Compounds for promoting tubulin degradation and application thereof

Abstract

The invention provides a compound for promoting tubulin degradation and application thereof, and belongs to the technical field of chemical drugs. The compound provided by the invention is a compound as shown in a formula I, and a salt, a stereoisomer, a crystal form, a hydrate and a solvate thereof. The invention provides a new anti-tumor drug which has an excellent anti-tumor effect, can effectively promote tubulin degradation, can overcome the drug resistance of tumors, is used for treating drug-resistant tumors, and has a good application prospect. # imgabs0 #

Description

Technical Field

[0001] The invention belongs to the technical field of chemical medicines, and in particular relates to a compound for promoting tubulin degradation and uses thereof. Background Art

[0002] Malignant tumors are one of the major diseases that currently endanger human health. In recent years, the incidence and mortality rates of most malignant tumors in the world have been on the rise. The latest global cancer research data for 2020 released by the World Health Organization's International Agency for Research on Cancer (IARC) show that the number of malignant tumor cases in the world is about 19.3 million (4.57 million in China), and the number of deaths is about 9.96 million (3 million in China); among urban residents, cancer ranks first among all causes of death. Whether it is the number of new cancer cases or the number of deaths, China ranks first in the world, and the incidence rate shows a clear trend of younger age. Therefore, it is of great social significance to find low-toxic and highly efficient active molecules for the treatment of malignant tumors.

[0003] Microtubules are important components of the cytoskeleton, mainly composed of α-tubulin and β-tubulin heterodimers, and are present in all eukaryotic cells. They are highly dynamic polymers, and their polymerization dynamics are strictly regulated in space and time. During mitosis, microtubules pull daughter chromosomes to the two poles in the form of spindles, and their morphology changes continuously with the progress of mitosis to ensure the smooth progress of mitosis. At the same time, microtubules play an important role in a series of processes such as cell movement regulation, cell signaling, cell morphology formation and maintenance, secretion, and intracellular transport. Since tumor cells have the characteristics of rapid division, anticancer drugs targeting microtubules can disrupt the normal polymerization or depolymerization of microtubules in tumor cells, prevent tumor cell mitosis, and cause them to stagnate in the G2 / M phase, thereby inducing tumor cell apoptosis. At the same time, this type of compound can also inhibit the function of microtubules, limit the proliferation, migration and angiogenesis of endothelial cells, and inhibit tumor angiogenesis.

[0004] Although classic anti-microtubule chemotherapy drugs such as vinorelbine, paclitaxel, and gemcitabine have been successfully used in clinical practice for about 30 years, their structural biology research is relatively lagging behind. It was not until 1998 that the first paclitaxel-tubulin crystal complex structure was obtained, which explained its binding mode and mechanism of action with tubulin at the molecular level. With the development of structural biology, seven different microtubule targeting agent binding sites have been discovered one by one: paclitaxel site, vincristine site, colchicine site, maytansine site, laulimalide / peloruside site, pironetin site, and cevipabulin site. The first five sites are located on β microtubules (paclitaxel site, vincristine site, colchicine site, maytansine site, and laulimalide / peloruside site), and the last two sites (pironetin site and cevipabulin site) are located on α microtubules. Microtubule targeting agents can be divided into three categories according to their different mechanisms of action: (1) Tubulin polymerizers that promote tubulin polymerization (inhibitors acting on the paclitaxel site and laulimalide / peloruside site); (2) Tubulin depolymerizers that inhibit tubulin polymerization (inhibitors acting on the vincristine site, maytansine site, pironetin site and most inhibitors acting on the colchicine site); (3) Tubulin degraders that promote tubulin degradation (a few covalent / non-covalent inhibitors acting on the colchicine site and inhibitors acting on the cevipabulin site). In recent years, several small molecule microtubule inhibitors acting on different binding sites have been approved by the US FDA for the treatment of malignant tumors, such as Ixabepilone (paclitaxel site), Cabazitaxel (paclitaxel site) and Eribulin mesylate (vincristine site). At the same time, the ClinicalTrials.gov website shows that more than a thousand microtubule-related clinical trials are underway. This proves that microtubules are an important target for anti-tumor drug research.

[0005] However, while existing anti-microtubule drugs have therapeutic effects, they also have significant toxic side effects on normal tissues and the nervous system, and are very likely to induce drug resistance in tumors. The main problems include: 1) chemotherapy resistance caused by overexpression of βⅢ tubulin and isomerism or mutation of amino acids in the binding site; 2) multidrug resistance caused by P-glycoprotein pumping substrate molecules (anti-tumor drugs) out of cells; 3) severe dose-related toxicity in peripheral neuritis, nerves, and blood. Among them, the drug resistance of tumors is the most serious problem, which determines the scope of use and clinical effectiveness of chemotherapy drugs. Many naturally derived chemotherapy drugs, such as alkaloid anticancer drugs (colchicine, vinblastine, harringtonine, and paclitaxel, etc.) and anthracycline anticancer antibiotics (doxorubicin and daunorubicin, etc.), are very likely to develop drug resistance. The mechanism of tumor resistance is quite complex and is the result of the combined action of multiple factors. Drug resistance caused by overexpression of βⅢ tubulin has long been confirmed in many clinical studies. Multiple subtypes of β-tubulin (including βI, βIIa, βIIb, βIII, βIVa, βIVb, βV, and βVI) are highly expressed in many types of cancer, such as lung cancer, breast cancer, prostate cancer, gastric cancer, and melanoma. In addition, overexpression of β-tubulin in different cancers is closely related to disease progression, overall survival, and resistance to chemotherapeutic drugs. Many clinical studies have found that overexpression of βIII tubulin in patient tumor tissues can lead to significant resistance to chemotherapy, and βIII tubulin overexpression has become a biomarker for clinical chemotherapy resistance. In-depth mechanistic studies have found that the mechanism by which βIII tubulin overexpression leads to chemotherapy resistance is that the amino acid at position 218 of βIII tubulin is alanine, while the amino acid at position 218 of other subtypes of β-tubulin is threonine. The change in this site significantly leads to a decrease in the affinity of tubulin to chemotherapeutic drugs, thereby leading to resistance. It has been more than 20 years since the first discovery that βIII tubulin overexpression is associated with clinical chemotherapy resistance. Although many studies have been devoted to directly inhibiting the expression of βIII tubulin or inhibiting its activity to overcome drug resistance, the results have been minimal, and the existence of drug resistance is still a major problem in clinical treatment. Therefore, it is of great clinical significance to develop anti-tumor microtubule drugs that are less toxic, more efficient, more economical, more targeted, and can overcome drug resistance. Summary of the invention

[0006] The purpose of the present invention is to provide a class of compounds for promoting tubulin degradation and uses thereof.

[0007] The present invention provides a compound represented by formula I, a salt thereof, a stereoisomer thereof, a crystal form thereof, a hydrate thereof, and a solvate thereof:

[0008]

[0009] in,

[0010] X is selected from CR5, N;

[0011] R5 is selected from hydrogen, C1-C6 alkyl;

[0012] R1 is selected from a 6- to 15-membered aryl group substituted by n R6 groups, and a 5- to 10-membered heteroaryl group substituted by n R6 groups;

[0013] n is an integer from 0 to 5;

[0014] Each R6 is independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, cyano, 6-15 membered aryl substituted by m R7, -C(O)R8, -NR9C(O)R8, -NR9R 10 、-O(CH2) a R 11 ;

[0015] m is an integer from 0 to 5;

[0016] Each R7 is independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, and cyano;

[0017] R8, R9, R 10 are independently selected from hydrogen, C1-C6 alkyl;

[0018] a is an integer from 0 to 10;

[0019] R 11 Selected from hydrogen, o R 12 Substituted 6- to 15-membered aryl;

[0020] o is an integer from 0 to 5;

[0021] Each R 12 Each independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, and cyano;

[0022] R2 is selected from hydrogen, -NR 13 R 14 ;

[0023] R 13 , R 14 are independently selected from hydrogen, C1-C6 alkyl;

[0024] R3 is selected from hydrogen, C1-C6 alkyl, C1-C6 alkenyl;

[0025] R4 is selected from (CH2) b R 15 ;

[0026] b is an integer from 1 to 10;

[0027] R 15 Selected from p R16 substituted 6- to 15-membered aryl, substituted by p R 16 Substituted 5- to 10-membered heteroaryl;

[0028] p is an integer from 0 to 5;

[0029] Each R 16 Each independently selected from C1-C6 alkyl, halogen, -OR 17 ;

[0030] R 17 Selected from C1-C6 alkyl, trifluoromethyl;

[0031] Or R3 and R4 are connected to form q R 18 Substituted 6- to 15-membered aromatic group, q R 18 Substituted 5- to 10-membered heteroaryl;

[0032] q is an integer from 0 to 5;

[0033] Each R 18 Each independently selected from C1-C6 alkyl, C1-C6 alkoxy, and halogen;

[0034] The compound is not

[0035] Furthermore, the compound is represented by Formula II:

[0036]

[0037] in,

[0038] X is selected from CR5, N;

[0039] R5 is selected from hydrogen, C1-C6 alkyl;

[0040] R1 is selected from a 6- to 15-membered aryl group substituted by n R6 groups, and a 5- to 10-membered heteroaryl group substituted by n R6 groups;

[0041] n is an integer from 0 to 5;

[0042] Each R6 is independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, cyano, 6-15 membered aryl substituted by m R7, -C(O)R8, -NR9C(O)R8, -NR9R 10 、-O(CH2) a R 11 ;

[0043] m is an integer from 0 to 5;

[0044] Each R7 is independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, and cyano;

[0045] R8, R9, R 10 are independently selected from hydrogen, C1-C6 alkyl;

[0046] a is an integer from 0 to 10;

[0047] R 11 Selected from hydrogen, o R 12 Substituted 6- to 15-membered aryl;

[0048] o is an integer from 0 to 5;

[0049] Each R 12 Each independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, and cyano;

[0050] R2 is selected from hydrogen, -NR 13 R 14 ;

[0051] R 13 , R 14 are independently selected from hydrogen, C1-C6 alkyl;

[0052] R3 is selected from hydrogen, C1-C6 alkyl, C1-C6 alkenyl;

[0053] The compound is not

[0054] Further,

[0055] X is selected from CH, N;

[0056] R1 is selected from the following groups substituted by n R6: phenyl, naphthyl, pyridyl, furyl,

[0057] n is 0, 1, 2, 3, 4;

[0058] Each R6 is independently selected from C1-C4 alkyl, C1-C4 alkoxy, halogen, cyano, phenyl substituted by m R7, -C(O)R8, -NR9C(O)R8, -NR9R10, -O(CH2)aR11;

[0059] m is 0, 1, 2, 3, or 4;

[0060] Each R7 is independently selected from C1-C4 alkyl, C1-C4 alkoxy, halogen, and cyano;

[0061] R8, R9, R 10 are independently selected from hydrogen, C1-C4 alkyl;

[0062] a is 0, 1, 2, 3, 4;

[0063] R 11 is selected from hydrogen, phenyl;

[0064] R2 is selected from hydrogen, -N(H)R13;

[0065] R 13 Selected from hydrogen, C1-C4 alkyl;

[0066] R3 is selected from hydrogen, C1-C3 alkyl, and C1-C3 alkenyl.

[0067] Furthermore, the compound is represented by formula III:

[0068]

[0069] in,

[0070] R1 is selected from the following groups substituted by n R6: phenyl, naphthyl, pyridyl, furyl,

[0071] n is 0, 1, 2, 3, 4;

[0072] Each R6 is independently selected from C1-C4 alkyl, C1-C4 alkoxy, halogen, cyano, phenyl substituted by m R7, -C(O)R8, -NR9C(O)R8, -NR9R10, -O(CH2) a R 11 ;

[0073] m is 0, 1, 2, 3, or 4;

[0074] Each R7 is independently selected from C1-C4 alkyl, C1-C4 alkoxy, halogen, and cyano;

[0075] R8, R9, and R10 are independently selected from hydrogen and C1-C4 alkyl;

[0076] a is 0, 1, 2, 3, 4;

[0077] R 11 is selected from hydrogen, phenyl;

[0078] The compound is not

[0079] Furthermore, the compound is represented by formula IV:

[0080]

[0081] in,

[0082] R 15 Selected from p R 16substituted 6- to 15-membered aryl, substituted by p R 16 Substituted 5- to 10-membered heteroaryl;

[0083] p is an integer from 0 to 5;

[0084] Each R 16 Each independently selected from C1-C6 alkyl, halogen, -OR 17 ;

[0085] R 17 Selected from C1-C6 alkyl, trifluoromethyl;

[0086] The compound is not

[0087] Further,

[0088] R 15 Selected from p R 16 Substituted with the following groups: phenyl, naphthyl, pyridyl, furyl,

[0089] p is 0, 1, 2, 3, 4;

[0090] Each R 16 Each independently selected from C1-C4 alkyl, halogen, -OR 17 ;

[0091] R 17 Selected from C1-C4 alkyl, trifluoromethyl.

[0092] Furthermore, the compound is represented by Formula V:

[0093]

[0094] in,

[0095] R3 is selected from C1~C3 alkyl and C1~C3 alkenyl.

[0096] Furthermore, the compound is represented by Formula VI:

[0097]

[0098] in,

[0099] R1 is selected from the following groups substituted by n R6: phenyl, naphthyl, pyridyl, furyl,

[0100] n is 0, 1, 2, 3, 4;

[0101] Each R6 is independently selected from C1-C4 alkyl, C1-C4 alkoxy, halogen, and cyano;

[0102] Alternatively, the compound is represented by formula VII:

[0103]

[0104] in,

[0105] R3 is selected from hydrogen;

[0106] R4 is selected from (CH2) b R 15 ;

[0107] b is 1;

[0108] R 15 Selected from

[0109] Or R3 and R4 are connected to form

[0110] Furthermore, the compound is one of the following structures:

[0111]

[0112]

[0113]

[0114]

[0115] The present invention also provides the use of the aforementioned compound, its salt, its stereoisomer, its crystal form, its hydrate, and its solvate in the preparation of a microtubule protein degrading agent.

[0116] The present invention also provides the use of the aforementioned compound, its salt, its stereoisomer, its crystal form, its hydrate, and its solvate in the preparation of a drug for preventing and / or treating cancer.

[0117] Furthermore, the cancer is cervical cancer, colorectal cancer, breast cancer, leukemia, lung cancer, cervical cancer, lymphoma, or ovarian cancer.

[0118] The present invention also provides the use of the aforementioned compound, its salt, its stereoisomer, its crystal form, its hydrate, and its solvate in the preparation of ADC antibody-drug conjugates.

[0119] Preferably, the aforementioned compound, its salt, its stereoisomer, its crystal form, its hydrate, or its solvate is a small molecule toxic compound in an ADC antibody-drug conjugate.

[0120] The present invention also provides a medicine, which is a preparation prepared with the aforementioned compound, its salt, its stereoisomer, its crystal form, its hydrate, or its solvate as an active ingredient and pharmaceutically acceptable excipients.

[0121] The present invention also provides an ADC antibody-drug conjugate, which is prepared using the aforementioned compound, its salt, its stereoisomer, its crystal form, its hydrate, or its solvate as a small molecule toxic compound.

[0122] The compounds and derivatives provided in the present invention can be named according to the IUPAC (International Union of Pure and Applied Chemistry) or CAS (Chemical Abstracts Service, Columbus, OH) nomenclature system.

