Synthesis and biological activity of conjugates between paclitaxel and the cell delivery vector penetratin

Article abstract of DOI:10.1016/j.bmcl.2006.02.035

Synthesis of paclitaxel-penetratin (pAntp) constructs, in which the 2′- or 7-position of paclitaxel was used as the attachment site for linker connecting the drug and peptide moieties, is described. Paclitaxel-2′-pAntp[43-58]-NH₂ 3b and paclita

Full text of DOI:10.1016/j.bmcl.2006.02.035

Bioorganic & Medicinal Chemistry Letters 16 (2006) 2628–2631
Synthesis and biological activity of conjugates between
paclitaxel and the cell delivery vector penetratin
Shudong Wang,* Nikolai Z. Zhelev, Susan Duff and Peter M. Fischer
Cyclacel Limited, James Lindsay Place, Dundee DD1 5JJ, Scotland, UK
Received 1 February 2006; revised 13 February 2006; accepted 14 February 2006
Available online 2 March 2006
Abstract—Synthesis of paclitaxel–penetratin (pAntp) constructs, in which the 20- or 7-position of paclitaxel was used as the attach-
ment site for linker connecting the drug and peptide moieties, is described. Paclitaxel–20-pAntp[43–58]-NH2 3b and paclitaxel–20-
pAntp[52–58]-NH2 3c showed excellent antitumour activity against human lung and breast cancer cell lines. These conjugates were
highly soluble and stable with a half-life of >8 h under cell culture conditions. The drug–peptide conjugates may be therapeutically
useful due to improved pharmaceutical properties.
Ó 2006 Elsevier Ltd. All rights reserved.
The pAntp peptide, known as PenetratinÒ, is a novel
cell-permeable peptide derived from the third helix of
the Drosophila antennapedia homeoprotein.1 It has been
used as a nonviral, nontoxic and highly efficient vector
for delivering bioactive substances, that by themselves
are membrane-impermeable, to the cytoplasm or nucle-
us of cells.2–6 It has been shown, for example, that doxo-
rubicin, when coupled covalently to penetratin, was
capable of crossing the blood–brain barrier in mice, thus
significantly increasing drug uptake in comparison with
free doxorubicin.6,7 It has also been demonstrated that
since doxorubicin–peptide conjugates bypassed P-glyco-
protein, increased cellular drug uptake could be
achieved, suggesting that vectorised drug conjugates
may be capable of overcoming multi-drug resistance.8
52RRMKWKK58-NH2 (pAntp[52–58]-NH2) of the
parent 16mer sequence was necessary and sufficient
for effective cell membrane translocation. In connec-
tion with a research programme directed at the assess-
ment of the efficacy of penetratin derivatives as vectors
for the delivery of poorly bioavailable therapeutic
agents, particularly those which are to be used for
cancer therapy, we designed and synthesised a series
of penetratin-drug constructs,10,11 in the hope that this
system may improve the pharmaceutical properties of
the drugs by, for example, improving solubility and
bioavailability, or by minimising toxicity and overcom-
ing drug resistance.
Paclitaxel 1, a diterpene natural product,12 has been
shown to be highly cytotoxic to cancer cells and is a po-
tent anticancer drug used in the clinic.13,14 Paclitaxel
acts by promoting the assembly of stable microtubules
from tubulin and it inhibits the disassembly process,
thus interfering with the G2- and M-phases of the cell
cycle.15,16 Therapeutic use of paclitaxel has, however,
several drawbacks. These problems are mainly associat-
ed with extremely low water solubility, poor bioavail-
ability and emergence of drug resistance in patients
treated with paclitaxel. FTo date a great deal of research
has been carried out to modify the chemical structure of
paclitaxel in order to improve its physicochemical prop-
erties.17–19 Among the most notable paclitaxel deriva-
tives synthesised and characterised so far are prodrugs
in which the 20- and 7-hydroxyl groups of the molecule
are engaged in a functional group that collapses, upon
in vivo activation, to release paclitaxel.20–22
The SARs of penetratin peptides in terms of minimum
active peptide length and relative importance of amino
acid residues required for membrane translocation
have been studied thoroughly.9 Cell penetration assays
using human cell cultures with a series of truncated
peptides revealed that the C-terminal segment
Keywords: Paclitaxel; Penetratin; Cell delivery vector; Drug–peptide
conjugate; Antitumour activity.
