Chemical studies on Taxus canadensis.

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)

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REVIEW Chemical Studies on Taxus canadensis by Yong Li a ), Fang Qin b ), Si-Ming Wang a ), Rui-Xia Guo b ), Yue-Feng Zhang* a ), Yu-Cheng Gu c ), and Qing-Wen Shi b ) a

) Department of Thoracic Surgery and Department of Pharmacy, The Fourth Hospital of Hebei Medical University, 12 Jiankang Lu, Shijiazhuang 050011, P. R. China (phone: þ 86-311-66696452; e-mail: [email protected]) b ) Department of Medicinal Natural Product Chemistry, School of Pharmaceutical Sciences, Hebei Medical University, 361 Zhongshan East Road, Shijiazhuang 050017, P. R. China c ) Syngenta Jealotts Hill International Research Centre, Berkshire, UK

A series of new taxanes, 1 – 93, have been isolated, together with 37 known taxoids including Taxol (paclitaxel) and cephalomannine, from the Canadian yew, Taxus canadensis (Taxaceae) in the past 30 years. These new taxoids possess various skeletons containing 5/7/6, 6/10/6, 6/5/5/6, 6/8/6, and 6/12 ring systems and six new taxanes with four novel skeletons, i.e., a taxane with a 6/6/8/6 ring system, a taxane with a [3.3.3] propellane skeleton, three taxanes with [3.3.3] [3.4.5] dipropellane sytems, as well as a novel taxane with a unique 5/5/4/6/6/6 hexacyclic skeleton, containing a unique [3.3.2] propellane, were isolated for the first time from natural sources. It should be emphasized that 13-acetyl-9-dihydrobaccatin III, a very useful starting material for the semisynthesis of Taxol and Taxotere, represents the most abundant taxane in the needles of this yew tree. These findings establish the above mentioned yew tree as significantly different from the remaining species. On the other hand, some chemical modifications on the taxanes isolated from this plant were carried out.

Contents 1. Introduction 2. Phytochemical Studies of Taxanes 3. Chemical Modification of Taxanes 4. Concluding Remarks

1. Introduction. – Natural products are abundant sources of diverse bioactive metabolites and continue to be one of the most important sources of pharmacologically active compounds in the quest for drugs against life-threatening diseases such as microbial infections, diseases of the heart and the circulatory system, cancer, and others. Chemical studies on various natural products have provided the impetus to great advances in organic chemistry, and led to discovery of diverse natural scaffolds which were used as lead compounds for the drug discovery. On the other hand, natural products have also provided insight into new mechanisms of action, and cancer treatment would be immeasurably poorer without the insights and the compounds derived from nature. Yews (Taxus spp., Taxaceae) are slow-growing evergreen, 2013 Verlag Helvetica Chimica Acta AG, Zrich

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gymnospermous shrubs commonly used for ornamental landscaping or arbor. Plants from the genus Taxus (Taxaceae) are a rich source of biologically active diterpenoids belonging to the unique structure class of taxanes [1] [2]. Taxol (paclitaxel, 1) is one of the most important anticancer drugs currently on the market for the treatment of ovarian and breast cancers, and showed promising effects for a variety of other cancers such as neck, lung, gastrointestinal, and bladder. Its unique mechanism of action, limited availability, and relative lipophilicity have generated worldwide interest, and extensive chemical studies have been carried out [1 – 6]. As a result, more than 500 taxane diterpenoids have meanwhile been isolated and identified [7]. Systemic phytochemical studies have shown that the needles of the yew are an important raw material for the pharmaceutical industry as a renewable source of the remarkable antitumor drug Taxol, and the suitable starting material (such as baccatin III, 10deacetylbaccatin III, and 13-acetyl-9-dihydrobaccatin III) for the practical semisynthesis of Taxol and Taxotere [1]. Structureactivity relationship and syntheticmodification studies have been aimed at increasing activity and solubility of new analogs. The genus Taxus, the only known source of taxane diterpenoids, comprises ca. 12 species distributed throughout northern hemisphere, with only one species, Canadian yew, Taxus canadensis (Fig. 1), being endemic to Canada. Taxus canadensis is a low-trailing shrub ubiquitous to the Quebec region, and its composition has been shown to deviate considerably from those of the other species [8 – 10]. Interest in the

Fig. 1. Needles and seeds of the Canadian yew, Taxus canadensis, taken from Montreal Plant Garden, Quebec, Canada


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Canadian yew was prompted by the discovery that its needles can be a rich source of 13acetyl-9-dihydrobaccatin III (5 – 7 times the amount of paclitaxel (Taxol ), depending on the collection site [8] [9], which is specific to this variety and has only been found as traces in the bark of Taxus chinensis [11]), which was used before as the starting material for the semisynthesis of Taxol and its analogs [12]. Previous studies on the Canadian yew have led to the identification of more than 50 taxanes with various skeletons [13 – 15]. Recent investigations resulted in the discovery of several new taxanes with three novel skeletons and of several taxane glycosides from the Canadian yew. In this review, we compile the work on the title plant in the past 20 years [16 – 19]. 2. Phytochemical Studies of Taxanes. – Phytochemical investigation on the Canadian yew was initiated in 1991, and the first report was published in 1992. Eight taxanes, including taxol, cephalomannine, 10-deacetylbaccatin III, 1-acetyl-10-deacetylbaccatin III, 5-decinnamate taxagifine, and three new taxanes, 2 – 4, were isolated from the needles and stems [8]. Simultaneously, 3 was also isolated by other scientist from the needles of the same plant [20]. Due to the magnetic anisotropy of the 4,20epoxy ring, HC(5) was observed at an unusual upfield resonance compared to that of 4,20-exomethylidenetaxane. Accordingly, compound 4 and some other taxanes with 4,20-epoxy groups were assigned incorrectly [21] [22]. Compound 4 was eventually revised to 5 [9]. Actually, 5 was isolated originally in trace amount from the bark of the Chinese yew, Taxus chinensis [11].

