CN109456279B

CN109456279B - Thiazolylaminobenzamide acetate derivatives and uses thereof

Info

Publication number: CN109456279B
Application number: CN201811187130.5A
Authority: CN
Inventors: 彭俊梅; 刘娟; 黄红林; 曹轩; 刘映; 李娜; 柯栢怡; 李博
Original assignee: University of South China
Current assignee: University of South China
Priority date: 2018-10-12
Filing date: 2018-10-12
Publication date: 2022-11-22
Anticipated expiration: 2038-10-12
Also published as: CN109456279A

Abstract

The invention discloses a thiazole aminobenzamide acetate derivative with antitumor activity, which can be used for preparing an anticancer drug, particularly as a Bcr-Abl tyrosine kinase inhibitor, and particularly as a T315I mutant Bcr-Abl tyrosine kinase inhibitor.

Description

Thiazolylaminobenzamide acetate derivatives and uses thereof

Technical Field

The invention relates to the field of medicinal chemistry, and particularly relates to a series of thiazole aminobenzamide acetate derivatives, and a preparation method and application thereof.

Background

Chronic Myelogenous Leukemia (CML), a malignant myeloproliferative disease derived from hematopoietic stem cells, accounts for 15% to 20% of all leukemias, and is the most common myeloproliferative disease. The pathogenesis of the tumor comes from a Bcr-Abl gene formed by fusing a proto-oncogene c-Abl on a chromosome 9 and a Bcr gene on a chromosome 22. Currently, bcr-Abl tyrosine kinase has been demonstrated to be the most ideal molecular target for the treatment of CML (Blood, 1996,87, 3036-3038 Blood,2000,96 343-356. Various efforts have been made by the pharmaceutical workers around the Bcr-Abl tyrosine kinase. Currently, the successfully marketed Bcr-Abl tyrosine kinase inhibitors are imatinib, nilotinib, ladotinib, dasatinib, shu Bo tinib and ponatinib.

Wherein, imatinib is a first generation Bcr-Abl tyrosine kinase inhibitor and is a first-line medicament for treating CML, but patients in different periods can generate part of drug resistance after long-term administration. Resistance may be caused by a variety of mechanisms, of which Bcr-Abl point mutations are the most common and most influential (Nat Rev Drug Discov,2007,6 (10): 834-848).

Nilotinib, ladotinib, dasatinib and bosutinib are second generation Bcr-Abl tyrosine kinase inhibitors. Compared with imatinib, the drug resistance produced by most mutants can be solved, but the drug resistance caused by the T315I mutation cannot be effectively solved (Ann Oncol,2007,18 (6): 42-46 Eur J cancer,2010,46 (10): 1781-1789, haematologica,2014,99 (7): 1191-1196.

Ponatinib is a third-generation Bcr-Abl tyrosine kinase inhibitor and can effectively solve drug resistance caused by T315I mutation (Cancer Cell,2009,16 (5): 401-412). However, ponatinib causes serious and fatal thrombosis and angiostenosis, and can induce complications such as heart disease, myocardial infarction, stroke, limb ischemia and even tissue necrosis (Chinese medicine alert, 2017,14 (4): 218-221).

The T315I mutation occurs in the Bcr-Abl kinase domain at position 315, referred to as "gatekeeper", and threonine (Thr) is replaced by isoleucine (Ile) in the wild-type Bcr-Abl. Thr315 in the wild-type Bcr-Abl can form a key hydrogen bond with imatinib, nilotinib and the like, and when the Thr315 is substituted by Ile315, the key hydrogen bond cannot be formed. In addition, ile315 after mutation sterically hinders imatinib, making it effective against wild-type Bcr-Abl and not against Bcr-ablT315I due to its large size (Cancer Cell,2002,2 (2): 117-125. The research on the effect of the ponatinib and Bcr-AblT315I shows that: the alkyne bond in ponatinib structure does not form hydrogen bonds with both Thr315 and Ile315, and does not sterically hinder Ile315 due to the smaller structure (pharmaceutical advances, 2014,38 (5): 333-339.

The inventor designs and synthesizes a series of 2-aminothiazole compounds in earlier work, and activity test results show that the compounds have good antitumor activity (CN 102319244A, CN102675303 and CN1080031152A, CN 107459513A). Therefore, the inventor thinks that the 2-aminothiazole group is combined into a molecular structure, and expects to obtain a Bcr-Abl tyrosine kinase inhibitor which can be used as a T315I mutant and reduce toxic and side effects to a human body.

Disclosure of Invention

The technical problem to be solved by the invention is as follows: a class of thiazolylaminobenzamide acetate derivatives are provided, which can be used as T315I mutant Bcr-Abl tyrosine kinase inhibitors.

In a first aspect, the present invention provides compounds of formula I and pharmaceutically acceptable salts thereof, having the following structure:

wherein: r ¹ Each independently selected from halogen, -OH, -NO ₂ 、-CN、C ₁ -C ₆ Alkyl radical, C ₁ -C ₆ Alkoxy radical, C ₁ -C ₆ Haloalkyl, C ₁ -C ₆ A haloalkoxy group;

R ² selected from hydrogen, C ₁ -C ₆ Alkyl radical, C ₁ -C ₆ Alkoxy radical, C ₁ -C ₆ Haloalkyl, C ₁ -C ₆ A haloalkoxy group;

R ³ selected from hydrogen, -OH, -NO ₂ CN, halogen, C ₁ -C ₆ Alkyl radical, C ₁ -C ₆ Alkoxy radical, C ₁ -C ₆ Haloalkyl, C ₁ -C ₆ Haloalkoxy, C ₆ -C ₁₀ Aryl radical C ₁ -C ₆ An alkyl group;

R ⁴ is selected from C ₁ -C ₆ Alkyl radical, C ₁ -C ₆ A haloalkyl group;

n is selected from 0, 1, 2,3 or 4.

