JPH0782280A - New prodrug and its production - Google Patents

Abstract

PURPOSE:To obtain a new prodrug high in safety, presenting antitumor effect through its selective activation in cancerous tissue in cancer therapies using a complex composed of cancer antibody and enzyme, by hydrogenolysis of a specific compound. CONSTITUTION:The objective prodrug, a compound of formula I (R is of formula II or III), e.g. N-[p-N,N-bis(2chloroethyl) aminobenzoyl]-O-phosphono-L-tyrosine, can be obtained by hydrogenolysis of a compound of formula IV (Bzl is benzyl), for example, in the presence of an organic solvent such as methanol and a catalyst such as palladium-carbon in a hydrogen atmosphere at room temperature to 50 deg.C.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel prodrug and a method for producing the same.

More specifically, the following formula (I)

[0003]

[Chemical 9] (In the formula, R is

[0004]

[Chemical 10] Or

[0005]

[Chemical 11] Indicates. And a pharmacologically acceptable salt thereof, and a method for producing the same.

[0006]

BACKGROUND OF THE INVENTION For the desired chemotherapy of cancer, drugs that act specifically only on cancer cells are needed. That is, cancer tissue does not consist, strictly speaking, of one type of homogeneous cell (eg G. Heppner, Cancer Res., 44 , 2
259-2265, 1984), so there is a need for a drug that kills various types of cancer cells in cancer tissues, but not normal cells.

At present, there are unsatisfactory antitumor agents effective against cancer, but most of these agents act on proliferating cells rather than on cancer cells. Therefore, if it proliferates, it acts not only on cancer cells but also on normal cells, and it is difficult to avoid side effects. That is, the anti-tumor agent actually used may be manageable in some cases because the anti-tumor effect and the side effect are found to be well balanced. If such an antitumor agent can be made to act more specifically on cancer tissues, cancer chemotherapy will be greatly improved.

Under these circumstances, the idea of an antitumor system using a prodrug of an antitumor agent and a cancer antibody-enzyme complex (Patent Application Publication No. 2-504630 and Patent Application Publication No. 2-504630) 223532) brings light to cancer chemotherapy.

In this system, first, a low-toxicity prodrug of an antitumor agent is prepared. Then, a complex (cancer antibody-enzyme complex) consisting of an antibody against an antigen on the surface of the cancer cell and an enzyme capable of activating the prodrug of this antitumor agent is administered, and this cancer antibody-enzyme complex forms an antigen. −
Localized on the surface of cancer cells by antibody reaction. Then, the prodrug of the antitumor agent is administered thereafter. By doing so, the prodrug of the administered antitumor agent spreads throughout the body, but it is converted to the parent compound antitumor agent by the cancer antibody-enzyme complex mainly localized on the surface of cancer cells. However, since it is difficult to convert into an antitumor agent in a place unrelated to the cancer tissue, the side effect is less than that in the case where the antitumor agent which is the parent compound is administered.

This system is based on the conventional system (Vitetta et al.), Which is premised on the administration of a complex consisting of a cancer antibody and a toxin (cancer antibody-toxin complex) and the specific incorporation into cancer cells. al., Science, 238 , 1098-1104,1
987) is fundamentally different from the fact that it uses an enzyme that can be used repeatedly. In this system, even if the number of cancer antibody-enzyme complex bound to cancer tissue is limited, it is possible to increase the amount of activated antitumor agent in the vicinity of cancer tissue by administering a large amount of prodrug. It has the advantage of being able to.

[0011]

However, since the enzymes in human blood have various physiological and pathological changes, the prodrugs of the above-mentioned cancer antibody-enzyme complex and an antitumor agent. When a prodrug that can be activated by only one kind of enzyme existing in human blood is used in the system using and, the enzyme that appears temporarily in a large amount in the blood causes the prodrug in the blood to grow. The weakness remains that the drug is easily activated and loses its specificity to cancer tissue. That is, the prodrug is activated in the blood by this enzyme and spreads throughout the body as an antitumor agent, so that side effects are highly likely to occur.

[0012] Japanese Patent Application Publication No. 2-504630 and Japanese Patent Application Publication No. 2-223532 disclose, for example, prodrugs of antitumor agents which can be activated only by the action of alkaline phosphatase. Children in pregnancy and pregnant women have high levels of alkaline phosphatase in their blood and also in liver and biliary tract disease (E.
J. King et al., Med.Bull., 9 , 160-164,1953), so side effects similar to conventional antitumor agent administration occur in such cases. Further, the above-mentioned publication also discloses a prodrug of an antitumor agent which can be activated only by the action of carboxypeptidase, but it is known that carboxypeptidase A has a high value, for example, in the case of pancreatic disease (Peterson et al., Analyt. Biochem., 125 , 42
0-426,1982), the safety of this prodrug becomes a problem in such cases.

Since the probability that two kinds of enzymes in blood simultaneously have abnormally high values is lower than the probability that only one enzyme has abnormally high values, a prodrug that is activated only by the action of two kinds of enzymes. Can reach cancer tissues more selectively than prodrugs that are activated by only one enzyme.

The object of the present invention is to treat cancer using a complex of a cancer antibody and an enzyme (cancer antibody-enzyme complex) when the blood concentration of alkaline phosphatase or carboxypeptidase is abnormally high. However, it is to provide a prodrug that is selectively activated in cancer tissues and exerts an antitumor effect, and a method for producing the same.

[0015]

As a result of examination, the present inventors found that the following formula (I)

[0016]

[Chemical 12] (In the formula, R is

[0017]

[Chemical 13] Or

[0018]

[Chemical 14] Indicates. ) The compound [hereinafter referred to as compound (I)] or a pharmaceutically acceptable salt thereof is not activated by alkaline phosphatase or carboxypeptidase alone, but is activated only when both are present, The present invention has been completed by discovering that it is a prodrug that is suitable for the purpose of the present invention.

The pharmacologically acceptable salts of compound (I) include alkali metal salts such as sodium salts and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, triethylamine salts, ethanolamine salts and diethanolamine salts. Amine salts such as

In the formula (I), R is

[0021]

[Chemical 15] The compound of the present invention [Compound (Ia)] or a pharmaceutically acceptable salt thereof can be produced by hydrogenolysis of Compound (II) according to the following reaction formula.

