2017 Volume 65 Issue 2 Pages 178-185
Fatty acid synthesis (FAS) is an essential metabolism during the whole growth and development process of the bacterial. Several key enzymes which involved in this biosynthetic pathway have been considered as useful targets for the development of new antibacterial agents. Among them, β-ketoacyl-acyl carrier protein synthase III (FabH) is the most magnetic target, since it is central to the initiation of fatty acid biosynthesis and is highly conserved of both Gram-positive and Gram-negative bacteria. Following the previous researches, Schiff-based derivatives with dioxygenated rings and N-heterocycle were synthesized in succession, and their biological activities as potential FabH inhibitors were evaluated in this paper. Among these 15 compounds, compound 2E exhibited the best antibacterial activities with minimum inhibitory concentration (MIC) values 1.56–3.13 mg/mL against the tested bacterial strains and showed the most powerful Escherichia coli (E. coli) FabH inhibitory activities with IC50 of 2.1 µM. Also the conceivable binding conformation of placing compound 2E into the E. coli FabH active site was affirmed docking simulation.
A number of antibiotics have been invented and applied to against diseases after Fleming found penicillin in 1928 and Florery used penicillin in the treatment of infectious diseases in 1940.1) Antibiotics have become one of the greatest achievements in the field of medicine and made great contribution for health of people in 20th Century. But many pathogenic bacteria showed drug resistances to antibiotics for the abuse used of antibiotics. The occurrence of drug resistance and the propagation speed of pathogenic bacteria are eye-popping.2) So looking for new antibacterial targets and develop new antibacterial agents is urgent. Fatty acid synthesis (FAS) play an effective role in the metabolic process of most organisms. Comparing the FAS I (exist in Mammals and yeast),3–5) FAS II exist in bacteria and plants, form by a series of separated small proteins. Each step of the FAS is catalyzed by specific monofunctional enzyme independent.6–8) So developing of new antibacterial agents targeting one or several monofunctional enzymes in bacterial fatty acid synthesis become quite feasible.9,10)
Among all enzymes of bacterial fatty acid synthesis, β-ketoacyl-acyl carrier protein (ACP) synthase III (FabH) shows some special characteristics11): (1) It catalyzes the starting step of FAS rotate; (2) FabH can be inhibited by the final product Palmitoyl–ACP and the whole loop terminates, so it plays a key role in regulation of FAS; (3) FabH has higher substrate specificity. The substrate of the FabH is only acetyl CoA, while both FabB and FabF use Acyl–ACP as their substrate; (4) FabH is prevalently existed in a large number of clinical pathogens, such as Gram-positive bacteria, Gram-negative bacteria, chlamydia, anaerobic bacteria, mycobacteria. FabH is also essential in their survival, while some enzymes, for example, FabA, FabB, FabI are not found in all pathogens.12)
The notable features of the FabH have attracted a great number of researchers and many pioneer studies focusing on FabH have been reported.13–15) FabH shows high conserved on gene sequences and three-dimensional structures of Gram-positive bacteria and Gram-negative bacteria, while any homologous proteins cannot be found in human. More importantly, amino acid residues at FabH active sites between Gram-positive and Gram-negative bacteria were no essential differences. FabH has been identified is the key target enzyme in the fatty acid synthesis of bacteria, and can be chosen as the new target for developing broad-spectrum antibiotics.16–18)
Many research groups including our group devoted to investigating antibacterial with FabH as target, and some potential FabH inhibitors with high antibacterial activities have been exploited19–24) (Fig. 1).
Four novel Schiff bases derived from YKAs3003 that retained cyclohexylamine moiety as skeleton structure were designed in the previous studies. Among them, seven-membered ring was taken as the most appropriate structure for developing new FabH inhibitor via receptor-oriented pharmacophore model and docking. A series of compounds were synthesized and evaluated against different bacterial and tested their Escherichia coli (E. coli) FabH inhibitory activities. Compound 10 containing 16 aliphatic substituents was found as the most potent E. coli FabH inhibitory activity with an IC50 value of 1.6 µM. During this process of developing antibiotic agents, we also found that the nitrogen heterocycle replacing at the right side showed better antibacterial activity than benzene ring, it could be another strategy to explore better antibacterial agents.25) We synthesize and evaluate a series of Schiff base with nitrogen heterocycle substitute and dioxygenated seven-membered fuse ring, and investigate their biological activities in this paper. The whole design strategy was showed in Fig. 2.
A lead compound (YKAs3003) initiates a series of novel antibiotic agents with nitrogen heterocycle.
The synthesis of targeted compounds 2A–O was presented in Fig. 3. The synthesis of intermediate aldehyde 1 was similar to previous article.25) For developing more effective antibiotics agents and discussing the different trends in structure–activity relationships (SARs) of the designed compounds handily, the substituents on the right hand were divided into two parts, one with heterocylic methanamine group, the other heterocycle amine. Dehydration reaction by different amines and intermediate aldehyde 1 produced the final designed analogous. The products 2A–O were obtained with 77–93% yields via flash chromatography. The structures of these compounds were fully characterized by spectroscopic methods and elemental analysis.
Reagents and conditions: a) Cs2CO3, DMF, 70°C, overnight; b) R-NH2, EtOH, RT, overnight.
