CDK6 / 9 dual inhibitor as well as preparation method and application thereof

Abstract

The invention relates to the field of medicinal chemistry, in particular to a CDK6 / 9 dual inhibitor and a preparation method and application thereof.The CDK6 / 9 dual inhibitor has CDK6 and CDK9 inhibitory activity at the same time, has an inhibitory effect on abnormal hyperplasia of a psoriasis cell model and inflammatory factor disorder and has good safety and selectivity in a CDK family; the inhibitor can be used as a therapeutic drug for psoriasis dermatitis, psoriatic arthritis and other autoimmune diseases.

Description

Technical Field

[0001] The present invention relates to the field of medicinal chemistry, and in particular to a CDK6 / 9 dual inhibitor and a preparation method and use thereof. Background Art

[0002] Psoriasis is an immune-mediated inflammatory disease of the skin. However, traditional anti-psoriatic drugs have poor efficacy, are prone to relapse and are highly toxic. Therefore, it is a huge challenge to develop a safe and long-term treatment for psoriasis.

[0003] Cyclin-dependent kinase 6 (CDK6) and cyclin-dependent kinase 9 (CDK9) can be used to understand psoriasis. CDK6-mediated inflammatory cell proliferation and CDK9-mediated inflammatory factor disorder are intertwined in the process of psoriasis and promote each other, maintaining and driving the progression of psoriasis. Studies have shown that inhibiting the CDK4 / 6-Rb-CDC6 signaling pathway can inhibit the inflammatory proliferation of psoriasis (Sun S, et al. Berberine downregulates CDC6 and inhibits proliferation via targeting JAK-STAT3 signaling in keratinocytes [J]. Cell Death Dis. 2019, 10 (4): 274); in 2024, we found that selective inhibition of CDK9 can alleviate psoriatic dermatitis by inhibiting the STAT3 pathway (Zhao F, et al. Cyclin-Dependent kinase 9 (CDK9) inhibitor Atuveciclibameliorates Imiqui mod-Induced Psoriasis-Likedermatitis in micebyinhibiting various inflammation factors via STAT3 signaling pathway. Int Immunopharmacol. 2024; 129: 111652).

[0004] Currently, CDK pan-inhibitors with CDK6 or CDK9 inhibitory activity are generally highly toxic and have limited clinical prospects; single-target CDK6 or CDK9 inhibitors are highly toxic and are mostly used in anti-tumor research; there are very few selective CDK6 / 9 dual inhibitors, and only one is used to treat triple-negative breast cancer, but this molecule is easily captured and excreted by p-gp and has poor anti-psoriatic activity. In other words, the existing selective CDK6 / 9 dual inhibitors are not suitable for anti-psoriatic treatment.

[0005] In summary, it is particularly important to develop an inhibitor that not only has good anti-psoriasis activity but also can reduce the toxicity of single-target inhibition. Summary of the invention

[0006] The purpose of the present invention is to provide a CDK6 / 9 dual inhibitor and its preparation method and use, which has CDK6 and CDK9 inhibitory activity, inhibits abnormal proliferation and inflammatory factor disorder in psoriasis cell models, has good safety and selectivity within the CDK family, and can be used as a therapeutic drug for psoriatic dermatitis, psoriatic arthritis and other autoimmune diseases.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] A compound structure is shown in Formula 1;

[0009]

[0010] Wherein R1 is any one of the following groups;

[0011]

[0012] A pharmaceutical preparation comprising the compound according to claim 1 or a pharmaceutically acceptable salt or deuterated product thereof.

[0013] Furthermore, the pharmaceutically acceptable salt includes: hydrochloride, methanesulfonate, fumarate, tartrate, citrate, maleate, sodium salt or potassium salt.

[0014] Furthermore, the pharmaceutical preparation also includes a carrier or an excipient.

[0015] Furthermore, the pharmaceutical preparation also includes a therapeutic agent, and the therapeutic agent includes a therapeutic agent for autoimmune diseases, and the therapeutic agent may be methylprednisolone, cyclophosphamide or azathioprine.

[0016] Furthermore, the dosage form of the pharmaceutical preparation is a clinically or pharmaceutically acceptable dosage form; the dosage form includes oral preparations, rectal preparations, nasal preparations, topical preparations, vaginal preparations or parenteral preparations.

[0017] Specifically, the above-mentioned pharmaceutical preparation is a CDK6 / 9 dual inhibitor.

