Disclosure of Invention
The invention aims to provide a naphthalene urea-piperidine compound, and a preparation method and application thereof.
Based on the above purpose, the invention adopts the following technical scheme:
the structural formula of the naphtyl urea-piperidine compounds is shown as a general formula I:
wherein R1 is selected fromSpecifically, the compound has the following structure:
the preparation process of the compound IY220816B-1 is as follows:
the preparation method comprises the following specific steps:
(1) Dissolving a compound 1A, boc2 O and Pd/C in methanol, and stirring under hydrogen to react completely; filtering the reaction solution by using diatomite, leaching by using ethyl acetate, and carrying out organic phase spin-drying column chromatography to obtain an intermediate 2A, wherein the mol ratio of the compound 1A, boc2 O to Pd/C is 1:1:0.1;
(2) Compound 2A, hbtu and DIEA were dissolved in DMF and stirred at room temperature to complete the reaction; diluting the reaction solution with ethyl acetate, washing with saturated saline, and performing organic phase drying spin-drying column chromatography to obtain a compound 3A; the molar ratio of compound 2A, HBTU and DIEA is 1:1:1.2:1.2;
(3) Dissolving the compound 3A in methanol, adding an ethyl acetate solution of hydrogen chloride, and stirring at room temperature to react completely; directly spin-drying the reaction solution; the molar ratio of the compound 3A to the hydrogen chloride is 1.29:40;
(4) Dissolving a compound 4A and a compound 5 in tetrahydrofuran, and stirring at 110-130 ℃ to react completely; and (3) directly spin-drying the reaction solution for column chromatography to obtain the IY220816B-1, wherein the molar ratio of the compound 4A to the compound 5 is 1:1.
The synthesis of compound ID220825A-1 was as follows:
the preparation method comprises the following specific steps:
(1) Dissolving the compound 1a and potassium tert-butoxide in tetrahydrofuran, stirring for 20-40 min, adding the compound 1B, and continuing stirring at room temperature to react completely; directly spin-drying the reaction solution for column chromatography to obtain a compound 2B, wherein the molar ratio of the compound 1B to the compound 1a to the potassium tert-butoxide is 1:1.2:1.2;
(2) Dissolving the compound 2B in ethanol, heating to 45-55 ℃, adding iron powder at 45-55 ℃ and continuously stirring for complete reaction; filtering the reaction solution by using diatomite, and directly spin-drying the filtrate by column chromatography to obtain a compound 3B, wherein the molar ratio of the compound 2B to the iron powder is 1:5;
(3) Dissolving a compound 3B and a compound 5 in tetrahydrofuran, and stirring at 110-130 ℃ to react completely; and (3) directly spin-drying the reaction solution by column chromatography to obtain the ID220825A-1, wherein the molar ratio of the compound 3B to the compound 5 is 1:1.
The preparation process of the compound IY220825A-1 is as follows:
(1) Dissolving a compound 1C and a compound 5 in tetrahydrofuran, and stirring at 110-130 ℃ to react completely; directly spin-drying the reaction solution for column chromatography to obtain a compound 2C, wherein the molar ratio of the compound 1C to the compound 5 is 1:1;
(2) Dissolving a compound 2C, a compound 2b, a compound K2CO3 and Pd (dppf) Cl2 in a mixture of 1, 4-dioxane and water, and stirring at 100-110 ℃ to react completely; and (3) carrying out direct spin-drying column chromatography on the reaction solution to obtain the IY220825A-1, wherein the mol ratio of the compound 2C to the compound 2b to the compound K2CO3 to Pd (dppf) Cl2 is 1:1.2:1.2:0.1, and the volume ratio of the 1, 4-dioxane to water is 10:1. The preparation process of the compound IY221116B-1 is as follows:
(1) Dissolving compound 1D, compound 1b, HBTU and DIEA in DMF, stirring at room temperature for reacting for a period of time (0.5-1.5 h), and then reacting at 110-130 ℃ completely; directly spin-drying the reaction solution for column chromatography to obtain a compound 2D, wherein the molar ratio of the compound 1D to the compound 1b to the HBTU to the DIEA is 1:1:1.2:1.2;
(2) Dissolving a compound 2D, a compound 2c, BINAP, pd (OAc)2 and Cs2CO3 in toluene, and stirring at 110-120 ℃ under the protection of nitrogen to react completely; directly spin-drying the reaction solution for column chromatography to obtain an intermediate 3D, wherein the molar ratio of the compound 2D to the compound 2c, BINAP, pd (OAc)2 to the Cs2CO3 is 1:1:0.1:0.05:1.2;
(3) Dissolving the compound 3D in dichloromethane, dropwise adding trifluoroacetic acid at the temperature of 0 ℃, and stirring to react completely; directly spin-drying the reaction solution, diluting with ethyl acetate, adjusting the pH to 8 with saturated sodium bicarbonate aqueous solution, and performing organic phase drying spin-drying column chromatography to obtain a compound 4D;
The molar volume ratio of compound 3D to trifluoroacetic acid was 0.88mmol:5mL;
(4) Dissolving a compound 4D and a compound 5 in tetrahydrofuran, and stirring at 110-130 ℃ to react completely; and (3) directly spin-drying the reaction solution for column chromatography to obtain the IY221116B-1, wherein the molar ratio of the compound 4D to the compound 5 is 1:1.
