Antitumor drug and use thereof

Abstract

The present invention discloses an anti-tumor drug and its use. The present invention provides a compound as shown in formula I or a pharmaceutically acceptable salt thereof. The compound provided by the present invention has excellent agonist activity and can enter cells independently of hENT1 as a transporter, which is of great significance for the treatment of patients with hENT1 deficiency.

Description

Technical Field

[0001] The invention relates to an anti-tumor drug and application thereof. Background Art

[0002] Gemcitabine, chemical name: 2',2'-difluoro-2'-deoxycytidine hydrochloride, is an anti-tumor drug launched by Eli Lilly and Company in the United States. It has been approved for the treatment of pancreatic cancer, breast cancer, non-small cell lung cancer and other tumors. Gemcitabine is one of the most effective first-line drugs for the treatment of non-small cell lung cancer and pancreatic cancer. It has the advantages of high remission rate, prolonged survival and low side effects.

[0003] The clinical effect of gemcitabine is limited by a variety of intrinsic and acquired resistance mechanisms, among which the reduced expression of nucleoside transporters, especially human equilibrative nucleoside transporter 1 (hENT1), which is required for uptake by cancer cells, is the main cause of gemcitabine resistance. This is because, similar to other nucleoside drugs, gemcitabine is a hydrophilic compound that cannot enter the cell by passive diffusion through the cell membrane, so it requires specific transporters to be transported into tumor cells. hENT1 is an important transporter discovered so far that transports gemcitabine into tumor cells, which leads to a significant reduction in the uptake of gemcitabine in cells lacking human equilibrative nucleoside transporter 1 (hENT1), which also leads to the easy development of gemcitabine resistance in patients with such transporter deficiency.

[0004] Therefore, there is still a need in this field to further develop anti-tumor drugs that do not rely on hENT1 as a transporter. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide an anti-tumor drug that does not rely on hENT1 as a transporter in view of the deficiencies of the prior art. The present invention provides an anti-tumor drug and its use. The compound drug provided by the present invention has excellent agonist activity and can enter cells independently of hENT1 as a transporter, which is of great significance for the treatment of patients with hENT1 deficiency.

[0006] The present invention solves the above technical problems through the following technical solutions.

[0007] The present invention provides a compound as shown in Formula I or a pharmaceutically acceptable salt thereof,

[0008]

[0009] in,

[0010] R1 is C8-C 24 alkyl;

[0011] R2 and R3 are each independently hydrogen or C1-C4 alkyl;

[0012] R4 is hydrogen;

[0013] The carbon atom with "#1" is a chiral carbon atom or an achiral carbon atom. When it is a chiral carbon atom, it is in S configuration and / or R configuration.

[0014] In certain preferred embodiments of the present invention, certain groups in the compound of formula I or a pharmaceutically acceptable salt thereof are defined as follows, and the unmentioned groups are the same as those described in any embodiment of the present invention (referred to as "in some embodiments of the present invention").

[0015] In some embodiments of the present invention, R1 is C 15 -C 19 alkyl.

[0016] In some embodiments of the present invention, each of the C1-C4 alkyl groups is independently methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl; preferably methyl.

[0017] In some embodiments of the present invention, R2 is hydrogen or methyl.

[0018] In some embodiments of the present invention, R3 is hydrogen or methyl.

[0019] In some embodiments of the invention, the structural unit Selected from , , , , and .

[0020] In some embodiments of the present invention, the compound as shown in Formula I is any of the following compounds:

[0021] , , , , and .

[0022] The present invention provides a pharmaceutical composition, comprising:

[0023] (1) the compound of formula I or a pharmaceutically acceptable salt thereof, and

[0024] (2) Pharmaceutically acceptable excipients.

[0025] The present invention also provides the use of the compound as shown in formula I or its pharmaceutically acceptable salt, and the pharmaceutical composition in the preparation of drugs, wherein the drugs are used to treat pancreatic cancer, breast cancer or non-small cell lung cancer; preferably, they are used to treat pancreatic cancer, breast cancer or non-small cell lung cancer with nucleoside transporter 1 (hENT1) deficiency.

