Synthesis and biological evaluation of 2'-carbamate-linked and 2'-carbonate-linked prodrugs of paclitaxel selective activation by the tumor-associated protease plasmin

Article abstract of DOI:10.1021/jm0009078

The nontoxic paclitaxel-2'-carbamate prodrugs 2-5 and paclitaxel-2'-carbonate prodrug 6 were synthesized and tested for activation by the tumor-associated enzyme plasmin. A generally applicable method for the synthesis of paclitaxel-2'-carbamates was deve

Full text of DOI:10.1021/jm0009078

J . Med. Chem. 2000, 43, 3093-3102
3093
Syn th esis a n d Biologica l Eva lu a tion of 2′-Ca r ba m a te-Lin k ed a n d
2′-Ca r bon a te-Lin k ed P r od r u gs of P a clita xel: Selective Activa tion by th e
Tu m or -Associa ted P r otea se P la sm in
Franciscus M. H. de Groot, Leon W. A. van Berkom, and Hans W. Scheeren*
Department of Organic Chemistry, NSR-Center for Molecular Structure, Design and Synthesis, University of Nijmegen,
Toernooiveld 1, 6525 ED Nijmegen, The Netherlands
Received J anuary 14, 2000
The nontoxic paclitaxel-2′-carbamate prodrugs 2-5 and paclitaxel-2′-carbonate prodrug 6 were
synthesized and tested for activation by the tumor-associated enzyme plasmin. A generally
applicable method for the synthesis of paclitaxel-2′-carbamates was developed. In buffer solution,
prodrug 2, which contained an unsubstituted ethylenediamine spacer, was not stable, whereas
prodrugs 3-6 were highly stable. Prodrugs 3-6 showed on average a decrease in cytotoxicity
of more than 8000-fold in comparison with the parent drug in seven human tumor cell lines.
Prodrugs 5 and 6 are the most nontoxic prodrugs of paclitaxel that yield the free parent drug
upon selective activation currently reported. Enzyme hydrolysis and spacer elimination rates
were determined by incubation of prodrugs 5 and 6 in the presence of human plasmin. From
these results, prodrug 6 was selected as the promising prodrug for further in vivo studies.
In tr od u ction
paclitaxel begs development of an improved delivery
system.11 The conversion of paclitaxel into prodrugs that
are hydrolyzed by the tumor-associated protease plas-
min might yield more selective derivatives that, in
addition, show improved water solubility.
The lack of selectivity of chemotherapeutic anticancer
agents such as paclitaxel (1) (Chart 1) is still a serious
drawback in conventional cancer chemotherapy. To
render anticancer drugs more selective for tumor cells,
the existence of tumor-associated enzymes can be
exploited to convert a nontoxic prodrug into the biologi-
cally active parent compound in a site-specific manner.
A high level of a cytotoxic anticancer drug in tumor
tissue can be generated via this concept.
The serine protease plasmin plays a key role in tumor
invasion and metastasis.1,2 The proteolytically active
form of plasmin is localized at the tumor level because
it is formed from its inactive proenzyme form plas-
minogen by urokinase-type plasminogen activator, pro-
duced by cancer and/or stroma cells.3 Active plasmin
is very rapidly inhibited by inhibitors such as R2-anti-
plasmin in the blood circulation. Thus, plasmin is a very
promising enzyme for exploitation in a tumor-specific
prodrug approach and can serve as a suitable target for
prodrug monotherapy.4-6 In a preceding paper,6 we
reported the first anthracycline prodrugs that were
efficiently activated by plasmin. The first paclitaxel
prodrugs designed for activation by plasmin are now
described.
Paclitaxel (Taxol)7 is a chemotherapeutic agent with
promising antitumor activity,8 especially against ovar-
ian, breast, and lung cancers.9 Although paclitaxel, like
other cytotoxic agents, shows severe side effects, rela-
tively few efforts have been made to develop selective
antitumor prodrugs of paclitaxel. An additional problem
for clinical application of this compound is its low water
solubility, and for administration it must be dissolved
in Chremophor EL, which is believed to cause hyper-
sensitivity reactions.10 The low therapeutic index of
A majority of the efforts for derivatization of paclitaxel
with peptides were performed to obtain derivatives with
improved water solubility,12-16 and only a few examples
of the targeting of paclitaxel have been reported.17-19
Recently, peptide conjugates of paclitaxel18,19 have been
reported which were claimed to be extracellularly stable
and that contained a monoclonal antibody which was
needed for tumor specificity. After internalization by the
cell, the dipeptide needed to be cleaved intracellularly
by lysosomal enzymes (cathepsin B).
It was reasoned that 2′-carbamate prodrugs of pacli-
taxel would best fulfill the requirements of an ideal
prodrug, i.e., low cytotoxicity for healthy tissue, high
stability against unspecific enzymes, and fast hydrolysis
by the tumor-associated enzyme. By modification of the
2′-hydroxyl functionality the prodrug could be expected
to lose its cytotoxic activity.20-22 It is important to note
that linkage of the promoiety to the 2′-position of
paclitaxel is only feasible if the resulting bond is
biologically stable and does not lead to premature drug
release. In literature several paclitaxel-2′-esters and
paclitaxel-2′-carbonates have been reported that were
hydrolyzed at the 2′-position by nonspecific enzymes.23,24
Very recently, two papers appeared in which paclitaxel
prodrugs were reported containing a labile 2′-ester
linkage.25,26 In the first report,25 a peptide derivative of
paclitaxel was described which was designed for binding
to a cell surface receptor. Although the paclitaxel
conjugate was shown to retain receptor binding proper-
ties, the conjugate might be unspecifically cleaved at
the paclitaxel-2′-succinate linkage by ubiquitous extra-
cellular esterases. In the second paper,26 a tetrasaccha-
ride conjugate of paclitaxel was presented in which the
* To whom correspondence should be addressed. Tel: +31-(0)24-
3652331. Fax: +31-(0)24-3652929. E-mail: jsch@sci.kun.nl.
10.1021/jm0009078 CCC: $19.00 © 2000 American Chemical Society
Published on Web 07/19/2000

3094 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 16
de Groot et al.
Ch a r t 1
Sch em e 1
promoiety was linked to the 2′-position of paclitaxel via
a labile 2′-ester linkage. This paclitaxel conjugate was
designed for binding to hyaluronic acid receptors.
A high priority in the design of our paclitaxel prodrugs
is that they should show a marked decrease in cyto-
toxicity, relative to paclitaxel. By linking the specifier
to the drug via a 2′-carbamate, the resulting bond is
expected to be stable against unspecific hydrolysis by
esterases, proteases, and other enzymes distributed
ubiquitously throughout the body. None of the paclitaxel
derivatives reported until now, designed for exerting
increased tumor selectivity, contain a paclitaxel-2′-
carbamate linkage.
On the basis of the above rationale, paclitaxel car-
bamate prodrugs 2-5 (Chart 1) were synthesized. After
plasmin hydrolysis the spacer should spontaneously
cyclize to yield a cyclic urea derivative, thereby releasing
free paclitaxel, as depicted in Scheme 1.27 The diamine
spacers were selected because they are appropriate for
attachment to the tripeptide via an amide bond and
attachment to the drug via a carbamate bond. In
addition, the incorporation of a spacer might enhance
the rate of plasmin hydrolysis. Since it has been
reported that a N,N′-dimethyl-substituted ethylenedi-
amine spacer gives a faster cyclization reaction than the
unsubstituted spacer,27 this entity was also incorporated
in paclitaxel tripartate prodrugs.
Although a carbonate linkage between drug and
promoiety would be expected to be less stable against
unspecific enzymes than a carbamate linkage, it would
probably be much less susceptible to esterase cleavage
than an ester linkage. The paclitaxel-2′-carbonate pro-
drug 6 which contains a 1,6-elimination spacer was then
synthesized (Chart 1). This aromatic spacer proved to
be a versatile self-eliminating connector in our plasmin
prodrugs of anthracyclines.6 If cleavage of the tripeptide
by plasmin should occur, then spacer elimination is
assured.28
The synthesized prodrugs were characterized biologi-
cally by determination of (i) stability during 3 days of
incubation in buffer solution, (ii) kinetics of enzymatic
activation and spacer cyclization during incubation in
the presence of plasmin, and (iii) cytotoxicity in seven
human tumor cell lines compared with the cytotoxicity
of the parent drug, paclitaxel.
