SMTP-7 derivative and application thereof

Abstract

The invention relates to an SMTP-7 derivative and application thereof. Specifically, provided is a compound represented by formula I or a pharmaceutically acceptable salt thereof, in which each group is as defined in the specification.

Description

[0001] This application is a divisional application of the Chinese Patent Application No. 202280008550.0, filed on February 10, 2022, with the title of “SMTP-7 Derivative and Use Thereof”. TECHNICAL FIELD

[0002] The present disclosure belongs to the field of medicine, and relates to a SMTP-7 derivative and use thereof. BACKGROUND

[0003] SMTP-7 (TMS-007, Stachybotrys microspore triprenyl phenol-7) was extracted from a special mold (Stachybotrys microspora) on the leaves of a special tree on the island of Xitang, Okinawa in 2000, and is a small molecule plasminogen activator with a structure similar to vitamin E. It has a novel mechanism of action in breaking down blood clots, and is also believed to inhibit local inflammation at the site of thrombus. In addition, SMTP-7 also has anti-tumor angiogenesis activity, antioxidant activity and activity in promoting tissue regeneration (WEIMIN H, SHIGEKIO, et al. J. Antibiot., 2000, 53(3): 241-247).

[0004]

[0005] Plasminogen is the precursor of plasmin, which can be activated to generate plasmin. It is a protease that can hydrolyze many proteins, including thrombus proteins. SMTP-7 binds to plasminogen and changes its molecular conformation, making it easier to be activated by plasminogen activators. Therefore, SMTP-7 itself has no function of activating plasminogen, but only makes the activation process easier. This unique combination of SMTP-7 makes SMTP-7 a promising best-in-class thrombolytic drug for treating acute ischemic stroke (AIS), and has the potential to extend the treatment window compared to existing standard thrombolytic drugs (although many antihypertensive, lipid-lowering and anticoagulant drugs can prevent stroke, the only therapeutic drug for ischemic stroke is recombinant tissue plasminogen activator (rt-PA, alteplase), which is a major component of glycoprotein containing 526 amino acids).

[0006] SMTP molecules induce conformational changes in plasminogen, resulting in an increased rate of plasminogen binding to fibrin and ultimately activation to plasmin. In addition, SMTP induces autocleavage of plasmin to provide an angiogenic human angiostatin-like fragment. This activity is thought to be the mechanism of the anti-angiogenic and anti-tumor effects of SMTP molecules. Furthermore, the increase in activated plasmin induced by SMTP can control local extracellular proteolysis, leading to tissue remodeling, wound healing and tissue regeneration. SUMMARY

[0007] The disclosure provides a compound represented by Formula I or a pharmaceutically acceptable salt thereof,

[0008]

[0009] wherein 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 , R47 , 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 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 , R50 , 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 is deuterium.

[0010] In some embodiments, the compound of Formula I, or a pharmaceutically acceptable salt thereof, is wherein R 27 is selected from deuterium, R 28 is selected from deuterium, R 36 is selected from deuterium, R 37 is selected from deuterium.

[0011] In some embodiments, the compound of Formula I, or a pharmaceutically acceptable salt thereof, is wherein R 26 is selected from deuterium, R 38 is selected from deuterium.

[0012] In some embodiments, the compound of Formula I, or a pharmaceutically acceptable salt thereof, is wherein R 24 is selected from deuterium, R 25 is selected from deuterium, R 39 is selected from deuterium, R 40 is selected from deuterium.

[0013] In some embodiments, the compound of Formula I, or a pharmaceutically acceptable salt thereof, is wherein R 29 is selected from deuterium.

[0014] In some embodiments, the compound of Formula I, or a pharmaceutically acceptable salt thereof, is wherein R 30 is selected from deuterium, R 31 is selected from deuterium.

[0015] In some embodiments, the compound of Formula I, or a pharmaceutically acceptable salt thereof, is wherein R 34 is selected from deuterium, R 35 is selected from deuterium.

[0016] In some embodiments, the compound of Formula I, or a pharmaceutically acceptable salt thereof, is wherein R 16 is selected from deuterium, R 17 is selected from deuterium, R 47 is selected from deuterium, R 48 is selected from deuterium.

[0017] In some embodiments, the compound of Formula I, or a pharmaceutically acceptable salt thereof, is wherein R 12 is selected from deuterium, R 13 is selected from deuterium, R 14selected from deuterium, R

[0018] In some embodiments, the compound of Formula I, or a pharmaceutically acceptable salt thereof, is 50 selected from deuterium, R 51 selected from deuterium, R 52 selected from deuterium.

[0019] In some embodiments, the compound of Formula I, or a pharmaceutically acceptable salt thereof, is 23 selected from deuterium, R 41 selected from deuterium.

[0020] In some embodiments, the compound of Formula I, or a pharmaceutically acceptable salt thereof, is 32 selected from deuterium, R 33 selected from deuterium.

[0021] In some embodiments, the compound of Formula I, or a pharmaceutically acceptable salt thereof, is 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, the compound of Formula I, or a pharmaceutically acceptable salt thereof, is 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.

[0023] In some embodiments, the compound of Formula I, or a pharmaceutically acceptable salt thereof, is 1 selected from deuterium, R 2 selected from deuterium, R 3 selected from deuterium.

[0024] In some embodiments, the compound of Formula I, or a pharmaceutically acceptable salt thereof, is 4 selected from deuterium, R 5 selected from deuterium, R 6 selected from deuterium.

[0025] In some embodiments, the compound of Formula I, or a pharmaceutically acceptable salt thereof, is 58 selected from deuterium, R 59 selected from deuterium, R 60 selected from deuterium.

