RU2785715C1 - USE OF MONOCYCLIC β-LACTAM COMPOUND IN PHARMACY - Google Patents

Abstract

FIELD: pharmaceutics.

SUBSTANCE: invention relates to the use of therapeutically effective amount of a compound represented by the formula (I) or its pharmaceutically acceptable salt for the production of a drug for the treatment of pneumonia, while pneumonia is caused by infection with gram-negative bacteria, in particular Pseudomonas aeruginosa, a pharmaceutical composition, and a method for the treatment of such pneumonia.

EFFECT: treatment of pneumonia caused by infection with gram-negative bacteria, in particular Pseudomonas aeruginosa.

14 cl, 1 dwg, 3 tbl, 2 ex

Description

[0001] The present application claims priority on the following application:

[0002] CN 201811549551.8 filed December 18, 2018

FIELD OF TECHNOLOGY

[0003] The present invention relates to the use of the compound represented by formula (I) and its pharmaceutically acceptable salts in the field of pharmacy.

BACKGROUND OF THE INVENTION

[0004] Experts and public health officials generally consider the emergence and spread of drug-resistant bacteria to be one of the major public health challenges of the 21st century. The incidence of antibiotic resistance and its association with severe infectious diseases is increasing at an alarming rate. Of particular concern is the growing prevalence of nosocomial resistance to pathogenic microorganisms. Of the more than two million nosocomial infections that occur annually in the United States, 50% to 60% are caused by antibiotic-resistant bacteria. High levels of resistance to commonly used antimicrobials increase the morbidity, mortality and costs associated with nosocomial infections. The number of patients who died from incurable nosocomial infections continues to grow. Antibiotic resistance is estimated to kill 700,000 people worldwide every year, and some experts predict that number will reach 10 million by 2050 unless efforts are made to develop new therapeutics or therapeutic schedules (Nature, 2017, 543, 15). Treatment options for infections caused by multidrug-resistant gram-negative bacteria, including Enterobacteriaceae and non-fermentative bacteria, are particularly limited; even more serious is that the pipeline of development of the pharmaceutical industry includes few compounds that can break through the resistance of bacteria (Clin. Inf. Dis., 2009, 48, 1-12).

[0005] Over the past few decades, a very successful and well-tolerated class of β-lactam antibiotics has been the mainstay for the treatment of infections caused by gram-negative pathogens. Of these, third-generation cephalosporins, carbapenems, and monocyclic lactams are especially widely used to treat infections caused by Gram-negative bacteria. However, the emergence of an increasing number of β-lactamases and other resistance mechanisms is seriously threatening the medium-term availability of current compounds in these subclasses, especially extended-spectrum lactamases (ESBLs) and carbapenemases are important drivers of drug resistance; therefore, there is an urgent need for new β-lactam antibiotics that can break through drug resistance to fill this gap.

[0006] Since aztreonam is the only FDA-approved monocyclic β-lactam used worldwide, and the second analogue (tigemonam) is sold only in the Japanese market, the value of aztreonam as a monocyclic β-lactam antibiotic is far from fully understood (Rev. Infect. Dis. , 1985, 7, 579-593). On the other hand, bacterial resistance impairs aztreonam permeability, increases exocytosis, and reduces the antibacterial spectrum. To increase the permeability of monocyclic β-lactams to bacteria, a series of iron carrier trapping systems for monocyclic β-lactam molecules have been introduced at Basilea (WO 2007065288), Naeja Pharmaceuticals (WO 2002022613) and Squibb&Sons (AS 5290929, EP 531976, EP 484881). Pfizer recently re-examined a monocyclic β-lactam bearing a sulfonamidocarbonyl activating group at the N1 position (WO 2010070523). In addition, in WO 2008116813 Basilea describes a combination therapy using monocyclic β-lactams and carbapenems. AiCuris (WO 2013110643) and Novartis (WO 2015148379) reported studies on enhancing activity by modifying substituents on the aztreonam molecules, respectively, with compounds of the formula below, wherein group A is an aromatic ring structure to which amidino and guanidine groups are attached. Novartis (WO 2017050218) also reports salt forms of one of the compounds currently in pre-clinical or clinical development.

SHORT DESCRIPTION

[0007] The present invention provides the use of a compound represented by formula (I) or a pharmaceutically acceptable salt thereof for the preparation of a medicament for the treatment of pneumonia.

[0009] In some embodiments of the present invention, where the pneumonia in the application described above is caused by Pseudomonas aeruginosa infection.

[0010] The present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of a compound represented by formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient, and one or more pharmaceutically acceptable excipients and/or pharmaceutically acceptable carriers.

[0011] In some embodiments of the present invention, the adjuvant in the composition described above is a surface stabilizer, solubilizer, buffer, opacifier, binder, disintegrant, or lubricant.

[0012] In some embodiments of the present invention, the surface stabilizer in the composition described above comprises an amphoteric surfactant, a nonionic surfactant, a cationic surfactant, or an anionic surfactant, or a combination thereof.

[0013] In some embodiments of the present invention, the pharmaceutical composition in the case of the composition described above, is for oral use.

[0014] In some embodiments of the present invention, the pharmaceutical composition in the case of the composition described above is a tablet or capsule.

[0015] In some embodiments of the present invention, the pharmaceutical combination in the case of the composition described above is in the form of an injectable composition or inhalation composition.

[0016] According to the present invention, the use of the composition described above for the preparation of a medicament for the treatment of pneumonia is provided.

[0017] In some embodiments of the present invention, pneumonia, in which the composition is used, as described above, is caused by infection with gram-negative bacteria.

[0018] In some embodiments of the present invention, the pneumonia in which the composition is used as described above is caused by Pseudomonas aeruginosa infection.

TECHNICAL RESULT

[0019] The compound of the present invention has good antibacterial activity against Gram-negative bacteria, and in particular, it has excellent antibacterial activity against Pseudomonas aeruginosa.

