Preparation of phenolic paclitaxel metabolites

Article abstract of DOI:10.1021/jm960142x

The synthesis and biological evaluation of the two known phenolic metabolites of paclitaxel are described. The C3'-phenolic metabolite 2 of paclitaxel was prepared from 7-(triethylsilyl)-baccatin III (8) and enantioenriched N-benzoyl-2-azetidinone 7. The C2-phenolic metabolite 3 was synthesized from paclitaxel (1a) via selective C2 debenzoylation and reacylation.

Full text of DOI:10.1021/jm960142x

J . Med. Chem. 1996, 39, 2705-2709
2705
P r ep a r a tion of P h en olic P a clita xel Meta bolites
Haeil Park,† Michael Hepperle,† Thomas C. Boge,† Richard H. Himes,‡ and Gunda I. Georg*,†
Departments of Medicinal Chemistry and Biochemistry, University of Kansas, Lawrence, Kansas 66045
Received February 16, 1996X
The synthesis and biological evaluation of the two known phenolic metabolites of paclitaxel
are described. The C3′-phenolic metabolite 2 of paclitaxel was prepared from 7-(triethylsilyl)-
baccatin III (8) and enantioenriched N-benzoyl-2-azetidinone 7. The C2-phenolic metabolite 3
was synthesized from paclitaxel (1a ) via selective C2 debenzoylation and reacylation.
In tr od u ction
Paclitaxel (1a ; Figure 1) is regarded as one of the most
promising new agents for the treatment of drug-refrac-
tory ovarian cancer and metastatic breast cancer.1,2
Clinical evaluations of its potential for the chemo-
therapy of other cancers have revealed impressive
response rates for head and neck cancer, lung cancer,
and esophageal carcinomas.1,2 Paclitaxel is a mitotic
spindle poison with a unique mechanism of action,
promoting the formation of stable microtubules.3
Although paclitaxel has undergone detailed clinical,
biochemical, chemical, and structure-activity investiga-
tions,4 its in vivo drug disposition is not yet fully
understood.5,6 Since early pharmacological studies re-
vealed insignificant urinary excretion of paclitaxel, it
F igu r e 1. Structures of paclitaxel (1a ) and docetaxel (1b) and
the phenolic paclitaxel metabolites 2 and 3.
became clear that protein binding, metabolism, and
biliary excretion must play a significant role in its
disposition.5
human bile provided evidence for the formation of five
to seven metabolites in addition to unchanged paclitaxel
(3-5%).5,9 The major human metabolite is 6R-hydroxy-
paclitaxel (12-16%).5,9,10 6R-Hydroxypaclitaxel is also
the major metabolite in human liver microsomes.11-13
Metabolite 2 was found in 2% and other products in
about 3% in human bile as percent of the total admin-
istered dose, including the dihydroxylated paclitaxel
metabolite 3′-dephenyl-3′-(4-hydroxyphenyl)-6R-hydroxy-
paclitaxel.5,6 3′-Hydroxyphenyl metabolite 2 was also
identified as a metabolism product in human liver
microsomes.13,14 The identities of other metabolites in
human liver microsomes and additional metabolites
found in plasma5,9,15 are not known.
It is interesting to note that human and rat biliary
metabolites of docetaxel (1b; Figure 1), a semisynthetic
analogue of paclitaxel, are quite different from metabo-
lites of paclitaxel.6,16 Apparently, oxidative metabolism
takes place exclusively at the N-tert-butoxycarbonyl
group of docetaxel in both humans and rats. No
docetaxel metabolites modified at the 3′-phenyl group
or at the taxane ring system have been identified.
Although the chemical synthesis of docetaxel metabo-
lites has been described,17 the preparation of paclitaxel
metabolites has not been reported yet. Herein we wish
to detail the synthesis of the two known phenolic
paclitaxel metabolism products 2 and 3, through meth-
odology developed in our laboratory for the preparation
of 3′-phenyl-modified18 and 2-benzoate-modified19 pacli-
taxel analogues.
A paclitaxel metabolism study, conducted in rats,
investigated the elimination of nonradioactive paclitaxel
in bile and urine.7 Only about 10% of paclitaxel was
recovered in the urine of the rats. Biliary excretion
accounted for 12% of unchanged paclitaxel and 29% of
metabolites. Nine metabolites were detected, four of
which could be identified, but the identity of the other
five is still unknown.6 Two phenolic compounds were
found as the principal metabolites. The major metabo-
lite (13%) carries a hydroxyl group at the para position
of the 3′-phenyl group in the C13 side chain (2; Figure
1). The second phenolic metabolite, accounting for 5%
of the administered dose, is hydroxylated at the meta
position of the 2-benzoate group (3; Figure 1). Baccatin
III, which is obtained after hydrolysis of the C13-ester
group in paclitaxel, and a C19-hydroxylated product
were identified among the minor metabolites accounting
for less than 10%. The metabolites 2 and 3, identified
in these in vivo studies, were also detected when
metabolism assays were carried out with rat hepato-
cytes.8 Cytochrome P450 3A enzymes appear to effect
the observed aromatic hydroxylation.8
Biliary excretion is also the major pathway for human
paclitaxel clearance; however, the metabolic products
are different. In one metabolism study, 10% of the
administered paclitaxel dose was recovered unchanged
from urine and 20% from bile.5 In another disposition
study, about 14% of paclitaxel and an unknown major
polar metabolite were found in urine, yet the total fecal
excretion amounted to over 71%.9 Examination of
Resu lts a n d Discu ssion
† Department of Medicinal Chemistry.
The 3′-phenyl-hydroxylated paclitaxel metabolite was
synthesized by sodium hydride-mediated acylation of
‡ Department of Biochemistry.
