Elongated multiple electronic cascade and cyclization spacer systems in activatible anticancer prodrugs for enhanced drug release

Article abstract of DOI:10.1021/jo0158884

The design and synthesis of several novel elongated self-elimination spacer systems for application in prodrugs is described. These elongated spacer systems can be incorporated between a cleavable specifier and the parent drug. Naphthalene- and biphenyl-containing spacers were synthesized but did not eliminate. Prodrugs of the anticancer agents doxorubicin and paclitaxel are reported that contain two or three electronic cascade spacers. A novel catalytic application of HOBt was found for the synthesis of N-aryl carbamates through reacting a 4-nitrophenyl carbonate with an aniline derivative, to connect the 1,6-elimination spacers via a carbamate linkage. In addition, a double spacer-containing paclitaxel prodrug was synthesized, comprising a 1,6-elimination spacer and a bis-amine linker connected to paclitaxel via a 2′-carbamate linkage. Prodrugs in which the novel spacer systems were incorporated between a specific tripeptide specifier and the parent drug doxorubicin or paclitaxel proved to be significantly faster activated by plasmin in comparison with prodrugs containing conventional spacer systems. It is expected that the generally applicable novel spacer systems reported herein will contribute to future development of improved enzymatically activated prodrugs.

Full text of DOI:10.1021/jo0158884

J . Org. Chem. 2001, 66, 8815-8830
8815
Elon ga ted Mu ltip le Electr on ic Ca sca d e a n d Cycliza tion Sp a cer
System s in Activa tible An tica n cer P r od r u gs for En h a n ced Dr u g
Relea se
Franciscus M. H. de Groot, Walter J . Loos,‡ Ralph Koekkoek, Leon W. A. van Berkom,
Guuske F. Busscher, Antoinette E. Seelen, Carsten Albrecht, Peter de Bruijn,‡ 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, and Department of Medical
Oncology, Rotterdam Cancer Institute (Daniel den Hoed Kliniek) and University Hospital Rotterdam,
P.O. Box 5201, 3008 AE Rotterdam, The Netherlands
jsch@sci.kun.nl
Received J uly 3, 2001
The design and synthesis of several novel elongated self-elimination spacer systems for application
in prodrugs is described. These elongated spacer systems can be incorporated between a cleavable
specifier and the parent drug. Naphthalene- and biphenyl-containing spacers were synthesized
but did not eliminate. Prodrugs of the anticancer agents doxorubicin and paclitaxel are reported
that contain two or three electronic cascade spacers. A novel catalytic application of HOBt was
found for the synthesis of N-aryl carbamates through reacting a 4-nitrophenyl carbonate with an
aniline derivative, to connect the 1,6-elimination spacers via a carbamate linkage. In addition, a
double spacer-containing paclitaxel prodrug was synthesized, comprising a 1,6-elimination spacer
and a bis-amine linker connected to paclitaxel via a 2′-carbamate linkage. Prodrugs in which the
novel spacer systems were incorporated between a specific tripeptide specifier and the parent drug
doxorubicin or paclitaxel proved to be significantly faster activated by plasmin in comparison with
prodrugs containing conventional spacer systems. It is expected that the generally applicable novel
spacer systems reported herein will contribute to future development of improved enzymatically
activated prodrugs.
In tr od u ction
monotherapy3) or the enzyme can be targeted to tumor
tissue in an additional step preceding prodrug adminis-
tration (for example via antibody-directed enzyme pro-
drug therapy, ADEPT4).
One very important criterion for the development of a
successful in vivo selective prodrug is that the prodrug
must be efficiently activated by the site-specific enzyme
under physiological conditions.5 In this paper, several
elongated self-elimination spacer systems are presented
which enable fast prodrug activation.
Several structurally different enzymes have been
shown to cleave anticancer prodrugs containing a self-
elimination spacer much more readily than the corre-
sponding prodrugs lacking a spacer. In some cases with
prodrugs lacking a spacer, specifier removal did not occur
at all. This clearly demonstrates the beneficial effect of
spacer incorporation on prodrug activation characteris-
tics. Evidence for this recurring theme was obtained for
example with prodrug substrates for enzymes such as
â-glucuronidase6-9 and cathepsin.10 In our laboratory it
was found that if a specific tripeptide is coupled to an
anthracycline via a self-elimination spacer, the resulting
Drug delivery of therapeutic parent drugs via prodrugs
can offer multiple advantages over administration of the
free parent drug. One advantage is that an inactive
prodrug can be site-specifically activated to release the
active drug, which then exerts its biological function at
the target site. Target-selective delivery of biologically
active compounds is expected to become increasingly
important for application in diseases, like cancer, where
selectivity of existing drugs for the diseased area is
desired.1,2 Most chemotherapeutic agents used for treat-
ment of cancer have a small therapeutic window and
cause severe, sometimes life-threatening side effects,
while little therapeutic effect is seen.
Many reported prodrugs consist of three components:
a specifier, a spacer, and a parent drug. The specifier,
which is a substrate for a site-specific enzyme, is often
connected to the parent drug via a self-elimination
spacer. The spacer is incorporated to facilitate enzymatic
cleavage of the specifier. After specifier removal, the
spacer must spontaneously eliminate to release free drug.
The existence of tumor-associated enzymes can be ex-
ploited to cleave the specifier of the prodrug (prodrug
(3) de Groot, F. M. H.; Damen, E. W. P.; Scheeren, H. W. Curr. Med.
Chem. 2001, 8, 1093-1122.
* To whom correspondence should be addressed. Tel: +31-(0)24-
3652331. Fax: +31-(0)24-3652929.
(4) Senter, P. D. Faseb J . 1990, 188-193.
(5) (a) Connors, T. A. Biochemie 1978, 60, 979-987. (b) Carl, P. L.
Development of Target-Oriented Anticancer Drugs; Raven Press: New
York, 1983; pp 143-155.
‡ Daniel den Hoed Kliniek, Rotterdam, The Netherlands.
(1) (a) Dubowchik, G. M.; Walker, M. A. Pharmacol. Ther. 1999, 83,
67-123. (b) Huang, P. S.; Oliff, A. Curr. Opin. Gen. Dev. 2001, 11,
104-110.
(6) Haisma, H. J .; van Muijen, M.; Pinedo, H. M.; Boven, E. Cell
Biophys. 1994, 24/ 25, 185-192.
(2) Damen, E. W. P.; de Groot, F. M. H.; Scheeren, H. W. Exp. Opin.
Ther. Patents 2001, 11, 651-666.
(7) Leenders, R. G. G.; Scheeren, H. W.; Houba, P. H. J .; Boven, E.;
Haisma, H. J . Bioorg. Med. Chem. Lett. 1995, 5, 2975-2980.
10.1021/jo0158884 CCC: $20.00 © 2001 American Chemical Society
Published on Web 11/27/2001

8816 J . Org. Chem., Vol. 66, No. 26, 2001
de Groot et al.
Sch em e 1
prodrug is efficiently hydrolyzed by plasmin,11,12 whereas
a plasmin substrate directly coupled to an anthracycline
amino group is not activated by the enzyme.13,14
to be only marginal.18 In addition, electron-withdrawing
substituents on the phenyl ring may affect spacer frag-
mentation rates.19 It can be questioned whether major
improvement of drug release from prodrugs containing
electronic cascade spacers can be achieved by means of
introducing spacer substituents.
Two types of self-elimination spacers can be distin-
guished: (i) cyclization spacers, and (ii) electronic cascade
spacers. The most prominent example of an electronic
cascade spacer is the 1,6-elimination spacer developed
by Carl et al. After unmasking the aromatic amine or
hydroxyl function of these spacers, the amine or hydroxyl
group becomes electron-donating and initiates an elec-
tronic cascade that leads to the expulsion of the leaving
group, which releases the free drug after elimination of
carbon dioxide (Scheme 1).16
In general, electronic cascade spacers eliminate faster
upon unmasking than most cyclization spacers, for which
elimination is often characterized by long half-lives. In
our laboratory, p-aminobenzyl oxycarbonyl (PABC) elec-
tronic cascade spacer systems are used because they
eliminate virtually instantaneously upon unmasking of
the amine.16,17 If the spacer rapidly eliminates after
prodrug activation, it is the enzymatic prodrug activation
itself that determines the efficiency of drug release.
Most of the research (including that in our own
laboratory) to enhance drug release from prodrugs has
been conducted to increase the rate of enzymatic activa-
tion by placing electron-withdrawing substituents at
aromatic positions of self-immolative electronic cascade
linkers. However, the beneficial effect on prodrug activa-
tion of spacers containing electron-withdrawing substit-
uents over unsubstituted spacers in most cases proved
We hypothesized that elongated spacer systems could
decrease steric hindrance to a larger extent than con-
ventional spacers by virtue of the enlarged spacing
between the parent drug and the specifier. Indeed, in
several electronic cascade spacer-containing prodrugs
differences in enzymatic activation rates can still be
observed when different parent drugs are connected with
the same promoiety () part of prodrug that is coupled to
parent drug) or when a parent drug is connected to the
same promoiety via a different site of the drug. For
example, â-glucuronidase cleaves the glucuronide from
a â-glucuronide-cyclization spacer promoiety much slower
when paclitaxel is the parent drug20 in comparison with
the prodrug containing doxorubicin as the parent drug.8a
In another example, a dipeptide derivative of paclitaxel,
linked via a PABC spacer was more readily cleaved by
cathepsin B when paclitaxel was linked via its 7-position
than via its 2′-position.10b In addition, half-lives of cathe-
psin B cleavage of electronic cascade spacer-containing
prodrugs of doxorubicin or mitomycin C were much
shorter than the half-life of the corresponding prodrugs
with paclitaxel as the parent drug.10b Finally, plasmin
cleaves the tripeptide from an electronic cascade spacer-
containing doxorubicin prodrug much more readily than
the tripeptide from the corresponding paclitaxel pro-
drug.13,21
(8) (a) Leenders, R. G. G.; Gerrits, K. A. A.; Ruijtenbeek, R.;
Scheeren, H. W.; Haisma, H. J .; Boven, E. Tetrahedron Lett. 1995, 26,
1701-1704. (b) Leenders, R. G. G.; Damen, E. W. P.; Bijsterveld, E. J .
A.; Scheeren, J . W.; Houba, P. H. J .; van der Meulen-Muileman, I. H.;
Boven, E.; Haisma, H. J . Bioorg. Med. Chem. 1999, 7, 1597-1610.
(9) Madec-Lougerstay, R.; Florent, J .-C.; Monneret, C. J . Chem. Soc.,
Perkin Trans. 1 1999, 1369-1375.
Thus, in several prodrug systems the parent drug still
exerts a significant effect on the rate of enzymatic acti-
vation, even though the prodrugs mentioned contained
a spacer.
(10) (a) Dubowchik, G. M.; Firestone, R. A. Bioorg. Med. Chem. Lett.
1998, 8, 3341-3346. (b) Dubowchik, G. M.; Mosure, K.; Knipe, J . O.;
Firestone, R. A. Bioorg. Med. Chem. Lett. 1998, 8, 3347-3352.
(11) Yamashita, Y.-I.; Ogawa, M. Int. J . Oncol. 1997, 10, 807-813.
(12) Irigoyen, J . P.; Mun˜oz-Ca´noves, P.; Montero, L.; Koziczak, M.;
Nagamine, Y. Cell. Mol. Life Sci. 1999, 56, 104-132.
In the synthetic studies presented herein, the design,
synthesis, and kinetic properties of novel improved linker
(18) A chloro substituent on an aminobenzyl spacer only marginally
enhanced the rate of enzymatic prodrug activation by plasmin.13 In
the case of aminobenzyl spacer-containing anthracycline prodrugs for
activation by â-glucuronidase, a variety of substituents showed no
beneficial or only a marginal effect8b and anthracycline prodrugs
containing a spacer without substituents were used for further in vivo
evaluation: (a) Houba, P. H. J .; Boven, E.; van der Meulen-Muileman,
I. H.; Leenders, R. G. G.; Scheeren, J . W.; Pinedo, H. M.; Haisma, H.
J . Br. J . Cancer 2000, 84, 1-8. (b) Houba, P. H. J .; Boven, E.; van der
Meulen-Muileman, I. H.; Leenders, R. G. G.; Scheeren, J . W.; Pinedo,
H. M.; Haisma, H. J . Int. J . Cancer 2001, 91, 550-554.
(13) de Groot, F. M. H.; de Bart, A. C. W.; Verheijen, J . H.; Scheeren,
H. W. J . Med. Chem. 1999, 42, 5277-5283.
(14) Chakravarty, P. K.; Carl, P. L.; Weber, M. J .; Katzenellenbogen,
J . A. J . Med. Chem. 1983, 26, 638-644.
(15) (a) Nicolaou, M. G.; Yuan, C.-S.; Borchardt, R. T.; J . Org. Chem.
1996, 61, 8636-8641. (b) Wang, B.; Gangwar, S.; Pauletti, G. M.;
Siahaan, T. J .; Borchardt, R. T. J . Org. Chem. 1997, 62, 1363-1367.
(16) Carl, P. L.; Chakravarty, P. K.; Katzenellenbogen, J . A. J . Med.
Chem. 1981, 24, 479-480.
(17) In contrast to aminobenzyl spacer systems, hydroxybenzyl
electronic cascade spacers need electron-withdrawing substituents on
the phenyl part of the spacer in order to let spacer elimination take
place: (a) Monneret, C.; Florent, J .-C.; Gesson, J .-P.; J acquesy, J .-C.;
Tillequin, F.; Koch, M. ACS Symp. Ser. 1995, 574, 78-99. (b) Florent,
J .-C.; Dong, X.; Gaudel, G.; Mitaku, S.; Monneret, C.; Gesson, J .-P.;
J acquesy, J .-C.; Mondon, M.; Renoux, B.; Andrianomenjanahary, S.;
Michel, S.; Koch, M.; Tillequin, F.; Gerken, M.; Czech, J .; Straub, R.;
Bosslet, K. J . Med. Chem. 1998, 41, 3572-3581. (c) Platel, D.; Bonoron-
Ade`le, S.; Dix, R. K.; Robert, J . Br. J . Cancer 1999, 81, 24-27.
(19) Electron-donating substituents on the phenyl ring may acceler-
ate fragmentation because of stabilization of the developing positive
charge on the benzylic carbon: Hay, M. P.; Sykes, B. M.; Denny, W.
A.; O’Connor, C. J . J . Chem. Soc., Perkin Trans. 1 1999, 2759-2770.
(20) de Bont, D. B. A.; Leenders, R. G. G.; Haisma, H. J .; van der
Meulen-Muileman, I.; Scheeren, H. W. Bioorg. Med. Chem. 1997, 5,
405-414.
(21) de Groot, F. M. H.; van Berkom, L. W. A.; Scheeren, H. W. J .
Med. Chem. 2000, 43, 3093-3102.