[0020] Definitions and Description

[0021] In the present description, the following terms and phrases have the following meanings, unless otherwise indicated. A particular term or phrase, unless specifically defined, should not be construed as vague or obscure, but should be construed in a general sense. Where trade names are used in this specification, they are intended to refer to the respective products or their active ingredients. The term “pharmaceutically acceptable” is used herein to refer to those compounds, materials, compositions and/or dosage forms as follows: within the scope of sound medical judgement, suitable for use in contact with human and animal tissues without undue toxicity, irritation, allergic reaction, and /or other problems or complications, corresponding to a reasonable benefit/risk ratio.

[0022] The term "pharmaceutically acceptable salts" refers to salts of compounds according to the present invention, which are obtained from compounds containing these substituents proposed in the present invention, and relatively non-toxic acids or bases. In the case where the compounds of the present invention contain relatively acidic functionalities, base addition salts can be prepared by contacting the neutral form of such compounds with a sufficient amount of a base in pure solution or in a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic ammonia or magnesium salts, or similar salts. Where compounds of the present invention contain relatively basic functional groups, acid addition salts may be prepared by contacting the neutral form of such compounds with a sufficient amount of acid in pure solution or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts, including, for example, hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, hydrogen sulfate, hydroiodic acid, phosphorous acid, etc. P.; and organic acid salts, including acids such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p- toluenesulfonic acid, citric acid, tartaric acid, methanesulfonic acid, and the like; also include salts of amino acids such as arginine as well as salts of organic acids such as glucuronic acid (see Berge et al., "Pharmaceutical Salts", Journal of Pharmaceutical Science 66:1-19 (1977)). Some specific compounds of the present invention contain basic and acidic functional groups, so they can be converted to any base or acid addition salt.

[0023] Preferably, the neutral form of the compound is regenerated by contacting the salt with a base or acid in the usual manner and isolating the parent compound. The parent form of the compound differs from its various salt forms in certain physical properties, such as solubility in polar solvents.

[0024] As used herein, a "pharmaceutically acceptable salt" is a derivative of a compound of the present invention in which the parent compound has been modified by salting with an acid or base. Examples of pharmaceutically acceptable salts include, but are not limited to: a basic group such as salts of amines and inorganic or organic acids, acid radicals such as alkali or organic salts of carboxylic acids. Pharmaceutically acceptable salts include conventional non-toxic salts or quaternary ammonium salts of the parent compound, for example salts derived from non-toxic inorganic or organic acids. Conventional non-toxic salts include, but are not limited to, salts derived from inorganic and organic acids selected from 2-acetoxybenzoic acid, 2-hydroxyethanesulfonic acid, acetic acid, ascorbic acid, benzosulfonic acid, benzoic acid, bicarbonate radical, carbonic acid, citric acid, ethylenediaminetetraacetic acid, ethanedisulfonic acid, ethanesulfonic acid, fumaric acid, glucoheptose, gluconic acid, glutamic acid, glycolic acid, hydrobromic acid, hydrochloric acid, hydroiodide, hydroxy, hydroxynaphthalene, 2-hydroxyethanesulfonic acid, lactic acid, lactose, dodecylsulfonic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, nitric acid, oxalic acid, pamo acid, pantothenic acid, phenic acid, phosphoric acid, polygalactopropionic acid, hydrochloric acid, stearic acid, acetic acid, succinic acid, sulfamic acid, p-am inobenzoic acid, sulfuric acid, tannin, tartaric acid and p-toluenesulfonic acid.

[0025] Pharmaceutically acceptable salts according to the present invention can be synthesized from the parent compound containing an acid radical or a basic group using conventional chemical methods. Typically, such salts are prepared by reacting these compounds in their free acid or free base form with a stoichiometric amount of an appropriate base or acid in water or an organic solvent, or mixtures thereof. Generally, non-aqueous media such as ether, ethyl acetate, ethanol, isopropyl alcohol, or acetonitrile are preferred.

[0026] In addition to salt forms, the compounds of the present invention exist in the form of prodrugs. Prodrugs of the compounds of the present disclosure are readily converted to the compounds of the present invention by chemical changes under physiological conditions. In addition, prodrugs can be converted to the compounds of the present invention by chemical or biochemical means in an in vivo environment.

[0027] Some compounds of the present invention may exist in unsolvated or solvated forms, including hydrated forms. In general, the solvated forms are equivalent to the unsolvated forms and are intended to be included within the scope of the present invention.

[0028] Some compounds according to the present invention may contain asymmetric carbon atoms (optical centers) or double bonds. Racets, diastereomers, geometric isomers and individual isomers are included within the scope of the present invention.

и клиновидными пунктирными связями

при этом клиновидные сплошные связи

или клиновидные пунктирные связи

обозначены волнистыми линиями

а относительная конфигурация стереоцентра обозначена связями в виде сплошных линий

и связями в виде пунктирных линий

В случае, когда соединения согласно настоящему описанию содержат олефиновые двойные связи или другие центры геометрической асимметрии, они включают геометрические изомеры Е, Z, если не указано иное. Аналогично, все таутомерные формы включены в объем настоящего изобретения.[0029] Unless otherwise indicated, the absolute configuration of the stereocenter is indicated by wedge-shaped solid bonds.

and wedge-shaped dotted bonds

while wedge-shaped continuous bonds

or wedge-shaped dotted connections

marked with wavy lines

and the relative configuration of the stereocenter is indicated by connections in the form of solid lines

and links in the form of dotted lines

When the compounds according to the present description contain olefinic double bonds or other centers of geometric asymmetry, they include the geometric isomers E, Z, unless otherwise indicated. Likewise, all tautomeric forms are included within the scope of the present invention.

[0030] The compounds of the present invention may exist in particular geometric or stereoisomeric forms. The present invention includes all such compounds, including cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)-isomers, (b)-isomers, and also racemic and other mixtures thereof, such as enantiomerically or diastereomerically enriched mixtures, and all such mixtures are within the scope of the present invention. Additional asymmetric carbon atoms may be present on the substituents, such as alkyl groups. All such isomers and mixtures thereof are included within the scope of the present invention.

