Enhanced cytotoxicity of a polymer-drug conjugate with triple payload of paclitaxel

Article abstract of DOI:10.1016/j.bmc.2009.05.028

The development of targeting approaches to selectively release chemotherapeutic drugs into malignant tissue is a major challenge in anticancer therapy. We have synthesized an N-(2-hydroxypropyl)-methacrylamide (HPMA) copolymer-drug conjugate with an AB

Full text of DOI:10.1016/j.bmc.2009.05.028

Bioorganic & Medicinal Chemistry 17 (2009) 4327–4335
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Enhanced cytotoxicity of a polymer–drug conjugate with triple payload
of paclitaxel
Rotem Erez a, Ehud Segal b, Keren Miller b, Ronit Satchi-Fainaro b, Doron Shabat a,
*
a Department of Organic Chemistry, School of Chemistry, Raymond and Beverly Sackler Faculty of Exact Sciences, Tel Aviv University, Tel Aviv 69978, Israel
b Department of Physiology and Pharmacology, Sackler Faculty of Medical Sciences, Tel Aviv University, Tel Aviv 69978, Israel
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 29 January 2009
Revised 6 May 2009
Accepted 11 May 2009
Available online 18 May 2009
The development of targeting approaches to selectively release chemotherapeutic drugs into malignant
tissue is a major challenge in anticancer therapy. We have synthesized an N-(2-hydroxypropyl)-meth-
acrylamide (HPMA) copolymer–drug conjugate with an AB3 self-immolative dendritic linker. HPMA
copolymers are known to accumulate selectively in tumors. The water-soluble polymer–drug conjugate
was designed to release a triple payload of the hydrophobic drug paclitaxel as a result of cleavage by the
endogenous enzyme cathepsin B. The polymer–drug conjugate exhibited enhanced cytotoxicity on mur-
ine prostate adenocarcinoma (TRAMP C2) cells in comparison to a classic monomeric drug–polymer
conjugate.
Keywords:
Prodrug
Conjugate
HPMA copolymer
Self-immolative
Paclitaxel
Ó 2009 Elsevier Ltd. All rights reserved.
Polymer therapeutics
1. Introduction
adaptor that amplifies an enzymatic single cleavage event into
the release of three reporter units.7–9 A compound constructed of
Selective chemotherapy remains a key issue for successful
treatment of cancer. The use of targeting approaches and selective
release of chemotherapeutic drugs in the malignant tissue is con-
sequently needed.1 We and two other groups have recently re-
ported the design and synthesis of dendrimers that disassemble
into their building blocks in a self-immolative manner to release
the tail-group units after fragmentation has been initiated by a
triggering reaction.2–4 The unique disassembly of these molecules
was harnessed for the construction of self-immolative dendritic
prodrugs with single or multiple triggering modes of activation.5,6
The basic building block of a first-generation dendrimer is usually
referred to as an ABn unit, in which A is the head and B is the tail.
We have developed an efficient AB3 self-immolative dendritic
an AB3 dendritic system bearing three hydrophobic drug molecules
as tail-groups is not soluble under aqueous conditions and, there-
fore, cannot function as a drug delivery system. However, when
conjugated to the water-soluble N-(2-hydroxypropyl)-methacryla-
mide (HPMA) copolymer via an enzyme-cleavable linker, the
hydrophobic dendritic platform becomes a water-soluble drug
delivery system (Fig. 1, conjugate 1a). Here we report the synthe-
sis, characterization, and in vitro evaluation of an AB3 dendritic
paclitaxel prodrug conjugated with HPMA copolymer.
2. Results and discussion
2.1. Design of AB3 dendritic-prodrug conjugate
HPMA copolymers are water-soluble, biocompatible, non-
immunogenic, and non-toxic carriers that enable specific delivery
into tumor tissue.10–12 These macromolecules do not diffuse
through normal blood vessels but rather accumulate selectively
in tumors due to the enhanced permeability and retention (EPR)
effect.13 This phenomenon of passive diffusion through the hyper-
permeable neovasculature and localization in the tumor interstit-
ium is observed for this type of macromolecular agents and for
lipids.14 Furthermore, conjugation to HPMA copolymer should re-
strict the passage of drug through the blood brain barrier. It should
also prolong the circulating half-life of the conjugated drug
Abbreviations: AcOH, acetic acid; Alloc, allyl alcohol; Boc, butyl-O-carbonyl;
Bu3SnH, tributyltinhydride; DCM, dichloromethane; DIPEA, diisopropylethylene-
amine; DMAP, dimethylaminopyridine; DMF, dimethyl formamide; Et3N, triethyl-
amine; EtOAc, ethylacetate; Gly, glycine; Hex, hexane; HPMA, N-(2-
hydroxypropyl)methacrylamide; Leu, leucine; Lys, lysine; MeOH, methanol; NaOH,
sodium hydroxide; NH4Cl, ammonium chloride; NMM, N-methylmorpholine; ONp,
O-nitrophenyl; PABC, p-aminobenzyl carbonate; PABOH, p-aminobenzyl alcohol;
Pd(PPh3)4, tetrakis(triphenylphosphine)palladium; Phe, phenylalanine; PNP, p-
nitrophenyl; PTX, paclitaxel; TFA, trifluoroacetic acid; THF, tetrahydrofuran; TLC,
thin layer chromatography; TRAMP, transgenic adenocarcinoma of the mouse
prostate.
