JPH08134093A - Carcinostatic agent - Google Patents

Abstract

PURPOSE: To obtain a new compound consisting of 2',3'-dideoxy-3'-N-substituted- L-phenylalanylamide-5-fluorocytidine, having a cancer cell multiplication- inhibiting activity, reduced in side effects and toxicity, good in stability, and useful for carcinostatic agents, etc. CONSTITUTION: This new compound, a 2',3'-dideoxy-3'-N-substituted-L- phenylalanylamide-5-fluorocytidine of formula I (R is an amino-protecting group), has a cancer multiplication-inhibiting activity, realizes the improvement of bioavailability, the reduction in side effects and toxicity, the improvement of stability, etc., and is useful as a prodrug producing an excellent carcinostatic activity, etc. The compound is obtained by dissolving 3'-amino-2',3'-dideoxy-5- fluorocytidine of formula II in dimethylformamide, and subsequently reacting the dissolved compound of formula II with an N-substituted-L-phenylalanine derivative of formula III (R<1> is pentachlorophenyl, p-nitrophenyl, etc.,) in the presence of triethylamine at room temperature.

Description

Detailed Description of the Invention

[0001]

The present invention relates to 2 ', 3'-dideoxy-3'-N-substituted-L-phenylalanylamide-5-
TECHNICAL FIELD The present invention relates to fluorocytidine, a method for producing the same, and uses thereof.

[0002]

2. Description of the Related Art Drugs that have low activity or are inactive by themselves, but are metabolized into active form, that is, the fact that drugs are metabolized in the living body Drugs that aim to improve various actions by modifying the molecule are called prodrugs.

The purpose of prodrug formation is to prolong the action,
It has been broadly divided into the improvement of bioavailability such as increased water solubility and change of dosage form, reduction of side effects and toxicity, stabilization, and solution of formulation problems such as improvement of taste and odor. The drug is being developed as a drug.

Recently, prodrugs designed to preferentially cleave the active ingredient in cancer cells have been designed. 3′-Amino-2 ′, 3′-dideoxycytidine is known as a compound, and its effect on L1210, a mouse leukemia cell, has been reported [TSLin, W.
R. Mancini; J. Medicinal Chemistry, 26, 544 (198
3)]. It is a prodrug belonging to a new category, which is expected to release active nucleotides into cells by chymotrypsin, which is known to amplify in cancerous cells.

2 ', 3'-dideoxy-3'-N-substituted-L
-The parent compound of phenylalanylamido cytidine, 3'-amino-2 ', 3'-dideoxycytidine, is phosphorylated by deoxycytidine kinase in cells and further becomes its 5'-triphosphate, resulting in the DNA polymerase group. Inhibits the growth of cancer cells by inhibiting
By inhibiting not only S-phase-specific DNA polymerase α but also β, it is expected to be effective over a wider range of tumors. In addition, since deoxycytidine kinase plays an important role in the initial activation (phosphorylation) of prodrug by using 2'-deoxycytidine as a substrate, a substrate that is more easily phosphorylated than 2'-deoxycytidine is used. It would be desirable to use to develop compounds that are specific for inhibiting the growth of cancer cells.

[0006] Furthermore, 3'-amino-2 ', 3'-dideoxycytidine is effective at the cellular level (in vitro), but has a short half-life in blood and it is difficult to maintain its concentration in blood. It is desired to develop a drug that prolongs the pharmacological effect by further extending the blood half-life.

[0007]

DISCLOSURE OF THE INVENTION The present invention provides 3'by chymotrypsin in cancer cells in vivo.
-Amino-2 ', 3'-dideoxycytidine, which prolongs the blood half-life of a drug and maintains a high blood concentration, thereby providing a prodrug which has an excellent anticancer activity. To aim.

[0008]

Means for Solving the Problems As a result of intensive studies for solving the above problems, the present inventors have found that 2 ', 3'-dideoxy-3'-N-substituted-L-phenylalanylamide. It was found that the 5-fluoro derivative of cytidine has excellent antitumor activity, and the present invention has been completed. That is, the present invention provides the following formula (I):

[0009]

[Chemical 6]

2 ', 3'-dideoxy-3'-N-substituted-L-phenylalanylamido-5-fluorocytidine represented by the formula (wherein R represents an amino-protecting group).