Two Gram-negative bacterial strains, E. coli and P. fluorescence, and the two Gram-positive bacterial strains, B. subtilis and S. aureus, were used to evaluate the antibacterial activity of all compounds (2A–O), with the 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The minimum inhibitory concentrations (MIC) of these compounds against these bacteria were presented in Table 1. As the positive control, kanamycin B was also evaluated its antibacterial activity in the same condition with other synthesized compounds (2A–O).
| Compd.a) | A log Pb) | Minimum inhibitory concentrations [µg/mL] | |||
|---|---|---|---|---|---|
| Gram-negative | Gram-positive | ||||
| E. coli | P. aeruginosa | B. subtilis | S. aureus | ||
| 2A | 1.940 | 50 | >100 | >100 | >100 |
| 2B | 2.986 | 25 | 50 | >100 | >100 |
| 2C | 2.346 | 25 | 50 | 50 | 50 |
| 2D | 3.004 | 6.25 | 12.5 | 25 | 25 |
| 2E | 2.642 | 1.56 | 1.56 | 3.13 | 3.13 |
| 2F | 0.841 | 50 | 25 | >100 | >100 |
| 2G | 1.496 | >100 | >100 | >100 | >100 |
| 2H | 2.124 | 25 | 12.5 | 25 | 50 |
| 2I | 2.663 | 50 | 50 | >100 | >100 |
| 2J | 3.327 | 6.25 | 12.5 | 25 | 50 |
| 2K | 3.605 | 6.25 | 3.13 | 12.5 | 6.25 |
| 2L | 2.542 | 12.5 | 12.5 | 12.5 | 25 |
| 2M | 3.149 | 6.25 | 12.5 | 6.25 | 12.5 |
| 2N | 3.327 | 12.5 | 6.25 | 25 | 12.5 |
| 2O | 3.149 | 6.25 | 6.25 | 12.5 | 25 |
| Kanamycin B | −7.144 | 3.13 | 3.13 | 1.56 | 1.56 |
a) The compounds tested for antibacterial activity are consistent withthe description in Experimental. b) Calculated with discovery studio 3.1.26)
For discussing the SARs of these compounds, we divided the compounds into two groups: compounds (2A–D) which bear heterocyclic methanamine and compounds (2E–O) which bear heterocyclic amine. The methylene between imine and the heterocyclic show no effect on the activities, which meant length of the linker between N atom and N-heterocyclic (with none or one-carbon-atom gaps) was not the quite important factor of the antibacterial activities. However, Schiff-base compounds 2A, F, G with lower predicted lipophilicity (A log P<2) values exhibited poor activities. Compounds with substituent groups on the heterocyclic (2D, E, J, K, M–O) seemed to enhance the antibacterial activities than others with no substituent groups (2B, C, H, I). Compound 2D represented an exception, probably because the benzene of the 3H-indole could make up for the loss of substituent groups on N-heterocyclic caused by the length of the linker. No significant difference could be found between electron withdrawing substituents (2D, J–L, N) or electron-donating substituents (2M, O) on the N-heterocyclic. The position of the substituents at 4- or 5-pyridine didn’t obviously enhance the antibacterial activities. Regarding the substituent of pyridine, trifluoromethyl did better than other moieties (2K). The last noticeable discovery from compound 2E with tert-butyl piperidine carboxylate was the highest activitity, that the piperidine ring replacing another N-heterocyclic might provide one more tactics to develop better antibacterial agents.
Then we evaluated the E. coli FabH inhibiton of these synthetic compounds with potential antibacterial activities (2D, E, H, J–O), and the results were performed in Table 2. All compounds presented potent E. coli FabH inhibition, and the compound 2E with tert-butyl piperidine carboxylate showed the best activity against E. coli FabH with the IC50 value of 2.1 µM. This result indicated the potent antibacterial activities of compound 2E being the most potent FabH inhibitor. Compound 2K with trifluoromethyl substituent of the pyridine also showed higher activities than others compounds. This due to the trifluoromethyl not only caused the compound with highest A log P value (A log P 3.605) but also was a quite important substituent group in drug metabolism. Since the molecular polar surface area (PSA) is a considerable parameter in predicting drug transport, we calculated PSA in Table 2 too, which would relate the the binding affinity and the cell permeability of FabH inhibitors.27) The molecular PSA values of these compounds were in the range of 40–64 Å2, and therefore, this molecular property cannot significantly account for the results obtained. To sum up, the results of the E. coli FabH inhibitory activity of the test compounds described above extensively conformed to the SARs of their antibacterial activities. This exhibited that the efficient antibacterial activities of these compounds might be from their FabH inhibitory activities.
| Compd.a) | E.coli FabH IC50 [µM] | PSAb) [Å2] | Hemolysis LCc) [mg/mL] | Cytotoxicity IC50 [µM] |
|---|---|---|---|---|
| 2D | 6.7 | 40.41 | >10 | 148.7 |
| 2E | 2.1 | 58.77 | >10 | 189.5 |
| 2H | 14.5 | 40.44 | >10 | 138.1 |
| 2J | 12.8 | 40.44 | >10 | 178.4 |
| 2K | 3.8 | 40.44 | >10 | 198.6 |
| 2L | 9.9 | 63.38 | >10 | 154.9 |
| 2M | 12.3 | 40.44 | >10 | 201.3 |
| 2N | 15.1 | 40.44 | >10 | 180.4 |
| 2O | 7.4 | 40.44 | >10 | 125.6 |
a) The compounds tested for antibacterial activity are consistent with the description in Experimental. b) The PSA was calculated by discovery studio 3.1.26) c) Lytic concentration 30%.
