4.1. chemistry
High resolution mass spectra (HRMS) were recorded on Agilent 6520 Q-TOF LC/MS or Varian 7.0T FTMS (MALDI). 1H NMR spectra and 13C NMR were acquired on a Mercury Vx-300 (300 MHz) or Bruker AVANCE III (400 MHz) and referenced to tetramethylsilane (TMS). The residual solvent line was used as an internal standard and the chemical shift was reported in ppm (δ), Compounds were purified by silica gel column (200 - 300 mesh). The reaction process was detected by thin-layer chromatography (TLC) on silica gel GF254 plates (Qingdao Haiyang Chemical Plant, Qingdao, China) and the spots were observed with iodine or UV lamps (λ = 254 nm, 365 nm), The purity of the compounds was tested over 95.00% by HPLC (Waters 2695 Alliance system), with Kromasil 100-5-C18 column 4.6 × 250 mm, eluted by A/B = 32:68 (A, acetonitrile: methanol = 70:30; B, water: methanol = 95:5) containing 0.1% TFA at a flow rate of 1 mL/min.
4.2. Synthesis of tri-tert-butyl 2,2',2''-(1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate (2)
10.0 g (1 eq, 58.05 mmol) cyclen (1,4,7,10-tetraazacyclododecane) and 14.3 g (3 eq, 174.14 mmol sodium acetate were dissolved in 134 mL DMA, stirred in an ice water bath at 0 °C for 30 minutes, then 26 mL (3 eq, 174.14 mmol) tert-butyl bromoacetate in 50 mL DMA was added dropwise in 30 minutes. The slurry was stirred at room temperature for 5 days. 10.0 g (1.5 eq, 87.07 mmol) KBr in 660 mL of deionized water was heated to 50 °C, then was added to the slurry forming a yellow color solution. The pH of the solution was adjusted to 8-9 with Na2CO3. After filtration, the filter cake was washed with deionized water, then dried under a vacuum condition to give 26.5 g white solid powder, in a yield of 89%. 1H NMR (300 MHz, CDCl3) δ 3.37 (s, 4H), 3.28 (s, 2H), 3.09 - 3.08 (m, 4H), 2.91 - 2.88 (m, 12H), 2.17 (s, 1H), 1.46 (s, 27H). HRMS (MALDI) m/z 515.3806 [M+H]+, calcd. for [C26H51N4O6]+, 515.3809.
4.3. Synthesis of compound 2a-g
0.32 g (1.0 eq, 1 mmol) 1a was dissolved in 10 mL water-free DMF, and 150 μL (1.5 eq, 1.5 mmol) TEA and 169 μL (2.2 eq, 2.2 mmol) chloroacetyl chloride were added in sequence. The solution was stirred at room temperature for 24 h, After the reaction completion detected by TLC, 30 mL of deionized water was added. After filtration, the filter cake was washed with deionized water and dried overnight in the vacuum chamber, 0.27 g of the target compound 2a was obtained in a yield of 73%.
Compound 2b-g was synthesized in the same way as compound 2a in yields of 70 - 88%.
4.3.1. 7-(4-(2-chloroacetyl)piperazin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinoline
-3-carboxylic acid (2a)
Compound 2a, 0.27 g was obtained, white solid, yield 73%, 1H NMR (300 MHz, CDCl3), δ 14.86 (s, 1H, -COOH ), 8.79 (s, 1H, -C2H), 8.07 (d, J =12.48 Hz, 1H, -C5H), 7.39 (d, J = 6.99 Hz, 1H, -C8H), 4.14 (s, 2H, -CH2Cl ), 3.91 - 3.88 (m, 2H, 2’-piperazine-H ), 3.82 - 3.79 (m, 2H, 2’’-piperazine-H), 3.56, 3.44 - 3.41 (m, 2H, 3’-piperazine-H ), 3.35 - 3.33 (m, 2H, 3’’-piperazine-H), 1.44 - 1.42 (m, 2H, -NCHCH2), 1.29 - 1.26 (m, 1H, -NCH), 1.24 - 1.23 (m, 2H, -NHCHCH2). HRMS (ESI) m/z 408.1116 [M+H]+, calcd. for [C19H20ClFN3O4]+, 408.1126.
