JPS6310799A - Prodrug compound, production thereof and long-acting drug containing same - Google Patents

Abstract

NEW MATERIAL:The compound of formula I (A is residue of pharmaceutical compound containing >NH or -NH2 group in the molecule; R is residue of steroid compound; m is 1-5; n is 0 or 1) and its salt. EXAMPLE:[ 1aS-(1aalpha,8beta,8aalpha,8balpha) ]-6-amino-8-carbamoyloxymethyl-N'-[N-(cholest- 5-ene-3beta-oxycarbonyl)glycyl]-1,1a,2,8,8a,8b-hexahydro-8a-methoxy-5- methylaziridi no[2',3':3,4]pyrrolo[1,2-a]indole-4,7-dione. USE:Antitumor agent. PREPARATION:The compound of formula II or its reactive derivative at the carboxyl group or their salt is made to react with the compound of formula A-H or its salt.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] This invention relates to a novel prodrug compound for pharmaceuticals, a method for producing the same, and a long-acting preparation containing the same.

[Conventional techniques - $1 So-called prodrugs with various intramolecular modifications have been studied for the purpose of sustained release of drugs in vivo. For example, 5-fluorouracil, an anti-inflammatory agent, and cholesterol It is known that by synthesizing a prodrug bound via a spacer and incorporating it into ribosomes, it can be gradually converted into the crude compound 5-fluorouracil in a buffer solution (Pharmaceutical Society of Japan Vol.
2005 Meeting Lecture Abstracts (1985), Presentation No., 5%J
1-24, Title: 5 using cholesterol as a carrier
-Synthesis of FU prodrug and its application to lipid dispersion formulations).

Regarding mitomycin C, an antitumor agent, attempts have also been made to synthesize cholesteryloxycarbonylmitomycin C, a mitomycin C derivative bound to cholesterol via a spacer [-CO-], in the same manner as above [Chemical & Co., Ltd. Pharmaceutical Bulletin (Chew.

Pharm, Bull, ) 31 (11)
4083-4090 (1983) Ko.

[Problems to be Solved by the Invention] The above-mentioned mitomycin C derivatives have the problem that they can hardly be converted into the parent compound mitomycin C due to the stability of the bond between mitomycin C and cholesterol against chemical and oxygen hydrolysis. There was a point.

[Means for Solving the Problems] As a result of intensive research aimed at overcoming the above-mentioned problems, the inventors of the present invention combined mitomycin C with a steroid compound via various novel spacers. By synthesizing the derivative, when this prodrug is administered to a living body, the parent compound mitomycin C is released in the blood.
By creating a ribosomal formulation in which this prodrug is incorporated into ribosomes, the incorporation of the derivative into ribosomes reduces the conversion rate to mitomycin C, and as a result, mitomycin C
It has been found that the blood concentration of the compound can be maintained for a long period of time, and toxicity can also be reduced compared to when the crude compound itself is administered.

Furthermore, the present inventors applied this technique to 4-
Formyl-6,9-dihydroxy-14-year-old xar 1.
11-Diazatetracyclo-2,710,12[7,4,1,0,0]tetrazocar 2.4゜6-drien-8-ylmethylcarbamate (hereinafter referred to as FR900
482 substance), and bis(2-
It was discovered that a similar effect can be obtained by applying it to chloroethyl)amine (hereinafter referred to as nitrogen mustard), and furthermore, it was found that a similar effect can be obtained by applying it to 1-β-D-arabinofuranosylcytosine (
As a result of applying the present invention to cytarabine (hereinafter referred to as cytarabine), the present invention was completed based on the discovery that, in addition to the above-mentioned effects, the anti-malignant tumor effect was significantly improved.

The prodrug compounds of this invention are novel and have the general formula % formula %: ] (wherein A is a group in the molecule) NH or -NH2, a residue of a pharmaceutical compound, R is a residue of a steroid compound, m is an integer from 1 to 5, and n means 0 or 1, respectively).

In the prodrug compound [1], “
Examples of the pharmaceutical compound in the residue j of the pharmaceutical compound having a group NH in the molecule include antitumor agents such as mitomycin C, nitrogen mustard, and substance R900482. Examples of the pharmaceutical compound "residue of a pharmaceutical compound having -NH2 group in one molecule" include cytarabine, aminopterin, azacytidine, doxorubicin, daunorubicin, and the like. The term "residue of these pharmaceutical compounds" means a residue obtained by removing a hydrogen atom from the group >> or -NH2 in the molecule of the pharmaceutical compound.

Further, examples of the residue of the steroid compound include a group obtained by removing a hydrogen atom from the hydroxyl group at the 3-position of a steroid compound such as cholesterol, furestanol, lanosterol, ergosterol, and lidofolic acid.

The prodrug compound [1] of this invention and its salts can be produced by one of the following methods.

