CZ305660B6 - Oxytocin analogs - Google Patents

Abstract

The present invention relates to oxytocin analogs, which are characterized in that they have the structural formula A–M–B, which is expressed by the structural formulae I, II and III. These novel oxytocin analogs are suitable for visualization of oxytocin receptors.

Description

Oxytocin analogs

Field of technology

The invention relates to new oxytocin analogues, in particular for the fluorescent visualization of receptors, in particular oxytocin, in mammalian tissues.

Prior art

Nanapetide oxytocin (OT) is a hormone with peripheral and central effects. It is known primarily as an inducer of uterine contraction and milk ejection, but recently its effects have also been found in other peripheral tissues, such as the heart, bone, kidneys and other organs. In recent decades, the remarkable regulatory role of OT in the brain has been revealed, where this peptide acts as a neurotransmitter and neuromodulator (Klenerová V., Hynie S., The central regulatory role of oxytocin. Psychiatry 12: 4-10, 2008). It is involved in the process of learning, memory, social behavior, building relationships, maternal behavior and stress. OT acts through one type of oxytocin receptor (OTR) coupled to G-regulatory proteins. The multiplicity of oxytocin functions induced by stimulation of this receptor is studied not only under physiological conditions, but also in a number of diseases and mental disorders (Klenerová V., Hynie S., Some recent findings on oxytocin receptor signaling. Čs. Fyziologie 57: 78-82, 2008). In most cases, the functionality of these receptors has been demonstrated, at least by demonstration of stimulation of signaling molecules. Current research aims to reveal the mechanisms of action of OT at the molecular and cellular level with the aim of therapeutic use of this knowledge. The key issue is knowledge of the location and density of OTR in individual organs and tissues. Experimental molecular biology approaches are used, such as OTR mRNA expression and OTR protein expression. The presence of OTR protein is determined by immunohistochemical methods using specific primary antibodies. However, this method is very demanding not only technically, time-wise, but also economically, which is disadvantageous.

The essence of the invention

The disadvantages of the prior art methods used to detect the expression of oxytocin receptors are eliminated by the novel oxytocin analogs of the invention, which are based on compounds of formula IV.

A - M - B (IV), in which

A is [N, N-azidopentanoylcysteine] oxytocin,

B is difluoro {2- [phenyl (2H-pyrrol-2-ylidene-N, N-methyl] -1H-pyrrolatophosphorus,

M represents a bridge from the 1H-1,2,3-triazol-4-ylmethanol group of the 5- [4- (hydroxymethyl) -1H-1,2,3-triazol-1-yl] -N- (17H- 1,2,3-triazol-1-ylmethyl) pentanamide groups or 1- [4- (hydroxymethyl) -1H-1,2,3-triazol-1-yl] -N- (1H-1,2,3-triazole -4-ylmethyl) -3,6,9,12-tetraoxapentadecan-15-amide groups, said oxytocin analogs having the following formulas I, II, III

- 1 CZ 305660 B6

(AND)

(ID

-2EN 305660 B6

Oxytocin (OT) analogues according to the invention, conjugated with a green emitting fluorescent dye, the so-called fluorophore (B) {2- [phenyl (2,7-pyrrol-2-ylidene-KA-methyl] -1H-pyrrolato-KA} boron (Bodipy), were prepared by a 1,3-dipolar cycloadditions, wherein the compound (A) [N ^{-azidopentanoylcystein]} oxytocin was used as starting material for the synthesis of analogues of general formula (IV). conjugates OT-Bodipy different length are combined bridge (M), which allowed the formation of compounds with different affinities for oxytocin receptors.

The analogs of the invention of formula (I) to (III) were prepared such that the oxytocin (OT) was converted to ^[A -azidopentanoy Icy Stein-oxytocin action of 5-azidopentanové acid and a condensing agent N, N iisopropylkarbodiimidu in A-methylpyrrolidone.

