CN1315869C

CN1315869C - Osteoporosis resistant estrogen-RGD peptide conjugate and its application in medicine

Info

Publication number: CN1315869C
Application number: CNB2004100971952A
Authority: CN
Inventors: 彭师奇; 王超; 赵明; 熊瑜
Original assignee: Capital Medical University
Current assignee: Capital Medical University
Priority date: 2004-12-13
Filing date: 2004-12-13
Publication date: 2007-05-16
Anticipated expiration: 2024-12-13
Also published as: CN1789278A

Abstract

The present invention discloses a series of new estrogens, namely RGD peptide conjugated compounds with good anti-osteoporosis activity and a preparing method thereof. The estrogen peptide conjugated compounds of the present invention are prepared by the following method comprising: the third position or the seventeenth position of estrone or estradiol is introduced on a connecting arm; then, a protection intermediate synthesized by a liquid phase method is introduced on the connecting arm and is deprotected. As proved by an animal model test, the anti-osteoporosis activity of the compounds of the present invention is better than that of the estradiol and the estrone, and the compounds have no obvious side effect.

Description

Have the oestrogenic hormon-RGD peptide conjugate of function of resisting osteoporosis and the application in medical science

Technical field

The present invention relates to a kind of oestrogenic hormon conjugate, relate in particular to a kind of oestrogenic hormon peptide conjugate, its preparation method and application.

Background technology

Osteoporosis is a kind of many reasons metabolic osteopathy, is reduced to feature with the sclerotin diffusivity.Menopausal women more than 50 years old has fracture (the Davidson M that causes through having osteoporosis all through the ages in certain period of life more than 50%, DeSimone M E.Confronting osteoporosis:what we know, wherewe ' re headed.Clin Rev; 12,76-82,2002).Cause among the women of fracture in osteoporosis, the rehabilitation that will take long to more than 50%, this just keeps away some sequela of unavoidable generation and comprises death.According to estimates, meet with the women who fractures and have 20% meeting dead (Michaelsson K, Baron J A, et al., Influenceof parity and lactation on hip fracture risk.Am J Epidemiol at least; 153,1166-1172,2001).Preliminary investigation to Beijing and Shanghai shows that the osteoporosis of female morbidity is 40-50% more than 60 years old.Osteoporosis becomes an internationally recognized health problem just day by day.In current common disease, osteoporosis is positioned at top ten (Chen Xing violent wind etc., motion prevents the progress of post-menopausal osteoporosis, foreign medical science, geriatrics fascicle, 23 (2), 85-88,2002).

As far back as nineteen forty-one, Albright finds that osteoporosis is common in the postmenopausal women, and the main diseases that proposes estrogen deficiency and be postmenopausal osteoporosis is because of (Albright F, Smith P H, et al, Postmenopausalosteoporosis.JAMA, 116,2465-2474,1941).The mean age of postmenopausal women is 51 years old.This just means that the women will spend the time more than 1/3 after climacteric, just the women will be at (Mikinlay SM, the Brambilla DJ more than 30 years that live under the environment of estrogen deficiency, The normal menopansetransition, Maturitas, 14,103-15,1992).If there is not hormone replacement therapy (HRT), these women not only will be in the state of incapability of work, and will experience the illness that causes because of osteoporosis on some bodies, as cardiovascular disorder etc., or even lethality (Michael J.S, Selective estrogenreceptor modulators:the ideal estrogen replacement, 8,25-30,2001).Women throughout one's life the probability of osteoporosis fracture is 11-18%, wherein per 6 just have a meeting because of dead (the Spencer C P. Morris E P of the complication of osteoporosis, et al., Selective estrogen receptormodulators:women ' s penacea for the next millennium? Am J obstet Gynecol, 180,763-770,1999).

Controversies in hormone replacement in the elderly (ERT) can alleviate the symptom of menopausal women, can reduce cardiovascular disease risk, can stop bone loss (Stampter M, Colditz.G., et al., Estrogen replacementtherapy and coronary heart disease:a quantitative assessment of theepidimiological evidence.Prev Med, 20,47-63,1991).Use oestrogenic hormon to treat and to suppress bony defect and bone metabolism significantly, increase the density (BMD) of bone mineral material significantly, when the early stage women of menopause uses oestrogenic hormon to treat, oestrogenic hormon can make increase in density 3～4% (the Hosking D of vertebrae mineral, Chilrers C E D, et al.Prevention of Bone loss withAlendronate in Postmenopausal Women under 60 Years of Age, N Engl J Med, 338,485-492,1998).Those use hormone (estrogenic/progestogenic) to carry out the patient of alternative medicine (HRT) or carry out the patient of the HRT treatment in 5 years at least by a definite date in nearest 5～9 years, the risk of its fracture has reduced (Michealsson K more than 50%, Baron J A., et al., Hormone replacementtherapy and risk of hip fracture:Population based case-control study.The SwedishHip Fracture Study Group, BMJ, 316,1858-1863,1998).Although oestrogenic hormon be now till treatment postmenopausal women the most effective osteoporotic medicine, but life-time service hormone replacement therapy or estrogen replacement therapy, can cause some complication inevitably, as the bone forming surface is significantly descended, cause endometrial hyperplasia or tumour, cause vaginal hemorrhage, make the breast deliquescing, increase the danger of mammary cancer, cause hypertension or original hypertension is worsened, can obviously increase (the Colditz G A such as danger of venous thromboembolism disease, Hankinson S E, et al., The use of estrogen and progestin and the riskof breast cancer in postmenopausal women.N Engl J.Med.332,1589-93,1995), the estrogenic usage period is no more than 3 years usually (Scalley E K, Henrich J B, et al.; An overview ofestrogen replacement therapy in postmenopausal women., J.Women Health, 2,289～294,1993; Witt D M, Lousberg T R..Controversies surrounding estrogen usein postmenopausal women.Ann Pharmaeother, 31,745～755,1997; Josse R G., Effect of ovarian hormonal therapy on skeletal and estraskeletal tissues in women.Can Med Assoc J, 155,929～934,1996).

Summary of the invention

Technical problem to be solved by this invention provides a kind of anti-osteoporosis activity height, the low oestrogenic hormon peptide conjugate of side effect.

A kind of oestrogenic hormon peptide conjugate of the present invention has the structure of following general formula I:

Wherein, work as R ₃During for H, R ₁And R ₂One is H, and one is-OCOCH ₂CH ₂CO-Arg-Gly-Asp-Ser-OH, CH ₂CO-Arg-Gly-Asp-Val-OH ,-OCOCH ₂-CH ₂CO-Arg-Gly-Asp-Phe-OH, Ser-Asp-Gly-Arg-H ,=NNH-Val-Asp-Gly-Arg-H or=NNH-Phe-Asp-Gly-Arg-H; Work as R ₁And R ₂One is H, one when being OH, and R ₃Be selected from-CH ₂CO-Arg-Gly-Asp-Ser-OH ,-CH ₂CO-Arg-Gly-Asp-Val-OH ,-CH ₂CO-Arg-Gly-Asp-Phe-OH ,-CH ₂CONHNH-Ser-Asp-Gly-Arg-H ,-CH ₂CONH-NH-Val-Asp-Gly-Arg-H or-CH ₂CONHNH-Phe-Asp-Gly-Arg-H; Work as R ₁And R ₂When being ketonic oxygen jointly, R ₃Be selected from-CH ₂CO-Arg-Gly-Asp-Ser-OH ,-CH ₂CO-Arg-Gly-Asp-Val-OH or-CH ₂CO-Arg-Gly-Asp-Phe-OH.

Another technical problem to be solved by this invention provides a kind of preparation method of oestrogenic hormon peptide conjugate.

The preparation method of oestrogenic hormon peptide conjugate of the present invention may further comprise the steps:

Introduce connecting arm (COCH for 3 or 17 with oestrone and estradiol ₂CH ₂CO, CH ₂CO or CH ₂CONH), then the protection intermediate of liquid phase process synthetic Arg-Gly-Asp-Ser-OH, Arg-Gly-Asp-Val-OH or Arg-Gly-Asp-Phe-OH is incorporated on the connecting arm, more promptly through deprotection.

Series compound of the present invention confirms to have good anti-osteoporosis activity through test.The present invention utilize oestrogenic hormon to the osteoporotic obvious therapeutic action of menopause type, utilize RGD (Arg-Gly-Asp) peptide target, utilize the RGD tripeptide sequence to osteoclast and the adherent restraining effect of bone surface, RGD peptide and oestrogenic hormon are puted together, and therefore resulting oestrogenic hormon conjugate has better anti-osteoporosis activity than oestrogenic hormon.

The cell adhesion process of mediated by integrin is associated with the pathology of a series of important diseases.The for example integrin receptor on the surface behind the platelet activation and the Fibrinogen in the blood, Fibronectin or VWF adhesion forms thrombus.Plain and the healthy tissues adhesion of the integration on cancer cells surface causes metastasis of cancer.Plain and the bone matrix protein adhesion of the integration on osteoclast surface causes osteoporosis.Integrin receptor can be divided into the cell surface part and stride the hinge fraction that film enters cytoplasmic domain.The cell adhesion of mediated by integrin is participated in conjunction with the territory by aglucon.Combining the adherent proteic common sequences in territory with the aglucon of integrating element in metastasis of cancer, thrombosis and osteoporosis morbidity is RGD.

The formation of bone resorption device depends on the interaction between the plain and special bone matrix protein composition of osteoclast surface integration.The integration element that identifies on the osteoclast film mainly contains 1 three kinds of av β 3, a2 β 1 and av β.Wherein av β 3 is main integration elements of osteoclast identification ground substance of bone.With av β 3 bonded specificity bone matrix protein osteopontin (osteopontin, OPN), bone sialoprotein (bone sialoptein, BSP-II) and fibronectin (fibronectin FN) all contains the RGD tripeptide sequence.The osteoclast adhesion process of mediated by integrin, for osteoclast differentiation, ripe, raise, activation and bone resorption be very important.Experiment in vitro shows, integrates osteopontin, RGD tripeptide sequence, rgd peptide or the RGD peptide section of plain av β 3 monoclonal antibodies, synthetic, can suppress the osteoclast bone resorption.

There is " permission effect " between two main bodys of polypeptide and steroid hormone." permission effect " is meant that steroid hormone can play synergism to the biological effect of polypeptide and other mediator, and vice versa.Along with development of molecular biology, the adjusting of information transmitter substance behind acceptor of pointing out its possible mechanism mainly to show other hormone is acted on and the acceptor, and directly or indirectly act on the target cell gene.When the contriver has the endogenous small molecules dipeptides kyotorphin of strong analgesic activity in research, find that the analgesic activity of the conjugate of they and hydrocortisone and oestrone can strengthen.

Another object of the present invention is to provide a kind of pharmaceutical composition, compound and the cooperation of pharmaceutically acceptable carrier by structure shown in the general formula (I) form, general formula (I) compound that is about to pharmaceutically acceptable consumption is with after pharmaceutically acceptable carrier cooperates, and by the formulation method of this area routine it is prepared into any one appropriate drug composition.Usually said composition is suitable for oral administration and drug administration by injection, also is fit to other medication.

Said composition can be liquid preparation forms such as tablet, capsule, pulvis, particle, lozenge, suppository, or oral liquid or aseptic parenteral suspension.

According to different medications, O.1% composition can contain～active substance of 99% weight, the active substance of preferred 10～60% weight.

Embodiment

Specify the present invention below with reference to embodiment and test example, embodiments of the invention and test example only are used to technical scheme of the present invention is described, and non-limiting essence of the present invention.

Raw material and reagent:

Various protection amino acid and estradiol, oestrone are available from sigma company; Used amino acid is the L-configuration; Tlc silica gel GF254, column chromatography 200-300 order silica gel and silica gel H are that Haiyang Chemical Plant, Qingdao produces; Other reagent is domestic commercially available analytical pure except that indicating.Anhydrous tetrahydro furan, filters gained filtrate and becomes blue with sodium Metal 99.5 backflow (benzophenone is an indicator) after dry 24 hours with an amount of Vanadium Pentoxide in FLAKES, and 65-66 ℃ cut is collected in air distillation.

Absolute ethanol prepares according to a conventional method, the iodine that promptly in the round-bottomed flask of 1.5L, adds clean MAGNESIUM METAL of 5g and 0.5g, add 50～75ml dehydrated alcohol then, being heated to iodine disappears, continuing to be heated to magnesium all changes fully, add the 900ml dehydrated alcohol again, backflow 30min, 80 ℃ cut is collected in air distillation.Sealing is preserved.

Anhydrous dimethyl formamide prepares according to a conventional method, it is commercially available dimethyl formamide, the strong acid type ion exchange column of super-dry, the benzene that the back adds its 1/3 volume distills, after boiling off benzene substantially, decompression steams the cut of dimethyl formamide (1 ℃ of boiling range), and adding 4A molecular sieve is an amount of, and sealing is preserved standby.Pyridine refining: pyridine potassium hydroxide drying, after distilling, collect 114～116 ℃ cut.

4N HCl/ ethyl acetate prepares according to a conventional method, and promptly commercially available ethyl acetate, is filtered after dry 24 hours with Anhydrous potassium carbonate, gets the exsiccant ethyl acetate.With NaCl vitriol oil generation hydrogen chloride gas, gas feeds in the ethyl acetate solution behind vitriol oil wash bottle and calcium chloride drying tower and absorbs.Till the 4N that increases weight, the solution sealing is preserved.