[0018] The present invention also protects a method for preparing the above compound, comprising the following steps:

[0019] Preparation of the first intermediate: p-fluoronitrobenzene reacts with a hydroxyl-containing nitrogen heterocycle to obtain the first intermediate;

[0020] Preparation of the second intermediate: the first intermediate is reduced by zinc to obtain the second intermediate;

[0021] Preparation of the third intermediate: reacting the chloropyrimidine with the second intermediate to obtain the third intermediate;

[0022] Preparation of the compound of formula 1: The third intermediate is reacted with 4-(4-methylpiperazine)aniline to obtain the compound of formula 1.

[0023] Furthermore, in the preparation of the first intermediate, the solvent is DMF and the catalyst is K2CO3.

[0024] Furthermore, in the preparation of the third intermediate, the solvent is DMSO and the catalyst is K2CO3.

[0025] The present invention also protects the use of the above-mentioned pharmaceutical preparation in drugs for autoimmune diseases.

[0026] In a preferred embodiment of the present invention, the autoimmune disease is psoriasis.

[0027] The present invention also provides a method for preparing the compound of formula 1, and the preparation route thereof is as follows:

[0028]

[0029] In route 1: wherein R1 is any one of the following groups;

[0030]

[0031] a is a nitrogen heterocycle containing a hydroxyl group, K2CO3 (potassium carbonate), DMF (N,N-dimethylformamide), 70°C, wherein the nitrogen heterocycle is any one of the following:

[0032]

[0033] b is Zn (zinc powder), HAc (glacial acetic acid), 0℃-50℃;

[0034] c is K2CO3 (potassium carbonate), DMSO (dimethyl sulfoxide);

[0035] d is 4-(4-methylpiperazine)aniline, HCl (concentrated hydrochloric acid), IPA (isopropyl alcohol), 90°C.

[0036] In addition, the present invention also provides a use of a CDK6 / 9 dual inhibitor composition, and the use of the CDK6 / 9 dual inhibitor composition in the preparation of drugs against psoriasis and other autoimmune diseases.

[0037] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs.

[0038] The term "inhibitor" as used herein means that the enzymatic activity of cyclin-dependent kinases 6 and 9 is inhibited.

[0039] The above-mentioned carrier is a "pharmaceutical carrier", which specifically refers to conventional drug carriers in the pharmaceutical field, including conventional diluents, excipients (such as water, etc.), fillers (such as starch, etc.), binders (such as cellulose derivatives, gelatin, etc.), wetting agents (such as glycerol, etc.), disintegrants (such as agar, calcium carbonate, etc.), absorption promoters (such as quaternary ammonium compounds, etc.), surfactants (such as hexadecanol, etc.), adsorption carriers (such as kaolin and soap clay, etc.), lubricants (such as talc, etc.), and flavoring agents, sweeteners, etc. may also be added if necessary.

[0040] The "pharmaceutically acceptable dosage form" is suitable for administration by any appropriate route, such as oral (including buccal or sublingual administration), rectal administration, nasal administration, topical administration (including buccal, sublingual or transdermal administration), vaginal administration or parenteral administration (including subcutaneous injection, intramuscular injection, intravenous injection or intradermal injection). These preparations can be prepared by any method known in the field of pharmacy. For example, by mixing the active ingredient with a carrier or excipient.

[0041] Beneficial Effects

[0042] The CDK6 / 9 dual inhibitor obtained by the present invention has significant CDK6 and CDK9 inhibitory activity, significantly inhibits the inflammatory proliferation of human skin keratinocytes (HaCaT cells) induced by inflammatory factors and inhibits the phosphorylation of STAT3 in HaCaT cells jointly induced by IFN-γ / TNF-α, blocks the key pathway for the progression of psoriasis and significantly inhibits the expression level of major psoriasis-related inflammatory factors in HaCaT cells jointly induced by IFN-γ / TNF-α, and inhibits psoriatic dermatitis; the CDK6 / 9 dual inhibitor obtained by the present invention has a significant inhibitory effect on psoriasis inflammatory proliferation and has no significant toxicity.

