CN110551102B

CN110551102B - ALK covalent inhibitors and uses thereof

Info

Publication number: CN110551102B
Application number: CN201810555626.7A
Authority: CN
Inventors: 李锐; 魏于全
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2018-06-01
Filing date: 2018-06-01
Publication date: 2021-01-12
Anticipated expiration: 2038-06-01
Also published as: CN110551102A

Abstract

The present invention relates to ALK covalent inhibitionAn agent and application thereof, belonging to the technical field of targeted drugs. The invention aims to provide a compound capable of serving as an ALK covalent inhibitor. The invention designs and synthesizes a series of molecules by taking cysteine at the periphery of the binding pocket as a covalent site, and the structural formula of the molecules is shown as a formula I. The compound molecule can target cysteine residues outside an ALK active pocket, is connected with cysteine Cys1259 around the active pocket through covalent coupling by a linker, has strong selectivity, higher binding capacity and better biological activity, and can reduce the frequency of taking medicines, thereby increasing the medication compliance of patients.

Description

ALK covalent inhibitors and uses thereof

Technical Field

The invention relates to an ALK covalent inhibitor and application thereof, belonging to the technical field of targeted drugs.

Background

The general mechanism of inhibiting enzymes or receptors by traditional small molecule drugs is to reduce their activity by binding the small molecule drug to the target protein. The typical mode of binding of a drug to a target protein is through non-covalent interactions. Non-covalent interactions (electrostatic interactions, van der waals interactions, hydrogen bonding, and hydrophobic interactions) can improve the affinity and specificity of a ligand for a receptor, enzyme, or ion channel. Typically, the maximum binding energy of a small molecule to a protein through non-covalent interactions is 6.3 kj/mole per non-hydrogen atom. However, these non-covalent bindings are reversible and can be competed by endogenous substrates, affecting drug efficacy.

Covalent drugs are a class of drugs that exert their biological effects by binding to the protein of interest through covalent interactions. The covalent drug has strong binding force and long binding time, thereby having strong biological activity, reducing the dosage and the administration frequency, and further enhancing the compliance of patients. In addition to the corresponding non-covalent interactions, these drugs contain groups (including acrylamides, epoxides, α -haloketones, methylesterketones, aziridines, vinylsulfones, activated alkynes) that chemically react with specific amino acid residues of the target protein (including cysteine, serine, tyrosine, threonine, lysine, glutamic acid, aspartic acid) to form covalent bonds that are non-reversibly bound, such as C — N of 305 kj/mole, much larger than the non-covalent interactions, and that cannot be competed by endogenous substrates. Therefore, the covalent small molecule inhibitor has more advantages.

There are a number of covalent drugs available, most of which are discovered by chance. Covalent drug molecules are mainly covalently coupled with groups on catalytic sites in the binding pocket, and the binding method has the disadvantages that obvious drug tolerance is brought when the groups on the covalent coupling sites are mutated, and the risk of side reactions is high. A new covalent drug design concept has emerged in recent years: the design idea of targeting cysteine of a non-catalytic site in a binding pocket has the advantages that even if the catalytic site is mutated, obvious drug tolerance is not brought when the cysteine of a covalent coupling site is not mutated, the molecular activity is superior to that of a non-covalent inhibitor with the same structure, but if no amino acid residue capable of forming covalent coupling exists in the binding pocket of a target, a covalent drug cannot be designed aiming at the target.

Anaplastic Lymphoma Kinase (ALK) is a receptor tyrosine Kinase associated with three major types of tumors, blood, stromal and solid. About 3-7% of patients with non-small cell lung cancer (NSCLC) have tumor chromosome EML4 gene exon and ALK gene exon fused to form EML4-ALK fusion tyrosine kinase, and EML4-ALK fusion variant has high carcinogenicity and ALK is highly expressed in various tumor cells. Therefore, ALK becomes an attractive cancer therapeutic target.

LDK378, common name: ceritinib, trade name: zykadia, an ALK inhibitor currently on the market, is suitable for the treatment of crizotinib-progressing or intolerant non-small cell lung cancer (NSCLC) patients with Anaplastic Lymphoma Kinase (ALK) -positive metastasis. The chemical structural formula of the main components is as follows:

the structure of the LDK378 and ALK protein crystal complex is shown in figure 1. Since there are no covalently bondable residues within the binding pocket of the target protein, LDK378 binds to the target protein in a non-covalent form, has limited binding capacity, and has become clinically resistant.

Disclosure of Invention

In view of the above drawbacks, the technical problem to be solved by the present invention is to provide a compound that can be used as an ALK covalent inhibitor.

The structural formula of the compound is shown as the formula I:

wherein X is a linker moiety and Y is a covalent reaction target; and the compound can be covalently bonded with cysteine residue Cys1259 at the periphery of an ALK active pocket through a covalent reaction target head.

Preferably, X is

Wherein, is N in the ceritinib piperidine ring, and Z is-CH₂-、-NH-、-O-、-S-、

Z₁is-CH₂-、-NH-、-O-、-S-、

Z₂is-CH₂-, -NH-, -O-or-S-, m0 is any integer of 0 to 15, m1 is any integer of 0 to 8, m2 is any integer of 0 to 8, m3 is any integer of 0 to 8, m4 is any integer of 0 to 15, and m5 is any integer of 0 to 15; m is H or 1-8 saturated alkane or cycloalkane;

y is

A is H, saturated or unsaturated alkane, aromatic ring, heterocycle, carbonyl, halogen, nitryl or cyano.

Wherein, preferably, Z is

Z₁is-CH₂-, -NH-, -O-or-S-.

Preferably, Y is

More preferably Y is

Preferably, m0 is 1 or 2.

More preferably, the structural formula is shown in formula II:

r is

Or

n1 is 1 or 2, n2 is 0-8An integer, n3 is any one of 0-8, D is-CH₂-, -NH-, -O-or-S-, E is-NH-, -O-or-S-.

Preferably, the structural formula is shown as ConA-3, ConA-4, ConB-1 or ConB-3:

the invention also provides application of the compound in preparation of an ALK inhibitor.

The compound molecule of the invention can be used as an ALK inhibitor, and can be particularly used for preparing an ALK covalent inhibitor. As shown in fig. 3, the compound molecule of the present invention is linked to the cysteine around the active pocket by covalent coupling, and the advantages of this mode of action are: firstly, compound molecules are coupled with target proteins through covalent, so that the binding capacity is greatly improved, and stronger biological activity can be brought; secondly, the mother-core structure of the inhibitor molecule is not changed, the combination mode is not influenced, and better selectivity is still kept.

The invention also provides a pharmaceutical composition for treating tumors, which comprises a therapeutically effective amount of the compound and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier of the present invention is a conventional pharmaceutical carrier in the pharmaceutical field, such as diluent, excipient, filler, etc.

The invention also provides application of the compound in preparing a medicament for treating tumors.

The compound can be used for preparing medicaments for treating tumors. Preferably, the tumor is liver cancer, lung cancer or cervical cancer.

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

the compound molecule can target cysteine residues outside an ALK active pocket, is connected with cysteine around the active pocket through covalent coupling by a linker, has strong selectivity and higher binding capacity, can reduce off-target effect brought by a common covalent mode, has longer drug action time and can reduce the frequency of taking the drug.

