Synthesis, evaluation and 3D QSAR analysis of novel estradiol-RGD octapeptide conjugates with oral anti-osteoporosis activity

European Journal of Medicinal Chemistry 44 (2009) 1689–1704

Contents lists available at ScienceDirect

European Journal of Medicinal Chemistry

journal homepage: h ttp://www.elsevier.com/locate/ejmech

Original article

Synthesis, evaluation and 3D QSAR analysis of novel estradiol–RGD

octapeptide conjugates with oral anti-osteoporosis activity

,

a,b,

*

Jiangyuan Liu ^a, Xiaoyi Zhang ^b, Ming Zhao ^b, Shiqi Peng

**

^aCollege of Pharmaceutical Sciences, Peking University, Beijing 100083, PR China

^bCollege 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:

Received 13 May 2008

Received in revised form 11 August 2008

Accepted 11 September 2008

Available online 7 October 2008

To enhance the potency, reduce the side effects and improve oral property of estradiol in estrogen

replacement therapy (ERT), 6 novel estradiol–RGD octapeptide conjugates have been prepared. In an

ovariectomized mouse osteoporotic model, at an oral dosage of 110.3 nmol/kg per day, their anti-oste-

oporosis activity was signiﬁcantly higher than that of estradiol and estradiol–RGD tetrapeptide conju-

gates, and their risks of thrombogenesis and endometrial hyperplasia were signiﬁcantly lower than that

of estradiol and estradiol–RGD tetrapeptide conjugates. Using QSAR module of Cerius2, the 3D QSAR was

performed for both femur weights and femur ash weights of estradiol–RGD peptide conjugates receiving

mice. The r²of the 3D QSAR equations up to 0.995 and 0.988 indicates that they are capable of predicting

a comparatively exact anti-osteoporosis activity for a conjugate.

Keywords:

Estradiol

RGD octapeptide

Conjugate

Anti-osteoporosis

3D QSAR

1. Introduction

It is commonly accepted that osteoporosis is related to both the

decrease of bone formation modulated by osteoblast and the

The ovarium atrophy leads almost all postmenopausal women

confronting with osteoporosis [1]. More than 50% of these osteo-

porosis women suffered from fracture and some of them died of

secondary affection. Therefore osteoporosis is being a crushing

pressure for our society [2,3]. It is well known that the lack of

estrogen resulted from ovarium atrophy is responsible for post-

menopausal women developing osteoporosis. Consequently

estrogen replacement therapy (ERT) or estrogen–progestogen

replacement therapy (HRT) is clinically used for preventing osteo-

porosis [4]. In fact, ERT or HRT not only inhibits bone loss but also

decreases the risk of postmenopausal women developing coronary

heart disease [5,6]. It was found that a ﬁve-year HRT could decrease

the fracture risk of postmenopausal women by 50% [7]. In spite of

the efﬁcacy of ERT or HRT, a long-term therapy may induce a series

of dose-related side effects such as breast cancer, endometrial

hyperplasia and the thromboembolic events [8–12]. To improve the

clinical use of ERT or HRT structure modiﬁcations of estrogen, such

as conjugating estrogen with peptides, have attracted a lot of

interests [13–15].

increase of bone resorption modulated by osteoclast. In ERT or HRT

the role of estrogen is up-modulating the activity and proliferation

of osteoblast, and regulating the gene expression in osteoblast and

osteoclast [16–18]. Bone resorption is regulated by osteoclast

binding to bone surface, and depends on osteoclast adhering to

bone surface, which was mediated by integrin recognizing RGD

containing protein of osteoclasts [19]. This knowledge lead to

a hypothesis that the activity and proliferation of osteoblast and the

adhesion of osteoclast could be simultaneously upregulated and

downregulated by estrogen–RGD peptide conjugates. As a practice

a series of estrogen–RGD tetrapeptide conjugates were prepared

and bioassayed. On osteoporotic female mouse model, the conju-

gates possessed signiﬁcantly higher potency and lower side effects

than both estrogen and RGD tetrapeptides. On the other hand

however, the comparisons of the administration routes explored

that even though both oral and intraperitoneal administrations of

the conjugates gave similarly low side effects, the oral potency was

signiﬁcantly lower than that of intraperitoneal one, for instance the

femur ash weights, one of the important bone parameters, of orally

and intraperitoneally treated mice ranged from 28.7 mg to 33.7 mg

and from 34.4 mg to 40.0 mg, respectively [20]. These data suggest

that the conjugates’ oral potency needs to be increased and a cor-

responding strategy could be extending the peptide chain.

* Corresponding author. College of Pharmaceutical Sciences, Capital Medical

University, Beijing 100069, PR China. Tel./fax: þ86 10 8391 1528.

** Corresponding author. Tel./fax: þ86 10 83911535.

As a perspective experiment of this study the probability increasing

the oral stability via extending RGD peptide chain was evaluated by

pepsin promotion in vitro hydrolysis, for which RGDF and RGDFRGDF

E-mail addresses: mzhao@mail.bjum.edu.cn (M. Zhao), sqpeng@mail.bjum.

edu.cn (S. Peng).

doi:10.1016/j.ejmech.2008.09.036

1690

J. Liu et al. / European Journal of Medicinal Chemistry 44 (2009) 1689–1704

were selected as the substrates. Brieﬂy, the solution of 3.2 mg pepsin,

2.0 mg NaCl, 7 l concentrated hydrochloric acid and 1 ml water was

centrifuged at 0 ^ꢀC and 3500g for 5 min and the upper layer was stored

at 4 ^ꢀC as pepsin stock solution. To 100

l of pepsin stock solution 49

(0.1 mol) of RGDF or 97 g (0.1 mol) of RGDFRGDF was added, the

Asp(OBzl)-Ser(Bzl)-OBzl (1), HCl$H-Arg(NO₂)-Gly-Asp(OBzl)-Val-

OBzl (2), HCl$H-Arg(NO₂)-Gly-Asp(OBzl)-Phe-OBzl (3), HCl$H-

Arg(NO₂)-Gly-Asp(OBzl)-Ser(Bzl)-Arg(NO₂)-Gly-Asp(OBzl)-Ser(Bzl)-

OBzl (4), HCl$H-Arg(NO₂)-Gly-Asp(OBzl)-Val-Arg(NO₂)-Gly-Asp

(OBzl)-Val-OBzl (5), HCl$H-Arg(NO₂)-Gly-Asp(OBzl)-Phe-Arg(NO₂)-

Gly-Asp(OBzl)-Phe-OBzl (6) in 89%, 76%, 81%, 82%, 66% and 69% yield,

respectively. After hydrogenation 4–6 were converted into the cor-

responding peptides RGDSRGDS (7), RGDVRGDV (8) and RGDFRGDF

(9) in 86%, 73% and 76% yield respectively.

m

mg

m

formed mixture was incubated at 37 ^ꢀC for 4 h and the incubator was

monitored by HPLC–ESI–MS at 30 min intervals for 6 h. Our unpub-

lished MS data explored that under pepsin promotion RGDF was

completely degraded within 90 min, while in the 4 h incubation

system of RGDFRGDF/pepsin the molecule ion of RGDFRGDF was still

testable. Besides, in the literature it was pointed out that using artiﬁcial

extracellular matrix proteins containing a repetitive Arg-Gly-Asp the

cell adhesion ability was enhanced without increasing side effects [21].

These observations imply that the modiﬁcation of estrogen with

repetitive Arg-Gly-Asp peptide chain is worthwhile. Thus, in the

present paper 6 novel estradiol–RGD octapeptide conjugates were

designed and synthesized. Their oral anti-osteoporosis activity,

endometrial hyperplasia risk and thromboembolic side effect were

evaluated on osteoporotic female mouse model using 6 estradiol–RGD

tetrapeptide conjugates as the reference compounds. Besides,

dependence of their oral anti-osteoporosis activity on their structure

was rationalized with 3D QSAR.

2.1.2. Preparing estradiol-3-RGD peptide conjugates

In the preparations of estradiol-3-RGD tetrapeptide conjugates

and estradiol-3-RGD octapeptide conjugates the 3-hydroxyl group

of estradiol was ﬁrstly alkylated by ethyl bromoacetate to provide

ethyl estradiol-3-oxylacetate (10) in 83% yield. By saponiﬁcation 10

was converted into the acid 11 (96% yield), to which HCl$H-

Arg(NO₂)-Gly-Asp(OBzl)-AA-OBzl (1–3, AA ¼ Ser(Bzl), Val and Phe)

or

HCl$H-Arg(NO₂)-Gly-Asp(OBzl)-AA-Arg(NO₂)-Gly-Asp(OBzl)-

AA-OBzl (4–6) was introduced and the corresponding estradiol-3-

Arg(NO₂)-Gly-Asp(OBzl)-AA-OBzl (12–14, AA ¼ Ser(Bzl), Val and

Phe) and estradiol-3-Arg(NO₂)-Gly-Asp-(OBzl)-AA-Arg(NO₂)-Gly-

Asp(OBzl)-AA-OBzl [15–17, AA ¼ Ser(Bzl), Val and Phe] were

obtained in 53%, 37%, 61%, 71%, 62% and 71% yield, respectively.

Removing all protective groups by catalytic hydrogenation estra-

diol-3-Arg-Gly-Asp-AA (18–20, AA ¼ Ser, Val and Phe) and estra-

diol-3-Arg-Gly-Asp-AA-Arg-Gly-Asp-AA (21–23, AA ¼ Ser, Val and

Phe) were obtained in 63%, 91%, 48%, 41%, 52% and 71% yield,

respectively (Scheme 2).

2. Results and discussion

2.1. Preparing estradiol–RGD peptide conjugates

To develop orally active estrogen–RGD peptide conjugates

estradiol–RGD octapeptide conjugates were prepared via intro-

ducing the building blocks RGD octapeptides on to the 3- and 17-

2.1.3. Preparing estradiol-17b-RGD peptide conjugates

Treating estradiol with succinic anhydride the estradiol-17-

monoester of succinic acid (24) was obtained in 95% yield. Coupling

b

positions of estradiol. On the other hand RGD octapeptides and

estradiol–RGD tetrapeptide conjugates, which were prepared via

introducing the building blocks RGD tetrapeptides on to the 3- and

17-b positions of estradiol, were used as the reference compounds

24 and 1–3 estradiol-17

AA ¼ Ser(Bzl), Val and Phe] were obtained in 48%, 45% and 42%

yield, respectively. Coupling 24 and 4–7 estradiol-17 -Arg(NO₂)-Gly-

b-Arg(NO₂)-Gly-Asp(OBzl)-AA-OBzl [25–27,

b

of estradiol–RGD octapeptide conjugates.

Asp(OBzl)-AA-Arg(NO₂)-Gly-Asp(OBzl)-AA-OBzl [28–30, AA ¼ Ser(Bzl),

Val and Phe] were obtained in 48%, 45%, 42%, 71%, 62% and 71% yield,

respectively. Removing all protective groups by catalytic hydroge-

nation estradiol-17-Arg-Gly-Asp-AA–OH (31–33, AA ¼ Ser, Val and

Phe) and estradiol-17-Arg-Gly-Asp-AA-Arg-Gly-Asp-AA (34–36,

AA ¼ Ser, Val and Phe) were obtained in 79%, 84%, 86%, 41%, 52% and

71% yield, respectively (Scheme 3).

2.1.1. Preparation of RGD octapeptide conjugates

RGD tetrapeptides and octapeptides were prepared via liquid

phase method according to the route depicted in Scheme 1. The

coupling of Boc-Asp(OBzl) with L-Ser(Bzl)-OBzl, L-Val-OBzl, L-Phe-

OBzl and removal of Boc group provided three C-terminal fragments

HCl$H-Asp(OBzl)-Ser(Bzl)-OBzl, HCl$H-Asp(OBzl)-Val-OBzl and

HCl$H-Asp(OBzl)-Phe-OBzl in 94%, 92% and 87% yield, respectively.

The coupling of Boc-Asp(OBzl) with

L

-Ser(Bzl),

L

-Val and

L

-Phe

2.2. Evaluating oral anti-osteoporosis activity of estradiol–RGD

conjugates

provided another three tetrapeptides Boc-Asp(OBzl)-Ser(Bzl), Boc-

Asp(OBzl)-Val and Boc-Asp(OBzl)-Phe in 74%, 71% and 80% yield,

respectively. The coupling of Boc-Arg(NO₂) with Gly provided Boc-

Arg(NO₂)-Gly in 84% yield. The condensation of these dipeptide

fragments and removal of Boc group provided HCl$H-Arg(NO₂)-Gly-

To estimate the oral anti-osteoporosis activity of estradiol–

ꢁRGD octapeptide conjugates the ovariectomized mouse osteo-

porotic model was used. After oral administration of estradiol, RGD

i

ii

H-AA-OBzl

Boc-Asp(OBzl)-AA-OBzl

i

HCl H-Asp(OBzl)-AA-OBzl

i

Boc-Arg(NO₂)

Boc-Arg(NO₂)-Gly

ii

HCl H-Arg(NO₂)-Gly-Asp(OBzl)-AA-OBzl

Boc-Arg(NO₂)-Gly-Asp(OBzl)-AA-OBzl

i

1-3

i

Boc-Asp(OBzl)

Boc-Asp(OBzl)-AA

ii

HCl H-Asp(OBzl)-AA-Arg(NO₂)-Gly-Asp(OBzl)-AA-OBzl

Boc-Asp(OBzl)-AA-Arg(NO₂)-Gly-Asp(OBzl)-AA-OBzl

HCl H-Arg(NO₂)-Gly-Asp(OBzl)-AA-Arg(NO₂)-Gly-Asp(OBzl)-AA-OBzl

Boc-Arg(NO₂)-Gly

i

4-6

iii

ii

H-Arg-Gly-Asp-AA-Arg-Gly-Asp-AA

Boc-Arg(NO₂)-Gly-Asp(OBzl)-AA-Arg(NO₂)-Gly-Asp(OBzl)-AA-OBzl

7-9

Scheme 1. Preparation of RGD tetrapeptides and octapeptides, in 1 and 4 AA ¼ Ser(Bzl), 2 and 5 AA ¼Val, 3 and 6 AA ¼ Phe, 7 AA ¼ Ser, 8 AA ¼Val, 9 AA ¼ Phe; (i) DCC, HOBt and

NMM; (ii) hydrogen chloride in ethyl acetate; (iii) Pt/H₂.

J. Liu et al. / European Journal of Medicinal Chemistry 44 (2009) 1689–1704

1691

OH

ii

i

C H O CCH O

HO

HOCOCH O

2

5

2

iii

11

10

OH

iv

R COCH O

2

1

2

18-23

12-17

Scheme 2. Preparation of estradiol-3-RGD tetrapeptide and octapeptide conjugates, in 12 R₁¼ -Arg(NO₂)-Gly-Asp(OBzl)-Ser(Bzl)-OBzl, in 13 R₁¼ -Arg(NO₂)-Gly-Asp(OBzl)-Val-

OBzl, in 14 R₁¼ -Arg(NO₂)-Gly-Asp(OBzl)-Phe-OBzl, in 15 R₁¼ -Arg(NO₂)-Gly-Asp(OBzl)-Ser(Bzl)-Arg(NO₂)-Gly-Asp(OBzl)-Ser(Bzl)-OBzl, in 16 R₁¼ -Arg(NO₂)-Gly-Asp(OBzl)-Val-

Arg(NO₂)-Gly-Asp(OBzl)-Val-OBzl, in 17 R₁¼ -Arg(NO₂)-Gly-Asp(OBzl)-Phe-Arg(NO₂)-Gly-Asp(OBzl)-Phe-OBzl, in 18 R₂¼ -Arg-Gly-Asp-Ser, in 19 R₂¼ -Arg-Gly-Asp-Val, in 20 R₂¼ -

Arg-Gly-Asp-Phe, in 21 R₂¼ -Arg-Gly-Asp-Ser-Arg-Gly-Asp-Ser, in 22 R₂¼ -Arg-Gly-Asp-Val-Arg-Gly-Asp-Val, in 23 R₂¼ -Arg-Gly-Asp-Phe-Arg-Gly-Asp-Phe; (i) BrCH₂CO₂C₂H₅and

NaOC₂H₅; (ii) NaOH in methanol; (iii) DCC, HOBt, NMM and 1–6; (iv) Pt/H₂.

octapeptides (7–9), estradiol-3-RGD tetrapeptide conjugates (18–

20), estradiol-17 -RGD tetrapeptide conjugates (31–33), estradiol-

3-RGD octapeptide conjugates (21–23), estradiol-17 -RGD octa-

peptide conjugates (34–36), the serum calcium, phosphorous and

ALP, the femur calcium, phosphorous and mineral, the weights of

dry femurs and femur ash, and the lengths of the femurs were

measured to know their anti-osteoporosis potencies.

To assign a proper oral dose for estradiol–RGD octapeptide

conjugates RGDFRGDF and estradiol–RGDFRGDF were selected as

model compounds and as the pre-treatment a series doses (0.01,

decreased the content of serum calcium. However the decreased

extent of serum calcium by conjugates 21–23 and 34–36 was

signiﬁcantly higher than that decreased by conjugates 18–20 and

31–33. Though estradiol, octapeptides 7–9, the mixture of estradiol

and 7–9, conjugates 18–23 and 31–36 decreased the content of ALP

the decreasing extent of conjugates 21–23 and 34–36 was signiﬁ-

cantly higher than that of the other conjugates. On the other hand

all of them did not affect the content of serum phosphorous of the

mice.

b

0.11, 0.15, 0.30, 0.50, 1.0, 2.0 and 4.0

m

mol/kg) of them were orally

2.2.2. Estradiol–RGD conjugates increasing the weights of dry

femurs and femur ashes

administered to osteoporotic female mice to determine their ex

vivo anti-aggregation activities and anti-osteoporosis activities.

The ex vivo anti-platelet aggregation assay explored that for ADP

and PAF induced mouse platelet aggregation RGDFRGDF had

In order to evaluate the inﬂuence of estradiol–RGD conjugates

on the bone loss, the femurs of estradiol, octapeptides 7–9, the

mixture of estradiol and 7–9, conjugates 18–23 and 31–36

receiving mice were collected, treated and measured following the

procedure of Section 4.7 to record the weights of dry femurs and

their ashes and femur lengths, the data are listed in Table 2. The

measurements indicated that conjugates 18–23 and 31–36

increased the weights of dry femurs and their ashes, and the

increased extents by conjugates 21–23 and 34–36 were signiﬁ-

cantly higher than those by conjugates 18–20 and 31–33. On the

other hand neither octapeptides (7–9) nor the mixture of estradiol

and 7–9 increased the weights of dry femurs and femur ashes.

However estradiol, octapeptides 7–9, the mixture of estradiol and

7–9, conjugates 18–23 and 31–36 did not affect the length of mouse

femur.

0.31

estradiol–RGDFRGDF had 0.15

values, respectively. The anti-osteoporosis assay explored that

4.0 mol/kg RGDFRGDF exhibited no anti-osteoporosis activity,

whereas 0.11 mol/kg estradiol–RGDFRGDF exhibited signiﬁcant

anti-osteoporosis activity. All assays gave no evidence for side

m

mol/kg and 0.26

mmol/kg IC₅₀values, respectively, while

m

mol/kg and 0.11 mol/kg IC₅₀

m

effects. Thus 0.11

experiments.

mmol/kg was selected as general dose in all

2.2.1. Estradiol–RGD conjugates decreasing the content of calcium

and ALP in mouse serum

In order to evaluate the inﬂuence of estradiol–RGD octapeptides

on the bone loss the serum of estradiol, RGD octapeptides, estra-

diol–RGD tetrapeptide conjugates and estradiol–RGD octapeptide

conjugates receiving mice were collected, treated and measured

following the procedure of Section 4.7 to determine the calcium

and alkaline phosphatase (ALP) contents of the serum. The data are

listed in Table 1, which indicated that the contents of serum calcium

was decreased by estradiol–RGD conjugates 18–23 and 31–36, and

neither RGD octapeptides 7–9 nor the mixture of estradiol and 7–9

2.2.3. Estradiol–RGD conjugates increasing the femur calcium,

phosphorous and mineral

In order to evaluate the inﬂuence of estradiol–RGD octapeptides

on the bone loss the femurs of estradiol, octapeptides 7–9, the

mixture of estradiol and 7–9, conjugates 18–23 and 31–36

receiving mice were collected, treated and measured following the

procedure of Section 4.7 to record the calcium, phosphorous and

OCOCH CH COR

2

OCOCH CH COR

OCOCH CH CO H

OH

2

1

2

i

ii

HO

iii

HO

24

31-36

25-30

Scheme 3. Preparation of estradiol-17-RGD tetrapeptide and octapeptide conjugates, in 25 R₁¼ -Arg(NO₂)-Gly-Asp(OBzl)-Ser(Bzl)-OBzl, in 26 R₁¼ -Arg(NO₂)-Gly-Asp(OBzl)-Val-

OBzl, in 27 R₁¼ -Arg(NO₂)-Gly-Asp(OBzl)-Phe-OBzl, in 28 R₁¼ -Arg(NO₂)-Gly-Asp(OBzl)-Ser(Bzl)-Arg(NO₂)-Gly-Asp(OBzl)-Ser(Bzl)-OBzl, in 29 R₁¼ -Arg(NO₂)-Gly-Asp(OBzl)-Val-

Arg(NO2)-Gly-Asp(OBzl)-Val-OBzl, in 30 R₁¼ -Arg(NO₂)-Gly-Asp(OBzl)-Phe-Arg(NO₂)-Gly-Asp(OBzl)-Phe-OBzl, in 31 R₂¼ -Arg-Gly-Asp-Ser, in 32 R₂¼ -Arg-Gly-Asp-Val, in 33

R₂¼ -Arg-Gly-Asp-Phe, in 34 R₂¼ -Arg-Gly-Asp-Ser-Arg-Gly-Asp-Ser, in 35 R₂¼ -Arg-Gly-Asp-Val-Arg-Gly-Asp-Val, in 36 R₂¼ -Arg-Gly-Asp-Phe-Arg-Gly-Asp-Phe; (i) succinic

anhydride; (ii) DCC, HOBt, NMM and 1–6; (iii) Pt/H₂.

