CN115010711A - Pteridine 7(8H) -ketone compound and application thereof in pharmacy - Google Patents

Abstract

The invention discloses a pteridine 7(8H) -ketone compound and application thereof in pharmacy, belonging to the field of medicines. The pteridine 7(8H) -ketone derivative or the stereoisomer or the pharmaceutically acceptable salt thereof has a structure shown in a general formula (I) or (II), has better inhibitory activity on CDK2 kinase, has a certain degree of anti-tumor cell proliferation effect on HCT 116 and MV4-11 cell lines, can provide reference for CDK2 small molecule inhibitor research, and has very wide application prospect.

Description

Pteridine 7(8H) -ketone compound and application thereof in pharmacy

Technical Field

The invention relates to a pteridine 7(8H) -ketone compound and application thereof in pharmacy, belonging to the field of medicines.

Background

Cancer is one of the most serious life-threatening diseases, and the cause thereof is complicated, and it is likely to be induced by damage or mutation of cellular genetic material caused by genetic factors and external environmental factors. The treatment methods of cancer can be classified into conventional therapies represented by radiotherapy, chemotherapy, surgical treatment or combination of the three therapies and modern therapies represented by targeted therapy and immunotherapy. In the last decades, the efficiency of traditional treatment is improved with the development of science and technology, but the clinical application of traditional treatment is limited due to the defects of high toxic and side effects, poor tolerance of patients, adverse prognosis effects and the like. Therefore, the research on clinical drugs with more specific effects and fewer side effects is continuously conducted, and with the deep research and understanding on the pathogenesis of cancer, a targeted therapy specific to the key protein of cancer is generated. Traditional chemotherapy usually targets rapidly proliferating cancer cells by interfering with cell division, but this also targets rapidly dividing healthy cells non-specifically, creating well-known chemotherapy side effects; the molecular targeted therapy is used as a revolutionary therapy, and aims to block a specific biological transduction pathway or key cancer protein which participates in the growth and development of tumors, intervene in the regulation of cell cycles or induce cell death, and achieve the anti-tumor effect. Drugs for molecular targeted therapy are mainly classified into small molecules, monoclonal antibodies, immunotherapeutic cancer vaccines and gene therapy. Molecularly targeted drugs often exhibit different functions and properties, depending on the target, they act on cell surface antigens, growth factors, receptors or signal transduction pathways, regulating cell cycle progression, apoptosis, metastasis or angiogenesis. The identification of an ideal target is therefore crucial for the successful development of cancer molecule-targeted therapies. The mechanism of tumor development and development is continuously studied and understood, and the molecular and gene level has been deeply reached, so that the target for molecular targeted therapy is various and increasing, and mainly comprises cyclin, growth factors, signal molecules, apoptosis regulating factors, angiogenesis promoting molecules and the like.

The cell cycle, which is a fundamental process in the operation of cell life, is regulated by a variety of protein molecules. The most prominent pathological manifestation of cancer is that cell cycle regulation is disturbed, resulting in uncontrolled cell differentiation and apoptosis, resulting in unlimited proliferation of cells. With the intensive research and understanding on the molecular mechanism of cell cycle regulation, among many targets involved in regulating the cell cycle of tumors, Cyclin Dependent Kinases (CDKs) that promote cell cycle transition become key cancer therapeutic targets.

As a member of the CDKs family, cyclin dependent kinase 2(CDK2) binding to cyclins may be directly involved in regulating the cell cycle. In eukaryotic cells, CDK2 is a key cell cycle regulator; retinoblastoma protein (pRb) which inhibits the transcriptional activity of transcription factor (E2Fs) is a key substrate of CDK2 during G1/S phase. Late in G1 (after restriction enzyme cleavage), the activated CDK2-cyclin E complex and CDK 4/6-cyclin D complex together phosphorylate pRb, releasing and activating E2Fs, which initiates transcription of genes required for S phase. Throughout the S phase, CDK2-cyclinA complex and CDK1-cyclinA/B complex continued to maintain pRb phosphorylation, ensuring cell cycle progression. When the cells are ready to exit S phase, CDK2, upon binding to cyclin a, phosphorylates the transcription factor E2Fs, inactivating it, thereby preventing apoptosis triggered by sustained activity of E2 Fs. CDK2 controls the phosphorylation of various transcription factors in addition to pRb, such as SMAD3, FOXM1, FOXO1, as well as Upstream Binding Factors (UBF) and Myc proto-oncogene proteins, etc., which are involved in cell cycle progression at various levels. In addition to these cell cycle targets, CDK2 is involved in mammalian DNA replication, adaptive immune responses, cell differentiation, and apoptosis. There is increasing evidence that CDK2 is a key regulator of a variety of oncogenic signals. CDK2 is statistically overexpressed in about 86% of cancers, including liver, stomach, breast, colon, leukemia, prostate, and other tumors. Studies on the sensitivity of various human cancers with specific molecular highlights to CDK2 inhibition revealed that CDK2 might be an excellent therapeutic target. For example, CDK2 may be a therapeutic target in ovarian cancer cells with overexpression of cyclin E1, MYCN-expanded neuroblastoma cells, KRAS-mutated lung cancer cells, and various cancer cells with FBW7 mutations and overexpression of cyclin E1. CDK2 is highly expressed and essential for tumor cell proliferation in B cell lymphomas and glioblastomas. CDK2 was found to be significantly associated with metastasis of cancer cells in prostate cancer-related studies. CDK2 may also play a role in the development of breast cancer by phosphorylating and activating kinase receptors. Whereas inhibition of CDK2 induced differentiation in primary samples and cell lines of AML patients. In the process that cyclin D participates in normal ubiquitination as a proteasome degradation target, ubiquitin ligase (E3) takes AMBRA1 protein as a substrate receptor. In human tumor cells, the AMBRA1 protein is often mutated. Maiani E et al demonstrated through a series of molecular biology, cell biology and genetic experiments that deletion of AMBRA1 protein leads to increase of cyclin D level and promotes formation of cyclin D-CDK2 complex. Over time, the cytostatic effects of marketed CDK4/6 inhibitors are inevitably limited by primary and acquired resistance; recent research shows that CDK2, cyclin E and cyclin D are combined to form a complex as a compensation mechanism to activate a downstream signal path, and theoretically, specific targeting CDK2 can achieve the effect of resisting tumor cell proliferation. These all fully suggest that CDK2 is an important target for the study of antitumor drugs.

With the intensive research and understanding on the structure and function of CDK2 and the discovery of the potential capability of CDK2 in overcoming the drug resistance problem of CDK4/6 inhibitors in recent years, the design and development work of CDK2 small-molecule targeted inhibitors is greatly promoted. Although no specifically targeted CDK2 inhibitors are currently officially approved for marketing, several small molecule CDK2 inhibitors are already in clinical research. Most of the CDK2 inhibitors currently in clinical research belong to non-selective ATP competitive inhibitors, which act mainly on the ATP binding site of CDK2 and competitively bind CDK2 with ATP, thereby inhibiting its activity, i.e., effectively preventing tumor cell proliferation or promoting its apoptosis. Representative drugs include aminopurines, pyridopyrimidines, aminopyrazoles and pyrazoloquinazolines, which have all achieved some success in the treatment of cancer. In addition, there are many compounds which are in preclinical research stage and have high selective inhibitory activity against CDK2, mainly purines, thiazoles and indoles.

Based on the importance of CDK2 activity in cell cycle regulation and the critical role of this signaling pathway in the development of carcinogenesis, targeted inhibition of CDK2 activity is a potential therapeutic modality. In order to better meet the clinical requirements and treat cancers, a new CDK2 inhibitor with better curative effect and lower toxic and side effects needs to be developed.

Disclosure of Invention

In order to solve the above problems, the present invention is to provide a CDK2 inhibitor having a novel structure, and find that compounds having such a structure show excellent effects and actions, providing more therapeutic approaches for cancer treatment.

The first purpose of the invention is to provide pteridine 7(8H) -ketone derivatives with the structure shown in the general formula (I) or the general formula (II) or stereoisomers or pharmaceutically acceptable salts thereof,

or

Wherein R is ₁ 、R ₁ ' are each independently selected from unsubstituted or halogen-substituted C1-C8 straight or branched chain alkyl, unsubstituted or halogen-substituted C3-C6 cycloalkyl;

R ₂ is hydroxy, methoxy, -NHR _a Or are each

R _a Is H, C1-8 alkyl, NH ₂ ；R ₄ ' is C1-C8 alkyl, C1-C8 alkyl-substituted primary or secondary amino, C3-C6 cycloalkyl;

R ₃ 、R ₃ ' Each is independently selected from-NHR _b 、

R _b Is H, C1-8 alkaneRadical, NH ₂ ；R ₄ And R ₄ ' are each independently selected from C1-C8 alkyl, C1-C8 alkyl substituted primary or secondary amino, C3-C6 cycloalkyl.

In one embodiment of the invention, R ₁ 、R ₁ ' Each independently preferably represents a butyl group, a pentyl group, a pyranyl group, a pyrrolidinyl group, a cyclopentanol group, or a cyclopentyl group.