Examples of the amino group-protecting group represented by R include aliphatic acyl groups having 6 to 20 carbon atoms and aromatic acyl groups having 20 or less carbon atoms. Furthermore, the present invention provides the following formula (II):

[0012]

[Chemical 7]

3'-amino-2 ', 3'-dideoxy-5-fluorocytidine represented by the following formula (III):

[0014]

Embedded image

(Wherein R is the same as defined above, and R ¹ represents pentachlorophenyl, p-nitrophenyl, N-hydroxysuccinimide, N-hydroxyphthalimide or hydroxytriazole).
The following formula (I) characterized by reacting with an L-phenylalanine derivative:

[0016]

[Chemical 9]

A method for producing 2 ', 3'-dideoxy-3'-N-substituted-L-phenylalanylamide-5-fluorocytidine represented by the formula (wherein R is as defined above). Furthermore, the present invention provides the following formula (I):

[0018]

[Chemical 10]

2 ', 3'-dideoxy-3'-N-substituted-L-phenylalanylamide-5-fluorocytidine represented by the formula (wherein R is the same as above) is contained as an active ingredient. It is a cancer drug. Hereinafter, the present invention will be described in detail. The compound represented by the formula (I) is a 3'-amino- which is specifically cleaved and activated by chymotrypsin.
It is a prodrug of 2 ', 3'-dideoxy-5-fluorocytidine.

Compound of the present invention (hereinafter referred to as the present compound)
Can be obtained as follows. That is, the active ester of acylphenylalanine or phenylalanine, which is a phenylalanine with its amino group protected, and 3'-amino-2 ', 3'-dideoxy-5-fluorocytidine are reacted, for example, at room temperature for 10 to 30 hours. (Condensation)
It is obtained by The amino group-protecting group is preferably an aliphatic acyl group or an aromatic acyl group having 6 to 20 carbon atoms, such as a hexanoyl group, a benzoyl group, a naphthoyl group and a nicotinoyl group.

Examples of the active ester include pentachlorophenyl ester, p-nitrophenyl ester, N-hydroxysuccinimide ester, N
Examples thereof include hydroxyphthalimide ester and hydroxytriazole ester, and pentachlorophenyl ester is preferable. As the condensation method, the active ester method described above is used, but in addition, a mixed acid anhydride method,
N, N'-dicyclohexylcarbodiimide method (DCC
Method), an acid azide method and the like can also be used.

The present compound is specifically cleaved and activated by a protease in cancer cells. That is, in cancer cells, 3'-amino-2 ', 3'-dideoxy-5 was detected by chymotrypsin.
-Fluorocytidine is preferentially cleaved. Especially 2 ',
Activation is facilitated in the 5-fluoro derivative of 3'-dideoxy-3'-N-substituted-L-phenylalanylamide.

Therefore, the present compound has a particularly remarkable antitumor effect on chymotrypsin-producing cancer cells. Moreover, since this compound remains inactive until 3'-amino-2 ', 3'-dideoxy-5-fluorocytidine is cleaved by chymotrypsin, it is possible to prolong the half-life in blood. Medium concentration can be maintained high. this is,
It has different characteristics from a mere ester or amide type prodrug. In addition, when the present compound is administered as a carcinostatic agent, it is effective mainly against leukemia, lung cancer system and the like.

The method of administering the present compound may be oral or parenteral, and oral administration includes sublingual administration. For parenteral administration, injection, such as subcutaneous, intraperitoneal, intramuscular, intravenous injection,
Besides drips, suppositories etc. are included. In addition, the dose varies depending on the age of the administration subject, the administration route, and the number of administrations, and can be widely varied. In this case, the effective amount administered as a composition of an effective amount of the present compound and a suitable diluent and a pharmacologically usable carrier is 10 to 800 mg / kg body weight / day,
It is administered once a day to several times over 5 days.

When the present compound is orally administered, it may be used as tablets, granules, fine granules, powders and the like applicable thereto, and especially granules, fine granules and powders are capsules if necessary. As a unit dose dosage form. These solid preparations for oral administration usually contain additives such as binders, inclusion agents, excipients, lubricants, disintegrants, and wetting agents, which are commonly used in formulation, in their compositions. The liquid preparation for oral use may be in any form such as an aqueous solution, suspension, emulsion and syrup for internal use, or may be a dry product to be redissolved when used. Further, the composition may contain either an additive or a preservative.

