ANTIBACTERIAL AMINOGLYCOSIDE DERIVATIVES
20220227802 · 2022-07-21
Inventors
- Dongdong TANG (Shanghai, CN)
- Zhigang HUANG (Shanghai, CN)
- Cheng Li (Shanghai, CN)
- Charles Z. Ding (Shanghai, CN)
- Shuhui Chen (Shanghai, CN)
Cpc classification
C07H15/224
CHEMISTRY; METALLURGY
Y02A50/30
GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
International classification
Abstract
Disclosed are a new class of antibacterial aminoglycoside derivatives, pharmaceutical compositions containing such compounds, and application thereof in the preparation of drugs for treating diseases related to bacterial infections. Specifically disclosed are a compound represented by formula (II), pharmaceutically acceptable salts thereof, and isomers thereof.
##STR00001##
Claims
1. A compound represented by formula (II), a pharmaceutically acceptable salt thereof, or an isomer thereof: ##STR00021## wherein, ##STR00022## L is —O—CH.sub.2—CH.sub.2— or —CH.sub.2—; R.sub.1 is H or C.sub.1-3 alkyl; R.sub.2 is H, C.sub.1-3 alkyl or ##STR00023## wherein C.sub.1-3 alkyl is optionally substituted by 1, 2 or 3 substituent(s) independently selected from the group consisting of F, Cl, Br, I, —OH, —OCH.sub.3—CN, —NH.sub.2 and —NO.sub.2; R.sub.a and R.sub.b each independently is H, —C(═O)—NH.sub.2, —C(═O)—C.sub.1-3 alkyl or C.sub.1-3 alkyl, wherein —C(═O)—C.sub.1-3 alkyl and C.sub.1-3 alkyl are optionally substituted by 1, 2 or 3 R; and each R is independently F, Cl, Br, I, —OH, —OCH.sub.3, —CN or —NH.sub.2.
2. The compound, the pharmaceutically acceptable salt thereof, or the isomer thereof according to claim 1, wherein the compound has a structure represented by formula (I): ##STR00024## wherein R.sub.1 is H or C.sub.1-3alkyl; R.sub.2 is H, C.sub.1-3 alkyl or ##STR00025## wherein C.sub.1-3 alkyl is optionally substituted by 1, 2 or 3 substituent(s) independently selected from the group consisting of F, Cl, Br, I, —OH, —OCH.sub.3, —CN, —NH.sub.2 and —NO.sub.2; R.sub.a and R.sub.b each independently is H, —C(═O)—NH.sub.2, —C(═O)—C.sub.1-3 alkyl or C.sub.1-3 alkyl, wherein —C(═O)—C.sub.1-3 alkyl and C.sub.1-3 alkyl are optionally substituted by 1, 2 or 3 R; and each R is independently F, Cl, Br, I, —OH, —OCH.sub.3, —CN or —NH.sub.2.
3. The compound, the pharmaceutically acceptable salt thereof, or the isomer thereof according to claim 2, wherein the compound has a structure represented by formula (I-1): ##STR00026## wherein R.sub.a, R.sub.b and R.sub.1 are as defined in claim 2.
4. The compound, the pharmaceutically acceptable salt thereof, or the isomer thereof according to claim 1, wherein R.sub.1 is H or —CH.sub.3.
5. The compound, the pharmaceutically acceptable salt thereof, or the isomer thereof according to claim 4, wherein the compound has a structure represented by formula (I-2): ##STR00027## wherein R.sub.a and R.sub.b are as defined in claim 4.
6. The compound, the pharmaceutically acceptable salt thereof, or the isomer thereof according to claim 1, wherein each R is independently F or Cl.
7. The compound, the pharmaceutically acceptable salt thereof, or the isomer thereof according to claim 1, wherein R.sub.a and R.sub.b each independently is H, —C(═O)—NH.sub.2, —C(═O)—CH.sub.3, —CH.sub.3 or —CH.sub.2CH.sub.3, and wherein —C(═O)—CH.sub.3, —CH.sub.3 and —CH.sub.2CH.sub.3 are optionally substituted by 1, 2 or 3 R.
8. The compound, the pharmaceutically acceptable salt thereof, or the isomer thereof according to claim 7, wherein R.sub.a and R.sub.b each independently is H, —C(═O)—NH.sub.2, —C(═O)—CH.sub.3, —CH.sub.3, —CH(R).sub.2, —CH.sub.2CH.sub.3 or —CH.sub.2CH(R).sub.2.
9. The compound, the pharmaceutically acceptable salt thereof, or the isomer thereof according to claim 8, wherein R.sub.a and R.sub.b each independently is H or ##STR00028##
10. The compound, the pharmaceutically acceptable salt thereof, or the isomer thereof according to claim 1, wherein R.sub.2 is H, —CH.sub.3, —CH.sub.2CH.sub.3 or ##STR00029## and wherein —CH.sub.3 and —CH.sub.2CH.sub.3 are optionally substituted by 1, 2 or 3 substituent(s) independently selected from the group consisting of F, Cl, Br, I, —OH, —OCH.sub.3, —CN, —NH.sub.2 and —NO.sub.2.
11. The compound, the pharmaceutically acceptable salt thereof, or the isomer thereof according to claim 10, wherein R.sub.2 is ##STR00030##
12. The compound, the pharmaceutically acceptable salt thereof, or the isomer thereof according to claim 1, wherein the structure unit ##STR00031##
13. The compound, the pharmaceutically acceptable salt thereof, or the isomer thereof according to claim 12, wherein the structure unit ##STR00032##
14. A compound of the following formula, the pharmaceutically acceptable salt thereof or the isomer thereof: ##STR00033##
15. A pharmaceutical composition, comprising a therapeutically effective amount of the compound, the pharmaceutically acceptable salt thereof, or the isomer thereof according to claim 1 as an active ingredient, and a pharmaceutically acceptable carrier.
16. Use of the compound, the pharmaceutically acceptable salt thereof, or the isomer thereof according to claim 1 in the manufacture of a medicament for the treatment of bacterial infection-related diseases.
