WO2012115226A1 - Therapeutic agent for cancer - Google Patents

Therapeutic agent for cancer Download PDF

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WO2012115226A1
WO2012115226A1 PCT/JP2012/054553 JP2012054553W WO2012115226A1 WO 2012115226 A1 WO2012115226 A1 WO 2012115226A1 JP 2012054553 W JP2012054553 W JP 2012054553W WO 2012115226 A1 WO2012115226 A1 WO 2012115226A1
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compound
cancer
carboxamide
phenyl
indazol
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PCT/JP2012/054553
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French (fr)
Japanese (ja)
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淑美 ▲徳▼田
高橋　健
康雄 渡部
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協和発酵キリン株式会社
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
    • C07D409/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings
    • C07D409/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings linked by a chain containing hetero atoms as chain links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

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  • the present invention relates to a cancer therapeutic agent containing an indazole derivative or a pharmacologically acceptable salt thereof or a hydrate thereof as an active ingredient.
  • Type I insulin-like growth factor receptor (type I-insulin-like growth factor receptor, hereinafter also referred to as IGF-1R) is a receptor-type tyrosine kinase that is very similar in structure to the insulin receptor (insulin receptor). Yes, it is a heterotetramer consisting of two extracellular ⁇ subunits and two transmembrane ⁇ subunits [EMBO Journal, 5, 2503 (1986); Annual Review ⁇ Annual Review of Biochemistry, 69, 373 (2000)]. Its ligand type I or type II insulin-like growth factor-1 or 2 binds to the ⁇ subunit, thereby activating the ⁇ subunit having the kinase domain, As a result, IGF-1R is activated.
  • IGF-1R Increased or activated expression of IGF-1R in many cancer tissues (eg, lung cancer, colon cancer, pancreatic cancer, breast cancer, prostate cancer, liver cancer, melanoma, brain tumor, multiple myeloma, leukemia, etc.) [Endocrine Reviews, 21, 215 (2000); Nature Reviews Cancer, 4, 505 (2004)]. In rare cases, amplification of a chromosome in which IGF-1R is present is known in breast cancer and melanoma [Genes Chromosomes Cancer, Vol. 11, p. 63 (1994)] .
  • IGF-1R is considered to be an effective target for cancer treatment, and IGF-1R inhibitors are considered useful as therapeutic agents for various cancers.
  • a compound having an indazole skeleton represented by the following formula (I) and having an IGF-1R inhibitory activity is known as an antitumor agent (Patent Document 1).
  • R 1 represents cyano, —NR 4 R 5 (wherein R 4 represents a hydrogen atom, R 5 represents a substituted or unsubstituted lower alkyl, etc.) and the like, R 2 and R 3 Are the same or different and each represents a hydrogen atom, a halogen, a substituted or unsubstituted heterocyclic group, etc.]
  • An object of the present invention is to provide a therapeutic agent for cancer selected from colorectal cancer, lung cancer, sarcoma, and pancreatic cancer containing an indazole derivative or a pharmacologically acceptable salt thereof or a hydrate thereof as an active ingredient. It is to provide.
  • the present invention relates to the following (1) to (8).
  • a therapeutic agent for cancer selected from colorectal cancer, lung cancer, sarcoma, and pancreatic cancer, comprising thiophene-2-carboxamide or a pharmacologically acceptable salt thereof or a hydrate thereof as an active ingredient.
  • p-toluenesulfonic acid salt means para-toluenesulfonic acid salt, and may be described as tosylate salt hereinafter.
  • certain indazole derivatives or pharmacologically acceptable salts thereof or hydrates thereof such as (E) -N- ⁇ 5- [4- (2-hydroxyacetyl) piperazin-1-ylmethyl ] -2- [2- (1H-indazol-3-yl) vinyl] phenyl ⁇ -3-methylthiophene-2-carboxamide or its p-toluenesulfonate or its hydrate as an active ingredient
  • a therapeutic agent for cancer selected from lung cancer, sarcoma, and pancreatic cancer is provided.
  • compound (Ia) (Hereinafter also referred to as compound (Ia)), and compound (Ia) has been reported to have a potent IGF-1R inhibitory activity (WO 2006/080450 pamphlet). ).
  • the pharmacologically acceptable salt of carboxamide include pharmacologically acceptable acid addition salts, metal salts, ammonium salts, organic amine addition salts, amino acid addition salts and the like.
  • Acid addition salts include inorganic acid salts such as hydrochloride, sulfate, phosphate, acetate, trifluoroacetate, maleate, fumarate, tartrate, citrate, lactate, aspartate, Examples include organic acid salts such as glutamate and p-toluenesulfonate (tosylate).
  • metal salts include alkali metal salts such as sodium salt and potassium salt, and alkaline earth metal salts such as magnesium salt and calcium salt.
  • ammonium salts include salts such as ammonium and tetramethylammonium
  • organic amine addition salts include addition salts such as morpholine and piperidine
  • amino acid addition salts include lysine.
  • addition salts such as glycine and phenylalanine.
  • Test Example 1 In vitro growth inhibition test by tosylate of compound (Ia) in human colorectal cancer cell line Human colorectal cancer cell line HT-29 and its green fluorescent protein (hereinafter referred to as GFP) )
  • GFP green fluorescent protein
  • McCOY's 5A medium containing 1 vol% or 10 vol% inactivated fetal bovine serum (hereinafter referred to as FBS) (Invitrogen) was used. All cell lines were cultured and grown at 37 ° C. in a 5% carbon dioxide incubator.
  • FBS inactivated fetal bovine serum
  • All cell lines were cultured and grown at 37 ° C. in a 5% carbon dioxide incubator.
  • HT-29 strain or HT-29 / GFP at 1 ⁇ 10 3 / well when 10 vol% FBS is added to 96-well F bottom plate (Nalge Nunc), 5 ⁇ 10 3 / well when 1 vol% FBS is added And cultured at 37 ° C. for 24 hours in a 5% carbon dioxide incubator.
  • a solution containing the tosylate salt of compound (Ia) prepared by serial dilution with dimethyl sulfoxide (hereinafter referred to as DMSO) was added (the final concentration of DMSO in the well was 0.1 vol%), and again 5
  • the cells were cultured at 37 ° C. for 72 hours in a% carbon dioxide incubator.
  • cell growth reagent WST-1 (4- [3- (4-Iodophenyl) -2- (4-nitrophenyl) -2H-5-tetrazolio] -1,3-benzene disulfonate, Roche Diagnostics) and react for 2 hours
  • the absorbance (450 nm) reflecting the number of viable cells in the well was measured.
  • Tosylate of compound (Ia) is HT-29 derived from human colorectal cancer and its GFP expressing strain HT-29 / GFP, both in the presence of 1% FBS, in the presence of 45 nmol / L, 10% FBS.
  • GI 50 values of 143 and 272 nmol / L, respectively, indicate that the tosylate salt of Compound (Ia) has a growth-inhibiting effect on colon cancer cells and is useful as a therapeutic agent for colon cancer. It was suggested that there is.
  • Test Example 2 In vitro growth inhibition test by tosylate of compound (Ia) in human lung cancer cell lines Compound (Ia) against human lung cancer cell lines NCI-H526, NCI-H727, NCI-H358 and CaLu-6
  • the GI 50 value which is an indicator of the concentration showing 50% growth inhibition by tosylate, was calculated by the following method.
  • NCI-H526, NCI-H727 and NCI-H358 cultures were 1 vol% or 10 vol% FBS (Invitrogen), 10 mmol / L HEPES (Invitrogen), 1 mmol / L pyruvate (Invitrogen), and 4.5 g / Roswell Park Memorial Institute's Medium (RPMI) 1640 medium (Invitrogen) containing L glucose (Sigma) was used.
  • Modified Eagle Medium (MEM) medium (Invitrogen) containing 1 vol% or 10 vol% FBS and 0.1 mmol / L Non-Essential Amino Acids (Invitrogen) was used. All cell lines were cultured and grown at 37 ° C. in a 5% carbon dioxide incubator.
  • 96 well F bottom plate or 96 well U bottom plate NCI-H526 8 ⁇ 10 3 / well, NCI-H727 5 ⁇ 10 3 / well, NCI-H358 and CaLu-6 2 ⁇ 10 3 cells / well (both 10% FBS and 1% FBS) were seeded and cultured at 37 ° C. for 24 hours in a 5% carbon dioxide incubator.
  • the tosylate salt of compound (Ia) was found in lung cancer cells. It has been shown to have a growth-inhibiting effect on the lung cancer, suggesting that it is useful as a lung cancer therapeutic agent.
  • Test Example 3 In vitro growth inhibition test by tosylate of compound (Ia) in human sarcoma cell line TC-71, SK- which are human sarcoma cell lines with EWS / FLI-1 translocation ES-1, SK-N-MC and A-673, EWS / FLI-1 RD and RH-30 without translocation are indicators of concentration indicating 50% growth inhibition by tosylate of compound (Ia) A certain GI 50 value was calculated by the following method.
  • Iscove's Modified Dulbecco's Medium (IMDM) medium (Invitrogen) containing 1 vol% or 10 vol% FBS (Invitrogen) was used.
  • MaCOY's 5A medium (Invitrogen) containing 1 vol% or 15 vol% FBS was used for the culture of SK-ES-1.
  • RPMI1640 medium containing 10 mmol / L HEPES containing 1 vol% or 10 vol% FBS, 1 mmol / L pyruvate, and 4.5 g / L glucose was used for SK-N-MC culture.
  • Dulbecco's Modified Eagle Medium (DMEM) medium (Invitrogen) containing 1 vol% or 10 vol% FBS and 4.5 g / L glucose was used.
  • RPMI1640 medium (Invitrogen) containing 1 vol% or 10 vol% FBS was used for RH-30 culture. All cell lines were cultured and grown at 37 ° C. in a 5% carbon dioxide incubator.
  • TC-71 is 5 ⁇ 10 2 / well (10% FBS) or 2 ⁇ 10 3 / well (1% FBS)
  • RD is 1 ⁇ 10 3 / well ( 10 vol% FBS) or 2 ⁇ 10 3 / well (1 vol% FBS)
  • A-673 is 2 ⁇ 10 3 / well (10 vol% FBS) or 5 ⁇ 10 3 / well (1 vol% FBS)
  • SK-ES-1 is 8 ⁇ 10 3 / well (15 vol%, 1 vol% FBS)
  • SK-N-MC is 8 ⁇ 10 3 / well (10 vol% FBS, 1 vol% FBS) RH-30 was seeded at 2 ⁇ 10 3 cells / well (both 10% FBS and 1% FBS) and cultured at 37 ° C.
  • the GI 50 value was calculated as the concentration showing the growth inhibition rate.
  • Table 3 shows the GI 50 value of the tosylate salt of compound (Ia) in each cell.
  • the tosylate salt of compound (Ia) is TC-71, SK-N-MC and A-673 derived from human sarcoma with EWS / FLI-1 translocation, respectively, under 1 vol% FBS conditions. GI 50 values of ⁇ 3.2, ⁇ 3.2 and 20 nmol / L were shown.
  • GI 50 values were 11, 3.2 and 21 nmol / L, respectively.
  • a GI 50 value of 10 nmol / L was exhibited under 10 vol% FBS conditions.
  • RD and RH-30 without EWS / FLI-1 translocation show GI 50 values of 22 and 30 nmol / L, respectively, under 1 vol% FBS conditions, and under 10 vol% FBS conditions.
  • GI 50 values were 220 and 130 nmol / L, respectively, indicating that the tosylate salt of Compound (Ia) has a growth-inhibiting effect on sarcoma, and treated with sarcoma. It was suggested that it is useful as an agent.
  • Test Example 4 Inhibition of proliferation of compound (Ia) by tosylate in human pancreatic cancer cell line in vitro Growth inhibition of compound (Ia) by tosylate against human pancreatic cancer cell lines KP-3L and AsPC-1
  • the GI 50 value which is an index of concentration indicating 50%, was calculated by the following method.
  • RPMI Roswell Park Memorial Institute's Medium
  • Invitrogen containing 1 vol% or 10 vol% FBS (Invitrogen) was used.
  • the tosylate salt of Compound (Ia) has an inhibitory effect on the growth of pancreatic cancer cells. It was shown to be useful as a therapeutic agent for pancreatic cancer.
  • Test Example 5 Antitumor effect of compound (Ia) tosylate in human colon cancer cell line HT-29 / GFP transplanted mouse model HT-29 / GFP, a GFP expression strain of human colon cancer cell line HT-29
  • HT-29 / GFP transferred from Anticancer
  • McCOY's 5A medium Invitrogen
  • FBS Invitrogen
  • penicillin G 100 units / mL
  • streptomycin Invitrogen
  • the major axis and minor axis of the tumor grown subcutaneously with calipers 6 days after the transplantation were measured, and the tumor volume was determined according to the following formula.
  • Each group was divided into the following administration groups so that the average tumor volume and average body weight were uniform, and administration was started on this day as the start of the administration test.
  • Tosylate of compound (Ia) is suspended in 0.5 w / v% methylcellulose solution (Wako Pure Chemical Industries, Ltd.) at a concentration of 4 mg / mL, once a day from the start of administration to day 13 once daily
  • Oral administration was performed using an oral sonde at a dose of 0.01 mL (40 mg / kg) per gram of body weight.
  • Negative control group (Control): 0.5 w / v% methylcellulose solution
  • Compound (Ia) tosylate administration group 40 mg / kg (once a day x 14 days) From day 0, tumor volume was measured twice a week. The determination of the antitumor effect was performed by calculating the average value of the tumor volumes of each group and comparing the change in tumor volume (V / V 0 ) when the tumor volume on day 0 was V 0 .
  • FIG. 1 shows V / V 0 of each group measured over time.
  • (E) -N- ⁇ 5- [4- (2-hydroxyacetyl) piperazin-1-ylmethyl] -2- [2- (1H-indazol-3-yl) [Vinyl] phenyl ⁇ -3-methylthiophene-2-carboxamide or a pharmacologically acceptable salt thereof or a hydrate thereof is a treatment of cancer selected from colorectal cancer, lung cancer, sarcoma, and pancreatic cancer It was suggested that it is useful as an agent.
  • compound (Ia) or a pharmacologically acceptable salt thereof or a hydrate thereof can be administered alone as it is, it is usually desirable to provide it as various pharmaceutical preparations. These pharmaceutical preparations are used for animals or humans.
  • the pharmaceutical preparation according to the present invention may contain Compound (Ia) or a pharmacologically acceptable salt thereof as an active ingredient alone or as a mixture with any other active ingredient for treatment.
  • These pharmaceutical preparations are well known in the technical field of pharmaceutics by mixing the active ingredient together with one or more pharmacologically acceptable carriers (for example, diluents, solvents, excipients, etc.). It is produced by any known method.
  • oral or parenteral such as intravenous administration
  • examples of the dosage form include tablets and injections.
  • tablets suitable for oral administration can be produced using excipients such as lactose, disintegrants such as starch, lubricants such as magnesium stearate, binders such as hydroxypropylcellulose, and the like.
  • an injection suitable for parenteral administration can be produced using a diluent or a solvent such as a salt solution, a glucose solution, or a mixed solution of a saline solution and a glucose solution.
  • a diluent or a solvent such as a salt solution, a glucose solution, or a mixed solution of a saline solution and a glucose solution.
  • the dose and frequency of administration of compound (Ia) or a pharmacologically acceptable salt thereof or a hydrate thereof vary depending on the administration form, patient age, body weight, nature or severity of symptoms to be treated, etc. However, in the case of oral administration, it is usually administered once or several times a day in the range of 0.01 to 1000 mg, preferably 0.05 to 100 mg per adult. In the case of parenteral administration such as intravenous administration, 0.001 to 1000 mg, preferably 0.01 to 100 mg, is usually administered once or several times a day for each adult. However, the dose and the number of doses vary depending on the various conditions described
  • Comparative Example 1 Calculation Test for Human Liver Metabolic Clearance Pooled human liver microsomes (derived from 50 donors) were used in this study. A solution containing 0.2 mg protein / mL human liver microsome (Xenotech), 1 mmol / L ⁇ -NADPH (Wako Pure Chemical Industries) and 1 ⁇ mol / L test compound (containing 0.01 vol% DMSO and 0.5 vol% acetonitrile) was reacted at 37 ° C. for 30 minutes. Two volumes of acetonitrile were added and centrifuged (approximately 3000 ⁇ g, 5 minutes). The obtained supernatant was analyzed by LC / MS (Measurement A).
  • human serum albumin (Sigma Aldrich) having the same protein concentration was used instead of human liver microsome, and the same measurement as described above was performed (Measurement B).
  • the peak area ratio of the test compound of measurement A to measurement B was multiplied by 100 to calculate the residual ratio (%) of the test compound.
  • the reaction residual ratio at 0 min (100%) and the residual ratio in the reaction 30 minutes to natural-log transformed, an inclination obtained by plotting was elimination rate constant (k e) the reaction time.
  • the human liver metabolic intrinsic clearance (CL int ) was calculated by the following formula.
  • liver Ms protein amount / liver weight is 32.3 mg / g (contents described in Xenobiotica, Vol. 18, page 849 (1988)), liver weight / body weight is 25.7 g / kg (Pharmceutical Research, 10 Volume, page 1093 (1993)).
  • Table 5 shows CL ints of compounds (Ia), (II), (III) and (IV) calculated as described above. This result suggests that compound (Ia) has a lower CL int and is more metabolically stable than other compounds (II) to (IV) having similar chemical structures. In general, a compound serving as an active ingredient of a medicine is required to have little toxicity (side effects) and excellent pharmacokinetics. Therefore, it is considered that the compound (Ia) having a low CL int is superior as an active ingredient of a medicine compared with other compounds (II) to (IV).
  • Tablet (Compound (Ia) A tablet having the following composition is prepared by a conventional method.
  • Compound (Ia) 40 g, lactose 286.8 g and potato starch 60 g are mixed, and 10% aqueous solution of hydroxypropylcellulose 120 g is added thereto.
  • the obtained mixture is kneaded by a conventional method, granulated and dried, and then sized to obtain granules for tableting.
  • Formulation Compound (Ia) 20 mg Lactose 143.4 mg
  • Potato starch 30 mg Hydroxypropylcellulose 6 mg
  • Magnesium stearate 0.6 mg 200 mg
  • Compound (Ia) An injection having the following composition is prepared by a conventional method. Compound (Ia), 1 g, is added to and mixed with distilled water for injection. Further, hydrochloric acid and aqueous sodium hydroxide solution are added to adjust the pH to 7, and the total volume is made up to 1000 mL with distilled water for injection. The obtained mixture is aseptically filled into glass vials by 2 mL to obtain an injection (containing 2 mg of active ingredient per vial).
  • -Boc represents a tert-butoxycarbonyl group (-COC (CH 3 ) 3 ).
  • ⁇ Step 1> (E) -N- ⁇ 5-hydroxymethyl-2- [2- (1H-indazol-3-yl) vinyl] phenyl ⁇ -3-methylthiophene-2-carboxamide obtained in Example 108 of WO2006 / 080450 ( Compound 4, 1.8 g, 4.6 mmol) was dissolved in N, N-dimethylformamide (18 mL), methanesulfonyl chloride (0.68 mL, 8.8 mmol) was added, and the mixture was stirred at room temperature for 4 hours.
  • -Boc represents a tert-butoxycarbonyl group (-COC (CH 3 ) 3 ).
  • Compound 5 (4.0 g, 6.7 mmol) obtained in Reference Example 1 was added to a mixture of methanol (0.16 L), water (32 mL), and concentrated hydrochloric acid (8 mL), and the mixture was stirred at 70 ° C. for 1.5 hours.
  • the reaction solution was cooled to 4 ° C, 4 mol / L aqueous sodium hydroxide solution (26 mL) was slowly added to adjust to pH 11, methanol was distilled off under reduced pressure, ethanol (40 mL) was added, Water (40 mL) was added dropwise.
  • Acetoxyacetyl chloride (0.141 mL, 1.31 mmol, 1.1 equivalents) was added to a solution of compound 6 (500 mg, 1.09 mmol) obtained in Reference Example 2 in N, N-dimethylacetamide (10 mL), and the mixture was stirred for 30 minutes. Water (60 mL) and saturated aqueous sodium hydrogen carbonate solution (80 mL) were added, and the mixture was extracted with ethyl acetate (100 mL). The organic layer was washed with saturated brine (100 mL), and dried over anhydrous sodium sulfate.
  • a therapeutic agent for cancer containing an indazole derivative or a pharmacologically acceptable salt thereof or a hydrate thereof as an active ingredient can be provided.

