WO2007015632A1 - Atm and atr inhibitor - Google Patents

Abstract

The present invention relates to a urea compound of formula (1) and a pharmaceutically acceptable salt thereof having inhibitory activity of ATM and ATR. According to the present invention, the urea compound of formula (1) selectively binds to ATM and ATR, and thus specifically suppresses the function of ATM and ATR as protein kinase. Therefore, the urea compound according to the present invention can be used for controlling cell function and treating diseases, in connection with function abnormality in ATM and ATR

Description

ATM and ATR INHIBITOR

TECHNICAL FIELD

The present invention relates to a urea compound of formula (1) having an inhibitory activity of ATM and ATR, its derivatives and pharmaceutically acceptable salts thereof.

BACKGROUND ART

The genome of eukaryotic organisms is exposed to external damage factors such as ultraviolet rays, reactive compounds, ionic radiation etc., as well as by-products of metabolism such as inaccurately replicated DNA or reactive oxygen intermediates. If genomic DNA is damaged by the above factors, the survival of cells is threatened. Furthermore, pathological symptoms such as tissue damage, immune deficiency, cell aging, and development of cancer arise. [Abraham, R. T. (2001) Cell cycle checkpoint signaling through the ATM and ATR kinases. Genes & Dev. 15, 2177-2196]. In a normal cell, there is a mechanism called "the cell-cycle checkpoint" by which damage of DNA is observed and modified so that normal DNA can be maintained during the cell cycle. Thus, even though the cell cycle is repeated, the normal genome can be preserved.

A protein family called "PIKKs" (phosphoinositide 3-kinase related kinases) is known as one of major enzymes that take part in the cell-cycle checkpoint. In particular, among them, ATM (ataxia-telangiectasia mutated) protein and ATR (ATM and Rad 3-related) protein are known to play an important part in early signal transduction through the total process of cell-cycle checkpoint. ATM is a gene product of ataxia telangiectasia mutated polypeptide having about

350 kDa [Savitsky, K. et al. (1995) A single ataxia telangiectasiz gene with a product similar to PI-3kinase. Science 268, 1749-1753] and a serine/threonine protein phosphatase [Jung, M. et al. (1997) ATM gene product phosphorylates I kappa B-alpha. Cancer Res. 57, 24-27]. It phosphorylates proteins such as p53, NFKBIA, BRCAl, CTIP, NIBRIN (NBSl), TERFl, RAD9 and so on.

A cloned ATR protein analogous to the PIKK protein family [Cimprich, K. A. et al. (1996) cDNA cloning and gene mapping of a candidate human cell cycle checkpoint protein. Proc. Natl. Acad. Sci. 93, 2850-2855] as a serine/threonine protein phosphatase having about 300 kDa phosphorylates proteins such as BRCAl, CHEKl, H2AFX, MCM2, RAD 17, RPA2, SMCl and p53 to inhibit DNA replication and mitosis, and promote DNA repair, recombination, and apoptosis.

Both ATM and ATR protein are activated by DNA damage, but what proteins respond to any damages is not exactly known [Yang, J. et al. (2003) ATM, ATR and DNA-PK: initiators of the cellular genotoxic stress responses. Carcinogenesis 24, 1571- 1580]. It was just reported that ATM responds to double-strand breaks induced by ionic radiation, and ATR mainly responds to ultraviolet rays or stalled replication forks [See Lowndes, N. F. and Murguia, J. R. (2000) Sensing and responding to DNA damage. Curr. Opin. Genet. Dev. 10, 17-25; Abraham, R. T. (2001) Cell cycle checkpoint signaling through the ATM and ATR kinases. Genes Dev. 15, 2177-2196]. When gamma-radiation or neocarzinostatin that is a radiation-imitating medicine is treated to normal human cells, the amount of ATM protein does not change, but that of p53 protein increases [Brown, K. D. et al. (1997) The ataxia-telangiectasia gene product, a constitutively expressed nuclear protein that is not up-regulated following genome damage. Proc. Nat. Acad. Sci. 94, 1840-1845]. The increase of the amount of p53 protein induced by ionic radiation is mediated by specifically phosphorylating 15^th serine residue (Serl5) of p53 protein by ATM [Banin, S. et al. (1998) Enhanced phosphorylation of p53 by ATM in response to DNA damage. Science 281, 1674-1677; Canman, C. E. et al. (1998) Activation of the ATM kinase by ionizing radiation and phosphorylation of p53. Science 281, 1677-1679]. ATR protein also induces the phosphorylation of p53 protein resulted from damaging agents of DNA such as UV to increase the amount of p53 protein. [Tibbetts, R. S. et al. (1999) A role for ATR in the DNA damage-induced phosphorylation of p53. Genes Dev. 13, 152-157]. Therefore, inhibitors having low molecular weight that are specific for ATM/ ATR can be used for controlling the activity of p53 protein.

Caffeine and wortmannin that are non-specific inhibitors to ATM/ ATR can be used as addictives for increasing sensitivity in radiation therapy or chemotherapy of cancer which is performed by induction of DNA damage [Sarkaria, J. N. et al. (1998) Inhibition of phosphoinositide 3-kinase related kinases by the radiosensitizing agent wortmannin. Cancer Res. 58, 4375-4382; Nghiem, P. et al. (2001) ATR inhibition selectively sensitizes Gl checkpoint deficient cells to lethal premature chromatin condensation. Proc. Natl. Acad. Sci. 98, 9092-9097]. However, it is not clear whether - A -

the reaction is carried out by selective inhibition of ATM/ ATR or not since caffeine and wortmannin also react on protein kinases other than ATM/ATR proteins. However, it is clear that inhibitors having low molecular weight that are specific for ATM/ATR can be used as addictives for increasing cellular sensitivity in the ionizing radiation therapy and chemotherapy for cancer treatment [Anderson, H. J. et al. (2003) Inhibitors of the G2 DNA damage checkpoint and their potential for cancer therapy. Prog. Cell Cycle Res. 5, 423-430; Hickson, I. et al. (2004) Identification and characterization of a novel and specific inhibitor of the ataxia-telangiectasia mutated kinase ATM. Cancer Res. 64, 9152-9159]. The function of ATM is tissue-specific for ionizing radiation. In an Atm-/- mouse in which the ATM gene is removed by homologous recombination, Atm-/- thymocyte has resistance against apoptosis induced by gamma radiation in comparison with thymocytes having normal ATM, whereas the other cells show sensitivity to gamma radiation. [Xu, Y. and Baltimore, D. (1996) Dual roles of ATM in the cellular response to radiation and in cell growth control. Genes Dev. 10, 2401-2410]. In addition, in the central nervous system of Atm -/- mouse, apoptosis induced by genotoxic stress did not occur. [Chong, M. J. et al. (2000) Atm and Bax cooperate in ionizing radiation-induced apoptosis in central nervous system. Proc. Natl. Acad. Sci. 97, 889-894]. It was reported that ATM-dependent apoptosis induced by genotoxic stress at the nervous system in duration of development requires p53 protein and is varied according to the stage of cell differentiation [Lee, Y. et al. (2001) Ataxia telangiectasia mutated- dependent apoptosis after genotoxic stress in the developing nervous systems is determined by cellular differentiation status. J. Neurosci. 21, 6687-6693]. It was also reported that the cell death dependent on p53 is activated, when an Ab peptide known as a major virulence factor of Alzheimer's disease is treated to nerve cells [Kruman, 1. 1. et al. (2004) Cell cycle activation linked to neuronal cell death initiated by DNA damage. Neuron 41, 549-561]. According to a recent report, it is known that p53 protein is excessively expressed by mutant Huntington's protein, i.e., a virulence factor of Huntington's disease so as to lead to apoptosis [Bae, B. -I. et al. (2005) p53 mediated cellular dysfunction and behavioral abnormalities in Huntington's disease. Neuron 47, 29-41]. Overdose of glutamate or hypoxia causing nerve cell apoptosis is also known to activate p53 protein [Greig, N. H. et al. (2004) New therapeutic strategies and drug candidates for neurodegenerative disease: p53 and TNF-α inhibitors, and GLP-I receptor agonist. Ann. N. Y. Acad. Sci. 1035, 290-315]. In addition, it is known that excessive expression or activation of p53 protein causes apoptosis in Parkinson's disease [Blum, D. et al. (1997) p53 and Bax activation in 6-hydroxydopamine-induced apoptosis in PC12 cells. Brain Res. 751-139-142], stroke [Li, Y. et al. (1994) ρ53- immunoreactive protein and p53 mRNA expression after transient middle cerebral artery occlusion in rats. Stroke 25, 849-855], traumatic brain injury [Napieralski, J. A. et al. (1999) The tumor suppressor gene, p53, is induced in injured brain regions following experimental traumatic brain injury. MoI. Brain Res. 71, 78-86], ischemic insult and excitotoxic insult [Sakhi, S. et al. (1997) Induction of tumor suppressor p53 and DNA fragmentation in organotypic hippocampal cultures following excitotoxin treatment. Exp. Neurol. 145, 81-88; Uberti, D. et al. (1998) Induction of tumour-suppressor phosphoprotein p53 in the apoptosis of cultured rat cerebellar neurons triggered by excitatory amino acids. Eur. J. Neurosci. 10, 246-254; Culmsee, C. et al. (2001) A synthetic inhibitor of p53 protects neurons against death induced by ischemic and excitotoxic insults, and amyloid β-peptide. J. Neurochem. 77, 220-228; Crumrine, R. C. et al. (1994) Attenuation of p53 expression protects against focal ischemic damage in transgenic mice. J. Cereb. Blood Glow Metab. 14, 887-891]. In particular, it is reported that ATM/ATR-dependent DNA damage reaction occurs in degenerative brain diseases such as SBMA (spinobulbar muscular atrophy) caused by polyQ expansion, Huntington's disease, DRPLA (dentatorubral pallidolusian atrophy), and SCA (six spinocerebellar ataxias) [Giuliano, P. et al. (2003) DNA damage induced by polyglutamine-expanded proteins. Human MoI. Genet. 12, 2301-2309]. Therefore, ATM inhibitors can protect cells from genotoxicity and neurotoxicity at specific tissues or cells.

Most of somatic cells in normal human bodies have limited life span in vitro. [Hayflick, L. and Moorhead, P. S. (1961) The limited in vitro lifetime of human diploid cell strains. Exp. Cell Res. 25, 585-621]. The symptom that proliferation ability of cells decreases with time is called 'cellular senescence.' Further, in the cellular senescence, there are replicative senescence caused by the shortening of telomere, and premature senescence caused by various stresses or unbalance of signal system. Recently, it was reported that the shortening of telomere causes the cellular senescence initiated by ATM, and the inhibition of ATM protein results in restarting a cell cycle [Herbig, U. et al. (2004) Telomere shortening triggers senescence of human cells through a pathway involving ATM, p53, ρ21CIPl, but not ρl6INK4a. MoI. Cell 14, 501-513]. Various stresses that induce DNA damage such as radiation or oxidative stress cause the premature senescence, which is similar to the replicative senescence [Ben-Porath, I. and Weinberg, R. A. (2004) When cells get stressed: an integrative view of cellular senescence. J. Clin. Invest. 113, 8-13]. Even though all intracellular or extracelluar stresses do not cause ATM/ATR-dependent cellular senescence, it was reported that ATM/ ATR plays an important role in the cellular senescence process. In addition, it was reported that if the function of ATM/ ATR is blocked, the cell cycle resumes [d¹ Adda di Fagagna, F. et al. (2003) A DNA damage checkpoint response in telomere-initiated senescence. Nature 426, 194-198]. Therefore, ATM/ ATR inhibitors can be used for controlling abnormal symptoms induced by the cellular senescence.

