Tetracycline derivatives and uses thereof

Abstract

The application discloses a kind of tetracycline derivatives and purposes thereof.The tetracycline derivatives in the application can inhibit platelet aggregation, and the compounds have wide application prospects in the preparation of drugs for preventing or treating cerebral apoplexy.

Description

Technical Field

[0001] This invention belongs to the pharmaceutical field and provides a class of tetracycline derivatives and their uses. These compounds can inhibit platelet aggregation and can be used to prepare drugs for the treatment or prevention of stroke. Background Technology

[0002] Inhibiting platelet aggregation to prevent and treat thrombosis is a crucial approach in the prevention and treatment of thrombotic diseases. During physiological hemostasis and arterial thrombosis, platelets adhere to the exposed subendothelial matrix of blood vessels, leading to platelet activation, which is the initiation stage of thrombosis. Subsequently, platelet aggregation and release reactions occur, ultimately resulting in thrombosis. Post-adhesion signal transduction and platelet activation depend on the collagen-platelet glycoprotein receptor VI (GPVI) axis. Inhibiting this pathway can prevent platelet activation at an earlier stage, thereby inhibiting thrombosis. In vitro and in vivo experiments have shown that inhibitors or antibodies targeting GPVI can inhibit thrombosis and its inflammatory response without interfering with normal hemostasis, demonstrating both safety and efficacy. This helps address the shortcomings of current clinical medications, namely, the increased risk of bleeding associated with increased antiplatelet aggregation activity. This has spurred the development of various agents that regulate or inhibit GPVI-mediated platelet activation and thrombosis. Currently, these drugs are mainly divided into four categories: competitive inhibitors of GPVI, such as GPVI-Fc; anti-GPVI antibodies, such as antibodies that induce GPVI endocytosis or enzymatic cleavage (consumable antibodies) including JAQ1, mF1201, mF1232, and cF1232; GPVI blockers, which are antibodies that inhibit the function of GPVI, ranging from monoclonal antibodies (mAbs) to their antigen-binding fragments (Fabs) such as 9O12.3, 204-11, OM2, OM4, m-Fab-F, and 1G5; and the last category mainly targets the GPVI signaling pathway, such as curcumin and losartan. Among them, the GPVI competitive inhibitor soluble GPVI-Fc dimer protein (Revacept) has completed phase 1 clinical trials, and the results show that it can safely and effectively inhibit collagen-induced platelet aggregation without prolonging bleeding time.

[0003] Sareccycline is a novel, narrow-spectrum oral tetracycline antibiotic. Its tablet form was approved in the United States in October 2018, primarily for the treatment of non-nodular moderate-to-severe acne vulgaris in patients aged 9 years and older. The exact mechanism of action is not fully understood. Sareccycline is a tetracycline ribosomal protein inhibitor that inhibits protein synthesis through interaction with 70S bacterial ribosomes. However, unlike other tetracyclines, sareccycline's unique C7 extension into the messenger RNA (mRNA) channel directly interacts with the A-codon, thereby interfering with mRNA movement through this channel and / or disrupting the A-codon / anticodon interaction. Its pharmacological effects include in vitro efficacy against *Propionibacterium acnes* and other Gram-positive bacteria, and it also exhibits in vitro anti-inflammatory activity. In clinical trials, two phase III, double-blind, randomized controlled trials evaluated the efficacy of sareccycline in treating moderate-to-severe acne vulgaris, demonstrating that the drug is safe, effective, and well-tolerated.

[0004] This invention provides a class of tetracycline derivatives that can inhibit collagen-mediated platelet aggregation. These compounds have broad application prospects in the preparation of drugs for the treatment or prevention of stroke. Summary of the Invention

[0005] Technical problem solved: This invention provides a class of tetracycline derivatives and their uses. These compounds can inhibit collagen-induced platelet aggregation and can be used to prepare drugs for the treatment or prevention of stroke.