Elongated Spacer Systems for Enhanced Drug Release
Sch em e 2
J . Org. Chem., Vol. 66, No. 26, 2001 8817
systems, which are generally applicable in prodrugs to
facilitate release of free parent compound, are reported.
The chemotherapeutic agents doxorubicin and paclitaxel
serve as parent drugs.
case the parent drug is released after specifier removal
and two or more subsequent spacer eliminations (type
B, Scheme 2). Especially in the case of aminobenzyl
oxycarbonyl spacers this could also be a feasible approach
under physiological conditions, because (i) these spacers
eliminate rapidly upon unmasking,16 and (ii) PABC
spacers can be connected to one another via a carbamate
linkage, which is expected to be relatively stable against
ubiquitous enzymes.
Prodrugs of parent drugs that contain a hydroxyl group
(such as paclitaxel) can be improved by incorporation of
an elongated spacer system that contains one or more
electronic cascade spacers between the specifier and a
bis-amine cyclization spacer (type C, Scheme 2). A major
advantage of the use of type C elongated spacer systems
is that these promoieties can be linked to the OH-drug
via a relatively stable carbamate linkage.
To deliver proof of principle for the proposed 1,8- and
1,10-elimination processes (type A, Scheme 2), allyl
oxycarbonyl (Aloc) protected naphthalene and biphenyl
spacer systems were synthesized that contained benzyl-
amine as a model compound22 for an amine-containing
drug (Scheme 3).
The commercially available dimethyl ester analogues
1a ,b of the desired naphthalene and biphenyl compounds
were reduced to the mono-alcohols 2a ,b using LiAlH4.
Subsequent saponification of the esters to carboxylic
acids 3a ,b , followed by protection using tert-butyl di-
methylsilyl chloride yielded silyl ethers 4a ,b. The key
step of the synthesis of the model compounds was the
conversion of carboxylic acids 4a ,b into the corresponding
Syn th esis
Three possibilities that have been explored for creating
elongated electronic cascade spacer systems are depicted
in Scheme 2. First, additional aromatic groups can be
incorporated in conjugation with the phenyl group present
in existing 1,6-elimination spacers (type A). Second, two
or more electronic cascade spacers can be incorporated
between the specifier and the parent drug (type B). To
our knowledge, no papers describing either of these pos-
sibilities have been published before. Finally, prodrugs
containing a cyclization spacer can be improved by com-
bination of the cyclization spacer in one spacer system
with (an) additional electronic cascade spacer(s) (type C).
Type C spacer systems may be particularly suitable for
application in prodrugs of parent drugs that contain a
hydroxyl group.
The incorporation of additional aromatic groups in
conjugation with the phenyl group present in existing
electronic cascade spacers (type A, Scheme 2) was
explored by synthesizing spacer systems that consist of
N-capped naphthyl or biphenyl derivatives that contain
functionalized hydroxymethylene groups. These elon-
gated aromatic spacers are designed to eliminate accord-
ing to the mechanism depicted in Scheme 2. After
unmasking the primary amine, a 1,8-elimination or a
1,10-elimination electronic cascade should lead to expul-
sion of the leaving group and subsequent drug release.
More than one electronic cascade spacer can be incor-
porated between a specifier and the parent drug. In that
(22) Griffin, R. J .; Evers, E.; Davison, R.; Gibson, A. E.; Layton, D.;
Irwin, W. J . J . Chem. Soc., Perkin Trans. 1 1996, 1205-1211.

8818 J . Org. Chem., Vol. 66, No. 26, 2001
de Groot et al.
Sch em e 3
Sch em e 4
Aloc protected amines in a one-pot procedure. First, the
carboxylic acid was reacted with diphenyl phosphoryl
azide at room temperature. This resulted in the formation
of the corresponding acyl azide. Second, allyl alcohol was
added, and the reaction mixture was heated to a tem-
perature of 85 °C. The acyl azide rearranged into the
isocyanate that was immediately trapped by allyl alcohol
to lead to the Aloc protected amines. In case of naphtha-
lene spacer 4a , the Aloc was introduced and the silyl
protecting group was removed in one pot, affording 6a .
In case of biphenyl spacer 4b, the silyl group was not
removed yielding 5b. After deprotection of the silyl group
of 5b, activation of the resulting alcohols 6a ,b to the
corresponding 4-nitrophenyl carbonates, and subsequent
substitution with benzylamine, the naphthalene and
biphenyl model compounds 7a ,b were obtained.
F igu r e 1. Double spacer-containing model compound 9.
carbamate is reported, in which an aniline was reacted
with a 4-nitrophenyl carbonate at 60 °C for 10 h.23
An attempt was then made to couple a model com-
pound, N-acetyl-4-aminobenzyl alcohol, with a carbonyl
diimidazole (CDI) activated O-TBDMS protected 4-ami-
nobenzyl alcohol spacer. This reaction succeeded, and the
desired double spacer-containing model compound (9,
Figure 1) could be isolated. Disappointingly, when the
same CDI activated spacer was reacted with the tripep-
tide-spacer conjugate, no coupling reaction was ob-
served.
In another attempt to couple both spacers, the tripep-
tide-spacer conjugate was added to an isocyanate de-
rivative of the spacer,22 formed via a modified Curtius
rearrangement. Even under high-pressure conditions at
15 kbar, formation of the desired compound was not
observed.
Exploration of type B elongated spacer systems (Scheme
2) was performed by synthesizing plasmin-activated
prodrugs that contained more than one 1,6-elimination
spacer. As a plasmin substrate, the specific tripeptide
D-Ala-Phe-Lys was employed as a specifier. The synthesis
of the doubly Aloc protected tripeptide Aloc-D-Ala-Phe-
Lys(Aloc)-OH and the corresponding activated tripep-
tide-spacer conjugate 8 (Scheme 4) was described in a
previous paper.13
The possibility of synthesizing the N-aryl carbamate
via the benzyl chloroformate derivative of the tripeptide-
spacer conjugate was not investigated, as it appears both
from the literature24 and our own experience, that it is
difficult to obtain the benzylic chloroformate without
decomposition to the benzyl chloride.
The key step in the synthesis of these derivatives was
coupling a second spacer to the benzylic alcohol function
of a tripeptide-spacer conjugate. Several attempts were
unsuccessful. The activated 4-nitrophenyl carbonate
derivative 8 of the tripeptide-spacer conjugate did not
react with 4-aminobenzyl alcohol at room temperature,
whereas at 60 °C the starting material was degraded. In
the literature an example of formation of an N-aryl
(23) Obi, K.; Ito, Y.; Terashima, S. Chem. Pharm. Bull. 1990, 38,
917-924.
(24) Niculescu-Duvaz, D.; Niculescu-Duvaz, U.; Friedlos, F.; Martin,
J .; Spooner, R.; Davies, L.; Marais, R.; Springer, C. J . J . Med. Chem.
1998, 41, 5297-5309.

Elongated Spacer Systems for Enhanced Drug Release
Sch em e 5
J . Org. Chem., Vol. 66, No. 26, 2001 8819
At this point we started to look for a catalyst that could
catalyze the attack of an aromatic amine on an activated
nitrophenyl carbonate (Scheme 4). When the 4-nitrophe-
nyl carbonate activated tripeptide-spacer conjugate 8
was reacted with 4-aminobenzyl alcohol in the presence
of diphenyl phosphinic acid,25 at 40 °C, and the reaction
mixture was worked up after 6 days, the desired product
10 was isolated in a yield of 16%.
Then, hydroxy benzotriazole (HOBt) was tested for its
catalytic properties. It was previously reported that this
alcohol was able to catalyze the coupling reaction of an
aziridine nitrogen with a 4-nitrophenyl carbonate.26 We
hypothesized that it might also be an effective catalyst
for the synthesis of N-aryl carbamates. In the presence
of 0.2-0.3 equiv of HOBt, the desired N-aryl carbamate
10 was obtained at room temperature in a high yield
(97%) (Scheme 4). To our knowledge, this is the first
report describing the use of HOBt as a catalyst for
coupling an aromatic amine to an activated carbonate.
Both doxorubicin and paclitaxel prodrugs containing
two aminobenzyl oxycarbonyl spacers were synthesized
starting from the double spacer-containing conjugate 10
(Scheme 5). Activation of benzylic alcohol 10 to carbonate
11 and subsequent coupling with the amine of doxoru-
bicin or the 2′-hydroxyl group of paclitaxel led to pro-
tected prodrugs 12 and 14. Deprotection in the presence
of palladium catalyst and tributyltin hydride27 afforded
prodrugs 13 and 15.
F igu r e 2. Double spacer-containing doxorubicin prodrug 16
with a tryptophan residue in the tripeptide sequence.
A double 1,6-elimination spacer-containing doxorubicin
prodrug with a tryptophan residue at the P2 position
instead of a Phe (H-D-Ala-Trp-Lys-PABC-PABC-Doxoru-
bicin, 16, Figure 2) was also synthesized, because tryp-
tophan is presumed to improve plasmin substrate prop-
erties.28
(25) Aresta, M.; Berloco, C.; Quaranta, E. Tetrahedron 1995, 51,
8073-8088.
(26) Dubowchik, G. M.; King, H. D.; Pham-Kaplita, K. Tetrahedron
Lett. 1997, 38, 5261-5264.
(27) Dangles, O.; Guibe´, F.; Balavoine, G.; Lavielle, S.; Marquet, A.
J . Org. Chem. 1987, 52, 4984-4993.

8820 J . Org. Chem., Vol. 66, No. 26, 2001
de Groot et al.
Sch em e 6
To extend the scope of elongated spacer systems and
to reveal information on the optimal number of spacers
incorporated, a doxorubicin prodrug was prepared that
contained three 1,6-elimination spacers. Starting from
carbonate 11, a third spacer molecule was linked to the
peptide double spacer conjugate employing again HOBt
as a catalyst, smoothly affording N-aryl carbamate 17
(Scheme 6). Activation to carbonate 18, coupling with
doxorubicin to yield 19 and deprotection afforded the
triple spacer-containing prodrug 20.
Paclitaxel served as OH-drug for investigation of type
C elongated spacer systems (Scheme 2). A route to
synthesize paclitaxel-2′-carbamate prodrugs was previ-
ously reported.21 In the literature, there exists one
example of a prodrug of a phenol derivative that contains
a combined electronic cascade spacer/cyclization spacer
system, although elongation of the spacer system was not
mentioned as a purpose.29
A paclitaxel prodrug containing both a 1,6-elimination
PABC spacer and an N,N′-dimethyl ethylenediamine
spacer may possess desirable properties such as the
elongated nature of the spacer system and the nature of
the linkage between promoiety and drug being a car-
bamate.21,30 After proteolysis, paclitaxel would be released
following 1,6-elimination and subsequent intramolecular
cyclization (type C, Scheme 2).
The paclitaxel prodrug 26 was prepared in a reason-
able yield through a convergent synthetic approach
(Scheme 7). A deprotected spacer-drug conjugate 24
was synthesized by coupling a benzyl oxycarbonyl (Z)
monoprotected spacer 22 to the previously reported
2′-[4-nitrophenyl carbonate]-paclitaxel (21)21 to afford
23, followed by hydrogenolysis using H2, Pd/C in the
presence of acetic acid to yield the protonated amine 24.
This amine was coupled with the activated specifier-
spacer conjugate 8 to afford protected prodrug 25, by
adding base to a concentrated solution of 24 and 8 in
order to favor the intermolecular coupling reaction over
premature intramolecular spacer cyclization of 24. De-
protection of 25 yielded the desired paclitaxel prodrug
26. Another synthetic route, in which a Boc-protected
cyclization spacer was coupled to the activated tripep-
tide-spacer conjugate 8, failed, because after deprotec-
tion of the Boc group using 0.5 M hydrochloric acid/ethyl
acetate, an undesired product was isolated.31 Coupling
of two separate fragments according to the strategy
depicted in Scheme 7, in which the chemical link between
the two spacers was established in the final stage,
(29) Lougerstay-Madec, R.; Florent, J .-C.; Monneret, C.; Nemati, F.;
Poupon M. F. Anti-Cancer Drug Des. 1998, 13, 995-1007.
(30) Saari, W. S.; Schwering, J . E.; Lyle, P. A.; Smith, S. J .;
Engelhardt, E. L. J . Med. Chem. 1990, 33, 97-101.
(31) When the Boc group was deprotected from the tripeptide double
spacer conjugate, the corresponding tripeptide aminobenzyl chloride
conjugate (MS (FAB) m/e 656 (M + H)+, 678 (M + Na)+) was obtained,
presumably formed upon acidic cleavage of the benzyl carbamate. In
(28) Tryptophan instead of phenylalanine may fit better into the
S2 pocket of plasmin, a pocket specific for aromatic amino acids. In a
study where a library of inhibitors was screened, Trp provided up to
a 80-fold increase in affinity in comparison with Phe. (a) Backes, B.
J .; Harris, J . L.; Leonetti, F.; Craik, C. S.; Ellman, J . A. Nature Biotech.
2000, 18, 187-193. (b) Abato, P.; Conroy, J . L.; Seto, C. T. J . Med.
Chem. 1999, 42, 4001-4009.
the literature a low yield (30%) was reported for
a similar Boc
deprotection.29 The strategy depicted in Scheme 7 may provide a more
efficient synthesis of type
prodrugs.
C elongated spacer system-containing