In one embodiment of the invention, R ₂ The specific preference is as follows: methylamino, ethylamino, hydroxy, methoxy, difluoroethylamino, hydrazino-NH ₂ 、

In one embodiment of the invention, R ₃ 、R ₃ ' is preferably selected from:

in one embodiment of the invention, R ₄ 、R ₄ ' preferably C1-C8 alkyl.

In one embodiment of the invention, when R is ₃ And R ₃ ' is a

When R is ₄ Is methyl;

when R is ₃ And R ₃ ' is a

When R is ₄ ' is C1-C8 alkyl, C1-C8 alkyl-substituted primary or secondary amino, C3-C6 cycloalkyl.

In one embodiment of the invention, the pteridine 7(8H) -one derivatives may be selected from the compounds specified in table 1 below:

TABLE 1

In one embodiment of the invention, the pharmaceutically acceptable salt comprises: potassium salt, sodium salt, hydrochloride, formate, trifluoroacetate, phosphate, sulfate, and the like.

The second purpose of the invention is to provide a composition containing the pteridine 7(8H) -ketone derivative or the stereoisomer or the pharmaceutically acceptable salt thereof.

In one embodiment of the present invention, the composition contains the pteridine 7(8H) -ketone derivative or its stereoisomer or its pharmaceutically acceptable salt, and pharmaceutically acceptable excipients.

In one embodiment of the present invention, the pharmaceutically acceptable carrier refers to pharmaceutical excipients conventional in the pharmaceutical field, such as: diluents, excipients, fillers, binders, wetting agents, absorption enhancers, water and the like, the fillers being: starch, sucrose, lactose, microcrystalline cellulose, and the like; binders such as cellulose derivatives, alginates, gelatin, and polyvinylpyrrolidone; humectants such as glycerol; disintegrating agents such as sodium carboxymethyl starch, hydroxypropylcellulose, agar, calcium carbonate and sodium bicarbonate; absorption enhancers such as quaternary ammonium compounds; surfactants such as cetyl alcohol, sodium lauryl sulfate.

In one embodiment of the present invention, the composition comprises the pteridine derivatives or the stereoisomers or the pharmaceutically acceptable salts thereof as described above and at least one pharmaceutically acceptable excipient or carrier.

In one embodiment of the present invention, the pteridine 7(8H) -one derivative or a stereoisomer thereof or a pharmaceutically acceptable salt thereof is prepared by the following reaction formula:

wherein, in the above reaction formula, R ₁ And R ₁ ’、R ₂ 、R ₃ And R ₃ ’、R ₄ And R ₄ ' same as defined above; r is C1-C8 alkane.

The third object of the present invention is to provide a use of the pteridine 7(8H) -one derivatives or stereoisomers thereof or pharmaceutically acceptable salts thereof in the preparation of CDK2 inhibitors.

The fourth purpose of the invention is to provide the use of the pteridine 7(8H) -ketone derivatives or the stereoisomers or the pharmaceutically acceptable salts thereof in preparing the medicines for preventing or treating cancers.

In one embodiment of the invention, the cancer is selected from breast cancer, colon cancer, rectal cancer, kidney cancer, epidermal cancer, liver cancer, lung cancer, esophageal cancer, gallbladder cancer, ovarian cancer, pancreatic cancer, stomach cancer, cervical cancer, thyroid cancer, nasal cancer, head and neck cancer, prostate cancer, skin cancer, hematopoietic tumors of myeloid lineage, follicular thyroid cancer, tumors derived from mesenchymal cells, tumors of the central or peripheral nervous system, melanoma, glioma, seminoma, teratoma, osteosarcoma, xeroderma pigmentosum, keratoblastoma, follicular thyroid cancer, or kaposi's sarcoma.

Has the advantages that:

the pteridine derivatives or the stereoisomers or the pharmaceutically acceptable salts thereof have very good CDK2 inhibitory activity on CDK2, and can be used as a high-efficiency CDK2 inhibitor. The compounds claimed in the present invention have strong potency and selectivity for CDK2, which is advantageous in the development of a pharmaceutical suitable for use as CDK2 inhibitors, with a very broad potential for use.

Detailed Description

The term "alkyl" as used herein refers to a straight or branched chain saturated hydrocarbon group. In some embodiments, the alkyl group can have 1 to 10 carbon atoms (e.g., 1 to 8 carbon atoms). Examples of alkyl groups include methyl (Me), ethyl (Et), propyl (e.g., n-propyl and isopropyl), butyl (e.g., n-butyl, isobutyl, sec-butyl, tert-butyl), pentyl groups (e.g., n-pentyl, isopentyl, neopentyl), hexyl (e.g., n-hexyl and its isomers), and the like. The lower alkyl group typically has up to 4 carbon atoms. Examples of lower alkyl groups include methyl, ethyl, propyl (e.g., n-propyl and isopropyl), and butyl groups (e.g., n-butyl, isobutyl, sec-butyl, tert-butyl). In one embodiment one alkyl group or two or more alkyl groups may form a bridged alkyl group. I.e. wherein the alkyl groups are linked via another group (shown specifically as a cyclic group), are bridged by an alkyl chain to form a ring, i.e. to form a bridged fused ring.

As used herein, "cycloalkyl" refers to non-aromatic carbocyclic groups and includes cyclic alkyl, alkenyl, and alkynyl groups. Cycloalkyl groups can be monocyclic (e.g., cyclohexyl) or polycyclic (e.g., containing fused, bridged, and/or spiro ring systems) in which the carbon atoms are located inside or outside of the ring system. The cycloalkyl group as a whole can have 3 to 14 ring atoms (e.g., 3 to 8 carbon atoms for a monocyclic cycloalkyl group and 7 to 14 carbon atoms for a polycyclic cycloalkyl group). Any suitable on-ring position of the cycloalkyl group can be covalently linked to the defined chemical structure. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclopentenyl, cyclohexenyl, cyclohexadienyl, cycloheptatrienyl, bornyl, norpinyl, nortaryl, adamantyl, and spiro [4.5] decyl, and homologs, isomers, and the like thereof.

The compounds of general formula (I) and general formula (II) according to the present invention also include all pharmaceutically acceptable isotopically-labelled compounds, in which one or more atoms are replaced by an atom having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Isotopes suitable for inclusion in compounds of formula (I) and formula (II) of the invention include isotopes of hydrogen, for example ² H and ³ isotopes of H, carbon, e.g. ¹¹ C、 ¹³ C and ¹⁴ isotopes of C, nitrogen, e.g. ¹³ N and ¹⁵ isotopes of N, oxygen, e.g. ¹⁵ O、 ¹⁷ O and ¹⁸ O。

with heavier isotopes such as deuterium ² H substitution may provide certain therapeutic advantages in that,it has better metabolic stability, e.g., increased in vivo half-life or reduced dosage requirements, and is therefore preferred in some cases.

The synthesis method of the compound of the present invention will be described in detail below by way of examples.

Preparation of intermediate A-0

2, 4-dichloro-5-nitropyrimidine (30g, 154.7mmol) was dissolved in a mixed solution of DCM and MeOH (300mL, volume ratio 1: 1), iron powder (52g, 928.6mmol) and NH4Cl (49.7g, 929.1mmol) were added in portions, the temperature was raised to 50 ℃ and the reaction was stirred for 2 h. TLC monitoring reaction completion, hot with diatomite suction filtration, cooling to room temperature. The reaction solution was adjusted to pH 9 with saturated NaHCO3 solution, then water (150mL) was added and extracted three times with DCM (150mL), the organic phases were combined, then washed with saturated aqueous NaCl solution, dried with Na2SO4, filtered with suction, and the filtrate was concentrated under reduced pressure to give the crude product which was chromatographed over silica gel column [ eluent: DCM: MeOH ═ 100: 1(v/v) ] to give A-0 as a pale yellow solid (21g, yield 83.6%). MS-ESI (M/z) 163.983[ M + H ] +; 1H NMR (400MHz, DMSO-d 6). delta.8.14 (s,1H),6.12(s, 2H).

