Visualisation agents

FIELD: chemistry.

SUBSTANCE: invention concerns compounds of the formula (Ia) and their application in radiological pharmaceutical compositions for linking to receptors associated with angiogenesis.

EFFECT: possible application in diagnostics or therapy, eg for malignant or cardiac diseases, endometriosis, inflammatory diseases, rheumatoid arthritis and sarcoma Kaposi.

 

The present invention relates to new compounds based on peptides and their use in diagnostic methods of imaging such as single photon emission computed tomography (SPECT) or positron emission tomography (PET). More specifically the invention relates to the use of such compounds based on peptides as target vectors that bind to receptors associated with angiogenesis, in particular integrin receptors, such as receptor αβ3-integrin. Thus, such compounds can be used for diagnosis or therapy, for example, cancer, heart disease, endometriosis, inflammatory diseases, rheumatoid arthritis and Kaposi's sarcoma.

New blood vessels can be formed through two different mechanisms: vasculogenesis or angiogenesis. Angiogenesis is the formation of new blood vessels by a branch from the existing vessels. The primary stimulus for this process can serve as an insufficient supply of nutrients and oxygen (hypoxia) of the cells in the tissue. Cells can respond by secreting angiogenic factors, of which there are many; one example, which is often referred to, is the growth factor of vascular endothelium (VGF). These factors initiate the secretion of proteolytic enzymes, which break down proteins of the basal membrane, as well as inhibitors that limit the effect of these potentially harmful enzymes. Another notable impact of angiogenic factors is that they cause the migration and division of endothelial cells.

Endothelial cells, which are attached to the basal membrane, which forms a continuous layer around the blood vessels on the outer side, do not undergo mitosis. The combined effect of the weakening of the pins and signals from receptors for angiogenic factors causes endothelial cells to move, reproduce and evolve, and eventually to synthesize basement membrane around new blood vessels.

Angiogenesis is prominent in the growth and reconstruction of tissues, including wound healing and inflammatory processes. Tumor must initiate angiogenesis, when they reach a millimeter in size, in order to maintain its growth rate.

Angiogenesis is accompanied by characteristic changes in the endothelial cells and their environment. The surface of these cells is transformed in preparation for migration, and cryptic patterns reveal where the basement membrane is destroyed, in addition to many proteins that are involved in the implementation of the representation and regulation of proteolysis. In the case of tumors of the resulting network of blood vessels tend to be disorganized with the formation of sharp bends, and arteriovenous shunts. It is also believed that inhibition of angiogenesis is a promising strategy for anticancer therapy. Transformation accompanying angiogenesis, are also very promising for the diagnosis, and one example is a malignant disease, but this principle also shows great prospects for inflammation and many inflammatory diseases, including atherosclerosis, and macrophages early atherosclerotic lesions are potential sources of angiogenic factors.

Many of the ligands involved in cell adhesion, contain Tripeptide sequence arginine-glycine-aspartic acid (RGD). It turns out that the RGD-sequence acts as a primary site of recognition between ligands, representing the sequence, and receptors on the cell surface. In General I believe that the secondary interactions between the ligand and the receptor increases the specificity of the interaction. These secondary interactions can occur between groups of the ligand and the receptor, which is directly adjacent to the RGD sequence or on the websites which are located away from RGD.

Effective targeting and visualization of integrin receptors associated with angiogenesis in vivo, therefore requires selective vector-based RGD with high affinity, which is chemically stable. Moreover, the rate of excretion is an important factor when designing agents for visualization in order to reduce problems with background radiation.

In WO 03/006491 described connection-based peptides to target are the integrin receptors associated with angiogenesis. However, there is a need for more such compounds based on peptides that are useful for diagnostic visualization techniques, such as SPECT and PET, as well as for therapeutic treatment. In particular, there is a need for compounds based on peptides with a higher stability under reaction conditions used to activate the reporter group, such as a radionuclide.

Thus, according to the first aspect of the invention proposed compound of formula (I)

where R2represents a

where b is an integer from 0 to 10;

R3represents a C1-4alkalinity or2-4alkenylamine bridge;

W1is absent or is a spacer elements is ruppirovka, which is a C1-30hydrocarbonous group, possibly comprising 1 to 10 heteroatoms selected from oxygen, nitrogen and sulfur, and preferably is a derivative of glutaric and/or succinic acid and/or part of a molecule polyethyleneglycol and/or part of a molecule of the formula

Z1is an antineoplastic agent, chelating agent or a reporter group.