Further investigation on the needles of T. canadensis led to the isolation of 14 taxanes, including four new taxanes, 6 – 9, as well as ten known taxanes, i.e., taxol, 10deacetyltaxol, 7-epitaxol, cephalomannine, 10-deacetylbaccatin III, 7-epi-10-deacetylbaccatin III, 13-acetyl-9-dihydrobaccatin II, 5-decinnamate taxagifine, 2-deacetyl-5decinnamoyltaxinine J, and 1b-hydroxy-7,9-deacetylbaccatin I (5). The HMBC and NOE (Fig. 2) studies were carried out to establish that an Ac group was connected to C(5) and the epoxy group was b-oriented. For HC(5), the vicinal coupling constants with its neighboring HC(6) H-atoms are smaller (J ¼ 3.0 Hz), indicating that HC(5) adopted a pseudo-equatorial orientation. The NOEs observed for HC(5) were not very informative, since this H-atom interacted only with the vicinal HC(6) H-atoms and with HbC(20) [9]. All of these taxanes have similar substitution patterns only different in O-functions; these findings suggested that the esterification pattern might have a great relevance in the biogenesis of taxanes. One attractive possibility is that acetylation and benzoylation play a role in the trafficking of intermediates between cytosolic and membranous sites of biosynthesis [23].

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Fig. 2. Conformation of 4,20-epoxide taxane

During the studies on the biosynthesis of taxol (1), it was considered that geranylgeranyl diphosphate was first cyclized into a verticillene as an intermediate [24], but this bicyclic taxane was not isolated from yew trees until 1995. Zamir et al. reported the first 3,8-secotaxane (bicyclic taxane), canadensene (10), which was considered as a putative biogenetic precursor [25]. Unfortunately, the effort to transform the bicyclic taxane to tricyclic taxane failed. These results promoted us to speculate that bicyclic taxanes might have been derived from tricyclic taxanes after opening the C(3)C(8) bond. Three year later, an analog of canadensene (10), 5-epicanadensene (11), together with three 3,11-cyclic taxanes, 12 – 14, were isolated from the needle of this yew. Analogously, tetracyclic taxanes have been reported before from other yews but with some different substituents [1] [2]. It is the first time that 3,11-cyclic taxanes have been isolated from Taxus canadensis. Compound 12 was also the first compound of that series with a OH group at C(7) at that time [1] [2].

It is of great interest that canadensene (10) was the first and the only bicylic taxane with a b substitution at C(5). 5-Epicanadensene (11) was originally isolated from the Chinese yew, T. chinensis, and was erronoeusly considered as canadensene (10), although differences in both 1H- and 13C-NMR data were observed [26]. On the basis of the molecular-modeling work, the distances between the O-atoms of HOC(5) and HOC(20) were determined as 4.27 for canadensene (10) and 3.08 for the 5-


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epicanadensene (11; Fig. 3). Compound 11 seems to be more prone to cyclization to form an oxetane because of the proximity of the two OH groups. It is tempting to assume that one of them is used for the formation of a taxane, whereas the other acts as a dead-end metabolite. Preliminary studies by Zamir et al. showed that the isolation of the different stereoisomers must be dependent on the season of the plant collection. The biosynthetic puzzle is still unsolved. Why do only very few yew species (Taxus canadensis and T. chinensis needles, and Taxus cuspidata stems) produce these compounds? Why do both stereoisomers occur only in the Canadian yew? It is doubtful that the bicyclic taxanes originate from the opening of a fully oxygenated tricyclic taxane [27]; indeed, such a transformation by chemical reactions or in vivo has not been reported so far. The diversity of compounds obtained from the Canadian yew might perhaps hint to two possible pathways to form taxanes [28].

Fig. 3. Molecular models of the lowest-energy structures of canadensene (10) and 5-epicanadensene (11) illustrating the distances (in ) between the HOC(5) H-atom and the neighboring H-atoms

Further investigation on the needles (4.7 kg) of the Canadian yew resulted in the isolation of 19 known taxanes, i.e., taxuspine C, taxin B, taxinine, taxinine A, taxinine B, 10-deacetyltaxinine B, taxinine E, 10-deacetyltaxinine E, taxinine J, 2-deacetyltaxinine J, 2-deacetoxy-7-deacetyltaxinine J, 2,7-deacetoxytaxinine J, taxezopidine G, and taxuspine D, together with four new taxanes, 15 – 18. As in the other yew species, taxinine and taxinine E were found to be the major taxanes in the Canadian yew [29].

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Canadian yew has been shown to contain taxanes, which are not often found in the other yew trees. Thorough studies led to the isolation of 24 minor taxanes including 16 new compounds, 19 – 34, as well as eight known ones, i.e., taxacusin, taxtaxine, 2deacetyl-5-deamino-7,10-diacetylacyltaxine, N-debenzoyl-N-hexanoyl-7-epitaxol, 10deacetylcephalomanine, 10-deacetyl-10-oxo-7-epitaxol, 10-deacetyl-10-oxobaccatin V, and wallifoliol [10] [15] [30]. Of the new compounds, 19 represents a rare taxane with a


