FLUORINE-CONTAINING HETEROCYCLIC DERIVATIVES WITH MACROCYCLIC STRUCTURE AND USE THEREOF
20230044722 · 2023-02-09
Inventors
- Anle YANG (Chengdu, Sichuan, CN)
- Haoxi HUANG (Chengdu, Sichuan, CN)
- Tonghui CHEN (Chengdu, Sichuan, CN)
- Jie LIANG (Chengdu, Sichuan, CN)
- Dewei ZHANG (Chengdu, Sichuan, CN)
- Jingen DENG (Chengdu, Sichuan, CN)
- Hongbo LI (Chengdu, Sichuan, CN)
- Junjie PU (Chengdu, Sichuan, CN)
Cpc classification
A61K31/519
HUMAN NECESSITIES
A61P29/00
HUMAN NECESSITIES
A61P25/28
HUMAN NECESSITIES
C07D498/22
CHEMISTRY; METALLURGY
International classification
Abstract
Disclosed are a macrocyclic fluorine-containing heterocyclic derivative of formula (I), a preparation method thereof, and a use thereof in treatment of TRK kinase-mediated diseases.
##STR00001##
Claims
1-38. (canceled)
39. A compound of formula (I), a tautomer, a mesomer, a racemate, an enantiomer, a diastereomer or a mixture thereof, or a pharmaceutically acceptable salt thereof: ##STR00109##
40. The compound of formula (I), the tautomer, the mesomer, the racemate, the enantiomer, the diastereomer or the mixture thereof, or the pharmaceutically acceptable salt thereof according to claim 39, wherein the compound is in a form of a compound of formula (Ia) or (Ib), a tautomer, a mesomer, a racemate, an enantiomer, a diastereomer or a mixture thereof, or a pharmaceutically acceptable salt thereof: ##STR00110##
41. A pharmaceutical composition, comprising the compound, the tautomer, the mesomer, the racemate, the enantiomer, the diastereomer or the mixture thereof, or the pharmaceutically acceptable salt thereof according to claim 39, and a pharmaceutically acceptable diluent or carrier.
42. A compound of formula Ib-a or Ib-b or a salt thereof, or a compound of formula Ib-c or Ib-d or a salt thereof, ##STR00111## in the compound of formula Ib-a and Ib-b, wherein R.sub.1 is C.sub.1-C.sub.3 alkyl, and preferably, R.sub.1 is methyl or ethyl; in the compound of formula Ib-c and Ib-d, wherein R.sub.1 is C.sub.1-C.sub.3 alkyl, and X is halogen or ##STR00112## wherein R is selected from substituted or unsubstituted C.sub.1-C.sub.4 alkyl and a benzene ring, and the substituted C.sub.1-C.sub.4 alkyl or the benzene ring is substituted by one or more (for example, one to five) substituents selected from C.sub.1-C.sub.4 alkyl, nitryl, halogen and methoxy; and preferably, R.sub.1 is methyl or ethyl, and X is halogen or ##STR00113## wherein R is methyl or p-tolyl.
43. The compound according to claim 42, wherein the compound is selected from: ##STR00114##
44. A preparation method of a compound of formula Ib-a, comprising: subjecting a compound A and a compound B to a nucleophilic substitution reaction to obtain the compound of formula Ib-a: ##STR00115## wherein R.sub.1 is C.sub.1-C.sub.3 alkyl; and preferably, R.sub.1 is methyl or ethyl.
45. The method according to claim 44, wherein the nucleophilic substitution reaction is carried out in the presence of alkali; preferably, the alkali is selected from one or more of TEA, DIPEA and DMAP; and further preferably, the nucleophilic substitution reaction is carried out in a solvent, and preferably, the solvent is acetonitrile; and/or wherein the nucleophilic substitution reaction is carried out at a temperature higher than 30° C., preferably, the nucleophilic substitution reaction is carried out at a temperature of 50° C. to 60° C., and more preferably, the nucleophilic substitution reaction is carried out at a temperature of 60° C.
46. A preparation method of a compound of formula Ib-b, comprising: hydrolyzing a compound of formula Ib-a to obtain the compound of formula Ib-b, ##STR00116## wherein R.sub.1 is C.sub.1-C.sub.3 alkyl; and preferably, R.sub.1 is methyl or ethyl.
47. The method according to claim 46, wherein the hydrolysis reaction is carried out in the presence of alkali, preferably, the alkali is NaOH; and further preferably, the hydrolysis reaction is carried out in a solvent, and preferably, the solvent is an EtOH/H.sub.2O mixed solution, wherein V.sub.(EtOH)/V.sub.(H2O)=⅓.
48. A preparation method of a compound of formula Ib-c or a salt thereof, comprising: subjecting a compound of formula Ib-b and a compound C to an acid-amine condensation reaction to obtain the compound of formula Ib-c, wherein the compound C is ##STR00117## or a salt formed by the compound C and an acid, ##STR00118## wherein R.sub.1 is C.sub.1-C.sub.3 alkyl, and X is halogen or ##STR00119## wherein R is selected from substituted or unsubstituted C.sub.1-C.sub.4 alkyl and a benzene ring, and the substituted C.sub.1-C.sub.4 alkyl or the benzene ring is substituted by one or more (for example, one to five) substituents selected from C.sub.1-C.sub.4 alkyl, nitryl, halogen and methoxy; and preferably, R1 is methyl or ethyl, and X is halogen or ##STR00120## wherein R is methyl or p-tolyl.
49. The method according to claim 48, wherein a molar mass ratio of the compound of formula Ib-b to the compound C is 1: (1 to 2); more preferably 1: (1 to 1.5); and further preferably 1:1.1; and/or wherein the acid-amine condensation reaction is carried out in the presence of alkali and a condensing agent; further preferably, the alkali is selected from one or more of DIPEA, DMAP, TEA, N-methylmorpholine and pyridine; and further preferably, the condensing agent is selected from one or more of CDI, HBTU, BOP, PyBOP, DCC, HOBT, EDCI and HATU; and/or wherein the acid-amine condensation reaction is carried out in a solvent, and preferably, the solvent is selected from one or more of DCM, DMF, toluene, THF and acetonitrile.
50. The method according to claim 49, wherein a molar mass ratio of the compound of formula Ib-b to the alkali is 1: (2 to 5); and for example, 1: (2 to 3); and/or wherein a molar mass ratio of the compound of formula Ib-b to the condensing agent is 1: (1 to 3); and for example, 1: (1 to 2).
51. A preparation method of a compound of formula Ib-d, having the following route: ##STR00121## wherein R.sub.1 is C.sub.1-C.sub.3 alkyl, and X is halogen or ##STR00122## wherein R is substituted or unsubstituted C.sub.1-C.sub.4 alkyl or a benzene ring, and the substituted C.sub.1-C.sub.4 alkyl or the benzene ring is substituted by one or more (for example, one to five) substituents selected from C.sub.1-C.sub.4 alkyl, nitryl, halogen and methoxy; and preferably, R.sub.1 is methyl or ethyl, and X is halogen or ##STR00123## wherein R is methyl or p-tolyl.
52. The method according to claim 51, comprising: mixing a compound of formula Ib-c, a solvent, a dioxane solution of HCl or an ethyl acetate solution of HCl for reaction; wherein, further preferably, a concentration of the dioxane solution of HCl or the ethyl acetate solution of HCl ranges from 3 mol/L to 5 mol/L, and is preferably 4 mol/L; further preferably, a volume ratio of the solvent to the dioxane solution of HCl is 1: (0.8 to 1.2), and preferably 1:1; further preferably, a reaction temperature ranges from 50° C. to 55° C.; and further preferably, the solvent is acetonitrile.
53. A preparation method of a compound of formula (Ib), having the following route: ##STR00124## wherein X is halogen or ##STR00125## wherein R is substituted or unsubstituted C.sub.1-C.sub.4 alkyl or a benzene ring, and the substituted C.sub.1-C.sub.4 alkyl or the benzene ring is substituted by one or more (for example, one to five) substituents selected from C.sub.1-C.sub.4 alkyl, nitryl, halogen and methoxy; and preferably, X is halogen or ##STR00126## wherein R is methyl or p-tolyl.
54. The method according to claim 53, wherein a ring-closing reaction is carried out in the presence of alkali; further preferably, the alkali is selected from organic alkali and/or inorganic alkali; further preferably, the organic alkali is selected from one or more of DBU, sodium tert-butoxide, potassium tert-butoxide, sodium methoxide or sodium ethoxide, and the inorganic alkali is selected from one or more of potassium carbonate, sodium carbonate, sodium bicarbonate or cesium carbonate; and/or wherein a reaction temperature of the ring-closing reaction ranges from 20° C. to 50° C.; and for example, the reaction temperature is 30° C.
55. The method according to claim 54, wherein the ring-closing reaction is carried out in a solvent, and the solvent is selected from one or more of DMF, DCM, THF, acetonitrile and toluene.
56. The method according to claim 55, comprising: mixing the compound of formula Ib-d and the solvent, and then adding the alkali into a reaction system in batches for reaction, and/or wherein the alkali is the inorganic alkali, and the inorganic alkali is added into the reaction system at a temperature of 5° C. to 10° C.; further preferably, the inorganic alkali is potassium carbonate, and after the potassium carbonate is completely added, the reaction system is heated to 30° C. to 35° C. for reaction; or the inorganic alkali is sodium bicarbonate, and after the sodium bicarbonate is completely added, the reaction system is heated to 30° C. to 35° C. for reaction.
57. A method of inhibiting an activity of a TRK kinase, comprising administrating the compound, the tautomer, the mesomer, the racemate, the enantiomer, the diastereomer or the mixture thereof, or the pharmaceutically acceptable salt thereof according to claim 39, or the pharmaceutical composition thereof to a subject in need thereof.
58. A treatment method of a disease or an illness, comprising: using the compound, the tautomer, the mesomer, the racemate, the enantiomer, the diastereomer or the mixture thereof, or the pharmaceutically acceptable salt thereof according to claim 39, or the pharmaceutical composition thereof in a subject in need thereof, wherein the disease or the illness is selected from pain, cancer, inflammation and neurodegenerative diseases; preferably, the method comprises administering an effective amount of the compound, the tautomer, the mesomer, the racemate, the enantiomer, the diastereomer or the mixture thereof, or the pharmaceutically acceptable salt thereof according to claim 39, or the pharmaceutical composition thereof to the subject in need thereof; preferably, the disease or the illness is the cancer; and preferably, the cancer is selected from neuroblastoma, ovarian cancer, colorectal cancer, melanoma, head and neck cancer, gastric cancer, lung cancer, breast cancer, glioblastoma, medulloblastoma, secretory breast cancer, salivary gland carcinoma, papillary thyroid cancer, adult myeloid leukemia, pancreatic cancer, prostate cancer, appendix cancer, cholangiocarcinoma, gastrointestinal stromal tumor and infantile fibrosarcoma.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0101] The foregoing summary, as well as the following detailed description of the preferred invention, will be better understood when read in conjunction with the appended drawing:
[0102]
DETAILED DESCRIPTION OF THE INVENTION
[0103] In order to further illustrate the present application, a compound used as a TRK inhibitor, a preparation method thereof and an application thereof provided by the present application are described in detail hereinafter with reference to the following examples.
[0104] The following abbreviations have the following meanings:
[0105] HATU represents 2-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate;
[0106] HBTU represents O-benzotriazole-N,N,N′,N′-tetramethyluronium-hexafluorophosphate;
[0107] DBU represents 1,8-diazabicycloundec-7-ene;
[0108] PyBOP represents benzotriazol-1-yl-oxytripyrrolidino-phosphonium hexafluorophosphate;
[0109] EDCI represents 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide;
[0110] Cs.sub.2CO.sub.3 represents cesium carbonate;
[0111] THF represents tetrahydrofuran;
[0112] TEA represents triethylamine;
[0113] POCl.sub.3 represents phosphorus oxychloride;
[0114] DMAP represents 4-dimethylaminopyridine;
[0115] NMP represents n-methylpyrrolidone;
[0116] BOP represents a BOP reagent, which is benzotriazol-1-yloxytris(dimethylamino)-phosphonium hexafluorophosphate;
[0117] DCC represents dicyclohexylcarbodiimide;
[0118] HOBT represents 1-hydroxybenzotriazole;
[0119] SOCl.sub.2 represents thionyl chloride;
[0120] (COCl).sub.2 represents oxalyl chloride;
[0121] DCM represents dichloromethane;
[0122] CDI represents N,N′-Carbonyldiimidazole;
[0123] DMF represents N,N-Dimethylformamide;
[0124] NaOH represents sodium hydroxide;
[0125] DIPEA or DIEA represents N,N-Diisopropylethylamine;
[0126] H.sub.2O represents water;
[0127] TMSI represents trimethylsilyl iodide;
[0128] HCl/Dioxane represents a dioxane solution of hydrogen chloride;
[0129] rt represents a reaction temperature, which is a room temperature;
[0130] DIAD represents diisopropyl azodicarboxylate;
[0131] N,N-Diethylaniline represents N,N-Diethylaniline;
[0132] CH.sub.3CN or ACN represents acetonitrile;
[0133] Zn represents zinc powder;
[0134] NH.sub.4Cl represents ammonium chloride;
[0135] CH.sub.3MgBr represents methylmagnesium bromide;
[0136] LiOH represents lithium hydroxide;
[0137] PPh.sub.3 represents triphenylphosphine;
[0138] K.sub.2CO.sub.3 represents potassium carbonate;
[0139] EtOH represents ethanol;
[0140] V:V represents a volume ratio;
[0141] Pd/C represents palladium carbon;
[0142] H.sub.2 represents hydrogen;
[0143] Pd(dppf)Cl.sub.2 represents [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium;
[0144] XPhos represents 2-dicyclohexylphosphino-2,4,6-triisopropylbiphenyl;
[0145] OTs represents p-toluenesulfonyloxy,
##STR00033##
and
[0146] ee represents enantiomeric excess.