Preferably, R ¹ Each independently selected from halogen, -OH, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 haloalkyl, more preferably methyl, chloro, fluoro, hydroxy, trifluoromethyl; n is selected from 0, 1 or 2, preferably 0 or 1.

Preferably, R ² Selected from hydrogen, C1-C4 alkyl, C1-C4 haloalkyl, more preferably methyl, ethyl, trifluoromethyl.

Preferably, R ³ Selected from hydrogen, -OH, halogen, C ₁ -C ₄ Alkyl radical, C ₁ -C ₆ Alkoxy radical, C ₁ -C ₄ Haloalkyl, C ₁ -C ₄ Haloalkoxy, benzyl, more preferably hydrogen, benzyl.

Preferably, R ⁴ Is selected from C ₁ -C ₄ Alkyl radical, C ₁ -C ₄ The haloalkyl group is more preferably a methyl group or an ethyl group.

More preferably, the compounds of formula I according to the invention are selected from the following compounds:

in another aspect of the invention there is provided a process for the preparation of a compound of formula I, the reaction scheme being as follows:

wherein R is ¹ 、R ² 、R ³ 、R ⁴ And n is as defined above.

The preparation method of the invention specifically comprises the following reaction steps:

the method comprises the following steps: adding ammonium thiocyanate and acetone into a reactor, uniformly stirring, then dropwise adding benzoyl chloride, heating the solution to reflux after the solution is changed into white turbid liquid from clear, adding m-aminobenzoic acid in batches, cooling after the reaction is finished, filtering, and drying the obtained solid to obtain 3- (3-benzoyl thiourea) benzoic acid;

step two: adding 3- (3-benzoyl thiourea) benzoic acid and an alkaline aqueous solution into a reaction bottle to ensure that the pH is =13, stirring, heating and refluxing until the reaction is finished, cooling to room temperature, adding dilute hydrochloric acid, adjusting the pH to 2, standing for 24h, separating out a solid, filtering, and drying the solid to obtain 3-carboxyphenyl thiourea;

step three: adding 3-carboxyphenylthiourea, substituted 2-Br-1-phenylalkyl ketone and glacial acetic acid into a reaction bottle, uniformly stirring, heating to reflux, removing insoluble solids in the reaction bottle while the reaction bottle is hot after the reaction is finished, carrying out rotary evaporation on part of the solvent, cooling for 24 hours at normal temperature in a ventilated kitchen, separating out solids, filtering, and drying the obtained solids to obtain an intermediate thiazolaminobenzoic acid derivative;

step four: adding intermediate thiazole aminobenzoic acid derivative, EDCI, HOBT and absolute ethyl alcohol into a reaction bottle under the ice bath condition, reacting for 2-4h, and adding

Continuously carrying out ice-bath reaction on hydrochloride, DIPEA, DMAP and DMF for half an hour, then changing to room temperature reaction until the reaction is finished, slowly adding ice water while stirring until the solution is changed from clear to turbid, stirring for 0.5-1.5h at room temperature, then putting the solution into a refrigerator to separate out white solid, filtering, and drying the obtained solid to obtain the compound of the formula I.

Preferably, the molar ratio of m-aminobenzoic acid, ammonium thiocyanate and benzoyl chloride of step one is 1:1-1.5, preferably 1;

preferably, the step two alkaline aqueous solution is 10% of an NaOH aqueous solution, the dilute hydrochloric acid concentration is 4mol/L;

preferably, the molar ratio of the 3-carboxyphenylthiourea and the substituted 2-Br-1-phenylalkylketone of step three is 1:1-1.2, preferably 1:1;

preferably, step four

And

is 1 to 2:1, preferably 1.5

In another aspect of the present invention, a pharmaceutical composition is provided, which comprises the compound represented by formula I or a pharmaceutically acceptable salt thereof described in the present invention, and a pharmaceutically acceptable carrier or excipient.

In another aspect, the invention relates to the use of a compound according to the invention or a pharmaceutical composition comprising said compound for the manufacture of a medicament for the treatment of a cancer associated with a Bcr-Abl tyrosine kinase, in particular a cancer targeted against a T315I mutated Bcr-Abl tyrosine kinase.

Preferably, the cancer is selected from human chronic myeloid leukemia, liver cancer (such as HepG-2 cell strain), non-small cell lung cancer (such as A549 cell strain), more preferably human chronic myeloid leukemia (such as K562 cell strain), K562 imatinib-resistant cell strain (K562/R cell strain).

