3D QSAR of novel estrogen-RGD peptide conjugates: Getting insight into structural dependence of anti-osteoporosis activity and side effect of estrogen in ERT

Article abstract of DOI:10.1016/j.bmc.2009.03.057

To explore the structural dependence of the oral potency and side effect of estrogen-RGD peptide conjugates, here six novel conjugates were prepared via introducing RGD-tetrapeptides into both 3- and 17-positions of estradiol, and introducing RGD-octapept

Full text of DOI:10.1016/j.bmc.2009.03.057

Bioorganic & Medicinal Chemistry 17 (2009) 3680–3689
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
3D QSAR of novel estrogen–RGD peptide conjugates: Getting insight
into structural dependence of anti-osteoporosis activity and side effect
of estrogen in ERT
*
Ming Zhao, Jiangyuan Liu, Xiaoyi Zhang, Li Peng, Chunyu Li, Shiqi Peng
College of Pharmaceutical Sciences, Capital Medical University, Beijing 100069, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
To explore the structural dependence of the oral potency and side effect of estrogen–RGD peptide conju-
gates, here six novel conjugates were prepared via introducing RGD-tetrapeptides into both 3- and 17-
positions of estradiol, and introducing RGD-octapeptides into 3-position of estrone. In an ovariectomized
mouse model they exhibited higher anti-osteoporosis activity and lower side effect than estrogen. For 3D
QSAR analysis the anti-osteoporosis activities of nine known conjugates estrogen–RGD tetrapeptide con-
jugates were also provided. Using Cerius2 module their 3D QSAR analysis was performed, four equations
with high r2 values were established, and the structural dependence of the oral potency and side effect of
them was elucidated.
Received 12 February 2009
Revised 27 March 2009
Accepted 27 March 2009
Available online 5 April 2009
Keywords:
Estrogen
Estrogen–RGD peptide
Anti-osteoporosis
Side effect
Ó 2009 Elsevier Ltd. All rights reserved.
3D QSAR
1. Introduction
to the increase of the resorption osteoclast-modulated bone. The
role of estrogen is directly up-modulating osteoblast activity and
Osteoporosis, the most frequent bone remodeling disease and
defined as skeletal disorder, is characterized by a low bone mass
and a high risk of fractures and has been a major health problem
for elderly women. Ovarium atrophy leads postmenopausal wo-
men confronting with different level of osteoporosis.1 Since more
than 50% of the osteoporosis women may suffer from fracture
and some will die, osteoporosis has being crushing pressure for
our society.2,3 It is well known that estrogen lack results from ove-
rium atrophy is responsible for the osteoporosis development of
postmenopausal women, and estrogen replacement therapy
(ERT) or estrogen/progestogen replacement therapy (HRT) is
widely used to inhibit bone loss and consequently to decrease
the risk of coronary heart disease for postmenopausal women.4–7
The fracture risk of the postmenopausal women who receives
HRT for at least 5 years could be decreased by 50%.8 On the other
hand however, when the dose-related side effect such as endome-
trial hyperplasia and the thromboembolic events are considered
the efficacy of a long term ERT or HRT is usually discredited.9–13
To improve the efficacy a lot of efforts have being given to the
administration and/or the modification of estrogen.14–22
proliferation, and/or regulating the gene expression in osteoblasts
and osteoclasts.23–26 Bone resorption is regulated by osteoclasts
binding to bone surface and depends on the adhesiveness of osteo-
clast, which is mediated by the transmembrane vitronectin recep-
tor
a
vb3 integrin and its recognition to RGD containing protein of
osteoclast.27 Since upregulation of
a
vb3 integrin has been associ-
ated with osteoporosis, RGD peptides have been recognized as
emerging potential therapeutic agents. This knowledge implies
that the activity and proliferation of osteoblasts and the adhesive-
ness of osteoclasts can be simultaneously upregulated with estro-
gen and down-regulated with RGD peptide, respectively.
In this context, here six novel estrogen–RGD peptide conju-
gates were prepared by introducing RGD-tetrapeptides in to both
3- and 17-positions of estradiol, and by introducing RGD-octa-
peptides into 3-position of estrone. Based on the need of 3D QSAR,
9 known conjugates, estradiol-3-RGD tetrapeptides, estradiol-17-
RGD tetrapeptides and estrone-3-RGD tetrapeptides, were also
prepared as the reference conjugates. In an ovariectomized mouse
model, these novel conjugates, reference conjugates, estradiol and
estron (estrogen controls) were orally administered, and the
important parameters reflected the bone health of the treated
mice, such as the weight of femur and femur ash, calcium and
phosphorous in femur and blood, the level of serum ALP, bleeding
time and uterine weight, were measured. Using Cerius2 module,
the 3D QSAR analyses were performed, and four equations
It is well established that osteoporosis relates not only to the
decrease of the formation of osteoblast-modulated bone but also
* Corresponding author. Tel./fax: +86 1083911528.
E-mail address: sqpeng@bjmu.edu.cn (S. Peng).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.03.057

M. Zhao et al. / Bioorg. Med. Chem. 17 (2009) 3680–3689
3681
capable of predicting a comparatively exact anti-osteoporosis
activity and side effect for estrogen–RGD peptide conjugates were
established.28
Ser; estradiol-3V,17V: AA = Val and estradiol-3F,17F: AA = Phe)
were obtained in 38–88% yield.
To 8 HClꢀArg(NO2)-Gly-Asp(OBzl)-AA-OBzl [1–3, AA = Ser(Bzl),
Val and Phe] were introduced and the corresponding estradiol-3-
Arg(NO2)-Gly-Asp(OBzl)-AA-OBzl 11, 12 and 13 [AA = Ser(Bzl),
Val and Phe] were obtained in 53%, 37% and 61% yield, respectively.
Removing all protective groups by catalytic hydrogenation three
reference conjugates estradiol-3-Arg-Gly-Asp-AA (estradiol-3S,
AA = Ser; estradiol-3V, AA = Val and estradiol-3F AA = Phe) were
obtained in 48–91% yield.
