TWI462738B - Salt of n-[4-(1-cyanocyclopentyl)phenyl]-2-(4-pyridinemethyl)amino-3-pyridinecarboxamide - Google Patents

Description

Salt of N-[4-(1-cyanocyclopentyl)phenyl]-2-(4-pyridylmethyl)amino-3-pyridinecarboxamide

The present invention relates to a pharmaceutically acceptable salt of N-[4-(1-cyanocyclopentyl)phenyl]-2-(4-pyridylmethyl)amino-3-pyridinecarboxamide.

In the growth and metastasis of malignant tumors, neovascularization of tumors plays a very important role. When the tumor grows in excess of 1 mm ³ , new blood vessels need to be generated or budded from existing blood vessels to create a branch of blood vessels to provide sufficient blood to support the survival of the tumor cells. Tumor growth rate and metastatic propensity are related to the level of pro-angiogenic factors and the number of new microvessels. Since Folkman's hypothesis of "anti-angiogenic therapy" in the early 1970s, people's understanding of this field has made great progress, and inhibition of tumor angiogenesis has been recognized as a new anti-cancer strategy.

Tyrosine kinase vascular endothelial growth factor (VEGF) and its receptor (VEGFR) play an important role in tumor angiogenesis and are important targets in blocking tumor angiogenesis. Vascular endothelial growth factor (VEGF) is the most important factor in promoting angiogenesis in the body. Binding of VEGF to endothelial cell-derived vascular endothelial growth factor receptor (VEGFR) results in a variety of angiogenic responses, such as cell proliferation, migration, increased vascular permeability, and removal of endothelial cell precursor cells from the bone marrow. Members of the VEGFR family include: VEGFR1 (Flt-1), VEGFR2 (KDR/Flk-1), VEGFR3 (Flt-4). Pro-angiogenesis is primarily mediated by the binding of VEGF to VEGFR2 (KDR/Flk-1). A large number of human tumors show higher levels of VEGFR. At present, more than 40 monoclonal antibodies against VEGF and its receptor VEGFR and small inhibitors of VEGFR tyrosine kinase have entered the clinical study. Atechtin, a VEGF monoclonal antibody developed by Genetech after more than a decade, was approved for marketing in 2004. The combination of Avastin for colon cancer, lung cancer, and breast cancer confirms that its mechanism as an anti-VEGF drug is feasible, and it also contributes significantly to the mechanism of angiogenesis as an anti-cancer target.

Small molecule inhibitors of VEGFR The most notable drugs in recent years include the VAR inhibitor Vatalanib (PTK787) developed by Novartis/Schering for the treatment of colorectal cancer, and the treatment of AstraZeneca. VEGFR and epidermal growth factor receptor (EGFR) dual-target inhibitor Zactima (ZD-6474) in relapsed/refractory non-small cell lung cancer. VEGF inhibitors have become a very promising new non-cytotoxic antitumor drug. Compared with traditional cytotoxic drugs that inhibit tumor growth, therapeutic drugs targeting neovascularization have higher specificity, lower toxicity, and are advantageous for overcoming tumor resistance, and can be used for the treatment of various tumors.

N-[4-(1-Cyanocyclopentyl)phenyl]-2-(4-pyridylmethyl)amino-3-pyridinecarboxamide (hereinafter referred to as "Compound A") is a new generation of tyrosine A kinase inhibitor whose structure is represented by the following structural formula (I):

The above-mentioned compounds are described in Chinese Patent Application No. 02138671.4, the disclosure of which is hereby incorporated by reference. Compound A has been found to have a strong selective inhibition of VEGFR-2 in different laboratory tyrosine kinase receptor enzyme levels, with an IC ₅₀ of about 1 nM. In addition, Ret, VEGFR-1, PDGFR-β, c-kit, cSRC and other kinases also have a certain selective inhibitory activity. The pharmacodynamic study of human tumor xenografts in nude mice found that compound A was more effective than PTK787 in colon cancer Ls174t nude mice xenografts; compound A combined with oxaliplatin improved drug efficacy, but did not increase toxicity; The efficacy was better than that of PTK787. The efficacy of non-small cell lung cancer A549 nude mice xenografts was significantly stronger than that of PTK787, and the maximum efficacy was comparable to that of ZD6474 at conventional doses. In terms of toxicity, Compound A was well tolerated in the largest dose of 400 mg/kg nude mice.