Defining:

"alkyl" means consisting solely of carbon and hydrogen atoms, containing no unsaturation, and may be C1-6 alkyl. In some embodiments, the alkyl group has 1 to 6 or 1 to 4 carbon atoms. Representative saturated straight chain alkyl groups include, but are not limited to-methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, and-n-hexyl; and saturated branched alkyl groups include, but are not limited to-isopropyl, -sec-butyl, -isobutyl, -tert-butyl, -isopentyl, 2-methylbutyl, 3-methylbutyl, 2-methyl-pentyl, 3-methylpentyl, 4-methylpentyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2,3-dimethyl-butyl, and the like. The alkyl group is attached to the parent molecule by a single bond. Unless stated otherwise in the specification, an alkyl group is optionally substituted with one or more substituents independently including: acyl, alkyl, alkenyl, alkynyl, alkoxy, alkylaryl, cycloalkyl. In a non-limiting embodiment, the substituted alkyl group can be selected from the group consisting of fluoromethyl, difluoromethyl, trifluoromethyl, 2-fluoroethyl, 3-fluoropropyl, hydroxymethyl, 2-hydroxyethyl, 3-hydroxypropyl, benzyl, and phenethyl.

"alkoxy" means an "alkyl" group attached to the parent molecule through an oxygen atom, wherein "alkyl" has the meaning described above.

"haloalkyl" refers to an alkyl group wherein all hydrogen atoms are partially or fully replaced with a halogen selected from fluoro, chloro, bromo, and iodo. In some embodiments, all hydrogen atoms are each replaced with fluoro groups. In some embodiments, all hydrogen atoms are each replaced with a chloro group. Examples of haloalkyl groups include-CF 3, -CF2CF2CF3, -CFCl2, -CF2Cl, and the like.

In certain embodiments, the pharmaceutically acceptable form is a pharmaceutically acceptable salt, which is well known in the art. Examples of pharmaceutically acceptable salts are such as hydrochloric, hydrobromic, phosphoric, sulfuric, perchloric, acetic, oxalic, maleic, tartaric, citric, succinic or malonic, acetic, propionic, glycolic, pyruvic, oxalic, lactic, trifluoroacetic, methanesulfonic, ethanesulfonic, p-toluenesulfonic, salicylic, and the like.

"pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" includes any and all solvents, dispersion media, coating agents, isotonic and absorption delaying agents and the like. Pharmaceutically acceptable carriers or excipients do not destroy the pharmacological activity of the disclosed compounds and are non-toxic when administered in doses sufficient to deliver a therapeutic amount of the compound. The use of such media and agents for pharmaceutically active substances is well known in the art.

Compared with the prior art, the invention has the beneficial effects that:

(1) The invention provides a new thiazole amino benzamide acetic ester compound with anticancer activity, which widens the range of the existing anticancer compound and can be continuously optimized as a lead compound;

(2) The compound has good inhibition effect on CML cell strains K562 and K562 imatinib-resistant cells (K562/R), human liver cancer cells (HepG-2) and human non-small cell lung cancer cells (A549), has lower toxicity on human normal liver cells L02, and can avoid or reduce toxic and side effects on human bodies while inhibiting cancer cells;

(3) The compounds of the invention are capable of interacting with the target tyrosine kinases AbI1 and AbIl ^T315I Effective knotIt has good action intensity and can inhibit Bcr-AbIl ^T315I The activity of (3), and further effectively solves the problem of drug resistance caused by the T315I mutation, and can be used as a novel Bcr-Abl tyrosine kinase inhibitor for resisting the T315I mutation.

Detailed Description

The present invention will be described in detail with reference to examples. In the present invention, the following examples are provided to better illustrate the present invention and are not intended to limit the scope of the present invention. The materials, reagents and the like used in the examples are commercially available unless otherwise specified.

Example 1 (3- ((5-methyl-4-phenylthiazol-2-yl) amino) benzoyl) glycine methyl ester

A, step a:

11.4341g (0.12 mol) ammonium thiocyanate and 20mL acetone were added to a 100mL slant-ended reaction flask with mechanical stirring and condenser, and stirred well by mechanical stirring. 16.8034g (0.13 mol) benzoyl chloride was added dropwise (after 10min drop off) and the solution turned from clear to a white turbid solution. The reaction was heated to reflux, 14.1147g (0.10 mol) m-aminobenzoic acid was added in 4 portions, the progress of the reaction was monitored by TLC (ethyl acetate: petroleum ether = 4:1), and the reaction was completed for 8 h. Cooling, filtering and drying the solid to obtain 28.0041g of 3- (3-benzoyl thiourea) benzoic acid as light yellow powder at m.p.184-186 ℃.

A100 mL slant-ended reaction flask equipped with a condenser was charged with 0.9913g (0.12 mol) of 3- (3-benzoylthiourea-based) benzoic acid and 33mL 10% NaOH, and pH =13 was measured, and magnetic stirring was performed, heating and refluxing were performed, and TLC (ethyl acetate: petroleum ether = 4:1) was used to monitor the progress of the reaction and completion of the reaction for 4h. Cooling to room temperature, adding a proper amount of dilute hydrochloric acid of 4mol/L, adjusting the pH to 2, standing for 24 hours, precipitating a solid, filtering, drying the obtained solid, and weighing 0.6142g of white powder, wherein the yield is 86.72 percent and the m.p.186-187 ℃. ¹ H NMR(DMSO-D ₆ ，400MHz),δ：2.51(s，1H，NH),3.36(s,2H,NH ₂ ),7.43-8.03(m,4H,C ₆ H ₄ )，9.89(s,1H,COOH)。

