Triazine compound for reversing low molecular heparin anticoagulant activity, preparation method and application thereof

Abstract

The application belongs to the field of medicine, and discloses a compound or a pharmaceutically acceptable salt, a stereoisomer, a solvate thereof, the structure of which is shown in formula I. In vitro binding efficacy experiments and toxicity experiments of low molecular heparin and the triazine compound of the application show that the triazine compound can produce good combination with low molecular heparin, and can reverse the anticoagulation of heparin, is a safe and low-toxic small molecule compound, and can be used for preparing a medicine for reversing the anticoagulation activity of heparin, treating and / or preventing adverse reactions and diseases caused by excessive heparin anticoagulation. The application discloses the use of the triazine compound or the pharmaceutically acceptable salt, the stereoisomer, the solvate thereof in preparing an anticoagulant reversing agent. The application discloses the use of the triazine compound or the pharmaceutically acceptable salt, the stereoisomer, the solvate thereof in preparing a medicine for treating side effects of excessive anticoagulation caused by using heparin to treat diseases, and preparing a medicine for treating surgical operations in which the anticoagulation activity of heparin needs to be reversed.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of medicine, and relates to a triazine compound capable of reversing the anticoagulant activity of low molecular weight heparin, a preparation method and application thereof, in particular to a spermine-substituted triazine compound capable of reversing the anticoagulant activity of low molecular weight heparin, a preparation method and application thereof, and a pharmaceutical composition containing the spermine-substituted triazine compound. BACKGROUND

[0002] Heparin is a linear glycosaminoglycan and is the largest natural polyanion known in biological systems in terms of charge density. Clinically, heparin is an important anticoagulant that mainly inhibits blood coagulation by activating the antithrombin-dependent pathway to produce an anticoagulant effect. In the medical field, heparin is widely used in the prevention of thrombotic complications, cardiovascular surgery, hemodialysis, etc. Systemic heparinization is a common anticoagulation procedure in cardiac surgery and heart surgery and hemodialysis, but systemic heparinization has some adverse reactions, such as bleeding and allergy, etc.

[0003] Low molecular weight heparin (LMWH) is a derivative of heparin with a molecular weight of 3500-8000 Da. Compared with ordinary heparin, low molecular weight heparin has less negative charge and fewer side effects. Low molecular weight heparin has more advantages in preventing thrombotic complications and may have a lower risk of bleeding. Low molecular weight heparin has been widely used in anticoagulant therapy for pulmonary embolism, deep vein thrombosis and cardiac surgery, etc. Low molecular weight heparin can reduce the incidence of venous thromboembolism in hospitalized patients without increasing the risk of serious bleeding. At the end of the operation, in order to reduce the patient's possible hypotension, bradycardia and bleeding risk, it is necessary to monitor the heparin titer and use heparin reversal agent in time.

[0004] Protamine sulfate (PS) is the only approved heparin reversal agent in China. Protamine sulfate can quickly reverse the effect of heparin, but the use of protamine sulfate may cause adverse reactions, including allergic reactions, respiratory problems and serious cardiovascular reactions such as hypotension and bradycardia, which can cause coronary thrombosis and myocardial infarction in severe cases. It has been reported that protamine sulfate can only partially reverse the anticoagulant effect of low molecular weight heparin, and the FDA has not approved protamine sulfate for reversing low molecular weight heparin. In addition, the metabolite of protamine, propylamine, is a highly toxic substance. Environmental pollution, seasonal shortage and other factors further limit the availability and application of protamine. Therefore, it is necessary to develop a safe heparin reversal agent with clear efficacy.

[0005] In order to reverse the anticoagulation effect of heparin, Andexanet Alfa and small molecule drugs Delparantag and Ciraparantag have been developed as heparin reversal agents. Andexanet Alfa may cause procoagulant effects and thromboembolic events, and the treatment cost is high, which is difficult to promote in clinical practice. Delparantag is a class of small molecule salicylic acid derivatives, which has good neutralizing effect as a heparin reversal agent in vivo and in vitro, but the project was terminated due to low blood pressure cases in clinical phase II. Ciraparantag is a small molecule cation that binds to heparin through hydrogen bonding and charge interaction, and is currently in clinical phase II.

[0006] At present, it is still necessary to develop compounds with heparin anticoagulation reversal activity or better pharmacodynamic performance. SUMMARY

[0007] The purpose of the present application is to provide a novel class of triazine compounds or pharmaceutically acceptable salts, stereoisomers and solvates thereof with heparin anticoagulation reversal activity and better pharmacodynamic performance.

[0008] The purpose of the present application is achieved by the following technical solutions:

[0009] The triazine compound or pharmaceutically acceptable salt, stereoisomer and solvate thereof having the structure as shown in formula I:

[0010]

[0011] Among them:

[0012] R1, R2, R3, R4, which are the same or different from each other, can each be independently selected from hydrogen, optionally substituted alkyl connected to N atom by or without any number of alkyl chains, alkylamino, alkoxy, monocyclic or polycyclic aromatic ring group, monocyclic or polycyclic aliphatic ring group, polycyclic aromatic ring and aliphatic ring group; or can be directly connected to the nitrogen atom by R1 and R2, R3 and R4 to form an optionally substituted aromatic ring or aliphatic ring or aromatic ring and aliphatic ring;

[0013] R5, R6, R7, R8, R9, which are the same or different from each other, can each be independently selected from hydrogen, optionally substituted alkyl connected to N atom by or without any number of alkyl chains, alkylamino, alkoxy, monocyclic or polycyclic aromatic ring group, monocyclic or polycyclic aliphatic ring group, polycyclic aromatic ring and aliphatic ring group;

[0014] In the optional substitution, the substitution means being substituted by one or more substituents selected from halogen, C 1-6 alkyl, hydroxyl, cycloalkyl, heterocycloalkyl substituted or not substituted by heteroatoms, alkoxy, carbonyl, aryl, etc.

[0015] Furthermore, R1, R2, R3, and R4 may be the same as or different from each other, and can be independently selected from hydrogen, passing through or not passing through C. 1-3 The alkyl chain is optionally substituted with an alkylamino group, alkoxy group, monocyclic or polycyclic aromatic ring group, monoalicylic ring group, aromatic ring fused alicylic ring group, etc., connected to an N atom via R1 and R2, R3 and R4. It can also be optionally substituted with a piperazine group, morpholino group, piperidinyl group, imidazopiperidine group, etc. by direct connection with the nitrogen atom.

[0016] R5, R6, R7, R8, and R9 may be the same as or different from each other, and can be independently selected from hydrogen and C. 1-6 Alkyl, C 1-6 Alkylamino, C 1-6 Alkoxy;

[0017] In the optional substitutions described above, substitution refers to being replaced by one or more of the following substituents, selected from: halogens, C... 1-6 Alkyl, hydroxyl, cycloalkyl, heterocyclic alkyl with or without heteroatom substitution, alkoxy, carbonyl, aryl, etc.

[0018] Furthermore, the formed R1-N-R2 and R3-N-R4 groups may be identical or different from each other, and are independently selected from the following groups:

[0019]

[0020] m is an integer from 1 to 3, and n is an integer from 1 to 3;

[0021] R5, R6, R7, R8, and R9 are each independently selected from hydrogen.

[0022] As the most preferred technical solution of the present invention, a triazine compound with the structure shown in Formula I, or a pharmaceutically acceptable salt, stereoisomer, or solvate thereof:

[0023]

[0024] in:

[0025] R1-N-R2 is selected from the following groups:

[0026] R3-N-R4 is selected from the following groups: m is an integer from 1 to 3, and n is an integer from 1 to 3;

[0027] R5, R6, R7, R8, and R9 are each independently selected from hydrogen.

[0028] More specifically, the triazine compound of the present application is selected from the compounds shown in the following structures:

[0029] Table 1. Small molecule triazine compounds of the present application

[0030]

[0031]

[0032]

[0033] The pharmaceutically acceptable salt is an acid addition salt of the compound of formula I with hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, citric acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, maleic acid, benzenesulfonic acid, succinic acid, fumaric acid, salicylic acid, phenylacetic acid or mandelic acid. In addition, acid salts of inorganic bases such as alkali metal cations, alkaline earth metal cations, ammonium cations are also included.

[0034] The solvate is a hydrate of the triazine compound of formula I.

[0035] Another object of the present application is to provide a synthesis method of the triazine compound, the synthesis route is as follows:

[0036]

[0037] wherein An is Bm is Cy is R1, R2, R3, R4, R5, R6, R7, R8, R9 are as described above.

