KR20000025145A - Thioacetyl dipeptide derivatives and bifunctional ligand manufactured therefrom and preparation method thereof - Google Patents

Abstract

PURPOSE: A new thiopeptide derivatives and bifunctional ligands prepared therefrom and a preparation method thereof are provided which can produce a large amount of N3S thioacetyl dipeptide derivatives easily. CONSTITUTION: A new thiopeptide derivatives of formula I in the form of active ester as an intermediate useful for manufacturing a tetracoordinative chelate compound containing a sulfur atom which can be used widely for a metal chelation reaction including a radioactive nuclide of bifunctional ligands and bifunctional ligands manufactured therefrom and preparation method thereof are described. Various tetracoordinative chelate compounds of a N3S group which can be used effectively for diagnosing and treating a disease such as cancer can be manufactured in the case of using the compound I. In formula, T is an ester or acetyl group as a sulfur atom protecting group, R1,R2 are each independently H or amino acid side chain, X is -Cl, -O-(CO)-OR0, or -O-A in which R0 is ethyl, isobutyl, isovaleryl or t-butyl, A is 1-8C alkyl, alkenyl, alkinyl, alkoxy, aryl, aryloxy, alkylcarbonyl, pentachlorophenyl or pentafluorophenyl.

Description

Thioacetyl Dipeptide Derivatives, Bifunctional Ligands Prepared Therefrom, and Methods for Preparing the Same

The present invention relates to a thioacetyl dipeptiide derivative as an intermediate of a bifunctional ligand and a method for preparing the same, and more particularly, 4 containing a sulfur atom which can be widely used in the chelation reaction of metals including radionuclides. Thioacetyl dipeptide derivatives as intermediates useful in the preparation of coordination chelating agents, bifunctional ligands prepared therefrom, and methods for their preparation.

Radioisotopes tend to selectively accumulate only in specific areas with biological properties, such as cancerous tissues, inflammatory tissues, kidneys, heart, etc., in the field of nuclear medicine. It is utilized as a preparation for. Typically, it is used as a chelation compound using various ligands to improve stability and selectivity as a medicament, which is useful in that it can reduce unnecessary exposure and ensure high precision of diagnosis and treatment. As one of the methods for enhancing affinity for a specific site or tissue for imaging, the use of biochemical reactions between biomolecules in vivo has recently attracted attention. For example, in radioimmunodiagnostics and treatment using antigen-antibody reactions, it may be useful to label radioisotopes to biomolecules such as antibodies or oligopeptides, but considering the effects on biochemical stability, bifunctional ligands May be particularly useful.

Currently, chelated compounds labeled with radionuclides are useful in both diagnosis and treatment of diseases such as cancer, and their applications are also expanding. The most important properties required for radioisotope labeling compounds as such final drugs are, as described above, highly specific activity and excellent stability in-vivo and in-vitro. In order to meet these conditions, it has been desired in the art to develop bifunctional ligands having excellent chelation effects while having little effect on the properties of the biological molecules themselves.

In response to these demands, studies are being actively conducted to label biochemical or medically useful biological molecules such as antibodies and DNA with radioisotopes such as ^99m Tc or ¹⁸⁸ Re for diagnosis and treatment of diseases.

Conventionally, chelating compounds such as ethylenediaminetetraacetate (EDTA) or diethylenetriaminepentaacetate (DTPA) labeled with radionuclides have been reported to be useful for the evaluation of renal function (Klingquensmith et al., J. Nucle. Med. 23: 377 (1982)), and radiotechnetium chelate compounds of diamidodimercaptide have also been reported to be promising kidney drugs (J. Med. Chem. 29, 1933 (1986)).

European Patent Publication No. 0 329 481 also discloses a mercaptoacetylglyciglyciglycine (MAG ₃ , Fritzberg et al., J. Nucle. Med 27: 2) as a bifunctional ligand as an intermediate that can be used to prepare radionuclide labeled chelate compounds. 111-116 (1986)), where it is reported that MAG ₃ can be used as a diagnostic agent for renal function administered as it is labeled with a radionuclide such as ^99m Tc.

Compounds of this kind serve to safely link radionuclides to various target-specific biomolecules, such as carrier molecules such as proteins, antibodies or antibody fragments, DNA and lipids. These bifunctional chelating agents allow for the preparation of highly specific and stable biological molecules, the structure of which is four ligands comprising 1-2 thiols as S-donors and amine or amido forms as N-donors In addition, it is possible to form stable chelating compounds with radionuclides such as ^99m Tc or ¹⁸⁸ Re, and is commonly referred to as N ₂ S ₂ or N ₃ S ligand. The common structure for this type of ligand, on the other hand, is the N-terminus substituted with the S-protecting mercaptoacetyl group and C- as a useful moiety for the introduction of biological molecules such as proteins, lipids, or antibodies. It has a terminal. Such C-terminus is usually present in the form of an active ester to form a covalent bond directly with a free amine or sulfur atom in the biological molecule.

