HU221307B1 - Decapeptides having lhrh-antagonist activity, process for their preparation and pharmaceutical compositions comprising thereof - Google Patents

Abstract

The present invention relates to LHRH antagonist decapeptides having high ovulation inhibitory activity (AOA) and low histamine release activity (HRA). According to the present invention, [NAc - D2Nal1, DpClPhe2, D3Pal3, Ser4, Tyr5, DArg6, Leu7, Arg8, Pro9, DAla10] NH2 starting peptides are prepared by corresponding amino acid substitutions of basic and lipophilic regions. The present invention also relates to pharmaceutical compositions comprising adecapeptides for the treatment of disorders of the reproductive endocrine system, birth control as a contraceptive, as well as for the diagnosis and treatment of infertility. ŕ

Description

The present invention relates to novel decapeptides having LHRH antagonistic activity and their derivatives, to their preparation, to their use in the manufacture of pharmaceutical compositions and to pharmaceutical compositions containing them.

Hypothalamic luteinising hormone releasing hormone (LHRH) acts on the anterior pituitary lobe to stimulate the secretion of luteinising hormone (LH) and follicular stimulating hormone (FSH). The LHRH antagonist analogue acts rapidly on the anterior pituitary gland, has a long duration of action and is safe and reversible for use in contraception or suppression of gonadotropin secretion. LHRH antagonists are excellent antagonists for this type of application. To date, more than two thousand LHRH analogs have been designed and synthesized.

K. Liu et al., J. Med. Peptide Protein Rés. 35 (2): 157-160 (1990) disclose LHRH antagonists in which the amino acid at position 6 of LHRH has been replaced by phenylalanine derivatives.

Karten MJ et al. (Endocrine Reviews 7 (1), 44-66 (1986)) describe modified GnRH analogs at positions 2, 3, 6 or 10 and state that increasing the number of amino acid substitutions alone does not lead to necessarily for a bigger effect.

K. Folkers et al., Z. Naturforsch. 41C, 1087-1091 (1986)] synthesized LysH and ArgH LHRH antagonists in which substitutions at positions 3, 5, 6 and 8 were made. Two of the decapeptides produced showed good ovulation inhibiting activity.

Oeske, R.W., et al. H. Vickery and J. J. Nestor (eds.), "LHRH Analogs: Contraceptive and Therapeutic Appplication," MTP Press, Boston, 17-24 (1987)] describe LHRH analogs at positions 5 and 6, respectively. They contain MePal and / or Arg.

EP-A 0 277 829 discloses LHRH analogs in which the 8-position arginine side chain is alkylated.

The documents described above show that minor modifications in the protein sequence of LHRH lead to quite drastic changes in pharmacological properties.

However, among the LHRH analogs, the "Nal-Arg" analogue also exhibited very strong histamine-releasing activity (HRA). Administration of a dose 50 to 100 times the therapeutic dose caused transient edema in the rat and limbs in rats. The clinical trial showed histamine-like systemic effects. Other LHRH antagonists containing DArg ⁶ and DLys ⁶ showed similar side effects, with ED ₅₀ values below 1 pg / ml for HRA.

The present invention provides LHRH antagonists that have very high antiovulatory activity (AOA) and very low histamine release activity (HRA) and their side effects are negligible.

The topological similarity between the parent compound, the molecule represented by Formula II, and a neuropeptide, the substance P (SP), was investigated and found to be modified by the basic and lipophilic area of the parent compound molecule with high anti-ovulatory activity. AOA) and new antagonists with low histamine release activity (HRA). The term "modification" refers to the conversion or substitution of amino acids at the C-terminus of the molecule of Formula II to Tyr ⁵ -DArg ⁶ -Arg ⁸ and aromatic amino acids at the N-terminus. Preferably, the modification relates to the introduction of a basic group and the substitution of non-natural amino acids at positions 2, 3, 5, 6, 8 of the molecule of formula (II).

Preferred examples of modifications of the invention include:

1. In the molecule of formula (II), DArg ⁶ is replaced by D3Pal, which is an aromatic amino acid of appropriate basicity. This gives the molecule of formula (III).

2. Substituting Tyr ⁵ for Arg ^{5 in} molecule (III) yields molecule (IV).

3. Substitution of DPal ³ in DP IV with DPhe ³ or DXCH ₂ Phe derivatives of formula (IV) yields V or DXCH ₂ Phe ³ analogs thereof.

