Improved anti-osteoporosis potency and reduced endometrial membrane hyperplasia during hormone replacement therapy with estrogen-RGD peptide conjugates

DOI: 10.1021/jm070242a

Source and publish data:

Journal of Medicinal Chemistry p. 3340 - 3353 (2007)

Update date:2022-07-29

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Authors:

Xiong, Yu

Zhao, Ming

Wang, Chao

Heng, Wei Chang

Peng, Shiqi

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Article abstract of DOI:10.1021/jm070242a

To improve the specificity and potency of estrogen replacement therapy therapeutics while also minimizing the side effects such as bone resorption and thickening of the uterine wall, a series of novel estrogen-derived conjugates estradiol-3-RGD, estradiol

Full text of DOI:10.1021/jm070242a

3340

J. Med. Chem. 2007, 50, 3340-3353

Improved Anti-Osteoporosis Potency and Reduced Endometrial Membrane Hyperplasia During

Hormone Replacement Therapy with Estrogen-RGD Peptide Conjugates

Yu Xiong,^†Ming Zhao,*^,‡Chao Wang,^†Heng Wei Chang,*^,§and Shiqi Peng*^,‡

College of Pharmaceutical Sciences, Peking UniVersity, Beijing 100083, People’s Republic of China, College of Pharmaceutical Sciences,

Capital UniVersity of Medical Sciences, Beijing, 100069, People’s Republic of China, and C S Bio Company, 20 Kelly Court,

Menlo Park, California 94025

ReceiVed March 4, 2007

To improve the specificity and potency of estrogen replacement therapy therapeutics while also minimizing

the side effects such as bone resorption and thickening of the uterine wall, a series of novel estrogen-

derived conjugates estradiol-3-RGD, estradiol-17-RGD, and estrone-3-RGD peptides have been prepared.

In a mouse model, intraperitoneal (i.p.) administration of these estrogen-RGD peptide conjugates resulted

in decreased serum concentrations of calcium and alkaline phosphatase, as well as increased levels of calcium,

phosphorus, and minerals in the mouse femur. Furthermore, the anti-osteoporosis action of these conjugates

followed a dose-dependent manner and was accompanied with no observable effects on endometrial cell

hyperplasia. In addition to all of these compounds exhibiting biological activity when administered by the

i.p. route, we were particularly pleased to note that the estradiol-3-RGD and estradiol-17-RGD conjugates

were both orally active.

1. Introduction

osteoperotic effects of estradiol, estrone, and growth hormone

releasing peptides (GHRPs) were enhanced.¹⁴

Owing to ovarium atrophy, almost all postmenopausal women

over 55 years of age confront osteoporosis to varying extents,

with over 50% suffering from bone fractures and secondary

complications.^1-3Because the lack of estrogen resulting from

ovarium atrophy is responsible for the onset and progression

of osteoporosis, many postmenopausal women undergo estrogen

replacement therapy (ERT^a) or estrogen/progesterone replace-

ment therapy (HRT).⁴For postmenopausal women, ERT or HRT

not only inhibit bone loss,⁵but also decrease the risk of coronary

heart disease.⁶Michealsson et al. have shown that if postmeno-

pausal women receive HRT for at least 5 years their fracture

risk will be decreased by about 50%;⁷however, long-term

therapy has been linked to a series of dose-related side effects

such as breast cancer and hyperplasia of the uterine membrane.^8-11

In previous work, we have demonstrated that the preparation

of conjugates comprised of steroids and peptides can result in

synergism of both molecular “partners”. For example, in the

case of hydrocortisone-KTP and estrone-KTP conjugates, the

analgesic activities of hydrocortisone, estrone, and kyotorphin

(KTP) were all enhanced.¹²In hydrocortisone-urotoxin and

prednisolone-urotoxin conjugates, the immunosuppressive ac-

tivities of hydrocortisone, prednisolone, and the urotoxins were

all enhanced.¹³In estrogen-GHRPs conjugates, the anti-

It is commonly accepted that osteoporosis relates not only to

a decrease in bone formation modulated by osteoblasts, but also

to an increase in bone resorption modulated by osteoclasts. In

ERT and HRT, estrogen is used to treat the decrease in skeletal

muscle and bone by direct modulation of osteoblastic activity

and proliferation or by regulation of gene expression in

osteoblasts and osteoclasts.^15-17Bone resorption is regulated

by the binding of osteoclasts to the bone surface and is,

therefore, dependent upon osteoclast adhesiveness. This bone

adhesion process is mediated by the Arg-Gly-Asp (RGD)-

binding cell-surface integrin receptor.¹⁸

Based on this information, we surmised that a “two-pronged”

approach, combining the effects of estrogen on upregulation of

osteoblastic activity and proliferation with the adhesion proper-

ties of the RGD peptide to downregulate osteoclast adhesiveness,

and the synergism for both estrogen and peptide resulted from

their conjugation may prove useful in the design of more

efficacious osteoperosis inhibitors. Herein, we report our

preliminary work in this area, which includes the synthesis and

in vivo characterization of three kinds of estrogen-peptide

conjugates, wherein the RGD peptide is incorporated at the 3-

or 17-position of estradiol and the 3-position of estrone.

* To whom correspondence should be addressed. Tel.: (650) 322-1111

(H.C.); +86 10 8280 2482 (M.Z.); 86-10-8391-1528 (S.P.). Fax: (650)

322-2278 (H.C.); +86 10 8280 2482 (M.Z.); 86-10-8391-1528 (S.P.).

E-mail: chang@csbio.com (H.C.); mzhao@mail.bjum.edu.cn (M.Z.);

sqpeng@mail.bjum.edu.cn (S.P.).

2. Results and Discussion

2.1. Preparation of RGD Peptides. Boc-Arg(NO₂)-Gly-Asp-

(OBzl)-Ser(Bzl)-OBzl, Boc-Arg(NO₂)-Gly-Asp(OBzl)-Val-

OBzl, and Boc-Arg(NO₂)-Gly-Asp(OBzl)-Phe-OBzl were pre-

pared via the solution-phase method according to the route

depicted in Scheme 1. The stepwise synthesis was carried out

from C-terminal to N-terminal with L-Ser(Bzl)-OBzl, L-Val-

OBzl, and L-Phe-OBzl as the C-terminal residue, respectively.

Total yields were in a range of 76-82%. Upon removal of the

N-terminal Boc groups, the building blocks HCl‚H-Arg(NO₂)-

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

(OBzl)-Val-OBzl (2), and HCl‚H-Arg(NO₂)-Gly-Asp(OBzl)-

Phe-OBzl (3) were obtained in 93, 94, and 93% yield. Treating

^†Peking University.

^‡Capital University of Medical Sciences.

^§C S Bio Co.

^aAbbreviations: ERT, estrogen replacement therapy; ALP, alkaline

phosphatase; HRT, hormone replacement therapy; ca., circa; KTP, kyotor-

phin; GHRPs, growth hormone releasing peptides; AA, amino acid; DCC,

N,N′-dicyclohexylcarbodiimide; HOBt, 1-hydroxybenzotriazole hydrate; i.p.,

intraperitoneal; µL, microlitre; wt, weight; ESI-MS, electrospray ionization

mass spectrometery; OVX, ovariotomy; FAB-MS, fast atom bombardment

mass spectrometery; Boc, butyloxycarbonyl group; Bzl, benzyl group; THF,

tetrahydrofuran; ESI-MS, electrospray ionization mass spectrometery; Anal.

Calcd, analytical calculated; DMSO-d₆, dimethyl-d₆sulfoxide; HPLC, high

performance liquid chromatography; CMC, carboxymethylcellulose.

Published on Web 06/16/2007

HRT with Estrogen-RGD Peptide Conjugates

Scheme 1. Preparation of RGD Peptides^a

Journal of Medicinal Chemistry, 2007, Vol. 50, No. 14 3341

yield. Coupling of 7 and HCl‚H-Arg(NO₂)-Gly-Asp(OBzl)-AA-

OBzl (1-3) the corresponding estradiol-17-Arg(NO₂)-Gly-

Asp(OBzl)-AA-OBzl (8-10) were obtained in 61, 53, and 53%

yield, respectively. Following global deprotection by catalytic

hydrogenation, estradiol-3-Arg-Gly-Asp-AA-OH (11-13) were

obtained in 62, 68, and 58% yield, respectively. The yields

indicate that with the carbonylethylcarbonyl group as the linker

RGD peptides can be smoothly introduced into the 17-position

of estradiol.

^aFor H-AA-OBzl, AA ) Ser(Bzl), Val, and Phe; for 1, AA ) Ser(Bzl),

for 2 and 5, AA ) Val, for 3 and 6, AA ) Phe; and for 4, AA ) Ser.

Reagents: (I) DCC, HOBt, and Boc-Gly-OH; (II) hydrogen chloride in ethyl

acetate; (III) DCC, HOBt, and Boc-Asp(OBzl)-OH; (IV) DCC, HOBt, and

Boc-Arg(NO₂)-OH; (V) Pt/H₂.

2.4. Preparation of Estrone-3-RGD Peptide Conjugates.

As outlined in Scheme 3, the preparation of the estrone 3-RGD

peptide conjugates commenced with the alkylation of the C₃-

hydroxyl group of estrone using ethyl bromoacetate to give alkyl

ether 22 in 90% yield. Saponification of 22 yielded acid 23

(96% yield) to which HCl‚H-Arg(NO₂)-Gly-Asp(OBzl)-AA-

OBzl (1-3) were coupled to give the corresponding estrone-

3-Arg(NO₂)-Gly-Asp(OBzl)-AA-OBzl conjugates 24-26 in 62,

68, and 70% yield, respectively. Following global deprotection

by catalytic hydrogenation, estrone-3-Arg-Gly-Asp-AA-OH

conjugates 27-29 were obtained in 57, 76, and 56% yield,

respectively. The yields demonstrate that through the same linker

RGD peptides can also be smoothly introduced into the

3-position of estrone.

1-3 with Pd/H₂removed all protecting groups and the RGD

peptides (4-6) were obtained in 89-92% yield.

2.2. Preparation of Estradiol-3-RGD Peptide Conjugates.

The estradiol-3-RGD peptide conjugates were prepared as

outlined in Scheme 2. Thus, the C₃-hydroxyl group of estradiol

was first alkylated with ethyl bromoacetate to provide ethyl

estradiol-3-oxylacetate (14) in 83% yield. Saponification of 14

resulted in acid 15 in excellent yield (96%). Conjugation of 14

with HCl‚H-Arg(NO₂)-Gly-Asp(OBzl)-AA-OBzl (1-3) then

yielded the corresponding estradiol-3-Arg(NO₂)-Gly-Asp-

(OBzl)-AA-OBzl derivatives 16-18 in 93, 74, and 61% yield,

respectively. Following global deprotection by catalytic hydro-

genation, estradiol-3-Arg-Gly-Asp-AA-OH (19-21) were ob-

tained in 70, 73, and 77% yield, respectively. The yields indicate

that with the methylcarbonyl group as the linker RGD peptides

can be smoothly introduced into the 3-position of estradiol.

2.3. Preparation of Estradiol-17-RGD Peptide Conju-

gates. The estradiol-17-monoester of succinic acid (7) was first

prepared by treating estradiol with succinic anhydride in 95%

2.5. Estrogen-RGD Peptide Conjugates Inhibit Body

Weight Increase of Mice. To develop osteoporosis, female

Kuiming mice weighing 30.7 ( 3.1 g were subjected to

abdominal ovariotomy (OVX) according to standard proce-

dures.¹⁹To compensate for possible effects of the surgical

procedure, the mice of a sham group were subjected to an

abdominorotomy. On the fifth day after surgery, the mice then

received an intraperitoneal (i.p.) injection of an aqueous solution

of carboxymethylcellulose (CMC; 2 µL, 0.5 wt %), containing

Scheme 2. Preparation of 3- and 17-RGD Peptide Conjugates of Estradiol^a

^aReagents: (I) succinic anhydride; (II) DCC, HOBt, 1, 2, or 3; (III) Pt/H₂; (IV) ethyl bromoacetyl acetate; (V) NaOH. In 8 and 16, R₁) Arg(NO₂)-

Gly-Asp(OBzl)-Ser-OBzl; in 9 and 17, R₁) Arg(NO₂)-Gly-Asp(OBzl)-Val-OBzl; in 10 and 18, R₁) Arg(NO₂)-Gly-Asp(OBzl)-Phe-OBzl; in 11 and 19,

R₂) Arg-Gly-Asp-Ser-OH; in 12 and 20, R₂) Arg-Gly-Asp-Val-OH; and in 13 and 21, R₂) Arg-Gly-Asp-Phe-OH.

Scheme 3. Preparation of Estrone-3-RGD Peptide Conjugates^a

^aReagents: (I) ethyl 2-bromoacetate; (II) NaOH; (III) DCC, HOBt, 1, 2, or 3; (IV) H₂/Pd. In 24, AA ) Ser(Bzl); in 25 and 28, AA ) Val; in 26 and

29, AA ) Phe; and in 27, AA ) Ser.

3342 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 14

Xiong et al.

Table 1. Effect of i.p. Injection of Estrogen-RGDAA on Body Weight

Table 2. Effect of i.p. Injection of Estrogen-RGD Peptide Conjugates

on Serum ALP Level, Serum Calcium Concentration, and Serum

Phosphorus Concentration of Mice^a

of Mice^a

before

treatment

(g)

after

treatment

(g)

calcium

(mmol/L)

phosphorous

(mmol/L)

ALP

(U/L, king)

group

OVX

sham

estradiol

RGDS

estradiol + RGDS

RGDV

estradiol + RGDV

RGDF

estradiol + RGDF

estrone

estrone + RGDS

estrone + RGDV

estrone + RGDF

31.1 ( 2.2

30.0 ( 1.5

31.0 ( 2.6

30.7 ( 2.5

30.6 ( 2.7

30.3 ( 1.8

30.9 ( 1.5

30.3 ( 1.5

30.7 ( 2.0

30.4 ( 1.9

30.5 ( 2.6

29.8 ( 1.7

30.5 ( 2.3

29.8 ( 2.9

30.1 ( 2.0

30.4 ( 2.5

30.6 ( 2.1

29.9 ( 1.9

30.1 ( 2.4

30.3 ( 2.1

31.0 ( 2.2

30.4 ( 2.2

37.2 ( 2.6

34.5 ( 2.9^b

34.1 ( 2.3^b

34.0 ( 2.4^b

33.9 ( 2.7^c

35.4 ( 2.7^b

35.2 ( 1.6^b

33.4 ( 2.5^c

33.6 ( 2.3^c

34.5 ( 2.7^b

35.2 ( 2.5^b

33.3 ( 2.0^c

33.9 ( 1.9

34.8 ( 2.7^b

33.1 ( 2.2^c

34.3 ( 2.1^c

34.4 ( 2.2^c

32.9 ( 2.3^c

33.5 ( 2.5^c

34.1 ( 2.6^b

33.4 ( 2.3^c

34.5 ( 2.8^b

2.039 ( 0.0445

2.024 ( 0.0783

2.013 ( 0.0382

2.013 ( 0.0433

1.601 ( 0.00268

1.602 ( 0.00519

1.601 ( 0.00521

1.602 ( 0.00408

40.086 ( 2.884

34.171 ( 3.341^c

35.286 ( 1.733^c

36.328 ( 2.113^c

sham

estradiol

estradiol +

RGDS

2.010 ( 0.0417

2.002 ( 0.0271^b

2.004 ( 0.0347^b

2.012 ( 0.0389

1.603 ( 0.00371

1.602 ( 0.00385

1.599 ( 0.00487

1.603 ( 0.00417

36.643 ( 1.787^c

32.771 ( 2.050^d

31.429 ( 1.837^d

36.005 ( 1.772^c

estradiol +

RGDV

2.011 ( 0.0181

2.005 ( 0.0258^b

2.003 ( 0.0270^b

2.011 ( 0.0367

1.605 ( 0.00669

1.605 ( 0.00405

1.600 ( 0.00492

1.601 ( 0.00520

35.614 ( 1.486^c

31.057 ( 0.996^d

30.514 ( 1.301^d

36.084 ( 1.890^c

estradiol +

RGDF

2.012 ( 0.0324

2.005 ( 0.0258^b

2.005 ( 0.0160^b

2.012 ( 0.0175

2.013 ( 0.0441

1.599 ( 0.00733

1.605 ( 0.00498

1.605 ( 0.00537

1.601 ( 0.00417

1.603 ( 0.00439

35.643 ( 2.756^c

31.486 ( 1.285^d

31.529 ( 1.676^d

36.271 ( 3.228^c

36.204 ( 1.913^c

estrone

estrone +

RGDS

estrone +

RGDV

estrone +

RGDF

2.000 ( 0.0284^b

2.010 ( 0.0395

1.604 ( 0.00268

1.603 ( 0.00550

32.371 ( 1.910^e

35.970 ( 1.983^c

^aDose ) 110.3 × 10^-3mmol/kg, n ) 12. OVX ) ovariotomy. The

statistical analysis of the data was carried out by the use of an ANOVA

test, and P < 0.05 was considered significant. ^bCompared to OVX, P <

0.05. ^cCompared to OVX, P < 0.01.

2.008 ( 0.0179^b

2.013 ( 0.0382

1.602 ( 0.00560

1.601 ( 0.00488

32.229 ( 1.467^e

36.433 ( 1.936^c

2.008 ( 0.0344^b

1.602 ( 0.00315

32.407 ( 2.137^e

estradiol, estrone, RGDS, RGDV, RGDF, a mixture of estradiol

and RGDS, a mixture of estradiol and RGDV, a mixture of

estradiol and RGDF, a mixture of estrone and RGDS, a mixture

of estrone and RGDV, a mixture of estrone and RGDF,

estradiol-17-RGD peptide conjugates (11-13), estradiol-3-

RGD peptide conjugates (19-21), and estrone-3-RGD peptide

conjugates (27-29; each at 110.3 × 10^-3mmol/kg). The mice

were injected once daily for 4 weeks. On the day of the last

administration, the mice were weighed and the body weights

were recorded (Table 1). As shown, the data demonstrate that

the surgical operation and the treatment with estrogen and

estrogen-RGD peptide conjugates inhibits mouse body weight

increase, which suggests that the inhibition of the increase of

the body weight of mice is independent of the conjugations.

