Synthesis and anti-tubulin activity of a 3′-(4-Azidophenyl)-3′- dephenylpaclitaxel photoaffinity probe

Article abstract of DOI:10.1021/jm030581d

The synthesis and biological evaluation of a novel paclitaxel photoaffinity probe is described. The synthesis involved the preparation of an azide-containing C13 side chain through a Staudinger cycloaddition followed by a lipase-mediated kinetic resolution to obtain the azetidinone in 99% ee. Coupling of the enantiopure side chain precursor to 7-TES-baccatin III and subsequent silyl ether deprotection afforded 3′-(4-azidophenyl)-3′- dephenylpaclitaxel, which was shown to be as active as paclitaxel in tubulin assembly and cytotoxicity assays.

Full text of DOI:10.1021/jm030581d

J. Med. Chem. 2004, 47, 6459-6465
6459
Articles
Synthesis and Anti-Tubulin Activity of a
3′-(4-Azidophenyl)-3′-dephenylpaclitaxel Photoaffinity Probe
Jared T. Spletstoser,† Patrick T. Flaherty,† Richard H. Himes,‡ and Gunda I. Georg*,†
Department of Medicinal Chemistry, University of Kansas, 1251 Wescoe Hall Drive, Lawrence, Kansas 66045-7582, and
Department of Molecular Biosciences, University of Kansas, 1200 Sunnyside Avenue, Lawrence, Kansas 66045-7534
Received November 19, 2003
The synthesis and biological evaluation of a novel paclitaxel photoaffinity probe is described.
The synthesis involved the preparation of an azide-containing C13 side chain through a
Staudinger cycloaddition followed by a lipase-mediated kinetic resolution to obtain the
azetidinone in 99% ee. Coupling of the enantiopure side chain precursor to 7-TES-baccatin III
and subsequent silyl ether deprotection afforded 3′-(4-azidophenyl)-3′-dephenylpaclitaxel, which
was shown to be as active as paclitaxel in tubulin assembly and cytotoxicity assays.
Introduction
Paclitaxel (1, Taxol; Figure 1) is regarded as one of
the clinically most effective chemotherapeutic agents.
It is currently employed in the treatment of a variety
of cancers including drug-refractory ovarian cancer,
Kaposi’s sarcoma, small-cell lung cancer, and metastatic
breast cancer.1 Paclitaxel is believed to block mitosis
by suppressing microtubule dynamics that leads to
apoptosis.2 Even though paclitaxel is known to bind to
the â-subunit of tubulin, little is known of its interac-
tions at the molecular level. Insight into specific residue
interaction has been gained through the use of photo-
affinity probes,3-8 fluorescent probes,9 electron crystal-
lography,10 tubulin mutations,11 and computational
studies.12-14
Recently, there has been much discussion on the
biologically active orientation of paclitaxel bound to
tubulin. Three hypotheses have emerged with respect
to the bioactive conformation of the highly flexible C2
and C3′ side chains. The first involves an aggregation
where the groups of the C2 benzoyl and C3′ benzamide
side chains are in proximity to one another as is seen
in the solid state.15 The second conformation aligns the
C2 benzoyl and C3′ phenyl side chains as is observed
by NMR in polar solvents and is termed “hydrophobic
collapse”.12,16 A third hypothesis orients the side chains
of paclitaxel in a T-shape with the C2 benzoyl group
equidistant to both the phenyl rings at C3′ and the
N-benzoyl group.13
Figure 1. Structure of paclitaxel and 3′-azidophenylpacli-
taxel.
(along with the electron crystallograph)10 to the pacli-
taxel binding site. We hypothesized that photolabeling
experiments with a photoaffinity probe at the 3′-phenyl
group should provide additional detailed information on
the paclitaxel binding site and the bound conformation
of the phenylisoserine side chain.
On the basis of known paclitaxel SAR,18 we expected
that placing an azide group at the para position of the
C3′ phenyl ring should retain activity of the analogue
while allowing for satisfactory labeling of the tubulin
protein. Herein, we report the synthesis and biological
evaluation of the first paclitaxel photoaffinity label
carrying an azide moiety at the C3′ phenyl group.
Chemistry and Synthesis
The semisynthesis of paclitaxel and analogues re-
quires the asymmetric synthesis of the phenylisoserine
side chain and its coupling to naturally occurring bac-
catin III.19,20 Synthetic routes to this side chain include
chiral ester-imine cyclocondensation reactions21-24 and
the asymmetric aminohydroxylation of trans-cinnamic
esters.25 The stability of the azide functionality to these
reactions was of major concern, and attempts to syn-
thesize an azide-containing phenylisoserine using these
methodologies were unsuccessful.
Since its first use by Knowles et al. in 1969,17 the
arylazide moiety has become one of the most commonly
used photoactivatable functionalities. Azide-based pa-
clitaxel photoaffinity analogues with the labels attached
to paclitaxel via an ester or amide linkage through the
N3′,3,4,6 O2,4,7 and O75,6 positions have provided insight
Requiring a milder route to the side chain, we turned
to the Staudinger reaction, a [2 + 2] ketene-imine
cycloaddition.26,27 At the time of this project’s undertak-
ing, there were few reports of catalytic, asymmetric
* To whom correspondence should be addressed. Phone: 785-864-
4498. Fax: 785-864-5326. E-mail: georg@ku.edu.
† Department of Medicinal Chemistry.
‡ Department of Molecular Biosciences.
10.1021/jm030581d CCC: $27.50 © 2004 American Chemical Society
Published on Web 11/11/2004

6460 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 26
Spletstoser et al.
Scheme 1a
The Staudinger cyclization was envisioned to occur
by generating the ketene in situ from 8 (Scheme 2). This
was accomplished with the addition of a 2 M solution
of oxalyl chloride in the presence of a catalytic amount
of DMF to form the acid chloride.33 Ketene formation
and spontaneous cyclization ensued, following cannu-
lation of the acid chloride solution to a mixture of
triethylamine and imine 6. The crude solid was taken
up into methanol and aqueous citric acid, deprotecting
the TMS ether, affording â-lactam 10 in excellent yield.
The predicted cis relationship at C3 and C4 was
material in setting the required configuration of the
phenylisoserine side chain. This stereochemical outcome
was anticipated on the basis of the conrotatory [2 + 2]
pathway predicted for the Staudinger cyclization.34
None of the trans isomer was detected.35 Acetylation of
10 with acetyl chloride provided 11, setting the stage
for the lipase-mediated kinetic resolution. A more direct
route to 11 involved ketene generation from com-
mercially available acetoxyacetyl chloride and subse-
quent Staudinger cyclization with imine 6 in 83%
yield.36 Both methods proved to be valid entries to the
(()-lactam 11.
The resolution proceeded smoothly using Pseudomo-
nas cepacia lipase in a 0.2 M phosphate buffer with 10%
acetonitrile as a cosolvent,32 affording 12 in >99% yield,
based on the desired enantiomer, and 99% enantiomeric
excess as determined by HPLC. Saponification of the
enantiopure ester 12 with lithium hydroxide and sub-
sequent protection of the resultant alcohol with TBSCl
gave (+)-15 in 84% over two steps (Scheme 3).
Oxidative deprotection of the p-methoxyphenyl group
with ammonium cerium(IV) nitrate (CAN)37 provided
compound 16 in 78% yield.38 Imidation of the lactam
nitrogen of 16 with benzoyl chloride22 served to activate
the lactam toward nucleophilic attack from baccatin III.
Azetidinone 17 was obtained in 73% yield after chro-
matography and used immediately in the coupling with
7-TES-baccatin III (19) (Scheme 4).
a (a) (i) H2SO4, NaNO2, H2O; (ii) NaN3, H2O; (b) PCC, CH2Cl2;
(c) p-anisidine, EtOH.
Staudinger (or Staudinger-like) reactions;28 however,
ample research on chiral auxiliary mediated versions
had been published.27,29,30 Chiral auxiliaries connected
to the nitrogen of the imine generally proceed with lower
levels of diastereoselectivity compared to those found
on the carbon portion of the imine or on the ketene
itself.30 However, specific substitutions required at C2′
and C3′ of paclitaxel (corresponding to C3 and C4 of the
azetidinone precursor, respectively) allowed only the use
of N-imine auxiliaries. One example of a chiral ben-
zaldimine from enantiopure (S)-(-)-1-(p-methoxyphen-
yl)propyl-1-amine was reported in good chemical yield
and, after recrystallization, excellent enantiopurity
(>99%).31 Notably this N-benzyl derivative could be
cleaved under oxidative conditions.32 This system was
examined using the acid chloride derived from bis-
(trimethylsilyl)glycolate 8 (vide infra), and analysis of
the products indicated a 1:1 mixture of inseparable
diastereomers.
We therefore turned to resolution techniques reported
previously by Brieva et al.32 They reported the use of
lipases to resolve racemic 3-hydroxy-4-aryl â-lactams as
a route to the enantiopure paclitaxel side chain.32 The
high stereochemical and substrate specificity reported
for this class of enzymes was an attractive solution to
our problem.
The Staudinger cyclization precursor 6 was made via
known synthetic transformations as outlined in Scheme
1. 4-Aminobenzyl alcohol was converted to the azide by
diazotization of amine 3, followed by displacement with
sodium azide. The resultant azido alcohol 4 was oxidized
to the aldehyde with PCC followed by addition of freshly
sublimed p-anisidine, producing imine 6 in excellent
yield.
The absolute stereochemistry of the azetidinone was
determined by X-ray crystallography of the chloroacetyl
derivative 18. An ORTEP drawing (Figure 2) shows the
R,S configuration of C3 and C4, respectively.
Coupling procedures for N-acylated â-lactams typi-
cally involve strong lithiated amine salts serving as the
base for baccatin III deprotonation.39 The stability of
the azide to these nucleophilic amines was of concern.
We therefore turned to coupling procedures that do not
involve the use of nucleophilic bases.23,24 An amount of
50 equiv of sodium hydride23 was added to a solution of
To obtain the other Staudinger reactant, an amount
of 2 equiv of TMSCl was added to glycolic acid (7) to
give bis(trimethylsilyl)-glycolate (8) (Scheme 2). This
ester was not isolated but was used immediately in the
next step.
Scheme 2a
a (a) TMSCl, DMAP, pyridine, CH2Cl2; (b) (i) (ClCO)2, DMF, CH2Cl2, 0 °C; (ii) 0.2 equiv of 6, TEA, CH2Cl2, 0 °C to room temp; (iii)
MeOH, aqueous citric acid; (c) 6, TEA, CH2Cl2, 0 °C to room temp; (d) AcCl, DMAP, TEA, THF; (e) 0.2 M NaH2PO4 buffer solution with
pH at 7.00/CH3CN, Pseudomonas cepacia lipase.

A Paclitaxel Photoaffinity Probe
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 26 6461
Scheme 3a
a (a) LiOH, H2O/acetone 0 °C; (b) TBDMSCl, DMAP, imidazole, CH2Cl2; (c) CAN, CH3CN/H2O; (d) benzoyl chloride, TEA, CH2Cl2, 0 °C
to room temp.
Scheme 4a
icity and microtubule assembly studies indicate that a
photoaffinity analogue has been obtained that is of
similar potency compared to paclitaxel. The photolabel-
ing studies will be performed and presented in due
course. Information obtained from these studies is
expected to provide details on the paclitaxel binding site
and to give insight into the tubulin-bound conformation
of the phenylisoserine side chain of paclitaxel.
a (a) NaH, 17, THF, 0 °C, room temp; (b) HF/pyridine, pyridine,
0 °C to room temp.
Experimental Section
(4-Azidophenyl)methan-1-ol (4). Alcohol 3 (1.55 g, 12.6
mmol) was taken up in 4 N H2SO4 (15 mL) at 0 °C, affording
a reddish suspension. NaNO2 (1.30 g, 18.9 mmol) was added
as a solution in water (10 mL) open to the air. The reaction
mixture was maintained at 0 °C for 15 min with the solution
clearing. NaN3 (1.23 g, 18.9 mmol) in water (10 mL) was added
slowly with gas evolution. The reaction mixture was stirred
at room temperature for 1 h, resulting in a brownish-white
precipitation. The reaction mixture was extracted with Et2O,
and the organic layers were dried with MgSO4 and concen-
trated under reduced pressure. Flash chromatography with
EtOAc/hexanes (30% f 40%) afforded 1.69 g (90%) of a
1
yellowish solid: mp 27-28 °C; IR (neat) 3333, 2107 cm-1; H
NMR (500 MHz, CDCl3) δ 4.69 (d, J ) 5.68 Hz, 2H), 7.04 (d,
J ) 8.38 Hz, 2H), 7.38 (d, J ) 8.27 Hz, 2H); 13C NMR (125
MHz, CDCl3) δ 64.6, 119.0, 128.5, 137.5, 139.3; HRMS (FAB+)
m/z calcd for C7H7N3O [M+] 149.0589, found 149.0567.
4-Azidobenzaldehyde (5). To a solution alcohol 4 (1.69 g,
11.3 mmol) in CH2Cl2 (50 mL) was added PCC (4.15 g, 19.3
mmol) in one portion. The resulting mixture was stirred at
room temperature for 1 h. The reaction mixture was then
filtered over a pad of silica gel on a fritted funnel. The solvent
was removed, affording 1.53 g (92%) of a yellow liquid: IR
(neat) 2119, 1696 cm-1; 1H NMR (400 MHz, CDCl3) δ 7.17 (d,
Figure 2. Structure and ORTEP drawing of derivative 18.
7-TES-baccatin III19 and 17, affording the protected
paclitaxel derivative 20 (Scheme 4) in 74% yield. Depro-
tection of the silyl protecting groups in 20 under
standard conditions gave 2 as a white powder in 83%
yield.
J ) 8.54 Hz, 2H), 7.89 (d, J ) 10.62 Hz, 2H), 9.95 (s, 1H); 13
C
NMR (125 MHz, CDCl3) δ 119.4, 131.5, 133.2, 146.2, 190.5;
HRMS (FAB+) m/z calcd for C7H5N3O [M+] 147.0433, found
147.0439.
Anti-Tubulin Activity
Compound 2 was evaluated for its ability to interact
with tubulin in a microtubule assembly assay and was
found to be as active as the parent compound 1 with an
effective dose ratio (ED50/ED50(paclitaxel)) of 1.1. An in vitro
study against MCF-7 and MCF7-ADR (multidrug-
resistant phenotype) breast cancer cell lines showed
activity similar to that of paclitaxel with an ED50/
ED50(paclitaxel) of 2.7 and 1.5, respectively.40 These results
showed that compound 2 had activity comparable to
that of the parent paclitaxel. These findings suggest that
2 holds promise for future photolabeling studies.
(4-Azidobenzylidene)-(4-methoxyphenyl)amine (6). To
a solution of freshly sublimed p-anisidine (4.01 g, 31.3 mmol)
in EtOH (7 mL) at room temperature open to the air, was
added a solution of aldehyde 5 (3.41 g, 23.1 mmol) in EtOH (2
mL) in one portion. A dense white precipitate formed im-
mediately. The reaction mixture was stirred for an additional
hour. The solid was collected by vacuum filtration, affording
5.34 g (93%) of bright-yellow crystals: mp 121-123 °C; IR
1
(neat) 3418, 2120, 1620 cm-1; H NMR (500 MHz, CDCl3) δ
8.46 (s, 1H), 7.91 (d, J ) 8.50 Hz, 2H), 7.26 (d, J ) 11.90 Hz,
2H), 7.13 (d, J ) 8.46 Hz, 2H), 6.96 (d, J ) 8.71 Hz, 2H), 3.86
(s, 3H); 13C NMR (125 MHz, CDCl3) δ 55.4, 114.3, 119.2, 122.1,
130.0, 133.4, 142.5, 144.6, 156.8, 158.3; HRMS (FAB+) m/z
calcd for C14H13N4O [MH+] 253.1089, found 253.1093.
Conclusions
(+)-4-(4-Azidophenyl)-3-hydroxy-1-(4-methoxyphenyl)-
azetidin-2-one (10). A 0.27 M solution of bis-silylester 8 (15.1
mmol) in CH2Cl2 was cooled to 0 °C, and a catalytic amount
of DMF (0.328 g, 4.50 mmol) was added followed by dropwise
addition of oxalyl chloride as a 2 M solution in CH2Cl2 (16.0
mmol). The reaction mixture was stirred 1 h at 0 °C and 1 h
at room temperature. Meanwhile, a solution of triethylamine
An efficient synthesis of the 3′-(4-azidophenyl)-3′-
dephenylpaclitaxel photoaffinity probe was achieved.
The azide moiety survived all synthetic transformations.
A lipase kinetic resolution was used to resolve the
enantiomers of the phenylisoserine precursor. This
resolution is scalable to gram quantities. The cytotox-

6462 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 26
Spletstoser et al.
(3.60 g, 35.6 mmol) and imine 6 (0.892 g, 3.35 mmol) in CH2-
Cl2 (13 mL) was cooled to 0 °C. The imine solution was then
cannulated to the acid chloride solution (recooled to 0 °C). The
reaction mixture was allowed to warm to room temperature
over 4 h. The reaction mixture was then poured into 100 mL
of water and extracted with CH2Cl2. The combined organic
layers were washed with NaHCO3 and brine. The organic
fraction was then concentrated under reduced pressure. The
resulting residue was taken up in methanol (50 mL), and to it
was added citric acid (614 mg, 6.67 mmol) as a solution in
water. The resulting mixture was allowed to react 30 min at
room temperature. The methanol was removed under reduced
pressure, and the residue was taken up in CH2Cl2 (50 mL)
and washed with NaHCO3 and brine. The organic fraction was
concentrated and the crude residue was subjected to column
chromatography in EtOAc/hexanes (3:7), affording 925 mg
(89%) of an off-white solid: mp 135-137 °C; IR (neat) 3432,
mg (99%) of a white powder, and spectral data are found in
the Supporting Information.
(4S,3R)-4-(4-Azidophenyl)-3-hydroxy-1-(4-methoxyphen-
yl)azetidin-2-one (14). To a stirred solution of 12 (0.025 g,
0.07 mmol) in ACS grade acetone (2 mL) at 0 °C open to the
air was added a 2 N solution of LiOH (0.14 mmol) in water.
The reaction mixture was allowed to warm to room temper-
ature over 1 h. The reaction mixture was then diluted with
EtOAc (12 mL) and washed with water. The organic layer was
washed with brine, dried with MgSO4, and concentrated under
reduced pressure. Flash chromatography with EtOAc/hexanes
(1.5:5) afforded 21.7 mg (>99%) of an off-white solid: mp 132-
1
134 °C; [R]22 +220 (c 1.0, CHCl3). H and 13C NMR, IR, and
D
HRMS data are identical to 10 and are found in the Supporting
Information.
(4S,3R)-4-[4-(Azidophenyl)]-3-tert-butyldimethylsiloxy-
1-(4-methoxyphenyl)azetidin-2-one (15). A solution of 14
(0.113 g, 0.365 mmol), TBSCl (0.149 g, 0.986 mmol), imidazole
(0.067 g, 0.986 mmol), and DMAP (0.121 g, 0.986 mmol) in
CH2Cl2 (3 mL) was stirred under argon at room temperature
for 1 h. The reaction was quenched with water (15 mL) after
the mixture was diluted with CH2Cl2. The aqueous layer was
extracted with CH2Cl2, and the combined organic layers were
washed with brine, dried over MgSO4, and concentrated under
reduced pressure. Flash chromatography with EtOAc/hexanes
(1:5) afforded 130 mg (84%) of an off-white solid: mp 117-
1
2124, 2097, 1723 cm-1; H NMR (500 MHz, CDCl3) δ 3.05 (d,
J ) 6.93 Hz, 1H) 3.78 (s, 3H), 5.22 (br s, 1H), 5.26 (d, J ) 5.06
Hz, 1H), 6.83 (d, J ) 8.92 Hz, 2H), 7.09 (d, J ) 8.36 Hz, 2H),
7.29 (d, J ) 8.87 Hz, 2H), 7.36 (d, J ) 8.33 Hz, 2H); 13C NMR
(125 MHz, CDCl3) δ 55.4, 61.7, 77.6, 114.4, 118.7, 119.6, 129.0,
129.8, 130.2, 140.7, 156.5, 165.3; HRMS (FAB+) m/z calcd for
C16H15N4O3 [MH+] 311.1139, found 311.1139.
(+)-4-(4-Azidophenyl)-1-(4-methoxyphenyl)-2-oxoaze-
tidin-3-yl Acetate (11). Procedure A from 10. To a solution
of DMAP (0.910 g, 7.45 mmol), 10 (2.11 g, 6.81 mmol), and
triethylamine (3.71 g, 36.7 mmol) in THF (60 mL) was added
dropwise neat acetyl chloride (2.21 g, 28.1 mmol), immediately
forming a slurry. The ice bath was removed, and the reaction
mixture was stirred at room temperature for 1 h, then poured
into NaHCO3 (50 mL) and extracted with Et2O. The combined
organic extracts were washed with brine and dried over
MgSO4, and the solvent was removed under reduced pressure.
Column chromatography with EtOAc/hexanes (3:7) gave 2.174
g of a white solid (91%).
Procedure B from Commercially Available Acetoxy-
acetyl Chloride (9). To a solution of 6 (0.042 g, 0.166 mmol)
in CH2Cl2 (1 mL) was added a solution of 9 (0.033 g, 0.240
mmol) and TEA (0.028 g, 0.280 mmol) in CH2Cl2 (1 mL) via
cannula at 0 °C. The yellow solution was allowed to warm to
room temperature over 12 h. The solution was quenched with
water and poured into NaHCO3 and CH2Cl2. The aqueous layer
was extracted with CH2Cl2 three times. The combined organic
layers were washed with brine, dried over MgSO4, and
concentrated under reduced pressure. Flash chromatography
with EtOAc/hexanes (3:7) afforded 49 mg (83%) of a white
1
118 °C; IR (neat) 2121, 2098, 1732 cm-1; H NMR (500 MHz,
CDCl3) δ -0.09 (s, 3H), 0.11 (s, 3H), 0.71 (s, 9H), 3.75 (s, 3H),
5.12-5.13 (overlapped, 2H), 6.80 (d, J ) 9.02 Hz, 2H), 7.03
(d, J ) 8.49, 2H), 7.27 (d, J ) 9.01, 2H), 7.33 (d, J ) 8.51,
2H); 13C NMR (125 MHz, CDCl3) δ -5.4, -4.9, 17.8, 25.2, 55.3,
62.2, 77.6, 114.3, 118.6, 118.8, 129.7, 130.7, 130.9, 140.0, 156.2,
165.2; HRMS (FAB+) m/z calcd for C22H29N4O3Si [MH+]
425.2009, found 425.2010; [R]22 +115 (c 1.05, CHCl3).
D
(4S,3R)-4-[4-(Azidophenyl)]-3-tert-butyldimethylsi-
loxyazetidin-2-one (16). A solution of 15 (0.104 g, 0.245
mmol) in HPLC-grade acetonitrile (6 mL) was cooled to -20
°C (external temperature) for 30 min. An ice-cold solution of
CAN (0.390 g, 0.711 mmol) in water (2.5 mL) was added with
fast dropwise addition, carefully ensuring that the internal
temperature did not rise above -10 °C. The reaction mixture
was maintained at -15 to -10 °C for 2 h. The aqueous layer
was extracted three times with Et2O. The combined organic
extracts were washed with water, NaHCO3, and brine and
dried over MgSO4, and the solvent was removed under reduced
pressure. Flash chromatography with EtOAc/hexanes (1:5)
afforded 61 mg (78%) as an off-white solid: mp 80-81 °C; IR
(neat) 2127, 2094, 1750, 1712 cm-1; 1H NMR (500 MHz, CDCl3)
δ -0.12 (s, 3H), 0.07 (s, 3H), 0.69 (s, 9H), 4.80 (d, J ) 4.71 Hz,
1H), 5.07 (dd, J ) 4.68 and 2.86 Hz, 1H), 6.39 (s, 1H), 7.04 (d,
J ) 8.50 Hz, 2H), 7.32 (d, J ) 8.45 Hz, 2H); 13C NMR (125
MHz, CDCl3) δ -5.4, -5.0, 17.8, 25.2, 58.6, 79.5, 118.5, 129.4,
133.1, 140.0, 169.8; HRMS (FAB+) m/z calcd for C15H23N4O2-
Si [MH+] 319.1590, found 319.1574; [R]22D +45.1 (c 1.10, CH2-
Cl2).
solid: mp 159-160 °C; IR (neat) 2130, 2100, 1757, 1603 cm-1
;
1H NMR (400 MHz, CDCl3) δ 1.74 (s, 3H), 3.75 (s, 3H), 5.32
(d, J ) 4.79 Hz, 1H), 5.90 (d, J ) 4.80 Hz, 1H), 6.80 (d, J )
8.96 Hz, 2H), 7.01 (d, J ) 8.45 Hz, 2H), 7.26 (d, J ) 8.95 Hz,
2H), 7.30 (d, J ) 8.45 Hz, 2H); 13C NMR (100 MHz, CDCl3) δ
20.3, 55.8, 61.3, 76.9, 114.9, 119.2, 119.5, 129.4, 129.9, 130.5,
141.0, 157.0, 161.5, 169.6; HRMS (FAB+) m/z calcd for
C18H17N4O4 [MH+] 353.1250, found 353.1247.
(4S,3R)-4-[4-(Azidophenyl)]-3-tert-butyldimethylsiloxy-
1-(phenylcarbonyl)azetidin-2-one (17). A solution of 16
(0.025 g, 0.079 mmol), DMAP (0.003 g, 0.024 mmol), and TEA
(0.016 g, 0.158 mmol) in CH2Cl2 (1 mL) was cooled to 0 °C
under argon for 5 min. Neat benzoyl chloride (0.017 g, 0.120
mmol) was added in one portion, and the ice bath was removed.
After 90 min, another 1.5 equiv of benzoyl chloride was added
and the reaction mixture was allowed to stir at room temper-
ature. The reaction was quenched with ice-cold brine after
another 90 min. The reaction mixture was diluted with CH2-
Cl2, washed three times with brine, and dried over MgSO4,
and the solvent was removed under reduced pressure. Flash
chromatography with EtOAc/hexanes (1:10) afforded 24 mg
(73%) as an off-white solid: mp 80-83 °C; IR (neat) 2125, 1800,
(4S,3R)-4-(4-Azidophenyl)-1-(4-methoxyphenyl)-2-
oxoazetidin-3-yl Acetate (12). A 0.2 M NaH2PO4 buffer
solution was prepared (Millipore water), and the pH was
adjusted to 7.00 using 0.2 M Na2HPO4. Racemic lactam 11
(1.60 g, 4.54 mmol) was dissolved in a buffer/acetonitrile mix
(150 mL/25 mL) open to the air. Pseudomonas cepacia lipase
was added in one portion (1.78 g), and the mixture was stirred
vigorously for 24 h. The aqueous mixture was extracted with
EtOAc, and the combined organic layers were washed with
brine, dried over MgSO4, and concentrated under reduced
pressure. Flash chromatography with EtOAc/hexanes (3:7)
afforded 799 mg (>99% yield of enantiomer 12) of a white
solid: mp 120-121 °C; [R]22 +22 (c 0.55, CHCl3); HPLC,
D
1
1677 cm-1; H NMR (500 MHz, CDCl3) δ -0.06 (s, 3H), 0.12
Chiralpak AD-RH, 20% i-PrOH/hexane, 0.5 mL/min, 254 nm,
retention time for the major isomer ) 16.7 min, 99% ee,
(s, 3H), 0.75 (s, 9H), 5.15 (d, J ) 6.13 Hz, 1H), 5.41 (d, J )
6.11 Hz, 1H), 7.06 (d, J ) 8.5 Hz, 2H), 7.39 (d, J ) 8.48 Hz,
2H), 7.51 (t, J ) 7.93 Hz, 2H), 7.62 (t, J ) 7.47 Hz, 1H), 8.05
(d, J ) 7.2 Hz, 2H); 13C NMR (125 MHz, CDCl3) δ -5.4, -5.0,
1
retention time for the minor isomer ) 15.6 min. H and 13C
NMR, IR, and HRMS data are identical to those of 11 and are
found in the Supporting Information. 13 was isolated as 790

A Paclitaxel Photoaffinity Probe
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 26 6463
17.9, 25.2, 60.1, 76.3, 118.7, 128.1, 129.5, 129.8, 130.6, 131.8,
133.4, 140.0, 164.9, 166.2; HRMS (FAB+) m/z calcd for
C22H27N4O3Si [MH+] 423.1852, found 423.1847; [R]22D +135 (c
1.40, CHCl3).
6.93 Hz, 1H), 5.81 (d, J ) 8.85 Hz, 1H), 6.26-6.30 (overlapped,
2H), 6.98 (d, J ) 8.91 Hz, 1H), 7.10 (d, J ) 8.27 Hz, 2H), 7.41-
7.44 (m, 2H), 7.51-7.56 (m, 5H), 7.63-7.66 (m, 1H), 7.75 (1/2
ABq, J ) 7.89 Hz, 2H), 8.16 (1/2 ABq, J ) 7.94, 2H); 13C (125
MHz) δ 9.5, 14.9, 20.8, 21.6, 22.6, 26.8, 35.6, 43.1, 45.6, 54.2,
58.6, 72.2, 72.4, 72.9, 74.8, 75.5, 79.0, 81.2, 84.3, 119.5, 127.0,
128.7, 129.0, 130.1, 132.0, 133.3, 133.7, 134.7, 140.1, 141.7,
166.8, 167.0, 170.3, 171.2, 172.4, 203.5; HRMS (FAB+) m/z
(4S,3R)-Chloroacetic Acid 2-(4-Azidophenyl)-1-(4-meth-
oxyphenyl)-4-oxoazetidin-3-yl Ester (18). A solution of
chloroacetyl chloride (0.0096 g, 0.090 mmol) in THF (1 mL) at
room temperature was added to a solution of DMAP (0.0010
g, 0.0040 mmol), TEA (0.020 g, 0.20 mmol), and alcohol 18
(0.012 g, 0.040 mmol) via syringe. The reaction mixture was
stirred for 1 h, and the reaction was monitored by TLC. The
solution was quenched with NH4Cl, and the aqueous layer was
extracted with CH2Cl2. The combined organic layers were
washed with NaHCO3 and brine. The organic layer was dried
with brine and concentrated under reduced pressure. Flash
chromatography with EtOAc/hexanes (1:5) gave 5 mg (33%,
unoptimized) as a white solid: mp 140-143 °C; IR (neat) 2125,
2095, 1757, 1634 cm-1; 1H NMR (400 MHz, CDCl3) δ 3.60 (1/2
ABq, J ) 15.21 Hz, 1H), 3.78 (s, 3H), 3.87 (1/2 ABq, J ) 15.21
Hz, 1H), 5.39 (d, J ) 4.82 Hz, 1H), 6.00 (d, J ) 4.84 Hz, 1H),
6.83 (d, J ) 9.04 Hz, 2H), 7.04 (d, J ) 8.54 Hz, 2H), 7.27 (d,
J ) 7.27 Hz, 2H), 7.32 (d, J ) 8.51 Hz, 2H); 13C NMR (100
MHz, CDCl3) δ 40.2, 55.9, 61.2, 77.5, 114.9, 119.2, 119.7, 128.7,
129.8, 130.3, 141.4, 157.3, 160.4, 166.2; HRMS (FAB+) m/z
calcd for C47H51N4O14 [MH+] 895.3402, found 895.3418; [R]22
D
-21 (c 1.0, CH2Cl2); HPLC, IProSIL C18 AQ, 1:1 CH3CN/H2O,
1.0 mL/min, 254 nm, retention time ) 5.5 min, 97.5% purity.
Tubulin Assembly Assay. The ED50 value is the concen-
tration of the compound that causes 50% of the maximum
reduction in supernatant protein concentration. Maximum
reduction was obtained using 25 µM paclitaxel. A mixture of
10 µM tubulin bovine brain (1 mg/mL) with varying concentra-
tions of 2 in 100 µL of buffer (with 4% DMSO from the
analogue solution) containing 0.1 M Pipes, pH 6.9, 1 mM
MgSO4, 1 mM EGTA, and 0.5 mM GTP at 37 °C was incubated
for 15 min, and the samples were centrifuged in a Beckman
TL-100 ultracentrifuge for 4 min at 35 000g. Protein concen-
trations of the supernates were determined, and under these
conditions, tubulin did not assemble in the absence of pacli-
taxel or 2.
Cell Cytotoxicity Assay. MCF-7 and MCF7-ADR human
breast cancer cells were plated in 96-well culture plates at a
density near 2000 cells per well in Dulbecco’s MEM/F12
containing 10% fetal calf serum. The cells were incubated for
24 h, after which the medium was replaced with fresh medium
with 2 at varying concentrations. The cultures were grown an
additional 72 h, and the degree of proliferation was measured
using sulforhodamine B.
calcd for C18H15N4O4Cl [MH+] 387.0860, found 387.0854; [R]22
D
+4.0 (c 0.25, CHCl3). An X-ray crystal structure has been
obtained confirming the absolute configuration. See Supporting
Information.
3′-(4-Azidophenyl)-2′-tert-butyldimethylsiloxy-3′-dephen-
yl-7-triethylsiloxypaclitaxel (20). To a cold (0 °C) solution
of 7-TES-baccatin III (19, 0.027 g, 0.038 mmol) and 17 (0.024
g, 0.057 mmol) in THF (1 mL) was added NaH (0.046 g, 1.90
mmol) in one portion. The reaction mixture was stirred at 0
°C for 1 h and then warmed to room temperature and stirred
for an additional 3 h. The mixture was then cooled to 0 °C
and quenched with slow, dropwise addition of acetic acid. The
solution was extracted with ether, and the combined organic
layers were dried over MgSO4 and concentrated under reduced
pressure. Flash chromatography with EtOAc/hexanes (1:5)
gave 32 mg (74%) as a white solid: mp 186-189 °C; IR (neat)
Acknowledgment. This work was supported by the
National Institutes of Health, Grant CA-82801. The
authors thank Dr. Doug Powell of the University of
Kansas for X-ray structure determination, NaPro Bio-
therapeutics, Inc., Boulder, CO, for providing the taxane
starting material, and Amano Pharmaceutical Co., Ltd.,
Nagoya, Japan, for the gift of the lipase. J.T.S. is a
recipient of an American Foundation for Pharmaceutical
Education predoctoral fellowship and a Department of
Defense predoctoral fellowship (Grant DAMD17-99-1-
9243).
3426, 2122, 1725, 1665 cm-1 1H NMR (400 MHz, CDCl3) δ
;
-0.23 (s, 3H), 0.03 (s, 3H), 0.58-0.63 (m, 6H), 0.85 (s, 9H),
0.95 (t, J ) 7.90 Hz, 9H), 1.20 (s, 3H), 1.28 (s, 3H), 1.72 (s,
3H), 1.90-1.97 (m, 1H) 2.04 (s, 3H), 2.07-2.13 (m, 2H), 2.19
(s, 3H), 2.37-2.43 (m, 1H), 2.58 (s, 3H), 3.85 (d, J ) 6.96 Hz,
1H), 4.22 (1/2 ABq, J ) 8.42 Hz, 1H), 4.34 (1/2 ABq, J ) 8.37
Hz, 1H), 4.47-4.52 (m, 1H), 4.66 (br s, 1H), 4.97 (d, J ) 8.6
Hz, 1H), 5.69-5.73 (overlapped, 2H), 6.28 (t, J ) 8.85 Hz, 1H),
6.47 (s, 1H), 7.06-7.1 (m, 3H), 7.35 (d, J ) 8.39 Hz, 2H), 7.41-
7.45 (m, 2H), 7.50-7.56 (m, 3H), 7.61-7.65 (m, 1H), 7.75 (1/2
ABq, J ) 7.46 Hz, 2H), 8.15 (1/2 ABq, J ) 7.48 Hz, 2H); 13C
NMR (100 MHz) δ -5.2, -4.7, 5.6, 7.7, 10.5, 14.7, 18.6, 21.3,
21.9, 23.5, 25.6, 26.9, 30.1, 35.9, 38.2, 44.6, 47.1, 55.8, 58.8,
71.9, 72.6, 75.3, 79.2, 81.7, 84.6, 119.7, 127.5, 128.5, 129.2,
130.6, 132.3, 134.1, 134.2, 135.7, 140.2, 140.5, 167.3, 167.6,
169.7, 170.5, 171.7, 202.1; HRMS (FAB+) m/z calcd for
C59H78N4O14Si2 [MH+] 1123.5232, found 1123.5132; [R]22D -55
(c 0.75, CH2Cl2).
Supporting Information Available: General methods,
experimental procedures, and spectral data for those com-
pounds not provided in the Experimental Section and X-ray
structural information for compound 18. This material is
available free of charge via the Internet at http://pubs.acs.org.
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