Chemotherapeutic bone-targeted bisphosphonate prodrugs with hydrolytic mode of activation

Article abstract of DOI:10.1016/j.bmcl.2007.11.029

Osseous tissues are considered to be limited as therapeutic target sites due to their biological properties. We have designed and synthesized two kinds of hydrolytically activated chemotherapeutic prodrugs containing bisphosphonate, a bone-targeting moiet

Full text of DOI:10.1016/j.bmcl.2007.11.029

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 816–820
Chemotherapeutic bone-targeted bisphosphonate prodrugs
with hydrolytic mode of activation
Rotem Erez,a Sharon Ebner,b Bernard Attalib and Doron Shabata,*
aDepartment of Organic Chemistry, School of Chemistry, Raymond and Beverly Sackler Faculty of Exact Sciences,
Tel-Aviv University, Tel Aviv 69978, Israel
bDepartment of Physiology and Pharmacology, Sackler Faculty of Medical Sciences, Tel-Aviv University, Tel Aviv 69978, Israel
Received 24 August 2007; revised 6 November 2007; accepted 9 November 2007
Available online 17 November 2007
Abstract—Osseous tissues are considered to be limited as therapeutic target sites due to their biological properties. We have designed
and synthesized two kinds of hydrolytically activated chemotherapeutic prodrugs containing bisphosphonate, a bone-targeting moi-
ety. The first can be conjugated to drug molecules with an available hydroxy group; the drug is attached to the bisphosphonate com-
ponent through an ester-labile linkage. The second is for use with drug molecules with amine functional group. In this case, a self-
immolative linker is used to attach the drug to the bisphosphonate component through a carbonate-labile linkage. The concept was
demonstrated using the drugs camptothecin, which has a hydroxy functional group, and tryptophan, which is a model molecule for
a drug with amine functionality. Both prodrugs showed significant binding capability to hydroxyapatite, the major component of
bone, and were hydrolytically activated under physiological conditions.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Chemotherapeutic prodrugs with capability to target
certain tissues or cell types may enhance the potency
or eliminate the side effects of drugs.1–4 Although many
prodrug approaches have been demonstrated, osseous
tissues are considered to be limited as target sites due
to their biological properties. In contrast to other tis-
sues, the blood flow rate in bones is very low, because
they mainly consist of an inorganic compound Ca10(-
PO4)6(OH)2, termed hydroxyapatite (HAP). Recently a
promising drug delivery system with a bisphosphonate
(BP) moiety for targeting osseous tissues was proposed.5
Bisphosphonates have high affinity for HAP and calci-
fied tissues are the main targets for accumulation after
administration of bisphosphonates into the body.6,7 Sev-
eral examples of anticancer drugs linked to a bisphosph-
onate moiety have been reported in the scientific
literature.8 Most of them failed to show any improved
anticancer effect, however, when tested in vivo in
mice.8,9 In these compounds, the chemotherapeutic drug
was linked to the bisphosphonate through a stable
chemical linkage that did not allow release of the active
drug. Interestingly, when anti-inflammatory drugs were
conjugated to a bisphosphonate moiety through an ester
linkage, promising results were observed in animal-mod-
el experiments.10–12 Therefore, we assume that a bis-
phosphonate moiety conjugated to a chemotherapeutic
agent through a hydrolyzable linker will target the drug
conjugate to the osseous tissue, where the active parent
drug will be slowly released through the hydrolysis of
the linkage. Here we report the design, synthesis, and
in vitro evaluation of two new prodrugs, based on
known chemotherapeutic drugs, attached to bisphosph-
onate moiety through a hydrolyzable linkage. The link-
age was spontaneously hydrolyzed at a physiological pH
(half-life time of hours to days) and active drug was re-
leased. Both prodrugs showed significant binding capa-
bility to hydroxyapatite, the major component of bone.
In order to demonstrate our concept, we chose to conju-
gate a bisphosphonate moiety to the known chemother-
apeutic drug camptothecin.13 In clinical trials,
camptothecin was not effective due to its extremely
low aqueous solubility and severe toxic side effects.14
Several new chemical derivatives of camptothecin that
overcome some of the disadvantages of the parent
compound are now being evaluated in clinical trials.2
Camptothecin is an ideal candidate for conjugation
with bisphosphonate as this moiety will impart both
water solubility and target the compound to bone.
Keywords: Bisphosphonate; Prodrug; Cancer; Bone targeting.
*
Corresponding author. Tel.: +972 (0) 3 640 8340; fax: +972 (0) 3 640
9293; e-mail: chdoron@post.tau.ac.il
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.11.029

