LHRH-ANTAGONISTS

Abstract: This invention consists of two aspects: 1) the method of design and synthesis of LHRH antagonists; 2) the products thereafter obtained by using the above method. Taking (NAc-D2Na1<1>, DpClPhe<2>, D3Pal<3>, Ser<4>, Tyr<5>, DArg<6>, Leu<7>, Arg<3>, Pro<9>, DAla<10>)NH2 as the parent compound, a series of new analogs expressed as (NAc-D2Nal<1>, AA<2>, AA<3>, Ser<4>, AA<5>, AA<6>, Leu<7>, AA<3>, Pro<9>, DAla<10>)NH2 are obtained by fine modification of both lipophilic area and alkaline area of the molecule of the parent compound. In this way, the high antiovulatory activity of the parent compound can be maintained and the histamine releasing activity can be reduced to the level so as to meet the clinical requirement.

Description: LHRH-ANTAGONISTS t Sped f 1 cat i o n The products and their application of method for design and synthesis of Iute1n1z1ng hormone releasing hormone antagonists The present 1nvet1on relates to novel peptides and their derivatives having exact chemical structure. The Invention 1s also directed to the methods of their preparations and applications. Hypothalamlc lutelnlzlng hormone releasing hormone (LHRH) acts on the anterior pituitary gland to stimulate the secretion of lutelnlzlng hormone (LH) and folllcular stimulating hormone (FSH) . The antagonistic analog of LHRH acts on anterior pituitary rapidly, lasts a long duration, can be safely and reverslbly used for contraception or selectively suppression of gonadotropln secretion. For such kind of application, LHRH antagonists are superior to agonists. Up to now, there are more than two thousands of LHRH analogs have been designed and synthesized, among which "Nal-Arg" analog showed fairly high ant1 fertl 11 ty activity. However, "Nal-Arg" analog showed also very strong hi staml ne-rel easl ng activity (HRA) . It caused transient edema of the face and extremities in rats when administrated at a dosage as high as 50-100 times of therapeutic dose. The result of clinical trial demonstrated hi staml ne-rel ated systemic effects. Other LHRH antagonists containing DArg 6 or DLys 6 showed similar side effects, their ED 50 for HRA were below SUBSTITUTE SHEET 1 μg/ml. The present invention provides new LHRH antagonists which have very high antiovulatory activity (AOA) and very low hi staml ne-rel easi ng activity HRA) and negligible side effects. The contents and examples of this Invention are as fol 1 ows : The design methodology of this Invention- 1s based on the topologlcal similarity between the molecule of parent compound [NAc-D2Kal 1 , DpClPhe 2 , D3Pal 3 , Ser 4 , Tyr 5 , DArg 6 , Leu 7 , Arg 8 , Pro 9 , DAla 10 ]NH 2 (ID and a neoropeptl de , Substance P, which features the modification of both alkaline and lipophilic area in t molecular of the parent compound to give new antagonis having both high AOA and low HRA. The term "modification" hereof 1s adjusting or substitution of the amid adds in he area of Tyr 5 -DArg 6 -Arg 8 in C-term1nus and the aromatic acids 1n N-term1nus of (II More specifically, the design 1s Introduction of suita alkaline group and substitutions of unnatural amino aslds 1n position 2, 3, 5, 6, 8 of (ID. The following are also the methods and examples of this 1 nventi on . 1. Substitution of D3pal which is a aromatic amino add having suitabele basicity for DArg 6 in (II) t obtain analog (III): [NAc-D2Nal - , DpClPhe 2 , D3Pal 3 Ser 4 , Tyr 5 , D3Pal 6 , Leu 7 , Arg 8 , Pro 9 , OAla 10 ]NH 2 Substitution of Arg 5 for Tyr 5 In (III) to obtain (IV): [NAc-D2Nal *, DpClPhe 2 , D3Pal 3 , Ser 4 , Arg 5 , D3Pal 6 , Leu 7 , Arg 8 , Pro 9 , DAla 10 ]NH 2 SUBSTITUTE SHEET Substitution of Dphe 3 or Its derivatives DXCH 2 Phe for D3pal 3 In (IV) to obtain (V) : [ NAc-D2Nal x , DpClPhe 2 , DPhe 3 , Ser 4 , Arg 5 , D3Pal 6 , Leu 7 , Arg 8 , Pro y , DAla 1 l 0 u ]NH 2 or Its (DXCH 2 Phe°) analogs Substitutions of Dphe 3 or Its derivates for D3pal 3 1n (III) to obtain (V): [ NAc-D2Nal * , DpClPhe 2 , DPhe 3 , Ser 4 , Tyr 5 , D3Pal 6 , Leu 7 , Arg 8 , Pro 9 , DAla 10 ]NH 2 or Its (DXCH 2 Phe 3 ) analogs. A series of new LHRH antagonists