[0022]

[Chemical 16] (In the formula, Bzl represents a benzyl group.) That is, the hydrogenolysis is dissolved in an organic solvent in which the compound (II) can be dissolved, for example, methanol or ethanol,
It is usually carried out by stirring at room temperature to 50 ° C. under a hydrogen atmosphere in the presence of a catalyst. The catalyst is palladium-
Any catalyst used in conventional hydrogenation may be used, such as carbon or palladium black.

The compound (II) is a novel compound and can be produced by the following reaction formula.

[0024]

[Chemical 17] (In the formula, Bzl represents a benzyl group.) That is, first, the compound (V) is dissolved in an ether solvent such as diethyl ether or tetrahydrofuran,
0.9 to 1.2 equivalents of sodium hydride is usually added to 0 to 15
After reacting at 0 ° C, 2 to 3 equivalents of dibenzyl phosphorochloridate is usually reacted at 0 to 15 ° C to obtain the compound (VI). Next, the compound (VI) is treated with an ether solvent (eg, diethyl ether, tetrahydrofuran,
The compound (V) was dissolved in dioxane) and treated with hydrogen chloride to eliminate the t-butyloxycarbonyl group.
II) is obtained. Finally, 2 to compound (VII)
0.8 to 1.1 equivalents of p- [N, N, -bis (2-chloroethyl) amino] in the presence of 5 equivalents of triethylamine
Compound (II) is obtained by reacting benzoyl chloride at 0 ° C to room temperature.

In the formula (I), R is

[0026]

[Chemical 18] The compound [compound (Ib)] of the present invention represented by can be produced according to the following reaction scheme.

[0027]

[Chemical 19] (In the formula, X represents a halogen atom.) That is, the compound (VIII) is dissolved in an organic solvent capable of dissolving it, for example, N, N-dimethylaminoacetamide, and 1 to 1.5 equivalents of 6-halogeno is used. Methyl-2,4
-The compound (Ib) is obtained by reacting the pteridinediamine (IX) at room temperature to 60 ° C.

The compound (VIII) can be produced according to the following reaction formula.

[0029]

[Chemical 20] (In the formula, Bzl represents a benzyl group, and X represents a halogen atom.) That is, first, the compound (X) is dissolved in an ether solvent such as diethyl ether or tetrahydrofuran, and
Reacting with 1 to 1.2 equivalents of isobutyl chloroformate in the presence of 2-3 equivalents of triethylamine at 30--10 ° C, followed by 0.9-1.2 equivalents of compound (VII).
Is added and reacted at −30 ° C. to 30 ° C. to give compound (XI)
To get Then, the compound (XI) is dissolved in an ether solvent (for example, diethyl ether, tetrahydrofuran, dioxane) and treated with hydrogen chloride to eliminate the t-butyloxycarbonyl group to obtain a compound (XII). Then, in a halogen-based solvent (eg chloroform, methylene chloride), in the presence of 2 to 4 equivalents of diisopropylethylamine with respect to compound (XII), 0.9 to 1.
Compound (XIII) is obtained by reacting 2 equivalents of 4- [N- (benzyloxycarbonyl) methylamino] benzoyl halide usually at 0 to 15 ° C.

Finally, the compound (XIII) is dissolved in an organic solvent in which it can be dissolved, for example, methanol or ethanol, and usually 10 to 30 in the presence of a catalyst such as palladium-carbon or palladium black in a hydrogen atmosphere. Compound (VIII) is obtained by hydrogenolysis with stirring at ° C.

Compound (I) of the present invention produced by the above method
Can be converted to its pharmacologically acceptable salt by a conventional method, or can be converted to another salt by a conventional salt exchange.

The compound (I) of the present invention or a pharmaceutically acceptable salt thereof is used together with a cancer antibody-enzyme complex in the treatment of cancer, and a cancer antibody-alkaline phosphatase complex and a cancer antibody-carboxypeptidase complex are used. Administered with both. The cancer antibody-alkaline phosphatase complex includes, for example, a complex in which alkaline phosphatase is bound to L6 which is an antibody against human lung cancer (Senter, PD et al., Proc. Natl. Acad. Sci. USA, 85 ,
4842-4846,1988) can be used. The cancer antibody-carboxypeptidase complex includes, for example, a complex obtained by binding carboxypeptidase A to KS1 / 4 which is an antibody against human lung cancer (Edgar haenseler et al., Biochemistry,
31 , 891-897, 1992) can be used.

The compound (I) of the present invention or a pharmaceutically acceptable salt thereof is usually administered in the form of an injection. This injection is, for example, an isotonicity agent (eg glucose, D
-Sorbitol, etc.), a preservative (for example, benzyl alcohol, chlorobutanol, etc.) and a buffer (phosphate buffer, sodium acetate buffer) and the like can be mixed and produced by a conventional method.

The compound (I) of the present invention or a pharmaceutically acceptable salt thereof is usually administered intravenously, and the dose is usually 1 to 500 mg / kg body weight of the compound (I). Is administered once a day or in 2 to 3 divided doses.

[0035]

When the compound (I) of the present invention or a pharmaceutically acceptable salt thereof is not activated by alkaline phosphatase or carboxypeptidase alone and both alkaline phosphatase and carboxypeptidase act. It is activated only by and is restored to an antitumor agent and expresses its activity. And the toxicity of the compound (I) of the present invention or a pharmacologically acceptable salt thereof is low (see Test Examples 1 to 3 below).

Therefore, the compound (I) of the present invention or a pharmacologically acceptable salt thereof, even when the alkaline phosphatase has a high value (for example, a child in a bone growing stage, a pregnant woman and a liver disease), It is useful as an antitumor prodrug that can be safely used even when carboxypeptidase has a high value (for example, in pancreatic disease).