Testing tool

Nuclear magnetic resonance spectrometer device model is VXR-300S (300MHz) or INOVA-500 (500MHz).The mass spectrometer model is the ZAB-MS of FAB-MS with Britain VG company, the Trace MS System mass spectrograph and the MALDI-TOF-MS of U.S. Thermo Finnigan company.The fusing point instrument is the desk-top micro-fusing point instrument of XT5 heat that instrument electric light instrument plant of Beijing section produces, and temperature is not proofreaied and correct.Polarimeter is the POLAARTRONIC D type trace polarimeter of SCHMIDT-HAENSCH company, and sample pool is 5 centimetres, volume 0.7ml.

Embodiment 1 Boc-Ser's (Bzl)-OBzl (compound 1) is synthetic

1.0g (3.386mmol) Boc-Ser (Bzl)-OH is dissolved in the 2.5ml dehydrated alcohol, adds 0.557g (1.693mmol) Cs ₂CO ₃The aqueous solution, room temperature reaction is after 2 hours, is evaporated to driedly, and residue adds dehydrated alcohol, be evaporated to dried, 3 times repeatedly, colorless solid.Put dried overnight in the moisture eliminator.This colorless solid is dissolved in the 2ml dry DMF, adds 0.402ml (3.386mmol) bromobenzyl, and reaction is 16 hours about 55 ℃.Reaction solution poured in the culture dish dry up solvent with blower.The residue acetic acid ethyl dissolution filters insolubles, and filtrate is used saturated NaHCO successively ₃The aqueous solution, the saturated NaCl aqueous solution, 5%KHSO ₄The aqueous solution, the saturated NaCl aqueous solution were respectively given a baby a bath on the third day after its birth all over (aforesaid operations is referred to as conventional processing hereinafter).The organic layer anhydrous sodium sulfate drying filters, be evaporated to dried, colourless thick product, with ethyl acetate-sherwood oil recrystallization, pure title compound 1.223g (94%).

Embodiment 2 HClH-Ser's (Bzl)-OBzl (compound 2) is synthetic

500mg (1.299mmol) Boc-Ser (Bzl)-OBzl is dissolved in the 3ml 4NHCl/ ethyl acetate, and room temperature reaction 1.5 hours, TLC show that raw material point disappears.Reaction mixture is evaporated to dried, and residue is used acetic acid ethyl dissolution again, is evaporated to dried.This operation 4 times repeatedly, residue is washed with the anhydrous diethyl ether bubble, gets title compound, is directly used in the next step.ESI-MS：286.6[M+H] ⁺，571.2[2M+H] ⁺.

Embodiment 3 Boc-Asp (OBzl)-Ser (Bzl)-OBzl's (compound 3) is synthetic

A) 420mg (1.299mmol) Boc-Asp (OBzl)-OH is dissolved among the anhydrous THF of 2ml, and ice bath adds the anhydrous THF solution of the DCC of 175.5mg (1.299mmol) HoBt and 268mg (1.299mmol) down, and 0 ℃ was reacted 30 minutes.

B) 417.6mg (1.299mmol) HClH-Ser (Bzl)-OBzl is dissolved among the anhydrous THF of 2ml, and ice bath adds 155 μ l N-methylmorpholines down, reacts after 10 minutes, and this solution is joined in the top A reaction solution.Add about pH=8～9 that N-methylmorpholine makes both mixed reaction solutions, room temperature reaction is after 8 hours, and TLC shows raw material point disappearance.Filter out insolubles, filtrate decompression is concentrated into dried, and residue is used the acetic acid ethyl dissolution residue after washing 6 times with sherwood oil and anhydrous diethyl ether bubble, and filtrate is used saturated NaHCO successively ₃The aqueous solution, the saturated NaCl aqueous solution, 5%KHSO ₄The aqueous solution, the saturated NaCl aqueous solution are respectively given a baby a bath on the third day after its birth time.The organic layer anhydrous sodium sulfate drying filters, and filtrate decompression is concentrated into dried, gets colorless product.With ethyl acetate-sherwood oil recrystallization.Get title compound 722.3mg (94.3%).

Embodiment 4 HClH-Asp (OBzl)-Ser (Bzl)-OBzl's (compound 4) is synthetic

500mg (1.299mmol) Boc-Asp (OBzl)-Ser (Bzl)-OBzl is dissolved in the 3ml 4NHCl/ ethyl acetate, and room temperature reaction 2 hours, TLC show that raw material point disappears.Reaction mixture is evaporated to dried, and residue is used acetic acid ethyl dissolution again, be evaporated to dried, this operation repeatedly 3 times, residue is washed with the anhydrous diethyl ether bubble, title compound, be directly used in the next step.ESI-MS：491.7[M+H] ⁺，981.2[2M+H] ⁺.

Embodiment 5 Boc-Gly-Asp (OBzl)-Ser (Bzl)-OBzl's (compound 5) is synthetic

Adopt the operation of embodiment 3, make 494mg (90.5%) title compound by 114mg (0.8432mmol) Boc-Gly-OH and 444.5mg (0.8432mmol) HClH-Asp (OBzl)-Ser (Bzl)-OBzl.ESI-MS?648.4[M+H] ⁺.

Embodiment 6 HClH-Gly-Asp (OBzl)-Ser (Bzl)-OBzl's (compound 6) is synthetic

Adopt the operation of embodiment 4, make 347.5mg (96%) title compound by 400mg (0.6179mmol) Boc-Gly-Asp (OBzl)-Ser (Bzl)-OBzl.ESI-MS：548.7[M+H] ⁺，1095.6[2M+H] ⁺.

Embodiment 7 Boc-Arg (NO ₂)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl's (compound 7) is synthetic

Adopt the operation of embodiment 3, by 197mg (0.6197mmol) Boc-Arg (NO ₂)-OH and 363mg (0.6179mmol) HClH-Asp (OBzl)-Ser (Bzl)-OBzl makes 466mg (88.7%).Mp72-74℃，[a] ²⁰ _D＝+5.0°(c＝1，CHCl ₃：MeOH＝10∶1)，FAB-MS(m/e)848.1[M-H] ⁺.

Embodiment 8 HClH-Arg (NO ₂)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl's (compound 8) is synthetic

Adopt the operation of embodiment 4, by 400mg (0.4710mmol) Boc-Arg (NO ₂)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl makes title compound 370mg (92.7%), FAB-MS (m/e) 750.3[M+H] ⁺

Embodiment 9 HClH-Arg-Gly-Asp-Ser-OH's (compound 9) is synthetic

100mg (0.1273mmol) HClH-Arg (NO ₂)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl is dissolved in methanol=3/2 mixed solvent, adds 10mg10%Pd/C, logical H ₂, carry out catalytic hydrogenolytic cleavage.Room temperature reaction 5 hours stops reaction, filters, and filtrate decompression is concentrated into dried, and residue is worn away for several times with anhydrous diethyl ether, gets 54mg (90.2%) title compound, is the colorless solid powder.mp183-187℃，[a] ²⁰ _D＝+6°(c＝1，H ₂O)，ESI-MS：434.6[M+2H] ⁺.

Embodiment 10 Boc-Val-OBzl's (compound 10) is synthetic

Boc-Val-OH 1g (4.608mmol) is dissolved in the 2.5ml dehydrated alcohol, adds Cs ₂CO ₃0.750g the aqueous solution (2.304mmol), room temperature reaction is after 2 hours, and reaction mixture is evaporated to dried, and residue adds dehydrated alcohol again, be evaporated to again dried, 3 times repeatedly, colorless solid.Put dried overnight in the moisture eliminator.The gained colorless solid is dissolved in the 3ml dry DMF, adds 0.547ml (4.608mmol) bromobenzyl, reaction is 16 hours about 55 ℃.Reaction mixture is evaporated to dried.Residue is used acetic acid ethyl dissolution again, filters out insolubles, and filtrate is used saturated NaHCO successively ₃The aqueous solution, the saturated NaCl aqueous solution, 5%KHSO ₄The aqueous solution, the saturated NaCl aqueous solution are respectively given a baby a bath on the third day after its birth time.The organic layer anhydrous sodium sulfate drying filters, be evaporated to dried, colourless thick product, with ethyl acetate-sherwood oil recrystallization, pure title compound 1.34g (94.7%).

Embodiment 11 HClH-Val-OBzl's (compound 11) is synthetic

Adopt the operation of embodiment 4, get 311mg (98%) title compound by 400mg (1.303mmol) Boc-Val-OBzl.ESI-MS：208.5[M+H] ⁺，415.4[2M+H] ⁺.

Embodiment 12 Boc-Asp's (OBzl)-Val-OBzl (compound 12) is synthetic

Adopt the operation of embodiment 3, make 634mg (95.0%) title compound, be colorless solid, FAB-MS:513.4[M+H] by 422mg (1.303mmol) Boc-Asp (OBzl)-OH and 317mg (1.303mmol) HClH-Val-OBzl ⁺

Embodiment 13 HClH-Asp's (OBzl)-Val-OBzl (compound 13) is synthetic

Adopt the operation of embodiment 4, get 254.9mg (97%) title compound by 300mg (0.5854mmol) Boc-Asp (OBzl)-Val-OBzl.

Embodiment 14 Boc-Gly-Asp's (OBzl)-Val-OBzl (compound 14) is synthetic

Adopt the operation of embodiment 3, make 319mg (95.7%) title compound by 102mg (0.5854mmol) Boc-Gly-OH and 263mg (0.5854mmol) HClH-Asp (OBzl)-Val-OBzl.mp?77-79℃。ESI-MS：570.5[M+H] ⁺，592.6[M+Na] ⁺，1161.2[2M+Na] ⁺.

Embodiment 15 HClH-Gly-Asp's (OBzl)-Val-OBzl (compound 15) is synthetic

Adopt the operation of embodiment 4, get 261mg (96%) title compound, ESI-MS:470.7[M+H] by 300mg (0.5268mmol) Boc-Gly-Asp (OBzl)-Val-OBzl ⁺

Embodiment 16 Boc-Arg (NO ₂)-Gly-Asp's (OBzl)-Val-OBzl (compound 16) is synthetic

Adopt the operation of embodiment 3, by 167mg (0.5268mmol) Boc-Arg (NO ₂)-OH and 266.5mg (0.5268mmol) HClH-Asp (OBzl)-Val-OBzl makes 405.8mg (88.9%) title compound.mp95-97°，[a] ²⁰ _D＝+7(c＝1，CHCl ₃∶MeOH＝10∶1)，FAB-MS(m/e)771.3[M+H] ⁺.809.3[M+K] ⁺.。

Embodiment 17 HClH-Arg (NO ₂)-Gly-Asp's (OBzl)-Val-OBzl (compound 17) is synthetic

Adopt the operation of embodiment 4, by 500mg (0.6489mmol) Boc-Arg (NO ₂)-Gly-Asp (OBzl)-Val-OBzl gets 470mg (94%) title compound, FAB-MS (m/e) 671.5[M+H] ⁺

Embodiment 18 HClH-Arg-Gly-Asp-Val-OH's (compound 18) is synthetic

Adopt the operation of embodiment 9, by 100mg (0.1298mmol) HClH-Arg (NO ₂)-Gly-Asp (OBzl)-Val-OBzl gets 55mg (88.7%) title compound, is colourless powder.mp180-182℃，[a] ²⁰ _D＝+3°(c＝1，H ₂O)，ESI-MS：445.4[M+H] ⁺

Embodiment 19 Boc-Phe-OBzl's (compound 19) is synthetic

Boc-Phe-OH 1g (3.774mmol) is dissolved in the 3ml dehydrated alcohol, adds Cs ₂CO ₃0.621g the aqueous solution (1.887mmol), room temperature reaction are after 2 hours, the evaporated under reduced pressure solvent adds dehydrated alcohol again, and evaporated under reduced pressure 3 times repeatedly, gets white solid.Put dried overnight in the moisture eliminator.Above-mentioned gained colorless solid is dissolved in the 3ml dry DMF, adds 0.448ml (3.774mmol) bromobenzyl, reaction is 16 hours about 55 ℃.Reaction solution poured in the culture dish dry up solvent with blower.And then use acetic acid ethyl dissolution, and filtering out insolubles, filtrate is used saturated NaHCO successively ₃The aqueous solution, the saturated NaCl aqueous solution, 5%KHSO ₄The aqueous solution, the saturated NaCl aqueous solution are respectively given a baby a bath on the third day after its birth time.The organic layer anhydrous sodium sulfate drying filters, and is spin-dried for, and gets colourless thick product, with ethyl acetate-sherwood oil recrystallization, gets the pure title compound of 1.26lg (95%).Rf＝0.72(A).ESI-MS：378.4[M+Na] ⁺.。

Embodiment 20 HClH-Phe-OBzl's (compound 20) is synthetic

Adopt the operation of embodiment 4, get 470mg (94%) title compound, be directly used in the next step by 500mg (1.407mmol) Boc-Phe-OBzl.

