Accepted Manuscript Synthesis, molecular docking and biological evaluation of coumarin derivatives containing piperazine skeleton as potential antibacterial agents She-Feng Wang, Yong Yin, Xun Wu, Fang Qiao, Shao Sha, Peng-Cheng Lv, Jing Zhao, Hai-Liang Zhu PII: DOI: Reference:
S0968-0896(14)00702-0 http://dx.doi.org/10.1016/j.bmc.2014.09.048 BMC 11835
To appear in:
Bioorganic & Medicinal Chemistry
Received Date: Revised Date: Accepted Date:
5 July 2014 6 September 2014 23 September 2014
Please cite this article as: Wang, S-F., Yin, Y., Wu, X., Qiao, F., Sha, S., Lv, P-C., Zhao, J., Zhu, H-L., Synthesis, molecular docking and biological evaluation of coumarin derivatives containing piperazine skeleton as potential antibacterial agents, Bioorganic & Medicinal Chemistry (2014), doi: http://dx.doi.org/10.1016/j.bmc.2014.09.048
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Synthesis, molecular docking and biological evaluation of coumarin derivatives containing piperazine skeleton as potential antibacterial agents She-Feng Wang +, Yong Yin+, Xun Wu, Fang Qiao, Shao Sha, Peng-Cheng Lv*, Jing Zhao *, Hai-Liang Zhu*.
State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, People’s Republic of China + *
These authors contributed equally to this work.
Corresponding authors. Tel.: +86 25 83592572; Fax: +86 25 83592672. E-mail address: [email protected] (H.-L. Zhu).
1
Abstract A series of 4-hydroxycoumarin derivatives were designed and synthesized in order to find some more potent antibacterial drugs. Their antibacterial activities against
Escherichia
coli,
Pseudomonas
aeruginosa,
Bacillus
subtilis
and
Staphylococcus aureus were tested. These compounds showed good antibacterial activities against Gram-positive strains. Compound 4g represented the most potent antibacterial activity against Bacillus subtilis and S.aureus with MIC of 0.236, 0.355 µg/mL, respectively. What’s more, it showed the most potent activity against SaFabI with IC50 of 0.57 µM. Molecular docking of 4g into S.aureus Enoyl-ACP-reductase active site were performed to determine the probable binding mode, while the QSAR model was built to check the previous work as well as to introduce new directions.
Keywords Coumarin derivatives Antibacterial activities SaFabI Molecular docking 3D-QSAR
2
1. Introduction
The emergence of bacterial resistance to most of the antibacterials is becoming a serious global healthcare problem.
1
Particularly, the spread of multiresistant
Gram-positive bacteria, such as methicillin resistant Staphylococcus aureus (MRSA), 2
was fast. It was only two years after the introduction of the antibiotic methicillin in
1959 that resistant strains of the gram-positive human pathogen Staphylococcus aureus emerged. Since then, methicillin-resistant S. aureus (MRSA) infections have become an increasing problem in hospitals worldwide. However, antibiotic resistance has drastically outpaced new antibiotic discovery, which flourished between the 1940s and 1960s. Only a few novel scaffolds were approved between 1962 and 2000, such as the carbapenems. It is an urgent to elaborate new, highly potent antibiotics with alternative modes of actions. In this regard, bacterial fatty acid biosynthesis (FAS) is an attractive target. 3 Fatty acid synthesis (FAS) pathways are divided into two distinct forms: FAS-I and FAS-II.
4
Enzymes of FAS-II pathway have been selectively targets for the
development of novel antibacterial agents.
5
The FAS-II pathway is consist of some
distinct and highly conserved enzymes: β-hydroxacyl-ACP-dehydratase (FabZ), β-ketoacyl-ACP-reductase (FabG), and enoyl-ACP-reductase (FabI, also known as ENR). For most bacterias, FabI, the unique ENR, is responsible for the concluding reduction step of each elongation cycle. It represents a key physiological regulator of fatty acid biosynthesis, and it has been carried out to be an important drug target for the development of novel antibacterials and antimalarials.
6-8
Furthermore, it has been
proved that the sequences of human FAS have many similarities with some other creatures.9 This has led to the pursuit of specific FabI inhibitors as novel antibacterial agents which would not be expected to interfere with comparable human biochemical processes. 10 The antibacterial target of isoniazid (Scheme 1), 11 used in the treatment of tuberculosis for 50 years, and triclosan (Scheme 1),
12
the broad spectrum biocide
in a wide range of consumer goods, have been determined to be the enoyl-ACP reductase (FabI). Here we describe a series of novel FabI inhibitors. 3
Coumarins are a class of compounds found widely in nature, they show a broad spectrum of activities and are frequently associated with low toxicity,
13
and they can
be considered as a privileged scaffold and an ideal framework for the design of compounds that can interact with different targets as their inherent affinity for several biological targets.