17. The use according to claim 16, wherein the bacteria are Carbapenem-resistant Enterobacteriaceae.
18. A pharmaceutical composition, comprising a therapeutically effective amount of the compound, the pharmaceutically acceptable salt thereof, or the isomer thereof according to claim 14 as an active ingredient, and a pharmaceutically acceptable carrier.
19. Use of the compound, the pharmaceutically acceptable salt thereof, or the isomer thereof according to claim 14 in the manufacture of a medicament for the treatment of bacterial infection-related diseases.
20. Use of the pharmaceutical composition according to claim 15 in the manufacture of a medicament for the treatment of bacterial infection-related diseases.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED EMBODIMENTS
[0084] The present invention is described in detail through the following examples, which are not meant any adverse limitation to the present invention. While the present invention are described in detail herein with its specific embodiments being disclosed, various changes and improvements made thereto will be obvious for those skilled in the art without departing from the spirit and scope of the present invention.
Example 1: Compound 1
[0085] ##STR00018##
[0086] Diphenylphosphinyl hydroxylamine (10 g, 42.88 mmol, 1 eq), Compound 1-1 (13.65 g, 128.64 mmol, 12.19 mL, 3 eq) and sodium tert-butoxide (4.95 g, 51.46 mmol, 1.2 eq) were dissolved in tetrahydrofuran (100 mL), stirred and reacted at 5-15° C. for 16 hours. The reaction liquid was filtered, and the filtrate was concentrated to obtain Compound 1-2.
[0087] Step 2:
[0088] Compound 1-2 (5.19 g, 42.84 mmol, 1 eq) obtained in the previous step in tetrahydrofuran (100 mL) and N,N-di-BOC-1H-pyrazole-1-carboxamidine (13.30 g, 42.84 mmol, 1 eq) were stirred and reacted at 66° C. for 16 hours. The reaction liquid was cooled to room temperature, and extracted with ethyl acetate (100 mL×2) after water (300 mL) was added.
[0089] The organic phases were combined, dried over sodium sulfate and filtered. The filtrate was concentrated to obtain a crude product, and the Compound 1-3 was obtained through column chromatography (silica, petroleum ether/ethyl acetate=20/1, 1/1 (v/v)).
[0090] Step 3:
[0091] Compound 1-3 (1 g, 2.75 mmol, 1 eq) and 2-iodoxybenzoic acid (847.59 mg, 3.03 mmol, 1.1 eq) were dissolved in dimethyl sulfoxide (10 mL), and the reaction liquid was stirred at 40° C. for reaction 1 hour. The reaction liquid was filtered, and the filtrate was extracted with tert-butyl methyl ether (20 mL×2 times) after water (40 mL) was added. The organic phases were combined and washed with saturated sodium thiosulfate (10 mL). The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated to obtain Compound 1-4.
[0092] Step 4:
[0093] Amberlite (ion exchange resin) IRA-402(OH) (500 g) was added to methanol (500 mL), and the solution was stirred at 20° C. for 1 hour. Then the mixture was filtered, the filter cake was added to methanol (500 mL), and then Compound 1-5 was added into this mixture. The mixture was stirred at 20° C. for 11 hours. During the reaction, Compound 1-5 dissolved. The reaction liquid was filtered, and the filtrate was concentrated to obtain Compound 1-6. LCMS (ESI) m/z: 448.4 (M+1).
[0094] Step 5:
[0095] Compound 1-6 (15 g, 33.52 mmol, 1 eq) was dissolved in methanol (150 mL), and then S-ethyl 2,2,2-trifluoroethyl thioester (4.24 g, 26.82 mmol, 0.8 eq) in methanol (150 mL) was added dropwise to the above methanol solution. The mixed solution was stirred at 20° C. for 16 hours. Then zinc acetate (14.72 g, 80.44 mmol, 2.4 eq) was added to the solution, and then (N-hydroxy-5-norbornene-2,3-dicarboxyl-imido)-tert-butyl ester (16.85 g, 60.33 mmol, 1.8 eq) and triethylamine (10.17 g, 100.55 mmol, 14.00 mL, 3 eq) in tetrahydrofuran (170 mL) were added dropwise to the mixed solution. The reaction liquid was stirred at 20° C. for 30 hours, then quenched with glycine (7 g), and then concentrated. The concentrated liquid was diluted with dichloromethane (1000 mL), and then washed twice with aqueous solution of (300 mL) (water:ammonia=7:3). The organic phase was concentrated. The crude product was purified by column chromatography (silica, dichloromethane/methanol=50/1-5/1 (v/v), containing a small amount of ammonia water) to obtain Compound 1-7. LCMS (ESI) m/z: 744.3 (M+1).
[0096] Step 6:
[0097] (2S)-4-(tert-butyloxycarbonylamino)-2-hydroxy-butyric acid (6.85 g, 31.26 mmol, 1.5 eq) was dissolved into N,N-dimethylformamide (150 mL) and N-hydroxy-5-norbornene-2,3-dicarboximide (5.60 g, 31.26 mmol, 1.5 eq) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (4.85 g, 31.26 mmol, 5.53 mL, 1.5 eq) were added to the solution. The reaction liquid was stirred at 20° C. for 2 hours, followed by adding Compound 1-7 (15.5 g, 20.84 mmol, 1 eq) therein. The reaction liquid was stirred at 20° C. for 16 hours, then diluted with water (200 mL), and extracted with ethyl acetate (50 mL×3). The combined organic phases were washed with saturated brine (100 mL), then dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated to obtain a mixture. The mixture was purified by column chromatography (silica, dichloromethane/methanol=50/1-10/1 (v/v)) to obtain Compound 1-8. LCMS (ESI) m/z: 945.5 (M+1).
[0098] Step 7:
[0099] Compound 1-8 (16.40 g, 17.35 mmol, 1 eq), di-tert-butyl dicarbonate (4.55 g, 20.83 mmol, 4.78 mL, 1.2 eq), DIEA (2.69 g, 20.83 mmol, 3.6 mL, 1.2 eq) were dissolved in tetrahydrofuran (170 mL). Nitrogen replacement was performed for three times. The reaction liquid was stirred at 20° C. for 16 hours. The reaction liquid was diluted with water (200 mL), and then extracted with dichloromethane (100 mL×2). The combined organic phases were washed successively with 0.1M hydrochloric acid (20 mL) and saturated brine (60 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated to obtain a solid mixture. The mixture is purified by column chromatography (silica, petroleum ether/ethyl acetate=15/1-0/1 (v/v)) to obtain the target Compound 1-9. LCMS (ESI) m/z: 1045.3 (M+1).
