CN1737009A - Polypeptide compound and medicinal use thereof - Google Patents

Abstract

The present invention relates to a class polypeptide compound and a medicinal use thereof, especially as bone formulation stimulant as the purposes aspect the osteoporosis in prevention or treatment.

Description

Polypeptide compound and medicinal use thereof

Invention field

The present invention relates to a class polypeptide compound and a medicinal use thereof, especially as bone formulation stimulant as the purposes aspect the osteoporosis in prevention or treatment.

Background technology

It is that effect is opposite, a pair of factor of balance regulation that scleroblast is grown with building in the process with osteoclast at osseous tissue again.Because the influence of astogeny and some drug side effects, the osteoclast overacfivity makes bone metabolism unbalance, finally causes osteoporosis.Replenish calcium or take the medicine that suppresses osteoclast activity, can only partly improve the consequence that osteoporosis causes.People wish to seek to have simultaneously and suppress osteoclast activity and promote active this material of scleroblast for this reason.This material will more help treating osteoporosis.

Treating osteoporotic medicine at present clinically has following several:

Diphosphonate, comprise that the pain that osteoporosis is caused has certain curative effect according to the directly formation of inhibition osteoclast such as risedronate sodium (Risedronate) for sodium phosphate (Etidronate), clodronate disodium (Clodronate), alendronate (Alendronate) and latest generation.But SM can reduce the bone transformation efficiency, increases bone fragility, has the danger of fracture.In addition, also can cause side effects such as side effect of digestive tract, soft tissue calcification, proteinuria, heating;

Estrogens comprises that nilestriol, Ethinylestradiol, medroxyprogestrone Acetate etc. all can be used for the treatment of postmenopausal osteoporosis cancer, can obviously alleviate quick bone loss, reduce symptoms such as fracture incidence and ostalgia.Their maximum deficiency is the risk that causes mammary gland and endometrial hyperplasia and cancer sometimes;

Thyrocalcitonin can effectively reduce serum calcium, suppress bone resorption.Mainly act on pain and scleromalacia that hypercalcinemia and osteoporosis cause.But because of its molecular weight greater than 3200, be a kind of macromole, therefore have the untoward reaction of M-band sample, as fash, urticaria, skin pruritus occur.Headache, dizziness, instability of gait, numb in every limb, tic etc. often appear aspect neural system.Poor appetite, nausea and vomiting, abdominal pain diarrhea aspect Digestive tract, can occur, have a stomach upset and constipation etc.;

The Chinese medicine of other drug such as fluorochemical, Calcium of intensifiens agent, isoflavones and nourishing disney and strengthening bone etc. are not affirmed its definite curative effect as yet.Seeking and develop new bone formulation stimulant or treatment medicine for treating osteoporosis thus is still and very needs.

Summary of the invention:

The inventor now finds based on following formula Oesteoblast growth peptide (OGP) C-terminal 10-14 fragment peptide (be called for short OGP5),

R ¹＝H，R ²＝OH，R ³＝H，R ⁴＝H，R ⁵＝-Gly-Gly-OH---------OGP5

Obtain a series of new polypeptide compounds, these polypeptide compounds demonstrate good bone forming hormesis, thereby can be used for prevention or treatment osteoporosis.

Therefore, first aspect present invention relates to the polypeptide compound of formula I

Wherein

R ¹＝H，Boc，ArSO ₂，Aba，Ac，Aca；

R ²＝H，OH；R ³＝H，NO ₂，OH；

R ⁴＝H，NO ₂，OH；

R ⁵＝Aba-OH，Aba-NH ₂，Aba-NHEt，Aba-NMe ₂， Aca-OH，Aca-NH ₂，Aca-NHEt，Aca-NME ₂，

Condition is that this compound is not R ¹=H, R ²=OH, R ³=H, R ⁴=H, R ⁵=-Gly-Gly-OH.

The invention still further relates to pharmaceutical composition, it comprises formula I compound and pharmaceutical carrier or vehicle.

The invention further relates to formula I compound and be used for the bone formulation stimulant purposes in preparation.

Further, formula I compound can be by in the following table 1 shown in the compound.

Table 1 formula I compound

G48B G48C H8A H8B H8C H8D H8E H8F H8G H9A H9B H9C H9D H9E

Tos HO-PhCO- H H H Aba- Aba- Aba- Aca- Aca- Aca- Aba- Aba- Aba-

OH OH OH OH OH H H H H H H NO 2 NO 2 NO 2

H H NO 2 NO 2 NO 2 H H H H H H H H H

NO 2 NO 2 H H H OH OH OH OH OH OH OH OH OH

-Aca-NHEt -Aca-NHEt -Aca-OH -Aca-OPac -Aca-NHEt -OH -NHEt -OPac -OH -OPac -NHEt -OPac -OH -NHEt

According to the present invention, formula I compound preferred G35I, G38I or G38K.

According to the present invention, bone formulation stimulant is said for example and be can be prevention or treat osteoporotic medicine among the present invention.

According to the present invention, pharmaceutical carrier described in the present invention or vehicle are carrier or vehicle commonly used in the pharmacy field.

Description of drawings

Fig. 1 is the effect of the G35I of different concns to the human osteoblast cell.

