JP2008255098A - Anilinoquinazoline compound and its use - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a compound showing anticancer and antitumor activity through a novel action mechanism, and a medicine composition containing the compound. <P>SOLUTION: This invention relates to an anilinoquinazoline compound represented by the formula or its salt, and this drug composition contains the compound or its salt. The compound exerts the anticancer activity by a novel action mechanism which is different from that of current compounds (for example, paclitaxel, docetaxel, gefitinib, and taxol). Therefore, it is possible to provide a composition for curing cancer having little side-effect by using the compound by itself or in combination with other anticancer drugs. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to an anilinoquinazoline-based compound and use thereof.

  Until now, many anticancer and antitumor agents having various action mechanisms have been developed. For example, paclitaxel and docetaxel inhibit mitosis and cell proliferation by binding to tubulin and inhibiting microtubule depolymerization, and are effective in tumors where other existing drugs do not work (eg, ovarian cancer and breast cancer). It is shown that there is. In addition, gefitinib has been shown to selectively inhibit tyrosine kinase of epidermal growth factor receptor and is effective for non-small cell lung cancer to which existing drugs do not work.

  Taxol and the like are useful as anticancer agents, but serious side effects such as bone marrow suppression and anaphylactic shock have been reported, and development of anticancer agents having a novel mechanism of action is desired.

  An object of the present invention is to provide a compound exhibiting an anticancer activity by a novel mechanism of action.

  The present inventors have found a compound exhibiting an anticancer activity by a mechanism different from that of an existing compound having an anticancer activity, and have completed the present invention.

［２］ ［１］に記載の化合物又はその塩を含む医薬組成物。
［３］ 抗癌剤である［２］に記載の医薬組成物。 The present invention provides the following.
[1] A compound represented by the following formula or a salt thereof.

[2] A pharmaceutical composition comprising the compound or a salt thereof according to [1].
[3] The pharmaceutical composition according to [2], which is an anticancer agent.

  A compound having an anticancer activity by a novel mechanism of action is provided. By using the compound of the present invention alone or in combination with other anticancer agents, a composition for cancer treatment with small side effects can be provided.

<1> Compound of the Present Invention The compound of the present invention is a compound represented by the following formula, and can be produced by the method described in Example 1 described later.

  The salt of the compound of the present invention is not particularly limited, and includes salts with organic substances and inorganic substances. When the compound of the present invention is used in a pharmaceutical composition, it is preferably a pharmaceutically acceptable salt.

  The compound of the present invention exhibits anticancer / antitumor activity by a novel mechanism of action, as shown in Examples below.

<2> Pharmaceutical composition of the present invention The pharmaceutical composition of the present invention comprises the compound of the present invention or a salt thereof as an active ingredient.
Usually, the compound of the present invention or a salt thereof as an active ingredient can be made into a preparation (pharmaceutical composition) using a pharmaceutically acceptable carrier. Examples of the pharmaceutically acceptable carrier include an excipient or a base. Moreover, the formulation may contain the additive used normally. The dosage form is appropriately selected depending on the administration route. Formulations include the active ingredient peptide and other anticancer agents that are separately packaged and integrated. Further, the dosage is appropriately selected depending on the intended anticancer drug treatment, the patient's condition, and the like. The pharmaceutical composition of the present invention is preferably an anticancer agent and can be administered to a patient who is receiving or intending to receive an anticancer agent treatment.

  Hereinafter, examples of the present invention will be specifically described. However, the following examples should be regarded as an aid for obtaining specific recognition of the present invention, and the scope of the present invention is limited by the following examples. It is not something.

Example 1 Synthesis of Compound Q15 and Biotinylated Q15

1-1 Synthesis of compound 2

To 7-chloroquinazoline 1 (212.9 mg, 0.603 mmol) were added n-butanol (6 mL) and piperazine (103.9 mg, 1.21 mmol), and the mixture was stirred overnight under reflux. The solvent was distilled off under reduced pressure, and ethyl acetate was added to the residue. After stirring, the product was collected by filtration to obtain the desired product (142 mg, yield 58%) as a pale yellow solid.
2: ¹ H NMR (DMSO-d ₆ , 400MHz) δppm: 9.24 (s, 1H), 8.65 (s, 1H), 8.19 (m, 1H), 7.81 (m, 1H), 7.47 (t, 1H), 7.38 (s, 1H), 3.34 (br s, 1H), 3.24 (m, 4H), 3.08 (m, 4H).