Embodiment 21 Boc-Asp's (OBzl)-Phe-OBzl (compound 21) is synthetic

Adopt the operation of embodiment 3, get 757mg (96%) title compound by 455mg (1.407mmol) Boc-Asp (OBzl)-OH and 516mg (1.407mmol) HClH-Phe-OBzl.mp88-90℃.ESI-MS：583.6[M+Na] ⁺，1143.0[2M+Na] ⁺

Embodiment 22 HClH-Asp's (OBzl)-Phe-OBzl (compound 22) is synthetic

Adopt the operation of embodiment 4, get 403mg (98%) title compound by 500mg (0.8919mmol) Boc-Asp (OBzl)-Phe-OBzl.ESI-MS：461.8[M+H] ⁺.

Embodiment 23 Boc-Gly-Asp's (OBzl)-Phe-OBzl (compound 23) is synthetic

Adopt the operation of embodiment 3, get 509mg (94.3%) title compound by 153mg (0.8746mmol) Boc-Gly-OH and 403mg (0.8746mmol) HClH-Asp (OBzl)-Phe-OBzl.mp?72-74℃。ESI-MS：618.5[M+H] ⁺，640.6[M+Na] ⁺，1257.0[2M+Na] ⁺.

Embodiment 24 HClH-Gly-Asp's (OBzl)-Phe-OBzl (compound 24) is synthetic

Adopt the operation of embodiment 4, make 411mg (98%) title compound, be directly used in the next step by 500mg (0.8097mmol) Boc-Gly-Asp (OBzl)-Phe-OBzl.ESI-MS：518.7[M+H] ⁺，1035.4[2M+H] ⁺.

Embodiment 25 Boc-Arg (NO ₂)-Gly-Asp's (OBzl)-Phe-OBzl (compound 25) is synthetic

Adopt the operation of embodiment 3, by 236mg (0.7412mmol) Boc-Arg (NO ₂)-OH and 411mg (0.7412mmol) HClH-Gly-Asp (OBzl)-Phe-OBzl makes 528.5mg (87%) title compound.mp148-150℃，[a] ²⁰ _D＝+7°(c＝1，CHCl ₃∶MeOH＝10∶1)，FAB-MS(m/e)818.6[M-H] ⁺.

Embodiment 26 HClH-Arg (NO ₂)-Gly-Asp's (OBzl)-Phe-OBzl (compound 26) is synthetic

Adopt the operation of embodiment 4, by 500mg (0.6101mmol) Boc-Arg (NO ₂)-Gly-Asp (OBzl)-Phe-OBzl makes 461mg (93.3%) title compound, is directly used in the next step, FAB-MS (m/e) 720.5[M+H] ⁺

Embodiment 27 HClH-Arg-Gly-Asp-Phe-OH's (compound 27) is synthetic

Adopt the operation of embodiment 9, by 100mg (0.1323mmol) HClH-Arg (NO ₂)-Gly-Asp (OBzl)-Phe-OBzl gets 64mg (92%) title compound, is colourless powder.mp?158-160℃，[a] ²⁰ _D＝+4°(c＝2，H ₂O)，ESI-MS：492.5[M+2H] ⁺

Synthesizing of embodiment 28 γ-(3-hydroxyl oestradiol-17-oxygen base) propionic acid (compound 28)

100mg (1mmol) Succinic anhydried is dissolved in the 1ml pyridine, treat warm dissolving after, add 100mg (0.3676mmol) estradiol, 90 ℃ of reactions are after 10 hours, thin layer detects, raw material point disappearance.Behind the stopped reaction, add 20ml frozen water and 75mg sodium-chlor, use ethyl acetate extraction after the vigorous stirring, merge organic layer, anhydrous Na _2sO ₄Drying is filtered, and the dried organic solvent of vacuum concentration gets yellow mucilage.This mucilage is dissolved in the 1ml methyl alcohol, adds an amount of cold 10%K ₂CO ₃Solution makes pH maintain 8.5-9, and room temperature was placed 18 hours, and back Dropwise 5 0% acetate after vacuum concentration is removed organic solvent, adds 2ml frozen water and 75mg sodium-chlor to pH=6, uses ethyl acetate extraction, and organic layer with icy water backwash several times.Anhydrous Na ₂SO ₄Drying is filtered, and the dried organic solvent of vacuum concentration gets light yellow mucilage.Put refrigerator overnight, separated out solid, and, got off-white powder in second day with a small amount of cold benzene washing.Column chromatography for separation, eluent are chloroforms: methyl alcohol=10: 1, get 137mg (94.5%) title compound, and be colourless powder.Rf＝0.33(C)。FAB-MS(m/e)373.6[M+H] ⁺。mp148-150℃。[a] ²⁰ _D＝+35°(c＝1，THF). ¹HNMR(DMSO-d6)δ?0.774(s，3H，18-CH ₃)；

4.618(t，1H，17-H)；6，400(s，1H，4-H)；6.517(d，1H，3-OH)；7.04(d，1H，1-H)；8.993(s，1H，3-OH)；12.192(s，1H，COOH)

Embodiment 29 N-[γ-(3-hydroxyl oestradiol-17-oxygen base) propionyl]-Arg (NO ₂)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl's (compound 29) is synthetic

Adopt the operation of embodiment 3, by 88mg (0.2355mmol) γ-(3-hydroxyl oestradiol-17-oxygen base) propionic acid (28) and 185mg (0.2355mmol) HClH-Arg (NO ₂)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl (8) gets the pure title compound of 158mg (60.8%), is colourless powder.Rf＝0.42(C)。mp143-145℃，[a] ²⁰ _D＝+20°(c＝1，CHCl ₃∶MeOH＝10∶1)，ESI-TOF：1104.4[M+H] ⁺

Embodiment 30 N-[γ-(3-hydroxyl oestradiol-17-oxygen base) propionyl]-Arg (NO ₂)-Gly-Asp's (OBzl)-Val-OBzl (compound 30) is synthetic

Adopt the operation of embodiment 3, by 121mg (0.3253mmol) γ-(3-hydroxyl oestradiol-17-oxygen base) propionic acid (28) and 230mg (0.3253mmol) HClH-Arg (NO ₂)-Gly-Asp (OBzl)-Val-OBzl (17) gets the pure title compound of 177mg (53.1%), is colourless powder.Rf＝0.53(C)。mp151-153℃，[a] ²⁰ _D＝+30°(c＝1，CHCl ₃∶MeOH＝10∶1)，ESI-TOF∶1025.5[M+H] ⁺

Embodiment 31 N-[γ-(3-hydroxyl oestradiol-17-oxygen base) propionyl]-Arg (NO ₂)-Gly-Asp's (OBzl)-Phe-OBzl (compound 31) is synthetic

Adopt the operation of embodiment 3, by 39mg (0.1041mmol) γ-(3-hydroxyl oestradiol-17-oxygen base) propionic acid (28) and 78mg (0.1041mmol) HClH-Arg (NO ₂)-Gly-Asp (OBzl)-Phe-OBzl (26) gets the pure title compound of 65mg (58.2%), is colourless powder.Rf＝0.51(C)mp156-158℃，[a] ²⁰ _D＝+23°(c＝2，CHCl ₃∶MeOH＝10∶1)，ESI-TOF：1074.2[M+H] ⁺

Embodiment 32 N-[γ-(3-hydroxyl oestradiol-17-oxygen base) propionyl]-Arg-Gly-Asp-Ser-OH (compound 32) synthetic

Adopt the operation of embodiment 9, by 100mg (0.09067mmol) N-[γ-(3-hydroxyl oestradiol-17-oxygen base) propionyl]-Arg (NO ₂)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl (29) gets 44mg (62%) target compound, is colourless powder.Rf＝0.47(D)，mp178-180℃，[a] ²⁰ _D＝+50°(c＝1，MeOH)，FAB-MS：788.4[M+H] ⁺，IR(KBr)3367cm ^-1(v?CO-NH)，1720，1658cm ^-1(vC＝O).

¹HNMR(ppm)δ?0.773(s，3H，18-CH ₃)；4.601(t，1H，17-H)；6.439(s，1H，4-H)；6.514(d，1H，2-H)；7.045(d，1H，1-H)；9.068(s，1H，3-OH)

Embodiment 33 N-[γ-(3-hydroxyl oestradiol-17-oxygen base) propionyl]-Arg-Gly-Asp-Val-OH (compound 33) synthetic

Adopt the operation of embodiment 9, by 100mg (0.09766mmol) N-[γ-(3-hydroxyl oestradiol-17-oxygen base) propionyl]-Arg (NO ₂)-Gly-Asp (OBzl)-Val-OBzl (30) gets 53.4mg (68.4%) target compound, is colourless powder.Rf＝0.50(D)，mp193-195℃，[a] ²⁰ _D＝+56°(c＝0.5，MeOH)，MALDI-TOF：800.4[M+H] ⁺.IR(KBr)3360cm ^-1(vCO-NH)，1659cm ^-1(v?C＝O). ¹HNMR(ppm)δ?0.754(s，3H，18-CH ₃)；0.850(d，6H，Val-2CH ₃)；

6.429(s，1H，4-H)；6.494(d，1H，2-H)；6.931(d，1H，1-H)；9.032(s，1H，3-OH)

Embodiment 34 N-[γ-(3-hydroxyl oestradiol-17-oxygen base) propionyl]-Arg-Gly-Asp-Phe-OH (compound 34) synthetic

Adopt the operation of embodiment 9, by 100mg (0.09320mmol) N-[γ-(3-hydroxyl oestradiol-17-oxygen base) propionyl]-Arg (NO ₂)-Gly-Asp (OBzl)-Phe-OBzl (31) gets 45.8mg (58%) target compound, is colourless powder.，Rf＝0.48(D)，mp196-198℃，[a] ²⁰ _D＝+45°(c＝0.5，MeOH)，MALDI-TOF：848.4[M+H] ⁺，870.4[M+Na] ⁺IR(KBr)3391cm ^-1(v?CO-NH)，1661cm ^-1(v?C＝O). ¹HNMR(ppm)δ?0.764(s，3H，18-CH ₃)；6.434(s，1H，4-H)；6.502(d，1H，2-H)；7.009(d，1H，1-H)；7.023(m，1H，aromatic?H?of?Phe)；7.075(m，2H，aromatic?H?ofPhe)；7.103(m，2H，aromatic?H?ofPhe)；9.045(s，1H，3-OH)

Synthesizing of the inferior methoxyl group-estradiol of embodiment 35 3-ethoxy carbonyls (compound 35)

500mg (1.85mmol) estradiol is dissolved among the anhydrous THF of 10ml, the ethanolic soln that adds the 1.32ml2M sodium ethylate, react after 30 minutes, add 0.612ml (5mmol) ethyl bromoacetate, 55 ℃ of reactions are after 16 hours, and reaction mixture is evaporated to dried, and residue is again with dissolution with solvents and be evaporated to dried, this operation 4 times repeatedly is so that remove residual ethyl bromoacetate.Residue column chromatographic isolation and purification (chloroform/ether, 100/3) gets the pure title compound of 550mg (83%), is colourless powder.Rf＝0.42(E)。mp88-90℃。[a] ²⁰ _D＝+60°(c＝1，THF)。

Synthesizing of the inferior methoxyl group-estradiol of embodiment 36 3-carboxylics (compound 36)

Inferior methoxyl group-the estradiol of 450mg (1.257mmol) 3-ethoxy carbonyl (35) is dissolved in the 3ml dehydrated alcohol, and ice bath drips an amount of 2NNaOH aqueous solution down, and room temperature reaction is after 2.5 hours, and thin layer detects raw material point and disappears, and adds an amount of KHSO ₄Solid makes reaction solution pH=7, and the reaction mixture concentrating under reduced pressure is added less water after removing organic solvent, uses an amount of KHSO again ₄Solid makes reaction solution pH=2, uses ethyl acetate extraction, anhydrous Na ₂SO ₄Drying is filtered, and concentrating under reduced pressure gets the pure title compound of 397mg (95.6%), is colourless powder.Rf＝0.27(B)。mp214-215℃。[a] ²⁰ _D＝+50°(c＝0.6，THF)。EI-MS(m/e)330[M] ⁺.

Embodiment 37 N-(estradiol-3-oxygen base methylene carbonyl)-Arg (NO ₂)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl's (compound 37) is synthetic

Adopt the operation of embodiment 3, by inferior methoxyl group-estradiol (36) of 50mg (0.1514mmol) 3-carboxylic and 119mg (0.1514mmol) HClH-Arg (NO ₂)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl (8) gets the pure title product of 150mg (93.2%), is colourless powder.Rf＝0.44(C)，mp114-116℃。[a] ²⁰ _D＝+55°(c＝0.5，MeOH)。ESI-MS?1062.5[M+H] ⁺

Embodiment 38 N-(estradiol-3-oxygen base methylene carbonyl)-Arg (NO ₂)-Gly-Asp's (OBzl)-Val-OBzl (compound 38) is synthetic

Adopt the operation of embodiment 3, by inferior methoxyl group-estradiol (36) of 50mg (0.1514mmol) 3-carboxylic and 107mg (0.1514mmol) HClH-Arg (NO ₂)-Gly-Asp (OBzl)-Val-OBzl (17) gets the pure title product of 111mg (73.5%), is colourless powder.Rf＝0.52(C)。mp130-132℃。[a] ²⁰ _D＝+60°(c＝1，MeOH)，FAB-MS(m/e)1025.9[M+H] ⁺.

