JP2003514867A - Urotensin-II cyclic analog - Google Patents

Abstract

  (57) [Summary] The present invention relates primarily to urotensin-II cyclic analogs and pharmaceutical compositions containing them.

Description

Detailed Description of the Invention

FIELD OF THE INVENTION The present invention relates generally to urotensin-II cyclic analogs and pharmaceutical compositions containing them.

BACKGROUND OF THE INVENTION Central control of cardiovascular homeostasis is achieved by a combination of both direct neural control and systemic neurohormonal activation. As a result, the resulting release of both contractile and relaxant factors is normally under stringent regulation, but deviations from this status can lead to pathologically causal cardiac hemodynamic dysfunction. . This neurohumoral axis, so-called angiotensin-II,
Major mammalian vasoactivators, including endothelin-1, norepinephrine, all function through interactions with specific G-protein coupled receptors (GPCRs). Urotensin-II, represents a novel member of this neurohumoral axis.

In fish, this peptide has the following significant hemodynamic and endocrine effects in various end organ systems and tissues: Smooth muscle contraction Smooth muscle preparation from the gastrointestinal tract, respiratory, and genitourinary tract Vascular and non-vascular smooth muscle contractions at the origin of inclusions. Both pressor and antihypertensive activity has been described following tissue administration of exogenous peptides. Osmotic regulation: effects including regulation of transepithelial ion (Na ⁺ , Cl ⁻ ) transport. Although diuretic effects are described, these effects are considered secondary to induce renal vascular effects (high GF).
R). Metabolism: Urotensin-II affects prolactin secretion and exhibits a lipolytic effect in fish (activating triacylglycerol lipase, leading to the mobilization of unesterified free fatty acids). (Pearson, et al. Proc. Natl. Acad. Sci. (USA) 1980, 77, 5021; Conlon, et al. J.
Exp. Zool. 1996, 275, 226.)

In the study of human urotensin-II, it is: an extremely potent and effective vasoconstrictor, a persistent contractile activity that is extremely resistant to outflow, and a detrimental effect on cardiac performance. It was found to have (myocardial contractility). Human urotensin-II was evaluated for contractile activity in aortas isolated from rats and found to be the most potent contractile agonist identified to date. The in vitro pharmacology and in vivo hemodynamic characteristics of human urotensin-II indicate that it plays a pathological role in cardiovascular disease characterized by excessive or abnormal vasoconstriction and myocardial dysfunction (Ames). Et al Nature 1999, 401
, 282).

Urotensin-II analogs are useful for identifying agonists and antagonists / inhibitors of urotensin II and for treating the conditions associated with human urotensin II imbalance. For example, it is useful to enhance the action of the U-II system by mimicking the agonist activity of U-II at its cognate receptor or by moderating the uptake / metabolism of U-II. These compounds include congestive heart failure, stroke, ischemic heart disease (angina, myocardial ischemia), cardiac arrhythmia, hypertension (essential and pulmonary), COPD, restenosis, asthma, (Hay DWP, Luttma).
nn MA, Douglas SA: 2000, Br J Pharmacol: volume 131, pages 10-12) useful in the treatment of neuroinflammatory and metabolic vascular disease, all of which are caused by abnormal vasoconstriction and / or myocardial dysfunction Characterized Both U-II and GPR14 are expressed in the mammalian CNS (Ames et al. Nature 1999, 401, 282) and therefore they are used to treat addiction, schizophrenia, impulse, anxiety, stress, depression, and neuromuscular function. May be useful. Functional U-II receptors are expressed in rhabdomyosarcoma cell lines and may therefore have tumor applicability. Urotensin may be involved in various metabolic disorders such as diabetes (Ames et al Nature 1999, 401, 282).
, Nothacker et al., Nature Cell Biology 1: 383-385, 1999).

SUMMARY OF THE INVENTION In one aspect, the invention provides urotensin II analogs and pharmaceutical compositions containing them. In a second aspect, the invention provides the use of urotensin analogs to identify agonists and antagonists of urotensin II, and inhibitors of urotensin II. In another aspect, the invention provides the use of urotensin analogs for treating the conditions associated with urotensin II imbalance. In still yet another aspect, the invention provides congestive heart failure, stroke, ischemic heart disease.
(Angina, myocardial ischemia), cardiac arrhythmia, hypertension (essential and pulmonary), COPD, restenosis,
Provided is the use of urotensin analogues for the treatment of asthma, neuroinflammation and metabolic angiopathy, addiction, schizophrenia, impulse, anxiety, stress, depression, neuromuscular function, and diabetes. Other aspects and advantages of the invention are further described in the detailed description of the preferred embodiments of the invention below.

