KR101401664B1 - Novel pyrimidine-2,4-diamine derivatives for differentiation of neural stem cells and medical use thereof - Google Patents

Novel pyrimidine-2,4-diamine derivatives for differentiation of neural stem cells and medical use thereof Download PDF

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KR101401664B1
KR101401664B1 KR1020110118910A KR20110118910A KR101401664B1 KR 101401664 B1 KR101401664 B1 KR 101401664B1 KR 1020110118910 A KR1020110118910 A KR 1020110118910A KR 20110118910 A KR20110118910 A KR 20110118910A KR 101401664 B1 KR101401664 B1 KR 101401664B1
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민경훈
김현정
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중앙대학교 산학협력단
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Abstract

본 발명은 신경줄기세포 분화조절제용 신규 피리미딘-2,4-디아민 유도체 및 이의 의학적 용도에 관한 것으로, 상기 피리미딘-2,4-디아민 유도체를 이용하여 신경줄기세포를 신경세포 특히 뉴런으로 분화시킬 수 있으므로, 상기 유도체를 포함한 조성물은 뇌졸중, 알츠하이머병, 파킨슨병 또는 척수 손상 질환 등을 포함하는 신경세포손상 질환의 치료 또는 예방에 유용하게 사용될 수 있다.The present invention relates to a novel pyrimidine-2,4-diamine derivative for regulating neural stem cell differentiation regulator and its medical use, wherein the pyrimidine-2,4-diamine derivative can be used to differentiate neural stem cells into neurons, Therefore, the composition containing the derivative may be useful for the treatment or prevention of neuronal cell damage diseases including stroke, Alzheimer's disease, Parkinson's disease, or spinal cord injury diseases.

Description

신경줄기세포 분화조절제용 신규 피리미딘-2,4-디아민 유도체 및 이의 의학적 용도 {Novel pyrimidine-2,4-diamine derivatives for differentiation of neural stem cells and medical use thereof}Novel pyrimidine-2,4-diamine derivatives for neural stem cell differentiation regulators and novel medicinal uses thereof

본 발명은 신경줄기세포 분화조절제용 신규 피리미딘-2,4-디아민 유도체 및 이의 의학적 용도에 관한 것이다.The present invention relates to novel pyrimidine-2,4-diamine derivatives for modulating neural stem cell differentiation and their medical uses.

뇌졸중(stroke), 알츠하이머병, 파킨슨병, 탈수초질환(demyelinating disease), 척추 손상(spinal cord injury) 등은 신경 세포 손상에 의해 신경기능에 이상이 생기는 질환으로, 상기 질환들로 인해 손상된 신경세포는 재생되지 않아 치료가 어렵고 대증요법으로 약물 또는 외과적 수술이 이루어지나 이러한 치료법은 일시적이거나 정상적인 세포에도 손상을 입혀 문제점으로 지적되고 있다.Stroke, Alzheimer's disease, Parkinson's disease, demyelinating disease, and spinal cord injury are disorders in which neuronal dysfunction is caused by nerve cell damage, Is difficult to treat because it is not regenerated, and the drug or surgical operation is performed with the symptom therapy. However, these treatments are pointed out as a problem due to damage to transient or normal cells.

이에 최근에는 질환에 의해 파괴되거나 손상된 세포를 외부로부터 공급해 주는 세포 치료제가 효과적인 것으로 제시되고 있다. 이러한 세포 치료제로는 신경줄기세포(Neural stem cells: NSCs)가 각광을 받고 있다.Recently, it has been suggested that a cell therapy agent that supplies cells destructed or damaged by diseases from outside is effective. Neural stem cells (NSCs) are in the spotlight as such cell therapy drugs.

신경줄기세포는 신경계에 존재하는 전구세포(progenitor cell)의　서브타입으로, 성상세포(astrocytes), 희돌기교세포(oligodendrocytes), 뉴런(neurons)으로 분화할 수 있는 능력을 가지고 있다. 중추신경계(CNS)와 말초신경계(PNS)에서 분리하여 다세포성 뉴로스피어(Multicellular neurospheres)를 만들 수 있고 이 세포가 각각의 조건에서 글리아(glia) 계통과 신경 계통으로 분화하게 된다(Sally Temple et al. 2001). Neural stem cells are subtypes of progenitor cells present in the nervous system and have the ability to differentiate into astrocytes, oligodendrocytes, and neurons. It can separate from the central nervous system (CNS) and the peripheral nervous system (PNS) to form multicellular neurospheres, which differentiate into glia and nervous systems at each condition (Sally Temple et al ., 2001).

뇌신경계 조직을 다른 조직과는 달리 면역거부반응이 거의 없기 때문에, 신경줄기세포를 이식하였을 때 이식된 세포의 장기간 생존을 기대할 수 있고, 따라서 신경세포 손상에 의해 유발되는 다양한 신경 질환에 대한 세포 치료제로서도 많은 연구가 이루어지고 있다. Since the cervical nervous system tissue has almost no immune rejection reaction unlike other tissues, long-term survival of the transplanted cells can be expected when the neural stem cells are transplanted, and as a cell therapy agent for various neurological diseases caused by nerve cell damage Much research has been done.

그러나, 세포 치료제로서의 신경줄기세포의 유용성을 높이기 위해서는 신경줄기세포를 효율적으로 특정 세포로 분화시키는 기술이 필요하다. However, in order to increase the utility of neural stem cells as a cell therapy agent, there is a need for a technique for efficiently differentiating neural stem cells into specific cells.

이에, 본 발명자들은 신규한 피리미딘-2,4-디아민 유도체가 신경줄기세포를 특정세포 즉, 뉴런으로 분화시키는 신경줄기세포 분화조절제인 것을 밝혀냄으로써 본 발명을 완성하였다.
Accordingly, the present inventors have completed the present invention by discovering that the novel pyrimidine-2,4-diamine derivative is a neural stem cell differentiation regulator that differentiates neural stem cells into specific cells, that is, neurons.

본 발명의 목적은 신경줄기세포 분화조절 활성을 갖는 신규 피리미딘-2,4-디아민 유도체 및 이의 제조방법을 제공하는 데에 있다.It is an object of the present invention to provide a novel pyrimidine-2,4-diamine derivative having neural stem cell differentiation-regulating activity and a process for producing the same.

또한, 본 발명의 다른 목적은 상기 피리미딘-2,4-디아민 유도체를 포함하는 신경줄기세포의 신경세포로의 분화유도용 조성물을 제공하는 데에 있다.Another object of the present invention is to provide a composition for inducing differentiation of neural stem cells comprising the pyrimidine-2,4-diamine derivative into neurons.

또한, 본 발명의 또다른 목적은 상기 조성물을 신경줄기세포와 함께 배양하는 단계를 포함하는, 신경줄기세포를 신경세포로 분화시키는 방법을 제공하는 데에 있다.It is still another object of the present invention to provide a method of differentiating neural stem cells into neural cells, comprising culturing the composition with neural stem cells.

또한, 본 발명의 또다른 목적은 상기 피리미딘-2,4-디아민 유도체를 포함하는 신경세포손상 질환 치료 또는 예방용 약학조성물을 제공하는 데에 있다.Still another object of the present invention is to provide a pharmaceutical composition for treating or preventing a neuronal cell damage disease comprising the pyrimidine-2,4-diamine derivative.

상기 목적을 달성하기 위하여, 본 발명은 하기 화학식 1로 표시되는 피리미딘-2,4-디아민 유도체 또는 그의 염을 제공한다. In order to achieve the above object, the present invention provides a pyrimidine-2,4-diamine derivative represented by the following formula (1) or a salt thereof.

[화학식 1][Chemical Formula 1]

Figure 112011090098428-pat00001
Figure 112011090098428-pat00001

상기 화학식 1에서, In Formula 1,

R1 및 R2는 각각 독립적으로 R 1 and R 2 are each independently

Figure 112011090098428-pat00002
또는
Figure 112011090098428-pat00003
Figure 112011090098428-pat00002
or
Figure 112011090098428-pat00003

이며, R1 및 R2중 적어도 어느 하나는

Figure 112012035776432-pat00050
이며, , And at least one of R 1 and R 2 is
Figure 112012035776432-pat00050
Lt;

상기 R3, R4 및 R6는 각각 독립적으로 수소, C1 -12 알킬, C1 -12 할로알킬 또는 C1 -12 헤테로알킬이며, Wherein R 3, R 4 and R 6 are each independently hydrogen, C 1 -12 alkyl, C 1 -12 alkyl, or a halo-C 1 -12 alkyl, heteroaryl,

R5는 수소, 히드록시기, 할로겐, C1 -12 알킬, C1 -12 할로알킬, C1 -12 헤테로알킬, C1 -12 알콕시, C1 -16 아릴알킬 또는 C1 -16 헤테로아릴알킬일 수 있다. R 5 is hydrogen, hydroxy, halogen, C 1 -12 alkyl, C 1 -12 alkyl, halo, C 1 -12 alkyl, heteroaryl, C 1 -12 alkoxy, C 1 -16 alkyl, aryl or C 1 -16 alkyl, heteroaryl days .

또한 본 발명은 화학식 1로 표시되는 피리미딘-2,4-디아민 유도체 또는 그의 염을 포함하는 신경줄기세포의 신경세포로의 분화유도용 조성물을 제공한다.The present invention also provides a composition for inducing the differentiation of neural stem cells into neurons comprising the pyrimidine-2,4-diamine derivative represented by the formula (1) or a salt thereof.

또한, 본 발명은 상기 조성물을 신경줄기세포와 함께 배양하는 단계를 포함하는, 신경줄기세포를 신경세포로 분화시키는 방법을 제공한다.The present invention also provides a method for differentiating neural stem cells into neural cells, comprising culturing the composition together with neural stem cells.

또한, 본 발명은 화학식 1로 표시되는 피리미딘-2,4-디아민 유도체 또는 그의 염을 포함하는 신경세포손상 질환 치료 또는 예방용 약학조성물을 제공한다.The present invention also provides a pharmaceutical composition for treating or preventing a neuronal cell damage disease comprising the pyrimidine-2,4-diamine derivative represented by the formula (1) or a salt thereof.

본 발명에 따른 피리미딘-2,4-디아민 유도체를 이용하여 신경줄기세포를 신경세포 특히 성상세포로 분화시킬 수 있으므로, 상기 유도체를 포함한 조성물은 뇌졸중, 알츠하이머병, 파킨슨병 또는 척수 손상 질환 등을 포함하는 신경세포손상 질환의 치료 또는 예방에 유용하게 사용될 수 있다.Since the neural stem cells can be differentiated into neurons, particularly astrocytes, by using the pyrimidine-2,4-diamine derivatives according to the present invention, the compositions containing the derivatives include stroke, Alzheimer's disease, Parkinson's disease or spinal cord injury diseases Which is useful for the treatment or prevention of neuronal damage diseases.

도 1은 본 발명에 따른 피리미딘-2,4-디아민 유도체에 의한 신경줄기세포의 뉴런으로의 유도를 관찰한 것으로, 뉴런 마커(TuJ1) 및 성상세포 마커(GFAP)에 대한 면역형광 이미지이다.FIG. 1 shows the induction of neural stem cells into neurons by the pyrimidine-2,4-diamine derivative according to the present invention, which is an immunofluorescent image for neuron marker (TuJ1) and astrocyte marker (GFAP).

이하, 본 발명을 보다 상세하게 설명한다.Hereinafter, the present invention will be described in more detail.

본 발명은 하기 화학식 1로 표시되는 피리미딘-2,4-디아민 유도체 또는 그의 염을 제공한다:The present invention provides a pyrimidine-2,4-diamine derivative represented by the following formula (1) or a salt thereof:

[화학식 1][Chemical Formula 1]

Figure 112011090098428-pat00005
Figure 112011090098428-pat00005

상기 화학식 1에서, In Formula 1,

R1 및 R2는 각각 독립적으로 R 1 and R 2 are each independently

Figure 112011090098428-pat00006
또는
Figure 112011090098428-pat00007
Figure 112011090098428-pat00006
or
Figure 112011090098428-pat00007

이며, R1 및 R2중 적어도 어느 하나는

Figure 112012035776432-pat00051
이며, , And at least one of R 1 and R 2 is
Figure 112012035776432-pat00051
Lt;

상기 R3, R4 및 R6는 각각 독립적으로 수소, C1 -12 알킬, C1 -12 할로알킬 또는 C1 -12 헤테로알킬이며, Wherein R 3, R 4 and R 6 are each independently hydrogen, C 1 -12 alkyl, C 1 -12 alkyl, or a halo-C 1 -12 alkyl, heteroaryl,

R5는 수소, 히드록시기, 할로겐, C1 -12 알킬, C1 -12 할로알킬, C1 -12 헤테로알킬, C1 -12 알콕시, C1 -16 아릴알킬 또는 C1 -16 헤테로아릴알킬일 수 있다. R 5 is hydrogen, hydroxy, halogen, C 1 -12 alkyl, C 1 -12 alkyl, halo, C 1 -12 alkyl, heteroaryl, C 1 -12 alkoxy, C 1 -16 alkyl, aryl or C 1 -16 alkyl, heteroaryl days .