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C(12)¼C(13) bond, whereas both 22 and 27 are taxanes with a HOCH2 group at C(4). Compound 24 is an 11(15 ! 1)abeo-taxane with vicinal C¼O groups at C(9) and C(10), and also the first example with a BzO group at C(15). It was reported that 11(15 ! 1)abeo-taxane with two vicinal C¼O groups at C(9) and C(10) could be transformed easily to wallifoliol. Compounds 28 and 31 closely resemble to taxol (1) with exception that an Ac group replaced the Bz group in the side chain at C(13) in 28, while HOC(7) group was acetylated in 31. In vitro studies of cytotoxicity to the breast adenocarcinoma cell line MCF7 revealed that 31 is cytotoxic (IC50 10 nm), similar to cephalomannine (IC50 6 nm), and little less cytotoxic compared to taxol (IC50 2 nm). Compound 33 and 10-deacetyl-10-oxo-7-epitaxol had IC50 values of 80 and 64 nm, respectively, indicating that a C(10)¼O group reduces the bioactivity. Reinvestigation on the minor taxanes from the needles of the Canadian yew (4.0 kg) led to the discovery of 27 new taxanes, 35 – 61 [11] [31 – 34]. Among them, 39, 44, and 45 have C(4)¼C(5) bonds, while both 39 and 44 have cinnamoyloxy (CinnO) groups at C(20), and 44 had additionally a C(11),C(12)-epoxy ring. Croteau and coworkers have demonstrated that taxoid with a C(4)¼C(5) bond, not that containing a 4(20),11(12)-diene moiety, which has been proposed as one crucial intermediate in the taxane biosynthesis, is the first intermediate in the biosynthesis of taxol [1 – 4]; thus, the isolation of these compounds from the natural source will provide some clues to understand the biosynthesis and the biogenesis of taxoids in the yew trees [35 – 38]. Compound 40 had a CinnO group at C(13) with a rare (Z)-configuration. The characteristic coupling constant of HC(2’) and HC(3’) of a (Z)-cinnamoyl group is

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ca. 12.0 Hz, and their chemical shifts are shifted upfield, compared to the corresponding (E)-cinnamoyl group (HC(2’) and HC(3’) resonated at ca. 6.8 and 7.8 ppm, respectively, and the coupling constant is ca. 16.0 Hz). Compound 42 was the first example of a C(3),C(11)-cyclic taxane with a 7-epi-substituent. Compounds 52 – 54 were taxane glucosides isolated together with a known taxaneglucoside, taxa-4(20), 11-diene-2a,5a-diacetate 14b-(2’S,3’R)-3’-hydroxy-2’-methylbutyrate-10-b-glucoside, which was the first reported example of a taxane with a glucosyl substituent on ring B [39]. It should be noted that, when the sample of taxaneglucoside was solubilized in CHCl3 , it solidified as a gel giving rise to broad signals in the NMR spectrum, and two isomers exhibited two doublets at d(H) 4.35 (major peak) and d(H) 4.42 ppm (minor


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peak) for the anomeric H-atom of the sugar. However, when it was dissolved in acetone, sharp signals were, detected only one peak for HC(1’’) confirming the purity [39]. Compound 55 was the first taxane with rare conjugated C(4)¼C(20) and C(5)¼C(6) bonds. Compounds 46 and 56 – 63 were taxanes with amino-containing side chains at C(5), while 59 was a rare example of taxane with a HOC(17) group, and 62 and 63 were pseudo-alkaloid taxanes with a C(3),C(11)-cyclic 6/5/5/6 ring system and rearranged 2(3 ! 20)abeo-taxane skeleton, respectively. Although several taxanes with an aminocontaining side chain at C(5) have been reported from different yew trees [1] [2], this is the first report of their occurrence in the Canadian yew since 1992. Mass spectra can provide useful information about the difference of the amino-containing side chain at C(5) (Fig. 4).

Fig. 4. Fragmentation pattern observed in positive-ion FAB-MS/MS spectra of taxanes with different amino-containing side chains at C(5)

The bicyclic taxane canadensene (10) was first isolated from the needles of the Canadian yew in 1995. Further study on the needles yielded five additional analogs of 10. They are taxachitriene B, 2-deacetyltaxachitriene A, 13,20-dideacetyltaxachitriene A, 5-deacetyltaxachitriene B, and a new bicyclic taxane with a CinnO goup at C(20) in 64 [40]. The first example of conformational exchange in the natural taxane enol acetate 65 was reported in [41]. The structure of two stable conformers were established using a

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combination of 1D- and 2D-NMR techniques, including gs-HMQC, gs-HMBC, NOESY, and T-ROESY. The Gaussian 03 program was used to confirm the conformers deduced from the NMR experiments (Figs. 5 and 6). Taxane 65 consists of three rings. The central eightmembered ring is connected with two six-membered rings. Approximately 30 different isomeric structures resulted using simple optimization on the STO-3G level. Eighteen isomers were fully optimized using B3LYP/6-31G* basis set. The main objective for the first optimization for the 18 isomers was to determine the lowest-energy form of the three rings such as chair or boat form. The calculation indicated that the boat form had the lowest energy. On the basis of this information, we started to optimize the central ring. The two conformers with lowest energies were determined with correct axial and equatorial side-chain positions. The optimized structures of the crown (C) and boatchair (BC) forms are shown in Figs. 5 and 6, and some structural parameters are compiled in Table 1. The energy difference between the two forms was 0.00944 Hartree (0.92 and 0.22 kcal/mol). The short distance between C(11)OH and C(10)O in the BC conformer (1.74 ) is in agreement with the NMR findings that there must be a Hbond stabilizing this conformation as compared to the C form (3.11 ). As predicted by our NMR spectra, the dihedral angle between HC(9) and HC(10) is quite small (51.78) in the C form and large (156.48) in the BC form. The calculation of the eightmembered ring is in agreement with the NMR finding that there is a H-bond between HOC(11) and HOC(10) in the BC but not in the C form. The change in dihedral angle in the fragment OC(10)C(9)OAc (Table 1) suggests that the exchange between the two conformers proceeds by pseudorotation around the C(10)C(9) bond.

