CN103524599B - Cyclic peptide lhrh antagonist derivative and pharmaceutical use thereof - Google Patents

Abstract

The present invention relates to the Cyclopeptide derivatives with LHRH receptor antagonist activity shown in general formula I, its preparation method, containing their pharmaceutical composition and they row gland cancer, carcinoma of endometrium, ovarian cancer, mammary cancer and other sexual hormoue relevant with reproduction rely on the purposes in relative disease, contraception etc. before the treatment.

Description

Cyclic peptide lhrh antagonist derivative and pharmaceutical use thereof

Technical field

The present invention relates to and there is luteinizing hormone releasing hormone (LHRH) receptor antagonist activity, there is the Cyclopeptide derivatives suppressing the effect of pituitary gonad-stimulating hormone, the effect of inhibition glandular secretion steroid hormone, its preparation method, containing their pharmaceutical composition and they row gland cancer, carcinoma of endometrium, ovarian cancer, mammary cancer and other sexual hormoue relevant with reproduction rely on the purposes in relative disease, contraception etc. before the treatment.

Background technology

LHRH is also known as GnRH(gonadotropin-releasing hormone), its main physiological function is the sex hormone level in control agent.LHRH in body is mainly by hypothalamus (Hypothalamus) neuronal cell synthesis secretion, discharge to hypophysis (Pituitary) portal vein (Portalveins) with about one hour pulse-repetition once, by with special LHRH receptors bind on hypophysis, impel hypophysis in blood circulation, secrete luteotropic hormone (LH) and follicle-stimulating hormone (FSH) two kinds of gonad-stimulating hormone.LH and FSH arrives Male sexual body of gland testis (Testis) with blood circulation and plays short spermatogenesis (Spermatogenesis) effect and short testosterone (Testosterone, T) Synthesis; Or arrive Female sexual body of gland ovary (Ovary) and play short Folliculogenesis effect (Folliculogenesis) and short oestrogenic hormon (Estrone, E) and progesterone (Progesterone, P) Synthesis.Finally, steroid sex hormone T, E and P feed back to hypothalamus and hypophysis again.Therefore visible, play keying action in the physiological function of LHRH sex hormone level in hypothalamus control agent.

LHRH is made up of ten amino-acid residues, and C-end is containing amide structure.The primary structure of LHRH is as follows:

p-Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH ₂

Lhrh antagonist, by blocking the release of LHRH effect and then suppression LH, therefore may be used for the treatment of sex hormone related condition as diseases such as prostate cancers.Compared with agonist, lhrh antagonist have effective speed fast, without recovering after upper punch phenomenon, drug withdrawal soon, to advantages such as serum androgen controlled level are strong.Can expect, the curative effect of lhrh antagonist treatment prostate cancer is better than agonist, is more easily easily accepted by the patient, has larger application prospect than agonist.

Although the lhrh antagonist developed at present is existing a lot, as a kind of peptide medicament, great majority still exist that bioavailability is low, and Half-life in vivo is short, the high weak point of histamine release amount.Similar to other peptide medicament, lhrh antagonist medicine is also difficult to pass through oral absorption.As far as we know, the lhrh antagonist medicine of listing is at present all non-oral administration.Compared with linear peptides, cyclic peptide has better enzyme stability, and some cyclic peptide is because lipotropy strengthens, oral absorption ability can also be strengthened, therefore, linear LHRH decapeptide antagonist derivatives is converted into cyclic peptide by object of the present invention exactly, wish keep former activated while, improve its enzyme stability and permeable membrane receptivity, thus obtain novel lhrh antagonist medicine that even can be oral.

Summary of the invention

The present inventor has now found that formula I Cyclopeptide derivatives after deliberation

Or its steric isomer or its salt without physiological-toxicity, there is good LHRH antagonistic activity and higher enzyme stability, therefore can be used as medicine relies on treating correlative diseases purposes for prostate cancer, carcinoma of endometrium, ovarian cancer, mammary cancer and other sexual hormoue.

