CN116813692A - Phosphorus-containing compounds, pharmaceutical compositions thereof and uses thereof - Google Patents

Abstract

The invention discloses a phosphorus-containing compound, a pharmaceutical composition thereof and application thereof. Specifically, the invention discloses a compound shown as a formula I or pharmaceutically acceptable salt thereof. The phosphorus-containing compound has better inhibition activity on MAP4K3 or HPK1 kinase。

Description

Phosphorus-containing compounds, pharmaceutical compositions thereof and uses thereof

Technical field

The present invention relates to phosphorus-containing compounds, pharmaceutical compositions thereof and uses thereof.

Background technique

HPK1 is a serine/threonine protein kinase that uses ATP or other nucleotides as a phosphate donor. HPK1 plays a key role in negative regulation of immune function. HPK1 is a key negative feedback regulator of T cell receptors and B cell receptors, which can negatively regulate the functions of T cells and B cells; inhibiting HPK1 can enhance anti-tumor immune responses. Multiple studies have found that HPK1 is a potential tumor immunotherapy target, and HPK1 inhibitors can become useful therapeutic drugs for cancer immunotherapy.

MAP4K3 is also a serine/threonine kinase and belongs to the mammalian Ste20-like kinase family. Overexpression of MAP4K3 can induce autoimmune diseases and cancer metastasis. Inhibiting MAP4K3 signaling can reduce the development of several diseases, including autoimmune diseases and cancer metastasis. Therefore, inhibiting MAP4K3 could be a useful therapeutic approach to treat autoimmune diseases as well as cancer recurrence without inducing an autoimmune response.

Contents of the invention

The technical problem to be solved by the present invention is to overcome the shortcomings of limited types of kinase inhibitors in the prior art and provide a new type of phosphorus-containing compound, its pharmaceutical composition and its use. The compound of the present invention has good inhibitory activity against MAP4K3 or HPK1 kinase.

The present invention solves the above technical problems through the following technical solutions.

The present invention provides a compound represented by formula I or a pharmaceutically acceptable salt thereof,

Where n is 3, 4 or 5.

In a certain embodiment of the present invention, the compound represented by formula I is the following compound:

The present invention also provides a pharmaceutical composition, which contains substance X and pharmaceutically acceptable excipients, where the substance

The present invention also provides a use of the above-mentioned substance X or the above-mentioned pharmaceutical composition in the preparation of HPK1 inhibitors.

The present invention also provides an application of the above-mentioned substance cancer or liver cancer.

The present invention also provides a use of the above-mentioned substance X or the above-mentioned pharmaceutical composition in preparing a MAP4K3 inhibitor.

The present invention also provides an application of the above-mentioned substance Examples include psoriatic arthritis, rheumatoid arthritis, or systemic lupus erythematosus.

The present invention also provides an application of the above-mentioned substance Diseases are, for example, psoriatic arthritis, rheumatoid arthritis or systemic lupus erythematosus.

In the present invention, the "inhibitor" can be used in vivo in mammals; it can also be used in vitro, mainly for experimental purposes, for example, as a standard sample or control sample to provide comparison, or to prepare reagents according to conventional methods in this field. box.

In the present invention, "pharmaceutically acceptable" refers to substances (such as pharmaceutical excipients) that do not affect the biological activity or properties of the compounds of the invention and are relatively non-toxic, that is, the substances can be administered to individuals without causing adverse biological effects. React or interact in an adverse manner with any component contained in the composition.

In the present invention, the term "pharmaceutically acceptable salt" refers to a salt obtained by reacting a compound with a pharmaceutically acceptable (relatively non-toxic, safe, and suitable for patient use) acid or base. When the compound contains relatively acidic functional groups, base addition salts can be obtained by contacting the free form of the compound with a sufficient amount of a pharmaceutically acceptable base in a suitable inert solvent. When the compound contains relatively basic functional groups, acid addition salts can be obtained by contacting the free form of the compound with a sufficient amount of a pharmaceutically acceptable acid in a suitable inert solvent.