Using variously designed bridges, preferably of the type 1H-1,2,3-triazol-1-ylmethanol or 5- [4- (hydroxymethyl) -1H-1,2,3-triazol-1-yl] -A - (1H-1,2,3-triazol-4-ylmethyl) pentanamide or 1- [4- (hydroxymethyl) -1H-1,2,3-triazol-1-yl] -N- (127-1) , 2,3-triazol-4-ylmethyl) -3,6,9,12-tetraoxapentadecan-15-amide, was a 1,3-dipolar cycloaddition reaction catalyzed by copper iodide to oxytocin in the presence of tris [(1-benzyl- 12 / -1,2,3-triazol-4-yl) methyl] amine (Rostovtsev VV, Green LG, Fokin VV, Sharpless KB A stepwise Huisgen cycloaddition process: Copper (I) -catalyzed regioselective „ligation“ of azides and terminal alkynes, AngewChem Int Ed. 41: 2596-2599, 2002; Hein JE, Tripp JC, Krasnova LB, Sharpless KB, Fokin VV Copper (I) -Catalyzed Cycloaddition of Organic Azides and 1-lodoalkynes Angew. Chem. Int. 48: 8018-8021, 2009.) attached fluorescent dye difluoro {2- [phenyl (2H-pyrrol-2-ylidenea]) methyl] -1,27-pyrrolato-KA] boron.

Due to the cost-effectiveness of current immunohistochemical methods and not always sufficient reproducibility of the binding of previously used antibodies to detect OTR, the inventors were looking for the possibility to visualize OTR by binding with oxytocin labeled with a fluorescent marker, without the need for primary antibodies. New alternative methods for binding oxytocin derivatives conjugated to green emitting fluorescent dye to OTR have been developed. These new analogs of the invention have enabled easy, rapid, unambiguous and inexpensive detection of oxytocin receptors and their screening prior to the use of hitherto demanding and expensive immunohistochemical methods.

Explanation of drawings

In the enclosed FIG. 1 shows the structural formulas (I) to (III) of the novel analogues according to the invention, OT-Bodips I, II, III. In FIG. 2 are microscopic images showing that the fluorophore used binds to the OTR and the immunohistochemical reaction is therefore negative. Giant. 3 shows an diam

-3 CZ 305660 B6 oxytocin receptor damage in the uterus by immunohistochemical reaction with primary anti-OTR antibody, further binding of OT-Bodipy analogues (I), (II), (III) to OTR and colocalization of oxytocin receptor and BT-Bodipy analogues bound to OTR. In FIG. 4 shows the competition of OTBodips conjugates (I), (II), (111) and oxytocin for epitopes on the OTR.

The analogues according to the invention were prepared by the inventors in the laboratories of the University of Chemical Technology in Prague and successfully verified in practice at the workplace of the 1st Medical Faculty of Charles University in Prague. The following examples merely illustrate but do not limit these analogs.

Examples of embodiments of the invention

Example 1

The analog of formula (I) was prepared by dissolving oxytocin acetate (518 mg, 0.48 mmol) in N-methylpyrrolidone (3 mL). 5-Azidopentanoic acid (315 mg, 2.2 mmol) and N, N-diisopropylcarbodiimide (620 μΐ, 4 mmol) were added to the solution at 18-23 ° C. The medium was adjusted to pH 8 with Α, Α, Α-triethylamine and the mixture was stirred at 18-23 ° C for 60 hours. The reaction was quenched by the addition of water (50 mL) and the emulsifying solution was repeatedly shaken with diethyl ether. The emulsion had to be filtered repeatedly. The aqueous solution was lyophilized three times with the addition of dioxane (20 mL). The crude product [Λ “azidopentanoylcysteine]] oxytocin (140 mg, 0.12 mmol) was obtained in a yield of 25% by weight, the purity of which (80%) was verified by HPLC (Kromasil Cl8 column, 5x250 mm) in a gradient of 10 to 80% B over 30 minutes, BA, acetonitrile - 0.04% trifluoroacetic acid in water at a flow rate of 1 ml / min and detection at 220 nm. The infrared spectrum of the ATR product was measured by the KBr crystal technique, confirming the presence of the azide group by valence vibration at 2098 cm ¹ . HRMS-ESI: monoisotopic mass. 1131.49537 Da, found (m / z) 1132.50518 (calculated 1132.50265) corresponds to ion [M + H] ⁺ , 1154.48514 (calculated 1154.48459) corresponds to ion [M + Na] ⁺ , 566.75496 (calculated 566.75496) corresponds to ion [M + H] ^{2 +} , 577.74561 (calculated 577.74593) corresponds to the ion [M + Na] ^{2+ of the} prepared substance.