The preparation process of the compound ID221028A-1 is as follows:
(1) Dissolving a compound 1C and a compound 5 in tetrahydrofuran, and stirring at 110-130 ℃ to react completely; directly spin-drying the reaction solution for column chromatography to obtain a compound 2C, wherein the molar ratio of the compound 1C to the compound 5 is 1:1;
(2) And (3) dissolving the compound 2C, the compound 2d and Pd (PPh3)Cl2, cuI and triethylamine in DMF, heating to 45-55 ℃ and stirring to react completely, and carrying out direct spin-drying column chromatography on the reaction solution to obtain the ID221028A-1, wherein the molar ratio of the compound 2C to the compound 2d to Pd (PPh3)Cl2, cuI and triethylamine is 1:1:0.1:0.1:4).
The preparation process of the compound IY220831B-1 is as follows:
(1) Dissolving a compound 2A, a compound 1c and CDI in tetrahydrofuran, and stirring at 70-75 ℃ to react completely; directly spin-drying and mixing the reaction solution, and passing through a column to obtain a compound 2F, wherein the molar ratio of the compound 2A to the compound 1c to the CDI is 1:1:1;
(2) Dissolving compound 2F in methanol, and adding ethyl acetate solution of hydrogen chloride; stirring at room temperature to react completely; directly spinning the reaction solution to obtain a compound 3F, wherein the molar ratio of the compound 2F to hydrogen chloride is 1:40;
(3) Dissolving a compound 3F and a compound 5 in tetrahydrofuran, and stirring at 110-130 ℃ to react completely; and (3) directly spin-drying the reaction solution for column chromatography to obtain the IY220831B-1, wherein the molar ratio of the compound 3F to the compound 5 is 1:1.
A biologically acceptable salt of the above described naphthylurea-piperidines with at least one of acetic acid, dihydrofolic acid, benzoic acid, citric acid, sorbic acid, propionic acid, oxalic acid, fumaric acid, maleic acid, hydrochloric acid, malic acid, phosphoric acid, sulfurous acid, sulfuric acid, vanillic acid, tartaric acid, ascorbic acid, boric acid, lactic acid and ethylenediamine tetraacetic acid.
The application of the naphthyridine urea-piperidine compounds and the biologically acceptable salts thereof in preparing anti-tumor drugs, wherein the anti-tumor drugs are drugs for treating tumors related to STAT3 signaling.
Preferably, the antitumor drug is a drug for treating breast cancer, liver cancer, lung cancer, drug-resistant lung cancer, colon cancer, leukemia, gastric cancer, pancreatic cancer, prostate cancer and esophageal cancer.
It is another object of the present invention to provide a class of small molecule compounds with targeted antitumor activity.
The tumor may be specifically a STAT3 high-expression or constitutively activated tumor including, but not limited to, breast cancer, liver cancer, lung cancer, drug-resistant lung cancer, colon cancer, leukemia, stomach cancer, pancreatic cancer, prostate cancer, esophageal cancer, and the like.
Specifically, the invention synthesizes a class of naphthylurea-piperidine compounds IY220816B-1, ID 2208235A-1, IY221116B-1, ID221028A-1 and IY220831B-1 with brand new structures. The proliferation inhibition effect of the compounds on tumor cells is detected by an MTT method, the influence of the compounds on the cell cycle and apoptosis of the tumor cells is detected by a flow cytometry method, and the inhibition effect of the compounds on JAK2/STAT3 signals is clarified by immunoblotting and other methods.
The results show that the compounds IY220816B-1, ID 2208235A-1, IY221116B-1, ID221028A-1 and IY220831B-1 can effectively inhibit proliferation of cells such as breast cancer, liver cancer, lung cancer, drug-resistant lung cancer, colon cancer, leukemia, gastric cancer, pancreatic cancer, prostate cancer, esophageal cancer and the like, and inhibit activation of STAT3 signal channels.
The tumor cells are breast cancer cells MDA-MB-468, liver cancer cells HepG2, HCC97H, lung cancer cells PC9, octreotide-resistant lung cancer cells PC9-AR, colon cancer cells HT-29, leukemia cells Jurkat, gastric cancer cells HGC27, pancreatic cancer cells BxPC-3, prostate cancer cells Lncap and esophageal cancer cells KYSE450.
In summary, the present invention provides a novel naphthylurea-piperidine compound and its derivatives for use in tumor therapy and potential molecular mechanisms.
Detailed Description
In order to make the technical purpose, technical scheme and beneficial effect of the present invention more clear, the technical scheme of the present invention is further described below with reference to the accompanying drawings and specific embodiments.
