Heparin small molecule antagonist-quaternary ammonium salt macrocyclic compound and preparation method thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a heparin small molecule antagonist-quaternary ammonium salt macrocyclic compound and a preparation method thereof. The quaternary ammonium salt macrocyclic compound disclosed by the invention comprises three piperazine functionalized quaternary ammonium salt macrorings and four methylated quaternary ammonium salt macrorings; the quaternary ammonium salt macrocyclic compound can be used for preparing a heparin small molecule antagonist. The compound shows excellent antagonism efficiency on ungraded heparin and low-molecular-weight heparin, and shows a wide therapeutic dose window; and the antagonism efficiency and dosage window of the protamine are superior to those of clinically used protamine. Besides, in-vivo and in-vitro safety evaluation proves that the quaternary ammonium salt macrocycle has good biocompatibility, and shows clinical transformation potential as a protamine substitute as a small molecule antagonist.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a heparin small molecule antagonist—a quaternary ammonium salt macrocyclic compound and its preparation method. Background Technology

[0002] Heparin, as a highly effective anticoagulant, has played an irreplaceable role in cardiovascular interventional surgery, hemodialysis, and the prevention and treatment of thrombotic diseases since its first clinical application in 1935. However, its potent anticoagulant properties are accompanied by a significant bleeding risk, especially in complex procedures such as percutaneous coronary intervention (PCI) and transcatheter aortic valve replacement (TAVR). Patients often face serious bleeding complications due to heparinization, severely impacting postoperative recovery and treatment safety. Protamine sulfate, currently the only widely used heparin antagonist in clinical practice, is a cationic polypeptide rich in arginine. It binds to the highly sulfated polysaccharide chains in heparin through electrostatic interactions, forming a stable complex that neutralizes the anticoagulant activity of heparin. However, protamine sulfate has several limitations in practical applications: while it has a high neutralizing capacity for unfractionated heparin (UFH), its antagonistic efficiency against low molecular weight heparin (LMWHs) is only about 60%, which is insufficient to meet clinical needs. More seriously, as a heterologous protein drug, it poses a significant risk of immunogenicity, which can cause adverse reactions such as allergic reactions, hypotension, and pulmonary hypertension, and even endanger life. Therefore, developing a novel heparin antagonist that combines high efficiency, broad spectrum, and good biocompatibility has become one of the urgent problems to be solved in the field of anticoagulation therapy.

[0003] In recent years, researchers have focused on developing various heparin antagonists, including small molecules, polymers, peptides, and proteins, in an attempt to overcome the limitations of protamine. Despite some progress, no drug can currently completely replace protamine in clinical practice. From a mechanistic perspective, existing strategies largely rely on the electrostatic attraction between the cationic groups in the antagonist and the polyanionic chains of heparin. While reasonable and universal, these strategies still have shortcomings in terms of binding strength, specificity, and safety. Against this backdrop, quaternary ammonium macrocyclic compounds, as an emerging molecular platform, exhibit unique heparin binding potential due to their combination of the strong positive charge of quaternary ammonium salts and the cavity recognition capability of the macrocyclic matrix. Summary of the Invention

[0004] The purpose of this invention is to provide a class of small molecule antagonists with high antagonistic efficiency and wide therapeutic window against heparin—quaternary ammonium macrocyclic compounds, which exhibit antagonistic efficiency and dose window far superior to protamine sulfate used clinically.

[0005] The quaternary ammonium salt macrocyclic compounds (also called quaternary ammonium salt macrocyclic molecules) provided by this invention are two types of quaternized cationic macrocycles, namely piperazine-functionalized quaternary ammonium salt macrocycles and methylated quaternary ammonium salt macrocycles, denoted as X. Specifically, they include seven structural formulas, denoted as compound 1, compound 2, compound 3, compound 4, compound 5, compound 6, and compound 7; compounds 1-3 are piperazine-functionalized quaternary ammonium salt macrocycles, and compounds 4-7 are methylated quaternary ammonium salt macrocycles; their structural formulas are as follows:

[0006]

[0007] The quaternary ammonium salt macrocyclic compound provided by this invention is a piperazine-functionalized and methylated cationic macrocyclic compound, which has the advantages of water solubility, high stability and low toxicity.

