JPH0656802A - Tetraazacyclododecane derivative and its use - Google Patents

Abstract

(57) [Summary] (Modified) [Objective] To provide a bifunctional complexing agent capable of forming a metal complex useful for medical imaging and treatment. 【Constitution】 , A bifunctional tetraazacyclododecane derivative, a bifunctional tetraazacyclododecane derivative-a metal complex complex obtained by complexing a functional substance and a metal ion, and nuclear magnetic resonance containing the metal complex complex , X-ray or radiodiagnostic agents and radiotherapy agents. (N is an integer of 1 to 6; R ₁ and R ₂ are H, an alkyl group; A is H,
(Haloacetyl group, etc.)

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention has a strong complex-forming ability with respect to various metal ions, and is useful as a metal carrier for in vivo application such as medical image diagnosis and treatment. Tetraazacyclododecane derivative, more specifically 1,4,7,10-tetraazacyclododecane-4,7,1
The present invention relates to a novel difunctional complexing agent having a 0-triacetic acid (abbreviated as DO3A) skeleton and its use.

[0002]

2. Description of the Related Art In the fields of medical diagnostic imaging and therapy, the effectiveness of safely introducing various metal ions into a living body is gradually being established. Furthermore, in recent years, in order to obtain physiological biological information such as in vivo reaction, for example, metabolism, enzyme reaction, receptor binding, antigen-antibody reaction, or characteristic in vivo behavior, proteins, peptides, antibodies, carbohydrates, Attempts are rapidly progressing in combination with substances that interact with biological substances or biological systems such as lipids (collectively referred to as functional substances) and these metal ions. Therefore, as one of the technical means for binding such a metal ion and a functional substance, it has been conventionally expected to apply a bifunctional complexing agent as its bonding factor.

The medically desirable properties required of a bifunctional complexing agent depend on the diagnostic or therapeutic purpose. However, the desired properties that are commonly required for all uses are:
The high complex stability of the metal ion complexed with the complexing agent and the activity retention of the functional substance after the binding of the complexing agent and the functional substance. The high complex stability suppresses the release of metal ions from the complexing agent and minimizes the induction of side effects due to metal toxicity (especially a problem for compounds with long residence time in the body).
The good activity retention of the functional substance can control the in-vivo behavior of the metal complex complex compound composed of the metal-bifunctional complexing agent-functional substance according to the purpose. Moreover, when a metal complex is used as a contrast agent for nuclear magnetic resonance diagnosis or X-ray diagnosis, the properties desired for the bifunctional complexing agent become more severe. Due to the increased dosage for humans, high water solubility, osmotic pressure close to human body fluid as a complex and ease of synthesis are required. High water solubility is a high-concentration solution formulation (for example, about 0.
It is 5M. ) Can be prepared, and the low osmotic pressure reduces the load on the volume of the circulatory system or the fluid equilibrium upon in vivo administration. In addition, the ease of synthesis makes the overall synthesis extremely efficient, leading to a reduction in manufacturing cost. Thus, in view of these requirements, a bifunctional complexing agent has been newly developed,
Various innovations have been made.

A classic and representative bifunctional complexing agent is a diethylenetriaminepentaacetic acid (abbreviated as DTPA) type.
[Tokutei Hira 2-501385 / Brechbiel MW, In
org. Chem. , 25, 2772 (1986) / Esteb
an JM et al. Nucl. Med. , 28, 861 (19
1987) / Westerberg DA et al., J. Med. Chem. ,
32, 236 (1989)] or ethylenediaminetetraacetic acid (abbreviated as EDTA) type [Goodwin DA et al.,
J. Med. Chem. , 17, p. 1304 (1974) /
Yeh SM et al., Anal. Biochem. , Pages 100, 152
(1979)], so-called chain bifunctional complexing agents were first developed. However, such a metal complex complex compound with a chain-type bifunctional complexing agent is the result of in-vivo acid-catalyzed decomplexation or competitive complexation with Ca ²⁺ or Zn ^2+. Or as a competition with transferrin [Moerlein SM et al., Int. J. Nucl. Med. Bio
l. , 8, 277 (1981)], the complex tends to be unstable. The decrease in the stability of the complex promotes the release of metal ions from the metal complex complex compound, which contributes to the occurrence of side effects due to the toxicity of free metal ions, and also in terms of diagnostic imaging, the disordered behavior of free metal ions As a result, the signal / noise ratio is significantly reduced, and accurate information cannot be obtained. Furthermore, these complexing agents often take a trivalent metal ion (many metal ions that are useful in medical imaging and treatment are often trivalent).
When it is complexed, it will have a negative charge of -2 or -1 per complex unit, and will contain a harmful effect factor on the living body such as an increase in osmotic pressure. Also, it is not always easy to synthesize [Westerberg D
A et al. Med. Chem. , 32, 236 (1989)
Year)].

Therefore, recently, as a next-generation compound following the chain type bifunctional complexing agent, the bifunctional 1,4,
7,10-Tetraazacyclododecane-aminopolyacetic acid has received attention. These complexing agents were developed with a particular focus on improving the stability of the complex and are capable of forming a more dynamically inactive complex with metal ions than conventional chain bifunctional complexing agents. Is said to be. For example, Gd ion (III) and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) which are in the spotlight as metal ions for nuclear magnetic resonance diagnosis are abbreviated. ) And the complex formed by Gd-DTPA or Gd
-Since it has extremely high complex stability as compared with EDTA [Moi MK et al., Cancer. Res. (Suppl.), 5
0,789s page (1990)], the improvement of tolerability to living organisms is clear. Therefore, the use of bifunctional complexing agents with 1,4,7,10-tetraazacyclododecane-aminopolyacetic acid as the metal complexing site holds promise.