* Corresponding author. Tel.: +972 (0) 3 640 8340; fax: +972 (0) 3 640 9293.
E-mail address: chdoron@post.tau.ac.il (D. Shabat).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.05.028

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R. Erez et al. / Bioorg. Med. Chem. 17 (2009) 4327–4335
O
O
O
DRUG
O
H
N
H
HPMA copolymer
Enzyme substrate
N
DRUG
O
O
O
O
O
O
O
DRUG
1a
n
Figure 1. Schematic structure of an AB3 dendritic prodrug conjugated with HPMA copolymer via an enzyme-cleavable linker.
compared to that of the free drug, hence more effectively inhibiting
the growth of tumor endothelial and tumor epithelial cells.15–18
The taxane paclitaxel (PTX) is a potent anti-neoplastic agent.
PTX is highly effective as anti-neoplastic in a monotherapy dosing
schedule and in combination therapy for the treatment of meta-
static prostate and breast cancers.19–21 The primary mode of action
of PTX is to promote and stabilize microtubulin assembly; this
inhibition of microtubule dynamics19,22,23 causes impaired mitosis,
leading to cell cycle arrest and finally to apoptosis. Despite its
strong anticancer activity, PTX is poorly water-soluble and has
serious dose-limiting toxicities and causes hypersensitivity reac-
tions in its commercial formulation. The side effects originate from
the formulating vehicle, a polyethoxylated castor oil known as
cremophor EL, and the absence of selectivity for target tissue.24
Therefore, we sought to construct a water-soluble, copolymer-
based dendritic prodrug system for tissue-specific delivery of PTX
based on the design presented in Figure 1.
Conjugate 1 (Fig. 2) is constructed of HPMA copolymer linked to
an AB3 self-immolative dendritic system with three hydrophobic
PTX molecules as tail groups. The enzymatic substrate linker was
designed to be cleaved by the lysosomal enzyme cathepsin B.
Cleavage should occur upon internalization of the conjugate in
cells and will result in release of three PTX molecules per
dendrimer.
phenylalanine-lysine-p-aminobenzyl
linker
(Phe-Lys-PABC).
Cleavage at this linker by the lysosomal enzyme cathepsin B
will result in disassembly (Fig. 3). The six-amino-acid peptide
was used in order to provide convenient conjugation chemis-
try, a longer spacer, and higher probability of cleavage. Enzy-
matic cleavage at the Phe-Lys will trigger disassembly of
conjugate 1. The free amine intermediate (PABC-dendrimer)
will undergo 1,6-elimination and decarboxylation, followed by
triple elimination to release the three PTX molecules from each
dendritic platform.
2.2. Synthesis of copolymer-dendritic-prodrug conjugate 1
The synthesis of conjugate 1 is outlined in Figure 4. Previously
synthesized L L-Lys(alloc)-OH to
-Boc-Phe-ONp25 was reacted with
give dipeptide 3. The latter was conjugated with 4-aminobenzyl
alcohol to generate alcohol 4. Isocyanate 58 was reacted with ben-
zyl alcohol 4 to give carbamate 6, followed by deprotection with
amberlyst 15 to generate triol 7. Activation with 4-nitrophenyl
chloroformate afforded tricarbonate 8, which was reacted with
three equivalents of PTX to yield compound 9. Deprotection of
the terminal Boc group with TFA allowed the conjugation with
HPMA copolymer-Gly-Phe-Leu-Gly-ONp. The remaining activated
sites of the copolymer were conjugated with excess aminoethanol
to generate compound 10. Deprotection of the Lys alloc residue of
10 afforded the desired conjugate 1.
The dendritic platform is attached to the HPMA copolymer-
Gly-Phe-Leu-Gly via
a
carbamate linkage to the dipeptide
Paclitaxel
O
O
O
HO
O
O
O
O
N
O
OH
O
H
H
O
O
O
H
O
OH
O
O
O
O
O
O
HO
O
Paclitaxel
O
O
O
O
O
O
O
O
X=90%
Y=2.7%
Gly-Phe-Leu-Gly
O
Phe-Lys
HN
PABC
HN
Y
O
O
O
Ph
O
O
O
O
O
H
H
H
H3C
N
N
N
N
N
N
H
N
H
H
H
O
O
O
CH2
X
O
O
OH
NH3
HPMA copolymer
O
O
H
O
H
O
C
O
N
H2
C
H
H
H3C
N
C
CH3
O
O
OH
HO
O
O
CH2
O
Paclitaxel
1
Figure 2. Chemical structure of HPMA copolymer AB3 dendritic-prodrug conjugate 1. The dipeptide linker (blue) is a substrate for cathepsin B. All amino acids used have
natural -configuration.
L

R. Erez et al. / Bioorg. Med. Chem. 17 (2009) 4327–4335
4329
O
OH
O
O
O
H
O
O
O
O
HO
O
H2N
O
O
O
HN
O
O
O
O
O
O
O
HO
HO
O
NH
O
O
O
O
O
H
OH
O
H
O
O
O
H
O
3 X
N
O
Cathepsin B
37oC, pH 5.5
O
H2O
O
OH
O
O
O
O
Conjugate 1
OH
O
O
O
O
O
O
O
O
NH2
O
O
HO
Paclitaxel
CO2
HO
OH
+ PABOH
+
O
HPMA copolymer
O
NH
H
O
H
N
+
HO
O
O
Phe-Lys
OH
O
O
O
PABC-dendrimer
Figure 3. Disassembly mechanism of conjugate 1.