4. Substitution of DPal ³ in DP III with DPhe ³ or DXCH ₂ Phe derivatives thereof to give V or DXCH ₂ Phe ³ analogues thereof.

A series of novel LHRH antagonists of formula (I) are synthesized in which

AA ² = DpCIPhe;

AA ³ = D3Pal, DPhe ;;

AA ⁵ = Arg, Tyr, Mop;

AA ⁶ = D 3 Pal;

AA ⁸ = Arg.

Amino acids are commonly used in the art and are well known in the art. In the description and examples, abbreviations other than those generally known are those accepted in the art.

For ease of understanding, the following is a list of all abbreviations used in this description and their explanations:

Ac acetyl below alanine AOA antiovulatory activity Arg arginine Bap [(Dibutylamino) methyl] phenylalanine BOC tert-butoxycarbonyl BuOAc butyl acetate BuOH butanol CAT cutaneous anaphylactoid test DCC dicyclohexylcarbodiimide DCM methylene chloride (dichloromethane)

HU 221 307 Bl

D2Nal D-P- (2-naphthyl) alanine D3Pal D-3- (3-pyridyl) alanine DpClPhe p-chloro-D-phenylalanine DpFPhe p-fluoro-D-phenylalanine D6Qal D-3- (6-quinolyl) alanine DMF Ν, Ν-dimethylformamide eap [(Diethylamino) methyl] phenyl-alanine ed ₅₀ Dose corresponding to 50% response EtOAc ethyl acetate FSH follicle stimulating hormone Glu glutamic acid Gly glycine His histidine HOBT 1-Hydroxybenzotriazole HPLC High Performance Liquid Chromatography HRA histamine-releasing activity HRT histamine-releasing test IEN ion exchange chromatography with post-column IPA isopropyl alcohol LH luteinising hormone LHRH luteinising hormone releasing hormone Leu leucine Lys lysine map [(Dimethylamino) methyl] phenyl-alanine Met methionine Mop (Morpholino-methyl) -phenylalanine NS normal saline Priest [(Dipropylamino) methyl] phenylalanine Phe phenylalanine Pip (Piperidino-methyl) -phenylalanine PIPES piperazine-N, N'-bis (2-ethanesulfonic acid acid) Pro proline Rf retention factor NEITHER standard error Ser serine TFA trifluoroacetic acid TLC thin layer chromatography (thin layer chromatography) TR retention time Trp tryptophan Tyr tyrosine tear (Tetrahidropirrolil-methyl) -phenylalanine. Above using the method described

LHRH antagonists are useful as peptide drugs for the treatment of reproductive endocrine disorders such as endometriosis, premature puberty in children, and for the treatment of prostate and breast cancer, and as male or female contraceptives for birth control. They can also be used to diagnose and treat infertility. Such peptide pharmaceutical compositions may be formulated as standard injectable formulations, injectable capsules or other formulations suitable for practical use.

The neuropeptide substance P (SP) plays a very important role in the natural course of histamine release in the brain; The ED ₅₀ value for HRA is 5-15 μΜ. Substance P (SP can be represented by the formula H-ArgPro-Lys-Pro-Gln-Gln-Phe-Gly-Ala-Met-NH _2. Examination of its structure to the HRA showed that the Arg at the N-terminus of the SP molecule. '-Pro ² -Lys ^{3 is} essential for the histamine-releasing activity (HRA) of the molecule since deletion of these three amino acids resulted in the complete elimination of HRA, whereas in the C-terminus, deletion of one, two or three amino acids retains the original One-fourth of the activity of the SP molecule, with the further deletion of Phe ⁸ and Phe ^7. The HRA was reduced to 4% and 0.57% of the original activity of the SP molecule, respectively. The further deletion of Gin ⁶ and Gin ⁵ did not in the above data suggest that the lipophilic area around phe ⁷ · ⁸ in terms of determining the value of HRA,., this space SP molecule involved in mast cell (mast cell) receptor with binding.

As previously mentioned, the LHRH D2Nal ', DArg ⁶ analogs showed very high histamine-releasing activity, their molecular structure was topologically similar to SP; the DArg ⁶ -Leu ⁷ -Arg ⁸ sequence fragment in the former appears to correspond to the Arg'-Pro ² -Lys ^{3 in} the latter, each containing a pair of highly basic amino acid residues with only one neutral amino acid residue, that is, LHRH both - D2Nal * and DArg ⁶ - analogs and SP also contain two highly basic amino acid residues which are 1,3-positions relative to each other. On the other hand, the enrichment of aromatic amino acid residues in the former is considered to be equivalent to the Phe ⁷ ' ⁸ area in the SP for the determination of the HRA.