Embodiment

The following examples are to further describe of the present invention, but it does not mean any limitation of the invention.

Embodiment 1

G35I's is synthetic

Synthetic route:

Reaction conditions:

a)1.NaOH/H ₂O，2.ClCOOiBu/NMM，-20℃，3.1NHCl/H ₂O

b)1.NaOH，2.(Boc) ₂O/tBuOH，3.1NHCl/H ₂O

c)BrCH ₂CO-Ph/TEA d)3.5NHCl/HOAc e)EDC/HOBt/DIEA/DIEA

f)Boc-Tyr-OH/EDC/HOBt/DIEA g)Zn/HOAc

Operational condition:

The preparation of Boc-Gly-Phe-OH (1):

3.504g (20mmol) Boc-Gly-OH is dissolved in the 50mL exsiccant tetrahydrofuran (THF), in-20 ℃ and following 2.34mL (20mmol) NMM and 2.7mL (20mmol) isobutyl chlorocarbonate of adding of stirring, 2.5 add 4.956g (30mmol) H-Phe-OH/1.2g (30mmol) the NaOH/ aqueous solution of precooling after minute, behind the low-temp reaction 30 minutes, disappear in room temperature reaction to raw material again, stopped reaction, steam and remove tetrahydrofuran (THF), thin up, with twice of ethyl acetate extraction, again with water backwash ethyl acetate layer, combining water layer, transferring pH with 1NHCl under ice-water bath and stirring is 2, solution becomes turbid, with ethyl acetate extraction sour water layer twice, organic layer is successively with 1NHCl (* 2), H ₂O (* 2), saturated brine (* 2) washing are with anhydrous Na ₂SO ₄Drying, the filtering siccative, filtrate evaporate to dryness yellowly oily matter, it is molten entirely gradually to add anhydrous diethyl ether, becomes turbid immediately, and precipitation capacity increases step by step, places in refrigerator, leaches white precipitate, and it is a single-point that TLC detects.The heavy 5.393g of product, yield 83.7%, mp:137-139 ℃, Rf=0.2 (PE: EtOAc: HOAc=3: 2: 0.1).

¹H-NMR (300MHz, CDCl ₃) δ (ppm): 1.436 (s, 9H, (CH ₃) ₃C), 3.191 (m, 2H, Ar-CH ₂), 3.884,3.695 (dd, each 1H, J=16.8Hz, AB system, NHCH ₂CO), 4.873 (br, 1H, NHCHCH ₂Ar), and 7.141-7.278 (m, 5H, Ar-H).

The preparation of Boc-Aca-OH (2):

6.559g (50mmol) Aca-OH and 2g (50mmol) NaOH are placed 100mL eggplant type bottle, add 12.5mL H ₂The dissolving of O-15mL t-BuOH dropwise added 12.004g (55mmol) (Boc) in 30 minutes ₂The 12.5mL t-BuOH liquid of O, insulation adds the 75mL trimethyl carbinol more a little, spend the night in powerful stirring of room temperature, add the dilution of 250mL water, with sherwood oil (* 3) washing, water layer is under ice-water bath and powerful the stirring, add 625mL EtOAC, and, tell EtOAc with 1NHCl accent pH to 2, the sour water layer is with EtOAc extraction three times, merge EtOAc, with H ₂O (* 3), salt solution (* 3) washing is with anhydrous sodium sulfate drying, the filtering siccative, colourless EtOAc evaporated under reduced pressure yellowly oil, room temperature is placed and is separated out white tiny crystallization, add petroleum ether, leach white crystals, detect through TLC, iodine is smoked to be a single-point, the heavy 8.339g of product, yield 72.20%, mp:31-33 ℃, Rf=0.6 (PE: EtOAc: HOAc=2: 1: 0.1).

¹H-NMR(300MHz，CDCl ₃)δppm：1.439(s，9H，(CH ₃) ₃C)，1.348-1.521(m，4H，CH ₂CH ₂CH ₂CH ₂COOH)，1.652(m，2H，J＝7.5Hz，NH(CH ₂) ₃CH ₂)，2.354(t，2H，J＝7.5Hz，CH ₂COOH)，3.108(t，2H，J＝6.5Hz，NHCH ₂)。

The preparation of Boc-Aca-OPac (3):

Take by weighing 1.288 g (6.47mmol) bromoacetophenone and in 100ml eggplant type bottle, add acetic acid ethyl dissolution, in 0 ℃ and stir under add the ethyl acetate solution of 1358mg (5.878mmol) Boc-Aca-OH (2) and 0.92mL (6.47mmol) TEA, room temperature reaction to raw material disappears, add the dilution of 150mL ethyl acetate, ethyl acetate layer is successively with saturated NaHCO ₃(* 2), H ₂O (* 2), saturated NaCl (* 2) washing, anhydrous sodium sulfate drying, the filtering siccative, filtrate decompression evaporate to dryness yellowly oil, solidify a moment, adds petroleum ether, leaches precipitation, and drying obtains product, and it is a single-point that TLC detects.The heavy 1.992g of product, yield 97.10%, mp:65-67 ℃, Rf=0.41 (PE: EtOAc=3: 1), FAB-MS (m/z): 350[M+H] ⁺

HCl.NG ₂The preparation of-Aca-OPac (4):

The Boc-Aca-OPac (3) of 9.608g (27.53mmol) is placed 250ml eggplant type bottle, drip 3.5NHCl/HOAc to molten entirely in ice-water bath and under stirring, under room temperature, react again, remove solvent under reduced pressure to the raw material disappearance, obtain yellow oil, add anhydrous diethyl ether and grind, washing is filtered, obtain white solid, leach, product is heavy: 7.543g, yield: 95.97%.