4.3.2. 7-(4-(2-chloroacetyl)piperazin-1-yl)-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-
carboxylic acid (2b)
Compound 2b, 0.29 g was obtained , white solid, yield 83%, 1H NMR (300 MHz, CDCl3), δ 14.92 (s, 1H, -COOH), 8.70 (s, 1H, -C2H), 8.13 (d, J = 11.1 Hz, 1H, -C5H), 6.85 (d, J = 7.5 Hz, 1H, -C8H), 4.36 - 4.33 (m, 2H, -N-CH2), 4.14 (s, 2H, -CH2Cl), 3.91 - 3.88 (m, 2H, 2’, -piperazine-H), 3.82 - 3.78 (m, 2H, 2’’-piperazine-H), 3.43 - 3.37 (m, 2H, 3’-piperazine-H), 3.34 - 3.30 (m, 2H, 3’’, -piperazine-H), 1.28 - 1.27(m, 3H, - NCH2CH3). HRMS (ESI) m/z 396.1120 [M+H]+, calcd. for [C18H20ClFN3O4]+, 396.1126.
4.3.3. 7-(4-(2-chloroacetyl)-3-methylpiperazin-1-yl)-1-cyclopropyl-6-fluoro-8-methoxy-4-
oxo-1,4-dihydroquinoline-3-carboxylic acid (2c)
Compound 2c, 0.30 g was obtained, white solid, yield 71%, 1H NMR (300 MHz, CDCl3), δ 14.67 (s, 1H, -COOH), 8.81 (s, 1H, -C2H), 7.88 (d, J = 11.9 Hz, 1H, -C5H), 4.13 (s, 2H, -CH2Cl), 4.06 - 3.99 (m, 1H, -NCH), 3.75 (s, 3H, -OCH3), 3.74 - 3.71 (m, 2H, 3’’, -piperazine-H), 3.53 - 3.48 (m, 2H, 2’, -piperazine-H), 3.39 - 3.35 (m, 2H, 2’’, -piperazine-H), 3.31 - 3.27 (m, 1H, 3’, -piperazine-H), 1.46 - 1.40 (m, 2H, -NCHCH2), 1.26 - 1.20 (m, 2H, -NCHCH2), 1.08 - 0.96 (m, 3H, 3’, -piperazine-CHCH3 ). HRMS (ESI) m/z 452.1380 [M+H]+, calcd. for [C21H24ClFN3O5]+, 452.1389.
4.3.4. 7-(4-(2-chloroacetyl)piperazin-1-yl)-1-ethyl-6-fluoro-4-oxo-1,4-dihydro-1,8-naphthyri
dine-3-carboxylic acid (2d)
Compound 2d, 0.32 g was obtained, white solid, yield 81%, 1H NMR (300 MHz, CDCl3), δ 14.83 (s, 1H), 8.69 (s, 1H), 8.15 (d, J = 12.9 Hz, 1H), 4.44 - 4.36 (m , 2H), 4.11 (s, 2H), 3.93 - 3.85 (m,4H), 3.83 - 3.73 (m, 4H), 1.53 - 1.48 (t, J = 7.1 Hz, 3H). HRMS (ESI) m/z 397.1072 [M+H]+, calcd. for [C17H19ClFN4O4]+, 397.1079.
4.3.5. 7-(3-(2-chloro-N-methylacetamido)piperidin-1-yl)-1-cyclopropyl-6-fluoro-8-methoxy-4-
oxo-1,4-dihydroquinoline-3-carboxylic acid (2e)
Compound 2e, 0.41 g was obtained, white solid, yield 88%, 1H NMR (300 MHz, CDCl3), δ 14.76 (s, 1H), 8.79 (dd, J = 3 Hz, 1H), 7.86 - 7.80 (m, 1H), 4.69 - 4.62 (m, 1H), 4.07 - 4.02 (m, 1H), 3.85 (s, 2H), 3.50 - 3.47 (br, 3H), 3.36n - 3.26 (m, 1H), 3.23 - 3.20 (m, 1H), 3.05 (s, 3H), 2.07 - 1.74 (m, 6H), 1.27 - 1.21 (m, 2H), 1.06 - 0.97 (m,2H). HRMS (ESI) m/z 466.1547 [M+H]+, calcd. for [C22H26ClFN3O5]+, 466.1545.