In the case of parenteral administration, stabilizers, buffers, preservatives, isotonic agents, and other additives are included, and usually provided in a unit dose ampoule or multi-dose container. .
The composition may be in powder form for reconstitution with a suitable carrier, eg, pyrogen-free, sterile water, when used. Here, the pharmacological effect (anticancer activity) using the present compound will be described by giving test examples of the present compound as follows.

[Test Example 1] Antitumor activity of the present compound on mouse leukemia cells The present compound includes 2 ', 3'-dideoxy-3'-N-hexanoyl-L-phenylalanyl-amino-5-fluorocytidine. (Hereinafter referred to as "hexanoyl body") and 2 ',
3'-dideoxy-3'-N-benzoyl-L-phenylalanyl-amino-5-fluorocytidine (hereinafter referred to as "benzoyl compound") was used. As a comparative compound, 3'-amino-2 ', 3'-dideoxy-5-fluorocytidine (hereinafter referred to as "3'-amino-FCdR"), 2', 3'-dideoxy-3'-N. -Acetyl-L-
Phenylalanyl-amino-5-fluorocytidine (hereinafter referred to as "acetyl compound") and 2 ', 3'-dideoxy-
3′-Nt-butoxycarbonyl-L-phenylalanyl-amino-5-fluorocytidine (hereinafter “t-Bo
"C-body") was used.

10 ⁶ of mouse leukemia cells, P388, were transplanted into the abdominal cavity of mice (CDF ₁ , female, 8 weeks old) (1).
5 per group). The present compound and comparative compound were dissolved or suspended in physiological saline and administered once a day continuously from day 1 to day 5 after tumor cell transplantation in the amount shown in FIG. 1 into the abdominal cavity of mice. . In addition, a group in which only physiological saline was administered by the same method (control group) was similarly tested.

The mice were observed for 90 days after the tumor cell transplantation, and their survival time was measured. In addition, the change in body weight of the mice 17 days after the tumor cell transplantation was measured. Further, with the administration group of the present compound and the comparative compound as T and the control group as C, the ratio of the survival days of the administration group to the control group (T / C (%)) was determined, and this was used as an index of antitumor activity. .

FIG. 1 shows the results. From FIG. 1, the benzoyl compound, which is a compound of the present invention, showed remarkable antitumor activity.

[Test Example 2] Antitumor activity of this compound against Lewis lung cancer In the same manner as in Test Example 1, the antitumor activity against Lewis lung cancer was measured. 3 × 10 ⁵ Lewis lung cancer, mouse (BDF1
System, female, 8 weeks old, subcutaneously transplanted (5 per group)
). The above compound is dissolved in physiological saline or suspended in physiological saline containing carboxymethylcellulose sodium salt, and is continuously administered from day 1 to day 5 after tumor transplantation for 1 day per day.
Once, a predetermined amount (see Table 1) was administered to the abdominal cavity of the mouse.
In addition, a group in which only physiological saline was administered by the same method (control group) was similarly tested. The results are shown in Table 1. From Table 1, the compounds of the present invention, hexanoyl form and benzoyl form, showed remarkable anticancer activity.

[0032]

[Table 1]

Each numerical value shown in the item of "Survival time" in Table 1 indicates the average number of survival days ± standard error per 5 mice.

[0034]