X Abstract published in Advance ACS Abstracts, J une 1, 1996.
S0022-2623(96)00142-2 CCC: $12.00 © 1996 American Chemical Society

2706 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 14
Park et al.
Sch em e 1a
Sch em e 2a
a
(a) LDA, THF, -78 °C to room temperature, 12 h; (b) BzCl,
Et3N, DMAP, 1 h; (c) 7-(triethylsilyl)baccatin III (8), NaH, THF,
35 °C, 4 h; (d) pyridinium hydrogen fluoride, pyridine, 0 °C to room
temperature, 8 h.
a
(a) Imidazole, TBSCl, DMF, room temperature, 8 h, then
TESCl, 5 h; (b) t-BuOK, H2O, THF, -40 to -15 °C, 24 h; (c)
3-(benzyloxy)benzoic acid, DMAP, DCC, toluene, 55 °C, 20 h; (d)
pyridinium hydrogen fluoride, pyridine, 0 °C to room temperature,
6 h; (e) Pd/C, cyclohexene:ethanol ) 1:1, 60 °C, 2 h.
protected baccatin III 8 with enantioenriched (3R,4S)-
N-benzoyl-2-azetidinone 7 (Scheme 1). The asymmetric
ester enolate-imine cyclocondensation reaction of gly-
colate 420 and N-(trimethylsilyl)benzaldimine 5 resulted
in the formation of â-lactam 6 in 32% yield and 80% ee.
Acylation of 6 with benzoyl chloride in the presence of
triethylamine and a catalytic amount of N,N-(dimethyl-
amino)pyridine (DMAP) provided N-benzoyl â-lactam 7
in 60% yield. Reaction of the sodium alkoxide of
7-(triethylsilyl)baccatin III (8)21 with compound 7 af-
forded protected taxane 9 in 80% yield. Due to kinetic
resolution in the acylation step, only one diastereo-
isomer of 9 was formed. Simultaneous deprotection of
all three silyl groups with pyridinium hydrofluoride
produced a 90% yield of paclitaxel metabolite 2.
12 in 82% yield. Treatment of 12 with pyridinium
hydrofluoride in pyridine removed the silyl protecting
groups and provided C2-modified paclitaxel 13 in 51%
yield. Hydrogen transfer conditions, generated by cyclo-
hexene and 10% Pd/C in ethanol, converted compound
13 into metabolite 3.
Metabolites 2 and 3 were characterized by positive
FAB mass spectrometry as well as 1H and 13C NMR
spectroscopy. Both derivatives show a molecular ion
peak at m/ z ) 870 (MH+), consistent with the addition
of one hydroxyl group to the paclitaxel molecule (m/ z
1
) 853). The H NMR spectral data are very similar to
The paclitaxel metabolite 3, hydroxylated at the
2-benzoyl group, was prepared via DCC/DMAP-promot-
ed coupling of 2-debenzoylpaclitaxel 11 with 3-(benzyl-
oxy)benzoic acid (Scheme 2). The two secondary hy-
droxyl groups of paclitaxel were selectively protected in
a one-flask procedure to give compound 10 in excellent
yield by adding first tert-butyldimethylsilyl chloride to
silylate the C2′-hydroxyl group followed by triethylsilyl
chloride to silylate the C7-hydroxyl group. Protected
paclitaxel 10 could now easily be debenzoylated at C2
with anhydrous KOH to give 11 in high yield.22 The
bulky tert-butyldimethylsilyl protecting group at C2′ is
necessary to prevent the hydrolytic cleavage of the C13
side chain under the strong basic reaction conditions,23
and the C7-hydroxyl group must be protected to avoid
base-catalyzed C7 isomerization as well as acylation in
the following step.24 Esterification of compound 11 and
3-(benzyloxy)benzoic acid25 in the presence of DCC and
DMAP in refluxing methylene chloride gave reacylated
the ones reported by Monsarrat et al. for 2 and 3.7 Since
the para position of the 3′-phenyl group in 2 is hydroxyl-
ated, two doublets for the ortho and meta protons at
7.23 and 6.75 ppm are observed. The introduction of a
m-hydroxyl group at the C2-benzoate moiety causes the
resonance of one of the ortho protons to appear as a
singlet at 7.73 ppm. The resonance of the phenolic
proton in 3 is detected at 9.05 ppm. The benzylic
methylene protons in 13 are identified as a nicely
separated quartet at 5.16 ppm.
