Mechanistic considerations pertaining to the solvolysis of paclitaxel analogs bearing ester groups at the C2′ position

Article abstract of DOI:10.1016/S0040-4039(01)01643-4

Dilute solutions of paclitaxel-related derivatives having chloroacetyl esters in the C2′ position undergo ready methanolysis according to pseudo first-order kinetics while more concentrated solutions appear to be stabilized, possibly by the formation of h

Full text of DOI:10.1016/S0040-4039(01)01643-4

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 42 (2001) 7747–7750
Mechanistic considerations pertaining to the solvolysis of
paclitaxel analogs bearing ester groups at the C2% position
Wieslaw A. Klis,* Jeffrey G. Sarver and Paul W. Erhardt
Center for Drug Design and Development, The University of Toledo College of Pharmacy, Toledo, OH 43606-3390, USA
Received 19 June 2001; accepted 21 August 2001
Abstract—Dilute solutions of paclitaxel-related derivatives having chloroacetyl esters in the C2% position undergo ready
methanolysis according to pseudo first-order kinetics while more concentrated solutions appear to be stabilized, possibly by the
formation of hydrophobic aggregates that tend to bury this reaction center. Methanolysis is also attenuated in the presence of
weak acid, suggesting that paclitaxel’s neighboring benzamide nitrogen may be participating in the reaction by serving as an
assisting nucleophile. © 2001 Elsevier Science Ltd. All rights reserved.
Selective manipulation of the hydroxyl groups present
in paclitaxel (PAC) and in 10-deacetylpaclitaxel (DAP)
(Fig. 1) to produce stable analogs and water-soluble
prodrugs, has received considerable attention over the
course of the last twenty years of PAC-related
research.1–11 Deutsch et al.3 have shown that among all
of the hydroxyl groups present in this family of com-
pounds, the C2% OH is the most reactive toward
acylation. When PAC is treated with carbonyldiimida-
zole, the resulting C2% acylated intermediate can addi-
tionally form an oxazolone derivative, an interesting
side-reaction more recently encountered by de Groot et
al.4 as well. Amino acid derivatives connected via ester
linkages at the C2% position are considerably less stable
than when connected at C7 such that the C2% arrange-
ment has been extensively pursued during prodrug
strategies. Likewise, Mathew et al.5 has exploited the
instability of C2% esters to produce C7-amino acid esters
of PAC by partial, selective hydrolysis of the 2%,7-bis-
substituted PAC analogs at pH 7.4. Harada et al.6 have
speculated that the instability of the C2% amino acid
esters is due to steric repulsion of the bulky groups
attached to C2% and C3% as well as to an electronic effect
from these types of esters% amino groups. The latter has
also been previously implicated by Zhao et al.7 and
more recently by Pendri et al.8 who suggested more
specifically that protonation of the amino group could
serve to assist attack of the C2% acyl functionality by
external nucleophiles due to a simple inductive effect.
Other investigators have further postulated that C2%
esters of PAC are particularly susceptible to cleavage
by various hydrolytic enzymes present in vivo.9,10
In an attempt to better understand the interaction of
PAC and related compounds with various components
within cancer cells,12,13 we have recently had occasion
to deploy monochloroacetyl (CAC) protection of both
PAC and DAP. During development of an HPLC
Figure 1. Structures of paclitaxel (PAC), 10-deacetylpaclitaxel
(DAP) and selected derivatives. For PAC, R=COCH3; For
DAP, R=H; For the monochloroacetyl (CAC) derivatives, a
ClCH2CO-adduct replaces one or more of the hydroxy group
hydrogens located at positions C2%, C7 and C10.
method
to
purify
2%,7-bis-monochloroacetyl-10-
deacetylpaclitaxel (2%,7-bis-CAC-DAP), we observed
that the compound was unstable in methanol solution
at a concentration of 1 mg/ml. After confirming this
observation and characterizing the breakdown product
Keywords: paclitaxel-C2% esters; methanolysis; hydrophobic aggre-
gates; neighboring group catalysis.
* Corresponding author.
0040-4039/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(01)01643-4