A-H or a reactive derivative thereof at the carboxy group or a salt thereof A-CO(CH2)rn(NHCO)n-R[I or a salt thereof (wherein A, R, m and n are each as defined above) In other words, the prodrug compound [1] and its salts can be produced by reacting the compound [I] or its reactive derivative at the carboxy group or its salt with the compound [I] or its salt. .

Suitable salts of compounds [rl, [1] and [II[] include organic acid salts such as acetate, maleate, tartrate, methanesulfonate, benzenesulfonate, toluenesulfonate, etc. Acid addition salts such as inorganic acid salts such as hydrochlorides, hydrobromides, sulfates, phosphates; metal salts such as sodium, potassium, calcium, and magnesium salts; ammonium salts; trimethylamine salts, triethylamine salts , organic amine salts such as dicyclohexylamine salts, and the like.

Suitable reactive derivatives of the carboxy group of compound [1[] include acid halides, acid anhydrides, activated amides, activated esters, and the like.

Suitable examples include acid chlorides, acid azides, substituted phosphoric acids such as dialkyl phosphoric acids, phenyl phosphoric acids, and halogenated phosphoric acids; aliphatic carboxylic acids such as pivalic acid and pentanoic acid; and aromatic carboxylic acids such as benzoic acid. : mixed acid anhydrides with acids such as riluponic acid; symmetrical acid anhydrides; activated amides with imidazole, 4-substituted imidazole, dimethylpyrazole, triazole or tetrazole; activated esters such as cyanomethyl ester, methoxymethyl ester, etc. , N
, N-dimedylhydroxyamine, 1-hydroxy-2
-(IH)-pyridone, N-hydroxysuccinimide, N-hydroxyphthalimide, 1-hydroxy-6-
Examples include esters with N-hydroxy compounds such as chloro-IH-benzotriazole. These reactive derivatives can be arbitrarily selected from them depending on the type of compound [11] to be used.

The reaction usually involves water, methanol, ethanol, acetone,
The reaction is carried out in a conventional solvent such as dioxane, acetonitrile, chloroform, methylene chloride, ethylene chloride, tetrahydrofuran, ethyl acetate, N,N-dimethylformamide, pyridine, but any solvent that does not adversely affect the reaction may be used. , or any other solvent. These conventional solvents may be used in combination with water.

When compound [I[] is used in the reaction in the form of free acid or salt, N,N'-dicyclohexylcarbodiimide;N-cyclohexyl-N'-morpholinoethylcarbodiimide-N'-(4- diethylaminocyclohexyl)carbodiimide, N. N'-diethylcarbodiimide;N-ethyl-N'-(3-dimethylaminepropyl)
Carbodiimide; 1-ethyl-3-(3-dimethyl)
Carbodiimide hydrochloride, N. N-carbonylbis(2-
Methylimidazole); trialkyl phosphite: polyethyl phosphate; phosphorus oxychloride (phosphoryl chloride); thionyl chloride, oxalyl chloride; 2-ethyl-7-hydroxybenzisooquinazolium salt; 1-(1)-chlorobenzene sulfate ;1)-6-chloro-IH-benzotriazole; produced by the reaction of dimethylformamide with thionyl chloride, phosgene, phosphorus oxychloride, etc. in the presence of a conventional condensing agent such as the so-called Vilsmeier reagent, etc. It is preferable to do this.

This reaction also applies to alkali metal bicarbonates, tri(lower)alkylamines (e.g., trimethylamine, triethylamine, etc.), pyridine, N-(lower)alkylmorpholines, N,N-di(lower)alkylbenzylamines, etc. The reaction temperature, which may be carried out in the presence of an inorganic or organic base, is not particularly limited, and the reaction is usually carried out under cooling or at room temperature.

target compound CI] and starting compounds [1] and [1
1 based on asymmetric carbon atoms, double bonds, etc. in the molecule in 111
When stereoisomers such as optical isomers or geometric isomers are included, the compounds [I], [I[] and [1+
1] as well as mixtures thereof are intended to be included within the scope of this invention.

The prodrug compound [1] of this invention and its salts are present in the blood as mitomycin C, FR900482 substance,
These parent compounds are converted into parent compounds such as nitrogen mustard or cytarabine, which have antitumor effects and are effective against various cancers (e.g., gastric cancer, lung cancer, lung adenocarcinoma, liver cancer, rectal cancer, pancreatic cancer, etc.). It is useful in the treatment of breast cancer, uterine cancer, etc.), leukemia (eg lymphocytic leukemia, myeloid leukemia, etc.), and the like.