*
Corresponding author at present address: Centre for Biomolecular
Sciences, School of Pharmacy, University of Nottingham, University
Park, Nottingham NG7 2RD, UK. Tel.: +44 115 8466863; fax: +44
115 9513412; e-mail: shudong.wang@nottingham.ac.uk
Present address: Centre for Biomolecular Sciences, School of
Pharmacy, University of Nottingham, University Park, Nottingham
NG7 2RD, UK.
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.02.035

S. Wang et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2628–2631
2629
As a part of our vectorised drug conjugation pro-
gramme10,11 we synthesised a number of paclitaxel–pep-
tide penetratin (paclitaxel–pAntp) constructs. Three
forms of penetratin peptides, 43RQIKIWFQNRRMK
WKK58-OH(pAntp[43–58]-OH), 43RQIKIWFQNRR
MKWKK58-NH2 (pAntp[43–58]-NH2) and 52RRMK
WKK58-NH2 (pAntp[52–58]-NH2), were used as deliv-
ery vectors.9 Paclitaxel was attached through the 20- or
7-hydroxyl positions to the end of the peptides via a
succinimidopropionyl-sulfide linker, using chemo-selec-
tive maleimide-thiol chemistry. The synthesis and bio-
logical activities of these compounds are summarised
herein.
The pAntp peptides, paclitaxel–20-pAntp 3a–3c and pac-
litaxel–7-pAntp 6a, were tested for their antitumour ef-
fects against human lung and breast carcinoma cell
lines using a standard cellular proliferation MTT assay
(Table 1).24 The free peptides were inactive up to
20 lM concentration in this assay. The constructs 3b
and 3c were shown to be as potent as the unconjugated
parent compound paclitaxel. The peptide–acid conju-
gate 3a was modestly cytotoxic in A549 cells with an
IC50 value of 0.27 lM. Paclitaxel–7-Antp[43–58]-OH
6a showed significantly reduced potency compared to
the 20-Antp[43–58]-OH 3a counterpart. These results
are in line with what has been found with other paclit-
axel 20- and 7-OH ester prodrugs, for example, the ace-
tates25 and succinates,19 where the 7-OH-conjugated
isomers were generally found to be less cytotoxic than
the 20-OH conjugates.17,26,27 The inactivity of the 7-
OH esters presumably emanates from the much higher
hydrolytic stability of the sterically hindered 7-OH es-
ters. Esterification of both the 20- and 7-OH groups ap-
pears to reduce or abolish innate paclitaxel activity and
only the 20-linked conjugates release paclitaxel intracel-
lularly at a productive rate. It should be noted, however,
that some non-dissociated 7-OH paclitaxel ester conju-
gates have been reported to retain the ability to induce
microtubule assembly in vitro.25,26
Paclitaxel–pAntp constructs were prepared as outlined
in Scheme 1. Selective acylation of the sterically less
hindered secondary 20-hydroxyl of paclitaxel with 3-
maleimidopropionic anhydride in pyridine at room tem-
perature gave paclitaxel-20-maleimidopropionate 2 in
good yield. Cysteine was introduced as an N-terminal
residue of the pAntp peptides in order to facilitate
unambiguous conjugation. Reaction of paclitaxel-20-
maleimidopropionate with Cys-pAntp peptides proceed-
ed smoothly in the presence of base to afford 3.
