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Phenanthroid constituents from Aristolochia argentina
Biochemical Systematics and Ecology 46 (2013) 83–87
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Phenanthroid constituents from Aristolochia argentina Horacio A. Priestap* Department of Biological Sciences, Florida International University, 11200 S.W. 8th Street, HLS1-313, Miami, FL 33199, United States
a r t i c l e i n f o Article history: Received 3 July 2012 Accepted 9 September 2012 Available online 16 October 2012 Keywords: Aristolochia argentina Aristolochiaceae Aristothiolide Aristololide 9-ethoxyaristolactam IV 9-ethoxyaristolactam I 4,5-dioxodehydroasimilobine
1. Subject and source The genus Aristolochia (Aristolochiaceae) consists of approximately 600 species largely distributed in tropical and warmtemperate regions of the world (Cronquist, 1988). Aristolochia argentina Gris., a twining plant growing in central and north Argentina, is popularly used as medicinal plant to treat arthritis and other ailments (Dominguez, 1928). Underground parts of A. argentina were collected at Cruz Alta, Tucuman. A voucher specimen (BACP 4602) is deposit at the Herbarium of the “Centro de Estudios Farmacologicos y Botanicos”, Argentina. 2. Previous work Earlier studies on A. argentina revealed the occurrence of numerous aristolochic acids and aristolactams on the underground parts of the plant (Priestap, 1985, 1987). The aerial parts of A. argentina also contain aristolochic acids (Priestap et al., 2012). 3. Present study In continuation of earlier investigations on the chemical composition of A. argentina, it is now reported on the identification of the new compounds aristothiolide (1) and 9-ethoxyaristolactam IV (2a), along with the known compounds 9-ethoxyaristolactam (2b), aristololide (3) and 4,5-dioxodehydroasimilobine (4). 9-Ethoxyaristolactam I (2b) was previously reported for Aristolochia mollisima (Lou et al., 1989). Aristololide (3) is a phenanthroid lactone found in the roots of Aristolochia
* Tel.: þ1 305 348 0375. E-mail addresses: priestap@fiu.edu, [email protected]. 0305-1978/$ – see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bse.2012.09.015
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indica (Achari et al., 1983) and Aristolochia elegans (Shi et al., 2004). The 4,5-dioxodehydroasimilobine (4) belongs to the family of dioxoaporphine alkaloids. It was first isolated from Aristolochia indica (Achari et al., 1982) and also reported for related families (Kumar et al., 2003).
O
O
4
O
O
2 3
O NH
S O
10
O O
9
5
OCH2 CH3
8
6 CH3O
O
O
1
7
OCH3
OCH3
R
OCH3
2a R = OCH3 2b R = H
1
3
O HO
O NH
CH3O
COOH
O
O
COOH
NO2
O
NO2
O
OCH3
R 4
5
6a R = H 6b R = OCH3
Dried underground parts of A. argentina were sequentially extracted with petrol and EtOH as previously reported (Priestap, 1987). The petrol extract from 23 kg of plant material gave an oil (ca. 1 kg) which was submitted CC and prep. TLC on silica gel to yield 30 mg of aristololide (3) and 2 mg of aristothiolide (1). A similar amount of compounds 1 and 3 is also present in the EtOH extract. The ethanolic extract (ca. 2 kg) from 13.77 kg of plant material was partitioned between water and ethyl ether. The ethereal extracts (247 g) were fractionated as previously reported (Priestap, 1985) to give 1 mg of 9-ethoxyaristolactam IV (2a), 49 mg of 9-ethoxyaristolactam I (2b) and 79 mg of 4,5-dioxodehydroasimilobine (4). TLC was carried out as follows: silica gel (Rf), (1) hexane–C6H6, 1:1 (3, 0.66; 1, 0.58); (2) C6H6–MeCOEt, 17:3 (2b, 0.41; 2a, 0.37); (3) CHCl3–EtOH, 9:1 (4, 0.48); Al2O3, (4) CHCl3–EtOH, 100:1 (2b, 0.78; 2a, 0.74); (3) CHCl3–EtOH, 95:5 (4, 0.48). Compounds were detected as yellow or red (4) spots and also by fluorescence at 360 nm. HPLC was carried out in a ThermoFinnigan chromatograph (Thermo Electron Corporation) using a C18 RP Hypersil GOLD column (RP5, 250 4.6 mm, pore size 5 mm, Thermo Electron Corporation). The eluting systems consisted of 60% of water and 40% of 1% HOAc in MeOH, isocratic (Rt: 4, 4.9; 2b, 15.3; 2a, 16.1; 3, 34.5; 1, 36.3 min). Flow rate, 1 ml/min. Detection at 254 nm. 1H NMR spectra were measured at 80 MHz and 13C NMR spectra at 20 MHz in a Varian FT-80A spectrometer. Electron impact mass spectra were recorded in a Varian Mat CH7A instrument at 70 eV. ESI(þ)-TOFMS-HRMS analysis was performed by flow injection into an Agilent Technologies 6210 TOF/LCMS instrument operated in the (þ)-ESI-MS mode. Aristothiolide (1) is a sulfur-containing compound that exhibits similar chromatographic properties than aristololide (3) co-occurring in the same plant. ESI(þ)-TOFMS-HRMS afforded m/z 341.0500 for [M þ H]þ implying the molecular formula of C18H12O5S. Aristothiolide (1) showed an absorption maximum at 251 nm, typical of the phenanthrene chromophore, and a carbonyl absorption at n 1784 cm1. The 1H NMR showed the usual singlets of the 3,4-methylenedioxy group, H-2, H-9 and two MeO groups. In addition, compound 1 exhibits two doublets, one of them at low field (H-5), with characteristic metacoupling (J5,7 ¼ 2.5 Hz) consistent with the 6,8-dioxy-substitution observed in aristolochic acids and aristolactams. Interestingly, H-2 is significantly shifted to low fields in aristothiolide (1). For example, H-2 absorbs at d 8.17 whereas it absorbs at d 7.86 in aristololide (3). It is likely that H-2 suffers deshielding because of the anisotropic effect from diamagnetic circulations at the carbonyl group. The observed deshielding (0.31 ppm) may be due to the bulk sulfur atom that forces the carbonyl group to a position closer to this proton. A similar effect is observed between 1-indanone and a-tetralone, and may also occur in dioxoaporphines. Thus, H-2 in 4,5-dioxodehydroasimilobine (4) absorbs at d 8.09, i.e. at 0.47 ppm lower fields than the same proton in the corresponding aristolactam (aristolactam AII) (d 7.62). Another remarkable example is provided by 9-methoxyaristolochic acid II (5) isolated from Aristolochia ponticum (Houghton and Ogutveren, 1991a). The signal for H-2 at d 8.35 is much more downfield (0.54 ppm) than that recorded for the equivalent proton in aristolochic acid II (6a) (d 7.81).