2.6. Estrogen-RGD Peptide Conjugates Decrease the

Serum Levels of Calcium and ALP in Mice. To explore the

effect of estrogen-RGD peptide conjugates on the content of

calcium and ALP in the serum, blood was drawn by retrorbital

puncture on the day after the last day of the 4 week dosing

regime. The blood was allowed to stand at room temperature

for 30 min and was then centrifuged (3000 g for 20 min). The

serum was removed by pipet and stored at -20 °C and serum

calcium content was measured using o-methylphenolphthalein,

as previously described²⁰(Table 2). The serum phosphorus

content was measured using molybdenum blue, as previously

described.²¹The serum alkaline phosphatase (ALP) level was

measured using a functional assay with disodium phenylphos-

phate as the substrate. The data listed in Table 2 indicate that

the content of calcium in the serum is significantly decreased

after administration of 11-13, 19-21, and 27-29, but is

unchanged by treatment with estradiol, estrone, the mixtures

of estradiol and RGD peptides, and the mixture of estrone and

RGD peptides. Serum ALP levels were clearly reduced by the

administration of estradiol, estrone, the mixtures of estradiol

and RGD peptides, and the mixture of estrone and RGD

peptides. However, the conjugates 11-13, 19-21, and 27-29

were far more effective at reducing ALP levels. The significantly

^aDosage ) 110.3 × 10^-3mmol/kg, n ) 12. OVX ) ovariotomy. The

statistical analysis of the data was carried out by the use of an ANOVA

test, and p < 0.05 was considered significant. ^bCompared to OVX, P <

0.05. ^cCompared to OVX, P < 0.01. ^dCompared to OVX, estradiol, and

RGD peptides, P < 0.01. ^eCompared to OVX, estrone, and RGD peptides,

P < 0.01.

higher inhibition of serum calcium and ALP levels in mice for

estrogen-RGD peptide conjugates demonstrates that the con-

jugation of estrogen and RGD peptides is helpful for enhancing

their corresponding inhibitions.

2.7. Estrogen-RGD Peptide Conjugates Inhibit Bone Loss

of Mice. To evaluate the direct influence of the estrogen-RGD

peptide conjugates on bone loss, the weights of dry femurs, the

weights of femur ashes, and the lengths of femurs from the mice

of all groups were measured. After body weighing and blood

drawing, the mice were euthanized by anesthetization (pento-

barbital sodium, 40.0 mg/kg, i.p.) followed by removal of the

left femur. After complete removal of the muscle, the femur

length was measured and the femurs were then defatted by

immersion in a solution of chloroform-methanol (2:1) for 3 h

(repeated 2 times). The femurs were then heated at 120 °C for

6 h, cooled, and weighed to record the dry weight. The femurs

were then incinerated in a furnace at 800 °C for 8 h, cooled,

and the residual ash was weighed. Dry femur weights listed in

Table 3 indicate that estradiol, estrone, the mixtures of estradiol

and RGD peptides, and the mixture of estrone and RGD peptides

did not inhibit bone loss in the recipient mice. The weights of

dry femurs from mice receiving estradiol, estrone, the mixtures

of estradiol and RGD peptides, and the mixture of estrone and

RGD peptides are significantly lower than that of the abdomi-

norotomy-receiving mice (sham group). However, estrogen-

RGD peptide conjugates did inhibit bone loss in the recipient

mice, as the weights of dry femurs of the mice receiving 11-

13, 19-21, and 27-29 are significantly higher than that of

vehicle-receiving mice (OVX group) and no significant differ-

ence is found between the weights of dry femurs of the mice

receiving estrogen-RGD peptide conjugates and the weight of

HRT with Estrogen-RGD Peptide Conjugates

Journal of Medicinal Chemistry, 2007, Vol. 50, No. 14 3343

Table 3. Effect of i.p. Injection of Estrogen-RGD Peptide Conjugates

on the Length of Femur and the Weight of Dry Femur and Femur Ash

of Mice^a

Table 4. Effect of i.p. Injection of Estrogen-RGD Peptide Conjugates

on the Calcium, Phosphorus, and Mineral Content of Mouse Femurs^a

calcium

(%)

phosphorous

(%)

mineral

(ratio)

wt of dry

femur (mg)

wt of femur

ash (mg)

length of femur

(cm)

group

OVX

group

39.545 ( 2.551

53.213 ( 2.365^c

44.466 ( 3.041^c

43.903 ( 2.402^c

22.951 ( 1.960

24.811 ( 1.049^c

24.995 ( 2.307^b

24.891 ( 2.067^b

0.304 ( 0.0272

0.487 ( 0.0922^c

0.496 ( 0.0945^c

0.413 ( 0.0557^c

OVX

sham

estradiol

estradiol + RGDS

RGDS

58.5 ( 3.94

74.0 ( 8.32^c

57.7 ( 6.96

58.2 ( 6.33

59.8 ( 4.65

66.4 ( 5.38^d

63.4 ( 6.39^e

59.0 ( 6.37

58.8 ( 4.65

62.6 ( 3.68^e

63.7 ( 6.27^e

59.5 ( 6.55

60.0 ( 6.49

66.0 ( 5.48^d

64.2 ( 5.32^f

61.1 ( 5.87

60.8 ( 6.19

66.9 ( 5.70^d

58.6 ( 5.89

63.9 ( 7.10^e

59.7 ( 6.24

65.9 ( 5.37^d

17.8 ( 2.17

42.9 ( 4.83^c

28.5 ( 6.80^c

27.9 ( 5.10^c

27.8 ( 4.06^c

34.4 ( 5.65^g

37.0 ( 6.25^g

28.1 ( 3.60^c

27.8 ( 3.53^c

35.8 ( 3.13^g

40.0 ( 4.81^g

28.2 ( 3.59^c

22.5 ( 7.43^b

34.8 ( 4.67^g

35.8 ( 4.37^g

20.8 ( 4.30^b

23.4 ( 4.50^b

22.6 ( 2.97^c

26.2 ( 4.48^b

21.9 ( 6.32^b

25.5 ( 6.01^b

24.7 ( 4.53^h

1.597 ( 0.0390

1.646 ( 0.0438^c

1.630 ( 0.0646

1.620 ( 0.0540

1.617 ( 0.0397

1.634 ( 0.0358^b

1.642 ( 0.0500^b

1.618 ( 0.0523

1.614 ( 0.0455

1.655 ( 0.0490^c

1.640 ( 0.0306^c

1.621 ( 0.0451

1.620 ( 0.0343

1.635 ( 0.0273^b

1.635 ( 0.0474^b

1.606 ( 0.0317

1.600 ( 0.0522

1.634 ( 0.0459^b

1.604 ( 0.0320

1.628 ( 0.0309^b

1.606 ( 0.0340

1.639 ( 0.0456^b

sham

estradiol

estradiol +

RGDS

43.480 ( 2.331^c

51.312 ( 2.602^d

54.885 ( 1.668^d

43.009 ( 2.055^c

24.888 ( 2.059^b

24.920 ( 1.623^b

24.949 ( 2.221^b

24.823 ( 2.217^b

0.396 ( 0.0460^c

0.520 ( 0.0819^c

0.582 ( 0.0512^f

0.402 ( 0.0406^c

estradiol + RGDV

RGDV

estradiol + RGDF

RGDF

estrone

estrone + RGDS

estrone + RGDV

estrone + RGDF

estradiol +

RGDV

42.739 ( 1.816^c

53.078 ( 2.349^d

58.350 ( 0.926^d

44.103 ( 3.121^c

24.759 ( 2.267^b

24.867 ( 1.536^b

24.991 ( 1.338^b

24.765 ( 2.258^b

0.394 ( 0.0411^c

0.625 ( 0.0399^d

0.630 ( 0.0690^d

0.407 ( 0.0411^c

estradiol +

RGDF

43.815 ( 1.792^c

52.798 ( 1.376^d

56.690 ( 1.474^d

43.840 ( 2.659^c

43.466 ( 2.450^c

24.585 ( 1.204^b

24.665 ( 1.069^b

25.460 ( 1.265^c

23.979 ( 1.087

24.756 ( 2.260^b

0.423 ( 0.0685^c

0.533 ( 0.0921^c

0.585 ( 0.0555^f

0.430 ( 0.0522^c

0.426 ( 0.0548^c

estrone

estrone +

RGDS

estrone +

RGDV

estrone +

RGDF

47.613 ( 1.891^e

43.634 ( 2.569^c

24.399 ( 1.110^b

24.767 ( 1.309^b

0.438 ( 0.0388^c

0.422 ( 0.0681^c

^aDosage ) 110.3 × 10^-3mmol/kg, n ) 12. OVX ) ovariotomy. The

statistical analysis of the data was carried out by the use of an ANOVA

test, and p < 0.05 was considered significant. ^bCompared to OVX, P <

0.05. ^cCompared to OVX, P < 0.01. ^dCompared to OVX and estradiol, P

< 0.01, to estrone and RGD peptides, P < 0.05. ^eCompared to OVX,

estradiol, and RGD peptides, P < 0.05. ^fCompared to OVX, P < 0.01, to

estradiol and RGD peptides, P < 0.05. ^gCompared to OVX and estradiol,

P < 0.01, and to RGD peptides, P < 0.05. ^hCompared to OVX, P < 0.01,

and to estrone, P < 0.05.

47.173 ( 1.772^e

43.722 ( 2.490^c

24.750 ( 1.083^b

24.766 ( 2.249^b

0.429 ( 0.0387^c

0.420 ( 0.0556^c

49.550 ( 2.436^e

24.588 ( 1.080^b

0.462 ( 0.0510^c

^aDosage ) 110.3 × 10^-3mmol/kg, n ) 12. OVX ) ovariotomy. The

statistical analysis of the data was carried out by the use of an ANOVA

test, and p < 0.05 was considered significant. ^bCompared to OVX, P <

0.05. ^cCompared to OVX, P < 0.01. ^dCompared to OVX, estradiol, and

RGD peptides, P < 0.01. ^eCompared to OVX, estrone, and RGD peptides,

P < 0.01. ^fCompared to OVX and RGD peptides, P < 0.01, and to estradiol,

P < 0.05.

dry femurs of sham group mice. The data in Table 3 indicate

that though the femur ash weights of mice receiving estradiol,

estrone, the mixtures of estradiol and RGD peptides, and the

mixture of estrone and RGD peptides are significantly higher

than that of vehicle receiving mice (OVX group), they are

significantly less than that of mice receiving 11-13 and 19-

21. Similarly, except the femur length of the mice receiving

estrone, the mixtures of estrone and RGD peptides, estradiol,

and the mixtures of estradiol and RGD peptides, the femur

length of the mice receiving 11-13, 19-21, and 27-29 are

significantly higher than that of the vehicle-receiving mice

(OVX group). All of the data listed in Table 3 suggest that

estrogen-RGD peptide conjugates 11-13, 19-21, and 27-

29 are able to inhibit bone loss in OVX mice.

2.8. Effect of Estrogen-RGD Peptide Conjugates on Bone

Calcium, Phosphorus Content, and Mineral Content. The

femurs were then incinerated in a furnace at 800 °C for 8 h,

cooled, and weighed to record the ash weight and calculate the

ratio of the ash weight to dry femur weight (i.e., the mineral

content of the femur). To investigate the influence of estrogen-

RGD peptide conjugates on the content of calcium, phosphorus,

and minerals in the mouse femurs, the weighed ashes of the

left femurs were dissolved in hydrochloric acid (6 N, 0.5 mL)

and diluted with ultrapure water (4.5 mL). An aliquot (0.05 mL)

of the solution was drawn and diluted to 1 mL with ultrapure

water. The calcium content of the aqueous solution was

measured using o-methylphenolphthalein. The phosphorus

content of the aqueous solution was measured by the method

of molybdenum blue.²²Based on the measured data, the ratios

of calcium and phosphorus in the ash were calculated. The data

are listed in Table 4. As shown, the calcium, phosphorus, and

mineral content in the femurs of the mice in the sham and the

drug treatment groups are significantly higher than that of the

mice in OVX group. When compared to the mice receiving

estradiol, estrone, the mixtures of estradiol and RGD peptides,

and the mixtures of estrone and RGD peptides, the femurs of

the mice receiving 11-13, 19-21, and 27-29 show signifi-

cantly higher contents of calcium and the femurs of the mice

receiving 12, 19, and 21 show significantly higher contents of

mineral. Thus, the estrogen-RGD peptide conjugates substan-

tially increase the content of calcium, phosphorus, and mineral

in the femur. This is not only consistent with their inhibition of

bone loss, but also with the advantage that resulted from the

conjugation of estrogen and RGD peptides.

2.9. Effect of Estrogen-RGD Peptide Conjugates on

Organ Weights. To examine the effect of estrogen-RGD

peptide conjugates on organ weights, the removed lungs, livers,

spleens, and uteri were weighed directly and the weights are

listed in Table 5. The data indicate that though the liver weight

of the mice receiving estradiol is occasionally higher than that

of the mice in the OVX group, the weights of lungs, livers, and

spleens of the mice in all groups show no significant difference.

However, the weights of the uteri of mice receiving estradiol,

estrone, the mixtures of estradiol and RGD peptides, and the

mixtures of estrone and RGD peptides were significantly heigher

than that of OVX, sham and estrogen-RGD peptide conjugates

receiving mice. These results indicate that estrogen-RGD

peptide conjugates exhibited no observable influence on en-

dometrial cell hyperplasia, which reveals that the conjugation

of estrogen and RGD peptides is helpful for eliminating the

dose-related side effects of estrogen.

2.10. Dose-Related Inhibition of Mouse Bone Loss by

Estrogen-RGD Peptide Conjugates 19-21. To evaluate the

effect of the dose of estrogen-RGD peptide conjugates on bone

3344 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 14

Xiong et al.

Table 5. Effect of i.p. Injection of Estrogen-RGD Peptide Conjugates on the Weight of Lung, Liver, Spleen, and Uterus of Mice^a

lung

(mg)

liver

(g)

spleen

(mg)

uterus

(mg)

group

OVX

sham

estradiol

estradiol + RGDS

RGDS

163.0 ( 23.1

164.0 ( 24.7

168.0 ( 30.8

165.0 ( 26.1

168.0 ( 22.7

151.0 ( 18.8

173.0 ( 20.1

167.0 ( 24.7

171.0 ( 25.9

167.0 ( 27.9

162.0 ( 19.0

165.0 ( 24.4

163.0 ( 17.7

164.0 ( 24.5

169.0 ( 30.3

176.0 ( 25.9

170.0 ( 24.9

173.0 ( 16.4

172.0 ( 23.7

168.0 ( 10.7

169.0 ( 22.3

179.0 ( 16.0

1.329 ( 0.156

1.367 ( 0.198

1.565 ( 0.199^b

1.372 ( 0.190

1.295 ( 0.186

1.371 ( 0.178

1.377 ( 0.185

1.370 ( 0.187

1.153 ( 0.110^b

1.353 ( 0.221

1.363 ( 0.133

1.422 ( 0.183

1.413 ( 0.122

1.391 ( 0.129

1.395 ( 0.160

1.318 ( 0.113

1.331 ( 0.159

1.347 ( 0.146

1.387 ( 0.155

1.389 ( 0.139

1.410 ( 0.187

1.458 ( 0.199

130.0 ( 31.9

151.0 ( 41.0

151.0 ( 35.7

148.0 ( 32.3

138.0 ( 37.6

130.0 ( 37.2

124.0 ( 36.4

129.0 ( 33.6

127.0 ( 33.0

143.0 ( 36.8

132.0 ( 45.0

130.0 ( 25.8

134.0 ( 25.5

142.0 ( 32.0

131.0 ( 34.4

150.0 ( 31.0

146.0 ( 32.3

129.0 ( 26.8

144.0 ( 33.0

131.0 ( 37.2

139.0 ( 40.7

140.0 ( 41.1

89.0 ( 24.9^c

91.5 ( 30.0^c

177.1 ( 43.6^b

178.0 ( 42.5^b

71.0 ( 52.2^c

55.5 ( 34.4^d

42.5 ( 18.7^d

177.7 ( 42.7^b

61.1 ( 28.7^d

69.4 ( 33.6^d

50.3 ( 31.1^d

178.2 ( 44.1^b

69.8 ( 41.9^d

71.2 ( 26.0^d

55.4 ( 28.8^d

141.9 ( 45.7^b

143.2 ( 46.0^b

63.3 ( 43.4^d

145.5 ( 47.0^b

51.9 ( 32.0^d

143.7 ( 41.2^b

55.8 ( 32.6^d

estradiol + RGDV

RGDV

estradiol + RGDF

RGDF

estrone

estrone + RGDS

estrone + RGDV

estrone + RGDF

^aDosage ) 110.3 × 10^-3mmol/kg, n ) 12. OVX ) ovariotomy. The statistical analysis of the data was carried out by the use of an ANOVA test, and

p < 0.05 was considered significant. ^bCompared to OVX, P < 0.01. ^cCompared to estradiol and estrone, P < 0.01. ^dCompared to OVX, estradiol, and

estrone, P < 0.01.

Table 6. Effect of 19-21 at Different Doses (i.p.) on the Femur

Length, the Dry Femur Weight, and the Femur Ash Weight of Recipient

Mice^a

higher than that of the mice receiving a low dose of 19-21.