4.3.6. 7-(1-(2-chloroacetyl)octahydro-6H-pyrrolo[3,4-b]pyridin-6-yl)-1-cyclopropyl-6-fluoro-
8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (2f)
Compound 2f, 0.41 g was obtained, white solid, yield 86%, 1H NMR (300 MHz, CDCl3), δ 14.94 (s, 1H), 8.76 (s, 1H), 7.78 (d, J = 13.8 Hz, 1H), 5.24 - 5.16(m, 1H), 4.62 - 4.68 (m, 1H), 4.15 - 4.14 (m, 2H), 4.00 (s, 2H), 3.90 - 3.83 (m, 2H), 3.60 (s, 3H), 3.40 - 3.28 (m, 2H), 2.41 - 2.31 (m, 1H), 1.93 - 1.89 (m, 2H), 1.68 -1.56 (m, 2H), 1.31 (br, 2H), 1.12 (br, 2H). HRMS (ESI) m/z 478.1547[M+H]+, calcd. for [C23H26ClFN3O5]+, 478.1545.
4.3.7. 10-(1-(2-chloroacetamido)cyclopropyl)-9-fluoro-3-methyl-7-oxo-2,3-dihydro-7H-
[1,4]oxazino[2,3,4-ij]quinoline-6-carboxylic acid (2g)
Compound 2g, 0.33 g was obtained, white solid, yield 84%, 1H NMR (300 MHz, CDCl3), δ 14.75 (s, 1H), 8.70 (s, 1H), 7.67 (d, J = 10 Hz, 1H), 7.36 (s, 1H), 4.64 - 4.60 (m, 2H), 4.53 - 4.49 (m, 1H), 3.94 (s, 2H), 1.66 (d, J = 6.6 Hz, 3H), 1.48 -1.41 (m, 2H), 1.38 - 1.37 (m, 2H). HRMS (ESI) m/z 395.0807 [M+H]+, calcd. for [C18H17ClFN2O5]+, 395.0810.
4.4 Synthesis of compound 3a-g
0.22 g (1.1 eq, 0.42 mmol) compound 2 and 0.11 g (2 eq, 0.76 mmol) K2CO3 in 20 mL acetonitrile were added to a 50 mL round bottom flask, stirred at room temperature for 10 min, and then 0.15 g (1 eq, 0.378 mmol) compound 2a was added, the reaction proceeded at 50 °C for 24 h. The reaction was quenched and separated on silica gel column eluted by CH2Cl2-MeOH (in a volume ratio 20:1) gave 0.29 g of 3a in a yield of 81%.
Compound 3b-g was synthesized in the same way as compound 3a, in the yields of 80 - 90%.
4.4.1. 1-cyclopropyl-6-fluoro-4-oxo-7-(4-(2-(4,7,10-tris(2-(tert-butoxy)-2-oxoethyl)-1,4,7,10-
tetraazacyclododecan-1-yl)acetyl)piperazin-1-yl)-1,4-dihydroquinoline-3-carboxylic acid (3a)
Compound 3a, 0.29 g was obtained, light yellow oil, yield 81%, 1H NMR (300 MHz, CDCl3), δ 8.56 (s, 1H), 7.77 (d, J = 13.1 Hz, 1H), 7.40 (br, 1H), 4.97 (s, 1H), 3.72 (br, 4H), 3.39 (br, 8H), 3.29 (d, J = 7.5 Hz, 4H), 3.06 - 2.75 (m, 16H), 1.40 (s, 27H), 1.30 (s, 1H), 1.20 (s, 1H), 0.99 (s, 2H). HRMS (ESI) m/z 886.5084 [M+H]+, calcd. for [C45H69FN7O10]+, 886.5090.