Elongated Spacer Systems for Enhanced Drug Release
Sch em e 7
J . Org. Chem., Vol. 66, No. 26, 2001 8821
Sch em e 8
provided the most efficient route to the paclitaxel pro-
drug.
P r oof of P r in cip le a n d Kin etic Stu d ies. The Aloc
functions of the naphthalene- and biphenyl-containing
spacers 7a ,b were deprotected, to generate free amines
27a ,b. The deprotected products should subsequently
undergo spontaneous spacer elimination to release free
benzylamine and regenerate amino-naphthyl methanol
(28a ) or amino-biphenyl methanol (28b), as depicted in
Scheme 8.
F igu r e 3. Biphenyl spacer-containing model compound 29
with an aromatic amine in the leaving group.
Ta ble 1. P la sm in Activa tion Ra tes a n d Sp a cer
Cycliza tion Ra te of Sin gle a n d Dou ble
Sp a cer -Con ta in in g P a clita xel P r od r u gs
T1/2 act.
(min)
T1/2 cycl
(min)
Formation of a new and more polar product was
observed; however, no benzylamine or amino alcohols
28a ,b were detected at room temperature. Even heating
of the reaction mixtures up to 100 °C under basic
conditions did not lead to the desired elimination reac-
tion. Both amines 27a ,b were isolated. To verify whether
lack of elimination could be partly ascribed to the leaving
group ability of the N-benzyl carbamic acid, the N-phenyl
leaving group analogue 29 was synthesized, because of
its assumed improved leaving group ability, again using
HOBt as a catalyst (Figure 3).
Even with this improved leaving group, the desired
1,10-elimination of the biphenyl part of 29 occurred
neither at room temperature nor at 100 °C.
The synthesized elongated spacer system-containing
prodrugs (types B and C) were subjected to incubation
spacer system
3021
26
15
1 × PABC
42
4
7.5
1 × PABC, 1 × cyclization
2 × PABC
47
experiments in the presence of the protease plasmin, to
determine the rate of enzymatic prodrug activation.
In the case of the paclitaxel prodrugs, incubation was
performed at 37 °C in 0.1 M Tris/hydrochloric acid buffer
(pH 7.3), at a prodrug concentration of 200 µM and 0.03
U/mL human plasmin. Using capillary electrophoresis,
half-lives of plasmin activation and in case of paclitaxel
prodrug 26 also the half-life of spacer cyclization were
determined (Table 1). Both double spacer-containing
paclitaxel prodrugs were converted to yield the corre-
sponding parent drug. A plasmin activated paclitaxel

8822 J . Org. Chem., Vol. 66, No. 26, 2001
de Groot et al.
F igu r e 4. Reference prodrugs 30 and 31 that contain a single spacer.
F igu r e 5. Release of free parent drug doxorubicin from elongated spacer system-containing prodrugs 13 (B), 16 (C), and 20 (D)
in comparison with single spacer-containing prodrug 31 (A).
prodrug containing one 1,6-elimination spacer (H-D-Val-
Leu-Lys-PABC-Paclitaxel, 30, Figure 4) was reported
previously.21 This prodrug contains a D-Val-Leu-Lys
sequence, which shows plasmin substrate properties
comparable to D-Ala-Phe-Lys.32
The doxorubicin prodrugs containing multiple spacer
systems were incubated at 37 °C in 0.1 M Tris/hydro-
chloric acid buffer (pH 7.3), at an expected maximally
approachable doxorubicin concentration of approximately
1 µM in the presence of 0.0025 U/mL human plasmin,
on three or four separate occasions. Using a fully vali-
dated HPLC method for detection of doxorubicin,33 the
rates of enzymatic activation by plasmin of the multiple
electronic cascade spacer system-containing doxorubicin
prodrugs 13, 16, and 20 were determined. The plasmin-
activated doxorubicin prodrug containing one 1,6-elimi-
nation spacer (H-D-Ala-Phe-Lys-PABC-Doxorubicin, 31,
Figure 4) was reported previously.13 In Figure 5 the
doxorubicin concentration after addition of plasmin (at
(33) de Bruijn, P.; Verweij, J .; Loos, W. J .; Kolker, H. J .; Planting,
A. S. T.; Nooter, K.; Stoter, G.; Sparreboom, A. Anal. Biochem. 1999,
266, 216-221.
(32) Eisenbrand, G.; Lauck-Birkel, S.; Tang, W. C. Synthesis 1996,
1246-1258.

Elongated Spacer Systems for Enhanced Drug Release
J . Org. Chem., Vol. 66, No. 26, 2001 8823
Ta ble 2. Su m m a r y of Con sta n ts of th e Hill Equ a tion for
th e Differ en t Doxor u bicin P r od r u gs
and biphenyl spacer systems (type A, Scheme 2) unfor-
tunately were resistant to the proposed 1,8- and 1,10-
elimination reactions, respectively. This observation may
be explained by the fact that in both cases aromaticity
of two ring systems must be sacrificed and possibly this
energy barrier for dearomatization of the bicyclic spacers
is too high. An additional drawback for elimination of
the biphenyl system in comparison with the naphthalene
spacer is the repulsion of the biphenyl ortho hydrogens
when both phenyl groups leave their tilted orientation
to reach a flat structure to make the electronic cascade
possible.
Mu ltip le P ABC Sp a cer System s. The synthesized
doxorubicin and paclitaxel prodrugs contain linker sys-
tems comprising two (13, 15, and 16) or three (20)
electronic cascade PABC spacers (type B, Scheme 2). To
the best of our knowledge, linker systems comprising two
or more connected electronic cascade spacers have not
been published up till now. Release of the leaving group
(the drug) occurred after two or three subsequent elec-
tronic cascade spacer eliminations. The half-lives for
enzymatic prodrug activation were significantly lower
when elongated spacer systems were incorporated.
The synthesis of multiple PABC spacer systems was
enabled through a novel synthetic application of HOBt
as a catalyst. The HOBt-catalyzed coupling appears to
be a fruitful reaction mainly when an alcohol that must
be converted to an N-aryl-carbamate is sterically hin-
dered. It has been shown that HOBt can be employed to
covalently link aminobenzyl oxycarbonyl electronic cas-
cade spacers via carbamate linkages.
Plasmin hydrolysis of the double and triple 1,6-
elimination spacer-containing doxorubicin prodrugs is, at
the plasmin and prodrug concentrations used, at least
two times faster than plasmin hydrolysis of the corre-
sponding single spacer-containing prodrug,13 reflected
both in T50 and γ values. The triple spacer-containing
prodrug 20 shows the highest enzymatic activation rate.
Additional incubation experiments would be necessary
to enable more significant discrimination between the
double and triple spacer systems. The Hill constants γ
indicate that the initial plasmin activation rate is higher
for the double spacer-containing prodrugs 13 and 16 than
for single spacer-containing prodrug 31, whereas the pro-
drug containing the longest spacer system (triple spacer-
containing prodrug 20) shows the highest initial rate of
doxorubicin release.
spacer system
T50 (h)
γ
r
p
3113
13
1 × PABC
2 × PABC
2 × PABC
3 × PABC
1.66 ( 0.23 1.18 ( 0.085 0.991 <0.001
0.82 ( 0.28 0.72 ( 0.093 0.978 <0.001
0.64 ( 0.062 0.72 ( 0.039 0.958 <0.001
0.57 ( 0.11 0.43 ( 0.031 0.949 <0.001
16a
20
a
Contains a D-Ala-Trp-Lys specifier.
T ) 0) is depicted for doxorubicin prodrugs 31, 13, 16,
and 20.
The constants of the Hill equation34 that follow from
the data depicted in Figure 5 are outlined in Table 2.
The time needed to reach a 50% conversion to doxorubicin
is T50, whereas γ (the Hill constant) represents the
sigmoidicity of the curve. Since the hydrolysis of the
prodrugs started directly after the addition of plasmin,
the Hill constant is representative for the initial prodrug
conversion rate. A lower γ means a steeper curve, and a
higher initial prodrug conversion rate. An r-value close
to 1 and a p-value close to 0 indicate that all data fit
significantly in the Hill curve.
As shown in Figure 5 and Table 2, all curves could be
significantly fitted using the Hill equation. The time
needed to reach a 50% conversion to doxorubicin was in
the following order: 31 > 13 > 16 > 20.
All prodrugs were incubated in 0.1 M Tris/HCl buffer
(pH 7.3) for 3 days at 37 °C and showed no formation of
degradation products. Because a markedly decreased
cytotoxicity of a prodrug in comparison with the parent
drug is a requirement of a successful prodrug, all pro-
drugs were tested for their cytotoxicity in a panel of seven
human tumor cell lines. Paclitaxel prodrugs 15 and 26
showed an average decrease of in vitro cytotoxicity of
respectively 3.7-fold and 73-fold in comparison with
parent paclitaxel upon incubation for 5 days. Doxorubicin
prodrugs 13, 16, and 20 showed an average decrease of
in vitro cytotoxicity of 30-fold, 24-fold, and 22-fold,
respectively, when compared with parent doxorubicin
under similar conditions.
Discu ssion a n d Con clu sion s
Incorporation of elongated spacer systems in plasmin-
activated prodrugs resulted in increased enzymatic ac-
tivation rates when compared to prodrugs containing a
conventional spacer. At the prodrug and enzyme concen-
trations used, elongated spacer-containing doxorubicin
prodrugs were activated with a 2- to 3-fold higher rate,
whereas elongated spacer-containing paclitaxel prodrugs
showed a 6- to 10-fold higher activation rate. Application
of elongated spacer systems resulted in major improve-
ments of enzymatic activation rates, particularly when
a bulky drug, such as paclitaxel, was the parent drug. It
is feasible to assume that there exists an optimum for
the length of spacer systems required for ensuring a
maximal enzymatic prodrug activation rate. This opti-
mum will be determined by several parameters, including
the enzyme and the parent drug that is used.
In case of prodrugs with paclitaxel as the parent drug,
the double electronic cascade spacer-containing prodrug
15 released the parent drug with a 6-fold increased rate
when compared to single spacer-containing prodrug 30,
upon incubation with plasmin.
Com bin a tion of P ABC Sp a cer (s) w ith a Bisa m in e
Cycliza t ion Sp a cer . Prodrugs of parent drugs that
are coupled via a hydroxyl group may be improved by
attaching the specifier to the drug via both one or more
electronic cascade spacers and a bisamine cyclization
spacer, which is connected to the OH-drug via a carbam-
ate function (type C, Scheme 2). Applying this approach
Na p h th a len e a n d Bip h en yl Sp a cer System s. Al-
though several examples in the literature suggest that
the (nonaromatic) structures that were hypothesized to
be generated are not inconceivable,35 the naphthalene
(35) (a) Biewer, M. C.; Biehn, C. R.; Platz, M. S.; Despre´s, A.;
Migirdicyan, E. J . Am. Chem. Soc. 1991, 113, 616-620. (b) Fukushima,
T.; Okazeri, N.; Miyashi, T.; Suzuki, K.; Yamashita, Y.; Suzuki, T.
Tetrahedron Lett. 1999, 40, 1175-1178. (c) Minabe, M.; Mochizuki,
H.; Yoshida, M.; Toda, T. Bull. Chem. Soc. J pn. 1989, 62, 68-72. (d)
Szunerits, S.; Utley, J . H. P.; Nielsen, M. F. J . Chem. Soc., Perkin
Trans. 2 2000, 669-675.
(34) Holford, N. H. G.; Sheiner, L. B. Clin. Pharmacokin. 1981, 6,
429-453.