Ch em istr y
The prodrugs in the present paper were prepared by
the coupling reactions between the drug and the spacer
end of the tripeptide spacer moieties. D-Ala-Phe-Lys and
D-Val-Leu-Lys were selected as tripeptides, since they
can serve as suitable plasmin substrates.29
Since 2′-carbamate prodrugs of paclitaxel have not
been reported, we developed a route to selectively
convert the 2′-hydroxyl group into the corresponding
carbamate. It was thought that the most feasible route
for the preparation of the 2′-carbamate was to react the
amino group of the tripeptide spacer moiety with a 2′-
activated paclitaxel derivative. During its synthesis
however, the 2′-activated paclitaxel derivative should
not react with the 3′-nitrogen in a cyclization reaction
to give the corresponding oxazolone which is formed
easily.12,24 When the activation of the 2′-hydroxyl func-
tion was attempted with carbonyldiimidazole (CDI), the
undesired ring-closed product was obtained. Reaction
of paclitaxel with 4-nitrophenyl chloroformate at 0 °C
also led to the undesired 2′,7-disubstituted product.

2′-Carbamate/ Carbonate-Linked Prodrugs of Paclitaxel
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 16 3095
Sch em e 2
Sch em e 3
When paclitaxel was treated with 10 equiv of 4-nitro-
phenyl chloroformate at -50 °C in the presence of
pyridine, the desired activated 2′-carbonate 7 was
formed in a reasonable yield (Scheme 2). No oxazolone
was detected, and the 7-hydroxyl group remained unaf-
fected.
spacer was substituted with a Z group, probably via an
intermolecular reaction, to give a fully protected tri-
peptide spacer moiety. The spacer was monoprotected
by reaction with excess of spacer with di-tert-butyldi-
carbonate prior to coupling to prevent this. Compound
17 was then connected to both tripeptides 12 and 13 to
yield protected promoieties 18 and 19, and the Boc
group was removed using hydrochloric acid/ethyl acetate
resulting in the formation of ammonium salts 21 and
22. The final coupling step was performed by addition
of 21 or 22 to a solution of 2′-activated paclitaxel 7 and
base to yield both protected prodrugs 24 and 25.
Hydrogenolysis in the presence of acetic acid yielded
paclitaxel prodrugs 3 and 4. The synthesis of prodrug
5 containing the monomethyl spacer was achieved in a
similar manner. Protected tripeptide 13 was reacted
with N-Boc-N-methylethylenediamine (16) to yield com-
pound 20. The Boc-substituted amine was then depro-
tected and reacted with activated paclitaxel derivative
7. Removal of both Z groups in the usual manner yielded
paclitaxel prodrug 5.
The synthesis of the D-Ala-Phe-Lys tripeptide was
described in a preceding paper;6 D-Val-Leu-Lys was
prepared via well-established peptide chemistry (Scheme
3). Allyloxycarbonyl (Aloc) or benzyloxycarbonyl (Z) was
chosen as the protecting group. Doubly protected tri-
peptides 11 and 12 were synthesized starting with the
coupling of H-Lys(Boc)-OBu to the hydrazide of Boc-
protected D-Val-Leu via in situ formation of the corre-
sponding azide (Scheme 3). Removal of the Boc groups
and the tert-butyl ester protection under acidic condi-
tions yielded deprotected tripeptide 10, and following
protection of the amines using Aloc-ONSu or Z-ONSu
under basic conditions yielded the doubly protected
tripeptides 11 and 12. The protection of D-Ala-Phe-Lys6
with Z groups yielding Z-D-Ala-Phe-Lys(Z)-OH (13) was
performed in a similar manner.
For the construction of paclitaxel prodrug 6, 4-amino-
benzyl alcohol was first coupled to the tripeptide Aloc-
D-Val-Leu-Lys(Aloc)-OH (11) (Scheme 5) using isobutyl
chloroformate, and the resultant benzylic alcohol 27 was
activated by treatment with 4-nitrophenyl chlorofor-
mate, to give carbonate 28. Reaction of the 2′-hydroxyl
of paclitaxel with this activated carbonate derivative in
the presence of DMAP led to the protected tripartate
prodrug 29. The Aloc protecting groups were removed
using tributyltin hydride in the presence of a palladium
catalyst.30 Final treatment with hydrochloric acid in
ethyl acetate provided the corresponding paclitaxel
prodrug 6 as its double ammonium salt.
Ethylenediamine was then coupled to the Z-protected
tripeptide 13 using isobutyl chloroformate, and the
obtained tripeptide spacer was isolated as its acetic acid
salt (14, Scheme 4). This salt was added to paclitaxel-
2′-carbonate (7) in the presence of a base to yield
protected prodrug 15. The Z groups were removed by
hydrogenolysis resulting in the formation of prodrug 2.
The presence of 5% acetic acid in the solvent was
essential to prevent formation of baccatin III.
The attachment of the N,N′-dimethyl-substituted
ethylenediamine spacer to the tripeptide was attempted
in a manner similar to that for the unsubstituted spacer.
However, under basic conditions the amino group of the

3096 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 16
de Groot et al.
Sch em e 4
Ta ble 1. Cytotoxicity (ID50 values,a,b ng/mL) of Paclitaxel (1)
and the 2′-Modified Paclitaxel Prodrugs 2-6 in Various Tumor
Cell Lines32
half-lives of enzymatic hydrolysis of prodrugs 5 and 6
were determined by capillary electrophoresis.31 The
prodrugs were incubated at a concentration of 200 µM
with 100 µg/mL human plasmin in a Tris buffer solution
(pH 7.3). The half-life of enzymatic hydrolysis for
prodrug 6 was 42 min, while prodrug 5 was hydrolyzed
with a half-life of 3.5 min. As expected the 1,6-elimina-
tion of the spacer of prodrug 6 was instantaneous,
whereas the half-life of spacer cyclization of prodrug 5,
after peptide cleavage, was 23 h.
compd
MCF-7 EVSA-T WIDR IGROV M19 A498 H226
paclitaxel (1)
prodrug 2c
prodrug 3c
prodrug 4c
prodrug 5c
prodrug 6c
<3
16
6.6
6.9
>63
9.2
<3
11
7.2
5.2
>63
9.3
<3
13
10
11
47
11
10
19
11
26
45
27
<3
45
17
24
47 >63
20 >60
<3
40
38
45
<3
41
21
25
48
16
a
Drug dose that inhibited cell growth by 50% compared to
untreated control cultures. SRB cell viability test. ×103.
b
c
All prodrugs 2-6 showed a strongly reduced cytotox-
icity in seven well-characterized human tumor cell lines
compared with paclitaxel (Table 1).
Biologica l Da ta
An average 8000-fold reduction in cytotoxicity was
observed, ranging from 1100-20000 times. Even pacli-
taxel-2′-carbonate prodrug 6 proved to be highly stable
in these cell assays. From these results it can be
concluded that prodrugs 5 and 6 are, to our knowledge,
the most nontoxic prodrugs of paclitaxel reported until
now, which do yield the free parent drug upon selective
activation.
The prodrugs 3-6 were highly stable in a Tris buffer
solution (pH 7.3). After 3 days of incubation no degrada-
tion products were detected. However, prodrug 2 de-
composed completely within 24 h, baccatin III being one
of the degradation products.