In the process according to the invention for the synthesis of the compounds of the formula I, the various starting materials used for the reaction are preparable by the person skilled in the art according to the prior art, or can be prepared by methods known from the literature, or can be obtained commercially. The intermediates, raw materials, reagents, reaction conditions and the like used in the above reaction schemes may be appropriately changed according to the knowledge already known to those skilled in the art.
In the present invention, unless otherwise specified, wherein: (i) The temperature is expressed in degrees centigrade (DEG C), and the operation is performed in a room temperature environment; more specifically, the room temperature is 20-30 ℃; (ii) Drying the organic solvent by a common drying method, evaporating the solvent by a rotary evaporator under reduced pressure, wherein the bath temperature is not higher than 50 ℃; the volume ratio of the developing agent to the eluent is equal; (iii) the reaction process is followed by Thin Layer Chromatography (TLC); (iv) The final product had satisfactory proton nuclear magnetic resonance (1 H-NMR).
EXAMPLE 1 Synthesis of IY220816B-1 Compound
The compound IY220816B-1, named 4- (2- (piperidin-1-yl) ethoxy) -N- (4- (3- (pyridin-4-ylmethyl) ureido) naphthalen-1-yl) benzamide,
The synthetic route is as follows:
Tert-butyl (4-aminonaphthalen-1-yl) carbamate (intermediate 2A)
Raw material 1A (1 g,5.31mmol,1.0 eq), boc2 O (1.16 g,5.31mmol,1.0 eq) and Pd/C (100 mg,0.1 eq) were dissolved in 150ml methanol and reacted under hydrogen for 12 hours with stirring; TLC (PE/ea=2/1, rf/product=0.25) showed that the starting material was reacted completely, with a new point; the reaction solution was filtered through celite, rinsed with ethyl acetate, and the organic phase was spin-dry stirred through the column and rinsed with (PE/ea=10/1 to 1/1) to give intermediate 2A (1.05 g, 76.6%) as a yellow solid.
Step 2.Tert-butyl (4- (4- (2- (piperidin-1-yl) ethoxy) benzamido) naphthalen-1-yl) carbamate (intermediate 3A)
Raw material 2A (400 mg,1.55mmol,1.0 eq), raw material 2A (383 mg,1.55mmol,1.0 eq), HBTU (704 mg,1.86mmol,1.2 eq) and DIEA (240 mg,1.86mmol,1.2 eq) were dissolved in 10ml DMF and stirred at room temperature for 12 hours; TLC (DCM/meoh=20/1, rf/product=0.35) showed that the starting material was reacted completely, with a new point; the reaction was diluted with 100ml ethyl acetate and washed 3 times with saturated brine (100 ml x 3), the organic phase was dried, spin-dried and column-passed, and rinsed with (DCM/meoh=100/1-20/1) to give intermediate 3A (580 mg, 76.5%) as a yellow solid.
Step 3N- (4-aminonaphthalen-1-yl) -4- (2- (piperidin-1-yl) ethoxy) benzamide (intermediate 4A)
Raw material 3A (630 mg,1.29mmol,1.0 eq) was dissolved in 20ml of methanol, and 10ml of 4mol/L ethyl acetate solution of hydrogen chloride was added thereto, followed by stirring at room temperature for reaction for 2 hours; TLC (DCM/meoh=20/1, rf/product=0) indicated that the starting material was reacted completely, a new spot was generated; the reaction solution was directly dried by spin to give 4A (600 mg, 97%) as a white solid.
Step (a) 4.4-(2-(piperidin-1-yl)ethoxy)-N-(4-(3-(pyridin-4-ylmethyl)ureido)naphthalen-1-yl)benzamide(IY220816B-1)
Raw material 4A (100 mg,0.22mmol,1.0 eq) and raw material 5 (44 mg,0.22mmol,1.0 eq) were dissolved in 20ml tetrahydrofuran and reacted for 10 hours with stirring at 120 ℃. TLC (DCM/meoh=10/1, rf/product=0.2) showed complete reaction of starting material, new spot was generated; the reaction was directly spin-dried through the column and rinsed with (DCM/meoh=50/1 to 10/1) to give IY220816B-1 (55 mg, 48.7%) as a yellow solid.
1H NMR(CDCl3,300MHz)δ:10.16(s,1H),8.82(s,1H),8.54(d,J＝6.0Hz,2H),8.07(d,J＝9.0Hz,1H),7.95(d,J＝9.0Hz,2H),7.60(d,J＝9.0Hz,2H),7.55-7.47(m,2H),7.37(d,J＝6.0Hz,2H),7.10(d,J＝6.0Hz,2H),4.43(d,J＝6.0Hz,2H),4.18(t,J＝6.0Hz,2H),2.70(t,J＝6.0Hz,2H),2.51(t,J＝6.0Hz,2H),1.52(t,J＝6.0Hz,2H),1.25(m,2H).