of the formula [NAc-D2Nal 1 , AA 2 , AA 3 , Ser 4 , AA 5 , AA 6 , Leu 7 , AA 8 , Pro 9 DAla 10 ]NH 2 have been synthesized, where AA are natura or unnatural amino adds which are expressed as D- or L-ArAla. More specifically herein, AA' D-pClPhe, D-ArAla, DPhe, Ar-Ala, DXCH 2 Phe; AA 3 = D3Pal, Ar-Ala, D-ArAla, DPhe, D-XCH 2 Phe; AA 5 - Arg, DMap, P1p, Tyr, Pal , Mop, Tep, Map, Phe, Eap, Pap, Bap, DMop; AA" D3Pal , D-Ar-Ala, D-XCH 2 Phe; AA ( P1p, Mop, Tep, Map, Eap, Pap, Bap, Arg; in which SUBSTITUTE SHEET - H C 1 n whi ch and R' 2 N-, RR'N- 1 n whi ch R — CH3— i CH CH — , C Hy— , C^Hg— , H— j R — CH - , CH CH 2 — , C Hy—, CiH - , H— ; The LHRH antagonists obtained by using the above described method, as a kind of peptide medicine, can be used to treat the disorder of reproductive endocrine system, such as edometri osi s , precocious puberty of childeren and to treat prostate cancer and SUBSTITUTE SHEET breast cancer as well as used as male or female contraceptives for birth control, or used 1n the diagnosis and treatment of 1ntert1l1ty, etc. Such peptide medicine can be- prepared as normal Injection Injectable capsules or other formul atat1 ons for real appl 1 catl on . Further description of this inventation is as follows: In the natural course of histamine releasing in the body, neuropeptide substance P plays a very Important role, Its ED 50 for HRA is 5-15 μM. The chemical structure of SP is [Arg 1 , Pro 2 , Lys 3 , Pro 4 , Gin 5 , Gin 6 Phe 7 , Phe 8 , Gly 9 , Leu 10 , Met 11 ]NH 2 . The study on the relationship between its structure and HRA showed that Arg 1 -Pro 2 -Lys 3 1n the N-term1 nus 1n the molecule of SP 1s essential for Its HRA because deletion of these three amino adds from the molecule entirely abolished Its HRA. By contrast, deletion of one two or three amino adds 1n C-terminus remained HRA as high as one fourth as HRA o Itself. Further deletion of Phe 8 and Phe 7 , HRA reduced 4 % and 0,57 % of those of SP. Further deletion of Gin 5 -6 did not cause significant change of HRA. The above data Implies that the !1phoph1l1c area around phe 7,8 determines the value of HRA, this area Involves in the binding of molecule with the receptor of mast cell. As mentioned previously, (D2Nal 1 , DArg 6 ) analogs of LHRH showed very high HRA, its molecular structure has topological similarity with SP: DArg 6 -Leu 7 -Arg 8 In the former appears to be corresponding to Arg 1 -Pro 2 -Lys 3 in the latter, both consist of a pair of strongly basic amino add residues between which only one neutral amino add residue 1s present, 1. e. both [D2Nal 1 ,DArg analog of LHRH and SP contains two SUBSTITUTE SHEET strongly basic amino add residues which are In 1,3 posltlton relation ship. On the other hand, a clutter of aromatic amino add residues 1n the former is considered to be corresponding to Phe 7*8 area In SP 1n terms of determination of he magnitude of HRA. The design of this invention consists of two aspects: one 1s modifying Tyr 5 -DArg 6 -Arg 8 area in C-term1nus, the other is fine adjusting the aromatic adds after optimizing the modlficatin of the alkalious area in C-term1nus. [NAc-D2Nal x , DpClPhe 2 , D3Pal 3 , Ser 4 , Tyr 5 , DArg 6 , Leu 7 , Arg 8 , Pro 9 , DAla 10 ]NH 2 (II) 1s used as parent compund, which showed AOA 100 % at 0,5 μg 1n coin oil, 57 % at 0,25 μg. First, DArg 6 1n (ID could be replaced by weekly basic or neutral aromatic adds, such as D3Pal , D6Qal , tetrahydrotryptophan, methyl tryptophan. [NAc-D2Nal 1 , DpClPhe 2 , D3Pal 3 - 6 *, DAI a 10 1LHRH ( III ) was optalned when D3Pal 6 was substituted for DArg 6 1n (ID. (Ill) showed AOA 100 % at 3 μg, 83 % at 1 μg (1n corn dl), and Its ED 50 for HRA was 9.8 μg/ml, much better than that of "Nal-Arg" analog ED 50 for HRA was less than 1 μg/ml. It seems that the basicity of the whole molecule should equal to or closed to that of a pair of arginlne in order to obtain high AOA. Because position 5 , like position 6, does not involve in the receptor binding, a wide variety of amino add including arglnine can be Inserted in position 5. A series of new analogs were designed. For