DOI: 10.1002/slct.

201901391 Full Papers

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2 z Medicinal Chemistry & Drug Discovery
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Synthesis, Antimicrobial Activity and 3D-QSAR Study of
6 Hybrid Oxazine Clubbed Pyridine Scaffolds
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8 Nisheeth C. Desai,*[a] Nayan B. Bhatt,[a] Surbhi B. Joshi,[a] Krunalsinh A. Jadeja,[a] and
9 Vijay M. Khedkar[b]
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Compounds 1-((1-(4-(2H-benzo[e][1,3]oxazin-3(4H)-yl)phenyl) antimicrobial activity of the titled compounds. Statistically
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ethylidene)amino)-6-((arylidene)amino)-4-(4-chlorophenyl)-2- significant 3D-QSAR (CoMFA and CoMSIA) models were created
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oxo-1,2-dihydropyridine-3,5-dicarbonitriles (4a-j) were pre- which could provide valuable information about the structural
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pared, characterized and screened for antimicrobial activity. modification desired to improvise the biological activity. Addi-
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3D-quantitative structure activity relationship (3D-QSAR) was tionally, the predictions based on the molecular docking study,
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explained by CoMFA and CoMSIA models to rationalize the against microbial DNA gyrase, were carried out.
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Hybrid scaffolds were useful to enhance the biological activity
20 Introduction
as it increases the number of binding sites. The shining
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Antimicrobial agents are used from the last 70 years to treat example of a hybrid drug is cadazolid. It was synthesized by
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the infections caused by microbes.[1] In recent times, microbes fusing ciprofloxacin and tedizolid. Heterocycles such as the six
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have developed resistance to antimicrobials developed to kill and five-membered rings are kinetically favorable and can be
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them, making the existing medicines less effective. Further- prepared easily in high yields. In this paper, we have tested the
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more, overuse or misuse of antimicrobial drugs can develop effect of electron-withdrawing and electron donating groups
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resistance faster.[2] As per the data of Centers for Disease on antimicrobial activity. Previously our research group synthe-
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Control and Prevention, 23 million people die each year sized and screened the antimicrobial activity of pyridine and 2-
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because of antimicrobial resistance in the United States.[1] amino-4-methylthiazole-5-carboxylate based hybrid bio-active
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Although some species can still be treated with the traditional compounds .[7–15] 3D-QSAR study is supportive to compare
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treatments such as the diseases caused by E. coli bacteria, no between antimicrobial activity and chemical structure.[16,17] The
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specific treatment is available to prevent symptoms.[3] For P. contour plots derived from CoMFA and CoMSIA models provide
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aeruginosa infections, treatment of hospitalized patients is the information about the electrostatic, steric properties
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more difficult because of increasing medicinal resistance.[4] The (CoMFA), hydrophobicity and hydrogen bonding (CoMSIA)
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solution of this worldwide problem is to synthesis new effective which influencing the antimicrobial activity. We also performed
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antimicrobials which are not yet resistant towards microbes. exploratory molecular docking studies on the microbial DNA
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The study of hybrid heterocyclic compounds has garnered gyrase. It reorganized in vitro biological data and demonstrated
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much attention in medicinal chemistry.[5,6] This group of the mechanism of an antimicrobial activity for the further
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compounds is instrumental in fighting against bacterial resist- research permanence is on-going.
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ance. In continuation, we have adopted a hybrid approach for
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the development of novel antimicrobials. The synthesis of these
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scaffolds is based on the fusion of two or more than two drugs Results and Discussion:
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or two or more bioactive moieties with the goal of retaining
43 Chemistry
the biological action of principal fragments.
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We selected two important heteromeric moieties oxazine The synthetic pathway of 1-((1-(4-(2H-benzo[e][1,3]oxazin-
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and pyridine as a linker for the synthesis of bioactive molecules. 3(4H)-yl)phenyl)ethylidene)amino)-6-((arylidene)amino)-4-(4-
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chlorophenyl)-2-oxo-1,2-dihydropyridine-3,5-dicarbonitriles
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(4 a-j) is described in the Scheme-1. N’-(1-(4-(2H-benzo[e][1,3]
48 [a] Prof. N. C. Desai, Dr. N. B. Bhatt, S. B. Joshi, K. A. Jadeja oxazin-3(4H)-yl)phenyl)ethylidene)-2-cyanoacetohydrazide (1),
49 Division of Medicinal Chemistry, Department of Chemistry (DST-FIST
refluxed with 2-(4-chlorobenzylidene)malononitrile (2) in the
50 Sponsored & UGC NON-SAP), Mahatma Gandhi Campus, M. K. Bhavnagar
University, Bhavnagar-364002, India presence of piperidine catalyst, produced intermediate (3).
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E-mail: dnisheeth@rediffmail.com Intermediate 3 is generated by nucleophilic attack of the
52 [b] Dr. V. M. Khedkar nitrogen on the carbon of the nitrile group followed by a
53 Department of Pharmaceutical Chemistry, Shri Vile Parle Kelavani
proton transfer and a tautomerization. Intermediate 3 refluxed
54 Mandal’s Institute of Pharmacy, Mumbai - Agra National Hwy, Dhule,
Maharashtra 424001, India with different derivatives of benzaldehyde for 8–10 h and
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Supporting information for this article is available on the WWW under produced reported compounds (4 a-j).
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https://doi.org/10.1002/slct.201901391
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23 Scheme 1. Preparation of newly synthesized compounds (4a-j)
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26 Table 1. Results of Antimicrobial activities of compounds (4a-j)
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Sr. No. -R Minimum inhibitory concentration (MIC) in μg/mL Minimum inhibitory concentration (MIC) in μg/mL
28 E. c. MTCC (443) P. a. MTCC (1688) S. a. MTCC (96) S. p. MTCC (442) C. a. MTCC (227) A. n. MTCC (282) A. c. MTCC (1323)