Embodiment 39 N-(estradiol-3-oxygen base methylene carbonyl)-Arg (NO ₂)-Gly-Asp's (OBzl)-Phe-OBzl (compound 39) is synthetic

Adopt the operation of embodiment 3, by inferior methoxyl group-estradiol (36) of 50mg (0.1514mmol) 3-carboxylic and 119mg (0.1514mmol) HClH-Arg (NO ₂)-Gly-Asp (OBzl)-Phe-OBzl (26) gets the pure title product of 96.5mg (60.9%), is colourless powder.Rf＝0.55(C)。mp136-138℃。[a] ²⁰ _D＝+55°(c＝1，MeOH)，FAB-MS(m/e)1048.2[M+H] ⁺.

Embodiment 40 N-(estradiol-3-oxygen base methylene carbonyl)-Arg-Gly-Asp-Ser-OH's (compound 40) is synthetic

Adopt the operation of embodiment 9, by 100mg (0.09421mmol) N-(estradiol-3-oxygen base methylene carbonyl)-Arg (NO ₂)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl (37) gets the pure title compound of 49.1mg (70%), is colourless powder.Rf＝0.41(D)，mp155-157℃。[a] ²⁰ _D＝+39°(c＝0.5，MeOH)，ESI-TOF：746.3[M+H] ⁺?IR(KBr)3387cm ^-1(v?CO-NH)，1659cm ^-1(v?C＝O). ¹HNMR(ppm)δ＝0.663(s，3H，18-CH ₃)；4.513(s，2H，3-O-CH ₂)；6.645(s，1H，4-H)；6.701(d，1H，2-H)；7.156(d，1H，1-H)；

Embodiment 41 N-(estradiol-3-oxygen base methylene carbonyl)-Arg-Gly-Asp-Val-OH's (compound 41) is synthetic

Adopt the operation of embodiment 9, by 100mg (0.09757mmol) N-(estradiol-3-oxygen base methylene carbonyl)-Arg (NO ₂)-Gly-Asp (OBzl)-Val-OBzl (38) gets the pure title compound of 53.9mg (73%), is colourless powder.Rf＝0.43(D)，mp174-176℃。[a] ²⁰ _D＝+45°(c＝0.5，MeOH)，FAB-MS：757.9[M+H] ⁺。IR(KBr)3402cm ^-1(v?CO-NH)，1660cm ^-1(v?C＝O). ¹HNMR(DMSO-d6)δ＝0.646(s，3H，18-CH ₃)；0.731(d，6H，Val-2CH ₃)；3.006(d，1H，Val-CH-CO)；4.505(s，2H，3-O-CH ₂-CO)；6.596(s，1H，4-H)；6.949(d，2H，2-H)；7.136(d，2H，1-H)。

Embodiment 42 N-(estradiol-3-oxygen base methylene carbonyl)-Arg-Gly-Asp-Phe-OH's (compound 42) is synthetic

Adopt the operation of embodiment 9, by 100mg (0.09549mmol) 17 β-OH-estradiol-3-O-ethanoyl-Arg (NO ₂)-Gly-Asp (OBzl)-Phe-OBzl (39) gets the pure title compound of 59.2mg (77%), is colourless powder.Rf＝0.45(D)，mp180-182℃。[a] ²⁰ _D＝+48°(c＝0.5，MeOH)，，FAB-MS：806.3[M+H] ⁺IR(KBr)3405cm ^-1(v?CO-NH)，1660cm ^-1(v?C＝O). ¹HNMR(ppm)δ0.659(s，3H，18-CH ₃)；6.645(s，1H，4-H)；6.667(d，1H，2-H)；7.063(d，1H，1-H)；7.079(m，1H，aromatic?H?of?Phe)；7.127(m，2H，aromatic?H?of?Phe)；7.275(m，2H，aromatic?H?ofPhe).

Synthesizing of the inferior methoxyl group-estradiol of embodiment 43 3-diazanyls (compound 43)

Inferior methoxyl group-the estradiol of 200mg (0.5587mmol) 3-ethoxy carbonyl (35) is dissolved in 3ml methyl alcohol, drip the 1ml80% hydrazine hydrate down in 55 ℃, react after 4 hours, stopped reaction, refrigerator are placed and are spent the night, and second day with the solid filtering of separating out, washing, drying gets 184mg (96%) title compound, is no powder.Rf＝0.21(C)。mp153-155℃，FAB-MS(m/e)345.5[M+H] ⁺

Synthesizing of the inferior methoxyl group-estradiol of embodiment 44 Boc-Ser (Bzl)-3-diazanyl (compound 44)

Adopt the operation of embodiment 3, get the pure title compound of 305mg (89.1%), be the colorless solid powder by 163mg (0.5523mmol) BocSer (Bzl) OH and the inferior methoxyl group-estradiol of 190mg (0.5523mmol) 3-diazanyl (43).Rf＝0.34(B).FAB-MS(rm/e)620.6[M+H] ⁺.

Synthesizing of the inferior methoxyl group-estradiol of embodiment 45 Boc-Val-3-diazanyls (compound 45)

Adopt the operation of embodiment 3, get the pure title compound of 288mg (96%), be the colorless solid powder by 120mg (0.5523mmol) BocValOH and the inferior methoxyl group-estradiol of 190mg (0.5523mmol) 3-diazanyl (43).mp173-174℃，Rf＝0.35(B).FAB-MS(m/e)543[M] ⁺.

Synthesizing of the inferior methoxyl group-estradiol of embodiment 46 Boc-Phe-3-diazanyls (compound 46)

Adopt the operation of embodiment 3, get the pure title product of 73mg (85%), be the colorless solid powder by 39mg (0.1453mmol) BocPheOH and the inferior methoxyl group-estradiol of 50mg (0.1453mmol) 3-diazanyl (43).Rf＝0.31(B).FAB-MS(m/e)592[M+H] ⁺

Synthesizing of the inferior methoxyl group-estradiol of embodiment 47 HCl Ser (Bzl)-3-diazanyl (compound 47)

Adopt the operation of embodiment 4, get 79mg (94%) title compound, be colourless powder by the inferior methoxyl group-estradiol of 100mg (0.1615mmol) Boc-Ser (Bzl)-3-diazanyl (44).

Synthesizing of the inferior methoxyl group-estradiol of embodiment 48 HClVal-3-diazanyls (compound 48)

Adopt the operation of embodiment 4, get 110.8mg (97%) title compound, be colourless powder by the inferior methoxyl group-estradiol of 140mg (0.2579mmol) Boc-Val-3-diazanyl (45).

Synthesizing of the inferior methoxyl group-estradiol of embodiment 49 HClPhe-3-diazanyls (compound 49)

Adopt the operation of embodiment 4, get 78.8mg (95%) title compound, be colourless powder by the inferior methoxyl group-estradiol of 100mg (0.1691mmol) Boc-Phe-3-diazanyl (46).

Synthesizing of the inferior methoxyl group-estradiol of embodiment 50 BocAsp (OBzl)-Ser (Bzl)-3-diazanyl (compound 50)

Adopt the operation of embodiment 3, get 115mg (85%) title compound, be colourless powder by 52mg (0.1615mmol) BocAsp (OBzl) OH and the inferior methoxyl group-estradiol of 84mg (0.1615mmol) HCl Ser (Bzl)-3-diazanyl (47).Rf＝0.53(B)。FAB-MS(m/e)826.3[M+H] ⁺

Synthesizing of the inferior methoxyl group-estradiol of embodiment 51 Boc-Asp (OBzl)-Val-3-diazanyl (compound 51)

Adopt the operation of embodiment 3, get 174mg (90%) title compound, be colourless powder by 83.4mg (0.2579mmol) BocAsp (OBzl) OH and the inferior methoxyl group-estradiol of 114.2mg (0.2579mmol) HClVal-3-diazanyl (48).Rf＝0.48(B)。FAB-MS(m/e)749.7[M+H] ⁺

Synthesizing of the inferior methoxyl group-estradiol of embodiment 52 BocAsp (OBzl)-Phe-3-diazanyl (compound 52)

Adopt the operation of embodiment 3, get 185mg (89%) title compound, be colourless powder by 55mg (0.1691mmol) BocAsp (OBzl) OH and the inferior methoxyl group-estradiol of 83mg (0.1691mmol) HClPhe-3-diazanyl (49).mp110-112℃，Rf＝0.56(B)。FAB-MS(m/e)797.4[M] ⁺

Synthesizing of the inferior methoxyl group-estradiol of embodiment 53 HClAsp (OBzl)-Ser (Bzl)-3-diazanyl (compound 53)

Adopt the operation of embodiment 4, get the pure title compound of 184.5mg (96%), be directly used in the next step by the inferior methoxyl group-estradiol of 219mg (0.2650mmol) BocAsp (OBzl)-Ser (Bzl)-3-diazanyl (50).

Synthesizing of the inferior methoxyl group-estradiol of embodiment 54 HClAsp (OBzl)-Val-3-diazanyl (compound 54)

Adopt the operation of embodiment 4, get the pure title compound of 79.7mg (92%), be directly used in the next step by the inferior methoxyl group-estradiol of 100mg (0.1335mmol) Boc-Asp (OBzl)-Val-3-diazanyl (51).

Synthesizing of the inferior methoxyl group-estradiol of embodiment 55 HClAsp (OBzl)-Phe-3-diazanyl (compound 55)

Adopt the operation of embodiment 4, get the pure title compound of 80.3mg (94%), be directly used in the next step by the inferior methoxyl group-estradiol of 100mg (0.1255mmol) BocAsp (OBzl)-Phe-3-diazanyl (52).

Embodiment 56 HClH-Gly-OCH ₃Synthesizing of (compound 56)

The 25ml anhydrous methanol is cooled to-10 ℃, stirs slowly to add 6.5mlSOCl down ₂, after 10 minutes, add the 2g glycine.Behind the room temperature reaction 2 days, concentrating under reduced pressure adds 12ml methyl alcohol again, concentrates secondary repeatedly.Use recrystallizing methanol.Get product 2.75g (95%).

Embodiment 57 BocArg (NO ₂)-Gly-OCH ₃Synthesizing of (compound 57)

500mg (1.5660mmol) BocArg (NO ₂) OH is dissolved among the anhydrous THF of 7ml, cryosel is bathed and is added 0.1863ml (1.5660mmol) NMM down, behind the several minutes, add chloro-butyric acid isobutyl ester 0.295ml (1.879mmol), add the anhydrous THF solution of 225mg (1.5660mmol) compound (56) and 0.1863ml (1.5660mmol) NMM behind the several minutes again.Stop reaction after 3 hours, conventional processing gets the pure title product of 525.6mg (86%), is colourless powder.

Embodiment 58 BocArg (NO ₂)-Gly-OH's (compound 58) is synthetic

Adopt the operation of embodiment 36, by 200mg (0.4935mmol) BocArg (NO ₂)-Gly-OCH ₃(57) get the pure title product of 151mg (81%), be colourless powder.Rf＝0.21(C)。

Embodiment 59 BocArg (NO ₂Synthesizing of the inferior methoxyl group-estradiol of)-Gly-Asp (OBzl)-Ser (Bzl)-3-diazanyl (compound 59)

Adopt the operation of embodiment 3, by 100mg (0.2650mmol) BocArg (NO ₂Inferior methoxyl group-the estradiol of)-Gly-OH and 202mg (0.2650mmol) HClAsp (OBzl)-Ser (Bzl)-3-diazanyl (53) gets the pure title compound of 204mg (71%), is colourless powder.Rf＝0.23(C)。FAB-MS(m/e)：1085.3[M+H] ⁺

Embodiment 60 BocArg (NO ₂Synthesizing of the inferior methoxyl group-estradiol of)-Gly-Asp (OBzl)-Val-3-diazanyl (compound 60)

Adopt the operation of embodiment 3, by 50mg (0.1335mmol) BocArg (NO ₂Inferior methoxyl group-the estradiol of)-Gly-OH and 92mg (0.1335mmol) HClAsp (OBzl)-Val-3-diazanyl (54) gets the pure title compound of 93.4mg (69.5%), is colourless powder.Rf＝0.20(C)。FAB-MS(m/e)：1007.6[M+H] ⁺。

Embodiment 61 BocArg (NO ₂Synthesizing of the inferior methoxyl group-estradiol of)-Gly-Asp (OBzl)-Phe-3-diazanyl (compound 61)

Adopt the operation of embodiment 3, by 46mg (0.1255mmol) BocArg (NO ₂Inferior methoxyl group-the estradiol of)-Gly-OH and 89.8mg (0.1255mmol) HClAsp (OBzl)-Phe-3-diazanyl (55) gets the pure title compound of 92.6mg (70%), is colourless powder.Rf＝0.27(C)。FAB-MS(m/e)：1055.5[M+H] ⁺。

Inferior methoxyl group-the oestrone (compound 62) of embodiment 62 3-ethoxy carbonyls

Adopt the operation of embodiment 35, get the pure title compound of 593mg (90%), be colourless powder by 500mg (1.8491mmol) oestrone and 0.750ml (5.5mmol) ethyl bromoacetate.Rf＝0.53(E)，mp98-100℃，[a] ²⁰ _D＝+138°(c＝0.5，THF)，FAB-MS(m/e)357.3[M+H] ⁺.