example, substitution of Arg 5 for Tyr 5 In (III) gave [NAc-DNal 1 , DpClPhe 2 , D3Pal 3 - 6 ', Arg 5 , DAla 10 ] LHRH (IV) . Both (IV) and (ID contained two arglnlnes, but the distance between two arginines in (IV), whose geometric relationship became 1, 4, 1. e. there were two other amino acids between these two SUBSTITUTESHEET arglnlne, was larger than that 1n (ID . Therefore, HRA would be reduced and, on the other hand, because of th presence of two arglnlne, AOA should not be lower than that of (II) . The bloassay result of (IV) showed that ED 50 for HRA was 3,5 μg/ml , while AOA was 60 % at 0, 12 μg (corn oil) , 85 % at 0,25 μg, 100 % at 0,5 μg . This was the first time for LHRH antagonists to achieve ED 50 for AOA which was equal or less than 0,125 μg. Therefore further design was based on the structure of (IV) . There are four alkallons residues, D3Pal 3 ' 6 and Arg 5 ' 8 1n the molecule (IV) , while (II) contains only three alkallnous. Therefore, 1t 1s resonable to replace one D3Pal by a neutral amino add; on the other hand, (IV) showed very strong hydrophi 11 d ty and reducing the hydrophi 11 d ty by the substitution of a hydrophoblc amino add for DPal 1n (IV) would be beneficial to the retention of the drug 1n the body and then to the extension of the effective duration. A new series of analogs were then designed by substitution for D3Pal 3 . (V) showed 100 % of AOA at 1 μg (1n saline) , equal to that of parent compund (IV) , while HRA redued by a half: EDJJQ for HRA was 7,4 μg/ml . Further substitution of DPhe 2 for DClPhe 2 reduced the 11 pophi 11 ci ty of this area in the molecule and reduced HRA Arg 5 -D3Pal 6 -Leu 7 -Arg 8 1n the C-term1nus of (IV) seems to play a major role 1n triggering histamine releasing. D3Pal combines aromadty, basicity and hydrophi 11 d ty 1n one molecule, 1t 1s also stero-acceptabl e 1n LHRH antagonists for receptor binding. S1m1l1arly, design of new series of unnatural amino acids prosessing the same SUBSTITUTESHEET character as D3Pal may lead to better LHRH antagonists that (IV) or (V). Modification of natural, lipophilic, aromatic amino add e. g. phenyl al ani ne, for example, by means of the method described below in The Synthels of Novel Unnatural Amino Adds, lead to a series of novel amino adds which combine aromadty, hydrophi! 1 d ty and basicity 1n one melecule and can expressed the as formula: R 1 R NCH 2 C 6 H CH 2 CH(NH)C0 2 H (VI), were R j and R 2 may be the same or may differ each other, may be chain-like or cyclic, may also contain hetero-atom. With the R j and R 2 change, a series of amino acid can be obtained, which show systemically changed basicity, hydrophi 11 dty and stero-character . Introduction of those amino adds In position 5, 6, 8 of (IV) have given three series of new antagonists of LHRH. The bloassay results showed that each series gave at least one new antagonist showing 100 % AOA at 1 μg, similar to that of- (IV), while HRA was si nificantly reduced. An example was (VII): [NAc-D2Nal 1 , DpClphe 2 , D3Pal 3 , Ser 4 , Mop 5 , D3Pal 6 , Leu 7 , Arg 8 , Pro 9 , DA!a 10 ]-NH 2 , which showed 100 % of AOA at 1 μg, ED 50 14,7 μg/ml for HRA, appeared better than (V). When substitution of (VI) for Arg 8 In (IV), the extent of HRA decreae was positively correlated to the length of R In (VI), so ED 50 for HRA could be higher than 200 μg/ml, such kind of compounds can be easily dissolved in aqueous solution and expected to be utilized clinically without formulation problems. The results demonstrated that Arg 8 or Lys 8 was not essential for hi ghly. potent LHRH antagonists. Suitable alkaline center 1n position 8 can ensure high AOA, meanwhile activity inducing mast cell to release histamine was remarkablely reduced when the basic enter mentioned above posseslng significant stero-hi nder. SUBSTITUTESHEET s This Invention combining the modification In both N- and C-term1nus lead to better LHRH antagonists. The process of synthesis are Illustrated as follows: 1. The synthesis of Novel Unnatural Amino Adds Over 60 series and nonserles, D- or L-am1no adds are designed and synthesized