PROBE FOR DETECTING CARBAPENEM-RESISTANT BACTERIA AND USE THEREOF

Abstract

The present disclosure relates to a compound represented by Chemical Formula 1, a probe for detecting antibiotic-resistant bacteria, which includes the compound, a composition containing the compound, a kit including the compound and a method for detecting antibiotic-resistant bacteria. A compound probe having a carbapenem structure and including a linker and a fluorophore can detect beta-lactamase or carbapenemase at high sensitivity and, therefore, can be applied to various biochemical researches. In addition, the compound probe can clinically detect carbapenemase-producing antibiotic-resistant bacteria and allows molecular diagnosis of antibiotic-resistant bacterial infectious diseases and analysis of antibiotic-resistant bacteria from a target sample at high sensitivity. Therefore, it can be effectively used for medicinal uses such as in-vitro diagnosis.

Claims

1. A compound represented by Chemical Formula 1: ##STR00055## wherein R is a hydrogen atom or C.sub.1-C.sub.2 alkyl; L is substituted or unsubstituted vinyl, substituted or unsubstituted aryl, substituted or unsubstituted carbamate, substituted or unsubstituted thiocarbamate, substituted or unsubstituted amine, or substituted or unsubstituted pyridinium; and Z is a fluorescent dye.

2. The compound according to claim 1, wherein the L is any one selected from a group consisting of —(CH═CH).sub.nCH.sub.2—, —(C≡C).sub.nCH.sub.2—, —(CH═CH).sub.nCH.sub.2—O—Ar—CH.sub.2—, —(C≡C).sub.nCH.sub.2—O—Ar—CH.sub.2—, —(CH═CH).sub.nCH.sub.2—S—Ar—CH.sub.2—, —(C≡C).sub.nCH.sub.2—S—Ar—CH.sub.2—, —(CH═CH).sub.nCH.sub.2—NH—Ar—CH.sub.2—, —(C≡C).sub.nCH.sub.2—NH—Ar—CH.sub.2—, —(CH═CH).sub.nCH.sub.2—OCON—, —(C≡C).sub.nCH.sub.2—OCON—, —(CH═CH).sub.nCH.sub.2—OCSN—, ##STR00056## and —(C≡C).sub.nCH.sub.2—OCSN—; the n is 0, 1, 2, 3 or 4; the Ar is ##STR00057## and in the ##STR00058## Y is O, NH or S.

3. The compound according to claim 1, wherein the fluorescent dye is any one selected from a group consisting of coumarin, umbelliferone, aminocoumarin, fluorescein, resorufin, carboxyrhodamine, rhodamine, naphthalimide, cyanine, luciferin, CR110, EvoBlue, Alexa Fluor, Flamma, Indocyanine green, 2-((E)-2-((E)-2-(4-(2-carboxyethyl)phenoxy)-3-((E)-2-(3,3-dimethyl5-sulfonato-1-(3-(tri-methylamino)-propyl)indolin-2-ylidene)ethylidene)cyclohex-1-enyl)vinyl)-3,3-dimethyl-1-(3-(trimethylamino)-propyl)-3H-indolium-5-sulfonate disodium bromide and BODIPY.

4. The compound according to claim 3, wherein the BODIPY is any one selected from a group consisting of pyridyl BODIPY, BODIPY FL, BODIPY R6G, BODIPY TMR, BODIPY 581/591, BODIPY TR, BODIPY 630/650, BODIPY 650/665, BODIPY 558/568, BODIPY 564/570 and a combination thereof.

5. The compound according to claim 1, wherein the Z exhibits fluorescence as it is cleaved from Chemical Formula 1 together with the L by β-lactamase or carbapenemase.

6. The compound according to claim 1, wherein the compound represented by Chemical Formula 1 is selected from a group consisting of the following compounds; ##STR00059## ##STR00060## wherein TBS is t-butyldimethylsilyl and PNB is p-nitrobenzyl.

7. A probe for detecting antibiotic-resistant bacteria, comprising the compound according to claim 1.

8. The probe according to claim 7, wherein the antibiotic is a carbapenem-based antibiotic.

9. The probe according to claim 7, wherein the antibiotic-resistant bacteria are bacteria expressing carbapenemase.

10. The probe according to claim 8, wherein the carbapenem-based antibiotic is one or more antibiotic selected from a group consisting of imipenem, meropenem, ertapenem and doripenem.

11-14. (canceled)

15. A method for detecting antibiotic-resistant bacteria, comprising: (a) a step of contacting the compound according to claim 1 with a target sample; and (b) a step of detecting fluorescence from the compound according to claim 1 from the target sample.

16. The method according to claim 15, wherein the sample is one or more selected from a group consisting of a cell culture, blood, saliva, sputum, cerebrospinal fluid, urine, feces and a combination thereof.

17-20. (canceled)

Description

BEST MODE

[0053] In the present disclosure, it was confirmed that a broad spectrum of carbapenem-resistant bacteria can be detected with high sensitivity by contacting a library of carbapenem-linker-fluorophore represented by Chemical Formula 1 with a substrate of carbapenemase.

[0054] Accordingly, in an aspect, the present disclosure relates to a compound represented by Chemical Formula 1.

##STR00009##

[0055] In the above formula,

[0056] R is a hydrogen atom or C.sub.1-C.sub.2 alkyl;

[0057] L is substituted or unsubstituted vinyl; substituted or unsubstituted aryl; substituted or unsubstituted carbamate; or substituted or unsubstituted thiocarbamate; substituted or unsubstituted amine; substituted or unsubstituted pyridinium; and

[0058] Z is a fluorescent dye.

[0059] In the present disclosure, the L may be any one selected from a group consisting of —(CH═CH).sub.nCH.sub.2—, —(C≡C).sub.nCH.sub.2—, —(CH═CH).sub.nCH.sub.2—O—Ar—CH.sub.2—, —(C≡C).sub.nCH.sub.2—O—Ar—CH.sub.2—, —(CH═CH).sub.nCH.sub.2—S—Ar—CH.sub.2—, —(C≡C).sub.nCH.sub.2—S—Ar—CH.sub.2—, —(CH═CH).sub.nCH.sub.2—NH—Ar—CH.sub.2—, —(C≡C).sub.nCH.sub.2—NH—Ar—CH.sub.2—, —(CH═CH).sub.nCH.sub.2—OCON—, —(C≡C).sub.nCH.sub.2—OCON—, —(CH═CH).sub.nCH.sub.2—OCSN—,

##STR00010##

and —(C≡C).sub.nCH.sub.2—OCSN— (Ar=

##STR00011##

[0060] In the present disclosure, the L is a linker which connects the core structure of Chemical Formula 1 with Z.

[0061] In the present disclosure, the fluorescent dye may be any one selected from a group consisting of coumarin, umbelliferone, aminocoumarin, fluorescein, resorufin, carboxyrhodamine, rhodamine, naphthalimide, cyanine, luciferin, CR110, EvoBlue, Alexa Fluor, Flamma, Indocyanine green, 2-((E)-2-((E)-2-(4-(2-carboxyethyl)phenoxy)-3-((E)-2-(3,3-dimethyl5-sulfonato-1-(3-(tri-methylamino)-propyl)indolin-2-ylidene)ethylidene)cyclohex-1-enyl)vinyl)-3,3-dimethyl-1-(3-(trimethylamino)-propyl)-3H-indolium-5-sulfonate disodium bromide and BODIPY. However, the fluorescent dye is not limited as long as it emits light in the fluorescence region when it is cleaved from the β-lactam ring structure.

[0062] In the present disclosure, the EvoBlue may be EvoBlue 10 or EvoBlue 30, the Alexa Fluor may be one selected from a group consisting of Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750 and Alexa Fluor 790, and the Flamma may be one selected from a group consisting of Flamma496, Flamma507, Flamma 530, Flamma 552, Flamma 560, Flamma 575, Flamma 581, Flamma 648, Flamma 675, Flamma 749, Flamma 774 and Flamma 775.

[0063] In addition, in the present disclosure, the BODIPY may be any one selected from a group consisting of pyridyl BODIPY, BODIPY FL, BODIPY R6G, BODIPY TMR, BODIPY 581/591, BODIPY TR, BODIPY 630/650, BODIPY 650/665, BODIPY 558/568, BODIPY 564/570 and a combination thereof.

[0064] In the present disclosure, the “fluorescent dye” refers to a fluorescent moiety, and the fluorescent moiety may mean a fluorescent molecule which absorbs light of a specific frequency (e.g., UV light) and generates a fluorescence signal when cleaved from Chemical Formula 1, a derivative thereof or a conjugate thereof. For example, the fluorescent moiety may be a quencher dye. Non-limiting examples of the fluorescent moiety may include phenolic dyes such as umbelliferone, fluorescein and resorufin; aromatic amines; other compounds such as rhodamine, etc. In addition, the fluorescent moiety may be, for example, coumarin and related dyes; xanthene dyes such as fluorescein, rhodol and rhodamine; resorufin; cyanine dyes; bimane dyes; acridines; isoindole; dansyl dyes; aminophthalic hydrazides such as luminol and isoluminol derivatives; aminophthalimide; aminonaphthalimide; aminobenzofuran; aminoquinoline; dicyanohydroquinone; BODIPY; or europium and terbium complexes and related compounds.

[0065] In addition, the fluorescent moiety may further include a free carboxyl group, an ester (e.g., N-hydrosuccinimide (NHS) ester) or a maleimide derivative, and may also include a streptavidin, biotin, phalloidin, amine, azide or iodoacetamide conjugate.

[0066] In the present disclosure, the fluorescent moiety may be bound to the carbapenem-based compound via the linker. Accordingly, the fluorescent moiety according to a specific exemplary embodiment may exhibit fluorescence as it is cleaved from Chemical Formula 1 together with the linker (L) by β-lactamase or carbapenemase.

[0067] In the present disclosure, fluorescence may be emitted as Z is cleaved from Chemical Formula 1 together with L by β-lactamase or carbapenemase.

[0068] In an exemplary embodiment of the present disclosure, a broad spectrum of carbapenem-resistant bacteria can be detected using a substrate of carbapenemase using a library of the carbapenem-linker (red rectangle)-fluorophore.

##STR00012## ##STR00013##

[0069] In another aspect, the present disclosure relates to a probe for detecting antibiotic-resistant bacteria, which includes the compound.

[0070] In the present disclosure, the antibiotic may be a carbapenem-based antibiotic, and the antibiotic-resistant bacteria may be carbapenemase-expressing bacteria.

[0071] In the present disclosure, the carbapenem-based antibiotic may be selected from a group consisting of imipenem, meropenem, ertapenem and doripenem, although not being limited thereto.

[0072] The following chemical formulas show the chemical structures of imipenem, meropenem, doripenem and ertapenem.

##STR00014##

[0073] In another aspect, the present disclosure relates to a reagent composition for detecting antibiotic-resistant bacteria, which contains the compound.

[0074] In another aspect, the present disclosure relates to a kit for diagnosing infection by antibiotic-resistant bacteria, which includes the compound.

[0075] The fluorescence emitted from the compound represented by Chemical Formula 1 may be an indicator of the presence of ESBL, particularly carbapenemase and/or bacteria expressing the same, in a sample, and may be used for diagnosis of infectious diseases caused by the bacteria.

[0076] In the present disclosure, the bacterial infection may be infection by carbapenemase-expressing bacteria.