[0038] comprising the following steps:

[0039] Step (1), using tetrahydrofuran as the reaction solvent, using N,N-diisopropyl ethylamine as the acid binding agent, cyanuric chloride and An are reacted at a temperature of -20°C to obtain intermediate III; wherein the molar ratio of cyanuric chloride and An is 1:1; the molar ratio of cyanuric chloride and N,N-diisopropyl ethylamine is 1:1-1:1.2;

[0040] Step (2), using dichloromethane as the reaction solvent, using N,N-diisopropyl ethylamine as the acid binding agent, intermediate III and Bm are fed at a temperature of -20°C, and then reacted at a temperature of 0°C to obtain intermediate II; the molar ratio of intermediate III and Bm is 1:1; the molar ratio of intermediate III and N,N-diisopropyl ethylamine is 1:1-1:3.2;

[0041] Step (3), intermediate II and Cy are reacted under reflux condition with dimethyl sulfoxide as reaction solvent and triethylamine as acid-binding agent to obtain a triazine compound with structure shown in formula I; the molar ratio of intermediate II and Cy is 1:1-1:3; the molar ratio of intermediate II and triethylamine is 1:1-1:5.

[0042] In step (1), after the reaction is completed, water is added to the reaction solution to quench the reaction, dichloromethane is used for extraction, the dichloromethane layers are combined, the dichloromethane layers are sequentially washed with saturated sodium chloride solution, dried with anhydrous sodium sulfate, concentrated under reduced pressure, and then separated and purified by silica gel column chromatography to obtain intermediate III.

[0043] In step (2), after the reaction is completed, silica gel column chromatography is used for separation and purification to obtain intermediate II.

[0044] In step (2), after the reaction is completed, hydrochloric acid-ethyl acetate is added to the reaction solution to adjust pH≤1, acetone is used for washing, suction filtration is performed, the filter cake is resuspended with methanol, sodium bicarbonate is added to adjust pH≥8, concentrated under reduced pressure, separated and purified by preparative thin layer chromatography, concentrated under reduced pressure, and then freeze-dried to obtain a triazine compound with structure shown in formula I.

[0045] Another object of the present application is to provide a pharmaceutical composition, which comprises the triazine compound or a pharmaceutically acceptable salt, stereoisomer or solvate thereof as an effective component or a main effective component, and a pharmaceutically acceptable carrier.

[0046] The in vitro binding efficacy experiment and toxicity experiment of low molecular heparin and the small molecule triazine compound of the present application show that the triazine compound of the present application can produce good combination with low molecular heparin, and reverse the anticoagulation of heparin, is a safe and low-toxic small molecule compound, and can be used for preparing a drug for reversing the anticoagulation activity of heparin, treating and / or preventing adverse reactions and diseases caused by excessive heparin anticoagulation, and has a good clinical application prospect.

[0047] Another object of the present application is to provide the use of the triazine compound or a pharmaceutically acceptable salt, stereoisomer or solvate thereof or the pharmaceutical composition in the preparation of an anticoagulant reversing agent.

[0048] Another object of the present application is to provide the use of the triazine compound or a pharmaceutically acceptable salt, stereoisomer or solvate thereof or the pharmaceutical composition in the preparation of a drug for treating adverse reactions caused by excessive anticoagulation during the treatment of diseases with heparin, and in the preparation of a drug for reversing the anticoagulation activity of heparin during a surgical operation.

[0049] The diseases treated with heparin are thromboembolic diseases; the embolic diseases are myocardial infarction, thrombotic phlebitis and pulmonary embolism; and the surgical operation is hemodialysis, extracorporeal circulation, catheterization and microvascular surgery. Attached Figure Description

[0050] Figure 1 The absorption spectra of azure A were obtained by adding different concentrations of compound 20 to the system in the presence of enoxaparin sodium. Detailed Implementation

[0051] The technical solution of the present invention will be further described in detail below through embodiments. The provided embodiments are intended to provide those skilled in the art with instructions on how to prepare the compounds for which protection is claimed in this invention, but the embodiments are merely examples and are not intended to limit the scope of protection of the present invention.

[0052] The compounds of the present invention can be prepared according to conventional methods in the art, using suitable reagents, raw materials, and purification methods known to those skilled in the art. The compounds of the present invention can also be conveniently prepared by optionally combining various synthetic methods described herein or known in the art, such combinations being readily performed by those skilled in the art.

[0053] The structure of the compounds of this invention was determined by mass spectrometry (MS) and / or proton nuclear magnetic resonance (NMR) spectroscopy. 1 Determined by H NMR.

[0054] Example 1

[0055] Preparation of N 2 -(3-((4-((3-aminopropyl)amino)butyl)amino)propyl)-N 4 -(2-(pyrrolid-1-yl)ethyl)-N 6 -((tetrahydro-2H-pyran-4-yl)methyl)-1,3,5-triazine-2,4,6-triamine (compound 1)

[0056]

[0057] Step 1: Synthesis of 4,6-dichloro-N-((tetrahydro-2H-pyran-4-yl)methyl)-1,3,5-triazine-2-amine (intermediate I-A2)

[0058] To a 100 mL flask was added 3,5-dichloro-4-hydroxy-1,3,5-triazine (1.00 g, 5.97 mmol) and 30 mL of tetrahydrofuran, at temperature -20 °C, N,N-diisopropyl ethylamine (0.82 g, 6.42 mmol) was added, then 4-aminomethyl tetrahydro pyran (compound A2, 0.75 g, 6.42 mmol) was dissolved in 10 mL of tetrahydrofuran, and added dropwise to the above solution at temperature -20 °C, after addition, the reaction was stirred for 2 h, TLC monitoring reaction was complete. To the reaction system was added 100 mL of water to quench the reaction, dichloromethane (100 mL x 2) was extracted, the dichloromethane layer was combined, washed with saturated sodium chloride solution (100 mL x 3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 10:1 V / V) to obtain 1.20 g of the product as a yellow solid, 4,6-dichloro-N-((tetrahydro-2H-pyran-4-yl)methyl)-1,3,5-triazin-2-amine.

[0059] LC-MS (ESI): m / z (M+H) + Calcd for C9H 13 Cl2N4O: 263.04, Found: 263.12.

[0060] Step two: 6-chloro-N 2 -(2-(pyrrolidin-1-yl)ethyl)-N 4 Synthesis of 6-chloro-N

[0061] To a 50 mL flask was added 4,6-dichloro-N-((tetrahydro-2H-pyran-4-yl)methyl)-1,3,5-triazin-2-amine (1.20 g, 4.56 mmol), 15 mL of dichloromethane and N,N-diisopropyl ethylamine (1.89 g, 14.59 mmol), stirred to dissolve, at temperature -20 °C, 1-(2-aminoethyl)pyrrolidine (compound B1, 521 mg, 4.56 mmol) was added, and the reaction was carried out at 0 °C for 2 h, TLC monitoring reaction was complete. Purified by silica gel column chromatography (dichloromethane:methanol = 20:1 V / V) to obtain 698 mg of white solid, intermediate I-A2B1.

[0062] 1H NMR (300 MHz, Chloroform-d) δ 3.79-3.58 (m, 5H), 3.50 (dd, J = 5.7, 2.9 Hz, 2H), 3.41 (dt, J = 15.9, 6.0 Hz, 1H), 2.74-2.60 (m, 6H), 1.88 (dt, J = 6.0 Hz, 1H), 1.86-1.73 (m, 6H), 1.56 (td, J = 5.8, 2.9 Hz, 2H).

[0063] Step three: N 2 -(3-((4-((3-aminopropyl)amino)butyl)amino)propyl)-N 4 -(2-(pyrrolidin-1-yl)ethyl)-N 6 Synthesis of (2-(4-methylpiperazin-1-yl)ethyl)-N

[0064] Into a 10 mL vial, 6-chloro-N 2 -(2-(pyrrolidin-1-yl)ethyl)-N 4 Into a 10 mL vial, 6-chloro-N

[0065] 1 H NMR (300 MHz, Chloroform-d) δ 3.79-3.58 (m, 5H), 3.50 (dd, J = 5.7, 2.9 Hz, 2H), 3.41 (dt, J = 15.9, 6.0 Hz, 1H), 2.74-2.60 (m, 6H), 1.88 (dt, J = 6.0 Hz, 1H), 1.86-1.73 (m, 6H), 1.56 (td, J = 5.8, 2.9 Hz, 2H).

[0066] Into a 10 mL vial, 6-chloro-N 2 -(3-((4-((3-aminopropyl)amino)butyl)amino)propyl)-N 4 -(2-(4-methylpiperazin-1-yl)ethyl)-N6 -((tetrahydro-2H-pyran-4-yl)methyl)-l,3,5-triazine-2,4,6-triamine (Compound 2)

[0067]

[0068] Following the procedure of Example 1, 1-(2-aminoethyl)pyrrolidine was replaced with an equivalent amount of 1-(2-aminoethyl)-4-methylpiperazine to give 263 mg of a pure white solid, Compound 2, in 82.5% yield.

