Design, synthesis, and biological evaluation of novel estradiol-bisphosphonate conjugates as bone-specific estrogens

Article abstract of DOI:10.1016/j.bmc.2009.12.041

Bone deficiency causes osteoporosis and often decreases quality of life in patients with rheumatoid arthritis. Estrogens are known to protect elderly women from bone loss. Synthesis of new estradiol-bisphosphonate conjugates (E₂-BPs) was accomplished and their in vivo activity as bone-specific estrogens were examined. Among them, MCC-565 showed selective estrogenic activity in bones; but it showed little estrogenic activity in the uterus. We also found that the linker moiety in E₂-BPs was essential for the absorption and specificity of the conjugates.

Full text of DOI:10.1016/j.bmc.2009.12.041

Bioorganic & Medicinal Chemistry 18 (2010) 1143–1148
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis, and biological evaluation of novel estradiol–bisphosphonate
conjugates as bone-specific estrogens
Masahiko Morioka a,d, Akihito Kamizono b, Hirosato Takikawa c, Akihisa Mori a, Hiroaki Ueno a,
Shu-ichiro Kadowaki b, Yoshihide Nakao b, Kuniki Kato d, Kazuo Umezawa d,
*
a Medicinal Chemistry Laboratory, Research Division, Mitsubishi-Tanabe Pharma Corporation, 1000, Kamoshida-cho, Aoba-ku, Yokohama 227-0033, Japan
b Pharmacology Laboratory, Research Division, Mitsubishi-Tanabe Pharma Corporation, 1000, Kamoshida-cho, Aoba-ku, Yokohama 227-0033, Japan
c Department of Agrobioscience, Graduate School of Agricultural Sciences, Kobe University, 1-1 Rokkodai, Nada-ku, Kobe 657-8501, Japan
d Department of Applied Chemistry, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama 223-0061, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Bone deficiency causes osteoporosis and often decreases quality of life in patients with rheumatoid
arthritis. Estrogens are known to protect elderly women from bone loss. Synthesis of new estradiol–bis-
phosphonate conjugates (E2–BPs) was accomplished and their in vivo activity as bone-specific estrogens
were examined. Among them, MCC-565 showed selective estrogenic activity in bones; but it showed lit-
tle estrogenic activity in the uterus. We also found that the linker moiety in E2–BPs was essential for the
absorption and specificity of the conjugates.
Received 26 October 2009
Revised 11 December 2009
Accepted 15 December 2009
Available online 22 December 2009
Keywords:
Osteoporosis
Estrogen
Ó 2009 Elsevier Ltd. All rights reserved.
Bisphosphonate
1. Introduction
pain.11 Developing a drug delivery system targeting bones would
be a unique approach for the therapy of postmenopausal bone loss,
Bone deficiency causes osteoporosis and often decreases quality
of life in patients with rheumatoid arthritis. Bones are always
metabolized by osteoclasts and osteoblasts. Osteoclasts absorb
bones and osteoblasts replace new bone materials. The increase
of osteoclast activity or the decrease of osteoblast activity cause
bone loss. One of the hormones that increases bone mass is estro-
gens including estradiol (E2) having the steroid structure. Estrogen
deficiency caused by menopause induces bone loss in about two-
thirds of women. Therefore, it would be possible to treat osteopo-
rosis by estrogen administration as a hormone replacement ther-
apy, which was initiated about 10 years ago.1,2 Although estrogen
undoubtedly increases the bone mass, it often shows adverse ef-
fects such as abnormal uterine bleeding and generation of breast
cancer and/or uterus cancer. Estrogen receptor agonists such as
raloxifene are also employed to preserve bones in osteoporosis,3–
especially if the estrogens could be delivered selectively to the
bone. For this purpose various estrogen conjugates have been pre-
pared, and stable binding between E2 and the bisphosphonate moi-
ety have been mainly employed. Although several estrogen/
bisphosphonate conjugates have been reported,12–17 none of them
has been used clinically for the treatment of osteoporosis.
As the previously prepared estrogen/bisphosphonate com-
pounds showed stable binding between E2 and the bisphosphonate
moiety, the local release of E2 from these compounds on the bone
was found to be difficult. In 1996, Bauss et al. prepared substituted
estradiols having a bisphosphonate moiety attached at the 17 posi-
tion via a linker.18 They suggested that the conjugate compounds
mightbe hydrolyzed to releaseactive estradiolinto thebloodstream.
OH
17
5
but its activity toward bone tissue is not prominent compared
with that of estrogen therapy.6 On the other hand, bisphosphonate
compounds tend to be accumulated on the bone surface, increasing
the bone mass; therefore, bisphosphonate-including compounds
such as etidronate and alendronate were tested for use in osteopo-
rosis therapy.7–9 These compounds were clinically used; however,
they showed toxicity toward the jawbone10 and caused muscle
3
HO
Estradiol (E2)
Driven by the suggestion of Bauss et al., we sought to synthesize
novel estradiol–bisphosphonate conjugates (E2–BPs) in order to
develop a useful anti-osteoporosis medication (Fig. 1).
* Corresponding author.
