3-O-Phosphate ester conjugates of 17-β-O-{1-[2-carboxy-(2-hydroxy-4- methoxy-3-carboxamido)anilido]ethyl}1,3,5(10)-estratriene as novel bone-targeting agents

Article abstract of DOI:10.1016/j.bmcl.2010.10.023

A series of 3-O-phosphorylated analogs (4-10) of a novel bone-targeting estradiol analog (3) were synthesized after a thorough study of the reaction of 3 with a selection of phosphoryl chlorides under a variety of reaction conditions. Evaluation of these novel phosphate analogs for affinity for hydroxyapatite revealed that they bind with equal or higher affinity when compared to the bone tissue accumulator, tetracycline.

Full text of DOI:10.1016/j.bmcl.2010.10.023

Bioorganic & Medicinal Chemistry Letters 20 (2010) 7450–7453
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
3-O-Phosphate ester conjugates
of 17-b-O-{1-[2-carboxy-(2-hydroxy-4-methoxy-3-carboxamido)
anilido]ethyl}1,3,5(10)-estratriene as novel bone-targeting agents
Shama Nasim a, Ashish P. Vartak a, William M. Pierce Jr. b, K. Grant Taylor c, Ned Smith b, Peter A. Crooks a,
⇑
a Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, KY 40536, USA
b Department of Pharmacology and Toxicology, School of Medicine, University of Louisville, Louisville, KY 40292, USA
c Department of Chemistry, University of Louisville, Louisville, KY 40292, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 3-O-phosphorylated analogs (4–10) of a novel bone-targeting estradiol analog (3) were syn-
thesized after a thorough study of the reaction of 3 with a selection of phosphoryl chlorides under a vari-
ety of reaction conditions. Evaluation of these novel phosphate analogs for affinity for hydroxyapatite
revealed that they bind with equal or higher affinity when compared to the bone tissue accumulator,
tetracycline.
Received 30 August 2010
Accepted 5 October 2010
Available online 13 October 2010
Keywords:
Ó 2010 Elsevier Ltd. All rights reserved.
Phosphate esters
Bone-targeting agents
Hydroxyapatite binding
Osteoporosis
Osteoporosis is a condition that arises due to low mineral
density of bone, and results in skeletal fragility with increased risk
of bone fracture. At present, about ten million Americans suffer
from osteoporosis with about eight million being women. Another
thirty four million in the US population suffer from osteopenia,
which is a condition where bone mineral density (BMD) is lower
than normal, but not low enough to be classified as osteoporosis.1,2
Clinical osteoporosis, which presents as fragility fractures, is a
common condition in post-menopausal women; however, covert
or subclinical osteoporosis that has not yet presented fractures is
far more widespread, and is ultimately responsible for untold num-
bers of preventable bone fractures.3 Numerous factors, including
hyperthyroidism, menopause or ovariectomy, lead to conditions
that cause increased osteoclast activity in proportion to bone
growth.4 Agents currently available for prophylaxis or treatment
of osteoporosis include bis-phosphonates, calcitonin, selective
estrogen receptor modulators (SERMs), selective androgen recep-
tor modulators (SARMs), and parathyroid hormone (PTH). Estrogen
replacement therapy is often a method of choice in the clinical
treatment of osteoporosis that results from insufficient levels of
estrogen.5 The salubrious effect of estrogen on the resorption of
bone tissue arises from its anti-apoptotic effect on osteoclasts.
Unfortunately, direct treatment of osteoporosis and other related
disorders with estrogens, such as estradiol (1), is hampered by
the drug’s non-specific effects, for example, its estrogenic proper-
ties, where increased concentrations of estradiol cause undesirable
effects in males.6
Several years ago, we initiated a program aimed at restricting
the effect of estrogens to bone tissue. Our strategy focused on
the development of analogs of estradiol that would possess a
tendency to localize predominantly in bone tissue. One of the drug
design strategies we considered was the conjugation of an estra-
diol moiety to chemical entities that possessed high affinity for
structural elements commonly contained in bone (i.e., osteotropic
OH
O
OH
O
H
N
H2N
O
O
H3CO
HO
1
2
HO
H3CO
H2N
O
N
H
O
O
OH
HO
3
⇑
Corresponding author. Tel.: +1 859 257 1718; fax: +1 859 257 7585.