Therefore a first aspect of the present invention relates to the compound shown in formula i, or its steric isomer or the salt without physiological-toxicity,

Wherein,

Xaa ³for D-Phe or D-Pal;

Xaa ⁴for L or D type Cys, Asp, Glu or Lys;

Xaa ⁵for L or D type Aph (Hor) or Arg;

Xaa ⁶for L or D type Aph (Cbm) or Pal;

Xaa ⁸for Ilys or Arg;

Y is L or D type Cys-Ala, Asp-Ala, Glu-Ala, Lys-Ala, Pro-Cys, Pro-Asp, Pro-Glu or Pro-Lys, and wherein two amino acid can be L-type or D type independently of one another;

Wherein by the Cys side chain thiol of 4 and Y with disulfide linkage Cheng Huan, or by the amino acid Asp of 4 and Y, the side chains such as Glu, Lys are with amido linkage Cheng Huan.

The compound of any one according to a first aspect of the present invention, it is selected from following one or more:

(1) Xaa ³for D-Pal;

(2) Xaa ⁴for Cys;

(3) Xaa ⁵for D-Aph (Hor);

(4) Xaa ⁶for D-Aph (Cbm);

(5) Y is Cys-D-Ala or Pro-D-Cys.

The compound of any one, wherein Xaa according to a first aspect of the present invention ³for D-Pal.

The compound of any one, wherein Xaa according to a first aspect of the present invention ⁴for Cys.

The compound of any one, wherein Xaa according to a first aspect of the present invention ⁵for D-Aph (Hor).

The compound of any one, wherein Xaa according to a first aspect of the present invention ⁶for D-Aph (Cbm).

The compound of any one according to a first aspect of the present invention, wherein Y is Cys-D-Ala or Pro-D-Cys.

The compound of any one according to a first aspect of the present invention, it is selected from following compound:

I.e. compound 1,2,3,4,5,6,7.

Preferably, the compounds of this invention is selected from compound 2,3,4,5.

A second aspect of the present invention relates to pharmaceutical composition, and it contains the compound of any one of at least one first aspect present invention, and optional pharmaceutically acceptable auxiliaries, carrier or vehicle.

The compound that a third aspect of the present invention relates to any one of first aspect present invention is for the preparation of preventing and/or treating sex hormone related condition or for the purposes in the medicine of practising contraception.

Purposes according to a third aspect of the present invention, wherein said sex hormone related condition comprises sexual hormoue associated cancer, precocious puberty, hyperplasia of prostate, endometriosis, menoxenia (as amenorrhoea, dysfunctional uterine bleeding or ovulation failure) or Infertility etc.

Purposes according to a third aspect of the present invention, wherein said sexual hormoue associated cancer comprises prostate cancer, carcinoma of endometrium, mammary cancer or ovarian cancer etc.

A fourth aspect of the present invention relates to the purposes of compound in the medicine for the preparation of suppression pituitary gonad-stimulating hormone (such as LH and/or FSH) and/or inhibition glandular secretion steroid hormone (such as oestrogenic hormon, progestogen and/or testosterone) of any one of first aspect present invention.

A fifth aspect of the present invention relates to the preparation method of the compound of any one of first aspect present invention 1-3, it is characterized in that:

Take mbha resin as carrier, Boc-or Fmoc-Preservation tactics, DCC/HOBT or BOP/DIEA is condensation reagent, and HCl/ dioxane or piperidines/DMF are deprotecting regent, after amino acid condensation completes, use I ₂remove the Trt protecting group on sulfydryl, intramolecular two sulfhydryl oxidases are with disulfide linkage Cheng Huan simultaneously, cut down by Cyclopeptide derivatives after having reacted with liquid HF from mbha resin.

Except as otherwise noted, the term used in this application has following implication.

In the present invention, all amino acid configuration, except being labeled as D-type, are L-type.

In the present invention, described disulfide linkage refers to the-S-S-formed between halfcystine and cysteine side chain.

In the present invention, described amido linkage refers to the-CO-NH-formed between amino acid and amino acid.

