J. CHEM. SOC. PERKIN TRANS. I 1988 Leurosinone: A New Binary lndole Alkaloid from Catharanthusroseus Atta-ur-Rahman," Muzaffar Alam, lrshad Ali, Habib-ur-Rehman, and lntikhabul Haq H.E.J. Research Institute of Chemistry, University of Karachi, Karachi-32, Pakistan A new binary indole alkaloid, leurosinone, has been isolated from the leaves of Catharanthus roseus, and has been assigned structure (1) on the basis of spectral studies. Catharanthus roseus (L) G. Don (Apocynaceae) is widely distributed throughout Pakistan. A number of indole and binary indole alkaloids have previously been reported from this plant of which vinblastine (VLB) and vincristine play an im- portant part in human chemotherapeutic During studies on this plant, we have isolated a new binary alkaloid, leurosinone, to which structure (1)-has been 0 C0,Me assigned on the basis of n.m.r.studies including homo-decoupling experiments. Its stereochemistry has been determined by a series of n.0.e. difference measurements and I3C n.m.r. assignments made by DEPT pulse sequence and GASPE experiments. Results and Discussion The U.V. spectrum of compound (1) indicated the presence of both indole and dihydroindole chromophores with Amax.-the ion at m/z807 by linked scan measurements showed that the following ions arose directly from it: m/z766,749,648,455, and 351. The overall fragmentation pattern was very similar to that of leurosine.6 The ions at m/z 152.1068 (C9H14NO) ion (I) and at m/z 208.1327 (C,,H1amp;02) ion (II) are consistent with the presence of an epoxide function in the piperidine ring.More- over, ion (11) shows that the CH,Ac unit is attached near the piperidine unit. The possibility of the CH2Ac group being present in the vindoline half of the molecule was eliminated as the normal fragmentation of the vindoline 6-8 moiety with ions at m/z 296,282, 188, 174, 135, 122, and 107 was observed. The overall fragmentation pattern of leurosinone (1) is shown in Schemes 1 and 2. The 'H n.m.r. spectrum ofleurosinone (1) was consistent with the binary nature of the molecule. Analysis of the data showed that a vindoline moiety substituted at the 10-position was present. Two three-proton singlets at 6 3.78 and 2.12 were assigned to the methyl groups of the 16-methoxycarbonyl and 17-acetoxy groups, respectively.The 1 1-OMe group on the aromatic ring resonated as a three-proton singlet at 6 3.80 whereas the NMe protons appeared as another three-proton singlet at 6 2.70.9,'0 The methylene protons of the 20-ethyl group appeared as two multiplets centred at 6 1.33 (19a-H) and 1.85 (19P-H) indicating their non-equivalence due to their prochiral nature.' 'The 18-methyl protons appeared as a triplet =at 6 0.79 (J18,19a = J18,1987.2 Hz). Irradiation of the methyl protons at 6 0.79 resulted in both the 19-methylene protons signals at 6 1.33 and 1.85 collapsing into doublets, each showing geminal coupling only (Jiga,198 13.6 Hz). Reciprocal decoupling effects were also observed when the 19-methylene protons were irradiated.A doublet at 6 5.28 (J1s,1410.4 Hz) was assigned to the olefinic proton at C-15.9-10 The other olefinic proton at C-14 resonated at 6 5.84 and was coupled with the 3a- and 3P- (MeOH) 214, 260, and 296 nm, which typifies vinbla~tine.