[0006] Technical solution: A class of compounds as shown in Formula I, or pharmaceutically acceptable salts, hydrates, solvates, racemic mixtures, enantiomers, diastereomers, and tautomers thereof, characterized in that,

[0007]

[0008] in,

[0009] R1, R2, R3, R4, R5, R6, R7, R8, R9, R 10 R 11 R 12 R 13 R 14 R 15 R 16 and R 17 Each is independently selected from hydrogen or deuterium, and R1, R2, R3, R4, R5, R6, R7, R8, R9, R 10 R 11 R 12 R 13 R 14 R 15 R 16 Or R17 At least one of them is deuterium.

[0010] Preferably, R1, R2 and R3 are deuterium.

[0011] Preferably, R1, R2, R3, R4, R5 and R6 are deuterium.

[0012] Preferably, R1, R2, R3, R4, R5, R6, R 10 R 11 and R 12 It is deuterium.

[0013] Preferably, R1, R2, R3, R4, R5, R6, R 10 R 11 R 12 R 13 R 14 and R 15 It is deuterium.

[0014] Preferably, R4, R5 and R6 are deuterium.

[0015] Preferably, R4, R5, R6, R 10 R 11 and R 12 It is deuterium.

[0016] Preferably, R4, R5, R6, R 10 R 11 R 12 R 13 R 14 and R 15 It is deuterium.

[0017] Preferably, R 10 R 11 and R 12 It is deuterium.

[0018] Preferably, R 10 R 11 R 12 R 13 R 14 and R 15 It is deuterium.

[0019] Preferably, R9 is deuterium.

[0020] Preferably, R7 or R8 is deuterium.

[0021] More preferably, the compound is selected from:

[0022]

[0023] Compound S1, as shown above;

[0024]

[0025] Compound S2, as shown above;

[0026]

[0027] Compound S3 is shown above.

[0028] The present invention provides the use of a compound of Formula I or a pharmaceutically acceptable salt, hydrate, solvate, racemic mixture, enantiomer, diastereomer and tautomer thereof in the preparation of a medicament for the treatment and / or prevention of stroke.

[0029] The present invention provides a pharmaceutical composition comprising a compound of Formula I or a pharmaceutically acceptable salt, hydrate, solvate, racemic mixture, enantiomer, diastereomer, tautomer, and a pharmaceutically acceptable carrier thereof.

[0030] Beneficial effects:

[0031] The present invention describes a class of tetracycline derivatives, which are characterized by their ability to inhibit collagen-induced platelet aggregation and can be used to prepare drugs for the treatment or prevention of stroke. Detailed Implementation

[0032] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0033] Example 1: Synthesis of compound S1

[0034] Synthesis route:

[0035]

[0036] Synthesis process:

[0037] Intermediate B1: A1 (1 g, 2.41 mmol) was mixed with 8.25 kg of trifluoroacetic acid and stirred at ambient temperature under a nitrogen atmosphere until dissolved. The resulting solution was cooled to 0 °C, and then N-iodosuccinimide (0.625 g, 2.77 mmol) was added. The reaction was allowed to proceed at room temperature until HPLC confirmed completion. Next, the TFA was distilled, and the residue was cooled to 15–20 °C. 10 mL of isopropanol was slowly added, followed by approximately 90 mL of tetrahydrofuran, to precipitate the product. The precipitated product was cooled to -8 °C and maintained at this temperature for 2–8 hours. Finally, the precipitated product was filtered, washed with cold THF, and dried at no more than 30 °C to give B1 (500 mg, 38%).

[0038] Intermediate C1: B1 (500 mg, 0.93 mmol) was dissolved in NMP, and then Et3SiH (215 mg, 1.86 mmol), Pd(OAc)2 (11 mg, 0.04 mmol), and Xantphos (53 mg, 0.09 mmol) were added. The reaction was carried out under CO pressure at room temperature. After the reaction was detected by HPLC to be complete, the mixture was filtered and purified to obtain C1 (200 mg, 48%).

