Somatostatin agonists

FIELD: bioorganic chemistry.

SUBSTANCE: invention provides somatostatin agonists of general formula: A1-cyclo{Cys-A2-D-Trp-A3-A4-Cys}-A5-Y1 (I), wherein A1 represents aromatic D- or L-α-amino acid selected from Phe, D-Phe, Tyr, D-Tyr, β-Nal, D-β-Nal, Cha, or D-Cha; A2 aromatic α-amino acid selected from Phe, Tyr, β-Nal, and Cha; A3 Lys or Orn; A4 β-hydroxyvaline, Ser, hSer, or Thr; A5 β-hydroxyvaline, Ser, hSer, or Thr; and Y1 represents NH2; amide nitrogen atoms of peptide groups and amine group of A1 in compound I are optionally substituted by methyl group, provided that at least one methyl group is available and that compound I cannot have following formula: D-Phe-cyclo{Cys-Phe-D-Trp-Lys-(N-Me-Thr)-Cys}-Thr-NH-2. pharmaceutically acceptable salts of compound I are also claimed.

EFFECT: expanded synthetic possibilities in peptide synthesis.

24 cl, 2 tbl, 18 ex

 

Background of the invention

It has been shown that somatostatin (SRIF), tetradecapeptide found Brazeau et al., has a strong inhibitory action on various secretory processes in tissues such as the pituitary, pancreas and gastrointestinal tract. SRIF also acts as a neuromodulator in the Central nervous system. These biological actions of SRIF, all inhibitory in nature, induced through a series of related G-protein receptors, which were described five different types (SSTR-1 - SSTR-5). These five subtypes have similar affinity in relation to endogenous SRIF-ligands, but have a different distribution in different tissues. SRIF is associated with five different receptor subtypes (SSTR) with relatively high and equal affinity for each subtype. SRIF provides a variety of effects, including modulation of the release of hormones such as growth hormone, glucagon, insulin, Amylin, and neurotransmitter release. Some of these effects were associated with its binding to the specific receptor SRIF. For example, inhibition of growth hormone has been attributed to the somatostatin receptor type 2 (SSTR-2) (Raynor et al., Molecular Pharmacol. 43:838 (1993); Lloyd et al., Am. J. Physiol. 268:G102 (1995)), whereas inhibition of insulin has been attributed to receptor somatostatin type-5 (SSTR-5) (Coy, et al., 197:366-371 (1993)). Asset is the s types 2 and 5 was associated with suppression of growth hormone and more specifically GH-secreting adenomas (acromegaly) and TSH-secreting adenomas. Activation of type 2 but not type 5, was associated with the treatment of prolactin-secreting adenomas.

As is well known to specialists in this field, SRIF and its analogs applicable in the treatment of a large number of various diseases and/or conditions. Exemplary, but not exhaustive list of such diseases and/or conditions include: Cushing's syndrome (see Clark, R.V. et al., Clin. Res. 38, p. 943A, 1990); gonadotropinum (see Ambrosi B., et al., Acta Endocr. (Copenh.) 122, 569-576, 1990); hyperparathyroidism (see Miller, D., et al., Canal. Med. Ass. J., Vol. 145, pp. 227-228, 1991); Paget's disease (see Palmieri, G.M.A., et al., J. of Bone and Mineral Research, 7, (Suppl. 1), p. S240 (Abs. 591), 1992); vipoma (see Koberstein, B., et al., Z. Gastroenterology, 28, 295-301, 1990, Christensen, C., Acta Chir. Scand. 155, 541-543, 1989); nesidioblastosis and hyperinsulinism (see Laron, Z., Israel J. Med. Sci., 26., No. 1, 1-2, 1990, Wilson, D.C., Irish J. Med. Sci., 158, No. 1, 31-32, 1989 and Micic, D., et al., Digestion, 16, Suppl. 1.70. Abs. 193, 1990); gastrinoma (see Bauer, F.E., et al., Europ. J. Pharmacol., 183, 55, 1990); the syndrome of Zollinger-Ellison (see Mozell, E., et al., Surg. Gynec. Obstet., 170, 476-484, 1990); hypersecretory diarrhea associated with AIDS and other conditions caused by AIDS, see Cello, J.P., et al., Gastroenterology, 98, No. 5, Part 2, Suppl., A163 1990 due to the increased level of gastrin-releasing peptide, see Alhindawi, R., et al., Can. J. Surg., 33, 139-142, 1990; secondary graft versus host, see Bianco J.A., et al., Transplantation, 49, 1194-1195, 1990; diarrhea associated with chemotherapy, see Petrelli, N., et al., Proc. Amer. Soc. Clin. Oncol., Vol. 10, P 138, Abstr. No. 417 1991); SYN the rum irritation of the colon (see O'donnell, L.J.D., et al., Aliment oil displayed pure. Pharmacol. Therap., Vol. 4, 177-181, 1990); pancreatitis (see Tulassay, Z., et al., Gastroenterology, 98, No. 5, Part 2, Suppl., A238, 1990); Crohn's disease (see Fedorak, R.N., et al., Can. J. Gastroenterology, 3, No. 2, 53-57, 1989); systemic sclerosis (see Soudah, H., et al., Gastroenterology, 98, No. 5, Part 2, Suppl., A129, 1990); cancer of the thyroid gland (see Modigliani, E., et al., Ann. Endocr. (Paris), 50, 483-488, 1989); psoriasis (see Camisa, C., et al., Cleveland Clinic J. Med., 57, No. 1, 71-76, 1990); hypotension (see Hoeldtke, R.D., et al., Arch. Phys. Med. Rehabil., 69, 895-898, 1988 and Kooner, J.S., et al., Brit. J. Clin. Pharmacol., 28, 735P-736P, 1989); panic attacks (see Abelson, J.L., et al., Clin. Psychopharmacol., 10, 128-132, 1990); sclerodoma (see Soudah, H., Clin. Res., Vol. 39, p. 303A, 1991); obstruction of the small intestine (see Nott, D.M., et al., Brit. J. Surg., Vol. 77, p. A691, 1990); gastroesophagal reflux (see Branch, M.S., et al., Gastroenterology, Vol. 100, No. 5, Part 2, Suppl., p. A425, 1991); duodenogastric reflux (see Hasler, W., et al., Gastroenterology, Vol. 100, No. 5, Part 2, Suppl., p. A448, 1991); graves ' disease (see Chang, T.C., et al., Brit. Med. J., 304, p. 158, 1992); polycystic ovarian disease (see Prelevic, G.M., et al., Metabolism Clinical and Experimental, 41, Suppl. 2, pp. 76-79, 1992); bleeding of the upper part of the gastrointestinal tract (see Jenkins, S.A., et al., Gut, 33, pp. 404-407, 1992 and Arrigoni, A., et al., American Journal of Gastroenterology, 87, p. 1311, (abs. 275), 1992); false cyst and the pancreatic ascites (see Hartley, J.E., et al., J. Roy. Soc. Med., 85, pp. 107-108, 1992); leukemia (see Santini, et al., 78, (Suppl. 1), p. 429A (Abs. 1708), 1991); meningioma (see Koper, J.W., et al., J. Clin. Endocr, Metab., 74, pp. 543-547, 1992); and cancer cachexia (see Bartlett, D.L., et al., Surg. Forum., 42, pp. 14-16, 1991).

