An SMTP-7 derivative and its use

Abstract

The present disclosure relates to an SMTP-7 derivative and its use. Specifically, a compound of Formula I or a pharmaceutically acceptable salt thereof is provided, wherein each group is as defined in the specification. #imgabs0#

Description

Technical Field

[0001] The present invention belongs to the field of medicine and relates to an SMTP-7 derivative and its use. Background Art

[0002] SMTP-7 (TMS-007, Stachybotrys microspore triprenyl phenol-7) was extracted in 2000 from a fungus (Stachybotrys microspora) found on fallen leaves on Iriomote Island, Okinawa Prefecture. It is a small molecule plasminogen activator with a structure similar to vitamin E. It has a novel mechanism of action for dissolving blood clots and is believed to inhibit local inflammation at the site of thrombosis. SMTP-7 also exhibits anti-angiogenic, antioxidant, and tissue regeneration activities (WEIMIN H, SHIGEKI O, et al. J. Antibiot., 2000, 53(3):241-247).

[0003]

[0004] Plasminogen is a precursor to plasmin, which, upon activation, can generate plasmin. This protease hydrolyzes many proteins, including thrombin. SMTP-7 binds to plasminogen, altering its molecular conformation and making it more susceptible to activation by plasminogen activators. Therefore, SMTP-7 itself does not activate plasminogen; it simply facilitates the activation process. This unique combination of effects makes SMTP-7 a promising candidate for a best-in-class thrombolytic drug for the treatment of acute ischemic stroke (AIS), potentially extending the therapeutic window compared to existing standard thrombolytic drugs. (Although many antihypertensive, lipid-lowering, and anticoagulant drugs can prevent stroke, the only currently available therapeutic agent for ischemic stroke is recombinant tissue plasminogen activator (rt-PA, alteplase), a 526-amino acid glycoprotein.)

[0005] SMTP induces a conformational change in plasminogen, resulting in an increase in its binding to fibrin and ultimately in the activation of plasmin. SMTP also induces the autocleavage of plasmin to provide an angiogenic human angiostatin-like fragment. This activity is believed to underlie the antiangiogenic and antitumor effects of SMTP. Furthermore, the SMTP-induced increase in activated plasminogen may control local extracellular proteolysis, leading to tissue remodeling, wound healing, and tissue regeneration. Summary of the Invention

[0006] The present disclosure provides a compound represented by Formula I or a pharmaceutically acceptable salt thereof,

[0007]

[0008] Among them, R 1 、R 2 、R 3 、R 4 、R 5 、R 6 、R 7 、R 8 、R 9 、R 10 、R 11 、R 12 、R 13 、R 14 、R 15 、R 16 、R 17 、R 18 、R 19 、R 20 、R 21 、R 22 、R 23 、R 24 、R 25 、R 26 、R 27 、R 28 、R 29 、R 30 、R 31 、R[[ID=6⑦]] 32 、R 33 、R<⑪ 34 、R 35 、R 36 、R 37 、R 38 ]>、R 39 、R 40 、R 41 、R 42 、R 43 、R 44 、R 45 、R 46 、R 47 、R 48 、R 49 [[ID=⑪02]]、R 50 、R 51 、R 52 、R 53 、R 54 、R 55 、R 56 、R 57 [[ID=⑪18]]、R 58 、R 59 、R 60 、R 61 [[ID=⑪2⑥]]、R 62 and R 63 It should be noted that there may be some unclear or incorrect formatting in the original text. If possible, it is recommended to check and clarify the source for a more accurate translation.are each independently hydrogen or deuterium, and R 1 、R 2 、R 3 、R 4 、R 5 、R 6 、R 7 、R 8 、R 9 、R 10 、R 11 、R 12 、R 13 、R 14 、R 15 、R 16 、R 17 、R 18 、R 19 、R 20 、R 21 、R 22 、R 23 、R 24 、R 25 、R 26 、R 27 、R 28 、R 29 、R 30 、R 31 、R 32 、R 33 、R 34 、R 35 、R 36 、R 37 、R 38 、R 39 、R 40 、R 41 、R 42 、R 43 、R 44 、R 45 、R 46 、R 47 、R 48 、R 49 、R 50 、R 51 、R 52 、R 53 、R 54 、R 55 、R 56 、R 57 、R 58 、R 59 、R 60 、R 61 、R 62 and R 63 At least one of them is deuterium.

[0009] In some embodiments, R 27 Selected from deuterium, R 28 Selected from deuterium, R 36 Selected from deuterium, R 37 Selected from deuterium.

[0010] In some embodiments, R 26 Selected from deuterium, R 38 Selected from deuterium.

[0011] In some embodiments, R 24 Selected from deuterium, R 25 Selected from deuterium, R 39 Selected from deuterium, R 40 Selected from deuterium.

[0012] In some embodiments, R 29 Selected from deuterium.

[0013] In some embodiments, R 30 Selected from deuterium, R 31 Selected from deuterium.

[0014] In some embodiments, R 34 Selected from deuterium, R 35 Selected from deuterium.

[0015] In some embodiments, R 16 Selected from deuterium, R 17 Selected from deuterium, R 47 Selected from deuterium, R 48 Selected from deuterium.

[0016] In some embodiments, R 12 Selected from deuterium, R 13 Selected from deuterium, R 14 Selected from deuterium.

[0017] In some embodiments, R 50 Selected from deuterium, R 51 Selected from deuterium, R 52 Selected from deuterium.