The loading of conjugate 1 with drug was determined by UV
spectroscopy; the PTX-chromophore absorbs at a wavelength of
254 nm. The spectroscopic measurements indicated that at maxi-
mum loading, about 15 units of PTX (5 of AB3 units) were attached
per molecule of HPMA copolymer.
Figure 8. Twenty-four hours post-treatment, proliferation of
TRAMP C2 cells was inhibited by free PTX, conjugate 1, and conju-
gate 2 (at PTX-equivalent concentrations) with IC50s of 2, 10, and
>10 lM, respectively (Fig. 8a). The control conjugate bearing the
non-cleavable (Gly)4-PABC linker, exhibited less than 10% inhibi-
tion at all concentrations (Fig. 8a). Forty-eight hours post-treat-
ment, the differences among the inhibitory concentrations of
PTX, conjugate 1, and conjugate 2 were reduced significantly:
IC50s were 200, 300, and 350 nM, respectively (Fig. 8b). Although
the trends were similar to those observed at the 24 h time point,
the lower IC50 was comparable amongst the different treatments
due to the longer exposure of the cells to PTX. In contrast, at
48 h, the non-cleavable conjugate had an IC50 of greater than
2.3. Synthesis of copolymer–drug conjugate 2
To enable comparison of the activity of the dendritic-polymeric
conjugate to that of a classic copolymer–drug conjugate, we syn-
thesized conjugate 2 bearing one unit of PTX per polymer conjuga-
tion site (Fig. 5). PTX was conjugated to HPMA copolymer-Gly-Phe-
Leu-Gly through a carbonate linkage with the dipeptide Phe-Lys-
PABC as previously described.26 Like the release mechanism of con-
jugate 1, cleavage by cathepsin B will trigger the disassembly of
conjugate 2 through spontaneous 1,6-benzyl elimination and
decarboxylation, leading to the release of free PTX (Fig. 5).
The synthesis of conjugate 2 was achieved as presented in
Figure 6. Activation of alcohol 4 with 4-nitrophenyl chlorofor-
mate gave carbonate 11, which was reacted with PTX to yield
compound 12. Deprotection of Boc-Phe-Lys(alloc)-PABC-PTX 12
allowed the conjugation with HPMA copolymer-Gly-Phe-Leu-
Gly-ONp. The remaining activated sites of the copolymer were
conjugated with excess aminoethanol to generate compound
13. Deprotection of the Lys alloc residue of 13 afforded the de-
sired conjugate 2. The loading of PTX molecules in conjugate 2
was determined using UV spectroscopy via the chromophore of
PTX at a wavelength of 254 nm. The UV measurements were
performed in was water (10% acetonitrile as a cosolvent for
PTX). About seven units of PTX were attached per molecule of
HPMA copolymer.
10 lM (Fig. 8b); the minor growth inhibition observed may be
due to a small amount of spontaneous hydrolysis of the carbonate
linkage between PTX and the PABC linker.
The AB3 dendritic prodrug system used to link PTX to the water-
soluble HPMA copolymer fulfills two major functions. First, it in-
creases the loading of drug units onto the polymeric vehicle. The
dendritic linker allowed conjugation of 15 PTX-molecules per poly-
mer, whereas the monomeric linker permitted conjugation of only
seven PTX molecules. Second, the self-immolative dendron ampli-
fies a single cleavage event through a rapid triple-elimination reac-
tion to release the active PTX molecules. The effect of these two
functions was reflected by the IC50s in cytotoxicity assays compar-
ing the dendritic conjugate 1 and to the classic conjugate 2. After
24 h, conjugate 1 was 10-fold more toxic than conjugate 2 in a
standard cell-growth inhibition assay (IC30 of conjugate 1 was
1000 nM compared with IC30 of conjugate 2 of 10,000 nM, i.e., 1-
order of magnitude higher).
3. Conclusions
2.4. Cell-growth inhibition assays
In summary, we have synthesized a new HPMA copolymer con-
jugate with an AB3 dendritic linker bearing three molecules of the
highly hydrophobic drug paclitaxel. The water-soluble copolymer–
drug conjugate was designed for activation by cathepsin B and re-
leased a triple payload of PTX from each cleavage site. The AB3 self-
immolative dendritic linker acts as a molecular amplifier. This
strategy increased the loading capacity of drug molecules per a
polymer molecule to more than twice that of the classic conjugate.
The difference in cytotoxicity between the trimeric drug–copoly-
To evaluate the ability of dendritic conjugate 1 and conjugate 2
to inhibit cell proliferation in the presence of cathepsin B, we per-
formed a standard cell-growth inhibition assay using TRAMP C2
cells. In order to prove that the two conjugates are active only upon
the cleavage by cathepsin B, we synthesized a control conjugate
bearing the non-cleavable linker Gly-Gly-Gly-Gly-PABC (Fig. 7).