14
Like ensaculine, one kind of AChE inhibitors, a coumarin
derivative containing piperazine ring (Scheme 1). There have been performed a lot of efforts which aimed at designing functionalized synthetic coumarins as effective agents that can affect some cellular functions potently and selectively.
15–24
Since
piperazines and its derivatives are important pharmacophores, they are effective ingredients in many marketed drugs like olaparib and Piperazinyl-linked ciprofloxacin dimers which was reported as potent antibacterial agents (Scheme 1).
25-26
These
results promoted us to synthesize new derivatives of coumarins with piperazine skeleton for the sake of finding new effectively antibacterial agents. Based on these things mentioned above, we synthesized two series of novel coumarins derivatives owning piperazine skeleton (4a-4n) and (5a-5n) (Table 1), while compounds 5a, 5i, 5l and 5m were reported before.
27
We studied their antibacterial activities against
Bacillus subtilis (B. subtilis) and Staphylococcus aureus (S. aureus), Escherichia coli (E. coli), Pseudomonas aeruginosa (P. aeruginosa), and Staphylococcus aureus Enoyl-ACP-reductase (SaFabI) (PDB code: 4CV0) inhibitory activities. Docking simulation was performed using the X-ray crystallographic structure of SaFabI in complex with the most potent inhibitor to explore the binding mode of the compound at the active site. Additionally, we also used the results of molecular orbital calculations (3D-QSAR model) to study the structure–activity relationship and guide the further study.
2. Results and discussion
2.1. Chemistry
Compounds 4a-4n and 5a-5n were synthesized by the routes outlined in Scheme 4
2. They were first designed and synthesized from coumarin and piperazine derivatives except componds 5a, 5i, 5l and 5m. The chemical structures of these coumarin derivatives were summarized in Table 1. All of the synthetic compounds were characterized by 1H NMR, elemental analysis and mass spectrum, and they gave satisfactory analytical and spectroscopic data, which were in full accordance with their depicted structures.
2.2. Biological activity
2.2.1. Antibacterial activity
The synthesized compounds were tested for their antibacterial activities against two Gram-negative bacterial strains: E. coli and P. aeruginosa and two Gram-positive bacterial strains: B. subtilis and S. aureus by MTT method with MH medium (Muellere-Hinton medium: casein hydrolysate 17.5 g, soluble starch 1.5 g, beef extract 1000 mL). The MICs (minimum inhibitory concentrations) of the compounds against these bacteria were reported in Table 2, the activity of reference drug Penicillin G was also included. The results revealed that those compounds showed good activity against Gram-positive bacteria but inactive against Gram-negative bacteria. Most of the new compounds exhibited good inhibitory activities against both B. Subtilis and S. Aureus. As shown in Table 2. Compound 4g showed the most potent activities with MIC values of 0.236 and 0.355 µg/mL against B. subtilis and S. aureus, respectively, which were superior to the positive control Penicillin G with MIC values of 0.752 and 1.685 µg/mL. Compound 5g, with MIC values of 0.861, 0.327 µg/mL against B. subtilis and S. aureus, respectively, also displayed better activities than the positive control Penicillin G. 4f (MIC=0.612 µg/mL) and 4i (MIC=0.339 µg/mL) showed better inhibitory activities against B. subtilis than Penicillin G; 5i (MIC=1.857 µg/mL) and 5n (MIC=0.916 µg/mL) exhibited moderate inhibitory against B. subtilis; 4c (MIC=0.612 µg/mL), 4n (MIC=0.612 µg/mL), 5f (MIC=0.612 5