The preparation of Boc-Gly-Phe-Aca-OPac (5):

With 1.428g (5mmol) HCl.NH ₂-Aca-OPac (4) is in the 100mL eggplant-shape bottle, add an amount of exsiccant DMF dissolving, add 1.771g (5.5mmol) Boc-Gly-Phe-OH (1) and 919mg (6mm0l) HOBt again, bathe and stir the DMF mixed solution that slowly drips 1.150g (6mm0l) EDC.HCl and 1.05mL (6mmol) DIEA in following 20 minutes in cryosel, continue to stir 30 minutes, removing cryosel bathes, disappear in room temperature reaction to raw material, stopped reaction, add a large amount of distilled water dilutings down in stirring, with EtOAc aqueous layer extracted (* 2), the EtOAc layer is again with 1NHCl (* 3), H ₂O (* 3), saturated NaHCO ₃(* 3), H ₂O (* 3), saturated brine (* 3) washing, anhydrous Na ₂SO ₄Dry, the filtering siccative, filtrate evaporate to dryness yellowly oily matter adds anhydrous diethyl ether and occurs white precipitate immediately, spend the night in the refrigerator placement, leach white precipitate, TLC is a single-point, the heavy 2.486g of product, yield 89.91%, mp:74-76 ℃, and Rf=0.46 (PE: EtOAc=1: 4), FAB-MS (m/z): 554.3[M+H] ⁺

HCl.NH ₂The preparation of-Gly-Phe-Aca-OPac:

With the same HCl.NH of 1.748g (3.16mmol) Boc-Gly-Phe-Aca-OPac ₂The operation of-Aca-OPac (4) obtains white solid, and it is a single-point that TLC detects, the heavy 1.217mg of product, yield 82.15%, mp:109.7-112.6 ℃, Rf=0.20 (DCM: MeOH=10: 1).

The preparation of Boc-Tyr-Gly-Phe-Aca-OPac:

With 245mg (0.5mmol) HCl.NH ₂-Gly-Phe-Aca-OPac, 169mg (0.6mmol) Boc-Tyr-OH, 100mg (0.65mmol) HOBt, 125mg (0.65mmol) EDC.HCl and 0.114mL (0.65mmol) DIEA are with the operation of Boc-Gly-Phe-Aca-OPac, after the EtOAc layer drying, filtrate decompression concentrates evaporate to dryness, add anhydrous diethyl ether and separate out white precipitate, leach white precipitate, it is a single-point that TLC detects, the heavy 270mg of product, yield 75.42%, mp:83.3-86 ℃,, Rf=0.3 (PE: EtOAc=1: 2), FAB-MS (m/z): 717.4[M+H] ⁺

Amino acid analysis: Tyr 0.935 (1), and Gly 1.098 (1), and Phe 1.094 (1), Aca0.883 (1).

The preparation of Boc-Tyr-Gly-Phe-Aca-OH (6):

The Boc-Tyr-Gly-Phe-Aca-OPac of 300mg (0.42mmol) is placed bottle at the bottom of the garden of 100ml, add down about 25mlHOAc in 45 ℃ outer baths and dissolve, add 5g (76.9mmol) Zn powder in batches, continue to react under 45 ℃ to raw material and disappear, the filtering precipitation, use absolute ethanol washing, merging filtrate, evaporate to dryness, use acetic acid ethyl dissolution, move in the separating funnel, successively through 1NHCl (* 2), saturated brine (* 2) washing, through anhydrous Na ₂SO ₄After the drying, be evaporated to thick liquid under bathing outside 40 ℃, grind with anhydrous diethyl ether and wash, leach, drying obtains white powder.

Product is heavy: 180mg, yield: 71.8%.

HCl.NH ₂The preparation of-Tyr-Gly-Phe-Aca-OH (G35I):

Operation with the same compound of Boc-Tyr-Gly-Phe-Aca-OH (4) obtains white powder.Product is heavy: 136mg, and yield: 85%, FAB-MS:m/z 499[M+1].

Embodiment 2 G36H are synthetic

Synthetic route:

Reaction conditions:

a)1.NaOH/H ₂O，2.ClCOOiBu/NMM，-20℃，3.1NHCl/H ₂O

b)BrCH ₂CO-Ph/TEA c)3.5NHCl/HOAc

d)Boc-Phe-OH/EDC/HOBt/DIEA e)Boc-Gly-OH/EDC/HOBt/DIEA

f)Boc-Tyr-OH/EDC/HOBt/DIEA g)Zn/HOAc

Operational condition:

The preparation of Boc-Gly-Gly-OH:

With 3.504g (20mmol) Boc-Gly-OH, 6.006g (80mmol) H ₂N-Gly-OH is with the preparation manipulation of Boc-Gly Phe-OH (1), and after the EtOAc layer drying, filtrate decompression concentrates and obtains white solid, adds anhydrous Et ₂The O agitator treating leaches.It is a single-point that TLC detects.The heavy 3.262g of product, yield 70.3%, mp:121-123 ℃, Rf=0.2 (PE: EtOAc: HOAc=1: 1: 0.1).