The hemolytic activities of these selected compounds were observed. And the cytotoxic activities were also tested by mouse embryonic fibroblast cell line (NIH-3T3) using the MTT assay as well.28) These results were summarized in Table 2.
All the compounds displayed low hemolytic activities. Besides, the cytotoxicity assay determined the selectivity of our compounds for bacterial over mammalian cells. All these data suggested that the compounds with potent inhibitory activities were low toxic.
The E. coli FabH inhibitory activity of compound 2E is lower than compound 10, but the MIC values of compound 2E and compound 10 are both described as 1.56 µg/mL against E. coli in the same experimental condition. For deeper comparison of the biological activities between compound 10 and compound 2E, we have reset the gradients of these two compounds from 0 to 1.6 µg/mL per 0.1 µg/mL. Then the results showed MIC of compound 2E and compound 10 were 1.1 and 1.5 µg/mL, respectively. We did not raise this detailed data because the absorption, distribution, metabolism, excretion and toxicity (ADMET) properties were more directly to indicate the advantages of compound 2E in this manuscript. The MIC value in deeper comparison actually reflect the same point. The A log P value of compound 10 was 8.501, whereas that of compound 2E was 2.642. According to Lipinski’s Rule of Five, the range of A log P values for medicine should be −0.5–5.6. Thus, even compound 10 was slightly better than compound 2E in E. coli FabH inhibitory activity, when applied to practice, compound 2E could be more potential.
Molecular docking of the most active compound 2E and E. coli FabH was displayed for demonstrating the interaction binding mode between the target protein and small molecules, which used the he E. coli FabH–CoA complex structure (PDB: 1HNJ) as binding model.13) The accompanying results were shown in Fig. 4, composed of two interaction maps. The docking study was performed using the CDOCKER protocol.26) The three dimensional (3D) optimal conformation and the 2D diagram interacting with the FabH active site was presented in Figs. 4a and 4b, respectively. The main chain of ASN147 forms hydrogen bond (NH–N, 2.34 Å, 169.512°) with one nitrogen atom of compound 2E while ALA246 participate in a π–sigma interaction (2.81 Å) with the benzene ring of compound 2E.
The H-bond was displayed as dotted line and the amino acid it acts on is labeled. The π–sigma interaction was shown as straight line with correlated amino acid labeled.
These interactions were critical for the stabilization of the binding mode. Comparing with the original crystal conformation of substrate malonyl-CoA, compound 2E indicated more powerful interactions with the active site. Hence, compound 2E could be considered as a potential inhibitor of E. coli FabH with potent antibacterial activity.
In this paper, 15 novel Schiff base with different N-heterocyclic were synthesized and their antibacterial activities against E. coli FabH were evaluated firstly. Within our research, certain compounds (2D, E, H, J–O) showed anti-Gram-negative bacteria activities. Compound 2E exhibited the best antibacterial activity with MIC values of 1.56– 3.13 mg/mL against the tested bacterial strains and presented the most potent E. coli FabH inhibitory activity with IC50 of 2.1 µM. Initiatory structure–activity relationships and molecular modeling study provided further insight into interactions between the enzyme and its ligands. This study showed that 2E was a novel compound that could be potent antimicrobial inhibitor of FabH and provide useful advice for the designed of E. coli FabH inhibitors as an antibacterial agents. Moreover, developing novel compounds bearing similar structures of compound 2E will be investigated in future studies.
All chemicals (reagent grade) were purchased from Sigma-Aldrich. Separation of the compounds by column chromatography was carried out with silica gel 60 (200–300 mesh ASTM, Merck, Germany). The quantity of silica gel used was 50–100 times the weight charged on the column. Then, the eluates were monitored using TLC. Melting points (mp) were determined on a XT4 MP apparatus (Taike Corp., Beijing, China). Electrospray ionization (ESI) MS were obtained on a Mariner System 5304 mass spectrometer, and 1H- and 13C-NMR spectra were recorded on a Bruker DPX 400 spectrometer. Elemental analyses were performed on a Heraeus CHN-O-Rapid instrument and were within ±0.4% of the theoretical values.