µg/mL) and 5n (MIC=0.612 µg/mL) displayed better inhibitory activities against S. aureus than Penicillin G (MIC=1.685 µg/mL). According to these data, in 4a-4n, we found that compounds which have a methoxy group on the position of benzene ring showed higher antibacterial activity than other compounds. Especially compound 4g, which had a methoxy group on the 4-position of benzene ring, showed higher antibacterial activity against B. Subtilis and S. aureus with MIC values of 0.236 µg/mL and 0.355 µg/mL than compound 4e which had a methoxy group on the 2-position of benzene ring with MIC values of 3.883 µg/mL and 1.832 µg/mL and compound 4f which had a methoxy group on the 3-position of benzene ring with MIC values of 0.612 µg/mL and 2.523 µg/mL, the same trend was also found in compounds 5e-5g. Compounds 4i-4h and 5i-5h, which introduced two chlorine atoms at 2,3-position or 3,4-positon in benzene ring, showed that compounds with substitutes at 3,4-position displayed better inhibitory activity than substitutes at 2,3-postion. These results indicated that compounds with substitution at the para- position exhibited more potent antibacterial activities than compounds with substitution at the meta- and ortho-position. Compounds 4l-4m and 5l-5m which introduced fluorine atom at 2-position or 4-positon in benzene ring, displayed a dramatic loss in activity compared to compounds 4n and 5n with a trifluoromethyl at 3-position. Among compounds 4a-4d and 5a-5d the order of inhibitory activities showed the potency of 4c > 4d > 4a> 4b and 5d>5b>5c>5a which indicated the activity of compounds with substitutes were superior to that of without sbustitutes. Varieties of substitutes of phenyl like trifluoromethyl, methyl and halogen can lead to different antibacterial activities. For example, the derivatives which have electron-withdrawing substituents (like halogen and trifluoromethyl) displayed weaker activities against B. subtilis and S. aureus than those having electron-donating substituents (such as methyl and methoxyl), which suggested that substituent group can affect the inhibitory activities, and compounds with electron-donating substituents exhibited better inhibitory activities than those with electron-withdrawing substituents. 6
2.2.2. S. aureus FabI inhibitory activity
The ability of 4a-4n and 5a-5n to inhibit the recombinant SaFabI enzyme were examined. All data were shown in Table 3. Among compounds 4a-4n, 4d and 4f exhibited moderate SaFabI inhibition with IC50 values of 3.82 µM and 2.73 µM, respectively. While 4g and 4n showed the highest inhibitory activity with IC50 values of 0.57 µM and 0.97 µM, respectively. With compounds 5a-5n, all compounds in this subset displayed moderate activity toward SaFabI. With the exception of 5g featuring a p- methoxyl subunit, which turned out to be the most active compound within series II (IC50 = 0.83 µM). 5f and 5n inhibited SaFabI with IC50 values of 54.62 and 1.82 µM, respectively. As showed in Table 3, compounds with substituents on the position of benzene ring displayed better inhibitory activity than that with no substituents on the position of benzene ring (4a). Compounds 4g (IC50 =0.57 µM) and 5g (IC50 =0.83 µM) with p-methoxyl on the position of benzene ring showed the highest inhibitory activity. Compounds 4e-4g and 5e-5g, which introduced a methoxy group at 2-position, 3-positon or 4-positon in benzene ring, showed the SaFabI inhibitory activity trend: 4e (IC50 =5.54 µM)50 23.023 >50 >50 2.856 1.443 >50 >50 3.65 2.395 >50 33.028 3.883 2.523 >50 49.823 0.612 1.832 >50 17.059 0.236 0.355 >50 >50 18.169 25.956 >50 >50 0.339 >50 >50 >50 13.928 11.639 >50 >50 25.971 >50 40.57 >50 >50 >50 >50 >50 >50 >50 40.39 >50 1.078 0.957 >50 >50 >50 41.545 33.64 >50 15.749 >50 >50 >50 34.418 46.387 >50 >50 1.938 >50 >50 >50 10.955 8.186 >50 >50 6.957 0.837 >50 >50 0.861 0.327 >50 >50 4.512 23.221 >50 >50 1.857 >50 >50 48.07 7.183 28.482 >50 >50 2.938 >50 >50 46.019 31.321 >50 >50 >50 34.794 29.099 >50 >50 0.916 1.083 >50 >50 0.752 1.685 22.358 21.957
30
Table 3. S. aureus FabI inhibitory activity of synthetic compounds. compounds 4a 4b 4c 4d 4e 4f 4g 4h 4i 4j 4k 4l 4m 4n
S. aureus FabI IC50(µM) 47.79 41.26 6.23 3.82 5.54 2.73 0.57 12.51 22.27 17.66 31.39 71.09 62.85 0.97
compounds 5a 5b 5c 5d 5e 5f 5g 5h 5i 5j 5k 5l 5m 5n
31
S. aureus FabI IC50(µM) 45.51 35.49 42.52 11.37 7.15 4.62 0.83 8.41 19.75 21.94 26.57 43.22 37.57 1.82
Table 4. Hemolytic activities and cytotoxicity of the selected compounds. compounds 4c 4d 4e 4f 4g 4n 5e 5f 5g 5h 5n penicillin a Lytic concentration 30%.