Fig. 5. Crown (C; left) and boat-chair (BC; right) conformations of 65

Fig. 6. Optimized structure of the C (left) and BC conformation (right) of 65. Some nuclei of Ac groups and Me(18) have been removed for better viewing.


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Table 1. Optimized Structural Parameters of the Crown (C ) and Boat-Chair ( BC) Conformations of 65. d, Interatomic distances []; f, bond angle [8]; f, dihedral angle [8]; DE, energy difference [ Hartree].

d[C(11)OH · · · OC(10)] d[C(11)O · · · HOC(10)] d[C(11)O] d[C(10)O] d[C(1)C(2)] d[C(2)C(3)] d[C(3)C(8)] d[C(8)C(9)] d[C(9)C(10)] d[C(10)C(11)] d[C(11)C(15)] d[C(15)C(1)] f [ C(11)C(10)C(9)] f [ C(10)C(9)C(8)] f [ OC(10)C(9)OAc] f [ HC(10)C(9)H )] DE

C

BC

3.11179 2.1289 1.457 1.41644 1.56351 1.55322 1.59215 1.56920 1.55604 1.580 1.5662 1.56242 126.632 126.654 166.606 51.7043 1918.4660781

1.74103 2.9257 1.43203 1.44948 1.55794 1.55334 1.57658 1.57958 1.57662 1.62315 1.58196 1.56750 126.215 119.123 148.744 156.44017 1918.4566357

A breakthrough on the studies of T. canadensis needles was the isolation and structure determination of five novel taxanes, 66 – 70, with two new skeleton types [16 – 18]. A taxane with a 6/6/8/6 ring system, 66, a taxane-derived [3.3.3] propellane, 67, as well as three taxane-derived [3.3.3] [3.4.5] dipropellanes, 68 – 70, were identified by extensive 2D-NMR studies.

A putative biogenesis of the dipropellanes was depicted in Scheme 1 with taxinine A, previously isolated from the needles of the Canadian yew [13], as a starting material. The first step would be a rearrangement of the C(4)¼C(20) bond to a more stable 3,3,4,4-tetrasubstituted compound that we named 20-deoxytaxezopidine B, by analogy with taxezopidine B with a HOC(20) group, which has been found in the seeds of the Japanese yew [42]. Abstraction of an allylic H-atom from C(20) would lead to a 3,11cyclic intermediate with an enol form on C(12)C(13). The three-dimensional structure of taxanes assures the proximity of the C(4)¼C(20) and C(12)¼C(13) bonds, causing the cyclization to form a C(12)C(20) bond and restoring a C(13)¼O group. This reaction is promoted by an oxidation of the C(4)¼C(20) bond. The last cycle

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Scheme 1. Proposed Mechanism for the Biosyntheses of Three Dipropellane Taxanes 68 – 70

involves the attack of the HOC(4) group at C(13)¼O to form a stable complex molecule as shown in Scheme 1. These compounds are the first dipropellanes isolated from a natural source. One of these propellanes [3.3.3] is based on the C(3)C(11) bond which is shared by three cyclopentanes. The second one [3.4.5] has the C(3)C(4) bond shared by a cyclopentane, a cyclohexane, and an oxacyclohexane. As expected, these novel taxanes did not exhibit cytotoxicity, and they do not have any of the bioactivities associated with the parent compound Taxol. None of the required chemical groups associated with this activity (C(2)Bz, C(4)C(5)-oxetane, and C(13)-side chain) occur in these metabolites [4]. They are additional examples of the amazing diversity of secondary metabolites in nature. Studies on the rooted cuttings of Canadian yew resulted in the isolation of three new, i.e., 71 – 73, and 26 known taxanes [43]. The known taxanes were identified as taxuspine F, baccatin III, 5-decinnamoyltaxinine B 11,12-oxide, 5a-decinnamoyltaxinine J, taxayuntin, taxuspine W, baccatin IV, taxinine M, 2a,7b,9a-trideacetyl-1bhydroxybaccatin I, baccatin VI, 13a-acetyl-13a-decinnamoyltaxchinin B, taxuspine L, 7b,13a-dideacetyl-9a,10b-didebenzoyltaxchinin C, taxinine NN-3 (9a-deacetyltaxinine), paclitaxel, 10b-deacetylcephalomanine, 10b-deacetylbaccatin III, 13a-acetyl-9dihydrobaccatin III, taxinine, 2-deacetoxytaxinine J, cephalomannine, 5-decinnamoyltaxuspine D, taxinine A, 5-epicanadensene, 2a-deacetyl-5a-decinnamoyltaxinine J, and 5a-decinnamoyltaxigifine. Compound 71 is a rare 1-hydroxylated 3,11-cyclotaxane. It is interesting to note that HC(2) exhibited a long-range 1H,1H-COSY correlation with HbC(14) (J ¼ 1.8 Hz) in 1-hydroxylated 3,11-cyclotaxanes. The relative amount of these metabolites in the needles of the mature Canadian yew and in the rooted cuttings is surprising. In the mature Canadian yew, 13-acetyl-9-dihydrobaccatin III (3) is the most abundant metabolite, followed by taxinine and taxinine E. The three major metabolites in rooted cuttings of the Canadian yew are 5-decinnamoyltaxagifine, 10deacetylbaccatin III, and paclitaxel. The role of these secondary metabolites in the plant or in the rooted cuttings is presently unknown, but the differences between the metabolites from these various sources is intriguing.


