Synthesis of an analogue of the bisphosphonate drug Ibandronate for targeted drug-delivery therapeutic strategies

Article abstract of DOI:10.1039/b9nj00597h

An analogue of the bisphosphonate drug Ibandronate was prepared and coupled via a cleavable ester function to a bromoacetyl linker with specific reactivity for thiol groups. This compound should find useful applications in therapeutic strategies aiming to deliver bisphosphonate drugs specifically to cancer cells making use of proteins as vectors. The specific delivery of bisphosphonates to cancer cells instead of bone, the usual site of accumulation of these cytotoxic drugs, could greatly widen their therapeutic applications.

Full text of DOI:10.1039/b9nj00597h

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PAPER
www.rsc.org/njc | New Journal of Chemistry
Synthesis of an analogue of the bisphosphonate drug Ibandronate
for targeted drug-delivery therapeutic strategieswz
Nicolas Camper, Christopher J. Scott and Marie E. Migaud*
Received (in Montpellier, France) 26th October 2009, Accepted 7th December 2009
First published as an Advance Article on the web 27th January 2010
DOI: 10.1039/b9nj00597h
An analogue of the bisphosphonate drug Ibandronate was prepared and coupled via a cleavable
ester function to a bromoacetyl linker with specific reactivity for thiol groups. This compound
should find useful applications in therapeutic strategies aiming to deliver bisphosphonate drugs
specifically to cancer cells making use of proteins as vectors. The specific delivery of
bisphosphonates to cancer cells instead of bone, the usual site of accumulation of these
cytotoxic drugs, could greatly widen their therapeutic applications.
the treatment of bone metastases.21 The applications of
Introduction
bisphosphonates in anticancer therapy could be greatly
Bisphosphonates are the most commonly used drugs for
widened if a way to redirect bisphosphonates to cancer cells
the treatment of bone diseases.1,2 This specific therapeutic
localised to soft tissue instead of bone could be found.
application results from two particular properties. First,
Conjugating bisphosphonates to vectors specific to cancer
bisphosphonates have a cytotoxic activity.3–6 They are potent
biomarkers could be a way to achieve this goal. Vectorisation
inhibitors of farnesyl pyrophosphate synthase, an enzyme
strategies have proved very useful for rendering drug bio-
involved in the biosynthesis of isoprenoids.7 Inhibition of this
distribution more specific to the site of disease, especially for
cytotoxic anticancer drugs.22,23 Many of these drugs have
enzyme prevents the formation of farnesyl pyrophosphate and
geranylgeranyl pyrophosphate, two isoprenoids used for
a limited therapeutic usefulness because of their lipophilic
protein prenylation. In absence of prenylation, many proteins,
character. This lipophilicity leads to high levels of side-effects
in particular proteins involved in intracellular signalling
due to the accumulation of the drug in non-targeted tissues.
pathways, become mislocalised and non-functional, which
Conjugated to vectors specific for cancer cells, these drugs
leads to apoptosis of the cells.4,8 Secondly, bisphosphonates
have, however, limited non-specific biodistribution and
have a biodistribution specificity for bones.9 The characteristic
reduced side-effects.24–26
bisphosphonate moiety present in all of these drugs gives them
Among the different types of vectors investigated for
affinity for hydroxyapatite, the main component of bone
targeted drug-delivery strategies to cancer cells, proteins and
mineral.10
peptides are some of the most common. Many of the natural
Combined together, these two particular properties make
ligands of receptors overexpressed by cancer cells are proteins
bisphosphonates ideal drugs for the treatment of bone diseases
or peptides, which readily provides vectors with specificity for
as their cytotoxicity is restricted to bone cells. Osteoclasts, cells
these cells. Monoclonal antibodies specific to cancer bio-
involved in bone resorption, are the cells most exposed to
markers have also proved to be valuable vectors for the
targeting of cancer cells.27,28 Their usefulness as vectors for
bisphosphonates and bisphosphonates are thus extensively
used to prevent osteoporosis.11,12 The bone binding property
targeted-drug delivery strategies has been demonstrated by the
approval of Mylotargs, an immunoconjugate composed of
of the bisphosphonate moiety has also been used on its own to
target various molecules to bones. Drugs, hormones and
the cytotoxic drug calicheamicin attached to a monoclonal
growth factors promoting bone formation have been attached
antibody specific to the CD33 receptor, for the treatment of
to bisphosphonates in order to prevent osteoporosis.13–15
acute myeloid leukemia.29 In order to study the possibilities of
Bisphosphonate-conjugated fluorescent and radioactive labels
targeted delivery of bisphosphonates to cancer cells, we have
are also common clinical tools for bone imagery.9,16 On the
thus investigated the synthesis of bisphosphonates suitable for
other hand, the use of bisphosphonates for their cytotoxic
conjugation to proteins.
properties only has not been investigated to the same extent.
