Bioactive taxoids from the Japanese yew Taxus cuspidata

BioactiveTaxoids from theJapaneseYewTaxus cuspidata

Jun’ichi Kobayashi, Hideyuki Shigemori

Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, Japan

!

Abstract: A series of new taxoids, named taxuspines A–H and J–Z (1–25) and taxezopidines A–H

and J–L (26–36), have been isolated together with 37 known taxoids (37–73) including paclitaxel

(53) from the Japanese yew, Taxus cuspidata Sieb. et Zucc. (Taxaceae). These new taxoids possess

various skeletons containing 5/7/6, 6/10/6, 6/5/5/6, 6/8/6, or 6/12-membered ring systems. Among

the new taxoids, some non-taxol-type compounds remarkably reduced CaCl2-induced depoly-

merization of microtubules, or increased cellular accumulation of vincristine in multidrug-

resistant tumor cells as potent as verapamil. On the other hand, chemical derivatization of taxinine

(37), one of major taxoids obtained from this yew, led to the discovery of unusual reactions of

taxinine derivatives. Here we describe our recent results on the isolation, structure elucidation,

and bioactivity of these new and known taxoids and the formation of unexpected products of the

unusual reactions of taxinine. � 2002 Wiley Periodicals, Inc. Med Res Rev, 22, No. 3, 305–328, 2002;

Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/med.10005

Key words: taxoids; Taxus cuspidata; microtubule depolymerization; multidrug resistance;

unusual reactions

1 . I N T R O D U C T I O N

Since the discovery of the promising anticancer activity of paclitaxel (53) and some related

compounds, chemical studies on constituents of different yew trees have resulted in the isolation of a

large number of new taxoids.1–3 Paclitaxel (53) exhibits potent antitumor activity against different

cancers which have not been effectively treated by existing antitumor drugs. This is due to the

unique activity of paclitaxel (53) that it inhibits remarkably microtubule depolymerization process.

Many researchers have reported that an N-acylphenylisoserine group at C-13 and an oxetane ring

at C-4 and C-5 play important roles for this unique activity of paclitaxel (53). Recent reports4 on

clinical trials of paclitaxel (53), however, have disclosed that this hightly effective drug is inac-

tive against colon cancer and renal cell carcinoma, which are originated from tissues that express

constitutively the MDR1 gene. Thus, the observed lack of activity against these tumors could be

partly due to the fact that this drug is subjected to multidrug-resistance (MDR).4,5 In our search for

305

Correspondence to: Jun’ichi Kobayashi.E-mail: [email protected]

Contract grant sponsor:Ministryof Education,Science,Sports, andCulture of Japan.

Medicinal Research Reviews, Vol. 22, No. 3, 305^328, 2002

� 2002 Wiley Periodicals, Inc.

new bioactive taxoids, we examined extracts of the Japanese yew Taxus cuspidata Sieb. et Zucc.

(Taxaceae) and obtained new taxoids, taxuspines A–H and J–Z (1–25) and taxezopidines A–H and

J–L (26–36),6–16 together with known taxoids (37–73)17 containing paclitaxel (53) and paclitaxel-

type compounds (54–61) with an N-acylphenylisoserine group at C-13 and an oxetane ring at C-4

and C-5. Among the taxoids, non-paclitaxel-type compounds such as taxuspine D (4), taxezopidine

K (35), taxezopidine L (36), and taxagifine (49) were found to remarkably reduce CaCl2-induced

depolymerization of microtubules. On the other hand, some non-paclitaxel-type taxoids incre-

ased cellular accumulation of vincristine (VCR) in MDR tumor cells, while paclitaxel (53) and

paclitaxel-type compounds did not show such an activity. Taxuspine C (3), a non-paclitaxel-type

compound isolated from this yew, increased the cellular accumulation of VCR in multidrug-

resistant tumor cells as potently as verapamil enhanced the chemotherapeutic effect of VCR in

P388/VCR-bearing mice. On the other hand, from the chemical derivatization of taxinine (37),

one of major taxoids obtained from this yew, we found unusual reactions of taxinine derivatives.

The present review provides our recent results on the isolation, structure elucidation, and bioactivity

of these new taxoids and the formation of unexpected products of the unusual reactions of taxinine.

2 . I S O L A T I O N A N D S T R U C T U R E S O F N E W T A X O I D S

The methanolic extract of stems, leaves, and seeds of T. cuspidata collected at Sapporo, Japan

was partitioned between toluene and water, and then aqueous layer was partitioned with CHCl3.

The toluene and CHCl3 soluble portions were subjected to a silica gel column followed by reversed-

phase and silica gel column chromatographies and centrifugal partition chromatography to afford

taxuspines A–H and J–Z (1–25) and taxezopidines A–H and J–L (26–36) together with known

taxoids (37– 73) (Fig. 1).

Figure 1.

306 * KOBAYASHI AND SHIGEMORI

A. 11(15!1)-abeo-Taxanes (5/7/6-Membered Ring System)

Taxuspine A (1), C42H48O11, was isolated in 0.0017% w/w yield from stems of the Japanese yew.

Detailed analyses of the 2D NMR data (1H-1H COSY, HMQC, and HMBC) led to the structure of 1which was constructed from a 5/7/6-membered ring system with a hydroxyisopropyl, a cinnamoyl,

Figure 1. (Continued)

TAXOIDS FROM THE JAPANESE YEW * 307

a benzoyl, and three acetyl groups. The relative stereochemistry of a hydroxyisopropyl, a cinna-

moyl, three acetyl, and a benzoyl groups at C-1, C-5, C-7, C-9, C-10, and C-13 and the ring

conformation in 1 was elucidated on the basis of the NOESY data and 1H-1H coupling constants

(Fig. 2). Taxuspines J (9), M (12), O (14), and Y (24) were also assigned as 11(15!1)-abeo-taxanes

on the basis of 2D NMR data. Taxuspines J (9) and M (12) were 10-debenzoyl-10-acetyltaxuspine

A and 10-debenzoyltaxuspine A, respectively, while taxuspines O (14) and Y (24) contained a

carbonyl group at C-13. Taxuspine Q (16) was also an 11(15!1)-abeo-taxane with an oxetane ring

at C-4 and C-5 and a tiglic acid group at C-10.

B. 2(3!20)-abeo-Taxanes (6/10/6-Membered Ring Systems)

Taxuspine B (2), C35H42O10, was obtained in 0.00097% w/w yield, and its structure consisting of a

6/10/6-membered ring system with a cinnamoyl, a carbonyl, and three acetyl groups was elucidated

by applying several types of 2D NMR techniques. The relative stereochemistry of the functional

groups and the ring conformation in 2 were elucidated on the basis of the NOESY data and 1H-1H

coupling constants (Fig. 2). Taxuspine W (22) was assigned as a 5-decinnamoyltaxuspine B by

NMR data, possessing a marked caged conformation with an hydrogen bonding between the

hydroxy group at C-5 and the acetyl group at C-13.

C. 3,11-Cyclotaxanes (6/5/5/6-Membered Ring System)

Taxuspine C (3), C35H42O9, was obtained in 0.0017% w/w yield. The structure of 3 was interpreted

by the extensive analysis of its spectroscopic data and determined to be composed of a 6/5/5/

6-membered ring system containing a cinnamoyl, a carbonyl, and three acetyl groups. Relative

stereochemistry of 3 was elucidated by the NOESY spectrum and 1H-1H coupling constants (Fig. 2).

