KR860001360B1 - Process for the preparation of 1-(p-substituted or unsubstituted amino alkyl) phenyl propane-1,2-dione bis (thiosemicarbazone) derivatives - Google Patents

Abstract

The title compds. I [R,R',R1,R2=H, C1-3 alkyl; n=0-3! were prepd. by condensation of an appropriate oxime with a given thiosemicarbazide and used as carriers for radioactive elements and physiol. active substances, for noninvasive nuclear medicine diagnosis.

Description

Process for the preparation of 1- (p-substituted or unsubstituted aminoalkyl) phenylpropane-1,2-dione bis (thiosemicabazone) derivatives

The present invention relates to a process for the preparation of 1- (p-substituted or unsubstituted aminoalkyl) phenylpropane-1, 2-dione bis (thiocarbazone) derivatives (hereinafter referred to as "AAPT").

More specifically, the present invention is a general formula:

Method of preparing AAPT (wherein R, R ', R ¹ and R ² are each a hydrogen atom or a C ₁ -C ₃ alkyl group and n is an integer of 0 to 3) and a physiologically active substance carrier for these substances, It relates to the use as a carrier for radioactive metal elements.

Non-invasive nuclear medical diagnostics, such as the recording of blood circulation systems, dynamic studies and quantitative measurements, imaginary physiological abnormalities or the localization of abnormalities, ¹³¹ I-labeled serum albumin and ¹³¹ I-labeled iodine 131 ( ¹³¹ I Physiological agents, denoted by), have been widely used.

However, ¹³¹ I has a long half-life of about 8 days and releases beta rays so patients dosed with it are exposed to large amounts of radiation.

^In order to overcome the aforementioned deficiencies in ¹³¹ I-labeled physiological agents, plans have been made to provide radiodiagnostic agents of radioactive metal elements that are more advantageous than iodine-131 in combination with physiological agents. It has been. In such attempts, a labeling method is known in which a physiological agent that can be used as a radiodiagnostic agent is directly treated with a radioactive metal salt to produce a chelate compound. For example, human serum albumin is treated with human serum albumin with an aqueous solution containing pertechnetate-type technetium-99m ( ^99m Tc) in the presence of a reducing agent to obtain ^99m Tc-labeled human serum albumin. Furthermore, bleomycin is treated with an aqueous solution containing indium chloride type indium-111 ( ¹¹¹ In) to obtain ¹¹¹ In labeled bleomycin. However, the chelating properties of these physiological agents are not sufficient, and the chelate bonds produced once are easily separated. In practice, ^99m Tc labeled serum albumin and ¹¹¹ In labeled bleomycin have low stability after administration in vivo.

Thus, when administered in vivo, ^99m Tc and ¹¹¹ In are rapidly ^released , so the radioactive response in vivo is inconsistent with serum albumin or bleomycin, which are physiological agents. This is a fatal flaw for nuclear medical diagnosis based on accurate traces of radioactive reactions consistent with the response of physiological agents.

The inventors of the present invention have disclosed that 3-oxobutyralcarboxylic acid bis (4-methylthiosemicarb of 3-oxobutyralcarboxylic acid bis as a chelating agent that can be bound to a physiological agent (Japanese Patent Publication No. 34664, 34664). Bazon) has previously been proposed. However, the end groups of the aforementioned compounds are carboxyl groups which can be bound to physiological agents by dama amino bonds and are therefore limited in use.

The present inventors also proposed the use of 3-aminomethylene-2, 4-pentanedionebis (4-alkylthiosemicarbazone) (Japanese Patent Publication No. 1982, 102860). However, the end groups of these compounds are located at unsubstituted carbon atoms which reduce the electron density of amino nitrogen atoms, resulting in impaired reactivity.

As a result of extensive research, it is known that AAPT (I) has strong chelating properties and can bind to amino and / or carboxyl groups of physiological agents in mild conditions.

It is also known that chemical products containing metal elements bonded to AAPT (I) are sufficiently stable in vivo. In addition, it is known that chemical products containing physiological agents and radioactive metal elements, which are mediated by AAPT (I), are sufficiently stable in living bodies and that the reaction or radioactivity in living bodies matches those of physiological agents themselves. .

The present invention provides AAPT (I) useful as a chemical carrier for physiological agents and / or radioactive metal elements. Also provided are radioactive metal element binding compounds containing chelated AAPT (I) and radioactive metal elements useful as radiodiagnostic agents. Furthermore, there is provided a physiological agent binding AAPT (I) containing chemically bound AAPT (I) and a physiologically active agent, with or without a mediated binding aid useful as a non-radioactive carrier for use in nuclear medical diagnostics. Also provided are chelated physiological agent binding AAPT (I) useful as radiodiagnostic agents and radioactive metal element labeled physiological agent binding AAPT (I) containing radioactive metal elements.

AAPT (I) is new and also general formula:

The oxime of the compound of can be prepared by condensing with 4-R ³ -thiosemicarbazide, wherein R ³ is R ¹ or R ² .

Wherein R and R 'are each as defined above.