On the other hand, the binding between the ligand as a chelating agent and the biological molecule is based on the formation of a direct covalent bond as described above, and there is a method using a biotin- (strep) avidin system having a high binding affinity (DJ Hnatowich et al .: J. Nucl. Med., 28, 1294-1302).

It is known to form strong bonds between the biotin and (strep) avidin due to extremely complementary steric fit. This high affinity between avidin-biotin can be used for the diagnosis and treatment of cancer in two- or three-stage techniques in nuclear medicine using radionuclides. That is, a pretargettig tumor technique in which a biotinylated anticancer antibody is pre-administered and then radiolabeled biotinylated avidin or (strep) avidin is administered. It is one of the promising approaches that can improve the relative ratio between normal tissues.

Among the various methods for the preparation of bifunctional chelating agents having a structure having various moieties as described above, the only method for preparing N ₃ S-based ligands is the MAG ₃ proposed by Fritzberg et al. J. Nucl. Med. 27 : 111-116 (1986), Cancer, 80 : 2347-2353). This method is to react an active ester of S-protected thioglycolic acid with a tripeptide such as triglycine, as shown in the following Chemical Scheme 2.

[Scheme 6]

(Wherein Bz represents a benzoyl group)

This method appears to be simple on the surface, but has the following problems associated with using tripeptides directly. That is, when the tripeptide is used to prepare a bifunctional chelating agent having a structure having various moieties including biotin at the C-terminus of the peptide for binding to a biological molecule, the tripeptide is generally commercially available. Since it is not sold, there is a problem of synthesizing it using an organic chemical synthesis method or an enzymatic synthesis method. However, the preparation of tripeptides by organic chemical synthesis, which inevitably entails the attachment of a protecting group to the N- or C-terminus and the leaving of a protecting group, is much more complicated and yields much less than that of the preparation of dipeptides. There is a problem that there is a significant limitation in the manufacturing target range. On the other hand, when the tripeptide is prepared by enzymatic synthesis using a solid phase, it is not necessary to perform a complex and cumbersome protecting group attachment reaction and a protecting group leaving reaction on the N-terminus or C-terminus and While there is a relatively easy advantage, there is a problem that a large amount of production is difficult, and there are still disadvantages as mentioned above compared to the case of preparing dipeptides. Therefore, the production and use of tripeptides for the preparation of bifunctional chelating agents is problematic, as well as inefficient and cumbersome, resulting in low yields.

In order to solve the conventional problems as described above, the present invention obtains a dipeptide intermediate using an S-protected thioglycolic acid group as the N-terminal protecting group of the peptide and economical and various combinations of dipeptides, and then C-terminal. In the case of using conventional tripeptides by introducing donor units having various moieties such as β, γ-amine alkanoic acid, aminocyclo alkanoic acid, biotin, as well as conventional amino acids, the preparation was not easy. It is for easily providing a large amount of thioacetyl dipeptide derivative of the N ₃ S series.

Accordingly, the first object of the present invention is a novel thioacetyl dipeptide derivative useful as an intermediate of the N ₃ S family ligand required for the preparation of a 4-coordinate chelate compound widely used in the chelation reaction of metals including radionuclides and using the same. It is to provide a N ₃ S series ligand.

A second object of the present invention is to provide a method for preparing various compounds according to the first object described above using dipeptides.

The present invention provides novel thioacetyl dipeptide derivatives represented by the following formula [I] as intermediates for the preparation of N ₃ S series ligands required for the preparation of 4-coordinate chelate compounds widely used for the chelation reaction of metals including radionuclides and The present invention relates to a N ₃ S-based ligand and a method of preparing the same.

Formula I

In the food,

T is a sulfur atom protecting group, an ester group or an acetal group,

R ₁ and R ₂ are each independently H or an amino acid side chain,

X is -Cl, or -O- (CO) -OR ₀ , or -OA,

here,

R ₀ is an ethyl group, isobutyl group, isovaleryl group or t-butyl group,

A is o-nitrophenyl group, m-nitrophenyl group, p-nitrophenyl group, 2,4-dinitrophenyl group, succinimidyl group, tetrafluorophenyl group, pentachlorophenyl group, or pentafluorophenyl group.

The compound represented by Formula [I], [II], [III], and [IV] which concerns on this invention, and its manufacturing method are shown by following Reaction Formula (1).

As a starting material in the method of this invention, it is preferable to use the sulfur atom protective thioglycolic acid represented by following formula [VI].