The absolute stereochemistry of 3 was established by the following photochemical reaction18;

Figure 2. Stereostructures of taxuspines A�D (1� 5) and X (23) (Dottedarrowsdenote NOESYcorrelations).


irradiation of taxinine (37), of which absolute stereochemitry has been determined by X-ray

analysis,19 in dioxane with an Hg-lamp gave rise to compounds 3 and 22-Z-form of 3 (Fig. 3). All

spectral data ([a]D, 1H and 13C NMR, IR, UV, and EIMS) of 3 derived from taxinine (37) were

identical with those of taxuspine C (3). Thus, the absolute stereochemistry of taxuspine C (3) was

concluded to be the same as that of taxinine (37). The rearrangement, which formally involves a

hydrogen transfer from C-3 to C-12 and bond formation between C-3 and C-11, might be a triplet

reaction. A concerted ss2þ ps

2 route was proposed for the photochemical reaction.18 Taxuspine

H (8) was elucidated as a 5-decinnamoyl-5-(3-N,N-dimethylamino-3-phenylpropanoyl)taxuspine C

by NMR data and photochemical reaction from taxine II (41) into 8.

D. Taxanes (6/8/6-Membered Ring System)

Most taxoids have a 6/8/6-membered ring system. A known major taxoid, taxinine (37),20 has been

isolated in 0.041% w/w yield from the Japanese yew, T. cuspidata Sieb. et Zucc. The structures of

taxuspines F (6), G (7) and taxezopidines C (28), D (29), E (30) were elucidated by comparison of

the NMR data with taxinine (37). These compounds have a cyclohexenone (ring A) and C-4(20)-

exomethylene. Taxuspines D (4), P (15), and taxezopidine K (35) are the first examples of taxoids

involving an enol acetate moiety in ring A. The conformation of ring B in these compounds was

different from those of other taxoids having a 6/8/6-membered ring system, judging from

comparison of the 1H-1H coupling constants (J9,10¼ 4.4–4.8 Hz) of 4, 15, and 35 with those

(J9,10 ¼ ca. 10 Hz) of other taxanes such as taxinine (37) and 38–47 (Fig. 2 and 4). Taxuspines L (11)

and R (17) were rare example of taxoids involving a hydroxymethyl group at C-4. Taxuspine K (10)

was the first example of taxoids containing a 6/8/6-membered ring system with a tetrahydrofuran

ring at C-2, C-3, C-4, and C-20, while it has been reported that taxoids containing an oxetane ring

at C-4 and C-5 could be converted into taxoids with a tetrahydrofuran ring at C-2, C-3, C-4, and

C-20.21 A chair-like conformation of ring B in 10 was deduced from the coupling constant (4.5 Hz)

between H-9 and H-10 and NOESY correlations. Taxuspines S (18) and T (19), and taxezopidine

L (36) have a tetrahydrofuran ring in ring A, which were similar to taxagifine (49). The confor-

mation of ring B in 18, 19, and 36 was similar to those of taxuspines D (4) and P (15), judging from1H-1H coupling constant (J9,10¼ 2.8 Hz for 18, J9,10 ¼ 3.8 Hz for 19, and J9,10¼ 3.2 Hz for 36) and

Figure 3. Photochemical reactionof taxinine (37).


NOESY correlations. Taxuspines E (5) and N (13) possessed an oxetane ring between C-4 and C-5

whose structures were similar to that of 10-deacetylbaccatin III (70), while 5 and 13 differed in the

functional group at C-9 (a hydroxy and an acetoxy group for 5 and 13, respectively; a carbonyl

group for 10-deacetylbaccatin III (70). Taxuspine N (13) was the first example of taxoids containing

a 6/8/6-membered ring system with a 3-N,N-dimethylamino-3-phenylpropanoyl group at C-13 from

Taxus species. Taxuspines H (8), N (13), P (15), and Z (25) contained a 3-N,N-dimethylamino-3-

phenylpropanoyl group (Winterstein’s acid). The absolute stereochemistry of this group was

determined to be all R by chiral HPLC analysis (Sumichiral OA-5000) of the acid hydrolysates of

taxuspines H (8), N (13), P (15), and Z (25). The structure of taxuspine V was assigned to be 21 by

NMR data and acetylation of 21 into 1b-hydroxybaccatin I (48).22 Taxezopidines A (26) and J (34)

were only two example of taxane diterpenes containing an oxabicyclo[2.2.2]octene moiety from

yew trees. It was noted that 26 and 34 have a cage-like backbone conformation similar to usual

taxoids consisting of a 6/8/6-membered ring system. Taxezopidine B (27) was the first taxoid with

a double bond at C-3 and C-4.

E. Bicyclic Taxane Related Diterpenes (6/12-Membered Ring System)

Taxuspine U (20), C28H40O11, was isolated in 0.000017% w/w yield from stems of the Japanese

yew. Analysis of the 1H and 13C NMR data and HMQC spectrum of 20 provided four acetyls, five

oxymethines, one oxymethylene, one trisubstituted olefin, two tetrasubstituted olefins, and four

methyl groups. Since seven out of nine unsaturations were accounted for, 20 was inferred to

contain two rings. Detailed analysis of the 2D NMR data (1H-1H COSY, HMQC, and HMBC) led

to the structure of taxuspine U (20) as constructed from a 6/12-membered ring system. NOESY

correlations (Fig. 5) of H-1/H-14a, H-1/H3-17, H-13/H-14a, and H-13/H3-17 indicated a boat

conformation of ring A, while those of H-1/H-2 and H-2/H3-16 implied that a hydroxy group at C-2

was a-oriented. The a-orientation of H-10 was deduced from a NOESY correlation of H-10/H3-18.

The relative stereochemistries at C-5 and C-7 were investigated by combination of the NOESY data

Figure 4. Three-dimensional structures of 4, 37, and 49byMM2calculation.


and molecular mechanics calculations, in which four diastereomers (20a, 5S*7S*; 20b, 5S*7R*;

20c, 5R*7S*; 20d, 5R*7R*) were considered, and systematic conformational searching23 for each

diastereomer was carried out by using Macromodel program.24 The calculation of the distances

between H-7/H-10, H-7/H3-18, and H-5/H-20b of conformational isomers within 3 kcal/mol for

each diastereomer (20a*20d) was summarized in Table I. The distances of H-7/H-10, H-7/H3-18,

and H-5/H-20b for 20a were within 3.0 A, while those of 20b*20d were over 3.0 A. These results

suggested that relative stereochemistry of ring B in taxuspine U (20) was 2R*, 5S*, 7S*, and 10R*

(Fig. 5). The structure of taxuspine X (23) was similar to that of taxuspine U (20) except for acetyl

groups at C-2, C-7, and C-20 and a cinnamoyl group at C-5 in 23. It was easy to elucidate the relative

stereochemistry of 23 by NOESY correlations (Fig. 2). The skeletons of these compounds and

related taxoids are similar to that of verticillene (Fig. 6).