In order to prepare paclitaxel-7-maleimidopropionate,
the 20-hydroxyl was masked as a methoxyacetate ester.23
A solution of paclitaxel and DIEA in CH2Cl2was treat-
ed with methoxyacetic acid N-hydroxysuccinimidyl ester
to afford 20-methoxyacetyl paclitaxel, which was acylat-
ed at the 7-hydroxyl with 3-maleimidopropionic
anhydride in the presence of a catalytic amount of 4-
N,N-dimethylaminopyridine (DMAP) to provide 4
(Scheme 1). This was followed by coupling to Cys-
pAntp, then treatment with ethanolamine, to give the fi-
nal conjugates 6. p-Methoxytrityl as an alternative acid
labile protecting group was also introduced at the 20-po-
sition of paclitaxel in quantitative yield. Elimination of
this group, following acylation of the 7-OH to give 5,
was effected with dilute solution of chloroacetic acid/
CH2Cl2, using anisole to scavenge the trityl cation liber-
ated during the deprotection.
As expected, the aqueous solubility of the conjugates 3
and 6 was enhanced considerably (e.g., >10 mg/mL,
>4.5 mM for 3c) in comparison to unconjugated paclit-
axel (<0.01 mg/mL). The stability of the peptides and
constructs in tissue culture media was examined next.
Solutions of the compounds in Dulbecco’s modified Ea-
gle’s medium (DMEM) containing 10% foetal calf ser-
um at concentrations varying from 1 to 40 lM were
incubated at 37 °C. Samples were taken at various time
points and were analysed. Degradation of pAntp[43–
58]-OH was observed after incubating for periods as
short as 10 min (Fig. 1). Mass spectral analysis of the
degradation fraction, isolated by RP-HPLC, indicated
that cleavage of the C-terminal lysines had occurred.
Apparently removal of Lys58 is rapid, followed by fur-
ther C-terminal proteolytic truncation. It was, however,
O
O
OR2
7
O
O
AcO
O
Ph
A:
B:
Table 1. Antiproliferative activity of pAnp peptides and conjugates 3
and 6 against human tumour cell lines
N
N
H
O
NH
2'
O
O
O
a
Compound
IC50 (lM)
O
Ph
O
HO
AcO
OCOPh
MCF7b
A549c
OR1
NH2
R3
Antp(43-58)-OH
Antp(43-58)-NH2
na
na
na
na
S
1: R1 = R2 = H
a
2: R1 = A, R2 = H
3: R1 = B, R2 = H
4: R1 = COCH2OMe, R2 = A
5: R1 = CPh2-(p-OMe-Ph), R2 = A
6: R1 = H, R2 = B
Antp(52-58)-NH2
3a
na
nt
na
b
O
0.27
<0.015
<0.015
ꢀ10
<0.015
c
d
a: R3 = pAntp[43-58]-OH
b: R3 = pAntp[43-58]-NH2
c: R3 = pAntp[52-58]-NH2
3b
3c
<0.017
<0.015
nt
e
f
6a
Paclitaxel 1
<0.015
Scheme 1. Reagents: (a) 3-maleimidopropionic anhydride, pyridine,
CH2Cl2; (b) H-Cys-R3, Et3N, DMF; (c) i—N-hydrox-
ysuccinimidomethoxyacetate, DIEA, CH2Cl2; ii—3-maleimidoprop-
ionic anhydride, DMAP, CH2Cl2; (d) i—p-methoxytrityl chloride,
pyridine, CH2CH2; ii—3-maleimidopropionic anhydride, DMAP,
CH2Cl2; (e) i—H-Cys-R3, Et3N, DMF; ii—H2N(CH2)2OH, MeOH;
(f) i—chloroacetic acid, anisole, CH2Cl2; ii—H-Cys-R3, Et3N, DMF.
a Concentration of test compound producing 50% growth inhibition
using an MTT assay. Cells were treated with test compounds for 72 h.
The stated values are means of three independent experiments (na,
not active; nt, not tested).
b Human breast cancer cell line.
c Human lung cancer cell line.

2630
S. Wang et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2628–2631
MCF7 cells were treated with the constructs for 1 h
and 3 days, respectively, and quantified by MTT assay
(Table 2). When the tumour cells were exposed to 3b
and 3c for 1 h, a period during which the conjugates
were shown to be stable under the culture conditions,
both compounds exhibited excellent cytotoxicity with
IC50 values ranging from 0.043 to 0.618 lM. It was not-
ed that 3c showed activity comparable to that of paclit-
axel in A549 cells, although less potent in MCF7 cells.