Hemolysis LC30a (mg/mL) >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10
32
Cytotoxicity IC50 (µM) 127.37 152.61 255.83 178.25 198.42 172.26 164.38 141.26 191.53 223.16 188.44 185.72
Table 5. Experimental, predicted inhibitory activity of all synthesized compounds against S. aureus FabI by 3D-QSAR models based on active conformations selected from the preliminary molecular docking study. Compounds 4a 4ba 4c 4d 4e 4f 4g 4h 4i 4j 4k 4l 4m 4na 5a a 5b 5c 5da 5e 5f 5g 5ha 5i 5j 5k 5l 5m 5n a
Experimental pIC50 b 4.32 4.38 5.28 5.42 5.26 5.56 6.24 4.9 4.65 4.75 4.5 4.15 4.2 5.59 4.94 4.45 4.37 4.94 5.15 5.34 6.08 5.08 4.7 4.66 4.58 4.36 4.43 5.72
Predicted pIC50 4.49 4.46 5.28 4.97 5.01 5.70 6.15 4.77 4.67 4.73 4.63 4.07 4.13 5.68 5.05 4.75 4.63 5.24 5.11 5.29 5.94 4.92 5.22 4.59 4.33 4.31 4.57 5.73
Residual error -0.17 -0.08 0.00 0.45 0.25 -0.14 0.09 0.13 -0.02 0.02 -0.13 0.08 0.07 -0.09 -0.11 -0.30 -0.26 -0.30 0.04 0.05 0.14 0.16 -0.52 0.07 0.25 0.05 -0.14 -0.01
Compounds were selected as the test sets while the rest ones were in the training
sets. b
The IC50 values of the compounds against SaFabI (Table 3) were converted into
pIC50
values
by
using
(http://www.sanjeevslab.org/tools-IC50.html).
33
the
online
calculator
Figure 1 A. Binding mode of compound 4g with FabI from S. aureus. For clarity, only interacting residues were labeled. Hydrogen bonding interactions are shown in green dotted lines. This figure was made using DS.
34
Figure 1B. Binding mode of compound 4g with FabI. The enzyme is shown as surface; while 4g docked structures are shown as sticks. This figure was made using DS.
35
Figure 2 Linear fit curve of the observed pIC50 values versus the predicted values of the training set part and the test set part, R2= 0.88.
36
Figure 3 3D-QSAR of coumarin derivatives containing piperazine for SaFabI (pdb: 4CV0). (C) Iso-surface of the 3D-QSAR model coefficients on electrostatic potential grids. The blue triangle mesh represents positive electrostatic potential and the red area represents negative electrostatic potential. (D) Iso-surface of the 3D-QSAR model coefficients on Van der Waals grids. The green triangle mesh representation indicates positive coefficients; the yellow triangle mesh indicates negative coefficients. 37
Scheme 1 Chemical structure of drugs.
Scheme 2 Reagents and conditions: (a) Acetone, K2CO3, 50 ℃, 10-12 h; (b) K2CO3, DMF, 110 ℃, 20-24 h; (c) Cs2CO3, DMF,90 ℃, 8-10 h; (d) CH2Cl2, HOBt, EDC, 50℃, 8-12 h.
38
Synthesis, molecular docking and biological evaluation of coumarin derivatives containing piperazine skeleton as potential antibacterial agents She-Feng Wang +, Yong Yin+, Xun Wu, Fang Qiao, Shao Sha, Peng-Cheng Lv*, Jing Zhao *, Hai-Liang Zhu*.
State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, People’s Republic of China + *
These authors contributed equally to this work.
Corresponding authors. Tel.: +86 25 83592572; Fax: +86 25 83592672. E-mail address: [email protected] (H.-L. Zhu).
A series of 4-hydroxycoumarin derivatives were designed and synthesized in order to find some more potent antibacterial drugs. Their antibacterial activities against
Escherichia
coli,
Pseudomonas
aeruginosa,
Bacillus
subtilis
and
Staphylococcus aureus were tested. These compounds showed good antibacterial activities against Gram-positive strains. Compound 4g represented the most potent antibacterial activity against Bacillus subtilis and S.aureus with MIC of 0.236, 0.355 μg/mL, respectively. What’s more, it showed the most potent activity against SaFabI 39
with IC50 of 0.57 μM. Molecular docking of 4g into S.aureus Enoyl-ACP-reductase active site were performed to determine the probable binding mode, while the QSAR model was built to check the previous work as well as to introduce new directions.