Test Examples The effects of the present invention will be described below with reference to test examples. In the test examples, the following abbreviations mean the following compounds. BAM-Tyr-P ... N- [pN, N-bis (2-chloroethyl) aminobenzoyl] -O-phosphono-L-tyrosine (Compound of Example 1) BAM-Tyr-P.3Na .. ... N- [pN, N-bis (2-chloroethyl) aminobenzoyl] -O-phosphono-L-tyrosine trisodium salt (compound of Example 2) MTX-Tyr-P ... N- [ N- (4-amino-4-deoxy
-10-Methylpteroyl) -α-L-glutamyl] -O-phosphono
-L- tyrosine (compound of Example 3) BAM-Tyr ... N- [pN, N-bis (2-chloroethyl) aminobenzoyl] tyrosine (compound of Reference Example 1) BAM-Tyr.Na.・ ・ ・ ・ ・ N- [pN, N-bis (2-chloroethyl) aminobenzoyl] tyrosine sodium salt (Compound of Reference Example 1) MTX-Tyr ・ ・ ・ ・ ・ N- [N- (4-amino- 4-deoxy-10-
Methylpteroyl) -α-L-glutamyl] -L-tyrosine (Compound of Reference Example 2) BAM ... Bustonic acid mustard MTX .... Methotrexate

Test Example 1 Hydrolysis Test 1) Test Material a) Test Compound BAM-Tyr-P.3Na (Compound of Example 2) MTX-Tyr-P (Compound of Example 3) b) Enzyme Solution Alkaline Phosphatase solution: Alkaline phosphatase manufactured by Sigma was adjusted to a concentration of 100 units / ml with 0.1 M Tris-HCl buffer (pH 7.2).

Carboxypeptidase A solution: Carboxypeptidase A manufactured by Sigma is diluted with 0.1 M Tris-hydrochloric acid buffer solution (pH 7.2) to prepare BAM-Tyr-P.
For the hydrolysis of 3Na, a concentration of 500 units / ml,
100 units / m for hydrolysis of MTX-Tyr-P
The one adjusted to a concentration of 1 was used. 2) Test method BAM-Tyr-P.3Na is a 0.1 M tris-hydrochloric acid buffer solution (pH 7.
In 2), MTX-Tyr-P was dissolved in 0.1 M Tris-hydrochloric acid buffer solution (pH 7.2) and the concentration was 0.5 m.
g / ml solutions were prepared respectively. These solutions 400
50 μl of alkaline phosphatase solution and 50 μl of 0.1 M Tris-HCl buffer (pH 7.2),
50 μl of carboxypeptidase A solution and 50 μl of 0.1M Tris-hydrochloric acid buffer (pH 7.2) or 50 μl of alkaline phosphatase solution and 50 μl of carboxypeptidase A solution were added, and then 30 ° C. at 37 ° C.
Incubated for minutes. 1.5 ml of 1N-
After the reaction was stopped by adding hydrochloric acid, the reaction product and the remaining test compound were analyzed by high performance liquid chromatography.

The conditions of high performance liquid chromatography are as follows. Column: Inertsil ODS-2 (GL Science Co., Ltd.), 4.6 × 150 mm Column temperature: 40 ° C. Detection method: UV absorption at 320 nm Moving layer: Methanol-water-acetic acid mixed solvent (BAM-T)
Mixed ratio of 60: 40: 2 for analysis of yr-P.3Na and its reaction product, mixed ratio of 20: 80: 2 for analysis of MTX-Tyr-P and its reaction product) Flow rate: 1 ml / Min.

The BAM-T of Example 1 was used as a standard substance for identifying and quantifying reaction products and unreacted test compounds.
yr-P, BAM-Tyr of Reference Example 1, known BAM,
MTX-Tyr-P of Example 3, MTX-T of Reference Example 2
yr and the known MTX were used. The retention time of each compound was BAM-Tyr-P (12.1.
Min), BAM-Tyr (5.6 min), BAM (7.8)
Min), MTX-Tyr-P (8.6 min), MTX-Ty
r (6.9 min) and MTX (2.9 min). 3) Test results BAM-Tyr-P.3Na or MTX-Tyr-
When each enzyme was allowed to act on P, the kind of compound detected from each reaction solution and its amount (percentage of detected compound,
The values in parentheses are shown in Table 1. Compounds other than the compounds listed in Table 1 were not detected or were in a negligible amount even if they were detected.

[0042]

[Table 1] Enacted enzyme ^* Test compound APase CPaseA APase + CPaseA MTX-Tyr-P MTX-Tyr MTX-Tyr-P + MTX MTX (100) (99.4: 0.6) (100) BAM-Tyr-P BAM-Tyr BAM-Tyr -P + BAM BAM + BAM-Tyr-P (100) (97.8: 2.2) (99.1: 0.9) ^* APase acted on alkaline phosphatase, CPaseA acted on carboxypeptidase A, and APase + CPaseA acted simultaneously on these enzymes. Indicates.

From the above test results, the following is clear. The compound of the present invention (MTX-Tyr-P, BAM-Tyr
-P) or a pharmacologically acceptable salt thereof is treated with alkaline phosphatase to almost completely hydrolyze the phosphate group, but the parent compound (MTX,
BAM) is not generated. When carboxypeptidase A is allowed to act on the compound of the present invention, it remains almost unchanged. Only when alkaline phosphatase and carboxypeptidase A are allowed to act on the compound of the present invention at the same time, parent compounds (MTX, BAM) which are antitumor agents are produced.

[Test Example 2] Antitumor effect and toxicity test of MTX-Tyr-P To confirm that MTX-Tyr-P exerts an antitumor effect by the actions of both alkaline phosphatase and carboxypeptidase A, In addition, MTX-Tyr
-P toxicity, MTX-Tyr-P alkaline phosphatase product toxicity and MTX-Tyr-P
In order to investigate the toxicity of the carboxypeptidase A product of E. coli, it was tested using a cultured cancer cell line. 1) Test material a) Test compound MTX-Tyr-P (compound of Example 3) MTX (control compound) b) Enzyme solution Alkaline phosphatase solution: Alkaline phosphatase manufactured by Sigma Co. is used in 10 mM HPES buffer solution (p).
(H7.2) -containing serum-free RPMI1640 medium (manufactured by Nissui Pharmaceutical Co., Ltd.) and adjusted to a concentration of 25 units / ml was used.