3,4-Dihydro-2H-benzo[b][1,4]dioxepine-7-carbaldehyde (1)An 25-mL round flask was charged with 3,4-dihydroxybenzaldehyde (0.716 g, 5.20 mmol, 1.0 equiv.), Cs2CO3 (3.38 g, 10.4 mmol, 2.0 equiv.) and N,N-dimethylformamide (DMF) (8 mL). 1,3-Dibromopropane (2.10 g, 10.4 mmol, 2.0 equiv.) was added. The reaction mixture was refluxed at 70°C overnight. After cooled to the room temperature (RT), the reaction solution was filtered through a short pad of Celite and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography on silica gel to give compound 1 as a colorless oil. Yield: 88%. Rf=0.6 (4 : 1 hexane–EtOAc). 1H-NMR (400 MHz, CDCl3) δ: 9.85 (s, 1H), 7.47 (dd, J=7.2, 1.8 Hz, 2H), 7.06 (m, 1H), 4.36 (m, 2H), 4.30 (t, J=5.9 Hz, 2H), 2.26 (m, 2H). 13C-NMR (100 MHz, CDCl3) δ: 190.54, 156.44, 151.01, 131.93, 125.36, 122.74, 121.79, 70.28, 70.15, 30.73. MS (ESI): m/z 179.2 [M+H]+. Anal. Calcd for C10H10O3: C 67.41, H 5.66, O 26.94; Found: C 67.32, H 5.68, O 26.96.
Compounds 2Compound 1 (535 mg, 3.00 mmol, 1 equiv.) was dissolved in MeOH (3 mL). The corresponding amine (3.6 mmol) was added. The reaction mixture was stirred at RT for overnight. The solvent was removed under reduced pressure. The residue was purified by column chromatography (neutral Al2O3, EtOAc/hexane) to give pure compound 2.
(E)-N-((3,4-Dihydro-2H-benzo[b][1,4]dioxepin-7-yl)methylene)-1-(1H-pyrazol-3-yl)methanamine (2A)White solid; yield: 89%. Rf=0.5 (4 : 1 hexane–EtOAc); mp: 105.2–105.8°C. 1H-NMR (400 MHz, dimethyl sulfoxide (DMSO)-d6) δ: 8.31 (s, 2H), 8.05 (s, 1H), 7.14 (d, J=2.0 Hz, 1H), 7.07 (dd, J=8.3, 2.0 Hz, 1H), 6.69 (d, J=8.3 Hz, 1H), 4.80 (s, 2H), 4.27 (m, 4H), 2.21 (m, 2H); 13C-NMR (100 MHz, DMSO-d6) δ: 158.31, 157.08, 153.24, 139.37, 128.63, 125.56, 122.18, 121.74, 70.42, 70.39, 64.97, 31.59. MS (ESI): m/z 258.1 [M+H]+. Anal. Calcd for C14H15N3O2: C 65.35, H 5.88, N 16.33, O 12.44; Found: C 65.42, H 5.83, N 16.36, O 12.49.
(E)-N-((3,4-Dihydro-2H-benzo[b][1,4]dioxepin-7-yl)methylene)-1-(3H-indol-2-yl)methanamine (2B)White solid; yield: 91%. Rf=0.5 (4 : 1 hexane–EtOAc); mp: 145.7–146.3°C. 1H-NMR (400 MHz, DMSO-d6) δ: 8.24 (d, J=12.8 Hz, 2H), 7.87 (d, J=7.7 Hz, 1H), 7.55 (d, J=6.3 Hz, 2H), 7.48 (d, J=10.6 Hz, 1H), 7.40 (t, J=7.3 Hz, 1H), 7.23 (t, J=7.3 Hz, 1H), 6.99 (m, 2H), 4.25 (m, 4H), 3.16 (s, 2H), 2.21 (m, 2H); 13C-NMR (100 MHz, DMSO-d6) δ: 160.45, 153.38, 151.21, 136.36, 131.93, 127.67, 123.49, 122.33, 121.91, 121.64, 121.26, 119.26, 119.08, 114.09, 111.24, 61.95, 36.52, 31.05. MS (ESI): m/z 307.1 [M+H]+. Anal. Calcd for C19H18N2O2: C 74.49, H 5.92, N 9.14, O 10.44; Found: C 71.53, H 5.94, N 9.15, O 10.48.
(E)-N-((3,4-Dihydro-2H-benzo[b][1,4]dioxepin-7-yl)methylene)-1-(pyridin-3-yl)methanamine (2C)White solid; yield: 81%. Rf=0.5 (4 : 1 hexane–EtOAc); mp: 145.7–146.3°C. 1H-NMR (400 MHz, DMSO-d6) δ: 8.59 (s, H), 8.50 (dd, J=7.7, 1.7 Hz, 1H), 8.45–8.40 (m, 1H), 8.01 (s, 1H), 7.52–7.43 (m, 1H), 7.37 (q, J=4.9 Hz, 1H), 7.00 (dd, J=22.5, 8.5 Hz, 1H), 4.76 (s, 2H), 4.23–4.08 (m, 4H), 2.12 (dt, J=10.2, 5.2 Hz, 2H). 13C-NMR (100 MHz, DMSO-d6) δ: 161.85, 153.74, 150.63, 149.73, 148.71, 136.13, 128.80, 125.09, 123.84, 123.02, 122.21, 121.43, 70.88, 70.82, 61.49, 31.59. MS (ESI): m/z 269.3 [M+H]+. Anal. Calcd for C16H16N2O2: C 71.62, H 6.01, N 10.44, O 11.93; Found: C 71.57, H 5.99, N 10.49, O 11.91.