Synthesizing of the inferior methoxyestrones of embodiment 63 3-carboxylics (compound 63)

Adopt the operation of embodiment 36, be dissolved in the 3ml dehydrated alcohol by the inferior methoxyl group-oestrone of 500mg (1.4025mmol) 3-ethoxy carbonyl (62), ice bath drips an amount of 2NNaOH aqueous solution down, and room temperature reaction is after 2.5 hours, and thin layer detects raw material point and disappears, and adds an amount of KHSO ₄Solid makes reaction solution pH=7, behind the dried organic solvent of vacuum concentration, adds less water, uses an amount of KHSO again ₄Solid makes reaction solution pH=2, uses ethyl acetate extraction, anhydrous Na ₂SO ₄Drying is filtered, be evaporated to dried, 442mg (96%) title compound, be colourless powder.Rf＝0.42(B)。mp?214-215℃；[a] ²⁰ _D＝+159°(c＝0.45，THF)，FAB-MS(m/e)329.3[M+H] ⁺.

Embodiment 64 N-(oestrone-3-oxygen ethanoyl) Arg (NO ₂)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl's (compound 64) is synthetic

Inferior methoxyestrone (63) of 50mg (0.1514mmol) 3-carboxylic and 20mg (0.1514mmol) HoBt, be dissolved among the anhydrous THF of 1ml, ice bath adds down the DCC of 33mg (0.1514mmol), after 30 minutes reaction solution is joined 119mg (0.1514mmol) HClH-Arg (NO ₂In the anhydrous THF solution of)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl (8), and keep with NMM about the pH=8 of reaction solution.Behind the room temperature reaction 8 hours, tlc analysis raw material point is basic to disappear, conventional processing, and crude product gets 99mg (62%) title compound through column chromatographic isolation and purification (chloroform/methanol, 10/1), is colourless powder.Rf＝0.47(C)。mp133-135℃，[a] ²⁰ _D＝+32°(c＝0.5，CHCl ₃∶MeOH＝10∶1)，FAB-MS(m/e)1059.6[M] ⁺.

Embodiment 65 N-(oestrone-3-oxygen ethanoyl)-Arg (NO ₂)-Gly-Asp's (OBzl)-Val-OBzl (compound 65) is synthetic

Inferior methoxyestrone (63) of 50mg (0.1514mmol) 3-carboxylic and 20mg (0.1514mmol) HoBt, be dissolved among the anhydrous THF of 1ml, ice bath adds down the DCC of 33mg (0.1514mmol), after 30 minutes reaction solution is joined 107mg (0.1514mmol) HClH-Arg (NO ₂In the anhydrous THF solution of)-Gly-Asp (OBzl)-Val-OBzl (17), and keep with NMM about the pH=8 of reaction solution.Behind the room temperature reaction 8 hours, tlc analysis raw material point is basic to disappear, conventional processing, and crude product gets 101mg (68%) title compound through column chromatographic isolation and purification (chloroform/methanol, 10/1), is colourless powder.Rf＝0.56(C)。mp124-126℃[a] ²⁰ _D＝+26°(c＝0.5，CHCl ₃∶MeOH＝10∶1)，FAB-MS(m/e)981.5[M+H] ⁺.

Embodiment 66 N-(oestrone-3-oxygen ethanoyl)-Arg (NO ₂)-Gly-Asp's (OBzl)-Phe-OBzl (compound 66) is synthetic

Inferior methoxyestrone (63) of 50mg (0.1514mmol) 3-carboxylic and 20mg (0.1514mmol) HoBt, be dissolved among the anhydrous THF of 1ml, ice bath adds down the DCC of 33mg (0.1514mmol), after 30 minutes reaction solution is joined 119mg (0.1514mmol) HClH-Arg (NO ₂In the anhydrous THF solution of)-Gly-Asp (OBzl)-Phe-OBzl (26), and keep with NMM about the pH=8 of reaction solution.Behind the room temperature reaction 8 hours, tlc analysis raw material point is basic to disappear, conventional processing, and crude product gets 110mg (70.3%) title compound through column chromatographic isolation and purification (chloroform/methanol, 10/1), is colourless powder.Rf＝0.57(C)。mp130-132℃，[a] ²⁰ _D＝+28°(c＝0.5，CHCl ₃∶MeOH＝10∶1)，FAB-MS(m/e)1028.5[M-H] ⁺.

Embodiment 67 N-(oestrone-3-oxygen ethanoyl) Arg-Gly-Asp-Ser-OH's (compound 67) is synthetic

Adopt the operation of embodiment 9, by 100mg (0.0944mmol) N-(oestrone-3-oxygen ethanoyl) Arg (NO ₂)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl (64) gets 40mg (57%) target compound, is colourless powder.Rf＝0.55(D)，mp170-172℃，[a] ²⁰ _D＝+55°(c＝1，MeOH)，FAB-MS：743.5[M] ⁺? ¹HNMR(ppm)δ?0.827(s，3H，18-CH ₃)；4.534(s，2H，3-O-CH ₂)；6.646(s，1H，4-H)；6.700(d，1H，2-H)；7.174(d，1H，1-H)；

Embodiment 68 N-(oestrone-3-oxygen ethanoyl) Arg-Gly-Asp-Val-OH's (compound 68) is synthetic

Adopt the operation of embodiment 9, by 100mg (0.1020mmol) N-(oestrone-3-oxygen ethanoyl)-Arg (NO ₂)-Gly-Asp (OBzl)-Val-OBzl (65) gets 58.5mg (76%) target compound, is colourless powder.，Rf＝0.60(D)，mp164-1?66℃，[a] ²⁰ _D＝+44°(c＝0.5，MeOH)FAB-MS：756.3[M+H] ⁺? ¹HNMR(ppm)δ?0.747(d，6H，Val-2CH ₃)；0.829(s，3H，18-CH ₃)；4.531(s，2H，3-O-CH ₂)；6.640(s，1H，4-H)；6.700(d，1H，2-H)；7.171(d，1H，1-H)；

Embodiment 69 N-(oestrone-3-oxygen ethanoyl)-Arg-Gly-Asp-Phe-OH's (compound 69) is synthetic

Adopt the operation of embodiment 9, by 100mg (0.09713mmol) N-(oestrone-3-oxygen ethanoyl)-Arg (NO ₂)-Gly-Asp (OBzl)-Phe-OBzl (66) gets 43.7mg (56%) target compound, is colourless powder.Rf＝0.53(D)，mp167-169℃，[a] ²⁰ _D＝+32°(c＝0.5，MeOH)，FAB-MS：803.2[M] ⁺? ¹HNMR(ppm)δ?0.810(s，3H，18-CH ₃)；4.548(3-O-CH ₂)；6.653(s，1H，4-H)；6.708(d，1H，2-H)；7.092(d，1H，1-H)；7.113(m，1H，aromatic?H?of?Phe)；7.129(m，2H，aromatic?H?of?Phe)；7.151(m，2H，aromatic?H?of?Phe)；9.045(s，1H，3-OH)

Synthesizing of embodiment 70 17-diazanyl oestrone (compound 70)

100mg (0.37mmol) oestrone is dissolved in 0.5ml1, in the 4-dioxane, is heated to 60 ℃, adds the 1ml80% hydrazine hydrate again, and 60 ℃ are reacted after 4 hours down, put refrigerator and cooled but, filter and collect the solid matter of separating out.Get product 98mg (93%) title compound, be colourless powder.Rf＝0.52(B)，mp.240(dec)，[a] ²⁰ _D＝+102°(C＝1，DMF).EI-MS(m/e)284[M] ⁺.

Embodiment 71 H-Asp's (OBzl)-OH (compound 71) is synthetic

The benzylalcohol (50ml) that newly steams is joined in the mixing solutions of anhydrous diethyl ether (50ml) and the vitriol oil (5ml), after vacuum pumps ether, stir time add in batches aspartic acid (6.7g, 50mmol).Behind the room temperature reaction 24 hours,, under the vigorous stirring situation, drip 25ml pyridine neutralization reaction liquid again with the alcohol dilution reaction solution of 100ml95%.Reaction mixture is put refrigerator overnight.Filter and collect the solid of separating out.Fully steep with ether and to wash.With the hot water recrystallization that contains pyridine.Get 4.5g (40%) title compound, be colourless powder.mp.218-220℃。[a] ²⁰ _D＝+28°(C＝1，HCl)

Embodiment 72 BocGlyONp's (compound 72) is synthetic

Adopt the operation of embodiment 3,, get the pure title compound of 835mg (98.8%), be light yellow solid, Rf=0.64 (F) by 500mg (2.8571mmol) BocGlyOH and 405mg (2.9142mmol) HONP.

Embodiment 73 BocGly-Asp's (OBzl)-OH (compound 73) is synthetic

A) 230mg (0.7770mmol) compound (72) is dissolved among the 2mlTHF; B) dissolve 174mg (0.7770mmol) compound (71) and 65mg (0.7770mmol) NaHCO with less water ₃. the A reaction solution is joined in the B reaction solution, and room temperature reaction was added a little NMM and is made reaction pH maintain weakly alkaline after 24 hours in the reaction process.Reaction finishes final vacuum and concentrates dried organic solvent, adds the less water dissolution residual substance, with ether extraction earlier several times, and water layer solid K HSO ₄Be acidified to pH=2, use ethyl acetate extraction, anhydrous sodium sulfate drying filters, and vacuum concentration is done filtrate, gets 269mg (91%) title compound, is colorless solid.

Embodiment 74 17-Boc-Ser's (Bzl)-diazanyl oestrone (compound 74) is synthetic

Adopt the operation of embodiment 3, get the pure title compound of 455.5mg (79.9%), be colourless powder by 300mg (1.016mmol) BocSer (Bzl) OH and 17-diazanyl oestrone (70).Rf＝0.51(B)。FAB-MS(m/e)562.4[M+H] ⁺.mp?127-129℃.

Synthesizing of embodiment 75 17-Boc-Val-diazanyl oestrone (compound 75)

Adopt the operation of embodiment 3, get the pure title compound of 643mg (72.5%), be colourless powder by 400mg (1.8433mmol) BocValOH and 523.5mg (1.8433mmol) 17-diazanyl oestrone (70).Rf＝0.50(B)。mp131-133℃，FAB-MS(m/e)483.7[M+H] ⁺.

Synthesizing of embodiment 76 17-Boc-Phe-diazanyl oestrone (compound 76)

Adopt the operation of embodiment 3, get the pure title compound of 433mg (92.5%), be colourless powder by 234mg (O.8805mmol) BocPheOH and 250mg (0.8805mmol) 17-diazanyl oestrone (70).Rf＝0.62(B)。mp?125-127℃.FAB-MS(m/e)531.7[M+H] ⁺。

Synthesizing of embodiment 77 17-Ser (Bzl)-female keto hydrochloride of diazanyl (compound 77)

Adopt the operation of embodiment 4, get the pure title compound of 154mg (97%), be colourless powder, be directly used in the next step by 179mg (0.3189mmol) 17-Boc-Ser (Bzl)-diazanyl oestrone (74).

Synthesizing of the female keto hydrochlorides of embodiment 78 17-Val-diazanyls (compound 78)

Adopt the operation of embodiment 4, get the pure title compound of 122.4mg (94%), be colourless powder, be directly used in the next step by 150mg (0.3106mmol) 7-Boc-Val-diazanyl oestrone (75).

Synthesizing of the female keto hydrochlorides of embodiment 79 17-Phe-diazanyls (compound 79)

Adopt the operation of embodiment 4, get the pure title compound of 150.3mg (96%), be colourless powder, be directly used in the next step by 178mg (0.3350mmol) 17-Boc-Phe-diazanyl oestrone (76).

Embodiment 80 17-Boc-Gly-Asp (OBzl) Ser's (Bzl)-diazanyl oestrone (compound 80) is synthetic

Adopt the operation of embodiment 3, get 223mg (85%) title compound, be colourless powder by 121mg (0.3189mmol) BocGly-Asp (OBzl)-OH (73) and 154mg (0.3095mmol) 17-Ser (Bzl)-female keto hydrochloride of diazanyl (77).

Synthesizing of embodiment 81 17-Boc-Gly-Asp (OBzl)-Val-diazanyl oestrone (compound 81)

Adopt the operation of embodiment 3, get 213mg (92%) title compound, be colourless powder by 118mg (0.3106mmol) BocGly-Asp (OBzl)-OH (73) and 122.4mg (0.2920mmol) the female keto hydrochloride of 17-Val-diazanyl (78).

Synthesizing of embodiment 82 17-Boc-Gly-Asp (OBzl)-Phe-diazanyl oestrone (compound 82)

Adopt the operation of embodiment 3, get 242.6mg (91.3%) title compound by 127mg (0.3350mmol) BocGly-Asp (OBzl)-OH (73) and 150.3mg (0.3217mmol) the female keto hydrochloride of 17-Phe-diazanyl (79).ESI-TOF(m/e)794.6[M+H] ⁺，816.6[M+Na] ⁺.

Synthesizing of embodiment 83 17-Gly-Asp (OBzl)-Ser (Bzl)-female keto hydrochloride of diazanyl (compound 83)

Adopt the operation of embodiment 4, get 86.9mg (94%) title compound, be colorless solid, be directly used in the next step by 100mg (0.1214mmol) 17-Boc-Gly-Asp (OBzl) Ser (Bzl)-diazanyl oestrone (80).

Synthesizing of embodiment 84 17-Gly-Asp (OBzl)-female keto hydrochloride of Val-diazanyl (compound 84)

Adopt the operation of embodiment 4, get 85.7mg (93.5%) title compound, be colorless solid, be directly used in the next step by 100mg (0.1342mmol) 17-Boc-Gly-Asp (OBzl)-Val-diazanyl oestrone (81).