E-mail address: umezawa@applc.keio.ac.jp (K. Umezawa).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.12.041

1144
M. Morioka et al. / Bioorg. Med. Chem. 18 (2010) 1143–1148
presence of a catalytic amount of N,N-dimethylaminopyridine
2. Results and discussion
2.1. Synthesis of conjugates
(DMAP) afforded bisphosphonoamide 13 in 72% yield. Cleavage
of the methoxymethyl (MOM) group at 3-position of estradiol
and the tetra ethyl ester groups of phosphonate was successively
accomplished by the treatment with trimethylsilyl iodide (TMSI)
to provide conjugate compound 1 in 95% yield.
Conjugate 6 (MCC-565) was synthesized as shown in Scheme 2.
Curtius rearrangement reaction of succinic acid monobenzyl ester
14 using diphenylphosphoryl azide (DPPA) followed by the addi-
tion of estrone to the intermediate isocyanate provided the carba-
Conjugate 1 was synthesized as shown in Scheme 1. Monoben-
zyl esterification of Meldrum’s acid 7 provided monobenzylester 8
in 54% yield. By use of this half-ester, the ester linkage was in-
stalled in the requisite C17 position of 3-methoxymethyl-estradiol
9 to give benzylmalonate 10 with the condensation agent dic-
yclohehxylcarbodiimide (DCC) in 98% yield. The benzyl group of
10 was reductively cleaved to yield the corresponding acid 11 un-
der catalytic hydrogenation conditions. Coupling 10 with tetra-
ethyl aminomethylenebisphosphonate 1219 using DCC in the
mate, which was successively debenzylated by
a catalytic
hydrogenation to give the desired C-3 carbamate-rinked acid 15
in 28% overall yield. Then, condensation of 15 with 12 using
O
O
PO3HNa
PO3HNa
O
O
PO3HNa
PO3HNa
O
N
H
O
N
H
HO
HO
1
3
5
2
H
N
O
PO3HNa
PO3HNa
O
O
O
PO3HNa
PO3HNa
O
O
N
H
N
H
HO
HO
4
OH
OH
NaHO3P
NaHO3P
O
O
NaHO3P
NaHO3P
O
O
N
H
O
N
H
N
H
O
6: MCC-565
Figure 1. Structures of conjugate compounds.
Scheme 1. Reagents and conditions: (a) BnOH, toluene, reflux, 3 h; (b) DCC, DMAP, CH2Cl2, rt, 10 h; (c) 10% Pd-C, H2, EtOH–THF, rt, 2 h; (d) TMSI, CH2Cl2, ꢀ20 °C, 0.5 h, then
NaOAc, MeOH.

M. Morioka et al. / Bioorg. Med. Chem. 18 (2010) 1143–1148
1145
Scheme 2. Reagents and conditions: (a) (i) DPPA, TEA, toluene, then estorone, 100 °C, 1 h; (ii) Pd-C, H2, EtOH–THF, rt, 1 h; (b) 12, N-methylmorpholine, iso-
butylthioroformate, ꢀ10 °C, 0.5 h; (c) NaBH4, MeOH, 0 °C, 0.5 h; (d) TMSI, CH3CN, ꢀ20 °C, 0.5 h, then NaOAc, MeOH.
isobutylthioroformate to give 16 followed by reduction with so-
dium borohydride afforded 17b-alcohol 17. Finally, cleavage of
the tetra ethyl ester groups of 16 was accomplished by the treat-
ment with trimethylsilyl iodide (TMSI) to provide the conjugate
compound 6 in 84% yield.
120.0
100.0
80.0
60.0
40.0
20.0
0.0
BV/TV
UW/BW
Conjugates 2–5 were synthesized by the same procedure used
for conjugate 1 or 6.
Conjugate 1 having malonic acid as a spacer between E2 and the
bisphosphonate was designed, supposing that malonic ester group
might be more stable against enzymatic hydrolysis than the succi-
nic or glutaric ester group due to steric hindrance. In fact, conju-
gate 1 could not be hydrolyzed by esterase to release E2 after 8 h.
Conjugate 2 was even more sterically hindered with the gem-di-
methyl group, and it is considered to be more stable than conjugate
1. Conjugate 3 contained phthalic acid as a spacer. Conjugate 4
showed higher water solubility than the other derivatives, since
it contained two NH groups in the linkage. Conjugates 5 and 6 were
equipped with activated ester and carbamate at the 3-position,
respectively. Conjugate 6 (MCC-565) showed increased the solubil-
ity in water, since it also contained two NH groups in the linkage.
Esterase is known to have broad substrate specificity and there-
fore, is responsible for the hydrolysis of many exogenous com-
pounds.20 Cathepsin K is an essential protease for osteoporosis.21,22
It is a cysteine-protease, but it mayalso hydrolyze esters and amides.
Therefore, the free estrogen may be released from the bisphospho-
nate conjugates by esterase and/or proteases around the bone. In
addition, labile E2–BPs may also be cleaved under acidic conditions,
whichcanbe providedbytheprotonpumpintheosteoclasts.23 Thus,
E2–BPs are likely to be cleaved enzymatically or chemically around
osseous tissue.