4.4.2. 1-ethyl-6-fluoro-4-oxo-7-(4-(2-(4,7,10-tris(2-(tert-butoxy)-2-oxoethyl)-1,4,7,10-tetraaz
acyclododecan-1-yl)acetyl)piperazin-1-yl)-1,4-dihydroquinoline-3-carboxylic acid (3b)
Compound 3b, 0.29 g was obtained, light yellow oil, yield 88%, 1H NMR (300 MHz, CDCl3), δ 8.48(s, 1H, -C2H), 7.76 (d, J = 12.8 Hz, 1H, -C5H ),6.97 (s, 1H, -C8H) , 4.44 - 4.35 (m, 2H, -NCH2), 3.65 (br, 8H), 3.36 - 3.22 (m, 8H), 2.91 - 2.72 (m, 16H), 1.35 (s, 30H, -CH3). HRMS (ESI) m/z 896.4912 [M+Na]+, calcd. for [C44H68FN7NaO10]+, 896.4909.
4.4.3. 1-cyclopropyl-6-fluoro-8-methoxy-7-(3-methyl-4-(2-(4,7,10-tris(2-(tert-butoxy)-2-oxoe
thyl)-1,4,7,10-tetraazacyclododecan-1-yl)acetyl)piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-
carboxylic acid (3c)
Compound 3c, 0.27 g was obtained, light yellow oil, yield 88%, 1H NMR (300 MHz, CDCl3), δ 8.60 (s, 1H, -C2H ), 7.69 (d, J = 12.3 Hz, 1H, -C5H), 4.37 - 4.24 (m, 1H, -NCH), 4.17 - 4.07 (m, 1H,3’, -piperazine-H), 3.85 (s, 3H, -OCH3), 3.68 - 3.56 (m, 6H, 2’, 2’’,3’’, -piperazine-H), 3.38 (s, 8H, COCH2), 3.2 - 3.0(m, 8H, -NCH2CH2N-), 2.98 - 2.70 (m, 8H, -NCH2CH2N-), 1.40 (s, 27H, -CH3), 1.28 - 1.26 (m, 2H,-NCH2), 1.06 -1.04(m, 2H, -NCH2), 0.82 (s, 3H, -CH3). HRMS (ESI) m/z 952.5165 [M+Na]+, calcd. for [C47H72FN7NaO11]+, 952.5157.
4.4.4. 1-ethyl-6-fluoro-4-oxo-7-(4-(2-(4,7,10-tris(2-(tert-butoxy)-2-oxoethyl)-1,4,7,10-tetraaza
cyclododecan-1-yl)acetyl)piperazin-1-yl)-1,4-dihydro-1,8-naphthyridine-3-carboxylic acid (3d)
Compound 3d, 0.30 g was obtained, light yellow oil, yield 90%, 1H NMR (300 MHz, CDCl3), δ 15.01 (s, 1H), 8.68 (s, 1H), 8.08 (d, J = 13.1 Hz, 1H), 4.49 - 4.42 (m, 2H), 4.03 - 3.77 (m, 8H), 3.71 (s, 2H), 3.59 (br, 4H), 3.38 (s, 2H), 3.12 - 3.10 (m, 4H), 2.96 - 2.88 (m, 8H), 2.54 (br, 4H),1.46 (s, 18H), 1.25 (s, 9H), 0.87 (t, J = 6.7 Hz, 3H ). HRMS (ESI) m/z 875.5054 [M+H]+, calcd. for [C43H68FN8O10]+, 875.5042.
4.4.5 1-cyclopropyl-6-fluoro-8-methoxy-7-(3-(N-methyl-2-(4,7,10-tris(2-(tert-butoxy)-2-oxo
ethyl)-1,4,7,10-tetraazacyclododecan-1-yl)acetamido)piperidin-1-yl)-4-oxo-1,4-dihydroquinoline
-3-carboxylic acid (3e)
Compound 3e, 0.26 g was obtained, light yellow oil, yield 86%, 1H NMR (300 MHz, CDCl3), δ 14.76 (s, 1H), 8.72 (t, J = 2.8 Hz, 1H), 7.77 - 7.69 (m, 1H), 5.25 (br, 1H), 4.65 - 4.54 (br, 1H), 4.06 - 3.99 (m, 1H), 3.76 - 3.75 (m, 1H), 3.72 (s, 2H), 3.69 - 3.66 (m, 1H), 3.58 (br, 1H), 3.46 - 3.42 (br, 2H), 3.33 (s, 3H), 3.23 (s, 2H), 3.10 (br, 3H), 3.03 (s, 2H), 2.92 - 2.91 (br, 2H), 2.88 (s, 2H), 2.19 (br, 8H), 1.84 (br, 8H), 1.42 - 1.40 (m, 27H), 1.24 - 1.23 (m, 2H), 1.19 (br, 2H). HRMS (ESI) m/z 944.5515 [M+H]+, calcd. for [C48H75FN7O11]+, 944.5509.