DETAILED EXAMPLES
Preparation Example 1
Preparation of ethyl 5-chloro-6-fluoropyrazo[1,5-a]pyrimidine-3-carboxylate
[0147] ##STR00034##
Step 1: preparation of 2-fluoromalonic acid
[0148] ##STR00035##
[0149] At room temperature, diethyl 2-fluoromalonate (5.0 g) and sodium hydroxide (17.3 g) were weighed and dissolved in an ethanol/water (100 mL/100 mL) mixed solution, and reacted overnight. LCMS showed that the reaction was complete. The reaction solution was concentrated to remove ethanol, and added with water (50 mL). The pH was adjusted to about 1 with concentrated hydrochloric acid. The solution was extracted with methyl tert-butyl ether for four times, and organic phases were combined, dried with anhydrous sodium sulfate, filtered and concentrated to obtain 3.7 g of the title compound, which could be used for next reaction without purification.
[0150] MS (ESI) m/z (M−H).sup.+=121.1.
Step 2: preparation of ethyl 5,7-dichloro-6-fluoropyrazo[1,5-a]pyrimidine-3-carboxylate
[0151] ##STR00036##
[0152] At room temperature, 2-fluoromalonic acid (2.0 g) and ethyl 5-amino-1H-pyrazole-4-carboxylate (1.7 g) were weighed and dissolved in phosphorus oxychloride (20 mL), then added with N,N-Dimethylformamide (2 mL) and N,N-Diethylaniline (4.9 g), and heated to 110° C. for reaction for 3 hours. LCMS showed that the reaction was complete. The reaction solution was concentrated to remove phosphorus oxychloride, and then poured into a saturated sodium bicarbonate solution (100 mL) to keep the solution alkaline. The solution was extracted with ethyl acetate for three times, dried with anhydrous sodium sulfate, filtered and concentrated. The obtained crude product was purified by column chromatography, and the obtained solid was washed with petroleum ether, and dried to obtain 1.7 g of the title compound.
[0153] MS (ESI) m/z (M+H).sup.+=278.0.
[0154] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.80 (s, 1H), 4.33 (q, J=7.2 Hz, 2H), 1.32 (t, J=7.0 Hz, 3H).
Step 3: preparation of ethyl 5-chloro-6-fluoropyrazo[1,5-a]pyrimidine-3-carboxylate
[0155] ##STR00037##
[0156] Ethyl 5,7-dichloro-6-fluoropyrazo[1,5-a]pyrimidine-3-carboxylate (1.14 g) and ammonium chloride (800 mg) were weighed and dissolved in an ethanol/tetrahydrofuran/water (30 mL/10 mL/20 mL) mixed solution, added with zinc powder (1.3 g) during stirring for reaction for 5 minutes, and then the zinc powder was filtered and removed. The filter cake was washed with ethyl acetate, the filtrate was collected, dried with anhydrous sodium sulfate, filtered and concentrated, and the obtained crude product was purified by column chromatography to obtain 800 mg of the title compound.
[0157] MS (ESI) m/z (M+H).sup.+=244.0.
[0158] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 9.93 (d, J=4.4 Hz, 1H), 8.68 (s, 1H), 4.31 (q, J=7.2 Hz, 2H), 1.31 (t, J=7.2 Hz, 3H).
Preparation Example 2
Preparation of (R)-1-(5-fluoro-2-methoxypyridin-3-yl)ethyl-1-amine hydrochloride
[0159] ##STR00038##
Step 1: preparation of (R)—N-(((5-fluoro-2-methoxypyridin-3-yl)methylene)-2-methylpropane-2-sulfenamide
[0160] ##STR00039##
[0161] (R)-2-methylpropane-2-sulfenamide (12.9 g) was dissolved in tetrahydrofuran (100 mL), and then added with 5-fluoro-2-methoxynicotinaldehyde (15.0 g) and cesium carbonate (40.9 g) in sequence. The obtained mixture reacted at room temperature for 2 hours. TLC showed that raw materials were completely consumed. The reaction solution was subjected to suction filtration, the filter cake was washed with tetrahydrofuran for three times, and the obtained filtrate was washed with a saturated sodium chloride solution once, dried with anhydrous sodium sulfate, filtered and concentrated. The obtained crude product was purified by column chromatography to obtain 23.0 g of the title compound.
[0162] MS (ESI) m/z (M+H).sup.+=259.1.
[0163] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.67 (d, J=2.4 Hz, 1H), 8.42 (d, J=3.2 Hz, 1H), 8.14 (dd, J=8.4, 3.2 Hz, 1H), 3.98 (s, 3H), 1.18 (s, 9H).
Step 2: preparation of (R)—N—((R)-1-(5-fluoro-2-methoxypyridin-3-yl)ethyl)-2-methylpropane-2-sulfenamide
[0164] ##STR00040##
[0165] (R)—N-(((5-fluoro-2-methoxypyridin-3-yl)methylene)-2-methylpropane-2-sulfenamide (5.0 g) was weighed and dissolved in tetrahydrofuran (40 mL). The obtained mixture was cooled to −78° C., and dropwise added with methylmagnesium bromide (7.8 mL, 3 M) slowly, and the temperature was kept below −65° C. After the dropwise addition was complete, the temperature was recovered to room temperature naturally, and the reaction was continued for 1 hour. TLC showed that the reaction was complete. The reaction solution was poured into a saturated ammonium chloride solution (1 L), and extracted with ethyl acetate, and organic phases were combined, washed with a saturated sodium chloride solution, dried with anhydrous sodium sulfate, filtered and concentrated. The obtained crude product was purified by column chromatography to obtain 4.5 g of the title compound.
[0166] MS (ESI) m/z (M+H).sup.+=275.2.
[0167] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.04 (d, J=2.8 Hz, 1H), 7.74 (dd, J=9.2, 3.2 Hz, 1H), 5.80 (d, J=8.8 Hz, 1H), 4.57-4.50 (m, 1H), 3.88 (s, 3H), 1.33 (d, J=6.8 Hz, 3H), 1.11 (s, 9H).
Step 3: preparation of (R)-1-(5-fluoro-2-methoxypyridin-3-yl)ethyl-1-amine hydrochloride
[0168] ##STR00041##
[0169] At room temperature, (R)—N—((R)-1-(5-fluoro-2-methoxypyridin-3-yl)ethyl)-2-methylpropane-2-sulfenamide (4.5 g) was dissolved in a 1,4-dioxane solution of hydrogen chloride (4 M, 30 mL), and reacted overnight. LCMS showed that raw materials were completely consumed. The reaction solution was concentrated to obtain 3.1 g of crude product, wherein ee was higher than 95%, and the crude product could be directly used in the next step without purification.
[0170] MS (ESI) m/z (M+H).sup.+=171.2.
[0171] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.80-8.66 (m, 3H), 8.18 (d, J=2.8 Hz, 1H), 8.04-8.00 (m, 1H), 7.09-6.60 (m, 1H), 4.51-4.45 (m, 1H), 3.90 (s, 3H), 1.49 (d, J=6.4 Hz, 3H).
Preparation Example 3
preparation of (R)-5-fluoro-3-(1-(2,2,2-trifluoroacetyl)pyrrolidin-2-yl)pyridin-2-yl trifluoromethanesulfonate
[0172] ##STR00042##
Step A: preparation of (R)-2,2,2-trifluoro-1-(2-(5-fluoro-2-methoxypyridin-3-yl)pyrrolidin-1-yl)ethyl-1-one
[0173] ##STR00043##
[0174] (R)-5-fluoro-2-methoxy-3-(pyrrolidin-2-yl)pyridine (2.6 g) was weighed and dissolved in methanol (25 mL), added with triethylamine (2.0 g), and added with ethyl trifluoroacetate (2.8 g) at 0° C. After the addition was complete, the obtained mixture reacted at room temperature overnight. After LCMS showed that the reaction was complete, the reaction solution was poured into 20 mL of water, and extracted with ethyl acetate, and organic phases were combined, washed with saturated saline once, dried with anhydrous sodium sulfate, then filtered and concentrated. The obtained crude product was purified by column chromatography to obtain 2.0 g of the title compound.
[0175] MS (ESI) m/z (M+H).sup.+=293.1
Step B: preparation of (R)-2,2,2-trifluoro-1-(2-(5-fluoro-2-methoxypyridin-3-yl)pyrrolidin-1-yl)ethyl-1-one
[0176] ##STR00044##
[0177] (R)-2,2,2-trifluoro-1-(2-(5-fluoro-2-methoxypyridin-3-yl)pyrrolidin-1-yl)ethyl-1-one (2.0 g) was weighed and dissolved in acetonitrile (20 mL), and dropwise added with iodotrimethylsilane (14.0 g) at room temperature, and reacted at room temperature overnight after the addition was complete. After the reaction was complete, the reaction solution was poured into 1 M sodium thiosulfate solution (30 mL), and extracted with dichloromethane, and organic phases were combined, washed with a saturated saline solution once, dried with anhydrous sodium sulfate, filtered and concentrated. The obtained crude product was purified by column chromatography to obtain 1.7 g of the title compound.
[0178] MS (ESI) m/z (M+H).sup.+=279.1
Step C: preparation of (R)-5-fluoro-3-(1-(2,2,2-trifluoroacetyl)pyrrolidin-2-yl)pyridin-2-yl trifluoromethanesulfonate
[0179] ##STR00045##
[0180] (R)-2,2,2-trifluoro-1-(2-(5-fluoro-2-methoxypyridin-3-yl)pyrrolidin-1-yl)ethyl-1-one (1.2 g) was weighed and dissolved in dichloromethane (12 mL), added with triethylamine (1.0 g), and dropwise added with trifluoromethanesulfonic anhydride (2.2 g) slowly, and reacted at room temperature overnight after the addition was complete. After TLC showed that the reaction was complete, the reaction solution was poured into water (20 mL), and extracted with dichloromethane, and organic phases were combined, washed with a saturated saline solution once, dried with anhydrous sodium sulfate, filtered and concentrated. The obtained crude product was purified by column chromatography to obtain 1.5 g of the title compound.
[0181] MS (ESI) m/z (M+H).sup.+=411.1
Preparation Example 4
Preparation of (R)-1-(2-methoxypyridin-3-yl)ethyl-1-amine hydrochloride
[0182] ##STR00046##
Step 1: preparation of (R, E)-N-((2-methoxypyridin-3-yl)methylene)-2-methylpropane-2-sulfenamide
[0183] ##STR00047##
[0184] (R)-2-methylpropane-2-sulfenamide (4.2 g) was dissolved in tetrahydrofuran (50 mL), and then added with 2-methoxynicotinaldehyde (4.0 g) and cesium carbonate (14.2 g) in sequence. The obtained mixture reacted at room temperature for 2 hours. TLC showed that raw materials were completely consumed. The reaction solution was subjected to suction filtration, the filter cake was washed with tetrahydrofuran for three times, and the obtained filtrate was washed with a saturated sodium chloride solution once, dried with anhydrous sodium sulfate, filtered and concentrated. The obtained crude product was purified by column chromatography to obtain 6.1 g of the title compound.
[0185] MS (ESI) m/z (M+H).sup.+=241.1
Step 2: preparation of (R)—N—((R)-1-(2-methoxypyridin-3-yl)ethyl)-2-methylpropane-2-sulfenamide
[0186] ##STR00048##
[0187] (R,E)-N-((2-methoxypyridin-3-yl)methylene)-2-methylpropane-2-sulfenamide (6.1 g) was dissolved in tetrahydrofuran (60 mL). The obtained mixture was cooled to −78° C., and then dropwise added with methylmagnesium chloride (11.0 mL, 3 M) slowly, and the temperature was kept below −65° C. during dropwise adding. After the dropwise addition was complete, the temperature was recovered to room temperature naturally, and the reaction was continued for 1 hour. The reaction solution was poured into a saturated ammonium chloride solution (1 L), and extracted with ethyl acetate, and organic phases were washed with a saturated sodium chloride solution once, dried with anhydrous sodium sulfate, filtered and concentrated. The obtained crude product was purified by column chromatography and then pulped with petroleum ether to obtain 1.0 g of the title compound.
[0188] MS (ESI) m/z (M+H).sup.+=257.2
Step 3: preparation of (R)-1-(2-methoxypyridin-3-yl)ethyl-1-amine hydrochloride
[0189] ##STR00049##
[0190] (R)—N—((R)-1-(2-methoxypyridin-3-yl)ethyl)-2-methylpropane-2-sulfenamide (1.0 g) was dissolved in dioxane hydrochloride (20 mL), and the system reacted at room temperature overnight. LCMS showed that raw materials were completely consumed. The system was concentrated, and the obtained crude product (840 mg) could be directly used in the next step.