[0077] In the present disclosure, a disease caused by infection by carbapenemase-expressing bacteria may be one or more selected from a group consisting of skin and soft tissue infection, febrile neutropenia, respiratory tract infection, upper respiratory tract infection, bronchiolitis, (pathogenic) pneumonia, sepsis, encephalomeningitis, surgical infection, dysentery, infectious sinusitis, peritonitis, anthrax, Lyme disease, osteomyelitis, legionellosis, brucellosis, acute enteritis, community-acquired respiratory tract infection, trachoma, neonatal inclusion conjunctivitis, botulinum food poisoning, acute food poisoning, diarrhea, hemorrhagic colitis, bronchitis, gastric ulcer, endocarditis, salmonellosis, gastroenteritis, opportunistic infection, otitis media, paranasal sinusitis, pharyngitis, acne, keratosis pilaris, rosacea, harlequin ichthyosis, xeroderma pigmentosum, keratoderma, eczema and necrotizing fasciitis.

[0078] In the present disclosure, the “infectious disease” may refer to a disease or condition associated with the presence of an organism (infectious agent) in a subject or a patient. Particularly, it may refer to a “bacterial infectious disease”. For example, a “carbapenem-based antibiotic-resistant bacterial infectious disease” may mean an antibiotic-resistant bacterial infectious disease which is not effectively treated with carbapenem-based antibiotics.

[0079] In the present disclosure, “diagnosis” may include decision of the susceptibility of a subject to a specific disease or disorder, decision of the presence of a specific disease or disorder in a subject, decision of the prognosis of a subject having a specific disease or disorder (e.g., identification of an infectious disease or status or decision of the responsiveness to or effect of the treatment of the disease) or therametrics (e.g., monitoring of the status of a subject in order to provide information about therapeutic effects). The subject is not limited as long as it is a mammal suspected of a bacterial infectious disease. Specifically, it may be human.

[0080] In another aspect, the present disclosure relates to a method for detecting antibiotic-resistant bacteria, which includes: (a) a step of contacting the compound with a target sample; and (b) a step of detecting the intensity of fluorescence from the sample.

[0081] In the present disclosure, the sample may be one or more selected from a group consisting of a cell culture, blood, saliva, sputum, cerebrospinal fluid, urine feces and a combination thereof.

[0082] The step of contacting may include a step of adding the biological sample or a target sample, which has been pretreated, to a composition containing the compound of Chemical Formula 1. The pretreatment of the sample may be performed adequately by those skilled in the art depending on purposes.

[0083] An infectious disease caused by antibiotic-resistant bacteria may be diagnosed or antibiotic-resistant bacteria may be detected by contacting (reacting) with the target sample and measuring fluorescence emission due to hydrolysis of the compound of Chemical Formula 1 and, finally, analyzing the intensity of the fluorescence. The analysis of the fluorescence signal may be performed using various methods known in the art, and the result is interpreted and processed using an adequate apparatus available in the art. For example, protocols and processes known in the art, including a fluorescence analyzer, a microplate reader, an automated processing system using robotic devices, a laser scanning system, etc., may be used.

[0084] Hereinafter, the present disclosure will be described in more detail through examples. The following examples are provided only to illustrate the present disclosure, and it will be obvious to those having ordinary skill in the art that the scope of the present disclosure is not limited by the examples.

MODE FOR INVENTION

Example 1: Preparation of Probe for Detecting Antibiotic-Resistant Bacteria

1.1. Preparation of 4-nitrobenzyl (R)-4-((2R,3S)-3-((R)-1-((tert-butyldimethylsilyl)oxy)ethyl)-4-oxoazetidin-2-yl)-2-diazo-3-oxopentanoate (1)

[0085] ##STR00015##

[0086] After dissolving 1-hydroxyethyl azetidin-2-one (5.00 g, 10.8 mmol) in anhydrous dimethylformamide (42.0 mL) under nitrogen gas, imidazole (4.42 g, 64.8 mmol) and tert-butyldimethylsilyl chloride (7.72 g, 51.2 mmol) were added at 0° C. The reaction mixture was stirred at room temperature for 12 hours. After terminating reaction by adding an aqueous ammonium chloride solution, the reaction mixture was extracted with ethyl acetate and distilled water. The organic layer was dried by adding anhydrous MgSO.sub.4 and then filtered, and the filtrate was concentrated in vacuo. Compound 1 (6.16 g, 95%) was produced as white powder by purifying the resulting mixture on silica gel by column chromatography (ethyl acetate/n-hexane, 1:2 (v/v)).

[0087] .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 8.26 (d, J=8.6 Hz, 2H), 7.54 (d, J=8.6 Hz, 2H), 5.89 (s, 1H), 5.37 (d, J=13.2 Hz, 1H), 5.33 (d, J=13.2 Hz, 1H), 4.21-4.16 (m, 1H), 3.90-3.89 (m, 2H), 2.96 (d, J=2.9 Hz, 1H), 1.19 (d, J=6.0 Hz, 3H), 1.17 (d, J=5.9 Hz, 3H), 0.85 (s, 9H), 0.06 (s, 3H), 0.04 (s, 3H).

1.2. Preparation of 4-nitrobenzyl (4R,5R,6S)-6-((R)-1-((tert-butyldimethylsilyl)oxy)ethyl)-4-methyl-7-oxo-3-(((trifluoromethyl)sulfonyl)oxy)-1-azabicyclo[3.2.0]heptane-2-carboxylate (2)

[0088] ##STR00016##

[0089] After dissolving diazomethane (100 mg, 0.198 mmol) in dichloromethane (0.600 mL) in a sealed tube, rhodium acetate dimer (1 mg, 0.002 mmol) and zinc chloride (1 mg, 0.006 mmol) were added. The reaction mixture was warmed to 60° C. and then stirred for 1 hour. After slowly cooling a mixture of the ketone intermediate to −78° C. 2,2,6,6-tetramethylpiperidine (40 μL, 0.238 mmol) and N,N-diisopropylethylamine (17 μL, 0.100 mmol) were added and trifluoromethanesulfonic anhydride (37 μL, 1.68 mmol) was added dropwise over 5 minutes. After stirring for 1 hour, Compound 2 (117 mg, 97%) was produced as white powder by warming the mixture to room temperature and purifying on silica gel by column chromatography (dichloromethane).

[0090] .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 8.22 (d, J=8.7 Hz, 2H), 7.62 (d, J=8.8 Hz, 2H), 5.42 (d, J=13.6 Hz, 1H), 5.35 (d, J=13.6 Hz, 1H), 4.34 (dd, J=3.4, 11.0 Hz, 1H), 4.30-4.26 (m, 1H), 3.41-3.29 (m, 2H), 1.29 (d, J=7.3 Hz, 3H), 1.22 (d, J=6.2 Hz, 3H), 0.87 (s, 9H), 0.08 (s, 3H), 0.06 (s, 3H).

[0091] <Ketone Intermediate>

[0092] .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 8.23 (d, J=8.6 Hz, 2H), 7.52 (d, J=8.5 Hz, 2H), 5.31 (d, J=13.3 Hz, 1H), 5.25 (d, J=13.4 Hz, 1H), 4.71 (s, 1H), 4.32-4.28 (m, 1H), 4.24 (dd, J=2.4, 7.9 Hz, 1H), 3.22 (dd, J=2.4, 5.4 Hz, 1H), 2.82-2.74 (m, 1H), 1.26 (d, J=6.2 Hz, 3H), 1.20 (d, J=7.8 Hz, 3H), 0.87 (s, 9H), 0.09 (s, 3H), 0.07 (s, 3H).

1.3 Preparation of. (tributylstannyl)methanol (3)

[0093] ##STR00017##

[0094] After dissolving diisopropylamine (2.9 mL, 20.6 mmol) in anhydrous tetrahydrofuran (86 mL), n-butyllithium (1.6 M in hexane, 12.9 mL, 20.6 mmol) was added slowly at −78° C. After conducting reaction for 5 minutes, tributyltin hydride (5 g, 17.2 mmol) was added dropwise over 30 minutes. After stirring the mixture for 20 minutes, adding paraformaldehyde (722 mg, 24.1 mmol) and warming to room temperature, the mixture was stirred for 12 hours. After terminating reaction using an aqueous ammonium chloride solution, the reaction mixture was extracted with diethyl ether and an aqueous solution. The organic layer was dried by adding anhydrous MgSO.sub.4 and then filtered, and the filtrate was concentrated in vacuo. Compound 3 (3.05 g, 55%) was produced as colorless oil by purifying the resulting mixture on silica gel by column chromatography (diethyl ether/n-hexane, 1:3 (v/v)).

[0095] .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 4.01 (d, J=6.6 Hz, 1H), 4.00 (d, J=6.6 Hz, 1H), 1.55-1.47 (m, 6H), 1.35-1.25 (m, 6H), 0.93-0.67 (m, 15H).

1.4. Preparation of tert-butyldimethyl(prop-2-yn-1-yloxy)silane (4)

[0096] ##STR00018##

[0097] After dissolving propargyl alcohol (1.00 g, 17.8 mmol) in dichloromethane (42.0 mL), imidazole (1.82 g, 26.8 mmol) and tert-butyldimethylsilyl chloride (4.03 g, 26.8 mmol) were added at 0° C. The reaction mixture was warmed to room temperature and stirred for 12 hours. After terminating reaction using an aqueous ammonium chloride solution, the reaction mixture was extracted with diethyl ether and an aqueous solution. The organic layer was dried using anhydrous MgSO.sub.4 and then filtered, and the filtrate was concentrated in vacuo. Compound 4 (2.10 g, 69%) was produced as colorless oil by purifying the resulting mixture on silica gel by column chromatography (diethyl ether/n-hexane, 1:30 (v/v)).

[0098] .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 4.30 (d, J=2.4 Hz, 2H), 2.38 (d, J=2.4 Hz, 1H), 0.90 (s, 9H), 0.12 (s, 6H).

1.5. Preparation of (E)-3-(tributylstannyl)prop-2-en-1-ol (5)

[0099] ##STR00019##

[0100] Azobisisobutyronitrile (289 mg, 1.76 mmol) and tributyltin hydride (1.9 mL, 7.05 mmol) were added to the silyl ether compound (Compound 4, 1.00 g, 5.87 mmol). The reaction mixture was warmed to 80° C. and then stirred for 2 hours. After cooling the reaction mixture to 0° C. and dissolving in anhydrous tetrahydrofuran (11.7 mL), 1.0 M tetrabutylammonium fluoride dissolved in tetrahydrofuran (11.7 mL, 11.7 mmol) was added and the mixture was stirred for 2 hours. After terminating reaction using an aqueous ammonium chloride solution, the reaction mixture was extracted with diethyl ether and an aqueous solution. The organic layer was dried using anhydrous MgSO.sub.4 and then filtered, and the filtrate was concentrated in vacuo. Compound 5 (1.34 g, 66%) was produced as colorless oil by purifying the resulting mixture on silica gel by column chromatography (diethyl ether/n-hexane, 1:10 (v/v)).

[0101] .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 6.25-6.08 (m, 2H), 4.15 (bs, 2H), 1.50-1.44 (m, 6H), 1.32-1.26 (m, 6H), 0.90-0.85 (m, 15H).

