WO2018128407A1 - Novel quinolinone derivative and pharmaceutical composition for preventing or treating allergic disease such as asthma or atopy comprising same as active ingredient - Google Patents

Novel quinolinone derivative and pharmaceutical composition for preventing or treating allergic disease such as asthma or atopy comprising same as active ingredient Download PDF

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WO2018128407A1
WO2018128407A1 PCT/KR2018/000165 KR2018000165W WO2018128407A1 WO 2018128407 A1 WO2018128407 A1 WO 2018128407A1 KR 2018000165 W KR2018000165 W KR 2018000165W WO 2018128407 A1 WO2018128407 A1 WO 2018128407A1
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PCT/KR2018/000165
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변영주
전영호
이기호
이기용
정용우
손상현
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고려대학교 세종산학협력단
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Priority to US16/473,362 priority Critical patent/US11168094B2/en
Priority to CN201880005933.6A priority patent/CN110177796B/en
Priority to EP18736592.9A priority patent/EP3567044A4/en
Priority claimed from KR1020180001019A external-priority patent/KR102009756B1/en
Publication of WO2018128407A1 publication Critical patent/WO2018128407A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D498/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D498/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
    • C07D498/04—Ortho-condensed systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents

Definitions

  • the present invention relates to a novel quinoline derivative, and more particularly, 4-substituted 2- that exhibits efficacy in preventing or treating allergic diseases such as asthma or atopy by controlling intracellular signal transduction by TSLP and IL-33.
  • It relates to a pharmaceutical composition for the prevention and treatment of allergic diseases such as asthma or atopic dermatitis.
  • Bronchodilators or anti-inflammatory drugs used in the treatment of allergic inflammatory diseases are performed mainly for symptomatic therapy, which may temporarily reduce the symptoms. However, they do not have fundamental control over allergic diseases. .
  • Th2 cells are well known to play a pivotal role in inducing allergic reactions.
  • CD4 T cells When CD4 T cells are stimulated by antigens in lymphocytes, they can be differentiated into several Th cells according to cytokines that are recognized at the same time, and the recognized cytokines are thymic stromal lymphoprotein (TSLP). Or in the case of type 2 cytokines such as IL-4, these cells differentiate into Th2 to induce an allergic reaction.
  • TSLP thymic stromal lymphoprotein
  • Interleukin-33 is a congenital cytokine produced mainly by various external stimuli in mucosal epithelial cells, and is known to play an important role in regulating the immune response of Th2 cell mediated allergic reactions such as asthma.
  • the IL-33 receptor complex for IL-33 mediated signaling consists of the ligand IL-33, the ligand conjugate ST2 (IL-1R4), and the signal receptor IL-1 receptor accessory protein (IL-1RAcP; IL-1R3). It is. Stimulation of IL-33 produces Th2 inflammatory cytokines and chemokines including IL-4, IL-5, IL-6, IL-13 and IL-8.
  • the IL-33 receptor complex is passed through sub-signals such as NF-kB and AP-1 via IL-1 receptor-associated kinase (IRAK), TNF receptor associated factor 6 (TRAF6) and / or MAPKs. Activate the molecules of the system.
  • sub-signals such as NF-kB and AP-1 via IL-1 receptor-associated kinase (IRAK), TNF receptor associated factor 6 (TRAF6) and / or MAPKs.
  • TSLP and IL-33 are cytokines that play an important role in the differentiation of Th2 cells and are expected to be able to treat allergic diseases by controlling them.
  • Non-Patent Document 2 S. Kamijo, H. Takeda, T. Tokura, M. Suzuki, K. Inui, M. Hara, H. Matsuda, A. Matsuda, K. Oboki, T. Ohno, H. Saito, S Nakae, K. Sudo, H. Suto, S. Ichikawa, H. Ogawa, K. Okumura, T. Takai, IL-33-mediated innate response and adaptive immune cells contribute to maximum responses of protease allergen-induced allergic airway inflammation , J Immunol, 190 (2013) 4489-4499.
  • the present invention is to provide a novel quinolinone derivative exhibiting efficacy in the prevention or treatment of allergic diseases such as asthma or atopy by controlling intracellular signal transduction by TSLP and IL-33.
  • compositions for the prevention or treatment of allergic diseases such as asthma or atopy containing the novel quinolinone derivatives as an active ingredient, and a pharmaceutically acceptable carrier.
  • the present invention provides a quinolinone derivative represented by the following [formula 1a] or [formula 1b] to solve the above problems.
  • the present invention contains a quinolinone derivative represented by the above [Formula 1a] or [Formula 1b] as an active ingredient, prevention or treatment of allergic diseases such as asthma or atopy, including a pharmaceutically acceptable carrier It provides a pharmaceutical composition.
  • the quinolinone derivative compound according to the present invention is capable of effectively inhibiting the formation of an inflammatory response of an allergic disease such as asthma or atopic dermatitis, and fundamentally prevents various allergic diseases such as asthma or atopy using a pharmaceutical composition comprising the same. It can be prevented or treated.
  • FIG. 1 is a graph showing the evaluation of TSLP-TSLPR interaction inhibition of the compound [Formula 2] according to the present invention using an ELISA.
  • Figure 2 is a graph showing the evaluation of TSLP-TSLPR interaction inhibition of the compound [Formula 7] according to the present invention using an ELISA.
  • Figure 3 is a graph showing the inhibition of IL-6 secretion by the IL-33 inhibition of the formula [10] according to the present invention.
  • Figure 6 is a graph showing the chemical shift perturbation of Thr 120 residues of IL-33 protein by the compound according to the present invention.
  • Figure 7 is a graph showing the chemical shift perturbation of the Glu 121 residue of the IL-33 protein by the compound according to the present invention.
  • the quinolinone derivatives represented by the following [Formula 1a] and [Formula 1b] is a key cytokine TSLP and IL-33, which is a key cytokine to induce allergic diseases such as asthma or atopy, between such cytokines and receptors It was confirmed that effectively inhibits the binding of the present invention was completed based on this.
  • One aspect of the present invention relates to a quinolinone derivative represented by the following [Formula 1a] or [Formula 1b], and controls the intracellular signal transduction by TSLP and IL-33, and the cytokine TSLP (thymic stromal lymphopoietin) It is characterized by inhibiting the binding between the TSLP receptor or the binding between the cytokine IL-33 and ST-2.
  • TSLP thymic stromal lymphopoietin
  • R 1 and R 2 is the same as or different from each other, and each independently an alkyl group having 1 to 4 carbon atoms, an alkylene group having 1 to 4 carbon atoms, and any one selected from the following [Formula 1].
  • Z is a hetero atom selected from O, S or N, or-(CH 2 ) m- (m is an integer of 0 to 5).
  • X and Y are each independently hydrogen (H), halogen group (fluor (F), chlorine (Cl), bromine (Br), iodine (I)), hydroxy group (OH), amino group (NH 2 ), nitro group ( NO 2 ), cyano group (CN), trifluoromethyl group (CF 3 ), CO-R ', NHR', NR'R ', NHCOR' and COOR '(any R' is 1 to 4 carbon atoms) Alkyl group).
  • p is an integer of 0-4
  • q is an integer of 0-5, and when said p and q are multiple, some X and some Y are each the same or different.
  • the quinolinone derivative represented by the above [Formula 1a] or [Formula 1b] is not limited by this, but may be specifically any one selected from the following [Formula 2] to [Formula 15].
  • the quinolinone derivatives according to the present invention can be prepared through a Sandmeyer reaction, a Suzuki-bond and a Heck reaction under a palladium-catalyst, a nitro group reduction reaction, a cyclization reaction and a substitution reaction under basic conditions, for example starting materials.
  • ethyl 2-aminooxazole-4-carboxylate as a starting material, various 2-aryloxazole-4-carboxylate derivatives can be synthesized using arylboronic acid substituted in ethyl 2-chlorooxazole-4-carboxylate obtained through Sandmeyer reaction.
  • 2-aryl-5-aryloxazole-4-carboxylate derivatives having various substituents introduced therein may be prepared using 2-iodo-1-nitrobenzene substituted in the Heck reaction. After the reduction of the nitro group through a hydrogenation reaction, it is possible to synthesize a quinolinone derivative through the intramolecular reaction of the amine group and the ester group under basic conditions.
  • Various basic quinolinone derivatives represented by the above [Formula 1a] or [Formula 1b] may be prepared by a substitution reaction with dialkylethyl bromide under basic conditions.
  • the compounds of [Formula 2] to [Formula 13] may be prepared through the synthesis process as follows, and the following [1] is quinolinone represented by [Formula 1a] or [Formula 1b] according to the present invention.
  • [2] and [3] are flow charts schematically showing the synthesis process of a compound wherein R 1 and R 2 are ethyl groups. .
  • Another aspect of the present invention relates to a pharmaceutical composition for preventing or treating allergic diseases, such as asthma or atopy, containing a quinolinone derivative represented by the above [Formula 1a] or [Formula 1b] as an active ingredient, TSLP And control intracellular signal transduction by IL-33, inhibit the binding between the cytokine thymic stromal lymphopoietin (TSLP) and the TSLP receptor or inhibit the binding between the cytokine IL-33 and ST-2.
  • allergic diseases such as asthma or atopy
  • a quinolinone derivative represented by the above [Formula 1a] or [Formula 1b] as an active ingredient
  • TSLP thymic stromal lymphopoietin
  • the pharmaceutical composition according to the present invention can be used for the prevention and treatment of a wide range of allergic diseases such as asthma or atopic dermatitis, but the allergic disease is atopic dermatitis, urticaria rhinitis, allergic rhinitis or asthma May be a disease.
  • composition according to the present invention may be administered in the form of a complex preparation together with other drugs known for the prophylaxis or treatment of allergic diseases such as asthma or atopy, or other components such as carriers, diluents, adjuvants and stabilizers, It may also include.
  • the form of the composition according to the present invention may be variously selected depending on the mode to be administered, but is not limited thereto, for example, solid phases such as tablets, pills, powders, capsules, gels, ointments, fluids or suspensions, and the like. It may be in the form of a semi-solid or liquid dosage form, may be administered in a unit dosage form suitable for the precise administration of the dosage alone, orally or parenterally, and in the case of parenteral administration, intravenous, subcutaneous, It can be administered by intramuscular injection.
  • solid phases such as tablets, pills, powders, capsules, gels, ointments, fluids or suspensions, and the like.
  • It may be in the form of a semi-solid or liquid dosage form, may be administered in a unit dosage form suitable for the precise administration of the dosage alone, orally or parenterally, and in the case of parenteral administration, intravenous, subcutaneous, It can be administered by intramuscular injection.
  • compositions may include, depending on the desired formulation, a pharmaceutically acceptable carrier, diluent, adjuvant, stabilizer, defined as an aqueous-based carrier commonly used in formulating pharmaceutical compositions for human administration.
  • Carrier means a substance that facilitates the addition of the compound into a cell or tissue, and is commonly used in the preparation, for example, carbohydrate compounds (eg lactose, amylose, dextrose, water) Cross, sorbitol, mannitol, starch, cellulose, etc.), acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, salt solution, alcohol, arabian Rubber, vegetable oils (e.g.
  • Diluent is defined as a substance that not only stabilizes the biologically active form of a compound of interest, but also is diluted in water to dissolve the compound.
  • examples of diluents include distilled water, physiological saline, Ringer's solution, glucose solution, and Hank's solution and the like.
  • Stabilizers can be selected from the group consisting of proteins, sugars, buffers and mixtures thereof.
  • lubricants, wetting agents, sweetening agents, flavoring agents, emulsifiers, suspending agents, preservatives and the like but also not limited thereto.
  • an effective amount of other components such as carriers, diluents, adjuvants and stabilizers and the like is an amount effective to obtain a pharmaceutically acceptable formulation in terms of solubility, biological activity, etc. of the component.
  • prevention has not been diagnosed as having a disease or a disease, but means inhibiting the occurrence of such a disease or a disease
  • treatment refers to the inhibition of the development of the disease or a disease, disease or disease It is meant to include the alleviation and elimination of diseases or diseases.
  • "contains as an active ingredient” means that the ingredient is included in an amount necessary or sufficient to realize a desired biological effect.
  • the determination of the amount to be included as an active ingredient is an amount for treating a subject disease, and may be determined in consideration of matters that do not cause other toxicity, for example, the disease or condition being treated, the form of the composition to be administered, It may vary depending on various factors such as the size of the subject or the severity of the disease or condition.
  • One of ordinary skill in the art can empirically determine the effective amount of an individual composition without undue experimentation.
  • pharmaceutically acceptable means a property that does not impair the biological activity and physical properties of the compound.
  • reaction mixture was reflected for 1 hour with stirring, cooled to room temperature, the reaction mixture was partitioned between ethyl acetate and 2M sodium hydroxide solution, and the aqueous layer was washed twice more with ethyl acetate. The organic layer was washed with brine, dried (MgSO 4 ) and concentrated in vacuo.
  • the product was purified by silica gel column chromatography (hexane / Et 2 O, 5: 1 to 3: 1, v / v) to give product 22b as a white solid (250 mg, 73%) in the shape of fluffy.
  • a palladium catalyst was added to a solution of compound 23b (192 mg, 0.54 mmol) and MeOH (15 mL), and stirred for 1 hour in a hydrogen (50 psi) atmosphere. After the reaction mixture was filtered through a celite bed, the volatiles were reduced to remove and 4a (145 mg, 82%) was obtained as a yellow solid.
  • ELISA was used to measure the response by HRP using FLAG-TSLP protein and TSLPR-His protein.
  • the TSLP-TSLPR interaction assay was used to evaluate the inhibitory activity.
  • ELISA analysis was performed on Ni-NTA HisSorb plates (Qiagen, Germany). Dispense 100 ⁇ L of the TSLPR solution with hexahistidine (His) tag at the C-terminus to each well of the plate and incubate for 2 hours at room temperature, then wash each well twice with 200 ⁇ L of PBS, 0.05% Tween-20. Then 100 ⁇ L of the compound solution (sample) according to the invention and 100 ⁇ L of TSLP with N-terminal FLAG tag were dispensed. After incubation overnight at 4 ° C., each well was washed twice and treated with 100 ⁇ L of blocking buffer (PBS-0.05% Tween 20 and 1% skim milk).
  • blocking buffer PBS-0.05% Tween 20 and 1% skim milk
  • the plates were washed twice and then coated with 100 ⁇ L of monoclonal anti-FLAG horseradish peroxidase antibody (Sigma-Aldrich Co., USA) for 2 hours, incubated at room temperature, and then each well was washed five times. Each well was treated with 200 ⁇ L of o-phenylenediaminedihydrochloride (Sigma-Aldrich Co., USA) solution and incubated for 30 minutes. After incubation, 1N HCl was added to each well.
  • Optical densities (ODs) were measured at 450 nm of a microplate spectrophotometer, and the TSLP-TSLPR interaction inhibitory effect was calculated using the following formula, and the results are shown in the following [Table 1].
  • Inhibition (inhibition) (%) (1-sample OD / control OD) ⁇ 100
  • HMC-1 a human mast cell line
  • IL-33 and the compound according to the present invention at a predetermined concentration were mixed with the cells, incubated at 37 ° C. for 24 hours, and then the supernatant was obtained by centrifugation before IL-6.
  • the secretion of IL-6 was analyzed by ELISA kit (Biolegend, USA), and the results are shown in the following [Table 2].
  • the quinolinone derivatives according to the present invention are the cytokines TSLP and TSLP receptors or the binding of IL-33 and ST-2, which play a key role in inducing allergic diseases such as asthma or atopy.
  • Splenocytes were isolated from DO11.10 mice and approximately 1 ⁇ 10 6 CD4 T cells were transferred intravenously to pure BALB / c mice.
  • CSPs Chemical shift perturbations
  • IL-33 was cloned by the expression vector pPROEX, an N-terminal His-tag fusion protein in E. coli BL21 (DE3), and cells were cultured at 0.5 mM IPTG (isopropyl-bD-) when the cell density (OD 600 ) reached 0.6. thiogalactoside) and further growth at 20 ° C. overnight.
  • IPTG isopropyl-bD-
  • thiogalactoside cell density
  • lysis buffer 0.1 M Tris pH 7.4, 0.3 M NaCl, 1 mM ⁇ -mercaptoethanol, 0.1% TritonX100, and 0.1 mM phenylmethylsulfonyl fluoride.
  • Cell lysis was sonicated in an ice bath, and cell lysates were centrifuged at 10000 ⁇ g, 4 ° C. for 25 minutes. The pellet was discarded and the supernatant was eluted on 5 mL of HisPur cobalt resin column Thermo Scientific Inc.
  • TEV protease was used to cleave the fusion protein overnight in dialysis buffer (Tris 20 mM (pH 7.4), sodium chloride 300 mM) and 4 ° C.
  • the IL-33 containing mixture was placed in a HisPur cobalt resin column and washed with Tris 0.1 M (pH 7.4), 0.3 M sodium chloride and 1 mM ⁇ -mercaptoethanol, and the bound protein was then tris 0.1 M (pH 7.4), sodium chloride 0.3 M and Elution with 1 mM (20 mL) ⁇ -mercaptoethanol.
  • the protein was loaded on a Superdex S75 gel filtration column (16/60 GE Healthcare) at 20 mM sodium phosphate (pH 6.8), sodium chloride 100 mM, BME 5 mM to obtain pure IL-33 protein.
  • the quinolinone derivative compound according to the present invention is capable of effectively inhibiting the formation of an inflammatory response of an allergic disease such as asthma or atopic dermatitis, and fundamentally prevents various allergic diseases such as asthma or atopy using a pharmaceutical composition comprising the same. It can be prevented or treated.

Abstract

The present invention relates to a novel quinolinone derivative compound which is efficacious for preventing or treating an allergic disease such as asthma or atopy by controlling intracellular signaling via SLP and IL-33. The quinolinone derivative compound according to the present invention is able to effectively inhibit the occurrence of an inflammatory reaction of an allergic disease such as asthma or atopy, and the pharmaceutical composition comprising the same can be used to fundamentally prevent or treat various allergic and asthmatic diseases.

Description

신규한 퀴놀리논 유도체 및 이를 유효성분으로 포함하는 천식 또는 아토피 등의 알러지성 질환의 예방 또는 치료용 약학 조성물Novel quinolinone derivatives and pharmaceutical compositions for the prevention or treatment of allergic diseases such as asthma or atopy comprising the same as an active ingredient
본 발명은 신규한 퀴놀린 유도체에 관한 것으로서, 더욱 상세하게는 TSLP와 IL-33에 의한 세포 내 신호 전달을 제어하여 천식 또는 아토피 등의 알러지성 질환의 예방 또는 치료에 효능을 나타내는 4-substituted 2-aryl-oxazolo[4,5-c] quinolin-4(5H)-one 또는 5-substituted 2-aryl-oxazolo[4,5-c]quinolin-4(5H)-one 유도체 화합물 및 이를 유효성분으로 함유하는 천식 또는 아토피 등의 알러지성 질환의 예방 및 치료용 약학 조성물에 관한 것이다.The present invention relates to a novel quinoline derivative, and more particularly, 4-substituted 2- that exhibits efficacy in preventing or treating allergic diseases such as asthma or atopy by controlling intracellular signal transduction by TSLP and IL-33. aryl-oxazolo [4,5-c] quinolin-4 (5H) -one or 5-substituted 2-aryl-oxazolo [4,5-c] quinolin-4 (5H) -one derivative compound and containing it as an active ingredient It relates to a pharmaceutical composition for the prevention and treatment of allergic diseases such as asthma or atopic dermatitis.
알러지성 염증 질환의 치료에 사용되는 기관지 확장제 혹은 항염증제는 대증 요법 위주로 수행되어 증상 완화에 일시적으로는 효과를 볼 수 있지만, 알러지 질환의 근본적인 제어가 불가능하여 근원적으로 질병을 치료하지 못한다는 단점을 지닌다.Bronchodilators or anti-inflammatory drugs used in the treatment of allergic inflammatory diseases are performed mainly for symptomatic therapy, which may temporarily reduce the symptoms. However, they do not have fundamental control over allergic diseases. .