Estradiol was treated with succinic anhydride and 17-b-carb-
oxyethylcarbonyl-oxyestradiol (10) was offered in 95% yield. Cou-
pling 10 with 1, 2 and 3 estradiol-17b-Arg(NO2)-Gly-Asp(OBzl)-
AA-OBzl 17, 18 and 19 [AA = Ser(Bzl), Val and Phe] were obtained
in 48%, 45% and 42% yield, respectively. Removing all protective
groups by catalytic hydrogenation another three reference conju-
gates estradiol-17-Arg-Gly-Asp-AA (estradiol-17S: AA = Ser; estra-
diol-17V: AA = Val; estradiol-17F: AA = Phe) were obtained in 79–
86% yield. The procedure, synthetic data, and the chemical physical
data of all estradiol-3,17-di(Arg-Gly-Asp-AA) conjugates are given
in a SI file.
2. Results and discussion
2.1. Preparation of RGD peptide moieties
The tetrapeptides and octapeptides of RGD used for preparing
the conjugates were synthesized via solution method according
to the route depicted in Scheme 1. Upon the coupling of Boc-As-
p(OBzl) with L-Ser(Bzl)-OBzl, L-Val-OBzl and L-Phe-OBzl, as well
as then removing Boc group HClꢀAsp(OBzl)-Ser(Bzl)-OBzl,
HClꢀAsp(OBzl)-Val-OBzl and HClꢀAsp(OBzl)-Phe-OBzl were ob-
tained in 94%, 92% and 87% yield, respectively. Upon coupling
Boc-Arg(NO2) and Gly Boc-Arg(NO2)-Gly was obtained in 84% yield.
After the corresponding coupling and deprotection reactions
HClꢀArg(NO2)-Gly-Asp(OBzl)-Ser(Bzl)-OBzl (1), HClꢀArg(NO2)-Gly-
Asp(OBzl)-Val-OBzl (2), HClꢀArg(NO2)-Gly-Asp(OBzl)-Phe-OBzl (3),
HClꢀArg(NO2)-Gly-Asp(OBzl)-Ser(Bzl)-Arg(NO2)-Gly-Asp(OBzl)-Ser
(Bzl)-OBzl (4), HClꢀArg(NO2)-Gly-Asp(OBzl)-Val-Arg(NO2)-Gly-As-
p(OBzl)-Val-OBzl (5) and HClꢀArg(NO2)-Gly-Asp(OBzl)-Phe-Arg
(NO2)-Gly-Asp(OBzl)-Phe-OBzl (6) were obtained in 89%, 76%,
81%, 82%, 66% and 69% yield, respectively. The procedure, syn-
thetic data, and chemical physical data of all peptides are given
in a SI file.
2.3. Preparing novel estrone-17–RGD octrapeptide conjugates
The preparation of novel estrone-17–RGD octrapeptide conju-
gates is shown with Scheme 3. The 3-hydroxyl group of estrone
was first alkylated by ethyl bromoacetate to provide ethyl es-
trone-3-oxylacetate in 94% yield. By saponification ethyl estrone-
3-oxylacetate was converted to estrone-3-oxylacetic acid (20,
96% yield), to which HClꢀArg(NO2)-Gly-Asp(OBzl)-AA-Arg(NO2)-
Gly-Asp(OBzl)-AA-OBzl (4–6) were introduced to form estrone-3-
Arg(NO2)-Gly-Asp-(OBzl)-AA-Arg(NO2)-Gly-Asp(OBzl)-AA-OBzl 24,
25 and 26 [AA = Ser(Bzl), Val and Phe] in 71%, 82% and 80% yield,
respectively. Removing all protective groups 3 novel estrone-3-
Arg-Gly-Asp-AA-Arg-Gly-Asp-AA (estrone-3SS: AA = Ser; estrone-
3VV: AA = Val; estrone-3FF: AA = Phe) were provided in 41–71%
yield.
2.2. Preparing novel conjugates estradiol-3,17-di(Arg-Gly-Asp-
AA)
The preparation of novel estradiol-3,17-di(RGD tetrapeptide)
conjugates is shown with Scheme 2. Estradiol was alkylated with
ethyl bromoacetate to provide 3-ethoxycabonylmethoxyestradiol
(7) in 83% yield. By saponification 7 was converted into 3-carb-
oxylmethoxyestradiol (8, 96% yield). Treating 8 with succinic
anhydride 3-carboxymethoxyl-17b-carboxyethylcarbonyloxyest-
radiol (9) was provided in 81% yield, to which 1–3 were intro-
duced and corresponding estradiol-3,17-di[Arg(NO2)-Gly-Asp
(OBzl)-AA-OBzl] 14, 15 and 16 [AA = Ser(Bzl), Val and Phe] were
obtained in 71%, 82% and 80% yield, respectively. Removing all
protective groups by catalytic hydrogenation three novel conju-
gates estradiol-3,17-di(Arg-Gly-Asp-AA) (estradiol-3S,17S: AA =
To 20 HClꢀArg(NO2)-Gly-Asp(OBzl)-AA-OBzl [1-3, AA = Ser(Bzl),
Val and Phe] were introduced to form the corresponding estrone-
3-Arg(NO2)-Gly-Asp(OBzl)-AA-OBzl 21, 22 and 23 [AA = Ser(Bzl),
Val and Phe] in 62%, 68% and 61% yield, respectively. Removing
all protective groups from 21–26 by catalytic hydrogenation 3 ref-
Scheme 1. Preparation of RGD tetrapeptides and octapeptides, in 1 and 4 AA = Ser(Bzl), 2 and 5 AA = Val, 3 and 6 AA = Phe; (i) DCC, HOBt and NMM; (ii) hydrogen chloride in
ethyl acetate (4 N).