through the four synthetic routes outlined 1n the schema below. The structure of these unnatural amino adds are shown with the general structural formulas listed In the same schema. Some of these amino adds have alkalinity, hydrophi 11dty and aromatldty respectively, while the others have them all together 1n the molecule. SUBSTITUTESHEET ID Path 1 Path 2 ArNH- ArCH 2 Cl + HC(COOEt) 2 HCl NH-Ac ArN 2 Cl ArCH-,C(C00Et)p I CH 2 =CH00H NH-Ac NaOH/EtOH d,l- ArCH d,l- ArCH 2 CHC00H Ac 2 0 COOH Ar-CH -C-COOEt I NH-Ac d,l- ArCH 2 CHC00H NH-Ac S0U 2 /Et0H SUBSTITUTE SHEET d,l- ArCH CHCOOEt I NH-Ac d-ArCHn d-ArCH 2 d-ArCHpCHCOOH 1- ArCH,CHCOOH I NH-Boc NH-Boc SUBSTITUTESHEET /I N H-Bo c Path I D or L-Ar-CH2-CH-C00H wherein • ©- Path II D or L-ArCH2-CH-C00H wherein SUBSTITUTE SHEET SUBSTITUTE SHEET IV NHCOCH3 EtOH Path III SOCl 2 NHCOCH3 Chymotrypsi n g NHCOCH- SUBSTITUTE SHEET NHCCH- ■ CH 2 CHCOOEt HC NHCOCH- d- XCH- HCl Secondary EtOH d- XCH 2 - 1 -XCH 2 - ~CH 2 CHC00Et NH-Boc NHCOCH 3 HCl 1) dlcarbonate I 2) H + -H,-© -^..... NH-Boc wherein X = - 0 N- N , R 2 N- , RR'N- SUBSTITUTE SHEET IL HCO- Ar-CH0+C 6 H 5 -C0-NHCH 2 C0 2 H Ar-CH=C-C Ac 2 0 \ Path IV N = C \ C 6 H 5 coned. HCl Ar-CH=C-CO->H Ar-CH 2 -CH-C0 2 H I Pd/C NH-C0-C 6 H 5 NH-CO-C 6 H 5 SOC1 subti 11 si n Ar-CH 2 CHC0 2 CH 3 MeOH | pH 7,6 NHC0C 6 H 5 Ar-CH 2 CHC0 2 CH 3 Ar-CH 2 CHC0 2 H NHC0C 6 H 5 NHC0C 6 H 5 D-1 somer L-i somer I 6NHC1 I 6NHC1 Ar SUBSTITUTE SHEET Path IV D or L Ar-CH 2 -CH-C00H, wherein NH, Y = (CH 3 ) 2 N-, (CH 3 CH 2 ) 2 N-, ( CH 3 CH 2 CH 2 ) 2 N- , N-, (CH 3 CH 2 CH 2 CH 2 ) 2 N- SUBSTITUTE SHEET IS 2. Synthesis of Peptide The synthesis begins from the C-term1nus of the peptide on benzhydryl ami ne hydrochloride resin (BHA resin) utilizing the method of solid-phase peptide synthesis Introduced by Merr1f1eld. It is a three-step process Including anchor, coupling and cleavage. D1 chl oromethane (DCM) is the major solvent used for washing between each step of reaction while isopropanol alcohol (IPA) and N, N-d1methyl formami de (DMF) are also used when it is necessary. Catalyzed by excessive d1 cycl ohexyl carbodl 1ml de (DCC), coupling reaction is carried out, while adequate amount of 1-hydroxybenzotr1azol e (HOBT) 1s added. The degree of the coupling reaction 1s monitored with alses nlnhydrln method. The second coupling reaction would be carried out 1f it gives a positive result in Kalses test. The peptide chain Is cleaved from the resin using anhydrous hydrogen fluoride (HF) in the presence of an1 sole after the completion of all reactions necessary on the resin, all of the temporary protecting group are deprotected at the same time. After washed by ethyl acetate or ether crude products of LHRH antagonists are obtained by aqueous acetic add extraction followed by lyophi 11 zati on . The yield is over 50 %. Purification of Peptide (1) The peptide 1s purified by gel permeation chromatography or silica partition chromatography through a column as high as 60 - 100 cm with the aid of UV/TLC monitoring. The LHRH antastlts SUBSTITUTESHEET IS purified once are obtained after lyoph1liz1ng the major fractions. The yield 1s 50 - 90 % and the purity can be over 90 %. (2) The peptide then is further purified on Waters high performance liquid chromatography (HPLC) instrument using reverse phase C18 column (7,8 x 300 mm) (μ-Bondapak 84176) . The yield of this step 1s 20-50 % while the purity is no less than 99 %. 4. Purity Analysis of Peptide (1) TLC analysis It 1s carried out on a plastic sheet coated by silica gel 60 F254 of 5 - 10 cm height. They all shows a single spot when developed 1n five different solvent systems. (2) HPLC analysis They all shows a single peak when eluted with two kinds of solvent system, respectively, utilizing Waters HPLC Instrument on a analytic column (μ-Bondapak 27324) when monitored by UV 210. The sample size is 10 - 200 μg. 5. Amino Add Analysis of Peptide: According to the PICO-TAG method developed by Waters Company, 50 μg of sample which have been dried under vacuum over 2 hours is weighed accurately on a 10~ g scale balance. After