기관지 천식, 아토피성 피부질환, 알러지성 비염 등의 환경질환은 면역 질환으로 알려져 있고, Th2 세포는 알러지 반응을 유발하는데 중추적인 역할을 하는 것으로 잘 알려져 있다. CD4 T 세포가 림프구에서 항원에 자극을 받을 때 동시에 인식하는 사이토카인 (cytokine)에 따라서 여러 종류의 Th 세포로 분화가 가능하며, 이때 인식하는 사이토카인이 흉선 기질 림프단백질 (thymic stromal lymphoprotein, TSLP) 또는 IL-4 등과 같은 타입 2 사이토카인일 경우에 이러한 세포들은 Th2로 분화하여 알러지 반응을 유발한다.Environmental diseases such as bronchial asthma, atopic dermatitis and allergic rhinitis are known as immune diseases, and Th2 cells are well known to play a pivotal role in inducing allergic reactions. When CD4 T cells are stimulated by antigens in lymphocytes, they can be differentiated into several Th cells according to cytokines that are recognized at the same time, and the recognized cytokines are thymic stromal lymphoprotein (TSLP). Or in the case of type 2 cytokines such as IL-4, these cells differentiate into Th2 to induce an allergic reaction.
인터루킨-33 (IL-33)은 주로 점막상피세포에서 외부의 다양한 자극에 의해 생성이 되는 선천성 싸이토카인으로, 주로 천식과 같은 Th2 세포 매개의 알러지 반응의 면역 반응을 조절하는 중요한 역할을 하는 것으로 알려져 있다. IL-33 매개 신호전달을 위한 IL-33 수용체 복합체는 리간드인 IL-33, 리간드 결합체인 ST2 (IL-1R4)와 신호전달체인 IL-1 수용체 부속 단백질 (IL-1RAcP;IL-1R3)로 구성되어 있다. IL-33의 자극에 의하여 IL-4, IL-5, IL-6, IL-13 및 IL-8을 포함하는 Th2 염증성 사이토카인 및 케모카인이 만들어진다. IL-33이 결합하게 되면 IL-33 수용체 복합체는 IRAK (IL-1 receptor-associated kinase), TRAF6 (TNF receptor associated factor 6) 및/또는 MAPKs를 통하여 NF-kB 및 AP-1과 같은 하위신호전달체계의 분자들을 활성화한다.Interleukin-33 (IL-33) is a congenital cytokine produced mainly by various external stimuli in mucosal epithelial cells, and is known to play an important role in regulating the immune response of Th2 cell mediated allergic reactions such as asthma. . The IL-33 receptor complex for IL-33 mediated signaling consists of the ligand IL-33, the ligand conjugate ST2 (IL-1R4), and the signal receptor IL-1 receptor accessory protein (IL-1RAcP; IL-1R3). It is. Stimulation of IL-33 produces Th2 inflammatory cytokines and chemokines including IL-4, IL-5, IL-6, IL-13 and IL-8. When IL-33 binds, the IL-33 receptor complex is passed through sub-signals such as NF-kB and AP-1 via IL-1 receptor-associated kinase (IRAK), TNF receptor associated factor 6 (TRAF6) and / or MAPKs. Activate the molecules of the system.
종합하면, TSLP와 IL-33은 Th2 세포의 분화에 중요한 역할을 하는 사이토카인으로서, 이를 제어함으로써 알러지 질환의 근본적 치료가 가능할 것으로 기대된다.Taken together, TSLP and IL-33 are cytokines that play an important role in the differentiation of Th2 cells and are expected to be able to treat allergic diseases by controlling them.
[선행기술문헌][Preceding technical literature]
비특허문헌 1 : Julia V, Macia L, Dombrowicz D. Nat Rev Immunol. 2015;15:308-322.[Non-Patent Document 1] Julia V, Macia L, Dombrowicz D. Nat Rev Immunol. 2015; 15: 308-322.
비특허문헌 2 : S. Kamijo, H. Takeda, T. Tokura, M. Suzuki, K. Inui, M. Hara, H. Matsuda, A. Matsuda, K. Oboki, T. Ohno, H. Saito, S. Nakae, K. Sudo, H. Suto, S. Ichikawa, H. Ogawa, K. Okumura, T. Takai, IL-33-mediated innate response and adaptive immune cells contribute to maximum responses of protease allergen-induced allergic airway inflammation, J Immunol, 190 (2013) 4489-4499.Non-Patent Document 2: S. Kamijo, H. Takeda, T. Tokura, M. Suzuki, K. Inui, M. Hara, H. Matsuda, A. Matsuda, K. Oboki, T. Ohno, H. Saito, S Nakae, K. Sudo, H. Suto, S. Ichikawa, H. Ogawa, K. Okumura, T. Takai, IL-33-mediated innate response and adaptive immune cells contribute to maximum responses of protease allergen-induced allergic airway inflammation , J Immunol, 190 (2013) 4489-4499.
이에 따라 본 발명은 TSLP와 IL-33에 의한 세포내 신호 전달을 제어하여 천식 또는 아토피 등의 알러지성 질환의 예방 또는 치료에 효능을 나타내는 신규한 퀴놀리논 유도체를 제공하고자 한다.Accordingly, the present invention is to provide a novel quinolinone derivative exhibiting efficacy in the prevention or treatment of allergic diseases such as asthma or atopy by controlling intracellular signal transduction by TSLP and IL-33.
또한, 상기 신규한 퀴놀리논 유도체를 유효성분으로 함유하고, 약제학적으로 허용되는 담체를 포함하는 천식 또는 아토피 등의 알러지성 질환의 예방 또는 치료용 약학 조성물을 제공하고자 한다.In addition, to provide a pharmaceutical composition for the prevention or treatment of allergic diseases, such as asthma or atopy containing the novel quinolinone derivatives as an active ingredient, and a pharmaceutically acceptable carrier.
본 발명은 상기 과제를 해결하기 위하여, 하기 [화학식 1a] 또는 [화학식 1b]로 표시되는 퀴놀리논 유도체를 제공한다.The present invention provides a quinolinone derivative represented by the following [formula 1a] or [formula 1b] to solve the above problems.
[화학식 1a][Formula 1a]
Figure PCTKR2018000165-appb-I000001
Figure PCTKR2018000165-appb-I000001
[화학식 1b][Formula 1b]
Figure PCTKR2018000165-appb-I000002
Figure PCTKR2018000165-appb-I000002
상기 [화학식 1a] 및 [화학식 1b]의 구조 및 구체적인 치환기 및 이의 구체적인 퀴놀리논 유도체에 대해서는 후술한다.The structures of Formulas [1a] and [Formula 1b], specific substituents, and specific quinolinone derivatives thereof will be described later.
또한, 본 발명은 상기 [화학식 1a] 또는 [화학식 1b]로 표시되는 퀴놀리논 유도체를 유효성분으로 함유하고, 약제학적으로 허용되는 담체를 포함하는 천식 또는 아토피 등의 알러지성 질환의 예방 또는 치료용 약학 조성물을 제공한다.In addition, the present invention contains a quinolinone derivative represented by the above [Formula 1a] or [Formula 1b] as an active ingredient, prevention or treatment of allergic diseases such as asthma or atopy, including a pharmaceutically acceptable carrier It provides a pharmaceutical composition.
본 발명에 따른 퀴놀리논 유도체 화합물은 천식 또는 아토피 등의 알러지성 질환의 염증 반응 형성을 효과적으로 억제할 수 있는 것으로서, 이를 포함하는 약학 조성물을 이용하여 다양한 천식 또는 아토피 등의 알러지성 질환을 근본적으로 예방하거나 치료할 수 있다.The quinolinone derivative compound according to the present invention is capable of effectively inhibiting the formation of an inflammatory response of an allergic disease such as asthma or atopic dermatitis, and fundamentally prevents various allergic diseases such as asthma or atopy using a pharmaceutical composition comprising the same. It can be prevented or treated.
도 1은 ELISA를 이용한 본 발명에 따른 [화학식 2] 화합물의 TSLP-TSLPR 상호작용 저해 평가를 나타낸 그래프이다.1 is a graph showing the evaluation of TSLP-TSLPR interaction inhibition of the compound [Formula 2] according to the present invention using an ELISA.
도 2는 ELISA를 이용한 본 발명에 따른 [화학식 7] 화합물의 TSLP-TSLPR 상호작용 저해 평가를 나타낸 그래프이다.Figure 2 is a graph showing the evaluation of TSLP-TSLPR interaction inhibition of the compound [Formula 7] according to the present invention using an ELISA.
도 3은 본 발명에 따른 [화학식 10]의 IL-33 저해에 의한 IL-6 분비 저해 평가를 나타낸 그래프이다.Figure 3 is a graph showing the inhibition of IL-6 secretion by the IL-33 inhibition of the formula [10] according to the present invention.
도 4 및 도 5는 본 발명에 따른 화합물의 효과를 확인하기 위한 HDM (house dust mite, 집먼지진드기) 유발 알러지 기도 염증 마우스 모델 실험결과로서, 본 발명에 따른 [화학식 5] (KB-1517), [화학식 10] (KB-1518) 200 ㎍을 처리한 마우스의 기도 내 총 세포수 및 호산구 (eosinophils) 수를 나타낸 그래프이다.4 and 5 are the results of HDM (house dust mite) induced allergic airway inflammation mouse model experiment to confirm the effect of the compound according to the present invention, [Formula 5] (KB-1517), (KB-1518) (KB-1518) It is a graph showing the total cell number and eosinophils number in the airway of mice treated with 200 μg.
도 6은 본 발명에 따른 화합물에 의한 IL-33 단백질의 Thr 120 잔기의 화학적 이동 변동(chemical shift perturbation)을 나타낸 그래프이다.Figure 6 is a graph showing the chemical shift perturbation of Thr 120 residues of IL-33 protein by the compound according to the present invention.
도 7은 본 발명에 따른 화합물에 의한 IL-33 단백질의 Glu 121 잔기의 화학적 이동 변동(chemical shift perturbation)을 나타낸 그래프이다.Figure 7 is a graph showing the chemical shift perturbation of the Glu 121 residue of the IL-33 protein by the compound according to the present invention.
이하, 본 발명을 더욱 상세하게 설명하기로 한다.Hereinafter, the present invention will be described in more detail.
본 발명의 발명자들은 하기 [화학식 1a]와 [화학식 1b]로 표시되는 퀴놀리논 유도체가 천식 또는 아토피 등의 알러지성 질환을 유도하는 핵심적인 싸이토카인인 TSLP와 IL-33, 이러한 사이토카인과 수용체 사이의 결합을 효과적으로 저해함을 확인하였으며, 이를 기반으로 본 발명을 완성하게 되었다.The inventors of the present invention, the quinolinone derivatives represented by the following [Formula 1a] and [Formula 1b] is a key cytokine TSLP and IL-33, which is a key cytokine to induce allergic diseases such as asthma or atopy, between such cytokines and receptors It was confirmed that effectively inhibits the binding of the present invention was completed based on this.
본 발명의 일 측면은 하기 [화학식 1a] 또는 [화학식 1b]로 표시되는 퀴놀리논 유도체에 관한 것으로서, TSLP와 IL-33에 의한 세포내 신호 전달을 제어하고, 싸이토카인 TSLP(thymic stromal lymphopoietin)와 TSLP 수용체 사이의 결합을 저해하거나 싸이토카인 IL-33과 ST-2 사이의 결합을 저해하는 것을 특징으로 한다.One aspect of the present invention relates to a quinolinone derivative represented by the following [Formula 1a] or [Formula 1b], and controls the intracellular signal transduction by TSLP and IL-33, and the cytokine TSLP (thymic stromal lymphopoietin) It is characterized by inhibiting the binding between the TSLP receptor or the binding between the cytokine IL-33 and ST-2.
[화학식 1a][Formula 1a]
Figure PCTKR2018000165-appb-I000003
Figure PCTKR2018000165-appb-I000003
[화학식 1b][Formula 1b]
Figure PCTKR2018000165-appb-I000004
Figure PCTKR2018000165-appb-I000004
상기 [화학식 1a] 및 [화학식 1b]에서,In [Formula 1a] and [Formula 1b],
R1 및 R2는 서로 동일하거나 상이하고, 각각 독립적으로 탄소수 1 내지 4의 알킬기, 탄소수 1 내지 4의 알킬렌기 및 하기 [구조식 1] 중에서 선택되는 어느 하나이다.R 1 and R 2 is the same as or different from each other, and each independently an alkyl group having 1 to 4 carbon atoms, an alkylene group having 1 to 4 carbon atoms, and any one selected from the following [Formula 1].
[구조식 1][Formula 1]
-Z-알킬기-Z-alkyl group
상기 [구조식 1]에서, Z는 O, S 또는 N으로부터 선택되는 이종원자이거나, 또는 -(CH2)m-이다(상기 m은 0 내지 5 의 정수임).In [Formula 1], Z is a hetero atom selected from O, S or N, or-(CH 2 ) m- (m is an integer of 0 to 5).
X 및 Y는 각각 독립적으로 수소(H), 할로겐기(플루오르(F), 클로린(Cl), 브롬(Br), 요오드(I)), 히드록시기(OH), 아미노기(NH2), 니트로기(NO2), 시아노기(CN), 트리플루오르메틸기(CF3), CO-R', NHR', NR'R', NHCOR' 및 COOR' 중에서 선택되는 어느 하나이다(상기 R'는 탄소수 1 내지 4의 알킬기임).X and Y are each independently hydrogen (H), halogen group (fluor (F), chlorine (Cl), bromine (Br), iodine (I)), hydroxy group (OH), amino group (NH 2 ), nitro group ( NO 2 ), cyano group (CN), trifluoromethyl group (CF 3 ), CO-R ', NHR', NR'R ', NHCOR' and COOR '(any R' is 1 to 4 carbon atoms) Alkyl group).
p는 0 내지 4의 정수이고, q는 0 내지 5의 정수이며, 상기 p 및 q가 복수인 경우 복수의 X 및 복수의 Y는 각각 서로 동일하거나 상이하다.p is an integer of 0-4, q is an integer of 0-5, and when said p and q are multiple, some X and some Y are each the same or different.
상기 [화학식 1a] 또는 [화학식 1b]로 표시되는 퀴놀리논 유도체는 이에 의해서 그 범위가 제한되는 것은 아니나, 구체적으로 하기 [화학식 2] 내지 [화학식 15] 중에서 선택되는 어느 하나일 수 있다.The quinolinone derivative represented by the above [Formula 1a] or [Formula 1b] is not limited by this, but may be specifically any one selected from the following [Formula 2] to [Formula 15].
[화학식 2] [화학식 3][Formula 2] [Formula 3]
Figure PCTKR2018000165-appb-I000005
Figure PCTKR2018000165-appb-I000006
Figure PCTKR2018000165-appb-I000005
Figure PCTKR2018000165-appb-I000006
[화학식 4] [화학식 5][Formula 4] [Formula 5]
Figure PCTKR2018000165-appb-I000007
Figure PCTKR2018000165-appb-I000008
Figure PCTKR2018000165-appb-I000007
Figure PCTKR2018000165-appb-I000008
[화학식 6] [화학식 7][Formula 6] [Formula 7]
Figure PCTKR2018000165-appb-I000009
Figure PCTKR2018000165-appb-I000010
Figure PCTKR2018000165-appb-I000009
Figure PCTKR2018000165-appb-I000010
[화학식 8] [화학식 9][Formula 8] [Formula 9]
Figure PCTKR2018000165-appb-I000011
Figure PCTKR2018000165-appb-I000012
Figure PCTKR2018000165-appb-I000011
Figure PCTKR2018000165-appb-I000012
[화학식 10] [화학식 11][Formula 10] [Formula 11]
Figure PCTKR2018000165-appb-I000013
Figure PCTKR2018000165-appb-I000014
Figure PCTKR2018000165-appb-I000013
Figure PCTKR2018000165-appb-I000014
[화학식 12] [화학식 13][Formula 12] [Formula 13]
Figure PCTKR2018000165-appb-I000015
Figure PCTKR2018000165-appb-I000016
Figure PCTKR2018000165-appb-I000015
Figure PCTKR2018000165-appb-I000016
[화학식 14] [화학식 15][Formula 14] [Formula 15]
Figure PCTKR2018000165-appb-I000017
Figure PCTKR2018000165-appb-I000018
Figure PCTKR2018000165-appb-I000017
Figure PCTKR2018000165-appb-I000018
본 발명에 따른 퀴놀리논 유도체는 Sandmeyer 반응, palladium-촉매하에서 Suzuki-결합과 Heck반응, 니트로기 환원반응, 염기 조건하에서 고리화 반응 및 치환반응을 통하여 제조할 수 있는 바, 예를 들어 출발 물질로 ethyl 2-aminooxazole-4-carboxylate를 출발 물질로 하여 Sandmeyer반응을 통하여 얻어진 ethyl 2-chlorooxazole-4-carboxylate에 치환된 arylboronic acid를 사용하여 다양한 2-aryloxazole-4-carboxylate 유도체를 합성할 수 있다. 또한, Heck 반응시 치환된 2-iodo-1-nitrobenzene을 사용하여 다양한 치환기가 도입된 2-aryl-5-aryloxazole-4-carboxylate 유도체를 제조할 수 있다. 수소화 반응을 통하여 니트로기를 환원한 후 염기 조건에서 아민기와 에스테르기의 분자내 반응을 통하여 퀴놀리논 유도체를 합성할 수 있다. 염기 조건하에서 dialkylethyl bromide와 치환 반응을 통하여 상기 [화학식 1a] 또는 [화학식 1b]로 표시되는 다양한 퀴놀리논 유도체를 제조할 수 있다.The quinolinone derivatives according to the present invention can be prepared through a Sandmeyer reaction, a Suzuki-bond and a Heck reaction under a palladium-catalyst, a nitro group reduction reaction, a cyclization reaction and a substitution reaction under basic conditions, for example starting materials. By using ethyl 2-aminooxazole-4-carboxylate as a starting material, various 2-aryloxazole-4-carboxylate derivatives can be synthesized using arylboronic acid substituted in ethyl 2-chlorooxazole-4-carboxylate obtained through Sandmeyer reaction. In addition, 2-aryl-5-aryloxazole-4-carboxylate derivatives having various substituents introduced therein may be prepared using 2-iodo-1-nitrobenzene substituted in the Heck reaction. After the reduction of the nitro group through a hydrogenation reaction, it is possible to synthesize a quinolinone derivative through the intramolecular reaction of the amine group and the ester group under basic conditions. Various basic quinolinone derivatives represented by the above [Formula 1a] or [Formula 1b] may be prepared by a substitution reaction with dialkylethyl bromide under basic conditions.
보다 구체적으로 하기와 같은 합성과정을 통하여 [화학식 2] 내지 [화학식 13] 화합물을 제조할 수 있으며, 하기 [1]은 본 발명에 따른 [화학식 1a] 또는 [화학식 1b]로 표시되는 퀴놀리논 유도체 중에서 R1과 R2가 메틸기인 화합물의 합성과정을 개략적으로 도시한 흐름도이고, 하기 [2] 및 [3]은 R1과 R2가 에틸기인 화합물의 합성과정을 개략적으로 도시한 흐름도이다.More specifically, the compounds of [Formula 2] to [Formula 13] may be prepared through the synthesis process as follows, and the following [1] is quinolinone represented by [Formula 1a] or [Formula 1b] according to the present invention. In the derivative, a flow chart schematically showing the synthesis process of a compound wherein R 1 and R 2 are methyl groups, and [2] and [3] are flow charts schematically showing the synthesis process of a compound wherein R 1 and R 2 are ethyl groups. .