[0026] In some embodiments, the compound of Formula I, or a pharmaceutically acceptable salt thereof, is 61 selected from deuterium, R 62selected from deuterium, R 63 selected from deuterium.

[0027] In some embodiments, the compound of Formula I, or a pharmaceutically acceptable salt thereof, is 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 61 selected from deuterium, R 62 selected from deuterium, R 63 selected from deuterium.

[0028] In some embodiments, the compound of Formula I, or a pharmaceutically acceptable salt thereof, is 7 selected from deuterium, R 57 selected from deuterium.

[0029] In another aspect, some embodiments provide a compound of Formula I, or a pharmaceutically acceptable salt thereof, wherein

[0030]

[0031] Exemplary compounds of Formula I include, but are not limited to:

[0032]

[0033]

[0034] Also provided in the present disclosure is a pharmaceutical composition comprising at least one therapeutically effective amount of a compound of Formula I, or a pharmaceutically acceptable salt thereof, as described above, and a pharmaceutically acceptable excipient.

[0035] In some embodiments, the unit dose of the pharmaceutical composition is 0.001 mg to 1000 mg.

[0036] In certain embodiments, the pharmaceutical composition contains 0.01-99.99% of a compound of Formula I, or a pharmaceutically acceptable salt thereof, as described above, based on the total weight of the composition. In certain embodiments, the pharmaceutical composition contains 0.1-99.9% of a compound of Formula I, or a pharmaceutically acceptable salt thereof, as described above. In certain embodiments, the pharmaceutical composition contains 0.5%-99.5% of a compound of Formula I, or a pharmaceutically acceptable salt thereof, as described above. In certain embodiments, the pharmaceutical composition contains 1%-99% of a compound of Formula I, or a pharmaceutically acceptable salt thereof, as described above. In certain embodiments, the pharmaceutical composition contains 2%-98% of a compound of Formula I, or a pharmaceutically acceptable salt thereof, as described above.

[0037] In certain embodiments, the pharmaceutical composition contains 0.01-99.99% of the pharmaceutically acceptable excipient, based on the total weight of the composition. In certain embodiments, the pharmaceutical composition contains 0.1-99.9% of the pharmaceutically acceptable excipient. In certain embodiments, the pharmaceutical composition contains 0.5-99.5% of the pharmaceutically acceptable excipient. In certain embodiments, the pharmaceutical composition contains 1-99% of the pharmaceutically acceptable excipient. In certain embodiments, the pharmaceutical composition contains 2-98% of the pharmaceutically acceptable excipient.

[0038] The present disclosure also provides a method for preventing and / or treating a cardiovascular and cerebrovascular disease by administering to the patient a therapeutically effective amount of the compound of the aforementioned Formula I or a pharmaceutically acceptable salt thereof. In some embodiments, the disease is selected from a thromboembolic disease. In some embodiments, the disease is selected from myocardial infarction, angina pectoris, restenosis and reocclusion after angioplasty or aortocoronary bypass, disseminated intravascular coagulation, stroke, transient ischemic attack, peripheral arterial occlusive disease, pulmonary embolism, or deep vein thrombosis.

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

[0040] The present disclosure also provides the compound of the aforementioned Formula I or a pharmaceutically acceptable salt thereof for use in preventing and / or treating a cardiovascular and cerebrovascular disease. In some embodiments, the disease is selected from a thromboembolic disease. In some embodiments, the disease is selected from myocardial infarction, angina pectoris, restenosis and reocclusion after angioplasty or aortocoronary bypass, disseminated intravascular coagulation, stroke, transient ischemic attack, peripheral arterial occlusive disease, pulmonary embolism, or deep vein thrombosis.

[0041] In another aspect, the pharmaceutically acceptable salt of the compound described in the present disclosure is selected from an inorganic salt or an organic salt.

[0042] In another aspect, the compounds of the present disclosure can exist in particular geometric or stereoisomeric forms. The present disclosure contemplates all such compounds, including cis- and trans-forms, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)- isomers, (L)-isomers, as well as the racemic mixtures and other mixtures thereof, such as those that are enantiomeric or diastereomeric mixtures. Additional asymmetric carbon atoms can be present in a substituent group. All such isomers, as well as mixtures thereof, are included within the scope of the present disclosure.

[0043] Optically active (R)- and (S)-isomers, as well as D and L isomers, can be prepared by chiral synthesis or by chiral reagents or other conventional techniques. If desired, one enantiomer of a compound of the present disclosure can be obtained by asymmetric synthesis or derivatization with a chiral auxiliary, separation of the resulting diastereomeric mixture, and cleavage of the auxiliary to provide the pure desired enantiomer. Alternatively, when a basic functional group (such as an amino group) or an acidic functional group (such as a carboxyl group) is present in the molecule, diastereomeric salts can be formed with an appropriate optically active acid or base, and the diastereomeric salt separated by conventional means, and the pure enantiomer recovered by treating with base or acid, as appropriate. Additionally, separation of the enantiomers and diastereomers is typically accomplished by chromatography using a chiral stationary phase, optionally in combination with chemical derivatization (e.g., formation of carbamates from amines).