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Abstract

Provided is a therapeutic agent for cancer that is selected from among colon cancer, lung cancer, sarcoma and pancreatic cancer, said therapeutic agent comprising, as the active ingredient, (E)-N-{5-[4-(2-hydroxyacetyl)piperazin-1-ylmethyl]-2-[2-(1H-indazol-3-yl)vinyl]phenyl}-3-methylthiophene-2-carboxamide, a pharmacologically acceptable salt thereof or a hydrate of the same.

Description

癌治療剤Cancer treatment
　本発明は、インダゾール誘導体もしくはその薬理学的に許容される塩またはその水和物を有効成分として含有する癌治療剤に関する。 The present invention relates to a cancer therapeutic agent containing an indazole derivative or a pharmacologically acceptable salt thereof or a hydrate thereof as an active ingredient.
　I型インスリン様増殖因子受容体(type I insulin-like growth factor receptor、以下、IGF-1Rと表すこともある)は、インスリン受容体(insulin receptor)に構造が非常に類似した受容体型チロシンキナーゼであり、2個の細胞外αサブユニットと2個の細胞膜貫通型βサブユニットからなるヘテロ4量体である[エンボ・ジャーナル(EMBO Journal)、5巻、2503頁(1986年);　アニュアル・レビュー・オブ・バイオケミストリー(Annual Review of Biochemistry)、69巻、373頁(2000年)]。そのリガンドであるI型またはII型のインスリン様増殖因子(insulin-like growth factor-1, 2)が、そのαサブユニットに結合することにより、キナーゼドメインをもつβサブユニットが活性化され、その結果、IGF-1Rが活性化される。 Type I insulin-like growth factor receptor (type I-insulin-like growth factor receptor, hereinafter also referred to as IGF-1R) is a receptor-type tyrosine kinase that is very similar in structure to the insulin receptor (insulin receptor). Yes, it is a heterotetramer consisting of two extracellular α subunits and two transmembrane β subunits [EMBO Journal, 5, 2503 (1986); Annual Review・ Annual Review of Biochemistry, 69, 373 (2000)]. Its ligand type I or type II insulin-like growth factor-1 or 2 binds to the α subunit, thereby activating the β subunit having the kinase domain, As a result, IGF-1R is activated.
　多くの癌組織(例えば、肺癌、大腸癌、膵癌、乳癌、前立腺癌、肝癌、黒色腫、脳腫瘍、多発性骨髄腫、白血病等)で、IGF-1Rの発現の上昇または活性化がみられることが知られている[エンドクリン・レビューズ(Endocrine Reviews)、21巻、215頁(2000年);　ネイチャー・レビューズ・キャンサー(Nature Reviews Cancer)、4巻、505頁(2004年)]。また、稀ではあるが、乳癌および黒色腫において、IGF-1Rが存在する染色体の増幅が知られている[ジーンズ・クロモソームズ・キャンサー(Genes Chromosomes Cancer)、11巻、63頁(1994年)]。 Increased or activated expression of IGF-1R in many cancer tissues (eg, lung cancer, colon cancer, pancreatic cancer, breast cancer, prostate cancer, liver cancer, melanoma, brain tumor, multiple myeloma, leukemia, etc.) [Endocrine Reviews, 21, 215 (2000); Nature Reviews Cancer, 4, 505 (2004)]. In rare cases, amplification of a chromosome in which IGF-1R is present is known in breast cancer and melanoma [Genes Chromosomes Cancer, Vol. 11, p. 63 (1994)] .
　故に、IGF-1Rは、癌治療の有効な標的と考えられ、IGF-1R阻害薬は様々な癌の治療薬として有用であると考えられる。
　下記の式(I)で示されるインダゾール骨格を有し、IGF-1R阻害活性を有する化合物が抗腫瘍剤として知られている(特許文献1)。 Therefore, IGF-1R is considered to be an effective target for cancer treatment, and IGF-1R inhibitors are considered useful as therapeutic agents for various cancers.
A compound having an indazole skeleton represented by the following formula (I) and having an IGF-1R inhibitory activity is known as an antitumor agent (Patent Document 1).
Figure JPOXMLDOC01-appb-C000001
Figure JPOXMLDOC01-appb-C000001
[式中、R1はシアノ、-NR4R5 (式中、R4は水素原子等を表し、R5は置換もしくは非置換の低級アルキル等を表す)等を表し、R2およびR3は同一または異なって、水素原子、ハロゲン、置換もしくは非置換の複素環基等を表す] [Wherein R 1 represents cyano, —NR 4 R 5 (wherein R 4 represents a hydrogen atom, R 5 represents a substituted or unsubstituted lower alkyl, etc.) and the like, R 2 and R 3 Are the same or different and each represents a hydrogen atom, a halogen, a substituted or unsubstituted heterocyclic group, etc.]
国際公開第2006／080450号パンフレットInternational Publication No. 2006/080450 Pamphlet
　本発明の目的は、インダゾール誘導体もしくはその薬理学的に許容される塩またはその水和物を有効成分として含有する大腸癌、肺癌、サルコーマ(sarcoma)、および膵臓癌から選ばれる癌の治療剤を提供することにある。 An object of the present invention is to provide a therapeutic agent for cancer selected from colorectal cancer, lung cancer, sarcoma, and pancreatic cancer containing an indazole derivative or a pharmacologically acceptable salt thereof or a hydrate thereof as an active ingredient. It is to provide.
　本発明は、以下の(1)～(8)に関する。
(1) (E)-N-{5-[4-(2-ヒドロキシアセチル)ピペラジン-1-イルメチル]-2-[2-(1H-インダゾール-3-イル)ビニル]フェニル}-3-メチルチオフェン-2-カルボキサミドもしくはその薬理学的に許容される塩またはその水和物を有効成分として含有する、大腸癌、肺癌、サルコーマ(sarcoma)、および膵臓癌から選ばれる癌の治療剤。
(2) (E)-N-{5-[4-(2-ヒドロキシアセチル)ピペラジン-1-イルメチル]-2-[2-(1H-インダゾール-3-イル)ビニル]フェニル}-3-メチルチオフェン-2-カルボキサミドもしくはその薬理学的に許容される塩またはその水和物が、(E)-N-{5-[4-(2-ヒドロキシアセチル)ピペラジン-1-イルメチル]-2-[2-(1H-インダゾール-3-イル)ビニル]フェニル}-3-メチルチオフェン-2-カルボキサミドもしくはそのp-トルエンスルホン酸塩またはその水和物である(1)記載の癌の治療剤。
(3) (E)-N-{5-[4-(2-ヒドロキシアセチル)ピペラジン-1-イルメチル]-2-[2-(1H-インダゾール-3-イル)ビニル]フェニル}-3-メチルチオフェン-2-カルボキサミドもしくはそのp-トルエンスルホン酸塩またはその水和物が、(E)-N-{5-[4-(2-ヒドロキシアセチル)ピペラジン-1-イルメチル]-2-[2-(1H-インダゾール-3-イル)ビニル]フェニル}-3-メチルチオフェン-2-カルボキサミドもしくはそのp-トルエンスルホン酸塩である(2)記載の癌の治療剤。
(4) (E)-N-{5-[4-(2-ヒドロキシアセチル)ピペラジン-1-イルメチル]-2-[2-(1H-インダゾール-3-イル)ビニル]フェニル}-3-メチルチオフェン-2-カルボキサミドもしくはそのp-トルエンスルホン酸塩が(E)-N-{5-[4-(2-ヒドロキシアセチル)ピペラジン-1-イルメチル]-2-[2-(1H-インダゾール-3-イル)ビニル]フェニル}-3-メチルチオフェン-2-カルボキサミドのp-トルエンスルホン酸塩である(3)記載の癌の治療剤。 The present invention relates to the following (1) to (8).
(1) (E) -N- {5- [4- (2-Hydroxyacetyl) piperazin-1-ylmethyl] -2- [2- (1H-indazol-3-yl) vinyl] phenyl} -3-methyl A therapeutic agent for cancer selected from colorectal cancer, lung cancer, sarcoma, and pancreatic cancer, comprising thiophene-2-carboxamide or a pharmacologically acceptable salt thereof or a hydrate thereof as an active ingredient.
(2) (E) -N- {5- [4- (2-hydroxyacetyl) piperazin-1-ylmethyl] -2- [2- (1H-indazol-3-yl) vinyl] phenyl} -3-methyl Thiophene-2-carboxamide or a pharmacologically acceptable salt thereof or a hydrate thereof is (E) -N- {5- [4- (2-hydroxyacetyl) piperazin-1-ylmethyl] -2- [ The therapeutic agent for cancer according to (1), which is 2- (1H-indazol-3-yl) vinyl] phenyl} -3-methylthiophene-2-carboxamide or a p-toluenesulfonic acid salt thereof or a hydrate thereof.
(3) (E) -N- {5- [4- (2-hydroxyacetyl) piperazin-1-ylmethyl] -2- [2- (1H-indazol-3-yl) vinyl] phenyl} -3-methyl Thiophene-2-carboxamide or its p-toluenesulfonate or hydrate thereof is (E) -N- {5- [4- (2-hydroxyacetyl) piperazin-1-ylmethyl] -2- [2- The therapeutic agent for cancer according to (2), which is (1H-indazol-3-yl) vinyl] phenyl} -3-methylthiophene-2-carboxamide or a p-toluenesulfonate thereof.
(4) (E) -N- {5- [4- (2-hydroxyacetyl) piperazin-1-ylmethyl] -2- [2- (1H-indazol-3-yl) vinyl] phenyl} -3-methyl Thiophene-2-carboxamide or its p-toluenesulfonate is (E) -N- {5- [4- (2-hydroxyacetyl) piperazin-1-ylmethyl] -2- [2- (1H-indazole-3 The therapeutic agent for cancer according to (3), which is p-toluenesulfonate of -yl) vinyl] phenyl} -3-methylthiophene-2-carboxamide.
(5) (E)-N-{5-[4-(2-ヒドロキシアセチル)ピペラジン-1-イルメチル]-2-[2-(1H-インダゾール-3-イル)ビニル]フェニル}-3-メチルチオフェン-2-カルボキサミドのp-トルエンスルホン酸塩を有効成分として含有する大腸癌の治療剤。
(6) (E)-N-{5-[4-(2-ヒドロキシアセチル)ピペラジン-1-イルメチル]-2-[2-(1H-インダゾール-3-イル)ビニル]フェニル}-3-メチルチオフェン-2-カルボキサミドのp-トルエンスルホン酸塩を有効成分として含有する肺癌の治療剤。
(7) (E)-N-{5-[4-(2-ヒドロキシアセチル)ピペラジン-1-イルメチル]-2-[2-(1H-インダゾール-3-イル)ビニル]フェニル}-3-メチルチオフェン-2-カルボキサミドのp-トルエンスルホン酸塩を有効成分として含有するサルコーマ(sarcoma)の治療剤。
(8) (E)-N-{5-[4-(2-ヒドロキシアセチル)ピペラジン-1-イルメチル]-2-[2-(1H-インダゾール-3-イル)ビニル]フェニル}-3-メチルチオフェン-2-カルボキサミドのp-トルエンスルホン酸塩を有効成分として含有する膵臓癌の治療剤。 (5) (E) -N- {5- [4- (2-hydroxyacetyl) piperazin-1-ylmethyl] -2- [2- (1H-indazol-3-yl) vinyl] phenyl} -3-methyl A therapeutic agent for colorectal cancer comprising p-toluenesulfonate of thiophene-2-carboxamide as an active ingredient.
(6) (E) -N- {5- [4- (2-hydroxyacetyl) piperazin-1-ylmethyl] -2- [2- (1H-indazol-3-yl) vinyl] phenyl} -3-methyl A therapeutic agent for lung cancer comprising p-toluenesulfonate of thiophene-2-carboxamide as an active ingredient.
(7) (E) -N- {5- [4- (2-hydroxyacetyl) piperazin-1-ylmethyl] -2- [2- (1H-indazol-3-yl) vinyl] phenyl} -3-methyl A therapeutic agent for sarcoma containing p-toluenesulfonate of thiophene-2-carboxamide as an active ingredient.
(8) (E) -N- {5- [4- (2-hydroxyacetyl) piperazin-1-ylmethyl] -2- [2- (1H-indazol-3-yl) vinyl] phenyl} -3-methyl A therapeutic agent for pancreatic cancer comprising p-toluenesulfonate of thiophene-2-carboxamide as an active ingredient.
　なお、p-トルエンスルホン酸塩は、パラ-トルエンスルホン酸塩を意味し、以下、トシル酸塩と表記することもある。 In addition, p-toluenesulfonic acid salt means para-toluenesulfonic acid salt, and may be described as tosylate salt hereinafter.
　本発明により、特定のインダゾール誘導体またはその薬理学的に許容される塩またはそれらの水和物、例えば、(E)-N-{5-[4-(2-ヒドロキシアセチル)ピペラジン-1-イルメチル]-2-[2-(1H-インダゾール-3-イル)ビニル]フェニル}-3-メチルチオフェン-2-カルボキサミドもしくはそのp-トルエンスルホン酸塩またはその水和物を有効成分として含有する大腸癌、肺癌、サルコーマ(sarcoma)、および膵臓癌から選ばれる癌の治療剤等が提供される。 According to the present invention, certain indazole derivatives or pharmacologically acceptable salts thereof or hydrates thereof, such as (E) -N- {5- [4- (2-hydroxyacetyl) piperazin-1-ylmethyl ] -2- [2- (1H-indazol-3-yl) vinyl] phenyl} -3-methylthiophene-2-carboxamide or its p-toluenesulfonate or its hydrate as an active ingredient A therapeutic agent for cancer selected from lung cancer, sarcoma, and pancreatic cancer is provided.