[0039] Compound S1: C1 (230 mg, 519.87 μmol) was dissolved in DMA (2 mL), then D1 (69.79 mg, 1.04 μmol) was added. The mixture was stirred at 25 °C for 10 min, then NaBH3CN (39.20 mg, 623.84 μmol) was added to the above solution. The mixture was stirred at 25 °C for 20 min. After the reaction was confirmed to be complete by HPLC, the reaction solution was quenched with water. The solution was purified by preparative high performance liquid chromatography (HPLC conditions: column: Phenomenex luna C18100*40mm*5um; mobile phase: [H2O(0.04% HCl)-ACN]; gradient: 1%-30% B over 8.0 min) to obtain the product. A yellow solid compound S1 (80.0 mg, 28.12%) was obtained.

[0040] ESI-MS: 494.2 [M+H] + ;

[0041] 1H NMR(400MHz,DMSO-d6)δ14.80(s,1H),11.81(br s,1H),10.55(br s,1H),9.59(br s,1H),9.06(s,1H),7.54(d,J=8.6Hz,1H),6.86(d,J=8.6Hz,1H),4.35(s,1H),4.09-3.66(m,2H),3.24(br dd,J=4.0,15.6Hz,1H),3.03(d,J=12.0Hz,1H),2.99-2.94(m,1H),2.93-2.84(m,6H),2.35(br t,J=14.8Hz,1H),2.28-2.19(m,1H),1.55-1.40(m,1H)

[0042] Example 2: Synthesis of compound S2

[0043] Synthesis route:

[0044]

[0045] Synthesis process:

[0046] Compound S2: C1 (240 mg, 542.47 μmol) and D1 (69.55 mg, 1.08 mmol) were dissolved in DMA (2 mL), and the mixture was stirred at 25 °C for 10 min. NaBH3CN (40.91 mg, 650.97 μmol) was added to the above solution. The solution was stirred at 25 °C for 20 min. After the reaction was confirmed to be complete by HPLC, the reaction solution was quenched with water. The solution was purified by preparative high performance liquid chromatography (HPLC conditions: column: Phenomenex luna C18 100*40 mm*5 μm; mobile phase: [H2O (0.04% HCl)-ACN]; gradient: 1%-30% B over 8.0 min) to obtain the product. A yellow solid compound S2 (112.1 mg, 38.81%) was obtained.

[0047] ESI-MS: 491.2 [M+H] + ;

[0048] 1H NMR(400MHz,DMSO-d6)δ14.81(s,1H),11.78(br s,1H),10.47(br s,1H),9.57(s,1H),9.06(s,1H),7.50(d,J=8.4Hz,1H),6.84(d,J=8.4Hz,1H),4.33(s,1H),3.95-3.77(m,2H),3.30(s ,3H),3.26-3.16(m,1H),3.02(d,J=13.5Hz,1H),2.99-2.94(m,1H),2.93-2.81(m,6H),2.34(t,J=14.8Hz,1H),2.23(br d,J=13.8Hz,1H),1.55-1.40(m,1H).

[0049] Example 3: Synthesis of compound S3

[0050] Synthesis route:

[0051]

[0052] Synthesis process:

[0053] Compound S3: C1 (230 mg, 519.87 μmol) and D3 (66.65 mg, 1.04 mmol) were dissolved in DMA (2 mL), and the solution was stirred at 25 °C for 10 min. Then, NaBH3CN (39.20 mg, 623.84 μmol) was added to the above solution. The mixture was stirred at 25 °C for 20 min. After the reaction was confirmed to be complete by HPLC, the reaction solution was quenched with water. The solution was purified by preparative high performance liquid chromatography (HPLC conditions; column: Phenomenex luna C18 100*40 mm*5 μm; mobile phase: [H2O (0.04% HCl)-ACN]; gradient: 1%-30% B over 8.0 min) to obtain the product. A yellow solid compound S3 (73.2 mg, 26.25%) was obtained.

[0054] ESI-MS: 491.2 [M+H] + ;

[0055] 1H NMR(400MHz,DMSO-d6)δ14.84(s,1H),11.77(br s,1H),10.30(br s,1H),9.58(s,1H),9.08(s,1H),7.48(d,J=8.6Hz,1H),6.84(d,J=8.6Hz,1H),4.31(s,1H),3.91-3.63(m,2H),3.25(br dd,J=4.4,15.6Hz,1H),3.02(d,J=14.2Hz,1H),2.97(d,J=2.0Hz,1H),2. 94-2.78(m,6H),2.41-2.29(m,1H),2.26-2.17(m,1H),1.57-1.44(m,1H).