The term "pharmaceutically acceptable excipients" refers to the excipients and additives used in the production of pharmaceuticals and formulations. They are all substances other than active ingredients included in pharmaceutical preparations. Please refer to the Pharmacopoeia of the People's Republic of China (2020 Edition), Part IV, or the Handbook of Pharmaceutical Excipients (Raymond C Rowe, 2009 Sixth Edition).

The term "prevention" refers to the reduction of the risk of acquiring or developing a disease or disorder.

The term "treatment" refers to therapeutic therapy or palliative measures. When referring to a specific condition, treatment means: (1) alleviating the disease or one or more biological manifestations of the condition, (2) interfering with (a) one or more points in the biological cascade that causes or causes the condition or (b) ) one or more biological manifestations of a condition, (3) amelioration of one or more symptoms, effects, or side effects associated with the condition, or one or more symptoms, effects, or side effects associated with the condition or its treatment, or (4) slow the progression of a condition or one or more biological manifestations of a condition. "Treatment" may also refer to prolonging survival compared to expected survival without treatment.

On the basis of not violating common sense in the field, the above preferred conditions can be combined arbitrarily to obtain preferred examples of the present invention.

The reagents and raw materials used in the present invention are all commercially available.

The positive and progressive effect of the present invention is that the compound of the present invention has good inhibitory activity against MAP4K3 or HPK1.

Detailed ways

The present invention is further described below by means of examples, but the present invention is not limited to the scope of the described examples. Experimental methods that do not indicate specific conditions in the following examples should be selected according to conventional methods and conditions, or according to product specifications.

An exemplary method for preparing a high performance liquid chromatograph is as follows:

Method A:

Chromatographic column: Gilson2 Xbrige C18 19*150mm, 5μm; mobile phase: acetonitrile aqueous solution (0.1% ammonium bicarbonate) 20% to 60% gradient elution, flow rate 15mL/min.

Representative methods of high performance liquid chromatography-mass spectrometry analysis:

method 1:

Instrument: Agilent 1200_series HPLC-6120MS; Chromatographic column: Waters Xbridge C18 column (1.8 μm, 4.6*50mm); column temperature: 40°C; mobile phase: acetonitrile aqueous solution (containing 0.02% ammonium acetate) 5% to 95% gradient elution , running time 6.5 minutes; flow rate: 1.5mL/min.

Method 2:

Instrument: Agilent 1200_series HPLC-6120MS; Chromatographic column: Waters Xbridge C18 column (1.8 μm, 4.6*50mm); column temperature: 40°C; mobile phase: acetonitrile aqueous solution (containing 0.1% trifluoroacetic acid) 5% to 95% gradient wash Take off, run time 6.5 minutes; flow rate: 1.5mL/min.

Method 3:

Instrument: Agilent 1260_series HPLC-6120MS; Chromatographic column: Diamonsil Plus C18 column (1.8μm, 4.6*30mm); Column temperature: 40°C; Mobile phase: acetonitrile aqueous solution (containing 0.02% ammonium acetate) 5% to 95% gradient Elution, running time 2.5 minutes; flow rate: 1.5mL/min.

Example 1: Preparation of Compound 1

The synthesis route is as follows:

Step 1. Synthesis of intermediate 1-2

Compound 1-1 (135 mg, 0.29 mmol, CAS 2227609-24-5), 14-(tosyloxy)-3,6,9,12-tetraoxatetradecanoic acid tert-butyl ester (136 mg, 0.29 mmol, CAS 169751-73-9) and N,N-diisopropylethylamine (76 mg, 0.58 mmol) were dissolved in 2 mL DMF and stirred at 60°C for 16 hours. The reaction was cooled to room temperature, 10 mL of water was added to the reaction solution under stirring conditions, and the solid was filtered and dried to obtain yellow solid compound 1-2 (200 mg, yield 91%). The crude product was used directly in the next reaction without further purification. LC-MS (ESI, method 3) t _R =1.61min, m/z (M+H) ⁺ =748.7.

Step 2. Synthesis of intermediates 1-3

Compound 1-2 (110 mg, 0.75 mmol) was dissolved in 5 mL of 4M hydrochloric acid ethyl acetate solution, and stirred at room temperature for 2 hours. The generated solid was filtered and dried to obtain pale white solid compound 1-3 (57 mg, yield 56%). The crude product was used directly in the next reaction without further purification. LC-MS (ESI, method 3) t _R =1.21min, m/z (M+H) ⁺ =692.6.