The prodrug compound [11] of the present invention is prepared by a conventional method into a solubilized preparation, an emulsion preparation, a ribosome preparation, etc., and then administered by injection (e.g., intravenous injection, intramuscular injection, intratumoral injection, etc.), oral administration, or direct administration. It is administered into the living body by administration, etc. Then, it is gradually converted into the parent compound (for example, mitomycin C%FR900482 substance, nitrogen mustard, or cytarabine) in the blood, and the blood concentration of the compound is maintained for a long time compared to when the crude compound itself is administered. Moreover, it can improve the antitumor effect and reduce toxicity. Furthermore, among the various preparations mentioned above, especially when used as a ribosome preparation, the blood concentration of R-a can be maintained the highest.

Phospholipids used as membrane materials when manufacturing ribosome preparations include those derived from egg yolk, soybeans, and other animal tissues, such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, and sphingomyelin, and mixtures thereof. Examples include synthetic lecithins such as egg yolk lecithin, soybean lecithin, dipalmitoyl lecithin, and distearoyl lecithin. These phospholipids are supplemented with the usual additives such as cholesterol, dicetyl phosphate or alpha
- Tofuferol and the like may be added as appropriate.

Ribosome preparations can be produced by known methods. That is, the prodrug compound [! Dissolve Co or its salts and phospholipids in a suitable solvent such as chloroform, methanol, or ethanol, place this in a suitable container, and remove the solvent under reduced pressure. It can be produced by adding an aqueous solution (for example, phosphate buffered saline, etc.), shaking and solubilizing it, and then removing the surfactant from this solution using a surfactant removal device.

In addition, after dissolving phospholipids in an organic solvent such as chloroform or ethanol, placing the solution in a suitable container, and distilling off the solvent under reduced pressure to form a thin film of phospholipids on the inner surface of the container,
It can also be produced by adding an aqueous solution of the prodrug compound [11 or a salt thereof, followed by shaking or ultrasonication.

Furthermore, they can be produced by conventional methods such as ether injection, and the method for producing ribosome preparations is not particularly limited.

The prodrug compound [1] of this invention and its salts are usually administered at a unit dosage of 1 mg = 1000 mg once a day.
Although the drug is administered 4 times, the dose may be increased or decreased as appropriate depending on the type of parent compound, patient's age, weight, symptoms, administration method, co-administration with other anti-Ilium agents, etc. In addition, in the case of injection administration,
The above dosages may be administered, for example, once or twice a week or over a period of 1 to 3 weeks.

[Effect of the invention] The effects of the present invention will be explained below using test examples.

1 Trial 1 A chloroform solution containing 84 μmole of egg yolk lecithin, 36 μmole of egg yolk soup, 36 μmole of ingomyelin, and 6 μmole each of the mitomycin C prodrug obtained in Example 1 or Example 2 described later was dispensed into a round bottom flask. , chloroform was distilled off. After adding 258 mg of sodium chlorate and 4.8 mg of pH 7.4 phosphate-strengthened saline (hereinafter referred to as PBS), it was further shaken to obtain a purple clear liquid. Sodium cholate was removed from this solution using a drug removal device (trademark, manufactured by Dianorm) to obtain a ribosome suspension containing mitomycin C prodrug.

On the other hand, a chloroform solution containing 70 μmole of egg yolk cytin, 730 II mole of egg yolk sphingomylic acid, and 5 μmole of the prodrug of FR900482 substance (Example 6) was dispensed into a round bottom flask, and the FR900482 substance was prepared in the same manner as described above. A prodrug-containing ribosome suspension was obtained.

The above two types of mitomycin C prodrug-containing ribosome suspensions were each diluted 2.5 times with PBS. Further, the ribosome suspension containing the FR900482 substance prodrug has a FR900482 substance prodrug concentration of 0.
Dilute with PBS to 5μmole/-, ml
lax-GV (trademark, manufactured by Millibore) filter (
0.22μ).

Mouse, rat and human serum (4,51+111
．． After adding the above ribosome suspension and 0.511111 of an ethanol solution of each prodrug to the solution at 37°C, samples were taken over time, and the prodrug concentration in the sample was determined by high performance liquid chromatography.

The conversion half-life (t) of each prodrug to the parent compound in various serums was calculated from the remaining amount of each prodrug at each time point.

Ward! Exhibit 1 Table 1 shows the conversion half-life (in t) of each prodrug to the parent compound in various sera.

Rooster-1 From the above test results, it is clear that the prodrug of this invention
It can be seen that it is converted to the parent compound in vitro blood and that the incorporation of each prodrug into ribosomes increases its conversion half-life.

Conversion test 2 to compound A chloroform-methanol mixture (8:2 v/v) containing 70 μmoles of egg yolk lecithin, 30 μmol of egg yolk sphingomyelin, and 30 μmol of the cytarabine prodrug (Example 8) obtained in Example 8 below was added to a round bottom. The mixture was fractionated into a flask, and the solvent was distilled off. Add to this 53.8 mg of sodium cholate and 5 PBS, Qml! A colorless and clear liquid was obtained after adding the turmeric.