These results show that cytotoxicity is not due to liber-
ation of free paclitaxel extracellularly, but is mediated
by membrane translocation of the intact conjugates,
which is rapid, followed by intracellular release of bioac-
tive paclitaxel.9,28
Examination of cancer cell line cultures showed that
induction of cell death was very similar in paclitaxel-
and conjugate-treated samples. In both cases, character-
istic accumulation of cells in the G2/M-phase was ob-
served and induction of apoptosis (by TUNEL assay)
was extensive at 24 h post-treatment.
Figure 1. Degradation of pAntp[43-58]-OH in tissue culture medium.
See text for conditions. Aliquots were withdrawn and analysed by RP-
HPLC. The elution positions of the intact peptide and the C-terminal
Lys-truncation product are indicated, as is the incubation time.
discovered that proteolysis of these lysine residues was
blocked when the terminal lysine was present as the car-
boxamide rather than the carboxylate. Thus pAntp-NH2
peptides were stable with half-lives of >12 h under the
same conditions. These findings are significant, since
presence of the terminal Lys58 residue in penetratin vari-
ants is required absolutely for membrane translocation
activity, as we have shown.9 Penetratin is very common-
ly employed as a cellular delivery vector for a variety of
cell biology applications and it is frequently unclear if
the peptide acid or amide has been used.28 Clearly use
of the peptide acid severely limits the cell delivery poten-
tial of penetratin peptides.
Furthermore, conjugates 3b and 3c were assessed for
their ability to affect microtubule polymerisation
(Fig. 2). Presumably the observed activity of the conju-
gates, which was only somewhat lower than that of pac-
litaxel itself, is a result of liberation of paclitaxel or
bioactive metabolites from the conjugates under the
physiological conditions of the assay.
In summary, the chemistry for the synthesis of paclit-
axel–pAntp constructs and cellular biological properties
of the conjugates were described.32 Paclitaxel–20-
Antp[43–58]-NH2 3b and paclitaxel–20-Antp[52–58]-
We next looked at the stability of the bioactive paclit-
axel–peptide conjugates in tissue culture medium under
similar conditions. The stability of each conjugate corre-
lated to that of its peptide counterpart. Thus, paclitaxel–
20-pAntp[43–58]-NH2 3b and paclitaxel-20-pAntp[52–
58]-NH2 3c were found to be stable with half-lives of
>8 h by RP-HPLC and mass spectrometric assessment.
Again this is in agreement with the reported hydrolytic
stability at pH 7.4 of other paclitaxel prodrugs with sim-
ilar ester functions.19,27,29
Paclitaxel–pAntp conjugates 3b and 3c were further sub-
jected to time-exposure cellular experiments. A549 and
Table 2. Effect of exposure time on antiproliferate activity of paclitaxel
1 and conjugates 3 (IC50, lM)a
Compound
1-h treatment
72-h treatment
MCF-7b
A549b
MCF7c
A549c
3b
3c
0.202
0.325
0.043
0.618
0.043
0.028
<0.017
<0.015
<0.015
<0.015
<0.015
<0.015
Paclitaxel 1
a Concentration of test compound producing 50% growth inhibition
using an MTT assay. Cells were treated with test compounds for 72 h.
The stated values are means of three independent experiments (na:
not active; nt: not tested).
Figure 2. In vitro microtubule stabilization using a rhodamine–tubulin
assay.30,31 Dose–response curves are shown in the upper panel for
paclitaxel 1 (j) and the pAntp conjugates 3b (d) and 3c (s). Low-
level fluorescence micrographs are shown in the lower panel (a, 0.5 lM
1; b, 0.5 lM 3c and c, negative control).
b 1 h treatment.
c 72 h treatment.