[0043] The CDK6 / 9 dual inhibitor can be obtained by the preparation method of the CDK6 / 9 dual inhibitor provided by the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0045] Figure 1 The inhibitory effect of compound 2 on inflammatory proliferation of HaCaT cells in the specific embodiment of the present application;

[0046] Figure 2 This is a graph showing that compound 2 in a specific embodiment of the present application inhibits the phosphorylation of STAT3 in HaCaT cells induced by IFN-γ / TNF-α. Figure 2 a is a protein blot. Figure 2 b is the quantitative graph of phosphorylation;

[0047] Figure 3 This is a graph showing that compound 2 reduces the expression of multiple inflammatory factors in HaCaT cells induced by IFN-γ / TNF-α in a specific embodiment of the present application. Figure 3 a is the expression diagram of inflammatory factor IL-1β, Figure 3 b is the expression diagram of inflammatory factor IL-6, Figure 3 c is the expression graph of inflammatory factor IL-8. DETAILED DESCRIPTION

[0048] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0049] Example 1

[0050] Synthesis of 1-{4-[(5-chloro-2-{[4-(4-methylpiperazin-1-yl)phenyl]amino}pyrimidin-4-yl)amino]phenyl}azetidin-3-ol (1):

[0051] (1) Synthesis of 1-(4-nitrophenyl)azetidin-3-ol (5):

[0052]

[0053] In a round-bottom flask, p-fluoronitrobenzene (450 mg, 3.19 mmol, 1.0 eq), 3-hydroxyazetidine hydrochloride (419.3 mg, 3.83 mmol, 1.2 eq) and K2CO3 (1322.4 mg, 9.57 mmol, 1.5 eq) were added in sequence, dissolved in DMF (8.00 mL), and stirred at 70°C for 4 h. After TLC detection (petroleum ether: ethyl acetate = 10:1) showed that the reaction was completed, the mixture was concentrated and extracted with ethyl acetate. The organic phase was washed with water and saturated sodium chloride solution in sequence, dried over anhydrous Na2SO4, evaporated to dryness under reduced pressure, and the residue was purified by column chromatography (eluent: petroleum ether: ethyl acetate = 4:1) to obtain a yellow solid (5) with a yield of 86%.

[0054] (2) Synthesis of 1-(4-aminophenyl)azetidin-3-ol (6):

[0055]

[0056] In a round-bottom flask, intermediate 5 (408.8 mg, 2.11 mmol, 1.0 eq), zinc powder (2065.8 mg, 31.59 mmol, 15.0 eq) were added in sequence, and glacial acetic acid (8.00 mL) was dissolved, placed in an ice bath for 5 min, protected by nitrogen, and stirred at 50° C. for 8 h. After TLC detection of the reaction (dichloromethane: anhydrous methanol = 20:1), the reaction was detected, and the filter cake was washed with ethyl acetate several times. The filtrate was concentrated, and then the remaining filtrate was acid-base neutralized in an ice bath, and the glacial acetic acid was neutralized with a saturated sodium bicarbonate solution to a pH between 7 and 8, extracted with ethyl acetate, and the aqueous phase was added with a saturated sodium bicarbonate solution and washed with ethyl acetate several times, dried over anhydrous Na2SO4, and evaporated to dryness under reduced pressure. The residue was purified by column chromatography (eluent: dichloromethane: anhydrous methanol = 20:1) to obtain a gray-black solid (6) with a yield of 64%.

[0057] (3) Synthesis of 1-{4-[(2,5-dichloropyrimidin-4-yl)amino]phenyl}azetidin-3-ol (8):

[0058]

[0059] 2,4,5-trichloropyrimidine (367.1 mg, 2.00 mmol, 1.2 eq), intermediate 6 (273.7 mg, 1.67 mmol, 1.0 eq) and K2CO3 (691.5 mg, 5.00 mmol, 3.0 eq) were added to a round-bottom flask in sequence, dissolved in DMSO (5.00 mL), and stirred at room temperature for 2 h. After TLC detection (petroleum ether: ethyl acetate = 4:1) showed that the reaction was completed, it was concentrated and extracted with ethyl acetate. The organic phase was washed with water and saturated sodium chloride solution in sequence, dried over anhydrous Na2SO4, evaporated to dryness under reduced pressure, and the residue was purified by column chromatography (eluent: petroleum ether: ethyl acetate = 4:1) to obtain a yellow solid (8) with a yield of 74%.

