Kinase-mediated trapping of bi-functional conjugates of paclitaxel or vinblastine with thymidine in cancer cells

Article abstract of DOI:10.1016/j.bmcl.2006.07.003

In the present work, we explore the possibility of introducing selectivity to existing chemotherapeutics via the design of non-pro-drug, bi-functional molecules comprising a microtubule-binding agent and a substrate for a disease-associated kinase. The design, synthesis, and in vitro biological evaluation of paclitaxel-thymidine and vinblastine-thymidine bi-functional conjugates are reported here. This work provides the first account of 'kinase-mediated trapping' of cancer therapeutics.

Full text of DOI:10.1016/j.bmcl.2006.07.003

Bioorganic & Medicinal Chemistry Letters 16 (2006) 5194–5198
Kinase-mediated trapping of bi-functional conjugates
of paclitaxel or vinblastine with thymidine in cancer cells
Simon E. Aspland,a,* Carlo Ballatore,a Rosario Castillo,a Joel Desharnais,a
Trisha Eustaquio,a Philip Goelet,a Zijian Guo,a Qing Li,a David Nelson,a
Chengzao Sun,a Angelo J. Castellinoa and Michael J. Newmanb
aAcidophil LLC, 2330 W. Joppa Road, Suite 330, Lutherville, MD 21093, USA
bDihedron Corporation (Absorbed by Acidophil LLC during 2005), USA
Received 30 May 2006; revised 3 July 2006; accepted 5 July 2006
Available online 25 July 2006
Abstract—In the present work, we explore the possibility of introducing selectivity to existing chemotherapeutics via the design of
non-pro-drug, bi-functional molecules comprising a microtubule-binding agent and a substrate for a disease-associated kinase. The
design, synthesis, and in vitro biological evaluation of paclitaxel–thymidine and vinblastine–thymidine bi-functional conjugates are
reported here. This work provides the first account of ‘kinase-mediated trapping’ of cancer therapeutics.
Ó 2006 Elsevier Ltd. All rights reserved.
One of the most important problems associated with
cancer chemotherapy is the poor selectivity exhibited
by the majority of the commonly employed cytotoxic
agents, which results in a narrow therapeutic index. Sev-
eral attempts have been made to improve selectivity of
such agents, such as by incorporating a targeting moiety
(e.g., Mylotarg),1 or using pro-drug approaches de-
signed to exploit differences between tumor and normal
cells.2 Over the past several years, advances in elucidat-
ing the complex chain of events that take part during the
onset and progression of cancer have clearly highlighted
the critical role played by protein and small molecule ki-
nases. The level of expression and/or activation of sever-
al of these enzymes accounts for some of the most
important intracellular differences between normal and
malignant cells. Indeed, a number of kinases, including
small molecule kinases such as hexokinase 2 (HK2),3
sphingosine kinase 1 (SK1)4, and thymidine kinase 1
(TK1),5 are known to be either over-expressed or aber-
rantly activated in tumor cells. Interestingly, a growing
body of evidence clearly suggests that substrates of
HK2,6 SK17, and TK18 selectively accumulate in cancer
cells, presumably due to the negative charges being add-
ed through phosphorylation. This phenomenon, which
we refer to as kinase-mediated trapping (KMT), has
proven very useful for tumor imaging. For example,
TK1-mediated trapping of the thymidine analog 30-de-
oxy-30-[18F]fluorothymidine ([18F]FLT) has been
exploited in positron emission topography (PET) for tu-
mor imaging.9 Based on these observations, we became
interested in exploring KMT as a novel mechanism for
increasing the selectivity of existing chemotherapeutic
agents, by conjugation to substrates of tumor-specific
kinases (Fig. 1).
In order to test this approach, several issues need to be
considered, namely: (i) the selection of a kinase and its
substrate, (ii) the selection of a cytotoxic drug to be
paired with this kinase substrate, (iii) the method for
linking these components in such a manner that both
retain their activities, and (iv) the selection of appropri-
ate assays to evaluate these bi-functional molecules.
We reasoned that TK1 would provide a good trapping
mechanism since TK1 has been shown to be over-
expressed in ovarian10 and colorectal11 carcinomas as
well as certain leukemias,12 and since [18F]FLT has been
exploited in tumor imaging by PET. Furthermore,
modification of the N3 position of thymidine was
explored based on the reports of Tjarks and co-workers,
which indicated that relatively large molecules could be
attached to this position without drastic reduction in the
rate of TK1-mediated phosphorylation.13 In order to
Keywords: Kinase-mediated trapping; Thymidine kinase 1; Thymidine;
Paclitaxel; Vinblastine; Fluorescein.
*
Corresponding author. Tel.: +1 443 279 9971; fax: +1 410 494
4247; e-mail: saspland@acidophil.com
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.07.003