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Since the 9-MeO and H-2 are quite apart in 5, it is likely that, by introduction of the 9-MeO group in the molecule, crowding of the substituents at positions 9, 10 and 1 of the phenanthrene nucleus forces the carboxyl group to move to a position closer to H-2, thus causing deshielding of this proton. The electron ionization mass spectra of aristothiolide (1) exhibit sets of fragment ions which indicate the successive losses of neutral fragments (CH3, CO, HCO, H2CO and H), also observed in aristolactams and aristololide (3), e.g., fragment ions at m/z 340, 325, 297, 282, 254, 224, 196 and 168 suggest the degradation pathway [M]þ–CH3–CO–CH3–CO–H2CO–2CO. The above considerations lead to the conclusion that aristothiolide (1) is related to aristololide (3) in which the C-10 oxygen has been replaced by a sulfur atom, and an extra MeO group is located at C-6. Aristothiolide can then be formulated as 1 (8,10-dimethoxy-5H-thiofuro-[40 ,30 ,20 :1,10]phenanthro[3,4-d]-1,3-dioxol5-one). 9-ethoxyaristolactam IV (2a) (C20H17NO6, Mþ 367) was isolated from fractions containing non-phenolic aristolactams. The 1 H NMR spectrum of 9-ethoxyaristolactam IV resembles that of aristolactam IV. The most relevant features in the spectrum is the absence of the signal for H-9 and the appearance of signals typical of the ethoxy group. The resonance of H-5 of 2a, and in minor extent that of H-7, are shifted to lower fields (0.18 ppm), presumably due to the fact that the MeO-8 group is forced out of the plane by the peri EtO substituent at C-9. The position of the methoxy groups at d 3.90 and d 3.93 indicates that the compound is 6,8-dimethoxy-9-ethoxy substituted since 9-methoxy groups appear at d 3.82–3.84 in 9-methoxyaristolactams (Houghton and Ogutveren, 1991b). Primary fissions in 9-ethoxyaristolactam IV (2a) involve losses of ethylene and the CH3CH2 radical from the EtO group. Main fragment ions indicate the degradation sequences [M]þ–C2H5–CO– H2CO–CH3–CO and [M]þ–C2H4–H2CO–HCO. The above findings, together with the co-occurrence of the 9-ethoxyaristolactam I (2b) in the same plant, indicate that the novel aristolactam from A. argentina is the 9-ethoxy derivative of aristolactam IV, named 9-ethoxyaristolactam IV (2a) [7-ethoxy-8,10-dimethoxy-benzo[f]-1,3-benzodioxolo[6,5,4-cd]indol-5(6H)-one]. 8,10-dimethoxy-5H-thiofuro[40 ,30 ,20 :1,10]phenanthro[3,4-d]-1,3-dioxol-5-one, aristothiolide (1): Mp 250 (CHCl3-n-PrOH); UV lmax nm: 251, 263 sh, 293, 308, 342, 357, 414 sh, 421; IR nmax cm1: 1684, 1677, 1628, 1370, 1285, 1160, 1063. 1H NMR (DMSO-d6) d 3.97 (3H, s, MeO-6), 4.04 (3H, s, MeO-8), 6.57 (2H, s, CH2O2), 6.95 (1H, d, J5,7 ¼ 2.5 Hz, H-7), 7.73 (1H, s, H-9), 7.90 (1H, dd, J5,7 ¼ 2.5 Hz, J5,9 ¼ 0.7 Hz, H-5), 8,17 (1H, s, H-2); EIMS m/z (rel. int.): 342 P þ 2 (9.2) (calcd. for C18H12O5S, 7.2%), 341 (25.2), 340 [M]þ (100), 327 m þ 2 (2.8) (calcd. for C17H9O5S, 2.9%), 326 (7.3), 325 (41.5), 299 m þ 2 (1.7) (calcd. for C16H9O4S, 2.0%), 298 (4.6), 297 (30.1), 284 m þ 2 (2.3) (calcd. for C15H6O4S, 2.2%), 283 (5.9), 282 (34.0), 254 (12.2), 224 (3.5), 196 (4.9), 170 [M]þþ (23.8), 168 [C11H4S]þ (4.0), 111 (4.8), 85 (9.6), 71 (15.2), 69 (10.2), 57 (21.3); ESI(þ)-TOFMS-HRMS (m/z), 341.0500 [M þ H]þ (calcd. for C18H13O5S, 341.0478; Dm 6.4 ppm); 363.0282 [M þ Na]þ (calcd. for C18H12O5SNa, 363.0298; Dm 4.4 ppm); 703.0672 [2M þ Na]þ (calcd. for C36H24O10S2Na, 703.0703; Dm 6.7 ppm). 