¹H-NMR(300MHz，CD ₃COCD ₃)δppm：1.405(9H，s，(CH ₃) ₃C)，3.755(2H，d，NHCH ₂CONH，J＝6.0Hz)，3.959(2H，d，J＝5.4Hz，NHCH ₂COOH)。

The preparation of Boc-Gly-Gly-OPac:

Synthetic operation with 3.012g (12.98mmol) Boc-Gly-Gly-OH and 2.842g (14.28mmol) bromoacetophenone Boc-Aca-OPac (3), after the EtOAc layer drying, filtrate decompression concentrates and obtains oily matter, adds the sherwood oil grinding and obtains white powder, leach, it is a single-point that TLC detects.The heavy 4.206g of product, yield 92.58%, mp:56.8-57.4 ℃, Rf=0.4 (PE: EtOAc=2: 1).

¹H-NMR(300MHz，CDCl ₃)δppm：1.457(9H，s，(CH ₃) ₃C)，3.875(2H，d，NHCH ₂CONH，J＝5.7Hz)，4.291(2H，d，J＝5.5Hz，NHCH ₂COO)，5.430(2H，s，COOCH ₂CO)，6.606(1H，br，NH)，7.481-7.919(5H，m，Ar-H)。

The preparation of HCl.Gly-Gly-OPac:

With the same HCl.NH of 3.976g (11.36mmol) Boc-Gly-Gly-OPac ₂-Aca-OPac (4) operation, reaction leaches insolubles after finishing, with anhydrous Et ₂The O thorough washing leaches and obtains white powder, and it is a single-point that TLC detects.The heavy 3.251g of product, yield 99.89%, mp:1181.8-184.6 ℃, Rf=0.75 (DCM: MeOH=2.5: 1).

The preparation of Boc-Phe-Gly-Gly-OPac:

Preparation manipulation with 859mg (3mmol) HCl.Gly-Gly-OPac and 817mg (3.08mmol) Boc-Phe-OH, 505mg (3.3mmol) HOBt, 634mg (3.3mmol) EDC.HCl and the same Boc-Gly-Phe-Aca-OPac of 0.58mL (3.3mmol) DIEA (5), after the EtOAc layer drying, filtrate decompression concentrates, obtain white powder, add the abundant agitator treating of anhydrous diethyl ether, leach.It is a single-point that TLC detects.The heavy 1.398g of product, yield 93.76%, mp:140.8-141.9 ℃, Rf=0.27 (PE: EtOAc=2: 3), FAB-MS (m/z): 498[M+H] ⁺, 398[M+H-Boc] ⁺

HCl.H ₂The preparation of N-Phe-Gly-Gly-OPac:

With the same HCl.NH of 1.256g (2.527mmol) Boc-Phe-Gly-Gly-OPac ₂-Aca-OPac (4) operation leaches the white precipitate in the reaction solution, and add anhydrous diethyl ether and be washed till neutrality, oven dry, it is a single-point that TLC detects, the heavy 1.075mg of product, yield 98.2%, mp:111.4-115.3 ℃, Rf=0.6 (DCM: MeOH=5: 1).

The preparation of Boc-Gly-Phe-Gly-Gly-OPac:

With 1.095g (2.53mmol) HCl.H ₂N-Phe-Gly-Gly-OPac and 487mg (2.78mmol) Boc-Gly-OH, 465mg (3.036mmol) HOBt, 583mg (3.036mmol) EDC.HCl and the same Boc-Gly-Phe-Aca-OPac of 0.54mL (3.036mmol) DIEA (5) operation, after the EtOAc layer drying, filtrate decompression concentrates, obtain white precipitate, add the anhydrous diethyl ether agitator treating, leach, it is a single-point that TLC detects.The heavy 1.085g of product, yield 77.41%, mp:147.1-148.3 ℃, Rf=0.3 (DCM: MeOH=20: 1), FAB-MS (m/z): 555.3[M+H] ⁺

HCl.H ₂The preparation of N-Gly-Phe-Gly-Gly-OPac:

With the same HCl.NH of 1.027g (1.854mmol) Boc-Gly-Phe-Gly-Gly-OPac ₂-Aca-OPac (4) operation leaches the white precipitate in the reaction solution, and add ethyl acetate and be washed till neutrality, oven dry, it is a single-point that TLC detects.The heavy 857mg of product, yield 94.24%, mp:174.2-178 ℃, Rf=0.4 (DCM: MeOH=5: 1).