[0056] Example 4: Study on the in vitro antiplatelet aggregation activity of sarrencycline, compound S1, compound S2, and compound S3 1. Materials and Methods

[0057] 1.1 Animals

[0058] Sprague-Dawley (SD) rats, Shanghai Slack Laboratory Animal Co., Ltd.

[0059] 1.2 Reagents and Consumables

[0060]

[0061] 1.3 Preparation and Grouping of Dosing Formulations

[0062] One day prior to the experiment, accurately weigh salpercycline, compound S1, compound S2, and compound S3 powders, dissolve them in DMF to prepare a 120 mM stock solution, and vortex and pipette to ensure complete dissolution until clear. Dilute with DMF to prepare gradient solutions of 40 mM, 12 mM, 4 mM, 1.2 mM, 0.4 mM, and 0.12 mM. Seven concentrations were set for each drug, grouped as follows: blank control group, 0.3 μM, 1 μM, 3 μM, 10 μM, 30 μM, and 100 μM, with each group tested at least three times.

[0063] 1.4 Preparation of platelet-rich plasma

[0064] Rats were intraperitoneally injected with xylazine hydrochloride (10 mg / kg) and 50 mg / kg injection solution was used, with the depth of anesthesia adjusted to a level that elicited a mild response from the animal even with severe stimulation. The abdominal aorta was dissected, and any residual fluid and mucous membrane were wiped dry. Blood was collected using a venous lancet and sodium citrate anticoagulant tubes (1:9, BD, 2.7 mL). The first tube of blood was discarded, and two middle tubes were collected. The tubes were gently shaken upside down for no more than 5 minutes to prevent platelet activation. The blood was centrifuged at 180g for 15 minutes at room temperature. The middle portion of the supernatant was carefully transferred to an EP tube using a pipette, avoiding aspiration of red blood cells. The resulting turbid supernatant is platelet-rich plasma (PRP), which was labeled and numbered. Physiological saline was used as platelet-poor plasma (PPP) for zeroing and diluting PRP. Blood samples should be stored at room temperature (15–25°C) after collection, as low temperatures can activate platelets and increase their aggregation. Testing should be completed within 3 hours of blood collection.

[0065] 1.5 Determination of platelet aggregation rate

[0066] Connect the semi-automatic platelet aggregator to the power supply and preheat for about 30 minutes. Once the temperature reaches approximately 37℃ and stabilizes, proceed with the experiment. Dilute physiological saline and PRP solution at a 1:3 ratio (total volume 200μL). Add a stir bar to the test cup and preheat in the thermostat port for 3-5 minutes. Set the instrument to TEST mode. Place the physiological saline test cup in the test channel and press the ENT key. The instrument will automatically detect the zero point, and the window will display a value, for example, P40. After this value stabilizes, press the corresponding channel key, remove the physiological saline test cup, and place the PRP test cup in it. The window will display another value, for example, R30. Press the corresponding channel key, and the window will display the inducing agent. Use a pipette to add 15μL of the inducing agent to the bottom of the cup. Press the corresponding channel key, and the instrument will enter the platelet aggregation testing state. The window will display the timer. Press the corresponding channel key again, and the window will display the current platelet aggregation rate. After completion, the window will display the maximum platelet aggregation rate within 5 minutes. Record the result and replace with the next test cup.

[0067] The final concentration of COL collagen was 7 μg / mL.

[0068] 1.6 Calculation of Platelet Aggregation Inhibition Rate

[0069] Based on the measured maximum aggregation rate (MAR) of the platelet aggregation samples, the platelet aggregation inhibition rate of sarrencycline at each concentration compared to the blank control group was calculated. Calculation formula:

[0070] Platelet aggregation inhibition rate (%) = (MAR) 空白对照组 -MAR 测试药物组 ) / MAR 空白对照组 ×100%.

[0071] 1.7 Data Statistics

[0072] Experimental data are expressed as mean ± standard deviation (Mean ± SD). One-way ANOVA with Dunnett's method was used to test differences between groups. P < 0.05 was considered statistically significant.