According to the present invention, Cyclopeptide derivatives shown in formula I and steric isomer thereof not only demonstrate good result with it without physiological-toxicity salt in animal androgen antagonist secretion experiment, and demonstrate higher enzyme stability and lipotropy in measuring in vitro, therefore can be used as amcinonide for animal, be preferred for Mammals, particularly people.

Therefore the present invention also relates to containing the Cyclopeptide derivatives shown at least one formula I as the effective dose of activeconstituents and/or its steric isomer or its pharmaceutical composition without physiological-toxicity salt and customary pharmaceutical excipients or auxiliary material.Here " customary pharmaceutical excipients or auxiliary material " comprises any one or all solvents, dispersion medium, dressing, antiseptic-germicide or anti-mycotic agent, isotonic and sustained release dosage, and similar physiology compatibility agent, with applicable intravenous injection, intramuscular injection, subcutaneous injection, or other administering mode, as oral administration.According to the mode of administration, can by active compound dressing with protect compound from acid or the impact of other natural condition inactivation.

Term used herein " salt without physiological-toxicity " refers to and can retain parent compound expection physiologically active and can not produce salt or their composition of any unexpected toxic side effect.Such as: hydrochloride, hydrobromate, vitriol, phosphoric acid salt, nitrate, and acetate, oxalate, tartrate, succinate, malate, benzoate, embonate, alginates, mesylate, naphthalenesulfonate etc.Positively charged ion according to containing in salt can be again: sylvite, lithium salts, zinc salt, mantoquita, barium salt, bismuth salt, and the inorganic salt such as calcium salt also can be the organic salts such as such as trialkyl ammonium salts.

Formula (I) Cyclopeptide derivatives and steric isomer thereof or the pharmaceutical composition containing it can with known any mode administrations, as oral, muscle, subcutaneous etc., form of administration is tablet, capsule, buccal tablet, chewable tablet, elixir, suspensoid, transdermal agent, micro-capsule embedding medium, implants, syrup etc. such as.Can be ordinary preparation, sustained release preparation, controlled release preparation and various particulate delivery system.In order to unit dosage forms for administration is made tablet, various biodegradable or physiologically acceptable carrier well known in the art can be widely used.About the example of carrier, as saline based and various aqueous buffer solution, ethanol or other polyvalent alcohol, liposome, poly(lactic acid), vinyl-acetic ester, polyanhydride, polyglycolic acid, collagen, poe etc.

The dosage of the Cyclopeptide derivatives shown in formula I or its steric isomer depends on many factors, such as to prevent or the character of disease therapy and severity, the sex of patient or animal, age, body weight, susceptibility and individual reaction, particular compound used, route of administration, administration number of times and desired by the result for the treatment of etc. that reaches.Above-mentioned dosage can single dose form or be divided into several, such as two, three, four dosage forms for administration.Single maximal dose is generally no more than 30mg/Kg body weight, such as 0.001-30mg/Kg, preferred 0.01-5mg/Kg, and better dosage range is 0.5-2mg/Kg body weight.But, in some cases, the single dosage of more than 30mg/Kg body weight or below 0.001mg/Kg also may be used.

Embodiment

The abbreviation used in the present invention has implication below:

Ac – ethanoyl

Ala-L-Ala,

Arg-arginine,

Aph-4-amino phenylalanine

Hor-whey acyl group

Cbm-formamyl

Boc-tertbutyloxycarbonyl,

BOP-benzotriazole-1-oxygen-three (dimethylamino) phosphorus phosphofluoric acid,

Cys-halfcystine

DCC-dicyclohexylcarbodiimide,

DIEA-diisopropylethylamine,

HOBt-1-hydroxybenzotriazole,

Leu-leucine,

ILys-sec.-propyl Methionin

MBHA-phenylamino methyl resin,

Nal-naphthylalanine,

Pal-3-pyrazoleahtnine,

Phe-phenylalanine,

RP-HPLC-RPLC

Pro-proline(Pro),

Ser-Serine,

TEA-triethylamine,

Cpa-4-chlorophenylalanine

Below in conjunction with embodiment, embodiment of the present invention are described in detail, but it will be understood to those of skill in the art that the following example only for illustration of the present invention, and should not be considered as limiting scope of the present invention.Unreceipted actual conditions person in embodiment, the condition of conveniently conditioned disjunction manufacturers suggestion is carried out.Agents useful for same or the unreceipted production firm person of instrument, being can by the conventional products of commercial acquisition.