[0008] Leveraging the highly sulfonated and carboxylated polyanionic properties of heparin, cation-rich compounds are designed and synthesized to form stable complexes with heparin through electrostatic interactions, thereby antagonizing heparin. This is currently the main method for achieving highly efficient heparin antagonism. The quaternary ammonium macrocycle (X) provided in this invention (which is a piperazine-functionalized quaternary ammonium macrocycle) forms a multi-non-covalent bond synergistic binding mode through the synergistic cooperation of hydrogen bond networks, electrostatic interactions, and macrocyclic cavity recognition functions, achieving highly specific recognition and efficient neutralization of heparin molecules. This design strategy based on the supramolecular host-guest recognition principle not only provides a new approach to overcoming the limitations of traditional heparin antagonists but also demonstrates the application potential of macrocyclic compounds in the biomedical field.

[0009] In this invention, the quaternary ammonium macrocycle also includes other functionalized quaternary ammonium macrocycle molecules that can antagonize heparin.

[0010] In this invention, the quaternary ammonium macrocyclic salt exhibits good biocompatibility within the effective antagonistic dose range and has no significant risk of acute toxicity, hematologic toxicity, or tissue damage.

[0011] In this invention, the human plasma used in the experiment of quaternary ammonium macrocyclic heparin antagonism was obtained from the Shanghai Blood Center.

[0012] In this invention, the biological experimental protocols used in the quaternary ammonium salt macrocyclic antagonistic heparin experiment all comply with ethical requirements.

[0013] This invention also provides a method for preparing the aforementioned quaternary ammonium salt macrocyclic compound, which involves reacting a piperazine-functionalized tertiary amine monomer or a methylated tertiary amine monomer with a specific aliphatic chain length and a corresponding dibenzyl bromide monomer to generate the corresponding target macrocyclic compound. The specific steps are as follows:

[0014] (1) Using 1-(tert-butoxycarbonyl)piperazine or dimethylamine as raw materials, react with dibromo compounds with three or seven carbon aliphatic chains to obtain piperazine-functionalized tertiary amines and methylated tertiary amines with specific aliphatic chain lengths;

[0015] (2) A five-carbon aliphatic chain-long piperazine-functionalized tertiary amine was prepared by reducing and amination of 1-(tert-butyloxycarbonyl)piperazine with glutaraldehyde;

[0016] (3) Using piperazine-functionalized tertiary amines or methyl-functionalized tertiary amines as raw materials, the corresponding dibenzyl bromide monomers were reacted to prepare the corresponding quaternary ammonium salt macrocyclic compounds.

[0017] This invention investigated activated partial thromboplastin time (aPTT) and mouse tail docking experiments on the quaternary ammonium macrocyclic compound. Combined in vitro and in vivo experiments demonstrated that the piperazine-functionalized quaternary ammonium macrocyclic compound exhibits excellent neutralization efficacy and a wide therapeutic window against both unfractionated heparin (UFH) and low molecular weight heparin (LMWHs). Related animal experiments demonstrated that the piperazine-functionalized quaternary ammonium macrocyclic compound possesses good biocompatibility and low biotoxicity. In the heparin antagonism test, compound 1 showed the best effect.

[0018] The above experimental results demonstrate that this class of macrocyclic compounds exhibits excellent (superior to existing technologies) antagonistic activity and a wide dose window against both UFH and LMWHs, especially the piperazine-functionalized quaternary ammonium macrocyclic compounds, whose activity far exceeds that of protamine sulfate used clinically. This invention provides a candidate molecule with clinical application potential for the development of a new generation of small molecule heparin antagonists. Attached Figure Description

[0019] Figure 1 This is the synthetic route for the quaternary ammonium salt macrocyclic compounds of this invention.

[0020] Figure 2 To investigate the neutralization effects of piperazine functionalized molecules 1, 2, and 3 and protamine on unfractionated heparin (UFH, concentration 2 IU / mL) in an activated partial thromboplastin time (aPTT) assay.

[0021] Figure 3 To investigate the neutralizing effect of piperazine functionalized molecules 1, 2, and 3 and protamine on dalte (2 IU / mL) in an activated partial thromboplastin time (aPTT) assay.

[0022] Figure 4 To investigate the neutralizing effects of piperazine functionalized molecules 1, 2, and 3 and protamine on enoxaparin (2 IU / mL) in an activated partial thromboplastin time (aPTT) assay.