[0123] Definitions of terms used in the present invention: Unless otherwise stated, the initial definitions provided for groups or terms in this document apply to the groups or terms throughout the specification; for terms that are not specifically defined in this document, the meaning that a person skilled in the art can give them should be given based on the disclosure and context.

[0124] "Substitution" refers to the replacement of a hydrogen atom in a molecule by another different atom or molecule.

[0125] The minimum and maximum carbon atom content in a hydrocarbon group is indicated by a prefix, for example, the prefix C a ~C b Alkyl refers to any alkyl group containing from "a" to "b" carbon atoms. Thus, for example, "C1-C6 alkyl" refers to an alkyl group containing 1 to 6 carbon atoms; "C1-C6 alkoxy" refers to an alkoxy group containing 1 to 6 carbon atoms.

[0126] "Alkyl" refers to a saturated hydrocarbon chain having a specified number of carbon atoms. For example, C1-C6 alkyl refers to an alkyl group having 1 to 6 carbon atoms, i.e., 1, 2, 3, 4, 5 or 6 carbon atoms. Alkyl groups can be straight or branched. Representative branched alkyl groups have one, two or three branches. Alkyl groups include methyl, ethyl, propyl (n-propyl and isopropyl), butyl (n-butyl, isobutyl and tert-butyl), pentyl (n-pentyl, isopentyl and neopentyl) and hexyl, etc.

[0127] "Halogen" is fluorine, chlorine, bromine or iodine.

[0128] "Cycloalkyl" refers to a saturated or unsaturated all-carbon monocyclic or polycyclic ring (including fused rings, spiro rings or bridged rings) without a conjugated π electron system, such as but not limited to: The 3- to 12-membered cycloalkyl group refers to a cycloalkyl group which has 3 to 12 carbon atoms and forms a closed structure.

[0129] "Heterocycloalkyl" means that at least one carbon atom in the cycloalkyl ring is replaced by a heteroatom, and the heteroatom is O, N or S, such as but not limited to: The 3- to 12-membered heterocycloalkyl group refers to a heterocycloalkyl group which has 3 to 12 atoms and forms a closed structure.

[0130] "Aryl" refers to an all-carbon monocyclic or polycyclic ring (including fused rings, spirocyclic rings or bridged rings) with a conjugated π electron system, such as but not limited to phenyl, naphthyl, phenanthrenyl, anthracenyl, fluorenyl and indenyl. The aromatic ring may be fused to other cyclic groups (including saturated and unsaturated rings), but cannot contain heteroatoms such as O, N or S, and the point of attachment to the parent must be on a carbon atom on the ring with a conjugated π electron system, such as but not limited to The 6- to 12-membered aryl group refers to an aryl group having 6 to 12 carbon atoms to form a closed structure.

[0131] "Heteroaryl" refers to an aromatic group in which at least one carbon atom in the conjugated π-electron system is replaced by a heteroatom, wherein the heteroatom is O, N or S, for example, including but not limited to:

[0132]

[0133] The 5- to 12-membered heteroaryl group refers to a heteroaryl group which has 5 to 12 atoms to form a closed structure.

[0134] Compared with the prior art, the present invention has the following beneficial effects:

[0135] The present invention provides a new class of anti-tumor drugs, which have excellent anti-tumor effects, can effectively promote the degradation of tubulin, can overcome the drug resistance of tumors, are used for the treatment of drug-resistant tumors, and have good application prospects.

[0136] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0137] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0138] Figure 1 This is the design idea of ​​compound 8 and the structures of compounds 10, 13, 18-21.

[0139] Figure 2 The results showed that the compound promoted the degradation of tubulin in a concentration-dependent manner.

[0140] Figure 3 The antitumor activity of compound 21 and paclitaxel in the A2780T xenograft model: Dosing cycle: 21 once every two days, paclitaxel once a week; Dosing method: 21 tail vein injection, paclitaxel intraperitoneal injection; A is the effect of compound 21 on inhibiting tumor growth in the A2780T xenograft model; Tumor volume was measured once every two days, and the volume size was calculated as follows: V (mm 3 )=(π / 6)×length×width×width; B is the effect of compound 21 on the body weight of mice, and the body weight of mice was measured every two days; C is the effect of compound 21 on tumor weight; D is the macroscopic effect of compound 21 on tumor volume. DETAILED DESCRIPTION

[0141] The raw materials and equipment used in the specific embodiments of the present invention are all known products and are obtained by purchasing commercially available products.

[0142] Preparation of the compounds of the present invention:

[0143] (1) Compounds 2a-y and 3a-h were synthesized by replacing the 3-methoxybenzene ring with different substituted benzene rings and aromatic rings. The synthetic route is shown in Scheme 1. Commercially available 2-amino-4,6-dichloropyrimidine was reacted with 3,4-methylenedioxybenzylamine to obtain intermediate 1, which was then subjected to Suzuki reaction with different boronic acids to obtain target compounds 2a-y and 3a-h.

[0144] (2) Different aromatic benzylamines are used to replace 3,4-methylenedioxybenzylamine and react with 2-amino-4,6-dichloropyrimidine to obtain intermediates 4a-j. Intermediates 4a-j are then subjected to Suzuki reaction with 3-methoxyphenylboronic acid to obtain target compounds 5a-j. The synthetic route is shown in Scheme 2.

[0145] (3) BML284 was prepared according to the synthetic route 3 and then reacted with a halide to produce compounds 6a-d.

[0146] (4) Compound 8 was prepared by the method shown in Synthesis Scheme 4 and was used to investigate the effect of the linker cyclization of 5e on its activity.

[0147] (5) Compound 10 was prepared by the method shown in Synthesis Scheme 5 to investigate the effect of the amino group on the activity.

[0148] (6) Compound 13 was prepared by the method shown in Synthesis Scheme 6 to investigate the effect of the nitrogen atom at position 1 on the activity.

[0149] (7) Compound 15 was prepared by the method shown in Synthesis Scheme 7.

[0150] (8) Compounds 18 and 19 were prepared by the method shown in Synthesis Scheme 8.

[0151] Example 1. Synthesis Route 1: Synthesis of Compounds 2a-y, 3a-h

[0152]

[0153] Reagents and conditions: a) 3,4-methylenedioxybenzylamine, K2CO3, DMF, 80°C; b) substituted boronic acid, Pd(dppf)Cl2, K2CO3, 1,4-dioxane: water = 4:1 (volume ratio), reflux.

[0154] (a) Synthesis of intermediate compound 1

[0155] To a 25 mL round-bottom flask containing 164 mg (1.0 mmol, 1.0 eq.) of 4,6-dichloro-2-aminopyrimidine, 207 mg (1.5 mmol, 1.5 eq.) of K2CO3 was added, and then 8 mL of DMF was added. The mixture was stirred at room temperature until the compound was completely dissolved. Then, 166 mg (1.1 mmol, 1.1 eq.) of 3,4-methylenedioxybenzylamine was added in batches. The reaction was continued by stirring at 80°C and monitored by TLC. After the reaction of 4,6-dichloro-2-aminopyrimidine was completed, the reaction solution was poured into 40 mL of water and extracted three times with 10 mL of ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, mixed, and rapidly separated and purified to obtain compound 1.

[0156] N 4 -(Benzo[d][1,3]dioxolan-5-ylmethyl)-6-chloro-pyrimidine-2,4-diamine (1):

[0157]

[0158] 1 H NMR (400MHz, DMSO-d6) δ7.53(t,J=6.0Hz,1H),6.90–6.82(m,2H),6.77(dd,J=7.9,1.7Hz,1H),6.44(s,2H),5.98(s,2H),5.76(s,1H),4.36(s,2H).

[0159] (b) Synthesis of target compounds 2a-y, 3a-h

[0160] Into a 50 mL round-bottom flask were added 278 mg (1.0 mmol, 1.0 eq.) of intermediate 1, 1.2 mmol of substituted boronic acid, 207 mg (1.5 mmol, 1.5 eq.) of K2CO3, 30 mg (0.04 mmol, 0.04 eq.) of Pd(dppf)Cl2, and 20 mL of a mixed solution of 1,4-dioxane / water (4:1, v / v). The mixture was stirred at reflux for 2 h under nitrogen protection. After the reaction was completed, the mixture was cooled and filtered through diatomaceous earth. The filter cake was washed three times with 10 mL of ethyl acetate. The filtrate was evaporated and concentrated, the sample was mixed, and the target compounds 2a-y and 3a-h were obtained.

[0161] The structural formula and characterization data of target compounds 2a-y and 3a-h are as follows:

[0162] 1-(3-(2-amino-6-((benzo[d][1,3]dioxolan-5-ylmethyl)amino)pyrimidin-4-yl)phenyl)ethyl-1-one (2a)

[0163]

[0164] 1 H NMR (400MHz, DMSO-d6) δ8.46(t,J=1.8Hz,1H),8.13(dt,J=7.8,1.5Hz,1H),8.02(dt,J=7.7,1.5Hz,1H),7.60(t,J=7.7Hz,1H),7 .39(s,1H),6.93(d,J=1.5Hz,1H),6.89–6.80(m,2H),6.36(s,1H),6.13(s,2H),5.97(s,2H),4.44(d,J=6.0Hz,2H),2.63(s,3H). 13 C NMR(101MHz,DMSO-d6)δ198.30,164.51,164.03,147.68,146.44,139.20,137.45,134.45,1 31.19,129.80,129.37,126.12,120.92,108.48,101.23,27.30,27.28.HRMS-ESI:Calcd.For C 20 H 19 N4O3[M+H + ]363.1458,found:363.1453.

[0165] N 4 -(Benzo[d][1,3]dioxolan-5-ylmethyl)-6-(3-isopropylphenyl)pyrimidine-2,4-diamine (2b)

[0166]

[0167] 1 H NMR(400MHz, DMSO-d6)δ7.77(d,J=1.9Hz,1H),7.67(dt,J=7.5,1.5Hz,1H),7.40–7.27(m,3H),6.92(s,1H),6.88–6.79 (m,2H),6.26(s,1H),6.02(s,2H),5.97(s,2H),4.42(d,J=6.0Hz,2H),2.93(hept,J=7.0Hz,1H),1.23(d,J=6.9Hz,6H). 13 C NMR(101MHz,DMSO-d6)δ164.45,163.92,148.87,147.68,146.42,138.86,134.57,128.8 2,127.85,124.69,124.32,120.88,108.46,101.22,34.01,24.39.HRMS-ESI:Calcd.For C 21 H 23 N4O2[M+H + ]363.1822,found:363.1821.

[0168] N-(3-(2-amino-6-((benzo[d][1,3]dioxolan-5-ylmethyl)amino)pyrimidin-4-yl)phenyl)acetamide (2c)

[0169]

[0170] 1 H NMR(400MHz,DMSO-d6)δ10.03(s,1H),8.10(s,1H),7.67–7.60(m,1H),7.57–7.49(m,1H),7.43–7.30(m,2H) ,6.91(d,J=1.6Hz,1H),6.89–6.78(m,2H),6.21(s,1H),6.06–5.94(m,4H),4.46–4.38(m,2H),2.05(s,3H). 13C NMR(101MHz,DMSO-d6)δ168.83,164.42,163.90,147.68,146.42,139.89,139.43,134.5 3,129.12,121.51,120.88,120.51,117.58,108.48,101.22,24.48.HRMS-ESI:Calcd.For C 20 H 20 N5O3[M+H + ]378.1567,found:378.1565.

[0171] 3-(2-Amino-6-((benzo[d][1,3]dioxolan-5-ylmethyl)amino)pyrimidin-4-yl)benzonitrile (2d)

[0172]

[0173] 1 H NMR (400MHz, DMSO-d6) δ8.34(s,1H),8.21(d,J=8.0Hz,1H),7.89(d,J=7.7Hz,1H),7.67(t,J=7.8Hz,1H),7.45( d,J=7.6Hz,1H),6.94(s,1H),6.91–6.80(m,2H),6.38(s,1H),6.19(s,2H),5.98(s,2H),4.46(d,J=6.0Hz,2H). 13 C NMR(101MHz,DMSO-d6)δ163.99,147.67,139.89,134.38,133.30,131.27,130.27,120.93,119.22,112.06,108.49,101.24.HRMS-ESI:Calcd.For C 19 H 16 N5O2[M+H + ]346.1305,found:346.1296.

[0174] N 4 -(Benzo[d][1,3]dioxolan-5-ylmethyl)-6-(3-dimethylaminophenyl)pyrimidine-2,4-diamine (2e)

[0175]

[0176] 1H NMR (400MHz, DMSO-d6) δ7.31–7.18(m,3H),7.13(d,J=8.0Hz,1H),6.91(d,J=1.5Hz,1H),6.87–6.80(m,2 H),6.77(dd,J=8.1,2.6Hz,1H),6.22(s,1H),5.97(d,J=5.5Hz,4H),4.41(d,J=6.0Hz,2H),2.92(s,6H). 13 C NMR(101MHz,DMSO-d6)δ164.40,163.84,150.92,147.67,146.41,139.56,134.61,12 9.30,120.89,114.95,113.97,110.66,108.46,101.22,40.70.HRMS-ESI:Calcd.For C 20 H 22 N5O2[M+H + ]364.1774,found:364.1766.

[0177] 6-([1,1'-biphenyl]-3-yl)-N 4 -(Benzo[d][1,3]dioxolan-5-ylmethyl)pyrimidine-2,4-diamine (2f)

[0178]

[0179] 1 H NMR (400MHz, DMSO-d6) δ8.17(s,1H),7.87(d,J=7.7Hz,1H),7.76–7.66(m,3H),7.52(dt,J=14.7,7.6Hz,3H),7.44–7. 38(m,1H),7.34(s,1H),6.93(s,1H),6.91–6.80(m,2H),6.37(s,1H),6.07(s,2H),5.97(s,2H),4.44(d,J=6.0Hz,2H). 13 C NMR(101MHz,DMSO-d6)δ164.52,163.94,147.68,146.44,140.85,140.61,139.42,134.53,129.54 ,129.45,128.24,128.07,127.25,125.78,125.14,120.92,108.48,101.23.HRMS-ESI:Calcd.ForC 24 H 21 N4O2[M+H+ ]397.1665,found:397.1654.

[0180] N 4 -(Benzo[d][1,3]dioxolan-5-ylmethyl)-6-(3-(benzyloxy)phenyl)pyrimidine-2,4-diamine (2 g)

[0181]

[0182] 1 H NMR (400MHz, DMSO-d6) δ7.56(t,J=2.1Hz,1H),7.52–7.44(m,3H),7.40(t,J=7.4Hz,2H),7.34(td,J=7.5,7.1,4.6Hz,3H),7.06(dd ,J=8.1,2.7Hz,1H),6.92(s,1H),6.84(t,J=7.1Hz,2H),6.26(s,1H),6.04(s,2H),5.97(s,2H),5.15(s,2H),4.42(d,J=6.1Hz,2H). 13 C NMR(101MHz,DMSO-d6)δ164.51,163.86,158.95,147.68,140.37,137.55,134.53,129.94,128.9 3,128.31,128.17,120.91,119.28,116.21,113.22,108.49,101.24,69.70.HRMS-ESI:Calcd.For C 25 H 23 N4O3[M+H + ]427.1771,found:427.1760.