Mutation of ATM/ ATR in a mammalian cell results in the shortening of telomere [Pnadita, T. K. (2002) ATM function and telomere stability. Oncogene 21, 611-618]. It is known that the telomere is the terminal structure of a linear chromosome in a eukaryotic cell, and the essential structure for consistently maintaining the linear chromosome. In a normal cell, the telomere becomes shorter for every cell division. If the telomere becomes shorter to excess, cells no longer divide. It has been reported that the telomere becomes extremely shorter in various tissues of an Atm -/- mouse [Hande, M. P. et al. (2001) Extra-chromosomal telomeric DNA in cells from Atm -/- mice and patients with ataxia-telangiectasia. Hum. MoI. Genet. 10, 519-528]. Thus, ATM/ ATR inhibitors can be used as therapeutic agents for treating cellular proliferative diseases caused by the abnormal elongation of the telomere. The ATM/ATR is related with a response to oxidative stress. It was reported that

ATR responds to hypoxia. It was also reported that the ATM responds to reoxygenation so as to show its activity [Watters, D. J. (2003) Oxidative stress in ataxia telangiectasia. Redox. Rep. 8, 23-29; Hammond, E. M. et al. (2003) Hypoxia links ATR and p53 through replication arrest. MoI. Cell. Biol. 22, 1834-1843; Hammond, E. M. (2003) ATR/ATM targets are phosphorylated by ATR in response to hypoxia and ATR in response to reoxygenation. J. Biol. Chem. 278, 12207-12213]. Therefore, ATM/ATR inhibitors can be potentially used for controlling the abnormality in cell function caused by any oxidative stress.

The stress occurred in the chronic inflammation is caused by free radicals such as NO. The p53 protein is phosphorylated by the free radicals such as NO, wherein the ATM/ATR protein is concerned with the posttranslational modification of the p53 protein [Hofseth, L. J. et al. (2002) Nitric oxide-induced cellular stress and p53 activation in chronic inflammation. Proc. Natl. Acad. Sci. 100, 143-148]. In the chronic inflammation, the free radicals such as NO have been known to continually cause genetic damage. Thus, ATM/ATR inhibitors can be used for controlling cytotoxicity derived from the free radicals.

The ATM protein was reported to contribute to the stabilization of p53 protein by heat shock [Wang, C. and Chen, J. (2003) Phosphorylation and hsp90 binding mediate heat shock stabilization of p53. J. Biol. Chem. 278, 2066-2071]. The stabilization of p53 protein by heat shock causes cell cycle arrest or apoptosis [Nitta, M. et al. (1997) Heat shock induces transient p53-dependent cell cycle arrest at Gl/S. Oncogene 15, 561-568; Ohnishi, T. et al. (1996) p53-dependent induction of WAFl by heat treatment in human glioblastoma cells. J. Biol. Chem. 271 , 14510- 14513] . Therefore, ATM/ATR inhibitors can be used for controlling the abnormality in cells induced by heat shock.

AT patients are characterized by hypoplasia of thymus and deficiency in immune mechanism. An Atm-/- mouse was reported to show various immunodeficiencies similar to those as shown in AT patients [Xu, Y. and Baltimore, D. (1996) Dual roles of

ATM in the cellular response to radiation and in cell growth control. Genes Dev. 10,

2401 -2410] . Thus, ATM/ ATR inhibitors can be used for controlling immune function.

The ATM/ ATR plays a significant role in diseases caused by retroviruses. It was disclosed that the ATM/ ATR is required to stably introduce the DNA of a retrovirus into the genome of a host cell [Daniel, R. et al. (2001) Wortmannin potentiates integrase- mediated killing of lymphocytes and reduces the efficiency of stable transduction by retroviruses. MoI. Cell. Biol. 21, 1164-72; Daniel, R. et al. (2003) Evidence that the retroviral DNA integration process triggers an ATR-dependent DNA damage response. Proc. Natl. Acad. Sci. 100, 4778-4783; Roshal, M. et al. (2003) Activation of the ATR- mediated DNA damage response by the HIV-I viral protein R. J. Biol. Chem. 278, 25879-25886]. In addition, it was recently reported that ATM inhibitors suppress HIV- 1 infection [Lau, A. et al. (2005) Suppression of HIV-I infection by a small molecule inhibitor of the ATM kinase. Nat. Cell Biol. 7, 493-500]. Therefore, ATM/ATR inhibitors can be used for treating retrovirus-mediated diseases such as HIV infection and AIDS; and Human T-cell Lymphotropic Virus (HTLV) infection, HTLV associated Adult T-cell Leukemia/Lymphoma (ATLL), and Tropical Spastic Paraparesis/HTLV-1 Associated Myelopathy (TSP/HAM).

DETAILED DESCRIPTION OF THE INVENTION

TECHNICAL SUBJECT The present invention provides a urea compound of formula (1), its derivatives and pharmaceutically acceptable salts thereof that specifically inhibit the function of protein kinases of ATM and ATR by selectively binding to ATM and ATR.

In addition, the objective of the present invention is to provide a process for preparing a urea compound of formula (1), its derivatives and pharmaceutically acceptable salts thereof, and a pharmaceutical composition comprising them as an effective ingredient.

TECHNICAL SOLUTION

We have completed the invention and confirmed its significant effect of inhibiting activity of ATM/ ATR by introducing various substituents into the basic structure of the urea compound.

EFFECT OF THE INVENTION

Urea compounds according to the present invention can be used for controlling cellular function and treating diseases, in connection with the abnormality in the function of ATM and ATR.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a western blot picture showing that phosphorylation of p53 protein by ATR protein is inhibited by the urea compounds.

Fig. 2 is a western blot picture showing that phosphorylation of p53 protein by ATM protein is inhibited by the urea compounds. Fig. 3 is a western blot picture showing that phosphorylation of Serl5 of p53 protein by ATM protein is inhibited by the urea compounds in RKO cells and GM847 cells.

Fig. 4 is an electrophoresis picture showing that phosphorylation of p53 protein by ATM and ATR protein is inhibited by the urea compounds in vitro. Fig. 5 is a graph showing that activity of ATM and ATR is inhibited by the urea compounds depending on the concentration thereof in vitro.

Fig. 6 is an electrophoresis picture showing that phosphorylation of p53 protein by other protein kinases is not inhibited by the urea compounds in vitro.

Fig. 7 is a graph showing that apoptosis of human cancer cells by chemotherapy is increased by addition of the urea compounds. The numbers in the small squares represent the treatment concentration (μM) of the compounds.

Fig. 8 is a graph showing that apoptosis of RKO cells by doxorubicin is inhibited by the urea compounds depending on the concentration thereof.

Fig. 9 shows the analysis result that the suppression of the cell cycle in the RKO cells by doxorubicin is inhibited by the urea compounds.

Fig. 10 is a growth graph showing that the urea compounds inhibit replicative senescence; BJ cells were continuously subcultured to reach replicative senescence (as indicated by asterisk); then treated by the urea compounds (as indicated by an inverted triangle) and continuously subcultured; thereafter subcultured again without the urea compounds (as indicated by a triangle); and subcultured by treating the cells again with the urea compounds.

Fig. 11 is a cell picture showing that SA-β-gal dyeing is inhibited by the urea compounds.

Fig. 12 is a cell picture showing that SA-β-gal dyeing formed as a result of premature senescence is inhibited by the urea compounds.

BEST MODE FOR CARRYING OUT THE INVENTION

To achieve the above objective, the present invention provides a urea compound of formula (1), its derivatives and pharmaceutically acceptable salts thereof, as follows: Formula 1

wherein,

R₁ is any one of the following structures:

R₂ is any one of the following structures:

X is H, CH₃, CF₃, or CCl₃; and Y is O or S.

Hereinafter, the present invention will be described in more detail.

The above urea compounds of formula (1) may contain optical isomers, and may exist in free form or in the form of an acid or base addition salt thereof. The preferable acid addition salt may be, without limitation, hydrochloric acid, sulphuric acid, acetic acid, trifluoracetic acid, phosphoric acid, fumaric acid, maleic acid, citric acid, or lactic acid.

Preferably, in the above compound of formula (1),

R₂ is any one of the following structures:

X is CF₃, or CCl₃; and

Y is O, or S.

More preferably, in the above compound of formula (1),

R₁ is any one of the following structures:

R₂ is any one of the following structures;

X is CCl₃; and Y is S. The term "a urea compound(s)" used herein means a compound(s) that can inhibit the activity of ATM or ATR protein, and, in more detail, contains a prodrug thereof and all compounds having unique inhibition activity, wherein the prodrug itself has a little activity or no unique inhibition activity.

Some analysis methods that can be used for determining the decrement in activity of ATM or ATR protein by certain compounds of the present invention are described in the following examples.

The present invention provides a method of inhibiting the activity of ATM or ATR protein in cells, which comprises contacting the effective amount of the urea compounds, preferably in the form of a pharmaceutically acceptable composition, with cells. For example, cells (e.g., tumor cells or normal cells) that may be cultured in vitro is contacted with the urea compounds and a medicine having the known therapeutic effect, and so increased therapeutic effect of the compounds on the cells is observed.

The present invention provides a method of inhibiting the activity of ATM or ATR protein in vivo or in vitro comprising contacting the effective amount of the urea compounds with cells, and also provides the urea compounds inhibiting the activity of ATM or ATR protein.

MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in more detail with reference to the following examples, but the scope of the present invention should not be construed to be limited thereby in any manner since the following examples are described only for specifying the present invention. That which can be inferred from the detailed description and examples of the present invention by a skilled person in the art should be interpreted as being within the scope of the present invention. The references cited in the present invention are incorporated in the present invention for reference.

The present invention also provides a process of preparing the urea compound of formula (1). The urea compound of the above formula (1) can be synthesized by the following reaction formula (1). The compounds of the present invention may be in the form of optical isomers or diastereomers, and can be isolated and collected by the conventional technique.

A) Chemical example

The compound of formula (1) according to the present invention is prepared by synthesis process of the following reaction scheme (1). Reaction scheme (1)

wherein, R₁, R₂, X and Y are the same as defined in the above. In the reaction scheme (1), after the acid compound of formula (2) is stirred together with BoC₂O, ethyl chloroformate and isobutyl chloroformate in the presence of a suitable base such as triethylamine and pyridine, under nitrogen, NH₃ and NH₄HCO₃ are added thereto, and then the compound of formula (3) is synthesized. The reaction is completed when all of the compound of formula (2) has been consumed, which can be easily confirmed by thin-layer chromatography. The reaction solvents are preferably dichloromethane, dioxane and so on. The reaction temperature is O ^°C to room temperature. The reaction time is suitably 12 to 36 hours.

The amide compound of formula (3) is reacted with chloral hydrate, trifluoroacetaldehyde hemiacetal or acetaldehyde and with benzene or toluene to synthesize the compound of formula (4). If necessary, the addition of benzotriazole can promote the reaction. The reaction temperature is room temperature under reflux condition. The reaction time is suitably 12 to 36 hours. The compound of formula (4) is reacted with SOCl₂, PCl₅ or oxalylchloride and with benzene or toluene to synthesize the compound of formula (5). The reaction temperature is room temperature under reflux condition. The reaction time is suitably 1 to 24 hours. The compound of formula (5) is reacted with KSCN or KOCN and with dichloromethane, dioxane or acetonitrile, and then suitable amine derivatives (R₂NH₂) are added thereto, and thus the compound of formula (1) is synthesized. The reaction temperature is room temperature under reflux condition. The reaction time is suitably 1 to 24 hours. Aqueous hydrogen peroxide is added to the compound of formula (1), wherein

Y=S, under the acetic acid conditions, and thus the compound of formula (1), wherein Y=O, can be synthesized. The reaction temperature is room temperature under reflux condition. The reaction time is suitably 1 to 24 hours.