S. E. Aspland et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5194–5198
5195
negative charge on
phosphate traps drug
in diseased cell
which comprise either fluorescein or a cytotoxic agent
(PXL or VBL) conjugated to THY, the natural substrate
for TK1, through hydrolytically and enzymatically sta-
ble linkers. The synthetic strategy entailed coupling of
THY-linker building blocks with appropriately protect-
ed drug derivatives. THY-linkers 2–5 (Scheme 1) were
prepared via N3-alkylation of a- or b-THY with activat-
ed linkers of general structure 1.
D = Drug
S = Substrate
O
D
S
O
O
Cytoplasm
P
D
S
↑
Kinase
B
Nucleus
D
S
A
A 50-phosphorylated THY-linker 7 was also prepared
(Scheme 2) for the synthesis of selected phosphorylated
cytotoxic-THY conjugates (vide infra, A9 and C7, Table
1). For the fluorescently labeled thymidines, b- and a-
THY linkers 2d and 4d were reacted with fluorescein–
thioisocyanate to give 8 and 9, respectively (Scheme 2).
Interestingly initial studies with these fluorescein–THY
conjugates demonstrated that b-D-THY 8 but not its
non-phosphorylatable a-anomer 9 leads to differential
accumulation of fluorescence in cells as a function of
their total TK1 activity level (Fig. 2).
Tumor cell or tumor-associated endothelial cell
Figure 1. Kinase-mediated trapping of bi-functional molecules: phos-
phorylation of conjugates by an over-expressed or aberrantly activated
kinase selectively traps cytotoxic drugs in tumor cells in preference to
normal cells where the same kinase has normal levels of activity. (A)
Unphosphorylated conjugate is able to diffuse in and out of all cells;
(B) Intracellular phosphorylation of the conjugate mediated by the
disease-associated kinase leads to a selective accumulation/retention of
the conjugate inside cancer cells.
test TK1 as a drug trapping mechanism we investigated
the cellular accumulation of thymidine (THY) conjugat-
ed fluorescein (used as a detectable drug surrogate) and
the selective cytotoxicity of THY conjugated paclitaxel
(PXL) and vinblastine (VBL).
For PXL and VBL conjugates, reported structure–activ-
ity relationship studies identified sites expected to be tol-
erant of modification. Thus, C7 and C10 of PXL17 as
well as C3 of VBL18 were explored as points of attach-
ment in constructing drug–thymidine bi-functional mol-
ecules. Examples of non-phosphorylatable conjugates
of PXL or VBL and a-THY were also synthesized for
In the present study, we report the design, synthesis and
in vitro biological evaluation of bi-functional molecules
a
b
R
LinkerNHCbz
1
HO LinkerNH2
HO LinkerNHCbz
R = OTs or Br
THY-PEG Linkers
THY-Alkane Linkers
O
O
O
N
NH
2
N
NH
2
n
n
N
O
N
O
HO
HO
HO
HO
O
O
Cpd
2a
n
1
2
3
4
6
β−D-THY
or
3
2
OH
OH
Cpd
3f, 5f
3g, 5g
n
1
2
c, d
2b
α−D-THY
2c
O
O
2d, 4d
2e
N
O
O
OH
N
O
O
OH
4
N
NH
5
2
N
NH
2
n
O
n
Scheme 1. Synthesis of thymidine-linker building blocks. Reagents: (a) Cbz-Cl, TEA, MeOH; (b) TsCl, pyridine or PPh3, CBr4; (c) 1, K2CO3 (KI),
acetone/DMF; (d) H2, Pd/C.
O
N
O
N
H
N
NH
2
N
CBz
N
O
P
O
O
a, b, c
HO
O
HO
O
O
OH
OH
OH
7
6
O
H
N
H
HO
FLUX
N
O
α
N
S
N
O
N
O
O
OH
HO
S
O
N
β
FLUX
N
N
9
8
H
H
OH
FLUX = Fluorescein
Scheme 2. Reagents and conditions: (a) POCl3/TEA, TMP ꢀ10 °C; (b) ice/water; (c) H2, Pd/C.