The modification according to the invention has two important aspects: one is the modification of the Tyr ⁵ DArg ⁶ -Arg ⁸ region at the C-terminus and the other is the fine modification of the aromatic acids after optimization of the modification of the C-terminal basic region. The peptide of formula (II) is used as starting material which exhibited 100% ovulation inhibition (AOA) at 0.5 pg (corn oil) and 57% AOA at 0.25 pg.

In compound 11, DArg ^{6 can be} replaced by weakly basic or neutral aromatic acids such as D3Pal, D6Qal, tetrahydrotriptophane, methyl tryptophan. LHRH of Formula III [NAc-DNal ¹ , DpCIPhe ² , D3Pal ³ ' ⁶ , DAla ¹⁰ ] is obtained by replacing DArg ⁶ with D ³ Pal ⁶ in the compound of Formula II. Compound III showed 100% ovulation inhibition (AOA) at 3 pg, 83% at 1 pg (corn oil), and ED ₅₀ HRA at 9.8 pg / ml, much better , such as the Nal-Arg analogue (less than 1 pg / ml for ED ₅₀ HRA). The basicity of the whole molecule seems to be equal to or nearly equal to a couple of arginines in order to obtain a high level of ovulation inhibition (AOA). Since 5th place3

As in position 6, Bleach is not involved in receptor binding, a wide variety of amino acids, including arginine, can be introduced at position 5. A series of new analogs were designed. For example, in the molecule of formula (III), Tyr ^{5 is} replaced by five Arg ^{5 to form} [NAc-DNal ^! , DpCIPhe ² , D3Pal ³ · ⁶ , Arg ³ , DAla ¹⁰ ] LHRH is obtained. Both the compound (IV) and the compound (II) contain two arginines, but the distance between the two arginines in the compound (IV) has a geometric proportionality

1-4, i.e. two other amino acids between these two arginines, i.e. the distance is greater than in the compound of formula II. Therefore, HRA is reduced and on the other hand, due to the presence of the two arginines, the AOA (anti-ovulation effect) should not be less than that of the compound of formula (II). The bioassay obtained with the compound of formula (IV) showed that ED _{50 for} HRA was 3.5 µg / ml and AOA was 6% at 0.125 µg (administered in corn oil), 85% at 0.25 µg / ml and 100% at 0.5 gg.

This was the first time that for LHRH antagonists, an ED _{50 of} 0.125 µg or less was achieved for AOA. Therefore, further design was based on the compound of formula (IV).

The molecule of formula IV contains four basic residues, the D3Pal ³ · ⁶ and Arg ⁵ · ⁸ , whereas the molecule of formula II contains only three basic residues. Therefore, it is reasonable to replace a D3Pal with a neutral amino acid; on the other hand, compound (IV) showed very strong hydrophilicity, and a decrease in hydrophilicity in compound (IV) by replacing D3Pal with a hydrophobic amino acid would be beneficial in retaining the drug in the body and thereby increasing the duration of action. A series of new analogs were designed by replacing D3Pal ^{3 in} compound (IV). Most preferred was LHRH of formula [NAc-D2NaE, DpCIPhe ² , DPhe ³ , Arg ⁵ , D3Pal ⁶ , DAla ¹⁰ ], which contained DPhe ³ instead of D3Pal ³ . Compound (V) showed 100% AOA at a dose of 1gg (in saline), equivalent to the parent compound of Formula (IV) while decreasing by half with HRA; ED ₅₀ HRA 7.4 µg / ml.

Further substitution of DpCIPhe ^{2 for} DPhe ² reduced the lipophilicity of this region of the molecule and reduced HRA.

Arg ⁵ -D ³ Pal ⁶ -Leu ⁷ -Arg ^{8 at} the C-terminus of peptide (IV) appears to play a major role in inducing histamine release. D3Pal combines aromatic character, basicity and hydrophilicity within an amino acid molecule and is also spatially acceptable for receptor binding in LHRH antagonists. Designing a series of novel peptides containing non-natural amino acids with similar properties to D3Pal may lead to better LHRH antagonists than the compounds of formulas IV and V.