dissolved in water, 10 μg of aliquot 1s added to a reaction tube In which 1:1 hydrochloride add (containing 1 % phenol ) was added according to the manual . SUBSTITUTE SHEET The reaction lasts 22-24 hours at 105°C in a sealed container which had been filled with nitrogen and pumped to vacuum to remove the oxygen In reaction tube. Phenol 1sothl ocyanate 1s added to derive the amino group after evaporating of excessive hydrochloride add. Then it was analyzed with the HPLC-1 nstrument equipped with PICO-TAG amino add analytical column and monitored by UV254. The content of each amino acid and the relative mole ratio were calculated to give the amino add composition of the sample based on the comparison of the Integrated area of each amino add to that of H-standard sample of Waters. The classical 1 on-exchange-ni nhydri n derivation method (IEN) was also used as control which gave the same results. But 1t needed ten times more sample to get a satisfied result. 6. Evaluation of biological activity: Corbln's rat antiovulati on method is used. The healthy, adult, female SD rats (BW 200-250 g) are used in this experiment. All animals are maintained at 22-24 °C and on 14 h/10 h (light/dark) schedule. They are given standard food, and water ad libitum. The rats showing at least two consecutive 4-day estrous cycles in vaginal smear examination can be used in this experiment. The rats are given peptides (LHRH antagnists) at noon of proestrous with different dose 1n saline solution. The rats are sacrificed next day, their oviduct of two sides are exa ed under a dissecting microscope to determine the ovum number. The rats were divided into several groups according to the dosing, each group SUBSTITUTE SHEET consists of about 10 rats, and the control group in which the rats are given equal amount of saline consists of 9-10 rats. The antiovulatory activity (AOA) 1s shown in the following equation: number of unovulated rats A0A= X 100 % Total number of treated rats Evaluation of Histamine Releasing Activity: (1) . Histamine releasing test (HRT) in vitro The healthy, adult, male SD rats (BW 200-250 g) housing in the above same conditions are used in this experiment. After anesthetized by C0 2 the peritoneal cavity 1s washed with 50 ml of PIPES AC medium containing 20 units of heparin. Following centr1 fugatl on at 200xg for 8 min at 4 °C, cells are washed again and finally resuspended to a concentration of 8 to 24xl0 5 total 1 eucocyted/ml in PIPAS AC. This suspension contains approximately 5-10 % mast cells. Washed cells are used Immediately after collection and are prewarmed for 5 m1n at 37 °C prior to pipetting 0,3 ml aliquots into polystyrene tubes containing 0,3 ml of diluted peptide. The mixtures are Incubated for 15 min at 37 °C and the reaction stopped by centr1 fugati on at 400 xg for 15 min at 4 °C. The supernatans are assayed for histamine content by manual fluorometric assay method after successively extraction with n-butanol and n-heptane. The histamine content can be obtained from the histamine standard curve (see below) . The percentage of histamine release can be calculted from the following equation: SUBSTITUTE SHEET 2--. E-B H i s tam i n e r e l eas e ( % ) = X 100 % C -B where E is the fluorometric reading of experimental sample, B 1s the fluorometric reading of samples with cells and buffer only, and C 1st the fluorometric reading of "complete" (cells treated with HCI0 4 ) . The standard curve can be obtained by plotting the OD values on a fluorometer at 350 nm/450 nm (activation/fluorescent) against the concentrations of serially diluted solution of accurately weighted histamine hydrochloride. The relative parameter r of the histamine standard curve can be 0.9998, and the lowest detectable concentration of histamine is 0,5 ng/ml . The ED 50 value of peptide can be gotten from the dose response curves obtained by plotting the histamine release versus the peptide concentration on semi 1 ogari hmi c paper. All peptide samples should be tested with mast cells from a minimum of 3 different rats. (2). Cutaneous anphylactoid activity test (CAT): The healthy, adult, female SD rats (BW 250 g) are used 1n this experiment. The rats are injected SUBSTITUTE SHEET ~Z Intravenously with Evan's blue (1ml of 0,05 % solution). Immediately after that, the 0,05 ml of peptide solution (5, 0,5 and 0,05 μg/ml respectively) and saline (control) are Injected 1 ntradermal 1y Into a shaved section on the back of the animals. 