본 발명의 한 구체예에서, 상기 R3 및 R4 는 각각 독립적으로 수소, 메틸, 에틸일 수 있으나 이에 제한되는 것은 아니며, R6는 C1 -16 아미노알킬, 보다 구체적으로 2-모르폴리노에톡시, 2-피페리딜에톡시 또는 2-디에틸아미노에톡시일 수 있으나 이에 제한되는 것은 아니다. In one embodiment of the invention, R < 3 > and R < 4 > R 6 may be C 1 -16 aminoalkyl, more particularly 2-morpholinoethoxy, 2-piperidylethoxy or 2-di Ethylaminoethoxy, but is not limited thereto.

보다 구체적으로, 본 발명에 따른 화학식 1의 바람직한 화합물로는 이에 제한되는 것은 아니나, N2-(3,4-디메톡시페닐)-N4-(4-(2-모르폴리노에톡시)페닐)피리미딘-2,4-디아민, N4-(3,4-디메톡시페닐)-N2-(4-(2-모르폴리노에톡시)페닐)피리미딘-2,4-디아민 또는 N2,N4-비스(3,4-디메톡시페닐)피리미딘-2,4-디아민 등이 이에 해당할 수 있다. More specifically, preferred compounds of Formula I according to the present invention include, but are not limited to, N 2 - (3,4-dimethoxyphenyl) -N 4 - (4- (2-morpholinoethoxy) phenyl ) pyrimidine-2,4-diamine, N 4 - (3,4- dimethoxyphenyl) -N 2 - (4- (2- morpholino-ethoxy) phenyl) pyrimidine-2,4-diamine or N 2 , N 4 -bis (3,4-dimethoxyphenyl) pyrimidine-2,4-diamine, and the like.

본 발명에 따른 피리미딘-2,4-디아민 유도체는 공지의 방법에 의해 상응하는 염으로 제조할 수 있다. 이러한 염은 독성이 없는 수용성의 것이 바람직하다. 바람직한 염으로는 칼륨, 나트륨 등 등과 같은 알칼리 금속의 염; 칼슘, 마그네슘 등과 같은 알칼리토류 금속의 염; 그리고 테트라메틸 암모늄, 트리에틸아민, 메틸아민, 디메틸아민, 시클로펜틸아민, 벤질아민, 페네틸아민, 피페리딘, 모노에탄올아민, 디에탄올아민, 트리스(히드록시메틸)아민, 라이신, 아르기닌, N-메틸-D-글루카민 등과 같은 약학적으로 허용 가능한 아민의 염을 들 수 있다.The pyrimidine-2,4-diamine derivative according to the present invention can be prepared by a known method as a corresponding salt. Such salts are preferably water-soluble without toxicity. Preferred salts include salts of alkali metals such as potassium, sodium and the like; Salts of alkaline earth metals such as calcium, magnesium and the like; And amines such as tetramethylammonium, triethylamine, methylamine, dimethylamine, cyclopentylamine, benzylamine, phenethylamine, piperidine, monoethanolamine, diethanolamine, tris (hydroxymethyl) amine, lysine, arginine, And salts of pharmaceutically acceptable amines such as N-methyl-D-glucamine and the like.

본 발명에 사용되는 피리미딘-2,4-디아민 유도체는 공지의 방법에 의해 상응하는 산 부가염으로 제조할 수 있다. 이러한 산 부가염은 독성이 없는 수용성인 것이 바람직하다. 바람직한 산 부가염으로는 염산염, 브롬화수소산염, 요오드화수소산염, 황산염, 인산염 및 질산염과 같은 무기산염, 또는 아세트산염, 젖산염, 주석산염, 옥살산염, 푸마르산염, 말레인산염, 구연산염, 벤조산염, 메탄술폰산염, 에탄술폰산염, 벤젠술폰산염, 톨루엔술폰산염, 이세티온산염, 글루쿠론산염 및 글루콘산염과 같은 유기산염을 들 수 있다.The pyrimidine-2,4-diamine derivative used in the present invention can be prepared by a known method as the corresponding acid addition salt. These acid addition salts are preferably water-soluble without toxicity. Preferred acid addition salts include inorganic acid salts such as hydrochloride, hydrobromide, hydroiodide, sulfate, phosphate and nitrate, or organic acid salts such as acetate, lactate, tartrate, oxalate, fumarate, maleate, citrate, benzoate, methane Organic acid salts such as sulfonate, ethanesulfonate, benzenesulfonate, toluenesulfonate, isethionate, glucuronate and gluconate.

본 발명의 사용되는 피리미딘-2,4-디아민 유도체 또는 그의 염은 공지의 방법에 의해 수화물로 제조할 수 있다.The pyrimidine-2,4-diamine derivative or salt thereof used in the present invention can be prepared as a hydrate by a known method.

이하의 표 1 및 도 1에 도시된 바와 같이, 본 발명에 따른 피리미딘-2,4-디아민 유도체 또는 그의 염을 처리한 실험군은 대조군에 비해 5배 내지 8배 세포 분화 유도 효과가 탁월하며, 특히 DMSO 가 처리된 대조군에 비해 뉴런으로의 분화를 현저히 촉진시킬 수 있다. 따라서, 본 발명에 따른 피리미딘-2,4-디아민 유도체 또는 그의 염은 신경줄기세포를 신경세포, 특히 뉴런으로 분화를 촉진함으로써 신경세포들 간의 균형을 맞추어 신경세포손상 질환의 치료 또는 예방에 유용하게 사용될 수 있다.As shown in the following Table 1 and FIG. 1, the experimental group treated with the pyrimidine-2,4-diamine derivative or its salt according to the present invention is superior to the control group in inducing 5 to 8 fold cell differentiation, In particular, DMSO can significantly promote neuronal differentiation compared to the control group treated. Accordingly, the pyrimidine-2,4-diamine derivative or its salt according to the present invention can promote the differentiation of neural stem cells into neurons, particularly neurons, thereby balancing the neurons and thus being useful for the treatment or prevention of neuronal cell damage diseases Can be used.

또한, 본 발명은 하기 화학식 1로 표시되는 피리미딘-2,4-디아민 유도체 또는 그의 염을 유효성분으로 포함하는, 신경줄기세포의 신경세포로의 분화유도용 조성물을 제공한다:The present invention also provides a composition for inducing the differentiation of neural stem cells into neural cells, comprising the pyrimidine-2,4-diamine derivative represented by the following formula (1) or a salt thereof as an active ingredient:

[화학식 1][Chemical Formula 1]

Figure 112011090098428-pat00009
Figure 112011090098428-pat00009

상기 화학식 1에서, In Formula 1,

R1 및 R2는 각각 독립적으로 R 1 and R 2 are each independently

Figure 112011090098428-pat00010
또는
Figure 112011090098428-pat00011
Figure 112011090098428-pat00010
or
Figure 112011090098428-pat00011

이며, R1 및 R2중 적어도 어느 하나는

Figure 112012035776432-pat00052
이며, , And at least one of R 1 and R 2 is
Figure 112012035776432-pat00052
Lt;

상기 R3, R4 및 R6는 각각 독립적으로 수소, C1 -12 알킬, C1 -12 할로알킬 또는 C1 -12 헤테로알킬이며, Wherein R 3, R 4 and R 6 are each independently hydrogen, C 1 -12 alkyl, C 1 -12 alkyl, or a halo-C 1 -12 alkyl, heteroaryl,

R5는 수소, 히드록시기, 할로겐, C1 -12 알킬, C1 -12 할로알킬, C1 -12 헤테로알킬, C1 -12 알콕시, C1 -16 아릴알킬 또는 C1 -16 헤테로아릴알킬일 수 있다.
R 5 is hydrogen, hydroxy, halogen, C 1 -12 alkyl, C 1 -12 alkyl, halo, C 1 -12 alkyl, heteroaryl, C 1 -12 alkoxy, C 1 -16 alkyl, aryl or C 1 -16 alkyl, heteroaryl days .

특히, 본 발명에 따른 조성물은 상기 피리미딘-2,4-디아민 유도체 또는 그의 염을 1 내지 10 μM의 양으로 포함하는 것이 바람직하다.In particular, the composition according to the present invention preferably contains the pyrimidine-2,4-diamine derivative or its salt in an amount of 1 to 10 μM.

본 발명에 의해 분화될 수 있는 신경줄기세포는 특정한 신경계 세포를 만들어내는 신경전구세포의 단계를 거쳐서 신경세포로 분화하게 된다. 따라서, 미분화 상태이면서, 무한정 증식 및 신경세포로의 분화기능을 갖는 세포라면 특별히 제한되지는 않는다. 예를 들어, 배아 신경줄기세포, 성체 신경줄기세포, 배아생식 신경줄기세포 및 배아종양 신경줄기세포 등으로부터 선택될 수 있다.The neural stem cells that can be differentiated according to the present invention are differentiated into neural cells through the steps of neural progenitor cells that produce specific neural cells. Therefore, it is not particularly limited as long as it is an undifferentiated cell and has a function of infinite proliferation and differentiation into neurons. For example, embryonic neural stem cells, adult neural stem cells, embryonic germinal neural stem cells, and embryonic tumor neural stem cells.

상기 신경줄기세포로부터 분화된 신경세포는 뉴런(neuron), 성상세포(astrocyte), 희돌기교세포(oligodendrocyte) 또는 소교세포(microglia cell)일 수 있다.The neurons differentiated from the neural stem cells may be neurons, astrocyte, oligodendrocytes or microglia cells.

또한, 본 발명은 상기 조성물을 신경줄기세포와 함께 배양하는 단계를 포함하는, 신경줄기세포를 신경세포로 분화시키는 방법을 제공한다.The present invention also provides a method for differentiating neural stem cells into neural cells, comprising culturing the composition together with neural stem cells.

상기 배양은 DMEM(Dulbecco's modified Eagle's medium, Hyclone)/F12 배지와 같은 세포배양용 배지에서 수행될 수 있으며, 배양배지에는 B27, 섬유아세포성장인자(fibroblast-derived growth factor, FGF), 상피세포 성장인자(epidermal growth factor, EGF) 등과 같은 성장인자 등이 추가로 첨가될 수 있다. 배양기간은 3 내지 10일이 바람직하다.The culture may be performed in a cell culture medium such as DMEM (Dulbecco's modified Eagle's medium, Hyclone) / F12 medium. The culture medium may include B27, fibroblast-derived growth factor (FGF) a growth factor such as an epidermal growth factor (EGF), and the like may be further added. The incubation period is preferably 3 to 10 days.

또한, 본 발명은 하기 화학식 1로 표시되는 피리미딘-2,4-디아민 유도체 또는 그의 염을 유효성분으로 포함하는, 신경세포손상 질환 치료 또는 예방용 약학조성물을 제공한다:The present invention also provides a pharmaceutical composition for treating or preventing a neuronal cell damage disease, which comprises a pyrimidine-2,4-diamine derivative represented by the following formula (1) or a salt thereof as an active ingredient:

[화학식 1][Chemical Formula 1]

Figure 112011090098428-pat00013
Figure 112011090098428-pat00013

상기 화학식 1에서, In Formula 1,

R1 및 R2는 각각 독립적으로 R 1 and R 2 are each independently

Figure 112011090098428-pat00014
또는
Figure 112011090098428-pat00015
Figure 112011090098428-pat00014
or
Figure 112011090098428-pat00015

이며, R1 및 R2중 적어도 어느 하나는

Figure 112012035776432-pat00053
이며, , And at least one of R 1 and R 2 is
Figure 112012035776432-pat00053
Lt;

상기 R3, R4 및 R6는 각각 독립적으로 수소, C1 -12 알킬, C1 -12 할로알킬 또는 C1 -12 헤테로알킬이며, Wherein R 3, R 4 and R 6 are each independently hydrogen, C 1 -12 alkyl, C 1 -12 alkyl, or a halo-C 1 -12 alkyl, heteroaryl,

R5는 수소, 히드록시기, 할로겐, C1 -12 알킬, C1 -12 할로알킬, C1 -12 헤테로알킬, C1 -12 알콕시, C1 -16 아릴알킬 또는 C1 -16 헤테로아릴알킬일 수 있다. R 5 is hydrogen, hydroxy, halogen, C 1 -12 alkyl, C 1 -12 alkyl, halo, C 1 -12 alkyl, heteroaryl, C 1 -12 alkoxy, C 1 -16 alkyl, aryl or C 1 -16 alkyl, heteroaryl days .