Crude [N-azidopentanoylcysteine] ioxytocin (39 mg, 0.34 mmol) and difluoro (2 - {[4- (prop-2-yn-yloxy) phenyl] (2H-pyrrol-2-ylidene-KA) methyl } -177-pyrrolato-KA ^r ) boron (12 mg, 0.7 mmol) was weighed into a microwave flask and dissolved in dry propan-2-ol (2.5 mL). Copper iodide (1 mg, 5.25 mmol) and tris [(1-benzyl-1H-1,2,3-triazol-4-yl) methyl] amine (2 mg, 3.7 mmol) were added and the mixture was placed in a microwave reactor and stirred at 50 ° C for 2 hours. The crude product (48 mg) was dissolved in 5 ml of 20% acetic acid, the mechanical impurities were centrifuged and the mixture after the "click" reaction was applied to a Vydac Cl8 column (250x10 mm). Chromatography was performed on a gradient of 5 to 100% of the organic acetonitrile modifier (B), the aqueous component A was 0.04% trifluoroacetic acid (gradient time 95 min). Detection at 235 nm. Individual fractions were analyzed by HPLC on a YMC-PackPro Cl8 5 μm, 120 A (50x4.6 mm) column at a flow rate of 1.5 ml / min. A gradient of 5 to 85% A, B over 20 min was used. (A = 0.04% trifluoroacetic acid in water, B = acetonitrile), detection at 220 nm - diode array detector. Fractions containing sufficiently pure material were combined and lyophilized (10 mg was obtained). HRMS-ESI: monoisotopic mass. 1453.60427 Da, found (m / z) 1457.61387 (calculated 1454.61154) corresponds to the ion [M + H] ⁺ and 727.80962 (calculated 727.80941) corresponds to the ion [M] ²⁺ H of the prepared analogue of formula (I).

Example 2

The preparation of the analogue according to the invention of formula (II) was carried out in such a way that difluoro (2 - {[4- (prop-2-ynyloxy) phenyl] (2H-pyrrol-2-ylidene-N) methyl} -1. N-pyrrolato-K / V) boron (100 mg, 0.31 mmol) and 5-azidopentanoic acid (49 mg, 0.34 mmol) were weighed into a microwave flask and dissolved

-4CZ 305660 B6 in dry N, N-dimethylformamide (2 ml). Tris [(1-benzyl-1H-1,2,3-triazol-1-yl) methyl] amine (16 mg, 0.031 mmol), sodium ascorbate (7.9 mg, 0.04 mmol) and sulfate pentahydrate were added. copper CuSO ₄ -5H ₂ O (7.7 mg, 0.031 mmol) and the mixture was placed in a microwave reactor and stirred at 60 ° C for 60 minutes. The solvents were evaporated and the product was purified on a silica gel column (gradient, CH ₂ Cl ₂ -MeOH 100: 1 → 25: 1). The product difluoro {5- [4 ({4 - [(1H-pyrrol-2-yl-K7V) (2H] -pyrrol-2-ylidene-N7V) methyl] phenoxy} methyl) -1H-1 was isolated. , 2,3-triazol-1-yl] pentanoate} boron (127 mg, 0.27 mmol) in 88% yield.