[0191] MS (ESI) m/z (M+H).sup.+=153.2
Preparation Example 5
Preparation of 5-chloro-2-fluoropyrazo[1,5-a]pyrimidine-3-carboxylic acid
[0192] ##STR00050##
Step 1: preparation of 2-fluoropyrazo[1,5-c]pyrimidine-5-ol
[0193] ##STR00051##
[0194] 3-fluoro-1H-pyrazole-5-amine (2.1 g) and (E)-ethyl 3-ethoxyacrylate (4.5 g) was dissolved in N,N-Dimethylacetamide (50 mL), and then added with cesium carbonate (20.3 g). The obtained mixture reacted at 100° C. overnight. TLC showed that raw materials were completely consumed. The reaction solution was poured into 100 mL of water, and extracted with ethyl acetate for three times, organic phases were combined, washed with a saturated sodium chloride solution, dried with anhydrous sodium sulfate, filtered and concentrated. The obtained crude product was purified by column chromatography to obtain 2.7 g of the title compound.
[0195] MS (ESI) m/z (M+H).sup.+=154.2
Step 2: preparation of 5-chloro-2-fluoropyrazo[1,5-c]pyrimidine-3-formaldehyde
[0196] ##STR00052##
[0197] 2-fluoropyrazo[1,5-a]pyrimidine-5-ol (2.7 g) was dissolved in N,N-Dimethylacetamide (50 mL), and then added with phosphorus oxychloride (13.5 g). The obtained mixture reacted at 100° C. for 3 hours. TLC showed that raw materials were completely consumed. The reaction solution was poured into 50 mL of water for quenching, and extracted with ethyl acetate for three times, and organic phases were combined, washed with a saturated sodium chloride solution, dried with anhydrous sodium sulfate, filtered and concentrated. The obtained crude product was purified by column chromatography to obtain 3.3 g of the title compound.
[0198] MS (ESI) m/z (M+H).sup.+=200.1
Step 3: preparation of 5-chloro-2-fluoropyrazo[1,5-c]pyrimidine-3-carboxylic acid
[0199] ##STR00053##
[0200] Sodium chlorite (4.5 g) and sodium dihydrogen phosphate (3.0 g) were dissolved in water (15 mL), and slowly added into a dioxane (40 mL) solution of 5-chloro-2-fluoropyrazo[1,5-a]pyrimidine-3-formaldehyde (1.0 g) and 2-methyl-2-butene (9 mL) at 0° C. After the addition was complete, the obtained mixture reacted at room temperature for 5 hours. TLC showed that raw materials were completely consumed. The reaction solution was poured into 100 mL of water, and extracted with ethyl acetate for three times, and organic phases were combined, washed with a saturated sodium chloride solution, dried with anhydrous sodium sulfate, filtered and concentrated. The obtained crude product was purified by column chromatography to obtain 521 mg of the title compound.
[0201] MS (ESI) m/z (M+H).sup.+=216.2
Example 1
Preparation of (1.SUP.3.E,1.SUP.4.E,2.SUP.2.R,6R)-1.SUP.6.,3.SUP.5.-difluoro-6-methyl-7-aza-1(5,3)-pyrazolo[1,5-a]pyrimidine-3(3,2)-pyrazolo-2(1,2)-pyrrolidinyl cyclooctane-8-one
[0202] ##STR00054## ##STR00055##
Step 1: preparation of tert-butyl(R)-4-(5-fluoro-3-((R)-1-(2,2,2-trifluoroacetyl)pyrrolidin-2-yl)pyridin-2-yl)but-3-yn-2-yl carbamate
[0203] ##STR00056##
[0204] In argon atmosphere, (R)-5-fluoro-3-(1-(2,2,2-trifluoroacetyl)pyrrolidin-2-yl)pyridin-2-yl trifluoromethane sulfonate (2.68 g) was dissolved in acetonitrile (25 mL), and added with (R)-tert-butyl but-3-alkyn-2-yl carbamate (1.6 g), [1,1′-bis(diphenylphosphino)ferrocene]palladium dichloride (0.5 g), 2-dicyclohexyl phosphorus-2,4,6-triisopropyl biphenyl (0.6 g) and cesium carbonate (6.3 g) in sequence. The obtained mixture was heated to 80° C. for reaction for 2 hours. TLC showed that the reaction was complete. The reaction solution was cooled to room temperature and filtered, the filtrate was concentrated, and the obtained crude product was purified by column chromatography to obtain 1.9 g of the title compound.
[0205] MS (ESI) m/z (M+H).sup.+=430.2.
Step 2: preparation of tert-butyl(R)-4-(5-fluoro-3-((R)-1-(2,2,2-trifluoroacetyl)pyrrolidin-2-yl)pyridin-2-yl)butyl-2-yl)carbamate
[0206] ##STR00057##
[0207] In hydrogen atmosphere, tert-butyl(R)-4-(5-fluoro-3-((R)-1-(2,2,2-trifluoroacetyl)pyrrolidin-2-yl)pyridin-2-yl)but-3-yn-2-yl carbamate (1.6 g) was dissolved in anhydrous methanol (15 mL), and added with palladium/carbon (160 mg, 10% w/w). The obtained mixture reacted at room temperature for 4 hours. TLC showed that the reaction was complete. The reaction solution was filtered, the filtrate was concentrated, and the obtained crude product was purified by column chromatography to obtain 1.1 g of the title compound.
[0208] MS (ESI) m/z (M+H).sup.+=434.2.
Step 3: preparation of (R)-4-(5-fluoro-3-((R)-pyrrolidin-2-yl)pyridin-2-yl)but-2-amine hydrochloride
[0209] ##STR00058##
[0210] Tert-butyl(R)-4-(5-fluoro-3-((R)-1-(2,2,2-trifluoroacetyl)pyrrolidin-2-yl)pyridin-2-yl)butyl-2-yl)carbamate(600 mg) was dissolved in anhydrous ethanol (5 mL), and added with a sodium hydroxide solution (2 M, 3 mL). The obtained mixture was heated to 60° C. for reaction for 2 hours. TLC showed that the reaction was complete. The reaction solution was cooled to room temperature, added with a 1,4-dioxane solution of hydrogen chloride (4 M, 7 mL), and continuously stirred at room temperature until the reaction was complete. The system was directly concentrated to obtain 2.1 g of crude product, which could be directly used for next reaction without purification.
[0211] MS (ESI) m/z (M+H).sup.+=238.1.
Step 4: preparation of ethyl 5-((R)-2-(2-(R)-3-aminobutyl)-5-fluoropyridin-3-yl)-pyrrol-1-yl)-6-fluoropyrazo[1,5-c]pyrimidine-3-carboxylate
[0212] ##STR00059##
[0213] At room temperature, (R)-4-(5-fluoro-3-((R)-pyrrolidin-2-yl)pyridin-2-yl)but-2-amine hydrochloride (2.1 g) was dissolved in acetonitrile (10 mL), ethyl 5-chloro-6-fluoropyrazo[1,5-c]pyrimidine-3-carboxylate (369 mg) and N,N-Diisopropylethylamine (1.6 g) were added in sequence, and an obtained mixture was heated to 60° C. for reaction. After the reaction was complete, the reaction solution was filtered, the filtrate was concentrated, and the obtained crude product was purified by column chromatography to obtain 1.6 g of the title compound.
[0214] MS (ESI) m/z (M+H).sup.+=445.2.
Step 5: preparation of 5-((R)-2-(2-(R)-3-aminobutyl)-5-fluoropyridin-3-yl)-pyrrol-1-yl)-6-fluoropyrazo[1,5-c]pyrimidine-3-carboxylic acid lithium salt
[0215] ##STR00060##
[0216] At room temperature, ethyl 5-((R)-2-(2-((R)-3-aminobutyl)-5-fluoropyridin-3-yl)-pyrrol-1-yl)-6-fluoropyrazo[1,5-c]pyrimidine-3-carboxylate (400 mg) was dissolved in tetrahydrofuran (5 mL), and added with a lithium hydroxide solution (2 M, 2.7 mL). The obtained mixture was heated to 55° C. for reaction. After the reaction was complete, the reaction solution was concentrated to remove tetrahydrofuran, and freeze-dried to obtain 390 mg of crude product, which could be directly used for next reaction without purification.
[0217] MS (ESI) m/z (M+H).sup.+=417.2.
Step 6: preparation of (1.SUP.3.E,1.SUP.4.E,2.SUP.2.R,6R)-1.SUP.6.,3.SUP.5.-difluoro-6-methyl-7-aza-1(5,3)-pyrazolo[1,5-c]pyrimidine-3(3,2)-pyrazolo-2(1,2)-pyrrolidinyl cyclooctane-8-one
[0218] ##STR00061##
[0219] At room temperature, 5-((R)-2-(2-((R)-3-aminobutyl)-5-fluoropyridin-3-yl)-pyrrol-1-yl)-6-fluoropyrazo[1,5-c]pyrimidine-3-carboxylic acid lithium salt (340 mg) was dissolved in tetrahydrofuran (25 mL), added with 2-(7-benzotriazole oxide)-N,N,N′,N′-tetramethylurea hexafluorophosphate (800 mg) and N,N-Diisopropylethylamine (782 mg) in sequence, and stirred for reaction for 3 hours. TLC showed that the reaction was complete. The reaction solution was directly filtered, the solid was washed with ethyl acetate, the filtrate was concentrated to obtain 1.2 g of crude product, and the crude product was purified by column chromatography to obtain 9 mg of the title compound, wherein ee value was higher than 99%.
[0220] MS (ESI) m/z (M+H).sup.+=399.2.
[0221] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 9.20 (d, J=8.0 Hz, 1H), 8.37 (d, J=4.0 Hz, 1H), 8.13 (d, J=8.0 Hz, 1H), 8.05 (s, 1H), 7.59 (dd, J=8.0, 4.0 Hz, 1H), 5.59 (t, J=8.0 Hz, 1H), 4.29-4.23 (m, 1H), 4.20-4.10 (m, 1H), 4.07-4.03 (m, 1H), 2.86-2.68 (m, 3H), 2.49-2.43 (m, 1H), 2.23-2.18 (m, 1H), 2.10-1.99 (m, 2H), 1.76-1.69 (m, 1H), 1.21 (d, J=4.0 Hz, 3H).
Example 2
Preparation of ((1.SUP.3.E,1.SUP.4.E,2.SUP.2.R,5.SUP.1.S,5.SUP.3.S)-1.SUP.6.,3.SUP.5.-difluoro-4-oxa-6-aza-1(5,3)-pyrazolo[1,5-a]pyrimidine-3(3,2)-pyridine-2(1,2)-pyrrolidine-5(1,3)-cyclobutancycloheptan-7-one
[0222] ##STR00062## ##STR00063##
Step 1: preparation of ethyl (R)-6-fluoro-5-(2-(5-fluoro-2-methoxypyridin-3-yl)pyrrolidin-1-yl)pyrazolo[1,5-c]pyrimidine-3-carboxylate
[0223] ##STR00064##
[0224] (R)-5-fluoro-2-methoxy-3-(pyrrolidin-2-yl)pyridine hydrochloride (334 mg) was dissolved in acetonitrile (10 mL), and then added with N,N-Diisopropylethylamine (789 mg) and ethyl 5-chloro-6-fluoropyrazo[1,5-c]pyrimidine-3-carboxylate (350 mg) in sequence. The obtained mixture reacted at 60° C. for 3 hours. TLC showed that the reaction was complete. The reaction solution was poured into water (50 mL), and extracted with dichloromethane, and organic phases were combined, washed with a saturated sodium chloride solution, dried with anhydrous sodium sulfate, filtered and concentrated. The obtained crude product was purified by column chromatography to obtain 570 mg of the title compound.
[0225] MS (ESI) m/z (M+H).sup.+=404.2.
Step 2: preparation of (R)-6-fluoro-5-(2-(5-fluoro-2-methoxypyridin-3-yl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid
[0226] ##STR00065##
[0227] Ethyl (R)-6-fluoro-5-(2-(5-fluoro-2-methoxypyridin-3-yl)pyrrolidin-1-yl)pyrazolo[1,5-c]pyrimidine-3-carboxylate (570 mg) was dissolved in an ethanol/water (5 mL/15 mL) mixed solution, and then added with sodium hydroxide (282 mg). The obtained mixture reacted at 50° C. overnight. TLC showed that the reaction was complete. The reaction solution was poured into water (30 mL), The pH was adjusted to about 5 with a hydrogen chloride solution (2 M). The solution was extracted with dichloromethane, and organic phases were combined, washed with a saturated sodium chloride solution, dried with anhydrous sodium sulfate, filtered and concentrated to obtain 450 mg of the title compound, which could be directly used in the next step without purification.
[0228] MS (ESI) m/z (M+H).sup.+=376.2.