EXAMPLE 2 Synthesis of ID2208235A-1 Compound
The compound ID220825A-1, named 1- (4- (1- (4- (2- (piperidin-1-yl) ethoxy) phenyl) ethoxy) naphthalen-1-yl) -3- (pyridin-4-ylmethyl) urea, was synthesized as follows:
Step 1.1- (2- (4- (1- ((4-nitronaphthalen-1-yl) oxy) ethyl) phenyl) ethyl) piperidine (intermediate 2B)
Raw material 1a (1.57 g,6.28mmol,1.2 eq) and potassium tert-butoxide (704 mg,6.28mmol,1.2 eq) were dissolved in 20ml tetrahydrofuran, after stirring for 30 minutes raw material 1B (1.0 g,5.23mmol,1.0 eq) was added and the reaction was continued at room temperature for 30 minutes; TLC (DCM/meoh=20/1, rf/product=0.45) showed that the starting material was reacted completely, with a new point; the reaction was directly spun-dried over the column and rinsed with DCM/meoh=50/1 to 10/1 to give intermediate 2B (1.7 g, 77.3%) as a yellow oily liquid.
Step 2.4- (1- (4- (2- (piperidin-1-yl) ethoxy) phenyl) ethoxy) naphthalen-1-amine (intermediate 3B)
Raw material 2B (1 g,2.38mmol,1.0 eq) was dissolved in ethanol, warmed to 50℃and iron powder (664 mg,11.89mmol,5.0 eq) was added and the reaction stirred for 30 minutes at 50℃further; TLC (DCM/meoh=20/1, rf/product=0.25) showed that the starting material was reacted completely, with a new point; the reaction was filtered through celite and the filtrate was directly spin-dried through the column and rinsed with DCM/meoh=50/1-10/1 to give intermediate 3B (750 mg, 80.8%) as a reddish brown oil.
Step 3.1- (4- (1- (4- (2- (piperidin-1-yl) ethoxy) phenyl)
ethoxy)naphthalen-1-yl)-3-(pyridin-4-ylmethyl)urea(ID220825A-1)
Raw material 3B (200 mg,0.51mmol,1.0 eq) and raw material 5 (103 mg,0.51mmol,1.0 eq) were dissolved in 20ml tetrahydrofuran and reacted at 120℃for 10 hours with stirring; TLC (DCM/meoh=10/1, rf/product=0.2) showed complete reaction of starting material, new spot was generated; the reaction was directly spin-dried through the column and rinsed with (DCM/meoh=50/1 to 10/1) to give ID220825A-1 (120 mg, 44.7%) as a white solid.
1H NMR(CDCl3,300MHz)δ:8.0(d,J＝9.0Hz,2H),6.93(d,J＝9.0Hz,2H),4.17(t,J＝6.0Hz,2H),3.90(q,1H),2.82(t,J＝6.0Hz,2H),2.58-2.55(m,4H),1.66-1.61(m,4H),1.50(t,J＝3.0Hz,2H),1.25(d,J＝6.0Hz,3H).
EXAMPLE 3 Synthesis of IY 2208235A-1 Compound
The compound IY220825A-1, named 1- (4- (4- (2- (piperidin-1-yl) ethoxy) phenyl) naphthalen-1-yl) -3- (pyridin-4-ylmethyl) urea, was synthesized as follows:
Step 1.1- (4-bromonaphthalen-1-yl) -3- (pyridin-4-ylmethyl) urea (intermediate 2C)
Raw material 1C (1 g,4.5mmol,1.0 eq) and raw material 5 (910 mg,4.5mmol,1.0 eq) were dissolved in 100ml tetrahydrofuran and reacted at 120℃for 10 hours with stirring; TLC (DCM/meoh=10/1, rf/product=0.6) showed complete reaction of starting material, new spot was generated; the reaction was directly spin-dried through the column and rinsed with (DCM/meoh=50/1 to 30/1) to give intermediate 2C (810 mg, 50.6%) as a white solid.
Step 2.1- (4- (4- (2- (piperidin-1-yl) ethoxy) phenyl) naphthalen-1-yl) -3- (pyridin-4-ylmethyl) urea (IY 220825A-1)
Raw material 2C (200 mg,0.56mmol,1.0 eq), raw material 2b (186 mg,0.56mmol,1.2 eq), K2CO3 (93 mg,0.67mmol,1.2 eq) and Pd (dppf) Cl2 (12 mg,0.056mmol,0.1 eq) were dissolved in 1, 4-dioxane (20 ml) and water (2 ml) and reacted with stirring at 100℃for 3 hours; TLC (DCM/meoh=10/1, rf/product=0.2) showed complete reaction of starting material, new spot was generated; the reaction was directly spin-dried through the column and rinsed with (DCM/meoh=50/1 to 10/1) to give IY220825A-1 (220 mg, 81.5%) as a white solid.
1H NMR(CDCl3,300MHz)δ:8.0(d,J＝9.0Hz,2H),6.93(d,J＝9.0Hz,2H),4.17(t,J＝6.0Hz,2H),2.82(t,J＝6.0Hz,2H),2.58-2.55(m,4H),1.66-1.61(m,4H),1.50(t,J＝3.0Hz,2H).