[1][One]
Figure PCTKR2018000165-appb-I000019
Figure PCTKR2018000165-appb-I000019
[2][2]
Figure PCTKR2018000165-appb-I000020
Figure PCTKR2018000165-appb-I000020
[3][3]
Figure PCTKR2018000165-appb-I000021
Figure PCTKR2018000165-appb-I000021
본 발명의 다른 일 측면은 상기 [화학식 1a] 또는 [화학식 1b]로 표시되는 퀴놀리논 유도체를 유효성분으로 함유하는 천식 또는 아토피 등의 알러지성 질환의 예방 또는 치료용 약학 조성물에 관한 것으로서, TSLP와 IL-33에 의한 세포내 신호 전달을 제어하고, 싸이토카인 TSLP(thymic stromal lymphopoietin)와 TSLP 수용체 사이의 결합을 저해하거나 싸이토카인 IL-33과 ST-2 사이의 결합을 저해하는 것을 특징으로 한다.Another aspect of the present invention relates to a pharmaceutical composition for preventing or treating allergic diseases, such as asthma or atopy, containing a quinolinone derivative represented by the above [Formula 1a] or [Formula 1b] as an active ingredient, TSLP And control intracellular signal transduction by IL-33, inhibit the binding between the cytokine thymic stromal lymphopoietin (TSLP) and the TSLP receptor or inhibit the binding between the cytokine IL-33 and ST-2.
한편, 본 발명에 따른 약학 조성물은 천식 또는 아토피 등의 광범위한 알러지성 질환의 예방 및 치료에 사용 가능한 바, 이에 제한되는 것은 아니지만, 상기 알러지성 질환은 아토피성 피부염, 두드러기 비염, 알러지성 비염 또는 천식 질환일 수 있다.Meanwhile, the pharmaceutical composition according to the present invention can be used for the prevention and treatment of a wide range of allergic diseases such as asthma or atopic dermatitis, but the allergic disease is atopic dermatitis, urticaria rhinitis, allergic rhinitis or asthma May be a disease.
또한, 본 발명에 따른 약학 조성물은 천식 또는 아토피 등의 알러지성 질환의 예방 또는 치료용으로 알려진 다른 약물과 함께 복합 제제의 형태로 투여되거나, 또는 담체, 희석제, 보조제 및 안정화제 등과 같은 기타 성분을 포함할 수도 있다.In addition, the pharmaceutical composition according to the present invention may be administered in the form of a complex preparation together with other drugs known for the prophylaxis or treatment of allergic diseases such as asthma or atopy, or other components such as carriers, diluents, adjuvants and stabilizers, It may also include.
또한, 본 발명에 따른 조성물의 형태는 투여하고자 하는 모드에 따라서 다양하게 선택될 수 있으며, 이에 제한되는 것은 아니지만, 예를 들어 정제, 환약, 분말, 캡슐, 겔, 연고, 유체 또는 현탁액 등의 고상, 반고상 또는 액상의 투약 형태일 수 있고, 정확한 투약량의 단독 투여에 적절한 단위 투약 형태로 투여될 수 있으며, 경구 또는 비경구로 투여할 수 있고, 비경구 투여인 경우에는 정맥내 주입, 피하 주입, 근육 주입 등으로 투여할 수 있다.In addition, the form of the composition according to the present invention may be variously selected depending on the mode to be administered, but is not limited thereto, for example, solid phases such as tablets, pills, powders, capsules, gels, ointments, fluids or suspensions, and the like. It may be in the form of a semi-solid or liquid dosage form, may be administered in a unit dosage form suitable for the precise administration of the dosage alone, orally or parenterally, and in the case of parenteral administration, intravenous, subcutaneous, It can be administered by intramuscular injection.
또한, 상기 조성물은 인간 투여를 위한 약학 조성물을 제형화하는데 일반적으로 사용되는 수성-기제 운반제로 정의되는 약학적으로 허용 가능한 담체, 희석제, 보조제, 안정화제를 원하는 제형에 의존하여 포함할 수 있다.In addition, the compositions may include, depending on the desired formulation, a pharmaceutically acceptable carrier, diluent, adjuvant, stabilizer, defined as an aqueous-based carrier commonly used in formulating pharmaceutical compositions for human administration.
담체(carrier)라 함은 세포 또는 조직 내로 화합물의 부가를 용이하게 하는 물질을 의미하고, 예를 들어, 제제시에 통상적으로 이용되는 것으로서, 탄수화물류 화합물 (예: 락토스, 아밀로스, 덱스트로스, 수크로스, 솔비톨, 만니톨, 전분, 셀룰로스, 등), 아카시아 고무, 인산 칼슘, 알기네이트, 젤라틴, 규산 칼슘, 미세결정성 셀룰로스, 폴리비닐피롤리돈, 셀룰로스, 물, 시럽, 염 용액, 알코올, 아라비아고무, 식물성 기름 (예: 옥수수 기름, 목화 종자유, 두유, 올리브유, 코코넛유), 폴리에틸렌 글리콜, 메틸 셀룰로스, 메틸히드록시 벤조에이트, 프로필히드록시 벤조에이트, 활석, 스테아르산 마그네슘 및 미네랄 오일 등을 포함하나, 이에 한정되는 것은 아니다. 희석제(diluent)라 함은 대상 화합물의 생물학적 활성 형태를 안정화시킬 뿐만 아니라, 화합물을 용해시키는 물에서 희석되는 물질로 정의되는 것으로 예를 들어, 희석제로는 증류수, 생리 식염수, 링거액, 포도당 용액, 및 행크스(Hank's) 용액 등일 수 있다. 안정화제는 단백질, 당질, 완충제 및 그 혼합물로 이루어진 군으로부터 선택될 수 있다. 또한, 상기 성분들 이외에 윤활제, 습윤제, 감미제, 향미제, 유화제, 현탁제, 보존제 등을 추가로 포함하나, 이에 한정되는 것은 아니다.Carrier means a substance that facilitates the addition of the compound into a cell or tissue, and is commonly used in the preparation, for example, carbohydrate compounds (eg lactose, amylose, dextrose, water) Cross, sorbitol, mannitol, starch, cellulose, etc.), acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, salt solution, alcohol, arabian Rubber, vegetable oils (e.g. corn oil, cotton seed oil, soy milk, olive oil, coconut oil), polyethylene glycol, methyl cellulose, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate and mineral oil However, the present invention is not limited thereto. Diluent is defined as a substance that not only stabilizes the biologically active form of a compound of interest, but also is diluted in water to dissolve the compound. Examples of diluents include distilled water, physiological saline, Ringer's solution, glucose solution, and Hank's solution and the like. Stabilizers can be selected from the group consisting of proteins, sugars, buffers and mixtures thereof. In addition to the above components, lubricants, wetting agents, sweetening agents, flavoring agents, emulsifiers, suspending agents, preservatives and the like, but also not limited thereto.
또한, 이러한 담체, 희석제, 보조제 및 안정화제 등과 같은 기타 성분의 유효량은 성분의 용해성, 생물학적 활성 등으로 환산하여 약학적으로 허용 가능한 제형을 획득하는데 유효한 양이다.In addition, an effective amount of other components such as carriers, diluents, adjuvants and stabilizers and the like is an amount effective to obtain a pharmaceutically acceptable formulation in terms of solubility, biological activity, etc. of the component.
본 명세서에서, "예방"은 질환 또는 질병을 보유하고 있다고 진단된 적은 없으나, 이러한 질환 또는 질병의 발생을 억제하는 것을 의미하고, "치료"라 함은 질환 또는 질병의 발전의 억제, 질환 또는 질병의 경감 및 질환 또는 질병의 제거 등을 포함하는 의미이다.In the present specification, "prevention" has not been diagnosed as having a disease or a disease, but means inhibiting the occurrence of such a disease or a disease, "treatment" refers to the inhibition of the development of the disease or a disease, disease or disease It is meant to include the alleviation and elimination of diseases or diseases.
또한, "유효성분으로 포함"은 원하는 생물학적 효과를 실현하는데 필요하거나 또는 충분한 양으로 해당 성분이 포함되는 것을 의미한다. 실제 적용에 있어서 유효 성분으로 포함되는 양의 결정은 대상 질병을 치료하기 위한 양으로서, 다른 독성을 야기하지 않는 사항을 고려해서 결정될 수 있으며, 예를 들어 치료되는 질병 또는 병태, 투여되는 조성물의 형태, 피험체의 크기, 또는 질병 또는 병태의 심각도 등과 같은 다양한 인자에 따라서 변화될 수 있다. 본 발명이 속하는 분야에서 통상의 기술을 지닌 기술자라면 과도한 실험을 동반하지 않고 개별적 조성물의 유효량을 경험적으로 결정할 수 있다.In addition, "contains as an active ingredient" means that the ingredient is included in an amount necessary or sufficient to realize a desired biological effect. In practical applications, the determination of the amount to be included as an active ingredient is an amount for treating a subject disease, and may be determined in consideration of matters that do not cause other toxicity, for example, the disease or condition being treated, the form of the composition to be administered, It may vary depending on various factors such as the size of the subject or the severity of the disease or condition. One of ordinary skill in the art can empirically determine the effective amount of an individual composition without undue experimentation.
또한, "약제학적으로 허용되는"이라 함은 화합물의 생물학적 활성과 물성을 손상시키지 않는 성질을 의미한다.In addition, "pharmaceutically acceptable" means a property that does not impair the biological activity and physical properties of the compound.
기타 본 명세서에서 사용된 용어와 약어들은 달리 정의되지 않는 한 본 발명이 속하는 기술분야의 당업자에게 통상적으로 이해되는 의미로서 해석될 수 있다.Other terms and abbreviations used herein may be interpreted as meanings commonly understood by those skilled in the art to which the present invention belongs unless otherwise defined.
이하, 실시예를 통해서 본 발명을 더욱 구체적으로 설명하기로 하되, 하기 실시예는 본 발명의 이해를 돕기 위한 것일 뿐, 본 발명의 범위를 제한하는 것은 아니다.Hereinafter, the present invention will be described in more detail with reference to Examples, but the following Examples are provided only to assist the understanding of the present invention and do not limit the scope of the present invention.
합성예 : 본 발명에 따른 퀴놀리논 유도체의 합성Synthesis Example Synthesis of Quinolinone Derivatives According to the Present Invention
상기 화합물의 합성과정 흐름도 [1], [2], [3]에 따라 본 발명에 따른 [화학식 2] 내지 [화학식 15]의 퀴놀리논 유도체들을 하기 방법에 의해서 합성하였다.Synthesis of the compound According to the flow chart [1], [2], [3], the quinolinone derivatives of [Formula 2] to [Formula 15] according to the present invention were synthesized by the following method.
(a) 중간체 Ethyl 2-chlorooxazole-4-carboxylate (21)(a) Intermediate Ethyl 2-chlorooxazole-4-carboxylate (21)
에틸 2-아미노옥사졸-4-카르복실레이트 (468 mg, 3 mmol)를 t-부틸나이트라이트 (540 μl, 0.45 mmol), 염화구리 (600 mg, 4.5 mmol) 및 아세토니트릴 (22 ml)내에 60 ℃에서 첨가하고, 상기 혼합물을 80 ℃에서 1시간 동안 가열하였다. 혼합물을 냉각시킨 후에 디클로로메탄, 얼음, 염산에서 분배시켜서 수성층을 디클로로 메탄으로 추가로 추출하고 유기물을 염수로 세척, 건조(MgSO4), 증발시켰다. 생성물을 실리카겔 컬럼크로마토그래피 (hexane/Et2O, 7 : 1 내지 4 : 1, v/v)로 정제하여 솜털 형상의 백색 고체 (338 mg, 64 %)로서 생성물 21을 수득하였다.Ethyl 2-aminooxazole-4-carboxylate (468 mg, 3 mmol) was added to t-butylnitrite (540 μl, 0.45 mmol), copper chloride (600 mg, 4.5 mmol) and acetonitrile (22 ml). Add at 60 ° C. and heat the mixture at 80 ° C. for 1 h. After cooling the mixture was partitioned in dichloromethane, ice, hydrochloric acid, the aqueous layer was further extracted with dichloromethane and the organics were washed with brine, dried (MgSO 4 ) and evaporated. The product was purified by silica gel column chromatography (hexane / Et 2 O, 7: 1 to 4: 1, v / v) to give product 21 as a white fluffy white solid (338 mg, 64%).
Rf = 0.38 (hexane/Et2O = 2:1, v/v). 1H NMR (300 MHz, CDCl3) δ ppm 8.20 (s, 1H), 4.40 (q, J = 7.2 Hz, 2H), 1.39 (t, J = 6.9 Hz, 3H). LRMS (ESI) m/z 176.1 [M + H]+. R f = 0.38 (hexane / Et 2 O = 2: 1, v / v). 1 H NMR (300 MHz, CDCl 3 ) δ ppm 8.20 (s, 1H), 4.40 (q, J = 7.2 Hz, 2H), 1.39 (t, J = 6.9 Hz, 3H). LRMS (ESI) m / z 176.1 [M + H] + .
(b) (b) Suzuki반응을Suzuki Reactions 통한 중간체 ethyl 2- Through intermediate ethyl 2- aryloxazolearyloxazole -4--4- carboxylatecarboxylate 유도체 합성 방법 Derivative synthesis method
(1) 에틸 2-클로로옥사졸-4-카르복실레이트 (257 mg, 1.47 mmol), 4-플루오로페닐보론산 (252 mg, 1.8 mmol, 1.2 eq.) 및 테트라키스(트리페닐포스핀) 팔라듐(0) (85 mg, 0.07 mmol, 0.05 eq.)를 질소 분위기 실온에서 톨루엔 (20 mL) 및 2 M 탄산칼륨용액 (2.0 mL, 4.0 mmol)에 용해시켰다. 반응 혼합물을 교반하면서 1 시간 동안 리플렉싱하고, 실온에서 냉각시킨 후, 반응 혼합물을 에틸아세테이트와 2M 수산화나트륨 용액에 분배시켜, 수성층을 에틸아세테이트로 2회 더 세정하였다. 유기층을 염수로 세척, 건조(MgSO4)시키고, 진공에서 농축시켰다. 생성물을 실리카겔 컬럼크로마토그래피 (hexane/Et2O, 5 : 1 내지 3 : 1, v / v)로 정제하여 솜털 형상의 백색 고체 (250 mg, 73 %)로 생성물 22b를 수득하였다.(1) ethyl 2-chlorooxazole-4-carboxylate (257 mg, 1.47 mmol), 4-fluorophenylboronic acid (252 mg, 1.8 mmol, 1.2 eq.) And tetrakis (triphenylphosphine) Palladium (0) (85 mg, 0.07 mmol, 0.05 eq.) Was dissolved in toluene (20 mL) and 2 M potassium carbonate solution (2.0 mL, 4.0 mmol) at room temperature under nitrogen atmosphere. The reaction mixture was reflected for 1 hour with stirring, cooled to room temperature, the reaction mixture was partitioned between ethyl acetate and 2M sodium hydroxide solution, and the aqueous layer was washed twice more with ethyl acetate. The organic layer was washed with brine, dried (MgSO 4 ) and concentrated in vacuo. The product was purified by silica gel column chromatography (hexane / Et 2 O, 5: 1 to 3: 1, v / v) to give product 22b as a white solid (250 mg, 73%) in the shape of fluffy.
Rf = 0.30 (hexane/Et2O = 2:1, v/v). 1H NMR (300 MHz, CDCl3) δ ppm 8.25 (s, 1H), 8.11 (dd, J = 5.4 and 8.9 Hz, 2H), 7.16 (t, J = 9.0 Hz, 2H), 4.42 (q, J = 7.2 Hz, 2H), 1.40 (t, J = 7.2 Hz, 3H); 13C NMR (75 MHz, CDCl3) δ ppm 166.2, 162.9, 161.7, 161.3, 143.7, 134.7, 129.2, 129.1, 122.8, 122.8, 116.3, 116.1, 116.0, 115.7, 61.4, 14.3. LRMS (ESI) m/z 257.8 [M + Na]+. HRMS (ESI) m/z calculated for C12H10FNO3Na+ [M + Na]+: 258.0537; found: 258.0528.R f = 0.30 (hexane / Et 2 O = 2: 1, v / v). 1 H NMR (300 MHz, CDCl 3 ) δ ppm 8.25 (s, 1H), 8.11 (dd, J = 5.4 and 8.9 Hz, 2H), 7.16 (t, J = 9.0 Hz, 2H), 4.42 (q, J = 7.2 Hz, 2H), 1.40 (t, J = 7.2 Hz, 3H); 13 C NMR (75 MHz, CDCl 3 ) δ ppm 166.2, 162.9, 161.7, 161.3, 143.7, 134.7, 129.2, 129.1, 122.8, 122.8, 116.3, 116.1, 116.0, 115.7, 61.4, 14.3. LRMS (ESI) m / z 257.8 [M + Na] + . HRMS (ESI) m / z calculated for C 12 H 10 FNO 3 Na + [M + Na] + : 258.0537; found: 258.0528.
(2) Ethyl 2-phenyloxazole-4-carboxylate (22a)(2) Ethyl 2-phenyloxazole-4-carboxylate (22a)
Rf = 0.30 (hexane/Et2O = 2:1, v/v). 1H NMR (300 MHz, CDCl3) δ ppm 8.28 (s, 1H), 8.12 (dd, J = 2.1 and 7.2 Hz, 2H), 7.16 (dd, J = 1.8 and 5.3 Hz, 2H), 4.43 (q, J = 7.2 Hz, 2H), 1.41 (t, J = 7.2 Hz, 3H). LRMS (ESI) m/z 218.0 [M + H]+ and 239.9 [M + Na]+. R f = 0.30 (hexane / Et 2 O = 2: 1, v / v). 1 H NMR (300 MHz, CDCl 3 ) δ ppm 8.28 (s, 1H), 8.12 (dd, J = 2.1 and 7.2 Hz, 2H), 7.16 (dd, J = 1.8 and 5.3 Hz, 2H), 4.43 (q , J = 7.2 Hz, 2H), 1.41 (t, J = 7.2 Hz, 3H). LRMS (ESI) m / z 218.0 [M + H] + and 239.9 [M + Na] + .
(3) Ethyl 2-(4-chlorophenyl)oxazole-4-carboxylate (22c)(3) Ethyl 2- (4-chlorophenyl) oxazole-4-carboxylate (22c)
Rf = 0.38 (hexane/Et2O = 2:1, v/v). 1H NMR (300 MHz, CDCl3) δ ppm 8.27 (s, 1H), 8.05 (d, J = 8.4 Hz, 2H), 7.45 (d, J = 7.2 Hz, 2H), 4.43 (q, J = 7.2 Hz, 2H), 1.40 (t, J = 7.2 Hz, 3H). LRMS (ESI) m/z 251.8 [M + H]+ and 273.8 [M + Na]+.R f = 0.38 (hexane / Et 2 O = 2: 1, v / v). 1 H NMR (300 MHz, CDCl 3 ) δ ppm 8.27 (s, 1H), 8.05 (d, J = 8.4 Hz, 2H), 7.45 (d, J = 7.2 Hz, 2H), 4.43 (q, J = 7.2 Hz, 2H), 1.40 (t, J = 7.2 Hz, 3H). LRMS (ESI) m / z 251.8 [M + H] + and 273.8 [M + Na] + .
(4) Ethyl 2-(4-(trifluoromethyl)phenyl)oxazole-4-carboxylate (22d)(4) Ethyl 2- (4- (trifluoromethyl) phenyl) oxazole-4-carboxylate (22d)
Rf = 0.38 (hexane/Et2O = 2:1, v/v). 1H NMR (300 MHz, CDCl3) δ ppm 8.32 (s, 1H), 8.24 (d, J = 8.1 Hz, 2H), 7.74 (d, J = 8.1 Hz, 2H), 4.45 (q, J = 7.2 Hz, 2H), 1.42 (t, J = 7.2 Hz, 3H). LRMS (ESI) m/z 286.0 [M + H]+ and 308.1 [M + Na]+. R f = 0.38 (hexane / Et 2 O = 2: 1, v / v). 1 H NMR (300 MHz, CDCl 3 ) δ ppm 8.32 (s, 1H), 8.24 (d, J = 8.1 Hz, 2H), 7.74 (d, J = 8.1 Hz, 2H), 4.45 (q, J = 7.2 Hz, 2H), 1.42 (t, J = 7.2 Hz, 3H). LRMS (ESI) m / z 286.0 [M + H] + and 308.1 [M + Na] + .