[0031] Optically active (R)- and (S)-isomers, as well as D- and L-isomers can be obtained by chiral synthesis or using chiral reagents or other conventional methods. The desired enantiomer of the compound of the present invention can be obtained by asymmetric synthesis or derivatization using chiral auxiliary agents, where the resulting mixture of diastereomers is separated and the auxiliary group is cleaved off to obtain 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, diastereomeric salts are formed with an appropriate optically active acid or base, followed by separation of the diastereomers by conventional methods known in the art, and subsequent isolation to obtain pure enantiomers. In addition, enantiomers and diastereomers are typically separated by chromatography using a chiral stationary phase, optionally in combination with chemical derivatization (eg, to form a carbamate from an amine).

[0032] A compound of the present invention may contain unnaturally occurring atomic isotope ratios of one or more of the atoms constituting the compound. For example, compounds can be labeled with radioisotopes such as tritium ( ³ H), iodine -125 ( ¹²⁵ I) or C-14 ( ¹⁴ C). All isotopic variants of the compounds of the present invention, whether or not radioactive, are included within the scope of the present invention.

[0033] The term "pharmaceutically acceptable carrier" refers to any formulation or vehicle capable of delivering an effective amount of the active agent of the present invention without interfering with the biological activity of the active agent and without causing toxic or side effects to the host or patient. Typical carriers include water, oils, botanicals and minerals, creams, lotion bases, ointment bases, and the like. They contain suspending agents, tackifiers, skin penetration enhancers, and the like. Additional information on carriers can be found in Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott, Williams & Wilkins (2005), the content of said source is incorporated herein by reference.

[0034] The term "adjuvant" usually refers to a carrier, diluent and/or medium necessary for the preparation of an effective pharmaceutical composition.

[0035] For a drug or pharmacologically active agent, the term "effective amount" or "therapeutically effective amount" refers to an amount of the drug or agent that is not toxic but sufficient to achieve the desired effect. In the case of dosage forms for oral administration according to the present invention, an "effective amount" of one active substance in a composition refers to the amount necessary to achieve the desired effect in combination with another active substance in the composition. The determination of an effective amount varies from person to person depending on the age and general condition of the recipient and also on the particular active substance, and suitable effective amounts in each case can be determined by a person skilled in the art according to established experiments.

[0036] The terms "active ingredient", "therapeutic agent", "active substance" or "active agent" refer to a chemical compound that is effective in treating the disorder, disease, or condition of interest.

[0037] The solvents used according to the present invention are commercially available.

[0038] The following abbreviations are used in the present invention:

[0039] aq. is water; min represents minutes; FA is formic acid; m-CPBA is 3-chloroperoxybenzoic acid; equiv. is an equivalent amount; DCC is N,N'-dicyclohexylcarbodiimide; DXM is dichloromethane; PE is petroleum ether; DIAD is diisopropyl azodicarboxylate; DMF is N,N-dimethylformamide; BH ₃ ⋅SMe ₂ is borane dimethyl sulfide; DMSO is dimethyl sulfoxide; EtOAc is ethyl acetate; EtOH is ethanol; MeOH is methanol; Cbz is a benzyloxycarbonyl, amino protecting group; Voe is t-butoxycarbonyl, an amino protecting group; HOAc is acetic acid; ACN is acetontrile; BH ₃ is sodium cyanoborohydride; k.t. is room temperature; THF is tetrahydrofuran; Boc ₂ O is di-tert-butyl dicarbonate; TFA is trifluoroacetic acid; DIPEA is diisopropylethylamine; SOCl ₂ is thionyl chloride; iPrOH is 2-propanol; mp is the melting point; LDA is lithium diisopropylamide; TEMPO is 2,2,6,6-tetramethylpiperidine-1-oxyl radical or 2,2,6,6-tetramethylpiperidine oxide; NaClO is sodium hypochlorite; NaClO ₂ is sodium chlorite; HOBt is 1-hydroxybenzotriazole; psi is pounds per square inch; DMF⋅SO ₃ is N,N-dimethylformamide sulfotrioxide; KH ₂ PO ₄ is potassium dihydrogen phosphate; Bu ₄ HSO ₄ is tetrabutylammonium hydrogen sulfate; PPh ₃ is triphenylphosphine; NH ₂ NH ₂ ⋅H ₂ O is hydrazine hydrate; dppf is 1,1'-bis(diphenylphosphino)ferrocin; Pd ₂ (dba ₃ ) is tris(dibenzylideneacetone)dipalladium (0); MIC is the minimum inhibitory concentration; DMAP is 4-dimethylaminopyridine; BnBr is benzyl bromide; H ₂ O ₂ is hydrogen peroxide.

BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In FIG. 1 shows results of lung bacterial load in the treatment of Pseudomonas aeruginosa lung infection in immunosuppressed mice.

DETAILED DESCRIPTION

[0041] The following examples describe the present invention in detail, but are not intended to impose any unfavorable limitation on the present invention. The present invention has been described in detail in the present description, and its specific embodiments are also disclosed. It will be apparent to those skilled in the art that various changes and modifications may be made to specific embodiments without departing from the spirit and scope of the present invention.

[0042] Synthesis of Intermediate A1:

[0043]

[0044] Step 1: CTHF (1.50 L) compound A1_1 (100.00 g, 642.76 mmol, 1.00 eq.) and triethylamine (136.59 g, 1.35 mol, 187.10 ml) were added , 2.10 eq.), and the resulting mixture was cooled to 0°C; Boc ₂ O (154.31 g, 707.03 mmol, 162.43 ml, 1.10 eq.) in THF (500.00 ml) was added dropwise at this temperature, the reaction mixture was heated to 10°C and stirred at this temperature for 10 hours and then filtered; the filtrate was concentrated under reduced pressure to give a crude product, to which was added a saturated sodium bicarbonate solution (300 ml) and the mixture was extracted with ethyl acetate (500 ml*2); the combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give compound A1_2.

