Patent Application
20200087250
This application claims, under 35 U.S.C. § 119, the priority of Korean Patent Application No. 10-2018-0112056 filed on Sep. 19, 2018 in the Korean Intellectual Property Office, the disclosure of which is incorporated herein by reference in its entirety.
The present disclosure relates to an anticancer pharmaceutical composition and an ANO1 antagonist.
ANO1 is a calcium-activated chloride channel (CaCC) expressed in various cells and tissues. It performs physiological functions such as the secretion of epithelial fluid, contraction of smooth muscle, cellular growth, and sensory nerve signaling.
According to recent studies, it is reported that an ANO1 inhibitor may be useful in the treatment of ANO1-related diseases such as inflammatory airway disease, hypertension and pain (Proc. Natl. Acad. Sci. USA 109(2012) 16354-16359; Nat. Neurosci. 15 (2012) 1015-1021).
Recently, it was found that ANO1 is overexpressed in various cancer cells such as breast cancer, pancreatic cancer, head and neck cancer, gastrointestinal cancer and prostate cancer. ANO1 plays an important role in cellular growth, tumorigenesis and progression of cancer, and the inhibition of ANO1 significantly decreases the proliferation and migration of breast cancer, pancreatic cancer and prostate cancer cells (Mol. Cancer. 16 (2017) 152).
ANO1 and ANO2 function as CaCCs and have high structural similarities. ANO2 is overexpressed in olfactory sensory neurons and plays an important role in olfactory signal transduction (J. Gen. Physiol. 148 (2016) 293-311). In addition, it was found that ANO2 is involved in reduced spike generation in the thalamic cortex and hippocampal neurons and that motor dysfunction and motor learning disorder are induced in ANO2-knockout mice (Cerebellum. 16 (2017) 929-937).
Accordingly, development of an inhibitor that selectively inhibits ANO1 over ANO2 may provide a more appropriate therapy for ANO1-related diseases than existing ANO1 inhibitors.
The ANO1 inhibitors identified until now are CaCCinh-A01, T16Ainh-A01, MONNA, Ani9, AACT (10 bm), etc. Among them, Ani9 is the most specific ANO1 inhibitor which does not inhibit ANO2.
However, there is a need for novel compounds with an increased inhibitory effect for ANO1 without inhibiting ANO2.
Korean Patent Publication No. 10-2014-0112489.
Korean Patent Registration No. 1719541.
The present disclosure is directed to providing an anticancer pharmaceutical composition, which contains a novel compound effective in inhibiting the expression of ANO1 (TMEM16A) or a pharmaceutically acceptable salt thereof as an active ingredient.
The present disclosure is also directed to providing an anticancer tablet containing the pharmaceutical composition.
The present disclosure is also directed to providing an anticancer capsule containing the pharmaceutical composition.
The present disclosure is also directed to providing an anticancer syrup containing the pharmaceutical composition.
The present disclosure is also directed to providing a method for treating cancer, which may include administering the novel compound to a mammal.
The present disclosure is also directed to providing a pharmaceutical composition for preventing or treating smooth muscle contraction disorder, which contains the novel compound or a pharmaceutically acceptable salt thereof as an active ingredient.
The present disclosure is also directed to providing a method for treating smooth muscle contraction disorder, which may include administering the novel compound to a mammal.
The present disclosure is also directed to providing a pharmaceutical composition for preventing or treating pain, which contains the novel compound or a pharmaceutically acceptable salt thereof as an active ingredient.
The present disclosure is also directed to providing a method for treating pain, which may include administering the novel compound to a mammal.
The present disclosure is also directed to providing an ANO1 (TMEM16A) antagonist containing the novel compound.
The present disclosure is also directed to providing a method for inhibiting the expression of ANO1 (TMEM16A) by treating with the novel compound.
The anticancer pharmaceutical composition of the present disclosure may contain one or more compound selected from a group consisting of compounds represented by [Chemical Formula 1] through [Chemical Formula 4] or a pharmaceutically acceptable salt thereof as an active ingredient:
wherein
each of R1 and R2, which are identical to or different from each other, is independently selected from hydrogen, a halogen, C1-C6 alkyl and C1-C6 alkoxy;
each of R3 and R4, which are identical to or different from each other, is independently selected from hydrogen, a halogen, C1-C6 alkyl and C1-C6 alkoxy;
R5 is selected from C, N, S and O;
R6 is selected from
R7 is selected from
R8 is selected from hydrogen, a halogen, C1-C6 alkyl and C1-C6 alkoxy;
R9 is selected from
each of R10 and R11, which are identical to or different from each other, is independently selected from hydrogen, a halogen, C1-C6 alkyl, carbonyl and C1-C6 alkoxy;
each of X1 through X3, which are identical to or different from each other, is independently selected from C, N, O and S;
X4 is selected from C, N, O and S; and
each of X5 and X6, which are identical to or different from each other, is independently selected from hydrogen, a halogen, C1-C6 alkyl, carbonyl and C1-C6 alkoxy.
The one or more compound selected from a group consisting of compounds represented by [Chemical Formula 1] through [Chemical Formula 4] may have an IC50 [μM] for ANO1 of 0.01-65, specifically 0.01-0.08.
The one or more compound selected from a group consisting of compounds represented by [Chemical Formula 1] through [Chemical Formula 4] may inhibit the expression of ANO1 (TMEM16A) present in a cancer cell or a tumor cell.
The salt may be in one or more form selected from a group consisting of a hydrate, a solvate and a crystal.
A disease for which an anticancer effect is achieved by the pharmaceutical composition may be one or more selected from a group consisting of prostate cancer, thyroid cancer, stomach cancer, colon cancer, lung cancer, breast cancer, liver cancer, pancreatic cancer, testicular cancer, oral cancer, basal cell carcinoma, brain tumor, gallbladder cancer, cholangiocarcinoma, laryngeal cancer, retinoblastoma, cancer of the ampulla of Vater, bladder cancer, peritoneal cancer, adrenal cancer, non-small cell lung cancer, tongue cancer, small cell lung cancer, small intestine cancer, meningioma, esophageal cancer, renal pelvic/ureteral cancer, kidney cancer, malignant bone tumor, malignant soft tissue tumor, malignant lymphoma, malignant melanoma, eye neoplasm, urethral cancer, stomach cancer, sarcoma, pharynx cancer, cervical cancer, endometrial cancer, uterine sarcoma, metastatic brain tumor, colorectal cancer, vaginal cancer, spinal tumor, salivary gland cancer, tonsillar cancer, squamous cell carcinoma and anal cancer.
The anticancer tablet of the present disclosure may contain the pharmaceutical composition.
The anticancer capsule of the present disclosure may contain the pharmaceutical composition.
The anticancer syrup of the present disclosure may contain the pharmaceutical composition.
The method for treating cancer of the present disclosure may include administering the one or more compound selected from a group consisting of compounds represented by [Chemical Formula 1] through [Chemical Formula 4] to a mammal.
The pharmaceutical composition for preventing or treating smooth muscle contraction disorder of the present disclosure may contain the one or more compound selected from a group consisting of compounds represented by [Chemical Formula 1] through [Chemical Formula 4] or a pharmaceutically acceptable salt thereof as an active ingredient.
The smooth muscle contraction disorder may be one or more selected from a group consisting of inflammatory airway disease, hypertension and asthma.
The method for treating smooth muscle contraction disorder of the present disclosure may include administering the one or more compound selected from a group consisting of compounds represented by [Chemical Formula 1] through [Chemical Formula 4] to a mammal.
The pharmaceutical composition for preventing or treating pain disorder of the present disclosure may the one or more compound selected from a group consisting of compounds represented by [Chemical Formula 1] through [Chemical Formula 4] or a pharmaceutically acceptable salt thereof as an active ingredient.
The pain may be one or more selected from a group consisting of inflammatory nociceptive pain, inflammatory pain and neuropathic pain.
The method for treating pain of the present disclosure may include administering the one or more compound selected from a group consisting of compounds represented by [Chemical Formula 1] through [Chemical Formula 4] to a mammal.
The ANO1 (TMEM16A) antagonist of the present disclosure may be an ANO1 (TMEM16A) antagonist containing one or more compound selected from a group consisting of compounds represented by [Chemical Formula 1] through [Chemical Formula 4], and the antagonist may inhibit the expression of ANO1 (TMEM16A) in vitro.
The method for inhibiting the expression of ANO1 (TMEM16A) of the present disclosure may include a step of inhibiting the expression of ANO1 (TMEM16A) in vitro by treating with a substance containing one or more compound selected from a group consisting of compounds represented by [Chemical Formula 1] through [Chemical Formula 4].