Drawings

FIG. 1 shows the structure of LDK378 and ALK protein crystal complex.

FIG. 2 shows the position relationship between Cys1259 and LDK378 binding pocket.

FIG. 3 shows the positional relationship between ConB-1 and ALK active pocket according to the present invention.

FIG. 4 shows the IC of ConB-1 vs ALK₅₀。

FIG. 5 is a graph of tumor volume as a function of time of administration.

FIG. 6 shows the body weight of mice as a function of the administration time.

Detailed Description

The inventor of the invention researches and finds that the Cys1259 position is at the periphery of a binding pocket, and the positional relationship between the Cys1259 and the LDK378 binding pocket is shown in figure 2. A series of covalent drugs are designed and synthesized according to the relative positions of Cys1259 and LDK378, and the structural formula of the covalent drugs is shown in a formula I:

wherein X is a linker moiety and Y is a covalent reaction target; and the compound can be covalently bonded with cysteine residues at the periphery of an ALK active pocket. According to the binding conformation of ceritinib and a target protein and the position relation between the piperidine tail of the ceritinib and the corresponding cysteine amino residue Cys1259 at the periphery of an ALK pocket, a linker with a proper chain length and a proper composition is selected, and then a target head capable of forming covalent reaction with the Cys1259 is coupled, so that a novel covalent compound capable of being covalently bound with the Cys1259 at the periphery of an active pocket of the ALK is obtained.

Preferably, X is

Z₁is-CH₂-、-NH-、-O-、-S-、

y is

More preferably, Z is

Z1 is-CH 2-, -NH-, -O-, or-S-; namely, the preferred structural formula is as follows:

wherein m0 is any integer of 0-15, m1 is any integer of 0-8, m2 is any integer of 0-8, m3 is any integer of 0-8A number, m4 is any integer of 0 to 15, m5 is any integer of 0 to 15, Z₁is-CH₂-, -NH-, -O-or-S-, Z₂is-CH₂-、-NH-、-O-、-S-。

Preferably, Y is

More preferably, Y is

Preferably, m0 is 1 or 2.

More preferably, acrylamide is used as a reaction target, and the structural formula of the acrylamide is shown as a formula II.

R is

Or

n1 is 1 or 2, n2 is any integer of 0-8, n3 is any integer of 0-8, D is-CH₂-, -NH-, -O-or-S-, E is-NH-, -O-or-S-.

Preferably, n1 is 1 or 2. More preferably, n1 is 2, and the inventors have found that when n1 is 2, the resulting compound molecule has a high drug effect and a high tumor cell inhibition rate.

More preferably, n2 is 1,2,4,6 or 8 and n3 is 1,2 or 3.

Preferably, the formula is as follows:

more preferably, it has the formula shown in ConA-3, ConA-4, ConB-1 or ConB-3:

the compound molecule of the invention can be used as an ALK inhibitor, and can be particularly used for preparing an ALK covalent inhibitor. As shown in fig. 3, the compound molecule of the present invention is linked to the cysteine around the active pocket by covalent coupling, and the advantages of this mode of action are: firstly, the compound molecules are coupled with target proteins through covalent bonds, so that the binding capacity is greatly improved, and stronger biological activity can be brought.

The following examples are provided to further illustrate the embodiments of the present invention and are not intended to limit the scope of the present invention.

EXAMPLE 1 Synthesis of Compound ConA-1

1. Synthesis of intermediate 1

LDK378 is used as a raw material (330mg,0.6mmol), dissolved in 50mL of methanol, added with methyl acrylate (78mg,0.9mmol) and triethylamine (240mg,2.4mmol), stirred at normal temperature for 8h, and the progress of the reaction is detected by TLC. The treatment method comprises the following steps: distilling under reduced pressure to remove methanol, adding 150mL ethyl acetate, backwashing the organic solution with water for three times, removing water with anhydrous sodium sulfate, concentrating under reduced pressure, and performing column chromatography with PE/EA separation system to obtain white solid with yield of about 90%.

¹H NMR(400MHz,CDCl₃)δ＝9.49(s,1H),8.58(d,J＝8.4,1H),8.15(s,1H),7.99(s,1H),7.93(d,J＝8.0,1H),7.62(t,J＝7.9,1H),7.53(s,1H),7.24(d,J＝7.7,1H),6.80(s,1H),4.53(dt,J＝12.1,6.0,1H),3.71(s,3H),3.32–3.21(m,1H),3.06(d,J＝9.6,2H),2.77(d,J＝6.7,2H),2.72–2.52(m,3H),2.15(s,4H),1.76(s,4H),1.34(dd,J＝15.0,6.5,12H).¹³C NMR(101MHz,CDCl₃)δ＝157.52,155.39,155.33,144.72,138.52,134.65,131.25,127.46,126.87,124.91,123.71,123.09,120.63,110.82,105.72,71.43,55.45,54.30,53.90,51.71,37.94,32.77,22.27,18.93,15.37.HRMS(DART-TOF)calculated for C₃₂H₄₃ClN₅O₅S[M+H]⁺m/z 644.2673,found 644.2673.

2. Synthesis of intermediate 2

Intermediate 1(320mg, 0.5mmol) was dissolved in 50mL methanol and water 1: 1, adding sodium hydroxide (200mg, 5mmol), stirring at 60 ℃ for 10h, clarifying the reaction solution to become turbid, and monitoring the reaction by TLC. Reaction treatment: methanol was removed by rotary evaporation under reduced pressure and the pH of the aqueous layer was adjusted to 2 with dilute hydrochloric acid. And extracting with EA for 3 times, backwashing the oil layer with saturated salt solution for 1 time, drying with anhydrous sodium sulfate, and concentrating under reduced pressure to obtain white powder with yield of about 95%.

¹H NMR(400MHz,CDCl₃)δ＝9.51(s,1H),8.58(d,J＝8.4,1H),8.16(d,J＝1.2,1H),8.03(d,J＝3.2,1H),7.93(d,J＝7.9,1H),7.62(dd,J＝11.3,4.4,1H),7.58–7.51(m,1H),7.27(d,J＝6.5,1H),6.75(d,J＝37.6,1H),4.56(dt,J＝11.1,5.6,1H),3.51–3.14(m,3H),2.81(ddd,J＝47.2,24.3,12.3,3H),2.17(d,J＝8.1,3H),1.34(dd,J＝14.8,6.5,13H).HRMS(DART-TOF)calculated for C₃₁H₄₁ClN₅O₅S[M+H]⁺m/z 630.2517,found 630.2508.

3. Synthesis of intermediate 3

LDK378(560mg, 1mmol) was weighed out, dissolved in 50mL acetonitrile, tert-butyl bromoacetate (195mg, 1mmol), potassium carbonate (414mg, 3mmol) was added, refluxed for 5h and the reaction monitored by TLC. After completion of the reaction, 30mL of water was added to quench the reaction, and the mixture was extracted 3 times with 100mL of EA and then back-washed 3 times with water. The organic layer was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and subjected to column chromatography using PE/EA as a mobile phase. A yellow solid was obtained with a yield of about 76%.