The sodium cholate was then removed using riboprebs to obtain a ribosome suspension containing the cytarabine prodrug. Ribosome-suspended Ar& has a drug concentration of 5 μmo
Dilute with PBS to give le/mQ, 0.2
．． 0.1 and 0.08μ polycarbonate membranes (manufactured by Euclepore), followed by ml
Filtered through ex-GV filter (0.22μ).

After adding 0.08 mQ of the above ribosome suspension and cytarabine prodrug ethanol solution to rat serum (4.0 mQ, 37°C), samples were taken over time, and the cytarabine prodrug concentration in the sample was measured by high performance liquid chromatography. It was quantified by

The half-life of conversion to the parent compound (< ) was calculated from the remaining amount of cytarabine prodrug at each time point.

Ward! Table 2 shows the conversion half-life (numbered) from the prodrug to the parent compound.

From the above test results, it is clear that the prodrug of this invention
It can be seen that it is converted to the parent compound in vitro blood and that the incorporation of the prodrug into ribosomes increases its conversion half-life.

Chloroform l containing 126 μmole of egg yolk lecithin, 54 μmole of egg yolk sphingomyelin and 24 μmole of mitomycin C prodrug (Example 1)
14.3 fflQ of liquid d was fractionated into a round bottom flask, and chloroform was distilled off for 1 hour. In this, sodium cholate 1
After adding 94 mg and 6 ml of Pus, the mixture was shaken, and a ribosome suspension containing phytomycin C prodrug was obtained in the same manner as described above.

The suspended ribosome suspension was diluted with PB5 so that the mitomycin C prodrug concentration was 3.0 μa + ole/−.
and filtered through polycarbonate membrane (0.1μ). SD male and female rats 7 withdrawal, weight 30
0 g, 4 animals per group) containing a PBS solution of mitomycin C and a mitomycin C prodrug prepared by the above method! ! A cloudy solution 1.0IIIQ (both containing 3.0 μmole gold as mitomycin C) was injected intravenously, and blood was collected from the supraclavicular vein over time. The concentration of mitomycin C in the blood was determined by centrifuged plasma after protein removal. , measured by high-performance liquid chromatography.In addition, the concentration of mitomycin C prodrug in blood was
Blood was lyophilized, extracted with ethanol, and measured using high performance liquid chromatography.

Table 3 shows the blood concentrations of mitomycin C and mitomycin C prodrug (Example 1) as the average value above the standard deviation of 4 rats.

The above test results show that the ribosomal suspension containing the prodrug maintains the 11:I medium concentration of the parent compound (mitomycin C) for a long time.

Suction and Expulsion Test 2 L: The mitomycin C prodrug (Example 1)-containing ribosome suspension prepared in Suction and Exhaust Test 1 was diluted with PBS so that the mitomycin C prodrug concentration was 2.5 μmole/mQ. A PBS solution of mitomycin C and a drug-containing ribosomal suspension containing mitomycin C were added to ICR female mice (8 incubations, weight 25 g, 3 mice per group) filtered through Plan (0.1μ). 2rMi was injected intravenously, and after a certain period of time, blood was collected from the heart under ether anesthesia. The concentrations of 1ffiJ& of mitomycin C and mitomycin C prodrug in the blood were measured in the same manner as in Suction and Expulsion Test 1.

Table 4 shows the blood concentrations of mitomycin C and mitomycin C prodrug (Example 1) as the average value and standard deviation of three mice.

- The above test results show that the ribosome suspension containing the prodrug maintains the blood concentration of the parent compound (mitomycin C) for a long time.

Isamu Chichiyoshi J Mitomycin C prodrug (Example 1) 4 μmol
e, Vanasate 800 (trademark, manufactured by NOF Corporation)
40mg, egg yolk lecithin 14mg and) IcO-6
0<Trademark, manufactured by Nikko Chemicals Co., Ltd.) A chloroform solution containing 8 mg each of 1 was combined in a round bottom flask,
Chloroform was distilled off. To this was added 1 PB51mQ, and an emulsion preparation of mitomycin C prodrug was obtained using a sonicator. On the other hand, mitomain C prodrug (Example 1) 4 μmole J'J and HCO-
Each chloroform solution containing 6024 mg was combined in a round bottom flask, and the chloroform was distilled off. PB the residue
Disperse in S1- and stir vigorously to obtain a clear solution.

Q4ml of each of the two types of preparations obtained in this way (
41 mole/m of each drug! 1) In the same manner as in Inhalation and Exhaust Test 2, mice were intravenously injected, and the concentration of mitomycin C and mitomycin C prodrug in the blood were measured.

Table 5 shows the blood concentrations of mitomycin C and mitomycin C prodrug when mice were intravenously injected with preparations containing two types of mitomycin C prodrugs (Example 1), and the standard deviation above the average value of 3 mice. Shown as

The above test results show that emulsion or solubilized prodrugs maintain the blood concentration of the crude compound (mitomycin C) for a long time.