The anticancer pharmaceutical composition of the present disclosure may selectively inhibit the activity of ANO1 (TMEM16A) over ANO2 most strongly. It can inhibit the proliferation of PC-3 cells in a dose-dependent manner and exhibits very superior effect of reducing the ANO1 protein as compared to the existing Ani9 as well as superior plasma stability.
The ANO1 (TMEM16A) is a kind of CaCC present in a cancer cell or a tumor, and the inhibition of the expression of ANO1 (TMEM16A) is known to have anticancer activity. Therefore, the anticancer pharmaceutical composition according to the present disclosure achieves anticancer effect by containing a compound effective in inhibiting the activity of ANO1 (TMEM16A). In addition, an ANO1 (TMEM16A) antagonist can be provided using a compound which inhibits the expression of ANO1 (TMEM16A).
The present disclosure relates to a novel anticancer pharmaceutical composition which is effective in inhibiting the expression of ANO1 (TMEM16A).
Hereinafter, the present disclosure is described in detail.
The novel anticancer pharmaceutical composition of the present disclosure contains one or more compound selected from a group consisting of compounds represented by [Chemical Formula 1] through [Chemical Formula 4] or a pharmaceutically acceptable salt thereof as an active ingredient:
wherein
each of R1 and R2, which are identical to or different from each other, is independently selected from hydrogen, a halogen, C1-C6 alkyl and C1-C6 alkoxy;
each of R3 and R4, which are identical to or different from each other, is independently selected from hydrogen, a halogen, C1-C6 alkyl and C1-C6 alkoxy;
R5 is selected from C, N, S and O;
R7 is selected from
R8 is selected from hydrogen, a halogen, C1-C6 alkyl and C1-C6 alkoxy;
R9 is selected from
each of R10 and R11, which are identical to or different from each other, is independently selected from hydrogen, a halogen, C1-C6 alkyl, carbonyl and C1-C6 alkoxy;
each of X1 through X3, which are identical to or different from each other, is independently selected from C, N, O and S;
X4 is selected from C, N, O and S; and
each of X5 and X6, which are identical to or different from each other, is independently selected from hydrogen, a halogen, C1-C6 alkyl, carbonyl and C1-C6 alkoxy.
The C1-C6 alkyl may be any one selected from a group consisting of methyl, ethyl, isopropyl, propyl, butyl, isobutyl, t-butyl, pentyl and hexyl, the C1-C6 alkoxy may be any one selected from a group consisting of methoxy, ethoxy, isopropoxy, propoxy, butoxy, isobutoxy, t-butoxy, pentoxy and hexoxy, and the halogen may be any one selected from a group consisting of fluorine, chlorine, bromine, iodine and astatine.
Specifically, in the one or more compound selected from a group consisting of compounds represented by [Chemical Formula 1] through [Chemical Formula 4], R6 may be any one selected from a group consisting of
R7 may be any one selected from a group consisting of
and
R9 may be any one selected from a group consisting of
Specifically, the one or more compound selected from a group consisting of compounds represented by [Chemical Formula 1] through [Chemical Formula 4]having the above-described structure may be any one selected from a group consisting of compounds represented by [Chemical Formula 5] through [Chemical Formula 18], although not being limited thereto.
In the present disclosure, the one or more compound selected from a group consisting of compounds represented by [Chemical Formula 1] through [Chemical Formula 4] has an IC50 [μM] of 0.01-65, specifically 0.01-0.08.
Specifically, the pharmaceutical composition may contain a pharmaceutically acceptable salt of the one or more compound selected from a group consisting of compounds represented by [Chemical Formula 1] through [Chemical Formula 4], although not being specially limited thereto. For example, the pharmaceutically acceptable salt may be any one if the solubility of the one or more compound selected from a group consisting of compounds represented by [Chemical Formula 1] through [Chemical Formula 4] can be increased. In a specific exemplary embodiment, it may be an acid addition salt.
The pharmaceutically acceptable salt should be less toxic to the human body and should not negatively affect the biological activity and physicochemical properties of the parent compound. A free acid that may be used to prepare the pharmaceutically acceptable salt may be classified into an inorganic acid and an organic acid. As the inorganic acid, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, perchloric acid, bromic acid, etc. may be used. As the organic acid, acetic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, fumaric acid, maleic acid, malonic acid, phthalic acid, succinic acid, lactic acid, citric acid, citric acid, gluconic acid, tartaric acid, salicylic acid, malic acid, oxalic acid, benzoic acid, embonic acid, aspartic acid, glutamic acid, etc. may be used. An organic base that may be used to prepare an organic base addition salt includes tris(hydroxymethyl)methylamine, dicylcohexylamine, etc. An amino acid that may be used to prepare an amino acid addition salt is an natural amino acid such as alanine, glycine, etc. In addition, the one or more compound selected from a group consisting of compounds represented by [Chemical Formula 1] through [Chemical Formula 4] according to the present disclosure may also include, in addition to the pharmaceutically acceptable salt, any hydrate or solvate. The hydrate or solvate may be crystallized or rectrystallized after dissolving the one or more compound selected from a group consisting of compounds represented by [Chemical Formula 1] through [Chemical Formula 4] in a water-miscible solvent such as methanol, ethanol, acetone or 1,4-dioxane and then adding a free acid or a free base. In this case, a solvate (particularly, hydrate) may be formed. Therefore, in addition to various water-containing compounds that may be prepared with the one or more compound selected from a group consisting of compounds represented by [Chemical Formula 1] through [Chemical Formula 4] of the present disclosure by a method such as lyophilization, stoichiometric solvates including hydrate may be included as well.
The one or more compound selected from a group consisting of compounds represented by [Chemical Formula 1] through [Chemical Formula 4] or a pharmaceutically acceptable salt thereof may be contained in an amount of specifically 1-80 parts by weight, more specifically 20-60 parts by weight, based on 100 parts by weight of the total composition, although not being specially limited thereto. If the content of the one or more compound selected from a group consisting of compounds represented by [Chemical Formula 1] through [Chemical Formula 4] or a pharmaceutically acceptable salt thereof is less than 1 part by weight, it is difficult to sufficiently achieve the anticancer activity to be accomplished in the present disclosure. And, if the one or more compound selected from a group consisting of compounds represented by [Chemical Formula 1] through [Chemical Formula 4] or a pharmaceutically acceptable salt thereof exceeds 80 parts by weight, the contents of other substances used to prepare a pharmaceutical product may be limited disadvantageously.
Although the administration method of the anticancer pharmaceutical composition is not specially limited, it may be administered specifically intraarterially, intravenously, subcutaneously, intrarectally, intranasally, or via any other parenteral route. More specifically, it may be administered intraarterially, intravenously, orally, or directly into muscle cells.
The administration dosage of the composition will vary depending on the activity of the compound, administration route, severity of the condition to be treated, and previous medical history. However, increasing the administration dosage gradually starting from a level lower than required to achieve the desired therapeutic effect until the desired effect is achieved is within the level of the related art, and the desirable administration dosage may be determined depending on age, sex, somatotype and body weight. The composition may be processed further until it is prepared into a pharmaceutically acceptable formulation. The formulation of the composition may be determined generally although it will vary depending on pathological conditions and patients to be treated.
A disease for which an anticancer effect is achieved by the pharmaceutical composition may be any disease for which an anticancer effect is achieved by inhibiting the expression of ANO1 (TMEM16A) present in a cancer cell or a tumor cell. Specifically, an anticancer effect may be achieved for one or more disease selected from a group consisting of prostate cancer, thyroid cancer, stomach cancer, colon cancer, lung cancer, breast cancer, liver cancer, pancreatic cancer, testicular cancer, oral cancer, basal cell carcinoma, brain tumor, gallbladder cancer, cholangiocarcinoma, laryngeal cancer, retinoblastoma, cancer of the ampulla of Vater, bladder cancer, peritoneal cancer, adrenal cancer, non-small cell lung cancer, tongue cancer, small cell lung cancer, small intestine cancer, meningioma, esophageal cancer, renal pelvic/ureteral cancer, kidney cancer, malignant bone tumor, malignant soft tissue tumor, malignant lymphoma, malignant melanoma, eye neoplasm, urethral cancer, stomach cancer, sarcoma, pharynx cancer, cervical cancer, endometrial cancer, uterine sarcoma, metastatic brain tumor, colorectal cancer, vaginal cancer, spinal tumor, salivary gland cancer, tonsillar cancer, squamous cell carcinoma and anal cancer.
A method for treating cancer of the present disclosure may include administering the one or more compound selected from a group consisting of compounds represented by [Chemical Formula 1] through [Chemical Formula 4] to a mammal.