The present invention comprises a pharmaceutical composition, for example, a composition useful for inhibiting the activity of ATM/ ATR, comprising a pharmaceutically acceptable carrier or a diluent and the effective amount of compounds of formula (1) or their derivatives or pharmaceutically acceptable acid addition salts thereof.

The present invention provides a method of inhibiting the activity of ATM/ ATR comprising administering therapeutically effective amount of compounds of formula (1) or their derivatives or pharmaceutically acceptable acid addition salts thereof, to a mammal that requires inhibition of ATM/ ATR.

Further, the present invention provides a method for treating a certain disease, which includes administering therapeutically effective amount of compounds of formula (1) or their derivatives or pharmaceutically acceptable acid addition salts thereof, to a mammal that requires treatment of diseases mediated by ATM/ ATR. The compounds of the present invention can be used as additives in combination with radiation therapy and chemotherapy for increasing their sensitivity in treating ATM/ATR mediated diseases, for example, various solid cancers and hematologic malignancy. Further, the present invention provides a method of treating degenerative brain diseases in which apoptosis occurs by genotoxicity and neurotoxicity (e.g., Alzheimer's disease, Huntington's disease, hypoxia, Parkinson's disease, stroke, traumatic brain injury, ischemic insult and excitotoxic insult, spinobulbar muscular atrophy, DRPLA, SCA and so on), abnormal symptoms caused by cellular senescence such as replicative senescence or premature senescence, cellular proliferative diseases, abnormality in cellular function caused by oxidative stress, chronic inflammation, cellular abnormality induced by heat shock, and retrovirus-mediated diseases, and controlling immune function.

The present invention also relates to the use of the compounds of the present invention or their derivatives or pharmaceutically acceptable salts thereof, in preparing a medicine for preventing and treating diseases or disorders, for example, those related to increment of the amount of ATM/ATR. Representative structure and NMR spectrum data of the urea compounds having formula (1) according to the present invention are shown in the following Table 1. Table 1. Structure and activity of urea compounds

The names of each of the compounds corresponding to the compound numbers in table 1 are as follows: 1) 2,2-diphenyl-N-{2,2,2-trichloro-l-[3-(3-nitro-phenyl)-thioureido]- ethyl } -acetamide

2) 3 -methyl-N- { 2,2,2-trichloro- 1 - [3-(2-nitro-phenyl)-thioureido] -ethyl } - benzamide

3) 3-nitro-N-{2,2,2-trichloro-l-[3-(4-nitro-phenyl)-thioureido]-ethyl}- benzamide

4) 4-nitro-N-[2,2,2-trichloro-l-(3-phenyl-thioureido)-ethyl]-benzamide

5) 2-phenyl-N-{2,2,2-trichloro-l-[3-(4-nitro-phenyl)-thioureido]-ethyl}- acetamide 6) 2-phenyl-N-{2,2,2-trichloro-l-[3-(3-nitro-phenyl)-thioureido]-ethyl}- acetamide

7) 2-naphthalene- 1 -yl-N- {2,2,2-trichloro- 1 -[3-(4-nitro-phenyl)- thioureido] -ethyl } -acetamide

8) 2-naphthalene- 1 -yl-N- {2,2,2-trichloro- 1 - [3 -(3 -nitro-phenyl)- thioureido] -ethyl} -acetamide

9) 2-naphthalene-l-yl-N-{2,2,2-trichloro-l-[3-(2-nitro-phenyl)- thioureido] -ethyl} -acetamide

10) 2-methyl-4-nitro-penta-2,4-dienoic acid {2,2,2-trichloro-l-[3-(3-nitro- phenyl)-thioureido] -ethyl } -amide 12) 3-methyl-N- {2,2,2-trichloro- 1 -[3-(3-nitro-phenyl)-thioureido]-ethyl} - benzamide

13) 3,5-dinitro-N-[2,2,2-trichloro-l-(3-phenyl-thioureido)-ethyl]- benzamide

14) N- {2,2,2-trichloro- 1 - [3-(4-nitro-phenyl)-thioureido] -ethyl} -benzamide 15) 2-nitro-N-[2,2,2-trichloro-l-(3-phenyl-thioureido)-ethyl]-benzamide

16) 2-nitro-N-[2,2,2-trichloro-l-(3-m-tolyl-thioureido)-ethyl]-benzamide

17) 2-nitro-N-{2,2,2-trichloro-l-[3-(3-nitro-phenyl)-thioureido]-ethyl}- benzamide 18) 3,5-dinitro-N-[2,2,2-trichloro-l-(3-o-tolyl-thioureido)-ethyl]- benzamide

19) 3-nitro-N-{2,2,2-trichloro-l-[3-(2,5-dimethyl-phenyl)-thioureido]- ethyl}-benzamide 20) N-{ l-[3-(4-acetylamino-phenyl)-thioureido]-2,2,2-trichloro-ethyl}-2- nitro-benzamide

21) 2,2-diphenyl-N-[2,2,2-trichloro-l-(3-phenyl-thioureido)-ethyl]- acetamide

22) 2,2-diphenyl-N-{2,2,2-trichloro-l-[3-(2-nitro-phenyl)-thioureido]- ethyl} -acetamide

23) 2,2-diphenyl-N-{2,2,2-trichloro-l-[3-(lH-[l,2,4]triazol-3-yl)- thioureido] -ethyl } -acetamide

24) 2,2-diphenyl-N-{2,2,2-trichloro-l-[3-(2-methoxy-5-nitro-ρhenyl)- thioureido] -ethyl } -acetamide 25) furan-2-carboxylic acid {2,2,2-trichloro-l-[3-(4-nitro-phenyl)- thioureido] -ethyl} -amide

27) 2-phenyl-3-vinyl-pent-3-enoic acid {2,2,2-trichloro-l-[3-(3-hydroxy- phenyl)-thioureido] -ethyl } -amide

28) 2,2-diphenyl-N-{2,2,2-trichloro-l-[3-(3-cyano-phenyl)-thioureido]- ethyl} -acetamide

29) 3-[3-(2,2,2-trichloro-l-diphenylacetylamino-ethyl)-thioureido]- benzamide

30) N-{l-[3-(3-acetylamino-phenyl)-thioureido]-2,2,2-trichloro-ethyl}-2,2- diphenyl-acetamide

31) N-{ l-[3-(3-amino-phenyl)-thioureido]-2,2,2-trichloro-ethyl}-2,2- diphenyl-acetamide

32) 2,2-diphenyl-N-{2,2,2-trichloro-l-[3-(4-hydroxy-3-nitro-ρhenyl)- thioureido] -ethyl }-acetamide

33) 2,2-diphenyl-N-{2,2,2-trichloro-l-[3-(4-fluoro-3-nitro-phenyl)- thioureido] -ethyl } -acetamide

34) 2,2-diphenyl-N-{2,2,2-trifluoro-l-[3-(3-nitro-phenyl)-thioureido]- ethyl } -acetamide 35) N-{ 1 -[3-(3-cyano-phenyl)-thioureido]-2,2,2-trifluoro-ethyl}-2,2- diphenyl-acetamide

36) 9H-xanthen-9-carboxylic acid {2,2,2-trichloro-l-[3-(3-nitro-phenyl)- thioureido] -ethyl } -amide

37) 9H-xanthen-9-carboxylic acid {2,2,2-trichloro-l-[3-(3-cyano-phenyi)- thioureido] -ethyl} -amide

38) 2,2-diphenyl-N-{2,2,2-trichloro-l-[3-(3-nitro-phenyl)-ureido]-ethyl}- acetamide

39) 2,2-diphenyl-N-{2,2,2-trichloro-l-[3-(3-cyano-phenyl)-ureido]-ethyl}- acetamide 40) N- { l-[3-(3-nitro-phenyl)-thioureido]-ethyl}-2,2-diphenyl-acetamide

41) N- { 1 - [3-(3 -cyano-phenyl)-thioureido] -ethyl } -2,2-diphenyl-acetamide

The urea compounds of formula (1) of the present invention, prodrugs, optical isomers, diastereomers, their derivatives and pharmaceutically acceptable salts thereof show inhibitory activity of ATM/ATR, and thus the pharmaceutical composition comprising at least one of them as an effective ingredient(s) is useful as an agent for controlling the cellular function and treating diseases in connection with the abnormality in the function of ATM and ATR.

Therefore, the present invention provides a pharmaceutical composition for controlling the cellular function and treating diseases in connection with the abnormality in the function of ATM and ATR, comprising at least one of the above urea compounds of formula (1), prodrugs, optical isomers, diastereomers, their derivatives or salts thereof, as an effective ingredient(s).

The pharmaceutical composition of the present invention can be provided in various oral dosage forms or parenteral dosage formulations. The examples of oral dosage formulations are tablets, pills, hard and soft capsules, solutions, suspensions, emulsions, syrup, granules, elixirs, etc., wherein these formulations contain diluents (e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycine), or lubricants (e.g., silica, talc, stearic acid and magnesium or calcium salt thereof, and/or polyethylene glycol) as well as the effective ingredient(s). In addition, the tablets may contain a binding agent(s) such as magnesium aluminium silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidine, and in some cases, may further contain disintegrant or boiling mixture such as starch, agar, arginic acid or sodium salt thereof, and/or absorbent, colorant, flavoring agent, and sweetening agent.

Further, the pharmaceutical composition comprising the above compound of formula (1) as an effective agent can be parenterally administered, wherein the parenteral administration is performed by subcutaneous injection, intravenous injection, intramuscular injection or intrathoracic injection. In order to prepare the parenteral dosage formulations, the above compounds of formula (1) or their derivatives or pharmaceutically acceptable salts thereof are mixed with stabilizer or buffer in water to produce their solution or suspension, and then put into an ampule or vial in unit dosage form.

The above composition may be sterilized and/or contain preservatives, stabilizers, hydration agents or emulsification promoters, supporting agents such as salt for controlling osmotic pressure and/or buffers, and therapeutically useful substances, and can be formulated according to conventional methods such as mixing, granulation or coating.

The compound of formula (1) as an effective ingredient can be orally or parenterally administered to a mammal including a human at a dosage of 0.1 to 500 ing/ kg (on the basis of body weight), preferably, 0.5 to 100 mg/kg (on the basis of body weight), 1 time a day or with certain intervals.

Hereinafter, the present invention will be described in more detail with reference to the following examples, but the scope of the present invention should not be construed to be limited thereby in any manner.

Example 1: Synthesis of compound No. 1 of Table 1 (2,2-diphenyl-N- (2.2.2- trichloro-l-r3-(3-nitro-phenyl)-thioureidol-ethvU-acetamide) (Step 1) Synthesis of 2,2-diphenyl-acetamide

10 g (47.1 mmol) of diphenyl acetic acid and 12.34 g (56.5 mmol, 1.2 eq) of (Boc)₂O were added. 2.2 g (28.3 mmol, 0.6 eq) of pyridine and 5.6 g (70.7 mmol, 1.5 eq) Of NH₄HCO₃ were added thereto. Then, 100 ml of 1,4-dioxane was added thereto to dissolve them. Thereafter, the solution was stirred for 12 hours at room temperature. After the reaction was completed, the reactant was extracted with an organic layer by using EA/H₂O. The organic layer was concentrated, and then diethyl ether was added for crystallization thereto. The resultant solution was stirred for 1 hr and then filtered to obtain 9.49 g (95%) of 2,2-diphenyl-acetamide. ¹H NMR (200 MHz, CDCl₃) δ 7.35-7.27 (m, 10H) 5.99 (brs, IH) 5.60 (brs, IH)

(Step 2) Synthesis of 2,2-diphenyl-N-(2,2,2-trichloro-l-hydroxy-ethyI)- acetamide

4 g (18.9 mmol) of 2,2-diphenyl-acetamide and 3.4 g (20.7 mmol, 1.1 eq) of chloral hydrate were added, and 30 ml of benzene was added thereto to dissolve them. Then, for 12 hours, it was refluxed and stirred. After the reaction was completed, the reactant was concentrated. The residual starting materials were crystallized by adding diethyl ether, and then filtered and removed. The reaction solution was concentrated to obtain 6.13 g (91%) of 2,2-diphenyl-N-(2,2,2-trichloro-l-hydroxy-ethyl)-acetamide.

