Furanone derivative

Abstract

To provide a novel furanone derivative, and a medicine including the same. The furanone derivative is represented by the formula (I):

##STR00001##
wherein A represents —COOR1 or a hydrogen atom; R1 represents a hydrogen atom, an optionally substituted hydrocarbon group, or an optionally substituted heterocycle; R2 and R3 are the same or different and each independently represent a hydrogen atom, an optionally substituted hydrocarbon group, an optionally substituted phenyl group, an optionally substituted heterocycle, an optionally substituted heterocyclic fused ring, or an optionally substituted amino group; or alternatively, R2 and R3, taken together with the nitrogen atom to which they are attached, may form an optionally substituted heterocycle or an optionally substituted heterocyclic fused ring; and R4 represents a hydrogen atom or a halogen atom; with the proviso that when A represents —COOR1, R2 and R3 are not optionally substituted amino groups at the same time, and when A represents a hydrogen atom, R3 represents a hydrogen atom.

Description

wherein A denotes P/(P+S) for a blank well, B denotes P/(P+S) for a solvent well, and C denotes P/(P+S) for a compound-added well.

The IC₅₀ value of a tested compound was calculated by regression analysis of the percent inhibition values versus the (logarithmic) concentrations of the tested compound.

The IC₅₀ values of several compounds of the present invention against Cdc7 were all less than 1 μM, as shown in Table 4 below.

Test Example 2

[Inhibitory Effect on Phosphorylation Using Cells]

Cultured Cells

COLO205 cells derived from human colon adenocarcinoma (RCB2127, RIKEN BRC) were cultured in 10 cm dishes using RPMI-1640 medium (SIGMA, R8758) supplemented with 10% FCS (Equitech-bio) and 5% penicillin/streptomycin (GIBCO, 15140). After the cells were cultured to a confluence of 70 to 90%, the medium was removed, and the cells were treated with trypsin (2 mL, TrypLE Express, GIBCO, 12604-021) and then harvested using a new medium.

Addition of Drugs to Cultured Cells

COLO205 cells were seeded in 6-well plates (FALCON, 35046) to 2.5×10⁵ cells (1 ml) per well and cultured overnight in a 5% CO₂ incubator. A drug solution, which was prepared by adding 1.5 μl of a 0.1-mM stock solution in DMSO of a test compound to 500 μl of medium, was added to each well (to a final drug concentration of 0.1 μM), and then cultured for another 24 hours.

Determination of Inhibitory Effect on MCM2 Phosphorylation

Cells were cultured for 24 hours in the presence of a test compound, harvested using a similar way as described above, washed with PBS, and then pelletized and stored at −80° C. The frozen cells were thawed, subsequently suspended immediately in 2×SDS-PAGE electrophoresis buffer (1×10⁵ cells per 10 μl), and heated at 95° C. for 10 minutes, leading to the dissolution and denaturation of the proteins. The resulting sample solutions were subjected to SDS-PAGE using a 5-20% gradient acrylamide gel (e-PAGEL (5-20%), ATTO, E-T520L). After the electrophoresis was completed, the gel was immersed in tris-glycine buffer supplemented with 20% methanol, and the proteins in the gel were transferred onto a PVDF membrane (MILLIPORE, Immobilon-P, IPVH00010) using a semi-dry transfer apparatus (TRNS-BLOT SD SEMI-DRY TRANSFER CELL, BIO RAD).

The transferred PVDF membrane was blocked with 5% skim milk (Difco Skim Milk, BD, 232100), and then subjected to reaction with an anti-MCM2 goat antibody (N-19, Santa Cruz, sc-9839) or anti-phosphorylated MCM2 rabbit antibody (S53) (Bethyl, A300-756A) as a primary antibody and additionally with an anti-α tubulin mouse antibody (Clone DM 1A, SIGMA, 9026) as an internal control. Each band was detected by chemiluminescence using an HRP-labeled anti-goat IgG donkey antibody (Santa Cruz, sc2020), anti-rabbit IgG sheep antibody (Roche, 12015218001), or anti-mouse IgG donkey antibody (Jackson ImmunoResearch, 715-035-151) as a secondary antibody. The combinations of and the diluted concentrations of the primary and secondary antibodies were as indicated below.

TABLE 3
	Primary antibody	Secondary antibody
	(diluted concentration)	(diluted concentration)
1	Anti-MCM2 goat antibody	Anti-goat IgG donkey
	(1/500)	antibody (1/10000)
2	Anti-phosphorylated MCM2	Anti-rabbit IgG sheep
	rabbit antibody (S53)	antibody (1/5000)
	(1/500)
3	Anti-α-tubulin mouse	Anti-mouse IgG donkey
	antibody (1/1000)	antibody (1/100000)

The bands which were detected were normalized using the amount of endogenous α-tubulin as a reference, and the percent phosphorylation of MCM2 was calculated for each of the tested compounds. Their inhibitory effects on MCM2 phosphorylation were indicated by a triple asterisk (***) in the case where the percent phosphorylation of MCM2 was less than 20%, by a dual asterisk (**) in the case where the percent phosphorylation of MCM2 was not less than 20% and less than 50%, and by a single asterisk (*) in the case where the percent phosphorylation of MCM2 was not less than 50% and less than 70%.

It was found from this testing that as shown in Table-4, the tested compounds of the present invention inhibited the phosphorylation of MCM2 at a concentration of 0.1 μM.

