CN109734660B - Tert-butyl substituted serine-containing peptide-like compound and preparation method and application thereof - Google Patents
Tert-butyl substituted serine-containing peptide-like compound and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a peptide-like compound containing tert-butyl substituted serine, a preparation method and application thereof, wherein target compounds are synthesized by utilizing reactions such as acylation, Suzuki coupling, condensation and the like, and a compound library is constructed, and the compound is a Bcr-Abl small molecular tyrosine kinase inhibitor with a novel molecular structure.
Description
Technical Field
The invention relates to a tert-butyl substituted serine-containing peptide-like compound, and a preparation method and application thereof.
Background
Chronic myeloid leukemia (CM L) is a malignant clonal proliferative disease occurring in myeloid hematopoietic stem cells, and accounts for 15-20% of adult leukemia patients, and is characterized in that a Ph chromosome can be detected in CM L patients, the Ph chromosome is a breakpoint aggregation cluster-Elbeson (BCR-AB L) fusion gene formed by mutual translocation of normal chromosome 22 and chromosome 9 of human bodies, and the fusion gene codes for Bcr-Abl fusion protein with continuously activated tyrosine kinase activity.
Disclosure of Invention
The invention aims to provide a tert-butyl substituted serine-containing peptoid compound and a preparation method and application thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
a peptide-like compound containing tert-butyl substituted serine has the following structural formula:
wherein R is
-NH2、
A preparation method of a peptoid compound containing tert-butyl substituted serine comprises the following steps:
1) acylation reaction of 5-bromo-2-aminopyridine with an acyl chloride compound to produce an acylated 5-bromo-2-aminopyridine;
2)N2under protection, 5-Reacting the bromonicotinic acid, thionyl chloride and amine compounds to prepare ammoniated 5-bromonicotinic acid;
3) under the catalysis of palladium tetratriphenylphosphine, carrying out Suzuki coupling reaction on acylated 5-bromo-2-aminopyridine or aminated 5-bromonicotinic acid and carboxyphenylboronic acid to obtain a biphenyl compound;
4) Fmoc-O-tert-butyl-L-serine was condensed with 4-chloro-3-trifluoromethylaniline to give (9H-fluoren-9-yl) methyl (S) -3- (tert-butoxy) -1- ((4-chloro-3- (trifluoromethyl) phenyl) amino) -1-oxopropan-2-yl) carbamate;
5) (9H-fluoren-9-yl) methyl (S) -3- (tert-butoxy) -1- ((4-chloro-3- (trifluoromethyl) phenyl) amino) -1-oxopropan-2-yl) carbamate the Fmoc protecting group was removed to give (S) -2-amino-3- (tert-butoxy) -N- (4-chloro-3- (trifluoromethyl) phenyl) propanamide;
6) (S) -2-amino-3- (tert-butoxy) -N- (4-chloro-3- (trifluoromethyl) phenyl) propanamide is condensed with a biphenyl compound to produce a peptoid compound containing tert-butyl substituted serine.
The further improvement of the invention is that the specific process of the step 1) is as follows: dissolving 5-bromo-2-aminopyridine in anhydrous dichloromethane, adding triethylamine, dropwise adding an acyl chloride compound under an ice bath condition, removing the ice bath after dropwise adding is finished, heating to room temperature, reacting for 12h, and performing post-treatment to obtain acylated 5-bromo-2-aminopyridine.
The further improvement of the invention is that the specific process of the step 2) is as follows: in N2Under protection, dropwise adding thionyl chloride into 5-bromonicotinic acid, heating and refluxing for 2-3h after dropwise adding is finished until the solution is clear, removing thionyl chloride under reduced pressure to obtain a light yellow solid, dissolving the light yellow solid into anhydrous dichloromethane, and then dropwise adding the light yellow solid into a dichloromethane solution of an amine compound under an ice bath condition; after the dropwise addition, the temperature is raised to room temperature for reaction for 12 hours, and after the reaction is finished, post-treatment is carried out to obtain the ammoniated 5-bromo-nicotinic acid.
The further improvement of the invention is that the specific process of the step 3) is as follows: adding acylated 5-bromo-2-aminopyridine and p/m-carboxyphenylboronic acid to a reaction vessel, or adding aminated 5-bromo-nicotinic acid and p/m-carboxyphenylboronic acidAdding the mixture into a reaction container, and sequentially adding cesium carbonate and palladium tetratriphenylphosphine; then adding a mixed solution of acetonitrile and water, N2Protecting, heating to 90 ℃ and reacting for 48 hours; after the reaction is finished, post-treatment is carried out to obtain the biphenyl compound.
