Taxus canadensis abundant taxane: Conversion to paclitaxel and rearrangements

Bioorganic & Medicinal Chemistry 8 (2000) 1269±1280

Taxus canadensis Abundant Taxane: Conversion to Paclitaxel and

Rearrangements

Anastasia Nikolakakis,^aGaetan Caron,^aAlice Cherestes,^aFrancoise Sauriol,^b

Orval Mamer^cand Lolita O. Zamir^a,*

a

Â Â

Universite du Quebec, INRS-Institut Armand-Frappier, 531 boul. des Prairies, Laval, Quebec, Canada H7V 1B7

^bDepartment of Chemistry, Queen's University, Kingston, Ontario, Canada K7L 3N6

^cBiomedical Mass Spectrometry Unit, McGill University, 1130 Pine Ave. West, Montreal, Quebec, Canada H3A 1A3

Received 18 November 1999; accepted 17 January 2000

AbstractÐAn ecient conversion of Taxus canadensis abundant taxane, 9-dihydro-13-acetylbaccatin III to baccatin III is descri-

bed. Since the synthesis of paclitaxel from baccatin III has been reported, this work can be used for additional supply of this

powerful anticancer drug. In addition, new taxanes derived from skeletal rearrangements originating from oxidation±reduction

The Canadian yew (Taxus canadensis), a low trailing bush

very common in Quebec, is an interesting plant with

unusual taxanes speci®c to this yew.^{1 7}Its major taxane,

isolated from the needles, was identi®ed as 9-dihydro-l3-

acetylbaccatin III 1 in 1992 simultaneously by our

group¹and Gunawardana et al.⁸(Fig. 1). Depending on

the collection site, it is 5 to 7 times more abundant than

its co-metabolite paclitaxel 2. 9-Dihydro-13-acetyl-

baccatin III 1 has only been found as traces in the bark

of one other yew, Taxus chinensis.⁹Since T. canadensis

is more widespread¹⁰than Taxus brevifolia, from which

paclitaxel was originally isolated, a conversion of 1 to 2

would be of immense value. Acid and base rearrange-

ment products of taxane 1 emphasize its unusual

chemistry.^{11 13}The structure of 1 is very similar to the

core skeleton of paclitaxel, with a 9a-hydroxyl group

instead of a ketone and a C-13-acetyl group instead of

the side chain. Baccatin III 3 has been chemically con-

verted to paclitaxel.^14a,bA priori, the conversion of

taxane 1 to baccatin III 3 or paclitaxel 2 seems trivial. It

would require protecting the 7-hydroxyl, oxidizing C-9,

deacetylating C-13 and either deprotecting C-7 to form

3 or attaching the C-13-side chain and then deprotecting

the C-7 to form 2. This approach was only partly fea-

sible, and the ecient conversion of 1 to 3 followed

a dierent route, which is described in this publication.

In addition, we learned more about the chemistry of

this unique natural taxane from some rearrangement

reactions.

Results and Discussion

Conversion of 9-dihydro-13-acetylbaccatin III 1 to baccatin

III 3 or paclitaxel 2: approach 1

The ®rst approach of the conversion of 9-dihydro-13-

acetylbaccatin III 1 to baccatin III 3 (and hence to

paclitaxel 2) depended on the relative reactivities of C-7

and C-9. A mild oxidizing agent such as pyridinium

dichromate (PDC) oxidized the 7b-OH preferentially

over the 9a-OH of 9-dihydro-13-acetylbaccatin III 1 to

give taxane 4 (Scheme 1). Upon puri®cation of taxane 4,

it was unstable which led to opening of the oxetane, C-4

to C-20 acetyl migration and dehydration to give the d-

seco derivative 5, 9-dihydro-l3-acetyl-d-seco-5,6-dehy-

drobaccatin III. The instability of taxane 4 is reminis-

cent of the results obtained by Kingston¹⁵and Potier¹⁶

on the oxidation products of paclitaxel. Under Jones'

oxidation conditions, both C-7 and C-9 hydroxyls of

taxane 1 are oxidized to give taxane 6. Taxane 6 is

unstable during chromatography on silica and converts

to the d-seco derivative, 13-acetyl-d-seco-5,6-dehy-

drobaccatin III 7 (Scheme 1, Table 1). Most alkylating

reagents preferentially attack C-7. Acetylation of 1 with

acetic anhydride in pyridine and Jones' oxidation gave

7,13-diacetylbaccatin III 8. Unfortunately, selective

deacetylation of 8 to give 3 was unsuccessful. We were

*Corresponding author. Tel.: +1-450-687-5010, ext. 4260; fax:

(oce): +1-450-686-5501; (residence): +1-514-481-2797; e-mail: loli-

ta.zamir@iaf.uquebec.ca

PII: S0968-0896(00)00056-0

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A. Nikolakakis et al. / Bioorg. Med. Chem. 8 (2000) 1269±1280

Figure 1. Some metabolites in the Canadian yew: the abundant taxane 9-dihydro-13-acetylbaccatin III 1, paclitaxel 2 and baccatin III 3.

Scheme 1. Conversion of 9-dihydro-13-acetylbaccatin III 1 to taxanes 4±15. TES: triethylsilyl; TIPS: triisopropylsilyl; TBDPS: tert-butyldiphe-

nylsilyl. Reagents and conditions: (a) pyridinium dichromate, CH₂Cl₂, 23 ^ꢀC, 4.5 h, 13%; (b) CrO₃in concentrated H₂SO₄:H₂O (3:7), acetone, 23 ^ꢀC,

30 min, 69%; (c) silica gel chromatography (EtOAc), 100%; (d) i. Ac₂O, pyridine, 23 ^ꢀC, 18 h; ii. CrO₃in concentrated H₂SO₄:H₂O (3:7), acetone,

0 ^ꢀC, 1 h, 54%; (e) Benzyl bromide, Ag₂O, 23 ^ꢀC, 18 h, 47% for 9a, 6% for 9b; (f) triethylsilyl chloride, pyridine, 23 ^ꢀC, 72 h, 75%; (g) CrO₃in

concentrated H₂SO₄:H₂O (3:7), acetone, 23 ^ꢀC, 30 min; (h) triisopropylsilyl chloride, imidazole, DMF, 23 ^ꢀC, 72 h, 77% based on recovered starting

material; (i) t-butyldiphenylsilyl chloride, imidazole, DMF, 23 ^ꢀC, 72 h, 81% based on recovered starting material; (j) CrO₃in concentrated

H₂SO₄:H₂O (3:7), acetone, 23 ^ꢀC, 30 min, 21%; (k) CrO₃in concentrated H₂SO₄:H₂O (3:7), acetone, 23 ^ꢀC, 30 min, 77%; (l) (Method A): n-BuLi,

THF, 44 ^ꢀC, 2.5 h, 46% based on recovered starting material, (method B): NaBH₄, THF/0.05 M KPO₄buer, pH 7, 2:1, 23 ^ꢀC, 8 h, 66%.

surprised to ®nd that benzylation of taxane 1 proceeds

predominantly on C-9 instead of C-7 to give 9a as a

major product and 9b as a minor (Scheme 1). These

compounds could not be used eciently for the conver-

sion of 1 to baccatin III 3 or to paclitaxel 2. Silylating

reagents can react preferentially only with the C-7

hydroxyl and not with the C-9 (Scheme 1).¹⁷Indeed, 9-

dihydro-13-acetylbaccatin III 1 with triethylsilyl chloride

in pyridine¹⁷gave 7-triethylsilyl-9-dihydro-13-acetyl-

baccatin III 10. Similarly, 7-triisopropylsilyl-9-dihydro-

13-acetylbaccatin III 11 and 7-t-butyldiphenylsilyl-9-

dihydro-13-acetylbaccatin III 12 were obtained from

A. Nikolakakis et al. / Bioorg. Med. Chem. 8 (2000) 1269±1280

Table 1. ¹H and ¹³C NMR data for taxane 7 in CDCl₃

1271

Position

d ¹H mult.^a(J in Hz)

d

¹³C^b

HMBC

NOESY^c

1

2

3

4

77.6

74.2

53.9

73.7

5.58 d (6.3)

4.06 d (6.3)

1, 3, 8, 14, 166.7

1, 4, 8, Me-19, 20

3^w, Me-17^s, Me-19^s, 20b^w

2^m, 10^w, 14a^s, 18^s, OH-4^m

OH-4

5

6

7

8

3.39 s

6.76 d (10.5)

5.98 d (10.3)

3, 4, 5, 20

3^S, 5^w, 14a^s, 20b^s, Bz-O^w

153.4

123.7

197.3

63.0

3

8

6^s, 20b^w, OH-4^w

5^s

9

199.1

76.7

133.6

140.4

70.1

10

11

12

13

14a

14b

15

16

17

18

19

20a

20b

OAc

6.36 s

9, 11, 12, 15, 168.9

3^w, Me-18^s

5.88 br m

2.80 dd (15.1; 4.1)

2.47 dd (15.2; 10.5)

11, 12, 170.2

1,2, 13, 15

1, 2, 12, 13

14b^s, Me-16^s, Me-18^w

3^w, 13^w, 14b^s, OH-4^w

13^m, 14a^s

35.7

42.3

27.6

18.5

15.3

17.8

68.3

1.12 s

1.22 s

1.85 br s

1.59 s

4.35 d (12.2)

4.31 d (12.2)

2.23 s

1, 11, 15, Me-17

1, 11, 15, Me-16

11, 12, 13

13^s, 14b^w, 17^w

2^s, 16^m

3^w, 10^s

3, 7, 8, 9

4, 5, 171.0

171.0

2^s, 14b^w, Me-17^m, 20b^s

2^w, Me-19^s, OH-4^w

19^s

20.7, 168.9

21.0, 170.2

20.0, 171.0

166.7

129.9

128.7

2.17 s

1.82 s

OBz:

ortho

meta

para

C-1

7.95 d (7.6)

7.45 t (7.8)

7.56 t (7.6)

133.0

131.5

^aMult., multiplicity: s, singlet; d, doublet; t, triplet; dd, doublet of doublets; br, broad; m, multiplet; o, overlapping. The precision of the coupling

constants is 0.5 Hz.

