JPH0560479B2 - - Google Patents

Description

[Detailed description of the invention]

(Technical Field) The present invention relates to a novel ascamycin derivative, a method for synthesizing the same, and an anticancer agent containing the derivative as an active ingredient. (Background of the Invention) Conventionally, alkylating agents (nitrodiene mustards, ethyleneimines,
sulfonic acid esters), antimetabolites (folate antagonists, purine antagonists, pyrimidine antagonists), plant fission toxins (colcemid, vinblastine, etc.),
Antibiotics (sarcomycin, carcinophilin,
mitomycin, etc.), hormones (adrenal steroids, male hormones, female hormones), and porphyrin complex salts (marphyrin, copp), etc. are used. First, the present inventors searched for substances with excellent anticancer activity, and as a result of intensive research on the physiological activities of metabolites of microorganisms belonging to the genus Streptomyces, they discovered a new antibiotic.
We discovered that the RK-647A substance (ascamycin) has excellent anticancer activity, and obtained the knowledge that this substance can exert a remarkable effect on cancer treatment, and completed a new anticancer drug (Unexamined Japanese Patent Publication No. Publication No. 198981; The Journal of Antibiotics, vol.37,
No. 6, pp670-672 (1984); see Japanese Patent Application Laid-open No. 62-56500). Therefore, the present inventors continued their research with the aim of clarifying the structures of various amino acid analogs of ascamycin and their activity relationships, and as a result, succeeded in synthesizing a new amino acid analog compound of ascamycin. Furthermore, we have discovered that these ascamycin derivatives have excellent anticancer activity, and have completed the present invention with the knowledge that these substances can exhibit remarkable effects in cancer treatment. be. (Object of the Invention) An object of the present invention is to provide a novel ascamycin derivative and a method for synthesizing the same. Another object of the present invention is to provide an anticancer agent containing a novel ascamycin derivative as an active ingredient. (Structure of the invention) The present invention is based on the general formula: (In the formula, R ¹ is L-phenylalanine, L-
an aminoacyl group derived from an amino acid selected from the group consisting of proline and D-alanine, R ²
represents a hydrogen atom or a t-butyloxycarbonyl group, and R ³ represents a hydrogen atom or two R ^3s jointly represent an isopropylidene group. However, R ¹ never represents an L-alanine group. The present invention provides an ascamycin derivative compound represented by the following formula, a method for producing the same, and an anticancer agent containing the compound as an active ingredient. The starting material of the present invention is 2-chloro-9-(2',
3'-O-isopropylidene-5'-O-sulfamoyl-β-D-ribofuranosyl)adenine (E), which can be obtained, for example, by the following method. That is, 2-chloroadenosine (C) is obtained in two steps from 2,6-dichloropurine (A) and β-D-ribofuranose-1,2,3,5-tetraacetate (B) (Montgomery). , J.A. Hewson, K. (1964), Journal of Heterocyclic Chemistry (J.Heterocycle.
Chem.), 1, 213-214). The obtained 2-chloroadenosine (C) is converted into a 2',3'-isopropylidene acetal (D), and then treated with sulfamoyl chloride in the presence of sodium hydride to obtain the starting material (E) ( Gough, JC, Penglis-Caredes, F., Maguire, MH (1978) Journal of Medical Chemistry (J.Med.Chem.) 21,
See 520-525. ). The above steps are as follows. However, in the above method, the yield of starting material (E) is insufficient (about 40% compared to compound (D)), so
The inventors have developed an improved method. That is, by reacting compound (D) with sulfamoyl chloride in the presence of bis(tri-n-butyltin) oxide, starting material (E) can be obtained in good yield (approximately 80% or more).
Obtainable. (Reference example 1) Dissolve 68.2 mg of compound (D) in 6 ml of dry benzene,
Bis(tri-n-butyltin) oxide 0.33ml