1.6. Preparation of 4-nitrobenzyl (4S,5R,6S)-6-((R)-1-((tert-butyldimethylsilyl)oxy)ethyl)-3-(hydroxymethyl-4-methyl-7-oxo-1-azabicyclo[3.2.0]heptane-2-carboxylate (6)

[0102] ##STR00020##

[0103] After dissolving tri(2-furyl)phosphine (11.4 mg, 0.049 mmol), tris(dibenzylideneacetone)dipalladium-chloroform adduct (25.4 mg, 0.025 mmol) and zinc chloride (22.4 mg, 0.164 mmol) in anhydrous hexamethylphosphoric triamide (0.5 mL), the triflate compound (Compound 2, 100 mg, 0.164 mmol) and the stannane compound (Compound 3, 211 mg, 0.656 mmol) dissolved in hexamethylphosphoric triamide (0.1 mL) were slowly added to the mixture at 70° C. After stirring for 2 hours and cooling to 5° C., reaction was terminated using an aqueous ammonium chloride solution. The reaction mixture was extracted using ethyl acetate and an aqueous solution. The organic layer was dried using anhydrous MgSO.sub.4 and then filtered, and the filtrate was concentrated in vacuo. Compound 6 (42.0 mg, 52%) was produced as white powder by purifying the resulting mixture on silica gel by column chromatography (ethyl acetate/n-hexane, 1:8 to 1:2 (v/v)).

[0104] .sup.1H-NMR (400 MHz, CDCl3) δ 8.22 (d, J=8.7 Hz, 2H), 7.66 (d, J=8.7 Hz, 2H), 5.46 (d, J=13.8 Hz, 1H), 5.27 (d, J=13.9 Hz, 1H), 4.54 (dd, J=6.5, 14.8 Hz, 1H), 4.39 (dd, J=5.6, 14.8 Hz, 1H), 4.27-4.22 (m, 2H), 3.28-3.22 (m, 2H), 3.08-3.05 (m, 1H), 1.24 (d, J=6.2 Hz, 3H), 1.21 (d, J=7.4 Hz, 3H), 0.86 (s, 9H), 0.08 (s, 3H), 0.06 (s, 3H).

1.7. Preparation of 4-nitrobenzyl (4S,5R,6S)-6-((R)-1-((tert-butyldimethylsilyl)oxy)ethyl)-3-((E)-3-hydroxyprop-1-en-1-yl)-4-methyl-7-oxo-1-azabicyclo[3.2.0]heptane-2-carboxylate (7)

[0105] ##STR00021##

[0106] After dissolving tri(2-furyl)phosphine (19.1 mg, 0.082 mmol), tris(dibenzylideneacetone)dipalladium-chloroform adduct (42.5 mg, 0.041 mmol) and zinc chloride (10.9 mg, 0.182 mmol) in anhydrous N-methyl-2-pyrrolidinone (10.3 mL), the triflate compound (Compound 2, 500 mg, 0.821 mmol) and the stannane compound (Compound 5, 428 mg, 1.232 mmol) were added and the mixture was stirred at room temperature for 12 hours. After the stirring, the mixture was cooled to 5° C. and reaction was terminated using an aqueous ammonium chloride solution. The reaction mixture was extracted using ethyl acetate and an aqueous solution. The organic layer was dried using anhydrous MgSO.sub.4 and then filtered, and the solvent was removed from the filtrate in vacuo. Compound 7(315 mg, 74%) was produced as white powder by purifying the resulting mixture on silica gel by column chromatography (ethyl acetate/n-hexane, 1:4 to 1:2 (v/v)).

[0107] .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 8.22 (d, J=8.8 Hz, 2H), 7.67 (d, J=8.8 Hz, 2H), 7.31 (d, J=16.3 Hz, 1H), 6.20 (dt, J=5.5, 16.2 Hz, 1H), 5.44 (d, J=13.9 Hz, 1H), 5.27 (d, J=13.9 Hz, 1H), 5.27 (d, J=13.9 Hz, 1H), 4.32 (bs, 2H), 4.29-4.23 (m, 1H), 4.20 (dd, J=2.7, 9.4 Hz, 1H), 3.41-3.33 (m, 1H), 3.23 (dd, J=2.7, 5.6 Hz, 1H), 1.27 (d, J=6.2 Hz, 3H), 1.22 (d, J=7.3 Hz, 3H), 0.86 (s, 9H), 0.07 (s, 3H), 0.05 (s, 3H).

1.8. Preparation of 4-nitrobenzyl (4S,5R,6S)-6-((R)-1-((tert-butyldimethylsilyl)oxy)ethyl)-4-methyl-7-oxo-3-((E)-3-((2-oxo-2H-chromen-7-yl)oxy)prop-1-en-1-yl)-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylate (8)

[0108] ##STR00022##

[0109] After dissolving the alcohol compound (Compound 7, 100 mg, 0.194 mmol) in toluene (3.9 mL), umbelliferone (34.6 mg, 0.213 mmol) and triphenylphosphine (68.0 mg, 0.233 mmol) were added at 0° C. After stirring for 2 minutes, diisopropyl azodicarboxylate (49 μL, 0.290 mmol) was added to the mixture. The reaction mixture was warmed to room temperature and then stirred for 30 minutes. After terminating reaction using an aqueous ammonium chloride solution, the reaction mixture was extracted with ethyl acetate and distilled water. The organic layer was dried using anhydrous MgSO.sub.4 and then filtered, and the filtrate was concentrated in vacuo. Compound 8 (119 mg, 91%) was produced as white powder by purifying the resulting mixture on silica gel by column chromatography (ethyl acetate/n-hexane, 1:2 (v/v)).

[0110] .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 8.22 (d, J=8.6 Hz, 2H), 7.67 (d, J=8.7 Hz, 2H), 7.64 (d, J=9.6 Hz, 1H), 7.47 (d, J=8.3 Hz, 1H), 7.38 (d, J=8.6 Hz, 1H), 6.85 (dd, J=2.3, 8.6 Hz, 1H), 6.81 (d, J=7.0 Hz, 1H), 6.28-6.17 (m, 2H), 5.45 (d, J=13.8 Hz, 1H), 5.28 (d, J=14.0 Hz, 1H), 4.74 (d, J=5.8 Hz, 2H), 4.27-4.21 (m, 2H), 3.42-3.37 (m, 1H), 3.25 (dd, J=2.6, 5.4 Hz, 1H), 1.26 (d, J=6.2 Hz, 6H), 0.85 (s, 9H), 0.08 (s, 3H), 0.06 (s, 3H).

1.9. Preparation of 4-nitrobenzyl (4S,5R,6S)-6-((R)-1-hydroxyethyl)-4-methyl-7-oxo-3-((E)-3-((2-oxo-2H-chromen-7-yl)oxy)prop-1-en-1-yl)-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylate (9)

[0111] ##STR00023##

[0112] After dissolving the silyl ether compound (Compound 8, 40.1 mg, 0.059 mmol) in a 1:3 mixture of N-methyl-2-pyrrolidinone and dimethylformamide (1.2 mL), ammonium hydrogen fluoride (13.6 mg, 0.238 mmol) was added. The reaction mixture was stirred at room temperature for 30 hours. After terminating reaction with an aqueous sodium bicarbonate solution, the reaction mixture was extracted with ethyl acetate and distilled water. The organic layer was dried by adding anhydrous MgSO.sub.4 and then filtered, and the filtrate was concentrated in vacuo. Compound 9 (24.0 mg, 75%) was produced as white powder by purifying the resulting mixture on silica gel by column chromatography (ethyl acetate/n-hexane, 3:1 (v/v)).

[0113] .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 8.21 (d, J=8.7 Hz, 2H), 7.66 (d, J=8.7 Hz, 2H), 7.63 (d, J=9.6 Hz, 1H), 7.45 (d, J=16.3 Hz, 1H), 7.37 (d, J=8.6 Hz, 1H), 6.83 (dd, J=2.4, 8.6 Hz, 1H), 6.80 (d, J=2.3 Hz, 1H), 6.28 (d, J=4.8 Hz, 1H), 6.20 (dt, J=5.7, 16.3 Hz, 1H), 5.49 (d, J=13.8 Hz, 1H), 5.25 (d, J=13.8 Hz, 1H), 4.73 (d, J=5.8 Hz, 2H), 4.26-4.23 (m, 2H), 3.47-3.43 (m, 1H), 3.28 (dd, J=2.6, 6.9 Hz, 1H), 1.37 (d, J=6.2 Hz, 3H), 1.25 (d, J=7.2 Hz, 3H).

1.10. Preparation of (4S,5R,6S)-6-((R)-1-hydroxyethyl)-4-methyl-7-oxo-3-((E)-3-((2-oxo-2H-chromen-7-yl)oxy)prop-1-en-1-yl)-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid (OMCL01202)

[0114] ##STR00024##

[0115] After dissolving Compound 9 (36.0 mg, 0.066 mmol) and 5% Rh/C (4.2 mg) in a 2:1 mixture of tetrahydrofuran and an aqueous solution (2.1 mL) at 0° C., the mixture was warmed to room temperature. After stirring for 2 hours under hydrogen gas, the reaction mixture was filtered using a PTFE syringe filter. Compound OMCL01202 (12.0 mg, 44%) was produced as white powder by purifying the filtrate by preparative RP-HPLC (acetonitrile/aqueous solution, 2:8 to 100:0 (v/v)) and freeze-drying under reduced pressure.

[0116] .sup.1H-NMR (400 MHz, CD.sub.3OD) δ 7.87 (d, J=9.5 Hz, 1H), 7.52 (d, J=8.6 Hz, 1H), 7.46 (d, J=16.3 Hz, 1H), 6.97 (dd, J=2.4, 8.6 Hz, 1H), 6.93 (d, J=2.3 Hz, 1H), 6.22 (d, J=9.5 Hz, 1H), 6.03 (dt, J=6.1, 16.2 Hz, 1H), 4.74 (d, J=6.0 Hz, 2H), 4.10-4.05 (m, 2H), 3.37-3.26 (m, 1H), 3.15 (dd, J=2.4, 7.5 Hz, 1H), 1.28 (d, J=6.3 Hz, 3H), 1.27 (d, J=7.2 Hz, 3H).

1.11. Preparation of tert-butyl(4-((tert-butyldimethylsilyl)oxy)benzyl)oxy)dimethylsilane (11)

[0117] ##STR00025##

[0118] After dissolving hydroxybenzyl alcohol (500 mg, 4.03 mmol) in anhydrous dimethylformamide (20.2 mL), imidazole (2.02 g, 16.1 mmol) and tert-butyldimethylsilyl chloride (2.42 g, 16.1 mmol) were added at 0° C. The reaction mixture was warmed to room temperature and then stirred for 12 hours. After terminating reaction using an aqueous ammonium chloride solution, the reaction mixture was extracted with ethyl acetate and an aqueous solution. The organic layer was dried using anhydrous MgSO.sub.4 and then filtered, and the filtrate was concentrated in vacuo. Compound 11 (1.40 g, 99%) was produced as colorless oil by purifying the resulting mixture on silica gel by column chromatography (ethyl acetate/n-hexane, 1:8 to 1:4 (v/v)).

[0119] .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 7.17 (d, J=8.4 Hz, 2H), 6.79 (d, J=8.5 Hz, 2H), 4.67 (s, 2H), 0.98 (s, 9H), 0.93 (s, 9H), 0.18 (s, 6H), 0.08 (s, 6H).

1.12. Preparation of (4-((tert-butyldimethylsilyl)oxy)phenyl)methanol (12)

[0120] ##STR00026##

[0121] After dissolving the silyl ether compound (Compound 11, 35.3 mg, 0.10 mmol) in methanol (0.3 mL) at room temperature, N-iodosuccinimide (1.1 mg, 0.005 mmol) was added. After stirring for 12 hours and terminating reaction using an aqueous ammonium chloride solution, the reaction mixture was extracted with ethyl acetate and an aqueous solution. The organic layer was dried using anhydrous MgSO.sub.4 and then filtered, and the filtrate was concentrated in vacuo. Compound 12 (21.0 mg, 88%) was produced as colorless oil by purifying the resulting mixture on silica gel by column chromatography (ethyl acetate/n-hexane, 1:4 (v/v)).