1692

J. Liu et al. / European Journal of Medicinal Chemistry 44 (2009) 1689–1704

Table 1

Table 3

Effect of estradiol–RGD conjugates on calcium, phosphorous and ALP of mouse

serum.

Effect of estradiol–RGD octapeptides on calcium, phosphorous and mineral in mouse

femur.

Group^a

Calcium (mM)

Phosphorous (mM)

ALP

Group^a

Calcium (%)

Phosphorous (%)

Mineral (%)

OVX

Sham

Estradiol

7

3.39 ꢂ 0.15

3.30 ꢂ 0.11

3.24 ꢂ 0.24

3.27 ꢂ 0.25

3.29 ꢂ 0.11

3.23 ꢂ 0.20^c

3.18 ꢂ 0.17^b

3.01 ꢂ 0.18^d

3.00 ꢂ 0.20^d

3.28 ꢂ 0.19

3.21 ꢂ 0.32

3.22 ꢂ 0.19^c

3.26 ꢂ 0.19^c

3.04 ꢂ 0.14^d

3.02 ꢂ 0.09^d

3.27 ꢂ 0.18

3.27 ꢂ 0.22^c

3.24 ꢂ 0.10^c

3.21 ꢂ 0.15^c

3.05 ꢂ 0.09^d

3.01 ꢂ 0.15^d

3.18 ꢂ 0.28

2.82 ꢂ 0.29^h

3.01 ꢂ 0.27

3.13 ꢂ 0.20

3.29 ꢂ 0.14

2.98 ꢂ 0.20

3.01 ꢂ 0.27

2.91 ꢂ 0.49

2.60 ꢂ 0.29^h

2.94 ꢂ 0.35

3.12 ꢂ 0.41

2.99 ꢂ 0.23

3.04 ꢂ 0.12

2.70 ꢂ 0.34^h

3.04 ꢂ 0.12

3.24 ꢂ 0.52

3.33 ꢂ 0.60

3.19 ꢂ 0.61

3.01 ꢂ 0.28

3.01 ꢂ 0.40

3.01 ꢂ 0.28

28.78 ꢂ 2.74

22.97 ꢂ 1.71^e

26.22 ꢂ 2.92^f

25.45 ꢂ 2.34^f

26.29 ꢂ 2.58^f

26.62 ꢂ 2.12^f

23.93 ꢂ 2.90^e

20.27 ꢂ 2.88^g

20.18 ꢂ 2.55^g

25.12 ꢂ 2.44^f

26.19 ꢂ 2.48^f

26.01 ꢂ 2.71^f

22.96 ꢂ 2.96^e

20.64 ꢂ 2.08^g

19.56 ꢂ 1.96^g

26.09 ꢂ 2.09^f

25.99 ꢂ 1.67^f

25.42 ꢂ 1.35^f

23.18 ꢂ 2.79^e

20.06 ꢂ 2.15^g

20.18 ꢂ 2.19^g

OVX

Sham

Estradiol

7

54.45 ꢂ 1.35

62.08 ꢂ 2.40^b

57.28 ꢂ 0.87^b

55.61 ꢂ 1.22^b

57.58 ꢂ 1.28^b

58.76 ꢂ 2.13^b

59.34 ꢂ 1.22^b

63.12 ꢂ 3.15^c

65.16 ꢂ 2.42^c

56.45 ꢂ 2.01^b

56.91 ꢂ 1.24^b

57.84 ꢂ 1.76^b

57.49 ꢂ 0.98^b

64.22 ꢂ 0.99^c

67.49 ꢂ 0.98^c

57.57 ꢂ 3.47^b

57.67 ꢂ 3.29^b

57.42 ꢂ 1.04^b

59.36 ꢂ 1.17^b

63.92 ꢂ 2.14^c

64.36 ꢂ 1.17^c

19.31 ꢂ 1.16

20.16 ꢂ 0.19^d

19.81 ꢂ 0.21

18.58 ꢂ 0.65

18.91 ꢂ 0.95

20.24 ꢂ 0.04^d

20.37 ꢂ 0.12^d

21.56 ꢂ 0.44^e

21.40 ꢂ 0.23^e

19.85 ꢂ 0.26

19.78 ꢂ 0.04

20.24 ꢂ 0.42^d

20.14 ꢂ 0.28^d

21.12 ꢂ 0.18^e

21.94 ꢂ 0.28^e

19.43 ꢂ 0.84

19.39 ꢂ 1.18

20.09 ꢂ 0.19^d

20.56 ꢂ 0.26^d

21.33 ꢂ 0.68^e

21.56 ꢂ 0.26^e

0.603 ꢂ 0.011

0.633 ꢂ 0.017^f

0.608 ꢂ 0.018

0.610 ꢂ 0.017

0.607 ꢂ 0.032

0.605 ꢂ 0.011

0.604 ꢂ 0.010

0.626 ꢂ 0.014^g

0.625 ꢂ 0.010^g

0.572 ꢂ 0.039

0.591 ꢂ 0.021

0.611 ꢂ 0.022

0.612 ꢂ 0.007

0.624 ꢂ 0.009^g

0.624 ꢂ 0.007^g

0.583 ꢂ 0.044

0.592 ꢂ 0.022

0.596 ꢂ 0.027

0.605 ꢂ 0.013

0.623 ꢂ 0.011^g

0.625 ꢂ 0.013^g

Estradiol þ 7

18

31

21

34

18

31

21

34

8

Estradiol þ 8

19

32

22

35

19

32

22

35

9

Estradiol þ 9

20

33

23

36

20

33

23

36

a

Dosage ¼ 110.3 nmol/kg, n ¼ 12.

Compare to ovariotomy P < 0.01.

Compare to ovariotomy P < 0.05.

Compare to ovariotomy P < 0.01, to 18–20 and 31–33 P < 0.05.

Compare to ovariotomy P < 0.01.

Compare to ovariotomy P < 0.05.

Compare to ovariotomy P < 0.01, to 18–20 and 31–33 P < 0.01.

Compare to ovariotomy P < 0.01.

Dosage ¼ 110.3 nmol/kg, n ¼ 12.

b

c

b

c

d

e

f

Compare to ovariotomy P < 0.01.

Compare to ovariotomy P < 0.01, to 18–20 and 31–33 P < 0.01.

Compare to ovariotomy P < 0.01.

Compare to ovariotomy P < 0.01, to 18–20 and 31–33 P < 0.01.

Compare to ovariotomy P < 0.01.

d

e

f

g

h

g

Compare to ovariotomy P < 0.01, to 18–20 and 31–33 P < 0.05.

extents of femur phosphorous by conjugates 21–23 and 34–36

were signiﬁcantly higher than those increased by conjugates 18–23

and 31–33. The contents of femur mineral were increased by

conjugates 31–36 only.

mineral contents in the femurs and the data are listed in Table 3,

which indicated that the content of femur calcium was increased by

octapeptides 7–9, the mixture of estradiol and 7–9, conjugates 18–

23 and 31–36, and the increased extent of femur calcium by

conjugates 21–23 and 34–36 was signiﬁcantly higher than that

increased by 7–9, the mixture of estradiol and 7–9, the conjugates

18–20 and 31–33. The contents of femur phosphorous were

increased only by conjugates 18–23 and 31–36, and neither octa-

peptides 7–9 nor the mixture of estradiol and 7–9 increased the

contents of femur phosphorous. On the other hand the increased

2.3. Side effects of estradiol–RGD octapeptide conjugates receiving

mice

To estimate the side effects of orally administration of estradiol–

RGD conjugates the osteoporosis mouse model was also used. After

oral administration of estradiol, octapeptides 7–9, conjugates 18–

23 and 31–36 the weights of the organs and the coagulation were

tested.

Table 2

Effect of estradiol–RGD conjugates on the length and the weight of mouse femur.

Group^a

Weight of dry

femur (mg)

Weight of femur

ash (mg)

Length of

femur (cm)

2.3.1. Estradiol–RGD conjugates affecting no kidneys, lungs, livers,

and spleens of the mice

OVX

Sham

Estradiol

7

51.77 ꢂ 2.50

53.77 ꢂ 2.04^b

52.35 ꢂ 2.22

50.98 ꢂ 2.11

52.12 ꢂ 2.36

53.89 ꢂ 2.20^b

53.78 ꢂ 2.08^b

56.60 ꢂ 2.70^c

56.41 ꢂ 2.82^c

50.44 ꢂ 2.69

49.78 ꢂ 2.47

53.97 ꢂ 2.31^b

54.01 ꢂ 2.67^b

57.98 ꢂ 2.48^c

57.31 ꢂ 2.67^c

53.17 ꢂ 2.89

52.98 ꢂ 2.97

53.87 ꢂ 2.15^b

54.00 ꢂ 2.55^b

56.96 ꢂ 2.22^c

56.60 ꢂ 2.55^c

31.57 ꢂ 2.11

34.11 ꢂ 2.66^d

32.22 ꢂ 2.02

29.10 ꢂ 2.69

30.10 ꢂ 2.04

34.08 ꢂ 2.94^d

34.17 ꢂ 2.23^d

36.42 ꢂ 2.73^e

36.48 ꢂ 2.05^e

29.92 ꢂ 2.19

32.12 ꢂ 2.40

33.99 ꢂ 2.92^d

33.70 ꢂ 2.14^d

36.19 ꢂ 2.35^e

36.70 ꢂ 2.94^e

30.90 ꢂ 2.70

31.80 ꢂ 2.11

33.56 ꢂ 2.42^d

33.87 ꢂ 2.04^d

36.74 ꢂ 2.32^e

36.87 ꢂ 2.04^e

1.641 ꢂ 0.235

1.644 ꢂ 0.209

1.642 ꢂ 0.284

1.6.21 ꢂ 0.228

1.622 ꢂ 0.241

1.646 ꢂ 0.202

1.659 ꢂ 0.283

1.657 ꢂ 0.254

1.665 ꢂ 0.223

1.622 ꢂ 0.231

1.640 ꢂ 0.264

1.670 ꢂ 0.268

1.672 ꢂ 0.243

1.671 ꢂ 0.298

1.672 ꢂ 0.243

1.661 ꢂ 0.204

1.636 ꢂ 0.241

1.669 ꢂ 0.201

16.40 ꢂ 0.256

1.641 ꢂ 0.224

16.40 ꢂ 0.256

In order to evaluate the side effects of estradiol–RGD conjugates,

the kidneys, lungs, livers, and spleens of estradiol, octapeptides 7–

9, the mixture of estradiol and 7–9, conjugates 18–23 and 31–36

receiving mice were collected and measured following the proce-

dure of Section 4.7 to record their morphology and weights which

are listed in Table 4. It was found that oral administration of octa-

peptides 7–9, the mixture of estradiol and 7–9, the conjugates 18–

23 and 31–36 changed neither the morphology nor the weights of

the kidneys, lungs, livers, and spleens of the mice.

Estradiol þ 7

18

31

21

34

8

Estradiol þ 8

19

32

22

35

2.3.2. Estradiol–RGD conjugates increasing no risks of coagulation

and uterine weight getting of mice

9

In order to further evaluate the side effects of estradiol–RGD

conjugates the tail bleeding time and uterine weight of estradiol,

octapeptides 7–9, conjugates 18–23 and 31–36 receiving mice were

measured following the procedure of Section 4.8, which are listed

in Table 5. The data indicate that excepting estradiol and the

mixture of estradiol and 7–9, which shortened the tail bleeding

time, oral administration of estradiol–RGD conjugates 18–23 and

31–36 did not change the tail bleeding time. On the other hand the

uterine weights of the osteoporosis mice receiving estradiol and the

Estradiol þ 9

20

33

23

36

a

Dosage ¼ 110.3 nmol/kg, n ¼ 12.

b

c

Compare to ovariotomy P < 0.05.

Compare to ovariotomy P < 0.01, to 18–20 and 31–33 P < 0.05.

Compare to ovariotomy P < 0.05.

Compare to ovariotomy P < 0.01, to 18–20 and 31–33 P < 0.05.

d

e

J. Liu et al. / European Journal of Medicinal Chemistry 44 (2009) 1689–1704

1693

Table 4

activity parameter for 3D QSAR analysis. By using QSAR module of

Cerius²and following a standard procedure two equations were

established to reﬂect the relationships of the femur weights and the

femur ash weights and the structures. The resulted equations with

0.995 and 0.988 of r²demonstrated that both the femur weights

and the femur ash weights of the mice receiving 18–23 and 31–36

quantitatively correlated with their structures, suggesting these

equations are capable of exactly predicting the anti-osteoporosis

activity for unknown estradiol–RGD peptide conjugate.

Effect of estradiol–RGD conjugates on mouse kidney, lung, liver, and spleen.

Group^a

Spleen weight

(g)

Lung weight

(g)

Kidney weight Liver weight

(g)

OVX

0.145 ꢂ 0.018

0.106 ꢂ 0.013^b

0.144 ꢂ 0.017

0.120 ꢂ 0.044

0.121 ꢂ 0.015

0.107 ꢂ 0.005^c

0.120 ꢂ 0.016

0.114 ꢂ 0.009

0.118 ꢂ 0.009

0.128 ꢂ 0.013

0.120 ꢂ 0.010

0.110 ꢂ 0.009^d

0.116 ꢂ 0.013

0.105 ꢂ 0.004^c

0.109 ꢂ 0.007^d

0.109 ꢂ 0.014

0.125 ꢂ 0.011

0.118 ꢂ 0.028

0.125 ꢂ 0.011

0.113 ꢂ 0.024

0.109 ꢂ 0.010^d

0.112 ꢂ 0.009

0.109 ꢂ 0.011^d

0.119 ꢂ 0.007

0.109 ꢂ 0.011^d

0.168 ꢂ 0.022

0.168 ꢂ 0.015

0.173 ꢂ 0.020

0.169 ꢂ 0.027

0.169 ꢂ 0.014

0.193 ꢂ 0.010^e

0.182 ꢂ 0.015

0.170 ꢂ 0.009

0.174 ꢂ 0.014

0.156 ꢂ 0.007

0.160 ꢂ 0.016

0.167 ꢂ 0.014

0.178 ꢂ 0.013

0.164 ꢂ 0.023

0.178 ꢂ 0.013

0.167 ꢂ 0.018

0.157 ꢂ 0.005

0.159 ꢂ 0.017

0.160 ꢂ 0.009

0.169 ꢂ 0.017

0.160 ꢂ 0.009

1.490 ꢂ 0.177

1.342 ꢂ 0.117^f

1.552 ꢂ 0.101

1.425 ꢂ 0.142

1.506 ꢂ 0.119

1.492 ꢂ 0.145

1.537 ꢂ 0.227

1.448 ꢂ 0.162

1.479 ꢂ 0.130

1.398 ꢂ 0.208

1.360 ꢂ 0.198

1.400 ꢂ 0.053

1.550 ꢂ 0.155

1.469 ꢂ 0.121

1.550 ꢂ 0.155

1.319 ꢂ 0.136^f

1.235 ꢂ 0.133^g

1.336 ꢂ 0.182^f

1.266 ꢂ 0.201^f

1.439 ꢂ 0.099

1.266 ꢂ 0.201^f

Sham

Estradiol

7

Estradiol þ 7 0.125 ꢂ 0.023

18

31

21

34

0.142 ꢂ 0.029

0.150 ꢂ 0.033

0.133 ꢂ 0.029

0.149 ꢂ 0.026

0.122 ꢂ 0.014^b

0.114 ꢂ 0.016^b

0.133 ꢂ 0.017

0.149 ꢂ 0.027

0.145 ꢂ 0.034

0.149 ꢂ 0.027

0.122 ꢂ 0.016^b

0.117 ꢂ 0.020^b

0.177 ꢂ 0.008^b

0.135 ꢂ 0.032

0.131 ꢂ 0.020

0.135 ꢂ 0.032

2.4.1. Alignment of estradiol–RGD conjugates 18–23 and 31–36

To establish the valid 3D QSAR models, a proper alignment

procedure of estradiol–RGD conjugates 18–23 and 31–36 was

practiced using the target model align strategy in the align module

within Cerius². Based on the assumption that each structure of

estradiol–RGD conjugates 18–23 and 31–36 exhibits activity at the

same binding site of the receptor, they were aligned in a pharma-

cological active orientation. To obtain a consistent alignment, the

estradiol ring, the common pharmacophore, was selected as the

template for superposing estradiol–RGD conjugates 18–23 and 31–

36. The method used for performing the alignment was the

maximum common subgraph (MCS) [22]. 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

estradiol ring that is shared by estradiol–RGD conjugates 18–23 and

31–36. This subset was used for the alignment. A rigid ﬁt of atom

pairings was performed to superimpose each structure onto the

target model estradiol ring. Stereoview of aligned estradiol–RGD

conjugates 18–23 and 31–36 used for molecular ﬁeld generation is

shown in Fig. 1. The alignment stereoview explores that to super-

impose onto the estradiol ring, the RGD peptide chain of each

structure has to take individual conformation. This individual RGD

peptide chain conformation will affect the anti-osteoporosis

activity.

8

Estradiol þ 8

19

32

22

35

9

Estradiol þ 9

20

33

23

36

a

Dosage ¼ 110.3 nmol/kg, n ¼ 12.

Compare to ovariotomy P < 0.01.

Compare to ovariotomy P < 0.05.

Compare to ovariotomy P < 0.01.

Compare to ovariotomy P < 0.05.

Compare to ovariotomy P < 0.01.

b

c

d

e

f

g

mixture of estradiol and 7–9 were signiﬁcantly higher than those of

OVX, sham, 7–9, 18–23 and 31–36.

2.4. 3D QSAR analysis of the in vivo activity of estradiol–RGD

conjugates

2.4.2. QSAR module of Cerius²based MFA of estradiol–RGD

conjugates 18–23 and 31–36

After energy minimization using the MMFF94 (Merck Molecular

Force Field), the Molecular Field Analysis (MFA) was performed for

estradiol–RGD conjugates 18–23 and 31–36 using the QSAR module

of Cerius²[23]. A ﬁve-step-procedure consisted of generating

To gain insight into the correlation between the anti-osteopo-

rosis activity with the structure of estradiol–RGD conjugate, the

femur weights and femur ash weights of the mice receiving

estradiol–RGD conjugates 18–23 and 31–36 were selected as the

Table 5

Effect of estradiol–RGD conjugates on tail bleeding time and uterine weights.

Group^a

Bleeding time (s) after

last administration

Uterine

weight (g)

30 min

90 min

OVX

Sham

Estradiol

7

542 ꢂ 50

562 ꢂ 53

417 ꢂ 57^b

551 ꢂ 55

422 ꢂ 53^b

582 ꢂ 50

575 ꢂ 51

564 ꢂ 53

562 ꢂ 54

541 ꢂ 53

412 ꢂ 43^b

569 ꢂ 54

551 ꢂ 48

566 ꢂ 54

581 ꢂ 61

555 ꢂ 49

412 ꢂ 43^b

557 ꢂ 45

576 ꢂ 59

592 ꢂ 49

584 ꢂ 45

548 ꢂ 49

568 ꢂ 59

441 ꢂ 43^b

548 ꢂ 59

426 ꢂ 49^b

577 ꢂ 52

561 ꢂ 54

559 ꢂ 45

556 ꢂ 43

549 ꢂ 50

416 ꢂ 40^b

577 ꢂ 52

560 ꢂ 51

558 ꢂ 81

580 ꢂ 64

551 ꢂ 48

416 ꢂ 40^b

560 ꢂ 51

568 ꢂ 61

585 ꢂ 55

571 ꢂ 51

0.081 ꢂ 0.022

0.086 ꢂ 0.026

0.108 ꢂ 0.021^c

0.084 ꢂ 0.012

0.110 ꢂ 0.015^c

0.064 ꢂ 0.015^d

0.063 ꢂ 0.015^d

0.062 ꢂ 0.014^d

0.065 ꢂ 0.011^d

0.079 ꢂ 0.024

0.116 ꢂ 0.028^c

0.064 ꢂ 0.011^d

0.064 ꢂ 0.013^d

0.061 ꢂ 0.018^d

0.064 ꢂ 0.013^d

0.076 ꢂ 0.012

0.118 ꢂ 0.023^c

0.067 ꢂ 0.021

0.060 ꢂ 0.028

0.061 ꢂ 0.018^d

0.060 ꢂ 0.018^d

Estradiol þ 7

18

31

21

34

8

Estradiol þ 8

19

32

22

35

9

Estradiol þ 9

20

33

23

36

a

Dosage ¼ 110.3

m

mol/kg, n ¼ 12.

b

c

Compare to ovariotomy P < 0.01.

Compare to ovariotomy P < 0.05.

Fig. 1. Stereoview of the alignment of estradiol–RGD peptide conjugates 18–23 and

31–36 for generating molecular ﬁeld.

d

1694

J. Liu et al. / European Journal of Medicinal Chemistry 44 (2009) 1689–1704

conformers, energy minimization, matching atoms and aligning

molecules, setting preferences, and regression analysis was auto-

matically practiced in MFA. Molecular electrostatic and steric ﬁelds

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 ꢂ30.0 kcal/mol was set for

both electrostatic and steric ﬁelds. The total grid points generated

were 672. Though the 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 the 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 performed with the leave-one-out procedure.

PLS analysis was scaled, with all variables normalized to a variance

of 1.0.

The regions where variations in the steric or electrostatic

features of estradiol–RGD peptide conjugates (18–23 and 31–36) in

the training set lead to increase or decrease femur weights were

speciﬁed. Proton descriptor with positive coefﬁcient indicates

a region favorable for electropositive group, while negative coefﬁ-

cient indicates electronegative 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 hydrogen bond forming

group, while negative coefﬁcient indicates hydrogen bond forming

group not required at this position.