7-Ethoxy-8,10-dimethoxy-benzo[f]-1,3-benzodioxolo[6,5,4-cd]indol-5(6H)-one, 9-ethoxyaristolactam IV (2a): Mp >280 (HOAc-n-PrOH); UV lmax nm (log ε): 242 (4.45), 267 (4.29), 289 (4.19), 301 sh (4.15), 335 (3.87), 347 (3.83), 414 (3.78); 1H NMR (DMSO-d6) d 1.39 (3H, t, J ¼ 7.5 Hz, CH3CH2O-9), 3.92 (3H, s, MeO-8), 3.95 (3H, s, MeO-6), 4.00 (2H, q, J ¼ 7.5 Hz, CH3CH2O-9), 6.42 (2H, s, CH2O2), 6.81 (1H, d, J5,7 ¼ 2.5 Hz, H-7), 7.56 (1H, s, H-2), 7.70 (1H, d, J5,7 ¼ 2.5 Hz, H-5), 10.76 (1H, s, NH). EIMS m/z (rel. int.): 368 (20.3), 367 [M]þ (100), 339 (18.4), 338 (64.1), 324 (12.3), 320 (7.5), 310 (11.9), 309 (23.3), 308 (9.0), 295 (11.4), 294 (9.4), 283 (9.8), 282 (17.8), 280 (20.0), 267 (7.0), 265 (8.8), 250 (6.5), 169 (9.6). 7-ethoxy-8-methoxy-benzo[f]-1,3-benzodioxolo[6,5,4-cd]indol-5(6H)-one, 9-ethoxyaristolactam I (2b): Mp 290 (n-PrOH); UV lmax nm (log ε): 243 (4.50), 266 (4.44), 293 sh (4.24), 303 (4.27), 330 (4.01), 342 sh (3.93), 410 (3.97); IR nmax cm1: 3257, 1684, 1477, 1406, 1362, 1304, 1288, 1085, 1049, 750; 1H NMR (DMSO-d6): d 1.38 (3H, t, J ¼ 7.5 Hz, CH3CH2O-9), 3.94 (3H, s, MeO8), 4.00 (2H, q, J ¼ 7.5 Hz, CH3CH2O-9), 6.43 (2H, s, CH2O2), 7.19 (1H, dd, J6,7 ¼ 8.5 Hz, J5,7 ¼ 2.5 Hz, H-7), 7.48 (1H, t, J5,6 ¼ J6,7 ¼ 8.5 Hz, H-6), 7.56 (1H, s, H-2), 8.17 (1H, dd, J5,6 ¼ 8.5 Hz, J5,7 ¼ 2.5 Hz, H-5), 10.86 (1H, s, NH); 13C NMR (DMSO-d6): d 167.5 (C]O), 157.0 (C-8), 148.2 (C-3), 146.9 (C-4), 134.5 (C-4b), 128.2 (C-8a), 126.4 (C-9), 125.7 (C-6), 120.7 (C-10), 119.6 (C-5), 118.5 (C-1), 111.0 (C-7), 109.0 (C-4a), 105.7 (C-2), 103.1 (CH2O2), 70.1 (CH3CH2O), 56.5 (MeO-8), 15.3 (CH3CH2O) (C-10a nonrecorded). EIMS m/z (rel. int.): 338 (20.9), 337 [M]þ (100), 309 (25.4), 308 (80.0), 294 (20.3), 291 (8.0), 290 (18.1), 280 (7.4), 279 (5.1), 278 (13.1), 265 (12.3), 262 (6.4), 252 (7.8), 251 (9.3), 249 (19.9), 237 (8.4), 222 (10.6), 194 (6.7), 179 (5.1), 167 (5.9), 166 (7.2), 164 (14.2), 154 (5.9), 151 (9.9), 139 (9.3), 137 (5.2), 125 (7.0). 8-methoxy-5H-furo[40 ,30 ,20 :1,10]phenanthro[3,4-d]-1,3-dioxol-5-one, aristololide (3): Mp 260 (n-PrOH); UV lmax nm (log ε): 234 (4.50), 255 (4.36), 280 (4.22), 286 sh (4.19), 322 (3.91), 391 (3.80), 401 sh (3.79); IR nmax cm1: 1790, 1483, 1416, 1372, 1299, 1279, 1057, 991, 936, 817, 748; 1H NMR (DMSO-d6) d 4.02 (3H, s, MeO-8), 6.54 (2H, s, CH2O2), 7.24 (1H, dd, J6,7 ¼ 8.5 Hz, J5,7 ¼ 2.5 Hz, H-7), 7.54 (1H, s, H-9), 7.60 (1H, t, J5,6 ¼ J6,7 ¼ 8.5 Hz, H-6), 7.86 (1H, s, H-2), 8.08 (1H, dd, J5,6 ¼ 8.5 Hz, J5,7 ¼ 2.5 Hz, H-5). EIMS m/z (rel. int.): 296 (3.0), 295 (22.3), 294 [M]þ (100), 280 (16.6), 279 (88.9), 252 (4.2), 251 (26.7), 223 (4.7), 195 (7.1), 193 (3.2), 167 (3.0), 165 (6.9), 152 (5.2), 147 [M]þþ (7.7), 139 (13.2), 137 (14.3). ESI(þ)-TOFMS-HRMS (m/z), 295.0587 [M þ H]þ (calcd. for C17H11O5, 295.0606; Dm 6.6 ppm). 2-hydroxy-1-methoxy-4H-dibenzo[de,g]quinoline-4,5(6H)-dione, 4,5-dioxodehydroasimilobine (4): Mp >250 (MeOH– CHCl3); UV lmax nm (log ε): 239 sh (4.54), 245 (4.56), 303 (4.11), 316 (4.13), 390 sh (3.70), 445 (4.03), 458 sh (4.00); EtOH–KOH, 240 (4.57), 255 (4.5), 318 (4.07), 331 (4.11), 385 (3.69), 502 (4.04); IR nmax cm1: 3367, 1675, 1661, 1621, 1592, 1570, 1408, 1383, 1299, 1284, 957, 887, 748; 1H NMR (DMSO-d6) d 4.06 (3H, s, MeO-4), 7.47 (1H, s, H-9), 7.64 (2H, m, H-6, H-7), 7.92 (1H, m, H-8), 8.09 (1H, s, H-2), 9.44 (1H, m, H-5), 11.75 (1H, s, NH); 13C NMR (DMSO-d6) (carbons numbered to correspond phenanthrene derivatives) d 177.2 (C]O), 155.8 (C]O), 153.1 (C-4), 151.4 (C-3), 132.5 (C-8a), 130.3 (C-10), 128.8 (C-8), 128.5 (C-7), 127.8 (C-5), 127.3 (C-4b), 126.7 (C-6), 126.3 (C-1), 124.7 (C-4a), 117.3 (C-2), 112.4 (C-9), 59.7 (MeO-4) (C-10a non-recorded) (coherent with Achenbach et al., 1991; spectrum determined in C5H5N); EIMS m/z (rel. int.): 294 (21.3), 293 [M]þ (100), 265 (34.2), 250 (59.0), 222 (24.4), 169 (11.0), 166 (27.5), 164 (10.2), 155 (15.7) 141 (14.9), 139 (19.9).