The same dose-related effects of 19-21 on the serum calcium

content, the serum phosphorus content, and the serum ALP

activity are also revealed in the data of Table 8. All the data

support that the effect of 19-21 on bone loss follows a dose-

dependent manner.

dosage

(mmol/kg)

femur weight

(mg)

femur ash

weight (mg)

femur length

(cm)

group

110.3 × 10^-363.4 ( 6.25

37.0 ( 6.25

1.642 ( 0.0500

55.15 × 10^-356.3 ( 6.99^b31.6 ( 4.43^b1.622 ( 0.0430

11.03 × 10^-348.8 ( 4.97^c

18.1 ( 4.23^c1.642 ( 0.0421

40.0 ( 4.81

2.11. Dose Effects of Estradiol-RGD Peptide Conjugates

19-21 on Uterine Weights of Mice. To examine the risk of

of dose-related side effects, the weight of the liver, the lung,

the spleen, and especially the uterine of the mice receiving 19-

21, the estradiol-RGD peptide conjugates with a higher anti-

osteoporosis potency, at a dose of 110.3 × 10^-3mmol/kg (high

dose), 55.15 × 10^-3mmol/kg (moderate dose), and 11.03 ×

10^-3mmol/kg (low dose), respectively, were measured. The

data are listed in Table 9. It is clear that the weight of the liver,

the lung, the spleen, and the uterine of the mice receiving high,

moderate, and low doses of 19-21 show no significant

difference. The results indicate that the anti-osteoporosis action

of estradiol-RGD peptide conjugates followed a dose-dependent

manner and was accompanied with no observable effects on

endometrial cell hyperplasia.

110.3 × 10^-363.7 ( 6.27

1.640 ( 0.0306

55.15 × 10^-357.5 ( 4.87^b32.3 ( 4.70^b1.643 ( 0.0300

11.03 × 10^-350.9 ( 6.75^c

20.6 ( 7.33^c1.618 ( 0.0520

35.8 ( 4.37

110.3 × 10^-361.2 ( 6.74

1.623 ( 0.0475

55.15 × 10^-353.9 ( 4.08^b31.6 ( 2.87^b1.624 ( 0.0580

11.03 × 10^-346.9 ( 6.74^c

20.8 ( 7.11^c1.647 ( 0.0414

^an ) 12. The statistical analysis of the data was carried out by the use

of an ANOVA test, and p < 0.05 was considered significant. ^bCompared

to 110.3 × 10^-3mmol/kg, P < 0.05. ^cCompared to 55.15 × 10^-3mmol/

kg, P < 0.01.

loss, the weights of dry femurs, weights of femur ashes, the

lengths of femurs, the calcium content of femurs, the phosphorus

content of femurs, the mineral content of femurs, the serum

calcium content, the serum phosphorus content of and the serum

ALP activity of the mice receiving 19-21, the estradiol-RGD

peptide conjugates with higher anti-osteoporosis potency at a

dose of 110.3 × 10^-3mmol/kg (high dose), 55.15 × 10^-3mmol/

kg (moderate dose) and, 11.03 × 10^-3mmol/kg (low dose),

respectively, were measured. The data are listed in Tables 6-8.

The data in Table 6 demonstrate that the femur length, the dry

femur weight, and the femur ash weight of the mice receiving

a high dose of 19-21 are significantly higher than that of the

mice receiving a moderate dose of 19-21. On the other hand,

the femur length, the dry femur weight, and the femur ash weight

of the mice receiving a moderate dose of 19-21 are also

significantly higher than that of the mice receiving a low dose

of 19-21. Similarly, the data in Table 7 demonstrate that the

femur calcium content, the femur phosphorus content, and the

femur mineral content of the mice receiving a high dose of 19-

21 are significantly higher than that of the mice receiving a

moderate dose of 19-21, and the femur calcium content the

femur phosphorus content and the femur mineral content of the

mice receiving a moderate dose of 19-21 are significantly

2.12. Oral Administration of Estrogen-RGD Peptide

Conjugates Inhibited Bone Loss of Mice. To explore if

estrogen-RGD peptide conjugates are orally active, 11-13 and

19-20 were administered orally. The female Kuiming mice

weighing 30.7 ( 3.1 g were subjected to abdominal OVX, as

mentioned above. To compensate for the possible effects of the

surgical procedure, the mice of a sham group were subjected

to an abdominorotomy. On the fifth day after surgery, the mice

then received an oral administration of an aqueous solution of

CMC (0.2 mL, 0.5 wt %), containing estradiol, a mixture of

estradiol and RGDS, estradiol-17-RGD peptide conjugates

(11-13) and estradiol-3-RGD peptide conjugates (19-21; each

at 110.3 × 10^-3mmol/Kg). The mice were orally administered

once daily for 4 weeks. On the day of the last administration,

the blood of the mice was drawn by retrorbital puncture. The

blood was allowed to stand at room temperature for 30 min

and was then centrifuged (3000 g for 20 min). The serum was

removed by pipet and stored at -20 °C and the serum calcium

HRT with Estrogen-RGD Peptide Conjugates

Journal of Medicinal Chemistry, 2007, Vol. 50, No. 14 3345

Table 7. Effect of 19-21 at Different Doses (i.p.) on Femur Calcium Content, Femur Phosphorus Content, and Femur Mineral Content of Mice^a

dosage

(mmol/kg)

calcium

(%)

phosphorous

(%)

mineral

(ratio)

group

110.3 × 10^-3

55.15 × 10^-3

11.03 × 10^-3

110.3 × 10^-3

55.15 × 10^-3

11.03 × 10^-3

110.3 × 10^-3

55.15 × 10^-3

11.03 × 10^-3

54.885 ( 1.668

52.076 ( 2.399^c

41.548 ( 3.792^d

58.353 ( 0.926

57.130 ( 1.113^c

42.110 ( 3.759^d

56.693 ( 1.474

54.493 ( 1.381^c

42.855 ( 3.447^d

24.949 ( 2.221

24.274 ( 2.394

23.984 ( 2.711

24.991 ( 2.338

24.315 ( 2.123

23.978 ( 3.076

25.460 ( 3.265

24.845 ( 2.214

24.347 ( 1.869

0.582 ( 0.0512

0.479 ( 0.0686^c

0.310 ( 0.0776^d

0.630 ( 0.0690

0.539 ( 0.0973^b

0.345 ( 0.0120^d

0.585 ( 0.0555

0.504 ( 0.0401^c

0.338 ( 0.0121^d

^an ) 12. The statistical analysis of the data was carried out by the use of an ANOVA test, and p < 0.05 was considered significant. ^bCompared to 110.3

× 10^-3mmol/kg, P < 0.05. ^cCompared to 110.3 × 10^-3mmol/kg, P < 0.01. ^dCompared to 55.15 × 10^-3mmol/kg, P < 0.01.

Table 8. Effect of 19-21 at Different Doses (i.p.) on Serum ALP Level, the Serum Calcium Concentration, and Serum Phosphorus Concentration of

Mice^a

dosage

(mmol/kg)

calcium

(mmol/L)

phosphorous

(mmol/L)

ALP

(U/L, king)

group

110.3 × 10^-3

55.15 × 10^-3

11.03 × 10^-3

110.3 × 10^-3

55.15 × 10^-3

11.03 × 10^-3

110.3 × 10^-3

55.15 × 10^-3

11.03 × 10^-3

2.014 ( 0.0347

2.047 ( 0.0198^b

2.087 ( 0.0254^e

2.005 ( 0.0270

2.084 ( 0. 0483^c

2.117 ( 0.0115^d

2.0150 ( 0.016

2.040 ( 0.0369^b

2.093 ( 0.0176^e

1.599 ( 0.00487

1.607 ( 0.00356

1.611 ( 0.00265

1.600 ( 0.00492

1.586 ( 0.00274

1.605 ( 0.00324

1.605 ( 0.00537

1.602 ( 0.00314

1.600 ( 0.00264

31.429 ( 1.837

33.616 ( 2.643^b

40.327 ( 1.526^e

30.514 ( 1.301

32.786 ( 2.143^c

38.457 ( 1.546^e

31.529 ( 1.676

33.746 ( 1.724^c

38.643 ( 1.204^e

^an ) 12. The statistical analysis of the data was carried out by use of an ANOVA test, and p < 0.05 was considered significant. ^bCompared to 110.3

× 10^-3mmol/kg, P < 0.05. ^cCompared to 110.3 × 10^-3mmol/kg, P < 0.01. ^dCompared to 55.15 × 10^-3mmol/kg, P < 0.05. ^eCompared to 55.15 × 10^-3

mmol/kg, P < 0.01.

Table 9. Effect of 19-21 at Different Doses (i.p.) on the Liver Weight, Lung Weight, Spleen Weight, and Uterine Weight of the Mice^a

dosage

(mmol/kg)

liver weight

(g)

lung weight

(mg)

spleen weight

(mg)

uterine weight

(mg)

group

110.3 × 10^-3

55.15 × 10^-3

11.03 × 10^-3

110.3 × 10^-3

55.15 × 10^-3

11.03 × 10^-3

110.3 × 10^-3

55.15 × 10^-3

11.03 × 10^-3

1.377 ( 0.185

1.397 ( 0.231

1.424 ( 0.179

1.363 ( 0.133

1.462 ( 0.165

1.477 ( 0.227

1.395 ( 0.160

1.407 ( 0.227

1.575 ( 0.239

173.0 ( 20.1

167.0 ( 33.3

181.0 ( 24.9

182.0 ( 19.0

179.0 ( 33.2

188.0 ( 56.0

169.0 ( 30.3

155.0 ( 45.7

174.0 ( 33.3

124.0 ( 36.0

136.0 ( 46.3

125.0 ( 22.9

132.0 ( 45.0

140.0 ( 40.6

156.0 ( 53.0

131.0 ( 34.0

123.0 ( 36.8

145.0 ( 62.0

42.5 ( 18.7

48.6 ( 28.2

52.8 ( 38.8

50.3 ( 31.1

57.6 ( 43.9

55.7 ( 35.5

55.4 ( 28.8

55.3 ( 19.1

60.6 ( 47.3

^an ) 12; the statistical analysis of the data was carried out by the use of an ANOVA test, and p < 0.05 was considered significant.

Table 10. Effect of Orally Administered Estradiol-RGD Peptide

Conjugates on Femur Weight, Femur Ash Weight and Femur Length of

Mice^a

40.0 mg/kg, i.p.), followed by removal of the left femur. After

the treatment of the left femurs by use of the same procedure

as mentioned above, dry femur weights, femur ash weights,

femur length, femur calcium content, femur phosphorus content,

and femur mineral content of the mice receiving vehicle (CMC),

estradiol, a mixture of estradiol and RGD peptides, 11-13 and

19-21 were tested. The data are listed in Tables 10-12. The

data of the tables indicate that though oral administration of

estradiol is unable to inhibit the bone loss of the mice, the oral

administration of 11-13 and 19-21 is able to inhibit the bone

loss of the mice. The femur weight, femur ash weight, femur

length, femur calcium content, femur phosphorus content, and

femur mineral content of the mice orally administered with 11-

13 and 19-21 are significantly higher than that of the mice

orally administered with vehicle (OVX group), estradiol, and a

mixture of estradiol and RGD peptides. The results suggest that

the estradiol-3-RGD peptide conjugates and estradiol-17-RGD

peptide conjugates are both orally active.

weight of

dry femur

(mg)

weight of

femur ash

(mg)

length of

femur

(cm)

group

OVX

sham

estradiol

estradiol + RGDS

estradiol + RGDV

estradiol + RGDF

58.8 ( 4.15

73.3 ( 7.18^c

63.1 ( 6.54

62.9 ( 5.20

68.8 ( 6.42^d

63.0 ( 5.14

68.2 ( 5.32^d

62.6 ( 5.24

62.7 ( 4.77^b

68.6 ( 5.67^d

68.4 ( 4.68^d

63.1 ( 5.15^b

18.0 ( 2.20

42.4 ( 4.31^c

22.2 ( 4.23

22.3 ( 3.97

29.3 ( 4.54^e

22.0 ( 4.30

33.0 ( 5.81^e

21.7 ( 4.11

30.0 ( 4.67^e

30.4 ( 6.18^e

33.7 ( 7.46^e

28.7 ( 3.89^e

1.601 ( 0.0394

1.642 ( 0.0440^c

1.622 ( 0.0447

1.620 ( 0.0512

1.654 ( 0.0550^c

1.621 ( 0.0509

1.645 ( 0.0470^c

1.610 ( 0.0439

1.638 ( 0.0420^b

1.635 ( 0.0362^b

1.614 ( 0.0329

1.626 ( 0.0296

^aDosage ) 110.3 × 10^-3mmol/kg, n ) 12. OVX ) ovariotomy. The

statistical analysis of the data was carried out by the use of an ANOVA

test, and p < 0.05 was considered significant. ^bCompared to OVX, P <

0.05. ^cCompared to OVX, P < 0.01. ^dCompared to OVX, P < 0.01, and

to estradiol, P < 0.05. ^eCompared to OVX and estradiol, P < 0.01.

2.13. Oral Administration of Estrogen-RGD Peptide

Conjugates Gave No Influence on the Uterus of the Mice.

To examine the effect of oral administration of estradiol-RGD

peptide conjugates on the weights of organs, the removed lungs,

livers, spleens, and uteri of the mice orally receiving 110.3 ×

content, the serum phosphorus content, and the serum ALP level

were measured as mentioned above. After blood drawing, the

mice were euthanized by anesthetization (pentobarbital sodium,

3346 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 14

Xiong et al.

Table 11. Effect of Orally Administered Estradiol-RGD Peptide

Conjugates on Femur Calcium Content, Femur Phosphorus Content, and

Femur Mineral Content of Mice^a

2.14. Conclusion. In conclusion, through the conjugation of

estrogen and RGD peptides, the present paper provides a

modification method for estrogen to improve the efficacy of

HRT. By the in vivo assay data of nine novel estrogen-RGD

peptide conjugates, it is recognized that this kind of conjugation

is a promising new approach by which anti-osteoperotic drugs

with improved therapeutic efficacy might be developed for use

in HRT. For instance, in a mouse model, i.p. administration of

the estradiol-3-RGD, estradiol-17-RGD, and estrone-3-RGD

peptide conjugates resulted in decreased serum concentrations

of calcium and the level of ALP, as well as increased levels of

calcium, phosphorus, and minerals in the femur. Furthermore,

the anti-osteoporosis action of these compounds followed a dose-

dependent manner and was accompanied with no observable

effects on endometrial cell hyperplasia. In addition to these

compounds all exhibiting biological activity when administered

by the i.p. route, we were particularly pleased to note that the

estradiol-3-RGD and estradiol-17-RGD peptide conjugates

were both orally active.

calcium

(%)

phosphorous

(%)

mineral

(ratio, %)

group

OVX

sham

estradiol

estradiol +

RGDS

estradiol +

RGDV

estradiol +

RGDF

39.606 ( 2.563

53.400 ( 2.566^d

41.729 ( 2.670

41.699 ( 2.701

22.972 ( 1.958

25.101 ( 1.925^c

24.530 ( 2.173

24.499 ( 2.007

0.371 ( 0.0779

0.520 ( 0.0811^d

0.411 ( 0.0822

0.413 ( 0.0746

51.201 ( 2.653^d

41.694 ( 2.658

24.885 ( 2.300^b

24.532 ( 2.187

0.515 ( 0.0807^c

0.408 ( 0.0818

52.003 ( 2.776^d

41.705 ( 2.712

24.879 ( 1.963^b

24.465 ( 2.185

0.525 ( 0.0795^d

0.409 ( 0.0791

51.903 ( 2.822^d

51.988 ( 2.689^d

52.197 ( 2.765^d

53.001 ( 2.699^d

24.869 ( 2.126^b

24.994 ( 2.211^b

24.997 ( 2.312^b

25.532 ( 2.209^c

0.516 ( 0.0812^d

0.522 ( 0.0783^d

0.519 ( 0.0794^d

0.525 ( 0.0859^d

^aDosage ) 110.3 × 10^-3mmol/kg, n ) 12. OVX ) ovariotomy. The

statistical analysis of the data was carried out by the use of an ANOVA

test, and p < 0.05 was considered significant. ^bCompared to OVX, P <

0.05. ^cCompared to OVX, P < 0.01. ^dCompared to OVX and estradiol, P

< 0.01.

3. Experimental Section

3.1. Chemical Synthesis. The protected amino acids (AAs) with

L-configuration were purchased from Sigma Chemical Co. All

coupling and deprotection reactions were carried out under anhy-

drous conditions. Chromatography was performed on Qingdao silica

gel H. The purity of the intermediates and the products was

confirmed by TLC (Merck silica gel plates of type 60 F₂₅₄, 0.25

mm layer thickness) and HPLC (Waters, C₁₈column 4.6 × 150

mm). The AA analysis was determined by Hitachi 835-50 instru-

ment. FAB-MS were determined by VG-ZAB-MS high-resolution

GC/MS/DS and HP ES-5989x. Optical rotations were determined

on Schmidt+Haensch Polartronic D instrument.

3.1.1. General Procedure for Removal of the Boc of the

C-Terminal Component. The solution of 0.20 mmol of Boc-

protected compound in 2 mL of hydrogen chloride in ethyl acetate

(4 mol/L) was stirred at room temperature for 3 h. The reaction

mixture was evaporated to remove the solvent. The residue was

dissolved in 10 mL of ethyl acetate, and the solution was evaporated

to dryness. The resultant solid was used directly for a subsequent

coupling reaction.

Table 12. Effect of Oral Estradiol-RGD Peptide Conjugates on Serum

Calcium Content, Serum Phosphorus Content, and ALP Level of Mice^a

calcium

(%)

phosphorous

(%)

ALP

(U/L, king)

group

OVX

sham

estradiol

estradiol +

RGDS

estradiol +

RGDV

2.026 ( 0.0450

2.033 ( 0.0482

2.022 ( 0.0469

2.030 ( 0.0671

1.604 ( 0.00471

1.606 ( 0.00519

1.605 ( 0.00488

1.602 ( 0.00530

40.223 ( 2.947

34.691 ( 3.155^b

37.705 ( 3.221

38.114 ( 3.196

2.040 ( 0.0490

2.029 ( 0.0477

1.604 ( 0.00497

1.605 ( 0.00525

35.400 ( 3.134^b

38.006 ( 3.200

2.033 ( 0.0474

2.029 ( 0.0465

1.606 ( 0.00499

1.603 ( 0.00519

33.411 ( 2.780^c

37.995 ( 3.100

estradiol +

RGDF

2.036 ( 0.0476

2.032 ( 0.0489

2.035 ( 0.0491

2.041 ( 0.0526

1.602 ( 0.00590

1.604 ( 0.00438

1.606 ( 0.00496

1.603 ( 0.00319

35.511 ( 2.939^b

35.211 ( 2.896^b

34.521 ( 2.488^d

33.700 ( 2.682^c

3.1.2. Boc-Arg(NO₂)-Gly-Asp(OBzl)-Ser(Bzl)-OBzl. (1) Boc-

Asp(OBzl)-Ser(Bzl)-OBzl. To a solution of 420 mg (1.30 mmol)

of Boc-Asp(OBzl)-OH in 2 mL of anhydrous THF, 176 mg (1.30

mmol) of HOBt and 268 mg (1.30 mmol) of DCC were added.