[0023] Figure 5To investigate the neutralizing effect of piperazine functionalized molecules 1, 2, and 3 and protamine on nadrone (2 IU / mL) in an activated partial thromboplastin time (aPTT) assay.

[0024] Figure 6 In vivo evaluation of the antagonistic effect of compound 1 against the anticoagulant effect of unfractionated heparin (UFH): effect on total bleeding time in ICR mice (n=6). *p<0.05, ****p<0.001, ns(P>0.05).

[0025] Figure 7 In vivo evaluation of the antagonistic effect of compound 1 against the anticoagulant effect of dalte: effect on total bleeding time in ICR mice (n=6). *p<0.05, ****p<0.001, ns(P>0.05).

[0026] Figure 8 In vivo evaluation of the antagonistic effect of compound 1 against the anticoagulant effect of unfractionated heparin (UFH): effect on total bleeding in ICR mice (n=6). *p<0.05, ****p<0.001, ns(P>0.05).

[0027] Figure 9 In vivo evaluation of the antagonistic effect of compound 1 against the anticoagulant effect of dalte: effect on total bleeding in ICR mice (n=6). *p<0.05, ****p<0.001, ns(P>0.05).

[0028] Figure 10 Acute toxicity test in mice: ICR mice were subjected to an acute toxicity test for 14 days with quaternary ammonium macrocyclic compound 1 at doses of 0, 4, 8 and 12.5 mg / kg.

[0029] Figure 11 The hemolytic experiment of quaternary ammonium macrocyclic compound 1 on rat erythrocytes.

[0030] Table 1 summarizes the maximum antagonistic efficiencies of quaternary ammonium macrocyclic compounds 1–7 and protamine in human plasma against UFH and LMWHs in the activated partial thromboplastin time (aPTT) assay.

[0031] Table 2 summarizes the antagonistic dose windows of quaternary ammonium macrocyclic compounds 1-3 and protamine in human plasma for UFH and LMWHs in the activated partial thromboplastin time (aPTT) assay.

[0032] Table 3 summarizes the bleeding time and amount in the blank control group and negative control group in the mouse tail docking experiment.

[0033] Table 4 summarizes the bleeding time and amount in the heparin+1 group and the saline+1 group of mice in the tail docking experiment.

[0034] Table 5 summarizes the bleeding time and amount in the heparin + protamine group of mice in the tail docking experiment. Detailed Implementation

[0035] The present invention will be further described below with reference to the embodiments and accompanying drawings, but should not be construed as limiting the present invention.

[0036] Example 1: Preparation of quaternary ammonium salt macrocycles and their monomers.

[0037] The quaternary ammonium salt macrocycles provided by this invention are divided into two categories: one containing piperazine units and the other containing methyl units. The preparation routes are as follows: Figure 1 As shown: The corresponding tertiary amine monomers are prepared from 1-(tert-butyloxycarbonyl)piperazine or dimethylamine, and then reacted with the corresponding dibenzyl bromide monomers to obtain the target macrocyclic molecule. The specific operation is as follows:

[0038] Synthesis of Compound 1: 4,4'-Di(bromomethyl)diphenyl ether (0.2 g, 0.56 mmol) and Compound 8 (0.23 g, 0.56 mmol) were dissolved in acetonitrile and reacted at 90 °C for 72 h with stirring. After the reaction was complete, the mixture was cooled to room temperature, and the solvent was removed to obtain a white solid. The solid was redissolved in methanol and ion-exchanged with ammonium hexafluorophosphate to obtain a white powder 1-Boc. The protecting group of the obtained compound 1-Boc was removed with trifluoroacetic acid, followed by two ion-exchanges with ammonium hexafluorophosphate and tetrabutylammonium chloride to obtain white powder 1. 1-Boc (0.35 g, 34%): 1 H NMR (400MHz, Methanol-d4) δ7.72(d,J=8.3Hz,8H),7.25(d,J=8.2Hz,8H),4.93(s,8H),4. 15–4.02(m,8H),3.88-3.61(m,24H),3.57–3.45(m,8H),2.81–2.62(m,4H),1.48(s,36H). 1(0.21g,79%): 1 H NMR (400MHz, Deuterium Oxide) δ7.55(d,J=8.1Hz,8H),7.18(d,J=7.9Hz,8H),4.84(s,8H),3.90(d,J=11.2Hz,8H),3.82–3.72(m,32H),2.63–2.53(m,4H).