상기 신경세포손상 질환으로는 파킨슨씨병, 알츠하이머, 피크병(Pick's disease), 헌팅톤병(Huntington's disease), 근위축성 측면 경화증(amyotriophic lateral sclerosis), 허혈성 뇌질환(stroke), 탈수초질환(demyelinating disease) 및 척추 손상(spinal cord injury)으로 이루어진 군에서 선택될 수 있다.The neuronal cell damage diseases include Parkinson's disease, Alzheimer's disease, Pick's disease, Huntington's disease, amyotriophic lateral sclerosis, ischemic brain disease, demyelinating disease, And spinal cord injury. ≪ Desc / Clms Page number 2 >

보다 상세하게는, 상기 신경세포를 치료학적으로 유효한 양으로 신경세포손상 질환의 치료가 필요한 대상의 손상부위에 투여할 경우, 주변 중추신경계 또는 말초신경계의 신경전구세포 또는 신경줄기세포를 신경세포로 분화시켜 손상된 신경 기능을 회복시키고 신경 손상 질환을 치료할 수 있다. 이때 상기 대상은 인간을 포함하는 포유동물일 수 있다.More specifically, when the neuron is administered to a damaged area of a subject in need of treatment of neuronal damage disease with a therapeutically effective amount, neuronal progenitor cells or neural stem cells of peripheral nervous system or peripheral nervous system are differentiated into neuronal cells To restore damaged nerve function and to treat nerve damage diseases. Wherein the subject may be a mammal, including a human.

본 발명의 약학조성물은 유효 성분 이외에 약학적으로 허용되는 첨가제를 추가로 포함할 수 있다.The pharmaceutical composition of the present invention may further comprise pharmaceutically acceptable additives in addition to the active ingredient.

본 발명의 약학 조성물은 약학 분야에서 통상의 방법에 따라 환자의 신체 내 투여에 적합한 단위 투여형의 제제로 제형화될 수 있다. 이러한 목적에 적합한 제형으로는 비경구투여 제제로서 주사제 또는 구소 투여용 제제 등이 바람직하다. 이때, 일반적으로 사용되는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 함께 사용할 수 있다.The pharmaceutical composition of the present invention can be formulated into a unit dosage form suitable for administration to a patient in the body according to a conventional method in the field of pharmacy. As formulations suitable for this purpose, injectable preparations or preparations for enteral administration are preferred as parenteral administration preparations. At this time, a commonly used diluent such as a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant, or an excipient may be used together.

그리고, 신경세포의 1회 투여량은 1 x 105 세포/kg(체중), 바람직하게는 1회 또는 수회로 나누어 투여할 수 있다. 그러나, 유효 성분의 실제 투여량은 분화 및 증식하고자 하는 신경세포의 양, 투여경로, 환자의 체중, 연령 및 성별 등 여러 관련 인자를 고려하여 결정할 수 있으며, 따라서, 상기 투여량은 어떠한 형태로든 본 발명의 범위를 한정하는 것은 아니다.
Then, the single dose of the nerve cells is 1 x 10 5 cells / kg (body weight), and preferably may be administered once or several dividing circuit. However, the actual dosage of the active ingredient may be determined taking into account various relevant factors such as the amount of neuron to be differentiated and proliferated, the route of administration, the weight of the patient, age and sex, And are not intended to limit the scope of the invention.

이하, 본 발명의 이해를 돕기 위하여 실시예를 들어 상세하게 설명하기로 한다. 다만 하기의 실시예는 본 발명의 내용을 예시하는 것일 뿐 본 발명의 범위가 하기 실시예에 한정되는 것은 아니다. 본 발명의 실시예는 당업계에서 평균적인 지식을 가진 자에게 본 발명을 보다 완전하게 설명하기 위해 제공되는 것이다.
BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended to illustrate the contents of the present invention, but the scope of the present invention is not limited to the following examples. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

<< 제조예Manufacturing example 1> 2-(4- 1 > 2- (4- 니트로페녹시Nitrophenoxy )에탄올(1) 의 제조 ) Preparation of ethanol (1)

4-니트로페놀 (1.55g, 11.15mmol), 2-브로모에탄올 (1.583mL, 22.3mmol), 탄산칼륨 (7.7g, 55.7mmol)을 DMF (3mL)에 용해시킨 후, 80 ℃에서 반응혼합물을 밤새도록 교반하였다. 이후 에틸 아세테이트로 희석하고 물 및 염수로 세척한 후, 무수황산마그네슘으로 건조하고, 감압 하에서 농축하였다. 농축된 산물은 실리카 겔 컬럼 크로마토그래피 (EtOAc/헥산 = 1:1)로 정제되었고, 흰색 고체로서 2-(4-니트로페녹시)에탄올(1)을 얻을 수 있었다(1.38g, 7.53mmol, 수득률 67.6%). After dissolving 4-nitrophenol (1.55 g, 11.15 mmol), 2-bromoethanol (1.583 mL, 22.3 mmol) and potassium carbonate (7.7 g, 55.7 mmol) in DMF (3 mL) And stirred overnight. It was then diluted with ethyl acetate, washed with water and brine, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The concentrated product was purified by silica gel column chromatography (EtOAc / hexane = 1: 1) to give 2- (4-nitrophenoxy) ethanol (1) as a white solid (1.38 g, 7.53 mmol, 67.6%).

1H NMR (CDCl3): δ= 8.24 (dd, J = 9, 2Hz, 2H), 7.01 (dd, J= 9, 2Hz, 2H), 4.21 (m, 2H), 4.04 (m, 2H). 1 H NMR (CDCl 3): δ = 8.24 (dd, J = 9, 2Hz, 2H), 7.01 (dd, J = 9, 2Hz, 2H), 4.21 (m, 2H), 4.04 (m, 2H).

제조예 1의 반응식을 이하에 나타내었다. The reaction formula of Production Example 1 is shown below.

[반응식 1][Reaction Scheme 1]

Figure 112011090098428-pat00017

Figure 112011090098428-pat00017

<< 제조예Manufacturing example 2> 2-(4- 2 > 2- (4- 니트로페녹시Nitrophenoxy )) 에틸메탄설포네이트Ethyl methanesulfonate (2) 의 제조(2)

상기 2-(4-니트로페녹시)에탄올(1)(400mg, 2.18mmol), 트리에틸아민(3mL, 21.8mmol)을 THF (20mL)에 용해하고, 메탄설포닐클로라이드 (254.6μL, 3.27mmol)를 0℃ 에서 적하하였다. 반응 혼합물은 0℃에서 1시간동안 교반되었다. 그 후 에틸 아세테이트로 희석하였으며, 물 및 염수로 세척한 후, 무수황산마그네슘으로 건조하고, 감압 하에서 농축하였다. 농축된 산물은 실리카 겔 컬럼 크로마토그래피 (MeCN/CHCl3= 1:20)로 정제되었고, 2-(4-니트로페녹시)에틸메탄설포네이트(2) 을 얻을 수 있었다(566mg, 2.17mmol, 수득률 99 %). (400 mg, 2.18 mmol) and triethylamine (3 mL, 21.8 mmol) were dissolved in THF (20 mL), methanesulfonyl chloride (254.6 μL, 3.27 mmol) Was added dropwise at 0 占 폚. The reaction mixture was stirred at 0 &lt; 0 &gt; C for 1 hour. It was then diluted with ethyl acetate, washed with water and brine, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The concentrated product was purified by silica gel column chromatography (MeCN / CHCl 3 = 1 : 20) to give 2- (4-nitrophenoxy) ethyl methanesulfonate (2) (566 mg, 2.17 mmol, 99% yield).

1H NMR (CDCl3): δ= 8.21 (dd, J= 9, 2Hz, 2H), 6.97 (dd, J= 9, 2Hz, 2H), 4.59-4.62 (m, 2H), 4.33-4.36 (m, 2H), 3.10(s, 3H) 1 H NMR (CDCl 3): δ = 8.21 (dd, J = 9, 2Hz, 2H), 6.97 (dd, J = 9, 2Hz, 2H), 4.59-4.62 (m, 2H), 4.33-4.36 (m , &Lt; / RTI &gt; 2H), 3.10 (s, 3H)

제조예 2의 반응식을 이하에 나타내었다. The reaction formula of Production Example 2 is shown below.

[반응식 2][Reaction Scheme 2]

Figure 112011090098428-pat00018

Figure 112011090098428-pat00018

<< 제조예Manufacturing example 3> 2-(3- 3 > 2- (3- 니트로페녹시Nitrophenoxy )에탄올의 제조) Preparation of ethanol

3-니트로페놀 (180mg, 1.294mmol), 2-브로모에탄올 (110.24 μL, 1.553mmol), 탄산칼륨 (894mg, 6.47mmol)을 DMF (1mL)에 용해시킨 후, 80 ℃에서 반응혼합물을 밤새도록 교반하였다. 이후 에틸 아세테이트로 희석하고 물 및 염수로 세척한 후, 무수황산마그네슘으로 건조하고, 감압 하에서 농축하였다. 농축된 산물은 톨루엔으로 재결정되었고, 흰색 막대 결정으로서 2-(3-니트로페녹시)에탄올(3)을 얻을 수 있었다(140mg, 0.764mmol, 수득률 59%). After dissolving 3-nitrophenol (180 mg, 1.294 mmol), 2-bromoethanol (110.24 μL, 1.553 mmol) and potassium carbonate (894 mg, 6.47 mmol) in DMF (1 mL) Lt; / RTI &gt; It was then diluted with ethyl acetate, washed with water and brine, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The concentrated product was recrystallized from toluene to give 2- (3-nitrophenoxy) ethanol (3) as white rod crystals (140 mg, 0.764 mmol, 59% yield).

1H NMR (CDCl3): δ= 8.24 (dd, J = 9, 2Hz, 2H), 7.01 (dd, J= 9, 2Hz, 2H), 4.21 (m, 2H), 4.04 (m, 2H). 1 H NMR (CDCl 3): δ = 8.24 (dd, J = 9, 2Hz, 2H), 7.01 (dd, J = 9, 2Hz, 2H), 4.21 (m, 2H), 4.04 (m, 2H).

제조예 3의 반응식을 이하에 나타내었다. The reaction formula of Production Example 3 is shown below.

[반응식 3][Reaction Scheme 3]

Figure 112011090098428-pat00019

Figure 112011090098428-pat00019

<< 제조예Manufacturing example 4> 2-(3- 4 > 2- (3- 니트로페녹시Nitrophenoxy )) 에틸메탄설포네이트Ethyl methanesulfonate (4) 의 제조(4)

상기 2-(3-니트로페녹시)에탄올(3)(140mg, 0.764mmol), 트리에틸아민(1mL, 7.64mmol)을 THF (20mL)에 용해하고, 메탄설포닐클로라이드 (90μL, 1.147mmol)를 0℃ 에서 적하하였다. 반응 혼합물은 0℃에서 1시간동안 교반되었다. 그 후 에틸 아세테이트로 희석하였으며, 물 및 염수로 세척한 후, 무수황산마그네슘으로 건조하고, 감압 하에서 농축하였다. 농축된 산물은 실리카 겔 컬럼 크로마토그래피 (MeCN/CHCl3= 1:30)로 정제되었고, 2-(3-니트로페녹시)에틸메탄설포네이트(4) 를 얻을 수 있었다(196mg, 0.75mmol, 수득률 98%). (140 mg, 0.764 mmol) and triethylamine (1 mL, 7.64 mmol) were dissolved in THF (20 mL), and methanesulfonyl chloride (90 μL, 1.147 mmol) Followed by dropwise addition at 0 ° C. The reaction mixture was stirred at 0 &lt; 0 &gt; C for 1 hour. It was then diluted with ethyl acetate, washed with water and brine, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The concentrated product was purified by column chromatography on silica gel (MeCN / CHCl 3 = 1:30) , 2- (3- nitrophenoxy) ethyl methanesulfonate (4) (196 mg, 0.75 mmol, 98% yield).