Other indications associated with Akti is the situation of SRIF receptor subtypes, are the inhibition of insulin and/or glucagon and, more specifically, diabetes mellitus, angiopathy, proliferative retinopathy, the phenomenon down and nephropathy; inhibition of gastric secretion of acid and, more specifically, peptic ulcers, enterocoely and pancreatology fistula syndrome irritation of the colon, Dumping syndrome, watery diarrhoeal syndrome, AIDS-related diarrhea induced by chemotherapy diarrhea, acute or chronic pancreatitis and tumor secreting gastrointestinal hormone; cancer treatment, such as hepatoma; inhibition of angiogenesis; the treatment of inflammatory disorders such as arthritis, retinopathy, chronic graft rejection; angioplasty; prevention of bleeding vascular graft and gastrointestinal bleeding.

It is preferable to have similar, which is selective in relation to specific subtype or specific SRIF receptor subtypes responsible for the desired biological response, to reduce, thus, interaction with other receptor subtypes, which could lead to undesirable side effects. In addition, due to the short half-existence of native SRIF have been developed various analogs of SRIF, for example, for the treatment of acromegaly (Raynor, et al., Molecular Pharmacol. 43:838 (1993)). Development of si is enogastromy SRIF agonists smaller led to the discovery of distinct affinely various truncated ligands against different subtypes. Apparently, the residue Trp8-Lys9often present in the ligands that are recognized by this receptor. The residue Trp8-Lys9forms part β-bending, which is usually stabilized by substitution of L-Trp, D-Trp, cyclization of the skeleton, by a disulfide bridge or all of the restrictions. One unintended consequence of this structural simplification, conducted before the discovery of multiple receptor subtypes, was the loss of affinity of binding a wide range. An example of this is the high affinity type 2, but low affinity of types 1, 3, 4 and 5 peptides in a series of OCTREOTIDE®. Thus, many basic biological research with this type of analogue could not find the effects mediated by all subtypes, but not one subtype of SRIF receptor.

The authors of this invention have found that the restriction of the peptide skeleton can be introduced N-alkylation. This modification greatly limits the subject of this action the residue and the preceding him amino acid to an extended conformation and additionally blocks the intramolecular potential sites for the formation of hydrogen bonds, as well as the sites of cleavage by proteolytic enzymes, increasing, thus, the pharmacokinetic properties of the peptide. Commercially available are only a small number of N-methyl is of minislot, and their synthesis is difficult. However, in another aspect of the present invention, the authors describe the procedure for N-methylation shortened analogs of SRIF in any amino acid residue using solid-phase procedure, borrowed from a procedure reported by Miller and Scanlan (J. Am. Chem. Soc. 1997, 119, 2301-2302).

In one aspect this invention relates to a peptide in accordance with formula (I):

And1-cyclo{Cys-A2-D-Trp-A3-A4-Cys]-A5-Y1

(I)

where:

And1denotes optionally substituted D - or L-aromatic α-amino or optionally substituted D - or L-cyclo(C3-6)alkilany;

And2denotes optionally substituted aromatic α-amino or optionally substituted cyclo(C3-6)alkilany;

And3represents Lys or Orn;

And4means β-hydroxylamin, Ser, hSer, or Thr;

And5means β-hydroxylamin, Ser, hSer, or Thr

Y1IT denotes, NH2or other1where R1means (C1-6)alkyl;

where each specified optionally substituted aromatic α-amino acid and each specified optionally substituted cyclo(C3-6)alkilany is optionally substituted by one or more substituents, each of which is independently selected from the group consisting of g is lagena, NO2, OH, CN, (C1-6)alkyl, (C2-6)alkenyl, (C2-6)quinil, (C1-6)alkoxy, Bzl, O-Bzl and NR9R10where R9and R10each independently represent H or (C1-6)alkyl; and

where the amine nitrogen of each of the peptide bond and the amino group And1formula (I) optionally substituted methyl group, provided that there is at least one specified a methyl group;

and, in addition, provided that the specified connection is not D-Phe-cyclo{Cys-Phe-D-Trp-Lys-(N-Me-Thr)-Cys}-Thr-NH2;

or its pharmaceutically acceptable salt.

A preferred group of compounds of formula (I) are compounds in which:

And1denotes Phe, D-Phe, Tyr, D-Tyr, β-Nal, D-β-Nal, Cha or D-Cha;

And2represents Phe, Tyr, β-Nal or Cha and

Y1indicates HE or NH2;

or their pharmaceutically acceptable salt.