OVX (n=7)
sham (n=8)
Estradiol (n=8)
conjugate1 (nco=n8j)ugate2 (cno=n8j)ugate3 (nc=on8j)ugate4 (nco=n8j)ugate5 (cno=n8j)ugate6 (n=8)
Figure 2. Estrogenic effects on bone and uterus of estradiol and each conjugate
compounds in ovariectomized mice (Sham: ovary was exteriorized and interiorized
again without ovariectomy. BV: bone volume, TV: tissue volume, UW: uterus
weight, BW: body weight).
slightly decreased bone weight. E2 increased both the bone mass
and uterus weight, indicating that it caused systemic hormonal ef-
fects without local selectivity. None of the synthesized compounds
increased uterus weight. There was a significant difference of bone
mass between estradiol and conjugate 6.
Conjugate 6 increased the bone mass even more effectively than
E2 without increasing the uterine weight. Bone loss was optimally
prevented by conjugate 6 given at 0.1 mg/kg/4 weeks. Moreover,
conjugates 6 showed no bisphosphonate activity in rats. Bis-
phosphonate activity was measured by the suppression of Ca2+
ion release from the bone by PTHrp treatment. Conjugate 6 did
not suppress the release Ca2+ ion from the bone (data not shown).
The estrogenic activity of conjugate 6 was studied by use of a com-
petitive estrogen receptor binding assay employing estrogen
receptors purified from rat uterus. Conjugate 6 exhibited 2000
times weaker binding activity toward the estrogen receptor than
E2 (Fig. 3).
Conjugate 6 was not hydrolyzed by esterase (Sigma: E2884, po-
sitive control: phenylpropionic acid ethylester in PBS, 37 °C) or
amidase (Sigma: acylamide amidohydrolase, positive control: ben-
zoyl butylamide in PBS, 37 °C) within 8 h, nor was it hydrolyzed at
pH 4 (buffer solution HPCE pH 4.0 solution) even after 1 month.
However, E2 was released from the conjugate into the rat blood-
stream from 8 h, time dependently (TLC plate analyzed). Therefore,
conjugate 6 is likely to be delivered to the bone, where it would
slowly release E2.
Contamination of a very small amount of estradiol in conjugate
6 may be responsible for the effect at a high dose of the conjugate.
Therefore we concluded that conjugate 6 could show selective
estrogenic effects on bones.
2.2. Bioactivity
Next, we studied whether the pharmacological effects on bones
were due to the estrogenic activity of the conjugates. Fulvestrant is
a known anti-estrogen.24 As shown in Figure 4, administration of
this chemical clearly suppressed the effect of conjugate 6 on bone.
Considering that the uterus weight of the conjugate 6 treated rats
did not increase (Fig. 2) Therefore, it is likely that conjugate 6 effec-
tively released estradiol around the bones. It is possible that the 3-
The pharmacological effects of conjugates 1–6 were examined
in ovariectomized rats. Synthesized E2–BPs were given to SD fe-
male rats at 0.1 mg/kg, which is the optimized amount, by intrave-
nous administration. After the treatment, the uterus weight and
femur bone mass were scored. As shown in Figure 2, ovariecto-
mized rats showed a dramatically reduced uterus weight and a

1146
M. Morioka et al. / Bioorg. Med. Chem. 18 (2010) 1143–1148
120
100
80
60
40
20
0
tra were obtained with a HITACHI M2000A (positive-SIMS mode).
Column chromatography was carried out on Silica Gel 60 (230–
400 mesh ASTM; Merck). High Preparative Liquid Column chroma-
tography (HPLC) was carried out on COSMOSIL (C-18
20 ꢁ 250 mm, Nakalai Tesque). Elemental analyses were per-
formed with a PERKIN ELMER 240C Analyzer and were within
0.4% of the theoretical values.
Estradiol
Comp. 6
3.1. Malonic acid monobenzyl ester (8)
Dexamethasone
A solution of Meldrum’s acid 7 (2.32 g) and benzyl alcohol
(1.83 ml) in toluene (11.6 ml) was refluxed for 3 h. After removal
of the solvent in vacuo, the residue was purified by chromatogra-
phy over silica gel, with elution with CHCl3–MeOH (10:1), to give
8 (1.7 g, yield: 54%): 1H NMR (CDCl3), d 10.25 (br s, 1H), 7.37–
7.34 (m, 5H), 5.20 (s, 2H), 3.48 (s, 2H). Elemental Anal. (%) Calcd
for C10H10O4: C, 61.85; H, 5.19. Found: C, 61.73; H, 5.28.
Competiter Conc. (M)
Figure 3. Comparison of estrogen receptor binding activity of conjugate 6 with that
of estradiol by use of the competitive receptor binding assay. The total radioactivity
of bound [3H]-estradiol (3 nM) was 4810.2 cpm. Non-specific binding was deter-
mined by radioactivity of bound [3H]-estradiol (3 nM) in the presence of excess
non-radiolabeled estradiol (30 nM). The control value was 234.2 cpm. Percent
binding of [3H]-estradiol in the presence of ligand was calculated by the following
formula: Percent binding = [(B-NSB)/(Bo-NSB)] ꢁ 100 (%).