The preparation of Boc-Tyr-Gly-Phe-Gly-Gly-OPac:

With 245mg (0.5mmol) HCl.H ₂N-Gly-Phe-Gly-Gly-OPac and 155mg (0.55mmol) Boc-Tyr-OH, 92mg (0.6mmol) HOBt, 115mg (0.6mmol) EDC.HCl and the same Boc-Gly-Phe-Aca-OPac of 0.11mL (0.6mmol) DIEA (5) operation are through DCM/MeOH/Et ₂O is refining to obtain white powder, adds the anhydrous diethyl ether agitator treating, leaches, and it is a single-point that TLC detects.The heavy 267g of product, yield 74.48%, mp:112-114 ℃, Rf=0.6 (DCM: MeOH=10: 1), FAB-MS (m/z): 718.3[M+H] ⁺

G36H's is synthetic:

Operation with the same Boc-Tyr-Gly-Phe-Aca-OPac of 181mg (0.25mmol) Boc-Tyr-Gly-Phe-Gly-Gly-OPac (6), separate through silica gel column layer, with DCM: MeOH: HOAc (20: 1: 0.1) wash-out, obtain white powder, it is a single-point that TLC detects.The heavy 90mg of product, yield 60%, mp:136.5-138.7 ℃, Rf=0.4 (DCM: MeOH: HOAc=20: 1: 0.1), FAB-MS (m/z): 622.1[M+H+Na] ⁺, 522.1[M+H+Na-Boc] ⁺, 638.1[M+H+K] ⁺

Amino acid analysis: Gly 3.015 (3), and Phe 1.016 (1), and Tyr 0.969 (1).

Embodiment 3 G36E's is synthetic:

Synthetic route:

Reaction conditions:

a)1.NaOH/H ₂O，2.ClCOOiBu/NMM，-20℃，3.1NHCl/H ₂O

b)1.NaOH，2.(Boc) ₂O/tBuOH，3.1NHCl/H ₂O

c)BrCH ₂CO-Ph/TEA d)3.5NHCl/HOAc

e)EDC/HOBt/DIEA f)Boc-Tyr-OH/EDC/HOBt/DIEA

Operational condition:

The preparation of Boc-Aba-OH:

With 5.155g (50mmol) H ₂N-Aba-OH adds 12.5ml water, 2g (50mmol) NaOH and 15ml t-BuOH dissolving in 100ml eggplant type bottle, in 30 minutes, dropwise add 12.005g (55mmol) (Boc) under stirring ₂The 12.5ml t-BuOH liquid of O after the insulation, adds the 75ml trimethyl carbinol a little, the powerful stirring spent the night under room temperature, the operation of the same Boc-Aca-OH of aftertreatment (2) adds the sherwood oil grinding and white solid occurs, leaches the white solid thing, detect through TLC, iodine is smoked to be a single-point, the heavy 8.417g of product, yield 82.93%, mp:40.8-42.7 ℃, Rf=0.3 (PE: EtOAc: HOAc=2: 1: 0.1).

¹H-NMR(300MHz，CDCl ₃)δppm：1-440(s，9H，(CH ₃) ₃C)，1.823(m，2H，J＝6.9Hz，CH ₂CH ₂CH ₂)，2.401(t，2H，J＝7.2Hz，CH ₂COOH)，3.184(br，2H，NHCH ₂)，4.665(br，1H，NH)。

The preparation of Boc-Aba-OPac:

With 1.095g (5.5mmol) bromoacetophenone, 1.015g (5mmol) Boc-Aba-OH and the same Boc-Aca-OPac of 0.79mL (5.5mmol) TEA (3) operation, after the EtOAc layer drying, filtrate decompression obtains white solid after concentrating, and adds petroleum ether, leaches white powder, oven dry, it is a single-point that TLC detects, the heavy 1551mg of product, yield 96.82%, mp:98.2-99.4 ℃, Rf=0.5 (PE: EtOAc=2: 1).

¹H-NMR(300MHz，CDCl ₃)δppm：1.439(s，9H，(CH ₃) ₃C)，1.908(m，2H，CH ₂CH ₂CH ₂)，2.547(t，2H，J＝7.2Hz，CH ₂COO)，3.229(q，2H，J＝6.6Hz，NHCH ₂)，5.356(s，2H，CH ₂COAr)，7.465-7.922(m，5H，Ar-H)。

HCl.NH ₂The preparation of-Aba-OPac (7):

With the same HCl.NH of 2.603g (8.109mmol) Boc-Aba-OPac ₂-Aca-OPac (4) operation removes solvent under reduced pressure, obtains white solid, with the anhydrous diethyl ether thorough washing, leach white solid, it is a single-point that TLC detects, the heavy 1949mg of product, yield 93.34%, mp:126.1-129.2 ℃, Rf=0.4 (DCM: MeOH=8: 1).

The preparation of Boc-Gly-Phe-Aba-OPac:

With 771mg (3mmol) HCl.NH ₂-Aba-OPac (7), 1.014g (3.15mmol) Boc-Gly-Phe OH (1), 504mg (3.3mmol) HOBt, 633mg (3.3mmol) EDC.HCl and the same Boc-Gly-Phe-Aca-OPac of 0.58mL (3.3mmol) DIEA (5) operation, filtrate decompression concentrates and obtains white powder, with the anhydrous diethyl ether thorough washing, leach white powder, it is a single-point that TLC detects.The heavy 1.474g of product, yield 93.58%, mp:93.7-95.6 ℃, Rf=0.3 (PE: EtOAc=1: 4).FAB-MS(m/z)：526.1[M+H] ⁺。

HCl.NH ₂The preparation of-Gly-Phe-Aba-OPac:

With the same HCl.NH of 1.395g (2.657mmol) Boc-Gly-Phe-Aba-OPac ₂The operation of-Aca-OPac (4) behind the stopped reaction, solid occurs with the reaction solution concentrating under reduced pressure, with the anhydrous diethyl ether thorough washing, leaches precipitation, obtains white powder.It is a single-point that TLC detects, the heavy 1.173g of product, yield 95.66%, mp:174-178.8 ℃, Rf=0.4 (DCM: MeOH=6: 1).