[0100] Step 8:
[0101] Compound 1-9 (15.00 g, 14.35 mmol, 1 eq) and ammonia water (63.70 g, 1.82 mol, 70 mL, 126.62 eq) were dissolved in methanol (80 mL), and the mixture was stirred at 20° C. for 16 hours. The reaction liquid was concentrated to remove the solvent, diluted with water (100 mL), and extracted with dichloromethane (100 mL×3 times). The combined organic phases were washed with saturated brine (200 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated, and the concentrated mixture was purified by column chromatography (silica, first petroleum ether/ethyl acetate=10/1-0/1 (v/v), followed by dichloromethane/methanol=6/1 (v/v), the eluent contained a small amount of ammonia water) to obtain Compound 1-10. LCMS (ESI) m/z: 949.3 (M+1).
[0102] Step 9:
[0103] Compound 1-4 (50.63 mg, 0.15 mmol) and Compound 1-10 (145.00 mg, 0.15 mmol) were dissolved in methanol (5.00 mL), and then 4A molecular sieve (0.5 g) was added. The mixture was stirred for 0.5 hour at 18° C. under nitrogen atmosphere. Then sodium cyanoborohydride (19.20 mg, 0.30 mmol) was added and stirred for 1 hour. The completion of the reaction was detected by LCMS. The reaction liquid was filtered and concentrated, and separated by preparative-IPLC: Phenomenex Synergi C18 150×25 mm×10 μm; mobile phase: [water (0.225% formic acid)-acetonitrile]; acetonitrile %: 60%-90%, for 10 min to obtain Compound 1-11. LCMS (ESI) m/z: 1294.7 (M+1).
[0104] Step 10:
[0105] Compound 1-11 (91.00 mg, 71.97 mol) was dissolved in anhydrous dichloromethane (2.00 mL), cooled to 0° C. under nitrogen atmosphere. Trifluoroacetic acid (1.54 g, 13.51 mmol) was added and the reaction liquid was stirred at 0-19° C. for 9 hours, concentrated at room temperature, slurried with acetonitrile/methyl tert-butyl ether (4 mL, 1/3), filtered and concentrated to obtain Compound 1.
[0106] .sup.1H NMR (400 MHz, D.sub.2O) δ (ppm): 5.62 (s, 1H), 5.24 (s, 1H), 5.08 (s, 1H), 4.09-4.06 (m, 1H), 3.98-3.96 (m, 2H), 3.94-3.93 (m, 5H), 3.77-3.73 (m, 4H), 3.24 (s, 1H), 3.22-3.10 (m, 6H), 2.83 (s, 3H), 2.61-2.14 (m, 2H), 2.04-2.14 (m, 6H), 1.26 (s, 3H); LCMS (ESI) m/z: 664.5 (M+1).
Example 2: Compound 2
[0107] ##STR00019##
[0108] Step 1:
[0109] Compound 2-1 (1 g, 12.19 mmol, 71.94 μL, 1.2 eq), N,N-bis-BOC-1H-pyrazole-1-carboxamidine (3.15 g, 10.16 mmol, 1 eq) and triphenylphosphine (3.20 g, 12.19 mmol, 1.2 eq) were dissolved in tetrahydrofuran (40 mL), DIAD (2.46 g, 12.19 mmol, 2.37 mL, 1.2 eq) was added dropwise at 0° C. Then the mixture was heated to 20° C. and stirred for 12 hours. Water (100 mL) was added to the reaction solution, which was then extracted with ethyl acetate (50 mL, 3 times). The combined organic phases were washed with water (30 mL, 3 times), dried over anhydrous sodium sulfate, filtered and concentrated to obtain a crude product. The crude product was purified by chromatography column (silica, petroleum ether/ethyl acetate=50/1 to 20/1 (v/v)) to obtain Compound 2-2.
[0110] Step 2:
[0111] Compound 2-2 (3.25 g, 8.67 mmol, 0.5 eq) was added to Compound 1-2 (2.1 g, 17.34 mmol, 1 eq) in tetrahydrofuran (50 mL) at 20° C., and the reaction liquid was stirred at 67° C. for 12 hours. Water (100 mL) was added to the reaction liquid, which was then extracted with ethyl acetate (50 mL×3). The combined organic phases were washed with water (30 mL, 3 times), dried over anhydrous sodium sulfate, filtered and concentrated to obtain a crude product, which was purified by chromatography column (silica, petroleum ether/ethyl acetate=10/1 to 1/1 (v/v)) to obtain Compound 2-3.
[0112] Step 3:
[0113] 2-Iodoxy benzoic acid (108.09 mg, 386.02 μmol, 1.1 eq) was added to Compound 2-3 (150 mg, 350.93 μmol, 1 eq) in dimethyl sulfoxide (3 mL) at 40° C. The reaction liquid was stirred at 40° C. for 2 hours. Saturated sodium bicarbonate/sodium thiosulfate (30 mL, (v/v)) was added to the reaction liquid, and the reaction liquid was extracted with ethyl acetate (20 mL×2). The combined organic phases were washed with saturated sodium bicarbonate/sodium thiosulfate (10 mL×3 times (1/1, v/v)), dried over anhydrous sodium sulfate, filtered and concentrated to obtain Compound 2-4.
[0114] Step 4:
[0115] Compound 2-5 (118 mg, 277.37 mol, 1.1 eq) and 4A molecular sieve (300 mg) were added to Compound 1-10 (239.32 mg, 252.16 mol, 1 eq) in 1,2-dichloroethane (2 mL) at 20° C. the mixture was stirred for 1 hour, and then sodium acetate borohydride (64.13 mg, 302.59 mol, 1.2 eq) was added. The reaction liquid was stirred at 20° C. for 12 hours. with Water (20 mL) was added to the reaction liquid, which then was extracted with dichloromethane (20 mL×3). The combined organic phases were washed with water (10 mL×3), dried over anhydrous sodium sulfate, filtered and concentrated to obtain a crude product, which was then purified by preparative HPLC (column: Phenomenex Synergi C18 150×25 mm×10 μm; mobile phase: [water (0.225% formic acid)-acetonitrile]; acetonitrile %: 35%-56%, 7 min) to obtain Compound 2-6. LCMS (ESI) m/z: 1358.7 (M+1).