The compound represented by the formula [VI] is prepared by making thioglycolic acid into an alkali metal salt such as an alkali metal salt such as Na or K and then attaching various sulfur atom protecting groups. This reaction is carried out by addition of alkali metal hydroxide (M-OH) in benzoyl chloride. This protecting group prevents side reactions caused by sulfur atoms in each reaction step during the synthesis, stabilizes the chelating agent after the synthesis is completed, and can be easily removed for chelation with radionuclides. As such a sulfur atom protecting group, an ester group or an acetal group is preferable, and a benzoyl, acetyl, tetrahydro pyranyl, ethoxy ethyl, etc. are mentioned specifically ,.

The prepared S-protected thioglycolic acid is dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide, tetrahydrofuran (THF) or dimethoxyethane (DME) according to conventional methods in the art. Reaction at low temperature (0-5 ° C.) under anhydrous solvent such as) affords the compound of formula [VII] in the form of an active ester.

[Chemical Scheme 1]

According to the method of the present invention, the above-mentioned compound of formula [VII] in the form of an active ester is mixed in an aqueous alkali solution of an amino acid dipeptide or in an aqueous dimethylformamide solution, and then stirred for about 1 to 2 hours to give S- of formula [VIII]. Obtain protective thiacetyldipeptide.

R ₁ and R ₂ in the amino acid dipeptide are each independently H or an alkyl group having 1 to 4 carbon atoms which may be substituted. When R ₁ and R ₂ are each H, it is a glycine dipeptide. As the amino acid dipeptide usable in the present invention, dipeptides of monoamino monocarboxylic amino acids are preferable, and dipeptides consisting of glycine, alanine, valine, leucine and combinations thereof are preferable.

T and R ₁ and R ₂ in the above-mentioned S-protected thioacetyl dipeptide of formula [VIII] are as defined above and may have a protecting group if necessary.

The above-mentioned S-protected cheoacetyl dipeptides of the formula [VIII] have a carboxyl group for the formation of amide bonds, wherein the carboxyl group is one of the target compounds according to the invention represented by the formula [I] in the form of acid chlorides, mixed anhydrides and active esters. Obtain dipeptide derivatives.

First, when the compound of formula [I] is an acid chloride whose X = Cl, it is prepared by applying thionyl chloride, phosgene or phosphorus pentachloride on anhydrous solvent,

Secondly, when the compound of formula [I] is a mixed anhydride with X = -O- (CO) -OR ₀ , -20 ° C to 0 ° C in the presence of tertiary amines such as N-methyl morpholine or triethylamine It is obtained by reacting with an alkyl chloroformate at a low temperature of, wherein the alkyl group R ₀ used here is an ethyl group, isobutyl group, isovaleryl group, t-butyl group and the like.

Third, when the compound of formula [I] is in the form of an active ester wherein X = -OA, the S-protecting dipeptides are prepared in a suitable solvent, preferably dimethylformamide (DMF) and dimethoxyethane (DME), or After dissolving in a mixed solvent such as tetrahydrofuran (THF), an alcohol represented by A-OH in the presence of a condensing agent such as dicyclohexylcarbodiimide (DCC) or benzotriazole, wherein A may be substituted. , Succinimidyl group, tetrafluorophenyl group, o-nitrophenyl group, m-nitrophenyl group, p-nitrophenyl group, pentachlorophenyl group or pentafluorophenyl group, and the like. , Alkynyl, alkoxy, alkoxyalkyl, aryl, aryloxy, arylamino, alkylcarbonyl, or arylcarbonyl, heterocycle, or halogen). All innumerable The reaction temperature is low temperature, preferably in the range of -10 ° C to 15 ° C.

Compounds of formula [I] that can be produced by any of the three reactions described above are useful intermediates of the N ₃ S family ligands.

In said formula [I], T, R <_1> and R <_2> , are as defined above.

As used throughout this specification, the term "active ester" means an ester that is very reactive in nucleophilic substitution reactions and should be relatively stable in consideration of hydrolysis in aqueous solution.

Such active esters are highly reactive with amino groups or nucleic acid bases of biological molecules such as lipids, DNA, etc., having proteins or organic amino groups, thereby binding chelating agents containing these active esters to the biological molecules described above. These active esters need to be sufficiently electron-withdrawing so that the ester carbonyl is susceptible to attack by nucleophilic groups on biological molecules such as proteins, lipids or DNA. In other words, the kinetics of this reaction should allow the ester to react rapidly with amino groups such that unreacted free esters, which are susceptible to hydrolysis, can only be present for a short time.

Preferred such esters in the present invention include o-nitrophenyl, p-nitrophenyl, 2-chloro-4-nitrophenyl, cyanomethyl, 2-mercaptopyridyl, hydroxybenzotriazole and N-hydroxysuccinate. Imides, trichlorophenyl, tetrafluorophenyl, 2-fluorophenyl, 4-fluorophenyl, 2,4-difluorophenyl, o-nitro-p-sulfophenyl, N-hydroxyphthalimide, Tetrafluorothiophenyl etc. are mentioned. Most of the esters suitable for the present invention are activated phenols, in particular nitro-activated phenols and hydroxylamine based cycle compounds. Particularly preferred is 2,3,5,6-tetrafluorophenyl ester, and exhibits high reactivity and good stability.