Step 3: preparation of cyclobutyl ((1R,3r)-3-(6-fluoro-5-((R)-2-(5-fluoro-2-methoxypyridin-3-yl)pyrrolidin-1-yl)pyrazolo[1,5-c]pyrimidine-3-carboxamido)-4-methylbenzenesulfonate
[0229] ##STR00066##
[0230] (R)-6-fluoro-5-(2-(5-fluoro-2-methoxypyridin-3-yl)pyrrolidin-1-yl)pyrazolo[1,5-c]pyrimidine-3-carboxylic acid (300 mg), 2-(7-azobenzotriazole)-N,N,N′,N′-tetramethylurea hexafluorophosphate (456 mg) and N,N-Diisopropylethylamine (258 mg) were dissolved in dried tetrahydrofuran (10 mL) for reaction at room temperature for 1 hour, and then added with tert-butyl (3-hydroxycyclobutyl)carbamate hydrochloride (231 mg) for continuous reaction for 1 hour. TLC showed that the reaction was complete. The reaction solution was poured into water (30 mL), and extracted with ethyl acetate, and organic phases were combined, washed with a saturated sodium chloride solution, dried with anhydrous sodium sulfate, filtered and concentrated. The obtained crude product was purified by column chromatography to obtain 350 mg of the title compound.
[0231] MS (ESI) m/z (M+H).sup.+=599.2.
Step 4: preparation of cyclobutyl ((1R,3R)-3-(6-fluoro-5-((R)-2-(5-fluoro-2-hydroxypyridin-3-yl)pyrrolidin-1-yl)pyrazolo[1,5-c]pyrimidine-3-carboxamido)-4-methylbenzenesulfonate
[0232] ##STR00067##
[0233] Cyclobutyl ((1R,3R)-3-(6-fluoro-5-((R)-2-(5-fluoro-2-methoxypyridin-3-yl)pyrrolidin-1-yl)pyrazolo[1,5-c]pyrimidine-3-carboxamido)-4-methylbenzenesulfonate (300 mg) was weighed and added into 1,4-dioxane of hydrogen chloride (4 M, 10 mL). The obtained mixture reacted at 55° C. overnight. TLC showed that the reaction was complete. The reaction solution was concentrated to remove most of the 1,4-dioxane to obtain 300 mg of crude product, which could be directly used in the next step without purification.
[0234] MS (ESI) m/z (M+H).sup.+=585.2.
Step 5: preparation of ((1.SUP.3.E,1.SUP.4.E,2.SUP.2.R,5.SUP.1.S,5.SUP.3.S)-1.SUP.6.,3.SUP.5.-difluoro-4-oxa-6-aza-1(5,3)-pyrazolo[1,5-c]pyrimidine-3(3,2)-pyridine-2(1,2)-pyrrolidine-5(1,3)-cyclobutancycloheptan-7-one
[0235] ##STR00068##
[0236] Cyclobutyl ((1R,3R)-3-(6-fluoro-5-((R)-2-(5-fluoro-2-hydroxypyridin-3-yl)pyrrolidin-1-yl)pyrazolo[1,5-c]pyrimidine-3-carboxamido)4-methylbenzenesulfonate (150 mg) was dissolved in N,N-Dimethylformamide (6 mL), and then added with potassium carbonate (116 mg). The obtained mixture reacted at room temperature for 5 hours. TLC showed that the reaction was complete. The reaction solution was purified by a preparative high performance liquid chromatography (HPLC) to obtain 44 mg of the title compound, wherein ee value was higher than 99%.
[0237] MS (ESI) m/z (M+H).sup.+=413.2.
[0238] .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 9.23 (1H, d, J=8.0 Hz), 8.73 (1H, d, J=10.4 Hz), 8.13 (1H, s), 8.03 (1H, d, J=3.2 Hz), 7.80 (1H, dd, J=10.0, 4.0 Hz), 5.93-5.90 (1H, m), 5.14-5.11 (1H, m), 4.69-4.65 (1H, m), 4.38-4.34 (1H, m), 3.95-3.91 (1H, m), 3.09-3.02 (1H, m), 2.88-2.84 (1H, m), 2.38-2.27 (2H, m), 2.16-2.06 (2H, m), 1.77-1.66 (2H, m).
Example 3
Preparation of (3.SUP.1.S,3.SUP.3.S,6.SUP.3.E,6.SUP.4.E,8R)-1.SUP.5.,6.SUP.6.-difluoro-8-methyl-2-oxo-4,7-diaza-6(3,5)-pyrazolo[1,5-a]pyrimidine-1(2,3)-pyridine-3(1,3)-cyclobutylcyclooctane-5-one (compound Ib)
[0239] ##STR00069## ##STR00070##
Step 1: preparation of ethyl (R)-6-fluoro-5-((1-(5-fluoro-2-methoxypyridin-3-yl)ethyl)amino)pyrazolo[1,5-c]pyrimidine-3-carboxylate
[0240] ##STR00071##
[0241] (R)-1-(5-fluoro-2-methoxypyridin-3-yl)ethyl-1-amine hydrochloride (1.0 g) was dissolved in acetonitrile (20 mL), and then added with N,N-Diisopropylethylamine (1.9 g) and ethyl 5-chloro-6-fluoropyrazo[1,5-c]pyrimidine-3-carboxylate (1.2 g) in sequence. The obtained mixture reacted at 60° C. for 3 hours. TLC showed that the reaction was complete. The reaction solution was poured into water (50 mL), and extracted with dichloromethane, and organic phases were combined, washed with a saturated sodium chloride solution, dried with anhydrous sodium sulfate, filtered and concentrated. The obtained crude product was purified by column chromatography to obtain 1.1 g of the title compound.
[0242] MS (ESI) m/z (M+H).sup.+=378.2.
[0243] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 9.15 (d, J=6.4 Hz, 1H), 8.49 (d, J=8.0 Hz, 1H), 8.18 (s, 1H), 8.02 (d, J=3.2 Hz, 1H), 7.67 (dd, J=9.0, 3.0 Hz, 1H), 5.60-5.52 (m, 1H), 4.18-4.10 (m, 2H), 3.93 (s, 3H), 1.50 (d, J=6.8 Hz, 3H), 1.22 (t, J=7.0 Hz, 3H).
Step 2: preparation of (R)-6-fluoro-5-(((1-(5-fluoro-2-methoxypyridin-3-yl)ethyl)amino)pyrazolo[1,5-c]pyrimidine-3-carboxylic acid
[0244] ##STR00072##
[0245] At room temperature, ethyl (R)-6-fluoro-5-((1-(5-fluoro-2-methoxypyridin-3-yl)ethyl)amino)pyrazolo[1,5-c]pyrimidine-3-carboxylate (1.1 g) was dissolved in an ethanol/water (5 mL/15 mL) mixed solution, and then added with sodium hydroxide (584 mg). The obtained mixture reacted at 50° C. overnight. TLC showed that the reaction was complete. The reaction solution was concentrated to remove ethanol, and the residual solution was poured into water (20 mL). The pH was adjusted to about 5 with a hydrogen chloride solution (2 M). The solution was extracted with dichloromethane, and organic phases were combined, washed with a saturated sodium chloride solution, dried with anhydrous sodium sulfate, filtered and concentrated to obtain 800 mg of crude product, which could be directly used in the next step without purification.
[0246] MS (ESI) m/z (M+H).sup.+=350.1.
[0247] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 11.68 (s, 1H), 9.13 (d, J=6.0 Hz, 1H), 8.51 (d, J=7.6 Hz, 1H), 8.14 (s, 1H), 8.01 (d, J=2.8 Hz, 1H), 7.72 (dd, J=9.0, 3.0 Hz, 1H), 5.59-5.52 (m, 1H), 3.92 (s, 3H), 1.50 (d, J=6.8 Hz, 3H).
Step 3: preparation of cyclobutyl ((1R,3R)-3-(6-fluoro-5-(((R)-1-(5-fluoro-2-methoxypyridin-3-yl)ethyl)amino)pyrazolo[1,5-c]pyrimidine-3-carboxamido)-4-methylbenzenesulfonate
[0248] ##STR00073##
[0249] (R)-6-fluoro-5-(((1-(5-fluoro-2-methoxypyridin-3-yl)ethyl)amino)pyrazolo[1,5-c]pyrimidine-3-carboxylic acid (800 mg), 2-(7-azobenzotriazole)-N,N,N′,N′-tetramethylurea hexafluorophosphate (1.0 g) and N,N-Diisopropylethylamine (886 mg) were dissolved in dried tetrahydrofuran (10 mL). The obtained mixture reacted at room temperature for 1 hour, and then added with tert-butyl (3-hydroxycyclobutyl)carbamate hydrochloride (953 mg) for continuous reaction for 1 hour. TLC showed that the reaction was complete. The reaction solution was poured into water (30 mL), and extracted with ethyl acetate, and organic phases were combined, washed with a saturated sodium chloride solution, dried with anhydrous sodium sulfate, filtered and concentrated. The obtained crude product was purified by column chromatography to obtain 800 mg of the title compound.
[0250] MS (ESI) m/z (M+H).sup.+=573.2.
[0251] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 9.21 (d, J=6.0 Hz, 1H), 8.57 (d, J=7.6 Hz, 1H), 8.09 (s, 1H), 8.05 (d, J=2.8 Hz, 1H), 7.80-7.78 (m, 2H), 7.66 (dd, J=8.8, 2.8 Hz, 1H), 7.60 (d, J=6.8 Hz, 1H), 7.47 (d, J=8.0 Hz, 2H), 5.44-5.37 (m, 1H), 4.96-4.92 (m, 1H), 4.33-4.28 (m, 1H), 3.80 (s, 3H), 2.47-2.38 (m, 5H), 2.24-2.18 (m, 1H), 2.12-2.08 (m, 1H), 1.52 (d, J=6.8 Hz, 3H).
Step 4: preparation of cyclobutyl ((1R,3R)-3-(6-fluoro-5-(((R)-1-(5-fluoro-2-hydroxypyridin-3-yl)ethyl)amino)pyrazolo[1,5-a]pyrimidine-3-carboxamido)-4-methylbenzenesulfonate
[0252] ##STR00074##
[0253] Cyclobutyl (1R,3R)-3-(6-fluoro-5-(((R)-1-(5-fluoro-2-methoxypyridin-3-yl)ethyl)amino)pyrazolo[1,5-c]pyrimidine-3-carboxamido)-4-methylbenzenesulfonate (800 mg) was dissolved 1,4-dioxane of hydrogen chloride (4 M, 10 mL). The obtained mixture reacted at 55° C. overnight. TLC showed that the reaction was complete. The reaction solution was directly concentrated to remove most of the 1,4-dioxane to obtain 600 mg of crude product, which could be directly used in the next step without purification.
[0254] MS (ESI) m/z (M+H).sup.+=559.2.
Step 5: preparation of (3.SUP.1.S,3.SUP.3.S,6.SUP.3.E,6.SUP.4.E,8R)-1.SUP.5.,6.SUP.6.-difluoro-8-methyl-2-oxo-4,7-diaza-6(3,5)-pyrazolo[1,5-c]pyrimidine-1(2,3)-pyridine-3(1,3)-cyclobutylcyclooctane-5-one
[0255] ##STR00075##
[0256] Cyclobutyl (1R,3R)-3-(6-fluoro-5-(((R)-1-(5-fluoro-2-hydroxypyridin-3-yl)ethyl)amino)pyrazolo[1,5-c]pyrimidine-3-carboxamido)-4-methylbenzenesulfonate (250 mg) was dissolved in N,N-Dimethylformamide (6 mL), and then added with potassium carbonate (232 mg). The system reacted at room temperature for 5 hours. TLC showed that the reaction was complete. The system was poured into water (10 mL), and extracted with ethyl acetate, and organic phases were combined, washed with a saturated sodium chloride solution, dried with anhydrous sodium sulfate, filtered and concentrated. The obtained crude product was purified by preparative thin-layer chromatography and purified by preparative high-performance liquid chromatography to obtain 58.0 mg of the title compound, wherein ee value was higher than 99.5%. .sup.1H NMR data of the compound is shown in
[0257] MS (ESI) m/z (M+H).sup.+=387.1.
[0258] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 9.22-9.16 (m, 1H), 9.15 (d, J=12.0 Hz, 1H), 8.93 (d, J=8.0 Hz, 1H), 8.10 (s, 1H), 8.05 (d, J=4.0 Hz, 1H), 7.87 (dd, J=8.8, 3.0 Hz, 1H), 5.79-5.67 (m, 1H), 5.15-5.12 (m, 1H), 4.70-4.63 (m, 1H), 3.10-3.04 (m, 1H), 2.92-2.86 (m, 1H), 2.18-2.12 (m, 1H), 1.69-1.63 (m, 1H), 1.54 (d, J=8.0 Hz, 3H).
Example 4
Preparation of (3.SUP.1.S,3.SUP.3.S,6.SUP.3.E,6.SUP.4.E,8R)-8-methyl-2-oxo-4,7-diaza-6(3,5)-pyrazolo[1,5-a]pyrimidine-1(2,3)-pyridine-3(1,3)-cyclobutylcyclooctane-5-one
[0259] ##STR00076## ##STR00077##
Step 1: preparation of ethyl (R)-5-((1-(2-methoxypyridin-3-yl)ethyl)amino)pyrazolo[1,5-c]pyrimidine-3-carboxylate
[0260] ##STR00078##
[0261] (R)-1-(2-methoxypyridin-3-yl)ethyl-1-amine hydrochloride (360 mg) was dissolved in acetonitrile (20 mL), and then added with N,N-Diisopropylethylamine (831 mg) and ethyl 5-chlorofluoropyrazo[1,5-c]pyrimidine-3-carboxylate (398 mg) in sequence. The obtained mixture reacted at 60° C. for 3 hours. TLC showed that raw materials were completely consumed. The reaction solution was poured into 50 mL of water, and extracted with dichloromethane, and organic phases were washed with a saturated sodium chloride solution, dried with anhydrous sodium sulfate, filtered and concentrated. The obtained crude product was purified by column chromatography to obtain 240 mg of the title compound.