A chloroform-methanol mixture (8:2 v/v, 12 ml) containing 147 μmoles of egg yolk lecithin, 63 μmoles of egg yolk sphincominilin, and 42 μmoles of cytarabine prodrug (Example 8) was dispensed into a round-bottomed flask. The solvent was evaporated at C for 10 minutes.

Add 262.5μl of sodium cholate to this.
Then shake 7ml of PBS and shake as described above.
The best way to get rid of the soil! A ribosome suspension solution having a cytarabine prodrug content of 5 μmole/m was obtained in the same manner as described in Section 4.

An aqueous solution of cytarabine and a ribosome suspension containing a cytarabine prodrug prepared by the above method were administered to SD male rats (7 rats, body weight approximately 250 g, 4 rats per group).1. off
1Q (both containing 45 μmole/ml) was injected intravenously, and blood was collected from the jugular vein over time.

The concentrations of cytarabine and cytarabine prodrug in blood were each measured by high performance liquid chromatography.

Table 6 shows the blood concentrations of cytarabine and cytarabine prodrug (Example 8) as the mean value ± standard deviation of 4 rats.

The results of the two-leg test show that the ribosomal suspension containing the prodrug maintains the blood concentration of the parent compound (cytarabine) for a long time.

Toxicity test Egg yolk lecithin 1 68 phosphorus 54 μmole and mitomycin C prodrug (Example 1) at 3.75, 24, 37, 5 μm, respectively
The chloroform solution containing mole was combined in a round bottom flask, the chloroform was distilled off, and a ribosome suspension containing mitomycin C prodrug was obtained in the same manner as above. This ribosome suspension was prepared at concentrations of mitomycin C prodrug of 0, 15, 0.96, and 1.5, respectively.
Qmgi equivalent to tomycin C/PB to be mQ
Diluted with S, polycarbonate membrane run (01μ)
It was filtered by

Mitomycin C was administered to ICR male mice (6 weeks old).
PBS solution (dose: 1.0 and 6.4 mg/kg/
) and the above mitomycin C prodrug-containing ribosomal suspension (dosage: 1.5 as mitomycin C).
0, 6, 4, 10 mg/kg/day) were administered to mice by intravenous injection three times on the 1st, 3rd, and 5th days, and the body weights were measured from the 1st day to the 15th day of administration. It was measured.

Human Narrow Base 3 Table 7 shows the changes in body weight of mice when each preparation was administered by intravenous injection as the average value and standard deviation of five mice.

It can be seen that the ribosomal suspension containing the capsule-1 prodrug is less toxic than the aqueous solution of the parent compound.

245 μmole of egg yolk lecithin, 105 μmole of egg yolk sphingomyelin and 87.5 μmole of cytarabine prodrug (Example 8), respectively.
Chlorophorhe methanol solution containing (8:2v/
v) in a round-bottomed flask, and the same method as above was used to adjust the cytarabine prodrug concentration to 10 and 20 μm.
A ribosome suspension of ole/mQ was obtained.

Mouse leukemia cells L1210 (105 cells) were transferred to BD
It was intraperitoneally transplanted into F-strain female mice (7 weeks old). After transplant 2
4 hours, cytarabine aqueous solution (dose: too and 3000 μmole/kg), and the above cytarabine prodrug-containing ribosomal suspension (dose = 100 μmole/kg).
and 300 μmole/kg), and an aqueous solution (dose: 1
00 and 300 μmole/kg) per 20 breaths of mouse body weight, respectively. 2mjl and Q, 3m1
1 (Cytarabine Prodrug 7g 300 μmole/kg
Administered by intravenous injection at a rate of Table 8 shows the results of the 7BHAC ○ I test to determine whether the mice were alive or dead after administration.

Meat-1 It can be seen that the ribosome suspension containing the prodrug of this invention has superior antitumor effects compared to the aqueous solution of its parent compound and the aqueous BHAC solution.

Antitumor Test 2 245 μmol of egg yolk lentin, 105 μmole of egg yolk sphingomyelin, and 87.5 and 157.5 μmol of cytarabine prodrug (Example 8), respectively.
Chloroform-methanol solution (3:lv) containing e.
/v) in a round-bottomed flask, and carry out the following antitumor test 1.
In the same way as in the case of cytarabine prodrug concentration is 10
and a ribosome suspension of 18 μmole/mA was obtained.

Sections of human lung adenocarcinoma A349 were isolated from BDF 1 female mice (
The kidney was transplanted under the kidney capsule at 8 days (appropriate cooling).

On days 1.5 and 9 after transplantation, aqueous cytarabine solution (dose 1000 μmole/kg) and the above cytarabine prodrug-containing ribosome suspension, suspension (dose rl:
: too and 180 μmole/kg) was administered intravenously at a dose of 02 mQ per 20 g of mouse body weight.