3 . B I O G E N E T I C C O N S I D E R A T I O N

Since the new taxoids, taxuspines A–H and J–Z (1–25) and taxezopidines A–H and J–L (26–36),

from the Japanese yew T. cuspidata possessed various skeletons, we wish to propose the plausible

biogenesis of these taxoids. The biosynthesis of taxane skeleton (6/8/6-membered ring system) has

recently been suggested to involve cyclization of geranylgeranyl diphosphate to taxa-4(5),11-diene

by Croteau et al.,25 while the pathway of the 3,11-cyclotaxanes (6/5/5/6-membered ring system)

has been proposed to be derived from taxanes by concerted ss2þ ps

2 route.18 The 11(15!1)-abeo-

taxanes (5/7/6-membered ring system) may be biogenetically generated through a Wagner–

Meerwein rearrangement of ring A in a 6/8/6-membered ring system.26 On the other hand, the

Figure 5. Relative stereochemistryof taxuspineU (20).

Table I. Molecular Mechanics Calculations for Diastereomers 20a–d

Distancea

Diastereomer H-7/H-10 (A) H-7/H-18 (A) H-5/H-20b (A)

20a (5R* 7S*) 2.42 2.98 2.23

20b (5R* 7R*) 3.61 3.70 2.43

20c (5S* 7S*) 2.45 2.66 3.61

20d (5S* 7R*) 3.70 3.97 3.42

aDistances for the lowest energy conformers.


2(3!20)-abeo-taxanes (6/10/6-membered ring system) seem to be derived from verticillene

through the cyclization of D4(20),7-verticillene.27 The bicyclic taxane-related compounds (6/12-

membered ring system) might be derived from verticillene (Fig. 6).28

4 . B I O A C T I V I T I E S

A. Inhibitory Activity of Ca2þ -Induced Microtubule Depolymerizationby Taxoids

Paclitaxel (53) is unique among antimitotic drugs in that it promotes the polymerization of tubulin

a,b-heterodimers by binding to and stabilizing the resulting microtubule polymer. It differs from

antimitotic drugs such as colchicine, podophyllotoxin, and the Vinca alkaloids, which inhibit

microtubule assembly. Microtubules polymerized in the presence of taxol are resistant to

depolymerization by Ca2þ ions.29 The effect of taxoids (1–73) was examined against the CaCl2-

induced depolymerization of microtubules.

Microtubule proteins were polymerized under normal polymerization conditions30 in the

absence and the presence of paclitaxel (53) or taxoids (1–52) and (54–73), and, after 30 min

incubation, CaCl2 was added. Microtubule polymerization and depolymerization were monitored by

the increase and the decrease in turbidity. The results were summarized in Fig. 7 as the changes in

the relative absorbance at 400 nm. The CaCl2-induced depolymerization of microtubules (shown as

control) was completely inhibited by 10 mM of paclitaxel (53). Among the tested taxoids, taxuspine

Figure 6. Plausiblebiogenesis of taxoids.


D (4), taxezopidine K (35), taxezopidine L (36), and taxagifine (49) reduced the depolymerization

process in a remarkable fusion, suggesting that these compounds have paclitaxel-like activity to

microtubule systems. The potency of 4, 35, 36, and 49 in inhibition of the depolymerization process

corresponded to half to one-third of that of paclitaxel (53). Compounds 2, 39, and 62 exhibited

moderate activity, while the other compounds showed little or no such effect. On the other hand,

paclitaxel-type compounds 54–61 inhibited the depolymerization process as potently as pacli-

taxel (53). It is noted that compounds 4, 35, 36, and 49 lacking both an oxetane ring and an N-

acylphenylisoserine moiety exhibited potent activity. Since these active compounds 4, 35, 36, and

49 possessed a cinnamoyl group at C-5, the cinnamoyl group may play an important role for binding

to microtubules like the acyl group at C-13 in paclitaxel (53). In addition, the acetoxy group at C-10

and the hydroxy group at C-11 in 4, 35, 36, and 49 may be important in the inhibition of microtubule

depolymerization like the oxetane moiety at C-4 and C-5 and the acetoxy group at C-4 in paclitaxel

(53). It is noted that the conformation of 4 is very similar to that of 49 by macromodel calculations

(Fig. 4), while the conformation of 37 is different from those of 4, 35, 36, and 49. These results

suggested that it may be important for inhibition of microtubule depolymerization by both func-

tional groups and the conformation of 4, 35, 36, and 49.31

B. Effect of Taxuspine D (4) on Mitotic Spindle Formation in SeaUrchin Embryos

The mitotic spindle formation of the taxuspine D (4)-treated sea urchin eggs was investigated under

a polarization microscope. When the fertilized eggs were treated with 2.5 mg/ml of 4, the normal

spindles were not seen at the metaphase and overstabilized spindles with high birefringence density

were observed (Fig. 8), and the following egg divisions were completely suppressed. This mode of

Figure 7. Effects of taxoids on Ca2þ

-induced Microtubule Depolymerization.The temperature was held at 37�Cand changes in

turbidity weremonitoredat 400 nm.For the drug-protein studies,10 nMofdrugdissolved in DMSOwasadded to1ml buffer solution

containing2mgmicrotubuleprotein.ThefinalDMSOconcentrationwaslessthan1%.After30minincubationofthetestmixture,4mM

CaCI2 was added, and themixture was further incubated foranother 30 min.The turbidity changesweremonitored throughout the

incubationtime.


operation was almost identical with that of paclitaxel (53) at 10 mg/ml.16 Since taxuspine D (4) and

paclitaxel (53) induced overstabilized spindles with high birefringence density, they were shown to

act on components of the mitotic apparatus and to effectivly inhibit cell division.

C. Increased Cellular Accumulation of Vincristine in Multidrug-ResistantCells by Taxoids

The cellular accumulation of vincristine (VCR) is reduced in multidrug-resistant (MDR) tumor cells

as compared with parental cells.32 MDR-reversing agents such as verapamil increase the reduced

accumulation of antitumor agents in MDR cells and overcome multidrug resistance.33,34 The effect

of taxoids (1–73) on the cellular accumulation of VCR in multidrug-resistant human ovarian cancer

2780AD cells was examined and the results were shown in Table II. Verapamil at 1 and 10 mg/ml

increased the VCR accumulation in a dose-dependent manner. Compounds 2, 3, 9, 23, 25, 41, 42,

44, and 45 increased the VCR accumulation as potently as verapamil. Compounds 12, 25, 37–40,

43, 50, 62, 65, and 71 increased moderately the accumulation, while paclitaxel (53) and

cephalomannine (55) decreased the VCR accumulation in 2780AD cells. It is noted that (i) the

potent active compounds 2, 3, 9, 23, 25, 41, 42, 44, and 45 possessed a cinnamoyl or 3-N,N-

dimethylamino-3-phenypropanoyl group at C-5; (ii) these compounds have neither an oxetane ring

nor a phenylisoserine group at C-13. These results suggested that many taxoids could be substrates

of P-glycoprotein and some of them might be useful for overcoming multidrug resistance.31,35–37

