) 3.78 dd (16.0,3.9)
— 2•83b
9.14s 5.59d(11.2) 5.82d(17.5)
7
8
5.OOd(br,— 13) 3.07 dt (12.4,4.5) 5.18d (br, 12.4) 2.98 m 7.56 d (7.7)
4.96d(br— 13)
7.20t(7.8) 7.40d(7.5)
7.13t(7.7) 7.21t(7.7) 7.40d(7.7)
7.14t(7.8) 7.20t(7.8) 7.37d(7.8)
3.74 dd (16.5,3.9)
2.91 dd (16.3,13.2) 4.06 dd (16.3,3.7)
2.86 dd (16.5, 13.0) 3.76 dd (16.5,3.6)
9.lOs 1.28t(7.5)
9.14s 1.59d(6.7)
9.08s 1.51d(6.7)
type alkaloids isolated from Nauclea orientallsa.
Proton at carbon:
3
a 5b
6a,b
11
12
14 '4b 17 18a
'8b 19 21
3.07 dt (12.3,4.2) 5.16 d(br, —12) 2.97 m
2.98 dt (12.6,4.5) 5.13d (br, 12.6) 2.92 m 7.54 d (7.8)
—
—
—
6.92 dd (17.5, 11.2)
2.81 qb (7.5)
5.07 q (6.7)
5.20 q (6.7)
8.74s
8.48s
8.57s
8.75s
Spectra were measured at 300.13 MHz in CDCI3-CD3OD (4:1); data given in ppm relative to TMS; in brackets i-values in Hz. Signals overlapped.
In its El-mass spectrum, compound 2 showed a molecular ion at in/z 315, two mass units more than that of angustine (1), corresponding to a molecular formula C20H17N30. Its 1H-NMR spectrum was very similar to that of 1, except for the vinylic resonances which were absent. A triplet at ó = 1.36 ppm (H-18), accounting for three protons, and a quartet at 5 = 2.92 ppm (H-19) for two protons coupled to the protons at = 1.36 ppm, indicated that 2 bore an ethyl side chain at C-20. Compound 2 thus was identified as 18,19-dihydroangustine, for the first time reported here from a natural source.
As a further, unknown angustine-type compound, 9 was isolated. Its FAB-MS revealed a [M + 1] ion at ,n/z 330, analyzing for C20H15N302. The 1H-NMR spectrum of 9 again was very similar ro that of angustine (1). In con-
trast to the spectrum of 1, the 1H-NMR spectrum of 9 showed two signals, a doublet at 6 = 7.30 ppm (H-12) and a doublet of doublets at 6 = 6.90 ppm (H-li), for two ortho-
coupled aromatic protons. Additionally, the doublet of
doublets showed a meta-coupling (2.0 Hz) to a proton with resonance at 6= 6.97 ppm (H-9). This was consistent with a phenolic hydroxy group at C-loin the molecule of 9. Hence, isolate 9 was iO-hydroxyangustine, an alkaloid which has not been described in the literature thus far.
The compounds 3, 4, 7, and 8 were identified as 3,14-dihydroangustine-type constituents. The 1HNMR spectra of these compounds show marked differences when compared to those of the angustine-type constituents. The latter group shows an olefinic singlet for the proton at C-i4 in the region 6 = 6.6—7.2 ppm, and two triplets for the methylene protons at C-5 and C-6, respectively. In the 3,14dihydro series, a broad doublet for H-3 appears in the region 6 = 4.9—5.0 ppm, and a doublet of doublets for each of the two geminal protons at C-14. In addition, in the 3,14-dihydro series, the two geminal protons at C-5 are not equiva-
lent, as opposed to the angustine-type compounds, and show markedly different chemical shifts, H5a at 5 = 2.9 3.1 ppm, and H-5b between 6 = 5.1 and 5.2 ppm. Corn-
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a Spectra were measured at 300.13 MHz in CDCI3-CD3OD (4:1); data given in ppm relative to TMS; in brackets J-values in Hz.
46 Planta Med. 58(1992)
C. A. J. Erdelmeier et al.
pound 3 displayed spectroscopic properties very close to data published for 3,14-dihydroangustine (11, 12).