Suitable chelating agents Z1include agents of formula

where

each R1A, R2A, R3Aand R1Aindependently represents a group RA,

each group RAindependently represents N or C1-10alkyl, C3-10alkylaryl,2-10alkoxyalkyl, C1-10hydroxyalkyl, C1-10alkylamino, C1-10foralkyl, or 2 or more groups RAtogether with the atoms to which they are attached, form a carbocyclic, heterocyclic, saturated or unsaturated ring,

or Z1may be a chelating agent represented by the formula (i), (ii), (iii) or (iv)

A preferred example of a chelating agent represented by formula (v)

The compounds of formula (I)containing chelating agents of the formula And may be labeled with radioactive isotopes, showing good radiochemical purity (RHC)at room temperature in aqueous conditions at a pH close to neutral.

The role of spacer elements grouping W1is that Z1were at a certain distance from the active site of the peptide component. For example, spacer elements grouping W1can separate bulky antitumor agent or chelating agent from the active site peptide.

Additional examples of suitable chelating agents Z1disclosed in US-A-4647447, WO 89/00557, US-A-5367080, US-A-5364613 and additionally include the agents described in the table.

In one aspect of the present invention Z1presents antitumor agent. In this aspect, the compound of formula (I) will focus on angiogenic site associated with cancer, and to deliver anticancer agent to the affected area. The antitumor agent can be represented by cyclophosphamide, hlorambuzila, busulfan, methotrexate, citarabinom, fluorouracil, vinblastine, paclitaxel, doxorubicin, duno what Ubichinon, etoposide, teniposide, cisplatin, amsacrine, docetaxel, and can also be used in a wide range of other anticancer agents.

Reporter groups (Z1in the compounds of formula (I) can be any group that is detectable, either directly or indirectly in a diagnostic imaging procedure in vivo. Are preferred reporter groups, which emit or may cause the emission of detectable radiation (e.g., a radionuclide, such as positron emitting radionuclide).

For visualization by magnetic resonance (Mr) reporter group must be either an isotope with non-zero nuclear spin (such as19F) or a substance having spins of unpaired electrons and hence paramagnetic, super-paramagnetic, ferrimagnetic or ferromagnetic properties; for light imaging reporter group should be svetorasseivateley (for example, painted or unpainted particle), setpagetitle or light emitter; for magnetometric imaging group must have detectable magnetic properties; for electrical impedance imaging of reporter group should affect the electrical impedance; and for scintigraphy, spet as, PET and the like reporter group should be a radionuclide.

In General determined that the reporter group can be either (1) a chelating agent, as defined above, chelated with metal or polyatomic metal-containing ion (i.e. TSS, and so forth), where the metal is a metal with a high atomic number (for example, with an atomic number of more than 37), paramagnetic radical (for example, a transition metal or lanthanide), or a radioactive isotope, (2) covalently linked non-metallic radical, which is the site of unpaired electrons (e.g., oxygen or carbon in the form of a stable free radical), a nonmetal with high atomic number or a radioisotope, (3) polyatomic cluster or crystal containing atoms of high atomic number, demonstrating cooperative magnetic characteristics (e.g., superparamagnetism, ferrimagnetism or ferromagnetism)or containing radionuclides.

Chelated with metal reporter group, preferably selected from the group90Y99mTc111In47Sc,68Ga68Ga51Cr177mSn62Cu167Tm97EN,188Re,177Lu,199Au,203Pb and141Ce and chelated with a chelating group, as defined above.

Methods of metallation any) is appropriate chelating agents known to specialists in this field of technology. Metals can be included in the chelating agent through any of the three common methods: direct inclusion, matrix synthesis and/or Parametrierung. Direct connection is preferred.

Thus, it is desirable that metal ions easily form a complex with a chelating agent, for example, by simple exposure or mixing an aqueous solution containing a chelating agent group metal salt in aqueous solution, preferably having a pH in the range of from about 4 to about 11. Salt can be any salt, but salt is preferably water-soluble salt of a metal, such as halogenated salt, and more preferably such salts are selected so as not to interfere with the binding of a metal ion with a chelating agent. Containing chelating agent group is preferably in aqueous solution at pH from about 5 to about 9, more preferably pH from about 6 to about 8. Containing chelating agent group may be mixed with a buffer salt such as citrate, carbonate, acetate, phosphate and borate with optimum pH. Preferably, the buffer salt is selected so as not to interfere with the subsequent binding of a metal ion with a chelating agent.

The following isotopes or isotope pairs can be IP is alsomany as for visualization, and for therapy without requiring changes to methods of introducing a radioactive label, or chelating agent:47Sc21;141Ce58;188Re75;177Lu71;199Au79;47Sc21;131I53;67Cu29;131I53and123I53;188Re75and99mTc43;90Y39and87Y39,47Sc21and44Sc21;90Y39and123I53;146Sm62and153Sm62; and90Y39and111In39.