[0116] Step 5:
[0117] Compound 2-8 (40 mg, 29.44 μmol, 1 eq) was dissolved in dichloromethane (1 mL), and trifluoroacetic acid (1.54 g, 13.51 mmol, 1 mL, 458.70 eq) was added at 0° C. The reaction liquid was heated to 20° C. and stirred for 2 hours, and then cooled to 0° C. Methyl tert-butyl ether (15 mL) was added and the mixture was filtered, washed with methyl tert-butyl ether (2 mL×3), and dried with an oil pump at 40° C. to obtain Compound 2.
[0118] .sup.1H NMR (400 MHz, D.sub.2O) δ=6.18-5.87 (m, 1H), 5.63 (s, 1H), 5.28-5.22 (m, 1H), 5.08 (d, J=3.8 Hz, 1H), 4.23-4.16 (m, 1H), 4.09-4.03 (m, 3H), 3.98-3.89 (m, 2H), 3.83 (br t, J=5.2 Hz, 1H), 3.79-3.68 (m, 8H), 3.46-3.38 (m, 1H), 3.33 (br d, J=13.0 Hz, 1H), 3.26-3.22 (m, 2H), 3.16-3.07 (m, 2H), 2.83 (s, 3H), 2.69-2.55 (m, 1H), 2.42-2.29 (m, 1H), 2.19-2.06 (m, 2H), 1.95-1.85 (m, 1H), 1.83-1.71 (m, 1H), 1.29-1.23 (m, 3H).
[0119] LCMS (ESI) m/z: 758.3 (M+1).
Example 3: Compound 3
[0120] ##STR00020##
[0121] Step 1:
[0122] Compound 3-1 (16.00 g, 115.12 mmol) was dissolved in acetonitrile (200.00 mL), and N—BOC-hydroxylamine (15.33 g, 115.12 mmol) and DBU (19.28 g, 126.63 mmol) were added successively. The mixture was reacted at 11° C.-25° C. for 16 hours, and then concentrated. The residue was diluted with ethyl acetate (350 mL), washed with water (100 mL×3), washed with saturated brine (100 mL) once, dried over anhydrous sodium sulfate and concentrated. The residue was separated by column chromatography (filler: silica gel powder, eluent: ethyl acetate/petroleum ether=0-1/1 (v/v)) to obtain Compound 3-2.
[0123] Step 2:
[0124] Compound 3-2 (1.00 g, 5.23 mmol) and a solution of hydrogen chloride in dioxane (10 mL, 4 mmol) were mixed together and stirred at 20° C. for 16 hours, and then concentrated under reduced pressure to obtain Compound 3-3.
[0125] Step 3:
[0126] Compound 3-3 (581.27 mg, 6.38 mmol) and N,N-bis-Boc-1-guanylpyrazole (1.80 g, 5.8 mmol) were dissolved in tetrahydrofuran (20 mL) and triethylamine (1 mL) was added. The solution was stirred at 80° C. for 16 hours, and the completion of the reaction was detected by LCMS. The mixture was poured into water (100 mL), extracted with ethyl acetate (100 mL×3). The combined organic phases were washed with saturated brine (30 mL), dried over anhydrous sodium sulfate and concentrated. The residue was separated by column chromatography (filler: silica gel powder, eluent: ethyl acetate/petroleum ether=50/1-20/1 (v/v)) to obtain Compound 3-4.
[0127] Step 4:
[0128] 2-Iodoxy benzoic acid (0.34 g, 1.2 mmol) was added to Compound 3-4 (0.40 g, 1.50 mmol) in dimethyl sulfoxide (5.00 mL), and the mixture was stirred at 40° C. for 3 hours under nitrogen atmosphere. The reaction liquid was diluted with ethyl acetate (100 mL), washed with water (50 mL×2) and saturated brine (50 mL) and concentrated. The residue was separated by column chromatography (filler: silica gel powder, eluent: ethyl acetate/petroleum ether=0-1/1) to obtain Compound 3-5.
[0129] Step 5:
[0130] 4A molecular sieve (0.5 g) was added to Compound 3-5 (50.63 mg, 0.15 mmol) and Compound 1-10 (145.00 mg, 0.15 mmol) in methanol (5.00 mL). The mixture was stirred for 0.5 hour at 18° C. under nitrogen atmosphere, and then stirred for another 1 hour after sodium cyanoborohydride (19.20 mg, 0.30 mmol) was added. The completion of reaction was detected by LCMS. The reaction liquid was filtered and concentrated, and separated by preparative-HPLC: Phenomenex Synergi C18 150×25×10 μm; mobile phase: [water (0.225% formic acid))-acetonitrile]; acetonitrile %: 60%-90%, for 10 minutes to obtain Compound 3-7.
[0131] Step 6:
[0132] Compound 3-7 (91.00 mg, 71.97 mol) was dissolved in anhydrous dichloromethane (2.00 mL), cooled to 0° C. under nitrogen atmosphere. Trifluoroacetic acid (1.54 g, 13.51 mmol) was added, and the reaction liquid was stirred at 0-19° C. for 9 hours, concentrated at room temperature, and the residue was washed with acetonitrile/methyl tert-butyl ether (4 mL, 1/3) to obtain Compound 3.
[0133] .sup.1H NMR (400 MHz, D.sub.2O) δ (ppm): 5.62 (s, 1H), 5.24 (s, 1H), 5.08 (s, 1H), 4.09-4.06 (m, 1H), 3.98-3.96 (m, 2H), 3.94-3.93 (m, 5H), 3.77-3.73 (m, 4H), 3.24 (s, 1H), 3.22-3.10 (m, 6H), 2.83 (s, 3H), 2.61-2.14 (m, 2H), 2.04-2.14 (m, 6H), 1.26 (s, 3H): LCMS (ESI) m/z: 664.5 (M+1).
