A CDK6 / 9 dual inhibitor and its preparation method and use

Abstract

The present invention relates to the field of medicinal chemistry, and specifically to a CDK6 / 9 dual inhibitor, a preparation method, and an application thereof. This inhibitor has both CDK6 and CDK9 inhibitory activity, has an inhibitory effect on abnormal proliferation and inflammatory factor disorders in psoriasis cell models, and has good safety and selectivity within the CDK family. This inhibitor can be used as a therapeutic drug for psoriatic 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 skin disease. However, traditional anti-psoriatic drugs are ineffective, prone to relapse and highly toxic. Therefore, developing a safe and long-term treatment for psoriasis is a huge challenge.

[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, promoting 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 dermatocytes [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 Imiquimod-Induced Psoriasis-Likedermatitis in miking 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, with only one used in the treatment of 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 purposes.

[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 present invention aims to provide a dual CDK6 / 9 inhibitor, its preparation method, and use. This inhibitor possesses both CDK6 and CDK9 inhibitory activity, suppresses abnormal proliferation and inflammatory cytokine disturbances in psoriasis cell models, and exhibits good safety and selectivity within the CDK family. It can be used as a therapeutic agent 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] Formula 1;

[0011] Wherein R1 is any one of the following groups;

[0012] 、 、 .

[0013] A pharmaceutical preparation comprising the above compound or its pharmaceutically acceptable salt or deuterated substance.

[0014] Furthermore, the pharmaceutically acceptable salts include: hydrochloride, methanesulfonate, fumarate, tartrate, citrate, maleate, sodium salt or potassium salt.

[0015] Furthermore, the pharmaceutical preparation also includes a carrier or excipient.

[0016] Furthermore, the pharmaceutical preparation also includes a therapeutic agent, which includes a therapeutic agent for autoimmune diseases, and the therapeutic agent may be methylprednisolone, cyclophosphamide or azathioprine.

[0017] Furthermore, the dosage form of the pharmaceutical preparation is a clinically or pharmaceutically acceptable dosage form; the dosage form includes oral administration preparations, rectal administration preparations, nasal administration preparations, topical administration preparations, vaginal administration preparations or parenteral administration preparations.

[0018] Specifically, the above-mentioned pharmaceutical preparation is a CDK6 / 9 dual inhibitor.

[0019] The present invention also protects a method for preparing the above-mentioned compound, comprising the following steps:

[0020] Preparation of the first intermediate: p-fluoronitrobenzene reacts with a hydroxyl-containing nitrogen heterocycle to obtain the first intermediate;

[0021] Preparation of the second intermediate: the first intermediate is reduced by zinc to obtain the second intermediate;

[0022] Preparation of the third intermediate: reacting the chloropyrimidine with the second intermediate to obtain the third intermediate;

[0023] Preparation of the compound represented by Formula 1: The third intermediate reacts with 4-(4-methylpiperazine)aniline to obtain the compound represented by Formula 1.

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

[0025] Furthermore, in the preparation of the third intermediate, the solvent is DMSO and the catalyst is K2CO3.

[0026] The present invention also protects the use of the above-mentioned pharmaceutical preparation in autoimmune disease medicine.

[0027] In a preferred embodiment of the present invention, the autoimmune disease is psoriasis.

[0028] The present invention also provides a method for preparing the compound of formula 1, and the preparation route is as follows:

[0029]

[0030] Route 1

[0031] In route 1: wherein R1 is any one of the following groups;

[0032] 、 、 .

[0033] a is a nitrogen heterocycle containing a hydroxyl group, K2CO3 (potassium carbonate), DMF (N,N-dimethylformamide), 70°C, wherein the nitrogen heterocycle is any of the following:

[0034] 、 、 .

[0035] b is Zn (zinc powder), HAc (glacial acetic acid), 0℃-50℃;

[0036] c is K2CO3 (potassium carbonate), DMSO (dimethyl sulfoxide);

[0037] d is 4-(4-methylpiperazine)aniline, HCl (concentrated hydrochloric acid), IPA (isopropyl alcohol), 90°C.