TRAMP C2 cells were challenged for 24 h or 48 h with free PTX,
conjugate 1, conjugate 2, or the control conjugate over a range of
concentrations. Cell viability was measured using colorimetric as-
say based on the tetrazolium salt XTT. The data are presented in
mer conjugate and the monomeric one was reflected in
a
cell-growth inhibition assay. Our new dendritic drug–copolymer

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R. Erez et al. / Bioorg. Med. Chem. 17 (2009) 4327–4335
O
OTBS
O
NH
O
O
O
O
H
H
N
OCN
N
OTBS
O
N
OH
O
N
H
N
H
H
H2N
OH
O
O
5
OTBS
52%
O
H3N COO
83%
OH
O
NO2
O
N
H
O
HN
O
HN
O
82%
3
4
O
O
O
O
O
O
4-Nitrophenyl
chloroformate
H
H
OTBS
OH
OH
Amberlyst 15
71%
N
N
O
N
N
O
O
N
H
N
H
O
H
H
O
O
O
O
HN
HN
OTBS
OH
56%
HN
O
HN
O
OTBS
O
O
6
7
O
O
O
OH
O
O
N
O
H
O
H
O
HO
O
O
O
O
O
O
NO2
O
O
O
O
O
O
O
O
O
H
H
PTX
92%
N
N
O
N
N
H
O
N
H
N
H
H
O
O
O
O
O
HN
O
HN
O
HN
O
O
O
O
O
NO2
O
NH
O
NH
O
O
O
O
O
O
O
O
O
NO2
8
9
HO
O
O
O
O
O
HO
O
O
H
O
O
O
O
OH
H
O
H
N
O
O
CH3
CH3
H2
C
H2
C
O
O
X=90%
Y=2.7%
OH
O
O
X
Y
C
O
O
O
HN
NH
CH2
O
CHOH
CH3
NH
O
HN
O
NH
O
OH
O
O
O
O
H
O
HN
O
O
O
HN
O
O
O
HO
NH
O
O
AcOH
Bu SnH
HN
O
O
1) TFA
2) HPMA-ONp
3
Pd(PPh )
3 4
1
O
HN
O
3) Aminoethanol
O
O
HO
O
NH
O
O
O
H
O
O
O
O
O
O
O
O
OH
O
O
O
O
O
O
O
O
O
HO
O
O
NH
H
O
H
N
O
O
OH
O
O
10
O
Figure 4. Synthesis of dendritic-polymeric conjugate 1.
conjugate system may offer significant advantages in inhibition of
tumor growth relative to monomeric drug–polymer conjugates,
especially if the targeted or secreted enzyme that initiates drug re-
lease is present at relatively low levels in the malignant tissue.

R. Erez et al. / Bioorg. Med. Chem. 17 (2009) 4327–4335
4331
HPMA copolymer
CH
3
CH
3
H C
2
H C
2
H N
2
X=90%
Y
X
C
O
O
Y=8%
HN
NH
CH
2
O
CHOH
CH
O
NH
O
O
O
HO
3
Gly-Phe-Leu-Gly
Cathepsin B
O
O
O
O
O
O
H
o
O
H
N
37 C, pH 5.5
HN
O
O
O
NH
OH
O
O
HPMA copolymer
O
+
HN
HN
PABC-Paclitaxel
Phe-Lys
O
Cathepsin B
Phe-Lys
PABC
NH
H O
2
O
CO
2
H N
3
Cathepsin B
O
O
O
O
O
O
O
HO
O
HO
O
O
H
O
O
H
N
O
OH
H
+ PABOH
O
H
N
O
O
O
OH
O
O
O
O
OH
O
O
O
O
Paclitaxel
2
Paclitaxel
Figure 5. Disassembly mechanism of conjugate 2.
0.5:10:89.5). Upon completion of the reaction the solvent was re-
moved under reduced pressure and the crude product was purified
using column chromatography on silica gel (AcOH/MeOH/EtOAc,
0.5:10:89.5) to give compound 3 (107 mg, 83%) as a white solid.
1H NMR (200 MHz, CDCl3): d = 7.24–7.11 (5H, m); 5.83 (1H, m);
5.45–5.11 (3H, m); 4.50–4.48 (3H, m); 3.08–2.92 (4H, m); 1.84–
1.64 (2H, m); 1.44–1.36 (4H, m); 1.30 (9H, s). 13C NMR
(100 MHz, CDCl3): d = 177.09, 164.91, 158.52, 157.61, 138.45,
134.82, 131.26, 130.50, 128.83, 120.24, 82.27, 67.51, 57.59, 53.98,
42.37, 38.60, 33.53, 33.44, 30.14, 22.63. MS (FAB): m/z: 478.3
[M+H]+, 500.3 [M+Na]+.
4. Experimental
4.1. General
HPMA copolymer-Gly-Gly-p-nitrophenol (ONp) incorporating
5 mol % of the methacryloyl-Gly-Gly-p-nitrophenol ester monomer
units, and HPMA copolymer-Gly-Phe-Leu-Gly-ONp incorporating
10 mol % of the methacryloyl-Gly-Phe-Leu-Gly-p-nitrophenol ester
monomer units were obtained from Polymer Laboratories (Church
Stretton, UK). The HPMA copolymer-GFLG-ONp has a molecular
weight of 31,600 Da and a polydispersity of 1.66.
All reactions requiring anhydrous conditions were performed
under an Ar or N2 atmosphere. Chemicals and solvents were either
A.R. grade or purified by standard techniques. All reagents, includ-
ing salts and solvents, were purchased from Sigma–Aldrich. Thin
layer chromatography (TLC) silica gel plates (Merck 60 F254) were
visualized by irradiation with UV light and/or by treatment with
a solution of phosphomolybdic acid (20 wt % in ethanol) followed
by heating. Flash chromatography (FC) was performed on silica
gel (Merck 60, particle size 0.040–0.063 mm); eluent is given in
parentheses in sections on individual compounds. 1H NMR was
performed on either a Bruker AMX 200 or 400 instrument. The
chemical shifts are expressed in d relative to TMS (d = 0 ppm) and
the coupling constants J in hertz. The spectra were recorded in
CDCl3 or MeOD at room temperature. The 400 Mesh copper grid
was obtained from SPI Supplies.