^bThe ¹³C chemical shifts were extracted from the HMQC and HMBC experiments (for quaternary carbons) (0.2 ppm).

^cNOESY intensities are marked as strong (s), medium (m) or weak (w).

A. Nikolakakis et al. / Bioorg. Med. Chem. 8 (2000) 1269±1280

1275

very similar to the NMR data of the base rearrange-

ment of another d-seco derivative derived from 13-oxo-

baccatin III.¹⁶Indeed, the compounds dier only by the

substituent on C-l3 (an a-acetoxy-C-l3 in taxane 19 and

a keto-group in ref 16). Comparison of the respective

NMR data con®rm our structural determination. The

formation of taxane 19 can be explained by the same

mechanism shown by Potier's group.¹⁶High resolution

mass spectrometry con®rmed the elemental composition

of the sodiated quasimolecular ion of taxane 19.

and processed using standard procedures. For phase

sensitive 2D experiments (NOESY and HMQC), the

data were acquired using the hypercomplex phase mode.

The NOESY experiment was obtained using a mixing

time of 0.3 s and a relaxation delay of 1 s. The intensity

of the cross-peaks in the NOESY experiment is desig-

nated as strong (s), medium (m) and weak (w). Positive

ion Fast Atom Bombardment Mass Spectra (FAB MS)

were obtained with a Vacuum Generators ZAB-HS

double-focussing instrument using a xenon beam having

8 kV energy at 1 mA equivalent neutral current. Low

resolution mass spectra were obtained in glycerol. Sam-

ples were dissolved in 0.2 mL DMSO before addition of

0.5 mL glycerol. FABHRMS was similarly obtained in

glycerol±DMSO at a resolving power of 12,000.

Conclusion

The chemistry of the conversion of the Canadian yew

abundant taxane, 9-dihydro-13-acetylbaccatin III, to

baccatin III and hence to paclitaxel in four steps was

unexpected. These reactions could be used to increase

the supply of paclitaxel. Obviously the transformation

of T. baccata major taxane, 10-deacetylbaccatin III to

baccatin III^14ais preferable. Unfortunately, the supply

of the European yew is limited, while T. canadensis

seems to be widespread^10,27and additional supply of

paclitaxel and taxanes could be useful. The new skele-

tons obtained from rearrangement reactions will add to

the ongoing investigation of the roles of cycles B and C

in tubulin binding. In addition, these structures could be

used as a backbone to couple to inhibitors of cellular

targets which have been shown to confer resistance to

taxanes.

9-Dihydro-13-acetyl-^D-seco-5,6-dehydrobaccatin III 5. 9-

Dihydro-13-acetylbaccatin III 1 was isolated from the

needles of T. canadensis as previously described.^1,29-

Dihydro-13-acetylbaccatin III 1 (15 mg; 0.024 mmol) in

CH₂C1₂(3 mL) was treated with pyridinium dichromate

(29 mg; 0.077 mmol) at 23 ^ꢀC for 4.5 h. The mixture was

diluted in CH₂C1₂and washed with brine, dried, ®ltered

and evaporated in vacuo. The residue was puri®ed by

semi-preparative HPLC (25±100% CH₃CN in H₂O,

50min gradient at a ¯ow rate of 3 mL/min) aording 9-

dihydro-13-acetyl-d-seco-5,6-dehydrobaccatin III 5 (2 mg,

13%, R_t=32.97min). FABHRMS: C₃₃H₄₀O₁₂Na

[M+Na⁺] required 651.2418, found 651.2420; ¹H

NMR (CDCl₃, 500 MHz) d 8.10 (d, J=7.6 Hz, 2H, H-

2,6 of OBz), 7.61 (t, J=7.3 Hz, 1H, H-4 of OBz), 7.50 (t,

J=7.5 Hz, 2H, H-3,5 of OBz), 6.69 (d, J=10.3 Hz, 1H,

H-5), 6.20 (d, J=10.3 Hz, 1H, H-10), 5.88 (d,

J=10.8 Hz, 1H, H-6), 5.75 (br m, 1H, H-13), 5.73 (br d,

1H, H-2), 4.38 (d, J=12.2 Hz, 1H, H-20a), 4.37 (d,

J=12.7 Hz, 1H, H-20b), 4.32 (d, J=11.7 Hz, 1H, OH-

9), 4.19 (t, J=11.2 Hz, 1H, H-9), 3.42 (d, J=5.8 Hz, 1H,

H-3), 3.24 (s, 1H, OH-4), 2.73 (dd, J=15.1, 3.4 Hz, 1H,

H-14a), 2.46 (dd, J=15.6, 10.3 Hz, 1H, H-14b), 2.16 (s,

3H, OAc), 2.10 (s, 3H, OAc), 1.84 (s, 3H, OAc), 1.72 (s,

3H, H-18), 1.63 (s, 3H, H-19), 1.58 (s, 3H, H-17), 1.09

(s, 3H, H-16); ¹³C NMR (CDCl₃, HMQC data) d 206.1

(C-7), 170.9 (CO±OAc), 170.5 (CO±OAc), 169.9 (CO±

OAc), 166.8 (CO±OBz), 152.8 (C-5), 138.1 (C-12), 136.7

(C-11), 133.8 (C-4 of OBz), 130.1 (C-2,6 of OBz), 129.3

(C-1 of OBz), 128.9 (C-3,5 of OBz), 124.6 (C-6), 77.6 (C-

1), 75.2 (C-9), 74.8 (C-4), 73.3 (C-10), 72.3 (C-2), 71.2

(C-13), 69.0 (C-20), 53.9 (C-3), 51.3 (C-8), 41.8 (C-15),

36.5 (C-14), 29.3 (C-16), 21.3 (OAc), 21.1 (OAc), 20.3

(OAc), 20.2 (C-17), 19.5 (C-19), 15.7 (C-18).

Experimental

Instrumentation

Flash chromatography was performed on Silica Gel 60,

230±400 mesh (EM Science). Thin layer chromato-

graphy was conducted on Silica Gel 60 F₂₅₄pre-coated

TLC plates, 0.25 mm (EM Science). MgSO₄was the

drying agent used in all work up procedures. Analytical

HPLC was performed on a Waters 600^Edelivery system

coupled to a PDA 996 detector. Preparative and semi-

preparative HPLC were carried out on a Waters Delta

Prep 3000 instrument coupled to a UV 486 Tunable

Absorbance detector set at 227 nm (Waters, Montreal,

Quebec, Canada). Analytical HPLC was performed with

two Whatman partisil 10 ODS-2 analytical columns in

series (4.6Â250 mm). Semi-preparative HPLC was per-

formed with two Whatman partisil 10 ODS-2 Mag 9

semi-preparative columns in series (9.4Â250 mm). Pre-

parative HPLC was performed with one partisil 10

ODS-2 MAG-20 preparative column (22Â500 mm).