Carboxypeptidase A solution: Carboxypeptidase A manufactured by Sigma was added to 10 mM HEPES.
A serum-free RPMI1640 medium (manufactured by Nissui Pharmaceutical Co., Ltd.) containing a buffer solution (pH 7.2) and adjusted to a concentration of 50 units / ml was used.

C) Cancer cell lines: HeLa, KB and K
ATO III 2) Test method a) Preparation of test solution Test solution when enzyme is allowed to act: MTX-Tyr-P
Was dissolved in serum-free RPMI1640 medium (manufactured by Nissui Pharmaceutical Co., Ltd.) containing 10 mM HEPES buffer (pH 7.2) to prepare a solution having a concentration of 0.8 mg / ml. This 80
10 μl of alkaline phosphatase solution and 10 μl
Serum-free R containing mM HEPES buffer (pH 7.2)
10 μl of PMI1640 medium, 10 μl of carboxypeptidase A solution and 10 mM HEPES buffer (pH
10 μl of serum-free RPMI1640 medium containing 7.2),
Alternatively, add 10 μl of alkaline phosphatase solution and 10 μl of carboxypeptidase A solution, and add at 37 ° C.
Let react for minutes. The reaction solution was heat-treated at 90 ° C for 1 minute to inactivate the enzyme, and this was used as a test solution when the enzyme was allowed to act.

Test solution when no enzyme is allowed to act: MTX
-Tyr-P was added to 10 mM HEPES buffer (pH
It was dissolved in a serum-free RPMI1640 medium (manufactured by Nissui Pharmaceutical Co., Ltd.) solution containing 7.2) and the concentration was adjusted to 0.64 mg / ml, which was used as a test solution when the enzyme was not allowed to act. b) Measurement of toxicity Cells in HeLa, KB or KATOIII cell line culture dishes were dissociated with trypsin to give 10 mM HEPES.
Buffer solution (pH 7.2) and 10% fetal bovine serum (Hyclo
A cell suspension was prepared using a serum-free RPMI1640 medium solution containing Ne. 100 μl of this cell suspension (2000-3000 in terms of the number of cells) was put in each well of a 96-well microculture dish (Falcon 3075, Becton Dickinson). Each test solution is a cell culture solution (RP
MI1640 (manufactured by Nissui Pharmaceutical Co., Ltd.) was diluted 2-fold into each well, and the cells were cultured in a 37 ° C. carbon dioxide incubator (5% carbon dioxide concentration) at a sufficient humidity for 3 days.

Next, the living cells in the culture medium are
TT method (Green et al., J. Immunol. Methods, 70 , 257-268,
1984) to determine the toxicity to cells. That is, 10 μl of MTT solution (concentration, 5 mg / ml) was added to each culture and incubated at 37 ° C. for 4 hours to obtain 100 formazan produced from the MTT by the enzyme derived from living cells.
After dissolving with μl of dimethyl sulfoxide, the absorbance at 550 nm was measured with a spectrophotometer. The relationship between the dose of the test compound and the absorbance was plotted on a graph to determine the concentration of the test compound that suppressed the number of cultured cells to 50% (IC ₅₀ ).

For reference, methotrexate (M
A test solution of TX) was prepared in the same manner as 2) and a) above and tested according to b) above. 3) Test results Table 2 shows the test results (average value of the test results of two times).

[0050]

[Table 2] IC ₅₀ (pmol / ml) Enacted enzyme ^* HeLa strain KB strain No KATO III strain > 896 851> 896 APase 784 582> 896 Cpase 806 762> 896 APase + CpaseA 27 22 29 29 control (MTX IC ₅₀ ) 119 7 ^* Apase indicates that alkaline phosphatase, CPaseA indicates carboxypeptidase A, and APase + CPaseA indicates that these enzymes act simultaneously.

As shown in Table 2 above, when no enzyme was allowed to act on MTX-Tyr-P, when alkaline phosphatase was allowed to act, or carboxypeptidase A was used.
When each of the cells is acted on, the toxicity to each cell line is low. On the other hand, when alkaline phosphatase and carboxypeptidase A were allowed to act simultaneously on MTX-Tyr-P, a toxicity close to the cytotoxicity of MTX, that is, an antitumor effect was exhibited.

[Test Example 3] BAM-Tyr-P.3Na
Antitumor effect and toxicity test of 1) Test material a) Test compound BAM-Tyr-P.3Na (Compound of Example 2) BAM (Control compound) b) Enzyme solution Alkaline phosphatase solution: Alkaline phosphatase manufactured by Sigma Co., 10 mM A serum-free RPMI1640 medium (manufactured by Nissui Pharmaceutical Co., Ltd.) containing a HEPES buffer (pH 7.2) and adjusted to a concentration of 100 units / ml was used.

Carboxypeptidase A solution: Carboxypeptidase A manufactured by Sigma was added to 10 mM HEPES.
A serum-free RPMI1640 medium (manufactured by Nissui Pharmaceutical Co., Ltd.) containing a buffer solution (pH 7.2) and adjusted to a concentration of 500 units / ml was used.

C) Cancer cell line: HeLa 2) Test method Since the antitumor agent BAM shows extremely weak activity with respect to the KB strain and the KATO III strain, it was tested only with the HeLa strain. a) Preparation of test solution Test solution in the case of acting enzyme: In the same manner as in Test Example 2, BAM-Tyr-P.3Na was allowed to act on alkaline phosphatase, carboxypeptidase A or both of these enzymes, and then Centricon 10 ( Amic
on) to remove the enzyme, which was used as a test solution when the enzyme was allowed to act.

Test solution when no enzyme is allowed to act: BAM
-Tyr-P.3Na was dissolved in a serum-free RPMI1640 medium (manufactured by Nissui Pharmaceutical Co., Ltd.) solution containing 10 mM HEPES buffer (pH 7.2) to give a concentration of 0.64 mg / ml.
Was used as a test solution when no enzyme was allowed to act. b) Measurement of toxicity The measurement method is the same as the method described in Test Example 2. In addition,
For reference, BAM cytotoxicity was also tested. 3) Test results The results are shown in Table 3.