4.1.3. Compound 4
Compound 3 (832.1 mg, 1.74 mmol) was dissolved in dry THF
and the solution was cooled to À15 °C. NMM (0.19 mL,
1.74 mmol) and isobutyl chloroformate (0.27 mL, 2.09 mmol)
were added. The reaction was stirred for 20 min and a solution
of 4-aminobenzyl alcohol (321.85 mg, 2.61 mmol) in dry THF
was added. The reaction mixture was stirred for 2 h with pro-
gress monitored by TLC (100% EtOAc). Upon completion of the
reaction, the solvent was removed under reduced pressure and
the crude product was purified by using column chromatography
on silica gel (100% EtOAc) to give compound 4 (835 mg, 82%) as
a yellow solid.
1H NMR (200 MHz, MeOD): d = 7.56 (2H, d, J = 8 Hz); 7.29 (2H, d,
J = 8 Hz); 7.21–7.07 (5H, m); 5.86 (1H, m); 5.29–5.10 (2H, m); 4.83
(2H, s); 4.49–4.46 (4H, m); 3.17–3.08 (4H, m); 1.88–1.70 (2H, m);
1.44 (4H, m); 1.34 (9H, s). 13C NMR (100 MHz, CDCl3): d = 174.08,
171.45, 158.61, 157.79, 139.17, 138.87, 138.03, 134.79, 131.12,
130.71, 130.49, 129.90, 129.08, 122.15, 82.71, 67.48, 66.83, 58.08,
55.74, 42.09, 39.84, 32.70, 31.31, 30.16, 24.31. MS (FAB): m/z:
583.3 [M+H]+, 605.3 [M+Na]+.
4.1.1. L-Boc-Phe-ONp
L
-Boc-Phe-ONp was synthesized according to the procedure de-
scribed previously.25
4.1.2. Compound 3
L
-Boc-Phe-ONp (104.3 mg, 0.27 mmol) was dissolved in 2 mL
DMF. Commercially available -Lys(alloc)-OH (62 mg, 0.27 mmol)
and Et3N (100 L) were added. The reaction mixture was stirred
for 12 h and was monitored by TLC (AcOH/MeOH/EtOAc,
4.1.4. Compound 5
L
Compound 5 was synthesized according to the procedure de-
l
scribed previously.8

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R. Erez et al. / Bioorg. Med. Chem. 17 (2009) 4327–4335
O
O
HN
O
O
O
H
NH
N
O
N
H
N
O
O
H
O
4-Nitrophenyl
chloroformate
O
O
HN
O
HN
PTX
94%
NO
2
4
O
HN
O
79%
O
O
O
O
O
O
11
HO
O
O
O
H
O
H
N
O
O
OH
O
O
CH
CH
3
3
H
C
H
C
2
2
O
12
X=90%
Y=8%
X
Y
C
O
O
O
O
HN
NH
CH
2
O
O
CHOH
CH
NH
3
HN
AcOH
Bu3SnH
Pd(PPh3)4
NH
1) TFA
2) HPMA-ONp
2
3) Aminoethanol
HN
O
NH
O
HN
O
HN
O
O
O
O
O
HO
O
O
O
H
O
H
N
O
O
O
OH
O
O
O
13
Figure 6. Synthesis of monomeric drug–copolymer conjugate 2.
CH
3
CH
by TLC (EtOAc/Hex, 1:1). Upon completion of the reaction, the sol-
vent was removed under reduced pressure. The crude product was
purified by using column chromatography on silica gel (EtOAc/Hex,
1:1) to give compound 6 (196.7 mg, 52%) as a yellow solid.
1H NMR (200 MHz, CDCl3): d = 7.58 (2H, d, J = 8 Hz); 7.35–7.14
(9H, m); 5.87 (1H, m); 5.34 (1H, m); 5.24 (2H, m); 5.18 (1H, m);
4.86 (1H, m); 4.70–4.67 (8H, m); 4.56 (1H, m); 3.19–3.06 (4H,
m); 1.65–1.47 (6H, m); 1.25 (9H, s); 0.89 (27H, s); 0.05 (18H, s).
13C NMR (100 MHz, CDCl3): d = 176.2, 158.4, 151.7, 144.1, 139.8,
137.3, 136.5, 134.7, 130.5, 128.7, 128.4, 127.3, 126.4, 125.9,
124.7, 120.3, 116.4, 78.2, 76.5, 73.8, 73.6, 62.1, 54.7, 54.5, 40.5,
38.5, 31.0, 30.7, 25.1, 21.6, 20.3, 18.2, À6.2. MS (FAB): m/z:
1156.4 [M+Na]+.
3
H C
2
H C
2
X
X=95
Y=3
Y
C
O
O
O
O
HN
NH
CH
2
O
CHOH
CH
NH
NH
3
HN
HN
O
O
O
O
O
O
HO
O
4.1.6. Compound 7
O
H
O
H
N
O
Compound 6 (196.7 mg, 0.44 mmol) was dissolved in 6 mL
MeOH and Amberlyst-15 was added. The reaction was stirred in
room temperature for 1.5 h and progress was monitored by TLC
(EtOAc/MeOH, 95:5). Upon completion of the reaction, Amber-
lyst-15 was filtered out and the solvent was removed under re-
duced pressure. The crude product was purified by using column
chromatography on silica gel (EtOAc/MeOH 95:5) to give com-
pound 7 (97.7 mg, 71%) as a white solid.