(E)-N-{5-[4-(2-ヒドロキシアセチル)ピペラジン-1-イルメチル]-2-[2-(1H-インダゾール-3-イル)ビニル]フェニル}-3-メチルチオフェン-2-カルボキサミドのp-トルエンスルホン酸塩の抗腫瘍効果を表す。縦軸(V/Vo)は、0日目の腫瘍体積をV0としたときの腫瘍体積変化を表し、横軸は投与開始日からの日数を表す。○は陰性対照群(Control)、□を黒く塗りつぶしたものは化合物(Ia)のトシル酸塩投与群を表す。　**：P<0.01の有意差を表す((E)-N-{5-[4-(2-ヒドロキシアセチル)ピペラジン-1-イルメチル]-2-[2-(1H-インダゾール-3-イル)ビニル]フェニル}-3-メチルチオフェン-2-カルボキサミドのp-トルエンスルホン酸塩の投与群の陰性対照群比、Student's t-test)　***：P<0.001の有意差を表す((E)-N-{5-[4-(2-ヒドロキシアセチル)ピペラジン-1-イルメチル]-2-[2-(1H-インダゾール-3-イル)ビニル]フェニル}-3-メチルチオフェン-2-カルボキサミドのp-トルエンスルホン酸塩の投与群の陰性対照群比、Student's t-test)(E) -N- {5- [4- (2-hydroxyacetyl) piperazin-1-ylmethyl] -2- [2- (1H-indazol-3-yl) vinyl] phenyl} -3-methylthiophene-2 -Represents the antitumor effect of p-toluenesulfonate of carboxamide. The vertical axis (V / Vo) represents the change in tumor volume when the tumor volume on day 0 is V 0, and the horizontal axis represents the number of days from the start date of administration. ○ represents the negative control group (Control), and black squares represent the tosylate administration group of Compound (Ia). **: represents a significant difference of P <0.01 ((E) -N- {5- [4- (2-hydroxyacetyl) piperazin-1-ylmethyl] -2- [2- (1H-indazol-3-yl) ) Vinyl] phenyl} -3-methylthiophene-2-carboxamide p-toluenesulfonate treated group negative control group, Student's t-test) ***: P <0.001 (significant difference) ((E ) -N- {5- [4- (2-hydroxyacetyl) piperazin-1-ylmethyl] -2- [2- (1H-indazol-3-yl) vinyl] phenyl} -3-methylthiophene-2-carboxamide P-toluenesulfonate administration group negative control group ratio, Student's t-test)
　(E)-N-{5-[4-(2-ヒドロキシアセチル)ピペラジン-1-イルメチル]-2-[2-(1H-インダゾール-3-イル)ビニル]フェニル}-3-メチルチオフェン-2-カルボキサミドもしくはその薬理学的に許容される塩またはそれらの水和物は、公知の方法、例えば、国際公開第2006/080450号(WO2006/080450)パンフレット記載の方法またはそれに準じた方法で得ることができる。 (E) -N- {5- [4- (2-hydroxyacetyl) piperazin-1-ylmethyl] -2- [2- (1H-indazol-3-yl) vinyl] phenyl} -3-methylthiophene-2 -Carboxamide or a pharmacologically acceptable salt thereof or a hydrate thereof can be obtained by a known method, for example, the method described in WO 2006/080450 (WO2006 / 080450) pamphlet or a method analogous thereto. Can do.
　(E)-N-{5-[4-(2-ヒドロキシアセチル)ピペラジン-1-イルメチル]-2-[2-(1H-インダゾール-3-イル)ビニル]フェニル}-3-メチルチオフェン-2-カルボキサミドのトシル酸塩は、WO2006/080450記載の方法で得られる(E)-N-{5-[4-(2-ヒドロキシアセチル)ピペラジン-1-イルメチル]-2-[2-(1H-インダゾール-3-イル)ビニル]フェニル}-3-メチルチオフェン-2-カルボキサミドを常法によりp-トルエンスルホン酸塩化(トシル酸塩化)することにより得ることができるが、後記の参考例に記載の方法でも得ることができる。 (E) -N- {5- [4- (2-hydroxyacetyl) piperazin-1-ylmethyl] -2- [2- (1H-indazol-3-yl) vinyl] phenyl} -3-methylthiophene-2 -Carboxamide tosylate is obtained by the method described in WO2006 / 080450 (E) -N- {5- [4- (2-hydroxyacetyl) piperazin-1-ylmethyl] -2- [2- (1H- Indazol-3-yl) vinyl] phenyl} -3-methylthiophene-2-carboxamide can be obtained by p-toluenesulfonate formation (tosylate formation) by a conventional method. It can also be obtained by the method.
　(E)-N-{5-[4-(2-ヒドロキシアセチル)ピペラジン-1-イルメチル]-2-[2-(1H-インダゾール-3-イル)ビニル]フェニル}-3-メチルチオフェン-2-カルボキサミドは、式(Ia) (E) -N- {5- [4- (2-hydroxyacetyl) piperazin-1-ylmethyl] -2- [2- (1H-indazol-3-yl) vinyl] phenyl} -3-methylthiophene-2 -Carboxamide has the formula (Ia)
Figure JPOXMLDOC01-appb-C000002
Figure JPOXMLDOC01-appb-C000002
で表される化合物であり(以下、化合物(Ia)と表すこともある)、化合物(Ia)は強力なIGF-1R阻害作用を有することが報告されている(国際公開第2006/080450号パンフレット)。
　(E)-N-{5-[4-(2-ヒドロキシアセチル)ピペラジン-1-イルメチル]-2-[2-(1H-インダゾール-3-イル)ビニル]フェニル}-3-メチルチオフェン-2-カルボキサミドの薬理学的に許容される塩としては、例えば薬理学的に許容される酸付加塩、金属塩、アンモニウム塩、有機アミン付加塩、アミノ酸付加塩等が挙げられる。酸付加塩としては塩酸塩、硫酸塩、リン酸塩等の無機酸塩、酢酸塩、トリフルオロ酢酸塩、マレイン酸塩、フマル酸塩、酒石酸塩、クエン酸塩、乳酸塩、アスパラギン酸塩、グルタミン酸塩、p-トルエンスルホン酸塩(トシル酸塩)等の有機酸塩が挙げられ、金属塩としてはナトリウム塩、カリウム塩等のアルカリ金属塩、マグネシウム塩、カルシウム塩等のアルカリ土類金属塩、アルミニウム塩、亜鉛塩等が挙げられ、アンモニウム塩としてはアンモニウム、テトラメチルアンモニウム等の塩が挙げられ、有機アミン付加塩としてはモルホリン、ピペリジン等の付加塩が挙げられ、アミノ酸付加塩としてはリジン、グリシン、フェニルアラニン等の付加塩が挙げられる。 (Hereinafter also referred to as compound (Ia)), and compound (Ia) has been reported to have a potent IGF-1R inhibitory activity (WO 2006/080450 pamphlet). ).
(E) -N- {5- [4- (2-hydroxyacetyl) piperazin-1-ylmethyl] -2- [2- (1H-indazol-3-yl) vinyl] phenyl} -3-methylthiophene-2 Examples of the pharmacologically acceptable salt of carboxamide include pharmacologically acceptable acid addition salts, metal salts, ammonium salts, organic amine addition salts, amino acid addition salts and the like. Acid addition salts include inorganic acid salts such as hydrochloride, sulfate, phosphate, acetate, trifluoroacetate, maleate, fumarate, tartrate, citrate, lactate, aspartate, Examples include organic acid salts such as glutamate and p-toluenesulfonate (tosylate). Examples of metal salts include alkali metal salts such as sodium salt and potassium salt, and alkaline earth metal salts such as magnesium salt and calcium salt. , Aluminum salts, zinc salts, etc., ammonium salts include salts such as ammonium and tetramethylammonium, organic amine addition salts include addition salts such as morpholine and piperidine, and amino acid addition salts include lysine. And addition salts such as glycine and phenylalanine.
　以下の試験例1～5においては、(E)-N-{5-[4-(2-ヒドロキシアセチル)ピペラジン-1-イルメチル]-2-[2-(1H-インダゾール-3-イル)ビニル]フェニル}-3-メチルチオフェン-2-カルボキサミド(化合物(Ia))のトシル酸塩を用いた。
試験例1： in vitroでのヒト大腸癌細胞株における化合物(Ia)のトシル酸塩による増殖阻害試験
　ヒト大腸癌細胞株HT-29株およびその緑色蛍光たんぱく質(Green Fluorescent Protein、以下、GFPと表す)発現株であるHT-29/GFP(Anticancer社より譲渡)に対する化合物(Ia)のトシル酸塩による増殖阻害50%を示す濃度の指標であるGI50値を以下の方法で算出した。 In the following Test Examples 1 to 5, (E) -N- {5- [4- (2-hydroxyacetyl) piperazin-1-ylmethyl] -2- [2- (1H-indazol-3-yl) vinyl ] Tosylate salt of phenyl} -3-methylthiophene-2-carboxamide (compound (Ia)) was used.
Test Example 1: In vitro growth inhibition test by tosylate of compound (Ia) in human colorectal cancer cell line Human colorectal cancer cell line HT-29 and its green fluorescent protein (hereinafter referred to as GFP) ) The GI 50 value, which is an indicator of the concentration indicating 50% growth inhibition of the compound (Ia) by tosylate, against the expression strain HT-29 / GFP (assigned from Anticancer) was calculated by the following method.
　HT-29株およびHT-29/GFPの培養には1 vol%または10 vol%非働化牛胎児血清(以下、FBSと表す)(Invitrogen)を含むMcCOY's 5A培地(Invitrogen)を用いた。
　いずれの細胞株も5%炭酸ガスインキュベーター内で37 ℃にて培養し増殖させた。96ウェルF底プレート(Nalge Nunc)に10 vol% FBS添加時は1×103 個/well、1 vol% FBS添加時は5×103 個/wellのHT-29株またはHT-29/GFPを播種し、5%炭酸ガスインキュベーター内で37 ℃にて24時間培養した。ジメチルスルホキシド(以下、DMSOと表す)にて段階的に希釈して調製した化合物(Ia)のトシル酸塩を含む溶液をそれぞれ添加し(ウェル内でDMSOの終濃度は0.1 vol%)、再び5%炭酸ガスインキュベーター内で37 ℃にて72時間培養した。細胞増殖試薬WST-1(4-[3-(4-Iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate、Roche Diagnostics)を添加し、2時間反応させ、ウェル中の生細胞数を反映する吸光度(450 nm)を測定した。 For the culture of HT-29 strain and HT-29 / GFP, McCOY's 5A medium (Invitrogen) containing 1 vol% or 10 vol% inactivated fetal bovine serum (hereinafter referred to as FBS) (Invitrogen) was used.
All cell lines were cultured and grown at 37 ° C. in a 5% carbon dioxide incubator. HT-29 strain or HT-29 / GFP at 1 × 10 3 / well when 10 vol% FBS is added to 96-well F bottom plate (Nalge Nunc), 5 × 10 3 / well when 1 vol% FBS is added And cultured at 37 ° C. for 24 hours in a 5% carbon dioxide incubator. A solution containing the tosylate salt of compound (Ia) prepared by serial dilution with dimethyl sulfoxide (hereinafter referred to as DMSO) was added (the final concentration of DMSO in the well was 0.1 vol%), and again 5 The cells were cultured at 37 ° C. for 72 hours in a% carbon dioxide incubator. Add cell growth reagent WST-1 (4- [3- (4-Iodophenyl) -2- (4-nitrophenyl) -2H-5-tetrazolio] -1,3-benzene disulfonate, Roche Diagnostics) and react for 2 hours The absorbance (450 nm) reflecting the number of viable cells in the well was measured.
　DMSOを添加して72時間培養した後に測定した吸光度をコントロールの吸光度とし、DMSOを添加した直後に測定した吸光度の値をブランクプレートの吸光度として、次の数式を用いて化合物(Ia)のトシル酸塩による各細胞の細胞増殖阻害率((%) [inhibition(%)])を算出した。なお、以下の数式中、化合物(Ia)は被検化合物と表す。 The absorbance measured after adding DMSO for 72 hours was taken as the absorbance of the control, the absorbance measured immediately after adding DMSO as the absorbance of the blank plate, and the tosylic acid of compound (Ia) using the following formula: The cell growth inhibition rate ((%) [inhibition (%)]) of each cell by the salt was calculated. In the following formula, compound (Ia) is represented as a test compound.
Figure JPOXMLDOC01-appb-M000003
Figure JPOXMLDOC01-appb-M000003
　化合物(Ia)のトシル酸塩の濃度をX軸、各濃度での阻害率の平均値をY軸にプロットし、このプロットをXLfit4.1.1 (ID Business Solutions)のModel205にて近似し、50%の増殖阻害率を示す濃度としてGI50値を算出した。各細胞における化合物(Ia)のトシル酸塩のGI50値を表1に示した。化合物(Ia)のトシル酸塩はヒト大腸癌由来のHT-29株およびそのGFP発現株HT-29/GFPに対して、1% FBS存在下においていずれも45 nmol/L、10% FBS存在下において、それぞれ143および272 nmol/LのGI50値を示したことから、化合物(Ia)のトシル酸塩は大腸癌細胞に対する増殖抑制作用を有することが示され、大腸癌の治療剤として有用であることが示唆された。 Plotting the tosylate concentration of compound (Ia) on the X-axis and the mean inhibition rate at each concentration on the Y-axis, approximating this plot with Model 205 of XLfit4.1.1 (ID Business Solutions), 50% The GI 50 value was calculated as the concentration showing the growth inhibition rate. Table 1 shows the GI 50 value of the tosylate salt of compound (Ia) in each cell. Tosylate of compound (Ia) is HT-29 derived from human colorectal cancer and its GFP expressing strain HT-29 / GFP, both in the presence of 1% FBS, in the presence of 45 nmol / L, 10% FBS. GI 50 values of 143 and 272 nmol / L, respectively, indicate that the tosylate salt of Compound (Ia) has a growth-inhibiting effect on colon cancer cells and is useful as a therapeutic agent for colon cancer. It was suggested that there is.
Figure JPOXMLDOC01-appb-T000004
  