D. Competitive Binding of [3H]Azidopine and Taxoids toP-Glycoprotein

Many calcium-channel blockers reverse the multidrug-resistant phenotype and inhibit active

transport of cytotoxic drugs out of multidrug-resistant cells. Azidopine is a photoaffinity analog of

the dihydropyridine class of calcium channel blockers that specially labels P-glycoprotein. The

ability to inhibit photoaffinity labeling of P-glycoprotein by azidopine has frequently been used as

an indicator of whether a particular compound is a P-glycoprotein ‘‘substrate’’.38,39 In particular,

it is believed that compounds which compete with azidopine for a common binding site on the

multidrug transporter will be able to block photolabeling. As shown in Fig. 9, [3H]azidopine labeled

P-glycoprotein (170 kDa) that was present in adriamycin-resistant human leukemia K562/ADM

cells (lanes 1 and 10), and binding of [3H]azidopine to P-glycoprotein was significantly reduced by

the presence of verapamil (lanes 17 and 18). Compounds 2, 3, 41, 42, and 44 reduced binding of

[3H]azidopine to P-glycoprotein more potently than verapamil (lanes 2–5, 8, 9, and 11–14), while

Figure 8. EffectsofTaxuspineD (4) andPaclitaxel (53) onmitotic spindleformationinseaurchinembryo.A: TreatedwithtaxuspineD (4, 2.5 mg/ml), (B) treatedwith paclitaxel (53,10 mg/ml) at 35minafter fertilization, (C) control.Polarizationmicroscopy.


taxinine (37) did not show such activity (lanes 6 and 7) and paclitaxel (53) showed quite weak

reduction (lanes 15 and 16). Since compounds 2, 3, 41, 42, and 44 increased VCR accumulation as

potently as verapamil as described above, it is suggested that these taxoids bind to the same binding

site on P-glycoprotein as that of azidopine.31

E. Combined Chemotherapeutic Effect of VCR and Taxuspine C (3) onP388/VCR-Bearing Mice

Since taxuspine C (3) increased cellular accumulation of VCR in multidrug-resistant tumor cells as

potently as verapamil, and inhibited [3H]-azidopine photolabeling of P-glycoprotein efficiently as

described above, we examined combined chemotherapeutic effect of VCR and 3 on P388/VCR-

bearing mice. VCR (0.1 or 0.2 mg/kg) alone given i.p. daily for 5 days starting on day 1 had no

significant chemotherapeutic effect on P388/VCR-bearing mice (Table III). When taxuspine C (3,

200 mg/kg) together with VCR was given i.p. daily for 5 days, the life span of P388/VCR-bearing

mice was increased. The prominent result was observed at a taxuspine C (3) dose of 200 mg/kg

given daily with 0.2 mg/kg VCR, whereby the T/C value was 138%.40

Table II. Effects of Taxoids 1–73 on Accumulation of Vincristine (VCR) in Multidrug-Resistant 2780AD Cells

VCR accumulation (% of control) VCR accumulation (% of control)

with a taxoid concentration of with a taxoid concentration of

Compound 1 mg/ml 10 mg/ml Compound 1 mg/ml 10 mg/ml

1 142a

246 45 250 882

2 219 713 47 207 363

3 246 768 48 119 135

4 133 325 49 194 242

6 141 257 50 169 537

7 139 271 51 225 213

8 174 567 52 115 169

9 258 842 53 (paclitaxel) 83 56

10 167 234 55 88 93

11 177 264 60 192 218

12 176 584 61 132 138

14 151 191 62 219 639

15 133 358 63 143 192

16 145 206 64 158 313

17 147 208 65 175 466

18 136 190 66 142 198

19 196 308 67 147 235

23 158 774 68 171 240

24 106 550 69 119 298

25 173 784 70 153 271

37 195 571 71 205 439

38 153 461 72 152 216

39 116 438 73 173 372

40 167 436 Verapamil 254 739

41 177 704

42 233 841

43 102 517

44 266 798

aThe amounts of VCR accumulated in multidrug-resistant human ovarian cancer 2780AD cells were determined in the presence of 1and10 mg/mlof taxoids. The values represent means of triplicate determinations, and are expressed as the relative amounts of VCR accumulated in the cells as

comparedwith the control experiment.


F. Cytotoxicity Studies

Cytotoxic activity of all the taxoids (1–73) against murine leukemia L1210 cells and human

epidermoid carcinoma KB cells is shown in Table IV. Paclitaxel (53) and paclitaxel-type compounds

54–61 exhibited very potent cytotoxicity against KB cells (IC50 0.0015–0.086 mg/ml). Compounds

5, 16, 18, 19, 22, 49, 63, 72, and 73 also showed potent cytotoxicity against KB cells (IC50 0.08–0.86

mg/ml). Compound 5 possessing an oxetane ring, but no N-acylphenylisoserine group was the most

potent (IC50 0.08 mg/ml) among these taxoids. It was interesting that compounds 16, 18, 19, 22, 49,

63, 72, and 73 without an oxetane ring and an N-acylphenylisoserine moiety exhibited such

cytotoxicity. These cytotoxic compounds 16, 18, 19, 22, 49, 63, 72, and 73 possessed an acetoxy

group at C-2, while the other functional groups were different from one another. A combination of

the acetoxy group at C-2 and the other functional groups such as an oxetane ring may be important

for cytotoxicity against KB cells, as many researchers have pointed out that no northern part

(positions 7, 9, and 10) but the southern part (positions 1, 2, 4, and 5) of taxoids is intimately

associated with its cytotoxicity.1

Table III. Effect of Taxuspine C (3) on Antitumor Activity of Vincristine (VCR) in P388/VCR-Bearing Mice

Treatmenta n Medianb (days) Range (days) T/C (%)

Control 5 11.4 10 15 100

VCR (0.2mg/kg) 5 12.6 10 15 110

VCR (0.1mg/kg) 5 10.6 10 11 92

TaxuspineC (3) (200mg/kg) 5 15.8 12^20 138

þVCR (0.2mg/kg)

TaxuspineC (3) (200mg/kg) 5 12.0 10 15 105

þVCR (0.1mg/kg)

aCDF1mice were given i.p. implants of10

6P388/VCR leukemiacells on day 0. Taxuspine C (3) together withVCR was given i.p. daily for 5 days.

bT/C value: median survival time of treatedmice dividedby thatof controlmice.

Figure 9. Effect of taxoids on [3H]azidopine photoaffinity labeling of P-glycoprotein. Adriamycin-resistant human leukemia K562/

ADM cell membrane vesicles (50 mg of protein) were incubated with [3H]azidopine (200 nM) in the presence of taxoids (10 and

100 mM), taxuspine B (2, lanes 2 and 3), taxuspine C (3, lanes 4 and 5), taxinine (37, lanes 6 and 7), 2 0-desacetoxyaustrospicatine(42, lanes 8 and 9), 2-desacetoxytaxinine J (44, lanes11and12), taxine II (41, lanes13 and14), andpaclitaxel (53, lanes15 and16),andverapamil (lanes17and18). After1hat roomtemperature, thesamplesweresubsequently irradiatedwithUVlight for 30min. After

separationby SDS-polyacrylamide gel electrophoresis, the intensity of P-glycoproteinphotolabelingwas detectedby fluorography;

thebandwithamolecularmassof170 kDa isdue to P-glycoprotein.Taxoidconcentrations inmMare indicatedalongthe topofeach

gel.Thepositionofmolecularmass standards inkDaare indicatedat the leftofgel.