[0056]

[Table 3] Enacted enzyme ^* No IC ₅₀ (pmol / ml) for HeLa strain > 360 APase 202 Cpase> 360 APase + Cpase 126 Control (IC _{50 of} BAM) 129 ^* Apase is alkaline phosphatase, CPase A is carboxypeptidase A, Apase + CPaseA indicates that these enzymes acted simultaneously.

As shown in Table 3, BAM-Tyr-P.3Na
There was little effect on HeLa cells and the toxicity was low when the enzyme was not acted on or when carboxypeptidase A was acted on. When alkaline phosphatase was allowed to act, the cytotoxicity was slightly stronger than that when the enzyme was not applied, but the toxicity was lower than when alkaline phosphatase and carboxypeptidase A were acted simultaneously. On the other hand, when the alkaline phosphatase and carboxypeptidase A are simultaneously acted on, cytotoxicity is stronger, and BAM
It was comparable to cytotoxicity.

The results of Test Examples 1 to 3 above show that the compound of the present invention is activated and exhibits an antitumor effect only when both alkaline phosphatase and carboxypeptidase act together, and these enzymes act alone. When applied or when it does not act at all, it indicates low toxicity.

Accordingly, the compounds of the present invention can be administered only when co-administered with the cancer antibody-alkaline phosphatase complex and the cancer antibody-carboxypeptidase complex.
Since it is specifically activated in the vicinity of cancer cells, it can be advantageously used as a safe carcinostatic agent that does not show systemic toxicity. Of course, the compound of the present invention can also be used with a complex in which alkaline phosphatase and carboxypeptidase are bound to one cancer antibody.

[0060]

EXAMPLES The present invention will be described in detail below with reference to examples.

Example 1 N- [pN, N-bis (2-chloroethyl) aminobenzoyl]-
O-phosphono-L-tyrosine (Compound Ia) In the following steps (i) to (iv), N- [pN, N-bis (2-chloroethyl) aminobenzoyl] -O-phosphono-L-tyrosine was converted to Synthesized.

(I) Benzyl N-tert-butoxycarbonyl
-O-dibenzylphosphono-L-tyrosinate (Compound V
Synthesis of I) Benzyl N-tert-butoxycarbonyl-L-tyrosinate
(Fabwerke Hoechst AG, Brit.pat., 1201121, 7.92g,
0.021 mol) in anhydrous tetrahydrofuran (100 ml) with stirring under ice-cooling, sodium hydride (60 w / w% oily, 853 mg, 0.0
(21 mol) was added and the mixture was stirred for 20 minutes under ice cooling. Next, dibenzyl phosphorochloridate (Atherton et al., J. Chem. Soc., 2
20 , 1106-1111, 1948, 13.9g, 0.047mol) of carbon tetrachloride (5
The solution (0 ml) was added dropwise over 15 minutes under ice cooling, and after completion of the dropwise addition, the mixture was further stirred under ice cooling for 1 hour. Water (10 ml) was added to the reaction solution, and the solvent was evaporated under reduced pressure. Ethyl acetate (200 m
l) was added, and the mixture was washed successively with water, 1N-hydrochloric acid, water, saturated aqueous sodium hydrogen carbonate solution, water and saturated brine, and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography [developing solvent: n-hexane-ethyl acetate (7: 3)] to obtain 10.44 g (yield 78%) of the title compound as a colorless oil. . ¹ H-NMR (60MHz, CDCl ₃ )
δ: 1.39 (9H, s), 3,04 (2H, d, J = 6.0Hz), 4.3-4.9 (1H, m), 5.0
3 (2H, s), 5.11 (2H, s), 5.17 (2H, s), 6.97 (4H, s), 7.31 (15H,
s).

(Ii) benzyl O-dibenzylphosphono-L-
Synthesis of tyrosinate (compound VII) hydrochloride Benzyl N-tert-butoxycarbonyl-O-dibenzylphosphono-L-tyrosinate (compound VI) (10.44g, 0.017
mol) to 4N-HCl / dioxane solution (60 ml), and under ice cooling
Stir for 1.5 hours. After distilling off the solvent under reduced pressure, ether
(50 ml) was added and the mixture was allowed to stand at room temperature overnight, and the precipitated crystals were collected by filtration to give the title compound (7.41 g, yield 79%). ¹ H-NMR (60MHz, CDCl ₃ ) δ: 3.0-3.7 (2H, br), 4.1-4.8 (1H, b
r), 4.8-5.5 (6H, m), 6.9-7.8 (19H, m), 8.4-9.3 (3H, br).

(Iii) Benzyl N- [pN, N-bis (2-chloroethyl) aminobenzoyl] -O-dibenzylphosphono-L-
Synthesis of tyrosinate (Compound II) p- [N, N-bis (2-chloroethyl) amino] benzoic acid (Eld
erfield et al., J. Org. Chem., 26 , 4996-4997,1961, 0.5
g, 1.9 mmol) was placed in a flask, dissolved in anhydrous toluene (2 ml), and thionyl chloride (1 ml) was added. Then at 85 ℃ 25
The mixture was stirred for a minute, the solvent was distilled off under reduced pressure, and p- [N, N-bis (2-
Chloroethyl) amino] benzoic acid chloride was obtained. Anhydrous tetrahydrofuran (5 ml) was added to and dissolved in this, and benzyl O-dibenzylphosphono-L-tyrosinate hydrochloride (1
g, 1.9 mmol) and triethylamine (1 ml) were added, and the mixture was stirred at room temperature for 1 hr. The reaction solution was diluted with 1N-hydrochloric acid and then extracted with ethyl acetate. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was evaporated. The obtained residue was purified by silica gel column chromatography [developing solvent: chloroform-methanol (19: 1)] to obtain 1.28 g (yield 87%) of the title compound as a colorless oil. ¹ H-NMR (300MHz, CDCl ₃ ) δ: 3.07 (2H, m), 3.63 (4H, t), 3.76
(4H, t), 5.08 (1H), 5.17 (6H), 6.50 (1H, d, J = 7.43Hz), 6.91
(2H, d, J = 8.57Hz), 6.99 (2H, d, J = 8.07Hz), 7.31 (15H, br),
7.64 (2H, d, J = 8.88Hz).