Preferred non-metallic atomic reporter groups include radioisotopes, such as123I131I and18F, and the atoms are non-zero nuclear spin, such as19F, and heavy atoms, such as I.

In a preferred aspect of the invention Z1in the compound of formula (I) includes a positron emitting radionuclide that is included either as a prosthetic group, or by substitution reactions or accession, or by helatoobrazutee. Suitable positron emitting radionuclides for this purpose include11C,18F,15Oh,13N75Br,122I124I82Rb,68Ga and62Cu, of which11C and18F are preferred. Thus, the resulting compound of formula (I) can be used the ANO when imaging by positron emission tomography (PET).

Thus, according to preferred aspect of the invention proposed compound of formula (Ia)

where

R1is either a link, or

where a is an integer from 1 to 30;

R2represents a

where b is an integer from 0 to 10;

R3represents a C1-4alkalinity or2-4alkenylamine bridge;

The linker is a1-30hydrocarbonous group, possibly comprising 1 to 10 heteroatoms.

In the compounds of formula (Ia)

R3represents preferably1-4alkylene and more preferably

-CH2-;

and represents preferably an integer from 1 to 10 and most preferably represents 5;

b is preferably 1.

In the formula (Ia) Linker represents a C1-30hydrocarbonous group, possibly comprising 1 to 10 heteroatoms, such as oxygen or nitrogen, and may be selected to provide a good pharmacokinetics in vivo, such as suitable characteristics excretion. Suitable linker groups include alkyl, alkeline, alkyline chain, aromatic, polyaromatic and heteroaromatic rings and polymers, include the s etilenglikolevye, amino acid or carbohydrate subunit. The linker is preferably selected from (II), (III) and (IV):

where

n is an integer from 1 to 20;

m is an integer from 1 to 10;

p is an integer from 1 to 20;

q is an integer from 0 to 4;

r is an integer from 1 to 10.

In the formula (II) n typically represents from 2 to 6, preferably 3, and m typically represents from 1 to 4, preferably 2.

In the formula (III) p is typically represents from 1 to 6, preferably 3.

In the formula (IV) group -(CH2)q- preferably attached in the para-position relative to the amide group, q typically represents from 0 to 4, preferably 1, and r typically represents from 1 to 4, preferably 2.

As shown in the analysis of competitive binding in vitro, the compounds of formula (I) and (Ia) bind to receptors associated with angiogenesis. These compounds can thus be useful for the treatment, in vivo diagnostic and imaging of diseases and conditions associated with angiogenesis.

The term "diseases and conditions associated with angiogenesis" includes such diseases and conditions referenced below. This also is sent to WO 98/47541.

Diseases and conditions associated with angiogenesis include various forms of cancer and metastasis, such as cancer of the breast, skin, colon and rectum, pancreas, prostate, lung or ovary.

Other diseases and conditions associated with angiogenesis, are inflammation (e.g., chronic inflammation), atherosclerosis, rheumatoid arthritis and gingivitis.

Additional diseases and conditions associated with angiogenesis, are arteriovenous malformations, astrocytomas, choriocarcinoma, glioblastomas, gliomas, hemangioma (children, capillary), hepatoma, hyperplasia of the endometrium, itemizedoverlay myocardium, endometriosis, sarcoma, macular degeneration, melanoma, neuroblastoma, occlusive peripheral artery disease, osteoarthritis, psoriasis, retinopathy (diabetes, proliferative), scleroderma, seminoma and ulcerative colitis.

Thus, according to an additional aspect of the invention proposed compound of formula (I) or (Ia) for use in medicine, particularly in in vivo diagnosis or imaging, for example, by PET, disease or condition associated with angiogenesis.

Alternatively, a method of in vivo diagnosis or imaging of a disease or condition associated the angiogenesis, including the stage of introduction of the compounds of formula (I) or (Ia) in the human or animal with the subsequent generation of the image, preferably PET-images of part or all of the specified organism. Additionally, a method of treatment of a disease or condition associated with angiogenesis, comprising introducing a therapeutically effective amount of the compounds of formula (I) in the human or animal.