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The compounds described in this study are minor or trace constituents of A. argentina. They are biosynthetically derived from 1-benzyltetrahydroisoquinoline (1-BTIQ) alkaloids like other phenanthroid constituents usually found in the Aristolochiaceae (magnoflorine, aristolochic acids). 1-BTIQ alkaloids, which originate from the amino acid tyrosine, are responsible for the biosynthesis of several alkaloidal types including the aporphines, berberines, morphines and others (Mothes et al., 1985; Waterman, 1999). Most of the phenanthroid constituents found in members of the Aristolochiaceae are derived from aporphine alkaloids. Aristolochia plants characteristically contain aristolochic acids (AAs). These acids are rare outside the Aristolochiaceae [Cocculus triolobus (Menispermaceae) (Watanabe et al., 1988); Thecacoris annobonae (Euphorbiaceae) (Kuete et al., 2010)]. It was postulated that the 1-BTIQ alkaloids, via aporphine alkaloids, are oxidized 4,5-dioxoaporphines that can suffer net decarbonylation to give aristolactams (Castedo et al., 1976; Mothes et al., 1985), which in turn can be further oxidized to yield the AAs (Kumar et al., 2003; Lin et al., 1997; Mothes et al., 1985; Sharma et al., 1982). Aristothiolide (1) and aristololide (3) seem to be related compounds. Aristololide (3) is the lactonic form of a hypothetic 10-oxygenated 1-phenanthroic acid. It was suggested that aristololide (3) is a by-product of AA-I (6b) (Achari et al., 1983). Reduction of AA-I can give an amino acid, which, via the corresponding imine, is converted into the 10-hydroxy acid and then the lactone (3), or, alternatively, the lactone (3) can arise from AA-I by an intramolecular nucleophilic displacement of the nitro group by the carboxylate anion (Achari et al., 1983). 4. Chemotaxonomic significance Chemical constituents of plants have the potential to produce new insights into problems of angiosperm phylogeny and diversification (Fairbrothers et al., 1975; Grayer et al., 1999). The Aristolochiaceae seems to have evolved from the Magnoliales (Cronquist, 1988; Takhtajan, 1980), primitive angiosperms chemically typified by the presence of alkaloids derived from the 1-BTIQ skeleton (Cronquist, 1988; Waterman, 1999, 2007). 1-BTIQ-derived AAs, dioxoaporphines and aristolactams are typical constituents of the Aristolochiaceae. However, although the presence of AAs is almost entirely restricted to the Aristolochiaceae, dioxoaporphines and aristolactams are also ubiquitous in related families. The occurrence of dioxoaporphines and aristolactams in higher plants has been reviewed by Kumar et al. (2003). This review is illustrative about trends concerning the distribution of these compounds in angiosperms and has the potential to yield information of systematic importance. Dioxoaporphines and aristolactams accumulate in a chemosystematically significant manner in Annonaceae, Aristolochiaceae and Piperaceae, whereas they are rare in other groups of angiosperms. Thus, aristolactams were found in 34 species of the Aristolochiaceae (Aristolochia, Asarum), in 20 species of the Annonaceae (Fissistigma, Goniothalamus, Uvaria, etc.) and in 18 species of the Piperaceae–Saururaceae complex (Piper, Saururus, Houttuynia). Outside the above families, they were reported for the callus tissue of Stephania cepharantha (Menispermaceae; order Ranunculales) (Akasu et al., 1974) and Doryphora sassafras (Monimiaceae; order Laurales) (Mix et al., 1982). Inspection of the dioxoaporphines afforded a similar result. This type of compounds was found in 16 species of the Aristolochiaceae, 21 species of the Annonaceae and in 20 species of the Piperaceae–Saururaceae group. They were also recorded for 2 species of the Menispermaceae (Stephania, Telitoxicum) and 3 species of Papaveraceae–Fumariaceae complex (Glacium, Platycapnos, Sarcocapnos). The above analysis shows that dioxoaporphines and aristolactams are predominantly diffused in the Annonaceae, Aristolochiaceae and Piperaceae and consequently chemical links may exist among these families. The family Aristolochiaceae is usually treated as the distinct order Aristolochiales within the Subclass Magnoliidae (Cronquist, 1988; Takhtajan, 1980) or placed within the order Piperales together with the families Piperaceae, Saururaceae and Lactoridaceae (Judd et al., 2002). The distribution of dioxoaporphines and aristolactams in higher plants supports the close relationship between the Aristolochiaceae and Piperaceae previously inferred from morphological characters and DNA sequence data (Judd et al., 2002; Kelly and Gonzalez, 2003; Wanke et al., 2007a,b). Similarly, the consistent co-occurrence of these compounds in the Annonaceae suggests that this family is related to the Aristolochiaceae and Piperaceae. The connexion between Aristolochiaceae and Annonaceae is supported by many similarities between both families, e.g., sieve-element plastids of Aristolochia closely resemble those of Annona (Endress, 1994; Takhtajan, 1980). The Aristolochiales would be an advanced member because of its structurally advanced modification of 1-BTIQ alkaloids into AAs (Fairbrothers et al., 1975). The Magnoliales (Subclass Magnoliidae), including the Annonaceae, has often been considered part of the ancestral angiosperms and probably the most ancient types of existing angiosperms from which the Aristolochiales, the Piperales and other orders may have been derived (Cronquist, 1988; Takhtajan, 1980). The chemical data here discussed suggest that a significant degree of evolutionary divergence from a common ancestor occurred in the ancestral angiosperm complex, but some chemical characters were preserved in certain families. The problem of interpreting the occurrence and distribution of secondary metabolites in higher plants, as well as evolutionary advances through changes in biosynthetic pathways, is