3.2. Malonic acid benzyl ester 3-methoxymethoxy-1,3,5-
estratriene-17-yl ester (10)
To a solution of compound 9 (500 mg, 1.58 mmol) in CH2Cl2
(5.0 ml) were added DMAP (0.25 g, 2.05 mmol), DCC (0.42 g,
2.05 mmol), and 8 (0.31 g, 1.58 mmol). The resultant mixture was
stirred at room temperature for 10 h, and the mixture was then di-
luted with EtOAc (30 ml). The organic layer was washed with H2O
and saturated brine, dried over anhydrous magnesium sulfate, and
concentrated. The residue was purified by chromatography over
silica gel with hexane–EtOAc (5:1) as the eluent, to give 10
(640 mg, yield: 98%): 1H NMR (CDCl3), d 7.38–7.34 (m, 5H), 7.19
(d, 1H, J = 6.5 Hz), 6.83 (dd, 1H, J = 6.5 Hz, 2.0 Hz), 6.77 (d, 1H,
J = 2.0 Hz), 5.18 (d, 2H, J = 2.3 Hz), 5.15 (s, 2H), 4.74 (t, 1H,
J = 6.0 Hz), 3.47 (s, 3H), 3.43 (s, 2H), 2.90–2.80 (m, 2H), 2.30–2.10
(m, 3H), 1.90–1.65 (m, 3H), 1.60–1.20 (m, 7H), 0.73 (s, 3H). 13C
NMR (D2O), d 166.53, 166.43, 155.07, 137.97, 135.21, 133.72,
128.59, 128.52, 128.49, 126.38, 116.21, 113.77, 94.45, 83.85,
67.25, 55.90, 49.69, 43.78, 43.06, 41.85, 38.43, 36.71, 31.59,
29.71, 27.29, 27.16, 26.11, 23.21, 22.66, 14.13, 11.89. Elemental
Anal. (%) Calcd for C30H36O6: C, 73.15; H, 7.37. Found: C, 72.86;
H, 7.37.
position is better than the 17-position to obtain more effective
compounds. The linker structure of conjugate 6 is likely to be bet-
ter than that of others, perhaps because compound 6, having the
carbamate structure at the 3-position of E2, is more labile to biolog-
ical stimuli.
In conclusion, we designed and synthesized novel E2–BPs in
which the linkers were sensitive to esterases, proteases, and acidic
conditions on bones to act as bone-seeking E2 prodrugs. Among
them, conjugate 6 with an activated carbamate moiety was shown
to be the most effective and specific for bones.
3. Experimental
1H NMR spectra were recorded at ambient temperature on an
AC-250 Brucker spectrometer in CDCl3 or D2O. 13C NMR and 31P-
NMR spectra were recorded at ambient temperature on an Brucker
ADBNCE 400 spectrometer in CDCl3 or D2O. 13C NMR spectra of
conjugate 1 was recorded at ambient temperature on an Brucker
ADBNCE 600 spectrometer in D2O. The chemical shifts were given
in d (ppm), and coupling constants were reported in Hz. Mass spec-
3.3. Malonic acid mono-(3-methoxymethoxy-1,3,5-estratriene-
17-yl) ester (11)
A solution of 10 (640 mg, 1.58 mmol), 10% Pd-C (60 mg) in THF
(6.0 ml), and EtOH (6.0 ml) was stirred under a H2 atmosphere at
room temperature for 2 h. The reaction mixture was then filtered
through a Celite pad, and the filtrate was concentrated in vacuo
to give 11 (648 mg, yield: 99%) as white crystals: 1H NMR (CDCl3),
d 7.19 (d, 1H, J = 6.5 Hz), 6.82 (dd, 1H, J = 6.5 and 1.9 Hz), 6.77 (d,
1H, J = 1.9 Hz), 5.14 (s, 2H), 4.78 (t-like, 1H), 3.47 (s, 3H), 3.44 (br
s, 2H), 2.90–2.80 (m, 2H), 2.40–1.40 (m, 13H), 0.85 (s, 3H). 13C
NMR (D2O), d 170.42, 167.46, 155.06, 137.96, 133.65, 126.39,
116.23, 113.78, 94.44, 84.47, 55.91, 49.67, 43.78, 43.13, 40.56,
38.43, 36.78, 29.69, 27.34, 27.16, 26.09, 23.22, 11.99. Elemental
Anal. (%) Calcd for C23H30O6ꢂ0.2H2O: C, 68.03; H, 7.55. Found: C,
67.89; H, 7.60.
Effects on fermus
0.700
P< 0.05
0.680
0.660
*
**
0.640
0.620
0.600
0.580
**
3.4. N-(Bis-(diethoxyphosphoryl)-methyl)-malonamic acid (3-
methoxymethoxy-1,3,5-estratriene-17-yl) ester (13)
0
0.560
Conjugate6
(0.1 mg/kg)
-
0
-
+
0
+
0.02
OVX
+
+
-
FULVESTRANT
(mg/kg/day)
To a solution of 11 (620 mg, 1.54 mmol) in CH2Cl2 (7 ml) were
added DMAP (245 mg, 2.0 mmol), DCC (635 mg, 2.0 mmol) and tet-
raethyl aminomethylenebisphosphonate 12 (467 mg, 1.54 mmol).
After the resultant mixture had been stirred at room temperature
for 10 h, the reaction mixture was diluted with EtOAc (30 ml).
0
0.2
2.0
2.0
Sham
Sham
Figure 4. Estrogenic effect on bone of conjugate 6 was abolished by concurrent
administration of pure anti-estrogen: FULVAESTRANT.

M. Morioka et al. / Bioorg. Med. Chem. 18 (2010) 1143–1148
1147
The organic layer was washed with H2O and saturated brine, dried
over anhydrous magnesium sulfate, and concentrated in vacuo.