[0073] 2 Experimental Results

[0074] 2.1 Effect of sarrocycline on collagen COL-induced platelet aggregation

[0075] As shown in Table 1, compared with the blank control group, sarrecycline inhibited platelet aggregation in a concentration-dependent manner within the range of 1–100 μM. One-way ANOVA results showed that sarrecycline exhibited significant anti-platelet aggregation activity starting at 3 μM, with inhibition rates of 1.06%, 21.06%, 31.21%, 65.99%, and 84.12% at concentrations of 1, 3, 10, 30, and 100 μM, respectively. The half-maximal inhibitory concentration (IC50) of sarrecycline for inhibiting collagen-induced platelet aggregation was 17.1 μM, n = 4.

[0076] Table 1. Effects of sarrocycline on COL-induced platelet aggregation

[0077]

[0078] Data are expressed as Mean ± SD. Compared with the model group, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0079] 2.2 Effect of compound S1 on collagen COL-induced platelet aggregation

[0080] As shown in Table 2, compared with the blank control group, compound S1 inhibited platelet aggregation in a concentration-dependent manner within the concentration range of 0.1–100 μM. One-way ANOVA results showed that compound S1 at 0.3 μM had a significant anti-platelet aggregation effect, with inhibition rates of 0.55%, 22.74%, 37.96%, 62.71%, 65.04%, 73.70%, and 93.85% at concentrations of 0.1, 0.3, 1, 3, 10, 30, and 100 μM, respectively. The half-maximal inhibitory concentration (IC50) of compound S1 for inhibiting collagen-induced platelet aggregation was 1.033 μM, n = 3.

[0081] Table 2. Effects of compound S1 on COL-induced platelet aggregation

[0082]

[0083]

[0084] Data are expressed as Mean ± SD. Compared with the model group, **p < 0.01, ***p < 0.001.

[0085] 2.3 Effect of compound S2 on collagen COL-induced platelet aggregation

[0086] As shown in Table 3, compared with the blank control group, compound S2 inhibited platelet aggregation in a concentration-dependent manner within the range of 0.3–100 μM. One-way ANOVA results showed that compound S2 at 3 μM exhibited significant anti-platelet aggregation activity, with inhibition rates of 2.51%, 15.32%, 33.21%, 41.55%, 61.45%, and 90.59% at concentrations of 0.3, 1, 3, 10, 30, and 100 μM, respectively. The half-maximal inhibitory concentration (IC50) for compound S2 in inhibiting collagen-induced platelet aggregation was 16.69 μM (n = 3).

[0087] Table 3. Effects of compound S2 on COL-induced platelet aggregation

[0088]

[0089] Data are expressed as Mean ± SD. Compared with the model group, **p < 0.01, ***p < 0.001.

[0090] 2.4 Effect of compound S3 on collagen COL-induced platelet aggregation

[0091] As shown in Table 4, compared with the blank control group, compound S3 inhibited platelet aggregation in a concentration-dependent manner within the range of 1–300 μM. One-way ANOVA results showed that compound S3 exhibited significant anti-platelet aggregation activity starting at 10 μM, with inhibition rates of 4.49%, 17.98%, 43.16%, 50.035%, 59.09%, and 85.52% at concentrations of 1, 3, 10, 30, 100, and 300 μM, respectively. The half-maximal inhibitory concentration (IC50) for compound S3 in inhibiting collagen-induced platelet aggregation was 12.33 μM (n = 3).

[0092] Table 4. Effects of compound S3 on COL-induced platelet aggregation

[0093]

[0094]

[0095] Data are expressed as Mean ± SD. ***p < 0.001 compared to the model group.

[0096] Table 5 shows the in vitro antiplatelet aggregation IC50 values ​​of salpercycline, compound S1, compound S2, and compound S3 under COL induction.

[0097]

[0098] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention. No markings in the claims should be construed as limiting the scope of the claims.