[0262] MS (ESI) m/z (M+H).sup.+=342.2
Step 2: preparation of (R)-5-((1-(2-methoxypyridin-3-yl)ethyl)amino)pyrazolo[1,5-c]pyrimidine-3-carboxylic acid
[0263] ##STR00079##
[0264] Ethyl (R)-5-((1-(2-methoxypyridin-3-yl)ethyl)amino)pyrazolo[1,5-a]pyrimidine-3-carboxylate (240 mg) was dissolved in an ethanol/water (3 mL/9 mL) mixed solution, and then added with sodium hydroxide (140 mg) at room temperature. The obtained mixture reacted at 50° C. overnight. After the reaction was complete, the reaction solution was concentrated to remove ethanol, and then poured into water (20 mL). The pH was adjusted to about 5 with an aqueous hydrochloric acid solution (2 N). The solution was extracted with dichloromethane, and organic phases were washed with a saturated sodium chloride solution, dried with anhydrous sodium sulfate, filtered and concentrated to obtain 191 mg of the title compound, which could be directly used in the next step without purification.
[0265] MS (ESI) m/z (M+H).sup.+=314.1
Step 3: preparation of cyclobutyl (1R,3R)-3-(5-(((R)-1-(2-methoxypyridin-3-yl)ethyl)amino)pyrazolo[1,5-a]pyrimidine-3-carboxamido)4-methylbenzenesulfonate
[0266] ##STR00080##
[0267] (R)-5-((1-(2-methoxypyridin-3-yl)ethyl)amino)pyrazolo[1,5-c]pyrimidine-3-carboxylic acid (191 mg), O-(7-azabenzotriazole-1-yl)-N,N,N′N′-tetramethylurea hexafluorophosphate (255 mg) and N,N-Diisopropylethylamine (197 mg) were dissolved in dried tetrahydrofuran (10 mL). The obtained mixture reacted at room temperature for 1 hour, and then added with (1R,3R)-3-(4-methylphenylsulfonyloxy)cyclobutanamine hydrochloride (219 mg) for continuous reaction for 1 hour. The reaction solution was poured into 30 mL of water, and extracted with ethyl acetate for three times, and organic phases were washed with a saturated sodium chloride solution, combined, dried with anhydrous sodium sulfate, filtered and concentrated. The obtained crude product was purified by column chromatography to obtain 270 mg of the title compound.
[0268] MS (ESI) m/z (M+H).sup.+=537.2
Step 4: preparation of cyclobutyl (1R,3R)-3-(5-(((R)-1-(2-hydroxypyridin-3-yl)ethyl)amino)pyrazolo[1,5-a]pyrimidine-3-carboxamido)4-methylbenzenesulfonate
[0269] ##STR00081##
[0270] Cyclobutyl (1R,3r)-3-(5-(((R)-1-(2-methoxypyridin-3-yl)ethyl)amino)pyrazolo[1,5-a]pyrimidine-3-carboxamido)4-methylbenzenesulfonate (270 mg) was dissolved 1,4-dioxane of hydrogen chloride (4 M, 10 mL). The obtained mixture reacted at 55° C. overnight. After the reaction was complete, the reaction solution was concentrated to remove most of the 1,4-dioxane to obtain 250 mg of the title compound, which could be directly used for next reaction without purification.
[0271] MS (ESI) m/z (M+H).sup.+=523.2
Step 5: preparation of (3.SUP.1.S,3.SUP.3.S,6.SUP.3.E,6.SUP.4.E,8R)-8-methyl-2-oxo-4,7-diaza-6(3,5)-pyrazolo[1,5-a]pyrimidine-1(2,3)-pyridine-3(1,3)-cyclobutylcyclooctane-5-one
[0272] ##STR00082##
[0273] Cyclobutyl (1R,3R)-3-(5-(((R)-1-(2-hydroxypyridin-3-yl)ethyl)amino)pyrazolo[1,5-a]pyrimidine-3-carboxamido)4-methylbenzenesulfonate (130 mg) was dissolved in N,N-Dimethylformamide (6 mL), and then added with cesium carbonate (299 mg). After the addition was complete, the obtained mixture reacted at room temperature for 5 hours. TLC showed that the reaction was complete. The reaction solution was poured into 10 mL of water, and extracted with ethyl acetate for three times, and organic phases were combined, washed with a saturated sodium chloride solution, dried with anhydrous sodium sulfate, filtered and concentrated. The obtained crude product was purified by preparative HPLC to obtain 36.6 mg of the title compound.
[0274] MS (ESI) m/z (M+H).sup.+=351.2
[0275] .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 9.29 (1H, d, J=10.8 Hz), 8.84 (1H, d, J=7.2 Hz), 8.56 (1H, d, J=7.6 Hz), 8.04-8.02 (2H, m), 7.75 (1H, dd, J=7.6 Hz, 2.0 Hz), 7.03-7.00 (1H, m), 6.37 (1H, d, J=7.6 Hz), 5.69-5.62 (1H, m), 5.15-5.12 (1H, m), 4.69-4.62 (1H, m), 3.08-3.02 (1H, m), 2.91-2.85 (1H, m), 2.16-2.11 (1H, m), 1.70-1.65 (1H, m), 1.45 (3H, d, J=7.2 Hz).
Example 5
Preparation of (3.SUP.1.S,3.SUP.3.S,6.SUP.3.E,6.SUP.4.E,8R)-6.SUP.6.-fluoro-8-methyl-2-oxo-4,7-diaza-6(3,5)-pyrazolo[1,5-a]pyrimidine-1(2,3)-pyridine-3(1,3)-cyclobutylcyclooctane-5-one
[0276] ##STR00083## ##STR00084##
Step 1: preparation of ethyl (R)-6-fluoro-5-((1-(2-methoxypyridin-3-yl)ethyl)amino)pyrazolo[1,5-c]pyrimidine-3-carboxylate
[0277] ##STR00085##
[0278] (R)-1-(2-methoxypyridin-3-yl)ethyl-1-amine hydrochloride (390 mg) was dissolved in acetonitrile (20 mL), and then added with N,N-Diisopropylethylamine (898 mg) and ethyl 5-chloro-6-flupyrazolo[1,5-c]pyrimidine-3-carboxylate (508 mg) in sequence. The obtained mixture reacted at 60° C. for 3 hours. TLC showed that raw materials were completely consumed. The reaction solution was poured into 50 mL of water, and extracted with dichloromethane for three times, and organic phases were combined, washed with a saturated sodium chloride solution, dried with anhydrous sodium sulfate, filtered and concentrated. The obtained crude product was purified by column chromatography to obtain 420 mg of the title compound.
[0279] MS (ESI) m/z (M+H).sup.+=360.2
Step 2: preparation of (R)-6-fluoro-5-((1-(2-methoxypyridin-3-yl)ethyl)amino)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid
[0280] ##STR00086##
[0281] Ethyl (R)-6-fluoro-5-((1-(2-methoxypyridin-3-yl)ethyl)amino)pyrazolo[1,5-a]pyrimidine-3-carboxylate (420 mg) was dissolved in an ethanol/water (4 mL/12 mL) mixed solution, and then added with sodium hydroxide (187 mg) at room temperature. After the addition was complete, the obtained mixture reacted at 50° C. overnight. After the reaction was complete, the reaction solution was concentrated to remove ethanol, and then poured into water (20 mL). The pH was adjusted to about 5 with an aqueous hydrochloric acid solution (2 N). The solution was extracted with dichloromethane for three times, and organic phases were combined, washed with a saturated sodium chloride solution, dried with anhydrous sodium sulfate, filtered and concentrated to obtain 356 mg of the title compound, which could be directly used in the next step without purification.
[0282] MS (ESI) m/z (M+H).sup.+=332.1
Step 3: preparation of cyclobutyl (1R,3R)-3-(6-fluoro-5-(((R)-1-(2-methoxypyridin-3-yl)ethyl)amino)pyrazolo[1,5-a]pyrimidine-3-carboxamido)4-methylbenzenesulfonate
[0283] ##STR00087##
[0284] (R)-6-fluoro-5-((1-(2-methoxypyridin-3-yl)ethyl)amino)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid (356 mg), O-(7-azabenzotriazole-1-yl)-N,N,N′,N′-tetramethylurea hexafluorophosphate (494 mg) and N,N-Diisopropylethylamine (418 mg) were dissolved in dried tetrahydrofuran (10 mL). The obtained mixture reacted at room temperature for 1 hour, and then added with (1R,3R)-3-(4-methylphenylsulfonyloxy)cyclobutanamine hydrochloride (388 mg) for continuous reaction for 1 hour. The reaction solution was poured into 30 mL of water, and extracted with ethyl acetate for three times, and organic phases were combined, washed with a saturated sodium chloride solution, dried with anhydrous sodium sulfate, filtered and concentrated. The obtained crude product was purified by column chromatography to obtain 500 mg of the title compound.
[0285] MS (ESI) m/z (M+H).sup.+=555.2
Step 4: preparation of cyclobutyl (1R,3R)-3-(6-fluoro-5-(((R)-1-(2-hydroxypyridin-3-yl)ethyl)amino)pyrazolo[1,5-c]pyrimidine-3-carboxamido)4-methylbenzenesulfonate
[0286] ##STR00088##
[0287] Cyclobutyl (1R,3R)-3-(6-fluoro-5-(((R)-1-(2-methoxypyridin-3-yl)ethyl)amino)pyrazolo[1,5-c]pyrimidine-3-carboxamido)4-methylbenzenesulfonate (500 mg) were added into 1,4-dioxane of hydrogen chloride (4 M, 10 mL). The obtained mixture reacted at 55° C. overnight. After the reaction was complete, the reaction solution was concentrated to remove most of the 1,4-dioxane to obtain 280 mg of the title compound, which could be directly used for next reaction without purification.
[0288] MS (ESI) m/z (M+H).sup.+=541.2
Step 5: preparation of (3.SUP.1.S,3.SUP.3.S, 6.SUP.3.E,6.SUP.4.E,8R)-6.SUP.6.-fluoro-8-methyl-2-oxo-4,7-diaza-6(3,5)-pyrazolo[1,5-c]pyrimidine-1(2,3)-pyridine-3(1,3)-cyclobutylcyclooctane-5-one
[0289] ##STR00089##
[0290] Cyclobutyl (1R,3R)-3-(6-fluoro-5-(((R)-1-(2-hydroxypyridin-3-yl)ethyl)amino)pyrazolo[1,5-c]pyrimidine-3-carboxamido)4-methylbenzenesulfonate (150 mg) was dissolved in N,N-Dimethylformamide (6 mL), and then added with potassium carbonate (144 mg). After the addition was complete, the obtained mixture reacted at room temperature for 5 hours. The reaction solution was poured into 10 mL of water, and extracted with ethyl acetate for three times, and organic phases were combined, washed with a saturated sodium chloride solution, dried with anhydrous sodium sulfate, filtered and concentrated. The obtained crude product was purified by preparative HPLC to obtain 45.2 mg of the title compound.
[0291] MS (ESI) m/z (M+H).sup.+=369.2
[0292] .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 9.21-9.15 (2H, m), 9.02 (1H, d, J=7.6 Hz), 8.08 (1H, s), 8.04-8.02 (1H, m), 7.92 (1H, dd, J=7.6 Hz, 5.2 Hz), 7.05-7.02 (1H, m), 5.80-5.73 (1H, m), 5.17-5.13 (1H, m), 4.70-4.63 (1H, m), 3.09-3.03 (1H, m), 2.92-2.86 (1H, m), 2.17-2.12 (1H, m), 1.70-1.64 (1H, m), 1.52 (3H, d, J=7.2 Hz).
Example 6
Preparation of (3.SUP.1.S,3.SUP.3.S,6.SUP.3.Z,6.SUP.4.E,8R)-1.SUP.5.,6.SUP.2.-difluoro-8-methyl-2-oxo-4,7-diaza-6(3,5)-pyrazolo[1,5-a]pyrimidine-1(2,3)-pyridine-3(1,3)-cyclobutylcyclooctane-5-one
[0293] ##STR00090##
Step 1: preparation of (R)-2-fluoro-5-((1-(5-fluoro-2-methoxypyridin-3-yl)ethyl)amino)pyrazolo[1,5-c]pyrimidine-3-carboxylic acid
[0294] ##STR00091##
[0295] (R)-1-(5-fluoro-2-methoxypyridin-3-yl)ethyl-1-amine hydrochloride (702 mg) was dissolved in acetonitrile (20 mL), and then added with N,N-Diisopropylethylamine (1.2 g) and 5-chloro-2-flupyrazolo[1,5-c]pyrimidine-3-carboxylic acid (521 mg) in sequence. The obtained mixture reacted at 50° C. for 3 hours. TLC showed that raw materials were completely consumed. The reaction solution was poured into 50 mL of water, and extracted with ethyl acetate for three times, and organic phases were combined, washed with a saturated sodium chloride solution, dried with anhydrous sodium sulfate, filtered and concentrated to obtain 150 mg of crude product of the title compound.