However, in the course of drug research, the inventors found that N-[4-(1-cyanocyclopentyl)phenyl]-2-(4-pyridylmethyl)amino-3-pyridinecarboxamide is stable. , bioavailability and other aspects are still not satisfactory.

The inventors have found through long-term efforts that N-[4-(1-cyanocyclopentyl)phenyl]-2-(4-) is present in view of the stability, bioavailability and the like of the compound during use. The preparation of the corresponding pharmaceutically acceptable salts of pyridylmethyl)amino-3-pyridinecarboxamide can solve this problem.

A first aspect of the invention relates to a pharmaceutically acceptable salt of N-[4-(1-cyanocyclopentyl)phenyl]-2-(4-pyridylmethyl)amino-3-pyridinecarboxamide Wherein the pharmaceutically acceptable salt is a conventional inorganic or organic salt in the art, and further, the inorganic salt is preferably a hydrochloride, a hydrobromide, a sulfate, a nitrate or a phosphate. The organic salt is preferably methanesulfonate, maleate, tartrate, succinate, acetate, trifluoroacetate, fumarate, citrate, citrate, benzenesulfonate. Acid salt, benzoate, naphthalene sulfonate, lactate, malate. Particularly preferred pharmaceutically acceptable salts are the mesylate and the hydrochloride which are more advantageous in terms of stability, traits and bioavailability relative to other salts.

A second aspect of the invention relates to a pharmaceutically acceptable salt of N-[4-(1-cyanocyclopentyl)phenyl]-2-(4-pyridylmethyl)amino-3-pyridinecarboxamide For the preparation, the preparation of the compound can be carried out according to a salt forming method conventional in the art.

A third aspect of the invention relates to a pharmaceutical composition comprising a therapeutically effective amount of N-[4-(1-cyanocyclopentyl)phenyl]-2-(4-pyridylmethyl)amine A pharmaceutically acceptable salt of 3-pyridinecarbachamide, wherein the pharmaceutical composition may further comprise one or more pharmaceutically acceptable carriers.

A fourth aspect of the invention relates to a pharmaceutically acceptable salt of N-[4-(1-cyanocyclopentyl)phenyl]-2-(4-pyridylmethyl)amino-3-pyridinecarboxamide The use of a medicament for the treatment of a tumor.

1. Preparation of a pharmaceutically acceptable salt of Compound A Preparation Example 1: Preparation of hydrochloride salt of Compound A

5.049 g of Compound A (12.7 mmol) was suspended in 120 ml of ethanol, and a hydrochloric acid standard solution (0.5322 mol/L) of 23.89 ml was added dropwise, and the mixture was heated to reflux, and the solid was dissolved and cleaved (if it was insoluble, it was hot filtered), and it was naturally cooled to room temperature. (23 ° C) a crystalline solid precipitated. After suction filtration, the filter cake was washed with ethanol (20 ml × 2), dried, and transferred to a vacuum drying oven (CaCl ₂ ) at 80 ° C for 5 hours to obtain a hydrochloride of Compound A, 3.619 g (65.7%). Melting range: 200 to 202.5 ° C. Moisture 5.1%. The solvent remains 0.025%.