~.~ methylene protons (J14,3a = J14.38 The i.r. spectrum showed absorptions at 3 460 (NH and OH), 1 730 cm-' (saturated ester), and also an additional carbonyl at 1710cm-'. The high resolution mass spectrum showed the molecular ion at m/z 864.4349 (C4,H60N40,0). The M+ was further confirmed by FAB and FD mass spectrometry. Linked scan measurements on the molecular ion showed that the ion at m/z 807.3977 (C4,H,,N409) arose directly from it, indicating the loss of 57 a.m.u.(ie. -CH,Ac or its equivalent) from the molecular ion. An examination of the fragmentation pathway of = 3.6 Hz). The 3a-H resonated as a multiplet at 6 3.34 whereas the 3P-H appeared at 6 3.39 (J3a,38= 12.0 Hz). Irradiation at 6 3.34 resulted in the multiplet of the 14-H at 6 5.84 collapsing into a quartet (J1s,14 10.4, J14,3fi 3.6 Hz). Irradiation of 14-H at 6 5.84 led to the collapse of the multiplets at 6 3.39 and 3.34 into simple doublets, showing only geminal coupling between 3a- and 3P-protons. One-proton singlets were observed at 6 6.10 (1 2-H) and 6.59 (9-H), typical of shielded aromatic protons in a para relationship. This established that the point ofattachment of the indole moiety was at C-10, analogous to vinbla~tine.~*'~.'~ The 2176 J.CHEM. SOC. PERKIN TRANS. I 1988 aamp;-MeO,CO / 0 0 Me00-QOH rsquo;bsol;J Me! OAc Me I COzMeCOtMe m/z 864 m/z 807 m/z 351 1rsquo; COzMe m/z 455 Me Me ; m/z 648 CMe m/z 749 Scheme 1. splitting patterns of the C-18 and C-18rsquo; protons indicated that the two ethyl side-chains were unsubstituted. The principal rsquo;H n.m.r. resonances of the indole moiety were observed at 6 0.96 (1 8rsquo;-Me), 3.60 (16rsquo;-CO,Me), 7.07 (10rsquo;-H), 7.10 (11rsquo;-H), 7.15 (12rsquo;-H), and 7.44 (9rsquo;-H),13 with an exchangeable indole NH appearing at 6 7.96. The methylene protons of the 20rsquo;-ethyl group appeared as two multiplets centred at 6 1.30 (19rsquo;a-H) and 1.79 (19rsquo;P-H), indicating their non-equivalence. The 18rsquo;-methyl protons appeared as a triplet at60.96(Jl,,,,,., = J18,,19,B= 7.4Hz).Thepresenceofa 15rsquo;,20rsquo;- epoxide function was established by the n.m.r.signal at 6 3.29 (doublet, J14,,15r4.1 Hz), consistent with the presence of an epoxymethine 15rsquo;-proton coupled with the 14rsquo;-proton. The two most striking differences on comparison of the lsquo;H n.m.r. spectrum of leurosinone (1) with that of leurosine were the presence of an additional 3 H singlet at 6 2.09 assigned to the methyl of an acetyl group and the absence of the doublet for the Srsquo;P-proton at 6 3.67.14 The absence of this doublet suggested that the CH,Ac group was attached at this position in a p-configuration in leurosinone (1). A series of n.0.e. difference measurements were carried out to ascertain the position and stereochemistry of the CH,Ac in leurosinone (1).The n.0.e. results are presented in Table 1 and confirm the structure of leurosinone as (1). Support for the structure (1) was provided by the broad band lsquo;Gated spin echo,rsquo; (GASPE) and