(E)-1-(6-Chloropyridin-3-yl)-N-((3,4-dihydro-2H-benzo[b][1,4]dioxepin-7-yl)methylene)methanamine (2D)White solid; yield: 87%. Rf=0.5 (4 : 1 hexane–EtOAc); mp: 120.3–120.9°C. 1H-NMR (400 MHz, DMSO-d6) δ: 8.52 (s, 1H), 8.19 (d, J=2.3 Hz, 1H), 8.02 (d, J=8.2 Hz, 1H), 7.62 (d, J=8.3 Hz, 1H), 7.38 (d, J=8.0 Hz, 1H), 6.85 (s, 2H), 4.38 (s, 2H), 4.28 (dt, J=14.7, 5.7 Hz, 4H), 2.23 (m, 2H). 13C-NMR (100 MHz, DMSO-d6) δ: 161.89, 154.38, 150.28, 149.72, 148.16, 136.36, 128.31, 125.05, 123.94, 123.11, 122.40, 121.28, 70.42, 70.38, 62.28, 31.29. MS (ESI): m/z 303.4 [M+H]+. Anal. Calcd for C16H15ClN2O2: C 63.47, H 4.99, Cl 11.71, N 9.25, O 10.57; Found: C 63.54, H 5.02, Cl 11.65, N 9.25, O 10.48.
(E)-tert-Butyl 4-((3,4-Dihydro-2H-benzo[b][1,4]dioxepin-7-yl)methyleneamino)piperidine-1-carboxylate (2E)Brown solid; yield: 84%. Rf=0.6 (4 : 1 hexane–EtOAc); mp: 89.7–90.1°C.1H-NMR (400 MHz, DMSO-d6) δ: 8.30 (s, 1H), 7.47 (s, 1H), 7.14 (d, J=8.2 Hz, 1H), 6.70 (d, J=16.3 Hz, 1H), 4.31–4.26 (m, 4H), 4.22 (dd, J=10.1, 4.2 Hz, 4H), 3.90 (d, J=12.9 Hz, 1H), 2.28 (m, 2H), 2.18 (dt, J=9.8, 3.7 Hz, 4H), 1.42 (s, 9H). 13C-NMR (100 MHz, DMSO-d6) δ: 161.28, 157.48, 153.08, 151.13, 132.27, 123.32, 121.51, 121.08, 79.82, 70.43, 70.41, 69.79, 44.40, 31.54, 29.66, 24.82. MS (ESI): m/z 361.4 [M+H]+. Anal. Calcd for C20H28N2O4: C 66.64, H 7.83, N 7.77, O 17.76; Found: C 66.56, H 7.80, N 7.83, O 17.69.
(E)-5-((3,4-Dihydro-2H-benzo[b][1,4]dioxepin-7-yl)methyleneamino)pyrimidin-2(1H)-one (2F)White solid; yield 79%. Rf=0.3 (4 : 1 hexane–EtOAc); mp: 121.4–122.3°C. 1H-NMR (400 MHz, DMSO-d6) δ: 8.17 (s, 1H), 8.01 (s, 1H), 7.31 (d, J=7.0 Hz, 1H), 7.08 (d, J=8.0 Hz, 1H), 6.85 (s, 2H), 5.56 (d, J=7.0 Hz, 1H), 4.21 (dt, J=10.8, 5.6 Hz, 4H), 2.15 (dt, J=11.0, 5.6 Hz, 2H). 13C-NMR (100 MHz, DMSO-d6) δ: 163.72, 158.81, 155.19, 154.24, 148.90, 132.39, 128.93, 124.08, 122.26, 121.65, 105.49, 70.87, 70.85, 31.55. MS (ESI): m/z 272.3 [M+H]+. Anal. Calcd for C14H13N3O3: C 61.99, H 4.83, N 15.49, O 17.69; Found: C 62.04, H 4.83, N 15.41, O 17.75.
(E)-N-((3,4-Dihydro-2H-benzo[b][1,4]dioxepin-7-yl)methylene)pyrimidin-4-amine (2G)White solid; yield: 93%. Rf=0.4 (4 : 1 hexane–EtOAc); mp: 143.7–144.5°C. 1H-NMR (400 MHz, DMSO-d6) δ: 8.61 (s, 1H), 8.32 (s, 1H), 8.03 (d, J=5.9 Hz, 1H), 7.46 (d, J=2.1 Hz, 1H), 7.35 (dd, J=8.3, 2.0 Hz, 1H), 6.98 (d, J=8.2 Hz, 1H), 6.40 (dd, J=5.9, 1.3 Hz, 1H), 4.24 (dt, J=11.6, 5.7 Hz, 4H), 2.23 (m, 2H). 13C-NMR (100 MHz, DMSO-d6) δ: 167.20, 164.34, 158.36, 154.21, 143.07, 131.28, 129.20, 124.38, 122.20, 121.80, 115.01, 70.42, 70.38, 31.27. MS (ESI): m/z 256.1 [M+H]+. Anal. Calcd for C14H13N3O2: C 65.87, H 5.13, N 16.46, O 12.54; Found: C 65.91, H 5.10, N 14.48, O 12.59.