[0069] m.p. >250 °C. 1 H NMR (300 MHz, D20) δ 3.84 (d, J = 11.6 Hz, 2H), 3.47 - 2.45 (m, 31H),

[0070] 2.01 - 1.72 (m, 5H), 1.69 - 1.51 (m, 6H), 1.25 - 1.10 (m, 2H). HRMS (ESI): m / z [M+H] + Calculated MS for C 26 H 54 N 11 O = 536.4513; Found MS = 536.4495.

[0071] Example 3

[0072] Preparation of N 2 -(3-((4-((3-aminopropyl)amino)butyl)amino)propyl)-N 4 -(pyridin-3-ylmethyl)-N 6 -((tetrahydro-2H-pyran-4-yl)methyl)-l,3,5-triazine-2,4,6-triamine (Compound 3)

[0073]

[0074] Following the procedure of Example 1, 1-(2-aminoethyl)pyrrolidine was replaced with an equivalent amount of 1-(2-aminoethyl)-4-methylpiperazine to give 263 mg of a pure white solid, Compound 2, in 82.5% yield.

[0075] m.p. >250 °C. 1 H NMR (300 MHz, D20) δ 3.84 (d, J = 11.6 Hz, 2H), 3.47 - 2.45 (m, 31H),

[0076] (t, J = 6.9 Hz, 1H), 4.63 (s, 2H), 3.80 (dd, J = 35.9, 11.2 Hz, 2H), 3.34 (s, 2H), 3.15 (s, 2H), 3.07-2.85 (m, 11H), 2.64 (d, J = 25.1 Hz, 1H), 2.07-1.82 (m, 4H), 1.67 (s, 5H), 1.33 (d, J = 13.0 Hz, 1H), 1.18 (d, J = 13.7 Hz, 1H), 0.93 (s, 1H). HRMS (ESI): m / z [M+H] + Calculated MS for C 25 H 45 N 10 O = 501.3778; Found MS = 501.3763.

[0077] Example 4

[0078] Preparation of N 2 -(3-((4-((3-aminopropyl)amino)butyl)amino)propyl)-N 4 -(pyridin-2-ylmethyl)-N 6 -((tetrahydro-2H-pyran-4-yl)methyl)-1,3,5-triazine-2,4,6-triamine (Compound 4)

[0079]

[0080] Referring to the method of Example 1, replace 1-(2-aminoethyl)pyrrolidine with an equivalent amount of 2-aminomethylpyridine to obtain 264 mg of a pure white solid, which is Compound 4, with a yield of 80.2%.

[0081] m.p. > 250 °C. 1 H NMR (300 MHz, D20) δ 8.60 (d, J = 5.9 Hz, 1H), 8.46 (td, J = 8.1, 1.7 Hz,

[0082] 1H), 7.97-7.80 (m, 2H), 4.88 (s, 2H), 3.90-3.79 (m, 1H), 3.72 (dd, J = 12.0, 4.1 Hz, 1H), 3.39 (s, 2H), 3.14 (t, J = 11.6 Hz, 2H), 2.99 (ddd, J = 19.2, 13.2, 7.2 Hz, 11H), 2.59 (s, 1H), 2.05-1.82 (m, 4H), 1.68 (s, 5H), 1.31-1.05 (m, 2H), 0.92-0.76 (m, 1H). HRMS (ESI): m / z [M+H] +Calculated MS for C 25 H 44 N 10 O=501.3778;Found MS=501.3778.

[0083] Example 5

[0084] Preparation of N 2 -(3-((4-((3-aminopropyl)amino)butyl)amino)propyl)-N 4 -((tetrahydro-2H-pyran-4-yl)methyl)-6-(3,4,6,7-tetrahydro-5H-imidazo[4,5- c]pyridin-5-yl)-1,3,5-triazine-2,4-diamine (Compound 5)

[0085]

[0086] Referring to the method of Example 1, replace 1-(2-aminoethyl)pyrrolidine with equimolar amount of 4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridine dihydrochloride to obtain 227 mg of pure white solid, which is Compound 5, with a yield of 70.2%.

[0087] m.p. >250 °C. 1 H NMR (300 MHz, D20) δ 7.64 (s, 1H), 4.59 (s, 2H), 3.95-3.78 (m, 4H),

[0088] 3.43-2.76 (m, 16H), 2.59 (d, J = 5.8 Hz, 2H), 2.04-1.42 (m, 11H), 1.27-1.00 (m, 2H). HRMS (ESI): m / z [M+H] + Calculated MS for C 25 H 46 N 11 O = 516.3887; Found MS = 516.3872. Example 6

[0089] Preparation of N 2 -(2-(1H-imidazol-4-yl)ethyl)-N 4 -(3-((4-((3-aminopropyl)amino)butyl)amino)propyl)-N 6 -((tetrahydro-2H-pyran-4-yl)methyl)-1,3,5-triazine-2,4,6-triamine (Compound 6)

[0090]

[0091] Referring to the method of Example 1, replace 1-(2-aminoethyl)pyrrolidine with equimolar amount of histamine dihydrochloride to obtain 259 mg of pure white solid, which is compound 6, yield 79.4%.

[0092] m.p. >250 °C. 1 H NMR (400 MHz, D20) δ 7.61 (s, 1H), 6.81 (s, 1H), 4.24-2.62 (m, 24H),

[0093] 2.00-1.48 (m, 9H), 1.21 (p, J = 9.6, 6.8 Hz, 2H). HRMS (ESI): m / z [M+H] + Calculated MS for C 24 H 46 N 11 O = 504.3887; Found MS = 504.3866.

[0094] Example 7

[0095] Preparation of N 2 -(2-(1H-benzo[d]imidazol-2-yl)ethyl)-N 4 -(3-((4-((3-aminopropyl)amino)butyl)amino)propyl)-N 6 -((tetrahydrofuran-3-yl)methyl)-1,3,5-triazine-2,4,6-triamine (Compound 7)

[0096]

[0097] Referring to the method of Example 1, replace 1-(2-aminoethyl)pyrrolidine with equimolar amount of histamine dihydrochloride to obtain 259 mg of pure white solid, which is compound 6, yield 79.4%.

[0098] m.p. >250 °C. 1 H NMR (300 MHz, D20) δ 7.34 (dd, J = 6.1, 3.2 Hz, 2H), 7.07 (dd, J = 6.1,

[0099] 3.2 Hz, 2H), 3.60 (s, 5H), 3.35-2.73 (m, 18H), 1.91-1.41 (m, 10H). HRMS (ESI): m / z [M+H] + Calculated MS for C 27 H 46 N 11O = 540.3887; Found MS = 540.3871.

[0100] Example 8

[0101] N 2 -(2-(1H-Indol-2-yl)ethyl)-N 4 -(3-((4-((3-Aminopropyl)amino)butyl)amino)propyl)-N 6 -((Tetrahydro-2H-pyran-4-yl)methyl)-1,3,5-triazine-2,4,6-triamine (Compound 8)

[0102]

[0103] Following the procedure of Example 1, substituting 2-(1H-benzo-2-imidazolyl)- ethylamine for 1-(2-aminoethyl)pyrrolidine in an equivalent amount, afforded 317 mg of a pure white solid, Compound 8, in 78.9% yield.

[0104] m.p. > 250 °C. 1 H NMR (400 MHz, D20) δ 7.52 (s, 2H), 7.32 (s, 2H), 4.02-3.66 (m, 4H),

[0105] 3.45-2.66 (m, 16H), 2.64 (s, 2H), 2.08-0.99 (m, 13H). HRMS (ESI): m / z [M+H] + Calculated MS for C 28 H 48 N 11 O = 554.4043; Found MS = 554.4029.

[0106] Example 9

[0107] Preparation of N 2 -(3-((4-((3-Aminopropyl)amino)butyl)amino)propyl)-6-morpholino-N 4 -(2-(Pyrrolidin-1-yl)ethyl)-1,3,5-triazine-2,4-diamine (Compound 9)

[0108]

[0109] Following the procedure of Example 1, substituting morpholine for 4-aminomethyltetrahydropyran in an equivalent amount, afforded 187 mg of a pure white solid, Compound 9, in 55.7% yield.

[0110] m.p.: 232-234 °C. 1H NMR (400 MHz, D20) δ 3.62 (s, 10H), 3.29 (d, J = 23.9 Hz, 8H), 2.94

[0111] (q, J = 8.6, 7.7 Hz, 10H), 1.99 - 1.75 (m, 8H), 1.62 (s, 4H). HRMS (ESI): m / z [M+H] + Calculated MS for C 23 H 41 N 10 O = 479.3924; Found MS = 479.3950.