Embodiment solid-phase synthesized carrier mbha resin used is Tianjin Nankai synthesis responsibility company limited product; The natural amino acid of DCC, HOBT, BOP, DIEA and Boc-protection or Fmoc-protection is by gill biochemical corp, Shanghai and Chengdu Kai Tai New Technology Co., Ltd. product, and the alpha-non-natural amino acid of Boc-or Fmoc-protection provides by the synthesis of this laboratory except explanation.

Embodiment 1: the synthesis of cyclic peptide lhrh antagonist derivative

Taking 0.5gMBHA resin is placed in reactor, adds DCM(4-dicyanomethylene) swelling 30min, magnetic agitation, makes resin dispersed.Drain, with DCM, methyl alcohol (MeOH), DCM washing (3 × 2min) (lower same), drain.Add 10%DIEA/DCM neutralization (2 × 5min), dissociate amino, and washing resin is drained.(1) Fmoc-D-Ala-OH, 1.08mmolHOBt, 1.08mmolDIC(N of 1.08mmol is added, N '-DIC) at 2mLCH ₃room temperature reaction 4h in OH, 4mLDCM.(2) wash, drain, get a little resin triketohydrindene hydrate indicator and detect.Get a little resin in vial, add each two of three kinds of solution of triketohydrindene hydrate successively.110 DEG C of heating 5min.Positive: most of resin is blue or light red, solution au bleu; Negative: resin is water white transparency, and solution is that true qualities are light yellow.If positive, repeat 1.(3) if negative, according to the order of holding N to hold from C, successively by 10 amino acid couplings on resin.

(4) A: after all amino acid completes condensation, adds the I2 of 10 times amount in peptide resin, with DMF(dimethyl formamide) be solvent, room temperature reaction 4h becomes ring.After having reacted, HF cracking excision protecting group and resin, obtain thick peptide after lyophilize.Through in press chromatogram purification and HPLC to analyze pure peptide.Synthesize compound 5,6,7 in this way, i.e. compound

(4) B: after all amino acid completes condensation, removes Boc protecting group with 25%TFA, and washing, adds Hor, 1.08mmolHOBt, 1.08mmolDIC of 1.08mmol, room temperature reaction 4h.Detect, if the positive is reacted again, until be negative.And then add the I2 of 10 times amount, take DMF as solvent, room temperature reaction 4h becomes ring.After having reacted, HF cracking excision protecting group and resin, obtain thick peptide after lyophilize.Through in press chromatogram purification and HPLC to analyze pure peptide.Synthesize compound 1,2,3,4 in this way, i.e. compound

The MS/HPLC data of each compound more than synthesized are see table 1.

The MS/HPLC data of table 1 synthetic cyclic peptide

Embodiment 2: suppress testosterone effect experiment in rat body

Animal (SD male rat) body weight is claimed before experiment, glass capillary ball rear vein beard is taken a blood sample, chemiluminescence determination Testosterone Content in Serum is used after separation of serum, by testosterone concentration and body weight random packet, often organize 4 animals, respectively at the injection of disposable excess subcutaneous or gavage testing compound (various dose) 8,16,24 hours etc. afterwards, ball rear vein beard is taken a blood sample, the centrifugal 8min of 5000rpm, measures Testosterone Content in Serum by being separated the serum chemoluminescence method (U.S. BeckmanCoulter company AccessImmunoassaySystem Chemiluminescence Apparatus) obtained.Concrete outcome refers to table 2, can find out that compound of the present invention effectively can suppress the testosterone activity in rat body.