¹H NMR (200 MHz, CDCl₃) δ 7.41~7.23(m, 10H) 6.50~6.45(m, IH) 5.95~5.87(m, IH) 5.02(s, IH) 4.7(brs, IH)

(Step 3) Synthesis of 2,2-diphenyI-N-(l,2,2,2-tetrachloro-ethyL)-acetamide

3.4 g (9.37 mmol) of 2,2-diphenyl-N-(2,2,2-trichloro-l-hydroxy-ethyl)- acetylamide was added and 15 ml of benzene was added thereto. Then, 2.8 g (23.42 mmol, 2.5 eq) of SOCl₂ was added thereto, and refluxed and stirred for about 6 hours. After the reaction was completed, the reactant was completely concentrated, and crystallized by adding hexane, and then stirred for about 30 min. The solid materials were filtered to obtain 2.36 g (66.9 %) of 2,2-diphenyl-N-(l,2,2,2-tetrachloro-ethyl)- acetamide.

¹H NMR (200 MHz, CDCl₃) δ 7.43~7.25(m, 10H) 6.57(s, IH) 5.06(s, IH) (Step 4) Synthesis of 2,2-diphenyl-N-{2,2,2-trichloro-l-[3-(3-nitro-phenyl)- thioureido-ethy 1] -acetamide

1,000 mg (2.652 mmol) of 2,2-diphenyl-N-(l,2,2,2-tetrachloro-ethyl)-acetamide was added and 40 ml of CH₃CN was added thereto. Then, 284 mg (2.92 mmol, 1.1 eq) of KNCS was added thereto, and stirred for 1 hr. After the reaction was completed, the reactant was filtered, and thus remaining KNCS was removed. To the reaction solution thereof, 403 mg (2.92 mmol, 1 eq) of 3-nitroaniline was added, and stirred at room temperature for 12 hours. After the reaction was completed, the reactant was concentrated, and crystallized by adding diethyl ether thereto. Then, it was stirred at room temperature for about 30 min, and then filtered to obtain 800 mg (56%) of 2,2- diphenyl-N-{2,2,2-trichloro-l-[3-(3-nitro-phenyl)-thioureido]-ethyl}-acetamide.

Example 2: Synthesis of compound No. 27 of Table 1 (2.2-diphenyl-N- (2.2,2- trichloro- 1 - [3 -(3 -hydroxy-phenyl )- thioureidol -ethyl } -acetamide)

100 mg (0.27 mmol) of 2,2-diphenyl-N-(l,2,2,2-tetrachloro-ethyl)-acetamide obtained in Example 1 was added, and 7 ml of CH₃CN was added thereto. Then, 28 mg (0.28 mmol, 1.1 eq) of KNCS was added thereto and stirred for 1 hr. After the reaction was completed, the reactant was filtered, and thus remaining KNCS was removed. To the reaction solution thereof, 29 mg (0.27 mmol, 1 eq) of 3-hydroxy aniline was added, and stirred at room temperature for 12 hours. After the reaction was completed, the reactant was concentrated, and crystallized by adding diethyl ether thereto. Then, it was stirred at room temperature for about 30 min, and then filtered to obtain 41 mg (30%) of 2,2-diphenyl-N-{2,2,2-trichloro-l-[3-(3-hydroxy-phenyl)- thioureido] -ethyl-acetamide.

Example 3: Synthesis of compound No. 28 of Table 1 (2,2-diphenyl-N-{ 2,2,2- trichloro-1-[3-(3-cyano-phenyl)-thioureido]-ethyl} -acetamide)

100 mg (0.27 mmol) of 2,2-diphenyl-N-(l,2,2,2-tetrachloro-ethyl)-acetamide obtained in Example 1 was added, and 7 ml of CH₃CN was added thereto. Then, 28 mg (0.28 mmol, 1.1 eq) of KNCS was added thereto and stirred for 1 hr. After the reaction was completed, the reactant was filtered, and thus remaining KNCS was removed. To the reaction solution thereof, 31 mg (0.27 mmol, 1 eq) of 3- aminobenzonitrile was added, and stirred at room temperature for 12 hours. After the reaction was completed, the reactant was concentrated, and crystallized by adding diethyl ether thereto. Then, it was stirred at room temperature for about 30 min, and then filtered to obtain 99 mg (72%) of 2,2-diphenyl-N-{2,2,2-trichloro-l-[3-(3-cyano- phenyl)-thioureido] -ethyl-acetamide.

Example 4: Synthesis of compound No. 29 of Table 1 (3-[3-(2,2.2-trichloro-l- diphenylacetylamino-ethyl)-thioureido]-benzamide) 100 mg (0.27 mmol) of 2,2-diphenyl-N-(l,2,2,2-tetrachloro-ethyl)-acetamide obtained in Example 1 was added and 7 ml of CH₃CN was added thereto. Then, 28 mg (0.28 mmol, 1.1 eq) of KNCS was added thereto and stirred for 1 hr. After the reaction was completed, the reactant was filtered, and thus remaining KNCS was removed. To the reaction solution thereof, 36 mg (0.27 mmol, 1 eq) of 3- aminobenzamide was added, and stirred at room temperature for 12 hours. After the reaction was completed, the reactant was concentrated, and crystallized by adding diethyl ether thereto. Then, it was stirred at room temperature for about 30 min, and then filtered to obtain 82 mg (58%) of 3-[3-(2,2,2-trichloro-l-diphenylacetylamino- ethyl)-thioureido]-benzamide.

Example 5: Synthesis of compound No. 30 of Table 1 (TSf-I l-[3-(3-acetylamino- phenylVthioureido]-2,2,2-trichloro-ethyU-2,2-diphenyl-acetamide')

100 mg (0.27 mmol) of 2,2-diphenyl-N-(l,2,2,2-tetrachloro-ethyl)-acetamide obtained in Example 1 was added and 7 ml of CH₃CN was added thereto. Then, 28 mg (0.28 mmol, 1.1 eq) of KNCS was added thereto and stirred for 1 hr. After the reaction was completed, the reactant was filtered, and thus remaining KNCS was removed. To the reaction solution thereof, 40 mg (0.27 mmol, 1 eq) of 3- aminoacetanilide was added, and stirred at room temperature for 12 hours. After the reaction was completed, the reactant was concentrated, and crystallized by adding diethyl ether thereto. Then, it was stirred at room temperature for about 30 min, and then filtered to obtain 94 mg (65%) of N-{l-[3-(3-acetylamino-phenyl)-thioureido]- 2,2,2-trichloro-ethyl}-2,2-diphenyl-acetamide. Example 6: Synthesis of compound No. 31 of Table 1 (N -{ 1-[3-(3-amino- phenylVthioureidol-2,2,2-trichloro-ethvU-2,2-diphenyl-acetamide)

100 mg (0.27 mmol) of 2,2-diphenyl-N-(l,2,2,2-tetrachloro-ethyl)-acetamide obtained in Example 1 was added and 7 ml of CH₃CN was added thereto. Then, 28 mg (0.28 mmol, 1.1 eq) of KNCS was added thereto and stirred for 1 hr. After the reaction was completed, the reactant was filtered, and thus remaining KNCS was removed. To the reaction solution thereof, 29 mg (0.27 mmol, leq) of 3- phenylenediamine was added, and stirred at room temperature for 12 hours. After the reaction was completed, the reactant was concentrated, and crystallized by adding diethyl ether thereto. Then, it was stirred at room temperature for about 30 min, and then filtered to obtain 94 mg (65%) of N-{l-[3-(3-amino-phenyl)-thioureido]-2,2,2- trichloro-ethyl}-2,2-diphenyl-acetamide.

Example 7: Synthesis of compound No. 32 of Table 1 (2,2-diphenyl-N- (2,2,2- trichloro-l-[3-(4 -hvdroxy-3-nitro-phenyl)thioureidoi-ethvl}-acetamide)

100 mg (0.27 mmol) of 2,2-diphenyl-N-(l,2,2,2-tetrachloro-ethyl)-acetamide obtained in Example 1 was added and 7 ml of CH₃CN was added thereto. Then, 28 mg (0.28 mmol, 1.1 eq) of KNCS was added thereto and stirred for 1 hr. After the reaction was completed, the reactant was filtered, and thus remaining KNCS was removed. To the reaction solution thereof, 41 mg (0.27 mmol, leq) of 3-amino-2-nitro phenol was added, and stirred at room temperature for 12 hours. After the reaction was completed, the reactant was concentrated, and crystallized by adding diethyl ether thereto. Then, it was stirred at room temperature for about 30 min, and then filtered to obtain 77 mg (52.4%) of 2,2-diphenyl-N-{2,2,2-trichloro-l-[3-(4-hydroxy-3-nitro- phenyl)-thioureido]-ethyl}-acetamide.

Example 8: Synthesis of compound No. 33 of Table 1 (2,2-diρhenyl-N- (2,2,2- trichloro- 1 - [3 -(4-fluoro-3-nitro-phenyl)- thioureido] -ethyl} -acetamide)

100 mg (0.27 mmol) of 2,2-diρhenyl-N-(l,2,2,2-tetrachloro-ethyl)-acetamide obtained in Example 1 was added and 7 ml of CH₃CN was added thereto. Then, 28 mg (0.28 mmol, 1.1 eq) of KNCS was added thereto and stirred for 1 hr. After the reaction was completed, the reactant was filtered, and thus remaining KNCS was removed. To the reaction solution thereof, 41 mg (0.27 mmol, 1 eq) of 4-fluoro-2-nitro aniline was added, and stirred at room temperature for 12 hours. After the reaction was completed, the reactant was concentrated, and crystallized by adding diethyl ether thereto. Then, it was stirred at room temperature for about 30 min, and then filtered to obtain 71 mg (48%) of 2,2-diphenyl-N-{2,2,2-trichloro-l-[3-(4-fluoro-3-nitro-phenyl)- thioureido] -ethyl } -acetamide .

Example 9: Synthesis of compound No. 34 of Table 1 (2,2-diphenyl-N- (2,2,2- trifluoro-l-[3-(3-nitro-phenyl)-thioureido] -ethyl} -acetamide) (Step 1) Synthesis of 2,2-diphenyI-N-(2,2,2-trifluoro-l-hydroxy-ethyl)- acetamide

1.5 g (7.1 mmol) of 2,2-diphenyl-acetamide and 1.1 g (7.8 mmol, 1.1 eq) of trifluoroacetaldehyde ethyl hemiacetal were added, and 30 ml of 1,4-dioxane was added thereto to dissolve them. Then, it was refluxed and stirred for 48 hours. After the reaction was completed, the reactant was concentrated. The residual starting materials were crystallized by adding diethyl ether thereto, and then filtered and removed. The reaction solution was concentrated to obtain 6.13 g (91%) of 2,2-diphenyl-N-(2,2,2- trifluoro- 1 -hydroxy-ethyl)-acetamide.

¹H NMR (200 MHz, CDCl₃) δ 9.43~9.40(m, IH) 7.48~7.46(m, IH) 7.33~7.21(m, 10H) 5.73~5.66(m, IH) 5.13(s, IH)

(Step 2) Synthesis of N-(l-chloro-2,2,2-trifluoro-ethyl)-2,2-diphenyl- acetamide 1 g (3.23 mmol) of 2,2-diphenyl-N-(2,2,2-trifluoro-l-hydroxy-ethyl)-acetamide was added and 15 ml of benzene was added thereto. Then, 0.96 g (8.085mmol, 2.5 eq) of SOCl₂ was added thereto, and refluxed and stirred for about 6 hours. After the reaction was completed, the reactant was completely concentrated, and crystallized by adding hexane thereto, and then stirred for about 30 min. The solid materials were filtered to obtain 0.57 g (53.9 %) of N-(l-chloro-2,2,2-trifluoro-ethyl)-2,2-diphenyl- acetamide.