[0112] Example 10

[0113] Preparation of N 2 -(3-((4-((3-aminopropyl)amino)butyl)amino)propyl)-N 4 -(2-(4-methylpiperazin-l-yl)ethyl)-6-morpholino-l,3,5-triazine-2,4-diamine (Compound 10)

[0114]

[0115] Following the procedure of Example 1, substituting 4-aminomethyltetrahydropyran for an equivalent amount of morpholine and 1-(2-aminoethyl)pyrrolidine for an equivalent amount of 1-(2-aminoethyl)-4-methylpiperazine, 227 mg of a pure white solid was obtained, i.e., Compound 10, in 69.7% yield.

[0116] m.p.: 239-240 °C. 1 H NMR (400 MHz, D20) δ 3.71 - 3.27 (m, 13H), 3.02 - 2.52 (m, 19H), 2.41 (s, 3H), 1.98 - 1.76 (m, 4H), 1.62 (s, 4H). HRMS (ESI): m / z [M+H] + Calculated MS for C 24 H 50 N 11 O = 508.4200.; Found MS = 508.4199.

[0117] Example 11

[0118] Preparation of N 2 -(3-((4-((3-aminopropyl)amino)butyl)amino)propyl)-6-morpholino-N 4 -(pyridin-3-ylmethyl)-l,3,5-triazine-2,4-diamine (Compound 11)

[0119]

[0120] Following the procedure of Example 1, replace 4-aminomethyltetrahydropyran with equimolar amount of morpholine and 1-(2-aminoethyl)pyrrolidine with equimolar amount of 3-aminomethylpyridine to obtain 260 mg of pure white solid, which is compound 11, in 76.8% yield.

[0121] m.p. >250 °C. 1 H NMR (400 MHz, D20) δ 8.45 - 8.21 (m, 2H), 7.69 (d, J = 7.9 Hz, 1H), 7.28

[0122] (t, J = 6.4 Hz, 1H), 4.42 (s, 2H), 3.56 (s, 8H), 3.32 (s, 2H), 3.09 - 2.61 (m, 10H), 2.11 - 1.28 (m, 8H). 13 C NMR (101 MHz, D20) δ 165.77, 165.45, 164.27, 147.55, 147.12, 136.33, 135.75, 124.12, 66.27, 47.10, 46.84, 44.78, 43.39, 41.53, 38.67, 36.86, 26.05, 24.58, 23.31, 22.91. HRMS (ESI): m / z [M+H] + Calculated MS for C 23 H 41 N 10 O = 473.3465; Found MS = 473.3470.

[0123] Example 12

[0124] Preparation of N 2 -(3-((4-((3-aminopropyl)amino)butyl)amino)propyl)-6-morpholino-N 4 -(pyridin-2-ylmethyl)-1,3,5-triazine-2,4-diamine (Compound 12)

[0125]

[0126] Following the procedure of Example 1, replace 4-aminomethyltetrahydropyran with equimolar amount of morpholine and 1-(2-aminoethyl)pyrrolidine with equimolar amount of 2-aminomethylpyridine to obtain 241 mg of pure white solid, which is compound 12, in 71.2% yield.

[0127] m.p. >250 °C.1 H NMR (400 MHz, D20) δ 8.61 (d, J = 5.9 Hz, 1H), 8.46 (t, J = 7.9 Hz, 1H),

[0128] 7.97 (d, J = 8.1 Hz, 1H), 7.87 (t, J = 6.9 Hz, 1H), 4.91 (s, 2H), 3.78 - 3.41 (m, 10H), 3.05 (dt, J = 27.2, 9.0 Hz, 9H), 2.62 (s, 1H), 2.14 - 1.66 (m, 8H). HRMS (ESI): m / z [M+H] + Calculated MS for C 23 H 41 N 10 O = 473.3465; Found MS = 473.3458.

[0129] Example 13

[0130] Preparation of N1-(3-aminopropyl)-N 4 -(3-((4-morpholinyl-6-(3,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridin-5-yl)-1,3,5-triazin-2- yl)amino)propyl)butane-1,4-diamine (Compound 13)

[0131]

[0132] Following the procedure of Example 1, replacing 4-aminomethyltetrahydropyran with an equivalent amount of morpholine and 1-(2-aminoethyl)pyrrolidine with an equivalent amount of 4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridine dihydrochloride, 270 mg of a pure white solid was obtained, i.e. Compound 13, in 81.8% yield.

[0133] m.p.: 242-246 °C. 1 H NMR (400 MHz, D20) δ 7.48 (s, 1H), 4.52 (s, 2H), 3.90 - 3.30 (m, 11H), 3.03 - 2.64 (m, 10H), 2.52 (s, 2H), 1.91 - 1.75 (m, 5H), 1.70 - 1.31 (m, 5H). HRMS (ESI): m / z [M+H] + Calculated MS for C 23 H 42 N 11 O = 488.3574; Found MS = 488.3557.

[0134] Example 14

[0135] Preparation of N 2 -(2-(1H-Imidazol-4-yl)ethyl)-N 4 -(3-(4-((3-aminopropyl)amino)butyl)amino)propyl)-6-morpholino-1,3,5-triazine-2,4-diamine (Compound 14)

[0136]

[0137] Following the procedure of Example 1, replacing 4-aminomethyltetrahydropyran with equimolar morpholine, and 1-(2-aminoethyl)pyrrolidine with equimolar histamine dihydrochloride, afforded 245 mg of pure white solid, Compound 14, in 72.9% yield.

[0138] m.p. >250 °C. 1 H NMR (500 MHz, Chloroform-d) δ 8.39 (s, 1H), 4.55 (s, 2H), 3.90-3.66 (m,

[0139] 11H), 3.06-2.61 (m, 13H), 1.82-1.60 (m, 4H), 1.53-1.34 (m, 4H). HRMS (ESI): m / z [M+H] + Calculated MS for C 23 H 42 N 11 O = 476.3574; Found MS = 476.3563.

[0140] Example 15

[0141] N 2 -(3-((4-((3-aminopropyl)amino)butyl)amino)propyl)-N 4 -(4-morpholinophenyl)-N 6 -(2-(pyrrolidin-1-yl)ethyl)-1,3,5-triazine-2,4,6-triamine (Compound 15)

[0142]

[0143] Following the procedure of Example 1, replacing 4-aminomethyltetrahydropyran with equimolar 4-(4-morpholinophenyl)aniline, afforded 245 mg of pure white solid, Compound 15, in 79.1% yield.

[0144] m.p. >250 °C. 1H NMR (300 MHz, D2O) δ 7.54 - 6.89 (m, 4H), 3.94 - 2.63 (m, 29H), 2.02-

[0145] 1.35 (m, 12H). HRMS (ESI): m / z [M+H] + Calculated MS for C 29 H 52 N 11 O = 570.4356; Found MS = 570.4345.

[0146] Example 16

[0147] Preparation of N 2 -(3-((4-((3-aminopropyl)amino)butyl)amino)propyl)-N 4 -(2-(4-methylpiperazin-l-yl)ethyl)-N 6 -(4-morpholinophenyl)-l,3,5-triazine-2,4,6-triamine (Compound 16)

[0148]

[0149] Referring to the method of Example 1, 4-aminomethyltetrahydropyran was replaced with equimolar amount of 4-(4-morpholinyl)aniline, 1-(2-aminoethyl)pyrrolidine was replaced with equimolar amount of l-(2-aminoethyl)-4-methylpiperazine to give 205 mg of pure white solid, Compound 16, in 68.2% yield.

[0150] m.p. >250 °C. 1 H NMR (300 MHz, D2O) δ 7.43 - 6.77 (m, 4H), 3.96 - 3.70 (m, 4H), 3.34 (d,

[0151] J = 18.9 Hz, 5H), 3.03 - 2.53 (m, 26H), 2.02 - 1.72 (m, 4H), 1.57 (s, 4H). HRMS (ESI): m / z [M+H] + Calculated MS for C 30 H 55 N 12 O = 599.4622; Found MS = 599.4610.

[0152] Example 17

[0153] Preparation of N 2 -(3-((4-((3-aminopropyl)amino)butyl)amino)propyl)-N 4-(4-morpholinophenyl)-N 6 -(pyridin-3-ylmethyl)-1,3,5-triazine-2,4,6-triamine (Compound 17)

[0154]

[0155] Referring to the method of Example 1, 4-aminomethyltetrahydropyran was replaced with an equivalent amount of 4-(4-morpholinophenyl)aniline, and 1-(2- aminoethyl)pyrrolidine was replaced with an equivalent amount of 3-aminomethylpyridine to obtain 224 mg of a pure white solid, which is Compound 17, in a yield of 73.2%.

[0156] m.p. >250 °C. 1 H NMR (300 MHz, D20) δ 8.29 (s, 2H), 7.71 (s, 1H), 7.41-6.68 (m, 5H), 4.35 (s, 2H), 3.73 (s, 4H), 3.28 (s, 2H), 3.04-2.59 (m, 14H), 2.06-1.50 (m, 8H). HRMS (ESI): m / z [M+H] + Calculated MS for C 29 H 46 N 11 O = 564.3887; Found MS = 564.3863.