As one of the characteristic steps of the present invention, the thioacetyl dipeptide derivative according to the present invention represented by the above-mentioned formula [I] is reacted in a water-soluble medium with various compounds of the above-mentioned formula [V] having an amino group. The compound represented by [II] is produced. That is, an aqueous solution of the amino group-containing compound to be introduced is dissolved in a suitable solvent, preferably DMF and DME or a mixed solvent of DMF and THF, in the form of an active ester, as described above. The reaction mixture obtained by adding the mixture to the mixture at room temperature and stirring for further 2 hours was concentrated by distillation under reduced pressure, and then adjusted to pH at about 2-3 to obtain another target compound according to the present invention represented by the above formula [II]. .

Wherein T and R ₁ and R ₂ are as defined above,

B is C ₃ -C ₆ cycloalkane, or `

Is,

R ₃ is a side chain of a common amino acid such as R ₁ and R _2, and may be attached with a protecting group if necessary. In particular, when R ₃ is the side chain of lysine, the ε-amino group includes those in which amide is bonded to biotin.

When B is cycloalkane, B is 1,1-, 1,2-, 1,3-, 1,4-cyclohexyl, or 1,1-, 1,2-, 1,3-cyclopentyl, or 1,1-, 1,2-, 1,3-cyclobutyl, or 1,1-, 1,2-cyclopropyl.

n is an integer of 1-6 when R ₃ is H, and an integer of 1 when R ₃ is not H.

Compounds represented by the formula [II], which is one of the objective compounds of the present invention obtained according to the present invention, are all 4 coordination chelating agents of the N ₃ S series in which hetero atoms are composed of three nitrogen atoms and one sulfur atom, In which the sulfur atom is a divalent sulfur atom and the nitrogen atom is each independently an amide, and among the compounds [II] obtained according to the method of the present invention, a novel compound mercaptoacetyl glycylglyciyl-γ-aminobutyl Acids (MAG ₂ GABA) and mercaptoacetylglycidyl glyciobiotin (MAG ₂ Biocytin), mercaptoacetylglycidyl glycylglycine (MAG ₃ ), which is a conventionally known compound, and the like.

In the compound represented by the above formula [II], the carboxyl terminus may be activated in the form of an active ester represented by the above formula [III] for conjugation with a biological molecule, and this reaction is chelated with the radionuclide. Available both after and before chelation. That is, after dissolving the compound represented by the formula [II] in a mixed solution of a compound such as N-hydroxysuccinimide and a solvent such as DMF, THF or DME, adding DCC and stirring at room temperature for 10 to 20 hours A compound of the other active ester form according to the invention represented by the above formula [III] in a yield of 87.8% can be obtained.

In formula [III], A is as defined above and forms an active ester in the form of-(CO) -O-A.

Here, the active ester represented by-(CO) -O-A is an active ester such as the formula [I] described above, but an active ester under more demanding conditions. That is, this active ester group is one of the bifunctional groups possessed by the chelating agent mentioned in the present invention and becomes a conjugation group (conjugated group) for binding to proteins, lipids or DNA. In general, these proteins, lipids or DNAs serve to deliver the radionuclide complexed in the bound ligand to a desired target point in the body. These conjugation groups need to be capable of conjugation under mild conditions such that they can maintain the activity of biological molecules such as proteins in an aqueous medium. Proteins usually contain free amino groups or the like as functional groups that can be used for such conjugation.

When the active ester of the compound represented by the above formula [III] is reacted with biocithin, another compound according to the present invention according to the present invention is obtained. Z represents a biocitin group here.

The present invention will be described in more detail with reference to the following examples.

EXAMPLE

Example 1

Preparation of S-benzoylthioglycolic acid (compound of formula [VI])

Prepare 350ml aqueous solution of NaOH 44.4g (1.1moles) water in the flask, add 350ml of benzene, and add 47.5g (0.5 moles) of thioglycolic acid dropwise using an ice water bath so that the temperature of this mixture does not exceed 15 ℃. Salt solution was prepared. 64.4 ml (78.1 g, 0.55 moles) of benzoyl chloride was added dropwise thereto while being careful not to exceed 10 ° C., and the reaction mixture was further stirred at room temperature for 30 minutes. After taking only the water layer and washing two more times with 100ml of benzene to remove organic impurities and adjusted the pH to 1 with concentrated hydrochloric acid. The resulting white precipitate was filtered, washed well with cold water and dried to give 94.64 g of the desired product. The crude product was recrystallized from 50 ml of ethyl acetate to give 51.15 g (52.1% yield) of the title compound as a white crystalline solid.