Preparation of intermediate A-1

A-0(20g, 122.7mmol) was dissolved in anhydrous THF (200mL), a mixed solution of TEA (31.0g, 306.7mmol) and cyclopentylamine (26.1g, 306.7mmol) was added dropwise from a constant pressure base funnel at room temperature, and after dropping, the mixture was transferred to 60 ℃ and stirred under reflux for 4 h. TLC to monitor completion of the reaction, cool to room temperature, concentrate the reaction under reduced pressure to remove most of the solvent, add water (100mL) and extract with EA (150mL) three times, combine the organic phases, wash with saturated aqueous NaCl, add Na ₂ SO ₄ Drying, suction filtering, and concentrating the filtrate under reduced pressure to obtain light yellow solid A-1(23g, yield 88.4%). MS-ESI (M/z) 213.114[ M + H] ⁺ ； ¹ H NMR(400MHz,DMSO-d ₆ )δ7.35(s,1H),6.66(d,J＝6.8Hz,1H),4.95(s,2H),4.23(q,J＝6.8Hz,1H),1.95(dt,J＝12.6,6.4Hz,2H),1.68(tq,J＝10.5,3.8,3.3Hz,2H),1.60–1.52(m,2H),1.50–1.42(m,2H)。

Preparation of intermediate A-2

Intermediate a-2 was prepared as described for a-1, except that cyclopentylamine was replaced with butylamine to give a pale yellow solid (11.3g, 92.1% yield). MS-ESI (M/z) 201.099[ M + H] ⁺ ； ¹ H NMR(400MHz,DMSO-d ₆ )δ7.35(s,1H),6.54(d,J＝7.7Hz,1H),4.94(s,2H),3.99(p,J＝6.8Hz,1H),1.56–1.48(m,2H),1.13(d,J＝6.5Hz,3H),0.87(t,J＝7.4Hz,3H)。

Preparation of intermediate A-3

Preparation of intermediate A-3 referring to A-1, except that cyclopentylamine was replaced with pentylamine to give a pale yellow solid (11.6g, yield 88.2%. MS-ESI (M/z):215.116[ M + H:. RTM.: 215.116: (M + H))] ⁺ ； ¹ H NMR(400MHz,DMSO-d ₆ )δ7.35(s,1H),6.43(d,J＝8.2Hz,1H),4.94(s,2H),3.91(dt,J＝7.8,5.3Hz,1H),1.61–1.53(m,2H),1.46(dt,J＝13.6,7.4Hz,2H),0.85(t,J＝7.4Hz,6H)。

Preparation of intermediate A-4

Intermediate A-4 was prepared as described for A-1, except that cyclopentylamine was replaced with tetrahydro-2H-pyran-4-amine to give a pale yellow solid (11.9g, 85.2% yield). MS-ESI (M/z) 229.095[ M + H] ⁺ ； ¹ H NMR(400MHz,DMSO-d ₆ )δ7.39(s,1H),6.68(d,J＝7.2Hz,1H),4.96(s,2H),4.05–4.01(m,1H),3.90–3.85(m,2H),3.43(dd,J＝11.7,2.1Hz,2H),1.89–1.84(m,2H),1.48(td,J＝11.8,7.7Hz,2H)。

Preparation of intermediate A-5

Intermediate A-5 was prepared by reference to A-1, except that cyclopentylamine was replaced with tert-butyl 3-aminopyrrolidine-1-carboxylate to give a pale yellow solid (12.7g, 66.5% yield). MS-ESI (M/z) 314.154[ M + H] ⁺ ； ¹ H NMR(400MHz,DMSO-d ₆ )δ7.42(s,1H),6.89(d,J＝6.1Hz,1H),5.02(s,2H),4.48–4.38(m,1H),3.62–3.56(m,1H),3.44–3.34(m,2H),3.18–3.12(m,1H),2.18–2.12(m,1H),1.86(dt,J＝12.9,6.4Hz,1H),1.41(d,J＝3.4Hz,9H)。

Preparation of intermediate A-6

Intermediate A-6 was prepared by reference to A-1, except that cyclopentylamine was replaced with (1R,2R) -2-aminocyclopentan-1-ol to give a pale yellow solid (10.5g, 81.8% yield). MS-ESI (M/z) 229.095[ M + H] ⁺ ； ¹ H NMR(400MHz,DMSO-d ₆ )δ7.37(s,1H),6.65(d,J＝6.7Hz,1H),4.97(s,2H),4.82(d,J＝4.3Hz,1H),4.06–4.00(m,1H),3.95(d,J＝5.3Hz,1H),2.12–2.05(m,1H),1.87(ddd,J＝12.8,6.3,2.2Hz,1H),1.71–1.61(m,2H),1.53–1.37(m,2H)。

Preparation of intermediate A-7

A-1(15g, 70.1mmol) and K were added under nitrogen protection ₂ CO ₃ (19.5g, 141.5mmol) was dissolved in acetone (150mL), oxalyl chloride monocaproate was slowly added dropwise under ice-bath conditions, the temperature was controlled not to exceed 5 ℃ and the mixture was allowed to warm to room temperature for 2h after dropping. TLC monitoring reaction completion, suction filtration of reaction solution, washing of filter cake with acetone (50mL), filtrate reductionAfter concentration under reduced pressure, a tan oily liquid was obtained. The tan oil was dissolved in EtOH (100mL) and transferred to a sealed tube, TEA (8.5g, 84.1mmol) was added and the temperature was raised to 100 ℃ for 4 h. TLC monitored completion of the reaction, cooled to room temperature, solid precipitated, filtered and the filter cake dried to afford A-7 as a white solid (14.7g, 78.9% yield). MS-ESI (M/z) 267.090[ M + H] ⁺ ； ¹ H NMR(400MHz,DMSO-d ₆ )δ12.30(s,1H),8.29(s,1H),5.48(ddd,J＝9.6,7.5,1.9Hz,1H),2.13–2.05(m,2H),1.98(td,J＝6.0,5.6,3.1Hz,2H),1.83(td,J＝8.1,7.3,2.5Hz,2H),1.65–1.57(m,2H)。

Preparation of intermediate A-8

Intermediate A-8 was prepared as described for A-7, except that A-1 was replaced with A-2 to give a white solid (9.4g, 73.7% yield). MS-ESI (M/z) 255.076[ M + H] ⁺ ； ¹ H NMR(400MHz,DMSO-d ₆ )δ12.33(s,1H),8.29(s,1H),5.10(q,J＝7.2Hz,1H),2.11–2.04(m,1H),1.88(dt,J＝14.0,7.1Hz,1H),1.46(d,J＝6.9Hz,3H),0.82(t,J＝7.5Hz,3H)。

Preparation of intermediate A-9

Intermediate A-9 was prepared as described for A-7, except that A-1 was replaced with A-3 to give a white solid (9.7g, 77.5% yield). MS-ESI (M/z) 269.098[ M + H] ⁺ ； ¹ H NMR(400MHz,DMSO-d ₆ )δ12.39(s,1H),8.31(s,1H),4.95(s,1H),2.11(s,2H),1.90–1.79(m,2H),0.79(t,J＝7.5Hz,6H)。

Preparation of intermediate A-10

Preparation method of intermediate A-10 referring to A-7, the difference is that A-1 is replaced by A-4,white solid (9.6g, yield 77.6%) was obtained. MS-ESI (M/z) 283.068[ M + H] ⁺ ； ¹ H NMR(400MHz,DMSO-d ₆ )δ8.25(s,1H),5.21(td,J＝8.0,4.0Hz,1H),3.98(dd,J＝11.5,4.5Hz,2H),3.45–3.39(m,2H),2.66(d,J＝7.1Hz,2H),1.59–1.55(m,2H)。

Preparation of intermediate A-11

Intermediate A-11 was prepared as described for A-7, except that A-1 was replaced with A-5 to give a white solid (9.3g, 79.3% yield). MS-ESI (M/z) 368.059[ M + H] ⁺ ； ¹ H NMR(400MHz,DMSO-d ₆ )δ12.27(s,1H),8.30(s,1H),3.75–3.42(m,4H),3.38(t,J＝8.6Hz,1H),2.42(d,J＝9.6Hz,1H),2.18(dd,J＝8.2,5.2Hz,1H),1.41(d,J＝11.0Hz,9H)。

Preparation of intermediate A-12

A-6(8g, 35.1mmol) was dissolved in DCM (80mL) and imidazole (6g, 87.75mmol) and DMAP (0.5g, 4.1mmol) were added under nitrogen. The temperature is reduced to 0 ℃, tert-butyldiphenylchlorosilane (10.6g, 38.6mmol) is added dropwise, and after the dropwise addition is finished, the temperature is raised to room temperature and stirring is carried out for 4 hours. TLC monitoring reaction completion, adding water to reaction solution, extracting with DCM for three times, combining organic phases, washing with saturated NaCl aqueous solution, adding Na ₂ SO ₄ Drying, suction filtration, and vacuum concentration of the filtrate to obtain a crude product, which is subjected to silica gel column chromatography [ eluent: DCM: MeOH ═ 150: 1(v/v)]After isolation and purification, A-12 was obtained as a dark brown solid (9.5g, yield 58.1%). MS-ESI (M/z) 467.223[ M + H] ⁺ ； ¹ H NMR(400MHz,DMSO-d ₆ )δ7.62–7.59(m,4H),7.43–7.32(m,7H),6.55(d,J＝7.7Hz,1H),4.86(s,2H),4.49–4.44(m,1H),4.15(d,J＝4.5Hz,1H),2.12(dt,J＝8.9,4.5Hz,1H),1.74(s,1H),1.67–1.59(m,2H),1.52–1.46(m,2H),0.99(s,9H)。