4.4.6 1-cyclopropyl-6-fluoro-8-methoxy-4-oxo-7-(1-(2-(4,7,10-tris(2-(tert-butoxy)-2-oxoethyl)-
1,4,7,10-tetraazacyclododecan-1-yl)acetyl)octahydro-6H-pyrrolo[3,4-b]pyridin-6-yl)-1,4-dihydroquinoline-3-carboxylic acid (3f)
Compound 3f, 0.30 g was obtained, light yellow oil, yield 90%, 1H NMR (300 MHz, CDCl3), δ 8.74 (d, J = 4.2 Hz, 1H), 7.77 - 7.72 (m, 1H), 5.18 - 5.10 (m, 1H), 4.29 - 4.22(m, 1H), 4.00 (br, 3H), 3.69 (s, 2H), 3.58 (s, 2H), 3.55 (s, 2H), 3.35 (s, 2H), 3.28 (br, 2H), 3.12 - 3.09 (m, 2H), 3.09 - 3.00 (m, 2H), 2.44 - 2.00 (m, 16H), 2.71 - 1.70 (br, 1H), 1.85 - 1.81 (m, 4H), 1.45 - 1.43 (m, 27H), 1.13 - 1.06 (m, 2H), 0.83 - 0.77 (m, 2H). HRMS (ESI) m/z 956.5515 [M+H]+, calcd. for [C49H75FN7O11]+, 956.5509.
4.4.7 9-fluoro-3-methyl-7-oxo-10-(1-(2-(4,7,10-tris(2-(tert-butoxy)-2-oxoethyl)-1,4,7,10-
tetraazacyclododecan-1-yl)acetamido)cyclopropyl)-2,3-dihydro-7H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxylic acid (3g)
Compound 3g, 0.27 g was obtained, light yellow oil, yield 82%, 1H NMR (300 MHz, CDCl3), δ 14.86(s, 1H), 9.67 (s, 1H), 8.64 (d, J = 3.6 Hz, 1H), 7.60 (d, J = 10.1 Hz, 1H), 4.89 - 4.83 (m, 1H), 4.56 - 4.52 (m, 1H), 3.48 - 3.45 (m, 1H) 3.38 (s, 2H), 3.36 (s, 2H), 3.31 (s, 2H), 3.28 (s, 2H), 2.87 (br, 8H), 2.77 - 2.69 (m, 8H), 1.45 - 1.43 (m, 27H), 1.32 (br, 1H), 1.24 (br, 3H), 1.08 (br, 2H), 0.89 - 0.85 (m, 1H). HRMS (ESI) m/z 873.4769[M+H]+, calcd. for [C44H66FN6O11]+, 873.4774.
4.5 Synthesis of compound 4a-g
0.2 g (1 eq, 0.23 mmol) 3a was dissolved in 10 mL of TFA and stirred for about 40 - 48 h to remove the tert-butyl groups on the carboxylic acids in DO3A. Then the excess TFA was removed under vacuum. The 0.2 g final de-protected product 4a was precipitated with cold ether, filtered and dried under vacuum, in the yield of 80%.
Compound 4b-g was synthesized in the same way as compound 4a, in the yields of 70 - 90%.
4.5.1 2,2',2''-(10-(2-(4-(3-carboxy-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinolin-7-yl)pip
erazin-1-yl)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (4a)
Compound 4a, 0.13 g was obtained, powdery light yellow solid, yield 80%, 1H NMR (300 MHz, CD3OD), δ 8.73 (s, 1H), 7.86 (d, J = 13.1 Hz, 1H), 7.54 (br, 1H), 4.16 (s, 1H), 3.78 - 3.65 (br, 16H), 3.46 - 3.31 (br, 16H), 1.38 (br, 2H), 1.20 (br, 2H). HRMS (ESI) m/z 756.2663 [M+K]+, calcd. for [C33H44FKN7O10]+, 756.2771.