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Further work on the Canadian yew, Taxus canadensis, led to the isolation of a novel taxane with an unique 5/5/4/6/6/6 ring system, i.e. the unique [3.3.2]propellane 74 [44].

Compound 74 represents the first example of a novel C-atom framework with a rare 5/5/4/6/6/6 ring system, which represents the most complex skeleton of all natural taxanes [1 – 3]. The proposed hexacyclic structure embodying a unique [3.3.2]propellane ring system is entirely consistent with its spectral properties, and is chemically and biogenetically plausible. A biosynthetic pathway from 9-deacetyltaxinine A [32] to 74 was proposed in Scheme 2. It should be emphasized that intermediate C has a 6/8/6/6 tetracyclic skeleton, displaying a cage-like backbone, and a similar taxane has been isolated from this plant [16]. The C(3)¼C(4) and C(11)¼C(12) bonds were spatially close, and [2 þ 2] cycloaddition could occur to form the cyclobutane ring. The cooccurrence of the novel type taxane 74 and intermediate C implies that the former should be biosynthesized from a tetracyclotaxane-type precursor. The C-skeleton of 74 was a new addition to the architectural diversity of the taxane family. Scheme 2. Hypothetical Biosynthetic Route from 9-Deacetyltaxinine A to 74

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Rooted cuttings are used in garden nurseries for the production of ornamental yews. Usually, it is assumed that the taxane composition of rooted cuttings would provide insight on their biosynthesis in the plant. The taxane composition and their amounts, however, are very different when comparing rooted cuttings and needles of the same Canadian source, Taxus canadensis. Further investigation on the rooted cuttings of Taxus canadensis resulted in the isolation of a new taxane alkaloid with an N-formyl group in the side chain in 75 [45]. It should be noted that the side chain displayed signals in duplicate in the 1H- and 13C-NMR spectra, including 2D-NMR spectra, in CDCl3 . These spectra revealed that, in CDCl3 solution, 75 existed as a mixture of two rotamers, 75/76, in a ratio of ca. 2 : 1. Therefore, each of the signals of the two rotamers was assigned individually by interpretation of the 2D-NMR spectra (COSY, HSQC, and HMBC). Taxane 75 is the first example of an taxane alkaloid with a CHO instead of a Me group at the N-atom and exhibited cis- and trans-rotamers to the N-Me group. Recently, another new taxane alkaloid, 77, with an N-formyl-containing side chain was characterized from the rooted cuttings of Taxus canadensis [46] Further studies on Canadian yew have led to the isolation of a novel taxane with a unique 6/5/5/6/4/5 ring system ([3.3.2]propellane), named taxpropellane (78) [47].

Compound 78 was the first taxane with a novel 6/5/6/4/5 ring system, one of the most complex skeleton cores among the natural taxanes. It is unique in that it possesses an unusual hemiketal ring between C(13) and C(20), and C(4) is connected to C(12), and C(3) to C(11). The proposed hexacyclic structure containing a unique [3.3.2]propellane ring system is entirely consistent with its spectral properties, as well as chemically and biogenetically plausible [5]. A hypothetical biogenetic pathway from 9-deacetyltaxinine A to canataxapropellane B was proposed in Scheme 3. It should be emphasized


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Scheme 3. Hypothetical Biogenetic Route from 9-Deacetyltaxinine A to 78

that structure C in Scheme 3 has a 6/8/6/6 ring system, which showed a cage-like backbone, and a similar taxane has been isolated from this plant, corresponding to a key intermediate in the biogenensis of canataxapropellane B. The C-skeleton of 78 represents a new addition to the architectural diversity of the taxane family and highlights the rich variety of metabolites found within the genus Taxus. This compound is of interest for its biogenetic pathway. On the other hand, these canataxapropellanes are considered as chemotaxonomic markers for T. canadensis. The bioactivity of 78 was tested; unfortunately, no cytotoxicity was observed, and further investigations are not planned. Recent investigations on the needles of T. canadensis resulted in the isolation of new compounds, namely, 9a,10b-diacetoxy-5a-(cinnamoyloxy)-13(17)-epoxy-2a,13adihydroxytaxa-4(20),11-diene (79) and 2a,10b-diacetoxy-5a-(cinnamoyloxy)-13(17)epoxy-9a,13a-dihydroxytaxa-4(20),11-diene (80) [48]. A new taxane with an aminocontaining side chain at C(5) and a new 11(15 ! 1)abeo-taxane with a tetrahydrofuran ring composed of C(2), C(3), C(4), and C(20) were identified for the first time from the needles of the Canadian yew, Taxus canadensis [49]. Their structures were characterized as 2a,7b,9a,10b,13-pentaacetoxy-11b-hydroxy-5a-{[3-(dimethylamino)2-hydroxy-3-phenylpropanoyl]oxy}-11b-hydroxytaxa-4(20),12-diene (81) and 13a,20bdiacetoxy-2a,20-epoxy-5a,7b,9a,10b-tetrahydroxy-11(15 ! 1)abeo-taxa-11,15-diene (82) on the basis of 1D- and 2D-NMR evidence, and HR-FAB-MS analysis. Taxane 81 contains a rare C(12)¼C(13) bond and a basic side chain, while taxane 82 bears a rare isopropenyl group at C(1). Two new 11(15 ! 1)abeo-taxanes with tetrahydrofuran rings along C(2), C(3), C(4), and C(20) were also identified for the first time from the needles of the Canadian yew, Taxus canadensis [50]. The structures were characterized as 4a,10b,13a-triacetoxy-15-(benzoyloxy)-2a,20b-epoxy-11(15 ! 1)abeo-tax-11-ene5a,7b,9a-triol (83) and 4a,7b,9a,10b,15-pentaacetoxy-2a,20b-epoxy-11(15 ! 1)abeotax-11-ene-5a,13a-diol (84) on the basis of 1D- and 2D-NMR evidence, and HR-FABMS analysis. Compound 83 showed weak growth inhibitory activities against T-98 and MM1-CB cells in vitro [50].