The further improvement of the invention is that the specific process of the step 4) is that 4-chloro-3-trifluoromethylaniline, HOBT and HATU are dissolved in anhydrous dichloromethane, then DIPEA is dripped, after the dripping is finished, dichloromethane solution of Fmoc-O-tert-butyl-L-serine is dripped, the reaction is carried out for 5H at room temperature, and the after-treatment is carried out, thus obtaining (9H-fluoren-9-yl) methyl (S) -3- (tert-butoxy) -1- ((4-chloro-3- (trifluoromethyl) phenyl) amino) -1-oxoprop-2-yl) carbamate.
The further improvement of the invention is that the specific process of the step 5) is as follows: (9H-fluoren-9-yl) methyl (S) -3- (tert-butoxy) -1- ((4-chloro-3- (trifluoromethyl) phenyl) amino) -1-oxopropan-2-yl) carbamate was dissolved in anhydrous dichloromethane at 0 ℃ and a 20% by volume solution of piperidine in DMF was added dropwise to the solution, followed by reaction for 1 hour and workup to give (S) -2-amino-3- (tert-butoxy) -N- (4-chloro-3- (trifluoromethyl) phenyl) propionamide.
The further improvement of the invention is that the specific process of the step 6) is as follows: dissolving a biphenyl compound and PyBOP in anhydrous DMF, dropwise adding a DIPEA solution, reacting at room temperature for 5min, adding (S) -2-amino-3- (tert-butoxy) -N- (4-chloro-3- (trifluoromethyl) phenyl) propionamide, reacting at room temperature for 12h, and carrying out post-treatment to obtain the peptoid compound containing tert-butyl substituted serine.
A peptoid compound containing tert-butyl substituted serine is applied to preparation of drugs for inhibiting the activity of Abl kinase and T315I mutant Abl kinase.
A peptide-like compound containing tert-butyl substituted serine, and its application in preparing antineoplastic agent are disclosed.
Compared with the prior art, the invention has the advantages that the invention synthesizes target compounds by utilizing reactions such as acylation, Suzuki coupling, condensation and the like, constructs a compound library, the compounds are Bcr-Abl small molecule tyrosine kinase inhibitors with novel molecular structures, and characterizes the structures of the target compounds by means of MS, NMR and the like, the invention adopts a fragment-based drug design strategy, takes diphenylpyridine as a hinge region binding fragment, introduces tert-butyl substituted L-serine as flexible L inker, constructs a peptide-like small molecule compound library with kinase inhibitory activity, and discovers a tyrosine kinase inhibitor with Bcr-Abl kinase inhibitory activity by ADP-Glo kinase activity screening, kinase screening tests show that the compounds have certain inhibitory activity on Abl kinase and T315I mutant Abl kinase, and structure-activity relationship analysis shows that derivatives of the introduced tert-butyl substituted L-serine have good spatial matching with the Abl kinase sites, the action mode is consistent with reference small molecule imatinib L, and the introduction of the tert-butyl substituted tyrosine kinase has important inhibitory activity on ABr kinase and the side chain inhibitory activity of a novel ATP-tyrosine kinase receptor with improved side chain effect.
Drawings
FIG. 1 is a synthetic route diagram of the present invention.
Detailed Description
The present invention will be described in detail with reference to the accompanying drawings.
Referring to fig. 1, the structural formula of a peptoid compound containing tert-butyl substituted serine of the present invention is:
wherein, R is specifically as follows, see Table 1:
groups represented by R in Table 1
The following description will be made by way of specific examples.
Referring to fig. 1, the preparation method of the peptoid compound containing tert-butyl substituted serine with the structure comprises the following steps:
1) acylating 5-bromo-2-aminopyridine with a corresponding acyl chloride compound to prepare acylated 5-bromo-2-aminopyridine;
the specific process of the step 1) is as follows: dissolving 5-bromo-2-aminopyridine in anhydrous dichloromethane, adding triethylamine, slowly dropwise adding the corresponding acyl chloride compound into the solution under the ice bath condition, and after dropwise adding, removing the ice bath, heating to room temperature, and reacting for 12 h. After the reaction is finished, dichloromethane is added for dilution, washing is carried out, saturated sodium bicarbonate is washed, saturated sodium chloride is washed, anhydrous sodium sulfate is dried, reduced pressure distillation and column chromatographic separation are carried out, and a white solid, namely acylated 5-bromo-2-aminopyridine, is obtained.