[0060] (4) Synthesis of 1-{4-[(5-chloro-2-{[4-(4-methylpiperazin-1-yl)phenyl]amino}pyrimidin-4-yl)amino]phenyl}azetidin-3-ol (3):

[0061]

[0062] In a round-bottom flask, intermediate 8 (242.2 mg, 0.78 mmol, 1.0 eq), 4-(4-methylpiperazine)aniline (149.3 mg, 0.78 mmol, 1.0 eq) and concentrated hydrochloric acid (0.1 mL) were added in sequence, dissolved in isopropanol (6.00 mL), and stirred at 90°C for 9 h. After TLC detection of the reaction (petroleum ether: ethyl acetate = 2:1), the reaction was completed, and ethyl acetate was extracted. The organic phase was washed with water and saturated sodium bicarbonate solution in sequence, neutralized with HCl with saturated sodium bicarbonate solution, and dried with anhydrous Na2SO4, evaporated to dryness under reduced pressure, and the residue was purified by column chromatography (eluent: petroleum ether: ethyl acetate = 2:1) to obtain a brown solid (1) with a yield of 70%.

[0063] The hydrogen spectrum data of the brown solid (1):

[0064] 1 H NMR(600MHz,DMSO-d6)δ8.95(s,1H),8.50(s,1H),7.97(s,1H),7.43(d,J=8.5Hz, 2H),7.25(d,J=8.2Hz,2H),6.73(d,J=8.5Hz,2H),6.66-6.60(m,2H),4.22(t,J=6. 5Hz, 1H), 3.89 (p, J=5.4Hz, 1H), 3.68 (ddd, J=52.2, 11.1, 5.0Hz, 2H), 3.15 (ddt, J= 78.7,12.5,6.0Hz,2H),3.02(t,J=4.9Hz,4H),2.45(t,J=4.9Hz,4H),2.22(s,3H). 13 C NMR (151MHz, DMSO) δ167.44,158.39,156.84,154.31,146.39,146.23,133.36,132.17,131.99,129.13,127.81,126. 05,120.54,116.13,112.24,69.38,65.50,55.17,49.38,48.50,47.34,46.21,30.48,19.13,14.03.ESI-HRMS:calcd for C 24 H 28 ClN7O[M+H] + :466.2122; found:466.2132.

[0065] Embodiment 2:

[0066] Synthesis of (3R)-1-{4-[(5-chloro-2-{[4-(4-methylpiperazin-1-yl)phenyl]amino}pyrimidin-4-yl)amino]phenyl}tetrahydropyrrol-3-ol (2):

[0067] (1) Synthesis of (3R)-1-(4-nitrophenyl)tetrahydropyrrol-3-ol (9):

[0068]

[0069] Compound 9 was prepared according to step (1) of Example 1, except that 3-hydroxyazetidine hydrochloride was replaced by (R)-3-pyrrolidinol.

[0070] (2) Synthesis of (3R)-1-(4-aminophenyl)tetrahydropyrrol-3-ol (10):

[0071]

[0072] Compound 10 was prepared according to step (2) of Example 1, except that 1-(4-nitrophenyl)azetidin-3-ol was replaced by (3R)-1-(4-nitrophenyl)pyrrol-3-ol.

[0073] (3) Synthesis of (3R)-1-{4-[(2,5-dichloropyrimidin-4-yl)amino]phenyl}tetrahydropyrrole-3-ol (11):

[0074]

[0075] Compound 11 was prepared according to step (3) of Example 1, except that 1-(4-aminophenyl)azetidin-3-ol was replaced by (3R)-1-(4-aminophenyl)tetrahydropyrrol-3-ol.

[0076] (4) Synthesis of (3R)-1-{4-[(5-chloro-2-{[4-(4-methylpiperazin-1-yl)phenyl]amino}pyrimidin-4-yl)amino]phenyl}tetrahydropyrrol-3-ol (2):

[0077]

[0078] Compound 2 was prepared according to step (4) of Example 1, except that 1-{4-[(2,5-dichloropyrimidin-4-yl)amino]phenyl}azetidin-3-ol was replaced with (3R)-1-{4-[(2,5-dichloropyrimidin-4-yl)amino]phenyl}tetrahydropyrrol-3-ol.