40
S0968-0896(14)00702-0 http://dx.doi.org/10.1016/j.bmc.2014.09.048 BMC 11835
To appear in:
Bioorganic & Medicinal Chemistry
Received Date: Revised Date: Accepted Date:
5 July 2014 6 September 2014 23 September 2014
Please cite this article as: Wang, S-F., Yin, Y., Wu, X., Qiao, F., Sha, S., Lv, P-C., Zhao, J., Zhu, H-L., Synthesis, molecular docking and biological evaluation of coumarin derivatives containing piperazine skeleton as potential antibacterial agents, Bioorganic & Medicinal Chemistry (2014), doi: http://dx.doi.org/10.1016/j.bmc.2014.09.048
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Synthesis, molecular docking and biological evaluation of coumarin derivatives containing piperazine skeleton as potential antibacterial agents She-Feng Wang +, Yong Yin+, Xun Wu, Fang Qiao, Shao Sha, Peng-Cheng Lv*, Jing Zhao *, Hai-Liang Zhu*.
State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, People’s Republic of China + *
These authors contributed equally to this work.
Corresponding authors. Tel.: +86 25 83592572; Fax: +86 25 83592672. E-mail address: [email protected] (H.-L. Zhu).
1
Abstract A series of 4-hydroxycoumarin derivatives were designed and synthesized in order to find some more potent antibacterial drugs. Their antibacterial activities against
Escherichia
coli,
Pseudomonas
aeruginosa,
Bacillus
subtilis
and
Staphylococcus aureus were tested. These compounds showed good antibacterial activities against Gram-positive strains. Compound 4g represented the most potent antibacterial activity against Bacillus subtilis and S.aureus with MIC of 0.236, 0.355 µg/mL, respectively. What’s more, it showed the most potent activity against SaFabI with IC50 of 0.57 µM. Molecular docking of 4g into S.aureus Enoyl-ACP-reductase active site were performed to determine the probable binding mode, while the QSAR model was built to check the previous work as well as to introduce new directions.
Keywords Coumarin derivatives Antibacterial activities SaFabI Molecular docking 3D-QSAR
2
1. Introduction
The emergence of bacterial resistance to most of the antibacterials is becoming a serious global healthcare problem.
1
Particularly, the spread of multiresistant
Gram-positive bacteria, such as methicillin resistant Staphylococcus aureus (MRSA), 2
was fast. It was only two years after the introduction of the antibiotic methicillin in
1959 that resistant strains of the gram-positive human pathogen Staphylococcus aureus emerged. Since then, methicillin-resistant S. aureus (MRSA) infections have become an increasing problem in hospitals worldwide. However, antibiotic resistance has drastically outpaced new antibiotic discovery, which flourished between the 1940s and 1960s. Only a few novel scaffolds were approved between 1962 and 2000, such as the carbapenems. It is an urgent to elaborate new, highly potent antibiotics with alternative modes of actions. In this regard, bacterial fatty acid biosynthesis (FAS) is an attractive target. 3 Fatty acid synthesis (FAS) pathways are divided into two distinct forms: FAS-I and FAS-II.
4
Enzymes of FAS-II pathway have been selectively targets for the
development of novel antibacterial agents.
5
The FAS-II pathway is consist of some
distinct and highly conserved enzymes: β-hydroxacyl-ACP-dehydratase (FabZ), β-ketoacyl-ACP-reductase (FabG), and enoyl-ACP-reductase (FabI, also known as ENR). For most bacterias, FabI, the unique ENR, is responsible for the concluding reduction step of each elongation cycle. It represents a key physiological regulator of fatty acid biosynthesis, and it has been carried out to be an important drug target for the development of novel antibacterials and antimalarials.
6-8
Furthermore, it has been
proved that the sequences of human FAS have many similarities with some other creatures.9 This has led to the pursuit of specific FabI inhibitors as novel antibacterial agents which would not be expected to interfere with comparable human biochemical processes. 10 The antibacterial target of isoniazid (Scheme 1), 11 used in the treatment of tuberculosis for 50 years, and triclosan (Scheme 1),
12
the broad spectrum biocide
in a wide range of consumer goods, have been determined to be the enoyl-ACP reductase (FabI). Here we describe a series of novel FabI inhibitors. 3
Coumarins are a class of compounds found widely in nature, they show a broad spectrum of activities and are frequently associated with low toxicity,
13
and they can
be considered as a privileged scaffold and an ideal framework for the design of compounds that can interact with different targets as their inherent affinity for several biological targets.