2)N2Under protection, 5-bromonicotinic acid reacts with thionyl chloride and corresponding amine compounds to prepare ammoniated 5-bromonicotinic acid;
the specific process of the step 2) is as follows: in N2Under protection, dropwise adding thionyl chloride into 5-bromonicotinic acid, heating and refluxing for 2-3h after dropwise adding is finished until the solution is clear, removing thionyl chloride by rotary decompression to obtain light yellow solid, dissolving the light yellow solid into anhydrous dichloromethane, and slowly dropwise adding the active intermediate solution into dichloromethane solution of corresponding amine compounds under an ice bath condition; after the dropwise addition, the temperature is raised to room temperature for reaction for 12 hours, and after the reaction is finished, K is added into the reaction system2CO3Separating the solution to obtain dichloromethane phase, extracting the water phase with dichloromethane, combining the organic phase and anhydrous Na2SO4Drying; separating and purifying by column chromatography to obtain white solid, i.e. ammoniated 5-bromo-nicotinic acid.
3) Under the catalysis of palladium tetratriphenylphosphine, carrying out Suzuki coupling reaction on acylated 5-bromo-2-aminopyridine or aminated 5-bromonicotinic acid and p/m-carboxyphenylboronic acid to obtain a biphenyl compound;
the specific process of the step 3) is as follows: adding acylated 5-bromo-2-aminopyridine and p/m-carboxyl phenylboronic acid into a reaction vesselIn a reactor, or ammoniated 5-bromo-nicotinic acid and p/m-carboxyphenylboronic acid are added into a reaction container, and then cesium carbonate and palladium tetratriphenylphosphine are sequentially added; to the mixture was added a mixed solution of acetonitrile/water, N2Protecting, heating in oil bath to 90 ℃ for reaction for 48 h; after the reaction, the reaction solution was cooled to room temperature and filtered. Adjusting the pH value of the filtrate to 4 by using hydrochloric acid, separating out a white solid, performing suction filtration, and performing vacuum drying on a filter cake to obtain a product, namely the biphenyl compound.
4) Fmoc-O-tert-butyl-L-serine was condensed with 3-trifluoromethyl-4-chloroaniline to give (9H-fluoren-9-yl) methyl (S) -3- (tert-butoxy) -1- ((4-chloro-3- (trifluoromethyl) phenyl) amino) -1-oxopropan-2-yl) carbamate;
the specific process of the step 4) is that 4-chloro-3-trifluoromethylaniline, HOBT and HATU are dissolved in anhydrous dichloromethane, then DIPEA is dripped, after the dripping is finished, solution of Fmoc-O-tert-butyl-L-serine dissolved in anhydrous dichloromethane is dripped, the reaction is carried out for 6 hours at room temperature, after the reaction is finished, water is added to stop the reaction, dichloromethane is used for extraction, organic phases are combined, saturated NaCl is used for washing, and anhydrous NaSO is used for washing4Drying and separating by column chromatography to obtain (9H-fluoren-9-yl) methyl (S) -3- (tert-butoxy) -1- ((4-chloro-3- (trifluoromethyl) phenyl) amino) -1-oxopropan-2-yl) carbamate;
5) (9H-fluoren-9-yl) methyl (S) -3- (tert-butoxy) -1- ((4-chloro-3- (trifluoromethyl) phenyl) amino) -1-oxopropan-2-yl) carbamate the Fmoc protecting group was removed to give (S) -2-amino-3- (tert-butoxy) -N- (4-chloro-3- (trifluoromethyl) phenyl) propanamide;
the specific process of the step 5) is as follows: (9H-fluoren-9-yl) methyl (S) -3- (tert-butoxy) -1- ((4-chloro-3- (trifluoromethyl) phenyl) amino) -1-oxoprop-2-yl) carbamate was dissolved in anhydrous dichloromethane at 0 ℃ and a 20% by volume solution of piperidine in DMF was added dropwise, after 1H of reaction, work-up was carried out. To obtain (S) -2-amino-3- (tert-butoxy) -N- (4-chloro-3- (trifluoromethyl) phenyl) propanamide.
6) (S) -2-amino-3- (tert-butoxy) -N- (4-chloro-3- (trifluoromethyl) phenyl) propanamide is condensed with a biphenyl compound to produce a peptoid compound containing tert-butyl substituted serine.
The specific process of the step 6) is as follows: the biphenyl compound and PyBOP were dissolved in anhydrous DMF, and DIPEA solution was added dropwise thereto, followed by reaction at room temperature for 5min, followed by addition of (S) -2-amino-3- (tert-butoxy) -N- (4-chloro-3- (trifluoromethyl) phenyl) propionamide. Reacting at room temperature for 12h, and performing post-treatment to obtain the peptide-like compound containing serine.
The peptoid compound containing tert-butyl substituted serine prepared by the method is applied to inhibiting the activity of Abl kinase and T315I mutant Abl kinase.
The compounds have anti-tumor effect and can be applied to preparation of anti-tumor drugs.