Oximes wherein R and R 'are each C ₁ -C ₃ alkyl group are as follows:

Can be obtained as

In the above process, compound (III) is C-acylated with propionyl chloride according to the known method (Yu V Markova: Chem. Abst., 63, 17951f (1965)) to obtain compound (IV), which is essentially By conventional methods (Nathan Levin et al., Org. Syn, Coll., Vol. 3, p. 191, published 1955), isopropyl or nitrite isoamylonitriated to obtain oxime (V).

Oxime, wherein R and R 'are each hydrogen atoms, is the following process:

Can be obtained.

In the above process, compound (VI) is N-acylated with a trifluoroacetylating agent to obtain an N-protecting compound (VII), which is the same method as in the C-acylation of dialkylamino compound (III). C-acylated. The obtained compound (v) was obtained by removing the N-protecting group according to the method of Haward Newman (J. Org. Chem., Vol. 30, p. 1289 (published in 1965)) to obtain compound (IX), which is a dialkylamino compound. Nitrosolation is carried out in the same manner as in nitrosification of (IV) to obtain oxime (X).

Oxime condensation of compound (II) with 4-R ³ -thiosemicarbazide can be made in a single process or in two processes.

When R ¹ and R ² produce the same AAPT (I), condensation is usually carried out by reacting the oxime of compound (II) with 4-R ³ -thiosemicarbazide in a molar ratio of 1: 2 or more. Is made of. When R ¹ and R ² produce different AAPT (I), condensation usually reacts the oxime of compound (II) with 4-R ¹ -or 4-R ² -thiosemicarbazide in approximately equimolar ratios and then It consists of two processes in which the produced monothio semicarbazone is reacted with 4-R ² or 4-R ¹ -thiosemicarbazide in approximately equimolar ratios.

In general, condensation takes place in the presence of an acidic catalyst such as hydrochloric acid, hydrobromic acid or sulfuric acid, preferably in an inert solvent such as methanol or ethanol.

The AAPT (I) thus produced can be bound to a carboxyl or amino group of a physiologically active substance, with or without all binding aids, in a mild state, to produce chelates and aminos that leave these physiologically active substances unchanged. It has two thiosemicarbazone groups that can hold. Therefore, it is useful as a carrier for radioactive metal elements and physiological agents.

AAPT (I) is treated with a physiological agent to produce a physiological agent binding AAPT (I) as a non-radioactive carrier.

In order to prepare a radioactive metal element-labeled physiological agent binding AAPT (I) as a radiodiagnostic agent of the present invention, AAPT (I) is usually first bound to a physiological agent, and the binding product is then expressed as a radioactive metal element. do.

The term "physiological agent" refers to any substance that exhibits a specific accumulation at a particular site or tissue or diseased location or exhibits a specific response to a particular physiological condition. Traces of the response in vivo can provide useful information for diagnosis. Such physiological agents having carboxyl groups or amino groups can be advantageously used in the present invention. Even when no carboxyl group or amino group is present, the carboxyl group and the amino group can be introduced and used in advance. Specific examples of physiological agents include blood proteins (eg, human serum albumin, fibrinogen), enzymes (eg, urokinase, streptokinase), hormones (eg, thyrotropin, parathyroid hormone), immune antibodies (eg, , IgG), antibiotics (eg bleomycin, kanamycin), sugars, fatty acids, amino acids, and the like.

The combination of AAPT (I) and the physiological agent can be accomplished according to all methods conventionally employed to bind an amino group to a carboxyl or amino group. Examples of such methods include carbodiimide methods, glutaraldehyde methods and the like.

According to the carbodiimide method, physiological agents having AAPT (I) having a primary amino group and a carboxyl group are 1-cyclohexyl-3- (2-morpholinyl-4-ethyl) carbodiimide or 1-ethyl- It condenses in the presence of carbodiimide, such as 3- (3-dimethylaminopropyl) carbodiimide, to form a carbonamide bond between the amino group and the carboxyl group. Under the glutaraldehyde method, AAPT (I) having a primary amino group and physiological agents having a primary amino group are reacted in the presence of glutaraldehyde as a binding aid, either released or liberated as described above. The seed base is reduced with a reducing agent such as sodium borohydride. In this product, two amino groups are bonded through a pentamethylene bond. These binding methods are wholly advantageous for the completion of binding in a mild state, so that no deactivation, denaturation or degradation of physiological agents occurs physically.

If desired, the physiological agent binding thus prepared (hereinafter referred to as "PAS-binding") AAPT (I) is essentially used to remove impurities such as unreacted reagents by known methods such as separation, gel filtration or tube chromatography. It can be purified. As a result, the bond product is usually frozen into an aqueous solution which can be used to represent radioactive metal elements.

Alternatively, this aqueous solution is subjected to lyophilization, evaporation or other treatment under low temperature and reduced pressure to obtain a dry product, which can also be used as a solution for drying or display purposes. Depending on the use, aqueous solutions or dry products may be combined with additives such as PH regulators (e.g. acids, bases, buffers), stabilizers (e.g. ascorbic acid), isotonic agents (e.g. sodium chloride) or preservatives (e.g. benzyl alcohol). Let's do it. In contrast, the above-described aqueous solution or dry product may contain any reducing or oxidizing agent, which will act on the radioactive metal element to be displayed to obtain a stable chelate product as described below. Furthermore, PAS-bonded AAPT (I) is intrinsically extremely stable and can be easily labeled as a radioactive metal element in the simple manner described below, and therefore can be stored and supplied on demand as well. (I) and manufacture from physiological agents can be saved by the practitioner.