[0122] .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 7.22 (d, J=8.5 Hz, 2H), 6.82 (dt, J=2.3, 9.0 Hz, 2H), 4.60 (s, 2H), 0.98 (s, 9H), 0.19 (s, 6H).

1.13. Preparation of 7-((4-((tert-butyldimethylsilyl)oxy)benzyl)oxy)-2H-chromen-2-one (13)

[0123] ##STR00027##

[0124] After dissolving the benzyl alcohol compound (Compound 12, 1.5 g, 5.34 mmol) in toluene (108 mL), umbelliferone (961 mg, 5.927 mmol) and triphenylphosphine (1.89 g, 6.47 mmol) were added at 0° C. After stirring for 2 minutes, diisopropyl azodicarboxylate (1.37 mL, 8.08 mmol) was added to the mixture. The reaction mixture was warmed to room temperature and then stirred for 30 minutes. After terminating reaction using an aqueous ammonium chloride solution, the reaction mixture was extracted with ethyl acetate and distilled water. The organic layer was dried using anhydrous MgSO.sub.4 and then filtered, and the filtrate was concentrated in vacuo. Compound 13 (2.02 g, 98%) was produced as white powder by purifying the resulting mixture on silica gel by column chromatography (ethyl acetate/n-hexane, 1:8 (v/v)).

[0125] .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 7.63 (d, J=9.5 Hz, 1H), 7.37 (d, J=8.4 Hz, 1H), 7.29 (d, J=8.4 Hz, 2H), 6.91 (d, J=2.3 Hz, 1H), 6.88 (d, J=8.7 Hz, 2H), 6.86 (d, J=8.4 Hz, 1H), 6.25 (d, J=9.4 Hz, 1H), 5.03 (s, 2H), 0.98 (s, 9H), 0.20 (s, 6H).

1.14. Preparation of 7-((4-hydroxybenzyl)oxy)-2H-chromen-2-one (14)

[0126] ##STR00028##

[0127] After dissolving Compound 13 (1.00 g, 2.61 mmol) in anhydrous tetrahydrofuran (26.1 mL) under argon gas, acetic acid (1.05 mL, 18.3 mmol) and tetrabutylammonium fluoride (1 M solution in THF, 13 mL, 13 mmol) were sequentially added slowly at 0° C. The reaction mixture was warmed to room temperature and then stirred for 3 hours. Compound 14 (635 mg, 91%) was produced as white powder by concentrating the reaction mixture in vacuo and purifying on silica gel by column chromatography (acetone/n-hexane, 1:2 (v/v)).

[0128] .sup.1H-NMR (400 MHz, Acetone-De) δ 7.89 (d, J=9.5 Hz, 1H), 7.68 (d, J=9.3 Hz, 1H), 7.35 (d, J=8.5 Hz, 2H), 6.98-6.96 (m, 2H), 6.87 (d, J=8.5 Hz, 2H), 6.21 (d, J=9.5 Hz, 1H), 5.13 (s, 2H).

1.15. Preparation of 4-nitrobenzyl (4S,5R,6S)-6-((R)-1-((tert-butylmethylsilyl)oxy)ethyl)-4-methyl-7-oxo-3-((4-(((2-oxo-2H-chromen-7-yl)oxy)methyl)phenoxy)methyl)-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylate (15)

[0129] ##STR00029##

[0130] After dissolving the alcohol compound (Compound 6, 129 mg, 0.263 mmol) in toluene (5.3 mL), the phenol compound (Compound 14, 77.6 mg, 0.289 mmol) and triphenylphosphine (92.2 mg, 0.316 mmol) were added at 0° C. After stirring for 2 minutes, diisopropyl azodicarboxylate (67 μL, 0.394 mmol) was added to the mixture. The reaction mixture was warmed to room temperature and then stirred for 30 minutes. After terminating reaction using an aqueous ammonium chloride solution, the reaction mixture was extracted with ethyl acetate and distilled water. The organic layer was dried by adding anhydrous MgSO.sub.4 and then filtered, and the filtrate was concentrated in vacuo. Compound 15 (76 mg, 46%) was produced as white powder by purifying the resulting mixture on silica gel by column chromatography (ethyl acetate/n-hexane/chloroform, 2:5:4 (v/v)).

[0131] .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 8.21 (d, J=8.7 Hz, 2H), 7.66 (d, J=9.0 Hz, 2H), 7.63 (d, J=9.8 Hz, 1H), 7.37 (d, J=8.5 Hz, 1H), 7.35 (d, J=8.6 Hz, 2H), 6.93 (d, J=8.7 Hz, 2H), 6.91-6.87 (m, 2H), 6.25 (d, J=9.4 Hz, 1H), 5.47 (d, J=14.6 Hz, 1H), 5.47 (d, J=13.8 Hz, 1H), 5.27 (d, J=13.8 Hz, 1H), 5.05 (s, 2H), 4.72 (d, J=15.1 Hz, 1H), 4.29-4.25 (m, 2H), 3.50-3.42 (m, 1H), 3.30 (dd, J=3.2, 5.2 Hz, 1H), 1.24 (d, J=6.2 Hz, 6H), 0.86 (s, 9H), 0.09 (s, 3H), 0.07 (s, 3H).

1.16. Preparation of 4-nitrobenzyl (4S,5R,6S)-6-((R)-1-hydroxyethyl)-4-methyl-7-oxo-3-((4-(((2-oxo-2H-chromen-7-yl)oxy)methyl)phenoxy)methyl)-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylate (16)

[0132] ##STR00030##

[0133] After dissolving the silyl ether compound (Compound 15, 95.0 mg, 0.128 mmol) in a 1:3 mixture of N-methyl-2-pyrrolidinone and dimethylformamide (2.4 mL), ammonium hydrogen fluoride (29.3 mg, 0.513 mmol) was added. The reaction mixture was stirred at room temperature for 30 hours. After terminating reaction with an aqueous sodium bicarbonate solution, the mixture was extracted with ethyl acetate and distilled water. The organic layer was dried by adding anhydrous MgSO.sub.4 and then filtered, and the filtrate was concentrated in vacuo. Compound 16 (58.0 mg, 72%) was produced as white powder by purifying the resulting mixture on silica gel by column chromatography (ethyl acetate/n-hexane, 3:1 (v/v)).

[0134] .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 8.22 (d, J=8.8 Hz, 2H), 7.67-7.62 (m, 3H), 7.38-7.33 (m, 3H), 6.92-6.89 (m, 3H), 6.86 (d, J=2.2 Hz, 1H), 6.25 (d, J=9.4 Hz, 1H), 5.52 (d, J=13.6 Hz, 1H), 5.47 (d, J=14.8 Hz, 1H), 5.26 (d, J=13.8 Hz, 1H), 5.06 (s, 2H), 4.74 (d, J=14.8 Hz, 1H), 4.29-4.24 (m, 2H), 3.52-3.44 (m, 1H), 3.33 (dd, J=3.0, 6.3 Hz, 1H), 1.35 (d, J=6.2 Hz, 3H), 1.25 (d, J=1.2 Hz, 3H).

1.17. Preparation of (4S,5R,6S)-6-((R)-1-hydroxyethyl-4-methyl-7-oxo-3-((4-(((2-oxo-2H-chromen-7-yl)oxy)methyl)phenoxy)methyl)-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid (OMCL01203)

[0135] ##STR00031##

[0136] After dissolving Compound 16 (26.0 mg, 0.041 mmol) and 5% Rh/C (2.6 mg) in a 2:1 mixture of tetrahydrofuran and an aqueous solution (1.4 mL) at 0° C., the mixture was warmed to room temperature. After stirring for 2 hours under hydrogen gas, the reaction mixture was filtered using a PTFE syringe filter. Compound OMCL01203 (10.0 mg, 50%) was produced as white powder by purifying the filtrate by preparative RP-HPLC (acetonitrile/aqueous solution, 2:8 to 100:0 (v/v)) and then freeze-drying under reduced pressure.

[0137] .sup.1H-NMR (400 MHz, CD.sub.3OD) δ 7.87 (d, J=9.6 Hz, 1H), 7.52 (d, J=9.3 Hz, 1H), 7.35 (d, J=8.5 Hz, 2H), 6.98-6.96 (m, 4H), 6.23 (d, J=9.5 Hz, 1H), 5.54 (d, J=13.5 Hz, 1H), 5.08 (s, 2H), 4.68 (d, J=13.4 Hz, 1H), 4.08-4.02 (m, 2H), 3.47-3.46 (m, 1H), 3.17 (dd, J=2.8, 7.5 Hz, 1H), 3.12-3.11 (m, 1H), 1.25 (d, J=6.3 Hz, 3H), 1.17 (d, J=7.1 Hz, 3H).

1.18. Preparation of 4-nitro-(4S,5R,6S)-6-((R)-1-((tert-butylmethylsilyl)oxy)ethyl)-4-methyl-7-oxo-3-((E)-3-(4-(((2-oxo-2H-chromen-7-yl)oxy)methyl)phenoxy)prop-1-en-1-yl)-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylate (18)

[0138] ##STR00032##

[0139] After dissolving the allyl alcohol compound (Compound 7, 20 mg, 0.039 mmol) in toluene ((0.8 mL), the phenol compound (Compound 14, 11.5 mg, 0.043 mmol) and triphenylphosphine (13.7 mg, 0.047 mmol) were added at 0° C. After stirring for 2 minutes, diisopropyl azodicarboxylate (9.8 μL, 0.058 mmol) was added to the mixture. The reaction mixture was warmed to room temperature and then stirred for 30 minutes. After terminating reaction using an aqueous ammonium chloride solution, the reaction mixture was extracted with ethyl acetate and distilled water. The organic layer was dried by adding anhydrous MgSO.sub.4 and then filtered. The solvent was removed from the filtrate in vacuo. Compound 18 (22 mg, 74%) was produced as white powder by purifying the resulting mixture on silica gel by column chromatography (ethyl acetate/n-hexane/chloroform, 2:5:4 (v/v)).

[0140] .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 8.21 (d, J=8.7 Hz, 2H), 7.66 (d, J=8.7 Hz, 2H), 7.63 (d, J=9.5 Hz, 1H), 7.43 (d, J=16.3 Hz, 1H), 7.38-7.34 (m, 3H), 6.93-6.84 (m, 4H), 6.25 (d, J=9.5 Hz, 1H), 6.24-6.19 (m, 1H), 5.44 (d, J=13.9 Hz, 1H), 5.27 (d, J=13.9 Hz, 1H), 5.05 (s, 2H), 4.69 (d, J=5.7 Hz, 2H), 4.27-4.24 (m, 1H), 4.20 (dd, J=2.6, 9.4 Hz, 1H), 3.43-3.35 (m, 1H), 3.24 (dd, J=2.6, 5.6 Hz, 1H), 1.26 (d, J=6.2 Hz, 3H), 1.23 (d, J=7.3 Hz, 3H), 0.86 (s, 9H), 0.08 (s, 3H), 0.06 (s, 3H).