A preferred group of compounds of the immediately preceding group of compounds are compounds in which And1represents D-Phe, or Tyr; or in which And2denotes Phe; or in which And3denotes Lys; or in which And4represents Thr; or in which And5represents Thr; or their pharmaceutically acceptable salt.

In an even more preferred embodiment, this invention describes a compound of formula (I)where the specified connection has the formula:

or its pharmaceutically acceptable salt.

In another aspect of this invention describes a compound in accordance with formula (II)

where:

And1denotes a D - or L-isomer of Ala, Leu, Ile, Val, Nle, Thr, Ser, β-Nal, β-Pal, Trp, Phe, 2,4-dichloro-Phe, pendaftar-Phe, p-X-Phe, or o-X-Phe, where X denotes CH3, Cl, Br, F, OH, OCH3or NO2;

And2represents Ala, Leu, Ile, Val, Nle, Phe, β-Nal, pyridyl-Ala, Trp, 2,4-dichloro-Phe, pendaftar-Phe, o-X-Phe or p-X-Phe, where X denotes CH3, Cl, Br, F, OH, OCH3or NO2;

And3denotes pyridyl-Ala, Trp, Phe, β-Nal, 2,4-dichloro-Phe, pendaftar-Phe, o-X-Phe or p-X-Phe, where X denotes CH3, Cl, Br, F, OH, OCH3or NO2;

And6denotes Val, Ala, Leu, Ile, Nle, Thr, Abu, or Ser;

And7represents Ala, Leu, Ile, Val, Nle, Phe, β-Nal, pyridyl-Ala, Trp, 2,4-dichloro-Phe, pendaftar-Phe, o-X-Phe or p-X-Phe, where X denotes CH3, Cl, Br, F, OH, OCH3or NO2;

And8denotes a D - or L-isomer of Ala, Leu, Ile, Val, Nle, Thr, Ser, Phe, β-Nal, pyridyl-Ala, Trp, 2,4-dichloro-Phe, pendaftar-Phe, p-X-Phe, or o-X-Phe, where X denotes CH3, Cl, Br, F, OH, OCH3or NO2;

each R1and R2independently represents H, lower acyl or lower alkyl; and R3indicates HE or NH2; where the amine nitrogen of each of the amide-peptide bonds and N-terminal amino group optionally substituted methyl group is,

provided that there is at least one specified methyl group in the compound of formula (II); that at least one of the A1and8and one of the A2and7must be an aromatic amino acid; and a1And2And7and8can't be all aromatic amino acids;

or its pharmaceutically acceptable salt.

In one embodiment, this invention describes a compound in accordance with formula (II)where the specified connection selected from the list consisting of:

or its pharmaceutically acceptable salt.

In another embodiment, this invention describes a peptide selected from the list of peptides identified by the "group III"consists of:

(amide bridge formed between Lys* Asp);

or its pharmaceutically acceptable salt, where in the compounds having two Cys residue, there is a disulfide bond, and where the amine nitrogen of each amide-peptide bond and the amino group of the N-terminal amino acids optionally substituted methyl group, PR is the condition, there is at least one specified methyl group in this connection.

The following aspect of this invention describes the SRIF agonists containing N-methylated analogs of SRIF agonists covered formulas, or agonists described in the publications below.

Application EP No. P5 164 EU (Inventor: G. Keri);

Van Binst, G. et al. Peptide Research 5:8 (1992);

Horvath, A. et al. Abstract, "Conformations of Somatostatin Analogs Having Antitumor Activity", 22nd European peptide Symposium, September 13-19, 1992, Interlaken, Switzerland;

U.S. patent No. 6001801 (1999);

U.S. patent No. 4904642 (1990);

U.S. patent No. 4871717 (1989);

U.S. patent No. 4853371 (1989);

U.S. patent No. 4725577 (1988);

U.S. patent No. 4684620 (1987);

U.S. patent No. 4650787 (1987);

U.S. patent No. 4603120 (1986);

U.S. patent No. 4585755 (1986);

Application EP 0203031 A2 (1986);

U.S. patent No. 4522813 (1985);

U.S. patent No. 4486415 (1984);

U.S. patent No. 4485101 (1984);

U.S. patent No. 4435385 (1984);

U.S. patent No. 4395403 (1983);

U.S. patent No. 4369179 (1983);

U.S. patent No. 4360516 (1982);

U.S. patent No. 4358439 (1982);

U.S. patent No. 4328214 (1982);

U.S. patent No. 4316890 (1982);

U.S. patent No. 4310518 (1982);

U.S. patent No. 4291022 (1981);

U.S. patent No. 4238481 (1980);

U.S. patent No. 4235886 (1980);

U.S. patent No. 4224190 (1980);

U.S. patent No. 4211693 (1980);

U.S. patent No. 4190648 (1980);

U.S. patent No. 4146612 (1979);

U.S. patent No. 4133782 (1979);

U.S. patent No. 5506339 (1996);

U.S. patent No. 4261885 (1981);

U.S. patent No. 4728638 (1988);

U.S. patent No. 4282143 (1981);

U.S. patent No. 421039 (1980);

U.S. patent No. 4209426 (1980);

U.S. patent No. 4190575 (1980);

Application EP 0363589 A2 (1990);

EP patent No. 0389180 (1990);

Application EP No. 0505680 (1982);

Application EP No. 0083305 (1982);

Application EP No. 0030920 (1980);

PCT application number WO 97/01579 (1997);

PCT application number WO 91/18016 (1991);

PCT application number WO 91/09056 (1991);

PCT application number WO 90/12811 (1990);

PCT application number WO 88/05052 (1988);

Application for patent of great Britain No. GB 2095261 (1981); and

Application for French patent No. FR 2522655 (1983).

The compounds of formula (I), formula (II) and group (III) of this application is applicable for the same applications that SRIF, depending on the binding specificity, or lack thereof, as may be determined is described here analyses of the binding.