In designating AAPT (I) or PAS-bonded AAPT (I) as a non-radioactive carrier as a radioactive metal element, AAPT (I) or PAS-bonded AAPT (I) can be treated with a radioactive metal element of a suitable type.

The term "radioactive metal element" means any radioactive metal element with physical properties suitable for nuclear medical diagnosis. Examples of specific radioactive metal elements include gallium-67 ( ⁶⁷ Ga), gallium-68 ( ⁶⁸ Ga), thallium- ²⁰¹ ( ²⁰¹ Tl), ¹¹¹ In, ^99m Tc and the like.

They are usually used in salt form, in particular in water soluble salt form.

Depending on the type or state of the radioactive metal element, they adopt two different marking methods. When the radioactive metal element is in an electron state that does not require reduction or oxidation to produce a stable chelate compound, the AAPT (I) or PAS-bonded AAPT (I) is in contact with the radioactive metal element in an aqueous medium to display the radioactive metal element AAPT ( I) or radioactive metal element labeled PAS bond AAPT (I). This display method is applied to ⁶⁷ Ga, ¹¹¹ In and so on.

In the case of a valence radioactive metal element that is required for reduction or oxidation to produce a stable chelate compound, AAPT (I) or PAS-bonded AAPT (I) is contacted with a radioactive metal element in an aqueous medium in the presence of a reducing or oxidizing agent. Obtain radioactive metal element labeled AAPT (I) or radioactive metal element labeled PAS bond AAPT (I). This marking method can be applied to ^99m Tc or the like.

As the reducing agent, a monobasic tin chloride salt, i.e. divalent tin ions, is usually used. Specific examples include stannous halides (eg, stannous chloride, stannous fluoride), stannous sulfate, stannous nitrate, stannous acetate, stannous citrate, and the like. Resin with an ion-exchange resin saturated with Sn ⁺⁺ ions, such as Sn ⁺⁺ ions may also be used.

For example, when the radioactive metal element is ^99m Tc, AAPT (I) or PAS-bonded AAPT (I) is treated with ^99m Tc of pertechnetate type in an aqueous medium in the presence of a reducing agent such as stannous salt. There is no particular restriction on the order of introduction of the reagents described above into the reaction system. Typically, however, first of all, mixing of stannous salt with pertechnetate should be avoided in an aqueous medium. The stannous salt may be used in an amount sufficient to sufficiently reduce the pertechnetate.

Radiodiagnostics should have sufficient radiation and radioactivity concentrations to ensure reliable diagnosis.

For example, when the radioactive metal element is ^99m Tc, the amount of 0.1-50mCi may be generally contained in about 0.5-5.0 ml at the time of administration.

The amount of AAPT (I) or PAS bound AAPT (I) is sufficient to produce stable chelate compounds with radioactive metal elements.

The radioactive metal element marker AAPT (I) or radioactive metal element marker PAS-bonded AAPT (I) thus produced is stable enough to be a radioactive diagnostic agent, and can thus be stored and supplied on demand. If desired, the radiodiagnostic agent may contain additives such as pH adjusters (eg, acids, bases, buffers), stabilizers (eg, ascorbic acid) or isotonic agents (eg, sodium chloride).

The radioactive metal element labeled AAPT (I) and the radioactive metal element labeled PAS bond AAPT (I) of the present invention are useful for nuclear medical diagnosis. For example, ^99m Tc labeled AAPT (I) or ⁶⁷ Ga labeled AAPT (I) can be used for recording and functional measurements of myocardium. Also, for example, ^99m Tc-labeled human serum albumin-binding AAPT (I) can be used intravenously in the human body for recording, functional studies and quantitative measurements of the blood circulation.

Furthermore, for example, ^99m Tc-labeled fibrinogen-bound AAPT (I) or ^99m Tc-labeled utokinase-binding AAPT (I) can be used for minimizing thrombosis simultaneously with detection and recording of thrombosis because they are deposited at thrombosis sites.

In addition, eg ^99m Tc-labeled streptokinase binding AAPT (I) is useful for locating myocardial infarction. In addition, ^99m Tc-labeled thyroid stimulating hormone-binding AAPT (I) is useful for detecting and recording cancer in the thyroid gland.

The radiodiagnostic agents of the present invention can typically be administered to a patient in an amount sufficient to yield the radioactivity necessary for the diagnosis of an organ or tissue by a suitable route via the intravenous route. For example, intravenous administration of about 1 to 3 ml of ^99m Tc-labeled radiodiagnostic agent with radioactivity of about 1 to 20mCi is entirely suitable for diagnostic purposes.

The advantages of PAS binding AAPT (I) as a nonradioactive carrier are summarized as follows.

(A) Long term stability after manufacture.

(B) Undesirable side reactions such as deactivation, denaturation or degradation do not occur physically to the physiological agent, as they can be prepared in mild conditions.

(C) Physiologically active substances having carboxyl or amino groups can be used as starting materials.

(D) The introduction of such groups into physiological agents, even if no carboxyl or amino groups are present, makes them useful as starting materials.