[0157] Example 18

[0158] Preparation of N 2 -(3-((4-((3-aminopropyl)amino)butyl)amino)propyl)-N 4 -(4-morpholinophenyl)-N 6 -(pyridin-2-ylmethyl)-1,3,5-triazine-2,4,6-triamine (Compound 18)

[0159]

[0160] Referring to the method of Example 1, 4-aminomethyltetrahydropyran was replaced with an equivalent amount of 4-(4-morpholinophenyl)aniline, and 1-(2- aminoethyl)pyrrolidine was replaced with an equivalent amount of 3-aminomethylpyridine to obtain 224 mg of a pure white solid, which is Compound 17, in a yield of 73.2%.

[0161] m.p. >250 °C. 1H NMR (400 MHz, D20) δ 8.29 (s, 1H), 7.57 (s, 1H), 6.90 (d, J = 188.5 Hz, 6H), 4.39 (s, 2H), 3.70 (s, 4H), 3.34 - 2.56 (m, 16H), 1.95 - 1.30 (m, 8H). 13 C NMR (101 MHz, D20) δ 181.41, 175.00, 165.42, 163.60, 160.38, 158.01, 148.13, 138.05, 132.34, 122.66, 120.78, 117.52, 66.20, 50.18, 47.03, 46.65, 45.27, 44.64, 36.69, 36.29, 26.14, 24.25, 23.24, 23.09. HRMS (ESI): m / z [M+H] + Calculated MS for C 29 H 46 N 11 O2 = 564.3887; Found MS = 564.3867.

[0162] Example 19

[0163] Preparation of N 2 -(3-((4-((3-aminopropyl)amino)butyl)amino)propyl)-N 4 -(4-morpholinophenyl)-6-(3,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridin-5-yl)-1,3,5-triazine-2,4-diamine (Compound 19)

[0164]

[0165] Following the procedure of Example 1, 4-aminomethyltetrahydropyran was replaced with equimolar amount of 4-(4-morpholinyl)aniline, 1-(2-aminoethyl)pyrrolidine was replaced with equimolar amount of 4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridine dihydrochloride to give 262 mg of pure white solid, Compound 19, in 85.3% yield.

[0166] m.p. > 250 °C. 1 H NMR (400 MHz, D20) δ 8.46 (s, 1H), 7.28 (s, 2H), 6.92 (s, 2H), 4.00 - 2.60 (m, 26H), 2.05 - 1.43 (m, 8H). HRMS (ESI): m / z [M+H] + Calculated MS for C 29 H47 N 12 O = 579.3996; Found MS = 579.3986.

[0167] Example 20

[0168] Preparation of N 2 -(2-(1H-Imidazol-4-yl)ethyl)-N 4 -(3-((4-((3-Aminopropyl)amino)butyl)amino)propyl)-N 6 -(4-Morpholinophenyl)-1,3,5-triazine-2,4,6-triamine (Compound 20)

[0169]

[0170] Following the procedure of Example 1, substituting 4-aminomethyltetrahydropyran with equimolar amount of 4-(4-morpholinyl)aniline, 1-(2-aminoethyl)pyrrolidine with equimolar amount of histamine dihydrochloride, afforded 256 mg of pure white solid, Compound 20, yield 82.6%.

[0171] m.p. >250 °C. 1 H NMR (400 MHz, D20) δ 8.22 (d, J = 42.6 Hz, 1H), 7.53-6.87 (m, 5H), 4.01-2.54 (m, 25H), 2.03-1.32 (m, 8H). HRMS (ESI): m / z [M+H] + Calculated MS for C 28 H 47 N 12 O = 567.3996; Found MS = 567.3986.

[0172] Example 21

[0173] Preparation of N 2 -(3-((4-((3-Aminopropyl)amino)butyl)amino)propyl)-N 4 ,N 4 -Bis(2-methoxyethyl)-N 6 -(2-(Pyrrolidin-1-yl)ethyl)-1,3,5-triazine-2,4,6-triamine (Compound 21)

[0174]

[0175] Following the procedure of Example 1, substituting 4-aminomethyltetrahydropyran with equimolar amount of bis(2-methoxyethyl)amine, afforded 148 mg of pure white solid, Compound 21, yield 46.4%.

[0176] m.p. >250 °C. 1 H NMR (400 MHz, D20) δ 3.95 - 3.22 (m, 23H), 3.09 - 2.90 (m, 11H), 2.13 - 1.84 (m, 8H), 1.74 - 1.55 (m, 4H). HRMS (ESI): m / z [M+H] + Calculated MS for C 25 H 53 N 12 O2= 525.4353; Found MS = 525.4332.

[0177] Example 22

[0178] Preparation of N 2 -(3-((4-(3-aminopropyl)amino)butyl)amino)propyl)-N 4 ,N 4 -bis(2-methoxyethyl)-N 6 -(2-(4-methylpiperazin-l-yl)ethyl)-l,3,5-triazine-2,4,6-triamine (Compound 22)

[0179]

[0180] Referring to the method of Example 1, 4-aminomethyltetrahydropyran was replaced by equimolar amount of bis(2-methoxyethyl)amine, 1-(2-aminoethyl)pyrrolidine was replaced by equimolar amount of l-(2-aminoethyl)-4-methylpiperazine to obtain 240 mg of pure white solid, which was the target compound 22 with a yield of 76.9%.

[0181] m.p. >250 °C. 1 H NMR (400 MHz, D20) δ 3.79 - 3.24 (m, 23H), 3.11 - 2.86 (m, 12H), 2.74 (d, J = 12.6 Hz, 6H), 2.04 (q, J = 8.1 Hz, 2H), 1.90 (q, J = 10.8, 6.2 Hz, 2H), 1.76 - 1.59 (m, 4H). HRMS (ESI): m / z [M+H] + Calculated MS for C 26 H 56 N 11 O2= 554.4618; Found MS = 554.4604.

[0182] Example 23

[0183] Preparation of N 2-(3-((4-((3-aminopropyl)amino)butyl)amino)propyl)-N 4 ,N 4 - bis(2-methoxyethyl)-N 6 -(pyridin-3-ylmethyl)-1,3,5-triazine-2,4,6-triamine (Compound 23)

[0184]

[0185] Referring to the method of Example 1, 4-aminomethyltetrahydropyran was replaced by equimolar amount of bis(2-methoxyethyl)amine, 1-(2-aminoethyl)pyrrolidine was replaced by equimolar amount of 3-aminomethylpyridine to obtain 282 mg of pure white solid, i.e. Compound 23, in 87.3% yield.

[0186] m.p. >250 °C. 1 H NMR (400 MHz, D20) δ 8.37 - 8.20 (m, 2H), 7.63 (d, J = 7.9 Hz, 1H), 7.25 (s, 1H), 4.39 (s, 2H), 4.14 - 2.73 (m, 26H), 1.92 - 1.37 (m, 8H). 13 C NMR (101 MHz, D20) δ 165.75, 165.03, 163.86, 147.12, 146.94, 136.30, 135.84, 124.12, 69.72, 57.97, 47.16, 46.88, 46.18, 45.50, 44.85, 41.49, 36.95, 26.14, 24.89, 23.52, 23.01. HRMS (ESI): m / z [M+H] + Calculated MS for C 25 H 47 N 10 O2 = 519.3883; Found MS = 519.3899.

[0187] Example 24

[0188] Preparation of N 2 -(3-((4-((3-aminopropyl)amino)butyl)amino)propyl)-N 4 ,N 4 - bis(2-methoxyethyl)-N 6 -(pyridin-2-ylmethyl)-1,3,5-triazine-2,4,6-triamine (Compound 24)

[0189]

[0190] Following the procedure of Example 1, replace 4-aminomethyltetrahydropyran with equimolar amount of bis(2-methoxyethyl)amine, replace l-(2-aminoethyl)pyrrolidine with equimolar amount of 2-aminomethylpyridine to give 260 mg of pure white solid, which is compound 24, yield 80.6%.

[0191] m.p. >250 °C. 1 H NMR (400 MHz, D20) δ 8.40 (d, J = 5.3 Hz, 1H), 7.91 (s, 1H), 7.43 (dd, J = 29.0, 7.4 Hz, 2H), 4.61 (s, 2H), 3.70 - 3.34 (m, 8H), 3.22 (s, 4H), 3.03 (ddd, J = 18.9, 15.4, 8.4 Hz, 14H), 2.05 - 1.96 (m, 2H), 1.89 (t, J = 7.8 Hz, 2H), 1.75 - 1.60 (m, 4H). HRMS (ESI): m / z [M+H] + Calculated MS for C 25 H 47 N 10 O2 = 519.3883; Found MS = 519.3861.