Figure JPOXMLDOC01-appb-T000004
  
試験例2： in vitroでのヒト肺癌細胞株における化合物(Ia)のトシル酸塩による増殖阻害試験
　ヒト肺癌細胞株NCI-H526、NCI-H727、NCI-H358およびCaLu-6株に対する化合物(Ia)のトシル酸塩による増殖阻害50%を示す濃度の指標であるGI50値を以下の方法で算出した。
　NCI-H526、NCI-H727およびNCI-H358の培養には1 vol%または10 vol% FBS(Invitrogen)、10 mmol/L HEPES(Invitrogen)、1 mmol/L ピルビン酸(Invitrogen)、および4.5 g/L グルコース(Sigma)を含むRoswell Park Memorial Institute's Medium(RPMI)1640培地(Invitrogen)を用いた。 Test Example 2: In vitro growth inhibition test by tosylate of compound (Ia) in human lung cancer cell lines Compound (Ia) against human lung cancer cell lines NCI-H526, NCI-H727, NCI-H358 and CaLu-6 The GI 50 value, which is an indicator of the concentration showing 50% growth inhibition by tosylate, was calculated by the following method.
NCI-H526, NCI-H727 and NCI-H358 cultures were 1 vol% or 10 vol% FBS (Invitrogen), 10 mmol / L HEPES (Invitrogen), 1 mmol / L pyruvate (Invitrogen), and 4.5 g / Roswell Park Memorial Institute's Medium (RPMI) 1640 medium (Invitrogen) containing L glucose (Sigma) was used.
　CaLu-6の培養には1 vol%または10 vol% FBS、および0.1 mmol/L Non-Essential Amino Acids(Invitrogen)を含むModified Eagle Medium(MEM)培地(Invitrogen)を用いた。
　いずれの細胞株も5%炭酸ガスインキュベーター内で37 ℃にて培養し増殖させた。96ウェルF底プレートまたは96ウェルU底プレート(Nalge Nunc)にNCI-H526は8×103 個/well、NCI-H727は5×103 個/well、NCI-H358およびCaLu-6は2×103 個/well(10% FBS、1% FBSとも)を播種し、5%炭酸ガスインキュベーター内で37 ℃にて24時間培養した。DMSOにて段階的に希釈して調製した化合物(Ia)のトシル酸塩を含む溶液をそれぞれ添加し(ウェル内でDMSOの終濃度は0.1 vol%)、再び5%炭酸ガスインキュベーター内で37 ℃にて72時間培養した。細胞増殖試薬WST-1(4-[3-(4-Iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate、Roche Diagnostics)を添加し、2時間反応させ、ウェル中の生細胞数を反映する吸光度(450 nm)を測定した。 For culture of CaLu-6, Modified Eagle Medium (MEM) medium (Invitrogen) containing 1 vol% or 10 vol% FBS and 0.1 mmol / L Non-Essential Amino Acids (Invitrogen) was used.
All cell lines were cultured and grown at 37 ° C. in a 5% carbon dioxide incubator. 96 well F bottom plate or 96 well U bottom plate (Nalge Nunc) NCI-H526 8 × 10 3 / well, NCI-H727 5 × 10 3 / well, NCI-H358 and CaLu-6 2 × 10 3 cells / well (both 10% FBS and 1% FBS) were seeded and cultured at 37 ° C. for 24 hours in a 5% carbon dioxide incubator. A solution containing the tosylate salt of Compound (Ia) prepared by serial dilution in DMSO was added (the final concentration of DMSO in the well was 0.1 vol%), and again in a 5% carbon dioxide incubator at 37 ° C. For 72 hours. Add cell growth reagent WST-1 (4- [3- (4-Iodophenyl) -2- (4-nitrophenyl) -2H-5-tetrazolio] -1,3-benzene disulfonate, Roche Diagnostics) and react for 2 hours The absorbance (450 nm) reflecting the number of viable cells in the well was measured.
　DMSOを添加して72時間培養した後に測定した吸光度をコントロールの吸光度とし、DMSOを添加した直後に測定した吸光度の値をブランクプレートの吸光度として、次の数式を用いて化合物(Ia)のトシル酸塩による各細胞の細胞増殖阻害率((%) [inhibition(%)])を算出した。なお、以下の数式中、化合物(Ia)は被検化合物と表す。 The absorbance measured after adding DMSO for 72 hours was taken as the absorbance of the control, the absorbance measured immediately after adding DMSO as the absorbance of the blank plate, and the tosylic acid of compound (Ia) using the following formula: The cell growth inhibition rate ((%) [inhibition (%)]) of each cell by the salt was calculated. In the following formula, compound (Ia) is represented as a test compound.
Figure JPOXMLDOC01-appb-M000005
  
Figure JPOXMLDOC01-appb-M000005
  
　化合物(Ia)のトシル酸塩の濃度をX軸、各濃度での阻害率の平均値をY軸にプロットし、このプロットをXLfit4.1.1 (ID Business Solutions)のModel205にて近似し、50%の増殖阻害率を示す濃度としてGI50値を算出した。各細胞における化合物(Ia)のトシル酸塩のGI50値を表2に示した。化合物(Ia)のトシル酸塩はヒト肺癌由来のNCI-H526、NCI-H727、NCI-H358、およびCaLu-6に対して1 vol% FBS条件下でそれぞれ、9.4、34、32未満、および97 nmol/LのGI50値を示した。さらに、10 vol% FBS条件下においても増殖抑制活性を示し、GI50値はそれぞれ、190、46、150、および370 nmol/Lであったことから、化合物(Ia)のトシル酸塩は肺癌細胞に対する増殖抑制作用を有することが示され、肺癌治療剤として有用であることが示唆された。 Plotting the tosylate concentration of compound (Ia) on the X-axis and the mean inhibition rate at each concentration on the Y-axis, approximating this plot with Model 205 of XLfit4.1.1 (ID Business Solutions), 50% The GI 50 value was calculated as the concentration showing the growth inhibition rate. Table 2 shows the GI 50 value of the tosylate salt of compound (Ia) in each cell. The tosylate salt of Compound (Ia) is less than 9.4, 34, 32, and 97 respectively under 1 vol% FBS conditions against NCI-H526, NCI-H727, NCI-H358, and CaLu-6 from human lung cancer. A GI 50 value of nmol / L was shown. Furthermore, since it exhibited growth inhibitory activity even under 10 vol% FBS conditions, and the GI 50 values were 190, 46, 150, and 370 nmol / L, respectively, the tosylate salt of compound (Ia) was found in lung cancer cells. It has been shown to have a growth-inhibiting effect on the lung cancer, suggesting that it is useful as a lung cancer therapeutic agent.
Figure JPOXMLDOC01-appb-T000006
  