[0026] The term "pharmaceutically acceptable" means relatively non-toxic, safe, and suitable for use by patients.

[0027] The term "pharmaceutically acceptable salt" refers to a salt obtained by reacting a compound with a pharmaceutically acceptable acid or base. When the compound contains a relatively acidic functional group, a base addition salt can be obtained by contacting the compound with a sufficient amount of a pharmaceutically acceptable base in a suitable inert solvent. When the compound contains a relatively basic functional group, an acid addition salt can be obtained by contacting the compound with a sufficient amount of a pharmaceutically acceptable acid in a suitable inert solvent. For details, please refer to Handbook of Pharmaceutical Salts: Properties, Selection, and Use (P.Heinrich Stahl, Camille G. Wermuth, 2011, 2nd Revised Edition).

[0028] The term "pharmaceutically acceptable excipients" refers to all substances contained in pharmaceutical preparations other than active pharmaceutical ingredients, which are generally divided into two categories: excipients and additives. For details, please refer to the Pharmacopoeia of the People's Republic of China (2020 Edition) and Handbook of Pharmaceutical Excipients (Paul J Sheskey, Bruno C Hancock, GaryP Moss, David J Goldfarb, 2020, 9th Edition).

[0029] The term "treating" refers to removing the cause or alleviating the symptoms.

[0030] The term "prevention" refers to reducing the risk of developing a disease.

[0031] The term "alkyl" refers to a linear or branched, saturated, monovalent hydrocarbon group having a specified number of carbon atoms (e.g., C1-C8, C4-C8, etc.). Alkyl includes, but is not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl, etc.

[0032] Without violating the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0033] The reagents and raw materials used in the present invention are commercially available.

[0034] The positive and progressive effects of the present invention are:

[0035] (1) The compounds provided by the present invention have excellent agonist activity;

[0036] (2) The compounds provided by the present invention can enter cells independently of hENT1 as a transporter, which is of great significance for the treatment of patients with hENT1 deficiency. DETAILED DESCRIPTION

[0037] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0038] Example compounds:

[0039] ;

[0040] Comparison compounds:

[0041]

[0042] Example 1: Synthesis of Compound 2

[0043]

[0044] Step 1

[0045] Under N2 atmosphere, compound 2-2 (7.1 g, 55.6 mmol, 1.5 eq) was added dropwise to a stirred solution of compound 2-1 (10.0 g, 37.0 mmol, 1.0 eq) and pyridine (6.4 g, 81.5 mmol, 2.2 eq) in cyclohexane (100 mL) at 0 °C for 10 minutes. The resulting mixture was stirred at 25 °C for 16 hours under N2 atmosphere. The resulting mixture was diluted with H2O (100 mL) and extracted with EtOAc (100 mL x 3). The combined organic layer was washed with brine and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography to obtain compound 2-3 (10.0 g, 75%) as a white solid.

[0046] 1H NMR (400 MHz, CDCl3) δ 5.73 (s, 2H), 4.22 (t, J = 6.7 Hz, 2H), 1.73–1.66 (m, 2H), 1.30–1.23 (m, 30H), 0.87 (t, J = 6.3 Hz, 3H).

[0047] Step 2

[0048] To a stirred solution of compound s1-1 (10.3 g, 39.0 mmol, 1.0 eq) in TMP (100 mL) was added POCl3 (35.6 g, 234.0 mmol, 6.0 eq) dropwise at 0 °C under N2 atmosphere for 30 minutes. The resulting mixture was stirred at 25 °C for 2 hours under N2 atmosphere. The reaction was quenched with ice water (20 mL) at 0 °C. The mixture was neutralized to pH = 7-8 with NaOH (2M). The resulting mixture was extracted with DCM (200 mL x 5). The aqueous layer was concentrated under reduced pressure. The residue was dissolved in MeOH (100 mL). The resulting mixture was filtered and the filter cake was washed with MeOH (30 mL x 3). The filtrate was concentrated under reduced pressure. The residue was dissolved in MeOH (40 mL). The product was precipitated by adding EtOH (400 mL). The precipitated solid was collected by filtration and washed with EtOH (30 mL x 3). The product was concentrated in vacuo to give compound s1 (8.8 g, 66%) as a white solid.