EXAMPLE 4 Synthesis of IY221116B-1 Compound
The name of the compound IY221116B-1 is 1-(4-(3-(4-(2-(piperidin-1-yl)ethoxy)phenyl)-1,2,4-oxadiazol-5-yl)naphthalen-1-yl)-3-(pyridin-4-ylmethyl)urea,
The synthetic route is as follows:
Step 1.5- (4-bromonaphthalen-1-yl) -3- (4- (2- (piperidin-1-yl) ethoxy) phenyl) -1,2,4-oxadiazole (intermediate 2D)
Raw material 1D (1 g,3.98mmol,1.0 eq), raw material 1b (1.05 g,3.98mmol,1.0 eq), HBTU (1.8 g,4.8mmol,1.2 eq) and DIEA (618 mg,4.8mmol,1.2 eq) were dissolved in 100ml DMF and reacted for 1 hour at room temperature with stirring, then for 10 hours at 120 ℃. TLC (DCM/meoh=10/1, rf/product=0.35) showed that the starting material was reacted completely, a new spot was generated; the reaction was directly spin-dried through the column and rinsed with (DCM/meoh=50/1 to 10/1) to give intermediate 2D (1.7 g, 89.1%) as an off-white solid.
Step 2N- (2, 4-dimethoxybenzyl) -4- (3- (4- (2- (piperidin-1-yl) ethoxy)
Phenyl) -1,2, 4-oxadiazol-yl naphthalen-1-amine (intermediate 3D)
Raw material 2D (1 g,2.09mmol,1.0 eq), raw material 2c (349 mg,2.09mmol,1.0 eq), BINAP (130 mg,0.209mmol,0.1 eq), pd (OAc)2 (24 mg,0.104mmol,0.05 eq) and Cs2CO3 (817 mg,2.51mmol,1.2 eq) were dissolved in 100ml toluene and reacted under nitrogen at 110℃for 12 hours with stirring; TLC (DCM/meoh=10/1, rf/product=0.25) showed complete reaction of starting material, new spot was generated; the reaction was directly spin-dried through the column and rinsed with (DCM/meoh=50/1 to 10/1) to give intermediate 3D (650 mg, 55.1%) as a white solid.
Step 3.4- (3- (4- (2- (piperidin-1-yl) ethoxy) phenyl) -1,2, 4-oxadiazol-5-yl) naphthalen-1-amine (intermediate 4D)
Raw material 3D (500 mg,0.88mmol,1.0 eq) was dissolved in 20ml dichloromethane, 5ml trifluoroacetic acid (67 mmol) was added dropwise at 0℃and the reaction was stirred at room temperature for 1 hour; TLC (DCM/meoh=10/1, rf/product=0.25) showed complete reaction of starting material, new spot was generated; the reaction was dried directly by spin-drying, diluted with ethyl acetate (150 ml), ph=8 was adjusted with saturated aqueous sodium bicarbonate, the organic phase was dried by spin-drying and applied to the column, and rinsed with (DCM/meoh=50/1-10/1) to give intermediate 4D (350 mg, 95.4%) as a yellow solid.
Step 4.1- (4- (3- (4- (2- (piperidin-1-yl) ethoxy) phenyl) -1,2, 4-oxadiazol-5-yl)
naphthalen-1-yl)-3-(pyridin-4-ylmethyl)urea(IY221116B-1)
Raw material 4D (200 mg,0.48mmol,1.0 eq) and raw material 5 (97 mg,0.48mmol,1.0 eq) were dissolved in 20ml tetrahydrofuran and reacted at 120℃for 10 hours with stirring; TLC (DCM/meoh=10/1, rf/product=0.1) showed complete reaction of starting material, new spot was generated; the reaction was directly spin-dried through the column and rinsed with (DCM/meoh=50/1 to 10/1) to give IY221116B-1 (102 mg, 39.2%) as a yellow solid.
1H NMR(CDCl3,300MHz)δ:8.02(d,J＝9.0Hz,2H),6.90(d,J＝9.0Hz,2H),4.12(t,J＝6.0Hz,2H),2.82(t,J＝6.0Hz,2H),2.58-2.53(m,4H),1.66-1.60(m,4H),1.51(t,J＝3.0Hz,2H).
EXAMPLE 5 Synthesis of ID221028A-1 Compound
The compound ID221028A-1, named 1- (4- ((4- (2- (piperidin-1-yl) ethoxy) phenyl) ethynyl) naphthalen-1-yl) -3- (pyridin-4-ylmethyl) urea, was synthesized as follows:
Step 1.1- (4-bromonaphthalen-1-yl) -3- (pyridin-4-ylmethyl) urea (intermediate 2C)
See example 3, step1 for a specific preparation procedure.