[0296] MS (ESI) m/z (M+H).sup.+=350.2
Step 2: preparation of cyclobutyl (1R,3R)-3-(2-fluoro-5-(((R)-1-(5-fluoro-2-methoxypyridin-3-yl)ethyl)amino)pyrazolo[1,5-a] pyrimidine-3-carboxamido)4-methylbenzenesulfonate
[0297] ##STR00092##
[0298] (R)-2-fluoro-5-((1-(5-fluoro-2-methoxypyridin-3-yl)ethyl)amino)pyrazolo[1,5-c]pyrimidine-3-carboxylic acid (150 mg, crude product), O-(7-azabenzotriazole-1-yl)-N,N,N′,N′-tetramethylurea hexafluorophosphate (70 mg) and N,N-Diisopropylethylamine (222 mg) were dissolved in dried tetrahydrofuran (10 mL). The obtained mixture reacted at room temperature for 1 hour, and then added with (1R,3R)-3-(4-methylphenylsulfonyloxy)cyclobutanamine hydrochloride (119 mg) for continuous reaction for 1 hour. The reaction solution was poured into 30 mL of water, and extracted with ethyl acetate for three times, and organic phases were combined, washed with a saturated sodium chloride solution, dried with anhydrous sodium sulfate, filtered and concentrated. The obtained crude product was purified by column chromatography to obtain 240 mg of the title compound.
[0299] MS (ESI) m/z (M+H).sup.+=573.2
Step 3: preparation of cyclobutyl (1R,3R)-3-(2-fluoro-5-(((R)-1-(5-fluoro-2-hydroxypyridin-3-yl)ethyl)amino)pyrazolo[1,5-a]pyrimidine-3-carboxamido)4-methylbenzenesulfonate
[0300] ##STR00093##
[0301] Cyclobutyl (1R,3R)-3-(2-fluoro-5-(((R)-1-(5-2-methoxypyridin-3-yl)ethyl)amino)pyrazolo[1,5-c]pyrimidine-3-carboxamido)4-methylbenzenesulfonate (240 mg) was dissolved in acetonitrile (10 mL), and then added with 1,4-dioxane of hydrogen chloride (4 M, 5 mL). The obtained mixture reacted at 60° C. overnight. After the reaction was complete, the reaction solution was concentrated to remove dioxane to obtain 154 mg of the title compound, which could be directly used for next reaction without purification.
[0302] MS (ESI) m/z (M+H).sup.+=559.2
Step 4: preparation of (3.SUP.1.S,3.SUP.3.S,6.SUP.3.Z,6.SUP.4.E,8R)-1.SUP.5.,6.SUP.2.-difluoro-8-m ethyl-2-oxo-4,7-diaza-6(3,5)-pyrazolo[1,5-a]pyrimidine-1(2,3)-pyridine-3(1,3)-cyclobutylcyclooctane-5-one
[0303] ##STR00094##
[0304] Cyclobutyl (1R,3r)-3-(2-fluoro-5-(((R)-1-(5-fluoro-2-hydroxypyridin-3-yl)ethyl)amino)pyrazolo[1,5-c]pyrimidine-3-carboxamido)4-methylbenzenesulfonate (154 mg) was dissolved in N,N-Dimethylformamide (6 mL), and then added with cesium carbonate (144 mg). After the addition was complete, the obtained mixture reacted at room temperature for 3 hours. The reaction solution was poured into 10 mL of water, and extracted with ethyl acetate for three times, and organic phases were combined, washed with a saturated sodium chloride solution, dried with anhydrous sodium sulfate, filtered and concentrated. The obtained crude product was purified by preparative HPLC to obtain 28.0 mg of the title compound.
[0305] MS (ESI) m/z (M+H).sup.+=387.1
[0306] .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 9.11 (1H, d, J=10.8 Hz), 8.90 (1H, d, J=7.2 Hz), 8.46 (1H, d, J=7.6 Hz), 8.05 (1H, d, J=2.8 Hz), 7.68 (1H, dd, J=8.8 Hz, 3.2 Hz), 6.43 (1H, d, J=7.6 Hz), 5.62-5.59 (1H, m), 5.14-5.10 (1H, m), 4.66-4.64 (1H, m), 3.08-3.03 (1H, m), 2.90-2.85 (1H, m), 2.16-2.11 (1H, m), 1.69-1.64 (1H, m), 1.47 (3H, d, J=6.8 Hz).
Testing Example 1
kinase Inhibitory Activity
[0307] Sundia Meditech (Shanghai) Co., Ltd. was commissioned to complete the testing example.
1. Experimental Purpose
[0308] Inhibitory activities of the compound of the present application on three kinases TRKa, TRKA(G595R) and TRKC(G623R) were measured.
2. Experimental Materials
[0309] 2.2.1 Reagents and Consumables
TABLE-US-00001 Name of reagent Supplier Article number Batch number TRKa Carna 08-186 13CBS-0565G TRKA (G595R) signalchem N16-12BG-100 H2714-7 TRKC(G623R) signalchem N18-12CH-100 D2567-8 Kinase substrate 22 GL 112393 P190329-SL112393 DMSO Sigma D8418-1L SHBG3288V 384-well plate PerkinElmer 6007290 810712 (white)
[0310] 2.2.2 Instruments
[0311] Centrifuge (manufacturer: Eppendorf, model number: 5430); microplate reader (manufacturer: Perkin Elmer, model number: Caliper EZ ReaderII); and Echo 550 (manufacturer: Labcyte, model number: Echo 550)
3. Experimental Method
[0312] 3.1 A tested compound was accurately weighed and dissolved in 100% DMSO to prepare into a 10 mM solution.
[0313] 3.2 Kinase Reaction Process
[0314] 3.2.1 1×kinase buffer was prepared.
[0315] 3.2.2 Preparation of compounds of gradient concentrations: the tested compound had an initial concentration of 1000 nM, and was diluted into a 100% DMSO solution of 100 times final concentration in the 384-well plate, and then the 100% DMSO solution was diluted by 3 times to obtain DMSO solutions of the compound with 10 concentrations. 250 nL of the compound of 100 times final concentration was transferred to a reaction plate OptiPlate-384F by a dispenser Echo 550.
[0316] 3.2.3 A kinase solution of 2.5 times final concentration was prepared with 1×kinase buffer.
[0317] 3.2.4 10 μL of kinase solution of 2.5 times final concentration was added into a compound well and a positive control well respectively; and 10 μL of 1×kinase buffer was added into a negative control well.
[0318] 3.2.5 The reaction plate was shaken and mixed evenly, and then incubated at room temperature for 10 minutes.
[0319] 3.2.6 Mixed solutions of ATP and kinase substrate 22 of 5 times and 3 times final concentrations were prepared with 1×kinase buffer.
[0320] 3.2.7 15 μL of mixed solution of ATP and substrate of 5 times or 3 times final concentration was added.
[0321] 3.2.8 The 384-well plate was centrifuged at 1,000 rpm for 30 seconds, shaken and mixed evenly, and then incubated at room temperature for corresponding time.
[0322] 3.2.9 The kinase reaction was terminated.
[0323] 3.2.10 A conversion rate was read by the microplate reader Caliper EZ Reader.
[0324] 4. Data Analysis
[0325] Calculation formula
Inhibition rate %=(conversion rate % maximum−conversion rate % sample)/(conversion rate % maximum−conversion rate % minimum)×100
[0326] wherein the conversion rate % sample was: a reading number of a conversion rate of a sample; the conversion rate % minimum was: an average value of the negative control well, which represented a reading number of a conversion rate without an enzyme activity well; and the conversion rate % maximum was: an average value of a ratio of the positive control well, which represented a reading number of a conversion rate without a compound inhibition well.
[0327] Fitting of dose-effect curve: a log value of concentration was used as an X axis, a percentage inhibition rate was used as a Y axis, and the dose-effect curve was fitted by a log (inhibitor) vs. response-variable slope of the analysis software GraphPad Prism 5 (four-parameter model fitting), so as to obtain an IC.sub.50 value of each compound on enzyme activity.
[0328] Results are shown in the following Table 1:
TABLE-US-00002 TABLE 1 IC.sub.50 values of inhibitory activities of the compound of the present application on three kinases Serial number TRKa TRKA(G595R) TRKC(G623R) of example IC.sub.50 (nM) IC.sub.50 (nM) IC.sub.50 (nM) 1 0.62 2.50 3.70 LOXO-195 0.47 1.60 2.50 2 0.42 1.20 1.80 Positive control 1 0.39 0.72 1.70 3 0.36 0.89 1.50 Positive control 2 0.43 0.97 1.40 4 320 >1000 >1000 5 127 561 >1000 6 0.52 2.90 8.20
[0329] LOXO-195 is a second generation TRK inhibitor, with the following chemical structure:
##STR00095##
[0330] The positive control 1 is the compound in example 5 of patent WO2019210835, with the following chemical structure:
##STR00096##
[0331] The positive control 2 is the compound in example 6 of patent WO2019210835, with the following chemical structure:
##STR00097##
[0332] From biological activity data of the compound in the specific examples, the compound of the present application has high inhibitory activities on three kinases TRKa, TRKA(G595R) and TRKC(G623R). Examples 3 to 6 show that effects of fluorine substitution at different positions on the inhibitory activities on three kinases are quite different, and the fluorine substitution at different positions produces unexpected technical effects.
Testing Example 2
In-Vitro Cell Viability
[0333] Hefei Precedo Biomedical Technology Co., Ltd. was commissioned to complete the testing example, and NTRK mutant cells used were constructed by this company.
1. Experimental Purpose
[0334] Inhibitory effects of the compound of the present application on growth of three NTRK mutant cells (Ba/F3 LMNA-NTRK1-G667C, Ba/F3 EVT6-NTRK3-G623R and Ba/F3 LMNA-NTRK1-G595R) were measured.
2. Reagents and Consumables
Cell Lines:
[0335]
TABLE-US-00003 Quantity of Cell line Cell type cells/well Medium Ba/F3 LMNA- Suspension 3000 RPMI-1640 + NTRK1-G667C 10% FBS Ba/F3 EVT6- Suspension 3000 RPMI-1640 + NTRK3-G623R 10% FBS Ba/F3 LMNA- Suspension 3000 RPMI-1640 + NTRK1-G595R 10% FBS
Reagents:
[0336] Fetal calf serum FBS (GBICO, Cat#10099-141); CellTiter-Glo® Luminescent Cell Viability Assay (CTG, Promega, Cat#G7573); 96-well transparent flat-bottom black wall panel (Thermo®, Cat#165305); and RPMI-1640 (Hyclone, Cat#SH30809.01).
3. Experimental Process
[0337] 3.1 Cell Culture and Inoculation:
[0338] Cells in logarithmic growth phase were collected and counted by a blood platelet counter, a cell concentration was adjusted to 3 to 6×10.sup.4 cell/mL, 90 μL of cell suspension was added into a 96-well plate, and cells in the 96-well plate were cultured at 37° C. under 5% CO.sub.2 and 95% humidity overnight.
[0339] 3.2 Drug Dilution and Addition:
[0340] The to-be-tested compound was prepared into a 10 times drug solution in a medium containing 1% DMSO, with a highest concentration of 10 and the drug solution was diluted by 3 times in sequence to obtain drug solutions with 9 concentrations in total. More than 10 μL of prepared drug solution was added into each well of the 96-well plate in which the cells were inoculated. Then, 90 μL of medium containing 1% DMSO was added to obtain a drug solution with a highest concentration of 1 μM, and the drug solution was diluted by 3 times in sequence to obtain drug solutions with 9 concentrations in total. A final concentration of DMSO in each well was 0.1%, and three multiple wells were provided for the drug of each concentration. The cells in the 96-well plate in which the drug has been added were cultured at 37° C. under 5% CO.sub.2 and 95% humidity for 72 hours, and then subjected to CTG analysis.
[0341] 3.3 End-Point Plate Reading
[0342] An equal volume of CTG solution was added into each well, the 96-well plate was placed at room temperature for 20 minutes to stabilize a luminescence signal, and a luminescence value was read.
4. Data Processing
[0343] Data was analyzed by GraphPad Prism 7.0 software, and fitted by nonlinear S-curve regression to obtain a dose-effect curve, from which an IC.sub.50 value was calculated.
Cell survival rate (%)=(Lum to-be-tested drug−Lum medium control)/(Lum cell control−Lum medium control)×100%.
[0344] Experimental results are shown in the following Table 2:
TABLE-US-00004 TABLE 2 IC.sub.50 values of inhibitory activities of the compound of the present application on three cells Ba/F3 LMNA- Ba/F3 EVT6- Ba/F3 LMNA- Number of NTRK1-G595R NTRK3-G623R NTRK1-G667C example IC.sub.50 (nM) IC.sub.50 (nM) IC.sub.50 (nM) 1 13 190 5.9 LOXO-195 12 145 6.3 2 2.3 14 3.0 Positive 1.8 18 1.8 control 1 3 4.5 25 3.2 Positive 13 107 6.5 control 2 4 >1000 >1000 >1000 5 >1000 >1000 >1000 6 116 536 40
[0345] LOXO-195 is a second generation TRK inhibitor, with the following chemical structure:
##STR00098##
[0346] The positive control 1 is the compound in example 5 of patent WO2019210835, with the following chemical structure:
##STR00099##
[0347] The positive control 2 is the compound in example 6 of patent WO2019210835, with the following chemical structure:
##STR00100##
[0348] From biological activity data of the compound in the specific examples, the compound of the present application has strong growth inhibition effects on three NTRK mutant cells (Ba/F3 LMNA-NTRK1-G667C, Ba/F3 EVT6-NTRK3-G623R and Ba/F3 LMNA-NTRK1-G595R). The inhibitory activities of the compounds in example 1 and example 2 on three NTRK mutant cells are not significantly different from those of the corresponding positive compound, but the inhibitory activities of the compound in example 3 of the present application on three NTRK mutant cells are significantly higher than those of the corresponding positive control 2. Examples 3 to 6 show that effects of fluorine substitution at different positions on the inhibitory activities on three NTRK mutant cells are quite different, and the fluorine substitution at different positions produces unexpected technical effects.