Thus, in another aspect, this invention describes a method of binding one or more somatostatin receptors human subtypes 1, 2, 3, 4 and 5, which provides for the stage of introduction of one or more compounds of the formula (I) and/or formula (II) and/or group (III) or pharmaceutically acceptable salts (salts) of such compounds or of such compounds to a recipient in need of such introduction.

In a preferred variant of the method, directly above described method of inducing actions of somatostatin agonist which the stages of introduction of one or more compounds of the formula (I) and/or formula (II) and/or group (III) or pharmaceutical preparations is automatic acceptable salts (salts) of such compounds or of such compounds to the recipient, needy in this introduction.

In a more preferred variant of the method, directly above described method of treating diseases or conditions of human or other animal in need thereof which the stages of introduction of one or more compounds of the formula (I) and/or formula (II) and/or group (III) or pharmaceutically acceptable salts (salts) of such compounds or those compounds specified person or animal, where the disease or condition selected from the group consisting of Cushing's syndrome, gonadotropinum, hyperparathyroidism, Paget's disease, vipoma, nesidioblastosis and hyperinsulinism, gastrinoma syndrome Zollinger-Ellison, hypersecretory diarrhea associated with AIDS and other conditions, syndrome of irritation of the colon, pancreatitis, Crohn's disease, systemic sclerosis, thyroid cancer, psoriasis, hypotension, panic attacks, sclerodoma, obstruction of the small intestine, gastroesophagal reflux, duodenogastric reflux, graves ' disease, polycystic disease of the ovary, bleeding of the upper part of the gastrointestinal tract, false cysts of the pancreas, pancreatic ascites, leukemia, meningioma, cancer cachexia, acromegaly, restenosis, hepatoma, lung cancer, melanoma, inhibition of accelerated growth of a solid tumor, is Eisenia body weight, treatment of insulin resistance, syndrome X, prolongation of survival of pancreatic cells, fibrosis, hyperlipidemia, hyperammonemia, hyperprolactinemia and prolactinomas.

Except for the N-terminal amino acids, all abbreviations (e.g., for Phe And1) amino acids in this description represent the structure-NH-CH(R)-CO-, where R in the preceding formula represents a side chain amino acids (for example, CH3for Ala). For N-terminal amino acids of this abbreviation refers to the structure (R1R2)-N-CH(R)-CO-, where R is a side chain of amino acids and R1and R2have the above values.

Nomenclature for subtypes of somatostatin receptors is in accordance with the recommendations of IUPHAR, where SSTR-4 refers to the receptor, originally cloned Bruno et al., and SSTR-5 refers to the receptor, cloned O'carroll et al. The usual abbreviations of amino acids are in accordance with the recommendations of the IUPAC-IUB. The following are abbreviations of certain amino acids which may be found here:

Abu = α-aminobutyric acid;

Aib = α-aminoadamantane acid;

β-Ala = β-alanine;

Amp = 4-aminophenylalanine;

Ava = 5-aminosalicilova acid;

Cha = cyclohexylamine;

Gaba = γ-aminobutyric acid;

Lys = lysine;

β-Nal = β-(2-naphthyl)alanine;

Nle = norley is in;

Nva = Norvaline;

Orn = ornithine;

Pal = β-(3-pyridinyl)alanine;

Phe = phenylalanine;

Ser = serine;

hSer = homoserine;

Thr = threonine and

Tyr = tyrosine.

Additional abbreviations include:

DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene;

DCM (DHM), dichloromethane;

DIC, dicyclohexylcarbodiimide;

DIEA, diisopropylethylamine;

DMF (DMF), dimethyl formamide;

MTBD, 1,3,4,6,7,8-hexahydro-1-methyl-2H-pyrimido[1,2-a]pyrimidine;

NPS 2-nitrophenyloctyl;

TBTU, O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium-tetrafluoroborate; and

TFA (TFU), triperoxonane acid.

The compound of this invention or its pharmaceutically acceptable salt may be administered orally, parenterally (e.g., intramuscular, intraperitoneal, intravenous or subcutaneous injection or introduction of the implant), nazalnam, vaginal, rectal, sublingual, or local way of introduction, and may be prepared with pharmaceutically acceptable carriers to provide dosage forms appropriate for each route of administration.

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In such solid dosage forms the active substance is mixed with at least one inert pharmaceutically acceptable carrier such as sucrose, lactose or starch. the such dosage forms can also comprise, that is normal practice, additional substances other than inert diluents, e.g., lubricating agents such as magnesium stearate. In the case of capsules, tablets and pills, the dosage forms may also contain a buffer means. Tablets and pills can also be prepared with intersolubility coatings.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, elixirs containing inert diluents commonly used in this field, such as water. Besides such inert diluents, compositions can also include excipients, such as moistening agents, emulsifying and suspendresume agents and sweeteners, flavours and flavouring agents.

The preparations of this invention for parenteral administration include sterile aqueous and non-aqueous solutions, suspensions or emulsions. Examples of nonaqueous solvents or media are propylene glycol, polyethylene glycol, vegetable oils such as olive oil and corn oil, gelatin and suitable for injectable organic esters, such as etiloleat. Such dosage forms may also contain excipients, for example preservatives, moisturizing, emulsifying and d is spalinowa agents. They can be sterilized, for example, by filtration through a retaining bacteria filter, by incorporation of sterilizing agents into the compositions, by irradiating the compositions, or by heating the compositions. They can also be prepared in the form of sterile solid compositions which can be dissolved in sterile water or some other sterile environment for injection immediately before use.

Compositions for rectal or vaginal injection are preferably suppositories which may contain, in addition to the active substance, excipients such as cocoa butter or wax for suppositories.

Compositions for nasal or sublingual introduction also prepared with standard fillers are well known in this field.