[0045] ¹ H NMR (400 MHz, CDCl ₃ ) δ (ppm): 5.51 (br s, 1H), 4.46-4.31 (m, 1H), 4.03-3.86 (m , 2H), 3.83-3.72 (m, 3H), 2.64 (br s, 1H), 1.46 (s, 9H).

[0046] Step 2: A1_2 was dissolved in THF (2000 ml), the mixture was stirred at -50°C for 10 minutes, then MeMgBr (3M, 638.59 ml, 6.00 eq.) was added dropwise over 20 minutes at -50°C. The resulting mixture was stirred at 25°C for 60 minutes, after which the reaction mixture was quenched at 0°C by adding dilute hydrochloric acid (2000 ml, 0.5 M), then the resulting mixture was extracted with ethyl acetate (500 ml *2); the combined organic layers were washed with saturated aqueous sodium chloride, dried over anhydrous sodium sulfate and filtered, the filtrate was concentrated under reduced pressure to give a crude product, which was washed with petroleum ether/ethyl acetate (70 ml, 10/1) with stirring and purified with using column chromatography (SiO ₂ , petroleum ether/ethyl acetate = 10/1 to 1/1 (v/v)) to obtain Compound A1_3.

[0047] ¹ H NMR (400 MHz, CDCl ₃ ) δ (ppm): 5.41-5.23 (m, 1H), 3.96 (br d, J=11.2 Hz, 1H), 3, 79-3.70 (m, 1H), 3.40 (br d, J=8.3 Hz, 1H), 2.53-2.39 (m, 2H), 1.39 (s, 9H), 1.28(s, 3H), 1.18(s, 3H).

[0048] Step 3: A1_3 (30 r, 136.81 mmol, 1.00 eq.) was dissolved in a mixed solution of sodium phosphate buffer (540.00 ml, 0.7 M, 2.76 eq.) and acetonitrile ( 300 ml), then TEMPO (2.15 g, 13.68 mmol, 0.10 eq.), NaClO (81.47 g, 5.47 mmol, 67.33 ml, 0.5% purity, 0.04 eq.) and NaClO ₂ (98.99 g, 1.09 mol, 8.00 eq.) in water (300 ml) by stirring at 35°C; the resulting mixture was stirred at 35° C. for 12 hours, then cooled to room temperature and citric acid (10 g) was added; the resulting mixture was extracted with ethyl acetate (500 ml*4), the combined organic layers were washed with saturated aqueous sodium chloride solution (100 ml), dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated under reduced pressure. To the obtained crude product was added aqueous sodium carbonate (300 ml, 2 M) and washed with ethyl acetate (200 ml*2); the aqueous layer was cooled to 0° C. and adjusted to pH 3.0 with dilute hydrochloric acid (1 M); the aqueous solution was saturated with sodium chloride and the resulting mixture was extracted with ethyl acetate (500 ml*4); the combined organic layers were washed with saturated aqueous sodium chloride (50 ml), dried over anhydrous sodium sulfate, and filtered, and the filtrate was concentrated under reduced pressure to give compound A1_4.

[0049] ^d6 NMR (400 MHz, CDCl ₃ ) δ (ppm): 5.42 (br d, J=7.8 Hz, 1H), 4.18 (br d, J=8.4 Hz, 1H) , 1.39 (s, 9H), 1.30 (s, 3H), 1.22 (s, 3H).

[0050] Step 4: Al_4 (48 r, 205.78 mmol, 1.00 eq) was dissolved in DMF (700 mL) then DCC (84.92 g, 411.56 mmol, 83.25 mL, 2 00 eq.) and HOBt (55.61 g, 411.56 mmol, 2 eq.), the mixture was stirred at 10°C for 0.5 h, then O-benzylhydroxylamine hydrochloride (39.41 g, 246.93 mmol, 1.20 eq.) and sodium bicarbonate (69.15 g, 823.11 mmol, 32.01 ml, 4 eq.) in water; the resulting mixture was stirred at 10°C for 1.5 h, then the reaction mixture was filtered and the filtrate was concentrated under reduced pressure; the crude product was diluted with water (400 ml) and subjected to extraction with ethyl acetate (500 ml*2). The combined organic layers were washed with saturated aqueous sodium chloride, dried over anhydrous sodium sulfate and filtered; the filtrate was concentrated under reduced pressure, and the resulting residue was purified by column chromatography (SiO ₂ , petroleum ether/ethyl acetate = 6/1 to 3/1 (v/v)) to give Compound A1_5.

[0051] ¹ H NMR (400 MHz, DMSO-d ₆ ) δ (ppm): 11.06 (s, 1H), 7.45-7.32 (m, 5H), 6.45 (br d, J =9.2 Hz, 1H), 4.80 (d, J=2.6 Hz, 2H), 4.65 (s, 1H), 4.04 (d, J=7.0 Hz, 1H), 3.77 (br d, J=9.2 Hz, 1H), 1.40 (s, 9H), 1.11 (s, 3H), 1.08 (s, 3H);

[0052] LC-MS (ESI) m/z: 283 (M-56+1).

[0053] Step 5: A1_5 (57 g, 168.44 mmol, 1 eq) was dissolved in pyridine (600 ml) by stirring at 55° C. for 12 h and pyridine sulfur trioxide (187.67 g, 1.18 mol, 7 eq.); then the reaction mixture was concentrated under reduced pressure and the resulting solid was dissolved in ethyl acetate (800 ml); aqueous potassium carbonate (816.94 ml, 2 M, 9.7 eq.) was added to the solid at 0° C. and the resulting mixture was stirred at 100° C. for 2 h; then the reaction mixture was cooled to room temperature and was extracted with ethyl acetate (400 ml*3); the combined organic layers were dried over sodium sulfate, filtered and concentrated under reduced pressure; the resulting crude product was purified by column chromatography (SiO ₂ , petroleum ether/ethyl acetate = 12/1 to 9/1 (v/v)) to give Compound A1_6.