(5) Ethyl 2-(4-methoxyphenyl)oxazole-4-carboxylate (22e)(5) Ethyl 2- (4-methoxyphenyl) oxazole-4-carboxylate (22e)
Rf = 0.13 (hexane/Et2O = 2:1, v/v). 1H NMR (300 MHz, CDCl3) δ ppm 8.24 (s, 1H), 8.06 (d, J = 9.0 Hz, 2H), 6.98 (d, J = 9.0 Hz, 2H), 4.43 (q, J = 7.2 Hz, 2H), 1.42 (t, J = 7.2 Hz, 3H). LRMS (ESI) m/z 247.7 [M + H]+ and 269.9 [M + Na]+.R f = 0.13 (hexane / Et 2 O = 2: 1, v / v). 1 H NMR (300 MHz, CDCl 3 ) δ ppm 8.24 (s, 1H), 8.06 (d, J = 9.0 Hz, 2H), 6.98 (d, J = 9.0 Hz, 2H), 4.43 (q, J = 7.2 Hz, 2H), 1.42 (t, J = 7.2 Hz, 3H). LRMS (ESI) m / z 247.7 [M + H] + and 269.9 [M + Na] + .
(C) (C) Heck반응을Heck reaction 통한 중간체 ethyl 2-aryl-5- Intermediate intermediate ethyl 2-aryl-5- aryloxazolearyloxazole -4--4- carboxylatecarboxylate 유도체 합성 방법 Derivative synthesis method
(1) 22b (192 mg, 0.8 mmol), 2-요오도니트로벤젠 (398 mg, 1.6 mmol, 2.0 eq.), 팔라듐 아세테이트 (11.2 mg, 0.05 mmol, 0.06 eq.), 트리페닐포스핀 (21 mg, 0.08 mmol, 0.1 eq.), 탄산세슘 (651.6 mg, 2.0 mmol, 2.5 eq.) 및 DMF (4 mL)의 혼합물을 질소로 플러시하고, 140 ℃에서 3 시간동안 가열한 후에, 냉각된 혼합물을 에틸아세테이트로 희석하고 물, 염수로 세척하고, 건조(MgSO4)시킨 후에, 진공에서 농축시켰다. 생성물을 실리카겔 컬럼크로마토그래피 (hexane/ Et2O, 5 : 1 내지 1 : 1, v/v)로 정제하여 황색의 니들형상의 결정 (192 mg, 67 %)으로 생성물 23b를 수득하였다.(1) 22b (192 mg, 0.8 mmol), 2- iodonitrobenzene (398 mg, 1.6 mmol, 2.0 eq.), Palladium acetate (11.2 mg, 0.05 mmol, 0.06 eq.), Triphenylphosphine (21 A mixture of mg, 0.08 mmol, 0.1 eq.), cesium carbonate (651.6 mg, 2.0 mmol, 2.5 eq.) and DMF (4 mL) was flushed with nitrogen and heated at 140 ° C. for 3 hours, then the cooled mixture Was diluted with ethyl acetate, washed with water, brine, dried (MgSO 4 ) and concentrated in vacuo. The product was purified by silica gel column chromatography (hexane / Et 2 O, 5: 1 to 1: 1, v / v) to give product 23b as yellow needle crystals (192 mg, 67%).
Rf = 0.35 (hexane/Et2O = 1:1, v/v). 1H NMR (300 MHz, CDCl3) δ ppm 8.20 (d, J = 8.1 Hz, 1H), 8.14 (q, J = 8.4 Hz, 1H), 8.18-8.09 (m, 1H), 7.83-7.65 (m, 3H), 7.19 (t, J = 8.7 Hz, 2H), 4.34 (q, J = 7.2 Hz, 2H), 1.29 (t, J = 7.2 Hz, 3H); 13C NMR (75 MHz, CDCl3) δ ppm 166.3, 161.3, 160.6, 151.0, 148.5, 132.9, 132.6, 131.3, 130.2, 129.3, 129.2, 124.9, 122.6, 122.5, 122.5, 116.3, 116.0, 61.6, 14.0. LRMS (ESI) m/z 357.4 [M + H]+, 379.0 [M + Na]+, and 395.0 [M + K]+. HRMS (ESI) m/z calculated for C18H14FN2O5 + [M + H]+: 357.0881; found: 357.0865.R f = 0.35 (hexane / Et 2 O = 1: 1, v / v). 1 H NMR (300 MHz, CDCl 3 ) δ ppm 8.20 (d, J = 8.1 Hz, 1H), 8.14 (q, J = 8.4 Hz, 1H), 8.18-8.09 (m, 1H), 7.83-7.65 (m , 3H), 7.19 (t, J = 8.7 Hz, 2H), 4.34 (q, J = 7.2 Hz, 2H), 1.29 (t, J = 7.2 Hz, 3H); 13 C NMR (75 MHz, CDCl 3 ) δ ppm 166.3, 161.3, 160.6, 151.0, 148.5, 132.9, 132.6, 131.3, 130.2, 129.3, 129.2, 124.9, 122.6, 122.5, 122.5, 116.3, 116.0, 61.6, 14.0. LRMS (ESI) m / z 357.4 [M + H] + , 379.0 [M + Na] + , and 395.0 [M + K] + . HRMS (ESI) m / z calculated for C 18 H 14 FN 2 O 5 + [M + H] + : 357.0881; found: 357.0865.
(2) Ethyl 5-(2-nitrophenyl)-2-phenyloxazole-4-carboxylate (23a)(2) Ethyl 5- (2-nitrophenyl) -2-phenyloxazole-4-carboxylate (23a)
Rf = 0.23 (hexane/Et2O = 1:1, v/v). 1H NMR (300 MHz, CDCl3) δ ppm 8.19 (d, J = 7.8 Hz, 1H), 8.16-8.08 (m, 1H), 8.12 (d, J = 7.8 Hz, 1H), 7.76-7.66 (m, 1H), 7.77 (d, J = 4.2 Hz, 2H), 7.55-7.43 (m, 3H), 4.32 (q, J = 7.2 Hz, 2H), 1.27 (t, J = 7.2 Hz, 3H). LRMS (ESI) m/z 339.3 [M + H]+, 361.1 [M + Na]+, and 377.0 [M + Na]+. HRMS (ESI) m/z calculated for C18H14N2O5Na+ [M + Na]+: 361.0795; found: 361.0778.R f = 0.23 (hexane / Et 2 O = 1: 1, v / v). 1 H NMR (300 MHz, CDCl 3 ) δ ppm 8.19 (d, J = 7.8 Hz, 1H), 8.16-8.08 (m, 1H), 8.12 (d, J = 7.8 Hz, 1H), 7.76-7.66 (m , 1H), 7.77 (d, J = 4.2 Hz, 2H), 7.55-7.43 (m, 3H), 4.32 (q, J = 7.2 Hz, 2H), 1.27 (t, J = 7.2 Hz, 3H). LRMS (ESI) m / z 339.3 [M + H] + , 361.1 [M + Na] + , and 377.0 [M + Na] + . HRMS (ESI) m / z calculated for C 18 H 14 N 2 0 5 Na + [M + Na] + : 361.0795; found: 361.0778.
(3) Ethyl 2-(4-(3) Ethyl 2- (4- chlorophenylchlorophenyl )-5-(2-) -5- (2- nitrophenylnitrophenyl )) oxazoleoxazole -4--4- carboxylatecarboxylate (23c) (23c)
Rf = 0.30 (hexane/Et2O = 1:1, v/v). 1H NMR (300 MHz, CDCl3) δ ppm 8.18 (d, J = 8.1 Hz, 1H), 8.05 (dd, J = 4.2 and 10.1 Hz, 1H), 8.05 (d, J = 8.7 Hz, 1H), 7.81-7.65 (m, 3H), 7.46 (d, J = 8.7 Hz, 1H), 7.46 (dd, J = 4.2 and 9.3 Hz, 1H), 4.31 (q, J = 7.2 Hz, 2H), 1.26 (t, J = 7.2 Hz, 3H); 13C NMR (75 MHz, CDCl3) δ ppm 161.2, 160.5, 151.1, 148.5, 137.6, 132.8, 132.6, 131.3, 130.3, 129.2, 128.6, 124.9, 124.6, 122.6, 61.6, 29.7, 14.0. LRMS (ESI) m/z 373.1 [M + H]+, 395.0 [M + Na]+, and 411.0 [M + k]+. HRMS (ESI) m/z calculated for C18H14ClN2O5 + [M + H]+: 373.0586; found: 373.0589.R f = 0.30 (hexane / Et 2 O = 1: 1, v / v). 1 H NMR (300 MHz, CDCl 3 ) δ ppm 8.18 (d, J = 8.1 Hz, 1H), 8.05 (dd, J = 4.2 and 10.1 Hz, 1H), 8.05 (d, J = 8.7 Hz, 1H), 7.81-7.65 (m, 3H), 7.46 (d, J = 8.7 Hz, 1H), 7.46 (dd, J = 4.2 and 9.3 Hz, 1H), 4.31 (q, J = 7.2 Hz, 2H), 1.26 (t , J = 7.2 Hz, 3H); 13 C NMR (75 MHz, CDCl 3 ) δ ppm 161.2, 160.5, 151.1, 148.5, 137.6, 132.8, 132.6, 131.3, 130.3, 129.2, 128.6, 124.9, 124.6, 122.6, 61.6, 29.7, 14.0. LRMS (ESI) m / z 373.1 [M + H] + , 395.0 [M + Na] + , and 411.0 [M + k] + . HRMS (ESI) m / z calculated for C 18 H 14 ClN 2 O 5 + [M + H] + : 373.0586; found: 373.0589.
(4) Ethyl 5-(2-(4) Ethyl 5- (2- nitrophenylnitrophenyl )-2-(4-() -2- (4- ( trifluoromethyltrifluoromethyl )phenyl)) phenyl) oxazoleoxazole -4--4- carbcarb oxylate (23d) oxylate (23d)
Rf = 0.20 (hexane/Et2O = 1:1, v/v). 1H NMR (300 MHz, CDCl3) δ ppm 8.24 (d, J = 8.1 Hz, 2H), 8.21 (d, J = 10.5 Hz, 1H), 7.86-7.68 (m, 5H), 4.32 (q, J = 7.2 Hz, 2H), 1.27 (t, J = 7.2 Hz, 3H); 13C NMR (75 MHz, CDCl3) δ ppm 166.1.1, 159.9, 151.7, 148.5, 133.0, 132.6, 127.3, 126.0, 125.9, 124.9, 61.8, 14.0. LRMS (ESI) m/z 407.0 [M + H]+, 428.7 [M + Na]+, and 445.3 [M + K]+. HRMS (ESI) m/z calculated for C18H14F3N2O5 + [M + H]+: 407.0849; found: 407.0809.R f = 0.20 (hexane / Et 2 O = 1: 1, v / v). 1 H NMR (300 MHz, CDCl 3 ) δ ppm 8.24 (d, J = 8.1 Hz, 2H), 8.21 (d, J = 10.5 Hz, 1H), 7.86-7.68 (m, 5H), 4.32 (q, J = 7.2 Hz, 2H), 1.27 (t, J = 7.2 Hz, 3H); 13 C NMR (75 MHz, CDCl 3 ) δ ppm 166.1.1, 159.9, 151.7, 148.5, 133.0, 132.6, 127.3, 126.0, 125.9, 124.9, 61.8, 14.0. LRMS (ESI) m / z 407.0 [M + H] + , 428.7 [M + Na] + , and 445.3 [M + K] + . HRMS (ESI) m / z calculated for C 18 H 14 F 3 N 2 O 5 + [M + H] + : 407.0849; found: 407.0809.
(5) Ethyl 2-(4-(5) Ethyl 2- (4- methoxyphenylmethoxyphenyl )-5-(2-) -5- (2- nitrophenylnitrophenyl )) oxazoleoxazole -4--4- carboxylatecarboxylate (23e) (23e)
Rf = 0.20 (hexane/Et2O = 1:1, v/v). 1H NMR (300 MHz, CDCl3) δ ppm 8.18 (d, J = 8.7 Hz, 1H), 8.07 (d, J = 8.7 Hz, 2H), 7.82-7.64 (m, 3H), 7.00 (t, J = 9.0 Hz, 2H), 4.33 (q, J = 7.2 Hz, 2H), 1.28 (t, J = 7.2 Hz, 3H); 13C NMR (75 MHz, CDCl3) δ ppm 162.1, 161.5, 161.5, 150.4, 148.5, 132.8, 132.6, 131.1, 130.0, 128.8, 124.8, 122.8, 118.8, 114.3, 61.5, 55.5, 29.7, 14.1. LRMS (ESI) m/z 369.1 [M + H]+, 391.1 [M + Na]+, and 407.1 [M + K]+. HRMS (ESI) m/z calculated for C19H17N2O6 + [M + H]+: 369.1081; found: 369.1090.R f = 0.20 (hexane / Et 2 O = 1: 1, v / v). 1 H NMR (300 MHz, CDCl 3 ) δ ppm 8.18 (d, J = 8.7 Hz, 1H), 8.07 (d, J = 8.7 Hz, 2H), 7.82-7.64 (m, 3H), 7.00 (t, J = 9.0 Hz, 2H), 4.33 (q, J = 7.2 Hz, 2H), 1.28 (t, J = 7.2 Hz, 3H); 13 C NMR (75 MHz, CDCl 3 ) δ ppm 162.1, 161.5, 161.5, 150.4, 148.5, 132.8, 132.6, 131.1, 130.0, 128.8, 124.8, 122.8, 118.8, 114.3, 61.5, 55.5, 29.7, 14.1. LRMS (ESI) m / z 369.1 [M + H] + , 391.1 [M + Na] + , and 407.1 [M + K] + . HRMS (ESI) m / z calculated for C 19 H 17 N 2 O 6 + [M + H] + : 369.1081; found: 369.1090.
(d) 수소화 반응을 통한 중간체 아민 유도체 합성 방법(d) Synthesis of Intermediate Amine Derivatives by Hydrogenation
(1) Ethyl 5-(2-(1) Ethyl 5- (2- aminophenylaminophenyl )-2-(4-) -2- (4- fluorophenylfluorophenyl )) oxazoleoxazole -4--4- carboxylatecarboxylate (24b) (24b)
화합물 23b (192 mg, 0.54 mmol), MeOH (15 mL)의 용액에 팔라듐 촉매를 넣고, 수소 (50 psi) 분위기에서 1 시간 동안 교반하였다. 반응 혼합물을 셀라이트베드를 통해 여과한 후에 휘발성 물질을 환원하여 제거하고 4a (145 mg, 82 %)를 황색 고체로서 수득하였다.A palladium catalyst was added to a solution of compound 23b (192 mg, 0.54 mmol) and MeOH (15 mL), and stirred for 1 hour in a hydrogen (50 psi) atmosphere. After the reaction mixture was filtered through a celite bed, the volatiles were reduced to remove and 4a (145 mg, 82%) was obtained as a yellow solid.
Rf = 0.30 (hexane/Et2O = 1:1, v/v). 1H NMR (300 MHz, CDCl3) δ ppm 8.18-8.07 (m, 1H), 8.12 (q, J = 8.4 Hz, 1H), 7.43 (dd, J = 1.2 and 7.8 Hz, 1H), 7.29 (td, J = 8.1 and 1.5 Hz, 1H), 7.22-7.08 (m, 1H), 7.16 (t, J = 8.4 Hz, 1H), 6.84 (td, J = 7.8 and 1.2 Hz, 1H), 6.82 (d, J = 8.1 Hz, 1H), 4.39 (q, J = 7.2 Hz, 2H), 4.16 (bs, 2H), 1.34 (t, J = 7.2 Hz, 3H); 13C NMR (75 MHz, CDCl3) δ ppm 166.2, 162.8, 162.1, 159.8, 154.4, 145.7, 131.8, 131.6, 129.8, 129.1, 129.0, 122.8, 122.8, 118.2, 116.7, 116.3, 116.0, 112.7, 61.5, 14.2. LRMS (ESI) m/z 327.1 [M + H]+ and 349.1 [M + Na]+. HRMS (ESI) m/z calculated for C18H16FN2O3 + [M + H]+: 327.1139; found: 327.1133.R f = 0.30 (hexane / Et 2 O = 1: 1, v / v). 1 H NMR (300 MHz, CDCl 3 ) δ ppm 8.18-8.07 (m, 1H), 8.12 (q, J = 8.4 Hz, 1H), 7.43 (dd, J = 1.2 and 7.8 Hz, 1H), 7.29 (td , J = 8.1 and 1.5 Hz, 1H), 7.22-7.08 (m, 1H), 7.16 (t, J = 8.4 Hz, 1H), 6.84 (td, J = 7.8 and 1.2 Hz, 1H), 6.82 (d, J = 8.1 Hz, 1H), 4.39 (q, J = 7.2 Hz, 2H), 4.16 (bs, 2H), 1.34 (t, J = 7.2 Hz, 3H); 13 C NMR (75 MHz, CDCl 3 ) δ ppm 166.2, 162.8, 162.1, 159.8, 154.4, 145.7, 131.8, 131.6, 129.8, 129.1, 129.0, 122.8, 122.8, 118.2, 116.7, 116.3, 116.0, 112.7, 61.5, 14.2. LRMS (ESI) m / z 327.1 [M + H] + and 349.1 [M + Na] + . HRMS (ESI) m / z calculated for C 18 H 16 FN 2 O 3 + [M + H] + : 327.1139; found: 327.1133.
(2) Ethyl 5-(2-aminophenyl)-2-phenyloxazole-4-carboxylate (24a)(2) Ethyl 5- (2-aminophenyl) -2-phenyloxazole-4-carboxylate (24a)
Rf = 0.40 (hexane/Et2O = 1:2, v/v). 1H NMR (300 MHz, CDCl3) δ ppm 8.22-8.11 (m, 1H), 8.15 (d, J = 7.8 Hz, 1H), 7.54-7.41 (m, 4H), 7.31 (td, J = 8.7 and 1.5 Hz, 1H), 6.87 (t, J = 7.5 Hz, 1H), 6.84 (d, J = 8.1 Hz, 1H), 4.42 (q, J = 7.2 Hz, 2H), 4.25 (bs, 2H), 1.38 (t, J = 7.2 Hz, 3H). LRMS (ESI) m/z 309.3 [M + H]+, 331.2 [M + Na]+, and 347.0 [M + K]+. HRMS (ESI) m/z calculated for C18H16N2O3Na+ [M + Na]+: 331.1053; found: 331.1051.R f = 0.40 (hexane / Et 2 O = 1: 2, v / v). 1 H NMR (300 MHz, CDCl 3 ) δ ppm 8.22-8.11 (m, 1H), 8.15 (d, J = 7.8 Hz, 1H), 7.54-7.41 (m, 4H), 7.31 (td, J = 8.7 and 1.5 Hz, 1H), 6.87 (t, J = 7.5 Hz, 1H), 6.84 (d, J = 8.1 Hz, 1H), 4.42 (q, J = 7.2 Hz, 2H), 4.25 (bs, 2H), 1.38 (t, J = 7.2 Hz, 3H). LRMS (ESI) m / z 309.3 [M + H] + , 331.2 [M + Na] + , and 347.0 [M + K] + . HRMS (ESI) m / z calculated for C 18 H 16 N 2 O 3 Na + [M + Na] + : 331.1053; found: 331.1051.