1H NMR (CDCl3): δ= 8.21 (dd, J= 9, 2Hz, 2H), 6.97 (dd, J= 9, 2Hz, 2H), 4.59-4.62 (m, 2H), 4.33-4.36 (m, 2H), 3.10(s, 3H) 1 H NMR (CDCl 3): δ = 8.21 (dd, J = 9, 2Hz, 2H), 6.97 (dd, J = 9, 2Hz, 2H), 4.59-4.62 (m, 2H), 4.33-4.36 (m , &Lt; / RTI &gt; 2H), 3.10 (s, 3H)

제조예 4의 반응식을 이하에 나타내었다. The reaction formula of Production Example 4 is shown below.

[반응식 4][Reaction Scheme 4]

Figure 112011090098428-pat00020
Figure 112011090098428-pat00020

<< 제조예Manufacturing example 5> 2-(4- 5 > 2- (4- 니트로페녹시Nitrophenoxy )) 에틸메탄설포네이트Ethyl methanesulfonate (2)의 (2) 아미노화Amination 반응 reaction

2-(4-니트로페녹시)에틸메탄설포네이트(2)(0.727mmol) 또는 2-(3-니트로페녹시)에틸메탄설포네이트(1) , 탄산칼륨(502.4mg, 3.64mmol), 및 부합하는 2차 아민 (2.18mmol)을 DMF(1mL)에 용해한 후, 70℃에서 24시간 교반하였다. 그 후 반응 혼합물을 에틸 아세테이트로 희석하였으며, 여과하고, 여과물을 감압 하에서 농축하였다. 농축된 산물은 실리카 겔 컬럼 크로마토그래피 (MeOH/디클로로메탄 = 1:15~1:20)로 정제되었다. A mixture of 2- (4-nitrophenoxy) ethyl methanesulfonate (2) (0.727 mmol) or 2- (3-nitrophenoxy) ethyl methanesulfonate (1), potassium carbonate (502.4 mg, 3.64 mmol) (2.18 mmol) was dissolved in DMF (1 mL), and the mixture was stirred at 70 占 폚 for 24 hours. The reaction mixture was then diluted with ethyl acetate, filtered and the filtrate was concentrated under reduced pressure. The concentrated product was purified by silica gel column chromatography (MeOH / dichloromethane = 1: 15-1: 20).

4-(2-(4-4- (2- (4- 니트로페녹시Nitrophenoxy )모르폴린 (5a)) Morpholine (5a)

1H NMR (CDCl3): δ= 8.17 (dd, J= 9, 2Hz, 2H), 6.97 (dd, J= 9, 2Hz, 2H), 4.17-4.21(m, 2H), 3.71-3.74 (m, 4H), 2.81-2.85 (m, 2H), 2.56-2.59 (m, 4H) 1 H NMR (CDCl 3): δ = 8.17 (dd, J = 9, 2Hz, 2H), 6.97 (dd, J = 9, 2Hz, 2H), 4.17-4.21 (m, 2H), 3.71-3.74 (m , 4H), 2.81-2.85 (m, 2H), 2.56-2.59 (m, 4H)

1-(2-(4- 1- (2- (4- 니트로페녹시Nitrophenoxy )에틸)피페리딘 (5b) ) Ethyl) piperidine &lt; RTI ID = 0.0 &gt; (5b)

1H NMR (CDCl3): δ= 8.19 (d, J = 9.3Hz, 2H), 6.57 (d, J = 9.3Hz, 2H), 4.19 (t, J = 6.0Hz, 2H), 2.80 (t, J = 6.0Hz, 2H), 2.45-2.55 (m, 4H), 1.57-1.65 (m, 4H), 1.45-1.50 (m, 2H) 1 H NMR (CDCl 3): δ = 8.19 (d, J = 9.3Hz, 2H), 6.57 (d, J = 9.3Hz, 2H), 4.19 (t, J = 6.0Hz, 2H), 2.80 (t, J = 6.0Hz, 2H), 2.45-2.55 (m, 4H), 1.57-1.65 (m, 4H), 1.45-1.50 (m, 2H)

N,N-N, N- 디에틸Diethyl -2-(4- -2- (4- 니트로페녹시Nitrophenoxy )) 에탄아민Ethanamine (5c)  (5c)

1H NMR (CDCl3): δ= 8.19 (d, J = 9.0Hz, 2H), 6.97 (d, J = 9.0Hz, 2H), 4.13 (t, J = 6.0Hz, 2H), 2.90 (t, J = 6.0Hz, 2H), 2.65 (q, J = 7.2Hz, 4H), 1.08 (t, J = 7.2Hz, 6H) 1 H NMR (CDCl 3): δ = 8.19 (d, J = 9.0Hz, 2H), 6.97 (d, J = 9.0Hz, 2H), 4.13 (t, J = 6.0Hz, 2H), 2.90 (t, J = 6.0Hz, 2H), 2.65 (q, J = 7.2Hz, 4H), 1.08 (t, J = 7.2Hz, 6H)

4-(2-(3- 4- (2- (3- 니트로페녹시Nitrophenoxy )에틸)모르폴린 (5d)) Ethyl) morpholine (5d)

1H NMR (CDCl3): δ= 7.83 (dd, J = 7.8, 1.5Hz, 1H), 7.64 (t, J = 2.1Hz, 1H), 7.43 (t, J = 7.8Hz, 1H), 7.35 (dd, J = 7.8 2.1Hz, 1H), 4.19 (t, J = 5.7Hz, 2H), 3.75 (t, J = 4.5Hz, 4H), 2.85 (t, J = 5.7Hz, 2H), 2.56 (t, J = 4.5Hz, 4H)
 1 H NMR (CDCl 3): δ = 7.83 (dd, J = 7.8, 1.5Hz, 1H), 7.64 (t, J = 2.1Hz, 1H), 7.43 (t, J = 7.8Hz, 1H), 7.35 ( dd, J = 7.8 2.1Hz, 1H ), 4.19 (t, J = 5.7Hz, 2H), 3.75 (t, J = 4.5Hz, 4H), 2.85 (t, J = 5.7Hz, 2H), 2.56 (t , J = 4.5 Hz, 4H)

제조예 5의 각각의 반응식을 이하에 나타내었다. The reaction formulas of Production Example 5 are shown below.

[반응식 5][Reaction Scheme 5]

Figure 112011090098428-pat00021
Figure 112011090098428-pat00021

<< 제조예Manufacturing example 6>  6> 니트로기의Nitro group 아미노기로의 환원반응 Reduction reaction to amino group

상기 제조예 6의 반응산물(0.22mmol), 포름산 암모늄(과량), Pd/C (10 %)을 THF(4mL)에 용해하고 상온에서 1시간동안 교반하였다. 반응 시간은 Pd/C (10 %) 촉매의 양에 의존적이었고, TLC를 매 10분간 체크하였다. 잔여 포름산 암모늄 및 Pd/C가 여과되었고, 여과액은 감압 하에서 농축되었다. 이렇게 얻어진 비정제 산물을 추가적인 정제없이 다음 단계에서 사용되었다. The reaction product (0.22 mmol), ammonium formate (excess) and Pd / C (10%) of Preparation 6 were dissolved in THF (4 mL) and stirred at room temperature for 1 hour. The reaction time was dependent on the amount of Pd / C (10%) catalyst and TLC was checked every 10 minutes. The residual ammonium formate and Pd / C were filtered, and the filtrate was concentrated under reduced pressure. The crude product thus obtained was used in the next step without further purification.

4-(2-4- (2- morpholinoethoxy모르프올inoethoxy )) anilineaniline (6a) (6a)

4-(2-(4- (2- ( piperidinpiperidine -1--One- ylyl )) ethoxyethoxy )) anilineaniline (6b) (6b)

4-(2-(4- (2- ( diethylaminodiethylamino )) ethoxyethoxy )) anilineaniline (6c)(6c)

3-(2-3- (2- morpholinoethoxy모르프올inoethoxy )) anilineaniline (6d) (6d)

제조예 6의 각각의 반응식을 이하에 나타내었다. Each reaction formula of Production Example 6 is shown below.

[반응식 6][Reaction Scheme 6]

Figure 112011090098428-pat00022
Figure 112011090098428-pat00022

<< 제조예Manufacturing example 7> 2- 7> 2- 클로로Chloro -N-(4-(2--N- (4- (2- 모르폴리노에톡시Morpholinoethoxy )) 페닐Phenyl )피리미딘-4-아민 (7) 의 제조 ) Pyrimidin-4-amine (7)

4-(2-모르폴리노에톡시)아닐린 (6a) (434mg, 1.35mmol)을 n-BuOH(10mL)에 용해하고, 2,4-디클로로피리미딘(223mg, 1.49mmol) 및 트리에틸아민(1.35mmol)을 한방울씩 적하하였다. 혼합용액은 환류조건에서 24시간동안 가열되었다. 그 후 디클로로메탄으로 희석하고, 물 및 염수로 세척하였으며, 무수황산마그네슘으로 건조하고, 감압 하에서 농축하였다. 농축된 산물은 DCM / 메탄올 (20 : 1)을 사용하는 컬럼에서 정제되었고, 용리액에서 2-클로로-N-(4-(2-모르폴리노에톡시)페닐)피리미딘-4-아민 (7)을 얻었다(수득률 55%).(434 mg, 1.35 mmol) was dissolved in n-BuOH (10 mL) and 2,4-dichloropyrimidine (223 mg, 1.49 mmol) and triethylamine 1.35 mmol) was added dropwise. The mixed solution was heated under reflux conditions for 24 hours. It was then diluted with dichloromethane, washed with water and brine, dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The concentrated product was purified on a column using DCM / methanol (20: 1) and eluted with 2-chloro-N- (4- (2-morpholinoethoxy) phenyl) pyrimidin- ) (Yield 55%).

1H NMR (CDCl3): δ= 8.06 (d, J = 6Hz, 1H), 7.18 (d, J= 9, 2Hz, 2H), 6.93 (d, J= 9, 2Hz, 2H), 6.91(s, 1H), 6.40(d, J = 6 Hz, 1H), 4.11-4.15 (m, 2H), 3.73-3.76 (m, 4H), 2.80-2.84 (m, 2H), 2.58-2.61 (m, 4H) 1 H NMR (CDCl 3): δ = 8.06 (d, J = 6Hz, 1H), 7.18 (d, J = 9, 2Hz, 2H), 6.93 (d, J = 9, 2Hz, 2H), 6.91 (s , 1H), 6.40 (d, J = 6 Hz, 1H), 4.11-4.15 (m, 2H), 3.73-3.76 (m, 4H), 2.80-2.84 (m, 2H), 2.58-2.61 (m, 4H )

제조예 7의 반응식을 이하에 나타내었다. The reaction formula of Production Example 7 is shown below.

[반응식 7][Reaction Scheme 7]

Figure 112011090098428-pat00023
Figure 112011090098428-pat00023

<< 제조예Manufacturing example 8> N-(2- 8 > N- (2- 클로로피리미딘Chloropyrimidine -4-일)Yl) 벤조Benzo [d]티아졸-6-[d] thiazol-6- 아민(8)의Of amine (8) 제조 Produce

2,4-디클로로피리미딘 (113mg, 0.76mmol), 6-아미노벤조티아졸 (136mg, 91mmol) 및 트리에틸아민 (0.76mmol) 을 n-BuOH (10ml)에 용해하고, 60℃ 에서 12시간도안 교반하였다. 그 후 반응 혼합물을 디클로로메탄으로 희석하고, 물 및 염수로 세척하였으며, 무수황산마그네슘으로 건조하고, 감압 하에서 농축하였다. 농축된 산물은 EtOAc / 헥산 (1 : 1)을 사용하는 컬럼에서 정제되었고, 용리액에서 N-(2-클로로피리미딘-4-일)벤조[d]티아졸-6-아민(8) 을 얻었다(수득률 29%).(13 mg, 0.76 mmol), 6-aminobenzothiazole (136 mg, 91 mmol) and triethylamine (0.76 mmol) were dissolved in n-BuOH (10 ml) Lt; / RTI &gt; The reaction mixture was then diluted with dichloromethane, washed with water and brine, dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The concentrated product was purified on a column using EtOAc / hexane (1: 1) and N- (2-chloropyrimidin-4-yl) benzo [d] thiazol- (Yield: 29%).