[0192] Example 25

[0193] Preparation of N 2 -(3-((4-((3-aminopropyl)amino)butyl)amino)propyl)-N 4 ,N 4 -bis(2-methoxyethyl)-6-(3,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridin-5-yl)-1,3,5-triazine-2,4-diamine (compound 25)

[0194]

[0195] Following the procedure of Example 1, replace 4-aminomethyltetrahydropyran with equimolar amount of bis(2-methoxyethyl)amine, replace l-(2-aminoethyl)pyrrolidine with equimolar amount of 2-aminomethylpyridine to give 260 mg of pure white solid, which is compound 24, yield 80.6%.

[0196] m.p. >250 °C. 1H NMR (300 MHz, D20) δ 8.47 (d, J = 5.0 Hz, 1H), 3.63 (q, J = 6.4, 5.0 Hz, 6H), 3.29 (d, J = 4.9 Hz, 6H), 3.06 (dq, J = 22.5, 7.5 Hz, 15H), 2.62 (s, 5H), 1.19 (q, J = 7.9, 7.3 Hz, 8H). 13 C NMR (400 MHz, D20) δ 168.82, 166.94, 166.31, 134.50, 130.15, 122.69, 69.64, 58.51, 49.10, 49.05, 48.44, 47.57, 47.43, 44.81, 44.40, 40.55, 38.61, 33.59, 28.67, 27.24, 23.32. HRMS (ESI): m / z [M+H] + Calculated MS for C 25 H 48 N 11 O2= 534.3992; Found MS = 533.3982.

[0197] Example 26

[0198] In vitro binding force qualitative test of triazine compounds with enoxaparin sodium

[0199] The size and potential of the complex formed by small molecule triazine compounds and enoxaparin sodium in aqueous solution were measured by dynamic light scattering.

[0200] Experimental instruments: Zetasizer Nano ZS nanoparticle size potential instrument, from Malvern Instruments Co., Ltd. in the United Kingdom; particle size sample pool, from Malvern Instruments Co., Ltd. in the United Kingdom (model: DTS0012); potential sample pool, from Malvern Instruments Co., Ltd. in the United Kingdom (model: DTS1070).

[0201] Experimental materials: protamine sulfate, from TCI (product number P0675); Ciraparantag, from PeptiBio; low molecular weight heparin (LMWH) selected enoxaparin sodium injection (hereinafter referred to as enoxaparin solution), from Sanofi-Aventis (40 mg: 4000 AXa IU, product number CS543A); Tris, from GBCBIO (product number G3470); compounds 1-25.

[0202] The specific experimental steps are as follows: before each use of the particle size sample cell or the potential sample cell, clean the sample cell with distilled water three times, and rinse the sample cell with the sample to be tested. (1) Determination of the size of the complex: 100 mg / mL enoxaparin solution is diluted with distilled water to 0.1 mg / mL, and the test compound or control (protamine sulfate) is prepared into 0.1, 1, 10 mg / mL with distilled water. The diluted enoxaparin solution is mixed with the test compound or control in Tris-HCl buffer (pH 7.4), and after each preparation, the sample is allowed to equilibrate for 2 min to prepare the sample for size measurement. Each measurement is performed in triplicate, and all titration points are measured twice. (2) Zeta potential measurement: 5 mg / mL enoxaparin solution and 5 mg / mL test substance (compound 1-compound 25, LMWH, protamine sulfate) solution are mixed in Tris-HCl buffer (pH 7.4), and the final volume of the sample is 0.8 mL. In the sample, the concentration of enoxaparin solution is 0.1 mg / mL, and the concentration of the test compound or control is 0.05, 0.1, 0.15, 0.2, or 0.3 mg / mL. After each preparation, the sample is allowed to equilibrate for 2 min, and each measurement is performed three times.

[0203] All measurements are performed at a polymer concentration high enough to bind at least 90% of the enoxaparin sodium, and the results are expressed in terms of average particle size / nm, polydispersity index, and Zeta potential. The test results are shown in Table 2 (the results are expressed as mean ± standard deviation).

[0204] The Zeta potential of the complex of the small molecule compound and enoxaparin demonstrates that the binding of the compound to enoxaparin is based on charge interaction. The particle size of the complex formed by the binding of enoxaparin sodium and the small molecule triazine compound is about 100 nm and is tightly bound, and the average size of the complex formed by protamine sulfate and enoxaparin is about 1000 nm. The small size of the heparin-reversal agent complex is beneficial for reducing postoperative complications in clinical patients with intravenous injection of heparin. The polydispersity coefficient of the complex formed by the binding of the small molecule heparin reversal agent and enoxaparin is basically less than 0.3, indicating that the distribution of the complex formed in the system is relatively uniform.

[0205] Table 2. Size distribution and Zeta potential of the complex of LMWH and reversal agent and LMWH

[0206] Number Average particle size / nm Polydispersity index Zeta potential / mV LMWH — — -8.20±0.75 Protamine sulfate 1421.92±257.16 0.455±0.020 13.54±0.26 1 132.63±14.84 0.208±0.015 -0.66±0.46 3 163.20±42.52 0.186±0.010 -1.76±1.32 6 128.00±26.03 0.332±0.025 -4.13±1.05 16 146.35±8.65 0.258±0.012 0.00±0.01 17 89.39±20.29 0.165±0.023 -0.37±0.18 18 105.56±17.48 0.119±0.031 -0.11±0.03 19 88.03±3.64 0.189±0.052 -0.21±0.05 20 106.11±16.85 0.132±0.087 -0.14±0.03 21 116.10±26.68 0.133±0.016 -0.18±0.58 22 82.94±16.92 0.137±0.008 -1.15±0.43 23 100.06±26.53 0.290±0.015 -0.62±0.47 24 87.38±13.48 0.052±0.001 -0.21±0.14

[0207] Example 27

[0208] Quantitative test of competitive binding of small molecule triazine compounds and azure A to enoxaparin sodium

[0209] The cationic dye azure A colorimetric method was used to quantitatively evaluate the relative ability of the small molecule heparin reversing agent to bind enoxaparin. Heparin forms a complex with azure A dye, and the small molecule heparin reversing agent reverses the enoxaparin-azure A complex to different degrees, and the system is accompanied by an increase in the intensity of the 600 nm / 630 nm absorption band and a decrease in the intensity of the 510 nm absorption band.

[0210] Experimental instruments: SpectraMax iD3 microplate reader.

[0211] Experimental materials: Protamine sulfate from Sigma (product number P0675); Ciraparantag from PeptiBio; low molecular weight heparin selected enoxaparin sodium injection from Sanofi-Aventis (40 mg: 4000 AXa IU, product number CS543A); azure A from Shanghai McLean Biochemical Technology Co., Ltd. (product number C14252971); compounds 1-25.

[0212] The specific experimental steps are as follows: according to the mass ratio of enoxaparin sodium to azure A is 1:2, enoxaparin solution and azure A are resuspended with distilled water, and the enoxaparin-azure A mixed solution is prepared by shaking and mixing in a centrifuge tube. At a temperature of 20°C, the ratio of λ(600nm) / λ(510nm) absorbance is used to evaluate the competitive binding ability of triazine compounds to enoxaparin sodium, and each compound is determined in triplicate.

[0213] The results are shown in Table 3. In the quantitative test of competitive binding with azure A, the triazine compounds of the present application can reverse the competitive binding ability of azure A to enoxaparin sodium to different degrees, and the reversal effect of compounds 19, 20 and 21 is better than that of other compounds, with reversal rates of 79.56%, 74.38% and 81.82%, respectively. The reversal rate of Ciraparantag to azure A is 79.15%. Protamine sulfate has a higher positive charge and a relatively rigid protein structure, so it has a stronger competitive effect on enoxaparin under the same conditions.

[0214] The absorption spectrum between 400 nm and 750 nm of a 96 transparent hole plate was measured to determine the amount of compound required for complete displacement of azure A. As shown in Figure 1 , compound 20 was used as an example, m 化合物20 : m LMWH ≥1.5, enoxaparin sodium in the system is complexed with the compound, indicating that the compound binds to enoxaparin sodium in a dose-dependent manner.

[0215] Table 3. Quantitative test of competitive binding of small molecule triazine compounds and azure A to enoxaparin

[0216] Compound number Reversal rate of Azure A Compound number Reversal rate of Azure A Compound number Reversal rate of Azure A 1 67.08％ 10 67.69％ 19 79.56％ 2 67.52％ 11 62.67％ 20 74.38％ 3 61.92％ 12 47.15％ 21 81.82％ 4 62.15％ 13 61.12％ 22 52.34％ 5 72.50％ 14 55.98％ 23 53.65％ 6 69.82％ 15 68.42％ 24 62.66％ 7 62.01％ 16 57.88％ 25 47.31％ 8 67.53％ 17 68.26％ Ciraparantag 79.15％ 9 64.64％ 18 64.25％ Protamine sulfate 99.89％

[0217] Example 28

[0218] Small molecule heparin reversal agent coagulation factor activity test

[0219] Enoxaparin sodium exerts its anticoagulant effect primarily through the anti-thrombin dependent mechanism to form an anti-thrombin-heparin-coagulation factor Xa ternary complex. Anti-Xa assay is an enzyme activity test to test the ability of a compound to reverse heparin in clinical and scientific research.