Preparation Example 2: Sulfate preparation of Compound A

3.092 g of Compound A (7.778 mmol) was suspended in 120 ml of ethanol, and 14.89 ml (7.793 mmol) of sulfuric acid standard solution (0.5234 mol/L) was added dropwise, and heated to reflux. The solid was dissolved and clarified (if there is insoluble matter, it can be filtered by heat) It was concentrated to 100 ml by pressure, and naturally cooled to room temperature (23 ° C) to precipitate a crystalline solid. After suction filtration, the filter cake was washed with ethanol (8 ml×2), drained and transferred to a vacuum drying oven (CaCl ₂ ) and pumped at 80 ° C for 5 hours to obtain 2.662 g of sulfate of compound A. The rate is 57.7%). Melting range: 199.5 to 230 ° C (unmelted).

Preparation Example 3: Phosphate Preparation of Compound A

1.910g of compound A (4.805mmol) and 225ml of ethanol, phosphoric acid standard solution (0.5008mol / L) 9.29ml (4.803mmol), heated to reflux, after 4 hours, the solid dissolved and clarified, naturally cooled to room temperature (25 ° C) crystal The solid precipitated. After suction filtration, the filter cake was washed with ethanol (5 ml × 2), drained and transferred to a vacuum drying oven (CaCl ₂ ) at 80 ° C for 6 hours to obtain a compound A phosphate 1.150 g (calculated according to the free base content) 46.1%). Melting range: 205 to 258 ° C (unmelted).

Preparation Example 4: Methanesulfonate salt of Compound A

In a 5 L reaction flask, 170 g (0.428 mol) of compound A, 42.5 g (0.442 mol) of methanesulfonic acid, and 2.55 L of a 95% aqueous solution of isopropanol were added, and the mixture was heated to complete dissolution under nitrogen protection and protected from light. The yellow transparent solution was filtered while hot, cooled to room temperature, filtered, washed with isopropyl alcohol, and dried in vacuo to give white crystals (180.2 g, 0.365 mol). In a 5 L reaction flask, 180.2 g of the methanesulfonate salt of Compound A and 2.52 L of a 95% aqueous solution of isopropanol were added, and the mixture was heated under nitrogen to be completely dissolved in the dark, stirred, filtered while hot, and the filtrate was cooled to room temperature. The mixture was filtered, washed with isopropyl alcohol and dried in vacuo to give white crystals (161.5 g). Melting range: 193.5 to 195 °C.

Preparation Example 5: Citrate of Compound A

2.886 g of compound A free base, 0.522 g of citric acid and 80 mL of ethanol were mixed and heated to be nearly boiling. The solid was dissolved and cleaved into a colorless clear solution, cooled to room temperature, and a white crystalline solid was precipitated, suction filtered, and the filter cake was washed with ethanol (3 mL×2). The mixture was pumped in a vacuum oven at 80 ° C for 6 hours to obtain 2.283 g of a white needle-like solid, yield 79%. The melting range is 160.5 to 162.0 °C.

Preparation Example 6: Maleate of Compound A

2.508 g of compound A free base, 0.351 g of maleic acid and 110 mL of ethanol are mixed and heated to reflux. The solid is dissolved and cleaved into a pale yellow solution. A small amount of flocculent insoluble matter is added to the activated carbon and then boiled, and then filtered off by hot filtration. The filtrate is slightly concentrated to ~90 mL, and cooled to room. The precipitate was pale yellow crystals, suction filtered, and the filter cake was washed with a small amount of ethanol. The mixture was vacuumed in a vacuum oven at 80 ° C for 6 hours to give a pale yellow needle solid 1.09 g, yield 40%. The melting range is 115 to 160 °C.

Preparation Example 7: Succinate of Compound A

2.827 g of Compound A free base, 0.401 g of succinic acid and 70 mL of ethanol were mixed and heated to reflux. The solid was dissolved and clarified. A small amount of flocculent insoluble matter was added to the activated carbon for 10 minutes, then filtered off by hot filtration. The filtrate was concentrated to ~25 mL, cooled to room temperature, and precipitated. The white crystalline solid was suction filtered, and the filter cake was washed with a small amount of ethanol, and the mixture was stirred at 80 ° C for 6 hours in a vacuum drying oven to obtain a light yellow needle solid 1.09 g, yield 77%. The melting range is 117 to 161.5 °C.

