JPWO2014061772A1 - Novel NK3 receptor agonist - Google Patents

Abstract

An object of the present invention is to provide a novel peptide having an excellent neurokinin 3 receptor agonist activity. Formula (I) [Each symbol in the formula is as described in the specification. Or a salt thereof.

Description

  The present invention relates to a peptide having a neurokinin (NK) receptor agonistic action and use thereof. More specifically, the present invention relates to peptides having selective and strong binding affinity to NK3 receptor and agonistic action, and uses thereof.

GPR54 receptor ligands, which are GPCRs reported as receptors for kisspeptin involved in reproductive physiology, have been created (Patent Documents 1 to 3, Non-Patent Documents 1 to 6). The kisspeptin neuron population in front of the hypothalamus is an ovulation center that regulates the surge-like secretion of gonadotropin-releasing hormone (GnRH) by the positive feedback action of estrogen, and kisspeptins are expected as new drugs that regulate ovulation induction Has been.
On the other hand, in recent years, new findings regarding pulsatile secretion of GnRH, which are thought to be involved in follicular development, have been found one after another, and kisspeptin neurons in the hypothalamic arcuate nucleus serve as the center that promotes pulsatile secretion of GnRH. It is functioning, and neurokinin and its receptor are expressed at the same time in arcuate nucleus kisspeptin neurons, and pulsed secretion of GnRH is induced by the autocrine action of neurokinin. It has been reported (Non-Patent Document 7). Furthermore, it has been found that the firing activity in this neuron is completely synchronized with the LH pulse (Non-Patent Documents 8 and 9).
From these findings, it is assumed that NK3 receptor ligand (agonist) known as a neurokinin receptor can be a drug that regulates GnRH pulse and LH pulse and controls follicular development, and various NK3 receptors. Attempts have been made to create selective agonists. For example, [MePhe ⁷ ] -NKB and senktide are known as NK3 receptor selective agonists (Non-patent Documents 10 and 11). These peptides that are already on the market as research reagents have been used for various basic studies involving tachykinins and neurokinin receptors.
All of these peptides are excellent NK3 receptor-selective ligands, but there is still a need for novel NK3 receptor-selective ligands that are expected to be highly active, highly selective, and have a sustained action in vivo. This is the current situation.
In some infertility patients, reports of abnormal GnRH pulses due to NK3 receptor mutations indicate that the NK3 receptor is expected to be a new target for the treatment of infertility involving follicular growth failure Has been.

WO2004 / 060264 WO2007 / 125619 WO2009 / 139298

Niida A, Wang Z, Tomita K, Oishi S, Tamamura H, Otaka A, Navenot JM, Broach JR, Peiper SC, Fujii N. Bioorg. Med. Chem. Lett. 16 (1) 134-137 (2006) Tomita K, Niida A, Oishi S, Ohno H, Cluzeau J, Navenot JM, Wang Z, Peiper SC, Fujii N. Bioorg. Med. Chem. 14 (22) 7595-7603 (2006) Tomita K, Narumi T, Niida A, Oishi S, Ohno H, Fujii N. Biopolymers, 88 (2) 272-278 (2007) Tomita K, Oishi S, Cluzeau J, Ohno H, Navenot JM, Wang Z, Peiper SC, Akamatsu M, Fujii N. J. Med. Chem. 50 (14) 3222-3228 (2007) Tomita K, Oishi S, Ohno H, Fujii N. Biopolymers, 90 (4) 503-511 (2008) Tomita K, Oishi S, Ohno H, Peiper SC, Fujii N. J. Med. Chem. 51 (23) 7645-7649 (2008) Maeda K-I, Ohkura S, Uenoyama Y, Wakabayashi Y, Oka Y, Tsukamura H, Okamura H. Brain Res, 1364, 103-115 (2010) Ohkura S, Takase K, Matsuyama S, Mogi K, Ichimaru T, Wakabayashi Y, Uenoyama Y, Mori Y, Steiner RA, Tsukamura H, Maeda KI, Okamura H. J. Neuroendocrinol. 21, 813-821 (2009) Wakabayashi Y, Nakada T, Murata K, Ohkura S, Mogi K, Navarro VM, Clifton DK, Mori Y, Tsukamura H, Maeda KI, Steiner RA, Okamura H. J. Neurosci. 30, 3124-3132 (2010) Drapeau G, D'Orleans-Juste P, Dion S, Rhaleb NE, Rouissi NE, Regoli D. Neuropeptides. 10 (1) 43-54 (1987) Laufer R, Gilon C, Chorev M, Selinger Z. J Biol Chem. 261: 10257-10263 (1986)

  An object of this invention is to provide the novel peptide which has a NK3 receptor agonist effect, and its use.

Genomic information of various animal species including lower organisms has been clarified, and it has been clarified that endogenous tachykinins having different peptide sequences exist for each animal. However, there has been no example of analyzing the effects of these tachykinins having various sequences on various neurokinin receptors of humans or mammals to which drug development is applied.
Based on the peptide chain sequence of tachykinins assumed from the genome information of various organisms reported so far, the present inventors are required for a neurokinin receptor (NK3 receptor) selective ligand ( Structural requirements, receptor selectivity, etc.) were scrutinized. That is, using the sequence information of tachykinins derived from various animals, a candidate neurokinin receptor selective agonist compound consisting of 5 to 12 residues is designed and synthesized, and receptor binding activity and agonist activity are obtained by an in vitro evaluation system. Quantitatively evaluated.
As a result, many tachykinins derived from various animals show non-selective binding affinity to three types of neurokinin receptors (NK1, NK2, and NK3 receptors), whereas neurokinin B A derivative obtained by converting the fifth Phe (Phe5) into Asp, Asn, Gln, Gly, Val, Leu, or Glu and the seventh Val (Val7) into MePhe is selective only for the NK3 receptor. It was found to show binding affinity at low concentrations. All of these derivatives induced an increase in intracellular Ca ²⁺ concentration in the NK3 receptor-expressing cell line, and were confirmed to be potent NK3 receptor agonists. It was also clarified that the peptide chain of 5 residues on the C-terminal side shows agonist activity. The present invention has been completed based on these findings.
That is, the present invention is as follows.
[1] Formula (I)