[0054] ¹ H NMR (400 MHz, CDCl ₃ ) δ (ppm): 7.41 (br d, J=1.0 Hz, 5H), 5.02-4.97 (m, 2H), 4, 32 (d, .7=6.7 Hz, 1H), 1.50-1.43 (m, 9H), 1.34 (s, 3H), 1.11 (s, 3H);

[0055] LC-MS (ESI) m/z: 321.1 (M+1).

[0056] Step 6: A1_6 (31 r, 96.76 mmol, 1.00 eq.) was dissolved in methanol (620 ml) and Pd/C (3 g, 10%) was added under nitrogen, then the reaction tube was replaced with nitrogen three times, then the reaction tube was saturated with hydrogen gas at 20°C and reacted at a pressure of 50 psi (about 345 kPa) for 1 hour, then the reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to obtain compound A1_7.

[0057] Step 7: To a solution of A1_7 (22 g, 95.54 mmol, 1.00 eq.) in DMF (220 ml) was added DMF⋅SO ₃ (17.56 g, 114.65 mmol, 1.2 eq. .); the mixture was stirred at 0°C for 1 h and then diluted with saturated KH ₂ PO ₄ (200 ml); the resulting mixture was extracted with ethyl acetate (100 ml) and Bu ₄ HSO ₄ (38.93 g, 114.65 mmol, 1.20 eq.) was added to the combined aqueous layers for 20 min at 10°C and the resulting aqueous phase was extracted with EtOAc ( 350 ml*4); the combined organic phases were concentrated under reduced pressure to give Compound A1_8.

[0058] Stage 8: A1_8 (68 g, 123.24 mmol, 1.00 eq.) was added to trifluoroacetic acid (300 ml) and the mixture was stirred at 15° C. for 4 hours under nitrogen atmosphere; the reaction mixture was diluted with dichloromethane (350 ml) and filtered and the filtrate was concentrated under reduced pressure to obtain compound A1.

[0059] ₁ H NMR (400 MHz, DMSO-d ₆ ) δ (ppm): 8.79 (br s, 3H), 4.18 (br s, 1H), 1.46-1.38 (m, 6H).

[0060] Synthesis of intermediate A2:

[0061]

[0062] Step 1: BOC-ONB (29.80 g, 106.69 mmol , 2 eq.) and Et ₃ N (11.34 g, 112.03 mmol, 15.59 ml, 2.1 eq.) in THF (330 ml) at 20°C, and the resulting mixture was stirred at 20°C for 11 h, filtered and the filtrate was concentrated under reduced pressure, the resulting residue was diluted with a solution of potassium carbonate (100 ml, 2 M) and subjected to extraction with ethyl acetate (100 ml * 2); the organic phases were combined and concentrated under reduced pressure to give Compound A2_2.

[0063] Step 2: To a solution of A2_2 (15 g, 45.26 mmol, 1 eq.) in MeOH (150 ml) was added BrCN (7.86 g, 74.21 mmol, 5.46 ml, 1.64 eq. .) and sodium acetate (7.43 g, 90.51 mmol, 2 eq.) at 0°C, the mixture was stirred at room temperature for 2 h, then diluted with saturated sodium carbonate solution (300 ml) and subjected to extraction with ethyl acetate ( 100 ml); the organic phases were concentrated under reduced pressure, and the residue was purified by column chromatography (SiO ₂ , petroleum ether/ethyl acetate = 5/1 to 1/1) to give compound A2_3.

[0064] Step 3: Compound A2_3 (4.2 g, 11.78 mmol, 1 eq.) and pyrazole hydrochloride (1.23 g, 11.78 mmol, 1 eq.) were added to THF (40 ml) and replaced nitrogen three times, then the mixture was stirred at 75°C for 12 h; the reaction mixture was cooled to room temperature, diluted with ethyl acetate (100 ml), filtered and the filter cake was collected to give compound A2_4 after drying.

[0065] LCMS (ESI) m/z: 425.4 (M+1).

[0066] Step 4: To a solution of Compound A2_4 (2.1 g, 4.56 mmol, 1 eq.) in DCM (20 mL) at 0°C was added TFAA (765.41 mg, 3.64 mmol, 506, 89 μl, 0.8 eq.) and triethylamine (1.01 g, 10.02 mmol, 1.39 ml, 2.2 eq.), the mixture was stirred at 0°C for 20 min, then diluted with water (20 ml), the resulting mixture was subjected to extraction with DCM (50 ml*2), the organic layers were combined and concentrated under reduced pressure to obtain Compound A2.

[0067] Synthesis of intermediate A3:

[0068]

[0069] Step 1: Compound A3_1 was added to a mixed solution of 1,1,1,3,3,3-hexafluoro-2-(trifluoromethyl)propan-2-ol (10.16 g, 43.06 mmol, 10 eq. ) and DCM (20 ml), the reagent was stirred at room temperature for 45 min (20-25°C) and concentrated under reduced pressure to obtain compound A3.

[0070] Example 1: Preparation of the compound represented by formula (I)

[0071]

[0072] Step 1: To a solution of compound 11 (29 g, 128.30 mmol, 1 eq.) in THF (300 ml) was added BH ₃ ⋅ SMe ₂ (10 M, 38.49 ml, 3 eq.). The mixture was reacted at 80° C. for 12 hours, then cooled to 0° C. and quenched with methanol (100 ml); then dilute hydrochloric acid (90 ml, 1 M) was added by stirring at 80° C. for 1 hour, and the mixture was concentrated under reduced pressure to remove the solvent; the residue was diluted with water (100 ml) and extracted with ethyl acetate (150 ml*2); the aqueous layer was then adjusted to pH=10-11 with aqueous sodium hydroxide (1 M) and the resulting aqueous phase was extracted with ethyl acetate (150 ml*2); the combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give Compound 12.