The prodrugs 3-6 were incubated with human plas-
min in a Tris buffer solution (pH 7.3) in order to
determine whether the enzyme was able to hydrolyze
them to yield free paclitaxel. Prodrugs 3 and 4 were not
converted into paclitaxel; they remained stable in the
presence of plasmin for at least 3 days. Prodrugs 5 and
6 produced paclitaxel under these circumstances. The
Discu ssion
It is interesting to note that paclitaxel-2′-carbamate
prodrug 2 is not stable in buffer solution at pH 7.3,

2′-Carbamate/ Carbonate-Linked Prodrugs of Paclitaxel
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 16 3097
Sch em e 5
Sch em e 6
whereas prodrugs 3 and 4 both proved to be stable in
this buffer for 3 days. It was rationalized that the
carbamate nitrogen of prodrug 2 could account for the
formation of baccatin III by assisting in a kind of
addition-elimination sequence, as depicted in Scheme
6. The methyl substituent on the carbamate nitrogen
in prodrugs 3 and 4 would hinder this undesired
reaction. Unfortunately, the N,N′-dimethyl-substituted
prodrugs are not hydrolyzed by plasmin, probably
because of the steric hindrance imposed by the methyl-
substituted amide bond on the enzyme active site.
Therefore, the prodrug 5 was synthesized. The methyl
substituent on the carbamate nitrogen should prevent
fragmentation, and the absence of the methyl group on
the amide nitrogen might make enzymatic cleavage by
plasmin possible. Both of these hypotheses proved to
be valid, since prodrug 5 was stable in buffer solution
and was hydrolyzed by plasmin, to yield paclitaxel.
The enzymatic activation rates of the paclitaxel
prodrugs are comparable to the rates of the anthra-
cycline prodrugs reported in the previous paper.6 Pro-
teolysis of the anthracycline prodrugs proceeded with
half-lives of 7-19 min at a plasmin concentration of 50
µg/mL, while the paclitaxel prodrugs of the current
paper are hydrolyzed with half-lives of 3.5 and 42 min
at a plasmin concentration of 100 µg/mL. This enzyme
concentration is not necessarily the in vivo concentra-
tion needed for the concept to work, although from a
physiological point of view it can be considered rather
high. However, it was demonstrated that the above-
mentioned doxorubicin prodrugs were equally cytotoxic
to free doxorubicin in u-PA-transfected cells. Appar-
ently, these cells produced sufficient plasmin for com-
plete activation of the anthracycline prodrugs.
When the plasmin hydrolysis rates of prodrugs 5 and
6 are compared to one another, it is expected that the

3098 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 16
de Groot et al.
under an argon atmosphere was added pyridine (4 drops). At
-50 °C, 275 mg (6.0 equiv) of 4-nitrophenyl chloroformate
dissolved in dry dichloromethane was added. The reaction
mixture was stirred at -50 °C and after 4 h 4-nitrophenyl
chloroformate (4.2 equiv) was added. After 1 h the mixture
was diluted with dichloromethane and washed with 0.5 N
potassium bisulfate and brine and dried over anhydrous
sodium sulfate. After evaporation of the solvents the residual
yellow film was purified by means of column chromatography
(ethyl acetate-hexane, 1:1), to yield 133 mg of activated
paclitaxel 7 (78%, 73% conversion): mp 161 °C; 1H NMR (300
MHz, CDCl3) δ 1.15 (s, 3H, 17), 1.26 (s, 3H, 16), 1.69 (s, 3H,
19), 1.92 (s, 3H, 18), 2.22 (s, 3H, 10-OAc), 2.49 (s, 3H, 4-OAc),
2.55 (m, 1H, 6a), 3.82 (d, 1H, J ) 7.0 Hz, 3), 4.21 (d, 1H, J )
8.4 Hz, 20b), 4.33 (d, 1H, J ) 8.4 Hz, 20a), 4.42 (m, 1H, 7),
4.96 (bd, 1H, J ) 7.7 Hz, 5), 5.53 (d, 1H, J ) 2.7 Hz, 2′), 5.70
(d, 1H, J ) 7.1 Hz, 2), 6.10 (dd, 1H, J ) 2.6 Hz, J ) 9.4 Hz,
3′), 6.29 (s, 1H, 10), 6.34 (m, 1H, 13), 6.90 (d, 1H, J ) 9.4 Hz,
N-H), 7.34 (d, 2H, J ) 9.1 Hz, nitrophenyl), 7.37-7.65
(m, 11H, aromatic), 7.75 (d, 2H, J ) 7.2 Hz, aromatic), 8.15
(d, 2H, J ) 7.2 Hz, aromatic), 8.26 (d, 2H, J ) 9.1 Hz,
nitrophenyl); MS (FAB) m/e 1020 (M + H)+, 1042 (M + Na)+.
Anal. (C54H54N2O18‚1.5H2O) C, H, N.
Boc-D-Va l-Leu -Lys(Boc)-OBu (9). To a solution of 2.92 g
(8.50 mmol) of Boc-D-Val-Leu-N2H3 (8) in ethyl acetate under
an argon atmosphere at a temperature of -30 °C was added
dropwise 7.5 mL of a 3.2 M hydrochloric acid-ethyl acetate
solution. The reaction mixture was kept at -30 °C and tert-
butylnitrite (1.2 mL, 1.2 equiv) was added. After 60 min the
solution was neutralized by addition of Et3N (3.4 mL, 24.5
mmol), then H-Lys(Boc)-OBu‚HCl (3.06 g, 1.06 equiv) was
added. Again Et3N (1.4 mL, 10 mmol) was added dropwise
to the reaction mixture. The reaction mixture was stirred at
4 °C for 20 h. The mixture was washed with 10% citric acid
and sodium bicarbonate. The organic layer was dried over
anhydrous sodium sulfate and evaporated in vacuo. The
product was purified by means of column chromatography
(ethyl acetate-hexane, 1:1), obtaining 3.11 g (60%) of the
desired product 9: mp 175-176 °C; 1H NMR (300 MHz, CDCl3)
δ 0.90-1.00 (m, 12H, 2 CH3-Val and 2 CH3-Leu), 1.20-1.75
(m, 6H, 3 CH2-Lys), 1.44 (m, 27H, 6 CH3 -Boc and 3 CH3 -OBu),
1.80 (m, 1H, CH-Leu), 2.15 (m, 1H, CH-Val), 3.09 (m, 2H,
N-CH2-Lys), 3.94 (m, 1H, HR), 4.45 (m, 2H, 2 HR); MS
(FAB) m/e 1616 (M + H)+, 637 (M + Na)+. Anal. (C31H58N4O8)
C, H, N.
H-D-Va l-Leu -Lys-OH (10). To a solution of 3.71 g (6.03
mmol) of compound 9 in dichloromethane (20 mL) was added
3.2 M hydrochloric acid-ethyl acetate (100 mL). After 60 min
the solution was concentrated to dryness in vacuo. tert-Butyl
alcohol was added and evaporated and the resulting solid (2.60
g, 100%) was freeze-dried in dioxane-water and used without
further purification: 1H NMR (300 MHz, CDCl3/CD3OD) δ 0.92
(d, 3H, J ) 5.9 Hz, CH3-Leu), 0.98 (d, 3H, J ) 5.9 Hz, CH3-
Leu), 1.08 (d, 3H, J ) 6.8 Hz, CH3-Val), 1.09 (d, 3H, J ) 6.8
Hz, CH3-Val), 1.40-2.00 (m, 9H, 3 CH2-Lys, CH-Leu and CH2-
Leu), 2.26 (m, 1H, CH-Val), 3.00 (m, 2H, N-CH2-Lys), 3.98
(m, 1H, HR), 4.30-4.50 (m, 2H, 2 HR), 8.09 (d, 1H, J ) 7.8
Hz, N-H), 8.81 (d, 1H, J ) 7.4 Hz, N-H); MS (FAB) m/e 359
(M + H)+, 381 (M + Na)+.
Gen er a l P r oced u r e for Z-P r otection of Un p r otected
Tr ip ep tid es. Z-D-Va l-Leu -Lys(Z)-OH (12). To solution of 977
mg (2.27 mmol) of H-D-Val-Leu-Lys-OH (10) in water-aceto-
nitrile was added triethylamine until a pH of 9.5 was reached.
Then a solution of 1.15 g (2.04 equiv) of Z-ONSu in acetonitrile
was added and the reaction mixture was kept basic at a pH of
9.0 by adding triethylamine. After the pH of the mixture did
not alter anymore, the solution was stirred for 0.5 h. The
solution was evaporated in vacuo to remove acetonitrile and
the resulting solution was acidified with 1.0 M hydrochloric
acid until a pH of 1.5 was reached. A small amount of methanol
was added and the water layer was extracted with chloroform.