(Where
A1 represents an aspartic acid residue or is deleted;
A2 represents a pyroglutamic acid, lysine or valine residue or is deleted;
A3 represents a proline, alanine or aspartic acid residue or is deleted;
A4 represents a serine, lysine, asparagine, aspartic acid, arginine, threonine or methionine residue or is deleted;
A5 represents or is missing a lysine, serine, proline, glycine or histidine residue;
A6 represents an asparagine or aspartic acid residue or is deleted;
A7 represents any α-amino acid residue or is deleted;
A8 represents an isoleucine, valine or N-methylphenylalanine residue;
Phe represents a phenylalanine residue;
Gly represents a glycine residue;
Leu represents a leucine residue;
Met-NH ₂ represents methionine amide) or a salt thereof.
[2] The peptide or salt thereof according to the above [1], wherein the peptide or salt thereof is any one peptide or salt thereof represented by the following sequence;
pGlu-Pro-Asn-Pro-Asp-Glu-Phe-MePhe-Gly-Leu-Met-NH ₂ (SEQ ID NO: 2)
pGlu-Pro-Asn-Pro-Asp-Asp-Phe-MePhe-Gly-Leu-Met-NH ₂ (SEQ ID NO: 3)
pGlu-Pro-Asp-Pro-Asn-Asp-Phe-MePhe-Gly-Leu-Met-NH ₂ (SEQ ID NO: 4)
pGlu-Pro-Ser-Lys-Asp-Asp-Phe-MePhe-Gly-Leu-Met-NH ₂ (SEQ ID NO: 5)
H-Asp-Val-Pro-Lys-Ser-Asp-Asp-Phe-MePhe-Gly-Leu-Met-NH ₂ (SEQ ID NO: 6)
H-Lys-Pro-Arg-Pro-Asp-Asp-Phe-MePhe-Gly-Leu-Met-NH ₂ (SEQ ID NO: 7)
pGlu-Ala-Asp-Pro-Asn-Asp-Phe-MePhe-Gly-Leu-Met-NH ₂ (SEQ ID NO: 8)
H-Asp-Thr-Gly-Asp-Asp-Phe-MePhe-Gly-Leu-Met-NH ₂ (SEQ ID NO: 9)
pGlu-Pro-Ser-Lys-Asp-Ala-Phe-MePhe-Gly-Leu-Met-NH ₂ (SEQ ID NO: 10)
pGlu-Pro-Ser-Lys-Asp-Asp-Phe-Ile-Gly-Leu-Met-NH ₂ (SEQ ID NO: 11)
H-Asp-Val-Pro-Lys-Ser-Asp-Gln-Phe-MePhe-Gly-Leu-Met-NH ₂ (SEQ ID NO: 12)
H-Asp-Val-Pro-Lys-Ser-Asp-Asp-Phe-Val-Gly-Leu-Met-NH ₂ (SEQ ID NO: 13)
H-Met-His-Asp-Phe-Phe-MePhe-Gly-Leu-Met-NH ₂ (SEQ ID NO: 14)
H-His-Asp-Phe-Phe-MePhe-Gly-Leu-Met-NH ₂ (SEQ ID NO: 15)
H-Asp-Phe-Phe-MePhe-Gly-Leu-Met-NH ₂ (SEQ ID NO: 16)
H-Phe-Phe-MePhe-Gly-Leu-Met-NH ₂ (SEQ ID NO: 17)
H-Phe-MePhe-Gly-Leu-Met-NH ₂ (SEQ ID NO: 18)
H-Asp-Met-His-Asp-Asp-Phe-MePhe-Gly-Leu-Met-NH ₂ (SEQ ID NO: 19)
H-Asp-Met-His-Asp-Ala-Phe-MePhe-Gly-Leu-Met-NH ₂ (SEQ ID NO: 20)
H-Asp-Met-His-Asp-Glu-Phe-MePhe-Gly-Leu-Met-NH ₂ (SEQ ID NO: 21)
H-Asp-Met-His-Asp-Gly-Phe-MePhe-Gly-Leu-Met-NH ₂ (SEQ ID NO: 22)
H-Asp-Met-His-Asp-His-Phe-MePhe-Gly-Leu-Met-NH ₂ (SEQ ID NO: 23)
H-Asp-Met-His-Asp-Ile-Phe-MePhe-Gly-Leu-Met-NH ₂ (SEQ ID NO: 24)
H-Asp-Met-His-Asp-Lys-Phe-MePhe-Gly-Leu-Met-NH ₂ (SEQ ID NO: 25)
H-Asp-Met-His-Asp-Leu-Phe-MePhe-Gly-Leu-Met-NH ₂ (SEQ ID NO: 26)
H-Asp-Met-His-Asp-Met-Phe-MePhe-Gly-Leu-Met-NH ₂ (SEQ ID NO: 27)
H-Asp-Met-His-Asp-Asn-Phe-MePhe-Gly-Leu-Met-NH ₂ (SEQ ID NO: 28)
H-Asp-Met-His-Asp-Pro-Phe-MePhe-Gly-Leu-Met-NH ₂ (SEQ ID NO: 29)
H-Asp-Met-His-Asp-Gln-Phe-MePhe-Gly-Leu-Met-NH ₂ (SEQ ID NO: 30)
H-Asp-Met-His-Asp-Arg-Phe-MePhe-Gly-Leu-Met-NH ₂ (SEQ ID NO: 31)
H-Asp-Met-His-Asp-Ser-Phe-MePhe-Gly-Leu-Met-NH ₂ (SEQ ID NO: 32)
H-Asp-Met-His-Asp-Thr-Phe-MePhe-Gly-Leu-Met-NH ₂ (SEQ ID NO: 33)
H-Asp-Met-His-Asp-Val-Phe-MePhe-Gly-Leu-Met-NH ₂ (SEQ ID NO: 34)
H-Asp-Met-His-Asp-Trp-Phe-MePhe-Gly-Leu-Met-NH ₂ (SEQ ID NO: 35)
H-Asp-Met-His-Asp-Tyr-Phe-MePhe-Gly-Leu-Met-NH ₂ (SEQ ID NO: 36)
[3] A medicament comprising the peptide according to [1] or [2] above, or a salt thereof.
[4] The medicament according to the above [3], which is a neurokinin receptor agonist.
[5] The medicament according to [4] above, wherein the neurokinin receptor is an NK3 receptor.
[6] The medicament according to [3] above, wherein tachykinins and / or neurokinin receptors are therapeutic agents for diseases associated with the onset and progression thereof.
[7] The medicament according to [6] above, wherein the disease is sexual dysfunction associated with pain or excessive / deficiency of sex hormone secretion.
[8] Tachykinins and / or neurokinin receptors are involved in the onset and progression thereof, comprising administering an effective amount of the peptide or salt thereof according to [1] or [2] to a patient in need thereof To treat the disease.
[9] The method according to [8] above, wherein the disease is sexual dysfunction associated with pain or excessive / deficiency of sex hormone secretion.
[10] A reagent comprising the peptide of [1] or [2] or a salt thereof.
[11] The peptide according to [1] or [2] above, which is used for treatment of a disease in which tachykinins and / or neurokinin receptors are involved in the onset and progression thereof.
[12] The peptide according to [11] above, wherein the disease is sexual dysfunction associated with pain or excessive / deficiency of sex hormone secretion.

Several derivatives have been found that exhibit receptor binding and agonist activity for the NK3 receptor that is approximately equivalent to or better than [MePhe ⁷ ] -NKB. In addition to elucidating the biological activity of tachykinins across animal species, a strong activity was also observed in derivatives of amino acid residues converted to be essential for biological activity in [MePhe ⁷ ] -NKB. New compounds were obtained that could not be predicted by application of the prior art.
The highly active and highly selective NK3 receptor agonist obtained by the present invention is a therapeutic agent for diseases associated with excess or deficiency of tachykinins secretion, such as pain, sexual dysfunction associated with excess or deficiency of sex hormone secretion. It is useful as a drug for improvement and as a reagent for basic science experiments for developing therapeutics for these diseases.

Hereinafter, the definition of each symbol used in this specification will be described in detail.
In the present specification, amino acids and the like are represented by abbreviations based on abbreviations by IUPAC-IUB Commission on Biochemical Nomenclature or conventional abbreviations in the field, examples of which are described below. In addition, when there are optical isomers for amino acids, “L-” indicates L-form (for example, “L-Ala” is L-form Ala), and “D-” indicates “D-”. (For example, “D-Ala” is D-form Ala), and “DL-” is D-form and L-form racemate (eg “DL-Ala” is D-form) A racemic mixture of Ala and L Ala). In the present invention, the amino acid residue may be D-form or L-form, but is preferably L-form.
Gly or G: Glycine Ala or A: Alanine Val or V: Valine Leu or L: Leucine Ile or I: Isoleucine Ser or S: Serine Thr or T: Threonine Cys or C: Cysteine Met or M: Methionine Glu or E: Glutamic acid Asp or D: Aspartic acid Lys or K: Lysine Arg or R: Arginine His or H: Histidine Phe or F: Phenylalanine Tyr or Y: Tyrosine Trp or W: Tryptophan Pro or P: Proline Asn or N: Asparagine Gln or Q: Glutamine pGlu: pyroglutamic acid MePhe: N-methylphenylalanine

  The present invention provides a peptide having an agonistic action specific to a neurokinin receptor, specifically, an NK3 receptor. Specifically, the formula (I)

(Where
A1 represents an aspartic acid residue or is deleted;
A2 represents a pyroglutamic acid, lysine or valine residue or is deleted;
A3 represents a proline, alanine or aspartic acid residue or is deleted;
A4 represents a serine, lysine, asparagine, aspartic acid, arginine, threonine or methionine residue or is deleted;
A5 represents or is missing a lysine, serine, proline, glycine or histidine residue;
A6 represents an asparagine or aspartic acid residue or is deleted;
A7 represents any α-amino acid residue or is deleted;
A8 represents an isoleucine, valine or N-methylphenylalanine residue;
Phe represents a phenylalanine residue;
Gly represents a glycine residue;
Leu represents a leucine residue;
Met-NH ₂ represents a peptide represented by methionine amide (also referred to herein as peptide (I)) or a salt thereof.