For example, modification of natural lipophilic aromatic amino acids such as phenylalanine as described in the "Synthesis of New Non-Natural Amino Acids" section below, leads to a series of new amino acids that combine aromatic, hydrophilic, and basicity within a molecule that It may be represented by the general formula (VI) in which R ¹ and R ^{2 may be the} same or different, may be linked or cyclic, and may contain a heteroatom. By varying the groups R ¹ and R ^2, a series of amino acids is obtained in which the basicity, hydrophilicity and space structure are systematically altered. The introduction of these amino acids at positions 5, 6, 8 of the molecule of formula IV results in three sets of new LHRH antagonists. Biological test results showed that each series had at least one new antagonist which, like Compound IV, exhibited 100% AOA at 1 µg, whereas HRA was significantly reduced. An example of compound (VII), which had 100% AOA at 1 µg, was an ED50 HRA of 14.7 µg / ml and appeared to be better than compound (V). When Arg ^{8 in} the compound of formula (IV) was replaced by an amino acid of formula (VI), the decrease in HRA was directly proportional to the length of the R group in formula (VI), so that the ED50 for HRA was 200 µg / ml. higher than that. Such compounds are readily soluble in aqueous solution and are expected to be clinically useful without formulation problems. The results showed that Arg ⁸ or Lys ^{8 is} not essential for highly potent LHRH antagonists. The appropriate basic center at position 8 can provide high AOA, while the mast cell inducing histamine release activity is significantly reduced when the above-mentioned basic center was significantly inhibited.

The present invention, which combines modifications at both the N- and C-terminus, results in improved LHRH antagonists.

The synthesis is described in detail below:

1. Synthesis of new non-natural amino acids

More than 60 D- or L-amino acids have been designed and synthesized in Schemes I-IV shown in Schemes [A] to [C]. by four types of synthesis pathways. The structure of these unnatural amino acids is represented by the general structural formulas shown in the Schemes. Some of these amino acids are basic, hydrophilic or aromatic, while others combine these properties in the molecule.

In the general formulas used in the synthesis of Reaction Scheme I in Scheme [A], Ar is 1-naphthyl, 2-naphthyl, 4-chlorophenyl or 4-fluorophenyl.

In Scheme [A], Scheme II. in the general formula (I), Ar is 1-naphthyl, 2-naphthyl, 4-chlorophenyl, 4-fluorophenyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-quinolyl, 3-quinolyl, 4-quinolyl, 1-imidazolyl, 4-pyridazinyl, 5-pyridazinyl, 2-piperazinyl,

A 2-oxo-4-dihydronaphthyl group or a group of formula (A) wherein A is piperidino, morpholino, 1-pyrrolidinyl or R ' _{2 is} N or RR'N.

In Scheme [B], Scheme III. wherein X is piperidino, morpholino, 1-pyrrolidinyl, or R ' ₂ N, or RR' N, wherein R 'and R are as defined in formula (Γ).

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In Scheme [C], IV. in the general formulas of the synthesis route are aryl, naphthyl, 2-naphthyl, 4-chlorophenyl, 4-fluorophenyl, 2-pyridyl,

3-pyridyl, 4-pyridyl, 2-quinolyl, 3-quinolyl, 4-quinolyl, 1imidazolyl, 4-pyridazinyl, 5-pyridazinyl, 2-piperazinyl, 2-oxo-4-dihydronaphthyl or a 4-substituted Y-substituted phenyl group wherein Y is dimethylamino, diethylamino, dipropylamino, diisopropylamino, dibutylamino, piperidino, morpholino or 1-pyrrolidinyl.

2. Synthesis of the peptide

Using the solid phase peptide synthesis method introduced by Merrifield, synthesis on benzhydrylamine hydrochloride resin (BHA resin) begins at the C-terminus of the peptide. The process consists of three main steps, end group attachment, coupling and cleavage. Methylene dichloride is the main solvent used for washing between each reaction step, although isopropyl alcohol (IPA) and dimethylformamide (DMF) are also used if necessary. The coupling reaction is carried out with excess dicyclohexylcarbodiimide (DCC) by addition of a sufficient amount of 1-hydroxybenzotriazole (HOBT). The progress of the coupling reaction is monitored by the Kaiser ninhydrin method. The second coupling reaction can only be performed if a positive result is obtained in the Kaiser test. The peptide chain is cleaved from the resin after completion of all necessary reactions on the resin using anhydrous hydrogen fluoride (HF) in the presence of anisole; however, all temporary protecting groups are cleaved. After washing with ethyl acetate or diethyl ether, extraction with aqueous acetic acid solution followed by lyophilization gave the crude LHRH antagonist product. Yield over 50%.