O
O
OH
O
O
O
Figure 7. Structure of the non-cleavable HPMA copolymer-Gly-Gly-Gly-Gly-PABC-
PTX conjugate.26
4.1.5. Compound 6
1H NMR (200 MHz, MeOD): d = 7.57 (2H, m); 7.43–7.38 (4H, m);
7.23–7.15 (5H, m); 5.86 (1H, m); 5.30 (1H, m); 5.24 (2H, m); 5.12
(1H, m); 4.87–4.63 (9H, m); 4.52 (1H, m); 3.15–2.95 (4H, m); 1.65–
1.40 (6H, m); 1.28 (9H, s). 13C NMR (100 MHz, MeOD): d = 172.3,
A solution of compound 4 (235.6 mg, 0.40 mmol) in 3 mL THF,
lL NEt3 was added to the isocyanate residue 5 (185.74 mg,
0.33 mmol). The reaction was stirred for 24 h and was monitored
100

R. Erez et al. / Bioorg. Med. Chem. 17 (2009) 4327–4335
4333
172.0, 157.3, 156.4, 155.4, 144.2, 138.4, 138.1, 137.9, 136.4, 136.2,
130.1, 128.4, 128.1, 127.6, 126.3, 125.9, 118.2, 117.1, 79.4, 67.3,
67.0, 66.7, 58.6, 55.1, 54.1, 42.3, 37.2, 31.1, 28.7, 28.2, 22.4. MS
(FAB): m/z: 814.3 [M+Na]+.
1H NMR (200 MHz, CDCl3): d = 8.29–8.27 (6H, m); 7.58 (2H, m);
7.37 (6H, m); 7.24–7.14 (9H, m); 5.86 (1H, m); 5.34–5.30 (7H, s);
5.24 (2H, m); 5.18 (1H, m); 4.56–4.28 (4H, m); 3.17–3.14 (4H,
m); 1.76–1.33 (6H, m); 1.28 (9H, s). 13C NMR (100 MHz, CDCl3):
d = 172.0, 169.3, 156.61, 155.1, 154.4, 152.3, 145.5, 138.2, 135.8,
134.6, 132.7, 130.7, 129.0, 128.7, 127.1, 125.2, 121.6, 119.9,
117.5, 80.8, 69.5, 67.0, 65.4, 56.2, 53.8, 30.1, 29.4, 28.1, 22.3. MS
(FAB): m/z: 1287.0 [M+H]+, 1309.0 [M+Na]+.
4.1.7. Compound 8
Compound 7 (97.7 mg, 0.12 mmol) was dissolved in 3 mL dry
THF and a catalytic amount of pyridine (10 lL) was added. The
solution was cooled to À20 °C and a solution of PNP-chloroformate
(372.9 mg, 1.85 mmol) in 2 mL of dry THF was added dropwise.
The temperature was not allowed to exceed À10 °C. The mixture
was stirred for 3 h and reaction progress was monitored by HPLC
and by TLC (EtOAc/Hex, 2:1). Upon completion, the reaction was
diluted with EtOAc and was washed with saturated NH4Cl solution.
The organic layer was dried over magnesium sulfate and the
solvent was removed under reduced pressure. The crude product
was purified by using column chromatography on silica
gel (EtOAc/Hex, 2:1) to give compound 8 (88 mg, 56%) as a white
solid.
4.1.8. Compound 9
Compound 8 (22 mg, 17.09
Paclitaxel (52.60 mg, 61.55
l
mol) was dissolved in dry DCM.
lmol) and DMAP (7.52 mg,
61.55 lmol) were added. The reaction mixture was stirred for
24 h and progress was monitored by TLC (EtOAc/Hex, 8:1). Upon
completion of the reaction, the solvent was removed under re-
duced pressure and the crude product was purified by using col-
umn chromatography on silica gel (EtOAc/Hex, 8:1) to give
compound 9 (54.6 mg, 92%) as a white solid. MS (FAB): m/z:
3432.3 [M]+, 3433.1 [M+H]+, 3455.3 [M+Na]+.
4.1.9. Compound 10
4.1.10. Compound 1
Compound 9 (51.1 mg, 14.67
l
mol) was dissolved in 1.8 mL TFA
Compound 10 (71.4 mg, 23.8
(1.5 mL). Acetic acid (14.01 L, 118.9
142.62 mol), and a catalytic amount of Pd(PPh3)4 were added.
The reaction mixture was stirred for 2 h and was then
concentrated under reduced pressure, followed by addition of
10 mL of acetone. The precipitate was filtered out and was
washed with acetone several times. The crude product was
purified by using Sephadex LH20 column chromatography
(100% MeOH, 1 mL/min) to give compound 1 (26.9 mg) as a
white solid.
l
mol) was dissolved in DMF
and stirred for 2 min at 0 °C. The excess acid was removed under
reduced pressure and the crude amine salt was dissolved in 2 mL
DMF.HPMAcopolymer-Gly-Phe-Leu-Gly-ONp(44 mg,ONp = 14.67 l-
l
l
mol), Bu3SnH (76.40 L,
l
l
mol) was added followed by the addition of Et3N (100 lL). The
reaction mixture was stirred for 12 h and then excess aminoetha-
nol was added. Most of the solvent was removed under reduced
pressure, followed by addition of 10 mL of acetone. The precipitate
was filtered out and was washed with acetone several times to give
compound 10 (71.4 mg) as a white solid.