Preparation of intermediate A-13

Intermediate A-13 was prepared as described for A-7, except that A-1 was replaced with A-12 to give a pale yellow solid (5.6g, 62.7% yield). MS-ESI (M/z) 521.176[ M + H] ⁺ ； ¹ H NMR(400MHz,DMSO-d ₆ )δ8.17(s,1H),7.43–7.37(m,4H),7.32–7.23(m,5H),7.12(s,1H),5.45–5.40(m,1H),4.86(s,1H),2.08–2.04(m,1H),1.91(d,J＝7.4Hz,2H),1.87–1.83(m,1H),1.77(d,J＝5.6Hz,2H),0.92(s,9H)。

Preparation of intermediate A-14

Preparation method of intermediate A-14 referring to I-2, except that A-13 is used as raw material and 4-methylsulfoaniline is replaced by 1-methylsulfonyl 4-aminopiperidine to obtain white solid (0.8g, yield 62.8%). MS-ESI (M/z) 663.308[ M + H] ⁺ ； ¹ H NMR(400MHz,DMSO-d ₆ )δ11.71(s,1H),7.97(s,1H),7.41(dd,J＝7.3,5.8Hz,3H),7.33–7.25(m,5H),7.15(d,J＝7.6Hz,2H),5.52(s,1H),4.87(s,1H),3.67(s,1H),3.52(d,J＝12.1Hz,2H),2.88(s,3H),2.88–2.71(m,3H),2.09–1.96(m,2H),1.84(d,J＝44.0Hz,6H),1.52(d,J＝11.8Hz,2H),0.91(s,9H)。

Preparation of intermediate A-15

A-2(5g, 18.8mmol), POCl ₃ (20mL) were added to the reaction flask in order and the reaction was refluxed for 4 h. TLC monitoring reaction completion, cooling to room temperature, decompression concentrating to remove most solvent, slowly dropping residual reaction liquid into ice water, separating out solid, suction filtering, drying filter cake to obtain white solid A-15(4.2g, yield 78.4%). MS-ESI (M/z) 285.044[ M + H] ⁺ ； ¹ H NMR(400MHz,CDCl3)δ8.95(s,1H),5.84(ddd,J＝9.6,7.6,1.9Hz,1H),2.27–2.21(m,2H),2.19–2.11(m,2H),2.05–1.98(m,2H),1.78–1.71(m,2H)。

Preparation of intermediate A-16

A-15(2g, 7mmol) was dissolved in EtOH (20mL), 1-methylsulfonyl 4-aminopiperidine (1.2g, 7mmol) was added, and the mixture was stirred at 40 ℃ for 2 h. TLC monitoring reaction completion, reaction liquid cooling to room temperature, solid precipitation, suction filtration, filter cake drying to obtain white solid A-16(1.8g, yield 60.1%). MS-ESI (M/z) 427.146[ M + H] ⁺ ； ¹ H NMR(400MHz,CDCl ₃ )δ8.64(s,1H),6.53(d,J＝7.9Hz,1H),5.81(t,J＝8.4Hz,1H),4.17–4.11(m,1H),3.83(d,J＝12.4Hz,2H),2.99(dd,J＝12.6,10.0Hz,2H),2.85(s,3H),2.23(dd,J＝13.2,4.3Hz,4H),2.16–2.11(m,2H),2.03–1.97(m,2H),1.77–1.71(m,4H)。

Preparation of intermediate A-17

Preparation of Compound A-17 referring to A-16, except that 1-methylsulfonyl 4-aminopiperidine was replaced with 4-amino-N, N-dimethylpiperidine-1-sulfonamide, a white solid was obtained (1.6g, yield 51.1%). MS-ESI (M/z) 456.180[ M + H] ⁺ ； ¹ H NMR(400MHz,CDCl ₃ )δ8.60(s,1H),6.47(s,1H),5.75(s,1H),4.08(s,1H),3.70(s,2H),3.06(d,J＝20.0Hz,2H),2.82(s,6H),2.10(s,4H),1.95(s,2H),1.64(d,J＝23.8Hz,4H)。

Example 18-cyclopentyl-2- ((4- (methylsulfonyl) phenyl) amino) -5, 8-dihydropterin-6, 7-dione (I-1)

A-2(1g, 3.8mmol) was dissolved in n-butanol (10mL) and 4-methylsulfoaniline (0.7g, 4.2mmol) and p-toluene were added in orderSulfonic acid monohydrate (0.7g, 3.8mmol), heating to 120 ℃, and refluxing and stirring for reaction for 12 h. TLC monitored the reaction completion, the reaction was cooled to room temperature and filtered to give a yellow solid which was slurried with PE to give compound I-1 as a pale yellow solid (0.7g, 45.9% yield). MS-ESI (M/z) 402.142[ M + H] ⁺ ； ¹ H NMR(400MHz,DMSO-d ₆ )δ10.11(s,1H),8.24(s,1H),7.94(d,J＝8.9Hz,2H),7.83–7.80(m,2H),5.65(d,J＝8.7Hz,1H),3.34(s,1H),3.15(s,3H),2.17(dd,J＝12.5,7.5Hz,2H),1.96(d,J＝8.4Hz,2H),1.89–1.82(m,2H),1.63(q,J＝6.1Hz,2H)。

Example 28-cyclopentyl-6-hydroxy-2- ((1- (methylsulfonyl) piperidin-4-yl) amino) pterin-7 (8H) -one (I-2)

A-2(5g, 18.8mmol) is dissolved in DMSO (50mL), DIPEA (2.9g, 22.6mmol) and 1-methylsulfonyl-4-aminopiperidine (4.7g, 26.3mmol) are sequentially added, the temperature is raised to 100 ℃ for reaction for 6h, and then the temperature is raised to 110 ℃ for further reaction for 6 h. TLC monitoring reaction completion, cooling to room temperature, adding water, extracting three times with DCM, combining the organic phases, then washing with saturated aqueous NaCl solution, adding Na ₂ SO ₄ Drying, suction filtration, and silica gel column chromatography of the crude product obtained from the filtrate under reduced pressure [ eluent: DCM: MeOH ═ 100: 1(v/v)]After isolation and purification, the compound I-2 was obtained as a white solid (4.2g, yield 54.7%). MS-ESI (M/z) 409.150[ M + H] ⁺ ； ¹ H NMR(400MHz,DMSO-d ₆ )δ11.74(s,1H),8.03(s,1H),7.13(d,J＝7.4Hz,1H),5.60(t,J＝8.7Hz,1H),3.80(s,1H),3.54(dt,J＝12.7,4.2Hz,2H),2.88(s,3H),2.84(dd,J＝11.5,2.7Hz,2H),2.18(t,J＝7.3Hz,2H),1.98–1.92(m,4H),1.77(d,J＝9.3Hz,2H),1.57(dd,J＝17.2,8.0Hz,4H)。

Example 38-cyclopentyl-2- ((1- (ethylsulfonyl) piperidin-4-yl) amino) -6-hydroxypterin-7 (8H) -one (I-3)

Preparation method of Compound I-3 referring to Compound I-2, except that 1-methylsulfonyl 4-aminopiperidine was replaced with 1- (ethylsulfonyl) piperidin-4-amine hydrochloride, the compound I-3 was obtained as a white solid (0.76g, yield 47.4%). MS-ESI (M/z) 423.127[ M + H] ⁺ ； ¹ H NMR(400MHz,DMSO-d ₆ )δ11.75(s,1H),8.03(s,1H),7.15(s,1H),5.60(t,J＝8.8Hz,1H),3.81(s,1H),3.62–3.56(m,2H),3.05(t,J＝7.4Hz,2H),2.97–2.90(m,2H),2.17(d,J＝10.3Hz,2H),1.96–1.89(m,4H),1.81–1.74(m,2H),1.60–1.49(m,4H),1.22(s,3H)。

Example 48-cyclopentyl-2- ((1- (cyclopropylsulfonyl) piperidin-4-yl) amino) -6-hydroxypterin-7 (8H) -one (I-4)

Preparation of Compound I-4 referring to Compound I-2, except that 1-methylsulfonyl 4-aminopiperidine was replaced with 1- (cyclopropylsulfonyl) piperidin-4-amine hydrochloride, the compound I-4 was obtained as a white solid (0.7g, yield 43.2%). MS-ESI (M/z) 435.206[ M + H] ⁺ ； ¹ H NMR(400MHz,DMSO-d ₆ )δ11.74(s,1H),8.03(s,1H),7.13(s,1H),5.62–5.56(m,1H),3.81(s,1H),3.60(d,J＝12.0Hz,2H),2.97(d,J＝11.8Hz,2H),2.17(d,J＝7.9Hz,2H),1.94(s,4H),1.78(s,2H),1.60–1.53(m,4H),1.30(s,2H),0.99(d,J＝7.8Hz,2H)。

Example 54- ((8-cyclopentyl-6-hydroxy-7-oxo-7, 8-dihydropterin-2-yl) amino) -N, N-dimethylpiperidine-1-sulfonamide (I-5)