The compounds of formula (I) or (Ia) is preferably introduced into the composition of the radiopharmaceutical. "Radiopharmaceutical drug" is defined in the present invention as a drug containing the compound of formula (I) or (Ia) in a form suitable for administration to a mammal, such as man. The introduction is preferably carried out by injection of the radiopharmaceutical in the form of an aqueous solution. This radiopharmaceutical preparation may contain additional ingredients, such as buffers, pharmaceutically acceptable soljubilizatory (e.g., cyclodextrins or surfactants such as pluronic, twin, or phospholipids), pharmaceutically acceptable stabilizers and / or antioxidants (such as ascorbic acid, gentisic acid or para-aminobenzoic acid) or fillers for lyophilization (such as sodium chloride or m is NIT). Radiopharmaceutical drug is administered in an amount which gives a reliable image, taking into account the nature of the studied disease or condition, the weight of the patient and other factors that will be apparent to the person skilled in the art. Where the reporter group contains a metal, usually a dose of from 0.001 to 5.0 mmol chelate imaging metal ion per kilogram of body weight of the patient is effective to achieve reliable image. To PET a suitable amount of the compounds of formula (I) or (Ia) is from 0.1 to 100 MCI, preferably from 1 to 20 MCI.

Thus, in an additional aspect of the invention proposed radiopharmaceutical preparation containing the compound of formula (I) or (Ia) and one or more than one pharmaceutically acceptable excipient. The present invention additionally offered a pharmaceutical preparation containing a therapeutically effective amount of the compounds of formula (I) or salts thereof together with one or more pharmaceutically acceptable adjuvant, excipient or diluent.

The effective therapeutic dose of a compound of formula (I) will depend on the condition and the patient being treated, but generally will be in the range from 1 pmol/kg to 1 mmol/kg body weight.

The compounds of formula (I) can be obtained and the use of methods of organic synthesis, including solid-phase method of Merrifield, using an automatic peptide synthesizer (J. Am. Chem. Soc., 85:2149 (1964)), and methods similar to those described in WO 03/006491. The compounds of formula (I) can be purified using high performance liquid chromatography (HPLC).

The compound of formula (Ia) can be obtained from the corresponding compounds of formula (V)

where R1, R2and R3are as defined for compounds of formula (I), and X represents a leaving group selected from chloro, bromo and iodo, and preferably represents chloro;

by interacting with the corresponding compound of formula (VI)

where the Linker is as defined for compounds of formula (I).

The compounds of formula (V) are novel and thus constitute an additional aspect of the present invention.

The interaction of compounds of formulas (V) and (VI) can be carried out using methods described in international patent application WO 03/080544. In General, the interaction may be carried out in a suitable solvent, for example in aqueous buffer at pH in the range from 5 to 11, and non-extremal temperatures from 5 to 70°C., preferably at ambient temperature.

The connection form is s (V) can be obtained by standard methods of peptide synthesis, for example, solid-phase peptide synthesis, for example as described in Atherton, E. and Sheppard, R.C.; "Solid Phase Synthesis"; IRL Press: Oxford, 1989. In WO 03/006491 also described the synthesis of analogous peptides and in this respect are included in this description by reference. Enable bridging group, R3can be carried out by reacting the corresponding peptide containing two free tirinya group, with a suitable dichloramine or dichloramine (such as dichloromethane, when R3must be a methylene). The inclusion of the group X-CH2C(O)-" in the compound of formula (V) can be achieved by the interaction of N-terminal or aminecontaining amino acid, preferably lysine of the peptide with a reagent of formula (VII)

under standard conditions for the formation of a peptide bond; where X is as defined for compounds of formula (V), a Z is a HE or a suitable activating group such as chloro, bromo, fluorescent, -OC(O)CH2-X, where X is as defined for compounds of formula (V), or when Z represents-HE, the acid may be activated in situ by the use of agents such as hexaphosphate 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium (HBTU), or N-oxide hexaphosphate N-[(dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridine-1-ylmethylene]-N-methylmethane the deposits (HATU).

The compounds of formula (VI) can be obtained by standard methods such as those described in the international patent application WO 03/080544, for example, from corresponding compounds of formula (VIa)

where L represents a leaving group such as para-toluensulfonate, triftorbyenzola or methanesulfonate, and the Linker is as defined for compounds of formula (VI), and R represents hydrogen or a protective group of the thiol;

by interaction with water [18F]-fluoride obtained in the cyclotron, suitably pre-activated by evaporation from a base (for example, from tetrabutylammonium or2CO3/Kryptofix-222 (Kryptofix-222), in a suitable solvent, such as acetonitrile, N,N-dimethylformamide or dimethylsulfoxide, typically at elevated temperature, for example 60 to 120°C, followed by removal of any protective group of the thiol using standard methods.

The compounds of formula (VI), in which the Linker represents the formula (II)can be obtained from the corresponding compounds of formula (VIb)

where L represents a leaving group such as para-toluensulfonate, triftorbyenzola or methanesulfonate, and n and m are as defined for four the uly (II), and R represents hydrogen or a protective group of the thiol;

by interaction with water [18F]-fluoride obtained in the cyclotron, suitably pre-activated by evaporation from a base (for example, from tetrabutylammonium or2CO3/Kryptofix-222), in a suitable solvent, such as acetonitrile, N,N-dimethylformamide or dimethylsulfoxide, typically at elevated temperature, for example 60 to 120°C, followed by removal of any protective group of the thiol using standard methods.