8824 J . Org. Chem., Vol. 66, No. 26, 2001
de Groot et al.
Sa p on ifica tion of 2a to 3a . To a solution of 10 equiv of
NaOH (3.05 g) in 50 mL of water was added 1.64 g (7.6 mmol)
of 2a . After 40 min, 2 mL of MeOH was added to dissolve the
ester. MeOH was evaporated after 3 h, and 1 M HCl solution
was added until pH 3 was reached. The mixture was filtered,
and the white solid was dissolved in a mixture of CH2Cl2 and
MeOH. After drying over anhydrous Na2SO4, the organic layer
was evaporated to dryness, yielding 1.44 g (94%) of the desired
product 3a . Mp 234 °C; 1H NMR (300 MHz, DMSO) δ 4.69 (s,
2H), 7.54 (d, 1H, J ) 8.5 Hz), 7.90 (s, 1H), 7.96 (s, 2H), 8.05
(d, 1H, J ) 8.5 Hz), 8.56 (s, 1H); MS (EI) m/e 202 (M)+. Anal.
(C12H10O3) calculated C 71.28%, H 4.98%, measured C 71.00%,
H 5.04%.
to paclitaxel yielded prodrug 26, which showed a mark-
edly decreased cytotoxicity, and a 10-fold increased
plasmin hydrolysis rate when compared to the prodrug
containing a single spacer (30).
P r od r u g Sta bility. Several conclusions can be drawn
from the cytotoxicity data. Single-, double-, and triple-
spacer-containing doxorubicin prodrugs 31, 13, 16, and
20, show, respectively, 17-fold,13 30-fold, 24-fold, and 22-
fold reduced cytotoxicity in comparison with doxorubicin.
The fact that the number of spacers does not show a
distinct effect on prodrug cytotoxicity may imply that the
carbamate bond connecting two PABC spacers is rela-
tively stable, at least in vitro, against ubiquitous en-
zymes.
Silyl P r otection of 3a to 4a . A solution of 2.5 equiv (1.04
g) of tert-butyl dimethylsilyl chloride and 3.0 equiv (0.466 g)
of imidazole in dry THF was stirred under an argon atmo-
sphere. After 15 min, 1.40 g (6.9 mmol) 3a in dry THF was
added dropwise. The reaction mixture was stirred overnight,
THF was evaporated, and CH2Cl2 was added. The organic
layer was washed with 0.5 M KHSO4 and brine and evaporated
to yield a solid compound, which was dissolved in THF. To
this mixture was added saturated NaHCO3 to hydrolyze the
silyl ester. After stirring for 1 h, THF was evaporated and the
solution was neutralized using 0.5 M KHSO4. The product was
extracted with ether and washed with brine. The organic layer
was dried over anhydrous Na2SO4 and evaporated to dryness
yielding 0.82 g (37%) of the desired product 4a , which was used
When considering that double spacer-containing pa-
clitaxel-2′-carbonate prodrug 15 shows much higher in
vitro cytotoxicity than single spacer-containing paclitaxel-
2′-carbonate prodrug 30,21 one can imagine that the
enlarged spacing between specifier and drug not only
facilitates the desired prodrug activation, but may also
facilitate ubiquitous cleavage of the promoiety-drug
bond, depending on the chemical nature of this linkage.
The 2′-carbonate linked paclitaxel prodrug 15 shows
20-fold higher in vitro cytotoxicity than the 2′-carbamate
linked paclitaxel prodrug 26. The markedly lower cyto-
toxicity of the double spacer-containing 2′-carbamate
paclitaxel prodrug 26 shows that linkage of a promoiety
to paclitaxel via a 2′-carbamate function may increase
prodrug stability against ubiquitous enzymes. Thus,
combination of electronic cascade spacer(s) with a bisamine
cyclization spacer (type C) seems to yield a spacer system
that is particularly useful for incorporation in prodrugs
that are coupled via a hydroxyl group of the parent drug.
In vivo studies with prodrugs that contain the elon-
gated spacer systems described are currently in progress.
It is anticipated that the elongated spacer systems
reported herein can be employed in prodrugs or biocon-
jugates that are designed for enzymatic activation. Use
of these spacer systems is not limited to anticancer
prodrugs, as they can serve also for targeting to other
diseases or diseased areas where a specific disease-
associated or targeted enzyme is present.
1
without further purification. Mp 168 °C; H NMR (300 MHz,
CDCl3) δ 0.14 (s, 6H), 0.97 (s, 9H), 4.92 (d, 2H, J ) 4.8 Hz),
7.51 (d, 1H, J ) 8.5 Hz), 7.84-7.96 (m, 3H), 8.10-8.13 (m,
1H), 8.70 (s, 1H).
Cu r tiu s Rea r r a n gem en t a n d Aloc P r otection To Give
6a . To a solution of 242 mg (0.765 mmol) of 4a in dry toluene
under an argon atmosphere were added diphenylphosphoryl
azide (198 µL, 1.2 equiv) and Et3N (129 µL, 1.2 equiv). After
stirring overnight at room temperature, allyl alcohol (1.5 mL)
was added and the mixture was stirred at 85 °C for 5 h, and
then CHCl3 was added. The organic layer was washed with
saturated NaHCO3, 0.5 M KHSO4, 10% citric acid and brine,
dried over anhydrous Na2SO4 and evaporated. The residual
crude product was purified by means of column chromatog-
raphy (EtOAc-hexane 2:5) to afford 96 mg (49%) of the Aloc-
protected desilylated product 6a . Mp 112 °C; 1H NMR (300
MHz, CDCl3) δ 4.70-4.72 (m, 2H), 4.83 (s, 2H), 5.27-5.43 (m,
2H), 5.94-6.07 (m, 1H), 6.85 (s, 1H), 7.37-7.47 (m, 2H), 7.74
(s, 2H), 7.77 (s, 1H), 7.99 (s, 1H). MS (EI) m/e 257 (M)+. Anal.
(C15H15O3N) calculated C 70.02%, H 5.88%, measured C
69.82%, H 5.95%.
Activa tion of 6a a n d Cou p lin g of Ben zyla m in e To Give
7a . To a solution of 88 mg of 6a (0.34 mmol) in dry THF under
an argon atmosphere were added 4-nitrophenyl chloroformate
(138 mg, 2.0 equiv) and dry pyridine (83 µL, 3.0 equiv). The
reaction mixture was stirred at room temperature and after
4, 19, and 21 h were added, respectively, 3 equiv (112 mL), 3
equiv (112 mL), and 1.5 equiv (56 mL) of benzylamine. After
23 h, THF was evaporated and EtOAc was added. The organic
layer was washed with 0.1 M NaOH, 10% citric acid, and brine,
dried over anhydrous Na2SO4, and evaporated to dryness,
yielding 135 mg (100%) of the desired product 7a . Mp 163 °C;
1H NMR (300 MHz, DMSO) δ 4.22 (d, 2H, J ) 6.2 Hz), 4.66
(d, 2H, J ) 5.4 Hz), 5.18 (s, 2H), 5.24-5.37 (m, 2H), 5.95-
6.08 (m, 1H), 7.21-7.34 (m, 4H), 7.44 (d, 1H, J ) 8.7 Hz),
7.55-7.58 (m, 1H), 7.77-7.87 (m, 4H), 8.08 (s, 1H), 9.97 (s,
1H); MS (EI) m/e 390 (M)+. Anal. (C23H22O4N2(‚1/2H2O))
calculated C 69.16%, H 5.80%, N 7.01%, measured C 69.20%,
H 5.86%, N 6.75%.
Red u ction of 1b to 2b. To a solution of 9.99 g (37.0 mmol)
of dimethyl 4,4′-biphenyldicarboxylate 1b in dry CH2Cl2 (75
mL) under an argon atmosphere was added LiAlH4 (1.053 g,
0.75 equiv). The reaction mixture was stirred at room tem-
perature. Subsequently were added, after 2 h, dry THF (7 mL),
dry CH2Cl2 (40 mL), and 0.5 equiv of LiAlH4 (708 mg), after
24 h, CH2Cl2 (40 mL) and 0.3 equiv of LiAlH4 (420 mg), and
after 25, 26, and 27 h, respectively, 0.3 equiv (429 mg), 0.5
equiv (710 mg), and 0.25 equiv (355 mg) of LiAlH4. After 28 h,
Exp er im en ta l Section
Mass spectra were recorded with a MAT 9005, using FAB
and EI modes. For other analytical instruments used, see ref
21. All solvents were, if necessary, distilled and dried prior to
use, following standard procedures. Thin-layer chromatogra-
phy was performed using Merck precoated silica gel (60F254
)
plates and compounds were detected with UV light, am-
monium molybdate solution, or with a Chloro-TDM test.
Column chromatography was performed using Baker silica gel
in the solvents indicated. Purchased compounds were used
without further purification.
Red u ction of 1a to 2a . To a solution of 6.04 g (24.7 mmol)
of dimethyl 2,6-naphthalenedicarboxylate 1a in dry THF under
an argon atmosphere was added LiAlH4 (1.17 g, 1.25 equiv).
The reaction mixture was stirred at room temperature and
after 1 and 2 h 0.5 equiv of LiAlH4 were added. After 3 h the
organic layer was evaporated and the product was purified
by means of column chromatography (EtOAc-hexane 1:1)
yielding 1.65 g (31%) of the desired product 2a . Mp 113 °C;
1H NMR (300 MHz, CDCl3) δ 2.08 (s, 1H), 3.98 (s, 3H), 4.88
(d, 2H, J ) 3.6 Hz), 7.50-7.54 (m, 1H), 7.82-7.85 (m, 2H),
7.92 (d, 1H, J ) 8.5 Hz), 8.02-8.06 (m, 1H), 8.57 (s, 1H); MS
(EI) m/e 216 (M)+. Anal. (C13H12O3) calculated C 72.21%, H
5.59%, measured C 72.08%, H 5.59%.

Elongated Spacer Systems for Enhanced Drug Release
J . Org. Chem., Vol. 66, No. 26, 2001 8825
CH2Cl2 and 10% citric acid were added, and the mixture was
filtered over Hyflo. The organic layer was washed with 10%
citric acid and brine, dried over anhydrous Na2SO4, and
evaporated yielding the crude product. The product was
purified by means of column chromatography (CHCl3, CHCl3-
MeOH 9:1; respectively) to afford 2.00 g (25%, conversion
88%) of the desired product 2b . Mp 170 °C; 1H NMR (300
MHz, CDCl3) δ 3.94 (s, 3H), 4.77 (d, 2H, J ) 3.3 Hz), 7.47 (d,
2H, J ) 8.1 Hz), 7.63 (d, 2H, J ) 8.5 Hz), 7.66 (d, 2H, J ) 8.6
Hz), 8.11 (d, 2H, J ) 8.3 Hz); MS (EI) m/e 242 (M)+. Anal.
(C15H14O3) calculated C 74.36%, H 5.82%, measured C 74.01%,
H 6.06%.
Sa p on ifica tion of 2b to 3b. To a solution of 10 equiv
NaOH (325 mg) in a water/THF (50 mL/40 mL) mixture was
added 1.97 g (32.9 mmol) of 2b. After 2 h, 30 mL of MeOH
was added. MeOH was evaporated after 4.5 h, and 1 M HCl
solution was added until a pH of 3 was reached. After filtration
of the mixture, the residue was washed with 10% citric acid
and water and dissolved in MeOH. The organic layer was dried
over anhydrous Na2SO4 and evaporated to dryness yielding
1.855 g (100%) of the desired product 3b. Mp 247 °C; 1H NMR
(300 MHz, DMSO) δ 4.56 (d, 2H, J ) 4.8 Hz), 7.44 (d, 2H, J )
8.0 Hz), 7.70 (d, 2H, J ) 8.1 Hz), 7.80 (d, 2H, J ) 8.3 Hz),
8.02 (d, 2H, J ) 8.2 Hz); MS (EI) m/e 228 (M)+. Anal. (C14H12O3‚
1/4 MeOH) calculated C 72.44%, H 5.55%, measured C 72.20%,
H 5.30%.
Silyl P r otection of 3b to 4b. A solution of 2.5 equiv (1.65
g) of tert-butyl dimethylsilyl chloride and 3.0 equiv (0.895 g)
of imidazole in dry THF was stirred under argon atmosphere.
After 15 min, 1.01 g (4.43 mmol) 3b in dry THF was added.
After stirring overnight the THF was evaporated and CH2Cl2
was added. The organic layer was washed with 0.5 M KHSO4
and brine and evaporated to yield a solid compound, which
was dissolved in THF. To this mixture was added saturated
NaHCO3, to saponify the silyl ester. After stirring for 1 h, THF
was evaporated, and the solution was neutralized with 0.5 M
KHSO4. The product was extracted with CH2Cl2 and washed
with brine. The organic layer was dried over anhydrous Na2-
SO4 and evaporated to dryness yielding 1.44 g (95%) of the
desired product 4b. Mp 227 °C; 1H NMR (300 MHz, CDCl3/
CD3OD) δ 0.14 (s, 6H), 0.97 (s, 9H), 4.81 (s, 2H), 7.43 (d, 2H,
J ) 8.0 Hz), 7.62 (d, 2H, J ) 8.0 Hz), 7.68 (d, 2H, J ) 8.2 Hz),
8.11 (d, 2H, J ) 8.2 Hz); MS (EI) m/e 342 (M)+. Anal. (C20H26O3-
Si) calculated C 70.14%, H 7.65%, measured C 70.47%, H
7.69%.
Activa tion of 6b a n d Cou p lin g of Ben zyla m in e To Give
7b. To a solution of 41 mg of 6b (0.14 mmol) in dry THF under
an argon atmosphere were added 4-nitrophenyl chloroformate
(31 mg, 1.0 equiv) and dry Et3N (30 µL, 1.5 equiv). The reaction
mixture was stirred at room temperature and after 1 and 2 h
were added, respectively, 1.0 equiv (20 µL) and 3.0 equiv (60
µL) of Et3N. After 3 and 4 h was added 2 equiv (56 mg) of
4-nitrophenyl chloroformate. After 19, 21, and 23 h were
added, respectively, 1.2 equiv (19 µL), 2.4 equiv (38 µL), and
2.4 equiv (38 µL) of benzylamine. After 40 h, THF was
evaporated and EtOAc was added. The organic layer was
washed with 0.1 M NaOH, 10% citric acid, and brine, dried
over anhydrous Na2SO4, and evaporated to dryness, yielding
the desired product 7b quantitatively. Mp 154 °C; 1H NMR
(300 MHz, CDCl3/CD3OD) δ 4.35 (s, 2H), 4.65 (m, 2H), 5.15
(s, 2H), 5.24-5.41 (m, 2H), 5.94-6.03 (m, 1H), 7.21-7.33 (m,
8H), 7.40-7.46 (m, 1H), 7.52-7.57 (m, 4H). MS (EI) m/e 416
(M)+. Anal. (C25H24O4N2) calculated C 72.10%, H 5.81%,
measured C 72.24%, H 6.09%.
Activa tion of 6b a n d Cou p lin g of 4-Am in oben zyl
Alcoh ol To Give 29. To a solution of 40 mg of 6b (0.14
mmol) in dry THF under an argon atmosphere were subse-
quently added in a period of 64 h: 180 mg of 4-nitrophenyl
chloroformate (6.0 equiv), Et3N (10 drops), 4-aminobenzyl
alcohol (63 mg, 3.6 equiv), HOBt (12 mg, 0.6 equiv), DIPEA (5
drops). After 64 h, dry DMF was added and THF was, under
an argon atmosphere, evaporated. Subsequently, 8 mg of
HOBt (0.4 equiv) and 5 drops of DIPEA were added. After 88
h DMF was evaporated, and EtOAc (50 mL) was added. The
organic layer was washed with 0.1 M NaOH, 10% citric acid,
and brine, dried over anhydrous Na2SO4, and evaporated. The
residual crude product was purified by means of column
chromatography (CHCl3-MeOH 9:1) to afford 32 mg (52%)
of the desired product 29. Mp 182 °C; 1H NMR (300 MHz,
CDCl3/CD3OD) δ 4.58 (s, 2H), 4.68 (d, 2H, J ) 5.6 Hz),
5.22 (s, 2H), 5.25-5.41 (m, 2H), 5.92-6.03 (m, 1H), 7.28
(d, 2H, J ) 8.5 Hz), 7.36-7.58 (m, 10H); MS (EI) m/e 432
(M)+, 283 (MAloc-spacer)+. Anal. (C25H24O5N2) calculated
C
69.43%, H 5.59%, N 6.48%, measured C 68.95%, H 5.58%, N
6.37%.
Aloc Dep r otection To Give 27a . To a solution of 10 mg
(0.026 mmol) of 7a in dry THF under an argon atmosphere
were added AcOH (7.0 µL, 5.0 equiv), tributyltin hydride (21.0
µL, 3.0 equiv) and a catalytic amount of palladium tetrakis-
(triphenylphosphine). After 18 h, the THF was evaporated and
the crude product was purified by means of column chroma-
tography (EtOAc/heptane 2/5) followed by preparative TLC
(EtOAc/heptane 1.5/1) to afford 8 mg (100%) of the spacer-
leaving group conjugate 27a . Mp 130 °C; 1H NMR (300 MHz,
CDCl3) δ 4.40 (d, 2H, J ) 5.9 Hz), 5.23 (s, 2H), 6.93-6.97 (m,
2H), 7.24-7.38 (m, 6H), 7.57-7.68 (m, 3H); MS (EI) m/e 306
(M)+.
Aloc Dep r otection To Give 27b. To a solution of 8.0 mg
(0.019 mmol) of 7b in dry THF under an argon atmosphere
were added AcOH (5.5 µL, 5.0 equiv), tributyltin hydride (16.0
µL, 3.0 equiv), and a catalytic amount of palladium tetrakis-
(triphenylphosphine). After 1 h, another catalytic amount of
palladium tetrakis(triphenylphosphine) was added. After 2 h,
THF was evaporated and the crude product was purified by
means of preparative TLC (EtOAc/heptane 1.5/1) to afford 4
mg (63%) of the spacer-leaving group conjugate 27b. Mp 128
°C; 1H NMR (300 MHz, CDCl3) δ 4.40 (d, 2H, J ) 5.8 Hz),
5.16 (s, 2H), 7.26-7.43 (m, 11H), 7.52 (d, 2H, J ) 8.1 Hz); MS
(EI) m/e 332 (M)+.
Aloc Dep r otection of 29 To Give th e Cor r esp on d in g
Bip h en yl Sp a cer Am in oben zyl Alcoh ol Con ju ga te. To a
solution of 15 mg (0.035 mmol) of 29 in dry THF under an
argon atmosphere were added AcOH (10.0 µL, 5.0 equiv),
tributyltin hydride (28.0 µL, 3.0 equiv), and a catalytic amount
of palladium tetrakis(triphenylphosphine). After 1 h, the THF
was evaporated and the crude product was purified by means
of preparative TLC (EtOAc/heptane 1.5/1) to afford 8 mg (66%)
of the spacer-leaving group conjugate. Mp 121 °C; 1H NMR
(300 MHz, CDCl3) δ 4.59 (s, 2H), 5.21 (s, 2H), 7.28-7.64 (m,
12H); MS (EI) m/e 348 (M)+.
Cu r tiu s Rea r r a n gem en t a n d Aloc P r otection To Give
5b. To a solution of 306 mg (0.893 mmol) of 4b in dry toluene
under an argon atmosphere were added diphenylphosphoryl
azide (227 µL, 1.2 equiv) and Et3N (148 µL, 1.2 equiv). The
reaction mixture was stirred at room temperature, and after
15 and 21 h, respectively, 0.4 equiv (76 µL) and 0.5 equiv (95
µL) of diphenylphosphoryl azide were added. After 39 h, allyl
alcohol (3 mL) was added and the mixture was stirred at 85
°C for 3 h, then CHCl3 was added. The organic layer was
washed with saturated NaHCO3, 10% citric acid, and brine,
dried over anhydrous Na2SO4, and evaporated. The residual
crude product was purified by means of column chromatog-
raphy (EtOAc-hexane 1:9) to afford 292 mg (82%) of the
1
desired product 5b. Mp 78 °C; H NMR (300 MHz, CDCl3) δ
0.12 (s, 6H), 0.96 (s, 9H), 4.68-4.70 (m, 2H), 4.78 (s, 2H), 5.26-
5.41 (m, 2H), 5.92-6.08 (m, 1H), 6.68 (s, 1H), 7.25-7.28 (m,
1H), 7.38 (d, 2H, J ) 8.1 Hz), 7.45 (d, 2H, J ) 8.6 Hz), 7.51-
7.56 (m, 3H); MS (EI) m/e 397 (M)+.
Dep r otection of 5b to 6b. To a solution of 274 mg (0.689
mmol) of 5b in dry THF under an argon atmosphere was added
tetrabutyl ammoniumfluoride (1.03 mL, 1.5 equiv). After
stirring for 1 h, EtOAc (50 mL) was added and THF was
evaporated. The mixture was washed with 10% citric acid,
saturated NaHCO3, and brine, dried over anhydrous Na2SO4,
and evaporated to dryness yielding 158 mg (81%) of the desired
product 6b. Mp 146 °C; 1H NMR (300 MHz, CDCl3/CD3OD)
4.67-4.68 (2 × s, 4H), 5.25-5.41 (m, 2H), 5.93-6.06 (m, 1H),
7.41 (d, 2H, J ) 7.6 Hz), 7.48-7.57 (m, 4H); MS (EI) m/e 283
(M)+. Anal. (C17H17O3N) calculated C 72.07%, H 6.05%, N
4.94%, measured C 71.53%, H 6.06%, N 4.88%.