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4a -H 200 1000 500 62.5 > 1000 > 1000 > 1000
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4b -2-OH 200 25 50 200 > 1000 > 1000 > 1000
31 4c -4-OH 100 250 500 125 200 200 500
32 4d -3-NO2 500 100 1000 1000 500 > 1000 > 1000
33 4e -4-NO2 250 1000 1000 500 500 > 1000 > 1000
4f -4-OCH3 500 1000 1000 500 50 100 100
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4g -3,4,5-(OCH3)3 500 125 500 500 1000 62.5 100
35 4h -4-OH-3-OCH3 25 500 1000 500 500 > 1000 25
36 4i -N,N’-(CH3)2 125 1000 125 500 100 > 1000 > 1000
37 4j -4-Cl 500 250 500 500 500 > 1000 > 1000
Ampiciline 100 100 250 100 - - -
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Grisiofulvin - - - - 500 100 100
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E. c. Escherichia coli, P. a. Pseudomonas aeruginosa, S. a. Staphylococcus aureus, S. p. Streptococcus pyogenes, C.a. Candida albicans, A. n. Aspergillus niger, A. c.
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Aspergillus clavatus
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mL. Compound 4 f (-4-OCH3) has shown good activity against
45 Determination of antibacterial and antifungal activities
the C. albicans at MIC 50 μg/mL. When the functional group
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Antimicrobial activity data is reported in (Table 1). Compound like 4 i (-N,N’-(CH3)2) was introduced the resulting compounds
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4 h (-4-OH-3-OCH3) showed very good activity against E.c at showed moderate activity against C. albicans at MIC 100 μg/
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MIC 25 μg/mL. Compound 4 c (-3-NO2) demonstrated moderate mL. Compound 4 g (-3,4,5-(OCH3)3 possessed good activity
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activity against E. coli at MIC 100 μg/mL. Amongst the against A. niger at MIC 62.5 μg/mL. Introduction of group like
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synthesized compounds, 4 b (-2-OH) was found to be the most (-4-OH-3-OCH3) in compound 4 h gave inhibition against A.
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potent as compared to standard drug at MIC 25 μg/mL against clavatus at MIC 25 μg/mL. Compound 4 f (-4-OCH3) and 4 g
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P. aeruginosa. Compound 4 d (-3-NO2) showed moderate (-3,4,5-(OCH3)3) exhibited moderate activity against A. clavatus
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activity against P. aeruginosa at MIC 100 μg/mL. Highest at MIC 100 μg/mL.
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inhibition is possessed by compound 4 b (-2-OH) against
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S. aureus at MIC 50 μg/mL. While the compound 4 a (-H)
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exhibited good activity against the S. pyogenes at MIC 62.5 μg/
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 Full Papers
3D QSAR of titled compounds considered as the largest out linear (they were, difference
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Alignment rule between actual value and predicted value is � 0.08).
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In 3D QSAR [Comparative Molecular Field Analysis (CoMFA) and
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Comparative Molecular Similarity Indices Analysis (CoMSIA)]
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studies, the 3D structures of the presented derivatives were
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aligned in the as shown in figure below (Figure no. 1). This
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alignment is calculated as per the suitable conformational
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model that would be assumed as a bioactive conformation.
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Because there is no information about ligand-receptor com-
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plexes, the lowest energy conformer of all derivatives was
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considered as a bioactive. The hypothesis for molecular
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recognition is that the most highly halogenated motif would
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be the preferred plane for asymmetrically substitutions.[18] The
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IUPAC numbering scheme is accepted in the original model[19]
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to simplify a rational and repeatable alignment technique
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Figure 1.
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33 Figure 2. Fig. (A) to (H) showed plots of observed vs. predicted activities of
34 modified model for both CoMFA and CoMSIA.
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Figure 1. Alignment of structures
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Obtained from CoMFA model and CoMSIA was 1.709 (4 b)
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and 1.680 (4 b) respectively. However, these models did not
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predict the log value of the biological activity accurately for
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In the present protocol, we have considered all models as compounds 4 i (CoMSIA) and hence, they were considered as
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best because it gave us information about all the possible the largest out linear.
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binding interactions. To extend the predictability for diverse Obtained from CoMFA model and CoMSIA was 2.067 (4 b)
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variety of chemical classes, we included all original training set and 2.013 (4 b) respectively. However, these models did not
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and prepared test set, which were randomly selected. We also predict the log value of the biological activity accurately for
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checked the significance of the model with the combined set compounds 4 c, 4 d, 4 g, 4 j (CoMFA) and 4 d, 4 f, 4 g, 4 h
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and obtain good results (Table 2). The results of CoMFA and (CoMSIA) and hence, they were considered as the largest out
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CoMSIA based 3D-QSAR analyses are presented in Table 3. linear.
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Obtained from CoMFA model and CoMSIA was 2.709 (4 a)
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and 2.666 (4 a) respectively. However, these models did not
50 Results and discussion on predictive log values of E. coli, P.
predict the log value of the biological activity accurately for
51 aeruginosa, S. aureus and S. pyogenes are as under.
compounds 4 j (CoMFA) and 4 f, 4 h (CoMSIA) and hence, they
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Obtained from CoMFA and CoMSIA models was 1.822 (4 h) and were considered as the largest out linear.
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1.826 (4 h) respectively. However, these models did not predict
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the log value of the biological activity accurately for com-
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pounds 4 g, 4 j (CoMFA) and 4 g (CoMSIA) and hence, were
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1 Table 2. significance of the CoMFA and CoMSIA models