5196
S. E. Aspland et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5194–5198
Table 1
comparison. PXL–THY conjugates were obtained by
Yielda (%)
reacting the amino group of the THY-linker inputs 2–
5, and 7 with either 1219,20 or 1321 to give the PXL C-
7 or C-10 conjugates of general structures A and B,
respectively (Scheme 3). Likewise VBL–THY conjugates
were prepared by reacting the THY-linkers 2–4, and 7
with 4-deacetyl-3-demethoxy-3-azidovinblastine, pre-
pared in situ from 1422 (Scheme 4).
Compound
Thy-linker
A1
A2
A3
A4
A5
A6
A7
A8
A9
B1
B2
B3
B4
B5
B6
B7
B8
C1
C2
C3
C4
C6
C7
2a
2b
2c
2d
2e
3f
63
69
95
68
40
70
20
40
10
36
40
10
45
13
65
52
20
8
3g
4d
7
Stability of key conjugates was assessed in tissue culture
medium containing 10% fetal calf serum and was found
to be comparable or better than the stability of parent
drugs (data not shown). All PXL and VBL conjugates
were evaluated for their ability to promote or inhibit
2a
2c
2d
3f
the polymerization of tubulin in a cell-free system.23
A
3g
4d
5f
TK1 assay was also employed to determine the phos-
phorylation rates of the conjugates in comparison with
THY.24
5g
2a
2b
2d
3f
18
15
39
40
45
In addition, all but the chemically phosphorylated con-
jugates were evaluated in a cytotoxicity assay using three
tumor cell lines: K562 leukemia, HT29 colorectal carci-
noma, and MCF7 mammary carcinoma. A summary of
the data obtained from the in vitro assays is shown in
Table 2.
4d
7
a Overall yield after HPLC purification.
Based on the data shown in Table 2 a number of
conclusions can be made: (i) With the exception of the
non-phosphorylatable analogs, many conjugates were
bi-functional in the biochemical assays with phosphoryl-
ation rates ranging from 13% to 68% of that of thymi-
dine, and exhibited high levels of drug activity in bio-
chemical assays. Remarkably, several conjugates (e.g.,
B1, B3, B4, C2, and C4) maintained microtubule
(MT)-binding ability, comparable to the parent drugs
(i.e., PXL or VBL). Among PXL conjugates, the C10
series exhibited greater MT-stabilizing ability than the
analogous C7 conjugates. (ii) When assayed for their
ability to alter the polymerization of tubulin, the chem-
ically phosphorylated conjugates (A9 and C7) exhibited
comparable activity to their non-phosphorylated
counterparts (A4 and C3), indicating the phosphoryla-
tion step does not alter the MT-binding ability of the
Accumulation of Fluorescein Thymidine Conjugates
100
90
80
70
60
50
40
30
20
10
0
-THY
-THY
CEM TK-
CEM
K562
Cell Line
Figure 2. Selective accumulation of fluorescently labeled thymidines in
TK1 expressing cells: leukemic cell lines expressing various levels of
TK1 (i.e., CEM TK-, (no TK1),14,15 CEM (medium TK1), and K562
(high TK1)) were incubated with 20 lM of the fluorescein–THY
conjugates for 16 h. Cells were then washed and fluorescence was
determined by flow cytometry.16
Thy Linker
NH
O
O
AcO
O
O
O
AcO
O
NO
2
O
O
O
O
O
a, b
O
O
O
H
N
O
H
N
H
H
OAc
HO OBz
OAc
HO OBz
OH
OCBz
A
12
N
H
Linker Thy
N
O
N
O
O
O
OH
O
O
OTES
O
O
O
c, d
O
O
H
HO OBz OAc
O
O
N
O
OTBS
H
N
H
H
OAc
OH
HO OBz
B
13
Scheme 3. Reagents and conditions: (a) Thy-linker-amine, DIEA, DCM; (b) H2, Pd/C; (c) Thy-linker-amine, IPA or t-BuOH, 82 °C; (d) HF/Py.