E-mail address: pcrooks@email.uky.edu (P.A. Crooks).
Figure 1. Estradiol (1) and bone-targeting analogs 2 and 3.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.10.023

S. Nasim et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7450–7453
7451
moieties).6–8 We have recently reported on the design, synthesis
and pharmacology of compounds in which Ca2+-chelators have
been conjugated to an estradiol moiety through succinoyl linker
units (Fig. 1, compound 2).8 These compounds were subsequently
found to possess poor hydrolytic stability in vivo, and were cleaved
prior to their arrival at the site of action. To overcome this problem,
we turned our attention to the design and synthesis of second gen-
eration analogs of 2 in which the succinoyl linker is replaced with a
more stable carboxyethyl linker (Fig. 1, compound 3).9 Although
this analog cannot be considered as an estradiol prodrug, due to
its inability to be hydrolytically cleaved to estradiol, it did retain
both the bone-targeting and anabolic properties of 2.
With regard to the current communication, it is well docu-
mented that phosphate groups have a strong affinity for Ca2+ and
other divalent metal ions, as well as affording better hydrophilicity
and water-solubility when incorporated into the structure of drug
molecules.10 To improve the bone-targeting capabilities of our sec-
ond generation analogs, we have prepared a series of phosphate
analogs of 3. It has been reported that phosphate esters can be
cleaved enzymatically, releasing phosphate in the body.11 Thus,
phosphorylated analogs of 3 would also serve as supplements to
dietary phosphorus, which are frequently utilized in popular
osteoporosis therapy regimens.
chloride itself, as evidenced by a rapid darkening of an admixture
of triethylamine and dibutylphosphoryl chloride, we sought to
generate the required phenoxide anion by deprotonating 3 with
an alkali metal base. Amongst various bases employed, (i.e., NaH,
NaHMDS, KHMDS, LHMDS and n-BuLi, all in THF), n-BuLi was
found to afford a clean white precipitate. The precise identity of
this precipitate was not determined, however, quenching of this
solution with silica gel led to the recovery of unchanged 3, signify-
ing that negligible degradation had occurred during the likely
deprotonation step. Treatment of the suspension of this precipitate
with dibutylphosphoryl chloride yielded product 5, which exhib-
ited NMR spectral characteristics consistent with the site of phos-
phorylation being at the 3-position of the estradiol moiety. This
compound was stable to air, and could be isolated in a pure form
after silica gel column chromatography.
Regarding the alkali metal amide bases that were utilized (i.e.,
LDA, LHMDS, KHMDS, etc.), the initial precipitate obtained had
characteristics similar to that produced by n-BuLi and also afforded
pure 3 when quenched, but the subsequent phosphorylation affor-
ded very complicated reaction mixtures. This is likely due to the
reaction of the conjugate acids (diisopropylamine and hexa-
methyl-disilazane, liberated during proton exchange) with dibu-
tylphosphoryl chloride. n-BuLi on the other hand, produces
butane gas as the conjugate acid, which possesses no nucleophilic
character under the reaction conditions employed. In the optimiza-
tion of the reaction conditions for the synthesis of phophorylated
analogs of 3, 1.05 equiv of n-BuLi were found to work best. The
use of more than 1.05 equiv of phosphoryl chloride offered no
advantage in the conversion ratio of 3 to phosphate esters 5–10,
which was generally around 70% in the best runs. Having opti-
mized the conditions for this chemical transformation, a total of
six phosphate esters, 5–10, were synthesized (Scheme 1) and fully
characterized.14
When considering the incorporation of a phosphate moiety into
the structure of compound 3, the potential sites of attachment of
the phosphate ester are at the 3-O-position of the estradiol moiety
and the phenolic oxygen of the aromatic Ca2+-chelating moiety.
The latter position was not considered to be a feasible strategy,
since the free phenoxide of the aromatic moiety is necessary for
its chelation with calcium ion. Thus, phosphate analogs of 3 were
synthesized with the phosphate moiety conjugated at the 3-O-po-
sition of the estradiol moiety.