(3.25 equivalents) was added to a 4 Å molecular sieve as a dehydrating agent, and the mixture was heated under reflux for 2 hours. After cooling the reaction solution to 5° C., 76.4 mg (4 equivalents) of sulfamoyl chloride dissolved in 3 ml of dry 1,4-dioxane was added dropwise. After reacting for 3 hours at room temperature and 10 hours at 5°C, sulfamoyl chloride solution (4 equivalents) was added dropwise again. After reacting at room temperature for 22 hours and concentrating under reduced pressure, the residue was extracted with hot hexane, ethyl acetate was added to the residue, and the mixture was washed successively with a saturated aqueous potassium fluoride solution and saturated brine, and the ethyl acetate layer was dried over magnesium sulfate. Then, concentrate under reduced pressure and perform TLC on silica gel.
The starting material (E) was obtained (yield: 80%). The obtained starting material (E) can be processed through the following steps to obtain the desired ascamycin derivative (). That is, compound (E) is reacted with di-t-butyl dicarbonate,
N ⁶ -t-butyloxycarbonyl compound (F) is obtained.
This reaction involves NaH, trimethylsilyl chloride,
This can be carried out in the presence of n-butyllithium, lithium diisopropylamine, bis(tri-n-butyltin) oxide, or the like. As the solvent, dimethylformamide, dioxane, tetrahydrofuran, benzene, etc. can be used. The reaction temperature and reaction time are −78 to 100, respectively.
°C for 1 to 20 hours is appropriate. Next, the obtained N ⁶ -t-butyloxycarbonyl compound (F) is coupled with an active ester of an amino acid selected from the group consisting of L-phenylalanine, L-propyline, and D-alanine to obtain a coupled compound (F). G) is obtained. This reaction is
It can be carried out in the presence of cesium carbonate, sodium hydride, trimethylsilyl chloride, n-butyllithium, lithium diisopropylamine, bis(tri-n-butyltin) oxide, or the like. As the solvent, dimethylformamide, dioxane, tetrahydrofuran, benzene, etc. can be used. The reaction temperature and reaction time are −78 to 100, respectively.
°C for 1 to 20 hours is appropriate. The amino acid used is one in which the amino group in the molecule is protected. Furthermore, as the active ester of the amino acid, those activated as active esters, the azide method, the mixed acid anhydride method, or acid halides used in ordinary peptide synthesis can be used. For example, in addition to carbonylimidazole, N-ethyl-5-phenylisoxazolium-3'-sulfonate (reagent "K"), N-ethyl-2'-hydroxybenzisoxazolium trifluoroborate salt, 1-ethoxycarbonyl-2-ethoxy-1,2-dihydroxyquinoline (EEDQ), 1-isobutyloxycarbonyl-2-isobutyloxy-1,2-dihydroxyquinoline (IIDQ), benzotriazolyl-N-hydroxy trisdimethylaminophosphonium hexafluorophosphate salt (Bop reagent),
Diphenylphosphoryl azide (DPPA) or the like or an acid chloride activated with amino acid carbonyl can be used. The ratio of compound (F):catalyst:active ester of aminocarboxylic acid is preferably approximately 1:1:1.5 (equivalent ratio). The obtained compound is deprotected (removal of isopropylidene and t-butyloxycarbonyl) to obtain the desired ascamycin derivative ().
This reaction involves trifluoroacetic acid, trifluoromethanesulfonic acid, methanesulfonic acid, HF,
This can be carried out using HBr/acetic acid, HCl/acetic acid, HCOOH, acetic acid, or a mixture of these acids and water. The reaction temperature and reaction time are −78 to 100, respectively.
°C for 0.5 to 20 hours is appropriate. Thus, the desired ascamycin derivative ()
An example of the process using t-butyloxycarbonyl-L-phenylalanyl imidazole is shown below. Examples of the ascamycin derivative () of the present invention thus obtained include, for example, the following. [-a]: 9-β-[5-O-(N-L-phenylalanyl)sulfamoyl-D-ribofuranosyl]-2-chloroadenine [Physical properties of (-a)] [α] ²⁰ _D = -4.47° (C = 0.745, H ₂ O-MeOH
(2:1)) ¹ H-NMR: δ ^D2O ppm (400MHz) 8.5 (1H, s, C8-H), 7.4 (5H, m, phenyl), 6.21 (1H, d, J = 5.25Hz, C1' −H), 4.8(1H, dd, J=5.25Hz, 5.3Hz, C2−
H), 4.56 (1H, dd, J=5.3Hz, 5.3Hz, C3′−
H), 4.51 (1H, m, C4′-H), 4.49, 4.42 (each 1H, each dd, C5′-H), 4.1 (1H, dd, J=7.88Hz, 6.3Hz,