[0039] Synthesis of Compound 2: The synthesis method was the same as that of Compound 1. A crude product was obtained by reacting 4,4'-di(bromomethyl)diphenyl ether (0.2 g, 0.56 mmol) and Compound 9 (0.25 g, 0.56 mmol). After ion exchange purification, a white powder, compound 2-Boc, was obtained. Compound 2-Boc (0.5 g, 0.27 mmol) was deprotected in the presence of trifluoroacetic acid, followed by ion exchange to obtain a white powder, compound 2. 2-Boc (0.29 g, 28%): 1 ¹H NMR (400MHz, Acetonitrile-d³) δ 7.45 (d, J = 8.1 Hz, 8H), 7.15 (d, J = 8.0 Hz, 8H), 4.45 (s, 8H), 3.97 (s, 4H), 3.93 (s, 4H), 3.63–3.44 (m, 8H), 3.42–3.24 (m, 16H), 3.17–3.02 (m, 8H), 1.88–1.76 (m, 8H), 1.46 (s, 36H), 1.35–1.28 (m, 4H). Compound 2 (0.23 g, 82%): 1 H NMR (400MHz, Deuterium Oxide) δ7.31(d,J=8.1Hz,8H),7.01(d,J=8.0Hz,8H),4.14(s,8H),3.31–3.9(m,32H),2.93–2.82(m,8H),1.67–1.62(m,8H),1.19–1.15(m,4H).

[0040] Synthesis of Compound 3: The synthesis method was the same as that of Compound 1. A crude product was obtained by reacting 4,4'-di(bromomethyl)diphenyl ether (0.2 g, 0.56 mmol) with Compound 10 (0.26 g, 0.56 mmol). After ion exchange, a white powder, compound 3-Boc, was obtained. Compound 3-Boc (0.5 g, 0.26 mmol) was deprotected in the presence of trifluoroacetic acid, and then purified by ion exchange to obtain the white powder, compound 3. Compound 3-Boc (0.37 g, 35%): 1 ¹H NMR (400MHz, Acetonitrile-d³) δ 7.46 (d, J = 8.3 Hz, 8H), 7.16 (d, J = 8.1 Hz, 8H), 4.45 (s, 8H), 3.95 (s, 4H), 3.92 (s, 4H), 3.60–3.43 (m, 8H), 3.40–3.25 (m, 16H), 3.23–3.03 (m, 8H), 1.86–1.74 (m, 8H), 1.47–1.26 (m, 48H). Compound 3 (0.24 g, 86%): 1H NMR (400MHz, Deuterium Oxide) δ7.61(d,J=8.5Hz,8H),7.27(d,J=8.1Hz,8H),4.83(s,8H),3.90–3.74(m,32H),3.61–3.48(m,8H),2.08–1.92(m,8H),1.64–1.41(m,12H).

[0041] Synthesis of Compound 4: Compound 4,4'-di(bromomethyl)diphenyl ether (0.2 g, 0.56 mmol) and Compound 11 (73 mg, 0.56 mmol) were dissolved in acetonitrile and reacted at 90 °C for 72 h with stirring. After the reaction was complete, the mixture was cooled to room temperature, and the white solid was collected by filtration. The solid was purified by column chromatography using methanol / water / saturated ammonium chloride aqueous solution (v / v 6:3:1). The purified solid was then subjected to two ion exchanges with ammonium hexafluorophosphate and tetrabutylammonium chloride to give compound 4 (0.12 g, 27%). 1 HNMR (400MHz, Deuterium Oxide) δ7.48 (d, J = 8.3 Hz, 8H), 7.10 (d, J = 8.2 Hz, 8H), 4.50 (s, 8H), 3.19–3.07 (m, 32H), 2.20–2.08 (m, 4H).