[0183] N 4 -(Benzo[d][1,3]dioxolan-5-ylmethyl)-6-(pyridin-4-yl)pyrimidine-2,4-diamine (2h)

[0184]

[0185] 1H NMR (400MHz, DMSO-d6) δ8.68–8.62(m,2H),7.80(d,J=5.4Hz,2H),7.59–7.46(m,2H),6.92(d,J= 1.6Hz,1H),6.84(q,J=8.0Hz,2H),6.37(s,1H),6.17(s,2H),5.97(s,2H),4.43(d,J=6.0Hz,2H). 13 C NMR(101MHz,DMSO-d6)δ164.10,150.56,147.68,146.47,145.97,134.30,120.98,108.50,101.24.HRMS-ESI:Calcd.For C 17 H 16 N5O2[M+H + ]322.1305,found:322.1296.

[0186] N 4 -(Benzo[d][1,3]dioxolan-5-ylmethyl)-6-(4-fluorophenyl)pyrimidine-2,4-diamine (2i)

[0187]

[0188] 1 H NMR(400MHz,DMSO-d6)δ7.94(dd,J=8.6,5.8Hz,2H),7.34(s,1H),7.30–7.21(m,2H),6.91(d,J =1.6Hz,1H),6.89–6.78(m,2H),6.24(s,1H),6.05(s,2H),5.97(s,2H),4.42(d,J=6.0Hz,2H). 13 C NMR(101MHz,DMSO-d6)δ164.64,164.50,163.92,147.68,146.44,135.23,135.20,13 4.51,128.90,128.82,120.90,115.78,115.57,108.47,101.23.HRMS-ESI:Calcd.for C 18 H 16 FN4O2[M+H + ]339.1257,found:339.1252.

[0189] N 4 -(Benzo[d][1,3]dioxolan-5-ylmethyl)-6-(p-methylphenyl)pyrimidine-2,4-diamine (2j)

[0190]

[0191] 1 H NMR (400MHz, DMSO-d6) δ7.79(d,J=8.0Hz,2H),7.28(t,J=5.5Hz,1H),7.23(d,J=8.0Hz,2H),6.91(d,J=1 .5Hz,1H),6.87–6.78(m,2H),6.24(s,1H),6.00(s,2H),5.97(s,2H),4.42(d,J=6.0Hz,2H),2.33(s,3H). 13 C NMR(101MHz,DMSO-d6)δ164.46,163.90,147.66,146.41,139.38,135.98,134.6 0,129.42,126.62,120.86,108.47,101.22,21.35,21.33.HRMS-ESI:Calcd.For C 19 H 19 N4O2[M+H + ]335.1509,found:335.1504.

[0192] N 4 -(Benzo[d][1,3]dioxolan-5-ylmethyl)-6-(4-isopropylphenyl)pyrimidine-2,4-diamine (2k)

[0193]

[0194] 1 H NMR (400MHz, DMSO-d6) δ7.81(d,J=8.2Hz,2H),7.29(d,J=8.1Hz,3H),6.91(s,1H),6.88–6.79(m,2H),6. 23(s,1H),5.97(d,J=4.8Hz,4H),4.41(d,J=6.0Hz,2H),2.91(hept,J=6.7Hz,1H),1.21(d,J=6.9Hz,6H). 13 C NMR(101MHz,DMSO-d6)δ164.46,163.90,150.24,147.67,146.41,136.42,134.6 0,126.74,126.72,120.89,108.46,101.22,33.71,24.24.HRMS-ESI:Calcd.For C 21 H 23N4O2[M+H + ]363.1822,found:363.1808.

[0195] N 4 -(Benzo[d][1,3]dioxolan-5-ylmethyl)-6-(4-tert-butylphenyl)pyrimidine-2,4-diamine (2l)

[0196]

[0197] 1 H NMR (400MHz, DMSO-d6) δ7.81 (dd, J=8.5, 1.7Hz, 2H), 7.45 (dd, J=8.3, 1.6Hz, 2H), 7.36–7.26 (m, 1H), 6.91 (d,J=1.8Hz,1H),6.88–6.78(m,2H),6.23(s,1H),6.05–5.91(m,4H),4.41(d,J=6.0Hz,2H),1.30(s,9H). 13 C NMR(101MHz,DMSO-d6)δ164.43,163.87,152.48,147.67,146.42,135.96,134.5 7,126.46,125.59,120.91,108.47,101.22,34.89,31.53.HRMS-ESI:Calcd.for C 22 H 25 N4O2[M+H + ]377.1978,found:377.2001.

[0198] N 4 -(Benzo[d][1,3]dioxolan-5-ylmethyl)-6-(4-ethoxyphenyl)pyrimidine-2,4-diamine (2m)

[0199]

[0200] 1 H NMR (400MHz, DMSO-d6) δ7.89–7.79(m,2H),7.24(t,J=6.0Hz,1H),7.00–6.93(m,2H),6.91(d,J=1.5Hz,1H),6.87–6 .78(m,2H),6.20(s,1H),5.96(d,J=4.8Hz,4H),4.41(d,J=6.0Hz,2H),4.06(q,J=7.0Hz,2H),1.34(t,J=7.0Hz,3H). 13C NMR(101MHz,DMSO-d6)δ164.45,163.84,160.12,147.67,146.40,134.65,130.9 3,128.10,120.85,114.57,108.46,101.21,63.56,15.11.HRMS-ESI:Calcd.for C 20 H 21 N4O3[M+H + ]365.1614,found:365.1612.

[0201] 6-([1,1'-biphenyl]-4-yl)-N 4 -(Benzo[d][1,3]dioxolan-5-ylmethyl)pyrimidine-2,4-diamine (2n)

[0202]

[0203] 1 H NMR (400MHz, DMSO-d6) δ8.00(d,J=8.1Hz,2H),7.74(dd,J=10.5,7.9Hz,4H),7.49(t,J=7.5Hz,2H),7.38(dd,J=14.3 ,7.2Hz,2H),6.94(d,J=1.5Hz,1H),6.90–6.81(m,2H),6.33(s,1H),6.06(s,2H),5.98(s,2H),4.44(d,J=6.0Hz,2H). 13 C NMR(101MHz,DMSO-d6)δ163.96,147.68,146.43,141.45,140.02,137.78,134.55,12 9.46,128.17,127.29,127.12,127.10,120.92,108.49,101.23.HRMS-ESI:Calcd.For C 24 H 21 N4O2[M+H + ]397.1665,found:397.1655.

[0204] N 4 -(Benzo[d][1,3]dioxolan-5-ylmethyl)-6-(4-phenoxyphenyl)pyrimidine-2,4-diamine (2o)

[0205]

[0206] 1H NMR (400MHz, DMSO-d6) δ7.99–7.87(m,2H),7.46–7.39(m,2H),7.36–7.28(m,1H),7.18(td,J=7.4,1.2Hz,1H),7.13– 7.01(m,4H),6.92(d,J=1.5Hz,1H),6.89–6.79(m,2H),6.24(s,1H),6.02(s,2H),5.97(s,2H),4.43(d,J=6.1Hz,2H). 13 C NMR(101MHz,DMSO-d6)δ164.48,163.90,158.45,156.60,147.67,146.42,134.57,133.76 ,130.63,128.51,124.35,120.89,119.63,118.40,108.48,101.22.HRMS-ESI:Calcd.For C 24 H 21 N4O3[M+H + ]413.1614,found:413.1603.

[0207] N 4 -(Benzo[d][1,3]dioxolan-5-ylmethyl)-6-(3,4-dimethoxyphenyl)pyrimidine-2,4-diamine (2p)

[0208]

[0209] 1 H NMR (400MHz, DMSO-d6) δ7.50(d,J=2.0Hz,1H),7.45(dd,J=8.4,2.0Hz,1H),7.24(t,J=6.1Hz,1H),7.01(d,J=8.4Hz,1H ), 6.92 (s, 1H), 6.84 (q, J = 7.9Hz, 2H), 6.23 (s, 1H), 5.97 (d, J = 1.3Hz, 4H), 4.42 (d, J = 6.0Hz, 2H), 3.80 (d, J = 4.4Hz, 6H). 13 C NMR(101MHz,DMSO-d6)δ164.42,163.81,150.47,148.93,147.66,146.40,134.65,131.4 1,120.87,119.42,111.83,110.15,108.46,101.22,55.98,55.90.HRMS-ESI:Calcd.ForC 20 H 21N4O4[M+H + ]381.1564,found:381.1552.

[0210] N 4 -(Benzo[d][1,3]dioxolan-5-ylmethyl)-6-(3,5-dimethoxyphenyl)pyrimidine-2,4-diamine (2q)

[0211]

[0212] 1 H NMR (400MHz, DMSO-d6) δ7.30(t,J=6.1Hz,1H),7.03(d,J=2.3Hz,2H),6.92(s,1H),6.83(q,J=7.9Hz,2 H), 6.55 (d, J = 2.3Hz, 1H), 6.23 (s, 1H), 6.03 (s, 2H), 5.97 (s, 2H), 4.41 (d, J = 5.9Hz, 2H), 3.78 (s, 6H). 13 C NMR(101MHz,DMSO-d6)δ164.45,163.84,160.91,147.67,146.42,141.12,134.5 3,120.91,108.47,104.72,101.77,101.23,55.72,55.69.HRMS-ESI:Calcd.For C 20 H 21 N4O4[M+H + ]381.1564,found:381.1553.

[0213] N 4 -(Benzo[d][1,3]dioxolan-5-ylmethyl)-6-(3,4,5-trimethoxyphenyl)pyrimidine-2,4-diamine (2r)

[0214]

[0215] 1 H NMR (400MHz, DMSO-d6) δ7.28(t,J=6.2Hz,1H),7.18(s,2H),6.92(d,J=1.5Hz,1H),6.89–6.79( m,2H),6.26(s,1H),6.01(s,2H),5.97(s,2H),4.42(d,J=6.0Hz,2H),3.83(s,6H),3.70(s,3H). 13C NMR(101MHz,DMSO-d6)δ164.44,163.81,153.23,147.66,146.41,139.14,134.5 9,134.45,120.93,108.47,104.06,101.23,60.53,56.30.HRMS-ESI:Calcd.For C 21 H 23 N4O5[M+H + ]411.1669,found:411.1659.

[0216] N 4 -(Benzo[d][1,3]dioxolan-5-ylmethyl)-6-(2-fluoro-3-methoxyphenyl)pyrimidine-2,4-diamine (2s)

[0217]

[0218] 1 H NMR (400MHz, DMSO-d6) δ7.44(s,1H),7.38(q,J=5.7Hz,1H),7.22–7.14(m,2H),6.91(d,J=1.6Hz,1H),6.86(d,J= 7.9Hz,1H),6.84–6.78(m,1H),6.19(s,1H),6.06(s,2H),5.98(d,J=2.4Hz,2H),4.50–4.31(m,2H),3.86(s,3H). 13 C NMR(101MHz,DMSO-d6)δ163.98,163.76,149.00,148.11,148.00,147.66,146.44,134.41,127.77,12 7.69,124.34,124.29,121.51,121.49,120.95,114.44,108.48,101.23,56.61.HRMS-ESI:Calcd.forC 19 H 18 FN4O3[M+H + ]369.1363,found:369.1352.

[0219] N 4 -(Benzo[d][1,3]dioxolan-5-ylmethyl)-6-(3'-chloro-4',6-difluoro-[1,1'-biphenyl]-3-yl)pyrimidine-2,4-diamine (2t)

[0220]

[0221] HRMS-ESI:Calcd.for C 24 H 18 ClF2N4O2[M+H + ]467.1087,found:467.1074.

[0222] 6-(Benzo[d][1,3]dioxolan-5-yl)-N 4 -(Benzo[d][1,3]dioxolan-5-ylmethyl)pyrimidine-2,4-diamine (2u)

[0223]

[0224] 1 H NMR (400MHz, DMSO-d6) δ7.46(d,J=9.0Hz,2H),7.26(t,J=6.2Hz,1H),6.97(d,J=8.0Hz,1H),6.92(d,J= 1.5Hz,1H),6.88–6.80(m,2H),6.19(s,1H),6.06(s,2H),5.98(d,J=6.1Hz,4H),4.41(d,J=6.0Hz,2H). 13 C NMR(101MHz,DMSO-d6)δ164.49,163.75,148.79,147.98,147.66,146.41,134.60 ,133.04,120.87,108.55,108.46,106.80,101.74,101.22.HRMS-ESI:Calcd.for C 19 H 17 N4O4[M+H + ]365.1250,found:365.1286.

[0225] N 4 -(Benzo[d][1,3]dioxolan-5-ylmethyl)-6-(2,3-dihydrobenzo[b][1,4]dioxan-6-yl)pyrimidine-2,4-diamine (2v)

[0226]

[0227] 1 H NMR (400MHz, DMSO-d6) δ7.45–7.34(m,2H),7.23(s,1H),6.94–6.78(m,4H),6.17(s,1H),5.96(d,J=2.6Hz,4H),4.40(d,J=6.0Hz,2H),4.26(s,4H).13 C NMR(101MHz,DMSO-d6)δ164.45,163.79,147.66,146.40,145.10,143.65,134.61,132.0 2,120.87,119.76,117.33,115.39,108.46,101.21,64.71,64.51.HRMS-ESI:Calcd.for C 20 H 19 N4O4[M+H + ]379.1407,found:379.1394.

[0228] N 4 -(Benzo[d][1,3]dioxolan-5-ylmethyl)-6-(indolin-5-yl)pyrimidine-2,4-diamine (2w)

[0229]

[0230] 1 H NMR (400MHz, DMSO-d6) δ7.62(s,1H),7.53(dd,J=8.2,1.8Hz,1H),7.09(s,1H),6.90(d,J=1.5Hz,1H),6.88–6.78(m,2H),6.47 (d,J=8.2Hz,1H),6.10(s,1H),5.96(s,2H),5.81(s,2H),4.39(d,J=6.1Hz,2H),3.47(t,J=8.5Hz,2H),2.94(t,J=8.5Hz,2H). 13 CNMR(101MHz,DMSO-d6)δ164.40,163.64,154.56,147.65,146.36,134.82,129.10,127.16,126. 48,122.89,120.78,108.45,108.37,107.74,101.20,46.95,43.59,29.28.HRMS-ESI:Calcd.for C 20 H 20 N5O2[M+H + ]362.1618,found:362.1604.

[0231] N 4 -(Benzo[d][1,3]dioxolan-5-ylmethyl)-6-(2-methyl-2-hydrogen-indazol-4-yl)pyrimidine-2,4-diamine (2x)

[0232]

[0233] 1 H NMR (400MHz, DMSO-d6) δ8.86(s,1H),7.64(d,J=8.5Hz,1H),7.54(d,J=7.0Hz,1H),7.35–7.25(m,2H),6.94(s ,1H),6.90–6.81(m,2H),6.37(s,1H),6.09(s,2H),5.97(d,J=0.8Hz,2H),4.43(d,J=6.0Hz,2H),4.19(s,3H). 13 C NMR(101MHz,DMSO-d6)δ164.42,163.88,149.36,147.70,146.44,134.56,131.94,127.02,125.44,120.90,118.78,108.51,101.24.HRMS-ESI:Calcd.for C 20 H 19 N6O2[M+H + ]375.1569,found:375.1565.