S. E. Aspland et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5194–5198
5197
OH
OH
N
N
N
H
N
H
CO2Me
CO2Me
N
N
H
H
a, b
OH
OH
MeO
N
OH
MeO
N
OH
H
O
H
O
Linker Thy
N
NHNH2
H
C
14
Scheme 4. Reagents: (a) NaNO2, HCl, MeOH; (b) Thy-linker-amine, DMF/DCM, DIEA.
Table 2
TK1 activitya
MPAb
K562 EC50
HT29EC50
MCF7 EC50
clogPd
c
c
c
Compound
PXL
A1
A2
A3
A4
A5
A6
A7
A8
A9
B1
B2
B3
B4
B5
B6
B7
B8
VBL
C1
C2
C3
C4
C5
C6
C7
N/A
35
100
66
0.014
2.065
2.875
1.588
0.227
0.207
4.475
3.213
0.553
—
0.006
3.000
2.614
0.875
0.191
0.074
2.000
3.640
1.417
—
0.036
2.030
1.716
0.787
0.096
0.075
1.720
1.600
0.296
—
4.77
2.4
68
29
56
78
2.94
3.46
3.99
5.05
1.51
1.23
4.77
—
26
13
83
59
16
48
35
44
NDe
N/A
46
74
73
105
86
0.217
0.221
0.091
0.763
1.150
0.181
0.314
1.531
0.001
0.043
0.030
0.010
0.016
0.020
0.014
—
0.114
0.173
0.091
0.145
1.025
0.268
0.182
1.150
0.002
0.106
0.049
0.047
0.021
0.021
—
0.149
0.107
0.099
0.143
0.265
0.114
0.112
0.390
0.003
0.100
0.151
0.166
0.038
0.076
—
1.83
2.84
3.42
1.51
1.23
3.42
1.51
1.23
5.23
1.59
2.12
2.82
1.51
1.24
2.82
—
49
45
100
102
91
48
34
NDe
NDe
NDe
N/A
15
92
87
89
100
93
49
27
107
80
23
97
84
37
NDe
N/A
93
97
—
—
a Phosphorylation rates expressed as % of THY.
b Ability to promote (PXL) or inhibit (VBL) the polymerization of tubulin, expressed as % of the parent drug.
c Concentration of test compound (lM) required to inhibit cell proliferation by 50%, average of at least three assays.
d Partition coefficient between water/n-octanol.
e Not detectable (ND).
conjugates. (iii) Although most conjugates were consid-
erably less potent cytotoxics than the parent drugs,
selected compounds (A4, A5, B3, and C3) did retain
low nanomolar EC50 values (10–100 nM). Interestingly,
the cell cytotoxicity data show a much broader variabil-
ity between conjugates (with EC50s ranging from 10 nM
to 4 lM) than predicted by the data obtained from the
cell-free assays. One possible explanation for the differ-
ences between the cell-based and the cell-free results
can be found in the different physical–chemical proper-
ties of the conjugates (e.g., lipophilicity) that could ac-
count for substantial changes in the conjugates’ ability
to diffuse through cellular membranes. Indeed, the cellu-
lar EC50 values seem to correlate with the calculated
partition coefficients (clogP), whereby the most potent
conjugates have a clogP of 3 or greater. (iv) The com-
parison of EC50 values of corresponding pairs of
phosphorylatable and non-phosphorylatable analogs
(A4/A8; B3/B6; B4/B7; B5/B8; and C3/C6) does not
indicate any overt correlation between potency and
TK1-mediated phosphorylation. This could be ex-
plained by an insufficient level of intracellular phosphor-
ylation, even though the conjugates were found to be
TK1 substrates in the cell-free assay. However, it is also
plausible that in vitro cytotoxicity assays may not be the
optimal setting for evaluating selectivity enhancement of
the conjugates. Therefore, it is possible that only appro-
priately designed in vivo models will enable the determi-
nation of the advantage of KMT of these bi-functional
molecules.

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S. E. Aspland et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5194–5198
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In conclusion, a set of conjugates comprising fluoresce-
in, PXL or VBL conjugated to thymidine have been de-
signed and synthesized. Collectively, the data presented
here indicate that fluorescent conjugates of thymidine
accumulate in cells in a manner that is consistent with
KMT. Cytotoxic drug-containing conjugates are bi-
functional in the biochemical assays and some of these
display acceptable cellular potency. Additional studies
will be needed to evaluate a gain in selectivity by KMT.
15. The authors would like to thank Professor Staffan
Eriksson for the kind gift of CEM and CEM TK-.
16. They would further like to thank Carina Rumold of the
SKCC, La Jolla, for all her help with FACS.
Acknowledgment
17. Kingston, D. G.; Jagtap, P. G.; Yuan, H.; Samala, L.
Fortschr. Chem. Org. Naturst. 2002, 84, 53.
The authors would like to thank Dr. Sydney Brenner for
the original idea behind this project.
18. Lavielle, G.; Hautefaye, P.; Schaeffer, C.; Boutin, J. A.;
Cudennec, C. A.; Pierre, A. J. Med. Chem. 1991, 34, 1998.
19. Castellino, A. J.; Ballatore, C.; Aspland, S. E.; Desharnais,
J.; Newman, M. J.; Sun, C.; Wirsching, P. U.S. Patent
Application 20050148534A1.
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23. The microtubule polymerization assay was done using a
fluorescence-based kit (Cytoskeleton) using the manufac-
turers’ recommended protocol. For VBL-containing con-
jugates, the assay was done in the presence of 20%
glycerol, which induces polymerization of tubulin.
24. C-terminal His-tagged human TK1 fusion protein was
produced then purified on a His-bind nickel-affinity
column (Novagen). Using this TK1, kinase activity is
measured with a coupled enzyme assay that follows the
production of ADP, via the conversion of b-NADH to
b-NAD+. Initial velocities for test compounds are divided
by velocities for the ATP-negative controls and then
compared to the velocity of thymidine.
10. Fujiwaki, R.; Hata, K.; Nakayama, K.; Moriyama, M.;
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