Since compound 3 has two potential phenolic sites for phos-
phorylation and several base-labile protons, we decided to attempt
the use of bases of varied strengths and under a variety of reaction
conditions. An attempt at the phosphorylation of 3 with diethyl
phosphoryl iodide (formed in situ through the oxidation of tri-
ethylphosphite with iodine) in the presence of pyridine as a nucle-
ophilic catalyst was unsuccessful, since unchanged 3 was isolated.
It was noticed in the original literature report that this method was
found to be successful with alcohols,12 although one phenolic
phosphorylation (with 4-hydroxy acetophenone, whose phenolic
group is extremely labile to deprotonation by pyridine) was re-
ported. This led us to suspect that the basicity of pyridine might
be insufficient to deprotonate the phenolic 3-hydroxy function of
3. Substitution of pyridine with triethylamine did not alter the
reaction course, but this could have been due to the fact that phos-
phoryl iodides can react with triethylamine itself.13 We next
attempted the use of the less reactive diethylphosphoryl chloride
in the presence of triethylamine. In this reaction, we observed
traces of the desired product in the crude reaction mixture, and
NMR spectral analysis of the reaction product showed a character-
istic upfield shift of the C-3 signal of the estradiol moiety from 154
to 148 ppm, which strongly suggests the presence of the phos-
phoryl moiety. A downfield shift of the aromatic proton resonances
of the ‘A’ ring of 3 at the 2 and 4-positions from 6.62 to 6.97 ppm
was also observed. Unfortunately, this product (4) was found to be
labile to silica gel chromatography. Attempts at purification of 4
through crystallization from various solvents led to degradation,
and therefore 4 was not considered a viable phosphate analog for
biological evaluation. However, when the same reaction was con-
ducted with dibutylphosphoryl chloride, the product was found
to be relatively stable, although the presence of numerous side-
products in the reaction mixture prevented its isolation in a pure
form. Since one of the reasons for the presence of side-products
could be the reaction of triethylamine with dibutylphosphoryl
These phosphate esters were found to be stable during silica gel
chromatographic purification. The conversion of 3 to analogs 5–10
was found to be clean, with estradiol (1) being the only significant
side-product. Since 3 and its derivatives have previously been
found to be highly susceptible to loss of the
oxycarbonyl unit,9 it is assumed that the formation of 1 in this
reaction is caused by deprotonation of the -proton of 3 by n-BuLi,
a-proton on the b-alk-
a
followed by elimination to form an olefin (acrylamide). The short-
chain aliphatic and benzyl phosphates 5–8 were isolated as solids,
while the long-chain hydrocarbon phosphates 9 and 10 were found
to be waxy semi-solids. All the phosphate esters were found to un-
dergo slight degradation (about 10–20% conversion to estradiol
over several weeks at ambient temperature in air). In contrast,
compound 3, upon storage at ambient temperature (in air), exhib-
ited a significantly greater amount (40–50%) of hydrolytic degrada-
tion at the amidic linkage of the osteotropic aromatic moiety
compared to its phosphoryl derivatives 5–10.
H3CO
H2 N
H3CO
H2N
O
O
N
H
N
H
O
O
O
OH
O
n-BuLi, THF
OH
(RO)2POCl,
O
P
0
ºC
rt RO
HO
O
RO
3
4
: R = Et (unstable)
5 : R = -Bu
n
6 : R = i-Bu
7 : R = i-Pr
8 : R = Bn
9 : R = (2-ethyl) hexyl
10 : R = n-dodecyl
Scheme 1. Phosphorylation conditions for the synthesis of compound 3.