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M. Zhao et al. / Bioorg. Med. Chem. 17 (2009) 3680–3689
OCO(CH2)2COR2
OCO(CH2)2COR1
OCO(CH2)2CO2H
v
iv
HOCOCH2O
R2COCH2O
Estradiol-3S,17S; 3V,17V; 3F,17F
R1COCH2O
9
14-16
iii
iv
OH
OH
OH
i
ii
HO
HO
C2H5O2CCH2O
HOCOCH2O
7
8
iii
iv
OH
OH
OCO(CH2)2CO2H
v
R2COCH2O
R1COCH2O
10
11-13
Estradiol-3S, 3V, 3F
OCO(CH2)2COR2
OCO(CH2)2COR1
v
HO
Estradiol-17S, 17V, 17F
HO
17-19
Scheme 2. Route for preparing estradiol–RGD conjugates, in 11 R1 = -Arg(NO2)-Gly-Asp(OBzl)-Ser(Bzl)-OBzl, in 12 R1 = -Arg(NO2)-Gly-Asp(OBzl)-Val-OBzl, in 13 R1
-Arg(NO2)-Gly-Asp(OBzl)-Phe-OBzl; in Estradiol-3S R2 = -Arg-Gly-Asp-Ser, in Estradiol-3V R2 = -Arg-Gly-Asp-Val, in Estradiol-3F R2 = -Arg-Gly-Asp-Phe; in 14 R1 = R2
=
=
-Arg(NO2)-Gly-Asp(OBzl)-Ser(Bzl)-OBzl, in 15 R1 = R2 = -Arg(NO2)-Gly-Asp(OBzl)-Val-OBzl, in 16 R1 = R2 = -Arg(NO2)-Gly-Asp(OBzl)-Phe-OBzl; in Estradiol-3S,17S R1 = R2
= -Arg-Gly-Asp-Ser, in Estradiol-3V,17V R1 = R2 = -Arg-Gly-Asp-Val, in Estradiol-3F,17F R1 = R2 = -Arg-Gly-Asp-Phe; in 17 R1 = -Arg(NO2)-Gly-Asp(OBzl)-Ser(Bzl)-OBzl, in 18
R1 = -Arg(NO2)-Gly-Asp(OBzl)-Val-OBzl, in 19 R1 = -Arg(NO2)-Gly-Asp(OBzl)-Phe-OBzl; in Estradiol-17S R1 = -Arg-Gly-Asp-Ser, in Estradiol-17V R1 = -Arg-Gly-Asp-Val, in
Estradiol-17F R1 = -Arg-Gly-Asp-Phe; (i) BrCH2CO2C2H5 and NaOC2H5; (ii) NaOH in methanol; (iii) DCC, HOBt, NMM and 4–6; (iv) DCC, HOBt, NMM and 1–3; (v) Pt/H2.
O
O
v
R3COCH2O
R4COCH2O
Estrone-3SS, 3VV, 3FF
24-26
iv
O
O
O
ii
HOCOCH2O
i
HO
C2H5O2CCH2O
20
iii
O
O
v
R2COCH2O
R1COCH2O
21-23
Estrone-3S, 3V, 3F
Scheme 3. Route for preparing Estrone–RGD conjugates, in 21 R1 = -Arg(NO2)-Gly-Asp(OBzl)-Ser(Bzl)-OBzl, in 22 R1 = -Arg(NO2)-Gly-Asp(OBzl)-Val-OBzl, in 23 R1
=
-Arg(NO2)-Gly-Asp(OBzl)-Phe-OBzl; in Estrone-3S R2 = -Arg-Gly-Asp-Ser, in Estrone-3V R2 = -Arg-Gly-Asp-Val, in Estrone-3F R2 = -Arg-Gly-Asp-Phe; in 24 R3 = -Arg(NO2)-Gly-
Asp(OBzl)-Ser(Bzl)-Arg(NO2)-Gly-Asp(OBzl)-Ser(Bzl)-OBzl, in 25 R3 = -Arg(NO2)-Gly-Asp(OBzl)-Val-Arg(NO2)-Gly-Asp(OBzl)-Val-OBzl, in 26 R3 = -Arg(NO2)-Gly-Asp(OBzl)-
Phe-Arg(NO2)-Gly-Asp(OBzl)-Phe-OBzl; in Estrone-3SS R4 = -Arg-Gly-Asp-Ser-Arg-Gly-Asp-Ser, in Estrone-3VV R4 = -Arg-Gly-Asp-Val-Arg-Gly-Asp-Val, in Estrone-3FF R4
=
-Arg-Gly-Asp-Phe-Arg-Gly-Asp-Phe; (i) BrCH2CO2C2H5 and NaOC2H5; (ii) NaOH in methanol; (iii) DCC, HOBt, NMM and 1–3; (iv) DCC, HOBt, NMM and 4–6; (v) Pt/H2.
erence conjugates, estrone-3-Arg-Gly-Asp-AA (estrone-3S: AA =
Ser; estrone-3V: AA = Val; estrone-3F: AA = Phe), were provided
in 57–76% yield. The procedure, synthetic data, and chemical phys-
ical data of all estrone-17–RGD octrapeptide conjugates are given
in a SI file.
2.4. Serum calcium and ALP of mice receiving estrogen–RGD
conjugates
The influence of estrogen–RGD peptide conjugates on the bone
loss may be embodied in serum calcium, phosphorous and ALP lev-

M. Zhao et al. / Bioorg. Med. Chem. 17 (2009) 3680–3689
3683
Table 1
Table 2
Calcium, phosphorous and ALP in serum of mice receiving conjugates
Weights of dry femurs and femur ashes of mice receiving conjugates
Groupa
Calcium
Phosphorous
ALP
Groupa
Dry femur
Femur ash
Femur length (cm)
OVX
3.39 0.15
3.30 0.11
3.34 0.24
3.35 0.26
3.33 0.20
3.32 0.19
3.34 0.10
3.35 0.17
3.36 0.19
3.34 0.25
3.22 0.16c
3.17 0.29c
3.19 0.19c
3.33 0.29
3.33 0.12
3.44 0.22
3.08 0.16b
3.00 0.14b
3.15 0.19c
3.18 0.28
2.82 0.29
3.01 0.27
3.13 0.43
2.98 0.20
2.99 0.23
3.19 0.61
3.01 0.27
3.04 0.12
3.01 0.28
2.99 0.29
2.84 0.20b
3.09 0.32
2.98 0.20
2.96 0.26
3.17 0.34
2.96 0.13c
3.22 0.21
3.01 0.40
28.78 2.74
22.97 1.71
27.22 2.92
27.97 2.86
26.62 2.12c
26.01 2.71c
25.42 1.35b
23.93 2.90b
22.96 2.96d
23.18 2.79d
19.61 5.98e
19.94 2.77e
15.80 2.63e
16.62 4.12e
17.42 2.17e
16.42 2.78e
13.80 2.57e
13.27 1.55e
15.06 2.95e
OVX
Sham
Estradiol
Estrone
51.77 2.50
53.77 2.04
52.35 2.22
53.03 2.32
53.89 2.20c
53.97 2.31c
53.87 2.15c
53.98 2.08c
54.01 2.27c
54.00 2.15c
55.22 2.02b
56.46 2.03b
55.46 2.01b
54.29 2.20c
54.12 1.62c
53.99 2.56c
55.90 2.49b
55.39 2.65b
55.26 2.22b
31.57 2.11
34.11 2.66
32.22 2.02
32.08 2.23
34.08 2.24e
34.29 2.22e
33.99 2.12e
34.17 2.23e
34.70 2.14d
34.87 2.04d
35.60 2.26d
35.75 2.27d
35.22 2.23d
34.38 2.94e
33.99 1.59e
33.85 2.01e
35.78 2.31d
35.29 2.28d
35.24 2.22d
1.64 0.24
1.64 0.21
1.64 0.28
16.45 0.42
1.65 0.20
1.67 0.27
1.67 0.20
1.66 0.28
1.67 0.24
16.40 0.26
16.39 0.52
16.56 0.49
16.18 0.37
16.46 0.50
16.60 0.25
16.47 0.34
16.73 0.42
16.55 0.41
16.41 0.24
Sham
Estradiol
Estrone
Estradiol-3S
Estradiol-3V
Estradiol-3F
Estradiol-17S
Estradiol-17V
Estradiol-17F
Estradiol-3S,17S
Estradiol-3V,17V
Estradiol-3F,17F
Estrone-3S
Estrone-3V
Estrone-3F
Estrone-3SS
Estrone-3VV
Estrone-3FF
Estradiol-3S
Estradiol-3V
Estradiol-3F
Estradiol-17S
Estradiol-17V
Estradiol-17F
Estradiol-3S,17S
Estradiol-3V,17V
Estradiol-3F,17F
Estrone-3S
Estrone-3V
Estrone-3F
Estrone-3SS
Estrone-3VV
Estrone-3FF
In calcium content column: bCompared with ovariotomy P < 0.01, with estradiol
and estrone P < 0.05. cCompared with ovariotomy P < 0.05.