The pharmaceutical composition of the present disclosure may prevent or treat smooth muscle contraction disorder, and the smooth muscle contraction disorder may be one or more selected from a group consisting of inflammatory airway disease, hypertension and asthma.
A method for treating smooth muscle contraction disorder of the present disclosure may include administering the one or more compound selected from a group consisting of compounds represented by [Chemical Formula 1] through [Chemical Formula 4] to a mammal.
The pharmaceutical composition of the present disclosure may prevent or treat pain, and the pain may be one or more selected from a group consisting of inflammatory nociceptive pain, inflammatory pain and neuropathic pain.
A method for treating pain of the present disclosure may include administering the one or more compound selected from a group consisting of compounds represented by [Chemical Formula 1] through [Chemical Formula 4] to a mammal.
According to recent studies, it is reported that an ANO1 inhibitor may be useful in the treatment of ANO1-related diseases such as inflammatory airway disease, hypertension and pain (Proc. Natl. Acad. Sci. USA 109(2012) 16354-16359; Nat. Neurosci. 15 (2012) 1015-1021).
The ANO1 inhibitor may provide an advantageous effect for inflammatory airway disease by inhibiting mucus hypersecretion from the airway epithelium and inhibiting the contraction of smooth muscle (Proc. Natl. Acad. Sci. U.S.A. 109 (2012) 16354-16359), and remarkably decreases the blood pressure of hypertensive mouse (Am. J. Physiol. 303 (2012) C1229-C1243; J. Mol. Cell. Cardiol. 82 (2015) 22-32). In addition, the inhibition of ANO1 activity in a mouse thermal pain model significantly reduces the pain of mouse and also reduces capsaicin-induced pain-related behavior (Proc. Natl. Acad. Sci. U.S.A. 112 (2015) 5213-5218; Mol. Pain 11 (2015 Sep. 12) 55).
Meanwhile, the pharmaceutical composition according to the present disclosure may be used as being added to a tablet, a capsule or a syrup.
A method for preparing the tablet is not particularly limited. In a specific exemplary embodiment, it is prepared by sieving a pharmaceutical composition containing the one or more compound selected from a group consisting of compounds represented by [Chemical Formula 1] through [Chemical Formula 4] or a pharmaceutically acceptable salt thereof, mixing with lactose, starch and pregelatinized corn starch, granulating the resulting powder by adding purified water, drying the resulting granule, mixing with magnesium stearate, and compressing the resulting mixture. Preferred ingredients of the tablet and their contents are 5.0 mg of the pharmaceutical composition according to the present disclosure, 150.0 mg of lactose BP, 30.0 mg of starch BP, 15.0 mg of pregelatinized corn starch BP and 1.0 mg of magnesium stearate.
A method for preparing the capsule is not particularly limited. In a specific exemplary embodiment, it is prepared by sieving a pharmaceutical composition containing the one or more compound selected from a group consisting of compounds represented by [Chemical Formula 1] through [Chemical Formula 4] or a pharmaceutically acceptable salt thereof, mixing with an excipient, and filling the resulting mixture in a gelatin capsule. Preferred ingredients of the capsule and their contents are 5.0 mg of the pharmaceutical composition according to the present disclosure, 100.0 mg of starch and 1.0 mg of magnesium stearate BP.
A method for preparing the syrup is not particularly limited. In a specific exemplary embodiment, it is prepared by dissolving white sugar in 500 mL of purified water, dissolving sodium carboxymethyl cellulose in 400 mL of purified water in a separate container, mixing the white sugar dissolved in purified water with the sodium carboxymethyl cellulose dissolved in purified water, adding and dissolving methylparaben and propylparaben, adding ethanol and then adding purified water to make the total volume of the solution 1,000 mL, and then suspending a pharmaceutical composition containing the one or more compound selected from a group consisting of compounds represented by [Chemical Formula 1] through [Chemical Formula 4] or a pharmaceutically acceptable salt thereof therein. Preferred ingredients of the capsule and their contents are 5.0 g of the pharmaceutical composition according to the present disclosure, 637.5 g of white sugar, 2.0 g of sodium carboxymethyl cellulose, 0.28 g of methylparaben, 0.12 g of propylparaben, 20 mL of ethanol and purified water as balance.
The present disclosure also provides an ANO1 (TMEM16A) antagonist.
The ANO1 (TMEM16A) antagonist of the present disclosure contains the one or more compound selected from a group consisting of compounds represented by [Chemical Formula 1] through [Chemical Formula 4].
The antagonist is an ANO1 (TMEM16A) antagonist which inhibits the activity of ANO1 (TMEM16A) in vitro.
By containing the one or more compound selected from a group consisting of compounds represented by [Chemical Formula 1] through [Chemical Formula 4], the ANO1 (TMEM16A) antagonist according to the present disclosure inhibits or decreases the activity of ANO1 (TMEM16A). As a result, the ANO1 (TMEM16A) antagonist according to the present disclosure inhibits or decreases the expression of ANO1 (TMEM16A) and may achieve an anticancer effect when applied into the body (in vivo). The inventors of the present disclosure have noticed the effect of the one or more compound selected from a group consisting of compounds represented by [Chemical Formula 1] through [Chemical Formula 4] of inhibiting the activity of ANO1 (TMEM16A), and claim the ANO1 (TMEM16A) antagonist according to the present disclosure in order to seek protection for the effect of inhibiting the activity of ANO1 (TMEM16A) in vitro under Article 2(1) of the Korean Patent Act, in addition to the medicinal invention.
The inhibition or decrease of the expression of ANO1 (TMEM16A) is achieved by the one or more compound selected from a group consisting of compounds represented by [Chemical Formula 1] through [Chemical Formula 4].
The present disclosure also provides a method for inhibiting the expression of ANO1 (TMEM16A).
The method for inhibiting the expression of ANO1 (TMEM16A) of the present disclosure includes a step of inhibiting the expression of ANO1 (TMEM16A) in vitro by treating with a substance containing the one or more compound selected from a group consisting of compounds represented by [Chemical Formula 1] through [Chemical Formula 4].
Hereinafter, specific examples will be provided to help understanding of the present disclosure. However, the following examples are for provided only as examples for illustrating the present disclosure and it will be apparent to those skilled in the art that various changes and modifications can be made within the spirit and scope of the disclosure and such changes and modifications belong to the scope of the appended claims.
2-(4-Chloro-2-methylphenoxy)acetohydrazide (100 mg, 0.47 mmol) and 2-(trifluoromethoxy)benzaldehyde (79 μL, 1.2 eq.) were added to EtOH (2.5 mL) and the resulting mixture was stirred at 80° C. for 16 hours. The precipitated product was filtered, collected and dried, affording a white solid represented by [Chemical Formula 5].
White solid, mp: 157.2-158.0° C.; 1H NMR (400 MHz, DMSO-d6) δ 11.83 (s, 1H, isomer b), 11.76 (s, 1H, isomer a), 8.57 (s, 1H, isomer b), 8.27 (s, 1H, isomer a), 8.06 (d, J=7.6 Hz, 1H, isomer a), 8.02 (d, J=8.0 Hz, 1H, isomer b), 7.62-6.54 (m, 3H, isomer b), 7.52-7.43 (m, 3H, isomer a), 7.28-7.12 (m, 3H, isomer a), 6.92-6.84 (m, 3H, isomer b), 5.2 (s, 2H, isomer a), 4.71 (s, 2H, isomer b), 2.24 (s, 3H, isomer b), 2.21 (s, 3H, isomer a); 13C NMR (100 MHz, DMSO-d6) δ 169.0, 155.2, 140.8, 137.2, 131.6, 130.1, 129.9, 128.4, 128.1, 128.0, 127.0, 126.9, 126.6, 126.4, 126.2, 124.0, 121.8, 113.1, 67.0, 65.3, 15.9; HRMS (ESI-QTOF) m/z [M+H]+ calcd for C17H15ClF3N2O3 387.0718. found 387.0719.
After adding Et3SiH (19.2 μL, 2 eq.) to a solution of Ani9 (20 mg, 0.06 mmol, Korean Patent Registration No. 1719541) in TFA (1 mL) at 0° C. and stirring for 1 hour at the same temperature, the reaction mixture was diluted with 15% aq. HCl (1 mL) and washed with hexane (1 mL). Then, the aqueous layer was cautiously basified with KOH pellets and extracted with CH2Cl2 (1 mL×2). The organic layer was dried with MgSO4 and concentrated under reduced pressure. The obtained crude residue was purified by silica gel column chromatography (hexane:ethyl acetate:dichloromethane=10:1:2, volume ratio), affording a [Chemical Formula 6] compound as a white solid.