Step (a) 2.1-(4-((4-(2-(piperidin-1-yl)ethoxy)phenyl)ethynyl)naphthalen-1-yl)-3-(pyridin-4-ylmethyl)urea(ID221028A-1)
Raw material 2C (200 mg,0.56mmol,1.0 eq), raw material 2d(129mg,0.56mmol,1.0eq),Pd(PPh3)Cl2(24mg,0.056mmol,0.1eq),CuI(10.69mg,0.056mmol,0.1eq) and triethylamine (227 mg,2.25mmol,4 eq) were dissolved in 10ml DMF and heated to 50℃and stirred for 2 hours; TLC (DCM/meoh=10/1, rf/product=0.2) showed that the starting material was reacted completely, a new spot was generated; the reaction was directly spin-dried through the column and rinsed with (DCM/meoh=50/1 to 10/1) to give ID221028a-1 (80 mg, 28.5%) as a grey solid.
1H NMR(CDCl3,300MHz)δ:8.10(d,J＝9.0Hz,2H),6.99(d,J＝9.0Hz,2H),4.25(t,J＝6.0Hz,2H),2.85(t,J＝6.0Hz,2H),2.56-2.53(m,4H),1.63-1.60(m,4H),1.52(t,J＝3.0Hz,2H).
EXAMPLE 6 Synthesis of IY220831B-1 Compound
The compound IY220831B-1, named 1- (4- (2- (piperidin-1-yl) ethoxy) phenyl) -3- (4- (3- (pyridin-4-ylmethyl) ureido) naphthalen-1-yl) urea, was synthesized as follows:
Step 1.Tert-butyl (4- (3- (4- (2- (piperidin-1-yl) ethoxy) phenyl) ureido) naphthalen-1-yl) carbamate (intermediate 2F)
Raw material 2A (1 g,3.87mmol,1.0 eq), raw material 1c (0.85 g,3.87mmol,1.0 eq) and CDI (0.63 g,3.87mmol,1.0 eq) were dissolved in 50ml tetrahydrofuran and reacted with stirring at 70℃for 10 hours; TLC (DCM/meoh=10/1, rf/product=0.3) showed complete reaction of starting material, new spot was generated; the reaction was directly spin-dried through the column and rinsed with (DCM/meoh=50/1 to 10/1) to give intermediate 2F (1.02 g, 52.3%) as a yellow solid.
Step 2.Tert-butyl (4- (3- (4- (2- (piperidin-1-yl) ethoxy) phenyl) ureido) naphthalen-1-yl) carbamate (intermediate 3F)
Raw material 2F (1 g,1.98mmol,1.0 eq) was dissolved in 20ml of methanol, and a 20ml, 4mol/L solution of hydrogen chloride in ethyl acetate was added; stirring at room temperature for reaction for 2 hours; TLC (DCM/meoh=10/1, rf/product=0) indicated that the starting material was reacted completely, a new spot was generated; the reaction solution was directly poured to give intermediate 3F (800 mg, 99%) as a white solid.
Step (a) 3.1-(4-(2-(piperidin-1-yl)ethoxy)phenyl)-3-(4-(3-(pyridin-4-ylmethyl)ureido)naphthalen-1-yl)urea(IY220831B-1)
Raw material 3F (200 mg,0.49mmol,1.0 eq) and raw material 5 (99 mg,0.49mmol,1.0 eq) were dissolved in 20ml tetrahydrofuran and reacted at 120℃for 10 hours with stirring; TLC (DCM/meoh=10/1, rf/product=0.1) showed complete reaction of starting material, new spot was generated; the reaction was directly spin-dried through the column and rinsed with (DCM/meoh=50/1 to 10/1) to give IY220831B-1 (101 mg, 39.1%) as a yellow solid.
1H NMR(CDCl3,300MHz)δ:8.05(d,J＝9.0Hz,2H),6.93(d,J＝9.0Hz,2H),4.15(t,J＝6.0Hz,2H),2.85(t,J＝6.0Hz,2H),2.55-2.53(m,4H),1.63-1.60(m,4H),1.51(t,J＝3.0Hz,2H).
Example 7: proliferation inhibiting effect of IY220816B-1, ID 2208235A-1, IY221116B-1, ID221028A-1 and IY220831B-1 on tumor cells such as breast cancer, liver cancer and lung cancer
Tumor cells in the logarithmic growth phase were collected separately, the cell suspension concentration was adjusted to 5X 104 cells/mL, and 96-well cell culture plates were added with a volume of 100ul per well. The DMSO is used as solvent for comparison, and the accepted STAT3 signal inhibitor WP1066 (Chinese name, (2E) -3- (6-bromo-2-pyridyl) -2-cyano-N- [ (1S) -1-phenylethyl ] -2-acrylamide, CAS:857064-38-1, has the structure ofAs positive control, the novel naphthyridine-piperidine compounds IY220816B-1, ID 2208235A-1, IY221116B-1, ID221028A-1, IY220831B-1 and the like are diluted by DMSO and added into culture wells, so that the final concentrations of the compounds in the system are respectively 0.1, 0.3, 1, 3, 10, 30, 100 and 300 (mu mol/L). After further culturing for 48h, 10. Mu.L of MTT solvent (5 mg/ml) was added to each well, incubated for 4h at 37℃and the culture supernatant was aspirated off, 150. Mu.L of DMSO was added to each well, shaking and decolorizing was performed for 10min on a shaker, reading was performed on a microplate reader, the OD at an absorbance wavelength of 490nm was measured, the results were recorded, and the cell growth curve was drawn with the dose of the compound as the abscissa and the absorbance as the ordinate. The statistical results of half inhibition rates (IC 50 values) of IY220816B-1, ID 2208235A-1, IY221116B-1, ID221028A-1, IY220831B-1 and the like on tumor cells are shown in FIG. 1.