4334
R. Erez et al. / Bioorg. Med. Chem. 17 (2009) 4327–4335
154.36, 147.32, 140.70, 137.91, 134.76, 131.75, 131.53, 131.08,
130.78, 129.17, 127.21, 123.72, 122.05, 119.61, 82.86, 72.65,
67.50, 58.17, 55.84, 41.95, 39.71, 32.42, 31.40, 30.16, 24.27. MS
(FAB): m/z: 770.4 [M+Na]+.
4.1.12. Compound 12
Compound 11 (360.3 mg, 0.48 mmol) was dissolved in dry
DCM. Paclitaxel (494.06 mg, 0.57 mmol) and DMAP (70.61 mg,
0.57 mmol) were added. The reaction mixture was stirred for 8 h
and progress was monitored by TLC (100% EtOAc). Upon comple-
tion of the reaction, the solvent was removed under reduced pres-
sure and the crude product was purified by using column
chromatography on silica gel (100% EtOAc) to give compound 12
(662 mg, 94%) as a white solid. MS (FAB): m/z: 1463.7 [M]+,
1486.9 [M+Na]+.
4.1.13. Compound 13
Compound 12 (29.36 mg, 20.06 lmol) was dissolved in 1.5 mL
TFA and stirred for 2 min at 0 °C. The excess acid was removed un-
der reduced pressure and the crude amine salt was dissolved in
2 mL DMF. HPMA copolymer-Gly-Phe-Leu-Gly-ONp (60.2 mg,
ONp = 20.06
lmol) was added, followed by the addition of Et3N
(100 L). The reaction mixture was stirred for 12 h and then excess
l
aminoethanol was added. Most of the solvent was removed under
reduced pressure, followed by addition of 10 mL of acetone. The
precipitate was filtered out and was washed with acetone several
times to give compound 13 (74.8 mg) as a white solid.
4.1.14. Compound 2
Compound 13 (74.8 mg, 24.9
(1.5 mL). Acetic acid (14.67 L, 124.5
149.4 mol), and a catalytic amount of Pd(PPh3)4 were added.
l
mol) was dissolved in DMF
l
l
mol), Bu3SnH (80.03 L,
l
l
The reaction mixture was stirred for 2 h and was then concentrated
under reduced pressure, followed by addition of 10 mL of acetone.
The precipitate was filtered out and was washed with acetone sev-
eral times. The crude product was purified by using Sephadex LH20
column chromatography (100% MeOH, 1 mL/min) to give com-
pound 2 (33.2 mg) as a white solid.
Figure 8. Inhibition of proliferation of TRAMP C2 cells by conjugate 1 and conjugate
2. (a) At 24 h post-treatment, free PTX (closed squares), conjugate
triangles), and conjugate 2 (closed circles) inhibited the proliferation of TRAMP C2
cells with IC50s of 2, 10, and >10 M, respectively. The conjugate bearing the non-
1 (closed
4.2. Cell-growth inhibition assays
l
cleavable (Gly)4-PABC linker (open circles) showed modest growth inhibition of less
than 10% inhibition at all concentrations. (b) At 48 h post-treatment, free PTX
(closed squares), conjugate 1 (closed triangles), and conjugate 2 (closed circles) had
IC50s of 200, 300, and 350 nM, respectively, whereas the non-cleavable conjugate
TRAMP C2 cells were kindly provided by Prof. Norman
Greenberg (Fred Hutchinson Cancer Research Center, Seattle,
Washington, USA). Cells were cultured in complete growth med-
ium: Dulbecco’s modified Eagle’s medium (DMEM) supplemented
had an IC50 of >10
logarithmic scale.
lM. Data represent mean SD. X axis values are presented at a
with 10% fetal bovine serum (FBS), 100
mL streptomycin 12.5 U/mL nystatin, 1
1.5 g/L sodium bicarbonate, 100 mM androstan, and 2 mM
l
g/mL penicillin, 100 U/
M sodium pyruvate,
-gluta-
l
L
4.1.11. Compound 11
mine (Biological Industries, Israel). Cells were grown at 37 °C in 5%
CO2.
Compound 4 (353.6 mg, 0.60 mmol) was dissolved in dry THF
and the solution was cooled to 0 °C. DIPEA (0.42 mL, 2.42 mmol),
PNP-chloroformate (367 mg, 1.82 mmol), and a catalytic amount
of pyridine were added. The reaction was stirred for 2 h and pro-
gress was monitored by TLC (EtOAc/Hex, 3:1). Upon completion
of the reaction, the solvent was removed under reduced pressure.
The crude product was diluted with EtOAc and washed with satu-
rated NH4Cl. The organic layer was dried over magnesium sulfate
and the solvent was removed under reduced pressure. The crude
product was purified by using column chromatography on silica
gel (EtOAc/Hex, 3:1) to give compound 11 (453.2 mg, 79%) as a
white solid.