제조예 8의 반응식을 이하에 나타내었다. The reaction formula of Production Example 8 is shown below.

[반응식 8][Reaction Scheme 8]

Figure 112011090098428-pat00024
Figure 112011090098428-pat00024

<< 제조예Manufacturing example 9> 2- 9> 2- 클로로Chloro -N-(3,4--N- (3,4- 디메톡시페닐Dimethoxyphenyl )피리미딘-4-) Pyrimidin-4- 아민(9)의Of amine (9) 제조 Produce

3,4-데미톡시아닐린(100mg, 0.653mmol), 2,4-디클로로피리미딘 (116.7mg, 0.783mmol) 및 소듐 아세테이트 무수물 (80.3mg, 0.979mmol) 을 THF/물 (1:1) 용액에 용해하고 60℃에서 두시간동안 교반하였다. 그 후 반응 혼합물을 에틸 아세테이트로 희석하고 물 및 염수로 세척하였으며, 무수황산마그네슘으로 건조하고, 진공 조건 하에서 농축하였다. 농축된 산물은 MeCN/CHCl3 = 1:10 을 사용하는 실리카 겔 크로마토그래피로 정제되었고, 2-클로로-N-(3,4-디메톡시페닐)피리미딘-4-아민(9)을 얻었다(71.8mg, 0.27mmol, 수득률 41.4%).(1: 1) solution of 3,4-dimethoxy aniline (100 mg, 0.653 mmol), 2,4-dichloropyrimidine (116.7 mg, 0.783 mmol) and sodium acetate anhydride (80.3 mg, 0.979 mmol) Lt; RTI ID = 0.0 &gt; 60 C &lt; / RTI &gt; for two hours. The reaction mixture was then diluted with ethyl acetate, washed with water and brine, dried over anhydrous magnesium sulfate and concentrated under vacuum conditions. The concentrated product was MeCN / CHCl 3 = was purified by silica gel chromatography using 1: 10, 2-Chloro -N- (3,4- dimethoxy-phenyl) pyrimidin-4-amine (9) ( 71.8 mg, 0.27 mmol, yield 41.4%).

제조예 9의 반응식을 이하에 나타내었다. The reaction formula of Production Example 9 is shown below.

[반응식 9][Reaction Scheme 9]

Figure 112011090098428-pat00025

Figure 112011090098428-pat00025

<< 제조예Manufacturing example 10> 1- 10> 1- 메톡시Methoxy -4-니트로벤젠 (10)의 제조 -4-nitrobenzene (10)

4-니트로페놀 (280mg, 2.013mmol), 요오드화메틸 (150μL, 2.4mmol) 및 탄산칼륨(과량)을 아세톤에 용해하고 3시간동안 환류시켰다. 반응 혼합물을 에틸 아세테이트로 희석하고, 여과한 후, 여과액을 진공 조건 하에서 농축하였다. 농축된 산물은 EtOAc/헥산 = 1:5 을 사용하는 실리카 겔 크로마토그래피로 정제되었고, 1-메톡시-4-니트로벤젠 (10)을 얻었다(287.7mg, 1.878mmol, 수득률 94 %).4-Nitrophenol (280 mg, 2.013 mmol), methyl iodide (150 μL, 2.4 mmol) and potassium carbonate (excess) were dissolved in acetone and refluxed for 3 hours. The reaction mixture was diluted with ethyl acetate, filtered and the filtrate was concentrated under vacuum conditions. The concentrated product was purified by silica gel chromatography using EtOAc / hexane = 1: 5 to give 1-methoxy-4-nitrobenzene (10) (287.7 mg, 1.878 mmol, 94% yield).

제조예 10의 반응식을 이하에 나타내었다. The reaction formula of Production Example 10 is shown below.

[반응식 10][Reaction Scheme 10]

Figure 112011090098428-pat00026

Figure 112011090098428-pat00026

<< 제조예Manufacturing example 11> 4- 11> 4- 메톡시아닐릴(11)의The methoxyanilyl (11) 제조  Produce

1-메톡시-4-니트로벤젠(10)(287.7mg, 1.878mmol), 활성 탄소 상의 팔라듐 5% (촉매량) 및 포름산 암모늄(과량)의 혼합물이 상온에서 2시간동안 교반되었다. 상기 혼합물에서, 메탄올이 증발되었고, 에틸 아세테이트로 희석되었다. 상기 혼합물이 여과되었고, 여과액은 진공 조건 하에서 농축되었다. 이렇게 얻어진 비정제 산물을 추가적인 정제없이 다음 단계에서 사용되었다. A mixture of 1-methoxy-4-nitrobenzene (10) (287.7 mg, 1.878 mmol), 5% palladium on activated carbon and catalytic amount of ammonium formate (excess) was stirred at room temperature for 2 hours. In the mixture, methanol was evaporated and diluted with ethyl acetate. The mixture was filtered, and the filtrate was concentrated under vacuum conditions. The crude product thus obtained was used in the next step without further purification.

제조예 11의 반응식을 이하에 나타내었다. The reaction formula of Production Example 11 is shown below.

[반응식 11][Reaction Scheme 11]

Figure 112011090098428-pat00027

Figure 112011090098428-pat00027

<< 제조예Manufacturing example 12> 2- 12> 2- 클로로Chloro -N-(4--N- (4- 메톡시페닐Methoxyphenyl )피리미딘-4-) Pyrimidin-4- 아민(12)의The amine (12) 제조  Produce

2,4-디클로로피리미딘(280mg, 1.878mg), 4-메톡시아닐린(11)(1,878mmol), 및 트리에틸아민(785μL, 5.63mmol)을 1-부탄올에 용해하고 40℃에서 10시간동안 교반하였다. 반응 혼합물을 에틸 아세테이트로 희석하고, 포화 NaHCO3( aq ) 로 중화한 후, 유기층을 에틸아세테이트로 추출하였다. 유기층을 염수로 세척하고, 무수황산마그네슘으로 건조하고, 진공 조건 하에서 농축하였다. 디클로로메탄을 가한 후, 부유액을 여과하고, 디클로로메탄으로 세척하여 비정제 산물을 얻었고, 이를 통해 2-클로로-N-(4-메톡시페닐)피리미딘-4-아민(12)을 얻었다(188mg, 0.798mmol, 수득률 42.5%). 4-methoxyaniline (11) (1,878 mmol), and triethylamine (785 μL, 5.63 mmol) were dissolved in 1-butanol and the mixture was stirred at 40 ° C. for 10 hours Lt; / RTI &gt; The reaction mixture was diluted with ethyl acetate and neutralized with saturated NaHCO3 ( aq ) , then the organic layer was extracted with ethyl acetate. The organic layer was washed with brine, dried over anhydrous magnesium sulfate, and concentrated under vacuum conditions. After adding dichloromethane, the supernatant was filtered and washed with dichloromethane to give the crude product which gave 2-chloro-N- (4-methoxyphenyl) pyrimidin-4-amine (12) , 0.798 mmol, yield 42.5%).

제조예 12의 반응식을 이하에 나타내었다. The reaction formula of Production Example 12 is shown below.

[반응식 12][Reaction Scheme 12]

Figure 112011090098428-pat00028

Figure 112011090098428-pat00028

<< 제조예Manufacturing example 13> 2- 13> 2- 클로로Chloro -N-(2,4--N- (2,4- 디메톡시페닐Dimethoxyphenyl )피리미딘-4-아민 (13)) Pyrimidin-4-amine (13)

2,4-디메톡시아닐린 (280.4mg, 1.830mmol), 및 2,4-디클로로피리미딘(300mg, 2.013mmol)을 이소프로판올에 용해시키고, 19시간동안 상온에서 교반하였다. 상기 혼합 용액으로부터 용매를 증발시키고, 에틸 아세테이트로 희석한 후, 물 및 염수로 세척하고 무수 황산마그네슘으로 건조한 후 진공조건 하에서 농축하였다. 비정제 산물은 실리카 겔 컬럼 크로마토그래피로 정제되었고, 2-클로로-N-(2,4-디메톡시페닐)피리미딘-4-아민 (13)을 얻었다(153.1mg, 0.576mmol, 수득률 31.5%).2,4-dimethoxyaniline (280.4 mg, 1.830 mmol) and 2,4-dichloropyrimidine (300 mg, 2.013 mmol) were dissolved in isopropanol and stirred at room temperature for 19 hours. The solvent was evaporated from the mixed solution, diluted with ethyl acetate, washed with water and brine, dried over anhydrous magnesium sulfate, and concentrated under vacuum conditions. The crude product was purified by silica gel column chromatography to give 153.1 mg (0.576 mmol, Yield: 31.5%) of 2-chloro-N- (2,4-dimethoxyphenyl) pyrimidin- .

제조예 13의 반응식을 이하에 나타내었다. The reaction formula of Production Example 13 is shown below.

[반응식 13][Reaction Scheme 13]

Figure 112011090098428-pat00029

Figure 112011090098428-pat00029

<< 제조예Manufacturing example 14> 2- 14> 2- 클로로Chloro -4-(3,4--4- (3,4- 디메톡시페녹시Dimethoxyphenoxy )피리미딘(14)의 제조 ) &Lt; / RTI &gt; pyrimidine (14)

3,4-디메톡시페놀 (100mg, 0.649mmol), 2,4-디클로로피리미딘 (115.97mg, 0.778mmol), 및 탄산칼륨(과량)을 DMF(1mL)에 용해하고, 24시간동안 80℃에서 교반하였다. 반응 혼합물은 에틸 아세테이트로 희석되었으며, 여과 후 여과물은 진공조건 하에서 농축되었다. 비정제 산물은 실리카 겔 크로마토그래피로 정제되었고, 이를 통해 2-클로로-4-(3,4-디메톡시페녹시)피리미딘(14)을 얻었다(166.5mg, 0.624mmol, 수득률 96 %).Dissolve 3,4-dimethoxyphenol (100 mg, 0.649 mmol), 2,4-dichloropyrimidine (115.97 mg, 0.778 mmol), and potassium carbonate (excess) in DMF (1 mL) Lt; / RTI &gt; The reaction mixture was diluted with ethyl acetate, and after filtration, the filtrate was concentrated under vacuum conditions. The crude product was purified by silica gel chromatography to obtain 2-chloro-4- (3,4-dimethoxyphenoxy) pyrimidine (14) (166.5 mg, 0.624 mmol, yield 96%).

제조예 14의 반응식을 이하에 나타내었다. The reaction formula of Production Example 14 is shown below.

[반응식 14][Reaction Scheme 14]

Figure 112011090098428-pat00030

Figure 112011090098428-pat00030

<< 실시예Example 1>  1> NN 22 -(3,4-- (3,4- 디메톡시페닐Dimethoxyphenyl )-) - NN 44 -(4-(2-- (4- (2- 모르폴리노에톡시Morpholinoethoxy )) 페닐Phenyl )피리미딘-2,4-) Pyrimidine-2,4- 디아민Diamine (15a) 의 제조  (15a)

2-클로로-N-(4-(2-모르폴리노에톡시)페닐)피리미딘-4-아민(7) (63 mg, 0.20 mmol), 및 3,4-디메톡시아닐린(35mg, 0.23mmol)을 n-부탄올(3mL)에 용해시키고 170℃에서 25분간 마이크로웨이브에서 교반하였다. 그 후 반응 혼합액은 디클로로메탄으로 희석되었고, 물 및 염수로 세척되었으며, 무수 황산 마그네슘으로 건조되었다. 감압 조건 하에서 용매의 증발이 이루어졌으며, 비정제 산물을 얻었다. 비정제 산물은 그 후 DCM/메탄올 (20:1) 을 사용하는 컬럼에서 정제되었고, 용리액에서 N2-(3,4-디메톡시페닐)-N4-(4-(2-모르폴리노에톡시)페닐)피리미딘-2,4-디아민 (15a)을 얻을 수 있었다(수득률 20%).Amine ( 7) (63 mg, 0.20 mmol), and 3,4-dimethoxyaniline (35 mg, 0.23 mmol) were added to a solution of 2-chloro-N- ) Was dissolved in n-butanol (3 mL) and stirred in a microwave at 170 째 C for 25 minutes. The reaction mixture was then diluted with dichloromethane, washed with water and brine, and dried over anhydrous magnesium sulfate. The solvent was evaporated under reduced pressure and the crude product was obtained. The crude product was then purified on a column using DCM / methanol (20: 1) and eluted with N 2 - (3,4-dimethoxyphenyl) -N 4 - (4- (2- Phenyl) pyrimidine-2,4-diamine (15a) was obtained (yield 20%).