4.5.2. 2,2',2''-(10-(2-(4-(3-carboxy-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinolin-7-yl)piperazin
-1-yl)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (4b)
Compound 4b, 0.12 g was obtained, powdery light yellow solid, yield 75%, 1H NMR (300 MHz, CD3OD), δ 8.79 (s, 1H), 7.92 (brs, 1H), 7.14 (br, 1H), 4.16 (br, 2H), 3.76 (br, 16H), 3.31 (br, 16H), 1.52 (br, 3H). HRMS (ESI) m/z 744.2667 [M+K]+, calcd. for [C32H44FKN7O10]+,744.2771.
4.5.3. 2,2',2''-(10-(2-(4-(3-carboxy-1-cyclopropyl-6-fluoro-8-methoxy-4-oxo-1,4-dihydroquinol
in-7-yl)-2-methylpiperazin-1-yl)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (4c)
Compound 4c, 0.13 g was obtained, powdery light yellow solid, yield 79%, 1H NMR (400 MHz, DMSO) δ 14.87 (s, 1H), 8.69 (s, 1H), 7.72 (d, J = 11.9 Hz, 1H), 4.62 - 4.60 (m, 1H), 4.16 (brs, 3H), 4.07 (br, 2H), 3.73 (br, 4H) 3.54 (br, 1H), 3.45 (brs, 8H), 3.37 - 3.34 (m, 4H), 3.17 (s, 10H), 3.03 (s, 2H), 1.30 - 1.28 (m, 2H), 1.13 - 1.12 (m, 2H), 1.02 (brs, 3H). 13C NMR (101 MHz, DMSO) δ 176.38, 176.36, 172.87, 165.70, 158.57, 158.25, 150.70, 146.23, 139.70, 139.59, 134.23, 121.37, 118.38, 115.41, 106.77, 64.96, 63.72, 54.75, 54.71, 54.51, 54.05, 53.09, 52.94, 51.65, 50.95, 50.83, 50.49, 50.30, 50.15, 45.49, 37.18, 15.90, 15.19, 9.10, 9.03. HRMS (ESI) m/z 800.2921 [M+K]+, calcd. for [C35H48FKN7O11]+, 800.3033.
4.5.4. 2,2',2''-(10-(2-(4-(6-carboxy-8-ethyl-3-fluoro-5-oxo-5,8-dihydro-1,8-naphthyridin-2-yl)
piperazin-1-yl)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (4d)
Compound 4d, 0.13 g was obtained, powdery light yellow solid, yield 81%, 1H NMR (300 MHz, CD3OD), δ 8.82 (s, 1H), 8.03 (d, J= 11.1 Hz, 1H), 4.49 (br, 2H), 4.12 (br, 4H), 3.92 (br, 4H), 3.73 (br, 8H), 3.48 - 3.41(m, 8H), 3.25 - 3.14 (m, 8H), 1.46 (br, 3H). HRMS (ESI) m/z 745.2623 [M+K]+, calcd. for [C31H43FKN8O10]+, 745.2723.
4.5.1.5. 2,2',2''-(10-(2-((1-(3-carboxy-1-cyclopropyl-6-fluoro-8-methoxy-4-oxo-1,4-dihydroquin
olin-7-yl)piperidin-3-yl)(methyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (4e)
Compound 4e, 0.12 g was obtained, powdery light yellow solid, yield 73%, 1H NMR (300 MHz, CD3OD), δ 8.76(d, J = 2.0 Hz, 1H), 7.71 - 7.63 (m, 1H), 4.50 (br, 1H), 4.14 (s, 3H), 4.02 - 3.99 (m, 1H), 3.81 (s, 2H), 3.75 (s, 2H), 3.71 (s, 2H), 3.61- 3.32 (m, 16H), 3.21 (br, 2H), 3.01 (br, 2H), 2.93 (s, 3H), 1.84 (br, 4H), 1.26 - 1.25(m, 1H), 1.19 - 1.15 (m, 2H), 1.13 - 1.10 (m, 1H), 1.02 - 0.99 (m, 2H). HRMS (ESI) m/z 814.3093 [M+K]+, calcd. for [C36H50FKN7O11]+, 814.3189.