Figure JPOXMLDOC01-appb-T000006
  
試験例3： in vitroでのヒトサルコーマ(sarcoma)細胞株における化合物(Ia)のトシル酸塩による増殖阻害試験
　EWS/FLI-1 転座を有するヒトサルコーマ(sarcoma)細胞株であるTC-71、SK-ES-1、SK-N-MCおよびA-673、EWS/FLI-1 転座をもたないRDおよびRH-30に対する化合物(Ia)のトシル酸塩による増殖阻害50%を示す濃度の指標であるGI50値を以下の方法で算出した。 Test Example 3: In vitro growth inhibition test by tosylate of compound (Ia) in human sarcoma cell line TC-71, SK- which are human sarcoma cell lines with EWS / FLI-1 translocation ES-1, SK-N-MC and A-673, EWS / FLI-1 RD and RH-30 without translocation are indicators of concentration indicating 50% growth inhibition by tosylate of compound (Ia) A certain GI 50 value was calculated by the following method.
　TC-71の培養には1 vol%または10 vol% FBS(Invitrogen)を含むIscove's Modified Dulbecco's Medium(IMDM)培地(Invitrogen)を用いた。
　SK-ES-1の培養には1 vol%または15 vol% FBSを含むMaCOY's 5A培地(Invitrogen)を用いた。
　SK-N-MCの培養には1 vol%または10 vol% FBSを含む10 mmol/L HEPES、1 mmol/L ピルビン酸、および4.5 g/L グルコースを含むRPMI1640培地を用いた。 For TC-71 culture, Iscove's Modified Dulbecco's Medium (IMDM) medium (Invitrogen) containing 1 vol% or 10 vol% FBS (Invitrogen) was used.
MaCOY's 5A medium (Invitrogen) containing 1 vol% or 15 vol% FBS was used for the culture of SK-ES-1.
RPMI1640 medium containing 10 mmol / L HEPES containing 1 vol% or 10 vol% FBS, 1 mmol / L pyruvate, and 4.5 g / L glucose was used for SK-N-MC culture.
　A-673およびRDの培養には1 vol%または10 vol% FBS、および4.5 g/L グルコースを含むDulbecco's Modified Eagle Medium(DMEM)培地(Invitrogen)を用いた。
　RH-30の培養には1 vol%または10 vol% FBSを含むRPMI1640培地(Invitrogen)を用いた。
　いずれの細胞株も5%炭酸ガスインキュベーター内で37 ℃にて培養し増殖させた。96ウェルF底プレート(Nalge Nunc)にTC-71は5×102 個/well(10％ FBS)または2×103 個/well(1％ FBS)、RDは1×103 個/well(10 vol% FBS)または2×103 個/well(1 vol% FBS)、A-673は2×103 個/well(10 vol% FBS)または5×103 個/well(1 vol% FBS)、SK-ES-1は8×103 個/well(15 vol%、1 vol% FBSとも)、SK-N-MCは8×103 個/well(10 vol% FBS、1 vol% FBSとも)、RH-30は2×103 個/well(10% FBS、1% FBSとも)を播種し、5%炭酸ガスインキュベーター内で37 ℃にて24時間培養した。DMSOにて段階的に希釈して調製した化合物(Ia)のトシル酸塩を含む溶液をそれぞれ添加し(ウェル内でDMSOの終濃度は0.1 vol%)、再び5%炭酸ガスインキュベーター内で37 ℃にて72時間培養した。細胞増殖試薬WST-1(4-[3-(4-Iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate、Roche Diagnostics)を添加し、2時間反応させ、ウェル中の生細胞数を反映する吸光度(450 nm)を測定した。 For culture of A-673 and RD, Dulbecco's Modified Eagle Medium (DMEM) medium (Invitrogen) containing 1 vol% or 10 vol% FBS and 4.5 g / L glucose was used.
RPMI1640 medium (Invitrogen) containing 1 vol% or 10 vol% FBS was used for RH-30 culture.
All cell lines were cultured and grown at 37 ° C. in a 5% carbon dioxide incubator. 96-well F bottom plate (Nalge Nunc), TC-71 is 5 × 10 2 / well (10% FBS) or 2 × 10 3 / well (1% FBS), RD is 1 × 10 3 / well ( 10 vol% FBS) or 2 × 10 3 / well (1 vol% FBS), A-673 is 2 × 10 3 / well (10 vol% FBS) or 5 × 10 3 / well (1 vol% FBS) ), SK-ES-1 is 8 × 10 3 / well (15 vol%, 1 vol% FBS), SK-N-MC is 8 × 10 3 / well (10 vol% FBS, 1 vol% FBS) RH-30 was seeded at 2 × 10 3 cells / well (both 10% FBS and 1% FBS) and cultured at 37 ° C. for 24 hours in a 5% carbon dioxide incubator. A solution containing the tosylate salt of Compound (Ia) prepared by serial dilution in DMSO was added (the final concentration of DMSO in the well was 0.1 vol%), and again in a 5% carbon dioxide incubator at 37 ° C. For 72 hours. Add cell growth reagent WST-1 (4- [3- (4-Iodophenyl) -2- (4-nitrophenyl) -2H-5-tetrazolio] -1,3-benzene disulfonate, Roche Diagnostics) and react for 2 hours The absorbance (450 nm) reflecting the number of viable cells in the well was measured.
　DMSOを添加して72時間培養した後に測定した吸光度をコントロールの吸光度とし、DMSOを添加した直後に測定した吸光度の値をブランクプレートの吸光度として、次の数式を用いて化合物(Ia)のトシル酸塩による各細胞の細胞増殖阻害率((%) [inhibition(%)])を算出した。なお、以下の数式中、化合物(Ia)は被検化合物と表す。 The absorbance measured after adding DMSO for 72 hours was taken as the absorbance of the control, the absorbance measured immediately after adding DMSO as the absorbance of the blank plate, and the tosylic acid of compound (Ia) using the following formula: The cell growth inhibition rate ((%) [inhibition (%)]) of each cell by the salt was calculated. In the following formula, compound (Ia) is represented as a test compound.
Figure JPOXMLDOC01-appb-M000007
  
Figure JPOXMLDOC01-appb-M000007
  
　化合物(Ia)のトシル酸塩の濃度をX軸、各濃度での阻害率の平均値をY軸にプロットし、このプロットをXLfit4.1.1 (ID Business Solutions)のModel205にて近似し、50%の増殖阻害率を示す濃度としてGI50値を算出した。各細胞における化合物(Ia)のトシル酸塩のGI50値を表3に示した。化合物(Ia)のトシル酸塩はEWS/FLI-1 転座を有するヒトサルコーマ(sarcoma)由来のTC-71、SK-N-MCおよびA-673に対して、1 vol% FBS条件下でそれぞれ、3.2未満、3.2未満および20 nmol/LのGI50値を示した。さらに、10 vol% FBS条件下においても増殖抑制活性を示し、GI50値はそれぞれ、11、3.2未満および21 nmol/Lであった。SK-ES-1に対しては、10 vol% FBS条件下で10 nmol/LのGI50値を示した。一方、EWS/FLI-1 転座をもたないRDおよびRH-30に対して、1 vol% FBS条件下でそれぞれ、22および30 nmol/LのGI50値を示し、10 vol% FBS条件下においても、GI50値はそれぞれ、220および130 nmol/Lであったことから、化合物(Ia)のトシル酸塩はサルコーマ(sarcoma)に対する増殖抑制作用を有することが示され、サルコーマ(sarcoma)治療剤として有用であることが示唆された。 Plotting the tosylate concentration of compound (Ia) on the X-axis and the mean inhibition rate at each concentration on the Y-axis, approximating this plot with Model 205 of XLfit4.1.1 (ID Business Solutions), 50% The GI 50 value was calculated as the concentration showing the growth inhibition rate. Table 3 shows the GI 50 value of the tosylate salt of compound (Ia) in each cell. The tosylate salt of compound (Ia) is TC-71, SK-N-MC and A-673 derived from human sarcoma with EWS / FLI-1 translocation, respectively, under 1 vol% FBS conditions. GI 50 values of <3.2, <3.2 and 20 nmol / L were shown. Furthermore, it showed growth inhibitory activity even under 10 vol% FBS conditions, and the GI 50 values were 11, 3.2 and 21 nmol / L, respectively. For SK-ES-1, a GI 50 value of 10 nmol / L was exhibited under 10 vol% FBS conditions. On the other hand, RD and RH-30 without EWS / FLI-1 translocation show GI 50 values of 22 and 30 nmol / L, respectively, under 1 vol% FBS conditions, and under 10 vol% FBS conditions. GI 50 values were 220 and 130 nmol / L, respectively, indicating that the tosylate salt of Compound (Ia) has a growth-inhibiting effect on sarcoma, and treated with sarcoma. It was suggested that it is useful as an agent.
Figure JPOXMLDOC01-appb-T000008
Figure JPOXMLDOC01-appb-T000008
試験例4： in vitroでのヒト膵臓癌細胞株における化合物(Ia)のトシル酸塩による増殖阻害試験
　ヒト膵臓癌細胞株KP-3LおよびAsPC-1に対する化合物(Ia)のトシル酸塩による増殖阻害50%を示す濃度の指標であるGI50値を以下の方法で算出した。
　KP-3LおよびAsPC-1の培養には1 vol%または10 vol% FBS(Invitrogen)を含むRoswell Park Memorial Institute's Medium(RPMI) 1640培地(Invitrogen)を用いた。 Test Example 4: Inhibition of proliferation of compound (Ia) by tosylate in human pancreatic cancer cell line in vitro Growth inhibition of compound (Ia) by tosylate against human pancreatic cancer cell lines KP-3L and AsPC-1 The GI 50 value, which is an index of concentration indicating 50%, was calculated by the following method.
For culture of KP-3L and AsPC-1, Roswell Park Memorial Institute's Medium (RPMI) 1640 medium (Invitrogen) containing 1 vol% or 10 vol% FBS (Invitrogen) was used.
　いずれの細胞株も5%炭酸ガスインキュベーター内で37 ℃にて培養し増殖させた。96ウェルF底プレート(Nalge Nunc)にKP-3Lは2.5×103 個/well(10% FBS)または5×103 個/well(1% FBS)、AsPC-1は2×103 個/well(10 vol% FBS)または4×103 個/well(1 vol% FBS)を播種し5%炭酸ガスインキュベーター内で37 ℃にて24時間培養した。DMSOにて段階的に希釈して調製した化合物(Ia)のトシル酸塩を含む溶液をそれぞれ添加し(ウェル内でDMSOの終濃度は0.1 vol%)、再び5%炭酸ガスインキュベーター内で37 ℃にて72時間培養した。細胞増殖試薬WST-1(4-[3-(4-Iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate、Roche Diagnostics)を添加し、2時間反応させ、ウェル中の生細胞数を反映する吸光度(450 nm)を測定した。 All cell lines were cultured and grown at 37 ° C. in a 5% carbon dioxide incubator. 96 well F bottom plate (Nalge Nunc) KP-3L 2.5 × 10 3 / well (10% FBS) or 5 × 10 3 / well (1% FBS), AsPC-1 2 × 10 3 / Well (10 vol% FBS) or 4 × 10 3 cells / well (1 vol% FBS) was seeded and cultured in a 5% carbon dioxide incubator at 37 ° C. for 24 hours. A solution containing the tosylate salt of Compound (Ia) prepared by serial dilution in DMSO was added (the final concentration of DMSO in the well was 0.1 vol%), and again in a 5% carbon dioxide incubator at 37 ° C. For 72 hours. Add cell growth reagent WST-1 (4- [3- (4-Iodophenyl) -2- (4-nitrophenyl) -2H-5-tetrazolio] -1,3-benzene disulfonate, Roche Diagnostics) and react for 2 hours The absorbance (450 nm) reflecting the number of viable cells in the well was measured.
　DMSOを添加して72時間培養した後に測定した吸光度をコントロールの吸光度とし、DMSOを添加した直後に測定した吸光度の値をブランクプレートの吸光度として、次の数式を用いて化合物(Ia)のトシル酸塩による各細胞の細胞増殖阻害率((%) [inhibition(%)])を算出した。なお、以下の数式中、化合物(Ia)は被検化合物と表す。 The absorbance measured after adding DMSO for 72 hours was taken as the absorbance of the control, the absorbance measured immediately after adding DMSO as the absorbance of the blank plate, and the tosylic acid of compound (Ia) using the following formula: The cell growth inhibition rate ((%) [inhibition (%)]) of each cell by the salt was calculated. In the following formula, compound (Ia) is represented as a test compound.
Figure JPOXMLDOC01-appb-M000009
  
Figure JPOXMLDOC01-appb-M000009
  
　化合物(Ia)のトシル酸塩の濃度をX軸、各濃度での阻害率の平均値をY軸にプロットし、このプロットをXLfit4.1.1 (ID Business Solutions)のModel205にて近似し、50%の増殖阻害率を示す濃度としてGI50値を算出した。各細胞における化合物(Ia)のトシル酸塩のGI50値を表4に示した。化合物(Ia)のトシル酸塩はヒト膵臓癌由来のKP-3LおよびAsPC-1に対して1 vol% FBS条件下でそれぞれ、150および9.0 nmol/LのGI50値を示した。さらに、10 vol% FBS条件下においても増殖抑制活性を示し、GI50値はそれぞれ、260および38 nmol/Lであったことから、化合物(Ia)のトシル酸塩は膵臓癌細胞の増殖抑制作用を有することが示され、膵臓癌治療剤として有用であることが示唆された。 Plotting the tosylate concentration of compound (Ia) on the X-axis and the mean inhibition rate at each concentration on the Y-axis, approximating this plot with Model 205 of XLfit4.1.1 (ID Business Solutions), 50% The GI 50 value was calculated as the concentration showing the growth inhibition rate. Table 4 shows the GI 50 value of the tosylate salt of compound (Ia) in each cell. The tosylate salt of Compound (Ia) showed GI 50 values of 150 and 9.0 nmol / L for KP-3L and AsPC-1 derived from human pancreatic cancer under 1 vol% FBS conditions, respectively. Furthermore, it showed growth-inhibitory activity even under 10 vol% FBS conditions, and the GI 50 values were 260 and 38 nmol / L, respectively. Therefore, the tosylate salt of Compound (Ia) has an inhibitory effect on the growth of pancreatic cancer cells. It was shown to be useful as a therapeutic agent for pancreatic cancer.
Figure JPOXMLDOC01-appb-T000010
Figure JPOXMLDOC01-appb-T000010
試験例5： ヒト大腸癌細胞株HT-29/GFP移植マウスモデルにおける化合物(Ia)のトシル酸塩による抗腫瘍効果
　ヒト大腸癌細胞株HT-29株のGFP発現株であるHT-29/GFPをマウスに移植した場合における化合物(Ia)のトシル酸塩による抗腫瘍効果を以下の方法で検討した。
　HT-29/GFP(Anticancer社より譲渡)を、10 vol% FBS(Invitrogen)、100単位/mL ペニシリンGおよび100 μg/mLストレプトマイシン(Invitrogen) を含むMcCOY's 5A培地(Invitrogen)中、5%炭酸ガスインキュベーター内で37 ℃にて培養し増殖させた。 Test Example 5: Antitumor effect of compound (Ia) tosylate in human colon cancer cell line HT-29 / GFP transplanted mouse model HT-29 / GFP, a GFP expression strain of human colon cancer cell line HT-29 The antitumor effect of Tosylate of Compound (Ia) when transplanted into mice was examined by the following method.
HT-29 / GFP (transferred from Anticancer) was transferred to 5% carbon dioxide in McCOY's 5A medium (Invitrogen) containing 10 vol% FBS (Invitrogen), 100 units / mL penicillin G and 100 μg / mL streptomycin (Invitrogen). The cells were grown at 37 ° C. in an incubator.
　移植前にバリカンを用いて雄性C.B-17/Icr-scid/scidJclマウス(日本クレア)の腹側部の毛を剃り、リン酸緩衝生理食塩液(PBS)中に懸濁したHT-29/GFP(6×107 個/mL)を剃毛した腹部の皮下に0.05 mL 移植した(3×106 cells/0.05 mL/個体)。
　移植6日後にノギスにて皮下で増殖した腫瘍の長径・短径を測定し、以下の式に従って腫瘍体積を求めた。 HT-29 / GFP suspended in phosphate buffered saline (PBS) after shaving the ventral hair of male CB-17 / Icr-scid / scidJcl mice (Claire Japan) using clippers before transplantation 0.05 mL was transplanted subcutaneously into the shaved abdomen (6 × 10 7 cells / mL) (3 × 10 6 cells / 0.05 mL / individual).
The major axis and minor axis of the tumor grown subcutaneously with calipers 6 days after the transplantation were measured, and the tumor volume was determined according to the following formula.
Figure JPOXMLDOC01-appb-M000011
  