(3) Ethyl 5-(2-(3) Ethyl 5- (2- aminophenylaminophenyl )-2-(4-) -2- (4- chlorophenylchlorophenyl )) oxazoleoxazole -4--4- carboxylatecarboxylate (24c) (24c)
Rf = 0.23 (hexane/Et2O = 1:1, v/v). 1H NMR (300 MHz, CDCl3) δ ppm 8.20 (d, J = 8.1 Hz, 1H), 8.14 (dd, J = 5.4 and 8.9 Hz, 2H), 7.83-7.65 (m, 3H), 7.19 (t, J = 8.7 Hz, 2H), 4.44 (q, J = 7.2 Hz, 2H), 1.29 (t, J = 7.2 Hz, 3H); 13C NMR (75 MHz, CDCl3) δ ppm 166.3, 161.3, 160.6, 151.0, 148.5, 132.9, 132.6, 131.3, 130.2, 129.3, 129.2, 124.9, 122.6, 122.5, 122.5, 116.3, 116.0, 61.6, 14.0. LRMS (ESI) m/z 343.1 [M + H]+, 365.1 [M + Na]+. HRMS (ESI) m/z calculated for C18H115ClN2O3Na+ [M + Na]+: 365.0663; found: 365.0678R f = 0.23 (hexane / Et 2 O = 1: 1, v / v). 1 H NMR (300 MHz, CDCl 3 ) δ ppm 8.20 (d, J = 8.1 Hz, 1H), 8.14 (dd, J = 5.4 and 8.9 Hz, 2H), 7.83-7.65 (m, 3H), 7.19 (t , J = 8.7 Hz, 2H), 4.44 (q, J = 7.2 Hz, 2H), 1.29 (t, J = 7.2 Hz, 3H); 13 C NMR (75 MHz, CDCl 3 ) δ ppm 166.3, 161.3, 160.6, 151.0, 148.5, 132.9, 132.6, 131.3, 130.2, 129.3, 129.2, 124.9, 122.6, 122.5, 122.5, 116.3, 116.0, 61.6, 14.0. LRMS (ESI) m / z 343.1 [M + H] + , 365.1 [M + Na] + . HRMS (ESI) m / z calculated for C 18 H 1 15 ClN 2 O 3 Na + [M + Na] + : 365.0663; found: 365.0678
(4) Ethyl 5-(2-(4) Ethyl 5- (2- aminophenylaminophenyl )-2-(4-() -2- (4- ( trifluoromethyltrifluoromethyl )phenyl)) phenyl) oxazoleoxazole -4-car boxylate (24d)-4-car boxylate (24d)
Rf = 0.29 (CH2Cl2-MeOH = 20:1, v/v). 1H NMR (300 MHz, CDCl3) δ ppm 8.25 (d, J = 8.1 Hz, 2H), 7.74 (d, J = 8.1 Hz, 2H), 7.44 (t, J = 7.8 Hz, 1H), 7.31 (t, J = 7.8 Hz, 1H), 6.86 (t, J = 7.5 Hz, 1H), 6.83 (d, J = 7.8 Hz, 1H), 4.40 (q, J = 7.2 Hz, 2H), 4.17 (s, 2H), 1.29 (t, J = 7.2 Hz, 3H); 13C NMR (75 MHz, CDCl3) δ ppm 162.0, 159.2, 155.1, 132.0, 131.6, 127.1, 126.0, 125.9, 118.2, 116.9, 112.4, 61.7, 14.2. LRMS (ESI) m/z 356.8 [M + H]+ and 398.9 [M + Na]+. HRMS (ESI) m/z calculated for C18H16F3N2O3 + [M + Na]+: 377.1108; found: 377.1094.R f = 0.29 (CH 2 Cl 2 -MeOH = 20: 1, v / v). 1 H NMR (300 MHz, CDCl 3 ) δ ppm 8.25 (d, J = 8.1 Hz, 2H), 7.74 (d, J = 8.1 Hz, 2H), 7.44 (t, J = 7.8 Hz, 1H), 7.31 ( t, J = 7.8 Hz, 1H), 6.86 (t, J = 7.5 Hz, 1H), 6.83 (d, J = 7.8 Hz, 1H), 4.40 (q, J = 7.2 Hz, 2H), 4.17 (s, 2H), 1.29 (t, J = 7.2 Hz, 3H); 13 C NMR (75 MHz, CDCl 3 ) δ ppm 162.0, 159.2, 155.1, 132.0, 131.6, 127.1, 126.0, 125.9, 118.2, 116.9, 112.4, 61.7, 14.2. LRMS (ESI) m / z 356.8 [M + H] + and 398.9 [M + Na] + . HRMS (ESI) m / z calculated for C 18 H 16 F 3 N 2 O 3 + [M + Na] + : 377.1108; found: 377.1094.
(5) Ethyl 5-(2-(5) Ethyl 5- (2- aminophenylaminophenyl )-2-(4-) -2- (4- methoxyphenylmethoxyphenyl )) oxazoleoxazole -4--4- carboxylatcarboxylat e (24e) e (24e)
Rf = 0.28 (CH2Cl2-MeOH = 20:1, v/v). 1H NMR (300 MHz, CDCl3) δ ppm 8.07 (d, J = 8.7 Hz, 2H), 7.44 (d, J = 7.5 Hz, 1H), 7.28 (t, J = 8.1 Hz, 2H), 6.98 (d, J = 8.7 Hz, 1H), 6.85 (t, J = 7.5 Hz, 1H), 6.81 (d, J = 7.8 Hz, 1H), 4.39 (q, J = 7.2 Hz, 2H), 4.18 (bs, 2H), 1.34 (t, J = 7.2 Hz, 3H); 13C NMR (75 MHz, CDCl3) δ ppm 162.8, 162.3, 157.8, 151.9, 138.4, 130.2, 129.3, 122.7, 121.6, 119.1, 115.4, 114.4, 111.6, 61.4, 55.4, 14.2. LRMS (ESI) m/z 339.1 [M + H]+ and 361.1 [M + Na]+. HRMS (ESI) m/z calculated for C19H19N2O4 + [M + H]+: 339.1339; found: 339.1452.R f = 0.28 (CH 2 Cl 2 -MeOH = 20: 1, v / v). 1 H NMR (300 MHz, CDCl 3 ) δ ppm 8.07 (d, J = 8.7 Hz, 2H), 7.44 (d, J = 7.5 Hz, 1H), 7.28 (t, J = 8.1 Hz, 2H), 6.98 ( d, J = 8.7 Hz, 1H), 6.85 (t, J = 7.5 Hz, 1H), 6.81 (d, J = 7.8 Hz, 1H), 4.39 (q, J = 7.2 Hz, 2H), 4.18 (bs, 2H), 1.34 (t, J = 7.2 Hz, 3H); 13 C NMR (75 MHz, CDCl 3 ) δ ppm 162.8, 162.3, 157.8, 151.9, 138.4, 130.2, 129.3, 122.7, 121.6, 119.1, 115.4, 114.4, 111.6, 61.4, 55.4, 14.2. LRMS (ESI) m / z 339.1 [M + H] + and 361.1 [M + Na] + . HRMS (ESI) m / z calculated for C 19 H 19 N 2 O 4 + [M + H] + : 339.1339; found: 339.1452.
(e) 고리화 반응을 통한 퀴놀리논 유도체 합성 방법(e) Method of synthesizing quinolinone derivatives through cyclization reaction
(1) 2-(4-fluorophenyl)oxazolo[4,5-c]quinolin-4(5H)-one (25b)(1) 2- (4-fluorophenyl) oxazolo [4,5-c] quinolin-4 (5H) -one (25b)
24b (95 mg, 0.27 mmol), DME (7 mL) 및 2 M 탄산칼륨 용액 (0.5 mL, 1.0 mmol) 혼합물을 12 시간동안 가열 환류시키 후에, 고체를 여과로 수집하고, 냉각된 EtOH로 세척한 후에 진공하에서 건조시켜 5a (45 mg, 60 %)를 수득하였다.After 24 b (95 mg, 0.27 mmol), DME (7 mL) and 2 M potassium carbonate solution (0.5 mL, 1.0 mmol) were heated to reflux for 12 hours, the solids were collected by filtration and washed with cold EtOH. Drying in vacuo then gave 5a (45 mg, 60%).
Rf = 0.18 (CH2Cl2-MeOH = 1:1, v/v). 1H NMR (300 MHz, DMSO-d6) δ ppm 11.19 (bs, 1H), 7.40 (d, J = 9.0 Hz, 1H), 7.38 (d, J = 9.0 Hz, 1H), 7.14 (d, J = 7.8 Hz, 1H), 6.71 (t, J = 7.2 Hz, 1H), 6.35 (d, J = 7.2 Hz, 1H), 6.59 (d, J = 8.7 Hz, 1H), 6.47 (t, J = 7.8 Hz, 1H); 13C NMR (75 MHz, CDCl3) δ ppm 166.3, 161.3, 160.6, 151.0, 148.5, 132.9, 132.6, 131.3, 130.2, 129.3, 129.2, 124.9, 122.6, 122.5, 122.5, 116.3, 116.0, 61.6, 14.0. LRMS (ESI) m/z 302.7 [M + Na]+ and 318.8 [M + K]+. HRMS (ESI) m/z calculated for C16H10FN2O2 + [M + H]+: 281.0721; found: 281.0713.R f = 0.18 (CH 2 Cl 2 -MeOH = 1: 1, v / v). 1 H NMR (300 MHz, DMSO-d 6 ) δ ppm 11.19 (bs, 1H), 7.40 (d, J = 9.0 Hz, 1H), 7.38 (d, J = 9.0 Hz, 1H), 7.14 (d, J = 7.8 Hz, 1H), 6.71 (t, J = 7.2 Hz, 1H), 6.35 (d, J = 7.2 Hz, 1H), 6.59 (d, J = 8.7 Hz, 1H), 6.47 (t, J = 7.8 Hz, 1H); 13 C NMR (75 MHz, CDCl 3 ) δ ppm 166.3, 161.3, 160.6, 151.0, 148.5, 132.9, 132.6, 131.3, 130.2, 129.3, 129.2, 124.9, 122.6, 122.5, 122.5, 116.3, 116.0, 61.6, 14.0. LRMS (ESI) m / z 302.7 [M + Na] + and 318.8 [M + K] + . HRMS (ESI) m / z calculated for C 16 H 10 FN 2 O 2 + [M + H] + : 281.0721; found: 281.0713.
(2) 2-Phenyloxazolo[4,5-c]quinolin-4(5H)-one (25a)(2) 2-Phenyloxazolo [4,5-c] quinolin-4 (5H) -one (25a)
Rf = 0.30 (CH2Cl2-MeOH = 20:1, v/v). 1H NMR (300 MHz, DMSO-d6) δ ppm 12.08 (bs, 1H), 8.29-8.18 (m, 2H), 8.05 (d, J = 7.8 Hz, 1H), 7.72-7.56 (m, 4H), 7.52 (d, J = 8.1 Hz, 1H), 7.37 (t, J = 7.8 Hz, 1H); 13C NMR (75 MHz, CDCl3) δ ppm 166.3, 161.3, 160.6, 151.0, 148.5, 132.9, 132.6, 131.3, 130.2, 129.3, 129.2, 124.9, 122.6, 122.5, 122.5, 116.3, 116.0, 61.6, 14.0. LRMS (ESI) m/z 262.8 [M + H]+, 285.2 [M + Na]+, and 300.6 [M + K]+. HRMS (ESI) m/z calculated for C16H11N2O2 + [M + H]+: 263.0815; found: 263.0845. R f = 0.30 (CH 2 Cl 2 -MeOH = 20: 1, v / v). 1 H NMR (300 MHz, DMSO-d 6 ) δ ppm 12.08 (bs, 1H), 8.29-8.18 (m, 2H), 8.05 (d, J = 7.8 Hz, 1H), 7.72-7.56 (m, 4H) , 7.52 (d, J = 8.1 Hz, 1H), 7.37 (t, J = 7.8 Hz, 1H); 13 C NMR (75 MHz, CDCl 3 ) δ ppm 166.3, 161.3, 160.6, 151.0, 148.5, 132.9, 132.6, 131.3, 130.2, 129.3, 129.2, 124.9, 122.6, 122.5, 122.5, 116.3, 116.0, 61.6, 14.0. LRMS (ESI) m / z 262.8 [M + H] + , 285.2 [M + Na] + , and 300.6 [M + K] + . HRMS (ESI) m / z calculated for C 16 H 11 N 2 O 2 + [M + H] + : 263.0815; found: 263.0845.
(3) 2-(4-Chlorophenyl)oxazolo[4,5-c]quinolin-4(5H)-one (25c)(3) 2- (4-Chlorophenyl) oxazolo [4,5-c] quinolin-4 (5H) -one (25c)
Rf = 0.32 (CH2Cl2-MeOH = 20:1, v/v). 1H NMR (300 MHz, DMSO-d6) δ ppm 8.24 (d, J = 8.4 Hz, 2H), 8.01 (d, J = 7.5 Hz, 1H), 7.02 (t, J = 8.7 Hz, 2H), 7.56 (d, J = 7.5 Hz, 1H), 7.51 (t, J = 8.4 Hz, 1H), 7.31 (t, J = 7.5 Hz, 1H); 13C NMR (75 MHz, CDCl3) δ ppm 166.3, 161.3, 160.6, 151.0, 148.5, 132.9, 132.6, 131.3, 130.2, 129.3, 129.2, 124.9, 122.6, 122.5, 122.5, 116.3, 116.0, 61.6, 14.0. LRMS (ESI) m/z 335.2 [M + K]+. HRMS (ESI) m/z calculated for C16H9ClN2O2Na+ [M + Na]+: 319.0245; found: 319.0276.R f = 0.32 (CH 2 Cl 2 -MeOH = 20: 1, v / v). 1 H NMR (300 MHz, DMSO-d 6 ) δ ppm 8.24 (d, J = 8.4 Hz, 2H), 8.01 (d, J = 7.5 Hz, 1H), 7.02 (t, J = 8.7 Hz, 2H), 7.56 (d, J = 7.5 Hz, 1H), 7.51 (t, J = 8.4 Hz, 1H), 7.31 (t, J = 7.5 Hz, 1H); 13 C NMR (75 MHz, CDCl 3 ) δ ppm 166.3, 161.3, 160.6, 151.0, 148.5, 132.9, 132.6, 131.3, 130.2, 129.3, 129.2, 124.9, 122.6, 122.5, 122.5, 116.3, 116.0, 61.6, 14.0. LRMS (ESI) m / z 335.2 [M + K] + . HRMS (ESI) m / z calculated for C 16 H 9 ClN 2 O 2 Na + [M + Na] + : 319.0245; found: 319.0276.
(4) 2-(4-(Trifluoromethyl)phenyl)oxazolo[4,5-c]quinolin-4(5H)-one (25d)(4) 2- (4- (Trifluoromethyl) phenyl) oxazolo [4,5-c] quinolin-4 (5H) -one (25d)
Rf = 0.38 (CH2Cl2-MeOH = 20:1, v/v). 1H NMR (300 MHz, DMSO-d6) δ ppm 8.44 (d, J = 8.1 Hz, 2H), 8.08 (d, J = 7.8 Hz, 1H), 8.03 (d, J = 8.4 Hz, 2H), 7.63 (t, J = 7.2 Hz, 1H), 7.53 (d, J = 8.1 Hz, 1H), 7.38 (t, J = 7.5 Hz, 1H); 13C NMR (75 MHz, CDCl3) δ ppm 166.3, 161.3, 160.6, 151.0, 148.5, 132.9, 132.6, 131.3, 130.2, 129.3, 129.2, 124.9, 122.6, 122.5, 122.5, 116.3, 116.0, 61.6, 14.0. LRMS (ESI) m/z 353.5 [M + Na]+ and 369.0 [M + K]+. HRMS (ESI) m/z calculated for C17H10F3N2O2 + [M + H]+: 331.0689; found: 331.0682. R f = 0.38 (CH 2 Cl 2 -MeOH = 20: 1, v / v). 1 H NMR (300 MHz, DMSO-d 6 ) δ ppm 8.44 (d, J = 8.1 Hz, 2H), 8.08 (d, J = 7.8 Hz, 1H), 8.03 (d, J = 8.4 Hz, 2H), 7.63 (t, J = 7.2 Hz, 1H), 7.53 (d, J = 8.1 Hz, 1H), 7.38 (t, J = 7.5 Hz, 1H); 13 C NMR (75 MHz, CDCl 3 ) δ ppm 166.3, 161.3, 160.6, 151.0, 148.5, 132.9, 132.6, 131.3, 130.2, 129.3, 129.2, 124.9, 122.6, 122.5, 122.5, 116.3, 116.0, 61.6, 14.0. LRMS (ESI) m / z 353.5 [M + Na] + and 369.0 [M + K] + . HRMS (ESI) m / z calculated for C 17 H 10 F 3 N 2 O 2 + [M + H] + : 331.0689; found: 331.0682.
(5) 2-(4-Methoxyphenyl)oxazolo[4,5-c]quinolin-4(5H)-one (25e)(5) 2- (4-Methoxyphenyl) oxazolo [4,5-c] quinolin-4 (5H) -one (25e)
Rf = 0.28 (CH2Cl2-MeOH = 20:1, v/v). 1H NMR (300 MHz, DMSO-d6) δ ppm 11.18 (bs, 1H), 7.38-7.31 (m, 1H), 7.33 (d, J = 9.0 Hz, 1H), 7.19 (dd, J = 1.2 and 8.1 Hz, 1H), 6.76 (td, J = 8.4 and 1.5 Hz, 1H), 6.68 (d, J = 9.0 Hz, 1H), 6.53 (td, J = 8.1 and 1.2 Hz, 1H), 6.41-6.33 (m, 1H), 6.35 (d, J = 9.0 Hz, 1H); 13C NMR (75 MHz, CDCl3) δ ppm 166.3, 161.3, 160.6, 151.0, 148.5, 132.9, 132.6, 131.3, 130.2, 129.3, 129.2, 124.9, 122.6, 122.5, 122.5, 116.3, 116.0, 61.6, 14.0. LRMS (ESI) m/z 293.1 [M + H]+, 314.9 [M + Na]+, and 331.0 [M + K]+. HRMS (ESI) m/z calculated for C17H13N2O3 + [M + H]+: 293.0921; found: 293.0919.R f = 0.28 (CH 2 Cl 2 -MeOH = 20: 1, v / v). 1 H NMR (300 MHz, DMSO-d 6 ) δ ppm 11.18 (bs, 1H), 7.38-7.31 (m, 1H), 7.33 (d, J = 9.0 Hz, 1H), 7.19 (dd, J = 1.2 and 8.1 Hz, 1H), 6.76 (td, J = 8.4 and 1.5 Hz, 1H), 6.68 (d, J = 9.0 Hz, 1H), 6.53 (td, J = 8.1 and 1.2 Hz, 1H), 6.41-6.33 ( m, 1H), 6.35 (d, J = 9.0 Hz, 1H); 13 C NMR (75 MHz, CDCl 3 ) δ ppm 166.3, 161.3, 160.6, 151.0, 148.5, 132.9, 132.6, 131.3, 130.2, 129.3, 129.2, 124.9, 122.6, 122.5, 122.5, 116.3, 116.0, 61.6, 14.0. LRMS (ESI) m / z 293.1 [M + H] + , 314.9 [M + Na] + , and 331.0 [M + K] + . HRMS (ESI) m / z calculated for C 17 H 13 N 2 O 3 + [M + H] + : 293.0921; found: 293.0919.
(f) 알킬화 반응을 통한 퀴놀리논 유도체 최종 화합물 합성 방법(f) Method for synthesizing quinolinone derivative final compound through alkylation reaction
(1) 5-(2-((1) 5- (2- ( dimethylaminodimethylamino )ethyl)-2-) ethyl) -2- (4-fluorophenyl)oxazolo(4-fluorophenyl) oxazolo [4,5-c][4,5-c] quinolinquinolin -4(5H)-one (3) -4 (5H) -one (3)
화합물 25b (43 mg, 0.153 mmol), 2-브로모-N, N-다이메틸에탄아민하이드로브로마이드 (80 mg, 0.31 mmol, 1.2 eq.), 칼륨카보네이트 (63 mg, 0.46 mmol, 3.0 eq.)에 DMF (5 mL)를 넣고, 130 ℃에서 3 시간 동안 교반하고 실온으로 냉각시키고, 이후 용매를 진공하에서 제거하하고, Et2O-MeOH (10 : 1 내지 3 : 1, v/v)를 용리액으로 사용하는 실리카겔 컬럼크로마토그래피로 정제하여 [화학식 3]을 수득하였다.Compound 25b (43 mg, 0.153 mmol), 2-bromo-N, N-dimethylethanaminehydrobromide (80 mg, 0.31 mmol, 1.2 eq.), Potassium carbonate (63 mg, 0.46 mmol, 3.0 eq.) DMF (5 mL) was added, stirred at 130 ° C. for 3 hours, cooled to room temperature, then the solvent was removed in vacuo, and Et 2 O-MeOH (10: 1 to 3: 1, v / v) was added to the eluent. Purification by silica gel column chromatography was used to obtain [Formula 3].