30 minutes after the injection, the rats are sacrificed and the dorsal skin was reflected. The diameters of the lesions are measurd in millimeters 1n two perpendicular directions with a vernier caliper. The diameter of control 1s usually less than 5,5 mm. The amount of Evan's blue permeating Into the skin from the blood vessel can be spectrophometrl cal ly measured, too. The skin corresponding to the lesion area is cut down and Immersed 1n a mixture of acetone/saline (7:3, Vol/Vol) overnight. After centr1 fugatl on next day, the content of Evan's blue 1 n the supernatant 1s measured with a spectrophotometer (UV-260) at 610 nm against reference solutin of acetone/saline (7:3). Each peptide were tested in a minimum of 3 different rats . A variety of new LHRH antagnlsts were designed and systheslzed by means of the method described above. In brief, the new structure of LHRH antagonists was obtained by single or multiple substitution of the various natural and unnatural amino acids listed in the previous paragraphs. A part of examples of new LHRH antagonists obtained thereupon are illustrated in table 1 SUBSTITUTESHEET Table 1 : A Part of Examples Related to This Invention Analogue AA 1 AA 2 AA 3 AA8 AA9 AA 10 Parent NAc-D2NaDpCl Phe D3Pal Arg Pro DA1a-NH2 P1 p P1p Mop DPip P 1 p CO Pap c O P 1 p CO Pap H C Eap H rπ Mop O X DPhe Map m m DPClPhe Map H DPhe Mop Pep Mop DpFPhe D3Pal Mop DPhe Eap Pap Table 1 : A Part of Examples Related to This Invention, Page 2 Analogue AA 1 AA 2 AA 3 Parent NAc-D2NaDpC1 Phe D3Pa1 DPClPhe DPClPhe DPhe DFPhe DFPhe DFPhe Hep The Applications of This Invention: 1. After finishing the prec!1n1cal pharmacology and toxicology study, we can apply these new LHRH antagonists which have high therapeutic effectiveness and low side effect In clinic so as to develop new medicine for treating the endometrl os1 s and their disorder In reproductive endocrine system including precious puperty of children, prostate cancer and breast cancer. Since they suppress the secretion of gonadotropln through competing receptor with endogenous LHRH, and act rapidly reverslbly and safely, they can be further developed as new type of contraceptives for male or female. Besides, they can be also used In treatment of Infertility and for selectively and reverslbly abolishing the function of pitutary gland 1n terms of secreting gonadotropi n . Being a kind of peptide medicine, the LHRH antagonists desrcibed herein are unlikly to be administrated orally. But they can be easily made into lyophilized powder which are ready to dissolve 1n saline for Injecting iv, sc or im. Morover, long-actin delivery systems, such as biodegradable, Injectable capsules are studied. The capsules can be implanted subcutaneously by a special syringe and would be adsorbed by the tissue after release of all peptide contents and do not need to remove surgically. The long-acting delivery system 1s specially useful for long-term admi nstrati on of LHRH analogues 1n clinic. SUBSTITUTE SHEET The following are the analyses results of the examples (taking three analogues IV, V, VII as typical exampl es) : (1) The Purity Thin layer chromatography (TLC): There is only a single spot 1n each of the chromatogram developed in five different solvent systems . High performance liquid chromatography (HPLC): There 1s only a single peak 1n each of the chromatogram eluted with two different sovent systems . The values of Rf and retention time TR are shown 1n Table 2, also with reference of Figure 1-4. SUBSTITUTE SHEET Table 2: The chromatographic analysis results of LHRH antagonists HPLC TLC Analogs TR1 TR2 Rfl Rf2 Rf3 Rf4 Rf5 (min) IV 7.55 5.26 0.23 0.21 0.31 0.19 0.65 V 7.90 8.11 0,32 0.30 0.35 0.30 0.69 VII 16.19 9.58 0.17 