Synthesizing of embodiment 85 17-Gly-Asp (OBzl)-female keto hydrochloride of Phe-diazanyl (compound 85)

Adopt the operation of embodiment 4, get 51.7mg (92%) title compound by 61mg (0.07688mmol) 17-Boc-Gly-Asp (OBzl)-Phe-diazanyl oestrone (82),, be directly used in the next step.

Embodiment 86 17-Boc-Arg (NO ₂Synthesizing of)-Gly-Asp (OBzl)-Ser (Bzl)-diazanyl oestrone (compound 86)

Adopt the operation of embodiment 3, by 39mg (0.1214mmol) BocArg (NO ₂) OH and 92.3mg (0.1214mmol) 17-Gly-Asp (OBzl)-Ser (Bzl)-female keto hydrochloride of diazanyl (83) 84.6mg (68%) title compound, be colorless solid.Rf＝0.43(C)。FAB-MS(m/e)1026[M+H] ⁺.

Embodiment 87 17-Boc-Arg (NO ₂Synthesizing of)-Gly-Asp (OBzl)-Val-diazanyl oestrone (compound 87)

Adopt the operation of embodiment 3, by 43mg (0.1342mmol) BocArg (NO ₂) OH and 91.6mg (0.1342mmol) 17-Gly-Asp (OBzl)-female keto hydrochloride of Val-diazanyl (84) 92.7mg (73%) title compound.mp137-139℃，FAB-MS(m/e)947.4[M+H] ⁺

Embodiment 88 17-Boc-Arg (NO ₂Synthesizing of)-Gly-Asp (OBzl)-Phe--diazanyl oestrone (compound 88)

Adopt the operation of embodiment 3, by 25mg (0.07688mmol) BocArg (NO ₂) OH and 56.2mg (0.07688mmol) 17-Gly-Asp (OBzl)-female keto hydrochloride of Phe-diazanyl (85) 61mg (80%) title compound.ESI-TOF(m/e)995.7[M+H] ⁺，1017.7[M+Na] ⁺.

The anti-osteoporosis activity test of test example 1 The compounds of this invention

One, material and reagent

1, for test agent: the respective compound 9,18,27,67,68,69,32,33,34,40,41 and 42 that the embodiment of the invention 9,18,27,67,68,69,32,33,34,40,41 and 42 is prepared.

2, positive control medicine: estradiol, oestrone.

3, laboratory animal: the female mouse of Kunming kind in 7 ages in week, body weight 30.7 ± 3.1g, portion provides by the Department Of Medicine, Peking University animal.

4, reagent preparation:

1) take by weighing 32.5mg OCPC (OCPC), the 1.1g8-hydroxyquinoline adds water 250ml, adds concentrated hydrochloric acid 7.5ml, fully after the stirring and dissolving, adds ultrapure water and gets the OCPC test solution to 500ml.Measuring diethylamine 21ml adds ultrapure water and is diluted to 500ml and gets the diethylamine test solution.

2) precision takes by weighing the Carbon Dioxide calcium 2.5g of new oven dry, adds ultrapure water 100ml, drips concentrated hydrochloric acid 5ml, is diluted to 1000ml and gets standard calcium liquid (1mg/ml).

3) take by weighing Tricholroacetic Acid 50g, thiocarbamide 5g with the dissolving of 300ml ultrapure water, adds ferrous ammonium sulphate 20g again, makes dissolving, is diluted to 500ml and gets ferrous-Tricholroacetic Acid reagent.

4) slowly add in the cold ultrapure water of vitriol oil 45ml to 200ml, the limit edged stirs, and is cooled to room temperature, takes by weighing ammonium molybdate 22g, is dissolved in the 200ml ultrapure water, mixes and be diluted to 500ml with sulphuric acid soln to get ammonium molybdate reagent.

5) take by weighing anhydrous potassium dihydrogenphosphate 109.9mg, be dissolved in the 500ml ultrapure water, add the anticorrosion phosphorus reference liquid (50mg/L) that gets of 2ml chloroform.

6) take by weighing anhydrous sodium carbonate 6.36g, sodium bicarbonate 3.36g, 4-aminoantipyrene 1.5g, be dissolved in the 1000ml ultrapure water carbonate buffer solution (pH=10), in brown bottle, preserve.

7) boil ultrapure water 500ml earlier, the elimination of micro-organisms adds disodium phenyl phosphate (containing two molecular crystal water) 2.54g rapidly, after the cooling, adds the anticorrosion disodium phenyl phosphate substrate solution (20mmol/L) that gets of 2ml chloroform, and refrigerator is preserved.

8) take by weighing Tripotassium iron hexacyanide 2.5g and be dissolved in the 400ml ultrapure water, take by weighing boric acid 17g and be dissolved in the 400ml ultrapure water, mix two liquid, be diluted to 1000ml and get potassium ferricyanide solution, put in the brown bottle and keep in Dark Place.

9) precision takes by weighing 50mg phenol, and ultrapure water is diluted to the preparation (0.05g/L) that 1000ml gets the phenol standardized solution.

10) take by weighing bovine serum albumin 0.5g, add ultrapure water 20ml mixing, insulation is 1 hour in 56 ℃ of water-baths, takes out to put coldly, and it is standby to put in the refrigerator quick-frozen.Face with preceding 37 ℃ of water-baths of reserve liquid are melted, pour in the aqueous solution that contains the 5g sodium-acetate, add concentrated hydrochloric acid 3.5ml again, ultrapure water is diluted to 500ml, and surveying pH=4.0 is the serum reference substance.

Two, test method and result

With 192 7 the week age female Kunming mouse be divided into 16 groups at random, i.e. oophorectomize group (OVX), sham operated rats (sham), oophorectomize+17 beta estradiol treatment groups, oophorectomize+estrone group, oophorectomize+compound 8 treatment groups, oophorectomize+compound 17 treatment groups, oophorectomize+compound 26 treatment groups, oophorectomize+compound 67 treatment groups, oophorectomize+compound 68 treatment groups, oophorectomize+compound 69 treatment groups, oophorectomize+compound 32 treatment groups, oophorectomize+compound 33 treatment groups, oophorectomize+compound 34 treatment groups, oophorectomize+compound 40 treatment groups, oophorectomize+compound 41 treatment groups, oophorectomize+compound 42 treatment groups.

Press the dosage intraperitoneal injection of anesthesia mouse of 40mg/Kg with 2% vetanarcol, cut off mouse peritoneal, find the uterine tube of both sides, oviducal cecum finds ovary in both sides, remove the mouse bilateral ovaries, tubal ligation (if mouse comes to life in the surgical procedure, then with cotton dip in get an amount of anhydrous diethyl ether place the nose place of mouse make it anesthesia).After ligation is good, drip several penicillin, again mouse peritoneal is sewed up, coat an amount of 0.1% Morpan BB thimerosal in the wound outside, to avoid wound infection to mouse peritoneal.

Postoperative beginning in 5 days administration.Oophorectomize group and sham operated rats abdominal injection every day blank solution (5 ‰ carboxymethyl cellulose aqueous solution), other organizes medicine of abdominal injection every day (dosage is 0.1103 μ mol/Kg).After around the administration, weigh, mouse is extractd eyeball and gets blood and put to death, leave standstill 30min after, 3000g/ divides centrifugal 20min, serum, in-20 ℃ of preservations.Measuring blood calcium with the OCPC method, measure serium inorganic phosphorus content with molybdenum blue method, is the content that substrate is measured alkaline phosphatase (ALP) with disodium phenyl phosphate.Lung, liver, spleen, the uterus of separating mouse get their net weight.Get mouse left side femur, reject clean muscle tissue, with the dissection length of vernier callipers mensuration femur, promptly two ankle length.And then use chloroform: methyl alcohol (2: 1) soaks twice (each 3 hours), carries out degreasing.Femur after the degreasing was placed 120 ℃ of baking ovens 6 hours, and the cooling back claims dry weight.To measure the femur of dry weight, place 800 ℃ of muffle furnace calcinings 8 hours, the cooling back claims ash heavy.The calculating ash weighs the ratio (g/g) with dry weight, obtains bone mineral content (BMC).With the femur of the ashing dissolving with hydrochloric acid with 0.5ml 6N, ultrapure water is diluted to 5ml, gets this solution of 0.05ml again, is diluted to 1ml, and is standby.

Blood calcium determination

Operate by table 1.After shaking up, measure optical density at the 570nm place, on the standard calcium curve, find corresponding calcium ion concn with 721 type ultraviolet-visible pectrophotometers.The standard calcium liquid of preparation is diluted to 2.5mg/ml, 3.0mg/ml, 3.5mg/ml, 4.0mg/ml, 4.5mg/ml, 5.0mg/ml, 5.5mg/ml, 6.0mg/ml, 8.0mg/ml.With the optical density of OCPC method mensuration 570nm, drawing standard curve.

Table 1 OCPC method is measured serum calcium ion concentration ^*

Reagent	Control tube	Measure pipe
Reagent	Control tube	Measure pipe	Serum calcium developer serum reference substance	?- ?4ml ?50μl	?50μl ?4ml ?-

^*Blood calcium concentration (mmol/L)=0.5 ^*Measure calcium ion concn

Serium inorganic phosphorus is measured

Operate by table 2.After shaking up, measure optical density at 660nm with 721 type ultraviolet-visible pectrophotometers.

Table 2 ferrous ammonium sulphate method is measured serium inorganic phosphorus content ^*

Reagent (ml)	U (measuring pipe)	S (standard pipe)	B (blank pipe)
Reagent (ml)	U (measuring pipe)	S (standard pipe)	B (blank pipe)	Serum 50mg/L phosphorus reference liquid ultrapure water is ferrous-Tricholroacetic Acid reagent	0.2ml - - 4.8ml	- 0.2ml - 4.8ml	- - 0.2ml 4.8ml
Mixing left standstill 10 minutes, centrifugation 10 minutes					0.2ml - - 4.8ml	- 0.2ml - 4.8ml	- - 0.2ml 4.8ml
				Supernatant liquor 4.0ml ammonium molybdate reagent	0.5ml	4.0?ml 0.5?ml	4.0?ml 0.5ml

Serum inorganic phosphorus (mmol/L)=Au * 1.615/As

Serum alkaline phosphatase determination

Operate by table 3.After shaking up,, measure optical density at the 510nm place with 721 type ultraviolet-visible pectrophotometers with the distilled water zeroising.With (A _U-A _C) difference looks into the phenol typical curve, obtains enzyme activity unit.

Table 3 colorimetric method for determining serum alkaline phosphatase (King unit) ^*

Reagent	Measure pipe (A _U)	Control tube (A _c)
Reagent	Measure pipe (A _U)	Control tube (A _c)	The substrate solution potassium ferricyanide solution serum reference substance that the pre-temperature of serum paraoxonase phthalate buffer is 37 ℃	0.1ml 1.0ml 1.0ml 3.0ml -	37 ℃ of water-bath 15min of 37 ℃ of water-bath 5min of-1.0ml 1.0ml 0.1ml

The mensuration of table 4 phenol typical curve

Reagent	B	?1	2	?3	?4	?5	?6	?7	?8	?9	?10
Reagent	B	?1	2	?3	?4	?5	?6	?7	?8	?9	?10	Phenol reference liquid (μ l) ultrapure water (ml) carbonate buffer solution (ml)	- 1.1 1.0	?2 ?1.098 ?1.0	5 1.095 1.0		?8 ?1.092 ?1.0		?10 ?1.09 ?1.0		?12 ?1.088 ?1.0		?15 ?1.085 ?1.0		?18 ?1.082 ?1.0	?20 ?1.08 ?1.0	?22 ?1.078 ?1.0	?25 ?1.075 ?1.0
	37 ℃ of water-bath 5min												- 1.1 1.0	?2 ?1.098 ?1.0	5 1.095 1.0		?8 ?1.092 ?1.0		?10 ?1.09 ?1.0		?12 ?1.088 ?1.0		?15 ?1.085 ?1.0		?18 ?1.082 ?1.0	?20 ?1.08 ?1.0	?22 ?1.078 ?1.0	?25 ?1.075 ?1.0
	37 ℃ of water-bath 5min											Potassium ferricyanide solution (ml)	3.0	?3.0	3.0	3.0		?3.0		?3.0		?3.0		3.0		?3.0	?3.0	?3.0

Bone calcium is measured

Operate by table 5.After shaking up, measure optical density at the 570nm place, on the standard calcium curve, find corresponding calcium ion concn with 721 type ultraviolet-visible pectrophotometers.

Table 5 OCPC method is measured calcium content of bone ^*

Reagent	Control tube	Measure pipe
Reagent	Control tube	Measure pipe	Solution H calcium developer ultrapure water	- 4ml 50μl	50μl 4ml -

^*Calcium content of bone=2 ^*It is heavy to measure calcium concn/bone ash

Bone phosphorus is measured

Operate by table 6.After shaking up, measure optical density at the 660nm place with 721 type ultraviolet-visible pectrophotometers.