The dosage of the active ingredient in the compositions of this invention may vary; however, it is necessary that the amount of active ingredient was such that there was obtained a suitable dosage form. The selected dosage depends upon the desired therapeutic effect, the route of administration and duration of treatment. Usually the dose levels of 25 mg/kg/day to 100 mg/kg/day (kg body weight) administered daily as a single dose or in divided multiple doses to humans and other animals, for example mammals, for the floor is placed the desired therapeutic effect.

Preferred the usual dose range is 250 mcg/kg/day to 5 mg/kg/day (per kg of body weight daily which can be administered in a single dose or in divided multiple doses.

Further, the compound of this invention or its pharmaceutically acceptable salt can be administered in a composition with a prolonged action, such as described in the following patents. Among these forms are usually preferred form for slow release within 14 days or 28 days. U.S. patent No. 5672659 describes the prolonged release composition containing a peptide and a complex polyester. U.S. patent No. 5595760 describes the prolonged release composition containing the peptide in gel form. U.S. patent No. 5821221 describes polymer compositions prolonged release containing peptide and chitosan. U.S. patent No. 5915883 describes the prolonged release composition containing a peptide and a cyclodextrin. International patent application number PCT/US99/01180 (publication number WO 99/38536, August 5, 1999) describes absorbable composition of the peptide prolonged action. The contents of the previous patents and applications incorporated herein by reference.

Application of the compositions of the immediate or extended release depends on the type of indications. If the indication is acute is or Verhoture violation, will be the preferred treatment is a form of immediate release and not the composition of extended release. On the contrary, for preventive or long-term treatments will be preferred composition sustained release.

Detailed description of the invention

On the basis of the specialist in this field will be able to use this invention in its full extent. Thus, the following private options should be considered only as illustrations of the present invention and should not be construed as limiting the full scope of this invention.

Synthesis

Resin hydrochloride 4-methylbenzhydrylamine (0.25 or 0.5 mEq/g) was obtained from Advanced ChemTech Inc., Louisville, KY. Nα-tert-butyloxycarbonyl (BOC)-protected amino acids were purchased from Bachem Inc., Torrance, CA, Advanced ChemTech Inc. and Synthetech Inc., Albany, OR. The reactive side chains of the amino acids was masked by one of the following groups: Cys, 4-methylbenzyloxycarbonyl; Lys, 2-chlorobenzenesulfonyl; Thr, O-benzyl; Tyr, O-2,6-dichlorobenzyl. All reagents and solvents were ACS purity or higher, and they were used without additional purification.

The compounds of this invention, for example, the compounds of formula (I), was synthesized on functionalized 4-methylbenzhydrylamine, 1% crosslinked polystyrene resin 0.25, or 0.5 mEq/g), 0.25 mmol-scale synthesizer Advanced ChemTech model 200) using the following Protocol: release, 40% TFU (2 min, 20 min); the cycle of leaching DCM (three washes); neutralization, 10% DIEA (1 min, 5 min); washing cycle DMF; leaching cycle DCM (twin flush); double binding; first with 1,3-diisopropylcarbodiimide esters (3 EQ.), 30 min in DCM; DCM washing (three washes); the second linking with prior esters of TBTU (3 EQ.), 90 min in DMF, with a catalytic amount of DIEA; DMF washing (one flushing; flushing DCM (three washes). Binding assays were subjected to qualitative monitoring using ninhydrin test.

Nα-protection. After removal of the protective group of amino group in a desirable site of methylation the resin suspended in DCM (20 ml). To this suspension was added kallidin (3 EQ.) and about nitrobenzenesulfonamide (3 EQ.) and this mixture was shaken using a synthesizer Advanced ChemTech model 200) for 2 hours. Then the resin was subjected to washing DCM (2 rinsing and washing DMF (3 washes). Protection was subjected to qualitative monitoring using ninhydrin test.

Nα-methylation. Secure on-nitrobenzenesulfonamide resin is suspended in DMF (20 ml)to which was added MTBD (3 EQ.) and 4-nitrobenzenesulfonate or dimethylsulfate (Cys11). The mixture was shaken with IP is the use of the synthesizer Advanced ChemTech model 200) for 0.5 h and the resin was subjected to washing DMF (4 washes).

Remove protection Nα-IU. After methylation the desired balance of the resin is again suspended in DMF (20 ml). To this suspension was added DBU (3 equiv.) and 2-mercaptoethanol (3 EQ.) and the mixture was shaken for 0.5 h using a synthesizer Advanced ChemTech model 200). Then, the resin is thoroughly washed with DMF (5 washes).

Cleavage of the peptide. The peptides were tsalala from resin carrier with simultaneous removal of the protective groups of the side chains by acidolysis using anhydrous hydrogen fluoride containing acceptor anisole (˜30% og/og)for 45 min at 0°C. the Peptides were cyclically in 90% acetic acid (˜600 ml) with a small excess of I2(15 min). Then the excess of I2was removed by the addition of ascorbic acid.

The cleaning. The crude peptides were purified preparative RP-HPLC on associated with C-18 silica gel using axial compression column (Dynamax-300A, 5 or 8 μm, with 21.4 x 250 mm). Used system elution with a linear gradient at a speed of DC 20 ml/min: A; 0.1% OF TFU,; 0,1% TFU in 80% MeCN, 20% - 50% at 1% per minute. The separation was monitored using analytical RP-HPLC at 215 nm. The fractions containing the product were combined, concentrated in vacuo and subjected to lyophilization. Each peptide was obtained as a fluffy white powder constant weight by lyophilization from aqueous acetic acid, the Purity of the final product was assessed by analytical RP-HPLC at 215 nm. Analytical RP-HPLC were recorded using media Vydac C-18 (4,6×250 μm, pore size 300 Å, Liquid Separation Group). The linear gradient used when the speed of a current of 1.5 ml/min: HPLC-1 AND 0.1% OF TFU; B, 0.1% OF TFU in 80% MeCN; 20% - 50% at 1% per minute; HPLC-2, C, 5% MeCN in TEAP (0.1 M, pH 3); D, 20% in MeCN, 10% D - 70% D at 1% per minute. Eluent from the column were monitored at 215 nm. The retention time and the purity of each peptide was evaluated using a Rainin Dynamax HPLC Method Manager.