[0234] N 4 -(Benzo[d][1,3]dioxolan-5-ylmethyl)-6-(1H-indol-4-yl)pyrimidine-2,4-diamine (2y)

[0235]

[0236] 1 H NMR (400MHz, DMSO-d6) δ11.20(s,1H),7.45(dd,J=16.8,7.7Hz,2H),7.39(t,J=2.8Hz,1H),7.36–7.26(m,1H),7.13( t,J=7.7Hz,1H),6.93(d,J=1.5Hz,1H),6.91–6.74(m,3H),6.33(s,1H),5.97(d,J=3.5Hz,4H),4.42(d,J=5.9Hz,2H). 13 C NMR(101MHz,DMSO-d6)δ164.22,163.81,147.71,146.43,137.12,134.62,131.23,126.14,12 5.85,121.02,120.85,118.91,112.87,108.51,108.41,102.14,101.23.HRMS-ESI:Calcd.for C 20 H18 N5O2[M+H + ]360.1460,found:360.1465.

[0237] N 4 -(Benzo[d][1,3]dioxolan-5-ylmethyl)-6-phenylpyrimidine-2,4-diamine (3a)

[0238]

[0239] 1 H NMR (400MHz, DMSO-d6) δ7.93–7.84(m,2H),7.47–7.40(m,3H),7.33(s,1H),6.92(d,J=1.6 Hz,1H),6.88–6.79(m,2H),6.26(s,1H),6.03(s,2H),5.97(s,2H),4.42(d,J=6.0Hz,2H). 13 C NMR(101MHz,DMSO-d6)δ164.48,163.95,147.68,146.43,138.80,134.55,129.84,128.84,126.70,120.90,108.48,101.23.HRMS-ESI:Calcd.for C 18 H 17 N4O2[M+H + ]321.1352,found:321.1353.

[0240] N 4 -(Benzo[d][1,3]dioxolan-5-ylmethyl)-6-(furan-3-yl)pyrimidine-2,4-diamine (3b)

[0241]

[0242] 1 H NMR (400MHz, DMSO-d6) δ8.08(d,J=1.8Hz,1H),7.71(t,J=1.8Hz,1H),7.25(s,1H),6.90(d,J= 1.6Hz,1H),6.87–6.76(m,3H),6.03(s,1H),5.97(s,2H),5.93(s,2H),4.38(d,J=6.0Hz,2H). 13C NMR(101MHz,DMSO-d6)δ164.15,163.83,147.65,146.40,144.47,142.39,134.59,126.87,120.86,109.06,108.45,108.42,101.21.HRMS-ESI:Calcd.forC 16 H 15 N4O3[M+H + ]311.1144,found:311.1140.

[0243] N 4 -(Benzo[d][1,3]dioxolan-5-ylmethyl)-6-(naphthalen-1-yl)pyrimidine-2,4-diamine (3c)

[0244]

[0245] 1 H NMR(400MHz, DMSO-d6)δ8.21–8.10(m,1H),7.95(ddd,J=7.3,5.2,1.7Hz,2H),7.59–7.45(m,4H),7.4 0(t,J=6.0Hz,1H),6.95(s,1H),6.92–6.81(m,2H),6.10(s,2H),5.98(d,J=7.2Hz,3H),4.44(s,2H). 13 C NMR(101MHz,DMSO-d6)δ163.67,147.70,146.47,138.60,134.45,133.75,130.79,12 8.90,128.60,126.52,126.47,126.28,125.75,108.52,101.25.HRMS-ESI:Calcd.for C 22 H 19 N4O2[M+H + ]371.1509,found:371.1498.

[0246] N 4 -(Benzo[d][1,3]dioxolan-5-ylmethyl)-6-(naphthalen-2-yl)pyrimidine-2,4-diamine (3d)

[0247]

[0248] 1H NMR(400MHz,DMSO-d6)δ8.48(d,J=1.6Hz,1H),8.06–7.90(m,4H),7.55(dt,J=6.2,3.4Hz,2H),7.38(s,1H ),6.93(d,J=1.4Hz,1H),6.89–6.81(m,2H),6.43(s,1H),6.08(s,2H),5.97(s,2H),4.45(d,J=6.0Hz,2H). 13 C NMR(101MHz,DMSO-d6)δ164.54,164.01,147.69,146.44,136.25,134.57,133.98,133.25,129.04 ,128.30,127.99,127.13,126.86,126.06,124.55,120.90,108.49,101.23.HRMS-ESI:Calcd.for C 22 H 19 N4O2[M+H + ]371.1509,found:371.1496.

[0249] N 4 -(Benzo[d][1,3]dioxolan-5-ylmethyl)-6-(6-methoxypyridin-3-yl)pyrimidine-2,4-diamine (3e)

[0250]

[0251] 1 H NMR (400MHz, DMSO-d6) δ8.68(d,J=2.4Hz,1H),8.16(dd,J=8.7,2.5Hz,1H),7.34(s,1H),6.92(d,J=1.5 Hz,1H),6.90–6.80(m,3H),6.24(s,1H),6.05(s,2H),5.97(s,2H),4.42(d,J=6.1Hz,2H),3.90(s,3H). 13 C NMR(101MHz,DMSO-d6)δ164.77,164.34,163.90,147.66,146.42,145.65,137.55,134.51,128.01,120.92,110.63,101.22,53.87.HRMS-ESI:Calcd.for C 18 H 18 N5O3[M+H + ]352.1410,found:352.1399.

[0252] N 4 -(Benzo[d][1,3]dioxolan-5-ylmethyl)-6-(9,9-dimethyl-9H-fluoren-2-yl)pyrimidine-2,4-diamine (3f)

[0253]

[0254] 1 H NMR(400MHz, DMSO-d6)δ8.06(s,1H),7.92–7.83(m,3H),7.58(dd,J=6.1,2.4Hz,1H),7.35(qd,J=7.6,3.8Hz,3H), 6.96–6.93(m,1H),6.89–6.82(m,2H),6.36(s,1H),6.08(s,2H),5.98(s,2H),4.46(d,J=5.9Hz,2H),1.47(s,6H). 13 C NMR(101MHz,DMSO-d6)δ164.48,163.85,154.26,153.81,147.68,146.43,140.36,138.54,137.99,134.58,1 28.17,127.63,125.98,123.33,121.03,120.89,120.50,108.48,101.23,46.95,27.37.HRMS-ESI:Calcd.for C 27 H 25 N4O2[M+H + ]437.1978,found:437.1966.

[0255] N 4 -(Benzo[d][1,3]dioxolan-5-ylmethyl)-6-(1-methyl-1H-indol-4-yl)pyrimidine-2,4-diamine (3 g)

[0256]

[0257] 1 H NMR (400MHz, DMSO-d6) δ7.49(dd,J=14.1,7.7Hz,2H),7.37(d,J=3.1Hz,1H),7.31(t,J=6.0Hz,1H),7.20(t,J=7. 8Hz,1H),6.93(d,J=1.5Hz,1H),6.89–6.78(m,3H),6.31(s,1H),5.98(s,4H),4.42(d,J=5.9Hz,2H),3.81(s,3H).

[0258] N 4 -(Benzo[d][1,3]dioxolan-5-ylmethyl)-6-(1H-indol-5-yl)pyrimidine-2,4-diamine (3h)

[0259]

[0260] 1 H NMR (400MHz, DMSO-d6) δ11.18(s,1H),8.15(d,J=1.7Hz,1H),7.67(dd,J=8.6,1.7Hz,1H),7.41(d,J=8.6Hz,1H),7.36(t,J=2.7Hz,1H), 7.23(s,1H),6.93(d,J=1.5Hz,1H),6.89–6.80(m,2H),6.50(t,J=2.5Hz,1H),6.28(s,1H),5.96(d,J=8.5Hz,4H),4.43(d,J=6.1Hz,2H).

[0261] Example 2, Synthesis Route 2: Synthesis of Compounds 5a-j

[0262]

[0263] Reagents and conditions: a) substituted benzylamine, K2CO3, DMF, 80°C; b) 3-methoxyphenylboronic acid, Pd(dppf)Cl2, K2CO3, 1,4-dioxane:water=4:1 (volume ratio), reflux.

[0264] 4,6-Dichloro-2-aminopyrimidine reacts with substituted benzylamine to obtain intermediates 4a-j, and the synthesis method is the same as the preparation of intermediate compound 1 in Example 1. 3-Methoxyphenylboronic acid reacts with intermediates 4a-j to obtain compounds 5a-j, and the synthesis method is the same as the preparation of compounds 2a-y in Example 1.

[0265] The structural formula and characterization data of target compounds 5a-j are as follows:

[0266] N 4 -Benzyl-6-(3-methoxyphenyl)pyrimidine-2,4-diamine (5a)

[0267]

[0268] 1H NMR(400MHz,DMSO-d6)δ7.47–7.42(m,2H),7.40–7.30(m,6H),7.26–7.19(m,1H), 7.01–6.96(m,1H),6.27(s,1H),6.02(s,2H),4.53(d,J=6.0Hz,2H),3.79(s,3H). 13 CNMR(101MHz,DMSO-d6)δ163.90,159.83,140.63,140.37,129.90,128.73,127.73,127.13,119.01,115.47,112.04,55.57.HRMS-ESI:Calcd.for C 18 H 19 N4O[M+H + ]307.1559,found:307.1556.

[0269] N 4 -(3-Fluorobenzyl)-6-(3-methoxyphenyl)pyrimidine-2,4-diamine (5b)

[0270]

[0271] 1 H NMR(400MHz, DMSO-d6)δ7.45(dd,J=7.5,1.5Hz,3H),7.40–7.32(m,2H),7.21–7.12(m,2H),7.05(td, J=8.7,2.6Hz,1H),7.02–6.96(m,1H),6.28(s,1H),6.05(s,2H),4.55(d,J=6.1Hz,2H),3.80(s,3H). 13 C NMR(101MHz,DMSO-d6)δ163.89,161.51,159.84,143.91,143.84,140.32,130.70, 130.62,123.68,119.04,115.52,114.41,114.20,113.95,113.74,112.05,55.57.

[0272] N 4 -(3,5-difluorobenzyl)-6-(3-methoxyphenyl)pyrimidine-2,4-diamine (5c)

[0273]

[0274] 1H NMR (400MHz, DMSO-d6) δ7.53–7.41(m,3H),7.35(t,J=8.0Hz,1H),7.12–6.95(m,4H),6.29(s,1H),6.08(s,2H),4.55(d,J=6.1Hz,2H),3.80(s,3H). 13 C NMR(101MHz,DMSO-d6)δ164.43,164.13,164.00,163.86,161.68,161.55,159.84,145.86,140.23,12 9.92,119.06,115.58,112.07,110.70,110.45,102.74,102.48,102.23,55.55.HRMS-ESI:Calcd.for C 18 H 17 F2N4O[M+H + ]343.1371,found:343.1371.

[0275] N 4 -(2,4-difluorobenzyl)-6-(3-methoxyphenyl)pyrimidine-2,4-diamine (5d)

[0276]

[0277] 1 H NMR (400MHz, DMSO-d6) δ7.52–7.43(m,3H),7.41–7.31(m,2H),7.24–7.17(m,1H),7.04(td,J=8.6,2. 6Hz, 1H), 6.99 (dd, J = 8.1, 2.6Hz, 1H), 6.32 (s, 1H), 6.10 (s, 2H), 4.54 (d, J = 5.9Hz, 2H), 3.79 (s, 3H). 13 C NMR(101MHz,DMSO-d6)δ164.42,163.87,159.84,140.30,131.30,129.92,128.98,126.82,123.45,11 9.01,116.23,115.52,112.03,111.76,111.53,104.33,104.07,103.81,55.58.HRMS-ESI:Calcd.for C 18 H 17 F2N4O[M+H + ]343.1370,found:343.1371.

[0278] 6-(3-methoxyphenyl)-N 4 -(4-(Trifluoromethoxy)benzyl)pyrimidine-2,4-diamine (5e)

[0279]

[0280] 1 H NMR (400MHz, DMSO-d6) δ7.51–7.42(m,5H),7.39–7.30(m,3H),6.99(ddd,J=8.2 ,2.5,1.2Hz,1H),6.28(s,1H),6.05(s,2H),4.56(d,J=6.1Hz,2H),3.80(s,3H). 13 C NMR(101MHz,DMSO-d6)δ164.50,163.91,159.84,147.56,140.33,140.28,129.9 0,129.54,121.38,119.03,115.50,112.06,55.57,55.54.HRMS-ESI:Calcd.for C 19 H 18 F3N4O2[M+H + ]391.1383,found:391.1371.

[0281] N 4 -(3,4-dimethoxybenzyl)-6-(3-methoxyphenyl)pyrimidine-2,4-diamine (5f)

[0282]

[0283] 1 H NMR (400MHz, DMSO-d6) δ7.49–7.42(m,2H),7.34(t,J=7.9Hz,1H),7.31–7.25(m,1H),6.98(ddd,J=8.1,2.5,0.9Hz ,2H),6.92–6.83(m,2H),6.27(s,1H),6.02(s,2H),4.43(d,J=5.9Hz,2H),3.79(s,3H),3.74(s,3H),3.71(s,3H). 13C NMR (101MHz, DMSO-d6) δ164.52,163.89,159.83,149.10,148.17,140.39,132.92,129. 90,119.93,119.01,115.45,112.19,112.04,56.01,55.90,55.55.HRMS-ESI:Calcd.for C 20 H 23 N4O3[M+H + ]367.1770,found:367.1765.

[0284] N 4 -(4-methoxybenzyl)-6-(3-methoxyphenyl)pyrimidine-2,4-diamine (5 g)

[0285]

[0286] 1 H NMR(400MHz,DMSO-d6)δ7.47–7.41(m,2H),7.37–7.25(m,4H),6.98(dd,J=8.1,2.6Hz,1H),6 .92–6.86(m,2H),6.26(s,1H),6.02(s,2H),4.45(d,J=6.0Hz,2H),3.79(s,3H),3.72(s,3H). 13 C NMR(101MHz,DMSO-d6)δ164.50,163.88,159.82,158.62,140.39,132.46,129.8 9,129.07,119.00,115.45,114.14,112.03,55.55,55.52.HRMS-ESI:Calcd.for C 19 H 21 N4O2[M+H + ]337.1665,found:337.1667.

[0287] 6-(3-methoxyphenyl)-N 4 -(3,4,5-Trimethoxybenzyl)pyrimidine-2,4-diamine (5h)

[0288]

[0289] 1H NMR(400MHz, DMSO-d6)δ7.49–7.42(m,2H),7.32(dt,J=12.1,6.9Hz,2H),7.01–6.96(m,1H),6.70 (s,2H),6.29(s,1H),6.04(s,2H),4.43(d,J=5.9Hz,2H),3.79(s,3H),3.75(s,6H),3.62(s,3H). 13 C NMR(101MHz,DMSO-d6)δ163.83,159.84,153.25,140.30,136.77,136.14,129.91,119.02 ,115.50,112.05,105.38,60.46,60.44,56.31,56.29,55.59,55.55.HRMS-ESI:Calcd.for C 21 H 25 N4O4[M+H + ]397.1876,found:397.1872.