[0220] Experimental instruments: SpectraMax iD3 microplate reader.

[0221] Experimental reagents: Anti-Xa kit, commercial two-stage kit Biophen Heparin Anti-Xa (product number 221005) from HYPHEN BioMed, France; anhydrous citric acid from Greagent (product number P1508679).

[0222] The experimental method is as follows: first, reconstitute the Biophen Heparin Anti-Xa kit from HYPHEN BioMed, in ultrapure water, incubate the triazine compound and enoxaparin sodium at a mass ratio of 2:1 at 37°C for 10 min, dilute to the appropriate concentration to prepare the sample, take 40 μL of the reconstituted sample and add it to the 96-well plate, add 40 μL of antithrombin to each well, shake well and incubate at 37°C for 2 min. Add 40 μL of coagulation factor Xa to each well, shake well and incubate at 37°C for 2 min. Add 40 μL of coagulation factor Xa specific chromogenic substrate factor to each well, shake well and incubate at 37°C for exactly 2 min. Finally, add 80 μL of 2% citric acid solution to each well, shake well, and immediately use the microplate reader to read the absorbance at 405 nm. Three replicate wells are set up for each test compound. The anti-coagulation effect of the small molecule triazine compound in reversing heparin is reflected by the anti-Xa factor neutralization rate.

[0223] Anti-Xa factor neutralization rate (%) = {(OD 待测化合物 - OD 肝素对照 ) / (OD 无肝素对照 - OD 肝素对照 )} x 100%

[0224] As shown in Table 4, under the same conditions, protamine sulfate cannot completely reverse the anticoagulant effect of enoxaparin, and the anti-Xa factor activity of compounds 1-25 is better than that of protamine sulfate. Ciraparantag can be combined with chelating agents, therefore, the anti-Xa factor test is not suitable for clinical monitoring of Ciraparantag. The anti-Xa factor activity test shows the excellent ability of the spermine-substituted triazine compounds of the present application to reverse the anticoagulant effect of heparin.

[0225] Table 4. Anti-Xa factor activity of heparin reversal agents

[0226] Compound number Neutralization rate of anti-Xa factor Compound number Neutralization rate of anti-Xa factor Compound number Neutralization rate of anti-Xa factor 1 71.08％ 10 57.66％ 19 80.15％ 2 64.57％ 11 65.95％ 20 84.29％ 3 70.76％ 12 46.62％ 21 80.69％ 4 65.42％ 13 58.66％ 22 72.20％ 5 61.53％ 14 54.86％ 23 84.04％ 6 73.28％ 15 66.07％ 24 86.17％ 7 66.36％ 16 72.00％ 25 67.79％ 8 64.65％ 17 78.70％ Protamine sulfate 45.49％ 9 61.37％ 18 82.21％ Ciraparantag 9.62％

[0227] Example 29

[0228] Red blood cell hemolysis toxicity experiment of small molecule heparin reversal agent

[0229] After the drug is injected intravenously, it directly contacts the blood tissue. The cell membrane of red blood cells is negatively charged, and cationic compounds have the possibility of directly inducing red blood cell rupture, i.e. hemolysis. Hemolysis experiment is a pre-experiment of cytotoxicity of medical materials, and is a necessary characterization of blood contact with medical materials.

[0230] Experimental instruments: the centrifuge is Titan DMC-12K; the enzyme label instrument is SpectraMax iD3.

[0231] Experimental reagents: PBS buffer (Bi Yun Tian Biotechnology Co., Ltd., product number C0221A).

[0232] Fresh blood comes from human volunteers. After successful venous blood sampling, the blood is immediately placed in a blue sodium citrate coagulation test tube and stored at 4°C.

[0233] The test steps are as follows: according to the required working concentration, the compound is dissolved and prepared into a compound solution with a concentration of 2.5 mg / mL using PBS buffer. The compound sample solution and fresh blood are added to the centrifuge tube at a volume ratio of 9:1, inverted and mixed, and three parallel experiments are set for each test compound. The mixed sample is incubated in a 37°C constant temperature incubator for 1 hour; then centrifuged at a speed of 3500 rpm for 15 minutes, the supernatant is aspirated, and the absorbance is measured at 575 nm using an enzyme label instrument.

[0234] Hemolysis rate = [A575nm-Mean(NC)] / [Mean(PC)-Mean(NC)]x100%

[0235] Wherein, A 575 nm: the absorbance value of the compound sample at 575 nm; Mean(NC): the mean value of the negative control hole; Mean(PC): the mean value of the positive control hole. The absorbance of the sample with 100% hemolysis in ultrapure water is the positive control, and the absorbance in PBS is the negative control.

[0236] The hemolytic toxicity primary screening results of the triazine compounds are shown in Table 5. According to GB / T 14233.2-2005, the general judgment index for qualified blood direct contact material is hemolysis rate < 5%. Both protamine sulfate and Ciraparantag have different degrees of hemolytic toxicity, and their strong alkalinity destroys the stability of the outer membrane of red blood cells. Under the action of the triazine compounds of the present application, the hemolysis rate is all < 2%, indicating that the triazine compounds have good potential for intravenous application and are safe and low-toxicity heparin reversal agents.

[0237] Table 5. Hemolysis caused by heparin reversal agents after incubation in PBS at 37℃ for 1h

[0238] Compound number Hemolysis rate Compound number Hemolysis rate Compound number Hemolysis rate 1 ＜2％ 10 ＜2％ 19 ＜2％ 2 ＜2％ 11 ＜2％ 20 ＜2％ 3 ＜2％ 12 ＜2％ 21 ＜2％ 4 ＜2％ 13 ＜2％ 22 ＜2％ 5 ＜2％ 14 ＜2％ 23 ＜2％ 6 ＜2％ 15 ＜2％ 24 ＜2％ 7 ＜2％ 16 ＜2％ 25 ＜2％ 8 ＜2％ 17 ＜2％ Protamine sulfate 9.62％ 9 ＜2％ 18 ＜2％ Ciraparantag 2.66％

[0239] Example 30

[0240] Effect of small molecule heparin reversal agent on the proliferation activity of human umbilical vein endothelial cells

[0241] Vascular endothelial cells are involved in the release of substances, the regulation of blood clotting function, the formation of blood vessels, and the inflammatory and immune processes. Heparin intravenous injection affects the growth and metabolism of vascular endothelium. Small molecule heparin reversal agents targeting heparin can directly bind to the cell membrane surface through charge interaction while reversing the anticoagulation effect of heparin. Testing the basic cytotoxicity of heparin reversal agents on endothelial cells is a common way to evaluate toxicity. Human umbilical vein endothelial cells (HUVEC) were incubated with the test compounds at the working concentration for 48 hours, and the possible toxicity of small molecule heparin reversal agents was studied by colorimetric MTT staining.

[0242] Experimental instruments: super clean bench (Thermo); centrifuge is Dalong DM0412; cell counter is Thermo Countess3 automatic cell counter; CO2 incubator from Thermo; enzyme label instrument is SpectraMax iD3.

[0243] Experimental materials: human umbilical vein endothelial cells (HUVEC, from American model culture collection); DMEM medium, fetal bovine serum (Gibco); trypsin (Amresco); DMSO (Sigma product number D1435); MTT (Shanghai Yuanye Biotechnology Co., Ltd., product number S19063); compounds 1-25.

[0244] HUVEC was cultured in DMEM + 10% FBS complete medium and adherent growth cultured at 37℃, 5% CO2, 95% humidity.

[0245] Experimental method: HUVEC in logarithmic growth phase was digested by trypsin and centrifuged to obtain cell precipitate, which was resuspended with fresh culture medium, stained with trypan blue, diluted to appropriate concentration, and 50 μL of the cell suspension was taken and seeded in 96-well plates at 4000 cells per well. The 96-well plates were placed in a carbon dioxide incubator overnight for culture. Preparation of the mother liquor of the test compound: all compounds were prepared into a 5 mg / mL compound mother liquor using ultrapure water with 5% DMSO as a dissolving aid, which was stored at -80°C and used after being divided into small portions. The compound mother liquor was diluted with culture medium to an appropriate concentration, so that the final concentration of the test was 50 μg / mL. 50 μL of the test compound solution was added to the 96-well plate, and three replicate wells were set for each test compound. The 96-well plate was placed in a carbon dioxide incubator for further culture for 1 day to allow the cells to grow adherently. 20 μL of 5 mg / mL MTT reagent was added to each well, and after 4 hours of incubation, 150 μL of DMSO was added to each well and mixed well for 5 minutes of shaking. Finally, the absorbance value was read at 580 nm by a microplate reader, and the cell growth inhibition efficiency was calculated.