[0073] Step 2: To a solution of compound 12 (6 g, 30.29 mmol, 1 eq.) in dichloromethane (50 ml) was added Boc ₂ O (6.61 g, 30.29 mmol, 6.96 ml, 1 eq.) and triethylamine (6.13 g, 60.59 mmol, 8.43 ml, 2 eq.); the mixture was stirred at 20°C for 12 h and concentrated under reduced pressure to remove the solvent; the residue was diluted with water (100 ml) and subjected to extraction with ethyl acetate (50 ml*3); the combined organic layers were concentrated under reduced pressure to give a residue, which was purified by column chromatography (SiO ₂ , petroleum ether/ethyl acetate = 1/0 to 10/1 (v/v)) to give compound 1_3.

[0074] Step 3: to Compound 1_3 (9 g, 30.18 mmol, 1 eq) and 4,4,4',4',5,5,5',5'-octamethyl-2,2'- bis(1,3,2-dioxaborane) (15.33 g, 60.37 mmol, 2 eq.) in DMSO (150 ml) was added Pd(dppf)Cl ₂ ⋅CH ₂ Cl ₂ (2.46 g, 3 .02 mmol, 0.1 eq.) and potassium acetate (11.85 g, 120.73 mmol, 4 eq.); the mixture was replaced with nitrogen three times and stirred at 90° C. for 12 hours; the reaction mixture was diluted with water (200 ml) and subjected to extraction with ethyl acetate (150 ml*3); the combined organic layers were filtered and the filtrate was concentrated under reduced pressure, and the residue was purified by column chromatography (SiO ₂ , petroleum ether/ethyl acetate = 100/1 to 20/1 (v)) to give Compound 1_4.

[0075] Step 4: To a solution of Compound 1_4 (11 g, 31.86 mmol, 1 eq.) in THF (100 ml) was added H ₂ O ₂ (86.69 g, 764.69 mmol, 73.47 ml, 30% purity, 24 eq.) and acetic acid (9.95 g, 165.68 mmol, 9.48 ml, 5.2 eq.); the mixture was stirred at 20°C for 12 h, then quenched with saturated sodium carbonate (30 ml) and the resulting mixture was diluted with water (10 ml) and subjected to extraction with ethyl acetate (20 ml * 2); the combined organic layers were concentrated under reduced pressure to give a residue, which was purified by column chromatography (SiO ₂ , petroleum ether/ethyl acetate = 15/1-7/1 (v/v)) to give Compound 1_5. LC-MS (ESI) m/z: 180 (M-56+1). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ (ppt): 7.09 (t, J=6.3 Hz, 1H), 6.72-6.65 (m, 2H), 4.47 ( brt, J=12.7 Hz, 4H), 1.45 (s, 9H).

молекулярных сит (8 г) в МТВЕ (10 мл) добавляли катализатор A3 (673,34 мг, 800,64 мкмоль, 0,03 экв.), смесь трижды заменяли азотом и перемешивали при 20°С в течение 12 ч; реакционную смесь разбавляли этилацетатом (30 мл), фильтровали и фильтрат концентрировали при пониженном давлении и очищали с помощью колоночной хроматографии (SiO₂, петролейный эфир/этилацетат = от 6/1 до 3/1 (об./об.)) с получением Соединения 16.[0076] Step 5: to a solution of intermediate 1_5 (6.8 g, 26.69 mmol, 1 eq.), ethyl oxirane-2-carboxylate (7.75 g, 66.72 mmol, 2.5 eq.) and

molecular sieves (8 g) in MTBE (10 ml) was added catalyst A3 (673.34 mg, 800.64 μmol, 0.03 eq.), the mixture was replaced with nitrogen three times and stirred at 20°C for 12 h; the reaction mixture was diluted with ethyl acetate (30 ml), filtered, and the filtrate was concentrated under reduced pressure and purified by column chromatography (SiO ₂ , petroleum ether/ethyl acetate = 6/1 to 3/1 (v/v)) to give Compound 16.

[0077] Step 6: To a solution of compound 16 (6.3 g, 17.32 mmol, 1 eq.) in DCM (20 ml) was added TFA (14.88 g, 130.51 mmol, 9.66 ml, 7 .53 eq.) at 0°C, the mixture was stirred at 20°C for 1 h and concentrated under reduced pressure to give the trifluoroacetic acid salt of Compound 1_7.

[0078] Step 7: To a solution of intermediate A2 (3.8 g, 7.30 mmol, 1 eq.) in DMFA (30 ml) was added triethylamine (2.95 g, 29.20 mmol, 4.06 ml , 4 eq.) and the trifluoroacetic acid salt of Compound 1_7 (5.33 g, 14.60 mmol, 2 eq.); the mixture was stirred at 45°C for 2 hours, then concentrated under reduced pressure to remove DMF, the residue was diluted with water (50 ml) and was extracted with ethyl acetate (50 ml); the combined organic layers were washed with saturated aqueous sodium chloride (25 ml), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give a residue which was purified by column chromatography (SiO ₂ , petroleum ether/ethyl acetate = 1/1 to 0/1) to get Compound 1_8. LCMS (ESI) m/z: 704.4 (M+1).

[0079] Step 8: To a solution of Compound 1_8 (3.3 g, 4.50 mmol, 1 eq.) in MeOH (20 ml) was added NaOH (378.39 mg, 9.46 mmol, 2.1 eq.) ; after stirring the mixture at 20° C. for 17 h, the reaction mixture was adjusted to pH=3-4 with dilute hydrochloric acid (2 M) and concentrated under reduced pressure, the residue was diluted with methanol (20 ml) to dissolve, then filtered and concentrated at reduced pressure to obtain Compound 19. LCMS (ESI) m/z: 580.5 (M+1).