(E)-N-((3,4-Dihydro-2H-benzo[b][1,4]dioxepin-7-yl)methylene)pyridin-3-amine (2H)White solid; yield 90%. Rf=0.4 (4 : 1 hexane–EtOAc); mp: 135.9–136.4°C. 1H-NMR (400 MHz, DMSO-d6) δ: 8.58 (s, 1H), 8.46 (ddd, J=6.2, 3.6, 1.0 Hz, 2H), 7.67 (ddd, J=8.1, 2.6, 1.5 Hz, 1H), 7.59–7.52 (m, 2H), 7.48–7.39 (m, 1H), 7.13–7.06 (m, 1H), 4.22 (dt, J=13.1, 5.8 Hz, 4H), 2.20–2.12 (m, 2H). 13C-NMR (100 MHz, DMSO-d6) δ: 162.13, 154.58, 151.41, 147.70, 147.31, 143.38, 131.70, 128.09, 124.84, 124.47, 122.38, 122.33, 70.92, 70.84, 31.44. MS (ESI): m/z 255.3 [M+H]+. Anal. Calcd for C15H14N2O3: C 70.85, H 5.55, N 11.02, O 12.58; Found: C 70.76, H 5.59, N 11.16, O 12.41.
(E)-N-((3,4-Dihydro-2H-benzo[b][1,4]dioxepin-7-yl)methylene)pyridin-2-amine (2I)White solid; yield: 84%. Rf=0.4 (4 : 1 hexane–EtOAc); mp: 145.3–145.7°C. 1H-NMR (400 MHz, DMSO-d6) δ: 9.01 (s, 1H), 8.39 (dd, J=8.3, 1.7 Hz, 1H), 7.53 (ddd, J=8.2, 5.9, 3.1 Hz, 1H), 7.36–7.26 (m, 2H), 7.19–7.11 (m, 1H), 7.08–6.94 (m, 1H), 6.89–6.81 (m, 1H), 4.23 (dd, J=11.0, 5.5 Hz, 4H), 2.17 (td, J=9.8, 5.5 Hz, 2H). 13C-NMR (100 MHz, DMSO-d6) δ: 162.24, 156.68, 154.34, 151.47, 150.29, 145.68, 142.84, 137.90, 129.22, 127.22, 125.03, 122.97, 115.22, 70.92, 70.79, 30.95. MS (ESI): m/z 255.3 [M+H]+. Anal. Calcd for C15H14N2O3: C 70.85, H 5.55, N 11.02, O 12.58; Found: C 70.92, H 5.54, N 11.08, O 12.50.
(E)-5-Chloro-N-((3,4-dihydro-2H-benzo[b][1,4]dioxepin-7-yl)methylene)pyridin-2-amine (2J)White solid; yield: 85%. Rf=0.4 (4 : 1 hexane–EtOAc); mp: 138.2–138.8°C. 1H-NMR (400 MHz, DMSO-d6) δ: 9.03 (s, 1H), 8.52 (d, J=2.6 Hz, 1H), 7.62 (dd, J=7.5, 1.7 Hz, 2H), 7.36 (d, J=8.5 Hz, 1H), 7.13–7.07 (m, 1H), 6.45 (dd, J=8.8, 0.6 Hz, 1H), 4.30–4.15 (m, 4H), 2.16 (dq, J=11.6, 5.9 Hz, 2H). 13C-NMR (100 MHz, DMSO-d6) δ: 162.63, 158.98, 151.42, 147.68, 146.11, 138.75, 137.17, 131.25, 125.53, 122.77, 121.17, 117.79, 70.87, 70.83, 31.35. MS (ESI): m/z 289.2 [M+H]+. Anal. Calcd for C15H13ClN2O2: C 62.40, H 4.54, Cl 12.28, N 9.70, O 11.08; Found: C 62.47, H 4.55, Cl 12.34, N 9.62, O 11.08.
(E)-N-((3,4-Dihydro-2H-benzo[b][1,4]dioxepin-7-yl)methylene)-5-(trifluoromethyl)pyridin-2-amine (2K)White solid; yield: 89%. Rf=0.5 (4 : 1 hexane–EtOAc); mp: 127.5–128.1°C. 1H-NMR (400 MHz, DMSO-d6) δ: 8.51 (s, 1H), 8.03–7.96 (m, 1H), 7.70 (dd, J=8.9, 2.4 Hz, 1H), 7.08–6.92 (m, 2H), 6.73 (d, J=8.9 Hz, 1H), 6.62 (dd, J=5.4, 1.7 Hz, 1H)4.11 (dd, J=8.4, 5.3 Hz, 4H), 2.10 (dd, J=10.9, 5.5 Hz, 2H). 13C-NMR (100 MHz, DMSO-d6) δ: 162.61, 159.95, 156.70, 151.26, 150.94, 146.17, 134.37, 132.28, 125.66, 122.98, 122.15, 120.13, 114.13, 70.82, 70.76, 31.96. MS (ESI): m/z 322.3 [M+H]+. Anal. Calcd for C16H13F3N2O2: C 59.63, H 4.07, F 17.68, N 8.69, O 9.93; Found: C 59.68, H 4.09, F 17.53, N 8.73, O 9.89.