2. Related properties of pharmaceutically acceptable salts of Compound A

4, stability

Stability of different kinds of pharmaceutically acceptable salts of Compound A under different conditions

(Starting purity of raw material: 99.6%, content determined by HPLC)

Conclusion: From the results of the above stability test, it can be seen that the stability of the hydrochloride and the methanesulfonate is most satisfactory, and in particular, the methanesulfonate has very good stability.

3. Pharmacological activity of pharmaceutically acceptable salts of Compound A Example 1: Inhibition of the receptor protein tyrosine kinase by the mesylate salt of Compound A

(a) method

ELISA method (I Posner et al, J. Biol. Chem., Oct, 1992, Vol. 267, Issue 29, 20638-20647): an enzyme well plate coated with an enzyme reactant Poly (Glu, Tyr) _4:1 , Add enzyme, sample, ATP and other reactions, detect substrate phosphorylation with monoclonal antibody against phospho-tyrosine (PY99), and add HRP-labeled goat anti-mouse IgG, OPD color detection to detect the phosphorylation degree of the reactant; A control group without tyrosine kinase and a control well of the corresponding DMSO concentration; a reaction was stopped by adding 2 M H ₂ SO ₄ 50 μl/well, and read with a tunable wavelength microplate spectrometer VERSAmax (Sunnyvale, CA, USA), colorimetric The reaction was observed to observe the OD490nm value.

The relative inhibition rate of the drug to the tyrosine kinase protein was determined.

The half-inhibitory concentration IC ₅₀ was calculated by the LOGIT method according to the respective concentration inhibition rates. Each of the above experiments was repeated 3 times, and the average IC ₅₀ value of the 3 experiments was determined as the final index of the inhibition ability.

(2) Results

The results of the detection of the inhibition of eight tyrosine kinases by the mesylate salt of Compound A and the positive control compound PTK787 are shown in Table 1. The results showed that the mesylate salt of compound A significantly inhibited the kinase activities of KDR, Flt1, PDGFRβ, c-Kit and c-Src at the molecular level, and the half-inhibitory concentration (IC ₅₀ ) was 2.43 nM and 70.08 nM, respectively. , 537.31nM, 420.31nM, 348.53nM; positive control compound PTK787 on KDR, Flt1, PDGFRβ, c- Kit kinase inhibition activity IC ₅₀ were 33.30nM, 84.69nM, 416.51nM, 606.11nM. The results showed that the mesylate salt of compound A had a strong inhibitory effect on the kinase activity of vascular endothelial growth factor receptor 1, 2 (Flt1/VEGFR1, KDR/VEGFR2), and the inhibitory effect on KDR kinase was strong. It inhibited Flt1 and the IC ₅₀ of KDR kinase was 13.7 times lower than that of the positive control compound, that is, the methanesulfonate of Compound A inhibited KDR more strongly than PTK787. At the same time, the mesylate salt of compound A also has a considerable inhibitory effect on the other members of the third type of receptor tyrosine kinase, platelet-derived growth factor receptor β (PDGFRβ) and stem cell growth factor receptor (c-Kit), but weak. Its inhibition of vascular endothelial growth factor receptor. The positive compound PTK787 had no inhibitory effect on the non-receptor tyrosine kinase c-Src when the concentration was raised to 10 ⁴ nM, while the methanesulfonate of Compound A inhibited the c-Src kinase activity by an IC ₅₀ of 348.53 nM. When the concentration rose to 10 ⁴ nM, the mesylate salt of Compound A did not inhibit the kinase activity from other family kinases such as epidermal growth factor receptor EGFR1 and ErbB2, and fibroblast growth factor receptor FGFR1. At the same time, the experimental results also showed that the methanesulfonate of compound A inhibited KDR kinase activity at the molecular level stronger than the positive compound PTK787, and the inhibition of tyrosine kinases Flt1, PDGFR and c-Kit was basically the same. At the same level of strength, the mesylate salt of Compound A is more selective than PTK787 in selectivity, and it also inhibits the kinase activity of the non-receptor tyrosine kinase c-Src. In summary, the mesylate salt of Compound A is a tyrosine kinase inhibitor with significant selective inhibition of KDR, accompanied by inhibition of Flt1, PDGFR, c-Kit, c-Src and other kinases. .