[0349] From the point of view of clinical medication, according to data of Ba/F3 EVT6-NTRK3-G623R in Table 2, the inhibitory activity of the compound in example 3 of the present application is 4 times higher than that of the positive control compound 2, and only a lower dosage is needed clinically for a patient with NTRK3-G623R mutation.
Testing Example 3
Stability Test in Liver Microsomes
1. Experimental Purpose
[0350] A stability of the compound of the present application in liver microsomes of human, rat and mouse was measured.
2. Experimental Materials and Instruments
Reagents and Consumables:
[0351]
TABLE-US-00005 Name of reagent Supplier Item number Batch number Liver microsome of human BiolVT X008070 SDL Liver microsome of rat BiolVT M00001 TIQ Liver microsome of mouse Biopredic MIC255037 BQM
3. Experimental Steps
[0352] 3.1 A buffer and a liver microsome were prepared into an incubation solution according to the following table:
TABLE-US-00006 Reagent Concentration Volume Phosphate buffer 100 mM 216.25 μL Liver microsome 20 mg/mL 6.25 μL
[0353] 3.2 The following two experiments were carried out respectively: a) incubation system added with coenzyme factor NADPH: 25 μL of NADPH (10 mM) was added into an incubation solution (mainly comprising a liver microsome and a phosphate buffer), so that final concentrations of the liver microsome and the NADPH were 0.5 mg/mL and 1 mm respectively; and b) incubation system without coenzyme factor NADPH: 25 μL of phosphate buffer (100 mM) was added into the incubation solution, so that the final concentration of the liver microsome was 0.5 mg/mL. The incubation systems above were preheated at 37° C. for 10 minutes respectively.
[0354] 3.3 2.5 μL of positive control compound or a tested compound solution (100 μM) was added into the incubation systems described in “step 3.2” above respectively for reaction, and the positive control compound was verapamil (purchased from Sigma), so that a final concentration of the tested compound of the present application or the positive control compound was 1 μM. The incubation solution added with the compound was incubated in water at 37° C. in batches.
[0355] 3.4 30 μL of aliquot was taken from a reaction solution after 0.5 minute, 5 minutes, 15 minutes, 30 minutes and 45 minutes respectively, and the reaction was terminated by adding 5 times volume of cold acetonitrile containing 200 nM caffeine and 100 nM tolbutamide. The aliquot was centrifuged at a gravity acceleration of 3,220 g for 40 minutes, and 100 μL of supernatant was mixed with 100 μL of ultrapure water, and then used for LC-MS/MS analysis.
[0356] 3.5 Data Analysis
[0357] A peak area was determined by an extracted ion chromatogram. A slope value k was determined by linear regression of a remaining percentage of a parent drug relative to a natural logarithm of an incubation time curve.
[0358] In-vitro half-life periods (t.sub.1/2) were respectively calculated and determined according to the slope value, and an average value of the in-vitro half-life periods was converted into an in-vitro intrinsic clearance rate (CLint, represented by μL/min/mg protein).
[0359] Experimental results are shown in the following Table 3:
TABLE-US-00007 TABLE 3 Stability data of the compound of the present application in liver microsomes of human, rat and mouse t.sub.1/2 (min) CL.sub.int (μL/min/mg) % Remaining(45 min) Species Example Human Rat Mouse Human Rat Mouse Human Rat Mouse 1 8.17 7.35 16.76 169.73 188.59 82.72 4.24 3.30 16.15 LOXO-195 16.78 9.12 7.45 82.65 152.03 186.01 16.23 4.88 1.98 2 16.11 6.50 13.03 86.01 213.62 106.46 14.38 2.37 9.22 Positive control 1 15.02 11.27 17.63 92.30 123.02 78.63 12.43 9.03 16.90 3 127.85 60.77 43.21 11.11 22.81 32.10 69.89 51.22 39.49 Positive control 2 84.82 46.29 18.94 16.36 29.96 73.23 67.39 40.95 11.64
[0360] LOXO-195 is a second generation TRK inhibitor, with the following chemical structure:
##STR00101##
[0361] The positive control 1 is the compound in example 5 of patent WO2019210835, with the following chemical structure:
##STR00102##
[0362] The positive control 2 is the compound in example 6 of patent WO2019210835, with the following chemical structure:
##STR00103##
[0363] From data of the compound in the specific examples, the compound of the present application has a good stability in the liver microsomes of human, rat and mouse. The stabilities of the compounds in example 1 and example 2 in the liver microsomes of human, rat and mouse are not significantly different from that of the corresponding positive compound, or the stabilities of the compounds in example 1 and example 2 in the liver microsomes of most species are worse. However, the stability of the compound in example 3 in the microsomes of human, rat and mouse liver is obviously better than that of the positive control 2.
Testing Example 4
In-Vivo Pharmacokinetic Study on SD Rats Intravenously and Orally Administrated with Tested Compound
1. Experimental Animals
[0364] Species: SD rats, SPF level. Source: the animals were transferred from the animal repository (999M-017) of the experimental institution of Shanghai Sippe-Bk Lab Animal Co., Ltd. Quantity: 3 rats for each formulation.
2. Preparation of Test Samples
[0365] 2.1 An appropriate amount of test samples were accurately weighed, added to a final volume of 5% DMSO, 10% polyethylene glycol-15 hydroxystearate and 85% normal saline, and fully mixed by vortex or ultrasound to obtain 0.2 mg/mL administration solution for intravenous administration.
[0366] 2.2 An appropriate amount of test samples were accurately weighed, added to a final volume of 5% DMSO, 10% polyethylene glycol-15 hydroxystearate and 85% normal saline, and fully mixed by vortex or ultrasound to obtain 0.5 mg/mL administration solution for oral and intragastric administration.
3. Experimental Design
[0367]
TABLE-US-00008 Administration Test Quantity Dosage Concentration volume Administration Sample Group sample (male) (mg/kg) (mg/mL) (mL/kg) mode collection 1 Example 2 3 1 0.2 5 IV Plasma 2 3 5 0.5 10 PO Plasma 3 Positive 3 1 0.2 5 IV Plasma 4 control 1 3 5 0.5 10 PO Plasma 5 Example 3 3 1 0.2 5 IV Plasma 6 3 5 0.5 10 PO Plasma 7 Positive 3 1 0.2 5 IV Plasma 8 control 2 3 5 0.5 10 PO Plasma
4. Administration Mode
[0368] Weighing was carried out before administration, and the dosage was calculated according to the weight. Intravenous administration, or oral intragastric administration was carried out.
5. Time Point of Blood Sampling
[0369] The blood sampling was carried out before administration and 0.083 hour, 0.25 hour, 0.5 hour, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, and 24 hours after administration.
6. Sample Collection and Disposition
[0370] The blood sampling was carried out through a jugular vein or other suitable modes, and about 0.20 mL of each sample was collected. Heparin sodium was used for anticoagulation, the collected blood samples were placed on ice, and plasma was centrifuged within 2 hours (the centrifugation was carried out by a centrifugal force of 6,800 g for 6 minutes at 2° C. to 8° C.). The collected plasma samples were stored in a refrigerator at −80° C. before analysis, and the remaining plasma samples after analysis were continuously stored in the refrigerator at −80° C. for temporary storage.
7. Biological Analysis and Data Processing
[0371] A plasma concentration of a test subject was detected, a plasma concentration-time curve was drawn, and BLQs were all recorded as 0. When pharmacokinetic parameters were calculated, a concentration before administration was calculated as 0; BLQ appearing before C.sub.max (comprising “No peak”) was calculated as 0; and BLQ appearing after C.sub.max (comprising “No peak”) did not participate in the calculation. According to blood concentration data at different time points, the pharmacokinetic parameters such as AUC(0-t), T.sub.1/2 and C.sub.max were calculated by WinNonlin.
TABLE-US-00009 TABLE 4 Data of in-vivo pharmacokinetic study on SD rats intravenously and orally administrated with tested compound Positive Compound Example 2 control 1 IV @ Half-life period t.sub.1/2(h) 0.33 0.45 1 mg/kg Concentration to peak 875.13 1163.29 C.sub.max (nM) Area under curve 422.34 605.23 AUC.sub.(0-t)(ng .Math. h/mL) Apparent clearance rate 39.40 27.54 Cl(mL/min .Math. kg) PO @ Half-life period t.sub.1/2(h) 0.55 0.68 5 mg/kg Concentration to peak 527.18 932.14 C.sub.max (nM) Area under curve 1497.55 2273.35 AUC.sub.(0-t)(ng .Math. h/mL) Bioavailability F % 70.92 75.12 Positive Compound Example 3 control 2 IV @ Half-life period t.sub.1/2(h) 5.00 4.54 1 mg/kg Concentration to peak 573.49 780.63 C.sub.max (nM) Area under curve 1477.45 1008.75 AUC.sub.(0-t)(ng .Math. h/mL) Apparent clearance rate 11.23 16.44 Cl(mL/min .Math. kg) PO @ Half-life period t.sub.1/2(h) 3.01 3.00 5 mg/kg Concentration to peak 1297.63 632.71 C.sub.max (nM) Area under curve 8769.13 3345.13 AUC.sub.(0-t)(ng .Math. h/mL) Bioavailability F % 118.71 66.32
[0372] The positive control 1 is the compound in example 5 of patent WO2019210835, with the following chemical structure:
##STR00104##
[0373] The positive control 2 is the compound in example 6 of patent WO2019210835, with the following chemical structure:
##STR00105##
[0374] From data of the compound in the specific examples, the in-vivo pharmacokinetic parameters (AUC/CL/F %) of the compound in example 2 on the SD rats are worse than those of the corresponding positive compound, but the in-vivo pharmacokinetic parameters (AUC/CL/F %) of the compound in example 3 on the SD rats are unexpectedly and significantly higher than those of the positive control 2.
[0375] Compared with the positive control compound 2, efficacies and pharmacokinetics of the compound in example 3 of the present application on mutated NTRK3-G623R and NTRK1-G667C are significant improved, which can realize clinical benefits of low dosage and low administration frequency.
Testing Example 5
Bidirectional Permeability Study on Test Compound on MDCK-MDR1 Cell Line
1.1 Materials
[0376] MDCK-MDR1 cells were purchased from Netherlands Cancer Institute, and cells between 10 and 20 generations were used.
1.2 Experimental Design
1.2.1 Cell Culture and Plate Inoculation
[0377] 1) Before cell inoculation, 50 μL of cell medium was added into each well of an upper chamber of Transwell, and 25 mL of cell medium was added into a lower culture plate. The culture plate was placed in an incubator at 37° C. under 5% CO.sub.2 for incubation for 1 hour, and then could be used for inoculating cells.
[0378] 2) The MDCK-MDR1 cells were resuspended with the medium, so that a final concentration was 1.56×10.sup.6 cells/mL. A cell suspension was added into an upper chamber of a 96-well Transwell culture plate according to 50 μL per well. The incubator was set at 37° C. under 5% CO.sub.2, and was ensured to have a relative humidity of 95% for culturing for 4 days to 8 days. The medium was replaced 48 hours after inoculation, the culturing lasted for 4 days to 8 days, and the medium was replaced every other day.
1.2.2 Evaluation of Integrity of Cell Monolayer Film
[0379] 1) The original medium in the lower culture plate was removed, and fresh preheated medium was added into the upper chamber.
[0380] 2) A resistance of the monolayer film was measured by a resistance meter (Millipore, USA), and a resistance of each well was recorded.
[0381] 3) After finishing measuring, the Transwell culture plate was put back into the incubator.
[0382] 4) Calculation of resistance value: measured resistance value (ohms)×film area (cm.sup.2)=TEER value (ohm cm.sup.2), and if the TEER value was less than 42 ohms cm.sup.2, the well could not be used for a penetration experiment.
1.2.3 Solution Preparation
[0383] 1) The to-be-tested compound was prepared into 10 mM mother solution with DMSO.
[0384] 2) The positive control compound was prepared into 10 mM mother solution with DMSO.
[0385] 3) Metoprolol (purchased from China National Institutes for Food and Drug Control) and Digoxin (purchased from Tianjin Yifang Technology Co., Ltd.) were positive control compounds in this experiment.
1.2.4 Drug Penetration Experiment
[0386] 1) The MDCK-MDR1 Transwell culture plate was taken from the incubator. The cell monolayer film was moistened and washed twice with a preheated HBSS (25 mM HEPES, pH 7.4) buffer, and incubated at 37° C. for 30 minutes.
[0387] 2) A stock solution of the control compound and the test compound was diluted in DMSO to obtain a 200 μM solution, and then diluted with HBSS (10 mM HEPES, pH 7.4) to obtain a 1 μM working solution. A final concentration of DMSO in the incubation system was 0.5%.