(Iv) N- [pN, N-bis (2-chloroethyl) aminobenzoyl] -O-phosphono-L-tyrosine (compound I-
Synthesis of a) benzyl N- [pN, N-bis (2-chloroethyl) aminobenzoyl] -O-dibenzylphosphono-L-tyrosinate (0.74
g, 0.95 mmol), and 10% palladium-carbon (35 mg)
Was placed in a flask, and methanol (10 ml) was added. The inside of the flask was replaced with hydrogen, and the mixture was stirred under a hydrogen atmosphere at room temperature for 1 hour. After filtering the reaction solution, the solvent was distilled off under reduced pressure,
430 mg (92% yield) of the title compound was obtained. ¹ H-NMR (300MHz, DMSO-d ₆ ) δ: 3.04 (2H, m), 3.75 (8H, m), 4.
54 (1H, m), 6.78 (2H, d, J = 8.96Hz), 7.05 (2H, d, J = 7.73Hz),
7.26 (2H, d, J = 8.51Hz), 7.71 (2H, d, J = 8.86Hz), 8.37 (1H, d,
J = 8.11Hz).

Example 2 N- [pN, N-bis (2-chloroethyl) aminobenzoyl]-
O-phosphono-L-tyrosine trisodium salt (compound I-
a sodium salt of a) N- [pN, N-bis (2-chloroethyl) aminobenzoyl]-
O-phosphono-L-tyrosine (431 mg) was dissolved in methanol (10 ml), and 1N-sodium hydroxide (3 ml) was added. The solvent was evaporated, and the obtained residue was mixed with water-methanol mixed solvent (1:
Recrystallization from 1) gave 396 mg (yield 82%) of the title compound as a white powder. Elemental analysis (as _{C 20 H 21 N 2 O 7} Cl 2 PNa 3 · 2H 2 O): Calculated (%) C, 39.50; H , 4.14; N, 4.61 Found (%) C, 39.45; H , 3.98; N, 4.42

Example 3 N- [N- (4-amino-4-deoxy-10-methylpteroyl) -α
-L-glutamyl] -O-phosphono-L-tyrosine (compound I-
b) N- [N- (4-amino-4-deoxy-10-methylpteroyl) -α-L-glutamyl] -O-phosphono-L-tyrosine was added in the following steps (i) to (iii). Synthesized.

(I) Dibenzyl N- (N-tert-butoxycarbonyl-α-L-glutamyl) -O-dibenzylphosphono-L-
Chiroshineto (Compound XI) Synthesis of γ- benzyl N-tert-butoxycarbonyl -L- glutamate (compound X, CH Li et al., J.Org.Chem., 28, 178-
181,1963, 2.49g, 7.4mmol) of tetrahydrofuran (40
(ml) solution under stirring at -15 ° C with triethylamine (1.64 g, 16.
2mmol) and isobutyl chloroformate (1.11g, 8.1mmo
l) was added and the mixture was stirred at -15 ° C for 15 minutes. Then benzyl O-
Dibenzylphosphono-L-tyrosinate hydrochloride (compound V
II (4.19 g, 7.4 mmol) was added, the reaction temperature was raised from -15 ° C to room temperature over 3 hours with stirring, and the mixture was further stirred at room temperature for 18 hours. The solvent was distilled off under reduced pressure, and ethyl acetate (70 ml) was added to the residue.
Was added, and the mixture was washed successively with water, 1N-hydrochloric acid, water, saturated aqueous sodium hydrogen carbonate solution and saturated brine, and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography [developing solvent: n-hexane-ethyl acetate (6: 4)] to give the title compound as a colorless oil.
3.84 g (yield 61%) was obtained. ¹ H-NMR (60MHz, CDCl ₃ ) δ: 1.40 (9H, s), 1.7-2.7 (4H, m), 3.0
2 (2H, d, J = 6.0Hz), 3.9-4.5 (1H, m), 4.6-5.3 (1H, m), 5.00 (2
H, s), 5.06 (4H, s), 5.15 (2H, s), 6.97 (4H, s), 7.28 (20H, s).

(Ii) Dibenzyl N- [N- [4- [N- (benzyloxycarbonyl) methylamino] benzoyl] -α-L-
Synthesis of Glutamyl] -O-dibenzylphosphono-L-tyrosinate (Compound XIII) Dibenzyl N- (N-tert-butoxycarbonyl-α-L-glutamyl) -O-dibenzylphosphono-L-tyrosinate (1.9
4N-HCl / dioxane solution (20 ml) was added to 4 g, 2.3 mmol,
The mixture was stirred under ice cooling for 1 hour. The solvent was distilled off under reduced pressure to obtain 1.80 g (yield 100%) of dibenzyl N-α-L-glutamyl-O-dibenzylphosphono-L-tyrosinate hydrochloride (Compound XII).

This compound (1.80 g, 2.3 mmol) was dissolved in methylene chloride (30 ml), and diisopropylethylamine (0.88 g, 6.8 mmol) and 4-[(benzyloxycarbonyl) methylamine] benzoyl chloride were stirred under ice cooling. (Piper et a
L., J. Med. Chem., 25 , 182-187, 1982, 0.69 g, 2.3 mmol) was added, and the mixture was stirred for 45 minutes under ice cooling. The solvent was evaporated under reduced pressure, ethyl acetate (100 ml) was added to the residue, and the mixture was washed successively with water, 1N-hydrochloric acid, water, saturated aqueous sodium hydrogen carbonate solution and saturated brine, and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography [developing solvent: chloroform-methanol (100: 1) then chloroform-methanol (50: 1)] to give the title compound.
1.29 g (yield 55%) was obtained. ¹ H-NMR (60MHz, CDCl ₃ ) δ: 1.9-2.8 (4H, m), 2.9-3.2 (2H, m),
3.33 (3H, s), 4.5-5.4 (12H, m), 6.99 (4H, s), 7.0-8.0 (4H,
m), 7.35 (25H, s).

