Quassinoids and Other Constituents from crenata H. C. Krebs3, P. J. Schilling3, R. Wartchowb, and M. Boltec a Zentrum für Lebensmittelwissenschaften, ZA für Chemische Analytik und Endokrinologie, Haus 123, Tierärztliche Hochschule, Bischofsholer Damm 15, D-30173 Hannover, Germany b Institut für Anorganische Chemie, Universität Hannover, Callinstr. 9, D-30167 Hannover, Germany c Institut für Organische Chemie, J. W. Goethe-Universität, Marie Curie-Str. 11, D-60439 Frankfurt/Main, Germany Reprint request to Prof. H. C. Krebs. Fax: +49-(0)511-8567690. E-mail: [email protected] Z. Naturforsch. 56b, 315-318 (2001); received September 1, 2000 Picrasma crenata, , Quassinoids Two new, 16-/?-0-methylneoquassin and 16-ß-O-ethylneoquassin, and four known quassi­ noids have been isolated together with coniferyl aldehyde, coniferin, cantin-6-one, 4,5-di- methoxycantin-6-one and (+)-neo-olivil from the wood of Picrasma crenata. Their structures were determined on basis of spectroscopic and X-ray analysis.

1. Introduction 2. Results and Discussion Picrasma crenata (Simaroubaceae) is a Brazilian Ground wood of P. crenata has been extracted which is used in traditional medicine to treat with ethanol/water and the extract was separated Diabetes mellitus [1]. To this end, a decoction of between chloroform/water, butanol/water and by ground wood is prepared. of the family Sim­ chromatography to yield six quassinoid com­ aroubaceae are well known to contain quassinoids pounds. Four of them showed identical spectra as and alkaloids. Some quassinoids have received at­ described for quassin [11,12], neoquassin (1) [13], tention due to their biological activity as potential parain [10], and ll-dihydro-12-norneoquassin antitumor [2-7] as well as antiulcer agents [8]. Po­ [12,14], lonsky and Lederer [9] isolated quassin from the NMR spectra of neoquassin (1) were almost in wood of P. crenata. Furthermore, parain, isopar- accordance with literature [13], Several proton ain, and 12-norquassin have been found in the and carbon nuclei gave rise to pairs of signals. It bark [10]. We re-investigated this plant and iso­ is therefore evident that both the 16a- and 16/?- lated quassin, neoquassin (1), parain, 11-dihydro- OH hemiacetal forms are present. Integrals in the 12-norneoquassin in addition to two new quassi­ NMR as well as peak heights in the 13C NMR noids, 16-/?-0-methyl- (2b) and 16-ß-O-ethylneo- spectrum showed that the ratio was 25% of a-neo- quassin (3). Furthermore, we obtained coniferyl quassin (la) and 75% of ß-neoquassin (lb). NM R aldehyde, coniferin, cantin-6-one, 4,5-dimethoxy- signals could be assigned unequivocally by two- cantin-6-one, and (-i-)-neo-olivil. dimensional experiments; as a result, the 13C chemical shifts of the 4- and 8-methyl groups as given by De Beilis and Lovati [13] have to be re­ versed. Preliminary results in a diabetes test showed positive reactions which will be pub­ lished elsewhere. OMe la: R1 = H, R2 = OH NMR data of 2b were similar to the results re­ lb: R1 = OH, R2 = H ported for 16-a-methylneoquassin (2a) by Barbetti 2a: R1 = H, R2 = OCH3 et al. [12]. H R EIM S of 2b gave the same molecular 2b: R1 = o c h 3, r 2 = h formula, C23H320 6, as 2a. NMR data, the assign­ H J,S R1 ment of which were made by two-dimensional ex­ 3: R1 = OC2H5, Rs = H t ° '' R2 periments, showed that there were three methoxy