a
Dosage = 110.3 nmol/kg, n = 12, weights of dry femurs and femur ashes are
In phosphorous content column: bCompared with ovariotomy P < 0.01. cCom-
pared with ovariotomy P < 0.05.
represented as X SD mg, femur length is represented as X SD cm.
b
Compared to ovariotomy, estradiol and estrone P < 0.01.
Compared to ovariotomy P < 0.05.
Compared to ovariotomy P < 0.01, to estradiol and estrone P < 0.05.
Compared to ovariotomy P < 0.05.
c
In ALP column: bCompared with ovariotomy P < 0.01; cCompared with ovariot-
omy P < 0.05. dCompared with ovariotomy P < 0.01, with estradiol and estrone
P < 0.05. eCompared with ovariotomy, estradiol and estrone P < 0.01.
d
e
a
Dosage = 110.3 nmol/kg; n = 12; serum calcium and phosphorous are repre-
sented as X SD mM; Serum ALP is represented as X SD U/L king
parison does imply that introducing RGD tetrapeptides into both
the 3- and 17-positions of estradiol and introducing RGD octapep-
tides into the 3-position of estrone could be an effective strategy to
increase the oral activity of inhibiting bone loss and/or enhancing
bone forming for ovariectomized mice. The procedure of the assay
is given in a SI file.
els. To explore the effect of oral estrogen–RGD peptide conjugates
on serum calcium, phosphorous and ALP levels, mice were orally
administrated 110.3 nmol/kg of the novel conjugates estradiol-
3S,17S, estradiol-3V,17V, estradiol-3F,17F, estrone-3SS, estrone-
3VV and estrone-3FF, and 110.3 nmol/kg of reference conjugates
(estradiol-3S, estradiol-3V, estradiol-3F, estradiol-17S, estradiol-
17V, estradiol-17F, estrone-3S, estrone-3V and estrone-3F),
110.3 nmol/kg of estradiol or estrone (estrogen control) and aque-
ous solution of 0.5% carboxymethylcellulose (vehicle control) fol-
lowing the procedure of item 4.6. The data are listed in Table 1,
which indicate that the serum calcium content and ALP value of
the mice of vehicle control, estrogen control and reference conju-
gates are significantly higher than those of the mice receiving
estradiol-3S,17S, estradiol-3V,17V, estradiol-3F,17F, estrone-3SS,
estrone-3VV and estrone-3FF. This comparison indicates that
introducing RGD tetrapeptides into both the 3- and 17-positions
of estradiol and introducing RGD octapeptides into the 3-position
of estrone leads to the inhibition of the bone loss of ovariectomized
mice, therefore the conjugation could be an effective strategy to in-
crease the oral activity of estrogen for ovariectomized mice. The
procedure of the assay is given in a SI file.
2.6. Femur calcium, phosphorous and mineral of mice
receiving estrogen–RGD conjugates
The influence of estrogen–RGD peptide conjugates on the bone
forming and bone loss may be also embodied in femur calcium,
phosphorous and mineral levels. To further explore the effect of
estrogen–RGD conjugates on femur calcium, mice were orally
administrated 110.3 nmol/kg of novel conjugates estradiol-3S,17S,
estradiol-3V,17V, estradiol-3F,17F, estrone-3SS, estrone-3VV and
estrone-3FF, 110.3 nmol/kg of reference conjugates, 110.3 nmol/
kg of estrogen, and vehicle following the procedure of item 4.7.
The data are listed in Table 3, which indicate that femur calcium
and phosphorous of the mice receiving vehicle and estrogen are sig-
nificantly lower than those of the mice receiving estradiol-3S,17S,
estradiol-3V,17V, estradiol-3F,17F, estrone-3SS, estrone-3VV and
estrone-3FF. The comparison again implies that introducing RGD
tetrapeptides into both the 3- and 17-positions of estradiol and
introducing RGD octapeptides into the 3-position of estrone could
be an effective strategy to increase the oral activity of inhibiting
bone loss and/or enhancing bone forming for ovariectomized mice.
The procedure of the assay is given in a SI file.