Preparation method of Compound I-5 referring to Compound I-2, except that 1-methylsulfonyl 4-aminopiperidine was replaced with 4-amino-N, N-dimethylpiperidine-1-sulfonamide hydrochloride, the compound I-5 was obtained as a pale yellow solid (0.74g, yield 45.1%). MS-ESI (M/z) 438.191[ M + H] ⁺ ； ¹ H NMR(400MHz,CDCl ₃ )δ8.22(s,1H),5.65(d,J＝8.8Hz,1H),3.91(s,1H),3.69(d,J＝12.9Hz,2H),3.02(d,J＝10.6Hz,2H),2.85(s,6H),2.26(d,J＝9.2Hz,2H),2.12(d,J＝10.3Hz,4H),1.92–1.88(m,2H),1.69(d,J＝8.5Hz,4H)。

Example 68- (sec-butyl) -6-hydroxy-2- ((1- (methylsulfonyl) piperidin-4-yl) amino) pterin-7 (8H) -one (I-6)

Preparation of Compound I-6 referring to Compound I-2, except that A-8 was used as the starting material, Compound I-6 was obtained as a pale yellow solid (0.8g, yield 51.3%). MS-ESI (M/z) 397.182[ M + H] ⁺ ； ¹ H NMR(400MHz,DMSO-d ₆ )δ11.81(s,1H),8.05(s,1H),7.17(s,1H),5.22(s,1H),3.77(s,1H),3.54(dd,J＝11.9,3.7Hz,2H),2.88(s,3H),2.88–2.82(m,2H),2.26–2.05(m,2H),2.00–1.77(m,4H),1.55(t,J＝11.0Hz,2H),1.46(d,J＝6.8Hz,4H)。

Example 76-hydroxy-2- ((1- (methylsulfonyl) piperidin-4-yl) amino) -8- (pentan-3-yl) pterin-7 (8H) -one (I-7)

Preparation of Compound I-7 referring to I-2, except that A-9 was used as the starting material, the compound I-7 was obtained as a pale yellow solid (1g, yield 52.2%). MS-ESI (M/z) 411.203[ M + H] ⁺ ； ¹ H NMR(400MHz,DMSO-d ₆ )δ11.87(s,1H),8.06(s,1H),7.18(s,1H),5.19(s,1H),3.74(s,1H),3.54(d,J＝11.8Hz,2H),2.88(s,5H),2.18–1.80(m,6H),1.55(d,J＝11.3Hz,2H)。

Example 86-hydroxy-2- ((1- (methylsulfonyl) piperidin-4-yl) amino) -8- (tetrahydro-2H-pyran-4-yl) pterin-7 (8H) -one (I-8)

The preparation method of the compound I-8 refers to I-2, and the difference is that A-10 is taken as a raw material to obtain a light yellow solid I-8 compoundThis substance (0.9g, yield 48.3%). MS-ESI (M/z) 425.217[ M + H] ⁺ ； ¹ H NMR(400MHz,DMSO-d ₆ )δ10.80(s,1H),8.05(s,1H),7.18(s,1H),5.29(q,J＝10.4,9.3Hz,1H),3.99(dd,J＝11.5,4.5Hz,2H),3.76(s,1H),3.56(dd,J＝10.1,6.1Hz,2H),3.42–3.36(m,2H),2.88(s,3H),2.87–2.82(m,2H),2.74(dt,J＝13.6,6.8Hz,2H),2.02–1.95(m,2H),1.58–1.50(m,4H)。

Example 96-hydroxy-2- ((1- (methylsulfonyl) piperidin-4-yl) amino) -8- (pyrrolidin-3-yl) pterin-7 (8H) -monohydrochloride (I-9)

Dissolving A-10(1g, 2.7mmol) in DMSO (10mL), sequentially adding DIPEA (0.4g, 3.3mmol) and 1-methylsulfonyl-4-aminopiperidine (0.7g, 3.8mmol), heating to 100 ℃ for reaction for 6h, then heating to 110 ℃ for further reaction for 6 h. TLC monitoring reaction completion, cooling to room temperature, adding water, extracting three times with DCM, combining the organic phases, then washing with saturated aqueous NaCl solution, Na ₂ SO ₄ Dried and concentrated under reduced pressure, and the resulting crude product was purified by silica gel column chromatography [ eluent: DCM: MeOH, 50: 1(v/v)]Separating and purifying to obtain an intermediate; the intermediate was dissolved in HCl-MeOH (5mL) and stirred at room temperature overnight. TLC monitored completion of the reaction and concentrated under reduced pressure to remove the solvent to give compound I-9 as a white solid (0.4g, yield 33.3%). MS-ESI (M/z) 410.179[ M + H] ⁺ ； ¹ H NMR(400MHz,Deuterium Oxide)δ7.98(s,1H),6.04–5.99(m,1H),3.88(d,J＝12.0Hz,1H),3.55–3.47(m,4H),3.29–3.20(m,2H),2.92–2.86(m,2H),2.83(s,3H),2.44(dh,J＝13.5,4.2Hz,2H),2.19(s,2H),1.97(s,1H),1.62–1.55(m,2H)。

Example 106-hydroxy-8- ((1R,2R) -2-hydroxycyclopentyl) -2- ((1- (methylsulfonyl) piperidin-4-yl) amino) pterin-7 (8H) -one (I-10)

A-14(0.5g, 0.8mmol) was dissolved in THF (5mL) and tetramethyl was addedAmine fluoride (0.2g, 2mmol) was stirred at room temperature overnight. TLC monitoring reaction completion, reaction liquid decompression concentration to remove most solvent, adding water, DCM extraction three times, organic phase combination, then saturated NaCl aqueous solution washing, adding Na ₂ SO ₄ Drying, suction filtration, and silica gel column chromatography of the crude product obtained from the filtrate under reduced pressure [ eluent: DCM: MeOH ═ 100: 1(v/v)]Isolation and purification gave compound I-10 as a white solid (0.22g, yield 62.5%). MS-ESI (M/z) 425.173[ M + H] ⁺ ； ¹ H NMR(400MHz,DMSO-d ₆ )δ11.75(s,1H),8.06(s,1H),7.15(s,1H),5.35–5.28(m,1H),4.79(s,1H),4.76(d,J＝7.0Hz,1H),3.78(s,1H),3.58–3.53(m,2H),2.89(s,3H),2.83(dt,J＝11.9,3.2Hz,2H),2.15–2.02(m,2H),1.99–1.93(m,2H),1.84(p,J＝5.1,4.4Hz,2H),1.73(s,1H),1.55(dq,J＝18.2,7.2,6.3Hz,3H)。

Example 118-cyclopentyl-6- (methylamino) -2- ((1- (methylsulfonyl) piperidin-4-yl) amino) pterin-7 (8H) -one (II-11)

Mixing I-2(0.5g, 1.2mmol) and POCl ₃ (5mL) are added into the reaction flask in turn, and the reaction is performed under reflux for 4 h. Monitoring the completion of the reaction by TLC, cooling to room temperature, concentrating under reduced pressure to remove most of the solvent, slowly dripping the residual reaction solution into ice water, separating out solids, and filtering to obtain a filter cake; adding the filter cake and 30% methylamine alcohol solution (3mL) into a reaction bottle, and carrying out reflux reaction for 6 h. TLC to monitor completion of the reaction, cool to room temperature, add water (10mL) to the reaction, extract three times with DCM (15mL), combine the organic phases, wash with saturated aqueous NaCl, add Na ₂ SO ₄ Drying, suction filtration, and silica gel column chromatography of the crude product obtained from the filtrate under reduced pressure [ eluent: DCM: MeOH ═ 200: 1(v/v)]After isolation and purification, the compound II-11 was obtained as a yellow solid (0.34g, yield 65.9%). MS-ESI (M/z) 422.143[ M + H] ⁺ ； ¹ H NMR(400MHz,DMSO-d ₆ )δ8.34(d,J＝1.0Hz,1H),7.42(d,J＝5.1Hz,1H),7.11(d,J＝7.8Hz,1H),5.74(t,J＝8.7Hz,1H),3.84(s,1H),3.55(d,J＝11.9Hz,2H),2.88(d,J＝0.9Hz,3H),2.85(dd,J＝11.4,3.6Hz,5H),2.22(s,2H),1.97(d,J＝11.5Hz,4H),1.81(d,J＝10.3Hz,2H),1.65–1.54(m,4H)。

Example 128-cyclopentyl-2- ((1- (ethylsulfonyl) piperidin-4-yl) amino) -6- (methylamino) pterin-7 (8H) -one (II-12)

Preparation of Compound II-12 with reference to Compound II-11, except that I-3 was used as the starting material, yellow solid II-12 compound (0.09g, yield 29.2%) was obtained. MS-ESI (M/z) 436.239[ M + H] ⁺ ； ¹ H NMR(400MHz,CDCl ₃ )δ8.48(s,1H),6.21(d,J＝5.5Hz,1H),5.79–5.73(m,1H),5.13(s,1H),4.05–3.97(m,1H),3.80(dd,J＝12.5,4.5Hz,2H),3.07(d,J＝5.0Hz,3H),3.06–2.98(m,4H),2.34(t,J＝9.6Hz,2H),2.21–2.15(m,2H),2.05(s,2H),1.91(t,J＝4.9Hz,2H),1.73–1.65(m,4H),1.27(s,3H)。