[0042] Synthesis of compound 5: The synthesis method was the same as that of compound 4. The crude product was obtained by reacting 1,4-di(bromomethyl)benzene (0.2 g, 0.76 mmol) and compound 11 (98 mg, 0.76 mmol). The crude product was then subjected to column chromatography and two ion exchanges to obtain compound 5 (0.25 g, 54%) as a white solid. 1 H NMR (400MHz, Deuterium Oxide) δ7.64(s,8H),4.68(s,8H),3.20(s,32H),2.31–2.19(m,4H).

[0043] Synthesis of compound 6: The synthesis method was the same as that of compound 4. The crude product was obtained by reacting 4,4'-dibromomethylbiphenyl (0.2 g, 0.59 mmol) and compound 11 (76 mg, 0.59 mmol). The crude product was then subjected to column chromatography and two ion exchanges to obtain a white solid compound 6 (0.23 g, 63%). 1 H NMR (400MHz, Deuterium Oxide) δ7.70 (d, J = 8.3 Hz, 8H), 7.47 (d, J = 8.6 Hz, 8H), 4.44 (s, 8H), 3.17–3.03 (m, 32H), 2.15–1.99 (m, 4H).

[0044] Synthesis of compound 7: The synthesis method was the same as that of compound 4. The crude product was obtained by reacting 4,4'-dibromomethylbiphenyl (0.2 g, 0.59 mmol) and compound 12 (0.11 g, 0.59 mmol). The crude product was then subjected to column chromatography and two ion exchanges to obtain compound 7 (0.28 g, 65%) as a white solid. 1 H NMR (400MHz, Deuterium Oxide) δ7.64(d,J=7.8Hz,8H),7.48(d,J=7.8Hz,8H),4.34(s,8H),3.09–3.00(m,8H),2.97(s,24H),1.80–1.67(m,8H),1.33–1.19(m,12H).

[0045] Synthesis of Compound 8: 1,3-Dibromopropane (3.0 g, 14.9 mmol), 1-(tert-butyloxycarbonyl)piperazine (6.1 g, 32.7 mmol), and potassium carbonate (8.2 g, 59.6 mmol) were dissolved in acetonitrile and heated at 90 °C for 48 h. The reaction was stopped, and the mixture was filtered while hot. The filtrate was collected, cooled, and a white solid precipitated. Filtering yielded a white solid (5.8 g, 95%). 1 H NMR (400MHz, Chloroform-d) δ3.39 (t, J = 4.1Hz, 8H), 2.42–2.26 (m, 12H), 1.68–1.61 (m, 2H), 1.42 (s, 18H).

[0046] Synthesis of compound 9: 1,5-glutaraldehyde (1.08 mL, 50 wt.% in water, 5.37 mmol), 1-(tert-butyloxycarbonyl)piperazine (2 g, 10.75 mmol) and sodium triacetoxyborohydride (3.68 g, 17.2 mmol) were dissolved in dichloroethane and reacted at room temperature for 24 h. The reaction was quenched with water, and the aqueous phase was collected by separation. The addition of sodium bicarbonate solid resulted in the precipitation of a white solid. The mixture was filtered to give a white solid (2.3 g, 49%). 1 H NMR (600MHz, Chloroform-d) δ3.42 (t, J = 5.0 Hz, 8H), 2.36 (t, J = 4.9 Hz, 8H), 2.34–2.30 (m, 4H), 1.53–1.47 (m, 4H), 1.45 (s, 18H), 1.33–1.28 (m, 2H).

[0047] Synthesis of compound 10: The synthetic method is the same as that of compound 8. It was obtained by reacting compound 1,7-dibromoheptane (3.0 g, 11.6 mmol) with 1-(tert-butyloxycarbonyl)piperazine (4.76 g, 25.6 mmol) to give a white solid (5.1 g, 94%). 1HNMR (400MHz, Chloroform-d) δ3.42 (t, J = 5.1 Hz, 8H), 2.35 (t, J = 5.1 Hz, 8H), 2.32–2.27 (m, 4H), 1.49–1.42 (m, 22H), 1.32–1.25 (m, 6H).

[0048] Synthesis of Compound 11: 1,3-Dibromopropane (3 g, 14.8 mmol) and dimethylamine (15 mL, 40 wt% in H2O) were reacted with chloroform as solvent at room temperature for 48 h. After removing the solvent, the mixture was redissolved in dichloromethane, and the organic phase was washed with 2 M NaOH. The mixture was separated, the organic phase was collected, dried, and the solvent was removed to obtain a yellow oily liquid (1.7 g, 98%). 1 H NMR (400MHz, Chloroform-d) δ2.24–2.19(m,4H),2.14(s,12H),1.61–1.52(m,2H).