Yield: 39%. Rf = 0.15 (EtO2-MeOH = 5:1, v/v). 1H NMR (300 MHz, CDCl3) δ ppm 8.33-8.22 (m, 1H), 8.29 (dd, J = 5.4 and 8.7 Hz, 1H), 8.03 (dd, J = 1.5 and 8.0 Hz, 1H), 7.64 (td, J = 7.2 and 1.2 Hz, 1H), 7.57 (d, J = 8.4 Hz, 1H), 7.38 (t, J = 6.9 Hz, 1H), 7.22 (t, J = 8.4 Hz, 1H), 7.27-7.16 (m, 1H), 4.58 (t, J = 7.8 Hz, 2H), 2.66 (t, J = 7.8 Hz, 2H), 2.41 (s, 6H); 13C NMR (75 MHz, CDCl3) δ ppm 166.5, 163.1, 161.7, 157.3, 152.3, 130.5, 129.8, 129.7, 129.6, 122.8, 122.8, 121.9, 116.4, 116.1, 115.4, 111.6, 56.3, 45.9, 40.8, 29.7, 22.7. LRMS (ESI) m/z 352.2 [M + H]+ and 374.0 [M + Na]+. HRMS (ESI) m/z calculated for C18H19FN3O2 + [M + H]+: 352.1456; found: 352.1496.Yield: 39%. R f = 0.15 (EtO 2 -MeOH = 5: 1, v / v). 1 H NMR (300 MHz, CDCl 3 ) δ ppm 8.33-8.22 (m, 1H), 8.29 (dd, J = 5.4 and 8.7 Hz, 1H), 8.03 (dd, J = 1.5 and 8.0 Hz, 1H), 7.64 (td, J = 7.2 and 1.2 Hz, 1H), 7.57 (d, J = 8.4 Hz, 1H), 7.38 (t, J = 6.9 Hz, 1H), 7.22 (t, J = 8.4 Hz, 1H), 7.27 -7.16 (m, 1H), 4.58 (t, J = 7.8 Hz, 2H), 2.66 (t, J = 7.8 Hz, 2H), 2.41 (s, 6H); 13 C NMR (75 MHz, CDCl 3 ) δ ppm 166.5, 163.1, 161.7, 157.3, 152.3, 130.5, 129.8, 129.7, 129.6, 122.8, 122.8, 121.9, 116.4, 116.1, 115.4, 111.6, 56.3, 45.9, 40.8, 29.7, 22.7. LRMS (ESI) m / z 352.2 [M + H] + and 374.0 [M + Na] + . HRMS (ESI) m / z calculated for C 18 H 19 FN 3 O 2 + [M + H] + : 352.1456; found: 352.1496.
(2) 5-(2-((2) 5- (2- ( dimethylaminodimethylamino )ethyl)-2-) ethyl) -2- phenyloxazolo[4,5-c]quinolinphenyloxazolo [4,5-c] quinolin -4(5H)-one (2)-4 (5H) -one (2)
Yield: 39%. Rf = 0.25 (Et2O-MeOH = 5:1, v/v). 1H NMR (300 MHz, CDCl3) δ ppm 8.34-8.27 (m, 2H), 8.07 (dd, J = 1.5 and 7.8 Hz, 1H), 7.69-7.51 (m, 5H), 7.38 (td, J = 7.2 and 1.2 Hz, 1H), 4.59 (t, J = 7.5 Hz, 2H), 2.68 (t, J = 7.5 Hz, 2H), 2.41 (s, 6H); 13C NMR (75 MHz, CDCl3) δ ppm 162.6, 157.4, 152.3, 137.9, 131.6, 130.5, 130.0, 129.0, 127.4, 126.5, 122.6, 122.0, 115.4, 111.8, 56.3, 45.9, 40.9. LRMS (ESI) m/z 333.4 [M + H]+ and 355.3 [M + Na]+. HRMS (ESI) m/z calculated for C20H20N3O2 + [M + H]+: 334.1550; found: 334.1557.Yield: 39%. R f = 0.25 (Et 2 O-MeOH = 5: 1, v / v). 1 H NMR (300 MHz, CDCl 3 ) δ ppm 8.34-8.27 (m, 2H), 8.07 (dd, J = 1.5 and 7.8 Hz, 1H), 7.69-7.51 (m, 5H), 7.38 (td, J = 7.2 and 1.2 Hz, 1H), 4.59 (t, J = 7.5 Hz, 2H), 2.68 (t, J = 7.5 Hz, 2H), 2.41 (s, 6H); 13 C NMR (75 MHz, CDCl 3 ) δ ppm 162.6, 157.4, 152.3, 137.9, 131.6, 130.5, 130.0, 129.0, 127.4, 126.5, 122.6, 122.0, 115.4, 111.8, 56.3, 45.9, 40.9. LRMS (ESI) m / z 333.4 [M + H] + and 355.3 [M + Na] + . HRMS (ESI) m / z calculated for C 20 H 20 N 3 0 2 + [M + H] + : 334.1550; found: 334.1557.
(3) 2-(4-(3) 2- (4- chlorophenylchlorophenyl )-5-) -5- (2-(dimethylamino)ethyl)oxazolo[4,5-c]quinolin(2- (dimethylamino) ethyl) oxazolo [4,5-c] quinolin -4(5H)-one (4) -4 (5H) -one (4)
Yield: 26%. Rf = 0.17 (Et2O-MeOH = 5:1, v/v). 1H NMR (300 MHz, CDCl3) δ ppm 8.24 (d, J = 8.7 Hz, 2H), 8.04 (d, J = 8.1 Hz, 1H), 7.96 (d, J = 8.4 Hz, 1H), 7.68 (td, J = 7.5 and 1.5 Hz, 1H), 7.53 (d, J = 8.7 Hz, 2H), 7.42 (t, J = 7.8 Hz, 1H), 4.43 (t, J = 5.1 Hz, 2H), 2.86 (t, J = 5.1 Hz, 2H), 2.41 (s, 6H); 13C NMR (75 MHz, CDCl3) δ ppm 151.6, 138.1, 136.9, 130.6, 129.4, 129.0, 128.7, 127.5, 123.5, 121.44, 113.7, 109.5, 57.3, 53.4, 45.8, 29.7. LRMS (ESI) m/z 368.0 [M + H]+ and 390.2 [M + Na]+. HRMS (ESI) m/z calculated for C20H19ClN3O2 + [M + H]+: 368.1160; found: 368.1202.Yield: 26%. R f = 0.17 (Et 2 O-MeOH = 5: 1, v / v). 1 H NMR (300 MHz, CDCl 3 ) δ ppm 8.24 (d, J = 8.7 Hz, 2H), 8.04 (d, J = 8.1 Hz, 1H), 7.96 (d, J = 8.4 Hz, 1H), 7.68 ( td, J = 7.5 and 1.5 Hz, 1H), 7.53 (d, J = 8.7 Hz, 2H), 7.42 (t, J = 7.8 Hz, 1H), 4.43 (t, J = 5.1 Hz, 2H), 2.86 ( t, J = 5.1 Hz, 2H), 2.41 (s, 6H); 13 C NMR (75 MHz, CDCl 3 ) δ ppm 151.6, 138.1, 136.9, 130.6, 129.4, 129.0, 128.7, 127.5, 123.5, 121.44, 113.7, 109.5, 57.3, 53.4, 45.8, 29.7. LRMS (ESI) m / z 368.0 [M + H] + and 390.2 [M + Na] + . HRMS (ESI) m / z calculated for C 20 H 19 ClN 3 O 2 + [M + H] + : 368.1160; found: 368.1202.
(4) 5-(2-((4) 5- (2- ( dimethylaminodimethylamino )ethyl)-2-) ethyl) -2- (4-(trifluoromethyl)(4- (trifluoromethyl) phenyl)phenyl) oxazolo[4,5 -c]quinolinoxazolo [4,5-c] quinolin -4(5H)-one (5)-4 (5H) -one (5)
Yield: 37%. Rf = 0.13 (Et2O-MeOH = 5:1, v/v). 1H NMR (300 MHz, CDCl3) δ ppm 8.43 (d, J = 8.1 Hz, 2H), 8.09 (dd, J = 1.2 and 7.8 Hz, 1H), 7.81 (d, J = 8.4 Hz, 2H), 7.68 (dddd, J = 1.5, 7.2, and 8.7 Hz, 1H), 7.60 (d, J = 8.4 Hz, 1H), 7.41 (td, J = 7.8 and 0.9 Hz, 1H), 4.60 (t, J = 7.5 Hz, 2H), 2.68 (t, J = 7.8 Hz, 2H), 2.42 (s, 6H); 13C NMR (75 MHz, CDCl3) δ ppm 161.0, 157.3, 152.8, 138.1, 131.0, 127.7, 126.1, 126.1, 126.0, 126.0, 122.8, 122.1, 115.5, 111.5, 56.3, 45.9, 37.6. LRMS (ESI) m/z 402.1 [M + H]+ and 424.1 [M + Na]+. HRMS (ESI) m/z calculated for C21H19F3N3O2 + [M + H]+: 402.1424; found: 402.1426.Yield: 37%. R f = 0.13 (Et 2 O-MeOH = 5: 1, v / v). 1 H NMR (300 MHz, CDCl 3 ) δ ppm 8.43 (d, J = 8.1 Hz, 2H), 8.09 (dd, J = 1.2 and 7.8 Hz, 1H), 7.81 (d, J = 8.4 Hz, 2H), 7.68 (dddd, J = 1.5, 7.2, and 8.7 Hz, 1H), 7.60 (d, J = 8.4 Hz, 1H), 7.41 (td, J = 7.8 and 0.9 Hz, 1H), 4.60 (t, J = 7.5 Hz, 2H), 2.68 (t, J = 7.8 Hz, 2H), 2.42 (s, 6H); 13 C NMR (75 MHz, CDCl 3 ) δ ppm 161.0, 157.3, 152.8, 138.1, 131.0, 127.7, 126.1, 126.1, 126.0, 126.0, 122.8, 122.1, 115.5, 111.5, 56.3, 45.9, 37.6. LRMS (ESI) m / z 402.1 [M + H] + and 424.1 [M + Na] + . HRMS (ESI) m / z calculated for C 21 H 19 F 3 N 3 O 2 + [M + H] + : 402.1424; found: 402.1426.
(5) 5-(2-((5) 5- (2- ( dimethylaminodimethylamino )ethyl)-2-) ethyl) -2- (4-methoxyphenyl)oxazolo[4,5-c]quinol(4-methoxyphenyl) oxazolo [4,5-c] quinol in-4(5H)-one (6) in-4 (5H) -one (6)
Yield: 18%. Rf = 0.08 (Et2O-MeOH = 5:1, v/v). 1H NMR (300 MHz, CDCl3) δ ppm 8.24 (d, J = 9.0 Hz, 2H), 8.04 (d, J = 8.4 Hz, 1H), 7.64-7.60 (m, 2H), 7.40-7.33 (m, 1H), 7.04 (d, J = 8.7 Hz, 2H), 4.62 (t, J = 7.5 Hz, 2H), 3.09 (s, 3H), 2.72 (t, J = 7.8 Hz, 2H), 2.44(s, 6H); 13C NMR (75 MHz, CDCl3) δ ppm 162.8, 162.4, 157.5, 152.0, 137.6, 130.1, 129.9, 129.4, 122.6, 121.8, 119.1, 115.4, 114.4, 111.8, 56.0, 55.5, 45.6, 40.5, 29.7. LRMS (ESI) m/z 364.1 [M + H]+ and 387.1 [M + Na]+. HRMS (ESI) m/z calculated for C21H22N3O3 + [M + H]+: 364.1656; found: 364.1694.Yield: 18%. R f = 0.08 (Et 2 O-MeOH = 5: 1, v / v). 1 H NMR (300 MHz, CDCl 3 ) δ ppm 8.24 (d, J = 9.0 Hz, 2H), 8.04 (d, J = 8.4 Hz, 1H), 7.64-7.60 (m, 2H), 7.40-7.33 (m , 1H), 7.04 (d, J = 8.7 Hz, 2H), 4.62 (t, J = 7.5 Hz, 2H), 3.09 (s, 3H), 2.72 (t, J = 7.8 Hz, 2H), 2.44 (s , 6H); 13 C NMR (75 MHz, CDCl 3 ) δ ppm 162.8, 162.4, 157.5, 152.0, 137.6, 130.1, 129.9, 129.4, 122.6, 121.8, 119.1, 115.4, 114.4, 111.8, 56.0, 55.5, 45.6, 40.5, 29.7. LRMS (ESI) m / z 364.1 [M + H] + and 387.1 [M + Na] + . HRMS (ESI) m / z calculated for C 21 H 22 N 3 O 3 + [M + H] + : 364.1656; found: 364.1694.
(6) (6) N,NN, N -- dimethyldimethyl -2-((2--2-((2- phenyloxazolo[4,5-c]quinolinphenyloxazolo [4,5-c] quinolin -4--4- ylyl )oxy)) oxy) ethanamineethanamine (7) (7)
Yield: 9%. Rf = 0.36 (Et2O-MeOH = 5:1, v/v). 1H NMR (300 MHz, CDCl3) δ ppm 8.38-8.31 (m, 2H), 8.16 (dd, J = 0.9 and 8.1 Hz, 1H), 7.96 (d, J = 8.4 Hz, 1H), 7.66 (td, J = 7.2 and 1.5 Hz, 1H), 7.59-7.48 (m, 4H), 4.84 (t, J = 6.0 Hz, 2H), 2.94 (t, J = 6.0 Hz, 1H), 2.42 (s, 6H); 13C NMR (75 MHz, CDCl3) δ ppm 163.0, 154.7, 153.9, 144.2, 131.6, 129.1, 129.0, 127.9, 127.6, 126.7, 126.5, 124.7, 120.3, 114.7, 64.1, 57.9, 45.9. LRMS (ESI) m/z 333.9 [M + H]+ and 356.9 [M + Na]+. HRMS (ESI) m/z calculated for C20H20N3O2 + [M + H]+: 334.1550; found: 334.1552.Yield: 9%. R f = 0.36 (Et 2 O-MeOH = 5: 1, v / v). 1 H NMR (300 MHz, CDCl 3 ) δ ppm 8.38-8.31 (m, 2H), 8.16 (dd, J = 0.9 and 8.1 Hz, 1H), 7.96 (d, J = 8.4 Hz, 1H), 7.66 (td , J = 7.2 and 1.5 Hz, 1H), 7.59-7.48 (m, 4H), 4.84 (t, J = 6.0 Hz, 2H), 2.94 (t, J = 6.0 Hz, 1H), 2.42 (s, 6H) ; 13 C NMR (75 MHz, CDCl 3 ) δ ppm 163.0, 154.7, 153.9, 144.2, 131.6, 129.1, 129.0, 127.9, 127.6, 126.7, 126.5, 124.7, 120.3, 114.7, 64.1, 57.9, 45.9. LRMS (ESI) m / z 333.9 [M + H] + and 356.9 [M + Na] + . HRMS (ESI) m / z calculated for C 20 H 20 N 3 0 2 + [M + H] + : 334.1550; found: 334.1552.
(7) 2-((2-(7) 2-((2- (4-fluorophenyl)oxazolo(4-fluorophenyl) oxazolo [4,5-c][4,5-c] quinolinquinolin -4--4- ylyl )oxy)-) oxy)- N,NN, N -dimethylethanamine (8)-dimethylethanamine (8)
Yield: 9%. Rf = 0.30 (Et2O-MeOH = 5:1, v/v). 1H NMR (300 MHz, CDCl3) δ ppm 8.41-8.28 (m, 1H), 8.29 (dd, J = 5.4 and 9.0 Hz, 1H), 8.14 (d, J = 8.1 Hz, 1H), 7.96 (d, J = 8.1 Hz, 1H), 7.67 (ddd, J = 1.5, 7.1, and 8.4 Hz, 1H), 7.52 (td, J = 8.0 and 0.9 Hz, 1H), 7.29-7.18 (m, 2H), 4.83 (t, J = 6.0 Hz, 2H), 2.93 (t, J = 6.0 Hz, 2H), 2.41 (s, 6H); 13C NMR (75 MHz, CDCl3) δ ppm 166.6, 163.2, 161.8, 154.6, 153.9, 144.2, 129.9, 129.8, 128.0, 126.4, 124.8, 123.0, 123.0, 120.2, 116.4, 116.1, 114.6, 64.1, 57.9, 45.8, 29.7. LRMS (ESI) m/z 352.2 [M + H]+ and 374.0 [M + Na]+. HRMS (ESI) m/z calculated for C20H19FN3O2 + [M + H]+: 352.1456; found: 352.1498.Yield: 9%. R f = 0.30 (Et 2 O-MeOH = 5: 1, v / v). 1 H NMR (300 MHz, CDCl 3 ) δ ppm 8.41-8.28 (m, 1H), 8.29 (dd, J = 5.4 and 9.0 Hz, 1H), 8.14 (d, J = 8.1 Hz, 1H), 7.96 (d , J = 8.1 Hz, 1H), 7.67 (ddd, J = 1.5, 7.1, and 8.4 Hz, 1H), 7.52 (td, J = 8.0 and 0.9 Hz, 1H), 7.29-7.18 (m, 2H), 4.83 (t, J = 6.0 Hz, 2H), 2.93 (t, J = 6.0 Hz, 2H), 2.41 (s, 6H); 13 C NMR (75 MHz, CDCl 3 ) δ ppm 166.6, 163.2, 161.8, 154.6, 153.9, 144.2, 129.9, 129.8, 128.0, 126.4, 124.8, 123.0, 123.0, 120.2, 116.4, 116.1, 114.6, 64.1, 57.9, 45.8, 29.7. LRMS (ESI) m / z 352.2 [M + H] + and 374.0 [M + Na] + . HRMS (ESI) m / z calculated for C 20 H 19 FN 3 O 2 + [M + H] + : 352.1456; found: 352.1498.
(8) 2-((2-(8) 2-((2- (4-chlorophenyl)oxazolo(4-chlorophenyl) oxazolo [4,5-c][4,5-c] quinolinquinolin -4--4- ylyl )oxy)-) oxy)- N,NN, N -dimethylethanamine (9)-dimethylethanamine (9)
Yield: 8%. Rf = 0.28 (Et2O-MeOH = 5:1, v/v). 1H NMR (300 MHz, CDCl3) δ ppm 8.27 (d, J = 8.4 Hz, 2H), 8.14 (d, J = 8.1 Hz, 1H), 7.96 (d, J = 8.1 Hz, 1H), 7.67 (t, J = 7.5 Hz, 1H), 7.53 (d, J = 8.1 Hz, 3H), 4.84 (t, J = 5.1 Hz, 2H), 2.94 (t, J = 5.1 Hz, 2H), 2.42 (s, 6H); 13C NMR (75 MHz, CDCl3) δ ppm 161.7, 154.6, 153.9, 144.3, 138.0, 129.4, 129.3, 128.4, 128.0, 126.4, 125.2, 124.8, 120.2, 114.6, 64.8, 57.9, 53.4, 45.8, 31.9. LRMS (ESI) m/z 368.2 [M + H]+ and 390.2 [M + Na]+. HRMS (ESI) m/z calculated for C20H19ClN3O2 + [M + H]+: 368.1160; found: 368.1195.Yield: 8%. R f = 0.28 (Et 2 O-MeOH = 5: 1, v / v). 1 H NMR (300 MHz, CDCl 3 ) δ ppm 8.27 (d, J = 8.4 Hz, 2H), 8.14 (d, J = 8.1 Hz, 1H), 7.96 (d, J = 8.1 Hz, 1H), 7.67 ( t, J = 7.5 Hz, 1H), 7.53 (d, J = 8.1 Hz, 3H), 4.84 (t, J = 5.1 Hz, 2H), 2.94 (t, J = 5.1 Hz, 2H), 2.42 (s, 6H); 13 C NMR (75 MHz, CDCl 3 ) δ ppm 161.7, 154.6, 153.9, 144.3, 138.0, 129.4, 129.3, 128.4, 128.0, 126.4, 125.2, 124.8, 120.2, 114.6, 64.8, 57.9, 53.4, 45.8, 31.9. LRMS (ESI) m / z 368.2 [M + H] + and 390.2 [M + Na] + . HRMS (ESI) m / z calculated for C 20 H 19 ClN 3 O 2 + [M + H] + : 368.1160; found: 368.1195.