7452
S. Nasim et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7450–7453
temperature for 18 h before being evaporated to dryness on
Table 1
a rotary
evaporator (note: bath temperature not exceeding 30 °C). The resulting crude
residue was purified by silica gel flash chromatography, utilizing mixtures of
CH2Cl2 and CH3OH as eluting solvent. The compounds were loaded onto the
column either as solutions in benzene or as dispersions on silica gel (three
times the weight of the compound). Dialkylphosphoryl chlorides were
prepared in accordance with a previously reported method.17
Relative binding of 17-b-estradiol and analogs 5–10 to
hydroxyapatite compared to tetracycline binding
Compound
Binding index (%)
relative to tetracycline
17-b-Estradiol
Tetracycline
5
6
7
8
9
10
0
100
100
180
200
190
180
160
17-b-O-{1-[2-Carboxy-(2-hydroxy-4-methoxy-3-carbox-amido)anilido]ethyl}1,3,5-
estratriene-3-dibutyl phosphate (5): White solid; Rf = 0.3 (CH2Cl2/MeOH, 20:1);
mp = 78–80 °C; 1H NMR (300 MHz, CDCl3) d 14.4 (s, 1H), 8.72 (s, 1H), 8.46 (d,
J = 9.0 Hz, 1H), 8.20 (br s, 1H), 7.24 (d, J = 8.2 Hz, 1H), 6.96 (app d, J = 11.3 Hz,
2H), 6.41 (d, J = 9.0 Hz, 1H), 5.76 (s, 1H), 4.13 (m, 4H), 3.95 (s, 3H), 3.82 (m, 2H),
3.50 (t, J = 8.1 Hz, 1H), 2.83 (m, 2H), 2.66 (m, 2H), 2.3–2.0 (m, 2H), 1.70–1.22
(m, 19H), 0.91 (t, J = 7.2 Hz, 6H), 0.84 (s, 3H) ppm; 13C NMR (75 MHz, CDCl3) d
172.5, 170.2, 154.2, 148.6, 138.5, 137.1, 126.7, 124.3, 122.5, 120.0, 117.1, 102.6,
100.1, 89.9, 68.4, 66.1, 56.5, 50.6, 44.2, 43.6, 39.1, 38.5, 38.2, 32.5, 29.8, 28.2,
28.1, 27.2, 26.5, 23.3, 18.9, 13.9, 11.9 ppm; ESI-MS m/z 701 (M+H)+. Anal. Calcd
for C37H53N2O9P: C, 63.41; H, 7.62; N, 4.00. Found: C, 63.21; H, 7.49; N, 4.29.
17-b-O-{1-[2-Carboxy-(2-hydroxy-4-methoxy-3-carboxamido)anilido]ethyl}1,3,5-
estratriene-3-isobutyl phosphate (6): White solid; Rf = 0.35 (CH2Cl2/MeOH,
20:1), mp = 84–86 °C; 1H NMR (300 MHz, CDCl3) d 14.4 (s, 1H), 8.68 (s, 1H),
8.43 (d, J = 9.0 Hz, 1H), 8.15 (br s, 1H), 7.19 (d, J = 7.8 Hz, 1H), 6.93 (app d,
J = 11.4, 2H), 6.37 (d, J = 9.0 Hz, 1H), 5.86 (br s, 1H), 3.90 (s, 3H), 3.88–3.80 (m,
6H), 3.46 (t, J = 7.5 Hz, 1H), 2.80 (m, 2H), 2.65 (m, 2H), 2.25–1.16 (m, 16H), 0.94
(d, J = 6.6, 11H), 0.81 (s, 3H) ppm; 13C NMR (75 MHz, CDCl3) d 172.7, 170.4,
154.3, 148.6, 138.6, 137.2, 126.7, 124.4, 122.5, 120.1, 117.1, 102.6, 100.1, 89.9,
74.4, 66.1, 56.4, 50.5, 44.1, 43.5, 39.0, 38.4, 38.5, 38.1, 29.7, 29.3, 29.2, 28.1,
27.2, 26.4, 23.3, 18.8, 11.8 ppm; ESI-MS m/z 701 (M+H)+. Anal. Calcd for
As a predictive measure of the ability of this class of compound
to bind to bone tissue in vivo, the ability of compounds 5–10 to
bind to crystalline hydroxyapatite in vitro was determined using
UV–vis spectroscopy.15 These studies were conducted in parallel
with binding studies on tetracycline for comparison, as it is well
established that tetracycline accumulates preferentially in bone-
tissue because of a very strong affinity for hydroxyapatite.16 The
binding indices of the novel phosphate prodrugs are shown in
Table 1. Superior binding to hydroxyapatite is clearly evident when
compared to 17-b-estradiol alone, and is comparable to that of tet-
racycline. Within the phosphate prodrug series, increased lipophil-
icity or branching in the aliphatic substituents appears to improve
hydroxyapatite binding, since analogs 6–10 are somewhat more
potent than analog 5.