For a targeted drug delivery strategy to be successful, drug
Bisphosphonates have, however, an interesting anticancer
and vector must meet some requirements: the drug must be
activity.17–20 Some of them are even approved as drugs for
coupled to the protein without interfering with its target
recognition properties; a mechanism allowing the release of
the drug at the target site must be designed; the drug must
Center for Cancer Research and Cell Biology, Queen’s University of
finally be released in an active form. We tried to address all
these issues in the design of conjugatable bisphosphonates for
targeted drug delivery strategies. Cysteine residues were
selected as sites for the coupling of the bisphosphonate. These
amino acids are relatively rare in proteins, providing specific
Belfast, 97 Lisburn road, Belfast, UK BT9 7BL.
E-mail: m.migaud@qub.ac.uk; Fax: +44 2890972776;
Tel: +44 2890972793
w This article is part of a themed issue on Biophosphates.
z Electronic supplementary information (ESI) available: NMR data
for compounds 1 and 3. See DOI: 10.1039/b9nj00597h
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anchor points for the bisphosphonates. Using recombinant
DNA techniques, they can also be engineered at specific
positions in the protein to avoid interference with the target
binding properties of the protein.30 Finally, several thiol
reactive groups such as maleimides or haloesters allow the
specific conjugation of molecules to proteins.26,31 We also
anticipated an ester linkage to provide an appropriate stability
in vivo as well as a mechanism to release the bisphosphonates
inside cancer cells.13 Esters should be cleaved under the acidic
conditions of the endosomes after internalisation of the
vectorised drug. Finally, an analogue of the commercial drug
Ibandronate12 with an additional alcohol located at the end of
its side-chain was chosen for the vectorisation (Scheme 1). The
structure of this analogue presents many of the characteristic
elements of the pharmacophore required for FPPSase
inhibition. As such, it is anticipated that bisphosphonate 3
will inhibit FPPSase while its alcohol group will provide the
necessary functional group for the formation of an ester
linkage with the carrier protein.32,33
Scheme 2 Formation of the bisphosphonate moiety 6.
Its synthesis involved the formation of the bisphosphonate
moiety 6. Protection of the bisphosphonic acid moiety as a
bisphosphonate tetraester was required due to the difficulty of
purification of highly polar bisphosphonic acids. The aldehyde
6 was obtained in three steps from 2-bromoethyl-1,3-dioxolane
(Scheme 2). The Michaelis-Arbusov reaction of this product
with triethyl phosphite afforded the phosphonate 4 in 62%
yield after 24 h heating at 110 1C.34,35 To complete the
formation of the gem-bisphosphonate moiety, phosphonate 4
was treated with n-BuLi and the resulting carbanion reacted
with diethyl chlorophosphate to give the bisphosphonate
product 5 in 48% yield.35 As this phosphonoalkylation
reaction was gradually inactivated by the acido-basic reaction
between the phosphonate carbanion and the bisphosphonate
product 5, two equivalents of n-BuLi were required in total to
achieve good conversion rates. Deprotection of the cyclic
acetal 5 under acidic conditions (2 M HCl : acetone 10 : 1/
50 1C/3 h) gave the aldehyde 6 in 78% yield.
Results and discussion
Compound 10, a key intermediate in the synthesis of an
Ibandronate analogue attached to a linker with thiol specific
reactivity, was our first synthetic target.
The second part of the synthesis of compound 10
consisted of the formation of the side-chain of the Ibandronate
analogue and its coupling to the bisphosphonate 6. N-Methyl-
5-benzyloxypentamine
8 was synthesized in two steps
by Dess–Martin oxidation36 of 5-benzyloxypentanol into
5-benzyloxypentanal 7 (82%) followed by reductive amination
of this product with methylamine using sodium triacetoxy-
borohydride as a reducing agent (Scheme 3).37 N-Methyl-5-
benzyloxypentamine 8 was isolated in 51% yield after alumina
gel chromatography.
Similar reductive amination conditions were used to
couple N-methyl-5-benzyloxypentamine 8 to the aldehyde 6
(Scheme 4). Compound 9 was isolated in 74% yield after
purification by column chromatography. The alcohol 10 was
Scheme 1 Structure of Ibandronate and of its analogue functionalised
for targeted drug-delivery strategies.
Scheme 3 Synthesis of the side-chain of the Ibandronate analogue 1.
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the presence of pyridine and gave the ester 12 in 60% yield
after purification by silica gel chromatography (Scheme 6).