0932-0776/2001/0300-0315 $06.00 © 2001 Verlag der Zeitschrift für Naturforschung, Tübingen • www.znaturforsch.com D

Dieses Werk wurde im Jahr 2013 vom Verlag Zeitschrift für Naturforschung This work has been digitalized and published in 2013 by Verlag Zeitschrift in Zusammenarbeit mit der Max-Planck-Gesellschaft zur Förderung der für Naturforschung in cooperation with the Max Planck Society for the Wissenschaften e.V. digitalisiert und unter folgender Lizenz veröffentlicht: Advancement of Science under a Creative Commons Attribution Creative Commons Namensnennung 4.0 Lizenz. 4.0 International License. 316 H. C. Krebs-P. J. Schilling • Quassinoids and Other Constituents from Picrasma crenata groups in the molecule. So it was obvious that 2b 97.7 ppm), the orientation of the ethoxy group in is an isomer of 2a. To determine the structural iso­ 3 was also determined as ß. Thus, 3 is the new merism of 2b a X-ray crystal structure analysis was compound 16-ß-O-ethylneoquassin. Treatment of performed which showed the structure to be 16-/?- neoquassin (1) with methanol or ethanol under O-methylneoquassin. The absolute configuration chromatographic conditions did not furnish the ac- was determined, too, thus confirming the struc­ etals 2 and 3, respectively. tures given in [15] and [16]. Lang’at et al. have In addition to the quassinoids the ethanol/water used a mixture of 2a and 2b as an intermediate in extract furnished coniferyl aldehyde [17], coniferin their synthesis of derivatives of quassin, but no 13C [18], cantin-6-one [19], 4,5-dimethoxycantin-6-one NMR data were given and no crystal structure de­ [20] and (-i-)-neo-olivil [21] after chrom atography termination was performed [15]. Shing et al. have at silica gel, amberlite XAD 2 and sephadex LH used 2b as an intermediate in their synthesis of 20. Spectroscopic data were in accordance with lit­ quassin. They have determined the crystal struc­ erature. ture of quassin, but not of 2b [16]. The com parison of the two molecular structures shows a good con­ 3. Experimental gruence of the skeletons, except, of course, in the 3.1. General vicinity of C16. NMR spectra (!H: 300.13 MHz; 13C: 75.47 MHz) Com pound 3 has a molecular weight of 418, and recorded in pyridine-D5 (1 and 2b) or methanol- from HREIMS the molecular formula could be D 4 (3) soln with TMS as internal standard on a deduced as C24H340 6. A peak in the MS at m /z = Bruker AM 300 instrument. Chemical shifts (6) 373 pointed to the loss of an ethoxy group. NMR are expressed in ppm. MS were measured by di­ spectra of 3 and 2b were similar. The absence of rect inlet with 70 eV ionization at 150 °C on Finni- the peak for the 16-OMe group is characteristic; gan MAT 312 and MAT 95 spectrometers. instead of it the 13C NMR spectrum of 3 showed a peak for a methylene group at d - 63.6 ppm and 3.2. Plant material for a methyl group at 15.5 ppm. As the signal for Plant material was collected in September 1994 C16 of 3 (d = 97.4 ppm) agreed with that of 2b (<5 = in southern Brazil, near the border to Argentina. Botanical identification was made by Prof. Dr. Walter B. Mors, Universidade Federal do .