Example 138-cyclopentyl-2- ((1- (cyclopropylsulfonyl) piperidin-4-yl) amino) -6- (methylamino) pterin-7 (8H) -one (II-13)

Preparation of Compound II-13 referring to II-11, except that I-4 was used as the starting material, yellow solid II-13 compound (0.1g, yield 32.4%) was obtained. MS-ESI (M/z) 448.235[ M + H] ⁺ ； ¹ H NMR(400MHz,CDCl ₃ )δ8.48(s,1H),6.21(d,J＝5.3Hz,1H),5.79–5.74(m,1H),4.00(d,J＝8.1Hz,1H),3.82–3.77(m,2H),3.12–3.06(m,5H),2.19(dd,J＝13.1,3.8Hz,2H),2.10–2.01(m,4H),1.93–1.89(m,2H),1.71(dt,J＝6.9,2.8Hz,4H),1.23–1.21(m,2H),1.04–1.02(m,2H)。

Example 148-cyclopentyl-6- (ethylamino) -2- ((1- (methylsulfonyl) piperidin-4-yl) amino) pterin-7 (8H) -one (II-14)

Preparation of Compound II-14 referring to Compound II-11, except that the methylamine alcohol solution was replaced with ethylamine alcohol solution, a yellow solid of Compound II-14 (0.32g, yield 60.3%) was obtained. MS-ESI (M/z) 436.234[ M + H] ⁺ ； ¹ H NMR(400MHz,DMSO-d ₆ )δ8.33(s,1H),7.36(t,J＝5.9Hz,1H),5.75(dd,J＝10.5,7.0Hz,1H),3.55(d,J＝12.0Hz,2H),3.37–3.30(m,4H),2.88(s,3H),2.86(d,J＝2.7Hz,1H),2.25–2.17(m,2H),1.97(d,J＝11.9Hz,4H),1.81(t,J＝4.8Hz,2H),1.64–1.53(m,4H),1.15(t,J＝7.1Hz,3H)。

Example 158-cyclopentyl-6- ((2, 2-difluoroethyl) amino) -2- ((1- (methylsulfonyl) piperidin-4-yl) amino) pterin-7 (8H) -one (II-15)

Compound II-15 and preparation method reference compound II-11 except that the methylamine alcohol solution was replaced with difluoroethylamine solution to give compound II-15 as a white solid (0.19g, yield 55.4%). MS-ESI (M/z) 472.200[ M + H] ⁺ ； ¹ H NMR(400MHz,CDCl ₃ )δ8.45(s,1H),6.36(t,J＝6.5Hz,1H),5.76(d,J＝8.9Hz,1H),5.06(d,J＝7.8Hz,1H),4.02–3.96(m,1H),3.93–3.85(m,2H),3.80(d,J＝12.3Hz,2H),3.00–2.94(m,2H),2.85(s,3H),2.36–2.30(m,2H),2.23–2.18(m,2H),2.07(t,J＝8.0Hz,2H),1.96–1.90(m,2H),1.75–1.67(m,4H)。

Example 164- ((8-cyclopentyl-6- ((2, 2-difluoroethyl) amino) -7-oxo-7, 8-dihydropterin-2-yl) amino) -N, N-dimethylpiperidine-1-sulfonamide (II-16)

Preparation method of compound II-16 referring to II-11, except that I-5 is used as raw material, and methylamine alcohol solution is replaced by difluoroethylamine solution to obtain pale yellow solid compound II-16 (0.08g, yield 23.3%). MS-ESI (M/z) 501.224[ M + H] ⁺ ； ¹ H NMR(400MHz,DMSO-d ₆ )δ8.36(s,1H),7.67(d,J＝6.2Hz,1H),6.19(d,J＝4.3Hz,1H),5.74(t,J＝9.0Hz,1H),3.86(s,1H),3.72(p,J＝4.5Hz,2H),3.58(d,J＝12.5Hz,2H),2.97(t,J＝11.9Hz,2H),2.76(s,6H),2.24(s,2H),1.96(d,J＝15.9Hz,4H),1.84–1.78(m,2H),1.66–1.53(m,4H)。

Example 178-cyclopentyl-6-methoxy-2- ((1- (methylsulfonyl) piperidin-4-yl) amino) pterin-7 (8H) -one (II-17)

Mixing I-2(0.5g, 1.2mmol) and POCl ₃ (5mL) are added into the reaction flask in turn, and the reaction is performed under reflux for 4 h. Monitoring the completion of the reaction by TLC, cooling to room temperature, concentrating under reduced pressure to remove most of the solvent, slowly dripping the residual reaction solution into ice water, separating out solids, and filtering to obtain a filter cake; the filter cake and DMF-DMA (8mL) were added to the reaction flask and the temperature was raised to 120 ℃ for 4 h. TLC to monitor the completion of the reaction, cooled to room temperature, added the reaction solution dropwise to ice water, precipitated a solid, filtered and dried to obtain a white solid II-17 (0.29g, yield 56.1%). MS-ESI (M/z) 423.171[ M + H] ⁺ ； ¹ H NMR(400MHz,DMSO-d ₆ )δ8.47(s,1H),8.34(s,1H),5.70(s,1H),3.89(s,3H),3.57(d,J＝3.4Hz,2H),3.54(s,2H),2.88(d,J＝1.6Hz,3H),2.83(s,1H),2.22(s,2H),1.97(d,J＝10.8Hz,4H),1.79(s,2H),1.61(d,J＝9.5Hz,4H)。

Example 188- (sec-butyl) -6-methoxy-2- ((1- (methylsulfonyl) piperidin-4-yl) amino) pterin-7 (8H) -one (II-18)

Preparation of Compound II-18 referring to II-17, except that I-3 was used as the starting material, pale yellow solid compound II-18 (0.28g, yield 54.6%) was obtained. MS-ESI (M/z) 411.198[ M + H] ⁺ ； ¹ H NMR(400MHz,DMSO-d ₆ )δ8.47(s,1H),7.56(s,1H),5.33(s,1H),3.90(s,3H),3.84(s,1H),3.59–3.53(m,2H),2.89(s,5H),2.14–1.87(m,4H),1.61–1.54(m,2H),1.50(s,3H)。

Example 196-methoxy-2- ((1- (methylsulfonyl) piperidin-4-yl) amino) -8- (pentan-3-yl) pterin-7 (8H) -one (II-19)

Preparation of Compound II-19 referring to II-17, except that I-4 was used as the starting material, the compound II-19 was obtained as a pale yellow solid (0.22g, yield 53.2%). MS-ESI (M/z) 425.179[ M + H] ⁺ ； ¹ H NMR(400MHz,DMSO-d ₆ )δ8.49(s,1H),7.64(s,1H),5.33(s,1H),3.91(s,3H),3.56(d,J＝11.6Hz,2H),2.90(s,5H),2.34(s,2H),1.90(d,J＝66.9Hz,4H),1.58(d,J＝11.9Hz,2H)。

Example 206-methoxy-2- ((1- (methylsulfonyl) piperidin-4-yl) amino) -8- (tetrahydro-2H-pyran-4-yl) pterin-7 (8H) -one (II-20)

Preparation of Compound II-20 referring to II-17, except that I-5 was used as the starting material, pale yellow solid compound II-20 (0.23g, yield 55.7%) was obtained. MS-ESI (M/z) 439.136[ M + H] ⁺ ； ¹ H NMR(400MHz,DMSO-d ₆ )δ8.47(s,1H),7.60(d,J＝51.5Hz,1H),5.42(s,1H),4.00(dd,J＝11.2,4.4Hz,2H),3.89(s,4H),3.59(s,2H),3.42(d,J＝12.4Hz,2H),2.90(s,3H),2.85(d,J＝12.3Hz,4H),2.01(d,J＝19.3Hz,2H),1.58(t,J＝13.3Hz,4H)。

Example 218-cyclopentyl-6-hydrazino-2- ((1- (methylsulfonyl) piperidin-4-yl) amino) pterin-7 (8H) -one (II-21)

Preparation of Compound II-21 referring to Compound II-11, except that methylamine alcohol solution was replaced with hydrazine hydrate, yellow solid compound II-21 (0.24g, yield 46.4%) was obtained. MS-ESI (M/z) 423.214[ M + H] ⁺ ； ¹ H NMR(400MHz,DMSO-d ₆ )δ8.36(s,1H),7.17(d,J＝7.7Hz,1H),5.78–5.71(m,1H),3.85(s,1H),3.55(d,J＝12.2Hz,2H),2.89(s,3H),2.85(dd,J＝11.7,2.7Hz,2H),2.21(s,2H),1.97(dd,J＝10.3,5.2Hz,4H),1.84–1.78(m,2H),1.64–1.55(m,4H)。