The mixture was stirred at 0 °C for 0.5 h to provide solution A. At

0 °C, to a solution of 418 mg (1.30 mmol) of HCl‚H-Ser(Bzl)-

OBzl in 2 mL of anhydrous THF, 155 µL of N-methylmorpholine

was added, and the mixture was stirred for 10 min to provide

solution B. At 0 °C, solutions A and B were mixed. The solution

was adjusted with N-methylmorpholine to pH 8.5. The reaction

mixture was stirred at room temperature for 8 h, and TLC (CHCl₃/

CH₃OH, 10:1) indicated the complete disappearance of Boc-Asp-

(OBzl)-OH. On evaporation, the residue was dissolved in 50 mL

of ethyl acetate. The solution was washed successively with 5%

sodium bicarbonate, 5% citric acid, and saturated sodium chloride

and the organic phase was dried over anhydrous sodium sulfate.

Filtration, evaporation under reduced pressure, and purification by

chromatography (CHCl₃/CH₃OH, 30:1) provided 722 mg (94%)

of the title compound as a colorless powder. ESI-MS (m/e) 591

[M + H]⁺.

(2) HCl‚H-Asp(OBzl)-Ser(Bzl)-OBzl. A solution of 500 mg

(1.30 mmol) of Boc-Asp(OBzl)-Ser(Bzl)-OBzl in 3 mL of hydrogen

chloride in ethyl acetate (4 mol/L) was stirred at room temperature

for 2 h, and TLC (CHCl₃/CH₃OH, 10:1) indicated the complete

disappearance of Boc-Asp(OBzl)-Ser(Bzl)-OBzl. The reaction

mixture was evaporated to remove the solvent. The residue was

dissolved in 10 mL of ethyl acetate, and the solution was evaporated

to dryness. The resulting solid was used for a subsequent coupling

reaction directly. ESI-MS (m/e) 591 [M + H]⁺.

^aDosage ) 110.3 × 10^-3mmol/kg, n ) 12. OVX ) ovariotomy. The

statistical analysis of the data was carried out by the use of an ANOVA

test, and p < 0.05 was considered significant. ^bCompared to OVX, P <

0.01. ^cCompared to OVX and estradiol, P < 0.01. ^dCompared to OVX, P

< 0.01, and to estradiol, P < 0.05.

Table 13. Effect of Oral Estradiol-RGD Peptide Conjugates on the

Mice Uterus Weight

lung

(mg)

liver

(g)

spleen

(mg)

uterus

(mg)

group

OVX

sham

165.0 ( 23.9 1.332 ( 0.176 130.0 ( 31.9 89.0 ( 24.9

164.0 ( 24.2 1.337 ( 0.191 131.0 ( 41.5 41.5 ( 30.1

estradiol 168.0 ( 25.1 1.335 ( 0.198 141.0 ( 35.9 40.1 ( 18.4

161.0 ( 25.5 1.331 ( 0.189 135.0 ( 35.5 36.7 ( 13.0

167.0 ( 24.9 1.340 ( 0.188 133.0 ( 36.7 50.7 ( 31.9

167.0 ( 24.5 1.338 ( 0.199 142.0 ( 38.0 38.2 ( 10.8

166.0 ( 24.8 1.329 ( 0.194 138.0 ( 33.5 43.2 ( 23.6

163.0 ( 24.4 1.337 ( 0.199 134.0 ( 37.6 40.7 ( 19.6

165.0 ( 24.0 1.339 ( 0.188 135.0 ( 38.7 47.5 ( 30.5

^aDosage ) 110.3 × 10^-3mmol/kg, n ) 12. OVX ) ovariotomy.

10^-3mmol/kg of estradiol, 11-13, and 19-21 were directly

weighed and the data are listed in Table 13. As shown in Table

13, the weights of lungs, livers, spleens, and uteri of the mice

of all groups exhibit no significant difference. These results

suggest that the anti-osteoporosis action of oral administration

of estradiol-RGD peptide conjugates had no observable effects

on endometrial cell hyperplasia even though an obvious anti-

osteoporosis action was observed.

HRT with Estrogen-RGD Peptide Conjugates

Journal of Medicinal Chemistry, 2007, Vol. 50, No. 14 3347

(3) Boc-Gly-Asp(OBzl)-Ser(Bzl)-OBzl. When the same proce-

dure for preparing Boc-Asp(OBzl)-Ser(Bzl)-OBzl was used, from

227 mg (1.30 mmol) of Boc-Gly-OH and 767 mg (1.30 mmol) of

HCl‚H-Asp(OBzl)-Ser(Bzl)-OBzl, 765 mg (91%) of the title

compound was obtained as a colorless powder. ESI-MS (m/e) 648

[M + H]⁺.

(4) HCl‚H-Gly-Asp(OBzl)-Ser(Bzl)-OBzl. When the same

procedure for preparing HCl‚H-Asp(OBzl)-Ser(Bzl)-OBzl was used,

from 841 mg (1.30 mmol) of Boc-Gly-Asp(OBzl)-Ser(Bzl)-OBzl,

663 mg (93%) of the title compound was obtained. The resulting

solid was used for a subsequent coupling reaction directly. ESI-

MS (m/e) 548 [M + H]⁺.

Hz, 2H), 1.629 (m, J ) 6.37 Hz, 2H),1.015 (d, J ) 4.14 Hz, 6H),

1.388 (s, 9H). ESI-MS (m/e) 771 [M + H]⁺. [R]²⁰9.0 (c 1.0,

CHCl₃/MeOH 10:1). AA analysis: calcd, Arg/Gly/Asp/Val ) 1.00:

1.00:1.00:1.00; found, Arg/Gly/Asp/Val ) 0.97:1.00:0.98:0.98.

3.1.4. Boc-Arg(NO₂)-Gly-Asp(OBzl)-Phe-OBzl. (1) Boc-Asp-

(OBzl)-Phe-OBzl. When the same procedure for preparing Boc-

Asp(OBzl)-Ser(Bzl)-OBzl was used, from 420 mg (1.30 mmol) of

Boc-Asp(OBzl)-OH and 376 mg (1.30 mmol) of HCl‚H-Phe-OBzl,

626 mg (96%) of the title compound was obtained as a colorless

powder. Mp 88-90 °C, ESI-MS (m/e) 561 [M + H]⁺.

(2) HCl‚H-Asp(OBzl)-Phe-OBzl. When the same procedure for

preparing HCl‚H-Asp(OBzl)-Ser(Bzl)-OBzl was used, from 728 mg

(1.30 mmol) of Boc-Asp(OBzl)-Phe-OBzl, 571 mg (95%) of the

title compound was obtained as a colorless powder. The resulting

solid was used directly for subsequent coupling reaction. ESI-MS

(m/e) 461 [M + H]⁺.

(3) Boc-Gly-Asp(OBzl)-Val-OBzl. When the same procedure

for preparing Boc-Asp(OBzl)-Ser(Bzl)-OBzl was used, from 227

mg (1.30 mmol) of Boc-Gly-OH and 645 mg (1.30 mmol) of HCl‚

H-Asp(OBzl)-Val-OBzl, 754 mg (94%) of the title compound was

obtained as a colorless powder. Mp 72-74 °C. ESI-MS (m/e) 618

[M + H]⁺.

(5) Boc-Arg(NO₂)-Gly-Asp(OBzl)-Ser(Bzl)-OBzl. When the

same procedure for preparing Boc-Asp(OBzl)-Ser(Bzl)-OBzl was

used, from 415 mg (1.30 mmol) of Boc-Arg(NO₂)-OH and 758

mg (1.30 mmol) of HCl‚H-Gly-Asp(OBzl)-Ser(Bzl)-OBzl, 982 mg

(89%) of the title compound was obtained as a colorless powder.

IR (KBr) 3350, 3345, 3323, 3031, 1681, 1600, 1503, 1462, 1445,

1393, 1370, 740, 696 cm^-1. H NMR (DMSO-d₆) 8.951 (s, 1H),

8.401 (s, 1H), 8.237 (s, 1H), 8.110 (s, 1H), 8.010 (s, 1H), 7.350 (t,

J ) 7.86 Hz, 1H), 7.343 (t, J ) 7.88 Hz, 1H), 7.337 (t, J ) 7.84

Hz, 1H), 7.304 (t, J ) 7.88 Hz, 2H), 7.293 (t, J ) 7.88 Hz, 2H),

7.253 (t, J ) 7.84 Hz, 2H), 7.246 (d, J ) 7.84 Hz, 2H), 7.186 (d,

J ) 7.82 Hz, 2H), 7.175 (d, J ) 7.82 Hz, 2H), 7.038 (s, 1H), 6.746

(s, 1H), 5.166 (s, 2H), 5.149 (s, 2H), 5.135 (s, 2H), 4.902 (dt, J )

6.31 Hz, J ) 6.60 Hz, 1H), 4.714 (t, J ) 6.50 Hz, 1H), 4.703 (t,

J ) 6.44 Hz, 1H), 3.967 (s, 2H), 3.771 (d, J ) 6.44 Hz, 2H), 2.830

(d, J ) 6.60 Hz, 2H), 2.627 (t, J ) 6.48 Hz, 2H), 1.840 (m, J )

4.42 Hz, 2H), 1.627 (m, J ) 6.36 Hz, 2H), 1.386 (s, 9H). ESI-MS

(4) HCl‚H-Gly-Asp(OBzl)-Phe-OBzl. When the same procedure

for preparing HCl‚H-Asp(OBzl)-Ser(Bzl)-OBzl was used, from 802

mg (1.30 mmol) of Boc-Gly-Asp(OBzl)-Phe-OBzl, 633 mg (94%)

of the title compound was obtained as a colorless powder. The

resulting solid was used directly for a subsequent coupling reaction.

ESI-MS (m/e) 518 [M + H]⁺.

(5) Boc-Arg(NO₂)-Gly-Asp(OBzl)-Phe-OBzl. When the same

procedure for preparing Boc-Asp(OBzl)-Ser(Bzl)-OBzl was used,

from 415 mg (1.30 mmol) of Boc-Arg(NO₂)-OH and 719 mg (1.30

mmol) of HCl‚H-Gly-Asp(OBzl)-Phe-OBzl, 926 mg (87%) of the

title compound was obtained as a colorless powder. Mp 148-

150 °C. IR (KBr) 3350, 3342, 3326, 3030, 1682, 1601, 1500, 1463,

(m/e) 849 [M + H]⁺. Mp 72-74 °C. [R]²⁰5.0 (c 1.0, CHCl₃/

MeOH 10:1). AA analysis: calcd, Arg/Gly/Asp/Ser ) 1.00:1.00:

1.00:1.00; found, Arg/Gly/Asp/Ser ) 0.98:1.00:1.04:0.97.

3.1.3. Boc-Arg(NO₂)-Gly-Asp(OBzl)-Val-OBzl. (1) Boc-Asp-

(OBzl)-Val-OBzl. When the same procedure for preparing Boc-

Asp(OBzl)-Ser(Bzl)-OBzl was used, from 420 mg (1.30 mmol) of

Boc-Asp(OBzl)-OH and 315 mg (1.30 mmol) of HCl‚H-Val-OBzl,

632 mg (95%) of the title compound was obtained as a colorless

powder. ESI-MS (m/e) 513 [M + H]⁺.

1444, 1392, 1374, 748, 702 cm^-1. H NMR (DMSO-d₆) 8.933 (s,

1H), 8.375 (s, 1H), 8.222 (s, 1H), 8.084 (s, 1H), 8.001 (s, 1H),

7.338 (t, J ) 7.83 Hz, 1H), 7.333 (t, J ) 7.84 Hz, 1H), 7.328 (t,

J ) 7.82 Hz, 1H), 7.305 (t, J ) 7.83 Hz, 2H), 7.290 (t, J ) 7.82

Hz, 2H), 7.274 (t, J ) 7.84 Hz, 2H), 7.240 (d, J ) 7.84 Hz, 2H),

7.180 (d, J ) 7.83 Hz, 2H), 7.176 (d, J ) 7.82 Hz, 2H), 7.035 (s,

1H), 6.743 (s, 1H), 5.125 (s, 2H), 5.133 (s, 2H), 5.007 (dt, J )

6.30 Hz, J ) 6.56 Hz, 1H), 4.880 (t, J ) 6.51 Hz, 1H), 4.416

(t, J ) 6.40 Hz, 1H), 4.100 (d, J ) 4.70 Hz, 2H), 3.168 (d, J )

6.40 Hz, 2H), 2.827 (d, J ) 6.50 Hz, 2H), 2.618 (t, J ) 6.48 Hz,

2H), 1.844 (m, J ) 4.45 Hz, 2H), 1.622 (m, J ) 6.30 Hz, 2H),

(2) HCl‚H-Asp(OBzl)-Val-OBzl. When the same procedure for

preparing HCl‚H-Asp(OBzl)-Ser(Bzl)-OBzl was used, from 666 mg

(1.30 mmol) of Boc-Asp(OBzl)-Val-OBzl, 505 mg (94%) of the

title compound was obtained as a colorless powder. The resulting

solid was used directly for a subsequent coupling reaction. ESI-

MS (m/e) 413 [M + H]⁺.

(3) Boc-Gly-Asp(OBzl)-Phe-OBzl. When the same procedure

for preparing Boc-Asp(OBzl)-Ser(Bzl)-OBzl was used, from 227

mg (1.30 mmol) of Boc-Gly-OH and 582 mg (1.30 mmol) of HCl‚

H-Asp(OBzl)-Val-OBzl, 711 mg (96%) of the title compound was

obtained as a colorless powder. Mp 77-79 °C; ESI-MS (m/e) 571

[M + H]⁺.

1.374 (s, 9H). ESI-MS (m/e) 819 [M + H]⁺. [R]²⁰7.0 (c 1.0,

CHCl₃/MeOH ) 10:1). AA analysis: calcd, Arg/Gly/Asp/Phe )

1.00:1.00:1.00:1.00; found, Arg/Gly/Asp/Phe ) 0.98:1.00:1.04:0.97.

3.1.5. General Procedure for the Removal of NO₂and Bzl of

the Protective Peptides. A suspension of 1.00 mmol of NO₂and

Bzl protected peptides, 25 mg of Pd/C (5%), and 30 mL of formic

acid in methanol (4.4%) was agitated with hydrogen (0.02 Mpa) at

room temperature for 24 h. The reaction mixture was filtrated. The

filtrate was evaporated, the residue was triturated with ether, and

the resulting solid was purified on a Sephadex G-10 column with

water as the mobil phase. The collected fractions were lyophilized

to provide the corresponding peptide.

(4) HCl‚H-Gly-Asp(OBzl)-Val-OBzl. When the same procedure

for preparing HCl‚H-Asp(OBzl)-Ser(Bzl)-OBzl was used, from 741

mg (1.30 mmol) of Boc-Gly-Asp(OBzl)-Val-OBzl, 581 mg (95%)

of the title compound was obtained. The resulting solid was used

directly for

subsequent coupling reaction. ESI-MS

(m/e) 471 [M + H]⁺.

(5) Boc-Arg(NO₂)-Gly-Asp(OBzl)-Val-OBzl. When the same

procedure for preparing Boc-Asp(OBzl)-Ser(Bzl)-OBzl was used,

from 415 mg (1.30 mmol) of Boc-Arg(NO₂)-OH and 658 mg (1.30

mmol) of HCl‚H-Gly-Asp(OBzl)-Val-OBzl, 901 mg (90%) of the

title compound was obtained as a colorless powder. Mp 95-

97 °C. IR (KBr) 3347, 3340, 3323, 3030, 1680, 1603, 1504, 1460,

3.1.6. H-Arg-Gly-Asp-Ser-OH. (1) HCl‚H-Arg(NO₂)-Gly-Asp-

(OBzl)-Ser(Bzl)-OBzl. When the general Boc deprotection pro-

cedure was used, from 848 mg (1.00 mmol) of Boc-Arg(NO₂)-

Gly-Asp(OBzl)-Ser(Bzl)-OBzl, 710 mg (93%) of the title compound

was obtained. ESI-MS (m/e) 739 [M + H]⁺.

(2) H-Arg-Gly-Asp-Ser-OH (4). When the general procedure

for catalytic hydrogenation of the protected peptides was used, from

774 mg (1.00 mmol) of HCl‚H-Arg(NO₂)-Gly-Asp(OBzl)-Ser(Bzl)-

OBzl, 399 mg (92%) of the title compound was obtained as a

colorless powder. Mp 183-187 °C. IR (KBr) 3451, 3348, 3340,

3325, 2455, 1680 cm^-1. ¹H NMR (DMSO-d₆) 10.89 (s, 1H), 10.80

(s, 1H), 8.760 (s, 2H), 8.270 (s, 1H), 8.213 (s, 1H), 8.006 (s, 1H),

7.238 (s, 2H), 6.668 (s, 1H), 6.543 (s, 1H), 4.670 (t, J ) 6.62 Hz,

1H), 4.528 (t, J ) 6.52 Hz, 1H), 4.512 (t, J ) 6.40 Hz, 1H), 4.113

1446, 1395, 1372, 747, 696 cm^-1. H NMR (DMSO-d₆) 8.942 (s,

1H), 8.386 (s, 1H), 8.223 (s, 1H), 8.085 (s, 1H), 8.011 (s, 1H),

7.336 (t, J ) 7.85 Hz, 1H), 7.330 (t, J ) 7.86 Hz, 1H), 7.302

(t, J ) 7.85 Hz, 2H), 7.290 (t, J ) 7.86 Hz, 2H), 7.240 (d, J )

7.85 Hz, 2H), 7.184 (d, J ) 7.86 Hz, 2H), 7.035 (s, 1H), 6.743 (s,

1H), 5.130 (s, 2H), 5.129 (s, 2H), 5.001 (dt, J ) 6.30 Hz, J ) 6.61

Hz, 1H), 4.887 (d, J ) 6.48 Hz, 1H), 4.423 (d, J ) 6.42 Hz, 1H),

4.104 (d, J ) 4.70 Hz, 2H), 3.078 (m, J ) 4.14 Hz, 1H), 2.833 (d,

J ) 6.62 Hz, 2H), 2.631 (t, J ) 6.47 Hz, 2H), 1.843 (m, J ) 4.44

3348 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 14

Xiong et al.