14
Like ensaculine, one kind of AChE inhibitors, a coumarin
derivative containing piperazine ring (Scheme 1). There have been performed a lot of efforts which aimed at designing functionalized synthetic coumarins as effective agents that can affect some cellular functions potently and selectively.
15–24
Since
piperazines and its derivatives are important pharmacophores, they are effective ingredients in many marketed drugs like olaparib and Piperazinyl-linked ciprofloxacin dimers which was reported as potent antibacterial agents (Scheme 1).
25-26
These
results promoted us to synthesize new derivatives of coumarins with piperazine skeleton for the sake of finding new effectively antibacterial agents. Based on these things mentioned above, we synthesized two series of novel coumarins derivatives owning piperazine skeleton (4a-4n) and (5a-5n) (Table 1), while compounds 5a, 5i, 5l and 5m were reported before.
27
We studied their antibacterial activities against
Bacillus subtilis (B. subtilis) and Staphylococcus aureus (S. aureus), Escherichia coli (E. coli), Pseudomonas aeruginosa (P. aeruginosa), and Staphylococcus aureus Enoyl-ACP-reductase (SaFabI) (PDB code: 4CV0) inhibitory activities. Docking simulation was performed using the X-ray crystallographic structure of SaFabI in complex with the most potent inhibitor to explore the binding mode of the compound at the active site. Additionally, we also used the results of molecular orbital calculations (3D-QSAR model) to study the structure–activity relationship and guide the further study.
2. Results and discussion
2.1. Chemistry
Compounds 4a-4n and 5a-5n were synthesized by the routes outlined in Scheme 4
2. They were first designed and synthesized from coumarin and piperazine derivatives except componds 5a, 5i, 5l and 5m. The chemical structures of these coumarin derivatives were summarized in Table 1. All of the synthetic compounds were characterized by 1H NMR, elemental analysis and mass spectrum, and they gave satisfactory analytical and spectroscopic data, which were in full accordance with their depicted structures.
2.2. Biological activity
2.2.1. Antibacterial activity
The synthesized compounds were tested for their antibacterial activities against two Gram-negative bacterial strains: E. coli and P. aeruginosa and two Gram-positive bacterial strains: B. subtilis and S. aureus by MTT method with MH medium (Muellere-Hinton medium: casein hydrolysate 17.5 g, soluble starch 1.5 g, beef extract 1000 mL). The MICs (minimum inhibitory concentrations) of the compounds against these bacteria were reported in Table 2, the activity of reference drug Penicillin G was also included. The results revealed that those compounds showed good activity against Gram-positive bacteria but inactive against Gram-negative bacteria. Most of the new compounds exhibited good inhibitory activities against both B. Subtilis and S. Aureus. As shown in Table 2. Compound 4g showed the most potent activities with MIC values of 0.236 and 0.355 µg/mL against B. subtilis and S. aureus, respectively, which were superior to the positive control Penicillin G with MIC values of 0.752 and 1.685 µg/mL. Compound 5g, with MIC values of 0.861, 0.327 µg/mL against B. subtilis and S. aureus, respectively, also displayed better activities than the positive control Penicillin G. 4f (MIC=0.612 µg/mL) and 4i (MIC=0.339 µg/mL) showed better inhibitory activities against B. subtilis than Penicillin G; 5i (MIC=1.857 µg/mL) and 5n (MIC=0.916 µg/mL) exhibited moderate inhibitory against B. subtilis; 4c (MIC=0.612 µg/mL), 4n (MIC=0.612 µg/mL), 5f (MIC=0.612 5