7-Oxo-13-acetylbaccatin III 6. 9-Dihydro-13-acetyl-

baccatin III 1 (10 mg; 0.016 mmol) was dissolved in

acetone (1.5 mL) and treated with Jones' reagent (50 mL

of a 1 mL solution of CrO₃(0.20 g) in concentrated

H₂SO₄:water (3:7)), at 23 ^ꢀC for 30 min. The reaction

was quenched with KHCO₃, dried, and ®ltered through

dry silica gel. The column was eluted with ether and the

expected 7-oxo-13-acetylbaccatin III 6 was obtained

(6.9 mg, 69%). FABHRMS: C₃₃H₃₈O₁₂Na [M+Na⁺]

NMR and mass spectrometry measurement

All the NMR data were obtained at room temperature

on a Varian UNITY-500 spectrometer operating at

499.84 MHz for proton and at 125.70 MHz for carbon-

13. The NMR spectra were obtained on 5±10 mg dis-

solved in 0.3±0.4 mL of CDCl₃. The solvent was used as

an internal reference (7.25 ppm for proton and 77.0 ppm

for carbon-13). The various 2D spectra were acquired

1

required 649.2261, found 649.2261; H NMR (CDCl₃,

500 MHz) d 8.09 (d, J=7.8 Hz, 2H, H-2,6 of OBz), 7.63

(t, J=7.3 Hz, 1H, H-4 of OBz), 7.49 (t, J=7.6 Hz, 2H,

1276

A. Nikolakakis et al. / Bioorg. Med. Chem. 8 (2000) 1269±1280

H-3,5 of OBz), 6.46 (s, 1H, H-10), 6.12 (t, J=8.7 Hz,

1H, H-13), 5.75 (d, J=6.7 Hz, 1H, H-2), 5.09 (d,

J=6.3 Hz, 1H, H-5), 4.45 (d, J=8.4 Hz, 1H, H-20a),

4.31 (d, J=6.7 Hz, 1H, H-3), 4.27 (d, J=8.2 Hz, 1H, H-

20b), 3.06 (dd, J=18.9, 6.5 Hz, 1H, H-6a), 2.85 (d,

J=18.8 Hz, 1H, H-6b), 2.33 (s, 3H, OAc), 2.23 (om, 2H,

H-l4a/b), 2.21 (s, 3H, OAc), 2.18 (s, 3H, OAc), 2.05 (s,

3H, H-18), 1.85 (s, 3H, H-19). 1.19 (s, 3H, H-17), 1.16 (s,

3H, H-16); ¹³C NMR (CDCl₃, HMQC data) d 205.6 (C-

9), 199.8 (C-7), 170.1 (CO±OAc), 169.6 (CO±OAc), 169.0

(CO±OAc), 166.9 (CO±OBz), 141.5 (C-12), 133.9 (C-4

of OBz), 132.8 (C-11), 130.0 (C-2,6 of OBz), 129.0 (C-1

of OBz), 128.7 (C-3,5 of OBz), 84.6 (C-5), 79.0 (C-1),

78.6 (C-4), 77.2 (C-10), 76.3 (C-20), 74.0 (C-2), 69.4 (C-

13), 64.2 (C-8), 44.8 (C-3), 42.9 (C-15), 41.7 (C-6), 35.7

(C-14), 25.9 (C-l7), 22.2 (OAc), 21.2 (OAc), 20.9 (OAc),

20.8 (C-16), 14.5 (C-19), 14.2 (C-18).

¹³C NMR (CDCl₃, HMQC data) d 202.0 (C-9), 170.4

(CO±OAc), 170.2 (CO±OAc), 169.6 (CO±OAc), 168.9

(CO±OAc), 166.9 (CO±OBz), 141.2 (C-12), 133.7 (C-4

of OBz), 132.2 (C-11), 130.1 (C-2,6 of OBz), 128.8 (C-

3,5 of OBz), 83.8 (C-5), 80.7 (C-4), 78.6 (C-1), 76.2 (C-

20), 75.3 (C-10), 74.2 (C-2), 71.2 (C-7), 69.3 (C-13), 56.0

(C-8), 47.1 (C-3), 42.9 (C-15), 35.3 (C-14), 33.2 (C-6),

26.0 (C-16), 22.2 (OAc), 21.1 (OAc), 20.8 (OAc), 20.5

(C-17), 20.5 (OAc), 14.5 (C-18), 10.4 (C-19).

9-Benzyloxy-13-acetylbaccatin III 9a and 7-benzyloxy-

13 - acetylbaccatin III 9b. A solution of 9-dihydro-13-

acetylbaccatin III (0.15 g; 0.24 mmol) in DMF (9 mL)

was treated with freshly prepared Ag₂O (83 mg;

0.36 mmol). The solution was cooled to 0 ^ꢀC and benzyl

bromide (0.030 mL; 0.25 mmol) was added. The mixture

was stirred at 23 ^ꢀC for 18 h. The slurry was then ®ltered

through a bed of dry silica gel, and rinsed through with

ethyl acetate. The organic solution was then washed with

brine, dried, and evaporated. The residue was chroma-

tographed through silica gel (EtOAc:hexane, gradient of

60:40±75:25) to give 9a (80 mg, 47%) and 9b (11 mg;

6%). FABHRMS of 9a: C₄₀H₄₈O₁₂Na [M+Na⁺]

required 743.3044, found 743.3041; ¹H NMR of 9a

(CDCl₃, 500 MHz) d 8.10 (d, J=7.1 Hz, 2H, H-2,6 of

OBz), 7.62 (t, J=7.3 Hz, 1H, H-4 of OBz), 7.49 (t,

J=7.8 Hz, 2H, H-3,5 of OBz), 7.42±7.28 (m, 5H, ben-

zyl), 6.32 (d, J=11.0 Hz, 1H, H-10), 6.16 (t, J=8.8 Hz,

1H, H-13), 5.82 (d, J=5.6 Hz, 1H, H-2), 5.22 (s, 1H,

OH-7), 4.98 (d, J=10.7 Hz, 1H, CH₂of benzyl), 4.95 (d,

J=8.8 Hz, 1H, H-5), 4.85 (d, J=10.8 Hz, 1H, CH₂of

benzyl), 4.54 (d, J=11.0 Hz, 1H, H9), 4.32 (om, 1H, H-

7), 4.32 (od, J=8.3 Hz, 1H, H-20a), 4.13 (d, J=8.3 Hz,

1H, H-20b), 3.03 (d, J=5.8 Hz, 1H, H-3), 2.61 (dt,

J=15.1, 8.5, 8.5 Hz, 1H, H-6a), 2.27 (s, 3H, OAc), 2.19

(s, 3H, OAc), 2.22 (om, 2H, H-14a/b), 1.96 (s, 3H,

OAc), 1.83 (om, 1H, H-6b), 1.98 (s, 3H, H-18), 1.79 (s,

3H, H-17), 1.78 (s, 3H, H-19), 1.74 (s, 1H, OH-1), 1.27

(s, 3H, H-16); ¹³C NMR of 9a (CDCl₃, HMQC data) d

170.4 (CO±OAc), 169.3 (CO±OAc), 169.0 (CO±OAc),

167.1 (CO±OBz), 140.6 (C-12), 136.9 (C-1 of CH₂ben-

zyl), 133.8 (C-4 of OBz), 133.6 (C-11), 130.1 (C-2,6 of

OBz), 129.1 (C-1 of OBz), 128.8 (C-3,5 of OBz), 128.7

(C-3,5 of CH₂benzyl), 128.2 (C-4 of CH₂benzyl), 126.9

(C-2,6 of CH₂benzyl), 86.4 (C-9), 84.2 (C-5), 81.8 (C-4),

78.5 (C-1), 78.4 (CH₂of benzyl), 76.3 (C-20), 73.5 (C-2),

73.5 (C-10), 72.2 (C-7), 69.6 (C-l3), 46.8 (C-8), 46.5 (C-

3), 42.9 (C-15), 37.2 (C-6), 35.5 (C-14), 28.2 (C-16), 22.8

(OAc), 22.7 (C-17), 21.2 (OAc), 21.1 (OAc), 14.8 (C-18),

12.9 (C-19). FABHRMS of 9b: C₄₀H₄₈O₁₂Na [M+Na⁺]

required 743.3044, found 743.3042; ¹H NMR of 9b

(CDCl₃, 500 MHz) d 8.08 (d, J=7.3 Hz, 2H, H-2,6 of

OBz), 7.60 (t, J=7.6 Hz, 1H, H-4 of OBz), 7.47 (t,

J=7.6 Hz, 2H, H-3,5 of OBz), 7.41±7.33 (m, 5H, ben-

zyl), 6.21 (d, J=11.0 Hz, 1H, H-10), 6.16 (t, J=8.5 Hz,

1H, H-13), 5.74 (d, J=5.9 Hz, 1H, H-2), 5.15 (d,

J=10.2 Hz, 1H, OH-9), 4.98 (d, J=9.0 Hz, 1H, H-5),

4.69 (d, J=10.7 Hz, 1H, CH₂of benzyl), 4.63 (d,

J=10.5 Hz, 1H, CH₂of benzyl), 4.30 (ot, 1H, H-9), 4.31

(od, J=8.0 Hz, 1H, H-20a), 4.22 (dd, J=10.0, 7.1 Hz,

1H, H-7), 4.17 (d, J=8.3 Hz, 1H, H-20b), 3.07 (d,

J=5.9 Hz, 1H, H-3), 2.75 (ddd, J=14.6, 9.2, 7.0 Hz,

1H, H-6a), 2.28 (s, 3H, OAc), 2.19 (s, 3H, OAc), 2.18 (om,

13-Acetyl-^D-seco-5,6-dehydrobaccatin III 7. 9-Dihydro-

13-acetylbaccatin III 1 (10 mg; 0.016 mmol) was dis-

solved in acetone (1.5 mL) and treated with Jones'

reagent (50 mL of a 1 mL solution of CrO₃(0.20 g) in

concentrated H₂SO₄:water (3:7)), at 23 ^ꢀC for 30 min.