[Formula]) 3.2 (1H, dd, J=6.3Hz, 14Hz,

[Formula]) 3.05 (1H, dd, J=7.88Hz, 6.3Hz,

[Formula]) UV: 263nm (ε12300) IR: As shown in Figure 1. (KBr method) [-b]: 9-β-[5-O-(N-L-prolyl)sulfamoyl-D-ribofuranosyl]-2-chloroadenine [Physical properties of (-b)] [α] ²⁰ _D : = -13.5° (C = 0.84, H ₂ O) ¹ H-NMR: δ ^D2O ppm (400MHz) 8.35 (1H, s, C8-H) , 6.04 (1H, d, J=
5Hz, C1'-H) 6.04 (1H, d, J = 5Hz, C1'-H) 4.75 (1H, dd, J = 5Hz, 4.9Hz, C2'-H) 4.53 (1H, dd, J = 4.9Hz , 5.9Hz, C3'-H) 4.46 (1H, m, C4'-H), 4.42 (2H, m,
C5′−H ₂ ) 4.21 (1H, dd, J=7Hz, 9.6Hz,

【formula】) 3.38 (2H, m,

【formula】) 2.39 (1H, m,

【formula】) 2.08 (1H, m,

[Formula]) 2.01, 1.93 (each 1H, m,

[Formula]) UV: 263nm (ε12270) IR: As shown in Figure 2. (KBr method) [-c]:9-β-[5-O-(N-D-alanyl)sulfamoyl-D-ribofuranosyl]-
2-chloroadenine [Physical properties of (-c)] [α] ²⁰ _D : = -9.36° (C = 0.438, H ₂ O) ¹ H-NMR: δ ^D2O ppm (400MHz) 8.25 (1H, s, C8-H) 5.98 (1H, d, J = 6.5Hz, C1'-H) 4.62 (1H, t, J = 4.7Hz, C2'-H) 4.41 (1H, t, J = 4.7Hz, C3'-H) 4.34 ( 1H, m, C4'-H) 4.30 (2H, m, C5'-H) 3.73 (1H, q, J=7.0Hz,

[Formula]) 1.35 (3H, d, J = 7.0Hz, -CH ₃ ) UV: 263nm (ε12300) IR: As shown in Figure 3. (KBr method) The antibacterial activity (inhibition circle size: mm) of each compound against plant pathogenic bacteria by the agar plate method (using a circular filter paper with a diameter of 8 mm containing 8 nmol of each compound) was as follows. (Potato Seucloth medium was used as the medium.)

[Physical properties of compound (F)]

Mass spectrometry (SIMS): m/z 521 (MH) ^{+ 1} H-NMR: δ ^CDCl3 _TMS 8.12 (1H, s, C8-H) 6.17 (1H, d, J = 3Hz, C1'-H) 5.31 (1H , dd, J=3Hz, J=6.7Hz,
C2'-H) 5.07 (1H, dd, J=4Hz, J=6.7Hz,
C3'-H) 4.57 (1H, m, C4'-H) 4.39 (2H, m, C5'-H) UV: λ _nax (MeOH) 255 (11000 sh) 273 (18200) 280 (16100 sh) Example 1 N ⁶ -t-Butyloxycarbonyl-2-chloro-9-[5'-O-(N-t-butyloxycarbonyl-L-phenylalanyl)sulfamoyl-
β-D-ribofuranosyl]adenine (G-a) Compound (F) 35 mg (0.0667 mmole) anhydrous
Add to 22.5 mg (1 equivalent) of cesium carbonate dissolved in 0.72 ml of DMF and suspended in 0.5 ml of anhydrous DMF at room temperature.
Stir for 60 minutes. Add 25 mg (1.5 equivalents) of t-butyloxycarbonyl-L-phenylalanine to the reaction solution.
and N,N'-carbonyldiimidazole 16mg
(1.5 equivalents) was dissolved in anhydrous DMF and reacted at room temperature for 30 minutes, and a solution was added dropwise at -20°C. The reaction was carried out while gradually increasing the temperature from -20°C to room temperature over 6 hours. Ethyl acetate was added to the reaction solution, washed sequentially with 10% citric acid and saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified using silica gel TLC to obtain 42 mg of compound (G-a). (yield 82%). [Physical properties of (G-a)] Mass spectrometry (SIMS): m/z 768 (M+H) ^{+ 1} H-NMR: δ ^D2O ppm (400 MHz) 8.34 (1H, s, C8-H), 7.0 ~ 7.3
(5H, m, phenyl) 6.24 (1H, d, J=3Hz, C1'-H) 4.0 (1H, m,

[Formula]) 3.18, 3.60 (each 1H, dd,

[Physical properties of (G-b)]

Mass spectrometry (SIMS): m/z 718 (M+H) ^{+ 1} H-NMR: δ ^D2O ppm (400 MHz) 8.53 (1H, s, C8-H) 6.29 (1H, d, J = 3Hz, C1'-H ) 5.17 (1H, m, C2'-H) 5.06 (1H, dd, J=3Hz, 6Hz, C3'-
H) 4.50 (1H, m, C4'-H) 4.29, 4.18 (1H, ddd, C5'-H each) 4.50 (1H, m,

【formula】 3.32 (2H, m,

[Physical properties of (G-c)]