The bisphosphonate group of compound 12 was deprotected
by silyl-dealkylation (TMSBr (20 equiv.)/dry DCM/25 1C/72 h,
followed by methanolysis) to give almost quantitatively the
linker attached bisphosphonate 3.
No other side reaction was observed under these conditions.
Bisphosphonate 3 was, however, prone to degradation when
stored under non-buffered conditions, the acidity of the
bisphosphonic acid group causing the hydrolysis of the ester
group linking the drug to the bromoacetyl linker.
Conclusions
Scheme 4 Synthesis of compound 10, a key intermediate in the
synthesis of Ibandronate analogues functionalised for targeted drug
delivery.
We have developed an effective synthetic sequence that gives
access to conjugatable bisphosphonates and successfully
synthesised an activated bisphosphonate analogue of
Ibandronate. The conjugation of the bromoacetyl linker
coupled Ibandronate analogue 12 to a protein carrier and
the biological activity of both the free Ibandronate analogue 1
and its vectorised form will be reported in due course to
demonstrate the broad potential of vectorised bisphosphonates
in anticancer therapy.
finally obtained in 39% yield after hydrogenation with a
palladium catalyst.
Different approaches then had to be investigated in order to
couple this Ibandronate analogue to a thiol reactive linker.
The type of linker, the conditions used for the deprotection
of the bisphosphonate moiety and the order of the linker
coupling and bisphosphonate deprotection reactions were
varied in order to find conditions allowing the formation of
an Ibandronate analogue coupled to a thiol reactive linker.
In a first attempt, a linker with a maleimide group, a
frequently used thiol reactive moiety,24 was used. N-Maleimido-
propionic acid, a commercial product, was turned into its acyl
chloride derivative using thionyl chloride and reacted directly
with the alcohol 10 in the presence of triethylamine to form the
ester 11 in 70% yield (Scheme 5). The bisphosphonate group
of compound 11 could be deprotected by silyl-dealkylation
using a large excess of TMSBr (20 equiv.) in dry DCM for
72 h.38 However, using these deprotection conditions, an
addition of bromide also occurred on the maleimide
double bond.
Experimental
Chemicals were purchased from Sigma Aldrich. Solvents were,
when necessary, dried and stored for up to three weeks on
molecular sieves. DCM and Et3N were dried over CaCl2, and
THF over Na/benzophenone. DMF was dried by storing a
fresh bottle of solvent over activated molecular sieves under
argon. All reactions were done under argon atmosphere and
monitored on commercially available pre-coated TLC plates
(layer thickness 0.25 mm) of Kieselgel 60 F254. Compounds
were visualised by use of a UV lamp and/or a suitable
dipping solution and heating. Column chromatography was
performed either manually or using a Biotage System with
Merck 60 (40–60 mm) silica gel or aluminium oxide (activated,
neutral, Brockmann grade I) as the solid phase. Mass spectro-
metric data (MS) were obtained by electrospray (ES) on a
A different approach to the bisphosphonate deprotection
and the linker coupling was also investigated using the same
N-maleimidopropionic acid linker. In this approach, the
bisphosphonate group of the alcohol 10 was deprotected
under strong acidic conditions (6 M HCl /reflux /12 h)
(Scheme 5).39 If the bisphosphonate group of compound 10
was cleanly deprotected into a bisphosphonic acid, an
additional modification occurred again: about 60% of the
alcohol group was converted into a chloride. However, this
chloride could be completely converted back into an alcohol
by simply heating to reflux the mixture of alcohol and chloride
in distilled water for 24 h. The Ibandronate analogue 1 could
be obtained in near quantitative yields with this method.
However, attempts to esterify the deprotected Ibandronate
analogue 1 using conditions similar to the ones used for the
formation of amide bonds in water (HBTU (1.1 equiv.)/
triethylamine (2.2 equiv.)/dry DMF/r.t./O/N) remained
unsuccessful.40
Waters LCT Premier Mass Spectrometer. The 1H, 13C and 31
P
NMR spectra were recorded in CDCl3 and D2O on Bruker
Avance DRX 500 or DRX 300 spectrometers. TMS (0 ppm,
1H NMR), CHCl3 (77 ppm, 13C) and triethyl phosphate
(0.2 ppm, 31P NMR) were used as internal references. The
chemical shifts (d) are reported in ppm (parts per million) and
the coupling constants (J values) in Hz.
