Synthesis and hydroxyapatite binding activity of bisphosphonate-statin conjugates

Article abstract of DOI:10.2174/157017809788681356

To develop a methodology for delivering anabolic agents statins to bone, a bone targeting bisphosphonate was used as a carrier of drugs. Four statin-bisphosphonate conjugates were synthesized and examined for their bone mineral affinity in vitro by hydrox

Full text of DOI:10.2174/157017809788681356

Letters in Organic Chemistry, 2009, 6, 419-423
419
Synthesis and Hydroxyapatite Binding Activity of Bisphosphonate-Statin
Conjugates
Guangyu Xu*,a, Gaolei Zuoa, Shihua Zhonga and Wei Zhangb
aCollege of Chemistry and Chemical Engineering, Hunan Normal University, Changsha, China
bCollege of Medicine, Hunan Normal University, Changsha, China
Received December 29, 2008: Revised May 07, 2009: Accepted May 13, 2009
Abstract: To develop a methodology for delivering anabolic agents statins to bone, a bone targeting bisphosphonate was
used as a carrier of drugs. Four statin-bisphosphonate conjugates were synthesized and examined for their bone mineral
affinity in vitro by hydroxyapatite absorption method. The conjugates showed high binding activity with HAP, implying
the modified statins possess the potential of bone targeting property.
Keywords: Statins, bisphosphonates, conjugate, bone targeting, hydroxyapatite.
INTRODUCTION
tissue [4], have been used to target non-specific bone
therapeutic agents to bone [5].
The development of new anabolic agents for the treatment
of osteoporosis is an area of intense focus in drug discovery
[1]. Mundy G. and his colleagues reported that statins, 3-
Here we choose bisphosphonate as bone-targeting carrier
and plan to conjugate with statins through amide bond. The
structures of conjugates are shown in Fig. (1). Hydroxy-
apatite as the principal inorganic component of bone
represents a promising target for the selective drug delivery
to bone. In this paper a hydroxyapatite binding assay was
established and the affinity of the statin-bisphosphonate
congjugates for HAP is reported.
hydroxy-3-methylglutaryl coenzyme
A
(HMG CoA)
reductase inhibitor, can stimulate bone formation in vitro and
in vivo [2]. Though oral administration of simvastatin
seemed to be effective in rats, the statins now available are
probably unsuitable due to their low systemic bioavailability.
Statins do not localize preferentially to bones. After
F
O
ONa
ONa
ONa
P
P
O
OH
OH
O
O
ONa
P
P
OH
OH
O
n
ONa
ONa
N
N
N
H
H
n
ONa
N
O
H
N
ONa
O
2a n=0
2b n=2
1a n=0
1b n=2
F
Fig. (1). Chemical structure of statin-bisphosphonate conjugates.
absorption, most of them are biotransformed in the liver. As
a consequence, only trace amount of statins may finally
reach the bone after the hepatic first-pass metabolism [3]. If
bone-targeting moiety conjugated to statin could help deliver
the statin-conjugate to the osseous tissue, where statin will
be released through the hydrolysis of the linkage bond, the
concentration inside the bone could be improved and more
information about the effect of statins on bone could be
obtained.
RESULTS AND DISCUSSION
Initial attempts to synthesize amide 7 by direct
aminolysis of commercially available fluvastatin tertbutyl
ester 3 with aminobisphosphonate 4a [6, 7] or 4b [8] resulted
in little or no desired product. The alternative efforts to
condense fluvastatin acid 5 with 4 in the presence of N, N-
dicyclohexylcarbodiimide or N, N-carbonydiimidazole only
gave the dehydrated product of fluvastatin acid as major
product (Scheme 1).
Bisphosphonates (BPs), chemical analogues of pyrophos-
phonate with strong, near-irreversible affinity for the osseous
The successful synthesis of conjugates 1 is outlined in
Scheme 2. Firstly, fluvastatin acid 5 was converted into
lactone 6 in refluxing toluene [9]. However the chiral center
at the vinyl position undergoes epimerization by a Sn1
mechanism, the enantiomer that chiral center was not
changed can be separated by column chromatography.
*Address correspondence to this author at the College of Chemistry and
Chemical Engineering, Hunan Normal University, Changsha, 410081, P. R.
China; Fax: +86(731) 8872531; E-mail: gyxu@hunnu.edu.cn
1570-1786/09 $55.00+.00
© 2009 Bentham Science Publishers Ltd.

420 Letters in Organic Chemistry, 2009, Vol. 6, No. 5
Xu et al.