Biologica l Eva lu a tion . The paclitaxel metabolites
2 and 3 as well as the benzylated precursor 13 of
metabolite 3 were tested and compared with paclitaxel
for their ability to initiate the polymerization of tubulin
in the microtubule assembly assay and for their cyto-
toxicity against B16 melanoma cells (Table 1).26 Me-
tabolite 2 was about 2 times as active in the microtubule
assembly assay but about 7 times less cytotoxic against
B16 melanoma cells than paclitaxel. This result sug-

Phenolic Paclitaxel Metabolites
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 14 2707
Ta ble 1. In Vitro Biological Evaluation of Paclitaxel
Metabolites 2 and 3 and Precursor 13
(3R ,4S )-3-[(t er t -Bu t yld im e t h ylsilyl)oxy]-4-[4-[(t er t -
bu tyld im eth ylsilyl)oxy]p h en yl]-2-a zetid in on e (6). To a
stirred solution of diisopropylamine (0.38 mL, 2.69 mmol) in
THF (1 mL) was added a 2.5 M solution of n-butyllithium (1.08
mL, 2.69 mmol) in hexanes at -78 °C under argon gas. The
reaction mixture was stirred at 0 °C for 30 min and then
recooled to -78 °C. To the cold mixture was then added a
solution of the chiral glycolate 420 (1180 mg, 2.07 mmol) in
THF (2 mL) via cannula under argon gas. The reaction
mixture was stirred for 1 h at -78 °C followed by the addition
of benzaldimine 5 (826 mg, 2.69 mmol). The mixture was
stirred at -78 °C for 4 h and then at room temperature for 8
h. The reaction was quenched with the addition of a saturated
NH4Cl solution (5 mL); then the mixture was diluted with
diethyl ether (100 mL) and washed with brine (100 mL). The
organic layer was dried over anhydrous MgSO4 and concen-
trated under reduced pressure. Purification of the crude
material by silica gel flash chromatography (EtOAc:hexane )
1:9) gave 266 mg (32%) of â-lactam 6 as a white solid. HPLC
analysis for the determination of the enantiomeric excess of
azetidinone 6 was performed on a Waters Model 481 LC
spectrometer detector set at 254 nm equipped with a Waters
740 data module integrator using a chiral column (Diacel
Chiracel OD-H). The flow rate was 0.5 mL/min, utilizing a
20:1 hexane:2-propanol mobile phase; ee ) 80%: mp ) 97-
103 °C; [R]D +36° (c ) 0.32, CHCl3); 1H NMR (300 MHz, CDCl3)
δ -0.16 (s, 3H, SiCH3), 0.04 (s, 3H, SiCH3), 0.17 (s, 6H, 2 ×
SiCH3), 0.67 (s, 9H, (CH3)3), 0.98 (s, 9H, (CH3)3), 4.73 (d, J )
4.7 Hz, 1H, H4), 5.01 (dd, J ) 2.8, 4.6 Hz, 1H, H3), 6.10 (br s,
1H, NH), 6.82 (d, J ) 8.4 Hz, 2H, aryl), 7.17 (d, J ) 8.4 Hz,
2H, aryl); 13C NMR (125 MHz, CDCl3) δ -5.4, -4.9, -4.5, 25.4,
25.7, 58.8, 79.5, 119.8, 128.9, 129.2, 155.7, 169.7; FAB HRMS
m/ z calcd for (M + H)+ C21H38NO3Si2 408.2387, found 408.2366.
(3R,4S)-N-Ben zoyl-3-[(ter t-b u t yld im et h ylsilyl)oxy]-4-
[4-[(ter t-bu tyldim eth ylsilyl)oxy]ph en yl]-2-azetidin on e (7).
To a cold (0 °C) solution of â-lactam 6 (220 mg, 0.54 mmol),
triethylamine (0.5 mL, 2.5 mmol), and DMAP (catalytic
amount) in anhydrous CH2Cl2 (2 mL) was added benzoyl
chloride (0.3 mL, 1.8 mmol). The reaction mixture was stirred
for 1 h at room temperature, and then the reaction was
quenched with saturated aqueous NH4Cl solution (10 mL); the
mixture was diluted with CH2Cl2 (50 mL), washed with a
saturated aqueous NaHCO3 solution (20 mL) and brine (50
mL), and dried over anhydrous MgSO4. The desiccant was
removed by filtration and the solvent evaporated under
reduced pressure. Purification of the crude product by silica
gel flash chromatography (EtOAc:hexane ) 5:95) gave 60%
yield of compound 7 as a white solid: mp ) 85-88 °C; [R]D
ED50/ED50 (paclitaxel)
microtubule
compd
assembly assaya
B16 melanomab
2
3
13
0.60
2.0
24
6.8
>20
not evaluated
a
ED50 is the concentration which causes polymerization of 50%
b
of the tubulin present in 15 min at 37 °C. ED50 refers to the
concentration which produces 50% inhibition of proliferation after
40 h incubation.
gests that the phenolic hydroxyl group does not interfere
with the binding of 2 to microtubules but may reduce
cellular uptake because of the increased hydrophilicity.
Similar bioactivities were obtained by Monsarrat et al.,
who reported that 2 was as active as paclitaxel in a
microtubule disassembly assay and about 9 times less
cytotoxic than the parent against L1210 leukemia cells.7
Metabolite 3 was reported to have greatly reduced
activity (39 times) on L1210 leukemia cell growth but
showed paclitaxel-like activity in the microtubule dis-
assembly assay.7 The reported data are in accordance
with the results from our investigations, which demon-
strate paclitaxel-like activity of 3 in the microtubule
assembly assay yet significant loss of cytotoxicity against
B16 melanoma cells. The poor activity of 13 was
unexpected because other meta-substituted 2-aroyl-2-
debenzoylpaclitaxel analogues, carrying a 3-methoxy,27
3-chloro,19 or other meta substituents, display improved
or paclitaxel-like activity in tubulin assays.19,27 The
greatly reduced activity of 13 points to a severe interfer-
ence of the benzyl group with the paclitaxel binding site.
Su m m a r y
The chemical syntheses of the two known phenolic
paclitaxel metabolites 2 and 3 are reported for the first
time. The compounds were prepared in good overall
yields by semisynthesis from baccatin III and an N-acyl
â-lactam and by a selective deacylation-reacylation
protocol. The availability of these metabolites through
synthesis should facilitate further studies of the disposi-
tion of paclitaxel.
1
+130° (c ) 0.70, CHCl3); H NMR (300 MHz, CDCl3) δ -0.11
(s, 3H, SiCH3), -0.08 (s, 3H, SiCH3), 0.17 (s, 6H, 2 × SiCH3),
0.72 (s, 9H, (CH3)3), 0.97 (s, 9H, (CH3)3), 5.09 (d, J ) 6.1 Hz,
1H, H4), 5.36 (d, J ) 6.1 Hz, 1H, H3), 6.82 (d, J ) 8.5 Hz, 2H,
aryl), 7.22 (d, J ) 8.5 Hz, 2H, aryl), 7.50 (t, J ) 7.8 Hz, 2H,
aryl), 7.59 (t, J ) 7.8 Hz, 1H, aryl), 8.00 (d, J ) 7.1 Hz, 2H,
aryl); 13C NMR (125 MHz, CDCl3) δ -5.4, -5.0, -4.5, -4.5,
17.9, 18.2, 25.3, 25.7, 60.4, 76.4, 119.9, 126.5, 128.1, 129.3,
129.9, 132.1, 133.3, 155.8, 165.4, 166.3; FAB HRMS m/ z calcd
for (M + H)+ C28H41NO4Si2 511.2574, found 511.2551.