TRAMP C2 cells were plated at 2000 cells/well in DMEM supple-
mented with 5% FBS and incubated for 24 h (37 °C; 5% CO2). The
medium was then replaced with complete growth medium. Cells
were challenged with free PTX, compound 1, compound 2, or the
non-cleavable HPMA copolymer-(Gly)4-PABC-PTX over a range
of concentrations (1–10,000 nM paclitaxel-equivalent concentra-
tions) and incubated for 24 h or 48 h. Cell viability was measured
using XTT reagent. Absorbance was measured at a wavelength of
450–500 nm. A wavelength of 630–690 nm was also recorded.
4.3. Non-cleavable HPMA copolymer-Gly-Gly-Gly-Gly-PABC-PTX
1H NMR (200 MHz, CDCl3): d = 8.26 (2H, d, J = 8 Hz); 7.64 (2H, d,
J = 8 Hz); 7.40–7.34 (4H, m); 7.22–7.14 (5H, m); 5.83 (1H, m); 5.24
(2H, s); 5.18–5.06 (2H, m); 4.56–4.37 (4H, m); 3.19–3.05 (4H, m);
1.95–1.73 (2H, m); 1.59–1.46 (4H, m); 1.39 (9H, s). 13C NMR
(100 MHz, CDCl3): d = 174.11, 171.51, 158.63, 157.60, 157.46,
The non-cleavable conjugate was prepared by conjugation of
HPMA copolymer-Gly-Gly-ONp with Gly-Gly-PABC-PTX. Use of
the Gly-Gly rather than Phe-Lys dipeptide resulted in a conjugate
that could not be cleaved by cathepsin B.

R. Erez et al. / Bioorg. Med. Chem. 17 (2009) 4327–4335
12. Satchi, R.; Connors, T. A.; Duncan, R. Br. J. Cancer 2001, 85, 1070.
4335
References and notes
13. Maeda, H.; Wu, J.; Sawa, T.; Matsumura, Y.; Hori, K. J. Controlled Rel. 2000, 65,
271.
14. Takatsuka, S.; Yamada, N.; Sawada, T.; Ogawa, Y.; Maeda, K.; Ohira, M.;
Ishikawa, T.; Nishino, H.; Seki, S.; Hirakawa, Y. S. C. K. Int. J. Oncol. 2000, 17,
253.
15. Satchi-Fainaro, R. J. Drug Target 2002, 10, 529.
16. Duncan, R. Biochem. Soc. Trans. 2007, 35, 56.
17. Miller, K.; Satchi-Fainaro, R. In Wiley Encyclopedia of Chemical Biology; Civjan, N.
R., Ed.; John Wiley & Sons, 2009; Vol. 3, pp. 783-799.
18. Duncan, R. Nat. Rev. Cancer 2006, 6, 688.
19. Herbst, R. S.; Khuri, F. R. Cancer Treat. Rev. 2003, 29, 407.
20. Cetnar, J. P.; Malkowicz, S. B.; Palmer, S. C.; Wein, A. J.; Vaughn, D. J. Urology
2008, 71, 942.
1. Bagshawe, K. D.; Sharma, S. K.; Springer, C. J.; Rogers, G. T. Ann. Oncol. 1994, 5,
879.
2. Szalai, M. L.; Kevwitch, R. M.; McGrath, D. V. J. Am. Chem. Soc. 2003, 125, 15688.
3. de Groot, F. M.; Albrecht, C.; Koekkoek, R.; Beusker, P. H.; Scheeren, H. W.
Angew. Chem., Int. Ed. 2003, 42, 4490.
4. Amir, R. J.; Pessah, N.; Shamis, M.; Shabat, D. Angew. Chem., Int. Ed. 2003, 42,
4494.
5. Haba, K.; Popkov, M.; Shamis, M.; Lerner, R. A.; Barbas, C. F., 3rd; Shabat, D.
Angew. Chem., Int. Ed. 2005, 44, 716.
6. (a) Shamis, M.; Lode, H. N.; Shabat, D. J. Am. Chem. Soc. 2004, 126, 1726; (b)
Shabat, D. J. Polym. Sci., Part A: Polym. Chem. 2006, 44, 1569.
7. Adler-Abramovich, L.; Perry, R.; Sagi, A.; Gazit, E.; Shabat, D. Chembiochem
2007, 8, 859.
8. Sagi, A.; Segal, E.; Satchi-Fainaro, R.; Shabat, D. Bioorg. Med. Chem. 2007, 15,
3720.
9. Sella, E.; Shabat, D. Chem. Commun. 2008, 5701.
10. Satchi-Fainaro, R.; Puder, M.; Davies, J. W.; Tran, H. T.; Sampson, D. A.; Greene,
A. K.; Corfas, G.; Folkman, J. Nat. Med. 2004, 10, 255.
11. Satchi-Fainaro, R.; Hailu, H.; Davies, J. W.; Summerford, C.; Duncan, R.
Bioconjugate Chem. 2003, 14, 797.
21. Bhalla, K. N. Oncogene 2003, 22, 9075.
22. Parekh, H.; Simpkins, H. Gen. Pharmacol. 1997, 29, 167.
23. Engels, F. K.; Mathot, R. A.; Verweij, J. Anticancer Drugs 2007, 18, 95.
24. Gelderblom, H.; Verweij, J.; Nooter, K.; Sparreboom, A. Eur. J. Cancer 2001, 37,
1590.
25. Hohenlohe-Oehringen, K.; Call, L. Monatsh. Chem. 1968, 99, 1289.
26. Miller, K.; Erez, R.; Shabat, D.; Satchi-Fainaro, R. Angew. Chem. Int. Ed. Engl.
2009, 48, 2949.