Example 2: Effect of mesylate salt of compound A on human colon cancer Ls174t nude mice xenografts

(1) Experimental animals:

BALB/cA-nude nude mice, sputum, 5-6 weeks old, purchased from Shanghai Slack Laboratory Animals LLC. Certificate No.: SCXK (Shanghai) 2004-0005. Feeding environment: SPF level.

(2) Experimental steps:

After 1 week of adaptation, the animals were subcutaneously inoculated with human colon cancer Ls174t tumor tissue. After the tumors were grown to 100 to 300 mm ³ , the animals were randomly divided into groups (d0). The dose of the compound A was 50 mg/kg, 100 mg/kg, 200 mg/kg; PTK787 50 mg/kg, 100 mg/kg, 200 mg/kg. Oral administration (gavage), d0 to d13 days, once a day, a total of 14 times. The tumor volume was measured 2 to 3 times a week, and the rats were weighed and recorded. The tumor volume (V) is calculated as: V = 1/2 × a × b ² where a and b represent length and width, respectively.

(3) Results:

Enzymatic and cell level experiments demonstrated that the main target of the mesylate salt of Compound A was VEGFR2/KDR (IC ₅₀ was 2.43±1.30 nM); the internationally similar compound with the same target and the clinical trial started earlier was Novartis. PTK787 company (the KDR IC ₅₀ of 33.30 ± 14.45nM); therefore, this experiment as a positive compound PTK787. According to the preliminary test of the mesylate salt of Compound A and the literature report of PTK787 ^[1] , we used three doses of 50, 100, 200 mg/kg, and evaluated and compared the efficacy by the same dose and the same dosage regimen. The results are shown in Table 2. The results showed that the methanesulfonate of Compound A inhibited the growth of human colon cancer Ls174t in a dose-dependent manner; when administered at 200 mg/kg, the T/C% reached 16.3%; while the PTK787 200 mg/kg administration significantly inhibited Ls174t. Growth, but T/C% is only 60.2%, the effect is significantly less than the mesylate salt of Compound A. The literature (I Posner et al, J. Biol. Chem., Oct, 1992, Vol. 267, Issue 29, 20638-20647) reports that PTK787 75 mg/kg can achieve a T/C% of up to 40% when administered. Our results showed no significant effect on the administration of PTK787100 mg/kg, and the T/C% was only 71.5%. In the control literature, they found that the initial tumor volume at the time of administration was small (25-100 mm ³ ), which was at least 1.5 to 6 times smaller than the initial tumor volume when we started the administration; they were administered for 28 days or Above; and their T/C% is the best T/C% in the whole experiment, not the T/C% at the end of the experiment. The best T/C% of our experiment is after administration. On the 10th day, at this time, the T/C% of PTK787 100 and 200 mg/kg were 60.7% and 45.8%, respectively, which was close to the literature. In addition, it should be emphasized that because there are many factors affecting the efficacy of the experiment, only in the same system can be compared, the efficacy of PTK787 in this experiment is not consistent with the literature, but does not affect the mesylate salt of compound A and its efficacy. Comparison. From Table 2, it can be calculated that the ED50 of the mesylate salt of Compound A against colon cancer Ls174t is 97.2 mg/kg, and the ED50 of PTK787 is 458.7 mg/kg, indicating that the mesylate salt of Compound A is superior to Ls174t. PTK787.