8826 J . Org. Chem., Vol. 66, No. 26, 2001
de Groot et al.
Syn th esis of th e Dou ble Sp a cer -Con ta in in g Mod el
Com p ou n d 9. TBDMS protected spacer was synthesized by
adding imidazole (1.34 g, 3.1 equiv) to a solution of tert-butyl
dimethylsilyl chloride (2.48 g, 2.6 equiv) in dry THF. After 30
min, 4-aminobenzyl alcohol (780 mg, 6.33 mmol) was added
and the reaction mixture was stirred for 2 h. The solution was
concentrated to dryness, and the residual product was dis-
solved in CH2Cl2 and washed with saturated NaHCO3, and
the organic layer was dried over anhydrous Na2SO4 and
evaporated in vacuo. The crude product was subjected to
column chromatography (SiO2, CHCl3/MeOH 9/1) and freeze-
dried twice from dioxane to obtain 1.35 g (90%) of the desired
silyl protected spacer. 1H NMR (300 MHz, CDCl3) δ 0.07 (s,
6H), 0.92 (s, 9H), 3.60 (bs, 2H), 4.62 (s, 2H), 6.64 (d, 2H, J )
8.4 Hz), 7.11 (d, 2H, J ) 8.3 Hz) ppm.
(d, 2H, J ) 8.3 Hz), 8.27 (d, 2H, J ) 9.1 Hz) ppm; MS (FAB)
m/e 952 (M + H)+, 974 (M + Na)+. Anal. C40H46N6O12(‚1/4H2O)
calculated C 59.51%, H 5.81%, N 10.41%, measured C 59.52%,
H 5.54%, N 10.12%.
Doxor u bicin cou p lin g To Give Aloc-D-Ala -P h e-Lys-
(Aloc)-P ABC-P ABC-Doxor u bicin (12). The double spacer-
containing 4-nitrophenyl carbonate 11 (140 mg, 0.147 mmol)
and doxorubicin‚HCl (94.1 mg, 1.1 equiv) in N-methylpyrro-
lidinone were treated at room temperature with Et3N (22.5
µL, 1.1 equiv). The reaction mixture was stirred in the dark
for 72 h, again Et3N (1.1 equiv) was added and after an
additional 24 h the reaction mixture was diluted with 10%
2-propanol/EtOAc. The organic layer was washed with water
and brine and was dried (Na2SO4). After evaporation of the
solvents, the crude product was purified by means of column
chromatography (CHCl3-MeOH; 9:1) followed by circular
chromatography using a Chromatotron supplied with a 2 mm
silica plate (CHCl3-MeOH; 9:1), to yield 72 mg (36%) of
protected prodrug 12. Mp 129 °C; 1H NMR (300 MHz, CDCl3/
CD3OD) δ 1.22 (d, 3H, J ) 7.1 Hz), 1.27 (d, 3H, J ) 6.7 Hz),
1.25-2.00 (m, 8H), 2.15 (dd, 1H), 2.36 (bd, 1H), 3.04 (bd, 1H),
2.90-3.50 (m, 5H), 3.37 (bs, 1H), 3.58 (m, 1H), 3.85 (m, 1H),
4.08 (s, 3H), 4.14 (m, 1H), 4.29 (dd, 1H), 4.37-4.68 (m, 5H),
4.76 (s, 2H), 4.96 (s, 2H), 5.11 (s, 2H), 5.02-5.40 (m, 4H), 5.48
(bs, 1H), 5.61-6.00 (m, 3H), 7.08-7.39 (m, 9H), 7.33 (d, 2H, J
) 8.3 Hz), 7.42 (d, 1H, J ) 8.4 Hz), 7.62 (d, 2H, J ) 8.0 Hz),
7.80 (t, 1H, J ) 8.1 Hz), 8.03 (d, 1H, J ) 7.5 Hz) ppm; MS
(FAB) m/e 1378 (M + Na)+. Anal. C69H77N7O22(‚2H2O) calcu-
lated C 59.52%, H 5.86%, N 7.04%, measured C 59.34%, H
5.71%, N 6.66%.
N-Acetyl protected spacer was synthesized by adding
NaHCO3 (2.75 g, 1.1 equiv) and acetic anhydride (2.96 mL,
1.05 equiv) to a solution of 3.67 g of 4-aminobenzyl alcohol
(29.8 mmol). After 2 h, the solution was filtered and the filtrate
was evaporated to dryness. The crude product was subjected
to column chromatography (SiO2, CHCl3/MeOH 9/1) to obtain
4.30 g (87%) of the desired N-acetyl protected spacer as a white
1
solid. H NMR (300 MHz, CDCl3) δ 2.18 (s, 3H), 4.66 (s, 2H),
7.32 (d, 2H, J ) 8.4 Hz), 7.49 (d, 2H, J ) 8.4 Hz) ppm.
A solution of O-silyl protected spacer (218 mg, 0.918 mmol)
in dry THF was added dropwise to a solution of carbonyl
diimidazole (149 mg, 1.0 equiv) in dry THF, and the mixture
was stirred for 24 h. A solution of N-acetyl protected spacer
(152 mg, 1.0 equiv) and Et3N (127 µL, 1.0 equiv) in THF was
added dropwise, and the reaction mixture was allowed to stir
for 96 h. The solution was concentrated to dryness, and the
residual product was dissolved in CH2Cl2 and washed with
saturated NaHCO3, a 10% citric acid solution, and brine. The
organic layer was dried over anhydrous Na2SO4 and evapo-
rated in vacuo. The crude product was subjected to column
chromatography (SiO2, CHCl3/MeOH 20/1) to obtain 85 mg
Depr otection To Give pr odr u g H-D-Ala-P h e-Lys-P ABC-
P ABC-Doxor u bicin (13). To a solution of 48 mg (0.035 mmol)
of protected prodrug 12 in dry THF/CH2Cl2 under an argon
atmosphere was added morpholine (31 µL, 10 equiv) together
with a catalytic amount of Pd(PPh3)4. The reaction mixture
was stirred for 1 h in the dark. The red precipitate was
collected by means of centrifugation. EtOAc was added, and
the mixture was acidified using 1.0 mL of 0.5 M hydrochloric
acid/EtOAc. The precipitate was collected by means of cen-
trifugation and washed several times with EtOAc. tert-Butyl
alcohol was added and evaporated, and the resulting red film
was freeze-dried in water, yielding 37 mg (83%) of doxorubicin
prodrug 13. Mp > 300 °C; 1H NMR (300 MHz, CDCl3/CD3OD)
δ 1.20 (d, 3H, J ) 7.0 Hz), 1.27 (d, 3H, J ) 6.5 Hz), 1.38-2.05
(m, 8H), 2.18 (dd, 1H), 2.36 (bd, 1H), 2.82-3.41 (m, 6H), 3.37
(s, 1H), 3.60 (bs, 1H), 4.02 (m, 1H), 4.08 (s, 3H), 4.18 (m, 1H),
4.53 (dd, 1H), 4.66 (m, 1H), 4.77 (s, 2H), 4.95 (bs, 2H), 5.14 (s,
2H), 5.27 (bs, 1H), 5.48 (bs, 1H), 7.09-7.50 (m, 11H), 7.58 (d,
2H, J ) 8.4 Hz), 7.82 (t, 1H, J ) 8.0 Hz), 8.03 (d, 1H, J ) 7.6
Hz) ppm; MS (FAB) m/e 1188 (M + H)+, m/e 1210 (M + Na)+.
Anal. (duplo) C61H69N7O18(‚5.7HCl) calculated C 52.42%, H
5.39%, N 7.01%, measured C 52.38%, H 5.71%, N 7.14%.
1
(22%) of the desired model compound 9. H NMR (300 MHz,
CDCl3) δ 0.08 (s, 6H), 0.92 (s, 9H), 2.11 (s, 3H), 4.67 (s, 2H),
5.10 (s, 2H), 7.08 (bs, 1H), 7.25 (m, 4H), 7.35 (d, 2H, J ) 8.2
Hz), 7.45 (d, 2H, J ) 8.3 Hz), 7.80 (bs, 1H) ppm.
Cou p lin g of sp a cer to 8 to give Aloc-D-Ala -P h e-Lys-
(Aloc)-P ABC-P ABA (10). To a solution of 156 mg (0.194
mmol) of compound 8 and 26.3 mg (1.1 equiv) 4-aminobenzyl
alcohol in dry DMF under an argon atmosphere were added
DIPEA (34 µL, 1.0 equiv) and a catalytic amount of N-hydroxy
benzotriazole (7.9 mg, 0.3 equiv). The reaction solution was
stirred for 24 h after which it was diluted with 10% propanol-
2/EtOAc. The organic layer was washed with saturated
NaHCO3, 0.5 M KHSO4, and brine, dried over anhydrous
Na2SO4, and evaporated to dryness. The yellow residual film
was purified by means of column chromatography (SiO2,
CHCl3/MeOH 9/1) to yield 148 mg (97%) of the desired product
10. Mp 196-197 °C; 1H NMR (300 MHz, CDCl3) δ 1.20 (d, 3H,
J ) 6.4 Hz), 1.27-2.05 (m, 6H), 2.99-3.27 (m, 4H), 4.00-4.64
(m, 7H), 4.57 (s, 2H), 5.14 (s, 2H), 5.06-5.37 (m, 4H), 5.72 (m,
1H), 5.88 (m, 1H), 7.10-7.46 (m, 11H), 7.64 (d, 2H, J ) 8.3
P a clita xel Cou p lin g To Give 2′-[Aloc-D-Ala -P h e-Lys-
(Aloc)-P ABC-P ABC]-P a clita xel (14). 4-Nitrophenyl carbon-
ate 11 (47.4 mg, 0.0498 mmol) and paclitaxel (42.3 mg, 1.0
equiv) in dry THF/CH2Cl2 under an argon atmosphere were
treated at room temperature with N,N-dimethyl-4-aminopy-
ridine (6.7 mg, 1.1 equiv). The reaction mixture was stirred
in the dark for 48 h and was then concentrated to dryness.
The product was dissolved in CH2Cl2, and the organic layer
was washed with saturated NaHCO3, 0.5 M KHSO4 and brine
and dried over anhydrous Na2SO4. After evaporation of the
solvents the residual yellow film was purified by means of
column chromatography (SiO2, EtOAc/Hex/MeOH 5/5/1), to
yield 67.5 mg (82%) of protected paclitaxel prodrug 14. Mp
137-138 °C;1H NMR (300 MHz, CDCl3) δ 1.14 (s, 3H), 1.23
(s, 3H), 1.27 (d, 3H, J ) 7.1 Hz), 1.05-2.10 (m, 6H), 1.67 (s,
3H), 1.89 (s, 3H), 2.22 (s, 3H), 2.44 (s, 3H), 2.97-3.21 (m, 4H),
3.81 (d, 1H, J ) 7.0 Hz), 4.03 (m, 1H), 4.20 (d, 1H, J ) 8.4
Hz), 4.31 (d, 1H, J ) 8.4 Hz), 4.43 (m, 1H), 4.34-4.74 (m, 6H),
4.90-5.37 (m, 11H), 5.44 (d, 1H, J ) 2.9 Hz), 5.63 (m, 1H),
5.69 (d, 1H, J ) 7.1 Hz), 5.87 (m, 1H), 5.97 (bd, 1H, J ) 2.9
Hz, J ) 9.2 Hz), 6.26 (m, 1H), 6.29 (m, 1H), 7.05-7.80 (m,
26H), 8.14 (d, 2H, J ) 7.2 Hz) ppm; MS (FAB) m/e 1668 (M +
Hz) ppm; MS (FAB) m/e 809 (M + Na)+. Anal. C41H50N6O10
-
(‚1/2H2O) calculated C 61.87%, H 6.46%, N 10.56%, measured
C 61.84%, H 6.38%, N 10.38%.
Activa tion of 10 to Aloc-D-Ala -P h e-Lys(Aloc)-P ABC-
P ABC-P NP (11). A solution of 80.2 mg (0.102 mmol) of
compound 10, pyridine (25 µL, 3.0 equiv), and 4-nitrophenyl
chloroformate (44.3 mg, 0.220 mmol) was stirred in dry THF/
CH2Cl2 under an argon atmosphere at 0 °C for 2 h and
overnight at room temperature. The solution was evaporated
in vacuo, and the residual product was dissolved in CH2Cl2.
After washing the organic layer with brine and 0.5 M KHSO4,
the organic layer was dried over anhydrous Na2SO4 and
concentrated to dryness. The resulting crude product was
subjected to column chromatography (SiO2, CHCl3/MeOH 20/
1) to obtain 61.9 mg (84%) of compound 11. Mp 69-70 °C; 1H
NMR (300 MHz, CDCl3/CD3OD) δ 1.23 (d, 3H, J ) 7.0 Hz),
1.10-2.08 (m, 6H), 3.04-3.27 (m, 4H), 4.06 (m, 1H), 4.26 (m,
1H), 4.35-4.70 (m, 5H), 5.04-5.47 (m, 4H), 5.14 (s, 2H), 5.24
(s, 2H), 5.72 (m, 1H), 5.90 (m, 1H), 7.10-7.46 (m, 13H), 7.65