(E) It is possible to provide PAS-bonded AAPT (I) of radioactive metal element representations in such a simple way of contacting radioactive metal elements in aqueous media.

The advantages of radioactive metal labeling AAPT (I) as a radiodiagnostic agent are summarized as follows:

(A) Radioactive metal elements are completely chelated.

(B) Rapidly and highly deposited in the liver, kidneys and especially myocardial layers.

(C) It can be manufactured in an extremely simple process.

Likewise, the advantages of PAS binding AAPT (I) of radioactive metal element labeling as a radiodiagnostic agent are summarized as follows.

(A) Long term stability after manufacture.

(B) The display efficiency by radioactive metal element is extremely high (almost 100%).

(C) Labeling is extremely simple and undesirable side reactions such as deactivation, denaturation or degradation do not occur in physiological agents in combination with AAPT (I).

(D) Among the various radioactive metal elements, the most suitable one can be selected and used for diagnosis purposes. Therefore, the information for diagnosis is not only strengthened quantitatively but also qualitatively by reduction of the exposure dose.

Unless otherwise defined and practical embodiments of the present invention are described in detail in the following examples where% is by weight.

Example 1

Preparation of 1- (p-N, N-dimethylaminoethyl) phenylpropane-1,2, -dionebis (4-methylthiosemicarbazone)), hereinafter referred to as "DEPM"

(A) Anhydrous aluminum chloride (66 g) was added to a solution of N, N-dimethylphenethylamine (22.35 g) in dry carbon disulfide (225 mL). Pionyl chloride (15.27 g) was then added dropwise to the solution described above at 50-60 ° C. with stirring for at least about 1 hour, and the solution was refluxed with stirring for 4.5 hours.

Cold water was added to this solution to decompose the aluminum chloride. The solution was adjusted slightly alkaline with sodium hydroxide solution and extracted with ethyl ether. The ethereal extract was dried over anhydrous sodium sulfate, concentrated and distilled under reduced pressure to obtain p-N, N-dimethylamino ethylpropiophenone (25 g, 81%) as a fraction boiled at 115-117 ° C / 1-2 mmHg.

(B) Dry hydrogen chloride was bubbled into pure p-N, N-dimethylaminoethylpropiophenone solution (2.33 g) obtained in (A) from pure ethanol (75 mL).

Isopropyl nitrate (1.07 g) was added to this solution and stirred overnight at room temperature. Ethanol was removed from the solution by distillation to obtain a crude product, which was recrystallized from ethanol to obtain p-N and N-dimethylaminoethyl-2-hydroxyiminopropiophenone close to the theoretical yield.

Melting point: 220-222 ° C. (hydrochloride);

186-188 ° C. (freebase).

analysis :

Theory (%): C, 57.93; H, 7. 28; N, 10.56

Actual: C, 57.67; H, 7.07; N, 10.35

(C) pN, N-dimethylaminoethyl-2-hydroxyiminopropiophenone (2.34 g) and 4-methylthio semicarbazide (2.31 g) in 90% ethanol (12 mL) The solution was adjusted to pH 2 with concentrated hydrochloric acid and refluxed with stirring for 8 hours.

After cooling, the resulting crystals were recovered and recrystallized with ethanol to obtain DEPM (1.0 g, 25%).

Melting point: 230-232 ° C. (hydrochloride).

analysis :

Theory (%): C, 47.36; H, 6.65; N, 22.64

Actual: C, 47.38; H, 6.56; N, 22.80

Example 2

Preparation of 1- (p-aminomethyl) phenylpropane-1,2-dione bis (4-methylthiosemicarbazone) (hereinafter referred to as "AMPM"):

A solution of p-aminomethyl-2-hydroxyiminopropiophenone (2.06 g) and 4-methylthiosemicarbazide (2.31 g) in ethanol (10 mL) was adjusted to pH 2 with concentrated hydrochloric acid for 7 hours. It was refluxed while stirring. After cooling, the resulting crystals were recovered and recrystallized with ethanol to obtain AMPM (1.1 g, 31%).

Assay (hydrochloride):

Theory (%): C, 41.40; H, 5.88; N, 24.21

Actual: C, 41.45; H, 5.96; N 24.16

Example 3

Preparation of 1- (p-aminoethyl) phenylpropane-1, 2-dione bis (4-methylosemiccarbazone) (hereinafter referred to as "AEPM"):

(A) Trifluoroacetic anhydride (15 mL) was added to a solution of phenethylamine (12.2 g) in pyridine (30 mL). The solution was stirred for 12 hours at room temperature and then distilled under reduced pressure to give N-trifluoroacetylphenethylamine (16.5 g, 75%) as a fraction distilled at 103-104 ° C./4 mmHg.

(B) Anhydrous aluminum chloride (22 g) was added to a solution of N-trifluoroacetylphenethylamine (11.0 g) obtained in (A) in dry carbon disulfide (75 mL). Propionyl chloride (5.1 g) was then added dropwise to the above solution with stirring. The solution was refluxed with stirring for 4.5 hours and left overnight. Cold water was added to this solution to decompose the aluminum chloride. The solution was adjusted slightly alkaline with sodium hydroxide solution and extracted with ethyl acetate. The extract was dried over anhydrous sodium sulfide and concentrated to give a black residue, which was dissolved in warm methanol, treated with activated carbon and cooled to give p-N-trifluoroacetaminoethylpropiophenone (3.34 g, 24.5%).