(Iii) N- [N- (4-amino-4-deoxy-10-
Methylpteroyl) -α-L-glutamyl] -O-phosphono-L-
Synthesis of Tyrosine (Compound Ib) Dibenzyl N- [N- [4-[(benzyloxycarbonyl) methylamino] benzoyl] -α-L-glutamyl] -O-dibenzylphosphono-L-tyrosinate (290 mg, 0.28 mmol) to 10%
Palladium-carbon (25 mg) and methanol (15 ml) were added, and the mixture was stirred under a hydrogen atmosphere at room temperature for 2 hours. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to give N- [N- [4- (methylamino) benzoyl] -α-L-glutamyl] -O-phosphono-L-tyrosine (Compound VIII). This was dissolved in anhydrous N, N-dimethylacetamide (3 ml), and 6- (bromomethyl) -2,4-pteridinediamine hydrochloride (Piper et al., J.
Org.Chem., 42 , 208-211, 1977, 96 mg, 0.29 mmol),
The mixture was stirred at room temperature for 14 hours and further at 55 ° C for 10 hours. The solvent was distilled off under reduced pressure, and the residue was purified by preparative high performance liquid chromatography [column: YMC-Pack ODS (20x250mm), solvent: methanol-water-acetic acid (20: 80: 2)] and then with methanol. The crystals were washed to obtain 93 mg (yield 47%) of the title compound as a yellow powder. Melting point 195-220 ° C (decomposition) ¹ H-NMR (300MHz, DMSO-d ₆ + D ₂ O) δ: 1.7-2.1 (2H, m), 2.20 (2
H, t, J = 7.3Hz), 2.3-3.2 (2H, m), 3.25 (3H, s), 4.3-4.5 (2H,
m), 4.87 (2H, s), 6.82 (2H, d, J = 8.8Hz), 6.96 (2H, d, J = 8.0H
z), 7.04 (2H, d, J = 8.0Hz), 7.70 (2H, d, J = 8.8Hz), 8.58 (1H,
s). Elemental analysis values (as C ₂₉ H ₃₂ N ₉ O ₁₀ P ・ 5H ₂ O): Calculated value (%) C, 48.07; H, 4.87; N, 17.40 Measured value (%) C, 48.31; H, 4.81; N, 17.49

Reference Example 1 N- [pN, N-bis (2-chloroethyl) aminobenzoyl]
Tyrosine N- [pN, N-bis (2-chloroethyl) aminobenzoyl] tyrosine was synthesized by steps (1) and (2) below.

(1) Synthesis of benzyl N- [pN, N-bis (2-chloroethyl) aminobenzoyl] tyrosinate p- [N, N-bis (2-chloroethyl) amino] benzoic acid (0.5
g, 1.9 mmol) was placed in a flask and dissolved in anhydrous toluene (2 ml). Thionyl chloride (1.1 ml, 15 mmol) in this solution
Was added and the mixture was heated with stirring at 80 ° C. for 30 minutes. The solvent was evaporated under reduced pressure, and the residue was dissolved in anhydrous tetrahydrofuran (5 ml).
Benzyl-L-tyrosinate (590 mg, 1.9 mmo
l) and triethylamine (0.5 ml) were added, and the mixture was stirred at room temperature for 1 hr. 1N-hydrochloric acid was added to the reaction solution, the mixture was extracted with ethyl acetate, the organic layer was washed with saturated brine, and dried over anhydrous sodium sulfate. The solvent was evaporated, the residue was purified by silica gel column chromatography [developing solvent: chloroform-methanol (19: 1)], and the title compound was obtained as a colorless oil.
0.87 g (yield 87%) was obtained. ¹ H-NMR (300MHz, CDCl ₃ ) δ: 3.12 (2H, m), 3.61 (4H, t), 3.75
(4H, t), 5.07 (1H, t), 5.17 (2H, q), 6.56 (1H, d, J = 7.82Hz),
6.62 (2H, d, J = 7.85Hz), 6.66 (2H, d, J = 7.85Hz), 6.84 (2H, d,
J = 8.44Hz), 7.35 (5H, br), 7.63 (2H, d, J = 8.87Hz).

(2) Synthesis of N- [pN, N-bis (2-chloroethyl) aminobenzoyl] tyrosine Benzyl N- [pN, N-bis (2-chloroethyl) obtained above
Aminobenzoyl] tyrosinate (0.8 g, 1.6 mmol) and 10% palladium-carbon (80 mg) were placed in a flask and methanol (10 ml) was added. Replace the inside of the flask with hydrogen,
The mixture was stirred under a hydrogen atmosphere at room temperature for 1 hour. The reaction solution was filtered and the solvent was distilled off under reduced pressure to obtain 613 mg of the title compound (yield: 92
%) Obtained. ¹ H-NMR (300MHz, DMSO-d ₆ ) δ: 2.98 (2H, m), 3.76 (8H, m), 4.
49 (1H, m), 6.64 (2H, d, J = 8.42Hz), 6.78 (2H, d, J = 8.98Hz),
7.09 (2H, d, J = 8.44Hz), 7.71 (2H, d, J = 8.86Hz), 8.28 (1H, d,
J = 8.11Hz, -CONH), 9.18 (1H, br, -OH), 12.6 (1H, br, -COOH). This compound (610 mg) was dissolved in methanol (10 ml) to give 1N.
-Sodium hydroxide aqueous solution (1.5 ml) was added, the solvent was evaporated, and the residue was recrystallized from water-methanol (1: 1) to obtain 520 mg (yield 75%) of this sodium salt as a white powder. Elemental analysis value (as C ₂₀ H ₂₁ N ₂ O ₄ Cl ₂ Na ・ H ₂ O): Calculated value (%) C, 51.63; H, 4.98; N, 6.02 Measured value (%) C, 51.63; H, 4.94 ; N, 5.95

Reference Example 2 N- [N- (4-amino-4-deoxy-10-methylpteroyl) -α
-L-glutamyl] -L-tyrosine In the steps (1) to (3) below, N- [N- (4-amino-4-deoxy
-10-Methylpteroyl) -α-L-glutamyl] -L-tyrosine was synthesized.