Difluoro {5- [4 - ({4 - [(1 H -pyrrolo-2-yl-K / V) (2 // - pyrrol-2-ylidene-KJV) ^m ethyl] phenoxy} methyl) HL, 2,3-Triazol-1-yl] pentanoate} boron (100 mg, 0.21 mmol) and propargylamine hydrochloride (39 mg, 0.42 mmol) were dissolved in distilled dichloromethane (4 mL). N- (3-Dimethylaminopropyl) -N-ethylcarbodiimide (43 mg, 0.28 mmol), p- (dimethylamino) pyridine (34 mg, 0.28 mmol) and N-hydroxysuccinimide (18 mg, 0.16 mmol) were added and the reaction was stirred for 4 hours at 18-23 [deg.] C. The solvent was evaporated and the product was purified twice on a silica gel column (CHCl 3 -MeOH 100/1) to give the product difluoro {A- (prop-2-yn-1-yl) -. 5- [4 - ({4 - [(1 H -pyrrol-2-yl-N, N) (2H-pyridin-2-ylidene-N, N) methyl] phenoxy} methyl) -1 H -1,2,3-triazole -1-yl] pentanamidoboro (78 mg, 0.16 mmol) in 72% yield.

Difluoro {N- (prop-2-yn-1-yl) -5- [4 - ({4 - [(1H-pyrrol-2-yl-N-) (2H-pyrrol-2-ylidene- Ki) methyl] phenoxy} methyl) -1H-1,2,3-triazol-1-yl] pentanamidato} boron (17.1 mg, 0.34 mmol) and crude [N, N-azidopentanoylcysteine] oxytocin (35 mg, 0.31 mmol), prepared as described in Example 1, were weighed into a microwave flask and dissolved in dry N-dimethylformamide (2 mL). Copper iodide (1 mg, 5.25 mmol) and tris [(1-benzyl-1H-1,2,3-triazol-4-yl) methyl] amine (1.8 mg, 3.4 mmol) were added and the mixture was placed in a microwave reactor and stirred at 50 ° C for 2 hours. The crude product (45 mg) was dissolved in 5 ml of 20% acetic acid, the mechanical impurities were centrifuged and the mixture after pumping was pumped onto a Vydac Cl8 column (250x10 mm). Chromatography was performed on a gradient of 5 to 100% of the organic acetonitrile modifier (B), the aqueous component A was 0.04% trifluoroacetic acid (gradient duration was 95 min). Detection at 235 nm. Individual fractions were analyzed by HPLC on a YMC-PackPro C18 5 μm, 120 A (50x4.6 mm) column at a flow rate of 1.5 ml / min. A gradient of 5 to 85% A, B over 20 min was used. (A = 0.04% trifluoroacetic acid in water B = acetonitrile) detection at 220 nm - diode array detector. Fractions containing sufficiently pure material were combined and lyophilized (12 mg was obtained). HRMS-ESI: monoisotopic mass. 1633.70538 Da, found (m / z) 1634.71512 (calculated 1634.71266) corresponds to ion [M + H] ⁺ , 1656.69629 (calculated 1656.69460) corresponds to ion [M + Na] ⁺ , 817.86147 (calculated 817.85997) corresponds to ion [M] ²⁺ H and 828.85123 (calculated 828.85094) corresponds to the ion [M] ²⁺ Na of the prepared analogue of formula (II).

Example 3

The preparation of the analogue according to the invention of formula (III) was carried out such that difluoro (2 - {[4- (prop-2-yn-1-yloxy) phenyl] (2H-pyrrol-2-ylidene-N) methyl} -1H-difluoro N-pyrrolato-N-boron (322 mg, 1 mmol) and 1-azido-3,6,9,12-tetraoxapentacdecan-15-oic acid (291 mg, 1 mmol) were weighed into a microwave flask and dissolved in dry AA -dimethylformamide (3 ml). Tris [(1-benzyl-1H-1,2,3-triazol-4-yl) methyl] amine (27 mg, 0.05 mmol), sodium ascorbate (15 mg, 0.75 mmol), pentahydrate were added copper sulfate CuSO ₄ -5H ₂ O (12 mg, 0.05 mmol) and the mixture was placed in a microwave reactor and stirred at 60 ° C for 60 minutes. Toluene (5 ml) was added and the solvents were evaporated. The product was repeatedly purified on a silica gel column (gradient, CH ₂ Cl ₂ -MeOH 100: 1 → 25: 1). The product difluoro {1- [4 - ({4 - [(1H-pyrrol-2-yl-KA) (2H-pyrrol-2-ylidene-KA) methyl] phenoxy} methyl) -1H-isolated. 1,2,3-triazol-1-yl] -3,6,9,12-tetraoxapentadecane-15α-boron (490 mg, 0.8 mmol) in 80% yield.