7748
W. A. Klis et al. / Tetrahedron Letters 42 (2001) 7747–7750
as 7-CAC-DAP we suggested that such treatments
might represent a convenient, general method to selec-
tively remove ester functionalities from the 2%-position
during synthetic manipulations of the PAC frame-
work.14 Interestingly, we also found that more concen-
trated methanolic solutions, namely 20 mg/ml for use in
preparative HPLC, did not appear to undergo solvoly-
sis. Likewise, the use of methanol to quench unreacted
acylating reagent immediately after chloroacetylation
did not appear to disturb the C2% ester, the latter
reflecting a derivatized DAP concentration of 4 mg/ml
in total solvent having an acidic pH. Thus, we decided
to further investigate the apparent differences in the
stability of several PAC and DAP CAC esters relative
to concentration and acidity. The compounds we exam-
ined are shown in Table 1.
ied for up to 120 h, no significant breakdown to DAP
was detected.
For the more concentrated experiments, a 10 mg/ml
solution of 2%,7-bis-CAC-DAP was allowed to stand at
ambient temperature. 10 ml aliquots were withdrawn
and diluted with 90 ml of acetonitrile such that 20 ml
samples could be injected into the HPLC at the same
concentrations used in the previous assays. As before,
isolation and crystallization of the product followed by
complete characterization confirmed its structure as
7-CAC-DAP. It can be noted that the NMR chemical
shifts for the C2% proton among the various family
members are diagnostic for loss of the 2%-CAC esters
(Table 1). For the more concentrated case, however, the
apparent T1/2 was found to be 16.2 h suggesting that
increased sample concentration can have a protective
effect upon solvolysis. Likewise, when the 1 mg/ml
studies were repeated in the presence of 0.2% acetic
acid, the decay curves again suggested that there is a
protective effect on hydrolysis. For the weakly acidic
studies, less than 4% breakdown of 2%,7-bis-CAC-DAP
was observed over 48 h and the calculated T1/2 was
determined to be >1,000 h. Thus, the presence of
monochloroacetic acid after acetylation fortuitously
serves to protect the product when methanol is used to
quench this reaction.
A 1 mg/ml solution of 2%,7-bis-CAC-DAP in methanol
was allowed to stand at ambient temperature. 20 ml
samples were withdrawn at T=0, 2, 24 and 48 h.
Longer incubations were deployed for compounds
showing little breakdown. Aliquots were assayed
directly by HPLC after establishing that responses
obtained for serial dilutions of a freshly prepared 10
mg/ml stock solution of 2%,7-bis-CAC-DAP were linear
throughout the concentration range anticipated for the
samples. A decay curve was generated by following the
decrease in compound peak area versus time. An
apparent half-life (T1/2) was then calculated from a
semi-log plot using Microsoft Excel. The decay curve
for 2%,7-bis-CAC-DAP appears to follow pseudo first-
order kinetics typical for solvolysis reactions. The T1/2
value for conversion to 7-CAC-DAP is 5.5 h. The
product growth curve matches very closely with the
compound decay curve. Similar results were obtained
for all of the other 2%-chloroacetylated derivatives listed
in Table 1 except for 2%-CAC-DAP whose T1/2 appears
to be about three times longer (18 h). Nevertheless, this
derivative is still much more labile than the chloroacetyl
esters placed at either the 7- or 10-positions (T1/2 values
all >>72 h). For example, when 7-CAC-DAP was stud-
When identical functionalities attached to different
locations on the same molecular framework display
significantly different chemical reactivity, the involve-
ment of neighboring groups as either catalysts or
inhibitors of such reactions is likely to be operative.15
An especially important case for catalysis occurs when
the participating groups can adopt orientations
analogous to a favorable ring system while the reaction
traverses its transition state. In 2%,7-bis-paclitaxel-
related esters, the 2%-ester functionality resides in close
proximity to the 3%-amide group. The latter can provide
nucleophilic catalysis via either its carbonyl oxygen or
its nitrogen lone pair.16 Normally, the electron density
of the nitrogen lone pair is partially displaced by its
Table 1. Stability of PAC and DAP CAC analogs in methanol at 1 mg/ml
Compound
RT (min)
NMR C2%H
T1/2 (h)
5.6
Product
2%,7,10-Tri-CAC-DAPa
2%,7-Bis-CAC-DAPa
2%-CAC-DAP
2%,7-Bis-CAC-PACa
2%-CAC-PACb
7,10-Bis-CAC-DAP
7-CAC-DAP
7-CAC-PAC
DAP
PAC
17.7
12.2
6.0
16.6
8.3
14.2
8.2
12.7
4.2
5.58
5.59
5.55
5.59
5.55
4.80
4.78
4.81
4.78
4.78
7,10-Bis-CAC-DAP
7-CAC-DAP
DAP
7-CAC-PAC
5.5
18.1
6.4
4.3
PAC
\\72c
\\72c
\\72c
\\72c
\\72c
–
–
–
–
–
5.5
Compound abbreviations are provided in Fig. 1. RT=Retention times on an analytical HPLC (Waters) equipped with a reversed-phase column
(Supleco Discovery C18) using a gradient elution (40% acetonitrile in water going to 85% over a 45 min time period) at a flow rate of 1 ml/min
monitored by UV detection (225 nm). NMR (CDCl3) values for the C2% proton shifts are in ppm relative to TMS. T1/2=Apparent half-lives (see
text). All compounds were independently prepared and characterized by HPLC, NMR and elemental analyses (their syntheses and biological
properties will be reported elsewhere).
a Previously reported by Rao et al.
b Previously reported by Rimoldi et al.21
c Less than 5% breakdown after 72 h.