White solid, mp: 98.2-100.6° C.; 1H NMR (400 MHz, CDCl3) δ 7.73 (br s, 1H), 7.29 (t, J=7.0 Hz, 1H), 7.18 (dd, J=1.2, 7.6 Hz, 1H), 7.14-7.09 (m, 2H), 6.93-6.87 (m, 2H), 6.56 (d, J=8.4 Hz, 1H), 5.04 (s, 1H), 4.50 (s, 2H), 4.04 (s, 2H), 3.86 (s, 3H), 2.11 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 166.8, 158.1, 154.0, 131.0, 130.9, 129.5, 128.5, 126.9, 126.8, 125.1, 120.6, 112.5, 110.6, 67.5, 55.6, 51.7, 16.3; HRMS (ESI-QTOF) m/z [M+H]+ calcd for C17H20ClN2O3 335.1157. found 335.1154.
After adding iodobenzene diacetate (32 mg, 1.1 eq.) to a solution of Ani9 (30 mg, 0.090 mmol, Korean Patent Registration No. 1719541) in CH2Cl2 (1 mL) at room temperature and stirring at room temperature for 6 hours, the reaction mixture was concentrated under vacuum. The obtained crude residue was purified by silica gel column chromatography (hexane:ethylacetate:dichloroethane=20:1:2 to 10:1:2, volume ratio), affording a [Chemical Formula 7] compound as a white solid.
White solid, mp: 104.8-105.9° C.; 1H NMR (400 MHz, CDCl3) δ 7.93 (d, J=8.0 Hz, 1H), 7.52 (t, J=8.0 Hz, 1H), 7.16-7.04 (m, 4H), 6.95 (d, J=8.8 Hz, 1H), 5.231 (s, 2H), 3.95 (s, 3H), 2.23 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 164.7, 162.0, 158.1, 154.6, 133.6, 131.0, 129.5, 126.8, 126.7, 120.9, 113.2, 112.7, 112.1, 60.7, 56.1, 16.3; HRMS (ESI-QTOF) m/z [M+Na]+ calcd for C17H15ClN2NaO3 355.0820, found 355.0822.
After adding iodobenzene diacetate (33 mg, 1.1 eq.) to a solution of
(30 mg, 0.094 mmol) in CH2Cl2 (1 mL) at room temperature and stirring at room temperature for 16 hours, the reaction mixture was concentrated under vacuum. The obtained crude residue was purified by silica gel column chromatography (hexane:ethylacetate:dichloroethane=20:1:2, volume ratio), affording a [Chemical Formula 8] compound as a white solid.
White solid, mp: 96.8-98.7° C.; 1H NMR (400 MHz, CDCl3) δ 8.08 (t, J=7.6 Hz, 1H), 7.60-7.53 (m, 1H), 7.31 (t, J=7.6 Hz, 1H), 7.26 (t, J=9.2 Hz, 1H), 7.16-7.11 (m, 2H), 6.94 (d, J=9.2 Hz, 1H), 5.34 (s, 2H), 2.24 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 162.7, 161.5, 158.9, 154.5, 134.1, 134.0, 131.1, 130.0, 129.99, 129.6, 127.0, 126.7, 124.9, 124.86, 117.3, 117.1, 113.1, 60.5, 16.2; HRMS (ESI-QTOF) m/z [M+H]+ calcd for C16H13ClFN2O2 319.0644. found 319.0641.
[Chemical Formula 9]
After stirring a mixture of 2-(4-chloro-2-methylphenoxy)acetohydrazide (100 mg, 0.47 mmol) and 2-(trifluoromethyl)benzaldehyde (74 μL, 1.2 eq.) in EtOH (2.5 mL) at 80° C. for 12 hours, the precipitated product was filtered, collected and dried, affording a [Chemical Formula 9] compound as a white solid.
White solid, mp: 158.2-160.0° C.; 1H NMR (400 MHz, DMSO-d6) δ 11.95 (s, 1H, isomer b), 11.87 (s, 1H, isomer a), 8.66 (s, 1H, isomer b), 8.36 (s, 1H, isomer a), 8.22 (d, J=8 Hz, 1H, isomer a), 8.17 (d, J=8 Hz, 1H, isomer b), 7.83-7.7 (m, 3H, isomer a), 7.67-7.58 (m, 3H, isomer b), 7.27-7.12 (m, 3H, isomer a), 6.93-6.86 (m, 3H, isomer b), 5.23 (s, 2H, isomer a), 4.72 (s, 2H, isomer b), 2.24 (s, 3H, isomer b), 2.21 (s, 3H, isomer a); 13C NMR (100 MHz, DMSO-d6) δ 169.2, 164.6, 155.2, 155.0, 139.0, 132.8, 131.8, 130.1, 130.0, 129.9, 128.4, 127.1, 126.9, 126.4, 126.2, 125.9, 124.0, 113.3, 113.1, 67.0, 65.3, 15.9; HRMS (ESI-QTOF) m/z [M+H]+ calcd for C17H15ClF3N2O2 371.0769. found 371.0765.
After stirring a mixture of 2-(4-chloro-2-methylphenoxy)acetohydrazide (100 mg, 0.47 mmol) and 1-(2-(trifluoromethoxy)phenyl)ethan-1-one (45 μL, 1.2 eq.) in EtOH (2.5 mL) at 80° C. for 12 hours, the precipitated product was filtered, collected and dried for 5 hours, affording a [Chemical Formula 10] compound as a white solid.
White solid, mp: 119.2-121.0° C.; 1H NMR (400 MHz, CDCl3) δ 9.56 (s, 1H, isomer a), 9.35 (s, 1H, isomer b), 7.67 (d, J=7.6 Hz, 1H, isomer a), 7.48 (d, J=7.6 Hz, 1H, isomer b), 7.52-7.4 (m, 3H, isomer b), 7.37-7.28 (m, 3H, isomer a), 7.23-7.15 (m, 2H, isomer a), 7.12 (s, 1H, isomer b), 7.05 (dd, J=2.0, 8.6 Hz, 1H, isomer b), 5.09 (s, 2H, isomer b), 4.69 (s, 2H, isomer a), 2.34 (s, 3H, isomer a), 2.32-2.26 (m, 3H, isomer a, b), 2.21 (s, 3H, isomer b); 13C NMR (100 MHz, CDCl3) δ 170.4, 163.7, 155.1, 153.5, 152.4, 147.8, 146.9, 146.7, 131.0, 130.8, 130.8, 130.6, 130.5, 130.0, 127.1, 127.0, 127.96, 126.2, 1120.5, 112.6, 112.4, 67.4, 66.1, 16.4, 16.3, 16.2, 16.16; HRMS (ESI-QTOF) m/z [M+H]+ calcd for C18H17ClFN2O3 401.0874. found 401.0873.
After stirring a mixture of 2-(4-chloro-2-methylphenoxy)acetohydrazide (100 mg, 0.47 mmol), ethyl 2-fluoro-2-(2-formylphenoxy)acetate (126 mg, 1.2 eq.) and AcOH (0.25 mL, solvent, 10 vol %) at 80° C. for 12 hours, the precipitated product was filtered, collected and dried, affording a [Chemical Formula 11] compound as a white solid.
The ethyl 2-fluoro-2-(2-formylphenoxy)acetate was obtained as follows. After adding K2CO3 (679 mg, 3 eq.) to a mixture of 2-hydroxybenzaldehyde (200 mg, 1.64 mmol) and ethyl 2-bromo-2-fluoroacetate (230 μL, 1.2 eq.) in acetone (6 mL) and stirring at room temperature for 3 hours, the reaction mixture was concentrated under vacuum. The obtained crude residue was extracted with an organic mixture of CH2Cl2 and H2O. After drying the organic layer on MgSO4 and concentrating under vacuum, the residue was purified by silica gel column chromatography (hexane:ethylacetate:dichloroethane=20:1:2, volume ratio), affording ethyl 2-fluoro-2-(2-formylphenoxy)acetate as a colorless oil.