¹H NMR (200 MHz, CDCl₃) δ 7.43~7.23(m, 10) 6.39(s, IH) 5.03(s, IH) (Step 3) Synthesis of 2,2-diphenyI-N-{2,2,2-trifluoro-l-[3-(3-nitro-phenyl)- thioureido]-ethyl}-acetamide 225 mg (0.69 mmol) of N-(l-chloro-2,2,2-trifluoro-ethyl)-2,2-diphenyl-acetamide was added and 10 ml of CH₃CN was added thereto. Then, 74 mg (0.76 mmol, 1.1 eq) of KNCS was added thereto, and stirred for 1 hr. After the reaction was completed, the reactant was filtered, and thus remaining KNCS was removed. To the reaction solution thereof, 95 mg (0.69 mmol, leq) of 3-nitro aniline was added, and stirred at room temperature for 12 hours. After the reaction was completed, the reactant was concentrated, and crystallized by adding diethyl ether thereto. Then, it was stirred at room temperature for about 30 min, and then filtered to obtain 85 mg (25%) of 2,2- diphenyl-N-{2,2,2-trifluoro-l-[3-(3-nitro-phenyl)-thioureido]-ethyl}-acetamide.

Example 10: Synthesis of compound No. 35 of Table 1 (N- (l-[3-(3-cyano- phenyl)-thioureido1-2,2,2-trifluoro-ethyll-2,2-diphenyl-acetamide)

225 mg (0.69 mmol) of N-(l-chloro-2,2,2-trifluoro-ethyl)-2,2-diphenyl-acetamide obtained in Example 9 was added and 10 ml of CH₃CN was added thereto. Then, 74 mg (0.76 mmol, 1.1 eq) of KNCS was added thereto, and stirred for 1 hr. After the reaction was completed, the remaining KNCS was removed from the reactant by filtration and 82 mg (0.69 mmol, 1 eq) of 3-aminobenzonitrile was added to the reaction solution thereof, and then stirred at room temperature for 12 hours. After the reaction was completed, the reactant was concentrated, and crystallized by adding diethyl ether thereto. Then, it was stirred at room temperature for about 30 min, and then filtered to obtain 241 mg (75%) of N-{l-[3-(3-cyano-phenyl)-thioureido]-2,2,2-trifluoro-ethyl}- 2,2-diphenyl-acetamide.

Example 11 : Synthesis of compound No. 36 of Table 1 (N-(2,2,2-trichloro-l-(3-

Q-nitrophenyPthioureido^ethyD-gH-xanthen-g-carboxamide) (Step 1) Synthesis of 9H-xanthen-9-carboxamide 10 g (44.2 mmol) of xanthen-9-carboxylic acid and 11.6 g (53.0 mmol, 1.2 eq) of (Boc)₂O were added. Then, 2.1 g (26.5 mmol, 0.6 eq) of pyridine and 5.3 g (66.3 mmol, 1.5 eq) Of NH₄HCO₃ were added thereto. Then, 100 ml of 1,4-dioxane was added thereto to dissolve them. Thereafter, the solution was stirred for 12 hours at room temperature. After the reaction was completed, the reactant was extracted with an organic layer by using EA/H₂O. The organic layer was concentrated, and then diethyl ether was added for crystallization thereto. The resultant solution was stirred for 1 hr and then filtered to obtain 8.47 g (85%) of 9H-xanthen-9-carboxamide.

¹H NMR (200 MHz, CDCl₃) δ 7.8(brs, IH) 7.33~7.24(m, 4H) 7.13~7.05(m, 5H) 4.88(s, IH) (Step 2) Synthesis of N-(2,2,2-trichloro-l-hydroxyethyl)-9H-xanthen-9- carboxamide

1 g (4.44 mmol) of 9H-xanthen-9-carboxamide and 2.2 g (13.32 mmol, 3 eq) of chloral hydrate were added, and 30 ml of benzene was added thereto to dissolve them. Then, for 12 hours, it was refiuxed and stirred. After the reaction was completed, the reactant was concentrated. The residual starting materials were crystallized by adding diethyl ether thereto, and then filtered and removed. Then, the reaction solution thereof was concentrated to obtain 1.5 g (90%) of N-(2,2,2-trichloro-l-hydroxyethyl)- 9H-xanthen-9-carboxamide.

¹H NMR (200 MHz, CDCl₃) δ 7.43~7.26(m, 4H) 7.18~7.07(m, 4H) 6.25~(m, IH) 5.75~5.68(m, IH) 4.94(s, IH) 4.46~4.44(m, IH)

(Step 3) Synthesis of N-(l,2,2,2-tetrachloroethyl)-9H-xanthen-9-carboxamide

Ig (2.68 mmol) of N-(2,2,2-trichloro-l-hydroxyethyl)-9H-xanthen-9- carboxamide was added and 15 ml of benzene was added thereto. Then, 1.6 g (13.42 mmol, 5 eq) of SOCl₂ was added thereto, and refluxed and stirred for about 6 hours. After the reaction was completed, the reactant was completely concentrated, and crystallized by adding hexane thereto, and then stirred for about 30 min. The solid materials were filtered to obtain 0.65 g (62.2 %) of N-(l,2,2,2-tetrachloroethyl)-9H- xanthen-9-carboxamide.

¹H NMR (200 MHz, CDCl₃) δ 7.42~7.34(m, 4H) 7.22-7.13(m, 4H) 6.40(s, IH) 5.03(s, IH)

(Step 4) Synthesis of N-(2,2,2-trichIoro-l-(3-(3-nitrophenyl)thioureido)ethyI)- 9H-xanthen-9-carboxamide 150 mg (0.38mmol) of N-(l,2,2,2-tetrachloroethyl)-9H-xanthen-9-carboxamide was added and 7 ml of CH₃CN was added thereto. Then, 41 mg (0.42 mmol, 1.1 eq) of KNCS was added thereto, and stirred for 1 hr. After the reaction was completed, the reactant was filtered, and thus remaining KNCS was removed. To the reaction solution thereof, 53 mg (0.38 mmol, leq) of 3-nitro aniline was added, and stirred at room temperature for 12 hours. After the reaction was completed, the reactant was concentrated, and crystallized by adding diethyl ether thereto. Then, it was stirred at room temperature for about 30 min, and then filtered to obtain 142 mg (67%) of N- (2,2,2-trichloro-l-(3-(3-nitrophenyl)thioureido)ethyl)-9H-xanthen-9-carboxamide.

Example 12: Synthesis of compound No. 37 of Table 1 (N-(2,2,2-trichloro-l-(3-

(3-cvanophenvl)thioureido)ethyl)-9H-xanthen-9-carboxamide)

150 mg (0.38mmol) of N-(l,2,2,2-tetrachloroethyl)-9H-xanthen-9-carboxamide obtained in Example 11 was added and 7 ml of CH₃CN was added thereto. Then, 41 mg (0.42 mmol, 1.1 eq) of KNCS was added thereto, and stirred for 1 hr. After the reaction was completed, the reactant was filtered, and thus remaining KNCS was removed. To the reaction solution thereof, 46 mg (0.38 mmol, 1 eq) of 3-cyano aniline was added, and stirred at room temperature for 12 hours. After the reaction was completed, the reactant was concentrated, and crystallized by adding diethyl ether thereto. Then, it was stirred at room temperature for about 30 min, and then filtered to obtain 126 mg (62%) of N-(2,2,2-trichloro-l-(3-(3-cyanophenyl)thioureido)ethyl)-9H- xanthen-9-carboxamide .

Example 13: Synthesis of compound No. 38 of Table 1 (2,2-diphenyl-N- (2,2,2- trichloro- 1 - [3 -(3-nitro-phenyl)-ureidol -ethyl } -acetamide)

50 mg (0.09 mmol) of 2,2-diphenyl-N-{2,2,2-trichloro-l-[3-(3-nitro-phenyl)- thioureido] -ethyl }-acetamide obtained in Example 1 was added to 2 ml of acetic acid. To the resultant solution, the mixture of 3 ml of acetic acid and 2 ml of 30% hydrogen peroxide was added dropwise. Then, it was stirred at room temperature for 4 hours. After the reaction was completed, the reactant was filtered and washed with H₂O. The solid materials were dissolved in ethyl acetate, washed with salt water, and extracted with an organic layer. The organic layer was dried with MgSO₄ and filtered. Then, the reaction solution was concentrated. It was crystallized by adding diethyl ether thereto. Then, it was stirred at room temperature for about 30 min, and then filtered to obtain 3 mg (6%) of 2,2-diphenyl-N-{2,2,2-trichloro-l-[3-(3-nitro-phenyl)-ureido]- ethyl} -acetamide. Example 14: Synthesis of compound No. 39 of Table 1 f2.2-diphenyl-N- (2,2,2- trichloro- 1 - [3 -(3 -cyano-phenyl)- ureido] -ethyl ) -acetamidel

50 mg (0.09 mmol) of 2,2-diphenyl-N-{2,2,2-trichloro-l-[3-(3-cyano-phenyl)- thioureido] -ethyl }-acetamide obtained in Example 3 was added to 2 ml of acetic acid. To the resultant solution, the mixture of 3 ml of acetic acid and 2 ml of 30% hydrogen peroxide was added dropwise. Then, it was stirred at room temperature for 4 hours. After the reaction was completed, the reactant was filtered and washed with H₂O. The solid materials were dissolved in ethyl acetate, washed with salt water, and extracted with an organic layer. The organic layer was dried with MgSO₄ and filtered. Then, the reaction solution was concentrated. It was crystallized by adding diethyl ether thereto. Then, it was stirred at room temperature for about 30 min, and then filtered to obtain 3 mg (6%) of 2,2-diphenyl-N-{2,2,2-trichloro-l-[3-(3-cyano-phenyl)-ureido]- ethyl}-acetamide.

The molecular structure of the compound of formula (1) according to the present invention was confirmed by NMR, MS, and the comparing of the measured value with the calculated value in an elementary analysis of the representative compound.

The pharmaceutical composition of the present invention can be formulated in the following dosage forms, but the scope of the present invention is not limited thereby.

Formulation Example 1 : Preparation of syrup

A syrup containing 2% (w/v) of the urea compound of the present invention or a salt thereof, as an effective ingredient, was prepared by the following method.

Acid addition salt of the representative urea compound, 2,2-diphenyl-N-{ 2,2,2- trichloro-l-[3-(3-nitro-phenyl)-thioureido]-ethyl}-acetamide (compound No. 1) or 9H- xanthen-9-carboxylic acid { 2,2,2-trichloro- 1 - [3 -(3 -nitro-phenyl)-thioureido] -ethyl } - amide (compound No. 36), saccharide, and saccharin were dissolved in 80 g of warm water, and cooled, to which glycerine, saccharin, flavoring agent, sorbic acid and water were added to fill in a bottle. After water was added to this mixture, the total amount was 100 ml. The above acid addition salt may be substituted with another salt according to the above examples.

Formulation Example 2: Preparation of tablet A tablet containing 15 mg of the above effective ingredient was prepared by the following method. 25Og of HCl salt of 2,2-diphenyl-N-{2,2,2-trichloro-l-[3-(3-nitro- phenyl)-thioureido] -ethyl }-acetamide (compound No. 1) was mixed with 175.9 g of lactose, 18O g of potato starch, and 32 g of colloidal silicic acid. To this mixture, 10% gelatin solution was added, triturated, and passed through a sieve of 14 mesh. Then, it was dried, and 16O g of potato starch, 50 g of talc and 5 g of magnesium stearate were added thereto to produce a tablet.