1.19. Preparation of 4-nitrobenzyl (4S,5R,6S)-6-((R)-1-hydroxyethyl)-4-methyl-7-oxo-3-((E)-3-(4-(((2-oxo-2H-chromen-7-yl)oxy)methyl)phenoxy)prop-1-en-1-yl)-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylate (19)

[0141] ##STR00033##

[0142] After dissolving the silyl ether compound (Compound 18, 44.0 mg, 0.057 mmol) in a 1:3 mixture of N-methyl-2-pyrrolidinone and dimethylformamide (1:2 mL), ammonium hydrogen fluoride (13.1 mg, 0.229 mmol) was added. The reaction mixture was stirred at room temperature for 30 hours. After terminating reaction with an aqueous sodium bicarbonate solution, the mixture was extracted with ethyl acetate and distilled water. The organic layer was dried by adding anhydrous MgSO.sub.4 and then filtered, and the filtrate was concentrated in vacuo. Compound 19 (17.0 mg, 51%) was produced as white powder by purifying the resulting mixture on silica gel by column chromatography (ethyl acetate/n-hexane, 3:1 (v/v)).

[0143] .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 8.21 (d, J=8.6 Hz, 2H), 7.66 (d, J=8.8 Hz, 2H), 7.63 (d, J=9.7 Hz, 1H), 7.43 (d, J=16.5 Hz, 1H), 7.38-7.33 (m, 3H), 6.92-6.85 (m, 4H), 6.26-6.19 (m, 2H), 5.48 (d, J=13.8 Hz, 1H), 5.25 (d, J=13.8 Hz, 1H), 5.05 (s, 2H), 4.69 (d, J=5.4 Hz, 2H), 4.28-4.21 (m, 2H), 3.48-3.41 (m, 1H), 3.28 (dd, J=2.4, 6.7 Hz, 1H), 1.38 (d, J=6.2 Hz, 3H), 1.24 (d, J=7.5 Hz, 3H).

1.20. Preparation of (4S,5R,6S)-6-((R)-1-hydroxyethyl)-4-methyl-7-oxo-3-((E)-3-(4-(((2-oxo-2H-chromen-7-yl)oxy)methyl)phenoxy)prop-1-en-1-yl)-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid (OMCL01204)

[0144] ##STR00034##

[0145] After dissolving Compound 19 (36.0 mg, 0.066 mmol) and 5% Rh/C (4.2 mg) in a 2:1 mixture of tetrahydrofuran and an aqueous solution (2.1 mL) at 0° C., the mixture was warmed to room temperature. After stirring for 2 hours under hydrogen gas, the reaction mixture was filtered using a PTFE syringe filter. Compound OMCL01204 (12.0 mg, 44%) was produced as white powder by purifying the filtrate by preparative RP-HPLC (acetonitrile/aqueous solution, 2:8 to 100:0 (v/v)) and then freeze-drying under reduced pressure.

[0146] .sup.1H-NMR (400 MHz, CD.sub.3OD) δ 7.87 (d, J=9.4 Hz, 1H), 7.52 (d, J=9.2 Hz, 1H), 7.42 (d, J=16.2 Hz, 1H), 7.35 (d, J=8.4 Hz, 2H), 6.98-6.95 (m, 4H), 6.22 (d, J=9.5 Hz, 1H), 6.01 (dt, J=6.0, 16.1 Hz, 1H), 5.09 (s, 2H), 4.65 (d, J=5.4 Hz, 2H), 4.08-4.03 (m, 2H), 3.14-3.12 (m, 2H), 1.28 (d, J=6.3 Hz, 3H), 1.15 (d, J=7.2 Hz, 3H).

1.21. Preparation of 4-nitrobenzyl(4S,5R,6S)-6-((R)-1-((tert-butyldimethylsilyl)oxy)ethyl)-4-methyl-7-oxo-3-((((2-oxo-4-(trifluoromethyl)-2H-chromen-7-yl)carbamoyl)methyl)-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylate (21)

[0147] ##STR00035##

[0148] After dissolving 7-amino-4-trifluoromethylcoumarin (50.0 mg, 0.218 mmol) in anhydrous dichloromethane (2.2 mL), triethylamine (60 μL, 0.436 mmol) was added. After slowly adding triphosgene (65.0 mg, 0.218 mmol) 10 minutes later, the reaction mixture was stirred at 90° C. for 6 hours. The resulting mixture was concentrated in vacuo and used in the next reaction without purification. The isocyanate mixture, which is an intermediate, was dissolved in anhydrous tetrahydrofuran (0.5 mL) and the alcohol compound (Compound 6, 27.0 mg, 0.055 mmol) and triethylamine (23 μL, 0.165 mmol) were added. The reaction mixture was stirred at room temperature for 12 hours. Compound 21 (32.0 mg, 78%) was produced as bright yellow powder by concentrating the resulting mixture in vacuo and then purifying on silica gel by column chromatography (ethyl acetate/n-hexane/chloroform, 1:4:3 (v/v)).

[0149] .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 8.21 (d, J=8.7 Hz, 2H), 7.66-7.62 (m, 4H), 7.33 (dd, J=2.0, 8.9 Hz, 1H), 7.01 (s, 1H), 6.68 (s, 1H), 5.60 (d, J=14.1 Hz, 1H), 5.46 (d, J=13.8 Hz, 1H), 5.28 (d, J=13.8 Hz, 1H), 4.94 (d, J=14.1 Hz, 1H), 4.30-4.25 (m, 2H), 3.34-3.28 (m, 2H), 1.24-1.22 (m, 6H), 0.85 (s, 9H), 0.08 (s, 3H), 0.06 (s, 3H).

1.22. Preparation of 4-nitrobenzyl (4S,5R,6S)-6-((R)-1-hydroxyethyl)-4-methyl-7-oxo-3-((((2-oxo-4-(trifluoromethyl)-2H-chromen-7-yl)carbamoyl)oxy)methyl)-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylate (22)

[0150] ##STR00036##

[0151] After dissolving the silyl ether compound (Compound 21, 23.9 mg, 0.032 mmol) in a 1:3 mixture of N-methyl-2-pyrrolidinone and dimethylformamide (0.6 mL), ammonium hydrogen fluoride (7.4 mg, 0.120 mmol) was added. The reaction mixture was stirred at room temperature for 30 hours. After terminating reaction with an aqueous sodium bicarbonate solution, the mixture was extracted with ethyl acetate and distilled water. The organic layer was dried by adding anhydrous MgSO.sub.4 and then filtered, and the filtrate was concentrated in vacuo. Compound 22 (8.0 mg, 40%) was produced as white powder by purifying the resulting mixture on silica gel by column chromatography (ethyl acetate/n-hexane, 2:1 (v/v)).

[0152] .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 8.22 (d, J=8.6 Hz, 2H), 7.67-7.62 (m, 4H), 7.33 (dd, J=1.9, 8.8 Hz, 1H), 7.10 (s, 1H), 6.69 (s, 1H), 5.58 (d, J=14.2 Hz, 1H), 5.50 (d, J=13.6 Hz, 1H), 5.27 (d, J=13.7 Hz, 1H), 4.95 (d, J=14.2 Hz, 1H), 4.31-4.25 (m, 2H), 3.39-3.31 (m, 2H), 1.35 (d, J=6.2 Hz, 3H), 1.24 (d, J=7.2 Hz, 3H).

1.23. Preparation of (4S,5R,6S)-6-((R)-1-hydroxyethyl)-4-methyl-7-oxo-3-((((2-oxo-4-(trifluoromethyl)-2H-chromen-7-yl)carbamoyl)oxy)methyl)-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid (OMCL01205)

[0153] ##STR00037##

[0154] After dissolving Compound 22 (25.0 mg, 0.040 mmol) and 5% Rh/C (2.1 mg) in a 2:1 mixture of tetrahydrofuran and an aqueous solution (1.2 mL) at 0° C., the mixture was warmed to room temperature. After stirring for 2 hours under hydrogen gas, the mixture was filtered using a PTFE syringe filter. Compound OMCL01205 (7.0 mg, 35%) was produced as white powder by purifying the filtrate by preparative RP-HPLC (acetonitrile/aqueous solution, 2:8 to 100:0 (v/v)) and then freeze-drying under reduced pressure.

[0155] .sup.1H-NMR (400 MHz, CD.sub.3OD) δ 7.77 (d, J=2.1 Hz, 1H), 7.70-7.67 (m, 1H), 7.46 (dd, J=2.1, 8.9 Hz, 1H), 6.75 (s, 1H), 5.56 (d, J=13.5 Hz, 1H), 4.97 (d, J=13.4 Hz, 1H), 4.17-4.09 (m, 2H), 3.26-3.15 (m, 2H), 1.30 (d, J=6.3 Hz, 3H), 1.21 (d, J=7.3 Hz, 3H).

1.24. Preparation of 4-nitrobenzyl (4S,5R,6S)-6-((R)-1-((tert-butyldimethylsilyl)oxy)ethyl)-4-methyl-7-oxo-3-((E)-3-(((2-oxo-4-(trifluoromethyl)-2H-chromen-7-yl)carbamoyl)oxy)prop-1-en-1-yl)-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylate (24)

[0156] ##STR00038##

[0157] After dissolving 7-amino-4-trifluoromethylcoumarin (99.5 mg, 0.434 mmol) in anhydrous dichloromethane (4.4 mL), triethylamine (133 μL, 0.955 mmol) was added. After slowly adding triphosgene (128.8 mg, 0.434 mmol) 10 minutes later, the reaction mixture was stirred at 90° C. for 6 hours. The resulting mixture was concentrated in vacuo and used in the next reaction without purification. The isocyanate mixture, which is an intermediate, was dissolved in anhydrous tetrahydrofuran (1.0 mL) and then the allyl alcohol compound (Compound 7, 56.3 mg, 0.109 mmol) and triethylamine (46 μL, 0.327 mmol) were added. The reaction mixture was stirred at room temperature for 12 hours. Compound 24 (61.5 mg, 73%) was produced as bright yellow powder by concentrating the resulting mixture in vacuo and then purifying on silica gel by column chromatography (ethyl acetate/n-hexane/chloroform, 1:4:3 (v/v)).

[0158] .sup.1H-NMR (400 MHz, CD.sub.3CN) δ 8.33 (s, 1H), 8.11 (d, J=8.7 Hz, 2H), 7.63-7.59 (m, 4H), 7.33 (dd, J=2.1, 8.9 Hz, 1H), 7.27 (d, J=16.4 Hz, 1H), 6.67 (s, 1H), 6.20 (dt, J=5.6, 16.2 Hz, 1H), 5.37 (d, J=14.2 Hz, 1H), 5.24 (d, J=14.1 Hz, 1H), 4.78 (d, J=6.8 Hz, 2H), 4.25-4.18 (m, 2H), 3.46-3.42 (m, 1H), 3.28 (dd, J=3.0, 4.6 Hz, 1H), 1.17-1.14 (m, 6H), 0.80 (s, 9H), 0.05 (s, 3H), 0.01 (s, 3H).

1.25. Preparation of 4-nitrobenzyl (4S,5R,6S)-6-((R)-1-hydroxyethyl)-4-methyl-7-oxo-3-((E)-3-(((2-oxo-4-(trifluoromethyl)-2H-chromen-7-yl)carbamoyl)oxy)prop-1-en-1-yl)-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylate (25)

[0159] ##STR00039##

[0160] After dissolving the silyl ether Compound 24 (35.0 mg, 0.032 mmol) in a 1:3 mixture of N-methyl-2-pyrrolidinone and dimethylformamide (0.6 mL), ammonium hydrogen fluoride (7.4 mg, 0.120 mmol) was added. The reaction mixture was stirred at room temperature for 30 hours. After terminating reaction with an aqueous sodium bicarbonate solution, the mixture was extracted with ethyl acetate and distilled water. The organic layer was dried by adding anhydrous MgSO.sub.4 and then filtered, and the filtrate was concentrated in vacuo. Compound 25 (14.0 mg, 65%) was produced as white powder by purifying the resulting mixture on silica gel by column chromatography (ethyl acetate/n-hexane, 2:1 (v/v)).