¹H NMR(400MHz,CDCl₃)δ＝9.49(s,1H),8.58(d,J＝8.3,1H),8.14(s,1H),7.97(d,J＝12.7,1H),7.92(d,J＝7.8,1H),7.62(t,J＝7.7,1H),7.54(s,1H),7.26(dd,J＝13.1,5.4,1H),6.82(s,1H),4.51(dq,J＝11.9,6.0,1H),3.34–3.22(m,1H),3.18(s,2H),3.09(d,J＝10.9,2H),2.68(dd,J＝15.9,7.8,1H),2.32(t,J＝10.8,2H),2.16(s,3H),1.82(dt,J＝34.1,11.0,4H),1.49(s,10H),1.34(dd,J＝17.5,6.4,13H).HRMS(DART-TOF)calculated for C₃₄H₄₇ClN₅O₅S[M+H]⁺m/z 672.2986,found 672.2975.

4. Synthesis of intermediate 4

Intermediate 3 was dissolved in 20mL of trifluoroacetic acid and stirred at room temperature for 5 h. After completion of the reaction, 30mL of DCM was added to the reaction mixture, and concentration was carried out under reduced pressure to obtain a white powder with a yield of about 90%.

¹H NMR(400MHz,MeOD)δ＝8.29(d,J＝7.9,1H),8.23(s,1H),7.99(d,J＝7.9,1H),7.70(t,J＝7.7,1H),7.51(t,J＝7.6,1H),7.40(s,1H),6.92(s,1H),4.72–4.59(m,1H),4.13(s,2H),3.82(t,J＝18.3,2H),3.35(s,4H),3.13(t,J＝11.7,1H),2.18(d,J＝12.2,3H),2.13–1.92(m,4H),1.37–1.16(m,13H).HRMS(DART-TOF)calculated for C₃₀H₃₉ClN₅O₅S[M+H]⁺m/z 616.2360,found616.2347.

5. Synthesis of intermediate ConA-1M

Acryloyl chloride (181mg,2mmol) was weighed out, dissolved in 15mL THF in an ice bath, and N-t-butoxycarbonyl-1, 3-propanediamine (348mg, 2mmol) was added, DIPEA (417mg,3mmol) was added, and the reaction was followed by about TLC at 0 ℃ to monitor the progress of the reaction. After the reaction was completed, 30mL of water was added to quench the reaction, the aqueous layer was extracted with EA 4 times, the organic layers were combined, back-washed with saturated brine 2 times, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and subjected to column chromatography using a PE/EA separation system. The product was a white powder with a yield of about 71%.

¹H NMR(400MHz,CDCl₃)δ＝6.25–6.15(m,1H),6.09(dd,J＝17.0,9.9,1H),5.54(dd,J＝9.9,1.8,1H),3.28(td,J＝12.0,4.9,2H),3.10(dd,J＝11.8,5.8,2H),1.65–1.52(m,2H),1.36(s,9H).HRMS(DART-TOF)calculated for C₁₁H₂₁N₂O₃[M+H]⁺m/z 229.1552,found 229.1545.

6. Synthesis of target product ConA-1

ConA-1M (46mg,0.2mmol) was dissolved in 5mL of trifluoroacetic acid and stirred at 50 ℃ for 1 h. The reaction was stopped, 10ml of lcm was added to the reaction solution and concentrated under reduced pressure to give a brown oil. To this oil was added 10mL of DMF and dissolved, and intermediate 2(63mg, 0.1mmol), HATU (45.6mg, 0.12mmol), DIPEA (38.7mg, 0.3mmol) were added. The reaction was carried out at room temperature for 15h, and the progress of the reaction was monitored by TLC. After completion of the reaction, the reaction was quenched with 30mL of water, the aqueous layer was extracted 4 times with EA, the organic layers were combined, back-washed 2 times with saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and subjected to column chromatography with DCM/MeOH system. The product was a white powder with a yield of about 52%.

¹H NMR(400MHz,CDCl₃)δ＝9.50(s,1H),8.58(d,J＝8.4,1H),8.14(d,J＝8.9,2H),7.93(d,J＝7.9,1H),7.62(t,J＝7.8,1H),7.54(s,1H),7.27–7.22(m,1H),6.77(s,1H),6.35–6.22(m,1H),6.14(dd,J＝17.0,10.2,1H),5.63(d,J＝10.2,1H),4.61–4.48(m,1H),3.47–3.21(m,6H),3.12(d,J＝11.3,2H),2.78–2.64(m,3H),2.45(t,J＝6.2,2H),2.23–2.08(m,5H),1.83(d,J＝13.3,2H),1.77–1.60(m,4H),1.34(dd,J＝19.1,6.5,13H).¹³C NMR(101MHz,CDCl₃)δ＝173.40,165.87,157.49,155.38,155.34,144.69,138.52,137.19,134.64,131.25,127.79,127.17,126.01,124.90,123.66,123.11,120.74,111.00,105.82,71.74,55.47,54.38,54.01,38.10,35.77,35.60,32.85,32.69,29.89,22.26,18.93,15.37.HRMS(DART-TOF)calculated for C₃₇H₅₁ClN₇O₅S[M+H]⁺m/z 740.3361,found 740.3400.

EXAMPLE 2 Synthesis of Compound ConA-2

1. Synthesis of intermediate ConA-2M

The preparation method of the intermediate is the same as that of ConA-1M, the raw material is N-tert-butyloxycarbonyl-1, 4-butanediamine, and other raw materials and treatment methods are the same. The yield was 76%.

¹H NMR(400MHz,CDCl₃)δ＝6.27(dd,J＝17.0,1.6,2H),6.13(dd,J＝17.0,10.2,1H),5.62(dd,J＝10.2,1.6,1H),4.72(s,1H),3.35(q,J＝6.4,2H),3.13(s,2H),1.62–1.49(m,4H),1.44(s,9H).¹³C NMR(101MHz,CDCl₃)δ＝165.74,156.23,130.99,126.13,79.27,39.20,28.41,27.71,26.50.HRMS(DART-TOF)calculated for C₁₂H₂₃N₂O₃[M+H]⁺m/z 243.1709,found 243.1701.

2. Synthesis of target product ConA-2

The preparation method of the target product is the same as that of ConA-1, the raw material is ConA-2M, and other raw materials and treatment methods are the same. The yield was 67%.

¹H NMR(400MHz,CDCl₃)δ＝9.51(s,1H),8.58(d,J＝8.3,1H),8.16(s,1H),8.03(s,1H),7.93(dd,J＝8.0,1.4,1H),7.62(dd,J＝11.4,4.3,1H),7.54(s,1H),7.31–7.23(m,2H),6.75(s,1H),6.75(s,1H),6.33–6.22(m,1H),6.17–6.06(m,1H),4.55(dt,J＝12.1,6.1,1H),3.46–3.11(m,7H),2.90–2.68(m,3H),2.49(t,J＝5.9,2H),2.31(t,J＝11.4,2H),2.16(s,3H),1.88(d,J＝12.7,2H),1.76–1.67(m,2H),1.65–1.53(m,4H),1.35(dd,J＝20.1,6.5,12H).¹³C NMR(101MHz,CDCl₃)δ＝172.69,165.83,157.46,155.36,155.33,144.70,138.49,136.78,134.66,131.27,130.98,127.93,127.27,126.19,124.88,123.63,123.15,120.80,110.97,105.86,71.87,55.49,54.31,53.88,39.13,38.53,37.77,32.57,32.00,27.18,26.48,22.26,18.93,15.37.HRMS(DART-TOF)calculated for C₃₈H₅₃ClN₇O₅S[M+H]⁺m/z 754.3517,found 754.3531.