S. Wang et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2628–2631
2631
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Compound 2: white solid (76% yield); 1H NMR (CDCl3): d
1.13, 1.22, 1.68, 1.91 (s, each 3H, CH3), 2.23, 2.47 (s, each
3H, CH3), 2.35 (m, 2H), 2.78 (t, 4H, J = 5.40 Hz), 2.84 (m,
2H), 3.81 (m, 2H), 3.87 (m, 1H), 4.26 (m, 2H), 4.44 (dd, 1H,
J = 10.87, 4.25 Hz), 4.98 (d, 1H, J = 7.69 Hz), 5.47 (d, 1H,
J = 3.45 Hz), 5.68 (d, 1H, J = 7.09 Hz), 6.05 (dd, 1H,
J = 9.28, 5.86 Hz), 6.28 (s, 1H), 6.18 (t, 1H, J = 8.77 Hz),
6.49 (s, 2H), 8.16–7.34 (m, 15H, Ph-H). 13C NMR (CDCl3):
d 10.01, 15.20, 21.22, 22.54, 23.09, 27.18, 32.90, 33.71, 35.90,
43.54, 45.96, 52.86, 58.89, 72.18, 72.53, 74.86, 75.51, 76.02,
79.52, 81.42, 84.89, 126.94, 127.91, 128.94, 128.94, 129.14,
129.45, 129.59, 130.65, 132.39, 133.11, 133.85, 134.09,
134.46, 137.17, 143.25, 167.45, 168.01. 168.10, 169.77,
170.29, 171.10, 171.69 and 204.24.
Compound 3a: white solid (62% yield); Anal. RP-HPLC
tR = 17.4 min (0–60% MeCN, purity >97%); MS m/z 3355.9
[M+H]+ (C161H229N37O38S2requires 3354.9). Compound
3b: white solid (53% yield); Anal. RP- HPLC tR = 18.5 min
(0–60% MeCN, purity >97%); MS m/z 3353.6 [M+H]+
(C161H230N38O37S2 requires 3353.9). Compound 3c: white
solid (53% yield); Anal. RP-HPLC tR = 17.2 min (0–60%
MeCN, purity >97%); MS m/z 2211.7 [M+H]+
(C106H148N22O26S2 requires 2210.6).
9. Fischer, P. M.; Zhelev, N. Z.; Wang, S.; Melville, J. E.;
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Compound 4: white solid (76% yield); Anal. RP-HPLC
tR = 21.8 min (10–70% MeCN, purity >98%); 1H NMR
(CDCl3): d 1.15, 1.20, 1.79, 1.96 (s, each 3H, CH3), 2.20, 2.45
(s, each 3H, CH3), 2.34 (m, 2H), 2.63 (m, 4H), 3.40 (s, 3H,
OCH3), 3.73–3.94 (m, 3H), 4.16–4.21 (m, 2H), 4.97 (d, 1H,
J = 8.06 Hz),5.54–5.69 (m, 3H), 5.98 (m, 1H), 6.22 (s, 1H),
6.24 (m, 1H), 6.68 (s, 2H), 7.12–8.13 (m, 15H, Ph-H).
Compound 5: white solid (49% yield); 1H NMR (CDCl3): d
1.18, 1.12, 1.76, 1.96 (s, each 3H, CH3), 2.17, 2.26 (s, each
3H, CH3), 2.10, 2.34 (m, 2H), 2.62 (m, 4H), 3.75 (s, 3H,
OCH3), 3.73-3.79 (m, 3H), 4.06 (m, 2H), 4.61 (d, 1H,
J = 3.47 Hz), 4.76 (d, 1H, J = 9.52 Hz), 5.53 (m, 1H), 5.60
(d, 1H, J = 6.98 Hz), 5.71 (m, 1H), 6.14 (s, 1H), 6.60 (m,
3H), 6.75–7.79 (m, 29H, Ph-H).
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Compound 6a: white solid (48% yield); Anal. RP-HPLC
tR = 14.3 min (10–70% MeCN, purity >97%); MS m/z
3355.7 [M+H]+ (C161H229N37O38S2 requires 3354.9).