  In the above formula (I), A1 represents an aspartic acid residue or is deleted.

  In the above formula (I), A2 represents a pyroglutamic acid, lysine or valine residue or is deleted.

  In the above formula (I), A3 represents a proline, alanine or aspartic acid residue or is deleted.

  In the above formula (I), A4 represents a serine, lysine, asparagine, aspartic acid, arginine, threonine or methionine residue or is missing.

  In the above formula (I), A5 represents a lysine, serine, proline, glycine or histidine residue or is deleted.

  In the above formula (I), A6 represents an asparagine or aspartic acid residue or is deleted.

  In the above formula (I), A7 represents any α-amino acid residue or is deleted. Here, the arbitrary α-amino acid residues are usually 20 kinds of amino acid residues, namely Gly, Ala, Val, Leu, Ile, Ser, Thr, Cys, Met, Glu, Asp, Lys, Arg, His, Selected from Phe, Tyr, Trp, Pro, Asn, Gln residues.

  In the above formula (I), A8 represents an isoleucine, valine or N-methylphenylalanine residue.

Peptide (I) is preferably a peptide having the following amino acid sequence.
pGlu-Pro-Asn-Pro- Asp-Glu-Phe-MePhe-Gly-Leu-Met-NH 2 ( SEQ ID NO: 2; peptide 45)
pGlu-Pro-Asn-Pro- Asp-Asp-Phe-MePhe-Gly-Leu-Met-NH 2 ( SEQ ID NO: 3; peptide 46)
pGlu-Pro-Asp-Pro- Asn-Asp-Phe-MePhe-Gly-Leu-Met-NH 2 ( SEQ ID NO: 4; peptide 48)
pGlu-Pro-Ser-Lys- Asp-Asp-Phe-MePhe-Gly-Leu-Met-NH 2 ( SEQ ID NO: 5; peptide 50)
H-Asp-Val-Pro- Lys-Ser-Asp-Asp-Phe-MePhe-Gly-Leu-Met-NH 2 ( SEQ ID NO: 6; peptide 52)
H-Lys-Pro-Arg- Pro-Asp-Asp-Phe-MePhe-Gly-Leu-Met-NH 2 ( SEQ ID NO: 7; peptide 54)
pGlu-Ala-Asp-Pro-Asn-Asp-Phe-MePhe-Gly-Leu-Met-NH ₂ (SEQ ID NO: 8; Peptide 56)
H-Asp-Thr-Gly-Asp-Asp-Phe-MePhe-Gly-Leu-Met-NH ₂ (SEQ ID NO: 9; Peptide 58)
pGlu-Pro-Ser-Lys- Asp-Ala-Phe-MePhe-Gly-Leu-Met-NH 2 ( SEQ ID NO: 10; peptide 201)
pGlu-Pro-Ser-Lys-Asp-Asp-Phe-Ile-Gly-Leu-Met-NH ₂ (SEQ ID NO: 11; peptide 202)
H-Asp-Val-Pro- Lys-Ser-Asp-Gln-Phe-MePhe-Gly-Leu-Met-NH 2 ( SEQ ID NO: 12; peptide 203)
H-Asp-Val-Pro- Lys-Ser-Asp-Asp-Phe-Val-Gly-Leu-Met-NH 2 ( SEQ ID NO: 13; peptide 204)
H-Met-His-Asp- Phe-Phe-MePhe-Gly-Leu-Met-NH 2 ( SEQ ID NO: 14; peptide D55)
H-His-Asp-Phe- Phe-MePhe-Gly-Leu-Met-NH 2 ( SEQ ID NO: 15; peptide D56)
H-Asp-Phe-Phe-MePhe-Gly-Leu-Met-NH ₂ (SEQ ID NO: 16; peptide D57)
H-Phe-Phe-MePhe- Gly-Leu-Met-NH 2 ( SEQ ID NO: 17; peptide D58)
H-Phe-MePhe-Gly-Leu-Met-NH ₂ (SEQ ID NO: 18; peptide D59)
H-Asp-Met-His-Asp-Asp-Phe-MePhe-Gly-Leu-Met-NH ₂ (SEQ ID NO: 19; peptide 42)
H-Asp-Met-His-Asp-Ala-Phe-MePhe-Gly-Leu-Met-NH ₂ (SEQ ID NO: 20; peptide 301)
H-Asp-Met-His-Asp-Glu-Phe-MePhe-Gly-Leu-Met-NH ₂ (SEQ ID NO: 21; peptide 302)
H-Asp-Met-His-Asp-Gly-Phe-MePhe-Gly-Leu-Met-NH ₂ (SEQ ID NO: 22; peptide 303)
H-Asp-Met-His-Asp-His-Phe-MePhe-Gly-Leu-Met-NH ₂ (SEQ ID NO: 23; peptide 304)
H-Asp-Met-His-Asp-Ile-Phe-MePhe-Gly-Leu-Met-NH ₂ (SEQ ID NO: 24; peptide 305)
H-Asp-Met-His-Asp-Lys-Phe-MePhe-Gly-Leu-Met-NH ₂ (SEQ ID NO: 25; peptide 306)
H-Asp-Met-His-Asp-Leu-Phe-MePhe-Gly-Leu-Met-NH ₂ (SEQ ID NO: 26; peptide 307)
H-Asp-Met-His-Asp-Met-Phe-MePhe-Gly-Leu-Met-NH ₂ (SEQ ID NO: 27; peptide 308)
H-Asp-Met-His-Asp-Asn-Phe-MePhe-Gly-Leu-Met-NH ₂ (SEQ ID NO: 28; peptide 309)
H-Asp-Met-His-Asp-Pro-Phe-MePhe-Gly-Leu-Met-NH ₂ (SEQ ID NO: 29; peptide 310)
H-Asp-Met-His-Asp-Gln-Phe-MePhe-Gly-Leu-Met-NH ₂ (SEQ ID NO: 30; peptide 312)
H-Asp-Met-His-Asp-Arg-Phe-MePhe-Gly-Leu-Met-NH ₂ (SEQ ID NO: 31; peptide 313)
H-Asp-Met-His-Asp-Ser-Phe-MePhe-Gly-Leu-Met-NH ₂ (SEQ ID NO: 32; peptide 314)
H-Asp-Met-His-Asp-Thr-Phe-MePhe-Gly-Leu-Met-NH ₂ (SEQ ID NO: 33; peptide 315)
H-Asp-Met-His-Asp-Val-Phe-MePhe-Gly-Leu-Met-NH ₂ (SEQ ID NO: 34; peptide 316)
H-Asp-Met-His-Asp-Trp-Phe-MePhe-Gly-Leu-Met-NH ₂ (SEQ ID NO: 35; peptide 317)
H-Asp-Met-His-Asp-Tyr-Phe-MePhe-Gly-Leu-Met-NH ₂ (SEQ ID NO: 36; peptide 318)