3. Purification of the Peptide (1) The peptide is purified by gel filtration chromatography or silica gel partition chromatography on a 60-100 cm height column with UV detection / TLC. Once purified, LHRH antagonists are obtained after lyophilization of the major fractions. The yield may be 50-90% and purity above 90%.

(2) The peptide was further purified on a Waters High Performance Liquid Chromatography (HPLC) C18 reversed phase column (μ-Bondapak 84176). The yield of this step is 20-50% and the purity is at least 99%.

4. Peptide purity assay

1. TLC analysis

It is carried out on a 5 to 10 cm high plastic sheet covered with silica gel 60 F254. Developed in five different solvent systems, each showed one spot.

(2) HPLC analysis

Each peak was eluted with two types of solvent systems using a Waters HPLC apparatus and an analytical column (μ-Bondapak 27324) and tested at UV 210. The sample was 10-200 pg.

5. Amino acid analysis of the peptide

The PICO-TAG method developed by Waters Company accurately weighed 50 µg on a 10 ⁶ g graduated scale, which was dried in vacuo for 2 hours. After dissolving in water, an aliquot containing 10 µg of material is added to a reaction tube into which is added a 1: 1 dilute hydrochloric acid solution containing 1% phenol according to the manual.

The reaction is maintained at 105 ° C for 22-24 hours in a sealed vessel which is filled with nitrogen and suction vacuum to remove oxygen in the reaction tube. After removing the excess hydrochloric acid, phenyl isothiocyanate is added to the reaction tube to form the amino group. It was then analyzed on an HPLC equipped with a PICO-TAG amino acid column and checked at UV 254. Calculating the amount of each amino acid and the relative molar ratio from the integrated region of each amino acid relative to the Waters H standard sample gives the amino acid composition of the sample. The classical ion-exchange-ninhydrin derivative (IEN) method was also used for control and this gave the same results. However, this requires ten times the number of samples to obtain satisfactory results.

6. Evaluation of biological activity

The Corbin rat antiovulation method was used. Healthy adult female SD rats weighing 200-250 g were used in the experiment. The animals were maintained at 22-24 ° C for 14/10 hours in alternating light / dark conditions. Standard feed and water were provided as desired. Rats with at least two consecutive 4-day estrogen cycles may be used for vaginal smear testing. The rats were given various doses of peptides (LHRH antagonists) dissolved in saline on the day of the ester. The following day, the rats were sacrificed and their ovaries were counted under the microscope under autopsy. The rats were divided into several groups according to the dose; each group consisted of about 10 rats, the control group, in which rats were given the same amount of saline, consisted of 910 rats. The anti-ovulation activity (AOA) is calculated using the following formula:

number of non-ovulated rats

AOA = - _; -χ 100% of total treated rats

7. Evaluation of Histamine Release Activity (HRA) (1) In vitro histamine release test (HRT)

Healthy adult male SD rats weighing 200 to 250 g, maintained as described in the previous experiment, were used for this experiment. After stunning with carbon dioxide, the abdominal cavity is washed with 50 ml PIPES AC medium containing 20 units of heparin. Subsequently, after centrifugation at 200 χ g for 8 minutes at 4 ° C, the cells were washed again and resuspended in PIPES AC solution to a total concentration of 24 x ^{10 5} leukocytes / ml. This suspension is present in an amount of about 5-10%

Contains 221 307 tartalmaz1 zonal cells. Washed cells were used immediately after harvesting, heated at 37 ° C for 5 minutes, and pipetted into 0.3 ml aliquots into polystyrene tubes containing 0.3 ml peptide solution. The mixtures were incubated for 15 minutes at 37 ° C and the reaction was stopped by centrifugation at 400 ° C for 15 minutes at 4 ° C. After extensive extraction of the supernatant with butanol and heptane, the histamine content is determined by a manual fluorometric assay. The histamine content can be obtained from the standard histamine curve (see below). The percentage of histamine release (HR%) can be calculated from the following equation:

E-B Histamine Release (%) = - χ 100%

C-B where

E is the fluorometric reading of the test sample,

B is the fluorometric reading of the samples obtained with cells and buffer only, and

C is the fluorometric reading for "complete" (cells treated with perchloric acid).