4.5.6. 2,2',2''-(10-(2-(6-(3-carboxy-1-cyclopropyl-6-fluoro-8-methoxy-4-oxo-1,4-dihydroquino
lin-7-yl)octahydro-1H-pyrrolo[3,4-b]pyridin-1-yl)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (4f)
Compound 4f, 0.14 g was obtained, powdery light yellow solid, yield 85%, 1H NMR (300 MHz, CD3OD), δ 8.80 (s, 1H), 7.69 - 7.64 (m, 1H), 4.56 - 4.51 (m, 1H), 4.15 (br, 3H), 3.97 - 3.90 (m, 8H), 3.65 (br, 4H), 3.52 - 3.45 (m, 8H), 3.38 - 3.35 (m, 2H), 3.08 (br, 4H), 2.87 (br, 1H), 2.35 (br, 1H), 1.88 (br, 4H), 1.64 -1.59 (m, 2H), 1.34 - 1.30 (m, 2H), 1.21 - 1.16 (m, 2H), 1.10 - 1.06 (m, 1H), 0.93 - 0.87 (m, 1H). HRMS (ESI) m/z 826.3093 [M+K]+, calcd. for [C37H50FKN7O11]+, 826.3189.
4.5.7. 2,2',2''-(10-(2-((1-(6-carboxy-9-fluoro-3-methyl-7-oxo-2,3-dihydro-7H-[1,4]oxazino[2,3
,4-ij]quinolin-10-yl)cyclopropyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (4g)
Compound 4g, 0.13 g was obtained, powdery light yellow solid, yield 81%, 1H NMR (300 MHz, CD3OD), δ 8.79 (s, 1H), 7.91 (s, 1H), 7.53 (d, J = 10 Hz, 1H), 4.75 (br, 1H), 4.56 (br, 1H), 4.47 (br, 1H), 3.49 - 3.40 (m, 8H), 3.32 - 3.17 (m, 8H), 3.21 - 3.15 (m, 8H), 1.33 (br, 2H), 1.26 (s, 3H), 1.17 - 1.10 (m, 2H). HRMS (ESI) m/z 705.2880 [M+H]+, calcd. for [C32H42FN6O11]+,705.2896. HRMS (ESI) m/z 743.2354 [M+K]+, calcd. for [C32H41FKN6O11]+, 743.2454.
4.6. Biological activity
4.6.1. MIC and MBC determinations in vitro
MRSA (clinical isolates), P. aeruginosa (clinical isolates) and E. coli (clinical isolates) were used in this experiment, which was provided by the People's Liberation Army 304 Hospital.
The antimicrobial activities of these newly synthesized compounds were evaluated in vitro against MRSA, P.aeruginosa and E.coli, which are the representative of Gram-positive bacteria strains and Gram-negative bacterial strains. The standard two folds serial dilution method in 96-well micro-test plates was employed according to the National Committee for Clinical Laboratory Standards (NCCLS) [43]. MRSA interaction with compound 4c, as an example of screening method, was described as following in detail. The experiments were performed in 96-well flat bottom plates in a sterile workbench. Firstly a rough screening outlined an antibacterial concentration range, then a series of 4c solutions in different concentrations were prepared by two folds serial dilution method respectively. The final concentration of the MRSA, logarithmic growth phase bacteria, was diluted to 106 colony-forming units/mL (CFU/mL). Then, 180 μL of 4c solution in the above serial different concentrations and 20 μL of MRSA were added to each well and mixed, respectively. Three sets of independent experiments were performed. Finally, the samples of plates were incubated in the dark at 37 °C and CFU was evaluated after 24 h. The turbidity degree of the bacterial solution was eye observed. The minimum inhibitory concentration (MIC) is defined as the first drug concentration with reduced turbidity, clear and transparent solution. Then, 100 μL of the different gradient concentration drug and bacteria mixed solution in the 96-well micro-test plate were separately aspirated, and the mixture was uniformly coated on LB solid medium, and further incubated at 37 °C in the dark, and evaluated after 24 h. The number of colonies less than or equal to 5 is used as the minimum bactericidal concentration (MBC).