[0049] Synthesis of compound 12: The synthesis method is the same as that of compound 11. The yellow oily liquid (1.7 g, 91%) was obtained by reacting compound 1,7-dibromoheptane (2.6 g, 10 mmol) with dimethylamine (5.9 mL, 40 wt% in H2O). 1 H NMR (400MHz, Chloroform-d) δ2.27–2.16(m,16H),1.50–1.40(m,4H),1.34–1.24(m,6H).

[0050] Example 2: Activated partial thromboplastin time (aPTT) experiment of quaternary ammonium macrocyclic compounds.

[0051] This invention systematically compared the neutralization effects of quaternary ammonium macrocycles and protamine on unfractionated heparin (UFH) and low molecular weight heparin (LMWHs) in human anemic platelet-rich plasma (PPP) using an activated partial thromboplastin time (aPTT) assay. The neutralization effects of methylated quaternary ammonium macrocycles (compounds 4–7) on UFH and LMWHs were comparable to or slightly better than those of protamine. Compared to compounds 4–7, piperazine-functionalized quaternary ammonium macrocycles (compounds 1–3) showed significantly enhanced neutralization activity against both UFH and LMWHs. Specifically, the methylated quaternary ammonium macrocycles exhibited higher neutralization capacity only against UFH (maximum antagonistic efficiencies of 76%, 93%, 94%, and 92%, respectively), while their neutralization efficiency against LMWHs was generally below 90% (Table 1). In contrast, piperazine-functionalized quaternary ammonium macrocyclic compounds not only achieved neutralization efficiencies of over 95% against UFHs, but also demonstrated highly efficient antagonism of over 90% against all three LMWHs, exhibiting a wider effective concentration range (Table 2). Specifically, the heparin concentration ranges for compound 1 to achieve over 90% antagonism against Dalte, Enoxa, and Natro were 25-270 μg / mL, 13-240 μg / mL, and 33-180 μg / mL, respectively; the concentration windows for compound 2 against Dalte, Enoxa, and Natro were 60-240 μg / mL, 90-270 μg / mL, and 60-135 μg / mL, respectively; and the concentration windows for compound 3 against Dalte, Enoxa, and Natro were 135-300 μg / mL, 120-240 μg / mL, and 210-240 μg / mL, respectively. These results clearly demonstrate that the introduction of the piperazine moiety significantly enhances the neutralizing capacity and dose range of macrocyclic heparin drugs, highlighting its key role in improving the therapeutic window.

[0052] Furthermore, the aPTT experimental results further demonstrate that, while maintaining the same structural backbone (such as compounds 1 and 4), simply altering the hydrogen bond acceptor ability can significantly affect their antagonistic performance. For example, piperazine-functionalized molecule 1 exhibits neutralization efficiencies exceeding 90% against UFH and the three LMWHs, with a broad effective concentration window; while methylated molecule 4, with the same backbone, shows antagonistic efficiencies below 80% against both UFH and the three LMWHs. This comparative result clearly indicates that, in the heparin antagonism process, in addition to electrostatic interactions, the synergistic participation of hydrogen bonds is a key factor in achieving efficient and stable binding, further confirming the rationality of the synergistic design of multiple non-covalent interactions.

[0053] To evaluate the practical application potential of the compounds of this invention, compound 1 was used as an example, and compared with commercial protamine sulfate (PRTM) under the same conditions using an aPTT test. Figure 2-5As shown, PRTM only exhibited high neutralization efficiency against UFH, reaching 98% at 22 μg / mL, but its efficiency rapidly decreased with increasing concentration, indicating a narrow therapeutic window and implying that the risk of bleeding may be caused by inappropriate dosage. Furthermore, PRTM's highest antagonistic efficiencies against the three Dalte, Enoxa, and Nadro were 72%, 62%, and 80%, respectively, consistent with previous reports, further confirming its limited antagonistic ability against LMWHs. In contrast, compound 1 not only possessed high antagonistic efficiency (>95%) and a wide dose window (15-270 μg / mL) against UFH, but also showed neutralization efficiencies exceeding 90% against all three LMWHs, demonstrating broad-spectrum and highly effective heparin antagonistic properties, potentially compensating for PRTM's clinical limitations in low molecular weight heparin antagonism.