The preparation of Boc-Tyr-Gly-Phe-Aba-OPac (G36E):

With 692mg (1.5mmol) HCl.NH ₂The operation of-Gly-Phe-Aba-OPac and 456mg (1.62mmol) Boc-Tyr-OH, 248mg (1.62mmol) HOBt, 334mg (1.74mmol) EDC.HCl and the same B0c-Gly-Phe-Aca-OPac of 0.31mL (1.74mmol) DIEA (5), concentrating under reduced pressure, obtain white solid, with the anhydrous diethyl ether thorough washing, leach white powder, it is a single-point that TLC detects.The heavy 887g of product, yield 85.95%, mp:108-110 ℃, Rf=0.4 (DCM: MeOH=8: 1), FAB-MS (m/z): 689.3[M+H] ⁺

Amino acid analysis: Tyr 0.946 (1), and Gly 1.099 (1), and Phe 1.083 (1), Aba0.871 (1).

Embodiment 4 G36J's is synthetic

Synthetic route:

Operational condition:

HCl.NH ₂The preparation of-Tyr-Gly-Phe-Aba-OPac (G36J):

With the same HCl.NH of 350mg (0.509mmol) Boc-Tyr-Gly-Phe-Aba-OPac (G36E) ₂The operation of-Aca-OPac (4) behind the stopped reaction, obtains white solid with the reaction solution concentrating under reduced pressure, with the anhydrous diethyl ether thorough washing, leaches white powder, and it is a single-point that TLC detects.The heavy 308mg of product, yield 96.89%, mp:182-185 ℃, Rf=0.5 (DCM: MeOH=6: 1).

Embodiment 5 G48A's is synthetic

Synthetic route:

Reaction conditions:

a)Boc-Aca-OH，Cs ₂CO ₃，NaI，60℃ b)1.HCl/HOAc，2.Et ₃N

c)Boc-Phe(4-NO ₂)-OH/DCC/HOBt d)HCl/HOAc

e)Boc-Gly-OH/DCC/HOBtNMM f)Boc-Tyr-OH/DCC/HOBt/NMM

g)NH ₂C ₂H ₅/THF

Operational condition:

The preparation of Boc-Aca-O-resin (8)::

Get 5g (5.5mmol) Merrifield resin (replacing equivalent 1.1mmol/g), 3.465g (15mmol) Boc-Aca-OH, 2.5g (7.67mmol) Cs ₂CO ₃And 10mgNaI.2H ₂O is suspended among the 60mL exsiccant DMF, reaction is 28 hours on 60 ℃ of constant temperature air baths, then with following solvent filter wash successively: DMF * 5, and anhydrous EtOH * 2,1NHCl * 2,50%EtOH * 2,95%EtOH * 2, anhydrous diethyl ether * 1, drain, take out about 40 ℃ of air-dry constant weights under infrared lamp, the heavy 6.055g of resin, resin weightening finish 1.055g.Yield 98.6%, SD=0.91mmol/g.

H ₂The preparation of N-Aca-O-resin (9):

Get 2g (1.82mmol) resin (8) move into volume be 10ml the reactive polypeptide pipe in, add 3.5NHCl/HOAc, seal suitable for reading, in room temperature, shook 5 minutes, and extracted solution, add 3.5NHCl/HOAc again, in room temperature, shook again 35 minutes, extract acid solution, resin is used following solvent filter wash successively, each about 1 minute: DCM (* 2), 95%EtOH (* 5), DMF (* 1), 6%TEA/EtOAc (* 2), 95%EtOH (* 1), 50%EtOH (* 1), 95%EtOH (* 1), anhydrous diethyl ether (* 1) is drained.Get the 1mg resin and carry out the ninhydrin reagent detection, resin and supernatant liquor show that Boc removes when being blueness (positive).

Boc-Phe (4-NO ₂The preparation of)-Aca-O-resin (10):

Take by weighing 1.041g (4mmol) Boc-Phe (4-NO ₂)-OH, HOBt 612mg (4mmol) add 15ml exsiccant DMF and make molten entirely in the 50ml triangular flask.Under the frozen water cooling, add 2.5ml (4mmol) DCC/THF solution.Place filtering DCU precipitation after 1 hour, filtrate is added in the reactive polypeptide pipe of above-mentioned resin (9), at room temperature shakes subsequently 15 hours.Get the 1mg resin and carry out the ninhydrin reagent detection, resin and supernatant liquor show that condensation is complete when being faint yellow (feminine gender).Behind the filtering reaction solution, resin is used following solvent filter wash: DMF (* 5) successively, EtOH (* 3), DMF (* 3), EtOH (* 3), anhydrous Et ₂O (* 2).Drain solvent, get resin (10).