TABLE 4
			Test Example 2
		Test Example 1	Inhibitory effect on
	Example	Cdc7 IC50 value (μM)	MCM2 phosphorylation
	15	0.002	**
	22	0.010	*
	63	0.006	***
	68	0.004	***
	78	0.011	***
	81	0.005	***
	89	0.003	***
	91	0.004	***
	102	0.004	***
	126	0.003	***
	129	0.009	***
	142	0.003	**
	157	0.003	**
	173	0.003	***
	226	0.004	***
	233	0.002	**
	239	0.001	**
	242	0.001	***
	244	0.004	***
	245	0.008	***
	246	0.008	***

INDUSTRIAL APPLICABILITY

The compounds provided by the present invention are capable of controlling the growth of cells through their inhibitory effects on Cdc7. Therefore, the compounds of the present invention which have an inhibitory effect on Cdc7 will be useful as a medicine, especially an agent for the treatment of diseases derived from abnormal growth of cells, such as cancers.

Claims

1. A furanone derivative or a pharmaceutically acceptable salt thereof, represented by the formula (I):

[Chemical Formula 1]

2. The furanone derivative or a pharmaceutically acceptable salt thereof according to claim 1, wherein A is —COOR1.

3. The furanone derivative or a pharmaceutically acceptable salt thereof according to claim 1, wherein A is a hydrogen atom.

4. The furanone derivative or a pharmaceutically acceptable salt thereof according to claim 1, wherein the furanone derivative or a pharmaceutically acceptable salt thereof is represented by the formula (II-Z) or formula (II-E):

5. The furanone derivative or a pharmaceutically acceptable salt thereof according to claim 1, wherein any and all heterocyclic groups are optionally substituted 3 to 8 membered heterocycle groups that contain up to two heteroatoms selected from the group consisting of nitrogen, sulfur, and oxygen.

6. The furanone derivative or a pharmaceutically acceptable salt thereof according to claim 1, wherein any and all hydrocarbon groups are 1 to 6 carbon hydrocarbon groups; and wherein any and all heterocyclic groups are optionally substituted 3 to 8 membered heterocycle groups that contain up to two heteroatoms selected from the group consisting of nitrogen, sulfur, and oxygen.

7. The furanone derivative or a pharmaceutically acceptable salt thereof according to claim 4, wherein A and R4 are both a hydrogen atom and R2 and R3 form a 3 to 8 membered heterocycle.

8. The furanone derivative or a pharmaceutically acceptable salt thereof according to claim 4, wherein A is —COOR1 where R1 is an optionally substituted 1 to 6 carbon hydrocarbon group, R4 is a hydrogen atom and R2 and R3 form a 3 to 8 membered heterocycle containing up to two heteroatoms selected from the group consisting of nitrogen, sulfur, and oxygen.

9. The furanone derivative or a pharmaceutically acceptable salt thereof according to claim 1, wherein A is —COOR1 where R1 is an optionally substituted 1 to 6 carbon hydrocarbon group, R4 is a hydrogen atom and one of either of R2 and R3 is a hydrogen atom.

10. The furanone derivative or a pharmaceutically acceptable salt thereof according to claim 1, wherein A is —COOR1 where R1 is an optionally substituted 1 to 6 carbon hydrocarbon group, R4 is a fluorine atom and one of either of R2 and R3 is a hydrogen atom.

11. The furanone derivative or a pharmaceutically acceptable salt thereof according to claim 1, wherein A and R4 are both a hydrogen atom, and one of either of R2 and R3 is a hydrogen atom.

12. The furanone derivative or a pharmaceutically acceptable salt thereof according to claim 1, wherein A is —COOR1 where R1 is an optionally substituted 1 to 6 carbon hydrocarbon group, R4 is a hydrogen atom, one of either of R2 and R3 is a hydrogen atom and one of either of R2 and R3 is an optionally substituted amino group.

13. The furanone derivative or a pharmaceutically acceptable salt thereof according to claim 1, wherein A is —COOR1 where R1 is an optionally substituted 1 to 6 carbon hydrocarbon group, R4 is a hydrogen atom, one of either of R2 and R3 is an optionally substituted 1 to 6 carbon hydrocarbon group and one of either of R2 and R3 is a 3 to 8 membered heterocycle containing up to two heteroatoms selected from the group consisting of nitrogen, sulfur, and oxygen.

14. The furanone derivative or a pharmaceutically acceptable salt thereof according to claim 1, wherein A is —COOR1 where R1 is an optionally substituted 1 to 6 carbon hydrocarbon group, R4 is a hydrogen atom, one of either of R2 and R3 is a hydrogen atom and one of either of R2 and R3 is an optionally substituted 3 to 8 membered heterocyclic fused ring.

15. The furanone derivative or a pharmaceutically acceptable salt thereof according to claim 1, wherein A is —COOR1 where R1 is a 3 to 8 membered heterocycle containing up to two heteroatoms selected from the group consisting of nitrogen, sulfur, and oxygen, R4 is a hydrogen atom and one of either of R2 and R3 is a hydrogen atom.

16. The furanone derivative or a pharmaceutically acceptable salt thereof according to claim 1, wherein the furanone derivative or pharmaceutically acceptable salt thereof is selected from the group consisting of:

17. The furanone derivative or a pharmaceutically acceptable salt thereof according to claim 1, wherein the furanone derivative or pharmaceutically acceptable salt thereof is selected from the group consisting of:

18. The furanone derivative or a pharmaceutically acceptable salt thereof according to claim 1, wherein the furanone derivative or pharmaceutically acceptable salt thereof is selected from the group consisting of:

19. The furanone derivative or a pharmaceutically acceptable salt thereof according to claim 1, wherein the pharmaceutically acceptable salt is the hydrochloride or p-toluenesulfonate salt.

20. The furanone derivative or a pharmaceutically acceptable salt thereof according to claim 16, wherein the pharmaceutically acceptable salt is the hydrochloride or p-toluenesulfonate salt.