C
37H53N2O9P: C, 63.41; H, 7.62; N, 4.00. Found: C, 63.32; H, 7.53; N, 4.28. 17-b-
O-{1-[2-Carboxy-(2-hydroxy-4-methoxy-3-carboxamido)anilido]ethyl}1,3,5-
estratriene-3-isopropyl phosphate (7): White solid; Rf = 0.4 (CH2Cl2/MeOH, 15:1)
mp = 104–106 °C; 1H NMR (300 MHz, CDCl3) d 14.4 (s, 1H), 8.68 (s, 1H), 8.46 (d,
J = 9.0 Hz, 1H), 8.17 (br s, 1H), 7.20 (app. d, J = 9.0 Hz, 1H), 6.93 (m, 2H), 6.39 (d,
J = 9.0 Hz, 1H), 5.81 (br s, 1H), 4.74 (m, 2H), 3.93 (s, 3H), 3.81 (m, 2H), 3.48 (t,
J = 8.4 Hz, 1H), 2.81 (m, 2H), 2.62 (m, 2H), 2.27–0.84 (m, 19H), 1.32 (m, 6H),
0.82 (s, 3H) ppm; 13C NMR (75 MHz, CDCl3) d 172.8, 170.4, 154.4, 149.0, 138.6,
137.1, 126.6, 124.5, 122.7, 121.7, 120.2, 117.3, 100.2, 90.0, 74.3, 73.5, 66.1, 56.4,
50.5, 44.2, 43.5, 39.1, 38.6, 38.2, 29.7, 28.1, 27.2, 26.4, 23.8, 23.7, 23.2,
11.9 ppm; ESI-MS m/z 673 (M+H)+. Anal. Calcd for C35H49N2O9P: C, 62.49; H,
7.34; N, 4.16. Found: C, 62.21; H, 7.51; N, 4.34. 17-b-O-{1-[2-Carboxy-(2-
hydroxy-4-methoxy-3-carboxamido)anilido]ethyl}1,3,5-estratriene-3-dibenzyl
phosphate (8): White solid; Rf = 0.5 (hexanes/CH2Cl2/MeOH, 2:10:3);
mp = 140–142 °C; 1H NMR (300 MHz, CDCl3) d 14.41 (s, 1H), 8.73 (s, 1H),
8.48 (d, J = 9.0 Hz, 1H), 8.19 (br s, 1H), 7.32 (s, 10H), 7.17 (d, J = 8.7 Hz, 1H), 6.88
(dd, J = 8.7, 1.5 Hz, 2H), 6.41 (d, J = 9.0 Hz, 1H), 5.86 (br s, 1H), 5.13 (s, 2H), 5.10
(s, 2H), 3.94 (s, 3H), 3.79 (m, 2H), 3.48 (t, J = 8.1 Hz, 1H), 2.76 (m, 2H), 2.68 (m,
2H), 2.27–1.1 (m, 13H), 0.83 (s, 3H) ppm; 13C NMR (75 MHz, CDCl3) d 172.5,
170.2, 154.2, 148.3, 138.6, 137.3, 135.7, 135.6, 128.6, 128.1, 126.7, 124.3, 122.5,
120.1, 117.2, 102.6, 100.2, 89.9, 70.1, 66.1, 56.5, 50.6, 44.2, 43.6, 39.1, 38.5,
38.2, 31.9, 29.8, 28.2, 27.3, 26.5, 23.4, 22.9, 14.4, 11.9 ppm; ESI-MS m/z 769
(M+H)+. Anal. Calcd for C43H49N2O9P: C, 67.17; H, 6.42; N, 3.64. Found: C,
67.35; H, 6.61; N, 3.91. 17-b-O-{1-[2-Carboxy-(2-hydroxy-4-methoxy-3-
carboxamido)anilido]ethyl}1,3,5-estratriene-3-bis(2-ethylhexyl) phosphate (9):
Yellow oil; Rf = 0.5 (pentane/CH2Cl2/MeOH, 2:10:3); 1H NMR (300 MHz,
CDCl3) d 14.42 (s, 1H), 8.72 (s, 1H), 8.46 (d, J = 9.0 Hz, 1H), 8.18 (br s, 1H),
7.21 (d, J = 8.4 Hz, 1H), 6.92 (br d, J = 8.2 Hz, 2H), 6.39 (d, J = 9.0 Hz 1H), 5.90 (br