µg/mL) and 5n (MIC=0.612 µg/mL) displayed better inhibitory activities against S. aureus than Penicillin G (MIC=1.685 µg/mL). According to these data, in 4a-4n, we found that compounds which have a methoxy group on the position of benzene ring showed higher antibacterial activity than other compounds. Especially compound 4g, which had a methoxy group on the 4-position of benzene ring, showed higher antibacterial activity against B. Subtilis and S. aureus with MIC values of 0.236 µg/mL and 0.355 µg/mL than compound 4e which had a methoxy group on the 2-position of benzene ring with MIC values of 3.883 µg/mL and 1.832 µg/mL and compound 4f which had a methoxy group on the 3-position of benzene ring with MIC values of 0.612 µg/mL and 2.523 µg/mL, the same trend was also found in compounds 5e-5g. Compounds 4i-4h and 5i-5h, which introduced two chlorine atoms at 2,3-position or 3,4-positon in benzene ring, showed that compounds with substitutes at 3,4-position displayed better inhibitory activity than substitutes at 2,3-postion. These results indicated that compounds with substitution at the para- position exhibited more potent antibacterial activities than compounds with substitution at the meta- and ortho-position. Compounds 4l-4m and 5l-5m which introduced fluorine atom at 2-position or 4-positon in benzene ring, displayed a dramatic loss in activity compared to compounds 4n and 5n with a trifluoromethyl at 3-position. Among compounds 4a-4d and 5a-5d the order of inhibitory activities showed the potency of 4c > 4d > 4a> 4b and 5d>5b>5c>5a which indicated the activity of compounds with substitutes were superior to that of without sbustitutes. Varieties of substitutes of phenyl like trifluoromethyl, methyl and halogen can lead to different antibacterial activities. For example, the derivatives which have electron-withdrawing substituents (like halogen and trifluoromethyl) displayed weaker activities against B. subtilis and S. aureus than those having electron-donating substituents (such as methyl and methoxyl), which suggested that substituent group can affect the inhibitory activities, and compounds with electron-donating substituents exhibited better inhibitory activities than those with electron-withdrawing substituents. 6
2.2.2. S. aureus FabI inhibitory activity
The ability of 4a-4n and 5a-5n to inhibit the recombinant SaFabI enzyme were examined. All data were shown in Table 3. Among compounds 4a-4n, 4d and 4f exhibited moderate SaFabI inhibition with IC50 values of 3.82 µM and 2.73 µM, respectively. While 4g and 4n showed the highest inhibitory activity with IC50 values of 0.57 µM and 0.97 µM, respectively. With compounds 5a-5n, all compounds in this subset displayed moderate activity toward SaFabI. With the exception of 5g featuring a p- methoxyl subunit, which turned out to be the most active compound within series II (IC50 = 0.83 µM). 5f and 5n inhibited SaFabI with IC50 values of 54.62 and 1.82 µM, respectively. As showed in Table 3, compounds with substituents on the position of benzene ring displayed better inhibitory activity than that with no substituents on the position of benzene ring (4a). Compounds 4g (IC50 =0.57 µM) and 5g (IC50 =0.83 µM) with p-methoxyl on the position of benzene ring showed the highest inhibitory activity. Compounds 4e-4g and 5e-5g, which introduced a methoxy group at 2-position, 3-positon or 4-positon in benzene ring, showed the SaFabI inhibitory activity trend: 4e (IC50 =5.54 µM)50 23.023 >50 >50 2.856 1.443 >50 >50 3.65 2.395 >50 33.028 3.883 2.523 >50 49.823 0.612 1.832 >50 17.059 0.236 0.355 >50 >50 18.169 25.956 >50 >50 0.339 >50 >50 >50 13.928 11.639 >50 >50 25.971 >50 40.57 >50 >50 >50 >50 >50 >50 >50 40.39 >50 1.078 0.957 >50 >50 >50 41.545 33.64 >50 15.749 >50 >50 >50 34.418 46.387 >50 >50 1.938 >50 >50 >50 10.955 8.186 >50 >50 6.957 0.837 >50 >50 0.861 0.327 >50 >50 4.512 23.221 >50 >50 1.857 >50 >50 48.07 7.183 28.482 >50 >50 2.938 >50 >50 46.019 31.321 >50 >50 >50 34.794 29.099 >50 >50 0.916 1.083 >50 >50 0.752 1.685 22.358 21.957
30
Table 3. S. aureus FabI inhibitory activity of synthetic compounds. compounds 4a 4b 4c 4d 4e 4f 4g 4h 4i 4j 4k 4l 4m 4n
S. aureus FabI IC50(µM) 47.79 41.26 6.23 3.82 5.54 2.73 0.57 12.51 22.27 17.66 31.39 71.09 62.85 0.97
compounds 5a 5b 5c 5d 5e 5f 5g 5h 5i 5j 5k 5l 5m 5n
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S. aureus FabI IC50(µM) 45.51 35.49 42.52 11.37 7.15 4.62 0.83 8.41 19.75 21.94 26.57 43.22 37.57 1.82
Table 4. Hemolytic activities and cytotoxicity of the selected compounds. compounds 4c 4d 4e 4f 4g 4n 5e 5f 5g 5h 5n penicillin a Lytic concentration 30%.