Step 3. Synthesis of Compound 1

Compound 1-3 (55mg, 0.062mmol), compound 1-4 (27mg, 0.062mmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyl Urea hexafluorophosphate (95 mg, 0.25 mmol) and N,N-diisopropylethylamine (40 mg, 0.31 mmol) were dissolved in 0.6 mL DMF and stirred at room temperature for 1 hour. Add 30 mL of ethyl acetate to the reaction and wash three times with 10 mL of water. The separated organic phase was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to obtain a crude product. The crude product was separated and purified using preparative high-performance liquid chromatography (method A) to obtain compound 1 as a white solid (20 mg, yield 29%). LC-MS (ESI, method 1) t _R =3.49min, m/z (M+H) ⁺ =1104.5. ¹ H NMR (400MHz, DMSO-d ₆ ) δ11.28 (s, 1H), 9.92 (br s,1H),8.98(s,1H),8.58(t,J＝5.6Hz,1H),8.44-8.42(m,1H),8.31(s,1H),8.21(s,1H),7.70(s ,1H),7.63-7.58(m,1H),7.49(t,J＝7.6Hz,1H),7.44-7.39(m,5H),7.18(t,J＝6.4Hz,1H),6.89(s, 1H),4.57-4.22(m,8H),4.03(s,3H),3.67-3.42(m,18H),3.26-2.99(m,2H),2.87-2.82(m,2H),2.67(s, 3H),2.43-2.08(m,1H),1.93-1.86(m,1H),1.79(s,3H),1.76(s,3H),0.93(s,9H).

Example 2: Preparation of Compound 2

The synthesis route is as follows:

Step 1. Synthesis of Compound 2

Compound 2-1 (60mg, 0.14mmol), N,N-diisopropylethylamine (74.2mg, 0.1mL, 0.57mmol) and 2-(7-azobenzotriazole)-N,N , N', N'-tetramethylurea hexafluorophosphate (69 mg, 0.18 mmol) was dissolved in 2 mL DMF. After stirring at room temperature for 5 minutes, compound 1-1 (76 mg, 0.16 mmol) was added and continued at room temperature. Stir for 2 hours. Filter, and the filtrate is separated and purified using preparative high-performance liquid chromatography (method A) to obtain yellow-green solid compound 2 (47 mg, yield 38%). LC-MS (ESI, method 2) t _R =4.51min, m/z (M+H) ⁺ =855.5. ¹ H NMR (400MHz, DMSO-d ₆ ) δ11.09 (s, 1H), 11.04 (d ,J＝6.4Hz,1H),8.40-8.38(m,1H),8.14(s,1H),8.08-8.06(m,1H),7.59-7.54(m,3H),7.40(t,J＝9.6 Hz,1H),7.15(dd,J＝12.8,5.6Hz,1H),7.06(t,J＝8.0Hz,1H),7.00(d,J＝6.8Hz,1H),6.90-6.88(m,1H ),6.50-6.48(m,1H),5.03(dd,J＝12.8,5.6Hz,1H),4.61(s,1H),4.57(s,1H),3.78(s,3H),3.65-3.62( m,4H),3.30-3.23(m,2H),2.91-2.82(m,1H),2.68-2.66(m,1H),2.60-2.56(m,2H),2.38(t,J＝7.6Hz, 2H),1.77(s,3H),1.74(s,3H),1.58-1.51(m,4H),1.32-1.30(m,6H).

Effect Example: Kinase Activity Test

Purpose

Detect the IC ₅₀ value of the test compound on HPK1 and MAP4K3 kinase. The test method is Mobility shift assay. The starting concentration of the compound is 5000nM, 3-fold dilution, 10 concentration points, multiple well detection, and the final ATP concentration is 1mM.

Reagents and consumables are shown in the table below:

laboratory apparatus

Centrifuge (manufacturer: Eppendorf, model: 5430)

Microplate reader (manufacturer: Perkin Elmer, model: Caliper EZ Reader II)

Echo 550 (manufacturer: Labcyte, model: Echo 550)

experimental method

1 compound preparation

Prepare the compound powder into a 10mM or 20mM DMSO stock solution. Store in a nitrogen cabinet away from light.