[0018] In some embodiments, R 23 Selected from deuterium, R 41 Selected from deuterium.

[0019] In some embodiments, R32 Selected from deuterium, R 33 Selected from deuterium.

[0020] In some embodiments, R 30 Selected from deuterium, R 31 Selected from deuterium, R 32 Selected from deuterium, R 33 Selected from deuterium, R 34 Selected from deuterium, R 35 Selected from deuterium.

[0021] In some embodiments, R 29 Selected from deuterium, R 30 Selected from deuterium, R 31 Selected from deuterium, R 32 Selected from deuterium, R 33 Selected from deuterium, R 34 Selected from deuterium, R 35 Selected from deuterium.

[0022] In some embodiments, R 1 Selected from deuterium, R 2 Selected from deuterium, R 3 Selected from deuterium.

[0023] In some embodiments, R 4 Selected from deuterium, R 5 Selected from deuterium, R 6 Selected from deuterium.

[0024] In some embodiments, R 58 Selected from deuterium, R 59 Selected from deuterium, R 60 Selected from deuterium.

[0025] In some embodiments, R 61 Selected from deuterium, R 62 Selected from deuterium, R 63 Selected from deuterium.

[0026] In some embodiments, R 1 Selected from deuterium, R 2 Selected from deuterium, R 3 Selected from deuterium, R 4 Selected from deuterium, R 5 Selected from deuterium, R 6 Selected from deuterium, R 58 Selected from deuterium, R 59 Selected from deuterium, R 60 Selected from deuterium, R 61Selected from deuterium, R 62 Selected from deuterium, R 63 Selected from deuterium.

[0027] In some embodiments, R 7 Selected from deuterium, R 57 Selected from deuterium.

[0028] On the other hand, some embodiments provide a compound of formula I or a pharmaceutically acceptable salt thereof.

[0029]

[0030] Typical compounds represented by formula I include but are not limited to:

[0031]

[0032]

[0033] The present disclosure also provides a pharmaceutical composition comprising at least one therapeutically effective amount of the compound represented by the aforementioned formula I or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.

[0034] In some embodiments, the unit dose of the pharmaceutical composition is 0.001 mg-1000 mg.

[0035] In certain embodiments, the pharmaceutical composition contains 0.01-99.99% of the compound of the aforementioned formula I or a pharmaceutically acceptable salt thereof, based on the total weight of the composition. In certain embodiments, the pharmaceutical composition contains 0.1-99.9% of the compound of the aforementioned formula I or a pharmaceutically acceptable salt thereof. In certain embodiments, the pharmaceutical composition contains 0.5%-99.5% of the compound of the aforementioned formula I or a pharmaceutically acceptable salt thereof. In certain embodiments, the pharmaceutical composition contains 1%-99% of the compound of the aforementioned formula I or a pharmaceutically acceptable salt thereof. In certain embodiments, the pharmaceutical composition contains 2%-98% of the compound of the aforementioned formula I or a pharmaceutically acceptable salt thereof.

[0036] In certain embodiments, the pharmaceutical composition comprises 0.01% to 99.99% of a pharmaceutically acceptable excipient, based on the total weight of the composition. In certain embodiments, the pharmaceutical composition comprises 0.1% to 99.9% of a pharmaceutically acceptable excipient. In certain embodiments, the pharmaceutical composition comprises 0.5% to 99.5% of a pharmaceutically acceptable excipient. In certain embodiments, the pharmaceutical composition comprises 1% to 99% of a pharmaceutically acceptable excipient. In certain embodiments, the pharmaceutical composition comprises 2% to 98% of a pharmaceutically acceptable excipient.

[0037] The present disclosure also provides a method for preventing and / or treating cardiovascular and cerebrovascular diseases, which comprises administering to the patient a therapeutically effective amount of a compound of the aforementioned formula I or a pharmaceutically acceptable salt thereof. In some embodiments, the disease is selected from thromboembolic diseases. In some embodiments, the disease is selected from myocardial infarction, angina pectoris, reocclusion and restenosis after angioplasty or aortocoronary bypass surgery, disseminated intravascular coagulation, stroke, transient ischemic attack, peripheral arterial occlusive disease, pulmonary embolism, or deep vein thrombosis.

[0038] The present disclosure also provides the use of a compound as shown in the aforementioned formula I or a pharmaceutically acceptable salt thereof or the aforementioned pharmaceutical composition in the preparation of a medicament for preventing and / or treating cardiovascular and cerebrovascular diseases. In some embodiments, the disease is selected from thromboembolic diseases. In some embodiments, the disease is selected from myocardial infarction, angina pectoris, reocclusion and restenosis after angioplasty or aortocoronary bypass surgery, disseminated intravascular coagulation, stroke, transient ischemic attack, peripheral arterial occlusive disease, pulmonary embolism or deep vein thrombosis.

[0039] The present disclosure also provides a compound of the aforementioned formula I or a pharmaceutically acceptable salt thereof for use in preventing and / or treating cardiovascular and cerebrovascular diseases. In some embodiments, the disease is selected from thromboembolic diseases. In some embodiments, the disease is selected from myocardial infarction, angina pectoris, reocclusion and restenosis after angioplasty or aortocoronary bypass surgery, disseminated intravascular coagulation, stroke, transient ischemic attack, peripheral arterial occlusive disease, pulmonary embolism, or deep vein thrombosis.