1,4, which has been found to have medical utility
Bifunctional complexing agents of the 7,10-tetraazacyclododecane-aminopolyacetic acid type are generally classified into three groups. The first group is a 1,4,7,10-tetraacetic acid derivative having a reactive side chain attached to a carbon atom in the tetraazacyclododecane ring, which is involved in the binding with a functional substance. Moi MK et al. Am. Chem.
Soc. 110, 6266 (1988) / Deshpande
SV et al. Nucl. Med. , P. 31, 473 (1990
)], The second group consists of a reactive side chain attached to the carboxyl group and a functional substance attached to the exocyclic α carbon of the 1st nitrogen atom in the tetraazacyclododecane ring 1,
4,7,10-Tetraacetic acid derivative [EP-A035345
No. 0]. Since these are the above-mentioned DOTA derivatives, they are expected to have strong complex stability with Gd ions and the like, but they will be negatively charged per complex unit, resulting in an increase in osmotic pressure or a high concentration solution. Unnecessary addition of counterions during preparation is forced. Also, the first group is generally cumbersome to synthesize and the inclusion of intermolecular or intramolecular cyclization reaction steps renders the overall synthesis inefficient. The third group that follows is DO
3A-type bifunctional complexing agent in which the reactive side chain is substituted on the 1-position nitrogen atom in the tetraazacyclododecane ring 4,7,1
It is a 0-triacetic acid derivative. These are generally easy to synthesize and have the advantage of forming an uncharged complex. However, the complex stability is lower than that of the DOTA-type bifunctional complexing agent, and the above-mentioned chain-type bifunctional complexing agent D is used.
It is almost the same as the TPA derivative. Compounds of this system are disclosed in JP-A-63-41468 or JP-A-64-5276.
No. 4, etc., no special consideration was given to the decrease in complex stability due to the lack of one carboxyl group from the 1,4,7,10-tetraacetic acid type, and the functionality was not considered. It cannot be said that the reactive functional group end which participates in the binding with the substance is also well activated.

[0007]

DISCLOSURE OF THE INVENTION The present invention overcomes the above-mentioned problems found in DO3A type bifunctional complexing agents, and the formed metal complex has high stability, no charge and good solubility. It is an object of the present invention to provide a cyclic bifunctional complexing agent which has a physiologically acceptable and active reactive functional group end that easily binds to a functional substance. Further, for the purpose of providing a complex compound in which the bifunctional complexing agent is bound to a functional substance and a metal complex thereof, the metal complex complex compound is a drug for nuclear magnetic resonance, X-ray or radiodiagnosis. And can be used as a radiotherapeutic agent.

[0008]

In the present invention, the bifunctional complexing agent has a reactive functional side chain attached to the 1st nitrogen atom of DO3A as a part of its structure, and this structure has a difunctional structure. It advantageously acts to sterically hinder the conformational cleavage of the complex structure required to release the metal ion from the sex-complexing agent. However, using any of a variety of reactive functional group side chains does not necessarily meet this purpose. The present inventors have conducted various studies in order to achieve the objective of suppressing the release of metal ions from the bifunctional complexing agent to a minimum, and as a result, the reactive functional group located at the β-position of the 1-position nitrogen atom It has been found that the presence of an amide linking moiety to which a carbonyl group is bound in the base side chain can be beneficial. The reactive functional group side chain has a functional group having a sufficiently active reactivity that easily reacts directly with the functional substance to form a covalent bond at the terminal portion thereof. Such a reactive functional group easily reacts with a thiol group, an amino group, a carboxyl group, a hydroxyl group, an aldehyde group, an aromatic group (such as a phenyl group) or a heterocyclic group (such as an imidazolyl group) of a functional substance. The active groups are used so that no special reactants are required in the reaction with the functional substance. However, in consideration of solubility of the bifunctional complexing agent of the present invention, binding to blood proteins in the body, and excretion in urine, a linear or branched chain is included in the reactive functional group side chain structure. No aryl groups should be present. Many of the metal ions that are useful for medical imaging and treatment are trivalent (eg, Gd, Dy, Ho, Bi, Os, In, Ga, Tc, Y, S.
Specific examples include m. ), Such a metal ion and the bifunctional complexing agent of the present invention form an uncharged complex. This allows the preparation of highly physiologically tolerated concentrated solutions based on low osmolality. The present invention has been completed based on these findings.

The gist of the present invention is the formula 1:

[Chemical 3]

[Wherein n represents an integer of 1 to 6, R ₁ and R ₂ are the same or different, and are a hydrogen atom or C ₁
-C ₄ alkyl group, A is a hydrogen atom or with a reactive group in the terminal position, C ₁ -C ₄ linear or branched saturated may be interposed or unsaturated by a carbonyl group
It represents a reactive functional group consisting of an alkylene group. ], Which is a physiologically acceptable bifunctional complexing agent characterized by being capable of stably binding a metal ion and a functional substance.