The organic layer was dried over anhydrous sodium sulfate
and evaporated to dryness. The product was purified by
crystallization from methanol-diisopropyl ether to obtain 826
nature of the tripeptide is not responsible for the
different enzyme activation rates. Both D-Ala-Phe-Lys
and D-Val-Leu-Lys have shown similar rates of pro-
teolysis by plasmin.5 Therefore, it is tempting to suggest
that plasmin prefers a substrate in which a tripeptide
is linked to the more flexible ethylenediamine spacer
compared with the more rigid aromatic spacer of pro-
drug 6.
The rate of spacer cyclization of 2′-carbamate prodrug
5, however, is not ideal for application under physio-
logical conditions, as the drug-linker intermediate
might diffuse from the site of cleavage before the parent
drug is released. As expected, the elimination of the
spacer in prodrug 5 is slower than the half-life of
35 min, which is reported for a N,N′-dimethyl-substi-
tuted ethylenediamine spacer which releases 4-hydroxy-
anisole.27
From Table 1 it is clear that the cytotoxicity of the
carbamate prodrugs shows a marked decrease in com-
parison with paclitaxel. This is probably caused by the
stable 2′-carbamate linkage of prodrugs 3-5. Decom-
position of prodrug 2 according to the mechanism
depicted in Scheme 6 leads to nontoxic degradation
products such as baccatin III. It is remarkable that
prodrug 6 also shows an unambiguous reduction in
cytotoxicity, whereas several paclitaxel-2′-carbonates
reported in the literature were converted to the corre-
sponding parent drug.24 In the case of prodrug 6 the
presence of the tripeptide possibly causes shielding of
the 2′-position toward esterases.
In conclusion, the first paclitaxel prodrugs designed
for activation by the tumor-associated enzyme plasmin
are reported. The in vitro data for prodrug 6 show that
this paclitaxel derivative is very promising for further
in vivo studies, since it meets all criteria required for a
successful prodrug. The prodrug shows high stability
in buffer, is not toxic in seven human tumor cell lines,
and shows a reasonable rate of enzymatic activation by
plasmin. Furthermore, paclitaxel derivatives 2-5 are
the first prodrugs of paclitaxel that are connected via a
2′-carbamate functionality. Our studies have shown that
in the design of new generations of paclitaxel prodrugs,
incorporation of a 2′-carbamate bond can contribute to
stability against undesired enzymatic cleavage and thus
to low cytotoxicity. As a consequence of these results,
we are currently working on 2′-carbamate prodrugs of
paclitaxel with enhanced enzyme activation and spacer
elimination characteristics.
Exp er im en ta l Section
Ch em istr y. 1H NMR spectra were determined using a
Bruker AM-300 (300 MHz, FT) spectrometer in the given
solvent. Chemical shift values are reported as δ-values in parts
per million relative to TMS as an internal standard. Mass
spectra were determined using a double-focusing VG 7070E
spectrometer. Elemental analyses were performed in triplicate
on a Carlo Erba Instruments CHNSO EA 1108 element
analyzer and were within 0.4% of the theoretical values
calculated for C, H, and N. Melting points were measured on
a Reichert Thermopan microscope and are uncorrected. Thin-
layer chromatography was performed using precoated silica
gel (60F254) plates, and compounds were detected with UV
light, ammonium molybdate solution, or a Chloro-TDM test.
Column chromatography was performed using Baker silica gel
in the solvents indicated.
2′-[4-Nitr op h en yl-ca r bon a te]p a clita xel (7). To a solution
of 194 mg (0.227 mmol) paclitaxel (1) in dry dichloromethane

2′-Carbamate/ Carbonate-Linked Prodrugs of Paclitaxel
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 16 3099
mg (58%) of the protected tripeptide 12: mp 147 °C; 1H NMR
(300 MHz, CDCl3/CD3OD) δ 0.80-1.00 (m, 12H, CH3-Leu and
CH3-Val), 1.25-1.75 (m, 8H, 3 CH2-Lys and CH2-Leu), 1.87
(m, 1H, CH-Leu), 2.04 (m, 1H, CH-Val), 3.11 (m, 2H, N-CH2-
Lys), 3.95 (m, 1H, HR), 4.42 (m, 2H, HR), 5.06 (m, 4H, CH2-Z),
7.20-7.40 (m, 10H, aromatic); MS (FAB) m/e 627 (M + H)+,
649 (M + Na)+. Anal. (C33H46N4O8) C, H, N.
(m, 3H, 7 and 2 HR), 4.71 (d, 1H, HR), 4.90-5.20 (m, 6H, 2
CH2-Z, 2′ and 5), 5.62 (d, 1H, J ) 6.9 Hz, 2), 5.75 (bd, 1H, 3′),
6.05 (bt, 1H, 13), 6.13 (s, 1H, 10), 7.00-7.60 (m, 28H,
aromatic), 8.10 (d, 2H, J ) 7.5 Hz, aromatic); MS (FAB) m/e
1555 (M + H)+, 1577 (M + Na)+. Anal. (C84H95N7O22‚2H2O)
calcd C, H, N 6.16; found C, H, N 5.59.
Gen er a l P r oced u r e for R em ova l of Z P r ot ect in g
Gr ou p s fr om Dou bly P r otected P r od r u gs 15 a n d 24-26.
2′-[H-D-Ala -P h e-Lys-NH-(CH2)2-NHCO]p a clita xel (‚2HCl)
(2). To a solution of 33.2 mg (0.0213 mmol) of protected
prodrug 15 in 5% acetic acid-methanol was added a catalytic
amount of 10% Pd-C. The mixture was stirred for 90 min
under an H2 atmosphere. The Pd-C was removed by means
of centrifugation. Ethyl acetate was added and methanol was
evaporated in vacuo. The product was extracted with water
and after addition of 1 M hydrochloric acid (85 µL) the solution
was freeze-dried. tert-Butyl alcohol was added and removed
by evaporation in vacuo and the residue was freeze-dried to
obtain 24 mg (83%) of the desired product 2: mp >195 °C dec;
1H NMR (300 MHz, CDCl3/CD3OD) δ 1.07 (d, 3H, J ) 6.5 Hz,
CH3-Ala), 1.15 (s, 3H, 17), 1.19 (s, 3H, 16), 1.00-2.00 (m, 6H,
3 CH2-Lys), 1.68 (s, 3H, 19), 1.91 (s, 3H, 18), 2.07 (m, 1H, 6b),
2.21 (s, 3H, 10-OAc), 2.40 (s, 3H, 4-OAc), 2.48 (m, 1H, 6a),
2.85-3.50 (m, 8H, 2 CH2-spacer, N-CH2-Lys and benzylic),
3.78 (d, 1H, J ) 7.0 Hz, 3), 4.20-4.45 (m, 3H, 3 HR), 4.23 (d,
1H, J ) 8.2 Hz, benzylic), 4.31 (d, 1H, J ) 8.5 Hz, benzylic),
4.60 (dd, 1H, J ) 10.4 Hz, J ) 4.2 Hz, 7), 4.98 (bd, 1H, J ) 9.0
Hz, 5), 5.49 (d, 1H, J ) 4.4 Hz, 2′), 5.68 (d, 1H, J ) 6.8 Hz, 2),
5.86 (m, 1H, 3′), 6.13 (bt, 1H, 13), 6.32 (s, 1H, 10), 7.10-7.68
(m, 16H, aromatic), 7.73 (d, 2H, J ) 7.6 Hz, aromatic), 8.12
(d, 2H, J ) 7.7 Hz, aromatic); MS (FAB) m/e 1286 (M + H)+,
1309 (M + Na)+. Anal. (C68H83N7O18‚9HCl) C, H, N.33
Aloc-D-Va l-Leu -Lys(Aloc)-OH (11). To solution of 955 mg
(2.21 mmol) of D-Val-Leu-Lys-OH (10) in water-acetonitrile
was added triethylamine until a pH of 9-9.5 was reached.