1H NMR (CDCl3, 300 MHz): δ= 7.96 (d, J = 6.0Hz, 1H), 7.22-7.26 (m, 4H), 7.01 (dd, 1H, 2.4Hz, J = 8.4), 6.89 (d, J = 9.0Hz, 2H), 6.81 (d, J = 8.4Hz, 1H), 6.74 (s, 1H), 6.00 (d, J = 6.0Hz, 1H), 4.11 (t, J = 5.7Hz, 2H), 3.86 (s, 3H), 3.82 (s, 3H), 3.75 (t, J = 4.5Hz, 4H), 2.81 (t, J =5.7 Hz, 2H), 2.59 (t, J = 4.5Hz, 4H) 1 H NMR (CDCl 3, 300 MHz): δ = 7.96 (d, J = 6.0Hz, 1H), 7.22-7.26 (m, 4H), 7.01 (dd, 1H, 2.4Hz, J = 8.4), 6.89 ( d, J = 9.0Hz, 2H) , 6.81 (d, J = 8.4Hz, 1H), 6.74 (s, 1H), 6.00 (d, J = 6.0Hz, 1H), 4.11 (t, J = 5.7Hz, 2H), 3.86 (s, 3H ), 3.82 (s, 3H), 3.75 (t, J = 4.5Hz, 4H), 2.81 (t, J = 5.7 Hz, 2H), 2.59 (t, J = 4.5Hz, 4H)

실시예 1의 반응식을 이하에 나타내었다. The reaction formula of Example 1 is shown below.

[반응식 15][Reaction Scheme 15]

Figure 112011090098428-pat00031
Figure 112011090098428-pat00031

<< 비교예Comparative Example 1>  1> NN 44 -(- ( 벤조[d]티아졸Benzo [d] thiazole -6-일)--6-yl) - NN 22 -(4-(2-- (4- (2- 모르폴리노에톡시Morpholinoethoxy )) 페닐Phenyl )피리미딘-2,4-) Pyrimidine-2,4- 디아민Diamine (15b)의 제조  (15b)

4-(2-모르폴리노에톡시)아닐린 (6a) (45 mg, 0.22mmol), N-(2-클로로피리미딘-4-일)벤조[d]티아졸-6-아민 (8) (57mg, 0.22mmol) 및 염산 (1방울)을 n-부탄올 (3mL)에 용해하였고, 170℃ 에서 한시간동안 마이크로웨이브에서 교반하였다. 그 후 반응 혼합액은 디클로로메탄으로 희석되었고, 물 및 염수로 세척되었으며, 무수 황산 마그네슘으로 건조되었다. 감압 조건 하에서 용매의 증발이 이루어졌으며, 비정제 산물을 얻었다. 비정제 산물은 그 후 DCM/메탄올 (10:1) 을 사용하는 컬럼에서 정제되었고, 용리액에서 N4-(벤조[d]티아졸-6-일)-N2-(4-(2-모르폴리노에톡시)페닐)피리미딘-2,4-디아민 (15b) 을 얻을 수 있었다(수득률 10%).4-yl) benzo [d] thiazol-6-amine (8) (prepared according to the procedure described for the synthesis of 4- (2-morpholinoethoxy) aniline 6a (45 mg, 0.22 mmol) 57mg, 0.22mmol) and hydrochloric acid (1 drop) were dissolved in n-butanol (3mL) and stirred in a microwave at 170 ° C for one hour. The reaction mixture was then diluted with dichloromethane, washed with water and brine, and dried over anhydrous magnesium sulfate. The solvent was evaporated under reduced pressure and the crude product was obtained. Crude product was then DCM / methanol (10: 1) was purified on a column using, N 4 of the eluent - (benzo [d] thiazol-yl -6-) -N 2 - (4- ( 2- know Phenyl) pyrimidine-2,4-diamine (15b) (yield: 10%).

1H NMR (Acetone d6, 300 MHz): δ= 9.08 (s, 1H), 8.93 (d, J= 2.1Hz, 1H), 8.87 (s, 1H), 8.35 (s, 1H) 8.02 (d, J= 5.7Hz, 1H), 7.95 (d, J= 9Hz, 1H), 7.63 (d, J= 9Hz, 2H), 7.53 (dd, J= 6.6, 2.1Hz, 1H) 6.93 (d, J= 9Hz, 2H), 6.24 (d, J= 6Hz, 1H) 4.15 (t, J= 6Hz, 2H) 3.63 (t, J= 4.5Hz, 4H), 2.76 (t, J=5.7Hz, 2H) 2.55 (t, J= 4.5Hz, 4H) 1 H NMR (Acetone d6, 300 MHz): δ = 9.08 (s, 1H), 8.93 (d, J = 2.1Hz, 1H), 8.87 (s, 1H), 8.35 (s, 1H) 8.02 (d, J = 5.7Hz, 1H), 7.95 ( d, J = 9Hz, 1H), 7.63 (d, J = 9Hz, 2H), 7.53 (dd, J = 6.6, 2.1Hz, 1H) 6.93 (d, J = 9Hz, 2H), 6.24 (d, J = 6Hz, 1H) 4.15 (t, J = 6Hz, 2H) 3.63 (t, J = 4.5Hz, 4H), 2.76 (t, J = 5.7Hz, 2H) 2.55 (t, J = 4.5 Hz, 4H)

비교예 1의 반응식을 이하에 나타내었다. The reaction formula of Comparative Example 1 is shown below.

[반응식 16][Reaction Scheme 16]

Figure 112011090098428-pat00032
Figure 112011090098428-pat00032

<< 실시예Example 2, 3>  2, 3> NN 44 -(3,4-- (3,4- 디메톡시페닐Dimethoxyphenyl )-) - NN 22 -(4-(2-(피페리딘-1-일)- (4- (2- (piperidin-1-yl) 에톡시Ethoxy )) 페닐Phenyl )피리미딘-2,4-) Pyrimidine-2,4- 디아민Diamine (15c) 및  (15c) and NN 22 -(4-(2-(- (4- (2- ( 디에틸아미노Diethylamino )) 에톡시Ethoxy )) 페닐Phenyl )-) - NN 44 -(3,4-- (3,4- 디메톡시페닐Dimethoxyphenyl )피리미딘-2,4-) Pyrimidine-2,4- 디아민Diamine (15d) 의 제조  (15d)

2-클로로-N-(3,4-디메톡시페닐)피리미딘-4-아민 (9) (1.2 equiv.), 적합한 아닐린(6b-c, 4-메톡시아닐린/1.0 equiv.) 및 4M HCl 에 존재하는 1,4-다이옥산을 1-부탄올에 용해시키고 170℃에서 한시간동안 마이크로웨이브에서 교반하였다. 그 후 반응 혼합액은 디클로로메탄으로 희석되었고, 물 및 염수로 세척되었으며, 무수 황산 마그네슘으로 건조되고 진공 조건 하에서 농축되었다. 비정제 산물은 그 후 실리카 겔 크로마토그래피(메탄올/DCM=1:15~1:10) 로 정제되었고, 원하는 산물을 획득하였다. (1.2 equiv.), The appropriate aniline (6b-c, 4-methoxyaniline / 1.0 equiv.) And 4M HCl &lt; RTI ID = 0.0 & Was dissolved in 1-butanol and stirred in a microwave at 170 &lt; 0 &gt; C for one hour. The reaction mixture was then diluted with dichloromethane, washed with water and brine, dried over anhydrous magnesium sulfate and concentrated under vacuum conditions. The crude product was then purified by silica gel chromatography (methanol / DCM = 1: 15-1: 10) to give the desired product.

실시예Example 2:  2: NN 44 -(3,4-- (3,4- 디메톡시페닐Dimethoxyphenyl )-) - NN 22 -(4-(2-(피페리딘-1-일)- (4- (2- (piperidin-1-yl) 에톡시Ethoxy )) 페닐Phenyl )피리미딘-2,4-) Pyrimidine-2,4- 디아민Diamine (15c) (15c)

실리카 겔 컬럼 크로마토그래피 (MeOH/DCM = 1:15~1:10), 분리 수율 58.2 %Silica gel column chromatography (MeOH / DCM = 1:15 to 1:10), separation yield 58.2%

1H NMR (CDCl3, 300 MHz) δ 7.95 (d, J =6.0Hz, 1H), 7.81 (s, 1H), 7.41 (d, J = 9.3Hz, 2H), 7.36 (s, 1H), 6.77-6.89 (m, 5H), 6.02 (d, J =6.0Hz, 1H), 4.00 (t, J = 6.3Hz, 2H), 3.86 (s, 3H), 3.77 (s, 3H), 2.85 (t, J = 6.3Hz, 2H), 2.63 (q, J = 7.2Hz, 4H), 1.06 (t, J = 7.2Hz, 6H)
 1 H NMR (CDCl 3, 300 MHz) δ 7.95 (d, J = 6.0Hz, 1H), 7.81 (s, 1H), 7.41 (d, J = 9.3Hz, 2H), 7.36 (s, 1H), 6.77 -6.89 (m, 5H), 6.02 (d, J = 6.0Hz, 1H), 4.00 (t, J = 6.3Hz, 2H), 3.86 (s, 3H), 3.77 (s, 3H), 2.85 (t, J = 6.3Hz, 2H), 2.63 (q, J = 7.2Hz, 4H), 1.06 (t, J = 7.2Hz, 6H)

실시예Example 3:  3: NN 22 -(4-(2-(- (4- (2- ( 디에틸아미노Diethylamino )) 에톡시Ethoxy )) 페닐Phenyl )-N) -N 44 -(3,4-디- (3,4-di 메Me 톡시페닐)피리미딘-2,4-Methoxyphenyl) pyrimidine-2,4- 디아민Diamine (15d) (15d)

실리카 겔 컬럼 크로마토그래피 (MeOH/DCM = 1:10), 분리 수율 26.4 %Silica gel column chromatography (MeOH / DCM = 1:10), yield 26.4%

1H NMR (CDCl3, 300 MHz) δ 7.97 (d, J =5.7Hz, 1H), 7.43 (d, J = 9.0Hz, 2H), 7.17 (s, 1H), 6.80-6.89 (m, 6H), 6.03 (d, J =5.7Hz, 1H), 4.09 (t, J = 6.0Hz, 2H), 3.89 (s, 3H), 3.82 (s, 3H), 2.78 (t, J = 6.0Hz, 2H), 2.47-2.51 (m, 4H), 1.58-1.65 (m, 4H), 1.44-1.49 (m, 2H) 1 H NMR (CDCl 3, 300 MHz) δ 7.97 (d, J = 5.7Hz, 1H), 7.43 (d, J = 9.0Hz, 2H), 7.17 (s, 1H), 6.80-6.89 (m, 6H) , 6.03 (d, J = 5.7Hz , 1H), 4.09 (t, J = 6.0Hz, 2H), 3.89 (s, 3H), 3.82 (s, 3H), 2.78 (t, J = 6.0Hz, 2H) , 2.47-2.51 (m, 4H), 1.58-1.65 (m, 4H), 1.44-1.49 (m, 2H)

실시예 2, 3의 반응식을 이하에 나타내었다. The reaction formulas of Examples 2 and 3 are shown below.