The compounds of formula (VI), in which the Linker represents the formula (III)can be obtained from the corresponding compounds of formula (VIc)

where L represents a leaving group such as para-toluensulfonate, triftorbyenzola or methanesulfonate, and p is as defined for formula (III), and R represents hydrogen or a protective group of the thiol;

by interaction with water [18F]-fluoride obtained in the cyclotron, suitably pre-activated by evaporation from a base (for example, from tetrabutylammonium or2CO3/Kryptofix-222), in a suitable solvent, such as acetonitrile, N,N-dimethylformamide or dimethylsulfoxide, a typical value when the temperature for example from 60 to 120°C, followed by removal of any protective group of the thiol using standard methods.

The compounds of formula (VI), in which the Linker represents the formula (IV)can be obtained from the corresponding compounds of the formula (VId)

where L' is a leaving group such as iodo, para-toluensulfonate, triftorbyenzola or methanesulfonate, and when q is 0, L' may be a nitro or salt iodine or ammonium, and q and r are as defined for formula (IV), and R represents hydrogen or a protective group of the thiol;

by interaction with water [18F]-fluoride obtained in the cyclotron, suitably pre-activated by evaporation from a base (for example, from tetrabutylammonium or2CO3/Kryptofix-222), in a suitable solvent, such as acetonitrile, N,N-dimethylformamide or dimethylsulfoxide, typically at elevated temperature, for example 60 to 120°C, followed by removal of any protective group of the thiol using standard methods.

In formulas (VIa), (VIb), (VIc) and (VId) suitable protective groups include thiol (phenyl)3With-(trityl) and others, the description of which can be found in Protecting Groups in Organic Synthesis, Theodora W. Greene and Peter G. M. Wuts, published by John Wiley & ons Inc. Removal of such protective groups of the thiol can be carried out by standard methods such as those described in Greene. For example, when R represents trityl free thiol can be formed by treatment with dilute acid, for example triperoxonane acid in a chlorinated solvent such as dichloromethane.

In one preferred aspect of compounds of formula (VIa), (VIb), (VIc) and (VId) may be associated with a solid carrier, such as polymer granules or coatings, for example tritely or horrically polymer. In this aspect, the excess reagents or by-products of the reaction of radiotolerance may be separated from associated with the polymer product by washing. Using the methods of removing protection, as described above, carry out the separation of the compounds of formula (VI) from solid media. This method may be particularly suitable for automated acquisition of compounds of the formula (VI). Alternatively, the by-products of removing the protection of the thiol, when they are insoluble in the reaction mixture can be removed by filtering.

According to an additional aspect of the invention proposed a set to get radiotolerance peptide of formula (I)containing prosthetic group of the formula (VIa), (VIb), (VIc) or (VIa) and the activated peptide of formula (V).

When set with the unity of formula (VIa), (VIb), (VIc) or (VId) is transformed into the corresponding compound of formula (VI), using the methods described above. Preferably the compound of formula (VI) or any tilsammen predecessor can be separated from the waste chemicals by passing the reaction mixture through the cartridge for solid phase extraction (SPE). The cartridge SPE may contain graphite filler or carrier18. Any protective group of the thiol can be removed, for example, by adding acid, such as triperoxonane acid. When Tolna group in the compound of formula (VI) is protected by a hydrophobic group, such as triticina group, removing the protecting can be easily implemented on the cartridge SPE, resulting in a hydrophobic protective group of the thiol (such as trityl) remains associated with the media, while labeled prosthetic group of the formula (VI) elute with high purity and yield. The compound of formula (VI) is then added to the compound of formula (V), which can be suitably dissolved in an aqueous buffer (pH 7-11). After interaction with non-extremal temperature for from 1 to 60 min labeled peptide can be purified, for example by SPE and collected.