2.5. Weights of femurs and femur ashes of mice receiving
estrogen–RGD conjugates
The influence of estrogen–RGD peptide conjugates on the form
and loss of bone may be directly embodied in the weights of fe-
murs and femur ashes of mice. To explore the effect of estrogen-
RGD conjugates on the weights of dry femurs and femur ashes,
mice were orally administrated 110.3 nmol/kg of the novel conju-
gates estradiol-3S,17S, estradiol-3V,17V, estradiol-3F,17F, estrone-
3SS, estrone-3VV and estrone-3FF, 110.3 nmol/kg of reference con-
jugates, 110.3 nmol/kg of estrogen, and vehicle following the pro-
cedure of item 4.7. The data are listed in Table 2, which indicate
that the weights of both the femur and femur ash of the mice
receiving vehicle and estrogen controls are significantly lower than
that of the mice receiving estradiol-3S,17S, estradiol-3V,17V, estra-
diol-3F,17F, estrone-3SS, estrone-3VV and estrone-3FF. The com-
2.7. Side effect of mice receiving estrogen–RGD conjugates
Dose-related side effects commonly occurred in long term ERT
or HRT are endometrial hyperplasia and the thromboembolic
events. To estimate the endometrial hyperplasia and the thrombo-
embolic risks of oral administration of novel conjugates, mice were
orally administrated 110.3 nmol/kg of estradiol-3S,17S, estradiol-
3V,17V, estradiol-3F,17F, estrone-3SS, estrone-3VV and estrone-
3FF, 110.3 nmol/kg of reference conjugates, 110.3 nmol/kg of estro-
gen, and vehicle control following the procedure of item 4.8. The
weights of organ and body of the mice explored that novel conju-

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M. Zhao et al. / Bioorg. Med. Chem. 17 (2009) 3680–3689
Table 3
conjugates. The comparison implies that introducing RGD tetra-
peptides into both the 3- and 17-positions of estradiol and intro-
ducing RGD octapeptides into the 3-position of estrone could be
an effective strategy to limit the side effect of estrogen in ERT.
The procedure of the assay is given in a SI file.
Femur calcium, phosphorous and mineral of mice receiving conjugates
Groupa
Calcium
Phosphorous
Mineral
OVX
Sham
Estradiol
Estrone
54.45 1.35
62.08 2.40
57.28 1.87
56.20 2.56
58.76 2.13b
57.84 1.76c
57.42 1.04c
59.14 1.22b
57.49 1.98c
58.06 2.17c
61.05 1.79b
61.56 2.05b
61.17 2.09b
57.76 2.13c
57.24 1.71c
56.96 2.94d
59.95 1.95b
60.50 2.12b
59.92 2.04b
19.31 1.16
20.16 0.19
19.81 0.21
19.11 0.34
20.24 0.04f
20.24 0.42f
20.09 0.19f
20.37 0.12f
20.14 0.28f
20.56 0.26f
20.35 0.32f
20.94 0.10e
21.21 0.29e
20.44 0.04e
20.08 0.24f
20.32 0.66f
21.76 0.22e
21.84 0.23e
20.33 0.68f
0.60 0.01
0.63 0.02
0.61 0.02
0.61 0.03
0.60 0.01
0.61 0.02
0.60 0.03
0.60 0.01
0.61 0.03
0.61 0.02
0.62 0.01g
0.62 0.01g
0.61 0.01h
0.60 0.02
0.61 0.02
0.59 0.06
0.62 0.02g
0.62 0.01g
0.61 0.01h
2.8. 3D QSAR analyses of estrogen–RGD conjugates
Estradiol-3S
Estradiol-3V
Estradiol-3F
Estradiol-17S
Estradiol-17V
Estradiol-17F
Estradiol-3S,17S
Estradiol-3V,17V
Estradiol-3F,17F
Estrone-3S
Estrone-3V
Estrone-3F
Estrone-3SS
Estrone-3VV
Estrone-3FF
To reveal the dependence of increasing oral anti-osteoporosis
activity and decreasing side effect of estrogen–RGD conjugates on
their structure the weight of the femur and femur ash was selected
as the anti-osteoporosis activity, bleeding time and uterine weight
stand for side effect, and QSAR module of Cerius2 was followed
according to a standard procedure to establish the equations to cor-
relate the weight of the femur and femur ash, the bleeding time and
uterine weight with the structures of estrogen–RGD conjugates.
2.8.1. Alignment of estrogen and estrogen–RGD conjugates
To establish valid 3D-QSAR models a proper alignment proce-
dure of estrogen and estrogen–RGD conjugates was performed
using the target model align strategy in the align module within
Cerius2. With an assumption that each structure of estrogen and
estrogen–RGD conjugates binds the same site of the receptor and
exhibits activity, they were aligned in a pharmacological active ori-
entation. To obtain a consistent alignment the estrogen ring, the
common pharmacophore, was selected as the template for super-
posing estrogen–RGD conjugates. The method used for performing
the alignment was Maximum Common Subgraph (MCS).29 MCS
looks at molecules as points and lines, and uses the techniques
out of graph theory to identify the patterns. Then MCS finds the
largest subset of atoms in estrogen ring that shared by estrogen–
RGD conjugates. This subset was used for the alignment. A rigid
fit of atom pairings was performed to superimpose each structure
onto the target model estrogen ring. The stereoview of aligned
estrogen and estrogen–RGD conjugates used for molecular field
generation is shown in Figure 1. The alignment stereoview ex-
plores that to superimpose onto estrogen ring the RGD peptide
chain of each structure has to take individual conformation. This
chain conformation of individual RGD-peptide will affect on the
anti-osteoporosis activity and side effects.
a
Dosage = 110.3 nmol/kg, n = 12, femurs calcium, phosphorous and mineral are
represented as X SD%.
b
Compared to ovariotomy, estradiol and estrone P < 0.01.
Compared to ovariotomy P < 0.01.
Compared with ovariotomy P < 0.05.
Compared to ovariotomy, estradiol and estrone P < 0.01.
Compared to ovariotomy P < 0.05, with estradiol and estrone P < 0.01.
Compared with ovariotomy P < 0.01.
Compared with ovariotomy P < 0.05.
c
d
e
f
g
h
gates had no effect on the weights of the spleen, lung, kidney, liver
and body of the mice.