[반응식 17][Reaction Scheme 17]

Figure 112011090098428-pat00033

Figure 112011090098428-pat00033

<< 실시예Example 4>  4> NN 44 -(3,4-- (3,4- 디메톡시페닐Dimethoxyphenyl )-) - NN 22 -(4-(2-- (4- (2- 모르폴리노에톡시Morpholinoethoxy )) 페닐Phenyl )피리미딘-2,4-) Pyrimidine-2,4- 디아민Diamine (15e)의 제조  (15e)

4-(2-모르폴리노에톡시)아닐린(6a) (39mg, 0.18mmol), 2-클로로-N-(3,4-디메톡시페닐)피리미딘-4-아민(9) (52mg, 0.20mmol) 및 HCl (1 방울)을 자일렌(3mL)에 용해시켰고, 180℃ 에서 1시간동안 마이크로웨이브에서 교반하였다. 그 후 반응 혼합액은 디클로로메탄으로 희석되었고, 물 및 염수로 세척되었으며, 무수 황산 마그네슘으로 건조되었다. 그 후 감압 조건 하에서 용매의 증발을 통해 비정제 산물을 얻었다. 비정제 산물은 그 후 DCM / 메탄올 (10 : 1)을 용리액으로 사용하는 컬럼으로 정제되었으며, 이를 통해 N4-(3,4-디메톡시페닐)-N2-(4-(2-모르폴리노에톡시)페닐)피리미딘-2,4-디아민 (15e)을 획득하였다(수득률 18%). Amine (9) (52 mg, 0.20 mmol) was added to a solution of 4- (2-morpholinoethoxy) aniline (6a) (39 mg, 0.18 mmol) and 2-chloro- N- (3,4- dimethoxyphenyl) mmol) and HCl (1 drop) were dissolved in xylene (3 mL) and stirred in a microwave at 180 &lt; 0 &gt; C for 1 hour. The reaction mixture was then diluted with dichloromethane, washed with water and brine, and dried over anhydrous magnesium sulfate. The crude product was then obtained by evaporation of the solvent under reduced pressure. Crude product was then DCM / methanol (10: 1) was purified by column using as the eluent, This N 4 - (3,4- dimethoxyphenyl) -N 2 - (4- (2- morpholinyl Nonethoxy) phenyl) pyrimidine-2,4-diamine (15e) (yield 18%).

1H NMR (CDCl3): δ= 8.30 (s, 1H), 8.16 (s, 1H), 7.94 (d, J = 6Hz, 1H), 7.65 (dd, J = 9, 2Hz, 2H) 7.23(d, J = 2Hz, 1H), 7.09(d, J = 9Hz, 1H), 6.84-6.90 (m, 3H), 6.13-6.15(d, J = 6Hz, 1H), 4.07 (t, 2H), 3.79 (s, 3H), 3.73 (s, 3H), 3.60(t, 4H), 2.71(t, 2H), 2.51(t, 4H) 1 H NMR (CDCl 3): δ = 8.30 (s, 1H), 8.16 (s, 1H), 7.94 (d, J = 6Hz, 1H), 7.65 (dd, J = 9, 2Hz, 2H) 7.23 (d , J = 2Hz, 1H), 7.09 (d, J = 9Hz, 1H), 6.84-6.90 (m, 3H), 6.13-6.15 (d, J = 6Hz, 1H), 4.07 (t, 2H), 3.79 ( (s, 3H), 3.73 (s, 3H), 3.60 (t, 4H)

실시예 4의 반응식을 이하에 나타내었다. The reaction formula of Example 4 is shown below.

[반응식 18][Reaction Scheme 18]

Figure 112011090098428-pat00034
Figure 112011090098428-pat00034

<< 비교예Comparative Example 2, 3, 4, 5, 6> 15f-j 화합물의 제조  2, 3, 4, 5, 6> Preparation of 15f-j compound

2-클로로피리미딘(9, 12, 13, 14 화합물/1.0 equiv.), 적합한 아닐린(6a, 6d /1.0 equiv), 및 캄포설폰산 (0.2 equiv.) 를 이소프로판올에 용해하고, 120℃에서 1시간동안 마이크로웨이브에서 교반하였다. 혼합액은 디클로로메탄으로 희석되었고, 물 및 염수로 세척되었으며, 황산마그네슘으로 건조된 후 진공조건 하에서 농축되었다. 비정제 산물은 실리카 겔 컬럼 크로마토그래피 (MeOH/DCM = 1:30~1:15)로 정제되어 원하는 산물을 얻었다.The appropriate aniline (6a, 6d / 1.0 equiv.) And camphorsulfonic acid (0.2 equiv.) Were dissolved in isopropanol and treated with 1 &lt; RTI ID = 0.0 &gt;Lt; / RTI &gt; The mixture was diluted with dichloromethane, washed with water and brine, dried over magnesium sulphate and concentrated under vacuum. The crude product was purified by silica gel column chromatography (MeOH / DCM = 1: 30-1: 15) to give the desired product.

비교예Comparative Example 2:  2: NN 44 -(3,4-- (3,4- 디메톡시페닐Dimethoxyphenyl )-) - NN 22 -(3-(2-- (3- (2- 모르폴리노에톡시Morpholinoethoxy )) 페닐Phenyl )피리미딘-2,4-) Pyrimidine-2,4- 디아민Diamine (15f) (15f)

실리카 겔 컬럼 크로마토그래피 (MeOH/DCM = 1:20~1:15), 분리 수율 24.8 %Silica gel column chromatography (MeOH / DCM = 1:20 to 1:15), yield of 24.8%

1H NMR (CDCl3, 300 MHz):δ= 8.01 (d, J =6.0Hz, 1H), 7.37 (t, J =2.1Hz, 1H), 7.30 (s, 1H), 7.17 (t, J = 7.8Hz, 1H), 7.07 (d, J = 7.8Hz, 1H), 6.90 (s, 1H), 6.86 (s, 2H), 6.85 (s, 1H), 6.55 (dd, J = 7.8, 2.1Hz, 1H), 6.07 (d, J =6.0Hz, 1H), 4.09 (t, J = 5.7Hz, 2H), 3.90 (s, 3H), 3.83 (s, 3H), 3.73 (t, J = 4.5Hz, 4H), 2.79 (t, J = 5.7Hz, 2H), 2.56 (t, J = 4.5Hz, 4H). 1 H NMR (CDCl 3, 300 MHz): δ = 8.01 (d, J = 6.0Hz, 1H), 7.37 (t, J = 2.1Hz, 1H), 7.30 (s, 1H), 7.17 (t, J = 7.8Hz, 1H), 7.07 (d , J = 7.8Hz, 1H), 6.90 (s, 1H), 6.86 (s, 2H), 6.85 (s, 1H), 6.55 (dd, J = 7.8, 2.1Hz, 1H), 6.07 (d, J = 6.0Hz, 1H), 4.09 (t, J = 5.7Hz, 2H), 3.90 (s, 3H), 3.83 (s, 3H), 3.73 (t, J = 4.5Hz, 4H), 2.79 (t, J = 5.7 Hz, 2H), 2.56 (t, J = 4.5 Hz, 4H).

비교예Comparative Example 3:  3: NN 44 -(4--(4- 메톡시페닐Methoxyphenyl )-) - NN 22 -(4-(2-- (4- (2- 모르폴리노에톡시Morpholinoethoxy )) 페닐Phenyl )피리미딘-2,4-) Pyrimidine-2,4- 디아민Diamine (15g) (15 g)

실리카 겔 컬럼 크로마토그래피 (MeOH/DCM = 1:20~1:15), 분리 수율 39.4 %Silica gel column chromatography (MeOH / DCM = 1:20 to 1:15), yield 39.4%

1H NMR (CDCl3, 300 MHz): δ=7.95 (d, J =5.7Hz, 1H), 7.43 (d, J = 9.3Hz, 2H), 7.31 (s, 1H), 7.22 (d, J = 9.3Hz, 2H), 6.86-6.92 (m, 5H), 5.99 (d, J =5.7Hz, 1H), 4.09 (t, J = 5.7Hz, 2H), 3.81 (s, 3H), 3.74 (t, J = 4.5Hz, 4H), 2.79 (t, J = 6.0Hz, 2H), 2.57 (t, J = 4.5Hz, 4H) 1 H NMR (CDCl 3, 300 MHz): δ = 7.95 (d, J = 5.7Hz, 1H), 7.43 (d, J = 9.3Hz, 2H), 7.31 (s, 1H), 7.22 (d, J = 9.3Hz, 2H), 6.86-6.92 (m , 5H), 5.99 (d, J = 5.7Hz, 1H), 4.09 (t, J = 5.7Hz, 2H), 3.81 (s, 3H), 3.74 (t, J = 4.5 Hz, 4H), 2.79 (t, J = 6.0 Hz, 2H), 2.57 (t, J = 4.5 Hz,

비교예Comparative Example 4:  4: NN 44 -(2,4-- (2,4- 디메톡시페닐Dimethoxyphenyl )-) - NN 22 -(4-(2- - (4- (2- 모르폴리노에톡시Morpholinoethoxy )) 페닐Phenyl )피리미딘-2,4-) Pyrimidine-2,4- 디아민Diamine (15h) (15h)

실리카 겔 컬럼 크로마토그래피 (MeOH/DCM = 1:30~1:20), 분리 수율 9.4 %Silica gel column chromatography (MeOH / DCM = 1:30 to 1:20), yield 9.4%

1H NMR (Acetone d6, 300 MHz): δ=8.18 (s, 1H), 7.93 (d, J =6.0Hz, 1H), 7.66-7.97 (m, 3H), 6.85 (d, J = 9.0Hz, 2H), 6.63 (d, J = 2.7Hz, 1H), 6.51 (dd, J = 9.0, 2.7Hz 1H), 6.17 (d, J = 5.7Hz, 1H), 4.09 (t, J = 5.7Hz, 2H), 3.84 (s, 3H), 3.81 (s, 3H), 3.62 (t, J = 4.5Hz, 4H), 2.73 (t, J = 6.0Hz, 2H), 2.52 (t, J = 4.5Hz, 4H) 1 H NMR (Acetone d6, 300 MHz): δ = 8.18 (s, 1H), 7.93 (d, J = 6.0Hz, 1H), 7.66-7.97 (m, 3H), 6.85 (d, J = 9.0Hz, 2H), 6.63 (d, J = 2.7Hz, 1H), 6.51 (dd, J = 9.0, 2.7Hz 1H), 6.17 (d, J = 5.7Hz, 1H), 4.09 (t, J = 5.7Hz, 2H J = 4.5 Hz, 4H), 2.73 (t, J = 6.0 Hz, 2H), 2.52 (t, J = 4.5 Hz, 4H), 3.84 (s, 3H) )

비교예Comparative Example 5:  5: NN 44 -(2,4-- (2,4- 디메톡시페닐Dimethoxyphenyl )-) - NN 22 -(3,4-- (3,4- 디메톡시페닐Dimethoxyphenyl )피리미딘-2,4-) Pyrimidine-2,4- 디아민Diamine (15i) (15i)

실리카 겔 컬럼 크로마토그래피 (MeOH/DCM = 1:30), 분리 수율 50.7 %Silica gel column chromatography (MeOH / DCM = 1: 30), yield of 50.7%

1H NMR (Acetone d6, 300 MHz): δ= 8.20 (s, 1H), 7.90-7.96 (m, 2H), 7.68 (s, 1H), 7.50 (d, J = 2.4Hz, 1H), 7.28 (dd, J = 8.7, 2.4Hz, 1H), 6.84 (d, J = 9.0Hz, 1H), 6.63 (d, J = 2.7Hz, 1H), 6.49 (dd, J = 9.0, 2.7Hz, 1H), 6.17 (d, J = 6.0Hz, 1H), 3.83 (s, 3H), 3.81 (s, 3H), 3.77 (s, 3H), 3.73 (s, 3H).&Lt; 1 &gt; H NMR (Acetone d6, 300 MHz): [ delta] 8.20 (s, 1H), 7.90-7.96 (m, 2H), 7.68 (s, 1H), 7.50 (d, J = 2.4Hz, 1H), 7.28 (dd, J = 8.7, 2.4Hz, 1H), 6.84 (d, J = 9.0Hz, 1H ), 6.63 (d, J = 2.7Hz, 1H), 6.49 (dd, J = 9.0, 2.7Hz, 1H), 6.17 (d, J = 6.0Hz, 1H), 3.83 (s, 3H), 3.81 (s, 3H), 3.77 (s, 3H), 3.73 (s, 3H).