[0007] (Detailed Description of the Invention)   The present invention has the formula (I):

[Chemical 2] Wherein R ₁ is benzyl; R ₂ is 3-indolylmethyl, hydrogen; R ₃ is C _(1-6) alkyl-NH ₂ , C _(1-6) alkyl-NHC (= NH ) NH ₂ ; R ₄ is benzyl, 4-hydroxybenzyl; R ₅ is hydrogen or Me; X is C _(1-6) alkyl, —NR ₆ CR ₇ R ₅ C (═O) _{NR 6 CR 7 R 5 C (} = O) -
In it; hydrogen R ₆ is independently, Me, ring C _(3-5) against or is R _7, benzyl chains to form; R ₇ is independently benzyl or cyclic for R ₆ C _{(3- 5)} means a chain], and a pharmaceutical composition containing the urotensin II analogue represented by the formula or a pharmaceutically acceptable salt thereof, and a compound thereof.

[0008]   As used herein, the term “alkyl” and similar terms such as “alkoxy”
The term includes all straight chain and branched isomers. Typical examples are methyl, ethyl,
n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl
And t-butyl, n-pentyl and n-hexyl.   The compounds of the present invention may contain one or more asymmetric carbon atoms, including racemic and
And may exist in an optically active form. These compounds and their diastates
All rheomers are considered to be within the scope of the present invention. R₁Is preferably benzyl; Preferably one R₂Is (R-) or (S-) 3-indolylmethyl,
Other R₂Is hydrogen; R₃Is preferably aminobutyl; R_FourIs preferably 4-hydroxybenzyl; R_FiveIs preferably hydrogen; X is preferably C_(2-4)Alkyl, or -NR₆CR₇R₅C (= O) NR₆CR ₇ R₅C (= O)-; R₆Is preferably hydrogen or R₇To C₍₃₎Forming a chain; R₇Is preferably benzyl or R₆To C₍₃₎Form a chain.

[0009] Preferred compounds are: c (Phe-Trp-Lys-Tyr-Phe-Pro) (SEQ ID NO: 1); c (Phe-Trp-Lys-Tyr-D-Pro-Pro) (SEQ ID NO: 2); c (Phe-Trp-Lys-Tyr-β-Ala) (SEQ ID NO: 3); c (Phe-Trp-Lys-Tyr-GABA) (SEQ ID NO: 4); and c (Phe-Trp-Lys-Tyr-5-aminovaleric acid) (SEQ ID NO: 5) Is.

(Preparation) The peptide was added to tert-butoxycarbonyl-L-prolyl-O-resin
= 0.7 molar equivalent / g). Peptide preparation consists of a synthetic cycle, hydrofluoric acid cleavage, cyclization, and purification. All amino acids were used as alpha tert-butoxycarbonyl derivatives. The side chain protecting groups are as follows: Tryptophan N (indole) formyl tyrosine Br-Z lysine trifluoroacetyl.

EXPERIMENTAL Each synthetic cycle consisted of: a) Deblocking with trifluoroacetic acid The resin was treated with 50% TFA in methylene chloride (2 to 3 times the resin volume), stirred for 30 minutes at room temperature and discharged. Let The resin was washed once with an equal volume of isopropanol for 1 minute and then twice with an equal volume of methanol for 1 minute. b) Coupling The resin of (a) above was treated twice with an equal volume of 10% triethylamine in methylene chloride.
It was washed for 1 minute, then washed twice with an equal volume of methanol for 1 minute, and finally washed twice with an equal volume of methylene chloride for 1 minute. To the resin was added 3 equivalents of tert-butoxycarbonylamino acid (dissolved in methylene chloride or methylene chloride / N, N-dimethylformamide mixture) and 3 equivalents of 1-hydroxybenzotriazole hydrate (N
, 1M solution in N-dimethylformamide) and the resulting suspension was stirred for 1 min. To the mixture was added 3 equivalents of dicyclohexylcarbodiimide (1M solution in methylene chloride) and the reaction was stirred for 60-120 minutes. The resin was then washed twice with an equal volume of methanol and twice with an equal volume of methylene chloride. A small sample was used for the ninhydrin test: incomplete coupling, subcycle b
) Was repeated; for complete coupling, synthesis with subcycle c) was continued.