31–36 in terms of the descriptors proton, methyl and hydroxyl

groups is aligned as Fig. 2 and expressed by Eq. (1). The correlation

of the femur weight tested on the in vivo osteoporosis mouse

model and the femur weight calculated using Eq. (1) is explained by

Fig. 3.

Activity [ 53:3225 D 0:01235ðH^D=2949Þ L0:018056ðHO^L=2766Þ

D0:024866ðHO^L=3218Þ L0:036975ðH^D=3220Þ

D 0:072378ðCH₃=3760ÞD 0:0121532ðHO^L=2742Þ

L0:035892ðH^D=2659Þ L 0:067739ðHO^L=2633Þ

L0:04947ðH^D=1649ÞL 0:050404ðHO^L=1850Þ

(1)

In Eq. (1) the data points (n), correlation coefﬁcient (r), square

correlation coefﬁcient (r²), cross-validated correlation coefﬁcient

(r²cv), bootstrap correlation coefﬁcient (r²_BS) and least square error

(LSE) were 12, 0.995, 0.991, 0.104, 0.737 and 0.0383, respectively. This

equation contains term of 0.01235(H^þ/2949) with positive coefﬁ-

cient, which means that at this position an electron-releasing group

will affect on the femur weight positively, and terms of 0.036975(H^þ/

3220), 0.035892(H^þ/2659) and 0.04947(H^þ/1649) with negative

coefﬁcients, which means that at these positions electron-with-

drawing groups will affect on the femur weight positively, from

proton descriptor. Eq. (1) contains term of 0.072378(CH₃/3760) with

positive coefﬁcient, which means that at this position a large group

will affect on the femur weight positively, from methyl descriptor. Eq.

(1) contains also terms of 0.0121532(HO^ꢁ/2742) and 0.024866(HO^ꢁ/

3218) with positive coefﬁcients, which means that at these positions

electron-releasing group will affect the femur weight positively, and

terms

of

0.018056(HO^ꢁ/2766),

0.067739(HO^ꢁ/2633)

and

0.050404(HO^ꢁ1850) with negative coefﬁcients, which means that at

these positions electron-withdrawing groups will affect the femur

weight positively, from hydroxyl descriptor.

2.4.2.1. MFA model for femur weights of the mice receiving estradiol–

RGD conjugates 18–23 and 31–36. The MFA model for femur

weights of the mice receiving estradiol–RGD conjugates 18–23 and

As examples Fig. 4 gives two representatives 34 (a), which has

electron-withdrawing groups near H^þ/1649, H^þ/2949, H^þ/3220,

HO^ꢁ/1850 and HO^ꢁ/2742 and totally increasing its femur weight, as

well as 22 (b), which has electron-withdrawing groups near H^þ/

2949, H^þ/3220, HO^ꢁ/3218 and a larger group near CH₃/3760 and

also totally increasing its femur weight.

2.4.2.2. MFA model for femur ash weights of the mice receiving

estradiol–RGD conjugates 18–23 and 31–36. The MFA model for

femur weights of the mice receiving estradiol–RGD peptide

conjugates 18–23 and 31–36 in terms of the descriptors proton,

methyl and hydroxyl groups is aligned as Fig. 5 and expressed by

Eq. (2). The correlation of the femur ash weight tested on the in vivo

Fig. 2. Stereoview of the alignment of estradiol–RGD peptide conjugates 18–23 and

31–36 with the femur weight as the activity parameter.

Fig. 3. Graph of femur weights of the mice receiving estradiol–RGD peptide conjugates

18–23 and 31–36 against the predicted femur weights by 3D QSAR.

J. Liu et al. / European Journal of Medicinal Chemistry 44 (2009) 1689–1704

1695

Fig. 4. Electrostatic and environments of conjugates 34 (a) and 22 (b) within 3D grid of Eq. (1).

osteoporosis mouse model and the femur ash weight calculated

using equation Eq. (2) is explained by Fig. 6.

This equation contains term of 0.000807(H^þ/2418) with positive

coefﬁcient, which means that at this position an electron-releasing

group will affect the femur ash weight positively, and terms of

0.070947(H^þ/4346) and 0.050821(H^þ/3230) with negative coefﬁ-

cients, which means that at these positions electron-withdrawing

groups will affect the femur ash weight positively, from proton

descriptor. It contains terms of 0.059459(CH₃/4059) and

0.058069(CH₃/2741) with positive coefﬁcient, which means that at

these positions large groups will affect the femur ash weight

positively, and terms of 0.076576(CH₃/2721) and 0.021407(CH₃/

1679) with negative coefﬁcient, which means that at these posi-

tions small groups will affect the femur ash weight positively, from

methyl descriptor. Eq. (2) contains also term of 0.002937(HO^ꢁ/

2661) with positive coefﬁcient, which means that at this position

an electron-releasing group will be favorable for increasing femur

ash weight, and terms of 0.03634(HO^ꢁ/2648), 0.00753(HO^ꢁ/2770)

and 0.05163(HO^ꢁ/3188) with negative coefﬁcients, which means

that at these positions electron-withdrawing groups will be

favorable for increasing femur ash weight, from hydroxyl

descriptor.

Activity [ 33:3666 L0:070947ðH^D=4346Þ L 0:076576ðCH₃=2721Þ

D 0:059459ðCH₃=4059Þ D0:002937ðHO^L=2661Þ

L0:021407ðCH₃=1679ÞL 0:03634ðHO^L=2648Þ

D0:058069ðCH₃=2741ÞL 0:00753ðHO^L=2770Þ

L0:05163ðHO^L=3188Þ D 0:000807ðH^D=2418Þ

L 0:050821ðH^D=3230Þ

(2)

In Eq. (2) the data points (n), correlation coefﬁcient (r), square

correlation coefﬁcient (r²), cross-validated correlation coefﬁcient

(r²cv), bootstrap correlation coefﬁcient (r²_BS) and least square error

(LSE) were 12, 0.988, 0.976, 0.085, 0.863 and 0.007, respectively.

As examples Fig. 7 gives two representatives 34 (a), which has

electron-withdrawing group near HO^ꢁ/2770 and large group near

CH₃/2741 and totally increasing its femur ash weight, as well as 22

(b), which has electron-withdrawing groups near H^þ/4346 and

HO^ꢁ/2648 and a larger group near CH₃/2741 and CH₃/4059 and also

totally increasing its femur ash weight.

Fig. 5. Stereoview of the alignment of estradiol–RGD peptide conjugates 18–23 and

31–36 with the femur ash weight as the activity parameter.

Fig. 6. Graph of femur ash weights of the mice receiving estradiol–RGD peptide

conjugates 18–23 and 31–36 against the predicted femur ash weights by 3D QSAR.

1696

J. Liu et al. / European Journal of Medicinal Chemistry 44 (2009) 1689–1704

Fig. 7. Electrostatic and environments of conjugates 34 (a) and 22 (b) within 3D grid of Eq. (2).

3. Conclusion

of anhydrous tetrahydrofuran (THF) 453 mg (2.2 mmol) of dicy-

clohexylcarbodiimide (DCC) was added. The solution was stirred at

0 ^ꢀC for 30 min, to which a solution of 250 mg (2.0 mmol) of HCl$H-

Gly-OCH₃in 10 ml of anhydrous THF was added. At 0 ^ꢀC the reac-

tion mixture was adjusted to pH 8.5 with N-methylmorpholine

(NMM), then stirred at room temperature for 8 h, and TLC (chlo-

roform/methanol, 5:1) indicated the disappearance of Boc-

Arg(NO₂). The precipitates of dicyclohexylurea (DCU) were

removed by ﬁltration. The ﬁltrate was evaporated under reduced

pressure and the residue was dissolved in 50 ml of ethyl acetate.

The solution was washed successively with a 5% aqueous solution

of sodium bicarbonate, a 5% aqueous solution of citric acid and

a saturated aqueous solution of sodium chloride. The organic phase

was dried over anhydrous sodium sulfate. After ﬁltration and

evaporation under reduced pressure, 0.688 g (88%) of the title

Osteoporosis is being a crushing pressure for our society, and

the modiﬁcation of estradiol to improve the ERT or HRT is clinically

important. The observations provided here explored that the risks

of thrombogenesis and endometrial hyperplasia of the mice

receiving 110.3 nmol/kg of estradiol–RGD peptide conjugates were

signiﬁcantly lower than that of mice receiving 110.3 nmol/kg of

estradiol, which suggested that the use of estradiol–RGD peptide

conjugates can simultaneously up-regulate the activity and prolif-

eration of osteoblasts and down-regulate the adhesiveness of

osteoclasts. In addition, the development levels of thrombogenesis

of the mice receiving 110.3 nmol/kg of estradiol–RGD octapeptide

conjugates were signiﬁcantly lower than those of mice receiving

110.3 nmol/kg of RGD tetrapeptide conjugates, which suggested

that introducing repeated RGD sequence to estradiol is a useful

strategy to further increase the activity of these conjugates. The

0.995 and 0.988 of the r²of 3D QSAR analysis resulted equations of

estradiol–RGD tetrapeptide and octapeptide conjugates explained

that the anti-osteoporosis activity of unknown conjugate can be

exactly predicted.

compound was provided as colorless powder. Mp. 94–95 ^ꢀC,

20

[a

]

¼ 0.4 (c ¼ 1.07, CH₃OH), ESI-MS (m/z) 391 [M þ H]^þ.

D

4.2.2. Boc-Arg(NO₂)-Gly

At 0 ^ꢀC to the solution of 5.72 g (14.7 mmol) of Boc-Arg(NO₂)-

Gly-OCH₃in 70 ml of methanol 30 ml of aqueous NaOH (2 N) was

added. The reaction mixture was stirred at 0 ^ꢀC for 30 min, adjusted

to pH 5.5 with hydrochloric acid (2 N). After ﬁltration the ﬁltrate

was evaporated under reduced pressure. The residue was dissolved

in 30 ml of methanol and the solution was ﬁltered. The ﬁltrate was

evaporated under reduced pressure and the residue was solidiﬁed

4. Experimental section

4.1. General

All the reactions were carried out under nitrogen (1 bar).¹H (300

and 500 MHz) and ¹³C (75 and 125 MHz) NMR spectra were recor-

ded on Bruker AMX-300 and AMX-500 spectrometers for solution

DMSO-d₆or CDCl₃with tetramethylsilane as internal standard. IR

spectra were recorded with a Perkin–Elmer 983 instrument. FAB/

MS was determined on VG-ZAB-MS and TOF-MS was recorded on

MDS SCIEX QSTAR. Melting points were measured on a XT5 hot

in 10 ml of anhydrous ether to provide 5.28 g (96%) of the title

20

compound as colorless powder. Mp. 130–132 ^ꢀC, [

a

]

¼ ꢁ4.8

D

(c ¼ 1.13, CH₃OH), ESI-MS (m/z) 377 [M þ H]^þ.

4.2.3. Boc-Asp(OBzl)-Ser(Bzl)-OBzl

Using the same procedure as described for Boc-Arg(NO₂)-Gly-

OCH₃from 5.04 g (15.5 mmol) of Boc-Asp(OBzl), 2.31 g (16.5 mmol)

of HOBt, 3.52 g (17.1 mmol) of DCC and 5.00 g (15.6 mmol) of

stage microscope (Beijing key electro-optic factory). All L-amino

acids were purchased from China Biochemical Corp. TLC was made

with Qingdao silica gel GF₂₅₄. Chromatography was performed with

Qingdao silica gel H₆₀or Sephadex-LH₂₀. All solvents were distilled

and dried before use by reference to literature procedures. Optical

rotations were determined with a Jasco P-1020 Polarimeter at 20 ^ꢀC.

The statistical analysis of all the biological data was carried out by

use of ANOVA test, P < 0.05 is considered signiﬁcant.

HCl$H-Ser(Bzl)-OBzl 9.55 g (98%) of the title compound was

20

obtained as yellow syrup. [

a

]

¼ ꢁ12.1 (c ¼ 1.13, MeOH), ESI-MS

D

(m/z) 592 [M þ H]^þ.

4.2.4. HCl$H-Asp(OBzl)-Ser(Bzl)-OBzl

To 9.0 g (1.3 mmol) of Boc-Asp(OBzl)-Ser(Bzl)-OBzl 80 ml of 4 N

solution of hydrochloride in ethyl acetate was added. The reaction

mixture was stirred at room temperature for 60 min and TLC

(chloroform/methanol, 5:1) indicated the disappearance of Boc-

Asp(OBzl)-Ser(Bzl)-OBzl. The reaction mixture was evaporated

under reduced pressure, the residue was dissolved in 40 ml of ethyl

acetate, the solution was again evaporated under reduced pressure

and the residue was washed with anhydrous ether to provide 6.02 g

4.2. Preparing RGD peptides

4.2.1. Boc-Arg(NO₂)-Gly-OCH₃

At 0 ^ꢀC to the solution of 638 mg (2.0 mmol) of Boc-Arg(NO₂)

and 297 mg (2.2 mmol) of 1-hydroxybenzotriazole (HOBt) in 20 ml

J. Liu et al. / European Journal of Medicinal Chemistry 44 (2009) 1689–1704

1697

(94%) of the title compound as colorless powder. Mp. 105–106 ^ꢀC,

(13.1 mmol) of HCl$H-Asp(OBzl)-Val-OBzl, 1.95 g (13.9 mmol) of

20

[

a]

¼ ꢁ13.2 (c ¼ 1.27, MeOH), ESI-MS (m/z) 492 [M þ H]^þ.

HOBt and 2.97 g (14.5 mmol) of DCC 9.31 g (92%) of the title

D

compound was obtained as colorless powder. Mp 59–62 ^ꢀC,

20

4.2.5. Boc-Asp(OBzl)-Val-OBzl

[a

]

¼ ꢁ22.7 (c ¼ 1.00, CH₃OH), ESI-MS (m/z) 772 [M þ H]^þ.

D

Using the same procedure as described for Boc-Arg(NO₂)-Gly-

OCH₃, from 3.59 g (11.1 mmol) of Boc-Asp(OBzl), 1.57 g (11.2 mmol)

of HOBt, 2.39 g (11.7 mmol) of DCC and 4 g (10.5 mmol) of TosH$H-

4.2.14. HCl$H-Arg(NO₂)-Gly-Asp(OBzl)-Val-OBzl (2)

Using the same procedure as described for HCl$H-Asp(OBzl)-

Ser(Bzl)-OBzl, from 6.23 g of Boc-Arg(NO₂)-Gly-Asp(OBzl)-Val-OBzl

and 60 ml of 4 N solution of hydrochloride in ethyl acetate 6.69 g

Val-OBzl 5.15 g (90%) of the title compound was obtained as yellow

20

syrup. [

a]

¼ ꢁ33.3 (c ¼ 1.07, CH₃OH), ESI-MS (m/z) 514 [M þ H]^þ.

D

(total yield 76%) of the title compound was obtained as colorless

20

4.2.6. HCl$H-Asp(OBzl)-Val-OBzl

Using the same procedure as described for HCl$H-Asp(OBzl)-

Ser(Bzl)-OBzl, from 6.15 g of Boc-Asp(OBzl)-Val-OBzl and 80 ml of

powder. Mp 98–100 ^ꢀC, [

a]

¼ ꢁ12.4 (c ¼ 1.00, CH₃OH), ESI-MS (m/z)

D

672 [M þ H]^þ.

4 N solution of hydrochloride in ethyl acetate 4.01 g (total yield

4.2.15. Boc-Arg(NO₂)-Gly-Asp(OBzl)-Phe-OBzl

20

92%) title compound was obtained, Mp 105–107 ^ꢀC, [

a

]

D

¼ ꢁ18.6

Using the same procedure as described for Boc-Arg(NO₂)-Gly-

OCH₃, from 5.08 g (13.5 mmol) of Boc-Arg(NO₂)-Gly, 6.35 g

(12.8 mmol) of HCl$H-Asp(OBzl)-Phe-OBzl, 2.00 g (14.3 mmol) of

HOBt and 3.05 g (14.9 mmol) of DCC 10.95 g (99%) of the title

(c ¼ 1.20, CH₃OH), ESI-MS (m/z) 414 [M þ H]^þ.

4.2.7. Boc-Asp(OBzl)-Phe-OBzl

Using the same procedure as described for Boc-Arg(NO₂)-Gly-

OCH₃, from 2.28 g (7.0 mmol) of Boc-Asp(OBzl), 1.04 g (7.4 mmol) of

HOBt,1.59 g (7.8 mmol) of DCC and 3.00 g (7.0 mmol) of TosH$H-Phe-

compound was obtained as colorless powder. Mp 102–104 ^ꢀC,

20

[a

]

¼ ꢁ20.6 (c ¼ 1.20, CH₃OH), ESI-MS (m/z) 820 [M þ H].

D

OBzl 4.15 g of the title compound was obtained as yellow powder. Mp

4.2.16. HCl$H-Arg(NO₂)-Gly-Asp(OBzl)-Phe-OBzl (3)

¼ 0.4 (c ¼ 1.27, MeOH), ESI-MS (m/z)562 [M þ H]^þ.

Using the same procedure as described for HCl$H-Asp(OBzl)-

Ser(Bzl)-OBzl, from 10.95 g of Boc-Arg(NO₂)-Gly-Asp(OBzl)-Phe-

OBzl and 100 ml of 4 N solution of hydrochloride in ethyl acetate

20

81.9–82.8 ^ꢀC,[

a]

D

4.2.8. HCl$H-Asp(OBzl)-Phe-OBzl

Using the same procedure as described for HCl$H-Asp(OBzl)-

Ser(Bzl)-OBzl, from 4.15 g of Boc-Asp(OBzl)-Phe-OBzl and 60 ml of

4 N solution ofhydrochlorideinethyl acetate3.03 g(totalyield86.5%)

7.84 g (total yield 81%) was obtained as colorless powder. Mp 102–

¼ ꢁ9.3 (c ¼ 1.47, CH₃OH), ESI-MS (m/z) 720 [M þ H]^þ.

20

104 ^ꢀC, [

a

]

D

of the title compound was obtained as yellow powder. Mp 123.6–

4.2.17. Boc-Ser(Bzl)-Arg(NO₂)-Gly-Asp(OBzl)-Ser(Bzl)-OBzl

20

125.0 ^ꢀC, [

a]

D

¼ ꢁ17.1 (c ¼ 1.00, CH₃OH), ESI-MS (m/z) 462 [M þ H]^þ.

Using the same procedure as described for Boc-Arg(NO₂)-Gly-

OCH₃, from 1.13 g (3.8 mmol) of Boc-Ser(Bzl), 3.0 g (3.8 mmol) of

HCl$H-Arg(NO₂)-Gly-Asp(OBzl)-Ser(Bzl)-OBzl, 0.57 g (4.1 mmol) of

HOBt and 0.87 g (4.2 mmol) of DCC 3.87 g (99%) of the title

4.2.9. Boc-Asp(OBzl)-Val

Using the same procedure as described for Boc-Arg(NO₂)-Gly-

OCH₃, from 4.80 g (14.8 mmol) of Boc-Asp(OBzl), 1.04 g

compound was obtained as colorless powder. Mp 103–104 ^ꢀC,

20

(16.3 mmol)

of

N-hydroxyl-succinamide

(HOSu),

3.36 g

[a

]

¼ þ0.4 (c ¼ 0.77, CH₃OH), ESI-MS (m/z) 1027 [M þ H]^þ.

D

(16.4 mmol) of DCC and 1.90 g (16.3 mmol) of

L

-Val 4.419 g (71%) of

20

the title compound was obtained as colorless syrup. [

a

]

¼ ꢁ8.8

4.2.18. HCl$H-Ser(Bzl)-Arg(NO₂)-Gly-Asp(OBzl)-Ser(Bzl)-OBzl

Using the same procedure as described for HCl$H-Asp(OBzl)-

Ser(Bzl)-OBzl, from 3.87 g of Boc-Ser(Bzl)-Arg(NO₂)-Gly-Asp(OBzl)-

Ser(Bzl)-OBzl and 60 ml of 4 N solution of hydrochloride in ethyl

D

(c ¼ 0.73, CH₃OH), ESI-MS (m/z) 424 [M þ H]^þ.

4.2.10. Boc-Asp(OBzl)-Phe

Using the same procedure as described for Boc-Arg(NO₂)-Gly-

OCH₃, from 4.80 g (14.8 mmol) of Boc-Asp(OBzl), 1.04 g

(16.3 mmol) of HOSu, 3.36 g (16.4 mmol) of DCC and 2.69 g

acetate 3.31 g (95%) of the title compound was obtained as colorless

20

powder. Mp159–163 ^ꢀC, [

a]

D

¼ ꢁ0.3 (c ¼ 0.40, CH₃OH), ESI-MS(m/z)

927 [M þ H]^þ.

(16.3 mmol) of

L-Phe 5.5 g (80%) of the title compound was

20

obtained as colorless powder. Mp 80–81 ^ꢀC, [

a

]

D

¼ 6.3 (c ¼ 0.90,

4.2.19. Boc-Asp(OBzl)-Ser(Bzl)-Arg(NO₂)-Gly-Asp(OBzl)-Ser(Bzl)-

OBzl

Using the same procedure as described for Boc-Arg(NO₂)-Gly-

OCH₃, from 1.12 g (3.4 mmol) of Boc-Asp(OBzl), 3.31 g (3.4 mmol) of

CH₃OH), ESI-MS (m/z) 472 [M þ H]^þ.

4.2.11. Boc-Arg(NO₂)-Gly-Asp(OBzl)-Ser(Bzl)-OBzl

Using the same procedure as described for Boc-Arg(NO₂)-Gly-

OCH₃, from 2.62 g (7.0 mmol) of Boc-Arg(NO₂)-Gly-OH, 3.67 g

(7.0 mmol) of HCl$H-Asp(OBzl)-Ser(Bzl)-OBzl, 1.03 g (7.4 mmol) of

HCl$H-Ser(Bzl)-Arg(NO₂)-Gly-Asp(OBzl)-Ser(Bzl)-OBzl,

0.51 g

(3.6 mmol) of HOBt and 0.78 g (3.8 mmol) of DCC 4.16 g (98%) of the

title compound was obtained as colorless powder. Mp 123–124 ^ꢀC,

20

HOBt and 1.58 g (7.7 mmol) of DCC to provide 5.65 g (95%) of the

[a

]

¼ ꢁ8.0 (c ¼ 0.67, CH₃OH), ESI-MS (m/z) 1233 [M þ H]^þ.