Embodiment 3: to GnRH(gonadotropin releasing hormone) acceptor antagonistic activity measure

Adopt and the detection of GnRH function of receptors is carried out to the fluorescence dye of calcium ion sensitivity, after GnRH acceptor is by agonist agonizes, the release of calcium ion in endoplasmic reticulum can be caused, calcium ion can be combined with fluorescence dye and make it fluorescence intensity and strengthen, fluorescent signal show more by force part or agonist activity stronger, otherwise part or agonist activity more weak.Because antagonism compounds energy is competitive and GnRH receptors bind, cause the release of calcium ion in endoplasmic reticulum to reduce, fluorescent signal also decreases.The inhibit activities of compound to be detected to GnRH acceptor is characterized according to the degree that signal lowers.

CHO-K1/G α 15 cell (purchased from GenScript) of stably express GnRH acceptor is inoculated into 384 microwell plates, cellar culture, goes down to posterity.Add fluorescence dye, subsequently, by the testing compound solution that configures, (detectable level is: 1e-9M; 1e-10M; 1e-11M; 1e-12M; 1e-13M; 1e-14M; 1e-15M; 1e-16M; Corresponding DMSO contrast is 1%v/v.) be added in cell plate, hatch rear calcium ion fluorescence detection reagent kit to detect, instrument overall detection time is 120 seconds, automatically LHRH was joined the 21st second time in cell plate and detect (reference: JackJ.Chenetal, IdentificationofanewclassofsmallmoleculeC5areceptorantag onists, Bioorganic & MedicinalChemistryLetters20 (2010) 662 – 664).

Data acquisition and issuance uses Excel and GraphPadPrism4 software program.For each detect aperture, using the average fluorescent strength value of 1 to 20 seconds as baseline, the maximum fluorescence intensity value of 21 to 120 seconds deducts baseline value and is relative intensity of fluorescence value (△ RFU), can calculate suppression per-cent according to this numerical value.Use GraphPadPrism4 quadruplex parameters to process data, thus calculate EC50 or IC50 value.Concrete outcome refers to table 2, can find out that compound of the present invention has the antagonistic activity to GnRH acceptor.

Embodiment 4: Determination of oil-water partition coefficient measures

Take about 0.5mg sample (testing compound), use 0.5mL water dissolution, high speed centrifugation, get supernatant liquor and carry out HPLC detection; The retention time t of compound in different ratios methyl alcohol/phosphate buffered saline buffer (pH=7.4) is measured with RP-HPLC _r(retention time namely in table 2), uses NaNO ₂solution surveys dead time t ₀, rated capacity factor k=(t _r-t ₀)/t ₀.Being X-coordinate with methanol content, is that ordinate zou makes curve with logk, must indulge and cut a square logD _oct(being equivalent to the logk of compound when methanol content is 0) is Determination of oil-water partition coefficient value (see F.Lombardo, M.Y.Shalaeva, K.A.Tupper, etal.ElogPoct:atoolforlipophilicitydeterminationindrugdi scovery.Journalofmedicinalchemistry, 2000,43(15): 2922 ~ 2928; F.Lombardo, M.Y.Shalaeva, K.A.Tupper, etal.ElogDoct:Atoolforlipophilicitydeterminationindrugdi scovery.2.Basicandneutralcompounds [J] .Journalofmedicinalchemistry, 2001,44(15): 2490 ~ 2497).Concrete outcome refers to table 2, can find out that cyclic peptide compounds of the present invention has higher lipotropy than linear peptides contrast.

Embodiment 5: estimation of stability: the transformation period in simulated intestinal fluid measures

By (loyal and upright see money to the testing compound aqueous solution (0.2mg/mL) and simulated intestinal fluid, flush, kingdom is flourish. Chinese Pharmacopoeia second annex [M]. and the Chinese drug standard, 2005.158) equal-volume mixing, hatch in 37 DEG C of constant temperature oscillators, different time points (0,10,20,40,60,90,120min) take out, 10% trifluoroacetic acid (TFA) termination reaction; High speed centrifugation (10000r/min) 10min, gets supernatant liquor and carries out HPLC analysis, by its concentration of calculated by peak area.With time t for X-coordinate, the denary logarithm (logc) of concentration of substrate is ordinate zou mapping, obtains logc=-at+b, transformation period t _1/2=0.3/a.Concrete outcome refers to table 2, can find out that compound of the present invention has higher enzyme stability.