[0290] N 4 -((1H-indol-5-yl)methyl)-6-(3-methoxyphenyl)pyrimidine-2,4-diamine (5i)

[0291]

[0292] 1 H NMR (400MHz, DMSO-d6) δ11.01(s,1H),7.50(s,1H),7.47–7.40(m,2H),7.32(ddd,J=13.2,6.8,3.0Hz,4H),7.09(dd,J=8.4,1.7 Hz,1H),6.99(ddd,J=9.2,7.9,2.2Hz,1H),6.37(t,J=2.6Hz,1H),6.29(s,1H),6.03(s,2H),4.58(d,J=5.8Hz,2H),3.79(s,3H). 13 C NMR(101MHz,DMSO-d6)δ163.87,159.82,135.47,130.62,129.90,128.06,125.98 ,121.51,118.99,115.45,112.00,111.66,101.33,55.57.HRMS-ESI:Calcd.forC 20 H 20 N5O[M+H +]346.1668,found:346.1670.

[0293] 6-(3-methoxyphenyl)-N 4 -(Naphthalen-2-ylmethyl)pyrimidine-2,4-diamine (5j)

[0294]

[0295] 1 H NMR(400MHz,DMSO-d6)δ7.91–7.81(m,4H),7.54–7.43(m,6H),7.35(td,J=8.2,1.8Hz, 1H),7.01–6.96(m,1H),6.33(s,1H),6.05(s,2H),4.71(d,J=6.0Hz,2H),3.79(s,3H). 13 C NMR(101MHz,DMSO-d6)δ163.93,159.84,140.35,138.27,133.41,132.56,129.92,128.32,128.01,1 27.97,126.61,126.50,126.03,125.70,119.04,115.51,112.04,55.57,55.55.HRMS-ESI:Calcd.for C 22 H 21 N4O[M+H + ]357.1715,found:357.1714.

[0296] Example 3, Synthesis Route 3: Synthesis of Compounds 6a-d

[0297]

[0298] Reagents and conditions: a) 3,4-methylenedioxybenzylamine, K2CO3, DMF, 80°C; b) 3-methoxyphenylboronic acid, Pd(dppf)Cl2, K2CO3, 1,4-dioxane: water = 4:1 (volume ratio), reflux; c) NaH, DMF, halide, 0°C.

[0299] The intermediate compound 1 was prepared according to the method described in Example 1. The compound BML284 was further prepared according to the method described in Example 1.

[0300] Add 350 mg (1.0 mmol, 1.0 eq.) BML284 to a 25 mL round-bottom flask, then add 5 mL DMF, stir for 5 min in an ice bath, add 44 mg (1.1 mmol, 1.1 eq.) NaH (60%) in batches at this temperature, then dissolve the halide (1.0 eq.) in 2 mL DMF, and slowly drop into the reaction bottle. After the reaction, pour the reaction solution into 20 mL ice water to quench, extract with ethyl acetate, wash the organic phase with saturated brine, dry with anhydrous sodium sulfate, evaporate and concentrate, mix the sample, and quickly separate and purify to obtain compounds 6a-d.

[0301] N 4 -(Benzo[d][1,3]dioxolan-5-ylmethyl)-6-(3-methoxyphenyl)pyrimidine-2,4-diamine (BML284)

[0302]

[0303] 1 H NMR (400MHz, DMSO-d6) δ7.48–7.41(m,2H),7.33(q,J=7.9Hz,2H),7.02–6.96(m,1H),6.92(d,J=1.5H z,1H),6.88–6.79(m,2H),6.26(s,1H),6.03(s,2H),5.97(s,2H),4.42(d,J=6.0Hz,2H),3.80(s,3H). 13 C NMR(101MHz,DMSO-d6)δ164.46,163.89,159.83,147.67,146.42,140.39,134.53,129.9 0,120.90,119.02,115.47,112.04,108.48,101.22,55.57,55.55.HRMS-ESI:Calcd.for C 19 H 19 N4O3[M+H + ]351.1458,found:351.1445.

[0304] N 4 -(Benzo[d][1,3]dioxolan-5-ylmethyl)-6-(3-methoxyphenyl)-N 4 -Methylpyrimidine-2,4-diamine (6a)

[0305]

[0306] 1H NMR (400MHz, DMSO-d6) δ7.61–7.53(m,2H),7.34(t,J=7.9Hz,1H),7.00(dd,J=8.1,2.6Hz,1H),6.88–6.80 (m,2H),6.78–6.72(m,1H),6.43(s,1H),6.10(s,2H),5.97(s,2H),4.72(s,2H),3.80(s,3H),3.01(s,3H). 13 C NMR(101MHz,DMSO-d6)δ164.08,163.53,163.26,159.83,147.82,146.64,140.57,132.78,129.80,1 20.92,119.46,115.53,112.47,108.64,108.29,101.30,89.23,55.63,35.49.HRMS-ESI:Calcd.for C 20 H 21 N4O3[M+H + ]365.1614,found:365.1605.

[0307] N 4 -(Benzo[d][1,3]dioxolan-5-ylmethyl)-N 4 -ethyl-6-(3-methoxyphenyl)pyrimidine-2,4-diamine (6b)

[0308]

[0309] 1 H NMR (400MHz, DMSO-d6) δ7.53(d,J=8.7Hz,2H),7.34(t,J=7.9Hz,1H),7.02–6.97(m,1H),6.90–6.83(m,2H),6.77(d,J= 7.6Hz,1H),6.37(s,1H),6.07(s,2H),5.97(s,2H),4.68(s,2H),3.80(s,3H),3.56–3.48(m,2H),1.09(t,J=6.9Hz,3H). 13 C NMR (101MHz, DMSO-d6) δ163.69,163.33,163.23,159.81,147.80,146.59,140.66,133.30,129. 81,119.44,115.42,112.52,108.60,101.29,55.61,55.59,41.80,12.98.HRMS-ESI:Calcd.forC21 H 23 N4O3[M+H + ]379.1771,found:379.1757.

[0310] N 4 -(Benzo[d][1,3]dioxolan-5-ylmethyl)-N 4 -propyl-6-(3-methoxyphenyl)pyrimidine-2,4-diamine (6c)

[0311]

[0312] 1 H NMR (400MHz, DMSO-d6) δ7.52(d,J=8.1Hz,2H),7.34(t,J=7.9Hz,1H),6.99(ddd,J=8.3,2.5,1.1Hz,1H),6.85(d,J=8.0Hz,2H),6.75(dd,J=8 .0,1.7Hz,1H),6.35(s,1H),6.07(s,2H),5.97(s,2H),4.70(s,2H),3.80(s,3H),3.41(s,2H),1.56(h,J=7.5Hz,2H),0.87(t,J=7.3Hz,3H). 13 C NMR(101MHz,DMSO-d6)δ163.63,163.17,159.80,147.79,146.57,140.66,133.24,129.82, 119.42,115.39,112.52,108.59,101.29,55.59,48.87,20.80,11.70.HRMS-ESI:Calcd.for C 22 H 25 N4O3[M+H + ]393.1927,found:393.1920.

[0313] N 4 -allyl-N 4 -(Benzo[d][1,3]dioxolan-5-ylmethyl)-6-(3-methoxyphenyl)pyrimidine-2,4-diamine (6d)

[0314]

[0315] 1H NMR(400MHz, DMSO-d6)δ7.56–7.46(m,2H),7.33(t,J=8.0Hz,1H),7.04–6.95(m,1H),6.89–6.83(m,2H),6.77(dd,J=8.2,1.6Hz,1H),6 .36(s,1H),6.12(s,2H),5.97(s,2H),5.80(ddq,J=16.0,10.8,5.1Hz,1H),5.21–5.10(m,2H),4.67(s,2H),4.12(s,2H),3.79(s,3H). 13 C NMR (101MHz, DMSO-d6) δ163.71,163.61,163.27,159.82,147.83,146.66,140.55,129.84,119. 39,116.88,115.51,112.46,108.60,101.31,89.62,55.60,55.58,49.45.HRMS-ESI:Calcd.for C 22 H 23 N4O3[M+H + ]391.1771,found:391.1773.

[0316] Example 4, Synthesis Route 4: Synthesis of Compound 8

[0317]

[0318] Reagents and conditions: a) 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline, K2CO3, DMF, 80°C; b) 3-methoxyphenylboronic acid, Pd(dppf)Cl2, K2CO3, 1,4-dioxane:water=4:1 (volume ratio), reflux.

[0319] 4,6-Dichloro-2-aminopyrimidine reacts with 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline to obtain intermediate 7, and the synthesis method is the same as the preparation of compound 1 in Example 1. Intermediate 7 is then reacted with 3-methoxyphenylboronic acid to obtain compound 8, and the synthesis method is the same as the preparation of compounds 2a-y in Example 1.

[0320] 4-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(2H)-yl)-6-(3-methoxyphenyl)pyrimidin-2-amine (8)

[0321]

[0322] 1H NMR (400MHz, DMSO-d6) δ7.67–7.58(m,2H),7.36(t,J=7.9Hz,1H),7.01(dd,J=8.1,2.6Hz,1H),6.81(s,1H),6.77(s,1H), 6.60(s,1H),6.09(s,2H),4.69(s,2H),3.87(d,J=5.8Hz,2H),3.82(s,3H),3.74(d,J=6.1Hz,6H),2.78(t,J=5.8Hz,2H). 13 C NMR(101MHz,DMSO-d6)δ163.76,163.55,163.34,159.85,147.79,147.73,140.54,129.79,127.16,126 .08,119.52,115.51,112.58,112.35,110.36,89.69,55.99,55.67,45.93,28.10.HRMS-ESI:Calcd.for C 22 H 25 N4O3[M+H + ]393.1927,found:393.1923.

[0323] Example 5, Synthesis Route 5: Synthesis of Compound 10

[0324]

[0325] Reagents and conditions: a) 3,4-methylenedioxybenzylamine, K2CO3, DMF, 80°C; b) 3-methoxyphenylboronic acid, Pd(dppf)Cl2, K2CO3, 1,4-dioxane:water = 4:1 (volume ratio), reflux.

[0326] The synthesis of compound 9 is the same as the preparation of compound 1 in Example 1.

[0327] N-(Benzo[d][1,3]dioxolan-5-ylmethyl)-6-chloropyrimidin-4-amine (9)

[0328]

[0329] 1 H NMR (400MHz, DMSO-d6) δ8.28(s,1H),8.11(s,1H),6.92–6.84(m,2H),6.79(d,J=8.1Hz,1H),6.55(s,1H),5.98(s,2H),4.45(s,2H).

[0330] Synthesis of compound 10

[0331] The synthesis of compound 10 is the same as the preparation of compounds 2a-y in Example 1.

[0332] N-(Benzo[d][1,3]dioxolan-5-ylmethyl)-6-(3-methoxyphenyl)pyrimidin-4-amine (10)

[0333]

[0334] 1 H NMR(400MHz, DMSO-d6)δ8.51(s,1H),7.88(d,J=6.2Hz,1H),7.60–7.51(m,2H),7.41(t,J=8.1Hz,1H),7.08– 7.02(m,1H),6.95(d,J=18.9Hz,2H),6.85(d,J=3.9Hz,2H),5.98(s,2H),4.49(d,J=5.9Hz,2H),3.82(s,3H). 13 C NMR(101MHz,DMSO-d6)δ163.35,160.05,158.85,147.76,146.59,139.25,133.79,130.31,1 20.98,119.07,116.25,112.01,108.55,108.41,101.30,55.61.43.83.HRMS-ESI:Calcd.for C 19 H 18 N3O3[M+H + ]336.1349,found:336.1335.

[0335] Example 6, Synthesis Route 6: Synthesis of Compound 13

[0336]

[0337] Reagents and conditions: a) MnO2, DCM, room temperature; b) 3-methoxyphenylboronic acid, Pd(dppf)Cl2, K2CO3, 1,4-dioxane: water = 4:1 (volume ratio), reflux; c) 1) tetraisopropyl titanate, THF, room temperature; 2) NaBH4, EtOH, room temperature.

[0338] Synthesis of compound 11

[0339] To a 50 mL round-bottom flask, 300 mg (2.0 mmol, 1.0 eq.) of piperonyl alcohol, 348 mg (4.0 mmol, 2.0 eq.) of manganese dioxide, and 30 mL of dichloromethane were added in sequence and reacted at room temperature with stirring. After the reaction was completed, the mixture was filtered through diatomaceous earth, the filter cake was washed with dichloromethane, and the filtrate was evaporated and concentrated to obtain compound 11, which was directly used in the subsequent reaction without further treatment.

[0340] Benzo[d][1,3]dioxolane-5-carbaldehyde (11)

[0341]

[0342] 1 H NMR (400MHz, DMSO-d6) δ9.81(s,1H),7.54(dd,J=7.9,1.6Hz,1H),7.32(d,J=1.6Hz,1H),7.13(d,J=8.0Hz,1H),6.17(s,2H).

[0343] Synthesis of compound 12

[0344] The synthesis method of compound 12 is the same as the preparation of compounds 2a-y in Example 1.

[0345] 4-(3-Methoxyphenyl)pyridine-2,6-diamine (12)

[0346]

[0347] 1 H NMR (400MHz, DMSO-d6) δ7.35(t,J=7.9Hz,1H),7.09(dt,J=7.8,1.2Hz,1H),7. 02(t,J=2.1Hz,1H),6.97–6.91(m,1H),5.90(s,2H),5.45(s,4H),3.80(s,3H).

[0348] Synthesis of compound 13

[0349] In a 25 mL round-bottom flask, 150 mg (1.0 mmol, 1.0 eq.) of intermediate 11, 215 mg (1.0 mmol, 1.0 eq.) of intermediate 12, 426 mg (1.5 mmol, 1.5 eq.) of tetraisopropyl titanate, and 3 drops of tetrahydrofuran solution were added in sequence, and the mixture was stirred at room temperature for 2 hours. Then, 5 mL of anhydrous ethanol was added thereto, and 76 mg of sodium borohydride was added in batches. The mixture was reacted at room temperature for 15 minutes. After the reaction was completed, 2 mL of water was added to the reaction system to quench the mixture, and the mixture was filtered through diatomaceous earth. The filter cake was washed with ethyl acetate, the filtrate was evaporated and concentrated, the sample was mixed, and the mixture was separated and purified by column chromatography to obtain compound 13.

[0350] N 2 -(Benzo[d][1,3]dioxolan-5-ylmethyl)-4-(3-methoxyphenyl)pyridine-2,6-diamine (13)

[0351]

[0352] 1 H NMR (400MHz, DMSO-d6) δ7.34(t,J=7.9Hz,1H),7.08(d,J=7.7Hz,1H),7.01(d,J=2.3Hz,1H),6.97–6.91(m,2H),6.83 (s,2H),6.51(t,J=6.2Hz,1H),5.96(s,2H),5.90(d,J=4.2Hz,2H),5.51(s,2H),4.35(d,J=6.2Hz,2H),3.79(s,3H). 13 C NMR(101MHz,Chloroform-d)δ159.82,157.94,157.61,152.58,147.89,146.73,141.28,133.15,129.72,1 20.54,119.33,113.98,112.54,108.27,108.02,100.99,95.87,94.32,55.30,46.34.HRMS-ESI:Calcd.for C 20 H 20 N3O3[M+H + ]350.1505,found:350.1506.

[0353] Example 7, Synthesis Route 7: Synthesis of Compound 15

[0354]

[0355] Reagents and conditions: a) 1) 3,4-methylenedioxybenzylamine, tetraisopropyl titanate, THF, room temperature; 2) NaBH4, EtOH, room temperature; b) indole-4-boronic acid pinacol ester, Pd(dppf)Cl2, K2CO3, 1,4-dioxane: water = 4:1 (volume ratio), reflux.

[0356] The preparation method of compound 14 is the same as the synthesis method of 13.