2 Training set Test set Combined Model Training set Test set Combined
3 model
E. coli (CoMFA) E. coli (CoMSIA)
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No. of components (N) 5 3 5 5 3 5
5
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6 q 0.837 0.869 0.838 0.836 0.859 0.830
SEP 0.045 0.011 0.008 0.041 0.010 0.007
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r2 0.986 0.978 0.961 0.992 0.991 0.973
8 SEE 0.033 0.069 0.066 0.025 0.045 0.055
9 F 521.87 211.40 264.42 900.28 497.26 383.57
10 SD 0.292 0.047 0.063 0.275 0.041 0.053
11 P. aeruginosa (CoMFA) P. aeruginosa (CoMSIA)
12 No. of components (N) 5 3 5 5 3 5
13 q2
0.837 0.869 0.850 0.846 0.824 0.855
14 SEP 0.025 0.010 0.007 0.023 0.009 0.006
15 r2 0.983 0.978 0.972 0.993 0.985 0.980
16 SEE 0.049 0.069 0.060 0.030 0.043 0.050
F 407.97 211.40 368.85 1058.32 312.16 521.16
17 SD 0.162 0.043 0.056 0.150 0.039 0.048
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S. aureus (CoMFA) S. aureus (CoMSIA)
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No. of components (N) 5 3 5 5 3 5
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21 q2 0.704 0.841 0.723 0.714 0.802 0.721
SEP 0.008 0.006 0.013 0.009 0.007 0.013
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r2 0.976 0.982 0.946 0.982 0.988 0.955
23 SEE 0.072 0.043 0.095 0.061 0.034 0.087
24 F 287.70 259.39 190.02 400.73 397.68 229.21
25 SD 0.054 0.028 0.099 0.057 0.031 0.100
26 S. pyogenes (CoMFA) S. pyogenes (CoMSIA)
27 No. of components (N) 5 3 5 5 3 5
28 q2
0.838 0.850 0.850 0.846 0.868 0.857
29 SEP 0.006 0.005 0.007 0.005 0.004 0.006
30 r2 0.977 0.991 0.968 0.987 0.997 0.974
SEE 0.048 0.031 0.054 0.036 0.019 0.050
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F 300.28 507.26 328.15 544.52 1348.69 397.57
32 SD 0.038 0.020 0.052 0.033 0.017 0.047
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N-optimum number of components, q2-LOO (Leave one out) cross-validated correlation coefficient, r2-noncross-validated correlation coefficient, SEE-standard
34 error of estimation, F F-test value, SEP-standard error of prediction, SD-standard deviation.
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38 CoMFA and CoMSIA contour plots:
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For the properly aligned training sets, in their active conforma-
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tions, 3D QSAR model can be developed.[20–22] Consequently
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the contour map produced from these models provided some
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information on the molecular binding site of the receptor.[23]
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Here we used structurally diverse training set. As the bioactive
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conformation is unavailable; we considered the rational molec-
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ular geometries to obtain maximal superposition with respect
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to molecular similarity. The contour maps of this study
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proposed the steric effect, electrostatic effect, hydrogen-bond
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acceptor, hydrogen-bond donor and hydrophobic field require-
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ments for ligand binding.
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[CoMFA and CoMSIA contour maps depicted around 4 h Figure 3. 4 h CoMFA (E. coli)
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generated from CoMFA and CoMSIA models. For Figure 3,
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Figure 4, 6 and 7: Green and yellow regions showed steric
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fields, favorable or unfavorable respectively, while red and blue
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regions depict where electronegative or electropositive groups negatively charged groups increase the activity. For figure 5
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are favoured. Blue isopleths depict areas positively charged and 8: Cyan counters indicate areas, H-bond donor groups
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groups increase the activity and red isopleths indicate; favoured and purple counter indicates H-bond acceptor group
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1 Table 3. Predicted biological activity in μg/mL