difficult (for a discussion see Waterman, 2007). The consistent presence of dioxoaporphines and aristolactams in the Annonaceae, the Aristolochiales and the Piperales suggests that such chemical characters from a common ancestor were preserved in these groups, whereas they were partially or entirely lost in other Magnoliales-derived groups (Cronquist, 1988). The sporadic occurrence of dioxoaporphines and aristolactams in Doryphora in the Monimiaceae (Laurales), Stephania and Telitoxicum in the Menispermaceae (Ranunculales) and certain genera of the Papaverales (Glacium, Platycapnos, Sarcocapnos) can be interpreted as relic characters. These orders (Laurales, Ranunculales, Papaverales) in which there probably was a continuing evolutionary decrease in the production of dioxoaporphines and aristolactams, are on the other side prolific in the biosynthesis of other structurally advanced major classes of 1-BITQ alkaloids (protoberberines, morphinans, etc.) arising from newly evolved biosynthetic pathways (Mothes et al., 1985; Waterman, 1999).
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This study reports on the unusual phenanthrene derivatives 1, 2a, 2b and 3 identified in A. argentina. They were so far only documented for the Aristolochiaceae. The rare groups of aristololides and 9-ethoxyaristolactams are now further documented for the Aristolochiaceae, and, together with aristothiolide (1), the first member of a new family of compounds, may serve as additional chemotaxonomic marks to delimit the family. Acknowledgement I thank Dr. Jodie V. Johnson (Florida University, Gainesville, Florida) for (þ)ESI-TOFMS-HRMS measurements and Josefina Awruch (University of Buenos Aires) for NMR measurements. References Achari, B., Chakrabarty, S., Bandyopadhyay, S., Pakrashi, S.C., 1982. Heterocycles 19, 1203. Achari, B., Bandyopadhyay, S., Saha, C.R., Pakrashi, S.C., 1983. Heterocycles 20, 771. Achenbach, H., Frey, D., Waibel, R., 1991. J. Nat. Prod. 54, 1331. Akasu, M., Itokawa, H., Fujita, M., 1974. Tetrahedron Lett. 3609 Castedo, L., Suau, R., Mourino, A., 1976. Tetrahedron Lett. 501 Cronquist, A., 1988. The Evolution and Classification of Flowering Plants, second ed. The New York Botanical Garden, Bronx, New York. Dominguez, J.A., 1928. Contribuciones a la Materia Medica Argentina. Peuser, Buenos Aires, Argentina. Endress, P.K., 1994. Diversity and Evolutionary Biology of Tropical Flowers. University Press, Cambridge, United Kingdown. Fairbrothers, D.E., Mabry, T.J., Scogin, R.L., Turner, B.L., 1975. Ann. Missouri Bot. Gard. 62, 765. Grayer, R.J., Chase, M.W., Simmonds, M.S.J., 1999. Biochem. Syst. Ecol. 27, 369. Houghton, P.J., Ogutveren, M., 1991a. Phytochemistry 30, 717. Houghton, P.J., Ogutveren, M., 1991b. Phytochemistry 30, 253. Judd, W.S., Campbell, C.S., Kellogg, E., Stevens, P.F., Donoghue, M.J., 2002. Plant Systematics, second ed. Sinauer Associates, Inc., Sunderland, Massachusetts. Kelly, L.M., Gonzalez, F., 2003. Syst. Bot. 28, 236. Kuete, V., Poumale Poumale, H.M., Guedem, A.N., Shiono, Y., Randrianasolo, R., Ngadjui, B.T., 2010. S. Afr. J. Bot. 76, 536. Kumar, V., Poonam, Prasad, A.K., Parmar, V.S., 2003. Nat. Prod. Rep. 20, 565. Lin, W.-A., Fu, H.-Z., Hano, Y., Nomura, T., 1997. J. Chin. Pharm. Sci. 6, 8. Lou, F.C., Ding, L.S., Waterman, P.G., 1989. Chem. Abstr. 111, 130763. Mix, D.B., Guinaudeau, H., Shamma, M., 1982. J. Nat. Prod. 45, 657. Mothes, K., Schutte, H.R., Luckner, M., 1985. Biochemistry of Alkaloids. VEB Deutscher Verlag der Wissenschaften, Berlin. Priestap, H.A., Velandia, A.E., Johnson, J.V., Barbieri, M.J., 2012. Biochem. Syst. Ecol. 40, 126. Priestap, H.A., 1985. Phytochemistry 24, 849. Priestap, H.A., 1987. Phytochemistry 26, 519. Sharma, V., Jain, S., Bhakuni, D.S., Kapil, R.S., 1982. J. Chem. Soc. 1153 Shi, L.-S., Kuo, P.-C., Tsai, Y.-L., Damu, A.G., Wu, T.-S., 2004. Bioorg. Med. Chem. 12, 439. Takhtajan, A.L., 1980. Bot. Rev. 46, 225. Wanke, S., Jaramillo, M.A., Borsch, T., Samain, M.-S., Quandt, D., Neinhuis, C., 2007a. Mol. Phylogenet. Evol. 42, 477. Wanke, S., Vanderschaeve, L., Mathieu, G., Neinhuis, C., Goetghebeur, P., Samain, M.-S., 2007b. Ann. Bot. 99, 1231. Watanabe, M., Miyakado, M., Iwai, T., Izumi, K., Yanagi, K., 1988. Agric. Biol. Chem. 52, 1079. Waterman, P.J., 1999. Biochem. Syst. Ecol. 27, 395. Waterman, P.J., 2007. Phytochemistry 68, 2896.
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Phenanthroid constituents from Aristolochia argentina Horacio A. Priestap* Department of Biological Sciences, Florida International University, 11200 S.W. 8th Street, HLS1-313, Miami, FL 33199, United States
a r t i c l e i n f o Article history: Received 3 July 2012 Accepted 9 September 2012 Available online 16 October 2012 Keywords: Aristolochia argentina Aristolochiaceae Aristothiolide Aristololide 9-ethoxyaristolactam IV 9-ethoxyaristolactam I 4,5-dioxodehydroasimilobine
1. Subject and source The genus Aristolochia (Aristolochiaceae) consists of approximately 600 species largely distributed in tropical and warmtemperate regions of the world (Cronquist, 1988). Aristolochia argentina Gris., a twining plant growing in central and north Argentina, is popularly used as medicinal plant to treat arthritis and other ailments (Dominguez, 1928). Underground parts of A. argentina were collected at Cruz Alta, Tucuman. A voucher specimen (BACP 4602) is deposit at the Herbarium of the “Centro de Estudios Farmacologicos y Botanicos”, Argentina. 2. Previous work Earlier studies on A. argentina revealed the occurrence of numerous aristolochic acids and aristolactams on the underground parts of the plant (Priestap, 1985, 1987). The aerial parts of A. argentina also contain aristolochic acids (Priestap et al., 2012). 3. Present study In continuation of earlier investigations on the chemical composition of A. argentina, it is now reported on the identification of the new compounds aristothiolide (1) and 9-ethoxyaristolactam IV (2a), along with the known compounds 9-ethoxyaristolactam (2b), aristololide (3) and 4,5-dioxodehydroasimilobine (4). 9-Ethoxyaristolactam I (2b) was previously reported for Aristolochia mollisima (Lou et al., 1989). Aristololide (3) is a phenanthroid lactone found in the roots of Aristolochia