[0357] N 2 -Benzo[d][1,3]dioxolan-5-ylmethyl)-4-bromo-pyridine-2,6-diamine (14)

[0358]

[0359] 1 H NMR (400MHz, DMSO-d6) δ6.87(d,J=1.6Hz,1H),6.83(d,J=8.0Hz,1H),6.77(dd,J=7.9,1.7Hz,1 H),6.71(t,J=6.2Hz,1H),5.96(s,2H),5.82–5.77(m,2H),5.74(s,2H),4.28(d,J=6.1Hz,2H).

[0360] The preparation method of compound 15 is the same as that of 2a-y.

[0361] N 2 -Benzo[d][1,3]dioxolan-5-ylmethyl)-4-(1H-indol-4-yl)pyridine-2,6-diamine (15)

[0362]

[0363] 1 H NMR (400MHz, DMSO-d6) δ11.19(s,1H),7.40–7.33(m,2H),7.11(t,J=7.7Hz,1H),7.00(dd,J=7.3,1.0Hz,1H),6.92(d,J=1.4Hz,1H),6. 87–6.80(m,2H),6.50(t,J=6.2Hz,1H),6.47(d,J=2.7Hz,1H),5.96(s,3H),5.93(d,J=1.3Hz,1H),5.47(s,2H),4.34(d,J=6.2Hz,2H).

[0364] Example 8, Synthesis Route 8: Synthesis of Compounds 18, 19

[0365]

[0366] Reagents and conditions: a) CH3I, NaH, DMF, 0°C; b) indole-4-boronic acid pinacol ester, Pd(dppf)Cl2, K2CO3, 1,4-dioxane: water = 4:1 (volume ratio), reflux.

[0367] 278 mg (1.0 mmol, 1.0 eq.) of compound N was dissolved in 41.5 eq. of NaH was added in batches to a DMF solution of -(benzo[d][1,3]dioxolan-5-ylmethyl)-6-chloro-pyrimidine-2,4-diamine (1) at 0°C, and stirred at this temperature for 10 minutes. Then, 142 mg (1.0 mmol, 1.0 eq.) of iodomethane was added to the mixed system. After the reaction was completed, 20 mL of water was added to quench the reaction, and the mixture was extracted with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate and purified by rapid column separation to obtain compounds 16 and 17. Compounds 16 and 17 were used as raw materials and compounds 18 and 19 were prepared according to the synthesis method of compounds 2a-y.

[0368] N 4 -(Benzo[d][1,3]dioxolan-5-ylmethyl)-6-(1H-indol-4-yl)-N 4 -Methylpyrimidine-2,4-diamine (18)

[0369]

[0370] 1 H NMR (400MHz, DMSO-d6) δ11.20(s,1H),7.49(d,J=7.4Hz,1H),7.44(d,J=8.0Hz,1H),7.37(t,J=2.7Hz,1H),7.13(t,J=7.7 Hz,1H),6.91–6.81(m,3H),6.76(dd,J=7.9,1.6Hz,1H),6.35(s,1H),6.07(s,2H),5.98(s,2H),4.71(s,2H),3.04(s,3H).

[0371] N 4 -(Benzo[d][1,3]dioxolan-5-ylmethyl)-6-(1H-indol-4-yl)-N 2 ,N 4 -Dimethylpyrimidine-2,4-diamine (19)

[0372]

[0373] 1 H NMR (400MHz, DMSO-d6) δ11.42(s,1H),7.55(d,J=8.1Hz,1H),7.46(s,2H),7.20(t,J=7.8Hz,1H),6 .97–6.67(m,5H),6.46(s,1H),6.00(s,2H),4.79(s,2H),3.23–3.08(m,3H),2.91(d,J=4.6Hz,3H).

[0374] Example 9. Synthesis of Compounds 20 and 21

[0375] To a 25 mL round-bottom flask containing 164 mg (1.0 mmol, 1.0 eq.) of 4,6-dichloro-2-aminopyrimidine, add 207 mg (1.5 mmol, 1.5 eq.) of K2CO3, then add 8 mL of DMF, stir at room temperature until the compound is completely dissolved, then add 212 mg (1.1 mmol, 1.1 eq.) of 6.7-dimethoxy-1,2,3,4-tetrahydroisoquinoline in batches, continue stirring and react at 80°C, and monitor by TLC. After the reaction of 4,6-dichloro-2-aminopyrimidine is complete, pour the reaction solution into 40 mL of water, add 10 mL of ethyl acetate to extract three times, wash the organic phase with saturated brine, dry over anhydrous sodium sulfate, mix the sample, and quickly separate and purify to obtain the intermediate 4-chloro-6-(6,7-dimethoxy-3,4-dihydroisoquinoline)-2(1H)-yl)pyrimidin-2-amine. The intermediate was used as a raw material to synthesize the target product 20 using the same preparation method of compounds 2a-y.

[0376] 4-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)-6-(1H-indol-4-yl)pyrimidin-2-amine (20)

[0377]

[0378] 1 H NMR (400MHz, DMSO-d6) δ11.22(s,1H),7.54(dd,J=7.4,0.9Hz,1H),7.47(d,J=8.0Hz,1H),7.41(t,J=2.8Hz,1H),7.16(t,J=7.7Hz,1H),7.01(t,J= 2.7Hz,1H),6.84(s,1H),6.77(s,1H),6.53(s,1H),6.07(s,2H),4.68(s, 2H), 3.85 (t, J = 5.8Hz, 2H), 3.73 (d, J = 5.1Hz, 6H), 2.80 (t, J = 5.8Hz, 2H).

[0379] To a 25 mL round-bottom flask containing 164 mg (1.0 mmol, 1.0 eq.) of 4,6-dichloro-2-aminopyrimidine, add 207 mg (1.5 mmol, 1.5 eq.) of K2CO3, then add 8 mL of DMF, stir at room temperature until the compound is completely dissolved, then add 146 mg (1.1 mmol, 1.1 eq.) of indole-5-methylamine in batches, continue stirring and react at 80°C, monitor by TLC, after the reaction of 4,6-dichloro-2-aminopyrimidine is complete, pour the reaction solution into 40 mL of water, extract three times with 10 mL of ethyl acetate, wash the organic phase with saturated brine, dry over anhydrous sodium sulfate, mix the sample, and quickly separate and purify to obtain intermediate N 4 -((1H-indol-5-yl)methyl)-6-chloropyrimidine-2,4-diamine. The intermediate is then used as a raw material to synthesize the target product 21 in the same manner as the preparation method of compounds 2a-y.

[0380] N 4 -((1H-indol-5-yl)methyl)-6-(1H-indol-4-yl)pyrimidine-2,4-diamine (21)

[0381]

[0382] 1 H NMR (400MHz, DMSO-d6) δ11.19(s,1H),11.01(s,1H),7.51(s,1H),7.47(d,J=7.4Hz,1H),7.42(d,J=8.1Hz ,1H),7.37–7.29(m,3H),7.16–7.06(m,2H),6.82(s,2H),6.39(d,J=2.5Hz,2H),5.94(s,2H),4.58(s,2H).

[0383] The beneficial effects of the present invention are verified by means of test examples below.

[0384] Experimental Example 1: Verification of the Effect of the Compounds of the Invention

[0385] 1 Experimental instruments, materials, and methods

[0386] 1.1 Experimental Instruments

[0387] Flash separation column: Biotage, Germany;

[0388] Low-speed centrifuge: Varifuge 3.0; Megafuge 1.0, Heraeus, Germany;

[0389] Ultra-low temperature refrigerator: Japan SANYO (-80℃);

[0390] Ultracentrifuge: Centrifuge 5415C, Eppendorf, Germany;

[0391] Ultraspeed 4℃ centrifuge: Biofuge 28RS, Heraeus, Germany;

[0392] Digital inverted microscope: CKX41, Olympus, Japan;

[0393] Inverted fluorescence microscope: Zeiss, Axiovert200;

[0394] Constant temperature oscillator: Thermomixer comfort, Eppendorf, Germany;

[0395] CO2 incubator: MCO-18AIC, SANYO, Japan;

[0396] Ordinary optical microscope: CHS, Olympus, Japan;

[0397] Thermohygrometer: Wuqiang Thermohygrometer Manufacturing Center, Hengshui City, Hebei Province, GJWS-A5;

[0398] Vortex mixer: Type 16700Mixer, Thermolyne, USA;

[0399] Low-temperature centrifuge: Biofuge 28RS, Heraeus SepaTech;

[0400] Normal temperature centrifuge: Thermo Scientific, Thermo SORVALL ST16 Centrifuge;

[0401] Liquid nitrogen tank: American Thermo company product, CY50985-70;

[0402] Electrophoresis system: Bio-RAD;

[0403] Flow cytometer: FASC420, USA;

[0404] Constant temperature water bath: PolyScience 9505, USA;

[0405] pH meter: pH meter 345, Corning, USA;

[0406] Electrophoresis apparatus, glass plates, electrophoresis tanks, transfer boxes and gel imaging systems: Bio-RAD, USA;

[0407] UV spectrophotometer: SmartSpec 3000, Bio-RAD, USA;

[0408] Clean bench: Biological Safety Cabinets, Class II, NUAIR, SANYO, Japan;

[0409] High pressure sterilizer: LaboAutoclave, SANYO, Japan;

[0410] Water purifier: EASYpure UF 07421, Millipore, USA;

[0411] Ultrasonic crusher: VC50, Sonics&Materials Inc, USA;

[0412] Pathological tissue bleaching and drying apparatus: PHY-III, Changzhou Zhongwei Electronic Instrument Factory;

[0413] Cell counter: Nanjing Jiancheng Bioengineering Company;

[0414] Automatic biochemical analyzer: Roche, Germany;

[0415] Microplate reader: M5, Molecular Device, USA;

[0416] Cell culture plate: 96T / 6T, Corning, USA;

[0417] Pipette: eppendorf, tegent beta;

[0418] Variable speed horizontal shaker: Kylin-Bell lab instruments, TS-2ORBITAL SHAKER.

[0419] 1.2 Experimental Materials

[0420] MTT: Sigma;

[0421] Paclitaxel, vincristine, colchicine, doxorubicin, maytansine, Plinabulin, Nocodazole, Pironetin, EBI: Selleck;

[0422] RPMI-1640 culture medium, DMEM culture medium, and trypsin containing EDTA: Hyclone;

[0423] Fetal bovine serum (FBS): Prairie Green Company;

[0424] DAPI, penicillin-streptomycin mixture, DMSO, PI (propidium iodide): Sigma;

[0425] MTT (thiazolyl blue): Sigma, USA, prepared into 5 mg / mL storage solution with sterile saline and stored in a 4°C refrigerator away from light.

[0426] BCA quantitative kit, protein pre-stained marker: Bio-Rad Company;

[0427] Cell RIPA lysis buffer, SDS-PAGE protein loading buffer, Tween-80, Tris-base, SDS powder, glycine, skim milk powder, Western blot-anti-dilution buffer: Beyotime Biotechnology Research Institute;

[0428] Western blot ECL chemiluminescent developer: abbkine;

[0429] PMSF: Amresc;

[0430] TritonX-100: Farco Corporation;

[0431] α-tubulin antibody, β-tubulin antibody, GAPDH antibody: Santa Cruz Company;

[0432] Flag antibody: Proteintech;

[0433] GFP antibody: Cell Signaling Technology;

[0434] Cleaved caspase 3 antibody: ZENBIO;

[0435] PARP antibody, Ubiquitin antibody: Santa Cruz Biotechnology;

[0436] Mouse secondary antibody, horseradish peroxidase-labeled goat anti-mouse antibody, and normal goat serum for blocking: Zhongshan Golden Bridge Biotechnology Co., Ltd.;

[0437] Porcine brain tubulin: Cytoskeleton, dissolved in tubulin buffer (80 mM PIPES (pH 6.9); 2 mM MgCl2; 0.5 mM EGTA; 1 mM GTP) at 10 mg / mL and stored at -80°C until use;

[0438] PBS buffer powder, PIPES, EGTA, MgCl2, NaCl, glycerol and other conventional reagents: Kelun Pharmaceutical, Sangon Biotech;

[0439] RNase: Dalian Meilun Company;

[0440] Cell apoptosis detection kit (Annexin V & PI double staining): Invitrogen;

[0441] PVDF membrane (0.2 μM), disposable 0.22 μm filter: Millipore.

[0442] Culture medium DMEM: Gibco;

[0443] Dimethyl sulfoxide (DMSO): Chengdu Kelon Chemical;

[0444] Tumor cells were preserved for this experiment;

[0445] Matrigel: BD biochemical reagents;

[0446] PBS: NaH2PO4·2H2O 0.449 g, Na2HPO4·12H2O 6.76 g, NaCl 8.74 g, add Milli-Q H2O 900 mL, adjust pH to 7.2, make up to volume, and sterilize by high pressure;

[0447] Animals required for animal model: provided by the State Key Laboratory of Biotherapy, Sichuan University.

[0448] 1.3 Experimental methods

[0449] 1.3.1 MTT assay to detect the effect of compounds on tumor cell proliferation activity

[0450] Tumor cells HeLa, HCT-116, MCF-7, K562, Molm-13, and A2780S / T in the logarithmic growth phase were selected and digested with trypsin. The cells were then prepared with complete culture medium at a concentration of 5 × 10 3 The cell suspension of 100 μL / mL was inoculated into a 96-well plate, 200 μL per well, and different concentration gradients of candidate compounds were added after overnight culture. After treatment for 72 hours, the supernatant was carefully taken, and 200 μL of freshly prepared serum-free medium containing 0.2 mg / mL MTT was added to each well and cultured for 4 hours. The supernatant was carefully discarded, and 200 mL DMSO was added. After complete dissolution, the absorbance value at 570 nm was detected by a microplate reader. Repeat three times and take the average value. The inhibition rate of each experimental group was calculated according to the inhibition rate = 1-absorbance value of the control well / absorbance value of the experimental well.

[0451] After calculating the inhibition rate of each concentration of compound on cell proliferation activity, the compound treatment concentration was converted into logarithmic form, and the corresponding inhibition rate was fitted to the dose-response curve using Graphpad prism 5.0 software, and the IC 50 value.

[0452] 1.3.2 Protein degradation experiment

[0453] Breast cancer cell line MCF-7, cervical cancer cell line HeLa, colon cancer cell line HCT116, chronic leukemia cell line K562 and acute leukemia cell line Molm-13 in logarithmic growth phase were selected and digested with trypsin. The cells were then prepared with complete culture medium at a concentration of 5×10 5 Cell suspensions of 100 cells / mL were inoculated into 96-well plates, with 2 mL per well. After overnight culture, candidate compounds with different concentration gradients were added for different treatment times. Cells were collected, and total protein was extracted and then subjected to western experiments to examine the effects of the compounds on the expression levels of α-tubulin and β-tubulin.

[0454] 1.3.3 Western Blot Experiment

[0455] After treating the experimental cells according to the set treatment time, compound, and concentration, collect the supernatant culture fluid, digest the collected cells with trypsin, centrifuge the cell fluid and supernatant at 1800 rpm for 3 minutes, discard the supernatant after centrifugation, and the precipitate is the cell sample required for the experiment.

[0456] After washing the cell samples twice with cold PBS, add RIPA lysis buffer (strong) to lyse the cells. Vortex every 5 minutes. After lysing for 20 minutes, sonicate 5 times, each time for 3 seconds, with a rest period of 9 seconds.