[0246] Cytotoxicity = 100% x (Sample - Blank) / (Control - Blank)

[0247] Sample: the absorbance of the compound experimental group at 580 nm; Blank: the absorbance of the blank control group with only medium at 580 nm; Control: the absorbance of the control group with medium and cells at 580 nm.

[0248] As shown in Table 6, under the action of the triazine compounds of the present application, more than 80% of the compounds HUVEC activity ≥ 75%, indicating that the triazine compounds have no effect on the normal growth of HUVEC, and are safe and low-toxic heparin reversal agents.

[0249] Table 6. In vitro HUVEC proliferation activity test results of heparin reversal agents

[0250] Compound number Cell activity Compound number Cell activity Compound number Cell activity 1 74.37％ 10 101.50％ 19 103.83％ 2 81.99％ 11 77.19％ 20 86.92％ 3 119.50％ 12 131.77％ 21 124.78％ 4 126.55％ 13 62.94％ 22 109.47％ 5 82.53％ 14 107.04％ 23 109.28％ 6 117.39％ 15 117.08％ 24 97.41％ 7 77.73％ 16 79.55％ 25 94.35％ 8 70.16％ 17 114.98％ Protamine sulfate 91.54％ 9 71.54％ 18 92.33％ Ciraparantag 89.63％

[0251] Example 31

[0252] Small molecule heparin reversal agent single-dose acute toxicity test in mice

[0253] Cationic compounds can cause tissue damage in the body after intravenous injection into the body, leading to pulmonary congestion and mononuclear cell infiltration. To evaluate the potential clinical application and safety of spermine-substituted triazine compounds, a single tail vein injection of high-dose compounds in female C57 mice was performed: C57 healthy female mice (7-8 weeks old, body weight 18-20 g) were randomly divided into groups, and each group was injected with a compound or normal saline via the tail vein. The activity of the mice was observed at regular intervals, and the mice were dissected 24 hours after tail vein injection to observe and record the condition of each organ.

[0254] The specific experimental steps are as follows: weigh the compound (compound 20, protamine sulfate, Ciraparantag), add an appropriate amount of normal saline to dissolve it completely, and obtain a compound solution with a concentration of 1.5 mg / mL. Filter with a 0.22 μm filter membrane. Mice were injected with normal saline or compound solution (15 mg / kg) via the tail vein within 3 seconds. Immediately after administration, the mice were observed for 30 minutes, and then the physiological state, mental state, life and death, and activity of the mice were observed at regular intervals every 1 hour. The mice were dissected 24 hours after tail vein injection to observe the main organs: heart, lung, liver, spleen, and kidney for any abnormal changes in size, bleeding, texture, color, and other changes, and the condition of each organ was recorded.

[0255] As shown in Table 7, mice were injected with compound 20, protamine sulfate, and Ciraparantag at a dose of 15 mg / kg. After intravenous injection, some mice showed reduced activity, apathy, and no obvious response to external stimuli. A single mouse injected with compound 20 had slightly discolored kidneys, and the rest showed no signs of abnormal activity or severe post-mortem changes. After intravenous injection of Ciraparantag, a single mouse had lung tissue congestion and pulmonary edema. Intravenous injection of protamine sulfate affected the basic morphology of the mouse's kidneys, and the mouse's alveoli and pulmonary aorta showed moderate to severe changes. The lungs were foamy and white to varying degrees, and a single mouse's spleen turned from dark red to pink. After intravenous injection of protamine sulfate, a single mouse had mild nasal discharge. This indicates that the triazine compound has no effect on the normal activity of mice and is a safe and low-toxicity heparin reversal agent.

[0256] Table 7. Observation of C57 mice within 24 hours after a single tail vein injection of reversal agent and dissection of main organs 24 hours later

[0257] Observation item Compound 20 Ciraparantag Protamine sulfate Normal saline Injection concentration 15 mg / kg 15 mg / kg 15 mg / kg - Decreased activity, apathy 1 / 6 1 / 6 2 / 6 0 Mild nasal discharge 0 0 1 / 6 0 Pulmonary abnormalities 0 1 / 6 4 / 6 0 Spleen discoloration 0 0 1 / 6 0 Kidney discoloration 1 / 6 0 4 / 6 0

[0258] Although embodiments of the present application have been shown and described, it is to be understood that various modifications, substitutions, replacements and changes can be made to these embodiments without departing from the principles and spirit of the present application, and the scope of the present application is defined by the appended claims and their equivalents.

Claims

1. A triazine compound having a structure as shown in Formula I or a pharmaceutically acceptable salt thereof: ###0001### Formula I wherein: R1, R2, R3, R4 are independently selected from hydrogen; R5, R6, R7, R8, R9 are independently selected from hydrogen. wherein: R1-N-R2and R3-N-R4are the same as or different from each other and are each independently selected from the following groups: m is an integer from 1 to 3 and n is an integer from 1 to 3.

2. A triazine compound having a structure as shown in Formula I or a pharmaceutically acceptable salt thereof: ###0002### Formula I wherein: R1, R2, R3, R4 are independently selected from hydrogen; R5, R6, R7, R8, R9 are independently selected from hydrogen.

3. A triazine compound having a structure as shown in Formula I or a pharmaceutically acceptable salt thereof: ###0003### Formula I wherein: R1, R2, R3, R4 are independently selected from hydrogen; R5, R6, R7, R8, R9 are independently selected from hydrogen.

4. A synthetic route of the triazine compound as shown in Formula I: ###0004### Formula I comprising the following steps: Step (1), reacting cyanuric chloride and An in tetrahydrofuran at a temperature of -20°C to obtain intermediate III; wherein the molar ratio of cyanuric chloride to An is 1:1; the molar ratio of cyanuric chloride to N,N-diisopropyl ethylamine is 1:1-1:1.2; Step (2), reacting intermediate III and Bm in dichloromethane at a temperature of -20°C, and then at a temperature of 0°C to obtain intermediate II; wherein the molar ratio of intermediate III to Bm is 1:1; the molar ratio of intermediate III to N,N-diisopropyl ethylamine is 1:1-1:3.2; Step (3), reacting intermediate II and Cy in dimethyl sulfoxide at reflux to obtain the triazine compound having a structure as shown in Formula I; wherein the molar ratio of intermediate II to Cy is 1:1-1:3; the molar ratio of intermediate II to triethylamine is 1:1-1:

5. R1-N-R2is selected from the following groups: R3-N-R4is selected from the following groups: m is an integer from 1 to 3 and n is an integer from 1 to 3; 5. A pharmaceutical composition comprising the triazine compound or a pharmaceutically acceptable salt thereof as claimed in any one of claims 1-3 as an effective component or a main effective component, and a pharmaceutically acceptable carrier.

6. Use of the triazine compound or a pharmaceutically acceptable salt thereof as claimed in any one of claims 1-3 or the pharmaceutical composition as claimed in claim 5 in the preparation of an anticoagulant reversal agent. 25。 4. A process for the preparation of the triazine compound of claim 1, characterized by:

7. Use of the triazine compound or a pharmaceutically acceptable salt thereof as claimed in any one of claims 1-3 or the pharmaceutical composition as claimed in claim 5 in the preparation of a medicament for treating excessive anticoagulant side effects caused by using heparin to treat a disease, or a medicament for treating a surgical operation requiring reversal of heparin anticoagulant activity. wherein An is Bm is Cy is R1, R2, R3, R4, R5, R6, R7, R8, R9 are as described in claim 1; The disease treated by using heparin is myocardial infarction, thrombotic phlebitis, or pulmonary embolism; the surgical operation is microvascular surgery.

9. Use of the triazine compound or a pharmaceutically acceptable salt thereof as claimed in any one of claims 1-3 or the pharmaceutical composition as claimed in claim 5 in the preparation of a medicament for treating hemodialysis requiring reversal of heparin anticoagulant activity.

10. Use of the triazine compound or a pharmaceutically acceptable salt thereof as claimed in any one of claims 1-3 or the pharmaceutical composition as claimed in claim 5 in the preparation of a medicament for treating extracorporeal circulation requiring reversal of heparin anticoagulant activity.

11. Use of the triazine compound or a pharmaceutically acceptable salt thereof as claimed in any one of claims 1-3 or the pharmaceutical composition as claimed in claim 5 in the preparation of a medicament for treating catheterization requiring reversal of heparin anticoagulant activity.

5. A pharmaceutical composition, characterized by: ​ ​ ​ 8. Use according to claim 7, characterized in that: ​ ​ ​ ​

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