Difluoro {1- [4 - ({4 - [(1H-pyrrol-2-yl-K7V) (2H-pyrrol-2-ylidene-K7V) methyl] phenoxy} methyl) -17,2,2 , 3-triazol-1-yl] -3,6,9,12-tetraoxapentadecan-15-oato} boron (100 mg, 0.16 mmol) and propargyl

The amine hydrochloride (39 mg, 0.42 mmol) was dissolved in distilled dichloromethane (4 mL). They were added N- (3-dimethylaminopropyl) - / V'-ethylcarbodiimide hydrochloride (47 mg, 0.24 mmol) and /> - (di ^me amino) ^pyridin (29 mg, 0.24 mmol) and the reaction was stirred 4 hours at 18-23 ° C. The solvent was evaporated and the product was purified twice on a silica gel column (gradient, CHCl 3 -MeOH 100 / 1-> 50/1). The product difluoro {N- (prop-2-yn-1-yl) -1- [4 - ({4 [(1H-pyrrol-2-yl-N)] (2H-pyrrol-2-yl) was isolated. ylidene-N (N) methyl] phenoxy} methyl) -1H-1,2,3-triazol-1-yl] 3,6,9,12-tetraoxapentadecan-15-amidato} boron (80 mg, 0.12 mmol ) in a yield of 75%.

Difluoro {N- (prop-2-yn-1-yl) -1- [4 - ({4 - [(17H-pyrrol-2-yl-N])] 2H-pyrrol-2-ylidene N, N-methyl] phenoxy} methyl) -1H-1,2,3-triazol-1-yl] -3,6,9,12-tetraoxapentadecan-15-amidato} boron (33 mg, 0.51 mmol) and crude [N-azidopentanoylcysteine] oxytocin (30 mg, 0.46 mmol), prepared as described in Example 1, were weighed into a microwave flask and dissolved in dry N, N-dimethylformamide (2 mL). Copper iodide (1 mg, 5.25 mmol) and tris [(1-benzyl] -1,2,3-triazol-4-yl) methyl] amine (2.7 mg, 0.5 mmol) were added and the mixture was placed in a microwave reactor and stirred at 50 ° C for 2 hours. The crude product (80 mg) was dissolved in 5 ml of 20% acetic acid, the mechanical impurities were centrifuged and the mixture was pumped onto a Vydac C18 column (250x10 mm) after a click reaction. Chromatography was performed on a gradient of 5 to 100% of the organic acetonitrile modifier (B), the aqueous component A was 0.04% trifluoroacetic acid (gradient time was 95 min). Detection at 235 nm. The individual fractions were analyzed by HPLC on a YMC-PackPro Cl8 5 μm column, 120 Å (50x4.6 mm) flow rate 1.5 ml / min. A gradient of 5 to 85% A, B over 20 min was used. (A = 0.04% trifluoroacetic acid in water B = acetonitrile) detection at 220 nm - diode array detector. Fractions containing sufficiently pure material were combined and lyophilized (15 mg was obtained). (HRMS-ESI: monoisotopic mass ·. 1781.77894 Da, found (m / z) 1783.79187 (calculated 1782.78621) corresponds to ion [M + H] ⁺ , 1804.77136 (calculated 1804.76816) corresponds to ion [M + Na] ⁺ , 891.89907 (calculated 891.89675) ) corresponds to the ion [M] ²⁺ H and 902.88859 (calculated 902.88772) corresponds to the ion [M] ²⁺ -Na of the prepared analogue of formula (III).

The novel analogs of the invention are shown in the accompanying FIG. 1.

Example 4

Checking the validity of the proposed method by demonstrating the binding of OT-Bodipy conjugates to OTR in mammalian tissue.