The invention is illustrated by the following examples in which the following abbreviations:

DHM: DIH orotan

TFU: triperoxonane acid

THF: tetrahydrofuran

HBTU: hexaphosphate 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium

Boc: tert-butoxycarbonyl

Fmoc: 9-fluorenylmethoxycarbonyl

TIS: triisopropylsilane

Examples

Example 1: Obtain RGD-containing peptide, labeled 3-fluoro-propylsulfonyl

The connection specified in the header:

1A) Synthesis of 3-criticalfor-propan-1-ol

Trailhead (27.9 mg; 0.1 mmol) and triethylamine (49 μl; 0.5 mmol) was dissolved in DHM (2 ml) before addition of 3-mercapto-1-propanol (9 μl; 0.1 mmol). DHM evaporated under reduced pressure after 6 hours and the crude product was purified by reverse-phase preparative chromatography (column Vydac 218TP1022; solvents A=water/0.1% of TFU and=CH3CN/0.1% of TFU; gradient 30-70% b over 40 min; flow rate 10 ml/minute; detection at 254 nm). Received 6 mg of purified substances (analytical HPLC: column Phenomenex Luna C18,00-4251-E0: solvents: A=water/0.1% of TFU and=CH3CN/0.1% of TFU; gradient 30-70% b over 10 min; flow rate 1.0 ml/min; retention time 7,73 min recorded at 214 and 254 nm). The structure was confirmed by NMR.

1b) Synthesis of 3-criticalfor-propyl ether methanesulfonate acid

Methylchloride (6 μl; 0075 mmol) was added to a solution of 3-criticalfor-propan-1-ol (5 mg; 0.015 mmol) and triethylamine (32 μl; 0.23 mmol) in THF (1 ml). After 30 min, the THF evaporated under reduced pressure and the crude product was dissolved in DHM, washed with a saturated solution of sodium bicarbonate in water, a saturated solution of sodium chloride and dried MgSO4. Output 10 mg obtained after evaporation under reduced pressure (analytical HPLC: column Luna C18,00B-4251-E0: solvents: A=water/0.1% of TFU and=CH3CN/0.1% of TFU; gradient 40-80% b over 10 minutes; flow rate 1.0 ml/min; retention time for 7.12 min recorded at 214 and 254 nm). The structure was confirmed by NMR.

1C) Synthesis of (3-fluoro-propylsulfonyl)triphenylmethane

The potassium fluoride (1.4 mg; 0,024 mmol) and Kryptofix 222 (9.0 mg; 0,024 mmol) was dissolved in acetonitrile (0.2 ml) (heating). Added 3-criticalfor-propyl ether methanesulfonate acid (5 mg, 0.012 mmol) in acetonitrile (0.2 ml). The reaction mixture was heated up to 80 degrees for 90 minutes. The crude product was purified by reverse-phase preparative chromatography (column Vydac 218TP1022; solvents A=water/0.1% of TFU and=CH3ARTICLE/0,1% TFU; the gradient of 40-90% over 40 min; flow rate 10 ml/min; detection at 254 nm). Received 2 mg of purified material (analytical HPLC: column Phenomenex Luna C18,00B-4251-E0: solvents: A=water/0.1% of TFU and=CH2CN/0.1% of TFU; gradient 40-80 In for 10 minutes; the flow rate of 1.0 ml/min; retention time of 8.2 min recorded at 214 and 254 nm). The structure was confirmed by NMR.

1d) Synthesis of Fmoc-Lys(Boc)-Cys(StBu)-Arg(Pmc)-Gly-Asp(OtBu)-Cys(StBu)-Phe-Cys(Trt)-Rink Amide AM polymer

Specified in the header of the peptide sequence was synthesized on an automated peptide synthesizer ABI 433A, starting with Rink Amide AM resin dispenser 0.1 mmol, using 1 mmol amino acid cartridges. Amino acids pre-activated using HBTU before combination.

1E) Synthesis of Fmoc-Lys(Boc)-cyclo[Cys(CH2)-Arg(Pmc)-Gly-Asp(OtBu)-Cys]-Phe-Cys(Trt)-Rink Amide AM polymer

0.05 mmol peptidyl-polymer, obtained as described in (1d), was treated with a solution of 346 μl of tributylphosphine, 100 μl of water and 2 ml of dimethylformamide. The reagents were removed after 90 minutes and the polymer was washed with dimethylformamide and dichloromethane. The polymer is then treated with a solution of 63 mg of tetrabutylammonium and 2 ml of dichloromethane. The reagents were removed by filtration through 2 hours and the polymer was washed several times with dichloromethane.

1f) Synthesis of CI-CH2CO-Lys-cyclo[Cys(CH2)-Arg-Gly-Asp-Cys]-Phe-Cys-NH2

9-Fluorenylmethoxycarbonyloxy group was removed from the peptidyl-polymer (1E) and N-end was subjected to chlorotyrosine when using Chloroacetic anhydride acid. The simultaneous removal of peptide and the protective groups of the side chain is of the polymer is then conducted in 5 ml triperoxonane acid (TFU), containing 2.5% of triisopropylsilane and 2.5% water for 1 hour and 40 minutes

After processing received 27 mg of the crude peptide (analytical HPLC: gradient of 0-40% b over 10 min where A=N2O/0.1% of TFU and=CH3CN/0.1% of TFU; 1 ml/min; column, Phenomenex Luna 3µ, C18 (2) 50×4.6 mm; detection, UV 214 nm; retention time of the product 7,79 min).