In the compound of formula 1, R ₁ and R ₂ are the same or different and represent a hydrogen atom or a C ₁ -C ₄ alkyl group, wherein R ₁ is a methyl group, R ₂ group is a hydrogen atom, or R It is preferred that ₁ and R ₂ are methyl groups.
n means the length of the crosslinked chain, and is arbitrarily selected as a number that does not substantially affect the usefulness of the functional substance and the metal complex complex compound to be bonded. However, in order to maintain the water-soluble property, it is not preferable that the number is large. 1-6 are suitable. Of these, 1-3 are most suitable. The group represented by A is a thiol group, amino group, carboxyl group, carbonyl group, hydroxyl group, aldehyde group, aromatic group (phenyl group, etc.) or heterocyclic group of a functional substance.
A group that can easily react with (such as an imidazolyl group) is used. For example, a hydrogen atom (meaning that the end of the reactive side chain becomes an amino group), or a reactive end group, for example, a halogen atom such as chlorine, bromine or iodine atom, or a thiol group, a hydrazine group (-NHNH ₂ ) and its derivatives [eg, —NH (CH ₃ ) NH ₂ , —NH
CONHNH ₂ or —NHCSNHNH _2, etc. ], A group selected from --NCO and --COR ₃ [R ₃ is azido (N ₃ )]
Group, an alkoxy group composed of C _{1 to} C ₄ such as a methoxy group, an imidyloxy group (for example, a succinimidyloxy group, etc.) or an imidazoyloxy group. ],-
COX (X is a halogen atom such as chlorine or bromine),
It is a reactive functional group having a reactive terminal group arbitrarily selected from N-hydroxysuccinimide ester group and the like. The reactive functional groups of A are those reactive end groups and C linked thereto, which may be mediated by a carbonyl group.
_It is composed of _{1 to} C ₄ alkylene groups (eg, -COCH
_{2 -, - COCH 2 CH 2} CH 2 -, - CH 2 COCH 2 CH
_{2 -, - CH 2 CH 2} CH 2 -, - CH 2 CH 2 - , etc.), there must be no straight or branched chain aryl group and derivatives thereof into the structure of the chain portion. This allows
The metal complex complex compound of the present invention enables rapid excretion of the metal complex into urine even when it is decomposed and metabolized in the body. The substitution or intervening position of the carbonyl group is not particularly limited. Thus, examples of the reactive functional groups represented by _{A, -COCH 2 Cl, -COCH 2 CH} 2 C
_{H 2 Cl, -CH 2 COCH 2} CH 2 Cl, -CH 2 CH 2 CH
_{2 Cl, -CH 2 CH 2 COCl} , such as -CH ₂ CH ₂ SH and the like.

Further, the salt of the compound represented by the formula 1 includes
Neutral salts containing inorganic or organic physiologically harmless cations as carboxylic acid counterions are included. Typical examples of the counter ion include inorganic cations derived from sodium, potassium, lithium and the like, and organic cations derived from meglumine, dimeglumine, ethanolamine, diethanolamine, morpholine, lysine, arginine, ornithine and the like.

Yet another aspect of the invention is Formula 2:

[Chemical 4]

[In the formula, n represents an integer of 1 to 6, R ₁ and R ₂ are the same or different, and a hydrogen atom or C ₁
To C ₄ alkyl group, A ′ is a reactive group consisting of a saturated or unsaturated linear or branched C _{1 to} C ₄ alkylene group having a reactive group at the terminal position, which may be interposed by a carbonyl group. Represents a functional group residue, and Z represents a compound selected from functional substances. ] It is a physiologically acceptable complex compound and its salt shown by these. R ₁ , R ₂ ,
A ′ and n are R ₁ , R ₂ , A and n of the compound represented by the formula 1.
Is the same as.

The selection of the functional substance used in the synthesis of the complex compound of the present invention is not absolute. Depending on the medical imaging and therapeutic purposes, any desired functional agent can be used for the synthesis of the complex compound. The functional substance means a substance that interacts with a biological substance or a biological system as described above. Such suitable substances are, for example, amino acids, peptides, sugars,
It may be a small molecule such as a fatty acid, a steroid, a porphyrin or an organ-specific functional group, or a polymer such as a protein, an antibody, an antibody fragment, an enzyme, a polysaccharide or a synthetic polymer. The bond between A ', which is the terminal of the reactive functional group, and Z, which is a functional substance, is performed by a covalent bond. Further, it is understood that the complex compound and the metal complex thereof of the present invention may include one or more molecules of the compound of Formula 1 bound to any one molecule of the functional substance or the metal complex thereof. .

The metal ions forming a complex with the compound represented by the formula 2 of the present invention have atomic numbers 31, 39, 43 and 4
4, 49, 57 to 71, 76, 77, 79, 81 or 83 are selected according to the use of diagnostic imaging or radiotherapy. Usually, the ionic value of metal is +3
Are preferred. When the metal complex complex compound of the present invention is used for nuclear magnetic resonance diagnosis, the metal ion must be paramagnetic and is selected from the group consisting of ions of lanthanoid series elements having atomic numbers of 57 to 70, Preferred are Gd, Dy and Ho. When used for X-ray diagnosis, the metal ion is selected from the group consisting of ions of lanthanoid elements having atomic numbers 57 to 70 and ions of elements 76 and 83, with Bi or Os being preferred. When used for radioactive diagnosis, the metal ion must be radioactive, and radioactive metal ions of In, Ga, Tc, Y, Gd and Sm are suitable. Furthermore, when used as a drug for radiotherapy, Y, Sm and Bi radioactive metal ions are selected.