Peptide (I) may be in the form of a salt. Examples of such salts include metal salts, ammonium salts, salts with organic bases, salts with inorganic acids, salts with organic acids, salts with basic or acidic amino acids, and the like. Preferable examples of the metal salt include alkali metal salts such as sodium salt and potassium salt; alkaline earth metal salts such as calcium salt, magnesium salt and barium salt; aluminum salt and the like. Preferable examples of the salt with an organic base include, for example, trimethylamine, triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, N, N′-dibenzylethylenediamine. And the like. Preferable examples of the salt with inorganic acid include salts with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid and the like. Preferable examples of the salt with organic acid include formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, and benzenesulfone. And salts with acid, p-toluenesulfonic acid and the like. Preferable examples of salts with basic amino acids include salts with arginine, lysine, ornithine and the like, and preferable examples of salts with acidic amino acids include salts with aspartic acid and glutamic acid, for example. It is done.
Of these, pharmaceutically acceptable salts are preferred. For example, when the compound has an acidic functional group, an inorganic salt such as an alkali metal salt (eg, sodium salt, potassium salt), an alkaline earth metal salt (eg, calcium salt, magnesium salt, barium salt), When the ammonium salt has a basic functional group in the compound, for example, a salt with an inorganic acid accompanied by hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, or acetic acid, phthalic acid, fumaric acid Preferred are salts with organic acids such as oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, methanesulfonic acid and p-toluenesulfonic acid.

Peptide (I) can be produced according to a peptide synthesis method known per se. For peptide synthesis, for example, either solid phase synthesis or liquid phase synthesis may be used. That is, the target peptide can be produced by repeatedly condensing the partial peptide or amino acid that can constitute peptide (I) and the remaining portion in accordance with the desired sequence. When the product having the desired sequence has a protecting group, the target peptide can be produced by removing the protecting group. Examples of known condensation methods and protecting group elimination methods include the methods described in the following (1) to (5).
(1) M. Bodanszky and MA Ondetti, Peptide Synthesis, Interscience Publishers, New York (1966)
(2) Schroeder and Luebke, The Peptide, Academic Press, New York (1965)
(3) Nobuo Izumiya et al., Basics and Experiments of Peptide Synthesis, Maruzen Co., Ltd. (1975)
(4) Haruaki Yajima and Shunpei Sugawara, Biochemistry Experiment Course 1, Protein Chemistry IV, 205, (1977)
(5) Supervised by Yajima Haruaki, Development of follow-up medicines Volume 14: Peptide synthesis Hirokawa Shoten In addition, after the reaction, the present invention is combined with conventional purification methods such as solvent extraction, column chromatography, liquid chromatography, recrystallization, etc. Can be purified and isolated. When the peptide obtained by the above method is a free form, it can be converted into an appropriate salt by a known method. Conversely, when it is obtained as a salt, it can be converted into a free form by a known method. .
The raw material compound may be a salt, and examples of such a salt include the same as those described above as the salt of the peptide of the present invention.

  Regarding the condensation of protected amino acids or peptides, various activating reagents that can be used for peptide synthesis can be used, and triphosphonium salts, tetramethyluronium salts, carbodiimides, and the like are particularly preferable. Trisphosphonium salts include benzotriazol-1-yloxytris (pyrrolidino) phosphonium hexafluorophosphate (PyBOP), bromotris (pyrrolidino) phosphonium hexafluorophosphate (PyBroP), 7-azabenzotriazole- 1-yloxytris (pyrrolidino) phosphonium hexafluorophosphate (PyAOP), tetramethyluronium salts are 2- (1H-benzotriazol-1-yl) -1,1,3,3-hexafluoro Phosphate (HBTU), 2- (7-azabenzotriazol-1-yl) -1,1,3,3-hexafluorophosphate (HATU), 2- (1H-benzotriazol-1-yl) -1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU), 2- (5-norbornene-2,3-dicarboximide) -1,1,3,3-tetramethyluronium tetra Fluoroborate (TNT U), O- (N-succimidyl) -1,1,3,3-tetramethyluronium tetrafluoroborate (TSTU), carbodiimides include DCC, N, N'-diisopropylcarbodiimide (DIPCDI), N- And ethyl-N ′-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI · HCl). These condensations include racemization inhibitors such as N-hydroxy-5-norbornene-2,3-dicarboximide (HONB), 1-hydroxybenzotriazole (HOBt), 1-hydroxy-7-azabenzotriazole ( HOAt), 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (HOOBt) etc.) is preferred. The solvent used for the condensation may be appropriately selected from solvents that are known to be usable for peptide condensation reactions. For example, anhydrous or hydrous acid amides such as N, N-dimethylformamide, N, N-dimethylacetamide and N-methylpyrrolidone, halogenated hydrocarbons such as methylene chloride and chloroform, alcohols such as trifluoroethanol and phenol , Sulfoxides such as dimethyl sulfoxide, tertiary amines such as pyridine, ethers such as dioxane and tetrahydrofuran, nitriles such as acetonitrile and propionitrile, esters such as methyl acetate and ethyl acetate, or appropriate mixtures thereof Is used. The reaction temperature is appropriately selected from a range known to be usable for peptide bond formation reaction, and is usually appropriately selected from the range of about -20 ° C to 50 ° C. The activated amino acid derivative is usually used in an excess of 1.5 to 6 times. In the case of solid phase synthesis, as a result of a test using the ninhydrin reaction, if the condensation is insufficient, sufficient condensation can be performed by repeating the condensation reaction without removing the protecting group. If sufficient condensation cannot be obtained even after repeating the reaction, the unreacted amino acid can be acylated with acetic anhydride or acetylimidazole so as not to affect the subsequent reaction.

Examples of the protecting group for the amino group of the starting amino acid include benzyloxycarbonyl (Z), tert-butoxycarbonyl (Boc), tert-pentyloxycarbonyl, isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, Cl-Z , Br-Z, adamantyloxycarbonyl, trifluoroacetyl, phthaloyl, formyl, 2-nitrophenylsulfenyl, diphenylphosphinothioyl, 9-fluorenylmethyloxycarbonyl (Fmoc), trityl (Trt), etc. .
Examples of the protecting group for the carboxyl group of the raw material amino acid include tert-butyl (Bu ^t ) group, benzyl (Bzl) group, C _1-6 alkyl group, C _3-10 cycloalkyl group, C _7-14 aralkyl group. Other examples include allyl, 2-adamantyl, 4-nitrobenzyl, 4-methoxybenzyl, 4-chlorobenzyl, phenacyl and benzyloxycarbonyl hydrazide, tert-butoxycarbonyl hydrazide, trityl hydrazide and the like.
The hydroxyl groups of serine and threonine can be protected, for example, by esterification or etherification. Examples of the group suitable for esterification include a lower (C _2-4 ) alkanoyl group such as an acetyl group, an aroyl group such as a benzoyl group, and a group derived from an organic acid. Examples of groups suitable for etherification include benzyl, tetrahydropyranyl, Bu ^t , Trt and the like.
Examples of groups for protecting the phenolic hydroxyl group of tyrosine include Bzl, 2,6-di-chlorobenzyl, 2-nitrobenzyl, Br-Z, Bu ^t, and the like.
Examples of the protecting group for imidazole of histidine include p-toluenesulfonyl (Tos), 4-methoxy-2,3,6-trimethylbenzenesulfonyl (Mtr), 2,4-dinitrophenyl (DNP), benzyloxymethyl (Bom ), Tert-butoxymethyl (Bum), Boc, Trt, Fmoc and the like.
Examples of protecting groups for the guanidino group of arginine include Tos, Z, 4-methoxy-2,3,6-trimethylbenzenesulfonyl (Mtr), p-methoxybenzenesulfonyl (MBS), 2,2,5,7,8. -Pentamethylchroman-6-sulfonyl (Pmc), mesitylene-2-sulfonyl (Mts), 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf), Boc, Z, NO ₂ etc. Is mentioned.
Examples of the protecting group for the side chain amino group of lysine include Z, Cl-Z, trifluoroacetyl, Boc, Fmoc, Trt, Mtr, 4,4-dimethyl-2,6-dioxocyclohexylideneyl (Dde ) And the like.
Examples of indolyl protecting groups for tryptophan include formyl (For), Z, Boc, Mts, Mtr and the like.
Examples of protecting groups for asparagine and glutamine include Trt, xanthyl (Xan), 4,4′-dimethoxybenzhydryl (Mbh), 2,4,6-trimethoxybenzyl (Tmob) and the like.
Examples of the activated carboxyl group of the raw material include the corresponding acid anhydride, azide, active ester [alcohol (eg, pentachlorophenol, 2,4,5-trichlorophenol, 2,4-dinitrophenol, cyano Methyl alcohol, paranitrophenol, HONB, N-hydroxysuccinimide, HOBt, and HOAt)] and the like. Examples of the activated amino group of the raw material include a corresponding phosphite amide.