m.p = 106-108 ° C

¹ H NMR (CDCl ₃ )

δ 7.47-8.0 (m, 5H,) 3.92 (s, 2H,)

Example 2

Preparation of S-benzoylthioglycolic acid succinimidyl ester (compound of formula [VII])

S-benzoylthioglycolic acid (51.15 g, 0.26 mol) and N-hydroxysuccinimide (31.41 g, 1.05 eq) were dissolved in 300 ml of dimethoxyethane (DME), and the mixture was cooled to -5 to 0 ° C. After dicyclohexylcarbodiimide (DCC, 56.32 g, 1.05 eq) was added. The reaction mixture was stirred for 2 hours at 0 ° C. and 16 hours at room temperature, and then filtered to remove dicyclohexylurea. The residue obtained by distillation of the filtrate under reduced pressure was added to ethyl acetate and recrystallized three times to obtain the title compound 40.364 g (52.9%) was obtained.

m.p = 135-141 ° C

¹ H-NMR (DMSO-d ₆ )

δ 8.0-7.5 (m, 5H,) 4.45 (s, 2H,) 2.8 (s, 4H,)

Example 3

Preparation of N- (S-benzoylmercaptoacetyl) -glyciglycine (MAG ₂ : compound of formula [VIII])

A solution of S-benzoylthioglycolic acid succinimidyl ester (23.3 g, 0.0794 moles) in 250 ml of anhydrous acetone was dissolved in another 500 ml flask with 10.49 g (0.0794 mol, 2eq.) And 13.3 g (0.1588 mol) of NaHCO ₃ . ) Was added to an aqueous alkali solution of glycylglycine dissolved in 100 ml of distilled water. After 1.5 hours, acetone was removed and the remaining aqueous solution was adjusted to pH 3.0 with concentrated HCl, and the obtained solid was washed twice with distilled water, then washed with ethanol and dried to obtain 30.391 g (89.0% yield) of the title compound.

m.p = 190-191 ° C

¹ H-NMR (DMSO-d ₆ )

δ 7.42-7.99 (m, 5H) 8.23 (t, 1H) 8.54 (t, 1H)

3.90 (s, 2H) 3.78 (d, J = 5.8 Hz, 4H)

Example 4

Preparation of Succinimidyl-N- (S-benzoylmercaptoacetyl) glycine glycinate (compound of the present invention of formula [I])

Into the flask, N- (S-benzoylmercaptoacetyl) glycylglycine (18.61 g, 0.06 mol) and N-hydroxysuccinimide (7.12 g, 0.06 mol) were added to 200 ml of a 1: 1 mixed solvent of DMF and DME. Dissolved. After the reaction vessel was cooled in an ice water bath, DCC (12.5 g, 0.06 moles) was added dropwise in 30 ml of DME. After stirring the reaction mixture at 0 ° C. for 2 hours at room temperature for 16 hours, the resulting DCC-urea precipitate was filtered off and the filtrate was concentrated by distillation under reduced pressure. 50 ml of ethyl acetate was added thereto and crystallized to give 13.07 g of white crystals as the first product, and the filtrate was distilled under reduced pressure to obtain 8.3 g of the second product. The first and second products were combined and washed well with isopropanol to give 19.1 g (78.2% yield) of the title compound as desired.

m.p = 144-148 ° C.

¹ H-NMR (DMSO-d ₆ )

δ 8.60 (brt, 2H,) 4.30 (d, J = 5.12 Hz, 2H,) 3.91 (s, 2H,)

3.82 (d, J = 5.22 Hz, 2H,) 2.83 (s, 4H)

Example 5

Preparation of N- (S-benzoylmercaptoacetyl) -glycosyl glycyl γ-aminobutanoic acid (MAG ₂ GABA: compound of formula [II])

γ-aminobutanoic acid (GABA) (1.57 g, 0.015 mol, 1.01 eq.) and NaHCO ₃ (2.53 g, 0.03 moles, 2.0 eq.) were dissolved in 30 ml of distilled water. A solution dissolved in 30 ml of DME and 7 ml of DMF of the compound (6.11 g, 0.015 moles) of I] was slowly added. After stirring the reaction mixture for 2 more hours at room temperature, DME was removed by distillation under reduced pressure, and concentrated HCl was added to adjust the pH to 2.0, and the resulting white solid was filtered. To this was added 10 ml of acetonitrile and recrystallization to give 4.61 g (77.7%) of the title compound (MAG ₂ Gaba) as white crystals.

m.p = 158-165 ° C

¹ H-NMR (DMSO-d ₆ )

δ 12.5 (br, 1H) 8.51 (t, J = 5.5 Hz, 1H) 8.13 (t, J = 5.8 Hz)

7.94-7.54 (m, 5H) 7.74 (t, J = 5.5 Hz, 1H) 3.89 (s, 2H)