G. Modulation of Multidrug Resistance in Tumor Cells byTaxinine Derivatives

1. Derivatization of Taxinine

Taxinine (37), a major taxoid isolated from the Japanese yew T. cuspidata, was chemically modified

to yield compounds 74–103 as follows.

Methanolysis of taxinine (37) with K2CO3 in MeOH at room temperature for 80 min gave 9,10-

di-O-deacetyltaxinine (74, 91%), while the reaction when continued for 6 h afforded a mixture of 74(70%) and 2,9,10-tri-O-deacetyltaxinine (75, 30%), which were separated by silica gel column

chromatography (hexane/EtOAc, 2:1). The 9,10-acetonide (76) was derived from 75 with 2,2-

dimethoxypropane in CH2Cl2 containing pyridinium p-toluenesulfonate (PPTS). Esterification of

compounds 74, 75, and 76 with cinnamic acid and 1,3-dicyclohexylcarbodiimide (DCC) afforded

the 9,10-di-O-cinnamate of 74 (77, 28%), the 2,9,10-tri-O-cinnamate of 75 (79, 24%), and the 2-O-

cinnamate of 76 (80, 60%), respectively (Scheme 1). On the other hand, treatment of 74 and 76 with

benzoyl chloride in pyridine gave the 9,10-di-O-benzoate of 74 (78, 56%) and the 2-O-benzoate

of 76 (81, 96%), respectively (Scheme 1). Hydrolysis of taxinine (37) with hydroxylamine sulfate in

Table IV. Cytotoxicity of Taxoids 1–73 Against Murine Leukemia L1210 Cells and Human Epidermoid

Carcinoma KB Cells

L1210 KB L1210 KB

Compound IC50 (mg/ml)a IC50 (mg/ml)

a Compound IC50 (mg/ml)a IC50 (mg/ml)

a

1 4.2 > 10 43 9.5 8.2

2 > 10 > 10 44 4.9 > 10

3 5.8 > 10 45 > 10 > 10

4 3.0 1.8 46 > 10 > 10

5 0.27 0.08 47 > 10 > 10

6 > 10 > 10 48 > 10 > 10

7 > 10 > 10 49 1.3 0.86

8 > 10 1.6 50 > 10 > 10

9 > 10 > 10 51 > 10 > 10

10 4.5 8.8 52 > 10 9.4

11 10.0 4.5 53 (paclitaxel) 0.33 0.0088

12 1.2 5.8 54 0.88 0.015

13 7.8 > 10 55 0.25 0.086

14 > 10 > 10 56 0.95 0.0048

15 2.5 2.6 57 0.21 0.0053

16 4.0 0.3 58 0.24 0.0017

17 4.6 > 10 59 0.21 0.0016

18 3.8 0.26 60 0.71 0.013

19 1.0 0.06 61 0.026 0.0015

22 8.0 0.35 62 3.8 > 10

23 4.2 > 10 63 > 10 0.4

24 5.4 > 10 64 > 10 > 10

25 > 10 6.2 65 2.7 > 10

34 4.2 2.4 66 > 10 > 10

35 1.7 4.8 67 > 10 > 10

36 2.1 2.0 68 > 10 > 10

37 > 10 > 10 69 7.1 > 10

38 8.9 > 10 70 > 10 > 10

39 > 10 > 10 71 10.0 7.4

40 4.6 6.9 72 6.2 0.60

41 > 10 > 10 73 1.1 0.17

42 7.2 > 10


EtOH/THF/H2O (1:1:1) containing triethylamine afforded taxinine A (38, 80%).10 Protection of the

OH-5 group in 38 with triethylsilyl chloride (TESCl) and imidazole followed by treatment with

DIBAL-H in anhydrous THF afforded 2-O-deacetyl-5-O-TES-taxinine A (31%, 2 steps), which was

esterified with cinnamic acid and DCC followed by treatment with tetrabutylammonium fluoride

(TBAF) to yield compound 82 (40%, 2 steps) (Scheme 2). NaBH4 reduction of 38 in THF/MeOH

(1:1) gave a 1:1 mixture of the OH-13a (83, 50%) and OH-13b (84, 50%) derivatives, which were

separated on a silica gel column (hexane/EtOAc, 2:1). Esterification of 83 and 84 with cinnamic acid

and DCC afforded the 13a-O-cinnamate of 83 (85, 52%) and the 13b-O-cinnamate of 84 (88, 52%),

respectively. Treatment of 83 with benzoyl chloride in pyridine gave the 13a-O-benzoate (86, 59%)

and the 5-O-benzoate (87, 40%), and the benzoylation of 84 under the same condition yielded the

5,13-b-di-O-benzoate (90, 63%). Treatment of 83 with cinnamoyl chloride in pyridine containing

4-dimethylaminopyridine (DMAP) afforded the 5,13-b-di-O-cinnamate (89, 50%) (Scheme 3).

Methanolysis of taxinine A (38) with K2CO3 in MeOH/dioxane (1:5) yielded 9,10-di-O-

deacetyltaxinine A (91, 74%). The 5-O-benzoate of 38 (92), the 5,9,10-tri-O-benzoate of 91 (94),

and the 2,5-di-O-benzoate of 7 (95) were obtained by benzoylation of 38, 91, and taxuspine G (7),11

respectively (Scheme 4). Treatment of taxinine A (38) with benzyloxymethyl chloride (BOMCl) led

to compound 103 (78%). The structures of 74–94, and 103 were confirmed by the 1H and 13C NMR

and MS spectral data and comparison with those of known compounds, while compounds 95–102were synthesized from 38 (Fig. 10).

2. Effects of Taxoids (7, 37, 38, and 74–103) on the Accumulation of VCR

in MDR Cells

The cellular accumulation of VCR is reduced in MDR tumor cells as compared with the

parental cells. The effect of taxoids (7, 37, 38, 74–103) on the cellular accumulation of VCR in

Scheme 2. (a)NH2OH�H2SO4,Et3N,THF/EtOH/H2O(1:1:1), reflux,8 h,80%; (b) (i) TESCl, lmidazole,CH2Cl2, rt,4 h,100%; (ii)DIBAL-H,

THF, rt, 6 h, 31%; (iii)Cinnamicacid,DCC,CH2Cl2, rt,16 h, 62%; (iv) TBAF,THF, rt, 3 h, 64%.

Scheme 1. (a) K2CO3, MeOH, dioxane, rt [74 (91%) for 80min, 74 (70%) and 75 (30%) for 6 h]; (b) (CH3)2C(OMe)2, PPTS,

CH2Cl2Cl2, rt, 8 h, 60%; c) Cinnamic acid,DCC,CH2Cl2, rt [74 to 77 (28%) for1h; 75 to 79 (24%) for 6 h; 76 to 80 (60%) for12h,

d) BzCl, pyr., rt [74 to 78 (56%) for12h; 75 to 81 (96%) for13h].


multidrug-resistant human ovarian cancer 2780AD cells was examined and the results are shown in

Table V. Verapamil at 1 and 10 mg/ml increased the VCR accumulation in a dose-dependent manner.