[0457] The ultrasonic sample was centrifuged at 13000rpm and 4℃ for 20min. The supernatant was taken as the cell lysate sample. After the sample was quantified by Bradford method, SDS-PAGE protein electrophoresis loading buffer was added and placed in a water bath above 95℃ for 5min to inactivate the protein sample. The sample preparation was completed.

[0458] Gel preparation: Prepare SDS-PAGE gel of corresponding concentration according to the size of the separated target protein. Loading: For gels prepared with 15-channel combs, load 5-20μL of sample according to the concentration of the protein sample; for gels prepared with 10-channel combs, load 10-30μL of sample according to the concentration of the protein sample. Electrophoresis: Electrophoresis at 80V voltage until the sample passes through the concentrated gel, then switch to 120V voltage and continue electrophoresis until the bromophenol blue indicator band migrates to the lower end of the gel and stop electrophoresis. The gel can continue to be used for western blot experiments or Coomassie blue staining detection.

[0459] Place the gel separated by electrophoresis in the pre-prepared and pre-cooled transfer solution. Cut off the separation gel part and place it on the transfer clamp, cut the PVDF membrane of appropriate size, use 0.2μm pore size PVDF membrane to detect proteins with molecular weight <20kD; use 0.45μm pore size PVDF membrane to detect proteins with molecular weight >20kD. Soak the PVDF membrane in methanol for about 10s to activate it before use. Place the activated PVDF membrane on the gel and remove the bubbles between the gel and the membrane. Determine the transfer current conditions according to the size of the protein. For target proteins <20kD, transfer the membrane at a constant current of 350mA for 30min; for target proteins >20kD, transfer the membrane at a constant current of 250mA for 2h. After the transfer, remove the PVDF membrane and wash it briefly in TBS.

[0460] After the PVDF membrane was blocked in 5% skim milk for 1 hour, it was briefly washed in TBS / T, incubated with primary antibody at 4°C overnight, washed 3 times with TBS / T the next day, 10 minutes each time, and then incubated with secondary antibody at room temperature for 1 hour and washed several times with TBS / T, changing the solution in between, with a total washing time of more than 45 minutes. After washing, add developer and develop.

[0461] The image J software was used to analyze the grayscale values ​​of the western blot experimental results and perform quantitative analysis.

[0462] 1.3.4 Crystal structure experiments

[0463] Protein expression and purification were based on the reported literature steps. RB3-SLD was expressed in E. coli and purified by anion exchange chromatography, followed by gel filtration in 10 mM HEPES-NaOH (150 mM NaCl, 2 mM DTT, pH = 7.2). The purified RB3-SLD protein was concentrated to 10 mg / mL and stored in a -80 °C refrigerator. His-tagged TTL was also expressed in E. coli and purified by Ni 2+Purification was performed by affinity chromatography, followed by gel filtration in 20mM Bis propane buffer (200mMNaCl, 2.5mM MgCl2, 5mMβ-mercaptoethanol, 1% glycerol, pH=6.5). Finally, it was concentrated to 20mg / mL and stored in a -80℃ refrigerator. The purity of RB3 and TTL was detected by SDS-PAGE. The purchased porcine brain tubulin was dissolved in GTB buffer at a concentration of 10mg / mL and stored at -80℃. Tubulin (10mg / mL), TTL (20mg / mL) and RB3 (10mg / mL) were mixed together in a ratio of 2:1.2:1.3 (tubulin: RB3: TTL). 1mMAMPPCP, 5mM tyrosine and 10mM DTT were added thereto, and the resulting mixture was concentrated to 20mg / mL at 4℃. Crystallization was then performed by sitting drop crystallization. 1 μL of protein solution and 1 μL of solution containing 6% PEG 4000, 5% glycerol, 0.1 M MES, 30 mM CaCl2 and 30 mM MgCl2 pH = 6.7 were mixed and incubated at 20°C. The growth process of crystals was accelerated by the seeding method. Crystals appeared after incubation at 20°C and reached a length of 200-300 mm in 3-5 days. A volume of 0.1 μL of 10 mM compound (dissolved in DMSO) solution was dripped into the drop containing crystals under 20°C ambient conditions. The soaking time varied between 1 and 24 hours, and the crystals were inspected visually. The crystals were fished out with a loop soaked in cryoprotectant (8% PEG 4000, 30 mM MgCl2, 30 mM CaCl2. 0.1 M MES, pH = 6.7 and 20% glycerol). The crystals were then quickly placed in liquid nitrogen for storage. Crystal data were collected at the Shanghai Synchrotron Radiation Center, light speed line: BL19U1. Using an MX225 CCD (charge coupled device) detector, the wavelength is The data were processed using Xia2 software with reference to existing literature. The structure was solved by molecular replacement, and the model was searched by Phaser using the existing apolipoprotein structure T2R-TTL (without soaking ligand) (PDB code: 4I55). After manual modeling, it was refined using Coot and Refmac5.

[0464] 1.3.5 In vivo animal experiments

[0465] SPF Balb / c male nude mice, 6-8 weeks old and weighing 16-22g, were used to establish a breast cancer A2780S / T cell line tumor-bearing mouse model to investigate its anti-tumor effect. After subculture of the A2780S / T tumor cell line, cells with good growth status were collected, washed once with serum-free medium, counted, and the cell concentration was adjusted with serum-free medium. The counted tumor cells were inoculated subcutaneously in the right hind limb of the nude mice. When the tumor volume reached 100mm3 At 4 pm, the mice were randomly divided into groups. Paclitaxel (dissolved in 10% ethanol and 5% Tween-80 saline) was intraperitoneally injected at 24 mg / kg per week, or different doses of the compound (in saline containing 5% ethanol and 5% Tween-80) were intraperitoneally injected every two days. The tumor volume was recorded every two days.

[0466] 2 Experimental results and discussion

[0467] 2.1 Preliminary in vitro antitumor activity studies of compounds

[0468] First, the synthesized target compounds 2a-y were screened for their in vitro antitumor activity. Five different human tumor cell lines were selected: breast cancer cell line MCF-7, cervical cancer cell line HeLa, colon cancer cell line HCT116, chronic leukemia cell line K562 and acute leukemia cell line Molm-13, and screened by MTT method. The screening results are shown in Table 1.

[0469] Table 1. Activity of compounds 2a-y on five tumor cell lines

[0470]

[0471]

[0472]

[0473]

[0474] a The half-inhibitory concentration after 72 h of drug treatment. Data represent three independent experiments, and values ​​are expressed as mean ± SEM. PTX refers to paclitaxel.

[0475] In vitro activity studies have shown that when the methoxy group at position 3 is replaced by smaller substituents such as acetyl (2a), isopropyl (2b), acetylamino (2c) and cyano (2d) or larger substituents (2f, 2g), the activity is greatly reduced (>2μM), because N165 creates steric hindrance. The in vitro antitumor activity of the N,N-dimethyl substituted product (2e) is also somewhat reduced compared with BML284 (0.28-0.55μM). Since the two methyl groups on the nitrogen atom can rotate freely, the steric hindrance caused by N165 can be minimized, so its activity loss is less than that of 2a-d. When different substituents are introduced at position 4 of the benzene ring (2h-o), the activity is significantly reduced. When the carbon atom at position 4 is replaced by a nitrogen atom (2h), and when a smaller substituent such as a fluorine atom (2i) or methyl (2j) is introduced at position 4, the hydrophobic interaction between it and the surrounding amino acids is weakened, so the activity is reduced. When a larger substituent (2k-o) is introduced at the 4-position, the steric hindrance of Q134 increases, and the activity decreases. When multiple substituents (2p-t) are introduced at different positions of the benzene ring, only the activity of 2q is maintained. Compared with BML284, compound 2s only introduces a smaller fluorine atom at the 2-position of the benzene ring, and the activity decreases by 2-9 times. This is because the hydroxyl group of Y200 is closer to the fluorine atom than to the nitrogen atom at the 1-position of the pyrimidine ring ( vs ), preferentially forming hydrogen bonds with fluorine atoms, destroying the hydrogen bond chelation effect between the nitrogen atom at position 1 and the amino group at position 2 and E198 and Y200, thus reducing the activity. The 3,4-dimethoxy group of compound 2p was cyclized to obtain compounds 2u and 2v, and the activity was restored, but it was not improved compared with BML284. The 2- and 3-position cyclizations of the benzene ring gave 2w-y, and the activity of 2y was significantly increased by 5 to 20 times, the activity of 2w was maintained, and the activity of 2x was significantly reduced. Since the N-methyl group of pyrazole is sterically hindered by T166, the activity is greatly lost. The hydrogen atoms on the nitrogen in the saturated tetrahydropyrrole ring or the unsaturated pyrrole ring can form hydrogen bonds with the hydroxyl oxygen of Y200. Since the pyrrole ring extends to β-tubulin, it can form an edge-to-faceπ-π interaction with F167, so the activity of 2y is significantly improved.

[0476] Table 2. Activity of compounds 3a-h on five tumor cell lines

[0477]

[0478]

[0479] a The half-inhibitory concentration after 72 h of drug treatment. Data represent three independent experiments, and values ​​are expressed as mean ± SEM. PTX refers to paclitaxel.

[0480] As shown in Table 2, the removal of the methoxy group at the 3rd position of the benzene ring weakened the hydrophobic interaction with the surrounding amino acid residues, and the activity of 3a decreased by 1-5 times. Further replacing the benzene ring of 3a with a smaller furan ring (3b) or a larger naphthalene ring (3c, 3d), 9,9-dimethylfluorene ring (3f), the hydrophobic interaction between the furan ring and the surrounding amino acid residues was weakened, and the 1-naphthyl group offset each other under the dual effects of hydrophobic interaction and steric hindrance. Therefore, although the activity of 3b and 3c decreased, it was generally maintained. The 2-naphthyl and 9,9-dimethylfluorene groups were mainly affected by the steric hindrance from Q134, so the activity was greatly reduced. The methoxy group at the 2nd position of 3e is small and can rotate, and is less affected by the steric hindrance from Q134, so the activity is maintained. The activity of compound 3h was significantly improved.

[0481] Table 3. Activity of compounds 5a-j on five tumor cell lines

[0482]

[0483]

[0484] a The half-inhibitory concentration after 72 h of drug treatment. Data represent three independent experiments, and values ​​are expressed as mean ± SEM. PTX refers to paclitaxel.

[0485] In order to investigate the effect of hydrogen bonding between the oxygen atom of the piperonyl ring and T179 on the activity, the present invention synthesized compounds 5a-j. The activity of compound 5f with an open dioxolane ring and compound 5i with an indole ring replacing the piperonyl ring was significantly increased. The methoxy oxygen atom at position 3 of 5f can still form a hydrogen bond with the hydroxyl hydrogen of T179, and the methoxy group at position 4 increases the hydrophobic interaction between it and the surrounding amino acid residues. The hydrogen atom on the indole nitrogen of 5i can form a hydrogen bond with the carbonyl oxygen of T179, and the pyrrole ring extending to α-tubulin increases the hydrophobic interaction between it and the surrounding amino acid residues, so the activity is significantly improved. After replacing the dioxolane ring with one or more fluorine atoms and trifluoromethoxy, the fluorine atom can still form a hydrogen bond with the hydroxyl hydrogen of T179 (5b-e), so the activity has almost no significant change. After introducing 4-methoxyphenyl and 3,4,5-trimethoxyphenyl to replace the piperonyl ring, the activity has almost no change (5g, 5h). The activity of the dioxolane structure (5a) without the piperonyl ring is 2-5 times lower than that of BML284, indicating that the hydrogen bond between the oxygen atom of the piperonyl ring and T179 has a certain influence on the activity, but it is not the most important factor. The activity of the product 5j, in which the piperonyl ring is replaced by a naphthalene ring, is not much different from that of BML284, further proving that the hydrogen bond with T179 is not critical to the activity. At the same time, the activities of 5g, 5h and 5j are compared with those of BML284, indicating that the steric hindrance of the surrounding amino acid residues is not greatly affected by the small hydrophobic substituents connected to the 3, 4 and 5 positions of the benzene ring of the piperonyl ring.

[0486] Table 4 Activity of compounds 6a-d on five tumor cell lines

[0487]

[0488]

[0489] a The half-inhibitory concentration after 72 h of drug treatment. Data represent three independent experiments, and values ​​are expressed as mean ± SEM. PTX refers to paclitaxel.

[0490] The amino acid residues around the -NH group at position 4 of the pyrimidine ring of BML284 include V236, C239, I316 and I376. In order to enhance the hydrophobic interaction between it and the surrounding amino acid residues, the present invention introduces a smaller alkyl substituent on the nitrogen atom. The results show that the activities of compounds 6a-d are significantly improved (Table 4), especially compound 6b, whose activity is increased by 2-11 times. This proves that the introduction of a smaller hydrophobic substituent at this position to increase the hydrophobic interaction between it and the surrounding amino acid residues is beneficial to the improvement of the activity of BML284.

[0491] In view of the fact that the activities of compounds 5f and 6b are significantly improved compared with BML284, the present invention retains the 3,4-dimethoxyphenyl structure of 5f and cyclizes the N-substituted ethyl of 6b to the 6-position of the benzene ring ( Figure 1 ), compound 8 was synthesized. In order to investigate the effect of the amino group at position 2 and the nitrogen atom at position 1 of the pyrimidine ring of BML284 on the activity, the present invention synthesized compounds 10 and 13 ( Figure 1 ). Its in vitro antitumor activity is shown in Table 5.

[0492] Table 5 Activity of compounds 10, 13, 18-21 on five tumor cell lines

[0493]

[0494]

[0495] a The half-inhibitory concentration after 72 h of drug treatment. Data represent three independent experiments, and values ​​are expressed as mean ± SEM. PTX refers to paclitaxel.

[0496] As shown in Table 5, the in vitro antitumor activity of compound 8 was significantly lower than that of 5f and 6b, and its activity was roughly equivalent to that of BML284, which may be due to the fact that the piperidine ring after cyclization cannot form hydrophobic interactions with the surrounding amino acid residues. The in vitro activity of compound 10 without the 2-amino group was lost by nearly 30 times, indicating that the hydrogen bond interaction between the 2-amino group and E198 plays a decisive role in stabilizing the small molecule conformation, thereby directly affecting its activity. Compounds 13 and 15 completely lost their activity, indicating that in the hinge region, the hydrogen bond formed between the nitrogen atom at position 1 and E198 and Y200 also plays a key role in stabilizing the small molecule conformation, determining whether the small molecule can enter the binding pocket and the binding affinity in the binding pocket.

[0497] Through the above structure-activity relationship discussion, the present invention obtained compounds 2y, 3h, 5f, 5i, 6a, 6b, 6c and 21 with significantly improved in vitro activity compared with BML284. After comprehensively considering their physicochemical properties and pharmacokinetic properties, the present invention selected compound 21 for further study.