1-2 Synthesis of Compound 3

Piperazine 2 (403 mg, 1.0 mmol) was dissolved in tetrahydrofuran (12 mL), and p-phenoxyphenyl isocyanate (211 mg, 1.0 mmol) was added. The yellow precipitate was collected by filtration, and the residue was washed with ethyl acetate and dried to obtain the target compound 3 (529 mg, yield 86%).
3: ¹ H NMR (DMSO-d ₆ , 400 MHz) δ ppm: 9.18 (s, 1H), 8.64 (s, 1H), 8.17 (dd, 1H), 7.80 (m, 1H), 7.49-7.37 (m, 7H), 7.35 (s, 1H), 7.10 (t, 1H), 6.95 (d, 1H), 3.61
(brs, 1H), 3.55 (s, 8H).

1-3 Synthesis of Compound 4

3 (199 mg, 0.32 mmol) was dissolved in ethanol (10 mL), 2 drops of concentrated hydrochloric acid and water (7 mL) were added, and the mixture was heated on an oil bath at 90 ° C. To this was added a solution of tin (II) chloride dihydrate (289 mg, 1.28 mmol) in ethanol (2 mL) at 50-60 ° C. and heated on an oil bath at 90 ° C. for 2 hours. After allowing to cool, triethylamine was added to adjust pH = 7, ethyl acetate was added, and insoluble matters were removed by Celite filtration. The product was extracted with ethyl acetate, washed with aqueous sodium bicarbonate and then with brine, dried over sodium sulfate, and concentrated under reduced pressure. The residue was washed with ethanol (20 mL) and then filtered, and the filtrate was concentrated to obtain target product 4 (49 mg, yield 26%).
4: ¹ H NMR (DMSO-d ₆ , 400 MHz) δ ppm: 9.44 (s, 1H), 8.65 (s, 1H), 8.37 (s, 1H), 8.18 (dd, 1H), 7.80 (m, 1H ), 7.33 to 7.55 (m, 7H), 7.23 (s, 1H), 6.96 (m, 2H), 5.31
(s, 2H), 3.46 (s, 8H).

1-4 Synthesis of Bio-Q15

1N NaOH aqueous solution (19 μL) was added to NHS-PEO4-Biotin (10 mg, 17 μmol) in water (100 μL) and stirred at room temperature for 1 hour, and then 1N hydrochloric acid aqueous solution (22 μL) was added and concentrated. . The concentrate was repeatedly substituted and concentrated with dehydrated pyridine to obtain a crude product of hydrolyzate PEO4-Biotin. After adding phosphorus trichloride (2.3 mg, 17 μmol) to pyridine solution (500 μL) of compound Q15 (9.9 mg, 17 μmol) and stirring at 80 ° C. for 1 hour, PEO4-Biotin crude product obtained above Was dissolved in pyridine (100 μL) and added. After stirring at 80 ° C. overnight, the reaction mixture was concentrated by adding water and purified by HPLC (2 mg, yield 11%).
Mass (ESI): m / z = 1057 (M + 1)

  The conditions for LC-MS analysis and fractionation are as follows.

<LC analysis conditions>
Equipment used Waters 2695 (LC section), 2487 (UV detection section), ZQ (MS detection section)
Column YMC-Pack Pro C18 S-5um 50 × 4.6mm (YMC)
Column temperature 30 ° C
Carrier A: H2O / MeCN = 95/5 (0.05% TFA), B: H2O / MeCN = 5/95 (0.05% TFA), 3.0mL / min, Linear Gradient

<LC preparative conditions>
Equipment used Waters 2525 (LC Pump), 2487 (UV detector), ZQ (MS detector), 2767 (flare collector) column CombiPrep Pro C18 S-5um 50 × 20mm (YMC)
Column temperature rt
Carrier A: 100% H2O (0.05% TFA), B: 100% MeCN (0.05% TFA), 25.0mL / min, Linear Gradient

<Example 2> Growth suppression test of Q15 on SW480 colon cancer cells
The ability of test compounds to inhibit the growth of SW480 cells [human colon cancer-derived cultured cells obtained from American Type Culture Collection (ATCC)] was measured.