Amino acid analysis. Peptides hydrolyzed under vacuum (110°S; 20 h) in 4 M methanesulfonic acid containing 0.2% 3-(2-amino-ethyl)indole (Pierce). Amino acid analyses were carried out in the hydrolysates after derivatization on-italiandesigned reagent (Sigma Chemicals Co.) using automated HPLC system (Rainin Instrument Co.), equipped with axial compression column C-18 100×4.6 mm, 3 μm with an integral protective column (Microsorb AAAnalysis™, Type O; Rainin Instrument Co.). Derivationally primary amino acids were suirable using dual gradient buffer A; 0.10 M sodium acetate, containing 4.5% vol./about. methanol and 0.5% vol./about. tetrahydrofuran, at a pH of 7.2, and a buffer; methanol. Used the sequence of the gradient, where 0% at 0 min; 35% And at 16.5 min; 90% at 30 min and 90% And at 33 min with a flow rate of 1.0 ml / min at ambient temperature. Eluent was subjected to monitoring at 340 N. and integrated using a Dynamax HPLC Method Manager (Rainin). Standard retention times were as follows; Asp, and 6.6 min; Arg, to 19.9 min; Trp, 25,4 min and Lys and 29.5 minutes Each peptide of table I gave the expected analytical results for the primary amino acids. Cysteine was not determined quantitatively.

Mass-spectrometry. Peptides were analyzed by mass spectrometry with a time span defined in the laser desorption/ionization using a matrix, using a mass spectrometer LaserMat 2000 (Thermal Bioanalysis, San Jose, CA) using α-cyano-4-hydroxyanisol acid as a matrix with Substance P (1348,7 Yes) as internal standard. In each case the spectra consisted of the main peak of the ions M-N for the internal standard, the expected peak of the analyte M-N and a small number of peaks associated with the matrix (<500 Da). The magnitude of the mass thus obtained for representative compounds of the present invention, shown in detail in table 1.

Inhibition similar to SRIF release of GH. Gathered front lobe of the pituitary gland of adult male rats and was dispersively their previously described method using trypsin/Gnkazy (Murphy, W.A.; Taylor, J.; Moreau, J.-P. and Coy, D.H., Peptide Res. 1989, 2, 128-132). Dispersed cells were diluted with sterile filtered medium Needle, modified by way of Dulbecco (MEM, Gibco Laboratories, Grand Island, NY), which was supplemented with 2.5% fetal calf serum (Gibco), 3% is oradini serum (Gibco), 10% fresh rat serum (stored on ice for no longer than 1 hour) from donors ' pituitary, 1% MEM with nonessential amino acids (Gibco), gentamycin (10 ng/ml, Sigma) and nystatin (10000 u/ml, Gibco). Cells were sown randomly at a density of approximately 200,000 cells per well (Costar cluster 24; Rochester Scientific Co., Rochester, NY). Seeded cells were maintained in the above medium, Dulbecco in a humidified atmosphere of 95% air/5% CO2at 37°C for 4-5 days. When preparing for the introduction of the hormone, the cells were washed with medium 199 (Gibco, 3 x 1 ml). Each dose of the compounds of this invention were tested in three replicates (wells) in a total volume of 1 ml of medium 199 containing 1% BSA (fraction V; Sigma Chemical Co.). All wells contained GHRH(1-29)NH2(1 nm). After incubation in an atmosphere of air/carbon dioxide (95/5%, 3 h at 37° (C) the medium was removed and stored at -20°to analyze the content of the hormone. Growth hormone in the media was measured by standard RIA double antibodies using components provided by Dr. A.E. Parlow, in the framework of the International programme on the hormones and the pituitary gland (NHHP) Torrance, CA. IC50agonist was calculated using Sigmaplot (Jandel Scientific, San Rafael, CA). Values were expressed as the average IC50(nm) ± SEM (standard error of the mean) (n) separate curves dose-response.

Functional expression of the cloned receptor Comat the statin person. Genomic clones containing the somatostatin receptors person (hSSTR-1 - hSSTR-5) (Yamada, Y., et al., Proc. Natl. Acad. Sci. USA. 1992, 89, 251-255; Yasuda, K., et al., J. Biol. Chem. 1992, 267, 20422-20428; Yamada, Y., et al., Mol. Pharmacol. 1992, 42, 2136-2142; Rohrer, L., et al., Proc. Natl. Acad. Sci. USA. 1993, 90, 4196-4200), were provided by Dr. Graeme I. Bell from the University of Chicago. cDNA hSSTR-1, hSSTR-2, hSSTR-3, hSSTR-4 and hSSTR-5 was isolated in the form > PST -XmnI fragment of 1.5 TPN, BamHI-HindIII fragment of 1.7 TPN, NcoI-HindIII fragment of 2.0 TPN, NheI-NdeI fragment of 1.4 TPN and HindIII-XbaI fragment of 1.2 TPN, respectively, each of which contains the entire coding region of a full-sized receptors. These fragments were independently subcloned into the corresponding sites of restricted in expressing vector pCMV5 mammals, to the right of the promoter of cytomegalovirus (CMV) human, obtaining expression plasmid pCMV5/hSSTR-1, pCMV5/hSSTR-2, pCMV5/hSSTR-3, pCMV5/hSSTR-4 and pCMV5/hSSTR-5. For transfection into cells Cho-K1 were added to the plasmid, pRSV-neo from the American type culture Collection (Rockville, MD), bearing neomicina breeding marker of mammalian cells.