Step b:3- [ (5-Ethyl-4-phenylthiazol-2-yl) amino ] benzoic acid

3.9432g (0.02 mol) of 3-carboxyphenylthiourea, 4.5213g (0.02 mol) of 2-Br-1-phenylbutanone and 20mL of glacial acetic acid were added to a 100mL inclined-mouth reaction flask with a condenser tube, stirred uniformly, heated to reflux, and reacted for about 24 hours by monitoring the progress of the reaction by TLC (developing agent: ethyl acetate: petroleum ether = 4:1). The insoluble solid in the reaction flask was removed while hot and part of the solvent was rotary evaporated. Cooling in a ventilation kitchen for 24h at normal temperature, separating out solid, filtering, drying the obtained solid, weighing to obtain 5.6.163g of brown yellow powder, wherein the yield is 66.54 percent and m.p.229-231 ℃. ¹ H NMR(DMSO-D ₆ ，400MHz)，δ：1.25(t，3H，J＝8.0Hz，CH ₃ )，2.85(q，2H，J＝8.0Hz，CH ₂ )，7.32-8.27(m，9H，C ₆ H ₄ ，C ₆ H ₅ )，10.38(s，1H，COOH)。

Step c: (3- ((5-methyl-4-phenylthiazol-2-yl) amino) benzoyl) glycine methyl ester

0.4153g (0.003 mol) 3- [ (5-ethyl-4-phenylthiazol-2-yl) amino group were added under ice bath conditions]Benzoic acid, 0.5812g (0.003 mol) EDCI, 0.4154g (0.003 mol) HOBT and 18ml absolute ethyl alcohol were added to a three-neck flask, and after about 3 hours of reaction, 0.2502g (0.002 mol) glycine methyl ester hydrochloride, 0.8ml DIPEA, 0.0979g (0.0008 mol) DMAP and 18ml DMF were added to continue the ice bath reaction for half an hour, and then the reaction was changed to room temperature reaction, TLC (ethyl acetate: petroleum ether = 4:1) was used to monitor the progress of the reaction, and the reaction was completed for about 21 hours. Ice water was slowly added with stirring until the solution became cloudy, about 40ml, at room temperature for 30min, and then placed in a refrigerator to precipitate a white solid. Filtering, and drying the obtained solid to obtain 0.2112g of white powder，m.p.135～137℃。 ¹ H NMR(CDCl ₃ ，400MHz)，δ：1.30(t，J＝8.0Hz，3H，CH ₂ CH ₃ )，1.41(s，3H，OCH ₃ )，2.89(q，J＝8.0Hz，2H，CH ₂ CH ₃ )，4.39(d，J＝4.0Hz，2H，NHCH ₂ )，7.26-8.00(m，9H，C ₆ H ₅ ，C ₆ H ₄ )。

Example 2 (3- ((5-methyl-4- (p-tolyl) thiazol-2-yl) amino) benzoyl) glycine methyl ester

The operation was as above to obtain 0.3018g of white powder with a yield of 68.23%, m.p.187-188 ℃. ¹ H NMR(DMSO-D ₆ ，400MHz)，δ：2.36(s，3H，CH ₃ )，2.42(s，3H，CH ₃ )，3.67(s，3H，OCH ₃ )，4.02(d，J＝4.0Hz，2H，NHCH ₂ )，7.26-8.07(m，8H，2×C ₆ H ₄ )，8.90(t，J＝4.0，1H，CONH)，10.30(s，1H，NH)。

Example 3- ((4- (4-chlorophenyl) -5-methylthiazol-2-yl) amino) benzoyl) glycine methyl ester

The operation was as above to obtain 0.3016g of yellow powder, 3242% yield 70.44%, m.p.158-159 ℃. ¹ H NMR(CDCl ₃ ，400MHz)，δ：1.41(s，3H，CH ₃ )，2.44(s，3H，OCH ₃ )，4.39(d，J＝4.0Hz，2H，NHCH ₂ )，7.34-8.01(m，8H，2×C ₆ H ₄ )。

Example 4 (3- ((4- (4-chlorophenyl) -5-ethylthiazol-2-yl) amino) benzoyl) glycine methyl ester

The operation was the same as above to obtain 0.3812g of khaki powder with a yield of 81.29%, m.p. 187-189 deg.C. ¹ H NMR(CDCl ₃ ，400MHz)，δ：1.28(t，J＝8.0Hz，3H，CH ₂ CH ₃ )2.84(q，J＝8.0Hz，2H，CH ₂ CH ₃ )，3.80(s，3H，OCH ₃ )，4.26(d，J＝4.0Hz，2H，NHCH ₂ )，6.95(s，1H，NH)，7.26-7.82(m，8H，2×C ₆ H ₄ )。

Example 5 (3- ((4- (4-hydroxyphenyl) -5-methylthiazol-2-yl) amino) benzoyl) glycine methyl ester

The procedure was as above to give 0.2332g of yellow powder in 59.74% yield, m.p.174-175 deg.C. ¹ H NMR(CDCl ₃ ，400MHz)，δ：2.39(s，3H，CH ₃ )，3.82(s，3H，OCH ₃ )，4.26(d，J＝4.0Hz，2H，NHCH ₂ )，6.81-7.75(m，8H，2×C ₆ H ₄ )。

Example 6 methyl (3- ((4- (4-fluorophenyl) -5-methylthiazol-2-yl) amino) benzoyl) glycinate

The operation was as above to obtain 0.3503g of pale pink powder with a yield of 79.82%, m.p.197-199 ℃. ¹ H NMR(CDCl ₃ ，400MHz)，δ：2.43(s，3H，CH ₃ )，3.82(s，3H，OCH ₃ )，4.26(d，J＝4.0Hz，2H，NHCH ₂ )，6.85(s，1H，NH)，7.12-7.89(m，8H，2×C ₆ H ₄ )。