F
O
OEt
F
P
P
OEt
O
O
OEt
4a n=0
4b n=2
OEt
OEt
n
P
OH
OH
O
H2N
OEt
OH
OH
O
O
OEt
P
OEt
n
N
H
N
O
N
7a n=0
7b n=2
3
F
1) KOH
H
CDI
2)
DCC or
C
l
4
OH
OH
O
OH
N
5
Scheme 1.
F
F
O
OEt
P
P
OEt
4a n=0
4b n=2
OEt
OEt
n
H2N
OH
OH
O
O
OH
TsOH
OH
reflux
THF
N
O
N
5
O
6
F
F
O
O
O
ONa
OEt
P
P
P
P
OH
OH
O
OH
OH
O
1) TMSBr,
collidine
ONa
ONa
OEt
OEt
n
n
N
N
H
2) NaOH
H
N
N
ONa
OEt
O
7a n=0
7b n=2
1a n=0
1b n=2
Scheme 2.
O
OH
OH
O
N
O
H
OH
N
TsOH
reflux
1) KOH
2) HCl
4
N
O
N
O
H
THF
60 ˚C
O
8
9
F
F
O
ONa
ONa
ONa
O
OEt
OEt
OEt
P
P
O
OH
OH
O
P
P
O
OH
OH
O
1) TMSBr,
collidine
n
N
N
N
n
N
N
N
H
H
H
H
ONa
O
2) NaOH
OEt
O
2a n=0
2b n=2
10a n=0
10b n=2
F
F
Scheme 3.

Synthesis and Hydroxyapatite Binding Activity
Letters in Organic Chemistry, 2009, Vol. 6, No. 5
421
Table 1. Results of Affinity of Statin-Bisphosphonate Conjugates on HAP Powder
Wavelength
peaks
Before
absorption
After 5mg HA
addition
Absorption ratio
After 10mg HA
addition
Absorption Ratio
Compound
(%)
(%)
1a
1b
2a
2b
242nm
242nm
232nm
220nm
50 ꢀg/ml
50ꢀg/ml
50ꢀg/ml
50ꢀg/ml
22.5ꢀg/ml
26.7ꢀg/ml
14.2ꢀg/ml
19.3ꢀg/ml
55.0
46.6
71.6
61.4
11.4ꢀg/ml
13.1ꢀg/ml
8.7ꢀg/ml
77.2
73.8
82.6
77.4
11.3ꢀg/ml
Aminolysis of lactone 6 with aminobisphosphonates 4 in
tetrahydrofuran at 60oC gave the desired amides 7, which
were deprotected with trimethylsilylbromide (20 equivalent)
[10] in the presence of 2,4,6-collidine to afford the free
fluvastatin-bisphosphonic acids. Addition of excess of 2,4,6-
collidine was beneficial to the fully dealkylation [11]. The
bisphosphonic acids were converted into the corresponding
sodium salts 1a by addition of 4 equivalence of 0.25 M
NaOH. The Atorvastatin-bisphosphonates 2 were obtained
from atorvastatin lactone 9 and aminobisphosphonates 4
under similar conditions as shown in Scheme 3.
Waters Q-TOF mass spectrometer. Microanalyses were
carried on a Leco CHN-2000 elemental analyzer.
Tetraethyl N-((3R,5S,E)-7-(3-(4-fluorophenyl)-1-isopro-
pyl)-1H-indol-2-yl)- 3,5-dihydroxyhept-6-enoyl)aminome-
thylenebisphosphonate (7a)
A solution of statin lactone 6 (166mg, 0.4mmol) in 3ml
THF was treated with aminomethylenebisphosphonate 4a
(228mg, 0.75mmol) at room temperature. The mixture was
then heated to 60oC and stirred for 48h. The solvent was
evaporated under vacuum, and the residue was dissolved in
10 ml EtOAc. The organic phase was washed with diluted
HCl (5ꢀ3 ml) and water (5ꢀ3 ml) and dried with Na2SO4.