Then a solution of 965 mg (2.2 equiv) of Aloc-ONSu in
acetonitrile was added and the reaction mixture was kept basic
by adding triethylamine. After the pH of the mixture did not
alter anymore, a 0.5 M solution of hydrochloric acid was added
until a pH of 3 was reached. The mixture was thoroughly
extracted with dichloromethane. The organic layer was washed
with water and the water layer was extracted again with
dichloromethane. The organic layer was dried over anhydrous
sodium sulfate and evaporated to dryness to result in the
desired product 11 as a white solid (1.123 g, 96%): 1H NMR
(300 MHz, CDCl3/CD3OD) δ 0.80-1.05 (m, 12H, 4 CH3 Val and
Leu), 1.20-2.15 (m, 10H, CH2-Lys and CH Val and CH Leu
and CH2 Leu), 3.13 (m, 2H, N-CH2-Lys), 3.94 (t, 1H, HR), 4.41
(m, 2H, HR), 4.54 (m, 4H, Aloc), 5.15-5.35 (m, 4H, Aloc), 5.75-
6.10 (m, 2H, Aloc); MS (FAB) m/e 527 (M + H)+, 549 (M +
Na)+. Anal. (C25H42N4O8‚0.25H2O) calcd C, H, N 10.55; found
C, H, N 10.12.
Z-D-Ala -P h e-Lys(Z)-OH (13): yield 51%; mp 180 °C; 1H
NMR (300 MHz, DMSO-d6) δ 0.93 (d, 3H, J ) 7.1 Hz, CH3-
Ala), 1.07-1.83 (m, 6H, 3 CH2-Lys), 2.73 (dd, 1H, benzylic),
2.98 (m, 2H, N-CH2-Lys), 3.08 (dd, 1H, benzylic), 4.00 (m, 1H,
HR), 4.15 (m, 1H, HR), 4.58 (m, H, HR), 4.90-5.00 (m, 4H,
CH2-Z), 7.10-7.35 (m, 15H, aromatic), 8.09 (d, 1H, J ) 8.6
Hz, N-H), 8.19 (d, 1H, J ) 7.5 Hz, N-H); MS (FAB) m/e 633
(M + H)+, 655 (M + Na)+. Anal. (C34H40N4O8‚0.5H2O) C, H, N.
Gen er a l P r oced u r e for th e Boc P r otection of Su bsti-
tu ted Eth ylen ed ia m in e Sp a cer s. NH2-(CH2)2-N(Me)-Boc
(16). To a solution of 4.21 g (56.8 mmol, 10.8 equiv) of
N-methylethylenediamine in dry tetrahydrofuran under an
Gen er a l P r oced u r e for th e Cou p lin g of Eth ylen ed i-
a m in e Sp a cer s to P r otected Tr ip ep tid es. Z-D-Ala -P h e-
Lys(Z)-NH-(CH2)2-NH2 (‚AcOH) (14). To a solution of 303 mg
(0.479 mmol) of Z-D-Ala-Phe-Lys(Z)-OH (13) dissolved in dry
tetrahydrofuran under an argon atmosphere at -60 °C were
added Et3N (73 µL, 1.1 equiv) and isobutyl chloroformate (68
µL, 1.1 equiv). After 180 min the reaction mixture was added
dropwise to a solution of ethylenediamine (20 equiv) in dry
dichloromethane at -60 °C. After 90 min the solvent was
evaporated and to the resulting solid were added water and
dichloromethane. The product was obtained by filtration and
purified by means of column chromatography (chloroform-
methanol-acetic acid, 85:10:5) yielding 183 mg (52%) of the
desired product 14: mp 155-156 °C; 1H NMR (300 MHz,
DMSO-d6) δ 0.91 (d, 3H, J ) 6.8 Hz, CH3-Ala), 1.10-1.70 (m,
6H, 3 CH2-Lys), 2.52 (t, 2H, CH2-spacer), 2.73 (dd, 1H,
benzylic), 2.80-3.10 (m, 5H, and N-CH2-Lys, benzylic and
CH2-spacer), 3.95 (m, 1H, HR), 4.10 (m, 1H, HR), 4.45 (m, 1H,
HR), 4.94 (m, 4H, 2 CH2-Z), 7.05-7.48 (m, 15H, aromatic); MS
(FAB) m/e 675 (M + H)+, 697 (M + Na)+.
argon atmosphere at
a temperature of 0 °C was added
dropwise Boc-O-Boc (1.12 mL, 5.23 mmol) in dry tetrahydro-
furan. After 6 h, tetrahydrofuran was evaporated in vacuo and
the resulting mixture was dissolved in ethyl acetate and
washed with brine. The organic layer was dried (Na2SO4) and
concentrated in vacuo to dryness. The product was purified
by column chromatography (chloroform-methanol, 1:1), ob-
taining 467 mg (51%) of monoprotected N-methylethylene-
diamine 16 as an oil: 1H NMR (300 MHz, CDCl3) δ 1.23 (s,
2H, NH2), 1.46 (s, 9H, CH3-Boc), 2.83 (t, 2H, J ) 6.4 Hz, CH2-
NH2), 2.88 (s, 3H, CH3-N), 3.27 (t, 2H, J ) 6.3 Hz, CH2-N).
NH(Me)-(CH2)2-N(Me)-Boc (17): yield 72%; 1H NMR (300
MHz, CDCl3) δ 1.46 (s, 9H, CH3-Boc), 2.45 (s, 3H, CH3-N-
Boc), 2.73 (t, 2H, CH2-N-Boc), 2.88 (s, 3H, CH3-N), 3.33 (t,
2H, CH2-N).
Z-D-Ala -P h e-Lys(Z)-N(Me)-(CH2)2-N(Me)-Boc (19): yield
51%; mp 42 °C; 1H NMR (300 MHz, CDCl3) δ 1.16 (d, 3H, J )
7.0 Hz, CH3-Ala), 1.10-1.75 (m, 6H, 3 CH2-Lys), 1.43 (s, 9H,
CH3-Boc), 2.70-3.70 (m, 14H, N-CH2-Lys, benzylic, 2 CH3-
spacer and 2 CH2-spacer), 4.15 (m, 1H, HR), 4.60-4.90 (m, 2H,
2 HR), 5.06 (m, 4H, CH2-Z), 7.00-7.40 (m, 15H, aromatic); MS
(FAB) m/e 825 (M + Na)+. Anal. (C43H58N6O9‚1.5H2O) calcd
C, H, N 10.13; found C, H, N 9.65.
Gen er a l P r oced u r e for th e Rem ova l of Boc P r otection
fr om P r otected P r om oieties 18-20. Z-D-Ala -P h e-Lys(Z)-
N(Me)-(CH2)2-N(Me) (‚HCl) (22). Compound 19 (58.7 mg,
0.0730 mmol) was dissolved in ethyl acetate (4 mL) and 8 mL
of 4 M hydrochloric acid was added. After 4 h the mixture was
concentrated in vacuo and to the residual product tert-butyl
alcohol was added and evaporated. The product was freeze-
dried in dioxane to obtain 58.0 mg (100%) of the desired
product 22 that was used without further purification: 1H
NMR (300 MHz, CDCl3/CD3OD) δ 1.14 (d, 3H, CH3-Ala),
1.20-1.80 (m, 6H, CH2-Lys), 2.64 (m, 3H, CH3-spacer), 2.90-
3.30 (m, 8H, N-CH2-Lys, benzylic, CH2-N-Boc and CH2-
N), 4.13 (m, 1H, HR), 4.59 (m, 2H, HR), 5.06 (m, 4H, CH2-Z),
7.10-7.40 (m, 15H, aromatic); MS (FAB) m/e 703 (M + H)+,
725 (M + Na)+.
Gen er a l P r oced u r e for Cou p lin g P r om oieties 14 a n d
21-23 to Activa ted P a clita xel Der iva tive 7. 2′-[Z-D-Ala -
P h e-Lys(Z)-NH-(CH2)2-NHCO]p a clita xel (15). To a solution
of 26.0 mg (0.0255 mmol) of activated paclitaxel 7 in dry DMF
under an argon atmosphere was added Et3N (10.6 µL, 3 equiv).