[0044] In the chemical structures of the compounds of the present disclosure, the bond is not specified as to configuration, i.e., if chiral isomers are present in the chemical structure, the bond may be or or both configurations. In the chemical structures of the compounds of the present disclosure, the bond is not specified as to configuration, i.e., it can be in the Z configuration or the E configuration, or both configurations. The compounds and intermediates of the present disclosure can also exist in different tautomeric forms, and all such forms are embraced within the scope of the present disclosure. The term "tautomers" or "tautomeric forms" refers to different energy structures that are interconvertible by low energy barriers. For example, prototropic tautomers (also known as proton transfer tautomers) include interconversions via proton migration, such as keto-enol and imine-enamine, lactam-lactim isomerization. An example of a lactam-lactim equilibrium is between A and B as shown below.

[0045]

[0046] ​​

[0047] All compounds in the present disclosure can be drawn as Form A or Form B. All tautomeric forms are within the scope of the present disclosure. The naming of the compounds does not exclude any tautomer.

[0048] The present disclosure also includes certain isotopically-labeled compounds of the present disclosure which are identical to those recited herein, but for the fact that 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 found in the compounds of the present disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, 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, and the like.

[0049] Unless otherwise stated, when a position is designated specifically as deuterium (D), the position is understood to have deuterium in an abundance of at least 3000 times greater than the natural abundance of deuterium, which is 0.015% (i.e., at least 45% deuterium incorporation). In certain embodiments, the compounds of the present disclosure have an abundance of each designated deuterium atom that is at least 3500 times greater than the natural abundance of deuterium (52.5% deuterium incorporation at each designated deuterium atom), at least 4000 times greater (60% deuterium incorporation), at least 4500 times greater (67.5% deuterium incorporation), at least 5000 times greater (75% deuterium incorporation), at least 5500 times greater (82.5% deuterium incorporation), at least 6000 times greater (90% deuterium incorporation), at least 6333.3 times greater (95% deuterium incorporation), at least 6466.7 times greater (97% deuterium incorporation), at least 6600 times greater (99% deuterium incorporation), or at least 6633.3 times greater (99.5% deuterium incorporation). The present disclosure also includes various deuterated forms of the compounds of Formula (I). Each available hydrogen atom attached to a carbon atom can be independently replaced with a deuterium atom. One skilled in the art would be able to synthesize deuterated forms of the compounds of Formula (I) with reference to the relevant literature. Commercially available deuterated starting materials can be used in the preparation of deuterated forms of the compounds of Formula (I), or they can be synthesized using conventional techniques employing deuterated reagents, including but not limited to deuterated borane, trideuteroborane tetrahydrofuran solution, deuterated lithium aluminum hydride, deuterated ethyl iodide, and deuterated methyl iodide, among others.

[0050] "Pharmaceutical composition" means a mixture of one or more of the compounds described herein, or physiologically acceptable salts or prodrugs thereof, with other chemical components, such as physiologically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of the active ingredient to a subject, and to facilitate absorption into the subject's system to thereby yield the biological activity of the active ingredient.

[0051] "Pharmaceutically acceptable excipient" or "acceptable excipient" includes any of the following: any auxiliary, carrier, excipient, glidant, sweetening, diluting, preservative, dye / colorant, flavoring, surfactant, wetting, dispersing, suspending, stabilizing, isotonic, solvent, or emulsor agent that has been approved by the U.S. Food and Drug Administration as being acceptable for use in humans or domestic animals.

[0052] An "effective amount" or "effective therapeutic amount" described in the present disclosure includes an amount sufficient to ameliorate or prevent symptoms or conditions of a medical disorder. An effective amount also means an amount sufficient to allow or facilitate diagnosis. An effective amount for a particular patient or veterinary subject can vary depending on factors such as the condition to be treated, the overall health status of the patient, the method route and dosage of administration, and the severity of side effects. An effective amount can be the maximum dose or administration regimen that avoids significant side effects or toxic effects. DETAILED DESCRIPTION

[0053] The present disclosure is further described in conjunction with the following examples, which are not intended to limit the scope of the present disclosure.

[0054] Experimental methods in the examples of the present disclosure, for which specific conditions are not specified, are generally performed according to conventional conditions, or according to the conditions suggested by the manufacturer of the material or commodity. Reagents for which specific sources are not specified are conventional reagents purchased on the market.

[0055] The structure of the compound is determined by nuclear magnetic resonance (NMR) or / and mass spectrometry (MS). NMR shifts (δ) are given in units of 10 -6 (ppm). NMR is measured by a Bruker AVANCE-400 nuclear magnetic instrument, and the measuring solvent is deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3), deuterated methanol (CD3OD), and the internal standard is tetramethylsilane (TMS).

[0056] MS is measured by Agilent 1200 / 1290 DAD-6110 / 6120 Quadrupole MS liquid chromatograph-mass spectrometer (manufacturer: Agilent, MS model: 6110 / 6120 Quadrupole MS).

[0057] Waters ACQuity UPLC-QD / SQD (Manufacturer: waters, MS model: waters ACQuity Qda Detector / waters SQ Detector) THERMO Ultimate 3000-Q Exactive (Manufacturer: THERMO, MS model: THERMO Q Exactive)

[0058] High performance liquid chromatography (HPLC) analysis uses Agilent HPLC 1200DAD, Agilent HPLC 1200VWD and Waters HPLCe2695-2489 high pressure liquid chromatograph.

[0059] Chiral HPLC analysis determination uses Agilent 1260DAD high performance liquid chromatograph.

[0060] High performance liquid preparation uses Waters 2545-2767, Waters 2767-SQ Detecor2, Shimadzu LC-20AP and Gilson GX-281 preparative chromatograph.

[0061] Chiral preparation uses Shimadzu LC-20AP preparative chromatograph.

[0062] CombiFlash rapid preparation instrument uses Combiflash Rf200 (TELEDYNE ISCO).