비교예Comparative Example 6: 4-(3,4- 6: 4- (3,4- 디메톡시페녹시Dimethoxyphenoxy )-N-(3,4-) -N- (3,4- 디메톡시페녹시Dimethoxyphenoxy )피리미딘-2-아민 (15j)) Pyrimidin-2-amine (15j)

실리카 겔 컬럼 크로마토그래피 (MeOH/DCM = 1:40), 분리 수율 30.6 %Silica gel column chromatography (MeOH / DCM = 1: 40), yield 30.6%

1H NMR (Acetone d6, 300 MHz): δ= 8.42 (s, 1H), 8.27 (d, J = 5.7 Hz, 1H), 7.17 (dd, J = 8.7, 2.4 Hz, 1H), 7.0 (d, J = 8.7 Hz, 1H), 6.85 (d, J = 2.7 Hz, 1H), 6.70~6.77 (m, 2H), 6.30 (d, J = 5.7 Hz, 1H), 3.86 (s, 3H), 3.80 (s, 3H), 3.74 (s, 3H), 3.63 (s, 3H) &Lt; 1 &gt; H NMR (Acetone d6, 300 MHz): [ delta] 8.42 (s, 1H), 8.27 (d, J = 5.7 Hz, 1H), 7.17 (dd, J = 8.7, 2.4 Hz, 1H), 7.0 (d, J = 8.7 Hz, 1H), 6.85 (d, J = 2.7 Hz, 1H), 6.70 ~ 6.77 (m, 2H), 6.30 (d, J = 5.7 Hz, 1H), 3.86 (s, 3H), 3.80 (s, 3H), 3.74 (s, 3H), 3.63 (s, 3 H)

비교예 2, 3, 4, 5, 6의 반응식을 이하에 나타내었다. The reaction formulas of Comparative Examples 2, 3, 4, 5 and 6 are shown below.

[반응식 19][Reaction Scheme 19]

Figure 112011090098428-pat00035
Figure 112011090098428-pat00035

<< 실시예Example 5>  5> NN 22 ,, NN 44 -- 비스(3,4-디메톡시페닐)피리미딘Bis (3,4-dimethoxyphenyl) pyrimidine -2,4--2,4- 디아민Diamine (15k)의 제조  (15k)

2,4-디클로로피리미딘(51mg, 0.34mmol) 3,4-디메톡시아닐린 (128mg, 0.84mmol) 및 트리에틸아민 (0.70mmol)을 자일렌(3mL)에 용해시키고 19시간동안 환류시키며 교반하였다. 그 후 반응 혼합물은 디클로로메탄으로 희석되었고, 물 및 염수로 세척되었으며, 무수 황산마그네슘으로 건조되었다. . 그 후 감압 조건 하에서 용매의 증발을 통해 비정제 산물을 얻었다. 비정제 산물은 그 후 DCM / 메탄올 (20 : 1)을 용리액으로 사용하는 컬럼으로 정제되었으며, 이를 통해 N2,N4-비스(3,4-디메톡시페닐)피리미딘-2,4-디아민 (15k) 을 획득하였다(수득률 7%).3,4-dimethoxyaniline (128 mg, 0.84 mmol) and triethylamine (0.70 mmol) were dissolved in xylene (3 mL) . The reaction mixture was then diluted with dichloromethane, washed with water and brine, and dried over anhydrous magnesium sulfate. . The crude product was then obtained by evaporation of the solvent under reduced pressure. The crude product was then purified by column using DCM / methanol (20: 1) as eluent to give N 2 , N 4 -bis (3,4-dimethoxyphenyl) pyrimidine- (Yield: 7%).

1H NMR (CDCl3): δ= 8.29 (s, 1H), 8.13 (s, 1H), 7.95 (d, J = 6Hz, 1H), 7.48 (d, J = 2Hz, 1H) 7.22-7.29 (m, 2H), 7.09-7.14 (m, 1H), 6.87(d, J = 9Hz, 1H), 6.82(d, J = 9Hz, 1H) 6.14(d, J = 6Hz, 1H), 3.71-3.81(m, 9H) 1 H NMR (CDCl 3): δ = 8.29 (s, 1H), 8.13 (s, 1H), 7.95 (d, J = 6Hz, 1H), 7.48 (d, J = 2Hz, 1H) 7.22-7.29 (m , 2H), 7.09-7.14 (m, 1H), 6.87 (d, J = 9Hz, 1H), 6.82 (d, J = 9Hz, 1H) 6.14 (d, J = 6Hz, 1H), 3.71-3.81 (m , 9H)

실시예 5의 반응식을 이하에 나타내었다. The reaction formula of Example 5 is shown below.

[반응식 20][Reaction Scheme 20]

Figure 112011090098428-pat00036

Figure 112011090098428-pat00036

<< 시험예Test Example 1> 면역세포화학 분석 1> Immunocytochemical analysis

면역세포화학 분석을 위하여, 세포 배양물을 4% 파라포름알데히드에서 30분 동안 고정시키고 인산 완충액(PBS)으로 세정하였다. 고정된 세포를 5% 정상 염소 혈청 및 PBS에 용해된 0.2% 트리톤 X-100으로 브로킹하였으며, β-튜블린 III (TuJ1)(모노클로날; 1:1,000; Sigma-Aldrich, MO), 신경교섬유질산성단백질(glial fibrillary acidic protein, GFAP) (폴리클로날; 1:1,000; Dako, Carpinteria, CA)에 대한 제1항체와 배양하였다. PBS로 세정한 후, Cy3 (1:1000, Molecular Probes, CA)에 접합된 제2항체와 30분 동안 배양하였다. 핵 염색으로 제2항체 배양 완료 후, 4,6-디아미디노-2-페닐인돌(DAPI) (1:10,000 in PBS)을 5분 동안 첨가하였다. 역상 형광현미경(DMIL; Leica, Wetzlar)을 이용하여 이미지를 얻었다.For immunocytochemical analysis, the cell cultures were fixed in 4% paraformaldehyde for 30 minutes and washed with phosphate buffered saline (PBS). The fixed cells were broke with 5% normal goat serum and 0.2% Triton X-100 dissolved in PBS and incubated with β-tubulin III (TuJ1) (monoclonal; 1: 1,000; Sigma-Aldrich, MO) And cultured with primary antibody against glial fibrillary acidic protein (GFAP) (polyclonal; 1: 1,000; Dako, Carpinteria, CA). After washing with PBS, the cells were incubated with a second antibody conjugated to Cy3 (1: 1000, Molecular Probes, CA) for 30 minutes. After completion of incubation of the second antibody with nuclear staining, 4,6-diamidino-2-phenylindole (DAPI) (1: 10,000 in PBS) was added for 5 minutes. Images were obtained using reversed phase fluorescence microscopy (DMIL; Leica, Wetzlar).

편향된 결과를 방지하기 위해, 이미지를 무작위로 촬영하였으며, GFAP 또는 TuJ1 양성 세포는 3-6 의존적 결과로부터 계수되었다. 총 세포 수를 계산하기 위해, DAPI 염색된 핵이 사용되었다. GFAP-양성 세포 또는 TuJ1 양성 세포의 수는 DAPI 양성 세포에 의해 백분율을 구하기 위해 나누어졌다. 각각의 화합물이 처리된 군의 백분율 값은 증가 배수로 표현되기 위해 컨트롤의 값에 의해 나누어졌다. To avoid biased results, images were taken at random and GFAP or TuJ1 positive cells were counted from 3-6 dependent results. To calculate total cell number, DAPI stained nuclei were used. The number of GFAP-positive cells or TuJ1-positive cells was divided to obtain percentages by DAPI-positive cells. The percentage value of the group treated with each compound was divided by the value of the control to be expressed as an increasing multiple.

결과를 표 1 및 도 1에 나타내었다. The results are shown in Table 1 and Fig.

compoundcompound NoNo .. foldfold increaseincrease 실시예 1 (15a) (KHN01) Example 1 (15a) (KHN01) 5.25.2 비교예 1 (15b)Comparative Example 1 (15b) 2.62.6 실시예 2 (15c)Example 2 (15c) 2.02.0 실시예 3 (15d)Example 3 (15d) 1.71.7 실시예 4 (15e) (KHN02)Example 4 (15e) (KHNO2) 5.55.5 비교예 2 (15f)Comparative Example 2 (15f) 1.51.5 비교예 3 (15g)Comparative Example 3 (15 g) 1.41.4 비교예 4 (15h)Comparative Example 4 (15h) 2.02.0 비교예 5 (15i)Comparative Example 5 (15i) 2.22.2 비교예 6 (15j)Comparative Example 6 (15j) 1.61.6 실시예 5 (15k) (KHN03)Example 5 (15k) (KHNO3) 8.08.0

표 1을 참조하면, 실시예 화합물이 처리된 군, 특히 실시예 1, 4, 5 화합물이 처리된 군은 다른 비교군이 처리된 군에 비해, 뉴런 마커인 TuJ1 양성 세포의 수가 현저히 증가되어 있음을 확인할 수 있었다. Referring to Table 1, the number of TuJ1-positive cells, which are neuronal markers, is significantly increased in the group treated with the compound of the Example, particularly in the group treated with the compounds of Examples 1, 4 and 5, compared with the group treated with the other comparative group .

이에 따라 실시예 1, 2 및 3에 대한 면역형광 이미지를 나타낸 도 1을 참조하면, 실시예 1, 2, 및 3의 처리에 따라 뉴런 마커인 TuJ1은 DMSO 처리 대조군에 비해 현저히 증가함을 알 수 있고, 성상세포 마커인 GFAP는 현저히 감소됨을 알 수 있었다. 따라서 본 발명의 화합물은 신경줄기세포를 성상세포가 아닌 뉴런으로 분화시키는 효과가 우수함을 알 수 있다.
1, which shows immunofluorescence images for Examples 1, 2 and 3, it can be seen that according to the treatment of Examples 1, 2 and 3, TuJ1, a neuronal marker, is significantly increased compared to DMSO treated control And that the GFAP, an astrocytic marker, was significantly reduced. Therefore, it can be seen that the compound of the present invention has an excellent effect of differentiating neural stem cells into neurons rather than astrocytes.

이상으로 본 발명의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적 기술은 단지 바람직한 실시예일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다. While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that such detail is solved by the person skilled in the art without departing from the scope of the invention. will be. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (9)

삭제delete 삭제delete N2-(3,4-디메톡시페닐)-N4-(4-(2-모르폴리노에톡시)페닐)피리미딘-2,4-디아민, N4-(3,4-디메톡시페닐)-N2-(4-(2-모르폴리노에톡시)페닐)피리미딘-2,4-디아민 및 N2,N4-비스(3,4-디메톡시페닐)피리미딘-2,4-디아민으로 이루어진 군에서 선택된 피리미딘-2,4-디아민 유도체를 유효성분으로 포함하는, 신경줄기세포의 뉴런(neuron)으로의 분화유도용 조성물.
N 2 - (3,4- dimethoxy-phenyl) -N 4 - (4- (2- morpholino-ethoxy) phenyl) pyrimidine-2,4-diamine, N 4 - (3,4- dimethoxyphenyl ) -N 2 - (4- (2-morpholinoethoxy) phenyl) pyrimidine-2,4-diamine and N 2 , N 4 -bis (3,4-dimethoxyphenyl) pyrimidine- -Diamine as an active ingredient, wherein the neural stem cell is differentiated into neurons.
제3항에 있어서,
상기 피리미딘-2,4-디아민 유도체를 1 내지 10 μM의 양으로 포함하는, 신경줄기세포의 뉴런(neuron)으로의 분화유도용 조성물.
The method of claim 3,
A composition for inducing differentiation of neural stem cells into neurons, comprising the above pyrimidine-2,4-diamine derivative in an amount of 1 to 10 [mu] M.
제3항에 있어서,
상기 신경줄기세포가 배아 신경줄기세포, 성체 신경줄기세포, 배아생식 신경줄기세포 및 배아종양 신경줄기세포로 이루어진 군으로부터 선택되는 것을 특징으로 하는, 신경줄기세포의 뉴런(neuron)으로의 분화유도용 조성물.
The method of claim 3,
The composition for inducing differentiation of neural stem cells into neurons, wherein the neural stem cells are selected from the group consisting of embryonic stem cell, adult neural stem cells, embryonic germinal neural stem cells, and embryonic tumor neural stem cells.
삭제delete 제3항의 조성물을 신경줄기세포와 함께 배양하는 단계를 포함하는, 신경줄기세포를 뉴런(neuron)으로 분화시키는 방법.
A method for differentiating neural stem cells into neurons, comprising culturing the composition of claim 3 together with neural stem cells.
삭제delete 삭제delete
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