EXAMPLES The present invention will be described in more detail below with reference to examples. However, the present invention is not limited to these examples. Example 1 Synthesis of 2 ′, 3′-dideoxy-3′-N-acyl-L-phenylalanylamide-5-fluorocytidine. Regarding 3'-amino-2 ', 3'-dideoxy-5-fluorocytidine, known methods [TSLin et al., J. Med. C.
hem., 26, 1691 (1983)], 3'-amino-2 ', 3'.
-Dideoxy-5-fluorocytidine (243 mg) was dissolved in dimethylformamide (DMF) (5 ml) to give N-acyl-L.
-Phenylalanine pentachlorophenyl ester (t
-Boc body, acetyl body, hexanoyl body or benzoyl body) (1.5 equivalents), triethylamine 150 μL,
The mixture was stirred at room temperature for 50 hours. The solvent was distilled off under reduced pressure as much as possible, partitioned with 30 ml of saturated sodium hydrogen carbonate and 30 ml of ethyl acetate, and the organic layer was washed with distilled water. Next, it was dried over anhydrous magnesium sulfate, placed on a column of silica gel (10 g), eluted with chloroform-ethanol (9: 1), and the eluate was subjected to silica gel thin layer chromatography to obtain a fraction containing the target substance. collected. After distilling off the solvent, it was crystallized from a mixed solution of a small amount of ethanol and ether. The melting point is
t-Boc body is 145-146 ℃, acetyl body is 156-157
℃, hexanoyl body 66 ~ 68 ℃, benzoyl body 138
It was ~ 139 ° C.

(1) The results of NMR are as follows. 3′-amino-2 ′, 3′-dideoxy-5-fluorocytidine (3′-NH ₂ —FCdR): UV (0.01N HCl) λ _max 277 nm (ε1179)
0), λ _min 238nm; UV (0.01N NaOH) λ _max 268nm (ε7820),
λ _min 246nm; NMR (Me ₂ SO-d ₆ ) δ 2.0-2.2 (m, 2H, 2'-H), 3.
27-3.34 (d, 2H, 3'-NH ₂ , D ₂ O exchangeable) 3.9-4.4 (m, 3H,
4'-H, 5'-H), 4.94 (br s, 1H, 5'-OH, D ₂ O exchangeable) 4.98
(t, 1H, 3'-H), 6.16 (t, 1H, 1'-H), 7.06 (br d, 2H, 4-N
H ₂ , D ₂ O exchangeable), 7.87 (d, 1H, 6-H). Ninhydrin test; positive.

2 ', 3'-dideoxy-3'-(N-acetyl-L-phenylalanyl) -amino-5-fluorocytidine: UV (Me0H) λ _max 271; NMR (Me ₂ SO-d ₆ ). δ2.0-2.2 (m, 2H, 2'-
H), 3.3 (s, 3H, -CH ₃ ), 3.6 (dd, 2H, -CH _2- ) 3.9-4.4 (m, 3
H, 4'-H, 5'-H), 4.5 (m, 1H, -CH-), 4.94 (br s, 1H, 5'-
OH, D ₂ O exchangeable), 4.98 (t, 1H, 3'-H), 6.16 (t, 1H, 1 '
-H), 7.06 (br d, 2H, 4-NH ₂ , D ₂ O exchangeable), 7.1-7.3 (m,
5H, aromatic-H), 7.87 (d, 1H, 6-H), 8.4-8.6 (m, 2H, -NH
-, D ₂ O interchangeable).

2 ', 3'-dideoxy-3'-(Nt-butoxycarbonyl-L-phenylalanyl) -amino-
5-Fluorocytidine: UV (Me0H) λ _max 271 nm; NMR (CDCl ₃ -d ₆ ) δ 1.31 (s, 9H, -C
H ₃ ), 2.0-2.2 (m, 2H, 2'-H), 3.6 (dd, 2H, -CH _2- ) 3.9-4.4
(m, 3H, 4'-H, 5'-H), 4.5 (m, 1H, -CH-), 4.94 (br s, 1
H, 5'-OH, D ₂ O exchangeable), 4.98 (t, 1H, 3'-H), 6.16 (t, 1
H, 1'-H), 7.06 (br d, 2, 4-NH ₂ , D ₂ O exchangeable), 7.1-7.3
(m, 5H, aromatic-H), 7.87 (d, 1H, 6-H), 8.4-8.6 (m, 2H,
-NH-, D ₂ O interchangeable).