[0161] .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 8.21 (d, J=8.7 Hz, 2H), 7.67-7.62 (m, 4H), 7.39 (d, J=16.0 Hz, 1H), 7.34 (d, J=2.1 Hz, 1H), 6.67 (s, 1H), 6.14 (dt, J=5.9, 16.2 Hz, 1H), 5.48 (d, J=13.8 Hz, 1H), 5.25 (d, J=13.8 Hz, 1H), 4.83 (d, J=5.7 Hz, 2H), 4.28-4.22 (m, 2H), 3.46-3.39 (m, 1H), 3.28 (dd, J=2.6, 6.7 Hz, 1H), 1.37 (d, J=6.2 Hz, 3H), 1.23 (d, J=7.3 Hz, 3H).

1.26. Preparation of (4S,5R,6S)-6-((R)-1-((tert-butyldimethylsilyl)oxy)ethyl)-4-methyl-7-oxo-3-((E)-3-(((2-oxo-4-(trifluoromethyl)-2H-chromen-7-yl)carbamoyl)oxy)prop-1-en-1-yl)-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid

[0162] ##STR00040##

[0163] After dissolving Compound 25 (48.0 mg, 0.073 mmol) and 5% Rh/C (3.8 mg) in a 2:1 mixture of tetrahydrofuran and an aqueous solution (1.2 mL) at 0° C., the mixture was warmed to room temperature. After stirring for 2 hours under hydrogen gas, the mixture was filtered using a PTFE syringe filter. Compound OMCL01206 (17.0 mg, 45%) was produced as white powder by purifying the filtrate by preparative RP-HPLC (acetonitrile/aqueous solution, 2:8 to 100:0 (v/v)) and then freeze-drying under reduced pressure.

[0164] .sup.1H-NMR (400 MHz, MeOD) δ 7.75 (d, J=2.1 Hz, 1H), 7.64 (dd, J=1.8, 8.9 Hz, 1H), 7.41 (d, J=2.2 Hz, 1H), 7.39 (d, J=2.1 Hz, 1H), 6.71 (s, 1H), 5.97 (dt, J=5.8, 16.3 Hz, 1H), 4.76 (d, J=5.3 Hz, 2H), 4.09-4.03 (m, 2H), 3.34-3.33 (m, 1H), 3.15-3.11 (m, 1H), 1.28 (d, J=6.2 Hz, 3H), 1.17 (d, J=7.2 Hz, 3H).

1.27. Preparation of tert-butyl(2-((tert-butyldimethylsiloxy)benzyl)oxy)dimethylsilane (27)

[0165] ##STR00041##

[0166] After dissolving hydroxybenzyl alcohol (500 mg, 4.03 mmol) in anhydrous dimethylformamide (20.2 mL), imidazole (1.27 g, 10.1 mmol) and tert-butyldimethylsilyl chloride (1.52 g, 10.1 mmol) were added at 0° C. The reaction mixture was warmed to room temperature and then stirred for 12 hours. After terminating reaction using an aqueous ammonium chloride solution, the reaction mixture was extracted with ethyl acetate and an aqueous solution. The organic layer was dried using anhydrous MgSO.sub.4 and then filtered, and the filtrate was concentrated in vacuo. Compound 27 (1.08 g, 76%) was produced as colorless oil by purifying the resulting mixture on silica gel by column chromatography (ethyl acetate/n-hexane, 1:15 (v/v)).

[0167] .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 7.46 (dt, J=1.0, 9.4 Hz, 1H), 7.11 (td, J=2.0, 9.6 Hz, 1H), 6.97 (td, J=1.2, 9.3 Hz, 1H), 6.74 (dd, J=1.2, 10.0 Hz, 1H), 4.76 (s, 2H), 1.01 (s, 9H), 0.95 (s, 9H), 0.22 (s, 6H), 0.10 (s, 6H).

1.28. Preparation of (2-((tert-butyldimethylsilyl)oxy)phenyl)methanol (28)

[0168] ##STR00042##

[0169] After dissolving the silyl ether compound (Compound 27, 636 mg, 1.80 mmol) in acetonitrile (25.8 mL) at room temperature, cerium chloride heptahydrate (13.4 g, 3.61 mmol) was added. After stirring at 90° C. for 12 hours and then terminating reaction using an aqueous ammonium chloride solution, the reaction mixture was extracted with ethyl acetate and an aqueous solution. The organic layer was dried using anhydrous MgSO.sub.4 and then filtered, and the filtrate was concentrated in vacuo. Compound 28 (49.0 mg, 72%) was produced as colorless oil by purifying the resulting mixture on silica gel by column chromatography (ethyl acetate/n-hexane, 1:8 to 1:4 (v/v)). 15 .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 7.31 (dd, J=2.0, 9.3 Hz, 1H), 7.18 (td, J=2.1, 9.7 Hz, 1H), 6.96 (dt, J=1.2, 9.3 Hz, 1H), 6.82 (d, J=10.1 Hz, 1H), 4.68 (s, 2H), 1.02 (s, 9H), 0.26 (s, 6H).

1.29. Preparation of 7-((2-((tert-butyldimethylsilyl)oxy)benzyl)oxy)-2H-chromen-2-one (29)

[0170] ##STR00043##

[0171] After dissolving the benzyl alcohol compound (Compound 28, 68.0 mg, 0.285 mmol) in toluene (5.80 mL), umbelliferone (50.8 mg, 0.314 mmol) and triphenylphosphine (100 mg, 0.342 mmol) were added at 0° C. After stirring for 2 minutes, diisopropyl azodicarboxylate (0.072 mL, 0.428 mmol) was added to the mixture. The reaction mixture was warmed to room temperature and then stirred for 30 minutes. After terminating reaction using an aqueous ammonium chloride solution, the reaction mixture was extracted with ethyl acetate and distilled water. The organic layer was dried by adding anhydrous MgSO.sub.4 and then filtered, and the filtrate was concentrated in vacuo. Compound 29 (84.0 mg, 77%) was produced as white powder by purifying the resulting mixture on silica gel by column chromatography (ethyl acetate/n-hexane, 1:8 (v/v)).

[0172] .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 7.62 (d, J=11.8 Hz, 1H), 7.39 (dd, J=1.9, 9.5 Hz, 1H), 7.36 (d, J=10.5 Hz, 1H), 7.22 (td, J=2.0, 9.7 Hz, 1H), 6.97 (td, J=1.2, 9.4 Hz, 1H), 6.91-6.85 (m, 3H), 6.24 (d, J=11.8 Hz, 1H), 5.12 (s, 2H), 0.97 (s, 9H), 0.25 (s, 6H).

1.30. Preparation of 7-((2-hydroxybenzyl)oxy)-2H-chromen-2-one (30)

[0173] ##STR00044##

[0174] After dissolving Compound 29 (427 mg, 1.12 mmol) in anhydrous tetrahydrofuran (11.2 mL) under argon gas, acetic acid (0.447 mL, 7.81 mmol) and tetrabutylammonium fluoride (1 M solution in THF, 5.6 mL, 5.58 mmol) were slowly added sequentially at 0° C. The reaction mixture was warmed to room temperature and then stirred for 3 hours. Compound 30 (292 mg, 98%) was produced as white powder by concentrating the reaction mixture in vacuo and then purifying on silica gel by column chromatography (acetone/n-hexane, 1:2 (v/v)).

[0175] .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 7.89 (d, J=11.8 Hz, 1H), 7.58 (d, J=10.4 Hz, 1H), 7.41 (dd, J=1.8, 9.4 Hz, 1H), 7.20 (td, J=1.9, 9.6 Hz, 1H), 7.01-6.98 (m, 2H), 6.94 (d, J=9.4 Hz, 1H), 6.88 (td, J=1.1, 9.3 Hz, 1H), 6.21 (d, J=11.9 Hz, 1H), 5.25 (s, 2H).

1.31. Preparation of 4-nitrobenzyl (4S,5R,6S)-6-((R)-1-((tert-butylmethylsilyl)oxy)ethyl)-4-methyl-7-oxo-3-((2-(((2-oxo-2H-chromen-7-yl)oxy)methyl)phenoxy)methyl)-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylate (31)

[0176] ##STR00045##

[0177] After dissolving the alcohol compound (Compound 6, 117 mg, 0.238 mmol) in toluene (4.8 mL), the phenol compound (Compound 30, 70.4 mg, 0.262 mmol) and triphenylphosphine (83.5 mg, 0.286 mmol) were added at 0° C. After stirring for 2 minutes, diisopropyl azodicarboxylate (60 μL, 0.357 mmol) was added to the mixture. The reaction mixture was warmed to room temperature and then stirred for 30 minutes. After terminating reaction using an aqueous ammonium chloride solution, the reaction mixture was extracted with ethyl acetate and distilled water. The organic layer was dried by adding anhydrous MgSO.sub.4 and then filtered, and the filtrate was concentrated in vacuo. Compound 31 (87.6 mg, 50%) was produced as white powder by purifying the resulting mixture on silica gel by column chromatography (ethyl acetate/n-hexane/chloroform, 2:5:4 (v/v)).

[0178] .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 8.22 (d, J=10.8 Hz, 2H), 7.68-7.58 (m, 3H), 7.42 (d, J=9.5 Hz, 1H), 7.37 (d, J=11.6 Hz, 1H), 7.32 (t, J=9.1 Hz, 1H), 7.02 (t, J=9.2 Hz, 1H), 6.95-6.89 (m, 3H), 6.26 (d, J=11.8 Hz, 1H), 5.53 (d, J=18.2 Hz, 1H), 5.48 (d, J=17.2 Hz, 1H), 5.28 (d, J=17.2 Hz, 1H), 5.05 (s, 2H), 4.80 (d, J=18.4 Hz, 1H), 4.27-4.20 (m, 2H), 3.41-3.33 (m, 1H), 3.27 (t, J=5.0 Hz, 1H), 1.21 (d, J=5.8 Hz, 3H), 1.20 (d, J=4.3 Hz, 3H), 0.83 (s, 9H), 0.08 (s, 3H), 0.02 (s, 3H).

1.32. Preparation of 4-nitrobenzyl (4S,5R,6S)-6-((R)-1-hydroxyethyl)-4-methyl-7-oxo-3-((2-(((2-oxo-2H-chromen-7-yl)oxy)methyl)phenoxy)methyl)-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylate (32)

[0179] ##STR00046##

[0180] After dissolving the silyl ether compound (Compound 31, 69.9 mg, 0.094 mmol) in a 1:3 mixture of N-methyl-2-pyrrolidinone and dimethylformamide (1.9 mL), ammonium hydrogen fluoride (21.6 mg, 0.378 mmol) was added. The reaction mixture was stirred at room temperature for 30 hours. After terminating reaction with an aqueous sodium bicarbonate solution, the mixture was extracted with ethyl acetate and distilled water. The organic layer was dried by adding anhydrous MgSO.sub.4 and then filtered, and the filtrate was concentrated in vacuo. Compound 32 (28.1 mg, 48%) was produced as white powder by purifying the resulting mixture on silica gel by column chromatography (ethyl acetate/n-hexane, 3:1 (v/v)).