[0079] The hydrogen spectrum data of compound 2:

[0080] 1H NMR(600MHz,DMSO-d6)δ8.92(s,1H),8.54(s,1H),7.97(s,1H),7.35(dd,J=50.9,8.5H z,4H),6.70(d,J=8.6Hz,2H),6.53-6.50(d,2H),4.98(d,J=3.7Hz,1H),3.44(dd,J=10 .0,4.9Hz,1H),3.33(d,J=8.4Hz,1H),3.28(td,J=8.6,3.7Hz,1H),3.08(dd,J=9.9,2. 4Hz,1H),3.00(t,J=5.0Hz,4H),2.43(t,J=4.9Hz,4H),2.21(s,3H),2.10-1.87(m,2H). 13 C NMR (151MHz, DMSO) δ158.42,156.96,154.30,146.20,145.67,133.28,127.23,126.27,12 0.70,116.04,111.49,69.86,56.75,55.18,49.36,46.26,46.18,34.40.ESI-HRMS:calcd for C 25 H 30 ClN7O[M+H] + :480.2279; found:480.2267.

[0081] Embodiment 3:

[0082] Synthesis of (3S)-1-{4-[(5-chloro-2-{[4-(4-methylpiperazin-1-yl)phenyl]amino}pyrimidin-4-yl)amino]phenyl}tetrahydropyrrol-3-ol (3):

[0083] (1) Synthesis of (3S)-1-(4-nitrophenyl)tetrahydropyrrol-3-ol (12):

[0084]

[0085] Compound 12 was prepared according to step (1) of Example 1, except that 3-hydroxyazetidine hydrochloride was replaced by (S)-3-pyrrolidinol.

[0086] (2) Synthesis of (3S)-1-(4-aminophenyl)tetrahydropyrrol-3-ol (13):

[0087]

[0088] Compound 13 was prepared according to step (2) of Example 1, except that 1-(4-nitrophenyl)azetidin-3-ol was replaced by (3S)-1-(4-nitrophenyl)tetrahydropyrrol-3-ol.

[0089] (3) Synthesis of (3S)-1-{4-[(2,5-dichloropyrimidin-4-yl)amino]phenyl}tetrahydropyrrole-3-ol (14):

[0090]

[0091] Compound 14 was prepared according to step (3) of Example 1, except that 1-(4-aminophenyl)azetidin-3-ol was replaced by (3S)-1-(4-aminophenyl)tetrahydropyrrol-3-ol.

[0092] (4) Synthesis of (3S)-1-{4-[(5-chloro-2-{[4-(4-methylpiperazin-1-yl)phenyl]amino}pyrimidin-4-yl)amino]phenyl}tetrahydropyrrol-3-ol (3):

[0093]

[0094] Compound 3 was prepared according to step (4) of Example 1, except that 1-{4-[(2,5-dichloropyrimidin-4-yl)amino]phenyl}azetidin-3-ol was replaced by (3S)-1-{4-[(2,5-dichloropyrimidin-4-yl)amino]phenyl}tetrahydropyrrol-3-ol.

[0095] Proton spectrum data of compound 3:

[0096] 1 H NMR (600MHz, DMSO-d6) δ8.93(s,1H),8.55(s,1H),7.97(s,1H),7.40(d,J=8.5Hz,2H),7.31(d,J=8.3 Hz,2H),6.70(d,J=8.6Hz,2H),6.53-6.49(m,2H),4.98(d,J=3.7Hz,1H),3.44(dd,J=10.0,5.0Hz,1H) ,3.34(td,J=8.6,6.9Hz,2H),3.28(td,J=8.6,3.7Hz,1H),3.08(dd,J=10.0,2.4Hz,1H),3.00(t,J=5 .0Hz,4H),2.43(t,J=4.9Hz,4H),2.21(s,3H),2.09-2.02(m,1H),1.91(ddt,J=13.0,7.0,3.5Hz,1H). 13C NMR(151MHz,DMSO)δ157.96,156.49,153.83,145.73,145.20,132.82,126.77,125.82,12 0.23,115.58,111.03,69.41,56.29,54.72,48.90,45.80,45.72,33.94.ESI-HRMS:calcd for C 25 H 30 ClN7O[M+H] + :480.2279; found:480.2267.

[0097] Experimental Example 1 Detecting the CDK6 and CDK9 inhibitory activities of a CDK6 / 9 dual inhibitor.

[0098] Using CDK6 inhibitor Palbociclib and CDK9 inhibitor Atuveciclib as positive controls, the inhibitory activity of compounds 1-3 obtained in Examples 1-3 against CDK6 and CDK9, and the selectivity for CDK2 were evaluated. The results are shown in Table 1 below.