[0388] 3) A transfer rate of the compound from a top end to a base end was measured. 125 μL of working solution was added into the upper chamber (top end), then 50 μL of sample solution was immediately transferred from a lower chamber (base end) to a 96-well plate filled with 200 μL of acetonitrile containing an internal standard as a 0-minute administration sample (A-B) for detection, and 235 μL of HBSS (25 mM HEPES, pH 7.4) buffer was added into the lower chamber. The internal standard contained 100 nM alprazolam, 200 nM caffeine and 100 nM tolbutamide. The 50 μL of sample solution transferred above was vortexed at a speed of 1,000 rpm for 10 minutes.
[0389] 4) A transfer rate of the compound from the base end to the top end was measured. 285 μL of working solution was added into the lower chamber (base end), then 50 μL of sample solution was immediately transferred from the upper chamber (top end) to a 96-well plate filled with 200 μL of acetonitrile containing an internal standard as a 0-minute administration sample (B-A) for detection, and 75 μL of HB SS (25 mM HEPES, pH 7.4) buffer was added into the upper chamber. The internal standard contained 100 nM alprazolam, 200 nM caffeine and 100 nM tolbutamide. The 50 μL of sample solution transferred above was vortexed at a speed of 1,000 rpm for 10 minutes. The experiment from the top end to the base end and the experiment from the base end to the top end should be carried out simultaneously.
[0390] 5) After adding the buffers into the lower chamber and the upper chamber respectively, the MDCK-MDR1 Transwell culture plate was incubated at 37° C. for 2 hours.
[0391] 6) After finishing incubating, 50 μL of sample solutions were respectively taken from an administration side (the upper chamber: Ap.fwdarw.Bl flux, the lower chamber: Bl.fwdarw.Ap) and a receiving side (the lower chamber: Ap.fwdarw.Bl flux, the upper chamber Bl.fwdarw.Ap) and added into new 96-well plates, and 4 times volume of ethanol containing the internal standard was added into the well plates. The internal standard substance contained 100 nM alprazolam, 200 nM caffeine and 100 nM tolbutamide. The solution was vortexed for 10 minutes, and then centrifuged at 3,220 g for 40 minutes. 100 μL of supernatant was sucked and mixed with equal volume of ultrapure water, and then subjected to LC-MS/MS analysis.
[0392] 7) The integrity of the cell monolayer film after incubation for 2 hours was evaluated with the leakage of fluorescent yellow. A fluorescent yellow stock solution was diluted with HB SS (10 mM HEPES, pH 7.4) to a final concentration of 100 μM, 100 μL of fluorescent yellow solution was added into each well of the upper chamber (top end), and 300 μL of HBSS (25 mM HEPES, pH 7.4) was added into each well in a base of the lower chamber (base end). After incubation at 37° C. for 30 minutes, 80 μL of solution was respectively sucked from upper and lower layers of each well and added into a new 96-well plate. Fluorescence measurement was carried out under an excitation wavelength of 485 nm and an emission wavelength of 530 nm by a microplate reader.
1.2.5 Analysis Conditions
[0393] LC system: Shimadzu
[0394] MS analysis: Triple Quad 5500 instrument from AB Inc with an ESI interface
[0395] 1) LC Parameters
[0396] Column temperature: 40° C.
[0397] Chromatographic column: Waters XSelect HSS T3 C18, 2.5 μM, 2.1×50 mm
[0398] Mobile phase: 0.1% formic acid dissolved in water (A) and 0.1% formic acid dissolved in acetonitrile (B)
[0399] Sample injection volume: 5 μL
[0400] Elution rate: 0.6 mL/min
TABLE-US-00010 Time (min) 0 0.2 0.7 1.2 1.25 1.5 % B 5 5 95 95 5 5
[0401] 2) MS Parameters
[0402] Ion source: Turbo spray
[0403] Ionization model: ESI
[0404] Scan type: Multi-reaction monitoring (MRM)
[0405] Curtain gas: 35 L/min
[0406] Collision gas: 9 L/min
[0407] Carrier gas: 50 L/min
[0408] Auxiliary gas: 50 L/min
[0409] Temperature: 500° C.
[0410] Ionspray voltage+5500 V (positive)
1.3 Data Analysis
[0411] A peak area was calculated according to results of ion chromatography. An apparent permeability coefficient (Papp, unit: cm/s×10.sup.−6) of the compound was calculated by the following formula:
[0412] wherein V.sub.A was a volume of the solution at the receiving end (Ap.fwdarw.Bl was 0.3 mL, Bl.fwdarw.Ap was 0.1 mL); Area was a film area of the Transwell-96-well plate (0.143 cm.sup.2); time was an incubation time (unit: s); and [drug] was a drug concentration.
[0413] Experimental results are shown in the following Table 5:
TABLE-US-00011 TABLE 5 Data of bidirectional permeability study on test compound on MDCK-MDR1 cell line in the same batch Number of P.sub.app (A-B) P.sub.app (B-A) Excretion example (10.sup.−6, cm/s) (10.sup.−6, cm/s) ratio* Example 3 11.13 51.91 4.67 Positive control 2 3.31 64.41 19.42 *Excretion ratio = P.sub.app (B-A)/P.sub.app (A-B)
[0414] The positive control 2 is the compound in example 6 of patent WO2019210835, with the following chemical structure:
##STR00106##
[0415] The permeability (P.sub.app(A-B)) of the compound in example 3 is better than that of the positive control 2, which indicates that the compound in example 3 is easier to be absorbed into cells. Meanwhile, the compound in example 3 has a low excretion ratio, which indicates that the compound in example 3 is not easy to be excreted, thus maintaining a high drug concentration in cells and producing better efficacy. Therefore, in combination with the in-vivo pharmacokinetic study on the SD rats, the in-vivo pharmacokinetic parameters (AUC/CL/F %) of the compound in example 3 on the SD rats are unexpectedly and significantly higher than those of the positive control 2.
Testing Example 6
In-Vitro Cell Viability
[0416] Hefei Precedo Biomedical Technology Co., Ltd. was commissioned to complete the testing example, and the cell lines used were constructed by this company.
1. Experimental Purpose
[0417] In-vitro anti-proliferative effects of the compound of the present application on 6 BaF3 cell lines were measured.
2. Reagents and Consumables
Cell Line:
[0418]
TABLE-US-00012 Quantity of Cell line Cell type cells/well Medium Ba/F3-LMNA- Suspension 2000 RPMI 1640 + NTRK1 10% FBS + 1% PS Ba/F3-LMNA- Suspension 2000 RPMI 1640 + NTRK1-V573I 10% FBS + 1% PS Ba/F3-LMNA- Suspension 2000 RPMI 1640 + NTRK1-F589L 10% FBS + 1% PS Ba/F3-LMNA- Suspension 2000 RPMI 1640 + NTRK1-G667S 10% FBS + 1% PS Ba/F3-TEL- Suspension 2000 RPMI 1640 + NTRK2 10% FBS + 1% PS Ba/F3-TEL- Suspension 2000 RPMI 1640 + NTRK3 10% FBS + 1% PS
Materials:
[0419]
TABLE-US-00013 Catalogue Brand Item number Batch number RPMI 1640 BI 01-100-1acs 0023019 Fetal bovine serum BI BI04-001-1ACS 1616756 Penicillin-streptomycin Corning 30-002-CI 30002311 solution Interleukin-3 Novusbio NBP2-35123 N12103071 CellTiterGlo Promega G7573 347569 Trypanblue Solarbio C0040 20181024 96-well white plate Corning 3917 33917037 96-well drug plate Beaver 40196 18C006004 Loading slot Corning 4870 10519072 10 μl tip Axygen T-300 00518391 200 μl tip Sangon F600227 F617LA2491 1,000 μl tip Excell CS015-0031 190486 50 ml centrifuge tube NEST 602052 111918EE01 15 ml centrifuge tube NEST 601052 051819EE02 Dimethyl sulfoxide TCI D5293 64Y4N-DS
3. Experimental Process
[0420] Cell Processing and Administration
[0421] Cell Culture Conditions
[0422] 6 Ba/F3 cell lines were cultured with RPMI 1640 (Biological Industries, Israel) +10% fetal calf serum (Biological Industries, Israel) +1% double antibiotics (Penicillin Streptomycin solution, Coring, USA), and cells were cultured for two generations after resuscitation, to be tested.
[0423] Preparation of 1,000× Compounds
[0424] The to-be-tested compound was prepared into 10 mM mother solution with DMSO, and then diluted to 1 mM with DMSO. The solution was diluted by 3 times to prepare into 1.0000 mM, 0.3333 mM, 0.1111 mM, 0.03704 mM, 0.01235 mM, 0.00412 mM, 0.00137 mM, 0.00046 mM, 0.00015 mM, and 0.0005 mM, and stored in a 96-well drug plate (Beaver, Suzhou), with 10 concentration gradients in total. Meanwhile, an equal volume of DMSO solvent was used as a negative control.
[0425] Preparation of 20× Compounds
[0426] The prepared 1,000× compounds of 10 concentration gradients were respectively diluted by 50 times into 20× compounds with complete medium, and stored in a 96-well drug plate (Beaver, Suzhou), with 10 concentration gradients in total. Meanwhile, an equal volume of DMSO solvent was used as a negative control.
[0427] Cell Plating
[0428] Cell suspension in logarithmic growth phase was inoculated on a 96-well white cell culture plate (Corning 3917, NY, USA), and a volume of each well was 95 μl (about 2000 cells/well).
[0429] 5 μl of 20× to-be-tested compounds were respectively added into the culture plate containing 95 μl of cell suspension above, and mixed evenly, with 2 wells for each concentration gradient. Final concentrations of the to-be-tested compounds were 1.0000 μM, 0.3333 μM, 0.1111 μM, 0.03704 μM, 0.01235 μM, 0.00412 μM, 0.00137 μM, 0.00046 μM, 0.00015 μM, and 0.0005 μM respectively.
[0430] Incubation was carried out in an incubator at 37° C. under 5% CO.sub.2 for 72 hours.
[0431] Plate Reading
[0432] The following steps were carried out according to the instruction of Promega CellTiter-Glo Luminescence Cell Viability Assay Kit (Promega-G7573).
[0433] (1). A CellTiter-Glo buffer was melted and placed at room temperature.
[0434] (2). A CellTiter-Glo substrate was plated at room temperature.
[0435] (3). The Celtiter-Glo buffer was added into a bottle of Celtiter-Glo substrate to dissolve the substrate, thus preparing a Celtiter-Glo working solution.
[0436] (4). The substrate was fully dissolved by slow vortex oscillation.
[0437] (5). The cell culture plate was taken out and placed for 10 minutes to balance to room temperature.
[0438] (6). 50 μl of CellTiter-Glo working solution was added into each hole.
[0439] (7) The culture plate was shaken on an orbital shaker for 2 minutes to induce cell lysis.
[0440] (8). The culture plate was placed at room temperature for 10 minutes to stabilize a luminous signal.
[0441] (9). The luminous signal was detected on a MD SpectraMax Paradigm plate reader.
[0442] Data Analysis
[0443] A corresponding fluorescence value RLU of each well was obtained through reading by SpectraMax Paradigm. Cell viability data was processed by the following formula:
[0444] Cell viability(%)=(RLU.sub.Drug−RLU.sub.Min)/(RLU.sub.Max−RLU.sub.Min)*100%. Cell viabilities of compounds of different concentrations were calculated in EXCEL, then cell viability curves were made by GraphPad Prism software, and related parameters were calculated. The parameters comprised a maximum cell viability, a minimum cell viability, and an IC.sub.50 value.
[0445] Experimental results are shown in the following Table 6:
TABLE-US-00014 TABLE 6 IC.sub.50 values of inhibitory activities of the compound of the present application on 6 BaF3 cell lines in the same batch IC.sub.50 (nM) of compound Positive Positive Cell line Example 2 control 1 Example 3 control 2 Ba/F3-LMNA-NTRK1 0.7 0.5 1.6 4.1 Ba/F3-LMNA-NTRK1- 0.3 0.2 1.1 2.3 V573I Ba/F3-LMNA-NTRK1- 2.4 6.6 1.2 4.5 F589L Ba/F3-LMNA-NTRK1- 3.4 2.7 8.4 19.9 G667S Ba/F3-TEL-NTRK2 1.6 0.9 3.4 7.3 Ba/F3-TEL-NTRK3 1.7 0.9 5.0 9.8
[0446] The positive control 1 is the compound in example 5 of patent WO2019210835, with the following chemical structure:
##STR00107##
[0447] The positive control 2 is the compound in example 6 of patent WO2019210835, with the following chemical structure:
##STR00108##
[0448] From biological activity data of the compound in the specific examples, the compound of the present application has strong growth inhibition effects on 6BaF3 cell lines. The inhibitory activities of the compound in example 2 on 6BaF3 cell lines are not significantly different from those of the corresponding positive compound, but the inhibitory activities of the compound in examples 3 of the present application on 6BaF3 cell lines are significantly higher than those of the corresponding positive control 2.
[0449] It will be appreciated by those skilled in the art that changes could be made to the embodiment described above without departing from the broad inventive concept thereof. It is understood, therefore, that this invention is not limited to the particular embodiment disclosed, but it is intended to cover modifications within the spirit and scope of the present invention as defined by the appended claims.