Table 2

A: subcutaneous administration, 500ug/kg, detected testosterone concentration after 8 hours.What be suppressed to below 1ng/mL is complete castration.Be less than control group testosterone concentration, but do not arrive 1ng/mL for part castration;

Measure under b:77% methyl alcohol/phosphate buffered saline buffer (7.4) condition.Aglient1200,4.6*150mmSB-C18 post.

Wherein Degarelix is a kind of commercial gonadotropin releasing hormone for prostate cancer hormonotherapy (GnRH) blocker, and testosterone levels can be caused to continue to reduce.

Although the specific embodiment of the present invention has obtained detailed description, it will be understood to those of skill in the art that.According to disclosed all instructions, can carry out various amendment and replacement to those details, these change all within protection scope of the present invention.Four corner of the present invention is provided by claims and any equivalent thereof.

Claims (12)

1. the compound shown in formula I, or it is without the salt of physiological-toxicity,

Wherein,

Xaa ³for D-Phe or D-Pal;

Xaa ⁴for L or D type Cys;

Xaa ⁵for L or D type Aph (Hor) or Arg;

Xaa ⁶for L or D type Aph (Cbm) or Pal;

Xaa ⁸for Ilys or Arg;

Y is L or D type Cys-Ala or Pro-Cys, and wherein two amino acid can be L-type or D type independently of one another;

Wherein by the Cys side chain thiol of 4 and Y with disulfide linkage Cheng Huan.

2. the compound of claim 1, is characterized in that being selected from following one or more:

(1) Xaa ³for D-Pal;

(2) Xaa ⁴for Cys;

(3) Xaa ⁵for D-Aph (Hor);

(4) Xaa ⁶for D-Aph (Cbm); With

(5) Y is Cys-D-Ala or Pro-D-Cys.

3. the compound of claim 1, it is selected from following compound:

4. pharmaceutical composition, it contains the compound of any one of at least one claim 1-3, and optional pharmaceutically acceptable auxiliaries, carrier or vehicle.

5. the compound of any one of claim 1-3 is for the preparation of preventing and/or treating sex hormone related condition or for the purposes in the medicine of practising contraception.

6. the purposes of claim 5, wherein said sex hormone related condition comprises sexual hormoue associated cancer, precocious puberty, hyperplasia of prostate, endometriosis, menoxenia or Infertility.

7. the purposes of claim 6, described menoxenia is selected from amenorrhoea, dysfunctional uterine bleeding or ovulation failure.

8. the purposes of claim 6, wherein said sexual hormoue associated cancer comprises prostate cancer, carcinoma of endometrium, mammary cancer or ovarian cancer.

9. the purposes of compound in the medicine for the preparation of suppression pituitary gonad-stimulating hormone and/or inhibition glandular secretion steroid hormone of any one of claim 1-3.

10. the purposes of claim 9, described pituitary gonad-stimulating hormone is LH and/or FSH.

The purposes of 11. claims 9, described sexual gland secretion steroid hormone is oestrogenic hormon, progestogen and/or testosterone.

The preparation method of the compound of 12. any one of claim 1-3, is characterized in that:

Take mbha resin as carrier, Boc-or Fmoc-Preservation tactics, DCC/HOBT or BOP/DIEA is condensation reagent, and HCl/ dioxane or piperidines/DMF are deprotecting regent, after amino acid condensation completes, use I ₂remove the Trt protecting group on sulfydryl, intramolecular two sulfhydryl oxidases are with disulfide linkage Cheng Huan simultaneously, cut down by Cyclopeptide derivatives after having reacted with liquid HF from mbha resin.