EXAMPLE 3 Synthesis of Compound ConA-3

1. Synthesis of intermediate ConA-3M

The preparation method of the intermediate is the same as that of ConA-1M, the raw material is N-tert-butyloxycarbonyl-1, 6-hexanediamine, and other raw materials and treatment methods are the same. The yield was 68%.

¹H NMR(400MHz,CDCl₃)δ＝6.28(dd,J＝17.0,1.4,1H),6.12(dd,J＝17.0,10.2,1H),5.62(dd,J＝10.2,1.3,1H),3.32(dd,J＝13.1,6.7,2H),3.11(dd,J＝12.6,6.2,2H),1.60–1.41(m,13H),1.34(dd,J＝8.8,5.6,4H).HRMS(DART-TOF)calculated for C₁₄H₂₆N₂O₃Na[M+Na]⁺m/z293.1841,found 293.1856.

2. Synthesis of target product ConA-3

The preparation method of the target product is the same as that of ConA-1, the raw material is ConA-3M, and other raw materials and treatment methods are the same. The yield was 67%.

¹H NMR(400MHz,CDCl₃)δ＝9.52(s,1H),8.58(d,J＝8.4,1H),8.16(s,1H),8.04(d,J＝10.3,2H),7.93(dd,J＝7.9,1.3,1H),7.67–7.58(m,1H),7.52(s,1H),7.25(d,J＝9.7,1H),6.78(d,J＝16.4,1H),6.31–6.20(m,1H),6.09(dd,J＝17.0,10.2,1H),5.90(s,1H),5.64–5.54(m,1H),4.58–4.46(m,1H),3.27(tq,J＝13.9,6.8,5H),3.13(d,J＝11.3,2H),2.72(dd,J＝12.2,6.9,3H),2.43(t,J＝6.1,2H),2.27–2.13(m,5H),1.85(d,J＝12.6,2H),1.67(dd,J＝22.0,12.1,2H),1.60–1.47(m,4H),1.42–1.29(m,16H).¹³C NMR(101MHz,CDCl₃)δ＝172.49,165.61,157.47,155.37,144.65,138.50,137.10,134.63,131.29,131.01,127.95,127.44,126.09,124.91,123.62,123.13,120.84,111.20,105.90,72.02,55.49,54.36,53.87,39.11,38.58,37.94,32.84,32.37,29.43,29.39,26.22,26.10,22.29,18.93,15.37.HRMS(DART-TOF)calculated for C₄₀H₅₇ClN₇O₅S[M+H]⁺m/z 782.3830,found 782.3863.

EXAMPLE 4 Synthesis of Compound ConA-4

1. Synthesis of intermediate ConA-4M

The preparation method of the intermediate is the same as that of ConA-1M, the raw material is N-tert-butyloxycarbonyl-1, 8-octanediamine, and other raw materials and treatment methods are the same. The yield was 67%.

¹H NMR(400MHz,CDCl₃)δ＝6.27(dd,J＝17.0,1.2,1H),6.11(dd,J＝17.0,10.2,1H),5.62(dd,J＝10.2,1.1,1H),4.56(s,1H),3.09(dd,J＝12.6,6.2,2H),1.59–1.39(m,13H),1.28(d,J＝17.7,9H).HRMS(DART-TOF)calculated for C₁₆H₃₀N₂O₃Na[M+Na]⁺m/z 321.2154,found 321.2149.

2. Synthesis of target product ConA-4

The preparation method of the target product is the same as that of ConA-1, the raw material is ConA-4M, and other raw materials and treatment methods are the same. The yield was 67%.

¹H NMR(400MHz,CDCl₃)δ＝9.52(s,1H),8.58(d,J＝8.3,1H),8.16(s,1H),8.02(s,2H),7.93(dd,J＝8.0,1.4,1H),7.67–7.58(m,1H),7.53(s,1H),7.27(d,J＝6.0,1H),6.75(s,1H),6.26(ddd,J＝17.0,6.5,1.4,1H),6.10(td,J＝17.4,9.1,1H),5.66–5.53(m,1H),4.61–4.45(m,1H),3.39–3.09(m,8H),2.73(dd,J＝14.2,5.1,3H),2.46(t,J＝6.0,2H),2.24(t,J＝11.1,2H),2.17(s,3H),1.86(d,J＝12.1,2H),1.71(dd,J＝22.6,11.9,2H),1.60–1.43(m,5H),1.41–1.28(m,21H).¹³C NMR(101MHz,CDCl₃)δ＝172.20,165.51,157.47,155.36,144.70,138.50,134.64,131.29,131.00,127.90,127.29,126.09,124.91,123.61,123.14,120.82,110.98,105.92,71.84,55.49,54.33,53.85,39.51,39.03,37.80,32.67,29.47,29.40,29.09,29.05,26.85,26.75,22.28,18.93,15.37.HRMS(DART-TOF)calculated for C₄₂H₆₁ClN₇O₅S[M+H]⁺m/z 810.4143,found810.4150.

EXAMPLE 5 Synthesis of Compound ConA-5

The preparation method of the target product is the same as that of ConA-1, the raw materials are ConA-1M and an intermediate 4, and other raw materials and treatment methods are the same. The yield was 35%.

¹H NMR(400MHz,CDCl₃)δ＝9.51(s,1H),8.58(d,J＝8.2,1H),8.15(s,1H),8.01(s,1H),7.93(dd,J＝8.0,1.5,1H),7.66–7.60(m,2H),7.57(s,1H),7.27–7.21(m,1H),6.81(s,1H),6.72(t,J＝6.0,1H),6.28(dd,J＝17.0,1.5,1H),6.15(dd,J＝17.0,10.2,1H),5.64(dd,J＝10.2,1.5,1H),4.66–4.54(m,1H),3.38(td,J＝12.4,6.4,4H),3.27(dq,J＝13.7,6.8,1H),3.10(s,2H),2.99(t,J＝10.9,2H),2.39(d,J＝14.2,2H),2.16(s,3H),1.85–1.69(m,6H),1.35(dd,J＝23.4,6.5,12H).¹³C NMR(101MHz,CDCl₃)δ＝165.90,157.48,155.36,155.32,144.74,138.50,137.20,134.64,131.28,131.22,127.78,127.13,126.01,124.92,123.66,123.13,120.78,111.05,105.80,71.69,61.86,55.48,55.08,37.53,35.88,35.70,32.80,29.80,29.69,22.28,18.94,15.37.HRMS(DART-TOF)calculated for C₃₆H₄₉ClN₇O₅S[M+H]⁺m/z 726.3204,found 726.3223.