lsquo;Distortionless enhancement by polarisation transferrsquo; l6 (DEPT) rsquo;3C n.m.r. experiments. The l3C n.m.r. data of leurosinone (1) are presented in Table 2. The presence of a vindoline moiety in the binary alkaloid was apparent from the very close correspondence in chemical shifts of the carbon atoms in the vindoline moiety of leurosinone with those of leurosine and vinblastine.rsquo; 7,1 The rdquo;C chemical shifts also served to establish the expected linkage of the indole moiety at C- 10of vindoline. In the indole moiety, the chemical shifts of both the aromatic and aliphatic carbons corresponded closely with those of leurosine.rsquo; Also, the methoxycarbonyl group afforded characteristic resonances at 6 52.38 and 174.30.Moreover, the presence of an epoxide function could be deduced from the signal at 6 62.25 which is consistent with a quaternary carbon of an oxirane system and assignable to C-20rsquo;; the other carbon of the oxirane system resonated at 6 63.62 and was assigned to C- 15rsquo;. The presence of an epoxide unit at C-15rsquo;-C-20rsquo; in leurosinone (1) was substantiated by the y-effect, due to the J. CHEM. SOC. PERKIN TRANS. I 1988 l+ Table 1. Connectivities established by n.0.e. difference spectra Proton Chemical irradiated shifts (6) n.0.e.connectivity NMe 2.70 16-CO2Me, 2-H, 17-H 2-H 3.72 NMe, 60-H, 12-H 17-H 5.46 19a-H, 19p-H cop2 m/z 455 m/z 282 15-H 14-H 3a-H 3P-H 5.28 5.84 3.34 3.39 18-Me, 19a-H, 14-H 15-H, 3a-H, 3P-H 3a-H, 14-H 14-H, 3P-H, 21-H 5a-H 2.38 3a-H, 5P-H, 21-H l+ 50-H 2.80 5a-H 6a-H 2.55 6P-H, 9-H, 21-H 6P-H 2.06 6a-H, 2-H 9-H 6.59 N-H, 6a-H, 21-H, 3rsquo;P-H 12-H 6.10 2-H, ll-OMe, NMe Me 21-H 17rsquo;a-H 2.61 2.34 18-Me, 5a-H, 6a-H, 9-H, 3a-H 17rsquo;P-H, 14-H, 15rsquo;-H, 5rsquo;a-H 17rsquo;0-H 3.78 6rsquo;P-H, 17rsquo;a-H, 15rsquo;-H m/z 188 14lsquo;-H 15rsquo;-H 1.23 3.29 17rsquo;a-H, 15rsquo;-H, 3rsquo;a-H, 3rsquo;P-H 17rsquo;a-H, 17rsquo;P-H, 14rsquo;-H, 21rsquo;P-H 2 1 rsquo;a-H 2.77 21rsquo;P-H, 18rsquo;-Me 21lsquo;P-H 2.40 15rsquo;-H, 21rsquo;a-H, 19rsquo;P-H 5rsquo;a-H 3.22 6rsquo;a-H, 17rsquo;a-H 6rsquo;a-H 3.02 5rsquo;a-H, 6lsquo;P-H, 9rsquo;-H 6rsquo;P-H 3.18 6rsquo;a-H, 17rsquo;P-H, 5rsquo;-CH2Ac 9lsquo;-H 7.44 6rsquo;a-H, 10rsquo;-H, 5rsquo;-CH2Ac Me Me Indole NH 5rsquo;-CH ,Ac 7.96 2.09 18-Me, 9-H, 12rsquo;-H, 16rsquo;-CO,Me 3rsquo;P-H, 19rsquo;P-H 5rsquo;-CH,Ac 2.94 6rsquo;P-H, 9rsquo;-H m/z 296 bsol; m/z 174 Table 2.13C N.m.r. spectrum of leurosinone Vindoline unit Indole unit amp; amp; Chemical Chemicalbsol;+ Carbon shift (6) Carbon shift (6) 2 83.46 2rsquo; 131.18 3 50.5 1a 3lsquo; 43.30 5 50.68a 5lsquo; 56.28 6 44.63 6rsquo; 29.53 7 53.29 7rsquo; 116.87 8 123.19 8rsquo; 129.23 9 123.57 9rsquo; 118.41 10 120.98 10lsquo; 122.40 m/z 107 m/z 121 11 158.13 11rsquo; 119.01 12 94.36 12rsquo; 110.42 Scheme 2. 13 153.50 13rsquo; 134.92 14 124.44 14rsquo; 32.95 epoxide, which was exerted on the 18rsquo;-methyl in comparison 15 130.07 15rsquo; 63.62 with that on the corresponding carbon in vinblastine.A 2.5 16 79.67 16rsquo; 55.35 17 76.46 17lsquo; 28.87 p.p.m. reduction in the y-effect on C-18rsquo; in leurosinone (1) (6 6.7 18 8.44 18lsquo; 9.22 in VLB, 6 9.2 in leurosinone) parallels the effect observed in 19 30.89 19lsquo; 28.24 leurosine l7 and confirms the replacement of a 20rsquo;-hydroxy 20 42.85 20rsquo; 62.25 group by a 20rsquo;-ether linkage. 