(d, J ) 6.40 Hz, 2H), 3.816 (s, 2H), 2.793 (d, J ) 6.40 Hz, 2H),

2.633 (t, J ) 4.40 Hz, 2H), 2.049 (s, 1H), 1.768 (m, J ) 4.40 Hz,

2H), 1.605 (m, J ) 6.01 Hz, 2H). ESI-MS (m/e) 434 [M + H]⁺.

Hz, 2H), 2.910 (m, J ) 4.22 Hz, 1H), 2.883 (t, J ) 5.11 Hz, 2H),

2.780 (t, J ) 5.11 Hz, 2H), 2.028 (m, J ) 4.78 Hz, 2H), 2.015 (m,

J ) 4.28 Hz, 2H), 1.871 (m, J ) 4.20 Hz, 1H), 1.840 (m, J ) 4.42

Hz, 2H), 1.761 (m, J ) 4.55 Hz, 2H), 1.703 (m, J ) 4.282 Hz,

1H), 1.654 (m, J ) 4.72 Hz, 2H), 0.774 (s, 3H). ESI-MS(m/e) 373

[R]²⁰6.0 (c 1.0, H₂O). AA analysis: calcd, Arg/Gly/Asp/Ser )

1.00:1.00:1.00:1.00; found, Arg/Gly/Asp/Ser ) 0.96:1.00:1.05:0.99.

3.1.7. H-Arg-Gly-Asp-Val-OH. (1) HCl‚H-Arg(NO₂)-Gly-Asp-

(OBzl)-Val-OBzl. When the general Boc deprotection procedure

was used, from 770 mg (1.00 mmol) of Boc-Arg(NO₂)-Gly-Asp-

(OBzl)-Val-OBzl, 664 mg (94%) of the title compound was

obtained. ESI-MS (m/e) 671 [M + H]⁺.

[M + H]⁺. [R]²⁰35.0 (c 1.00, THF). Anal. Calcd for C₂₂H₂₈O₅:

C, 70.94; H, 7.58. Found: C, 71.10; H, 7.69.

3.1.10. Estradiol-17-â-O-carbonylpropionyl-Arg(NO₂)-Gly-

Asp(OBzl)-Ser(Bzl)-OBzl (8). At 0 °C, to the solution of 88 mg

(0.24 mmol) of estradiol-17-â-O-carbonylpropionic acid and 32 mg

(0.24 mmol) of HOBt in 5 mL of anhydrous THF, 49 mg (0.24

mmol) of DCC was added. The mixture was stirred at 0 °C for 30

min and then the solution of 185 mg (0.24 mmol) of HCl·H-Arg-

(NO₂)-Gly-Asp(OBzl)-Ser(Bzl)-OBzl was added. The reaction

mixture was adjusted to pH 8.0 and stirred at room temperature

for 8 h, and TLC (ethyl acetate/petroleum, 6:1) indicated complete

disappearance of HCl·H-Arg(NO₂)-Gly-Asp(OBzl)-Ser(Bzl)-OBzl.

The reaction mixture was filtered, and the filtrate was evaporated

under vacuum. The residue was dissolved in 20 mL of chloroform

and washed with citric acid aqueous solution (5%), sodium

bicarbonate aqueous solution (5%), and saturated NaCl aqueous

solution succesively, then dried with anhydrous Na₂SO₄. After

filtration and evaporation, the residue was purified by flash

chromatography (chloroform/methanol, 10:1) to give 158 mg (61%)

of the title compound as a colorless powder. Mp 143-145 °C. IR

(KBr) 3353, 3342, 3325, 3084, 3070, 3033, 1745, 1687, 1603, 1501,

1460, 1445, 1397, 1368, 742, 694 cm^-1. ¹H NMR (DMSO-d₆) 8.942

(s, 1H), 8.389 (s, 1H), 8.223 (s, 1H), 8.087 (s, 1H), 8.004 (s, 1H),

7.344 (t, J ) 7.88 Hz, 1H), 7.341 (t, J ) 7.90 Hz, 1H), 7.334

(t, J ) 7.86 Hz, 1H), 7.301 (t, J ) 7.90 Hz, 2H), 7.291 (t, J )

7.90 Hz, 2H), 7.250 (t, J ) 7.86 Hz, 2H), 7.243 (d, J ) 7.86 Hz,

2H), 7.185 (d, J ) 7.84 Hz, 2H), 7.172 (d, J ) 7.84 Hz, 2H),

7.046 (d, J ) 7.52 Hz, 1H), 7.035 (s, 1H), 6.744 (s, 1H), 6.515

(d, J ) 7.51 Hz, 1H), 6.403 (s, 1H), 6.372 (s, 1H), 5.164 (s, 2H),

5.147 (s, 2H), 5.132 (s, 2H), 4.900 (dt, J ) 6.30 Hz, J ) 6.62 Hz,

1H), 4.711 (t, J ) 6.54 Hz, 1H), 4.700 (t, J ) 6.47 Hz, 1H), 4.612

(t, J ) 4.76 Hz, 1H), 3.964 (d, J ) 6.02 Hz, 2H), 3.768 (d, J )

4.12 Hz, 2H), 2.974 (t, J ) 4.56 Hz, 2H), 2.915 (m, J ) 4.25 Hz,

1H), 2.880 (t, J ) 5.16 Hz, 2H), 2.824 (d, J ) 6.62 Hz, 2H), 2.783

(t, J ) 5.13 Hz, 2H), 2.624 (t, J ) 6.50 Hz, 2H), 2.024 (m, J )

4.76 Hz, 2H), 2.017 (m, J ) 4.31 Hz, 2H), 1.874 (m, J ) 4.22 Hz,

1H), 1.843 (m, J ) 4.40 Hz, 2H), 1.765 (m, J ) 4.52 Hz, 2H),

1.723 (m, J ) 6.32 Hz, 2H), 1.700 (m, J ) 4.28 Hz, 1H), 1.656

(m, J ) 4.70 Hz, 2H), 1.625 (m, J ) 6.38 Hz, 2H), 0.776 (s, 3H).

[R]²⁰_D20.0 (c 1.00, CHCl₃/MeOH, 10:1). ESI-MS 1103 [M + H]⁺.

Anal. Calcd for C₅₈H₇₀N₈O₁₄: C, 63.14; H, 6.40; N, 10.16. Found:

C, 63.40; H, 6.58; N, 10.00. AA analysis: calcd, Arg/Gly/Asp/Ser

) 1.00:1.00:1.00:1.00; found, Arg/Gly/Asp/Ser ) 0.97:1.00:1.07:

0.98.

(2) H-Arg-Gly-Asp-Val-OH (5). When the general procedure

for catalytic hydrogenation of the protected peptides was used, from

706 mg (1.00 mmol) of HCl‚H-Arg(NO₂)-Gly-Asp(OBzl)-Val-

OBzl, 396 mg (89%) of the title compound was obtained. Mp 180-

182 °C. IR (KBr) 3349, 3337, 3330, 2430, 1682, 1390, 1372 cm^-1

¹H NMR (DMSO-d₆) 10.899 (s, 1H), 10.880 (s, 1H), 8.661 (s, 2H),

8.299 (s, 1H), 8.217 (s, 1H), 8.053 (s, 1H), 7.234 (s, 2H), 6.700

(s, 1H), 6.537 (s, 1H), 5.003 (d, J ) 6.59 Hz, 1H), 4.837 (d, J )

6.46 Hz, 1H), 4.395 (d, J ) 6.40 Hz, 1H), 3.997 (d, J ) 4.73 Hz,

2H), 2.891 (m, J ) 6.37 Hz, 1H), 2.782 (d, J ) 6.40 Hz, 2H),

2.628 (t, J ) 6.45 Hz, 2H), 1.763 (m, J ) 6.44 Hz, 2H), 1.604 (m,

J ) 6.35 Hz, 2H), 1.009 (d, J ) 6.37 Hz, 6H). ESI-MS (m/e) 446

[M + H]⁺. [R]²⁰3.0 (c 1.0, H₂O). AA analysis: calcd, Arg/Gly/

Asp/Val ) 1.00:1.00:1.00:1.00; found, Arg/Gly/Asp/Val ) 0.96:

1.00:0.98:0.97.

3.1.8. H-Arg-Gly-Asp-Phe-OH. (1) HCl‚H-Arg(NO₂)-Gly-

Asp(OBzl)-Phe-OBzl. When the general Boc deprotection proce-

dure was used, from 834 mg (1.00 mmol) of Boc-Arg(NO₂)-Gly-

Asp(OBzl)-Phe-OBzl, 700 mg (93%) of the title compound was

obtained. ESI-MS (m/e) 718 [M + H]⁺.

(2) H-Arg-Gly-Asp-Phe-OH (6). When the general procedure

for catalytic hydrogenation of the protected peptides was used, from

734 mg (1.00 mmol) of Boc-Arg(NO₂)-Gly-Asp(OBzl)-Phe-OBzl,

454 mg (92%) of the title compound was obtained. Mp 158-

160 °C. IR (KBr) 3353, 3345, 3322, 2422, 1684, 1601, 1500, 1460,

1450, 700 cm^-^{1 1}H NMR (DMSO-d₆) 10.975 (s, 1H), 10.901

(s, 1H), 8.620 (s, 2H), 8.251 (s, 1H), 8.174 (s, 1H), 8.032 (s, 1H),

7.312 (s, 2H), 7.227 (t, J ) 7.80 Hz, 2H), 7.156 (d, J ) 7.80 Hz,

2H), 7.098 (t, J ) 7.80 Hz, 1H), 6.927 (s, 1H), 6.746 (s, 1H), 4.968

(t, J ) 6.47 Hz, 1H), 4.815 (d, J ) 6.40 Hz, 1H), 4.433 (t, J )

6.42 Hz, 1H), 4.102 (d, J ) 4.70 Hz, 2H), 3.009 (d, J ) 6.42 Hz,

2H), 2.801 (d, J ) 6.47 Hz, 2H), 2.614 (t, J ) 6.45 Hz, 2H), 1.825

(m, J ) 5.99 Hz, 2H), 1.617 (m, J ) 6.46 Hz, 2H). ESI-MS (m/e)

494 [M + H]⁺. [R]²⁰4.0 (c 2.0, H₂O). AA analysis: calcd, Arg/

Gly/Asp/Phe ) 1.00:1.00:1.00:1.00; found, Arg/Gly/Asp/Phe )

0.96:1.00:0.98:0.98.

3.1.9. Estradiol-17-â-O-carbonylpropionic Acid (7). To a warm

solution of 100 mg (1.0 mmol) of succinic anhydride 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

and the ethyl acetate phase was separated and dried with anhydrous

Na₂SO₄. After filtration, the filtrate was evaporation at reduced

pressure to give a yellow syrup, which was dissolved in 1 mL of

methanol. The solution was adjusted to pH 8.5-9.0 with cold

aqueous K₂CO₃(10%) and stirred at room temperature for 18 h.

The solution was adjusted to a pH of 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 three times and drying

with anhydrous Na₂SO₄, the ethyl acetate phase was evaporated to

give a yellowish syrup. The syrup was kept in a refrigerater 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, 1602, 1504,

3.1.11. Estradiol-17-â-O-carbonylpropionyl-Arg(NO₂)-Gly-

Asp(OBzl)-Val-OBzl (9). When the same procedure as that used

in the preparation of 8 was used, from 121 mg (0.33 mmol) of

estradiol-17-â-O-carbonylpropinic acid and 230 mg (0.33 mmol)

of HCl·H-Arg(NO₂)-Gly-Asp(OBzl)-Val-OBzl, 177 mg (53%) of

the title compound was obtained as a colorless powder. Mp 151-

153 °C. IR (KBr) 3350, 3344, 3326, 3087, 3071, 3032, 1746, 1684,

1601, 1500, 1462, 1448, 1386, 1370, 746, 698 cm^-1. H NMR

(DMSO-d₆) 8.940 (s, 1H), 8.385 (s, 1H), 8.220 (s, 1H), 8.083

(s, 1H), 8.007 (s, 1H), 7.338 (t, J ) 7.87 Hz, 1H), 7.332 (t, J )

7.88 Hz, 1H), 7.304 (t, J ) 7.87 Hz, 2H), 7.294 (t, J ) 7.86 Hz,

2H), 7.241 (d, J ) 7.87 Hz, 2H), 7.186 (d, J ) 7.85 Hz, 2H),

7.044 (d, J ) 7.54 Hz, 1H), 7.037 (s, 1H), 6.746 (s, 1H), 6.517

(d, J ) 7.53 Hz, 1H), 6.401 (s, 1H), 6.370 (s, 1H), 5.127 (s, 2H),

5.135 (s, 2H), 5.007 (dt, J ) 6.32 Hz, J ) 6.60 Hz, 1H), 4.892

(d, J ) 6.51 Hz, 1H), 4.534 (d, J ) 6.50 Hz, 1H), 4.421 (d, J )

6.45 Hz, 1H), 4.106 (d, J ) 4.73 Hz, 2H), 3.071 (m, J ) 4.12 Hz,

1H), 2.970 (t, J ) 4.54 Hz, 2H), 2.911 (m, J ) 4.26 Hz, 1H),

2.882 (t, J ) 5.15 Hz, 2H), 2.825 (d, J ) 6.60 Hz, 2H), 2.784

(t, J ) 5.14 Hz, 2H), 2.620 (t, J ) 6.51 Hz, 2H), 2.025 (m, J )

4.75 Hz, 2H), 2.014 (m, J ) 4.30 Hz, 2H), 1.870 (m, J ) 4.20 Hz,

1H), 1.844 (m, J ) 4.42 Hz, 2H), 1.763 (m, J ) 4.50 Hz, 2H),

1463, 1372, 876, 831 cm^-1. H NMR (DMSO-d₆) 12.192 (s, 1H),

8.993 (s, 1H), 7.043 (d, J ) 7.50 Hz, 1H), 6.517 (d, J ) 7.50 Hz,

1H), 6,400 (s,1H), 4.618 (t, J ) 4.78 Hz, 1H), 2.972 (t, J ) 4.55

HRT with Estrogen-RGD Peptide Conjugates

Journal of Medicinal Chemistry, 2007, Vol. 50, No. 14 3349

1.724 (m, J ) 6.33 Hz, 2H), 1.702 (m, J ) 4.29 Hz, 1H), 1.653

(m, J ) 4.72 Hz, 2H), 1.621 (m, J ) 6.35 Hz, 2H), 1.012 (d, J )

4.12 Hz, 6H), 0.779 (s, 3H). Anal. Calcd for C₅₃H₆₈N₈O₁₃: C, 62.09;

H, 6.69; N, 10.93. Found: C, 61.92; H, 6.51; N, 11.12. AA

analysis: calcd, Arg/Gly/Asp/Val ) 1.00:1.00:1.00:1.00; found,

Arg/Gly/Asp/Val ) 0.98:1.00:0.97:0.98. [R]²⁰_D30.0 (c 1.00, CHCl₃/

MeOH, 10:1). ESI-MS 1025 [M + H]⁺.

carbonylpropinyl-Arg(NO₂)-Gly-Asp(OBzl)-Val-OBzl (9), 54 mg

(68%) of the title compound was obtained as a colorless powder.