[0099] Table 1

[0100]

[0101]

[0102] Active CDK2 / CyclinA2, CDK6 / CyclinD3, and CDK9 / CyclinT1 kinases were purchased from Promega and assayed using ADP-Glo ​​according to the manufacturer's instructions. TM Kinase inhibition was detected by kinase assay (Promega, Madison, Wisconsin, USA).

[0103] For CDK2 / CyclinA2 and CDK6 / CyclinD3, the assay was directed against the substrate Histone H1 at a final concentration of 0.1 mg / mL; for CDK9 / CyclinT1, the assay was directed against the PDKtide substrate (KTFCGTPEYLAPEVRREPRILSEEEQEMFRDFDYIADWC at a final concentration of 0.1 mg / mL. The final ATP concentration for CDK2 / CyclinA2, CDK6 / CyclinD3, and CDK9 / CyclinT1 reactions was 100% of the corresponding kinase. Km, 50, 25 and 25 μM, respectively. The maximum detection concentration of the tested compounds was 2 μM, and the gradient was diluted 5 times. The kinase reactions of CDK2, CDK6 and CDK9 lasted for 20, 60 and 120 minutes at room temperature, respectively, and were finally detected by a microplate reader (SpectraMaxi3x, CA, USA). The positive controls Palbociclib (MedChemExpress) and Atuveciclib (Selleck, TX, USA) were diluted to 50 mM in DMSO and stored at -20 °C before use.

[0104] The results showed that compounds 1, 2, and 3 all had significant CDK6 and CDK9 inhibitory activity, and had a certain selectivity for CDK2. Among them, the inhibitory IC of CDK6 and 9 of molecule 2 with the best activity was 50 They are 0.013 and 0.022 μM respectively, which are significantly higher in activity than previously discovered molecules.

[0105] Experimental Example 2: Detection of the inhibitory effect of CDK6 / 9 dual inhibitors on psoriasis inflammatory hyperplasia:

[0106] This example mainly uses methotrexate as a positive control to evaluate the anti-proliferative activity and main mechanism of action of compound 2 on psoriasis cell models. The psoriasis cell model uses IFN-γ / TNF-α combined induced HaCaT cells to model, and the anti-proliferative activity is detected by MTT method, the effect of target molecules on STAT3 phosphorylation is detected by western blot method, and the expression level of inflammatory genes is detected by RT-qPCR method. Other compounds of the present invention have similar beneficial effects to the compounds listed below, but this should not be understood as the compounds of the present invention having only the following beneficial effects.

[0107] (1) MTT method to investigate the inhibitory activity of compounds on psoriasis cell models.

[0108] The MTT method was used to observe the effect of HaCaT cells on anti-inflammatory proliferation in vitro. HaCaT cells were cultured in DMEM medium and seeded in 96-well plates, with 5×10 3cells. The maximum concentration of the tested compound 2 and the positive control MTX was 2 μM, with 5-fold gradient dilution. After incubation with the compound for 2 h, TNF-α / IFN-γ (2 ng / ml) was added and incubated for another 24 h. At 37 °C, 0.5 mg / ml MTT was incubated for 4 h, the supernatant was discarded, and an appropriate amount of dimethyl sulfoxide (DMSO) (SparkJade, Shandong, China) was added. The absorbance was read at 492 nm using an ELISA reader, and the IC was calculated using SPSS17.0 50 .

[0109] Results Figure 1 Compound 2 can significantly inhibit the inflammatory proliferation of human skin keratinocytes (HaCaT cells) induced by inflammatory factors, with good inhibitory activity in the concentration range of 0.08 to 2 μM and no significant toxicity.

[0110] (2) Western blot was used to detect the effect of compounds on STAT3 phosphorylation in psoriasis cell models.

[0111] HaCaT cells (5×10 5 / well, 6-well plate) at 37°C, 5% CO2 and cultured overnight. Western blot was used to detect the ability of 2 to reduce the level of p-STAT3 (Y705). Cells were pretreated with compounds at the indicated concentrations for 2 hours. Then, the cells were further treated with TNF-α / IFN-γ (2ng / mL) for 24h. HaCaT cells were collected, digested and centrifuged, and then lysed with RIPA buffer (SparkJade, Shandong, China) containing 1mMPMSF (SparkJade, Shandong, China) and 2mM phosphatase inhibitor (Beyotime, Shandong, China). Proteins were quantified with BCA, in a 95°C water bath for 10min, and proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene fluoride (PVDF) membranes.