D

20

title compound as yellow powder. Mp 82–83 ^ꢀC, [

a

]

¼ ꢁ18.9

D

(c ¼ 1.00, CH₃OH), ESI-MS (m/z) 850 [M þ H]^þ.

4.2.20. HCl$H-Asp(OBzl)-Ser(Bzl)-Arg(NO₂)-Gly-Asp(OBzl)-

Ser(Bzl)-OBzl

4.2.12. HCl$H-Arg(NO₂)-Gly-Asp(OBzl)-Ser(Bzl)-OBzl (1)

Using the same procedure as described for HCl$H-Asp(OBzl)-

Ser(Bzl)-OBzl, from 4.16 g of Boc-Asp(OBzl)-Ser(Bzl)-Arg(NO₂)-Gly-

Asp(OBzl)-Ser(Bzl)-OBzl and 60 ml of 4 N solution of hydrochloride

Using the same procedure as described for HCl$H-Asp(OBzl)-

Ser(Bzl)-OBzl, from 6.23 g of Boc-Arg(NO₂)-Gly-Asp(OBzl)-Ser(Bzl)-

OBzl and 60 ml of 4 N solution of hydrochloride in ethyl acetate

in ethyl acetate 3.78 g (96%) of the title compound was obtained as

20

4.02 g (total yield 89%) of the title compound was obtained as

colorless powder. Mp 129–131 ^ꢀC, [

a

]

¼ ꢁ6.9 (c ¼ 1.00, CH₃OH),

D

20

colorless powder. Mp 106–108 ^ꢀC, [

a

]

¼ ꢁ4.3 (c ¼ 0.70, CH₃OH),

ESI-MS (m/z) 1133 [M þ H]^þ.

D

ESI-MS (m/z) 750 [M þ H]^þ.

4.2.21. Boc-Asp(OBzl)-Val-Arg(NO₂)-Gly-Asp(OBzl)-Val-OBzl

Using the same procedure as described for Boc-Arg(NO₂)-Gly-

OCH₃, from 2.11 g (5.0 mmol) of Boc-Asp(OBzl)-Val, 3.52 g

(5.0 mmol) of HCl$H-Arg(NO₂)-Gly-Asp(OBzl)-Val-OBzl, 0.74 g

4.2.13. Boc-Arg(NO₂)-Gly-Asp(OBzl)-Val-OBzl

Using the same procedure as described for Boc-Arg(NO₂)-Gly-

OCH₃, from 4.93 g (13.09 mmol) of Boc-Arg(NO₂)-Gly, 5.89 g

1698

J. Liu et al. / European Journal of Medicinal Chemistry 44 (2009) 1689–1704

(5.3 mmol) of HOBt and 1.13 g (5.5 mmol) of DCC 5.14 g (96%) of

4.2.29. Boc-Arg(NO₂)-Gly-Asp(OBzl)-Phe-Arg(NO₂)-Gly-Asp(OBzl)-

Phe-OBzl

Using the same procedure as described for Boc-Arg(NO₂)-Gly-

OCH₃, from 0.89 g (2.3 mmol) of Boc-Arg(NO₂)-Gly, 2.5 g

(3.4 mmol) of HCl$H-Asp(OBzl)-Phe-Arg(NO₂)-Gly-Asp(OBzl)-Phe-

OBzl, 0.34 g (2.4 mmol) of HOBt and 0.51 g (2.5 mmol) of DCC 2.50 g

the title compound was obtained as colorless powder. Mp 157–

20

158 ^ꢀC,

[a

]

¼ ꢁ23.6 (c ¼ 0.87, CH₃OH), ESI-MS (m/z) 1077

D

[M þ H]^þ.

4.2.22. HCl$H-Asp(OBzl)-Val-Arg(NO₂)-Gly-Asp(OBzl)-Val-OBzl

Using the same procedure as described for HCl$H-Asp(OBzl)-

Ser(Bzl)-OBzl, from 5.14 g of Boc-Asp(OBzl)-Val-Arg(NO₂)-Gly-

Asp(OBzl)-Val-OBzl and 80 ml of 4 N solution of hydrochloride in

(78%) of the title compound was obtained as colorless powder. Mp

20

124–126 ^ꢀC, [

a

]

¼ ꢁ12.0 (c ¼ 0.60, CH₃OH), ESI-MS (m/z) 1431

D

[M þ H]^þ.

ethyl acetate 4.33 g (90%) of the title compound was obtained as

20

colorless powder. Mp 116–118 ^ꢀC, [

a

]

¼ ꢁ11.9 (c ¼ 1.00, CH₃OH),

4.2.30. HCl$H-Arg(NO₂)-Gly-Asp(OBzl)-Phe-Arg(NO₂)-Gly-

Asp(OBzl)-Phe-OBzl (6)

Using the same procedure as described for HCl$H-Asp(OBzl)-

Ser(Bzl)-OBzl, from 2.50 g of Boc-Arg(NO₂)-Gly-Asp(OBzl)-Phe-

Arg(NO₂)-Gly-Asp(OBzl)-Phe-OBzl and 50 ml of 4 N solution of

D

ESI-MS (m/z) 977 [M þ H]^þ.

4.2.23. Boc-Asp(OBzl)-Phe-Arg(NO₂)-Gly-Asp(OBzl)-Phe-OBzl

Using the same procedure as described for Boc-Arg(NO₂)-Gly-

OCH₃, from 0.47 g (1.0 mmol) of Boc-Asp(OBzl)-Phe, 0.75 g

(1.0 mmol) of HCl$H-Arg(NO₂)-Gly-Asp(OBzl)-Phe-OBzl, 0.15 g

(1.1 mmol) of HOBt and 0.23 g (1.1 mmol) of DCC 1.18 g (99%) of the

hydrochloride in ethyl acetate 2.1 g (88%) of the title compound was

20

obtained as colorless powder. Mp 127–129 ^ꢀC,

[a

]

¼ ꢁ6.5

D

(c ¼ 1.07, CH₃OH), ESI-MS (m/z) 1331 [M þ H]^þ.

title compound was obtained as colorless powder. Mp 92–94 ^ꢀC,

20

[

a]

¼ ꢁ12.7 (c ¼ 1.00, CH₃OH), ESI-MS (m/z) 1173 [M þ H]^þ.

4.2.31. HCl$H-Arg-Gly-Asp-Ser-Arg-Gly-Asp-Ser (7)

D

To the suspension of 100 mg (0.07 mmol) of 4, 8 ml of methanol,

2 ml of water, 10 mg of Pd/C (10%) hydrogen gas was bubbled for

20 h and TLC indicates complete disappearance of 4. After ﬁltration

the ﬁltrate was evaporated under vacuum and the residue was

4.2.24. HCl$H-Asp(OBzl)-Phe-Arg(NO₂)-Gly-Asp(OBzl)-Phe-OBzl

Using the same procedure as described for HCl$H-Asp(OBzl)-

Ser(Bzl)-OBzl, from 1.18 g of Boc-Asp(OBzl)-Phe-Arg(NO₂)-Gly-

Asp(OBzl)-Phe-OBzl and 20 ml of 4 N solution of hydrochloride in

triturated with ether repeatedly to provide the title compound in

20

ethyl acetate 1.01 g (total yield 92%) of the title compound was

51 mg (86% yield) as colorless powder. Mp 117–119 ^ꢀC, [

a

]

D

¼ ꢁ37.0

obtained as colorless powder. Mp 145–146 ^ꢀC,

[a

]

¼ ꢁ5.4

(c ¼ 0.95, CH₃OH), ESI-MS 849 [M þ H]^þ. ¹H NMR (BHSC-300,

20

D

(c ¼ 0.63, CH₃OH), ESI-MS (m/z) 1073 [M þ H]^þ.

CDCl₃)

d

¼ 11.0 (s, 3H), 7.89 (d, J ¼ 5.2 Hz, 7H), 5.7 (s, 2H), 4.62

(q, J ¼ 6.3 Hz, 1H), 4.86 (q, J ¼ 6.2 Hz, 2H), 4.55 (q, J ¼ 6.3 Hz, 1H),

4.53 (t, J ¼ 6.3 Hz, 1H), 4.09 (d, J ¼ 6.2 Hz, 4H), 4.03 (d, J ¼ 7.0 Hz,

4H), 3.56 (q, J ¼ 6.5 Hz, 1H), 2.73 (t, J ¼ 6.6 Hz, 4H), 2.65 (q,

J ¼ 6.8 Hz, 4H), 2.0 (s, 10H), 1.79 (q, J ¼ 7.4 Hz, 4H), 1.55 (m, 4H).

Anal. Calcd for C₃₀H₅₂N₁₄O₁₅: C 42.45, H 6.17, N 23.10; Found C

42.64, H 6.30, N 23.28.

4.2.25. Boc-Arg(NO₂)-Gly-Asp(OBzl)-Ser(Bzl)-Arg(NO₂)-Gly-

Asp(OBzl)-Ser(Bzl)-OBzl

Using the same procedure as described for Boc-Arg(NO₂)-Gly-

OCH₃, from 1.42 g (3.8 mmol) of Boc-Arg(NO₂)-Gly, 4.42 g (3.8 mmol)

of HCl$H-Asp(OBzl)-Ser(Bzl)-Arg(NO₂)-Gly-Asp(OBzl)-Ser(Bzl)-OBzl,

0.56 g (4.0 mmol) of HOBt and 0.86 g (4.2 mmol) of DCC 5.14 g

(91%) of the title compound was obtained as colorless powder. Mp

4.2.32. HCl$H-Arg-Gly-Asp-Val-Arg-Gly-Asp-Val (8)

Using the same procedure as described for 7, from 100 mg

(0.07 mmol) of 5 48 mg (73%) of the title compound was obtained

20

134–136 ^ꢀC,

[a]

¼ ꢁ6.3 (c ¼ 1.07, CH₃OH), ESI-MS (m/z)

D

1491[M þ H]^þ.

20

as colorless powder. Mp 121–23 ^ꢀC, [

a]

¼ ꢁ69.0 (c ¼ 1.01, CH₃OH),

D

4.2.26. HCl$H-Arg(NO₂)-Gly-Asp(OBzl)-Ser(Bzl)-Arg(NO₂)-Gly-

Asp(OBzl)-Ser(Bzl)-OBzl (4)

Using the same procedure as described for HCl$H-Asp(OBzl)-

Ser(Bzl)-OBzl, from 2.50 g of Boc-Arg(NO₂)-Gly-Asp(OBzl)-Ser(Bzl)-

Arg(NO₂)-Gly-Asp(OBzl)-Ser(Bzl)-OBzl and 60 ml of 4 N solution of

ESI-MS 873 [M þ H]^þ. ¹H NMR (BHSC-300, CDCl₃)

d

¼ 11.0 (s, 3H),

7.89 (d, J ¼ 5.2 Hz, 7H), 5.70 (s, 2H), 4.86 (q, J ¼ 6.2 Hz, 2H), 4.53

(t, J ¼ 6.3 Hz, 2H), 4.45 (q, J ¼ 6.0 Hz, 1H), 4.09 (d, J ¼ 6.0 Hz, 4H),

3.56 (q, J ¼ 6.5 Hz, 1H), 2.78 (m, 1H), 2.73 (t, J ¼ 6.6 Hz, 4H), 2.68 (m,

1H), 2.65 (q, J ¼ 6.0 Hz, 4H), 2.00 (s, 8H), 1.79 (q, J ¼ 7.4 Hz, 4H), 1.55

(m, 4H), 1.01 (d, J ¼ 8.1 Hz, 12H). Anal. Calcd for C₃₄H₆₀N₁₄O₁₃: C

46.78, H 6.93, N 22.46; Found C 46.60, H 6.88, N 22.65.

hydrochloride in ethyl acetate 2.15 g (90%) of the title compound

20

was obtained as colorless powder. Mp. 157–159 ^ꢀC, [

a]

¼ ꢁ4.3

D

(c ¼ 0.60, CH₃OH), ESI-MS (m/z) 1391 [M þ H]^þ.

4.2.33. HCl$H-Arg-Gly-Asp-Phe-Arg-Gly-Asp-Phe (9)

4.2.27. Boc-Arg(NO₂)-Gly-Asp(OBzl)-Val-Arg(NO₂)-Gly-Asp(OBzl)-

Val-OBzl

Using the same procedure as described for Boc-Arg(NO₂)-Gly-

OCH₃, from 1.17 g (3.1 mmol) of Boc-Arg(NO₂)-Gly, 3.0 g (3.0 mmol)

of HCl$H-Asp(OBzl)-Val-Arg(NO₂)-Gly-Asp(OBzl)-Val-OBzl, 0.46 g

(3.3 mmol) of HOBt and 0.68 g (3.3 mmol) of DCC 2.73 g (69%) of

Using the same procedure as described for 7, from 100 mg

(0.07 mmol) of 6 55 mg (77%) of the title compound was obtained

20

as colorless powder. Mp 149–151 ^ꢀC,

[

a]

¼ ꢁ17.0 (c ¼ 1.06,

D

CH₃OH), ESI-MS 969 [M þ H]^þ. ¹H NMR (BHSC-300, CDCl₃)

¼ 11.0

d

(s, 3H), 7.89 (d, J ¼ 5.2 Hz, 7H), 7.21 (d, J ¼ 7.3 Hz, 4H), 7.12 (m, 4H),

7.08 (t, J ¼ 7.2 Hz, 2H), 5.70 (s, 2H), 4.92 (q, J ¼ 6.5 Hz, 1H), 4.86

(q, J ¼ 6.2 Hz, 3H), 4.53 (t, J ¼ 6.3 Hz, 1H), 4.09 (d, J ¼ 6.2 Hz, 4H),

3.56 (q, J ¼ 6.5 Hz, 1H), 3.05 (d, J ¼ 6.3 Hz, 4H), 2.73 (t, J ¼ 6.6 Hz,

4H), 2.65 (q, J ¼ 6.8 Hz, 4H), 2.00 (s, 8H), 1.79 (q, J ¼ 7.4 Hz, 4H), 1.55

(m, 4H). Anal. Calcd for C₄₂H₆₀N₁₄O₁₃: C 52.06, H 6.24, N 20.24;

Found C 51.87, H 6.38, N 20.07.

the title compound was obtained as colorless powder. Mp 129–

20

130 ^ꢀC,

[a

]

¼ ꢁ17.7 (c ¼ 1.00, CH₃OH), ESI-MS (m/z) 1435

D

[M þ H]^þ.

4.2.28. HCl$H-Arg(NO₂)-Gly-Asp(OBzl)-Val-Arg(NO₂)-Gly-

Asp(OBzl)-Val-OBzl (5)

Using the same procedure as described for HCl$H-Asp(OBzl)-

Ser(Bzl)-OBzl, from 2.50 g of Boc-Arg(NO₂)-Gly-Asp(OBzl)-Val-

Arg(NO₂)-Gly-Asp(OBzl)-Val-OBzl and 50 ml of 4 N solution of

4.3. Preparing N-(estradiol-3-oxyacetyl)-RGD octapeptide

conjugates

hydrochloride in ethyl acetate 2.25 g (95%) of the title compound

4.3.1. Ethyl estradiol-3-oxyacetate (10)

The solution of 1.00 g (3.7 mmol) of estradiol in 15 ml of

anhydrous THF and the solution of 7.40 ml of sodium ethoxide in

20

was obtained as colorless powder. Mp 135–137 ^ꢀC, [

a

]

D

¼ ꢁ33.2

(c ¼ 1.20, CH₃OH), ESI-MS (m/z) 1335 [M þ H]^þ.

J. Liu et al. / European Journal of Medicinal Chemistry 44 (2009) 1689–1704

1699

anhydrous ethanol (2 M) were mixed and stirred at room

4.3.4. N-(Estradiol-3-oxyacetyl)-Arg(NO₂)-Gly-Asp(OBzl)-Val-

Arg(NO₂)-Gly-Asp(OBzl)-Val-OBzl (16)

Using the same procedure as described for Boc-Arg(NO₂)-Gly-

OCH₃, from 0.19 g (0.60 mmol) of 11, 0.70 g (0.60 mmol) of 5, 0.09 g

(0.60 mmol) of HOBt and 0.13 g (0.60 mmol) of DCC 0.53 g (61.8%)

temperature for 30 min, and then 1.64 ml (d ¼ 1.514 g/ml,

12.2 mmol) of ethyl bromoacetate was added. The mixture was

stirred at 55 ^ꢀC for 16 h and TLC (ethyl acetate/petroleum, 6:1)

indicated complete disappearance of estradiol. The reaction

mixture was ﬁltered and the ﬁltrate was evaporated under

vacuum. The residue was dissolved in 15 ml of ethyl acetate and

evaporated under vacuum, which was repeated for 4 times to

completely remove the residual ethyl bromoacetate. The residue

was puriﬁed on silica gel column (chloroform/ether, 10:0.3) to

provide 1.22 g (92%) of the title compound as colorless powder. Mp

88–90 ^ꢀC; IR (KBr) 3352, 3031, 2864, 1745, 1604, 1502, 1464, 1370,

of the title compound was obtained as colorless powder. Mp 168–

20

172 ^ꢀC, ESI-MS (m/z) 1547 [M þ H]^þ, [

a]

¼ ꢁ4.4 (c ¼ 1, DMSO). ¹H

D

NMR (DMSO-d₆)

d

/ppm ¼ 8.48 (d, J ¼ 5.1 Hz, 2H), 8.46 (t, J ¼ 7.2 Hz,

2H), 8.34 (d, J ¼ 4.8 Hz, 2H), 8.28 (d, J ¼ 5.1 Hz, 2H), 8.16 (d,

J ¼ 4.5 Hz, 2H), 7.35 (s, 2H), 7.34 (s, 2H), 7.27–7.40 (m, 15H), 7.14 (d,

J ¼ 8.7 Hz, 1H), 6.68 (d, J ¼ 8.7 Hz, 1H), 6.62 (s, 1H), 5.11 (s, 2H), 5.09

(s, 2H), 5.07 (s, 2H), 5.05 (d, J ¼ 6.6 Hz, 1H), 5.03 (m, 1H), 5.03 (m,

1H), 4.52 (m, 2H), 4.51 (m, 2H), 4.11 (m, 2H), 4.10 (m, 2H), 3.61 (d,

J ¼ 2.1 Hz, 2H), 3.57 (d, J ¼ 2.1 Hz, 2H), 3.28 (d, J ¼ 3.3 Hz, 1H), 2.96

(d, J ¼ 2.4 Hz, 2H), 2.91 (d, J ¼ 2.7 Hz, 2H), 2.90 (d, J ¼ 1.8 Hz, 2H),

2.86 (d, J ¼ 2.7 Hz, 2H), 2.81 (m, 1H), 2.34 (m, 1H), 2.20 (m, 1H), 1.98

(m, 2H), 1.98 (m, 2H), 1.93 (m, 2H), 1.90 (m, 2H), 1.87 (m, 2H), 1.86

(m, 2H), 1.74 (m, 2H), 1.72 (m, 2H), 1.68 (m, 2H), 1.64 (m, 2H), 1.63

(m, 1H), 1.61 (m, 1H), 1.10 (s, 3H), 0.97 (d, J ¼ 1.8 Hz, 3H), 0.97 (d,

J ¼ 1.8 Hz, 3H), 0.96 (d, J ¼ 1.8 Hz, 3H), 0.96 (d, J ¼ 1.8 Hz, 3H). ¹³C

874, 832 cm^ꢁ1. ¹H NMR (DMSO-d₆)

d

/ppm ¼ 7.12 (d, J ¼ 7.5 Hz, 1H),

6.71 (s, 1H), 6.52 (d, J ¼ 7.5 Hz, 1H), 5.14 (s, 2H), 4.61 (q, J ¼ 4.8 Hz,

1H), 4.20 (q, J ¼ 4.4 Hz, 2H), 2.97 (t, J ¼ 4.6 Hz, 2H), 2.91

(m, J ¼ 4.2 Hz, 1H), 2.55 (s, 1H), 2.03 (m, J ¼ 4.8 Hz, 2H), 2.01

(m, J ¼ 4.3 Hz, 2H), 1.87 (m, J ¼ 4.2 Hz, 1H), 1.84 (m, J ¼ 4.4 Hz, 2H),

1.76 (m, J ¼ 4.6 Hz, 2H), 1.70 (m, J ¼ 4.3 Hz, 1H), 1.65 (m, J ¼ 4.7 Hz,

2H), 1.41 (t, J ¼ 4.4 Hz, 3H), 0.78 (s, 3H). ESI-MS (m/z) 359 [M þ H]^þ,

20

[

a]

¼ þ60.0 (c ¼ 1.00, THF).

D

NMR (DMSO-d₆)

d

/ppm ¼ 171.96, 171.49, 171.19, 171.04, 170.57,

4.3.2. Estradiol-3-oxyacetic acid (11)

170.49, 170.22, 169.27, 169.09, 168.52, 159.75, 157.09, 155.94, 137.99,

136.45, 136.27, 133.51, 128.84, 128.56,128.49,128.42, 128.33,126.64,

112.59, 114.92, 80.51, 67.48, 66.41, 58.25, 58.12, 50.01, 49.77, 49.54,

47.98, 43.98, 43.26, 37.04, 36.70, 33.81, 31.13, 30.86, 30.38, 30.24,

27.26, 25.79, 24.92, 23.23, 19.67, 19.34, 18.57, 18.37, 11.70.