White solid, mp: 100.3-101.5° C.; 1H NMR (400 MHz, CDCl3) δ 9.50 (s, 1H, isomer a), 9.03 (s, 1H, isomer b), 8.51 (s, 1H, isomer a), 8.20-8.15 (m, 1H, isomer a, b), 7.91 (d, J=7.2 Hz, isomer b), 7.47-7.40 (m, 1H, isomer a, b), 7.25-7.12 (m, 4H for isomer a, 3H for isomer b), 7.07 (dd, J=8.4 Hz, 1H, isomer b), 6.77 (d, J=8.4 Hz, 1H, isomer a), 6.71 (d, J=8.8 Hz, 1H, isomer b), 6.05-5.86 (m, 1H, isomer a, b), 5.13 (s, 2H, isomer b), 4.66 (s, 2H, isomer a), 4.41-4.32 (m, 2H, isomer a, b), 2.33 (s, 3H, isomer a), 2.30 (s, 3H, isomer b), 1.40-1.32 (m, 3H, isomer a, b); 13C NMR (100 MHz, CDCl3) δ 169.4, 164.1, 163.8, 155.1, 154.4, 153.9, 144.0, 139.5, 132.3, 132.0, 131.0, 130.7, 127.6, 127.0, 126.2, 125.0, 116.9, 116.7, 113.1, 112.7, 103.9, 103.8, 101.6, 101.56, 67.8, 66.2, 62.9, 62.8, 16.3, 16.2, 14.0, 13.99; HRMS (ESI-QTOF) m/z [M+Na]+ calcd for C20H20ClFN2NaO5 445.0937. found 445.0936.
After stirring a mixture of 2-(4-chloro-2-methylphenoxy)acetohydrazide (100 mg, 0.47 mmol) and furan-2-carbaldehyde (46 μL, 1.2 eq.) in EtOH (2.5 mL) at 80° C. for 18 hours, the precipitated product was filtered, collected and dried, affording a [Chemical Formula 12] compound as a white solid.
White solid, mp: 140.5-143.7° C.; 1H NMR (400 MHz, DMSO-d6) δ 11.57 (s, 1H, isomer a), 11.48 (s, 1H, isomer b), 8.18 (s, 1H, isomer b), 7.89 (s, 1H, isomer a), 7.86-7.81 (m, 1H, isomer a, b), 7.27-7.11 (m, 2H, isomer a, b), 6.93-6.90 (m, 1H, isomer a, b), 6.88 (d, J=8.8 Hz, 1H, isomer b), 6.8 (d, J=8.8 Hz, 1H, isomer a), 6.65-6.61 (m, 1H, isomer a, b), 5.09 (s, 2H, isomer a), 4.67 (s, 2H, isomer b), 2.23 (s, 3H, isomer b), 2.20 (s, 3H, isomer a); 13C NMR (100 MHz, DMSO-d6) δ 168.6, 164.1, 155.2, 154.9, 149.1, 149.0, 145.4, 145.1, 137.7, 134.0, 130.1, 129.9, 128.8, 128.4, 126.4, 126.3, 124.6, 124.0, 114.0, 113.8, 113.2, 113.0, 112.2, 112.19, 67.0, 65.0, 15.9; HRMS (ESI-QTOF) m/z [M+H]+ calcd for C14H14ClN2O3 293.0687. found 293.0687.
After adding iodobenzene diacetate (36 mg, 1.1 eq.) to a solution of the [Chemical Formula 12] compound (30 mg, 0.102 mmol) in CH2Cl2 (1 mL) at room temperature and then stirring at room temperature for 16 hours, the reaction mixture was concentrated under vacuum. The obtained crude residue was purified by silica gel column chromatography (hexane:ethylacetate:dichloroethane=20:1:2, volume ratio), affording a [Chemical Formula 13] compound as a white solid.
White solid, mp: 110.6-112.3° C.; 1H NMR (400 MHz, CDCl3) δ 7.66 (s, 1H), 7.21 (d, J=3.6 Hz, 1H), 7.16-7.10 (m, 2H), 6.92 (d, J=8.0 Hz, 1H), 6.62 (dd, J=3.2 Hz, 1H), 5.30 (s, 2H), 2.23 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 161.6, 158.7, 154.4, 146.2, 139.1, 131.1, 129.5, 127.0, 126.7, 115.0, 113.0, 112.4, 60.3, 16.2; HRMS (ESI-QTOF) m/z [M+H]+ calcd for C14H12ClN3O2 291.0531. found 291.0530.
After stirring a mixture of 2-(4-chloro-2-methylphenoxy)acetohydrazide (100 mg, 0.47 mmol) and picolinaldehyde (53 μL, 1.2 eq.) in EtOH (2.5 mL) at 80° C. for 12 hours, the precipitated product was filtered, collected and dried, affording a [Chemical Formula 14] compound as a white solid.
White solid, mp: 179.8-180.2° C.; 1H NMR (400 MHz, DMSO-d6) δ 11.81 (s, 1H, isomer a, b), 8.61 (s, 1H, isomer a, b), 8.31 (s, 1H, isomer b), 8.04 (s, 1H, isomer a), 8.02-7.80 (m, 2H, isomer a, b), 7.41 (s, 1H, isomer a, b), 7.28-7.10 (m, 2H, isomer a, b), 6.95-6.64 (m, 1H, isomer a, b), 5.21 (s, 2H), 4.72 (s, 2H), 2.29-2.15 (m, 3H, isomer a, b); 13C NMR (100 MHz, DMSO-d6) δ 169.1, 164.5, 155.2, 154.9, 152.8, 149.6, 149.5, 148.0, 144.3, 136.9, 136.8, 130.1, 129.9, 128.8, 128.4, 126.4, 126.3, 124.6, 124.4, 124.0, 120.0, 119.8, 113.3, 113.1, 66.9, 65.2, 15.9; HRMS (ESI-QTOF) m/z [M+H]+ calcd for C15H15ClN3O2 304.0847. found 304.0843.
After adding 2-methoxybenzylamine (42 μL, 1.2 eq.), EDC-HCl (78 mg, 1.5 eq.), DMAP (2 mg, 0.05 eq.) and Et3N (76 μL, 2 eq.) to a solution of 2-(4-chloro-2-methylphenoxy)acetic acid (53 mg, 0.26 mmol) in CH2Cl2 (1 mL) at 0° C. and stirring at 40° C. for 16 hours, the reaction mixture was diluted with CH2Cl2 (2 mL) and washed with 15% HCl (1 mL). The aqueous layer was extracted once more with CH2Cl2 (2 mL) and the organic layer was dried with MgSO4 and concentrated under reduced pressure. The obtained crude residue was purified by silica gel column chromatography (hexane:ethylacetate:dichloroethane=20:1:2 to 10:1:2, volume ratio), affording a [Chemical Formula 15] compound.
White solid, mp: 91.6-93.5° C.; 1H NMR (400 MHz, CDCl3) δ 7.31-7.24 (m, 2H), 7.20 (br s, 1H), 7.13 (s, 1H), 7.08 (d, J=8.4 Hz, 1H), 6.93 (t, J=7.4 Hz, 1H), 6.87 (d, J=8.4 Hz, 1H), 6.93 (d, J=8.8 Hz, 1H), 4.53 (d, J=6.0 Hz, 2H), 4.53 (s, 2H), 3.81 (s, 3H), 2.24 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 167.6, 157.7, 130.8, 129.8, 129.2, 128.2, 126.9, 126.5, 125.8, 120.9, 112.5, 110.3, 67.7, 55.3, 39.4, 16.2; HRMS (ESI-QTOF) m/z [M+H]+ calcd for C17H19ClNO3 320.1048. found 320.1045.
After adding 3-methoxybenzylamine (38 μL, 1.2 eq.), EDC-HCl (72 mg, 1.5 eq.), DMAP (1.5 mg, 0.05 eq.) and Et3N (70 μL, 2 eq.) to a solution of 2-(4-chloro-2-methylphenoxy)acetic acid (50 mg, 0.25 mmol) in CH2Cl2 (1 mL) at 0° C. and stirring at 40° C. for 16 hours, the reaction mixture was diluted with CH2Cl2 (2 mL) and washed with 15% HCl (1 mL). The aqueous layer was extracted once more with CH2Cl2 (2 mL) and the organic layer was dried with MgSO4 and concentrated under reduced pressure. The obtained crude residue was purified by silica gel column chromatography (hexane:ethylacetate:dichloroethane=20:1:2 to 10:1:2, volume ratio), affording a [Chemical Formula 16] compound as a white solid.
White solid, mp: 95.8-96.5° C.; 1H NMR (400 MHz, CDCl3) δ 7.26 (t, J=7.8 Hz, 1H), 7.16-7.10 (m, 2H), 6.88-6.78 (m, 4H), 6.70 (d, J=8.4 Hz, 1H), 4.55-4.51 (m, 4H), 3.78 (s, 3H), 2.20 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 168.1, 160.1, 154.1, 139.3, 131.0, 130.0, 128.5, 126.9, 119.8, 113.2, 113.2, 112.8, 68.0, 55.4, 43.1, 16.4; HRMS (ESI-QTOF) m/z [M+H]+ calcd for C17H17ClNO3 320.1048. found 320.1043.