21 66.1 1 21rsquo; 54.01 The most striking difference in the 13C n.m.r. of leurosinone C0,Me 170.83 C0,Me 174.30 (1) in comparison with that of leurosine was the the presence of C0,Me 52.17 C0,Me 52.38 an additional carbonyl carbon resonance at 6 209.40, a NMe 38.38 5rsquo;-CH,Ac 38.77 methylene carbon at 6 38.77, a low-field value of the methyl OCOMe 171.69 5rsquo;-CH,COMe 209.40 carbon at 6 31.66, and a methine carbon at 6 56.28.These data OCOMe 21.10 5rsquo;-CH,COMe 3 1.66 suggested the presence of a CH,Ac group at C-5rsquo;.The location ArOMe 55.88 of the substituent at C-5rsquo; rather than at any other site was a Assignments are interchangeable. indicated by the low-field value of the methine carbon, the chemical shift of which (6 56.28) was in agreement with it being adjacent to a nitrogen atom. natural products.rsquo; Thus, to establish that leurosinone (1) is not Leurosinone (1) probably arises from leurosine by oxidation formed during isolation, the extraction was carried out with the at the 5rsquo;-N bond to the corresponding immonium species, which rigorous exclusion of acetone, and leurosinone (1) was detect- is then trapped by a molecule of acetone. Acetone has previously able both in the fresh chloroform extracts as well as in the been shown to be involved in the biosynthesis of other complex purified fractions.2178 Experimental M.p.s were obtained on a Gallenkamp apparatus. lsquo;H (300.13 MHz) and 13C (75.47 MHz) N.m.r. spectra were recorded in CDCI, on a Bruker AM-300 FT spectrometer. Chemical shifts are in p.p.m. (6) from SiMe, as internal standard. The i.r. spectra were run on a JASCO-IR A-1 spectrophotometer in CHCl,, and U.V.spectra on a Shimadzu U.V. 240 spectrophotometer in MeOH. Optical rotations were measured on a Schmidt and Haensch polartronic-D electronic polarimeter in CHCl,. Mass spectra were obtained with Finnigan MAT 112 and Finnigan MAT 3 12 double-focussing mass spectrometers.Isolation of Leurosinone (l).-Air-dried leaves (20 kg) of Catharanthus roseus (L) G. Don were powdered and extracted with chloroform. The chloroform extracts were combined, filtered, and evaporated under reduced pressure to a gum. The gummy material (200 g) was partially dissolved in 2 aqueous tartaric acid (1 1). After the undissolved material was filtered off, the aqueous solution (pH 2.9) was extracted with chloroform (3 x 2 1). The organic extracts were removed, and the pH of the aqueous acidic phase was adjusted with aqueous ammonia (28) to pH 5.0. Further extraction with chloroform (3 x 2 1) afforded an alkaloidal fraction (60 g). This was chromatogra- phed on a column (60 mm diameter) packed with silica gel PF-254 (2 kg). Elution was carried out with increasing polarities of light petroleum-chloroform, chloroform, and chloroform-methanol. The eluates obtained from chloroform-methanol (9 : 1) afforded an alkaloidal mixture which was concentrated and separated by preparative t.1.c.on precoated silica gel plates (layer thickness 0.2 mm) in ethyl acetate-ethanol(8 :2). A faster moving band was scraped off and elution with methanol afforded a pure amorphous alkaloid (1) (30 mg) (RF 0.34); xi4 +86rdquo; (c 0.02 in CHCl,); h,,,.