At 0 ^ꢀC to the solution of 1.30 g (3.6 mmol) of 10 in 10 ml of

methanol 10 ml of NaOH aqueous solution (2 N) was added. The

reaction mixture was stirred at room temperature for 1.5 h and TLC

(ethyl acetate/petroleum, 6:1) indicated complete disappearance of

10. The pH of the reaction mixture was adjusted to 2 by adding

KHSO₄powder. The reaction mixture was extracted by ethyl acetate

and the ethyl acetate phase was dried over anhydrous Na₂SO₄. After

ﬁltration the ﬁltrate was evaporated under vacuum to provide

1.11 g (93%) of the title compound as colorless powder. Mp 214–

215 ^ꢀC. IR (KBr) 3350, 3033, 2866, 1726, 1602, 1501, 1463, 1372, 875,

4.3.5. N-(Estradiol-3-oxyacetyl)-Arg(NO₂)-Gly-Asp(OBzl)-Phe-

Arg(NO₂)-Gly-Asp(OBzl)-Phe-OBzl (17)

Using the same procedure as described for Boc-Arg(NO₂)-Gly-

OCH₃, from 0.18 g (0.54 mmol) of 11, 0.70 g (0.51 mmol) of 6, 0.08 g

(0.57 mmol) of HOBt and 0.12 g (0.59 mmol) of DCC 0.59 g (70.6%)

831 cm^ꢁ1

.

¹H NMR (DMSO-d₆)

d

/ppm ¼ 10.55 (s, 1H), 7.00 (d,

of the title compound was obtained as colorless powder. Mp 146–

20

J ¼ 7.5 Hz, 1H), 6.63 (s, 1H), 6.51 (d, J ¼ 7.5 Hz, 1H), 5.00 (s, 2H), 4.42

(q, J ¼ 4.8 Hz, 1H), 2.97 (t, J ¼ 4.6 Hz, 2H), 2.89 (m, J ¼ 4.3 Hz, 1H),

2.56 (s,1H), 2.02 (m, J ¼ 4.8 Hz, 2H), 2.01 (m, J ¼ 4.3 Hz, 2H),1.86 (m,

J ¼ 4.2 Hz, 1H), 1.84 (m, J ¼ 4.4 Hz, 2H), 1.75 (m, J ¼ 4.6 Hz, 2H),

148 ^ꢀC, ESI-MS (m/z) 1643 [M þ H]^þ, [

a

]

D

¼ þ 4.5^ꢀ(c ¼ 1, DMSO). ¹H

NMR (DMSO-d₆)

d

/ppm ¼ 8.40 (d, J ¼ 5.1 Hz, 2H), 8.34 (t, J ¼ 7.2 Hz,

2H), 8.23 (d, J ¼ 4.8 Hz, 2H), 8.13 (d, J ¼ 5.1 Hz, 2H), 8.05

(d, J ¼ 4.5 Hz, 2H), 7.34 (s, 2H), 7.30 (s, 2H), 7.13–7.28 (m, 25H), 7.14

(d, J ¼ 8.7 Hz, 1H), 6.67 (d, J ¼ 8.7 Hz, 1H), 6.65 (s, 1H), 5.10

(d, J ¼ 5.5 Hz,1H), 5.08 (s, 2H), 5.07 (s, 2H), 5.05 (s, 2H), 5.04 (m,1H),

5.03 (m, 1H), 4.79 (s, 2H), 4.60 (m, 2H), 4.54 (m, 2H), 3.75

(d, J ¼ 2.1 Hz, 2H), 3.74 (d, J ¼ 2.1 Hz, 2H), 3.20 (d, J ¼ 3.3 Hz, 1H),

3.10 (d, J ¼ 2.4 Hz, 2H), 2.79 (m, 1H), 2.79 (m, 2H), 2.58 (m, 2H), 2.58

(m, 2H), 1.95 (m, 2H), 1.92 (m, 2H), 1.92 (m, 2H), 1.84 (m, 2H), 1.82

(m, 2H), 1.81 (m, 2H), 1.81 (m, 2H), 1.71 (m, 2H), 1.71 (m, 2H), 1.69

(m, 2H), 1.68 (m, 1H), 1.64 (m, 1H), 1.48 (m, 2H), 1.42 (m, 2H), 0.91 (s,

1.70 (m, J ¼ 4.3 Hz, 1H), 1.65 (m, J ¼ 4.7 Hz, 2H), 0.783 (s, 3H). ESI-

20

MS (m/z) 331 [M þ H]^þ. [

a

]

¼ 50.0 (c ¼ 0.60, THF).

D

4.3.3. N-(Estradiol-3-oxyacetyl)-Arg(NO₂)-Gly-Asp(OBzl)-Ser(Bzl)-

Arg(NO₂)-Gly-Asp(OBzl)-Ser(Bzl)-OBzl (15)

Using the same procedure as described for Boc-Arg(NO₂)-Gly-

OCH₃, from 0.17 g (0.52 mmol) of 11, 0.70 g (0.49 mmol) of 4,

0.08 g (0.57 mmol) of HOBt and 0.12 g (0.57 mmol) of DCC 0.60 g

(71.4%) of the title compound was obtained as colorless powder.

3H). ¹³C NMR (DMSO-d₆)

d

/ppm ¼ 171.98, 171.46, 171.24, 170.87,

20

Mp 176–178 ^ꢀC, ESI-MS (m/z) 1703 [M þ H]^þ, [

a

]

¼ 3.0 (c ¼ 1,

170.57, 170.45, 170.21, 169.09, 168.52, 159.76, 157.09, 155.9, 137.98,

137.30,136.43, 136.08, 133.44, 129.70,129.57,128.85, 128.49, 128.35,

127.06, 126.70, 114.86, 112.56, 80.51, 67.48, 66.50, 54.38, 52.72,

52.42, 49.96, 49.62, 49.41, 47.96, 43.97, 43.25, 42.21, 37.57, 36.93,

33.81, 30.35, 29.67, 27.26, 26.44, 25.78, 24.93, 23.22, 16.40.

D

DMSO). ¹H NMR (DMSO-d₆)

d

/ppm ¼ 8.46 (d, J ¼ 5.1 Hz, 2H), 8.36

(t, J ¼ 7.2 Hz, 2H), 8.28 (d, J ¼ 4.8 Hz, 2H), 8.29 (d, J ¼ 5.1 Hz, 2H),

8.25 (d, J ¼ 4.5 Hz, 2H), 7.35 (s, 2H), 7.34 (s, 2H), 7.26–7.32 (m,

25H), 7.15 (d, J ¼ 8.7 Hz, 1H), 6.69 (d, J ¼ 8.7 Hz, 1H), 6.63 (s, 1H),

5.15 (s, 2H), 5.14 (s, 2H), 5.14 (s, 2H), 5.08 (d, J ¼ 5.1 Hz, 1H), 4.82

(s, 2H), 4.79 (s, 2H), 4.70 (s, 2H), 4.56 (m, 2H), 4.54 (m, 2H), 4.55

(m, 2H), 4.48 (m, 2H), 3.76 (d, J ¼ 2.1 Hz, 2H), 3.75 (d, J ¼ 2.1 Hz,

2H), 3.53 (d, J ¼ 3.3 Hz, 1H), 2.97 (d, J ¼ 2.4 Hz, 2H), 2.92 (d,

J ¼ 2.7 Hz, 2H), 2.91 (d, J ¼ 1.8 Hz, 2H), 2.82 (m, 1H), 2.74 (m, 2H),

2.73 (m, 2H), 1.95 (m, 2H), 1.94 (m, 2H), 1.82 (m, 2H), 1.82 (m, 2H),

1.81 (m, 2H), 1.80 (m, 2H), 1.73 (m, 2H), 1.72 (m, 2H), 1.69 (m, 2H),

1.67 (m, 1H), 1.65 (m, 1H), 1.43 (m, 2H), 1.41 (m, 2H), 0.87 (s, 3H).

4.3.6. N-(Estradiol-3-oxyacetyl)-Arg-Gly-Asp-Ser-Arg-Gly-Asp-Ser

(21)

Using the same procedure as described for 7, from 300 mg

(0.176 mmol) of 15 180 mg (88%) of the title compound was

20

obtained as colorless powder. Mp 250 ^ꢀC (decomp.), [

a

]

¼ ꢁ3.6

D

(c ¼ 1.48, DMSO), ESI-MS (m/z): 1161 [M þ H]^þ, 1057

[M ꢁ (Ser) þ H]^þ, 942 [M ꢁ (Asp-Ser) þ H]^þ, 885 [M ꢁ (Gly-Asp-

Ser) þ H]^þ, 729 [M ꢁ (Arg-Gly-Asp-Ser) þ H]^þ, 642 [M ꢁ (Ser-Arg-

Gly-Asp-Ser) þ H]^þ, 527 [M ꢁ (Asp-Ser-Arg-Gly-Asp-Ser) þ H]^þ,

470 [M ꢁ (Gly-Asp-Ser-Arg-Gly-Asp-Ser) þ H]^þ. ¹H NMR (DMSO-

¹³C NMR (DMSO-d₆)

d

/ppm ¼ 171.01, 170.79, 170.49, 170.20, 170.16,

169.21, 168.97, 159.80, 157.15, 138.51, 138.29, 136.47, 136.24,

128.83, 128.67, 128.41, 128.32, 128.15, 127.96, 127.86, 126.63,

112.63, 115.00, 80.54, 72.79, 72.69, 69.52, 67.34, 66.62, 66.24,

66.18, 53.55, 53.16, 52.82, 52.47, 50.06, 49.76, 49.51, 48.02, 43.99,

39.02, 37.08, 36.90, 36.71, 33.81, 30.42, 29.66, 26.48, 25.82, 24.91,

23.25, 11.70.

d₆)

d

/ppm ¼ 9.88 (s, 1H), 9.665 (s, 2H), 8.42 (d, J ¼ 5.4 Hz, 2H), 8.37

(t, J ¼ 6.8 Hz, 2H), 8.26 (d, J ¼ 4.8 Hz, 2H), 8.12 (d, J ¼ 5.4 Hz, 2H),

8.11 (d, J ¼ 4.5 Hz, 2H), 8.01 (s, 1H), 7.98 (s, 1H), 7.34 (s, 2H), 7.32

(s, 2H), 7.13 (d, J ¼ 8.7 Hz, 1H), 6.67 (d, J ¼ 8.7 Hz, 1H), 6.63 (s, 1H),

1700

J. Liu et al. / European Journal of Medicinal Chemistry 44 (2009) 1689–1704

4.60 (t, J ¼ 3.6 Hz, 1H), 4.58 (t, J ¼ 3.6 Hz, 1H), 4.64 (s, 2H), 4.63

(d, J ¼ 4.5 Hz, 1H), 4.62 (m, 1H), 4.52 (m, 1H), 4.51 (m, 1H), 4.50 (m,

1H), 4.41 (m, 2H), 3.88 (d, J ¼ 3.6 Hz, 2H), 3.67 (d, J ¼ 3.3 Hz, 2H),

3.62 (d, J ¼ 2.1 Hz, 2H), 3.60 (d, J ¼ 2.1 Hz, 2H), 3.58 (m, 1H), 3.54

(d, J ¼ 3.3 Hz, 2H), 3.42 (d, J ¼ 3.6 Hz, 2H), 2.86 (t, J ¼ 2.1 Hz, 2H),

2.74 (m, 1H), 2.47 (m, 2H), 2.10 (m, 2H), 1.98 (m, 2H), 1.87 (m, 2H),

1.75 (m, 2H), 1.73 (m, 2H), 1.62 (m, 2H), 1.60 (m, 2H), 1.52 (m, 2H),

1.46 (m, 2H), 1.36 (m, 2H), 1.28 (m, 1H), 1.26 (m, 1H), 1.24 (m, 2H),

1.61 (m, 2H), 1.60 (m, 2H), 1.50 (m, 2H), 1.47 (m, 2H), 1.36 (m, 2H),

1.35 (m,1H),1.34 (m,1H),1.27 (m, 2H), 0.78 (s, 3H). ¹³C NMR (DMSO-

d₆)

d

/ppm ¼ 175.89, 174.35, 172.64, 172.55, 171.42, 170.0, 169.03,

168.74, 168.33, 158.16, 156.03, 139.23, 138.81, 138.00, 133.46, 129.87,

128.49, 128.05, 126.00, 114.91, 112.60, 80.54, 67.15, 56.51, 55.75,

52.34, 50.31, 50.04, 48.01, 44.00, 43.28, 38.50, 37.06, 33.81, 30.40,

29.89, 29.41, 27.30, 25.81, 23.26, 11.72. Anal. Calcd for C₆₂H₈₄N₁₄O₁₆

C 58.11, H 6.61, N 15.30; Found C 58.30, H 6.78, N 15.49.

:

0.67 (s, 3H). ¹³C NMR (DMSO-d₆)

d/ppm ¼ 176.03, 175.29, 174.86,

173.54, 173.08, 172.61, 171.45, 170.83, 168.80, 168.39, 157.72, 157.10,

137.98, 133.37, 126.66, 114.87, 112.59, 80.52, 67.05, 62.77, 50.08,

56.50, 47.98, 44.00, 42.75, 42.59, 37.04, 35.61, 34.75, 33.82, 30.37,

29.70, 27.30, 26.48, 25.79, 24.93, 23.25, 15.38. Anal. Calcd for

C₅₀H₇₆N₁₄O₁₈: C 51.72, H 6.60, N 16.89; Found C 51.52, H 6.46, N

16.70.

4.4. Preparing estradiol-17b-O-carbonylpropinyl-RGD octapeptide

conjugates

4.4.1. Estradiol-17-b-O-carbonylpropinic acid (24)

To the warm solution of 100 mg (1.0 mmol) of succinic anhy-

drate in 1 ml of pyridine 100 mg (0.37 mmol) of estradiol was

added. The reaction mixture was stirred at 90 ^ꢀC for 10 h and TLC

(chloroform/methanol/acetic acid, 20:1:0.4) indicated complete

disappearance of estradiol. The reaction mixture was cooled to

room temperature, mixed with 20 ml of ice water and 75 mg of

sodium chloride, and stirred vigorously. The mixture was then

extracted with ethyl acetate, the ethyl acetate phase was separated

and dried with anhydrous Na₂SO₄. After ﬁltration the ﬁltrate was

evaporated at reduced pressure to give a yellow syrup. The syrup

was dissolved in 1 ml of methanol. The solution was adjusted to pH

8.5–9.0 with cold aqueous K₂CO₃(10%) and strolled at room

temperature for 18 h. The solution was adjusted to pH 6.0 with

acetic acid (50%) and evaporated at reduced pressure. The residue

was mixed with 2 ml of ice water and 75 mg of sodium chloride.

The mixture was extracted with 50 ml of ethyl acetate and the ethyl

acetate phase was separated. After washing with ice water for 3

times and drying with anhydrous Na₂SO₄. The ethyl acetate phase

was evaporated to give a yellowish syrup. The syrup was kept in

a refrigerator for 18 h to give 137 mg (95%) of the title compound as

a colorless powder. Mp 148–150 ^ꢀC, IR (KBr) 3253, 3035, 2867, 1742,

4.3.7. N-(Estradiol-3-oxyacetyl)-Arg-Gly-Asp-Val-Arg-Gly-Asp-Val

(22)

Using the same procedure as described for 7, from 300 mg

(0.194 mmol) of 16 140 mg (60.8%) of the title compound was

20

obtained as colorless powder. Mp 228–230 ^ꢀC,

[

a]

¼ ꢁ5.1

D

(c ¼ 1.80, DMSO), ESI-MS (m/z): 1186 [M þ H]^þ, 1069

[M ꢁ (Val) þ H]^þ, 954 [M ꢁ (Asp-Val) þ H]^þ, 897 [M ꢁ (Gly-Asp-

Val) þ H]^þ, 741 [M ꢁ (Arg-Gly-Asp-Val) þ H]^þ, 642 [M ꢁ (Val-Arg-

Gly-Asp-Val) þ H]^þ, 527 [M ꢁ (Asp-Val-Arg-Gly-Asp-Val) þ H]^þ,

470 [M ꢁ (Gly-Asp-Val-Arg-Gly-Asp-Val) þ H]^þ. ¹H NMR (DMSO-d₆)

d

/ppm ¼ 10.14 (s, 1H), 9.90 (s, 2H), 8.64 (d, J ¼ 5.4 Hz, 2H), 8.51

(t, J ¼ 7.2 Hz, 2H), 8.34 (d, J ¼ 5.1 Hz, 2H), 8.16 (d, J ¼ 4.5 Hz, 2H), 8.09

(d, J ¼ 4.5 Hz, 2H), 8.07 (s, 1H), 8.04 (s, 1H), 7.35 (s, 2H), 7.18

(d, J ¼ 8.1 Hz, 1H), 7.13 (s, 2H), 6.78 (d, J ¼ 8.7 Hz, 1H), 6.61 (s, 1H),

4.61 (d, J ¼ 3.3 Hz, 1H), 4.56 (s, 2H), 4.52 (m, 1H), 4.42 (m, 1H), 4.41

(m,1H), 4.26 (m,1H), 4.25 (m,1H), 4.23 (m,1H), 4.11 (m, 2H), 3.68 (d,

J ¼ 3.6 Hz, 2H), 3.59 (d, J ¼ 3.3 Hz, 2H), 3.27 (m, 1H), 3.21

(d, J ¼ 2.1 Hz, 2H), 3.20 (d, J ¼ 2.1 Hz, 2H), 2.87 (t, J ¼ 2.1 Hz, 2H), 2.84

(m, 1H), 2.60 (m, 2H), 2.58 (m, 2H), 2.57 (m, 1H), 2.20 (m, 2H), 2.099

(m, 2H),1.96 (m, 2H),1.87 (m,1H),1.78 (m, 2H),1.78 (m, 2H),1.66 (m,

2H), 1.58 (m, 2H), 1.50 (m, 2H), 1.41 (m, 2H), 1.36 (m, 1H), 1.32 (m,

1H),1.04 (s, 3H), 0.836 (d, J ¼ 1.8 Hz, 3H), 0.82 (d, J ¼ 1.8 Hz, 3H), 0.81

1602, 1504, 1463, 1372, 876, 831 cm^ꢁ^{1 1}H NMR (DMSO-d₆)

. d/

ppm ¼ 12.34 (s, 2H), 7.14 (d, J ¼ 8.7 Hz, 1H), 6.57 (d, J ¼ 2.4 Hz, 1H),

6.63 (s, 1H), 4.62 (t, J ¼ 9.0 Hz, 1H), 3.60 (s, 2H), 3.49 (t, J ¼ 8.7 Hz,

2H), 3.36 (dt, J ¼ 8.1 Hz, J ¼ 6.9 Hz, 1H), 2.11 (t, J ¼ 4.8 Hz, 2H), 2.06

(t, J ¼ 9.0 Hz, 2H), 1.78 (dt, J ¼ 4.8 Hz, J ¼ 3.3 Hz, 2H), 1.56

(dt, J ¼ 7.1 Hz, J ¼ 3.9 Hz, 2H), 1.51 (dt, J ¼ 3.6 Hz, J ¼ 2.7 Hz, 2H), 1.48

(t, J ¼ 6.6 Hz, 2H), 1.47 (dt, J ¼ 7.8 Hz, J ¼ 3.0 Hz, 1H), 1.38

(dt, J ¼ 6.3 Hz, J ¼ 4.5 Hz, 2H), 1.34 (dt, J ¼ 6.6 Hz, J ¼ 4.8 Hz, 1H), 1.17

(d, J ¼ 1.8 Hz, 3H), 0.80 (d, J ¼ 1.8 Hz, 3H). ¹³C NMR (DMSO-d₆)

d/

ppm ¼ 176.05, 174.01, 172.71, 172.53, 171.33, 170.76, 169.04, 168.89,

168.27, 168.27, 158.04, 157.90, 138.03, 133.56, 126.63, 112.65, 114.93,

80.54, 67.24, 58.58, 57.68, 52.17, 50.08, 48.02, 47.32, 44.01, 38.04,

37.08, 33.80, 31.11, 30.68, 30.41, 29.86, 27.30, 26.50, 25.81, 25.22,

24.91,19.66,19.47,18.40,17.97,15.35. Anal. Calcd for C₅₄H₈₄N₁₄O₁₆: C

54.72, H 7.14, N 16.54; Found C 54.53, H 7.30, N 16.35.

(s, 3H). ¹³C NMR (DMSO-d₆)

137.89, 133.19, 114.54, 112.30, 82.37, 64.79, 49.97, 49.48, 43.99,

d

/ppm ¼ 173.93, 172.33, 170.85, 155.95,

43.69, 37.02, 36.84, 30.35, 29.65, 27.52, 27.19, 26.47, 23.25, 12.31.

20

ESI-MS (m/z) 373 [M þ H]^þ. [

a

]

¼ 35.0 (c ¼ 1.00, THF).