[0040] In another aspect, the pharmaceutically acceptable salts of the compounds described in the present disclosure are selected from inorganic salts or organic salts.

[0041] On the other hand, the compounds of the present disclosure may exist in specific geometric or stereoisomeric forms. The present disclosure contemplates all such compounds, including cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, and racemic mixtures and other mixtures thereof, such as enantiomerically or diastereomerically enriched mixtures, all of which are within the scope of the present disclosure. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All of these isomers and their mixtures are included within the scope of the present disclosure.

[0042] Optically active (R)- and (S)-isomers, as well as D and L isomers, can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If one enantiomer of a compound of the present disclosure is desired, it can be prepared by asymmetric synthesis or derivatization with a chiral auxiliary, wherein the resulting diastereomeric mixture is separated and the auxiliary group is cleaved to provide the pure desired enantiomer. Alternatively, when the molecule contains a basic functional group (such as an amino group) or an acidic functional group (such as a carboxyl group), a diastereomeric salt is formed with an appropriate optically active acid or base, and then the diastereoisomers are separated by conventional methods known in the art, and then the pure enantiomer is recovered. In addition, the separation of enantiomers and diastereomers is typically accomplished using chromatography, which employs a chiral stationary phase and is optionally combined with a chemical derivatization method (e.g., carbamate formation from an amine).

[0043] In the chemical structures of the compounds disclosed herein, the bond Indicates that the configuration is not specified, that is, if chiral isomers exist in the chemical structure, the bond Can be or include both Two configurations. In the chemical structure of the compound disclosed in the present invention, the bond The configuration is not specified, that is, it can be Z configuration or E configuration, or contain both configurations.

[0044] Compounds and intermediates of the present disclosure can also exist in different tautomeric forms, and all such forms are included in the scope of the present disclosure. The term "tautomer" or "tautomeric form" refers to structural isomers of different energies that can interconvert via a low energy barrier. For example, proton tautomers (also referred to as prototransfer tautomers) include interconversions via proton migration, such as keto-enol and imine-enamine, lactam-lactim isomerization. The lactam-lactim equilibrium example is between A and B as shown below.

[0045]

[0046] All compounds in this disclosure can be drawn as either Form A or Form B. All tautomeric forms are within the scope of this disclosure. The naming of the compounds does not exclude any tautomers.

[0047] The present disclosure also includes isotopically labeled compounds of the present disclosure that are identical to those described herein, but where one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into the compounds of the present disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, such as 2 H. 3 H.11 C. 13 C. 14 C. 13 N. 15 N. 15 O. 17 O. 18 O. 31 P. 32 P. 35 S. 18 F. 123 I. 125 I and 36 Cl et al.

[0048] Unless otherwise noted, when a position is specifically designated as deuterium (D), the position is understood to have an abundance of deuterium at least 3000 times greater than the natural abundance of deuterium, which is 0.015% (ie, at least 45% deuterium incorporation). In certain embodiments, the abundance of the compounds of the present disclosure for each designated deuterium atom is at least 3500 times (52.5% deuterium incorporation at each designated deuterium atom), at least 4000 times (60% deuterium incorporation), at least 4500 times (67.5% deuterium incorporation), at least 5000 times (75% deuterium incorporation), at least 5500 times (82.5% deuterium incorporation), at least 6000 times (90% deuterium incorporation), at least 6333.3 times (95% deuterium incorporation), at least 6466.7 times (97% deuterium incorporation), at least 6600 times (99% deuterium incorporation), or at least 6633.3 times (99.5% deuterium incorporation) greater than the natural abundance of deuterium. The present disclosure also includes various deuterated forms of compounds of formula (I). Each available hydrogen atom attached to a carbon atom can be independently replaced by a deuterium atom. Those skilled in the art will be able to synthesize deuterated forms of compounds of formula (I) with reference to relevant literature. Commercially available deuterated starting materials may be used to prepare deuterated compounds of formula (I), or they may be synthesized using conventional techniques using deuterated reagents, including but not limited to deuterated borane, trideuterated borane in tetrahydrofuran, deuterated lithium aluminum hydride, deuterated iodoethane, and deuterated iodomethane.

[0049] A "pharmaceutical composition" refers to a mixture containing one or more compounds described herein, or their physiologically acceptable salts or prodrugs, together with other chemical components, as well as other components such as physiologically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to an organism, facilitating absorption of the active ingredients and thereby exerting their biological activity.

[0050] "Pharmaceutically acceptable excipients" or "acceptable excipients" include, but are not limited to, any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye / colorant, flavoring agent, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent or emulsifier that has been approved by the U.S. Food and Drug Administration as acceptable for use by humans or domestic animals.

[0051] As used herein, an "effective amount" or "therapeutically effective amount" encompasses an amount sufficient to ameliorate or prevent the symptoms or conditions of a medical condition. An effective amount also refers to an amount sufficient to permit or facilitate diagnosis. The effective amount for a particular patient or veterinary subject may vary depending on factors such as the condition to be treated, the patient's overall health, the route and dosage of administration, and the severity of side effects. An effective amount can be the maximum dose or dosage regimen that avoids significant side effects or toxic effects. DETAILED DESCRIPTION

[0052] The present disclosure is further described below with reference to examples, but these examples are not intended to limit the scope of the present disclosure.