Mp 193-195 °C. IR (KBr) 3360, 3357, 3348, 3030, 1689, 1603,

1501, 1460, 1445, 1382, 1373, 749, 703 cm^-1. ¹H NMR (DMSO-

d₆) 9.155 (s, 1H), 9.127 (s, 1H), 9.032 (s, 1H), 8.226 (s, 1H), 8.203

(s, 1H), 8.075 (s, 1H), 8.002 (s, 1H), 7.225 (s, 2H), 7.040 (d, J )

7.54 Hz, 1H), 7.039 (s, 1H), 6.722 (s, 1H), 6.512 (s, 1H), 6.449

(d, J ) 7.50 Hz, 1H), 4.867 (d, J ) 6.52 Hz, 1H), 4.611 (t, J )

6.32 Hz, 1H), 4.532 (d, J ) 6.51 Hz, 1H), 4.423 (d, J ) 6.43 Hz,

1H), 4.101 (d, J ) 4.74 Hz, 2H), 2.962 (t, J ) 4.56 Hz, 2H), 2.920

(m, J ) 4.13 Hz, 1H), 2.903 (m, J ) 4.27 Hz, 1H), 2.880 (t, J )

5.14 Hz, 2H), 2.822 (d, J ) 6.57 Hz, 2H), 2.780 (t, J ) 5.15 Hz,

2H), 2.614 (t, J ) 6.50 Hz, 2H), 2.027 (m, J ) 4.76 Hz, 2H),

2.016 (m, J ) 4.32 Hz, 2H), 1.866 (m, J ) 4.21 Hz, 1H), 1.845

(m, J ) 4.43 Hz, 2H), 1.760 (m, J ) 4.51 Hz, 2H), 1.720 (m, J )

6.30 Hz, 2H), 1.700 (m, J ) 4.29 Hz, 1H), 1.655 (m, J ) 4.70 Hz,

2H), 1.617 (m, J ) 6.34 Hz, 2H), 0.862 (d, J ) 4.15 Hz, 6H),

0.754 (s, 3H). Anal. Calcd for C₃₉H₅₇N₇O₁₁: C, 58.56; H, 7.18; N,

12.26. Found: C, 58.75; H, 7.32; N, 12.45. AA analysis: calcd,

Arg/Gly/Asp/Val ) 1.00:1.00:1.00:1.00; found, Arg/Gly/Asp/Val

3.1.12. Estradiol-17-â-O-carbonylpropionyl-Arg(NO₂)-Gly-

Asp(OBzl)-Phe-OBzl (10). When the same procedure as that used

in the preparation of 8 was used, from 39 mg (0.11 mmol) of

estradiol-17-â-O-carbonylpropionic acid and 78 mg (0.11 mmol)

of HCl·H-Arg(NO₂)-Gly-Asp(OBzl)-Phe-OBzl, 65 mg (53%) of the

title compound was obtained as a colorless powder. Mp 156-

158 °C. IR (KBr) 3352, 3345, 3323, 3084, 3068, 3030, 1744, 1680,

1603, 1502, 1465, 1446, 1390, 1364, 745, 701 cm^-1. H NMR

(DMSO-d₆) 8.935 (s, 1H), 8.377 (s, 1H), 8.225 (s, 1H), 8.087

(s, 1H), 8.002 (s, 1H), 7.340 (t, J ) 7.85 Hz, 1H), 7.336 (t, J )

7.86 Hz, 1H), 7.330 (t, J ) 7.84 Hz, 1H), 7.307 (t, J ) 7.85 Hz,

2H), 7.292 (t, J ) 7.84 Hz, 2H), 7.272 (t, J ) 7.80 Hz, 2H), 7.243

(d, J ) 7.86 Hz, 2H), 7.184 (d, J ) 7.83 Hz, 2H), 7.177 (d, J )

7.80 Hz, 2H), 7.040 (d, J ) 7.55 Hz, 1H), 7.032 (s, 1H), 6.741

(s, 1H), 6.509 (d, J ) 7.51 Hz, 1H), 6.403 (s, 1H), 6.368 (s, 1H),

5.124 (s, 2H), 5.131 (s, 2H), 5.011 (dt, J ) 6.33 Hz, J ) 6.58 Hz,

1H), 4.886 (t, J ) 6.50 Hz, 1H), 4.530 (t, J ) 6.51 Hz, 1H), 4.417

(t, J ) 6.42 Hz, 1H), 4.101 (d, J ) 4.72 Hz, 2H), 3.165 (d, J )

6.45 Hz, 2H), 2.967 (t, J ) 4.55 Hz, 2H), 2.907 (m, J ) 4.27 Hz,

1H), 2.879 (t, J ) 5.14 Hz, 2H), 2.822 (d, J ) 6.55 Hz, 2H), 2.780

(t, J ) 5.15 Hz, 2H), 2.611 (t, J ) 6.50 Hz, 2H), 2.027 (m, J )

4.73 Hz, 2H), 2.017 (m, J ) 4.32 Hz, 2H), 1.869 (m, J ) 4.22 Hz,

1H), 1.843 (m, J ) 4.43 Hz, 2H), 1.760 (m, J ) 4.52 Hz, 2H),

1.725 (m, J ) 6.30 Hz, 2H), 1.700 (m, J ) 4.27 Hz, 1H), 1.655

(m, J ) 4.70 Hz, 2H), 1.617 (m, J ) 6.32 Hz, 2H), 0.824 (s, 3H).

[R]²⁰_D23.0 (c 1.00, CHCl₃/MeOH, 10:1). ESI-MS 1073 [M + H]⁺.

Anal. Calcd for C₅₇H₆₈N₈O₁₃: C, 63.79; H, 6.39; N, 10.44. Found:

C, 63.52; H, 6.23; N, 10.61. AA analysis: calcd, Arg/Gly/Asp/

Phe ) 1.00:1.00:1.00:1.00; found, Arg/Gly/Asp/Phe ) 0.97:1.00:

1.05:0.98.

) 0.99:1.00:1.03:0.97. [R]²⁰56.0 (c 0.5, MeOH). ESI-MS (m/e)

800 [M + H]⁺.

3.1.15. Estradiol-17-â-O-carbonylpropionyl-Arg-Gly-Asp-

Phe-OH(13). When the same procedure as that used in the

preparation of 11 was used, from 100 mg (0.093 mmol) of estradiol-

17-â-O-carbonylpropionyl-Arg(NO₂)-Gly-Asp(OBzl)-Phe-OBzl (10),

46 mg (58%) of the title compound was obtained as a colorless

powder. Mp 196-198 °C. IR (KBr) 3391, 3382, 3377, 3032, 1681,

1605, 1505, 1462, 743, 700 cm^-1. H NMR (DMSO-d₆) 9.065

(s, 1H), 9.054 (s, 1H), 9.045 (s, 1H), 8.061 (s, 1H), 8.035 (s, 1H),

8.020 (s, 1H), 8.001 (s, 2H), 7.233 (s, 2H), 7.116 (t, J ) 7.81 Hz,

2H), 7.075 (d, J ) 7.80 Hz, 2H), 7.010 (t, J ) 7.79 Hz, 1H), 7.006

(s, 1H), 6.955 (d, J ) 7.56 Hz, 1H), 6.900 (s, 1H), 6.432 (d, J )

7.50 Hz, 1H), 4.910 (dt, J ) 6.30 Hz, J ) 6.55 Hz, 1H), 4.870

(t, J ) 6.52 Hz, 1H), 4.846 (t, J ) 6.50 Hz, 1H), 4.500 (t, J )

6.43 Hz, 1H), 4.122 (d, J ) 4.70 Hz, 2H), 3.042 (d, J ) 6.43 Hz,

2H), 2.963 (t, J ) 4.53 Hz, 2H), 2.910 (m, J ) 4.28 Hz, 1H),

2.872 (t, J ) 5.16 Hz, 2H), 2.820 (d, J ) 6.56 Hz, 2H), 2.776 (t,

J ) 5.16 Hz, 2H), 2.593 (t, J ) 6.52 Hz, 2H), 2.024 (m, J ) 4.70

Hz, 2H), 2.014 (m, J ) 4.34 Hz, 2H), 1.865 (m, J ) 4.24 Hz, 1H),

1.840 (m, J ) 4.42 Hz, 2H), 1.755 (m, J ) 4.53 Hz, 2H), 1.720

(m, J ) 6.32 Hz, 2H), 1.702 (m, J ) 4.26 Hz, 1H), 1.656 (m, J )

3.1.13. Estradiol-17-â-O-carbonylpropionyl-Arg-Gly-Asp-Ser-

OH (11). A suspension of 100 mg (0.091 mmol) of estradiol-17-

â-O-carbonylpropionyl-Arg(NO₂)-Gly-Asp(OBzl)-Ser(Bzl)-OBzl (8),

15 mg of Pd/C (10%), 3 mL of methanol, and 1 mL of THF were

stirred at room temperature. To this stirred suspension, hydrogen

was bubbled for 24 h and TLC (ethyl acetate/petroleum, 6:1)

indicated complete disappearance of 8. The reaction mixture was

filtered, and the filtrate was evaporated under vacuum. The residue

was triturated with ether and the colorless powder was purified on

a Sephadex LH 20 column (10-30% ethanol). The desirable

fraction was evaporated under vacuum to give 44 mg (62%) of the

title compound as a colorless powder. Mp 178-180 °C. IR (KBr)

3440, 3351, 3345, 3323, 3031, 1693, 1604, 1506, 1465, 1442, 746,

700 cm^-1. ¹H NMR (DMSO-d₆) 9.072 (s, 1H), 9.068 (s, 1H), 9.063

(s, 1H), 8.043 (s, 1H), 8.035 (s, 1H), 8.017 (s, 1H), 8.004 (s, 2H),

7.241 (s, 2H), 7.044 (d, J ) 7.50 Hz, 1H), 7.039 (s, 1H), 6.751

(s, 1H), 6.518 (d, J ) 7.50 Hz, 1H), 6.407 (s, 1H), 4.854 (dt, J )

6.32 Hz, J ) 6.60 Hz, 1H), 4.683 (t, J ) 6.53 Hz, 1H), 4.603 (t,

J ) 4.74 Hz, 1H), 4.590 (t, J ) 6.45 Hz, 1H), 4.021 (d, J ) 6.44

Hz, 2H), 3.960 (d, J ) 6.00 Hz, 2H), 2.972 (t, J ) 4.55 Hz, 2H),

2.913 (m, J ) 4.26 Hz, 1H), 2.879 (t, J ) 5.15 Hz, 2H), 2.820 (d,

J ) 6.60 Hz, 2H), 2.778 (t, J ) 5.12 Hz, 2H), 2.602 (t, J ) 6.52

Hz, 2H), 2.025 (m, J ) 4.75 Hz, 2H), 2.016 (m, J ) 4.32 Hz, 2H),

1.870 (m, J ) 4.23 Hz, 1H), 1.841 (m, J ) 4.42 Hz, 2H), 1.763

(m, J ) 4.53 Hz, 2H), 1.721 (m, J ) 6.30 Hz, 2H), 1.702 (m, J )

4.27 Hz, 1H), 1.653 (m, J ) 4.72 Hz, 2H), 1.622 (m, J ) 6.35 Hz,

4.71 Hz, 2H), 1.613 (m, J ) 6.30 Hz, 2H), 0.821 (s, 3H). [R]²⁰

45.0 (c 0.5, MeOH). ESI-MS (m/e) 848 [M + H]⁺. Anal. Calcd

for C₄₃H₅₇N₇O₁₁: C, 60.91; H, 6.78; N, 11.56. Found: C, 61.10;

H, 6.96; N, 11.37. AA analysis: calcd, Arg/Gly/Asp/Phe ) 1.00:

1.00:1.00:1.00; found, Arg/Gly/Asp/Phe ) 0.98:1.00:1.03:0.99.

3.1.16. Ethyl Estradiol-3-oxyacetate (14). A solution of 500

mg (1.85 mmol) of estradiol in 10 mL of anhydrous THF and 1.32

mL of sodium ethoxide in anhydrous ethanol (2 mmol/L) was mixed

and stirred at room temperature for 30 min, and then 0.612 mL

(5 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 four times to completely remove the

residual ethyl bromoacetate. The residue was purified by flash

chromatography (chloroform/ether, 10:0.3) to provide 550 mg

(83%) of the title compound as a colorless powder. Mp 88-90 °C.

IR (KBr) 3352, 3031, 2864, 1745, 1604, 1502, 1464, 1370, 874,

832 cm^-1. ¹H NMR (DMSO-d₆) 7.121 (d, J ) 7.52 Hz, 1H), 6.710

(s, 1H), 6.523 (d, J ) 7.52 Hz, 1H), 5.141 (s, 2H), 4.613 (q, J )

4.77 Hz, 1H), 4.203 (q, J ) 4.44 Hz, 2H), 2.974 (t, J ) 4.56 Hz,

2H), 2.907 (m, J ) 4.24 Hz, 1H), 2.551 (s, 1H), 2.025 (m, J )

4.76 Hz, 2H), 2.013 (m, J ) 4.26 Hz, 2H), 1.867 (m, J ) 4.22 Hz,

1H), 1.842 (m, J ) 4.43 Hz, 2H), 1.757 (m, J ) 4.56 Hz, 2H),

1.704 (m, J ) 4.28 Hz, 1H), 1.653 (m, J ) 4.73 Hz, 2H), 1.411

(t, J ) 4.44 Hz, 3H), 0.781 (s, 3H). ESI-MS(m/e) 359 [M + H]⁺.

[R]²⁰_D60.0 (c 1.00, THF). Anal. Calcd for C₂₂H₃₀O₄: C, 73.71; H,

8.44. Found: C, 73.58; H, 8.61.

2H), 0.773 (s, 3H). [R]²⁰50.0 (c 1.00, MeOH). ESI-MS 788 [M

+ H]⁺. Anal. Calcd for C₃₇H₅₃N₇O₁₂: C, 56.41; H, 6.78; N, 12.44.

Found: C, 56.60; H, 6.92; N, 12.26. AA analysis: calcd, Arg/Gly/

Asp/Ser ) 1.00:1.00:1.00:1.00; found, Arg/Gly/Asp/Ser ) 1.02:

1.00:1.01:0.97.

3.1.14. Estradiol-17-â-O-carbonylpropinyl-Arg-Gly-Asp-Val-

OH (12). When the same procedure as that used in the preparation

of 11 was used, from 100 mg (0.098 mmol) of estradiol-17-â-O-

3350 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 14

Xiong et al.

3.1.17. Estradiol-3-oxyacetic acid (15). At 0 °C to a solution

of 450 mg (1.26 mmol) of 3-ethyloxycarbonylmethylenoxylestradiol

(14) in 3 mL of anhydrous ethanol, 0.5 mL of NaOH aqueous

solution (2 mol/L) was added. The reaction mixture was stirred at

room temperature for 2.5 h and TLC (ethyl acetate/petroleum, 6:1)

indicated complete disappearance of 14. 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 397 mg (96%) of the title

compound as a colorless powder. Mp 214-215 °C. IR (KBr) 3350,

3033, 2866, 1726, 1602, 1501, 1463, 1372, 875, 831 cm^-1. ¹H NMR

(DMSO-d₆) 10.551 (s, 1H), 7.001 (d, J ) 7.50 Hz, 1H), 6.631

(s, 1H), 6.511 (d, J ) 7.51 Hz, 1H), 5.002 (s, 2H), 4.415 (q, J )

4.76 Hz, 1H), 2.966 (t, J ) 4.55 Hz, 2H), 2.889 (m, J ) 4.25 Hz,

1H), 2.560 (s, 1H), 2.020 (m, J ) 4.75 Hz, 2H), 2.010 (m, J )

4.25 Hz, 2H), 1.862 (m, J ) 4.23 Hz, 1H), 1.837 (m, J ) 4.44 Hz,

2H), 1.750 (m, J ) 4.55 Hz, 2H), 1.700 (m, J ) 4.25 Hz, 1H),

1.647 (m, J ) 4.74 Hz, 2H), 0.783 (s, 3H). ESI-MS (m/e) 331 [M

(m, J ) 6.32 Hz, 2H), 1.702 (m, J ) 4.26 Hz, 1H), 1.653 (m, J )

4.70 Hz, 2H), 1.627 (m, J ) 6.34 Hz, 2H), 0.816 (s, 3H), 0.780

(d, J ) 4.14 Hz, 6H). Anal. Calcd for C₅₁H₆₆N₈O₁₂: C, 62.31; H,

6.77; N, 11.40. Found: C, 62.14; H, 6.59; N, 11.57. AA analysis:

calcd, Arg/Gly/Asp/Val ) 1.00:1.00:1.00:1.00; found, Arg/Gly/Asp/

Val ) 0.99:1.00:0.98:0.97. [R]²⁰60.0 (c 1.00, MeOH). ESI-MS

983 [M + H]⁺.

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

Phe-OBzl (18). When the same procedure as that used in the

preparation of 8 was used, from 50 mg (0.15 mmol) of estradiol-

3-oxyacetic acid and 119 mg (0.15 mmol) of HCl·H-Arg(NO₂)-

Gly-Asp(OBzl)-Phe-OBzl, 97 mg (61%) of the title compound was

obtained as a colorless powder. Mp 136-138 °C. IR (KBr) 3354,

3343, 3326, 3081, 3065, 3031, 1742, 1683, 1601, 1500, 1466, 1445,

1392, 1362, 748, 700 cm^-1. H NMR (DMSO-d₆) 8.381 (s, 1H),

8.223 (s, 1H), 8.081 (s, 1H), 8.002 (s, 1H), 7.340 (t, J ) 7.82 Hz,

1H), 7.396 (t, J ) 7.82 Hz, 1H), 7.392 (t, J ) 7.81 Hz, 1H), 7.339

(t, J ) 7.82 Hz, 1H), 7.301 (t, J ) 7.83 Hz, 2H), 7.290 (t, J )

7.86 Hz, 2H), 7.251 (t, J ) 7.82 Hz, 2H), 7.207 (d, J ) 7.82 Hz,

2H), 7.184 (d, J ) 7.85 Hz, 2H), 7.176 (d, J ) 7.83 Hz, 2H),

7.121 (d, J ) 7.50 Hz, 1H), 7.033 (s, 1H), 6.705 (s, 1H), 6.521

(d, J ) 7.50 Hz, 1H), 6.411 (s, 1H), 6.377 (s, 1H), 5.160 (s, 2H),

5.144 (s, 2H), 5.011 (s, 2H), 4.900 (dt, J ) 6.30 Hz, J ) 6.62 Hz,

1H), 4.708 (t, J ) 6.51 Hz, 1H), 4.691 (t, J ) 6.45 Hz, 1H), 4.608

(t, J ) 4.75 Hz, 1H), 4.006 (s, 2H), 3.981 (d, J ) 6.03 Hz, 2H),

2.965 (t, J ) 4.56 Hz, 2H), 2.910 (m, J ) 4.28 Hz, 1H), 2.781

(t, J ) 5.14 Hz, 2H), 2.631 (t, J ) 6.50 Hz, 2H), 2.560 (s, 1H),

2.022 (m, J ) 4.75 Hz, 2H), 2.012 (m, J ) 4.35 Hz, 1H), 1.861

(m, J ) 4.26 Hz, 1H), 1.832 (m, J ) 4.43 Hz, 2H), 1.754 (m, J )

4.52 Hz, 2H), 1.720 (m, J ) 6.30 Hz, 2H), 1.700 (m, J ) 4.26 Hz,

1H), 1.650 (m, J ) 4.73 Hz, 2H), 1.625 (m, J ) 6.38 Hz, 2H),

+ H]⁺. [R]²⁰50.0 (c 0.60, THF). Anal. Calcd for C₂₀H₂₆O₄: C,

72.70; H, 7.93. Found: C, 72.54; H, 8.11.

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

Ser(Bzl)-OBzl (16). When the same procedure as that used in the

preparation of 8 was used, from 50 mg (0.15 mmol) of estradiol-

3-oxyacetic acid and 119 mg (0.15 mmol) of HCl·H-Arg(NO₂)-

Gly- Asp(OBzl)-Ser(Bzl)-OBzl, 150 mg (93.2%) of the title

compound was obtained as a colorless powder. Mp 114-116 °C.