After adding 5-chlorobenzo[d]oxazol-2-amine (50 mg, 1.2 eq.), EDC-HCl (72 mg, 1.5 eq.), DMAP (1.5 mg, 0.05 eq.) and Et3N (70 μL, 2 eq.) to a solution of 2-(4-chloro-2-methylphenoxy)acetic acid (50 mg, 0.25 mmol) in CH2Cl2 (1 mL) at 0° C. and stirring the mixture at 30° C. for 16 hours, the reaction mixture was diluted with CH2Cl2 (2 mL) and washed with 15% HCl (1 mL). The aqueous layer was extracted once more with CH2Cl2 (2 mL) and the organic layer was dried with MgSO4 and concentrated under reduced pressure. The obtained crude residue was purified by silica gel column chromatography (hexane:ethylacetate:dichloroethane=20:1:2, volume ratio), affording a [Chemical Formula 17] compound as a white solid.
White solid, mp: 165.5-168.2° C.; 1H NMR (400 MHz, CDCl3) δ 9.28 (br s, 1H), 7.61 (s, 1H), 7.42 (d, J=8.4 Hz, 1H), 7.27 (dd, J=2.0, 8.4 Hz, 1H), 7.20 (s, 1H), 7.16 (dd, J=2.0, 8.4 Hz, 1H), 6.75 (d, J=8.8 Hz, 1H), 4.75 (s, 2H), 2.34 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 165.2, 154.5, 153.5, 146.7, 131.3, 130.6, 128.7, 127.6, 127.0, 124.6, 119.2, 113.1, 111.1, 68.1, 29.7, 16.4; HRMS (ESI-QTOF) m/z [M+H]+ calcd for C16H13Cl2N2O3 351.0298. found 351.0296.
After adding 4-(ethoxycarbonyl)thiazol-2-aminium bromide (76 mg, 1.2 eq.), EDC-HCl (72 mg, 1.5 eq.), DMAP (1.5 mg, 0.05 eq.) and Et3N (70 μL, 2 eq.) to a solution of 2-(4-chloro-2-methylphenoxy)acetic acid (50 mg, 0.25 mmol) in CH2Cl2 (1 mL) at 0° C. and stirring at room temperature for 16 hours, the reaction mixture was diluted with CH2Cl2 (2 mL) and washed with 15% HCl (1 mL). The aqueous layer was extracted once more with CH2Cl2 (2 mL) and the organic layer was dried with MgSO4 and concentrated under reduced pressure. The obtained crude residue was purified by silica gel column chromatography (hexane:ethylacetate:dichloroethane=10:1:2, volume ratio), affording a [Chemical Formula 18] compound as a white solid.
White solid, mp: 154.0-155.0° C.; 1H NMR (400 MHz, CDCl3) δ 9.79 (br s, 1H), 7.90 (s, 1H), 7.19 (s, 1H), 7.13 (dd, J=2.4, 8.8 Hz, 1H), 6.72 (d, J=8.8 Hz, 1H), 4.73 (s, 2H), 4.41 (q, J=7.2 Hz, 2H), 2.31 (s, 3H), 1.4 (t, J=7.0 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ 166.9, 161.4, 156.6, 153.7, 142.1, 131.4, 129.1, 127.7, 127.0, 122.9, 113.2, 67.8, 61.7, 16.5, 14.5; HRMS (ESI-QTOF) m/z [M+Na]+ calcd for C15H15ClN2NaO4S 377.0333. found 377.0331.
White solid, mp: 193.0-196.0° C.; 1H NMR (400 MHz, DMSO-d6) δ 12.22 (s, 1H), 8.87 (s, 1H), 7.93 (s, 1H), 7.88 (d, J=7.6 Hz, 1H), 7.77-7.70 (m, 2H), 7.51 (dd, J=2.0, 8.8 Hz, 1H), 7.44 (t, J=7.8 Hz, 1H), 7.11 (d, J=8.4 Hz, 1H), 7.03 (t, J=7.6 Hz, 1H), 3.88 (s, 3H); 13C NMR (100 MHz, DMSO-d6) δ 157.9, 154.2, 152.9, 149.3, 144.3, 131.9, 128.6, 128.2, 127.1, 125.6, 122.3, 122.1, 120.8, 113.6, 111.9, 110.1, 55.7; HRMS (ESI-QTOF) m/z [M+H]+ calcd for C17H14ClN2O3 329.0687. found 329.0688.
Light yellow solid, mp: 156.4-159.0° C.; 1H NMR (400 MHz, CDCl3) δ 8.04 (d, J=7.6 Hz, 1H), 7.68 (d, J=1.6 Hz, 1H), 7.60-7.49 (m, 3H), 7.40 (dd, J=2.0, 8.8 Hz, 1H), 7.15-7.07 (m, 2H), 4.02 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 163.7, 158.2, 157.3, 154.1, 142.4, 133.7, 130.8, 129.8, 128.8, 127.4, 121.8, 121.0, 113.3, 112.4, 112.1, 109.3, 56.2; HRMS (ESI-QTOF) m/z [M+H]+ calcd for C17H12ClN2O3 327.0531. found 327.0532.
White solid, mp: 188.2-190.5° C.; 1H NMR (400 MHz, DMSO-d6) δ 11.64 (s, 1H, isomer a), 11.55 (s, 1H, isomer b), 8.26 (s, 1H, isomer b), 7.97 (s, 1H, isomer a), 7.45-7.1 (m, 4H, isomer a, b), 7.0 (s, 1H, isomer a, b), 6.95-6.75 (m, 1H, isomer a, b), 5.18 (s, 2H, isomer a) 4.69 (s, 2H, isomer b), 3.78 (s, 3H, isomer a, b), 2.30-2.13 (m, 3H, isomer a, b); 13C NMR (100 MHz, DMSO-d6) δ 169.0, 164.2, 159.5, 155.3, 154.9, 147.8, 143.7, 135.5, 135.4, 130.1, 130.0, 129.9, 128.8, 128.4, 126.4, 126.3, 124.5, 123.9, 120.1, 119.6, 116.4, 115.9, 113.2, 113.0, 111.5, 111.3, 66.9, 65.3, 55.2, 15.9; HRMS (ESI-QTOF) m/z [M+H]+ calcd for C17H18ClN2O3 333.1000. found 333.1003.
Light yellow solid, mp: 86.0-86.1° C.; 1H NMR (400 MHz, CDCl3) δ 7.64 (d, J=7.6 Hz, 1H), 7.59 (s, 1H) 7.42 (t, J=8.0 Hz, 1H), 7.17-7.07 (m, 3H), 6.94 (d, J=9.2 Hz, 1H), 5.31 (s, 2H), 3.88 (s, 3H), 2.24 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 165.9, 162.3, 160.1, 154.5, 131, 130.4, 129.5, 126.9, 126.7, 124.6, 119.6, 118.7, 113.1, 111.8, 60.6, 55.7, 16.2; HRMS (ESI-QTOF) m/z [M+H]+ calcd for C17H16ClN2O3 331.0844. found 331.0845.
White solid, mp: 200.3-201.8° C.; 1H NMR (400 MHz, DMSO-d6) δ 11.7 (s, 1H, isomer a, b), 8.29 (s, 1H, isomer b) 8.0 (s, 1H, isomer a), 7.61-7.43 (m, 3H, isomer a, b), 7.32-7.12 (m, 3H, isomer a, b), 6.91-6.85 (m, 1H, isomer a, b) 5.20 (s, 2H, isomer a), 4.70 (s, 2H, isomer b), 2.23 (s, 3H, isomer b), 2.2 (s, 3H, isomer a); 13C NMR (100 MHz, DMSO-d6) δ 169.1, 164.3, 163.6, 161.2, 155.3, 154.9, 142.4, 136.6, 130.9, 130.8, 130.1, 129.9, 128.4, 126.4, 126.3, 123.9, 123.6, 116.8, 113.1, 112.9, 112.7, 66.9, 65.3, 15.9; HRMS (ESI-QTOF) m/z [M+H]+ calcd for C16H15ClFN2O2 321.0801. found 321.0806.
White solid, mp: 85.2-87.8° C.; 1H NMR (400 MHz, CDCl3) δ 7.87 (d, J=8.0 Hz, 1H), 7.77 (d, J=9.2 Hz, 1H), 7.55-7.47 (m, 1H), 7.3-7.23 (m, 1H), 7.17-7.12 (m, 2H), 6.93 (d, J=8.4 Hz, 1H), 5.32 (s, 2H), 2.24 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 165.0, 164.96, 164.2, 162.6, 161.7, 154.5, 131.2, 131.1, 131.1, 129.5, 127.0, 126.7, 125.5, 125.4, 123.0, 122.98, 119.5, 119.3, 114.4, 114.2, 113.0, 60.5, 16.2; HRMS (ESI-QTOF) m/z [M+H]+ calcd for C16H13ClFN2O2 319.0644. found 319.0643.