R. Erez et al. / Bioorg. Med. Chem. Lett. 18 (2008) 816–820
817
Camptothecin can be esterified with bisphosphonate-bu-
tyric acid to form an esterolytic-activated prodrug, as
illustrated in Figure 1. The prodrug is termed Camdro-
nate; we chose the suffix ‘dronate’ since most bisphosph-
onate-based drugs follow this nomenclature.5
Since Camdronate is designed for bone targeting, we
sought for a simple model system to simulate bone tis-
sue. Hydroxyapatite (HAP) is a commercially avail-
able calcium mineral. A suspension of HAP in
aqueous media was previously used as a bone mod-
Camdronate was synthesized as illustrated in Figure 2.
Bisphosphonate carboxylic acid 1 was prepared as previ-
ously described.11 Chlorination of 1 with oxalyl chloride
generated acylchloride-tetraethyl-bisphosphonate 2.
Then camptothecin was acetylated with acylchloride 2
to give ester 3, which was deprotected with trim-
ethylsilylbromide to afford Camdronate. The phos-
phonic acid moiety was transformed into its water-
soluble sodium salt by the addition of an appropriate
amount of sodium hydroxide (until the pH of the solu-
tion reached 9).
Next, we evaluated the hydrolytic stability of the ester
linkage in Camdronate under physiological conditions.
Camdronate was incubated in RPMI cell-medium (pH
7.4) at 37 ꢁC and the release of free camptothecin was
monitored by RP-HPLC. Camdronate was gradually
hydrolyzed to release free camptothecin with a t1/2 of
40 h (Fig. 3).
Figure 3. Hydrolysis of Camdronate (blue) to release camptothecin
(red) in RPMI cell-medium, 37 ꢁC.
Camdronate was then evaluated by cell-growth inhibi-
tion assay using a cancerous acute lymphoblastic leuke-
mia (ALL) Jurkat cell line (Fig. 4). Camdronate was 10-
fold less active (IC50 2.1 · 10À8) than free camptothecin
(IC50 1.4 · 10À9). This was expected since the masking
of the 20-hydroxy group of camptothecin by an ester
generates a prodrug with reduced anticancer activity.
The prodrug can be recovered upon the hydrolysis of
the ester linkage. No cytotoxicity was detected for the
bisphosphonate-butyric acid moiety under the assay
conditions.
Figure 4. Growth inhibition activity of Camdronate (blue) and
camptothecin (red) on Jurkat cells after a 96-h incubation over a
range of concentrations of drug/prodrug.
O
N
O
N
N
N
O
H2O
O
O
O
O
O
P
HO
OH
OH
O
P
O
OH
OH
HO
OH
P
Camptothecin
Camdronate
OH
O
O
P
O
OH
OH
Figure 1. Hydrolysis of Camdronate under physiological conditions releases free, active camptothecin.
O
N
O
O
O
P
N
O
P
OEt
OEt
OEt
OEt
oxalyl
O
O
Cl
HO
chloride
TMSBr
94%
Camptothecin
57%
Camdronate
P
P
quant
O
O
OEt
OEt
O
OEt
OEt
O
P
3
2
1
O
OEt
OEt
P
O
OEt
OEt
Figure 2. Chemical synthesis of Camdronate.