3.76 (d, J = 5.59 Hz, 2H) 3.67 (d, J = 5.83 Hz, 2H)

3.06 (q, J = 6.5Hz, 2H) 2.20 (t, J = 7.46Hz, 2H)

1.62 (q, J = 7.20 Hz, 2H)

Example 6

Preparation of N- (S-benzoylmercaptoacetyl) -glycosyl glycylglycine (MAG ₃ : compound of formula [II])

The same procedure was repeated except that glycine was used instead of GABA (γ-aminobutanoic acid) in Example 5 to obtain the title compound.

m.p = 192-196 ° C. (decomposition)

¹ H-NMR (DMSO-d ₆ )

δ 12.5 (br, 1H) 8.51 (t, 1H) 8.15-8.25 (2t, 2H)

8.0-7.5 (m, 5H) 3.9 (s, 2H) 3.9-3.7 (m, 6H)

Example 7

Preparation of N- (S-benzoylmercaptoacetyl) -glycosyl glycyl 4-aminocyclohexylcarboxylic acid (compound of formula [II])

The same procedure was repeated except that cis-4-aminocyclohexylcarboxylic acid was used instead of glycine in Example 6 to obtain the title compound.

m.p = decomposition

¹ H-NMR (DMSO-d ₆ )

δ 12.5 (br, 1H) 8.51 (t, 1H) 8.15-8.25 (2t, 2H)

8.0-7.5 (m, 5H) 3.9 (s, 2H) 3.9-3.7 (m, 6H) 1-1.5 (m)

Example 8

Preparation of N- (S-benzoylmercaptoacetyl) glycyl glycyl-γ-aminobutanoic acid tetrafluorophenyl ester (compound of formula [III])

After dissolving N- (S-benzoylmercaptoacetyl) -glycosylsilyl-γ-aminobutyl acid (1 g, 2.5 mmol) and tetrafluorophenol (0.462 g, 0.0028 mol, 1.1 eq.) In 25 ml of DMF, DCC (0.57 g, 2.75 mmol) was added. The reaction mixture was stirred for 18 hours at room temperature, filtered and the filtrate was distilled under reduced pressure and the residue was recrystallized from ethyl acetate (60 ml) to give 381 mg (28.1% yield) of the title compound.

m.p = 174-180 ° C

¹ H-NMR (DMSO-d ₆ )

δ 8.53 (t, 1H) 8.16 (t, 1H) 7.94-7.54 (m, 5H)

7.93 (m, 1H) 7.80 (t, J = 5.66 Hz, 1H) 3.89 (s, 2H)

3.76 (d, J = 5.60Hz, 2H) 3.69 (d, J = 5.86Hz, 2H)

3.16 (q, J = 5.90Hz, 2H) 2.78 (t, J = 7.46Hz, 2H)

1.80 (q, J = 7.18Hz, 2H)

Example 9

Succinimidyl (S-benzoylmercaptoacetyl) glycylglycyl-γ-aminobutyrate (preparation of a compound of formula [III])

2.965 g (7.5 mmol) of (S-benzoylmercaptoacetyl) glycyl glycyl-γ-aminobutanoic acid and 0.89 g (7.5 mmol) of N-hydroxysuccinimide were dissolved in 35 ml of DMF and 7 ml of DME, followed by DCC (1.563 g, 7.5 mmol) of DME (5 ml) solution was added dropwise and stirred at room temperature for 15 hours. The resulting solid was filtered off and the residue obtained by distillation under reduced pressure was washed well with isopropanol to give 3.24 g (87.8% yield) of the title compound as a white solid.

m.p = 174-177 ° C. (with decomposition)

¹ H-NMR (DMSO-d ₆ )

δ 8.60 (t, 1H) 8.20 (t, 1H) 7.95-7.55 (m, 5H)

7.87 (t, J = 5.66 Hz, 1H) 3.91 (s, 2H) 3.78 (d, J = 5.62 Hz, 2H)

3.70 (d, J = 5.80Hz, 2H) 3.15 (q, J = 6.5Hz, 2H) 2.82 (s, 2H)

2.69 (t, J = 7.60, 2H) 1.77 (q, J = 7.2 Hz, 2H)

Example 10

(S-benzoylmercaptoacetyl) glycyl glycyl-gamma-aminobutyryl biocithin (preparation of a compound of formula [IV])

Succinimidyl ester of (S-benzoylmercaptoacetyl) glycylglycyl-γ-aminobutanoic acid (492.44 mg) in an aqueous solution of biocithin (372.5 mg, 1 mmol) and 5 ml of NaHCO ₃ (168 mg, 2 mmol) in distilled water. , 1 mmol) in 7 ml of DMF was added at once. The reaction mixture was stirred at room temperature for 15 hours, and then the pH was adjusted to 2.0. The resulting precipitate was filtered, washed well with water and acetonitrile sequentially and dried to give 660 mg (88% yield) of the title compound.