EXAMPLE 6 Synthesis of Compound ConA-6

The preparation method of the target product is the same as that of ConA-1, the raw materials are ConA-2M and an intermediate 4, and other raw materials and treatment methods are the same. The yield was 33%.

¹H NMR(400MHz,CDCl₃)δ＝9.51(s,1H),8.58(d,J＝8.2,1H),8.15(s,1H),8.06(d,J＝5.2,1H),8.02(s,1H),7.93(dd,J＝8.0,1.5,1H),7.68–7.59(m,1H),7.54(s,1H),7.30–7.22(m,1H),6.75(s,1H),6.33(t,J＝5.5,1H),6.25(dd,J＝17.0,1.6,1H),6.12(dd,J＝17.0,10.1,1H),5.60(dd,J＝10.1,1.6,1H),4.64–4.48(m,1H),3.44–3.15(m,7H),2.86–2.68(m,3H),2.47(t,J＝6.2,2H),2.28(t,J＝11.3,2H),2.16(s,3H),1.87(d,J＝12.5,2H),1.78–1.55(m,7H),1.34(dd,J＝20.3,6.5,12H).¹³C NMR(101MHz,CDCl₃)δ＝165.75,157.48,155.35,144.71,138.50,134.64,131.28,130.98,127.85,127.21,126.20,124.91,123.65,123.13,120.76,111.04,105.83,71.78,61.99,55.48,55.06,39.22,38.52,37.57,32.79,29.69,27.60,26.45,22.29,18.94,15.37.HRMS(DART-TOF)calculated for C₃₇H₅₁ClN₇O₅S[M+H]⁺m/z 740.3361,found 740.3348.

EXAMPLE 7 Synthesis of Compound ConA-7

The preparation method of the target product is the same as that of ConA-1, the raw materials are ConA-3M and an intermediate 4, and other raw materials and treatment methods are the same. The yield was 36%.

¹H NMR(400MHz,CDCl₃)δ＝9.51(s,1H),8.57(t,J＝7.1,1H),8.16(d,J＝3.0,1H),8.02(s,1H),7.93(d,J＝7.8,1H),7.62(t,J＝7.6,1H),7.56(d,J＝8.2,1H),7.25(d,J＝8.7,1H),6.79(s,1H),6.27(d,J＝17.0,1H),6.11(dd,J＝17.0,10.3,1H),5.82(s,1H),5.62(d,J＝10.2,1H),4.58(dt,J＝12.3,6.1,1H),3.29(dq,J＝20.8,6.7,5H),3.01(dd,J＝30.0,19.2,3H),2.67(dd,J＝23.3,12.7,1H),2.35(d,J＝9.6,2H),2.22–2.11(m,3H),2.05(d,J＝8.8,1H),1.77(dd,J＝25.4,11.2,4H),1.65–1.48(m,5H),1.46–1.29(m,15H).¹³C NMR(101MHz,CDCl₃)δ＝165.60,155.36,144.72,138.50,134.63,131.29,131.00,127.27,126.17,124.94,123.64,123.13,120.81,111.09,105.85,71.83,55.48,55.00,39.12,38.54,37.55,29.70,29.33,26.11,26.07,22.29,18.94,15.37.HRMS(DART-TOF)calculated for C₃₉H₅₅ClN₇O₅S[M+H]⁺m/z 768.3674,found 768.3659.

EXAMPLE 8 Synthesis of Compound ConA-8

The preparation method of the target product is the same as that of ConA-1, the raw materials are ConA-4M and an intermediate 4, and other raw materials and treatment methods are the same. The yield was 29%.

¹H NMR(400MHz,CDCl₃)δ＝9.51(s,1H),8.58(d,J＝8.2,1H),8.16(d,J＝3.2,1H),8.04(d,J＝18.4,1H),7.93(dd,J＝8.0,1.6,1H),7.66–7.59(m,1H),7.56(d,J＝11.3,1H),7.24(s,1H),6.80(d,J＝13.0,1H),6.30–6.23(m,1H),6.07(dd,J＝17.0,10.3,1H),5.68–5.58(m,2H),4.64–4.52(m,1H),3.39–3.24(m,5H),3.00(dd,J＝26.5,15.3,3H),2.34(t,J＝10.7,2H),1.75(dd,J＝32.2,10.2,4H),1.53(d,J＝6.6,5H),1.40–1.30(m,19H).¹³C NMR(101MHz,CDCl₃)δ＝165.52,157.48,155.36,144.71,138.50,134.63,131.29,130.96,127.86,127.32,126.18,124.93,123.64,123.14,120.83,111.12,100.00,71.85,62.05,55.49,55.00,39.54,38.90,37.60,32.88,29.70,29.49,29.02,26.75,22.30,18.93,15.37.HRMS(DART-TOF)calculated for C₄₁H₅₉ClN₇O₅S[M+H]⁺m/z 796.3987,found 796.3981.

EXAMPLE 9 Synthesis of Compound ConB-1

1. Synthesis of intermediate ConB-1M

The preparation method of the intermediate is the same as that of ConA-1M, the raw material is [2- (2-aminoethoxy) ethyl ] tert-butyl carbamate, and other raw materials and treatment methods are the same. The yield was 76%.

¹H NMR(400MHz,CDCl₃)δ＝6.22(dd,J＝17.0,2.0,1H),6.13(dd,J＝17.0,9.9,1H),5.55(dd,J＝9.9,2.0,1H),3.53–3.47(m,2H),3.46(s,4H),3.27–3.18(m,2H),1.37(s,9H).HRMS(DART-TOF)calculated for C₁₂H₂₃N₂O₄[M+H]⁺m/z 259.1658,found 259.1645.

2. Synthesis of target product ConB-1

The preparation method of the target product is the same as that of ConA-1, the raw material is ConB-1M, and other raw materials and treatment methods are the same. The yield was 42%.

¹H NMR(400MHz,CDCl₃)δ＝9.51(s,1H),8.58(d,J＝8.4,1H),8.16(s,1H),8.03(s,1H),7.98–7.90(m,1H),7.62(t,J＝7.3,1H),7.54(d,J＝10.5,1H),7.25(d,J＝8.8,2H),6.76(s,1H),6.28(dd,J＝17.0,1.3,1H),6.15(dd,J＝17.0,10.1,1H),5.62(dd,J＝10.2,1.3,1H),4.62–4.47(m,1H),3.68–3.41(m,8H),3.24(ddd,J＝37.5,22.1,8.9,3H),2.86–2.66(m,3H),2.49(t,J＝6.2,2H),2.26(t,J＝11.3,3H),2.16(s,3H),1.76(dt,J＝21.8,11.9,5H),1.34(dd,J＝19.4,6.5,13H).¹³C NMR(101MHz,CDCl₃)δ＝172.78,165.78,157.47,155.35,144.69,138.50,134.63,131.29,130.80,128.02,127.38,126.45,124.93,123.63,123.14,120.82,111.15,105.90,72.02,69.99,69.48,55.48,54.26,53.94,39.35,38.79,37.79,32.58,22.28,18.93,15.37.HRMS(DART-TOF)calculated for C₃₈H₅₃ClN₇O₆S[M+H]⁺m/z 770.3467,found 770.3418.