The tumor volume was measured on the 14th day after administration, and the growth inhibition rate was determined. (In this test, in order to clearly see the antitumor effect of each preparation, 23,25-dimethoxy-13,19,2L 27-tetramethyl-11
,28-dioxa-4-azatricyclo[22,3,1
,0'°9octacosu18-en-2 ,3.10
．． 1. After transplantation using 16-titraone as an immunosuppressant.
On days 2.5, 7.9 and 12, 32 mg/dose was administered subcutaneously. ) Test results Table 9 shows the results after transplantation when each formulation was administered by intravenous injection.
The tumor volume and tumor growth inhibition rate on day 4 are shown.

Table 1: It can be seen that the ribosome suspension containing the prodrug of the present invention has superior antitumor effects compared to the aqueous solution of its parent compound.

[Example] The present invention will be explained in more detail with reference to production examples and examples.

Production Example 1 Triethylamine (4.04 g) is added to a suspension of glycine (1.50 g) in water (100 ml). Dioxane (200ml) and cholesterol chloroformate (8.98g) were added to the resulting clear solution at 0°C.
The mixture is stirred for 3 hours. The reaction mixture is concentrated under reduced pressure and IN hydrochloric acid (23 mm) and chloroform (LOO ml+) are added to the residue. Separate the organic layer, wash with water and dry over magnesium sulfate. Distill the solvent away! Crystals are obtained, then washed with ethanol and collected by filtration to obtain white crystals of N-(cholest-5-ene-3β-oxydicarbonyl)glycine (6,64K).

Melting point: 175-177°C (decomposition) IR (Skyl): 3320. 1750. 1
665. 1570 cm-' Production Example 2 N-(frest-5-ene-3β-oxycarlevonyl)glycine (2,92 g) was mixed with dioxane (40 aQ
) and chloroform (6 relatives).
Hydroxysuccinimide (0,69 g) and dicyclohexylcarbodiimide (1,236 g) are added sequentially at 0° C. and the reaction mixture is left in the refrigerator for 16 hours.

The resulting precipitate was collected by filtration, and the filtrate was concentrated under reduced pressure to give N-
Succinimide ester of (cholest-5-ene-3β-oxydicarbonyl)glycine (3.23 g) is obtained.

Melting point: 159-164°C IR (chloroform): 3440. 1820
．． 1785. 1740°1720 Cm-1 Chapter 1 Week 1 β-Alanine (56 mg) and Sodium Bicarbonate (
Tetrahydrofuran (20 aQ ) and cholesterol chloroformate (4
49 mg) at 0°C and the mixture was stirred for 1 hour. The reaction mixture was concentrated under reduced pressure to give a residue of 0. IN hydrochloric acid (10
aQ ) and chloroform (40 ml).

The organic layer is separated, washed with water, and dried over magnesi cum sulfate. The solvent was distilled off to obtain a crude product, which was dissolved in chloroform and subjected to silica gel column chromatography (
40g). Elution with a mixture of chloroform and methanol yielded a white powder cy>N-(frest-5-en-3β-oxydicarbonyl)-β-alanine (1a7
mg).

Melting point: 127-130°C IR<')'3450. 170
5 cm-' Production Example 4 6-Amino-n-caproic acid (131 mg) and sodium hydrogen carbonate (84 mg) in tetrahydrofuran (l
Tetrahydrofuran (10 ml) and cholesterol chloroformate (449 ml) were added to a mixture of omQ ) and water (lQmQ ).
g) was added at 0°C, and the procedure was repeated in the same manner as in Production Example 3 to obtain N-(cholest-5-en-3β-oxydicarbonyl) (126 mg) as a white powder.

LR ('700) Am): 3440.
1700 cm-'Example 1 Mitomycin C (1.OOg) N. Triethylamine (0.33 g) and N-(cholest-5-ene-3) were added to N-dimethylformamide (zomQ) solution.
Succinimide ester of β-oxydicarbonyl)glycine (175 g) is added at room temperature and the reaction mixture is stirred at room temperature for 4 hours. Distill the solvent off, and add chloroform and water to the residue. Separate the organic layer, wash with water and dry over magnesium sulfate.

The solvent was distilled off to obtain a crude product, which was dissolved in chloroform and subjected to silica gel column chromatography (50 g). Elute with chloroform to form a dark purple powder [las
(laa, 8β.8aa, 8ba) ] −
6-amino-8-carbamoyloxymethyl-[N
-(Cholest-5-ene-3β-oxydicarbonyl)glycyl] 1 , 1a. 2, 8, 8a, 8b
- Hexahydro8a-methoxy-5-methylaziridino [2', 3': 3.4 Cobyro [1.
2-a Coindole-4,7-dione (1.74 g) is obtained.