Exp er im en ta l Section
Gen er a l. For general synthetic procedures, see ref 28.
4-[(tert-Butyldimethylsilyl)oxy]benzaldehyde was prepared by
standard silylation of 4-hydroxybenzaldehyde. All other start-
ing materials are commercially available, or their syntheses
have been described in the literature. For a description of the
biological assays, see refs 26 and 29.
4-[(t er t -Bu t yld im e t h ylsilyl)oxy]-N -(t r im e t h ylsilyl)-
ben zaldim in e (5).30 To 1,1,1,3,3,3-hexamethyldisilazane (57.5
mL, 27.5 mmol) was added slowly n-butyllithium (100 mL, 25
mmol, 2.5 M hexane solution). The reaction mixture was
cooled to 0 °C and diluted with THF (100 mL). After 20 min,
4-[(tert-butyldimethylsilyl)oxy]benzaldehyde (5.9 g, 25 mmol)
was added dropwise to the reaction mixture over 10 min. The
reaction was continued for 30 min at 0 °C and quenched with
trimethylsilyl chloride (23.5 mL). After 30 min, the solvent
was evaporated to one-half of the original volume. n-Pentane
(500 mL) was then added to the reaction mixture. The
precipitate was filtered using Celite, and removal of the solvent
in vacuo provided the imine in 95% (7.3 g) as a colorless liquid,
which was used without further purification: 1H NMR (300
MHz, CDCl3) δ -0.06 (s, 3H, SiCH3), -0.02 (s, 3H, SiCH3),
0.01 (s, 9H, Si(CH3)3), 0.74 (s, 9H, C(CH3)3), 6.63 (d, J ) 7.8
Hz, 2H, aryl), 7.46 (d, J ) 7.8 Hz, 2H, aryl), 8.67 (s, 1H,
CHdN).
2′-O-(ter t-Bu tyld im eth ylsilyl)-3′-[4-[(ter t-bu tyld im eth -
ylsilyl)oxy]p h en yl]-3′-d ep h en yl-7-O-(tr ieth ylsilyl)p a cli-
ta xel (9). 7-(Triethylsilyl)baccatin III (8; 44 mg, 0.063 mmol)
was dissolved in dry THF (1.5 mL). The solution was cooled
to 0 °C, and NaH (63 mg, 60% mineral oil dispersion, 1.57
mmol) was added. The suspension was stirred for 30 min at
0 °C, and then a solution of N-benzoyl-2-azetidinone 7 (89 mg,
0.18 mmol) in THF (2 mL) was added through a cannula. The
reaction mixture was stirred at 35 °C for 4 h, and then the
reaction was quenched with cold brine (5 mL); the mixture
was diluted with diethyl ether (70 mL), washed with brine (2
× 50 mL), and dried over anhydrous MgSO4. The suspension
was filtered and the filtrate evaporated to dryness under
reduced pressure. Purification by silica gel flash column
chromatography (EtOAc:hexane ) 1:9) provided 61 mg (80%
yield) of compound 9 as a white solid: mp ) 105-115 °C; [R]D
-48° (c ) 2.0, CHCl3); 1H NMR (500 MHz, CDCl3) δ -0.24 (s,
3H, SiCH3), -0.01 (s, 3H, SiCH3), 0.18 (s, 6H, 2 × SiCH3),

2708 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 14
Park et al.
0.55-0.62 (m, 6H, Si(CH2CH3)3), 0.82 (s, 9H, Si(CH3)3), 0.93
(t, J ) 7.8 Hz, 9H, Si(CH2CH3)3), 0.99 (s, 9H, Si(CH3)3), 1.17
(s, 3H, H17), 1.22 (s, 3H, H16), 1.70 (s, 3H, H19), 1.91 (m, 1H,
H6), 2.02 (s, 3H, H18), 2.13 (dd, J ) 9.4, 15.5 Hz, 1H, H14),
2.17 (s, 3H, 10-OAc), 2.40 (dd, J ) 9.6, 15.2 Hz, 1H, H14),
2.49-2.56 (m, 1H, H6), 2.57 (s, 3H, 4-OAc), 3.83 (d, J ) 6.9
Hz, 1H, H3), 4.22 (d, J ) 8.3 Hz, 1H, H20), 4.32 (d, J ) 8.3
Hz, 1H, H20), 4.47 (dd, J ) 6.8, 10.6 Hz, 1H, H7), 4.62 (d, J )
2.0 Hz, 1H, H2′), 4.95 (d, J ) 9.2 Hz, 1H, H5), 5.7 (m, 2H,
H3′, H2), 6.23 (t, J ) 9.1 Hz, 1H, H13), 6.45 (s, 1H, H10), 6.84
(d, J ) 8.4 Hz, 2H, m-aryl), 7.04 (d, J ) 9.0 Hz, 1H, NH), 7.17
(d, J ) 8.3 Hz, 2H, o-aryl), 7.39 (t, J ) 7.7 Hz, 2H, m-PhCON),
7.49 (t, J ) 7.4 Hz, 1H, p-PhCON), 7.51 (t, J ) 7.7 Hz, 2H,
m-PhCO2), 7.60 (t, J ) 7.3 Hz, 1H, p-PhCO2), 7.72 (d, J ) 7.0
Hz, 2H, o-PhCON), 8.13 (d, J ) 7.0 Hz, 2H, o-PhCO2); 13C NMR
(125 MHz, CDCl3) δ -5.3, -4.7, -3.9, 5.3, 6.7, 10.1, 14.2, 18.2,
18.2, 20.8, 21.5, 23.1, 25.6, 26.7, 26.6, 35.7, 37.3, 43.4, 46.7,
55.1, 58.5, 71.3, 72.3, 75.0, 75.2, 78.9, 81.27, 84.3, 120.3, 127.0,
127.5, 128.7, 128.7, 129.4, 130.2, 131.1, 131.7, 133.5, 133.8,
134.3, 140.2, 155.5, 166.9, 167.0, 169.2, 170.1, 171.4, 201.6;
FAB HRMS m/ z calcd for (M + H)+ C65H94NO15Si3 1212.5931,
found 1212.5946.