EXAMPLE 10 Synthesis of Compound ConB-2

1. Synthesis of intermediate ConB-2M

The preparation method of the intermediate is the same as that of ConA-1M, the raw material is tert-butyl 2- (2- (2-aminoethoxy) ethoxy) ethyl carbamate, and other raw materials and treatment methods are the same. The yield was 69%.

¹H NMR(400MHz,CDCl₃)δ＝6.30(dd,J＝17.0,1.3,1H),6.23–6.06(m,1H),5.64(d,J＝10.1,1H),3.70–3.51(m,9H),3.40–3.24(m,2H),1.46(d,J＝6.6,9H).HRMS(DART-TOF)calculated for C₁₄H₂₇N₂O₅[M+H]⁺m/z 303.1920,found 303.1893.

2. Synthesis of target product ConB-2

The preparation method of the target product is the same as that of ConA-1, the raw material is ConB-2M, and other raw materials and treatment methods are the same. The yield was 39%.

¹H NMR(400MHz,CDCl₃)δ＝9.51(s,1H),8.58(d,J＝8.3,1H),8.16(s,1H),8.02(s,1H),7.93(dd,J＝8.0,1.5,1H),7.67–7.59(m,1H),7.53(s,1H),7.31–7.26(m,1H),6.78(s,1H),6.28(dd,J＝17.0,1.6,2H),6.12(dd,J＝17.0,10.2,1H),5.68–5.56(m,1H),4.54(dt,J＝12.1,6.1,1H),3.69–3.41(m,11H),3.25(dq,J＝13.0,6.5,1H),3.14(d,J＝10.9,2H),2.69(dd,J＝26.2,14.6,3H),2.46(t,J＝6.2,2H),2.27–2.12(m,5H),1.78(dd,J＝33.0,11.0,5H),1.44–1.29(m,12H).¹³C NMR(101MHz,CDCl₃)δ＝172.45,165.61,157.48,155.36,144.69,138.51,134.62,131.29,130.86,127.90,127.28,126.38,124.94,123.64,123.12,120.77,111.20,71.87,70.43,70.12,69.86,55.48,54.25,53.97,39.27,38.99,32.62,29.70,22.27,18.94,15.37.HRMS(DART-TOF)calculated for C₄₀H₅₇ClN₇O₇S[M+H]⁺m/z 814.3729,found 814.3760.

EXAMPLE 11 Synthesis of Compound ConB-3

1. Synthesis of intermediate ConB-3M

¹H NMR(400MHz,CDCl₃)δ＝6.35–6.25(m,1H),6.24–6.08(m,1H),5.62(d,J＝10.1,1H),3.70–3.59(m,10H),3.58–3.50(m,4H),3.33(t,J＝13.0,2H),1.45(d,J＝8.4,9H).¹³C NMR(101MHz,CDCl₃)δ＝165.69,156.06,130.94,126.22,79.23,70.40,70.37,70.21,70.14,70.13,69.77,40.31,39.25,28.39.HRMS(DART-TOF)calculated for C₁₆H₃₁N₂O₆[M+H]⁺m/z 347.2182,found 347.2176.

2. Synthesis of target product ConB-3

The preparation method of the target product is the same as that of ConA-1, the raw material is ConB-3M, and other raw materials and treatment methods are the same. The yield was 42%.

¹H NMR(400MHz,CDCl₃)δ＝9.51(s,1H),8.59(d,J＝8.3,1H),8.20–8.14(m,1H),8.06–7.97(m,2H),7.93(dd,J＝8.0,1.5,1H),7.68–7.59(m,1H),7.53(s,1H),7.28–7.22(m,1H),6.78(s,1H),6.28(dd,J＝17.0,1.6,1H),6.14(dd,J＝17.0,10.2,1H),5.62(dd,J＝10.1,1.6,1H),4.63–4.48(m,1H),3.72–3.56(m,12H),3.55–3.43(m,4H),3.27(dq,J＝13.7,6.9,1H),3.12(d,J＝11.4,2H),2.78–2.64(m,3H),2.44(t,J＝6.4,2H),2.16(s,4H),1.86–1.65(m,5H),1.42–1.28(m,14H).¹³C NMR(101MHz,CDCl₃)δ＝172.69,165.72,157.49,155.35,144.69,138.51,137.29,134.64,131.28,130.92,127.80,127.23,126.33,124.90,123.62,123.12,120.75,111.11,105.84,71.77,70.43,70.38,70.24,70.10,69.96,55.48,54.31,54.00,39.30,39.01,38.03,32.73,29.69,22.28,18.94,15.37.HRMS(DART-TOF)calculated for C₃₀H₃₈Cl₂N₅O₄S[M+H]⁺m/z 634.2022,found 634.2013.

EXAMPLE 12 Synthesis of Compound ConB-4

The preparation method of the target product is the same as that of ConA-1, and the raw material is ConB-1M and an intermediate 4. Other raw materials and treatment methods are the same. The yield was 33%.

¹H NMR(400MHz,CDCl₃)δ＝9.51(s,1H),8.58(t,J＝6.3,1H),8.16(d,J＝2.6,1H),8.03(d,J＝6.3,1H),7.93(dd,J＝8.0,1.4,1H),7.66–7.59(m,1H),7.54(s,1H),7.46(t,J＝5.9,1H),7.25(d,J＝8.9,2H),6.77(s,1H),6.34–6.26(m,1H),6.23–6.11(m,1H),5.66–5.60(m,1H),4.65–4.49(m,1H),3.69–3.45(m,9H),3.26(dt,J＝13.7,6.8,1H),3.07(s,2H),2.98(d,J＝11.4,2H),2.32(dd,J＝11.7,9.6,2H),2.17(d,J＝13.0,3H),1.84–1.62(m,4H),1.43–1.28(m,12H).¹³C NMR(101MHz,CDCl₃)δ＝171.16,165.63,157.48,155.35,144.69,138.51,137.18,134.64,131.29,130.81,127.86,127.28,126.42,124.92,123.64,123.13,120.80,111.01,105.84,71.81,70.16,69.43,62.11,55.48,55.01,39.36,38.52,37.64,32.94,22.29,18.93,15.37.HRMS(DART-TOF)calculated for C₃₇H₅₁ClN₇O₆S[M+H]⁺m/z 756.3310,found 756.3319.

EXAMPLE 13 Synthesis of Compound ConB-5

The preparation method of the target product is the same as that of ConA-1, and the raw material is ConB-2M and an intermediate 4. Other raw materials and treatment methods are the same. The yield was 37%.