The results shown in FIG. 1 indicate that compared with the positive control medicines WP1066, IY220816B-1, ID 2208235A-1, IY221116B-1, ID221028A-1, IY220831B-1 and the like have better proliferation inhibition effects on breast cancer, liver cancer, lung cancer, drug-resistant lung cancer, colon cancer, leukemia, gastric cancer, pancreatic cancer, prostate cancer and esophageal cancer cells, and especially the IY220816B-1 has the strongest tumor inhibition activity on breast cancer. The application takes IY220816B-1 as an important research object, and further researches the anti-tumor effect and action mechanism of the compounds.
Example 8: inhibition of STAT3 signaling pathway in breast cancer cells by IY220816B-1
1. Cell culture and dosing a. MDA-MB-468 cells in logarithmic phase were digested with pancreatin, and single cell suspension with a density of 4X 105 cells/mL was prepared in L-15 medium containing 10% fetal bovine serum, and inoculated into 6-well plates with 2mL of cell suspension per well. Incubator incubation at 37℃ (MDA-MB-468 cell culture without CO2). After cell attachment, IY220816B-1 was added to give final concentrations of 0,0.015,0.03 and 0.06. Mu.M, respectively. c. After a further 48h of incubation, the cells were lysed with RIPA lysate and the proteins were collected.
2. Cell collection and lysis: a. the upper medium was discarded and the cells were washed twice with pre-chilled PBS. 100. Mu.L of precooled RIPA cell lysate (protease inhibitor and PMSF were added to the lysate at a ratio of 1:100) was added per well.
B. Lysing on ice for 3min, scraping off the cells with a cell scraper and collecting into a 1.5mL EP tube; the mixture was then placed on ice for 30min and vortexed once every 6 min. c.4℃and 12000g for 10min. d. Cell supernatants were transferred to new EP tubes. Cell supernatants are divided into two parts: 5 mu L of the sample is added into an EP tube of 1.5mL for BCA protein content measurement, and 45 mu L of 1 XPBS is added for even mixing; the remaining cell supernatant was quantified by taking 80. Mu.L, adding 5X SDS Loading Buffer. Mu.L, mixing well, boiling in boiling water for 10min, centrifuging, loading or storing in a refrigerator at-20deg.C. e. Protein concentration determination:
(1) BCA working solution preparation: the total required amount of A and B mixed working fluid is calculated according to the number of the standard substance and the sample to be tested. The volume ratio of the solid agent A to the solid agent B is 50:1, preparing working solution, and uniformly mixing by vortex oscillation for standby.
(2) 1 XPBS diluted protein standard:
| 7 pipe number | 1×PBS(μl) | BSA standard dosage | BSA standard (μg/ml) |
| A | 0 | 100 | 2000 |
| B | 200 | 200 | 1000 |
| C | 200 | 200 (From tube B) | 500 |
| D | 200 | 200 (Taken from the C-tube) | 250 |
| E | 200 | 200 (Taken from the D-tube) | 125 |
| F | 400 | 100 (Taken from E-tube) | 25 |
| G | 200 | 0 | 0 (Blank) |
(3) Protein standards and sample supernatants diluted with PBS (10-fold dilution) were each taken at 25 μl and added to a new 96-well plate. Then 200. Mu.L of BCA working solution prepared in advance was added respectively and mixed well. The bubbles are not generated by blowing, the cover of the 96-well plate is tightly covered, and the reaction is carried out for 30min in a constant temperature box at 37 ℃. (4) Taking out the 96-well plate, recovering to room temperature for 3-5 min, measuring the absorbance value of A562 on an enzyme label instrument, copying out the obtained value and storing the obtained value in an Excel table. A standard curve was made and the protein content of 1 μl per sample was calculated for protein loading.
3. SDS-PAGE: (1) The gel plate was fixed and 10% SDS-PAGE separating gel was prepared.
The release gel was prepared according to the following table: 10mL
(2) And (3) adding the mixed separating glue into 2 glue plates respectively, adding the glue plates to a position 1.0cm away from the top, filling the glue plates with absolute ethyl alcohol, and standing for 30-45 min. (3) After the gel is separated, the residual absolute ethyl alcohol is poured out, and the residual absolute ethyl alcohol is sucked clean by filter paper. (4) 5mL of 5% concentrated gel was prepared according to the following Table
| Deionized water | 2.77mL |
| 30％(m/v)Acrylamide | 830μL |
| 0.5M Tris-HCl (pH 6.8) buffer | 1.26mL |
| 10％(m/v)SDS | 50μL |
| 10％(m/v)APS | 50μL |
| TEMED | 5μL |
| Total | 5mL |
(5) Slowly adding the prepared concentrated glue into a glue plate to avoid generating bubbles, inserting a comb, and standing for 30-45 min.