Diethyl 2-(1,3-dioxolan-2-yl)ethylphosphonate 4
Triethyl phosphite (3.43 mL, 20 mmol, 1 equiv.) was added
dropwise to neat 2-(2-bromoethyl)-1,3-dioxolane (4.70 mL,
40 mmol, 2 equiv.) at r.t. under argon. The reaction mixture
was heated to 110 1C until the complete disappearance of
triethyl phosphite was confirmed by 31P NMR analysis of the
reaction mixture. Purification of the crude product by silica
gel column chromatography (MeOH–DCM, 0 : 100–5 : 95)
afforded an inseparable mixture of 4, diethyl ethylphosphonate
and diethyl H-phosphonate, which was distilled under reduced
pressure. Pure product 4 (2.93 g, 62%) was obtained as a pale
The use of a-haloacyl linkers, alternative linkers with thiol
specific reactivity,26 was considered as a possible solution to
attach an Ibandronate analogue to a thiol reactive linker. The
alcohol 10 was thus esterified with bromoacetyl bromide13 in
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Scheme 5 Strategies investigated for the coupling of a N-maleimidopropionic acid linker to the Ibandronate analogue 1.
3
OCH2CH3), 2.18 (2 H, tt, JP–C–C–H 17, J 6, P2CHCH2),
oil after vacuum distillation of diethyl H-phosphonate (46 1C
at 1 mmHg) and diethyl ethylphosphonate (60 1C at 1 mmHg).
dP(121 MHz; CDCl3; (EtO)3P(O)) +33.2; dH(300 MHz;
CDCl3; Me4Si) 1.32 (6 H, t, J 7, OCH2CH3), 1.79-2.00 (4 H,
m, PCH2CH2), 3.84-3.99 (4 H, m, OCH2CH2O), 4.06-4.15
(4 H, m, OCH2CH3), 4.95 (1 H, t, J 4, PCH2CH2CHO2);
dC(75.5 MHz; CDCl3; CHCl3) 16.8 (2 C, d, 3JP–O–C–C 6), 19.9
(d, 1JP–C 144), 27.3 (d, 2JP–C–C 4), 61.9 (2 C, d, 2JP–O–C 6), 65.5
2
2.63 (1 H, tt, JP–C–H 24, J 6, P2CHCH2), 3.85-3.99 (4 H, m,
OCH2CH2O), 4.13-4.24 (8 H, m, OCH2CH3), 5.25 (1 H, t, J 6,
P2CHCH2CHO2); dC(75.5 MHz; CDCl3; CHCl3) 16.8 (4 C, d,
2
1
3JP–O–C–C 7), 30.5 (t, JP–C–C 5), 32.8 (t, JP–C 134), 63.0
2
(2 C, d, JP–O–C 7), 63.1 (2 C, d, JP–O–C 7), 65.2 (2 C, s),
2
3
102.8 (t, JP–C–C–C 8); MS m/z (positive, ES) calcd for
C13H28O8NaP2 [M + Na]+ 397.1157 found 397.1171.
3
(2 C, s), 103.7 (d, JP–C–C–C 19); MS m/z (positive, ES) calcd
for C9H19O5NaP [M + Na]+ 261.0875 found 261.0868.
Tetraethyl 3-oxopropane-1,1-diyldiphosphonate 6
A solution of dioxolane 5 (2.68 g, 7.15 mmol) in a 10 : 1 2 M
HClaq : acetone mixture (55 mL) was heated to 50 1C for 3 h.
The reaction mixture was cooled down to r.t.. Volatiles were
evaporated in vacuo. The reaction mixture was then extracted
with DCM (3 ꢂ 50 mL). The organic layers were combined,
dried over MgSO4 and concentrated under vacuum to give the
pure aldehyde 6 (1.85 g, 78%) as a pale oil. dP(121 MHz;
CDCl3; (EtO)3P(O)) +23.3; dH(300 MHz; CDCl3; Me4Si) 1.32
(6 H, t, J 7, OCH2CH3), 1.32 (6 H, t, J 7, OCH2CH3), 2.93-
3.25 (3 H, m, P2CHCH2) 4.11-4.22 (8 H, m, OCH2CH3), 9.72
(1 H, t, J 1, C(O)H); dC(75.5 MHz; CDCl3; CHCl3) 16.6
Tetraethyl 2-(1,3-dioxolan-2-yl)ethane-1,1-diyldiphosphonate 5
n-BuLi (3.44 mL of a 1.6 M solution in hexane, 5.5 mmol,
1.1 equiv.) was added dropwise to a solution of phosphonate 4
(1.19 g, 5.0 mmol, 1 equiv.) in dry THF (30 mL) under argon,
cooled down to ꢁ78 1C. After stirring for 1 h at ꢁ78 1C,
diethyl chlorophosphate (759 mL, 5.25 mmol, 1.05 equiv.) was
added to the reaction mixture. After warming to r.t. overnight,
the reaction mixture was cooled down again to ꢁ78 1C. n-BuLi
(3.0 mL of a 1.6 M solution in hexane, 4.8 mmol, 0.95 equiv.)