818
R. Erez et al. / Bioorg. Med. Chem. Lett. 18 (2008) 816–820
el.16 An aqueous solution of Camdronate was vigor-
ously stirred with HAP for 1 h to allow binding of
the bisphosphonate moiety with the calcium mineral.
The mixture was filtered and the precipitate was
washed with water to remove any unbound Camdro-
nate. The precipitate (HAP and any bound Camdro-
nate) was then incubated in phosphate buffer saline,
pH 7.4 (PBS), at 37 ꢁC and the hydrolytic release of
camptothecin to the solution was monitored by RP-
HPLC. The camptothecin was slowly released through
hydrolysis over 300 h (Fig. 5, blue plot). In order to
ensure that the unconjugated camptothecin molecule
had no affinity for HAP, a control experiment was
performed in which the HAP suspension was mixed
with camptothecin and then filtered and washed. No
camptothecin was detected in the HAP precipitate
from this experiment (green). In contrast, when the
Camdronate/HAP ratio was doubled, the total amount
of free camptothecin released was correspondingly in-
creased (red).
Stable carbamate Linkage
Carbonate-Linkage
O
OMe
O
O
P
O
H
OH
OH
N
HN
O
OH
OH
Trypdronate
O
P
OMe
CO2H
O
Self-Immolative Linker
Figure 7. Chemical structure of Trypdronate.
moiety was linked through a labile carbonate bond to
the self-immolative moiety, 4-hydroxy-3,5-dimethoxy-
benzyl-alcohol, which was further attached through a
stable carbamate linkage to the amine group of
tryptophan.
The bisphosphonate moiety is designed to target the
drug conjugate to the osseous tissue, where the active
parent drug will be released through the hydrolysis of
the carbonate linkage and a spontaneous 1,6-elimination
followed by decarboxylation. A detailed proposed re-
lease mechanism is presented in Figure 8.
Chemotherapeutic drugs with an available amine func-
tional group, such as doxorubicin or melphalan, gener-
ally form hydrolytically stable derivatives upon
masking of their reactive amines through an amide or
a carbamate linkage. Such a doxorubicin-based prodrug
did not show any therapeutic effect when tested against
human tumor xenografts.9 In order to extend our con-
cept to additional drugs with an amine functional group,
we sought to link the bisphosphonate through a hydro-
lyzable bond to a short self-immolative linker for subse-
quent attachment to the amine group of the drug
(Fig. 6).
Trypdronate was synthesized as shown in Figure 9.
Tetrabenzyl-bisphosphonate 4 (prepared as published
before11) was reacted with protected bromohexanol
to generate compound 5, which was deprotected to
yield alcohol 6. Carbonate 9 was obtained by selective
silyl-protection of 7 to afford phenol 8, which was
acylated with 4-nitrophenyl chloroformate. Next, alco-
hol 6 was coupled with carbonate 9 to form com-
pound 10, which was deprotected in the presence of
amberlyst-15 to give alcohol 11. The latter was acyl-
ated with 4-nitrophenyl chloroformate to generate car-
bonate 12, which was coupled with tryptophan to
afford compound 13. Catalytic hydrogenation of 13
removed the benzyl protection groups of the bis-
phosphonate to afford Trypdronate.
As model molecule for a drug with an available amine
functional group, we chose the amino acid (L)-trypto-
phan. The chemical structure of a tryptophan prodrug
(Trypdronate) is shown in Figure 7. The bisphosphonate
The stability of Trypdronate was then evaluated under
physiological conditions. It was incubated in PBS (pH
7.4) at 37 ꢁC and the release of tryptophan was mon-
itored by RP-HPLC. Trypdronate was slowly hydro-
lyzed over seven days to release free tryptophan with
a t1/2 of 90 h (Fig. 10a). Next, we evaluated the bind-
ing ability of Trypdronate in our bone model. An
aqueous solution of Trypdronate was vigorously stir-
red with HAP for 1 h to allow binding of the bis-
phosphonate moiety with the calcium mineral. The
mixture was filtered and the precipitate was washed
with water to remove any unbound Trypdronate.
The precipitate was then incubated in PBS (pH 7.4)
at 37 ꢁC and the hydrolytic release of tryptophan to
the solution was monitored by RP-HPLC. The Try-
pdronate was slowly hydrolyzed over 170 h
(Fig. 10b, blue plot). Free tryptophan has no affinity
for HAP as shown by a control experiment in which
the HAP suspension was mixed with tryptophan and
then filtered and washed. No release of tryptophan
was observed from the HAP precipitate in the control
experiment (Fig. 10b, green). As observed with the
Camdronate, when the Trypdronate/HAP ratio was
Figure 5. Release of camptothecin from HAP-Camdronate under
physiological conditions.
DRUG
Self-immolative Linker
BP
N
H
stable bond
hydrolyzable bond
Figure 6. Schematic structure of a bisphosphonate prodrug of a drug
with an amine functional group.