N22
Isolate 4 showed similar spectral data as 3. Its El-mass spectrum revealed a molecular ion at m/z 317, two mass units higher than that of 3. The 1H-NMR spectrum of 4 was very similar to that of 3 except for a triplet at ó =
R'
________________________________ R2
name
H2c=cH—
H
angustine
2 5
H3c—cH2—
H
H
H
18,19 -dihydroangustine nauctefine
6
H3 C\ ,CH— HO
H
angustotine
9
HC=CH—
HO
lO-hydroxyangustine
R1
8 19
1
1.28 ppm
(J = 7.5 Hz, protons at C-18) and a quartet at
2.81 ppm (J = 7.5 Hz, protons at C-19), indicating an ethyl side chain in lieu of the vinylic group present in compound 3. Thus this constituent was identified as 3,14,18,19-tetrahydroangustine, previously not obtained from nature. The compounds 7 and 8 showed very similar spectroscopic data. Both displayed a molecular ion at m/z 333 in their El-mass spectra, accounting for a molecu-
data were very similar, though, not identical (Tables 2 and 3). In the 1H-NMR spectra of 7 and 8, prominent differences were observed for the signal of H-19 which was attached to one of the two asymmetric carbons in the molecule of these optical isomers. In the spectrum of 7, the H-19 quartet appeared at 6 = 5.07 ppm, whereas the respective proton in compound 8 resonated at 6 = 5.20 ppm, i.e. Aô = 0.13 ppm.
R
_________________________________________ name
R
3 4
18 19
I-f2c=cH— H3C—CH2—
3,11. -dihydroangustine 3,14, 18, 19-tetrahydrOangustine
In the 13C-NMR spectra of 7 and 8 the only significant difference is observed for the resonances of C-i 8 and C-i 9 (Table
3,14-dihydroangustoline, [e]0: —172.5°
3). The C-19 resonance in the spectrum of 7 is found at 6 = 67.2 ppm, shifted by Aô = 2ppm to higher frequencies when compared to the C-19 resonance in the spectrum of
3
7
,CH—
HO
HO
8
',CH—
isolate 8. Assuming that the alkaloids reported here are
3,11.-dihydroangustoline, [aID: —301.4°
formed from the strictosamide-type precursors recently described from Nauclea orientalis (2) which have 3a-H con-
H3c
Table 3 '3C-NMR spectral assignments of
Compound No.
C-Atom
2 3 5 6 7
8 9 10 11
12 13 14 15 16 17 18 19
20 21
22
3
4
6
7
8
127.2(H-6, H-14)
131.3'
132.2
137.1 (H-5, 1-1-14) 40.5(1-1-6) 19.3(1-1-5)
51.5 39.7 21.0 109.0 124.8 118.5 119.6 122.3 111.3 137.0 31.4 143.0 126.5 148.7** 120.2
51.7 39.8
127.5 137.4
132.0 51.4 39.6 20.9 108.8 126.4 118.4 119.6 122.2 111.3 136.9' 31.3 144.5 125.5
131.6 51.3 39.5 20.9 109.6 126.5 118.6 119.9 122.5 111.2 137.1'
149.1 23.1
149.3" 24.0 65.2
1b
115.0(H-5,H-6,H-9) 125.2(H-l0,H-12) 119.1(H-11) 119.8(H-12) 124.4(H-9) 111.5(H-10) 138.7(H-9,F-l.11) 94.1
139.8(H-17,H-21) 118.85(H-14,H-17) 149.3(H-21)
118.8
129.6(H-18b,H-21) 127.9(H-18,H-21) 146.4(H-17,H-19) 161.9
21.1 108.9 126.6 118.5 119.6 122.3 111.4 137.2
31.1 144.5
130.1
125.0 147.6 14.6 23.1
131.9'
136.4
149.5"
151.6 164.2
163.8
41.1 20.0 115.5
125.9 119.8 120.5 125.1 112.1 139.3 95.0 140.5 120.0 150.3 23.8 66.2 134.2 146.4 162.8
67.2 136.8' 150.0 163.7
31.4 142.7
124.6
136.8'
148.7" 163.4
75 MHz / solvent CDCI3-CD3OD (4: 1).
'H'3C long range correlations from HMBC experiment, delays optimized to observe J8 = 10 Hz. 'I" Assignments interchangeable within columns. §
Signals overlapped.
selected isolated alkaloidsa.
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lar formula of C20H19N302. As diastereoisomers, their NMR N
Indole Alkaloids with in vitro Antiproliferative Activity from the Ammoniacal Extract of Nauclea orientalis
Planta Med. 58(1992) 47
Fig. 1 Reverse (1H) detected 'H-'30 long range correlation spectrum of angustine 1 (pulse sequence optimized to observe c,H 10Hz; correlations of C-5 and 0-6 not shown(.