The rat uterus was used as a reference tissue containing OTR. The uterus was obtained from a female rat (Wistar, Czech Republic). Immediately after collection, it was embedded with cryoadhesive and frozen in isopentane cooled to -70 ° C. After freezing, the uterus was stored in a deep-freezing box at -80 ° C before slicing. Sections from 7 μm thick uterine horns were cut on a Leica CM 1850 cryostat. The slicing temperature was -24 ° C. Sections were collected on poly-Llysine-coated slides.

Before the actual demonstration of OT-Bodipy conjugate binding to OTR, it was necessary to 1) exclude the binding of fluorophore itself to uterine tissue (Fig. 2: I, II, III) and 2) determine the presence of oxytocin receptors in the reference tissue (Fig. 3a anti-OTR) .

Ad 1) In FIG. 2 are microscopic images of uterine sections showing that the Bodipy fluorophore used does not bind to the OTR and the immunohistochemical reaction is therefore negative. The above sections show the application of Bodipy diluted in various concentrations, where a higher dilution of Bodipy can reduce weak binding to a minimum.

Ad 2) Determination of the presence of OTR on tissue sections of the uterus was performed by an indirect two-step immunohistochemical method, based on immunological binding of labeled secondary

-6CZ 305660 B6 antibodies to primary antibody. An unlabeled primary antibody against the protein of interest (anti-OTR), i.e., oxytocin receptors, is first applied to the tissue section of the uterus, and then a fluorescently labeled secondary antibody is applied, which binds to the primary antibody. This is a relatively complicated and demanding method compared to using the new analogs of the invention.

A primary anti-oxytocin receptor antibody (Abeam Cat. No. abl 15664), diluted 1: 200, was used in the experiment with a blocking solution composed of: Bovine Serum Albumin (Sigma); Normal Goat Serum (Gibco); 10% aqueous solution of Triton X-100 (Pierce). Alexa Fluor® 546 (Invitrogen), diluted 1: 600, was used as a secondary antibody. The localization of uterine incision cell nuclei was determined using the intercalating fluorescent dye DAPI (4 ', 6-diamidine-2-phenylindole). The results of the OTR demonstration are shown in the determination of the binding of the individual structures of the oxytocin-Bodipy conjugate (Fig. 3a).

An experiment demonstrating the binding of OT-Bodipy conjugates to oxytocin receptors is shown in FIG. 3b. OT-Bodipy conjugates were diluted in methanol to a concentration of 0.5 μΜ, higher dilution of OT-Bodipy solutions increased the signal resolution of all three fluorescent conjugates. Figure 3b shows the binding OT-Bodips I, Π, III - green color.

After overlapping of the red-labeled oxytocin receptors with the primary antibody (anti-OTR) and the green-labeled binding of the OT-Bodipa analogs of the invention (I), (II), (III) to the OTR, the resulting orange-yellow color indicates colocalization of both ligands to the OTR. 3c).

Previous experiments show that OT-Bodipy conjugates (I), (II), III) bind to OTR located in the uterus. This binding can be competitively influenced by the addition of an excess of oxytocin. In all three cases, the binding of OT-Bodipy was significantly reduced by competition with OT, confirming the binding of OT-Bodipy conjugates to the oxytocin receptor (Fig. 4).

Industrial applicability

New oxytocin analogs for oxytocin receptor visualization allow rapid screening before using sophisticated and expensive immunohistochemical methods to detect OTR without the need for antibodies, which is of great importance for human and veterinary medicine.

Claims (1)

PATENT CLAIMS 1. Oxytocin analogs of general formula IV A-M-B in which (IV), A means [N'-azidopentanoylcysteine 'ioxytocin, B is difluoro {2- [phenyl (2H-pyrrol-2-ylidene-N, N) methyl] -1H-pyrrolato-KA} boron, M represents a bridge selected from the Z1 / Z1,2,3-triazol-4-ylmethanol group of the 5- [4- (hydroxymethyl) -1H-1,2,3-triazol-1-yl] -N- (1H- 1,2,3-triazol-4-ylmethyl) pentanamide groups or 1- [4-Ahydroxymethyl) -1H-1,2,3-triazol-1-yl] -N- (1H-1,2 , 3-triazol-4-ylmethyl) -3,6,9,12-tetraoxapentadecan-15-amide groups,