(9) (9) N,NN, N -- dimethyldimethyl -2-((2--2-((2- (4-(trifluoromethyl)(4- (trifluoromethyl) phenyl)phenyl) oxazolo[4,5-c]quinolinoxazolo [4,5-c] quinolin -4-yl)oxy)ethanamine (10) -4-yl) oxy) ethanamine (10)
Yield: 10%. Rf = 0.38 (Et2O-MeOH = 5:1, v/v). 1H NMR (300 MHz, CDCl3) δ ppm 8.47 (d, J = 8.1 Hz, 2H), 8.18 (d, J = 7.8 Hz, 1H), 7.98 (d, J = 8.4 Hz, 1H), 7.82 (d, J = 8.4 Hz, 2H), 7.69 (td, J = 7.2 and 1.5 Hz, 1H), 7.54 (td, J = 7.2 and 1.2 Hz, 1H), 4.49 (t, J = 6.3 Hz, 2H), 2.94 (t, J = 6.0 Hz, 2H), 2.42 (s, 6H); 13C NMR (75 MHz, CDCl3) δ ppm 166.1, 154.7, 154.1, 144.4, 129.8, 129.5, 128.0, 127.8, 126.4, 126.0, 126.0, 124.9, 120.4, 114.5, 64.2, 59.6, 45.9. LRMS (ESI) m/z 402.1 [M + H]+ and 424.1 [M + Na]+. HRMS (ESI) m/z calculated for C21H19F3N3O2 + [M + H]+: 402.1424; found: 402.1437.Yield: 10%. R f = 0.38 (Et 2 O-MeOH = 5: 1, v / v). 1 H NMR (300 MHz, CDCl 3 ) δ ppm 8.47 (d, J = 8.1 Hz, 2H), 8.18 (d, J = 7.8 Hz, 1H), 7.98 (d, J = 8.4 Hz, 1H), 7.82 ( d, J = 8.4 Hz, 2H), 7.69 (td, J = 7.2 and 1.5 Hz, 1H), 7.54 (td, J = 7.2 and 1.2 Hz, 1H), 4.49 (t, J = 6.3 Hz, 2H), 2.94 (t, J = 6.0 Hz, 2H), 2.42 (s, 6H); 13 C NMR (75 MHz, CDCl 3 ) δ ppm 166.1, 154.7, 154.1, 144.4, 129.8, 129.5, 128.0, 127.8, 126.4, 126.0, 126.0, 124.9, 120.4, 114.5, 64.2, 59.6, 45.9. LRMS (ESI) m / z 402.1 [M + H] + and 424.1 [M + Na] + . HRMS (ESI) m / z calculated for C 21 H 19 F 3 N 3 O 2 + [M + H] + : 402.1424; found: 402.1437.
(10) 2-((2-(10) 2-((2- (4-methoxyphenyl)oxazolo[4,5-c]quinolin(4-methoxyphenyl) oxazolo [4,5-c] quinolin -4--4- ylyl )oxy)-) oxy)- N,NN, N -- dimetdimet hylethanamine (11) hylethanamine (11)
Yield: 15%. Rf = 0.18 (Et2O-MeOH = 5:1, v/v). 1H NMR (300 MHz, CDCl3) δ ppm 8.26 (d, J = 8.1 Hz, 2H), 8.16 (d, J = 6.3 Hz, 1H), 7.96 (d, J = 8.4 Hz, 1H), 7.66 (dd, J = 6.9 and 14.3 Hz, 1H), 7.54 (dd, J = 7.8 and 16.2 Hz, 1H), 7.05 (d, J = 8.7 Hz, 2H), 4.86 (t, J = 6.3 Hz, 2H), 3.91 (s, 3H), 3.01 (t, J = 6.3 Hz, 2H), 2.47(s, 6H); 13C NMR (75 MHz, CDCl3) δ ppm 163.0, 162.5, 154.6, 153.6, 144.0, 129.4, 128.8, 127.9, 126.5, 124.7, 120.2, 119.2, 114.8, 114.5, 63.7, 57.8, 55.5, 45.6, 30.4. LRMS (ESI) m/z 364.5 [M + H]+ and 387.5 [M + Na]+. HRMS (ESI) m/z calculated for C21H22N3O3 + [M + H]+: 364.1656; found: 364.1693.Yield: 15%. R f = 0.18 (Et 2 O-MeOH = 5: 1, v / v). 1 H NMR (300 MHz, CDCl 3 ) δ ppm 8.26 (d, J = 8.1 Hz, 2H), 8.16 (d, J = 6.3 Hz, 1H), 7.96 (d, J = 8.4 Hz, 1H), 7.66 ( dd, J = 6.9 and 14.3 Hz, 1H), 7.54 (dd, J = 7.8 and 16.2 Hz, 1H), 7.05 (d, J = 8.7 Hz, 2H), 4.86 (t, J = 6.3 Hz, 2H), 3.91 (s, 3 H), 3.01 (t, J = 6.3 Hz, 2H), 2.47 (s, 6H); 13 C NMR (75 MHz, CDCl 3 ) δ ppm 163.0, 162.5, 154.6, 153.6, 144.0, 129.4, 128.8, 127.9, 126.5, 124.7, 120.2, 119.2, 114.8, 114.5, 63.7, 57.8, 55.5, 45.6, 30.4. LRMS (ESI) m / z 364.5 [M + H] + and 387.5 [M + Na] + . HRMS (ESI) m / z calculated for C 21 H 22 N 3 O 3 + [M + H] + : 364.1656; found: 364.1693.
(11) 5-(2-((11) 5- (2- ( diethylaminodiethylamino )ethyl)-2-) ethyl) -2- (4-fluorophenyl)oxazolo(4-fluorophenyl) oxazolo [4,5-c]quinolin- 4(5H)-one (12)[4,5-c] quinolin-4 (5H) -one (12)
Yield: 14%. Rf = 0.39 (Et2O-MeOH = 5:1, v/v). 1H NMR (300 MHz, CDCl3) δ ppm 8.35-8.26 (m, 1H), 8.30 (dd, J = 5.4 and 8.7 Hz, 1H), 8.03 (d, J = 7.5 Hz, 1H), 7.66-7.59 (m, 2H), 7.43-7.34 (m, 1H), 7.22 (t, J = 8.7 Hz, 2H), 4.55 (t, J = 7.8 Hz, 2H), 2.79 (t, J = 7.8 Hz, 2H), 2.68 (dd, J = 7.2 and 14.4 Hz, 4H), 1.09 (t, J = 7.2 Hz, 6H); 13C NMR (75 MHz, CDCl3) δ ppm 166.5, 163.2, 161.7, 157.4, 152.3, 138.0, 130.5, 129.8, 129.7, 129.6, 122.8, 122.8, 122.6, 121.8, 116.4, 116.1, 115.6, 111.6, 50.0, 47.6, 41.2, 12.0. LRMS (ESI) m/z 380.3 [M + H]+. HRMS (ESI) m/z calculated for C22H23FN3O2 + [M + H]+: 380.1769; found: 380.1817.Yield: 14%. R f = 0.39 (Et 2 O-MeOH = 5: 1, v / v). 1 H NMR (300 MHz, CDCl 3 ) δ ppm 8.35-8.26 (m, 1H), 8.30 (dd, J = 5.4 and 8.7 Hz, 1H), 8.03 (d, J = 7.5 Hz, 1H), 7.66-7.59 (m, 2H), 7.43-7.34 (m, 1H), 7.22 (t, J = 8.7 Hz, 2H), 4.55 (t, J = 7.8 Hz, 2H), 2.79 (t, J = 7.8 Hz, 2H) , 2.68 (dd, J = 7.2 and 14.4 Hz, 4H), 1.09 (t, J = 7.2 Hz, 6H); 13 C NMR (75 MHz, CDCl 3 ) δ ppm 166.5, 163.2, 161.7, 157.4, 152.3, 138.0, 130.5, 129.8, 129.7, 129.6, 122.8, 122.8, 122.6, 121.8, 116.4, 116.1, 115.6, 111.6, 50.0, 47.6, 41.2, 12.0. LRMS (ESI) m / z 380.3 [M + H] + . HRMS (ESI) m / z calculated for C 22 H 23 FN 3 O 2 + [M + H] + : 380.1769; found: 380.1817.
(12) (12) N,NN, N -diethyl-2-((2--diethyl-2-((2- (4-fluorophenyl)oxazolo(4-fluorophenyl) oxazolo [4,5-c][4,5-c] quinolinquinolin -4--4- ylyl )oxy) ethanamine (13)) oxy) ethanamine (13)
Yield: 8%. Rf = 0.51 (Et2O-MeOH = 5:1, v/v). 1H NMR (300 MHz, CDCl3) δ ppm 8.38-8.31 (m, 1H), 8.35 (dd, J = 5.4 and 9.0 Hz, 1H), 8.14 (d, J = 7.8 Hz, 1H), 7.96 (d, J = 8.4 Hz, 1H), 7.66 (ddd, J = 1.5, 7.1, and 8.4 Hz, 1H), 7.52 (t, J = 7.5 Hz, 1H), 7.28-7.19 (m, 2H), 4.81 (t, J = 6.9 Hz, 2H), 3.06 (t, J = 6.9 Hz, 2H), 2.72 (dd, J = 7.2 and 14.4 Hz, 4H), 1.14 (t, J = 7.2 Hz, 6H); 13C NMR (75 MHz, CDCl3) δ ppm 166.6, 163.2, 161.8, 154.6, 153.8, 144.2, 129.9, 129.8, 129.1, 128.0, 126.4, 124.7, 123.0, 123.0, 120.2, 116.5, 116.2, 114.6, 64.2, 51.0, 47.7, 29.7, 11.8. LRMS (ESI) m/z 380.3 [M + H]+. HRMS (ESI) m/z calculated for C22H23FN3O2 + [M + H]+: 380.1769; found: 380.1814.Yield: 8%. R f = 0.51 (Et 2 O-MeOH = 5: 1, v / v). 1 H NMR (300 MHz, CDCl 3 ) δ ppm 8.38-8.31 (m, 1H), 8.35 (dd, J = 5.4 and 9.0 Hz, 1H), 8.14 (d, J = 7.8 Hz, 1H), 7.96 (d , J = 8.4 Hz, 1H), 7.66 (ddd, J = 1.5, 7.1, and 8.4 Hz, 1H), 7.52 (t, J = 7.5 Hz, 1H), 7.28-7.19 (m, 2H), 4.81 (t , J = 6.9 Hz, 2H), 3.06 (t, J = 6.9 Hz, 2H), 2.72 (dd, J = 7.2 and 14.4 Hz, 4H), 1.14 (t, J = 7.2 Hz, 6H); 13 C NMR (75 MHz, CDCl 3 ) δ ppm 166.6, 163.2, 161.8, 154.6, 153.8, 144.2, 129.9, 129.8, 129.1, 128.0, 126.4, 124.7, 123.0, 123.0, 120.2, 116.5, 116.2, 114.6, 64.2, 51.0, 47.7, 29.7, 11.8. LRMS (ESI) m / z 380.3 [M + H] + . HRMS (ESI) m / z calculated for C 22 H 23 FN 3 O 2 + [M + H] + : 380.1769; found: 380.1814.
(13) 5-(2-((13) 5- (2- ( diethylaminodiethylamino )ethyl)-2-) ethyl) -2- (4-(trifluoromethyl)(4- (trifluoromethyl) phenyl)oxazolo[4,5-c]quinolin-4(5H)-one (14)phenyl) oxazolo [4,5-c] quinolin-4 (5H) -one (14)
Yield: 27%. Rf=0.39 (Et2O-MeOH = 5:1, v/v). 1H NMR (300 MHz, CDCl3)δppm 8.44 (d, J = 8.1 Hz, 2H), 8.08 (d, J = 7.8 Hz, 1H), 7.81 (d, J = 8.1 Hz, 2H), 7.71-7.60 (m, 2H), 7.70 (t, J = 7.8 Hz, 1H), 4.56 (t, J = 7.8 Hz, 2H), 2.80 (t, J = 8.1 Hz, 2H), 2.69 (dd, J = 7.2 and 14.4 Hz, 4H), 1.09 (t, J = 7.2 Hz, 6H).Yield: 27%. R f = 0.39 (Et 2 O-MeOH = 5: 1, v / v). 1 H NMR (300 MHz, CDCl 3 ) δ ppm 8.44 (d, J = 8.1 Hz, 2H), 8.08 (d, J = 7.8 Hz, 1H), 7.81 (d, J = 8.1 Hz, 2H), 7.71-7.60 (m, 2H), 7.70 (t, J = 7.8 Hz, 1H), 4.56 (t, J = 7.8 Hz, 2H), 2.80 (t, J = 8.1 Hz, 2H), 2.69 (dd, J = 7.2 and 14.4 Hz, 4H), 1.09 (t, J = 7.2 Hz, 6H).
(14) (14) N,NN, N -diethyl-2-((2--diethyl-2-((2- (4-(trifluoromethyl)(4- (trifluoromethyl) phenyl)phenyl) oxazolo[4,5-c]quinolinoxazolo [4,5-c] quinolin -4-yl)oxy)ethanamine (15)-4-yl) oxy) ethanamine (15)
Yield: 50%. Rf = 0.51 (Et2O-MeOH = 5 : 1, v/v). 1HNMR (300 MHz, CDCl3) δppm 8.45 (d, J = 8.1 Hz, 2H), 8.15 (d, J = 8.7 Hz, 1H), 7.95 (d, J = 8.4 Hz, 1H), 7.80 (d, J = 8.4 Hz, 2H), 7.68 (ddd, J = 1.5, 7.1, and 8.4 Hz, 1H), 7.53 (t, J = 7.2 Hz, 1H), 4.81 (t, J = 6.9 Hz, 2H), 3.06 (t, J = 7.2 Hz, 2H), 2.72 (dd, J = 7.2 and 14.4 Hz, 4H), 1.09 (t, J = 7.2 Hz, 6H).Yield: 50%. R f = 0.51 (Et 2 O-MeOH = 5: 1, v / v). 1 HNMR (300 MHz, CDCl 3 ) δ ppm 8.45 (d, J = 8.1 Hz, 2H), 8.15 (d, J = 8.7 Hz, 1H), 7.95 (d, J = 8.4 Hz, 1H), 7.80 (d, J = 8.4 Hz, 2H), 7.68 (ddd, J = 1.5, 7.1, and 8.4 Hz, 1H), 7.53 (t, J = 7.2 Hz, 1H), 4.81 (t, J = 6.9 Hz, 2H), 3.06 ( t, J = 7.2 Hz, 2H), 2.72 (dd, J = 7.2 and 14.4 Hz, 4H), 1.09 (t, J = 7.2 Hz, 6H).
실험예 1. ELISA를 이용한 TSLP-TSLPR 저해 효능 평가Experimental Example 1. Evaluation of TSLP-TSLPR Inhibition Effect by ELISA
본 발명에 따른 퀴놀리논 유도체 화합물이 TSLP와 TSLP 수용체 사이의 결합을 직접적으로 저해하는지를 확인하기 위해서 자체 제작한 FLAG-TSLP 단백질과 TSLPR-His 단백질을 활용하여, HRP에 의한 반응을 측정하는 ELISA를 이용한 TSLP-TSLPR 상호작용 assay를 구축하여, 저해 활성을 평가하였다.In order to confirm whether the quinolinone derivative compound according to the present invention directly inhibits the binding between TSLP and TSLP receptor, ELISA was used to measure the response by HRP using FLAG-TSLP protein and TSLPR-His protein. The TSLP-TSLPR interaction assay was used to evaluate the inhibitory activity.
ELISA 분석은 Ni-NTA HisSorb 플레이트 (Qiagen, Germany)에서 수행하였다. 플레이트의 각 웰에 C-말단에 헥사히스티딘(His) 태그를 갖는 TSLPR 용액 100 μL를 분배하고 실온에서 2시간 동안 인큐베이션 한 후, 각 웰을 PBS 200 μL, 0.05% 트윈-20으로 2회 세척한 후에 본 발명에 따른 화합물 용액(시료) 100 μL 및 N-말단 FLAG 태그를 갖는 TSLP 100 μL를 분배하였다. 4 ℃에서 밤새 항온 배양한 후에, 각 웰을 2회 세척하고 블로킹 완충액 (PBS-0.05 % Tween 20 및 탈지분유 1 %) 100μL로 처리하였다. 플레이트를 2회 세척한 후 monoclonal anti-FLAG horseradish peroxidase antibody (Sigma-Aldrich Co., USA) 100 μL로 2 시간 동안 코팅하고, 실온에서 배양한 후, 각 웰을 5회 세척하였다. 각각의 웰을 200μL의 o-페닐렌디아민디하이드로 클로라이드 (Sigma-Aldrich Co., USA) 용액으로 처리하고 30 분 동안 배양하였다. 배양 후, 각 웰에 1N HCl을 첨가하였다.ELISA analysis was performed on Ni-NTA HisSorb plates (Qiagen, Germany). Dispense 100 μL of the TSLPR solution with hexahistidine (His) tag at the C-terminus to each well of the plate and incubate for 2 hours at room temperature, then wash each well twice with 200 μL of PBS, 0.05% Tween-20. Then 100 μL of the compound solution (sample) according to the invention and 100 μL of TSLP with N-terminal FLAG tag were dispensed. After incubation overnight at 4 ° C., each well was washed twice and treated with 100 μL of blocking buffer (PBS-0.05 % Tween 20 and 1% skim milk). The plates were washed twice and then coated with 100 μL of monoclonal anti-FLAG horseradish peroxidase antibody (Sigma-Aldrich Co., USA) for 2 hours, incubated at room temperature, and then each well was washed five times. Each well was treated with 200 μL of o-phenylenediaminedihydrochloride (Sigma-Aldrich Co., USA) solution and incubated for 30 minutes. After incubation, 1N HCl was added to each well.
마이크로 플레이트 분광 광도계 450 nm에서 광학 밀도 (optical densities, ODs)를 측정하였으며, TSLP-TSLPR 상호작용 억제 효과는 하기 식을 이용하여 계산하였으며, 그 결과를 하기 [표 1]에 나타내었다.Optical densities (ODs) were measured at 450 nm of a microplate spectrophotometer, and the TSLP-TSLPR interaction inhibitory effect was calculated using the following formula, and the results are shown in the following [Table 1].