W. A. Klis et al. / Tetrahedron Letters 42 (2001) 7747–7750
7749
Figure 2. Mechanistic model for the methanolysis of PAC-related 2%-esters wherein the neighboring 3%-amide nitrogen atom
catalyzes the reaction by serving as a nucleophilic hydrogen bond/proton acceptor.
concentration-dependent aggregates in non-polar media
such as chloroform17 and to undergo hydrophobic-
driven, conformational collapse of the C13 side chain in
polar media.18 While the latter would presumably occur
in a concentration independent manner, another way
for the lipophilic C13 side chains to avoid protic, polar
solvents would be to cluster among two or more PAC
molecules, a process that would be promoted by
increasing concentration. Clustering of the C13 side
chains, in turn, would be expected to limit the access of
methanol to the 3%-amide-catalyzed, 2%-ester-reaction
area. Additional studies directed toward the further
elucidation of the proposed mechanism for solvolysis
are underway. The implications of our findings on the
pharmacological profiles and structure–activity rela-
tionships for the PAC-related family of compounds are
also important relative to recent models for PAC%s
association with microtubules.19,20 For example, Snyder
et al.20 has recently reported a binding conformation in
which b-tubulin’s His-229 imidazole ring appears to
become flanked by the phenyl rings from PAC’s 3%-
benzamido and 2-benzoyl moieties. The chemical model
proposed herein suggests that such a stacked arrange-
ment could be further stabilized by hydrogen bonding
or even proton transfer between the nucleophilic benza-
mide nitrogen and an imidazole N-H depending upon
the latter’s state of protonation.
resonance relationship with the carbonyl such that the
overall system is planar and the oxygen typically serves
as the predominant nucleophile. However, in this case
the adjacent phenyl ring also donates electrons to the
carbonyl such that there is a relative increase in the
localization of the lone pair on nitrogen, an increase in
the nitrogen%s tetrahedral character, and an increase in
its capacity to serve as a nucleophile.16 That this type of
catalysis would be expected to be very sensitive to
acidification compared to having the oxygen play such
a role, is in line with the experimental observation that
the overall solvolysis is dramatically attenuated in
weakly acidic solutions. As shown in Fig. 2, the 2%-ester,
the 3%-amide nitrogen, and one molecule of methanol
can be placed in an arrangement wherein the six atoms
that participate in the reaction (bolded) become ori-
ented in a spatial relationship that resembles a six-mem-
bered ring. In this model, the amide nitrogen serves as
a nucleophilic hydrogen bond acceptor to effect neigh-
boring group catalysis of the methanolysis reaction by
enhancing the nucleophilicity of the methanol oxygen.
While a concerted mechanism is also possible, the
resulting transesterification has been depicted as a two-
step process in order to emphasize the catalytic role
initially played by the nitrogen lone pair.
The concept of neighboring nucleophile-assisted hydrol-
ysis of 2%-PAC esters was used by Nicolaou et al.11 in
their design of PAC prodrugs. For example, the rate of
PAC release was found to increase across the series
HOOCCH2XCH2COO-2%-PAC according to the elec-
tron-withdrawing nature of the heteroatom systems
placed at X. In this case, however, the proposed mech-
anism initially involves complete removal of the car-
boxylic acid proton under basic conditions such that
the resulting anion can then effect nucleophilic attack
of the ester carbonyl located, by design, to be six atoms
away.
Acknowledgements
The authors thank Gunda Georg for her early discus-
sions about general PAC-related chemistry. This work
was funded from a research grant awarded to Paul
Erhardt by the Susan G. Koman Foundation.
The mechanism depicted in Fig. 2 would be expected to
be accompanied by pseudo first order kinetics wherein
the apparent half-life should be independent of sub-
strate concentration. While this is exactly what appears
to be happening in dilute solutions, we also observed
that more concentrated solutions have extended half-
lives. One possible explanation for this seeming para-
dox is that the PAC-related materials may be forming
aggregates as their concentrations are increased. In this
regard, PAC has been observed by others to form
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