Examples of methods for removing (eliminating) protecting groups include catalytic reduction in a hydrogen stream in the presence of a catalyst such as Pd black or Pd carbon, and anhydrous hydrogen fluoride, methanesulfonic acid, trifluoromethanesulfonic acid. , Acid treatment with trifluoroacetic acid, trimesylsilane bromide (TMSBr), trimethylsilyl trifluoromethanesulfonate, tetrafluoroboric acid, tris (trifluoro) boron, boron tribromide or a mixture thereof, diisopropylethylamine, triethylamine , Base treatment with piperidine, piperazine, etc., reduction with sodium in liquid ammonia, and the like. The elimination reaction by the acid treatment is generally performed at a temperature of −20 ° C. to 40 ° C. In the acid treatment, a cation scavenger such as anisole, phenol, thioanisole, metacresol, paracresol, dimethyl sulfide, 1 Addition of 1,4-butanedithiol, 1,2-ethanedithiol, etc. is effective. The 2,4-dinitrophenyl group used as the imidazole protecting group of histidine was removed by thiophenol treatment, and the formyl group used as the indole protecting group of tryptophan was the above 1,2-ethanedithiol and 1,4-butane. In addition to deprotection by acid treatment in the presence of dithiol or the like, it is also removed by alkali treatment with dilute sodium hydroxide, dilute ammonia or the like.
The protection of the functional group that should not be involved in the reaction of the raw material and the protection group, the elimination of the protective group, the activation of the functional group involved in the reaction, etc. can be appropriately selected from known protective groups or known means.

  Peptide (I) has methionine amide at the C-terminus. Peptide amides can be obtained by solid-phase synthesis using a resin for amide synthesis or by amidating the α-carboxyl group of the carboxyl-terminal amino acid and then bringing the peptide chain to the desired chain length on the amino group side. After the extension, a peptide excluding only the protecting group at the N-terminal α-amino group of the peptide chain and a peptide (or amino acid) excluding only the protecting group at the C-terminal carboxyl group were produced, Condensation in a mixed solvent as described above. The details of the condensation reaction are the same as described above. After purifying the protected peptide obtained by the condensation, all the protecting groups are removed by the above-described method to obtain the desired crude polypeptide. This crude peptide can be purified using various known purification means, and the main fraction can be lyophilized to obtain an amide of the desired peptide.

When peptide (I) exists as a configurational isomer (configuration isomer) such as enantiomer or diastereomer, conformer (conformation isomer), etc., these are also contained as peptide (I), If desired, each can be isolated by means known per se, the above-described separation and purification means. When peptide (I) is a racemate, it can be separated into an S form and an R form by a conventional optical resolution means.
When a stereoisomer exists in peptide (I), the case where this isomer is a single isomer or a mixture thereof is also included in peptide (I).
The peptide (I) may be a solvate (eg, hydrate) or a non-solvate (eg, non-hydrate).
Peptide (I) may be labeled with an isotope (eg, ³ H, ¹⁴ C, ³⁵ S, ¹²⁵ I) or the like.
Furthermore, peptide (I) may be a deuterium converter obtained by converting ¹ H to ² H (D).

  The peptide in the present specification has an N-terminus (amino terminus) at the left end and a C-terminus (carboxyl terminus) at the right end according to the convention of peptide notation. When "H-" is written in the N-terminal amino acid of the peptide, it indicates that the terminal amino group is not derivatized.

The peptide (I) may be a crystal, and the crystal form of the crystal may be single or plural. Crystals can be produced using a crystallization method known per se.
Peptide (I) may be a pharmaceutically acceptable cocrystal or cocrystal salt. Here, co-crystals or co-crystal salts are two or more unique at room temperature, each having different physical properties (eg structure, melting point, heat of fusion, hygroscopicity, solubility and stability). It means a crystalline substance composed of a simple solid. The cocrystal or cocrystal salt can be produced according to a cocrystallization method known per se.
Peptide (I) crystals have excellent physicochemical properties (eg, melting point, solubility, stability) and biological properties (eg, pharmacokinetics (absorbability, distribution, metabolism, excretion), expression of medicinal properties). As extremely useful.

Peptide (I) has an NK3 receptor agonistic action. From this action, peptide (I) can be used for mammals (eg, humans, monkeys, cats, pigs, horses, cows, mice, rats, guinea pigs, dogs, rabbits, etc.) tachykinins and / or neurokinin receptors. Is useful as a preventive or therapeutic agent for diseases and pathological conditions related to its onset and progression.
Examples of such diseases and pathologies include sexual dysfunction / developmental failure due to excessive or insufficient sex hormone secretion, benign tumors, malignant tumors, metabolic abnormalities, and the like.

  Peptide (I) can be used as a medicine by formulation according to a method known per se, for example, the method described in Japanese Pharmacopoeia, as it is or with a pharmacologically acceptable carrier.

The pharmaceutical comprising peptide (I) has low toxicity, and peptide (I) can be used as it is or pharmacologically acceptable carrier according to per se known means generally used in the preparation of pharmaceutical preparations. For example, tablets (including sugar-coated tablets, film-coated tablets, sublingual tablets, orally disintegrating tablets), powders, granules, capsules (including soft capsules and microcapsules), liquids, troches, and syrups , Emulsion, suspension, injection (eg, subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection, etc.), external preparation (eg, nasal preparation, transdermal preparation, ointment) , Suppositories (eg, rectal suppositories, vaginal suppositories), pellets, nasal preparations, pulmonary preparations (inhalants), drops, etc. Intravenous administration can be safely administered.
These preparations may be controlled-release preparations (eg, sustained-release microcapsules) such as immediate-release preparations or sustained-release preparations.
The content of peptide (I) in the pharmaceutical preparation is about 0.01 to about 100% by weight of the whole preparation.
The dose of peptide (I) is appropriately selected depending on the administration subject, symptoms, administration method and the like. For example, when peptide (I) is orally administered, the dose to humans (with a body weight of 60 kg) is about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg. When the peptide (I) is administered parenterally, the dose for humans (with a body weight of 60 kg) is about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.5 per day. -10 mg. This amount can be administered in one to several times a day.