4.6.2. The ADMET prediction
The ADMET (absorption, distribution, metabolism, excretion and toxicity) properties of the synthesized compounds were calculated using in-silico tools and web-based applications. Hydrogen bond donor (HBD), hydrogen bond acceptor (HBA), number of rotatable bonds (RBN), water accessible surface area (ASA), rule of five and total polar surface area (ASA_P) were calculated by MOE ver 2008.10 (Chemical Computing Group, Montreal, Canada). While, CPY2D6 inhibition (CYP2D6), Plasma protein binding (PPB), Carcinogenicity Mouse (Carcino_M) and hERG inhibition (hERG) were calculated via PreADMET ver 2.0 (Yonsei University, Seoul, Korea, https://preadmet.bmdrc.kr).
4.6.3. Docking study
A molecular docking study was performed using Glide modules of Schrödinger suite 2009 (Schrödinger LLC, NewYork) [44]. The X-ray structure of DNA gyrase complex with ciprofloxacin (PDB ID: 2XCT) and DNA topoisomerase IV with a novel fluoroquinolone (PDB ID: 4KPF) were retrieved from the protein data bank (PDB). Before docking, initial proteins were refined using protein preparation wizard to assign bond orders, add hydrogen atoms, treat metal ions, predict side chains and remove all water molecules [45, 46]. Subsequently, the proteins were optimized by reorienting the hydrogen bond network and energy minimized using OPLS2005 force field [47]. Receptor grid was generated around the co-crystallized ligands with the center coordinates 2XCT (x= 2.6951, y = 44.4391, z = 68.1055) and 4KPF (x = -40.3970, y = 78.2317, z = -10.9810), respectively. The size of the grid box was set to 20 Å and Vander Waals radius of receptor atoms was scaled to 1.00 with partial atomic charge less than 0.25. All the synthesized quinolones were constructed using the Maestro workspace build panel and prepared using the Ligprep application to generate variations on the ionization states, tautomers, stereochemistry and ring conformations. Then the molecular geometry of the structures was energy minimized using the OPLS2005 force field. With the default parameters, docking calculations were performed using Standard Precision (SP) mode [48]. Docking score, glide energy and glide ligand efficiency were together used to determine the best binding pose for each ligand. The Pose Viewer panel [49] was applied for visualization and distance measurement of the H-bond contacts between the ligands and active site residues of model proteins
4.6.4. Atomic force microscope assay of sample preparation
The prepared bacterial suspension was diluted to 107 CFU/mL with LB liquid medium and was mixed with the drug solution in an equal volume so that the concentration of 1c and 4c was 10 μmol/L respectively. The mixing solution was incubated at 37 °C for 30 min. The blank control group was also established at the same time. After that, the sample was centrifuged at 9000 RPM for 5 min and the supernatant was discarded. The precipitate was washed twice with physiological saline and centrifuged at 9000 RPM for 5 min. Finally, the bacterial samples were dropped onto the surface of the mica plate (about 0.5 cm2 ), dried naturally at ambient temperature, then scanned by an atomic force microscope (Veeco Multi Mode 8/B0021, Veeco German).
4.6.5. Cytotoxicity assay on the L-02, A549 and MCF-7 cells.
According to reports in the literature [50, 51], it is very meaningful to study compound 4c for the toxicity of human normal liver cells (L-02) and tumor cells (A549 and MCF-7). Here, L-02 cell was used as an example of the cytotoxicity assay in vitro [52]. L-02 cells in the log phase at the density 5×103 cells/well were collected and cultured in complete DMEM at 37 °C under 5% CO2 incubator for 24 h. After different concentrations of 1c and 4c were added to each well, L-02 cells were cultured for 24 h at 37 °C under 5% CO2. Then the medium was poured off before 100 μL of serum-free medium and 20 μL of MTS were added to each well, and the mixture was incubated for 1 h at 37 °C. Finally, the cell viability was measured by a microplate reader (Thermo, Varioskan Flash 3001, USA) at a wavelength of 490 nm, and calculated cell survival rate according to the following formula :
Cell survival rate (%) = (OD test - OD blank) / (OD control - OD blank) × 100%