Claims

1. A class of compounds of formula I or pharmaceutically acceptable salts, hydrates, solvates, racemic mixtures, enantiomers, diastereomers, and tautomers thereof, characterized in that, As shown in Equation I below: Among them, R1, R2, R3, R4, R5, R6, R7, R8, R9, R 10 R 11 R 12 R 13 R 14 R 15 R 16 and R 17 Each is independently selected from hydrogen or deuterium, and R1, R2, R3, R4, R5, R6, R7, R8, R9, R 10 R 11 R 12 R 13 R 14 R 15 R 16 Or R 17 At least one of them is deuterium.

2. The compound according to claim 1, or a pharmaceutically acceptable salt, hydrate, solvate, racemic mixture, enantiomer, diastereomer, and tautomer thereof, characterized in that, in, R1, R2, and R3 are deuterium.

3. The compound according to claim 1, or its pharmaceutically acceptable salt, hydrate, solvate, racemic mixture, enantiomer, diastereomer, and tautomer, characterized in that, in, R1, R2, R3, R4, R5, and R6 are deuterium.

4. The compound according to claim 1, or a pharmaceutically acceptable salt, hydrate, solvate, racemic mixture, enantiomer, diastereomer, and tautomer thereof, characterized in that, in, R1, R2, R3, R4, R5, R6, R 10 R 11 and R 12 It is deuterium.

5. The compound according to claim 1, or a pharmaceutically acceptable salt, hydrate, solvate, racemic mixture, enantiomer, diastereomer, and tautomer thereof, characterized in that, in, R1, R2, R3, R4, R5, R6, R 10 R 11 R 12 R 13 R 14 and R 15 It is deuterium.

6. The compound according to claim 1, or a pharmaceutically acceptable salt, hydrate, solvate, racemic mixture, enantiomer, diastereomer, and tautomer thereof, characterized in that, in, R4, R5, and R6 are deuterium.

7. The compound according to claim 1, or a pharmaceutically acceptable salt, hydrate, solvate, racemic mixture, enantiomer, diastereomer, and tautomer thereof, characterized in that, in, R4, R5, R6, R 10 R 11 and R 12 It is deuterium.

8. The compound according to claim 1, or a pharmaceutically acceptable salt, hydrate, solvate, racemic mixture, enantiomer, diastereomer, and tautomer thereof, characterized in that, in, R4, R5, R6, R 10 R 11 R 12 R 13 R 14 and R 15 It is deuterium.

9. The compound according to claim 1, or a pharmaceutically acceptable salt, hydrate, solvate, racemic mixture, enantiomer, diastereomer, and tautomer thereof, characterized in that, in, R 10 R 11 and R 12 It is deuterium.

10. The compound according to claim 1, or a pharmaceutically acceptable salt, hydrate, solvate, racemic mixture, enantiomer, diastereomer, and tautomer thereof, characterized in that, in, R 10 R 11 R 12 R 13 R 14 and R 15 It is deuterium.

11. The compound according to claim 1, or a pharmaceutically acceptable salt, hydrate, solvate, racemic mixture, enantiomer, diastereomer, and tautomer thereof, characterized in that, in, R9 is deuterium.

12. The compound according to claim 1, or a pharmaceutically acceptable salt, hydrate, solvate, racemic mixture, enantiomer, diastereomer, and tautomer thereof, characterized in that, in, R7 or R8 is deuterium.

13. The compound according to claim 1, or a pharmaceutically acceptable salt, hydrate, solvate, racemic mixture, enantiomer, diastereomer, and tautomer thereof, characterized in that, The compound is selected from: Compound S1, as shown above; Compound S2, as shown above; Compound S3, as shown above.

14. The compound according to any one of claims 1 to 13, or a pharmaceutically acceptable salt, hydrate, solvate, racemic mixture, enantiomer, diastereomer, and tautomer thereof, characterized in that, The use of the compound in the preparation of medicaments for the treatment and / or prevention of stroke.

15. A pharmaceutical composition, characterized in that, It comprises the compound of any one of claims 1 to 13 or a pharmaceutically acceptable salt, hydrate, solvate, racemic mixture, enantiomer, diastereomer, tautomer, and pharmaceutically acceptable carrier thereof.

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