[0063] Thin layer chromatography silica gel plate uses Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plate, the specification of silica gel plate used in thin layer chromatography (TLC) is 0.15mm-0.2mm, and the specification of thin layer chromatography separation and purification product is 0.4mm-0.5mm.

[0064] Silica gel column chromatography generally uses Yantai Huanghai silica gel 200-300 mesh silica gel as carrier.

[0065] Known starting materials of the present disclosure can be synthesized according to methods known in the art or purchased from ABCR GmbH&Co.KG, Acros Organics, Aldrich Chemical Company, AccelaChemBio Inc, Darui Chemicals and the like.

[0066] Unless otherwise specified, the reactions in the examples can be carried out under argon atmosphere or nitrogen atmosphere.

[0067] An argon or nitrogen atmosphere means that the reaction vessel is connected to an argon or nitrogen balloon of about 1 L in volume.

[0068] A hydrogen atmosphere means that the reaction vessel is connected to a hydrogen balloon of about 1 L in volume.

[0069] A pressurized hydrogenation reaction uses a Parr 3916EKX-type hydrogenation apparatus and a Qinglan QL-500-type hydrogen generator or a HC2-SS-type hydrogenation apparatus.

[0070] A hydrogenation reaction is usually vacuumed and filled with hydrogen repeatedly for 3 times.

[0071] A microwave reaction uses a CEM Discover-S 908860-type microwave reactor.

[0072] In the examples, a solution means an aqueous solution unless otherwise specified.

[0073] In the examples, a reaction temperature is room temperature, which is 20°C to 30°C unless otherwise specified.

[0074] In the examples, the monitoring of a reaction progress uses thin layer chromatography (TLC). The solvent ratio of a developing agent used in a reaction, an eluent used in column chromatography for purifying a compound, and a developing agent used in thin layer chromatography is adjusted according to the polarity of a compound. A small amount of a basic or acidic reagent such as triethylamine and acetic acid can be added for adjustment.

[0075] Example 1: Preparation of SMTP-7

[0076]

[0077] Stachybotrys microspora IFO30018 was inoculated into a seed culture medium (4% glucose, 0.5% soybean meal, 0.3% dried meat extract, 0.3% yeast extract, 0.01% antifoaming agent, pH 5.8) and cultured for 4 days. The seed culture medium was inoculated into a fermentation culture 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% antifoaming agent, pH 5.8), and cultured for 4 days. L-ornithine was added and the culture was further cultured for 1 day to complete the fermentation.

[0078] 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.

[0079] The solidification product is dissolved in methanol, pre-processed and prepared using reverse packing. After the steps of ethyl acetate extraction, etc., the target product is obtained.

[0080] Example 2: Preparation of δ-deuterated-L-ornithine (Compound 1e)

[0081]

[0082] (S)-2-(bis(tert-butoxycarbonyl)amino)-4-cyanobutanoic acid benzyl ester (Compound 1b)

[0083] Compound 1a (prepared according to Synlett, 2016, vol. 27, 2, 309-312, 74.8 g, 234.9 mmol) is dissolved in acetonitrile (750 mL), Boc20 (76.9 g, 352.4 mmol) is added, DMAP (2.9 g, 23.5 mmol) is added, and then reacted at 45°C for 1-1.5 hours. The solvent is removed by rotary evaporation, and the crude product is 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] + .

[0084] Compound 1c and 1d

[0085] Compound 1b (4.18 g, 10 mmol) is dissolved in EA (100 mL) (ultra dry) and D20 (20 mL), Pt02 (204 mg) is added, D2 is replaced, and the reaction is carried out under D2 protection at 30°C (external temperature) for 40 h. After the reaction is substantially complete, the liquid is separated, the aqueous phase is washed with EA, and then directly freeze-dried to obtain a solid 1.87 g. The solid is dissolved in acetonitrile (5 ml) and ethyl acetate (20 ml), stirred at room temperature, filtered, the filter cake is washed with ethyl acetate, and oil pump drying is performed to obtain a mixture of compounds 1c and 1d 1.54 g (yield 46%).

[0086] δ-deuterated-L-ornithine (Compound 1e)

[0087] The mixture of compounds 1c and 1d (195 mg, 0.58 mmol) is dissolved in 6M aqueous hydrochloric acid solution, and reacted at room temperature for 2 h. After the reaction is substantially complete, the solvent is removed by rotary evaporation, and oil pump drying is performed to constant weight to obtain 120 mg of a light yellow solid product compound 1e (yield 100%, purity 97.25%).

[0088] HNMR (D20, 400M): 1.95-1.94 (m, 4H), 2.91-2.98 (m, 0.027H), 3.96 (t, J = 6.4

[0089] Hz, 1H).

[0090] Example 3: Preparation of δ-deuterated SMTP-7 (Compound 1)

[0091]

[0092] Compound 1 was prepared according to the method of Example 1 using δ-deuterated L-ornithine (prepared according to Example 2).

[0093] Stachybotrys microspora IFO30018 was inoculated into a seed culture medium (4% glucose, 0.5% soy peptone, 0.3% dry broth, 0.3% yeast extract, 0.01% antifoam agent, pH 5.8) and incubated for 4 days. The seed culture was inoculated into a fermentation culture 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% antifoam agent, pH 5.8) and incubated for 4 days, and then δ-deuterated L-ornithine was added and incubated for 1 day to complete the fermentation.

[0094] 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.

[0095] The solidified product was dissolved in methanol and pre-treated and prepared using a reversed-phase filler. After the steps of ethyl acetate extraction and the like, the target product was obtained.