Example 1
A peptide-like compound containing tert-butyl substituted serine, R is
The preparation method comprises the following steps:
1) synthesis of N- (5-bromopyridin-2-yl) acetamide by dissolving 5-bromo-2-aminopyridine (5.19g, 30mmol) in 100ml of anhydrous dichloromethane, adding triethylamine 20ml, slowly dropping acetyl chloride (2.54ml) into the solution under ice bath condition, removing ice bath after dropping, raising the temperature to room temperature for reaction overnight (reaction 12h), after the reaction is finished, adding dichloromethane for dilution, washing with water (30ml × 3), and saturated NaHCO3Solution wash (30ml × 3), saturated NaCl wash (30ml), organic phase anhydrous Na2SO4And (5) drying. Column chromatography gave 5.65g of a white solid in 88% yield. Mp 78-81 deg.C; EI-MS (M/z):214[ M]+。
2) Synthesis of 4- (6- (acetylamino) pyridin-3-yl) benzoic acid: n- (5-Bromopyridin-2-yl) acetamide (4.30g,20mmol), m-carboxyphenylboronic acid (3.66g, 22mmol) were charged to a 250ml pear-shaped flask, followed by cesium carbonate (13.0g, 40mmol), and palladium tetrakistriphenylphosphine (1.2g, 1 mmol). To the mixture was added 200ml of acetonitrile/water (V: V ═ 3: 2). N is a radical of2Under protection, heating the oil bath to 90 ℃ for reaction for 48h, cooling the reaction solution to room temperature after the reaction is finished, carrying out suction filtration, adjusting the pH value of the filtrate to 4 by using 6 mol/L hydrochloric acid, separating out a white solid, carrying out suction filtration, and filtering a filter cakeVacuum drying to obtain 3.89g of product with 76% yield. Mp156-158 ℃; EI-MS (M/z) 256[ M [, M ]]+。
3) Dissolving 4-chloro-3-trifluoromethylaniline (1.07g, 5.46mmol), HOBT (1.92g, 11.7mmol) and HATU (4.45g, 11.7mmol) in anhydrous dichloromethane, dropwise adding DIPEA (3.02g, 23.4mmol), dropwise adding a solution of Fmoc-O-tert-butyl-L-serine (3.0g, 7.8mmol) in anhydrous dichloromethane after dropwise adding, reacting at room temperature for 6h, adding water to terminate the reaction after the reaction, extracting with dichloromethane (3 × 30ml), combining organic phases, washing with saturated NaCl (3 × 30ml), and washing with anhydrous NaSO4Drying and separating by column chromatography to obtain (9H-fluoren-9-yl) methyl (S) -3- (tert-butoxy) -1- ((4-chloro-3- (trifluoromethyl) phenyl) amino) -1-oxopropan-2-yl) carbamate; pale yellow oily solid (5.4 g).
4) (9H-fluoren-9-yl) methyl (S) -3- (tert-butoxy) -1- ((4-chloro-3- (trifluoromethyl) phenyl) amino) -1-oxoprop-2-yl) carbamate (5.4g, 9.6mmol) was dissolved in dry dichloromethane at 0 ℃ and a 20% by volume solution of piperidine in DMF (1ml) was added dropwise, after 1H of reaction and work-up. To obtain (S) -2-amino-3- (tert-butoxy) -N- (4-chloro-3- (trifluoromethyl) phenyl) propanamide. A pale yellow oily solid (3.04 g);
5) a biphenyl compound (0.3g, 1.17mmol) and PyBOP (0.51g, 0.98mmol) were dissolved in anhydrous DMF (10ml), a solution of DIPEA (0.5ml) was added dropwise, and after a reaction at room temperature for 5min, (S) -2-amino-3- (tert-butoxy) -N- (4-chloro-3- (trifluoromethyl) phenyl) propionamide (0.33g, 0.98mmol) was added. After 12h reaction at room temperature, the product was worked up to give 40.22g of t-butyl substituted serine-containing peptoid compound. White solid, yield 68%; EI-MS (M/z) 575[ M ] +; 577[ M ] -. MP: 102.08-104.7 ℃; 1H NMR (400MHz, DMSO)10.66(s,1H),10.65(s,1H),8.83(d, J ═ 7.3Hz,1H),8.74(s,1H), 8.29-8.18 (m,4H),7.92(t, J ═ 12.9Hz,4H),7.69(d, J ═ 8.9Hz,1H),7.60(t, J ═ 7.7Hz,1H),4.73(d, J ═ 7.0Hz,1H),3.73(dt, J ═ 15.5,8.8Hz,2H),2.77(t, J ═ 40.7Hz,1H),2.13(s,3H),1.14(s,9H).