White solid, mp: 70.8-72.0° C.; 1H NMR (400 MHz, CDCl3) δ 8.09-8.05 (m, 1H), 7.89-7.85 (m, 1H), 7.74-7.70 (m, 2H), 7.17-7.11 (m, 2H), 6.91 (d, J=8.4 Hz, 1H), 5.34 (s, 2H), 2.23 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 164.4, 163.5, 154.4, 132.4, 132.4, 132.1, 132.1, 132.1, 131.1, 129.6, 126.7, 112.8, 60.4, 16.1; HRMS (ESI-QTOF) m/z [M+H]+ calcd for C17H13ClF3N2O2 369.0612. found 369.0613.
Light yellow solid, mp: 85.6-86.6° C.; 1H NMR (400 MHz, CDCl3) δ 8.18 (d, J=7.2 Hz, 1H), 7.62 (t, J=7.2 Hz, 1H), 7.52-7.41 (m, 2H), 7.18-7.09 (m, 2H), 6.92 (d, J=8.0 Hz, 1H), 5.34 (s, 2H), 2.23 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 163.2, 163.0, 154.4, 146.6, 133.5, 131.0, 130.9, 129.5, 127.6, 126.9, 126.6, 122.5, 121.8, 119.2, 117.8, 112.8, 60.4, 16.0; HRMS (ESI-QTOF) m/z [M+H]+ calcd for C17H13ClF3N2O3 385.0561. found 385.0563.
White solid, mp: 176.4-179.2° C.; 1H NMR (400 MHz, DMSO-d6) δ 11.76 (s, 1H, isomer a), 11.70 (s, 1H, isomer b), 8.33 (s, 1H, isomer b), 8.03 (s, 1H, isomer a), 7.76-7.65 (m, 3H, isomer a), 7.62-7.53 (m, 3H, isomer b), 7.42 (t, J=8.4 Hz, 1H, isomer a, b), 7.28-7.12 (m, 3H, isomer a), 6.92-6.83 (m, 3H, isomer b), 5.20 (s, 2H, isomer a), 4.71 (s, 2H, isomer b), 2.24 (s, 3H, isomer b), 2.20 (s, 3H, isomer a); 13C NMR (100 MHz, DMSO-d6) δ 169.2, 164.4, 155.2, 154.9, 148.8, 146.1, 142.1, 136.6, 136.5, 131.0, 130.8, 130.1, 129.9, 128.7, 128.4, 126.4, 126.4, 126.2, 124.5, 123.9, 122.5, 122.1, 121.3, 118.8, 118.7, 113.2, 113.1, 67.0, 65.3, 15.9; HRMS (ESI-QTOF) m/z [M+H]+ calcd for C17H15ClF3N2O3 387.0718. found 387.0715.
White solid, mp: 80.3-80.8° C.; 1H NMR (400 MHz, CDCl3) δ 8.02 (d, J=8.0 Hz, 1H), 7.93 (s, 1H), 7.58 (t, J=8.0 Hz, 1H), 7.42 (d, J=8.4, 1H), 7.17-7.10 (m, 2H), 6.94 (d, J=8.0 Hz, 1H), 5.33 (s, 2H), 2.24 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 164.7, 162.7, 154.4, 149.8, 131.1, 131.0, 129.5, 127.1, 126.7, 125.5, 125.4, 124.6, 121.8, 119.7, 113.0, 60.5, 16.2; HRMS (ESI-QTOF) m/z [M+H]+ calcd for C17H13ClF3N2O3 385.0561. found 385.0568.
White solid, mp: 118.3-120.0° C.; 1H NMR (400 MHz, CDCl3) δ 8.80 (d, J=4.4 Hz, 1H), 8.27 (d, J=8.0 Hz, 1H), 7.95-7.87 (m, 1H), 7.50 (dd, J=5.2, 6.8 Hz, 1H), 7.16-7.10 (m, 2H), 6.94 (d, J=9.2 Hz, 1H), 5.35 (s, 2H), 2.23 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 164.9, 163.1, 154.3, 150.4, 143.1, 137.3, 130.9, 129.4, 126.7, 126.5, 126.2, 123.4, 112.7, 60.2, 16.1; HRMS (ESI-QTOF) m/z [M+H]+ calcd for C15H13ClN3O2 302.0691. found 302.0690.
Yellow solid, mp: 169.0-170.2° C.; 1H NMR (400 MHz, DMSO-d6) δ 11.81 (s, 1H, isomer b), 11.74 (s, 1H, isomer b), 9.70 (d, J=6.8 Hz, 1H, isomer b), 9.32 (d, J=6.4 Hz, 1H, isomer a), 9.21 (s, 1H, isomer b), 9.00 (s, 1H, isomer a), 7.73 (t, J=8 Hz, 1H, isomer a, b), 7.28-6.84 (m, 6H, isomer a, b), 5.24 (s, 2H, isomer a), 4.73 (s, 2H, isomer b), 4.38-4.27 (m, 2H, isomer a, b), 2.29-2.15 (m, 3H, isomer a, b) 1.41-1.28 (m, 3H, isomer a, b); 13C NMR (100 MHz, DMSO-d6) δ 168.4, 163.8, 155.3, 155.0, 140.1, 137.1, 134.5, 134.4, 130.1, 129.9, 128.2, 127.7, 126.4, 126.3, 121.7, 121.5, 120.2, 115.0, 113.2, 113.0, 104.1, 104.0, 66.9, 65.5, 60.5, 16.0, 14.2; HRMS (ESI-QTOF) m/z [M+H]+ calcd for C21H21ClN3O4 414.1215. found 414.1218.
Yellow solid, mp: 209.3-210.8° C.; 1H NMR (400 MHz, CDCl3) δ 13.81 (s, 1H, isomer a), 12.63 (s, 1H, isomer b), 7.83 (d, J=7.6 Hz, 2H, isomer a), 7.58 (d, J=7.2 Hz, 2H, isomer b), 7.41 (t, J=7.6 Hz, 1H, isomer a, b), 7.22-7.11 (m, 3H, isomer a, b), 6.88 (d, J=8 Hz, 1H, isomer a, b), 6.74 (d, J=8.4 Hz, 1H, isomer a, b), 5.24 (s, 2H, isomer b), 4.75 (s, 2H, isomer a), 3.27 (s, 3H, isomer a, b), 2.44 (s, 3H, isomer a), 2.31 (s, 1H, isomer b); 13C NMR (100 MHz, CDCl3) δ 166.1, 161.2, 153.9, 144.1, 138.9, 132.2, 131.2, 129.4, 126.9, 126.7, 123.7, 123.6, 122.4, 121.0, 119.5, 112.1, 109.1, 67.5, 66.0, 26.0, 25.6, 16.6, 16.4; HRMS (ESI-QTOF) m/z [M+H]+ calcd for C18H17ClN3O3 358.0953. found 358.0958.
White solid, mp: 215.4-217.5° C.; 1H NMR (400 MHz, DMSO-d6) (11.96 (s, 1H, isomer a), 10.62 (s, 1H, isomer b), 8.00-7.92 (m, 1H, isomer a, b), 7.35-7.12 (m, 3H, isomer a, b), 7.03-6.95 (m, 1H, isomer a, b), 6.90 (d, J=7.6 Hz, 2H, isomer, a), 6.83 (d, J=8.4 Hz, 2H, isomer b), 5.23 (s, 2H, isomer a), 4.76 (s, 2H, isomer b), 4.31-4.20 (m, 2H, isomer a, b), 2.89-2.80 (m, 2H, isomer a, b), 2.22 (s, 3H, isomer a, b); 13C NMR (100 MHz, DMSO-d6) δ 169.6, 164.1, 157.1, 156.9, 155.3, 154.9, 147.9, 142.8, 131.4, 131.0, 130.1, 129.9, 128.5, 128.3, 126.4, 126.2, 124.8, 124.7, 124.3, 123.9, 121.3, 120.3, 117.5, 117.4, 113.0, 66.5, 65.6, 64.5, 25.6, 25.1, 16.0; HRMS (ESI-QTOF) m/z [M+Na]+ calcd for C18H17ClN3NaO2 367.0820. found 367.0824.