[0096] 1 H NMR (400 MHz, DMSO-d6) δ 13.07 - 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, 5.7 Hz, 1H), 4.26 - 4.05 (m, 4H), 3.73 (dd, J = 13.0, 6.9 Hz, 2H), 2.82 (dt, J = 17.0, 4.8 Hz, 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).

[0097] Test Example 1: Pharmacokinetic study in rats

[0098] 1.1 Preparation of the test sample

[0099] Weigh appropriate amounts of SMTP-7 and compound 1, and add 2% DMSO and 98% physiological saline to each compound in sequence. After vortexing and sonicating to mix thoroughly, a clear solution with a concentration of 1 mg / mL is obtained for later use.

[0100] 1.2 Animals

[0101] SD rats, aged 6-8 weeks, weighing approximately 180-220g

[0102] 1.3 Scheme

[0103]

[0104] 1.4 Sample Collection

[0105] 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 after collection. Samples were collected at 10 time points: before drug administration and at 5 min, 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, 10 h, and 24 h after drug administration. The collected blood samples were placed in heparin-anticoagulated blood collection tubes and centrifuged to separate the plasma (centrifugation force 6800 g, 6 min, 2-8℃). Plasma samples were stored at -80℃ before analysis.

[0106] 1.5 Bioanalysis and Data Processing

[0107] The blood concentrations of each test substance were measured. While analyzing the test samples, the quality control samples were also analyzed, and the accuracy of more than 66.7% of the quality control samples was required to be between 80% and 120%.

[0108] When plotting plasma drug concentration-time curves, BLQ is always recorded as 0. When calculating pharmacokinetic parameters, the concentration before administration is calculated as 0; BLQ before Cmax (including "No peak") is calculated as 0; Cmax... max Subsequent BLQs (including "No peak") were not included in the calculation. Using blood drug concentration data at different time points, the following pharmacokinetic parameters were calculated using the non-compartmental model statistical moment method in Phoenix WinNonlin 7.0 software: AUC (0-t) AUC (0-∞) T 1 / 2 MRT, C max T max Parameters such as these.

[0109] Experimental results:

[0110] Group T 1 / 2 (h)]]> T max (h)]]> C max (ng / ml) AUC (0-t) (h*ng / ml) 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

[0111] Conclusion: Compared with SMTP-7, compound 1 showed a longer half-life, a lower C max .

[0112] Example 4: Preparation of a-deuterated-L-ornithine

[0113]

[0114] a-Deuterated-2-((tert-butoxycarbonyl)amino)-4-cyanobutyric acid (compound 2c)

[0115] Compound 1b (prepared according to Example 1, 25.0 g, 59.8 mmol) was stirred and dissolved in MeOD 125 mL under nitrogen protection, anhydrous potassium carbonate (41.3 g, 299.0 mmol) was added, and the reaction was carried out at 20-30 °C for 16 h. After the reaction was substantially completed, the system was directly concentrated to obtain crude compound 2c. MS (ESI) m / z 252.1 [M+Na] + .

[0116] Benzyl a-deuterated-2-((tert-butoxycarbonyl)amino)-4-cyanobutyrate (compound 2d)

[0117] Compound 2c crude (25.0 g, 59.8 mmol) was added to anhydrous acetonitrile 150 mL, BnBr (15.3 g, 89.7 mmol) was added, and the reaction was carried out at 20-30 °C for 6 h under nitrogen protection. After the reaction was substantially complete, the filter cake was washed with acetonitrile, and the filtrate was concentrated. The crude product was subjected to column chromatography to obtain 6.8 g of the target compound 2d, which was a racemate as detected by chiral HPLC (two-step yield 35.7 %), MS (ESI) m / z 343.1 [M+Na]+.

[0118] Benzyl a-deuterated-(S)-2-((tert-butoxycarbonyl)amino)-4-cyanobutyrate (compound 2e)

[0119] The 6.8 g of racemic product of compound 2d obtained by the above preparation was subjected to chiral resolution to obtain 3.6 g of compound 2e.

[0120] Instrument: MG II preparative SFC (SFC-14), chiral column: ChiralPak AD, 250 x 30 mm I.D., 5 μm, mobile phase A: carbon dioxide, mobile phase B: methanol (0.1 % ammonia water), gradient 15 %, flow rate: 60 ml / min, back pressure: 100 bar, column temperature: 38 °C, detection wavelength: 220 nm, separation time: about 6 min.

[0121] HNMR (CDC13, 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).

[0122] α-Deuterium-(S)-5-amino-2-((tert-butoxycarbonyl)amino)pentanoic acid (compound 2f)

[0123] Compound 2e (3.5 g, 10.9 mmol) was weighed, dissolved in 70 mL of ethyl acetate, 700 mL of pure water was added, PtO2(224 mg, 0.9 mmol) was added, and hydrogen was exchanged. The reaction was carried out at 20-30°C for 16 h. The reaction was substantially complete. Filtration was carried out, the filtrate was separated, the aqueous phase was collected, the aqueous phase was washed with water, and freeze-drying was carried out to obtain about 1.6 g of the crude product of the target compound 2f. 20 mL of ethyl acetate and 2 mL of acetonitrile were added, and the slurry was filtered. The solid was dried to obtain about 1.5 g of the pure product (yield 58.6%). MS-ESI: m / z 236.1 [M+H]+.

[0124] HNMR (D2O, 400M): 1.44 (s, 9H), 1.58~1.80 (m, 4H), 2.93~2.98 (m, 2H).