Example 2
A peptide-like compound containing tert-butyl substituted serine, R is
The preparation method comprises the following steps:
1) in N2Under protection, thionyl chloride (36ml, 494mmol) is added into 5-bromonicotinic acid (5.00g, 24.7mmol) dropwise, after dropwise addition is finished, heating reflux is carried out for 2-3h until the solution is clear, thionyl chloride is removed under reduced pressure in a rotary mode to obtain light yellow solid, the solid is dissolved into 30ml of anhydrous dichloromethane, the active intermediate solution is slowly added into a cyclopropylamine (3.77ml) dichloromethane (30ml) solution dropwise under ice bath conditions, after dropwise addition is finished, the temperature is increased to room temperature for reaction overnight, and after the reaction is finished, 2 mol/L K is added into the reaction system2CO3Solution 20 ml. dichloromethane phase was separated, aqueous phase was extracted with dichloromethane (15ml × 3), organic phases were combined, anhydrous Na2SO4And (5) drying. And (5) separating and purifying by column chromatography. (petroleum ether: ethyl acetate: 1) to give 5.27g of a white solid, yield 89%. Mp 140-142 ℃; EI-MS (M/z):240[ M]+。
Step 2) to step 5) were the same as in example 1 to give compound F10 as a white solid in 0.3g with a yield of 65%. EI-MS (M/z) 601[ M ] +; 603[ M ] -. MP: 90.1-91.3 ℃; 1H NMR (400MHz, DMSO)10.66(s,1H),9.11(t, J ═ 7.0Hz,1H),8.99(d, J ═ 1.8Hz,1H),8.89(d, J ═ 7.5Hz,1H),8.79(d, J ═ 4.1Hz,1H),8.50(t, J ═ 1.9Hz,1H),8.31(d, J ═ 13.8Hz,1H),8.27(t, J ═ 7.1Hz,1H), 8.00-7.95 (m,3H), 7.71-7.65 (m,2H),4.74(q, J ═ 6.8Hz,1H),3.74(dt, J ═ 9.0,5.8, 2H),2.51 (J ═ 3.5, 1H), 1.58 (m,1H), 14.78H, 1H, 14.7.78 (m,1H), 14.7.7.7.78 (m, 1H).
F1, F2, F5, F6, F7, F8 and F4 are synthesized in the same way;
f1, light yellow solid, yield 65%; EI-MS (M/z) 617[ M ] +; 616[ M ] -. MP: 50.1-52.3 ℃; 1HNMR (400MHz, DMSO)10.67(d, J ═ 10.1Hz,1H),10.02(d, J ═ 4.5Hz,1H), 8.77-8.68 (m,2H),8.26(d, J ═ 2.4Hz,1H),8.19(s,2H),8.04(d, J ═ 8.2Hz,2H), 7.97-7.85 (m,3H),7.70(t, J ═ 6.9Hz,1H),4.71(q, J ═ 6.4Hz,1H), 3.78-3.66 (m,2H),1.28(d, J ═ 12.2Hz,9H),1.14(s,9H).
F2, light yellow solid, yield 65%; EI-MS (M/z) 617[ M ] +; 616[ M ] -; 1H NMR (400MHz, DMSO)9.99(s,1H), 8.86-8.72 (m,2H), 8.31-8.17 (m,4H),7.91(ddd, J ═ 13.3,12.7,10.9Hz,4H), 7.74-7.57 (m,2H),4.68(dq, J ═ 13.6,6.5Hz,1H), 3.82-3.63 (m,3H),1.24(d, J ═ 18.5Hz,9H),1.14(d, J ═ 4.0Hz,9H).
F5, white solid, yield 56%; EI-MS (M/z) 533[ M ] +; 535[ M ] -. MP: 84.9. -86.7 ℃; 1HNMR (400MHz, DMSO)10.62(s,1H), 8.70-8.63 (m,3H),8.25(d, J ═ 2.4Hz,1H),7.97(t, J ═ 6.7Hz,2H),7.91(dd, J ═ 8.8,2.4Hz,1H),7.77(d, J ═ 8.5Hz,2H),7.69(d, J ═ 8.8Hz,1H),6.93(s,2H),4.69(q, J ═ 6.7Hz,1H),3.72(ddd, J ═ 24.1,9.2,6.8Hz,2H), 2.56-2.47 (m,2H),1.13(d, J ═ 7.8Hz,9H).