Example 7 (3- ((5-methyl-4-phenylthiazol-2-yl) amino) benzoyl) glycine methyl ester

The operation was as above to obtain 0.3903g of yellow powder with a yield of 86.66%, m.p.169-171 ℃. ¹ H NMR(CDCl ₃ ，400MHz)，δ：2.46(s，3H，CH ₃ )，3.80(s，3H，OCH ₃ )，4.26(d，J＝4.0Hz，2H，NHCH ₂ )，6.93(s，1H，NH)，7.31-7.81(m，9H，2×C ₆ H ₄ )。

Example 8 methyl (3- ((5-methyl-4- (4- (trifluoromethyl) phenyl) thiazol-2-yl) amino) benzoyl) glycinate

The operation was as above to obtain 0.3104g of light pink powder with a yield of 66.97%, m.p.211-213 ℃. ¹ H NMR(DMSO-D ₆ ，400MHz)，δ：2.43(s，3H，CH ₃ )，3.67(s，3H，OCH ₃ )，4.02(d，J＝4.0Hz，2H，NHCH ₂ )，7.26-8.07(m，8H，2×C ₆ H ₄ )，8.90(t，J＝4.0，1H，CONH)，10.30(s，1H，NH)。

Example 9 (3- ((5-Ethyl-4-phenylthiazol-2-yl) amino) benzoyl) phenylalanine methyl ester

The procedure was as above to give 0.2648g of yellow powder in 52.21% yield, m.p.111-113 ℃. ¹ H NMR(DMSO-D ₆ ，400MHz)，δ：1.24(t，J＝8.0Hz，3H，CH ₂ CH ₃ )2.84(q，J＝8.0Hz，2H，CH ₂ CH ₃ )，3.16(m，2H，CH ₂ C ₆ H ₅ )，3.64(s，3H，OCH ₃ )，4.68(m，1H，CH)，7.17-7.94(m，14H，2×C ₆ H ₅ ，C ₆ H ₄ )，8.82(d，J＝4.0，1H，CONH)，10.26(s，1H，NH)。

Example 10 (3- ((5-methyl-4- (p-tolyl) thiazol-2-yl) amino) benzoyl) phenylalanine methyl ester

The procedure was as above to give 0.2558g of brown powder in 66.97% yield m.p.113-115 ℃. ¹ H NMR(DMSO-D ₆ ，400MHz)，δ：2.34(s，3H，CH ₃ )，2.42(s，3H，CH ₃ )，3.12(m，2H，CH ₂ C ₆ H ₅ )，3.64(s，3H，OCH ₃ )，4.67(m，1H，CH)，7.24-7.94(m，13H，2×C ₆ H ₄ ，C ₆ H ₅ )，8.82(d，J＝4.0，1H，CONH)，10.19(s，1H，NH)。

Example 11 (3- ((4- (4-chlorophenyl) -5-methylthiazol-2-yl) amino) benzoyl) phenylalanine methyl ester

The operation is the same as above, 0.2648g of light pink powder is obtained, the yield is 51.72%, and m.p.166-167 ℃. ¹ H NMR(DMSO-D ₆ ，400MHz)，δ：2.44(s，3H，CH ₃ )，3.15(m，2H，CH ₂ C ₆ H ₅ )，3.66(s，3H，OCH ₃ )，4.69(m，1H，CH)，7.19-7.95(m，13H，2×C ₆ H ₄ ，C ₆ H ₅ )，8.81(d，J＝4.0，1H，CONH)，10.26(s，1H，NH)。

Example 12 methyl (3- ((4- (4-chlorophenyl) -5-ethylthiazol-2-yl) amino) benzoyl) phenylalanine

The procedure was as above to give 0.3618g of pale yellow powder in 66.23% yield, m.p.105-107 ℃. ¹ H NMR(DMSO-D ₆ ，400MHz)，δ：1.24(t，J＝8.0Hz，3H，CH ₂ CH ₃ )2.84(m，2H，CH ₂ CH ₃ )，3.11(m，2H，CH ₂ C ₆ H ₅ )，3.64(s，3H，OCH ₃ )，4.68(m，1H，CH)，7.19-7.95(m，13H，2×C ₆ H ₄ ，C ₆ H ₅ )，8.81(d，J＝4.0，1H，CONH)，10.26(s，1H，NH)。

Example 13 (3- ((4- (4-hydroxyphenyl) -5-methylthiazol-2-yl) amino) benzoyl) phenylalanine methyl ester

The procedure was as above to give 0.2316g of pale yellow powder with a yield of 44.91%, m.p.158-160 ℃. ¹ H NMR(CDCl ₃ ，400MHz)，δ：2.37(s，3H，CH ₃ )，3.23(m，2H，CH ₂ C ₆ H ₅ )，3.76(s，3H，OCH ₃ )，5.07(m，1H，CH)，6.71-7.68(m，13H，2×C ₆ H ₄ ，C ₆ H ₅ )。

Example 14 (3- ((4- (4-fluorophenyl) -5-methylthiazol-2-yl) amino) benzoyl) phenylalanine methyl ester