IR (KBr) 3355, 3340, 3322, 3086, 3074, 3031, 1748, 1683, 1601,

1500, 1462, 1443, 1395, 1366, 741, 692 cm^-1. ¹H NMR (DMSO-

d₆) 8.382 (s, 1H), 8.220 (s, 1H), 8.083 (s, 1H), 8.005 (s, 1H), 7.346

(t, J ) 7.85 Hz, 1H), 7.342 (t, J ) 7.88 Hz, 1H), 7.336 (t, J )

7.84 Hz, 1H), 7.303 (t, J ) 7.85 Hz, 2H), 7.292 (t, J ) 7.88 Hz,

2H), 7.254 (t, J ) 7.84 Hz, 2H), 7.245 (d, J ) 7.85 Hz, 2H), 7.186

(d, J ) 7.88 Hz, 2H), 7.175 (d, J ) 7.86 Hz, 2H), 7.123 (d, J )

7.51 Hz, 1H), 7.037 (s, 1H), 6.708 (s, 1H), 6.522 (d, J ) 7.51 Hz,

1H), 6.415 (s, 1H), 6.374 (s, 1H), 5.166 (s, 2H), 5.143 (s, 2H),

5.135 (s, 2H), 5.006 (s, 2H), 4.902 (dt, J ) 6.32 Hz, J ) 6.60 Hz,

1H), 4.713 (t, J ) 6.53 Hz, 1H), 4.705 (t, J ) 6.46 Hz, 1H), 4.615

(t, J ) 4.78 Hz, 1H), 3.965 (d, J ) 6.00 Hz, 2H), 3.769 (d, J )

4.15 Hz, 2H), 2.970 (t, J ) 4.55 Hz, 2H), 2.912 (m, J ) 4.26 Hz,

1H), 2.785 (t, J ) 5.15 Hz, 2H), 2.627 (t, J ) 6.52 Hz, 2H), 2.553

(s, 1H), 2.029 (m, J ) 4.74 Hz, 2H), 2.014 (m, J ) 4.33 Hz, 2H),

1.866 (m, J ) 4.25 Hz, 1H), 1.841 (m, J ) 4.42 Hz, 2H), 1.756

(m, J ) 4.53 Hz, 2H), 1.727 (m, J ) 6.34 Hz, 2H), 1.704 (m, J )

4.27 Hz, 1H), 1.652 (m, J ) 4.72 Hz, 2H), 1.629 (m, J ) 6.36 Hz,

0.786 (s, 3H). [R]²⁰51.0 (c 1.0, MeOH). ESI-MS 1031 [M +

H]⁺. Anal. Calcd for C₅₅H₆₆N₈O₁₂: C, 64.06; H, 6.45; N, 10.87.

Found: C, 64.22; H, 6.62; N, 10.70. AA analysis: calcd, Arg/Gly/

Asp/Phe ) 1.00:1.00:1.00:1.00; found, Arg/Gly/Asp/Phe ) 0.98:

1.00:0.97:0.99.

3.1.21. N-(Estradiol-3-oxyacetyl)-Arg-Gly-Asp-Ser-OH (19).

When the same procedure as that used in the preparation of 11

was used, from 100 mg (0.093 mmol) of estradiol-17-â-O-

carbonylpropinyl-Arg(NO₂)-Gly-Asp(OBzl)-Ser(Bzl)-OBzl, 49 mg

(70%) of the title compound was obtained as a colorless powder.

Mp 155-157 °C. IR (KBr) 3443, 3387, 3342, 3325, 3028, 1685,

1601, 1503, 1464, 1446, 745, 701 cm^-1. ¹H NMR (DMSO-d₆) 9.076

(s, 1H), 9.071 (s, 1H), 8.026 (s, 1H), 8.019 (s, 1H), 8.011 (s, 1H),

8.002 (s, 2H), 7.244 (s, 2H), 7.042 (d, J ) 7.51 Hz, 1H), 7.032

(s, 1H), 6.755 (s, 1H), 6.515 (d, J ) 7.51 Hz, 1H), 6.400 (s, 1H),

5.004 (s, 2H), 4.851 (dt, J ) 6.31 Hz, J ) 6.60 Hz, 1H), 4.677

(t, J ) 6.54 Hz, 1H), 4.600 (t, J ) 4.72 Hz, 1H), 4.579 (t, J )

6.42 Hz, 1H), 4.017 (d, J ) 6.42 Hz, 2H), 3.971 (d, J ) 6.03 Hz,

2H), 2.966 (t, J ) 4.52 Hz, 2H), 2.905 (m, J ) 4.27 Hz, 1H),

2.874 (t, J ) 5.12 Hz, 2H), 2.807 (d, J ) 6.61 Hz, 2H), 1.832 (m,

J ) 4.43 Hz, 2H), 1.762 (m, J ) 4.55 Hz, 2H), 1.725 (m, J ) 6.31

Hz, 2H), 1.700 (m, J ) 4.26 Hz, 1H), 1.655 (m, J ) 4.70 Hz, 2H),

1.629 (m, J ) 6.34 Hz, 2H), 1.600 (m, J ) 4.52 Hz, 2H), 1.579

(m, J ) 4.54 Hz, 2H), 1.544 (m, J ) 4.55 Hz, 2H), 0.733 (s, 3H).

[R]²⁰_D39.0 (c 0.50, MeOH). ESI-MS 745 [M + H]⁺. Anal. Calcd

for C₃₆H₅₂N₆O₁₁: C, 58.05; H, 7.04; N, 11.28. Found: C, 58.20;

H, 7.11; N, 11.11. AA analysis: calcd, Arg/Gly/Asp/Ser ) 1.00:

1.00:1.00:1.00; found, Arg/Gly/Asp/Ser ) 1.01:1.00:0.97:0.98.

3.1.22. N-(Estradiol-3-oxyacetyl)-Arg-Gly-Asp-Val-OH (20).

When the same procedure as that used in the preparation of 11

was used, from 100 mg (0.093 mmol) of estradiol-17-â-O-

carbonylpropionyl-Arg(NO₂)-Gly-Asp(OBzl)-Val-OBzl, 54 mg (73%)

of the title compound was obtained as a colorless powder. Mp 174-

176 °C. IR (KBr) 3402, 3355, 3343, 3032, 1687, 1601, 1500, 1462,

2H), 0.782 (s, 3H). [R]²⁰55.0 (c 0.5, MeOH). ESI-MS 1061 [M

+ H]⁺. Anal. Calcd for C₅₆H₆₈N₈O₁₃: C, 63.38; H, 6.46; N, 10.56.

Found: C, 63.22; H, 6.29; N, 10.71. AA analysis: calcd, Arg/Gly/

Asp/Ser ) 1.00:1.00: 1.00:1.00; found, Arg/Gly/Asp/Ser ) 0.99:

1.00:1.02:0.97.

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

Val-OBzl (17). When the same procedure as that used in the

preparation of 8 was used, from 50 mg (0.15 mmol) of estradiol-

3-oxyacetic acid and 107 mg (0.15 mmol) of HCl·H-Arg(NO₂)-

Gly-Asp(OBzl)-Val-OBzl, 111 mg (74%) of the title compound

was obtained as a colorless powder. Mp 130-132 °C. IR (KBr)

3355, 3342, 3328, 3089, 3073, 3034, 1745, 1682, 1600, 1505, 1463,

1446, 1388, 1375, 748, 699 cm^-1. H NMR (DMSO-d₆) 8.380

(s, 1H), 8.223 (s, 1H), 8.081 (s, 1H), 8.002 (s, 1H), 7.343 (t, J )

7.82 Hz, 1H), 7.337 (t, J ) 7.85 Hz, 1H), 7.301 (t, J ) 7.84 Hz,

2H), 7.290 (t, J ) 7.86 Hz, 2H), 7.241 (d, J ) 7.82 Hz, 2H), 7.183

(d, J ) 7.86 Hz, 2H), 7.121 (d, J ) 7.52 Hz, 1H), 7.039 (s, 1H),

6.711 (s, 1H), 6.520 (d, J ) 7.52 Hz, 1H), 6.418 (s, 1H), 6.376

(s, 1H), 5.164 (s, 2H), 5.140 (s, 2H), 5.003 (s, 2H), 4.900 (dt, J )

6.30 Hz, J ) 6.61 Hz, 1H), 4.715 (t, J ) 6.50 Hz, 1H), 4.707

(t, J ) 6.43 Hz, 1H), 4.611 (t, J ) 4.75 Hz, 1H), 3.968 (d, J )

6.02 Hz, 2H), 3.262 (m, J ) 4.12 Hz, 1H), 2.966 (t, J ) 4.58 Hz,

2H), 2.915 (m, J ) 4.25 Hz, 1H), 2.793 (t, J ) 5.16 Hz, 2H),

2.632 (t, J ) 6.55 Hz, 2H), 2.556 (s, 1H), 2.024 (m, J ) 4.76 Hz,

2H), 2.016 (m, J ) 4.30 Hz, 2H), 1.862 (m, J ) 4.26 Hz, 1H),

1.833 (m, J ) 4.46 Hz, 2H), 1.759 (m, J ) 4.54 Hz, 2H), 1.725

1446, 1380, 1375, 746, 700 cm^-1. H NMR (DMSO-d₆) 9.104

(s, 1H), 9.087 (s, 1H), 8.029 (s, 1H), 8.021 (s, 1H), 8.013 (s, 1H),

8.004 (s, 2H), 7.241 (s, 2H), 7.044(d, J ) 7.53 Hz, 1H), 7.030

(s, 1H), 6.754 (s, 1H), 6.512(d, J ) 7.53 Hz, 1H), 6.403 (s, 1H),

5.002 (s, 2H), 4.855 (dt, J ) 6.30 Hz, J ) 6.61 Hz, 1H), 4.673

(t, J ) 6.51 Hz, 1H), 4.574 (t, J ) 6.40 Hz, 1H), 4.602 (t, J )

HRT with Estrogen-RGD Peptide Conjugates

Journal of Medicinal Chemistry, 2007, Vol. 50, No. 14 3351

4.71 Hz, 1H), 3.979 (d, J ) 6.00 Hz, 2H), 2.963 (t, J ) 4.50 Hz,

2H), 2.902 (m, J ) 4.25 Hz, 1H), 2.876 (t, J ) 5.10 Hz, 2H),

2.801 (d, J ) 6.60 Hz, 2H), 1.837 (m, J ) 4.45 Hz, 2H), 1.768

(m, J ) 4.56 Hz, 2H), 1.722 (m, J ) 6.30 Hz, 2H), 1.704 (m, J )

4.28 Hz, 1H), 1.657 (m, J ) 4.71 Hz, 2H), 1.625 (m, J ) 6.35 Hz,

2H), 1.602 (m, J ) 4.51 Hz, 2H), 1.576 (m, J ) 4.52 Hz, 2H),

1.549 (m, J ) 4.56 Hz, 2H), 0.935 (d, J ) 4.25 Hz, 6H), 0.742

(s, 3H). [R]²⁰_D45.0 (c 0.50, MeOH). ESI-MS 758 [M + H]⁺. Anal.

Calcd for C₃₈H₅₆N₆O₁₀: C, 63.30; H, 7.46; N, 11.10. Found: C,

63.47; H, 7.57; N, 11.28. AA analysis: calcd, Arg/Gly/Asp/Val )

1.00:1.00:1.00:1.00; found, Arg/Gly/Asp/Val ) 0.98:1.00:0.98:0.97.

3.1.23. N-(Estradiol-3-oxyacetyl)-Arg-Gly-Asp-Phe-OH (21).

When the same procedure as that used in the preparation of 11

was used, from 100 mg (0.095 mmol) of estradiol-17-â-O-

carbonylpropionyl-Arg(NO₂)-Gly-Asp(OBzl)-Phe-OBzl, 59 mg (77%)

of the title compound was obtained as a colorless powder. Mp 180-

182 °C. IR (KBr) 3405, 3380, 3375, 3030, 1690, 1602, 1503, 1460,

745, 701 cm^-1. ¹H NMR (DMSO-d₆) 9.070 (s, 1H), 9.063 (s, 1H),

8.035 (s, 1H), 8.010 (s, 1H), 8.003 (s, 1H), 7.986 (s, 2H), 7.275

(t, J ) 7.80 Hz, 2H), 7.217 (s, 1H), 7.127 (d, J ) 7.80 Hz, 2H),

7.079 (d, J ) 7.85 Hz, 1H), 7.063 (d, J ) 7.50 Hz, 1H), 7.026

(s, 1H), 6.744 (s, 1H), 6.667 (d, J ) 7.51 Hz, 1H), 6.645 (s, 1H),

4.991 (s, 2H), 4.840 (dt, J ) 6.32 Hz, J ) 6.60 Hz, 1H), 4.670 (t,

J ) 6.52 Hz, 1H), 4.597 (t, J ) 4.70 Hz, 1H), 4.573 (t, J ) 6.40

Hz, 1H), 3.966 (d, J ) 6.00 Hz, 2H), 3.227 (t, J ) 4.72 Hz, 2H),

2.960 (t, J ) 4.54 Hz, 2H), 2.900 (m, J ) 4.28 Hz, 1H), 2.871 (t,

J ) 5.14 Hz, 2H), 2.800 (d, J ) 6.56 Hz, 2H), 1.840 (m, J ) 4.45

Hz, 2H), 1.766 (m, J ) 4.52 Hz, 2H), 1.728 (m, J ) 6.30 Hz, 2H),

1.670 (m, J ) 4.72 Hz, 2H), 1.626 (m, J ) 6.30 Hz, 2H), 1.604

(m, J ) 4.54 Hz, 2H), 1.584 (m, J ) 4.58 Hz, 1H), 1.572 (m, J )

4.54 Hz, 2H), 1.543 (m, J ) 4.52 Hz, 2H), 1.467 (m, J ) 4.56 Hz,

(t, J ) 7.82 Hz, 2H), 7.290 (t, J ) 7.84 Hz, 2H), 7.250 (t, J )

7.82 Hz, 2H), 7.242 (d, J ) 7.82 Hz, 2H), 7.183 (d, J ) 7.84 Hz,

2H), 7.172 (d, J ) 7.82 Hz, 2H), 7.120 (d, J ) 7.50 Hz, 1H),

7.034 (s, 1H), 6.710 (s, 1H), 6.520 (d, J ) 7.50 Hz, 1H), 6.412

(s, 1H), 6.376 (s, 1H), 5.167 (s, 2H), 5.145 (s, 2H), 5.136 (s, 2H),

5.002 (s, 2H), 4.900 (dt, J ) 6.30 Hz, J ) 6.58 Hz, 1H), 4.711 (t,

J ) 6.50 Hz, 1H), 4.702 (t, J ) 6.44 Hz, 1H), 4.613 (t, J ) 4.76

Hz, 1H), 3.964 (d, J ) 6.01 Hz, 2H), 3.767 (d, J ) 4.17 Hz, 2H),

2.972 (t, J ) 4.56 Hz, 2H), 2.919 (m, J ) 4.24 Hz, 1H), 2.790 (d,

J ) 5.16 Hz, 2H), 2.633 (t, J ) 6.50 Hz, 2H), 2.058 (t, J ) 4.76

Hz, 2H), 2.017 (m, J ) 4.36 Hz, 2H), 1.845 (m, J ) 4.46 Hz, 2H),

1.750 (m, J ) 4.55 Hz, 2H), 1.729 (m, J ) 6.32 Hz, 2H), 1.707

(m, J ) 4.29 Hz, 1H), 1.654 (m, J ) 4.75 Hz, 2H), 1.626 (m, J )

6.34 Hz, 2H), 0.792 (s, 3H). ESI-MS (m/e) 1059 [M + H]⁺. [R]²⁰

32.0 (c 0.5, CHCl₃.MeOH, 10:1). Anal. Calcd for C₅₆H₆₆N₈O₁₃:

C, 63.50; H, 6.28; N, 10.58. Found: C, 63.65; H, 6.35; N, 10.41.

AA analysis: calcd, Arg/Gly/Asp/Ser ) 1.00:1.00:1.00:1.00; found,

Arg/Gly/Asp/Ser ) 0.97:1.00:1.01:0.98.

3.1.27. Estrone-3-oxyacetyl-Arg(NO₂)-Gly-Asp(OBzl)-Val-

OBzl (25). When the same procedure as that used in the preparation

of 8 was used, from 50 mg (0.15 mmol) of estrone-3-oxyacetic

acid and 107 mg (0.15 mmol) of HCl·H-Arg(NO₂)-Gly-Asp(OBzl)-

Val-OBzl, 101 mg (68%) of the title compound was obtained as a

colorless powder. Mp 124-126 °C. IR (KBr) 3359, 3345, 3331,

3086, 3077, 3030, 1747, 1724, 1680, 1602, 1507, 1465, 1448, 1389,

1378, 744, 695 cm^-1. H NMR (DMSO-d₆) 8.377 (s, 1H), 8.220

(s, 1H), 8.078 (s, 1H), 8.000 (s, 1H), 7.345 (t, J ) 7.80 Hz, 1H),

7.339 (t, J ) 7.83 Hz, 1H), 7.305 (t, J ) 7.82 Hz, 2H), 7.294

(t, J ) 7.84 Hz, 2H), 7.245 (d, J ) 7.80 Hz, 2H), 7.185 (d, J )

7.84 Hz, 2H), 7.124 (d, J ) 7.50 Hz, 1H), 7.042 (s, 1H), 6.714

(s, 1H), 6.523 (d, J ) 7.50 Hz, 1H), 6.422 (s, 1H), 6.378 (s, 1H),

5.169 (s, 2H), 5.146 (s, 2H), 5.007 (s, 2H), 4.904 (dt, J ) 6.32 Hz,

J ) 6.60 Hz, 1H), 4.718 (t, J ) 6.52 Hz, 1H), 4.709 (t, J ) 6.41

Hz, 1H), 4.613 (t, J ) 4.76 Hz, 1H), 3.971 (d, J ) 6.00 Hz, 2H),

2.967 (t, J ) 4.55 Hz, 2H), 2.919 (m, J ) 4.28 Hz, 1H), 2.795 (t,

J ) 5.14 Hz, 2H), 2.634 (t, J ) 6.52 Hz, 2H), 2.027 (m, J ) 4.74

Hz, 2H), 2.019 (m, J ) 4.35 Hz, 2H), 1.866 (m, J ) 4.29 Hz, 1H),

1.835 (m, J ) 4.48 Hz, 2H), 1.762 (m, J ) 4.56 Hz, 2H), 1.733

(m, J ) 6.30 Hz, 2H), 1.706 (m, J ) 4.27 Hz, 1H), 1.656 (m, J )

4.72 Hz, 2H), 1.629 (m, J ) 6.35 Hz, 2H), 0.819 (s, 3H), 0.785 (d,

J ) 4.16 Hz, 6H). Anal. Calcd for C₅₁H₆₄N₈O₁₂: C, 62.43; H, 6.58;

N, 11.42. Found: C, 62.57; H, 6.50; N, 11.61. AA analysis: calcd,

Arg/Gly/Asp/Val ) 1.00:1.00:1.00:1.00; found, Arg/Gly/Asp/Val

1H), 0.752 (s, 3H). [R]²⁰48.0 (c 0.50, MeOH). ESI-MS 805 [M

+ H]⁺. Anal. Calcd for C₄₂H₅₆N₆O₁₀: C, 62.67; H, 7.01; N, 10.44.