D

4.3.8. N-(Estradiol-3-oxyacetyl)-Arg-Gly-Asp-Phe-Arg-Gly-Asp-Phe

(23)

4.4.2. Estradiol-17-O-b-carbonylpropionyl-Arg(NO₂)-Gly-

Using the same procedure as described for 7, from 300 mg

(0.194 mmol) of 17 110 mg (47.0%) of the title compound was

Asp(OBzl)-Ser(Bzl)-Arg(NO₂)-Gly-Asp(OBzl)-Ser(Bzl)-OBzl (28)

Using the same procedure as described for Boc-Arg(NO₂)-Gly-

OCH₃from 0.19 g (0.52 mmol) of 24, 0.70 g (0.49 mmol) of 5, 0.08 g

(0.57 mmol) of HOBt and 0.12 g (0.57 mmol) of DCC 0.55 g (65.3%)

20

obtained as colorless powder. Mp 210–212 ^ꢀC,

[

a

]

D

¼ ꢁ10.1

(c ¼ 1.64, DMSO), ESI-MS (m/z) 1281 [M þ H]^þ, 1117

[M ꢁ (Phe) þ H]^þ, 1002 [M ꢁ (Asp-Phe) þ H]^þ, 945 [M ꢁ (Gly-Asp-

Phe) þ H]^þ, 789 [M ꢁ (Arg-Gly-Asp-Phe) þ H]^þ, 642 [M ꢁ (Phe-Arg-

Gly-Asp-Phe) þ H]^þ, 527 [M ꢁ (Asp-Phe-Arg-Gly-Asp-Phe) þ H]^þ,

470 [M ꢁ (Gly-Asp-Phe-Arg-Gly-Asp-Phe) þ H]^þ. ¹H NMR (DMSO-

of the title compound was obtained as colorless powder. Mp 162–

164 ^ꢀC, ESI-MS (m/z): 1745 [M þ H]^þ, [

a

]

D

¼ 1.5 (c ¼ 1, DMSO). ¹H

20

NMR (DMSO-d₆)

d

/ppm ¼ 8.98 (s, 1H), 8.49 (d, J ¼ 5.1 Hz, 2H), 8.47

(t, J ¼ 7.2 Hz, 2H), 8.23 (d, J ¼ 5.4 Hz, 2H), 8.22 (d, J ¼ 5.1 Hz, 2H),

8.21 (d, J ¼ 4.5 Hz, 2H), 7.35 (s, 2H), 7.31 (s, 2H), 7.26–7.30 (m, 25H),

7.08 (d, J ¼ 8.7 Hz, 1H), 6.60 (d, J ¼ 8.7 Hz, 1H), 6.49 (s, 1H), 5.12

(s, 2H), 5.11 (s, 2H), 5.10 (s, 2H), 5.07 (t, J ¼ 5.1 Hz, 1H), 5.06

(t, J ¼ 5.1 Hz, 1H), 4.71 (s, 2H), 4.70 (s, 2H), 4.60 (m, 2H), 4.53 (m,

2H), 4.22 (m, 2H), 3.96 (t, J ¼ 2.1 Hz,1H), 3.76 (d, J ¼ 2.1 Hz, 2H), 3.74

(d, J ¼ 3.3 Hz, 1H), 3.46 (d, J ¼ 2.4 Hz, 2H), 3.41 (d, J ¼ 2.7 Hz, 2H),

2.91 (d, J ¼ 1. 8 Hz, 2H), 2.82 (m, 1H), 2.80 (t, J ¼ 2.4 Hz, 2H), 2.79

(t, J ¼ 2.4 Hz, 2H), 2.64 (m, 2H), 2.62 (m, 2H), 1.89 (m, 2H), 1.89

(m, 2H), 1.80 (m, 2H), 1.80 (m, 2H), 1.79 (m, 2H), 1.78 (m, 2H), 1.78

(m, 2H), 1.75 (m, 2H), 1.72 (m, 2H), 1.71 (m, 2H), 1.70 (m, 1H), 1.68

d₆)

d

/ppm ¼ 10.01 (s, 1H), 9.79 (s, 2H), 8.43 (d, J ¼ 5.4 Hz, 2H), 8.35

(t, J ¼ 6.8 Hz, 2H), 8.24 (d, J ¼ 4.8 Hz, 2H), 8.12 (d, J ¼ 5.4 Hz, 2H), 8.15

(d, J ¼ 4.5 Hz, 2H), 8.01 (s, 1H), 8.00 (s, 1H), 7.35 (s, 2H), 7.33 (s, 2H),

7.10–7.21 (m, 10H), 7.13 (d, J ¼ 8.7 Hz, 1H), 6.67 (d, J ¼ 8.7 Hz, 1H),

6.63 (s, 1H), 4.65 (s, 2H), 4.64 (d, J ¼ 4.5 Hz, 1H), 4.64 (m, 1H), 4.55

(m, 1H), 4.54 (m, 1H), 4.53 (m, 1H), 4.42 (m, 2H), 3.74 (d, J ¼ 3.6 Hz,

2H), 3.73 (d, J ¼ 3.3 Hz, 2H), 3.69 (d, J ¼ 2.1 Hz, 2H), 3.69

(d, J ¼ 2.1 Hz, 2H), 3.62 (m, 1H), 3.50 (d, J ¼ 3.3 Hz, 2H), 3.48

(d, J ¼ 3.6 Hz, 2H), 2.84 (t, J ¼ 2.1 Hz, 2H), 2.83 (m, 1H), 2.58 (m, 2H),

2.04 (m, 2H), 1.98 (m, 2H), 1.97 (m, 2H), 1.74 (m, 2H), 1.72 (m, 2H),

J. Liu et al. / European Journal of Medicinal Chemistry 44 (2009) 1689–1704

1701

20

(m, 1H), 1.39 (m, 2H), 1.36 (m, 2H), 1.04 (s, 3H). ¹³C NMR (DMSO-d₆)

obtained as colorless powder. Mp 250 ^ꢀC (decomp.), [

a

]

D

¼ ꢁ3.3

d/ppm ¼ 172.41, 171.82, 171.02, 170.80, 170.49, 170.21, 170.17, 169.79,

(c ¼ 1.50, DMSO), ESI-MS (m/z): 1203 [M þ H]^þ, 1099

[M ꢁ (Ser) þ H]^þ, 984 [M ꢁ (Asp-Ser) þ H]^þ, 927 [M ꢁ (Gly-Asp-

Ser) þ H]^þ, 771 [M ꢁ (Arg-Gly-Asp-Ser) þ H]^þ, 684 [M ꢁ (Ser-Arg-

Gly-Asp-Ser) þ H]^þ, 569 [M ꢁ (Asp-Ser-Arg-Gly-Asp-Ser) þ H]^þ,

512 [M ꢁ (Gly-Asp-Ser-Arg-Gly-Asp-Ser) þ H]^þ. ¹H NMR (DMSO-d₆)

169.33, 168.97, 159.79, 157.15, 138.52, 138.29, 136.47, 136.24, 128.84,

128.67, 128.41, 128.32, 128.15, 127.97, 127.86, 72.78, 70.02, 66.62,

66.23, 66.18, 53.54, 53.15, 53.02, 52.82, 51.74, 49.75, 49.50, 48.02,

42.44, 42.24, 38.80, 36.89, 36.66, 33.81, 30.25, 29.63, 29.25, 26.34,

24.91, 23.27, 14.51.

d

/ppm ¼ 10.11 (s, 1H), 9.84 (s, 2H), 8.86 (s, 1H), 8.63 (d, J ¼ 5.4 Hz,

2H), 8.53 (t, J ¼ 6.8 Hz, 2H), 8.24 (d, J ¼ 4.8 Hz, 2H), 8.12

(d, J ¼ 5.4 Hz, 2H), 8.12 (d, J ¼ 4.5 Hz, 2H), 8.00 (s, 1H), 7.85 (s, 1H),

7.32 (s, 2H), 7.23 (s, 2H), 7.13 (d, J ¼ 8.7 Hz, 1H), 6.76 (d, J ¼ 8.7 Hz,

1H), 6.61 (s, 1H), 4.65 (t, J ¼ 3.6 Hz, 1H), 4.64 (t, J ¼ 3.6 Hz, 1H), 4.57

(m, 1H), 4.50 (m, 1H), 4.48 (m, 1H), 4.47 (m, 1H), 4.25 (m, 2H), 3.91

(d, J ¼ 3.6 Hz, 2H), 3.76 (d, J ¼ 3.3 Hz, 2H), 3.52 (d, J ¼ 2.1 Hz, 2H),

3.53 (d, J ¼ 2.1 Hz, 2H), 3.53 (t, J ¼ 4.5 Hz, 1H), 3.46 (d, J ¼ 3.3 Hz,

2H), 3.34 (d, J ¼ 3.6 Hz, 2H), 2.94 (t, J ¼ 2.1 Hz, 2H), 2.72 (m, 1H),

2.24 (t, J ¼ 2.1 Hz, 2H), 2.16 (t, J ¼ 2.1 Hz, 2H), 2.14 (m, 2H), 2.10

(m, 2H), 1.98 (m, 2H), 1.78 (m, 2H), 1.71 (m, 2H), 1.67 (m, 2H), 1.64

(m, 2H), 1.59 (m, 2H), 1.46 (m, 2H), 1.32 (m, 2H), 1.31 (m, 2H), 1.27

(m, 1H), 1.20 (m, 1H), 1.18 (m, 2H), 0.77 (s, 3H). ¹³C NMR (DMSO-d₆)

4.4.3. Estradiol-17-O-b-carbonylpropionyl-Arg(NO₂)-Gly-

Asp(OBzl)-Val-Arg(NO₂)-Gly-Asp(OBzl)-Val-OBzl (29)

Using the same procedure as described for Boc-Arg(NO₂)-Gly-

OCH₃, from 0.22 g (0.58 mmol) of 24, 0.70 g (0.55 mmol) of 6, 0.09 g

(0.64 mmol) of HOBt and 0.13 g (0.64 mmol) of DCC 0.66 g (76.4%)

of the title compound was obtained as colorless powder. Mp 170–

20

173 ^ꢀC, ESI-MS (m/z) 1589 [M þ H]^þ, [

a

]

D

¼ þ 2.4 (c ¼ 1, DMSO). ¹H

NMR (DMSO-d₆)

d

/ppm ¼ 9.01 (s, 1H), 8.28 (d, J ¼ 5.1 Hz, 2H), 8.26

(t, J ¼ 7.2 Hz, 2H), 8.20 (d, J ¼ 5.4 Hz, 2H), 8.19 (d, J ¼ 5.1 Hz, 2H), 8.10

(d, J ¼ 4.5 Hz, 2H), 7.36 (s, 2H), 7.32 (s, 2H), 7.29–7.32 (m, 15H), 7.07

(d, J ¼ 8.7 Hz, 1H), 6.67 (d, J ¼ 8.7 Hz, 1H), 6.44 (s, 1H), 5.10 (s, 2H),

5.09 (s, 2H), 5.07 (d, J ¼ 6.6 Hz, 1H), 5.04 (m, 1H), 5.03 (m, 1H), 4.53

(m, 2H), 4.53 (m, 2H), 4.12 (m, 2H), 4.11 (m, 2H), 3.61 (d, J ¼ 2.1 Hz,

2H), 3.56 (d, J ¼ 2.1 Hz, 2H), 3.29 (d, J ¼ 3.3 Hz, 1H), 2.97 (d,

J ¼ 2.4 Hz, 2H), 2.92 (d, J ¼ 2.7 Hz, 2H), 2.91 (d, J ¼ 1.8 Hz, 2H), 2.87

(d, J ¼ 2.7 Hz, 2H), 2.85 (t, J ¼ 2.1 Hz, 2H), 2.84 (t, J ¼ 2.1 Hz, 2H), 2.81

(m, 1H), 2.40 (m, 1H), 2.36 (m, 1H), 1.99 (m, 2H), 1.97 (m, 2H), 1.94

(m, 2H), 1.93 (m, 2H), 1.88 (m, 2H), 1.86 (m, 2H), 1.75 (m, 2H), 1.73

(m, 2H), 1.69 (m, 2H), 1.66 (m, 2H), 1.63 (m, 1H), 1.62 (m, 1H), 1.11 (s,

3H), 0.90 (d, J ¼ 1.8 Hz, 3H), 0.87 (d, J ¼ 1.8 Hz, 3H), 0.86 (d,

d

/ppm ¼ 176.38, 175.74, 174.65, 173.33, 172.56, 172.09, 171.17, 170.43,

169.12, 157.93, 155.43, 138.97, 137.55, 130.66, 126.49, 113.25, 115.46,

82.35, 55.47, 53.15, 51.69, 49.60, 48.00, 43.72, 43.08, 38.82, 38.18,

36.71, 33.34, 31.41, 30.35, 29.88, 29.25, 27.12, 26.45, 23.19, 12.39.

Anal. Calcd for C₅₂H₇₈N₁₄O₁₉: C 51.91, H 6.53, N 16.30; Found C

51.74, H 6.38, N 15.77.

4.4.6. Estradiol-17b-O-carbonylpropionyl-Arg-Gly-Asp-Val-Arg-

Gly-Asp-Val (35)

Using the same procedure as described for 7, from 300 mg

(0.189 mmol) of 29 150 mg (64.6%) of the title compound was

J ¼ 1.8 Hz, 3H), 0.85 (d, J ¼ 1.8 Hz, 3H). ¹³C NMR (DMSO-d₆)

d/

ppm ¼ 172.62, 172.44, 171.98, 171.48, 171.22, 171.03, 170.58, 170.50,

170.24, 169.44, 169.12, 159.76, 157.09, 155.43, 137.56, 136.48, 136.29,

128.84, 128.55, 128.49, 128.42, 128.39, 128.33, 128.29, 126.50,

113.24, 115.43, 82.42, 67.50, 66.42, 58.31, 58.14, 49.58, 47.96, 43.72,

43.08, 38.79, 36.94, 36.70, 33.82, 30.85, 30.39, 30.27, 29.70, 25.57,

27.32, 25.60, 24.92, 23.26, 19.68, 19.34, 18.57, 18.40, 12.37.

20

obtained as colorless powder. Mp 213–216 ^ꢀC, [

a]

D

¼ ꢁ24.1 (c ¼ 1.55,

DMSO), ESI-MS (m/z): 1227 [M þ H]^þ, 1111 [M ꢁ (Val) þ H]^þ, 996

[M ꢁ (Asp-Val) þ H]^þ, 939 [M ꢁ (Gly-Asp-Val) þ H]^þ, 783 [M ꢁ (Arg-

Gly-Asp-Val) þ H]^þ, 684 [M ꢁ (Val-Arg-Gly-Asp-Val) þ H]^þ, 512

[M ꢁ (Gly-Asp-Val-Arg-Gly-Asp-Val) þ H]^þ. ¹H NMR (DMSO-d₆)

d/

ppm ¼ 10.17 (s, 1H), 9.90 (s, 2H), 8.99 (s, 1H), 8.62 (d, J ¼ 5.4 Hz, 2H),

8.47 (t, J ¼ 7.2 Hz, 2H), 8.33 (d, J ¼ 5.1 Hz, 2H), 8.14 (d, J ¼ 4.5 Hz, 2H),

8.04 (d, J ¼ 4.5 Hz, 2H), 8.02 (s, 1H), 7.98 (s, 1H), 7.34 (s, 2H), 7.17 (d,

J ¼ 8.1 Hz,1H), 7.13 (s, 2H), 6.79(d, J ¼ 8.7 Hz,1H), 6.65(s,1H), 4.62(m,

2H), 4.59(m,1H), 4.39(m,1H), 4.30(m,1H), 4.23(m,1H), 4.22(m,1H),

4.19 (m, 1H), 4.10 (m, 2H), 3.88 (d, J ¼ 3.6 Hz, 2H), 3.56 (d, J ¼ 3.3 Hz,

2H), 3.39 (t, J ¼ 3.6 Hz, 1H), 3.14 (d, J ¼ 2.1 Hz, 2H), 2.98 (d, J ¼ 2.1 Hz,

2H), 2.86 (t, J ¼ 2.1 Hz, 2H), 2.70 (m, 1H), 2.61 (t, J ¼ 2.1 Hz, 2H), 2.57

(m, J ¼ 2.1 Hz, 2H), 2.53 (m, 1H), 2.23 (m, 2H), 2.10 (m, 2H), 2.08 (m,

2H),1.95 (m, 2H),1.84 (m,1H),1.76 (m, 2H),1.73 (m, 2H),1.65 (m, 2H),

1.49 (m, 2H),1.49 (m, 2H),1.39 (m, 2H),1.31 (m,1H),1.29 (m,1H),1.09

(s, 3H), 0.84 (d, J ¼ 1.8 Hz, 3H), 0.83 (d, J ¼ 1.8 Hz, 3H), 0.82 (d,

4.4.4. Estradiol-17-O-b-carbonylpropionyl-Arg(NO₂)-Gly-

Asp(OBzl)-Phe-Arg(NO₂)-Gly-Asp(OBzl)-Phe-OBzl (30)

Using the same procedure as described for Boc-Arg(NO₂)-Gly-

OCH₃, from 0.20 g (0.54 mmol) of 24, 0.70 g (0.51 mmol) of 7, 0.07 g

(0.59 mmol) of HOBt and 0.12 g (0.59 mmol) of DCC 0.66 g (76.5%)

of the title compound was obtained as colorless powder. Mp 161–

20

163 ^ꢀC, ESI-MS (m/z) 1685 [M þ H]^þ, [

a]

¼ 3.5 (c ¼ 1, DMSO). ¹H

D

NMR (DMSO-d₆)

d

/ppm ¼ 8.99 (s, 1H), 8.63 (d, J ¼ 5.1 Hz, 2H), 8.53

(t, J ¼ 7.2 Hz, 2H), 8.43 (d, J ¼ 4.8 Hz, 2H), 8.21 (d, J ¼ 5.1 Hz, 2H), 8.13

(d, J ¼ 4.5 Hz, 2H), 7.40 (s, 2H), 7.38 (s, 2H), 7.19–7.35 (m, 25H), 7.07

(d, J ¼ 8.7 Hz, 1H), 6.67 (d, J ¼ 8.7 Hz, 1H), 6.65 (s, 1H), 5.08 (s, 2H),

5.07 (s, 2H), 5.04 (m, 1H), 5.03 (m, 1H), 4.78 (s, 2H), 4.51 (m, 2H),

4.50 (m, 2H), 3.74 (d, J ¼ 2.1 Hz, 2H), 3.73 (d, J ¼ 2.1 Hz, 2H), 3.21

(d, J ¼ 3.3 Hz, 1H), 3.13 (d, J ¼ 2.4 Hz, 2H), 2.86 (t, J ¼ 2.1 Hz, 2H),

2.85 (t, J ¼ 2.1 Hz, 2H), 2.78 (m, 1H), 2.78 (m, 2H), 2.57 (m, 2H), 2.56

(m, 2H), 1.98 (m, 2H), 1.97 (m, 2H), 1.94 (m, 2H), 1.89 (m, 2H), 1.81

(m, 2H), 1.85 (m, 2H), 1.87 (m, 2H), 1.78 (m, 2H), 1.73 (m, 2H), 1.69

(m, 2H), 1.63 (m, 1H), 1.61 (m, 1H), 1.49 (m, 2H), 1.43 (m, 2H), 0.90 (s,

J ¼ 1.8 Hz, 3H), 0.78 (d, J ¼ 1.8 Hz, 3H). ¹³C NMR (DMSO-d₆)

d/

ppm ¼ 176.12, 175.67, 174.27, 173.00, 172.78, 172.57, 171.39, 170.69,

169.02, 158.01, 155.42, 137.55, 130.64, 126.49, 113.25, 115.43, 82.34,

58.69, 57.61, 50.38, 49.60, 43.72, 43.08, 38.79, 38.19, 36.93, 31.12,

30.51, 30.25, 29.74, 29.55, 27.32, 26.35, 23.28,19.71,18.52,15.35. Anal.

Calcd for C₅₆H₈₆N₁₄O₁₇: C 54.80, H 7.06, N 15.98; Found C 54.61, H 7.19,

N 15.79.

3H). ¹³C NMR (DMSO-d₆)

d

/ppm ¼ 171.96, 171.41, 171.21, 170.81,

170.52, 170.44, 170.21, 169.93, 169.54, 168.95, 159.76, 157.12, 155.42

138.00, 137.56, 137.32, 136.47, 136.11, 130.66, 129.71, 129.56, 128.84,

128.81, 128.74, 128.48, 128.42, 128.33, 127.05, 126.51, 113.24, 115.42,

82.41, 67.50, 66.53, 54.47, 54.36, 49.57, 49.49, 48.00, 43.71, 43.08,

38.78, 37.01, 36.94, 36.80, 33.82, 29.63, 29.56, 27.60, 27.31, 25.60,

24.92, 23.26, 12.37.