Elongated Spacer Systems for Enhanced Drug Release
J . Org. Chem., Vol. 66, No. 26, 2001 8827
H)+, 1689 (M + Na)+. Anal. C89H99N7O25(‚2H2O) calculated C
62.78%, H 6.10%, N 5.76%, measured C 62.55%, H 5.82%, N
5.57%.
After the pH of the mixture did not alter any more, a 0.5 M
solution of hydrochloric acid in EtOAc was added until a pH
of 3 was reached. The mixture was thoroughly extracted with
CH2Cl2. The organic layer was washed with water, dried over
anhydrous Na2SO4, and evaporated. The product was purified
by means of column chromatography (SiO2, CHCl3/AcOH/
MeOH 85/10/5) to afford 302 mg (15%) of product as a cream
colored powder. Mp 116 °C; 1H NMR (300 MHz, CDCl3) δ 1.00-
1.80 (m, 9H), 2.80-3.35 (m, 4H), 4.13 (m, 1H), 4.30-4.95 (m,
6H), 5.01-5.40 (m, 4H), 5.70-6.30 (m, 3H), 6.90-7.70 (m, 5H)
ppm; MS (FAB) m/e 572 (M + H)+, 594 (M + Na)+, 1143 (2M
+ Na)+. Anal. C28H37N5O8(‚11/2H2O) calculated C 56.18%, H
6.44%, N 11.70%, measured C 56.07%, H 6.22%, N 11.21%.
Cou p lin g of Sp a cer To Give Aloc-D-Ala -Tr p -Lys(Aloc)-
P ABA. A solution of 239 mg (0.419 mmol) of Aloc-D-Ala-Trp-
Lys(Aloc)-OH was dissolved in dry THF under an argon
atmosphere and cooled to -40 °C. N-Methylmorpholine (48.3
µL, 1.05 equiv) and isobutyl chloroformate (57.0 µL, 1.05 equiv)
were added. The reaction mixture was stirred for 2 h at a
temperature below -30 °C. A solution of 4-aminobenzyl alcohol
(51.5 mg, 1.00 equiv) and N-methylmorpholine (50.6 µL, 1.1
equiv) in dry THF was added dropwise to the reaction mixture.
After 2 h, THF was evaporated and CH2Cl2 was added. The
organic layer was washed with saturated NaHCO3, a 0.5 M
KHSO4 solution and brine, dried over anhydrous Na2SO4, and
evaporated to afford 265 mg (94%) of the desired product as a
cream-colored powder, which was used without further puri-
fication. Mp 132 °C; 1H NMR (300 MHz, CDCl3/CD3OD) δ
1.00-1.62 (m, 9H), 2.90-3.70 (m, 4H), 4.10 (m, 1H), 4.48-
4.92 (m, 6H), 4.72 (s, 2H), 5.00-5.50 (m, 4H), 5.35-6.05 (m,
2H), 6.80-7.83 (m, 9H) ppm; MS (FAB) m/e 677 (M + H)+,
699 (M + Na)+.
Activa tion To Give Aloc-D-Ala -Tr p -Lys(Aloc)-P ABC-
P NP . To a solution of 384 mg (0.602 mmol) of Aloc-D-Ala-Trp-
Lys(Aloc)-PABA in dry THF/CH2Cl2 under an argon atmo-
sphere, 4-nitrophenyl chloroformate (182 mg, 1.50 equiv) and
dry pyridine (73 µL, 1.50 equiv) were added. The mixture was
stirred at room temperature for 48 h, and then EtOAc was
added. The organic layer was washed with 10% citric acid,
brine, and water, dried over anhydrous Na2SO4, and evapo-
rated yielding a yellow solid. The product was purified by
means of column chromatography (SiO2, CHCl3/MeOH 30/1)
to afford 324 mg (67%) of the product as a cream-colored
powder. Mp 100 °C;1H NMR (300 MHz, CDCl3) δ 1.00-2.10
(m, 9H), 2.90-3.70 (m, 4H), 3.81 (m, 1H), 4.10 (m, 1H), 4.38-
4.81 (m, 5H), 5.10-5.35 (m, 4H), 5.21 (s, 2H), 5.40-6.00 (m,
2H), 7.00-7.85 (m, 11H), 8.25 (d, 2H, J ) 8.1 Hz); MS (FAB)
m/e 842 (M + H)+, 864 (M + Na)+.
Dep r otection To Give P r od r u g 2′-[H-D-Ala -P h e-Lys-
P ABC-P ABC]-P a clita xel (15). To a solution of 51.4 mg
(0.0308 mmol) protected prodrug 14 in dry THF under an
argon atmosphere was added glacial AcOH (8.9 µL, 5 equiv)
together with tributyltin hydride (24.6 µL, 3 equiv) and a
catalytic amount of Pd(PPh3)4. After 30 min, 1 mL of 0.5 M
HCl/EtOAc was carefully added to the reaction solution. The
product was precipitated by addition of diethyl ether, and 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 HCl, and the
resulting product was dissolved in water and freeze-dried,
yielding 46.9 mg (100%) of prodrug 15. Mp > 192 °C (dec);1H
NMR (300 MHz, CDCl3/CD3OD): δ 1.15 (s, 3H), 1.21 (s, 3H),
1.10-2.00 (m, 9H), 1.67 (s, 3H), 1.90 (s, 3H), 2.20 (s, 3H), 2.43
(s, 3H), 2.85 (m, 4H), 3.80 (d, 1H, J ) 6.9 Hz), 4.24 (d, 1H, J
) 8.4 Hz), 4.31 (d, 1H, J ) 8.4 Hz), 4.39 (dd, 1H), 4.56 (m,
1H), 5.68 (m, 1H), 4.98 (d, 1H), 5.08 (m, 4H), 5.43 (d, 1H, J )
2.7 Hz), 5.70 (d, 1H, J ) 7.0 Hz), 5.97 (m, 1H), 6.22 (m, 1H),
6.32 (m, 1H), 7.05-7.68 (m, 24H), 7.71 (d, 1H, J ) 7.2 Hz),
8.14 (d, 2H, J ) 7.3 Hz) ppm; MS (FAB) m/e 1499 (M + H)+,
1521 (M + Na)+. Anal. C81H91N7O21(‚3.7HCl) calculated C
59.60%, H 5.85%, N 6.01%, measured C 59.60%, H 5.88%, N
5.98%.
Cou p lin g Rea ction To Give F m oc-Tr p -Lys(Boc)-OBu t.
To a solution of 3.00 g (5.73 mmol) Fmoc-Trp-ONSu (ONSu )
N-hydroxysuccinimide) in dry CH2Cl2 under an argon atmo-
sphere were added at 0 °C 0.791 mL (1.00 equiv) Et3N and
2.12 g (1.10 equiv) of H-Lys(Boc)-OBut‚HCl. The mixture was
stirred at room temperature for 5 h, CH2Cl2 was added, and
the organic layer was washed with 10% citric acid, saturated
NaHCO3, and water, dried over anhydrous Na2SO4, and
evaporated. The white solid (3.52 g, 86%) was used without
1
further purification. Mp 77 °C; H NMR (300 MHz, CDCl3) δ
1.10-1.92 (m, 24H), 2.80-3.20 (m, 3H), 3.52 (d, 1H), 4.19 (t,
1H), 4.29-4.82 (m, 5H), 6.54 (d, 1H), 7.06-7.76 (m, 12H) ppm;
MS (FAB) m/e 734 (M+ Na)+,1444 (2M + Na)+. Anal.
C
41H50N4O7(‚4H2O) calculated C 62.90%, H 6.30%, N 7.15%,
measured C 63.22%, H 6.49%, N 7.13%.
Dep r otection a n d Cou p lin g To Give Boc-D-Ala -Tr p -
Lys(Boc)-OBu t. 3.52 g (4.95 mmol) of Fmoc-Trp-Lys(Boc)-
OBut was dissolved in 100 mL of dioxane/MeOH/2 M NaOH
(70/25/5) and stirred at room temperature for 1 h. The mixture
was neutralized with AcOH (0.570 mL), and organic solvents
were evaporated. Water and dioxane were added, and the
solution was freeze-dried. Diisopropyl ether was added, and
after filtration the filtrate was evaporated. The product was
dissolved in dry CH2Cl2 and added at 0 °C to a solution of 1.41
g (4.93 mmol) of Boc-D-Ala-ONSu and 0.756 mL (1.10 equiv)
of Et3N in dry CH2Cl2. The mixture was stirred for 16 h after
which CH2Cl2 was added. The organic layer was washed with
10% citric acid, saturated NaHCO3, and water, dried over Na2-
SO4, and evaporated. The product was purified by means of
column chromatography ((SiO2, first EtOAc/heptane 1/1 and
then CHCl3/MeOH 9/1) to afford 2.26 g (3.42 mmol, 69%) of
the tripeptide as a white foam. Mp 117 °C; 1H NMR (300 MHz,
CDCl3) δ 0.99-1.90 (m, 36 H), 2.80-3.50 (m, 4H), 3.99 (m,
1H), 4.33 (m, 1H), 4.77 (bd, 1H), 6.90-7.72 (m, 5H) ppm; MS
(FAB) m/e 660 (M + H)+, 682 (M + Na)+. Anal. C34H53N5O8(‚
H2O) calculated C 60.25%, H 8.17%, N 10.33%, measured C
60.47%, H 8.08%, N 9.73%.
Dep r otection a n d Aloc P r otection To Give Aloc-D-Ala -
Tr p -Lys(Aloc)-OH. A 2.56 g (4.13 mmol) amount of Boc-D-
Ala-Trp-Lys(-Boc)-OBut was stirred in a solution of hydro-
chloric acid in EtOAc (3 M). After 5 h, the solvent was
evaporated, and tert-butyl alcohol was added and evaporated
twice to remove remaining hydrochloric acid. The product was
freeze-dried in dioxane/water to yield a brown colored powder.
To a solution of 1.71 g (3.62 mmol) D-Ala-Phe-Lys-OH in
water/CH3CN was added Et3N until a pH of 8 was reached.
Then a solution of 1.58 g (2.20 equiv) Aloc-ONSu in CH3CN
was added, and the mixture was kept basic by adding Et3N.
Cou p lin g of Sp a cer To Give Aloc-D-Ala -Tr p -Lys(Aloc)-
P ABC-P ABA. To a solution of 219 mg (0.260 mmol) of Aloc-
D-Ala-Trp-Lys(Aloc)-PABC-PNP and 35.2 mg (1.1 equiv) of
4-aminobenzyl alcohol in dry DMF under an argon atmosphere
were added DIPEA (45.3 µL, 1.00 equiv) and a catalytic
amount of N-hydroxybenzotriazole (10.5 mg, 0.30 equiv). The
reaction solution was stirred for 48 h after which it was diluted
with 10% propanol-2/EtOAc. The organic layer was washed
with saturated NaHCO3, 0.5 M KHSO4, and brine, dried over
anhydrous Na2SO4, and evaporated to dryness. The pale yellow
residual film was purified by means of column chromatography
(SiO2, CHCl3/MeOH 15/1) to yield 192 mg (89%) of the desired
1
product. Mp 287 °C; H NMR (300 MHz, CDCl3) δ 0.90-2.10
(m, 9H), 2.90-3.70 (m, 4H), 3.82 (m, H), 4.08 (m, 1H), 4.40-
4.86 (m, 5H), 4.59 (s, 2H), 4.90-5.40 (m, 4H), 5.08 (s, 2H), 5.50
(m, 1H), 5.92 (m, 1H), 6.72-7.82 (m, 13H) ppm; MS (FAB) m/e
848 (M + Na)+. Anal. C43H51N7O10(‚23/4H2O) calculated C
58.99%, H 6.50%, N 11.20%, measured C 59.15%, H 6.25%, N
11.15%.
Activa tion To Give Aloc-D-Ala -Tr p -Lys(Aloc)-P ABC-
P ABC-P NP . A solution of 70 mg (0.085 mmol) of Aloc-D-Ala-
Trp-Lys(Aloc)-PABC-PABA, pyridine (17 µL, 2.5 equiv), and
4-nitrophenyl chloroformate (34 mg, 2.0 equiv) was stirred
under an argon atmosphere at 0 °C for 2 h and for 24 h at
room temperature. The solution was evaporated in vacuo, and
the residual product was dissolved in CHCl3. After washing
the organic layer with brine and 0.5 M KHSO4, the organic