[0038] 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 for treating psoriasis and other autoimmune diseases.

[0039] 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.

[0040] The term "inhibitor" as used herein means that the enzymatic activities of cyclin-dependent kinases 6 and 9 are inhibited.

[0041] 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 glycerin, etc.), disintegrants (such as agar, calcium carbonate, etc.), absorption accelerators (such as quaternary ammonium compounds, etc.), surfactants (such as cetyl alcohol, etc.), adsorption carriers (such as kaolin and bentonite, etc.), lubricants (such as talc, etc.), and flavoring agents, sweeteners, etc. may also be added if necessary.

[0042] The term "any pharmaceutically acceptable dosage form" is suitable for administration by any appropriate route, such as oral (including buccal or sublingual administration), rectal, nasal, topical (including buccal, sublingual, or transdermal), vaginal, or parenteral (including subcutaneous, intramuscular, intravenous, or intradermal injection). These preparations can be prepared by any method known in the art of pharmacy, for example, by mixing the active ingredient with a carrier or excipient.

[0043] Beneficial effects

[0044] 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 levels of major psoriasis-related inflammatory factors in HaCaT cells jointly induced by IFN-γ / TNF-α, thereby inhibiting 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.

[0045] 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

[0046] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following is a brief introduction to 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 any creative work.

[0047] Figure 1 The inhibitory effect of compound 2 on inflammatory proliferation of HaCaT cells in the specific embodiment of this application;

[0048] Figure 2 This is a graph showing that compound 2 inhibits the phosphorylation of STAT3 in HaCaT cells induced by IFN-γ / TNF-α in a specific embodiment of the present application. Figure 2 a is a Western blot image, Figure 2 b is the quantitative graph of phosphorylation;

[0049] 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

[0050] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only 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 making creative efforts shall fall within the scope of protection of the present invention.

[0051] Example 1

[0052] Synthesis of 1-{4-[(5-chloro-2-{[4-(4-methylpiperazin-1-yl)phenyl]amino}pyrimidin-4-yl)amino]phenyl}azetidin-3-ol (1):

[0053] (1) Synthesis of 1-(4-nitrophenyl)azetidin-3-ol (5):

[0054]

[0055] 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 and dissolved in DMF (8.00 mL). The mixture was stirred at 70°C for 4 h. After TLC (petroleum ether:ethyl acetate = 10:1) showed that the reaction was complete, 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, and evaporated to dryness under reduced pressure. 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%.

[0056] (2) Synthesis of 1-(4-aminophenyl)azetidin-3-ol (6):

[0057]

[0058] In a round-bottom flask, intermediate 5 (408.8 mg, 2.11 mmol, 1.0 eq) and zinc powder (2065.8 mg, 31.59 mmol, 15.0 eq) were added in sequence and dissolved in glacial acetic acid (8.00 mL). The mixture was placed in an ice bath for 5 min, protected by nitrogen, and stirred at 50°C for 8 h. After TLC detection (dichloromethane:anhydrous methanol = 20:1) showed that the reaction was complete, the mixture was filtered, the filter cake was washed with ethyl acetate several times, the filtrate was concentrated, and the remaining filtrate was acid-base neutralized in an ice bath. The glacial acetic acid was neutralized with saturated sodium bicarbonate solution to a pH between 7 and 8, and the mixture was extracted with ethyl acetate. The aqueous phase was added with saturated sodium bicarbonate solution and washed with ethyl acetate several times. The mixture was 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%.

[0059] (3) Synthesis of 1-{4-[(2,5-dichloropyrimidin-4-yl)amino]phenyl}azetidin-3-ol (8):

[0060]

[0061] 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 (petroleum ether:ethyl acetate = 4:1) showed that the reaction was complete, the product 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, and evaporated to dryness under reduced pressure. 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%.