[0054] Based on the excellent performance of compound 1 in the aPTT test, this invention still uses compound 1 as an example to evaluate its in vivo activity.

[0055] Example 3: Mouse tail transverse section experiment.

[0056] Based on the aPTT in vitro experiment demonstrating the excellent heparin neutralizing properties of compound 1, this invention further evaluates the in vivo antagonistic effect of compound 1 against heparin using a mouse tail slit model. This model, a classic method for evaluating the in vivo antagonistic ability of compounds against heparin, allows for quantitative analysis of bleeding time and volume, directly reflecting the compound's ability to restore coagulation function. Based on the effective dose obtained from the aPTT experiment, this invention sets the in vivo dosage of compound 1 at 0.6 mg / kg and the heparin dosage at 200 IU / kg. ICR mice were randomly divided into five groups (n=6 per group, half male and half female) and injected twice via the tail vein at 0 minutes and 5 minutes, respectively. The specific groups were as follows: (a) UFH or LMWH (200 IU / kg) + macrocyclic compound 1 (0.6 mg / kg); (b) saline + macrocyclic compound 1 (0.6 mg / kg); (c) saline + saline (normal control); (d) UFH or LMWH (200 IU / kg) + saline (negative control); (e) UFH or LMWH (200 IU / kg) + protamine sulfate (2.6 mg / kg, positive control). After transverse cutting of the tail, the bleeding time from the start of bleeding to the point where no blood droplets formed was recorded. Bleeding samples were collected throughout the process using 1×1cm filter paper. After lysing the red blood cells with 10wt.% NaOH, the absorbance at 405nm was measured using an ELISA reader, and the total bleeding volume was calculated based on the standard curve. The relevant results are summarized in Tables 3 to 5.

[0057] like Figure 6-9As shown, the bleeding time and bleeding volume in the saline control group and the group treated with macrocyclic compound 1 alone were (12.9±5.1 min, 33.8±10.8 μL) and (13.8±4.2 min, 26.8±5.6 μL), respectively, with no significant difference between the two groups, indicating that compound 1 itself has no adverse effect on coagulation function. However, after injection of UFH or Dalte, both bleeding time and bleeding volume were significantly prolonged, with the UFH group showing 96.6±8.7 min (P=1.85×10⁻⁶). -9 ) and 506.8±109.3μL (P=9.74×10) -7 The Dalte group had 99.2 ± 11.1 min (P = 8.29 × 10⁻⁶). -9 ) and 478.5±142.7μL (P=1.82×10) -5 Notably, when compound 1 (0.6 mg / kg) was injected 5 minutes after UFH or Dalte, bleeding parameters returned to normal levels. In the UFH + compound 1 group, the bleeding time and amount were 12.9 ± 2.8 min (P = 7.01 × 10⁻⁶). -10 ) and 39.8±12.2μL (P=1.11×10 -6 There was no statistically significant difference between the Dalte + compound 1 group and the normal control group (P = 1.00, P = 0.39). The Dalte + compound 1 group also showed a similar antagonistic effect, with bleeding time and bleeding volume of 13.1 ± 2.7 min (P = 4.36 × 10⁻⁶). -9 ) and 29.1±7.2μL (P=1.64×10 -5 The results were comparable to those of the normal group (P = 1.00, P = 0.39). In contrast, the positive control protamine showed poor neutralization of UFH, with bleeding time and volume of 40.0 ± 11.1 min and 117.1 ± 53.6 μL, respectively, significantly higher than those of the compound 1 treatment group (12.9 ± 2.8 min, 39.8 ± 12.2 μL). The Dalte+PRTM group also showed a similar trend (bleeding time and volume of 40.2 ± 10.2 min and 100.7 ± 41.9 μL, respectively). These results consistently indicate that compound 1 not only has a highly effective antagonistic effect against UFH in vivo, but also exhibits significantly superior neutralizing activity against LMWHs compared to protamine, demonstrating broad prospects for clinical translation.