[0053] Experimental methods in the examples disclosed herein that do not specify specific conditions are generally performed under conventional conditions or according to the conditions recommended by the raw material or product manufacturers. Reagents that do not specify their specific sources are conventional reagents purchased from the market.

[0054] The structures of the compounds were determined by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS). -6 The unit of ppm is given. NMR measurements were performed using a Bruker AVANCE-400 NMR spectrometer. The solvents used were deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3), and deuterated methanol (CD3OD), and tetramethylsilane (TMS) was used as the internal standard.

[0055] MS was measured using an Agilent 1200 / 1290 DAD-6110 / 6120 Quadrupole MS liquid spectrometer-mass spectrometer (manufacturer: Agilent, MS model: 6110 / 6120 Quadrupole MS).

[0056] Waters ACQuity UPLC-QD / SQD (Manufacturer: Waters, MS Model: Waters ACQuity QdaDetector / Waters SQ Detector) THERMO Ultimate 3000-Q Exactive (Manufacturer: THERMO, MS Model: THERMO Q Exactive)

[0057] High performance liquid chromatography (HPLC) analysis was performed using Agilent HPLC 1200DAD, Agilent HPLC 1200VWD and Waters HPLC e2695-2489 high pressure liquid chromatographs.

[0058] Chiral HPLC analysis was performed using an Agilent 1260DAD high performance liquid chromatograph.

[0059] High performance liquid chromatography (HPLC) was performed using Waters 2545-2767, Waters 2767-SQ Detecor2, Shimadzu LC-20AP, and Gilson GX-281 preparative chromatographs.

[0060] Chiral preparations were performed using a Shimadzu LC-20AP preparative chromatograph.

[0061] The CombiFlash rapid preparation instrument used was Combiflash Rf200 (TELEDYNE ISCO).

[0062] The thin layer chromatography silica gel plate uses Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plate. The specification of the silica gel plate used in thin layer chromatography (TLC) is 0.15mm~0.2mm, and the specification used for thin layer chromatography separation and purification products is 0.4mm~0.5mm.

[0063] Silica gel column chromatography generally uses Yantai Huanghai Silica Gel 200-300 mesh silica gel as the carrier.

[0064] The known starting materials disclosed herein can be synthesized by methods known in the art, or can be purchased from ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, AccelaChemBio Inc, Darui Chemicals, and other companies.

[0065] Unless otherwise specified in the examples, all reactions can be carried out under an argon atmosphere or a nitrogen atmosphere.

[0066] Argon atmosphere or nitrogen atmosphere means that the reaction bottle is connected to an argon or nitrogen balloon with a capacity of about 1 L.

[0067] Hydrogen atmosphere means that the reaction bottle is connected to a hydrogen balloon with a capacity of about 1L.

[0068] The pressurized hydrogenation reaction uses a Parr 3916EKX hydrogenator and a Qinglan QL-500 hydrogen generator or an HC2-SS hydrogenator.

[0069] The hydrogenation reaction is usually carried out by evacuating the chamber and filling it with hydrogen, and the operation is repeated three times.

[0070] A CEM Discover-S 908860 microwave reactor was used for the microwave reaction.

[0071] Unless otherwise specified in the examples, the solution refers to an aqueous solution.

[0072] Unless otherwise specified in the examples, the reaction temperature is room temperature, 20°C to 30°C.

[0073] The reaction progress in the examples was monitored by thin layer chromatography (TLC). The developing solvent used in the reaction, the column chromatography eluent system used to purify the compound, and the developing solvent system for thin layer chromatography, the volume ratio of the solvent were adjusted according to the polarity of the compound, and a small amount of alkaline or acidic reagents such as triethylamine and acetic acid could be added for adjustment.

[0074] Example 1: SMTP-7 preparation

[0075]

[0076] Stachybotrys microspora IFO30018 was inoculated into a seed culture medium (4% glucose, 0.5% soybean pulp, 0.3% dried broth, 0.3% yeast extract, 0.01% defoamer, pH 5.8) and cultured for 4 days. The seed culture was then inoculated into a fermentation medium (5% sucrose, 0.1% yeast extract, 0.3% NaNO3, 0.1% K2HPO4, 0.05% MgSO4·7H2O, 0.05% KCl, 0.00025% CoCl2·6H2O, 0.0015% FeSO4·7H2O, 0.00065% CaCl2·2H2O, 0.01% defoamer, pH 5.8). After 4 days of culture, L-ornithine was added and cultured for another day to terminate the fermentation.

[0077] The fermentation broth was extracted with methanol. The extract was concentrated by rotary evaporation and then extracted with ethyl acetate. After dehydration with anhydrous sodium sulfate, the extract was filtered, concentrated, and dried to solidify.

[0078] The solidified material is dissolved in methanol, pretreated and prepared using reverse phase filler, and then extracted with ethyl acetate to obtain the target product.

[0079] Example 2: Preparation of δ-deuterated L-ornithine (Compound 1e)

[0080]

[0081] (S)-2-(bis(tert-butoxycarbonyl)amino)-4-cyanobutyric acid benzyl ester (Compound 1b)

[0082] Compound 1a (prepared according to Synlett, 2016, vol. 27, 2, 309–312, 74.8 g, 234.9 mmol) was dissolved in acetonitrile (750 mL), and Boc2O (76.9 g, 352.4 mmol) and DMAP (2.9 g, 23.5 mmol) were added. The mixture was reacted at 45°C for 1–1.5 hours. The solvent was removed by rotary evaporation, and the crude product was purified by column chromatography to obtain 95 g of the target compound 2b (purity 99.4%, yield 100%). MS (ESI) m / z 441.2 [M+Na] + .