(C) potassium carbonate (2.9 g) was added to a solution of pN-trifluoroacetaminoethylpropiophenone (2.73 g) in a mixture of methanol (30 mL) and water (12 mL), and the solution was Stir for rice. Potassium carbonate was decomposed by the addition of 5% hydrochloric acid. The solution was then made alkaline with 10% sodium hydroxide and extracted with ethyl ether.

The extract was dried over anhydrous sodium sulfate. Gas phase hydrogen chloride was bubbled into the extract to give p-aminoethylpropiophenone hydrochloride close to the theoretical yield.

analysis :

Theory (%): C, 74.36; H, 8. 60; N, 7.84

Actual: C, 74.54; H, 8.53; N, 7.90

(D) Dry hydrogen chloride was bubbled into a solution of p-aminoethylpropiophenone hydrochloride (2.14 g) in pure ethanol (50 mL).

Isopropyl nitrate (1.07 g) was added to this solution, which was stirred overnight at room temperature. The solvent was removed by distillation to give a crude product which was recrystallized from ethanol to give p-aminoethyl-2-hydroxyiminopropiophenone close to the theoretical yield.

(E) A solution of p-aminoethyl-2-hydroxyiminopropiophenone (1.22 g) and 4-methyl semicarbazide (1.6 g) obtained in (C) in 90% ethanol (10 mL) was prepared. Adjusted to 2 and refluxed with stirring for 7.5 h. After cooling, the resulting crystals were recovered and recrystallized with ethanol to give (0.65 g, 30%).

analysis :

Theory (%): C, 49.46; H, 6. 50; N, 26.30; S, 17.74

Actual: C, 49.29; H, 6. 34; N, 26.83; S, 17.54

Example 4

Preparation of Carrier Composition for Radiodiagnostics Containing DEPM:

A mixture of DEPM (3.9 mg), sodium hydroxide solution (1 mL), 0.1 M acetic acid buffer (pH 6.0) (3 mL) and 1N hydrochloric acid (1 mL) was prepared and left until a clear solution was obtained. All processes were carried out in aseptic conditions.

Example 5

Preparation of a Carrier Composition Containing Tin Tin Chloride:

The solution (1 mL) obtained in Example 4 was mixed with a solution of stannous chloride (0.1 mL) to shake the mixture for a short time to obtain a clear solution. The solution was stored in a capped vessel and the air in the vessel was converted to nitrogen gas.

Example 6

Preparation of a Carrier Composition Containing Stannous Ion Saturated with Ion Exchange Resin:

The solution (1 ml) obtained in Example 4 was mixed with an ion exchange resin (Dowex0W × 8, 3 mg) while absorbing stannous ions (5.5 μg, Sn ⁺² / mg resin). The mixture was shaken for a while and the stopper stored in a vessel, and the air in the vessel was changed to nitrogen gas. All processes were carried out in aseptic conditions.

Example 7

Preparation of DEPM-Containing Ga-labeled Radioactive Diagnostics:

The solution (1 ml) obtained in Example 4 was mixed with a potassium chloride ( ⁶⁷ Ga) solution (10 mCi / ml, 1 mCi), and the mixture was shaken for a while to obtain a ⁶⁷ Ga display radioactive diagnostic agent.

The process was carried out aseptically. This diagnostic demonstrated that the radioactive gallium was completely chelated and displayed a single radioactive spot of Rf = 0.55 in silica gel thin layer chromatography (solvent, 10% ammonium acetate: methanol = 1: 1).

Example 8

Preparation of ^99m Tc-labeled Radiodiagnostics Containing DEPM

A commercially available sodium pertechnetate solution ( ^{99 m} Tc) (1 mCi / ml, 1 mCi) was added to the carrier composition obtained in Example 6 and the mixture was slowly moved for about 3 minutes. Subsequently, the ion exchange resin was removed by filtration to obtain a ^99m Tc labeled radiopharmaceutical.

Example 9

Physical properties of ^99m Tc labeled radiodiagnostics containing DEPM:

The physical properties of the ^99m Tc labeled ^{radiodiagnostic} agent obtained in Example 8 were evaluated.

(1) pH = 6.0

(2) silica gel TLC

Chromatograms of the above-described labeled diagnostics, which developed a single radioactive peak with an isotropic mixture of 10% ammonium acetate and methanol, were detected by injection with a chromatogram syringe to Rf = 0.53-0.57. Pertechnetate ions ( ^99m Tc) were found to have an Rf value of 0.91 in the same chromatography system, demonstrating that the technetium -99m atom was completely chelated.

(3) electrophoresis

The electrophoresis of 0.1 m phosphate buffer (pH 7.0) was examined with a 500 V potential difference for 1 hour using a sheet of filter paper as an electrophoretic membrane. The monoradioactive peak was detected to be 0.9-1.0 cm at the negative side where the air dried membrane was injected using a chromatogram syringe. Since it is known that pertechnetate ( ^{99 m} Tc) ions travel 5.7-6.0 cm in both directions, it has been demonstrated by electrophoresis described above that the technetium -99 m atom is completely chelated and the chelated ions carry a positive charge.