It should also be noted that we have compared the dose of the mesylate salt of Compound A and PTK787 to 400 mg/kg, and found that although the mice were still tolerant to the above two compounds, the curative effect did not increase significantly. The dose-effect relationship is not very obvious, and the result is similar to another angiogenesis inhibitor SU11248. Therefore, in the subsequent experiments, we mainly used the dose of the mesylate of the compound A at the doses of 200, 100, 50 mg/kg to evaluate the therapeutic effect. .

According to the experimental protocol, the mice bearing the tumor were continuously administered for 14 days, and the mice were well tolerated by the two compounds without significant weight loss. Under the experimental protocol, the toxicity of the two compounds was not the same. Big.

Example 3: Effect of mesylate salt of compound A on human colon cancer HT-29 nude mice xenografts

(1) Experimental animals:

BALB/cA-nude nude mice, sputum, 5-6 weeks old, purchased from Shanghai Slack Laboratory Animals LLC. Certificate No.: SCXK (Shanghai) 2004-0005. Feeding environment: SPF level.

(2) Experimental steps:

After 1 week of adaptation, the animals were subcutaneously inoculated with human colon cancer HT-29 tumor tissue. After the tumors were grown to 100 to 300 mm ³ , the animals were randomly divided into groups (d0). The dose of the compound A was 50 mg/kg, 100 mg/kg, 200 mg/kg; PTK787 200 mg/kg. Oral administration (gavage), d0-d20, once a day for 21 times. The tumor volume was measured 2 to 3 times a week, and the rats were weighed and recorded. The tumor volume (V) is calculated as: V = 1/2 × a × b ² where a and b represent length and width, respectively.

(3) Results (see Table 3):

The results showed that the mesylate salt of Compound A significantly inhibited the growth of human colon cancer HT-29 in a dose-dependent manner. PTK787 also has better curative effect, but less than the mesylate salt of Compound A. When administered at 200 mg/kg, the T/C% of the mesylate salt of Compound A and PTK787 are 25.5% and 56.5%, respectively. The difference was statistically significant (P<0.01), indicating that the mesylate salt of Compound A was significantly better than PTK787 in the treatment of colon cancer HT-29. In addition, the tumor-bearing mice were well tolerated to the mesylate salt of Compound A and PTK787, and the toxicity was comparable.

Example 4: Study on oral bioavailability of Compound A

(1) Test animals

Male Sprague-Dawley (SD) rats (body weight: ~250g, experimental animal quality certificate number: 0006473) by Shanghai Slack Laboratory Animals Co., Ltd. (license number: SCXK [Shanghai] 2003-0003)) by Shanghai Provided by Lake Laboratory Animals LLC. After the arrival of the purchased SD rats, the relevant test materials and health conditions of the experimental animals were first examined. After passing the test, they entered the clean-grade rat room of the Animal Center of Shanghai Pharmaceutical Research Institute.

(2) Test equipment

The liquid chromatography-mass spectrometry system (LC/MS/MS) includes the American Agilent 1100 series binary pump, online degasser, autosampler, column oven, and the Thermo Finnigan TSQ Quantum triple quadrupole mass spectrometer. The instrument, the system working software is Xcalibur and Chemstation (USA). Other test instruments include: Techne nitrogen blow dryer (Germany); -80 °C ultra-low temperature SANY0 refrigerator (Japan); Vibrax VXR small shaker (Germany); MS1 vortex mixer (Germany); 92-2 timed thermostatic magnetic stirring (Shanghai); METTLER AE240 dual-range electronic analytical balance (0.01mg/41g, 0.1mg/205g) (Germany). EPPENDORF continuous dosing device (Germany) and so on.

(3) Experimental methods

1. LC/MS/MS analysis conditions

Liquid chromatography analysis conditions

Chromatography column: Agilent Zorbax SB-C18 column (50 mm x 2.1 mm ID); column temperature: 25 ° C; mobile phase: A: H2O-CH3CN (2:98, v/v), B: H2O-CH3CN (10:90, v/v) A: 25% + B: 75%, isocratic elution; flow rate: 0.25 mL/min; injection amount: 10 μL; analysis time: 3 minutes.