Examples of the pharmacologically acceptable carrier that may be used in the production of the medicament of the present invention include various organic or inorganic carrier substances that are conventionally used as a preparation material. For example, excipients, lubricants in solid preparations, Examples include binders and disintegrants, solvents in liquid preparations, solubilizers, suspending agents, tonicity agents, buffers, and soothing agents. If necessary, additives such as conventional preservatives, antioxidants, colorants, sweeteners, adsorbents, wetting agents and the like can be used in appropriate amounts.
Examples of the excipient include lactose, sucrose, D-mannitol, starch, corn starch, crystalline cellulose, light anhydrous silicic acid and the like.
Examples of the lubricant include magnesium stearate, calcium stearate, talc, colloidal silica and the like.
Examples of the binder include crystalline cellulose, sucrose, D-mannitol, dextrin, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, starch, sucrose, gelatin, methylcellulose, sodium carboxymethylcellulose and the like.
Examples of the disintegrant include starch, carboxymethyl cellulose, carboxymethyl cellulose calcium, carboxymethyl starch sodium, L-hydroxypropyl cellulose, and the like.
Examples of the solvent include water for injection, alcohol, propylene glycol, macrogol, sesame oil, corn oil, olive oil and the like.
Examples of the solubilizer include polyethylene glycol, propylene glycol, D-mannitol, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate and the like.
Examples of the suspending agent include surfactants such as stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, and glyceryl monostearate; for example, polyvinyl alcohol, polyvinylpyrrolidone, carboxy Examples include hydrophilic polymers such as sodium methylcellulose, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, and hydroxypropylcellulose.
Examples of the isotonic agent include glucose, D-sorbitol, sodium chloride, glycerin, D-mannitol and the like.
Examples of the buffer include buffer solutions of phosphate, acetate, carbonate, citrate and the like.
Examples of soothing agents include benzyl alcohol.
Examples of the preservative include parahydroxybenzoic acid esters, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid and the like.
Examples of the antioxidant include sulfite, ascorbic acid, α-tocopherol and the like.
Examples of the colorant include water-soluble edible tar dyes (eg, edible dyes such as edible red Nos. 2 and 3, edible yellows Nos. 4 and 5, edible blue Nos. 1 and 2), water-insoluble lake dyes (eg, And aluminum pigments of water-soluble edible tar pigments), natural pigments (eg, β-carotene, chlorophyll, bengara) and the like.
Examples of the sweetening agent include saccharin sodium, dipotassium glycyrrhizinate, aspartame, stevia and the like.
Examples of the adsorbent include porous starch, calcium silicate (trade name: Florite RE), magnesium aluminate metasilicate (trade name: Neusilin), and light anhydrous silicic acid (trade name: Cylicia).
Examples of the wetting agent include propylene glycol monostearate, sorbitan monooleate, diethylene glycol monolaurate, and polyoxyethylene lauryl ether.

  When producing an oral preparation, it may be coated for the purpose of taste masking, enteric properties or sustainability, if necessary.

  Examples of the coating base used for coating include a sugar coating base, a water-soluble film coating base, an enteric film coating base, and a sustained-release film coating base.

  As the sugar coating base, sucrose is used, and one or more selected from talc, precipitated calcium carbonate, gelatin, gum arabic, pullulan, carnauba wax and the like may be used in combination.

  Examples of the water-soluble film coating base include cellulose polymers such as hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxyethylcellulose, and methylhydroxyethylcellulose; polyvinyl acetal diethylaminoacetate, aminoalkyl methacrylate copolymer E [Eudragit E (trade name) ], Synthetic polymers such as polyvinylpyrrolidone; polysaccharides such as pullulan.

  Examples of the enteric film coating base include cellulose polymers such as hydroxypropylmethylcellulose phthalate, hydroxypropylmethylcellulose acetate succinate, carboxymethylethylcellulose and cellulose acetate phthalate; methacrylic acid copolymer L [Eudragit L (trade name) ], Acrylic acid polymers such as methacrylic acid copolymer LD [Eudragit L-30D55 (trade name)], methacrylic acid copolymer S [Eudragit S (trade name)], and natural products such as shellac.

  Examples of the sustained-release film coating base include cellulose polymers such as ethyl cellulose; aminoalkyl methacrylate copolymer RS [Eudragit RS (trade name)], ethyl acrylate-methyl methacrylate copolymer suspension [Eudragit Acrylic polymer such as NE (trade name)].

  Two or more of the coating bases described above may be mixed and used at an appropriate ratio. Moreover, you may use light-shielding agents, such as a titanium oxide, ferric oxide, etc. in the case of coating.

  The NK3 receptor is known to be involved in various in vivo phenomena. Therefore, the peptide (I) having an NK3 receptor agonistic action can be a useful tool for elucidating various in vivo phenomena involving the NK3 receptor. Therefore, the compound of the present invention is also useful as a research reagent.

  The present invention is further illustrated in detail by the following examples and test examples, which are merely implementations and are not intended to limit the present invention, and may be varied without departing from the scope of the present invention. May be.

Example 1 Synthesis of each peptide
Protected peptide by Fmoc solid phase synthesis method using HOBt · H ₂ O (11.5 mg, 0.075 mmol) and N, N′-diisopropylcarbodiimide (11.6 μL, 0.075 mmol) on Rink Amide resin (45.5 mg, 0.025 mmol) A resin was constructed. For the condensation of N-methylamino acid, HATU (27.6 mg, 0.073 mmol) / N, N-diisopropylethylamine (13 μL, 0.075 mmol) was used. The obtained resin was treated in a reaction solution of TFA / m-cresol / thioanisole / 1,2-ethanedithiol / H ₂ O (80: 5: 5: 5: 5) at room temperature for 2 hours to protect the side chain. Cleavage from the resin with group removal was performed. The resin was removed by filtration, and cold diethyl ether was added to the filtrate to precipitate the peptide. The obtained crude peptide was washed three times and then purified by HPLC to obtain the target peptide. The sequence and physical property data of the obtained peptide are shown in Table 1 below.

  PG-KII (SEQ ID NO: 38), PG-SP1 (SEQ ID NO: 39), Upperolein (SEQ ID NO: 40), Eledoisin (SEQ ID NO: 41), Kassinin (SEQ ID NO: 42), Bufokinin (SEQ ID NO: 43), Physalaemin (SEQ ID NO: 44) and Sialokinin II (SEQ ID NO: 45) are known tachykinins.

Test Example 1 Evaluation of NK3 receptor binding inhibitory activity of radioactive NKB Flp-In CHO cells in which NK3 receptor was forcibly expressed (produced by introducing a gene to stably express NK3 receptor in Flp-In CHO cells of Invitrogen) ([ ¹²⁵ I] His ³ , MePhe ⁷ ) -NKB receptor binding inhibitory activity of various peptides was evaluated using the receptor membrane fraction prepared from the above.
First, 50 μL of a compound (peptide) solution prepared using a buffer solution (50 mM HEPES, pH 7.4, 5 mM MgCl ₂ , 1 mM CaCl ₂ , 0.1% BSA) was added to each well. After adding 25 μL of a solution prepared so that the cell membrane fraction was 4 μL / well, 25 μL of 0.4 nM ([ ¹²⁵ I] His ³ , MePhe ⁷ ) -NKB solution was added and mixed. After incubation at room temperature for 1 hour, suction filtration was performed using a GF / B filter pretreated with 0.3% polyethyleneimine (PEI) and washed with PEI washing buffer (50 mM HEPES, pH 7.4, 0.5% BSA). Subsequently, after washing with a washing buffer (50 mM HEPES (pH 7.4), 500 mM NaCl, 0.1% BSA), the filter was sufficiently dried. Thereafter, 25 μL of MicroScint-O was added, and the radioactivity on the filter was measured at 1 min / well using TopCount NXT.

Test Example 2 Evaluation of NK3 Receptor Agonist Activity Changes in intracellular calcium concentration when Flp-In CHO cells forcibly expressing NK3 receptor were stimulated with various peptides were quantified.
First, NK3 receptor-expressing CHO cells were collected using trypsin and seeded in a 96-well plate at 4 × 10 ⁴ cells / well. After culturing overnight at 37 ° C., 50 μL of calcium indicator was added, and further cultured for 1 hour. Subsequently, 25 μL of the peptide solution prepared by 5-fold concentration was added dropwise, and the temporal change in intracellular calcium concentration was observed for 60 seconds by FlexStation.