저해도(억제효과) (%) = (1-시료 OD / 대조군 OD) × 100Inhibition (inhibition) (%) = (1-sample OD / control OD) × 100
화합물compound 저해도 (%)Inhibition (%)
0.1 mM0.1 mM 0.3 mM0.3 mM 1 mM1 mM
22 26.726.7 49.049.0 66.966.9
33 17.7 17.7 27.5 27.5 N/DN / D
44 28.8 28.8 46.6 46.6 N/DN / D
55 23.2 23.2 29.6 29.6 N/DN / D
66 25.8 25.8 54.7 54.7 N/DN / D
77 27.827.8 54.654.6 57.257.2
FLAG tag-free TSLP (1 mg/mL)FLAG tag-free TSLP (1 mg / mL) 40.740.7
N/D: Not determinedN / D: Not determined
실험예 2. IL-33 저해에 의한 IL-6 분비 저해 효능 평가Experimental Example 2. Evaluation of the inhibitory effect of IL-6 secretion by IL-33 inhibition
인간 비만 세포주 (Human mast cell line)인 HMC-1을 배양하여 96-웰 플레이트에 적정량의 세포를 유지하였다. IL-33과 소정 농도(0.03, 0.3, 3 μM)의 본 발명에 따른 화합물을 혼합하여 세포에 처리하고, 37 ℃에서 24시간 동안 인큐베이션한 후에, 원심분리에 의해서 상청액을 수득한 후에 IL-6 ELISA kit (Biolegend, USA)로 IL-6의 분비를 분석하고, 그 결과 하기 [표 2]에 나타내었다.HMC-1, a human mast cell line, was cultured to maintain the appropriate amount of cells in 96-well plates. IL-33 and the compound according to the present invention at a predetermined concentration (0.03, 0.3, 3 μM) were mixed with the cells, incubated at 37 ° C. for 24 hours, and then the supernatant was obtained by centrifugation before IL-6. The secretion of IL-6 was analyzed by ELISA kit (Biolegend, USA), and the results are shown in the following [Table 2].
화합물compound IL-6 분비 저해도 (%)IL-6 secretion inhibition (%)
0.03 uM0.03 uM 0.3 uM0.3 uM 3 uM3 uM
22 -- -11.8-11.8
33 -- 29.829.8
44 22.522.5 29.729.7
55 30.430.4 28.228.2
66 -- -57.8-57.8
77 -- 35.635.6
1010 32.732.7 49.349.3 51.351.3
1111 -- -216.4-216.4
IL-33 onlyIL-33 only 00
(-)값은 IL-6의 분비를 증가시켰음을 의미함Negative values indicate increased IL-6 secretion
상기 결과를 종합하면, 본 발명에 따른 퀴놀리논 유도체들은 천식 또는 아토피 등의 알러지성 질환을 유발하는데 핵심적인 역할을 하는 싸이토카인인 TSLP와 TSLP 수용체 사이의 결합 혹은 IL-33과 ST-2의 결합을 효과적으로 억제할 수 있고, 따라서 본 발명에 의하면 TSLP 또는 IL-33에 의해 매개되는 신호 전달을 억제함으로써, 천식 또는 아토피 등의 알러지성 질환의 근본적인 예방 및 치료가 가능하다.In summary, the quinolinone derivatives according to the present invention are the cytokines TSLP and TSLP receptors or the binding of IL-33 and ST-2, which play a key role in inducing allergic diseases such as asthma or atopy. Can be effectively inhibited, and according to the present invention, by inhibiting signal transduction mediated by TSLP or IL-33, it is possible to fundamentally prevent and treat allergic diseases such as asthma or atopy.
실험예Experimental Example 3. HDM (house dust mite, 집먼지진드기) 유발  3. Induced HDM (house dust mite) 알러지allergy 기도 염증 마우스 모델 Airway Inflammation Mouse Model
DO11.10 마우스에서 비장 세포를 분리하고, 약 1 × 106 CD4 T 세포를 정맥주사로 순수한 BALB/c 마우스에 옮겼다. 다음날부터 3일 동안 HDM (Dermatophagoides farinae 50 ㎍, Dermatophagoides pteronyssinus 50 ㎍, Greer Laboratories, Inc., USA) 및 OVA (Sigma-Aldrich Co., USA) 100 ㎍의 혼합물을 비강 경로를 통하여 마우스에 투여하였으며, 투여 후 2 일째부터 본 발명에 따른 화합물 200 ㎍ 또는 PBS (대조군 마우스)를 매일 3 회 복강 내로 처리하였다. 11 일째에 마우스를 안락사한 후에 기도 내의 총 세포와 호산구 (eosinophils) 수를 확인하였으며, 그 결과를 하기 도 4 및 도 5에 각각 나타내었다.Splenocytes were isolated from DO11.10 mice and approximately 1 × 10 6 CD4 T cells were transferred intravenously to pure BALB / c mice. A mixture of 50 μg of HDM (Dermatophagoides farinae, 50 μg of Dermatophagoides pteronyssinus, Greer Laboratories, Inc., USA) and 100 μg of OVA (Sigma-Aldrich Co., USA) was administered to the mice via the nasal route for 3 days from the next day. From day 2 after administration, 200 μg of compounds according to the invention or PBS (control mice) were treated intraperitoneally three times daily. After euthanizing mice on day 11, the total cells and eosinophils in the airways were confirmed, and the results are shown in FIGS. 4 and 5, respectively.
Figure PCTKR2018000165-appb-I000022
Figure PCTKR2018000165-appb-I000022
하기 도 4 및 도 5에서, 본 발명에 따른 화학식 5 (KB-1517), 화학식 10 (KB-1518) 200 ㎍을 처리한 마우스의 기도 내 총 세포와 호산구 (eosinophils) 수가 화합물을 처리하지 않은 경우에 비하여 유의적으로 감소하였음을 확인할 수 있다. 따라서, 본 발명에 따른 퀴놀리논 유도체를 이용하여 천식 또는 아토피 등의 알러지성 질환의 근본적인 예방 및 치료가 가능해진다.In the following Figures 4 and 5, the total cells and eosinophils in the airway of mice treated with 200 μg of Formula 5 (KB-1517) and Formula 10 (KB-1518) according to the present invention were not treated with the compound. It can be seen that significantly reduced compared to. Therefore, by using the quinolinone derivatives according to the present invention, it is possible to fundamentally prevent and treat allergic diseases such as asthma or atopy.
실험예 4. 2D NMR 분광분석Experimental Example 4 2D NMR Spectroscopy
Bruker 600 MHz NMR 분광계 (triple-resonance, pulsed field gradient probe (Bruker, Germany))를 이용하여 25 ℃에서 측정하였으며, IL-33의 2D 1H-15N HSQC 스펙트럼은 화합물 존재/부존재에서 각각 동일한 몰비로 측정 하였다. 2D 데이터는 탑스핀 3.1 (TopSpin 3.1, Bruker, Germany) 프로그램을 이용하여 처리 및 분석하였다.Measured at 25 ° C using a Bruker 600 MHz NMR spectrometer (triple-resonance, pulsed field gradient probe (Bruker, Germany)), the 2D 1 H- 15 N HSQC spectrum of IL-33 was the same molar ratio in the presence / absence of each compound Was measured. 2D data were processed and analyzed using the TopSpin 3.1 (Bruker, Germany) program.
화학적 이동 변화 (chemical shift perturbations, CSPs)는 다음의 방정식을 사용하여 계산하였다 (Δδ1H와 Δδ15N은 각각 1H, 15N으로 표지된 화학적 시프트 차이를 의미한다.)Chemical shift perturbations (CSPs) were calculated using the following equation (Δδ 1H and Δδ 15N represent chemical shift differences labeled 1 H and 15 N, respectively).
Figure PCTKR2018000165-appb-I000023
Figure PCTKR2018000165-appb-I000023
실험예 5. IL-33 단백질 발현 및 정제Experimental Example 5. IL-33 Protein Expression and Purification
IL-33은 E. coli BL21 (DE3)에서 N-말단 His-tag 융합 단백질인 발현 벡터 pPROEX에 의해서 클로닝되었으며, 세포 밀도 (OD600)가 0.6에 달했을 때 세포를 0.5 mM IPTG (isopropyl-b-D-thiogalactoside)로 유도한 후 20 ℃에서 하룻밤 동안 더 성장시켰다. 균일하게 표지된 15N IL-33을 얻기 위해, 박테리아 세포를 15N NH4Cl을 함유하는 M9 최소 배지에서 성장시켰다. 세포를 수득한 이후에 용균 완충액 (0.1 M Tris pH 7.4, 0.3 M NaCl, 1 mM β-mercaptoethanol, 0.1% TritonX100, and 0.1 mM phenylmethylsulfonyl fluoride)에 재현탁시켰다. 세포 용해는 얼음욕에서 초음파 처리를 수행하였으며, 세포 용해물을 10000 ×g, 4 ℃에서 25 분간 원심 분리하였다. 펠릿을 버리고 상등액을 인산나트륨 50 mM (pH 7.4), 염화나트륨 300 mM 및 β-mercaptoethanol 1 mM이 들어있는 HisPur 코발트 수지 컬럼 Thermo Scientific Inc.) 5 mL 상에서 용출한 후에, 버퍼를 인산나트륨 50 mM (pH 7.4), 염화나트륨 300 mM 및 β-mercaptoethanol 1 mM 및 이미다졸 250mM 로 교환하였다. TEV 프로테아제를 이용하여 dialysis buffer (Tris 20 mM (pH 7.4), 염화나트륨 300 mM) 및 4 ℃에서 밤새 융합 단백질을 절단하였다. IL-33 함유 혼합물을 HisPur 코발트 수지 칼럼에 넣고 Tris 0.1 M (pH 7.4), 염화나트륨 0.3 M 및 β-mercaptoethanol 1 mM로 세척한 이후에 결합된 단백질을 Tris 0.1 M (pH 7.4), 염화나트륨 0.3 M 및 β-mercaptoethanol 1 mM (20 mL)로 용출시켰다. 단백질을 인산나트륨 20 mM (pH 6.8), 염화나트륨 100 mM, BME 5 mM의 Superdex S75 겔 여과 컬럼 (16/60 GE Healthcare)에 로딩하여 순수한 IL-33 단백질을 얻었다.IL-33 was cloned by the expression vector pPROEX, an N-terminal His-tag fusion protein in E. coli BL21 (DE3), and cells were cultured at 0.5 mM IPTG (isopropyl-bD-) when the cell density (OD 600 ) reached 0.6. thiogalactoside) and further growth at 20 ° C. overnight. To obtain uniformly labeled 15 N IL-33, bacterial cells were grown in M9 minimal medium containing 15 N NH 4 Cl. Cells were obtained and then resuspended in lysis buffer (0.1 M Tris pH 7.4, 0.3 M NaCl, 1 mM β-mercaptoethanol, 0.1% TritonX100, and 0.1 mM phenylmethylsulfonyl fluoride). Cell lysis was sonicated in an ice bath, and cell lysates were centrifuged at 10000 × g, 4 ° C. for 25 minutes. The pellet was discarded and the supernatant was eluted on 5 mL of HisPur cobalt resin column Thermo Scientific Inc. containing 50 mM sodium phosphate (pH 7.4), 300 mM sodium chloride and 1 mM β-mercaptoethanol, and then the buffer was dissolved in 50 mM sodium phosphate (pH). 7.4), 300 mM sodium chloride and 1 mM β-mercaptoethanol and 250 mM imidazole. The TEV protease was used to cleave the fusion protein overnight in dialysis buffer (Tris 20 mM (pH 7.4), sodium chloride 300 mM) and 4 ° C. The IL-33 containing mixture was placed in a HisPur cobalt resin column and washed with Tris 0.1 M (pH 7.4), 0.3 M sodium chloride and 1 mM β-mercaptoethanol, and the bound protein was then tris 0.1 M (pH 7.4), sodium chloride 0.3 M and Elution with 1 mM (20 mL) β-mercaptoethanol. The protein was loaded on a Superdex S75 gel filtration column (16/60 GE Healthcare) at 20 mM sodium phosphate (pH 6.8), sodium chloride 100 mM, BME 5 mM to obtain pure IL-33 protein.
본 발명에 따른 퀴놀리논 유도체 화합물은 천식 또는 아토피 등의 알러지성 질환의 염증 반응 형성을 효과적으로 억제할 수 있는 것으로서, 이를 포함하는 약학 조성물을 이용하여 다양한 천식 또는 아토피 등의 알러지성 질환을 근본적으로 예방하거나 치료할 수 있다.The quinolinone derivative compound according to the present invention is capable of effectively inhibiting the formation of an inflammatory response of an allergic disease such as asthma or atopic dermatitis, and fundamentally prevents various allergic diseases such as asthma or atopy using a pharmaceutical composition comprising the same. It can be prevented or treated.

Claims (8)

  1. 하기 [화학식 1a] 또는 [화학식 1b]로 표시되는 퀴놀리논 유도체:A quinolinone derivative represented by the following [Formula 1a] or [Formula 1b]:
    [화학식 1a][Formula 1a]
    Figure PCTKR2018000165-appb-I000024
    Figure PCTKR2018000165-appb-I000024
    [화학식 1b][Formula 1b]
    Figure PCTKR2018000165-appb-I000025
    Figure PCTKR2018000165-appb-I000025
    상기 [화학식 1a] 및 [화학식 1b]에서,In [Formula 1a] and [Formula 1b],
    R1 및 R2는 서로 동일하거나 상이하고, 각각 독립적으로 탄소수 1 내지 4의 알킬기, 탄소수 1 내지 4의 알킬렌기 및 하기 [구조식 1] 중에서 선택되는 어느 하나이고,R 1 and R 2 is the same as or different from each other, and each independently an alkyl group having 1 to 4 carbon atoms, an alkylene group having 1 to 4 carbon atoms, and any one selected from the following [Formula 1],
    [구조식 1][Formula 1]
    -Z-알킬기-Z-alkyl group
    상기 [구조식 1]에서, Z는 O, S 또는 N으로부터 선택되는 이종원자이거나, 또는 -(CH2)m-이며(상기 m은 0 내지 5 의 정수임),In [Formula 1], Z is a hetero atom selected from O, S or N, or-(CH 2 ) m- (wherein m is an integer of 0 to 5),
    X 및 Y는 각각 독립적으로 수소, 할로겐기, 히드록시기, 아미노기, 니트로기, 시아노기, 트리플루오르메틸기, CO-R', NHR', NR'R', NHCOR' 및 COOR' 중에서 선택되는 어느 하나이고(상기 R'는 탄소수 1 내지 4의 알킬기임),X and Y are each independently selected from hydrogen, halogen, hydroxy, amino, nitro, cyano, trifluoromethyl, CO-R ', NHR', NR'R ', NHCOR' and COOR ' (Wherein R 'is an alkyl group having 1 to 4 carbon atoms),
    p는 0 내지 4의 정수이고, q는 0 내지 5의 정수이며, 상기 p 및 q가 복수인 경우 복수의 X 및 복수의 Y는 각각 서로 동일하거나 상이하다.p is an integer of 0-4, q is an integer of 0-5, and when said p and q are multiple, some X and some Y are each the same or different.
  2. 제1항에 있어서,The method of claim 1,
    상기 [화학식 1a] 또는 [화학식 1b]로 표시되는 퀴놀리논 유도체는 하기 [화학식 2] 내지 [화학식 15]로 표시되는 유도체 중에서 선택되는 어느 하나인 것을 특징으로 하는 퀴놀리논 유도체:A quinolinone derivative represented by [Formula 1a] or [Formula 1b] is any one selected from derivatives represented by the following [Formula 2] to [Formula 15]:
    [[화학식 2] [화학식 3][Formula 2] [Formula 3]
    Figure PCTKR2018000165-appb-I000026
    Figure PCTKR2018000165-appb-I000027
    Figure PCTKR2018000165-appb-I000026
    Figure PCTKR2018000165-appb-I000027
    [화학식 4] [화학식 5][Formula 4] [Formula 5]
    Figure PCTKR2018000165-appb-I000028
    Figure PCTKR2018000165-appb-I000029
    Figure PCTKR2018000165-appb-I000028
    Figure PCTKR2018000165-appb-I000029
    [화학식 6] [화학식 7][Formula 6] [Formula 7]
    Figure PCTKR2018000165-appb-I000030
    Figure PCTKR2018000165-appb-I000031
    Figure PCTKR2018000165-appb-I000030
    Figure PCTKR2018000165-appb-I000031
    [화학식 8] [화학식 9][Formula 8] [Formula 9]
    Figure PCTKR2018000165-appb-I000032
    Figure PCTKR2018000165-appb-I000033
    Figure PCTKR2018000165-appb-I000032
    Figure PCTKR2018000165-appb-I000033
    [화학식 10] [화학식 11][Formula 10] [Formula 11]
    Figure PCTKR2018000165-appb-I000034
    Figure PCTKR2018000165-appb-I000035
    Figure PCTKR2018000165-appb-I000034
    Figure PCTKR2018000165-appb-I000035
    [화학식 12] [화학식 13][Formula 12] [Formula 13]
    Figure PCTKR2018000165-appb-I000036
    Figure PCTKR2018000165-appb-I000037
    Figure PCTKR2018000165-appb-I000036
    Figure PCTKR2018000165-appb-I000037
    [화학식 14] [화학식 15][Formula 14] [Formula 15]
    Figure PCTKR2018000165-appb-I000038
    Figure PCTKR2018000165-appb-I000039
    Figure PCTKR2018000165-appb-I000038
    Figure PCTKR2018000165-appb-I000039
  3. 제1항에 있어서,The method of claim 1,
    상기 [화학식 1a] 또는 [화학식 1b]로 표시되는 퀴놀리논 유도체는 TSLP와 IL-33에 의한 세포내 신호 전달을 제어하는 것을 특징으로 하는 퀴놀리논 유도체.A quinolinone derivative represented by [Formula 1a] or [Formula 1b] is characterized in that the control of intracellular signal transduction by TSLP and IL-33.
  4. 제1항에 있어서,The method of claim 1,
    상기 [화학식 1a] 또는 [화학식 1b]로 표시되는 퀴놀리논 유도체는 싸이토카인 TSLP(thymic stromal lymphopoietin)와 TSLP 수용체 사이의 결합을 저해하거나 싸이토카인 IL-33과 ST-2 사이의 결합을 저해하는 것을 특징으로 하는 퀴놀리논 유도체.The quinolinone derivatives represented by the above [Formula 1a] or [Formula 1b] inhibit the binding between the cytokine thymic stromal lymphopoietin (TSLP) and the TSLP receptor or inhibit the binding between the cytokine IL-33 and ST-2. Quinolinone derivatives.
  5. 제1항에 따른 [화학식 1a] 또는 [화학식 1b]로 표시되는 퀴놀리논 유도체를 유효성분으로 함유하고, 약제학적으로 허용되는 담체를 포함하는 알러지성 질환의 예방 또는 치료용 약학 조성물.A pharmaceutical composition for preventing or treating allergic diseases, comprising a quinolinone derivative represented by [Formula 1a] or [Formula 1b] according to claim 1 as an active ingredient and a pharmaceutically acceptable carrier.
  6. 제5항에 있어서,The method of claim 5,
    상기 조성물은 TSLP와 IL-33에 의한 세포내 신호 전달을 제어하는 것을 특징으로 하는 알러지성 질환의 예방 또는 치료용 약학 조성물.The composition is a pharmaceutical composition for preventing or treating allergic diseases, characterized in that for controlling intracellular signal transduction by TSLP and IL-33.
  7. 제5항에 있어서,The method of claim 5,
    상기 조성물은 싸이토카인 TSLP(thymic stromal lymphopoietin)와 TSLP 수용체 사이의 결합을 저해하거나 싸이토카인 IL-33과 ST-2 사이의 결합을 저해하는 것을 특징으로 하는 알러지성 질환의 예방 또는 치료용 약학 조성물.The composition is a pharmaceutical composition for preventing or treating allergic diseases, characterized in that it inhibits the binding between the cytokine TSLP (thymic stromal lymphopoietin) and the TSLP receptor or inhibits the binding between cytokines IL-33 and ST-2.
  8. 제5항에 있어서,The method of claim 5,
    상기 알러지성 질환은 천식, 아토피성 피부염, 두드러기 비염 또는 알러지성 비염 질환인 것을 특징으로 하는 알러지성 질환의 예방 또는 치료용 약학 조성물.The allergic disease is asthma, atopic dermatitis, urticaria rhinitis or allergic rhinitis disease, characterized in that the pharmaceutical composition for the prevention or treatment of allergic diseases.
PCT/KR2018/000165 2017-01-06 2018-01-04 Novel quinolinone derivative and pharmaceutical composition for preventing or treating allergic disease such as asthma or atopy comprising same as active ingredient WO2018128407A1 (en)

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