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Two 3,11-bicyclic taxanes, 85 and 86, were isolated from the needles of Canadian yew [51]. Compound 86 bears a rare (Z)-cinnamoyl moiety at C(5). A new 6/8/6 ring system taxane was isolated from the needles of Taxus canadensis. The structure was characterized as 10b,13a-diacetoxy-5a,9a-dihydroxytaxa-4(20),11-diene (87) [52]. A new taxane peroxide, 4a,10b,13a-acetoxy-2a-(benzoyloxy)-7b,9a-epidioxy-5b,20-epoxytax-11-en-1b-ol (88) [53], was isolated and characterized for the first time in rooted cuttings of the Canadian yew. The 1H-NMR data of 88 resembled closely those of 3 (13acetyl-9-dihydrobaccatin III) except that HC(2) and HC(3) displayed little upfield shifts for 88. As the original NMR spectra of 88 and 3 were recorded in CDCl3 solution, to address whether HOC(7) and HOC(9) were involved in a peroxide ring, we recorded NMR data in (D6 )acetone, and signal of HOC(1), and not of HOC(7) and HOC(9) were observed for 88. On the other hand, signals corresponding to all of three free OH groups were observed for 3. A novel taxane with 6/8/6 ring system and a rare C(12)¼C(13) double bond, and a rare 2(3 ! 20)abeo-taxane with a C(13)¼O group were isolated from the needles of Taxus canadensis. Their structures were characterized as 7b,9a,10b-triacetoxytaxa4(20),12-diene-2a,5a,11b-triol (89) and 2a,7b,10b-triacetoxy-5a-hydroxy-2(3 ! 20)abeo-taxa-4(20),11-diene-9,13-dione (90) [54]. Most recently, a novel 6/12-ring bicyclic taxane with a 13,17-ether bridge, named canataxpyran A, was isolated from the needles of Taxus canadensis. Its structure was elucidated as (3E,8E)-7b,9,10b,20-tetraacetoxy-13b,17-epoxy-3,8-secotaxa-3,8,11-triene-2a,5a-diol (91). This bicyclic taxane gradually decomposed in CDCl3 to give the corresponding enones, i.e., (2E,7E)-10b,20-diacetoxy-13b,17-epoxy-4a,5a-dihydroxy-


1745

3,8-secotaxa-2,7,11-trien-9-one (canataxpyran B; 92) and (2E,7E)-5a,10b,20-triacetoxy-13b,17-epoxy-4a-hydroxy-3,8-secotaxa-2,7,11-trien-9-one (canataxpyran C, 93). Compound 91 is the first example of a natural 3,8-secotaxane with 13,17-O-bridge [55]. Unexpectedly, 91 was converted gradually to two products 92 (major) and 93 (minor) in CDCl3 during NMR experiments. The 2D-NMR spectrum of 91, kept for 2 d, indicated that most of 91 was converted to 92 and 93 (Scheme 4). Extensive NMR analysis of the solution revealed that these compounds were a,b-unsaturated ketones, one of the two major types of natural 3,8-secotaxanes. This would result from the elimination of the enol acetate moiety in 91. Under acidic conditions of CDCl3 , deacetylation at C(9) by nucleophilic attack of H2O (a) or 5-OH group (b), concomitant with the C¼C bond migration and elimination of 7-AcO group, formed Scheme 4. Transformations of 91 to 92 and 93

1746


the 7-en-9-one moieties. 9-O-Ac group migrated to 5-O-position in 93. In addition, migration of the (E)-C(3)¼C(4) bond to (E)-C(2)¼C(3) (d(C) 132.9 (C(2); 92) and 133.5 (C(3); 93) and hydroxylation at C(4) (d(HC(5) and d(HC(20)) were shifted upfield), i.e., allylic rearrangement, were observed for both 92 and 93. This skeleton has not been found in nature. The difference between 92 and 93 was in the chemical shift of HC(5) (d(H) 3.95 for 91 and 5.19 for 93), indicating 93 was 5-O-deacetyl derivative of 92. The structures of 92 and 93 were mentioned above and named canataxpyrans B and C, respectively. These transformations would demonstrate the biogenetic relationship of two representative 3,8-secotaxanes, from 3,8-secotaxa-3,8,11-trienes to 3,8-secotaxa3,7,11-trien-9-ones [55]. 3. Chemical Modifications of Taxanes. – Some chemical modifications on the taxanes were also carried out in our laboratory. 13-Acetyl-9-dihydrobaccatin III (3) was the major taxane isolated from the needles of Canadian yew, which is 5 – 7 times more abundant than taxol (1) in the bark of T. brevifolia. Different acidic rearrangements of 3 led to different abeo-taxanes, 94 and 95 (Scheme 5) [56] [57]. Scheme 5

Treatment of 13-acetyl-9-dihydrobaccatin III (3) with DMP/CSA, NaBH4 , Joness reagent, and MeONa sequentially resulted in the formation of a new taxane derivative, 97, with a novel skeleton in which ring A was opened, and ring C remained intact (Scheme 6) [58]. It was reported that taxuspine D, a taxane isolated from the Japanese yew needles, was found to promote the polymerization of tubulin with a potency corresponding to half of the activity of paclitaxel (1) [59] [60]. This result was surprising, since taxuspine D lacked all the key features essential for bioactivity: a side chain at C(13), a HOC(20) moiety, a BzO group at C(2), and an oxetane ring along C(4)C(5) [61]. Molecular-modeling studies indicated that the C(12)¼C(13) bond caused a substantial change in the conformation of the core skeleton, and the C(5)-cinnamoyl in taxuspine D was found to mimic part of the side chain at C(13) of paclitaxel (1) [62]. To further