0.08 0.16 0.40 0.12 Solution A + 80 % acetonltrlle Solvent System 2: Solution A 1s 0.01M KH 2 P0 4 aqueous solution (pH3) Solution B is 20 % solution A + 80 % acetonltrlle TLC solution system: 1. nBu0H/Et0AC/H0AC/H 2 0 (5:5:1:1) 2. nBu0AC/nBu0H/H0AC/H 2 0 (2:8:2:3) 3. nBuOAC/HOAC/H 2 0 (4:1:5), up phase 4. nBu0H/H0AC/H 2 0 (4:1:2) 5. nBu0H/Et0AC/H0AC/H 2 0 (1:1:1:1) SUBSTITUTE SHEET (2) Amino add analysis The analysis are carried out according to the method of classical IEN and new Pico-Tag, the results are shown in Table 3 and Figure 5,6. Table 3: The amino acid composition of LHRH antagonists ND: Not determined SUBSTITUTE SHEET 3o (3) The bioassay results The results of bioassays including antiovulatory activity at different doses and ED 50 for hi staml ne-real 1 s1 ng activity in vitro are illustrated 1n table 4 in which 26 antagonists are listed as exampl es . SUBSTITUTESHEET Table 4: Bioassay Results of New LHRH Antagonists based on Parent structure % AOA/μg HRA (μg/ml ) Subst1 tuted Ami no Ad ds 0.125 0.25 0.5 1.0 2.0 ED 50 ± SEM 17 The parent structure 1s: [NAc-D2Nal 1 , DpClphe 2 , D3Pal 3 , Ser 4 , Arg 5 , D3Pal 6 , Leu 7 , Arg 8 , Pro 9 , DAla 10 ]NH 2 SUBSTITUTE SHEET As Illustrated and described above, the LHRH antagonists designed and synthesized according to this Invention shows very good properties. They are pure in TLC or HPLC analysis. They are pure 1n TLC or HPLC analysis. Their compositions are correct, i. e., the same 1s designed. Their antlfertlle activity is high: they can Inhibit rat ovulation when Injected s. c. at the dosage of 0.1 to 2.0 μg at the noon of proestrus. Their histamine related side effect is low: their ED 50 for in vitro histamine releasing activity (the effective dose for rat mast cell to release 50 % of histamine) is ranged 5-300 μg/ml ,* the lesslon induced by them In the cutaneous anaphylact d test in rats 1s as small as required in clinic. Their warer-solub111ty is very good, all bioasays are carrld our in saline solution, so they are easy to formulated for Injection In clinic. They are also ready to formulated as long-acting delivery systems, among which injectable mi crocapsul es are most convenient for long-term suppression of gonadotropln and gonadal hormone. Therefore, they can be used as highly effective, reversible and safe contraceptives for both male and female. They can be also utilized for treatment of various diseases related to disorders of reproductive endocrine such as hormonedependent prostate cancer and breast cancer, endometr1 osi s , precoslous puberty of children. They are also useful in treatment of infertility. The new LHRH antagonists herein can also be utilized in the basic research of reproductive physiology and pharmacology, such as in the study on the function of plturtary gland, on the effect of gonadal hormones or gonadotrpins or LHRH on sexual behaviour, etc . SUBSTITUTE SHEET ABBREVIATIONS The following are abbreviations which have been used In the text of this patent application document. Ala al an1 ne AOA antiovulatory activity Arg argl n1 ne Bap dibutyl ami nomethyl phenyl al ani ne Boc t-butyloxyl carbonyl BuOAC butyl acetate CAT cutaneous anaphal actol d test DCC dicyclohexylcarbodi 1mide DCM d1 chl oromethane D2Nal D-β-(2-naphthyl ) alanine D3Pal D-β-(3-pyr1dyl ) alanine DpClPhe p-chloro-D-phenylalan1ne DpFPhe p-f l uoro-D-phenyl al an 1 ne D6Qal D-β- ( 6-qu1 nol yl ) al an i ne DMF N,N-d1methyl formamlde Eap di ethyl ami nomethyl phenyl al an1 ne ED 50 effective dose for 50 % response EtOAC ethyl acetate FSH fol 11 cl e-st1 mul at1 ng hormone Gl u gl utaml c add Gly glyd ne His hi st1 d1 ne HOBT l-hydroxybenzoltr1azole HPLC high performance liquid chromatography nlnhydrin derivation HRA hi staml ne-rel easi ng activity HRT hi stami ne-rel easi ng test IEN 1on exchange chromatography with post-col umn SUBSTITUTE SHEET 3-1 IPA isopropyl alcohol LH lutelnlzlng hormone LHRH lutelnlzlng hormone releasing hormone Leu leudne Lys lysine Map dimethyl ami nomethyl phenylalanine Met methionine Mop mophorl 1 nomethyl phenylalanine nBuOH n-butyl alcohol NS normal sal 1 ne Pap d1 propyl ami nomethyl phenylalanine Phe phenylalanine Pip piperi di nomethyl phenylalanine Pipes plperazlne-N, N ' -b1s[2-ethanesul foni c add] Pro proline Rf rate of flow SE standard error Ser serlne TFA tr1 f1 uoracet! c add TLC thin-layer chromatography TR retention time Trp tryptophan Tyr tyrosine Tep tetrahydroperrolyl methyl phenyl al an! ne SUBSTITUTE SHEET