Biological Activity Assay
Experimental Example 1: Detection of Antibacterial Effect of Compound (MIC)
[0134] Three strains Enterobacteriaceae E. coli ATCC 25922, E. coli ATCC BAA-2523, K. pneumonia ATCC BAA-1705 were used to determine the Minimum Inhibitory Concentration (MIC) of each compound by the micro-liquid dilution method according to the requirements of the Institute of Clinical and Laboratory Standard (CLSI). 2-fold series diluted compounds (with a final concentration range 0.125 g/mL-128 g/mL) were added to a round bottom 96-well plate (Catalog #3788, Corning). A single clone of fresh bacteria on the plate of Mueller Hinton II Agar (MHA, Cat. No. 211438, BD BBL™) after overnight culture was picked and suspended in sterile saline to adjust the concentration to 1×10.sup.8 CFU/mL, and then diluted to 5×10.sup.5 CFU/mL by Cation-Adjusted Mueller Hinton II Broth (MHB, Catalog #212332, BD BBL™), 100 μL of which was added to the round bottom 96-well plate containing the drug. The plate was inverted and incubated at 37° C. for 20-24 hours, and the MIC value was read. The lowest drug concentration that inhibits bacterial growth was defined as MIC. The results are shown in Table 1.
TABLE-US-00001 TABLE 1 Antibacterial effect detection data (MIC) of the compounds of the present invention MIC (μM) K. pneumoniae E. coli E. coli Strains ATCC BAA-1705 ATCC BAA-2523 ATCC 25922 Compound 1 4 2 2 Compound 2 4 4 2 Compound 3 0.25 1 0.5
[0135] Conclusion: The compounds of the present invention have good in vitro antibacterial activity.
Experimental Example 2: Evaluation of Pharmacokinetics in Rats
[0136] Purpose of the Experiment:
[0137] To test the pharmacokinetic parameters of the compound of the present invention in rats
[0138] Experimental Protocol:
[0139] 1) Experimental drug: Compound 1;
[0140] 2) Experimental animals: 3 male SD rats aged 7-9 weeks;
[0141] 3) Drug preparation: An appropriate amount of the drug was weighed and dissolved in saline to forma 60 mg/mL solution.
[0142] Experimental Operation:
[0143] Animals were administered the drug at a dose of 150 mg/kg and a concentration of 60 mg/mL by a single intravenous drop infusion via the tail vein for 30 minutes. Plasma samples were collected from the animals at 0, 0.0333, 0.0833, 0.25, 0.5, 1, 2, 4, 6, 8 and 24 hours after administration. The LC-MS/MS method was used to determine the drug concentration in the plasma sample, and the kinetic parameters of the tested drug are shown in Table 2:
TABLE-US-00002 TABLE 2 Pharmacokinetic evaluation results of the compound of the present invention in rats Clearance Maximum Volume of Area Rate Concen- Distri- Half- Under the Cl tration bution Life Curve (mL/ C.sub.max Vd T.sub.1/2 AUC Compound Kg/min) (nM) (L/Kg) (h) (nM .Math. h) Compound 1 9.48 315667 1.50 3.62 440373
[0144] Conclusion: The compound of the present invention has good pharmacokinetic properties in rats.
Experimental Example 3: Study on Pharmacokinetics in Mice
[0145] Purpose of the Experiment:
[0146] The purpose of this experiment is to evaluate the pharmacokinetic behavior of the compound after a single intravenous injection and intragastric administration, and to investigate the bioavailability after intragastric administration.
[0147] Experimental Operation:
[0148] CD-1 male mice aged 7 to 10 weeks were selected and treated by intravenous administration at the dose of 1 mg/kg. The mice were fasted for at least 12 hours before the administration, and resumed feeding 4 hours after the administration. The mice were free to drink during the entire experiment.
[0149] On the day of the experiment, the animals in the intravenous group were administered with corresponding compound by a single injection via tail vein with an administration volume of 5 mL/kg. The animals were weighed before the administration, and the administration volume was calculated based on the body weight. The sample collection time was: 0.083, 0.25, 0.5, 1, 2, 4, 8, and 24 h. Approximately 30 μL whole blood was collected through the saphenous vein at each time point to prepare plasma for high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) to determine the concentration.
[0150] All animals were subjected to euthanasia under CO.sub.2 anesthesia after the PK samples at the last time point were collected. The non-compartmental model of the pharmacokinetic software WinNonlin™ Version 6.3 (Pharsight, Mountain View, Calif.) was used to process the data of plasma concentration, and the linear log-trapezoidal method was used to calculate the pharmacokinetic parameters.
[0151] Experimental results: The evaluation results of PK properties in mice are shown in Table 3.
TABLE-US-00003 TABLE 3 Evaluation of the pharmacokinetic properties of the compound of the present invention in mice Clearance Maximum Volume of Area Rate Concen- Distri- Half- Under the Cl tration bution Life Curve (mL/ C.sub.max Vd T.sub.1/2 AUC Compound Kg/min) (nM) (L/Kg) (h) (nM .Math. h) Compound 1 15.1 7088 0.369 0.396 1924
[0152] Conclusion: The compound of the present invention has good pharmacokinetic properties in mice.
Experimental Example 4: Experimental Evaluation of Drug Efficacy in Mice (Mouse Thigh Muscle Model)
[0153] 12 female CD-1 mice were divided into 4 cages, 3 mice per cage, and were injected intraperitoneally with the immunosuppressant cyclophosphamide (150 mpk).
[0154] 24 hours later, 4 cages of mice were injected intraperitoneally again with the immunosuppressant cyclophosphamide (100 mpk). The strain E. coli ATCC-25922 (Enterobacteria ATCC-25922) was recovered on a MHA plate. The recovered colonies were picked and dissolved in saline to prepare E. coli ATCC-25922 bacterial solution with a concentration of 1.00E+07 CFU/mL for later use in mouse thigh muscle infection. The amount of bacterial solution injected into the thigh muscle of experimental mice was 100 L/mouse, that is, the inoculation amount was 1.00E+06 CFU/mouse. 2 h after infection, the thigh muscle tissue of the mice in control group was taken and placed in 10 mL saline, homogenized, and dotted on a plate with gradient dilution.