3.3. Extraction and isolation 3940 g wood without bark from Picrasma cren­ ata (Simaroubaceae) were ground and dried. 500 g portions were extracted at RT for 48 h each with 3 1 of petrolether (40-60 °C) followed by the same amount of ethyl acetate and ethanol/water (3:7). The ethanol/water extract was separated on am­ berlite XAD 2 by elution with water followed by methanol. The methanol eluate was partitioned between water and chloroform. After evaporation to dryness the chloroform fraction was chromato­ graphed using Sephadex LH 20 with methanol, sil­ ica gel with a gradient starting from petrolether via diethyl ether and ethyl acetate to methanol, Fig. 1. Molecular structure of 2b drawn with 30% ellip­ and silica gel with diethyl ether/ethyl acetate (9:1) soids using PLATON [22]. Selected bond lengths [A]: C2-C3 1.316(3), C12-C13 1.323(4), C7-017 1.440(3), to furnish 1 (131 mg), 2b (54 mg), and 3 (6 mg). 017-C16 1.413(3), C16-C15 1.498(3), C16-05 1.405(3), 05-C24 1.433(3). The second molecule in the asymmet­ 3.4. Neoquassin (1) ric unit differs from the first one mainly in one torsion angle: C13-C12-04-C23 121(1)°, C13'-C12'-04'-C23' Compound 1 was isolated as a mixture of 25% -92(1)°. of a-neoquassin (la) and 75% of ß-neoquassin H. C. Krebs-P. J. Schilling • Quassinoids and Other Constituents from Picrasma crenata 317

(lb) as deduced from NMR spectra. IR (KBr): v - 5.29 (d, / = 1 Hz, 1 H, 3-H). - 13C NMR (75.47 3446, 2942, 2879, 1691, 1633, 1449, 1378, 1354, MHz, C5 D 5N): see Table 1. - MS (EI, 70 eV): m / 1227, 1117, 1052 cm-1. - NMR (300.13 MHz, z (%) = 404 (100) [M+], 389 (28), 373 (15), 357 C5D 5N): <3 = 0.90 (d, J = 5 Hz, 4-Me lb), 0.95 (d, (13), 329 (22), 312 (21), 301 (27), 212 (39), 165 J = 5 Hz, 4-Me la), 1.00 (s, 8 -Me lb), 1.05 (s, 8 - (26), 152 (84), 127 (38), 91 (25), 69 (54). - Me la), 1.60 (s, 10-Me lb), 1.61 (s, 10-Me la), 1.64- H REIM S m /z = 404.219299 ([M +], calcd. 1.76 (m), 1.80 (s, 13-Me la), 1.83 (s, 13-Me lb), 404.219889 for C23H3206). 2.0-2.2 (m), 2.2-2.35 (m), 2.62-2.72 (m), 3.40 (m, 7- Crystal structure analysis of 2b: C23H320 6, M = H lb), 3.45 (s, 2-OM e la + b), 3.56 (m, 14-H la + 404.49 g/mol, recrystallized from acetone, col­ b), 3.68 (s, 12-OMe lb), 3.72 (s, 12-OMe la), 4.14 ourless fragment of a plate II (001), size 0.56 x 0.31 (m, 7-H la), 5.08 (m, 16-H lb), 5.22 (d, J = 1 Hz, x 0.13 mm, orthorhombic, space group P2 12121 3-H lb), 5.26 (d, / = 1 Hz, 3-H la), 5.65 (m, 16-H (No. 19), a = 8.376(2), b = 13.278(2), c = 38.471(4) la). - 13C NMR (75.47 MHz, C5D 5N): see Ta­ A, a = 90.00, ß = 90.00, y = 90.00°, V = 4278.6(13) ble 1. - MS (EI, 70 eV): m /z (% ) = 390 (100) [M+], Ä 3, Z = 8, Dx = 1.256 g/cm3, T = 300(2) K; Stoe 372 (35), 357 (18), 328 (18), 312 (13), 301 (47), 152 IPDS diffractometer, A(Mo-Ka) = 0.71073 A, (63), 127 (43), 91 (28), 69 (60). 0 max = 24.15°, 6802 unique (i?int = 0.0361) and 3857 observed ( l> 2 o \) reflections, 523 refined 3.5. 