[0181] .sup.1H-NMR (400 MHz, CDCl3) δ 8.21 (d, J=10.8 Hz, 2H), 7.67-7.59 (m, 3H), 7.42 (d, J=9.5 Hz, 1H), 7.37 (d, J=11.6 Hz, 1H), 7.32 (t, J=9.1 Hz, 1H), 7.01 (t, J=9.2 Hz, 1H), 6.96-6.89 (m, 3H), 6.25 (d, J=11.8 Hz, 1H), 5.52 (d, J=18.2 Hz, 1H), 5.48 (d, J=17.2 Hz, 1H), 5.28 (d, J=17.2 Hz, 1H), 5.06 (s, 2H), 4.79 (d, J=18.4 Hz, 1H), 4.28-4.21 (m, 2H), 3.41-3.43 (m, 1H), 3.28 (dd, J=3.0, 6.3 Hz, 1H), 1.35 (d, J=6.2 Hz, 3H), 1.25 (d, J=7.2 Hz, 3H).

1.33. Preparation of (4S,5R,6S)-6-((R)-1-hydroxyethyl)-4-methyl-7-oxo-3-((2-(((2-oxo-2H-chromen-7-yl)oxy)methyl)phenoxy)methyl)-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid (OMCL01207)

[0182] ##STR00047##

[0183] After dissolving Compound 32 (22.0 mg, 0.035 mmol) and 5% Rh/C (2.2 mg) in a 2:1 mixture of tetrahydrofuran and an aqueous solution (1.2 mL) at 0° C., the mixture was warmed to room temperature. After stirring for 2 hours under hydrogen gas, the mixture was filtered using a PTFE syringe filter. Compound OMCL01207 (5.4 mg, 31%) was produced as white powder by purifying the filtrate by preparative RP-HPLC (acetonitrile/aqueous solution, 2:8 to 10:0 (v/v)) and then freeze-drying under reduced pressure.

[0184] .sup.1H-NMR (400 MHz, CD.sub.3OD) δ 7.82 (d, J=9.5 Hz, 1H), 7.57 (d, J=11.6 Hz, 1H), 7.45 (d, J=9.1 Hz, 1H), 7.39 (d, J=9.2 Hz, 1H), 6.95-6.89 (m, 3H), 6.26 (d, J=11.8 Hz, 1H), 5.53 (d, J=18.2 Hz, 1H), 5.48 (d, J=17.2 Hz, 1H), 5.28 (d, J=17.2 Hz, 1H), 5.05 (s, 2H), 4.80 (d, J=18.4 Hz, 1H), 4.27-4.20 (m, 2H), 3.41-3.33 (m, 1H), 3.27 (t, J=5.0 Hz, 1H), 1.21 (d, J=5.8 Hz, 3H), 1.20 (d, J=4.3 Hz, 3H).

1.34. Preparation of 4-nitrobenzyl(4S,5R,6S)-6-((R)-1-((tert-butyldimethylsilyl)oxy)ethyl)-4-methyl-3-((((4-methyl-2-oxo-2H-chromen-7-yl)carbamoyl)oxy)methyl)-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylate (34)

[0185] ##STR00048##

[0186] After dissolving 7-amino-4-methylcoumarin (224 mg, 1.28 mmol) in anhydrous dichloromethane (6.4 mL), triethylamine (393 μL, 2.81 mmol) was added. After slowly adding triphosgene (129 mg, 1.28 mmol) 10 minutes later, the reaction mixture was stirred at 90° C. for 6 hours. The resulting mixture was concentrated in vacuo and used in the next reaction without purification. After dissolving the isocyanate mixture, which is an intermediate, in anhydrous tetrahydrofuran (1.6 mL), the alcohol compound (Compound 6,157 mg, 0.320 mmol) and triethylamine (134 μL, 0.960 mmol) were added. The reaction mixture was stirred at room temperature for 12 hours. Compound 34 (72.0 mg, 33%) was produced as white powder by concentrating the resulting mixture in vacuo and then purifying on silica gel by column chromatography (ethyl acetate/n-hexane/chloroform, 2:4:5 (v/v)).

[0187] .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 8.21 (dd, J=2.3, 8.6 Hz, 2H), 7.66 (d, J=10.9 Hz, 2H), 7.54 (d, J=10.9 Hz, 1H), 7.41 (d, J=10.1 Hz, 2H), 6.95 (s, 1H), 6.20 (d, J=1.4 Hz, 1H), 5.59 (d, J=17.7 Hz, 1H), 5.46 (d, J=17.2 Hz, 1H), 5.29 (d, J=17.3 Hz, 1H), 4.93 (d, J=17.8 Hz, 1H), 4.29-4.25 (m, 2H), 3.33-3.29 (m, 2H), 2.41 (d, J=1.3 Hz, 3H), 1.23 (d, J=7.6 Hz, 3H), 1.22 (d, J=3.1 Hz, 3H), 0.86 (s, 9H), 0.08 (s, 3H), 0.06 (s, 3H).

1.35. Preparation of 4-nitrobenzyl(4S,5R,6S)-6-((R)-1-hydroxyethyl)-4-methyl-3-((((4-methyl-2-oxo-2H-chromen-7-yl)carbamoyl)oxy)methyl)-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylate (35)

[0188] ##STR00049##

[0189] After dissolving the silyl ether compound (Compound 34, 82.0 mg, 0.119 mmol) in a 1:3 mixture of N-methyl-2-pyrrolidinone and dimethylformamide (2.4 mL), ammonium hydrogen fluoride (27.0 mg, 0.474 mmol) was added. The reaction mixture was stirred at room temperature for 30 hours. After terminating reaction with an aqueous sodium bicarbonate solution, the mixture was extracted with ethyl acetate and distilled water. The organic layer was dried by adding anhydrous MgSO.sub.4 and then filtered, and the filtrate was concentrated in vacuo. Compound 35 (35.0 mg, 51%) was produced as white powder by purifying the resulting mixture on silica gel by column chromatography (ethyl acetate/n-hexane, 3:1 (v/v)).

[0190] .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 8.23 (d, J=10.9 Hz, 2H), 7.66 (d, J=10.8 Hz, 2H), 7.54 (d, J=10.8 Hz, 1H), 7.41-7.40 (m, 2H), 6.96 (s, 1H), 6.20 (d, J=1.3 Hz, 1H), 5.58 (d, J=17.8 Hz, 1H), 5.51 (d, J=17.0 Hz, 1H), 5.25 (d, J=17.1 Hz, 1H), 4.93 (d, J=18.1 Hz, 1H), 4.31-4.26 (m, 2H), 3.40-3.26 (m, 2H), 2.38 (d, J=10.2 Hz, 3H), 1.35 (d, J=7.8 Hz, 3H), 1.25 (d, J=9.1 Hz, 3H).

1.36. Preparation of (4S,5R,6S)-6-((R)-1-hydroxyethyl)-4-methyl-3-((((4-methyl-2-oxo-2H-chromen-7-yl)carbamoyl)oxy)methyl)-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid (OMCL01208)

[0191] ##STR00050##

[0192] After dissolving Compound 35 (35.0 mg, 0.061 mmol) and 5% Rh/C (3.8 mg) in a 2:1 mixture of tetrahydrofuran and an aqueous solution (2.1 mL) at 0° C., the mixture was warmed to room temperature. After stirring for 2 hours under hydrogen gas, the mixture was filtered using a PTFE syringe filter. Compound OMCL01208 (7.0 mg, 26%) was produced as white powder by purifying the filtrate by preparative RP-HPLC (acetonitrile/aqueous solution, 2:8 to 10:0 (v/v)) and then freeze-drying under reduced pressure.

[0193] .sup.1H-NMR (400 MHz, CD.sub.3OD) δ 7.71 (d, J=11.0 Hz, 1H), 7.63 (d, J=2.4 Hz, 1H), 7.44 (dd, J=2.5, 10.8 Hz, 1H), 6.22 (s, 1H), 5.55 (d, J=16.9 Hz, 1H), 4.96 (d, J=17.0 Hz, 1H), 4.17-4.10 (m, 2H), 3.24-3.22 (m, 2H), 2.47 (s, 3H), 1.30 (d, J=7.9 Hz, 3H), 1.21 (d, J=9.1 Hz, 3H).

1.37. Preparation of 4-nitrobenzyl(4S,5R,6S)-6-((R)-1-tert-butyldimethylsilyl)oxy)ethyl)-3-formyl-4-methyl-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylate (37)

[0194] ##STR00051##

[0195] After dissolving the alcohol compound (Compound 6, 20.0 mg, 0.041 mmol) in anhydrous dichloromethane (0.3 mL) at 0° C., Dess-Martin periodinane (19.1 mg, 0.045 mmol) was added. The reaction mixture was stirred for 1 hour. After terminating reaction at 5° C. with an aqueous solution, the mixture was extracted with dichloromethane and distilled water. The organic layer was dried by adding anhydrous MgSO.sub.4 and then filtered, and the filtrate was concentrated in vacuo. Compound 37 (12.7 mg, 64%) was produced as bright yellow powder by purifying the resulting mixture on silica gel by column chromatography (ethyl acetate/n-hexane, 1:8 (v/v)).

[0196] .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 10.4, (s, 1H), 8.24 (d, J=10.8 Hz, 2H), 7.66 (d, J=10.8 Hz, 2H), 5.50 (d, J=17.1 Hz, 1H), 5.36 (d, J=17.1 Hz, 1H), 4.36 (dd, J=4.3, 13.0 Hz, 1H), 4.33-4.27 (m, 1H), 3.55-3.47 (m, 1H), 3.41 (t, J=5.0 Hz, 1H), 1.24 (d, J=9.4 Hz, 3H), 1.22 (d, J=8.0 Hz, 3H), 0.85 (s, 9H), 0.10 (s, 3H), 0.06 (s, 3H).

1.38. Preparation of 4-nitrobenzyl(4S,5R,6S)-6-((R)-1-((tert-butyldimethylsilyl)oxy)ethyl)-4-methyl-3-((((4-methyl-2-oxo-2H-chromen-7-yl)amino)methyl)-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylate (38)

[0197] ##STR00052##

[0198] After dissolving 7-amino-4-methylcoumarin (56.7 mg, 0.324 mmol) in anhydrous dichloromethane (1.7 mL), the aldehyde compound (Compound 37, 105.4 mg, 0.216 mmol) and acetic acid (2 μL, 0.216 mmol) were added. After stirring for 3 hours and adding sodium triacetoxyborohydride (137 mg, 0.648 mmol), the reaction mixture was stirred at room temperature for 12 hours. After terminating reaction with an aqueous sodium bicarbonate solution, the mixture was extracted with ethyl acetate and distilled water. The organic layer was dried by adding anhydrous MgSO.sub.4 and then filtered, the filtrate was concentrated in vacuo. Compound 38 (66.6 mg, 48%) was produced as yellow powder by purifying the resulting mixture on silica gel by column chromatography (ethyl acetate/n-hexane, 1:2 (v/v)).

[0199] .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 8.20 (d, J=10.7 Hz, 2H), 7.67 (d, J=10.6 Hz, 2H), 7.34 (d, J=11.2 Hz, 1H), 6.58-6.50 (m, 3H), 5.98 (s, 1H), 5.48 (d, J=17.4 Hz, 1H), 5.30 (d, J=17.4 Hz, 1H), 4.72 (d, J=20.4 Hz, 1H), 4.24 (t, J=7.1 Hz, 1H), 4.18 (dd, J=3.5, 12.8 Hz, 1H), 3.98 (d, J=20.5 Hz, 1H), 3.26-3.17 (m, 2H), 2.33 (s, 3H), 1.20 (d, J=8.2 Hz, 6H), 0.83 (s, 9H), 0.06 (s, 3H), 0.04 (s, 3H).