8828 J . Org. Chem., Vol. 66, No. 26, 2001
de Groot et al.
layer was dried over anhydrous Na2SO4 and concentrated to
dryness. The resulting crude product was subjected to column
chromatography (SiO2, CHCl3/MeOH 20/1) to obtain 54 mg
4-nitrophenyl chloroformate (25 mg, 2.0 equiv). After stirring
for 4.5 h at -40 °C and overnight at 6 °C, pyridine (10 µL, 2.0
equiv) and 4-nitrophenyl chloroformate (25 mg, 2.0 equiv) were
added again. This was repeated after 48 h stirring at 6 °C.
After another 48 h, the solution was evaporated in vacuo and
the residual product was dissolved in CHCl3. The organic layer
was washed with 10% citric acid, brine, and water, dried over
anhydrous Na2SO4, and evaporated, yielding a yellow solid.
The crude product was purified by means of column chroma-
tography (SiO2, CHCl3/MeOH 15/1) to give the desired product
18 quantitatively. 1H NMR (300 MHz, CDCl3/CD3OD) δ 1.12-
1.89 (m, 9H), 3.04 (m, 1H), 3.14 (m, 2H), 3.27 (bd, 1H), 4.09
(m, 1H), 4.28 (m, 1H), 4.34-4.68 (m, 5H), 5.02-5.40 (m, 4H),
5.14 (s, 2H), 5.21 (s, 2H), 5.31 (s, 2H), 5.72 (m, 1H), 5.90 (m,
1H), 7.10-7.52 (m, 17H), 7.63 (d, 2H, J ) 8.3 Hz), 8.27 (d,
2H, J ) 9.1 Hz) ppm; MS (FAB) m/e 1102 (M + H)+, 1124 (M
+ Na)+.
1
(64%) of the desired product as a pale yellow solid. H NMR
(300 MHz, CDCl3/CD3OD) δ 0.90-2.10 (m, 9H), 2.90-3.27 (m,
4H), 4.20 (m, 1H), 4.35-4.78 (m, 6H), 4.90-5.52 (m, 4H), 5.13
(s, 2H), 5.34 (s, 2H), 5.60 (m, 1H), 5.94 (m, 1H), 7.10-7.46 (m,
15H), 8.36 (d, 2H) ppm; MS (FAB) m/e 991 (M + H)+, 1013 (M
+ Na)+. Anal. C50H54N8O14(‚3/4H2O) calculated C 59.78%, H
5.57%, N 11.15%, measured C 60.12%, H 5.89%, N 10.76%.
Doxor u bicin Cou p lin g To Give Aloc-D-Ala -Tr p -Lys-
(Aloc)-P ABC-P ABC-Doxor u bicin . Aloc-D-Ala-Trp-Lys(Aloc)-
PABC-PABC-PNP (41 mg, 0.041 mmol) and doxorubicin‚HCl
(26 mg, 1.1 equiv) in N-methylpyrrolidinone were treated at
room temperature with Et3N (6.3 µL, 1.1 equiv). The reaction
mixture was stirred in the dark for 48 h, again Et3N (1.1 equiv)
was added, and after an additional 24 h the reaction mixture
was diluted with 10% 2-propanol/EtOAc. The organic layer was
washed with water and brine and was dried over anhydrous
Na2SO4. After evaporation of the solvents, the crude product
was purified by means of column chromatography (SiO2,
CHCl3/MeOH 9/1) followed by circular chromatography using
a Chromatotron supplied with a 2 mm silica plate (CHCl3-
MeOH 9/1), to yield 45 mg (78%) of the protected prodrug. Mp
Doxor u bicin Cou p lin g To Give Aloc-D-Ala -P h e-Lys-
(Aloc)-P ABC-P ABC-P ABC-Doxor u bicin (19). The 4-nitro-
phenyl carbonate 18 (69 mg, 0.063 mmol) and doxorubicin‚
HCl (40 mg, 1.1 equiv) in N-methylpyrrolidinone were treated
at room temperature with Et3N (9.7 µL, 1.1 equiv). The
reaction mixture was stirred in the dark for 24 h, and the
reaction mixture was diluted with 10% 2-propanol/EtOAc. The
organic layer was washed with water and brine and was dried
over anhydrous Na2SO4. After evaporation of the solvents, the
crude product was purified by means of column chromatog-
raphy (SiO2, CHCl3/MeOH 9/1) followed by circular chroma-
tography using a Chromatotron supplied with a 2 mm silica
plate (CHCl3/MeOH; 9/1), to yield 65 mg (71%) of the protected
1
130 °C; H NMR (300 MHz, CDCl3/CD3OD) δ 0.92-1.52 (m,
14H), 2.15 (dd, 1H), 2.36 (bd, 1H), 3.18 (bd, 1H), 2.90-3.10
(m, 6H), 3.59 (bs, 1H), 3.82 (m, 1H), 3.85 (m, 1H), 4.11 (s, 3H),
4.21 (m, 1H), 4.45 (dd, 1H), 4.30-4.62 (m, 5H), 4.76 (s, 2H),
4.96 (s, 2H), 5.11 (s, 2H), 5.13-5.40 (m, 4H), 5.48 (bs, 1H),
5.58 (m, 2H), 5.91(m, 1H), 6.70-7.39 (m, 11H), 7.41 (d, 1H, J
) 8.4 Hz), 7.63 (d, 2H), 7.78 (t, 1H), 8.03 (d, 1H, J ) 7.6 Hz)
ppm; MS (FAB) m/e 1417 (M + Na)+.
1
prodrug 19. H NMR (300 MHz, CDCl3/CD3OD) δ 1.10-1.80
(m, 14H), 2.14 (m, 1H), 2.36 (m, 1H), 2.82-3.41 (m, 6H), 3.37
(s, 1H), 3.60 (bs, 1H), 4.03 (m, 1H), 4.29 (m, 1H), 4.07 (s, 3H),
4.29 (dd, 1H), 4.37-4.68 (m, 5H), 4.76 (s, 2H), 4.95 (bs, 2H),
5.10 (s, 2H), 5.14 (s, 2H), 5.02-5.35 (m, 4H), 5.27 (bs, 1H),
5.47 (bs, 1H), 5.70 (m, 1H), 5.89 (m, 1H), 7.09-7.50 (m, 16H),
7.64 (d, 2H, J ) 8.4 Hz), 7.79 (t, 1H, J ) 8.1 Hz), 8.06 (d, 1H,
J ) 7.5 Hz) ppm; MS (FAB) m/e 1506 (M + H)+, 1528 (M +
Na)+.
Depr otection To Give P r odr u g H-D-Ala-Tr p-Lys-P ABC-
P ABC-Doxor u bicin (16). To a solution of 36 mg (0.026 mmol)
protected prodrug Aloc-D-Ala-Trp-Lys(Aloc)-PABC-PABC-
Doxorubicin in dry THF/CH2Cl2 under an argon atmosphere
was added morpholine (22 µL, 10 equiv) together with a
catalytic amount of Pd(PPh3)4. The reaction mixture was
stirred for 1 h in the dark. The red precipitate was collected
by means of centrifugation. EtOAc was added, and the mixture
was acidified using 0.5 mL of 1 M hydrochloric acid/EtOAc.
The precipitate was collected by means of centrifugation and
washed several times with EtOAc. tert-Butyl alcohol was added
and evaporated, and the resulting red film was freeze-dried
in water yielding 28 mg (72%) of the doxorubicin prodrug 16.
Mp 95 °C; 1H NMR (300 MHz, CDCl3/CD3OD) δ 1.10-1.96 (m,
14H), 2.09 (m, 1H), 2.35 (bd, 1H), 2.79-3.39 (m, 6H), 3.60 (s,
1H), 4.00 (bs, 1H), 4.09 (s, 3H), 3.95-4.15 (m, 2H), 4.29 (m,
1H), 4.51 (m, 1H), 4.77 (s, 2H), 4.86 (2 × d, 2H), 5.02 (s, CH2),
5.38 (bs, 1H), 5.48 (bs, 1H), 6.99-7.72 (m, 11H), 7.55 (d, 2H,
J ) 8.2 Hz), 7.62 (d, 1H, J ) 7.6 Hz), 7.83 (t, 1H), 8.05 (d, 1H,
Depr otection To Give P r odr u g H-D-Ala-P h e-Lys-P ABC-
P ABC-P ABC-Doxor u bicin (20). To a solution of 40 mg
protected prodrug 19 (0.027 mmol) in dry THF/CH2Cl2 under
an argon atmosphere were added morpholine (24 µL, 10 equiv)
and a catalytic amount of Pd(PPh3)4. The reaction mixture was
stirred for 1 h in the dark. The red precipitate was collected
by means of centrifugation and washed several times with
EtOAc. Water and dioxane were added and the mixture was
acidified using 4.3 mL of 0.125 mM hydrochloric acid. After
freeze-drying 26 mg (69%) of doxorubicin prodrug 20 was
obtained. Mp 66 °C; 1H NMR (300 MHz, CDCl3/CD3OD) δ 1.19
(d, 3H, J ) 6.9 Hz), 1.27 (d, 3H, J ) 6.6 Hz), 1.25-2.00 (m,
8H), 2.18 (dd, 1H), 2.33 (bd, 1H), 2.89-3.38 (m, 6H), 3.60 (s,
1H), 3.72 (m, 1H), 4.08 (s, 3H), 4.03-4.20 (m, 2H), 4.53 (dd,
1H), 4.66 (m, 1H), 4.77 (s, 2H), 4.96 (s, 2H), 5.11 (s, 2H), 5.17
(s, 2H), 5.27 (bs, 1H), 5.48 (bs, 1H), 7.05-7.35 (m, 16H), 7.52
(d, 2H, J ) 8.5 Hz), 7.84 (t, 1H), 8.01 (d, 1H, J ) 7.7 Hz) ppm.;
MS (FAB) m/e 1337 (M + H)+, 1359 (M + Na)+. Anal.
J
) 7.7 Hz) ppm; MS (FAB) m/e 1228 (M + H)+. Anal.
C63H70N8O18(‚7 1/2HCl) calculated C 50.42%, H 5.21%, N 7.47%,
measured C 50.56%, H 5.48%, N 7.35%.
Cou p lin g of a Th ir d Sp a cer to 11 To Give Aloc-D-Ala -
P h e-Lys(Aloc)-P ABC-P ABC-P ABA (17). A 100 mg (0.105
mmol) amount of Aloc-D-Ala-Phe-Lys(Aloc)-PABC-PABC-PNP
11 was dissolved in dry DMF under an argon atmosphere and
cooled to -8 °C. 4-Aminobenzyl alcohol (14.2 mg, 1.1 equiv),
DIPEA (18.3 µL, 1.0 equiv), and 1-hydroxybenzotriazole
(HOBt) (4 mg, 0.3 equiv) were added. The reaction mixture
was stirred for 48 h at room temperature and diluted with
10% 2-propanol/EtOAc. The organic layer was washed with
water, saturated NaHCO3, 0.5 M KHSO4, and brine, dried over
anhydrous Na2SO4, and evaporated to yield the desired product
17 as a cream-colored powder 86 mg (88%), which was used
C
69H76N8O20(‚61/2HCl) calculated C 52.64%, H 5.28%, N 7.12%,
measured C 52.66%, H 5.36%, N 6.78%.
Syn th esis of Z-P r otected Sp a cer 22. To a solution of 1.21
g (13.7 mmol) N,N′-dimethylethylenediamine in dry CH2Cl2
under an argon atmosphere at room temperature was added
dropwise a solution of Z-ONSu (338 mg, 1.36 mmol) in dry CH2-
Cl2. After stirring for 120 min, the solution was concentrated
in vacuo. The residual product was dissolved in EtOAc, and
the organic layer was washed with brine. The organic solvent
was dried over anhydrous Na2SO4 and evaporated to dryness.
The oily product was purified by means of column chroma-
tography (SiO2, CHCl3/MeOH 1/1) to obtain 249 mg (83%) of
the product 22 as an oil. 1H NMR (300 MHz, CDCl3) δ 2.42
(bd, 3H), 2.73 (m, 2H), 2.95 (s, 3H), 3.41 (bs, 2H), 5.13 (s, 2H),
7.25-7.40 (m, 5H) ppm.
1
without further purification. Mp 126 °C; H NMR (300 MHz
CDCl3) δ 0.95-2.05 (m, 9H), 2.88-3.11 (m, 4H), 3.95-4.62 (m,
7H), 4.75 (s, 2H), 5.12-5.21 (m, 6H), 5.09 (s, 2H), 5.65-6.00
(m, 2H), 6.79-7.41 (m, 15H), 7.62 (d, 2H) ppm; MS (FAB) m/e
959 (M + Na)+.
Activa tion of 17 to Aloc-D-Ala -P h e-Lys(Aloc)-P ABC-
P ABC-P ABC-P NP (18). To a solution of 59 mg (0.063 mmol)
of 17 in dry THF and CH2Cl2 under an argon atmosphere were
added at -40 °C, respectively, pyridine (13 µL, 2.5 equiv) and
Cou p lin g of 22 To Give 2′-[Z-N(Me)-(CH2)2-N(Me)CO]-
P a clita xel (23). To a solution of 114 mg (0.112 mmol) of 2′-
activated paclitaxel and 25 mg of Z-protected N,N′-dimethyl-