s, 1H), 4.03 (m, 4H), 3.93 (s, 3H), 3.81 (m, 2H), 3.48 (m, 2H), 2.83 (m, 2H), 2.63
(m, 2H), 2.26–2.02 (m, 4H), 1.90–0.86 (m, 38H), 0.81 (s, 3H) ppm; 13C NMR
(75 MHz, CDCl3) d 172.5, 170.2, 154.1, 148.6, 138.4, 137.0, 126.6, 124.3, 122.5,
120.0, 117.1, 102.5, 100.1, 89.9, 70.5, 66.1, 56.4, 50.5, 44.2, 43.6, 40.3, 40.2,
39.1, 38.5, 38.2, 30.1, 29.8, 29.1, 28.2, 27.3, 26.5, 23.5, 23.4, 23.2, 14.4, 11.9,
11.2 ppm; ESI-MS m/z 813 (M+H)+. Anal. Calcd for C45H69N2O9P: C, 66.48; H,
8.55; N, 3.45. Found: C, 66.21; H, 8.82; N, 3.75. 17-b-O-{1-[2-Carboxy-(2-
hydroxy-4-methoxy-3-carboxamido)anilido]ethyl}1,3,5-estratriene-3-
dodecylphosphate (10): Yellow oil: Rf = 0.6 (hexanes/CH2Cl2/MeOH, 2:10:3);
1H NMR (300 MHz, CDCl3) d 14.1 (s, 1H), 8.67 (s, 1H), 8.43 (d, J = 9.3 Hz, 1H),
8.14 (br s, 1H), 7.15 (d, J = 8.4 Hz, 1H), 6.93 (br d, 2H), 6.37 (d, J = 9.3 Hz, 1H),
5.87 (s, 1H), 4.10 (m, 4H), 3.90 (s, 3H), 3.79 (m, 2H), 3.46 (t, J = 8.1 Hz, 1H), 2.80
(m, 2H), 2.65 (m, 2H), 2.25–1.83 (m, 5H), 1.66 (m, 5H), 1.49–1.23 (m, 43H),0.86
(t, J = 7.2 Hz, 6H), 0.81 (s, 3H) ppm; 13C NMR (75 MHz, CDCl3) d 172.7, 170.4,
154.3, 148.8, 138.6, 137.2, 126.7, 124.4, 122.6, 120.1, 117.1, 102.6, 100.1, 90.0,
68.7, 66.2, 56.4, 50.5, 44.2, 43.6, 39.0, 38.5, 38.1, 32.1, 30.5, 30.4, 29.9, 29.8,
29.7, 29.4, 28.2, 27.2, 26.5, 25.7, 23.4, 23.0, 14.4, 11.9 ppm; ESI-MS m/z 925
(M+H)+. Anal. Calcd for C53H85N2O9P: C, 68.80; H, 9.26; N, 3.03. Found: C,
68.91; H, 9.47; N, 3.29.
In conclusion, we have developed a viable phosphorylation pro-
tocol for the regioselective synthesis of 3-O-phosphate analogs of
the bone-targeting estradiol analog 3. In vitro data indicate that
these analogs have bone-targeting properties comparable to tetra-
cycline, as indicated by their affinity for hydroxyapatite.
Acknowledgments
Financial support from Pradama, Inc., and the Kentucky
Science and Technology Corporation is gratefully acknowledged.
W.M.P., K.G.T. and P.A.C. have a financial interest in Pradama, Inc.
References and notes
1. Stoch, S. A.; Wagner, J. A. Clin. Pharmacol. Ther. 2008, 83, 172.
2. Assessment of fracture risk and its application to screening for postmenopausal
osteoporosis. Report of
a WHO Study Group. World Health Organization
technical report series, 2004, 843, pp 1–129.