¹H NMR(400MHz,CDCl₃)δ＝9.51(s,1H),8.58(t,J＝6.1,1H),8.16(d,J＝3.0,1H),8.02(s,1H),7.93(dd,J＝8.0,1.5,1H),7.67–7.59(m,1H),7.54(d,J＝6.3,1H),7.30–7.26(m,1H),6.79(s,1H),6.35–6.23(m,2H),6.13(dd,J＝17.0,10.2,1H),5.61(td,J＝9.8,3.4,1H),4.67–4.52(m,1H),3.66–3.48(m,12H),3.32–3.21(m,1H),3.06(s,2H),2.98(d,J＝11.4,2H),2.74–2.61(m,1H),2.31(dd,J＝11.5,9.2,2H),2.16(s,3H),1.82–1.67(m,4H),1.41–1.30(m,12H).¹³C NMR(101MHz,CDCl₃)δ＝170.89,165.58,157.48,155.36,144.69,138.50,137.32,134.63,131.29,130.85,127.83,127.29,126.41,124.92,123.63,123.13,120.79,111.10,105.85,71.82,70.43,70.13,70.09,69.84,62.10,55.48,54.99,39.26,38.80,37.60,32.94,22.30,18.94,15.37.HRMS(DART-TOF)calculated for C₃₇H₅₁ClN₇O₆S[M+H]⁺m/z 756.3310,found 756.3319.

EXAMPLE 14 Synthesis of Compound ConB-6

The preparation method of the target product is the same as that of ConA-1, and the raw material is ConB-3M and an intermediate 4. Other raw materials and treatment methods are the same. The yield was 21%.

¹H NMR(400MHz,CDCl₃)δ＝9.52(s,1H),8.58(t,J＝7.2,1H),8.16(d,J＝2.9,1H),8.02(s,1H),7.93(dd,J＝8.0,1.5,1H),7.67–7.60(m,1H),7.57–7.47(m,2H),7.27–7.23(m,1H),6.79(s,1H),6.28(dt,J＝7.7,2.7,1H),6.19–6.08(m,1H),5.67–5.58(m,1H),4.69–4.52(m,1H),3.68–3.49(m,16H),3.32–3.21(m,1H),3.08(d,J＝17.3,2H),2.98(d,J＝11.4,2H),2.30(dt,J＝11.3,5.7,2H),2.21–2.12(m,3H),1.82–1.65(m,4H),1.42–1.29(m,12H).¹³C NMR(101MHz,CDCl₃)δ＝170.82,165.57,157.48,155.35,144.70,138.50,137.36,134.64,131.29,130.93,127.78,127.23,126.28,124.91,123.63,123.13,120.78,111.07,105.84,71.74,70.56,70.50,70.24,70.19,69.83,62.09,55.48,54.99,39.29,38.82,37.62,32.93,29.69,22.30,18.94,15.37.HRMS(DART-TOF)calculated for C₄₁H₅₉ClN₇O₈S[M+H]⁺m/z 844.3834,found 844.3829.

Test example 1 molecular cell Activity test

The cell lines used in this experiment included: h3122 (human non-small cell lung cancer cell line), H2228 (human non-small cell lung cancer cell line), H1299 (human lung cancer cell line), A549 (human lung cancer cell line), and Hela (human cervical cancer cell line).

Experimental methods

Tumor cells in the logarithmic growth phase were taken, digested with trypsin, centrifuged and resuspended in fresh medium, and the cells were seeded at the appropriate density in 96-well plates at 100. mu.L per well of medium. After inoculation, the 96-well plate is placed in an incubator to continue culturing for 24h, so that the tumor cells adhere to the wall. And (3) culture environment: temperature 37 ℃ and 5% CO₂. When the cells adhere to the wall and reach the proper cell density, the medicine is added, the concentration of the primary screen is set to be 20, 10 and 5 mu M, and the concentration of the fine screen is designed to be 10, 5, 2.5, 1.25, 0.625 and 0.3 mu M. The experiment was stopped after 72h, 20. mu.L of MTT solution (5mg/mL) was added and placed in a cell incubator for 4 h. The well plate was removed, the supernatant was discarded, and 150. mu.L of LDMSO was added to each well to dissolve formazan. After the crystals were sufficiently dissolved, absorbance values (OD values) at 490 and 570nm were measured for each well using a microplate reader, and the inhibition rate of tumor cell proliferation in vitro and the cell survival rate in each experimental group were calculated. According to the formula: relative cell proliferation inhibition (%) (blank control-experimental group)/blank control group × 100%. Each set was provided with 3 parallel replicates and each experiment was repeated three times. Half maximal Inhibitory Concentration (IC) on different cells₅₀) See table 1, data units in table 1 are μ M.

TABLE 1 (IC)₅₀μM)

As can be seen from the table, the cell activity of conB-1 is improved by nearly 10 times compared with the activity of LDK378, and the molecule activity after covalent modification is far superior to that of the prototype drug, thus demonstrating the effectiveness of the covalent molecule design method.

Test example 2 kinase Activity test for ALK

Preparation of compound concentration gradient: test compounds were tested at 200nM and 20nM concentrations and diluted in 96-well plates to 100-fold final concentration in 100% DMSO, then each concentration of compound was further diluted with 1 x Kinase buffer to 5-fold final concentration of intermediate diluted solutions. Compound ConB-1 was tested at 200nM starting concentration, 5-fold diluted 10 concentrations, single well assay, again diluted in 96 well plates to 100-fold final 10 concentration gradient in 100% DMSO, then further diluted with 1 x kinese buffer at each concentration to 5-fold final intermediate dilution.

Respectively adding 5 mu L of the prepared compound solution into compound holes of a 384-hole plate, and testing each concentration single hole; mu.L of 5% DMSO was added to each of the negative and positive control wells.

A2.5 fold final concentration of Kinase solution was prepared using a 1 XKinase buffer.

Add 10. mu.L of 2.5 fold final concentration kinase solution to the compound well and positive control well, respectively; mu.L of 1 XKinase buffer was added to the negative control wells.

The mixture was centrifuged at 1000rpm for 30 seconds, shaken and mixed, and then incubated at room temperature for 10 minutes.

A2.5 fold final ATP and Kinase substrate22 mixture was made up using 1 XKinase buffer.

The reaction was initiated by adding 10. mu.L of a 2.5 fold final ATP and substrate mixture.

The 384 well plates were centrifuged at 1000rpm for 30 seconds, vortexed and incubated at 28 ℃ for 25 minutes.

Adding 25 μ L of termination detection solution to stop kinase reaction, centrifuging at 1000rpm for 30 s, shaking and mixing

The conversion was read using Caliper EZ Reader ii.

And (3) data analysis: the calculation formula of% Inhibition ═ 100%

Wherein: inhibition, Conversion% _ sample is the Conversion reading for the sample; conversion% _ min: negative control well mean, representing conversion readings without enzyme live wells; conversion% _ max: positive control wells are averaged for conversion readings in wells without compound inhibition.

The results of kinase activity on ALK for each compound are shown in table 2.

TABLE 2

IC for detecting ALK by ConB-1₅₀The results show that the IC50 for ConB-1 versus ALK is 0.5273nM, and the specific curves are shown in FIG. 4. IC of LDK378 on ALK₅₀At 0.2nM, the covalent binding mode had little effect on kinase activity.

Test example 3 inhibition test for LO2 cells

The experimental method is the same as MTT method. The inhibition rate of compound molecules on LO2 cells at 5 μ M concentration is shown in table 3.

TABLE 3

LO2 is human normal liver cells, and this experiment proves that the covalent molecules do not increase the toxicity of compound molecules to normal cells, and on the contrary, most molecules have less toxicity than positive molecules, thus showing better safety.