1.39. Preparation of 4-nitrobenzyl(4S,5R,6S)-6-((R)-1-hydroxyethyl)-4-methyl-3-((((4-methyl-2-oxo-2H-chromen-7-yl)amino)methyl)-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylate (39)

[0200] ##STR00053##

[0201] After dissolving the silyl ether compound (Compound 38,107 mg, 0.155 mmol) in a 1:3 mixture of N-methyl-2-pyrrolidinone and dimethylformamide (3.2 mL), ammonium hydrogen fluoride (35.4 mg, 0.621 mmol) was added. The reaction mixture was stirred at room temperature for 30 hours. After terminating reaction with an aqueous sodium bicarbonate solution, the mixture was extracted with ethyl acetate and distilled water. The organic layer was dried by adding anhydrous MgSO.sub.4 and then filtered, and the filtrate was concentrated in vacuo. Compound 39 (48.1 mg, 54%) was produced as bright yellow powder by purifying the resulting mixture on silica gel by column chromatography (ethyl acetate/n-hexane, 3:1 (v/v)).

[0202] .sup.1H-NMR (400 MHz, CDCl.sub.3) δ 8.18 (d, J=10.8 Hz, 2H), 7.65 (d, J=10.8 Hz, 2H), 7.30 (d, J=10.8 Hz, 1H), 6.48 (dd, J=2.8, 10.8 Hz, 1H), 6.42 (d, J=2.7 Hz, 1H), 5.95 (s, 1H), 5.51 (d, J=17.42 Hz, 1H), 5.25 (d, J=17.2 Hz, 1H), 4.71 (d, J=20.8 Hz, 1H), 4.19-4.15 (m, 2H), 3.95 (d, J=20.9 Hz, 1H), 3.25-3.18 (m, 2H), 2.33 (s, 3H), 1.29 (d, J=7.8 Hz, 3H), 1.18 (d, J=9.1 Hz, 3H). 1.40. Preparation of (4S,5R,6S)-6-((R)-1-hydroxyethyl)-4-methyl-3-((((4-methyl-2-oxo-2H-chromen-7-yl)amino)methyl)-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid (OMCL01209)

##STR00054##

[0203] After dissolving Compound 39 (40.5 mg, 0.076 mmol) and 5% Rh/C (6.4 mg) in a 2:1 mixture of tetrahydrofuran and an aqueous solution (2.4 mL) at 0° C., the mixture was warmed to room temperature. After stirring for 2 hours under hydrogen gas, the mixture was filtered using a PTFE syringe filter. Compound OMCL01209 (7.3 mg, 24%) was produced as white powder by purifying the filtrate by preparative RP-HPLC (acetonitrile/aqueous solution, 2:8 to 10:0 (v/v)) and then freeze-drying under reduced pressure.

[0204] .sup.1H-NMR (400 MHz, CD.sub.3OD) δ 7.46 (d, J=11.1 Hz, 1H), 6.67 (dd, J=2.5, 10.9 Hz, 1H), 6.48 (d, J=2.5 Hz, 1H), 5.91 (s, 1H), 5.48 (s, 1H), 4.78 (d, J=20.1 Hz, 1H), 4.04 (t, J=8.5 Hz, 1H), 3.98 (dd, J=3.4, 12.3 Hz, 1H), 3.88 (d, J=20.2 Hz, 1H), 3.13-3.05 (m, 2H), 2.36 (s, 3H), 1.24 (d, J=7.8 Hz, 3H), 1.14 (d, J=9.1 Hz, 3H).

Example 2: Detection of Antibiotic-Resistant Bacteria from Clinical Sample

[0205] The detection efficiency of carbapenemase-producing carbapenem-resistant bacteria of a control probe OMCL01201 (CPC-1 described in Chinese Patent Publication No. CN106279178A) and the OMCL01203 probe of the present disclosure was evaluated at Gangnam St. Mary's Hospital for the bacteria described in Table 1.

[0206] Briefly, 10 μL of a sample was lysed with a Tris-HCl buffer (B-PER, Thermo Scientific Pierce) and then vortexed for 1 minute. After culturing for 30 minutes at room temperature and centrifuging for 5 minutes (10,000 g), 30 μL of the supernatant was reacted with 100 μL of PBS and 13 μL of the fluorescent probe (1 mM). Then, fluorescence was measured for 50 minutes with 5-minute intervals using a fluorescence plate reader (Infinite F200pro, Tecan Group Ltd.).

TABLE-US-00001 TABLE 1 Carbapenemase No. of CPC-1 OMCL01203 class and gene isolates result(s) Pos. % result(s) Pos. % Ambler class A (39) KPC 33 15 45% 33 100% GES 2 0  0% 2 100% Ambler class B (17) NDM 9 8 89% 8  89% VIM 7 3 43% 7 100% IMP 1 0  0% 1 100% Ambler class D (6) OXA 6 1 17% 5  83% co-producing (3) KPC/OXA 2  0% 2 100% NDM/OXA 1 1 100%  1 100% CPE total 61 28 46% 59  97% Non-CP CRE (40) 40 0  0% 0  0%

[0207] As a result, the control probe CPC-1 determined 28 out of 61 CPE (carbapenemase-producing Enterobacteriaceae) species (46%) as positive, and determined all 40 non-CPE species as negative.

[0208] In contrast, the OMCL01203 probe determined 59 out of the 61 CPE species (97%) as positive, and determined all the 40 non-CPE species as negative.

[0209] In addition, the detection efficiency of the fluorescence detection method using the OMCL01203 probe was compared for carbapenemase-producing carbapenem-resistant bacteria (CP-CRE) and non-carbapenemase-producing carbapenem-resistant bacteria (non-CP-CRE) with mCIM, CarbaNP and CIT methods. The result is shown in Table 2.

[0210] As a result, the fluorescence detection method using the OMCL01203 probe determined only 3 out of 65 positive bacteria as false positive, and determined all negative bacteria as negative.

[0211] In contrast, the mCIM method showed a higher false-negative rate, the CarbNP method showed a higher false-positive rate, and the CIT method showed higher false-positive and false-negative rates, when compared with the fluorescence detection method using the OMCL01203 probe.

[0212] Accordingly, it was confirmed from the above results that the probe developed in the present disclosure has superior detection ability.

[0213] Although the specific exemplary embodiments of the present disclosure have been described in detail, it will be obvious to those having ordinary knowledge in the art that they are only preferred exemplary embodiments and the scope of the present disclosure is not limited by them. Thus, the substantial scope of the present disclosure is limited by the appended claims and their equivalents.

TABLE-US-00002 TABLE 2 Carbapenemase No. of Fluore mCIM CNP CIT class and gene isolates result result result result Carbapenemase No. of MICs (μg/ml) Fluore mCIM CNP CIT class and gene Species isolates IPM MEM ERT result result result result CPE (65) Ambler Class A (39) KPC (36) Citrobacter 1 >16 8 >16 Pos Pos Pos Pos-A freundii Escherichia 3 2 to 4 8 Pos Pos Pos Pos-A coli 8 Klebsiella 1 >16 16 >16 Pos Pos Pos Pos-A aerogenes Klebsiella 1 >16 >16 >16 Pos Pos Pos Pos-A oxytoca Klebsiella 30 4 to 8 to 16 to Pos Pos Pos Pos-A pneumoniae >16 >16 >16 GES (3) Escherichia 2 1 to 2 to 8 to Pos Neg Neg(2) Pos-A(1); coli >16 >16 >16 Neg(1) Klebsiella 1 >16 >16 >16 Neg(1) Neg Neg(1) Pos-A pneumoniae Ambler Class B (17) NDM (9) Citrobacter 1 8 16 >16 Pos Pos Pos-B Pos-B freundii Enterobacter 2 >16 >16 >16 Pos Pos Pos-B Pos-B cloacae Escherichia 4 8 to >16 >16 Pos Pos Pos-B Pos-B coli 16 Klebsiella 1 >16 >16 >16 Pos Pos Pos-B Pos-B pneumoniae Morganella 1 >16 4 2 Pos Pos Pos-B Pos-B morganii VIM (7) Citrobacter 4 0.5 to 16 to 2 to Pos Pos Pos-B(3); Pos-B(3); freundii >16 >16 >16 Neg(1) Neg(1) Enterobacter 1 4 2 1 Pos Pos Neg(1) Pos-B cloacae Klebsiella 1 2 1 0.5 Pos Pos Neg(1) Neg(1) oxytoca Klebsiella 1 >16 >16 >16 Pos Pos Pos-B Pos-B pneumoniae IMP (1) Escherichia 1 1 1 4 Pos Pos Neg(1) Neg(1) coli Ambler Class D (6) OXA (6) Citrobacter 1 2 0.5 1 Pos Pos Neg(1) Neg(1) freundii Escherichia 3 0.5 to 8 =0.25 to ≤=0.125 to Pos(1); Pos(2) Neg(3) Neg(3) coli 4 >16 Neg(2) Neg(1) Klebsiella 2 2 to 4 0.5 to 2 to Pos Pos Pos(1); Neg(2) pneumoniae 16 >16 Neg(1) co-producing (3) KPC/OXA (2) Klebsiella 2 4 to 16 4 to 8 to Pos Pos Pos Pos-A pneumoniae >16 >16 NDM/OXA (1) Klebsiella 1 4 4 16 Pos Pos Pos-B Pos-B pneumoniae non-CPE (154) non-CP-CRE Citrobacter 1 1 0.5 2 Neg Neg Neg Neg (48) freundii Enterobacter 4 8 to 2 to 16 to Neg Neg Neg Neg amnigenus 16 8 >16 Enterobacter 10 0.5 to =0.25 to 2 to Neg Neg Neg Pos-A(1); cloacae >16 >16 16 Neg(9) Escherichia 8 0.5 to =0.25 to 4 to Neg Neg Neg Neg coli 16 8 >16 Klebsiella 3 1 to 0.5 to 4 to Neg Neg Neg Neg aerogenes >16 16 >16 Klebsiella 22 0.5 to 0.5 to 4 to Neg Neg Neg Neg pneumoniae 16 8 >16 ESBL-producer (53) CTX-M (53) Escherichia 29 =0.25 to ≤=0.25 to ≤=0.125 to Neg Ind(2); Neg Neg coli 1 1 1 Neg(27) Klebsiella 24 =0.25 to ≤=0.25 ≤=0.125 to Neg Ind(2); Neg Neg pneumoniae 1 1 Neg(22) non-producer Escherichia 32 =0.25 to ≤=0.25 ≤=0.125 Neg Neg Neg Neg (53) coli 0.5 Klebsiella 21 =0.25 to ≤=0.25 ≤=0.125 Neg Neg Neg Neg pneumoniae 1 Abbreviations: CPE, carbapenemase-producing Enterobacteriaceae; ESBL, extended-spectrum-β-lactamase; MIC, minimum inhibitory concentration; IPM, imipenem; MEM, meropenem; ERT, ertapenem; Fluore, fluorogenic assay; mCIM, modified carbapenem inactivation method; CNP, Carba NP test; CIT, carbapenemase inhibition test. Abbreviations: Pos, positive; Neg, negative; Ind, indeterminate; Pos-A, Ambler class A carbapenemase detected; Pos-B, metallo-β-lactamase detected.

INDUSTRIAL APPLICABILITY

[0214] The present disclosure relates to a novel compound capable of detecting beta-lactamase and carbapenemase. The compound can detect beta-lactamase and carbapenemase with high sensitivity and, therefore, can be applied to various biochemical researches. In addition, the compound can clinically detect antibiotic-resistant bacteria and can be usefully used for in-vitro diagnosis of antibiotic-resistant bacterial infectious diseases.