Boc-Gly-Phe (4-NO ₂The preparation of)-Aca-O-resin (11):

Take by weighing 1.061g (4mmol) Boc-Gly-OH, 612mg (4mmol) HOBt adds 15ml exsiccant DMF and makes molten entirely in the 50ml triangular flask.Under the frozen water cooling, add 2ml (4mmol) DCC/THF solution.Place filtering DCU precipitation after a hour, filtrate for later use.The resin (10) that previous step is obtained takes off the Boc processing, and concrete operations are the same with the preparation of resin (9), and this step need not neutralize.After draining solvent, above-mentioned preliminary Boc-Gly-OH activation solution and NMM 0.12ml (1mmol) are added in the reaction tubes, mixed with resin.Stirred 16 hours in the room temperature.Get the 1mg resin and carry out the ninhydrin reagent detection, resin and supernatant liquor show that condensation is complete when being faint yellow (feminine gender).Filtering reaction solution, resin resin are used following solvent filter wash: DMF (* 5) successively, EtOH (* 3), DMF (* 3), EtOH (* 3), anhydrous Et ₂O (* 2).Drain solvent, resin is put under the infrared lamp, air-dry in 45 ℃ to constant weight.Claim to such an extent that peptide resin (11) weighs 2.25g, actual weightening finish 430mg, two steps connect peptide yield 95.3%.

Boc-Tyr-Gly-Phe (4-NO ₂The preparation of)-Aca-O-resin (12):

The operation of the consumption of each reagent and operation and resin (11) is identical.

H ₂N-Tyr-Gly-Phe (4-NO ₂The preparation of)-Aca-O-resin (13):

With the same H of resin (12) ₂The preparation manipulation of N-Aca-O-resin (9) is identical.

H ₂N-Tyr-Gly-Phe (4-NO ₂)-Aca-NHC ₂H ₅(G48A) preparation:

Getting 1.6g resin (0.97mmol) (13) mixes with 6mL70% ethamine/water and 6mLTHF, shook once in a while in the room temperature 24 hours behind the sealing orifice, collect filtrate, in 46 ℃ of water-baths, be evaporated to dried, residue grinds and filters through the 30mL anhydrous diethyl ether, obtain 480mg (theoretical amount 55.3mg) aminolysis yield: 86.8%, mp:109-111 ℃, FAB-MS (m/z): 571.4[M+H] ⁺

Embodiment 6 G35I are to the effect of vitro human activity of osteoblast proliferation

Method: press the method that people such as He Tao sets up (He Tao, Yang Qingming, Zheng Lianfu, the foundation and the evaluation of adult OC marrow culture method, Chinese journal of orthopedics, 1999,19:59) the thick loud, high-pitched sound cells in vitro that carries out people embryo long bone source is cultivated

The results are shown in Figure 1

Can see that by Fig. 1 the scleroblast under the effect of different treatment factor all reaches peak value at the 7th day cell quantity of vitro culture, cell quantity reduces gradually subsequently.In this culture system, the G35I of different concns all has the outgrowth effect of stimulation human osteoblast cell in various degree.At this point of peak value, this effect of G35I was the most remarkable when concentration was 10-7 and 10-8 mol.

Embodiment 7 G35I are to the bone formulation stimulant activity of spay rat

1. materials and methods

Laboratory animal is the Wistar female rats at 3 monthly ages, mean body weight 151.35 grams.Be divided into following 5 groups immediately:

OVX organizes spay, not drug treatment

S group normal control group

Comply with for sodium phosphate behind the Eti group removal ovary, 4mg/Kg/d. * 2 weeks, subcutaneous

Only give G35I 30nmol/d/ behind the G35I group removal ovary, subcutaneous

Comply with simultaneously for sodium phosphate and G35I behind the Eti+G35I group removal ovary, consumption is the same

Tetracycline marker: carry out tetracycline marker before putting to death animal, tsiklomitsin 50 mg/kg body weight/day are irritated stomach, continuous 2 days, irritate stomach femoral artery sacrificed by exsanguination rat after two days 3 days after 9 days again.

The enforcement of removal ovary operation: 0.5% vetanarcol, 20-30 mg/kg body weight abdominal injection.Behind the drug effect, in about 3-4 centimetre in back backbone escribe mouth, the passivity chorista enters and with mosquito forceps ovary is pulled to behind the peritonaeum externally, and the ordinary suture joint is pricked the back excision, will remain tissue and replace, and sews up a wound after giving penicillin in the part.

The preparation of undecalcified bone slice: bone specimen is through 10% formaldehyde fixed 2 days, and getting proximal tibia, to wear into 2-3mm with sand paper thick, exposes medullary space, places 0.5%Villanueva staining agent dyeing 72 hours.Under magnetic stirrer, use 75% ethanol successively, 80% ethanol, 95% ethanol, 100% ethanol, acetone and methymethacrylate dewatering and defatting, then sample is placed the plastic embedding box, carry out embedding with polymethylmethacrylate, it is dry to put into vacuum drying oven, treats to take out after the embedded block hardening.Be cut into the bone slice of 10-15 micron thickness with the Polycut slicing machine, under common and fluorescent microscope, measure its morphology parameter with the semi-automatic Quantimet of Opton Contron.