The bleeding time is listed in Table 4, which indicate that the
bleeding time of the mice receiving vehicle and estrogen–RGD con-
jugates is significantly longer than that of the mice receiving estra-
diol and estrone, which suggests that thromboembolic risk of
estradiol and estrone is avoided by use of novel conjugates. The
weights of the uterine are also listed in Table 4. The data indicate
that uterine weights of the mice receiving vehicle and the novel
conjugates are significantly lower than that of the mice receiving
estradiol and estrone, which suggests that the endometrial hyper-
plasia induced by estradiol and estrone is avoided by the novel
2.8.2. QSAR module of Cerius2 based MFA of estrogen and
conjugates
After energy-minimization using MMFF94 (Merck Molecular
Force Field), Molecular Field Analysis (MFA) was performed for
estrogen and estrogen–RGD conjugates using the QSAR module
of Cerius2.30 A five-step-procedure consisted of conformer genera-
Table 4
Bleeding time and uterine weights of mice receiving conjugates
Groupa
Bleeding time after last administration
Uterine weight
30 m
90 min
OVX
Sham
Estradiol
Estrone
542 50
562 53
417 57b
411 51b
582 50
569 54
557 45
575 51
551 48
576 59
580 52
591 86
522 38
579 52
563 40
581 49
588 71
582 67
579 46
548 49
568 59
441 43b
433 40b
577 52
577 52
560 51
561 54
560 51
568 61
570 52
532 20
524 27
568 44
571 57
576 55
516 39
514 58
574 49
0.081 0.022
0.086 0.026
0.108 0.021c
0.114 0.015b
0.064 0.015d
0.064 0.011d
0.067 0.021
0.063 0.015d
0.064 0.013d
0.060 0.028
0.050 0.013c
0.059 0.019d
0.050 0.010c
0.069 0.035
0.066 0.004d
0.060 0.007c
0.058 0.031d
0.059 0.012c
0.051 0.038d
Estradiol-3S
Estradiol-3V
Estradiol-3F
Estradiol-17S
Estradiol-17V
Estradiol-17F
Estradiol-3S,17S
Estradiol-3V,17V
Estradiol-3F,17F
Estrone-3S
Estrone-3V
Estrone-3F
Estrone-3SS
Estrone-3VV
Estrone-3FF
a
Dosage = 110.3 nmol/kg, n = 12; tail bleeding time is represented as X SD s
and uterine weight is represented as X SD g.
b
Compared to ovariotomy P < 0.01.
Compared to ovariotomy P < 0.01.
Compared to ovariotomy P < 0.05.
c
Figure 1. Alignment stereoview of estrogen and estrogen–RGD peptide conjugates
used for molecular field generation.
d

M. Zhao et al. / Bioorg. Med. Chem. 17 (2009) 3680–3689
3685
tion, energy minimization, atom match and molecular alignment,
preference setting, and regression analysis was automatically prac-
ticed in MFA. Electrostatic and steric fields of the molecules were
created by use of proton and methyl groups as probes, respectively.
These fields were sampled at each point of a regularly spaced grid
of 1 Å. An energy cutoff of 30.0 kcal/mol was set for both electro-
static and steric fields. Total grid points generated were 10,340.
Though spatial and structural descriptors such as dipole moment,
polarizability, radius of gyration, number of rotatable bonds,
molecular volume, principal moment of inertia, AlogP98, number
of hydrogen bond donors and acceptors, and molar refractivity
were also considered, only the highest variance holder proton,
methyl and hydroxyl descriptors were used. Regression analysis
was carried out using Genetic Partial Least Squares (G/PLS) method
consisting of 50,000 generations with a population size of 100. The
number of components was set to 5. Cross-validation was per-
formed with the leave-one-out procedure. PLS analysis was scaled
with all variables normalized to a variance of 1.0.
Figure 3. Graph of tested femur weight against predicted femur weight of mice
The regions where the variations of steric or electrostatic fea-
tures of estrogen and the conjugates in the training set lead to
the increase or decrease of femur weight were specified. Proton
descriptor with positive coefficient indicates a region favorable
for electropositive group, while negative coefficient indicates elec-
tronegative group required at this position. Methyl descriptor with
positive coefficient indicates a region favorable for large group,
while negative coefficient indicates small group required at this
position. Hydroxyl descriptor with positive coefficient indicates a
region favorable for the group to form hydrogen bond, while neg-
ative coefficient indicates the group to form hydrogen bond is not
required at this position.
receiving estrogen and estrogen–RGD peptide conjugates.
In Eq. 1 the data points (n), correlation coefficient (r) and square
correlation coefficient (r2) were 17, 0.998 and 0.989, respectively.
In Eq. 1 one term, H+/5867, has positive coefficient, which means
that at this position electron-withdrawing group increases femur
weight, four terms, CH3/4110, CH3/6984, CH3/7040 and CH3/
8295, have positive coefficient, which means that at these posi-
tions large group increases femur weight, one term, CH3/5371,
with negative coefficient, which means that at this position small
group decreases femur weight, four terms, HOꢁ/2193, HOꢁ/4227,
HOꢁ/7875 and HOꢁ/9071, have positive coefficient, which means
that at these positions hydrogen bond forming group increases fe-
mur weight, and two terms, HOꢁ/5313 and HOꢁ/7815, have nega-
tive coefficient, which means that at these positions hydrogen
bond forming group decreases femur weight. According to the va-
lue of term coefficient of the equation if a conjugate has its elec-
tron-withdrawing group near position H+/5867, hydrogen bond
forming group near positions HOꢁ/2193 and HOꢁ/4227, as well
as bulk group near position CH3/7040 it possess high anti-osteopo-
rosis activity.
According to Eq. 1 a favorable estrogen–RGD conjugate inhibit-
ing femur loss should have large group near CH3/6984–8295 region
and/or electron-withdrawing group near H+/5867 region and/or no
hydrogen bond forming group near HOꢁ/2193–4227 region. To ex-
plain this requirement representatives Estradiol-3v, Estradiol-17v,
and Estradiol-3v,17v are given in Figure 4a, as well as Estrone-3v
and Estrone-3vv are given in Figure 4b. Besides the same electro-
static and environments as estradiol ring, Estradiol-3v has a hydro-
gen bond forming group near HOꢁ/5313 and results in moderate
increase of femur weight, Estradiol-17v has small group near
CH3/7040 and electron-withdrawing group near H+/5867 thus to-
tally results in moderate increase of femur weight, while Estra-
diol-3v,17v has electron-withdrawing group near H+/5867,
hydrogen bond forming group near HO-/5315 and peptide chain
over CH3/7040 and thus results in high increase of femur weight.
Besides the same electrostatic and environments as estrone ring,
Estrone-3v has hydrogen bond forming group near HOꢁ/5315, pep-
tide chain over CH3/5313 and a small group near CH3/4110 and
thus totally results in low femur weight, Estrone-3vv has hydrogen
bond forming group near HOꢁ/2193 and thus resulting in high fe-
mur weight.
2.8.2.1. MFA model for femur weights of mice receiving estro-
gen and conjugates. MFA model for the femur weights of the mice
receiving estrogen or conjugates in terms of the descriptors proton,
methyl and hydroxyl groups is aligned as Figure 2 and expressed
by Eq. 1. The correlation of femur weights tested on the ovariecto-
mized mouse model and femur weights calculated from Eq. 1 is
shown in Figure 3.