1747

Scheme 6. New Skeleton 97 Formed from 13-Acetyl-9-dihydrobaccatin III (3)

test this hypothesis, different taxane core skeletons were constructed with side chains situated elsewhere than at C(13). The rearrangement reactions of a taxinine derivative, 7b,9a,10b-triacetoxy-5a-hydroxytaxa-4(20),11(12)-dien-13-one, when treated with activated Zn in glacial AcOH, was studied [63]. Taxanes with C(3)¼C(4) or C(10)¼C(11) bonds, as well as a C(11)C(12) epoxide were obtained (Scheme 7). Scheme 7

1748


In an attempt to rank the priority of compounds to be synthesized, a database of 28 virtual compounds was built, including the known bioactive compounds paclitaxel (1), docetaxel, and taxuspine D. The database was docked against the refined structure of btubulin using the DOCK program. According to the intermolecular energy dock score (Table 2), compounds 106 ( 38.4 kcal/mol) and 107 ( 36.1 kcal/mol) were predicted to be bioactive, since their dock scores were similar to that of docetaxel ( 37.3 kcal/ mol). In addition, compounds 108 ( 33.5 kcal/mol) and a virtual analog of compound 102 (Scheme 8), wherein the Cinn group had been replaced by the paclitaxel side chain, i.e., compound 109 ( 33.0 kcal/mol), had a dock score similar to that of taxuspine D ( 33.8 kcal/mol) and, therefore, were predicted to be bioactive. However, after synthesis of compounds 106, 107, and 108, the tubulin assembly assay revealed no bioactivity at 10-mm concentrations. This prompted us to investigate the reason for the false positives in the docking study. It is well-known that solvation free energies are omitted in the DOCK scoring function, although they can be taken into account implicitly. The solvation free energy was, therefore, calculated using the AM1SM5.4PDA model. To accelerate the process, only single-point calculations were performed on the docked conformations. Consequently, the changes in conformational energy on binding were omitted. The results compiled in Table 2 clearly show that solvation free enegies are an important factor in ligand binding. As indicated by the corrected dock scores, compounds 106 ( 7.4 kcal/mol), 107 ( 8.1 kcal/mol), and 108 ( 7.1 kcal/mol) should be inactive, since their corrected dock scores were much lower than that of taxuspine D ( 12.3 kcal/mol). Interestingly, the ranking of taxane 109 ( 14.5 kcal/mol) remained above that of taxuspine D after the correction of the solvation free energy. This virtual compound has a C(10)¼C(11) bond and a paclitaxel side chain at C(5). A multidisciplinary approach combining bioorganic mechanisms, synthetic chemistry, and molecular modeling led to interesting taxane structures with C(3)¼C(4) and C(10)¼C(11) bonds or with a C(11),C(12)-epoxy moiety. Introduction of side chains situated elsewhere than at C(13) produced very different conformations which may act on different cell targets. Table 2. Intermolecular-Energy Dock Score, the Solvation Free Energy (DGsolv ), and the Corrected Dock Score (kcal/mol) Compound

Dock score

DGsolv

Corrected dock score

Paclitaxel (1) Docetaxel Taxuspine D 106 107 108 109

44.4 37.3 33.8 38.4 36.1 33.5 33.0

26.1 22.5 21.5 31.0 28.0 26.4 18.5

18.3 14.8 12.3 7.4 8.1 7.1 14.5

Both taxuspine D and taxagifine have modified rings A, the former has a C(12)¼C(13) bond, while the latter has a C(16)C(12) ether linkage and a CinnO group at C(5), and both promoted the polymerization of tubulin but to ca. 1/3 that of paclitaxel (1) [64 – 66]. To test if the core skeleton of the taxane – that is a six-


1749

Scheme 8

a) AcCl, Pyridine, 48, 18 h; 104: 59%, 105: 36%. b) DCC, DMAP, CH2Cl2/toluene, 758, 5 h. c) TsOH, MeOH, 238; 106: 57%, 107: 58%. d) DCC, DMAP, CH2Cl2/toluene, 48, 18 h. e) TsOH, MeOH, 238, 18 h; 108: 58%.

membered ring fused to two eight- and six-membered rings – was essential, the natural canadensene analogs as well as a synthetic canadensene analog 110 were investigated (Scheme 9). It is assumed that the bicyclic structure of 5-epicanadensene (11), being very flexible, would enable the side chain at C(20) to reach the active site of b-tubulin, a known target of paclitaxel. Tubulin polymerization activities were investigated for 5epicanadensene (11), 20-(cinnamoyloxy)-5-epicanadensene (64), and docecanadensene (110) compared with Taxol (1). Whereas Taxol (1) induced the initial rate of tubulin polymerization almost twice as much as the control, the analogs only showed activities comparable to that of the control. We tested the in vitro cytotoxicities of taxanes 11, 64, and 110 against breast cancer cell lines MCF7 and MCF7-ADR (adriamycin-resistant), as compared with that of Taxol (1; Table 3). The IC50 values of

1750


Scheme 9

a) DCC, DMAP, CH2Cl2/toluene, 238, 2 h. b) TsOH, MeOH, 238, 45 min; 110: 67%. Table 3. Cytotoxicity of Canadensene Analogs (tumor cell cytotoxicity IC50 [nm] ) a ) Compound