At -30 °C a solution of 14 (18.7 mg, 1.0 equiv) and Et3N (170
µL) dissolved in dry DMF was added dropwise. The reaction
mixture was stirred at -20 °C for 2.5 h. The solution was
diluted with 10% 2-propanol-ethyl acetate and washed with
sodium bicarbonate, 0.5 N potassium bisulfate, brine and
water. The organic layer was dried (sodium sulfate) and
concentrated in vacuo. The residue was subjected to column
chromatography (ethyl acetate-hexane, 2:1) to give 23.5 mg
1
(59%) of compound 15: mp 134-137 °C; H NMR (300 MHz,
CDCl3) δ 1.10-2.00 (m, 15H, CH3-Ala, CH2-Lys, 16 and 17),
1.60 (s, 3H, 19), 1.88 (s, 3H, 18), 2.18 (s, 3H, 10-OAc), 2.34 (s,
3H, 4-OAc), 2.54 (m, 1H, 6a), 3.00-3.40 (m, 8H, 2 CH2-spacer,
N-CH2-Lys and benzylic), 3.67 (d, 1H, J ) 6.9 Hz, 3), 4.15 (d,
1H, J ) 8.4 Hz, 20b), 4.28 (d, 1H, J ) 8.4 Hz, 20a), 4.28-4.60

3100 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 16
de Groot et al.
2′-[Z-D-Ala -P h e-Lys(Z)-N(Me)-(CH 2)2-N(Me)CO]p a cli-
ta xel (24): the coupling reaction was performed in dichlo-
romethane; yield 52%; mp 116-117 °C; H NMR (300 MHz,
(d, 3H, J ) 7.00 Hz, CH3-Ala), 1.25-2.00 (m, 6H, CH2-Lys),
2.68 (s, 3H, CH3-spacer), 2.90-3.58 (m, 8H, N-CH2-Lys, 2
CH2-spacer and benzylic), 4.15 (m, 1H, HR), 4.24 (m, 1H, HR),
4.52 (m, 1H, HR), 5.06 (m, 4H, CH2-Z), 5.62 (m, 1H, NH), 7.10-
7.40 (m, 15H, aromatic). Anal. (C37H48N6O7‚1.5HCl) C, H, N.
1
CDCl3) δ 0.75-2.10 (m, 12H, CH3-Ala, 3 CH2-Lys and 17),
1.25 (s, 3H, 16), 1.68 (s, 3H, 19), 1.98 (s, 3H, 18), 2.20 (s, 3H,
10-OAc), 2.55 (s, 3H, 4-OAc), 2.70-3.30 (m, 12H, 2 CH3-spacer,
CH2-spacer, N-CH2-Lys and benzylic), 3.45 (m, 2H, CH2-
spacer), 4.05-4.90 (m, 6H, 3 HR, 20a, 20b and 7,), 4.95-5.15
(m, 5H, CH2-Z and 2′), 5.46 (d, 1H, 2), 5.67 (m, 1H, 3′), 6.15
(m, 1H, 13), 6.30 (s, 1H, 10), 7.05-7.95 (m, 23H, aromatic),
8.15 (m, 2H, aromatic); MS (FAB) m/e 1583 (M + H)+, 1606
(M + Na)+. Anal. (C86H99N7O22‚2.5H2O) C, H, N.
2′-[H -D-Ala -P h e-Lys-N(Me)-(CH2)2-N(Me)CO]p a clit a x-
el (‚2HCl) (3): yield 88%; mp >187 °C dec; 1H NMR (300 MHz,
CDCl3) δ 0.88 (d, 3H, CH3-Ala), 1.00-2.00 (m, 12H, CH2-Lys,
16 and 17), 1.52 (s, 3H, 19), 1.68 (s, 3H, 18), 1.95 (s, 3H, 10-
OAc), 2.20 (s, 3H, 4-OAc), 2.80-3.35 (m, 11H, CH3-spacer,
N-CH2-Lys, benzylic and 2CH2-spacer), 2.88 (s, 3H, CH3-
spacer), 3.45-4.90 (m, 7H, 3HR, 20a, 20b, 7 and 2′), 5.00 (m,
5H, 5), 5.38 (m, 1H, 2), 5.65 (m, 1H, 3′), 6.10 (m, 1H, 13), 6.40
(s, 1H, 10), 7.05-8.20 (m, 20H, aromatic); MS (FAB) m/e 1314
(M + H)+. Anal. (C70H87N7O18‚15HCl) calcd C 45.17, H 5.52,
N; found C 44.71, H 5.96, N.33
2′-[Z-D-Ala -P h e-Lys(Z)-NH -(CH 2)2-N(Me)CO]p a clit a x-
el (26): the coupling reaction was performed in dichloro-
methane-tetrahydrofuran-N-methylpyrrolidinone; yield 80%;
1
mp 132-133 °C; H NMR (300 MHz, CDCl3) δ 1.00-2.00 (m,
9H, CH3-Ala, 3 CH2-Lys), 1.12 (s, 3H, 17), 1.20 (s, 3H, 16),
1.68 (s, 3H, 19), 1.76 (s, 3H, 18), 2.21 (s, 3H, 10-OAc), 2.58 (s,
3H, 4-OAc), 2.79 (s, 3H, CH3-spacer), 2.80-3.30 (m, 8H, 2 CH2-
spacer, N-CH2-Lys and benzylic), 3.83 (d, 1H, J ) 6.9 Hz, 3),
4.23 (d, 1H, J ) 8.3 Hz, 20a), 4.31 (d, 1H, J ) 8.4 Hz, 20b),
4.40-4.60 (m, 2H, HR and 7), 4.80 (m, 1H, HR), 4.90-5.10 (m,
5H, 5 and CH2-Z), 5.45 (d, 1H, 2′), 5.69 (d, 1H, J ) 7.1 Hz, 2),
6.17 (m, 1H, 3′), 6.26 (bs, 1H, 13), 6.30 (s, 1H, 10), 7.10-7.65
(m, 26H, aromatic), 7.79 (d, 2H, J ) 7.7 Hz, N-benzoyl), 8.18
(d, 2H, J ) 7.3 Hz, O-benzoyl); MS (FAB) m/e 1568 (M + H)+,
1590 (M + Na)+. Anal. (C85H97N7O22‚0.75H2O) C, H, N.
2′-[H -D-Ala -P h e -Lys-NH -(CH 2)2-N(Me )CO]p a clit a xe l
(‚2HCl) (5): yield 100%; mp >202 °C dec; 1H NMR (300 MHz,
CDCl3/CD3OD) δ 1.05-1.20 (m, 9H, CH2-Ala, 16 and 17),
1.10-2.00 (m, 6H, 3 CH2-Lys), 1.69 (s, 3H, 19), 1.98 (s, 3H,
18), 2.21 (s, 3H, 10-OAc), 2.53 (s, 3H, 4-OAc), 2.91 (s, 3H, CH3-
spacer), 2.92-3.60 (m, 8H, 2 CH2-spacer, N-CH2-Lys and
benzylic), 3.82 (d, 1H, J ) 6.7 Hz, 3), 3.90-4.55 (m, 5H, 7,
20a, 20b, 2 HR), 4.70 (m, 1H, HR), 5.02 (m, 5H, 5), 5.39
(m, 1H, 2′), 5.71 (m, 1H, 3J ) 6.9, 2), 6.05 (m, 1H, 3′), 6.19 (m,
1H, 13), 6.36 (s, 1H, 10), 7.15-7.70 (m, 16H, aromatic), 7.80
(d, 2H, J ) 7.4 Hz, N-benzoyl), 8.15 (d, 2H, J ) 7.4 Hz,
Z-D-Va l-Leu -Lys(Z)-N(Me)-(CH2)2-N(Me)-Boc (18): yield
1
80%; mp 82 °C; H NMR (300 MHz, CDCl3) δ 0.90-1.05 (m,
12H, 2 CH3-Leu and 2 CH3-Val), 1.20-1.80 (m, 8H, 3 CH2-
Lys and CH2-Leu), 1.44 (s, 9H, CH3-Boc), 2.25 (m, 2H, CH-
Val and CH-Lys), 2.75-4.00 (m, 9H, N-CH2-Lys, CH3-spacer,
2 CH2-spacer), 3.05 (m, 3H, CH3-spacer), 3.96 (m, 1H, HR),
4.43 (m, 1H, HR), 4.84 (m, 1H, HR), 5.08 (m, 4H, CH2-Z), 7.25-
7.40 (m, 10H, aromatic); MS (FAB) m/e 797 (M + H)+, 819
(M + Na)+. Anal. (C42H64N6O9‚0.5H2O) C, H, N.