Figure JPOXMLDOC01-appb-M000011
  
　平均腫瘍体積と平均体重が均一になるように1群10匹ずつ以下のような投与群に分け、この日を投与試験開始0日として投与を開始した。
　化合物(Ia)のトシル酸塩は0.5 w/v%メチルセルロース溶液(和光純薬社製)に4 mg/mLの濃度で懸濁させ、投与開始0から13日目まで連日1日1回、マウス体重1 gあたり0.01 mL(40 mg/kg)の用量で、経口ゾンデを用いて経口投与した。 Each group was divided into the following administration groups so that the average tumor volume and average body weight were uniform, and administration was started on this day as the start of the administration test.
Tosylate of compound (Ia) is suspended in 0.5 w / v% methylcellulose solution (Wako Pure Chemical Industries, Ltd.) at a concentration of 4 mg / mL, once a day from the start of administration to day 13 once daily Oral administration was performed using an oral sonde at a dose of 0.01 mL (40 mg / kg) per gram of body weight.
A. 陰性対照群(Control)：0.5 w/v%メチルセルロース溶液
B. 化合物(Ia)のトシル酸塩投与群：40 mg/kg(1日1回×14日間)
　0日目以降、週に2回腫瘍体積の測定を行った。抗腫瘍効果の判定は各群の腫瘍体積の平均値を算出し、0日目の腫瘍体積をV0としたときの腫瘍体積変化(V/V0)の比較で行った。経日測定した各群のV/V0を図１に示す。 A. Negative control group (Control): 0.5 w / v% methylcellulose solution
B. Compound (Ia) tosylate administration group: 40 mg / kg (once a day x 14 days)
From day 0, tumor volume was measured twice a week. The determination of the antitumor effect was performed by calculating the average value of the tumor volumes of each group and comparing the change in tumor volume (V / V 0 ) when the tumor volume on day 0 was V 0 . FIG. 1 shows V / V 0 of each group measured over time.
　図１に示したように、化合物(Ia)のトシル酸塩投与により、陰性対照群に比べ、有意な抗腫瘍効果が認められた。
　14日目の化合物(Ia)のトシル酸塩投与群のV/V0を陰性対照群のV/V0で除した値(T/C)は、14日目において0.637であった。
　以上の結果より、化合物(Ia)のトシル酸塩は動物に移植した大腸癌細胞の増殖抑制作用を有することが示され、大腸癌治療剤として有用であることが示唆された。 As shown in FIG. 1, administration of the tosylate salt of Compound (Ia) showed a significant antitumor effect compared to the negative control group.
The value (T / C) obtained by dividing V / V 0 of the tosylate administration group of compound (Ia) on day 14 by V / V 0 of the negative control group was 0.637 on day 14.
From the above results, it was shown that the tosylate salt of Compound (Ia) has an inhibitory effect on the growth of colon cancer cells transplanted into animals, suggesting that it is useful as a therapeutic agent for colon cancer.
　以上、試験例1～5の結果により、(E)-N-{5-[4-(2-ヒドロキシアセチル)ピペラジン-1-イルメチル]-2-[2-(1H-インダゾール-3-イル)ビニル]フェニル}-3-メチルチオフェン-2-カルボキサミドもしくはその薬理学的に許容される塩またはそれらの水和物が、大腸癌、肺癌、サルコーマ(sarcoma)、および膵臓癌から選ばれる癌の治療剤等として有用であることが示唆された。 As described above, according to the results of Test Examples 1 to 5, (E) -N- {5- [4- (2-hydroxyacetyl) piperazin-1-ylmethyl] -2- [2- (1H-indazol-3-yl) [Vinyl] phenyl} -3-methylthiophene-2-carboxamide or a pharmacologically acceptable salt thereof or a hydrate thereof is a treatment of cancer selected from colorectal cancer, lung cancer, sarcoma, and pancreatic cancer It was suggested that it is useful as an agent.
　化合物(Ia)もしくはその薬理学的に許容される塩またはそれらの水和物は、そのまま単独で投与することも可能であるが、通常各種の医薬製剤として提供するのが望ましい。また、それら医薬製剤は、動物または人に使用されるものである。
　本発明に係わる医薬製剤は、活性成分として化合物(Ia)またはその薬理学的に許容される塩を単独で、または任意の他の治療のための有効成分との混合物として含有することができる。また、それら医薬製剤は、活性成分を薬理学的に許容される一種またはそれ以上の担体(例えば、希釈剤、溶剤、賦形剤等)と一緒に混合し、製剤学の技術分野においてよく知られている任意の方法により製造される。 Although compound (Ia) or a pharmacologically acceptable salt thereof or a hydrate thereof can be administered alone as it is, it is usually desirable to provide it as various pharmaceutical preparations. These pharmaceutical preparations are used for animals or humans.
The pharmaceutical preparation according to the present invention may contain Compound (Ia) or a pharmacologically acceptable salt thereof as an active ingredient alone or as a mixture with any other active ingredient for treatment. These pharmaceutical preparations are well known in the technical field of pharmaceutics by mixing the active ingredient together with one or more pharmacologically acceptable carriers (for example, diluents, solvents, excipients, etc.). It is produced by any known method.
　投与経路としては、治療に際し最も効果的なものを使用するのが望ましく、経口または、例えば静脈内等の非経口をあげることができる。
　投与形態としては、例えば錠剤、注射剤等があげられる。
　経口投与に適当な、例えば錠剤等は、乳糖等の賦形剤、澱粉等の崩壊剤、ステアリン酸マグネシウム等の滑沢剤、ヒドロキシプロピルセルロース等の結合剤等を用いて製造できる。 As the administration route, it is desirable to use the most effective one in the treatment, and oral or parenteral such as intravenous administration can be mentioned.
Examples of the dosage form include tablets and injections.
For example, tablets suitable for oral administration can be produced using excipients such as lactose, disintegrants such as starch, lubricants such as magnesium stearate, binders such as hydroxypropylcellulose, and the like.
　非経口投与に適当な、例えば注射剤等は、塩溶液、ブドウ糖溶液または塩水とブドウ糖溶液の混合液等の希釈剤または溶剤等を用いて製造できる。
　化合物(Ia)もしくはその薬理学的に許容される塩またはそれらの水和物の投与量および投与回数は、投与形態、患者の年齢、体重、治療すべき症状の性質もしくは重篤度等により異なるが、通常経口の場合、成人一人あたり、0.01～1000 mg、好ましくは0.05～100 mgの範囲で、1日1回ないし数回投与する。静脈内投与等の非経口投与の場合、通常成人一人あたり0.001～1000 mg、好ましくは0.01～100 mgを1日1回ないし数回投与する。しかしながら、これら投与量および投与回数に関しては、前述の種々の条件により変動する。 For example, an injection suitable for parenteral administration can be produced using a diluent or a solvent such as a salt solution, a glucose solution, or a mixed solution of a saline solution and a glucose solution.
The dose and frequency of administration of compound (Ia) or a pharmacologically acceptable salt thereof or a hydrate thereof vary depending on the administration form, patient age, body weight, nature or severity of symptoms to be treated, etc. However, in the case of oral administration, it is usually administered once or several times a day in the range of 0.01 to 1000 mg, preferably 0.05 to 100 mg per adult. In the case of parenteral administration such as intravenous administration, 0.001 to 1000 mg, preferably 0.01 to 100 mg, is usually administered once or several times a day for each adult. However, the dose and the number of doses vary depending on the various conditions described above.
　以下の比較例1においては、化合物(Ia)の他に、国際公開第2006/080450号パンフレット記載の以下の式(II)、(III)、(IV)で表される化合物(以下、化合物(II)、(III)、(IV)と表す)を用いた。 In Comparative Example 1 below, in addition to compound (Ia), compounds represented by the following formulas (II), (III) and (IV) described in International Publication No. 2006/080450 pamphlet (hereinafter referred to as compound ( II), (III) and (IV) are used.
Figure JPOXMLDOC01-appb-C000012
  
Figure JPOXMLDOC01-appb-C000012
  
比較例1： ヒト肝代謝固有クリアランスの算出試験
　本試験にはプールヒト肝ミクロソーム(50人のドナー由来)を用いた。0.2 mg protein/mLのヒト肝ミクロソーム(Xenotech)、1 mmol/Lのβ-NADPH(和光純薬工業)および1 μmol/Lの試験化合物を含む溶液(DMSO 0.01 vol%およびアセトニトリル 0.5 vol%含有)を、37 ℃にて、30分間反応させた。2倍量のアセトニトリルを添加し、遠心分離(約3000×g、5分間)した。得られた上清をLC/MSで分析した(測定A)。 Comparative Example 1: Calculation Test for Human Liver Metabolic Clearance Pooled human liver microsomes (derived from 50 donors) were used in this study. A solution containing 0.2 mg protein / mL human liver microsome (Xenotech), 1 mmol / L β-NADPH (Wako Pure Chemical Industries) and 1 μmol / L test compound (containing 0.01 vol% DMSO and 0.5 vol% acetonitrile) Was reacted at 37 ° C. for 30 minutes. Two volumes of acetonitrile were added and centrifuged (approximately 3000 × g, 5 minutes). The obtained supernatant was analyzed by LC / MS (Measurement A).
　また、ヒト肝ミクロソームの代わりに、同蛋白濃度のヒト血清アルブミン(シグマアルドリッチ)を用い、上記と同様の測定を行った(測定B)。
　測定Bに対する測定Aの試験化合物のピーク面積比に100を乗じ、試験化合物の残存率(%)を算出した。反応0分における残存率(100％)および反応30分における残存率を自然対数変換し、反応時間に対してプロットして得られた傾きを消失速度定数(ke)とした。この値を用い、以下の式によりヒト肝代謝固有クリアランス(CLint)を算出した。  Further, human serum albumin (Sigma Aldrich) having the same protein concentration was used instead of human liver microsome, and the same measurement as described above was performed (Measurement B).
The peak area ratio of the test compound of measurement A to measurement B was multiplied by 100 to calculate the residual ratio (%) of the test compound. The reaction residual ratio at 0 min (100%) and the residual ratio in the reaction 30 minutes to natural-log transformed, an inclination obtained by plotting was elimination rate constant (k e) the reaction time. Using this value, the human liver metabolic intrinsic clearance (CL int ) was calculated by the following formula.
Figure JPOXMLDOC01-appb-M000013
  
Figure JPOXMLDOC01-appb-M000013
  
　なお、上記式中、肝Ms蛋白量/肝重量は32.3 mg/g (Xenobiotica、18巻、849頁(1988年)記載の内容引用)、肝重量/体重は25.7 g/kg (Pharmceutical Research、10巻、1093頁(1993年)記載の内容引用)を用いて計算した。
　上記のようにして算出した、化合物(Ia)、(II)、(III)および(IV)のCLintを表5に示した。この結果より、化合物(Ia)は、その化学構造が類似した他の化合物(II)～(IV)に比べ、CLintが低く、より代謝安定であることが示唆された。一般に、医薬の活性成分となる化合物には、毒性(副作用)が少なく、薬物動態が優れていることが求められる。従って、CLintが低い化合物(Ia)は、他の化合物(II)～(IV)に比べ、医薬の活性成分として優れていると考えられる。 In the above formula, liver Ms protein amount / liver weight is 32.3 mg / g (contents described in Xenobiotica, Vol. 18, page 849 (1988)), liver weight / body weight is 25.7 g / kg (Pharmceutical Research, 10 Volume, page 1093 (1993)).
Table 5 shows CL ints of compounds (Ia), (II), (III) and (IV) calculated as described above. This result suggests that compound (Ia) has a lower CL int and is more metabolically stable than other compounds (II) to (IV) having similar chemical structures. In general, a compound serving as an active ingredient of a medicine is required to have little toxicity (side effects) and excellent pharmacokinetics. Therefore, it is considered that the compound (Ia) having a low CL int is superior as an active ingredient of a medicine compared with other compounds (II) to (IV).
Figure JPOXMLDOC01-appb-T000014
  