[0083] Compounds 1c and 1d

[0084] Compound 1b (4.18 g, 10 mmol) was dissolved in EA (100 mL) (ultra-dry) and D2O (20 mL), PtO2 (204 mg) was added to replace D2, and the reaction was carried out at 30°C (external temperature) under the protection of D2 for 40 h. The reaction was basically completed. The liquids were separated, and the aqueous phase was washed with EA and directly freeze-dried to obtain 1.87 g of a solid. The solid was dissolved in acetonitrile (5 ml) and ethyl acetate (20 ml), stirred at room temperature, filtered, and the filter cake was washed with ethyl acetate and dried with an oil pump to obtain 1.54 g of a mixture of compounds 1c and 1d (yield 46%).

[0085] δ-deuterated L-ornithine (Compound 1e)

[0086] A mixture of compounds 1c and 1d (195 mg, 0.58 mmol) was dissolved in 6 M aqueous hydrochloric acid solution and reacted at room temperature for 2 h. The reaction was almost complete, and the solvent was removed by rotary evaporation. The mixture was dried by oil pump to constant weight to obtain 120 mg of light yellow solid product compound 1e (yield 100%, purity 97.25%).

[0087] HNMR (D2O, 400M): 1.95~1.94(m,4H), 2.91~2.98(m,0.027H), 3.96(t,J=6.4Hz,1H).

[0088] Example 3: Preparation of δ-deuterated SMTP-7 (Compound 1)

[0089]

[0090] Compound 1 was prepared by using δ-deuterated L-ornithine (prepared according to Example 2) according to the method of Example 1.

[0091] Stachybotrys microspora IFO30018 was inoculated into a seed culture medium (4% glucose, 0.5% soy bean pulp, 0.3% dried broth, 0.3% yeast extract, 0.01% defoamer, pH 5.8) and cultured for 4 days. The seed culture was then inoculated into a fermentation medium (5% sucrose, 0.1% yeast extract, 0.3% NaNO3, 0.1% K2HPO4, 0.05% MgSO4·7H2O, 0.05% KCl, 0.00025% CoCl2·6H2O, 0.0015% FeSO4·7H2O, 0.00065% CaCl2·2H2O, 0.01% defoamer, pH 5.8). After 4 days of culture, δ-deuterated L-ornithine was added and cultured for another day to terminate the fermentation.

[0092] The fermentation broth was extracted with methanol. The extract was concentrated by rotary evaporation and then extracted with ethyl acetate. After dehydration with anhydrous sodium sulfate, the extract was filtered, concentrated, and dried to solidify.

[0093] The solidified material was dissolved in methanol, pretreated and prepared using reverse phase filler, and then extracted with ethyl acetate to obtain the target product.

[0094] 1 H NMR(400MHz,DMSO-d6)δ13.07–12.66(br.,1H),9.79(s,1H),9.73(s,1H),6.66(s,1H),6.62 (s,1H),5.28–5.09(m,3H),5.07–4.95(m,2H),4.72(dd,J=9.9,5.7Hz,1H),4.26–4.05(m,4H ),3.73(dd,J=13.0,6.9Hz,2H),2.82(dt,J=17.0,4.8Hz,2H),2.48–2.38(m,2H),2.17–2.05 (m,4H),2.04–1.95(m,4H),1.95–1.81(m,6H),1.66–1.46(m,23H),1.18(s,3H),1.15(s,3H).

[0095] Test Example 1: Pharmacokinetic Study in Rats

[0096] 1.1 Preparation of test samples

[0097] Appropriate amounts of SMTP-7 and compound 1 were weighed separately, and 2% DMSO and 98% saline were added in sequence. After thorough mixing by vortexing and sonication, a clear solution with a concentration of 1 mg / mL was obtained for later use.

[0098] 1.2 Animals

[0099] SD rats, aged 6-8 weeks, weighing approximately 180-220 g

[0100] 1.3 Plan

[0101]

[0102] 1.4 Sample collection

[0103] Blood samples were collected via the jugular vein or other suitable method, approximately 0.20 mL per sample, anticoagulated with sodium heparin, and immediately placed on ice. Blood samples were collected before administration and at 5 minutes, 0.25 hours, 0.5 hours, 1 hour, 2 hours, 4 hours, 6 hours, 10 hours, and 24 hours after administration, for a total of 10 time points. The collected blood samples were placed in heparinized blood collection tubes and centrifuged to separate the plasma (6800 g, centrifugation for 6 minutes, 2-8°C). Plasma samples were stored at -80°C until analysis.

[0104] 1.5 Bioanalysis and data processing

[0105] The blood concentration of each test substance is detected, and quality control samples are analyzed at the same time as the samples are analyzed. It is required that the accuracy of more than 66.7% of the quality control samples is between 80-120%.

[0106] When plotting the plasma drug concentration-time curve, BLQ is recorded as 0. When calculating pharmacokinetic parameters, the concentration before administration is calculated as 0; the BLQ before Cmax (including "No peak") is calculated as 0; C max The BLQ (including "No peak") that appears later is not included in the calculation. The following pharmacokinetic parameters are calculated using the non-compartmental statistical moment method of Phoenix WinNonlin7.0 software based on the blood drug concentration data at different time points: AUC (0-t) , AUC (0-∞) 、T 1 / 2 , MRT, C max 、T max and other parameters.