[0155] The specific administration of mice was as follows:
[0156] (1) 2 h after infection: At the end of 2 h infection, the thigh muscle tissue of the mice in the first cage was taken and placed in 10 mL saline, homogenized, and dotted on a plate with gradient dilution, two duplications for each mouse. The amount of bacteria loaded in the thigh muscle tissue of the mouse was counted. Mice in the third and fourth cages were injected respectively with 30 mpk Plazomicin and Compound 1 subcutaneously.
[0157] (2) 10 h after infection: Mice in the third and fourth cages were injected respectively with 30 mpk Plazomicin and Compound 1 subcutaneously. At the end of 24 h infection, the thigh muscle tissue of the mice in the second to fourth cages was taken and placed in 10 mL saline, homogenized, and dotted on a plate with gradient dilution, two duplications for each mouse. The amount of bacteria loaded in the thigh muscle tissue of the mouse was counted, and the experimental results were summarized and shown in
[0158] Conclusion: The results in
Experimental Example 5: Experimental Evaluation of Drug Efficacy in Mice (Mouse Pneumonia Model)
[0159] 21 CD-1 mice were divided into 7 cages, 3 mice per cage, and were injected intraperitoneally with the immunosuppressant cyclophosphamide (150 mpk) on the 4th day.
[0160] On the first day, 7 cages of mice were injected intraperitoneally again with the immunosuppressant cyclophosphamide (100 mpk). The strain Kpn ATCC-BAA-1705 (Klebsiella pneumoniae ATCC-BAA-1705) was recovered on a MHA plate. The recovered colonies were picked and dissolved in saline to prepare Kpn ATCC-BAA-1705 bacterial solution with a concentration of 4.00E+08 CFU/mL for later use in mouse lung infection. The amount of bacterial solution infected in the lung of experimental mice was 50 μL/mouse, that is, the inoculation amount was 2.00E+07 CFU/mouse. At 2 h and 24 h infection, the lung tissue of the mice in control group was taken and placed in 5 mL saline, homogenized, and dotted on a plate with gradient dilution.
[0161] The specific administration of mice was as follows:
[0162] (1) 2 h after infection: At the end of 2 h after infection, the lung tissue of the mice in the first cage was taken and placed in 5 mL saline, homogenized, and dotted on a plate with gradient dilution, two duplications for each mouse. The amount of bacteria loaded in the lung tissue of the mouse was counted. Mice in the third and fourth cages were injected respectively with 30 mpk and 10 mpk compound Plazomicin subcutaneously, mice in the fifth and sixth cages were injected respectively with 30 mpk and 10 mpk compound subcutaneously, and mice in the seventh cage were injected with 100 mpk meropenem subcutaneously.
[0163] (2) 10 h after infection: Mice in the third and fourth cages were injected respectively with 30 mpk and 10 mpk plazomicin subcutaneously, mice in the fifth and sixth cages were injected respectively with 30 mpk and 10 mpk compound 1 subcutaneously, and mice in the seventh cage were injected with 100 mpk meropenem subcutaneously. At the end of 24 h infection, the lung tissue of the mice in the second to seventh cages was taken and placed in 5 mL saline, homogenized, and dotted on a plate with gradient dilution, two duplications for each mouse. The amount of bacteria carried in the lung tissue of the mouse was counted, and the experimental results were summarized and shown in
[0164] Conclusion:
Experimental Example 6: Research Report for Auditory Safety of the New Aminoglycoside Antibiotic Drugs
[0165] Research Purposes:
[0166] To evaluate the effects of Compound 1 and the existing antibiotic plazomicin on auditory function in guinea pigs, and to evaluate the auditory toxicity of Compound 1.
[0167] Research Method:
[0168] Healthy adult guinea pigs (150-250 g) were employed as the research objects, and were randomly divided into saline control group, gentamicin group, compound Plazomicin group and Compound 1 group, with 8 animals in each group. Subcutaneous administration is used, and the following assays were carried out during and after the administration for 14 consecutive days:
[0169] 1. To analyze the effects of different drugs on the auditory function of guinea pigs, the compound action potential (CAP) of animals was recorded on the 14th day (29th day, i.e., 4 weeks) after administration. The results obtained were analyzed and the changes of the threshold shift, amplitude, latency and other indicators among different treatment groups were compared.
[0170] 2. After the different groups of animals were processed and the auditory function data thereof were collected, the cochlea of the animals was taken out for fixation and staining. Surface preparation of basilar membrane of the cochlea on one side was performed to count the loss of hair cells so as to make a cochlea map, and the cochlea on the other side was decalcified and frozen sectioned. The density of spiral ganglion neurons was counted and compared among groups.
[0171] Research Results:
[0172] 1. Administration Method and Treatment
[0173] Gentamicin from Dalian Meilun Biotechnology Co., Ltd., and Plazomicin and Compound Ifrom WuXi AppTec (WuHan) Co., Ltd. are used, the solution of which were prepared just before use each time by using saline for dissolution to the concentration of 50 mg/mL, and the injection dose was 100 mg/kg body weight. Method: subcutaneous injection, and confirmation of no liquid leakage after each injection.
[0174] 2. Analysis of Compound Action Potential (CAP)
[0175] The compound action potential (CAP) was tested and recorded when clicks and different frequencies of pure tones (1 KHz-32 kHz) were applied to each group of animals, and variations in amplitude and latency were mainly compared when clicks and medium and high frequency pure tones (16, 32 kHz) were applied. The magnitude of amplitude reflects the responsiveness of hair cells and auditory nerves. The larger the amplitude and the greater the slope of the I/O curve were, the better the responsiveness and the better the function were. In addition, the cochlea from apical turn to basal turn was responsive to sounds from low-frequency to high-frequency respectively, which is the frequency correspondence of the cochlear basilar membrane. The functional changes to different frequencies correspond to the different structural and functional changes of the cochlea fromapical turn to basal turn. The length of latency was also related to the function of the hair cells and auditory nerve response. Generally speaking, increasing of the threshold value when the cochlea was injured would inevitably lead to the extension of latency. In addition, the extension of latency when the threshold value did not change significantly also reflected the decrease in synchronicity of the auditory nerve discharge. In other words, the extension of latency reflected the decrease of response function. The previous ototoxicity of aminoglycoside antibiotics was mainly concentrated in the high-frequency area. In this study, the gentamicin group was consistent with previous results, as summarized below.