Formulation Example 3 : Preparation of solution for injection A solution for injection containing 10 mg of the above effective ingredient was prepared by the following method.

Ig of HCl salt of 2,2-diphenyl-N- {2,2,2-trichloro- l-[3-(3-nitro-phenyl)- thioureido] -ethyl }acetamide (compound No. 1), 0.6 g of sodium chloride and 0.1 g of ascorbic acid were dissolved in distilled water to form 100 ml of solution. This solution was filled in a bottle, and then heated at 20 ^°C for 30 min, and sterilized.

In addition, biological-pharmacological activity was examined to confirm that the urea compound of formula (1) of the present invention shows a superior effect as an inhibitor of ATM/ATR.

B) Acute toxicity test

Example 15: Acute toxicity test in a mouse

SPF-ICR mice (obtained from Orient) were adapted to a breeding room

environment for 1 week, in order to determine the acute toxicity for the single oral

administration of the urea compound No. 1. After the adaptation period, each group of

mice (5 mice/group) was moved into a cage, and the mice were raised at 22±2 ^°C , at a

relative humidity 40±20%, and in a light/dark (12hrs/12hrs) period using fluorescent

lamp. A tag indicating test date, animal LD. number and administration dose was

attached on the cage. The mice were allowed to have solid feed and drinking water

without limitation. Only 50% DMSO was administered to the mice from the control

group, while the urea compound No. 1 was administered at two different concentrations

to the mice from the test groups. 4-5 male mice were selected from each group, and

some of them were divided into high-dose test group (2,000 mg/kg) and medium-dose

test group (1,000 mg/kg) for the following tests. 50% DMSO was used as a solvent for administering the urea compound No. 1. The solution of the urea compound No. 1 was

orally administered to the mice in the following manner: for the medium-dose test group,

0.2 cc of solution / 25 g of mouse body weight; for high-dose test group, 0.4 cc of solution / 25 g of mouse body weight. All the test animals were monitored in regard to

abnormal symptoms every hour for 6 hours on the administration day. And the mice

were carefully monitored 1 time a day for 14 days from the next day, with regard to

movement, appearance, and autonomic nerve system symptom thereof, and with regard

to whether or not they survived. For all the test animals, the body weight was detected

on the administration day (0 day), on the 7^th day and on autopsy day (14^th day). The

autopsy was planned to carry out whenever a test animal died during this study. The

survived animals were killed using CO₂ gas, and the autopsy was carried out in order to

detect all the internal organs with the naked eye.

SPF-ICR mice (obtained from Samtaco) were adapted to a breeding room

environment for 1 week, in order to determine the acute toxicity for the single oral administration of the urea compound No. 33. After the adaptation period, each group of

mice (5 mice/group) was moved into a cage, and the mice were raised at 22±2 ^°C , at a

relative humidity 40±20%, and in a light/dark (12hrs/12hrs) period using fluorescent

lamp. A tag indicating test date, animal LD. number and administration dose was

attached on the respective cage. The mice were allowed to have solid feed and drinking water without limitation. 5 female mice and 5 male mice were selected from

each group, and some of them were allocated to high-dose test group (2,000mg/kg).

On the next day of the administration (1^st day), the mice were not allowed to have feed. And then, the urea compound No. 33 in corn oil (Sigma, C8267) was administered orally to the mice from the test group, while only corn oil was administered orally to the

mice from the control group. The general toxicity was monitored with the naked eye every 1 hour for 4 hours after the administration, and the mice were allowed to have

feed 4 hours later from the administration. The general toxicity was monitored every 24 hours for 14 days after the oral administration. The body weight of the mice was

detected on the 1^st day, on the 3^rd day, on the 7^th day and on the 14^th day after the

administration day (0 day). The autopsy was carried out for the mice on the 14^th day

after the administration day (0 day), and the internal organs thereof were detected

carefully with the naked eye.

Example 16: Results of oral acute toxicity test for the mice using urea compound

No. 1

Detection of clinical symptoms of dead animals:

All the mice survived at the end of the study period (Table 2), and no mouse

showed abnormal clinical symptoms.

Table 2. Survival rates of mice to which the urea compound No. 1 was

provided b sin le oral administration.

Detection of change of body weight of mice: All the animals gained weight

normally, and no animal lost weight (Table 3). Table 3. The change of body weight of mice to which the urea compound No.

1 was rovided b sin le oral administration.

S.D.: Standard Deviation

Autopsy opinion:

All the mice from the test groups and the control group survived. According to

the result of the autopsy for the survived mice, it was observed that internal organs

including spleen, gut, liver, kidney, lungs, heart and genital organs were normal in their

colors and size.

Example 17: Results of oral acute toxicity test for the mice using the urea

compound No. 33

Detection of clinical symptoms of dead animals:

All the mice subject to be tested survived at the end of the study period (6 days),

and no mouse showed abnormal clinical symptoms (Table 4). Table 4. Survival rates of mice to which the urea compound No. 33 was

Detection of change of body weight of mice: All the animals gained weight

normally, and no animal lost weight (Table 5).

Table 5. The change of weight of mice to which the urea compound No. 33

S.D.: Standard Deviation

In addition, the following biological tests were carried out, in order to detect pharmacological activity of the urea compound of formula (1) as an ATM/ ATR inhibitor.

C) Biological Example

Materials and Methods Example 18: Inhibition anaylsis of ATM and ATR in a cell

It is well known that phosphorylation of p53 protein for 15* residue (Serl5) depends on the activity of ATM or ATR protein [Canman, C. E. et al. (1998) Activation of the ATM kinase by ionizing radiation and phosphorylation of p53. Science 281, 1677-1679; Banin, S. et al. (1998) Enhanced phosphorylation of p53 by ATM in response to DNA damage. Science 281, 1674-1677; Tibbetts, R. S. et al. (1999) A role for ATR in the DNA damage-induced phosphorylation of p53]. Therefore, in order to analyze the change in the activity of ATM and ATR protein in a cell, phosphorylation of p53 protein for Serl5 was analyzed by using an RKO cell (purchased from ATCC) and a GM847 cell (purchased from ATCC). RKO cells (derived from a human large intestine cancer cell) were cultured in McCoy's 5 A medium (purchased from Invitrogen) containing 10% fetal bovine serum. GM847 cells of human fibroblasts, which have been immortalized by SV40 virus, were cultured in a DMEM medium containing 10% fetal bovine serum (purchased from Invitrogen).

The cultured RKO cells were pre-incubated for 2 hours by adding a medium containing the urea compound and then were incubated for 20 hours by adding 1 μM of doxorubicin. The cultured GM847 cells were pre-incubated for 2 hours by adding a medium containing the urea compound. The pre-incubated GM847 cells were additionally incubated for 2 hours after adding 1 μM of doxorubicin to them, or irradiating them with 30 J/m² of UV. After the incubated cells were retrieved and their cell lysates were retrieved, proteins were developed by SDS-PAGE and western blot analysis was performed for the proteins.

The western blot was carried out by anti-actin antibody (C-Il; purchased from Santa Cruz Biotechnology), anti-p53 antibody DO-I (purchased from Santa Cruz Biotechnology), and anti-p53 Serl5 antibody (purchased from Cell Signaling Technology). The result of densitometer analysis was defined as follows:

(Relative density for Serl5 of each compound) = (density of p53 Serl5)/(density of actin) (Inhibition intensity of phosphorylation for p53 Serl5)(%) = (relative density for

Serl5 of each compound)/(relative density for Serl5 in doxorubicin)* 100

Example 19: In vitro inhibition analysis of ATM and ATR

In vitro inhibition analysis of ATM and ATR was carried out according to the conventional method [Siato, S. et al. (2002) ATM mediates phosphorylation at multiple p53 sites, including Ser46, in response to ionizing radiation. J. Biol. Chem. 277, 12491- 12494; Tibbetts, R. S. et al. (1999) A role for ATR in the DNA damage-induced phosphorylation of p53. Genes Dev. 13, 152-157]. In order to analyze non-specific inhibition of protein kinases, in vitro analysis for protein kinases such as ERK, CKl, CDK2, JNK, PKC, CAK, Chk, PKR, p38, PI3K, etc. was carried out according to the conventional method [She, Q. -B. et al. (2000) ERKs and p38 kinase phosphorylate p53 protein at serine 15 in response to UV radiation. J. Biol. Chem. 275, 20444-20449; Knippschild, U. et al. (1997) p53 is phosphorylated in vitro and in vivo by the delta and epsilon isoforms of casein kinase 1 and enhances the level of caseine kinase 1 delta in response to topoisomerase-directed drugs. Oncogene 15, 1727-1736; Blaydes, J. P. et al. (2001) Stoichiometric phosphorylation of human p53 at ser315 stimulated p53- dependent transcription. J. Biol. Chem. 276, 4699-4708; Buschmann, T. et al. (2001) Jun NH₂-terminal kinase phosphorylation of p53 on Thr-81 is important for p53 stabilization and transcriptional activitires in response to stress. MoI. Cell. Biol. 21, 2743-2754; Baudier, J. et al. (1992) Characterization of the tumor suppressor p53 as a protein kinase C substrate and a SlOOb-binding protein. Proc. Natl. Acad. Sci. 89, 11627-11631; Ko, L. J. et al. (1997) p53 is phosphorylated by CDK7-cyclin H in a p36MATl -dependent manner. MoI Cell. Biol. 17, 7220-7229; Shieh, S.-Y. et al. (2000) The human hmologs of checkpoint kinases Chkl and Cdsl (Chk2) phosphorylate p53 at multiple DNA damage-inducible sites. Genes Dev. 14, 289-300; Cuddihy, A. R. et al. (1999) The double-stranded RNA activated protein kinase PKR physically associates with the tumor suppressor p53 protein and phosphorylates human p53 on serine 392 in vitro. Oncogene 18, 2690-2702; Huang, C. et al. (1999) ρ39 kinase mediates UV- induced phosphorylation of p53 protein at serine 389. J. Biol. Chem. 274, 12229-12235; Montmayeur, J.-P. et al. (1997) The platelet-derived growth factor β receptor triggers multiple cytoplasmic signaling cascades that arrive at the nucleus as distinguishable inputs. J. Biol. Chem. 272, 32670-32678]. By using protein kinases prepared by the above methods, the enzymatic analysis of protein phosphorylation was carried out as follows: prepared protein kinases were added to a buffer (10 mM Hepes (ρH7.5), 50 mM glycerophosphate, 50 mM NaCl, 10 niM MgCl₂, 10 mM MnCl₂, 10 μM ATP, and 1 mM dithiothreitol) containing 10 μCi [γ- ³²P]ATP and 1 βg GSTp53 protein (purchased from Santa Cruz Biotechnology), and allowed to be reacted for 30 mins at 30 ^°C . The protein kinases were reacted by adding the urea compound to the reactant so as to confirm its inhibition activity. After the reaction, proteins were developed by SDS-PAGE and the gel was dried and exposed to an X-ray film.

Example 20: Sensitivity analysis to chemotherapy in a cell HeLa cells (purchased from ATCC) of human cervical cancer cell line were incubated in a DMEM medium containing 10% bovine serum. VA- 13 cells (purchased from ATCC) which were immortalized by transforming WI-38 cells of normal human lung fibroblasts with SV40 virus, and MCF-7 cells (purchased from ATCC) of breast cancer cell line were incubated in a DMEM medium containing 10% fetal bovine serum. The agents used in the chemotherapy of human cancer are as follows: doxorubicin (purchased from Sigma), which is topoisomerase II inhibitor, was prepared at the concentration of 10 mM in DMSO (dimethyl sulfoxide); etopoxide (purchased from Calbiochem), which is topoisomerase II inhibitor, was prepared at the concentration of 40 mM in DMSO; and cisplatin (purchased from Calbiochem) of alkylating agent was prepared at the concentration of 30 mM in DMSO.