R. Erez et al. / Bioorg. Med. Chem. Lett. 18 (2008) 816–820
819
OMe
O
H2O
O
H
N
OH
O
OMe
O
P
OH
HN
O
OH
OH
O
P
O
P
CO2H
OH
OH
O
HO
P
OH
OH
Trypdronate
O
CO2
OMe
OH
OMe
HN
O
H2O
NH2
CO2H
Tryptophan
HN
HN
O
MeO
HO
MeO
CO2H
CO2
OH
Figure 8. Proposed mechanism of release of tryptophan via hydrolysis of the carbonate linkage of Trypdronate and spontaneous 1,6-elimination.
O
O
O
P
O
P
BnO
BnO
OTBS
BnO
BnO
P
OH
amberlyst 15
69%
BnO
BnO
OBn
OBn
OTBS
Br
P
BnO
BnO
61%
BnO
BnO
P
P
6
O
4
5
O
OMe
OH
OMe
O
OMe
OMe
OH
OMe
4-nitrophenyl
chloroformate
TBSO
HO
TBSO
TBSCl
84%
O
O
NO2
81%
OMe
8
9
7
OMe
OMe
O
O
O
P
OBn
amberlyst 15
86%
O
O
O
P
DMAP
74%
OBn
OBn
O
OBn
6
9
+
TBSO
OBn
OBn
HO
P
OBn
OBn
OMe
O
P
OMe
O
O
11
10
OMe
O
O
O
4-nitrophenyl
chloroformate
OBn
OBn
Tryptophan
82%
O
P
O2N
O
O
89%
OBn
OBn
O
P
OMe
O
12
OMe
O
O
P
H
OBn
OBn
H2
Pd/C
O
O
N
HN
O
Trypdronate
97%
OBn
O
P
OMe
CO2H
OBn
13
O
Figure 9. Chemical synthesis of Trypdronate.
doubled, the total amount of free tryptophan released
was correspondingly increased (red).
or increase the hydrolysis rate of the Trypdronate
prodrug. We recently demonstrated similar substitu-
ent effects on the disassembly rate of a self-immola-
tive molecular dendritic system.15 Tryptophan was
used as model for a drug with an available amine
functional group, since it is a simple molecule that
was applied before for such a purpose.17 Chemother-
apeutic drugs like doxorubicin would require modifi-
cation in the synthesis of their drug-bisphosphonate
conjugate, since the synthetic pathway presented in
Figure 9 is not entirely compatible with their struc-
ture (for example, the removal of the benzyl protect-
ing groups from 12 to yield 13 under catalytic
hydrogenation conditions will reduce the doxorubicin
The self-immolative linker used in the Trypdronate
design is based on 4-hydroxy-3,5-dimethoxybenzyl-
alcohol (7). This molecule is linked with the bis-
phosphonate component (the nature of the binding
site or its constant affinity is not known18) through
a carbonate linkage that hydrolyzed under physiolog-
ical conditions with a t1/2 of 90 h. The hydrolysis rate
of the carbonate bond can be controlled through the
nature of the substituent on the aromatic ring of the
self-immolative linker 7. Substituents of different elec-
tron-donating/withdrawing capabilities will decrease

820
R. Erez et al. / Bioorg. Med. Chem. Lett. 18 (2008) 816–820
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmcl.2007.11.029.
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Figure 10. (a) Hydrolysis of Trypdronate (blue) to release tryptophan
(red) in PBS (pH 7.4) at 37 ꢁC. (b) Release of tryptophan from HAP/
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molecules with an available hydroxy group. The drug
is attached to the bisphosphonate component through
an ester-labile linkage. The second option is suitable
for drug molecules with available amine functional
group. In this case, a self-immolative linker is used to
join the drug and the bisphosphonate component
through a carbonate-labile linkage. The concept was
demonstrated using camptothecin and tryptophan. Both
prodrugs bound to HAP, a model for bone, and were
hydrolytically activated under physiological conditions.
In the Jurkat cell line, the prodrug Camdronate was
10-fold less toxic than free camptothecin. We are cur-
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