Test example 4 Effect of covalent binding on molecular Activity

In order to prove the key role of covalent bond formation in the molecular activity of the compound, an acrylamide group in the molecule ConB-1 is replaced by propionamide, and the change of the molecular biological activity is observed, wherein the Reduced ConB-1 has the following molecular structural formula.

Synthesis of Reduced ConB-1 molecules

Propionic acid is used as a raw material, and other synthesis methods are the same as those of ConB-1, so that light yellow powder is obtained. The yield was about 30%.

¹H NMR(400MHz,CDCl₃)δ＝9.52(s,1H),8.57(d,J＝8.3,1H),8.16(s,1H),8.03(s,1H),7.93(dd,J＝7.9,1.4,1H),7.87–7.77(m,1H),7.67–7.59(m,1H),7.55(s,1H),7.24(s,1H),6.76(s,1H),6.22(d,J＝12.6,1H),4.54(dt,J＝12.1,6.1,1H),3.49(ddt,J＝16.2,10.5,5.2,8H),3.38–3.21(m,3H),3.00(t,J＝6.1,3H),2.63(t,J＝6.4,2H),2.50(t,J＝9.6,2H),2.35(dd,J＝15.1,7.6,1H),2.28–2.19(m,2H),2.16(s,3H),1.86(d,J＝22.6,5H),1.34(dd,J＝17.6,6.5,14H),1.13(t,J＝5.6,4H).HRMS(DART-TOF)calculated for C₃₈H₅₅ClN₇O₆S[M+H]⁺m/z 772.3623,found 772.3621.

The in vitro antiproliferative activity of the molecules on H3122 and H2228 cell lines was determined by the MTT method. The results are shown in Table 4.

TABLE 4IC₅₀(μM)

Numbering	H3122	H2228
			ConB-1	0.15	0.31
Reduced ConB-1	8.1	12.3
			LDK378	1.1	1.3

According to the inhibitory activity of the molecules on H3122 cell lines and H2228 cell lines, it was found that the molecular activity was reduced by 40 to 50 times after the reduction of the double bond of the acrylamide group in ConB-1, and was also reduced by 7 times or more as compared with LDK 378. After replacement of the acrylamide group by propionamide, little change in the chain length portion occurs. Theoretically, the binding pattern of the molecule to the binding site of the target protein is not altered, in part, by the inability of Cys1259 to form a covalent bond with the molecule. The key role of covalent bonding in molecular activity is fully illustrated by the large difference in molecular activity before and after double bond reduction.

Test example 5 in vivo antitumor Activity study of ConB-1 molecule

Based on the good in vitro anti-tumor activity and bioavailability of the ConB-1 molecule, the ConB-1 molecule is used for in vivo anti-tumor activity research, the selected cell strain is H3122, and the experimental animal is a Balb/c nude mouse. Experimental setup 2 groups: control and administration groups (ConB-110 mg/kg,20mg/kg,40mg/kg,100 mg/kg; positive control LDK 37820 mg/kg), 7 of which were administered per group.

The specific method comprises the following steps:

1) after H3122 tumor cells in logarithmic growth phase are digested by pancreatin, cells are collected by centrifugation; cells were washed 3 times with double-null (no antibiotics and serum) medium; adding a proper amount of double-culture-free basis suspension tumor cells, and carrying out density measurement on the tumor cells; according to the experimental requirements, the cell density is adjusted to 5X 10⁶one/mL.

2) The resuspended cells were 0.5X 10 cells per mouse⁶Individual cells (0.1mL) the cells were inoculated subcutaneously into the right axilla of mice (6-8 weeks old, 18-22g in weight).

3) About 10 days after inoculation, when the tumor at the inoculation part of the mice becomes tumor and the volume is 150-. Experimental setup 2 groups: control and administration groups (ConB-110 mg/kg,20mg/kg,40mg/kg,100 mg/kg; positive control LDK 37820 mg/kg), 7 of which were administered per group. The drug solvent is a mixed solvent of PEG300 and NMP 9, and each nude mouse is orally administered with 0.2mL per day on average.

And (3) recording experimental data: tumor volume and mouse body weight were measured every 3 days, including tumor major axis and minor axis perpendicular to major axis, in units: millimeters (mm); mouse body weight in grams (g). During the experiment, the health conditions of the mice, such as the animal activity, water intake and food intake, the glossiness and color of the hair of the mice, diarrhea, inflammation of tumor parts and the like, need to be observed.

Tumor volume (mm3) is tumor long diameter x short diameter/2

The tumor inhibition rate (%) is (mean tumor volume in control group-mean tumor volume in experimental group)/mean tumor volume in control group x 100%.

The experiment was carried out for 15 days.

The test results are shown in fig. 5 and 6. Wherein, FIG. 5 shows the change of tumor volume with the time of administration, and FIG. 6 shows the change of mouse body weight with the time of administration.

The health of the mice during the experiment was good: normal activity, normal water intake and food intake, normal skin glossiness and color of the mice, no diarrhea phenomenon and no inflammation at tumor parts. From the administration results, the compound ConB-1 can inhibit the tumor tissue growth compared with the control group, and the tumor inhibition rate is 55.5%, 63.7%, 68.7% and 78.1% at the administration dosage of 10mg/kg,20mg/kg,40mg/kg and 100 mg/kg; compared with the positive drug LDK378, the tumor inhibition rate of the positive drug LDK378 is 49.5% when the administration dose is 20 mg/kg. The in vivo antitumor activity experiment proves the superiority of the molecules, and the antitumor effect superior to that of the positive medicament can be still achieved under the condition of low administration dosage. The body weight of the mice slightly decreases in the early period of administration and gradually recovers in the later period.

In conclusion, the compound molecules of the invention can be used as ALK inhibitors, and the research shows that the binding mode of the molecules is different from that of positive drugs, the molecules are covalently bound, and the molecules are targeted to cysteine residues outside an active pocket. The newly designed compound molecules ConA-3, ConA-4, ConB-1 and ConB-3 have better cell activity than that of the positive drug LDK378, wherein the in vivo drug effect of the molecule ConB-1 is better than that of the positive drug LDK378 through in vivo anti-tumor activity research.

Claims

1. A compound having the structural formula I:

wherein X is a linker moiety and Y is a covalent reaction target; the compound can be covalently bonded with cysteine residue Cys1259 at the periphery of an ALK active pocket through a covalent reaction target head; and the structural formula is shown as formula II:

r is

2. The compound of claim 1, having the formula ConA-3, ConA-4, ConB-1 or ConB-3:

3. use of a compound according to claim 1 or 2 for the preparation of an ALK inhibitor.

4. Use of a compound according to claim 3 for the preparation of an ALK inhibitor, characterized in that: the ALK inhibitors are covalent inhibitors.

5. A pharmaceutical composition for treating tumors, characterized by: comprising a therapeutically effective amount of a compound of claim 1 or 2 and a pharmaceutically acceptable carrier.

6. Use of a compound according to claim 1 or 2 for the preparation of a medicament for the treatment of tumours.

7. The use of a compound according to claim 6 for the preparation of a medicament for the treatment of tumors, wherein: the tumor is liver cancer, lung cancer or cervical cancer.