[0058] Example 4: Biosafety Assessment

[0059] Based on previous experiments, macrocyclic molecule 1 has been confirmed to have high antagonistic efficiency and a wide dose window against both UFH and LMWHs in vitro and in vivo. This invention further evaluates its safety to comprehensively assess its potential for clinical translation. First, the maximum tolerated dose of compound 1 was determined using an ICR mouse model, and changes in vital signs were observed. The results showed that the maximum tolerated dose of compound 1 after a single intravenous injection in mice was 12.5 mg / kg, approximately 20.8 times its effective antagonistic dose in vivo (0.6 mg / kg), indicating a wide safety window. Based on this, we conducted acute toxicity experiments on mice for 14 days at doses of 0, 4, 8, and 12.5 mg / kg. Figure 10 As shown, during the two-week experiment, the mice exhibited a steady increase in body weight without death or obvious toxic symptoms, indicating that macrocyclic molecule 1 at doses below 12.5 mg / kg does not affect normal growth in mice. Furthermore, the rat erythrocyte hemolysis experiment showed that macrocyclic molecule 1 did not induce a significant hemolytic reaction at concentrations below 800 μg / mL. Figure 11 This demonstrates that it has good biocompatibility.

[0060] The above description is merely an optimized embodiment of the present invention and is not intended to limit the invention. All modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

[0061] Table 1. Maximum antagonistic efficiency of quaternary ammonium macrocyclic 1-7 and protamine against UFH and LMWHs in human plasma.

[0062]

[0063] Table 2. Antagonistic dose ranges of quaternary ammonium macrocyclic 1-3 and protamine in human plasma against UFH and LMWHs

[0064]

[0065] Table 3. Summary of bleeding time and amount in blank control (normal saline) and negative control (heparin, 200 IU / kg)

[0066] Table 4. Summary of bleeding time and bleeding volume in mice in the heparin + macrocyclic molecule group and the saline + macrocyclic molecule group.

[0067]

[0068] Table 5. Summary of bleeding time and bleeding volume in mice in the heparin + protamine group

[0069]

Claims

1. A small molecule heparin antagonist-quaternary ammonium salt macrocycle, characterized in that, The two types of quaternized cationic macrocycles are piperazine functionalized quaternary ammonium salt macrocycle and methylated quaternary ammonium salt macrocycle, and specifically include seven structural formulas, which are respectively denoted as compound 1, compound 2, compound 3, compound 4, compound 5, compound 6 and compound 7; the compound 1 to compound 3 are piperazine functionalized quaternary ammonium salt macrocycle, and the compound 4 to compound 7 are methylated quaternary ammonium salt macrocycle; the structural formulas are respectively as follows:

2. A process for the preparation of the quaternary ammonium salt macrocycle of claim 1, characterized in that, By using the highly sulfonated and carboxylated polyanion characteristics of heparin, a cation-rich compound is designed and synthesized, and a stable complex is formed with heparin through electrostatic interaction; through the synergistic cooperation of hydrogen bond network, electrostatic interaction and macrocyclic cavity recognition function, a multiple non-covalent bond synergistic binding mode is formed, and high specificity recognition and efficient neutralization of heparin molecules are realized; in particular, the piperazine functionalized tertiary amine monomer or the methylated tertiary amine monomer with a specific fatty chain length is used to react with the corresponding dibenzyl bromide monomer to generate the corresponding target macrocyclic compound, and the specific steps are as follows: (1) 1-(tert-butoxycarbonyl) piperazine or dimethylamine is used as a raw material to react with a three-carbon or seven-carbon fatty chain dibromide compound to obtain a piperazine functionalized tertiary amine and a methylated tertiary amine with a specific fatty chain length; (2) 1-(tert-butoxycarbonyl) piperazine is used as a raw material to react with glutaraldehyde to prepare a piperazine functionalized tertiary amine with a five-carbon fatty chain length; (3) the piperazine functionalized tertiary amine or the methyl functionalized tertiary amine is used as a raw material to react with the corresponding dibenzyl bromide monomer to prepare the corresponding quaternary ammonium salt macrocycle compound.

3. Use of the quaternary ammonium salt macrocycle compound of claim 1 in the preparation of a heparin small molecule antagonist.

4. The use of claim 1 has better antagonistic activity and a wide dose window than the prior art for ordinary heparin and low molecular weight heparin.

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