* Tel.: þ1 305 348 0375. E-mail addresses: priestap@fiu.edu, [email protected]. 0305-1978/$ – see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bse.2012.09.015
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indica (Achari et al., 1983) and Aristolochia elegans (Shi et al., 2004). The 4,5-dioxodehydroasimilobine (4) belongs to the family of dioxoaporphine alkaloids. It was first isolated from Aristolochia indica (Achari et al., 1982) and also reported for related families (Kumar et al., 2003).
O
O
4
O
O
2 3
O NH
S O
10
O O
9
5
OCH2 CH3
8
6 CH3O
O
O
1
7
OCH3
OCH3
R
OCH3
2a R = OCH3 2b R = H
1
3
O HO
O NH
CH3O
COOH
O
O
COOH
NO2
O
NO2
O
OCH3
R 4
5
6a R = H 6b R = OCH3
Dried underground parts of A. argentina were sequentially extracted with petrol and EtOH as previously reported (Priestap, 1987). The petrol extract from 23 kg of plant material gave an oil (ca. 1 kg) which was submitted CC and prep. TLC on silica gel to yield 30 mg of aristololide (3) and 2 mg of aristothiolide (1). A similar amount of compounds 1 and 3 is also present in the EtOH extract. The ethanolic extract (ca. 2 kg) from 13.77 kg of plant material was partitioned between water and ethyl ether. The ethereal extracts (247 g) were fractionated as previously reported (Priestap, 1985) to give 1 mg of 9-ethoxyaristolactam IV (2a), 49 mg of 9-ethoxyaristolactam I (2b) and 79 mg of 4,5-dioxodehydroasimilobine (4). TLC was carried out as follows: silica gel (Rf), (1) hexane–C6H6, 1:1 (3, 0.66; 1, 0.58); (2) C6H6–MeCOEt, 17:3 (2b, 0.41; 2a, 0.37); (3) CHCl3–EtOH, 9:1 (4, 0.48); Al2O3, (4) CHCl3–EtOH, 100:1 (2b, 0.78; 2a, 0.74); (3) CHCl3–EtOH, 95:5 (4, 0.48). Compounds were detected as yellow or red (4) spots and also by fluorescence at 360 nm. HPLC was carried out in a ThermoFinnigan chromatograph (Thermo Electron Corporation) using a C18 RP Hypersil GOLD column (RP5, 250 4.6 mm, pore size 5 mm, Thermo Electron Corporation). The eluting systems consisted of 60% of water and 40% of 1% HOAc in MeOH, isocratic (Rt: 4, 4.9; 2b, 15.3; 2a, 16.1; 3, 34.5; 1, 36.3 min). Flow rate, 1 ml/min. Detection at 254 nm. 1H NMR spectra were measured at 80 MHz and 13C NMR spectra at 20 MHz in a Varian FT-80A spectrometer. Electron impact mass spectra were recorded in a Varian Mat CH7A instrument at 70 eV. ESI(þ)-TOFMS-HRMS analysis was performed by flow injection into an Agilent Technologies 6210 TOF/LCMS instrument operated in the (þ)-ESI-MS mode. Aristothiolide (1) is a sulfur-containing compound that exhibits similar chromatographic properties than aristololide (3) co-occurring in the same plant. ESI(þ)-TOFMS-HRMS afforded m/z 341.0500 for [M þ H]þ implying the molecular formula of C18H12O5S. Aristothiolide (1) showed an absorption maximum at 251 nm, typical of the phenanthrene chromophore, and a carbonyl absorption at n 1784 cm1. The 1H NMR showed the usual singlets of the 3,4-methylenedioxy group, H-2, H-9 and two MeO groups. In addition, compound 1 exhibits two doublets, one of them at low field (H-5), with characteristic metacoupling (J5,7 ¼ 2.5 Hz) consistent with the 6,8-dioxy-substitution observed in aristolochic acids and aristolactams. Interestingly, H-2 is significantly shifted to low fields in aristothiolide (1). For example, H-2 absorbs at d 8.17 whereas it absorbs at d 7.86 in aristololide (3). It is likely that H-2 suffers deshielding because of the anisotropic effect from diamagnetic circulations at the carbonyl group. The observed deshielding (0.31 ppm) may be due to the bulk sulfur atom that forces the carbonyl group to a position closer to this proton. A similar effect is observed between 1-indanone and a-tetralone, and may also occur in dioxoaporphines. Thus, H-2 in 4,5-dioxodehydroasimilobine (4) absorbs at d 8.09, i.e. at 0.47 ppm lower fields than the same proton in the corresponding aristolactam (aristolactam AII) (d 7.62). Another remarkable example is provided by 9-methoxyaristolochic acid II (5) isolated from Aristolochia ponticum (Houghton and Ogutveren, 1991a). The signal for H-2 at d 8.35 is much more downfield (0.54 ppm) than that recorded for the equivalent proton in aristolochic acid II (6a) (d 7.81).
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Since the 9-MeO and H-2 are quite apart in 5, it is likely that, by introduction of the 9-MeO group in the molecule, crowding of the substituents at positions 9, 10 and 1 of the phenanthrene nucleus forces the carboxyl group to move to a position closer to H-2, thus causing deshielding of this proton. The electron ionization mass spectra of aristothiolide (1) exhibit sets of fragment ions which indicate the successive losses of neutral fragments (CH3, CO, HCO, H2CO and H), also observed in aristolactams and aristololide (3), e.g., fragment ions at m/z 340, 325, 297, 282, 254, 224, 196 and 168 suggest the degradation pathway [M]þ–CH3–CO–CH3–CO–H2CO–2CO. The above considerations lead to the conclusion that aristothiolide (1) is related to aristololide (3) in which the C-10 oxygen has been replaced by a sulfur atom, and an extra MeO group is located at C-6. Aristothiolide can then be formulated as 1 (8,10-dimethoxy-5H-thiofuro-[40 ,30 ,20 :1,10]phenanthro[3,4-d]-1,3-dioxol5-one). 