[0062] (4) Synthesis of 1-{4-[(5-chloro-2-{[4-(4-methylpiperazin-1-yl)phenyl]amino}pyrimidin-4-yl)amino]phenyl}azetidin-3-ol (3):

[0063]

[0064] 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 to a round-bottom flask in sequence, dissolved in isopropanol (6.00 mL), and stirred at 90°C for 9 h. The reaction was detected by TLC (petroleum ether:ethyl acetate = 2:1), indicating that the reaction was complete. The product was extracted with ethyl acetate, and the organic phase was washed with water and saturated sodium bicarbonate solution in sequence. HCl was neutralized with saturated sodium bicarbonate solution, and dried over anhydrous Na2SO4. The product was 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%.

[0065] Proton spectrum data of the brown solid (1):

[0066] 1 H NMR (600 MHz, DMSO- d 6) δ 8.95 (s, 1H), 8.50 (s, 1H), 7.97 (s, 1H),7.43 (d, J = 8.5 Hz, 2H), 7.25 (d, J = 8.2 Hz, 2H), 6.73 (d, J = 8.5 Hz, 2H), 6.66– 6.60 (m, 2H), 4.22 (t, J = 6.5 Hz, 1H), 3.89 (p, J= 5.4 Hz, 1H), 3.68 (ddd, J =52.2, 11.1, 5.0 Hz, 2H), 3.15 (ddt, J = 78.7, 12.5, 6.0 Hz, 2H), 3.02 (t, J = 4.9Hz, 4H), 2.45 (t, J = 4.9 Hz, 4H), 2.22 (s, 3H). 13 C NMR (151 MHz, 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.

[0067] Example 2:

[0068] Synthesis of (3R)-1-{4-[(5-chloro-2-{[4-(4-methylpiperazin-1-yl)phenyl]amino}pyrimidin-4-yl)amino]phenyl}tetrahydropyrrol-3-ol (2):

[0069] (1) Synthesis of (3R)-1-(4-nitrophenyl)tetrahydropyrrol-3-ol (9):

[0070]

[0071] Compound 9 was prepared according to step (1) of Example 1, except that 3-hydroxyazetidine hydrochloride was replaced by (R)-3-pyrrolidinol.

[0072] (2) Synthesis of (3R)-1-(4-aminophenyl)tetrahydropyrrol-3-ol (10):

[0073]

[0074] 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)tetrahydropyrrol-3-ol.

[0075] (3) Synthesis of (3R)-1-{4-[(2,5-dichloropyrimidin-4-yl)amino]phenyl}tetrahydropyrrole-3-ol (11):

[0076]

[0077] 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.

[0078] (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):

[0079]

[0080] 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.

[0081] Proton spectrum data of compound 2:

[0082] 1 H NMR (600 MHz, DMSO- d 6) δ 8.92 (s, 1H), 8.54 (s, 1H), 7.97 (s, 1H), 7.35 (dd, J = 50.9, 8.5 Hz, 4H), 6.70 (d, J = 8.6 Hz, 2H), 6.53 – 6.50 (d, 2H), 4.98 (d, J = 3.7 Hz, 1H), 3.44 (dd, J = 10.0, 4.9 Hz, 1H), 3.33 (d, J = 8.4 Hz,1H), 3.28 (td, J = 8.6, 3.7 Hz, 1H), 3.08 (dd, J = 9.9, 2.4 Hz, 1H), 3.00 (t,J =5.0 Hz, 4H), 2.43 (t, J = 4.9 Hz, 4H), 2.21 (s, 3H), 2.10 – 1.87 (m, 2H). 13 C NMR(151 MHz, DMSO) δ 158.42, 156.96, 154.30, 146.20, 145.67, 133.28, 127.23,126.27, 120.70, 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.

[0083] Example 3:

[0084] Synthesis of (3S)-1-{4-[(5-chloro-2-{[4-(4-methylpiperazin-1-yl)phenyl]amino}pyrimidin-4-yl)amino]phenyl}tetrahydropyrrol-3-ol (3):

[0085] (1) Synthesis of (3S)-1-(4-nitrophenyl)tetrahydropyrrol-3-ol (12):

[0086]

[0087] Compound 12 was prepared according to step (1) of Example 1, except that 3-hydroxyazetidine hydrochloride was replaced by (S)-3-pyrrolidinol.