SW480 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum, 2 mM glutamine, and non-essential amino acids at 37 ° C. in a 5% CO ₂ air incubator. Cells were collected from the petri dish using trypsin / ethylamine diamine tetraacetic acid (EDTA). Seed cells at a density of 2 x 10 ³ cells per well in a 96-well plate, 37 ° C, 5%
Standed for 24 hours in DMEM containing 10% fetal calf serum, 1 mM glutamine, and non-essential amino acids with CO ₂ . The cells were treated with or without the addition of compound Q15 at various concentrations (100, 50, 25, 12.5, 6.25, 3.125 μM in final concentration) in dimethyl sulfoxide (DMSO), and then incubated for 3 days. After this incubation period, the cell titer 96 AQueous One Solution Cell Proliferation Assay Kit (Promega Co., Ltd.) was used, and after treatment according to the method of the package insert, the absorbance was measured to determine the number of viable cells. The results are shown in FIG. 1, assuming that no drug addition (only DMSO added) is 100% and no cell is 0% cell viability.

The concentration of compound required to give 50% inhibition of growth (IC ₅₀ ) was 8.6 μM.

Example 3 Binding Protein Screening of Compound Q15 Screening was performed using the in vitro virus (hereinafter abbreviated as IVV) method [Horisawa, K et al, (2004), 32, e169 .: Reference 1], and IVV An IVV template RNA library for preparing the library was prepared from poly A + RNA (Premium Poly A + RNA, Takara Bio Inc.) obtained from SW480 cells according to Reference 1.

The screening method is as follows.
Biotinylated Q15 (2 mM DMSO solution, 1 μL) was added to magnetic streptavidin beads (Magnotex
SA, Takara Bio Inc., 0.67 mg) in an IPP150 buffer [10 mM Tris-HCl (pH 8.0), 150 mM sodium chloride, 0.1% NP-40, 0.2 mL] suspension and stirred at room temperature for 1 hour. After washing with IPP150 (0.2 mL x 3), suspend in IPP150 (0.2 mL), add 0.1 M biotin aqueous solution (20 μL), stir at room temperature for 1 hour, wash with IPP150 (0.2 mL x 3), and then wash with IPP150- Suspend in 2 buffer [10 mM Tris-HCl (pH 8.0), 150 mM sodium chloride, 0.1% NP-40, 1 mM dithiothreitol, 0.5 mg / mL bovine serum albumin, 20 μg / mL tRNA, 0.2 mL] Q15 fixed bead suspension.

  An IVV template RNA library (10 pmol) was translated with a wheat germ cell-free translation kit (Zoigene) under the composition and conditions shown in Table 3 to prepare an IVV library solution.

  The IVV library solution and the Q15 fixed bead suspension were mixed and stirred at 4 ° C. for 2 hours. After recovering the Q15 fixed beads, the beads were washed with IPP150-2 (0.8 mL × 3), and then the beads were suspended in pure water (0.1 mL). The bead suspension (1 μL) was reverse-transcribed and amplified with the One Step RT-PCR kit (Qiagen) under the composition shown in Table 4 and the conditions shown in Table 5 to obtain a cDNA library. Each primer sequence is shown in Table 6.

  The cDNA library was subjected to 2% agarose electrophoresis, and a fraction having a chain length of 700 to 1000 base pairs was recovered. The cDNA library reconstructed in this way was converted into an IVV template RNA library according to the method of Document 1, and used for the next screening.

  The above screening was repeated a total of 5 times, and the resulting cDNA library was cloned and sequenced. As a result, 2 clones out of 100 clones matched the sequence of Homo sapiens non-SMC condensin II complex, subunit G2 (NCAPG2, NM_017760).

<Example 4> Measurement of NCAPG2 gene amount in cDNA library Using a real-time PCR apparatus (LightCycler, Roche Diagnostics Inc.), before screening (round 0) and after repeating screening a total of 5 times ( Round 5) The number of molecules of NCAPG2 gene in the obtained cDNA library (5 ng each) was measured. The method used DNA primer 3 and DNA primer 4 (Table 7) that specifically amplify the NCAPG2 gene according to the package insert of LightCycler FastStart DNA Master SYBR Green I (Roche Diagnostics Inc.). The results and the calculated concentration rate are shown in Table 8.