C) Capping An equal volume of acetic anhydride (20% in methylene chloride) was added to the resin of (b) above,
The mixture was stirred for 5 minutes at room temperature. The resin was then washed twice with an equal volume of methanol and twice with an equal volume of methylene chloride. HF Cleavage: 1 ml of anhydrous anisole was added to 1.0 g of the resin of (c) above in a Teflon reactor. The bath was cooled with liquid nitrogen and charged with 10 ml hydrofluoric acid (distilled anhydride). The temperature was raised to 0 ° C. with ice water and the reaction was stirred for 1 hour. The hydrofluoric acid was distilled off at 0 ° C., the residue was washed with anhydrous ether, extracted with 1: 1 acetonitrile / water and lyophilized to give the cleaved linear peptide.

Cyclization: After extraction of the crude linear peptide from the cleaved resin, it was diluted with water to about 1 g / L. The pH was adjusted to 7.5-8.0 with ammonium hydroxide. The cloudy reaction mixture was stirred until no more shifts were detected by HPLC and / or the mixture was weak / negative by Ellman's test (2-3 days). The mixture was acidified to pH 4.0 and filtered to give crude cyclic disulfide peptide.

Purification: HPLC: Column Vydac C-18RP silica, 15-20 uM, diameter 2 inches The reaction mixture was loaded and the column was washed with tetraethylammonium phosphate buffer (pH 2.25). A linear gradient was used over 30 minutes (20% acetonitrile / 80% water to 80% acetonitrile / 20% water). 20-3
Fractions between 0 minutes were collected and analyzed for purity by analytical HPLC. Fractions greater than 95% pure were pooled, reloaded onto the same HPLC column for desalting with 0.1% trifluoroacetic acid "buffer" and lyophilized. The peptide sequence was confirmed using mass spectrum and amino acid analysis.

In order to use a compound of formula (I) or a pharmaceutically acceptable salt thereof for the treatment of humans and other mammals, the compound is usually formulated as a pharmaceutical composition according to standard pharmaceutical practice. Administering a compound of formula (I) or a pharmaceutically acceptable salt thereof in a standard manner for the treatment of indications, eg orally, parenterally, sublingually, transdermally, rectally, by inhalation or by buccal administration. You can

The compounds of formula (I) or their pharmaceutically acceptable salts, which are active when administered orally, can be formulated as syrups, tablets, capsules and lozenges. A syrup formulation will generally consist of a solution carrier of the compound or salt, for example ethanol, peanut oil, olive oil, glycerine or suspensions or solutions in water with flavoring or coloring agents. When the composition is in tablet form, any pharmaceutical carrier conventionally used in preparing solid formulations can be used. Examples of such carriers are magnesium stearate, terra alba, talc, gelatin,
Includes agar, pectin, acacia, stearic acid, starch, lactose and sucrose. When the composition is in the form of a capsule, any conventional encapsulation procedure using, for example, the above carriers in hard gelatin capsules is suitable.
When the composition is in the form of soft gelatin capsules, any pharmaceutical carrier conventionally used to prepare dispersions or suspensions may be considered, such as water soluble gums, celluloses, silicic acids or oils. , Can be incorporated into soft gelatin capsules. A typical parenteral composition comprises a solution or suspension of the compound or salt in a sterile aqueous or non-aqueous carrier, optionally a parenterally acceptable oil such as polyethyl glycol, polyvinylpyrrolidone, lectin, Peanut oil or sesame oil may be included.

Typical compositions for inhalation are in solution, suspension or emulsion form, to be administered as a dry powder or in the form of an aerosol using conventional propellants such as dichlorodifluoromethane or trichlorofluoromethane You can A typical suppository formulation may be a compound of formula (I) or a pharmaceutically acceptable salt thereof and a binding and / or lubricant such as a polymer glycol, gelatin, cocoa butter or Other low melting vegetable oils and fats or their synthetic analogues are included. A typical transdermal formulation may be a conventional aqueous or non-aqueous vehicle such as a cream,
It includes ointments, lotions or pastes, or is in the form of a medicated bandage, patch or film.

Preferably the composition is in unit dosage form, for example a tablet, capsule or metered aerosol dose, so that the patient may administer a single dose himself. These urotensin analogs are used to treat congestive heart failure, stroke, and ischemic heart disease (angina pectoris,
Myocardial ischemia), cardiac arrhythmia, hypertension (essential and pulmonary), COPD, restenosis, asthma, neuroinflammatory and metabolic vascular disease, addiction, schizophrenia, impulse, anxiety, stress, depression, neuromuscular function, And can be used to treat diabetes. No unacceptable toxicological effects are expected when the compounds of the present invention are administered in accordance with the present invention. The biological activity of the compounds of formula (I) is determined by the following test.