Example 228-cyclopentyl-6-hydrazino-2- ((4- (methylsulfonyl) phenyl) amino) pterin-7 (8H) -one (II-22)

Preparation of Compound II-22 referring to Compound II-11, the difference is that I-1 is the starting material and the methylamine alcohol solution is replaced with hydrazine hydrate to give the compound II-22 as a yellow solid (0.23g, yield 46.2%). MS-ESI (M/z) 416.174[ M + H] ⁺ ； ¹ H NMR(400MHz,DMSO-d ₆ )δ10.10(s,1H),8.55(s,1H),7.90(dd,J＝62.1,8.5Hz,4H),5.78(d,J＝18.0Hz,1H),4.53(s,1H),3.36(s,2H),3.16(s,3H),2.23(s,2H),1.95(d,J＝42.5Hz,4H),1.66(s,2H)。

Example 235-cyclopentyl-7- ((1- (methylsulfonyl) piperidin-4-yl) amino) - [1,2,4] triazolyl [4,3-f ] pterin-4 (5H) -one (II-23)

II-21(0.18g, 0.4mmol) was dissolved in triethyl orthoformate (2mL), warmed to 80 ℃ and reacted for 2h with stirring. TLC to monitor the completion of the reaction, cooled to room temperature, slowly added dropwise to ice water to precipitate a solid, filtered, and the filter cake was dried to obtain a white solid II-23 compound (0.12g, yield 65.2%). MS-ESI (M/z) 433.209[ M + H] ⁺ ； ¹ H NMR(400MHz,DMSO-d ₆ )δ9.69(s,1H),9.08(s,1H),8.39(s,1H),5.75(s,1H),3.85(s,1H),3.56(d,J＝11.9Hz,2H),2.92(d,J＝2.7Hz,1H),2.89(d,J＝3.4Hz,3H),2.86(d,J＝2.7Hz,1H),2.25(s,2H),1.98(d,J＝12.4Hz,4H),1.83(s,2H),1.60(d,J＝11.3Hz,4H)。

Example 245-cyclopentyl-7- ((4- (methylsulfonyl) phenyl) amino) - [1,2,4] triazolo [4,3-f ] pterin-4 (5H) -one (II-24)

Preparation of Compound II-24 referring to Compound II-23, except that II-22 was used as the starting material, yellow solid II-24 compound ((0.24g, yield 46.4%). MS-ESI (M/z):426.155[ M + H ])] ⁺ ； ¹ H NMR(400MHz,DMSO-d ₆ )δ10.48(s,1H),9.78(s,1H),9.31(s,1H),7.99–7.83(m,4H),5.83–5.75(m,1H),3.18(s,3H),2.27(q,J＝6.6Hz,2H),2.06–1.88(m,4H),1.66(q,J＝6.1Hz,2H)

Example 258-cyclopentyl-2- (ethylamino) -6- ((1- (methanesulfonyl) piperidin-4-yl) amino) pterin-7 (8H) -one (III-25)

A-16(0.5g, 1.2mmol) was dissolved in DMSO (5mL), and a solution of ethanamine (1mL) was added, warming to 80 ℃ and stirring for 6 h. TLC monitoring reaction completion, reaction liquid cooling to room temperature, dripping into ice water, separating out solid, suction filtering, drying filter cake to obtain white solid III-25 compound ((0.24, yield 45.8%). MS-ESI (M/z):436.252[ M + H ])] ⁺ ； ¹ H NMR(400MHz,DMSO-d ₆ )δ8.29(s,1H),7.25(d,J＝8.2Hz,1H),7.10(s,1H),5.76(t,J＝8.7Hz,1H),3.98–3.91(m,1H),3.54(dd,J＝9.8,6.4Hz,2H),3.30(dd,J＝7.3,5.9Hz,2H),2.90(d,J＝2.8Hz,1H),2.88(s,3H),2.84(d,J＝2.6Hz,1H),2.24(s,2H),2.00–1.91(m,4H),1.85–

1.78(m,2H),1.71–1.60(m,4H),1.13(t,J＝7.1Hz,3H)。

Example 268-cyclopentyl-2- ((2, 2-difluoroethyl) amino) -6- ((1- (methylsulfonyl) piperidin-4-yl) amino) pterin-7 (8H) -one (III-26)

Preparation method of compound III-26Reference was made to compound III-25 with the exception that the ethylamine alcohol solution was replaced with difluoroethylamine to give the compound III-26 as a yellow solid ((0.24g, 46.4% yield.) MS-ESI (M/z):472.218[ M + H)] ⁺ ； ¹ H NMR(400MHz,DMSO-d ₆ )δ8.34(s,1H),7.46(t,J＝6.2Hz,1H),7.37(d,J＝8.3Hz,1H),5.78–5.72(m,1H),4.01–3.93(m,1H),3.75–3.66(m,2H),3.55(dd,J＝9.7,6.5Hz,2H),2.92–2.89(m,1H),2.88(s,3H),2.87–2.84(m,1H),2.23(d,J＝10.5Hz,2H),1.96(tt,J＝12.2,8.0Hz,4H),1.87–1.80(m,2H),1.73–1.60(m,4H)。

Example 278-cyclopentyl-2-hydrazino-6- ((1- (methylsulfonyl) piperidin-4-yl) amino) pterin-7 (8H) -one (III-27)

Preparation of Compound III-27 referring to Compound III-25, except substituting ethylamine alcohol solution with hydrazine hydrate, yellow solid III-27 compound (0.22g, yield 43.3%) was obtained. MS-ESI (M/z) 423.212[ M + H] ⁺ ； ¹ H NMR(400MHz,DMSO-d ₆ )δ8.34(s,1H),8.14(s,1H),7.31(d,J＝8.2Hz,1H),5.84(t,J＝8.7Hz,1H),4.41(s,1H),3.99–3.93(m,1H),3.59–3.53(m,2H),2.90(d,J＝2.6Hz,1H),2.88(s,3H),2.84(d,J＝2.6Hz,1H),2.21(t,J＝9.9Hz,2H),2.02–1.91(m,4H),1.83(t,J＝5.1Hz,2H),1.71–1.60(m,4H)。

Example 284- ((8-cyclopentyl-2- (ethylamino) -7-oxo-7, 8-dihydropterin-6-yl) amino) -N, N-dimethylpiperidine-1-sulfonamide (III-28)

Preparation of Compound III-28 referring to Compound III-25, except that A-17 was used as the starting material, yellow solid III-28 (0.25g, yield 45.4%) was obtained. MS-ESI (M/z) 465.258[ M + H] ⁺ ； ¹ H NMR(400MHz,CDCl ₃ )δ8.43(s,1H),6.07(d,J＝7.8Hz,1H),5.85–5.79(m,1H),4.07–4.01(m,1H),3.75–3.69(m,2H),3.51–3.45(m,2H),3.11–3.04(m,2H),2.85(s,6H),2.34(dd,J＝12.4,7.4Hz,2H),2.15(dd,J＝13.1,3.7Hz,2H),2.06(dq,J＝7.9,3.5,2.3Hz,2H),1.90(q,J＝7.9,6.7Hz,2H)。

Example 294- ((8-cyclopentyl-2- ((2, 2-difluoroethyl) amino) -7-oxo-7, 8-dihydropterin-6-yl) amino) -N, N-dimethylpiperidine-1-sulfonamide (III-29)

Preparation of Compound III-29 referring to Compound III-25, the difference is that starting with A-17, the ethanamine alcohol solution is replaced with difluoroethanamine to give the compound III-29 as a yellow solid (0.28g, 46.7% yield). MS-ESI (M/z) 501.245[ M + H] ⁺ ； ¹ H NMR(400MHz,CDCl ₃ )δ8.46(s,1H),6.13(s,1H),5.78(t,J＝8.8Hz,1H),4.05(dt,J＝7.6,3.7Hz,1H),3.91–3.83(m,2H),3.75–3.70(m,2H),3.11–3.04(m,2H),2.85(s,6H),2.31(dd,J＝12.6,7.5Hz,2H),2.15(dd,J＝13.1,3.7Hz,2H),2.09–2.03(m,2H),1.95–1.88(m,2H),1.74–1.62(m,4H)。

Example 304- ((8-cyclopentyl-2-hydrazino-7-oxo-7, 8-dihydropterin-6-yl) amino) -N, N-dimethylpiperidine-1-sulfonamide (III-30)

Preparation of Compound III-30 referring to Compound III-25, the difference is that starting with A-17, the ethanamine alcohol solution is replaced with hydrazine hydrate to give the compound III-30 as a yellow solid (0.28g, yield 51.7%). MS-ESI (M/z) 452.240[ M + H] ⁺ ； ¹ H NMR(400MHz,CDCl ₃ )δ8.50(s,1H),6.41(s,1H),6.16(d,J＝7.8Hz,1H),5.84(t,J＝8.8Hz,1H),4.09–4.03(m,1H),3.73(dd,J＝10.7,6.8Hz,2H),3.10–3.04(m,2H),2.85(s,6H),2.30(dd,J＝12.4,7.5Hz,2H),2.16(dd,J＝13.2,3.7Hz,2H),2.09–2.03(m,2H),1.97–1.90(m,2H),1.64(dt,J＝10.7,2.2Hz,4H)。

EXAMPLE 31 Activity assay of Compounds

Evaluation of kinase inhibitory Activity of CDK2

Preparing a compound: compound powders were dissolved in 100% DMSO to make 10mM stock solutions. Test compound concentration was 500nM, and duplicate wells were tested. The stock was diluted 100-fold in 384-well plates with 100% DMSO. A100-fold final concentration of 250nL of compound was transferred to the target plate using a dispenser Echo 550. 250nL of 100% DMSO was applied to each of the negative and positive control wells.