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15(
154
I
21Q
21
I
-150
174
p0 ppm
a
p
i
p
5
a
3
hH
figuration, and this stereochemistry at C-3 is maintained in the 3,14-dihydroangustines, the NMR data of compounds 7 and 8 indicate that these two isolated differ only in their stereochemistry at C-19. Attempts to determine the absolute configuration at C-19 in 7 and 8 were unsuccessful. Table 3 shows the 13C-NMR data of selected
isolates, mainly those obtained in major quantities. Complete and unambiguous carbon NMR assignments were possible for the main constituent angustine (1) with the use of 2D reverse detected proton-carbon correlation spectro-
scopy. A 1H-detected heteronuclear multiple-quantum coherence experiment via direct coupling (HMQC) allowed all protonated carbons of 1, and thus those for 6, to be assigned. The same experiment performed on compound 7 permitted assignment of protonated carbons for this isolate and by analogy for the compounds 3, 4, and 8. Unambiguous assignments were not possible for C-2, C-20, and C-17, C-21 in 3, as well as for C-13 and C-20 in 7, and C-13, C-20, and C-i 7, C-2 1 in 8. For the 1H-detected heteronuclear multiple-bond correlation (HMBC) spectrum (Fig. 1) of 1, the pulse sequence was optimized to observe couplings in the range of 10Hz. The cEI and 2c,H couplings observed in this spectrum (Table 3) afforded the necessary information to assign all resonances for non-protonated carbons of 1. The spectrum was obtained with a 10mg sample of 1 in a total acquisition time of 9.5 hours.
All isolated alkaloids displayed in vitro antiproliferative activity against both T-24 and EGF depen-
dent MK cells (Table 4). Thus the hypothesis was confirmed
that, as with the well-known and structurally related antineoplastic agent camptothecin, angustine-type alkaloids
would also exhibit cytotoxicity. Very recently, 19-0methylangustoline was isolated from Camptotheca acuminata and shown to be cytotoxic when tested against P-388 leukemia cells (13). The biological test results are shown in Table 4 and permit some structure-activity relations to be concluded. There is no major difference in activity between the angustine-type and 3,14-dihydroangustine-type isolates. Presence of a C2 side chain at C-20 is not essential for the activity. However, introduction of a hydroxy substituent in the side chain at C-20, as is the case with the compounds 6, 7, and 8, results in an apparent loss of activity. As can be seen from Table 4, the stereochemistry at C-i9 in isolates 7 and 8 is not crucial for the antiproliferative activity. Compounds 1 and 9 show activities of the same
magnitude as 5, 3, and 4, in the T-24 test system, though, significantly stronger inhibition of EGF-dependent MK cell growth. In regard to the second objective of this investigation, the question whether or not these alkaloids are mere artifacts or do occur in the dried plant material, vari-
ous extractions of N. orientalis were carried out and chromatographically compared. Since the conventional TLC separation of these indole alkaloids proved to be dif-
ficult, overpressure layer chromatography (OPLC) was
employed in order to be able to identify individual alkaloids in the extracts. For the OPLC separation the continuous de-
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ii$ 20$ 191
48 Planta Med. 58(1992)
C. A. I Erdelmeier et al.
Table 4 In vitro antiproliferative activity of isolated alkaloids against human bladder carcinoma cells (T.24) and against EGF-dependent mouse keratinocytes (MK).
T.24a
MK
Angustine-type Isolates
1
3.3 (10.5) (29.2) (16.4) (99.4) (10.3)
0.4 (1.3)
4.3 (13.6)
1.1 (3.5) 1.2 (3.8)
2 9.2 5 4.7 6 32.9 9 3.4
3.4 (10.8)
3.6 (12.5) 4.2 (12.7) 0.5 (1.5)
reasoned that, whenever angustine-type constituents are found, the respective 3,14-dihydro compounds will also be
present.
3,14-Dihydroangustine-type Isolates 3,14.Dihydroangustine 3,14,18,19.Tetrahydroangustine 3,14-Dihydroangustoline([a}D.172.5°) 3,14-Dihydroangustoline([a]D-301.4°)
the precursors of the latter compounds. It can thus be
3
4 7 8
5.0 (15.8) 12.4 (37.2) 14.3 (42.9)
6.6 (19.8) 6.1 (18.3)
lC58-values in g/ml (j.tmol); serial dilutions of isolates were tested, isolates which could be tested twice gave reproducible results.
It can be concluded that the alkaloids found in this study do occur in dried Nauclea orientalis leaves in minor quantities. When ammonia is used in the extraction, there is a significant artificial increase in the formation of 3,1 4-dihydroangustine-type and angustine-type alkaloids from glycosidic precursors of the strictosamide-type. Alkaloid glycosides of the strictosamide-series cannot be detected in ammoniacal extracts ofN. orientalis, however, are
clearly present in such extracts prepared without ammonia.