Test Example 3 Evaluation of NK1 receptor binding inhibitory activity of radioactive substance P (SP) Flp-In CHO cells forcibly expressing NK1 receptor (genes that stably express NK1 receptor in Flp-In CHO cells of Invitrogen) Receptor membrane fractions prepared from the above (prepared by introduction) were used to evaluate the receptor binding inhibitory activity of [ ¹²⁵ I] -BH-SP of various peptides.
First, 50 μL of a compound prepared using a buffer solution (50 mM HEPES, pH 7.4, 5 mM MgCl ₂ , 1 mM CaCl ₂ , 0.1% BSA) was added to each well. After adding 25 μL of the solution prepared so that the cell membrane fraction was 4 μL / well, 25 μL of 0.4 nM [ ¹²⁵ I] -BH-SP solution was added and mixed. After incubation at room temperature for 1 hour, suction filtration was performed using a GF / B filter pretreated with 0.3% polyethyleneimine (PEI) and washed with PEI washing buffer (50 mM HEPES, pH 7.4, 0.5% BSA). Subsequently, after washing with a washing buffer (50 mM HEPES (pH 7.4), 500 mM NaCl, 0.1% BSA), the filter was sufficiently dried. Thereafter, 25 μL of MicroScint-O was added, and the radioactivity on the filter was measured at 1 min / well using TopCount NXT.

Test Example 4 Evaluation of NK2 receptor binding inhibitory activity of radioactive NKA Flp-In CHO cells in which NK2 receptor was forcibly expressed (produced by introducing genes into Invitrogen's Flp-In CHO cells so that the NK2 receptor is stably expressed) Using the receptor membrane fraction prepared from the above, the [ ¹²⁵ I] -NKA receptor binding inhibitory activity of various peptides was evaluated.
First, 50 μL of a compound prepared using a buffer (50 mM HEPES, pH 7.4, 5 mM MgCl ₂ , 1 mM CaCl ₂ , 0.1% BSA) was added to each well. After adding 25 μL of the solution prepared so that the cell membrane fraction was 4 μL / well, 25 μL of 0.4 nM [ ¹²⁵ I] -NKA solution was added and mixed. After incubation at room temperature for 1 hour, suction filtration was performed using a GF / B filter pretreated with 0.3% polyethyleneimine (PEI) and washed with PEI washing buffer (50 mM HEPES, pH 7.4, 0.5% BSA). Subsequently, after washing with a washing buffer (50 mM HEPES (pH 7.4), 500 mM NaCl, 0.1% BSA), the filter was sufficiently dried. Thereafter, 25 μL of MicroScint-O was added, and the radioactivity on the filter was measured at 1 min / well using TopCount NXT.

Test Example 5 In Vivo Activity Evaluation Five ovariectomized goats were used and surgically the recording electrode was placed in the vicinity of kisspeptin neurons in the hypothalamic arcuate nucleus. A recovery period of about 1 month, the neural activity is measured in real time as a multineuron firing activity (MUA) in an awake goat, and a transient increase in neural activity (MUA volley) that lasts about 2 minutes at a regular cycle I confirmed that I was awake. The MUA volley reflects the neural activity of kisspeptin neurons that secrete GnRH in pulses. In ovariectomized goats, the length of the endogenous MUA volley cycle is about 25 minutes (about ± 5 minutes), and varies somewhat depending on the individual, but within the same individual the cycle length is almost constant (within ± 2 minutes) ).
On the day of the test, the MUA measuring instrument was set and MUA was recorded continuously throughout the day. In addition, a jugular vein catheter for sample administration was placed. First, several endogenous MUA volleys occurring during the control time of about 2 hours were confirmed for each individual, and the average value (T) of the volley intervals (minutes) was calculated. Next, after the expression of an arbitrary MUA volley, the test sample solution was administered into the jugular vein at a timing of 1 / 2T. After administration, when MUA volley is expressed within 4 minutes, NK3 receptor agonist activity is positive (○), when it is expressed between 4 and 8 minutes, false positive (△), and when it occurs at more intervals, negative (×) was evaluated. The MUA volley confirmed in the negative sample is endogenous.
The test sample solution was prepared as follows. On the test day, 7.5 μL of a test sample prepared at a concentration of 10 mM in DMSO was dissolved in 4.5 mL of sterile distilled water. After confirming that it was completely dissolved without precipitation, 0.5 mL of sterile 9% NaCl was added, and the final concentration of the solution was 75 nmol / 5 mL, NaCl: 0.9%, DMSO: 0.15%. 2 mL (30 nmol) was taken from this into a syringe and used for activity evaluation. Moreover, in principle, activity evaluation was performed using arbitrary 2 of 5 animals per test sample.

  Table 2 shows the results of comparison of biological activities of tachykinins derived from various animals or derivatives thereof and the peptide (I) of the present invention based on Test Examples 1 to 5.

  Table 3 shows the results of comparing the biological activities of Eledoisin and Kassinin and the peptide (I) of the present invention based on Test Examples 1 to 4.

Table 4 shows the results of examining the biological activity of each peptide in which the N-terminal side of [MePhe ⁷ ] -NKB was deleted based on Test Examples 1 to 4.

In addition, the peptides D55, D56, D57, D58 and D59 were evaluated for stability in rat serum. The stability was evaluated by incubating each peptide obtained by solid phase synthesis at 37 ° C. in the presence of rat serum and analyzing the change with time by HPLC and ESI-MS. As a control, [MePhe ⁷ ] -NKB was used. Although [MePhe ⁷ ] -NKB was confirmed to disappear within approximately 6 hours, the peptides of the present invention showed slower degradation rates.

The results of examining the biological activity of each peptide in which the fifth amino acid of [MePhe ⁷ ] -NKB (corresponding to A7 in the peptide (I) of the present invention) was variously changed based on Test Examples 1 to 4 are shown. As shown in FIG.

[Sequence Listing Free Text]
SEQ ID NO: 1; peptide (I)
SEQ ID NO: 2; peptide 45
SEQ ID NO: 3; peptide 46
SEQ ID NO: 4; peptide 48
SEQ ID NO: 5; peptide 50
SEQ ID NO: 6; peptide 52
SEQ ID NO: 7; peptide 54
SEQ ID NO: 8; peptide 56
SEQ ID NO: 9; peptide 58
SEQ ID NO: 10; peptide 201
SEQ ID NO: 11; peptide 202
SEQ ID NO: 12; peptide 203
SEQ ID NO: 13; peptide 204
SEQ ID NO: 14; peptide D55
SEQ ID NO: 15; peptide D56
SEQ ID NO: 16; peptide D57
SEQ ID NO: 17; peptide D58
SEQ ID NO: 18; peptide D59
SEQ ID NO: 19; peptide 42
SEQ ID NO: 20; peptide 301
SEQ ID NO: 21; peptide 302
SEQ ID NO: 22; peptide 303
SEQ ID NO: 23; peptide 304
SEQ ID NO: 24; peptide 305
SEQ ID NO: 25; peptide 306
SEQ ID NO: 26; peptide 307
SEQ ID NO: 27; peptide 308
SEQ ID NO: 28; peptide 309
SEQ ID NO: 29; peptide 310
SEQ ID NO: 30; peptide 312
SEQ ID NO: 31; peptide 313
SEQ ID NO: 32; peptide 314
SEQ ID NO: 33; peptide 315
SEQ ID NO: 34; peptide 316
SEQ ID NO: 35; peptide 317
SEQ ID NO: 36; peptide 318
SEQ ID NO: 37; [MePhe ⁷ ] -NKB

  The highly active and highly selective NK3 receptor agonist obtained by the present invention is a therapeutic agent for diseases associated with excess or deficiency of tachykinins secretion, such as pain, sexual dysfunction associated with excess or deficiency of sex hormone secretion. It is useful as a drug for improvement and as a reagent for basic science experiments for developing therapeutics for these diseases.