  As a result of the IVV screening in Example 3, multiple clones of the NCAPG2 gene were cloned. In Example 4, quantification of the NCAPG2 gene in the cDNA library at the time of IVV screening was performed. When Q15 was used as a bait (in the case of Q15-fixed beads), NCAPG2 gene was screened 5 times before screening ( It was concentrated 705 times compared to round 0). On the other hand, mock (when no drug was fixed to the beads) was 0.13 times.

  Since IVV screening is a screening method using a molecule called IVV in which a protein and the gene that encodes it are bound, the enrichment of a specific gene in a cDNA library results from the interaction between the bait molecule and the protein. Is interpreted. Therefore, when the results of Examples 3 and 4 are combined, it is considered that the NCAPG2 protein was specifically interacted with compound Q15 and screened because the NCAPG2 gene was concentrated only in the case of Q15-fixed beads.

<Example 5> Pull-down assay of NCAPG2 protein in cell lysate In order to examine whether intracellular full-length NCAPG2 protein actually binds to compound Q15, a pull-down assay was performed using SW480 cell lysate and Q15-fixed beads.

(Method for preparing lysate) Dulbecco's modified eagle containing 10% fetal calf serum, 1 mM glutamine, and non-essential amino acids on SW480 cells on a 10cm culture plate (Corning) at 37 ° C in a 5% CO ₂ air incubator Cultured in medium (DMEM). SW480 cells that have become subconfluent (80-90%) are collected using a cell lifter (Corning) in the presence of PBS, and then the cells are sedimented at 1000 rpm. After removing the supernatant, the resulting cell sputum is subjected to cell extraction buffer (10 mM Tris-HCl, 150 mM NaCl, 1% NP-40, 0.1% deoxycholic acid, protease inhibitor cocktail from Roche Diagnostics) , pH 7.6) and let stand on ice for 30 minutes. The suspension is centrifuged at 15000 rpm for 10 minutes and the resulting supernatant is used as a cell lysate.

  The method for preparing Q15 fixed beads is as shown in Example 1. Q15-fixed beads (6 mg) were added with cell lysate (250 μL, total protein amount: 500 μg) and stirred at 4 ° C. for 2 hours. After collecting the beads, the beads were washed with IPP150 (0.5 mL × 3), and then suspended in an elution buffer (20 μL, composition is shown in Table 9). The suspension is heated at 96 ° C. for 10 minutes, and the supernatant is removed and washed with 5-20% SDS polyacrylamide electrophoresis gel (Wako Pure Chemical Industries, Ltd.) at 20 mA (constant current) for 90 minutes with transfer buffer ( The composition is shown in Table 10). The protein developed on the gel was transferred onto a polyvinylidene fluoride (PVDF) membrane and stained by Western blotting using an anti-NCAPG2 protein antibody (Bethyl Inc, Texas, USA). The results are shown in FIG. In FIG. 2, Mock represents a drug non-binding bead, and Iressa represents a biotinylated Iressa analog (following formula) -bound bead, which is a negative control.

  Examples 3 and 4 showed that compound Q15 interacts specifically with the NCAPG2 protein in the IVV molecule expressed in the in vitro translation system. In Example 5, it was examined whether NCAPG2 protein expressed in living cells specifically binds to compound Q15. Here, a pull-down assay, a general technique for examining binding, was performed using Q15 immobilized beads, Iressa immobilized beads and non-drug immobilized beads (mock). After the protein bound to each bead was eluted, it was separated by SDS polyacrylamide electrophoresis, transferred to a PVDF membrane, and stained with an anti-NCAPG2 protein antibody (Western blot method). As a result, only in the case of Q15-fixed beads, the NCAPG2 protein band was detected at a molecular weight of about 130k (the calculated molecular weight of NCAPG2 based on the amino acid sequence was 131k). In other words, not only the non-drug-immobilized beads but also the Iressa-immobilized beads similar to Q15, the NCAPG2 protein band is not detected, indicating that the binding of Q15 and NCAPG2 protein is very specific. Indicated.

  Condensin is an important protein complex involved in cell proliferation that is involved in chromosome aggregation and chromosome distribution during cell division [Hirano, T., Curr. Biol. (2005), 15, R265]. In addition, in cells in which NCAPG2 protein, which is a condensin constituent protein, is knocked down by RNA interference, it has been reported that the cell cycle is stopped in the M phase [Kittler, R. et al, Nature (2004), 432, 1036. The gene “FLJ2031” in this document is another name for NCAPG2.] Taking the above-described characteristics of the NCAPG2 protein and the results of this Example into consideration, Compound Q15 specifically binds to the NCAPG2 protein and suppresses cancer cell growth. That is, it was confirmed that the compound Q15 exhibits an anticancer action by an action mechanism different from that of the existing compound having an anticancer action. In addition, from the viewpoint of its mechanism of action, it is considered that it exhibits a wide range of anticancer / antitumor effects against various cancers.