The solution was diluted with ethyl acetate, washed with

a saturated solution of NaHCO₃and brine to neutrality,

dried, ®ltered and evaporated. Before puri®cation by

chromatography on silica gel, the product obtained, as

con®rmed by NMR, was 7-oxo-13-acetylbaccatin III 6.

Chromatography on silica gel (EtOAc) aorded 13-acetyl-

d-seco-5,6-dehydrobaccatin III

7

(10 mg, 100%).

FABHRMS: C₃₃H₃₈O₁₂Na [M+Na⁺] required 649.2261,

found 649.2261; ¹H and ¹³C NMR, HMBC and

NOESY (see Table 1).

7,13-Diacetylbaccatin III 8. To a solution of acetic

anhydride (60 mL) in pyridine (1.0 mL) was added,

dropwise over 20 min, a solution of 9-dihydro-13-

acetylbaccatin III 1(38 mg; 0.060 mmol) in pyridine

(1.0 mL). The mixture was stirred at 23 ^ꢀC for 18 h, then

diluted with ethyl acetate, washed with brine, dried, and

evaporated. The residue was dissolved in acetone

(2.0 mL) and treated with Jones' reagent (200 mL of a

1 mL solution of CrO₃(0.20 g) in concentrated

H₂SO₄:water (3:7)), at 0 ^ꢀC for 1 h. The solution was

diluted with ethyl acetate, washed with a saturated

solution of NaHCO₃and brine to neutrality, dried, ®l-

tered and evaporated. The residue was puri®ed by

chromatography on silica gel (EtOAc:hexane, 65:35)

aording 7,13-diacetylbaccatin III (taxane 8, 22 mg,

54% over two steps). FABHRMS: C₃₅H₄₂O₁₃Na

[M+Na⁺] required 693.2523, found 693.2526; ¹H

NMR (CDC1₃, 500 MHz) d 8.08 (d, J=7.3 Hz, 2H, H-

2,6 of OBz), 7.62 (t, J=7.1 Hz, 1H, H-4 of OBz), 7.49 (t,

J=7.8 Hz, 2H, H-3,5 of OBz), 6.26 (s, 1H, H-10), 6.17

(br t, J=8.6 Hz, 1H, H-13), 5.66 (d, J=7.1 Hz, 1H, H-

2), 5.59 (dd, J=10.5, 7.0 Hz, 1H, H-7), 4.97 (d,

J=8.5 Hz, 1H, H-5), 4.32 (d, J=8.3 Hz, 1H, H-20a),

4.16 (d, J=8.3 Hz, 1H, H-20b), 3.96 (d, J=6.6 Hz, 1H,

H-3), 2.61 (ddd, J=14.4, 8.1, 7.1 Hz, 1H, H-6a), 2.34 (s,

3H, OAc), 2.25 (d, J=9.0 Hz, 1H, H-14a), 2.21 (s, 3H,

OAc), 2.18 (s, 3H, OAc), 2.04 (s, 3H, OAc), 1.96 (br s,

3H, H-18), 1.83 (om, 1H, H-6b), 1.80 (s, 3H, H-19), 1.64

(s, 1H, OH-1), 1.21 (s, 3H, H-16), 1.16 (s, 3H, H-17);

A. Nikolakakis et al. / Bioorg. Med. Chem. 8 (2000) 1269±1280

1277

2H, H-14a/b), 2.15 (s, 3H, OAc), 1.97 (dd, J=14.2,

11.2 Hz, 1H, H-6b), 1.85 (s, 3H, H-19), 1.83 (s, 3H, H-

18), 1.70 (s, 3H, H-17), 1.25 (s, 3H, H-16); ¹³C NMR of

9b (CDCl₃, HMQC data) d 170.5 (CO±OAc), 170.5

(CO±OAc), 169.4 (CO±OAc), 167.0 (CO±OBz), 138.5

(C-12), 136.3 (C-1 of CH₂benzyl), 135.7 (C-11), 133.7

(C-4 of OBz), 130.1 (C-2,6 of OBz), 128.7 (C-2,6 of CH₂

benzyl), 128.6 (C-3,5 of CH₂benzyl), 128.5 (C-3,5 of

OBz), 128.4 (C-4 of CH₂benzyl), 83.7 (C-5), 82.0 (C-7),

78.8 (C-1), 76.6 (C-20), 76.3 (C-9), 73.3 (C-2), 72.5

(C-10), 71.5 (CH₂of benzyl), 68.8 (C-13), 47.8 (C-3),

45.2 (C-8), 43.1 (C-15), 35.2 (C-14), 33.9 (C-6), 28.3

(C-16), 22.9 (OAc), 22.5 (C-17), 21.4 (OAc), 21.3 (OAc),

14.8 (C-18), 13.0 (C-19).

and recovered 9-dihydro-13-acetylbaccatin III (1, 7 mg,

23%). FABHRMS: C₄₂H₆₂O₁₂Si+H⁺[M+H⁺] required

787.4089, found 787.4091; H NMR (CDCl₃, 500 MHz)

1

d 8.07 (d, J=7.6 Hz, 2H, H-2,6 of OBz), 7.59 (t,

J=7.2 Hz, 1H, H-4 of OBz), 7.46 (t, J=7.8 Hz, 2H, H-

3,5 of OBz), 6.15 (t, J=8.5 Hz, 1H, H-13), 6.10 (d,

J=11.0 Hz, 1H, H-10), 5.75 (d, J=5.3 Hz, 1H, H-2), 5.49

(d, J=9.5 Hz, 1H, OH-9), 4.91 (d, J=9.1 Hz, 1H, H-5),

4.69 (dd, J=10.3, 7.4 Hz, 1H, H-7), 4.37 (t, J=10.5 Hz,

1H, H-9), 4.30 (d, J=8.5 Hz, 1H, H-20a), 4.17 (d,

J=8.7 Hz, 1H, H-20b), 3.07 (d, J=5.7 Hz, 1H, H-3),

2.58 (ddd, J=12.9, 8.3, 7.7 Hz, 1H, H-6a), 2.26 (s, 3H,

OAc), 2.18 (s, 3H, OAc), 2.15 (om, 2H, H-14a/b), 2.10

(s, 3H, OAc), 2.07 (om, 1H, H-6b), 1.95 (s, 3H, H-18),

1.84 (s, 3H, H-19), 1.69 (s, 3H, H-17), 1.23 (s, 3H, H-

16), 1.12 (m, 21H, isopropyl-H); ¹³C NMR (CDCl₃,

HMQC data) d 170.4 (CO±OAc), 170.1 (CO±OAc),

169.4 (CO±OAc), 167.0 (CO±OBz), 138.6 (C-12), 135.6

(C-11), 133.6 (C-4 of OBz), 130.1 (C-2,6 of OBz), 129.3

(C-1 of OBz), 128.6 (C-3,5 of OBz), 83.7 (C-5), 81.7 (C-

4), 78.9 (C-1), 77.3 (C-7), 76.7 (C-20), 76.2 (C-9), 73.6

(C-2), 73.3 (C-10), 69.9 (C-13), 47.3 (C-3), 45.3 (C-8),

43.0 (C-15), 37.8 (C-6), 35.3 (C-14), 28.4 (C-16), 22.8

(OAc), 22.5 (C-17), 21.3 (OAc), 21.2 (OAc), 18.2 (iso-

propyl-CH₃), 18.1 (isopropyl-CH₃), 14.9 (C-18), 13.5

(C-Si), 12.8 (C-19).

7-Triethylsilyl-9-dihydro-13-acetylbaccatin III 10. 9-

Dihydro-13-acetylbaccatin III (35 mg; 0.056 mmol) in

pyridine (5 mL) was treated with triethylsilyl chloride

(0.19 mL; 1.1 mmol) at 4 ^ꢀC. The solution was allowed

to equilibrate to 23 ^ꢀC and was stirred for 72 h. The

reaction was quenched by addition of ice±water. The

pyridine was evaporated in vacuo and the aqueous resi-

due was extracted with EtOAc. The combined extracts

were dried, ®ltered and evaporated in vacuo. The residue

was puri®ed by chromatography on silica gel (EtOAc:

hexane, 45:55) aording 7-triethylsilyl-9-dihydro-13-

acetylbaccatin III (taxane 10, 31 mg, 75%). FABHRMS:

C₃₉H₅₆O₁₂Si+H⁺[M+H⁺] required 745.3619, found

745.3622; ¹H NMR (CDC1₃, 500 MHz) d 8.07 (d,

J=7.5 Hz, 2H, H-2,6 of OBz), 7.59 (t, J=7.6 Hz, 1H, H-

4 of OBz), 7.46 (t, J=7.5 Hz, 2H, H-3,5 of OBz), 6.16 (ot,

1H, H-13), 6.15 (od, J=10.7 Hz, 1H, H-10), 5.73 (d,

J=5.9 Hz, 1H, H-2), 5.32 (d, J=9.7 Hz, 1H, OH-9),

4.92 (d, J=9.2 Hz, 1H, H-5), 4.55 (dd, J=10.1, 7.1 Hz,

1H, H-7), 4.32 (t, J=10.5 Hz, 1H, H-9), 4.30 (d,

J=8.2 Hz, 1H, H-20a), 4.16 (d, J=8.3 Hz, 1H, H-20b),

3.05 (d, J=6.0 Hz, 1H, H-3), 2.48 (ddd, J=13.8, 8.2,

7.7 Hz, 1H, H-6a), 2.27 (s, 3H, OAc), 2.18 (s, 3H, OAc),

2.17 (om, 2H, H-14a/b), 2.11 (s, 3H, OAc), 1.98 (om,

1H, H-6b), 1.93 (s, 3H, H-18), 1.81 (s, 3H, H-19), 1.69

(s, 3H, H-17), 1.24 (s, 3H, H-16), 1.00 (t, J=8.0 Hz, 9H,

H-triethylsilyl-CH₃), 0.73 (q, J=7.9 Hz, 6H, H-triethyl-

silyl-CH₂); ¹³C NMR (CDCl₃, HMQC data) d 170.4

(CO±OAc), 170.3 (CO±OAc), 169.5 (CO±OAc), 167.0

(CO±OBz), 138.4 (C-12), 135.8 (C-11), 133.6 (C-4 of

OBz), 130.1 (C-2,6 of OBz), 129.3 (C-1 of OBz), 128.6

(C-3,5 of OBz), 83.8 (C-5), 81.9 (C-4), 78.9 (C-1), 76.5

(C-20), 76.3 (C-9), 76.3 (C-7), 73.5 (C-2), 72.6 (C-10),

69.9 (C-13), 47.4 (C-3), 44.9 (C-8), 43.1 (C-15), 38.0

(C-6), 35.3 (C-14), 28.4 (C-16), 22.8 (OAc), 22.6 (C-17),

21.3 (OAc), 21.3 (OAc), 14.9 (C-18), 12.6 (C-19), 6.7

(C-triethylsilyl-CH₃), 5.2 (C-triethylsilyl-CH₂).

7-t-Butyldiphenylsilyl-9-dihydro-13-acetylbaccatin III 12.

9-Dihydro-13-acetylbaccatin III 1 (92 mg; 0.15 mmol) in

DMF (1.4 mL) was treated with imidazole (0.16 g;

2.3 mmol) and t-butyldiphenylsilyl chloride (0.51 mL;

2.0 mmol) at 23 ^ꢀC for 72 h. The reaction mixture was

diluted in EtOAc and washed with brine, dried, ®ltered

and evaporated in vacuo. The residue was puri®ed by

chromatography on silica gel (gradient of EtOAc:hex-

ane, 50:50, to EtOAc, 100%) aording 7-t-butyldiphe-

nylsilyl-9-dihydro-13-acetylbaccatin III (12, 75 mg,

59%), and recovered 9-dihydro-13-acetylbaccatin III (1,

25 mg, 27%). FABHRMS: C₄₉H₆₀O₁₂Si+H⁺[M+H⁺]

1

required 869.3932, found 869.3936; H NMR (CDCl₃,

500 MHz) d 8.04 (d, J=7.5 Hz, 2H, H-2,6 of OBz), 7.90

(m, 2H, H-2,6 of Si-Ph₁), 7.64 (d, J=6.9 Hz, 2H, H-2,6 of

Si-Ph₂), 7.57 (t, J=7.1 Hz, 1H, H-4 of OBz), 7.50±7.30

(om, 8H, remaining aromatic protons of OBz, SiPh₁, Si-

Ph₂), 6.06 (t, J=8.6 Hz, 1H, H-13), 6.02 (d, J=11.2 Hz,

1H, H-10), 5.73 (d, J=6.0 Hz, 1H, H-2), 5.63 (d,

J=9.5 Hz, 1H, OH-9), 4.61 (d, J=9.1 Hz, 1H, H-5), 4.53

(dd, J=10.2, 7.0 Hz, 1H, H-7), 4.43 (t, J=10.4 Hz, 1H,

H-9), 4.20 (d, J=8.2 Hz, 1H, H-20a), 4.12 (d, J=8.3 Hz,

1H, H-20b), 2.89 (d, J=5.9 Hz, 1H, H-3), 2.18 (om, 1H,

H-6a), 2.14 (s, 3H, OAc), 2.13 (s, 3H, OAc), 2.11 (s, 3H,

OAc), 2.10 (om, 2H, H-14a/b), 2.01 (m, 1H, H-6b), 1.95

(s, 3H, H-19), 1.69 (s, 3H, H-18), 1.53 (s, 3H, H-17),

1.19 (s, 3H, H-16), 1.04 (s, 9H, H-Si-t-Bu); ¹³C NMR

(CDCl₃, HMQC data) d 170.4 (CO±OAc), 170.1 (CO±

OAc), 168.6 (CO±OAc), 167.0 (CO±OBz), 139.0 (C-12),

136.2 (C-2,6 of Si-Ph₁), 136.0 (C-2,6 of Si-Ph₂), 135.2

(C-11), 133.6 (C-4 of OBz), 130.1 (C-2,6 of OBz), 133.9,

130.9, 130.5, 129.8, 129.3, 128.6, 128.3, 127.5 (remaining

aromatic C of OBz, Si-Ph₁, Si-Ph₂), 83.6 (C-5), 81.5 (C-

4), 78.8 (C-1), 78.4 (C-7), 76.8 (C-20), 76.1 (C-9), 73.6 (C-

2), 73.6 (C-10), 69.7 (C-13), 47.4 (C-3), 45.3 (C-8), 42.9 (C-

15), 38.2 (C-6), 35.2 (C-14), 28.4 (C-16), 26.9 (C-Si-t-Bu),

7-Triisopropylsilyl-9-dihydro-13-acetylbaccatin III 11. 9-

Dihydro-13-acetylbaccatin III 1 (30 mg; 0.048 mmol) in

DMF (1.0 mL) was treated with imidazole (79 mg;

1.2 mmol) and triisopropylsilyl chloride (0.13 mL;

0.61 mmol) at 23 ^ꢀC for 72 h. The reaction mixture was

diluted in EtOAc and washed with brine, dried, ®ltered

and evaporated in vacuo. The residue was puri®ed by

chromatography on silica gel (gradient of EtOAc:hex-

ane, 50:50, to EtOAc, 100%) aording 7-triisopropylsi-

lyl-9-dihydro-13-acetylbaccatin III (11, 22 mg, 59%),

1278

A. Nikolakakis et al. / Bioorg. Med. Chem. 8 (2000) 1269±1280

22.7 (OAc), 22.4 (C-18), 21.2 (OAc), 21.2 (OAc), 19.0

(C-Si), 14.2 (C-17), 13.2 (C-19).

OAc), 2.17 (om, 2H, H-14a/b), 2.14 (s, 3H, OAc), 2.12

(s, 3H, OAc), 1.94 (m, 1H, H-6b), 1.83 (s, 3H, H-19),

1.58 (s, 3H, H-18), 1.23 (s, 3H, H-17), 1.10 (s, 3H, H-

16), 0.94 (s, 9H, H-Si-t-Bu); ¹³C NMR (CDCl₃, HMQC

data) d 202.1 (C-9), 170.1 (CO±OAc), 169.1 (CO±OAc),

168.9 (CO±OAc), 167.0 (CO±OBz), 141.4 (C-12), 136.1

(C-2,6 of Si-Ph₁), 136.0 (C-2,6 of Si-Ph₂), 134.4 (C-11),

133.6 (C-4 of OBz), 130.0 (C-2,6 of OBz), 132.8, 131.7,

129.6, 129.3, 128.6, 128.0, 127.3 (remaining aromatic C

of OBz, Si-Ph₁, Si-Ph₂), 84.0 (C-5), 80.7 (C-4), 78.9 (C-

1), 76.4 (C-20), 75.9 (C-10), 74.8 (C-2), 73.3 (C-7), 69.6

(C-13), 58.6 (C-8), 47.0 (C-3), 43.1 (C-15), 37.4 (C-6),

35.3 (C-14), 26.5 (C-16), 26.4 (C-Si-t-Bu), 22.4 (OAc),

21.2 (OAc), 20.9 (OAc), 20.6 (C-17), 19.0 (C-Si), 13.7

(C-18), 10.5 (C-19).

7-Triisopropylsilyl-13-acetylbaccatin III 13. 7-Triisopro-

pylsilyl-9-dihydro-13-acetylbaccatin III 11 (14 mg; 0.018

mmol) was dissolved in acetone (2.6 mL) and treated with

Jones' reagent (120mL of a 1 mL solution of CrO₃(0.20 g)

in concentrated H₂SO₄:water (3:7)), at 23^ꢀC for 30min.