[0088] (2) Synthesis of (3S)-1-(4-aminophenyl)tetrahydropyrrol-3-ol (13):

[0089]

[0090] 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.

[0091] (3) Synthesis of (3S)-1-{4-[(2,5-dichloropyrimidin-4-yl)amino]phenyl}tetrahydropyrrol-3-ol (14):

[0092]

[0093] 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.

[0094] (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):

[0095]

[0096] 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 with (3S)-1-{4-[(2,5-dichloropyrimidin-4-yl)amino]phenyl}tetrahydropyrrol-3-ol.

[0097] Proton spectrum data of compound 3:

[0098] 1 H NMR (600 MHz, DMSO- d 6) δ 8.93 (s, 1H), 8.55 (s, 1H), 7.97 (s, 1H), 7.40 (d, J = 8.5 Hz, 2H), 7.31 (d, J = 8.3 Hz, 2H), 6.70 (d, J = 8.6 Hz, 2H), 6.53– 6.49 (m, 2H), 4.98 (d, J = 3.7 Hz, 1H), 3.44 (dd, J = 10.0, 5.0 Hz, 1H), 3.34(td, J = 8.6, 6.9 Hz, 2H), 3.28 (td, J = 8.6, 3.7 Hz, 1H), 3.08 (dd, J = 10.0, 2.4Hz, 1H), 3.00 (t, J = 5.0 Hz, 4H), 2.43 (t, J = 4.9 Hz, 4H), 2.21 (s, 3H), 2.09 –2.02 (m, 1H), 1.91 (ddt, J = 13.0, 7.0, 3.5 Hz, 1H). 13C NMR (151 MHz, DMSO) δ157.96, 156.49, 153.83, 145.73, 145.20, 132.82, 126.77, 125.82, 120.23,115.58, 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.

[0099] Experimental Example 1 Detecting the CDK6 and CDK9 inhibitory activities of a CDK6 / 9 dual inhibitor.

[0100] Using CDK6 inhibitor Palbociclib and CDK9 inhibitor Atuveciclib as positive controls, the inhibitory activities of compounds 1-3 obtained in Examples 1-3 against CDK6 and CDK9, and their selectivity for CDK2 were evaluated. The results are shown in Table 1 below.

[0101] Table 1

[0102]

[0103] Active CDK2 / CyclinA2, CDK6 / CyclinD3, and CDK9 / CyclinT1 kinases were purchased from Promega, and kinase inhibition was detected using the ADP-Glo™ kinase assay (Promega, Madison, Wisconsin, USA) according to the manufacturer's instructions.

[0104] For CDK2 / CyclinA2 and CDK6 / CyclinD3, the assay was performed against the substrate Histone H1 at a final concentration of 0.1 mg / mL; for CDK9 / CyclinT1, the assay was performed 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 the K of the corresponding kinase. The kinase reactions for CDK2, CDK6, and CDK9 were performed at room temperature for 20, 60, and 120 minutes, respectively, and then detected using a microplate reader (SpectraMax i3x, CA, USA). Positive controls, palbociclib (MedChemExpress) and atuveciclib (Selleck, TX, USA), were diluted to 50 mM in DMSO and stored at −20°C until use.

[0105] 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 most active molecule 2 had an inhibitory IC of CDK6 and 9. 50 The activity of the two molecules is significantly improved compared with that of previously discovered molecules, which are 0.013 and 0.022 μM respectively.

[0106] Experimental Example 2: Detecting the inhibitory effect of CDK6 / 9 dual inhibitors on psoriasis inflammatory hyperplasia:

[0107] This example primarily uses methotrexate as a positive control to evaluate the anti-proliferative activity and primary mechanism of action of Compound 2 in a psoriasis cell model. The psoriasis cell model was established using HaCaT cells induced by a combination of IFN-γ and TNF-α. The anti-proliferative activity was assayed using the MTT assay, the effect of the target molecule on STAT3 phosphorylation was assayed using western blot, and the expression levels of inflammatory genes were assayed using RT-qPCR. Other compounds of the present invention have similar beneficial effects to the compounds listed below, but this should not be construed as indicating that the compounds of the present invention only have the following beneficial effects.