[0080] Step 9: To a solution of Compound 19 (2 g, 3.45 mmol, 1 eq.) in MeOH (20 ml) was added diphenyldiazomethane (1.34 g, 6.90 mmol, 2 eq.); the mixture was stirred at 20°C for 12 hours, then concentrated under reduced pressure and the residue was diluted with water (20 ml) and extracted with DCM (40 ml); the combined organic layers were washed with saturated aqueous sodium chloride solution (10 ml), dried over anhydrous sodium sulfate, then filtered and concentrated under reduced pressure, and the residue was purified using column chromatography (SiO ₂ , DCM/MeOH = 20/1 to 10/ 1 (v/v)) to give Compound 1_10. LCMS (ESI) m/z: 746.5 (M+1).

[0081] Step 10: to a solution of Compound 1_10 (1.2 g, 1.42 mmol, 1 eq.) and 2-hydroxyisoindoline-1,3-dione (278.65 mg, 1.71 mmol, 1.2 eq. .) in THF (12 ml) was added PPI13 (560.04 mg, 2.14 mmol, 1.5 eq.) and DIAD (431.75 mg, 2.14 mmol, 415.15 μl, 1.5 eq. ) at 0°С; the mixture was stirred at 20°C for 1 h, then concentrated under reduced pressure to remove THF, and the residue was purified using column chromatography (SiO ₂ , DCM/EtOH=20/1 to 10/1 (v/v)) to give Compound 111. LCMS (ESI) m/z: 891.5 (M+1).

[0082] Step 11: To a solution of Compound 1_11 (1 g, 1.10 mmol, 1 eq.) in EtOH (10 ml) was added NH ₂ NH ₂ ⋅H ₂ O (77.95 mg, 1.32 mmol, 75 .68 µl, 85% purity, 1.2 eq.); the mixture was stirred at 20°C for 30 min, filtered and the filtrate was concentrated under reduced pressure, the residue was diluted with water (10 ml) and extracted with DCM (20 ml), the combined organic layers were dried over anhydrous ammonium sulfate, filtered and concentrated under reduced pressure with receiving Connection 1_12. LCMS (ESI) m/z: 761.5 (M+1).

[0083] Step 12: To a solution of compound 1 12 (900 mg, 1.00 mmol, 1 eq.) in DCM (5 ml) and EJUN (5 ml) was added intermediate A 2 (416.01 mg, 1.00 mmol, 1 eq.), the mixture was stirred under nitrogen at 20°C for 1 h, then the reaction mixture was concentrated under reduced pressure and the residue was purified using column chromatography (SiO ₂ , DCM/MeOH = from 20/1 to 10/ 1 (v/v)) to give compound 1_13. LCMS (ESI) m/z: 1157.7 (M+1).

[0084] Step 13: To a solution of Compound 1_13 (200 mg, 163.39 µmol, 1 eq.) in DMF (2 ml) was added N,N'-diisopropylcarbodiimide (41.24 mg, 326.77 µmol, 2 eq. ) and HOBt (44.15 mg, 326.77 µmol, 2 eq.); after the mixture was stirred at 20° C. for 1 h, intermediate A1 (48.08 mg, 228.74 µmol, 1.4 eq.) and NaHCO ₃ (54.90 mg, 653.55 µmol, 25.42 µl, 4 eq.) and stirred at 20°C for 11 hours; the reaction mixture was diluted with water (8 ml) and the solid was collected by filtration to give Compound 1_14. LCMS (ESI) m/z: 1350.2 (M+1).

[0085] Step 14: To a solution of Compound 1_14 (220 mg, 163.01 µmol, 1 eq.) in DCM (1 ml) was added TFA (1.54 g, 13.51 mmol, 1 ml, 82.85 eq. ) at 0°C and stirred for 1 h; the reaction mixture was diluted with petroleum ether/ethyl acetate (10 ml, 4/1) and the solid was collected by filtration and purified by preparative HPLC (TFA, column: Phenomenex Synergi C18 150 mm×25 mm×10 μm; mobile phase: [water ( 0.1% TFA)-acetonitrile];% acetonitrile: 1%-30%, 9 min) to give Compound (I).

[0086] ¹ H NMR (400 MHz, D ₂ O) δ=7.23 (d, J=8.4 Hz, 1H), 7.10 (s, 1H), 6.93-6.85 (m , 2H), 5.19 (dd, J=2.0, 5.7 Hz, 1H), 4.87-4.76 (m, 4H), 4.64 (s, 1H), 4.54- 4.48 (m, 1H), 4.44-4.37 (m, 1H), 3.43 (br t, J=7.3 Hz, 4H), 3.04-2.91 (m, 4H ), 1.98 (quin, J=7.6 Hz, 4H), 1.41 (s, 3H), 0.97 (s, 3H) ppm; LC/MS (ESI) m/z: 741.3 (M+1).

[0087] Experimental Example 1: Study with the compound represented by formula (I) on Pseudomonas aeruginosa lung infection in mice

[0088] 1. Experimental strains

[0089] Pseudomonas aeruginosa RAI.

[0090] 2. Test drugs

[0091] 1) Test compound: The compound represented by formula (I),

[0092] (2) Reference compound: Substance according to AiCuris patent application WO 2018065636: reference compound I-g, aztreonam (product of Dalian Meilunbio Biotechnology Co., Ltd.).

[0093]

[0094] 3. Wednesday

[0095] Mueller-Hinton Agar (MHA) and TSA medium were purchased from BD.

[0096] 4. Experimental animals

[0097] 46 female CD-I (ICR) mice provided by Beijing Vital River Laboratory Animal Technology Co., Ltd., body weight 23-27 g, age 7 weeks.

[0098] 5. Experimental procedure:

[0099] (1) Immunosuppressive mice induced by intraperitoneal injection of cyclophosphamide

[0100] 46 immunosuppressive mice were obtained by intraperitoneal injection of cyclophosphamide 150 mg/kg on day 1 and day 4.

[0101] (2) Grouping in experiments

[0102] Seven groups were created, including groups with high, medium and low doses of the compound represented by formula (I), groups with high and medium doses of the compound I-g, the aztreonam group and the model group; there were 6 animals in each group, the remaining 4 animals were subjected to lung infection for 2 hours, and lung tissues were removed to calculate the bacterial load. See the table below for specific groups.