After the incubated cells were treated with trypsin and separated from incubation vessel, the number of cells was counted and then cells were cultured in a 96-well black plate having a transparent bottom. HeLa cells (-3,000 cells), MCF-7 cells (-6,000 cells), and VA- 13 cells (-6,000 cells) were cultured per well of a 96-well plate. The cells in a 96-well plate were cultured in a 37 ^°C incubator which was provided with 5% CO₂ for 24 hours and then were cultured again in a 37 ^°C incubator which was provided with 5% CO₂ for 2 hours by adding the urea compound. The cells to which the urea compound was added were treated with chemotherapy and they were cultured again in a 37 °C incubator which was provided with 5% CO₂ for 24 to 72 hours. As a negative control, the cells were cultured in DMSO that did not contain the urea compound or chemotherapy agents.

Cytotoxicity was analyzed by using a CellTiter-Glo Luminescent Cell Viability Assay reagent (purchased from Promega) according to the manual of the company. In brief, after the reagent was added to the cells treated with the urea compound and chemotherapy agents and hybridization reaction was carried out at room temperature for 10 mins, the luminescent intensity was calculated for luminescence generated by the reaction by using a luminescent detector (Wallac Victor V² Multi-reader). The cell mortality resulted from cellular toxicity was determined by comparing the luminescent intensity with the measurement from cells as a negative control.

Example 21 : Analysis of cell protection function induced by the urea compound

RKO cells of human large intestine cancer cells were cultured in McCoy's 5A medium containing 10% fetal bovine serum.

As an agent causing genotoxicity, doxorubicin of topoisomerase II inhibitor was prepared at the concentration of 10 mM in DMSO.

After the cultured cells were treated with trypsin and separated from the incubation vessel, the number of cells was counted and then the cells were cultured in a 96-well black plate having a transparent bottom wherein the number of cells is different in each well. After the cells were cultured in a 37 °C incubator which was provided with 5% CO₂ for 24 hours, they were cultured again by adding the urea compound in a 37 ^°C incubator which was provided with 5% CO₂ for 2 hours. The cells, to which the urea compound was added, were treated with doxorubicin and they were cultured in a 37 ^°C incubator which was provided with 5% CO₂ for 24 to 72 hours. As a negative control, the cells were cultured in DMSO that did not contain the urea compound or genotoxicity agents.

Cytotoxicity was analyzed by using CellTiter 96^® AQ_ueOus Non-Radioactive Cell Proliferation Assay reagent or MTT reagent (purchased from Promega) according to the manual of the company. In brief, after the reagent was added to the cells treated with the urea compound and genotoxicity agents and hybridization reaction was carried out at room temperature for 10 mins, the luminescent intensity was calculated for luminescence generated by the reaction by using a luminescent detector (Wallac Victor V² Multi-reader). The cell mortality resulted from cellular toxicity was determined by comparing the luminescent intensity with the measurement from cells as a negative control.

The change of cell cycle occurred during cell death by doxorubicin was analyzed using FACS (fluorescence activated cell sorter). The cells, which were treated with the compounds and doxorubicin, were isolated by applying trypsin from the incubation vessel and then were dyed with PI (propium iodide) solution (0.1 % sodium citrate, 0.1 % Triton X-IOO, 50 μg/ml propidium iodide and 1 mg/ml RNase A), and then the cell cycle was analyzed by FACSCalibur (purchased from Becton-Dickinson). Example 22: Analysis of inhibition of replicative senescence in a cell BJ cells (purchased from ATCC) of human prepuce cells were cultured in a DMEM medium containing 10% fetal bovine serum. BJ cells were subcultured in a ratio of 1 :4, and the number of cell population doubling (PD) was calculated with the cumulative number of cells obtained from the number of cells per subculture. The cells in replicative senescence were treated with 1 μM of the urea compound, and then the effect of the urea compound on the cells in senescence was analyzed by counting the number of cell population doubling in the same method as above.

The cells in senescence were positively dyed due to SA-β-galactosidase (SA-β- gal) characteristically expressed in them [Dimiri, G. D. et al. (1995) Proc. Natl. Acad.

Sci. 92, 9363-9367]. The incubated BJ cells were washed with PBS and then were fixed with PBS containing 2% formaldehyde and 0.2% glutaraldehyde for 5 mins. The cells fixed with PBS were washed, and then they were dyed for 12 hours by adding buffer containing 1 mg/ml of X-gal (5-bromo-4-chloro-3-indolyl β-D-galactoside) [150 niM NaCl, 2 mM MgCl₂, 5 mM K₃Fe(CN)₆, 5 mM K₄Fe(CN)₆, 40 mM citric acid and

Na₂HPO₄ (pH 6.0)].

Example 23: Analysis of inhibition of premature senescence in a cell

BJ cells (purchased from ATCC) of human prepuce cells were cultured in a DMEM medium containing 10% fetal bovine serum. BJ cells were subcultured in a ratio of 1 :4, and the number of cell population doubling (PD) was calculated with the cumulative number of cells obtained from the number of cells per subculture. The premature senescence in cells was induced with treatment of 100 μM of hydrogen peroxide (H₂O₂) to BJ cells in which the number of cell population doubling was 30 (PD30). The cells in premature senescence were treated with 1 μM of the urea compound, and then the effect of the urea compound on the cells in senescence was analyzed by counting the number of cell population doubling in the same method as above.

The incubated BJ cells were washed with PBS and then were fixed with PBS containing 2% formaldehyde and 0.2% glutaraldehyde for 5 mins. The fixed cells with

PBS were washed, and then they were dyed for 12 hours by adding buffer containing 1 mg/ml of X-gal (5-bromo-4-chloro-3-indolyl β-D-galactoside) [150 niM NaCl, 2 niM MgCl₂, 5 mM K₃Fe(CN)₆, 5 mM K₄Fe(CN)₆, 40 mM citric acid and Na₂HPO₄ (pH 6.0)].

Result

Inhibition of ATM and ATR protein activity in a cell

The inhibition of ATR activity by the urea compound in a cell was confirmed by using GM847 cells as mentioned in the part of "materials and methods". GM847 cells under incubation were pretreated with the urea compound for 2 hours and then were irradiated with 30 J/m² of UV. The cells were cultured again for 2 hours, and then phosphorylation of Serl5 of p53 protein in the cells was determined by performing western blot analysis. As shown in Fig. 1, it was observed that when GM847 cells were irradiated with UV, Serl5 of p53 protein was phosphorylated. It was reported that the phosphorylation of Ser 15 of p53 protein induced by UV irradiation did not lead to the change in the amount of p53 protein in GM847 cells and the phosphorylation was mediated by ATR protein [Lowndes, N. F. and Murguia, J. R. (2000) Sensing and responding to DNA damage. Curr. Opin. Genet. Dev. 10, 17-25; Abraham, R. T. (2001) Cell cycle checkpoint signaling through the ATM and ATR kinases. Genes Dev. 15, 2177-2196]. When GM847 cells were pretreated at the different concentration of the compound Nos. 28 and 34, it was confirmed that the phosphorylation of Ser 15 induced by UV irradiation was inhibited depending on the concentration of the compounds. In addition, it was confirmed that the inhibition of phosphorylation of Serl5 was not resulted from the change in the amount of p53 protein (Fig. 1).

The inhibition of ATM activity by the urea compound in a cell was also determined by using GM847 cells. After the GM847 cells in incubation were treated with the urea compounds for 2 hours, they were treated with 1 μM of doxorubicin. They were cultured again for 2 hours, and then the phosphorylation of Ser 15 of p53 protein was determined by performing western blot analysis. It was confirmed that Serl5 of p53 protein was specifically phosphorylated in the cells by doxorubicin, and the phosphorylation of Ser 15 was inhibited depending on the concentration of the urea compounds when GM847 cells were pretreated at the different concentration of the urea compound Nos. 28 and 34 (Fig. 2).

The inhibition of phosphorylation of Serl5 of p53 protein in a cell by the urea compound was determined by using GM847 cells and RKO cells. It was confirmed that when RKO cells were treated with 1 μM of doxorubicin in the same manner as GM847 cells, Serl5 of p53 protein was specifically phosphorylated in the RKO cells. It was confirmed that the compound Nos. 1, 27, 28, 29 and 31 inhibited the phosphorylation of Ser 15 of p53 protein induced by doxorubicin, depending on the concentration of the compounds. Specially, the compound Nos. 1, 27 and 28 inhibited the phosphorylation of Serl5 in both GM847 cells and RKO cells (Fig. 3). As shown in Fig. 3, the phosphorylation of Serl5 of p53 protein was changed in its intensity in RKO cells by treatment of the urea compounds.

Inhibition of ATM and ATR protein activity in vitro

The inhibition of ATM and ATR activity by the compounds was carried out in vitro and determined by using protein kinases prepared as mentioned in the above part of "materials and methods". As shown in Fig. 4, GST-p53 protein was specifically phosphorylated by wild type ATM and ATR proteins, while GST-p53 protein was not phosphorylated by mutant ATM and ATR proteins in which their protein kinase activity is eliminated. When the compound Nos. 28 and 33 were added to the reaction solution, the phosphorylation of GST-p53 protein by ATM and ATR was inhibited depending on the concentration of the compounds. LY294002(LY) compound (purchased from Calbiochem) used as a positive control also inhibited the phosphorylation of GST-p53 protein by ATM and ATR. The LY and the compound Nos. 28 and 33 could not inhibit the phosphorylation of GST-p53 protein by JNK and p38 protein kinases.

As a result of measurement of inhibition of ATM and ATR activity depending on the concentration of the compound No. 33, it was confirmed that the compound No. 33 inhibited the activity of ATM and ATR proteins at a lower concentration than LY294002 (Fig. 5).

The inhibitory activity of the urea compound was confirmed in vitro by using protein kinases known to phosphorylate p53 protein (Fig. 6). It was confirmed that the compound No. 33 did not inhibit the phosphorylation of GST-p53 protein by protein kinases other than ATM and ATR.

Increase of sensitivity to chemotherapy by ATM/ ATR inhibitor VA- 13 cell is a cell immortalized by transforming WI-38 cell of normal human lung fibroblast with SV40 virus. When VA- 13 cells were treated with cisplatin, etoposide, and doxorubicin used in cancer chemotherapy, the cell growth was inhibited depending on the concentration. It was confirmed that when the cells were treated with the compound No. 28 in combination with the chemotherapy agents, the cell growth was more strongly inhibited than when each of the chemotherapy agents was used alone (Fig. 7).

When HeLa cells originated from human uterine cancer were treated with the compound No. 28 together with cisplatin or doxorubicin, it was observed that the cell growth was more strongly inhibited than when each of the chemotherapy agents was used alone (Fig. 7). When MCF-7 cells of human uterine cancer cell line were treated with both doxorubicin and the compound No. 28, the cell growth was markedly inhibited (Fig. 7).

Protection of cells by ATM/ATR inhibitor

Doxorubicin is an inhibitor of topoisomerase II, and causes genotoxic stress to induce apoptosis of cells. When RKO cells were treated at the different concentration of the compound No. 28, the apoptosis induced by doxorubicin (Dox) was inhibited depending on the concentration of the compound No. 28 (Fig. 8). When RKO cells were treated with doxorubicin, the cell cycle was inhibited at the G2/M phase (see left- lower side in Fig. 9). When RKO cells were treated only with the compound No. 28, they showed the cell cycle that was similar to that as shown in the cells treated with DMSO as a negative control (see right-upper side in Fig. 9). When RKO cells was treated with doxorubicin after being treated with the compound No. 28, it was confirmed that the inhibition of the G2/M phase caused by doxorubicin was suppressed (see right-lower side in Fig. 9).