Table 6 ferrous ammonium sulphate method is measured the bone phosphorus content ^*

Reagent (ml)	U (measuring pipe)	S (standard pipe)	B (blank pipe)
Reagent (ml)	U (measuring pipe)	S (standard pipe)	B (blank pipe)	Solution H phosphorus standardized solution ultrapure water is ferrous-Tricholroacetic Acid reagent ammonium molybdate reagent	?0.2ml ?- ?- ?4.8ml ?0.625ml	?- ?0.2ml ?- ?4.8ml ?0.625ml	?- ?- ?0.2ml ?4.8ml ?0.625ml

^*Bone phosphorus content=5 ^*A _U/ A _S ^*Bone ash is heavy

The treatment result of intraperitoneal injection

Abdominal injection every day compound of the present invention 1 time (dosage is 0.1103 μ mol/Kg) treated for 4 weeks continuously, and the result of mensuration lists table 7-10 in.

The body weight of mouse before and after table 7 treatment

Group	First body weight (g)	End-body heavy (g)
Group	First body weight (g)	End-body heavy (g)	Oophorectomize group sham-operation group estradiol oestrone 9 18 27 67 68 69 32 33 34 40 41 42	31.1±2.2 30.0±1.5 31.0±2.6 30.4±2.5 30.7±2.5 30.3±1.5 29.8±1.7 29.9±1.9 30.3±2.1 30.4±2.2 30.3±1.8 30.4±1.9 29.8±2.9 30.9±1.5 30.5±2.6 30.1±2.0	37.2±2.6 34.5±2.9 ^a 36.1±2.3 ^h 34.3±2.1 ^a 34.0±3.4 ^a 33.4±2.5 ^d 33.3±1.8 ^d 32.9±2.2 ^d，h 34.1±2.6 ^a 34.5±2.8 ^a 35.8±2.7 ^a 34.5±2.8 ^a 34.8±2.7 ^a 35.2±1.6 ^a 35.2±2.5 ^a 33.1±1.9 ^d

A) with oophorectomize group ratio, P＜0.05; B) with estradiol group ratio, P＜0.05; C) with the estrone group ratio,

P＜0.05; D) with oophorectomize group ratio, P＜0.01; E) with estradiol group ratio, P＜0.01;

F) with estrone group ratio, P＜0.01; G) with sham operated rats ratio, P＜0.01; H) with the sham operated rats ratio,

P＜0.05；n＝12

Table 7 shows that the final body weight of each group of treatment all obviously increases than initial body weight.All there is not difference between the initial body weight of all groups.Sham operated rats compares with model control group that final body weight is obviously big, other respectively organizes final body weight and the final body weight of model control group is obviously little.Compare with sham operated rats, the final body weight body weight of estradiol group is obviously big, final the body weight final body weight and the sham operated rats final body heavy phase obviously little, other each group of 67 groups of compounds ought.

The lung of table 8 mouse, liver, spleen and uterus weight thereof

Group	Lung heavy (g)	Liver heavy (g)	Spleen heavy (g)		Uterus heavy (g)
Group	Lung heavy (g)	Liver heavy (g)	Spleen heavy (g)		Uterus heavy (g)	Oophorectomize group sham-operation group estradiol oestrone 9 18 27 67 68 69 32 33 34 40 41 42	0.163±0.0231 0.184±0.0277 0.168±0.0308 0.176±0.0259 0.168±0.0227 0.171±0.0259 0.163±0.0177 0.173±0.0164 0.0168±0.0107 0.179±0.0160 0.151±0.0188 0.167±0.0279 0.184±0.0245 ^a 0.173±0.0201 0.1?82±0.019 ^a 0.169±0.0303	1.329±0.156 ^e 1.367±0.198 ^b 1.565±0.199 ^a，h 1.318±0.113 ^d，e 1.295±0.186 ^e 1.15±0.110 ^{a，f，g，e} 1.413±0.122 ^b 1.347±0.146 ^e 1.389±0.139 ^b，c 1.458±0.199 ^b 1.371±0.178 ^b 1.353±0.221 ^b 1.391±0.129 ^b 1.377±0.185 ^b 1.363±0.133 ^e 1.395±0.160 ^b	0.130±0.0319 0.15?1?±0.041 0.151±0.0357 0.1?56±0.0510 0.138±0.0376 0.127±0.033 0.114±0.025 ^{a，e，f，g}0.123±0.026 ^b0.131±0.037 0.143±0.041 0.130±0.037 0.143±0.067 0.142±0.032 0.124±0.036 0.132±0.045 0.131±0.034		0.0390±0.0249 ^e，f，g 0.0915±0.0301 ^d，e，f 0.177±0.0436 ^c，d，g 0.141±0.0457 ^g 0.0710±0.0522 0.061?1±0.0287 ^a，h 0.0698±0.0419 ^d 0.0633±0.0434 ^f 0.0519±0.0320 ^g，f 0.0558±0.0326 ^h，f 0.0555±0.0344 ^h，e 0.0694±0.0336 ^a，e 0.0710±0.0260 ^d，e 0.0425±0.0187 ^e，g 0.0503±0.0311 ^e，g 0.0554±0.0288 ^e，g

A) with oophorectomize group ratio, P＜0.05; B) with estradiol group ratio, P＜0.05; C) with estrone group ratio, P＜0.05; D) with oophorectomize group ratio, P＜0.01; E) with estradiol group ratio, P＜0.01; F) with estrone group ratio, P＜0.01; G) with sham operated rats ratio, P＜0.01; H) with sham operated rats ratio, P＜0.05; N=12

As can be seen from Table 8, respectively organize lung and the spleen weight no significant difference of mouse after the treatment.Compare with sham operated rats and model control group, the liver weight of estradiol treatment group mouse is obviously big, P＜0.05.Other group Mouse Liver is heavy, and to compare difference not obvious with sham operated rats and model control group.The liver weight of compound 67,68,69,32,33,34,40,41 and 42 treatment group mouse is significantly less than the estradiol group.Prompting might cause fatty liver when using the estradiol treatment.

From mouse uterine weight as can be seen, the uterus weight of estradiol and oestrone treatment group mouse is obviously greater than sham operated rats, P＜0.01, and estradiol and oestrone treatment have tangible uterus hyperplasia phenomenon.Other group uterus weight presents in various degree less than estradiol and estrone group.The mouse uterine weight of model control group is significantly less than sham operated rats, P＜0.01, the obvious atrophy in uterus of prompting castration mouse.The same with model control group, 67,68,69,32,33,34,40,41 and 42 treatment group Mouse Uterus are obviously atrophy also.Prompting, compound of the present invention can not cause the uterus hyperplasia.

Table 9 mouse femur dry weight, ash is heavy, bone long, bone mineral content and ash weight/bone are long

Group	Femur dry weight (g)	Ash heavy (g)	Bone mineral content	Bone long (cm)	Ash weight/bone long (g/cm)
Group	Femur dry weight (g)	Ash heavy (g)	Bone mineral content	Bone long (cm)	Ash weight/bone long (g/cm)	Oophorectomize group sham-operation group estradiol oestrone 9 18 27 67 68 69 32 33 34 40 41 42	0.0585 ± 0.00394 ^g0.0740 ± 0.00832 ^d0.0577 ± 0.00696 ^g0.0611 ± 0.005?87 ^g0.0598 ± 0.00465 ^g0.0588 ± 0.00465 ^h0.0620 ± 0.00949 ^g0.0669 ± 0.00570 ^c，d，h0.0639 ± 0.00710 ^a，h0.0657 ± 0.00637 ^d，h0.0664 ± 0.00538 ^d，e，g0.0573 ± 0.00368 ^g0.0660 ± 0.00548 ^d，e，h0.0634 ± 0.00739 ^b，g0.0637 ± 0.00627 ^a，d，g0.0612 ± 0.00532 ^g	0.0178 ± 0.00217 ^g0.0429 ± 0.00483 ^d0.0285 ± 0.00680 ^d，g0.0208 ± 0.00430 ^a，g0.0178 ± 0.00406 ^g0.01?88 ± 0.00353 ^g0.0205 ± 0.00743 ^g0.0226 ± 0.00297 ^d，g0.0213 ± 0.00632 ^g0.0238 ± 0.00453 ^d，c，g0.0344 ± 0.00565 ^d，b，g0.0358 ± 0.003?13 ^d，e，g0.0348 ± 0.00467 ^b，d，g0.0370 ± 0.00625 ^d，e，h0.0400 ± 0.00481 ^d，e0.0358 ± 0.00437 ^d，e，g	0.304 ± 0.0272 ^g0.587 ± 0.0922 ^d0.496 ± 0.0945 ^d，h0.330 ± 0.0522 ^e，g0.296 ± 0.0460 ^g0.3?14 ± 0.0411 ^g0.323 ± 0.0685 ^g0.338 ± 0.0388 ^a，g0.329 ± 0.0687 ^g0.362 ± 0.05?10 ^d，g0.520 ± 0.0819 ^d0.625 ± 0.03?99 ^d，e0.533 ± 0.0921 ^d0.582 ± 0.0512 ^b，d0.630 ± 0.0690 ^d，e0.585 ± 0.0555 ^b，d	1.597 ± 0.0390 ^g1.646 ± 0.0438 ^d1.628 ± 0.0346 1.606 ± 0.0317 ^h1.617 ± 0.0397 1.614 ± 0.0455 1.623 ± 0.0343 1.614 ± 0.0559 1.626 ± 0.0609 1.639 ± 0.0456 ^a1.634 ± 0.0358 ^a1.615 ± 0.0490 1.635 ± 0.0273 ^a1.642 ± 0.0500 1.640 ± 0.0306 ^d1.623 ± 0.0474	0.0112±0.00148 ^g0.026?1±0.00295 ^d0.0175±0.00420 ^d，g0.0130±0.00280 ^e，g0.0110±0.00259 ^e，g0.0115±0.00222 ^e，g0.0126±0.00439 ^b，g0.0140±0.00202 ^d，g0.0132±0.00423 ^g0.0145±0.00281 ^d，g0.0210±0.00329 ^b，d，g0.0222±0.00220 ^d，e，g0.0213±0.00291 ^b，d，g0.0226±0.00405 ^d，e，h0.0243±0.00286 ^d，e0.0220±0.00270 ^d，e，g

Mouse femur dry weight, heavy, the bone of ash are long, bone mineral content and ash are heavy/and bone length sees Table 9.As can be seen from Table 9, model control group femur dry weight is significantly less than sham operated rats.Prompting, castration mouse is obviously lost the bone amount.Other group femur dry weight is obviously greater than model control group, and wherein the femur dry weight increase of compound 68,67,69,32,34,41 treatment group mouse is very obviously greater than model control group femur dry weight, P＜0.01.As seen, compound of the present invention and estradiol and oestrone can preventing osteoporosis.Compare with sham operated rats, it is obviously little that each organizes the mouse femur dry weight.Wherein the femur dry weight of model control group, estradiol group, estrone group, compound 9,27,32,33,40,41 and 42 treatment group mouse is significantly less than sham operated rats very much, P＜0.01, the femur dry weight of compound 17,67,68,69 and 34 treatment group mouse is significantly less than sham operated rats, P＜0.05.As seen, compound of the present invention and estradiol and oestrone can not reverse osteoporosis.Compare with estradiol treatment group, the femoral shaft representation of compound 32 and 34 treatment group mouse work big (P＜0.05), as seen, compound 32 of the present invention and 34 more outstanding than estradiol and oestrone.

Heavy as can be seen from the mouse femur of table 9 ash, except that compound 9,18,27,68, the grey weight average of each treatment group mouse femur is in various degree greater than model control group (P＜0.05-0.01).Heavy and sham operated rats does not have the significant difference except that compound 41 treatment group mouse femurs ashes, and other grey weight average of respectively organizing mouse femur has in various degree less than sham operated rats.32,34 groups of mouse femur ashes of compound are heavy obviously greater than the estradiol group, P＜0.05, and compound 33,40,41,42 treatment group mouse femurs ash is heavy very obviously greater than the estradiol group, P＜0.01.As seen, compound of the present invention 32,33,34,40,41 and 42 is more outstanding than estradiol and oestrone.

From table 9 mouse bone mineral content (BMC) as can be seen, the BMC of model control group is starkly lower than sham operated rats.The obvious osteoporosis of model control group mouse is described.Compare with model control group, estradiol group, compound 69,32,33,34,40,41 and 42 BMC are very obviously high, P＜0.01, and the BMC of compound 67 treatment group mouse is significantly high, P＜0.05.BMC and the model control group of oestrone, compound 9,18,27 and 68 treatment group mouse are suitable.Except that compound 33 and 41 treatment group mouse BMC, all the other are respectively organized mouse BMC and all are lower than sham operated rats in various degree, wherein model control group, estrone group, compound 9,18,27,68,69 treatment group mouse BMC highly significants are lower than sham operated rats, P＜0.01, the estradiol group significantly is lower than sham operated rats, there is not significant difference between compound 32,33,34,40,41 and 42 treatment group mouse BMC and the sham operated rats, approaching or suitable with normal value respectively.As seen, the clear and definite function of resisting osteoporosis of compound exhibits of the present invention.

The heavy ratio explanation with femur length of the ash of mouse in the table 9, compare the long ratio of the ash weight/bone of model control group mouse with sham operated rats obviously little.The long ratio of the ash weight/bones of estradiol group, compound 67,69,32,33,34,40,41 and 42 treatment group mouse is then obviously greater than model control group, P＜0.01, and all the other each groups are suitable with model control group.The ratio and the sham operated rats of the ash weight/bone length of compound 41 treatment group mouse are suitable, near normal value.The long ratio of the ash weight/bones of compound 40 treatment group mouse is significantly less than sham operated rats, P＜0.05.All the other respectively organize the long ratio highly significant of the ash weight/bone of mouse less than sham operated rats, P＜0.01.Except that the long ratio of the ash weight/bones of compound 32,33,34,40,41 and 42 treatment group mouse significantly greater than the estradiol group treatment group, the ratio that all the other each treatment group mouse ash weight/bones are grown is significantly less than the estradiol group.