[0103]

[0104] (3) Pseudomonas aeruginosa pulmonary infection

[0105] Mice were intratracheally injected with 50 μl of bacterial fluid (2×10 ³ cfu). Four mouse models were sacrificed 2 hours after infection.

[0106] (4) Introduction

[0107] 2 hours after infection, mice were intraperitoneally injected once at 2 hours, 4 hours, 6 hours and 8 hours according to the groups, a total of 4 times.

[0108] (5) Bacteria count

[0109] 24 hours after infection, mice in each group were sacrificed by cervical dislocation, and lung and kidney tissues were removed under aseptic conditions, then placed in a sterilized homogenate tube, weighed, the proper amount of saline (NS) was added, and homogenized with a homogenizer for 1 min; lung tissues in the model group were diluted 10 ⁴ , 10 ⁵ and 10 ⁶ times, lung tissues in each administration group were diluted 10 and 100 times, kidney tissues in the model group were diluted 10 ² , 10 ³ and 10 ⁴ times, and lung tissues in each group, the introduction was diluted 10 times; the serial diluent was seeded onto spiral coated TSA plates and incubated overnight at 37°C to determine the number of CFU using a colony counter.

[0110] (6) Body weight

[0111] Mice were weighed every day after the start of the test and recorded changes in body weight.

[0112] (7) Data processing

[0113] Plots of CFU distribution in lung tissue were made using Graphpad Prism mapping software. Mean CFUs and body weights were calculated using SPSS 19.0 software and differences between groups were analyzed using analysis of variance.

[0114] 6. Results of the experiment:

[0115] (1) Bacterial load in immunocompromised mice after pulmonary infection with Pseudomonas aeruginosa

[0116] Four immunosuppressive mice treated with cyclophosphamide twice intraperitoneally were infected with Pseudomonas aeruginosa PA14 at 1.06×10 ⁴ cfu; after 2 hours, lung tissues were removed and homogenized to determine the number of bacteria; calculated the bacterial load in mice with an average load in the range of 5.10×10 ³ CFU.

[0117] (2) Changes in body weight: The body weights of each group of animals are shown in Table 2.

[0118]

[0119] (3) Bacterial load in the lung tissue of mice after drug treatment

[0120] Mice were intraperitoneally injected with compounds of formula (I), compound Ig and aztreonam 2 hours, 4 hours, 6 hours and 8 hours after infection; animals were sacrificed 24 hours later, lung tissues were removed under aseptic conditions, immersed in saline (NS), homogenized, diluted appropriately, and then 50 μl of diluents were uniformly seeded onto TSA plates; the plates were incubated overnight in a 37° C. incubator to count the number of colonies, which were converted to cfu per milliliter using a dilution factor, and the base 10 bacterial load logarithmic value was calculated, and the mean and standard deviation were compared in each group. The results are shown in Table 3 and in FIG. 1. Bacterial load after 24 hours in the model group increased from 1.06×10 ⁴ CFU to 3.34×10 ⁸ CFU (LOG ₁₀ bacterial load was 8.14). The bacterial load of each administration group was significantly lower than that of the simulation group and was largely eliminated. The compound represented by formula (I) was completely eliminated in the high, medium and low dose groups.

[0121] Conclusions:

[0122] The compound represented by formula (I) has an in vivo curative effect on lung infection of Pseudomonas aeruginosa in immunosuppressed mice caused by cyclophosphamide, and can significantly reduce the bacterial load on lung tissues and eliminate Pseudomonas aeruginosa infected lungs. Meanwhile, the compound represented by the formula (I) completely eliminated the bacteria infected in the lung at the lowest dose. The body weight of the animals in the administration group did not change significantly, indicating that the compound represented by formula (I) is safe.

Claims (17)

1. Use of a therapeutically effective amount of a compound represented by formula (I) or a pharmaceutically acceptable salt thereof for the preparation of a medicament for the treatment of pneumonia, wherein the pneumonia is caused by infection with Gram-negative bacteria;

. 2. Use according to claim 1, wherein said pneumonia is caused by Pseudomonas aeruginosa infection. 3. A pharmaceutical composition for the treatment of pneumonia, containing a therapeutically effective amount of a compound represented by formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient and one or more pharmaceutically acceptable excipients and/or pharmaceutically acceptable carriers, wherein the pneumonia is caused by infection with Gram-negative bacteria;

. 4. The composition according to claim 3, characterized in that the auxiliary substance is a surface stabilizer, solubilizer, buffer, opacifier, binder, disintegrant or lubricant. 5. The composition of claim 4, wherein the surface stabilizer comprises an amphoteric surfactant, a nonionic surfactant, a cationic surfactant, an anionic surfactant, or a combination thereof. 6. Composition according to any one of paragraphs. 3-5, characterized in that the pharmaceutical composition is intended for oral administration. 7. Composition according to claim 6, characterized in that the pharmaceutical composition is a tablet or capsule. 8. The composition according to any one of paragraphs. 3-5, characterized in that the pharmaceutical combination is in the form of an injection preparation or an inhalation preparation. 9. The use of the composition according to any one of paragraphs. 3-8 to obtain a medicament for the treatment of pneumonia caused by infection with gram-negative bacteria. 10. Use according to claim 9, characterized in that the pneumonia is caused by Pseudomonas aeruginosa infection. 11. A method of treating pneumonia caused by infection with Gram-negative bacteria in a subject in need thereof, comprising administering to the subject a composition according to any one of paragraphs. 3-8. 12. The method of treating pneumonia according to claim 11, wherein the pneumonia is caused by Pseudomonas aeruginosa infection. 13. A method of treating pneumonia in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, wherein the pneumonia is caused by infection with Gram-negative bacteria;

. 14. The method of treating pneumonia according to claim 13, wherein the pneumonia is caused by Pseudomonas aeruginosa infection.

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