The preparation of the 1-substituted-1,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid type bifunctional complexing agent of the present invention is itself a bifunctional complexing process. Formula 3 useful as an agent:

[0018]

[Chemical 5]

[In the formula, n represents an integer of 1 to 6, R ₁ and R ₂ are the same or different, and a hydrogen atom or C ₁
~C represents an _alkyl group. ] It is desirable to obtain the compound represented by the formula (A is a compound represented by the formula 1 in which hydrogen atom) as a synthetic intermediate. By using the compound represented by the formula 3 as an intermediate raw material, a bifunctional complexing agent having various reactive groups capable of widely selecting a binding mode with a functional substance can be easily synthesized. The best method for preparing the compound of formula 3 is generally as follows. That is, 1,4,7,10-tetraazacyclododecane (DOT), which is known in the art, is added to the side chain moiety of the reactive functional group in the structure of Formula 3.
Abbreviated. ), Equation 4:

[0020]

[Chemical 6]

To give the desired 1-substituted-DOT, and further introduce a carboxylic acid moiety into the remaining three nitrogen atoms in the ring to give a difunctional complexing agent of formula 3 and a synthetic intermediate. Will be obtained.

DOT is generally commercially available in the form of a salt, but desalting operation is desirable to facilitate the reaction. Usually, the desalting operation is performed using an ion exchange resin. The reactive functional group side chain moiety in Formula 3 is represented by Formula 5:

[0023]

[Chemical 7]

[In the formula, R ₂ represents a hydrogen atom or a C ₁ -C ₄ alkyl group, X represents a halogen atom, and Y represents a halogen atom, a hydroxyl group or a C ₁ -C ₄ alkoxy group. ] The reactive carboxylic acid derivative shown by

[0025]

[Chemical 8]

[In the formula, n represents an integer of 1 to 6. ] It can manufacture by making it react with the diamine compound shown by these in the presence of bases, such as triethylamine.
This reaction may be carried out by a method known per se. For example, Y is a halogen atom [Fourie PJ et al., Eur. J. Nuc
l. Med. 4, 445 (1979)] or an alkoxy group [Washburn LC et al., Nucl. Med. Biol. 1
8, 313 (1991)], the reaction of the reactive carboxylic acid derivative of the formula 5 with the diamine compound of the formula 6 can be carried out by applying the known literatures described. Also, one amino group of the diamine compound of formula 6 may be protected with an amino protecting group commonly used in peptide synthesis. The amino protecting group can be easily converted into the compound of formula 3 by finally subjecting it to deprotection with an acid catalyst. The introduction of the reactive functional side chain moiety thus obtained into the nitrogen atom in the DOT ring is described in the prior art known in the art. Subsequent attachment of the carboxylic acid moiety to the remaining three nitrogen atoms in the DOT ring is shown in Formula 7:

[0027]

[Chemical 9]

[In the formula, R ₁ represents a hydrogen atom or a C ₁ -C ₄ alkyl group, X'represents a halogen atom, and Y'represents a hydroxyl group or a C ₁ -C ₄ alkoxy group. ] Alkylation is carried out using a halocarboxylic acid derivative represented by When a halocarboxylic acid derivative in which Y'is a hydroxyl group is used, it is preferably carried out in water having a pH of about 9-10. Adjust and maintain the reaction pH with an inorganic base such as an alkali metal hydroxide and warm to about 50-80 ° C. When a halocarboxylic acid derivative in which Y ′ is a C ₁ -C ₄ alkoxy group is used, an organic solvent such as acetonitrile, tetrahydrofuran or dimethylformamide is used in the presence of a base, for example, an inorganic base such as potassium carbonate or sodium carbonate. Among them, it is preferable to carry out at a high temperature, for example, a reflux temperature [EP-A0353450]. However, in this case, further saponification is required. The introduction of the carboxylic acid moiety into the nitrogen atom of the DOT ring may be performed prior to the introduction of the reactive functional group side chain moiety. In this case, it is suitable to protect one nitrogen atom in the DOT ring into which the side chain moiety of the reactive functional group is introduced with a benzyl group or the like [JP-A-63-41468].

For example, in the formula 1, R ₁ = CH ₃ , R ₂ = H, n
The compound of the present invention represented by A = 2 and A = H is prepared as follows. First, one amino group of ethylenediamine is protected with t-butyloxycarbonate by a method known per se, and then reacted with chloroacetyl chloride in ether under ice cooling (t-butyloxycarbonyl) -amino. Obtain ethylcarbamoylmethyl chloride. Subsequently, the desalted DOT is reacted with (t-butyloxycarbonyl) -aminoethylcarbamoylmethyl chloride in acetonitrile under reflux to introduce a reactive functional group side chain. Finally, after reacting ethyl 2-bromopropionate in acetonitrile in the presence of potassium carbonate to obtain a triester, deprotection of the t-butyloxycarbonyl group, which is an amino protecting group, using an acid solvent and Hydrolysis of the ester group yields the desired bifunctional complexing agent. In addition, a compound in which R ₁ is a hydrogen atom or a lower alkyl group excluding a methyl group is a monobromoacetic acid, a 2-bromobutanoic acid, and a 2-bromovaleric acid instead of the above-mentioned ethyl 2-bromopropionate, or those A desired compound can be produced by using the ester derivative of (the saponification is required in the case of the ester derivative). Similarly, instead of chloroacetyl chloride, 2-chloropropionyl chloride, 2-bromobutyryl bromide, ethyl-2
By using -bromovalerate, bromohexanoyl bromide, or the like, a compound in which R ₂ has a C _{1 to} C ₄ alkyl group can be obtained. While these compounds are useful as bifunctional complexing agents themselves, they also show promise as intermediate compounds for bifunctional complexing agents with various reactive ends, as described above. As an example,
By reacting the amino group at the terminal position of the reactive functional group of the present compound with chloroacetyl chloride in anhydrous dimethylformamide at room temperature, R ₁ = CH ₃ , R ₂ = H, n =
2, a compound represented by A = COCH ₂ Cl can be obtained. By applying the same method, a halogen atom, a thiol group, a hydrazine group and its derivative, -NCO, -COOR ₃ (R ₃ represents lower alkyl), an aldehyde group, -COX (X is chlorine) at the terminal position. Or a halogen atom such as bromine) or a reactive functional group A having an N-hydroxysuccinimide ester group is substituted.