[0107] Experimental results:

[0108] Group <![CDATA[T 1 / 2 (h)]]> <![CDATA[T max (h)]]> <![CDATA[C max (ng / ml)]]> <![CDATA[AUC (0-t) (h*ng / ml)]]> <![CDATA[MRT (0-t) ]]> 1 6.689±0.178 0.08±0.00 26,311.98±6,236.41 8,009.467±1,832.168 0.465±0.080 2 8.94±0.61 0.08±0.00 19,078.40±4,312.75 11,287.80±1,377.70 3.06±0.25

[0109] Conclusion: Compared with SMTP-7, compound 1 showed a longer half-life and lower C max .

[0110] Example 4: Preparation of α-deuterated L-ornithine

[0111]

[0112] α-Deuterated 2-((tert-butyloxycarbonyl)amino)-4-cyanobutanoic acid (Compound 2c)

[0113] Under nitrogen protection, compound 1b (prepared according to Example 1, 25.0 g, 59.8 mmol) was added to 125 mL of MeOD and stirred to dissolve. Anhydrous potassium carbonate (41.3 g, 299.0 mmol) was added and reacted at 20-30°C for 16 h. The reaction was almost complete and the system was directly concentrated to obtain crude compound 2c. MS (ESI) m / z 252.1 [M+Na] + .

[0114] α-Deuterated 2-((tert-Butoxycarbonyl)amino)-4-cyanobutyric acid benzyl ester (Compound 2d)

[0115] To the crude compound 2c (25.0 g, 59.8 mmol) was added 150 mL of anhydrous acetonitrile and BnBr (15.3 g, 89.7 mmol) under nitrogen protection. The reaction was carried out at 20-30°C for 6 h. The reaction was essentially complete. The mixture was filtered, the filter cake was washed with acetonitrile, and the filtrate was concentrated. The crude product was purified by column chromatography to give 6.8 g of the target compound 2d, which was detected by chiral HPLC as a racemate (two-step yield 35.7%). MS (ESI) m / z 343.1 [M+Na]+.

[0116] α-Deuterated (S)-2-((tert-Butoxycarbonyl)amino)-4-cyanobutyric acid benzyl ester (Compound 2e)

[0117] The racemic product of compound 2d (6.8 g) prepared above was subjected to chiral resolution to obtain compound 2e (3.6 g).

[0118] Instrument: MGⅡ preparative SFC (SFC-14), chiral column: ChiralPak AY, 250×30 mm ID, 5 μm, mobile phase A: carbon dioxide, mobile phase B: methanol (0.1% ammonia), gradient 15%, flow rate: 60 ml / min, back pressure: 100 bar, column temperature: 38°C, detection wavelength: 220 nm, separation time: approximately 6 min.

[0119] HNMR(CDCl3,400M): 1.44(s,9H),1.58~1.65(m,1H),1.97~2.04(m,1H),2. 23~2.28(m,1H),,2.38~2.46(m,1H),5.17~5.20(m,2H),7.36~7.38(m,5H).

[0120] α-Deuterated (S)-5-amino-2-((tert-butoxycarbonyl)amino)pentanoic acid (Compound 2f)

[0121] Compound 2e (3.5 g, 10.9 mmol) was weighed and dissolved in 70 mL of ethyl acetate. Purified water (700 mL) was added, followed by PtO (224 mg, 0.9 mmol). The hydrogen atmosphere was replaced and the reaction was allowed to proceed at 20-30°C for 16 h. The reaction was essentially complete. The filtrate was filtered, the filtrate was separated, and the aqueous phase was collected, washed with water, and lyophilized to obtain approximately 1.6 g of crude target compound 2f. 20 mL of ethyl acetate and 2 mL of acetonitrile were added, the mixture was slurried, filtered, and the solid was drained to obtain approximately 1.5 g of the pure product (yield 58.6%). MS-ESI: m / z 236.1 [M+H]+.

[0122] HNMR(D2O,400M):1.44(s,9H),1.58~1.80(m,4H),2.93~2.98(m,2H).

[0123] α-deuterated L-ornithine (Compound 2g)

[0124] Compound 2f (1.5 g, 6.4 mmol) was weighed and dissolved in approximately 10 mL of 6M HCl with stirring. The mixture was allowed to react at 20-30°C for 2 h. The reaction was nearly complete, and the reaction solution was lyophilized to obtain 1.2 g of crude compound 2f. 20 mL of acetonitrile was added to the mixture for slurrying, and the mixture was filtered to obtain 1.1 g of pure solid (yield 83.9%). MS-ESI: m / z 134.1 [M+H]+.

[0125] HNMR(D2O,400M):1.67~2.01(m,4H),2.98~3.01(m,2H).

[0126] Example 5: Preparation of α-deuterated SMTP-7 (Compound 2)

[0127]

[0128] The target product, Compound 2, was prepared by using α-deuterated L-ornithine (prepared according to Example 4) according to the method of Example 1.