9-ethoxyaristolactam IV (2a) (C20H17NO6, Mþ 367) was isolated from fractions containing non-phenolic aristolactams. The 1 H NMR spectrum of 9-ethoxyaristolactam IV resembles that of aristolactam IV. The most relevant features in the spectrum is the absence of the signal for H-9 and the appearance of signals typical of the ethoxy group. The resonance of H-5 of 2a, and in minor extent that of H-7, are shifted to lower fields (0.18 ppm), presumably due to the fact that the MeO-8 group is forced out of the plane by the peri EtO substituent at C-9. The position of the methoxy groups at d 3.90 and d 3.93 indicates that the compound is 6,8-dimethoxy-9-ethoxy substituted since 9-methoxy groups appear at d 3.82–3.84 in 9-methoxyaristolactams (Houghton and Ogutveren, 1991b). Primary fissions in 9-ethoxyaristolactam IV (2a) involve losses of ethylene and the CH3CH2 radical from the EtO group. Main fragment ions indicate the degradation sequences [M]þ–C2H5–CO– H2CO–CH3–CO and [M]þ–C2H4–H2CO–HCO. The above findings, together with the co-occurrence of the 9-ethoxyaristolactam I (2b) in the same plant, indicate that the novel aristolactam from A. argentina is the 9-ethoxy derivative of aristolactam IV, named 9-ethoxyaristolactam IV (2a) [7-ethoxy-8,10-dimethoxy-benzo[f]-1,3-benzodioxolo[6,5,4-cd]indol-5(6H)-one]. 8,10-dimethoxy-5H-thiofuro[40 ,30 ,20 :1,10]phenanthro[3,4-d]-1,3-dioxol-5-one, aristothiolide (1): Mp 250 (CHCl3-n-PrOH); UV lmax nm: 251, 263 sh, 293, 308, 342, 357, 414 sh, 421; IR nmax cm1: 1684, 1677, 1628, 1370, 1285, 1160, 1063. 1H NMR (DMSO-d6) d 3.97 (3H, s, MeO-6), 4.04 (3H, s, MeO-8), 6.57 (2H, s, CH2O2), 6.95 (1H, d, J5,7 ¼ 2.5 Hz, H-7), 7.73 (1H, s, H-9), 7.90 (1H, dd, J5,7 ¼ 2.5 Hz, J5,9 ¼ 0.7 Hz, H-5), 8,17 (1H, s, H-2); EIMS m/z (rel. int.): 342 P þ 2 (9.2) (calcd. for C18H12O5S, 7.2%), 341 (25.2), 340 [M]þ (100), 327 m þ 2 (2.8) (calcd. for C17H9O5S, 2.9%), 326 (7.3), 325 (41.5), 299 m þ 2 (1.7) (calcd. for C16H9O4S, 2.0%), 298 (4.6), 297 (30.1), 284 m þ 2 (2.3) (calcd. for C15H6O4S, 2.2%), 283 (5.9), 282 (34.0), 254 (12.2), 224 (3.5), 196 (4.9), 170 [M]þþ (23.8), 168 [C11H4S]þ (4.0), 111 (4.8), 85 (9.6), 71 (15.2), 69 (10.2), 57 (21.3); ESI(þ)-TOFMS-HRMS (m/z), 341.0500 [M þ H]þ (calcd. for C18H13O5S, 341.0478; Dm 6.4 ppm); 363.0282 [M þ Na]þ (calcd. for C18H12O5SNa, 363.0298; Dm 4.4 ppm); 703.0672 [2M þ Na]þ (calcd. for C36H24O10S2Na, 703.0703; Dm 6.7 ppm). 7-Ethoxy-8,10-dimethoxy-benzo[f]-1,3-benzodioxolo[6,5,4-cd]indol-5(6H)-one, 9-ethoxyaristolactam IV (2a): Mp >280 (HOAc-n-PrOH); UV lmax nm (log ε): 242 (4.45), 267 (4.29), 289 (4.19), 301 sh (4.15), 335 (3.87), 347 (3.83), 414 (3.78); 1H NMR (DMSO-d6) d 1.39 (3H, t, J ¼ 7.5 Hz, CH3CH2O-9), 3.92 (3H, s, MeO-8), 3.95 (3H, s, MeO-6), 4.00 (2H, q, J ¼ 7.5 Hz, CH3CH2O-9), 6.42 (2H, s, CH2O2), 6.81 (1H, d, J5,7 ¼ 2.5 Hz, H-7), 7.56 (1H, s, H-2), 7.70 (1H, d, J5,7 ¼ 2.5 Hz, H-5), 10.76 (1H, s, NH). EIMS m/z (rel. int.): 368 (20.3), 367 [M]þ (100), 339 (18.4), 338 (64.1), 324 (12.3), 320 (7.5), 310 (11.9), 309 (23.3), 308 (9.0), 295 (11.4), 294 (9.4), 283 (9.8), 282 (17.8), 280 (20.0), 267 (7.0), 265 (8.8), 250 (6.5), 169 (9.6). 7-ethoxy-8-methoxy-benzo[f]-1,3-benzodioxolo[6,5,4-cd]indol-5(6H)-one, 9-ethoxyaristolactam I (2b): Mp 290 (n-PrOH); UV lmax nm (log ε): 243 (4.50), 266 (4.44), 293 sh (4.24), 303 (4.27), 330 (4.01), 342 sh (3.93), 410 (3.97); IR nmax cm1: 3257, 1684, 1477, 1406, 1362, 1304, 1288, 1085, 1049, 750; 1H NMR (DMSO-d6): d 1.38 (3H, t, J ¼ 7.5 Hz, CH3CH2O-9), 3.94 (3H, s, MeO8), 4.00 (2H, q, J ¼ 7.5 Hz, CH3CH2O-9), 6.43 (2H, s, CH2O2), 7.19 (1H, dd, J6,7 ¼ 8.5 Hz, J5,7 ¼ 2.5 Hz, H-7), 7.48 (1H, t, J5,6 ¼ J6,7 ¼ 8.5 Hz, H-6), 7.56 (1H, s, H-2), 8.17 (1H, dd, J5,6 ¼ 8.5 Hz, J5,7 ¼ 2.5 Hz, H-5), 10.86 (1H, s, NH); 13C NMR (DMSO-d6): d 167.5 (C]O), 157.0 (C-8), 148.2 (C-3), 146.9 (C-4), 134.5 (C-4b), 128.2 (C-8a), 126.4 (C-9), 125.7 (C-6), 120.7 (C-10), 119.6 (C-5), 118.5 (C-1), 111.0 (C-7), 109.0 (C-4a), 105.7 (C-2), 103.1 (CH2O2), 70.1 (CH3CH2O), 56.5 (MeO-8), 15.3 (CH3CH2O) (C-10a nonrecorded). EIMS m/z (rel. int.): 338 (20.9), 337 [M]þ (100), 309 (25.4), 308 (80.0), 294 (20.3), 291 (8.0), 290 (18.1), 280 (7.4), 279 (5.1), 278 (13.1), 265 (12.3), 262 (6.4), 252 (7.8), 251 (9.3), 249 (19.9), 237 (8.4), 222 (10.6), 194 (6.7), 179 (5.1), 167 (5.9), 166 (7.2), 164 (14.2), 154 (5.9), 151 (9.9), 139 (9.3), 137 (5.2), 125 (7.0). 8-methoxy-5H-furo[40 ,30 ,20 :1,10]phenanthro[3,4-d]-1,3-dioxol-5-one, aristololide (3): Mp 260 (n-PrOH); UV lmax nm (log ε): 234 (4.50), 255 (4.36), 280 (4.22), 286 sh (4.19), 322 (3.91), 391 (3.80), 401 sh (3.79); IR nmax cm1: 1790, 1483, 1416, 1372, 1299, 1279, 1057, 991, 936, 817, 748; 1H NMR (DMSO-d6) d 4.02 (3H, s, MeO-8), 6.54 (2H, s, CH2O2), 7.24 (1H, dd, J6,7 ¼ 8.5 Hz, J5,7 ¼ 2.5 Hz, H-7), 7.54 (1H, s, H-9), 7.60 (1H, t, J5,6 ¼ J6,7 ¼ 8.5 Hz, H-6), 7.86 (1H, s, H-2), 8.08 (1H, dd, J5,6 ¼ 8.5 Hz, J5,7 ¼ 2.5 Hz, H-5). EIMS m/z (rel. int.): 296 (3.0), 295 (22.3), 294 [M]þ (100), 280 (16.6), 279 (88.9), 252 (4.2), 251 (26.7), 223 (4.7), 195 (7.1), 193 (3.2), 167 (3.0), 165 (6.9), 152 (5.2), 147 [M]þþ (7.7), 139 (13.2), 137 (14.3). ESI(þ)-TOFMS-HRMS (m/z), 295.0587 [M þ H]þ (calcd. for C17H11O5, 295.0606; Dm 6.6 ppm). 2-hydroxy-1-methoxy-4H-dibenzo[de,g]quinoline-4,5(6H)-dione, 4,5-dioxodehydroasimilobine (4): Mp >250 (MeOH– CHCl3); UV lmax nm (log ε): 239 sh (4.54), 245 (4.56), 303 (4.11), 316 (4.13), 390 sh (3.70), 445 (4.03), 458 sh (4.00); EtOH–KOH, 240 (4.57), 255 (4.5), 318 (4.07), 331 (4.11), 385 (3.69), 502 (4.04); IR nmax cm1: 3367, 1675, 1661, 1621, 1592, 1570, 1408, 1383, 1299, 1284, 957, 887, 748; 1H NMR (DMSO-d6) d 4.06 (3H, s, MeO-4), 7.47 (1H, s, H-9), 7.64 (2H, m, H-6, H-7), 7.92 (1H, m, H-8), 8.09 (1H, s, H-2), 9.44 (1H, m, H-5), 11.75 (1H, s, NH); 13C NMR (DMSO-d6) (carbons numbered to correspond phenanthrene derivatives) d 177.2 (C]O), 155.8 (C]O), 153.1 (C-4), 151.4 (C-3), 132.5 (C-8a), 130.3 (C-10), 128.8 (C-8), 128.5 (C-7), 127.8 (C-5), 127.3 (C-4b), 126.7 (C-6), 126.3 (C-1), 124.7 (C-4a), 117.3 (C-2), 112.4 (C-9), 59.7 (MeO-4) (C-10a non-recorded) (coherent with Achenbach et al., 1991; spectrum determined in C5H5N); EIMS m/z (rel. int.): 294 (21.3), 293 [M]þ (100), 265 (34.2), 250 (59.0), 222 (24.4), 169 (11.0), 166 (27.5), 164 (10.2), 155 (15.7) 141 (14.9), 139 (19.9).