[0125] α-Deuterium-L-ornithine (compound 2g)

[0126] Compound 2f (1.5 g, 6.4 mmol) was weighed, dissolved in 10 mL of 6M HCl, and stirred. The reaction was carried out at 20-30°C for 2 h. The reaction was substantially complete. The reaction solution was directly freeze-dried to obtain 1.2 g of the crude product of compound 2g. 20 mL of acetonitrile was added to the slurry, and filtration was carried out to obtain 1.1 g of the solid pure product (yield 83.9%). MS-ESI: m / z 134.1 [M+H]+.

[0127] HNMR (D2O, 400M): 1.67~2.01 (m, 4H), 2.98~3.01 (m, 2H).

[0128] Example 5: Preparation of α-deuterium-SMTP-7 (compound 2)

[0129]

[0130] The target product compound 2 was prepared by using α-deuterium-L-ornithine (prepared according to Example 4) according to the method of Example 1.

[0131] Example 6: Preparation of β-deuterium-L-ornithine (compound 3e)

[0132] β-deuterated-L-ornithine (compound 3e)

[0133]

[0134] Compound 3b

[0135] Compound 3a (3.8 g, 15.4 mmol, prepared according to Journal of the American Chemical Society, 2017, vol. 139, 39, 13830-13836), 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 reaction was carried out at 10-20 °C for 16 hours; filtration was performed to remove the insoluble matter, and the organic phase was washed with EA; the filtrate was concentrated to obtain a crude product, which was subjected to column chromatography (PE:EA = 10:1) to obtain 4.8 g of the target compound 3b (purity 93.3%, yield 97%).

[0136] MS-ESI: m / z 343.1 [M+Na] +

[0137] 1 HNMR (CDCI3, 400M): 1.45 (s, 9H), 4.36-4.40 (m, 1H), 5.17-5.24 (s, 2H), 7.27-7.40 (m, 5H).

[0138] Compound 3c

[0139] Compound 3b was a racemate, and after chiral preparation, 2.2 g of the target compound 3c in a single configuration was obtained.

[0140] Compound 3d

[0141] Compound 3c (2.2 g, 6.9 mmol) was stirred and dissolved in EA and water, and PtO2 (0.3 g, 1.3 mmol) was added; the system was replaced with hydrogen gas three times, and the reaction was carried out at 10-20 °C for 16 hours; filtration was performed, and the aqueous phase was separated and concentrated under reduced pressure; the crude product was obtained by drying with an oil pump; 20 mL of ethyl acetate and 2 mL of acetonitrile were added to the crude product, which was slurried at 10-20 °C; filtration was performed, and the solid was collected and dried to obtain 1.2 g of the target compound 3d.

[0142] MS-ESI: m / z 235.1 [M+H] +

[0143] Compound 3e

[0144] Compound 3d (1.2 g, 5.1 mmol) was dissolved in 12 mL 6M aqueous hydrochloric acid solution, reacted at 10-20 °C for 4 hours, and directly concentrated under oil pump reduced pressure to obtain 1.05 g of the target compound 3e (yield 100%).

[0145] MS-ESI: m / z 135.1 [M+H] +

[0146] 1 HNMR (CDC13, 400M): 1.67-1.81 (m, 2H), 1.91-1.93 (m, 0.1H), 2.95-2.32 (m, 2H), 3.98 (s, 1H).

[0147] Example 7: Preparation of β-deuterated SMTP-7 (Compound 3)

[0148]

[0149] The target product compound 3 was prepared using β-deuterated L-ornithine (prepared according to Example 6) according to the method of Example 1.

[0150] Example 8: Preparation of γ-deuterated-L-ornithine (Compound 4c)

[0151]

[0152] Compound 4b

[0153] 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), Boc2NH (2.1 g, 9.6 mmol) and triphenylphosphine (5.0 g, 19.1 mmol) were added, cooled to 0 °C, slowly added with DEAD (3.3 g, 19.1 mmol), after completion, returned to room temperature and stirred overnight, quenched with phosphonic acid buffer, extracted with ethyl acetate, combined the organic phase, washed with saturated brine, concentrated the organic phase and purified by column chromatography (EA: PE = 1:20) to obtain 1.7 g of the target product compound 4b (yield 50%).

[0154] 1H NMR (500 MHz, CDC13) δ 4.87 (dd, J = 9.5, 5.0 Hz, 1H), 3.70 (s, 3H), 3.59 (d, J = 3.1 Hz, 2H), 2.09 (dd, J = 14.2, 4.7 Hz, 1H), 1.88 - 1.81 (m, 1H), 1.49 (d, J = 3.3 Hz, 36H).

[0155] Compound 4c

[0156] 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 degrees (external temperature) for 16 hours. After cooling to room temperature, it was concentrated and dried by oil pump to give 500 mg of compound 4c (yield 92%).

[0157] MS-ESI: m / z 135.1 [M+H] +

[0158] 1 H NMR (500 MHz, D20) δ 3.99 (t, J = 6.3 Hz, 1H), 3.03 (s, 2H), 1.97 (qd, J = 14.5, 6.5 Hz, 2H).

[0159] Example 9: Preparation of γ-deuterated SMTP-7 (Compound 4)

[0160]

[0161] The target product compound 4 was prepared using γ-deuterated L-ornithine (prepared according to Example 8) according to the method of Example 1.

[0162] Test Example 2: Pharmacokinetic study in rats

[0163] 1.1 Preparation of test samples

[0164] An appropriate amount of SMTP-7, compound 2, compound 3 and compound 4, respectively, was weighed, physiological saline (2mM NaOH solution was added to adjust the pH to about 9.2), vortexed and ultrasonicated to give a clear solution with a concentration of 1 mg / mL, ready for use.