Figure JPOXMLDOC01-appb-T000014
  
　以下、本発明を実施例および参考例によりさらに具体的に説明するが、本発明の範囲はこれらの実施例に限定されることはない。 Hereinafter, the present invention will be described more specifically with reference to examples and reference examples, but the scope of the present invention is not limited to these examples.
錠剤(化合物(Ia))
　常法により、次の組成からなる錠剤を調製する。化合物(Ia)、40 g、乳糖286.8 gおよび馬鈴薯澱粉60 gを混合し、これにヒドロキシプロピルセルロースの10%水溶液120 gを加える。得られた混合物を常法により練合し、造粒して乾燥させた後、整粒し打錠用顆粒とする。これにステアリン酸マグネシウム1.2 gを加えて混合し、径8 mmの杵をもった打錠機(菊水社製RT-15型)で打錠を行って、錠剤(1錠あたり活性成分20 mgを含有する)を得る。
処方　化合物(Ia)　　　　　　　　　　　　 20　　mg
　　　乳糖　　　　　　　　　　　  　　　143.4　mg
　　　馬鈴薯澱粉　　　　　　　　　  　　 30　　mg
　　　ヒドロキシプロピルセルロース　  　　6　　mg
　　　ステアリン酸マグネシウム　　　　  　0.6　mg 
　　　　　　　　　　　　　　　　　　　　200　　mg Tablet (Compound (Ia))
A tablet having the following composition is prepared by a conventional method. Compound (Ia), 40 g, lactose 286.8 g and potato starch 60 g are mixed, and 10% aqueous solution of hydroxypropylcellulose 120 g is added thereto. The obtained mixture is kneaded by a conventional method, granulated and dried, and then sized to obtain granules for tableting. To this was added 1.2 g of magnesium stearate, mixed, and tableted with a tableting machine (RT-15 type, manufactured by Kikusui Co., Ltd.) with a 8 mm diameter punch to produce tablets (20 mg of active ingredient per tablet). Containing).
Formulation Compound (Ia) 20 mg
Lactose 143.4 mg
Potato starch 30 mg
Hydroxypropylcellulose 6 mg
Magnesium stearate 0.6 mg
200 mg
注射剤(化合物(Ia))
　常法により、次の組成からなる注射剤を調製する。化合物(Ia)、1 gを注射用蒸留水に添加して混合し、さらに塩酸および水酸化ナトリウム水溶液を添加してpHを7に調整した後、注射用蒸留水で全量を1000 mLとする。得られた混合液をガラスバイアルに2 mLずつ無菌的に充填して、注射剤(1バイアルあたり活性成分2 mgを含有する)を得る。
処方　化合物(Ia)　　　　　　　　　 　　   2　 mg
　　　塩酸　　　　　　　　　　　　　　　　　適量
　　　水酸化ナトリウム水溶液　　　　　　　  適量
　　　注射用蒸留水　　　　　　　　　　　　　適量　　　
　　　　　　　　　　　　　　　　　　　　　2.00　mL
参考例1
(E)-N-{2-[2-(1H-インダゾール-3-イル)ビニル]-5-[4-(tert-ブトキシカルボニル)ピペラジ-1-イルメチル]フェニル}-3-メチルチオフェン-2-カルボキサミド・塩酸塩(化合物5) Injection (Compound (Ia))
An injection having the following composition is prepared by a conventional method. Compound (Ia), 1 g, is added to and mixed with distilled water for injection. Further, hydrochloric acid and aqueous sodium hydroxide solution are added to adjust the pH to 7, and the total volume is made up to 1000 mL with distilled water for injection. The obtained mixture is aseptically filled into glass vials by 2 mL to obtain an injection (containing 2 mg of active ingredient per vial).
Formulation Compound (Ia) 2 mg
Hydrochloric acid appropriate amount Sodium hydroxide aqueous solution appropriate amount distilled water for injection appropriate amount
2.00 mL
Reference example 1
(E) -N- {2- [2- (1H-indazol-3-yl) vinyl] -5- [4- (tert-butoxycarbonyl) piperazin-1-ylmethyl] phenyl} -3-methylthiophene-2 -Carboxamide hydrochloride (Compound 5)
Figure JPOXMLDOC01-appb-C000015
Figure JPOXMLDOC01-appb-C000015
　上記式中、-Bocは、tert-ブトキシカルボニル基(-COC(CH3)3)を表す。
＜工程１＞
　WO2006/080450の実施例108で得られる(E)-N-{5-ヒドロキシメチル-2-[2-(1H-インダゾール-3-イル)ビニル]フェニル}-3-メチルチオフェン-2-カルボキサミド(化合物4, 1.8 g, 4.6 mmol)をN,N-ジメチルホルムアミド(18 mL)に溶解し、塩化メタンスルホニル(0.68 mL, 8.8 mmol)を加えて室温にて4時間撹拌した。反応終了後、テトラヒドロフラン(90 mL)を加えて飽和炭酸水素ナトリウム水溶液(18 mL)と飽和食塩水(18 mL)にて有機層を洗浄した。有機層を減圧濃縮することで粗(E)-N-{5-クロロメチル-2-[2-(1H-インダゾール-3-イル)ビニル]フェニル}-3-メチルチオフェン-2-カルボキサミド(2.6g)を得た。 In the above formula, -Boc represents a tert-butoxycarbonyl group (-COC (CH 3 ) 3 ).
<Step 1>
(E) -N- {5-hydroxymethyl-2- [2- (1H-indazol-3-yl) vinyl] phenyl} -3-methylthiophene-2-carboxamide obtained in Example 108 of WO2006 / 080450 ( Compound 4, 1.8 g, 4.6 mmol) was dissolved in N, N-dimethylformamide (18 mL), methanesulfonyl chloride (0.68 mL, 8.8 mmol) was added, and the mixture was stirred at room temperature for 4 hours. After completion of the reaction, tetrahydrofuran (90 mL) was added, and the organic layer was washed with saturated aqueous sodium hydrogen carbonate solution (18 mL) and saturated brine (18 mL). The organic layer was concentrated under reduced pressure to give crude (E) -N- {5-chloromethyl-2- [2- (1H-indazol-3-yl) vinyl] phenyl} -3-methylthiophene-2-carboxamide (2.6 g) was obtained.
＜工程２＞
　工程１で得られた粗(E)-N-{5-クロロメチル-2-[2-(1H-インダゾール-3-イル)ビニル]フェニル}-3-メチルチオフェン-2-カルボキサミド(2.6 g)にN,N-ジメチルアセトアミド(18 mL)、N,N-ジイソプロピルエチルアミン(0.79 mL, 4.4 mmol)、および1-(tert-ブトキシカルボニル)ピぺラジン(1.2 g, 6.6 mmol)を加えて80 ℃で2時間加熱還流した。反応終了後、室温まで冷却し、水(90 mL)を加えてから、酢酸エチル抽出(36 mL)を2回行った。有機層を飽和食塩水(10 mL)にて洗浄した後、無水硫酸ナトリウムで乾燥し、減圧濃縮した。得られた残渣にエタノール(72 mL)を加えて4 ℃に冷却後、濃塩酸(1.5 mL)を添加して2.5時間撹拌した。濾過して得られる固体を乾燥し、標記化合物5(2.1 g, 77%)を得た。
1H-NMR (DMSO-d6) δ 1.42 (s, 9H), 2.50 (s, 3H), 2.94-3.47 (m, 6H), 4.06 (m, 2H), 4.38 (brs, 2H), 7.07-7.14 (m, 2H), 7.39 (t, J = 7.5 Hz, 1H) , 7.50-7.70 (m, 6H), 8.03-8.08 (m, 2H), 10.0 (br, 1H), 13.2 (br, 1H).
ESI-MS (m/z); 558 [M+H]+
参考例2
(E)-N-{2-[2-(1H-インダゾール-3-イル)ビニル]-5-(ピペラジン-1-イルメチル)フェニル}-3-メチルチオフェン-2-カルボキサミド(化合物6) <Step 2>
Crude (E) -N- {5-chloromethyl-2- [2- (1H-indazol-3-yl) vinyl] phenyl} -3-methylthiophene-2-carboxamide (2.6 g) obtained in step 1 N, N-dimethylacetamide (18 mL), N, N-diisopropylethylamine (0.79 mL, 4.4 mmol), and 1- (tert-butoxycarbonyl) piperazine (1.2 g, 6.6 mmol) were added at 80 ° C. And heated at reflux for 2 hours. After completion of the reaction, the reaction mixture was cooled to room temperature, water (90 mL) was added, and ethyl acetate extraction (36 mL) was performed twice. The organic layer was washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Ethanol (72 mL) was added to the obtained residue, and the mixture was cooled to 4 ° C., concentrated hydrochloric acid (1.5 mL) was added, and the mixture was stirred for 2.5 hr. The solid obtained by filtration was dried to obtain the title compound 5 (2.1 g, 77%).
1 H-NMR (DMSO-d 6 ) δ 1.42 (s, 9H), 2.50 (s, 3H), 2.94-3.47 (m, 6H), 4.06 (m, 2H), 4.38 (brs, 2H), 7.07- 7.14 (m, 2H), 7.39 (t, J = 7.5 Hz, 1H), 7.50-7.70 (m, 6H), 8.03-8.08 (m, 2H), 10.0 (br, 1H), 13.2 (br, 1H) .
ESI-MS (m / z); 558 [M + H] +
Reference example 2
(E) -N- {2- [2- (1H-indazol-3-yl) vinyl] -5- (piperazin-1-ylmethyl) phenyl} -3-methylthiophene-2-carboxamide (Compound 6)
Figure JPOXMLDOC01-appb-C000016
Figure JPOXMLDOC01-appb-C000016
　上記式中、-Bocは、tert-ブトキシカルボニル基(-COC(CH3)3)を表す。
　参考例１で得られる化合物5(4.0 ｇ, 6.7 mmol)をメタノール(0.16 L)、水(32 ｍL)、および濃塩酸(8 mL)の混合液に加えて70 ℃で1.5時間撹拌した。反応溶液を4 ℃まで冷却し、4 mol/L 水酸化ナトリウム水溶液(26 mL)をゆっくりと加えてpH11とした後に、減圧下にてメタノールを留去し、エタノール(40 mL)を加えて、水(40 mL)を滴下した。濾過して得られる固体を減圧乾燥し、標記化合物6(3.1 g, 定量的)を得た。
1H-NMR (DMSO-d6) δ 2.32 (br, 4H), 2.50 (s, 3H), 2.68-2.72 (m, 4H), 3.45 (s, 2H), 7.05-7.09 (m, 2H), 7.24-7.39 (m, 3H), 7.47 (d, J = 16.6 Hz, 1H), 7.53 (d, J = 9.0 Hz, 1H), 7.60 (d, J = 16.6 Hz, 1H), 7.69 (d, J = 5.0 Hz, 1H), 7.89 (d, J = 8.1 Hz, 1H), 8.00 (d, J = 7.6 Hz, 1H), 9.85 (br, 1H), 13.2 (br, 1H).
参考例3
(E)-N-{5-[4-(2-ヒドロキシアセチル)ピペラジン-1-イルメチル]-2-[2-(1H-インダゾール-3-イル)ビニル]フェニル}-3-メチルチオフェン-2-カルボキサミド(化合物(Ia))の合成 In the above formula, -Boc represents a tert-butoxycarbonyl group (-COC (CH 3 ) 3 ).
Compound 5 (4.0 g, 6.7 mmol) obtained in Reference Example 1 was added to a mixture of methanol (0.16 L), water (32 mL), and concentrated hydrochloric acid (8 mL), and the mixture was stirred at 70 ° C. for 1.5 hours. The reaction solution was cooled to 4 ° C, 4 mol / L aqueous sodium hydroxide solution (26 mL) was slowly added to adjust to pH 11, methanol was distilled off under reduced pressure, ethanol (40 mL) was added, Water (40 mL) was added dropwise. The solid obtained by filtration was dried under reduced pressure to obtain the title compound 6 (3.1 g, quantitative).
1 H-NMR (DMSO-d 6 ) δ 2.32 (br, 4H), 2.50 (s, 3H), 2.68-2.72 (m, 4H), 3.45 (s, 2H), 7.05-7.09 (m, 2H), 7.24-7.39 (m, 3H), 7.47 (d, J = 16.6 Hz, 1H), 7.53 (d, J = 9.0 Hz, 1H), 7.60 (d, J = 16.6 Hz, 1H), 7.69 (d, J = 5.0 Hz, 1H), 7.89 (d, J = 8.1 Hz, 1H), 8.00 (d, J = 7.6 Hz, 1H), 9.85 (br, 1H), 13.2 (br, 1H).
Reference example 3
(E) -N- {5- [4- (2-hydroxyacetyl) piperazin-1-ylmethyl] -2- [2- (1H-indazol-3-yl) vinyl] phenyl} -3-methylthiophene-2 -Synthesis of carboxamide (compound (Ia))
Figure JPOXMLDOC01-appb-C000017
Figure JPOXMLDOC01-appb-C000017
　参考例2で得られる化合物6(500 mg, 1.09 mmol)のN,N-ジメチルアセトアミド(10 ｍL)溶液に塩化アセトキシアセチル(0.141 mL、1.31 mmol, 1.1 当量)を加えて30分間撹拌した。水(60 mL)と飽和炭酸水素ナトリウム水溶液(80 mL)を加え、酢酸エチル(100 mL)で抽出し、有機層を飽和食塩水(100 mL)で洗浄後、無水硫酸ナトリウムで乾燥した。
　減圧濃縮して得られる残渣(化合物(Ia)粗成物)にメタノール(5.0 mL)、水(1.0 mL)、および炭酸カリウム(75 mg, 0.501 mmol)を加えて1時間撹拌した。水(5 ml)を加えてからエタノール(1.0 mL)を加え、更に水(10 mL)を滴下した。混合液を40 ℃に加熱後、4 ℃まで冷却して1時間撹拌後に濾過することにより白色固体として、化合物(Ia)[化合物(Ia)エタノール結晶化試料］(535 mg, 95%)を得た。
1H-NMR (DMSO-d6) δ 2.40 (br, 4H), 2.51 (s, 3H), 3.49 (br, 4H), 3.54 (s, 2H), 4.07 (d, J = 5.1 Hz, 2H), 4.54 (t, J = 5.1 Hz, 1H), 7.05-7.11 (m, 2H), 7.27-7.39 (m, 3H), 7.48 (d, J = 16.7 Hz, 1H), 7.51-7.58 (m, 1H), 7.61 (d, J = 16.7 Hz, 1H), 7.70 (d, J = 5.0 Hz, 1H), 7.91 (d, J = 7.9 Hz, 1H), 8.00 (d, J = 8.6 Hz, 1H), 9.87 (br, 1H), 13.1 (br, 1H). Acetoxyacetyl chloride (0.141 mL, 1.31 mmol, 1.1 equivalents) was added to a solution of compound 6 (500 mg, 1.09 mmol) obtained in Reference Example 2 in N, N-dimethylacetamide (10 mL), and the mixture was stirred for 30 minutes. Water (60 mL) and saturated aqueous sodium hydrogen carbonate solution (80 mL) were added, and the mixture was extracted with ethyl acetate (100 mL). The organic layer was washed with saturated brine (100 mL), and dried over anhydrous sodium sulfate.
Methanol (5.0 mL), water (1.0 mL), and potassium carbonate (75 mg, 0.501 mmol) were added to the residue (compound (Ia) crude product) obtained by concentration under reduced pressure, and the mixture was stirred for 1 hour. Water (5 ml) was added, ethanol (1.0 mL) was added, and water (10 mL) was further added dropwise. The mixture was heated to 40 ° C, cooled to 4 ° C, stirred for 1 hour and filtered to obtain Compound (Ia) [Compound (Ia) ethanol crystallized sample] (535 mg, 95%) as a white solid. It was.
1 H-NMR (DMSO-d 6 ) δ 2.40 (br, 4H), 2.51 (s, 3H), 3.49 (br, 4H), 3.54 (s, 2H), 4.07 (d, J = 5.1 Hz, 2H) , 4.54 (t, J = 5.1 Hz, 1H), 7.05-7.11 (m, 2H), 7.27-7.39 (m, 3H), 7.48 (d, J = 16.7 Hz, 1H), 7.51-7.58 (m, 1H ), 7.61 (d, J = 16.7 Hz, 1H), 7.70 (d, J = 5.0 Hz, 1H), 7.91 (d, J = 7.9 Hz, 1H), 8.00 (d, J = 8.6 Hz, 1H), 9.87 (br, 1H), 13.1 (br, 1H).
参考例4
(E)-N-{5-[4-(2-ヒドロキシアセチル)ピペラジン-1-イルメチル]-2-[2-(1H-インダゾール-3-イル)ビニル]フェニル}-3-メチルチオフェン-2-カルボキサミド・p-トルエンスルホン酸塩(化合物(Ia)のp-トルエンスルホン酸塩)
　参考例3で得られた化合物(Ia)(100 mg, 0.194 mmol)にエタノール3 mLを加えて65 ℃に加熱し、得られた懸濁液にp-トルエンスルホン酸1.0 mol/L エタノール溶液を430 μL(0.427 mmol)加えた。得られた溶液を室温に冷却し、一晩撹拌後さらに冷蔵庫にて2時間以上撹拌を継続した。濾取により得られた結晶を、減圧下50 ℃にて乾燥し、化合物(Ia)のp-トルエンスルホン酸塩(104 mg, 収率77.9%)を得た。
1H NMR (DMSO-d6) δ (ppm): 2.21 (s, 3H), 2.44 (s, 3H), 2.80-4.50 (12H), 6.95-7.10 (m, 4H), 7.29-7.63 (m, 8H), 7.66 (d, J = 5.0 Hz, 1H), 7.95-8.05 (m, 2H), 9.80(brs, 1H), 9.95 (s, 1H), 13.17 (brs, 1H); 
元素分析： 計算値 C35H37N5O6S2として： C, 61.12; H 5.42; N 10.18. 実測値： C, 60.91; H, 5.30; N, 10.16. Reference example 4
(E) -N- {5- [4- (2-hydroxyacetyl) piperazin-1-ylmethyl] -2- [2- (1H-indazol-3-yl) vinyl] phenyl} -3-methylthiophene-2 -Carboxamide / p-toluenesulfonate (p-toluenesulfonate of compound (Ia))
3 mL of ethanol was added to the compound (Ia) (100 mg, 0.194 mmol) obtained in Reference Example 3 and heated to 65 ° C., and p-toluenesulfonic acid 1.0 mol / L ethanol solution was added to the resulting suspension. 430 μL (0.427 mmol) was added. The resulting solution was cooled to room temperature, stirred overnight, and further stirred in a refrigerator for 2 hours or more. The crystals obtained by filtration were dried at 50 ° C. under reduced pressure to obtain p-toluenesulfonate (104 mg, yield 77.9%) of compound (Ia).
1 H NMR (DMSO-d 6 ) δ (ppm): 2.21 (s, 3H), 2.44 (s, 3H), 2.80-4.50 (12H), 6.95-7.10 (m, 4H), 7.29-7.63 (m, 8H), 7.66 (d, J = 5.0 Hz, 1H), 7.95-8.05 (m, 2H), 9.80 (brs, 1H), 9.95 (s, 1H), 13.17 (brs, 1H);
Elemental analysis: Calculated C 35 H 37 N 5 O 6 S 2:. C, 61.12; H 5.42; N 10.18 Found: C, 60.91; H, 5.30 ; N, 10.16.
　本発明により、インダゾール誘導体もしくはその薬理学的に許容される塩またはその水和物を有効成分として含有する癌治療剤等を提供することができる。 According to the present invention, a therapeutic agent for cancer containing an indazole derivative or a pharmacologically acceptable salt thereof or a hydrate thereof as an active ingredient can be provided.