White solid, mp: 167.3-170.9° C.; 1H NMR (400 MHz, CDCl3) δ 9.70 (br s, 1H), 7.84 (d, J=8.0 Hz, 1H), 7.81 (d, J=8.4 Hz, 1H), 7.47 (t, J=7.6 Hz, 1H), 7.35 (t, J=7.6 Hz, 1H), 7.20 (s, 1H), 7.15 (d, J=8.8 Hz, 1H), 6.75 (d, J=8.8 Hz, 1H), 4.74 (s, 2H), 2.35 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 166.7, 156.4, 153.7, 148.4, 131.4, 129.0, 127.1, 126.6, 124.5, 121.6, 121.5, 113.1, 67.7, 16.5; HRMS (ESI-QTOF) m/z [M+H]+ calcd for C16H14ClN2O2S 333.0459. found 333.0461.
White solid, mp: 202.8-204.0° C.; 1H NMR (400 MHz, CDCl3) δ 9.55 (br s, 1H), 7.20 (s, 1H), 7.15 (dd, J=2.0, 8.8 Hz, 1H), 6.73 (d, J=8.4 Hz, 1H), 4.72 (s, 2H), 3.87 (s, 3H), 2.66 (s, 3H), 2.33 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 166.4, 163.1, 157.8, 156.9, 153.6, 131.4, 128.9, 127.8, 127.1, 116.6, 113.2, 67.7, 52.1, 17.3, 16.5; HRMS (ESI-QTOF) m/z [M+H]+ calcd for C15H16ClN2O4S 355.0514. found 355.0513.
White solid, mp: 147.5-149.1° C.; 1H NMR (400 MHz, CDCl3) δ 9.55 (s, 1H, isomer a), 9.15 (s, 1H, isomer b), 8.38 (s, 1H, isomer a), 8.20 (d, J=7.6 Hz, 1H, isomer a), 8.02 (s, 1H, isomer b), 7.84 (d, J=7.6 Hz, 1H, isomer b), 7.49-7.41 (m, 1H, isomer a, b), 7.36-7.30 (m, 1H, isomer a, b), 7.28-7.23 (m, 1H, isomer a, b), 7.19 (s, 1H, isomer a), 7.15-7.08 (m, 1H, isomer a, b), 7.02 (dd, J=8.6 Hz, 1H, isomer b), 6.78 (d, J=8.8 Hz, 1H, isomer a), 6.63 (d, J=8.4 Hz, 1H, isomer b), 5.55 (q, J=6.8 Hz, 1H, isomer b), 4.78 (q, J=6.8 Hz, 1H, isomer a), 2.31 (s, 3H, isomer a), 2.27 (s, 3H, isomer b), 1.71 (d, J=6.8 Hz, 3H, isomer b), 1.67 (d, J=6.8 Hz, 3H, isomer a); 13C NMR (100 MHz, CDCl3) δ 173.0, 168.4, 153.7, 142.9, 132.0, 131.7, 131.3, 130.9, 129.6, 127.9, 127.4, 127.35, 127.3, 127.2, 127.1, 126.3, 126.2, 120.9, 114.8, 76.3, 71.5, 19.0, 18.1, 16.6, 16.4; HRMS (ESI-QTOF) m/z [M+H]+ calcd for C18H17ClF3N2O3 401.0874. found 401.0873.
White solid, mp: 175.8-178.2° C.; 1H NMR (400 MHz, CDCl3) δ 9.93 (s, 1H), 8.42 (s, 1H), 8.21 (dd, J=1.2, 7.6 Hz, 1H), 7.43 (t, J=7.0 Hz, 1H), 7.33 (t, J=7.6 Hz, 1H), 7.26 (m, 1H), 7.18 (d, J=2.0 Hz, 1H), 7.05 (dd, J=2.4, 8.8 Hz, 1H), 6.81 (d, J=8.8 Hz, 1H), 2.26 (s, 3H), 1.61 (s, 6H); 13C NMR (100 MHz, CDCl3) δ 171.3, 151.2, 147.8, 142.3, 132.7, 131.8, 131.3, 128.4, 127.8, 127.3, 126.6, 126.4, 121.8, 121.0, 120.9, 119.2, 82.2, 25.3, 17.1; HRMS (ESI-QTOF) m/z [M+H]+ calcd for C19H19ClF3N2O3 415.1031. found 415.1035.
White solid, mp: 167.4-170.2° C.; 1H NMR (400 MHz, CDCl3) δ 9.53 (s, 1H), 8.58 (s, 1H), 8.23 (d, J=7.6 Hz, 1H), 7.51 (t, J=7.0 Hz, 1H), 7.38 (t, J=7.6 Hz, 1H), 7.30 (d, J=8.0 Hz, 1H), 7.28-7.25 (m, 1H), 7.19 (s, 2H), 2.32 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 155.6, 155.2, 148.0, 146.0, 145.3, 133.4, 132.5, 132.1, 131.3, 127.9, 127.3, 127.0, 125.5, 123.4, 120.9, 114.6, 16.5; HRMS (ESI-QTOF) m/z [M+H]+ calcd for C17H13ClF5N2O3 423.0529. found 423.0527.
As seen from Table 1, it was confirmed that the compounds of Examples 1-14 showed superior inhibitory activity selectively for ANO1 over ANO2. In contrast, the compounds of Comparative Examples 1-19 showed no inhibitory activity for ANO1 or ANO2.
In particular, the compound of Example 1 (Chemical Formula 5), which is an acylhydrazone having a trifluoromethoxy group at the ortho position showed the strongest ANO1 inhibitory activity, whereas the compound of Comparative Example 9 (Chemical Formula 27) having a trifluoromethoxy group at the meta position was found not to inhibit ANO1. This suggests that the orientation of the functional moiety is important.
In addition, it was confirmed that the compound of Example 6 (Chemical Formula 10) showed slightly weaker inhibitory activity than the compound of Example 1 (Chemical Formula 5).
Specifically, Fischer rat thyroid (FRT) cells expressing ANO1 were differentiated by culturing in DMEM/Ham's F12 medium in Transwell for 5 days and then short-circuit current was measured using an Ussing chamber. Then, a regular HCO3− solution (120 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2.5 mM HEPES, 1 mM CaCl2, 10 mM glucose, 25 mM NaHCO3) was added to a basolateral bath of the Ussing chamber, and a luminal bath was stabilized for 20 minutes by creating a Cl− gradient by maintaining at 70 mM Cl−. Then, after treating with the [Chemical Formula 5] compound at different concentrations (0, 0.003, 0.01, 0.03, 0.1, 0.3, 1.0 μM) and carrying out reaction for 20 minutes, ATP (100 μM) was added and the inhibitory effect for Cl− secretion was measured through ANO1 activity.
And,
As seen from
In addition, it was confirmed that 100 μM of the [Chemical Formula 5] compound did not inhibit the activity of CFTR which secretes Cl−.
These results show that the [Chemical Formula 5] compound selectively inhibits the activity of ANO1, 1000 times or higher, over the activity of ANO2 or CFTR.
In
In
As seen from
In
In
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In addition, it was confirmed that, whereas Ani9 (3, 10 μM) decreased the expression level of ANO1 in PC-3 cells (prostate cancer cells) expressing ANO1 by 5.2±6.3% and 29.2±3.4%, the [Chemical Formula 5] compound (3, 10 μM) decreased the expression level of ANO1 by 59.8±4.6% and 66.5±4.7%.
This reveals that the [Chemical Formula 5] compound (3, 10 μM) reduces the expression level of ANO1 about 11.5 times and 2.28 times as compared to Ani9.
As a result of the SAR assay, it was confirmed that some new compounds including the [Chemical Formula 5] compound (IC50=22 nM) can be used as strong ANO1 inhibitors. Through selectivity analysis, it was confirmed that the [Chemical Formula 5] compound is very selective for ANO1, with 1,000 time or higher selectivity over ANO2.
This result suggests that the [Chemical Formula 5] compound is the strongest selective ANO1 inhibitor at present. The [Chemical Formula 5] compound was found to inhibit the proliferation PC-3 cells in a dose-dependent manner. In addition, it was confirmed that the [Chemical Formula 5] compound exhibits a very high effect of reducing the ANO1 protein as compared to Ani9 and shows superior plasma stability.
In
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As seen from
This reveals that, although the [Chemical Formula 10] compound and the [Chemical Formula 17] compound (10 μM) exhibit relatively weaker effect of inhibiting the activity of the ANO1 than Ani9, they reduce the expression level of ANO1 by about 1.21 times and 2.05 times, respectively.
As seen from
This reveals that the [Chemical Formula 5] compound strongly inhibits the contraction of smooth muscle by inhibiting ANO1.
Accordingly, it can be used as a therapeutic agent for hypertension, which relaxes vascular smooth muscle, or as a therapeutic agent for asthma, which relaxes respiratory smooth muscle.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2018-0112056 | Sep 2018 | KR | national |