2. Rat test

The clean grade rat chamber is a 12/12 hour day/night cycle with humidity and temperature of 40 to 60% and 20 to 24 °C, respectively. Each 4 rats were housed in a stainless steel squirrel cage of size 36 x 24 x 19 ^cm3 . Drink water freely and feed a special rat feed once a day. Rats can be used to start drug metabolism animal experiments after 1 week of adaptation to the environment. Three Sprague-Dawley rats were orally administered with Compound A at a dose of 20 mg/kg.

Accurately weigh 24 mg of Compound A powder, first dissolve it with 4 mL of water, place it in a mortar, grind it evenly, and wash it with 8 mL of water into a 15 mL test tube to obtain a suspension with a concentration of 2 mg/mL for animal testing.

Blood was collected at 0 hours before administration and 0.083, 0.25, 0.5, 1.0, 2, 4, 6, and 8 hours after administration. After anesthesia with ether before blood collection (strictly controlled anesthesia), blood was collected from the posterior venous sinus 250 to 300 μL/time, and collected in a test tube previously added with heparin. After blood collection, plasma was prepared by centrifugation and divided into two portions at 50 μL, and stored at -70 ° C until analysis. The concentration of Compound A in blood samples at different time points of each animal was analyzed by LC/MS/MS method. The used rats were euthanized with CO ₂ gas.

The pharmacokinetic parameters of each group of animals tested were calculated using InnaPhase Kinetica ^(TM) software (USA).

3. Rat test results

Example 5: Comparison of oral bioavailability of four pharmaceutically acceptable salts of Compound A

(1) Test animals

Male Sprague-Dawley (SD) rats (body weight: ~250g, experimental animal quality certificate number: 0006473) by Shanghai Slack Laboratory Animals Co., Ltd. (license number: SCXK [Shanghai] 2003-0003)) by Shanghai Provided by Lake Laboratory Animals LLC. After the arrival of the purchased SD rats, the relevant test materials and health conditions of the experimental animals were first examined. After passing the test, they entered the clean-grade rat room of the Animal Center of Shanghai Pharmaceutical Research Institute.

(2) Test equipment

The liquid chromatography-mass spectrometry system (LC/MS/MS) includes the American Agilent 1100 series binary pump, online degasser, autosampler, column oven, and the Thermo Finnigan TSQ Quantum triple quadrupole mass spectrometer. The instrument, the system working software is Xcalibur and Chemstation (USA). Other test instruments include: Techne nitrogen blow dryer (Germany); -80 °C ultra-low temperature SANYO refrigerator (Japan); Vibrax VXR small shaker (Germany); MS1 vortex mixer (Germany); 92-2 constant temperature magnetic stirring (Shanghai); METTLER AE240 dual-range electronic analytical balance (0.01mg/41g, 0.1mg/205g) (Germany). EPPENDORF continuous dosing device (Germany) and so on.

(3) Experimental methods

1. LC/MS/MS analysis conditions

Liquid chromatography analysis conditions

Chromatography column: Agilent Zorbax SB-C18 column (50 mm x 2.1 mm ID); column temperature: 25 ° C; mobile phase: A: H20-CH3CN (2:98, v/v), B: H20-CH3CN (10:90, v/v) A: 25% + B: 75%, isocratic elution; flow rate: 0.25 mL/min; injection amount: 10 μL; analysis time: 3 minutes.

2. Rat test

The clean grade rat chamber is a 12/12 hour day/night cycle with humidity and temperature of 40 to 60% and 20 to 24 °C, respectively. Each 4 rats were housed in a stainless steel squirrel cage of size 36 x 24 x 19 ^cm3 . Drink water freely and feed a special rat feed once a day. Rats can be used to start drug metabolism animal experiments after 1 week of adaptation to the environment. Twelve Sprague-Dawley rats were divided into 4 groups, the number of animals in each group was 3, and the four groups were orally administered with hydrochloride, phosphate, maleate and methanesulfonate of Compound A at a dose of 20 mg/ Kg.