The operation was as above to obtain 0.2195g of white powder with a yield of 42.82%, m.p.245-248 ℃. ¹ H NMR(DMSO-D ₆ ，400MHz)，δ：2.45(s，3H，CH ₃ )3.15(m，2H，CH ₂ C ₆ H ₅ )，3.67(s，3H，OCH ₃ )，4.69(m，1H，CH)，7.15-87.96(m，13H，2×C ₆ H ₄ ，C ₆ H ₅ )，8.80(d，J＝4.0，1H，CONH)，10.22(s，1H，NH)。

Example 15 (3- ((5-methyl-4-phenylthiazol-2-yl) amino) benzoyl) phenylalanine methyl ester

The same procedure was followed to obtain 0.2706gWas obtained in 51.43% yield, m.p.128-131 ℃. ¹ H NMR(DMSO-D ₆ ，400MHz)，δ：2.45(s，3H，CH ₃ )，3.15(m，2H，CH ₂ C ₆ H ₅ )，3.67(s，3H，OCH ₃ )，4.69(m，1H，CH)，7.15-87.96(m，13H，2×C ₆ H ₅ ，C ₆ H ₄ )，8.80(d，J＝4.0，1H，CONH)，10.22(s，1H，NH)。

Example 16 (3- ((5-methyl-4- (4- (trifluoromethyl) phenyl) thiazol-2-yl) amino) benzoyl) phenylalanine methyl ester

The procedure is as above, giving 0.2706g of a pale yellow powder, yield 51.43%, m.p.120-122 ℃. ¹ H NMR(CDCl ₃ ，400MHz)，δ：2.44(s，3H，CH ₃ )，3.13(m，2H，CH ₂ C ₆ H ₅ )，3.64(s，3H，OCH ₃ )，4.66(m，1H，CHCH ₂ )，7.19-7.94(m，13H，2×C ₆ H ₄ ，C ₆ H ₅ )，8.80(d，J＝4.0，1H，CONH)，10.22(s，1H，NH)。

Example 17 Compounds and targets tyrosine kinases Abl (PDB: 2 GQG) and Bcr-Abl ^T315I (PDB: 3IK 3) docking and scoring

Molecular simulation software sybyl is utilized to carry out pairing on designed and synthesized compound and target Bcr-Abl ^T315I (PDB: 3IK 3) was subjected to docking. The stronger the docking strength, the better the inhibition effect on the target kinase. The testing steps are as follows (the specific operation of each step can be set according to the software requirement):

2.1 obtaining proteins

Landing site http:// www.rcsb.org/PDB/home. Do download protein (PDB: 3IK 3); the test compounds were saved in the SLN file in mol2 format with chemdream 3D pro.

2.2 introduction of proteins the proteins to be docked were introduced according to the software protocol.

2.3 protein preparation

2.4 analysis of protein Structure and preparation for docking

2.5 testing other settings

2.6 assignment of ligands to dock and submit work

2.7 browsing results of Surflex-Dock

After finishing all interaction docking operations, the scoring result will actively pop up in the Results Browser dialog box by Application>Docking Suite>The Analyze Results may be read in batch runs and the jobname selected in the upper right corner of the dialog box. Click View details these chemical structures with Abl and Bcr-Abl ^T315I Docking scheme for kinase.

The scoring results were as follows:

compound number	Abl(2GQG)	Abl ^T315I (3IK3)
			Example 1	5.8873	5.2744
Example 2	9.7379	6.8144
			Example 3	7.1443	4.2390
Example 4	8.9260	4.4084
			Example 5	9.5046	6.5603
Example 6	9.6977	4.9065
			Example 7	4.9506	5.2965
Example 8	11.1673	4.8349
			Example 9	10.5886	6.5007
Example 10	9.8738	7.6011
			Example 11	9.5625	5.7679
Example 12	9.2936	7.7459
			Example 13	10.0228	6.8300
Example 14	9.4023	6.4471
			Example 15	10.9034	4.8208
Example 16	12.2546	6.6120

The scoring results show that the compounds of the present application are useful for Abl kinase and Bcr-Abl ^T315I The kinase has good binding ability, and can inhibit Abl and Bcr-Abl ^T315I The activity of (3) and further effectively solving the problem of drug resistance caused by the T315I mutation, and can be used as a novel Bcr-Abl tyrosine kinase inhibitor for resisting the T315I mutation.

EXAMPLE 18 results of the protein kinase Activity test of interest

ADP-Glo ^TM Kinase detection

1 instruments and reagents

2 preparation of kinase assay buffer

The kinase buffer solution and/or the kinase detection reagent are prepared according to the instructions, and the kinase detection reagent is used immediately after being prepared or is stored at-20 ℃ after being subpackaged. The prepared reagent has no loss of detection signals after being frozen and thawed for several times.

3 preparation of ATP-ADP Standard conversion Curve

3.1 diluting Ultra Pure ATP and ADP provided by the kit with 1 Xkinase reaction buffer (5 Xkinase reaction buffer provided by the kit is required to be diluted in advance) to prepare 1ml of 1mM ATP and 500ul of 1mM ADP (1ml of 1mM ATP: 100ul ATP,900ul of 1X kinase buffer is prepared; 500ul of 1mM ADP: 50ul of ADP,450ul of 1X kinase buffer is prepared).

3.2 Add 1ml of the first prepared 1mM ATP and 500ul of 1mM ADP to the A1-A12 wells of 0.2ml EP tubes and mix well, which is the 1mM series.

3.3 dilution into 25 μ M series in B1-B12 wells was used to generate ATP-ADP conversion standard curves for ABL 1.