was added dropwise to the reaction mixture followed, after 1 h
stirring at –78 1C, by diethyl chlorophosphate (380 mL,
2.63 mmol, 0.53 equiv.). The reaction mixture was allowed to
warm to r.t. overnight. A saturated aqueous NH4Cl solution
(30 mL) was used to quench the reaction mixture. The aqueous
layer was extracted with diethyl ether (3 ꢂ 40 mL). The organic
layers were combined, dried over MgSO4 and concentrated
under vacuum to give 1.59 g of crude product. Purification
by silica gel column chromatography (MeOH–DCM,
1 : 99–5 : 95) afforded the pure bisphosphonate 5 (907 mg,
48%) as a yellow oil. dP(121 MHz; CDCl3; (EtO)3P(O))
+24.4; dH(300 MHz; CDCl3; Me4Si) 1.34 (12 H, t, J 7,
3
1
2
(4 C, d, JP–O–C–C 6), 30.2 (t, JP–C 140), 39.5 (t, JP–C–C 7),
2
2
63.2 (2 C, d, JP–O–C 7), 63.4 (2 C, d, JP–O–C 7), 197.7
3
(t, JP–C–C–C 8); MS m/z (positive, ES) calcd for C11H25O7P2
[M + H]+ 331.1076 found 331.1086; [M + Na]+ 353.0895
found 353.0911.
5-Benzyloxypentan-1-al 7
Dess–Martin periodinane (2.67 g, 6.3 mmol, 1.05 equiv.) was
added portionwise to a solution of 5-benzyloxypentan-1-ol
(1.16 mL, 6 mmol, 1 equiv.) in DCM (60 mL) at 0 1C. The
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was added after 4 d stirring at r.t. After another day stirring at
r.t., a 1 M NaOH aqueous solution (100 mL) was added to the
reaction mixture and stirred until complete dissolution of
all precipitates. The solution was extracted with diethyl ether
(3 ꢂ 50 mL). Organic layers were combined, dried over
MgSO4 and concentrated under vacuum to afford the crude
amine (1.68 g). Purification by alumina gel column chromato-
graphy (MeOH–DCM, 0 : 100–10 : 90) afforded the pure amine
8 (870 mg, 51%). dH(300 MHz; CDCl3; Me4Si) 1.35-1.56 (4 H,
m, CH2CH2), 1.64 (2 H, quintet, J 7, CH2), 2.42 (3 H, s, CH3),
2.57 (2 H, t, J 7, CH2N), 3.47 (2 H, t, J 7, OCH2), 4.50 (2 H, s,
PhCH2), 7.27-7.36 (5 H, m, Ph); dC(75.5 MHz; CDCl3; CHCl3)
22.9, 28.6, 35.4, 51.0, 69.3, 71.9, 126.5, 126.6 (2 C, s), 127.3
(2 C, s), 137.6; MS m/z (positive, ES) calcd for C13H22NO
[M + H]+ 208.1701 found 208.1698.
Tetraethyl 3-((5-(benzyloxy)pentyl)(methyl)amino)propane-1,1-
diyldiphosphonate 9
Sodium triacetoxyborohydride (1.34 g, 6.3 mmol, 1.5 equiv.)
was added portionwise to a stirred solution of 5-(benzyloxy)-
N-methylpentan-1-amine 8 (870 mg, 4.2 mmol, 1 equiv.) and
aldehyde 6 (1.45 g, 4.4 mmol, 1.1 equiv.) in dry DCM (50 mL)
under argon. After 4 h stirring at r.t., a 1 M NaOH aqueous
solution (50 mL) was added to the reaction mixture and stirred
until complete dissolution of all precipitates. The aqueous
layer was extracted with DCM (3 ꢂ 50 mL). Organic layers
were combined, dried over MgSO4 and concentrated under
vacuum to afford the crude amine (2.09 g). Purification by
alumina gel column chromatography (MeOH–DCM,
0 : 100–10 : 90) afforded the pure amine 9 (1.54 g, 74%).
dP(121 MHz; CDCl3; (EtO)3P(O)) +25.7; dH(300 MHz;
CDCl3; Me4Si) 1.33 (12 H, t, J 7, OCH2CH3), 1.37-1.52
(4 H, m, CH2CH2), 1.63 (2 H, quintet, J 7, CH2), 1.96-2.14
(2 H, m, P2CHCH2), 2.19 (3 H, s, CH3), 2.34 (2 H, t, J 7,
P2CHCH2CH2N), 2.54 (2 H, t, J 7, CH2N), 2.66 (1 H, tt,
2JP–C–H 24, J 6, P2CH), 3.46 (2 H, t, J 7, OCH2), 4.11-4.22
(8 H, m, OCH2CH3), 4.50 (2 H, s, PhCH2O), 7.27-7.37 (5 H, m,
Scheme 6 Coupling of a bromoacetic acid linker to the Ibandronate
analogue 1.