Melting point = 250°C250gss: 804 (M”) Legend 2 Cholest-5-ene-3β-oxyacetic acid [Australian Journal of Chemistry,
J, Chem, > 24.143-151 (197
1) of chloroform (667 mg)
N-hydroxysuccinimide (1 amu) solution
72 mg) and N,N'-dicyclohexylcarbodiimide (324 mg) are added at O''C and the mixture is stirred at room temperature overnight. The precipitate is filtered off and the filtrate is condensed under reduced pressure to give Cholest-5 Obtain the succinimide ester of -ene-3β-oxyacetic acid. Mitomycin C (502 mg)
To a solution of N,N-dimethylformamide (10ml) was added the above active ester and triethylamine (152ml).
g) and 4-dimethylaminopyridine (20+ng)
Add. The reaction mixture is stirred at 60° C. overnight and concentrated under reduced pressure. Add chloroform and water to the residue. Separate the organic layer and dry with magnesium sulfate. The solvent was distilled off to obtain a crude product, which was dissolved in a small amount of chloroform,
Subjected to silica gel column chromatography and eluted with chloroform, a dark purple powder [1aS-(laα,8β
, 8aα.

8bα)]-]6-amino-8-carbamoyloxymethyl N-(cholest-5-ene-3β-oquinapityl)-1,1a,2,8.8a,8b-hexahydro-88-methquin-5-methylaziridino[2 '.

3': 3.4] Pirolo [1,2-a Coin Dole-
4,7-dione (695 mg) is obtained.

Melting point: 145-150°C (decomposed).

IR (striped seal): 3430. 3320.
3200. 1710. 1650°1600, 15
50 cTn-1 Example 3 In the same manner as in Example 2, the α isomer at position 3 of cholesterol in Example 2 [1aS-(laα, 8β).

8aα, 8bα) co-6-amino-8-carbamoyl oxinemethyl-N-(cholest-5-ene-3α-oxyacefL) -1,1a, 2, 8.8a, 8b
-hexahydro-88-methoxy-5-methylaziridino[2',3':3.4]pyrrolo[1,2-acoindole-4,7-dione is obtained.

Melting point: 11g-120°C IR (Sujoor): 3420. 3320.
3200. 1700. 1600°1550 cm
-1 Example 4 Mitomycin C (37 mg) and triethylamine (1
3 mg) and 1-ethyl-3-(3-dimethyl)-carbodiimide hydrochloride (24 mg).

The reaction mixture is stirred at room temperature overnight and concentrated under reduced pressure. Add chloroform and water to the residue. Separate the organic layer;
Dry with magnesium sulfate. The solvent was removed to obtain a crude product, which was dissolved in a small amount of chloroform and subjected to silica gel column chromatography. [1aS-(
laα, 8β.

Baa, 8ba)]]6-amino-8-carbamoyl oxinemethyl-N'[N-(cholest-5-ene-3β-oxydicarbonyl)-β-alanyl]-1,
1a, 2, 8.8a, 8b - hexahyto 0-8a
-Methquin-5-methylaziridino [2', 3':
3,4] pyrrolo[1,2-a coindole-4,7-
Dione (22 mg) is obtained.

IR (chloroform): 3500. 3440
．． 3380. 1695. 1600°1565c
m-1 Harm J1 milk-mutant N-(cholest-5-ene-3β-oxydicarbonyl)
-6-Amino-n-capric acid (63 mg) in tetrahydrofuran (20 mM) and chloroform (5 m
i) Add mitomycin CC (37 mg), triethylamine (13 mg) and 1-ethyl-3-(3-dimethyl)-lupodiimide hydrochloride (24 mg) to the mixture,
Hereinafter, in the same manner as in Example 4, a dark purple powder [1aS -
(laα, 8β, 8aα, 8bα) two-6-amino-8
-carbamoyloxymethyl-[N-(cholesto-5-
En-3β-oxydicarbonyl 1a. 2.8.8a. 8b-hepsahydro 8g-methoxy-5-methylaziridino [2', 3':
3.4coviloro[1,2-mycoindole-4,7-dione (31 mg) is obtained.

IR (chloroform): 3500. 3
440, 3370. 1700. 1600.

1560 cm-1 Example 6 4-formyl-6,9-dihydroxy-14-oxa-
1,11-diazatetracyclo[7,4,1°02°7
．． Oto, 12 ]]tetrazocar 2.4.6-drien-8yl methyl carbamate (32 mg) with N,
Dissolve in N-methyformamide (1.0 m[).

Triethylamine (14s) and N-(
Succinimide ester of cholest-5-ene-3β-oxydicarbonyl)glycine (60 mg) is added. The mixture was stirred at room temperature for 6 hours and the solvent was distilled off under reduced pressure.

The residue was subjected to F[chromatography and eluted with a mixture of chloroform and methanol to give 11-[N-(cholest-5-ene-3β-oxydicarbonyl-6,9-dihydroxy-14-oxa- 1.11
-Diazatetracyclo[7.4.1. O2°7. 01
0°12Cotetradeca-2.Two isomers at the C-9 position of 4.6-drien-8-ylmethylcarbamate (9-
31 m α-OH isomers and 15 9-β-OH isomers
+ng).