The binding of the bifunctional complexing agent represented by the formula 1 to a desired functional substance can be carried out by a method known per se by a nucleophilic group of the functional substance, for example, a thiol group, an amino group, a carboxyl group, It is carried out by reacting a carbonyl group, a hydroxyl group, an aldehyde group, an aromatic group (such as a phenyl group), or a heterocyclic group (such as an imidazolyl group) with a terminal reactive group of the bifunctional complexing agent represented by Formula 1. . For example, the reaction between the bifunctional complexing agent represented by Formula 1 and the antibody is carried out by cooling to slight heating, for example, at room temperature for several minutes to several hours, for example, in an aqueous solvent such as a phosphate buffer. It can be performed by stirring for 30 to 60 minutes. It should be noted that, if necessary, the functional substance may be treated so as to generate a suitable reactive group for the reaction with the bifunctional complexing agent represented by the formula 1 before the coupling reaction. For example, in the case of a sugar, after activation with bromocyan, a compound of formula 1 having an amino group as a terminal reactive group is subsequently obtained.
The desired complex compound can be obtained by reacting a bifunctional complexing agent represented by

The salt of the compound represented by formula 1 or formula 2 is
It can be prepared by conventional means, eg by reaction with a suitable base or acid in a suitable aqueous solvent.

The selection of a suitable metal salt for any given metal ion is within the skill of the art. The metal ion used is the metal itself or its inorganic compound.
(Eg, chloride, oxide, etc.).
The complexing is carried out in an aqueous solution, preferably in a weak acid to weak base medium at a pH of about 4 to about 9 and any suitable reaction temperature of 0 to 100 ° C. is selected. The amount of metal ion used can be an amount equal to or more than the equimolar amount of the bifunctional complexing agent from the amount of the tracer. It should be noted that the step of complexing the metal is performed before the functional substance is bound to the bifunctional complexing agent represented by Formula 1.
Or later. The functional substance to be bound is selected within a range that does not cause denaturation.

The physiologically tolerable metal complex complex compound can be mixed with any pharmaceutically acceptable additive component by a conventional method to give a diagnostic contrast agent in any form. It is preferably dissolved in a physiologically acceptable aqueous solvent to give a solution or form of a diagnostic or therapeutic drug.

In order to use the metal complex complex compound of the present invention as a drug for nuclear magnetic resonance diagnosis, the amount of metal ion is generally 0.0001 to 10 mmol / kg, preferably 0.005 to 0.5 mmol / kg. It is administered in the amount of kg. For use as an X-ray diagnostic agent, the amount of metal ion is 0.01 to 20 mmol / kg, preferably 0.1 to 10 mmol / kg. Furthermore, in order to use as a drug for radiological diagnosis or treatment,
Radioactive doses of 370-18500 MBq are administered. It is usually administered intravenously, but in some cases it may be administered orally or intraarterially.

[0035]

EXAMPLES Next, the present invention will be described in more detail with reference to examples and test examples, but the technical scope of the present invention is not limited thereto. The abbreviations have the following meanings: DO3MA: 1,4,7,10-tetraazacyclododecane-1-aminoethylcarbamoylmethyl-4,7,10
-Tris [(R, S) -methylacetic acid] ClDO3MA: 1,4,7,10-tetraazacyclododecane [(N-chloroacetyl) aminoethylcarbamoylmethyl] -4,7,10-tris [(R, S) -Methylacetic acid] DNS-DO3MA: 1,4,7,10-Tetraazacyclododecane [(N-dansyl) aminoethylcarbamoylmethyl] -4,7,10-tris [(R, S) -methylacetic acid ] DOT: 1,4,7,10-tetraazacyclododecane

Example 1 (Synthesis of DO3MA) a) Synthesis of N- (t-butyloxycarbonyl) -ethylenediamine. 270 ml (4.04 mol) of ethylenediamine was slowly added dropwise to 500 ml of methanol under ice-water cooling. 100 g (0.46 mol) of di-t-butyl-dicarbonate dissolved in 140 ml of methanol was added dropwise thereto over 2 hours, and the mixture was stirred overnight at room temperature. The insoluble material was filtered off, the filtrate was concentrated, water was added to the residue, and the mixture was stirred at room temperature for 30 minutes.
The mixture was stirred for minutes and the insoluble material was filtered off. The filtrate was concentrated and extracted twice with ethyl acetate. The organic layers were collected, dried (anhydrous sodium sulfate was used), concentrated, and distilled under reduced pressure to obtain 56.5 g (yield 77%) of the desired product as an oil.