[0129] Example 6: Preparation of β-deuterated L-ornithine (Compound 3e)

[0130] β-deuterated L-ornithine (Compound 3e)

[0131]

[0132] Compound 3b

[0133] Compound 3a (3.8 g, 15.4 mmol, prepared according to Journal of the American Chemical Society, 2017, vol. 139, 39, 13830-13836) and anhydrous potassium carbonate (10.6 g, 77 mmol) were dissolved in 60 mL of anhydrous acetonitrile; BnBr (5.3 g, 30.8 mmol) was added, and the mixture was reacted at 10-20° C. for 16 hours; the mixture was filtered to remove insoluble matter, the organic phase was washed with EA, and the filtrate was concentrated to obtain a crude product. Column chromatography (PE:EA=10:1) gave 4.8 g of the target compound 3b (purity 93.3%, yield 97%).

[0134] MS-ESI: m / z 343.1[M+Na] +

[0135] 1 HNMR(CDCl3,400M):1.45(s,9H),4.36-4.40(m,1H),5.17-5.24(s,2H),7.27-7.40(m,5H).

[0136] Compound 3c

[0137] Compound 3b was a racemate, and after chiral preparation, 2.2 g of the target compound 3c was obtained as a configuration monomer.

[0138] Compound 3d

[0139] Compound 3c (2.2 g, 6.9 mmol) was dissolved in EA and water, and PtO2 (0.3 g, 1.3 mmol) was added. The system was evacuated and replaced with hydrogen three times. The reaction was carried out at 10-20°C for 16 hours, filtered, and about 50 mL of EA was added to the aqueous phase for separation. The aqueous phase was collected, concentrated under reduced pressure, and pumped dry to obtain a crude product. 20 mL of ethyl acetate and 2 mL of acetonitrile were added to the slurry at 10-20°C, filtered, washed with EA, and the solid was collected and dried to obtain 1.2 g of the target compound 3d.

[0140] MS-ESI: m / z 235.1[M+H] +

[0141] Compound 3e

[0142] Compound 3d (1.2 g, 5.1 mmol) was dissolved in 12 mL of 6 M aqueous hydrochloric acid, reacted at 10-20°C for 4 hours, and then concentrated under reduced pressure using an oil pump to obtain 1.05 g of the target compound 3e (yield 100%).

[0143] MS-ESI: m / z 135.1[M+H] +

[0144] 1 HNMR(CDCl3,400M):1.67-1.81(m,2H),1.91-1.93(m,0.1H),2.95-2.32(m,2H),3.98(s,1H).

[0145] Example 7: Preparation of β-deuterated SMTP-7 (Compound 3)

[0146]

[0147] The target product, Compound 3, was prepared by using β-deuterated L-ornithine (prepared according to Example 6) according to the method of Example 1.

[0148] Example 8: Preparation of γ-deuterated L-ornithine (Compound 4c)

[0149]

[0150] Compound 4b

[0151] Compound 4a (2.2 g, 6.4 mmol, prepared according to Journal of the American Chemical Society, 2018, vol. 140, 23, 7116–7126) was dissolved in tetrahydrofuran (50 mL), and Boc2NH (2.1 g, 9.6 mmol) and triphenylphosphine (5.0 g, 19.1 mmol) were added. The mixture was cooled to 0 ° C., and DEAD (3.3 g, 19.1 mmol) was slowly added dropwise. After completion, the mixture was returned to room temperature and stirred overnight. The reaction was quenched by adding phosphonic acid buffer, extracted with ethyl acetate, and the organic phases were combined, washed with saturated brine, and the organic phase was concentrated and purified by column chromatography (EA: PE = 1: 20) to obtain 1.7 g of the target product, compound 4b (yield 50%).

[0152] 1 H NMR(500MHz, CDCl3)δ4.87(dd,J=9.5,5.0Hz,1H),3.70(s,3H),3.59(d,J=3.1H z,2H),2.09(dd,J=14.2,4.7Hz,1H),1.88–1.81(m,1H),1.49(d,J=3.3Hz,36H).

[0153] Compound 4c

[0154] Compound 4b (1.7 g, 3.2 mmol) and 4M aqueous hydrochloric acid (30 mL) were added to a 100 mL sealed tube and heated to 90°C (external temperature) for 16 hours. The mixture was cooled to room temperature, concentrated, and dried by oil pump to obtain 500 mg of compound 4c (yield 92%).

[0155] MS-ESI: m / z 135.1[M+H] +

[0156] 1 H NMR (500MHz, D2O) δ3.99 (t, J=6.3Hz, 1H), 3.03 (s, 2H), 1.97 (qd, J=14.5, 6.5Hz, 2H).

[0157] Example 9: Preparation of γ-deuterated SMTP-7 (Compound 4)

[0158]

[0159] The target product, Compound 4, was prepared by using γ-deuterated L-ornithine (prepared according to Example 8) according to the method of Example 1.

[0160] Test Example 2: Pharmacokinetic Study in Rats

[0161] 1.1 Preparation of test samples

[0162] Appropriate amounts of SMTP-7, compound 2, compound 3, and compound 4 were weighed separately, and physiological saline (pH adjusted to about 9.2 with 2 mM NaOH solution) was added. After thorough mixing by vortexing and sonication, a clear solution with a concentration of 1 mg / mL was obtained for later use.

[0163] 1.2 Animals

[0164] SD rats, aged 6-8 weeks, weighing approximately 180-240 g

[0165] 1.3 Plan

[0166]

[0167] Detect the pharmacokinetic AUC of rats according to the method of Test Example 1 (0-t) , AUC (0-∞) 、T 1 / 2 , MRT, C max 、T max and other parameters.

[0168] Experimental results: After intravenous injection in rats, the T 1 / 2 There is little difference between the T and AUC of compound 4. 1 / 2 and AUC is lower than SMTP-7.