② preparing Kinase, preparing 1 XKinase buffer, and preparing 2.5 times final concentration Kinase solution by using the same.

③ adding 10 mu L of kinase solution with 2.5 times of final concentration into the compound hole and the positive control hole respectively; mu.L of a 2.5-fold Kinase buffer was added to the negative control wells. The mixture was centrifuged at 1000rpm for 30 seconds, shaken and mixed, and then incubated at room temperature for 10 minutes.

(iv) A mixed solution of ATP and Kinase substrate 22 was prepared at a final concentration of 25/15 times using 1 XKinase buffer, and 10. mu.L of the mixed solution was added to the well to initiate the reaction. The 384 well plates were centrifuged at 1000rpm for 30 seconds, shaken well and incubated at room temperature for 30 minutes.

And (5) stopping the reaction, namely adding 30 mu L of termination detection solution to stop the kinase reaction, centrifuging at 1000rpm for 30 seconds, shaking and uniformly mixing, and then reading the conversion rate by using an enzyme-labeling instrument. Calculating the formula: inhibition [ (% of conversion mean of positive control wells% of conversion mean of test compound) ]/(% of conversion mean of positive control wells% of conversion mean of negative control wells) ]) x 100. Dose-response curves were fitted using GraphPad Prism 5 software, with X-axis log [ concentration ]]Y-axis inhibition%, IC of compounds against CDK2-cyclin E kinase was calculated ₅₀ Value, fitting formula: y ═ Bottom + (Top-Bottom)/(1+10^ ((LogIC) ₅₀ -X)*HillSlope))。

Evaluation of cell proliferation inhibitory Activity of HCT 116 and MV4-11

HCT 116 cells: the inoculation density is 5X 10/ml ⁴ In each 96-well plate, a cell suspension (100. mu.L) was added and cultured in an incubator for 24 hours. Removing culture medium, adding diluted test compound solution (150 μ L) with different concentrations, setting blank control group (culture medium) and negative control group (cells and culture medium), and culturing in incubator for 48 hr. After removing the medium, MTT solution (0.5 mg. multidot.mL) was added ^-1 100 μ L) was incubated in the incubator for 4h, the MTT solution, DMSO (100 μ L) was removed, and the absorbance was read at 570nM using a microplate reader after shaking.

(ii) MV4-11 cells: the inoculation density is 1X 10 per ml ⁵ Separately, a cell suspension (100. mu.L) in a 96-well plate was added with a test compound (150. mu.L) diluted to different concentrations, while setting a blank control and a negative control, and cultured in an incubator for 72 hours. CCK-8 solution (10. mu.L) was added, incubated in an incubator for 4h, shaken and then read for absorbance at 450nM using a microplate reader.

Adopting GraphPad Prism 5 software to calculate inhibition rate under different concentrations, fitting a corresponding function according to an inhibition rate curve, and calculating IC ₅₀ The value is obtained. Calculating the formula: cell growth inhibition ═ 1- (experimental well absorbance-blank absorbance)/(negative control well absorbance-blank absorbance) × 100.

This example discloses the results of the in vitro CDK2 kinase inhibitory activity and partial inhibitory activity assay of compounds with better anti-tumor cell proliferation activity, as shown in tables 2 and 3:

TABLE 2 results of in vitro CDK2 kinase inhibitory activity assays for different pteridine 7(8H) -one compounds

Example 2 has an inhibitory activity against CDK2 that is superior to example 1, i.e. the introduction of piperidinamine at the 2-position of pyrimidine is more helpful in increasing the activity of the compound than aniline. Examples 4 and 5 showed that the inhibition rate of CDK2 kinase was lower than that of examples 2 and 3, and the effect of inhibiting the enzyme by introducing cyclopropyl or N, N-dimethyl into a sulfonyl group was far lower than that of a paraffin base. Examples 17-22 have good inhibitory activity on CDK2, wherein the best results of examples 17 and 21 are 82% and 86%, respectively. However, examples 25 to 30 all showed no high inhibitory activity against CDK 2.

Table 3 partial test results of compounds with better inhibitory activity on IC50 of CDK2 kinase and anti-tumor cell proliferation activity

The result of the cell antiproliferation experiment is consistent with the result of the kinase inhibitory activity, and basically forms a positive correlation relationship, namely, the compound with better inhibitory effect on CDK2-cyclin E kinase also has stronger proliferative inhibitory activity on tumor cells; the inhibition of CDK2 activation can inhibit cell proliferation to achieve the anti-tumor effect. The compounds tested showed varying degrees of inhibition of cell proliferation on both tumor cells, example 17 (IC) ₅₀ 1.93 μ M, 2.57 μ M), example 21 (IC) ₅₀ 1.62 μ M, 1.95 μ M) and example 22 (IC) ₅₀ 3.43 μ M, 1.17 μ M) has strong antiproliferative activity on human rectal cancer cell HCT 116 and human myeloid monocytic leukemia cell MV 4-11.

While the invention has been illustrated by the foregoing specific embodiments, it is not to be construed as being limited thereby; but that the present invention encompass the generic aspects previously disclosed. Various modifications and embodiments can be made without departing from the spirit and scope of the invention.

Claims (10)

1. Pteridine 7(8H) -ketone compounds with the structure shown in the general formula (I) or (II) or stereoisomers or pharmaceutically acceptable salts thereof,

or

R ₁ 、R ₁ ' are each independently selected from unsubstituted or halogen-substituted C1-C8 straight or branched chain alkyl, unsubstituted or halogen-substituted C3-C6 cycloalkyl;

R ₂ is hydroxy, methoxy, -NHR _a Or is

R _a Is H, C1-8 alkyl, NH ₂ ；R ₄ ' is C1-C8 alkyl, C1-C8 alkyl-substituted primary or secondary amino, C3-C6 cycloalkyl;

R ₃ 、R ₃ ' Each is independently selected from-NHR _b 、

R _b Is H, C1-8 alkyl, NH ₂ ；R ₄ And R ₄ ' are each independently selected from C1-C8 alkyl, C1-C8 alkyl substituted primary or secondary amino, C3-C6 cycloalkyl.

2. Pteridine 7(8H) -one compounds according to claim 1, or a stereoisomer thereof or a pharmaceutically acceptable salt thereof, wherein R is ₁ 、R ₁ ' are each independently selected from butyl, pentyl, pyranyl, pyrrolidinyl, cyclopentanol, cyclopentyl alkyl groups.

3. The pteridine 7(8H) -one compound or a stereoisomer or a pharmaceutically acceptable salt thereof according to claim 1, wherein R is ₂ Selected from methylamino, ethylamino, hydroxy, methoxy, difluoroethylamino, hydrazino, amino,

4. A pteridine 7(8H) -one compound according to claim 1, or a stereoisomer thereof or a pharmaceutically acceptable salt thereof, wherein the pharmaceutically acceptable salt comprises: potassium, sodium, hydrochloride, formate, trifluoroacetate, phosphate and sulfate.

5. A pharmaceutical composition comprising a pteridine 7(8H) -one compound of any one of claims 1-4, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

6. The pharmaceutical composition of claim 5, further comprising a pharmaceutically acceptable excipient.

7. The pharmaceutical composition of claim 6, wherein the pharmaceutically acceptable excipients comprise diluents, excipients, fillers, binders, wetting agents, absorption enhancers.

8. Use of a pteridine 7(8H) -one compound according to any one of claims 1 to 4, or a stereoisomer thereof or a pharmaceutically acceptable salt thereof, for the preparation of a CDK inhibitor.

9. Use of a pteridine-like compound according to any one of claims 1-4, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the prevention or treatment of cancer.

10. The use of claim 9, wherein cancer comprises: breast cancer, colon cancer, rectal cancer, kidney cancer, epidermal carcinoma, liver cancer, lung cancer, esophageal cancer, gallbladder cancer, ovarian cancer, pancreatic cancer, stomach cancer, cervical cancer, thyroid cancer, nasal cancer, head and neck cancer, prostate cancer, skin cancer, hematopoietic cell tumors of myeloid lineage, follicular thyroid cancer, tumors derived from mesenchymal cells, tumors of the central or peripheral nervous system, melanoma, glioma, seminoma, teratoma, osteosarcoma, xeroderma pigmentosum, keratoacanthoma, follicular thyroid cancer, kaposi's sarcoma.