Expression and transfection of the receptor. Transfection was performed by the method using calcium phosphate. Cells Cho-K1 was supported in α-minimum essential medium (α-MEM, Gibco), supplemented with 10% fetal calf serum, and were transfusional each of the expression plasmids using deposition of calcium phosphate. The clones, which is s inherits expression plasmid, selected in the α-MEM, supplemented with 500 μg/ml of geneticin (G418; Gibco). Independent clones of Cho-K1 collected consumerisim glass ring and were propagated in culture in selective medium. From the selected clones were obtained membrane, and the expression of hSSTR initially evaluated for binding with [125I]Tyr11-SRIF and [125I]MK-678 (SSTR-2).

Analyses linking radioligand. Cell membrane 5 cell types was obtained from homogenates (transmitter station, setting 6, 15 sec) of the respective cells Cho-K1 cooled on ice Tris-HCl (50 mm) and centrifuged (39000 g, 10 min × 2) intermediate resuspending in fresh buffer. The final precipitation resuspendable in Tris-HCl (10 mm) for analysis. Aliquots of membranes were incubated (30 min at 37aboutC) from 0.05 nm [125I]Tyr11-SRIF (types 1, 3, 4, 5) or [125I]MK-678 (type 2) 50 nm HEPES (pH 7.4)containing BSA (10 mg/ml); MgCl2(5 mm), Trasilol (200 CME/ml), bacitracin (0.02 mg/ml) and phenylmethanesulfonyl (0.02 mg/ml). The final volume of the reaction mixture for the assay was 0.3 ml, and incubation was stopped by rapid filtration through filters GF/C, pre-soaked in 0.3% of poly(ethylenimine), using the module for quick filtering Brandel. Then each tube and the filter was rinsed with aliquot of cold buffer (3 x 5 ml).

Specific binding is determined in a total amount associated radiali the Anda minus the number of ligand, linked in the presence of 1.0 μm SRIF. Using these analysis systems usually received the following total binding radioligand and nonspecific binding (nespec.): hSSTR-1, 7000 counts/min (total) against 3500 imp/min (nespec.); hSSTR-2, 9000 pulse/min (total) against 1000 imp/min (nespec.); hSSTR-3, 8000 pulses/min (total) against 1000 imp/min (nespec.); hSSTR-4, 6000 pulses/min (total) against 3500 imp/min (nespec.) and hSSTR-5, 7500 pulse/min (total) against 3500 imp/min (nespec.). The affinity of binding was expressed as values of Ki± SEM (nm) for each of the five receptor subtypes. Values of Kiobtained for representative compounds of the present invention, shown in detail in table 2.

Molecular modeling. All molecular modeling was performed on the computer Silicon Graphics Indigo2High Impact 10000 using SYBYL6.6 c force field of all atoms in Kollman. The PDB files for the three structures in solution NMR parent compound Sandostatin/OCTREOTIDE®; DPhe5-c[Cys6-Phe7-DTrp8-Lys9-Thr10-Cys11]-Thr12-Ola (1SOC and 2SOC) were obtained from the PDB. These structures were introduced in SYBYL6.6 and motivovany for the formation of N-methylated compounds on the basis of example 9. The partial atomic charges on Kollman was downloaded from the dictionary of monomers. These structures are optimized by annealing the mutated residue and then a full minimization of the energy with COI is whether the algorithm of conjugate gradient to the final gradient of the mean square values (rms) ≤ 0.01 kcal mol·And-1. Dependent on the distance of the dielectric function was used with default settings for all other options minimization.

Examples 9 and 18 alkilirovanie any remainder using a solid-phase procedure, collecting the resin methylbenzhydrylamine. After removal of the tert-butoxycarbonyl (BOC) group in the desired N-methylation of the free amine bound to the resin peptide was protected using o-nitrobenzenesulfonamide and collidine in dichloromethane. Then N-H amide o-nitrobenzenesulfonamide was selectively deprotonirovaniem strong, non-ionic blocked the basis of the MTBD and metilirovanie using methyl-p-nitrobenzenesulfonate in DMF. Methylated sulfonamide freed from the protective groups β-mercaptoethanol and DBU in DMF, and the reaction was easily monitored by the appearance of a bright yellow color in solution, indicating that the removal of on-nitrobenzenesulfonyl group of linked resin peptide. This removal of the protective groups were also slower, if N-sulfonic was not alkilirovanny, i.e. when the blocking realtimebondage peptide. The following amino acid linked twice using TBTU/DIPEA instead of DIC.

The sequence (o-NBS)HN-Cys11(4-MeZ)-Thr12(OBzl)-(R) failed to metilirovanie using methyl-about-nitrobenzenesulfonic the and. However, this problem was overcome by using dimethylsulfate as meteorologi agent instead of volume methyl-o-nitrobenzenesulfonate.

The affinity of binding (Kd, nm) of all SRIF analogues was assessed using them dependent on the concentration of eviction125I-radioactively labeled peptide ligands from membranes isolated from cells SNO, transfected with the corresponding receptor somatostatin person. As reference I used the affinity of binding SRIF-14 and SRIF-28 in the same system. SRIF-28 showed a particularly high affinity receptor type 5 in comparison with SRIF-14. Subject to the strong action that has the conformation of the side chain N-terminal amino acid on the biological activity of this type of analogue used two series of basic structures (compounds 9 and 18) for this study: one containing DPhe (similar to 9), and another containing the Tyr residue (similar to 18), with a total of 16 N-methylated analogues, structure and physico-chemical characteristics of which are given in table 1.

The compounds of this invention were synthesized as described above and/or as described in the various references cited here.

It should be clear that Ho is I this invention has been described together with its description, this detailed description is intended to illustrate but not to limit the scope of this invention. Other aspects, advantages and modifications presented in the claims. The content of each of the works cited here included as references in their entirety.