Melting point: 168-170°C (public order). (Mixture of 9-α-OH isomer and 9-β-OR isomer) 9-α-OH isomer IR (chloroform): 3340. 2
950. 1695. 1586.

1355 cm-' SIMS: 791 [M+H]", 813 [M
+Nal”9-β-OH isomer IR (chloroform): 3340.2
945. 1695. 1583.

1350 Cfl-1 SIMS: 791 [M+H]”, 82
9 [M+K]”Example 7 Bis(2-chloroethyl)amine hydrochloride (1.347g
) in 25% aqueous sodium hydroxide solution (1oomi)
Add dichloromethane (100 units) to the solution and shake. The organic layer is separated, dried over potassium carbonate, and filtered. Dichloromethane was distilled off under reduced pressure to obtain bis(2-chloroethyl)amine (0.52 g) as an oily substance. N-
Bis(2-chloroethyl)amine (85
．． 2 mg) and dicyclohequinlecarbodiimide (61.9 mg) and stirred for 6 hours. After standing in the refrigerator overnight (17 hours), filter and evaporate the solvent under reduced pressure. The residue was dissolved in chloroform-methanol (50:1
) Dissolved in a mixed solution and preparative layer chromatography (PLC)
Attach to. Developed with a mixture of chloroform-methanol (50:1), scraped off the desired part, extracted with a mixture of chloroform-methanol (4.1), filtered, and then distilled off the solvent under reduced pressure to obtain N- [N-(cholest-5-ene-3β-oxydicarbonyl)glyzylco-bis(2-
chloroethyl)amine (110 mg) is obtained.

Melting point J 98-100 ℃ Star! 1 N-(cholest-5-ene)-3β-oxydicarbonyl)glycine (2.74 g) and triethylamine (0
．． 78mQ) of tetrahydrofuran (40mQ)
) Add ethyl chloroformate (0.61 g) to the solution at -15°C.
and the reaction mixture was stirred at -15°C for 20 minutes.

1-β-D-arabinofuranosylcytosine (1.37 g) was added to the reaction mixture in dimethylformamide (30 m
) Add the solution dropwise at -15°C to -10°C. After addition, the mixture is stirred for 1 hour at 5° C. and then for 30 minutes at room temperature. The reaction mixture is concentrated under reduced pressure.

The residue was washed with methanol and dried to obtain the crude product, which was purified by silica gel column chromatography to obtain N'
-(N-(Cholest-5-ene-3β-oxycarbonyl)glycyl]-1-β-D-arabinofuranosylcytosine (1.8 g) is obtained as a white powder.

Melting point: 159-161°C (decomposition) IR (sudicol): 3330. 28
50, 1690, 1640.

1570 am-1

Claims (11)

[Claims] (1) General formula A-CO(CH_2)_m(NHCO)_n-R (wherein A is the residue of a pharmaceutical compound having a group NH or -NH_2 in the molecule, R is the residue of a steroid compound, m is 1~
(an integer of 5, n means 0 or 1, respectively) and salts thereof. (2) The prodrug compound and its salts according to claim 1, wherein the steroid compound is cholesterol. (3) The prodrug compound and its salts according to claim 1 or 2, wherein A is a residue of a pharmaceutical compound having a group NH in the molecule. (4) The prodrug compound and its salts according to claim 3, wherein the pharmaceutical compound is mitomycin C. (5) The prodrug compound and its salts according to claim 3, wherein the pharmaceutical compound is nitrogen mustard. (6) The prodrug compound and its salts according to claim 3, wherein the pharmaceutical compound is a FR900482 substance. (7) The prodrug compound and its salts according to claim 1 or 2, wherein A is a residue of a pharmaceutical compound having a group -NH_2 in the molecule. (8) The prodrug compound and its salts according to claim 7, wherein the pharmaceutical compound is cytarabine. (9) General formula HOOC(CH_2)_m(NHCO)_n-R (wherein R is the residue of the steroid compound, m is an integer from 1 to 5, n
means 0 or 1, respectively) or its reactive derivative at the carboxy group or its salt; A-CO(CH_2)_m(NHCO)_n-R (wherein A, R, m and n are each (same meaning as above) A method for producing a prodrug compound or a salt thereof. (10) General formula A-CO(CH_2)_m(NHCO)_n-R (wherein A is the residue of a pharmaceutical compound having a group NH or -NH_2 in the molecule, R is the residue of a steroid compound, m is 1~
(an integer of 5, n means 0 or 1, respectively) A long-acting preparation containing a prodrug compound or a salt thereof. (11) Claim 10 in which the split mold is a ribosome
Long-acting preparations as described in Section.