Claims (8)

  1. (E)-N-{5-[4-(2-ヒドロキシアセチル)ピペラジン-1-イルメチル]-2-[2-(1H-インダゾール-3-イル)ビニル]フェニル}-3-メチルチオフェン-2-カルボキサミドもしくはその薬理学的に許容される塩またはその水和物を有効成分として含有する、大腸癌、肺癌、サルコーマ(sarcoma)、および膵臓癌から選ばれる癌の治療剤。 (E) -N- {5- [4- (2-hydroxyacetyl) piperazin-1-ylmethyl] -2- [2- (1H-indazol-3-yl) vinyl] phenyl} -3-methylthiophene-2 -A therapeutic agent for cancer selected from colorectal cancer, lung cancer, sarcoma, and pancreatic cancer, containing carboxamide or a pharmacologically acceptable salt thereof or a hydrate thereof as an active ingredient.
  2. (E)-N-{5-[4-(2-ヒドロキシアセチル)ピペラジン-1-イルメチル]-2-[2-(1H-インダゾール-3-イル)ビニル]フェニル}-3-メチルチオフェン-2-カルボキサミドもしくはその薬理学的に許容される塩またはその水和物が、(E)-N-{5-[4-(2-ヒドロキシアセチル)ピペラジン-1-イルメチル]-2-[2-(1H-インダゾール-3-イル)ビニル]フェニル}-3-メチルチオフェン-2-カルボキサミドもしくはそのp-トルエンスルホン酸塩またはその水和物である請求項1記載の癌の治療剤。 (E) -N- {5- [4- (2-hydroxyacetyl) piperazin-1-ylmethyl] -2- [2- (1H-indazol-3-yl) vinyl] phenyl} -3-methylthiophene-2 -Carboxamide or a pharmaceutically acceptable salt thereof or a hydrate thereof is (E) -N- {5- [4- (2-hydroxyacetyl) piperazin-1-ylmethyl] -2- [2- ( The therapeutic agent for cancer according to claim 1, which is 1H-indazol-3-yl) vinyl] phenyl} -3-methylthiophene-2-carboxamide or a p-toluenesulfonate thereof or a hydrate thereof.
  3. (E)-N-{5-[4-(2-ヒドロキシアセチル)ピペラジン-1-イルメチル]-2-[2-(1H-インダゾール-3-イル)ビニル]フェニル}-3-メチルチオフェン-2-カルボキサミドもしくはそのp-トルエンスルホン酸塩またはその水和物が、(E)-N-{5-[4-(2-ヒドロキシアセチル)ピペラジン-1-イルメチル]-2-[2-(1H-インダゾール-3-イル)ビニル]フェニル}-3-メチルチオフェン-2-カルボキサミドもしくはそのp-トルエンスルホン酸塩である請求項2記載の癌の治療剤。 (E) -N- {5- [4- (2-hydroxyacetyl) piperazin-1-ylmethyl] -2- [2- (1H-indazol-3-yl) vinyl] phenyl} -3-methylthiophene-2 -Carboxamide or its p-toluenesulfonate or hydrate thereof is (E) -N- {5- [4- (2-hydroxyacetyl) piperazin-1-ylmethyl] -2- [2- (1H- 3. The cancer therapeutic agent according to claim 2, which is indazol-3-yl) vinyl] phenyl} -3-methylthiophene-2-carboxamide or a p-toluenesulfonate thereof.
  4. (E)-N-{5-[4-(2-ヒドロキシアセチル)ピペラジン-1-イルメチル]-2-[2-(1H-インダゾール-3-イル)ビニル]フェニル}-3-メチルチオフェン-2-カルボキサミドもしくはそのp-トルエンスルホン酸塩が(E)-N-{5-[4-(2-ヒドロキシアセチル)ピペラジン-1-イルメチル]-2-[2-(1H-インダゾール-3-イル)ビニル]フェニル}-3-メチルチオフェン-2-カルボキサミドのp-トルエンスルホン酸塩である請求項3記載の癌の治療剤。 (E) -N- {5- [4- (2-hydroxyacetyl) piperazin-1-ylmethyl] -2- [2- (1H-indazol-3-yl) vinyl] phenyl} -3-methylthiophene-2 -Carboxamide or its p-toluenesulfonate is (E) -N- {5- [4- (2-hydroxyacetyl) piperazin-1-ylmethyl] -2- [2- (1H-indazol-3-yl) 4. The therapeutic agent for cancer according to claim 3, which is p-toluenesulfonate of vinyl] phenyl} -3-methylthiophene-2-carboxamide.
  5. (E)-N-{5-[4-(2-ヒドロキシアセチル)ピペラジン-1-イルメチル]-2-[2-(1H-インダゾール-3-イル)ビニル]フェニル}-3-メチルチオフェン-2-カルボキサミドのp-トルエンスルホン酸塩を有効成分として含有する大腸癌の治療剤。 (E) -N- {5- [4- (2-hydroxyacetyl) piperazin-1-ylmethyl] -2- [2- (1H-indazol-3-yl) vinyl] phenyl} -3-methylthiophene-2 -A therapeutic agent for colorectal cancer comprising p-toluenesulfonate of carboxamide as an active ingredient.
  6. (E)-N-{5-[4-(2-ヒドロキシアセチル)ピペラジン-1-イルメチル]-2-[2-(1H-インダゾール-3-イル)ビニル]フェニル}-3-メチルチオフェン-2-カルボキサミドのp-トルエンスルホン酸塩を有効成分として含有する肺癌の治療剤。 (E) -N- {5- [4- (2-hydroxyacetyl) piperazin-1-ylmethyl] -2- [2- (1H-indazol-3-yl) vinyl] phenyl} -3-methylthiophene-2 -A therapeutic agent for lung cancer containing p-toluenesulfonate of carboxamide as an active ingredient.
  7. (E)-N-{5-[4-(2-ヒドロキシアセチル)ピペラジン-1-イルメチル]-2-[2-(1H-インダゾール-3-イル)ビニル]フェニル}-3-メチルチオフェン-2-カルボキサミドのp-トルエンスルホン酸塩を有効成分として含有するサルコーマ(sarcoma)の治療剤。 (E) -N- {5- [4- (2-hydroxyacetyl) piperazin-1-ylmethyl] -2- [2- (1H-indazol-3-yl) vinyl] phenyl} -3-methylthiophene-2 -A therapeutic agent for sarcoma containing p-toluenesulfonate of carboxamide as an active ingredient.
  8. (E)-N-{5-[4-(2-ヒドロキシアセチル)ピペラジン-1-イルメチル]-2-[2-(1H-インダゾール-3-イル)ビニル]フェニル}-3-メチルチオフェン-2-カルボキサミドのp-トルエンスルホン酸塩を有効成分として含有する膵臓癌の治療剤。 (E) -N- {5- [4- (2-hydroxyacetyl) piperazin-1-ylmethyl] -2- [2- (1H-indazol-3-yl) vinyl] phenyl} -3-methylthiophene-2 -A therapeutic agent for pancreatic cancer comprising p-toluenesulfonate of carboxamide as an active ingredient.
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006080450A1 (en) * 2005-01-27 2006-08-03 Kyowa Hakko Kogyo Co., Ltd. Igf-1r inhibitor
WO2008001886A1 (en) * 2006-06-30 2008-01-03 Kyowa Hakko Kirin Co., Ltd. Aurora inhibitor
WO2008001885A1 (en) * 2006-06-30 2008-01-03 Kyowa Hakko Kirin Co., Ltd. Abl KINASE INHIBITOR
WO2008111441A1 (en) * 2007-03-05 2008-09-18 Kyowa Hakko Kirin Co., Ltd. Pharmaceutical composition
WO2011158931A1 (en) * 2010-06-17 2011-12-22 協和発酵キリン株式会社 Useful salts of indazole derivative

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006080450A1 (en) * 2005-01-27 2006-08-03 Kyowa Hakko Kogyo Co., Ltd. Igf-1r inhibitor
WO2008001886A1 (en) * 2006-06-30 2008-01-03 Kyowa Hakko Kirin Co., Ltd. Aurora inhibitor
WO2008001885A1 (en) * 2006-06-30 2008-01-03 Kyowa Hakko Kirin Co., Ltd. Abl KINASE INHIBITOR
WO2008111441A1 (en) * 2007-03-05 2008-09-18 Kyowa Hakko Kirin Co., Ltd. Pharmaceutical composition
WO2011158931A1 (en) * 2010-06-17 2011-12-22 協和発酵キリン株式会社 Useful salts of indazole derivative

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