[0498] 2.2 Study on the activity of compounds in promoting tubulin degradation

[0499] BML284 can promote tubulin degradation. An oxygen atom on its piperine ring forms a hydrogen bond interaction with T179, the nitrogen atom at position 1 has hydrogen bond interactions with E198 and Y200, and the amino group at position 2 has hydrogen bond interactions with E198. The 3-methoxybenzene ring has hydrophobic interactions with the hydrophobic pocket composed of N165, F167, E198, Y200, V236 and L240. The previous QM / MM calculation study of the present invention showed that the low barrier hydrogen bond formed between the nitrogen atom at position 1 of the pyrimidine ring of BML284 and E198 may be the key group for promoting tubulin. In order to further explore whether this calculation result is correct, the present invention investigated the activity of compounds 21, 3a, 5a, 6a, 10 and 13 in promoting tubulin degradation. The results are as follows: Figure 2 As shown. Compound 21 significantly degrades tubulin at a concentration of 50 μM. 3a can promote tubulin degradation, indicating that the hydrophobic interaction between the 3-methoxybenzene ring and the hydrophobic pocket composed of N165, F167, E198, Y200, V236 and L240 is not a factor that promotes tubulin degradation. 5a can also promote tubulin degradation, indicating that the hydrogen bond interaction between the oxygen atom on the pepper ring and T179 is not a key factor in promoting tubulin degradation. 6a can also promote tubulin degradation, indicating that the introduction of a smaller hydrophobic alkyl group on the -NH group will not affect its activity in degrading tubulin. Compound 10 can still promote tubulin degradation at a high concentration (50 μM), indicating that the hydrogen bond between the amino group at position 2 of the pyrimidine ring and E198 is also not a key factor in promoting tubulin degradation. Compound 13 could not promote tubulin degradation even at a high concentration of 50 μM, which indicates that the hydrogen bond formed between the nitrogen atom at position 1 and E198 and Y200 is the key factor in promoting tubulin degradation.

[0500] 2.3 Study on the anti-resistance activity of compound 21 in vitro

[0501] At present, the phenomenon of multidrug resistance (MDR) generated during chemotherapy is the main reason for the failure of first-line chemotherapy drugs. The common resistance mechanisms identified in preclinical or clinical studies include two major categories: one is the transitional expression of a cell membrane protein called P-glycoprotein (P-gp); the other is the change in the expression level of different types of β-tubulin controlled by the β-Ⅲ gene. It is well known that paclitaxel is resistant to A549 / T (human lung cancer cell line) with overexpression of P-gp, vinblastine is resistant to HCT-8 / V (human colon cancer cell line) with overexpression of P-gp, doxorubicin is resistant to MCF-7 / ADR (human breast cancer cell line) with overexpression of P-gp, and paclitaxel is resistant to A2780 / T (human ovarian cancer cell line) with overexpression of β-tubulin III. 21 has shown good activity on non-resistant tumor cell lines (IC 50 =7.8±0.1 (HeLa), 3.2±0.0 (HCT116), 4.8±0.3 (MCF-7), 3.3±0.4 (K562), 6.5±0.3 (Molm-13 nM). The present invention further investigated whether the preferred compound 21 can overcome drug resistance. The investigation results are shown in Table 6.

[0502] Table 6 Proliferation inhibition activity of compound 21 on drug-resistant cells

[0503]

[0504] a The half-inhibitory concentration after 72 h of drug treatment. The data represent three independent experiments, and the values ​​are expressed as mean ± SEM. PTX refers to paclitaxel; b Drug resistance index: (IC on drug-resistant cells 50 Value / IC on drug-resistant cells 50 ); c ND means not tested

[0505] The results of multidrug resistance showed that 21 showed good cell activity in both sensitive and resistant cell lines. The IC 50The values ​​were 5.9±1.6, 8.0±1.9 and 6.8±nM, respectively. The resistance index of 21 was 0.9 (A2780S / T group), 0.8 (A549 / T group), and 0.7 (MCF-7 / ADR group), which were much smaller than 958.1 (A2780S / T group), 35.3 (A549 / T group), and 296.1 (MCF-7 / ADR group) of paclitaxel and 299.7 (MCF-7 / ADR group) of doxorubicin. These results indicate that 21 can overcome the resistance caused by overexpression of β-tubulin III and P-gp.

[0506] 2.4 In vivo antitumor activity study of compound 21

[0507] The pharmacokinetic study of compound 21 showed that tail vein administration was a relatively better administration method, so the present invention selected tail vein injection as the administration method when investigating the anti-tumor activity of different doses of 21 (30, 60 mg / kg) on ​​the A2780T model. Figure 3 shown.

[0508] Figure 3 A shows that the tumor inhibition rate of 21 at 20 mg / kg is 35.80%, showing a certain tumor inhibition activity. The tumor inhibition rate of 21 at 40 mg / kg reaches 64.33%, which is better than 10 mg / kg of paclitaxel (tumor inhibition rate 4.89%) administered once a week. This is because paclitaxel is resistant to A2780T (human ovarian cancer cell line) that overexpresses β-tubulin III, and its dosage of 10 mg / kg is relatively low, and the administration cycle is relatively long. Figure 3 As can be seen from B, there was no significant change in the body weight of mice after administration of 21 and paclitaxel, indicating that 21 was less toxic to mice at a higher dose of 40 mg / kg. Figure 3 As can be seen intuitively in C and D, at the two doses of 20 mg / kg and 40 mg / kg, 21 has a significant inhibitory effect on the volume and weight of the tumor compared with the blank control. The above in vivo experiments have proved that 21 can significantly inhibit the growth of A2780T tumors, that is, 21 can overcome the drug resistance caused by β-tubulin III overexpression, and its toxicity is relatively small.

[0509] 3 Summary

[0510] Anticancer drugs targeting microtubules can prevent the mitosis of tumor cells by destroying the normal polymerization or depolymerization of microtubules in tumor cells, causing them to stagnate in the G2 / M phase, thereby inducing apoptosis of tumor cells. At the same time, this type of compound can also inhibit the function of microtubules, limit the proliferation, migration and angiogenesis of endothelial cells, and inhibit tumor angiogenesis. Microtubules have always been an important target in the field of anti-tumor drug research. However, while existing anti-microtubule drugs achieve therapeutic effects, they will have significant toxic side effects on normal tissues and the nervous system, and are very likely to induce tumor resistance. Therefore, it is of great clinical significance to develop anti-tumor microtubule drugs that are low in toxicity, high in efficiency, economical, more targeted, and can overcome drug resistance.

[0511] Based on the crystal complex structure of BML284 and tubulin in previous studies, the present invention has carried out structural modification and equivalent replacement of the piperine ring, pyrimidine ring and benzene ring, synthesized a series of BML284 derivatives or analogs, carried out a more detailed structure-activity relationship discussion, and selected compound 21 through in vitro anti-tumor activity experiments. Further studies have shown that compound 21 can promote tubulin degradation through the ubiquitin-proteasome pathway, and in vitro anti-resistance experimental studies have shown that it can overcome drug resistance. In vivo experimental studies also show that 21 can overcome drug resistance. 40 mg / kg of 21 administered once every two days through the tail vein can effectively inhibit the growth of drug-resistant tumor A2780T, and the toxic side effects are relatively small. This provides a more reliable basis for further developing new tubulin degraders that can overcome drug resistance based on the colchicine site.

[0512] In summary, the present invention provides a new class of compounds having anti-tumor effects, among which compounds 2y, 3h, 5f, 5i, 6a, 6b, 6c and 21 have better anti-tumor effects than the existing compound BML284. Drug resistance experiments show that the compounds of the present invention (especially compounds 21, 3a, 5a, 6a) can effectively promote microtubule degradation and can be used to treat drug-resistant tumors. Further drug resistance experiments show that the compounds of the present invention (especially compound 21) have excellent therapeutic effects on drug-resistant tumors and can overcome the drug resistance caused by overexpression of β-tubulin III and P-gp.

Claims

1. The compound represented by formula I, its salt, its stereoisomer, its crystal form, its hydrate, and its solvate: in, X is selected from CR5, N; R5 is selected from hydrogen, C1-C6 alkyl; R1 is selected from a 6- to 15-membered aryl group substituted by n R6 groups, and a 5- to 10-membered heteroaryl group substituted by n R6 groups; n is an integer from 0 to 5; Each R6 is independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, cyano, 6-15 membered aryl substituted by m R7, -C(O)R8, -NR9C(O)R8, -NR9R 10 、-O(CH2) a R 11 ; m is an integer from 0 to 5; Each R7 is independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, and cyano; R8, R9, R 10 are independently selected from hydrogen, C1-C6 alkyl; a is an integer from 0 to 10; R 11 Selected from hydrogen, o R 12 Substituted 6- to 15-membered aryl; o is an integer from 0 to 5; Each R 12 Each independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, and cyano; R2 is selected from hydrogen, -NR 13 R 14 ; R 13 , R 14 are independently selected from hydrogen, C1-C6 alkyl; R3 is selected from hydrogen, C1-C6 alkyl, C1-C6 alkenyl; R4 is selected from (CH2) b R 15 ; b is an integer from 1 to 10; R 15 Selected from p R 16 substituted 6- to 15-membered aryl, with p R 16 Substituted 5- to 10-membered heteroaryl; p is an integer from 0 to 5; Each R 16 Each independently selected from C1-C6 alkyl, halogen, -OR 17 ; R 17 Selected from C1-C6 alkyl, trifluoromethyl; Or R3 and R4 are connected to form q R 18 Substituted 6- to 15-membered aromatic group, q R 18 Substituted 5- to 10-membered heteroaryl; q is an integer from 0 to 5; Each R 18 Each independently selected from C1-C6 alkyl, C1-C6 alkoxy, and halogen; The compound is not 2. The compound, salt, stereoisomer, crystal form, hydrate or solvate thereof according to claim 1, characterized in that: The compound is shown in formula II: in, X is selected from CR5, N; R5 is selected from hydrogen, C1-C6 alkyl; R1 is selected from a 6- to 15-membered aryl group substituted by n R6 groups, and a 5- to 10-membered heteroaryl group substituted by n R6 groups; n is an integer from 0 to 5; Each R6 is independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, cyano, 6-15 membered aryl substituted by m R7, -C(O)R8, -NR9C(O)R8, -NR9R 10 、-O(CH2) a R 11 ; m is an integer from 0 to 5; Each R7 is independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, and cyano; R8, R9, R 10 are independently selected from hydrogen, C1-C6 alkyl; a is an integer from 0 to 10; R 11 Selected from hydrogen, o R 12 Substituted 6- to 15-membered aryl; o is an integer from 0 to 5; Each R 12 Each independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, and cyano; R2 is selected from hydrogen, -NR 13 R 14 ; R 13 , R 14 are independently selected from hydrogen, C1-C6 alkyl; R3 is selected from hydrogen, C1-C6 alkyl, C1-C6 alkenyl; The compound is not 3. The compound, salt, stereoisomer, crystal form, hydrate or solvate according to claim 2, characterized in that: X is selected from CH, N; R1 is selected from the following groups substituted by n R6: phenyl, naphthyl, pyridyl, furyl, n is 0, 1, 2, 3, 4; Each R6 is independently selected from C1-C4 alkyl, C1-C4 alkoxy, halogen, cyano, phenyl substituted by m R7, -C(O)R8, -NR9C(O)R8, -NR9R 10 、-O(CH2) a R 11 ; m is 0, 1, 2, 3, or 4; Each R7 is independently selected from C1-C4 alkyl, C1-C4 alkoxy, halogen, and cyano; R8, R9, R 10 are independently selected from hydrogen, C1-C4 alkyl; a is 0, 1, 2, 3, 4; R 11 is selected from hydrogen, phenyl; R2 is selected from hydrogen, -N(H)R 13 ; R 13 Selected from hydrogen, C1-C4 alkyl; R3 is selected from hydrogen, C1-C3 alkyl, and C1-C3 alkenyl.

4. The compound, salt, stereoisomer, crystal form, hydrate or solvate thereof according to claim 3, characterized in that: The compound is shown in formula III: in, R1 is selected from the following groups substituted by n R6: phenyl, naphthyl, pyridyl, furyl, n is 0, 1, 2, 3, 4; Each R6 is independently selected from C1-C4 alkyl, C1-C4 alkoxy, halogen, cyano, phenyl substituted by m R7, -C(O)R8, -NR9C(O)R8, -NR9R 10 、-O(CH2) a R 11 ; m is 0, 1, 2, 3, or 4; Each R7 is independently selected from C1-C4 alkyl, C1-C4 alkoxy, halogen, and cyano; R8, R9, R 10 are independently selected from hydrogen, C1-C4 alkyl; a is 0, 1, 2, 3, 4; R 11 is selected from hydrogen, phenyl; The compound is not 5. The compound, salt, stereoisomer, crystal form, hydrate or solvate thereof according to claim 1, characterized in that: The compound is shown in formula IV: in, R 15 Selected from p R 16 substituted 6- to 15-membered aryl, with p R 16 Substituted 5- to 10-membered heteroaryl; p is an integer from 0 to 5; Each R 16 Each independently selected from C1-C6 alkyl, halogen, -OR 17 ; R 17 Selected from C1-C6 alkyl, trifluoromethyl; The compound is not 6. The compound, salt, stereoisomer, crystal form, hydrate or solvate according to claim 5, characterized in that: R 15 Selected from p R 16 Substituted with the following groups: phenyl, naphthyl, pyridyl, furyl, p is 0, 1, 2, 3, 4; Each R 16 Each independently selected from C1-C4 alkyl, halogen, -OR 17 ; R 17 Selected from C1-C4 alkyl, trifluoromethyl.

7. The compound, salt, stereoisomer, crystal form, hydrate or solvate thereof according to claim 1, characterized in that: The compound is represented by formula V: in, R3 is selected from C1~C3 alkyl and C1~C3 alkenyl.

8. The compound, salt, stereoisomer, crystal form, hydrate or solvate thereof according to claim 1, characterized in that: The compound is represented by formula VI: in, R1 is selected from the following groups substituted by n R6: phenyl, naphthyl, pyridyl, furyl, n is 0, 1, 2, 3, 4; Each R6 is independently selected from C1-C4 alkyl, C1-C4 alkoxy, halogen, and cyano; Alternatively, the compound is represented by formula VII: in, R3 is selected from hydrogen; R4 is selected from (CH2) b R 15 ; b is 1; R 15 Selected from Or R3 and R4 are connected to form 9. The compound, salt, stereoisomer, crystal form, hydrate or solvate thereof according to any one of claims 1 to 8, characterized in that: The compound is one of the following structures:

10. Use of the compound according to any one of claims 1 to 9, its salt, its stereoisomer, its crystal form, its hydrate or its solvate in the preparation of a microtubule protein degrading agent.

11. Use of the compound according to any one of claims 1 to 9, its salt, its stereoisomer, its crystal form, its hydrate or its solvate in the preparation of a medicament for preventing and / or treating cancer.

12. The use according to claim 11, characterized in that: The cancers are cervical cancer, colorectal cancer, breast cancer, leukemia, lung cancer, cervical cancer, lymphoma, and ovarian cancer.

13. Use of the compound, salt, stereoisomer, crystal form, hydrate or solvate of any one of claims 1 to 9 in the preparation of ADC antibody-drug conjugates; Preferably, the compound, salt, stereoisomer, crystal form, hydrate or solvate according to any one of claims 1 to 9 is a small molecule toxic compound in an ADC antibody-drug conjugate.

14. A drug, characterized in that: The invention relates to a preparation prepared by taking the compound, salt, stereoisomer, crystal form, hydrate or solvate as claimed in any one of claims 1 to 9 as active ingredients and adding pharmaceutically acceptable auxiliary materials.

15. An ADC antibody-drug conjugate, characterized in that: The invention is prepared by using the compound, salt, stereoisomer, crystal form, hydrate or solvate described in any one of claims 1 to 9 as a small molecule toxic compound.

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