The method for expressing of bone morphology parameter:

1. whole osseous tissue volume (Total Tissue Volume, TTV) (mm ³);

Comprise the volume of cortex of bone and the volume of spongy bone.

2. spongy bone volume (Sponge Bone Volume, SBV) (mm ³):

Medullary space and bone trabecular volume sum.

3. Trabecula Bone Volume (Trabecular Bone Volume, TBV) (mm ³):

The mineralising bone and the volume sum of the osteoid of mineralising not in the bone trabecula.

4. bone trabecula surface (Trabecular Bone Surface, TBS) (mm ²):

Bone trabecula surface-area in institute's survey scope.

5. the ratio (TBV/TTV) of Trabecula Bone Volume and whole osseous tissue volumes is (%):

Trabecula Bone Volume accounts for the per-cent of whole osseous tissue volumes in the useful range.

6. ratio (the S/V) (mm of bone trabecula surface and volume ²/ mm ³):

Be the ratio between the TBS/TBV, show bone surface that the unit Trabecula Bone Volume has what.

7. the ratio (TBV/SBV) of Trabecula Bone Volume and spongy bone volume is (%):

Trabecula Bone Volume accounts for the per-cent of spongy bone volume in the useful range.

8. average bone trabecula plate thickness (Mean Trabecularplate Thickness, MTPT) (μ m):

This parameter is to calculate according to Trabecula Bone Volume and bone trabecula table, promptly

$\mathrm{MTPT}=-\frac{\mathrm{TBV}}{\mathrm{TBS}}\×2000$

9. average bone trabecula plate density (Mean Trabecular Plate Density, MTPD) (/mm ²):

The every mm of this parameter indicating ²Bone trabecular quantity.

$\mathrm{MTPD}=\frac{\mathrm{TBV}}{\mathrm{SBV}}\×\frac{10}{\mathrm{MTPT}}$

10. average bone trabecula sheet separation (Mean Trabecular Plate Spacing, MTPS) (μ m):

$\mathrm{MTPS}=-\frac{1000}{\mathrm{MTPD}}-\mathrm{MTPT}$

Represent the distance between the bone trabecula plate.

2. result:

Table 2-1 G35I treatment group rat tibia morphology proximal mathematic(al) parameter (x ± s)

Parameter	The OVX group	The S group	The Eti group	The G35I group
					TBV/TTV(％) S/V(mm 2/mm 3) TBV/SBV(％) MTPT(μm) MTPD(/mm 2) MTPS(μm)	8.30±2.74 40.52±5.09 11.09±4.32 50.03±6.44 2.25±0.86 469.28±253.11	20.03±3.57 *e 36.12±2.32e 24.50±4.26 * 55.63±3.56e 4.40±0.63 * 175.77±35.01 *	11.55±3.55 44.16±5.66 14.36±4.26s 45.87±5.48 3.17±1.04 *s 301.92±122.34 *	14.64±1.32 *s 33.14±3.42 *e 18.58±2.00 *s 60.89±6.35 *e 3.06±0.29s 268.03±28.46 *

Table 2-2 G35I treatment group rat tibia morphology proximal mathematic(al) parameter (x ± s)

Number	The Eti+G35I group	F	p
				TBV/TTV(％) S/V(mm 2/mm 3) TBV/SBV(％) MTPT(μm) MTPD(/(mm 2) MTPS(μm)	15.18±3.28 *Se 37.72±1.77e 18.82±3.66 *S 53.12±2.64eg 3.56±0.77 * 238.91±61.13 *	12.67 6.81 10.67 7.33 6.39 4.28	＜0.01 ＜0.01 ＜0.01 ＜0.01 ＜0.01 ＜0.01

^*Representative is compared with OVX, and s, e, g represent the group with S respectively, and Eti group and G35I group be significance relatively

Can find that from the data of table 2 six bone morphology parameters of G35I all are better than positive treatment medicine (Eti group).Illustrate that G35I can prevent losing of trabecular bone to a certain extent and improve bone trabecular space structure, promptly increase bone trabecular thickness, reduce the distance between bone trabecula.These effects are removed the misery of sufferers of osteoporosis face to reducing patient's fracture, and it is significant to improve the quality of living.

Claims (5)

1. formula I polypeptide compound

Wherein

R ¹＝H，Boc，ArSO ₂，Aba，Ac，Aca；

R ²＝H，OH；R ³＝H，NO ₂，OH；

R ⁴＝H，NO ₂，OH；

R ⁵＝Aba-OH，Aba-NH ₂，Aba-NHEt，Aba-NMe ₂，

Aca-OH，Aca-NH ₂，Aca-NHEt，Aca-NME ₂；

Condition is that this compound is not R ¹=H, R ²=OH, R ³=H, R ⁴=H, R ⁵=-Gly-Gly-OH.

2. the compound of claim 1, its Chinese style I compound is selected from G35I, G38I or G38K.

3. pharmaceutical composition, it comprises compound and the pharmaceutical carrier or the vehicle of claim 1 or 2.

4. claim 1 or 2 compound purposes in the preparation bone formulation stimulant.

5. the purposes of claim 4, wherein said bone formulation stimulant is the medicine that is used to prevent or treat osteoporosis.

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