3.4 dilution into 5. Mu.M series in C1-C12 wells for ABL production ^T315I ATP-ADP conversion standard curve (2).

3.5 transfer 5ul of B1-B12 from the prepared 25. Mu.M, 5. Mu.M ATP + ADP series 0.2ml EP tube B1-B12 to A1-A12, B1-B12 of 384 well plates; 0.2ml of EP tube C1-C12 was transferred 5ul to C1-C12, D1-D12 of 384 well plates.

3.6 incubate 40min at room temperature.

3.7 5ul ADP-Glo per well of 384-well plate ^TM The reagent stops the kinase reaction while depleting unconverted ATP, leaving only ADP.

3.8 incubation at room temperature for a certain time.

3.9 Add 10ul kinase assay reagent to convert ADP to ATP and introduce luciferase and luciferin to detect ATP (384 well plates A1-A12, B1-B12 add 10ul kinase assay reagent per well.

Incubating at room temperature for 30-60min, wherein the incubation time depends on ATP concentration (30 min) used in kinase reaction process, and detecting with chemiluminescence apparatus.

Optimization of kinase reaction conditions

The amount of kinase can be used in the amounts described in the specification (ABL) ^T315I 1.4ng ABL1 is 1 ng).

5 by using ADP-Glo ^TM Kinase assay screening Compounds

5.1 reagent preparation

Drug dilution: taking 8 EP tubes of 1.5ml, adding double distilled water 600ul to No. 2-8, adding double distilled water 1110.18ul to No. 1 and testing compound 9.22ul, and diluting for half a time.

The drug concentration is: 248 16 32 64 128 (mu Mol/L)

Diluting with DMSO: diluting 8 EP tubes (1.5 ml), no. 2-8 with double distilled water 600ul, no. 1 with double distilled water 1110.18ul and DMSO9.22ul for half a time.

1X Buffer: adding 800ul of double distilled water, 200ul 5X buffer,0.5ul of DTT into a 1.5ml EP tube, and mixing uniformly (calculating the dosage before preparation).

62.5. Mu.M ATP: adding 397.5ul 1X buffer,2.5ul Ultra Pure ATP into 1.5ml of EP tube, and mixing.

12.5 μ M ATP, adding 200ul 1X Buffer into 1.5ml EP tube, adding 50ul prepared 25 μ M ATP, and mixing.

2.5ng/ul ABL1 protein kinase: adding 117.0ul 1X Buffer and 3.0ul ABL1, active into 1.5ml EP tube, and mixing.

3.5ng/ul ABL ^T315I Protein kinase: a1.5 ml EP tube was charged with 115.8ul 1X Buffer and 4.2ul ABL ^T315I And Active, and mixing uniformly.

5.2ABL ^T315I Kinase detection

1. Taking 0.2ml EP tube, marking as (1), adding 18ul 3.5ng/ul ABL without adding substrate ^T315I Protein kinase, 18ul 1X buffer,36ul 12.5 mu M ATP, and mixing uniformly for later use.

2. Taking 0.2ml EP tube, recording as (2), adding 90ul 3.5ng/ul ABLL ^T315I Protein kinase, 90ul substrate, 180ul12.5 mu M ATP, and mixing evenly for later use.

3.8 0.2ml EP tubes (EP tube No. 1) were taken, and 2ul DMSO was added from low to high

8 0.2ml EP tubes (EP tube No. 2) were taken, and 2ul DMSO was added from low to high

Taking 8 EP tubes (No. 3-6 EP tubes) of 0.2ml, adding 2ul of compound (4 compounds) from low to high

No. 3.1 EP tube (0.2 ml), 8ul of EP tube solution (1) (0.2 ml) per well, no. 2-6 EP tube (0.2 ml), 8ul of EP tube solution (2) ((0.2 ml) per well), mixing well, transferring to 384-well plate (5 ul per well).

5. Incubate at room temperature for 60min.

6. Add 5ul ADP-Glo per well ^TM And incubating for 40min.

7. Adding 10ul kinase detection reagent into each hole, incubating for 30min, and detecting.

Partial compound protein kinase Bcr-Abl ^T315I The activities are shown in the following table:

ND：Not detected。

example 19 in vitro antitumor Activity assay

The cell strain is selected from human chronic myelogenous leukemia cells (K562), K562 imatinib resistant cells (K562/R), human liver cancer cells (HepG-2), human non-small cell lung cancer cells (A549) and human normal liver cells (L02).

1) The K562 cells, K562/R cells, hepG-2 cells, A549 cells and L02 cells were cultured in 1640 medium containing 1% double antibody and 10% FBS, respectively, in 5% CO ₂ And subcultured once every 2 days in a saturated humidity incubator at 37 ℃. K562/R cells with 1640% containing 1% double antibody and 10% FBS, 4. Mu. Mol/L containing 5% CO ₂ And subculturing once in a saturated humidity incubator at 37 ℃ for 2-3 days.

Selecting K562 cells and K562/R cells in logarithmic growth phase, and preparing them in 1640 medium containing 10% FBS at a concentration of 4X 10 ⁴ The cells were suspended in a volume of one mL/mL and plated in 96-well plates, and 100. Mu.l of the cell suspension was added to each well. In 5% of CO ₂ After further incubation in a 37 ℃ saturated humidity incubator for 24 hours, 100. Mu.L of medium (provided with 3 secondary wells) with compound concentrations of 8, 16, 32, 64 and 128. Mu.M was added, followed by further incubation for 48 hours, 20. Mu.L of MTS was added, the resulting mixture was placed in the incubator and incubated for 4 hours, and the absorbance (OD) was measured at 490nm using a microplate reader. The replicates were repeated 3 times.