B) Synthesis of (t-butyloxycarbonyl) -aminoethylcarbamoylmethyl chloride. Nt-butyloxycarbonyl-ethylenediamine 5
6.5 g (0.35 mol) were dissolved in 300 ml ether.
Under ice-water cooling, triethylamine 70.8g (0.70mo
l) was added, and subsequently 39.6 g (0.35 mol) of chloroacetyl chloride dissolved in 300 ml of ether was added dropwise in 3.5 hours, and the mixture was stirred at room temperature for 2 hours. The precipitated crystals were collected by filtration, added with 300 ml of water, and mixed at room temperature. The crystals were collected again by filtration, dissolved in 600 ml of methylene chloride, and washed twice with water. The organic layers were collected, dried (using anhydrous sodium sulfate), concentrated, and dried under reduced pressure to give 50.2 g of the desired crystal (yield 61%).
Got

C) Synthesis of 1-[(t-butyloxycarbonyl) -aminoethylcarbamoylmethyl] -1,4,7,10-tetraazacyclododecane. 23 g of DOT.4 hydrochloride was passed through an ion exchange column (strongly basic resin: Diaion SA20A was used) for desalting to obtain 13 g (75.5 mmol) of DOT. To this was added 720 ml of acetonitrile and dissolved under reflux. 8.6 g (36.3 mmol) of (t-butyloxycarbonyl) -aminoethylcarbamoylmethyl chloride dissolved in 180 ml of acetonitrile was added dropwise in portions over 3 hours, then refluxed for 3 hours, and further stirred at room temperature overnight. did. The precipitate was filtered off, and the filtrate was concentrated and dried under reduced pressure to give an oily substance (20.6 g). This was passed through an ion exchange column (strongly basic resin: Diaion SA20A) for desalting, and then the eluate was concentrated. The residue was purified by silica gel column chromatography (eluent: chloroform: methanol: ammonia water = 8: 4: 1) to obtain the desired crystal 10.
0 g (74% yield) was obtained.

D) 1,4,7,10-Tetraazacyclododecane-1-aminoethylcarbamoylmethyl-4,7,10-tris
Synthesis of [(R, S) -methylacetic acid] -triethyl ester. 1-[(t-butyloxycarbonyl) -aminoethylcarbamoylmethyl] -1,4,7,10-tetraazacyclododecane (8.2 g, 22.0 mmol) in 800 m of acetonitrile
It dissolves in l and further 32.8 g (0.237 mo) of potassium carbonate
l) was suspended. Next, 47.6 g (0.263 mol) of ethyl 2-bromopropionate was added, and the mixture was stirred under reflux for 7 hours. The insoluble material was filtered off and concentrated. The residue was purified by silica gel column chromatography (eluent; methylene chloride: methanol = 9: 1) to obtain 12.0 g of the desired product (yield 81
%).

E) Synthesis of DO3MA.hydrochloride. 1,4,7,10-Tetraazacyclododecane-1-aminoethylcarbamoylmethyl-4,7,10-tris
[(R, S) -Methylacetic acid] -triethyl ester 12.0 g
(17.8 mmol) was added with 100 ml of acetic acid, and the mixture was stirred at room temperature. After adding 50 ml of concentrated hydrochloric acid little by little, the mixture was stirred at room temperature for 1 hour. Next, 100 ml of water was added and the mixture was refluxed for 4 hours and then concentrated. The residue was dissolved in 100 ml of water, treated with activated carbon, and filtered. The filtrate was concentrated and dried under reduced pressure to obtain 9.4 g (yield 100%) of the target product as slightly yellowish white crystals.

Proton-Nuclear Magnetic Resonance spectrum (solvent: D
₂ O, 270 MHz): 1.2 to 1.7 ppm (9 H, m), 2.
8-3.7ppm (22H, m), 4.2-4.6ppm (3H,
m).

FAB-mass spectrum (positive ion): m / z
489.5 [(M + H) ⁺ ], 511.5 [(M + Na) ⁺ ], 5
33.5 [(M + 2Na-H) ^<+ >].

Infrared absorption spectrum (potassium bromide): 14
00cm ^-1 (CH ₂ ), 1480cm ^-1 (CONH), 1620c
_{^{m -1 (NH 2), 1680cm}} -1 (COOH).

Example 2 (Synthesis of ClDO3MA) 2.0 g (4 mmol) of DO3MA was dissolved in 20 ml of anhydrous dimethylformamide, and then 1.67 ml of triethylamine.
(12 mmol) was added. 0.64 ml (8 mmol) of chloroacetyl chloride was added thereto, and the mixture was reacted at room temperature for 24 hours. The precipitate was filtered off and the filtrate was distilled off. The obtained oily substance was dissolved in water and stirred at room temperature for 1 hour. This solution was concentrated, and an ion exchange column (strongly basic resin: AG1-X4,
Eluent: water). The eluate was concentrated and passed through an ion exchange column (strongly acidic resin: AG50W-X8, eluent: water) again, and then the eluate was evaporated to dryness to give 1.24 g of the pale yellow semi-crystal target product (yield 50%).

Proton-Nuclear Magnetic Resonance spectrum (solvent:
D ₂ O, 270 MHz): 1.2 to 1.7 ppm (9 H, m),
2.8 ppm (2H, m), 3.0 ppm (2H, s), 3.1 to 3.
7ppm (18H, m), 4.2ppm (2H, s), 4.3ppm (3
H, s).

FAB-mass spectrum (positive ion): m / z
565 [(M + H) ⁺ ].

Infrared absorption spectrum (potassium bromide): 12
_{^{40cm -1 (CH 2 Cl),}} 1400cm -1 (CH 2), 1460
And 1550 cm ^-1 (CONH), 1660 cm ^-1 (COO
H).

Example 3 (Synthesis of DO3MA-Gd) 1.49 g (2.8 mmol) of DO3MA was dissolved in 15 ml of water, and 506.5 mg (1.4 mmo of gadolinium oxide) was dissolved therein.
l) was added. An appropriate amount of sodium hydroxide solution was added to adjust the pH to 8.0 to 9.0, and the mixture was reacted at 60 ° C for 24 hours. The precipitate formed was filtered off. The filtrate was concentrated. Dissolve the residue in water and ion-exchange column (strong acidic resin: AG50W-X
8, eluent: 1 M aqueous ammonia). The eluate was concentrated to obtain 1.04 g (yield 57%) of the target product as pale yellow crystals.