F6, white solid, yield 70%; EI-MS (M/z) 611[ M ] +; 613[ M ] -. MP: 202.2-203.1 ℃; 1HNMR (400MHz, DMSO)10.62(d, J ═ 24.5Hz,1H),8.81(d, J ═ 7.5Hz,1H),8.69(s,1H),8.18(ddd, J ═ 16.6,13.5,5.2Hz,3H), 7.96-7.81 (m,3H), 7.74-7.53 (m,2H),7.10(t, J ═ 9.5Hz,1H),4.73(q, J ═ 6.9, 1H), 3.79-3.66 (m,2H),3.01(td, J ═ 6.6,4.0Hz,1H),2.89(s,2H), 2.57-2.43 (m,2H), 1.77-1.67 (m,1H), 1.22-1.02 (m,9H).
F7, white solid, yield 70%; EI-MS (M/z) 611[ M ] +; 613[ M ] -. MP: 120.8-122.1 ℃; 1HNMR (400MHz, DMSO)10.62(s,1H), 8.75-8.63 (m,2H), 8.28-8.09 (m,3H),8.02(dt, J ═ 17.0,7.7Hz,3H),7.83(d, J ═ 8.4Hz,3H),7.10(d, J ═ 8.6Hz,1H), 4.79-4.60 (m,1H), 3.84-3.62 (m,2H),2.70(dd, J ═ 9.7,9.3Hz,3H), 1.17-1.09 (m,9H).
F8, white solid; the yield is 60%; EI-MS (M/z) 575[ M ] +; 577[ M ] -. MP: 104.8-107.4 ℃; 1HNMR (400MHz, DMSO)10.62(s,1H),8.60(d, J ═ 7.4Hz,1H),8.36(d, J ═ 2.4Hz,1H),8.25(d, J ═ 2.5Hz,1H), 7.99-7.93 (m,3H), 7.84-7.78 (m,1H),7.69(t, J ═ 8.2Hz,3H),6.55(d, J ═ 8.6Hz,1H),4.69(q, J ═ 6.8Hz,1H),3.72(ddd, J ═ 23.6,9.2,6.8Hz,2H),2.51(dd, J ═ 3.5,1.7Hz,1H), 1.77-1.70 (m,2H),1.13(d, 8H, 9.8 Hz, 1H).
F3, F9, F11, F12, F13 and F10 are synthesized in the same step;
f3, white solid, yield 60%; EI-MS (M/z) 601[ M ] +; 603[ M ] -. MP: 229.0-231.3 ℃; 1HNMR (400MHz, DMSO)11.05(s,1H),10.67(s,1H),9.08(s,2H),8.82(d, J ═ 7.5Hz,1H), 8.32-8.24 (m,2H),7.95(ddd, J ═ 18.3,9.6,7.2Hz,3H), 7.71-7.60 (m,2H),4.74(q, J ═ 6.9Hz,1H), 3.79-3.50 (m,3H), 2.22-2.11 (m,1H),1.14(s,9H),1.11(s,2H),0.86(s,2H).
F9, white solid, yield 65%; EI-MS (M/z) 601[ M ] +; 603[ M ] -. MP: 91.8-93.4 ℃; 1HNMR (400MHz, DMSO)10.63(s,1H),9.09(t, J ═ 9.0Hz,1H),8.99(d, J ═ 2.0Hz,1H), 8.79-8.75 (m,2H),8.49(t, J ═ 2.1Hz,1H),8.26(d, J ═ 2.5Hz,1H),8.08(d, J ═ 8.5Hz,2H),7.94(dd, J ═ 9.8,4.6Hz,2H),7.70(d, J ═ 8.8Hz,1H),4.71(q, J ═ 6.8Hz,1H), 3.79-3.66 (m,2H),2.74(s,2H), 1.78-1.68 (m,2H), 1.81.15 (s, 0.58H), 0.58-0H (m, 0H), 0.58(m, 1H).
F11, white solid, yield 65%; EI-MS (M/z) 617[ M ] +; 619[ M ] -. MP: 52.3-53.9 ℃; 1HNMR (400MHz, DMSO)10.64(s,1H),9.05(d, J ═ 2.1Hz,1H),8.79(dd, J ═ 21.0,7.5Hz,1H),8.60(t, J ═ 4.2Hz,1H),8.26(d, J ═ 2.4Hz,1H),8.17(dd, J ═ 4.5,2.3Hz,1H),8.08(s,1H), 7.97-7.93 (m,3H),7.68(d, J ═ 8.8Hz,1H),4.72(q, J ═ 6.7Hz,1H),3.73(dd, J ═ 10.1,4.4Hz,2H),3.49(d, J ═ 6.8, 2H),3.38(s, 1H), 3.19H, 3.6H, 6H, 3.6H, 3.19 (d, 6.8, 3.6H), 3.6H, 6, 1H), 3.6J ═ 6H, 1H, and 7H.