Example 6 Binding of Compound Q15 to Condensin II Complex
hCAP-G2 is one of the subunits of condensin II. Condensin is a protein complex consisting of two SMC (Structural Maintenance of Chromosome) proteins and three non-SMC subunits, and plays an important role in chromosome aggregation. SMC proteins SMC2 and SMC4 form a heterodimer and bind to DNA. Among the non-SMC subunits, CAP-H and CAP-H2 are proteins that bind to SMC dimers and are similar to each other. CAP-D2 and CAP-D3, CAP-G and CAP-G2 are also similar to each other, and there are repetitive sequences called HEAT repeats in the molecule. This HEAT repeat has been reported to be involved in protein-protein interactions. Since the region containing the HEAT repeat of hCAP-G2 was obtained by IVV screening of compound Q15, the effect of Q15 on the formation of condensin II complex was examined. Cell extraction buffer containing protease inhibitors (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% NP-40, 0.1%
DOC) was used to prepare a cell extract from SW480 cells. Q15-immobilized streptavidin magnetic beads (equivalent to 2 nmol in terms of biotin binding ability) were added to the cell extract with a total protein mass of 500 μg and reacted at 4 ° C. for 2 hours. After the reaction, the plate was washed 4 times with a washing buffer, and then bands of hCAP-G2 and SMC2 were confirmed by Western blotting. As a result, it was confirmed that hCAP-G2 and SMC2 were specifically bound to the drug Q15 (FIG. 3). From this, it was found that Q15 has a condensin II complex containing hCAP-G2 and SMC2.

Example 7 Growth Inhibition Test of Compound Q15 on Various Human Cultured Cancer Cells A growth inhibition test of compound Q15 on various human culture cancer cells was performed. The cultured human cancer cells used were breast cancer (Breast cancer, Br) 5 strains (HBC-4, BSY-1, HBC-5, NCF-7, MDA-MB-231), brain tumors (central nervous system cancer, Central Nerval System, CNS) 6 strains (U251, SF-268, SF-295, SF-539, SNB-75, SNB-78), colon cancer (Colon cancer, Co) 5 strains (HCC2998, KM-12, HT-29) HCT-15, HCT-116), lung cancer (Lung cancer, Lu) 7 strains (NCI-H23, NCI-H226, NCI-H522, NCI-H460, A549, DMS273, DMS114), melanoma (Melanoma, Me) 1 Strain (LOX-IMVI), ovarian cancer (Ovary cancer, Ov) 5 strains (OVCAR-3, OVCAR-4, OVCAR-5, OVCAR-8, SK-OV-3), renal cancer (Renal cancer, Re) 2 Strain (RXF-631F, ACHN), stomach cancer (Stomach cancer, St) 6 strains (St-4, MKN1, MKN7, MKN28, MKN45, MKB74), prostate cancer (Prostatic cancer, xPg) 2 strains (DU-145, PC) -3), a total of 39 shares. These were treated as one panel, and an in vitro drug sensitivity test was conducted to try to obtain a sensitivity pattern (Finger Print, described later) for that drug. The greatest merit of this system is that by comparing Finger Print with the data in the database, it is possible to select a compound having an action mode different from that of an existing anticancer agent, or to estimate the action mode of the compound. As a result of database analysis, antitumor activity can be evaluated in a xenograft system in which the cancer cells of the above panel are transplanted into nude mice for specimens evaluated as promising.

  The human cultured cancer cell panel is easy to understand when the entire panel is regarded as a single creature. When a drug is given to the panel as an input, what is obtained as an output is Finger Print. The feature of Finger Print is the key to evaluation, and the analysis is performed in a database. Since this database expands as screening is performed, it is expected that the information obtained with it will increase in mass. This is a new type of anticancer drug screening system suitable for the information age.

Cancer cells are seeded in a 96-well plate, and the sample solution (5 doses, usually 1 log interval from 10 ^-4 to 10 ^-8 M, the highest concentration is called High Conc) is added the next day, and cultured for 2 days. Thereafter, cell proliferation is measured colorimetrically with sulforhodamine B. Measurement results are input to a computer for data processing.