FAB-mass spectrum (positive ion): m / z
644 [(M + H) ⁺ ].

Infrared absorption spectrum (potassium bromide): 13
_{^{90cm -1 (CH 2), 1452cm}} -1 (CONH), 1601c
^{_{m -1 (NH 2, COO -}} ).

Example 4 (Synthesis of Cl-DO3MA-Gd) 642.9 mg (1 mmol) of DO3MA-Gd was suspended in 20 ml of anhydrous dimethylformamide, and 0.14 ml (1 mmol) of triethylamine was added. 0.4 ml (5 mmol) of chloroacetyl chloride was added dropwise thereto, and the mixture was reacted with stirring at room temperature for 24 hours. The reaction solution was concentrated, water was added to the residue to dissolve the residue, activated carbon was added, and the mixture was stirred overnight and filtered. The filtrate was concentrated and the ion exchange column (strongly basic resin: AG1
-X4, eluent: water). The eluate was concentrated to obtain 767 mg (yield 100%) of the target product as white crystals.

FAB-mass spectrum (positive ion): m / z
720 [(M + H) ⁺ ].

Infrared absorption spectrum (potassium bromide): 13
00 cm ^-1 (CH ₂ Cl), 1360 cm ^-1 (CH ₂ ), 1450
And 1490 cm ^-1 (CONH), 1600 cm ^-1 (CO
O ^-).

Example 5 (Synthesis of DO3MA-In-111) 20 mg (0.038 mmol) of DO3MA was dissolved by adding water, and then 0.5N sodium hydroxide solution was added thereto in an appropriate amount.
The H was adjusted to 8.5. 1 ml of this solution was taken, 0.1 ml of indium chloride solution (In-111, 1280 MBq) and then 0.1 ml of 1N sodium hydroxide solution were added, and the mixture was reacted for 10 minutes under pressure steam (1 kg / cm ² ). The resulting reaction solution was cation exchange cartridge (Seppack, eluent:
It was passed through water) to give DO3MA-In-111.

Radiochemical purity (thin layer chromatography): 96.2% (Rf 0.40).

Example 6 (Synthesis of DO3MA-Bi) Water was added to 105 mg (0.2 mmol) of DO3MA to dissolve it, 69 mg (0.22 mmol) of bismuth chloride was added, and a suitable amount of sodium hydroxide solution was added to adjust pH. It was adjusted to 8.0. This solution was reacted by stirring at 60 ° C. for 24 hours. The insoluble matter was filtered off, and the filtrate was concentrated to give 156 mg of the desired product as a white powder (yield 1
00%).

Proton-Nuclear Magnetic Resonance spectrum (solvent:
D ₂ O, 270 MHz): 1.4 ppm (6 H, m), 1.7 ppm
(3H, m), 2.9ppm (2H, m), 3.2ppm (6H, m),
3.5ppm (8H, m), 3.8ppm (4H, m), 4.1ppm (5
H, m).

FAB-mass spectrum (positive ion): m / z
695 [(M + H) ⁺ ], 717 [(M + Na-H) ⁺ ].

Infrared absorption spectrum (potassium bromide): 13
_{^{87cm -1 (CH 2), 1450cm}} -1 (CONH), 1593c
^{_{m -1 (NH 2, COO -}} ).

Example 7 (Synthesis of DNS-DO3MA-Gd) 1.323 g (2.1 mmol) of DO3MA-Gd was suspended in 20 ml of anhydrous dimethylformamide, and 2.832 g of dansyl chloride dissolved in 5 ml of anhydrous dimethylformamide was suspended therein. (10.5 mmol) was added dropwise. Further, 0.32 ml (2.3 mmol) of triethylamine was added and the mixture was allowed to stand at room temperature for 24
The reaction was allowed to stir for an hour. The reaction solution was filtered, the filtrate was concentrated, water was added, and the mixture was washed with ethyl acetate. Collect the water layer,
After adjusting the pH to 8, it was treated with activated carbon and concentrated. The residue is subjected to silica gel column chromatography (silica gel 60,
Eluent; chloroform: methanol: ammonia water = 2:
2: 1) and purification was performed to obtain 72.5 mg (yield 4%) of crystals of orange powder having fluorescence.

Infrared absorption spectrum (potassium bromide): 79
^{5cm -1 (Ar-H),} 1140cm -1 and 1389cm ^-1 (S
O ₂ NH): 1456 cm ^-1 (CH ₂ ): 1620 cm ^-1 (COO
^- ).

FAB-mass spectrum (positive ion): m / z
877 [(M + H) ⁺ ].

Example 8 (Liver MRI by DNS-DO3MA-Gd) Female ICR mouse (30) anesthetized with thiopental
g. 7-week-old) DNS-DO3MA-Gd solution (Gd concentration:
10 mmol) was administered into the tail vein (dosage: 50 mmol / kg). Immediately after the administration, the mouse was fixed in the prone position in the magnetic field of the nuclear magnetic resonance apparatus, and the abdominal region including the liver was imaged (coronal image). Further, as a control, before the administration of the present compound, a null value photograph of the same region of the same solid was taken.

The apparatus was CSI Omega (manufactured by GE), the magnetic field strength was 2T, and a birdcage type coil with a diameter of 4.5 cm was used as an imaging coil. The imaging conditions were 2mm slice thickness by spin echo method, 4 times of integration, resolution 256 ×
It was performed with 256 T1 emphasis (TR / TE: 500/100 msec).