(E)-6-((3,4-Dihydro-2H-benzo[b][1,4]dioxepin-7-yl)methyleneamino)nicotinonitrile (2L)Yellow solid; yield: 77%. Rf=0.4 (4 : 1 hexane–EtOAc); mp: 115.4–116.3°C. 1H-NMR (400 MHz, DMSO-d6) δ: 9.05 (s, 1H), 8.48 (ddd, J=4.8, 1.9, 0.8 Hz, 1H), 7.88 (dd, J=1.8, 1.2 Hz, 1H), 7.61 (s, 1H), 7.33–7.31 (m, 1H), 7.13–7.06 (m, 1H), 6.45 (ddd, J=7.1, 5.0, 1.0 Hz, 1H), 4.29–4.16 (m, 4H), 2.22 - 2.10 (m, 2H). 13C-NMR (101 MHz, DMSO-d6) δ: 161.82, 160.23, 154.80, 151.43, 149.25, 148.17, 139.01, 137.36, 131.52, 125.35, 122.61, 119.93, 112.23, 70.87, 70.76, 31.41. MS (ESI): m/z 280.1 [M+H]+. Anal. Calcd for C16H13N3O2: C 68.81, H 4.69, N 15.05, O 11.46; Found: C 68.87, H 4.73, N 14.96, O 11.37.
(E)-4-Chloro-N-((3,4-dihydro-2H-benzo[b][1,4]dioxepin-7-yl)methylene)pyridin-2-amine (2M)White solid; yield 92%. Rf=0.4 (4 : 1 hexane–EtOAc); mp: 105.8–106.4°C. 1H-NMR (400 MHz, DMSO-d6) δ: 9.03 (s, 1H), 8.46 (dd, J=4.3, 1.7 Hz, 1H), 7.63 (dd, J=6.9, 1.9 Hz, 2H), 7.11 (d, J=8.9 Hz, 1H), 6.66 (d, J=1.7 Hz, 1H), 6.61 (dd, J=5.5, 1.9 Hz, 1H), 4.23 (ddd, J=11.4, 8.4, 3.7 Hz, 4H), 2.22–2.12 (m, 2H). 13C-NMR (100 MHz, DMSO-d6) δ: 163.50, 161.40, 155.14, 151.40, 150.56, 149.87, 144.77, 128.80, 125.64, 122.86, 119.30, 112.17, 70.88, 70.73, 31.32. MS (ESI): m/z 289.4 [M+H]+. Anal. Calcd for C15H13ClN2O2: C 62.40, H 4.54, Cl 12.28, N 9.70, O 11.08; Found: C 62.45, H 4.57, Cl 12.23, N 9.68, O 11.12.
(E)-N-((3,4-Dihydro-2H-benzo[b][1,4]dioxepin-7-yl)methylene)-4-methylpyridin-2-amine (2N)White solid; yield: 88%. Rf=0.5 (4 : 1 hexane–EtOAc); mp: 153.3–153.6°C. 1H-NMR (400 MHz, DMSO-d6) δ: 9.05 (s, 1H), 8.33 (d, J=5.0 Hz, 1H), 7.75 (d, J=5.2 Hz, 1H), 7.61 (dd, J=6.9, 1.9 Hz, 2H), 7.18–7.03 (m, 1H), 6.31 (d, J=5.2 Hz, 1H), 4.23 (dt, J=13.6, 5.6 Hz, 4H), 2.36 (s, 3H), 2.15 (dd, J=11.2, 5.6 Hz, 2H). 13C-NMR (100 MHz, DMSO-d6) δ: 161.60, 160.33, 154.73, 151.43, 149.79, 148.85, 147.84, 131.59, 125.27, 122.51, 120.50, 113.81, 70.86, 70.74, 31.42, 21.01. MS (ESI): m/z 269.2 [M+H]+. Anal. Calcd for C15H16N2O2: C 71.62, H 6.01, N 10.44, O 11.93; Found: C 71.68, H 6.11, N 10.39, O 11.88.
(E)-N-((3,4-Dihydro-2H-benzo[b][1,4]dioxepin-7-yl)methylene)-3-methylpyridin-2-amine (2O)White solid; yield: 86%. Rf=0.5 (4 : 1 hexane–EtOAc); mp: 145.8–146.1°C. 1H-NMR (400 MHz, DMSO-d6) δ: 8.99 (s, 1H), 8.32–8.23 (m, 1H), 7.68 (ddd, J=7.4, 1.7, 0.7 Hz, 1H), 7.65–7.59 (m, 2H), 7.19 (dd, J=7.4, 4.8 Hz, 1H), 7.12 (dd, J=12.7, 8.2 Hz, 1H), 4.24–4.16 (m, 4H), 2.38 (s, 3H), 2.16 (dd, J=5.7, 3.3 Hz, 2H). 13C-NMR (100 MHz, DMSO-d6) δ: 160.74, 156.70, 151.47, 151.33, 146.54, 145.49, 139.47, 137.33, 132.28, 125.41, 122.50, 112.80, 70.88, 70.84, 30.96, 17.53. MS (ESI): m/z 269.2 [M+H]+. Anal. Calcd for C15H16N2O2: C 71.62, H 6.01, N 10.44, O 11.93; Found: C 71.70, H 5.97, N 10.48, O 11.87.