  This application is based on Japanese Patent Application No. 2012-231920 filed in Japan (filing date: October 19, 2012), the contents of which are incorporated in full herein.

Claims (14)

Formula (I)
(Where
A1 represents an aspartic acid residue or is deleted;
A2 represents a pyroglutamic acid, lysine or valine residue or is deleted;
A3 represents a proline, alanine or aspartic acid residue or is deleted;
A4 represents a serine, lysine, asparagine, aspartic acid, arginine, threonine or methionine residue or is deleted;
A5 represents or is missing a lysine, serine, proline, glycine or histidine residue;
A6 represents an asparagine or aspartic acid residue or is deleted;
A7 represents any α-amino acid residue or is deleted;
A8 represents an isoleucine, valine or N-methylphenylalanine residue;
Phe represents a phenylalanine residue;
Gly represents a glycine residue;
Leu represents a leucine residue;
Met-NH ₂ represents methionine amide) or a salt thereof. The peptide or salt thereof according to claim 1, wherein the peptide or salt thereof is any one peptide or salt thereof represented by the following sequence;
pGlu-Pro-Asn-Pro-Asp-Glu-Phe-MePhe-Gly-Leu-Met-NH ₂ (SEQ ID NO: 2)
pGlu-Pro-Asn-Pro-Asp-Asp-Phe-MePhe-Gly-Leu-Met-NH ₂ (SEQ ID NO: 3)
pGlu-Pro-Asp-Pro-Asn-Asp-Phe-MePhe-Gly-Leu-Met-NH ₂ (SEQ ID NO: 4)
pGlu-Pro-Ser-Lys-Asp-Asp-Phe-MePhe-Gly-Leu-Met-NH ₂ (SEQ ID NO: 5)
H-Asp-Val-Pro-Lys-Ser-Asp-Asp-Phe-MePhe-Gly-Leu-Met-NH ₂ (SEQ ID NO: 6)
H-Lys-Pro-Arg-Pro-Asp-Asp-Phe-MePhe-Gly-Leu-Met-NH ₂ (SEQ ID NO: 7)
pGlu-Ala-Asp-Pro-Asn-Asp-Phe-MePhe-Gly-Leu-Met-NH ₂ (SEQ ID NO: 8)
H-Asp-Thr-Gly-Asp-Asp-Phe-MePhe-Gly-Leu-Met-NH ₂ (SEQ ID NO: 9)
pGlu-Pro-Ser-Lys-Asp-Ala-Phe-MePhe-Gly-Leu-Met-NH ₂ (SEQ ID NO: 10)
pGlu-Pro-Ser-Lys-Asp-Asp-Phe-Ile-Gly-Leu-Met-NH ₂ (SEQ ID NO: 11)
H-Asp-Val-Pro-Lys-Ser-Asp-Gln-Phe-MePhe-Gly-Leu-Met-NH ₂ (SEQ ID NO: 12)
H-Asp-Val-Pro-Lys-Ser-Asp-Asp-Phe-Val-Gly-Leu-Met-NH ₂ (SEQ ID NO: 13)
H-Met-His-Asp-Phe-Phe-MePhe-Gly-Leu-Met-NH ₂ (SEQ ID NO: 14)
H-His-Asp-Phe-Phe-MePhe-Gly-Leu-Met-NH ₂ (SEQ ID NO: 15)
H-Asp-Phe-Phe-MePhe-Gly-Leu-Met-NH ₂ (SEQ ID NO: 16)
H-Phe-Phe-MePhe-Gly-Leu-Met-NH ₂ (SEQ ID NO: 17)
H-Phe-MePhe-Gly-Leu-Met-NH ₂ (SEQ ID NO: 18)
H-Asp-Met-His-Asp-Asp-Phe-MePhe-Gly-Leu-Met-NH ₂ (SEQ ID NO: 19)
H-Asp-Met-His-Asp-Ala-Phe-MePhe-Gly-Leu-Met-NH ₂ (SEQ ID NO: 20)
H-Asp-Met-His-Asp-Glu-Phe-MePhe-Gly-Leu-Met-NH ₂ (SEQ ID NO: 21)
H-Asp-Met-His-Asp-Gly-Phe-MePhe-Gly-Leu-Met-NH ₂ (SEQ ID NO: 22)
H-Asp-Met-His-Asp-His-Phe-MePhe-Gly-Leu-Met-NH ₂ (SEQ ID NO: 23)
H-Asp-Met-His-Asp-Ile-Phe-MePhe-Gly-Leu-Met-NH ₂ (SEQ ID NO: 24)
H-Asp-Met-His-Asp-Lys-Phe-MePhe-Gly-Leu-Met-NH ₂ (SEQ ID NO: 25)
H-Asp-Met-His-Asp-Leu-Phe-MePhe-Gly-Leu-Met-NH ₂ (SEQ ID NO: 26)
H-Asp-Met-His-Asp-Met-Phe-MePhe-Gly-Leu-Met-NH ₂ (SEQ ID NO: 27)
H-Asp-Met-His-Asp-Asn-Phe-MePhe-Gly-Leu-Met-NH ₂ (SEQ ID NO: 28)
H-Asp-Met-His-Asp-Pro-Phe-MePhe-Gly-Leu-Met-NH ₂ (SEQ ID NO: 29)
H-Asp-Met-His-Asp-Gln-Phe-MePhe-Gly-Leu-Met-NH ₂ (SEQ ID NO: 30)
H-Asp-Met-His-Asp-Arg-Phe-MePhe-Gly-Leu-Met-NH ₂ (SEQ ID NO: 31)
H-Asp-Met-His-Asp-Ser-Phe-MePhe-Gly-Leu-Met-NH ₂ (SEQ ID NO: 32)
H-Asp-Met-His-Asp-Thr-Phe-MePhe-Gly-Leu-Met-NH ₂ (SEQ ID NO: 33)
H-Asp-Met-His-Asp-Val-Phe-MePhe-Gly-Leu-Met-NH ₂ (SEQ ID NO: 34)
H-Asp-Met-His-Asp-Trp-Phe-MePhe-Gly-Leu-Met-NH ₂ (SEQ ID NO: 35)
H-Asp-Met-His-Asp-Tyr-Phe-MePhe-Gly-Leu-Met-NH ₂ (SEQ ID NO: 36)   A medicament comprising the peptide according to claim 1 or a salt thereof.   A medicament comprising the peptide according to claim 2 or a salt thereof.   The medicament according to claim 3, which is a neurokinin receptor agonist.   The medicament according to claim 4, which is a neurokinin receptor agonist.   The medicament according to claim 5, wherein the neurokinin receptor is an NK3 receptor.   The medicament according to claim 6, wherein the neurokinin receptor is an NK3 receptor.   The medicament according to claim 3, wherein tachykinins and / or neurokinin receptors are therapeutic agents for diseases associated with the onset and progression thereof.   The medicament according to claim 4, wherein tachykinins and / or neurokinin receptors are therapeutic agents for diseases associated with their onset and progression.   The medicament according to claim 9, wherein the disease is sexual dysfunction associated with pain or excess / deficiency of sex hormone secretion.   The medicament according to claim 10, wherein the disease is sexual dysfunction associated with pain or excess / deficiency of sex hormone secretion.   A reagent comprising the peptide according to claim 1 or a salt thereof.   A reagent comprising the peptide according to claim 2 or a salt thereof.