Additional product identification was performed using mass spectrometry with elektrorazpredelenie: expected M+H when 1018,3 detected by 1018,3.

1g) Synthesis of cyclo[CH2CO-Lys-cyclo[Cys(CH2)-Arg-Gly-Asp-Cys]-Phe-Cys-NH2

27 mg of the peptide of the product obtained as described in (1f), was dissolved in a mixture of water/acetonitrile. Brought the pH of the mixture to 8 with aqueous ammonia and stirred for 2 hours. After lyophilization received 26 mg of the desired product.

Purification by preparative HPLC (column Phenomenex Luna 5µ C18 (2) 250×21,20 mm) of the crude substance was performed using 0-30%, where

A=N2O/0.1% of TFU and=CH2CN/0.1% of TFU for 40 min at a flow rate of 10 ml/min. After lyophilization received 9 mg of pure substances. (Analytical HPLC: gradient 0-30% b over 10 min where A=N2O/0.1% of TFU and=CH3CN/0.1% of TFU; flow rate 1 ml/min; column, Phenomenex Luna 3µ C18 (2) 50×4.6 mm; detection, UV 214 nm; retention time of the product 7,00 min). Additionally the th product identification was performed using mass spectrometry with elektrorazpredelenie: expected M+H when 982,4, found at 982,3.

1h) Synthesis of cyclo[-CH2CO-LYs(CL-CH2CO-amino-PEG-cyclo[Cys(CH2)-Arg-Gly-Asp-Cys]-Phe-Cys-NH2

9 mg of the peptide obtained as described in (1g), 34 mg anhydride of BOC-amino PEG and 7 μl of N-methylmorpholine was dissolved in 1 ml of dimethylformamide and the mixture was stirred for 30 minutes. The reaction was suppressed by adding a solution of 25 μl of N-methylmorpholine and 2 ml of water (pH ~9). The mixture was stirred for 2 hours, then evaporated to dryness. The residue was treated with a solution of 5 ml of TFU, containing 2.5% of triisopropylsilane and 2.5% water for 1 hour. TFU evaporated under vacuum, was added to the residue, diethyl ether and the precipitate was washed with diethyl ether and dried in the air. The precipitate was dissolved in 3 ml of dimethylformamide together with 8 mg of Chloroacetic anhydride acid and 9 μl of N-methylmorpholine and the mixture was stirred for another 60 minutes, the Reaction mixture was evaporated to dryness.

Purification by preparative HPLC (column Phenomenex Luna 5µ C18 (2) 250×21,20 mm) of the crude substance was performed using 5-50%, where

A=N2O/0.1% of TFU and=CH3CM/0,1% TFU for 40 min at a flow rate of 10 ml/min. After lyophilization received 5 mg of pure substances. (Analytical HPLC: gradient, 5-50% b over 10 min where A=N2O/0.1% of TFU and=CH3CN/0.1% of TFU; flow rate 1 ml/min; column, Phenomenex Lna 3µ C18 (2) 50×4.6 mm; detection UV 214 nm; retention time of the product was 7.08 min). Additional product identification was performed using mass spectrometry with elektrorazpredelenie: expected M+H when 1436,5 detected by 1436,0.

1i) Site-specific conjugation to a chloroacetyl-modified peptide

Trityloxy group tsalala from (3-fluoro-propylsulfonyl)triphenylmethane (2.0 mg; 0,006 mmol)described in (1C), using TFU (100 μl) in the presence of TIS (10 ml) and water (10 μl) (5 min). The mixture was diluted with 250 μl of water and 250 μl of acetonitrile before adding solution c[CH2CO-Lys(CICH2CO-amino-PEG)-c[Cys(CH2)-Arg-Gly-Asp-Cys]-Phe-Cys]-NH2(4.0 mg; 0,003 mmol) of (1h) in 500 μl of water and 500 μl of acetonitrile and the pH was brought to 10 buffer solution of potassium carbonate (approximately 400 μl). The reaction mixture was heated to 60°C for 50 minutes, the Reaction mixture was extinguished TFU and purified using reverse-phase preparative chromatography (column Phenomenex, C18, OOG-4253-N0; solvents A=water/0.1% of TFU and=CH3CN/0.1% of TFU; gradient 10-50% b over 30 min; flow rate 5 ml/min; detection at 254 nm). Received 1.1 mg of purified substances (analytical HPLC: Vydac column TR: solvents; A=water/0.1% of TFU and=CH3CN/0.1% of TFU; gradient 10-50% b over 10 min; flow rate 1.0 ml/min; retention time 13,20 min, detection of the ri 214 and 254 nm). Further identification was performed using mass spectrometry, to obtain a value of m/z 1494,1 [M-H+]as expected for the desired product.