Hemolysis LC30a (mg/mL) >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10
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Cytotoxicity IC50 (µM) 127.37 152.61 255.83 178.25 198.42 172.26 164.38 141.26 191.53 223.16 188.44 185.72
Table 5. Experimental, predicted inhibitory activity of all synthesized compounds against S. aureus FabI by 3D-QSAR models based on active conformations selected from the preliminary molecular docking study. Compounds 4a 4ba 4c 4d 4e 4f 4g 4h 4i 4j 4k 4l 4m 4na 5a a 5b 5c 5da 5e 5f 5g 5ha 5i 5j 5k 5l 5m 5n a
Experimental pIC50 b 4.32 4.38 5.28 5.42 5.26 5.56 6.24 4.9 4.65 4.75 4.5 4.15 4.2 5.59 4.94 4.45 4.37 4.94 5.15 5.34 6.08 5.08 4.7 4.66 4.58 4.36 4.43 5.72
Predicted pIC50 4.49 4.46 5.28 4.97 5.01 5.70 6.15 4.77 4.67 4.73 4.63 4.07 4.13 5.68 5.05 4.75 4.63 5.24 5.11 5.29 5.94 4.92 5.22 4.59 4.33 4.31 4.57 5.73
Residual error -0.17 -0.08 0.00 0.45 0.25 -0.14 0.09 0.13 -0.02 0.02 -0.13 0.08 0.07 -0.09 -0.11 -0.30 -0.26 -0.30 0.04 0.05 0.14 0.16 -0.52 0.07 0.25 0.05 -0.14 -0.01
Compounds were selected as the test sets while the rest ones were in the training
sets. b
The IC50 values of the compounds against SaFabI (Table 3) were converted into
pIC50
values
by
using
(http://www.sanjeevslab.org/tools-IC50.html).
33
the
online
calculator
Figure 1 A. Binding mode of compound 4g with FabI from S. aureus. For clarity, only interacting residues were labeled. Hydrogen bonding interactions are shown in green dotted lines. This figure was made using DS.
34
Figure 1B. Binding mode of compound 4g with FabI. The enzyme is shown as surface; while 4g docked structures are shown as sticks. This figure was made using DS.
35
Figure 2 Linear fit curve of the observed pIC50 values versus the predicted values of the training set part and the test set part, R2= 0.88.
36
Figure 3 3D-QSAR of coumarin derivatives containing piperazine for SaFabI (pdb: 4CV0). (C) Iso-surface of the 3D-QSAR model coefficients on electrostatic potential grids. The blue triangle mesh represents positive electrostatic potential and the red area represents negative electrostatic potential. (D) Iso-surface of the 3D-QSAR model coefficients on Van der Waals grids. The green triangle mesh representation indicates positive coefficients; the yellow triangle mesh indicates negative coefficients. 37
Scheme 1 Chemical structure of drugs.
Scheme 2 Reagents and conditions: (a) Acetone, K2CO3, 50 ℃, 10-12 h; (b) K2CO3, DMF, 110 ℃, 20-24 h; (c) Cs2CO3, DMF,90 ℃, 8-10 h; (d) CH2Cl2, HOBt, EDC, 50℃, 8-12 h.
38
Synthesis, molecular docking and biological evaluation of coumarin derivatives containing piperazine skeleton as potential antibacterial agents She-Feng Wang +, Yong Yin+, Xun Wu, Fang Qiao, Shao Sha, Peng-Cheng Lv*, Jing Zhao *, Hai-Liang Zhu*.
State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, People’s Republic of China + *
These authors contributed equally to this work.
Corresponding authors. Tel.: +86 25 83592572; Fax: +86 25 83592672. E-mail address: [email protected] (H.-L. Zhu).
A series of 4-hydroxycoumarin derivatives were designed and synthesized in order to find some more potent antibacterial drugs. Their antibacterial activities against
Escherichia
coli,
Pseudomonas
aeruginosa,
Bacillus
subtilis
and
Staphylococcus aureus were tested. These compounds showed good antibacterial activities against Gram-positive strains. Compound 4g represented the most potent antibacterial activity against Bacillus subtilis and S.aureus with MIC of 0.236, 0.355 μg/mL, respectively. What’s more, it showed the most potent activity against SaFabI 39
with IC50 of 0.57 μM. Molecular docking of 4g into S.aureus Enoyl-ACP-reductase active site were performed to determine the probable binding mode, while the QSAR model was built to check the previous work as well as to introduce new directions.
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