[0165] 1.2 Animals

[0166] SD rats, age 6-8 weeks, body weight about 180-240g

[0167] 1.3 Protocol

[0168]

[0169] The pharmacokinetic AUC in rats was detected by referring to the method of Test Example 1 (0-t) , AUC (0-∞) , T 1 / 2 , MRT, C max , T max , and other parameters.

[0170] Experimental results: After intravenous administration in rats, the T 1 / 2 and AUC of SMTP-7, compound 2 and compound 3 had little difference, and the T 1 / 2 and AUC of compound 4 were lower than those of SMTP-7.

[0171] Test Example 3: Thromboembolism in rats

[0172] 1.1 Preparation of cerebral infarction model

[0173] The cerebral infarction model was prepared according to the literature method (J Cereb Blood Flow Metab, 1997, 17(2): 123-135.) to prepare a thrombotic stroke model. 0.1 mL of rat blood was immediately sucked into a PE50 tube, and after standing at room temperature for 2 h, it was stored at 4°C for 22 h. The thrombus was pushed out into 30 mL of normal saline and washed 3 times for 5 min each time. A 5 mm long embolus was cut and sucked into a specially designed PE50 tube at the end of the tube for standby.

[0174] After isoflurane gas anesthesia of the rats, they were fixed in a supine position on the operating table, the skin was incised in the middle of the neck, the right common carotid artery was isolated, and the internal carotid artery branch was separated and clamped with an artery clamp. A small incision was made at the common carotid artery, the embolus in the above-mentioned catheter was pushed into the intracranial with 0.4 mL of normal saline, and then the cannula was carefully pulled out, the common carotid artery was ligated, and the skin was sutured.

[0175] 2.2 Grouping and administration

[0176] Neurological function scoring was performed 1 h after modeling, and rats with a score of ≥8 were considered to be successfully modeled, and the rats were 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 with normal saline (2mM NaOH solution adjusted to pH about 9.2)), and the control drug group was given SMTP-7 (1 mg / ml, prepared with normal saline (2mM NaOH solution adjusted to pH about 9.2)). Intravenous administration was performed immediately after the end of modeling, and 10% of the drug solution was injected first, and the remaining 90% was infused for 30 min. The end of the test was 24 h after drug administration.

[0177] Neurological function scoring

[0178] The degree of animal behavior disorder was observed and scored at 24h after treatment. The scoring criteria were as follows:

[0179] MCAO rat neurological impairment score criteria

[0180]

[0181]

[0182] Cerebral hemorrhage assay

[0183] After blood sampling at 24h for scoring, the brain was taken by heart perfusion, and the brain tissue was frozen in a -20°C refrigerator. Then, the brain tissue was sliced from front to back, with each slice being 2mm thick. If a slice had hemorrhage, it was scored 1 point. The total score of each animal was the sum of the scores of all slices.

[0184] Cerebral infarction range assay

[0185] After blood sampling at 24h for scoring, the brain was taken by heart perfusion, and the brain tissue was frozen in a -20°C refrigerator. Then, the brain tissue was sliced from front to back, with each slice being 2mm thick. The brain tissue slices were placed in a 2% TTC solution and incubated at 37°C for 5min. The infarcted tissue was white, and the non-infarcted tissue was red. Image J software was used to measure the cerebral infarction area and calculate the percentage of the infarction area to the whole brain area.

[0186] Percentage of cerebral infarction area = cerebral infarction area / whole brain area x 100%

[0187] Data analysis method

[0188] Measurement data was represented by T-TEST was used for pairwise comparison, and P<0.05 was statistically different.

[0189] Experimental results:

[0190] Table 1: Behavioral score and cerebral infarction area

[0191]

[0192] Note: # indicates relative to the sham operation group, and * indicates relative to the model group.

[0193] Table 2: Cerebral hemorrhage score

[0194]

[0195]

[0196] In terms of behavior, both compound 1 and SMTP-7 improved the model group at the same dose (10 mg / kg), and the degree of improvement was comparable.

[0197] In terms of cerebral infarction area, both compound 1 and SMTP-7 significantly improved the model group, and compound 1 was better than SMTP-7 at a dose of 10 mg / kg (reduced by about 37%), and the probability of more severe infarction was lower.

[0198] In terms of cerebral hemorrhage, both compound 1 and SMTP-7 improved the model group at each dose, and 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%), and the occurrence of cerebral hemorrhage score ≥3 (10%) in SMTP-7 indicated that the brain had a higher risk of hemorrhagic transformation after administration of SMTP-7, which was not conducive to the treatment of ischemic stroke.

[0199] Conclusion: Compound 1 has an improving effect on neurological function and cerebral infarction area after cerebral infarction, and has a lower risk of hemorrhage.

Claims

1. A compound of formula I ###0001### or a pharmaceutically acceptable salt thereof. wherein 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 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 R is deuterium.

2. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein R 27 selected from deuterium, R 28 selected from deuterium, R 36 selected from deuterium, R 37 selected from deuterium.

3. The compound or pharmaceutically acceptable salt thereof according to claim 1 or 2, wherein R 26 is selected from deuterium, R 38 is selected from deuterium.

4. The compound or pharmaceutically acceptable salt thereof of any one of claims 1-3, wherein R 24 selected from deuterium, R 25 selected from deuterium, R 39 selected from deuterium, R 40 selected from deuterium.

5. The compound according to any one of claims 1-3, or a pharmaceutically acceptable salt thereof, wherein R 29 is selected from deuterium.