Example 2: Obtain RGD-containing peptide, labeled 3-[18F]-fluoro-propylsulfonyl

The connection specified in the header:

2A) Obtaining18F-sintana: (3-[18F]-fluoro-propylsulfonyl)-triphenylmethane

In a Wheaton vial (2 ml), filled Kryptofix® 222 (10 mg), potassium carbonate (1 mg dissolved in 50 μl water) and acetonitrile (0.8 ml), was added to water containing fluorine-18 (10 MCI; 1 ml). The solvent was removed by heating at 110°C for 1 hour in a stream of nitrogen. Added anhydrous acetonitrile (0.5 ml) and again evaporated as before. This step was repeated twice. The vial was cooled to room temperature, followed by the injection of a solution nelfinavir, obtained as described in Example (2b) (1 mg)in anhydrous dimethylsulfoxide (DMSO) (0.2 ml). The reaction mixture was stirred at 80°C for 5 min and analyzed by HPLC (gradient 1, radiochemical yield 90%).

The reaction mixture was diluted with a mixture of DMSO/water (1:1 vol./vol.; 0.15 ml) and was loaded in the SepPak cartridge-Plus (tC18, Waters), which was previously kondicionirovanie (10 ml of acetonitrile, 20 ml of water). The cartridge was washed in the Oh (10 ml) and the product was suirable, using acetonitrile. Radiochemical purity was 99%.

2b) Site-specific conjugation to a chloroacetyl-modified peptide

Unprotect (3-[18R]-fluoro-propylsulfonyl)triphenylmethane, obtained as described in (2A), and the subsequent interaction with chloroacetyl-modified peptide, obtained as described in (1h)was performed using methods similar to those described in Example (1i)

Biological data

Using known drugs of cell membrane expression of the receptor αvβ3-integrin, conducted research competitive binding when using125I-echistatin and F-labeled peptides as competing ligand. Received binding curves and expected values of Kiwhen using the software Prism™.

The compound of Example 1 showed a value of Ki7 nmol.

Example radiopharmaceutical composition

IngredientsNumber
1. The connection according to the invention10 MCI (quantity, suitable for positron emission tomography (PET))
2. Sterile pyrogen-free water50 ml
The source components are mixed to obtain an aqueous solution.

1. The compound of formula (Ia)
,
where R1represents a

where a is an integer from 1 to 10;
R2represents-NH2;
R3represents a C1-4Allenby bridge;
The linker represents -(CH2)p-where R is an integer from 1 to 20.

2. The compound of formula (Ia) according to claim 1, in which
R3represents-CH2-; and
but a 5.

3. The compound of formula (Ia) according to claim 1, which is a

4. The compounds of formula (Ia) according to any one of claims 1 to 3, bind to receptors associated with angiogenesis.

5. Radiopharmaceutical composition for binding to receptors associated with angiogenesis, containing the compound of formula (Ia) according to any one of claims 1 to 3 and one or more than one pharmaceutically acceptable excipient.

6. The method of obtaining the compounds of formula (Ia) according to any one of claims 1 to 3, including the interaction of the compounds of the formula (V)
,
where R1represents a
,
where a is an integer from 1 to 10;
R2represents-NH2;
R 3represents a C1-4Allenby bridge;
and X represents a leaving group selected from chloro, bromo and iodo, and is preferably chloro;
with a corresponding compound of formula (VI)
,
where the Linker is a -(CH2)p-where R is an integer from 1 to 20.

7. The compound of formula (V)
,
where R1represents a
,
where a is an integer from 1 to 10;
R2represents-NH2:
R3represents a C1-4Allenby bridge;
and X represents a leaving group selected from chloro, bromo and iodo, and is preferably chloro.

8. Set to obtain radiotolerance peptide of formula (Ia) according to any one of claims 1 to 3, containing:
1) compound of formula (VIa)

where L represents a leaving group such as para-toluensulfonate, triftorbyenzola or methanesulfonate;
The linker represents -(CH2)p-where R is an integer from 1 to 20;
R represents hydrogen or a protective group of the thiol;
and
2) the activated peptide of formula (V)
,
where R1represents a
,
where a representation is employed, an integer from 1 to 10;
R represents-NH2;
R3represents a C1-4Allenby bridge;
and X represents a leaving group selected from chloro, bromo and iodo, and is preferably chloro.



 

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