[0169] Test Example 3: Thromboembolism in rats

[0170] 1.1 Preparation of cerebral infarction model

[0171] An embolic stroke model was prepared according to the literature (J Cereb Blood Flow Metab, 1997, 17(2):123-135). 0.1 mL of rat blood was immediately drawn into a PE50 tube. After standing at room temperature for 2 hours, it was stored at 4°C for 22 hours. The thrombus was pushed out into 30 mL of normal saline and washed three times, each for 5 minutes. A 5 mm embolus was cut and drawn into a specially prepared PE50 tube at the end of the tube for later use.

[0172] Rats were anesthetized with isoflurane and placed in the supine position on the operating table. A midline skin incision was made, and the right common carotid artery was freed. The internal carotid artery branch was isolated and occluded with an artery clamp. A small incision was made in the common carotid artery, and the catheter-embedded embolus was injected into the skull with 0.4 mL of saline. The cannula was then carefully removed, the common carotid artery ligated, and the skin sutured.

[0173] 2.2 Grouping and Dosing

[0174] One hour after the modeling, neurological function scores were performed, and those with a score of ≥8 were considered successful models. The rats were then divided into a sham operation group, a model control group, a test drug group (5, 10, 20 mg / kg), and a control drug group (10 mg / kg), with 10 rats in each group. The sham operation group and the model control group were given normal saline, the test drug group was given compound 1 (1 mg / ml, prepared in normal saline (2 mM NaOH solution adjusted to pH 9.2)), and the control drug group was given SMTP-7 (1 mg / ml, prepared in normal saline (2 mM NaOH solution adjusted to pH 9.2)). Immediately one hour after the modeling, the drug was administered intravenously, with 10% of the drug volume initially injected, and the remaining 90% infused over 30 minutes. The endpoint of the experiment was 24 hours after the drug was administered.

[0175] Neurological function score

[0176] The degree of behavioral disturbance in the animals was observed and scored before administration and 24 hours after treatment. The scoring criteria are as follows:

[0177] Scoring criteria for neurological impairment in MCAO rats

[0178]

[0179]

[0180] Cerebral hemorrhage determination

[0181] After 24 hours of scoring and blood collection, the brain was harvested by cardiac perfusion and frozen at -20°C. The brain tissue was then sliced ​​from front to back, with each slice being 2 mm thick. Any bleeding in the slice was scored as 1 point, and the sum of the scores for each slice was the total bleeding score for each animal.

[0182] Determination of cerebral infarction area

[0183] After 24 hours of scoring and blood collection, the brain was harvested by cardiac perfusion and frozen at -20°C. The brain tissue was then sliced ​​from front to back, each slice 2 mm thick. The brain tissue sections were incubated in 2% tetrazolium tetrazolium (TTC) solution at 37°C for 5 minutes. Infarcted tissue appears white, while non-infarcted tissue appears red. Image J software was used to measure infarct area, and the percentage of infarcted area to total brain area was calculated.

[0184] Percentage of cerebral infarction area (%) = cerebral infarction area / total brain area × 100%

[0185] Data analysis methods

[0186] Measuring data The results showed that the difference was statistically significant when P < 0.05, using T-TEST for pairwise comparison.

[0187] Experimental results:

[0188] Table 1: Behavioral scores and cerebral infarction area

[0189]

[0190] Note: # indicates relative to the sham operation group, * indicates relative to the model group.

[0191] Table 2: Cerebral hemorrhage scores

[0192]

[0193]

[0194] In terms of behavior, at the same dosage (10 mg / kg), compound 1 and SMTP-7 showed improvement compared with the model group and the degree of improvement was comparable.

[0195] In terms of cerebral infarction area, compared with the model group, both compound 1 and SMTP-7 showed significant improvement, and compound 1 was superior to SMTP-7 at the same dose of 10 mg / kg (reduced by about 37%), and the probability of severe infarction was lower.

[0196] In terms of cerebral hemorrhage, compared with the model group, all dose groups of compound 1 and SMTP-7 showed improvement. At a dose of 10 mg / kg, the probability of cerebral hemorrhage after administration of compound 1 (10%) was lower than that of SMTP-7 (20%). In addition, SMTP-7 had a cerebral hemorrhage score ≥3 (10%), indicating that there is a higher risk of hemorrhagic transformation in the brain after administration of SMTP-7, which is not conducive to the clinical treatment of ischemic stroke.

[0197] Conclusion: Compound 1 can improve neurological function and cerebral infarction area after cerebral infarction, and has a lower risk of bleeding.

Claims

1. The following compound or a pharmaceutically acceptable salt thereof, 2. The compound or salt thereof according to claim 1, wherein the abundance of deuterium atoms is at least 4000 times the natural abundance of deuterium.

3. The compound or salt thereof according to claim 1, wherein the abundance of deuterium atoms is at least 5500 times the natural abundance of deuterium.

4. The compound or salt thereof according to claim 1, wherein the abundance of deuterium atoms is at least 6000 times the natural abundance of deuterium.

5. A pharmaceutical composition comprising a therapeutically effective amount of at least one compound according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.

6. Use of the compound according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 5, in the preparation of a medicament for preventing and / or treating cardiovascular and cerebrovascular diseases.

7. The use according to claim 6, wherein the disease is selected from thromboembolic diseases. The use according to claim 6 , wherein the disease is cerebral infarction.

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