2 Compound No. Actual Predicted Residual Actual Predicted Residual
3 CoMFA (E. coli) CoMSIA (E. coli)
4 4a 2.30 2.335 -0.035 2.30 2.267 0.033
5 4b 2.30 2.244 0.056 2.30 2.272 0.028
6 4c 2.40 2.395 0.005 2.40 2.373 0.027
4d 2.70 2.692 0.008 2.70 2.702 -0.002
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4e 2.40 2.379 0.021 2.40 2.383 0.017
8 4f 2.70 2.727 -0.027 2.70 2.671 0.029
9 4g 2.40 2.295 0.105 2.40 2.305 0.095
10 4h 1.80 1.822 -0.022 1.80 1.826 -0.026
4i 2.10 2.129 -0.029 2.10 2.107 -0.007
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4j 2.00 2.191 -0.191 2.00 2.035 -0.035
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13 CoMFA (P. aeruginosa) CoMSIA (P. aeruginosa)

14 4a 2.00 2.085 -0.085 2.00 1.999 0.001

15 4b 1.70 1.709 -0.009 1.70 1.680 0.020
16 4c 2.40 2.402 -0.002 2.40 2.395 0.005
4d 3.00 3.008 -0.008 3.00 3.040 -0.040
17 4e 3.00 2.987 0.013 3.00 2.996 0.004
18 4f 3.00 3.033 -0.033 3.00 3.037 -0.037
19 4g 2.10 2.079 0.021 2.10 2.092 0.008
20 4h 2.70 2.717 -0.017 2.70 2.665 0.035
4i 2.10 2.057 0.043 2.10 2.010 0.090
21 4j 2.40 2.379 0.021 2.40 2.335 0.065
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CoMFA (S. aureus) CoMSIA (S. aureus)
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24 4a 2.70 2.691 0.009 2.70 2.647 0.053
25 4b 2.00 2.067 -0.067 2.00 2.013 -0.013
4c 2.70 2.547 0.153 2.70 2.646 0.054
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4d 3.00 3.090 -0.090 3.00 3.103 -0.103
27 4e 3.00 2.989 0.011 3.00 2.971 0.029
28 4f 3.00 3.098 -0.098 3.00 3.351 -0.351
29 4g 1.80 1.981 -0.181 1.80 1.910 -0.110
4h 3.00 2.922 0.078 3.00 2.870 0.130
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4i 2.10 2.132 -0.032 2.10 2.154 -0.054
31 4j 2.70 2.474 0.226 2.70 2.653 0.047
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CoMFA (S. pyogenes) CoMSIA (S. pyogenes)
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34 4a 2.70 2.709 -0.009 2.70 2.666 0.034
4b 2.30 2.318 -0.018 2.30 2.302 -0.002
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4c 2.10 2.127 -0.027 2.10 2.055 0.045
36 4d 3.00 2.995 0.005 3.00 3.036 -0.036
37 4e 2.70 2.725 -0.025 2.70 2.720 -0.020
38 4f 2.70 2.642 0.058 2.70 2.615 0.085
4g 2.70 2.692 0.008 2.70 2.651 0.049
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4h 2.70 2.671 0.029 2.70 2.577 0.123
40 4i 2.70 2.714 -0.014 2.70 2.684 0.016
41 4j 2.70 2.510 0.190 2.70 2.644 0.056
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favoured. Magenta counter indicates electron acceptor group green contour is found near 5th position of pyridine, while
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more favoured and orange indicates less acceptor group. sterically disfavoured yellow contour near 4th position of
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Yellow and white contours indicate hydrophobic group pyridine ring. Thus, CoMFA and CoMSIA map emphasize that
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favoured and disfavoured respectively.] bulky groups near 5th, 6th, 7th and 8th positions of benzoxazine
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In figure 3 and 4, sterically favoured green regions are ring and 4th and 5th positions of pyridine are especially
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found near 5th, 6th, 7th and 8th positions of benzoxazine frame favorable for binding. So we can simply propose that any
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and at the same time small green isopleths near 4th position of substitution in lateral position evokes greater binding energy
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pyridine in CoMFA contour map. Blue contours near the 2nd relative to medial substitutions.
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and 4th positions of pyridine showed increase in positive charge In figure 3 and 4, positive charge favoured blue regions are
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and red contour near the 3rd position of pyridine indicates found respectively near 2nd and 3rd positions of pyridine. In
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decrease in positive charge leading to increase in biological general we can propose that positive charge is favoured on 2nd
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activities. Sterically disfavoured yellow contours are found near and 3rd positions of the pyridine ring. In CoMSIA, hydrophobic
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4th position of pyridine ring. In CoMSIA, sterically favoured contour maps showed the presence of one hydrophobic
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Figure 7. 4b CoMSIA (P. aeruginosa)
13 Figure 4. 4 h CoMSIA (E. coli)
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Figure 8. 4b CoMSIA (P. aeruginosa)
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positions of pyridine moiety represents the region where the
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hydrogen-bond donors are not expected on the receptor site
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and thus, a hydrogen-bond accepting substituent should be
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disfavoured.
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In figure 6 and 7 there are no sterically favoured green
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regions found near in CoMFA contour map. But, blue contour
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near 2nd position of pyridine showed increase in positive charge
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and red contour near 4th position of pyridine indicates decrease
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in positive charge leading to increase in biological activities.
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Figure 6. 4b CoMFA (P. aeruginosa) Sterically disfavoured yellow contours are also found near 2nd