Femur weight ¼ 53:59 þ 0:067ðHþ=5867Þ þ 0:0017ðCH3=4110Þ
ꢁ 0:0095ðCH3=5371Þ þ 0:035ðCH3=6984Þ
þ 0:0082ðCH3=7040Þ þ 0:0041ðCH3=8295Þ
þ 0:10ðHOꢁ=2193Þ þ 0:12ðHOꢁ=4227Þ
ꢁ 0:044ðHOꢁ=5313Þ ꢁ 0:14ðHOꢁ=7815Þ
þ 0:0086ðHOꢁ=7875Þ0:011ðHOꢁ=9071Þ
ð1Þ
2.8.2.2. MFA model for the weight of femur ash of mice
receiving estrogen and conjugates. MFA model for the weight
of the femur ash of the mice receiving estrogen and conjugates
in terms of the descriptors proton, methyl and hydroxyl groups is
Figure 2. Alignment stereoview of estrogen and estrogen–RGD peptide conjugates
affecting on the femur weight of mice.

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M. Zhao et al. / Bioorg. Med. Chem. 17 (2009) 3680–3689
Figure 4. Electrostatic and environments of Estradiol-3v, Estradiol-17v, and Estradiol-3v,17v (a) and Estrone-3v and Estrone-3vv (b) within the grid with 3D points of Eq. 1.
Femur ash weight ¼ 32:23 þ 0:043ðHþ=6960Þ þ 0:022ðCH3=1416Þ
þ 0:035ðCH3=1435Þ þ 0:046ðCH3=3696Þ
þ 0:005ðCH3=6301Þ þ 0:045ðCH3=6660Þ
þ 0:39ðCH3=9509Þ ꢁ 0:009ðHOꢁ=1720Þ
þ 0:071ðHOꢁ=4153Þ þ 0:046ðHOꢁ=4548Þ
þ 0:0062ðHOꢁ=5652Þ
ð2Þ
In Eq. 2 the data points (n), correlation coefficient (r) and square
correlation coefficient (r2) were 17, 0.998 and 0.990, respectively.
In Eq. 2 one term, H+/6960, has positive coefficient, which means
that at this position an electron-withdrawing group increases the
weight of the femur ash, six terms, CH3/1416, CH3/1435, CH3/
3696, CH3/6301, CH3/6660 and CH3/9509, have positive coefficient,
which means that at these positions large group increases the
weight of the femur ash, three terms, HOꢁ/4153, HOꢁ/4548 and
HOꢁ/5652, have positive coefficient, which means that at these
positions hydrogen bond forming group increases the weight of fe-
mur ash, and one term, HOꢁ/1720, has negative coefficient, which
means that at this position hydrogen bond forming group de-
creases the weight of the femur ash. According to the value of term
coefficient of the equation if a conjugate has its electron-with-
drawing group near H+/6960 and/or hydrogen bond forming group
near HOꢁ/4153–5652 and/or bulk group near CH3/6660–9509 it
will possess high anti-osteoporosis activity.
Figure 5. Alignment stereoview of estrogen and estrogen–RGD peptide conjugates
affecting on the femur ash weight.
aligned as Figure 5 and expressed by Eq. 2. The correlation of tested
weights of the femur ash and calculated weights is explained by
Figure 6.
To explain this requirement representatives Estradiol-3F, Estra-
diol-17F, and Estradiol-3F,17F are given in Figure 7a, as well as Es-
trone-3F and Estrone-3FF are given in Figure 7b. Besides the same
electrostatic and environments as estradiol ring Estradiol-3F has
no hydrogen bond forming group near HOꢁ/5652 and thus results
in no increase of the weight of femur ash, Estradiol-17F has a bulk
group near CH3/9509 and small group near CH3/6660 and totally
results in no increase of the weight of femur ash, while Estradiol-
3F,17F has an electron-releasing group near H+/6960, an elec-
tron-withdrawing group near HOꢁ/1720 and peptide chain over
CH3/3696 and thus results in high weight of femur ash. Estrone-
3F has hydrogen bond forming groups near HOꢁ/1720, HOꢁ/4153
and HOꢁ/4548, thus totally results in moderate weight of femur
ash, while Estrone-3FF has hydrogen bond forming group near
HOꢁ/4153 and bulk groups near CH3/1416, CH3/1435, CH3/6301
and CH3/6660, thus results in high weight of femur ash.
2.8.2.3. MFA model for bleeding time after mice receiving
estrogen and conjugates. The MFA model for bleeding time
90 min after the mice receiving estrogen and conjugates in terms
of the descriptors proton, methyl and hydroxyl groups is aligned
Figure 6. Graph of tested femur ash weight against predicted femur ash weight of
mice receiving estrogen and estrogen–RGD peptide conjugates.

M. Zhao et al. / Bioorg. Med. Chem. 17 (2009) 3680–3689
3687
Figure 7. Electrostatic and environments of Estradiol-3F, Estradiol-17F, and Estradiol-3F,17F (a) and Estrone-3F and Estrone-3FF (b) within the grid with 3D points of Eq. 2.
Bleeding time ¼ 404:62 þ 0:51ðHþ=3351Þ ꢁ 1:33ðHþ=6241Þ
ꢁ 1:3ðCH3=2976Þ ꢁ 0:74ðCH3=3696Þ
þ 5:22ðCH3=4152Þ ꢁ 1:2ðCH3=4254Þ
ꢁ 0:19ðCH3=6636Þ þ 5:46ðCH3=7440Þ
ꢁ 0:018ðCH3=7454Þ ꢁ 0:21ðHOꢁ=4531Þ
ꢁ 0:037ðHOꢁ=4933Þ
ð3Þ
In Eq. 3 the data points (n), correlation coefficient (r) and square
correlation coefficient (r2) were 17, 0.977 and 0.994, respectively.
In Eq. 3 one term, H+/3351, has positive coefficient, which means
that at this position electron-withdrawing group increases bleed-
ing time, one term, H+/6241, has negative coefficient, which means
that at this position electron-releasing group increases bleeding
time, two terms, CH3/4152 and CH3/7440, have positive coefficient,
which means that at these positions bulk group increases bleeding
time, five terms, CH3/2976, CH3/3696, CH3/4254, CH3/6636 and
CH3/7454 have negative coefficient, which means that at these
positions small group increases bleeding time, and two terms,
HO-/4531 and HO-/4933, have negative coefficient, which means
that at these positions hydrogen bond forming group increases
bleeding time. According to the value of term coefficient of the
equation if a conjugate has its electron-releasing group near H+/
6241 and/or small group near CH3/4152 and CH3/7440 it will pos-
sess low thromboembolic risk.