The standard curve is obtained by plotting OD values obtained on a fluorometer at 350 nm / 450 nm (activation / fluorescence) as a function of the various concentrations obtained with serial dilutions of accurately measured histamine hydrochloride solution. The relative parameter r of the standard histamine curve may be 0.9998 and the lowest detectable concentration of histamine is 0.5 ng / ml.

The ED ₅₀ value of a peptide is plotted against a dose-response curve plotted against histamine release (HR) on a semi-logarithmic paper.

Each peptide sample must be tested on mast cells from at least three different rats.

(2) Cutaneous anaphylactoid activity test (CAT)

In the experiment, healthy adult female rats weighing 250 g were used. The rats were injected intravenously with 1 ml of 0.05% Evans blue solution. Immediately thereafter, 0.05 ml (5, 0.5, and 0.05 μg / ml) peptide solution and saline (control) were injected intradermally into the shaved part of the back of the animals. 30 minutes after injection, the rats are sacrificed and the dorsal skin is examined. Using a fine-tuned measure, measure the diameter of the lesions in two orthogonal directions. The control diameter is generally less than 5.5 mm.

The amount of Evans blue penetrated from the vessel into the skin is also determined spectrophotometrically. The skin corresponding to the lesion is excised and immersed in acetone / saline (7: 3) overnight. After centrifugation the following day, the amount of Evans blue in the supernatant was determined using a UV-260 spectrophotometer at 610 nm using a 7: 3 acetone / saline control solution. Each peptide is tested in at least three different rats.

A series of novel LHRH antagonists were synthesized using the method described above. Thus, new LHRH antagonist constructs are obtained by single or multiple substitution with the various natural and non-natural amino acids listed above.

Exemplary LHRH antagonists of the present invention may be prepared as detailed in Steward and Young, "Solid Phase Peptide Synthesis". This work was published by Pierce Chemical Company (Rockford, Illinois, USA).

The results of these biological assays are summarized in Table 3.

Uses of the invention:

1. Following the completion of preclinical pharmacological and toxicological studies, these novel LHRH antagonists, which are highly therapeutically effective and have few side effects, may be used clinically in the treatment of endometriosis and other disorders of the reproductive endocrine system such as premature childhood puberty, prostate cancer and breast cancer. drug development. By suppressing gonadotropin secretion by competing with the receptor for endogenous LHRH, these agents can be rapidly, reversibly and safely transformed into new types of male and female contraceptives. In addition, they can be used in the treatment of infertility and for selective and reversible suppression of pituitary lobe function in gonadotropin secretion.

Because the LHRH antagonists described herein are peptide-type compounds, it is inadvisable to be administered orally. However, they are readily reconstituted into lyophilized powders which are readily dissolved in saline for intravenous, subcutaneous or intramuscular injection.

In addition, extended release systems, such as biodegradable, injectable capsules, have also been investigated. The capsules can be implanted with a special syringe under the skin and, once all peptide contents have been released, are absorbed into the tissues and do not require surgical removal. The extended release system is particularly suitable for long-term clinical administration of LHRH analogs.

In the following, the results of the analysis of the examples are given (three analogs of formula (IV), (V), (VII) are typical examples).

(1) Purity:

Thin layer chromatography (TLC):

Each chromatogram, developed in five different solvent systems, shows only one spot.

High Performance Liquid Chromatography (HPLC):

There is only one peak in each chromatogram eluted with two different solvent systems.

The retention factor (R _f ) and retention time (TR) values are given in Table 2. The thin-layer chromatograms are shown in Figure 1 and the HPLC diagrams are shown in Figure 2-4. Figures 3 to 5 are shown.

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Table 1

Results of chromatographic analysis of LHRH antagonists

analogs HPLC TLC TR1 TR2 rn r _g rn R (4 R (3 (minute) ARC 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

The solution is + 80% acetonitrile.

Solvent system 2:

moon: 0.01 M aqueous potassium dihydrogen phosphate solution (pH 3).

Solution B: 20% solution A + 80% acetonitrile.

Thin-layer chromatography solvent systems:

1. butanol / ethyl acetate / acetic acid / water (5: 5: 1: 3).