6. The compound according to any one of claims 1-5, or a pharmaceutically acceptable salt thereof, wherein R 30 is selected from deuterium, R 31 is selected from deuterium.

7. The compound according to any one of claims 1-6, or a pharmaceutically acceptable salt thereof, wherein R 34 is selected from deuterium, R 35 is selected from deuterium.

8. The compound or pharmaceutically acceptable salt thereof of any one of claims 1-7, wherein R 16 selected from deuterium, R 17 selected from deuterium, R 47 selected from deuterium, R 48 selected from deuterium.

9. The compound or pharmaceutically acceptable salt thereof of any one of claims 1-8, wherein R 12 selected from deuterium, R 13 selected from deuterium, R 14 selected from deuterium.

10. The compound or pharmaceutically acceptable salt thereof of any one of claims 1-9, wherein R 50 selected from deuterium, R 51 selected from deuterium, R 52 selected from deuterium.

11. The compound or pharmaceutically acceptable salt thereof of any one of claims 1-10, wherein R 23 is selected from deuterium, R 41 is selected from deuterium.

12. The compound or pharmaceutically acceptable salt thereof of any one of claims 1-11, wherein R 32 is selected from deuterium, R 33 is selected from deuterium.

13. The compound or pharmaceutically acceptable salt thereof of any one of claims 1-5 or 12, wherein 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.

14. The compound or pharmaceutically acceptable salt thereof of any one of claims 1-13, wherein R 1 selected from deuterium, R 2 selected from deuterium, R 3 selected from deuterium.

15. The compound or pharmaceutically acceptable salt thereof of any one of claims 1-14, wherein R 4 selected from deuterium, R 5 selected from deuterium, R 6 selected from deuterium.

16. The compound or pharmaceutically acceptable salt thereof of any one of claims 1-15, wherein R 58 selected from deuterium, R 59 selected from deuterium, R 60 selected from deuterium.

17. The compound or pharmaceutically acceptable salt thereof of any one of claims 1-16, wherein R 61 selected from deuterium, R 62 selected from deuterium, R 63 selected from deuterium.

18. The compound or pharmaceutically acceptable salt thereof of claim 17, wherein 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 61 selected from deuterium, R 62 selected from deuterium, R 63 selected from deuterium.

19. The compound or pharmaceutically acceptable salt thereof of any one of claims 1-18, wherein R 7 is selected from deuterium, R 57 is selected from deuterium.

20. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, which is ###0010### 21. A compound of formula I, or a pharmaceutically acceptable salt thereof, selected from the group consisting of: ###0011### ###0012### ###0013### ###0014### ###0015### ###0016### ###0017### ###0018### ###0019### ###0020### ###0021### ###0022### ###0023### ###0024### ###0025### ###0026### ###0027### ###0028### ###0029### ###0030### ###0031### ###0032### ###0033### ###0034### ###0035### ###0036### ###0037### ###0038### ###0039### ###0040### ###0041### ###0042### ###0043### ###0044### ###0045### ###0046### ###0047### ###0048### ###0049### ###0050### ###0051### ###0052### ###0053### ###0054### ###0055### ###0056### ###0057### ###0058### ###0059### ###0060### ###0061### ###0062### ###0063### ###0064### ###0065### ###0066### ###0067### ###0068### ###0069### ###0070### ###0071### ###0072### ###0073### ###0074### ###0075### ###0076### ###0077### ###0078### ###0079### ###0080### ###0081### ###0082### ###0083### ###0084### ###0085### ###0086### ###0087### ###0088### ###0089### ###0090### ###0091### ###0092### ###0093### ###0094### ###0095### ###0096### ###0097### ###0098### ###0099### ###0100### ###0101### ###0102### ###0103### ###0104### ###0105### ###0106### ###0107### ###0108### ###0109### ###0110### ###0111### ###0112### ###0113### ###0114### ###0115### ###0116### ###0117### ###0118### ###0119### ###0120### ###0121### ###0122### ###0123### ###0124### ###0125### ###0126### ###0127### ###0128### ###0129### ###0130### ###0131### ###0132### ###0133### ###0134### ###0135### ###0136### ###0137### ###0138### ###0139### ###0140### ###0141### ###0142### ###0143### ###0144### ###0145### ###0146### ###0147### ###0148### ###0149### ###0150### ###0151### ###0152### ###0153### ###0154### ###0155### ###0156### ###0157### ###0158### ###0159### ###0160### ###0161### ###0162### ###0163### ###0164### ###0165### ###0166### ###0167### ###0168### ###0169### ###0170### ###0171### ###0172### ###0173### ###0174### ###0175### ###0176### ###0177### ###0178### ###0179### ###0180### ###0181### ###0182### ###0183### ###0184### ###0185### ###0186### ###0187### ###0188### ###0189### ###0190### ###0191### ###0192### ###0193### ###0194### ###0195### ###0196### ###0197### ###0198### ###0199### ###0200### ###0201### ###0202### ###0203### ###0204### ###0205### ###0206### ###0207### ###0208### ###0209### ###0210### ###0211### ###0212### ###0213### ###0214### ###0215### ###0216### ###0217### ###0218### ###0219### ###0220### ###0221### ###0222### ###0223### ###0224### ###0225### ###0226### ###0227### ###0228### ###0229### ###0230### ###0231### ###0232### ###0233### ###0234### ###0235### ###0236### ###0237### ###0238### ###0239### ###0240### ###0241### ###0242### ###0243### ###0244### ###0245### ###0246### ###0247 ​ ​ ​ ​

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