(MeOH) 214, 260, and 296 nm; hmi,,,246 and 284 nm; v,,,.(CHCl,) 3 460 (NH, OH), 2 950 (CH), 1 730 (ester C=O), 1 710 (C=O), 1 615, (CX), 1 040 (CO), and 745 cm-rsquo; (ArCH); m/z 864 (Mf, 773,807 (93), 749 (8), 706 (24), 648 (4), 351 (7), 296 (2), 282 (3), 222 (6), 208 (14), 188 (1 l), 174 (4), 152 (12), 135 (61), 122 (47), 121 (39, and 107 (29); 6,(CDCl,, 300.13 MHz) 0.79 (3 H, t, Jlg,19 7.2 Hz, 18-Me), 0.96 (3 H, t, J,g,,,9, 7.4 Hz, 18rsquo;-Me), 2.09 (3 H, S, 5lsquo;-CH,Ac), 2.12 (3 H, s, 17-OAc), 2.70 (3 H, s, NMe), 3.60 (3 H, s, 16rsquo;-CO,Me), 3.72 (1 H, s, 2-H), 3.78 (3 H, s, 16-C02Me), 3.80 (3 H, s, 11-J.CHEM. SOC. PERKIN TRANS. I 1988 OMe), 5.28 (1 H, d, J,,,,, 10.4 Hz, 15-H), 5.46 (1 H, s, 17-H), 5.84(1 H,m, 14-H),6.10(1 H,s, 12-H),6.59(1 H,s,9-H), 7.08- 7.16(3H,m, lOrsquo;-,llrsquo;-,and12rsquo;-H),and7.44(1H,d,J7.5Hz,9rsquo;-H); 6,(CDCl,, 75.47 MHz) data are reported in Table 2. References 1 N. R. Chopra, S. L. Nayar, and I. C. Chopra, lsquo;Glossary of Indian Medicinal Plants,rsquo; C.S.I.R. Publication, New Delhi, 1956, p.255. 2 R. C. De Conti and W. A. Creasey, lsquo;The Catharanthus Alkaloid,rsquo; eds. W. I. Taylor and N. R. Farnsworth, Marcel Dekker, New York, N.Y., 1975, p. 237. 3 S. K. Carter and R. B. Livingston, Cancer Treatment Rep., 1976,60, 1141. 4 M. Hesse, lsquo;Indolalkaloide in Tabellen,rsquo; Springer-Verlag, Berlin, Germany, 1964. 5 M. Hesse, lsquo;Indolalkaloide in Tabellen, Ergansungswerk,rsquo; Springer- Verlag, Berlin, Germany, 1968. 6 D. J. Abraham and N. R. Farnsworth, J. Pharm. Sci., 1969,58,694. 7 B. K. Moza, 3. Trojanek, V. Hanus, and L. Dolejs, Collect. Czech. Chem. Commun., 1964, 29, 1913. 8 M. Gorman, N. Neuss, and K. Biemann, J. Am. Chem. Soc., 1962,84, 1058. 9 N. Neuss, M. Gorman, W. Hargrove, N. J. Cone, K. Biemann, G. Buchi, and R.E. Manning, J. Am. Chem. SOC., 1964,86, 1440. 10 A. De Bruyn, L. De Taeye, and R. Simonds, Bull. SOC. Chim. Belg., 1981, 90, 185. 11 Atta-ur-Rahman, I. Ali, and M. I. Choudhary, J. Chem. SOC.,Perkin Trans. I, 1986, 923. 12 A. De Bryun, L. De Taeye, and M. J. 0.Anteunis, Bull. SOC. Chim. Belg., 1980, 89, 629. 13 B. K. Hunter, L. D. Hall, and J. K. M. Sanders, J.Chem. SOC.,Perkin Trans. I, 1983, 657. 14 R. Simonds, A. De Bruyn, L. De Taeye, M. Verzele, and C. De Pauw, Planta Med., 1984, 50, 274. 15 G. A. Morris and R. Freeman, J. Am. Chem. Soc., 1979, 101, 760. 16 0.W. Sorensen and R. R. Ernst, J. Mugn. Reson., 1983, 51, 477. 17 E. Wenkert, E. W. Hagaman, B. Lal, G. E. Gutowski, A. S. Katner, J. C. Miller, and N. Neuss, Helv. Chim. Acta., 1975, 58, 1560. 18 D. E. Dorman and J. E. Paschal, Org. Mugn. Reson., 1976, 8 413. 19 G. Blasko, N. Murugesan, A. J. Freyer, M. Shamma, A. A. Ansari, and Atta-ur-Rahman, J. Am. Chem. SOC.,1982, 104,2039. Received 22nd October 1986; Paper 6/2059
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