The data of table 9 show that clearly compound of the present invention not only has good function of resisting osteoporosis, and wherein 32,33,34,40,41 and 42 function of resisting osteoporosis obviously is better than estradiol and oestrone, and all do not observe uterus hyperplasia side effect.

Table 10 explanation, compare model group mice serum alkaline phosphatase (ALP) with sham operated rats active obviously high, illustrates that the bone transformation of model control group mouse is stronger.Estradiol group, the active highly significant of 68,69,32,33,34,40,41 and 42 groups of mouse ALP of compound are lower than model control group, P＜0.01.Oestrone, compound 27 and 67 groups of mouse ALP activity significantly are lower than model control group, P＜0.05.Significantly be lower than the sham operated rats except that compound 9 and 18 groups of mouse ALP levels are significantly higher than sham operated rats and compound 33 and 41 groups of mouse ALP levels, other group is suitable with sham operated rats.These data declarations, bone transformation of mouse or reduction or approaching normal behind compounds for treating of the present invention.

Bone calcium, bone phosphorus, blood calcium, serium inorganic phosphorus and ALP after table 10. treatment

Group	Bone calcium (%)	Bone phosphorus (%)	Blood calcium (mmol/L)	Serium inorganic phosphorus (mmol/L)	ALP
Group	Bone calcium (%)	Bone phosphorus (%)	Blood calcium (mmol/L)	Serium inorganic phosphorus (mmol/L)	ALP	Oophorectomize group sham operated rats estradiol oestrone	39.545± 2.551 53.213± 2.365 ^d54.466± 3.0413 ^d43.84± 2.659 ^d，g，e	?22.951± ?1.960 ^g?24.811± ?1.049 ^d?24.695± ?2.307 ?23.979± ?1.087	2.015± 0.0545 2.064± 0.0783 2.020± 0.0382 2.050± 0.0175	1.601± 0.00268 1.602± 0.00519 1.601± 0.00521 1.601± 0.00417	40.086± 2.884 ^e，g 34.171±3.341 ^d 31.286±1.733 ^d 36.271± 3.228 ^a，e

9 18 27 67 68 69 32 33 34 40 41 42

?39.48± ?2.33?1 ^，g，e，f?40.139± ?1.816 ^g，e，f?37.815± ?1.792 ^g，e，f?41.61± ?1.891 ^{a，g，e，c}?41.173± ?1.772 ^g，e?39.550± ?2.436 ^g，e?51.312± ?2.602 ^d，b，?53.078± ?2.349 ^d，f?52.798± ?1.376 ^d，f?54.885± ?1.668 ^d?58.35± ?0.926 ^d，g，e?56.69± ?1.474 ^d，g，b

22.888± 2.059 ^h 22.759± 2.267 ^h 23.585± 2.284 23.279± 2.910 23.759± 1.923 23.288± 1.920 24.920± 1.623 ^a 24.867± 2.536 24.665± 3.069 24.949± 2.221 ^a 24.991± 2.338 ^a 25.460± 3.265 ^a

2.020± 0.0417 2.015± 0.0181 2.005± 0.0324 2.000± 0.0284 2.010± 0.0179 2.010± 0.0344 2.022± 0.0271 2.075± 0.0258 2.005± 0.0258 2.014± 0.0347 2.005± 0.0270 2.015±0.016

1.603± 0.00371 1.605± 0.00669 1.599± 0.00733 1.604± 0.00268 1.602± 0.00560 1.602± 0.00315 1.602± 0.00385 1.605± 0.00405 1.605± 0.00498 1.599± 0.00487 1.600± 0.00492 1.605± 0.00537

37.643± 1.787 ^e，h 38.614± 1.486 ^g，e 35.643±2.756 ^a 36.371± 1.910 ^a，e 36.229± 1.467 ^d，e 35.857± 2.337 ^d，e 32.771±2.050 ^d 31.057± 0.996 ^d，h 31.486±1.285 ^d 31.429±1，837 ^d 30.514± 1.301 ^d，h 31.529±1.676 ^d

Table 10 shows that also model control group mouse calcium content of bone significantly is lower than sham operated rats.Estradiol, oestrone, compound 67,32,33,34,40,41 and 42 treatment group mouse calcium content of bone highly significants are higher than model control group, P＜0.01-0.05, and all the other groups and model control group do not have significant difference.Compare the calcium content of bone highly significant low (P＜0.01) of model control group, oestrone, 9,18,27,67,68 and 69 groups of mouse of compound with sham operated rats.Except that the calcium content of bone highly significants of compound 32 treatment group mouse is lower than the estradiol group (P＜0.05), other compound and estradiol are suitable.Calcium content of bone and the sham operated rats of estradiol treatment group mouse are suitable.

The bone phosphorus content significance of compound 32,40,41 and 42 treatment group mouse is higher than model control group (P＜0.05).The bone phosphorus content highly significant of model control group mouse is lower than sham operated rats.Except that the bone phosphorus content significancees of oestrone and compound 8 treatment group mouse is lower than the sham operated rats (P＜0.05), other compounds for treating group is suitable with sham operated rats.This result has reflected the function of resisting osteoporosis of table invention compound from another angle.

The compounds of this invention oral administration activity

Irritate stomach every day and give compound 32,33,34,40,41,42 of the present invention and estradiol 1 time (dosage is 0.1103 μ mol/Kg), continuously 4 weeks of treatment, the uterus weight of mensuration, femur dry weight, ash weight, bone mineral content, bone length and bone ash weight/bone long line go into to show 11-13.

Uterus weight behind table 11. oral administration

Group	Uterus heavy (g)
Group	Uterus heavy (g)	Oophorectomize group sham-operation group estradiol 32 33 34 40 41 42	0.0390±0.0249 0.091?5±0.0301 0.0401±0.0184 0.0367±0.0130 0.0507±0.0319 0.0382±0.0108 0.0432±0.0236 0.0407±0.0196 0.0475±0.0305

n＝12

Table 11 explanation, compound 32,33,34,40,41,42 and estradiol all do not cause uterus hyperplasia side effect under the oral dosage that the present invention uses.

Femur dry weight behind table 12. oral administration, ash weight, bone mineral content, bone length and bone ash weight/bone are long

Group	Femur dry weight (g)	Femur ash heavy (g)	BMC	Bone long (cm)	Bone ash weight/bone long (g/cm)
Group	Femur dry weight (g)	Femur ash heavy (g)	BMC	Bone long (cm)	Bone ash weight/bone long (g/cm)	Oophorectomize estradiol 32	0.0585 ± 0.00394 0.063?1 ± 0.00654 0.0688 ± 0.00642 ^b	0.0178 ± 0.00217 0.0222 ± 0.00423 ^a0.0293 ± 0.00454 ^b，c	0.304 ± 0.0272 0.352 ± 0.0419 0.431 ± 0.0745 ^a	1.597 ± 0.0390 1.622 ± 0.0447 1.654 ± 0.0550 ^b	0.01?12 ± 0.00148 0.01?37 ± 0.00251 0.0177 ± 0.00309 ^b，c

33 34 40 41 42

0.0674 ± 0.00532 ^b 0.0627 ± 0.00477 0.0636 ± 0.00567 ^a 0.0684 ± 0.00468 ^b 0.0571 ± 0.00715

0.0330 ± 0.00581 ^b，d0.0300 ± 0.00467 ^b，c0.0304 ± 0.00618 ^b，c0.0337 ± 0.00746 ^b，d0.0287 ± 0.003?89 ^b，c

0.491 ± 0.083?5 ^b，d0.481 ± 0.0774 ^b，c0.478 ± 0.0678 ^b，c0.493 ± 0.0564 ^b，d0.503 ± 0.0521 ^b，d

1.645 ± 0.0470 ^b1.638 ± 0.0420 ^a1.635 ± 0.0362 ^a1.614 ± 0.0329 1.626 ± 0.0296

0.0201 ± 0.00327 ^b，d0.0183 ± 0.00274 ^b，d0.01?86 ± 0.00224 ^b，d0.0209 ± 0.00271 ^b，d0.0177 ± 0.00200 ^b，c

A) with oophorectomize group ratio, P＜0.05; B) with oophorectomize group ratio, P＜0.01; C) with estrone group ratio, P＜0.05; D) with estradiol group ratio, P＜0.01, n=12

Bone calcium, bone phosphorus and alkaline phosphatase behind table 13. oral administration

Group	Bone calcium (%)	Bone phosphorus (%)	ALP
Group	Bone calcium (%)	Bone phosphorus (%)	ALP	Oophorectomize group estradiol 32 33 34 40 41 42	?39.545±2.55?1 ?41.587±2.006 ^a?44.421±2.014 ^b?51.423±1.325 ^b，d?46.678±2.143 ^b，c?46.479±1.165 ^b，c?48.365±2.034 ^b，d?49.357±1.258 ^b，d	22.951±1.960 23.33?1±2.154 25.347±2.236 ^b，d 26.874±1.654 ^b，d 24.665±3.069 ^a，c 24.774±2.548 ^a，c 23.639±2.431 ^a 24.103±1.897 ^a	40.086±2.884 36.547±1.879 ^a 38.435±2.643 33.154±1.784 ^b，c 36.557±2.031 ^a 35.209±2.036 ^a 34.235±1.438 ^b 33.752±2.586 ^b，c

A) with oophorectomize group ratio, P＜0.05; B) with oophorectomize group ratio, P＜0.01; C) with the estrone group ratio,

P＜0.05：

D) with estradiol group ratio, P＜0.01, n=12

Table 12 and 13 explanations, compound 32,33,34,40,41,42 still shows tangible function of resisting osteoporosis under the oral dosage that the present invention uses, estradiol does not show tangible function of resisting osteoporosis.Compound 32,33,34,40,41 of the present invention and the 42 oral function of resisting osteoporosis that show make compound of the present invention have clear and definite application prospect.

Claims

1, a kind of oestrogenic hormon peptide conjugate with anti-osteoporosis activity is characterized in that having the structure of following general formula I:

General formula I

Work as R ₃During for H, R ₁And R ₂One is H, and one is-OCOCH ₂CH ₂CO-Arg-Gly-Asp-Ser-OH, CH ₂CO-Arg-Gly-Asp-Val-OH ,-OCOCH ₂-CH ₂CO-Arg-Gly-Asp-Phe-OH ,-Ser-Asp-Gly-Arg-H ,=NNH-Val-Asp-Gly-Arg-H or=NNH-Phe-Asp-Gly-Arg-H; Or work as R ₁And R ₂One is H, one when being OH, and R ₃Be selected from-CH ₂CO-Arg-Gly-Asp-Ser-OH ,-CH ₂CO-Arg-Gly-Asp-Val-OH ,-CH ₂CO-Arg-Gly-Asp-Phe-OH ,-CH ₂CONHNH-Ser-Asp-Gly-Arg-H ,-CH ₂CONH-NH-Val-Asp-Gly-Arg-H or-CH ₂CONHNH-Phe-Asp-Gly-Arg-H; Or work as R ₁And R ₂When being ketonic oxygen jointly, R ₃Be selected from-CH ₂CO-Arg-Gly-Asp-Ser-OH ,-CH ₂CO-Arg-Gly-Asp-Val-OH or-CH ₂CO-Arg-Gly-Asp-Phe-OH.

2, the preparation method of compound of Formula I may further comprise the steps:

General formula I

Work as R ₃During for H, R ₁And R ₂One is H, and one is-OCOCH ₂CH ₂CO-Arg-Gly-Asp-Ser-OH, CH ₂CO-Arg-Gly-Asp-Val-OH ,-OCOCH ₂-CH ₂CO-Arg-Gly-Asp-Phe-OH, Ser-Asp-Gly-Arg-H ,=NNH-Val-Asp-Gly-Arg-H or=NNH-Phe-Asp-Gly-Arg-H; Or work as R ₁And R ₂One is H, one when being OH, and R ₃Be selected from-CH ₂CO-Arg-Gly-Asp-Ser-OH ,-CH ₂CO-Arg-Gly-Asp-Val-OH ,-CH ₂CO-Arg-Gly-Asp-Phe-OH ,-CH ₂CONHNH-Ser-Asp-Gly-Arg-H ,-CH ₂CONH-NH-Val-Asp-Gly-Arg-H or-CH ₂CONHNH-Phe-Asp-Gly-Arg-H; Or work as R ₁And R ₂When being ketonic oxygen jointly, R ₃Be selected from-CH ₂CO-Arg-Gly-Asp-Ser-OH ,-CH ₂CO-Arg-Gly-Asp-Val-OH or-CH ₂CO-Arg-Gly-Asp-Phe-OH;

Introduce connecting arm: COCH for 3 or 17 with oestrone and estradiol ₂CH ₂CO, CH ₂CO or CH ₂CONH is incorporated into the protection intermediate of liquid phase process synthetic Arg-Gly-Asp-Ser-OH, Arg-Gly-Asp-Val-OH or Arg-Gly-Asp-Phe-OH on the connecting arm, again through deprotection promptly then.

3, the purposes of compound of Formula I in preparation treatment osteoporosis agents.