Acknowledgements This research was supported by a grant of the Swiss National Science Foundation. We wish to thank Dr. M. Baltis-
berger. ETH Zurich, for the preparation of the herbarium specimens and his assistance on the collecting trip. Our thanks are also due to Mr. Max Kuduk, University of Papua New Guinea for his val-
uable help in botanical matters. We gratefully acknowledge Ms. Claudia Oertel for technical assistance in the isolation work. We also thank Mr. H. Hafliger, ETH Zurich, for running the mass spectra and Dr. A. Wright for running 1D-NMR and 2D reverse detected 1H-13C short range correlation NMR spectra. Finally, we
wish to thank Dr. D. Moskau, Spectrospin AG, Fällanden, for measuring the 2D reverse detected 8H-13C long range correlation NMR spectrum of angustine.
References 1 Wolif-Eggert, R. (1977) Dissertation, Erlangen. 2 Erdelmeier, C. A. J., Wright, A. D., Orjala, J., Baumgartner, B., Hall, T., Sticher, 0. (1991) Planta Med. 57, 149—152. Fig. 2 Chromatograms of various extracts of Nauclea orientalis leaves, obtained by overpressure layer chromatography (OPLC). Continuous development for 45 mm (a) and 30 mm (b). Detection: Fluorescence at 366 nm. Samples were obtained by extraction of plant material, lixiviated with 10% ammonia (A) or with 10% sodium carbonate (B), followed by liquid-liquid partitioning. Samples C and D were obtained by extraction with 95% ethanol, and subjected to OPLC prior to (C) and after (D) liquid-liquid partitioning.
6
8
velopment technique was used to enhance resolution be-
tween components. Because of the typical long wave IJY fluorescence of the angustine-type compounds the detection was considerably facilitated. Fig. 2 shows a photograph of the OPLC separations of four different N. orientalis extracts, as 45 (Fig. 2a) and 30 mm (Fig. 2b) developments, respectively. The extract obtained with 10% ammonia and
subsequent alkaloid partitioning (A in Fig. 2) basically represents the extract prepared for the isolation and identification of individual alkaloids. All isolates of the angustine-
type series, angustine (1), 18,19-dihydroangustine (2),
Phillipson, J. D., Hemingway, S. H., Bisset, N. G., Houghton, P. J., Shellard, F. J. (1974) Phytochemistry 13. 973—978. Phillipson, J. D., Hemingway, S. R., Ridsdale, C. (1982) J. Nat. Prod. 45, 145—162. Zeches, M., Richard, B., Gueye-M'Bahia, L., Le Men-Olivier, L., Delaude, C. (1985) J. Nat. Prod. 48,42—46. Summers, M. F.. Marzilli, L. G., Bax, A. (1986) J. Am. Chem. Soc. 108,4285—4294. Baltisberger, M., Erdelmeier, C. A. J., Rali, T. (1989) Ber. Geobot. Inst. ETH 55, 252—259. Meyer, Th., Regenass, U., Fabbro, D., Alteri, E., ROsel, J.Müller, M., Carvatti, G., Matter, A. (1989) mt. J. Cancer 43, 851—856. Geissler, J. F., Traxier, P., Regenass, U., Murray, B. F., Roesel. J. L., Meyer, Th., McGlynn, E., Storni, A., Lydon, N. B. (1990) J. Biol. Chem. 265, 22255—22261.
° Nyiredy,
Sz., Dallenbach-Tölke, K., Sticher, 0. (1988) J. Planar
Chromatogr. 1, 336—342. Hotellier, F., Delaveau, P., Pousset, J. L. (1975) Phytochemistry 14, 1407— 1409. 12 13
Repke, D. B., Jahangir, Clark, R. D., Nelson, J. T., MacLean, D. B.
(1989) Tetrahedron 45, 2541—2550. Lin, L.-Z., Cordell, G. A. (1990) Phytochemistry 29, 2744—2746.
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Angustine 18,19-Dihydroangustine Nauclefine Angustoline 10-Hydroxyangustine
nauclefine (5), angustoline (6), and lO-hydroxyangustine (9) are detectable in alkaloidal extracts prepared in the absence of ammonia (Fig. 2). However, the 3,14-dihydroangustine-type compounds do not display UV fluorescence and are not visible under these conditions. Since the 3,14dihydroangustine-type alkaloids easily transform into their angustine-type derivatives, they most probably represent