reaction mixture was allowed to warm to r.t. and stirred for
2 h. A 1 M NaOH aqueous solution (60 mL) was then added
to the reaction mixture and stirred until complete dissolution
of all precipitates from the organic layer. The aqueous layer
was extracted with DCM (2 ꢂ 30 mL). The organic layers were
combined, washed with 1 M NaOHaq (30 mL) and brine
(2 ꢂ 30 mL), dried over MgSO4 and concentrated under
vacuum to afford the crude product (1.26 g) as a clear oil.
3
Ph); dC(75.5 MHz; CDCl3; CHCl3) 16.8 (4 C, d, JP–O–C–C 6),
2
1
23.6 (t, JP–C–C 5), 24.5, 27.6, 30.2, 33.7 (t, JP–C 134), 42.1,
56.1 (t, 3JP–C–C–C 7), 58.1, 62.7 (2 C, d, 2JP–O–C 7), 62.9 (2 C, d,
2JP–O–C 7), 70.8, 73.3, 127.9, 128.0 (2 C, s), 128.7 (2 ꢂ s), 139.1;
MS m/z (positive, ES) calcd for C24H46NO7P2 [M + H]+
522.2750 found 522.2742.
Pure aldehyde
7 (942 mg, 82%) was obtained after
purification by silica gel column chromatography (EtOAc : PE,
33 : 67–100 : 0). dH(300 MHz; CDCl3; Me4Si) 1.60-1.80 (4 H,
m, CH2CH2), 2.46 (2 H, td, J1 7, J2 2, CH2C(O)H), 3.49 (2 H,
t, J 6, OCH2), 4.50 (2 H, s, PhCH2), 7.27-7.38 (5 H, m, Ph),
9.76 (1H, t, J 2, C(O)H); dC(75.5 MHz; CDCl3; CHCl3) 19.4,
29.6, 44.0, 70.2, 73.4, 128.0, 128.0 (2 C, s), 128.8 (2 C, s),
138.9, 202.9.
Tetraethyl 3-((5-hydroxypentyl)(methyl)amino)propane-1,1-
diyldiphosphonate 10
10% Pd/C (6.12 g, 2.88 mmol, 1.5 equiv.) was added to a
stirred solution of the bisphosphonate 9 (1.0 g, 1.92 mmol,
1 equiv.) in EtOAc under argon. After three vacuum/H2 cycles
to remove argon from the reaction flask, 9 was hydrogenated
(balloon) at r.t. overnight under vigorous stirring. The reaction
mixture was then filtered on a celite pad and the filtrate
concentrated under vacuum to afford pure alcohol 10
(323 mg, 39%) as a pale oil. dP(121 MHz; CDCl3; (EtO)3P(O))
+25.7; dH(300 MHz; CDCl3; Me4Si) 1.34 (12 H, t, J 7,
OCH2CH3), 1.37-1.52 (4 H, m, CH2-CH2), 1.58 (2 H, quintet,
J 7, CH2), 2.06 (2 H, tq, 3JP–C–C–H 24, J 6.5, P2CHCH2), 2.19
(3 H, s, CH3), 2.35 (2 H, t, J 6.5, P2CHCH2CH2N), 2.55
5-(Benzyloxy)-N-methylpentan-1-amine 8
Sodium triacetoxyborohydride (2.42 g, 11.4 mmol, 1.4 equiv.)
was added portionwise to a stirred solution of 5-benzyloxy-
pentan-1-al 7 (1.57 g, 8.2 mmol, 1 equiv.) and methylamine
(41 mL of a 2 M solution in THF, 82 mmol, 10 equiv.) under
1
argon. Reaction progress was monitored by H NMR. More
sodium triacetoxyborohydride (2.42 g, 11.4 mmol, 1.4 equiv.)
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2
(2 H, t, J 7, CH2N), 2.71 (tt, JP–C–H 24, J 6.5, P2CH), 3.63
3.63 (2 H, t, J 7, CH2OH); dC(75.5 MHz; D2O) 21.0 (br),
1 2
(2 H, t, J 7, CH2OH), 4.11-4.22 (8 H, m, OCH2CH3); dC(75.5
MHz; CDCl3; CHCl3) 16.8 (4 C, d, JP–O–C–C 6), 22.6
22.5, 23.7, 31.1, 36.5 (t, JP–C 124), 40.0, 55.9 (t, JP–C–C 7),
56.6, 61.7; MS m/z (positive, ES) calcd for C9H23NO7P2Na
[M + Na]+ 342.0847 found 342.0840; calcd for CH22NO7P2Na2
[M ꢁ H + 2Na]+ 364.0667 found 364.0665.
3
2
(t, JP–C–C 4.5), 23.7, 26.0, 32.6, 33.9 (t, JP–C 134), 41.6,
1
3
55.3 (t, JP–C–C–C 7), 57.2, 62.7, 63.0 (2 C, d, JP–O–C 6.5Hz),
2
2
63.2 (2 C, d, JP–O–C 6.5); MS m/z (positive, ES) calcd for
C17H40NO7P2 [M + H]+ 432.2280 found 432.2269.
5-((3,3-Bis(diethoxyphosphoryl)propyl)(methyl)amino)-pentyl
2-bromoacetate 12
N-Maleimidopropionyl chloride
Bromoacetyl bromide (6.4 mL, 74 mmol, 1.1 equiv.) was added
dropwise to a stirred solution of the alcohol 10 (29 mg,
67 mmol, 1 equiv.) and pyridine (6.5 mL, 81 mmol, 1.2 equiv.)
in dry DCM (4 mL) cooled down to 0 1C under argon. The
reaction mixture was allowed to warm to r.t. and stirred for
1 h. The reaction mixture was concentrated under vacuum to
give the crude ester 12 (58 mg). Purification by silica gel
column chromatography (MeOH–DCM, 0 : 100–10 : 90)
afforded the pure ester 12 (22 mg, 60%). dP(121 MHz; CDCl3;
(EtO)3P(O)) +22.6; dH(300 MHz; CDCl3; Me4Si) 1.36 (12 H,
t, J 7, OCH2CH3), 1.46 (2 H, quintet, J 7, CH2), 1.73 (2 H,
quintet, J 7, CH2), 1.94 (2 H, br s, CH2), 2.46 (2 H, br s,
P2CHCH2), 2.62 (1 H, tt, 2JP–C–H 24, J 6, P2CH), 2.78 (3 H, s,
CH3), 2.93-3.15 (2 H, br m, P2CHCH2CH2N), 3.27-3.58
(2 H, br m, CH2N), 3.84 (2 H, s, BrCH2), 4.18 (2 H, t,
J 6, C(O)OCH2), 4.21 (8 H, quintet, J 7, OCH2CH3);
dC(75.5 MHz; CDCl3; CHCl3) 16.6 (4 C, d, 3JP–O–C–C 6), 20.7
Thionyl chloride (140 mL, 1.92 mmol, 8 equiv.) was added
dropwise to a solution of 3-maleimidopropionic acid (40 mg,
0.24 mmol, 1 equiv.) in dry DCM under argon. The reaction
mixture was heated to reflux for 20 h. Concentration of the
reaction mixture under vacuum afforded pure N-maleimido-
propionylchloride (44 mg, 100%) as a pale oil crystallizing
over time. dH(300 MHz; CDCl3; Me4Si) 3.25 (2 H, t, J 7,
CH2C(O)Cl), 3.86 (2 H, t, J 7, NCH2), 6.73 (2 H, s, CHQCH);
dC(75.5 MHz; CDCl3; CHCl3), 33.2, 45.0, 134.5 (2 C, s), 170.1
(2 C, s), 171.5.
5-((3,3-Bis(diethoxyphosphoryl)propyl)(methyl)amino)-pentyl
3-(2,5-dioxo-2H-pyrrol-1(5H)-yl) propanoate 11
N-Maleimidopropionyl chloride (24 mg, 0.129 mmol, 1.1 equiv.)
was added to a solution of the alcohol 10 (50 mg, 0.116 mmol,
1 equiv.) and triethylamine (49 mL, 0.349 mmol, 3 equiv.) in
dry DCM. The reaction mixture was stirred overnight at r.t.
Concentration of the reaction mixture under vacuum afforded
the crude ester 11 (113 mg). Purification by silica gel column
chromatography (MeOH–DCM, 0 : 100–10 : 90) afforded the
pure ester 11 (43 mg, 70%). dP(121 MHz; CDCl3; (EtO)3P(O))
+25.6; dH(300 MHz; CDCl3; Me4Si) 1.34 (14 H, t, J 7,
OCH2CH3+CH2), 1.48 (2 H, br s, CH2), 1.63 (2 H, quintet,
J 7, CH2), 2.08 (2 H, br s, P2CHCH2), 2.20 (3 H, br s, CH3),
2.34 (2 H, br s, CH2N), 2.55 (2 H, br s, P2CHCH2CH2N), 2.64
2
1
(t, JP–C–C 5), 23.4, 26.0, 27.0, 28.0, 34.3 (t, JP–C 134), 40.1,
54.4 (t, 3JP–C–C–C 6.5), 56.2, 63.5 (4 C, br s), 65.6, 167.4; MS m/z
(positive, ES) calcd for C19H41NO8P2Br [M + H]+ 552.1491
found 552.1533.
3-((5-(2-Bromoacetoxy)pentyl)(methyl)amino)propane-1,1-
diyldiphosphonic acid 3
Bromotrimethylsilane (120 mL, 0.91 mmol, 24 equiv.) was
added dropwise to a stirred solution of tetraethyl bisphos-
phonate 12 (21 mg, 38 mmol, 1 equiv.) in dry DCM (5 mL)
under argon. The reaction mixture was stirred at 25 1C in the
dark under argon for 3 d. The reaction mixture was then
concentrated under vacuum. The concentrate was solvolysed
with methanol (2 mL) at r.t. for 30 min and concentrated again
under vacuum. The concentrate was dissolved in distilled
water (2 mL). The aqueous layer was washed with DCM
(4 ꢂ 2 mL) and freeze-dried to afford the bisphosphonic acid
3 (17 mg, 95%) as a sticky gum. dP(121 MHz; D2O;
(EtO)3P(O)) +20.7; dH(300 MHz; D2O) 1.41 (2 H, quintet,
J 7, CH2), 1.64-1.77 (4 H, m, CH2 + CH2), 2.13-2.33 (3 H, br m,
P2CH + P2CHCH2), 2.83 (3 H, s, CH3), 3.11 (1 H, m,
CHAHBN), 3.19 (1 H, m, CHAHBN), 3.34 (1 H, m,
P2CHCH2CHACHBN), 3.44 (1 H, m, P2CHCH2CHACHBN),
3.99 (2 H, s, CH2Br), 4.24 (2 H, t, J 6, C(O)OCH2);
dC(75.5 MHz; D2O) 21.0 (br s), 22.6, 23.6, 26.9, 27.5, 36.4
2
(2 H, t, J 7, NCH2CH2C(O)O), 2.66 (1 H, tt, JP–C–H 24,
J 6, P2CH), 3.83 (2 H, t, J 7, NCH2CH2C(O)O), 4.06 (2 H, t,
J 7, C(O)OCH2), 4.12–4.23 (8 H, m, OCH2CH3), 6.72
(2 H, s, CHQCH); dC(75.5 MHz; CDCl3; CHCl3) 16.6
3
(4 C, d, JP–O–C–C 4), 23.3, 23.9, 27.1, 28.6, 33.1, 33.5
1
2
(t, JP–C 133), 33.8, 41.8, 55.9, 57.7, 62.5 (4 C, d, JP–O–C 24),
65.0, 134.4 (2 C, s), 170.5 (2 C, s), 170.9; MS m/z
(positive, ES) calcd for C24H45N2O10P2 [M + H]+ 583.2549
found 583.2560.
3-((5-Hydroxypentyl)(methyl)amino)propane-1,1-diyldi-phosphonic
acid 1
Tetraethyl bisphosphonate 10 (15 mg, 0.035 mmol, 1 equiv.)
was dissolved in a 6 M HCl aqueous solution (5 mL)
and heated to reflux for 20 h. The reaction mixture was
concentrated under vacuum. The concentrate was dissolved
in distilled water (5 mL) and heated to reflux for 20 h.
Concentration of the reaction mixture under vacuum afforded
pure bisphosphonic acid 1 (11 mg, 95%). dP(121 MHz; D2O;
(EtO)3P(O)) +21.2; dH(300 MHz; D2O) 1.45 (2 H, q, J 8,
CH2), 1.62 (quintet, J 7, CH2), 1.73-1.85 (2 H, br m, CH2),
2.36 (3 H, br s, P2CH + P2CHCH2), 2.91 (3 H, s, CH3), 3.13
(1 H, m, CHAHBN), 3.24 (1 H, m, CHAHBN), 3.42 (1 H, m,
P2CHCH2CHAHBN), 3.52 (1 H, m, P2CHCH2CHAHBN),
1
(t, JP–C 124), 40.0, 55.9 (br s), 56.5, 67.1, 170.6.
Acknowledgements
We are grateful to Prof. J. A. Johnston (Center for Cancer
Research and Cell Biology, Queen’s University of Belfast,
UK) for his valuable advice. This work was funded by the
European Social Fund.
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This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2010
954 | New J. Chem., 2010, 34, 949–955

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