The increased cellular accumulation of VCR by taxinine (37) and taxinine A (38) corresponded to

80 and 60%, respectively, of that of verapamil.6 For this activity, it was found that taxoids 99 and

103 were 1.4 and 1.5 times more potent than verapamil, respectively. Compounds 82, 85–87, 92, and

100–102 increased the VCR accumulation as potently as verapamil, while the activities of taxoids

74, 75, 83, 88, 90, 94, 95, 97, and 98 were comparable to those of taxinine (37) or taxinine A (38).

Figure 10.

Scheme 3. (a)NaBH4,THF/MeOH(1:1), rt,15min,83 (50%)and84 (50%); (b)Cinnamicacid,DCC,CH2Cl2 [83 to85 (52%), rt, 24 h;

84 to 88 (52%), rt,12 h]; (c) Cinnamoyl chloride,DMAP, pyr., 90�C, 24h, [84 to 89 (50%)]; d) BzCl, pyr., rt [83 to 86 (59%) and 87(40%), for 4 h; 84 to 90 (63%) for12h].

Scheme 4. (a) K2CO3,MeOH/dioxane (1:5), rt, 4 h,74%; (b) BzCl, pyr., rt, [91 to 93 (15%) for 6 h; 7 to 94 (67%) for 24h].


Compounds 7, 77–81, 84, 89, 93, and 96 showed weak activity. The activities of the 9,10-di-O-

deacetyl derivatives 74 and 75 of taxinine (37) were almost equal to that of 37, while the 9,10-di-O-

cinnamoyl (77 and 79), 9,10-di-O-benzoyl (78), and 9,10-O-acetonide (80 and 81) derivatives

showed less potent activity. The 2-O-cinnamoyl (82), 5-O-benzoyl (92), 13-O-cinnamoyl (85 and

88), 13-O-benzoyl (86), 5-O-TES (102), and 5-O-BOM (103) derivatives of taxinine A (38) were

more active than taxinine A (38), while the 5,13-di-O-cinnamoyl (89), 5,13-di-O-benzoyl (90), and

5,9,10-tri-O-benzoyl (93) derivatives showed less activity. Among compounds 83, 84, 85, and 88,

the taxoids (83 and 85) having an a-oriented functional group at C-13 were more active than those

(84 and 88) possessing the corresponding b-oriented functional group at C-13. The activity of

92 having a ketone group at C-13 was similar to that of 87 with a hydroxy group at C-13. For

compounds with an epoxide at C-4 and C-20 (95–101), the taxoids (99 and 100) having a cinnamoyl

or TES group at C-5 were as active as verapamil, while those (95–98) with a hydroxy or benzoyloxy

group at C-5 showed less activity. The activities of the a-epoxides (97 and 100) were almost equal to

those of the corresponding b-epoxides (98 and 101), respectively, while the a-epoxide (95) with

a hydroxy group at C-5 was more active than the corresponding b-epoxide (96).

In summary, some taxinine derivatives (82, 85, 86, 87, 92, 99, 102, and 103) containing a

cinnamoyloxy, a benzoyloxy, a TES, or a BOM group at C-2, C-5, or C-13 effectively increased

the cellular accumulation of VCR in MDR tumor cells, while another taxoids (77, 78, 79, 80, 81,

and 93) having cinnamoyloxy, benzoyloxy, or acetonide groups at both C-9 and C-10 showed

remarkable reduction of the activity. Since the 6/8/6-membered ring system of these taxinine

derivatives take commonly ‘‘cage’’-like backbone structures such as taxinine, the presence of the

bulky group at C-2, C-5, or C-13 oriented to inside of the ‘‘cage’’ structure may be important for its

Table V. Effects of Taxoids (7, 37, 38, 74–103, and 112–118) on the Accumulation of Vincristine (VCR) in

Multidrug-Resistant Cells

VCR accumulation (% of control) VCR accumulation (% of control)

with a taxoid concentration of with a taxoid concentration of

Compound 1 mg/ml 10 mg/ml Compound 1 mg/ml 10 mg/ml

37 (Taxinine) 195a

571 92 184 645

74 274 619 93 132 324

75 225 518 94 146 470

77 181 273 95 180 364

78 141 210 96 144 238

79 186 230 97 140 402

80 198 234 98 200 391

81 126 210 99 358 1027

38 (Taxinine A) 153 461 100 233 752

82 207 727 101 227 748

83 164 422 102 213 669

84 149 290 103 401 1110

85 249 699 112 149 310

86 185 643 113 143 184

87 151 649 116 129 190

88 213 551 117 161 292

89 157 234 118 203 308

90 197 391 Verapamil 254 739

7 (TaxuspineG) 139 271

aThe amounts of VCR accumulated in multidrug-resistant human ovarian cancer 2780AD cells were determined in the presence of 1and10 mg/mlof taxoids. The values represent means of triplicate determinations, and are expressed as the relative amounts of VCR accumulated in the cells as

comparedwith the control experiment.


effective binding to P-glycoprotein, whereas the existence of the bulky groups at C-9 and C-10

directed to outside of the ‘‘cage’’ structure may result in its less binding. It is noted that among these

taxinine derivatives (74*103) the two taxoids 99 and 103 were 1.4 and 1.5 times more potent than

verapamil, respectively. Compounds 99 and 103 showed weak or no cytotoxicity against murine

lymphoma L1210 cells with IC50 values of 3.0 and 8.0 mg/ml, respectively, and human epidermoid

carcinoma KB cells with IC50 values of > 10 and 9.0 mg/ml, respectively, in vitro, while the other

taxoids (74*98 and 100*102) also exhibited weak or no cytotoxicity. Thus, this study showed that

some taxinine derivatives are good modifiers of multidrug resistance in tumor cells.41

H. Concluding Remarks

It is noted that the non-paclitaxel-type taxoids, taxuspine D (4), taxezopidines K (35) and L (36),

taxagifine (49) exhibited potent inhibitory activity against Ca2þ -induced depolymerization of

microtubules, while another non-paclitaxel-type taxoid, taxuspine D (4), induced spindles with

strong birefringence like paclitaxel (53). Some taxoids (2, 3, 41, 42, and 44) inhibiting binding of

azidopine to P-glycoprotein in adriamycin-resistant K562/ADM cells increased the cellular

accumulation of vincristine in multidrug-resistant 2780AD cells as potently as verapamil. It is noted

that taxuspine C (3) given i.p. enhanced the chemotherapeutic effect of vincristine in P388/VCR-

bearing mice. These observations indicate that taxuspine C (3) interacts directly with P-glycoprotein

and inhibits the efflux of antitumor agents, thus overcoming multidrug resistance in vivo, like

verapamil. In addition, these taxoids exhibited weak or no cytotoxicity. From these results, it is

suggested that some taxoids can be a good modifier of multidrug resistance in cancer chemotherapy.

5 . R E A C T I O N S O F T A X I N I N E

A. Generation of a New Dimeric Compound of 5-Oxotaxinine A ThroughDiels-Alder Cycloaddition

Oxidation of taxinine A (38), which was derived from taxinine (37), with tetrapropylammonium

perruthenate (TPAP) yielded 5-oxotaxinine A (104, 80%). The structure of 104 was elucidated by

spectral data analysis including FDMS and 2D NMR. It was found that 104 was allowed to stand at

room temperature to occur a new dimeric compound 105 (Scheme 5). Compound 105 was generated

from 104 in benzene at 20–80�C in 50–75% yield, and the dimeric reaction without solvent at 80�C

proceeded quantitatively. Compound 105 was shown to have the molecular formula, C52H68O16, by

HRFABMS [m/z 949.4572 (MþH)þ , D�1.4 mmu], indicating that it was a dimer of 5-oxotaxinine

A (104). The 1H NMR spectrum of 105 showed proton signals due to each pair of three acetyl

methyls, three oxymethines, and four methyls. Each pair of these signals was assigned to to parts A

Scheme 5. (a) TPAP, 4-MethylmorpholineN-oxide,CH3CN,MS 4—, rt, 2 h, 80%; (b) neat, 80�C.