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position of oxazine and 5th position of pyridine frames. In
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CoMSIA sterically favoured green contours are found near 3rd
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and 5th positions of pyridine, while sterically disfavoured yellow
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favoured big yellow isopleth near to 3rd position of pyridine. contours near 1st position of benzoxazine and 2nd position of
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Small isopleths indicates their contribution in the hydrophobic pyridine. Thus, CoMFA and CoMSIA map emphasize that bulky
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interaction with the receptor. Steric contour showed that lateral groups near 1st position of benzoxazine ring and 3rd and 5th
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substitution induces the activity. Thus it can assume that any positions of pyridine are especially favorable for binding. So we
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lateral hydrophobic substitution may increase the binding can simply propose that any substitution in lateral position
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activity. In CoMSIA, H-bond donor and H-bond acceptor evokes greater binding energy relative to medial substitutions.
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contour map shows a big purple contour far from 1st position In figure 6 and 7, positive charge favoured blue regions are
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of pyridine, where proton acceptors are not expected in the found near 2nd position for pyridine. So we can propose that
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receptor. So a hydrogen-bond accepting substituent should be positive charge is favoured on 2nd position of the pyridine ring.
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favoured. In the contour plot for the hydrogen-bond acceptor In CoMSIA, hydrophobic contour maps show the presence of
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properties, the area highlighted in orange near the 4th and 5th one hydrophobic favoured big yellow isopleth near to 3rd and
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Figure 9. Binding mode of 4 a into the active site of DNA gyrase subunit b (on right side: green lines signify pi-pi stacking interactions, red line signify the
18 cation-pi interactions while the pink lines represent the hydrogen bonding interactions)
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34 Figure 10. Binding mode of Chlorobiocin into the active site of DNA gyrase subunit b (on right side: the pink lines represent the hydrogen bonding
interactions)
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4th positions of pyridine. Steric contour shows that any lateral cellular processes in bacteria (prokaryotes) but its absence in
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substitution induces the activity. Thus it can assume that any the higher eukaryotes makes it an attractive target for
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lateral hydrophobic substitution may increase the binding antibacterial medicinal chemistry research.[24–26]
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activity. In CoMSIA, H-bond donor and H-bond acceptor The results of the docking simulations for the oxazine
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contour map showed some big purple contours far from 2rd, 3th clubbed pyridine scaffold (4 a-j) revealed that the compounds
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and 5th positions of pyridine, where proton acceptors are not could snugly lock into the active of DNA gyrase with
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expected in the receptor and thus a hydrogen-bond accepting significantly high binding affinity. All of them adopted a
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substituent should be favoured. In this contour plot, there are homologous binding orientation and were found to be
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no contours for the hydrogen-bond acceptor properties, so stabilized within the active site via formation of several bonded
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prediction is not possible for this property. and non-bonded interactions. To obtain an indepth insight into
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the interactions (steric and electrostatic interactions) of these
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molecules with the enzyme active site, a detailed per-residue
50 Molecular Docking
interaction analysis was performed which is discussed for one
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With the aim of rationalizing the significant antimicrobial of the derivative 4 a and is summarized in Table 4 for 4 b-4 j.
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activity demonstrated by the oxazine clubbed pyridine scaffold The highest scored docked pose of 4 a (Figure 9) show that it
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(4 a-j) and to gain an insight into the molecular basis of their binds in the active site of DNA gyrase with a higher binding
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interactions with the biological target, a molecular docking affinity through multiple interactions with the active site
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study was carried out against bacterial DNA gyrase subunit b residues (docking score: 8.621; Glide energy: 49.786Kcal/
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(pdb id:1KZN) which is a very essential enzyme for almost all mol).
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Table 4. Quantitative per-residue interaction analysis of the Molecular docking study on DNA gyrase subunit b for the oxazine clubbed pyridine scaffold (4a-j).