O-benzoyl); MS (FAB) m/e 1301 (M + H). Anal. (C69H85N7O18
‚
8HCl) C, H, N.33
Z-D-Val-Leu -Lys(Z)-N(Me)-(CH2)2-N(Me) (‚HCl) (21): yield
100%; 1H NMR (300 MHz, CDCl3/CD3OD) δ 0.80-1.05 (m,
12H, 2CH3-Leu and 2 CH3-Val), 1.20-1.80 (m, 6H, 3 CH2-
Lys and CH2-Leu), 2.03 (m, 2H, CH-Val and CH-Lys), 2.60-
3.30 (m, 9H, N-CH2-Lys, CH3-spacer and 2 CH2-spacer), 3.08
(s, 3H, CH3-spacer), 3.85-4.65 (m, 3H, 3HR), 5.09 (m, 4H, CH2-
Z), 7.25-7.40 (m, 10H, aromatic); MS (FAB) m/e 697 (M + H)+,
719 (M + Na)+.
2′-[Z-D-Va l-Leu -Lys(Z)-N(Me)-(CH 2)2-N(Me)CO]p a cli-
ta xel (25): the coupling reaction was performed in dichloro-
methane; yield 33%; mp 118-120 °C; 1H NMR (300 MHz,
CDCl3) δ 0.70-1.05 (m, 12H, 2 CH3-Leu and 2 CH3-Val),
1.05-2.25 (m, 10H, CH2-Lys, CH2-Leu, CH-Val and CH-Leu),
1.13 (s, 3H, 17), 1.23 (s, 3H, 16), 1.69 (s, 3H, 19), 1.88 (s, 3H,
18), 2.22 (s, 3H, 10-OAc), 2.51 (s, 3H, 4-OAc), 2.70-3.60 (m,
12H, N-CH2-Lys, 2 CH3-spacer and 2 CH2-spacer), 3.82 (s,
1H, 3), 3.85-4.55 (m, 5H, 2 HR, 20a, 20b, 7), 4.80 (m, 1H, HR),
4.98 (m, 1H, 5), 5.06 (m, 4H, CH2-Z), 5.45 (bs, 1H, 2′), 5.67 (d,
1H, 2), 6.14 (dd, 1H, 3′), 6.26 (bs, 1H, 13), 6.30 (s, 1H, 10),
7.20-7.65 (m, 21H, aromatic), 7.78 (d, 2H, aromatic), 8.13 (m,
2H, aromatic); MS (FAB) m/e 1577 (M + H)+, 1599 (M + Na)+.
Anal. (C85H105N7O22‚5H2O) C, H, N.
2′-[H -D-Va l-Leu -Lys-N(Me)-(CH 2)2-N(Me)CO]p a clit a x-
el (‚2HCl) (4): yield 100%; mp >200 °C dec; 1H NMR (300
MHz, DMSO-d6) δ 0.80-1.10 (m, 12H, 2 CH3-Leu and 2 CH3-
Val), 1.10-2.00 (m, 10H, 3 CH2-Lys, CH2-Leu), 1.23 (m, 6H,
16 and 17), 1.49 (s, 3H, 19), 1.81 (s, 3H, 18), 2.10 (s, 3H, 10-
OAc), 2.15-2.40 (m, 2H, CH-Val and CH-Leu), 2.50 (s, 3H,
4-OAc), 2.60-3.50 (m, 12H, N-CH2-Lys, 2 CH3-spacer and 2
CH2-spacer), 3.75 (bs, 1H, 3), 3.90-4.80 (m, 4H, 2 HR, 20a and
20b), 4.55 (m, 1H, 7), 4.92 (m, 2H, 5 and HR), 5.10-5.90 (m,
3H, 2, 2′ and 3′), 5.83 (m, 1H, 13), 6.28 (s, 1H, 10), 7.35-8.40
(m, 15H, aromatic); MS (FAB) m/e 1308 (M + H)+.
Aloc-D-Va l-Leu -Lys(Aloc)-P ABA (27). A solution of 450
mg of protected tripeptide 11 (0.854 mmol) was dissolved in
dry tetrahydrofuran under an argon atmosphere and cooled
to -20 °C. N-Methylmorpholine (104 µL, 1.1 equiv) and
isobutyl chloroformate (122 µL, 1.1 equiv) were added. The
reaction mixture was stirred for 3 h at a temperature below
-20 °C. A solution of 4-aminobenzyl alcohol (132 mg, 1.25
equiv) and N-methylmorpholine (118 µL, 1.25 equiv) in dry
tetrahydrofuran was added dropwise to the reaction mixture.
The reaction mixture was allowed to come to room temperature
and was stirred for 16 h. Tetrahydrofuran was evaporated and
dichloromethane was added. The organic layer was washed
with saturated sodium bicarbonate, a 0.5 N potassium bisul-
fate solution and brine, dried over anhydrous sodium sulfate,
and evaporated. The residual crude product was purified by
means of column chromatography (chloroform-methanol, 9:1)
to afford 376 mg (70%) of the desired product 27 as a white
solid: mp 152 °C; 1H NMR (300 MHz, CDCl3/CD3OD) δ 0.75-
1.10 (m, 12H, 4 CH3 Val and Leu), 1.20-2.15 (m, 10H, CH2-
Lys and CH Val and CH Leu and CH2 Leu), 3.14 (m, 2H,
N-CH2-Lys), 3.81 (d, 1H, HR), 4.22 (m, 1H, HR), 4.25-4.70
(m, 5H, HR and Aloc), 4.58 (s, 2H, benzylic), 5.00-5.35 (m,
4H, Aloc), 5.55-6.10 (m, 2H, Aloc), 7.27 (d, 2H, aromatic), 7.61
(d, 2H, aromatic); MS (FAB) m/e 632 (M + H)+, 654 (M + Na)+.
Anal. (C32H49N5O8‚0.25H2O) calcd C, H, N 11.01; found C, H,
N 10.53.
Aloc-D-Va l-Leu -Lys(Aloc)-P ABC-P NP (28). To a solution
of 117 mg (0.185 mmol) of 27 in dry tetrahydrofuran-
dichloromethane under an argon atmosphere were added
4-nitrophenyl chloroformate (56 mg, 1.5 equiv) and dry pyri-
dine (23 µL, 1.5 equiv). The reaction mixture was stirred at
room temperature and after 24, 48 and 72 h were added
respectively 1, 1.5 and 1.5 equiv of both 4-nitrophenyl chloro-
formate and pyridine. After 96 h ethyl acetate was added. The
organic layer was washed with 10% citric acid, brine and
water, dried over anhydrous sodium sulfate and evaporated
yielding a yellow oil. The product was purified by means of
column chromatography (ethyl acetate-hexane, 1:1; chloro-
form-methanol, 30:1; respectively) to afford 112 mg (76%) of
the desired carbonate 28: mp 171 °C; 1H NMR (300 MHz,
CDCl3) δ 0.85-1.15 (m, 12H, 4 CH3 Val and Leu), 1.20-2.20
Z-D-Ala-P h e-Lys(Z)-NH-(CH2)2-N(Me)-Boc (20): yield 84%;
1
mp 52 °C; H NMR (300 MHz, CDCl3) δ 1.20 (d, 3H, J ) 6.9
Hz, CH3-Ala), 1.10-1.95 (m, 6H, 3 CH2-Lys), 1.42 (s, 9H,
CH3-Boc), 2.84 (s, 3H, CH3-spacer), 3.00-3.50 (m, 8H, N-CH2-
Lys, benzylic, 2 CH2-spacer), 4.00-4.45 (m, 2H, 2 HR), 4.62
(m, 1H, HR), 5.05 (m, 4H, CH2-Z), 7.10-7.40 (m, 15H,
aromatic). Anal. (C42H56N6O9) C, H, N.