(C-1-C-20) and B (C-1 0-C-20 0), corresponding to each half moiety of the dimer by 2D NMR (1H-1H

COSY, HMQC, and HMBC) data. HMBC correlations of H-3, H-6a, and H-20a to C-4 (d 101.7),

H-3 and H-6b to C-5 (d 146.9) indicated the presence of an enol (C-4, C-5, and O-5) in part A, while

the presence of an a-oxyketone (O-4 0, C-4 0, C-5 0, and O-5 0) was deduced from HMBC correlations

of H-3 0 and H-6 0b to C-4 0 (d 85.0), H-3 0, H2-6 0, and H-20 0 to C-5 0 (d 206.4) in part B. 1H-1H COSY

connectivities between H2-20 and H2-20 0 and HMBC correlations of H-20a to C-4 0 and H-20 0 to

C-4 revealed that 105 possessed a dihydropyran ring (C-4, C-5, O-5, C-4 0, C-20 0 and C-20), which

was supported by comparison with olefin chemical shifts (d 99.2 and 144.0) of a dihydropyran

reported.9 Thus the structure of 105 was assigned as a dimer of compound 104. The relative

stereochemistry at the spiro carbon (C-4 0) of 105 was deduced from the NOESY spectrum. The

cross peaks for H-6 0a/H3-19 0, H-6 0a/H2-20 0, and H3-19 0/H2-20 0 indicated that C-4 0 had R*

configuration (Fig. 11). Compound 105 was crystallized from i-PrOH and H2O to give prisms

of space group P212121. The crystal structure was solved by the direct method and refined by a full-

matrix least-squares method to R¼ 0.084 and RW¼ 0.249 using 2002 (I> 2s (I)) observed

reflections. The relative stereostructure of 105 was established by the X-ray analysis as shown in

Fig. 12, which corresponded to that elucidated by NMR data.

The formation of 105 from 104 is considered to be derived through regio- and stereo-specific

Diels–Alder cycloaddition between the enone (C-20, C-4, C-5, and O-5) of one molecule and

the exomethylene (C-4 0 and C-20 0) of another one, in which the exomethylene approached to the

enone. Similar dimerization has been also reported for formation of bistheonellasterone from

theonellasterone. Although many natural and derivatized taxoids have been reported, compound 105is the first example of dimeric taxoids. This type of dimeric reaction could be applied for other

taxoids possessing an exomethylene and a ketone at C-4 and C-5, respectively. Such dimerization

of other taxoids and bioactivities of the products are currently being investigated.42

Figure 11. Relative stereochemistryof the dimer (105) of 5-Oxotaxinine A (104).Dottedarrowsdenote NOESYcorrelations.

Figure 12. ORTEPdrawingof 105.


B. Stereoselective Epoxidation of 4(20)-Exomethylene inTaxinine Derivatives

About twenty taxoids with a 4(20)-epoxide moiety isolated from Taxus species so far all have been

reported to be b-epoxides, while chemical oxidation of taxoids with a 4(20)-exomethylene unit

using m-chloroperbenzoic acid (m-CPBA) or monoperphthalic acid4 has always given mainly the

a-epoxides. In fact, epoxidation of taxinine (37) and taxinine A (38), and taxinine derivative

110 with m-CPBA afforded the a-4(20)-epoxides selectively (a:b¼ 99:1). However, recently

we found that epoxidation of taxinine derivatives 110 and 111 with dimethyldioxirane (DMDO)

gave the b-4(20)-epoxides predominantly (a:b¼ 1:4–5).

In order to examine effects of oxidizing agents and the functional groups at C-5 in taxoids

on stereoselectivity of chemical epoxidation of the 4(20)-exomethylene moiety, several taxinine

derivatives such as 5-O-benzoyltaxinine A (107), 5-O-trifluoroacetyltaxinine A (108), 5-O-

pivaloyltaxinine A (109), 5-O-t-butyldimethylsilyltaxinine A (110), and 5-O-triethylsilyltaxinine A

(111) were prepared from taxinine A (38). Epoxidation of taxinine (37), taxinines A (38) and H

(106), and compounds 107–111 were carried out by using m-CPBA and DMDO and the results are

summarized in Table VI. In each case, a pair of oxidation products 37a, 38a, 107a–111a and 37b,38b, 107b–111b were obtained and separated by silica gel column chromatography. Epoxidation of

37, 38, and 110 with m-CPBA gave almost only the a-4(20)-epoxides, while m-CPBA epoxidation

of 106 and 111 afforded the a- and b-4(20)-epoxides in the ratio of 3:1 and 4:1, respectively.

Furthermore, treatment of 107–109 with m-CPBAyielded the a- and b-4(20)-epoxides in the ratio of

1:1. On the other hand, epoxidation of 110 and 111 with DMDO gave the a- and b-4(20)-epoxides in

the ratio of 1:5 and 1:4, respectively, while DMDO epoxidation of 38, 106, and 107 afforded the a-

and b-4(20)-epoxides in the ratio of 2:1. Treatment of 108 and 109 with DMDO yielded the a- and

b-4(20)-epoxides in the ratio of 1:1 and 1:2, respectively. The stereochemistry of the epoxide moiety

in 37a was elucidated to be a by the NOESY correlations, while that of 38a was deduced from the

fact that hydrolysis of 37a gave 38a. a-Orientation of the 4(20)-epoxides in 106a–111a was

assigned by the presence of an NOE between H3-19 and H-20a. The b-selectivity of epoxidation of

110 and 111 with DMDO may be explained by large steric hindrance between a silyl group at C-5

and DMDO. To our knowledge, this is the first stereoselective oxidation of taxoids with 4(20)-

exomethylene to give the b-4(20)-epoxides predominantly.43

Scheme 6.


C. Unusual Boron Trifluoride-Catalyzed Reactions of Taxinine DerivativesWith a- and b-4(20)-Epoxides

In our continuing chemical derivatization of taxinine (37), we found that boron trifluoride-catalyzed

reaction of b-4(20)-epoxy-5-O-triethylsilyl (TES) taxinine A (111b) yielded two unexpected com-

pounds, the 3,5-diene (112), the 3,8-cyclopropane (113), the cyclobutane (114), and the dioxane

(115) derivatives, while the similar reaction of the corresponding a-4(20)-epoxide (111a) afforded

the ring-contracted derivative (116), its hemiacetal dimer (117), and the orthoester (118) derivatives

as follows.

Treatment of the b-4(20)-epoxy-5-O-TES-taxinine A (111b), which was derived from taxinine

A, with boron trifluoride diethyl etherate (BF3*OEt2, 2 eq.) in CH2Cl2 at room temperature for 0.5 h

yielded the 3,5-diene (112, 42%), the 3,8-cyclopropane (113, 16%), the cyclobutane (114, 4%), and

the dioxane (115, 3%) derivatives (Scheme 7), while the reaction at 0�C afforded the 3,5-diene (112,

Scheme 7.