[0049] LCMS: [MS+H] + = 344.0

[0050] 1H-NMR (400 MHz, D2O) δ 7.93 (d, J = 8.0 Hz, 1H), 6.13 (d, J = 8.0Hz, 1H), 6.11–6.04 (m, 1H), 4.38–4.26 (m, 1H), 4.16–4.03 (m, 2H), 3.99–3.94(m, 1H).

[0051] Step 3

[0052] At 25°C, compound s1 (1.0 g, 2.92 mmol, 1.0 eq) and compound 2-3 (0.76 g, 7.30 mmol, 2.5 eq) were stirred and added to a DMF (30 mL) solution, and DIEA (2.64 g, 12.36 mmol, 6.0 eq) was added. The mixture was stirred at 57°C under N2 environment for 24 h, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with DCM:MeOH = 90:10~75:25, to obtain compound 2 as a white solid (106.4 mg, 5%).

[0053] LCMS: [MS+H] + = 670.2.

[0054] 1 H NMR (400 MHz, CD3OD) δ: 8.03 (d, J = 7.7 Hz, 1H), 6.28–6.19 (m,1H), 6.09 (d, J = 7.7 Hz, 1H), 5.54 (d, J = 11.8 Hz, 2H), 4.42–4.31 (m, 1H),4.30–4.24 (m, 1H), 4.17–4.13 (m, 1H), 4.13–4.08 (m, 2H), 4.05–4.00 (m, 1H),1.68–1.58 (m, 2H), 1.32–1.26 (m, 30H), 0.89 (t, J = 6.3 Hz 3H).

[0055] The following compounds were synthesized by similar methods:

[0056]

[0057] Comparative Example: Synthesis of Comparative Example 2 (Compound 10)

[0058]

[0059] Step 1

[0060] To a stirred solution of compound 10-2 (1.83 g, 22.9 mmol, 1.0 eq) in THF (50 mL) was added LDA (2 M, 28.6 mL, 57.2 mmol, 2.5 eq) at -20 °C under N2 atmosphere. The resulting mixture was stirred at 50 °C for 1 hour under N2 atmosphere. A solution of compound 10-1 (10.0 g, 26.3 mmol, 1.15 eq) in THF (40 mL) was added dropwise to the above mixture at -20 °C under N2 atmosphere. The resulting mixture was stirred at 50 °C for 2 hours under N2 atmosphere. The reaction was quenched with HCl (1M) at 0 °C. The resulting mixture was extracted with ethyl acetate (70 mL x3). The combined organic layers were washed with brine and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure and the residue was purified by silica gel column chromatography eluting with PE:EA (100:0~90:10) to obtain compound 10-3 (7.65 g, 85%) as a light yellow oil.

[0061] LCMS: [MH] + = 339.1.

[0062] Step 2

[0063] Compound 10-3 (7.65 g, 22.5 mmol, 1.0 eq), 10-4 (2.6 g, 2.25 mmol, 0.1eq) and K2CO3 (6.2 g, 45.0 mmol, 2.0 eq) were dissolved in DCM (70 mL) and H2O (70 mL) and stirred at 0°C for 5 minutes. Compound 10-4 (4.4 g, 27.0 mmol, 1.2 eq) was added dropwise to the above mixture at 0°C over 10 minutes. The resulting mixture was stirred at 25°C under N2 atmosphere for 16 hours and then extracted with DCM (100 mL x 3). The combined organic layer was washed with brine and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with PE:EA (100:0~98:2) to give compound 10-5 (4.75 g, 54%) as a white solid.

[0064] 1H NMR (400 MHz, CDCl3): δ 5.70 (s, 2H), 1.53–1.47 (m, 2H), 1.30–1.19 (m, 32H), 1.17 (s, 6H), 0.88 (t, J = 6.8 Hz, 3H).