Claims: C l a 1 ms 1. A method for designing and synthesizing LHRH antagonists by taking highly potent LHRH antagonist [ NAc-D2Nal * , DpClPhe 2 , D3Pal 3 , Ser 4 , Tyr 5 , Arg 6 , Leu 7 , Arg 8 , Pro 9 , DAla 10 ]NH 2 (II) as parent compound and modifying both alkalinous and lipophilic areas of the molecule of (II) , to obtain new LHRH antagonists having both high antiovulatory activity (AOA) and low hi staml nerel easi ng activity (HRA) based on Its topologlcal similarity with the molecule of a n1 uropeptl de, Substance P. 2. The method and process of design and synthesis based on claim 1 wherein Tyr 5 -DArg 6 -Arg 8 1n C-terminus and aromaric amino adds 1n N-term1nus In (ID is adjusted and replaced. The method of design and synthesis based on claim 1 and 2 wherein suitable alkalinous group is introduced into position 2,3,5,6,8 and unnatural amino add is Inserted in the above mentioned posi tions. SUBSTITUTE SHEET 3 4. The method of design and synthesis based on claim 1 wherin D3Pal having suitable basicity 1s substituted for DArg 6 1n (II) to give analog (III): [NAc-D2Nal 1 , DpClPHe 2 , D3Pal 3 , Ser 4 , Tyr 5 , D3Pal 6 , Leu 7 , Arg 8 , Pro 9 , DAla 10 ]NH 2 5. The method of design and synthesis based on claim 1 and 4 wherein Arg 5 1s substitued for Tyr 5 1n (III) to give (IV) [ NAc-D2Nal * , DpClPhe 2 , D3Pal 3 , Ser 4 , Arg 5 , D3Pal 6 , Leu 7 , Arg 8 , Pro 9 , DAla 10 ]NH-, The method and process of design and synthesis based on claim 5 wherein DPhe 3 is substituted for D3Pal 3 in (IV) to give (V): [Nac-D2Nal 1 , DpClPhe 2 , Dphe 3 , Ser 4 , Arg 5 , D3Pal 6 , Leu 7 , Arg 8 , Pro 9 , DAla 10 ]NH- The method and process of design and synthesis based on claim 4 wherin DPhe 3 1s substituted for D3Pal 3 1n (III) to give (V): [ NAc-D2Nal * , DpClPhe 2 , DPhe 3 , Ser 4 , Tyr 5 , D3Pal 6 , Leu 7 , Arg 8 , Pro 9 , DAla 10 ]NH-, A compound as described in claim 1 which 1s expressed as the formula, [ Ac-D2Nal 1 , AA 2 , AA 3 , Ser 4 , AA 5 , AA 6 , Leu 7 , AA 8 , Pro 9 , DAla 10 ]-NH 2 , 1n which AA are natural or unnatural amino adds on the formula D-or L-ArAla SUBSTITUTE SHEET where! n 1 n wh! ch and R 2 N-, RR'N-: 1 n wh1 ch R' = CH 3 -, CH 3 CH 2 -, C 3 H 7 -, C 4 H g -, H-; R = CH 3 -, CH 3 CH 2 -, C 3 H 7 -, C 4 H g -, H-; SUBSTITUTE SHEET The LHRH antagonists based on claim 8 AA' D-pClPhe, D-ArAla, DPhe, Ar-Ala, DXCH 2 Phe; AA : D3Pal , Ar-Ala, D-ArAla, DPhe, D-XCH 2 Phe; AA 5 - Arg, DMap, Pip, Tyr, Pal, Mop, Tep, Map, Phe, Eap, Pap, Bap, DMop; AA, D3Pal , D-Ar-Ala, D-XCH 2 Phe; AA 8 - Pip, Mop, Tep, Map, Eap, Pap, Bap, Arg; in whi ch SUBSTITUTE SHEET 1 n wh i c h an d R ' 2 N- , RR ' N- 1 n wh i ch R 1 = CH ~, CH CH 2 _ , C Hy—, C^Hg— , H—; R = CH _ , CH CH 2 _ , CJHT- , CjjHg— , H— ; 10. The application of LHRH antagonists as claimed In claim 8 odr 9 wherein the compound, as peptide medicine formulated as normal Injection, Injectable capsules and other pharmaceutical compositions is used for treating disorder 1n reproductive endocrinology system, including endometr1 os1 s , precocious puberty of children, prostate cancer and breast cancer, and for birth control as male or female contraceptive medicine or used for diagnosing and treating Infertility. 11. [N-AC-D2Nal - , P-Cl-D-Phe 2 , D3Pal 3 , Ser 4 , Mop 5 , D3Pal 6 , Leu 7 , Arg 8 , Pro 9 , D-Ala 10 ]NH 2 12. [N-AC-D-2Nal 1 , D-Phe 2 , D3Pal 3 , Ser 4 , Mop 5 , D3Pal 6 , Leu 7 , Arg 8 , Pro 9 , D-Ala 10 ]NH 2 13. [N-AC-D-2Nal - , P-Cl-D-Phe 2 , D3Pal 3 , Ser 4 , Arg 5 , D3Pal 6 , Leu 7 , Pap 8 , Pro 9 , D-Ala 10 ]NH 2 SUBSTITUTE SHEET

Inventors: XIAO SHAOBO, XIAO, SHAOBO
Applicants: ASTA MEDICA AG, ASTA MEDICA AKTIENGESELLSCHAFT
Application year: 1991
Main classification: A61K37
IPC codes: A61K38, C07K7