2 ', 3'-Dideoxy-3'-(N-hexanoyl-L-phenylalanyl) -amino-5-fluorocytidine: UV (Me0H) λ _max 271 nm; NMR (CDCl ₃ -d ₆ ) δ0 .8-0.9 (t, 3H,-
Cl ₃ ), 1.1-1.35 (m, 6H,-(CH ₂ ) _3- ), 1.5-1.6 (t, 2H, -CH ₂
-), 2.0-2.2 (m, 2H, 2'-H), 3.6 (dd, 2H, -CH _2- ) 3.9-4.4
(m, 3H, 4'-H, 5'-H), 4.5 (m, 1H, -CH-), 4.94 (br s, 1
H, 5'-OH, D ₂ O exchangeable), 4.98 (t, 1H, 3'-H), 6.16 (t,
1, 1'-H), 7.06 (br d, 2H, 4-NH ₂ , D ₂ O exchangeable), 7.1-7.
3 (m, 5H, aromatic-H), 7.87 (d, 1H, 6-H), 8.4-8.6 (m, 2H,
-NH-, D ₂ O interchangeable).

2 ', 3'-dideoxy-3'-(N-benzoyl-L-phenylalanyl) -amino-5-fluorocytidine: UV (Me0H) λ _max 271 nm; NMR (CDCl ₃ -d ₆ ) δ 2 .0-2.2 (m, 2H,
2'-H), 3.6 (dd, 2H, -CH _2- ) 3.9-4.4 (m, 3H, 4'-H, 5'-
H), 4.5 (m, 1H, -CH-), 4.94 (br s, 1H, 5'-OH, D ₂ O exchangeable), 4.98 (t, 1H, 3'-H), 6.16 (t, 1H , 1'-H), 7.06 (br
d, 2H, 4-NH ₂ , D ₂ O exchangeable), 7.1-7.3 (m, 5H, aromatic-
H), 7.4-7.53 (m, 5H, aromatic-H), 7.87 (d, 1H, 6-H), 8.4-
8.6 (m, 2H, -NH-, D ₂ O interchangeable).

(2) Partition coefficient (log P) in chloroform / water is shown in Table 2.

[0041]

[Table 2]

Table 2 shows the remarkable effect of imparting hydrophobicity to the hexanoyl compound and the benzoyl compound.

(3) N-acyl-phenylalanylamide-2 ', 3'-dideoxy-5-fluorocytidine γ
-Hydrolysis rate by chymotrypsin The hydrolysis rate constant (k) and elimination half-life (t1 / 2) of the enzyme (240 units / ml) are shown in Table 3 below.

[0044]

[Table 3]

As is clear from Table 3, the half-life of the benzoyl form is about 70 times that of the acetyl form, and a sustained release effect is expected.

INDUSTRIAL APPLICABILITY The present invention can provide a prodrug that exhibits excellent antitumor activity. Compounds of the invention 3'-N-acyl-phenylalanylamide-2 ', 3'-
Dideoxy-5-fluorocytidine is expected to suppress cancer cell proliferation by being specifically cleaved in cells by chymotrypsin to the parent compound and inhibiting DNA synthesis via phosphorylation, and also has a half-life. Since it has a long period of time, its activity (cancer cell growth inhibitory activity) can be enhanced and it is useful as an anticancer agent.

[Brief description of drawings]

FIG. 1 shows the antitumor activity of the present compound against mouse leukemia cells.

Claims (4)

[Claims] 1. The following formula (I): (In the formula, R represents a protecting group for an amino group),
3'-dideoxy-3'-N-substituted-L-phenylalanylamido-5-fluorocytidine. 2. The 2 ′, 3′-dideoxy-group according to claim 1, wherein the protective group for the amino group represented by R is an aliphatic acyl group having 6 to 20 carbon atoms or an aromatic acyl group. 3'-N
-Substituted-L-phenylalanylamide-5-fluorocytidine. 3. The following formula (II): 3′-amino-2 ′, 3′-dideoxy-5-fluorocytidine represented by the following formula (III): (In the formula, R is the same as the above, and R ¹ represents pentachlorophenyl, p-nitrophenyl, N-hydroxysuccinimide, N-hydroxyphthalimide, or hydroxytriazole.) N-substituted-L-phenylalanine The following formula (I) characterized by reacting with a derivative: (In the formula, R is the same as above), and 2 ′, 3′-
A method for producing dideoxy-3'-N-substituted-L-phenylalanylamido-5-fluorocytidine. 4. The following formula (I): (In the formula, R is the same as above), and 2 ′, 3′-
An anticancer agent containing dideoxy-3′-N-substituted-L-phenylalanylamido-5-fluorocytidine as an active ingredient.