16-ß-O-Methylneoquassin (2b) parameters, R gt(F) - 0.0353, w R (P 2) = 0.0578. Flack parameter x - -0.6(8). An additional mea­ 'H NMR (300.13 MHz, C5D 5N): <5 = 0.95 (d, J = surement (by M. B.) using Cu-Ka radiation 5 Hz, 3 H, 4-Me), 1.02 (s, 3 H, 8 -Me), 1.60 (s, 3 H, yielded Flack x= -0.03(19). Full crystallographic 10-Me), 1.62-1.96 (m, 4 H, 6 -H + 15-H), 1.76 (s, 3 details without structure factors have been depos­ H, 13-Me), 1.96-2.10 (m, 1 H, 5-H), 2.25-2.42 (m, ited at CCDC, No. 153139 & 153140. Files of struc­ 2 H, 4-H + 14-H), 3.30 (s, 3 H, 16-OMe), 3.44 (s, ture factors are available by R. W. 1 H, 9-H), 3.47 (s, 3 H, 2-O M e), 3.66 (m, 1 H, 7- H ), 3.70 (s, 3 H, 12-OMe), 4.79 (m, 1 H, 16-H),

Table 1. 13C NMR spectroscopic data of quassinoids 1-3 (d-values, TMS). c - la lb laa lba 2ab 2b 3 c 5D 5N c 5D 5N c d c i 3 CDCI3 c d c i 3 c 5d 5n c d 3o d 1 C 198.7 198.3 198.9 198.5 197.8 198.6 201.5 2 C 148.5 148.5 148.0 148.0 140.1 148.6 149.1 3 CH 116.4 116.4 116.3 116.3 116.1 116.5 118.6 4 CH 31.5 31.4 31.2 31.2 30.8 31.6 32.6 5 CH 44.3 44.3 43.6 43.7 42.9 44.5 45.7 6 c h 2 25.9 26.1 25.7 25.9 25.4 25.9 26.5 7 CH 68.8 77.1 69.0 77.4 68.4 69.4 70.6 8 C 38.6 38.3 38.4 38.2 36.5 38.5 39.6 9 CH 44.0 49.7 43.2 49.5 48.2 46.5 47.4 10 C 46.6 46.6 46.1 46.2 45.4 46.6 47.5 11 C 193.4 193.3 193.1 193.1 192.1 193.2 195.5 12 C 140.7 139.6 139.9 138.2 139.8 140.3 143.6 13 C 148.5 148.5 148.2 148.2 147.4 148.7 149.5 14 CH 46.3 47.0 45.8 46.5 45.8 44.3 45.4 15 c h 2 32.3 35.2 34.2 34.2 28.9 31.0 32.2 16 CH 90.6 96.2 90.7 95.7 96.6 97.7 97.4 4-Me c h 3 19.1 19.1 22.0 21.6 21.5C> 19.5 19.8 8-Me c h 3 20.0 21.4 19.4 19.5 19.2C> 22.0 22.4 10-Me c h 3 12.9 12.9 12.7 12.8 12.4 13.0 13.3 13-Me c h 3 15.1 15.0 15.2 15.1 14.8 15.2 15.4 2-O-Me c h 3 54.6 54.6 54.8 54.8 54.3 54.8 55.4 12-O-Me c h 3 59.1 59.1 59.1 59.1 58.6 59.2 59.8 16-O-Me c h 3 53.8 54.5 16-O-Et c h 2 63.6 16-O-Et c h 3 15.5 a De Beilis and Lovati [13]; b Barbetti et al. [12]; c may be reversed according to Barbetti et al. [12]. 318 H. C. Krebs-P. J. Schilling • Quassinoids and Other Constituents from Picrasma crenata

3.6. 16-ß-O-Ethylneoquassin (3) NMR (75.47 MHz. C5D5N): see Table 1. - MS (EI, 70 eV): m /z (%) = 418 (99) [M+], 404 (35), !H N M R (300.13 M Hz, C D 3OD): (5 = 1.10 (s, 3 373 (23), 357 (12), 329 (34), 313 (32), 303 (46), 226 H, 8-Me), 1.13 (d, / = 5 Hz, 3 H, 4-M e), 1.23 (t, (42), 207 (31), 165 (34), 152 (100), 127 (41), 91 J = 6 Hz, 3 H, C H 3 of 16-OEt), 1.52 (s, 3 H, 10- (30), 69 (51). - HREIMS m /z = 418.235168 ([M+], Me), 1.74-1.93 (m, 4 H, 6-H + 15-H), 1.88 (s, 3 H, calcd. 418.235539 for C24H340 6). 13-Me), 1.93-2.03 (m, 1 H, 5-H), 2.33-2.55 (m, 2 H, 4-H + 14-H), 3.20 (s, 1 H, 9-H), 3.42-3.53 (m, 1 H, Acknowledgements 7-H), 3.56 (s, 3 H, 2-OM e), 3.58 (s, 3 H, 12-OMe), We thank Prof. Dr. W. B. Mors, Universidade 3.64-3.80 (q, / = 6 Hz, 2 H, CH2 of 16-OEt), 4.98 Federal do Rio de Janeiro, for supplying the plant (m, 1 H, 16-H), 5.49 (d, J = 1 Hz, 1 H, 3-H). - 13C material and for botanical identification.