3′-Dep h en yl-3′-(4-h yd r oxyp h en yl)p a clita xel (2). The
protected taxane 9 (30 mg, 0.025 mmol) was dissolved in cold
(0 °C) pyridine (1 mL). To that solution was added pyridinium
hydrogen fluoride (0.2 mL) at 0 °C. The reaction mixture was
stirred for 3 h at 0 °C and for 5 h at room temperature. Excess
reagent was destroyed with saturated aqueous NaHCO3 (15
mL), and the reaction mixture was diluted with diethyl ether
(50 mL) and washed with an aqueous HCl solution (3%, 5 mL)
and brine (30 mL). The organic layer was separated, dried
over anhydrous MgSO4, filtered, and evaporated under reduced
pressure. Recrystallization (CH2Cl2/n-pentane) of the crude
product gave compound 2 in 91% yield (19 mg) as a colorless
solid: mp ) 184-189 °C; [R]D -54° (c ) 0.7, CHCl3); 1H NMR
(300 MHz, CDCl3 + D2O) δ 1.13 (s, 3H, H17), 1.20 (s, 3H, H16),
1.67 (s, 3H, H19), 1.76 (s, 3H, H18), 1.79-1.94 (m, 1H, H6),
2.22 (s, 3H, 10-OAc), 2.24-2.34 (m, 2H, H14), 2.35 (s, 3H,
4-OAc), 2.45-2.56 (m, 1H, H6), 3.76 (d, J ) 6.8 Hz, 1H, H3),
4.18 (d, J ) 8.3 Hz, 1H, H20), 4.28 (d, J ) 8.3 Hz, 1H, H20),
4.36 (dd, J ) 6.8, 10.7 Hz, 1H, H7), 4.71 (d, J ) 2.6 Hz, 1H,
H2′), 4.92 (d, J ) 9.3 Hz, 1H, H5), 5.63-5.67 (m, 2H, H3′, H2),
6.17 (t, J ) 8.4 Hz, 1H, H13), 6.26 (s, 1H, H10), 6.75 (d, J )
7.9 Hz, 2H, m-aryl), 7.23 (d, J ) 8.3 Hz, 2H, o-aryl), 7.38 (t, J
) 7.4 Hz, 2H, m-PhCON), 7.49 (t, J ) 7.8 Hz, 2H, m-PhCO2,
1H from p-PhCON), 7.59 (t, J ) 8.8 Hz, 1H, p-PhCO2), 7.73
(d, J ) 7.3 Hz, 2H, o-PhCON), 8.10 (d, J ) 7.4 Hz, 2H,
o-PhCO2); 13C NMR (125 MHz, CDCl3) δ 9.6, 14.8, 20.8, 21.7,
22.6, 26.9, 35.7, 35.8, 43.2, 45.8, 54.9, 58.6, 72.1, 72.2, 73.4,
75.0, 75.6, 79.0, 81.3, 84.4, 115.9, 127.1, 128.4, 128.7, 129.2,
129.5, 130.2, 132.0, 133.3, 133.6, 133.7, 141.9, 156.1, 167.0,
167.4, 170.6, 171.2, 172.6, 203.6; FAB HRMS m/ z calcd for
(M + H)+ C47H52NO15 870.3337, found 870.3327.
2′-O-(ter t-Bu tyld im eth ylsilyl)-7-O-(tr ieth ylsilyl)p a cli-
ta xel (10). Paclitaxel (500 mg, 0.58 mmol), DMAP (720 mg,
5.8 mmol), and TBSCl (883 mg, 5.8 mmol) were dissolved in
cold (0 °C) CH2Cl2 (5 mL) and stirred overnight at room
temperature. The solution was cooled in an ice bath while
TESCl (0.98 mL, 5.8 mmol) and DMAP (720 mg, 5.8 mmol)
were added. After 30 min at room temperature, the reaction
was quenched with brine (5 mL). The organic layer was
collected and washed with dilute HCl (10%, 5 mL) and
saturated aqueous NaHCO3 solution (5 mL) and dried (Na2SO4).