Based on the number of cells immediately before applying the drug (Time Zero) as a parameter for data analysis
GI50: Concentration that suppresses growth by 50% compared to control
TGI: Concentration that suppresses proliferation to the same number of cells as Time Zero (concentration that does not seem to increase or decrease the number of cells)
LC50: concentration that reduces the number of cells to 50% of Time Zero (the higher the cell killing effect, the lower this concentration)
Find the three types. Specifically, using “In Vitro Testing Results” data, the calculation is as follows.
When the average optical density [Mean Optical Density] (ODtest) at each concentration is greater than the OD value (Tz) of Time Zero (ODtest> Tz) Growth% [Percent Growth] (PG%) = ((ODtest-Tz) / (ODcontrol-Tz)) x 100
However, ODcontrol indicates the OD value of Control.
Conversely, when ODtest <Tz,
PG% = ((ODtest-Tz) / Tz) x 100
In this case, PG% is (-).

FIG. 4 shows dose response curves obtained by plotting the PG% value obtained above for each cell against the concentration (logarithm) for each organ cancer. Each curve is PG 50%,
Concentrations at the points where the 0% and −50% horizontal lines intersect correspond to Log GI50, Log TGI, and Log LC50, respectively. As can be seen from FIG. 4, compound Q15 has the most proliferative inhibitory effect on cultured cells of lung cancer (Lu) and colon cancer (Co). In lung cancer (Lu), GI50 of NCI-H522 strain is 4.2 × 10 ^−7. In M and colorectal cancer (Co), the GI50 of the HT-29 strain was 6.2 × 10 ⁻⁷ M, and a significant cancer cell growth inhibitory effect was observed on the nM order. Q15 strongly increases the growth of other cancer cell lines, such as breast cancer (Br), brain tumor (CNS), melanoma (Me), kidney cancer (Re), gastric cancer (St), etc. at concentrations of 10 ^-5 M or higher. Suppressed.

  Next, a sensitivity pattern “Finger Prints” (Mean Graphs) was created. The average value of Log GI50 is obtained for all tested strains, and this is referred to as Mean Graph Midpoint (MG_MID). Find the difference between this average value and the Log GI50 value for each individual cell (that is, the GI50 for each individual cell is a logarithmic value indicating how many times or a fraction of the average value is) Finger Print (Fig. 5) is a bar graph drawn left and right with the value at the center (scale 0). In FIG. 5, the bar graph on the left is GI50, the center bar graph is TGI, and the right bar graph is Finger Print on LC50. A strain with higher sensitivity (Differential Sensitivity) has a longer bar on the right side. One scale represents 1 log. The Delta displayed at the bottom of Finger Print indicates the difference between the most sensitive and the average value, and the Range below it indicates the difference in LogGI50 between the most sensitive and least sensitive strains. The same applies to Log TGI and Log LC50. When data is not obtained in the tested concentration range, the limit value (usually, upper limit value -4 or lower limit value -8) is substituted (this value is called False Values and In the "Summary of Evaluation" column, the number is shown under High for the upper limit and Low for the lower limit.) Therefore, the average value calculated here is not necessarily the true average value. Therefore, the expression mean midpoint (MG_MID) is used instead of mean. From FIG. 5, it was lung cancer (Lu), followed by colorectal cancer (Co) and brain tumor (CNS) that had many cultured cell lines (bar extends to the right) highly sensitive to compound Q15. For example, looking at the Log TGI in the middle of Figure 5, 5 out of 7 strains for lung cancer (Lu), 4 out of 5 strains for colorectal cancer (Co), and 4 out of 6 strains for brain tumor (CNS). Each bar graph extends to the right, indicating that it is highly sensitive.