Example 10

Response of DEPM-containing ^99m Tc-labeled Radiodiagnostics in Rats:

Each portion (0.1 mL) of the ^{99 m} Tc labeled ^{radiodiagnostic} agent prepared in Example 8 was administered to the tail vein of the rat, which was dissected at a fixed time and the radioactivity in each organ was evaluated.

Changes over time and standard deviations required for the distribution of radioactivity per unit weight of an engine are shown in the following table.

[Table 1]

Change in blood level in rats (% / g)

The above results clearly show that the radiopharmaceutical of the present invention rapidly accumulates at high concentrations in the liver, kidneys and myocardial layers. In particular, the accumulation in the myocardial layer is in contrast to the commonly used thallium chloride ( ²⁰¹ Tl). In addition, it has been demonstrated that the myocardial layer / blood ratio is more than 2 and can be used for nuclear medical diagnosis for the purpose of recording, dynamic research and functional testing of the myocardial layer.

Example 11

Response of DEPM-containing ⁶⁷ Ga labeled radiodiagnostics in rats:

The same results as in Example 10 were obtained by experiments using the ⁶⁷ Ga-labeled radiodiagnostic agent prepared in Example 7.

Example 12

Toxicity of ^99m Tc labeled radiodiagnostics containing DEPM:

Radiopharmaceuticals prepared in Examples 7 and 8 were subjected to radioactive decay to a moderate degree. Each of these products was administered intravenously to male and female rat groups at a dose of 1 ml per 100 g body weight (corresponding to 300 times the estimated dose for humans), and also for male and female rat groups consisting of five of each ICR lineage. Was administered a 0.5 ml dose (corresponding to 1500 times the estimated dose for humans) per 10 g of body weight. In contrast, the same amount of physiological saline as described above was administered intravenously to separate, identical animal groups as described above. These animals were kept for 10 days. The change in body weight was recorded daily.

No significant difference was noted between the treated and control rats. Ten days after dosing, all animals were dissected and abnormalities of various organs were examined. However, no abnormalities were observed in any animal.

From the above results, it can be said that the toxicity of the radiopharmaceutical according to the present invention is extremely low.

Example 13

Preparation of Human Serum Albumin Binding AEPM as Nonreflective Carrier:

Human serum albumin (freeze-dried; 75 mg) was dissolved in water (5 ml) to obtain a solution called "solution (A)" in the future. Separately, AEPM was dissolved in dimethylformamide at a concentration of 5 mg / ml. This solution (0.5 mL) was added to solution (A). The mixture thus produced is hereinafter referred to as "solution (B)".

Solution (B) was adjusted to pH 4.6 with 0.1N hydrochloric acid. An aqueous solution of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (10 mg / ml; 1.3 ml) was added to the solution (B) and adjusted to pH 4.6 with 0.1 N hydrochloric acid for about 10 hours. It stirred at the temperature below ° C. This mixture was dialyzed in a dialysis tube for 24 hours and dialyzed by a conventional method. Centrifugation and lyophilization gave human serum albumin-binding AEPM of white crystals. This crystal (67 mg) is dissolved in 0.2 M acetic acid buffer (pH 2.64; 5 mL), and the dissolved oxygen is replaced with nitrogen in advance. Aqueous stannous chloride solution (2.0 ml) and ascorbic acid (1.2 mg) were added. The solution thus obtained was passed through a 0.22-µm pore size filter. Each 1.5 ml portion of the filtrate was filled in a vial, and the inside thereof was washed with nitrogen to obtain a non-radioactive carrier of a slightly pale yellow transparent solution.

The above operation was carried out in aseptic conditions.

Example 14

Preparation of human serum albumin binding AMPM as nonradioactive carrier:

: AMPM (5 mg) was dissolved in dimethylformamide (2 mL). To this, an equimolar amount of glutaraldehyde was added to AMPM, and the resulting mixture was stirred at room temperature for 15 minutes to form a solution (A). Separately, human serum albumin (lyophilized; 100 mg) was dissolved in 0.01 M phosphate buffer-0.15 M sodium chloride solution (pH 7.4; 10 mL) to make solution (B). Solution A (1.0 mL) was added to Solution B with ice cooling, and the resulting mixture was stirred at the same temperature as described above for about 0.5 hours. After addition of sodium borohydride (1 mg), stirring was continued at 0-4 [deg.] C. temperature for about 1 hour to allow reduction to proceed. The mixture thus obtained was placed in a dialysis tube and dialyzed by conventional methods for 24 hours. The solution thus obtained was passed through a 0.22-µm pore size filter, and each 1.0 ml portion of the filtrate was placed in a vial and lyophilized to obtain a non-radioactive carrier. The above operation is carried out in aseptic conditions.

When dissolved in water, the non-radioactive carrier becomes a slightly pale yellow transparent solution.