Selecting HepG-2 cells, A549 cells and L02 cells in logarithmic growth phase, and preparing the cells in a medium containing 10% FBS at a concentration of 5X 10 ⁴ The cells were suspended in a volume of one mL/mL and plated in 96-well plates, and 100. Mu.l of the cell suspension was added to each well. In 5% of CO ₂ Culturing in 37 deg.C saturated humidity incubator for 24 hr, removing culture solution, adding culture medium 200 μ L (with 3 auxiliary wells) with compound concentration of 8, 16, 32, 64, and 128uM, culturing for 48 hr, adding MTT 20 μ L, and standingCulturing in incubator for 4 hr, discarding culture medium, adding 150uL DMAO into each well, shaking on shaking table at low speed for 10min in dark place to dissolve crystal completely, and measuring absorbance (OD) at 490nm with microplate reader. The replicates were repeated 3 times.

The cell viability (cell viability) calculation formula is as follows: cell bioavailability = (OD) _Drug -OD _Blank )/(OD _Control -OD _Blank )×100％

The test results were as follows:

the above table shows that the compound of the invention has good inhibitory action on CML cell strains K562 and K562 imatinib resistant cells (K562/R), namely has good inhibitory activity on K562 cells with imatinib resistance; meanwhile, the medicine has lower toxicity to normal human liver cells L02, and can avoid or reduce toxic and side effects on human bodies while inhibiting cancer cells.

Claims

1. A compound of formula I, or a pharmaceutically acceptable salt thereof, having the structure:

n is selected from 0, 1, 2,3 or 4.

2. A compound of formula I according to claim 1, or a pharmaceutically acceptable salt thereof, wherein R1 is each independently selected from halogen, -OH, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 haloalkyl; n is selected from 0, 1 or 2.

3. A compound of formula I according to claim 1, or a pharmaceutically acceptable salt thereof, wherein R1 is each independently selected from methyl, chloro, fluoro, hydroxy, trifluoromethyl; n is selected from 0 or 1.

4. A compound of formula I as claimed in any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, R ² Selected from hydrogen, C1-C4 alkyl, C1-C4 haloalkyl; r is ³ Selected from hydrogen, -OH, halogen, C ₁ -C ₄ Alkyl radical, C ₁ -C ₆ Alkoxy radical, C ₁ -C ₄ Haloalkyl, C ₁ -C ₄ Haloalkoxy, benzyl; r ⁴ Is selected from C ₁ -C ₄ Alkyl radical, C ₁ -C ₄ A haloalkyl group.

5. A compound of formula I according to claim 4, or a pharmaceutically acceptable salt thereof, R ² Selected from methyl, ethyl, trifluoromethyl; r ³ Selected from hydrogen, benzyl; r ⁴ Selected from methyl and ethyl.

6. A compound of formula I according to claim 1, selected from the following compounds:

7. a process for the preparation of a compound of formula I according to claim 1, which is represented by the reaction scheme:

wherein R is ¹ 、R ² 、R ³ 、R ⁴ And n is as defined in claim 1.

8. The method of claim 7, comprising the reaction steps of:

step four: adding intermediate thiazole aminobenzoic acid derivative, EDCI, HOBT and none under the ice bath conditionAdding water ethanol into a reaction bottle, reacting for 2-4h, adding

Continuously carrying out ice-bath reaction on hydrochloride, DIPEA, DMAP and DMF for half an hour, then changing to room temperature reaction until the reaction is finished, slowly adding ice water while stirring until the solution is changed from clear to turbid, stirring at room temperature for 0.5-1.5h, then putting the solution into a refrigerator to precipitate white solid, filtering, and drying the obtained solid to obtain the compound of the formula I.

9. The method of claim 8, wherein:

the mol ratio of m-aminobenzoic acid, ammonium thiocyanate and benzoyl chloride in the first step is 1:1-1.5;

step two the basic aqueous solution is 10% an NaOH aqueous solution, and the dilute hydrochloric acid concentration is 4mol/L;

the molar ratio of the 3-carboxyphenylthiourea to the substituted 2-Br-1-phenylalkyl ketone in step three is 1:1-1.2.

10. The method of claim 9, wherein:

the mol ratio of m-aminobenzoic acid, ammonium thiocyanate and benzoyl chloride in the first step is 1.2;

the molar ratio of the 3-carboxyphenylthiourea to the substituted 2-Br-1-phenylalkyl ketone in step three is 1:1.

11. A pharmaceutical composition comprising a compound of formula I as described in any one of claims 1-6, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, excipient.

12. Use of a compound according to any one of claims 1 to 6 or a pharmaceutical composition according to claim 11 for the manufacture of a medicament for the treatment of cancer which is a Bcr-Abl tyrosine kinase-targeted cancer.

13. The use according to claim 12, wherein the cancer is a cancer targeted against the T315I mutant Bcr-Abl tyrosine kinase.

14. Use according to claim 12 or 13, wherein the cancer is selected from human chronic myeloid leukemia, liver cancer, non-small cell lung cancer.

15. The use of claim 14, wherein the cancer is human chronic myeloid leukemia.