MCF 7-Wild type

MCF 7-ADR

Taxol (1) 11 64 110

2 1.52 750 5.38 655 1.42 740 3.99

> 5 b) 645 3.51 825 2.14 882 3.26

a

) Cytotoxicity of taxanes on the breast cancer cell lines MCF 7-wild type and MCF 7-ADR (adriamycin resistant). Exponentially growing cells were continuously exposed to taxanes at varying concentrations, and cell viability was evaluated by the MTT ( ¼ 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) metabolic assay. Data represent mean SEM of three separate experiments. IC50 is defined as the concentration of agent required to inhibit cell proliferation to 50% vs. untreated cells. b ) In mm.

compounds 11, 64, and 110 were comparable, ca. the 700 nm for the MCF7-wild-type cell line. However, Taxol (1) was more potent, as its IC50 value was in the 2-nm range. Similarly, for the MCF7-ADR cell line, the IC50 values for compounds 11, 64, and 110 were 645, 825, and 882 nm, respectively, compared with the IC50 value for 1, i.e., 5 mm. Unfortunately, these canadensene analogs 11, 64, and 110 were inactive in both the tubulin and the two cytotoxicity assays. 4. Concluding Remarks. – During the past 30 years, Taxus plants have constituted excellent targets for phytochemical investigation and led to the isolation of more than 550 taxane diterpenoids [67]. Now, it is hoped that continuing interest in structure modulation of these metabolites may provide novel potential medicinal agents. Of the taxanes isolated from the needles of Canadian yew, 6/8/6 ring system with exo-4(20)CH2 taxanes constituted the major components, with 6/8/6 ring systems with 20,5oxetane taxane and 6/5/5/6 ring system (3,11-cyclic) taxanes being less common. Approximately 20 new taxanes carried CinnO groups at C(5). The 6/8/6 ring systems with 4(20)-epoxy taxane, 11(15 ! 1)abeo-taxane and 2(3 ! 20)abeo-taxane were rather


1751

rare in the needles of this yew tree. On the other hand, two new taxanes with 12,13epoxy moieties and two new taxanes with C(12)¼C(13) bonds, both of them rare in the other yews [1 – 4] [67], were isolated from Canadian yew. Taxanes with propellane and dipropellane skeletons were only found in Canadian yew so far. It is of great interest to note that Canadian yew is the only species producing 13-acetyl-9-dihydrobaccatin III (3) as the most abundant taxane [8] [9] [20], which, as a starting material for the semisynthesis of Taxol (1), was only found in the Chinese yew as a trace component [11]. Bicyclic taxane canadensene (10) [25] was first isolated from this yew tree, and almost at the same time this bicyclic taxane was also reported from Chinese yew [68]. Interestingly, canadensene (10) was the first and the only taxane with a 5b-substituent. Molecular-modeling studies indicated that 10 adopted a striking U-shape in the 3Dmodel [28]. Three new taxanes were found that bear glycolic substituents C(O)CH2OH or C(O)CH2OAc (which was very rarely encountered in the other yews [1 – 4] [67]) at C(10), instead of a usual Ac or OH groups in Canadian yew. Such a difference may serve as a characteristic feature to distinguish Canadian (T. canadensis) from other yews. Further study is warranted to determine whether this plant belongs to a different chemotype. The needles of Canadian yew are also a good resource for Taxol (1), the content of which was higher (0.0477%) than in any other yew plants [69]. Relatively few bioassays were carried out for the isolated taxanes from this yew. Other compounds such as sesquiterpenoids, flavonoids, lignans, ecdysteroids, etc., which were reported from other yews, were rarely investigated. The authors gratefully acknowledge the financial support from the National Natural Science Foundation of China (81072551 and 81241101) and the Key Projects of Science & Technology of Hebei Province (11276103D-89). We also wish to extend our sincere thanks for the financial support from Syngenta Ltd. (2011-Hebei Medical University-Syngenta-03).

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[Studies on the backward-reactions in the xanthophyll-cycle of Chlorella, Spinacia and Taxus].

Chemical studies on a hamster sublingual glycoprotein.

Chemical composition and antimicrobial activity of solidago canadensis linn. Root essential oil.

Diuretic Properties and Chemical Constituent Studies on Stauntonia brachyanthera.

Chemical studies on the polysaccharides of Salicornia brachiata.

Chemical studies on tuberactinomycin. XV. Total synthesis of tuberactinomycin O.

Studies on residue-free decontaminants for chemical warfare agents.

Studies on the physico-chemical characteristics of polymorph migration stimulator.

Periodic quantum chemical studies on anhydrous and hydrated acid clinoptilolite.

Chemical and microbiological studies on leachates from a waste tip.

Comparative studies on microbial and chemical modifications of trichothecene mycotoxins.

Immunological and chemical studies on sub-fractions of carcinoembryonic antigen.

Quantum chemical studies of methadone.

Comparative proteomic analyses of two Taxus species (Taxus × media and Taxus mairei) reveals variations in the metabolisms associated with paclitaxel and other metabolites.

Chemical composition and anti-herpes simplex virus type 1 (HSV-1) activity of extracts from Cornus canadensis.

Spontaneous pasteurellosis in captive Rocky Mountain bighorn sheep (Ovis canadensis canadensis): clinical, laboratory, and epizootiological observations.

Psoroptic scabies in Rocky Mountain bighorn sheep (Ovis canadensis canadensis) from Wyoming.

A fatal case of Taxus poisoning.

Organogenesis in Taxus brevifolia tissue cultures.

Phenolic constituents of Taxus baccata leaves.

New Taxane Diterpenoids from Taxus yunnanensis.

Taxus spp. needles contain amounts of taxol comparable to the bark of Taxus brevifolia: analysis and isolation.

Quantum chemical studies of meperidine and prodine.

Chemical studies of enzyme active sites.