m.p = 194-196 (decomposition)

¹ H-NMR (DMSO-d ₆ )

δ 8.49 (t, 1H) 8.12 (t, 1H) 8.0 (d, J = 7.73 Hz, 1H)

7.95-7.56 (m, 5H) 7.75 (t, 1H) 6.38, 6.33 (s, 2H)

4.29 (m, 1 H) 4.13 (m, 1 H) 3.89 (s, 2 H)

3.77 (d, J = 5.63 Hz, 2H) 3.67 (d, J = 5.86 Hz, 2H)

3.10 (m, 1H) 3.05 (q, J = 6.7 Hz, 2H) 3.00 (q, J = 6.6 Hz)

2.83 (dd, J = 12.4, 5.11 Hz, 1H) 2.57 (d, J = 12.4 Hz, 1H)

2.12 (t, J = 7.50Hz, 2H) 2.04 (t, J = 7.4Hz) 1.2-1.6 (m, 14H)

MASS (ESI)

m / z 750.57 (M ⁺ +1)

Example 11

Succinimidyl N- (S-benzoylmercaptoacetyl) -triglycinate (compound of formula [III])

Using the same method as the preparation method of succinimidyl-MAG ₂ GABA of Example 9, it was recrystallized from isopropanol to obtain the title compound in 69.0% yield.

m.p = 146-149 ° C

¹ H-NMR (DMSO-d ₆ )

δ 8.5-8.6 (2t, 2H) 8.25-8.35 (t, 1H) 8.0-7.5 (m, 5H)

4.3 (d, 2H) 3.9 (s, 2H) 3.8 (m, 4H) 2.8 (s, 4H)

Example 12

Preparation of ^99m Tc Radioactive Isotope ^{Labels of} N- (S-benzoylmercaptoacetyl) -glyciglyciyl γ-aminobutanoic acid (III)

10 μl of MAG ₂ GABA (10 mg / ml in DMSO) obtained in Example 5 was added to the reaction tube, and 50 μl of 1 M sodium acetate was added, followed by 10 μl of Stannous tartrate (1 mg / ml). . 1 ml of 10 mCi pertechnetic acid was added thereto and reacted at 100 ° C. for 15 minutes. After the reaction, high performance liquid chromatography analysis by radioisotope detector yielded ^99m labeled MAG ₂ GABA with 90% label yield.

The present invention provides intermediates useful for the preparation of bifunctional ligands used for chelating various biological molecules such as proteins, antibodies, lipids and DNA into radionuclides and N ₃ S based bifunctional ligands using the same. Chelating compounds of the present invention using the same can be usefully used for radiological diagnosis and treatment of various diseases, specifically, using a chelate compound of the present invention to a monoclonal antibody derived from a specific cancer antigen ^99m Tc or ¹⁸⁸ A labeled compound labeled with a radioisotope such as Re may be administered to a patient to obtain a diagnostic image or useful as a pharmaceutical kit for treatment.

Claims (13)