The residue was then purified by chromatography over silica gel,
with elution with CHCl3–MeOH (10:1–1:1), to give 13 (504 mg,
yield: 72%): 1H NMR (CDCl3), d 7.77 (d, 1H, J = 7.5 Hz), 7.19 (d,
1H, J = 6.3 Hz), 6.83 (dd, 1H, J = 6.3 Hz, 2.0 Hz), 6.77 (d, 1H,
J = 2.0 Hz), 5.15 (s, 2H), 5.06 (dt, 1H, J = 16.2 and 7.5 Hz), 4.76 (t,
1H, J = 6.0 Hz), 4.30–4.10 (m, 8H), 3.47 (s, 3H), 3.41 (s, 2H), 2.90–
2.80 (m, 2H), 2.40–2.10 (m, 4H), 1.95–1.85 (m, 2H), 1.85–1.20
(m, 19H), 0.83 (s, 3H). HRMS (CI) calcd for C32H52NO11P2 [M+H]+:
688.3016. Found: 688.3013.
820, 792, 768. Elemental Anal. (%) Calcd for C22H27NO5: C, 68.55;
H, 7.06; N, 3.63. Found: C, 68.57; H, 7.03; N, 3.64.
3.7. ((3-(17-Oxo-1,3,5-estratrien-3-yloxycarbonylamino)
propionylamino)methyl)-bisphosphonic acid tetraethyl ester
(16)
A solution of 15 (231 mg, 0.6 mmol), iso-butylchloroformate
(0.086 ml), and N-methylmorpholine (0.073 ml) in THF (10 ml)
was stirred at ꢀ10 °C for 15 min. To this solution was added com-
pound 12 (0.20 g, 0.66 mmol) in THF (2.0 ml) at ꢀ10 °C, and the
reaction mixture was then stirred under cooling for 8 h. Next, the
reaction mixture was diluted with CHCl3 (40 ml), and washed with
H2O and saturated brine. After the organic layer had been dried
over anhydrous magnesium sulfate and concentrated in vacuo,
the residue was purified by chromatography over silica gel, with
elution with CHCl3–MeOH (10:1–1:1), to give 16 (440 mg, yield:
100%) as a light yellow syrup: 1H NMR (CDCl3), d 7.49 (br d, 1H,
J = 7.5 Hz), 7.24 (br d, 1H, J = 6.3 Hz), 6.85 (dd, 1H, J = 6.3 and
1.5 Hz), 6.83 (d, 1H, J = 1.5 Hz), 6.13 (t, 1H, J = 4.5 Hz), 5.26 (br s,
1H), 5.11 (dt, 1H, J = 16.5 and 7.5 Hz), 4.30–4.10 (m, 8H), 3.60–
3.50 (m, 2H), 2.95–2.80 (m, 2H), 2.70–2.58 (m, 2H), 2.55–1.95
(m, 7H), 1.70–1.30 (m, 18H), 0.90 (s, 3H). HRMS (CI) calcd for
C31H49N2O10P2 [M+H]+: 671.2862. Found: 671.2862.
3.5. N-(Bis-phosphono-methyl)-malonamic acid 3-hydroxy-
1,3,5-estratrien-17-yl ester 2 sodium salt (1)
To a solution of 13 (252 mg, 0.366 mmol) in CH3CN (10 ml) was
added trimethylsilyl iodide (0.313 ml) at ꢀ20 °C, and the resultant
mixture was stirred under cooling for 30 min. The reaction was
then quenched by the addition of CH2Cl2 (3.0 ml) and H2O
(0.5 ml). After evaporation of the solvent in vacuo, the residue
was diluted with MeOH (5.0 ml). To this reaction mixture was
added a solution of 1 M-CH3COONaꢂ3H2O in MeOH (5.0 ml), and
the resultant precipitate was collected by filtration and washed
with MeOH/H2O (5:1, 20 ml) to give the title compound
1
(201 mg, yield: 95.3%) as a white powder: 1H NMR (D2O), d 7.01
(d, 1H, J = 8.3 Hz), 6.48 (d, 1H, J = 8.3 Hz), 6.42 (s, 1H), 4.64 (t, 1H,
J = 8.8 Hz), 3.38 (s, 2H), 2.85–2.60 (m, 2H), 2.32–1.09 (m, 13H),
0.76 (s, 3H). 13C NMR (D2O), d 189.77, 188.63, 173.29, 158.52,
156.04, 141.92, 135.80, 129.84, 118.20, 115.84, 87.83, 52.19,
51.82, 51.35, 46.05, 45.74, 41.13, 39.21, 29.69, 29.44, 28.73,
3.8. ((3-(17-Hydroxy-1,3,5-estratrien-3-yloxycarbonylamino)
propionylamino)methyl)-bisphosphonic acid tetraethyl ester
(17)
To a solution of 16 (402 mg, 0.60 mmol) in MeOH (6.0 ml) was
added NaBH4 (34 mg, 0.90 mmol) at 0 °C under stirring, and the
resultant mixture was stirred for 30 min. The reaction mixture
was then poured into saturated aqueous NH4Cl (50 ml) and ex-
tracted with CHCl3 (30 ml ꢁ 2). The organic layer was concentrated
in vacuo, after which the residue was purified by chromatography
over silica gel with CHCl3–MeOH (10:1–1:1) as the eluent, to give
17 (242 mg, yield: 60%) as a pale yellow oil: 1H NMR (CDCl3): d 7.21
(d, 1H, J = 8.4 Hz), 6.81 (dd, 1H, J = 2.0 and 10.4 Hz), 6.77 (d, 1H,
J = 2.0 Hz), 6.02 (t, 1H, J = 6.2 Hz), 5.04 (dt, 1H, J = 16.5 and
7.5 Hz), 4.3–4.1 (m, 8H), 3.69 (t, 1H, J = 8.1 Hz), 3.62–3.50 (m,
2H), 2.85–2.70 (m, 2H), 2.60–2.50 (m, 2H), 2.35–1.10 (m, 13H),
1.31 (t, 12H), 0.73 (s, 3H). HRMS (CI) calcd for C31H51N2O10P2
[M+H ]+: 673.3019. Found: 673.3013.