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The compounds described in this study are minor or trace constituents of A. argentina. They are biosynthetically derived from 1-benzyltetrahydroisoquinoline (1-BTIQ) alkaloids like other phenanthroid constituents usually found in the Aristolochiaceae (magnoflorine, aristolochic acids). 1-BTIQ alkaloids, which originate from the amino acid tyrosine, are responsible for the biosynthesis of several alkaloidal types including the aporphines, berberines, morphines and others (Mothes et al., 1985; Waterman, 1999). Most of the phenanthroid constituents found in members of the Aristolochiaceae are derived from aporphine alkaloids. Aristolochia plants characteristically contain aristolochic acids (AAs). These acids are rare outside the Aristolochiaceae [Cocculus triolobus (Menispermaceae) (Watanabe et al., 1988); Thecacoris annobonae (Euphorbiaceae) (Kuete et al., 2010)]. It was postulated that the 1-BTIQ alkaloids, via aporphine alkaloids, are oxidized 4,5-dioxoaporphines that can suffer net decarbonylation to give aristolactams (Castedo et al., 1976; Mothes et al., 1985), which in turn can be further oxidized to yield the AAs (Kumar et al., 2003; Lin et al., 1997; Mothes et al., 1985; Sharma et al., 1982). Aristothiolide (1) and aristololide (3) seem to be related compounds. Aristololide (3) is the lactonic form of a hypothetic 10-oxygenated 1-phenanthroic acid. It was suggested that aristololide (3) is a by-product of AA-I (6b) (Achari et al., 1983). Reduction of AA-I can give an amino acid, which, via the corresponding imine, is converted into the 10-hydroxy acid and then the lactone (3), or, alternatively, the lactone (3) can arise from AA-I by an intramolecular nucleophilic displacement of the nitro group by the carboxylate anion (Achari et al., 1983). 4. Chemotaxonomic significance Chemical constituents of plants have the potential to produce new insights into problems of angiosperm phylogeny and diversification (Fairbrothers et al., 1975; Grayer et al., 1999). The Aristolochiaceae seems to have evolved from the Magnoliales (Cronquist, 1988; Takhtajan, 1980), primitive angiosperms chemically typified by the presence of alkaloids derived from the 1-BTIQ skeleton (Cronquist, 1988; Waterman, 1999, 2007). 1-BTIQ-derived AAs, dioxoaporphines and aristolactams are typical constituents of the Aristolochiaceae. However, although the presence of AAs is almost entirely restricted to the Aristolochiaceae, dioxoaporphines and aristolactams are also ubiquitous in related families. The occurrence of dioxoaporphines and aristolactams in higher plants has been reviewed by Kumar et al. (2003). This review is illustrative about trends concerning the distribution of these compounds in angiosperms and has the potential to yield information of systematic importance. Dioxoaporphines and aristolactams accumulate in a chemosystematically significant manner in Annonaceae, Aristolochiaceae and Piperaceae, whereas they are rare in other groups of angiosperms. Thus, aristolactams were found in 34 species of the Aristolochiaceae (Aristolochia, Asarum), in 20 species of the Annonaceae (Fissistigma, Goniothalamus, Uvaria, etc.) and in 18 species of the Piperaceae–Saururaceae complex (Piper, Saururus, Houttuynia). Outside the above families, they were reported for the callus tissue of Stephania cepharantha (Menispermaceae; order Ranunculales) (Akasu et al., 1974) and Doryphora sassafras (Monimiaceae; order Laurales) (Mix et al., 1982). Inspection of the dioxoaporphines afforded a similar result. This type of compounds was found in 16 species of the Aristolochiaceae, 21 species of the Annonaceae and in 20 species of the Piperaceae–Saururaceae group. They were also recorded for 2 species of the Menispermaceae (Stephania, Telitoxicum) and 3 species of Papaveraceae–Fumariaceae complex (Glacium, Platycapnos, Sarcocapnos). The above analysis shows that dioxoaporphines and aristolactams are predominantly diffused in the Annonaceae, Aristolochiaceae and Piperaceae and consequently chemical links may exist among these families. The family Aristolochiaceae is usually treated as the distinct order Aristolochiales within the Subclass Magnoliidae (Cronquist, 1988; Takhtajan, 1980) or placed within the order Piperales together with the families Piperaceae, Saururaceae and Lactoridaceae (Judd et al., 2002). The distribution of dioxoaporphines and aristolactams in higher plants supports the close relationship between the Aristolochiaceae and Piperaceae previously inferred from morphological characters and DNA sequence data (Judd et al., 2002; Kelly and Gonzalez, 2003; Wanke et al., 2007a,b). Similarly, the consistent co-occurrence of these compounds in the Annonaceae suggests that this family is related to the Aristolochiaceae and Piperaceae. The connexion between Aristolochiaceae and Annonaceae is supported by many similarities between both families, e.g., sieve-element plastids of Aristolochia closely resemble those of Annona (Endress, 1994; Takhtajan, 1980). The Aristolochiales would be an advanced member because of its structurally advanced modification of 1-BTIQ alkaloids into AAs (Fairbrothers et al., 1975). The Magnoliales (Subclass Magnoliidae), including the Annonaceae, has often been considered part of the ancestral angiosperms and probably the most ancient types of existing angiosperms from which the Aristolochiales, the Piperales and other orders may have been derived (Cronquist, 1988; Takhtajan, 1980). The chemical data here discussed suggest that a significant degree of evolutionary divergence from a common ancestor occurred in the ancestral angiosperm complex, but some chemical characters were preserved in certain families. The problem of interpreting the occurrence and distribution of secondary metabolites in higher plants, as well as evolutionary advances through changes in biosynthetic pathways, is difficult (for a discussion see Waterman, 2007). The consistent presence of dioxoaporphines and aristolactams in the Annonaceae, the Aristolochiales and the Piperales suggests that such chemical characters from a common ancestor were preserved in these groups, whereas they were partially or entirely lost in other Magnoliales-derived groups (Cronquist, 1988). The sporadic occurrence of dioxoaporphines and aristolactams in Doryphora in the Monimiaceae (Laurales), Stephania and Telitoxicum in the Menispermaceae (Ranunculales) and certain genera of the Papaverales (Glacium, Platycapnos, Sarcocapnos) can be interpreted as relic characters. These orders (Laurales, Ranunculales, Papaverales) in which there probably was a continuing evolutionary decrease in the production of dioxoaporphines and aristolactams, are on the other side prolific in the biosynthesis of other structurally advanced major classes of 1-BITQ alkaloids (protoberberines, morphinans, etc.) arising from newly evolved biosynthetic pathways (Mothes et al., 1985; Waterman, 1999).
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