BiologyBacterial suppressive assay: The antibacterial activity of the synthesized compounds was tested against E. coli ATCC 25922, P. aeruginosa ATCC 27853, S. aureus ATCC 6538 and B. subtilis ATCC 530 (kindly provided by pro Chang-Hong Liu, State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing, China) using Mueller–Hinton medium (MH medium: 17.5 g casein hydrolysate, 1.5 g soluble starch, 1000 mL beef extract). The MIC values of the tested compounds were determined by a colorimetric method using MTT. A stock solution of the test compound (100 µg/mL) in DMSO was prepared, and graded quantities were added to a specified volume of sterilized liquid MH medium. A specified volume of the compound-containing medium was then poured into microtiter plates. A suspension of the microorganism was prepared to contain approx. 105 cfu/mL and applied to microtiter plates with serially. diluted compounds in DMSO to be tested and incubated at 37°C for 24 h. After the MIC values were visually determined on each of the microtiter plates, phosphate buffered saline (PBS; 50 µL, 0.01 M, pH 7.4; 2.9 g Na2HPO4·12H2O, 0.2 g KH2PO4, 8.0 g NaCl, 0.2 g KCl, 1000 mL distilled H2O) containing MTT (2 mg/mL) was added to each well. Incubation was continued at RT for 4–5 h. The content of each well was removed, and 100 mL of isopropanol containing 5% HCl (final concentration 1 M) was added to extract the dye. After 12 h of incubation at RT, the optical density (OD) was measured with a microplate reader at 550 nm.
E. coli FabH inhibitory assay: Native E. coli FabH protein was overexpressed in E. coli DH10B cells using the pET30 vector (pET30 vector was kindly supplied by the State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University) and purified to homogeneity in three chromatographic steps (Q-Sepharose, MonoQ, and hydroxyapatite) at 4°C. The selenomethionine-substituted protein was expressed in E. coli BL21(DE3) cells and purified in a similar way. Harvested cells containing FabH were lysed by sonication in 20 mM Tris (pH 7.6) containing 5 mM imidazole and 0.5 M NaCl, and centrifuged (20000 rpm, 30 min, 4°C). The supernatant was applied to a Ni-NTA agarose column, washed, and eluted using a 5–500 mm imidazole gradient over 20 column volumes. Eluted protein was dialyzed against 20 mM Tris (pH 7.6) containing 1 mM dithiothreitol (DTT) and 100 mM NaCl. Purified FabHs were concentrated to 2 mg/mL and stored at −80°C in 20 mM Tris (pH 7.6) containing 100 mM NaCl, 1 mM DTT, and 20% glycerol for enzymatic assay.
In a final 20 µL reaction, 20 mM Na2HPO4 (pH 7.0) containing 0.5 mM DTT, 0.25 mM MgCl2, and 2.5 µM holo-ACP were mixed with 1 nM FabH, and H2O was added to 15 µL. After 1 min incubation, a 2 µL mixture of 25 µM acetyl-CoA, 0.5 mm reduced nicotinamide adenine dinucleotide (NADH), and 0.5 mM reduced nicotinamide adenine dinucleotide phosphate (NADPH) was added for FabH reaction for 25 min. The reaction was stopped by adding 20 µL of ice-cold 50% trichloroacetic acid (TCA), incubating for 5 min on ice, and centrifuging to pellet the protein. The pellet was washed with 10% ice-cold TCA and resuspended in 0.5 M NaOH (5 µL). The incorporation of the 3H signal in the final product was read by liquid scintillation. When determining the IC50 values, inhibitors were added from a concentrated DMSO stock such that the final concentration of DMSO did not exceed 2%.
Cytotoxicity test: The cytotoxic activity in vitro was measured against mouse fibroblast NIH-3T3 cells using the MTT assay. Cells were cultured in a 96-well plate at a density of 5×10−5 cells and different concentrations of compounds were respectively added to each well. The incubation was permitted at 37°C, 5% CO2 atmosphere for 24 h before the cytotoxicity assessments. Twenty microliters MTT reagent (4 mg/mL) was added per well 4 h before the end of the incubation. Four hours later, the plate was centrifugated at 1200 rcf for 5 min and the supernatants were removed, each well was added with 200 µL DMSO. The absorbance was measured at a wavelength of 570 nm (OD570 nm) on an enzyme-linked immunosorbent assay (ELISA) microplate reader. Three replicate wells were used for each concentration and each assay was measured three times, after which the average of IC50 was calculated. The cytotoxicity of each compound was expressed as the concentration of compound that reduced cell viability to 50% (IC50).
Docking StudyMolecular docking of compound 2E into the 3D Xray structure of E. coli FabH (PDB: 1HNJ)8) was carried out using Discovery Studio (v3.1) as implemented through the graphical user interface DS-CDOCKER protocol.21)
The 3D structures of the aforementioned compounds were constructed using Chem 3D ultra 12.0 (Chemical Structure Drawing Standard; Cambridge Soft Corporation, U.S.A.) and energy minimized by using MMFF94 with 5000 iterations and minimum RMS gradient of 0.10. The crystal structures of E. coli FabH (PDB: 1HNJ)8) complex were retrieved from the RCSB Protein Data Bank (http://www.rcsb.org/pdb/). All bound waters and ligands were eliminated from the protein and the polar hydrogen was added to the proteins.
This work was financed by China Postdoctoral Science Foundation.
The authors declare no conflict of interest.