Example 15

Preparation of Eurokinase Binding AMPM as Nonradioactive Carrier:

By cooling in an ice bath, the purified urokinase (freeze-dried; 50 mg) was dissolved in water to obtain a solution (A). Separately, AMPM was dissolved in dimethylformamide to a concentration of 5 mg / ml. The solution thus obtained (0.5 ml) was added to the solution (A) and the pH was adjusted to about 4.6 with 0.1 N hydrochloric acid to make solution (B). To the solution (B) was added an aqueous solution of 1-cyclohexyl-3- (2-morpholinyl-4-ethyl) carbodiimide (50 mg / ml; 1.5 mL). It was adjusted and stirred at a temperature of 5 ° C. or lower for about 2 hours. The reaction mixture was chromatographed with Sephadex G-50 (2 × 30 cm tube) and eluted with 0.01 M phosphate buffer -0.15 M sodium chloride solution (pH 7.4). This eluate was diluted with 0.01 M phosphoric acid-0.15 M sodium chloride solution to a concentration of 5.0 mg / ml urokinase.

The diluent was passed through a 0.22 μm pore size filter, and each 1.5 ml portion of the filtrate was filled in a vial to obtain a non-radioactive carrier which was a slightly pale yellow transparent solution.

The above operation was carried out in aseptic conditions.

Example 16

Preparation of ^99m Tc Marked Human Serum Albumin Binding AEPM as Radiodiagnostic Agent:

The human serum albumin binding AEPM (solution) obtained in Example 13 (1.0 mL) was mixed with physiological salt solution (0.5 mL) containing ^99m Tc (3mCi) in the form of pertechnetate, and stirred sufficiently to be useful as a radiodiagnostic agent. ^99m Tc labeled human serum albumin binding AEPM was obtained. This solution was pale yellow transparent and had a pH of about 3.0.

Example 17

Preparation of ⁶⁷ Ga Marked Human Serum Albumin Binding AMPM as Radiodiagnostic Agent:

Human serum albumin-bound AMPM (freeze-dried) obtained in Example 14 was dissolved in 0.2 M acetic acid buffer (pH 4.0; 1.0 ml), and 0.01 N hydrochloric acid (0.5 ml) containing ⁶⁷ Ga (2 mCi) of potassium chloride type was added thereto. Was added to obtain an aqueous solution containing ⁶⁷ Ga-labeled human serum albumin-binding AMPM useful as a radiodiagnostic agent. This solution was slightly pale yellow transparent and had a pH of about 3.7.

Example 18

Preparation of ^99m Tc Marked Human Serum Albumin Binding AEPM as Radiodiagnostic Agent:

In order to examine the labeling efficiency of the ^99m Tc labeled human serum albumin-bound AEPM obtained in Example 16, the aqueous solution was subjected to thin layer chromatography analysis using silica gel as a preservative and methyl ethyl ketone as a developing agent. Was carried out with a radiochromatography syringe. Radioactivity was recorded as a single peak of the original point. No peaks due to radioactive impurities such as free pertechnetate ions (Rf = 1.0) were observed.

The ^99m Tc-labeled human serum albumin-binding AEPM obtained in Example 16 was subjected to electrophoresis (1.7 mAcm; 15 minutes) using a veronal buffer (pH 8.6) as a developing solvent and a cellulose acetate membrane as an electrophoretic membrane. Injection was then carried out using a radio chromatograph syringe.

Radioactivity was recorded as a single peak at a location 1.8 cm from the baseline to the positive side. This position was the same position as the coloring band of human serum albumin by ponceau ³ R. As a result, it can be said that the ^99m Tc-labeled human serum albumin binding AEPM according to the present invention has a labeling efficiency close to 100% and the charge is substantially the same as that of human serum albumin.

Example 19

Response of ^99m Tc-labeled Human Serum Albumin-coupled AEPM in Rats:

^99m Tc-labeled human serum albumin binding AEPM (0.1 mL) obtained in Example 16 was administered to the tail vein of each female rat of SD lineage. Changes in blood levels over time were recorded. In contrast, the same test as described above was performed using conventional ^99m Tc labeled human serum albumin and conventional ¹³¹ I labeled human serum albumin.

The results are shown in a second table in which blood levels of the respective measurement times are expressed in absolute values (% of dose / g; average).

[Table 2]

Change in blood level in rats (% / g)

As a result, the ^99m Tc-labeled human serum albumin binding AEPM of the present invention can maintain a significantly high blood level for a long time compared with the conventional ^99m Tc-labeled human serum albumin and the conventional ¹³¹ I-labeled human serum albumin. Able to know. In addition, it can be seen that ^99m Tc-labeled human serum albumin binding AEPM is extremely stable in vivo. Thus, the ^99m Tc-labeled human serum albumin-coupled AEPM is well suited for use in nuclear medical diagnostics for the recording, identification, and quantitative measurement of the blood circulation system.

Example 20

Preparation of ⁶⁷ Ga Marked Human Serum Albumin Binding AMPM as Radiodiagnostic Agent:

In order to examine the labeling efficiency of ⁶⁷ Ga-labeled human serum albumin-bound AMPM obtained in Example 17, electrophoresis (1.7 mA / cm, 5 min) test was performed using Veronal buffer (pH 8.6) as a developing membrane, and injection was performed. Was carried out using a radio chromatograph syringe.

It was recognized that radioactivity was a single peak at a distance of 1.8 cm on both sides of the baseline. This position was identical to human serum albumin curling band by Ponso 3R.