This screening database has a function called “COMPARE Program” that compares Finger Prints between drugs and tests their similarity (Department of Molecular Pharmacology, Cancer Chemotherapy Center, Foundation for Cancer Research). Basically, it tests the statistical correlation between two drugs. If you enter Finger Print (Seed) for a sample drug, Finger
Drugs with Finger Print similar to the Seed are listed from the Print data in descending order of similarity. According to the results of the Finger Print analysis of more than 100 drugs including existing anticancer drugs by the COMPARE program, “Films with the same mechanism of action are highly correlated (similar to each other)
The very important fact of “showing nger print” is revealed. For example, DNA intercalators show very similar Finger Prints. In addition, it has been found that tubulin inhibitors, topoisomerase inhibitors, cisplatin analogues, 5-fluorouracil analogues, cytarabine analogues, methotrexate analogues, etc. also show similar finger prints within each group. Data analysis with the COMPARE program is particularly important as a sample evaluation criterion in HCC panel screening. If a sample with an unknown mechanism of action shows a finger print similar to that of any existing drug, the sample drug can be expected to have the same mechanism of action as the drug, and vice versa. This suggests that it has a unique mechanism of action. At present, the COMPARE program can be used at least as follows.

1) Selection of New Lead Select compounds that are not similar to Finger Print of any standard drug. Such a compound may be a new lead with a new type of mechanism of action (unknown).

2) Estimating the mechanism of action Even if the mechanism of action is completely unknown, if the Finger Print is analyzed with the COMPARE program and shows similarity to the Finger Print of any standard drug registered in the database, the standard It is suggested to have a mechanism of action close to that of the drug (otherwise, 1).

3) Search the database for more effective candidate compounds from the lead compound. Use the lead compound's Finger Print as the seed, extract substances with a high similarity finger print from the substances registered in the database, and determine the actual pharmacological activity. Find more effective compounds than research.

4) Structure-activity relationship From the group of structurally similar compounds, compound candidates that have the same mechanism of action as the parent compound are selected based on the similarity of Finger Print. Or conversely, a compound candidate having an action mechanism different from that of the mother compound is selected.

Delta and Range values that serve as criteria for Differential Activity (whether significant efficacy is seen against a specific cancer type or several cancer cell lines) were calculated from the data for Q15. As a result, the Delta value was 0.74 for GI50, 0.92 for TGI, and 0.97 for LC50. Regarding the range value, 1.31 was obtained for GI50, 1.79 for TGI, and 1.15 for LC50. Differential
As activity criteria, Delta ≥ 0.5 and Range ≥ 1, it can be judged that there is significant efficacy, so if compound Q15 shows significant efficacy against a specific cancer type or several cancer cell lines, it is differential I could conclude from the value of Activity.

  Next, the uniqueness of Finger Print was analyzed. As a result of comparing the finger print of the sample substance with all the existing anticancer drugs in the database by the COMPARE program, the r value of the anticancer drug (Drug A) that showed the highest value (the value closest to 1) Based on r value (rMAX), it evaluated as follows.

  (1) If rMAX <0.5 COMPARE Negative: There is no finger print similar to the sample substance in the existing anti-cancer drugs in the database → Expect new action mechanism.

  (2) If 0.5 ≤ rMAX <0.75 COMPARE Marginal: somewhat similar to Drug A → May have the same mechanism of action as Drug A, but may also have a new mechanism of action. Between (1) and (3). (There is something similar in the database)

  (3) If 0.75 ≤ rMAX COMPARE Positive: Similar to Drug A → Expected mechanism of action similar to Drug A.

  The rMAX value (Results of compare) of compound Q15 was calculated, and rMAX <0.5. As a result, it was clarified that there is no finger print similar to compound Q15 among the existing anticancer agents in the database, and a new mechanism of action can be expected.

  As mentioned above, when the analysis results of a series of human cultured cancer cell panels of compound Q15 are combined, compound Q15 has a novel chemical structure, and its effective concentration for inhibiting growth is sufficiently low for various cancer cells (also effective at nM level). Substances), Differential growth inhibition (especially high sensitivity in lung cancer and colon cancer cells) is recognized, and rMAX <0.5 (Results of compare), so it is an effective substance as a drug with an unprecedented mechanism of action It was concluded that

The result of the growth suppression test with respect to SW480 cell is shown. The result (electrophoresis photograph) of a pull-down assay is shown. The result (electrophoresis photograph) of the western blotting which shows the coupling | bonding of compound Q15 and a condensin II complex is shown. Figure 2 shows dose response curves of compound Q15 against various human cultured cancer cells. Figure 2 shows the sensitivity pattern of various human cultured cancer cells to compound Q15.

Claims (3)

A compound represented by the following formula or a salt thereof.

A pharmaceutical composition comprising the compound according to claim 1 or a salt thereof. The pharmaceutical composition according to claim 2, which is an anticancer agent.

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