[0112] Results Figure 2 Compound 2 can significantly inhibit the phosphorylation of STAT3 in HaCaT cells induced by the combination of IFN-γ / TNF-α, blocking this key pathway for the progression of psoriasis.

[0113] (3) RT-qPCR method was used to detect the inhibitory effect of the compounds on the expression of inflammatory factors in psoriasis cell models.

[0114] HaCaT cells (5×10 5 / well, 6-well plate) at 37°C, 5% CO2 overnight. RT-qPCR was used to detect the ability of 2 to reduce the levels of inflammatory factors IL-1β, IL-6, and IL-8. Cells were pretreated with compounds at specified concentrations for 2 hours. Then, cells were further treated with TNF-α / IFN-γ (2ng / mL) for 24h. After the culture, HaCaT cells were collected and the total intracellular mRNA was extracted using the SPARKeasyCellRNAkit kit; the protein nucleic acid quantification instrument was used for quantification and adjusted to an appropriate concentration; the extracted total mRNA was reverse transcribed into cDNA using the SPARKscriptⅡAll-in-oneRTSuperMixforQPCR kit; and the PCR reaction was performed using the 2×SYBRGreenqPCRMix kit. Results The target mRNA was calculated using the 2-ΔΔCt method. The calculation method is as follows: ΔCt = (target gene Ct value - reference gene Ct value); ΔΔCt = (target gene Ct value - average Ct of reference genes in each sample), and finally the relative expression of the target gene in each sample relative to the reference gene is obtained according to 2-ΔΔCt.

[0115] Results Figure 3 Compound 2 can significantly inhibit the expression levels of major psoriasis-related inflammatory factors in HaCaT cells induced by IFN-γ / TNF-α, and inhibit psoriatic dermatitis.

[0116] The above results show that the new 2,4-pyrimidinediamine derivative represented by compound 2 in the present invention has significant inhibitory activity against both CDK6 and CDK9, while the inhibitory activity against CDK2 is relatively weak, achieving selectivity within the CDK family. At the same time, the invented molecule has low cytotoxicity and significant anti-psoriatic effect in cell models, and is a potential therapeutic agent for relieving psoriasis and other immune inflammations.

Claims

1. A compound, characterized in that The compound structure is shown in Formula 1; Wherein R1 is any one of the following groups; 2. A pharmaceutical preparation, characterized in that: The pharmaceutical preparation comprises the compound according to claim 1 or a pharmaceutically acceptable salt or a deuterated substance thereof.

3. The pharmaceutical preparation according to claim 2, characterized in that The pharmaceutically acceptable salts include hydrochloride, methanesulfonate, fumarate, tartrate, citrate, maleate, sodium salt or potassium salt.

4. The pharmaceutical preparation according to claim 2, characterized in that The pharmaceutical preparation also includes a carrier or excipient.

5. The pharmaceutical preparation according to claim 2, characterized in that The pharmaceutical preparation includes a therapeutic agent for an autoimmune disease.

6. The pharmaceutical preparation according to claim 2, characterized in that The dosage form of the pharmaceutical preparation is a clinically or pharmaceutically acceptable dosage form, which includes oral administration preparations, rectal administration preparations, nasal administration preparations, topical administration preparations, vaginal administration preparations or parenteral administration preparations.

7. A method for preparing the compound of claim 1, comprising the following steps: Preparation of the first intermediate: p-fluoronitrobenzene reacts with a hydroxyl-containing nitrogen heterocycle to obtain the first intermediate; Preparation of the second intermediate: the nitro group of the first intermediate is reduced to an amino group by zinc powder to obtain the second intermediate; Preparation of the third intermediate: reacting the chloropyrimidine with the second intermediate to obtain the third intermediate; Preparation of the compound of formula 1: The third intermediate reacts with 4-(4-methylpiperazine)aniline to obtain the compound of formula 1; The hydroxyl-containing nitrogen heterocycle is any one of the following:

8. The method according to claim 7, characterized in that In the preparation of the first intermediate, the solvent is DMF and the catalyst is K2CO3.

9. The method according to claim 7, characterized in that: In the preparation of the third intermediate, the solvent is DMSO and the catalyst is K2CO3.

10. Use of the compound according to claim 1 or the pharmaceutical preparation according to any one of claims 2 to 6 in the preparation of drugs for autoimmune diseases.

Images