As a result of the foregoing, it can be said that the ⁶⁷ Ga-labeled human serum albumin binding AMPM according to the present invention has a display efficiency close to 100% and the charge is substantially the same as that of human serum albumin.

Example 21

Preparation of nonradioactive urokinase binding AMPM:

The enzymatic action of the non-radioactive urokinase-binding AMPM obtained in Example 15 was measured by ester degradation using N-α-acetyl-L-lysine methyl ester and was substantially the same as the purified urokinase used as starting material.

From the above results, it can be said that non-radioactive urokinase-binding AMPM can enzymatically act as starting purified urokinase.

In addition, the ¹¹¹ In-labeled urokinase-binding AMPM possessed an enzymatic action, indicating that there was practically no material difference from urokinase itself in the reaction in vivo.

Example 22

Stability of human serum albumin binding AEPM:

The human serum albumin binding AEPM obtained in Example 13 was stored in a refrigerator at 4 to 8 ° C. for 30 days and treated with ^{99 m} Tc according to the same method as in Example 16 to prepare an aqueous solution containing ^{99 m} Tc-labeled human serum albumin binding AEPM. Got it.

This solution was subjected to thin layer chromatography and electrophoresis by the same method as in Example 18, and the reaction in rats was examined by the same method as in Example 19. The result was practically the same as in Examples 18 and 19.

Thus, no material change occurred in the serum-serum albumin-coupled AEPM due to 30 human storage.

Example 23

Stability of Human Serum Albumin-Coupled AMPM:

Example 14 Human hayeoseo serum combined AMPM stored in the refrigerator for 30 days 4 to 8 ℃, examples treated in the same way 17 to ⁶⁷ Ga a solution containing the ⁶⁷ Ga show human serum albumin-combined AMPM obtained in. The solution was subjected to electrophoresis in the same manner as in Example 20. It was recognized that the radioactivity was a single peak, and the location was confirmed to be identical to human serum albumin by coloring of Ponso 3R. Thus, it can be said that no material changes occur in human serum albumin binding AMPM after 30 days of storage.

Example 24

Stability of ^99m Tc-labeled Human Serum Albumin-coupled AEPM:

An aqueous solution containing ^99m Tc labeled human serum albumin binding AEPM obtained in Example 16 was stored for 36 hours at room temperature (24-27 ° C). The solution was subjected to thin layer chromatography and electrophoresis in the same manner as in Example 18, and the reaction in rats was examined in the same manner as in Example 19.

The result was practically the same as in Examples 18 and 19. Therefore, it can be said that no material change occurs in ^99m Tc-labeled human serum albumin-bound AEPM after 36 hours of storage.

Example 25

Stability of ⁶⁷ Ga Marked Human Serum Albumin AMPM:

An aqueous solution containing Ga-labeled human serum albumin-binding AMPM obtained in Example 17 was stored at room temperature (24-27 ° C.) for 72 hours. Electrophoresis was carried out with this solution in the same manner as in Example 20. The radioactivity was recognized to be a single peak and the location was confirmed to be practically identical to human serum albumin by coloring of Ponsou 3R. Thus, it can be said that no change in material occurs with 72 hours of storage in ⁶⁷ Ga-labeled human serum albumin-bound AMPM.

Example 26

Toxicity of non-radioactive carriers:

Non-radioactive carriers obtained in Examples 13-15 (completely dissolved with 0.2 M acetic acid buffer in the case of non-radioactive carriers obtained in Example 14) were intravenously injected into male and female rat groups of 5 SD lineages in each group. Dose at a dose of 1 ml per 100 g body weight (corresponding to 600 times the estimated dose for humans) and 0.5 ml per 10 g body weight to male and female rat groups of 5 ICR lineages in each group (human Estimated dose for a dose corresponding to 3000 times). As a control, substantially the same amount of physiological saline as described above was administered intravenously to separate identical animal groups as described above.

These animals were kept for 10 days, and weight changes were recorded daily during this period.

Ten days after dosing, all animals were dissected for abnormal observation of various organs. However, no abnormalities were found in either animal.

In view of the foregoing, it can be said that the toxicity of the non-radioactive carriers of the present invention is extremely low.

Example 27

Toxicity of radiopharmaceuticals:

The ^99m Tc-labeled human serum albumin-binding AEPM obtained in Example 16 attenuated radioactivity to an appropriate degree, and the product was tested for toxicity in the same manner as in Example 26. No significant difference was noted between the dosing groups and the controls. No abnormalities were observed in their organs in all dissected animals 10 days after dosing. Thus, it can be said that no radiotoxicity occurs in test animals even when the radiodiagnostic agent of the present invention is administered at a large dose corresponding to 400 to 2000 times the estimated dose for humans.

Claims (4)

A method for preparing a compound of formula (I) by condensing an oxime of formula (II) with 4-R ³ -thiosemicarbazide, wherein R ³ is R ¹ or R ² .

Wherein R, R ', R ¹ and R ² are each a hydrogen atom or a C ₁ -C ₃ alkyl group and n is an integer of zero to zero. The process according to claim 1, wherein the condensation reaction is carried out in a one step process. The process according to claim 1, wherein the condensation reaction is carried out in a two step process. The method according to claim 1, wherein the oxime is monooxime.