[0108] (1) MTT method to investigate the inhibitory activity of compounds on the proliferation of psoriasis cell models.

[0109] The MTT assay 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 cells per well. 3cells. The maximum concentration of the test compound 2 and the positive control MTX was 2 μM, and the mixture was diluted 5-fold. 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 a microplate reader, and the IC was calculated using SPSS17.0. 50 .

[0110] Results see 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 0.08~2μM and no significant toxicity.

[0111] (2) Western blot was used to detect the effects of compounds on STAT3 phosphorylation in psoriasis cell models.

[0112] HaCaT cells (5×10 5 Cells were pretreated with the indicated compounds at the indicated concentrations for 2 h to assess their ability to reduce p-STAT3 (Y705) levels. HaCaT cells were harvested, digested, and centrifuged before being lysed with RIPA buffer (SparkJade, Shandong, China) containing 1 mM PMSF (SparkJade, Shandong, China) and 2 mM phosphatase inhibitors (Beyotime, Shandong, China). Proteins were quantified using BCA assay, incubated at 95°C in a water bath for 10 min, and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) before being transferred to polyvinylidene fluoride (PVDF) membranes.

[0113] Results see 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.

[0114] (3) RT-qPCR was used to detect the inhibitory effect of the compounds on the expression of inflammatory factors in psoriasis cell models.

[0115] HaCaT cells (5×10 5Cells were cultured overnight in 6-well plates (cells / well, 37°C, 5% CO2). RT-qPCR was used to assess the ability of 2 to reduce the levels of inflammatory factors IL-1β, IL-6, and IL-8. Cells were pretreated with the compounds at the indicated concentrations for 2 hours. Subsequently, cells were further treated with TNF-α / IFN-γ (2 ng / mL) for 24 hours. After culture, HaCaT cells were harvested and total cellular mRNA was extracted using the Spark Easy Cell RNA Kit. Total mRNA was quantified using a protein and nucleic acid quantifier and adjusted to an appropriate concentration. The extracted total mRNA was reverse transcribed into cDNA using the Spark Script II All-in-one RT Super Mix for qPCR Kit. PCR reactions were performed using the 2× SYBR Green qPCR Mix Kit. The results were calculated using the 2-ΔΔCt method for target mRNA. The calculation method is as follows: ΔCt = (Ct value of target gene - Ct value of internal reference gene); ΔΔCt = (Ct value of target gene - average Ct of internal reference gene of each sample). Finally, the relative expression level of target gene in each sample relative to the internal reference gene is obtained according to 2-ΔΔCt.

[0116] Results see Figure 3 Compound 2 can significantly inhibit the expression levels of major psoriasis-related inflammatory factors in HaCaT cells induced by the combination of IFN-γ / TNF-α, and inhibit psoriatic dermatitis.

[0117] These results demonstrate that the novel 2,4-pyrimidinediamine derivatives, represented by compound 2, exhibit significant inhibitory activity against both CDK6 and CDK9, while their inhibitory activity against CDK2 is relatively weak, demonstrating selectivity within the CDK family. Furthermore, these novel molecules exhibit minimal cytotoxicity and significant anti-psoriatic efficacy in cell models, making them potential therapeutic agents for alleviating psoriasis and other immune-inflammatory conditions.

Claims

1. A compound, characterized in that The compound structure is shown in Formula 1; 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 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 further includes a carrier or excipient.

5. The pharmaceutical preparation according to claim 2, characterized in that The pharmaceutical formulation 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, and the dosage form includes an oral administration preparation, a rectal administration preparation, a nasal administration preparation or a vaginal administration preparation.

7. A method for preparing the compound according to 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 using 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 The solvent for preparing the first intermediate is DMF and the catalyst is K2CO3.

9. The method according to claim 7, characterized in that The solvent for preparing the third intermediate 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.

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