Inhibition of replicative senescence in a cell by ATM/ ATR inhibitor

When BJ cells were continuously subcultured until the number of cell population doubling reached PD88, the replicative senescence occurred in the cells and so the cells did not show their growth. Then, when the cells were treated with compound No. 33, the cell division restarted in the treated cells to increase the number of the cells (Fig. 10). However, when the cells, which were in the process of the cell division restarted by the compound, were provided only with culture solution without supplying the compound to the cells, the cells stopped their cell division again not to increase the number of the cells. Thereafter, in case the cells were retreated with compound No. 33, their cell division was restarted to increase the number of the cells. It was observed that S A-β- galactosidase staining was inhibited by treatment of compound No. 33 and the inhibition of replicative senescence by compound No. 33 was reversible in the cells (Fig. 11).

Inhibition of premature senescence in a cell by ATM/ ATR inhibitor

When BJ cells were treated with 100 μM of hydrogen peroxide (H₂O₂) for 2 hours and then cultured for about a week, the premature senescence occurred in the cells and so the cells did not show their cell division. It was observed that there was a positive response to SA-β-gal staining in the premature senescence occurred by hydrogen peroxide, as shown in the replicative senescence. It was confirmed that when BJ cells in which premature senescence had occurred were treated with compound No. 33, the premature senescence was inhibited to show a negative response to SA-β-gal staining and to increase the number of the cells (Fig. 12).

INDUSTRIAL APPLICABILITY

A urea compound of formula (1) or a pharmaceutically acceptable salt thereof according to the present invention selectively binds to ATM and ATR so as to specifically suppress the function of ATM and ATR as protein kinase. Therefore, the urea compound and the pharmaceutical composition comprising it as an effective ingredient are very useful for controlling cell function and treating diseases, in connection with function abnormality in ATM and ATR.

Claims

WHAT IS CLAIMED IS

1. A urea compound of formula (1), its derivative or a pharmaceutically acceptable salt thereof:

Formula (1)

Wherein

R₁ is any one of the following structures:

R₂ is any one of the following structures:

X is H, CH₃, CF₃ or CCl₃; and Y is O or S.

2. The urea compound, its derivative or a pharmaceutically acceptable salt thereof according to claim 1, wherein R₂ is any one of the following structures:

3. The urea compound, its derivative or a pharmaceutically acceptable salt thereof according to claim 1, wherein R₂ is any one of the following structures:

4. The urea compound, its derivative or a pharmaceutically acceptable salt thereof according to claim 3, wherein R₁ is any one of the following structures:

5. The urea compound, its derivative or a pharmaceutically acceptable salt thereof according to claim 4, wherein R₂ is any one of the following structures:

6. The urea compound, its derivative or a pharmaceutically acceptable salt thereof according to claim 4, wherein R₂ is any one of the following structures:

7. The urea compound, its derivative or a pharmaceutically acceptable salt thereof according to claim 6, wherein X is CCl₃ or CF₃.

8. The urea compound, its derivative or a pharmaceutically acceptable salt thereof according to claim 6, wherein Y is S.

9. The urea compound, its derivative or a pharmaceutically acceptable salt thereof according to claim 8, wherein R₁ is any one of the following structures:

X is CCl₃.

10. The urea compound, its derivative or a pharmaceutically acceptable salt thereof according to claim 1, wherein the compound is any one selected from the group consisting of the following compounds:

2,2-diphenyl-N-{2,2,2-trichloro-l-[3-(3-nitro-phenyl)-thioureido]-ethyl}- acetamide;

3-methyl-N-{2,2,2-trichloro-l-[3-(2-nitro-phenyl)-thioureido]-ethyl}-benzamide; 3-nitro-N-{2,2,2-trichloro-l-[3-(4-nitro-phenyl)-thioureido]-ethyl}-benzamide;

4-nitro-N-[2,2,2-trichloro-l-(3-phenyl-thioureido)-ethyl]-benzamide; 2 -phenyl -N- {2,2,2-trichloro- 1 -[3-(4-nitro-phenyl)-thioureido] -ethyl} -acetamide; 2-phenyl-N- {2,2,2-trichloro- 1 -[3-(3-nitro-phenyl)-thioureido]-ethyl} -acetamide; 2-naphthalene- 1 -yl-N- {2,2,2-trichloro- 1 - [3-(4-nitro-phenyl)-thioureido] -ethyl } - acetamide;

2-naphthalene- 1 -yl-N- {2,2,2-trichloro- 1 - [3 -(3 -nitro-phenyl)-thioureido] -ethyl } - acetamide;

2-naphthalene- 1 -yl-N- {2,2,2-trichloro- 1 -[3-(2-nitro-phenyl)-thioureido] -ethyl } - acetamide; 2-methyl-4-nitro-penta-2,4-dienoic acid {2,2,2-trichloro-l-[3-(3-nitro-phenyl)- thioureido] -ethyl } -amide ;

3-methyl-N-{2,2,2-trichloro-l-[3-(3-nitro-phenyl)-thioureido]-ethyl}-benzamide; 3,5-dinitro-N-[2,2,2-trichloro-l-(3-phenyl-thioureido)-ethyl]-benzamide; N-{2,2,2-trichloro-l-[3-(4-nitro-phenyl)-thioureido]-ethyl}-benzamide; 2-nitro-N-[2,2,2-trichloro-l-(3-phenyl-thioureido)-ethyl]-benzamide;

2-nitro-N-[2,2,2-trichloro-l-(3-m-tolyl-thioureido)-ethyl]-benzamide; 2-nitro-N-{2,2,2-trichloro-l-[3-(3-nitro-phenyl)-thioureido]-ethyl}-benzamide; 3,5-dinitro-N-[2,2,2-trichloro-l-(3-o-tolyl-thioureido)-ethyl]-benzamide; 3-nitro-N-{2,2,2-trichloro-l-[3-(2,5-dimethyl-phenyl)-thioureido]-ethyl}- benzamide;

N- { 1 - [3-(4-acetylamino-phenyl)-thioureido] -2,2,2-trichloro-ethyl } -2-nitro- benzamide;

2,2-diphenyl-N-[2,2,2-trichloro- 1 -(3-phenyl-thioureido)-ethyl] -acetamide; 2,2-diphenyl-N-{2,2,2-trichloro-l-[3-(2-nitro-phenyl)-thioureido]-ethyl}- acetamide;

2,2-diphenyl-N-{2,2,2-trichloro-l-[3-(lH-[l,2,4]triazol-3-yl)-thioureido]-ethyl}- acetamide;

2,2-diphenyl-N- {2,2,2-trichloro- 1 -[3-(2-methoxy-5-nitro-phenyl)-thioureido]- ethyl} -acetamide; furan-2-carboxylic acid { 2,2,2-trichloro- 1- [3 -(4-nitro-phenyl)-thioureido] -ethyl }- amide;

2-phenyl-3-vinyl-pent-3-enoic acid {2,2,2-trichloro-l-[3-(3-hydroxy-phenyl)- thioureido] -ethyl } -amide ; 2,2-diphenyl-N-{2,2,2-trichloro-l-[3-(3-cyano-phenyl)-thioureido]-ethyl}- acetamide;

3-[3-(2,2,2-trichloro-l-diphenylacetylamino-ethyl)-thioureido]-benzamide;

N-{l-[3-(3-acetylamino-phenyl)-thioureido]-2,2,2-trichloro-ethyl}-2,2-diphenyl- acetamide;

N-{l-[3-(3-amino-phenyl)-thioureido]-2,2,2-trichloro-ethyl}-2,2-diphenyl- acetamide;

2,2-diphenyl-N- {2,2,2-trichloro- 1 -[3-(4-hydroxy-3-nitro-phenyl)-thioureido]- ethyl}-acetamide; 2,2-diphenyl-N-{2,2,2-trichloro-l-[3-(4-fluoro-3-nitro-phenyl)-thioureido]- ethyl } -acetamide ;

2,2-diphenyl-N-{2,2,2-trifluoro-l-[3-(3-nitro-phenyl)-thioureido]-ethyl}- acetamide;

N-{l-[3-(3-cyano-phenyl)-thioureido]-2,2,2-trifluoro-ethyl}-2,2-diphenyl- acetamide;

9H-xanthen-9-carboxylic acid {2,2,2-trichloro-l-[3-(3-nitro-phenyl)-thioureido]- ethyl} -amide;

9H-xanthen-9-carboxylic acid {2,2,2-trichloro-l-[3-(3-cyano-phenyl)-thioureido]- ethyl} -amide; 2,2-diphenyl-N-{2,2,2-trichloro-l-[3-(3-nitro-phenyl)-ureido]-ethyl}-acetamide;

2,2-diphenyl-N-{2,2,2-trichloro-l-[3-(3-cyano-phenyl)-ureido]-ethyl}-acetamide;

N-{l-[3-(3-nitro-phenyl)-thioureido]-ethyl}-2,2-diphenyl-acetamide; and

N-{l-[3-(3-cyano-phenyl)-thioureido]-ethyl}-2,2-diphenyl-acetamide.

11. An inhibitor of ATM and ATR comprising a urea compound of formula (1), its derivative or a pharmaceutically acceptable salt thereof, as defined in any one of claims 1 to 10.

12. The inhibitor of ATM and ATR according to claim 11, for controlling cellular function and treating diseases, in connection with functional abnormality in ATM and/or ATR.

13. The inhibitor of ATM and ATR according to claim 11, for increasing sensitivity to radiation therapy or chemotherapy in cells.

14. The inhibitor of ATM and ATR according to claim 11, for protecting cells from apoptosis caused by genotoxicity or neurotoxicity.

15. The inhibitor of ATM and ATR according to claim 11, for inhibiting replicative senescence in cells.

16. The inhibitor of ATM and ATR according to claim 11, for inhibiting premature senescence in cells.

17. The inhibitor of ATM and ATR according to claim 11, for inhibiting cellular proliferation caused by abnormal elongation of telomere.

18. The inhibitor of ATM and ATR according to claim 11 , for controlling abnormality in cellular function caused by oxidative stress.

19. The inhibitor of ATM and ATR according to claim 11, for controlling cytotoxicity caused by free radicals.

20. The inhibitor of ATM and ATR according to claim 11 , for controlling abnormality in cellular function caused by heat shock.

21. The inhibitor of ATM and ATR according to claim 11, for controlling immune function.

22. The inhibitor of ATM and ATR according to claim 11, for controlling retro virus- mediated diseases.

23. A pharmaceutical composition comprising: a urea compound of formula (1), its derivative or a pharmaceutically acceptable salt thereof, as defined in any one of claims 1 to 10; and a pharmaceutically acceptable carrier.

24. The pharmaceutical composition according to claim 23, for controlling cellular function and treating diseases, in connection with functional abnormality in ATM and /orATR.

25. The pharmaceutical composition according to claim 23, for treating cancer by increasing sensitivity to radiation therapy or chemotherapy in cells.

26. The pharmaceutical composition according to claim 23, for reducing genotoxicity or neurotoxicity by protecting cells from apoptosis caused by genotoxicity or neurotoxicity.

27. The pharmaceutical composition according to claim 23, for inhibiting replicative senescence or premature senescence in cells.

28. The pharmaceutical composition according to claim 23, for controlling retrovirus- mediated diseases.

29. The pharmaceutical composition according to any one of claims 23 to 28, wherein the composition is formulated in tablets, pills, hard and soft capsules, solutions, suspensions, emulsions, syrups, granules or elixirs.

30. The pharmaceutical composition according to claim 29, wherein the composition further comprises at least one substance selected from the group consisting of diluents, lubricants, preservatives, stabilizers, hydration agents, emulsification promoters, salts for controlling osmotic pressure and buffers.

31. An optical isomer or diastereomer of a urea compound of formula (1), its derivative or a pharmaceutically acceptable salt thereof, as defined in any one of claims 1 to 10.