F12, white solid; the yield is 65%; EI-MS (M/z) 617[ M ] +; 619[ M ] -. MP: 85.1-87.4 ℃; 1HNMR (400MHz, DMSO)10.66(s,1H),9.07(dd, J ═ 4.9,2.2Hz,1H),8.90(d, J ═ 7.5Hz,1H),8.61(d, J ═ 1.9Hz,1H),8.30(s,1H),8.26(d, J ═ 2.2Hz,1H),8.20(dd, J ═ 4.9,2.9Hz,1H),8.00(d, J ═ 7.8Hz,1H),7.96(d, J ═ 6.8Hz,1H),7.67(d, J ═ 6.9Hz,1H), 7.65-7.60 (m,1H), 4.79-4.73 (m,1H),3.73(dd, J ═ 1H), 3.5 (J ═ 1.5, 3.5 Hz,1H), 3.49(d, J ═ 2.8H, 1H), 3.09 (d, 1H, 3.3.3.3.3H), 3.49(d, J ═ 2H, 1H), 3.9H, 1H), 3.09 (d, 3.9H), 3.9H, 1H), 3.3.3.3.3.3.3.3.3.3.3.3.9H, 3.9H.
F13, light yellow solid, yield 60%; EI-MS (M/z) 617[ M ] +; 619[ M ] -. 1H NMR (400MHz, DMSO)10.66(s,1H),9.07(t, J ═ 3.7Hz,1H),8.90(d, J ═ 7.6Hz,1H),8.59(dd, J ═ 11.2,1.9Hz,1H), 8.32-8.21 (m,2H), 8.00-7.95 (m,2H),7.69(d, J ═ 8.8Hz,2H),7.61(dd, J ═ 4.5,2.2Hz,3H),7.56(dd, J ═ 4.8,2.3Hz,3H),4.75(q, J ═ 6.9Hz,1H), 3.79-3.68 (m,2H), 3.54-3.46 (m,2H),3.25(s, 2.68), 2.9H (89, 9H), 2.9H, 1H), 14H (m, 14H).
The Bcr-Abl kinase inhibitory activity screening was performed on the derivatives containing tert-butyl-substituted serine having antitumor activity prepared in the present invention.
The determination method specifically comprises the following steps:
the kinases AB L1, AB L (T315I) and substrate Abltide are available from Signal-Chem, and ADP-Glo from Promega is selectedTMThe enzyme inhibitory activity of the target compound is detected by the Kinase Assays detection kit, and the operation method is carried out according to the kit instructions.
In the Abl experiment, ATP (1mM) was applied to buffer (2 ×) (Tris 80mM, MgCl220mM, BSA 0.2mg/ml, DTT 2mM) diluted 80-fold to prepare ATP (125. mu.M) in buffer (2 ×), mixing the 125. mu.M ATP solution with the Abltide solution in a volume of 1:1 to prepare ATP (62.5. mu.M) -Abltide (0.5. mu.g/. mu.l) mixed solution for later use, and using buffer (1 ×) (Tris 40mM, MgCl 40 mM) for AB L1 kinase solution210mM, BSA 0.1mg/ml, DTT 1mM) was diluted 100-fold to make AB L1 (1 ng/. mu.l) in buffer (1 ×) for use.
ATP-Abltide in the Abl (T315I) experiment and AB L1 (T315I) were prepared as above, except that the ATP concentration was 12.5. mu.M and the AB L1 (T315I) concentration was 2 ng/. mu.l.
The target compound and the positive control drug (Imatinib) were formulated into 1.5 × 10 (1. 1 ×) using buffer (1 ×)-6The sample solution with mol/L concentration gradient is prepared by sequentially adding 2 mul ATP-Abltide mixed solution, 1 mul sample solution, 2 mul enzyme solution into each well of 384-well plate, adding 3 mul buffer solution and 2 mul ATP-Abltide mixed solution into blank wells, adding 2 mul ATP-Abltide mixed solution into control wells, 1 mul buffer solution, 2 mul enzyme solution, incubating at 30 deg.C for 60min after adding, adding 5 mul ADP-Glo reagent, incubating at 25 deg.C for 40min, adding Kinase detection reagent, incubating at 25 deg.C for 30min, measuring luminescence value of each well by using chemiluminescence module of Perkinmer multifunctional microplate reader, and calculating the inhibition rate of compound on Abl
The structural formula of the tert-butyl substituted serine-containing peptoid compound is as follows:
the kinase inhibitory activity of the t-butyl substituted serine-containing peptoid compounds of the above structural formula is shown in Table 2
TABLE 2 Tert-butyl substituted serine-containing peptoid pairs Bcr-Abl/Bcr-AblT315IInhibition ratio (%)
As can be seen from Table 2, most of the compounds have certain inhibitory activity on Bcr-Abl kinase and on Bcr-AblT315Kinase, most compounds have certain inhibitory activity, the inhibition rate is in the range of 40% to 100%, and the inhibition rate of partial compounds (F1, F9) on T315I mutant Abl kinase is above 90%. The activity is better. The activity results show that the difference of the substituent can directly influence the inhibition activity of the compound on the kinase.