4.4.7. Estradiol-17b-O-carbonylpropionyl-Arg-Gly-Asp-Phe-Arg-

Gly-Asp-Phe (36)

Using the same procedure as described for 7, from 300 mg

(0.178 mmol)of30120 mg(51.1%)ofthetitlecompoundwasobtained

20

as colorless powder. Mp 222–224 ^ꢀC, [

a]

D

¼ ꢁ25.8 (c ¼ 1.85, DMSO),

ESI-MS (m/z) 1323 [M þ H]^þ,1159 [M ꢁ (Phe) þ H]^þ, 1044 [M ꢁ (Asp-

Phe) þ H]^þ, 987 [M ꢁ (Gly-Asp-Phe) þ H]^þ, 831 [M ꢁ (Arg-Gly-Asp-

Phe) þ H]^þ, 684 [M ꢁ (Phe-Arg-Gly-Asp-Phe) þ H]^þ, 569 [M ꢁ (Asp-

Phe-Arg-Gly-Asp-Phe) þ H]^þ, 512 [M ꢁ (Gly-Asp-Phe-Arg-Gly-Asp-

4.4.5. Estradiol-17b-O-carbonylpropionyl-Arg-Gly-Asp-Ser-Arg-

Gly-Asp-Ser (34)

Using the same procedure as described for 7, from 300 mg

(0.172 mmol) of 28 110 mg (53.5%) of the title compound was

Phe) þ H]^þ.¹H NMR (DMSO-d₆)

/ppm ¼ 9.80(s,1H), 9.40(s, 2H), 8.73

d

(s, 1H), 8.51 (d, J ¼ 5.4 Hz, 2H), 8.32 (t, J ¼ 6.8 Hz, 2H), 8.21 (d,

1702

J. Liu et al. / European Journal of Medicinal Chemistry 44 (2009) 1689–1704

J ¼ 4.8 Hz, 2H), 8.14 (d, J ¼ 5.4 Hz, 2H), 8.11 (d, J ¼ 4.5 Hz, 2H), 7.94

(s, 1H), 7.46 (s, 1H), 7.34 (s, 2H), 7.34 (s, 2H), 7.04–7.19 (m, 10H), 7.13

(d, J ¼ 8.7 Hz, 1H), 6.49 (d, J ¼ 8.7 Hz, 1H), 6.44 (s, 1H), 4.61 (m, 1H),

4.59(m,1H),4.56(m,1H), 4.37(m,1H), 4.36(m, 2H), 4.10(d, J ¼ 3.6 Hz,

2H), 3.87 (d, J ¼ 3.3 Hz, 2H), 3.83 (d, J ¼ 2.1 Hz, 2H), 3.56 (d, J ¼ 2.1 Hz,

2H), 3.52 (t, J ¼ 4.5 Hz, 1H), 3.22 (d, J ¼ 3.3 Hz, 2H), 3.16 (d, J ¼ 3.6 Hz,

2H), 3.07 (t, J ¼ 2.1 Hz, 2H), 2.92 (m, 1H), 2.89 (m, 2H), 2.46 (t,

J ¼ 2.1 Hz, 2H), 2.34 (t, J ¼ 2.1 Hz, 2H), 2.08 (m, 2H), 1.90 (m, 2H), 1.88

(m, 2H), 1.80 (m, 2H), 1.73 (m, 2H), 1.65 (m, 2H),1.62 (m, 2H), 1.51 (m,

2H),1.49 (m, 2H),1.36 (m, 2H),1.35 (m,1H),1.31 (m,1H),1.28 (m, 2H),

(0.61 mmol) of HOBt and 0.13 g (0.63 mmol) of DCC 0.52 g (88.3%)

of the title compound was obtained as colorless powder. ESI-MS

20

(m/z) 1031 [M þ H]^þ. [

a

]

¼ 51.0 (c ¼ 1.0, CH₃OH). ¹H NMR (DMSO-

D

d₆)

d

/ppm ¼ 8.38 (s, 1H), 8.22 (s, 1H), 8.08 (s, 1H), 8.00 (s, 1H), 7.34

(t, J ¼ 7.8 Hz, 1H), 7.39 (t, J ¼ 7.8 Hz, 1H), 7.39 (t, J ¼ 7.8 Hz, 1H), 7.33

(t, J ¼ 7.8 Hz, 1H), 7.30 (t, J ¼ 7.8 Hz, 2H), 7.29 (t, J ¼ 7.8 Hz, 2H), 7.25

(t, J ¼ 7.8 Hz, 2H), 7.20 (d, J ¼ 7.8 Hz, 2H), 7.18 (d, J ¼ 7.8 Hz, 2H), 7.17

(d, J ¼ 7.8 Hz, 2H), 7.12 (d, J ¼ 7.5 Hz, 1H), 7.03 (s, 1H), 6.70 (s, 1H),

6.52 (d, J ¼ 7.5 Hz, 1H), 6.41 (s, 1H), 6.37 (s, 1H), 5.16 (s, 2H), 5.14

(s, 2H), 5.01 (s, 2H), 4.90 (dt, J ¼ 6.3 Hz, J ¼ 6.6 Hz, 1H), 4.70

(t, J ¼ 6.5 Hz, 1H), 4.69 (t, J ¼ 6.4 Hz, 1H), 4.60 (t, J ¼ 4.7 Hz, 1H), 4.00

(s, 2H), 3.98 (d, J ¼ 6.0 Hz, 2H), 2.96 (t, J ¼ 4.5 Hz, 2H), 2.91

(m, J ¼ 4.2 Hz, 1H), 2.78 (t, J ¼ 5.1 Hz, 2H), 2.63 (t, J ¼ 6.5 Hz, 2H),

2.56 (s, 1H), 2.02 (m, J ¼ 4.7 Hz, 2H), 2.01 (m, J ¼ 4.3 Hz, 1H), 1.86

(m, J ¼ 4.2 Hz, 1H), 1.83 (m, J ¼ 4.4 Hz, 2H), 1.75 (m, J ¼ 4.5 Hz, 2H),

1.72 (m, J ¼ 6.3 Hz, 2H), 1.70 (m, J ¼ 4.2 Hz, 1H), 1.65 (m, J ¼ 4.7 Hz,

2H), 1.62 (m, J ¼ 6.3 Hz, 2H), 0.78 (s, 3H).

0.76 (s, 3H). ¹³C NMR (DMSO-d₆)

d/ppm ¼ 175.81, 174.97, 174.19,

173.68, 173.01, 172.58, 171.42, 170.89, 170.61, 169.87, 169.04, 157.93,

155.43, 138.97, 138.66, 137.55, 130.66, 129.84, 129.52, 128.48, 128.17,

126.49, 113.25, 115.46, 82.35, 55.47, 53.65, 52.70, 49.60, 48.00, 43.72,

43.08,38.80,38.21, 37.80,36.93,34.78,32.15,30.27, 29.74,27.61, 26.34,

25.16, 23.28, 12.37. Anal. Calcd for C₆₄H₈₆N₁₄O₁₇: C 58.08, H 6.55, N

14.82; Found C 57.88, H 6.41, N 15.01.

4.5. Preparing N-(estradiol-3-oxyacetyl)-RGD tetrapeptide

4.5.4. N-(Estradiol-3-oxyacetyl)-Arg-Gly-Asp-Ser (18)

conjugates

Using the same procedure as described for 7, from 0.29 g

(0.27 mmol) of 12 0.13 g (63.0%) of the title compound was

4.5.1. N-(Estradiol-3-oxyacetyl)-Arg(NO₂)-Gly-Asp(OBzl)-Ser(Bzl)-

OBzl (12)

Using the same procedure as described for Boc-Arg(NO₂)-Gly-

OCH₃, from 0.17 g (0.52 mmol) of 11, 0.40 g (0.50 mmol) of 1, 84 mg

(0.60 mmol) of HOBt and 123 mg (0.60 mmol) of DCC 0.47 g (89%) of

obtained as colorless powder. ESI-MS (m/z): 745 [M þ H]^þ.

20

[a

]

¼ 39.0 (c ¼ 0.50, CH₃OH). ¹H NMR (DMSO-d₆)

/ppm ¼ 9.07

d

D

(s, 1H), 9.07 (s, 1H), 8.02 (s, 1H), 8.019 (s, 1H), 8.01 (s, 1H), 8.00 (s,

2H), 7.24 (s, 2H), 7.04 (d, J ¼ 7.5 Hz, 1H), 7.03 (s, 1H), 6.75 (s, 1H),

6.51 (d, J ¼ 7.5 Hz, 1H), 6.40 (s, 1H), 5.00 (s, 2H), 4.85 (dt,

J ¼ 6.3 Hz, J ¼ 6.6 Hz, 1H), 4.67 (t, J ¼ 6.5 Hz, 1H), 4.60 (t, J ¼ 4.7 Hz,

1H), 4.57 (t, J ¼ 6.4 Hz, 1H), 4.01 (d, J ¼ 6.4 Hz, 2H), 3.97 (d,

J ¼ 6.0 Hz, 2H), 2.96 (t, J ¼ 4.5 Hz, 2H), 2.90 (m, J ¼ 4.2 Hz, 1H), 2.87

(t, J ¼ 5.1 Hz, 2H), 2.80 (d, J ¼ 6.6 Hz, 2H), 1.83 (m, J ¼ 4.4 Hz, 2H),

1.76 (m, J ¼ 4.5 Hz, 2H), 1.72 (m, J ¼ 6.3 Hz, 2H), 1.70 (m, J ¼ 4.2 Hz,

1H), 1.65 (m, J ¼ 4.7 Hz, 2H), 1.62 (m, J ¼ 6.3 Hz, 2H), 1.60 (m,

J ¼ 4.5 Hz, 2H), 1.57 (m, J ¼ 4.5 Hz, 2H), 1.54 (m, J ¼ 4.5 Hz, 2H),

0.73 (s, 3H).

the title compound was obtained as colorless powder. ESI-MS (m/z)

20

1061 [M þ H]^þ. [

a

]

¼ 55.0 (c ¼ 0.5, CH₃OH). ¹H NMR (DMSO-d₆)

d/

D

ppm ¼ 8.38 (s, 1H), 8.20 (s, 1H), 8.08 (s, 1H), 8.00 (s, 1H), 7.34 (t,

J ¼ 7.8 Hz, 1H), 7.342 (t, J ¼ 7.8 Hz, 1H), 7.33 (t, J ¼ 7.8 Hz, 1H), 7.30 (t,

J ¼ 7.8 Hz, 2H), 7.29 (t, J ¼ 7.8 Hz, 2H), 7.25 (t, J ¼ 7.8 Hz, 2H), 7.24 (d,

J ¼ 7.8 Hz, 2H), 7.18 (d, J ¼ 7.8 Hz, 2H), 7.17 (d, J ¼ 7.8 Hz, 2H), 7.12 (d,

J ¼ 7.5 Hz,1H), 7.03 (s,1H), 6.70 (s,1H), 6.52 (d, J ¼ 7.5 Hz,1H), 6.41 (s,

1H), 6.374 (s,1H), 5.16 (s, 2H), 5.14 (s, 2H), 5.13 (s, 2H), 5.00 (s, 2H), 4.90

(dt, J ¼ 6.3 Hz, J ¼ 6.6 Hz,1H), 4.71 (t, J ¼ 6.5 Hz,1H), 4.70 (t, J ¼ 6.4 Hz,

1H), 4.61 (t, J ¼ 4.7 Hz, 1H), 3.96 (d, J ¼ 6.0 Hz, 2H), 3.76 (d, J ¼ 4.1 Hz,

2H), 2.97 (t, J ¼ 4.5 Hz, 2H), 2.91 (m, J ¼ 4.2 Hz, 1H), 2.78 (t, J ¼ 5.1 Hz,

2H), 2.62 (t, J ¼ 6.5 Hz, 2H), 2.55 (s, 1H), 2.02 (m, J ¼ 4.7 Hz, 2H), 2.01

(m, J ¼ 4.3 Hz,2H),1.86 (m, J ¼ 4.2 Hz,1H),1.84(m, J ¼ 4.4 Hz,2H),1.75

(m, J ¼ 4.5 Hz, 2H),1.72(m, J ¼ 6.3 Hz, 2H),1.70(m, J ¼ 4.2 Hz,1H),1.65

(m, J ¼ 4.7 Hz, 2H), 1.62 (m, J ¼ 6.3 Hz, 2H), 0.78 (s, 3H).

4.5.5. N-(Estradiol-3-oxyacetyl)-Arg-Gly-Asp-Val (19)

Using the same procedure as described for 7, from 0.22 g

(0.22 mmol) of 13 0.15 g (91%) of the title compound was obtained

20

as colorless powder. ESI-MS (m/z) 758 [M þ H]^þ. [

a

]

¼ 45.0

D

(c ¼ 0.50, CH₃OH). ¹H NMR (DMSO-d₆)

/ppm ¼ 9.10 (s, 1H), 9.08 (s,

d

1H), 8.02 (s, 1H), 8.02 (s, 1H), 8.01 (s, 1H), 8.00 (s, 2H), 7.24 (s, 2H),

7.04 (d, J ¼ 7.5 Hz, 1H), 7.03 (s, 1H), 6.75 (s, 1H), 6.51 (d, J ¼ 7.5 Hz,

1H), 6.40 (s,1H), 5.00 (s, 2H), 4.85 (dt, J ¼ 6.3 Hz, J ¼ 6.6 Hz,1H), 4.67

(t, J ¼ 6.5 Hz, 1H), 4.57 (t, J ¼ 6.4 Hz, 1H), 4.60 (t, J ¼ 4.7 Hz, 1H), 3.97

(d, J ¼ 6.0 Hz, 2H), 2.96 (t, J ¼ 4.5 Hz, 2H), 2.90 (m, J ¼ 4.2 Hz, 1H),

2.87 (t, J ¼ 5.1 Hz, 2H), 2.80 (d, J ¼ 6.6 Hz, 2H), 1.83 (m, J ¼ 4.4 Hz,

2H), 1.76 (m, J ¼ 4.5 Hz, 2H), 1.72 (m, J ¼ 6.3 Hz, 2H), 1.70 (m,

J ¼ 4.2 Hz, 1H), 1.65 (m, J ¼ 4.7 Hz, 2H), 1.62 (m, J ¼ 6.3 Hz, 2H), 1.60

(m, J ¼ 4.5 Hz, 2H), 1.57 (m, J ¼ 4.5 Hz, 2H), 1.54 (m, J ¼ 4.5 Hz, 2H),

0.93 (d, J ¼ 4.2 Hz, 6H), 0.74 (s, 3H).

4.5.2. N-(Estradiol-3-oxyacetyl)-Arg(NO₂)-Gly-Asp(OBzl)-Val-OBzl

(13)

Using the same procedure as described for Boc-Arg(NO₂)-Gly-

OCH₃, from 0.20 g (0.60 mmol) of 11, 0.40 g (0.60 mmol) of 2, 0.10 g

(0.70 mmol) of HOBt and 0.14 g (0.68 mmol) of DCC 0.55 g (88.7%) of

the title compound was obtained as colorless powder. ESI-MS (m/z)

20

1

983 [M þ H]^þ. [

a]

¼ 60.0 (c ¼ 1.00, MeOH). H NMR (DMSO-d₆)

d/

D

ppm ¼ 8.38 (s, 1H), 8.22 (s, 1H), 8.08 (s, 1H), 8.00 (s, 1H), 7.34 (t,

J ¼ 7.8 Hz, 1H), 7.33 (t, J ¼ 7.8 Hz, 1H), 7.30 (t, J ¼ 7.8 Hz, 2H), 7.29 (t,

J ¼ 7.8 Hz, 2H), 7.24 (d, J ¼ 7.8 Hz, 2H), 7.18 (d, J ¼ 7.8 Hz, 2H), 7.12 (d,

J ¼ 7.5 Hz,1H), 7.03 (s,1H), 6.71 (s, 1H), 6.520 (d, J ¼ 7.5 Hz,1H), 6.41 (s,

1H), 6.37 (s,1H), 5.16 (s, 2H), 5.14 (s, 2H), 5.00 (s, 2H), 4.90 (dt, J ¼ 6.3 Hz,

J ¼ 6.6 Hz, 1H), 4.71 (t, J ¼ 6.5 Hz, 1H), 4.70 (t, J ¼ 6.4 Hz, 1H), 4.61 (t,

J ¼ 4.7 Hz, 1H), 3.96 (d, J ¼ 6.0 Hz, 2H), 3.26 (m, J ¼ 4.1 Hz, 1H), 2.96 (t,

J ¼ 4.5 Hz, 2H), 2.91 (m, J ¼ 4.2 Hz, 1H), 2.79 (t, J ¼ 5.1 Hz, 2H), 2.63 (t,

J ¼ 6.5 Hz, 2H), 2.55 (s,1H), 2.02 (m, J ¼ 4.7 Hz, 2H), 2.01 (m, J ¼ 4.3 Hz,

2H),1.86 (m, J ¼ 4.2 Hz,1H),1.83 (m, J ¼ 4.4 Hz, 2H),1.75 (m, J ¼ 4.5 Hz,

2H),1.72 (m, J ¼ 6.3 Hz, 2H),1.70 (m, J ¼ 4.2 Hz,1H),1.65 (m, J ¼ 4.7 Hz,

2H), 1.62 (m, J ¼ 6.3 Hz, 2H), 0.81 (s, 3H), 0.78 (d, J ¼ 4.1 Hz, 6H).

4.5.6. N-(Estradiol-3-oxyacetyl)-Arg-Gly-Asp-Phe (20)

Using the same procedure as described for 7, from 0.35 g

(0.22 mmol) of 14 0.13 g (47.6%) of the title compound was obtained

20

as colorless powder. ESI-MS (m/z): 805 [M þ H]^þ. [

a]

¼ 48.0

D

(c ¼ 0.50, CH₃OH). ¹H NMR (DMSO-d₆)

/ppm ¼ 9.07 (s, 1H), 9.06 (s,

d

1H), 8.03 (s, 1H), 8.01 (s, 1H), 8.00 (s, 1H), 7.98 (s, 2H), 7.27 (t,

J ¼ 7.8 Hz, 2H), 7.21 (s, 1H), 7.12 (d, J ¼ 7.8 Hz, 2H), 7.07 (d, J ¼ 7.8 Hz,

1H), 7.06 (d, J ¼ 7.5 Hz, 1H), 7.02 (s, 1H), 6.74 (s, 1H), 6.66 (d,

J ¼ 7.5 Hz, 1H), 6.64 (s, 1H), 4.99 (s, 2H), 4.84 (dt, J ¼ 6.3 Hz,

J ¼ 6.6 Hz, 1H), 4.67 (t, J ¼ 6.5 Hz, 1H), 4.59 (t, J ¼ 4.7 Hz, 1H), 4.57 (t,

J ¼ 6.4 Hz, 1H), 3.96 (d, J ¼ 6.0 Hz, 2H), 3.22 (t, J ¼ 4.7 Hz, 2H), 2.96

(t, J ¼ 4.5 Hz, 2H), 2.90 (m, J ¼ 4.2 Hz, 1H), 2.87 (t, J ¼ 5.1 Hz, 2H),

2.80 (d, J ¼ 6.5 Hz, 2H), 1.84 (m, J ¼ 4.4 Hz, 2H), 1.76 (m, J ¼ 4.5 Hz,

2H), 1.72 (m, J ¼ 6.3 Hz, 2H), 1.67 (m, J ¼ 4.7 Hz, 2H), 1.62 (m,

J ¼ 6.3 Hz, 2H), 1.60 (m, J ¼ 4.5 Hz, 2H), 1.58 (m, J ¼ 4.5 Hz, 1H), 1.57

4.5.3. N-(Estradiol-3-oxyacetyl)-Arg(NO₂)-Gly-Asp(OBzl)-Phe-OBzl

(14)

Using the same procedure as described for Boc-Arg(NO₂)-Gly-

OCH₃, from 0.18 g (0.56 mmol) of 11, 0.40 g (0.53 mmol) of 3, 0.09 g

J. Liu et al. / European Journal of Medicinal Chemistry 44 (2009) 1689–1704

1703

(m, J ¼ 4.5 Hz, 2H), 1.54 (m, J ¼ 4.5 Hz, 2H), 1.46 (m, J ¼ 4.5 Hz, 1H),

J ¼ 6.5 Hz, 2H), 2.02 (m, J ¼ 4.7 Hz, 2H), 2.01 (m, J ¼ 4.3 Hz, 2H), 1.86

(m, J ¼ 4.2 Hz, 1H), 1.84 (m, J ¼ 4.4 Hz, 2H), 1.76 (m, J ¼ 4.5 Hz, 2H),

1.72 (m, J ¼ 6.3 Hz, 2H), 1.70 (m, J ¼ 4.2 Hz, 1H), 1.65 (m, J ¼ 4.7 Hz,

2H), 1.61 (m, J ¼ 6.3 Hz, 2H), 0.82 (s, 3H).

0.75 (s, 3H).

4.6. Preparing estradiol-17

conjugates

b-O-carbonylpropinyl-RGD tetrapeptide

4.6.4. Estradiol-17b-O-carbonylpropionyl-Arg-Gly-Asp-Ser (31)

4.6.1. Estradiol-17

b

-O-carbonylpropionyl-Arg(NO₂)-Gly-Asp(OBzl)-

Using the same procedure as described for 7, from 240 mg

Ser(Bzl)-OBzl (25)

(0.172 mmol) of 25 150 mg (79.2%) of the title compound was

20

Using the same procedure as described for Boc-Arg(NO₂)-Gly-

OCH₃from 0.20 g (0.53 mmol) of 24, 0.40 g (0.51 mmol) of 1, 0.08 g

(0.58 mmol) of HOBt and 0.12 g (0.58 mmol) of DCC 0.47 g (80.3%) of

obtainedascolorlesspowder. ESI-MS(m/z)788 [M þ H]^þ. [

a]

D

¼ 50.0

(c ¼ 1.00, CH₃OH). ¹H NMR (DMSO-d₆)

/ppm ¼ 9.07 (s, 1H), 9.06 (s,

d

1H), 9.06 (s,1H), 8.04 (s,1H), 8.03 (s,1H), 8.01 (s,1H), 8.00 (s, 2H), 7.24

(s, 2H), 7.04 (d, J ¼ 7.5 Hz, 1H), 7.03 (s, 1H), 6.75 (s, 1H), 6.51 (d,

J ¼ 7.5 Hz,1H), 6.40 (s, 1H), 4.85 (dt, J ¼ 6.3 Hz, J ¼ 6.6 Hz, 1H), 4.68 (t,

J ¼ 6.5 Hz, 1H), 4.60 (t, J ¼ 4.7 Hz, 1H), 4.59 (t, J ¼ 6.4 Hz, 1H), 4.02 (d,

J ¼ 6.4 Hz, 2H), 3.96 (d, J ¼ 6.0 Hz, 2H), 2.97 (t, J ¼ 4.5 Hz, 2H), 2.91 (m,

J ¼ 4.2 Hz, 1H), 2.87 (t, J ¼ 5.1 Hz, 2H), 2.82 (d, J ¼ 6.6 Hz, 2H), 2.77 (t,

J ¼ 5.1 Hz, 2H), 2.60(t, J ¼ 6.5 Hz, 2H), 2.02(m, J ¼ 4.7 Hz, 2H), 2.01 (m,

J ¼ 4.3 Hz, 2H),1.87 (m, J ¼ 4.2 Hz,1H),1.84(m, J ¼ 4.4 Hz, 2H),1.76 (m,

J ¼ 4.5 Hz, 2H),1.72 (m, J ¼ 6.3 Hz, 2H),1.70 (m, J ¼ 4.2 Hz,1H),1.65 (m,

J ¼ 4.7 Hz, 2H), 1.62 (m, J ¼ 6.3 Hz, 2H), 0.77 (s, 3H).

the title compound was obtained as colorless powder. ESI-MS (m/z)

20

1103 [M þ H]^þ. [

a

]

¼ 20.0 (c ¼ 1.00, CHCl₃:CH₃OH ¼ 10:1). ¹H NMR

D

(DMSO-d₆)

d

/ppm ¼ 8.94 (s, 1H), 8.38 (s, 1H), 8.22 (s, 1H), 8.08 (s,

1H), 8.00 (s,1H), 7.34 (t, J ¼ 7.8 Hz,1H), 7.34 (t, J ¼ 7.9 Hz,1H), 7.33 (t,

J ¼ 7.8 Hz, 1H), 7.30 (t, J ¼ 7.9 Hz, 2H), 7.29 (t, J ¼ 7.9 Hz, 2H), 7.25 (t,

J ¼ 7.8 Hz, 2H), 7.24 (d, J ¼ 7.8 Hz, 2H), 7.18 (d, J ¼ 7.8 Hz, 2H), 7.17 (d,

J ¼ 7.8 Hz, 2H), 7.04 (d, J ¼ 7.5 Hz, 1H), 7.03 (s, 1H), 6.74 (s, 1H), 6.51

(d, J ¼ 7.5 Hz, 1H), 6.40 (s, 1H), 6.37 (s, 1H), 5.16 (s, 2H), 5.14 (s, 2H),

5.13 (s, 2H), 4.90 (dt, J ¼ 6.3 Hz, J ¼ 6.6 Hz, 1H), 4.71 (t, J ¼ 6.5 Hz,

1H), 4.70 (t, J ¼ 6.4 Hz,1H), 4.61 (t, J ¼ 4.7 Hz,1H), 3.96 (d, J ¼ 6.0 Hz,

2H), 3.76 (d, J ¼ 4.1 Hz, 2H), 2.97 (t, J ¼ 4.5 Hz, 2H), 2.91 (m,

J ¼ 4.2 Hz,1H), 2.88 (t, J ¼ 5.1 Hz, 2H), 2.82 (d, J ¼ 6.6 Hz, 2H), 2.78 (t,

J ¼ 5.1 Hz, 2H), 2.62 (t, J ¼ 6.5 Hz, 2H), 2.02 (m, J ¼ 4.7 Hz, 2H), 2.01

(m, J ¼ 4.3 Hz, 2H), 1.87 (m, J ¼ 4.2 Hz, 1H), 1.84 (m, J ¼ 4.4 Hz, 2H),

1.76 (m, J ¼ 4.5 Hz, 2H), 1.72 (m, J ¼ 6.3 Hz, 2H), 1.70 (m, J ¼ 4.2 Hz,

1H), 1.65 (m, J ¼ 4.7 Hz, 2H), 1.62 (m, J ¼ 6.3 Hz, 2H), 0.77 (s, 3H).