Code Docking Glide Per-Residues interactions

Score Interaction Van der Waals (kcal/mol) Coulombic (kcal/mol) H-bonds(Å) Pi Pi
Energy (kcal/ Stacking (Å)
mol)
4a -8.621 -49.786 Thr165(-2.202), Arg136(-1.096), Ala96(-2.406), Ile90(-1.513), Pro79(-2.404), Ile78(-3.87), Thr165(-1.697), Arg136(-1.882), Arg76 Thr165(2.18), Gly77(2.32), Arg76
Gly77(-1.251), Arg76(-3.77), Asp73(-1.669), Glu50(-5.19), Ala47(-1.871), Asn46(-2.23), (-1.857), Glu50(-1.483), Asn46(-4.001) Asn46(1.78) (2.247)/
Val43(-1.494) Arg76
(2.247)

ChemistrySelect 2019, 4, 7541 – 7550

4b -8.774 -48.115 Thr165(-2.227), Arg136(-1.119), Ala96(-2.345), Ile90(-1.802), Pro79(-2.865), Ile78 Thr165(-2.02), Arg136(-2.208), Glu50(-1.214), Thr165(2.20),Gly77(2.33), Arg76
(-4.562), Gly77(-1.32), Arg76(-4.86), Arg76(-2.166), Asp73(-1.4), Glu50(-4.947), Ala47 Asn46(-4.734) Asn46(1.78) (2.266)/
(-1.959), Asn46(-1.865), Val43(-1.534) Arg76
(2.266)
4c -7.121 -44.640 Thr165(-2.171), Arg136(-1.211), Ala96(-2.234), Ile90(-1.544), Pro79(-2.673), Ile78 Thr165(-2.094), Arg136(-2.56), Ala96(-2.118), Thr165(2.18), Ala96(2.19), Arg76
(-4.415), Gly77(-1.294), Arg76(-4.399), Asp73(-1.291), Glu50(-4.748), Ala47(-1.918), Arg76(-1.616), Glu50(-1.232), Asn46(-4.279) Gly77(2.32), Asn46(1.78) (2.247)/
Asn46(-1.838), Val43(-1.528) Arg76
(2.247)
4d -7.808 -45.622 Thr165(-2.32), Arg136(-1.211), Ala96(-2.064), Ile90(-2.777), Pro79(-2.668), Ile78(-4.221), Thr165(-1.834), Arg136(-1.247), Ala96 Thr165(2.26), Gly77(2.35), Arg76
Gly77(-1.329), Arg76(-4.974), Asp73(-1.48), Glu50(-5.155), Ala47(-1.962), Val43(-1.527) (-0.942), Arg76(-1.116), Glu50(-2.034), Asn46 Asn46(1.69) (2.13)/
(-4.729) Arg76 (2.13)
4e -7.737 -44.211 Thr165(-2.301), Arg136(-1.113), Ala96(-2.336), Ile90(-2.575), Pro79(-3.533), Ile78 Thr165(-2.048), Arg136(-1.326), Glu50 Thr165(2.22), Gly77(2.34), Arg76
(-4.593), Gly77(-1.459), Arg76(-4.883), Asp73(-1.488), Glu50(-4.719), Ala47(-1.897), (-2.172), 49(-1.676), Asn46(-3.241) Asn46(2.02) (2.442)/
Asn46(-3.478), Val43(-1.516) Arg76
(2.442)
4f -8.685 -49.501 Thr165(-2.136), 136(-1.186), Ala96(-1.004), Ile90(-2.471), Pro79(-3.765), Ile78(-4.615), Thr165(-2.023), Arg136(-1.69), Glu50(-1.714), Thr165(2.22), Gly77(2.33), Arg76
Gly77(-1.35), Arg76(-4.926), Asp73(-1.556), Glu50(-4.654), Ala47(-1.704), Asn46(-3.534), Asp49(-1.032), Asn46(-3.232) Asn46(2.06) (2.292)/