Chioacetyl dipeptide derivatives represented by the following formula [I] in the form of active esters as intermediates of bifunctional ligands: Formula I In the food, T is a sulfur atom protecting group, an ester group or an acetal group, R ₁ and R ₂ are each independently H or an amino acid side chain, X is -Cl, -O- (CO) -OR ₀ , or -OA, here, R ₀ is an ethyl group, isobutyl group, isovaleryl group or t-butyl group, A may be substituted with an alkyl group, alkenyl group, alkynyl group, alkoxy, alkoxyalkyl, aryl, aryloxy, arylamino, alkylcarbonyl, or arylcarbonyl, heterocycle, or halogen having 1 to 8 carbon atoms, o -Nitrophenyl group, m-nitrophenyl group, p-nitrophenyl group, 2,4-dinitrophenyl group, succinimidyl group, tetrafluorophenyl group, pentachlorophenyl group, or pentafluorophenyl group. The method of claim 1, T is a group selected from the group consisting of benzoyl, acetyl, tetrahydro pyranyl and ethoxy ethyl, R ₁ and R ₂ are each independently H or an alkyl group of C _1-4 which may be substituted, A is a group selected from the group consisting of o-nitrophenyl, 2-chloronitrophenyl group, succinimidyl group and tetrafluorophenyl group Thioacetyl Dipeptide Derivatives. The method of claim 1, A thiacetyl dipeptide derivative wherein T is a benzoyl group, R ₁ and R ₂ are each H, and A is a succinimidyl group. N ₃ S -based bifunctional ligand represented by the following formula [II] to form a radionuclide and a chelate compound: Formula II In the food, T, R ₁ and R ₂ are each as defined in claim 1, B is C ₃ -C ₆ cycloalkane or Is, Wherein R ₃ is an amino acid side chain which may be substituted with a protecting group, n is an integer of 1-6 when R ₃ is H, and an integer of 1 when R ₃ is not H. The method of claim 4, wherein R ₁ and R ₂ are each H, B is R ₃ is-(CH ₂ ) ₄ -NH-biotin Bifunctional ligands. N ₃ S -based bifunctional ligand represented by the following formula [III] to form a radionuclide and a chelate compound: Formula III In the food, T, R ₁ and R _2, B are each as defined in claim 4, A is as defined in claim 1. The method of claim 6, R ₁ and R ₂ are each H, B is Wherein R ₃ is-(CH ₂ ) ₄ -NH-biotin, A is tetrafluorophenyl or succinimidyl Bifunctional ligands. N ₃ S -based bifunctional ligand represented by the following formula [IV] to form a radionuclide and a chelate compound: Formula IV In the food, T, R ₁ and R _2, B are each as defined in claim 4, Z is biocitin. The method of claim 8, R ₁ and R ₂ are each H, B is Where R ₃ is H, n is an integer of 3 Bifunctional ligands. Method for preparing a thioacetyl dipeptide derivative represented by the following formula [I] consisting of the following steps: (A) S-protected thioglycolic acid represented by the following formula [VI] is reacted in the presence of N-hydroxysuccinimide in anhydrous solvent of tetrahydrofuran or dimethoxyethane and represented by the following formula [VII] To produce a compound, (B) Next, the mixture is reacted with an aqueous alkali solution of amino acid dipeptides to produce a compound represented by the following formula [VIII], (C) ① When the compound of formula [I] is an acid chloride of X = Cl, the compound represented by the formula [VIII] and thionyl chloride, phosgene or phosphorus pentachloride are reacted with an anhydrous solvent phase, or (2) When the compound of formula [I] is a mixed anhydride with X = -O- (CO) -OR ₀ , it is reacted with alkyl chloroformate at low temperature of -20 ° C to 0 ° C in the presence of tertiary amine, (3) When the compound of formula [I] is an active ester with X = -OA, the S-protecting dipeptide is added to a mixed solvent such as dimethylformamide (DMF) and dimethoxyethane (DME) or tetrahydrofuran (THF). After dissolution, the reaction is carried out for 2-12 hours with an alcohol represented by A-OH in the presence of dicyclohexylcarbodiimide (DCC) or a benzotriazole condensing agent. [Chemical Scheme 2] In the food, T is a sulfur atom protecting group, an ester group or an acetal group, R ₁ and R ₂ are each independently H or an amino acid side chain, X is -Cl, -O- (CO) -OR ₀ , or -OA, here, R ₀ is an ethyl group, isobutyl group, isovaleryl group or t-butyl group, A may be substituted with an alkyl group, alkenyl group, alkynyl group, alkoxy, alkoxyalkyl, aryl, aryloxy, arylamino, alkylcarbonyl, or arylcarbonyl, heterocycle, or halogen having 1 to 8 carbon atoms, o -Nitrophenyl group, m-nitrophenyl group, p-nitrophenyl group, 2,4-dinitrophenyl group, succinimidyl group, tetrafluorophenyl group, pentachlorophenyl group, or pentafluorophenyl group. Method for producing a bifunctional ligand represented by the following formula [II], which is configured as follows: (A) The compound represented by the following formula [I] was dissolved in a solvent containing dimethylformamide, (B) added to an aqueous alkali solution of the compound represented by the following formula [V] and reacted; (C) pH is adjusted to 2-3. [Chemical Scheme 3] In the food, T, R ₁ and R ₂ , A are as defined in claim 10, B is C ₃ -C ₆ cycloalkane or Is, Wherein R ₃ is an amino acid side chain which may be substituted with a protecting group, n is an integer of 1-6 when R ₃ is H, and an integer of 1 when R ₃ is not H. Method for producing a bifunctional ligand represented by the following formula [III] composed as follows: (A) Mixed solvent of dimethylformamide with tetrahydrofuran or dimethoxyethane together with nitro-activated phenol or cyclic hydroxylamine before or after chelating the compound represented by the following formula [II] with the radionuclide After mixing in (B) dicyclohexylcarbodiimide is added and stirred at room temperature for 10-20 hours. [Chemical Scheme 4] In the food, T, R ₁ and R ₂ , A are as defined in claim 10, B is as defined in claim 11. The following formula [IV] which consists of adding the dimethylformamide solution which melt | dissolved the compound of following formula [III] to aqueous solution of biocitin, stirring, and drying from the filtrate which removed pH by adjusting pH to acidity. Method for producing a bifunctional ligand represented by. [Chemical Scheme 5] In the food, T, R ₁ and R ₂ , A are as defined in claim 10, B is as defined in claim 11, Z is biocitin.