The solution was diluted with ethyl acetate, washed with a

saturated solution of NaHCO₃and brine to neutrality,

dried, ®ltered and evaporated. Chromatography on silica

gel (EtOAc:hexane, 50:50) aorded 7-triisopropylsilyl-13-

acetylbaccatin III (taxane 13, 3 mg, 21%). FABHRMS:

C₄₂H₆₀O₁₂Si+H⁺[M+H⁺] required 785.3932, found

785.3930; ¹H NMR (CDCl₃, 500 MHz) d 8.07 (d,

J=7.6 Hz, 2H, H-2,6 of OBz), 7.59 (t, J=7.2 Hz, 1H,

H-4 of OBz), 7.46 (t, J=7.8 Hz, 2H, H-3,5 of OBz), 6.47

(s, 1H, H-10), 6.15 (br t, J=8.3 Hz, 1H, H-13), 5.67 (d,

J=7.1 Hz, 1H, H-2), 4.95 (br d, J=8.6 Hz, 1H, H-5),

4.62 (dd, J=10.7, 6.6 Hz, 1H, H-7), 4.31 (d, J=8.5 Hz,

1H, H-20a), 4.16 (d, J=8.3 Hz, 1H, H-20b), 3.84 (d,

J=7.1 Hz, 1H, H-3), 2.60 (ddd, J=14.4, 9.8, 6.6 Hz,

1H, H-6a), 2.33 (s, 3H, OAc), 2.23 (om, 2H, H-14a/b),

2.21 (s, 3H, OAc), 2.16 (s, 3H, OAc), 2.07 (s, 3H, H-18),

1.97 (ddd, J=13.4, 11.2,2.0 Hz, 1H, H-6b), 1.72 (s, 3H,

H-19), 1.23 (s, 3H, H-17), 1.16 (s, 3H, H-16), 1.04 (m,

21H, isopropyl-H).

7-t-Butyldiphenylsilylbaccatin III 15. (Method A): 7-t-

Butyldiphenylsilyl-13-acetylbaccatin III 14 (80 mg;

0.092 mmol) was dissolved in tetrahydrofuran (18 mL)

and cooled to 44 ^ꢀC. To this solution a 2.5 M solution

of n-BuLi in hexanes (0.12 mL; 0.29 mmol) was added

and the mixture was stirred at 44 ^ꢀC for 1 h. n-BuLi

(0.12 mL) was added again and the reaction was stirred

for an additional 1.5 h. The reaction was quenched with

brine and diluted in EtOAc. The organic layer was

separated, washed with brine to neutrality, dried, ®ltered

and evaporated in vacuo. The residue was puri®ed by

chromatography on silica gel (EtOAc:hexane, gradient

of 40:60 to 50:50) aording 7-t-butyldiphenylsilylbacca-

tin III (15, 22 mg, 29%) and recovered starting material,

7-t-butyldiphenylsilyl-13-acetylbaccatin III (14, 30 mg,

38%). (Method B): To a solution of 7-t-butyldiphenyl-

silyl-13-acetylbaccatin III 14 (24mg; 0.028 mmol) in THF

(0.9 mL) was added 0.05 M potassium phosphate buer,

pH 7.0 (0.45 mL). The opaque solution was treated with

NaBH₄(4Â4 mg; 0.11 mmol) at 23 ^ꢀC over a period of

8 h. The reaction was quenched by dilution with brine.

The mixture was diluted in EtOAc, washed with brine

to neutrality, dried, ®ltered and evaporated in vacuo.

The residue was puri®ed by chromatography as in

method A, aording 7-t-butyldiphenylsilylbaccatin III

(15, 15mg, 66%) and recovered starting material, 7-t-

butyldiphenylsilyl-13-acetylbaccatin III (14, 2 mg, 8%).

FABHRMS of 15: C₄₇H₅₆O₁₁Si+H⁺[M+H⁺] required

825.3670, found 825.3673; ¹H NMR (CDCl₃, 500 MHz) d

8.08 (d, J=6.3 Hz, 2H, H-2,6 of OBz), 7.73 (m, 2H, H-2,6

of Si-Ph₁), 7.58 (ot, 1H, H-4 of OBz), 7.57 (od, 2H, H-

2,6 of Si-Ph₂), 7.48 (om, 3H, H-3,4,5 of Si-Ph₁), 7.45 (ot,

J=7.5 Hz, 2H, H-3,5 of OBz), 7.38 (t, J=7.3 Hz, 1H,

H-4 of Si-Ph₂), 7.32 (t, J=7.6 Hz, 2H, H-3,5 of Si-Ph₂),

6.11 (s, 1H, H-10), 5.61 (d, J=7.1 Hz, 1H, H-2), 4.77

(om, 1H, H-13), 4.77 (om, 1H, H-5), 4.35 (dd, J=10.5,

6.8 Hz, 1H, H-7), 4.23 (d, J=8.5 Hz, 1H, H-20a), 4.12

(d, J=8.3 Hz, 1H, H-20b), 3.69 (d, J=6.9 Hz, 1H, H-3),

2.23 (ddd, J=14.6, 9.5, 6.6 Hz, 1H, H-6a), 2.18 (om,

2H, H-14a/b), 2.16 (s, 3H, OAc), 2.14 (s, 3H, OAc), 1.95

(ddd, J=15.1, 10.7, 2.0 Hz, 1H, H-6b), 1.88 (d,

J=4.9 Hz, 1H, OH-13), 1.84 (s, 3H, H-19), 1.69 (s, 3H,

H-18), 1.58 (s, 1H, OH-1), 1.21 (s, 3H, H-17), 0.97 (s, 3H,

H-16), 0.95 (s, 9H, H-Si-t-Bu); ¹³C NMR (CDCl₃, HMQC

data) d 202.4 (C-9), 170.0 (CO±OAc), 169.4 (CO±OAc),

167.1 (CO±OBz), 144.5 (C-12), 136.2 (C-2,6 of Si-Ph1),

136.2 (C-2,6 of Si-Ph₂), 133.7 (C-4 of OBz), 132.0

13-Acetylbaccatin III 17. 7-Triisopropylsilyl-13-acetyl-

baccatin III 13 was deprotected using the following

conditions: tetra-n-butylammonium ¯uoride in THF at

0 ^ꢀC for 18 h. The mixture was analyzed by analytical

HPLC (25±100% CH₃CN in H₂O, 50 min gradient at a

¯ow rate of 1 mL/min) and showed the formation of

taxane 17 (R_t32.78 min, 24%), taxane 16 (R_t37.66 min,

16%) and starting material 13 (R_t60.56 min, 43%). The

identity of taxane 17 was con®rmed by comparison with

standard 13-acetylbaccatin III²⁴with an additional gra-

dient (R_t38.70 min, analytical HPLC, 25±100% CH₃CN

in H₂O, 70 min gradient at a ¯ow rate of 1 mL/min).

7-t-Butyldiphenylsilyl-13-acetylbaccatin III 14. 7-t-Butyl-

diphenylsilyl-9-dihydro-13-acetylbaccatin III 12 (95 mg;

0.11 mmol) dissolved in acetone (16 mL) was treated

with Jones' reagent (0.79 mL of a 1 mL solution of CrO₃

(0.20 g) in concentrated H₂SO₄:water, 3:7) at 23 ^ꢀC for

30 min. The reaction mixture was diluted in ethyl acetate

and washed with a saturated solution of NaHCO₃and

with brine to neutrality. The organic phase was dried,

®ltered and evaporated in vacuo. The residue was pur-

i®ed by chromatography on silica gel (EtOAc:hexane,

40:60) aording 7-t-butyldiphenylsilyl-13-acetylbaccatin

III (14, 73 mg, 77%). FABHRMS: C₄₉H₅₈O₁₂Si+H⁺

1

[M+H⁺] required 867.3776, found 867.3772; H NMR

(CDCl₃, 500 MHz) d 8.04 (d, J=7.8 Hz, 2H, H-2,6 of

OBz), 7.72 (m, 2H, H-2,6 of Si-Ph₁), 7.58 (od, 2H, H-2,6

of Si-Ph₂), 7.58 (ot, 1H, H-4 of OBz), 7.50±7.30 (om,

8H, remaining aromatic protons of OBz, SiPh₁, Si-Ph₂),

6.15 (s, 1H, H-10), 6.03 (t, J=8.7 Hz, 1H, H-13), 5.64

(d, J=6.8 Hz, 1H, H-2), 4.69 (d, J=9.3 Hz, 1H, H-5),

4.34 (dd, J=10.4,6.6 Hz, 1H, H-7), 4.21 (d, J=8.3 Hz,

1H, H-20a), 4.11 (d, J=8.3 Hz, 1H, H-20b), 3.65 (d,

J=6.8 Hz, 1H, H-3), 2.23 (om, 1H, H-6a), 2.19 (s, 3H,

A. Nikolakakis et al. / Bioorg. Med. Chem. 8 (2000) 1269±1280

1279

(C-11), 130.1 (C-2,6 of OBz), 130.1 (C-3,5 of OBz),

129.6 (C-4 of SiPh₂), 128.5 (C-3,5 of Si-Ph₁), 127.9 (C-4

of Si-Ph₁), 127.4 (C-3,5 of Si-Ph₂), 83.8 (C-5), 80.5 (C-4),

78.6 (C-1), 76.4 (C-10), 76.4 (C-20), 74.5 (C-2), 73.2

(C-7), 67.7 (C-13), 58.5 (C-8), 47.1 (C-3), 42.5 (C-15),

37.8 (C-14), 37.3 (C-6), 26.3 (C-16), 26.3 (C-Si-t-Bu),

22.5 (OAc), 20.8 (OAc), 19.7 (C-17), 18.9 (C-Si), 13.7

(C-18), 10.1 (C-19).