The growth inhibitory activity of the t-butyl substituted serine-containing peptoid compounds on tumor cells was determined as follows. MTT method is adopted to test the growth inhibition activity of the peptoid compound containing the tert-butyl substituted serine on the tumor cells.
The peptoid compound containing tert-butyl substituted serine provided by the invention has an anti-tumor effect. Has effect in inhibiting proliferation activity of tumor cells in vitro and in human leukemia cells (K562 cells), and can be used for treating leukemia.
Taking human leukemia cells (K562 cells) in logarithmic growth phase, diluting with RPMI1640 medium to 104Cell solutions of the order of magnitude of one/ml were plated in parallel in 96-well plates (2000-4000/well) at a volume of 180. mu.l/well, 37 ℃ and 5% CO2Culturing for 12h in an incubator;
mu.l of test compound was added to each well at a different concentration to give a final concentration of 1.5 × 10-5mol/L, setting 3 multiple wells for each concentration, adding no compound into negative control and adding 6 multiple wells, setting nilotinib or imatinib as positive control, and continuing culturing for 48 h;
mu.l of MTT (5mg/ml) was added to each well to allow the wells to standMTT final concentration of 0.5mg/ml, incubating at 37 deg.C for 4h, carefully removing supernatant, adding DMSO 150 μ l into each well, shaking for 15min, measuring ultraviolet absorption (OD) at 490nm of each well with enzyme linked immunosorbent assay, calculating cell inhibition rate, and calculating IC of compound according to the inhibition rate by linear regression method50A value;
the formula for calculating the cell inhibition rate is as follows:
inhibition (% control well mean OD value-drug group mean OD value)/control well mean OD value × 100;
and (3) displaying a detection result: the alanine-containing peptoid compounds had different degrees of in vitro inhibition of the tumor cells compared to the negative control group, as shown in table 3.
K562 cell proliferation activity:
TABLE 3 inhibitory rate (%) of K562 cells by peptoid compounds containing tert-butyl-substituted serine
As can be seen from Table 3, the cell activity screening test shows that the compound has a certain inhibition rate of cell proliferation inhibition activity of 10.49% to 70% on K562 cells, wherein the compound with better activity has the best activity of F7, the inhibition rate reaches 68%, and the activity is equivalent to that of imatinib. For peptide-like compounds containing tert-butyl substituted serine, the influence on the biological activity is greatly different by introducing different substituents on the pyridine ring, and the influence on the biological activity is different by different positions of the substituents. Compounds F1, F4 and F9 against Bcr-AblT315IThe kinase inhibition activity is better; the compound F7 has better inhibitory activity on K562 cells, is close to that of imatinib, and is worthy of further research.
Based on the research on the previous Bcr-Abl tyrosine kinase inhibitor, the interaction analysis of Bcr-Abl protein and ligand and the like, the invention adopts a fragment-based drug design strategy, takes the diphenylpyridine as a hinge region binding fragment, introduces L-tert-butyl substituted serine as flexible L inker, and constructsA peptoid compound library with kinase inhibition activity, and a tyrosine kinase inhibitor with Bcr-Abl kinase inhibition activity is discovered by screening the kinase activity of ADP-Glo. The compound can be used for preparing anti-tumor (chronic granulocytic leukemia) medicine, and has effects of inhibiting Bcr-Abl and Bcr-AblT315IThe L-tert-butyl substituted serine structure is introduced, the structural diversity of the Bcr-Abl kinase inhibitor can be expanded, and meanwhile, an activity test shows that the tert-butyl substituted serine has an important effect on the inhibitory activity of a compound, can improve the affinity between a receptor and the compound, and can be used as a pharmacodynamic fragment of the Bcr-Abl tyrosine kinase inhibitor.
Claims (3)
1. A peptoid compound containing tert-butyl substituted serine is characterized in that the structural formula of the peptoid compound is as follows:
2. use of the tert-butyl substituted serine-containing peptoid compound of claim 1 in the preparation of a medicament for inhibiting the activity of Abl kinase, T315I mutant Abl kinase.
3. The use of a tert-butyl substituted serine containing peptoid according to claim 1 in the preparation of an anti-tumor medicament, wherein the tumor is leukemia.
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