Mass spectrometry (SIMS): m/z 692 (M+H) ^{+ 1} H-NMR: δ ^D2O ppm (400 MHz) 8.54 (1H, s, C8-H) 6.30 (1H, d, J = 3Hz, C1'-H ) 5.28 (1H, dd, C2'-H) 5.08 (1H, dd, C3'-H) 4.59 (1H, m, C4'-H) 4.40, 4.28 (2H, each dd, C5'-H) 4.05 ( 1H, m,

[Formula] 1.2 (3H, d, J=7Hz, -CH ₃ ) Example 4 Compound (-a) 16 mg of the compound (G-a) obtained in Example 1 was added to 90
% trifluoroacetic acid and reacted for 15 minutes at 0°C and for 1 hour at room temperature. The reaction solution was freeze-dried, and the residue was subjected to high-performance liquid chromatography (Sensyu Pack) using 30% methanol as a developing solvent.
ODS-H-14251) to purify the compound (-
a) 9 mg (yield: 82%) was obtained (optical yield of amino acid residue: 86%). Example 5 Compound (-b) 14 mg of the compound (G-b) obtained in Example 2,
After reacting in the same manner as in Example 4, purification was performed to obtain 7.4 mg (yield: 80%) of compound (-b) (optical yield of amino acid residue: 90%). Example 6 14 mg of the compound (G-c) obtained in Example 3 was
After reacting in the same manner as in Example 4, purification was performed to obtain 7.3 mg (yield: 80%) of compound (-c) (optical yield of amino acid residue: 86%). Formulation example 1 (injection/infusion) Add 5 g of powdered glucose to contain 10 mg of compound (-a) or (-b), dispense aseptically into vials, seal, and inert with nitrogen, helium, etc. Seal the gas and store in a cool, dark place. Before use, add 100 ml of 0.85% physiological saline to make an intravenous injection, and administer 10 to 100 ml per day by intravenous injection or drip depending on the symptoms. Formulation Example 2 (Injection/Drop) 2 mg of compound (-b) was prepared as an intravenous injection for mild symptoms in the same manner as Formulation Example 1, and administered for 1 day.
Administer 10 to 100 ml by intravenous injection or drip depending on the symptoms. Formulation Example 3 (Enteric-coated capsules) After thoroughly mixing 5 g of compound (-c), 2.46 g of lactose, and 0.04 g of hydroxypropyl cellulose, it was formed into granules according to a conventional method, and this was thoroughly dried. It is sieved to produce granules suitable for bottles, heat-seal packaging, etc. Next, 0.5 g of cellulose acetate phthalate and 0.5 g of hydroxypropyl methylcellulose phthalate are dissolved to form a coated base material, and the granules are suspended and flowed to coat this base material to form enteric-coated granules. This composition is filled into capsules to produce 100 enteric-coated capsule preparations. Test Example 1 (Anticancer Activity Test) Using compounds (-a), (-b), and (-c), the uptake rate of test cells was determined from the results obtained by the test method described above. The results are shown in Table 1.

[Table] From the above results, compounds (-a), (-b),
ID ₅₀ of (-c) was determined. The results are shown in Table 2. The compounds of the present invention are effective for Balb 3T3 (fibroblasts)
Since it inhibits the uptake of leucine into Ki 3T3 (cancer cells) at approximately the same concentration as ascamycin, it is found to be effective as an anticancer agent.

【table】 [Brief explanation of drawings]

FIG. 1 is a drawing showing the infrared absorption spectrum (IR) of compound (-a), FIG. 2 is a drawing showing the infrared absorption spectrum (IR) of compound (-b), and FIG. 3 is a drawing showing the infrared absorption spectrum (IR) of compound (-a). It is a drawing showing the infrared absorption spectrum of -c).

Claims (1)

[Claims] 1. General formula: (In the formula, R ¹ is L-phenylalanine, L-
an aminoacyl group derived from an amino acid selected from the group consisting of proline and D-alanine, R ²
represents a hydrogen atom or a t-butyloxycarbonyl group, and R ³ represents a hydrogen atom or two R ^3s jointly represent an isopropylidene group. ) Ascamycin derivative compound. 2 Structural formula: A compound represented by is reacted with di-t-butyl bicarbonate to form the structural formula: (In the formula, Boc represents t-butyloxycarbonyl group.) A compound represented by The general formula: (In the formula, R represents an aminoacyl group derived from an amino acid selected from the group consisting of L-phenylalanine, L-proline and D-alanine.) A method for synthesizing ascamycin derivatives. 3 General formula: (In the formula, R represents an aminoacyl group derived from an amino acid selected from the group consisting of L-phenylalanine, L-proline, and D-alanine.) An anticancer agent containing an ascamycin derivative compound represented by the following as an active ingredient: . 4. The anticancer agent according to claim 3 in a parenteral administration form. 5. The anticancer agent according to claim 3 in an oral administration form.