2. butyl acetate / butanol / acetic acid / water (2: 8: 2: 3). 20

3. butyl acetate / acetic acid / water (4: 1: 5) upper phase.

4. butanol / acetic acid / water (4: 1: 2).

5. butanol / ethyl acetate / acetic acid / water (1: 1: 1: 1).

Figure 1 shows a developed view of thin-layer chromatograms of the LHRH antagonists of formulas IV, V and VII in five different solvent systems.

Figure 2 shows the reverse phase HPLC spectrum of a pure sample of an LHRH antagonist of formula (IV).

(Capture conditions: 30 columns: μ-Bandapak Cl8 (3.9 mm x 30 cm); mobile phase: A: 0.1 M ammonium acetate, pH 7.0,

B: 20% A + 80% acetonitrile; gradient procedure: amount of B in 15 minutes 35

Increased from 10% to 100%; flow rate: 2 ml / min; detection: UV 229 nm.)

Figure 3 shows the HPLC spectrum of a pure sample of an LHRH antagonist of formula (V).

(Recording conditions:

Column: μ-Bondapak C18 (3.9 mm x 30 cm); mobile phase: A: 0.01 M potassium dihydrogen phosphate (pH = 3),

B: 20% A + 80% acetonitrile;

gradient procedure: increase of B from 40% to 100% in 15 minutes;

flow rate: 2 ml / min; detection: UV210nm.)

Figure 4 shows the HPLC spectrum of a pure sample of the LHRH antagonist of formula (VII).

(Recording conditions:

Column: μ-Bondapak C18 (3.9 mm x 30 cm); mobile phase: A: 0.01 M potassium dihydrogen phosphate (pH = 3),

B: 20% A + 80% acetonitrile;

gradient procedure: increase of B from 40% to 100% in 15 minutes;

flow rate: 2 ml / min; detection: UV 210 nm.) (2) Amino acid analysis:

The analysis was performed using the classical ion exchange ninhydrin derivative (IEN) and the new Pico-TAG method, the results are shown in Table 2 and Figures 5 and 6.

Table 2

Amino acid composition of LHRH antagonists

analogs Methods Ser Arg Ala Pro Leu Phe Paul PCIPhc At ARC IEN 0.86 2.05 1.01 0.99 1.13 + + NM Pico-TAG 0.92 2.25 0.91 1.01 0.91 + + + V IEN 0.81 2.02 1.03 1.03 1.12 0.99 + + + Pico-TAG 0.68 2.26 0.93 1.29 1.04 1.00 + + + VII IEN 0.91 0.91 1.00 1.00 1.09 + + NM |

NM: We didn't dare.

+: detected but not quantified.

Fig. 5 shows the PICO-55 TAGTM spectrum of the LHRH antagonist of formula IV.

Figure 6 shows the PICOTAG ™ spectrum of the LHRH antagonist of formula (V).

3. The results of the biological determinations shall:

The results of the biological assays, including the in vitro ovulation inhibitory activity (AOA) and in vitro ED ₅₀ values for the histamine release activity (HRA), are summarized in Table 3, which lists 26 antagonists.

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Table 3

Biological test results for novel LHRH antagonists relative to parent compound *)

substituted amino acids % AOA / pg HRA (pg / ml) ed ₅₀ ± sem 0,125 0.25 0.5 1.0 2.0 First starting compound ** 50 75 100 3.5 ± 0.38 Second D-Phe ³ > 29 60 100 7.4 ± 0.98 Third DPhe ² , DPhe ³ 0 18.5 ± 7.00 4th DTyr ⁵ , Lys ⁸ 40 5.0 ± 2.15 5th D-Phe ³ 60 35.0 ± 5.05 6th Map ⁵ 29 24.8 ± 4.47 1 7th Eap ⁵ 43 12.0 ± 0.50 8th Pap ⁵ 0 9.6 ± 0.19 9th Bap ⁵ 14 23.5 ± 5.78 10th D-Map ⁵ 12.5 18.3 ± 2.38 11th Tear ⁵ 14 36.8 ± 5.68 12th Pip ⁵ 17 33 71 100 9.4 ± 1.63 13th Mop5 * ") 25 100 14.7 ± 2.70 14th D-Map ⁶ 14 19.5 ± 2.50 15th D-Eap ⁶ 14 13.0 ± 1.00 16th D-Tep ⁶ 71 22.5 ± 3.25 17th D-Pip ⁶ 0 50 57 7.6 ± 2.48 18th D-Mop ⁶ 33 67 100 > 11 19th Map ⁸ 57 100 5.4 ± 1.22 20th Eap ⁸ 29 56.9 ± 15.1 21st Pap ^8th 50 88 70.4 ± 26.8 22nd Bap ⁸ 0 > 235 23rd Tear ⁸ 25 100 6.6 ± 2.13 24th Pip ⁸ 43 27.5 ± 2.50 25th Mop ⁸ 71 52.5 ± 17.5 26th D-Map ⁸ 0 28.0 ± 9.00