Figure 8. Alignment stereoview of estrogen and estrogen–RGD peptide conjugates
affecting on the bleeding time at 90 min after the mice were administratered.
as Figure 8 and expressed by Eq. 3. The correlation of the bleeding
time tested on the ovariectomized mouse model and the bleeding
time calculated from Eq. 3 is explained by Figure 9.
To explain this requirement representatives Estradiol-3S, Estra-
diol-17S, and Estradiol-3S,17S are given in Figure 10a, as well as Es-
trone-3F and Estrone-3FF are given in Figure 10b. Besides the same
electrostatic and environments as estradiol ring, Estradiol-3S has
small groups near CH3/3696 and CH3/4252 and thus results in
lengthening bleeding time, Estradiol-17S has bulk group near CH3/
7440 and resulting in lengthening bleeding time, while Estradiol-
3S,17S has electron-releasing group near H+/3351, small groups near
CH3/7454, CH3/6636 and CH3/3696, as well as bulk group near CH3/
4152 thus results in significant length of bleeding time. Estrone-3F
has small group near CH3/2976 and thus results in lengthening of
the bleeding time, while Estrone-3FF has small group near CH3/
2976, peptide chain over CH3/4152, electron-withdrawing groups
near H+/3351 and thus results in low thromboembolic risk.
2.8.2.4. MFA model for uterine weight of mice receiving estro-
gen and conjugates. The MFA model for uterine weight of the
mice receiving estrogen and conjugates in terms of the descriptors
proton, methyl and hydroxyl groups is aligned as Figure 11 and
Figure 9. Graph of tested bleeding time against predicted bleeding time of mice
receiving estrogen and estrogen–RGD peptide conjugates.

3688
M. Zhao et al. / Bioorg. Med. Chem. 17 (2009) 3680–3689
Figure 10. Electrostatic and environments of Estradiol-3S, Estradiol-17S, and Estradiol-3S,17S (a) as well as Estrone-3F and Estrone-3FF (b) within the grid with 3D points of
Eq. 3.
Figure 11. Alignment stereoview of estrogen and estrogen–RGD peptide conjugates
Figure 12. Graph of tested uterine weight against predicted uterine weight of mice
affecting on uterine weight.
receiving estrogen and estrogen–RGD peptide conjugates.
expressed by Eq. 4. The correlation of uterine weights tested on the
gen bond forming group increases uterine weight. According to
ovariectomized mouse model and uterine weights calculated from
the value of term coefficient of equation if a conjugate has its
Eq. 4 is explained by Figure 12.
electron-releasing group near H+/4323–7020 and/or small group
near CH3/5294–6642 it will possess low risk of endometrial
hyperplasia.
To explain this requirement representatives Estradiol-3S, Estra-
diol-17S, and Estradiol-3S,17S are given in Figure 13a, as well as
Uterine weight¼0:058þ0:000635ðHþ=4323Þ
þ0:000372ðHþ=4512Þþ0:000653ðHþ=7020Þ
þ0:000111ðCH3=5294Þþ0:00001ðCH3=5614Þ
Estrone-3F and Estrone-3FF are given in Figure 13b. Besides the
þ0:00097ðCH3=5712Þþ0:0021ðCH3=5826Þ
ꢁ0:0016ðCH3=6642Þꢁ0:000374ðHOꢁ=2669Þ
same electrostatic and environments as estradiol ring, Estradiol-
3S has electron-withdrawing and releasing groups near H+/4512
ꢁ0:0017ðHOꢁ=6579Þꢁ0:000374ðHOꢁ=7814Þ
ð4Þ
and thus results in no increase of uterine weight, Estradiol-17S
has electron-withdrawing and releasing groups near H+/7020 and
hydrogen bond forming group near HOꢁ/6579 and thus results in
the decrease of uterine weight, while Estradiol-3S,17S has elec-
tron-withdrawing and releasing groups near H+/7020 and peptide
chain over CH3/6642 and thus results in low uterine weight. Be-
sides the same electrostatic and environments as estrone, Es-
trone-3F has electron-releasing group near H+/4512 and thus
resulting in decrease of uterine weight, while Estrone-3FF has elec-
tron-releasing group near H+/4323 and hydrogen bond forming
group near HOꢁ/2669 thus results in significant decrease of uterine
weight.
In Eq. 4 the data points (n), correlation coefficient (r), square
correlation and coefficient (r2) were 17, 0.990 and 0.995, respec-
tively. In Eq. 4 three terms, H+/4323, H+/4512 and H+/7020, have
positive coefficient, which means that at these positions electron-
withdrawing group increases uterine weight, four terms, CH3/
5294, CH3/5614, CH3/5712 and CH3/5826, have positive coeffi-
cient, which means that at these positions bulk group increases
uterine weight, one term, CH3/6642, has negative coefficient,
which means that at this position small group increases uterine
weight, three terms, HOꢁ/2669, HO-/6579 and HOꢁ/7814, have
negative coefficient, which means that at these positions hydro-

M. Zhao et al. / Bioorg. Med. Chem. 17 (2009) 3680–3689
3689
Figure 13. Electrostatic and environments of Estradiol-3S, Estradiol-17S, and Estradiol-3S,17S (a) and Estrone-3F and Estrone-3FF (b) within the grid with 3D points of Eq. 4.
5. Takeo, C.; Ugai, K.; Araki, J.; Zhang, L.; Baba, M.; Ohashi, W.; Ueno, K.; Suzuki, Y.;
3. Conclusions
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604.
Osteoporosis has being crushing pressure for our society. The
modifications of estradiol and estrone aimed at the improvement
of ERT or HRT are clinically important. The observations provided
here explored that introducing RGD-tetrapeptide into both 3-posi-
tion and 17-position of estradiol, as well as introducing RGD-octa-
peptide into 3-position of estrone did result in the conjugate with
high oral anti-osteoporosis activity and lower risk of endometrial
hyperplasia and thromboembolic events. With the helps of the
3D QSAR analysis the conjugate met the requirements related to
high anti-osteoporosis activity and/or low side effect could be
designed.
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This work was finished in Beijing Area Major Laboratory of Pep-
tide and Small Molecular Drugs, supported by PHR (IHLB), Special
Project (2008ZX09401-002) and Natural Scientific Foundation of
China (30572235).
Supplementary data
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