[0176] Compound 1 only caused a decrease of CAP amplitude of the experimental group in the high frequency (32 kHz), suggesting hearing damage in the high frequency area, but the amplitude was still higher than that of the Gentamicin and Plazomicin groups. The damage of Plazomicin group occurred in a wider range, damaged at both 16 kHz and 32 kHz, and the damage at 32 kHz greater than that of Compound 1, but significantly lower than that of the Gentamicin group. The damage of Gentamicin to the experimental group was concentrated in the high frequency (32 kHz) area, the threshold shift at 32 kHz was obvious, and the damage was more serious than the drugs of the other two groups (see
[0177] 1) The amplitude results at 16 kHz showed that Compound 1 was no different from the Control group, while the Plazomicin group had 25.9% damage.
[0178] 2) The CAP amplitude results at 32 kHz showed that Compound 1, Plazomicin and Gentamicin caused 34.7%, 48.2% and 74.3% hearing damage at 32 kHz, respectively, that is, Compound 1 still caused hearing damage at 32 kHz, which however was reduced by 13.5% and 39.6% respectively compared to Plazomicin and Gentamicin.
[0179] 3) The CAP amplitude under click indicated that both Compound 1 and Gentamicin were consistent with the Control group, while Plazomicin caused hearing damage.
[0180] The specific values were as follows:
[0181] 1) CAP amplitude at 16 kHz: Two-way ANOVA (Holm-Sidak method) showed that there were differences between the four groups of animals, F3, 570=7.858, p<0.001. Among them, there was no statistical difference in hearing between animals in the Compound 1 group, the Control group, and the Gentamicin group. The hearing of animals in the Plazomicin group was lower than that in the Control group (t=4.566, p<0.001), Gentamicin group (t=4.099, p<0.001) and Compound 1 group (t=2.799, p=0.021) respectively. *: p<0.05. The response of each group was maximum at 90 dB, at which the hearing of animals in Compound 1 group (381.646±20.895 uv) was significantly higher than that in the Plazomicin group (282.058±22.569 uv, t=2.799, p=0.021) and not significantly different from the Control group (383.130±19.545) and Gentamicin group (373.329±15.332 uv). 2) CAP amplitude at 32 kHz: Two-way ANOVA (Holm-Sidak method) showed that there are differences between the four groups of animals, F3, 570=100.611, p<0.001. The hearing of animals in the Compound 1 group was lower than that in the Control group (t=5.019, p<0.001), higher than the Plazomicin group (t=3.128, p=0.002) and Gentamicin group (t=10.484, p<0.001). The response of each group was maximum at 90 dB, at which, the hearing of the animals in the Compound 1 group (79.420±7.000 uv) was lower than that in the Control group (121.608±6.548 uv, t=4.401, p<0.001), higher than that in the Gentamicin group (31.272±5.137 uv, t=5.545, p<0.001) and Plazomicin group (62.982±7.561 uv, t=1.595, p=0.111).
[0182] 3) CAP amplitude under click: Two-way ANOVA (Holm-Sidak method) showed differences among the four groups of animals, F3, 570=6.751, p<0.001. The hearing of animals in the Compound 1 group was significantly better than that in the Plazomicin group (t=3.493, p=0.003), and not significantly different between the Control group and the Gentamicin group.
[0183] In general, the results of CAP amplitude proved that the hearing damage of Compound 1 to experimental animals was significantly lower than that of Gentamicin and Plazomicin.
[0184] 3. Variations in the Number of Hair Cells
[0185] In order to compare the effects of different drugs on hair cells, hair cell staining and counting on the whole basilar membrane of the cochlea are performed. The results indicated that the Gentamicin group had 12-67.7% loss of outer hair cells in the medium and high frequency region (60-100% from the apical turn) and the loss was more obvious in the high frequency area. The Plazomicin group had 11.2-28.1% loss of outer hair cells in the high frequency (70-100% from the apical turn) and 16.7-24.2% loss of the outer hair cells at the beginning of the apical turn (10-20%). However, in the Compound 1 group, the loss of outer hair cells only occurred in the low-frequency region (from the top turn −40%), in which the loss rate of outer hair cells was about 2.5-11%, and the outer hair cells were relatively intact in the high frequency area (see
[0186] In the Compound 1 group, the inner hair cells were almost undamaged. Both the Plazomicin and Gentamicin groups had 3.5±3.0% and 9.3±4.1% loss of inner hair cells near the end of the basal turn (100% from the apical turn) respectively (see
[0187] In summary, the Compound group only had a slight loss of outer hair cells in the apical turn, and the rest part especially the basal turn and inner hair cells were preserved intact. The hair cell toxicity of Compound 1 was significantly lower than that of the Gentamicin and the Plazomicin.
[0188] 4. Variations of Spiral Ganglion Neurons
[0189] The cochlea of the guinea pig was defined as Turn 1, Turn 2, Turn 3, and Turn 4 from the basal to the apical turn. The spiral ganglion neurons (SGNs) were stained with TuJ on frozen sections, and the density thereof in a specific area were counted and compared between groups. There was no significant difference for the SGN density in each turn between the Compound 1 group and the Control group, that is, there was no damage to spiral ganglion neurons. The Gentamicin group had obvious damage in each turn. The Plazomicin group had a decrease in SGN density near the apical turn, but it was better than Gentamicin group. Through the two-way ANOVA, there was a significant difference between the groups, F(3, 74)=35.43, p<0.0001 (see
[0190] Conclusion: Based on the analysis of compound action potentials between different groups, it was confirmed that the Compound 1 group had hearing damage only at high frequency (32 kHz), which was better than the Plazomicin and Gentamicin groups. The observation of hair cells and spiral ganglion neurons confirmed that except for 2.5-11% loss of the outer hair cells near the apical turn, Compound 1 did not cause obvious damage to the outer hair cells in other areas, and the number of inner hair cells and the number of spiral ganglion neurons were not affected, which was significantly better than Gentamicin and Plazomicin groups. Therefore, Compound 1 was administered subcutaneously in animals (guinea pigs) for 14 consecutive days, and the ototoxicity thereof was less than that of the Plazomicin and Gentamicin after another 14 days. Based on the results of this study, it was confirmed that Compound 1 obtained by the present invention was better than Plazomicin and Gentamicin in terms of the auditory toxicity.