*) The starting compound is the compound of formula IV [Ac-D2Nal ¹ -DpClPhc ² -D3Pal ³ -Ser ⁴ -Arg ⁵ -D3Pal ⁶ -Lcu ⁷ -Arg ⁸ -Pro ⁹ -Ala ¹⁰ -NH ₂ ]. [Z. Naturforsch, 41c, 1087-1091 (1986)] **) Compound (V) ***) Compound (VII)

As illustrated and described above, the LHRH antagonists of the present invention exhibit very good properties. Thin layer chromatography and high performance liquid chromatography analysis. Their composition is correct, ie the same as the design. Their antiproliferative activity is high: they inhibit ratovulation by subcutaneous injection at a dose of 0.1 to 2.0 µg on the day of the ester. Histamine releasing side effect is low: their ED _{50 for} in vitro histamine releasing activity (effective dose to induce 50% histamine release in rat mast cell) 55 5-300 pg / ml; in the skin anaphylactic test, the lesion induced in rats is a clinically required low level. Their water solubility is very good and all the biological activity tests were carried out in saline so that they can be easily formulated as injectables. They can also be easily foamed into sustained release systems, of which injectable microcapsules are best suited to suppress gonadotropin or sex hormone secretion. Therefore, they can be used as very effective, reversible and safe contraceptives for both men and women. They can also be used to treat diseases associated with various disorders of the endocrine reproductive system, such as hormone dependent prostate and breast cancer, endometriosis, and early puberty in children. They can also be used to treat infertility. The novel LHRH antagonists described herein can also be used in basic reproductive physiological and pharmacological studies, such as studies of pituitary function and the effects of sex hormones or gonadotropins or LHRH on sexual behavior.

Claims (8)

PATENT CLAIMS 1. [NAc-D2Nal ¹ , DpClPhe ² , DPhe ³ , Ser ⁴ , Arg ⁵ , D ³ Pal ⁶ , Leu ⁷ , Arg ⁸ , Pro ⁹ , DAla ¹⁰ ] NH ₂ 2. [NAc-D2Nal ¹ , DpClPhe ² , DPhe ³ , Ser ⁴ , Tyr ⁵ , D ³ Pal ⁶ , 5 Leu ⁷ , Arg ⁸ , Pro ⁹ , DAla ¹⁰ ] NH ₂ 3. [NAc-D2Nal ¹ , DpClPhe ² , D3Pal ³ , Ser ⁴ , Mop ⁵ , D3Pal ⁶ , Leu ⁷ , Arg ⁸ , Pro ⁹ , DAla ¹⁰ ] NH ₂ 4. [NAc-DNal ¹ , DPhe ² , D3Pal ³ , Ser ⁴ , Mop ⁵ , D3Pal ⁶ , Leu ⁷ , Arg ⁸ , Pro ⁹ , DAla ¹⁰ ] NH ₂ 5. Use of the LHRH antagonists of claim 1 to prepare a medicament for the treatment of reproductive endocrine disorders and prostate and breast cancer. 6. Use of the LHRH antagonists of claim 1 as a male or female contraceptive for the manufacture of a medicament for the control of birth and for the diagnosis and treatment of infertility. 7. Procedure 1-4. LHRH antagonists according to claim 1, characterized in that [NAc-D2Nal ¹ , DpClPhe ² , D3Pal ³ , Ser ⁴ , Tyr ⁵ , DArg ⁶ , Leu ⁷ , Arg ⁸ , Pro ⁹ , DAla ¹⁰ ] NH _{2 are} basic and lipophilic. 10 of the region of the invention by appropriate amino acid substitution. A pharmaceutical composition according to any one of claims 1-4. The LHRH antagonist of claim 1, together with pharmaceutically acceptable carriers and / or excipients.