Elongated Spacer Systems for Enhanced Drug Release
J . Org. Chem., Vol. 66, No. 26, 2001 8829
ethylenediamine 22 in dry CH2Cl2 under an argon atmosphere
at -50 °C was added Et3N (20.0 µL, 0.144 mmol). The reaction
solution was stirred 7 h at -40 °C and then overnight at room
temperature. The solution was diluted with CH2Cl2 and
washed with saturated NaHCO3, brine, and 0.5 M KHSO4. The
organic layer was dried over anhydrous Na2SO4 and concen-
trated in vacuo to obtain a yellow film. The product was
purified by column chromatography (SiO2, EtOAc/Hex 2/1) to
obtain 113 mg (92%) of the desired product 23. Mp 130-131
°C; 1H NMR (300 MHz, CDCl3) δ 1.12 (s, 3H), 1.21 (s, 3H),
1.70 (s, 3H), 2.00 (s, 3H), 2.26 (s, 3H), 2.60 (s, 3H), 2.90 (s,
3H), 2.94 (s, 3H), 2.97 (m, 1H), 3.06 (m, 1H), 3.54 (m, 1H),
3.78 (m, 1H), 3.84 (d, 1H, J ) 7.2 Hz), 4.23 (d, 1H, J ) 8.4
Hz), 4.32 (d, 1H, J ) 8.4 Hz), 4.47 (m, 1H), 4.69 (d, 1H, J )
12.4 Hz), 4.85 (d, 1H, J ) 12.4 Hz), 5.01 (m, 1H), 5.47 (d, 1H,
J ) 2.9 Hz), 5.68 (d, 1H, J ) 7.0 Hz), 6.19 (dd, 1H, J ) 9.8 Hz,
J ) 2.9 Hz), 6.28 (s, 1H), 6.33 (m, 1H), 6.94-7.70 (m, 16H),
7.83 (d, 2H, J ) 7.3 Hz), 8.16 (d, 2H, J ) 7.1 Hz), 8.57 (d, 1H,
J ) 9.8 Hz) ppm; MS (FAB) m/e 1102 (M + H)+, 1124 (M +
Na)+. Anal. C60H67N3O17(‚H2O) calculated C 64.33%, H 6.21%,
N 3.73%, measured C 64.65%, H 6.11%, N 3.76%.
Dep r otection of 23 to 2′-[HN(Me)-(CH2)2-N(Me)CO]-
P a clita xel (24). To a solution of 61.8 mg (0.0561 mmol) 23 in
5% AcOH/MeOH was added a catalytic amount of 10% Pd/C.
The mixture was stirred for 1 h under a H2 atmosphere. The
Pd-C was removed by means of centrifugation. EtOAc was
added, and MeOH was evaporated in vacuo. The organic layer
was extracted with water. The water layer was freeze-dried
yielding 78.0 mg (100%) of the desired protonated product 24.
Cou p lin g of F r a gm en ts 8 a n d 24 to 2′-[Aloc-D-Ala -P h e-
Lys(Aloc)-P ABC-N(Me)-(CH2)2-N(Me)CO]-P a clita xel (25).
To a solution of 152 mg (0.0958 mmol) of paclitaxel-spacer 24
and 80.7 mg (0.101 mmol) of compound 8 in dry THF under
an argon atmosphere was added Et3N (200 µL, 1.44 mmol).
After 24 h the solution was concentrated to dryness and the
residual product was dissolved in CH2Cl2 and washed with
saturated NaHCO3 and brine. The organic layer was dried over
anhydrous Na2SO4 and evaporated in vacuo. The crude product
was subjected to column chromatography (SiO2, EtOAc/Hex/
MeOH 5/5/1) to obtain 113 mg (72%) of the protected prodrug
25. Mp 127-128 °C; 1H NMR (300 MHz, CDCl3) δ 1.13 (s, 3H),
1.22 (s, 3H), 1.27 (d, 3H, J ) 5.6 Hz), 1.04-2.00 (m, 6H), 1.69
(s, 3H), 2.00 (s, 3H), 2.22 (s, 3H), 2.59 (s, 3H), 2.90 (s, 3H),
2.91 (s, 3H), 2.76-3.46 (m, 6H), 3.54 (m, 1H), 3.74 (m, 1H),
3.84 (d, 1H, J ) 7.0 Hz), 4.00-5.00 (m, 3H), 4.23 (d, 1H, J )
8.4 Hz), 4.32 (d, 1H, J ) 8.4 Hz), 4.48 (m, 1H), 4.62 (d, 1H, J
) 12.3 Hz), 4.83 (d, 1H, J ) 12.4 Hz), 4.30-4.73 (m, 4H), 4.93-
5.39 (m, 5H), 5.48 (d, 1H, J ) 2.9 Hz), 5.69 (d, 1H, J ) 7.0
Hz), 5.54-5.78 (m, 1H), 5.88 (m, 1H), 6.18 (bd, 1H), 6.30 (s,
1H), 6.33 (m, 1H), 7.05-7.78 (m, 20H), 7.82 (d, 2H, J ) 7.4
Hz), 8.16 (d, 2H, J ) 7.2 Hz) ppm; MS (FAB) m/e 1653 (M +
Na)+. Anal. C86H102N8O24 calculated C 62.61%, H 6.35%, N
6.79%, measured C 62.40%, H 6.31%, N 6.36%.
Dep r otection To Give P r od r u g 2′-[H-D-Ala -P h e-Lys-
P ABC-N(Me)-(CH2)2-N(Me)CO]-P a clita xel (26). To a solu-
tion of 83.0 mg (0.0509 mmol) protected prodrug 25 in dry THF
under an argon atmosphere was added glacial AcOH (12 µL,
4.0 equiv) together with tributyltin hydride (41 µL, 3.0 equiv)
and a catalytic amount of Pd(PPh3)4. After 30 min the product
was precipitated by addition of diethyl ether. The white
precipitate was collected by means of centrifugation and
washed several times with diethyl ether. tert-Butyl alcohol was
added and evaporated again to remove an excess of HCl, and
the resulting product was dissolved in water/dioxane and
freeze-dried, yielding 56 mg (70%) of prodrug 26. Mp 142 °C;
1H NMR (300 MHz, CDCl3) δ 1.13 (s, 3H), 1.21 (s, 3H), 1.26
(d, 3H, J ) 6.6 Hz), 1.05-2.00 (m, 6H), 1.69 (s, 3H), 2.00 (s,
3H), 2.22 (s, 3H), 2.58 (s, 3H), 2.89 (s, 3H), 2.91 (s, 3H), 2.67-
3.64 (m, 3H), 2.95 (m, 1H), 3.07 (m, 2H), 3.15 (m, 1H), 3.78
(m, 1H), 3.83 (d, 1H, J ) 7.1 Hz), 4.10-5.05 (m, 2H), 4.22 (d,
1H, J ) 8.4 Hz), 4.32 (d, 1H, J ) 8.4 Hz), 4.46 (m, 1H), 4.60
(m, 1H), 4.65 (d, 1H, J ) 12.3 Hz), 4.80 (d, 1H, J ) 12.4 Hz),
4.99 (bd, 5H, J ) 7.4 Hz), 5.47 (d, 1H, J ) 2.9 Hz), 5.68 (d,
1H, J ) 6.9 Hz), 6.17 (bd, 1H, J ) 2.9 Hz, J ) 9.6 Hz), 6.30 (s,
1H), 6.31 (m, 1H), 7.05-7.70 (m, 20H), 7.82 (d, 2H, J ) 7.5
Hz), 8.16 (d, 2H, J ) 7.2 Hz), 8.54 (d, 1H, J ) 9.6 Hz) ppm;
MS (FAB) m/e 1463 (M + H)+, 1485 (M + Na)+. Anal.
C85H97N7O22(‚3AcOH) calculated C 61.04%, H 6.50%, N 6.71%,
measured C 60.91%, H 6.45%, N 7.10%.
Sta bility of th e Dou ble Sp a cer -Con ta in in g P a clita xel
P r od r u gs 15 a n d 26 in Bu ffer . The prodrugs were incubated
at a concentration of 150 µM in 0.1 M Tris/HCl buffer (pH 7.3)
for 72 h at 37 °C and showed no formation of degradation
products (TLC, RP18; CH3CN/H2O/AcOH 19/19/2).
Sta bility of th e Dou ble a n d Tr ip le Sp a cer -Con ta in in g
Doxor u bicin P r od r u gs 13, 16, a n d 20 in Bu ffer . The
prodrugs were incubated at a concentration of 100-135 µM
in 0.1 M Tris/HCl buffer (pH 7.3) for 72 h at 37 °C and showed
no formation of degradation products (TLC, RP18; CH3CN/H2O/
AcOH 19/19/2).
Cytotoxicity. The antiproliferative effect of prodrugs and
parent drugs was determined in vitro applying seven well-
characterized human tumor cell lines and the microculture
sulforhodamine B (SRB) test. The antiproliferative effects were
determined and expressed as ID50 values (ng/mL), which are
the (pro)drug concentrations that gave 50% inhibition when
compared to control cell growth after 5 days of incubation. Cell
lines: MCF-7; breast cancer. EVSA-T; breast cancer. WIDR;
colon cancer. IGROV; ovarian cancer. M19; melanoma. A498;
renal cancer. H226; nonsmall cell lung cancer.
cell line
MCF-7 EVSA-T WIDR IGROV M19 A498 H226
prodrug 13 242
prodrug 15 11
546
5
627
5
896
22
302
7
2303
25
503
7
prodrug 16 466
prodrug 20 481
448
244
119
<3.2
8
445
568
117
<3.2
11
234
115
499
<3.2
60
162
198
96
<3.2
16
631
354
681
<3.2 <3.2
90 199
857
907
62
prodrug 26
paclitaxel
60
<3.2
doxorubicin
10
En zym a tic Hyd r olysis of th e Elon ga ted Sp a cer Sys-
tem -Con ta in in g P a clita xel P r od r u gs by P la sm in . Enzy-
matic hydrolysis of the paclitaxel prodrugs was investigated
by incubation at a prodrug 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) (0.3 U/mg) at 37 °C. Both
double spacer-containing paclitaxel prodrugs were converted
to yield the corresponding parent drug. Capillary electrophore-
sis was carried out with a CE Ext. Light Path Capillary (80.5
cm, 50 µm), with 1:1 MeOH/0.05 M sodium phosphate buf-
fer (pH 7.0) as eluent. Detection was performed at 200 and
254 nm.
En zym atic Hydr olysis of th e Elon gated Spacer System -
Con ta in in g Doxor u bicin P r od r u gs by P la sm in . Enzy-
matic hydrolysis of the doxorubicin prodrugs was investigated
by incubation in a 0.1 M Tris/HCl buffer (pH ) 7.3) in the
presence of 0.0025 U/mL human plasmin at 37 °C, on three
or four separate occasions. The prodrugs were incubated at
concentrations, yielding a maximal doxorubicin concentra-
tion of approximately 1 µM in a total volume of 2 mL. At
1, 5, 10, 20, and 40 min and 1, 2, 4, and 8 h after the start of
the incubation an aliquot of 100 µL was withdrawn and added
to 900 µL of ice-cold human plasma in a glass tube, to which
a volume of 0.5 mL of acetone was added. After vigorous
mixing, the samples were stored at -80 °C until analysis. A
preincubation sample was taken in the absence of human
plasmin.
Doxor u bicin An a lysis. The concentration of doxorubicin,
released from the prodrugs, was determined by a validated
reversed-phase high-performance liquid chromatographic as-
say published by de Bruijn et al.33 In brief, after addition of
100 µL of the internal standard daunorubicin in acetone and
100 µL of aqueous zinc sulfate (70%), the samples were
vigorously mixed in a vortex for 10 min. Subsequently, the
samples were centrifuged for 15 min at 4000g. The clear
supernatant was transferred to a clean glass tube and evapo-
rated under a gentle stream of nitrogen for 45 min at 60 °C.
Aliquots of 100 µL of the residue were injected onto a HPLC.
Doxorubicin and daunorubicin were separated isocratically at

8830 J . Org. Chem., Vol. 66, No. 26, 2001
de Groot et al.
50 °C on a stainless steel column of 150 × 4.6 mm, packed
with Inertsil ODS 80A material (5 µm particle size). The
mobile phase was composed of water (pH ) 2.0)/CH3CN/THF
(76:24:0.5, v/v/v). The mobile phase was delivered at a flow
rate of 1.25 mL/min, and the column effluent was monitored
fluorimetrically at excitation and emission wavelengths of 480
and 560 nm, respectively.
model based on the modified Hill equation, as follows: C )
Cmin + Cmax * [(Tγ)/(Tγ + T50γ)], in which Cmin is the concentra-
tion of doxorubicin in the sample in the absence of plasmin,
Cmax is the maximum reachable doxorubicin concentration, T
is the time in hours, T50 is the time in hours needed to reach
50% of the maximum reachable doxorubicin concentration and
γ is the Hill constant describing the sigmoidicity of the curve.
Da ta An a lysis of Doxor u bicin In cu ba tion Exp er i-
m en ts. The data were fitted to a sigmoidal maximum effect
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