3. Vestergaard, P.; Mosekilde, L. Thyroid 2003, 13, 585.
4. Olson, E. J.; Lindgren, B. R.; Carlson, C. S. Bone 2008, 42, 907.
5. Rochira, V.; Marco, F.; Balestrieri, A.; Carani, C. J. Clin. Endo. Met. 2000, 85, 1841.
6. Pierce, William M. Jr.; Waite, Leonard, C.; Taylor, K. G.; Sato, Fumiyasu.;
Takahashi, Yoshio. PCT Int. Appl. 2000, 165 pp. CODEN: PIXXD2 WO
2000066613 A1 20001109.
7. Foye, W. O.; Duvall, R. N.; Talbot, M. H.; Zaim, R. H. J. Pharm. Sci. 1962, 51, 1012.
8. Neale, J. R.; Richter, N. B.; Merten, K. E.; Taylor, K. G.; Singh, S.; Waite, L. C.;
Emery, N. K.; Smith, B.; Cai, J.; Pierce, W. M., Jr. Bioorg. Med. Chem. Lett. 2009, 19,
680.
9. Nasim, S.; Vartak, A. P.; Pierce, W. M., Jr.; Taylor, K. G.; Crooks, P. A. Synth.
Commun. 2009, 17, 4321.
10. Monteil, M.; Guenin, E.; Migianu, E.; Lutomski, D.; Lecouvey, M. Tetrahedron
2005, 61, 7528.
11. Schultz, C. Bioorg. Med. Chem. 2003, 11, 885.
12. Stowell, J. K.; Widlanski, T. S. Tetrahedron Lett. 1995, 36, 1825.
13. Gubnitskaya, E. S.; Semashko, Z. T.; Parkhomenko, V. S.; Kirsanov, A. V. Zh. Obsh.
Khim. 1980, 50, 2171.
15. Hydroxyapatite binding assay. A 1 mM solution of each analog was prepared in
DMSO and diluted serially in 50 mM Tris–HCl buffer (pH 7, 1% DMSO), to attain
a final concentration of 10 nM. Tetracycline was utilized as a reference analyte
and ca. 50% bound to hydroxyapatite (HA) at a concentration of 10 nM. The HA
slurry was prepared as an admixture of 50 mg HA in 100 mL in 50 mM Tris–HCl
buffer containing 1% DMSO. For each analog, two samples were prepared. For
14. General procedure for the synthesis of 17-b-[[2-Carboxy(3-carboxamido-2-hydroxy-
4-methoxy)anilido]ethoxy]estra-1,3,5-triene-3-dialkyl phosphates (5–10).
n-Butyllithium (0.8 mL, 2.5 M solution in hexane) was diluted to 10 times its
volume with THF and the resulting mixture added drop-wise to a rapidly
stirring solution of 3 in THF (0.6 M) that was immersed in a salt-ice bath (bath
temperature À10 °C to À5 °C) under argon gas. The resulting white suspension
was stirred for 5 min and then the appropriate dialkylphosphoryl chloride
(1.0 equiv) was added to the reaction mixture in one portion. The suspension
one of the two samples, 1 mL of a 10 nM solution of the analog and 100 lL of
the HA slurry was pipetted into a microcentrifuge tube. The mixture was
agitated gently by inversion for 4 min and then centrifuged at 12,000g for
3 min to sediment the HA. The supernatant was decanted into another
microcentrifuge tube. An electronic spectral scan (UV–vis) was recorded for
became
a clear solution within 5–10 min and was stirred at ambient

S. Nasim et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7450–7453
7453
each analyte over the wavelength range 220–520 nm using a Varian Cary 300
Bio Scan spectrophotometer. The blank sample consisted of 50 mM Tris–HCl
buffer containing 1% DMSO. The kmax was determined and the extinction
coefficient (e) was then calculated. The absorbance of each analog after
incubation with HA was measured at the kmax, and the molar concentration of
the compound was then determined using the Beer–Lambert law and the
previously calculated extinction coefficient. The fraction adsorbed by HA for
each analog and for 17-b-estradiol was subsequently determined and
compared to that obtained for tetracycline to afford a binding index relative
to tetracycline binding.
16. Albert, A.; Rees, C. W. Nature 1956, 177, 433.
17. Gao, F.; Yan, X.; Shakya, T.; Baettig, O. M.; Ait-Mohand-Brunet, S.; Berghuis, A.
M.; Wright, G. D.; Auclair, K. J. Med. Chem. 2006, 49, 5273.