Table VI. Epoxidation of C-4(20)-Exomethylene of Taxinine (37),

Taxinine A (38), and Related Compounds (106–111)

m-CPBA DMDO

Compound a (a):b (b) Yield (%)a a (a):b (b) Yield (%)a

37 99 :1 99 99 :1 77b

38 99 :1 99 2 :1 66

106 3 :1 91 2 :1 86

107 1:1 84 2 :1 77

108 1:1 50 1:1 72

109 1:1 99 1: 2 63

110 99 :1 99 1: 5 69

111 4 :1 76 1:4 86

aIsolation yield.

b22(23)-double bondwas also epoxidized.


47%) but the 3,8-cyclopropane (113) was not obtained. When the reaction mixture was treated with

2,4-dinitrophenylhydrazine, formaldehyde-2,4-dinitrophenylhydrazone was obtained, suggesting

the liberation of formaldehyde in this reaction. On the other hand, treatment of the a-4(20)-epoxide

(111a), which was also derived from taxinine A, with BF3*OEt2 (2 eq.) in CH2Cl2 at 0�C for 0.5 h

afforded the cyclopentanecarbaldehyde (116, 41%), its hemiacetal dimer (117, 16%), and the

orthoester (118, 9%) derivatives (Scheme 8). The structures of 112–118 were elucidated by spectral

data including MS and 2D NMR.

The formation of the 3,5-diene (112), the 3,8-cyclopropane (113), the cyclobutane (114), and

the dioxane (115) derivatives from the b-4(20)-epoxide (111b) may be explained as follows. In the

first step, BF3*OEt2 induces the fission of C-4–O bond followed by 1,2-shift of hydride from C-3

to C-4, resulting in generation of the carbocation at C-3 (A). Liberation of formaldehyde and

dehydration of the allylic functional group at C-5 in A give the 3,5-diene (112). 1,4-Michael

addition between the a,b-unsaturated ketone (C-11–C-13) and the D3,4-double bond in 112 together

with cycloaddition among D5,6-double bond in 112 and two molecules of the liberated formaldehyde

from A affords compound 114, while the 3,8-cyclopropane (113) can arise from a trapping of the

carbocation center formed at C-3 (A) by the methyl (Me-19) followed by loss of a proton. On the

other hand, the fission of C-4–O bond of 111b by BF3*OEt2 followed by 1,2-shift of hydride from C-

20 to C-4 in the carbocation results in generation of an a-oriented formyl group at C-4 (B).

Furthermore, the concerted reaction (B) among the substituents at C-2, C-4, and C-5, and one

molecule of the liberated formaldehyde from A yields compound 115. In the case of the a-4(20)-

epoxide (111a), BF3*OEt2 induces the fission of C-4–O bond followed by ring contraction through

a pinacol-type rearrangement (B) to afford the cyclopentane-carbaldehyde (116), and then the

dimerization of 116 to 117. The pinacol-type rearrangement involving deprotection of the TES

group at C-5 accompanies with transfer of the C-6 carbon into C-4 position and generation of an a-

oriented formyl group. BF3*OEt2 induces the fission of C-4–O bond and then another concerted

reaction (C) among O-20, the acetoxy carbonyl at C-2, and the carbocation at C-4 gives the

orthoester (118) (Fig. 13).

It is noted that the orientation of the 4(20)-epoxides results in different types of the boron

trifluoride-catalyzed reactions.44,45

Scheme 8.


D. Bioactivities of Synthetic Taxoids

Cytotoxicity and multidrug resistant activity of some synthetic taxoids described above were

examined. Taxoids 105, 112–118 exhibited weak or no cytotoxicity against murine lymphoma

L1210 cells and human epidermoid carcinoma KB cells. Taxoids 112, 113, and 116–118 showed

weak activity against the cellular accumulation of VCR (Table V).46

A C K N O W L E D G M E N T

This work was partly supported by a Grant-in-Aid for Scientific Research from the Ministry of

Education, Science, Sports, and Culture of Japan.

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17. Known taxoids: taxinine (37), taxinine A (38), taxinine B (39), O-cinnamoyltaxacin I triacetate (40),

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1b-hydroxybaccatin I (48), taxagifine (49), taxacin (50), decinnamoyltaxagifine (51), taxinine M (52),

paclitaxel (53), 10-deacetyltaxol (54), cephalomannine (55), 10-deacetylcephalomannine (56), taxol

C (57), 10-deacetyltaxol C (58), taxol D (59), 7-epi-10-deacetyltaxol (60), 7-epi-taxol (61), taxchinin

B (62), brevifoliol (63), 13-acetylbrevifoliol (64), diacyltaxinine B (65), 19-debenzoyl-19-acetyltaxinine

M (66), taxacustin (67), taxayuntin (68), 7-O-acetyltaxine A (69), 10-deacetylbaccatin III (70),

2-deacetyl-5-decinnamoyltaxinine J (71), triacetyltaxicin I (72), and 2,9-dideacetyltaxinine (73).

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40. Kobayashi J, Hosoyama H, Wang X-X, Shigemori H, Sudo Y, Tsuruo T. Bioorg Med Chem Lett

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Jun’ichi Kobayashi was born in Hirosaki, Japan, and received his BS and MS degrees in pharmaceutical

sciences from Hokkaido University, studying synthetic methods of nucleosides and nucleotides. And then, he

joined the researcher at Mitsubishi-Kasei Institute of Life Science, Tokyo, and studied deuterium-labeled

peptide synthesis and its conformational analysis by NMR. After obtaining his PhD degree in pharmaceutical

sciences from Hokkaido University, he started investigations on isolation and structure elucidation of marine

natural products in Mitsubishi-Kasei Institute. He was a visiting scientist with Professor Kenneth Rinehart at

University of Illinois for 2 years. He came back as a senior researcher to Mitsubishi-Kasei Institute, continuing

his work in the marine natural product area. In 1989, he promoted a full professor at Graduate School of

Pharmaceutical Sciences, Hokkaido University. He pursued his interest in marine natural products chemistry,

while his research interests were expanded to biactive taxoids from yew trees, bioactive metabolites from

actinomycetes of the genus Nocardia, and unique alkaloids from higher plants. His laboratory has focused on

isolation, structure elucidation, stereochemistry, synthesis, biosynthesis, and bioactivity of unique natural

products from marine micro- and macro $B%__(B organisms as well as terrestrial microorganisms and higher

plants.

Hideyuki Shigemori was born in Hyogo, Japan, and received his BS in chemistry from Keio University in

Kanagawa. After obtaining his MSc and PhD degrees in natural products chemistry, he joined Professor

Jun’ichi Kobayashi at the Faculty of Pharmaceutical Sciences, Hokkaido University. He has isolated a large

number of new bioactive substances from plants, marine invertebrates, and micro-organisms. His research

interests are in the areas of medicinal and natural products chemistry. He is currently an associate professor at

the Graduate School of Pharmaceutical Sciences, Hokkaido University.


Documents

Bioactive taxoids from the Japanese yew Taxus cuspidata