Radioligand binding: HEK-293 cell membranes containing stable cloned human and rat GPR-14 (20ug / assay) were tested with 200pM [125I] h-U-II (200Ci / mmol- ¹ ). The final concentration of compound was 2 in the presence of increasing concentrations of compound in DMSO (0.1 nM to 10 uM)
Incubated in an incubation volume of 00 ul (20 mM Tris-HCl, 5 mM MgCl ₂ ). Incubation was carried out for 30 minutes at room temperature followed by filtration on a Brandel cell harvester through GF / B filters. ¹²⁵ I
Labeled U-II binding was quantified by gamma counting. Non-specific binding was defined by ¹²⁵ IU-II binding in the presence of 100 nM unlabeled human U-II. Data analysis was performed by non-linear least squares fitting. Ca ²⁺ mobilization: Microtiter plate-based Ca ²⁺ mobilization F for functional identification of ligands that activate HEK-293 cells expressing (stable) recombinant GPR-14
LIPR assay (Molecular Devices, Sunnyvale,
CA) was used. The day after trunkfection, cells were plated in poly-D-lysine coated 96-well black / colorless plates. After 18-24 hours, the medium was aspirated and Fluo3AM-loaded cells were exposed to various concentrations (10 nM to 30 uM) of test compound, followed by h-U-II. Fluorescence emission was read every 1 second for 1 minute, followed by every 3 seconds for the next 1 minute after the start of the assay. An inhibitory concentration of 50% (IC50) was calculated for various test compounds.

Inositol Phosphate Assay: HEK-293-GPR14 cells in T150 flasks were pre-labeled with 1 uCi myo- [ ³ H] inositol per ml of inositol-free Dulbecco's modified Eagle medium. After labeling the cells twice, Dulbecco's phosphate buffered saline (
DPBS) and then 3 minutes for 10 minutes in DPBS containing 10 mM LiCl.
Incubated at 7 ° C. Experiments in the absence of test compound and three different concentrations
Initiated by the addition of increasing concentrations of hU-II (1 pM to 1 μM) in the presence of (0.3, 1 and 10 uM), the incubation continued for another 5 minutes at 37 ° C., then 1
The reaction was stopped by the addition of 0% (final concentration) trichloroacetic acid and centrifugation. The supernatant was neutralized with 100 ul of 1 M Trizma base and the inositol phosphates were separated on an AG1-X8 column (0.8 ml packed, 100-200 mesh) in the formic acid phase. Inositol monophosphate was eluted with 8 ml of 200 mM ammonium formate. The combined inositol di and triphosphates were eluted with 4 ml of 1M ammonium formate / 0.1M formic acid. Eluted fractions were counted on a beta scintillation counter. The K _B was calculated based on the shift from the control curve.

Regulation of Neuroendocrine Factors Adult Sprague Dawley rats are surgically operated to guide the guide cannula to the lateral ventricles (demonstrated by the strength of the linking response to angiotensin II; 100 ng icv). After a 2-week recovery period, rats received human urotensin-I.
I, putative agonist ligand (1-10 ug, icv) or vehicle (0.9% saline solution) is given for 1 minute (90 seconds considering diffusion), and plasma changes of neuroendocrine factors are detected. Alternatively, human urotensin-II, a putative agonist ligand (100ug, injection iv) via an intravenous cannula located in the jugular vein of the left femur
Alternatively, anesthetized rats are prepared for acute systemic exposure to vehicle (0.9% saline solution). After 220 minutes, blood was placed in a suitable radioimmunoassay (RIAs
) For the subsequent assay of neuroendocrine markers.

Neuroendocrine markers include, but are not limited to: anterior (eg, ADH, OCT, ACTH) and posterior (eg, GH, TS).
H, LH, GSH, Prolactin) pituitary hormone; hypothalamic hormone (SST, GHRH, TRH, CRH); thyroid / parathyroid hormone (T4, T3, rT3, calcitocin, PTH); insulin, leptin, glucose; lipid Sex hormones (including release hormones); neurovascular hormones (ET, angiotensin II, aldosterone, NE);

EXAMPLES Examples 1-5 were prepared according to the general procedure outlined above using the appropriate starting materials:

[Table 1]