After removal of the solvent, the residue gum was purified
by chromatography on a silica gel column (EtOAc:PE:
CH3OH=4:4:1) to provide 131mg (47.0%) of pale yellow oil
of 7a. 1H NMR (CDCl3): ꢁ 7.52-7.08 (m, 8H, ArH), 6.69 (d,
1H, J=16.0 Hz, ArCH=CH), 5.71 (dd, 1H, J=6.9 Hz, 16 Hz,
ArCH=CH), 5.03 (dt, 1H, J=10.4 Hz, 22.0 Hz, CHP2), 4.89-
4.82 (m, 1H, CHOH), 4.49-4.48 (m, 1H, CHOH), 4.28-4.10
(m, 9H, CH (CH3)2, 4ꢂOCH2CH3), 2.51-2.38 (m, 2H, CH2),
1.66 (d, 6H, J=7.2 Hz, CH (CH3)2), 1.61-1.55 (m, 2H, CH2),
1.37-1.31 (m, 12H, 4ꢂOCH2CH3); 13C NMR (CDCl3): ꢁ
171.5, 161.5 (d, JC, F=243 Hz), 138.9, 135.0, 133.8, 132.0,
131.9, 131.7, 128.3, 121.6, 119.6, 119.3, 118.1, 115.3, 115.1,
114.4, 111.7, 72.0, 69.3, 63.9, 47.7, 43.4 (t, JC, P=125 Hz),
32.0, 29.7, 21.7, 16.4; 31P NMR (CDCl3): ꢁ 16.65 (dd,
J=67.5 Hz, 344.8 Hz); MS (TOF), m/z: 697.35 (M+1, 21),
679.34 (M+1-H2O, 55), 661.34 (M+1-2H2O, 100).
Hydroxyapatite (HAP) is commercially available calcium
mineral, and it is commonly studied as the simple model to
simulate bone tissue [5h, 12, 13]. To evaluate the bone
targeting capability of statin-bisphosphonate conjugates,
HAP was applied to simulate bone tissue in our studies. A
solution of statin-bisphosphonate conjugate in phosphate
buffer saline (pH 7.4) was vigorously stirred with HAP for
o
1h at 37 C to allow binding of the bisphosphonate moiety
with the calcium mineral. The mixture was centrifuged for
30 min at 4000rpm. Supernatant was separated and a UV
absorbance spectrum of the supernatant was recorded. The
corresponding values of peaks absorbance before and after
HAP treatment are listed in Table 1. In control binding
experiments, >99% of input parent (nonbisphosphonated)
fluvastatin and atorvastatin were recovered in the
supernatant. The results suggest that the synthetic statin-
bisphosphonate conjugates 1 and 2 have high bone affinity.
The overall trend can be seen that the binding activity with
HAP was decreased with increasing carbon chain length of
alkylenebisphosphonate. When the ratio of HAP/statin-
conjugate was doubled, the absorption efficiency was
correspondingly increased.
Compounds 7b, 10a, 10b were prepared following the
same procedure described for the preparation of 7a.
In summary, we synthesized four statin-bisphosphonate
conjugate compounds and studied their affinity to the
hydroxyapatite, a main component of bone. The high affinity
of the conjugates suggests that they have the potential ability
to deliver statins to the bone in vivo. The further defined
evaluation of the targeting ability of these compounds is
undergoing in our lab by using the animal model.
Tetraethyl 3-N-((3R,5S,E)-7-(3-(4-fluorophenyl)-1-isopro-
pyl)-1H-indol-2-yl)- 3,5-dihydroxyhept-6-enoyl)amino-
propane-1,1-bisphosphonate (7b)
1
Yield 80.4%. H NMR (CDCl3): ꢁ 7.55-7.00 (m, 8H,
ArH), 6.69 (d, 1H, J=16.2 Hz, ArCH=CH), 5.71 (dd, 1H,
J=5.1 Hz, 15.6 Hz, ArCH=CH), 4.91-4.82 (m, 1H, CHOH),
4.50-4.47 (m, 1H, CHOH), 4.23-4.12 (m, 9H, CH(CH3)2,
4ꢂOCH2CH3), 3.55-3.42 (m, 2H, NHCH2), 2.46 (tt, 1H,
J=5.7 Hz, 24.6 Hz, CHP2), 2.36-2.06 (m, 4H, CH2CHP2,
O=CCH2), 1.66 (d, 6H, J=6.9 Hz, CH(CH3)2), 1.62-1.43 (m,
2H, HOCHCH2), 1.35 (t, 12H, J=6.9 Hz, 4ꢂOCH2CH3); 13C
NMR (CDCl3): ꢁ 172.2, 161.3 (JC, F=243 Hz), 139.1, 134.9,
133.9, 132.0, 134.9, 131.7, 128.3, 121.6, 119.5, 119.3, 117.9,
115.3, 115.1, 114.3, 111.7, 72.2, 69.2, 63.0, 47.7, 42.7 (JC,
P=60 Hz), 38.6, 34.4, 29.7, 24.9, 21.7, 16.4; 31P NMR
(CDCl3): ꢁ 23.45 (d, J=37.5 Hz); MS (TOF), m/z: 725.39
EXPERIMENTAL
The starting materials were obtained from commercial
suppliers and used with or without purification as per need.
1
NMR, H (500MHz), 13C (125 MHz), and 31P spectra were
recorded on a Bruker-500 NMR spectrometer with TMS as
an internal standard (1H and 13C) and 85% H3PO3 as an
external standard (31P). Mass spectra were obtained on a

422 Letters in Organic Chemistry, 2009, Vol. 6, No. 5
Xu et al.
(M+1, 49), 707.38 (M+1-H2O, 45), 689.36 (M+1-2H2O,
100).
NMR (CF3CO2D): ꢀ 172.4, 159.8, 151.0, 138.7, 134.8,
133.5, 131.5, 131.2, 127.4, 124.3, 121.8, 119.6, 119.0, 115.0,
114.8, 113.5, 111.7, 69.7, 66.5, 47.5 (JC, P=65.0 Hz), 43.3,
42.6, 30.1, 20.8; 31P NMR (D2O): ꢀ 13.09; Anal. Calcd. for
C25H27FN2Na4O9P2: C, 44.66; H, 4.05; N, 4.17. Found: C,
44.18; H, 4.25; N, 4.01.
Tetraethyl (3R,5R)-(7-(3-(phenylcarbamoyl)-5-(4-fluoro-
phenyl)-2-isopropyl-4 -phenyl-1H-pyrrol-1-yl)-3,5-dihy-
droxyheptanoyl)aminomethylenebisphosphonate (10a)
Compounds 1b, 2a, 2b were prepared following the same
procedure described for the preparation of 1a.
1
Yield 62.3%. H NMR (CDCl3): ꢀ 7.21-6.87 (m, 14H,
ArH), 5.01 (dt, 1H, J=10.4 Hz, 22.0 Hz, CHP2), 4.26-4.07
(m, 10H, NCH2, 4ꢁOCH2CH3), 3.97-3.87 (m, 1H, CHOH),
3.74-3.52 (m, 2H, CHOH, CH(CH3)2), 2.47-2.30 (m, 2H,
O=CCH2), 1.54 (d, 6H, J=9.6 Hz, CH(CH3)2), 1.37-1.30 (m,
12H, 4ꢁOCH2CH3), 1.71-1.00 (m, 4H, HOCHCH2,
NCH2CH2); 13C NMR (CDCl3) ꢀ: 171.3, 164.0 (JC, F=200
Hz), 161.3, 141.5, 138.4, 134.7, 133.2, 133.2, 130.5, 128.7,
128.3, 127.9, 126.5, 123.5, 121.8, 119.6, 115.5, 115.3, 69.8,
69.6, 63.9, 63.8, 43.7, 41.7 (t, JC, P=67.5 Hz), 39.3, 26.1,
21.8, 21.7, 16.4; 31P NMR (CDCl3): ꢀ 16.16 (dd, J=67.5 Hz,
347.8 Hz); MS (TOF), m/z: 844.43 (M+1, 56), 751.37 (M+1-
PhNH2, 52), 707.38 (M-PhNH2-OEt, 47), 689.36 (M-PhNH2-
OEt-H2O, 100).
3-N-((3R,5S,E)-7-(3-(4-fluorophenyl)-1-isopropyl)-1H-indol-
2-yl)-3,5- dihydroxyhept-6-enoyl) aminopropane-1,1-
bisphosphonic acid tetrasodium (1b)
1
Yield 82%. H NMR (D2O): ꢀ 7.39-6.76 (m, 8H, ArH),
6.52 (d, 1H, J=15.5 Hz, ArCH=CH), 5.48 (dd, 1H, J=7.0 Hz,
16.5 Hz, ArCH=CH), 4.18 (t, 1H, J=6.5 Hz, CHOH), 3.83 (t,
1H, J=4.5 Hz, CHOH), 3.36-3.31 (m, 2H, CH2CHP2), 1.38
(d, 6H, J=6.5 Hz, CH(CH3)2), 2.25-1.36 (m, 7H, CH2CHP2,
HOCHCH2, NHCH2, O=CCH2); 31P NMR (D2O): ꢀ 19.78;
Anal. Calc. for C27H31FN2Na4O9P2: C, 46.30; H, 4.46; N,
4.00. Found: C, 46.02; H, 4.87; N, 4.03.
Tetraethyl (3R,5R)-3-N-(7-(3-(phenylcarbamoyl)-5-(4-
fluorophenyl)-2- isopropyl-4-phenyl-1H-pyrrol-1-yl)-3,5-
dihydroxyheptanoyl)aminopropane-1,1- bisphosphonate
(10b)
(3R,5R)-(7-(3-(Phenylcarbamoyl)-5-(4-fluorophenyl)-2-
isopropyl-4-phenyl-1H-pyrrol-1-yl)-3,5-dihydroxyhept-
anoyl)aminomethylenebisphosphonic acid tetrasodium
(2a)
Yield 66.8%. 1H NMR (CF3CO2D): ꢀ 7.58-7.06 (m, 14H,
ArH), 5.36 (t, 1H, J=22 Hz, CHP2), 4.59-3.41 (m, 5H, CH
(CH3)2, 2ꢁCHOH, NCH2), 2.88-2.73 (m, 2H, O=CCH2), 1.55
(d, 6H, J=6.4 Hz, CH(CH3)2), 2.40-1.00 (m, 4H,
1
Yield 96.3%. H NMR (CDCl3): ꢀ 7.20-6.87 (m, 14H,
ArH), 4.22-4.09 (m, 10H, NCH2, 4ꢁOCH2CH3), 3.96-3.88
(m, 1H, CHOH), 3.75-3.71 (m, 1H, CHOH), 3.60-3.38 (m,
3H, CH2CH2CP2, CH (CH3)2), 2.44 (t, 1H, J=25.6 Hz,
CHP2), 2.33-2.07 (m, 4H, CH2CP2, O=CCH2), 1.54 (d, 6H,
J=6.8 Hz, CH (CH3)2), 1.33 (t, 12H, J=6.8 Hz,
4ꢁOCH2CH3), 1.78-1.00 (m, 4H, HOCHCH2, NCH2CH2);
13C NMR (CDCl3): ꢀ 172.2, 164.0, 161.2, 141.4, 138.4,
134.6, 133.2, 133.2, 130.5, 128.7, 128.3, 127.8, 126.5, 123.5,
121.7, 119.6, 115.5, 115.2, 69.7, 69.6, 63.1, 62.9, 43.0, 41.7
(JC, P=81.3 Hz), 39.2, 38.6, 29.7, 26.1, 24.9, 21.8, 16.4; 31P
NMR (CDCl3) ꢀ: 23.46 (d, J=59.0 Hz); MS (TOF), m/z:
872.45 (M+1, 100), 779.40 (M+1-PhNH2, 51).
HOCHCH2CHOH,
NCH2CH2);
Anal.
Calc.
for
C34H36FN3Na4O10P2 : C, 49.83; H, 4.43; N, 5.13. Found: C,
50.25; H, 4.21; N, 4.53.
(3R,5R)-3-N-(7-(3-(Phenylcarbamoyl)-5-(4-fluorophenyl)-
2-isopropyl-4-phenyl-1H-pyrrol-1-yl)-3,5-dihydroxyhept-
anoyl)aminopropane-1,1-bisphosphonic acid tetrasodium
(2b)
Yield 85.9%. 1H NMR (CF3CO2D) ꢀ: 7.52-7.04 (m, 14H,
ArH), 4.46-3.38 (m, 7H, NCH2, 2ꢁCHOH, CH2CH2CP2,
CH(CH3)2), 3.03-2.48 (m, 5H, CHP2, CH2CH2CP2,
O=CCH2), 2.06-1.50 (m, 4H, HOCHCH2, NCH2 CH2 ), 1.53
(d, 6H, J=6.8 Hz, CH(CH3)2); 31P NMR (CF3CO2D) ꢀ: 13.67;
Anal. Calc. for C35H38FN3Na4O10P2: C, 50.43; H, 4.59; N,
5.04. Found: C, 50.56; H, 4.99; N, 4.62.
N-((3R,5S,E)-7-(3-(4-fluorophenyl)-1-isopropyl)-1H-indol-
2-yl)-3,5-dihydroxyhept-6-enoyl)aminomethylenebisphos-
phonic acid tetrasodium (1a)
A solution of fluvastatin-bisphosphonate conjugate 7a
(128mg, 0.186mmol) in anhydrous CH2Cl2 was cooled at
0ꢀ, then it was treated with 2,4,6-collidine (0.49 ml,
3.67mmol) and TMSBr (0.49 ml, 3.67mmol). After the
reaction mixture was stirred at 0ꢀ for 48h, 4ml toluene was
added into the solution. The volatiles were removed in
vacuum and the residue was dissolved in 0.25mol/L aqueous
NaOH solution (2.94 ml, 0.736mmol) at room temperature.
After 24h, acetone (20 ml) was added dropwise to give a
yellow precipitate, the solid was isolated by filtration and
washed with acetone (10 ml) and water (5 ml) to give 91mg
ACKNOWLEDGEMENTS
We thank the National Natural Science Foundation of
China (No. 20602010) and Hunan Provincial Natural
Science Foundation of China (No. 06JJ50024) for financial
support.
1
(73.6%) of pale yellow powder of 1a. H NMR (D2O): ꢀ
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