Found: C, 61.73; H, 7.07; N, 10.63. AA analysis: calcd, Arg/Gly/

Asp/Phe ) 1.00:1.00:1.00:1.00; found, Arg/Gly/Asp/Phe ) 0.98:

1.00:0.98:0.97.

3.1.24. Ethyl Estrone-3-oxyacetate (22). To the solution of 500

mg (1.85 mmol) of estrone in 10 mL of anhydrous THF, 1.4 mL

of sodium ethoxide in anhydrous ethanol (2.0 mol/L) was added.

The mixture was stirred for 0.5 h, to which 0.750 mL (5.5mmol)

of ethyl bromoacetate was added. The reaction mixture was stirred

at 56 °C for 16 h and TLC (CHCl₃/CH₃OH, 10:1) indicated the

complete disappearance of estrone. The reaction mixture was

evaporated to remove the solvent and excess of ethyl bromoacetate.

After purification on chromatography column of silica gel 593 mg

(90%) of the title compound was obtained as a colorless powder.

) 0.98:1.00:0.97:0.98. ESI-MS(m/e) 981 [M + H]⁺. [R]²⁰26.0

(c 0.5, CHCl₃/MeOH, 10:1).

3.1.28. Estrone-3-oxyacetyl-Arg(NO₂)-Gly-Asp(OBzl)-Phe-

OBzl (26). When the same procedure as that used in the preparation

of 8 was used, from 50 mg (0.15 mmol) of estrone-3-oxyacetic

acid and 119 mg (0.15 mmol) of HCl‚H-Arg(NO₂)-Gly-Asp(OBzl)-

Phe-OBzl, 110 mg (70%) of the title compound was obtained as a

colorless powder. Mp 130-132 °C. IR (KBr) 3357, 3346, 3328,

3083, 3062, 3029, 1745, 1720, 1681, 1600, 1504, 1467, 1443, 747,

703 cm^-1. ¹H NMR (DMSO-d₆) 8.385 (s, 1H), 8.227 (s, 1H), 8.084

(s, 1H), 8.010 (s, 1H), 7.342 (t, J ) 7.80 Hz, 1H), 7.399 (t, J )

7.80 Hz, 1H), 7.396 (t, J ) 7.77 Hz, 1H), 7.342 (t, J ) 7.80 Hz,

1H), 7.304 (t, J ) 7.81 Hz, 2H), 7.293 (t, J ) 7.84 Hz, 2H), 7.255

(t, J ) 7.80 Hz, 2H), 7.211 (d, J ) 7.80 Hz, 2H), 7.187 (d, J )

7.83 Hz, 2H), 7.177 (d, J ) 7.81 Hz, 2H), 7.124 (d, J ) 7.52 Hz,

1H), 7.036 (s, 1H), 6.707 (s, 1H), 6.525 (d, J ) 7.51 Hz, 1H),

6.414 (s, 1H), 6.382 (s, 1H), 5.163 (s, 2H), 5.147 (s, 2H), 5.014

(s, 2H), 4.903 (dt, J ) 6.32 Hz, J ) 6.64 Hz, 1H), 4.712 (t, J )

6.50 Hz, 1H), 4.694 (t, J ) 6.43 Hz, 1H), 4.002 (s, 2H), 3.985

(d, J ) 6.00 Hz, 2H), 2.962 (t, J ) 4.57 Hz, 2H), 2.913 (m, J )

4.27 Hz, 1H), 2.784 (t, J ) 5.16 Hz, 2H), 2.634(t, J ) 6.51 Hz,

2H), 2.025 (m, J ) 4.74 Hz, 2H), 2.014 (m, J ) 4.36 Hz, 1H),

1.865 (m, J ) 4.27 Hz, 1H), 1.835 (m, J ) 4.44 Hz, 2H), 1.756

(m, J ) 4.54 Hz, 2H), 1.723 (m, J ) 6.28 Hz, 2H), 1.706 (m, J )

4.27 Hz, 1H), 1.655 (m, J ) 4.74 Hz, 2H), 1.628 (m, J ) 6.36 Hz,

Mp. 98-100 °C. ESI-MS(m/e) 357 [M + H]⁺. [R]²⁰138.0

(c 0.50, THF).

3.1.25. Estrone-3-oxyacetic Acid (23). At 0 °C to the solution

of 500 mg (1.40 mmol) of ethyl estrone-3-oxyacetate in 3 mL of

anhydrous ethanol, 2 mL of aqueous solution of NaOH (2 mol/L)

was added dropwise. The reaction mixture was stirred at room

temperature for 2.5 h and TLC (CHCl₃/CH₃OH, 10:1) indicated

the complete disappearance of methyl estrone-3-oxyacetate. The

reaction mixture was adjusted with KHSO₄powder to pH 7.0 and

evaporated to remove the organic solvent. To the residue, 3 mL of

water was added and adjusted with KHSO₄powder to pH 2.0. The

mixture was extracted with ethyl acetate and dried over anhydrous

Na₂SO₄. After filtration the filtrate was evaporated to give 442 mg

(96%) of the title compound as a colorless powder. Mp 214-

215 °C. ESI-MS (m/e) 329 [M + H]⁺. [R]²⁰_D159.0 (c 0.45, THF).

3.1.26. Estrone-3-oxyacetyl-Arg(NO₂)-Gly-Asp(OBzl)-Ser-

(Bzl)-OBzl (24). When the same procedure as that used in the

preparation of 8 was used, from 50 mg (0.15 mmol) of estrone-3-

oxyacetic acid and 119 mg (0.15 mmol) of HCl·H-Arg(NO₂)-Gly-

Asp(OBzl)-Ser(Bzl)-OBzl, 99 mg (62%) of the title compound was

obtained as a colorless powder. Mp 133-135 °C. IR (KBr) 3352,

3343, 3326, 3084, 3072, 3028, 1749, 1722, 1680, 1603, 1505, 1460,

1445, 744, 693 cm^-1. H NMR (DMSO-d₆) 8.380 (s, 1H), 8.217

2H), 0.791 (s, 3H). ESI-MS (m/e) 1029 [M + H]⁺. [R]²⁰28.0

(s, 1H), 8.081 (s, 1H), 8.002 (s, 1H), 7.342 (t, J ) 7.82 Hz, 1H),

7.339 (t, J ) 7.84 Hz, 1H), 7.334 (t, J ) 7.82 Hz, 1H), 7.300

(c 0.5, CHCl₃/MeOH, 10:1). Anal. Calcd for C₅₅H₆₄N₈O₁₂: C,

64.19; H, 6.27; N, 10.89. Found: C, 64.34; H, 6.38; N, 10.71. AA

3352 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 14

Xiong et al.

analysis: calcd, Arg/Gly/Asp/Phe ) 1.00:1.00:1.00:1.00; found,

Arg/Gly/Asp/Phe ) 0.99:1.00:0.98:0.98.

62.65; H, 6.70; N, 10.66. AA analysis: calcd, Arg/Gly/Asp/Phe )

1.00:1.00:1.00:1.00; found, Arg/Gly/Asp/Phe ) 0.99:1.00: 0.97:

0.99.

3.1.29. Estrone-3-oxyacetyl-Arg-Gly-Asp-Ser-OH (27). When

the same procedure as that used in the preparation of 11 was used,

from 100 mg (0.094 mmol) of estrone-3-oxyacetyl-Arg(NO₂)-Gly-

Asp(OBzl)-Ser(Bzl)-OBzl (24), 40 mg (57%) of the title compound

were obtained as a colorless powder. Mp 170-172 °C. IR (KBr)

3449, 3392, 3347, 3329, 3031, 1726, 1682, 1604, 1505, 1462, 1445,

741, 700 cm^-1. ¹H NMR (DMSO-d₆) 9.073 (s, 1H), 9.067 (s, 1H),

8.023 (s, 1H), 8.015 (s, 1H), 8.006 (s, 1H), 8.000 (s, 2H), 7.241

(s, 2H), 7.038 (d, J ) 7.50 Hz, 1H), 7.029 (s, 1H), 6.751 (s, 1H),

6.512 (d, J ) 7.50 Hz, 1H), 6.404 (s, 1H), 5.008 (s, 2H), 4.847

(dt, J ) 6.30 Hz, J ) 6.58 Hz, 1H), 4.674 (t, J ) 6.52 Hz, 1H),

4.604 (t, J ) 4.70 Hz, 1H), 4.575 (t, J ) 6.40 Hz, 1H), 4.013 (d,

J ) 6.40 Hz, 2H), 3.968 (d, J ) 6.00 Hz, 1H), 2.962 (t, J ) 4.54

Hz, 2H), 2.902 (m, J ) 4.29 Hz, 1H), 2.872 (t, J ) 5.13 Hz, 2H),

2.811 (d, J ) 6.59 Hz, 2H), 1.830 (m, J ) 4.45 Hz, 2H), 1.766

(m, J ) 4.54 Hz, 2H), 1.728 (m, J ) 6.31 Hz, 2H), 1.708 (m, J )

4.25 Hz, 1H), 1.659 (m, J ) 4.72 Hz, 2H), 1.624 (m, J ) 6.30 Hz,

2H), 1.607 (m, J ) 4.50 Hz, 2H), 1.576 (m, J ) 4.52 Hz, 2H),

1.548 (m, J ) 4.56 Hz, 2H), 0.827 (s, 3H). ESI-MS (m/e) 744 [M

3.1.32. Bioassay In Vivo. The assessments described herein were

performed based on a protocol reviewed and approved by the ethics

committee of Peking 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% CMC just before use and kept in an ice

bath. Female Kuiming mice weighing 30.7 ( 3.1 g (7 weeks old,

purchased from Animal Center of Peking University) were anes-

thetized with pentobarbital sodium (40.0 mg/kg, i.p.). The mice of

OVX groups were given an abdominal OVX by the standard

procedure, and the mice of the sham group were given abdomi-

norotomy only. On the fifth day after the surgical operation, the

mice of the OVX and sham groups were intraperitoneally injected

with 2 µL of an aqueous solution of 0.5% CMC, the mice of the

treatment groups were intraperitoneally injected with 2 µL of the

solution of estrogen, the mixture of estrogen and RGD peptide, or

0.1103 µmol/Kg of the estrogen-RGD peptide in an aqueous

solution of 0.5% CMC 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 and blood

was drawn from the eye orbit. The mice were then anesthetized

with sodium pentobarbital (40.0 mg/kg, i.p.) and executed to remove

the lungs, livers, spleens, uteri, and left femurs.

After 30 min of standing, the blood was centrifugated at 3000 g

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-methylphenolphthalein complexing ketone. The phosphorus

content of the serum was measured by the method of molybdenum

blue. The ALP content of the serum was measured using disodium

phenylphosphate as the substrate.

The lungs, livers, spleens, and uteri were weighed directly. After

completely removing the muscle, the lengths of the left femurs were

measured and then immersed in a solution of chloroform-methanol

(2:1) two times (one time for 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 a furnace at 800 °C

for 8 h, cooled, weighed to record the ash weight and calculate the

rate of the ash weight to dry femur weight (namely, the mineral

content of the femur).

+ H]⁺. [R]²⁰55.0 (c 1.0, MeOH). Anal. Calcd for C₃₆H₅₀N₆O₁₁:

C, 58.21; H, 6.78; N, 11.31. Found: C, 58.40; H, 6.73; N, 11.13.

AA analysis: calcd, Arg/Gly/Asp/Ser ) 1.00:1.00:1.00:1.00; found,

Arg/Gly/Asp/Ser ) 1.02:1.00:0.98:0.98.

3.1.30. Estrone-3-oxyacetyl-Arg-Gly-Asp-Val-OH (28). When

the same procedure as that used in the preparation of 11 was used,

from 100 mg (0.102 mmol) of estrone-3-oxyacetyl-Arg(NO₂)-Gly-

Asp(OBzl)-Val-OBzl (25), 59 mg (76%) of the title compound was

obtained as a colorless powder. Mp 164-166 °C. IR (KBr) 3411,

3353, 3347, 3030, 1725, 1684, 1605, 1503, 1467, 1442, 1385, 1378,

748, 703 cm^-1. ¹H NMR (DMSO-d₆) 9.114 (s, 1H), 9.093 (s, 1H),

8.034 (s, 1H), 8.025 (s, 1H), 8.017 (s, 1H), 8.001 (s, 2H), 7.245

(s, 2H), 7.042 (d, J ) 7.50 Hz, 1H), 7.033 (s, 1H), 6.756 (s, 1H),

6.516 (d, J ) 7.50 Hz, 1H), 6.412 (s, 1H), 5.011 (s, 2H), 4.853

(dt, J ) 6.31 Hz, J ) 6.60 Hz, 1H), 4.675 (t, J ) 6.50 Hz, 1H),

4.572 (t, J ) 6.41 Hz, 1H), 4.610 (t, J ) 4.70 Hz, 1H), 3.974 (d,

J ) 6.01 Hz, 1H), 2.966 (t, J ) 4.52 Hz, 2H), 2.907 (m, J ) 4.28

Hz, 1H), 2.875 (t, J ) 5.10 Hz, 2H), 2.808 (d, J ) 6.62 Hz, 2H),

1.839 (m, J ) 4.48 Hz, 2H), 1.764 (m, J ) 4.53 Hz, 2H), 1.725

(m, J ) 6.27 Hz, 2H), 1.713 (m, J ) 4.26 Hz, 1H), 1.659 (m, J )

4.70 Hz, 2H), 1.628 (m, J ) 6.33 Hz, 2H), 1.613 (m, J ) 4.54 Hz,

2H), 1.578 (m, J ) 4.50 Hz, 2H), 1.545 (m, J ) 4.52 Hz, 2H),

0.938 (d, J ) 4.27 Hz, 6H), 0.747 (s, 3H). ESI-MS (m/e) 755 [M

The ashes of the left femurs were dissolved in 0.5 mL of

hydrochloric 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-methylphenol-

phthalein complexing ketone. The phosphorus content of the

aqueous solution was measured by the method of molybdenum blue.

+ H]⁺. [R]²⁰44.0 (c 0.5, MeOH). Anal. Calcd for C₃₈H₅₄N₆O₁₀:

C, 60.46; H, 7.21; N, 11.13. Found: C, 60.62; H, 7.30; N, 11.27.

AA analysis: calcd, Arg/Gly/Asp/Val ) 1.00:1.00:1.00:1.00; found,

Arg/Gly/Asp/Val ) 0.97:1.00:0.98:0.99.

3.1.31. Estrone-3-oxyacetyl-Arg-Gly-Asp-Phe-OH (29). When

the same procedure as that used in the preparation of 11 was used,

from 100 mg (0.097 mmol) of estrone-3-oxyacetyl-Arg(NO₂)-Gly-

Asp(OBzl)-Phe-OBzl (26), 44 mg (56%) of the title compound was

obtained as a colorless powder. Mp 164-166 °C; IR (KBr) 3408,

Acknowledgment. This work was supported by Beijing area

major laboratory of peptide and small molecular drugs and

National Natural Scientific Foundation of China (30572235).

We wish to express our deep appreciation to Professor Paul

Wentworth of the Department of Chemistry, the Scripps

Research Institute of U.S.A. for proofreading the manuscript.

3384, 3376, 3034, 1722, 1693, 1600, 1505, 1467, 748, 704 cm^-1

¹H NMR (DMSO-d₆) 9.075 (s, 1H), 9.068 (s, 1H), 8.037 (s, 1H),

8.014 (s, 1H), 8.006 (s, 1H), 7.982 (s, 2H), 7.271 (t, J ) 7.81 Hz,

2H), 7.213 (s, 1H), 7.120 (d, J ) 7.82 Hz, 2H), 7.072 (d, J ) 7.83

Hz, 1H), 7.060 (d, J ) 7.51 Hz, 1H), 7.022 (s, 1H), 6.741 (s, 1H),

6.663 (d, J ) 7.50 Hz, 1H), 6.643 (s, 1H), 4.993 (s, 2H), 4.843

(dt, J ) 6.30 Hz, J ) 6.58 Hz, 1H), 4.671 (t, J ) 6.50 Hz, 1H),

4.590 (t, J ) 4.71 Hz, 1H), 4.576 (t, J ) 6.42 Hz, 1H), 3.223

(t, J ) 4.74 Hz, 2H), 2.962 (t, J ) 4.55 Hz, 2H), 2.903 (m, J )

4.29 Hz, 1H), 2.873 (t, J ) 5.16 Hz, 2H), 2.802 (d, J ) 6.53 Hz,

2H), 1.843 (m, J ) 4.47 Hz, 2H), 1.763 (m, J ) 4.50 Hz, 2H),

1.721 (m, J ) 6.27 Hz, 2H), 1.674 (m, J ) 4.70 Hz, 2H), 1.623

(m, J ) 6.27 Hz, 2H), 1.602 (m, J ) 4.52 Hz, 2H), 1.582 (m, J )

4.55 Hz, 1H), 1.570 (m, J ) 4.56 Hz, 2H), 1.540 (m, J ) 4.50 Hz,

2H), 1.464 (m, J ) 4.53 Hz, 1H), 0.810 (s, 3H). ESI-MS

Supporting Information Available: Physical, analytical, and

spectrometric data of the target compounds (11-13, 19-21, and

27-29). This material is available free of charge via the Internet

at http://pubs.acs.org.

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JM070242A

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