4.6.5. Estradiol-17b-O-carbonylpropionyl-Arg-Gly-Asp-Val (32)

Using the same procedure as described for 7, from 260 mg

(0.26 mmol) of 26170 mg (84.0%) of the title compound was obtained

as colorless powder. ESI-MS (m/z) 800 [M þ H]^þ. [

a]

D

¼ 56.0 (c ¼ 0.5,

20

CH₃OH).¹H NMR (DMSO-d₆)

d

/ppm ¼ 9.15 (s,1H), 9.12 (s,1H), 9.03 (s,

1H), 8.22 (s,1H), 8.20 (s,1H), 8.07 (s,1H), 8.00 (s,1H), 7.22 (s, 2H), 7.04

(d, J ¼ 7.5 Hz, 1H), 7.03 (s, 1H), 6.72 (s, 1H), 6.51 (s, 1H), 6.44 (d,

J ¼ 7.5 Hz, 1H), 4.86 (d, J ¼ 6.5 Hz, 1H), 4.61 (t, J ¼ 6.3 Hz, 1H), 4.53 (d,

J ¼ 6.5 Hz, 1H), 4.42 (d, J ¼ 6.4 Hz, 1H), 4.10 (d, J ¼ 4.7 Hz, 2H), 2.96 (t,

J ¼ 4.5 Hz, 2H), 2.92 (m, J ¼ 4.1 Hz,1H), 2.90 (m, J ¼ 4.2 Hz,1H), 2.88 (t,

J ¼ 5.1 Hz, 2H), 2.82 (d, J ¼ 6.5 Hz, 2H), 2.78 (t, J ¼ 5.1 Hz, 2H), 2.61 (t,

J ¼ 6.5 Hz, 2H), 2.02 (m, J ¼ 4.7 Hz, 2H), 2.01 (m, J ¼ 4.3 Hz, 2H), 1.86

(m, J ¼ 4.2 Hz,1H),1.84 (m, J ¼ 4.4 Hz, 2H),1.76 (m, J ¼ 4.5 Hz, 2H),1.72

(m, J ¼ 6.3 Hz, 2H),1.70 (m, J ¼ 4.2 Hz,1H),1.65 (m, J ¼ 4.7 Hz, 2H),1.61

(m, J ¼ 6.3 Hz, 2H), 0.86 (d, J ¼ 4.1 Hz, 6H), 0.75 (s, 3H).

4.6.2. Estradiol-17b-O-carbonylpropionyl-Arg(NO₂)-Gly-Asp(OBzl)-

Val-OBzl (26)

Using the same procedure as described for Boc-Arg(NO₂)-

Gly-OCH₃, from 0.22 g (0.59 mmol) of 24, 0.40 g (0.57 mmol)

of 2, 0.09 g (0.65 mmol) of HOBt and 0.14 g (0.68 mmol) of DCC 0.49 g

(79.3%) of the title compound was obtained as colorless powder. ESI-

20

MS (m/z) 1025 [M þ H]^þ. [

a]

¼ 30.0 (c ¼ 1.00, CHCl₃:CH₃OH ¼ 10:1).

D

¹H NMR (DMSO-d₆)

d

/ppm ¼ 8.94(s,1H), 8.38(s,1H), 8.22(s,1H), 8.08

(s,1H), 8.00(s,1H), 7.33 (t,J ¼ 7.8 Hz,1H), 7.33 (t, J ¼ 7.8 Hz,1H), 7.30(t,

J ¼ 7.8 Hz, 2H), 7.29 (t, J ¼ 7.8 Hz, 2H), 7.24 (d, J ¼ 7.8 Hz, 2H), 7.18 (d,

J ¼ 7.8 Hz, 2H), 7.04 (d, J ¼ 7.5 Hz,1H), 7.037 (s,1H), 6.74 (s,1H), 6.51 (d,

J ¼ 7.5 Hz, 1H), 6.40 (s, 1H), 6.37 (s, 1H), 5.12 (s, 2H), 5.13 (s, 2H), 5.00

(dt, J ¼ 6.3 Hz, J ¼ 6.6 Hz, 1H), 4.89 (d, J ¼ 6.5 Hz, 1H), 4.53 (d,

J ¼ 6.5 Hz,1H), 4.42 (d, J ¼ 6.4 Hz,1H), 4.10 (d, J ¼ 4.7 Hz, 2H), 3.07 (m,

J ¼ 4.1 Hz, 1H), 2.97 (t, J ¼ 4.5 Hz, 2H), 2.91 (m, J ¼ 4.2 Hz, 1H), 2.88 (t,

J ¼ 5.1 Hz, 2H), 2.82 (d, J ¼ 6.6 Hz, 2H), 2.78 (t, J ¼ 5.1 Hz, 2H), 2.62 (t,

J ¼ 6.5 Hz, 2H), 2.02 (m, J ¼ 4.7 Hz, 2H), 2.01 (m, J ¼ 4.3 Hz, 2H), 1.87

(m, J ¼ 4.2 Hz,1H),1.84 (m, J ¼ 4.4 Hz, 2H),1.76 (m, J ¼ 4.5 Hz, 2H),1.72

(m, J ¼ 6.3 Hz, 2H),1.70 (m, J ¼ 4.2 Hz,1H),1.65 (m, J ¼ 4.7 Hz,2H),1.62

(m, J ¼ 6.3 Hz, 2H), 1.01 (d, J ¼ 4.1 Hz, 6H), 0.77 (s, 3H).

4.6.6. Estradiol-17b-O-carbonylpropionyl-Arg-Gly-Asp-Phe (33)

Using the same procedure as described for 7, from 240 mg

(0.22 mmol) of 27 160 mg (86.4%) of the title compound was

obtained as colorless powder. ESI-MS (m/z) 848 [M þ H]^þ.

20

[a]

¼ 45.0 (c ¼ 0.5, CH₃OH). ¹H NMR (DMSO-d₆)

/ppm ¼ 9.06 (s,

d

D

1H), 9.05 (s,1H), 9.04 (s,1H), 8.06 (s,1H), 8.03 (s,1H), 8.02 (s,1H), 8.00

(s, 2H), 7.23 (s, 2H), 7.11 (t, J ¼ 7.8 Hz, 2H), 7.07 (d, J ¼ 7.8 Hz, 2H), 7.01

(t, J ¼ 7.7 Hz, 1H), 7.00 (s, 1H), 6.95 (d, J ¼ 7.5 Hz,1H), 6.90 (s,1H), 6.43

(d, J ¼ 7.5 Hz, 1H), 4.91 (dt, J ¼ 6.3 Hz, J ¼ 6.5 Hz, 1H), 4.87 (t,

J ¼ 6.5 Hz, 1H), 4.84 (t, J ¼ 6.5 Hz, 1H), 4.50 (t, J ¼ 6.4 Hz, 1H), 4.12 (d,

J ¼ 4.7 Hz, 2H), 3.04 (d, J ¼ 6.4 Hz, 2H), 2.96 (t, J ¼ 4.5 Hz, 2H), 2.91 (m,

J ¼ 4.2 Hz, 1H), 2.87 (t, J ¼ 5.1 Hz, 2H), 2.82 (d, J ¼ 6.5 Hz, 2H), 2.77 (t,

J ¼ 5.1 Hz, 2H), 2.59 (t, J ¼ 6.5 Hz, 2H), 2.02 (m, J ¼ 4.7 Hz, 2H), 2.01

(m, J ¼ 4.3 Hz, 2H), 1.86 (m, J ¼ 4.2 Hz, 1H), 1.84 (m, J ¼ 4.4 Hz, 2H),

1.75 (m, J ¼ 4.5 Hz, 2H), 1.72 (m, J ¼ 6.3 Hz, 2H), 1.70 (m, J ¼ 4.2 Hz,

1H), 1.656 (m, J ¼ 4.7 Hz, 2H), 1.61 (m, J ¼ 6.3 Hz, 2H), 0.82 (s, 3H).

4.6.3. Estradiol-17b-O-carbonylpropionyl-Arg(NO₂)-Gly-Asp(OBzl)-

Phe-OBzl (27)

Using the same procedure as described for Boc-Arg(NO₂)-Gly-

OCH₃, from 0.21 g (0.56 mmol) of 24, 0.40 g (0.53 mmol) of 3, 85 mg

(0.61 mmol) of HOBt and 0.125 mg (0.61 mmol) of DCC 0.51 g (84.9%)

of the title compound was obtained as colorless powder. ESI-MS (m/

4.7. Anti-osteoporosis assay in vivo

z) 1073 [M þ H]^þ. [

a]

D

¼ 23.0 (c ¼ 1.00, CHCl₃:CH₃OH ¼ 10:1). ¹H

20

NMR (DMSO-d₆)

d/ppm ¼ 8.93 (s,1H), 8.37 (s,1H), 8.22 (s,1H), 8.08 (s,

The assessments described herein were performed based on

a protocol reviewed and approved by the ethics committee of

Capital Medical University. The committee assures the welfare of

the animals was maintained in accordance to the requirements of

the animal welfare act and according to the guide for care and use of

laboratory animals. The tested compound was dissolved in aqueous

solution of 0.5% carboxymethylcellulose just before use and kept in

an ice bath. ICR mice weighing 30.7 ꢂ 3.1 g (7 weeks old, purchased

from Animal Center of Peking University) were anesthetized with

pentobarbital sodium (40.0 mg/kg, ip). The mice of OVX groups

1H), 8.00 (s, 1H), 7.34 (t, J ¼ 7.8 Hz, 1H), 7.33 (t, J ¼ 7.8 Hz, 1H), 7.33 (t,

J ¼ 7.8 Hz, 1H), 7.30 (t, J ¼ 7.8 Hz, 2H), 7.29 (t, J ¼ 7.8 Hz, 2H), 7.27 (t,

J ¼ 7.8 Hz, 2H), 7.24 (d, J ¼ 7.8 Hz, 2H), 7.18 (d, J ¼ 7.8 Hz, 2H), 7.17 (d,

J ¼ 7.8 Hz, 2H), 7.04 (d, J ¼ 7.5 Hz,1H), 7.03 (s,1H), 6.74 (s,1H), 6.50 (d,

J ¼ 7.5 Hz, 1H), 6.40 (s, 1H), 6.36 (s, 1H), 5.12 (s, 2H), 5.13 (s, 2H), 5.01

(dt, J ¼ 6.3 Hz, J ¼ 6.5 Hz, 1H), 4.88 (t, J ¼ 6.5 Hz, 1H), 4.53 (t,

J ¼ 6.5 Hz, 1H), 4.41 (t, J ¼ 6.4 Hz, 1H), 4.10 (d, J ¼ 4.7 Hz, 2H), 3.16 (d,

J ¼ 6.4 Hz, 2H), 2.96 (t, J ¼ 4.5 Hz, 2H), 2.90 (m, J ¼ 4.2 Hz,1H), 2.87 (t,

J ¼ 5.1 Hz, 2H), 2.82 (d, J ¼ 6.5 Hz, 2H), 2.78 (t, J ¼ 5.1 Hz, 2H), 2.61 (t,

1704

J. Liu et al. / European Journal of Medicinal Chemistry 44 (2009) 1689–1704

were given abdominal ovariotomy by standard procedure and the

mice of sham group were given abdominorotomy only. On the 5th

day of surgical operation the mice of ovariotomy and sham groups

were orally administrated 0.2 ml of aqueous solution of 0.5%

carboxymethylcellulose, the mice of treatment groups were orally

administrated the solution of 110.3 nmol/kg estradiol, the mixture

of 110.3 nmol/kg estradiol and RGD peptide, 110.3 nmol/kg of the

estradiol–RGD tetrapeptides or estradiol–RGD octapeptides in

0.2 ml aqueous solution of 0.5% carboxymethylcellulose once a day.

All of the mice were treated according to the corresponding

procedure for 4 weeks. On the next day of the last administration

the mice were weighed, drawn blood via eye orbit, anesthetized

with pentobarbital sodium (40.0 mg/kg, ip) and executed to remove

the lungs, livers, spleens, uteri and left femurs.

After 30 min standing the blood was centrifuged at 3000g for

20 min and the serum was stored at ꢁ20 ^ꢀC before use. The calcium

content of the serum was measured by the method of o-methyl-

phenolphthalein complexing ketone. The phosphorous content of

the serum was measured by the method of molybdenum blue. The

alkaline phosphatase content of the serum was measured by using

disodium phenylphosphate as the substrate.

The lungs, livers, spleens and uteri were weighed directly. After

completely removing the muscle the lengths of left femurs were

measured and then immersed in the solution of chloroform–

methanol (2:1) for two times (one time 3 h). After defatting the left

femurs were heated at 120 ^ꢀC for 6 h, cooled, and weighed to record

the dry weight. The femurs were incinerated in mufﬂe furnace at

800 ^ꢀC for 8 h, cooled, weighed and recorded the ash weight and

calculate the rate of the ash weight to dry femur weight (namely

the mineral content of the femur).

The ashes of the left femurs were dissolved in 0.5 ml of hydro-

chloric acid (6 N) and diluted to 5 ml with ultrapure water, from

which 0.05 ml of the solution was drawn and diluted to 1 ml with

ultrapure water before use. The calcium content of the aqueous

solution was measured by the method of o-methylphenolphthalein

complexing ketone. The phosphorous content of the aqueous

solution was measured by the method of molybdenum blue.

References

[1] M. Davidson, M.E. DeSimone, Confronting osteoporosis: what we know, where

we’re headed, Clin. Rev. 12 (2002) 76–82.

[2] S. Amin, Y. Zhang, D.T. Felson, C.T. Sawin, M.T. Hannan, P.W.F. Wilson, Estradiol,

testosterone, and the risk for hip fractures in elderly men from the Framing-

ham study, Am. J. Med. 119 (2006) 426–433.

[3] G. Rajzbaum, Y. Be´zie, Postmenopausal osteoporosis and atheroma, Joint Bone

Spine 73 (2006) 610–613.

[4] J.S. Michael, Selective estrogen receptor modulators: the ideal estrogen

replacement, Primary Care Update OB/GYNS. 8 (2001) 25–30.

[5] J.C. Stevenson, HRT and the primary prevention of cardiovascular disease,

Maturitas 57 (2007) 31–34.

[6] A. Gebbie, Hormone replacement therapy, Medicine 35 (2007) 529–532.

[7] P. Vestergaard, L. Rejnmark, L. Mosekilde, Fracture reducing potential of

hormone replacement therapy on a population level, Maturitas 54 (2006)

285–293.

[8] J. Corte´s-Prieto, P. Juez-Martel, Incidences of breast cancer throughout long-

term hormone replacement therapy, J. Steroid Biochem. Mol. Biol. 104 (2007)

180–189.

[9] K. Brixen, B. Abrahamsen, M. Kassem, Prevention and treatment of osteopo-

rosis in women, Curr. Obstet. Gynaecol. 15 (2005) 251–258.

[10] M. Rees, Unravelling the confusion about HRT in women, J. Mens Health Gend.

2 (2005) 287–291.

[11] J.A. Raza, R.A. Reinhart, A. Movahed, Ischemic heart disease in women and the

role of hormone therapy, Int. J. Cardiol. 96 (2004) 7–19.

[12] M. Sgarabotto, M. Baldini, C.A. Dei, C. Manotti, A.L. Barilli, M. Rinaldi, L. Benassi,

A.B. Modena, Effects of raloxifene and continuous combined hormone therapy

on haemostasis variables: a multicenter, randomized, double-blind study,

Thromb. Res. 119 (2007) 85–91.

[13] C. Wang, M. Zhao, J. Yang, S.Q. Peng, Synthesis and analgesic effects of kyo-

torphin-steroid linkers, Steroids 66 (2001) 811–815.

[14] C. Wang, M. Zhao, S.Q. Peng, The synthesis and immunosuppressive activities

of steroid-urotoxin linkers, Bioorg. Med. Chem. 12 (2004) 4403–4421.

[15] C. Wang, W.N. Cui, M. Zhao, J. Yang, S.Q. Peng, Studies on the synthesis and

anti-osteoporosis of estrogen-GHRPS linkers, Bioorg. Med. Chem. Lett. 13

(2003) 143–146.

[16] D. Saintier, V. Khanine, B. Uzan, H.K. Ea, M.C. de Vernejoul, M.E. Cohen-Solal,

Estradiol inhibits adhesion and promotes apoptosis in murine osteoclasts in

vitro, J. Steroid Biochem. Mol. Biol. 99 (2006) 165–173.

[17] J.C. Gallagher, Advances in bone biology and new treatment for bone loss,

Maturitas 60 (2008) 65–69.

[18] T. Nakamura, Y. Imai, T. Matsumoto, S. Sato, K. Takeuchi, K. Igarashi, et al.,

Estrogen prevents bone loss via estrogen receptor and induction of Fas ligand

in osteoclasts, Cell 130 (2007) 811–823.

[19] X. Yan, L. Dong, The mechanism of RGD-insulin anti-osteoclastic bone

resorption, Bone 27 (2000) 32.

[20] Y. Xiong, M. Zhao, C. Wang, H.W. Chang, S. Peng, Improved anti-osteoporosis

potency and reduced endometrial membrane hyperplasia during hormone

replacement therapy with estrogen–RGD peptide conjugates, J. Med. Chem. 50

(2007) 3340–3353.

4.8. In vivo tail bleeding time assay [24,25]

[21] H. Kurihara, T. Nagamune, Cell adhesion ability of artiﬁcial extracellular matrix

proteins containing a long repetitive Arg-Gly-Asp sequence, J. Biosci. Bioeng.

100 (2005) 82–87.

[22] A.R. Shaikh, M. Ismael, C.A.D. Carpio, H. Tsuboi, M. Koyama, A. Endou, M. Kubo,

E. Broclawikc, A. Miyamoto, Three-dimensional quantitative structure–activity

relationship (3D-QSAR) and docking studies on (benzothiazole-2-yl)acetoni-

trile derivatives as c-Jun N-terminal kinase-3 (JNK3) inhibitors, Bioorg. Med.

Chem. Lett. 16 (2006) 5917–5925.

Thirty and ninety minutes later after the last administration, the

mouse was placed in a tube holder with its tail protruding, and a 2 mm

cut was made on the tail. Flowing blood was gently wiped away with

a tissue every 30 s until bleeding ceased and the time recorded. The

observed bleeding time at 30 min and 90 min after the ip injection or

orally administered mice was recorded to yield the bleeding time.

[23] M.R. Doddareddy, Y.S. Cho, H.Y. Koh, A.N. Pae, CoMFA and CoMSIA 3D QSAR

analysis on N1-arylsulfonylindole compounds as 5-HT6 antagonists, Bioorg.

Med. Chem. 12 (2004) 3977–3985.

[24] P. Maurice, V. Pires, C. Amant, A. Kauskot, S. Da Nascimento, P. Sonnet,

J. Rochette, C. Legrand, F. Fauvel-Lafeve, A. Bonnefoy, Antithrombotic effect of

the type III collagen-related octapeptide (KOGEOGPK) in the mouse, Vasc.

Pharmacol. 44 (2006) 42–49.

[25] A.S. William, E.S. Steven, E.S. Thomas, B.S. Anne, S.B. Jeffrey, S.H. Karen,

C.L. Eddie, L.O. Martin, Antithrombotic and hemostatic effects of a small

molecule factor XIa inhibitor in rats, Eur. J. Pharmacol. 570 (2007)

167–174.

Acknowledgements

This work supported by Beijing area major laboratory of peptide,

small molecular drugs, National Natural Scientiﬁc Foundation of

China (30572235) and Beijing Municipal Commission of Education

(KZ 200810025010).

Article Doi

Synthesis, evaluation and 3D QSAR analysis of novel estradiol-RGD octapeptide conjugates with oral anti-osteoporosis activity

DOI: 10.1016/j.ejmech.2008.09.036

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Authors:

Article abstract of DOI:10.1016/j.ejmech.2008.09.036

Full text of DOI:10.1016/j.ejmech.2008.09.036

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