25.59, 14.23. 31P NMR (D2O), d 12.71, 12.59. IR(cmꢀ1),
m 3286,
2925, 1717, 1644, 1543, 1500, 1450, 1346, 1287, 1166, 1055,
1007, 964, 889, 817, 785. Elemental Anal. (%) Calcd for
C22H31NO10P2Na2ꢂ2.5H2O: C, 42.59; H, 5.52; N, 2.26. Found: C,
42.48; H, 5.26; N, 2.23. HRMS (CI) calcd for C22H31NO10P2
[M+Na]+: 554.1319. Found: 554.1291.
3.6. 3-(17-oxo-1,3,5-estratrien-3-yloxycarbonylamino)-
propionic acid (15)
To a solution of succinic acid monobenzyl ester 14 (571 mg,
2.74 mmol) and triethylamine (458 mg, 3.29 mmol) in toluene
(5.7 ml) was added DPPA (0.62 ml, 2.88 mmol) at 0 °C, and the
reaction mixture was then stirred at 100 °C for 30 min. After the
gas evolution had ceased, the reaction mixture was cooled at
0 °C. To this reaction mixture was added estrone (740 mg,
2.74 mmol) in toluene (7.4 ml) dropwise, and the mixture was sub-
sequently stirred at 100 °C for 1 h. Then the reaction mixture was
allowed to cool to room temperature and diluted with chloroform
(40 ml). The organic layer was washed with H2O and saturated
brine and dried over anhydrous magnesium sulfate. After removal
of the solvent in vacuo, the residue was purified by chromatogra-
phy over silica gel, with CHCl3–MeOH (10:1) as the eluent, to give
colorless crystals (343 mg, yield: 26%). A solution of this compound
(343 mg), 10% Pd-C (34 mg) in EtOH (3.4 ml), and THF (3.4 ml) was
stirred under a H2 atmosphere for 1 h. The reaction mixture was
then filtered through a Celite pad, and the filtrate was concentrated
to give 15 (231 mg, yield: 83%) as a colorless crystal: 1H NMR
(CDCl3): d 7.66 (br s, 1H), 7.23 (d, 1H, J = 8.5 Hz), 6.90–6.80 (m,
3H), 5.62 (t, 1H, J = 6.2 Hz), 3.60–3.40 (m, 2H), 2.95–2.80 (m, 2H),
2.15–2.60 (m, 2H), 2.50–1.90 (m, 7H), 1.70–1.30 (m, 6H), 0.88 (s,
3H). 13C NMR (D2O), d 177.10, 154.94, 148.74, 137.86, 136.98,
126.30, 121.67, 118.83, 50.41, 48.00, 44.12, 38.01, 36.48, 35.44,
3.9. ((3-(17-Hydroxy-1,3,5-estratrien-3-yloxycarbonylamino)-
propionyl-amino)methyl)-bisphosphonic acid 2 sodium salts
(6)
To a solution of 17 (242 mg, 0.359 mmol) in CH3CN (2.3 ml) was
added trimethylsilyl iodide (0.31 ml, 2.18 mmol) under stirring at
ꢀ20 °C and the reaction mixture was then stirred for 30 min. Next,
the reaction was quenched by the addition of CH2Cl2 (3.0 ml) and
water (0.5 ml). After removal of the solvent in vacuo, the residue
was dissolved with MeOH (3.0 ml). To this reaction mixture was
added a solution of 1 M-CH3COONaꢂ3H2O in MeOH (3.0 ml), and
the resultant precipitate was collected by filtration and washed
with MeOH/H2O (5:1, 20 ml) to give the title compound
6
(164 mg, yield: 84%) as a white powder: 1H NMR (D2O): d 7.18
(d, 1H, J = 7.6 Hz), 6.7–6.4 (m, 2H), 4.23 (t, 1H, J = 18.6 Hz), 3.52
(t, 1H, J = 7.8 Hz), 2.80–2.60 (m, 6H), 2.2–0.9 (m, 13H), 0.53 (s,
3H). 13C NMR (D2O), d 173.54, 158.13, 149.06, 139.83, 139.37,
127.43, 122.61, 119.69, 82.28, 50.13, 48.87, 44.33, 43.54, 38.92,
37.01, 36.44, 29.71, 27.26, 26.60, 23.35, 11.44. 31P NMR (D2O), d
34.03, 31.52, 29.40, 26.35, 25.76, 21.59, 13.83. IR(cmꢀ1),
m
3368,
12.93, 12.81. IR(cmꢀ1),
m 3370, 3321, 2932, 2872, 1737, 1699,
2933, 1738, 1698, 1531, 1490, 1454, 1436, 1417, 1372, 1326,
1532, 1490, 1453, 1436, 1417, 1372, 1327, 1303, 1260, 1248,
1303, 1260, 1248, 1222, 1155, 1079, 1052, 1004, 965, 912, 896,
1211, 1156, 1079, 1052, 1006, 965, 911, 897, 884, 820, 792, 769.