1. The compound of formula (I),

And1-cyclo{Cys-A2-D-Trp-A3-A4-Cys}-A5-Y1,

(I)

where a1denotes a D - or L-aromatic α-amino acid selected from the group Phe, D-Phe, Tyr, D-Tyr, β-Nal, D-β-Nal, Cha or D-Cha;

And2denotes aromatic α-amino acid selected from the group Phe, Tyr, β-Nal or Cha;

And3represents Lys or Orn;

And4means β-hydroxylamin, Ser, hSer, or Thr;

And5means β-hydroxylamin, Ser, hSer, or Thr

Y1denotes NH2;

where the amine nitrogen of each of the peptide bond and the amino group And1formula (I) optionally substituted methyl group, provided that there is at least one specified a methyl group, and provided that the specified connection is not D-Phe-cyclo{Cys-Phe-D-Trp-Lys-(N-Me-Thr)-Cys}-Thr-NH2;

or its pharmaceutically acceptable salt.

2. The compound according to claim 1, where a1denotes Phe, D-Phe, Tyr, D-Tyr, β-Nal, D-β-Nal, Cha or D-Cha; And2represents Phe, Tyr, β-Nal and the and Cha and Y 1denotes NH2; or its pharmaceutically acceptable salt.

3. The compound according to claim 2, where a1represents D-Phe, or its pharmaceutically acceptable salt.

4. The compound according to claim 2, where a1represents Tyr, or its pharmaceutically acceptable salt.

5. The compound according to claim 2, where a2denotes Phe, or its pharmaceutically acceptable salt.

6. The compound according to claim 2, where a3represents Lys, or its pharmaceutically acceptable salt.

7. The compound according to claim 2, where a4represents Thr, or its pharmaceutically acceptable salt.

8. The compound according to claim 2, where a5represents Thr, or its pharmaceutically acceptable salt.

9. The compound according to claim 3 where the compound is a (N-Me-D-Phe)-{Cys-Phe-D-Trp-Lys-Thr-Cys}-Thr-NH2.

10. The compound according to claim 3 where the compound is a D-Phe-{(N-Me-Cys)-Phe-D-Trp-Lys-Thr-Cys}-Thr-NH2.

11. The compound according to claim 3 where the compound is a D-Phe-cyclo{Cys-(N-Me-Phe)-D-Trp-Lys-Thr-Cys}-Thr-NH2.

12. The compound according to claim 3 where the compound is a D-Phe-{Cys-Phe-(N-Me-D-Trp)-Lys-Thr-Cys}-Thr-NH2.

13. The compound according to claim 3 where the compound is a D-Phe-{Cys-Phe-D-Trp-(N-Me-Lys)-Thr-Cys}-Thr-NH2.

14. The compound according to claim 3 where the compound is a D-Phe-cyclo{Cys-Phe-D-Trp-Lys-Thr-(N-Me-Cys)}-Thr-NH2.

15. The compound according to claim 3 where the compound is a D-Phe-cyclo{Cys-Phe-D-Trp-Lys-Thr-Cys}-(N-Me-Thr)-NH 2.

16. The compound according to claim 4 where the compound is a (N-Me-Tyr)-{Cys-Phe-D-Trp-Lys-Thr-Cys}-Thr-NH2.

17. The compound according to claim 4, where the connection is a Tyr-{(N-Me-Cys)-Phe-D-Trp-Lys-Thr-Cys}-Thr-NH2.

18. The compound according to claim 4, where the connection is a Tyr-{Cys-(N-Me-Phe)-D-Trp-Lys-Thr-Cys}-Thr-NH2.

19. The compound according to claim 4, where the connection is a Tyr-{Cys-Phe-(N-Me-D-Trp)-Lys-Thr-Cys}-Thr-NH2.

20. The compound according to claim 4, where the connection is a Tyr-{Cys-Phe-D-Trp-(N-Me-Lys)-Thr-Cys}-Thr-NH2.

21. The compound according to claim 4, where the connection is a Tyr-{Cys-Phe-D-Trp-Lys-(N-Me-Thr)-Cys}-Thr-NH2.

22. The compound according to claim 4, where the connection is a Tyr-{Cys-Phe-D-Trp-Lys-Thr-(N-Me-Cys)}-Thr-NH2.

23. The compound according to claim 4, where the connection is a Tyr-{Cys-Phe-D-Trp-Lys-Thr-Cys}-(N-Me-Thr)-NH2.

24. A method of binding one or more somatostatin receptors subtypes 1, 2, 3, 4 and 5 person providing the stage of introduction of the compound according to claim 1 or its pharmaceutically acceptable salt to a recipient in need.

25. Method of inducing actions of somatostatin agonist which the stages of introducing a compound according to claim 1 or its pharmaceutically acceptable salt to a recipient in need.

26. A method of treating diseases or conditions of human or other animal in need thereof, predusmatrivayut compound according to claim 1 or its pharmaceutically acceptable salt to a specified mammal, where the specified disease or condition selected from the group consisting of Cushing's syndrome, gonadotropinum, hyperparathyroidism, Paget's disease, vipoma, nesidioblastosis, hyperinsulinism, gastrinoma syndrome Zollinger-Ellison, hypersecretory diarrhea associated with AIDS and other conditions, syndrome of irritation of the colon, pancreatitis, Crohn's disease, systemic sclerosis, thyroid cancer, psoriasis, hypotension, panic attacks, sclerodoma, obstruction of the small intestine, gastroesophagal reflux, duodenogastric reflux, graves ' disease, polycystic disease of the ovary, bleeding of the upper part of the gastrointestinal tract, false cysts of the pancreas, pancreatic ascites, leukemia, meningioma, cancer cachexia, acromegaly, restenosis, hepatoma, lung cancer, melanoma, inhibition of rapid growth in solid tumors, reduce body weight, treatment of insulin resistance, syndrome X, prolongation of survival of pancreatic cells, fibrosis, hyperlipidemia, hyperammonemia, hyperprolactinemia and prolactinomas.



 

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