The crude residue, obtained by removal of solvent under
reduced pressure, was purified by radial chromatography
(silica gel, 2 mm, 7:3 hexane:EtOAc) to give 565 mg (90%) of
the title compound as a colorless viscous oil: [R]D -54° (c )
1.1, CHCl3); 1H NMR (500 MHz, CDCl3) δ -0.32(s, 3H, SiCH3),
-0.22 (s, 3H, SiCH3), 0.55-0.61 (m, 6H, Si(CH2CH3)3), 0.80
(s, 9H, Si(CH3)3), 0.93 (t, J ) 8.1 Hz, 9H, Si(CH2CH3)3), 1.16
(s, 3H, H17), 1.22 (s, 3H, H16), 1.70 (s, 3H, H19), 1.89-1.94
(m, 1H, H6), 2.02 (s, 3H, H18), 2.10 (dd, J ) 9.0, 15.4 Hz, 1H,
H14), 2.17 (s, 3H, 10-OAc), 2.40 (dd, J ) 9.7, 15.3 Hz, 1H, H14),
2.50-2.55 (m, 1H, H6), 2.58 (s, 3H, 4-OAc), 3.84 (d, J ) 7.0
Hz, 1H, H3), 4.21 (d, J ) 8.4 Hz, 1H, H20), 4.32 (d, J ) 8.4
Hz, 1H, H20), 4.47 (dd, J ) 6.6, 10.4 Hz, 1H, H7), 4.67 (d, J )
2.0 Hz, 1H, H2′), 4.95 (d, J ) 8.3 Hz, 1H, H5), 5.70 (d, J ) 7.1
Hz, 1H, H2), 5.74 (d, J ) 8.8 Hz, 1H, H3′), 6.26 (t, J ) 8.9 Hz,
1H, H13), 6.45 (s, 1H, H10), 7.07 (d, J ) 8.9 Hz, NH), 7.30-
7.61 (m, 10H, aryl), 7.74 (d, J ) 7.7 Hz, 2H, aryl), 8.13 (d, J )
7.4 Hz, 2H, aryl); 13C NMR (125 MHz, CDCl3) δ -5.9, -5.2,
5.3, 6.7, 10.1, 14.3, 18.1, 20.9, 21.5, 23.1, 25.5, 25.7, 26.6, 35.6,
37.2, 43.3, 46.7, 55.6, 58.4, 71.4, 72.2, 74.9, 75.0, 75.1, 76.6,
78.8, 81.2, 84.2, 126.4, 127.0, 127.9, 128.7, 128.7, 129.2, 130.2,
131.8, 133.6, 133.7, 134.1, 138.3, 140.2, 166.9, 167.1, 169.3,
170.2, 171.4, 201.6; FAB HRMS m/ z calcd for (M + Li)+ C59H79
-
NO14Si2Li 1088.5199, found 1088.5243.
2′-O-(ter t-Bu tyld im eth ylsilyl)-2-O-d eben zoyl-7-O-(tr i-
eth ylsilyl)p a clita xel (11). A solution of anhydrous KOH was
prepared immediately prior to use by adding H2O (4 µL) to a
20-fold dilution of commercial t-BuOK in THF solution (0.05
M t-BuOK, 5 mL).31 An aliquot of this solution (1.6 mL, 0.072
M KOH) was then added to a solution of 10 (65 mg, 0.06 mmol)
in THF (4 mL) immersed in an CH3CN/CO2 bath. After the
addition, the temperature was raised to -15 °C for 12 h, the
reaction was quenched with a saturated aqueous NH4Cl
solution (5 mL), and then the mixture was warmed to room
temperature. Extraction between diethyl ether (50 mL) and
brine (30 mL) followed by drying (Na2SO4) of the collected
organic phase resulted in crude product which was subjected
to silica gel flash column chromatography (hexane:EtOAc )
4:1 to 1:1). Along with 15% (8 mg) of the dideacylated product
2′-O-(tert-butyldimethylsilyl)-4-O-deacetyl-2-O-debenzoyl-7-O-
(triethylsilyl)paclitaxel,23 compound 11 was isolated in 81%
yield (47 mg) as an amorphous solid: [R]D -54° (c ) 0.67,
1
CH2Cl2); H NMR (500 MHz, CD3OD) -0.12 (s, 3H, SiCH3),
-0.01 (s, 3H, SiCH3), 0.56-0.66 (m, 6H, SiCH2CH3), 0.83 (s,
9H, C(CH3)3), 0.95 (t, J ) 7.9 Hz, 9H, SiCH2CH3), 1.07 (s, 3H,
H17), 1.14 (s, 3H, H16), 1.63 (s, 3H, H19), 1.80-1.87 (m, 1H,
H6), 1.85 (s, 3H, H18), 1.99 (dd, J ) 8.8, 15.0 Hz, 1H, H14),
2.13 (s, 3H, 10-OAc), 2.24 (dd, J ) 9.5, 15.3 Hz, 1H, H14), 2.46
(s, 3H, 4-OAc), 2.52-2.58 (m, 1H, H6), 3.50 (d, J ) 6.7 Hz,
1H, H2), 3.88 (d, J ) 6.8 Hz, 1H, H3), 4.50 (dd, J ) 10.5, 6.6
Hz, 1H, H7), 4.63 (qab, J ) 8.9, 26 Hz, 2H, H20), 4.78 (d, J )
4.1 Hz, 1H, H2′), 5.01 (d, J ) 8.0 Hz, 1H, H5), 5.72 (d, J ) 4.0
Hz, 1H, H3′), 6.13 (t, J ) 9.1 Hz, 1H, H13), 6.39 (s, 1H, H10),
7.32-7.58 (m, 8H, Bz, Ph), 7.77-7.80 (m, 2H, Bz); FAB HRMS
m/ z calcd for (M + H)+ C52H75NO13Si2 978.4855, found
978.4870.
2-O-[3-(Ben zyloxy)b en zoyl]-2-O-(d eb en zoyl)p a clit a x-
el (13). To a stirring solution of 11 (43 mg, 0.044 mmol) in
methylene chloride (2 mL) was added, in the following order,
(3-benzyloxy)acetic acid (120 mg, 0.528 mmol), DMAP (64 mg,
0.528 mmol), and DCC (109 mg, 0.528 mmol). This solution
was refluxed for 5 h or until the disappearance of the starting
material. After cooling, the reaction mixture was diluted with
methylene chloride (50 mL) and filtered. The filtrate was
washed consecutively with 1 N HCl (10 mL), NaHCO3 (10 mL),
and brine (10 mL) and evaporated under reduced pressure.
The crude product was dissolved in pyridine (0.5 mL), and the
solution was cooled to 0 °C. To that mixture was added
pyridinium hydrogen fluoride (0.1 mL). The reaction mixture
was stirred at 0 °C for 3 h and at room temperature for another
5 h. Excess reagent was destroyed with saturated aqueous
NaHCO3 (5 mL), and the reaction mixture was diluted with
diethyl ether (50 mL) and washed with 3% aqueous HCl (10
mL) and brine (10 mL). The organic layer was separated, dried
over anhydrous MgSO4, filtered, and evaporated under reduced
pressure. Recrystallization (CH2Cl2/n-pentane) of the crude
product gave compound 13 in 51% yield as a white solid: mp
) 151-155 °C; [R]D -37° (c ) 0.4, CHCl3); 1H NMR (500 MHz,
CDCl3) δ 1.15 (s, 3H, H17), 1.23 (s, 3H, H16), 1.69 (s, 3H, H19),
1.80 (s, 3H, H18), 1.86-1.91 (m, H6), 2.22-2.38 (m, 2H, H14),
2.24 (s, 3H, 10-OAc), 2.36 (s, 3H, 4-OAc), 2.52-2.58 (m, 1H,
H6), 3.80 (d, J ) 7.0 Hz, 1H, H3), 4.20 (d, J ) 8.5 Hz, 1H,
H20), 4.35 (d, J ) 8.5 Hz, 1H, H20), 4.38-4.46 (m, 1H, H7),
4.79 (dd, J ) 2.5, 4.9 Hz, 1H, H2′), 4.95 (d, J ) 8.5 Hz, 1H,
H5), 5.11 (d, J ) 11.3 Hz, 1H, CH2Ph), 5.21 (d, J ) 11.3 Hz,
1H, CH2Ph), 5.67 (d, J ) 7.0 Hz, 1H, H2), 5.79 (d, J ) 8.9 Hz,
1H, H3′), 6.24 (t, J ) 9.0 Hz, 1H, H13), 6.27 (s, 1H, H10), 6.93
(d, J ) 8.9 Hz, NH), 7.21-7.23 (m, 1H, aryl), 7.34-7.43 (m,
14H, aryl), 7.71-7.79 (m, 4H, aryl); 13C NMR (125 MHz,

Phenolic Paclitaxel Metabolites
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 14 2709
(9) Walle, T.; Walle, U. K.; Kumar, G. N.; Bhalla, K. N. Taxol
Metabolism and Disposition in Cancer Patients. Drug Metab.
Dispos. 1995, 23, 506-512.
CDCl3) δ 9.6, 14.9, 20.9, 21.8, 22.6, 26.9, 35.7, 43.2, 45.6, 54.9,
58.6, 70.2, 72.2, 72.4, 73.0, 75.1, 75.6, 77.3, 79.0, 81.1, 84.4,
115.4, 121.2, 127.0, 127.6, 128.2, 128.3, 128.6, 128.7, 129.0,
129.8, 130.4, 132.0, 133.2, 133.6, 136.4, 137.9, 142.0, 158.9,
166.8, 167.0, 170.3, 171.3, 172.8, 203.6; FAB HRMS m/ z calcd
for (M + H)+ C54H58N1O15 960.3806, found 960.3850.
(10) Harris, J . W.; Katki, A.; Anderson, L. W.; Chmurny, G. N.;
Paukstelis, J . V.; Collins, J . M. Isolation, Structural Determi-
nation, and Biological Activity of 6-R-Hydroxytaxol, the Principle
Human Metabolite of Taxol. J . Med. Chem. 1994, 37, 706-709.
(11) Kumar, G. N.; Oatis, J . E., J r.; Thornburg, K. R.; Heldrich, F.
J .; Hazard, E. S., III; Walle, T. 6R-Hydroxytaxol: Isolation and
Identification of the Major Metabolite of Taxol in Human Liver
Microsomes. Drug Metab. Dispos. 1994, 22, 177-179.
(12) Kumar, G. N.; Walle, U. K.; Walle, T. Cytochrome P450 3A-
Mediated Human Liver Microsomal Taxol 6R-Hydroxylation. J .
Pharmacol. Exp. Ther. 1994, 268, 1160-1165.
(13) Cresteil, T.; Monsarrat, B.; Alvinerie, P.; Treluyer, J . M.; Vieira,
I.; Wright, M. Taxol Metabolism by Human Liver Microsomes:
Identification of Cytochrome P450 Isoenzymes Involved in Its
Biotransformation. Cancer Res. 1994, 54, 386-392.
(14) Harris, J . W.; Rahman, A.; Kim, B.-R.; Guengerich, F. P.; Collins,
J . M. Metabolism of Taxol by Human Hepatic Microsomes and
Liver Slices: Participation of Cytochrome P450 3A4 and an
Unknown P450 Enzyme. Cancer Res. 1994, 54, 4026-4035.
(15) Huizing, M. T.; Keung, A. C. F.; Rosing, H.; Van der Kuij, V.;
ten Bokkel Huinink, W. W.; Mandjes, I. M.; Dubbelman, A. C.;
Pinedo, H. L.; Beijnen, J . H. J . Clin. Oncol. 1993, 11, 2127.
(16) Monegier, B.; Gaillard, C.; Sable, S.; Vuilhorgne, M. Structures
of the Major Human Metabolites of Docetaxel (RP 56976 -
Taxotere). Tetrahedron Lett. 1994, 35, 3715-3718.
(17) Commerc¸on, A.; Bourzat, J . D.; Bezard, D.; Vuilhorgne, M.
Partial Synthesis of Major Human Metabolites of Docetaxel.
Tetrahedron 1994, 50, 10289-10298.
(18) Georg, G. I.; Cheruvallath, Z. S.; Harriman, G. C. B.; Hepperle,
M.; Park, H.; Himes, R. H. Synthesis and Biology of Substituted
3′-Phenyl Taxol Analogues. Bioorg. Med. Chem. Lett. 1994, 4,
2331-2336.
(19) Georg, G. I.; Ali, S. M.; Boge, T. C.; Datta, A.; Falborg, L.; Park,
H.; Mejillano, M.; Himes, R. H. Synthesis of Biologically Active
2-Benzoyl Paclitaxel Analogues. Bioorg. Med. Chem. Lett. 1995,
5, 259-264.
(20) For a detailed procedure for the synthesis of 4, see: Georg, G.
I.; Harriman, G. C. B.; Hepperle, M.; Clowers, J . S.; Vander
Velde, D. G.; Himes, R. H. Synthesis, Conformational Analysis
and Biological Evaluation of Heteroaromatic Taxanes. J . Org.
Chem. 1996, 61, 2664-2676.
2-O-Deben zoyl-2-O-(3-h yd r oxyben zoyl)p a clita xel (3).
To a solution of 13 (7 mg, 0.007 mmol) in a mixture of
cyclohexene (0.5 mL) and ethanol (0.5 mL) was added 10%
Pd/C (5 mg). The suspension was brought up to 60 °C and
stirred for 2 h. After cooling, the Pd/C was removed by
filtration through Celite and the solvent evaporated under
reduced pressure. Purification of the reaction mixture by flash
column chromatography on silica gel (EtOAc:hexane ) 6:4)
gave the final product 13 in quantitative yield (6.3 mg) as a
white solid: mp ) 169-172 °C; [R]D +56° (c ) 0.2, CHCl3); 1H
NMR (500 MHz, CDCl3) δ 1.09 (s, 3H, H17), 1.25 (s, 3H, H16),
1.71 (s, 3H, H19), 1.90 (s, 3H, H18), 2.07-2.12 (m, H6), 2.25
(s, 3H, 10-OAc), 2.46 (s, 3H, 4-OAc), 2.50-2.61 (m, 3H, H6,
H14), 3.83 (d, J ) 7.8 Hz, 1H, H3), 4.24 (d, J ) 8.4 Hz, 1H,
H20), 4.32 (d, J ) 8.4 Hz, 1H, H20), 4.44-4.46 (m, 1H, H7),
4.90 (d, J ) 7.7 Hz, 1H, H5), 4.95 (s, 1H, H2′), 5.54 (d, J ) 7.8
Hz, 1H, H2), 6.05 (d, J ) 10.1 Hz, 1H, H3′), 6.25 (s, 1H, H10),
6.48 (t, J ) 9.1 Hz, 1H, H13), 7.09 (dd, J ) 1.7, 8.1 Hz, 1H,
4-arylCO2), 7.13 (d, J ) 9.9, NH), 7.33 (t, J ) 8.0 Hz, 1H,
5-arylCO2), 7.38 (t, J ) 8.5 Hz, 1H, p-Ph), 7.40-7.45 (m, 4H,
o- and m-Ph), 7.47 (t, J ) 7.3 Hz, 2H, m-PhCON), 7.50-7.57
(m, 2H, 6-arylCO2, p-PhCON), 7.67 (d, J ) 7.2 Hz, 2H,
o-PhCON), 7.73 (s, 1H, 2-arylCO2), 9.05 (br s, 1H, aryl OH);
13C NMR (125 MHz, CDCl3) δ 9.8, 14.7, 20.9, 22.6, 23.0, 26.6,
29.7, 35.3, 36.3, 43.0, 45.7, 53.7, 58.3, 72.0, 72.3, 72.4, 75.3,
75.5, 80.1, 80.8, 84.4, 116.5, 121.4, 121.5, 126.5, 127.0, 128.4,
129.1, 130.0, 130.6, 132.6, 133.1, 133.4, 137.4, 141.5, 157.3,
1667.0, 169.7, 170.4, 171.3, 172.8, 203.5; HRMS m/ z calcd for
(M + H)+ C47H52N1O15: 870.3337, found 870.3325.
Ack n ow led gm en t. We gratefully acknowledge fi-
nancial assistance from the National Institute of Health.
A postdoctoral fellowship to T. C. Boge from the
Scientific Education Partnership of Hoechst Marion
Roussel Inc. is gratefully acknowledged. Paclitaxel was
provided to us for these studies by the National Cancer
Institute.
Su p p or tin g In for m a tion Ava ila ble: 1H NMR spectra for
compounds 2, 3, 5-7, 9-11, and 13 (9 pages). Ordering
information is given on any current masthead page.
(21) Ojima, I.; Habus, I.; Zhao, M.; Zucco, M.; Park, Y. H.; Sun, C.
M.; Brigaud, T. New and Efficient Approaches to the Semisyn-
thesis of Taxol and its C-13 Side Chain Analogs by Means of
â-Lactam Synthon Method. Tetrahedron 1992, 48, 6985-7012.
(22) Datta, A.; J ayasinghe, L. R.; Georg, G. I. Internal Nucleophile
Assisted Selective Deesterification Studies on Baccatin III.
Synthesis of 2-Debenzoyl- and 4-Deacetylbaccatin III Analogues.
J . Org. Chem. 1994, 59, 4689-4690.
(23) Georg, G. I.; Ali, S. M.; Boge, T. C.; Datta, A.; Falborg, L.; Himes,
R. H. Selective C-2 and C-4 Deacylation and Acylation of Taxol:
The First Synthesis of a C-4 Substituted Taxol Analogue.
Tetrahedron Lett. 1994, 35, 8931-8934.
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J M960142X