TABLE-US-00004 TABLE 4 Damage of outer ear hair cells on Day 29 (%, percentage) Damage percentage (%) Compound 1 Plazomicin Gentamicin 10 11.0 ± 1.3 24.4 ± 13.5 9.5 ± 2.9 20 6.3 ± 0.9 16.7 ± 13.4 4.1 ± 0.7 30 6.2 ± 1.1 10.5 ± 6.5 2.7 ± 0.7 40 2.5 ± 1.4 7.8 ± 4.0 3.2 ± 1.0 50 1.1 ± 0.8 7.2 ± 2.0 3.2 ± 1.5 60 1.4 ± 1.5 5.7 ± 5.1 12.6 ± 5.6 70 0.7 ± 0.3 11.2 ± 11.8 13.6 ± 6.1 80 0.5 ± 0.3 19.9 ± 15.7 12.0 ± 5.4 90 0.2 ± 0.3 20.7 ± 7.9 42.8 ± 8.2 100 0.9 ± 0.7 28.1 ± 10.1 67.7 ± 9.8
TABLE-US-00005 TABLE 5 Damage of inner hair cells on Day 29 (%, percentage) Damage percentage (%) Compound 1 Plazomicin Gentamicin 10 0.3 ± 0.3 0.5 ± 0.4 0 20 0 0.3 ± 0.3 0 30 0 0.5 ± 0.5 0 40 0.1 ± 0.1 0.2 ± 0.2 0 50 0 0 0 60 0 0.8 ± 0.8 0.4 ± 0.4 70 0.3 ± 0.3 1.3 ± 1.3 0.6 ± 0.6 80 0 0 0.2 ± 0.2 90 0 0 2.5 ± 1.7 100 0.7 ± 0.5 3.5 ± 3.0 9.3 ± 4.1
TABLE-US-00006 TABLE 6 Density variation of spiral ganglion neurons on Day 29 (n/10000 μm.sup.2) Turn Gentamicin Plazomicin Compound 1 Control Turn 4.407 ± 0.517 6.816 ± 0.852 8.230 ± 0.500 7.548 ± 0.534 1 Turn 5.540 ± 0.757 5.876 ± 0.536 8.282 ± 0.254 7.695 ± 0.298 2 Turn 4.604 ± 0.598 5.553 ± 0.793 8.136 ± 0.247 7.935 ± 0.292 3 Turn 5.259 ± 0.280 5.641 ± 0.767 7.469 ± 0.772 6.936 ± 0.205 4
Experimental Example 7: Toxicity Test of the Compounds of the Present Invention on HK-2 Cells
[0191] Cell Preparation:
[0192] On the day of the experiment, when the HK-2 cells in the culture flask reached 80%-90% confluent, the culture medium was discarded, the cells were washed twice with Dulbecco's phosphate buffered saline (DPBS) and digested for 1 to 2 minutes with 3 mL trypsin (T150 cell culture flask), and immediately 9 ml complete medium (RPMI1640+10% FBS) were added to terminate the digestion. After termination, single cells suspension were formed by pipetting gently, which were centrifuged at 1000 revolutions per second for 5 minutes. The supernatant was discarded, and fresh complete medium were added, and the cells were pipetted evenly. The actual cell density was measured according to the cell counter and the cell suspension was adjusted to 2.5×10.sup.5 cells/mL. 80 μL of cell suspension was drawn by a row pipettor and added into a 96-well black bottom plate (2×10.sup.4 cell/well), and then incubated in a carbon dioxide incubator for 4.5 hours, which was defined as a cell plate.
[0193] Preparation of Compounds:
[0194] a. The compound mother liquor was prepared according to the following table, with complete medium as the solvent;
TABLE-US-00007 TABLE 7 Compound Information Mass Purity Concentration Volume Compound (mg) (%) (mg/mL) (μL) Plazomicin 5.88 99.65 50 117.19 Compound 1 5.51 95 50 104.69 Netilmicin 5.00 — 50 100.00 Amikacin 5.74 — 50 114.80
[0195] b. 50 microliters of complete medium was added into columns 3-11 in the 96-v well plate;
[0196] c. 75 microliters of test compound (50 mg/mL) and positive control were added to the second column of the 96-v well plate;
[0197] d. 25 microliters of compound was drawn from the second column and added into the third column, blown and sucked a few times by a row pipettor, then 25 microliters of liquid was drawn from the third column and added to the fourth column, and subsequently subjected to a 3-fold series dilution until the 10th column. From column 2 to column 11, the compound concentration was 50, 16.67, 5.56, 1.85, 0.62, 0.21, 0.07, 0.02, 0.008, 0 mg/mL;
[0198] e. 20 μL compound solutions of various concentrations prepared were transferred by a row pipettor to the corresponding wells of the cell plate, which was defined as a testing plate.
[0199] Culture of the Testing Plate:
[0200] All the plates were incubated in an incubator at 37° C., 5% CO.sub.2 for 43 hours.
[0201] Reading:
[0202] After incubation, 10 microliters of Alma Blue was added to the testing plate. The testing plate was immediately incubated in an incubator at 37° C., 5% CO.sub.2 for 3 hours. Then the fluorescence value of each well of the testing plate was read by a microplate reader (wavelength Ex 540 nm/Em 585 nm). Then prism software was used to simulate the curve to calculate CC.sub.50 value.
[0203] Research Results:
TABLE-US-00008 TABLE 8 Experimental results and predicted CC.sub.50 CC.sub.50 of compound on CC.sub.50 of Maximum cells predicted by HK-2 cell inhibition software (Prism) Compound (mg/mL) rate (%) (mg/mL) Plazomicin >10 35.98 19.53 Compound 1 >10 21.96 113.9 Netilmicin 10.64 48.73 10.64 Amikacin 8.157 68.40 8.156
[0204] Conclusion: The toxicity of compound 1 and Plazomicin to HK-2 cells was significantly lower than that of Netilmicin and Amikacin. In combination with the toxicity regression curve and software prediction (