FIG. 1 is a morphological photograph of an organism showing an abdominal coronal fault including the liver before compound administration. Figure 2 shows the DNS-
It is a morphological photograph of the organism which shows the abdominal coronal fault including a liver about 55 minutes after DO3MA-Gd solution administration. The dansyl group is a functional functional group having liver tropism (Japanese Patent Application No. 3-29).
1260), excreted from the blood circulation system into the intestinal tract via the liver. It was revealed that this compound enhances the signal intensity of mouse liver, and that the functional substance can obviously control the in-vivo behavior of the present bifunctional complexing agent.

Test Example 1 (DO3MA-Gd in serum
Stability of DO3MA-Gd (24.0 mg, 37 mmol) was adjusted to exactly 10 ml with H ₂ O to obtain a 3.7 mM stock solution. SD rat (♀,
Serum 0.18 ml collected from 14-week-old) was added as a sample. As a control, 0.02 ml of H ₂ O was added to rat serum at a dose of 0.
Prepared by adding 18 ml.

Immediately after preparation of the above samples and controls, 37
Incubated in water bath. NMR for each
(0.5 T, FSE-60 pulse NMR apparatus, manufactured by JEOL Ltd.), relaxation time (T1) at 37 ° C. was measured 5 minutes after preparation, 1, 3, 6 and 24 hours after preparation.

As a result, the specimen was 363 msec after 5 minutes, 2
After 4 hours, it was slightly decreased to 343 msec. On the other hand, the control is 1970 msec after 5 minutes and 1870 ms after 24 hours.
ec and 100msec were also shortened. The results are shown in Table 1.

[0069]

Table 1 Relaxation time (T1; msec) Time (hr) of DO3MA-Gd Specimen control 0.083 363.1 1970 1 363.2 1930 3 356.4 1920 6 343.2 1860 24 342.6 1870

Then, the ratio (S / C,%) of the sample to the control was calculated at each time point, and it was 18.4% after 5 minutes and 18.3% after 24 hours, showing almost no difference. It was The calculation results are shown in Table 2.

[0071]

[Table 2]

If DO3MA-Gd is unstable in serum and Gd is easily released, the relaxation time is greatly shortened by the free Gd. However, even after 24 hours, DO
No reduction in relaxation time of 3MA-Gd was observed, and it was revealed that the complex stability of this compound in serum was high.

[0073]

INDUSTRIAL APPLICABILITY The present invention provides an active reactive functional group in which a metal complex formed is highly stable, has no charge, has good solubility, is physiologically acceptable, and easily binds to a functional substance. Provided is a DO3A-type cyclic bifunctional complexing agent having a group end. Also provided is a complex compound in which the bifunctional complexing agent is bound to a functional substance and a metal complex thereof,
The metal complex complex compound can be used as a nuclear magnetic resonance, radiation or X-ray diagnostic agent and radiotherapy agent.

[Brief description of drawings]

FIG. 1 is a morphological photograph of an organism showing an abdominal coronal fault including the liver before administration of a compound.

FIG. 2 is a morphological photograph of an organism showing an abdominal coronary fault including a liver about 55 minutes after administration of a DNS-DO3MA-Gd solution.

Front page continuation (72) Inventor Higashi Makoto 3 Kita-sode, Sodegaura-shi, Chiba Japan Medi-physics Co., Ltd. Central Research Laboratory

Claims (10)

[Claims] Claim 1: Formula 1: [In the formula, n represents an integer of 1 to 6, R ₁ and R ₂ are the same or different and represent a hydrogen atom or a C _{1 to} C ₄ alkyl group, and A represents a hydrogen atom or a reactive group at the terminal position. It represents a reactive functional group having a saturated or unsaturated linear or branched C ₁ -C ₄ alkylene group which may be interposed by a carbonyl group. ] The physiologically acceptable compound shown by these, and its salt. 2. The reactive group at the terminal position is a halogen atom, a thiol group, a hydrazine group and its derivatives, -NCO, -C.
OOR ₃ (R ₃ is C ₁ -C ₄ alkyl), an aldehyde group, -C
The compound according to claim 1, which is OX "(X is a halogen atom such as chlorine or bromine) or an N-hydroxysuccinimide ester group. 3. The compound according to claim 1, wherein R ₁ is methyl, R ₂ is hydrogen, and n is 1 to 3. 4. The compound according to claim 1, wherein R ₁ and R ₂ are methyl and n is 1 to 3. 5. The compound according to claim 1, wherein A is hydrogen. 6. The compound according to claim 1, wherein A is a haloacetyl group. 7. Formula 2: embedded image [In the formula, n represents an integer of 1 to 6, R ₁ and R ₂ are the same or different, represent a hydrogen atom or a C _{1 to} C ₄ alkyl group, and A ′ has a reactive group at a terminal position, Represents a reactive functional group residue consisting of a saturated or unsaturated linear or branched C ₁ -C ₄ alkylene group which may be interposed by a carbonyl group, and Z represents a compound selected from a functional substance. . ] The physiologically acceptable complex compound shown by these, and its salt. 8. A physiologically acceptable complex compound or a salt thereof according to claim 7, and an atomic number 31, 39, 43,
44, 49 or 57 to 71, 76, 77, 79, 8
A physiologically tolerable metal complex complex compound obtained by complexing at least one metal ion selected from the group of 1,83. 9. The metal ion is Gd, Dy, Ho, Bi, O.
The physiologically tolerable metal complex complex compound according to claim 8, which is s, In, Ga, Tc, Y or Sm. 10. A drug for nuclear magnetic resonance, X-ray or radiodiagnosis and a drug for radiotherapy, comprising at least one physiologically tolerable metal complex complex compound according to claim 8 or 9. .