(1) Synthesis of dibenzyl N- (N-tert-butoxycarbonyl-α-L-glutamyl) -L-tyrosinate γ-benzyl N-tert-butoxycarbonyl glutamate
(1.44g, 4.3mmol) in tetrahydrofuran (25ml) solution-
While stirring at 15 ° C, triethylamine (0.95g, 9.4mmol) and isobutyl chloroformate (0.58g, 4.2mmol) were added, and the mixture was stirred at -15 ° C for 15 minutes. Next, benzyl L-tyrosinate p-toluenesulfonate (1.89 g, 4.3 mmol) was added, the reaction temperature was raised from -15 ° C to room temperature over 1 hour with stirring, and the mixture was further stirred at room temperature for 30 minutes. The solvent was evaporated under reduced pressure, ethyl acetate (50 ml) was added to the residue, washed successively with water, 0.5N-hydrochloric acid, water, saturated aqueous sodium hydrogen carbonate solution and saturated brine, and dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography [developing solvent: n-hexane-ethyl acetate (6: 4) and then n-hexane-ethyl acetate (1: 1)] to give a colorless oil. 2.18 g (yield 87%) of the title compound was obtained. ¹ H-NMR (60MHz, CDCl ₃ ) δ: 1.39 (9H, s), 1.7-2.8 (4H, m), 2.8
-3.2 (2H, m), 4.0-4.5 (1H, m), 4.6-5.1 (1H, m), 5.06 (2H, s),
5.08 (2H, s), 5.55 (1H, d, J = 8Hz), 6.65 (2H, d, J = 9Hz), 6.87
(2H, d, J = 9Hz), 6.9-7.4 (1H, m), 7.30 (10H, s), 7.51 (1H,
s). (2) Synthesis of dibenzyl N- [N- (4-amino-4-deoxy-10-methylpteroyl) -α-L-glutamyl] -L-tyrosinate Dibenzyl N- (N-tert-butoxycarbonyl-α- L-glutamyl) -L-tyrosinate (727 mg, 1.2 mmol) in 4N-HCl /
Dioxane (10 ml) was added, and the mixture was stirred under ice cooling for 1 hour and 20 minutes. The solvent was distilled off under reduced pressure to obtain dibenzyl N-α-L-glutamyl-L-tyrosinate hydrochloride.

Diethylphosphonocyanidate (547 mg, 3.
4 mmol) in dimethylformamide (20 ml) and triethylamine (340 mg, 3.4 mmol) and 4-amino-4-deoxy-10-methylpteroic acid (Martinelli et al., J. Med. Chem., 22 , 869-
874, 1979, 364 mg, 1.1 mmol), and then at room temperature for 10 minutes,
The mixture was stirred at 80 ° C for 2 minutes. Next, triethylamine (125m
(g, 1.2 mmol) and the previously obtained dibenzyl N-α-L-glutamyl-L-tyrosinate hydrochloride were added, and the mixture was stirred at 80 ° C for 4 hours. The solvent was distilled off under reduced pressure, and chloroform (60 ml) was added to the residue.
And methanol (3 ml) were added, the mixture was washed with saturated aqueous sodium hydrogen carbonate solution, and dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and the residue was subjected to silica gel column chromatography [developing solvent: chloroform-methanol (10: 1).
], Followed by crystallization with methanol to give the title compound 3
44 mg (yield 39%) was obtained. ¹ H-NMR (300MHz, DMSO-d ₆ + D ₂ O) δ: 1.8-2.1 (2H, m), 2.38 (2
H, t, J = 7.5Hz), 2.8-3.0 (2H, m), 3.23 (3H, s), 4.3-4.5 (2H,
m), 4.81 (2H, s), 5.03 (2H, s), 5.05 (2H, s), 6.61 (2H, d, J = 8.
5Hz), 6.82 (2H, d, J = 8.9Hz), 6.96 (2H, d, J = 8.5Hz), 7.1-7.4
(10H, m), 7.70 (2H, d, J = 8.9Hz), 8.58 (1H, s).

(3) Synthesis of N- [N- (4-amino-4-deoxy-10-methylpteroyl) -α-L-glutamyl] -L-tyrosine Dibenzyl N- [N- (4-amino- 4-Deoxy-10-methylpteroyl) -α-L-glutamyl] -L-tyrosinate (130 mg,
0.16 mmol) in dimethylformamide (3 ml) in water (6 m
l) and barium hydroxide octahydrate (103 mg, 0.33 mmol) were added, and the mixture was stirred at room temperature for 16 hours. The solvent was distilled off under reduced pressure,
Water (8 ml) and sodium sulfate (46 mg, 0.32 mmol) were added to the residue, and the mixture was stirred at room temperature for 30 minutes. Insoluble matter is filtered off, and the filtrate is
The mixture was acidified by adding 10% acetic acid and left in the refrigerator for 3 days.
The precipitated solid was collected by filtration to give the title compound as a yellow powder (95%).
mg (yield 94%) was obtained. Melting point 190-210 ° C (decomposition) ¹ H-NMR (300MHz, DMSO-d ₆ + D ₂ O) δ: 1.8-2.1 (2H, m), 2.29 (2
H, t, J = 7.6Hz), 2.7-3.0 (2H, m), 3.23 (3H, s), 4.3-4.5 (2H,
m), 4.83 (2H, s), 6.62 (2H, d, J = 8.5Hz), 6.84 (2H, d, J = 9.0H
z), 7.00 (2H, d, J = 8.5Hz), 7.70 (2H, d, J = 9.0Hz), 8.56 (1H,
s) .Elemental analysis value (as C ₂₉ H ₃₁ N ₉ O ₇・ 1.5H ₂ O): Calculated value (%) C, 54.03; H, 5.32; N, 19.56 Measured value (%) C, 54.31; H, 5.31; N, 19.62

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI technical display area C07K 5/06 8318-4H

Claims (3)

[Claims] 1. The following formula (I): (In the formula, R is Or [Chemical 3] Indicates. ) Or a pharmacologically acceptable salt thereof. 2. The following formula (II): (In the formula, Bzl represents a benzyl group.) A compound represented by the following formula (I-
a) A method for producing the compound represented by or a pharmaceutically acceptable salt thereof. 3. The following formula (III): And a compound represented by the following formula (IV): (Wherein, X represents a halogen atom) is reacted with a compound represented by the following formula (Ib): A method for producing the compound represented by or a pharmaceutically acceptable salt thereof.