(6) The protein sample was removed and heated in a water bath at 100deg.C for 5min at 10000rpm and centrifuged for 5min. (7) The gel plate was fixed in an electrophoresis tank, SDS-PAGE electrophoresis buffer was added, the comb was pulled out, and the treated protein samples were sequentially added to the sample tank, 50. Mu.g per well of protein. (8) 80V electrophoresis for 40min. (9) Changing the voltage to 120V for electrophoresis for about 1.5 hours until bromophenol blue goes out of the colloid;
4. Western-blot: (1) And (3) placing the SDS-PAGE gel subjected to electrophoresis into TBST buffer solution for rinsing once, and placing the protein gel into transfer buffer solution for soaking. (2) Soaking a layer of cotton pad in a membrane transfer buffer solution, clamping onto a membrane transferring instrument by using forceps, placing a blackboard, the cotton pad, filter paper, albumin glue, PVDF membrane, filter paper, the cotton pad and a whiteboard in order, clamping, and placing into the membrane transferring instrument. If bubbles exist between each two layers, the bubbles are removed by using a glass solid tube to lightly roll. And (3) opening a film transfer instrument, and performing constant-current transfer printing for 80 minutes at 300 mA. (4) The membranes were placed in TBST buffer and rinsed 3 times for 8min each. (5) blocking with 20mL of 5% BSA-TBST blocking solution at room temperature for 2h. (6) Primary antibody was added and incubated at 4℃at 60rpm overnight. (7) The membranes were washed three times with TBST at room temperature, 60rpm, 10min each. (8) adding a secondary antibody, and incubating for 1h at room temperature. (9) The membranes were washed three times with TBST at room temperature, 60rpm, 10min each. (10) 1ml of each of chemiluminescent substrate solid solution A and solution B is taken and developed for 2min at room temperature. (11) the liquid on the membrane is sucked by filter paper, and the membrane is subjected to machine aeration.
5. And (3) preparation of a reagent:
(1) 10% SDS: 1g of high purity (electrophoretic grade) SDS was weighed into a 10mL centrifuge tube, added with about 8mL deionized water, dissolved by heating, and stored at room temperature to a volume of 10 mL.
(2) 10% Ammonium persulfate (Ammonium persulfate, AP): 1g of ammonium persulfate was weighed, dissolved by stirring after adding about 10mL of deionized water, and stored at 4 ℃.
(3) 5 Xrunning buffer: 15.1g of Tris, 94g of Glycine and 5.0g of SDS are weighed in a beaker, added with 1L of double distilled water for dissolution, stored at room temperature and diluted 5 times with time.
(4) Transfer buffer: 5.8g of Tris, 11.6g of glycine and 0.75g of SDS are weighed into a beaker, 700mL of double distilled water is added, the volume is fixed to 800mL after dissolution, and finally 200mL of methanol is added.
(5) 1.5Mol/L Tris-HCl,100ml: then 18.15gtris is dissolved in 80ml of water and adjusted to 8.8 by 4N HCl, and the volume is fixed to 100 ml.
(6) 0.5Mol/L Tris-HCl,1000ml: 60.5 g of tris base was weighed, water was added to 850ml, concentrated hydrochloric acid was added and stirred until all dissolved, the pH was adjusted to 6.8, and water was added to 1L.
(7) TBS buffer: 8.8g of NaCl was weighed out in 800mL of distilled water, dissolved, added with 10mL of 1mol/L Tris HCl (pH 7.5), and kept at room temperature to a volume of 1L.
(8) TBST buffer: to 1L TBS buffer, 500. Mu.L of 20% Tween20 was added to give a final concentration of 0.1% Tween20, and the mixture was prepared immediately.
(9) Blocking solution, antibody dilution: 5% skim milk powder or BSA was added to TBST buffer and ready-to-use.
As shown in FIG. 2, the Western blot results show that 0.06 mu M of IY220816B-1 can significantly inhibit the expression levels of STAT3 total protein (T-STAT 3), a key marker p-STAT3 for STAT3 activation (Tyr 705) and a downstream target protein Cyclin D1 of STAT3 in MDA-MB-468 cells.
In conclusion, the result shows that the piperazine compound represented by IY220816B-1 can effectively inhibit proliferation of various tumor cells, has obvious inhibition effect on STAT3 and related proteins, and shows good targeted anti-tumor effect. According to the general way of drug development (conventional anti-tumor in-vitro screening is carried out firstly and then targeted research is carried out), the compound can be applied to tumor therapeutic drugs related to abnormal cell proliferation, and the anti-tumor drugs can be prepared by salifying with human bodies or mixing with a medicinal carrier.
Finally, what should be said is: the above embodiments are only for illustrating the technical solution of the present invention, but any equivalent replacement of the present invention and modification or partial replacement without departing from the spirit and scope of the present invention should be covered in the scope of the claims of the present invention.