[0065] Step 3

[0066] Compound s1 (0.5 g, 1.46 mmol, 1.0 eq) was dissolved in DMF (20 mL). After stirring, compound 10-5 (1.13 g, 2.92 mmol, 2.0 eq), DIEA (940 mg, 7.29 mmol, 5.0 eq) and NaI (109 mg, 0.73 mmol, 0.5 eq) were added. Under N2 protection, the mixture was stirred at 70 °C for 16 h. The mixture was concentrated under reduced pressure and the residue was purified by silica gel column chromatography with DCM: MeOH (65: 35) as eluent to give compound 10 (39 mg, 4%) as a white solid.

[0067] LCMS: [M+H] + = 696.4.

[0068] 1 H NMR (400 MHz, CD3OD) δ 7.85 (d, J = 7.6 Hz, 1H), 6.26–6.19 (m, 1H), 5.97 (d, J = 7.5 Hz, 1H), 5.56–5.48 (m, 2H), 4.36–4.21 (m, 2H), 4.17–4.08 (m,1H), 4.02–3.92 (m, 1H), 1.55–1.45 (m, 2H), 1.30–1.19 (m, 32H), 1.15 (s, 6H), 0.88 (t, J = 6.8 Hz, 3H).

[0069] The following compounds were synthesized by similar methods:

[0070]

[0071] Activity detection method:

[0072] A549 and BxPC-3 cell survival assay

[0073] A549 (ATCC, Cat. No. CCL-185) and BxPC-3 (ATCC) cell lines were maintained in RPMI 1640 (Invitrogen) medium containing 20% ​​fetal bovine serum (ExCell Bio). Cells were centrifuged at 800 rpm for 5 min and resuspended in medium. The appropriate cell concentration was adjusted using a ViCell cell count (A549 400 cells / 50 μL, BxPC-3 2000 cells / 50 μL). 50 μL of the cell suspension was added to each well of the culture plate and incubated overnight at 37°C, 5% CO2. A series of designed concentrations of the Example and Comparative Example (gemcitabine) compounds dissolved in DMSO were added to each well. The final DMSO concentration was 0.2%. The corresponding blank controls were also run simultaneously (no cells and compounds, no compound). After culturing for 3 days at 37°C, 5% CO2, 25 μL CellTiter Glo reagent (Promega) was added to each well and centrifuged at 1000 rpm for 1 min. After equilibration at room temperature for 10 min to stabilize the light signal, EnVision detection was used. Data analysis was performed using XL-fit software (ID Business Solutions Ltd., version XL fit 5.0) to fit the inhibition rate and logarithmic transformed concentration. Inhibition rate % = (Max-sample) / Max*100, Max refers to the signal containing only cells without inhibitors. The inhibition rate was then used to perform nonlinear fitting (four-parameter method) analysis on the concentration to calculate the half effective concentration (EC 50 The corresponding results are shown in Table 1.

[0074] It can be seen from Table 1 that the compounds of the present invention have good inhibitory effects on both A549 and BxPC-3 tumor cells.

[0075] Efficiency of triphosphate conversion of active substances in A549 cells

[0076] A549 (ATCC, catalog number CCL-185) cells were used to evaluate the intracellular active substance triphosphate conversion efficiency of the compounds of the examples and comparative examples. 6 / mL density was resuspended in F-12K (Procell, PM150912) medium containing 10% PBS (ExCell, FND500). 1mL of cell suspension and 1.5mL of medium were added to a 6-well plate and cultured overnight in a 37°C / 5% CO2 incubator.

[0077] For wells that did not require the addition of inhibitors, the following procedures were performed: after cell attachment, the medium was discarded and medium containing 5 µM compound or positive control (gemcitabine) was added and cultured at 37°C / 5% CO2 for 1, 2, 4, or 6 hours.

[0078] For wells that required the addition of inhibitors, the following procedure was used: the human equilibrative nucleoside transporter 1 (hENT1) inhibitor dipyridamole (0.4 µg / mL) was pre-incubated with cells, the culture medium was discarded after 30 min, and the corresponding compound was added.