1148
M. Morioka et al. / Bioorg. Med. Chem. 18 (2010) 1143–1148
Elemental Anal. (%) Calcd for C23H32N2O10P2Na2ꢂ2H2O: C, 43.13; H,
5.45; N, 4.32. Found: C, 43.11; H, 5.45; N, 4.32. HRMS (CI) calcd for
C23H34N2O10P2 [M+Na]+: 583.1584. Found: 583.1614. Melting
Anal. (%) Calcd for C22H29NO10P2Na2ꢂ2H2O: C, 43.22; H, 5.44; N,
2.29. Found: C, 43.27; H, 5.21; N, 2.31. HRMS (CI) calcd for
C22H31NO10P2 [M+Na]+: 554.1319. Found: 554.1318.
point: >300 °C, specific rotation: ½a D25
¼ ꢀ1718 (c 0.085, H2O)
ꢃ
3.14. Animal experiments
3.10. N-(Bis-phosphono-methyl)-2,2-dimethyl-malonamic acid
3-hydroxy-1,3,5-estratrien-17-yl ester 2 sodium salt (2)
Estradiol was dissolved with 95% of corn oil and 5% of benzyl
alcohol. Conjugates 1–6 were dissolved in a mixture of 1% carboxy-
methyl cellulose and 1% citrate buffer. Female SD rats (7 weeks old)
were purchased from Japan SLC Co. Ltd (Shizuoka, Japan) and were
housed for 4 weeks in our laboratory. Before ovariectomy, each
animal was allocated to one of the study groups so that mean body
weight of each group would be equal. All animals except the sham-
operated controls were bilaterally ovariectomized. On the next
day, conjugates 1–6 were injected intravenously. The E2 group
was injected three times per week for 4 weeks on the first, second,
and fifth days. The total amount of E2 given was 560 mg/kg. The
estrogenic effects on bone were analyzed by measuring the bone
volume of the secondary spongiosa of the left tibia and by measur-
ing ash weight of the right femurs.
Compound 2 was prepared from compound 10 as a white pow-
der (yield: 52%) according to the similar procedure described for
compound 1: 1H NMR (D2O); d 7.04 (d, 1H, J = 8.2 Hz), 6.48 (d,
1H, J = 8.2 Hz), 6.45 (s, 1H), 4.07 (t, 1H, J = 18.6 Hz), 2.70–2.50 (m,
2H), 2.10–1.00 (m, 13H), 1.34 (s, 3H), 1.32 (s, 3H), 0.63 (s, 3H).
13C NMR (D2O), d 176.07, 174.05, 153.09, 138.91, 132.79, 126.87,
115.24, 112.87, 84.72, 51.21, 48.91, 48.37, 43.09, 42.98, 38.14,
36.34, 28.95, 26.65, 26.51, 25.77, 22.68, 22.48, 22.40, 11.44. 31P
NMR (D2O), d 12.70, 12.58. IR(cmꢀ1),
m 3210, 2927, 1708, 1617,
1536, 1288, 1078, 872, 816. HRMS (CI) calcd for C24H35NO10P2
[M+Na]+: 582.1632. Found: 582.1603.
3.11. N- (Bis-phosphono-methyl)-phthalamic acid 3-hydroxy-
1,3,5-estratrien-17-yl ester 2 sodium salt (3)
Acknowledgments
Compound 3 was prepared from Compound 9 and phthalic
anhydride as a white powder (yield: 11%) according to the similar
procedure described for compound 1: 1H NMR (D2O), d 7.80–7.50
(m, 3H), 7.50–7.30 (m, 1H), 7.09 (d, 1H, J = 8.2 Hz), 6.60–6.40 (m,
2H), 4.73 (t, 1H, J = 7.4 Hz), 4.53 (t, 1H, J = 19.8 Hz), 2.70–2.50 (m,
2H), 2.30–1.10 (m, 13H), 0.71 (s, 3H). Elemental Anal. (%) Calcd
for C27H31N2O10P2Na2ꢂ2.5H2O: C, 46.42; H, 4.93; N, 2.01. Found:
C, 46.42; H, 4.71; N, 1.83. HRMS (CI) calcd for C27H33NO10P2
[M+Na]+: 616.1475. Found: 616.1448.
We wish to thank Ms. Kaoru Ishizaki and Dr. Jun Ohgama for
assistance with the preparation of the manuscript, Mr. Toshihiko
Minegishi and Dr. Tetsuo Sekiya for assistance with the advice of
drug-design, and Dr. Akihiro Yamamoto, Mr. Haruki Inokawa and
Mr. Yoshiki Mori for examination with a synthetic route of MCC-
565.