[1] J. R. Pereyra, Ann. Fac. Med. Univ. Sao Paulo 14, [13] P. De Beilis, M. Lovati, Fitoterapia 65, 314 (1994). 269 (1938). [14] G. Grandolini, C. G. Casinovi, P. Barbetti, G. Far­ [2] J. Polonsky, Fortschr. Chem. Org. Naturst. 47, 221 della, Phytochemistry 26, 3085 (1987). (1985). [15] C. C. Lang’at, R. A. Watt, I. Toth, J. D. Phillipson, [3] T. Sakaki, S. Yoshimura, T. Tsuyuki, M. Ishibashi, Tetrahedron 54, 6841 (1998). Chem. Pharm. Bull. 32, 4702 (1984). [16] T. K.M. Shing, Q. Jiang, T. C. M. Mak, J. Org. [4] T. Sakaki, S. Yoshimura, T. Tsuyuki, M. Ishibashi, Chem. 63, 2056 (1998). Bull. Chem. Soc. Jpn. 58. 2673 (1985). [17] H. H. Barakat, M. A. M. Nawwar, J. Buddrus, M. [5] T. Sakaki, S. Yoshimura, T.Tsuyuki, M. Ishibashi, Linscheid, Phytochemistry 26, 1837 (1987). Bull. Chem. Soc. Jpn. 58, 2680 (1985). [18] N. G. Lewis, E. J. Inciong, H. Ohashi, G. H. N. Tow­ [6] T. Sakaki, S. Yoshimura, T. Tsuyuki, T. Takahashi, ers, E. Yamamoto, Phytochemistry 27, 2119 (1988). Tetrahedron Lett. 27, 593 (1986). [19] G. Zapesochnaya, V. Kurbin, V. Okhanov, A. Mir- [7] H. Morita, E. Kishi, K. Takeya, H. Itokawa, O. Ta­ oshnikov, Planta Med. 58, 192 (1992). naka, Chem. Lett. 5, 345 (1990). [20] E. Rodrigues, J. B. Fernandes, P. C. Vieira, M. F. G.F. [8] H. Tada, F. Yasuda, K. Otani, M. Doteuchi, Y. Ishi- Da Silva, Phytochemistry 31, 2499 (1992). hara, M. Shiro, Eur. J. Med. Chem. 26, 345 (1991). [21] M. Schöttner, J. Reiner, F. S. K. Tayman, Phyto­ [9] J. Polonsky, E. Lederer, Bull. Soc. Chim. Fr. 1959, chemistry 46. 1107 (1997). 1157 (1959). [22] A. L. Spek, PLATON, an integrated tool for the [10] J. C. Vitagliano, J. Comin, Phytochemistry 11, 807 analysis of the results of a single crystal structure (1972). determination. Acta Crystallogr. Suppl. A46, C-34 [11] M. Kim, K. Kawada, R. S. Gross, D. S. Watt, J. Org. (1990). Chem. 55, 504 (1990). [12] P. Barbetti, G. Grandolini, G. Fardella, I. Chiappini, Phytochemistry 32, 1007 (1993).