[0079] At the corresponding time point, the medium was discarded and the cells were washed twice with 3 mL PBS. 100 µL 0.25% trypsin-0.53 mM EDTA (Sigma-Aldrich, T4049) solution was added for 5 min of dissociation. After dissociation, 500 µL 70% methanol (pre-cooled at 4°C) was added to digest the cells. The digested cells were transferred to a 1.5 mL centrifuge tube and centrifuged at 10000 rpm and 4°C for 10 min. 500 µL of the supernatant was transferred to a 96-well plate and dried with nitrogen. 200 µL of the initial mobile phase (10% ACN in H2O) was added for reconstitution and transferred to LC-MSMS (Waters liquid chromatograph coupled to AB Sciex mass spectrometer) to detect the intracellular gemcitabine triphosphate concentration of the compound at each time point. The corresponding results are shown in Table 2.

[0080] As can be seen from Table 2, in A549 cells, in the presence of the hENT1 inhibitor, the compound of the present invention can still maintain a relatively high triphosphate conversion efficiency.

[0081] Efficiency of conversion of active substances into triphosphate in HepG2 cells

[0082] HepG2 (ATCC, catalog number HB-8065) ​​cells were used to evaluate the intracellular conversion efficiency of active substance triphosphate of the compounds of the examples and comparative examples. 6 / mL (HepG2) density was resuspended in MEME (Procell, PM150410) medium containing 10% PBS (ExCell, FND500). 1mL of cell suspension and 1.5mL of medium were added to a 6-well plate and cultured overnight in a 37°C / 5% CO2 incubator.

[0083] For wells that did not require the addition of inhibitors, the following procedures were performed: after cell attachment, the medium was discarded and medium containing 5 µM compound or positive control (gemcitabine) was added and cultured at 37°C / 5% CO2 for 1, 2, 4, or 6 hours.

[0084] For wells that required the addition of inhibitors, the following procedure was used: the human equilibrative nucleoside transporter 1 (hENT1) inhibitor dipyridamole (0.4 µg / mL) was pre-incubated with cells, the culture medium was discarded after 30 min, and the corresponding compound was added.

[0085] At the corresponding time point, the culture medium was discarded and the cells were washed twice with 3 mL PBS. 100 µL 0.25% trypsin-0.53 mM EDTA (Sigma-Aldrich, T4049) solution was added for 5 min of dissociation. After dissociation, 500 µL 70% methanol (pre-cooled at 4°C) was added to digest the cells. The digested cells were transferred to a 1.5 mL centrifuge tube and centrifuged at 10000 rpm and 4°C for 10 min. 500 µL of the supernatant was transferred to a 96-well plate and dried with nitrogen. 200 µL of the initial mobile phase (10% ACN in H2O) was added for reconstitution and transferred to LC-MSMS (Waters liquid chromatograph coupled to AB Sciex mass spectrometer) to detect the intracellular gemcitabine triphosphate concentration of the compound at each time point. The corresponding results are shown in Table 3.

[0086] As can be seen from Table 3, in HepG2 cells, in the presence of hENT1 inhibitor, the compound of the present invention can still maintain a relatively high triphosphate conversion efficiency.

[0087] Test results:

[0088] Table 1

[0089]

[0090] A:<100 nM, B:100~1000nM, C:>1000 nM;

[0091] Table 2

[0092]

[0093] C: <200nM, B: 200~500nM, A: >500nM;

[0094] Table 3

[0095]

[0096] C: <200nM, B: 200~500nM, A: >500nM.

Claims

1. A compound as shown in formula I or a pharmaceutically acceptable salt thereof, ; in, R1 is C 15 -C 19 alkyl; R2 and R3 are each independently hydrogen; R4 is hydrogen; the carbon atom with "#1" is an achiral carbon atom.

2. The compound of formula I or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The compound shown in formula I is any of the following compounds: and .

3. A pharmaceutical composition, characterized in that: The pharmaceutical composition comprises: (1) a compound of formula I according to any one of claims 1 to 2 or a pharmaceutically acceptable salt thereof, and (2) Pharmaceutically acceptable excipients.

4. Use of a compound of formula I as described in any one of claims 1 to 2 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition as described in claim 3 in the preparation of a drug, characterized in that: The medicine is used for treating pancreatic cancer, breast cancer or non-small cell lung cancer.

5. The use according to claim 4, characterized in that The drug is used for treating pancreatic cancer, breast cancer or non-small cell lung cancer with nucleoside transporter 1 deficiency.

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