References and notes
1. Eastell, R. N. Eng. J. Med. 1998, 338, 736.
2. Cauley, J. A.; Robinson, J.; Chen, Z.; Cummings, S. R.; Jacson, R. D.; LaCroix, A. Z.;
LeBoff, M.; Lewis, C. E.; McGowan, J.; Neuner, J.; Pettinger, M.; Stefanick, M. L.;
Wactawski-Wende, J.; Watts, N. B. JAMA 2003, 290, 1729.
3. Charalampos, D.; Maria, M.-T. Rev. Clin. Pharmacol. Pharm., Int. Ed. 2007, 21, 75.
4. Musiliyu, A.; Musa, M.; Omar, F. K.; John, S. C. Curr. Med. Chem. 2007, 14, 1249.
5. Inaba, M. Clin. Calcium 2004, 14, 27.
3.12. 3-(3-(Bis-phosphono-methyl)-ureido)-propionic acid 3-
hydroxy-1, 3, 5-estratrien-17-yl ester 2 sodium salt (4)
Compound 4 was prepared from compound 9 and succinic
anhydride as a white powder (yield: 32%) according to the similar
procedure described for compound 1: 1H NMR (D2O); d 7.18 (d, 1H,
J = 7.6 Hz), 6.7–6.4 (m, 2H), 4.23 (t, 1H, J = 18.6 Hz), 3.52 (t, 1H,
J = 7.8 Hz), 2.80–2.60 (m, 6H), 2.20–0.9 (m, 13H), 0.53 (s, 3H). 13C
NMR (D2O), d 153.06, 138.84, 135.44, 131.21, 129.47, 128.22,
126.81, 115.20, 112.82, 85.18, 48.84, 43.01, 38.17, 26.47, 25.79,
6. Cem, D.; Banu, D.; Ahmet, C.; Murat, E. Gynecol. Endocrinol. 2007, 23, 398.
7. Hosam, K.; Kamel, M. D. JAMDA 2007, 8, 434.
8. William, S.; Ronald, E.; Roberto, C. Drug Safety 2007, 30, 755.
9. Felicia, C.; Joao, L. C.; Manuel, D. C. Clin. Ther. 2007, 29, 1116.
10. Robert, E. M.; Yob, S. J. Oral Maxill. Surg. 2005, 63, 1567.
11. FDA ALERT (1/7/2008), Information for Healthcare Professionals.
12. Bosies, E.; Esswein, A.; Bauss, F. Ger. Offen., DE 4029499, 1990.
13. Saari, W. S.; Rodan, G. A.; Fisher, T. E.; Anderson, P. S. Eur. Pat. Appl., EP496520,
1992.
11.57. 31P NMR (D2O), d 12.68, 12.55. IR(cmꢀ1),
m 3269, 2923,
14. Sugioka, T.; Inazu, M. Eur. Pat. Appl., EP548884, 1993.
15. Ueno, H.; Kadowaki, S.; Kamizono, A.; Morioka, M.; Mori, A. Eur. Pat. Appl.,
EP555845, 1993.
16. Bauss, F.; Esswein, A.; Reiff, K.; Sponer, G.; Mueller-Beckmann, B. Calcified
Tissue Int. 1996, 59, 168.
2161, 1711, 1634, 1540, 1503, 1393, 1286, 1146, 1077, 889, 819,
785, 713. Elemental Anal. (%) Calcd for C23H32N2O10P2Na2ꢂ2H2O:
C, 43.13; H, 5.67; N, 4.37. Found: C, 43.28; H, 5.39; N, 4.36. HRMS
(CI) calcd for C23H34N2O10P2 [M+Na]+: 583.1584. Found: 583.1594.
17. Fujisaki, J.; Tokunaga, Y.; Takahashi, T.; Kimura, S.; Shimojo, F.; Hata, T. J. Drug
Targeting 1997, 5, 129.
3.13. N-(Bis-phosphono-methyl)-malonamic acid 17-hydroxy-
1,3,5-estratrien-3-yl ester 2 sodium salt (5)
18. Page Philip, C. B.; MacKenzie, M. J.; Gallagher, J. A. J. Org. Chem. 2001, 66, 3704.
19. Ludwig, M. Phosphorus, Sulfur, Relat. Elem. 1981, 11, 311.
20. Satoh, T.; Hosokawa, M. Annu. Rev. Pharmacol. Toxicol. 1998, 38, 257.
21. John, C. R.; Liao; Lenney, J. F. Biochem. Biophys. Res. Commun. 1984, 124, 909.
22. Inaoka, T.; Bilbe, G.; Ishibashi, O.; Tezuka, K.; Kokubo, T. Biochem. Biophys. Res.
Commun. 1995, 206, 89.
Compound 5 was prepared from estrone and compound 8 as a
white powder (yield: 33%) according to the similar procedure de-
scribed for compound 6: 1H NMR (D2O), d 7.26 (d, 1H, J = 8.4 Hz),
6.70–6.60 (m, 2H), 4.23 (t, 1H, J = 18.6 Hz), 3.51 (t, 1H, J = 7.5 Hz),
2.80–2.55 (m, 6H), 2.20–0.95 (m, 13H), 0.53 (s, 3H). Elemental
23. Vaes, G. Clin. Orthopedics Relat. Res. 1998, 231, 239.
24. Hermann, K. Ger. Offen. DE 4218743, 1993.