7548
Val43(-1.483) Arg76
(2.292)
4g -7.332 -42.853 Thr165(-2.297), Arg136(-1.118), Ala96(-1.21), Ile90(-1.667), Pro79(-2.3), Ile78(-3.346), Thr165(-1.76), Arg136(-2.541), Glu50(-2.989), Thr165(2.27), Gly77(2.35), Arg76
Gly77(-1.307), Arg76(-4.069), Asp73(-1.48), Glu50(-4.96), Asp49(-1.471), Ala47(-1.926), Asp49(-5.041), Asn46(-2.925), 45(-1.278) Asn46(2.00) (2.067)/
Asn46(-3.306), Val43(-1.531) Arg76
(2.067)
4h -8.168 -49.811 Thr165(-2.197), Arg136(-1.117), Ala96(-2.376), Ile90(-2.265), Pro79(-2.377), Ile78 Thr165(-2.285), Arg136(-1.597), Ala96 Thr165(2.13), Ala96(1.82), Arg76
(-4.406), Gly77(-1.269), Arg76(-3.543), Asp73(-1.165), Glu50(-4.35), Ala47(-2.024), (-2.218), Arg76(-2.227), Glu50(-1.011), Asp49 Gly77(2.32), Asn46(2.02) (2.432)/
Asn46(-3.619), Val43(-1.517) (-1.408), Asn46(-3.037) Arg76
(2.432)
4i -8.376 -48.023 Thr165(-2.009), Arg136(-1.108), Ala96(-2.223), Ile90(-2.067), Pro79(-3.06), Ile78(-4.464), Thr165(-2.079), Arg136(-2.104), Arg76 Thr165(2.19), Gly77(2.33), Arg76
Gly77(-1.294), Arg76(-4.221), Asp73(-1.688), Glu50(-4.7), Ala47(-1.746), Asn46(-3.066), (-1.341), Glu50(-1.56), Asp49(-1.325), Asn46 Asn46(1.94) (2.278)/
Val43(-1.357) (-3.714) Arg76
(2.278)
4j -7.721 -44.429 Thr165(-2.468), Arg136(-1.111), Ala96(-2.214), Ile90(-2.642), Pro79(-4.365), Ile78 Thr165(-1.741), Arg136(-2.32), Glu50(-2.114), Thr165(2.38), Gly77(2.39), Arg76
(-4.661), Gly77(-1.972), Arg76(-4.154), Asp73(-1.74), Glu50(-4.501), Ala47(-1.609), Asp49(-1.359), Asn46(-2.919) Asn46(2.11) (2.241)
Asn46(-3.477), Val43(-1.465)
Full Papers

© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

 Full Papers

1 Table 5. Data for the reference standard Chlorobiocin for DNA gyrase
2 Code Antibacterial Docking Glide Per-Residues interactions
3 activity (MIC Score Interaction
μg/ml) Energy
4
(kcal/mol) Van der Waals (kcal/mol) Coulombic (kcal/mol) H-bonds(Å)
5
6 Chlorobiocin - - - -9.902 -50.733 Val167(-2.896), Met166(-1.934), Thr165(-3.788), Lys139(-1.502), Arg136 Arg136(2.69),
Arg136(-1.951), Val120(-1.189), Ile90(-2.992), (-3.801), Asp74(-1.836), Arg136(2.11)
7
Pro79(-3.775), Ile78(-6.093), Gly77(-1.689), Asp73(-1.779), Asn46 Asn46(1.95),
8 Arg76(-3.38), Asp73(-2.924), Gln72(-1.898), (-4.776), Asp45(-2.008), Asn46(2.01),
9 Val71(-2.239), Glu50(-3.071), Asp49(-1.592), Asp73(2.19)
10 Ala47(-2.525), Asn46(-4.711), Val43(-2.847),
11
12
13
The Glide energy signifies how much the ligand is buried in revealed that the results given by electro-withdrawing groups
14
the cavity of the enzyme. That indicates the energy required showed antibacterial activity while electro-donating groups
15
for the formation of complex between the ligand and the displayed antifungal activity. Out of the ten derivatives, 4 a (-H),
16
enzyme. The per-residual interactions between the ligand and 4 b (-2-OH), 4 f (-4-OCH3), 4 g (-3,4,5-(OCH3)3) and 4 h (-4-OH-3-
17
the active site residues showed that the electrostatic inter- OCH3) exhibited good antimicrobial activity. 3D-QSAR data
18
actions are less prevalent over steric in its affinity towards DNA provides the idea for the derivatisation and changes to the
19
gyrase which is in harmony with the contour plots obtained for structure which improves activity results. Models of all CoMFA
20
3D-QSAR analysis. and CoMSIA gave more than 0.70 of q2 value and more than
21
It is observed that the most significant contribution to the 0.80 of r2 value. Furthermore molecular docking study could
22
binding of 4 a in the active site of DNA gyrase is the extensive help to establish a valuable link between the observed
23
network of favorable van der Waals (steric) interactions with antimicrobial activity and the binding affinity towards the DNA
24
Val43(-1.494 kcal/mol), Asn46(-2.23 kcal/mol), Ala47(-1.871 kcal/ gyrase enzyme. The insights obtained from the per-residue
25
mol), Glu50(-5.19 kcal/mol), Asp73(-1.669 kcal/mol), Arg76 interaction analysis in regards to the thermodynamic inter-
26
(-3.77 kcal/mol), Gly77(-1.251 kcal/mol), Ile78(-3.87 kcal/mol), actions (steric and electrostatic) were found to be in agreement
27
Pro79(-2.404 kcal/mol), Ile90(-1.513 kcal/mol), Ala96(-2.406 kcal/ with the contour plots of 3D-QSAR analysis. Overall, the
28
mol), Arg136(-1.096 kcal/mol) and Thr165(-2.202 kcal/mol) resi- docking simulations and the per-residue interaction analysis
29
dues in the active site of enzyme. The enhanced binding suggest that this scaffold has promising affinity towards the
30
affinity of 4 a towards DNA gyrase is also attributed to relatively bacterial DNA gyrase enzyme.
31
fewer but significant electrostatic (coulombic) interactions
32
observed Asn46(-4.001 kcal/mol), Glu50(-1.483 kcal/mol), Arg76
33 Supporting Information summary paragraph
(-1.857 kcal/mol), Arg136(-1.882 kcal/mol) and Thr165
34
(-1.697 kcal/mol) residues in the active site. The mechanical Supplementary data is equipped with materials and methods,
35
interlocking of 4 a was observed to be further enhanced by experimental procedures, spectroscopic data for characteriza-
36
three hydrogen bonds and one each pi-pi (π- π) and pi-cation tion, general information about CoMFA and CoMSIA 3D QSAR
37
stacking interactions. The three hydrogen bonding interactions models and details of Molecular Docking. Biological assays
38
were observed between Thr165, Gly77, Asn46 and nitrile were presented in the supplementary information.
39
functional groups with bonding distances of 2.18 Å, 2.32 Å and
40
1.78 Å respectively. Furthermore very prominent pi-pi stacking
41 Acknowledgement:
interaction was observed between Arg76 and the phenyl ring
42
in 4 a (2.247 Å). The same phenyl ring was also engaged in a Authors are grateful to the UGC, New Delhi and Department of
43
cation-pi interaction with Arg76 (2.247 Å). Such hydrogen Science and Technology, New Delhi (DST-FIST-SR/FST/CSI-212/
44
bonding and pi-pi/ pi- stacking interactions anchor the ligand 2010) for financial support under the NON-SAP and DST-FIST
45
into the active site to facilitate the steric and electrostatic programs respectively. Mr. Krunalsinh A Jadeja is thankful to
46
interactions. A similar per-residue interaction energy distribu- [UGC-MRP, F. No. 43-164/2014(SR)], University Grants Commis-
47
tion was observed for other oxazine clubbed pyridine deriva- sion, New Delhi for Project Fellowship. Authors thank to
48
tives (4 b-4 j) as well qualifying them as potential leads for Schrodinger Inc. for providing the Schrodinger molecular model-
49
structure-based lead optimization as DNA gyrase inhibitor. ling software (demo license) for performing the computational
50
studies.
51
52 Conclusion
53 Conflict of Interest
Synthesis of the 1-((1-(4-(2H-benzo[e][1,3]oxazin-3(4H)-yl)
54
phenyl)ethylidene)amino)-6-((arylidene)amino)-4-(4-chloro- The authors declare no conflict of interest.
55
phenyl)-2-oxo-1,2-dihydropyridine-3,5-dicarbonitriles was
56
achieved by the effective procedure. Antimicrobial activity data
57

ChemistrySelect 2019, 4, 7541 – 7550 7549 © 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
 Full Papers
Keywords: 3D-QSAR study · CoMFA · CoMSIA · Oxazine · [12] N. C. Desai, N. R. Shihory, G. M. Kotadiya, Chin. Chem. Lett. 2014, 25, 305–
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ChemistrySelect 2019, 4, 7541 – 7550 7550 © 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim