A facile route to paclitaxel C-10 carbamates

Article abstract of DOI:10.1016/j.bmcl.2005.03.072

A general protocol for the synthesis of paclitaxel C-10 carbamates is described. The method entails MeI-mediated activation of 2′-O-TBS-7-O-TES- 10-O-deacetyl-paclitaxel-10-O-carbonylimidazole prior to reaction with amines. This method is effective for the synthesis of paclitaxel C-10 derivatives, including bifunctional molecules.

Full text of DOI:10.1016/j.bmcl.2005.03.072

Bioorganic & Medicinal Chemistry Letters 15 (2005) 2477–2480
A facile route to paclitaxel C-10 carbamates
Carlo Ballatore,a,* Simon E. Aspland,a Rosario Castillo,a Joel Desharnais,a
Trisha Eustaquio,a Chengzao Sun,a Angelo J. Castellinoa,* and Amos B. Smith, IIIb,*
aAcidophil, LLC, Suite 250, 10835 Road to the Cure, San Diego, CA 92121, USA
bDepartment of Chemistry, University of Pennsylvania, 231 South 34th Street, Philadelphia, PA 19104-6323, USA
Received 31 January 2005; revised 17 March 2005; accepted 17 March 2005
Available online 12 April 2005
Abstract—A general protocol for the synthesis of paclitaxel C-10 carbamates is described. The method entails MeI-mediated
activation of 20-O-TBS-7-O-TES-10-O-deacetyl-paclitaxel-10-O-carbonylimidazole prior to reaction with amines. This method is
effective for the synthesis of paclitaxel C-10 derivatives, including bifunctional molecules.
ꢀ 2005 Elsevier Ltd. All rights reserved.
Paclitaxel (Fig. 1), a natural product isolated from
Taxus brevifolia,1 has been used widely to treat solid tu-
mours. The mechanism of action involves mitotic arrest
in cancer cells through stabilization of microtubules via
an inhibition of depolymerization.2 Recent pioneering
studies by Trojanowski and co-workers at the Univer-
sity of Pennsylvania have also shown that paclitaxel
may hold considerable promise for the treatment of neu-
ro- degenerative diseases, such as AlzheimerÕs disease,
by replacing the protein tau, which binds to and stabi-
lizes tubulin polymerization, and which is subject to mis-
folding.3 Other microtubule binding drugs such as the
epothilones, eleutherobine and discodermolide may hold
similar clinical potential.4
doses required, the lack of selectivity towards normal
and tumour cells, and the poor water solubility, com-
bined with the rapid onset of drug resistance and the
inability to cross the blood–brain barrier. In an at-
tempt to address these issues, numerous bifunctional
conjugates have been designed and synthesized over
the past few years to improve the overall biological
profile of paclitaxel by linking the taxane skeleton to
an auxiliary molecule. These molecules include, for
example, targeting moieties such as monoclonal anti-
bodies for selective delivery of the drug to tumours.5
Other tactics have been used to increase solubility,
and thereby the bioavailability of the drug, by attach-
ing PEGs, carbohydrates and/or phosphates.6–8 In
addition, fluorescent probe molecules have been linked
to paclitaxel to generate bifunctional conjugates to be
employed as imaging agents able to visualize micro-
tubules in living cells.9
Although an effective clinical agent in cancer chemo-
therapy, disadvantages associated with the use of
paclitaxel include significant toxicity due to the high
The vast majority of bifunctional taxane-conjugates
reported to date have been constructed exploiting
the C-20 or C-7 positions. Since the hydroxyl moiety at
C-20 is known to be essential for biological activity,
C-20 conjugates are typically designed to release the
parent drug. The C-7 position, on the other hand, is
known to be relatively tolerant to structural modifica-
tions, thus conjugates at C-7 can be designed to be
hydrolytically stable. However, the size of the substitu-
ents attached at C-7 may play a significant role in
determining the biological activity of the drug. For
example, hydrolytically stable conjugates containing
large substituents such as folic acid moieties were found
to be inactive.10
Figure 1.
*
Corresponding authors. Fax: +1 858 646 0999; e-mail: acastellino@
acidophil.com
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.03.072

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C. Ballatore et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2477–2480
bifunctional constructs with hydrolytically stable carb-
amate linkages. In 2004, Miller et al.11 reported an exam-
ple of direct carbamoylation of a paclitaxel analogue via
a C-10 carbonylimidazole intermediate.12 We have
taken a similar tack beginning with 20-O-TBS-7-O-
TES-10-O-deacetyl-paclitaxel (1),13 available in three
steps and in high overall yield from paclitaxel (Fig. 2).
Conversion of 1 into the C-10 carbonylimidazole deriv-
ative (2) was next achieved quantitatively upon reaction
with carbonyl-diimidazole (CDI) (Fig. 3). Compound 2
was obtained in high purity as determined by NMR and
LC–MS, and could be employed directly without purifi-
cation, or stored at ꢀ20 ꢁC for several weeks without
noticeable decomposition.14 For derivatization of 2, we
first explored a protocol, which called for addition of
5–7 equiv of various amines (Table 1) in tert-butanol
(0.04 M) at 82 ꢁC (Fig. 3, Method A). Greenwald
et al.6 had successfully employed nearly identical reac-
tion conditions for the synthesis of several paclitaxel
C-7 carbamates from the corresponding C-7 carbonyl-
imidazole. The data presented in Table 1 indicate this
protocol is, however, not generally applicable. For
example, carbamate formation was found to be depen-
dent upon concentration, temperature and amount of
amine employed. In addition, the nucleophilicity and
Figure 2. Reagents and conditions: (a) TBS–Cl, dimethylformamide,
imidazole, 60 ꢁC, 1.5 h; (b) hydrazine, ethanol, rt, 1.5 h; (c) TES–Cl,
DMAP, dichloromethane, rt, 1 h.
Recently, our group and others have examined the C-10
hydroxyl as an alternative point of attachment that
might be more tolerant of conjugation with large mole-
cules. We were particularly interested in developing a
general protocol that would allow easy access to C-10
Figure 3. Reagents and conditions: (a) dichloromethane/carbonyl-diimidazole, rt, 16 h; (b) 5–7 equiv of amine in tert-butanol, 82 ꢁC, 16 h;
(c) acetonitrile/pyridine (1:1), HF/Py, 0 ꢁC to rt, 4 h; (d) methyl iodide/acetonitrile, 55 ꢁC, 3 h, (e) 2–3 equiv amine, rt.

C. Ballatore et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2477–2480
Table 1. Examples of paclitaxel C-10 carbamates
2479
Entry
Compd
Amine
% Conversion
Method Aa
% Conversion
Method Bb
Yieldc (%)
MPAd (%)
H2N
O
CBz
1
2
5a
5b
>95
>95
59
57
81
75
O
N
H
2
O
H2N
O
OH
3
4
5c
5d
>95
<5e
57
45
67
O
O
NH2
O
OH
>95e
>95
117
HO
HO
NH2
O
OH
5
6
5e
5f
Undetectable
>95
42
80
92
59
HO
NH2
OH
N
H
O
7
8
5g
5h
Undetectable
Undetectablee
>95
69
59
94
60
N
H
N
>95e
N
H
9
5i
5j
Undetectable
Undetectablee
>95
45
71
65
NH2
O
OH
10
>95e
148
NH2
H2N
O
11
5k
Undetectablee
>95e
45
17
O
HO
O
OH
a Conversion of 2 to compounds of general structure 3 (Fig. 3), as determined by LC–MS.
b Conversion of 4 to compounds of general structure 3 (Fig. 3), as determined by LC–MS.
c Overall yield over two steps, after HPLC purification.
d Ability of the compounds of general structure 5 to promote polymerization of tubulin, expressed as percentage of the activity of the parent drug.
The microtubule polymerization assay was done using the fluorescence based kit (cat. # BK011) produced by Cytoskeleton (Denver, CO).
e Reaction carried out in slightly basic condition by addition of DIEA.
steric encumbrance of the amine was observed to play a
significant role in the success or failure of the
derivatization.
unsuccessful or proceeds too slowly to be synthetically
useful.
To improve the reaction protocol leading to these and
other ÔdifficultÕ to form carbamates, we treated carbon-
ylimidazole derivative 2 with methyl iodide (Fig. 3,
Method B). The activated C-10 carbonyl methyl-
imidazolinium salt (4) was obtained in near quantitative
yield.15 Reaction of 4 with 2–3 equiv of amine at room
temperature then furnished the desired adduct in good
to excellent yield. Moreover, all reactions were complete
within 20–30 min. Removal of the protecting groups in
situ led to the desired final adduct (e.g., 5h)16 after
HPLC purification17 (Table 1); overall yields were in
general very good.
Although primary aliphatic amines (entries 1–3) and
reactive secondary amines such as piperidine (entry 6)
provide the corresponding C-10 carbamates in high
yield, less reactive amines such as morpholine (entry 7)
fail to react. Likewise, aniline derivatives (entries 9–11)
and benzimidazole (entry 8) fail to produce the desired
carbamate analogues under the reaction conditions.
Moreover, if the amines are not completely soluble at
the relatively high concentration (0.2–0.3 M) required
for coupling (e.g., glutamic acid and glucosamine HCl,
entries 4 and 5, respectively), the reaction is either

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C. Ballatore et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2477–2480
temperature under nitrogen atmosphere, and then
extracted with water (5 mL). The organic layer was dried
over sodium sulfate, filtered and concentrated to give
890 mg (96% yield) of 2, which was subsequently used
The derived paclitaxel C-10 carbamates were subse-
quently evaluated for their ability to promote polymer-
ization and stabilize microtubules in vitro, in direct
comparison with paclitaxel (Table 1). With the excep-
tion of compound 5k (entry 11), the carbamates retain
good to excellent activity in the biochemical assay, with
two paclitaxel C-10 carbamates showing enhanced activ-
ity (entries 4 and 10). Taken together, these results sup-
port the thesis that the C-10 position is quite tolerant to
structural modifications.
1
without purification; H NMR (CDCl3, 300 MHz): d 8.26
(s, 1H), 8.18 (d, J = 9 Hz, 2H), 7.77 (d, J = 8 Hz, 2H), 7.53
(m, 11H), 7.14 (s, 1H), 7.09 (d, J = 9 Hz, 1H), 6.59 (s, 1H),
6.32 (app t, J = 9 Hz, 1H), 5.78 (m, 2H), 5.02 (d, J = 8 Hz,
1H), 4.72 (d, J = 2 Hz, 1H), 4.56 (m, 1H), 4.38 (d,
J = 8 Hz, 1H), 4.25 (d, J = 8 Hz, 1H), 3.88 (d, J = 7 Hz,
1H), 2.62 (s, 3H), 2.45 (m, 1H), 2.2 (m, 1H), 2.08 (s, 3H),
1.98 (m, 1H), 1.78 (s, 3H), 1.62 (s, 3H), 1.32 (m, 3H), 1.22
(s, 3H), 0.95 (m, 9H), 0.83 (s, 9H), 0.62 (m, 6H), 0.1 (s,
3H), ꢀ0.2 (s, 3H); Electrospray (LC–MS) m/z 1134
(M+H+, C61H80N3O14Si2 requires 1134). Retention time
9.92 min (1–99% B, Method 2).17
In summary, we demonstrate in this letter that activ-
ation of carbonylimidazole derivatives such as 2 with
methyl iodide (Method B) permits construction of steri-
cally encumbered C-10 paclitaxel carbamate analogues
under quite mild conditions, and as such can be em-
ployed for the construction of hydrolytically stable
bifunctional molecules possessing intricate architectures.
We further believe this activation protocol holds consid-
erable promise for the construction of sterically highly
encumbered carbamates in general.
15. Synthetic procedure for compound 4: to a solution of 20-
O-TBS, 7-O-TES, 10-O-deacetyl-paclitaxel-10-O-carbon-
yl-imidazole (2, 200 mg, 0.176 mmol) in acetonitrile
(2 mL) in a sealable tube was added methyl iodide
(1 mL) and the resulting mixture was stirred for 3 h at
55 ꢁC. A stream of nitrogen was then used to remove the
volatiles and the residue was held under high vacuum for
4 h. The residue was then redissolved in anhydrous DMSO
or another anhydrous aprotic solvent and used directly for
carbamate formation.
References and notes
16. Synthetic procedure for 5 h: To a solution of 20-O-TBS,
7-O-TES, 10-O-deacetyl-paclitaxel-10-O-carbonyl-methyl-
imidazolinium iodide (4, 45 mg, 0.035 mmol), in anhy-
drous DMSO (0.4 mL) was added benzimidazole (13 mg,
300 mol %) followed by DIEA (6.1 lL, 300 mol %). The
reaction mixture was stirred at room temperature for
30 min until complete decolouration. The mixture was
then diluted with pyridine (0.4 mL), cooled to 0 ꢁC and
treated with HF/pyridine (0.35 mL). The resulting solution
was stirred for 3 h allowing the temperature to rise to
room temperature. The mixture was then diluted with
EtOAc (10 mL) and extracted with a saturated solution of
CuSO4 (3 · 2 mL) and water (3 · 2 mL). The organic
solution was then dried over sodium sulfate, filtered and
concentrated. The residue so obtained was purified by
preparative RP-HPLC (Method 1).17 Fractions containing
the appropriate mass, as determined by analytical HPLC–
MS (Method 2) were pooled and CH3CN removed under
reduced pressure. The remaining aqueous mixture was
then lyophilized obtaining 20 mg of 5h (59% overall yield).
1HNMR (CDCl3, 300 MHz): d 8.62 (s, 1H), 8.18 (d,
J = 7 Hz, 2H), 8.06 (d, J = 7 Hz, 1H), 7.82 (d, J = 7 Hz,
1H), 7.77 (d, J = 7 Hz, 2H), 7.62 (m, 1H), 7.53 (m, 11H),
7.09 (d, J = 9 Hz, 1H), 6.59 (s, 1H), 6.32 (app t, J = 9 Hz,
1H), 5.78 (m, 2H), 5.02 (d, J = 8 Hz, 1H), 4.82 (d,
J = 3 Hz, 1H), 4.56 (m, 1H), 4.38 (d, J = 8 Hz, 1H), 4.25
(d, J = 8 Hz, 1H), 3.88 (d, J = 7 Hz, 1H), 2.62 (m, 1H),
2.38 (m, 5H), 1.95 (m, 4H), 1.78 (s, 3H), 1.38 (s, 3H), 1.22
(s, 3H); Electrospray (LC–MS) m/z 956 (M+H+,
C53H54N3O14 requires 956). Retention time 6.55 min (1–
99% B, Method 2).
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17. Preparative RP-HPLC purification was conducted on
YMC-Pack ODS-A columns (S-5 lM, 300 · 20 mm ID)
with gradient elution between 0% B and 50% B or 0% B
and 100% B (A = 0.05% TFA in H2O; B = 0.05% TFA in
CH3CN) with gradient times of 10 min and a flow rate of
25 mL/min with UV 220 nm detection (Method 1). Ana-
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Screen ODS-A column (S-5 lM, 50 · 4.6 mm ID) with
gradient elution of 0% B to 100% B (A = 0.05% TFA in
H2O; B = 0.05% TFA in CH3CN) with gradient times of
10 min and a flow rate of 3.5 mL/min with UV 220 nm and
electrospray MS detection (Method 2).
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14. Synthetic and selected spectroscopic data for compound 2:
To 20-O-TBS, 7-O-TES, 10-O-deacetyl-paclitaxel (1,
845 mg, 0.81 mmol), in anhydrous DCM (6 mL) was
added carbonyl-diimidazole (530 mg, 400 mol %). The
reaction mixture was allowed to stir for 16 h at room