1H, OH-7), 2.32 (s, 3H, OAc), 2.24 (om, 2H, H-14a/b),

2.24 (s, 3H, OAc), 2.20 (s, 3H, OAc), 1.90 (d, J=1.1 Hz,

3H, H-18), 1.67 (s, 3H, H-19), 1.23 (s, 3H, H-16), 1.13

(s, 3H, H-l7); ¹³C NMR (CDCl₃, HMQC data) d 203.4

(C-9), 170.9 (CO±OAc), 169.6 (CO±OAc), 169.2 (CO±

OAc), 166.6 (CO±OBz), 142.5 (C-12), 134.2 (C-4 of

OBz), 132.2 (C-11), 130.3 (C-2,6 of OBz), 128.8 (C-3,5

of OBz), 84.6 (C-5), 80.5 (C-4), 78.7 (C-1), 76.3 (C-20),

75.8 (C-10), 75.1 (C-2), 72.4 (C-7), 69.7 (C-13), 58.1 (C-

8), 45.6 (C-3), 42.6 (C-15), 35.6 (C-14), 35.6 (C-6), 26.7

(C-16), 22.4 (OAc), 21.4 (C-17), 21.1 (OAc), 20.9 (OAc),

15.0 (C-18), 9.4 (C-l9).

7-Epi-13-acetylbaccatin III 16. 7-Oxo-13-acetylbaccatin

III 6 (46mg; 0.073 mmol) was dissolved in methanol

(10 mL) and treated with NaBH₄(6mg; 0.16mmol) at

4 ^ꢀC, for 2 h. The reaction was quenched by dilution with

brine. The solution was extracted with dichloromethane,

and the combined organic layers were dried, ®ltered and

evaporated in vacuo. The residue was puri®ed by chro-

matography on silica gel (EtOAc:hexane, 50:50) aording

7-epi-13-acetylbaccatin III (16, 38mg, 82%). FABHRMS:

C₃₃H₄₀O₁₂Na [M+Na⁺] required 651.2418, found

651.2420; ¹H NMR (CDCl₃, 500 MHz) d 8.08 (dd,

J=7.8, 0.7 Hz, 2H, H-2,6 of OBz), 7.63 (t, J=7.3 Hz,

1H, H-4 of OBz), 7.50 (t, J=7.5 Hz, 2H, H-3,5 of OBz),

6.83 (s, 1H, H-10), 6.13 (br t, J=8.0 Hz, 1H, H-13), 5.75

(d, J=7.3 Hz, 1H, H-2), 4.95 (dd, J=9.3, 3.2 Hz, 1H, H-

5), 4.66 (d, J=11.7 Hz, 1H, OH-7), 4.40 (d, J=8.5 Hz,

1H, H-20a), 4.35 (d, J=8.5 Hz, 1H, H-20b), 3.96 (d,

J=7.3 Hz, 1H, H-3), 3.70 (ddd, J=11.7, 4.6, 1.7 Hz, 1H,

H-7), 2.41 (s, 3H, OAc), 2.36 (ddd, J=15.9, 9.5, 2.0 Hz,

1H, H-6a), 2.27 (om, 1H, H-6b), 2.27 (om, 2H, H-14a/

b), 2.21 (s, 3H, OAc), 2.20 (s, 3H, OAc), 1.87 (d,

J=1.2 Hz, 3H, H-18), 1.65 (s, 3H, H-19), 1.19 (s, 3H, H-

16), 1.15 (s, 3H, H-17); ¹³C NMR (CDCl₃, HMQC

data) d 207.3 (C-9), 171.6 (CO±OAc), 170.2 (CO±OAc),

169.4 (CO±OAc), 167.0 (CO±OBz), 140.6 (C-12), 133.9

(C-4 of OBz), 133.0 (C-11), 130.0 (C-2,6 of OBz), 129.3

(C-1 of OBz), 128.6 (C-3,5 of OBz), 82.5 (C-5), 82.1 (C-

4), 79.0 (C-1), 78.1 (C-10), 77.5 (C-20), 75.4 (C-7), 75.2

(C-2), 69.6 (C-13), 57.5 (C-8), 42.3 (C-15), 40.2 (C-3),

35.8 (C-14), 35.2 (C-6), 25.8 (C-16), 22.3 (OAc), 20.8

(OAc), 20.8 (OAc), 20.6 (C-17), 15.9 (C-19), 15.0 (C-18).

Baccatin III 3. To a solution of 13-acetylbaccatin III 17

(10 mg; 0.016 mmol) in THF (0.6 mL) was added 0.05 M

potassium phosphate buer, pH 7.0 (0.3mL). The opaque

solution was treated with NaBH₄(2Â5 mg; 0.13mmol) at

23^ꢀC over a period of 10 h. The reaction was quenched by

dilution with brine. The solution was extracted with di-

chloromethane, and the combined organic layers were

dried, ®ltered and evaporated in vacuo. The residue was

puri®ed by semi-preparative HPLC (25±100% CH₃CN

in H₂O, 50 min gradient at a ¯ow rate of 3 mL/min)

aording 3 (4.5 mg, 48%, R_t=28.45 min). FABHRMS:

C₃₁H₃₈O₁₁Na [M+Na⁺] required 609.2312, found

609.2313; The NMR spectroscopic properties of 3 were

identical to those of baccatin III.

Reduction of 7-oxo-13-acetylbaccatin III 6 with lithium

tri-tert-butoxyaluminohydride. Lithium tri-tert-butoxy-

aluminohydride (9 mg; 0.035 mmol) was added to THF

(0.875mL) at 4 ^ꢀC. To this cooled solution was added

dropwise 7-oxo-13-acetylbaccatin III 6 (5 mg; 0.008 mmol)

dissolved in THF (0.125 mL). The solution was stirred

at 4 ^ꢀC for 30 min and the reaction was quenched by

dilution with brine. The solution was diluted with ether,

washed with brine, dried, ®ltered and evaporated in

vacuo. The residue was puri®ed by chromatography on

silica gel (EtOAc:CH₂Cl₂, 20:80) aording 7 (1 mg,

20%) (Scheme 1) and 16 (1 mg, 20%) (Scheme 2).

13-Acetylbaccatin III 17. To a solution of 7-epi-13-acet-

ylbaccatin III 16 (7 mg; 0.011 mmol) in toluene (0.5 mL)

was added 1,8-diazabicyclo[5,4,0]undec-7-ene (55 mL;

0.37 mmol) and the mixture was stirred at 80 ^ꢀC for

1.5 h. The reaction mixture was then diluted in EtOAc

and washed with dilute HCl, a saturated solution of

NaHCO₃and brine. The organic layer was then dried,

®ltered and evaporated in vacuo. The residue was pur-

i®ed by semi-preparative HPLC (25±100% CH₃CN in

H₂O, 50 min gradient at a ¯ow rate of 3 mL/min) aord-

ing 13-acetybbaccatin III (17, 2 mg, 29%, R_t=33.58 min)

and recovered 7-epi-13-acetylbaccatin III (16, 4 mg,

57%, R_t=37.98 min). FABHRMS of 17: C₃₃H₄₀O₁₂Na

[M+Na⁺] required 651.2418, found 651.2420; ¹H NMR

(CDCl₃, 500 MHz) d 8.07 (d, J=7.6 Hz, 2H, H-2,6 of

OBz), 7.61 (t, J=7.5 Hz, 1H, H-4 of OBz), 7.48 (t,

J=7.4 Hz, 2H, H-3,5 of OBz), 6.30 (s, 1H, H-10), 6.18 (dd,

J=9.5, 8.2 Hz, 1H, H-13), 5.66 (d, J=7.2 Hz, 1H, H-2),

4.97 (dd, J=9.8, 1.6 Hz, 1H, H-5), 4.43 (br m, 1H, H-7),

4.30 (d, J=8.1 Hz, 1H, H-20a), 4.16 (d, J=8.5 Hz, 1H,

H-20b), 3.82 (d, J=6.9 Hz, 1H, H-3), 2.55 (ddd,

J=14.7, 9.4, 6.2 Hz, 1H, H-6a), 2.46 (br d, J=3.0 Hz,

Taxane 18. 7-Oxo-13-acetylbaccatin III 6 (15 mg;

0.024 mmol) was dissolved in CH₂Cl₂(1.5 mL). To this

solution was added n-Bu₄NBH₄(6 mg; 0.023 mmol) and

the mixture was stirred at 23 ^ꢀC for 30 min. The reaction

was stopped by addition of a potassium phosphate buf-

fer, pH 7.0. The solution was extracted with CH₂Cl₂and

the combined organic layers were washed with brine,

dried, ®ltered and evaporated in vacuo. The residue was

puri®ed by semi-preparative HPLC (25±100% CH₃CN

in H₂O, 70 min gradient at a ¯ow rate of 3 mL/min)

aording 18 (3 mg, 20%, R_t=43.93 min). FABHRMS:

C₃₃H₄₀O₁₂Na [M+Na⁺] required 651.2417, found

651.2420. For ¹H NMR and ¹³C NMR data see Table 2.

13-Acetyl-^D-seco-5,6-dehydro-20-deacetylbaccatin III 7a.