Example 6 Formulations for pharmaceutical use incorporating the compounds of the present invention can be prepared in various forms and with numerous excipients. Examples of such formulations are given below. Inhalation Formulation A compound of Formula I, (1 mg to 100 mg) is aerosolized from a metered dose inhaler to deliver the desired amount of drug per use. Tablets / ingredients per tablet 1. Active ingredient 40 mg (compound of formula I) 2. Corn starch 20 mg 3. Arginic acid 20 mg 4. Sodium alginate 20 mg 5. Magnesium stearate 1.3 mg 2.3 mg

Tablet Production: Step 1 Ingredients 1, 2, 3 and 4 are mixed in a suitable mixer / blender. Step 2 Add enough water to the mixture of Step 1 in small portions, carefully mixing after each addition. Addition and mixing of such water is until the mass has a viscosity which allows it to be converted into wet granules. Step 3 The wet mass is converted to granules by passing through a vibrating granulator using a # 8 mesh (2.38 mm) screen. Step 4 The wet granules are then dried in an oven at 140 ° F (60 ° C) until dry. Step 5 Lubricate the dried granules with ingredient number 5. Step 6 Compress smooth granules with a suitable tablet press.

The above description and examples generally demonstrate methods of making and using the compounds of the present invention. However, the invention is not limited to the particular embodiments described above, but includes all such modifications falling within the scope of the claims. Various references to magazines, patents, and other publications, described in this specification and constitute the state of the art, are incorporated herein by reference.

[Sequence list]

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 9/06 A61P 9/10 103 9/10 9/12 103 11/06 9/12 25/00 11 / 06 25/18 25/00 25/22 25/18 25/24 25/22 29/00 25/24 A61K 37/02 29/00 37/24 (72) Inventor Steven De Knight United States 19380 U Pennsylvania Est Chester, Cromwell Lane 115 (72) Inventor Gregory El Warren United States 19457 Parkerford, Pennsylvania Old Skylkill Road 1478 F Term (reference) 4C084 AA02 AA07 BA01 BA08 BA17 BA23 DB01 NA14 ZA012 ZA022 ZA122 ZA182 ZA362 ZA392 ZA402 ZA422 ZA452 ZA592 ZB112 ZC352 4H045 AA10 AA30 BA13 BA14 BA15 BA35 DA30 EA23 FA44 GA25 HA02

Claims (6)

[Claims] 1. Formula (I): Wherein R ₁ is benzyl; R ₂ is 3-indolylmethyl, hydrogen; R ₃ is C _(1-6) alkyl-NH ₂ , C _(1-6) alkyl-NHC (= NH ) NH ₂ ; R ₄ is benzyl, 4-hydroxybenzyl; R ₅ is hydrogen or Me; X is C _(1-6) alkyl, —NR ₆ CR ₇ R ₅ C (═O) _{NR 6 CR 7 R 5 C (} = O) -
In it; hydrogen R ₆ is independently, Me, ring C _(3-5) against or is R _7, benzyl chains to form; R ₇ is independently benzyl or cyclic for R ₆ C _{(3- 5} ) a chain] or a pharmaceutically acceptable salt thereof. 2. R ₁ is benzyl; one R ₂ is (R-) or (S-) 3-indolylmethyl, the other R ₂ is hydrogen; R ₃ is aminobutyl. ; be R ₄ is 4-hydroxybenzyl; R ₅ is hydrogen; X is C _(2-4) alkyl, or _{-NR 6 CR 7 R 5 C (} = O) NR 6 CR 7 R 5 C (
= O) -; or R ₆ is hydrogen, or cyclic C ₍₃ for R ₇₎ chain to form; and either R ₇ is benzyl or cyclic C ₍₃ for R _6,) chain formed Claim 1
The described compound. 3. c (Phe-Trp-Lys-Tyr-Phe-Pro); (SEQ ID NO: 1) c (Phe-Trp-Lys-Tyr-D-Pro-Pro); (SEQ ID NO: 2) c (Phe-Trp-Lys-Tyr-β-Ala); (SEQ ID NO: 3) c (Phe-Trp-Lys-Tyr-GABA); (SEQ ID NO: 4) and c (Phe-Trp-Lys-Tyr-5-aminovaleric acid) (SEQ ID NO: 5) The compound of claim 1 selected from the group consisting of: 4. A pharmaceutical composition comprising the compound of claim 1 and a pharmaceutically acceptable carrier. 5. A method of treating a condition associated with human urotensin II imbalance by administering to the subject in need thereof an effective amount of a compound of claim 1. 6. A method of treating stroke by administering an effective amount of a compound of claim 1 to a subject in need of treatment of stroke.