2 Kinase reaction process

(1) Prepare 1X kinase buffer.

(2) Preparation of compound concentration gradient: The starting concentration of the compound is 5000 nM, diluted 3 times to 10 concentration points, and diluted into a 100% DMSO solution with a final concentration of 100 times in a 384-well plate. Use the dispenser Echo550 to transfer 250 nL of the compound at 100 times the final concentration to the destination 384-well plate.

(3) Use 1X kinase buffer to prepare a kinase solution with 2.5 times the final concentration (final enzyme concentration: 0.625nM).

(4) Add 10 μL of 2.5 times the final concentration of kinase solution to the test compound wells and positive control wells, and add an equal volume of 1X kinase buffer to the negative control wells.

(5) Centrifuge at 1000 rpm for 30 seconds, shake and mix the reaction plate, and then incubate the reaction at room temperature for 10 minutes.

(6) Use 1X kinase buffer to prepare a mixed solution of 5 times the final concentration of ATP (5mM) and 3 times the final concentration of the kinase substrate solution (3μM).

(7) Add 15 μL of a mixed solution of ATP and substrate at 5/3 times the final concentration to start the reaction.

(8) Centrifuge the 384-well plate at 1000 rpm for 30 seconds, shake and mix, and incubate at room temperature for the corresponding time (HPK1 30min, MAP4K3 30min).

(9) Add 30 μL of stop detection solution (50 mM EDTA) to stop the kinase reaction, centrifuge at 1000 rpm for 30 seconds, and shake to mix.

(10) Use Caliper EZ Reader to read the conversion rate.

3Data analysis

Calculation formula:

% inhibition rate = (maximum conversion rate % - sample conversion rate %) * 100 / (maximum conversion rate % - minimum conversion rate %)

Among them: the sample conversion rate is the conversion rate reading of the sample, the minimum conversion rate is the average value of the negative control wells, which represents the conversion rate reading of the wells without enzyme activity; the maximum conversion rate refers to the conversion rate reading of the wells without compound inhibition.

Fitted dose-response curve

Taking the Log value of the concentration as the X-axis and the percentage inhibition rate as the Y-axis, use the Log(inhibitor) vs. response-Variable slope of the analysis software Graphpad Prism 5 to fit the dose-effect curve to obtain the IC ₅₀ of each compound on the enzyme activity. value. The calculation formula is Y=Bottom+(Top-Bottom)/(1+10^((LogIC50-X)*Hill slope)).

4Activity data are as follows:

From the above comparison, it can be seen that the inhibitory activity of Compound 1 on MAP4K3 and HPK1 is much better than that of Compound 2, which indicates that the compound of the present invention has good inhibitory activity on MAP4K3 and HPK1.

Claims (8)

1. A compound represented by formula I or a pharmaceutically acceptable salt thereof, Where n is 3, 4 or 5. 2. The compound represented by formula I or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that the compound represented by formula I is the following compound: 3. A pharmaceutical composition, which contains substance X and pharmaceutically acceptable excipients, and said substance . 4. The application of a substance X or a pharmaceutical composition as claimed in claim 3 in the preparation of HPK1 inhibitors, the substance Pharmaceutically acceptable salt. 5. Application of a substance For example, the compound represented by Formula I or a pharmaceutically acceptable salt thereof, preferably, the HPK1-related disease is cancer, such as colorectal cancer or liver cancer. 6. The application of a substance X or a pharmaceutical composition as claimed in claim 3 in the preparation of a MAP4K3 inhibitor, the substance Pharmaceutically acceptable salt. 7. Application of a substance Such as the compound represented by formula I or a pharmaceutically acceptable salt thereof, preferably, the disease related to MAP4K3 is an autoimmune disease, such as psoriatic arthritis, rheumatoid arthritis or systemic lupus erythematosus . 8. The use of a substance The autoimmune disease is, for example, psoriatic arthritis, rheumatoid arthritis or systemic lupus erythematosus, and the substance Take that with a grain of salt.