Z-D-Ala -P h e-Lys(Z)-NH-(CH 2)2-N(Me) (‚HCl) (23): yield
94%; mp 121 °C; 1H NMR (300 MHz, CDCl3/CD3OD) δ 1.19

2′-Carbamate/ Carbonate-Linked Prodrugs of Paclitaxel
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 16 3101
(m, 10H, CH2-Lys and CH Val and CH Leu and CH2 Leu),
2.90-3.40 (m, 2H, N-CH2-Lys), 3.82 (m, 1H, HR), 4.12 (m,
1H, HR), 4.30-4.65 (m, 5H, HR and Aloc), 5.23 (s, 2H,
benzylic), 5.00-5.45 (m, 4H, Aloc), 5.58 (m, 1H, Aloc), 5.87 (m,
1H, Aloc), 7.25-7.40 (m, 4H, aromatic), 7.75 (d, 2H, aromatic),
8.26 (d, 2H, aromatic); MS (FAB) m/e 797 (M + H)+, 819 (M +
Na)+. Anal. (C39H52N6O12) C, H.
was carried out with a CE Ext light path capillary (80.5 cm,
50 µm), with 1:1 methanol/0.05 M sodium phosphate buffer
(pH 7.0) as eluent. Detection was performed at 200 and 254
nm.
In Vit r o Cyt ot oxicit y in Seven Hu m a n Tu m or Cell
Lin es. The antiproliferative effect of prodrugs 2-6 and
paclitaxel was determined in vitro applying seven well-
characterized human tumor cell lines and the microculture
sulforhodamine B (SRB) test.34 The antiproliferative effects
were determined and expressed as ID50 values, which are the
(pro)drug concentrations that gave 50% inhibition when
compared to control cell growth after 5 days of incubation.
Results were averaged from experiments that were performed
in quadruplicate.
2′-[Aloc-D-Va l-Leu -Lys(Aloc)-P ABC]p a clita xel (29). p-
Nitrophenyl carbonate 28 (81 mg, 0.102 mmol) and paclitaxel
(87 mg, 1 equiv) in dry dichloromethane were treated at room
temperature with DMAP (14 mg, 1.1 equiv). The reaction
mixture was stirred in the dark for 48 h and was then diluted
with dichloromethane. The organic layer was washed with
saturated sodium bicarbonate, 0.5 N potassium bisulfate and
brine and dried over anhydrous sodium sulfate. After evapora-
tion of the solvents the residual white film was purified by
means of column chromatography (chloroform-methanol,
20:1), to yield 151 mg (99%) of protected paclitaxel prodrug
Ack n ow led gm en t. We are grateful to H. Adams
and Prof. Dr. G. I. Tesser for their contributions to the
peptide synthesis.
1
29: mp 143 °C; H NMR (300 MHz, CDCl3) δ 0.85-1.15 (m,
Su p p or tin g In for m a tion Ava ila ble: Elemental analyses.
This material is available free of charge via the Internet at
http://pubs.acs.org.
12H, 4 CH3 Val and Leu), 1.14 (s, 3H, 17), 1.24 (s, 3H, 16),
1.30-2.30 (m, 11H, CH2-Lys and CH Val and CH Leu and CH2
Leu and 6b), 1.69 (s, 3H, 19), 1.91 (s, 3H, 18), 2.23 (s, 3H, 10-
OAc), 2.44 (s, 3H, 4-OAc), 2.60 (m, 1H, 6a), 3.00-3.45 (m, 2H,
N-CH2-Lys), 3.72 (m, 2H, HR), 3.81 (d, 1H, 3), 4.06 (m, 1H,
HR), 4.20 (d, 1H, 20b), 4.32 (d, 1H, 20a), 4.23-4.65 (m, 5H,
Aloc and 7), 4.90-5.35 (m, 7H, benzylic spacer and 4 Aloc and
5), 5.44 (d, 1H, 2′), 5.52 (m, 1H, Aloc), 5.69 (d, 1H, 2), 5.88 (m,
1H, Aloc), 5.98 (dd, 1H, 3′), 6.27 (bt, 1H, 13), 6.30 (s, 1H, 10),
6.93 (d, 1H, NH), 7.25-7.55 (m, 12H, aromatic), 7.60 (d, 1H,
aromatic), 7.65-7.80 (m, 4H, aromatic), 8.14 (d, 2H, aromatic),
8.39 (bs, 1H, NH); MS (FAB) m/e 1512 (M + H)+, 1535
(M + Na)+. Anal. (C80H98N6O23‚1.75H2O) calcd C 62.27, H 6.63,
N; found C 61.83, H 6.13, N.
Refer en ces
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2′-[H-D-Va l-Leu -Lys-P ABC]p a clita xel (‚2HCl) (6). To a
solution of 56.4 mg (0.0373 mmol) of protected prodrug 29 in
dry tetrahydrofuran under an argon atmosphere was added
glacial acetic acid (11 µL, 5 equiv) together with tributyltin-
hydride (30 µL, 3 equiv) and a catalytic amount of Pd(PPh3)4.
After 45 min tributyltinhydride was added again (30 µL). After
90 min the reaction mixture carefully quenched with 1 mL
0.5 M hydrochloric acid-ethyl acetate. The white precipitate
was collected by means of centrifugation and washed several
times with ether. tert-Butyl alcohol was added and evaporated
again to remove an excess of hydrochloric acid and the
resulting product was dissolved in water and freeze-dried
yielding 36.6 mg (69%) of prodrug 6: mp 168 °C; 1H NMR (300
MHz, CDCl3/CD3OD) δ 0.92 (d, 3H, CH3-Leu), 0.98 (d, 3H, CH3-
Leu), 1.00-1.13 (m, 6H, 2 CH3 Val), 1.17 (s, 3H, 17), 1.22 (s,
3H, 16), 1.35-2.32 (m, 11H, CH2-Lys and CH Val and CH Leu
and CH2 Leu and 6b), 1.70 (s, 3H, 19), 1.85 (s, 3H, 18), 2.23 (s,
3H, 10-OAc), 2.44 (s, 3H, 4-OAc), 2.55 (m, 1H, 6a), 2.99 (bt,
2H, N-CH2-Lys), 3.81 (d, 1H, 3), 4.07 (m, 1H, HR), 4.26 (d,
1H, 20b), 4.33 (d, 1H, 20a), 4.36 (m, 2H, HR), 4.54 (m, 1H, 7),
5.00 (bd, 1H, 5), 5.13 (dd, 2H, benzylic spacer), 5.44 (d, 1H,
2′), 5.71 (d, 1H, 2), 5.97 (bd, 1H, 3′), 6.22 (bt, 1H, 13), 6.35 (s,
1H, 10), 7.20-7.70 (m, 15H, aromatic), 7.74 (d, 2H, aromatic),
8.14 (d, 2H, aromatic); MS (FAB) m/e 1343 (M + H)+, 1365 (M
+ Na)+. Anal. (C72H90N6O19‚3.4HCl) C, H, N.33
Biologica l Ch a r a cter iza tion . Sta bility of th e P r od r u g
in Bu ffer Solu tion . Prodrugs 3-6 were incubated at con-
centrations of 100-270 µM in 0.1 M Tris/hydrochloric acid
buffer (pH 7.3) for 3 days and showed no parent drug formation
(TLC). Under the same conditions prodrug 2 degraded com-
pletely within 24 h, yielding baccatin III.
Kin etics of En zym a tic Hyd r olysis. Hydrolysis of pro-
drugs 3-6 was investigated by incubation at a concentration
of 200 µM in 0.1 M Tris/hydrochloric acid buffer (pH 7.3) in
the presence of 100 µg/mL human plasmin (Fluka). Prodrugs
3 and 4 were not converted to paclitaxel by plasmin and
remained intact for 3 days. Prodrug 6 was converted to yield
paclitaxel immediately following enzymatic cleavage (t1/2 ) 42
min), while prodrug 5 showed formation of the intermediate
drug-linker moiety before paclitaxel was released (t1/2 activa-
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