Accurately weigh the hydrochloride, phosphate, maleate, methanesulfonate, powder 24mg of compound A, dissolve it in 4mL water, place it in a mortar, grind it evenly, and wash it with 8mL water into 15mL test tube. A suspension of 2 mg/mL was obtained for animal testing.

Blood was collected at 0 hours before administration and 0.083, 0.25, 0.5, 1.0, 2, 4, 6, and 8 hours after administration. After anesthesia with ether before blood collection (strictly controlled anesthesia), blood was collected from the posterior venous sinus 250 to 300 μL/time, and collected in a test tube previously added with heparin. After blood collection, plasma was prepared by centrifugation and divided into two portions at 50 μL, and stored at -70 ° C until analysis. The concentration of Compound A in blood samples at different time points of each animal was analyzed by LC/MS/MS method. The used rats were euthanized with CO ₂ gas.

The pharmacokinetic parameters of each group of animals tested were calculated using InnaPhase Kinetica ^(TM) software (USA).

3. Rat test results

The plasma concentrations of the salt, phosphate, maleate, and methanesulfonate groups of Compound A at different time points after oral administration of 20 mg/kg were listed in Tables 5 and 6, respectively. The drug concentration-time curves are shown in Figure 3, respectively. The pharmacokinetic parameters are shown in Tables 7 and 8.

Area under the time curve (0 to 8 hours); T _1/2 : half-life; Kel: elimination rate constant; MRT : average residence time of one molecule in vivo; CL : total plasma drug clearance; V _d : established in plasma concentration The pseudo-distributed volume on the basis.

Conclusion: By comparison with the bioavailability of Compound A as determined in Example 4, it was found that the salts of the compounds provided herein greatly improved the bioavailability of Compound A, particularly the hydrochloride and methanesulfonate salts.

Fourth, the preparation Preparation Example 1: Tablet

formula:

Preparation method: the pharmaceutically acceptable salt of compound A is passed through a sieve of 100 to 200 mesh, mixed with starch, granulated with 2% starch slurry, dried, mixed with magnesium stearate, compressed, tested, qualified, packaged .

Preparation Example 2: Capsule

formula:

Preparation methods: conventional granulation, capsule filling, testing, packaging.

Figure 1: Effect of mesylate salt of compound A and PTK787 on human colon cancer Ls174t nude mice xenografts.

Figure 2: Effect of mesylate salt of compound A and PTK787 on human colon cancer HT-29 nude mice xenografts.

Figure 3: Rat orally administered Compound A hydrochloride (rats 1 to 3), Compound A phosphate (rat 4 to 6), Compound A maleate (rat 7 to 9), and Mesylate salt of Compound A (rat 10 to 12) 20 Mg/kg drug-time curve.

The schema of the case is the result data, not the representative figure of the case. Therefore, there is no designated representative map in this case.

Claims (6)

A pharmaceutically acceptable salt of N-[4-(1-cyanocyclopentyl)phenyl]-2-(4-pyridylmethyl)amino-3-pyridinecarboxamide, which is selected from the group consisting of Methanesulfonate, maleate, phosphate and hydrochloride. The salt of claim 1, wherein the pharmaceutically acceptable salt is a methanesulfonate. A salt according to claim 1, wherein the pharmaceutically acceptable salt is a hydrochloride. A pharmaceutical composition comprising a therapeutically effective amount of a salt according to any one of claims 1 to 3 and one or more pharmaceutically acceptable carriers. A use of a salt according to any one of claims 1 to 3 for the preparation of a medicament for treating a tumor. A process for the preparation of a salt according to any one of claims 1 to 3, which comprises N-[4-(1-cyanocyclopentyl)phenyl]-2-(4-pyridylmethyl) a step of forming a salt of an amino-3-pyridinecarbendazim with a corresponding acid.