7-Oxo-13-acetylbaccatin III 6 (5 mg; 0.008 mmol) was

dissolved in THF (1 mL), and was reduced with a 1.0 M

solution of lithium tri-sec-butylborohydride (l-Selec-

tride¹) (10 mL; 0.010 mmol) at 4 ^ꢀC for 1 h. More l-

Selectride¹(10 mL; 0.010 mmol) was added and after

30 min at 4 ^ꢀC the reaction was quenched by dilution

with brine. The solution was diluted with ether, washed

1280

A. Nikolakakis et al. / Bioorg. Med. Chem. 8 (2000) 1269±1280

with brine, dried, ®ltered and evaporated in vacuo. The

residue was puri®ed by chromatography on silica gel

(EtOAc:hexane, 50:50) aording 7 (3 mg, 60%) and 7a

2. Zamir, L. O.; Nedea, M. E.; Zhou, Z.-H.; Belair, S.; Caron,

G.; Sauriol, F.; Jacqmain, E.; Jean, F. I.; Garneau, F.-X.;

Mamer, O. Can. J. Chem. 1995, 73, 655.

1

3. Zamir, L. O.; Zhou, Z.-H.; Caron, G.; Nedea, M. E.;

Sauriol, F.; Mamer, O. J. Chem. Soc., Chem. Commun. 1995, 529.

4. Zamir, L. O.; Nedea, M. E.; Zhou, Z.-H.; Caron, G.;

Sauriol, F.; Mamer, O. Phytochemistry 1996, 41, 803.

5. Zamir, L. O.; Zhang, J.; Kutterer, K.; Sauriol, F.; Mamer,

O.; Khiat, A.; Boulanger, Y. Tetrahedron 1998, 54, 15845.

6. Zamir, L. O.; Zhang, J.; Wu, J.; Sauriol, F.; Mamer, O. J.

Nat. Prod. 1999, 62, 1268.

7. Zhang, J.; Wu, J.; Sauriol, F.; Mamer, O.; Zamir, L. O.

Phytochemistry 2000, 54 (2), 221±230.

8. Gunawardana, G. P.; Premachandran, U.; Burres, N. S.;

Whittern, D. N.; Henry, R.; Spanton, S.; McAlpine, J. B. J.

Nat. Prod. 1992, 55, 1686.

9. Zhang, S.; Chen, W. M.; Chen, Y. H. Yaoxue Xuebao 1992,

27, 268.

10. Ter Haar, E. Exp. Opin. Ther. Patents 1998, 8, 571.

11. Zamir, L. O.; Zheng, Y. F.; Caron, G.; Sauriol, F.;

Mamer, O. Tetrahedron Lett. 1996, 37, 6435.

12. Zamir, L. O.; Balachandran, S.; Zheng, Y. F.; Nedea, M.

E.; Caron, G.; Nikolakakis, A.; Vishwakarma, R. A.; Sauriol,

F.; Mamer, O. Tetrahedron 1997, 53, 15991.

(2 mg, 43%). H NMR (CDCl₃, 500 MHz) d 7.84 (d,

J=7.6 Hz, 2H, H-2,6 of OBz), 7.61 (t, J=7.4 Hz, 1H,

H-4 of OBz), 7.45 (t, J=7.3 Hz, 2H, H-3,5 of OBz), 6.62

(d, J=9.5 Hz, 1H, H-5), 6.66 (s, 1H, H-10), 5.38 (d,

J=9.5 Hz, 1H, H-6), 5.89 (br m, 1H, H-13), 5.79 (d,

J=9.5 Hz, 1H, H-2), 4.76 (d, J=11.7 Hz, 1H, H-20a),

4.42 (od, 1H, H-20b), 4.40 (od, 1H, H-3), 2.46 (dd,

J=16.1, 10.0 Hz, 1H, H-14a), 2.36 (dd, J=16.3, 3.9 Hz,

1H, H-14b), 2.25 (s, 3H, OAc), 2.21 (s, 3H, OAc), 1.92

(br s, 3H, H-18), 1.38 (s, 3H, H-19), 1.14 (s, 3H, H-17),

1.12 (s, 3H, H-16); ¹³C NMR (CDCl₃, HMQC data)

d 206.3 (C-9), 201.7 (C-7), 170.2 (CO±OAc), 169.7 (CO±

OAc), 166.7 (CO±OBz), 143.7 (C-5), 139.3 (C-12), 135.7

(C-11), 134.3 (C-4 of OBz), 129.7 (C-1 of OBz), 129.5

(C-2,6 of OBz), 129.0 (C-3,5 of OBz), 119.9 (C-6), 79.1

(C-1), 78.5 (C-4), 76.4 (C-10), 75.8 (C-20), 73.6 (C-2),

69.5 (C-13), 63.9 (C-8), 47.3 (C-3), 41.8 (C-15), 36.3 (C-

14), 27.6 (C-16), 22.2 (C-19), 20.7 (OAc), 20.7 (OAc),

19.1 (C-17), 15.7 (C-18).

13. Zamir, L. O.; Cherestes, A. D.; Nikolakakis, A.; Sauriol,

F.; Mamer, O. Tetrahedron Lett. 1999, 40, 7917.

14. (a) Denis, J.-N.; Greene, A. E.; Guenard, D.; Gueritte-

Voegelein, F.; Mangatal, L.; Potier, P. J. Am. Chem. Soc.

1988, 110, 5917. (b) Baloglu, E.; Kingston, D. G. I. J. Nat.

Prod. 1999, 62, 1068.

15. Magri, N. F.; Kingston, D. G. I. J. Org. Chem. 1986, 51,

797.

16. Marder-Karsenti, R.; Dubois, J.; Chiaroni, A.; Riche, C.;

Guenard, D.; Gueritte, F.; Potier, P. Tetrahedron 1998, 54,

15833.

17. Klein, L. L.; Li, L.; Maring, C. J.; Yeung, C. M.; Thomas,

S. A.; Grampovnik, D. J.; Plattner, J. J. J. Med. Chem. 1995,

38, 1482.

18. Ley, S. V.; Norman, J.; Grith, W. P. Synthesis 1994, 639.

19. Klein, L. L. Tetrahedron Lett. 1993, 34, 2047.

20. Hanessian, S.; Lavallee, P. Can. J. Chem. 1975, 53, 2975.

21. Cainelli, G.; Manescalchi, F.; Panunzio, M. Synthesis

1976, 472

Taxane 19. A 1.0 M solution of lithium tri-sec-butyl-

borohydride in THF (32 mL, 0.032 mmol) was added to

THF (0.85 mL) at 4 ^ꢀC. To this cooled solution was

added dropwise 7-oxo-13-acetylbaccatin III 6 (5 mg;

0.008 mmol) dissolved in THF (165 mL). The solution

was stirred for 2 h at 4 ^ꢀC. The reaction was quenched

with brine and extracted with ether, dried, ®ltered and

evaporated in vacuo. The residue was puri®ed by semi-

preparative HPLC (25±100% CH₃CN in H₂O, 50 min

gradient at a ¯ow rate of 3 mL/min) aording 19

(1.45 mg, 30%, R_t=43.25 min) and 13-acetyl-d-seco-

5,6-dehydro-20-deacetylbaccatin III 7a (1.25 mg, 27%,

R_t=35.07 min). FABHRMS of 19: C₃₃H₃₈O₁₂Na

1

[M+Na⁺] required 649.2261, found 649.2260; For H

and ¹³C NMR data see Table 3.

22. Yuan, H.; Kingston, D. G. I. Tetrahedron Lett. 1998, 39,

4967.

Acknowledgements

23. Fang, W.-S.; Fang, Q.-C.; Liang, X.-T. Synthetic Commun.

1997, 27, 2305.

We thank the Natural Science and Engineering

Research Council of Canada for support via an operat-

ing grant to L. O. Z.

24. Ojima, I.; Bounaud, P.-Y.; Takeuchi, C.; Pera, P.; Ber-

nacki, R. J. Bioorg. Med. Chem. Lett. 1998, 8, 189.

25. Kingston, D. G. I.; Molinero, A. A.; Rimoldi, J. M. In

Progress in the Chemistry of Organic Natural Products; Herz,

W.; Kirby, G. W.; Moore, R. E.; Steglich, W.; Tamm, Ch.,

Eds.; Springer-Verlag: New York, 1993; Vol. 61, pp 1±206.

26. We thank a referee for making this interesting suggestion.

27. Hartzell, H. In The Yew Tree, a Thousand Whispers;

Hulogosi: Eugene, OR, 1991; pp 59, 73.

References

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Lett. 1992, 33, 5173.

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